Research and Hospital Divisions - NKI / AvL

Annual Report 1994 

Illustrations and unpublished data in these reports 

should not be used without permission ofthe author. 

Copyright ©: 

The N etherlands Cancer Institute 

Antoni van Leeuwenhoek Huis 

PIesmanlaan 121 

1066 CX Amsterdam 

The N etherlands 

Phone 31.20.5129111 

Fax 31.20.6172625 

ISSN 0376 - 7345

Contents 

Board Members 6 

Research and Hospital Divisions 8 

Introduction 13 

I Cell Biology 19 

11 Molecular Carcinogenesis 27 

111 Cellular Biochemistry 33 

IV Immunology 43 

V Molecular Biology 53 

VI Tumor Biology 63 

VII Molecular Genetics 71 

VIII Experimental Therapy 81 

IX Radiotherapy 91 

X MedicalOncology 101 

XI Surgical Oncology 113 

XII Psychosocial Research and Epidemiology 125 

Biometrics Department 131 

Biophysics Department 137 

Laboratory Animal Department 141 

Cancer Hospital 143 

Clinical W orking Parties 149 

Ongoing Trials 155 

Education in Oncology 177 

Projects 187 

Publications 195 

Author Index 227 

Personnel-Project Index 239

6 

Board Members 

International Scientific Advisory Board 

Jon J van Rood, Professor ofImmuno-Hematology, Leiden, president 

Joseph R Bertino, American Cancer Society Professor of Medicine and Pharmacology, New Haven, USA 

George Klein, Professor of Tumor Biology, Stockholm, Sweden 

Hilary Koprowski, Former Director of the Wistar Institute, Philadelphia, USA 

Susumu Tonegawa, Professor of Biology, MIT, Center of Cancer Research, Cambridge, Mass, USA 

I Bernard Weinstein, Professor of Medicine and Environmental Sciences, New Vork, USA 

Charles Weissmann, Professor of Molecular Biology, Zürich, Switzerland 

N ational Scientific Advisory board 

LA Aarden, Professor of Molecular Immunology (from February 1994) 

D Bootsma, Professor of Cell Biology and Genetics, Rotterdam 

AJ van der Eb, Professor of Fundamental Tumor Virology, Leiden 

CAM Haanen, Professor ofInternal Medicine, Nijmegen (till November 1994) 

SWJ Lamberts, Professor of Internal Medicine, Rotterdam 

HL Langevoort, Professor of Histology and Embryology, Amsterdam 

B Löwenberg, Professor of Hematology, Rotterdam (from February 1994) 

CJLM Meijer, Professor ofPathological Anatomy, Amsterdam 

CJM Melief, Professor ofImmunohematology, Leiden (from February 1994) 

J Oldhoff, Professor of General Surgery, Groningen 

HM Pinedo, Professor of Clinical Oncology, Amsterdam (from February 1994) 

11 van Rood, Professor ofImmuno-Hematology, Leiden 

E van der Schueren, Professor of Oncology, Leuven 

GNJ Tytgat, Professor of Gastro-enterology, Amsterdam 

PC van der Vliet, Professor of Physiological Chemistry, Utrecht (from February 1994)

Board of Directors 

P Borst, chairrnan and director of research 

L Neeleman, director organization and management 

A Roest, financial director 

clinical director, vacancy 

Laboratory Research Coordinator 

E Roos 

Board of Governors 

JD Hooglandt, president 

ML Frohn-de Winter, vice-president 

P den Tex, secretary 

o Hattink, treasurer 

AC van den Blink 

R Hazelhoff 

P van der Heyden (from 27.06.1994) 

PH Hugenholtz (till 27.06.1994) 

S van der Kooij 

J van der Meer 

JHM Temmink 

GNJ Tytgat 

MWM Vos-Van Gortel 

Ladies Committee 

MC Sickinge-van Eeghen, president 

YJ Engelsman-Prins, secretary 

D Jurgens-Kupsch, treasurer 

7

8 

Research and Hospital Divisions 

Research Divisions 

I Cel/ biology 

E Roos (head) 

J Calafat 

JG Collard 

CA FeItkamp 

A Sonnenberg 

Il Molecular carcinogenesis 

R Bernards (he ad) 

L den Engelse 

E Kriek (honorary staff member) 

E Scherer 

T Sixma (15.08.1994) 

J Westra (deceased 26.03.1994) 

III Cel/ular biochemistry 

WH MooIenaar (head) 

WJ van Blitterswijk 

J Borst 

A Tulp 

LN Vernie 

IV Immunology 

AM Kruisbeek (head) 

WR Gerritsen 

A Hekman 

EM Rankin 

H Spits 

CJM Vennegoor (stationed at the Free University) 

FA Vyth -Dreese 

K Weijer 

V Molecular biology 

RHA Plasterk (he ad) 

P Borst 

APM Jongsma 

HV Westerhoff (till November 1994) 

VI Tumor biology 

WJ Mooi (head) 

JH Daams 

AA van der Gugten 

PhC Hageman 

J Hilkens 

D Ivanyi 

RJAM Michalides 

M Sluyser 

AA Verstraeten 

VII Molecular Genetics 

AJM Berns (he ad) 

P Demant 

M Snoek 

MA van der Valk 

VIII Experimental therapy 

AC Begg (head) 

H Bartelink 

WJNooijen 

S Rodenhuis 

JH Schornagel 

LA Smets 

FA Stewart 

IX Radiotherapy 

JV Lebesque (head) 

H Bartelink 

RW de Boer 

J Borger 

IAD Bruinvis 

BNFM van Bunningen 

JMV Burgers 

EMFDamen 

LGH Dewit 

AAM Hart 

M van Herk 

RB Keus 

EAH Masselink 

BJ Mijnheer 

LMFMoonen 

SH Muller 

VJ de Ru 

NS RusselI 

CCE Schaake-Koning 

FW Wittkämper 

X Medical oncology 

R Somers (head till 01.09.1994) 

S Rodenhuis (head from 01.09.1994) 

JW Baars

PBaas 

JH Beijnen 

WW ten Bokkei Huinink 

JMG Bonfrer 

W Boogerd 

HBoot 

PF Bruning 

ME Craanen 

CC Delprat 

WR Gerritsen 

CA Hoefnagel 

SP Israëls 

C Mattern 

WJ Nooijen 

EM Rankin 

11 van der Sande 

JH Schornagel 

BGTaal 

o van Tellingen 

RA Valdés Olm os 

APE Vielvoye-Kerkmeer 

N van Zandwijk 

Research nurses 

D Batchelor 

AC Dubbelman 

ME Schot 

XI Surgical oncology 

JA van Dongen (head) 

EJ Aartsen (till 01.12.1994) 

AJM Balm 

F van Coevorden 

E Gortzak 

RT Gregor 

FJM Hilgers 

ThJM Helmerhorst 

S HorenbIas 

BBR Kroon 

W Meinhardt 

OE Nieweg 

EJTh Rutgers 

FAN Zoetmulder 

XII Psychosocial research and epidemiology 

NK Aaronson (head) 

FSAM van Dam 

FE van Leeuwen 

Biometrics department 

OB Dalesio (head) 

H van Tinteren 

JL te Velde 

B Schaefer (from 01.11.1994) 

Biophysics department 

GJF Blomrnestijn (head) 

LCJM Oomen 

HJ Stoffers 

Laboratory animal departments 

RGM ten Berg (head) 

Hospital Divisions 

Anesthesia 

M Hellendoorn-Smit (head) 

C Blackburn 

MKaag 

HVis 

Clinical chemistry and hematology 

WJ Nooijen (head) 

JMG Bonfrer 

Clinical research manager 

E Vos 

Gastro-enterology 

H Boot 

ME Craanen 

BGTaal 

Gynaecology 

ThJM Helmerhorst (head) 

EJ Aartsen (till 01.12.1994) 

P Kenemans 

Hospital pastors 

PG Kousemaker 

JE Ringrose-Wegman 

AH Tönis 

Internal medicine 

R Somers (head till 01.09.1994) 

S Rodenhuis (head from 01.09.1994) 

JW Baars 

EM Bais 

WW ten Bokkei Huinink 

PF Bruning 

CC Delprat 

WR Gerri tsen 

SP Israëls 

EM Rankin 

JH Schornagel 

Neurology 

W Boogerd 

11 van der Sande 

9

10 

Nuc/ear medieine 

CA Hoefnagel (head) 

SH Muller 

RA Valdés Olmos 

Nursing 

HIM Camerik (acting head) 

Otolaryngology - Head & Neek Surgery 

FJM Hilgers (head) 

AJM Balm 

R Fontijn 

S Gonggrijp 

RT Gregor 

APTimmers 

Pain Physieians 

C Mattern 

APE Vielvoye-Kerkmeer 

Pathology 

WJ Mooi (head) 

MPW Gallee 

P van Heerde 

D de Jong 

BM Loftus-Coll 

JL Peterse 

L van 't Veer 

Patient eounsellor 

M Keessen-de Vries 

Psych ia try 

LM Gualthérie van WeezeI 

Psyehosocial service 

DEEHahn 

Pulmonology 

P Baas 


Radiotherapy 

H Bartelink (head) 

RW de Boer 

JH Borger 

IAD Bruinvis 

BNFM van Bunningen 

JMV Burgers 

EMFDamen 

LGHDewit 

AAM Hart 

M van Herk 

RB Keus 

JV Lebesque 

EAH Masselink 

BJ Mijnheer 

LMF Moonen 

SH Muller 

VJ de Ru 

NS RusselI 

CCE Schaake-Koning 

FW Wittkämper 

Diagnostie radiology 

R Kröger (head) 

AP Besnard 

W Koops 

F de Leeuw 

Surgery 

JA van Dongen (head till 01.04.1994) 

BBR Kroon (head from 01.04.1994) 

F van Coevorden 

E Gortzak 

OE Nieweg 

EJTh Rutgers 

FAN Zoetrnulder 

Urology 

S HorenbIas 

W Meinhardt 

Consultant Staff 

Baeteriology 

WC van Dijk 

WPauw 

Dermatology 

H Neering 

General practitioner 

CC Delprat 

M axillo-facial surgery 

FJM Kroon 

Ophthalmology 

L Koornneeff 

Orthopedie surgery 

JW van der Eijken 

MW Fidler

Pediatrics 

PA Voûte 

Pharmacy 

JH Beijnen 

ACA Paal man 

Plastic surgery 

JB de Boer 

KBos 

Rehabilitation 

ELD Angenot 

Heads of General Services 

Audiovisual service 

JM Lomecky 

Central cancer library 

MBA Wilhelm-de Gouw 

Custodian research laboratory 

CA FeItkamp 

Financial administration 

JK Koppenol 

Housekeeping services 

RMD Schellens 

Medical administration 

JH Helversteyn 

Out-patient clinic 

R Pet, manager 

Personnel department 

JR de Bruijn 

Safety, health and welfare 

P de Lange 

Technical service 

TCM Wilmering 

II

Introduction 

The year 1994 was an important one for clinical 

research and the advanced patient care that provides the 

basis for clinical research. Our detailed plans for a new 

hospita1 building adjacent to the present buildings were 

submitted for approval to the government. To alleviate 

the most pressing space problems, we built an impressive 

interim outpatient clinic th at was nearly completed by the 

end of 1994. The reorganization of clinical departments 

into four main clusters was largely implemented in 1994 

and provides a more effective structure to direct clinical 

research. Notwithstanding the chilly Dutch research 

climate our research blossomed, clinical and basic, and 

the position of the Netherlands Cancer Institute as a 

national center of excellence was strengthened. 

Research highlights 

The list of research highlights that follows is a limited 

selection of the most significant discoveries of 1994 

suitable for presentation in simplified form. More 

complete and balanced overviews of all highlights of our 

research efforts can be found in the introductions of each 

of the 12 research divisions. 

The metastasis gene tiam-1 , discovered by 1 Collard 

and colleagues (Division I), became front-page news 

wh en a publication in Cell was picked up by the lay press 

in the Netherlands. More recent work is clarifying how 

abnormal expression of the tiam-1 results in invasion and 

metastasis of lymphomas. The Tiam-1 protein was found 

to activate a novel pathway for signal transduction 

between cell surface and the actin cytoskeleton, the Rac 

signaling pathway. 

E Roos and co-workers (Division I) used gene 

disruption in cultured cells to study the role of another gene 

in metastasis, the CD44 gene. In contrast to conclusions of 

other investigators they showed that CD44 did not appear 

to be essential for metastasis of a lymphoma. A 

Sonnenberg and co-workers (Division I) made important 

progress in the elucidation of the structure of the hernidesmosomes, 

cell surface organelles responsible for the 

proper attachment of epithelial cells to basement 

membranes. Loss of normal hemi-desmosomes is found in 

many carcinomas and is an important step in the transition 

of tissues fr om a benign into a malignant state. 

The group of Bernards (Division II) made progress in 

the study of the growth inhibitory function of the 

retinoblastoma protein-related pi 07. Two cellular targets 

of p107 were identified: the c-MYC oncoprotein and a 

newly isolated member of the E2F family of transcription 

factors, E2F -4. Bernards et al we re able to show that, like 

c-MYC, E2F-4 can stimulate cell cycle entry and has 

oncogenic activity. Interestingly, plO7 and the 

retinoblastoma protein are phosphorylated by different 

cyclin /cyclin dependent kinase complexes. These data 

demonstrate important functional differences between 

the various members of the retinoblastoma gene family. 

A Tulp, 1 Pieters and JJ Neefjes and their associates 

(Division III and IV) discovered a new intracellular 

compartment in skin cancer ceUs, using a sophisticated 

electrophoresis technique newly developed by Tulp. In 

this new compartment peptides are loaded onto class II 

MHC molecules, that present these peptides at the cell 

surface to the immune defense system. 

WH Mooienaar and associates (Division III) 

continued their analysis of lysophosphatidic acid (LPA), 

a new inter-cellular signal molecule, discovered by 

Mooienaar. In 1994 they showed that this rnitogenic 

phospholipid rapidly activates a chloride channel in 

quiescent fibroblasts and this may represent a new early 

signal in the proliferative response of fibroblasts. 

In a joint effort of the groups of P Borst (Division V) 

and AlM Berns (Division VII), a knock-out mouse was 

generated unable to make either of the two murine drugtransporting 

P-glycoproteins. These mice are healthy and 

fertile, showing that these P-glycoproteins are not 

required for normal development and health. These rnice 

are similar to patients with tumors resistant to natural 

product drugs in which the function of the MDRI 

P-glycoprotein is being blocked by inhibitors and they 

should be very useful for the study of alterations in drug 

metabolism that can be expected in these patients. 

RHA Plasterk and co-workers (Division V) continued 

their studies of the nematode C elegans, a simple model 

organism suitable for studies on the function of genes 

important in cancer research. In 1994 they showed that C 

elegans also uses P-glycoproteins to keep out drugs and 

that mutants in which the gene for one of these 

P-glycoproteins was disrupted became hypersensitive to 

chloroquine and co1chicine. This confirms the notion that 

P-glycoproteins protect organisms against toxins 

produced by plants and micro-organisms in their 

environment. 

RlAM Michalides (Division VI) and AlM Balm 

(Division XI) showed that overexpression of the cyclin 

DI gene is an unfavorable prognostic sign in he ad and 

neck cancer. 

The group of 1 Hilkens (Division VI) discovered that in 

an experimental model of pulmonary metastasis, 

expres sion of cell-surface glycoprotein episialin results in 

a greatly increased metastatic potential. In cancer cells, 

the very long and highly glycosylated extracellular 

13

AvL prizewinners: see honors 

T Sixma 

M Smeets 

Major discoveries are rare, however, and often it takes 

years to recognize that an interesting result is actually a 

major discovery. In the meantime, the public needs some 

indication that research money is being weil spent and 

research directors need objective parameters to measure 

achievement and distribute money accordingly. Two 

parameters that have found widespread acceptance are 

the citation frequency and impact of research 

publications. The impact measures the quality of the 

joumal in which an investigator publishes. High-quality 

joumals have a high impact. The citations measure the 

number of times an article is cited by other investigators. 

If an article is never cited, it is unlikely to contain a 

seminal discovery. The citation score of an article is like 

some wines: it gets better with age. Citations over a 30year 

period are obviously more informative than citations 

only in the year following publication. For practical 

purposes, however, only short-term citations can be used 

for judging active (rather than retired) scientists. 

Since 1982 we have recorded the short-term citations 

and impact of scientific articles published by the NKII 

AvL research staff. Table I presents the results of this 

analysis. The trend is up, confirming other indications 

L Boersma 

MFBG Gebbink 

Introduction 15 

that the NKII AvL is on the right track and Jives up to its 

reputation as a center of excellence. 

In 1991 an extern al site visit indicated that the Division 

of Chemical Carcinogenesis was losing some of its 

prominence and that an increased input of molecular 

biological know-how was required to bring this division 

back to the cutting edge of research in molecular 

carcinogenesis. This advice led to the strengthening of the 

division with molecular biologist René Bernards and A vL 

fellows Hein te Riele and Titia Sixma. Under a new name 

(Division of Molecular Carcinogenesis) and leadership, 

the division has rapidly regained its old momenturn. This 

is exemplified by the results of the last round of 

competitive grant applications to the Netherlands Cancer 

Foundation (KWF). Out of more than 200 applications, 

only two we re judged to be top quality (Al) and ten subtop 

(A2). Both Al and four A2 qualifications were given 

to NKII AvL projects. Both Al projects were entirely 

(Bernards) or partly (Van Lohuizen-Berns) from the 

Division of Molecular Carcinogenesis, plus two ofthe A2 

projects (Te Riele). This proves that the division is back 

on the [ast track and that the NKII A vL is able to 

revitalize its research, where necessary.

16 Introduction 

The Oncology Graduate School 

Amsterdam, European post-does and 

new NKI Professors 

Although the NKl / AvL has no formal teaching 

obligations, education is an essential part of the activities 

of the institute as detailed elsewhere in this report. In 

1993, 75 graduate students were doing research towards 

their PhD in the institute. Since 1989, the in-house 

teaching of these students has been formalized, initially in 

the NKl Graduate School, the first fully operational 

Graduate School in the Netherlands, and since 1991 in the 

Oncology Graduate School Amsterdam in which we 

collaborate with our colleagues of the University of 

Amsterdam and the Free University, Amsterdam. In 

1993, this Graduate School was formally recognized by 

the Royal Academy of the Arts and Sciences of the 

Netherlands and its activities were stimulated by a f1 1 

million grant fr om the Netherlands Organization for 

Scientific Research (NWO). We we re also recognized by 

the European Community as an institute for training 

post-docs from other European countries in the 

framework ofthe Human Capital and Mobility program. 

The NKl / AvL cannot award degrees, but many NKl / 

AvL staffmembers hold special part- time chairs in one of 

the Dutch universities, taking about 10% of their time. In 

1994, R Bernards was appointed part-time Professor of 

Molecular Carcinogenesis, JH Beijnen part-time 

Professor ofPharmacy, both at the University of Utrecht. 

HV Westerhoff became the part-time Professor of 

Mathematical Biochemistry at the University of 

Amsterdam. 

Some new developments in research 

New developments are taking place in every research 

division, but some of these deserve special mention. 

1) We set up a unit for structural analysis of proteins, 

headed by a new AvL fellow, Titia Sixma. The 

purchase of the expensive X-ray diffraction equipment 

for this unit was made possible by a large gift fr om the 

Table 2 

NKI research budgets, 1984 - 1994 (xl0 6 HFL) 

1984 1985 1986 

1) Annual program grants 

a) Department of health: 

- structural 15.4 16.3 15.0 

b) Dutch Cancer Society (KWF) 

- structural 5.2 4.8 4.3 

- incidental 0.2 

2) Large equipment and 

special facilities 1.5 2.0 1.8 

3) Project grants 6.7 7.7 8.9 

4) Yield bond issue 

5) Deficit 1.7 

* Estimate 

Nefkens Foundation and we are grateful to the 

Foundation for their generous and timely support. 

2) We started a sophisticated gene therapy trial in close 

collaboration with Somatix Therapy Corporation, 

California, USA. This phase-l trial was set up to test 

the side-effects of gene therapy with tumor cells taken 

from patients, modified by gene transfer to produce a 

growth factor, GM-CSF, and returned to the patients 

after irradiation to block their potential for 

multiplication. GM-CSF secretion makes the tumor 

cells more susceptible to attack by the host immune 

system. In mouse model systems, such modified cells 

lead to a massive response of the host T -cell defense 

and these activated T cells are also able to attack the 

original tumor cells, not producing GM-CSF. The 

re su lts of this trial can be expected in 1995. Gene 

therapy requires specialized facilities for culturing and 

handling patients cells under sterile conditions and we 

have optimized these facilities in 1994. These complex 

new therapeutic modalities require close collaboration 

between clinicians and basic scientists in immunology 

and molecular genetics. As an integrated cancer 

institute the NKV AvL provides a good environment 

for such sophisticated new treatments and we shall 

continue to give high priority to this interface between 

hospital and lab in the coming years. 

Research budgets 

Like most governments, the Dutch government 

struggles to balance the budget. Government spending 

rose by 47% between 1983 and 1992. This was clearly not 

caused by lavish support for cancer research, as our 

government co re grant rose from f115.9 to f116. 7 million 

between 1983 and 1992, a mere 5% (Tab Ie 2). Note th at 

these figures are not corrected for inflation. The 

purchasing power ofthis grant has fallen to less than 70% 

of its original value in these 10 years. We have only 

survived by rigorous cost-cutting and, in recent years, by 

the increasing support from our national cancer charity, 

the Dutch Cancer Society (KWF). 

In July 1992, the Secretary of Health announced his 

1987 1988 1989 1990 1991 1992 1993 1994* 

14.2 15.3 15.6 16.0 16.6 16.7 17.2 17.4 

4.1 4.1 4.1 4.2 6.0 7.6 9.1 11.1 

1.4 

2.2 2.9 2.3 3.5 3.5 3.5 3.5 4.0 

10.0 10.6 11.4 14.1 13.8 15.1 17.9 18.0 

0.9 1.7 1.7 1.7 1.7 1.7 1.7 

2.5 0.4

intention to cut the government core grant to the NKII 

AvL by 8%. In December 1992, however, we convinced 

the members of parliament that the cut should not be 

carried through. In July 1993, the Secretary of Health 

informed us ofhis plan to cut the NKII AvLcore grant by 

5% in 1994, and by an additionall0% in 1995. This would 

be devastating, as the core grants from the government 

and the Dutch Cancer Society pay for our staff salaries, 

our research costs and infrastructure. We also attract a 

large number of competitive project grants, as illustrated 

in Table 2, but these grants contain no overhead 

component. All overheads must be paid from co re grants. 

In December 1993 the members of parliament 

unanimously voted to overrule the 5% cut in our core 

grant. In July 1994, ho wever, the Department of Health 

announceda 15%cutinourcoregrantfor 1995. Weagain 

contacted parliament, but fortunately the new Health 

Minister completely retracted the cut before the matter 

was debated in parliament. 

Changes in personnel 

In March we were shocked by the sudden death of 

Gerard Westra, staff member of the Division of 

Molecular Carcinogenesis. Gerard was one of the most 

experienced staff members of our institute and an expert 

in chemical carcinogenesis. He worked in our institute for 

nearly 25 years and was known for his meticulous 

chemical studies of DNA adducts and their role in 

causing cancer. He was also known as a loyal friend with a 

keen eye for social injustice. He was the secretary of the 

staff assembly of the institute and his warm personality 

will be missed by his many friends in the lab and in the 

hospital. In November we also sadly lost Marieke Broug. 

For more than 12 years she worked as datamanager for 

the Department of Biometrics. 

Several staff members retired or left the institute to 

continue their care er elsewhere. Hennie Daams, staff 

member of the Division of Biology and one of the more 

artistic mavericks ofthe institute, retired early in 1994. He 

will continue some of his morphological studies on the 

development of breast cancer as an honorary staff 

member. Paul Co hen, staff radiologist, retired after a long 

and distinguished career in the institute. A wise and gentle 

man, he was a source of advice for many. He served for 

years on the governing board of the medical staff and he 

helped us out as interim head of the Radiology 

Department in the last phase of his career. 

Early in 1994 Carl Figdor, staff member of the Division 

of Immunology, left the institute to become the head of a 

new immunology group at the University of Nijmegen. 

Carl joined the institute in 1979 as a graduate student, 

working with Willie Bont on improved separation 

methods for blood components. Carl was trained as a 

biophysicist and he had a knack for developing 

instruments that were useful in biology. While his 

ingenuously designed machines produced purer and 

purer blood components, Carl became more and more 

interested in the ceUs he purified. Under the tutelage of 

Jan de Vries and Cees Melief he developed his 

immunological skiUs and when he left the institute after 

15 years, he had become an authority on monocytes, 

lntroduction 17 

leukocyte adhesion molecules and melanoma antigens. 

Enterprising, but gentle, he helped to shape tumor 

immunology and biological instrumentation in our 

institute. 

In October Hans Westerhoff left the institute to take 

over the chair of Microbial Physiology in the Free 

University in Amsterdam. Although Hans only spent 7 

years in the institute, he certainly left his mark, and a 

sizable group of students and post docs left with him for 

the Free University. A world expert on the application of 

irreversible thermodynamics to biological problems, 

Hans was a master modeler and provided advice to many 

in and outside the institute on the handling of quantitative 

biological data. His drive and enthusiasm will be missed. 

Evert Aartsen retired af ter a long and productive 

clinical period of28 years at the institute. A symposium in 

December 1994 and a liber amicorum honored his 

professional contributions and illustrated his popularity 

as friend and colleague. Dr Eric Tasseron left at the same 

time after 20 years as a voluntary part-time gynecologist. 

Two new AvL fellowsjoined the institute in 1994: Titia 

Sixma, a protein crystallographer, trained by Wim Hol in 

Groningen and Paul Sigler in Yale; and Hein te Riele, a 

molecular genetici st specialized in DNA recombination 

and tumor suppressor genes, trained in Groningen, the 

Pasteur Institute and in our Division of Molecular 

Genetics (Berns). Both fellows are stationed in the 

Division of Molecular Carcinogenesis. The AvL 

fellowship is a 5-year research career award for 

exceptionally gifted young scientists. It offers an 

independence rarely available to junior scientists in the 

N etherlands. 

Five new radiotherapists joined the institute in 1994, 

Th Schnabel, JSA Beiderbos, VJ de Ru, JG Salverda and 

BMP Aleman. OE Nieweg was appointed as staff 

surgeon, and W Meinhardt as part-time urologist. 

N ational and international activities 

In addition to their research and clinical activities, staff 

members of the Netherlands Cancer Institute fulfilled a 

large number of functions in internationalorganizations 

such as AACR, CIBA, EACR, EBCTCG, EMBO, 

EORTC, ESSO, ESTRO, ICRU, MRC, OECI, WHO. 

Staff members also served on boards of organizations 

such as the EOR TC cooperative groups, International 

Association for Breast Cancer Research, Nederlandse 

Commissie voor Stralingsdosimetrie, Nederlandse 

Werkgroep Hoofd-Halstumoren, Oog en Orbita 

Commissie, American Association for Psychological 

Oncology, European Community Committee on 

Palliative Cancer Care, Comprehensive Cancer Center 

Amsterdam, European Society for Therapeutic 

Oncology, the WHO Quality of Life Group, the 

International Academy of Pathology, the Gezondheidsraad, 

het NWO Gebiedsbestuur der Medische 

Wetenschappen, the European Cancer Center, National 

Advisory Board on AIDS, Netherlands Health Council 

Committee on Home Care for Cancer Patients, Scientific 

Council on Social Oncology (WRSQ) of the Dutch 

Cancer Society, Education and Psychosocial Research 

Committee of the Cancer Research Campaign, etc.

18 fnfroducfion 

Others served as editors of scientific books or served on 

editorial boards of journals (38 in total). Staff members 

we re also active in organizing national and international 

oncology meetings, workshops and congresses, and 

participated in teaching for the European School of 

Oncology and the ESTRO teaching course on Radiation 

Physics for Clinical Radiotherapy and the ESTRO 

teaching course on Basic Clinical Radiobiology. 

Outlook 

These are turbulent times for Dutch science. 

Government spending must be reduced, the Dutch 

economic position relative to its European partners is 

slowly but steadily going down and Dutch politicians 

tend to react with their standard solution: cut research, 

reduce long term investments in knowledge and 

infrastructure. The NKI/ A vL has thusfar survived the 

squeeze rather weil and we are confident that we shall 

retain the support for our activities if we continue to 

function efficiently and deliver the quality that may be 

expected from a center of excellence. 

We are fortunate to be helped in this task by so many 

competent and dedicated individuals, who give their time 

to our governing board and our national and 

international advisory boards. We are also indebted to 

the governing board and staff of the Dutch Cancer 

Society for their generous financial and moral support. 

We are grateful to all those individuals for their help 

and friendship and we hope that they will find inspiration 

in this report to continue their support of astrong and 

independent Netherlands Cancer Institute in 1995 and 

beyond. 

Piet Borst 

Director of Research

I Division of Cell Biology 

Head 

E Roos PhD 

Permanent academie staff 

J Calafat PhD, JG Collard PhD, CA Feitkamp PhD, A 

Sonnenberg PhD 

Other academie staff 

GO Delwel MSc, MHE Driessens MSc, A vd Flier MSc, 

CML Gimond PhD, GGM Habets MSc, 

H Kemperman MSc, FN van Leeuwen PhD, 

AA de Melker MSc, AML Meijne PhD, GAM Michiels 

PhD, R van der Neut PhD, CM Niessen MSc, 

G la Rivière PhD, MP Sanchez Aparicio MSc, JC Stam 

MSc, PJM Stroeken MSc 

Permanent technical staff 

JWRM Janssen, RA van der Kammen, IMM Kuikman, 

E Noteboom, CH Ong, NF Rodriguez Erena, 

EMF Ruuls-Van Stalle, YM Wijnands 

Other technical staff 

DLA Fles, EHM Huisman, AM Martinez de Velasco, 

PEM van Run, EAM van Rijthoven, SE Verbakel, 

AEM Zuurbier 

Students and trainee technicians 

IDamen, RGE Ebbers, B Franke, G Hassink, H Kain, 

I Kleine Budde, C de Lange, E Rots, GRuiter 

Guest 

L Sterk 

Secretary 

JAM van Niele-Pouw, VEM Akker 

19

20 Cel! Bio/ag)' 

Introduction 

In this division we focus on cell adhesion and motility, 

and their role in tumor progression and metastasis. We 

study the regulation of adhesion molecules and their 

interaction with the cytoskeleton. Furthermore, we aim 

to identify genes that con trol the invasive phenotype. The 

immuno-EM and F ACS facilities form part of this 

division. 

The major developments this year were: I) the Tiam-l 

gene, previously identified as an inducer of invasive and 

metastatic capacity in Iymphoma cells, was found to 

activate the Racl GTPase. Subsequently, a constitutively 

active mutant of Rac1 was indeed shown to induce 

invasion by itself. 2) We demonstrated the feasibility of 

targeted gene disruption as a tooi in metastasis research, 

by generating a CD44 double knock-out mutant of a 

metastatic Iymphoma. 3) The HDI hemidesmosomal 

protein was shown to bind the integrin rx6f34. This 

interaction is likely to be essential for hemidesmosome 

assembly. Furthermore, we found several integrins to be 

involved in interactions of tumor cells with hepatocytes 

and therefore potentially in liver metastasis; we 

demonstrated that a tumor cell surface mucin is a very 

effective inhibitor of adhesion; and we observed a novel 

adhesion structure in lymphoid cells adhering to laminin. 

Adhesion mechanisms in metastasis 

H Kemperman " MHE Driessens 2 ,3, G la Rivière 3 , 

AML Meijne 4 , NF Rodriguez Erena, 

YM Wijnands' , EAM van Rijthoven 2 , AEM Zuurbier 3 , 

PEM van Run 3.4, EMF Ruuls-Van Stalle, 

PJM Stroeken 2 , RGE Ebbers, I Kleine Budde, 

CA Feitkamp, E Roos 

Role of adhesion molecules in metastasis studied 

with knock-out mutants 

Targeted gene disruption in transgenie animals is now 

the method of choice to study the functions of proteins in 

vivo. If the same approach could be applied to metastatic 

tumor celllines, it would greatly facilitate the elucidation 

of the mechanisms of metastasis. We have therefore set 

out to disrupt potentially relevant gen es in the highly 

metastatic diploid lymphoma cell lines MDAY-D2 and 

ESb. So far, we have succeeded in the disruption of both 

alleles of CD44 in MDAY-D2. The screen for single 

knock-out clones was based on reduced CD44 surface 

expression, and double knock-out mutants were selected 

by F ACS sorting. The ratio of homologous recombination 

to random integration was of the order of 10- 3 . 

In human Iymphomas the degree of malignancy 

correlates with high levels ofCD44, i.e. the standard form 

not contammg alternatively spliced domains. 

Furthermore, there are indications that CD44 is required 

for s.c. growth and metastasis formation of lymphomas 

and melanomas in mice. Our in vivo studies with the CD44 

knock-out mutants are still preliminary but S.c. growth 

does occur and metastases do form, both after s.c. and i.V. 

injection. The results suggest quantitative differences, but 

this must be further established. 

Role of LFA-l andfibronectin receptors in 

lymphoma liver metastasis 

Metastatic lymphomas that invade diffusely into the 

liver, behave similarly in hepatocyte cultures. Previously, 

the adhesion molecule LF A-I was shown to be required 

for invasion by metastatic T-cell hybridomas and to bind 

to ICAM-I on hepatocytes which is concentrated on the 

lateral surfaces between which the tumor cells move. ESb 

cells invade similarly but the adhesion molecules involved 

are quite different. LF A-I levels are low but the f31 

integrin VLA-4 is expressed and may bind to fibronectin 

which is located on the dorsal surface of hepatocytes in 

vitro and on the sinusoidal surface in vivo. A combination 

of LF A- and VLA-blocking antibodies and peptides 

inhibited the interaction of ESb cells with hepatocytes, 

but not completely, suggesting th at still other adhesion 

mechanisms play a role. One of the candidates is the 

integrin rxEf37, which we found unexpectedly to be present 

on ESb cells. This is a marker for intestinallymphocytes 

and mediates their adhesion to intestinal epithelial cells. 

Preliminary results suggest that it is in fact involved in the 

interaction of ESb cells with hepatocytes. For MDA Y 

D2 cells, which express LFA-I and VLA-4 and the 

fibronectin receptor VLA-5 but not rxEf37, combinations 

of antibodies and peptides blocking the three expressed 

integrins had no substantial effect, therefore other 

adhesion molecules must be involved. 

Activation of integrins required for invasion 

Despite its involvement in invasion, LFA-I is not in an 

active state on the T-cell hybridomas, since the cells do 

not usually bind spontaneously to ICAM-l , but only in 

the presence of PMA or Mn2+ . Invasion is inhibited by 

pertussis toxin, due to interference with the LFA-Iactivating 

signal, since inhibition is reversed by PMA or 

Mn 2 + . This implies that the invaded tissues contain 

LF A-I-activating factors, the signals of which are 

transmitted by pertussis toxin-sensitive G proteins (see 

Figure 1.1). The fibronectin receptors mayalso require 

activation, since MDA Y -D2 cells do not ad here to 

fibronectin and ESb cells ad here poorly. Adhesion is 

induced or enhanced by PMA and, unexpectedly, by the 

9EG7 mAb against murine f31 integrins which was 

reported to block rather than to stimulate its function. 

Distribution of integrins studied with immuno-EM 

Using immuno-EM we have studied the distribution of 

adhesion molecules in cells subjected to wet-cleaving, a 

procedure that removes most of the cell body, leaving a 

substrate-adherent membrane and associated cytoskeleton. 

Such preparations can be studied by 

transmission EM. In focal contacts in fibroblasts, we 

found f31 integrins to be located in clusters at the edge 

rather than throughout the contact area. In T -cell 

hybridomas, LFA-l was always found in small clusters, 

even in suspended cells, and this did not change upon 

Mn 2 + -stimulated adhesion to ICAM-l. The cells spread 

extensively, and LFA-I was redistributed to the cell 

margins and in large amounts to the filopodia, but LFA-I

22 Cel! Biology 

a6f34 and the hemidesmosomal protein HDl , as 

determined in collaboration with C Niessen and A 

Sonnenberg of this division. Adhesion was also induced 

by treatment with an O-sialoglycoprotein endopeptidase, 

which c1eaved all epiglycanin from the surface. This also 

caused extensive aggregation, mediated by E-cadherin 

th at was fully functional in these suspension cens but was 

effectively masked, even by the small amounts of 

epiglycanin remaining after capping with the mAb. 

Adhesion to hepatocytes is similarly enhanced by 

capping and enzyme treatment but given the complete 

inhibition of matrix and intercellular adhesion by 

epiglycanin, it is remarkable that the cens bind to 

hepatocytes at all. However, we observed that epiglycanin 

is redistributed during the interaction with hepatocytes. 

This phenomenon may explain why cens expressing high 

levels of anti-adhesive mucins are still capable of 

metastasis formation. 

Carcinoma adhesion to hepatocy tes: role of 

fibronectin receptors including (X V integrins 

The other variant, T A3 / St, does not express 

epiglycanin and virtually no a6f34. Adhesion to 

hepatocytes is mediated by the f3I integrin VLA-5 that 

binds to fibronectin on the hepatocyte dorsal surface. The 

f31 subunit is hardly expressed by T A3 / Ha cells, which do 

not adhere to fibronectin, even after removal of 

epiglycanin. Rat fibronectin contains the OPAR 

carbohydrate epitope, which explains why the OPAR 

mAb inhibits the adhesion ofboth TA3 / St and TA3 / Ha 

cells. Fibronectin is located under the endothelium of 

liver microvessels, from which the tumor cells invade this 

organ, where no basement membrane is present as in 

other organs. For liver metastasis, adhesion to 

fibronectin may therefore be particularly relevant. Most 

carcinoma cells do not express VLA-5 but do adhere to 

fibronectin. Adhesion of HT29 human colon carcinoma 

cells, which metastasize to the liver upon intrasplenic 

injection into nude mice, was found to be mediated by aV 

integrins. The cells express both aVf35 and aVf36, and 

either one or both may be involved. The f35 and f36 

subunits are hardly expressed by normal cens in vivo but 

only by certain carcinomas, although they are present on 

normal epithelial cens in culture. Our results suggest th at 

these tumor integrins may be required for liver metastasis 

by certain carcinomas. 

Publieations 

Kemperman H et al. cen Adhesion Commun 1994; 2: 

45-58. 

Kemperman H et al. J cen Biol 1994; 127 (6, pt.2) (in 

press). 

Kemperman H et al. Invasion Metastasis (in press). 

Kemperman H et al. Clin Exp Metastasis (in press). 

Kemperman H et al. Cancer Surveys (in press). 

La Rivière AG et al. J cen Science 1994; 107: 551-9. 

Meijne AML et al. J cen Science 1994; 107: 1229-39. 

Meijne AML et al. J Cell Science 1994; 107: 2557-66. 

Notes 

1 Funding: Dutch Cancer Society, Project NKI 92-55. 




Invasion- and metastasis-inducing genes 

GGM Habets 1 , JC Stam 2 , GAM Michiels 3 , FN van 

Leeuwen4, RA van der Kammen, SE Verbakel4, 

B Franke, IDamen, H Kain, C de Lange, GRuiter, 

G Hassink, JG Conard 

Identification of the invasion and metastasis 

inducing Tiam-l gene 

The Tiaml gene was identified by proviral tagging in 

combination with in vitro selection for invasive 

T-Iymphoma variants. In 40 % of the invasive variants, 

proviral insertions were found within coding exons of the 

Tiaml gene, resulting in both truncated 5'-end and 3'-end 

transcripts that give rise to truncated N- and C-terminal 

Tiaml protein fragments, respectively. In one invasive 

variant, amplification of the Tiaml locus was observed 

with concomitant increase in the amount of normal 

Tiaml protein. This suggests that invasion can be induced 

by either increased amounts ofthe normal Tiaml protein 

or by protein truncation. Moreover, transfection of 

truncated Tiaml cDNAs into non-invasive Iymphoma 

cells made these cens invasive, establishing the invasioninducing 

capacity of Tiaml. 

Sequence homology and chromosomallocalization 

The Tiam 1 protein contains a domain with significant 

sequence similarity to the transforming and GDP-GTP 

exchange region of the proto-oncogene Db!. The Dblhomologous 

(DH) domain is shared with a number of 

structurally different genes inc1uding Dbl, Ber, CDC24, 

Vav and Eet-2. Most of the encoded proteins modulate 

the activity of Rho-like GTPases and induce malignant 

transformation after N-terminal truncation. Tiaml also 

contains two Pleckstrin homologous (PH) domains that 

have been proposed to mediate protein-protein 

interactions, similar to SH2 and SH3 domains. The 

sequence homology with other proto-oncogenes 

suggested that truncated Tiaml acts as an oncogene and 

that the encoded protein modulates the activity of Rho 

and/ or Rac proteins. These small GTPases transduce 

extracenular signals that affect cytoskeletal activities 

which determine the morphology, adhesion and motility 

of cens. Tiaml maps to the dis tal end of murine 

chromosome 16. The human homologue of Tiaml maps 

to chromosome 21 on band q22, centromeric of the Amll 

gene. 

LPA induces invasion 

Invasiveness of most retrovirally induced invasive 

T-Iymphoma variants is completely dependent on serum 

and recently we found th at the relevant serum component 

is lysophosphatidic acid (LPA). The LPA-induced 

invasion is inhibited by pertussis toxin (PT) suggesting 

that LP A acts through a PT -sensitive G protein, which 

has been demonstrated for some other effects of LPA. In

fibroblasts, LPA induces stress fibers, suggesting that 

RhoA is activated by LPA-induced signais. Whether and 

how LPA affects the Rho- and Rac-signaling pathways in 

the BW5147 lymphoma cells is currently being 

investigated. 

Tiaml conservation and expression pattern 

Tiaml is conserved amongst vertebrates. Mouse and 

human Tiaml are 95% identical at the amino acid level. 

The gene is highly expressed in brain and testis and 

moderately expressed in virtually all other murine tissues. 

Tiaml mRNA is also present in almost all of the forty 

tumor celllines that we have analyzed, including B- and 

T-cell lymphomas, melanomas, neuroblastomas and 

carcinomas of the breast, lung, ovary, bladder and 

pancreas. The evolutionary conservation as weIl as the 

broad expression pattern of Tiaml in normal tissues and 

tumorigenic cells suggests an important general function 

in cellular signaling processes. Whether Tiaml expres si on 

levels correlate with the invasive and metastatic capacity 

of tumorigenic cells and whether truncated Tiam 1 

proteins exist in human tumors is currently being 

investigated. 

Transforming and oncogenic potential 

The homology between Tiaml and the protooncogenes 

Dbl, Eet-2 and Vav suggests that Tiaml may 

act as an oncogene. We therefore determined its 

transforming and oncogenic potential by transfection of 

full-iength and truncated Tiaml cDNA constructs into 

NIH3T3 cells. Only the construct encoding the truncated 

C-terminal Tiaml protein, containing both PH domains 

and the DH domain, induced foci in NIH3T3 cells. 

Transfection of full-Iength Tiaml yielded morphologically 

similarly transformed cells but these were unable 

to develop foci as a result of polyploidization and growth 

arrest. Cells isolated from the foci induced by 

N-terminally truncated Tiaml produced tumors upon s.c. 

injection into nude mice in contrast to vector-transfected 

controls. Apparently, N-terminal truncation of the 

Tiam 1 protein activates its transforming and oncogenic 

potential, as has been documented for the Tiaml-related 

genes Dbl, Vav, and Eet2. 

Phosphorylation and turnover 

The Tiaml protein is predominantly phosphorylated 

on serine / threonine and not or hardlyon tyrosine 

residues. The normal Tiaml protein has a half-life of 

approximately 10 hours, whereas truncated Tiam 1 

proteins appear to be more stabie with a half-life of 

approximately 24 hours. In NIH3T3 cells, transfected 

with the construct encoding the N-terminally truncated 

Tiaml protein, an increase in tyrosine phosphorylation of 

P190 (RhoGAP) was found. Increased phosphorylation 

of this RasGAP-associated protein has been demonstrated 

upon growth stimulation by tyrosine kinase 

receptors and after oncogenic transformation of cells. 

This suggests that Tiaml is somehow able to cross-talk 

with the Ras-signaling pathway which might explain the 

established oncogenic activity of truncated Tiaml and 

Cell Biology 23 

our earlier finding that V12Ras-transfected BW5147 cells 

acquired invasive capacity. 

Tiaml affects cytoskeletal organization 

Upon growth stimulation of fibroblasts, Rac1 

stimulates the formation of actin filaments at the plasma 

membrane, leading to the formation ofmembrane ruffles, 

whereas RhoA regulates the assembly of focal adhesions 

and actin stress fibers. Immunohistochemical and 

confocal microscopic analyses revealed extensive 

membrane ruffling and a decreased number of stress 

fibers in Tiaml-transformed NIH3T3 cells. Based on 

these findings we proposed th at Tiam 1 is an activator 

(guanine nucleotide-dissociation stimulator) for Rac1 

and in some way down-modulates the activity of RhoA. 

Both the fulliength and the truncated Tiam 1 proteins are 

located in the cytoplasm. When membrane ruffles are 

induced, F -actin and Tiam 1 appear to accumulate and to 

co-Iocalize in these ruffles. Inactivation of RhoA by C3 

transferase does not affect the membrane ruffling, 

excluding the possibility that RhoA is involved in Tiamlinduced 

membrane ruffling. 

Cel! motility 

Tiaml-transfected NIH3T3 cells are less motile than 

untransfected controls or Vl2Ras- or V14Rhotransfected 

cells, as assessed by the phagokinesis- and 

wound migration assays. C3 transferase inhibited 

motility of all cell hnes tested. Others have shown that 

constitutively active RhoA is unable to induce motility, 

indicating th at active RhoA is required but not sufficient 

to induce cell motility. The low degree ofmotility (30% of 

controls) of Tiaml-transfectants might be explained by 

increased adhesion to the cell substrate, possibly by 

activation of integrins. This process is most likely 

regulated by Rho and Rac proteins. Alternatively, Tiamltransfected 

cells may have lost cell polarity which 

prevents directed motility. 

Tiaml activates Rac proteins 

The Tiaml effects on the cytoskeletal organization in 

NIH3T3 cells (i.e. induction of mem bra ne ruffling) 

suggested that this protein possesses guanine nucleotidedissociation 

stimulating activity towards Rac1. Results 

obtained in collaboration with A Verhoeven and B 

Boischer from the Central Laboratory ofthe Netherlands 

Red Cross Blood Transfusion Service in Amsterdam, 

indicate that Tiaml is able to activate the superoxidegenerating 

NADPH oxidase system of human 

neutrophils, a process regulated by Rac proteins. GTPySbound 

Rac1 stimulated the NADPH oxidase activity in 

the neutrophil in vitro assay much more than GDP-bound 

Rac1. Addition of GDP-bound Rac1 together with 

recombinant Tiam 1 resulted also in a time-dependent 

formation of superoxide comparable to GTPyS-bound 

Rac1. This substantiates our findings th at Tiaml 

activates the Rac signaling pathway.

24 Cell Bi%g)' 

Activated V12Racl induces invasion 

In earl ier studies we showed that Tiaml induces 

invasion in T-Iymphoma cells. Since Tiaml appears to be 

an activator ofRac1 , constitutively active Racl mayalso 

induce invasiveness by itself. Indeed, retroviral 

transduction of V] 2Racl into BW5147 T-Iymphomacells 

yielded invasive cells, whereas similarly transduced 

V14RhoA had no effect. The invasive cell c10nes expressed 

the exogenous V12Racl sequences whereas non-invasive 

c10nes did not. This indicates that invasiveness induced by 

Tiaml in BW5147 T-Iymphoma cells is primarily caused 

by the activation of Rac-like proteins. Inactivation of 

RhoA by C3-transferase treatment of cells abrogates 

invasion. Apparently, active RhoA is required but not 

sufficient to induce invasiveness in these Iymphoid cells. 

These studies implicate the Rac- and Rho-signaling 

pathways in processes of tumor progression, invasion and 

metastasis. 

Publications 

Collard JGNM Tijdschr Kanker 1994; 18 (4): 18-22. 

Habets GGM et al. Cell 1994; 77: 537-49. 

Habets GGM et al. Cytogenet Cell Genet 1994 (in 

press). 

Habets GGM et al. Oncogene 1994 (in press). 

Notes 

I Funding: Dutch Cancer Society, project NKI 91-15. 

2 Funding: Dutch Cancer Society, project NKI 92-52. 

3 Funding: Netherlands Organization for Scientific 

Research, Project 900-501-146. 

4 Funding: Dutch Cancer Society, project NKI 94-878. 

Receptors for matrix adhesion 

GO Delwel I, A van der Flier 2 , CML Gimond 3 , 

AM Martinez de Velasc0 4 , AA de Melker 5 , R van der 

Neut 6 , CM Niessen I, DLA Fles 5 , EHM Huisman I, 

I Kuikman, PW Modderman 7 , E Rots 8 , MP Sánchez 

Aparicio4, L Sterk 9 , A Sonnenberg 

The interaction of cells with extracellular matrix 

components influences their proliferation and 

differentiation. Cells adhere to extracellular matrix 

components via specific cell surface receptors, most of 

which belong to a family of structurally related molecules 

called integrins. Two integrins, rx6{31 and rx6{34, are 

strongly expressed on various epithelial cell types and 

mediate the adhesion of these cells to different laminin 

isoforms. In addition, they are involved in the interaction 

with the cytoskeleton: rx6{31 with the actin-cytoskeleton in 

focal contacts and rx6{34 with the keratin filaments in 

hemidesmosomes. We previously studied the ligand 

binding specificities of these integrins and their 

cytoplasmic interactions, as well as those of the rx3{31 

integrins. Our present investigations are aimed at 

studying the function of these integrins and their splice 

variants (both cytoplasmic and extracellular) in 

modulating extracellular signaling pathways and integrin 

activation. We have also extended our research to two 

other adhesion molecules, BP180, a transmembrane 

component ofhemidesmosomes, and P-selectin, a platelet 

and endothelial cell adhesion molecule. 

The cytoplasmic domain of a6A is a substrate for 

protein kinase C 

The A cytoplasmic variant of the rx6 subunit, but not 

the B variant, is phosphorylated after Phorbol myristoyl 

acetate (PMA) stimulation of many cell lines. In K562 

cells that had been transfected with cDNAs encoding the 

rx6A or rx6B integrin subunits, PMA also induced an 

increase in adhesion to laminin, the rx6B-transfected cells 

adhering better than the rx6A-transfected cells. 

Substitution of Ser 104 1 for Ala prevents phosphorylation 

of the rx6A integrin subunit, but not activation of rx6A{3I. 

In fact, this mutant integrin was activated more strongly 

than wild type rx6A{31 , suggesting th at phosphorylation 

prevents full activation of rx6A{31. Ser 1041 resides in a 

consensus sequence for protein kinase C (PKC) and 

alteration of this seq uence results in astrong reduction of 

PMA-induced phosphorylation. Synthetic peptides 

representing the cytoplasmic domain of the rx6A were 

phosphorylated in vitro by PKC and also by protein 

kinase A (PKA), but not by MAP kinase. The PKA 

phosphorylation site was found to be Ser 1048 • In intact 

cells, however, forskolin or dibutyryl-cAMP stimulation 

ofPKA did not induce phosphorylation ofthis site. These 

findings suggest th at PKC phosphorylates the rx6A 

integrin subunit and that rx6A is not an in vivo substrate 

for PKA. 

Distribution of the cytoplasmic variants of a3 and 

a6 in solid tumors 

In collaboration with 0 Ivanyi (Division of Tumor 

Biology) and C Patriarca (University of Milan), we have 

used monoclonal antibodies specific for the A and B 

cytoplasmic variants ofthe rx3 and rx6 integrin subunits to 

study expression of these variants in human tumors. The 

tissue specific expression of ecto- and cyto-domains of rx3 

and rx6 was found to be maintained in a subset of breast, 

colon, kidney and parotis tumors. In many breast, colon 

and parotis tumors one of the variants of rx6 was not 

expressed whereas they were both detectable in the 

corresponding normal tissues. In contrast, co-expression 

of the rx6 variants was found in some kidney tumors 

whereas only one variant was detected in the normal 

tissue. In a rninority of colon and kidney tumors, the 

cytodomains of rx3 and rx6 we re undetectable and absence 

of rx3 and rx6 was noted in a subset of breast, colon, kidney 

and parotis tumors. These observations show th at the 

expression of the integrin variants varies considerably 

and support the concept th at changes in their expression 

contribute to malignant behavior. Further studies may 

reveal the significance of these variants as possible 

markers for prognosis or differential diagnosis. 

Effects of mucins on integrin-mediated cell 

adhesion 

In collaboration with J Wesseling and J Hilkens 

(Division of Tumor Biology), we have studied the effect of 

expression ofepisialin on integrin-mediated cell adhesion.

A marked decrease in the adhesion of cells to extracellular 

matrix proteins was noted in cells that strongly express 

this mucin (see also Annual Report Division of Tumor 

Biology). Similar observations we re made for the 

expression of epiglycanin on adhesion of a.6f34 to laminin 

and kalinin, in collaboration with H Kemperman and E 

Roos (see also Adhesion mechanisms in metastasis). 

Interaction of integrin (1.6[34 with hemidesmosomal 

plaque protein HD1 

Hemidesmosomes are junctions that mediate adhesion 

of various epithelia to the underlying basement 

membrane and which serve as anchorage sites on the 

plasma membrane for keratin fïlaments. The a.6f34 

integrin is concentrated in hemidesmosomes and is 

thought to play an important role in their assembly, 

although the interactions required for assembly are far 

from dear. We have investigated the interaction of 

f34-cytoplasmic domain-GST fusion proteins with cellular 

proteins and demonstrated that a fragment of f34A , 

consisting of the two pairs of fibronectin type 111 repeats 

separated by the intervening segment, interacts 

specifically with the hemidesmosomal plaque protein 

HDI. 

In a splice variant of f34 , f34B, 53 amino acids are 

inserted in the region of the cytoplasmic domain required 

for localization in hemidesmosomes. f34B is a minor 

component in cells th at form hemidesmosomes, although 

it can be present in appreciable amounts in cells that 

contain HD 1 but do not form hemidesmosomes. We 

found that a GST-cytof34B fusion protein also bound to 

HD I. Furthermore, f34B , transfected into rat 804G 

bladder carcinoma cells, associated with endogenous a.6 

and was efficiently incorporated into hemidesmosomes 

where it co-localized with HDI. We suggest that HDI is 

involved in linking the a.6f34 integrin to the keratin 

fïlaments, although its presence together with a.6f34 is 

apparently not sufficient to ensure hemidesmosome 

formation. 

Generation of knock-out mice to study the in vivo 

function of the (1.6[31 and (1.6[34 integrins 

To study the role of the integrin subunit f34 in 

peripheral neurogenesis and skin development (two 

tissues that strongly express a.6(34) , we plan to generate 

mutant mice in which the f34 gene is inactivated by 

targeted gene disruption. Two overlapping dones were 

isolated from a 129Sv mouse genomic DNA library. A 

targeting vector was constructed by replacing the 

sequences comprising exons 1 and 2 by a hygromycin 

resistance gene. Mouse embryonic stem cells have 

recently been transfected with this construct. In addition, 

we will generate conditional knock-out mice in which the 

f34 gene is only inactivated in the epidermis or in Schwann 

cells (Cre-Lox P technique). The cre-recombinase will be 

expressed under the control of the keratin 14 or myelin 

basic protein (MBP) promotors. Targeting vectors for 

this approach are currently being made. 

In mouse embryo, a shift in expression from a.6A to a.6B 

occurs during development. Our aim is to study the role of 

these two cytoplasmic variants by preventing the shift 

Cel! Biology 25 

from a.6B to a.6A, thus leading to constitutive a.6B 

expression. Genomic dones containing the a.6A and the 

a.6B exons have been isolated and partially mapped and 

sequenced. Construction of a targeting vector lacking 

exon A is in progress. 

180-kD Bul/ous Pemphigoid Antigen (BP 180) is 

deficient in Generalized Atrophic Benign 

Epidermolysis Bul/osa 

Generalized atrophic benign epidermolysis bullosa 

(GABEB) is a form of non-Iethal junctional 

epidermolysis bullosa characterized by universal alopecia 

and atrophy of the skin. In collaboration with MCJM 

Jonkman (University Hospital, Groningen) we have 

found a deficiency of the 180kDa bullous pemphigoid 

antigen in th ree patients with GABEB from unrelated 

families . In their skin, no reactivity was seen with 

antibodies to BP 180, whereas staining for BP230 was 

normal. In the skin of six non-GABEB patients, 

expression of BP180 and BP230 appeared normal. 

Immunoblot analysis of cultured keratinocytes from one 

of the GABEB patients also failed to detect BPI80 

antigen, whereas BP230 was present in normal amounts. 

The amount of BP 180 mRNA was also reduced. In 

another GABEB patient, there were non-involved areas 

of skin, in which blistering could not be induced by 

rubbing, and these areas showed interrupted staining for 

BP180; there was no staining for BP180 in areas of 

dinically normal but involved skin of tbis patient. 

Molecular genetic studies are now in progress to identify 

the mutation(s) in the BP180 gene responsible for the 

abnormality. 

The role of tyrosine phosphorylationfor the 

function of P-selectin, the receptor of platelets and 

endothelial cells 

P-selectin is a 140 kDa adhesion molecule, located in 

secretion organelles in platelets and endothelial cells. 

U pon activation of these cells, P-selectin is translocated 

to the cell surface, where it can bind to ligand(s) present 

on most types ofleukocytes. Stimulation of platelets with 

thrombin induces transient phosphorylation ofP-selectin 

tyrosine (Tyr777) and threonine and sustained 

phosphorylation of serine (Ser788). We have previously 

shown that a fraction of imrnunoprecipitated platelet 

P-selectin is disulphide-linked to the abundant platelet 

tyrosine kinase, pp60 c - src , in al: 1 complex. In these 

complexes, P-selectin is phosphorylated on tyrosine but 

pp60 c - src autophosphorylation is inhibited. 

We have introduced point mutations into P-selectin 

cDNA th at result in the replacement of phosphorylated 

amino acids, i. e. Tyr777 -+Phe and Ser788 -+Ala. Wild 

type and mutant P-selectin cDNA's in pSVL-Hyg will be 

expressed in the murine pituitary tumor cellline, AtT-20, 

using hygromycin selection. Like platelets and 

endothelial cells, AtT-20 cells have regulated secretion 

(ACTH granules) and P-selectin has been shown to be 

targeted to the secretion granules in these cells. Targeting, 

secretion, endocytosis and binding to leukocytes of 

P-selectin and mutants will be studied by 

immunofluorescence and electron microscopy.

26 Cel! Biofogy 

Preliminary experiments indicate that wild-type and 

mutant P-selectin cDNA's in the pSVL-Hyg vector are 

expressed at appreciable levels after transient transfection 

of COS 7 cells. The functional significance of the 

P-selectin-pp60 c - src complex that is observed in platelet 

immunoprecipitates will be further explored by cotransfection 

of P-selectin and human pp60 c - src in COS 

cells. 

Publications 

Delwel GO et af. Mol Biol Cell1994; 5: 203-15. 

Delwel GO and Sonnenberg A. CRC press (in press). 

Durban EM et af. J Histochem Cytochem 1994; 42: 

185-96. 

Gimond C et af. Exp Cell Res (in press). 

Jonkman MF et af. J Clin Invest (in press). 

Kemperman H et af. J Cell Biol (in press). 

Modderman PW et af. Biochem J 1994; 299: 613-21. 

Niessen CM et af. J Cell Sci 1994; 107: 543-52. 

Niessen CM et af. Exp Cell Res 1994; 211: 360-67. 

Uematsu J et af. J Biochem 1994; 115: 469-76. 

Notes 

I Funding: Dutch Cancer Society, Project NKI 91-260. 

2 Funding: Yamanouchi Research Institute. 

3 Funding: EC Fellowship. 

4 Funding: EC Project 930246. 

5 Funding: Dutch Kidney Foundation, Project C 

91.1179. 


Research (NWO), Project 900-511-043. 

7 Funding: Netherlands Heart Foundation, Project 

93.054. Central Laboratory of the Netherlands Red 

Cross Blood Transfusion Service, Amsterdam. 

8 Undergraduate student. 

9 Guest, Department of Pathology, AMC, Amsterdam. 

Immuno-EM facility 

J Calafat, JWRM Janssen 

Intracellular localization of FcyRIII and the 

activation of neutrophils upon addition of 

granulocyte colony stimulatingfactor (G-CSF) 

Using albumin as a marker for secretory vesicles in 

neutrophils, we found co-Iocalization of FcyRIII and 

albumin but not of FcyRIII and lactoferrin, a marker of 

specific granules. The intracellular pool of FcyRIII is 

therefore 10cated in secretory vesicles. The effect of 

G-CSF on the mobilization of secretory vesicles was 

analyzed using semi-quantitative immuno-EM and 

compared with the effect of the chemotactic peptide 

FMLP. Like FMLP, G-CSF induced a decrease in the 

number of sm all secretory vesicles, the formation of these 

vesicles and the fusion of some of these large vesicles with 

the surface membrane. These results show that G-CSF is 

an activator of neutrophils. This study was performed in 

collaboration with M de Haas and J M Kerst of the 

Central Laboratory of the Netherlands Red Cross Blood 

Transfusion Service, Amsterdam. 

Ultrastructural distribution of E-selectin in 

cytokine-activated endothelial cells 

The fate of E-selectin expressed on (tumor necrosis 

factor)-activated monolayers of hu man endothelial cells 

(HUVEC) was investigated by confocal laser scanning 

microscopy. Cytokine-activated endothelial cells 

internalized mAb to E-selectin in a very rapid, energydependent 

fashion. By contrast, mAb against ICAM-l 

and VCAM-l remained surface-bound. Using peroxidase 

cytochemistry and EM, we studied the distribution of 

E-selectin after internalization. Numerous endosomal 

and tubular-shaped complexes were observed containing 

internalized E-selectin and these endosomal structures 

appeared to run from the cell surface deep into the 

cytoplasm. This result, together with the observations 

that upon internalization E-selectin co-Iocalizes with 

cathepsin B, indicate that the bulk of E-selectin was 

intracellularly degraded: This study was performed in 

collaboration with T W Kuijpers and D Roos of the 

Central Laboratory of the Netherlands Red Cross Blood 


Publications 

Cal afat J et af. J Cell Biology 1994;126: 967-77. 

De Haas M et al. Blood (in press). 

Kuijpers TW et af. J Immunol. 1994;152: 5060-69. 

Ligtenberg MJL et af. EMBO J 1994;13: 2565-73. 

Nijenhuis M et af. Eur J Immunol 1994;24: 873-83.

11 Division of Molecular Carcinogenesis 

Head 

R Bemards PhD 


L den Engelse PhD, E Scherer PhD, JG Westra PhD 

(deceased 25 March !994), TK Sixma PhD (from 

August), H te Riele PhD 

Honorary staff member 

E Kriek PhD 


RL Beijersbergen MSc, FA Blommaert MSc, 

G Hateboer MSc, RM Kerkhoven PhD, JC Klein MSc, 

CP Saris MSc, PM Voorhoeve MSc, NC Walworth 

PhD, N de Wind PhD 

Permanent technica! staff 

BGJ Floot (80%), AMC Gennissen (60%), WJ van Dijk, 

A Visser (50%), HHK Winterwerp (80%), HMJ Dekker 

VIaar 

Other technica! staff 

AMC van de Ende, EM Hijmans MSc, C Michael, 

C Schippers-Gillissen (40%) 

Guests 

KA Simmen PhD, B Smith-S0rensen MSc 

Under-graduate students 

L Carlée, EJW Dijkink, SF van Haren, Y Ramos 

Secretary 

MLRM Bresser MA (50%) 

27

28 Molecular Carcinogenesis 

Introduction 

In March of this year, our division was struck by the 

sudden death of one of our staff members, Gerard 

Westra. His enthusiasm for science and knowledge of 

nucleic acid chemistry will be sorely missed. 

Two new staff members joined the division in the past 

year. Hein te Riele adds important expertise in the area of 

mouse genetics. His research focuses on the role of DNA 

mismatch repair genes in cancer. Loss of mismatch 

repair-gene function has been implicated in hereditary 

non-polyposis colon cancer (NHPCC). A second line of 

research of the te Riele group concerns the role of the 

retinoblastoma gene family in mouse development and 

carcinogenesis. The second new staff member to join our 

division is Titia Sixma. She has a background in protein 

structure and will head a new protein structure group. A 

major investment in state of the art X-ray diffraction and 

computer equipment was made possible by a generous 

grant from the Nefkens Foundation. Apart from her own 

interest in proteins involved in signal transduction, she 

will collaborate with several groups in the institute to 

elucidate the th ree dimensional structure of proteins of 

interest. 

The research on cell cycle regulation has made 

significant progress in the past year. Several new genes 

have been isolated based on the ability of their encoded 

proteins to interact with known cell cycle regulatory 

proteins. In particular, the two new members of the E2F 

gene family turned out to be useful tools to study 

regulation of the retinoblastoma protein-related pI 07 and 

pl30. Through a collaboration with the two new staff 

members of the division, we hope that we will be able to 

study the function of cell cycle regulatory proteins, not 

only at the cellular level but also at the organism leve\. In 

collaboration with the new protein structure group, we 

hope to elucidate the three dimensional structure of some 

of these proteins. 

Functional analysis of dominant and 

recessive oncogene products 

R Bernards I , RL Beijersbergen I , AMC Gennissen, 

G Hateboer l , EM Hijmans, RM Kerkhoven l , 

KA Simmen, B Smith-S0rensen, PM Voorhoevel , 

NC Walworth 

Transcriptional control by MYC 

The product of the c-myc proto-oncogene, MYC, 

encodes a sequence-specific DNA binding protein with an 

amino-terminal transactivation domain and a carboxylterminal 

DNA-binding domain. MYC is essential for 

normal cell cycle progression. Little, however, is known 

about the regulation of MYC activity during the cell 

cycle. We have studied protein interactions of MYC and 

found that the retinoblastoma protein-related pl07 can 

form a specific complex with the MYC transactivation 

domain in vivo . Binding of pl07 to MYC significantly 

inhibits MYC transactivation. The domain of plO7 

involved in suppression of MYC activity coincides with 

the region of pl07 that is essential for growth 

suppression. Enforced expression of MYC overrides a 

pl07-induced cell cycle arrest, indicating that the decision 

to enter a cell cycle depends at least in part on the ratio of 

free MYC over pl07-bound MYC. We are currently 

studying whether the MYC/ pl07 complex can be 

dissociated by phosphorylation ofpl07 and/ or MYC, by 

cyclin-dependent kinases. 

We have also shown th at the MYC transactivation 

domain interacts directly with TATA-binding protein. To 

study whether this interaction is required for MYC 

transactivation, we have made random point mutants in 

the MYC transactivation domain. These mutants were 

selected in yeast for loss of transactivation ability. To 

date, we have isolated one amino acid substitution 

mutant of MYC that is defective for transactivation in 

yeast and mammalian cells. This mutant will help us 

identify which cellular proteins are involved in mediating 

MYC transactivation. 

Proteins that interact with pl07 

To further study the role of pl07 in cell cycle control, 

we have searched for additional cellular genes whose 

encoded proteins bind to p107. To date, we have isolated 

three novel cDNAs that encode plO7-interacting 

proteins. E2F-4 and E2F-5 are new members of the E2F 

gene family. Using specific sera, we have been able to 

show that E2F-4 interacts in vivo with both pl07 and the 

related pl30, whereas E2F-5 interacts only with pl30. 

Expression of E2F -4 stimulates the progression from G 1to 

S- and G2/ M phase of cell cycle, indicating that E2F-4 

is involved in cell cycle contro\. The role of E2F-4 in 

growth stimulation is further substantiated by the finding 

th at E2F-4 can cooperate with a ras oncogene in the 

transformation of rat embryo fibroblasts. 

We studied the effect of GI cyclin / cyclin-dependent 

kinase (cdk) complexes on the pl07 I E2F-4 complex and 

found that cyclin DI l cdk4 can dissociate the pI 07 I E2F-4 

complex, whereas the cyclin EI cdk2 complex was inactive 

in this assay. In contrast, the complex between pRb and 

E2F-I could be dissociated by both cyclin /cdk complexes 

(see Figure 11.1). That cyclin DlIcdk4 is a major kinase 

complex involved in the regulation of pl07 growth 

inhibitory activity, is also supported by our finding that a 

plO7-induced cell cycle arrest can be rescued by cyclin 

DlIcdk4 but not by cyclin E / cdk2. 

The third pl07-interacting protein that we have 

isolated in a yeast two hybrid screen encodes a novel 

subunit of protein phosphatase 2A (PP2A). The studies 

described above indicate that cyclin D I I cdk4 

phosphorylates and thereby inactivates p107. 

Significantly, cyclin Dl is implicated in the pathogenesis 

of a variety of human malignancies. Cyclin Dl may 

therefore derive (part of) its oncogenic activity by 

inactivating the growth inhibitory activity of p107. The 

identification of a PP2A subunit as a pl07 interacting 

protein suggests that PP2A may counteract (part of) the 

oncogenic activity of cyclin Dl by dephosphorylating 

pi 07. Experiments to test this model are in progress.

30 Mo/ecu/ar Carcinogenesis 

family members, spread over at least two generations, at 

least two having a first degree relationship and at least 

one diagnosed bel ow 50 years of age, accounts for 5-15% 

of colorectal cancers in the western world. The majority 

of tumors from HNPCC patients shows a decreased 

stability of the length of simple-nucleotide-sequence 

repeats (e.g. [CA]n)' This suggests that, early in their 

history, tumor cells have acquired some form of genetic 

instability, leading to acceleration of the evolutionary 

process of mutagenesis and selection, which underlies the 

development of canceL Recently, it has been 

demonstrated th at these tumors carry mutations in 

homologs of the bacterial genes mutS and mutL, which 

are essential for long-patch mismatch repair (LPMR). To 

date, one mutS homolog (MSH2) and three mutL 

homologs (MLHl, PMSl, PMS2) have been implicated 

in HNPCC. No relation has yet been observed between 

HNPCC and another mammalian mutS homo log, called 

Rep3. LPMR in bacteria contributes to genetic stability in 

two ways: it is anti-mutagenic and anti-recombinogenic. 

The existence of at least two mutS homologs in mammals 

may suggest that the mammalian genes have specialized 

functions. 

To provide a mouse model system for human HNPCC 

and to investigate the function of the mammalian mutS 

homologs, we started to generate mouse lines carrying 

defective Msh alleles in their germ line. The mouse version 

of MSH2 was cloned by N de Wind during a postdoctoral 

period in the laboratory of M Radman in Paris. Mouse 

Msh2, which is 93% identical at the protein level to its 

human counterpart, is ubiquitously expressed with high 

levels found in rapidly dividing embryonic stem cells. ES 

cells carrying a disruption in Msh2 we re obtained by 

homologous recombination. Chimeric mice derived from 

these cells are currently being tested for germ line 

transrnission of the mutation. Heterozygous Msh2 mice 

will be used to study the mechanism of mismatch repair 

deficiency, the tissue specificity of tumorigenesis and the 

(molecular) biology of mismatch repair-deficient tumors. 

In addition, an ES cell line was isolated carrying a 

disruption in both copies of Msh2. These cells are used to 

investigate the involvement of LPMR in mutagenesis, 

recombination and sensitivity to DNA damaging agents 

which affect proper base pairing. The function of Rep3, 

which is transcribed from the bidirectional DhJr promoter 

and is mainly expressed in testes, is being investigated 

along the same lines. 

The role of the retinoblastoma gene Rb-l and its 

homologs in development and tumorigenesis 

In the laboratory of A Berns at the NlG, several mouse 

models have been developed to study the role of Rb-l in 

murine development and tumorigenesis. Surprisingly, in 

hemizygous mice, the pituitary gland rather than the 

retina appeared susceptible to tumorigenesis by loss ofthe 

wild-type Rb alleIe. Yet, in chimeric mice carrying a high 

percentage of Rb-deficient cells, pRb deficiency was 

found to affect differentiation of at least one cell type of 

the developing retina and also differentiation of lens fiber 

cells, in both cases leading to apoptotic cell death. No 

defects we re seen in the chimeric CNS and skeletal 

muscle, which were expected to require a functional Rb 

gene for proper development. 

In a first approach to investigate whether additional 

mutations are required to reveal the involvement of Rb in 

tumorigenesis and development, knock-out mutations in 

both Rb-l and its homologpl07 have been introduced in 

ES cells. These cells will be used to generate chimeric mice 

which will be analysed for the phenotypic consequences 

of pR b / p 1 07 -deficiency. 

Publications 

De Wind N et al. Virology 1994; 200: 784-90. 

Kimman TG et al. Virology 1994; 205: 511-8. 

Robanus Maandag E et al. EMBO J 1994; 13: 4260-8. 

Note 

I Funding: EEC. 

Gene regulation in tissue-specific 

carcinogenesis 

E Scherer, HHK Winterwerp 

We use a molecular-biological approach to study the 

early events in tissue-specific carcinogenesis with 

pancreatic cancer as a model system. The expression of 

many tissue-specific genes changes during the multi-step 

development of canceL Decreased and increased 

expression as well as neo-expression of normally nonexpressed 

genes have been described. Some of these 

changes occur very early in the carcinogenic process and 

are, therefore, useful histochemical markers for the 

investigation of early tumor precursor stages. Several of 

the genes for pancreas-specific proteins contain E-box 

sequences (CANNTG) in their upstream regulatory 

sequences. The E-box sequence is recognized by a class of 

structurally related transcription factors th at all harbour 

a basic helix-loop-helix (bH LH) motif. E-boxes are 

frequently bound by heterodimeric protein complexes 

consisting of the product of the E2A gene and a second, 

tissue-specific bH LH protein. It is therefore likely that 

E-box-specific DNA binding factors play a role in the 

regulation of pancreatic gene expression as well as 

carcinogenesis. The first line of research therefore focuses 

on the cloning and characterization of pancreas-specific 

E-box DNA binding factors and the study of their 

expression in carcinogenesis. We are currently attempting 

to isolate genes encoding pan creatie bHLH proteins using 

degenerate PCR primers for the most conserved 

sequences ofthe bH LH domain to selectively amplify and 

clone such sequences from a pancreatic cDNA library. 

A second line of research concerns the gene encoding a 

pancreatic tumor marker. The earliest histochemical 

marker for foci of altered cells in azaserine-induced 

pancreatic carcinogenesis in rats is a zymogenmembrane-bound 

Mg + + -dependent ATPase (apyrase), 

which is increased in foci and differentiated tumors. The 

gene encoding this enzyme has not yet been cloned. A 

histochernically similar ATPase of rat liver, cloned 

recently, belongs to the family of cell adhesion molecules 

(CAM), functioning also as carcinoembryonic antigens 

(CEA). Using a 300 bp fragment from the conserved part 

of the first variabie Ig-like domain (ascribed to the ATP

inding fold) of the liver ecto-A TPase gene, we we re able 

to clone a 300 bp fragment of a new gene which differs 

from the liver ecto-A TPase fragment in 10 bp (8 aa 

substitutions, 4 of which are in, or close to, the A TPbinding 

domain). We are currently analyzing clones 

obtained by DNA hybridization screening from the rat 

pancreatic library, in order to generate the full-Iength 

sequence ofthe new CAM/ CEA gene. 

Publication 

Van Benthem J et al. Carcinogenesis 1994; 15: 

2023-2029. 

A 3 2 P-postlabeling assay for 

alkylphosphotriesters in DNA 

CP Saris', AMC van den Ende', JG Westra 

Alkylphosphotriesters (PTE) are persistent lesions, 

resulting from (random) alkylation of internucleotide 

phosphate oxygen atoms, which are believed to 

accumulate because of the lack of arepair mechanism. 

Resistance to enzymatic hydrolysis makes them easily 

obtainable as PTE dinucleotides. These properties make 

them attractive subjects for measuring DNA alkylation, 

either af ter short time exposure to high doses of 

chemotherapeutic alkylating agents or after long term 

exposure to low doses of environmental or endogenously 

formed alkylating agents. 

The 32P-postlabeling detection of alkyl PTE can be 

performed in two ways. The first method (referred to as 

the PDE method) is labeling of the di nucleoside 

monophosphate (dXpdY) that is formed upon alkali 

hydrolysis of a PTE. The second method (PTE) is direct 

labeling of the intact dinucleoside monoalkyl PTE 

(dXp(alkyl)dY). In contrast to the dinuc1eoside monophosphate 

dXpdY, not every di nucleoside monoalkyl 

PTE is a suitable substrate for labeling by polynucleotide 

kinase (PNK). Therefore, the PTE method has a lower 

recovery than the PDE method, but this is compensated 

by a lower background. When applied to a series of DNA 

samples of untreated animaIs, both methods appeared to 

correlate very weil (R=0.99, p=0.005). PTE levels were 

in the range of 10- 7 (calfthymus) to 5x10- 6 (rat liver). 

When rats we re treated with a single dose of the 

cytostatic drug procarbazine a linear dose-response 

relationship was found in the liver. We measured an 

increase of I methyl PTEIl06 nucleotides at a 

procarbazine dose of 1100 mg/ kg. In rats given a single 

dose of only 140 mg / kg of the liver carcinogen 

diethylnitrosamine (DEN), the increase in PTE was 25 

ethyl PTE/ 106 nucleotides. Apparently, PTE are formed 

more readily after exposure to DEN than procarbazine. 

We also monitored PTE content in granulocytes of 

Hodgkin's lymphoma patients that we re treated with 

procarbazine. The treatment consisted of procarbazine (1 

mg/ kg) combined with several non-methylating drugs, 

daily for a week per month. No significant increase in 

PTE levels was found. Given the results obtained with 

rats, this is hardly surprising, since the procarbazine dose 

required for a significant increase in PTE level is much 

higher than the dose given to Hodgkin's lymphoma 

M o/ecu/ar Carcinogenesis 31 

patients. Significant differences were, however, found 

between patients. Future studies wiJl focus on PTE as 

indicators of chronic exposure to low doses of either 

environmental or endogenous alkylating agents, rather 

than short term exposure to chemotherapeutic 

methylating agents. 

No te 

, Funding: Dutch Cancer Society, Project NKI 90-16. 

Oxidative DNA damage 

JC Klein', WJ van Dijk, E Kriek, JG Westra, 

MVM Lafleur 2 , J ReteJ2, A Teixeira 3 , 

G van de Werken 3 

Reactive oxygen species (ROS) are continuously 

produced in aerobic organisms as a byproduct of normal 

cellular oxygen metabolism or after exposure to certain 

exogenous agents. These species can induce a large variety 

of aberrant structures in DNA e.g. modified bases and 

strand breaks (single or double), some of which have 

al ready been shown to be mutagen ic in bacterial and 

mammalian cells. ROS, therefore, are considered to 

contribute significantly to processes of ageing, 

development of degenerative diseases and cancer. Many 

carcinogens and tumor promoting agents have been 

shown to induce increased amounts of oxidative damage 

in DNA. 

We studied the oxidative DNA damage induced in 

several target organs of rats after administration of 

aromatic amines (NOH-AAF, NOH-AABP, NOH 

MeAABP or PhIp). We focused on 7,8-dihydro-8-oxo-2'deoxyguanosine 

(8-0HdG), a chemically stabie moiety, 

which is considered to be representative for the amount of 

oxidative damage induced. A sensitive and highly 

reproducible method was developed, in which oxidized 

nucleosides are isolated, derivatized and subsequently 

analyzed by gas chromatography/mass spectrometry. 

With this technique we could measure differences in the 

8-0HdG content of colon and liver DNA and in the 

amounts of 8-0HdG excreted in urine. We found that the 

levels of 8-0HdG were approximately 50% decreased in 

liver and colon 24 hrs after adrninistration of a single dose 

of aromatic amines to the rats. Similar results we re 

obtained with 32P-postlabeling analysis, in which the 

(oxidized) nucleotides we re radioactively labeled and 

analyzed by 2-dimensional thin layer chromatography. 

Subsequent time course experiments with single doses 

ofPhIp revealed that the reduction in the 8-0HdG levels 

was only temporary, and that these levels we re restored to 

normal values 48 to 96 hrs after administration. These 

results we re in contrast with those reported by other 

investigators. To account for these unexpected effects, we 

studied the formation of oxididative DNA damage in the 

presence or absence of PhIp in an in vi/ro model system. 

The presence of PhIp protected 0XI74 DNA against 

damage induced by ionizing radiation. Hence, it was 

concluded that PhIp (and also other aromatic amines) 

prevents oxidation of DNA by trapping a substantial 

fraction of ROS th at otherwise could have attacked 

DNA.

32 M o/ecu/ar Carcinogenesis 

Publications 

Hermans RCA et al. J of Labeled Compounds and 

Radiopharmaceuticals 1993; XXXIV: 191-7. 

Klein JC et al. J Biol Chem (in press). 

Teixeira AJR et al. Ana1 Biochem 1993; 214: 474-83. 

Teixeira AJR et al. Anal Chem (in press). 

Teixeira AJR et al. Anal Biochem (in press). 

Notes 


2 Division of Molecular Toxico10gy, Free University of 

Amsterdam. 

3 National Institute of Hea1th and Environmental 

Protection, Bilthoven, The Netherlands. 

Platinum-DNA adducts 

FA Blommaert', C Michael' , CP Saris 2 , JH Schornagel, 

L Smets, AM Fichtinger-Schepman 3 

The anti-tumor drugs cis-diamminedichloroplatinum(II) 

(cisp1atin) and cis-diammine (1 , 1-cyclobutanedicarboxylato 

)platinum(II) (carboplatin) are 

widely used in the clinic as chemotherapeutic agents 

against a broad range of tumors. It is generally accepted 

th at the anti tumor effect of platinum compounds is based 

on its interaction with DNA, leading to the formation of 

various types of platinum-DNA adducts. In our studies 

we focus on the role of the formation and persistence of 

platinum-DNA adducts in cytotoxicity and tumor 

resistance against platinum compounds. 

The formation and persistence of carboplatin-DNA 

adducts in various tissues of rats were studied at the 

cellular level (using antiserum NKI-A59) and with a 

competitive ELISA, using antisera directed against the 

major platinum-DNA adducts (Pt-GG, Pt-AG, G-Pt-G 

and Pt-G). Adducts were observed in all tissues studied; 

high levels we re observed in kidney and liver, lower levels 

in the ovary and uterus, and minimallevels in the spleen 

and testis. The bifunctional adducts (Pt-GG, Pt-AG and 

G-Pt-G) were formed in equal amounts; a preference for 

GpG sequences, as is found for cisplatin (in vitro and in 

vivo), does not exist for carboplatin in vivo. Significant 

repair of the G-Pt-G and Pt-AG adducts, but hardly any 

repair of the Pt-GG adducts, was observed in the first 48 

hours. The ELISA results were in good agreement with 

previous imrnunocytochemica1 data. 

Our studies on the formation and persistence of 

cisplatin and carboplatin-DNA adducts in vitro and in 

vivo, indicated that an even more sensitive detection 

method was desired, particularly for carboplatin which 

gives much lower adduct levels. We developed a sensitive 

32P-postlabeling method for the detection of bifunctional 

intrastrand crosslinks Pt-GG and Pt-AG in DNA in vitro 

and in vivo. After enzymatic digestion of DNA, the 

positively charged platinum adducts we re purified from 

unplatinated products, using strong cation exchange 

chromatography. Subsequently the samples were 

deplatinated with cyanide, because platinated dinucleotides 

are very poor substrates for polynucleotide 

kinase. Analysis of the postlabeled mixture was 

performed by a combined TLC and HPLC-procedure. 

When applied to DNA platinated both in vitro and in vivo 

with cis- or carboplatin, good correlations with existing 

methods (AAS, immunocytochemistry and ELISA) were 

found. The detection limit ofthe assay was I adductll0 7 

nucleotides in a 10 J.Lg DNA sample. This postlabelling 

assay also allows the identification and quantitation of 

adducts formed by novel platinum compounds. We are 

currently investigating the identity and formation of 

DNA adducts fonned by the (promising) novel platinum 

drugs oxaliplatin and lobaplatin in vitro and in cells. 

Publication 

Blomrnaert FA et al. Cancer Research 1993; 53: 

5669-75. 

Notes 



3 TNO-MBL, Rijswijk.

III Division of Cellular Biochemistry 

Head 

WH MooIenaar PhD 


WJ van Blitterswijk PhD, J Borst PhD, JJe Neefjes 

PhD, A Tulp PhD, LN Vernie MSc 


J Alblas MSc, GS Brouns MSc, MCM van Dijk PhD, 

G Feiken MSc, MFBG Gebbink PhD, LA Gravestein, 

M Grommé MSc, PL Hordijk PhD, C Jalink PhD, 

A Khanum PhD, FR Postma MSc, GJA Ramakers 

PhD, D Schaap PhD, L Smit MSc, RW Wubbolts MSc, 

GCM Zondag MSc 


I van Etten (60%), GM Hengeveld, HAM Hilkmann 

(80%), EB Pastoors, 0 Tol, R van der Valk, I Verlaan, 

DJ Verwoerd, EFR de Vries, JMM de Widt, E Wojcik 

(60%) 


S Dusseljee, G van der Horst (60%), GM Koningstein, 

MCR Poland, L Srnith, JCM van der Wal (80%), 

X Zang MSc 

U nder-graduate students and trainee technicians 

JP Bayley, RR Bosch, R van Dijk, JH Hooijberg, 

B Houssa, RPC Oud, EAJ Reits, M Roesink 

Guests 

F Imamura PhD, FJG Muriana PhD, A Neisig MSc, 

F Sanderson MSc MD, M de Weers MSc 

Secretary 

JM Overwater (80%) 

33

34 Ce/lu/ar Biochemistry 

Introduction 

Major themes in the Division of Cellular Biochemistry 

are the mechanisms of signal transduction via cell surface 

receptors, the biochemistry of phospholipase action and 

various aspects of antigen presentation and intracellular 

peptide transport. 

The molecular basis of receptor-mediated signal 

transduction is a subject of great interest for 

understanding normal and deregulated cell behavior. The 

field covers both extracellular molecules, wruch mediate 

cell-cell communication, and the intracellular signaling 

pathways that underly their biological actions, 

particularly the control of cell growth and differentiation. 

Much progress has been made in recent years as new 

signaling cascades are being discovered and their 

biochemical details unraveled. Among our new findings, 

is that a naturally occurring phospholipid (LPA) acts on 

its own G-protein coupled receptor to activate the small 

GTP-binding proteins Ras and Rho, and thereby directs 

cell proliferation and cytoskeletal changes. In addition, 

electrophysiological approaches have revealed that the 

LPA receptor also activates a chloride channel in 

fibroblasts via a new signaling pathway. The resulting 

membrane depolarization may represent a novel early 

event in the proliferative response of quiescent cells. 

Another new finding is that newly generated 

diacylglycerol, derived from phosphatidylcholine 

through phospholipase C action, triggers the mitogenic 

Raf-MAP kinase pathway independently of Ras and 

protein kinase C. This may provide a clue to how Raf 

kinase becomes activated at the plasma membrane. 

Characterization of the mode of action and the 

biological function(s) of a recently cloned receptor-like 

tyrosine phosphatase (RPTPj.l) is a relatively new line of 

research in our division. Recent experiments support the 

view that RPTPj.l has a key role in cell-to-cell signalling 

and th us may contribute to the mechanisms of contact 

inhibition of cell growth. 

That similar signalling cascades are utilized by very 

different receptors, is exemplified by the finding that the 

activated B-cell receptor triggers complex formation of 

SH2-containing adaptor proteins, an event which was 

discovered earlier in growth factor-stimulated fibroblasts. 

Work on the early steps of antigen presentation by 

MHC molecules in our division has revealed the 

specificity and selectivity of peptide translocation via a 

specific transporter in the endoplasmic reticulum. A new 

endocytic compartment, where MHC class II molecules 

are loaded with peptides, has been further characterized 

and was found to be induced by MHC class II molecules 

themselves. Insight into the mechanism by which MHC 

molecules recognize and present antigenic peptides to 

cytotoxic T -celIs is of direct relevance to anti-tumor 

immune therapy. 

Signal transduction by G proteincoupled 

receptors 

WH Mooienaar, J Alblas l , I van Etten, GM Hengeveld, 

PL Hordijk 2 , F Imamura5, K Jalink 3 , A Khanurn, 

EB Pastoors, F Postma4, GJA Ramakers, I Verlaan 

Mode of action of lysophosphatidic acid 

Lysophosphatidic acid (LPA; l-acyl-sn-glycerol-3phosphate) 

is a water-soluble phospholipid with growth 

factor-like activities. LPA is produced and released by 

activated platelets (and presumably other cells) and is a 

normal constituent of serum but not plasma. In addition 

to stimulating the proliferation offibroblasts and smooth 

muscIe cells, LPA suppresses neurite outgrowth of 

neuroblastoma cells and thereby prevents their 

differentiation. Furthermore, it appears that LPA is the 

major serum factor responsible for stimulating tumor cell 

invasion in vitro. LPA activates its own G-proteincoupled 

receptor, which is present in numerous cell types. 

A 38-40 Da putative receptor for LPA has previously 

been identified by us using a photoreactive LPA analog. 

Our efforts to clone the LPA receptor cDNA using the 

COS cell expression cloning strategy have met with little 

success to date. In this procedure, pools of cDNA clones 

derived from a mouse neuroblastoma cD NA library we re 

transiently transfected into COS cells. Positive clones 

we re identified by enhanced binding of 32p_ LPA to single 

cells, as visualized by photoemulsion autoradiography 

and darkfield microscopy. A few cDNA clones that 

conferred enhanced LP A binding were isolated but, 

unfortunately, LPA binding to transfected cells was not 

specific since it could not be displaced by excess unlabeled 

LPA. Sequence analysis of partial cDNA dones did not 

reveal any homology to known proteins in the data bases. 

We must conclude that the COS cell expression cloning 

procedure, which works weil with peptide ligands, is not 

the method of choice of cloning receptors for lipophilic 

ligands like LPA. Alternative PCR-based procedures for 

cloning the LPA receptor are currently being pursued. 

Signal transduction through the LPA receptor focuses 

on two novel cascades that involve activation of the small 

GTP-binding proteins Ras and Rho, respectively. In 

fibroblasts, LP A activates p21 ras and its downstream 

effectors (Ras-MAP kinase cascade) via a pertussis-toxinsensitive 

G j protein. We found that th is pathway is also 

operational in COS cells transfected with a Myc-tagged 

MAP kinase construct. In these cells, LPA-induced MAP 

kinase activa ti on is fully blocked by pertussis toxin and an 

interfering mutant of p21 ras (N17-Ras). An, as yet 

unidentified, tyrosine kinase is thought to mediate the 

coupling between G j and Ras. An obvious candidate is 

the Src tyrosine kinase but overexpression of either 

normal c-src or a 'dominant-negative' src mutant did not 

affect LPA-induced activation of the Ras-MAP kinase 

pathway. From these and other experiments, we feel safe 

to conclude that Src does not function in the LPA 

receptor-Gj-Ras cascade. 

The COS cell system was also used to delineate which 

G-protein subunit(s) mediate(s) Ras activation. We 

found that overexpression of I3 1Y2 heterodimers 

stimulates the Ras-MAP kinase pathway, whereas free (Xj2 

subunits do not. These experiments, although 

preliminary, support the view that Gj-derived I3 1 Y 2 

subunits are responsible for triggering Ras activation by 

LPA. We are currently investigating the GDP/ GTP 

exchange mechanism that may be triggered by production 

of 13 1 Y 2 heterodimers. 

The second novel signaling pathway concerns LPA-

36 Cel/ular Biochemislry 

Introduction of a dominant-negative Src construct was 

found to completely inhibit LPA-induced membrane 

depolarization. On the other hand, expression of viral 

active v-Src results in sustained membrane 

depolarization. These results suggest a link between the 

LPA receptor, Src activation, the arachidonic acid 

cascade and chloride channel opening. The precise 

biochemical and biophysical details of this complex 

cascade are under further investigation. 

Stimulation of cel! growth and transformation by a 

truncated G protein-coupled receptor 

We have made C-terminal deletion mutants of the 

neurokinin A receptor, which serves as a model system for 

examining G-protein-mediated signaling pathways th at 

lead to cell growth and transformation. Unlike the 

wildtype neurokinin receptor, truncated receptors do not 

undergo desensitization in the continuous presence of 

Iigand. Lack of desensitization correlates weil with the 

removal of Ser/ Thr phosphorylation sites. Activation of 

these truncated receptors leads to prolonged activation of 

phospholipase C, enhanced tyrosine phosphorylation of 

cytoskeletal proteins and, most importantly, sustained 

activation of MAP kinase when compared to wild type 

receptors. Activation of MAP kinase is insensitive to 

pertussis toxin, unlike what is observed with LPA. 

Instead, a protein kinase C inhibitor (Ro 31-8220) 

completely inhibits MAP kinase activation. Ultimately, 

this prolonged MAP kinase activation leads to DNA 

synthesis and cell division. The truncated receptors also 

induce focus formation and anchorage-independent 

growth in a ligand-dependent manner (Figure III.l). The 

latter responses are not observed with peptide growth 

factors acting through receptor tyrosine kinases. Focus 

formation is partially inhibited by Ro 31-8220, at doses 

that fully inhibit DNA synthesis. Introduction ofC3 exoenzyme 

to inactivate the Rho proteins has no effect on 

focus formation. 

Focus formation, unlike enhanced DNA synthesis, 

may well result from the production of autocrine factors: 

we recently discovered that conditioned medium from 

fibroblasts expressing truncated neurokinin receptor 

contains a macromolecular polypeptide factor that may 

contribute to the induction of the transformed 

phenotype. This factor, which remains to be identified, 

triggers prominent tyrosine phosphorylation of a 90 kD 

protein in control fibroblasts. The 90 kD tyrosinephosphorylated 

substrate appears to be a member of the 

STAT family of cytosolic transcription factors that move 

into the nucleus upon phosphorylation to direct 

transcription. We are currently exarnining the identity of 

this new factor. Ultimately, these studies should help 

unravel and dissect G-protein-coupled signaling 

pathways that lead to (autocrine) cell growth and 

transformation. 

Publications 

Hordijk PL et al. J Biol Chem 1994; 269: 3534-8. 

Hordijk PL et al. J Biol Chem 1994; 269: 645-51. 

Jalink K et al. J Cell Biol 1994; 126: 801-10. 

Jalink K et al. Biochim Biophys Acta (in press). 

MooIenaar WH. Trends Cell Bio11994; 4: 213-9. 

MooIenaar WH et al. Curr Topics Membranes 1994; 40: 

439-50. 

MooIenaar WH. Curr Opin Cell Biol (in press). 

Notes 

) Funding: Netherlands Organization for Scientific 



3 Funding: Foundation for Biophysics, Project 

810-405-10.1. 

4 Funding: Foundation for Biophysics, Project 

810-405-10.2. 

5 Guest from the Center for Adult Diseases, Osaka, 

Japan. 

Characterization of a receptor-like 

protein tyrosine phosphatase, RPTP 11 

MFGB Gebbink 2 , GCM Zondag), G Koningstein) , 

E Feiken 2 , WH MooIenaar 

Receptor-like protein tyrosine phosphatases (receptor 

PTPs) represent a family of transmembrane proteins th at 

may transduce external signals by dephosphorylating 

phosphotyrosyl residues on intracellular substrates. We 

have cloned a cDNA encoding a new receptor-PTP, 

termed RPTP/-l, with two intracellular phosphatase 

domains. The ectodomain contains one Ig-like domain 

and four fibronectin type III-like repeats similar to those 

found in neural cell adhesion molecules. In addition 

RPTP/-l contains a newly discovered 'MAM' domain at its 

extreme N-terminal end. RPTP/-l transcripts are detected 

in all tissues, with the highest levels in 1ung, brain and 

heart. Using in situ hybridization techniques, RPTP/-l 

transcripts were found both in endothelial and smooth 

muscle cells. 

We have shown that RPTP/-l can act as a homophilic 

cell adhesion receptor by expressing the cDNA in insect 

Sf 9 cells using recom binant baculovirus, suggesting that 

RPTP /-l serve a normal physiological function in signaling 

cell-cell recognition. In a subsequent set of experiments 

we found th at RPTP/-l-expressing cells fail to ad here to 

cells ex pressing the closely related RPTP/-l molecule 

(experiments in collaboration with J Sap and J 

Schlessinger, New York University), suggesting that 

these molecules cannot mediate heterophilic cell-cell 

interactions. We have examined the contribution of the 

'MAM' domain to RPTP/-l mediated cell-cell adhesion. 

Deletion of the N-terminal 'MAM' domain of RPTP/-l 

domain abolishes cell-cell adhesion, suggesting th at this 

domain is essential for the adhesive function of RPTP/-l. 

Adhesion can be resto red by introducing the 'MAM' 

domain of RPTP/-l. Unexpectedly, this chimeric receptor 

fails to mediate adhesion with either RPTP/-l or RPTP/-l. 

These results indicate that the 'MAM' domain is not 

sufficient to determine the specificity of the interaction 

but that the Ig domain and fibronectin domains also 

contribute to cell-cell interaction. 

To generate anti-RPTP/-l monoclonal antibodies, we 

have developed a higWy efficient method to produce large 

amounts ofthe ectodomain of RPTP/-l for use as antigen. 

A construct in which the glutathion S-transferase (GST)

membrane signal transduction, the CD3 or CD79 

components, respectively. The general principle of 

antigen receptor-mediated signal transduction is that 

receptor triggering induces phosphorylation of the 

associated molecules on tyrosine residues within specific 

motif sequences, which is presumably mediated by srcrelated 

protein tyrosine kinases. Subsequently, these 

phosphorylated motifs become points of attachrnent for 

SH2 domain-containing enzymes, initiating signal 

transduction cascades. We are investigating the 

molecular basis of the differential signaling capacities of 

antigen receptor complexes. 

Previously, we have described the structure of the 

human pre-B-cell receptor complex, which consists of 

membrane J1 heavy chain, a pseudo light chain and 

CD79a (mb-I) and CD79b (B29) components. Given the 

role of the pre-BCR complex in the regulation of B-cell 

survival and differentiation, we investigated its potential 

for transmembrane signal transduction. However, cell 

surface expression of the pre-BCR complex appeared to 

be about lOO-fold lower than that ofthe BCR complex on 

mature B cells, which is due to reten ti on of the completely 

assembIed complex in the endoplasmic reticulum. 

Receptor proximal signalling events could not effectively 

be read out in pre-B-cell lines upon triggering of the 

receptor complex with specific antibodies. 

We found th at antigen receptor ligation on mature 

B-cells can induce tyrosine phosphorylation and an 

increase in kinase activity ofthe human Bruton's tyrosine 

kinase (Btk). This src-related protein tyrosine kinase is 

functionally deficient in X-linked agammaglobulinemia 

(XLA) , a severe immunodeficiency disease, hallmarked 

by a B-cell differentiation arrest at the pre-B-celllevel and 

impaired responses to antigen of the small population of 

residual mature B ceUs. Since BCR signalling is essen ti al 

in driving B-cell differentiation, our observation that Btk 

lies in a BCR-induced signal transduction pathway may 

explain the disease phenotype. In XLA patients a variety 

of mutations are found , not all of which affect the kinase 

activity of Btk. A single point mutation in the pleckstrin 

homology domain of Btk is sufficient to give the XLA 

phenotype in humans. Future work will therefore address 

the role of the various functional domains of Btk in 

coupling to upstream and downstream signalling 

components. 

To unravel BCR signaling we have synthesized fusion 

proteins containing the CD79a or CD79b cytoplasmic 

tails, which have been used to screen B-cell-derived 

cDNA expression libraries. The aim of this work was to 

identify novel signalling components, capable of 

associating with the CD79a/ b cytoplasrnic tails. To this 

end, the cytoplasmic tails have also been used as bait in 

the yeast two-hybrid system. Thus far, associating 

components have not been detected. One ofthe problems 

with the two-hybrid system is that CD79a tail mediates 

transactivation independent of protein-protein interaction. 

We are also looking at the mechanism of BCRmediated 

activation of the Ras signaling pathway. We 

have found that BCR triggering induces the formation of 

a complex containing tyrosine phosphorylated Shc and 

Grb2 proteins. In fibroblasts, tbis complex of adaptor 

proteins is involved in the activation of Ras, by means of 

Cellular Biochemistry 39 

the Grb2-associated guanine nucleotide exchange factor 

mSos. We have observed that mSos levels are low in 

human B lymphocytes compared to those of another 

exchange factor, C3G. We suggest that this, or other 

exchangers, is more important for Ras loading in B cells. 

We have studied the role of the pre-TCR in the 

regulation of T-cell differentiation. We found that 

thymocytes of RAG-l-/- mutant mice, which are 

differentiation-arrested at the pro-T -cell stage, carry very 

low levels of CD3 y and e components at the cell surface, 

in the absence of TCR chains, wbich cannot be formed in 

these recombination-deficient mice. Signal transduction 

function of these CD3 components could be induced by 

injection of RAG-l -/- mice with CD3e-specific antibody. 

Strikingly, tbis procedure induced T -ceB differentiation 

from the pro-T -cell stage to the CD4 + 8 + pre-T -cell stage 

and a fifty-fold expansion of the thymic compartment. 

We conclude that CD3ligation on pro-T-ceBs mimics the 

signal transduction function of the pre-TCR, which 

consists of TCRB chain and a novel component, gp33, 

and is known to induce pre-T-cell differentiation. 

Members ofthe Tumor necrosisfactor receptor 

( TNFR) family 

The TNFR farnily encompasses a number of 

transmembrane receptors with homology in the 

extracellular cysteine-rich ligand binding domain. 

Ligands of these receptors are either soluble or 

membrane-bound and related to TNF. TNFR family 

members have been implicated in the regulation of 

programmed cell death. Most dramatic are the apoptosisinducingcapacities 

ofTNFR and Apo-l / fas (CD95). The 

importance of regulation of ceB survival by these 

receptors in the immune system is illustrated by the lpr/ 

lpr and lpr cg mutant mice, which are defective in CD95 

signalling and suffer from aberrant lymphoproliferations 

and auto-immune disease. We are studying 

the function of CD27, which belongs to this family and is 

primarily expressed on T lymphocytes. We have isolated 

the murine CD27 cDNA and generated monoclonal 

antibodies to the murine CD27 (mCD27) protein. With 

these unique reagents, we have shown that mCD27 

cooperates with the TCR/ CD3 complex in inducing 

T-cell proliferation. Immunofluorescence analysis shows 

that the receptor is expressed on the majority of T 

lymphocytes and a small B-cell population. In contrast to 

the situation in humans, mCD27 is expressed throughout 

T -cell differentiation, fr om the pro-T -cell stage onwards. 

We aim to manipulate CD27 function in vitro and in vivo 

with the available monoclonal antibodies. In addition, we 

plan to make CD27-/- mutant mice to assess the 

contribution of CD27 to T-cell differentiation or mature 

T-cell function. To this end, sizable fragments of the 

mCD27 gene have been isolated. 

In collaboration with GT Williams 7 we also investigate 

the regulation of programmed cell death in T 

lymphocytes, using Jurkat T cells as a model system. This 

cellline expresses the TCR complex and the TNFR family 

members CD27, TNFRI and CD95. Jurkat is sensitive to 

TCR- and CD95-induced apoptosis. We have derived 

two series of variant subclones, JP and JA, from this line 

by culturing under selective pressure and repeated

40 Cellular Biochemistry 

limiting dilution. JP clones express TCR/ CD3 complex, 

but are defective in TCR-mediated signal transduction 

leading to apoptosis, as induced by anti-CD3 mAb. JA 

clones express CD95 but no longer undergo apoptosis 

up on triggering of this receptor with specific antibody. A 

number of JA clones are also defective in the TCRinduced 

apoptotic response, indicating that the TCR- and 

CD95-induced apoptosis pathways may have common 

aspects. We have also determined the response to the 

topoisomerase inhibitor and anti-cancer drug etoposide, 

which also induces apoptotic cell death. While wild type 

and JP clones are etoposide sensitive, eight out of ten JA 

clones undergo a cell cycle arrest in G2 upon etoposide 

treatment, but not the normally ensuing apoptosis. We 

are currently investigating the response of the Jurkat 

clones to X-irradiation, another apoptotic stimulus used 

in cancer treatment. This work wiIl aIlow us to define a 

number of complementation groups within the variant 

panel. We will choose clones disturbed in common 

signaling aspects leading to apoptosis as starting material 

for the isolation of cDNAs encoding proteins that confer 

apoptosis-resistance or -sensitivity. 

Publications 

Brouns GS et al. Int Immunol (in press). 

De Weers M et al. J Biol Chem 1994; 269: 23857-60. 

Gravestein LA et al. Int Immunol (in press). 

Jacobs H et al. Eur J Immuno11994; 24: 934-9. 

Smit L et al. J Biol Chem 1994; 269: 20209-12. 

Notes 

I Funding: Netherlands Organization for Scientific 






4 Immunohematology and Blood Bank, Academic 

Hospital, Leiden. 

5 Division of Molecular Genetics. 



7 Keele University, Staffordshire, UK. 

Antigen presentation by Major 

Histocompatibility Complex (MHC) 

molecules 

JJ Neefjes, A Neisig4, F Momburg 3 , GJ Hämmerling 3 , 

CJ Melief 4, XS Zang, E Wojcik-Jacobs, M Grommé I , 

S Dusseljee 2 , D Verwoerd, A Tulp, H Jansen 5 , 

J Calafat 5 , 0 Tol, F Sanderson 6 , J Trowsdalé, 

R Wubbolts 2 

Antigen presentation by MHC class I molecules 

MHC class I molecules present peptides, derived from 

cytosolic or nuclear antigens, to CD8 + T cells. We are 

studying those processes that precede peptide association 

with c1ass I molecules in order to better predict the 

peptides that are actually presented. We have thus 

established an in vitro assay in which the translocation of 

peptides fr om the cytosol to the ER lumen, by a 

heterodimeric multimembrane-spanning protein termed 

TAP, can be followed . By this method, we have 

established the fine specificity of peptide translocation. 

Depending on species, TAP preselects for C-terminal 

amino acids. Other positions do not show such a 

selectivity and all amino acids at position 1 to 8 in a model 

9-mer peptide are aIlowed. NotabIe exceptions are proline 

residues at position 2 or 3, th at negatively affect peptide 

translocation by TAP. Whereas MHC c1ass I molecules 

usually bind peptides of about 9 amino acids, TAP has a 

broader substrate specificity. TAPprefers peptides of 

9-13 amino acids but is able to translocate peptides of 

more than 40 amino acids, albeit with lower efficiency. 

We have studied the fate of peptides af ter their arrival 

in the ER lumen. Peptides are relatively stabIe in the ER 

and are slowly trimmed by peptidases. However, we 

noted that a majority of the peptides are rapidly released 

from the ER, in an ATP-dependent fashion, and appear 

in the cytosol where they are degraded by cytosolic 

peptidases. The nature of these peptidases is unknown 

and we are currently isolating them. However, a fraction 

of the peptides is escaping the cytosolic peptidase activity 

and reappears in the ER lumen. 

The importance of the respective pathways is studied 

by following a large set of peptides, known to be 

presented by MHC c1ass I molecules, for their ability to 

become translocated by TAP. Most, but not all, tested 

peptides we re good substrates for TAP. Four peptides 

were very inefficiently translocated. These peptides had 

one common denominator, a proline residue at position 

3. Repositioning this proline residue to position 4 or 5 by 

additional N-termina! amino acids, aIlowed efficient 

T AP-dependent peptide trans!ocation. Whether this 

reflects the natura! situation, is currently being analyzed. 

For this purpose, an assay is being established in which 

degradation of antigen in vitro is coupled to peptide 

translocation by TAP. 

Antigen presentation by MHC class II molecules 

MHC class II molecules present peptides to CD4 + 

T -ceIls. These peptides are generally generated in the 

endosomal system and we have shown th at class II 

molecules meet endocytosed antigen in a previously 

undescribed compartment. This compartment has a 

multilaminar morphology and a number of lysosomal 

characteristics and was termed MllC for MHC class II 

compartment. We have now shown that the multilaminar 

morphology of the MllC is caused by expression of c1ass 

II molecules. Apparently, information for the formation 

ofthis morphology is located in the transmembrane and/ 

or cytosplasmic region of class II molecules because 

multilaminar structures are not induced by class II 

molecules containing a TM / cytoplasmic region of class I 

molecules. We are currently analyzing in more detail the 

role ofthe tail ofMHC c1ass II molecules in the formation 

of MllC-like structures. 

It has recently been reported that a c1ass ll-like 

molecule, named DM, is a prerequisite for peptide 

loading of class II molecules. We have studied the 

intracellular localization of DM as a first step in

deterrnining how DM is involved in peptide loading of 

class II molecules. DM has an intracellular distribution 

that resembles that of lysosomal proteins; DM 

accumulates in MIIC-like structures. We are currently 

attempting to show how DM induces peptide loading of 

class II molecules. 

MHC class II molecules are able to present peptides 

derived from ER-retained proteins. We have followed 

degradation of ER-retained class II subunits. Both the 

intra- and extracellular region of the class II subunits 

determine the rate of degradation. We have now formally 

shown th at the endoplasmic reticulum contains all the 

ingredients necessary for degradation. 

Publications 

Calafat J et al. J Cell Biol 1994; 126: 967-77. 

Momburg F et al. Nature 1994; 367: 648-51. 

Momburg F et al. J Exp Med 1994; 179: 1613-23. 

Momburg F et al. Curr Op Immunol 1994; 6: 32-7. 

Neefjes J et al. In: 'MHC, a practical approach' (in 

press). 

Neisig A et al. J Immunol (in press). 

Nijenhuis M et al. Eur J Immunol 1994; 24: 873-83. 

Roelse J et al. J Exp Med 1994; 180: 1591-8. 

Sanderson F et al. Science 1994; 266: 1566-9. 

Notes 




3 DKFZ, Heidelberg, Germany. 

4 Academic Hospital, University of Leiden. 

5 Division of Cell Biology. 

6 ICRF, London, UK. 

Separation of eell organelles by physical 

methods 

A Tulp, D Verwoerd 

We have developed a density gradient electrophoresis 

(DGE) apparatus suitable for the separation of 

megadalton proteins, intact cells and subcellular 

organelles. Separation of components of the endosomal 

system, comprising early endosomes, return vesicles, 

endosome carrier vesicles, late endosomes and lysosomes, 

proved particularly rewarding. U sing DG E and pul sechase 

regimes of horse radish peroxidase endocytosis in 

MeI JuSo cells, we found that early and late endosomes 

are pre-existing organelles, which indicates that the preexisting 

compartment model rat her than the maturation 

model is the correct one. Moreover, relatively good 

separations were obtained between plasma membrane 

(read-out by 125I-transferrin bound to the transferrin 

receptor and cx-class I immuno-blotting) and early 

endosomes (probed by l25I-Tt). Using human hepatoma 

cells (HepG2), it was possible to separate vesicles th at 

return from the early sorting endosome to the plasma 

membrane, using l25I-Tf as a pro be under appropriate 

pulse-chase regimes. In collaboration with J Pieters 

(Division IV), the MIIC compartment, where class II 

molecules unite with peptides to form SDS-stable 

Cel/ular Biochemistry 41 

heterotrimers, was separated through a combination of 

DGE and equilibrium density centrifugation. In 

collaboration with J Neefjes the accessory HLA DM 

molecule was localized using DGE. 

A larger DGE apparatus incorporating a number of 

improvements was constructed and tested in 

collaboration with C Schlax and J Pieters. Furthermore, a 

flow-through cu vette was constructed by the Division of 

Biophysics (L Korbee, C Slee and GJF Blommestijn) so 

that cellular compartments could be detected by their 

absorbance when vesicles were emerging from the DGE 

apparatus. This powerful organel Ie separation technique 

will allow further elucidation of intracellular transport in 

relation to class II-restricted antigen presentation. 

Publications 

Calafat J et al. J Cell Bio11994; 126: 967-77. 

Sanderson F et al. Science 1994; 266: 1566-9. 

Tulp A et al. Nature 1994; 369: 120-6. 

Tulp A et al. Encyclopedia of Scientific Instrumentation 

(in press). 

Peptide synthesis 

LN Vernie, R van der Valk 

In 1993 a new peptide synthesizer was installed. For use 

with this synthesizer, the chemistry for peptide synthesis 

had to be changed from tBoc to Fmoc as protective 

moieties. These changes showed that, in general, the 

newly synthesized peptides are of a higher purity than in 

the former situation. 

In the past year, 134 peptides including 66 for research 

institutions and universities outside our institute, have 

been synthesized. These numbers are considerably higher 

than in the past, mainly thanks to the higher synthesizing 

capacity ofthe new equipment. Most ofthe peptides have 

been produced for raising antisera, or for use in biologica I 

studies. 

This year, we also got requests for peptides with special 

features. Most of these could be honored. These peptides 

we re used to study the translocation of peptides from the 

cytosol to the endoplasmic reticulum by the MHC 

encoded peptide transporters or were used to show the 

presence of free auto-antibodies against polymorphic 

epithelial mucin in serum from cancer patients. 

In the near future the technical possibilities of the 

peptide synthesizer will be used to incorporate special 

synthesized amino acids as UV -inducible crosslinkers and 

to set up a strategy for the automated synthesis of 

branched peptides. As part of routine quality control, 

each peptide is checked by HPLC for its purity and 

analyzed for amino acid composition. If necessary, a 

further purification of the peptides is performed by gel 

filtration and HPLC.

IV Division of Immunology 

Head 

AM Kruisbeek PhD 


WR Gerritsen MD PhD (50%), A Hekman MSc, 

EM Rankin MD PhD (50%), H Spits PhD, 

CGJM Vennegoor PhD, FA Vyth-Dreese PhD (60%), 

K Weijer DVM PhD (90%) 


DAmsen MSc, GM van Bleek PhD, B Blom MSc, 

M Haks MSc, M Hamel MSc, M Heemskerk PhD, 

o Huber MSc, D Izon PhD, MA Oosterwegel PhD, 

D Orsini PhD, JD Nieland MSc, PCM Res PhD, 

J Pieters PhD, C Schlax MSc, FJT Staal PhD, I Weirnar 

MSc 

Permanent techniea} staff 

P van den Berk, TAM Dellemijn (40%), 

W van de Kasteele, CJM Leupers (80%), A Pfauth, 

JJ Sein, AC Voordouw, P Weder 

Other technica] staff 

AQ Bakker, M Bakker, A Blom, K Liefkens, 

M Monnee, EMulIer 

Undergraduate students 

R Doumaid, A van der Goes, K Kieboom, P Koks, 

D Kramer, B Kremers, H Kreuwel, T Nieland, 

J Vermeulen, S Weijer 

Guests 

EE Eynon PhD, E Hooijberg MSc, AC Jaleco MSc, 

E Martinez Caceres PhD, C Revilla Calvo PhD, 

G Ruggiero PhD 

Secretaries 

GC Blankenzee (50%), MA van Halem 

43

44 Immunology 

Introduction 

The year 1994 brought the last of several major changes 

that have occurred in the Division of Immunology over 

the past 3 years. CG Figdor left the NKII A vL after a 

most productive and successful career in the field of 

regulation of adhesion and identification of melanomaassociated 

antigens. CG Figdor and his associates joined 

the Oncology / Hematology Division at the University 

Hospital Nijmegen, to establish a new Department of 

Tumor Immunology. The enthusiasm that C Figdor 

brought to his science has had a major impact on this 

division for many years and we look forward to 

continuing collaborations in the future. 

The research directions in the division have now been 

firmly established into four main areas: immunotherapy 

(at both the clinical and the pre-clinical level), 

mechanisms of antigen processing, regulation of 

hematopoiesis and T -cell development and regulation of 

T-cell activation. Together, these topics have generated a 

cohesive program in immunology, with multiple 

interactions between the groups participating in the 

different areas. For instance, the fundamental 

observation of G van Bleek that antigenic peptides 

representing major epitopes for CTL recognition can be 

eluted ofhsp96 isolated from certain tumor ceIls, is sure to 

have a major impact on the design of peptide vaccines 

aimed at generating T -cell responses against tumor 

antigens. The development of a human melanoma model 

in scid / scid and RAGI deficient mice by K Weijer and H 

Spits also impacts on the immunotherapy program ofthe 

division, as does the comparative study of genetically 

modified tumor cells transduced either with GM-CSF or 

various co-stimulatory and adhesion molecules by AM 

Kruisbeek and associates. Several associates in the 

division are involved in the T -cell function analysis of 

patients enrolled in the melanoma vaccination protocols, 

iniated by E Rankin and A Hekman, and the groups of H 

Spits and WR Gerritsen have started a collaboration on 

the generation of hu man T cells from peripheral blood 

stem cells. 

The direction of the T -cell development studies of the 

division has been guided by several recent findings. First, 

in human studies by F Staal, PRes and H Spits, it was 

demonstrated that a reporter gene could be successfully 

introduced into T cells developing from immature 

precursors with the help ofthe MFG retroviral vector. An 

analysis of the molecular regulation of human T-cell 

development will now be initiated, since introduction of 

genes selectively activating or inactivating expression of 

regulatory proteins is feasible. The method employed is 

highly efficient, with up to 40% of the progeny of 

transduced stem cells developing in fetal thymic organ 

cultures ex pressing the reporter gene. In a second 

development, a monoclonal antibody against mouse 

CD34 was generated, providing the opportunity to put 

mouse and human studies of early hematopoiesis in the 

same context. Human T cells deveJop from CD34 +CD38pluripotent 

stem ceUs and in the mouse, all T-cell 

progenitors also appeared to be contained (at least in the 

embryonic stage) in a CD34 + population of fetal liver 

cells. Finally, studies on the identification of molecules 

involved in thymocyte-stromal cell interactions took an 

unexpected turn wh en cloning of the first two of the 

cDNAs directing expression of thymic stromal antigens 

identified these proteins as members ofthe Ly-6 family of 

PI-anchored membrane proteins. The functional ability 

of this family of proteins has only been investigated on 

cells belonging to the T- or B-celIlineage, but no ligand(s) 

has been identified. We recently showed that expression 

of Ly-6 on thymic stromal cells can be regulated by 

several cytokines, and shall now focus on analyzing the 

functional role of these molecules in in vitro models of 

T -ceB differentiation. 

Identification of co-stimulating pathways other than 

those involving the dominant co-stimulatory interactions 

between CD28 and CTLA4 (on the T cell side) and B7-1 

(CD80) and B7-2 (on the APC side) has continued. CD40 

can function as a co-stimulatory molecule for the 

proliferation of CD4 T cells, but not for CD8 T cells. 

TCR induced apoptosis, however, could not be costimulated 

by CD40. While it has become c1ear th at 

negative selection of both human and mouse T cells does 

require co-stimulation, the molecules involved remain to 

be identified: our earlier studies (lones et al. Int Immunol 

1993; 5: 503-12), confirmed in other labs, appear to 

exclude an involvement of CD80 and the present studies 

found no role for CD40. A cDNA c10ning method 

developed for identification of molecules contributing to 

the co-stimulatory ability of T-lymphoma cells for CD8 

cells identified a new GTP binding protein. It is unc1ear 

how this protein transduces signals th at control costimulatory 

function, but recently derived stabie 

transfectants of cells lacking this function will allowan 

investigation of the mechanism(s) involved. 

Immunotherapy 

GM van Bleek l , PF Bruning 2 , WR Gerritsen, 

M Heemskerk 3 , A Hekman, E Hooijberg, 

AM Kruisbeek, D Orsini, EM Rankin, H Spits, 

FA Vyth-Dreese, K Weijer, I Weimar 4 , 

AEGKr Von dem Borne s , WM Dercksen S ,6, 

ClM Meliefl, GC de Gast 8 , W Schaesbergen S , 

E Van der SchootS, P van den Berk, A Blom 3 , 

TAM Dellemijn, W van de Kasteeie, M Monnee l , 

El Muller4, JJ Sein, P Weder 

In vitro and in vivo experiments using CD 19 and 

CD20 monoe/onal antibodies 

Previous experiments had shown that on normal and 

malignant B cells the density of CD20 antigens is higher 

than that of CD19 antigens. Moreover, CD20 antigen is 

not susceptible to antibody induced modulation. Using 

human target cells and interleukin-2 activated mouse NK 

cells as effectors, target cells sensitized with CD20 mAb 

were more efficiently killed than target cells coated with 

CD19mAb. 

We therefore investigated the effect of antigen density 

on the outcome of antibody dependent cellular 

cytotoxicity (ADCC) by using human B-cell targets 

transfected with cDNA encoding the human CD 19 

antigen (in collaboration with M Visseren, Leiden). The

expression of the CD 19 antigen on these transfectants 

reached the level of CD20 antigens. Using these 

transfectants, the level ofcytotoxicity reached with CD19 

mAb was the same as that with CD20 mAb, 

demonstrating the contribution of antigen density on 

target cells to susceptibility to ADCC. 

The efficacy of monoclonal anti body based 

immunotherapy of B-cell lymphoma has been tested in 

nude mice bearing large tumor burdens. Treatment 

started on day 18 after inoculation of Daudi tumor cells, a 

B-celllymphoma, when tumors had an average size of 81 

± 46 mm 3 . We tested the effect of B-cell specific 

monoclonal antibodies, one against CD19 and two 

against CD20, interleukin-2 (IL-2) and granulocyte/ 

macrophage colony-stimulating factor (GM-CSF) as 

single agents and various combinations of these agents. 

Treatment with anti-CD20, IL-2 and GM-CSF alone and 

the combinations (anti-CDI9 + IL-2), (anti-CD20 + 

IL-2) and (anti-CD20 + GM-CSF) all led to a significant 

reduction in the growth rates of tumors. Combination of 

a given mAb and a given cytokine always resulted in 

larger decreases in growth rates than either agent alone. 

Of all combinations tested the best results we re obtained 

with the combination of CD20 and IL-2; from 11 out of 

21 animals the tumor regressed completely. Cured 

animals from 2 experiments we re kept until day 90 or day 

120 aft er initial inoculation of tumor cells without 

recurrence. Thus, application of B-cell specific mAbs, 

together with certain cytokines, may be a powerful tooI 

for treatment of advanced B-celllymphomas. 

Following this premise, the Haematology Group 

Amsterdam (AMC, CLB, VU, AvL) has initiated a 

clinical trial using chimeric mouse/ human CD20 mAbs in 

combination with cytokines in the treatment ofmalignant 

lymphoma (collaboration with IDEC Pharmaceutical 

Corporation, San Diego, CA, USA). 

Functional expression of adhesion receptors and costimulatory 

molecules by fresh and immortalized 

B-cell non Hodgkin 's lymphoma cells 

To study the antigen presentation function of B-cell 

non-Hodgkin's lymphoma (B-NHL) cells, an in vitro 

model was set up in which the proliferative responses of 

nonnal donor T lymphocytes to B-NHL cells was 

measured. In this mixed lymphocyte-tumor cell culture 

system the stimulator capacity of resting B-NHL cells was 

compared to th at of short term anti-CD40 and rIL-4 

activated cells and long term cultured Epstein Barr virus 

immortalized malignant B-cell clones of the same patient 

(in co-operation with CA Feltkamp, Division I, JW van 

Oostveen, Molecular Pathology, Free University, 

Amsterdam, and M de Boer, Innogenetics, Gent, 

Belgium). Phenotypic analysis and blocking studies using 

monoclonal antibodies suggested an important role for 

accessory molecules, which we re highly expressed by the 

activated B-NHL cells and upregulated on resting 

B-NHL cells during co-cultivation with normal T cells. 

HLA-DR mediated T-cell proliferation, induced by the 

malignant B cells, required the involvement of adhesion 

receptors (LFA-1, activated LFA-l, LFA-3, ICAM-1) 

and co-stimulatory molecules (B7-lICD80, B7-2 / CD86, 

CT LA 4, CD40, CD40 ligand). A co-ordinate 

Immunology 45 

upregulation of accessory molecules during cocultivation 

on both B-NHL and T cells suggested that 

bidirectional signaling plays an important role in 

interactions between T lymphocytes and malignant B 

cells. These resting and activated clonal B-NHL cells will 

be useful in further dissecting the requirements for 

B-NHL co-stimulation. 

To examine the expression of co-stimulatory molecules 

in normal lymphoid tissues, immunoperoxidase staining 

was performed on human tonsil cryostat sections. In this 

study (in collaboration with D de Jong (Division VI), 

primary monoclonal antibodies we re used directed 

against various co-stimulatory molecules, including 

B7-lICD80, B7-2 / CD86, CD 28, CT LA 4, CD40 and 

CD40 ligand. All of these molecules are involved in 

cognate and contact dependent T -cell / B-cell interactions 

(Cl ark and Ledbetter. Nature 1994;367: 425-8). The 

localization of these molecules to distinct intra- or 

interfollicular areas of the tonsil germinal centers 

suggested specialized functions. The results from this 

study will form the basis for further immunohistochemical 

analysis of human B-NHL tumor tissues. 

Exploring autoimmunity against thyroid antigens 

for potential application in cancer immunotherapy 

Characterization of the antigens and the immune 

mechanism involved in thyroid degenerative conditions 

could give information relevant for the development of 

vaccines that may be applied in several forms of 

differentiated thyroid cancer. Two follicular thyroid 

carcinoma cell lines of human origin that produce the 

major thyroid antigens thyroid peroxidase (TPO) and 

thyroglobulin (Tg), were used to explore the development 

of anti-thyroid models in mice. One of these, a 

spontaneously outgrowing cell line containing a P53 

mutation, grows rapidly in scid/ scid and RAG 1 deficient 

mice. This tumor metastasizes to the lung, has a follicular 

morphology closely resembling nonnal thyroid tissue and 

expresses MHC class 11 molecules in situ. This RAG 1 

deficient or scid / scid tumor model will be used for 

adoptive transfer experiments with 11 human T-celllines 

and clones that have been established in vitro from tumor 

infiltrating lymphocytes, peripheral blood samples of 

Hashimoto patients, or thyroid infiltrating T cells from 

Grave's patients. Polyclonal expansion of those three 

categories of T cells has been perfonned. This material 

will be further expanded in an antigen specific way, using 

EBV B-celllines transfected with cD NA of human TPO 

or partial constructs of human Tg cDNA. Production of 

these celllines is underway. Once T-cell clones have been 

established that have anti-tumor effects in vitro and in vivo 

we will proceed to characterize the antigens th at are 

recognized. 

Immunotherapy in melanoma patients 

Two trials of active immunization of melanoma 

patients have been initiated this year (see Division X). In 

one study, patients who express the HLA-Al allotype and 

whose tumor expresses the MAGE 1 or MAGE 3 gene are 

vaccinated in a series of protocols using immunogens 

containing various fonns of the MAGE 1 or MAGE 3

population consisting of hemopoietic progenitor cells of 

all cell lineages, we analyzed the phenotype of the 

mobilized CD34 + peripheral blood progenitor cells 

(PBPC). The phenotyping revealed that a small 

proportion (1-5%) of the cells had the phenotype 

(MDR-l +, DR-, CD38-, CD45RO+) of very early 

hemopoietic progenitor cells. The majority (60-80%) of 

CD34 + eells had the phenotype of myeloid progenitor 

ce1ls (CD33 +, CD \3 +). 1-5% of the CD34 + cells coexpressed 

lymphoid antigens (CD7 +, CD 19 +), 

approximately 20% we re positive for the CD71 antigens 

(erythroid celllineage) and 5-20% had the CD4l or CD61 

molecule (megakaryocyte lineage) on their cell surface. 

Next, the corre\ation between the number of subpopulations 

of CD34 + cells l kg and early regeneration of 

peripheral blood counts was studied. It was hypothesized 

that the presence of committed progenitor cells in a PBPC 

graft would correlate with early recovery since these 

committed cells would only have to divide a few times 

before becoming a mature neutrophil or platelet. The 

number of CD34 + I CD41 + ceIls / kg (committed to 

megakaryocytic ce1l lineage) correlated very well with 

platelet recovery (r=0.81). However, early neutrophil 

recovery was not correlated at all with the presence of 

PBPC committed to the myeloid lineage (CD34 + I 

CD33 +, CD34 + l CD \3 +), although the number of 

CD34 + I CD33- cells / kg (phenotype of early progenitor 

cells) correlated weIl with early neutrophil recovery 

(r = 0.70). These results suggest th at ex vivo expansion of 

megakaryocytic progenitor cells would enhance recovery 

of platelets, but that ex vivo expansion of myeloid 

progenitor cells would not contribute to early recovery of 

neutrophils. 

Homing of PB PC involves the adhesion ofhemopoietic 

cells to vascular endothelial cells, migration through the 

vascular endothelium, entry in bone marrow and 

adherence to bone marrow stromal cells. Adhesion 

molecules play a crucial role in this process. We, 

therefore, investigated the expression of adhesion 

molecules on CD34 + cells. This analysis revealed a 

significant difference in expression of the VLA-4 

molecule on bone marrow CD34 + cells and PBPC 

CD34 + cells, but not for other adhesion molecules such 

as PECAM, HCAM, sialyl Lewis X, L-selectin, VLA-5 

and LFA-1. VLA-4 is expressed on approximately 60% of 

the CD34 + cells in steady state bone marrow or in bone 

marrow at the time of PBPC harvest, while only 20% of 

the PBPC CD34 + cells stain with the VLA-4 molecule. 

Additional studies on the role of adhesion molecules on 

CD34 + cells and their capacity for early engraftment, 

revealed the role of the L-selectin molecule. The number 

of CD34 + IL-selectin + ceIls / kg correlated both with 

platelet recovery (r = 0.80) and neutrophil recovery 

(r=0.71). 

In a multivariate analysis, the absence of the CD33 

molecule and the co-expression of the L-selectin molecule 

correlated better with neutrophil recovery than analysis 

of the number of CD34 + cells/ kg. The co-expression of 

the molecules CD41 and L-selectin gave additional 

information about the recovery of platelets. Further 

analysis suggest that additional phenotyping of CD34 + 

cells will help to identify PBPC grafts which contain a 

relatively low number of CD34 + cellsl kg but with a high 

potentialof early engraftment. 

Immun%gy 47 

Publications 

Dercksen WM et al. In: Leukocyte Typing V (in press). 

Gerritsen WR, O'Reilly RJ. Blood 1994; 84: 1906-12. 

Gerritsen WR et al. Bone Marrow Transpl 1994; 13: 

441. 

Hooijberg E et al. Cancer Res (in press). 

Vlasveld LT et al. Ann Onco11994; 5: 179-81. 

Vlasveld L T et al. Cancer Immunol Immunother (in 

press). 

Vyth-Dreese FA et al. Eur J Immunol 1993; 23: 3292-9. 

Notes 

I Funding: Dutch Caneer Society, Project NKI 93-528. 

2 Division of Medical Oncology. 



5 Central Laboratory of the Netherlands Red Cross 

Blood Transfusion Service, Amsterdam. 

6 Funding: European Cancer Center. 

7 Department of Immunohematology and Bloodbank, 

Academic Hospital Leiden. 

8 University Hospital, Utrecht. 

Antigen processing 

GM van Bleek l , J Pieters 2 , AM Kruisbeek, C Schlax 3 , 

K Liefkens, M Monnee l 

lntracellu/ar transport of MHC class IJ comp/exes 

Major histocompatibility complex (MHC) cJass II 

molecules present antigen ic peptides to CD4 + T cells at 

the plasma membrane of antigen presenting cells (APC). 

These antigenic peptides are believed to be derived from 

antigens that have been endocytosed by APC. The 

invariant chain (Ii) that is associated with MHC cJass II 

molecules intracellularly is believed to play an important 

role in the intracellular transport of MHC cJass II 

molecules. Our research focuses on 1) identification ofthe 

intracellular site where MHC cJass II molecules associate 

with antigenic peptides; 2) analysis of the transport 

pathways of MHC cJass II complexes and invariant 

chain; 3) dissection of the signals and mechanisms that 

mediate these transport pathways. 

We isolated (in collaboration with A Tulp and D 

Verwoerd, Division III) an intracellular compartment 

from human melanoma cells (MeI JuSo) enriched in 

MHC cJass II molecules. In this compartment, the cJass II 

associated invariant chain is degraded, while cJass II 

molecules have acquired peptides. This compartment 

therefore represents a novel stage of the endocyticl 

lysosomal pathway. Preliminary results suggest that a 

unique set of polypeptides is enriched in this 

compartment, the identity of which remains unknown. 

We are presently analyzing the composition of these 

MHC cJass II compartments in different cell types. In 

addition, we transfected MeI JuSo cells with HLA 

DRw51 molecules with or without an antigen from which 

peptides are presented (in collaboration with F 

Uytdehaag, Erasmus University Rotterdam). Since T-cell

48 Jmmun%gy 

lines specific for these peptides are available, these 

transfectants can be used to reconstitute antigen 

presentation in vitro using isolated MHC class II 

com partmen ts. 

An important function of the MHC class II associated 

invariant chain (Ii) is to target the class II molecules to the 

proper intracellular compartment where they can 

associate with antigenic peptides. It was shown previously 

that endosomal targeting information is present within 

the N-terminal cytoplasmic tail ofIi. We generated BHK 

cells transfected with wild type and a mutant Ii form 

lacking the endosomal targeting signals. These cells are 

currently used to dissect the molecular mechanism th at 

regulate targeting to endosomal compartments, using an 

improved procedure to isolate large quantities of 

endosomal/lysosomal organelles. This subcellular 

fractionation procedure is currently combined with 

biochemical approaches to gain further insight in the 

mechanism of endosomal targeting of Ii. 

Secretory mechanism of basic fibroblast growth 

factor 

Proteins destinated for export to the extracellular space 

are usually co-translationally translocated across the 

membrane of the endoplasmic reticulum by virtue of the 

presence of a hydrophobic signal peptide on the 

polypeptide. Basic fibroblast growth factor (bFGF) lacks 

such a signal sequence yet is secreted from cells. Our aim 

is to study the mechanism of secretion of th is protein. We 

showed that in a hepatoma cell line three translation 

products of bFGF mRNA are present, one of which can 

be found secreted in the culture medium. Morphological 

analysis at the light microscopy level indicates th at, 

intracellularly, bFGF is associated with vesicular 

structures. We are in the process of further characterizing 

these structures. 

Heat shock proteins as a tooI to identify tumor 

antigens and as an adjuvant for peptide vaccines 

Vaccination of mice with heat shock proteins (HSPs), 

gp96, hsp70 and hsp90, isolated from certain tumors can 

induce resistance to a subsequent transplant of these same 

tumors. This tumor specific immune response is mediated 

by CD8 + cells and is specific for the tumor fr om which 

the HSPs are isolated. The antigenicity of HSPs is not, as 

originally thought due to mutations in the heat shock 

proteins themselves but seems to be residing in the pool of 

short peptides that associate with HSPs. 

We recently started a project th at aims at elucidating 

the route of delivery of antigenic peptides by HSPs and 

characterization of the antigen presenting ceUs (APC) 

that play a role in the presentation of peptides th at are 

delivered by HSPs in vivo. Furthermore, we will 

characterize peptides th at are intracellularly associated 

with HSPs. We isolated the major ER residing stress 

protein gp96 from ceUs infected with vesicular stomatitis 

virus (VSV). In the complete mixture of peptides we 

detected the major antigenic peptide that is presented by 

the murine MHC molecule H-2Kb using a VSV specific 

CTL clone. We will characterize the product that 

associates with gp96 and compare this with the peptide 

th at is ultimately presented at the cell surface by H-2Kb. 

From hsp70 molecules we can elute the intracellularly 

bound peptides using A TP treatment. An iodinated 

peptide can be loaded efficiently onto these empty hsp70 

molecules. Such hsp/ single peptide complexes will be 

used to study the antigen presenting cells involved in the 

presentation of peptides delivered by HSPs and the 

efficiency of HSPs in improving peptide vaccines. 

Publication 

A Tulp et al. Nature 1994; 369: 120-6. 

Notes 

I Funding: Outch Cancer Society, Project NKI 93-528. 

2 Funding: NWO (Gebied Medische Wetenschappen), 

Project 900-509-175. 


Project 900-509-186. 

T -eell aetivation 

E Eynon I, MC Haks 2 , M Hamel, 0 Huber 3 , 

AM Kruisbeek, JO Nieland 4 , G Ruggier0 5 , H Spits, 

R Offringa 6 , T Rinke de Wit?, CJM Leupers 

Activation and reactivation potentialof T cells 

responding to Staphylococcal Enterotoxin B 

Superantigens (SAs) have in common biological 

features that distinguish them from conventional 

antigens: SAs induce vigorous proliferative responses in 

all T cells expressing particular V fJ chains, both in vitro 

and in vivo. In vivo these responses are followed by partial 

clonal deletion and induction of non-responsiveness in 

mature peripheral T cells. 

To elucidate the parameters that lead to superantigen 

induced non-responsiveness, an in vitro model for 

studying primary and secondary responses to the 

bacterial superantigen Staphylococcal Enterotoxin B 

(SEB) was established. In vitro SEB primed T cells show, 

upon reactivation with SEB, an early proliferative 

response that 'quenches' in time and is severely impaired 

three days after restimulation. Despite their overall 

impaired proliferative capacity and IL-2 production, 

these T cells are able to produce IFNy-and to upregulate 

activation markers C069 and IL-2Ra upon restimulation 

with SEB, illustrating that SEB-non-responsiveness is not 

absolute. Interestingly, T cells pretreated with platebound 

anti-C03e or anti-V fJ8 we re fully responsive to 

anti-CD3 and anti-V fJ8 restimulation but non-responsive 

to SEB restimulation. Thus, non-responsiveness to SEB 

seems to reflect an intrinsic inability of previously 

activated T cells to respond to SEB, probably reflecting 

differences in signal transduction pathways used in naive 

versus memory T cells. Whether these differences in 

responsiveness are related to the differential expression of 

isoforms of the membrane-bound tyrosine phosphatase 

C045 on naive and memory T cells is currently under 

investigation.

Alternative eo-stimulation 

Co-stimulatory signals can be generated as a 

consequence of interactions between the T-cell antigens 

CD28 and CTLA4 and their ligands B7-1 and B7-2. Yet, 

our earlier studies demonstrated that certain T 

lymphomas can provide co-stimulation for T cells, despite 

the absence of these dominant co-stimulatory molecules. 

To identify the molecules mediating co-stimulation by 

T lymphomas, we prepared a cD NA library ofRMA-S in 

pcDNA-l and set up a system to identify co-stimulatory 

molecules by functional screening. We screened about 

25,000 cDNA molecules in pools of 50, and isolated 12 

cDNA molecules, which upon COS transfection enabled 

COS ceUs to co-stimulate ConA or anti-CD3 activated 

T -cell responses. From the partial sequences we obtained 

thus far, we identified a protein L19 (0.4 Kb) known as a 

calmodulin binding protein; a molecule with homology 

with a GTP binding protein ofthe RAS gene superfamily 

(1.2 Kb); a molecule with weak homology with G-CSF 

(0.9 Kb); a molecule which is homologous with the gene 

LLREP3 (0.4 Kb) and one unknown cDNA (1.7 Kb). 

Interestingly, the GTP binding molecule is not expressed 

in RMA while it is expressed in RMA-S. This expression 

pattern correlates with differences in RMA-S versus 

RMA co-stimulation: RMA is not able to co-stimulate 

CD8 ceUs while RMA-S is. The defect in RMA can be 

reconstituted by transfecting the cD NA encoding the 

GTP-binding protein mentioned above. Sta bIe 

transfectants of RMA are currently being tested for 

protein analysis and for biological function. 

CD40 expressed on thymie epithelial eeUs is 

involved in eo-stimulation of anti-CD3 indueed 

proliferation of CD4 + thymoeytes but CD40 does 

not eo-stimulate aetivation indueed apoptosis 

Human thymic epithelial cells express CD40, an 

antigen involved in functional interactions between Tand 

B ceUs. In this study we examined the possible function of 

CD40 in TCR dependent thymocyte activation. We 

observed th at both CD4 +CD8- and CD4-CD8 + 

thymocytes could be stimulated to proliferate in vitro by 

anti-CD3 mAb in the presence of cultured thymic 

epitheIiaI cells. Co-stimulation of CD4 + thymocytes by 

thymic epithelial cells was partly inhibited by an anti 

CD40 mAb, while this mAb had no effect on costimulation 

of CD8 + thymocytes. The co-stimulatory 

ability of the CD40 molecule was confirmed by using 

murine P8I5 cells transfected with human CD40 antigen. 

The level of co-stimulation induced by P815 CD40 is 

comparable to that induced by P815 ceUs expressing 

CD80 (B7). 

We also examined whether CD40 and/ or CD80 are 

involved in activation induced apoptosis. Co-stimulation 

by CD80 strongly increases activation induced death of 

fetal thymocytes. However, co-stimulation by CD40 does 

not increase activation induced apoptosis of thymocytes. 

To confirm th at CD40 does not affect anti-CD3 induced 

cell death, a Jurkat leukemic cellline was established that 

constitutively expressed CD40L. The CD40L + Jurkat 

ceUs produce high levels of IL-2 upon activation with 

anti-CD3 in the presence of either P8l5-CD40 or P815- 

lmmunology 49 

CD80. However, in contrast to CD80, CD40 failed to 

increase anti-CD3 mediated apoptosis in CD40L + Jurkat 

cells. Thus, CD40 can co-stimulate TCR/ CD3 mediated 

activation of human thymocytes, but preferentially drives 

proliferation rather than apoptosis ofCD4 + thymocytes. 

Generating toleranee for self antigens: aetivation 

requirements 

Experimental allergic encephalomyelitis (EAE) is a 

model for cell mediated autoimmune disease which can be 

generated in certain strains of mice, including the H-2 s 

strain SJL/ J, by injection of myelin basic protein (MBP). 

We recently developed a model that wiU allow us to 

investigate why autoreactive, disease-inducing MPB 

reactive T cells have not been removed from the T-cell 

repertoire. MBP transgenic mice have been produced in 

which the transgene is expressed under the con trol of the 

immunoglobulin enhancer and c-myc promotor or under 

the control ofthe MHC class II promotor. At the moment 

these mice are screened for expression of MBP at the 

DNA, RNA and protein level. Another transgenic strain, 

in which the MBP transgene is linked to part of the gene 

encoding the invariant chain, under the control of the 

MHC class II promotor, is in preparation. By targeting 

the antigen to a compartment which is involved in the 

class II processing pathway, this form of MBP may be 

efficiently processed in the same way as an exogenous 

protein. U sing these transgenic mi ce we will analyse how 

the T -cell repertoire is affected by the presence of MBP in 

cell types and cellular compartments normally not 

expressing this protein (or peptides derived from it). In 

addition, several panels of T-cell hybridomas have been 

produced that are directed against peptides derived from 

either endogenous MBP or exogenous MBP. Together, 

these studies should help identify the condition for 

generating MBP specific tolerance. 

Notes 

I Funding: American Cancer Society. 


Project 900-507-178. 



Project 900-505-273. 

5 Funding: Associazone ltaliana Ricerca sul Cancro 

(AIRC), ltaly. 

6 Department of Immunohematology and Blood bank, 

University Hospital Leiden. 

7 Department of Immunology, Erasmus University, 

Rotterdam. 

T -eell differentiation 

DAmsen I, B Blom 2 , AC Jalec0 3 , EE Eynon4, 

MC Haks 5 , M Hamel, DJ Izon, AM Kruisbeek, 

E Mártinez-Cáceres 6 , M OosterwegeF, PCM Res 8 , 

H Spits, FJT Staal, K Weijer, PW Kincade 9 , D Graf 1o , 

RA Kroczek 10, AQ Bakker 2 , M BakkerS, A Pfauth, 

AC Voordouw

50 Immunology 

Characterization of the most primitive 

hematopoietic progenitor cell in the human thymus 

It is at present not known whether the progenitor cell 

th at colonizes the thymus is a pluripotent stem cell, a 

more differentiated but still multipotent progenitor cell, 

or a committed T-cell progenitor. To address this 

question we used a human/ mouse hybrid fetal thymic 

organ culture (FTOC), in which we can follow 

development of human T-cell precursors into more 

mature thymocytes in mouse fetal thymic lobes depleted 

of endogenous thymocytes. Fetal liver CD34 + cells were 

separated in CD38- cells (containing pluripotent stem 

cells) and CD38 + cells (containing more differentiated 

progenitor cells) and tested for their capacity to develop 

into T cells in mouse fetal thymic organs. It was found 

that only CD34 +CD38- fetalliver cells have the capacity 

to develop into T cells. The fetal and postnatal thymus 

contains a minute population ofCD34 + cells th at express 

low levels of CD38, representing less than 0.01 % of total 

thymocytes and 0.3 - 1 % of the total number of CD34 + 

thymocytes. The remaining 99% of the CD34 + 

thymocytes expresses CD38. Both CD34 +CD38- and 

CD34 +CD38 + thymocytes could develop into Tand NK 

cells in the human/mouse FTOC. Moreover, 20-30% of 

the CD34 +CD38- thymocytes can differentiate into 

dendritic cells in a combination of stem cell factor, TNFcx 

and GM-CSF. These findings suggest that CD34 +CD38thymic 

cells are the most primitive hematopoietic cells 

present in the thymus. To determine whether the 

CD34 +CD38- thymocytes contain pluripotent stem cells, 

we compared the phenotype of CD34 +CD38- cells from 

liver and thymus. It was found that CD34 + CD38thymocytes 

express CD45RA but not Thy-l. By contrast, 

pluripotent stem cells from the fetal liver express low 

levels of Thy-I and are negative for CD45RA. Our data 

therefore suggest that the most primitive hematopoietic 

cells in the thymus are multipotential, because they can 

differentiate into T, NK and dendritic cells, but are 

distinct from pluripotent stem cells. 

Phenotypic andfunctional characterization of 

mouse f etalliver derived stem cells 

Comparisons ofhuman and mouse T-cell development 

are complicated by the dissimilarity of the tools available 

for phenotypic characterization. The earliest intrathymic 

progenitor identified at this time in the mouse appears to 

be present in a c-KIT+CD44 +CD25-TN population that 

still has TCR genes in the germline configuration but 

al ready expresses RAG genes. In the human thymus, the 

earliest progenitor is contained within the CD34 + 

CD38-CD5- population. In preparation for a more 

adequate comparison between human and mouse T-cell 

development, we are now analyzing, with the help of a 

recently developed anti-mouse CD34 mAb, expression of 

mCD34 in the mouse during embryonic development. In 

addition, subpopulations of fetal liver (FL) derived 

CD34 + cells are tested in a recently developed in vitro 

T-cell differentiation assay in order to determine their 

repopulating capacity. In th is assay, FL cells are allowed 

to repopulate thymic stroma derived from RAG I 

deficient (and therefore Iymphocyte-deficient) mice and 

mltlatlOn of TCR-f3 chain gene rearrangements is 

analyzed by PCR. No T cells can be derived from the 

CD34- FL population, while both c-kit+ and c-kit 

CD34 + FL cells were capable of developing into T cells. 

Future studies will exarnine at which stage of their 

development CD34 + cells loose pluripotent stem cell 

function . 

Molecular regulation of early T-cell development in 

the mouse 

In our earl ier studies we observed th at CD3'}' is one of 

the earliest T -cell-specific genes expressed during T -cell 

development. Different CD3 proteins may affect T-cell 

development differentially; knockout mice lacking 

CD3'}', 15 and 8 exhibit an arrest at the pro-T-cell stage 

(personal communication B Malissen), while CD315 

knockout mice proceed to the DP stage of T-cell 

development (personal communication S Tonegawa). An 

analysis of the function of the expression of the CD3'}' 

gene in T-cell differentiation has been initiated. A 

genomic CD3'}' clone representing the first exon of this 

gene has been isolated. Mutants are currently being 

prepared and will be used to generate knockout mice. In a 

second strategy, isolation of genomic CD3'}' clones that 

represent the exon encoding for the transmembrane 

region of this protein (exon 4) have been isolated and 

knockout mice lacking this exon will also be prepared. 

The consequences of transmembrane signalling 

through the mature and immature CD3 complex of 

representative early and late T-cell lines, as well as fetal 

thymocytes, is currently being studied. Crosslinking with 

145-2Cll (anti-CD38) can induce IL-2 production and 

upregulation ofCD25 and CD69 in a day 14 fetal thyrnic 

cellline 34.1 L. This is consistent with our earl ier findings 

on induction of cytokine production in fresh fetal 

thymocytes. The 34.1 L cell line is CD3-TCRcxf3- by 

surface staining, although CD38 but not TCRf3 could be 

detected by intracellular staining. Northern blots reveal 

the presence of mRNA for all 3 CD3 proteins and an 

absence ofmRNA for TCRf3, indicating th at th is cellline 

represents the pro T-cell stage. Presence of CD44 

confirms this staging and anti-CD38 crosslinking induces 

transition of the CD44 +CD25- to the CD44 +CD25 + 

stage. Instead of cytokine production, cell death is 

induced by anti-CD38 crosslinking in a day 18 fetal 

thymic cell line 18.2 (CD3 + ,TCRcxf3 +). Additional 

parameters associated with T-cell maturation are 

currently being investigated in this cellline. 

Retroviral mediated gene transfer as a tooi to study 

human T-cell development 

Retroviral vectors have been used in most human gene 

therapy trials that have been undertaken thus far. Many 

of these therapies have focused on introduction of genes 

into hematopoietic stem cells with the goal to obtain 

expression in the mature T -lymphocytic progeny. It has 

proven difficult to achieve expression in the lymphoid 

lineage, although several groups have demonstrated low 

expression of the transduced gene in the myeloid lineage. 

We therefore decided to use an in vitro fetal thymic organ 

culture (FTOC), in which stem / progenitor cells can

develop into T cells to investigate the fate of a retrovirally 

introduced E Co/i LacZ gene in this system. The LacZ 

gene is useful in this respect because its expression can be 

measured on a Fluorescence Activated Cell Sorter 

(F ACS) in conjunction with cell surface markers. 

U sing the MFG-LacZ retrovirus, we successfully 

established conditions for introduction of the LacZ gene 

(which encodes p-galactosidase) into the human 

T -leukemia line Jurkat, three different antigen specific 

T-cell clones and immature CD3-4-8-TN- thymocytes. 

Eighty per cent of immature CD3-CD4-CD8- (triple 

negative TN) thymocytes transduced with MFG-LacZ 

retrovirus express p-galactosidase. Retroviral transduction 

of the LacZ gene does not impair the capacity of 

triple negative thymocytes to develop into single positive 

T cells in FTOC. Exactly like the non-transduced 

progenitors, the transduced cells develop into immature 

CD4 + and CD4 +8 + double positive (DP) thymocytes 

and into single positive thymocytes. Most importantly, 

p-galactosidase expression can be detected in 40% of the 

progeny of the transduced TN thymocytes. Those 

thymocytes that lost p-galactosidase expression during 

incubation in FTOC still carry the LacZ gene as 

determined with PCR. This experimental system, 

combining FTOC and retroviral transduction, pro vides a 

genetic tooI for study of human T -cell development. 

Identification of CD34 + subcapsular epithelial cells 

in the human thymus 

The most early hematopoietic progenitor cell in the 

thymus expresses high levels of the stem cell antigen 

CD34. In a further phenotypic analysis of CD34 high 

thymocytes we observed a population of CD34 high cells 

th at also express high levels of Thy-l. These cells we re not 

hematopoietic precursors as they did not express CD45, 

LFA-l or ICAM-3. Moreover, sorted CD34 high Thy-I hi gh 

cells contain cells that are clonogenic in vitro. The in vitro 

expanded cells express cytokeratin suggesting that they 

are of epithelial origin. CD34 high Thy-l hi gh cells express 

CDlO and MHC class I antigens and are heterogeneous 

with respect to expression of HLA-DR, CD40 and 

ICAM-l. Following culture in vitro, these cells loose 

expression of CD34 while maintaining Thy-l. Cultured 

cells express CDlO, CD40 and ICAM-l. Inspection of 

frozen sections of fetal and post-natal thymus samples 

indicates the presence of CD34 high Thy-l high cells in the 

subcapsular pericortical area. These cells may play an 

important role in T -cell development as the earliest 

hematopoietic progenitor cell passes the subcapsular 

layer before differentiating further into T cells within the 

thymus. Functional characterization of these cells 

including the capacity to produce cytokines is underway. 

Identification andfunctional analysis of a thymic 

stromal antigen 

We recently described a mAb (MTS23) reactive with a 

membrane Ag expressed on a subset of thymic medullary 

stromal cells. MTS23 also detects an antigen 

constitutively expressed at high levels on peripheral B 

cells, macrophages, and thymic and splenic dendritic cells 

of C57BL/ 6 mice but thymocytes and peripheral T cells 

Immunology SI 

do not express the antigen detected by MTS23. Although 

the antigen identified by MTS23 is absent from T cells 

and thymocytes, it can be upregulated within 24 hours 

af ter activation through TCR cross-linking. This may in 

part be due to cytokines produced by T cells as a 

consequence of TCR signalling, since recombinant 

IFNy-induced expression on peripheral T cells and on 

CD4 and (to a les ser extent) CD8 thymocytes. Other 

cytokines, such as IL-2 and IL-4, also upregulated 

expression on peripheral T cells, but not on thymocytes. 

Experiments are in progress to study whether 

transmembrane signalling events can be induced by 

MTS23 in both hematopoietic cells and in stromal cells. 

The molecule detected by MTS23 appears to be a 

member of the Ly-6 family of Pl-anchored membrane 

proteins. Treatment of stromal cells with PI-PLC before 

staining completely abolished expression. Using transient 

expression of 293T cells and a cDNA library of a bone 

marrow stromal cellline cloned into the pEF-BOS vector, 

a cDNA encoding the MTS23 target antigen was isolated. 

Partial sequencing and restriction enzyme mapping 

revealed that it represents the Ly-6A protein. MTS23 

therefore represents another mAb which detects members 

of the Ly-6 locus on bone marrow and thymic stromal 

cells. While the physiological significance of the presence 

ofLy-6 molecules on stromal cells is not clear, it has been 

known for some time that, at least in lymphocytes, 

cellular activation events can be induced upon Ly-6 

engagement. It will therefore be of interest to test the 

consequences of Ly-6 crosslinking on stromal cell 

function directly, as well as in assays that measure the 

effects of stromal cells on lymphopoiesis. 

Regulation of T-cell repertoire selection 

Both positive and negative selection involve interactions 

between the TCR on CD4 +CD8 + (DP) 

thymocytes and self peptides associated with MHC 

molecules on thymic stromal cells. How apparently 

similar TCR-MHC interactions give rise to distinct 

responses in DP thymocytes is not clear. Numerous data 

suggest that the overall avidity of the interaction, 

incJuding the affinity ofthe TCR for MHC + peptide and 

interactions with accessory molecules, might determine 

the fate of DP thymocytes. Our goals are to identify 

accessory molecules associated with positive or negative 

selection and to determine what their relative 

contribution is to the determination of the response of 

developing thymocytes. 

We developed an in vitro system for clonal deletion, 

utilizing thymocytes from mice transgenic for a TCR that 

recognizes the 88-104 COOH-terminal peptide from 

pigeon cytochrome C (P17) presented on I-Ek. 

Presentation of this peptide by an I-Ek transfected 

fibroblast cell line in an overnight co-culture results in 

strong deletion of these thymocytes as measured by 

ethidium bromide staining in m ulti-color flow-cytometry. 

ECDl fixation of the transfected fibroblasts abrogates 

their ability to induce deletion, but leaves their antigen 

presentation capacity intact, as addition of third party 

cells (that cannot themselves present P17) restores the 

deletional response. These results demonstrate that a 

signal through the TCR alone is insufficient for induction

52 Jmmunology 

of a deletional response and that an accessory signal is 

required that can be provided by, as yet unidentified, 

membrane bound molecules. We are currently testing a 

number of known co-stimulatory molecules, transfected 

into inert third party cells, for their ability to provide this 

accessory signal. These molecules include: CD40 ligand, 

CD30 ligand, 4-1 BB ligand, OX40 ligand, fas-Iigand, 

CD27ligand and pro-TNF, all ofwhich are members of 

the TNF receptor ligand family. Some of these molecules 

have al ready been found to be associated with apoptosis 

in other interactions than thymic selection. Furthermore, 

we are developing a short term model system for positive 

selection in which we aim to apply the same approach. 

Positive selection of human thymocy tes can occur 

in both the CD4 + CD8 + double positive and 

CD4 + CD8- and CD4-CD8 + single positive stages 

Positive selection in the thymus that eventually gives 

ri se to CD4 + and CD8 + single positive (SP) mature T 

cells is initiated at the CD4 +CD8 + DP stage. It is, 

however, controversial whether positive selection is 

required for downregulation of CD4 and CD8, 

respectively. To investigate this point, we started from the 

premise that completion of positive selection is required 

for the acquisition ofthe capacity to be expanded in vitro. 

Investigating the in vitro clonogenic capacity of 

thymocyte subpopulations, we found that cloned lines of 

DP thymocytes could be established from PHA-activated 

cultures. The frequency of clonogenic DP thymocytes was 

low (between land 3%). The clonogenic potentialof DP 

thymocytes is restricted to CD 1- DP cells. It was 

furthermore observed th at ± 50% of the SP cells express 

CD 1. These CD I + SP cells are not clonogenic. Assuming 

that T cells can only be cloned in vitro after being 

positively selected, our data indicate th at downregulation 

of CD 1 marks positive selection. The observation that 

part of the SP thymocytes expresses CD 1 and cannot be 

expanded in vitro implies that positive selection can occur 

not only at the DP but also at the SP stage. This notion is 

supported by the observation that human thymocytes, 

developed from immature CD4-CD8- thymocytes in a 

mouse thymic micro-environment, are arrested in the 

CD 1 + stage, although SP thymocytes are present. The SP 

recovered from the mouse fetal thymic organ culture 

thymocytes are not clonogenic in vitro, indicating that 

they were not positively selected by the mouse MHC 

molecules. Our results indicate th at CD4 and CD8 can be 

downregulated in the absence of positive selection and 

support a two-step process of positive selection. 

The Jurkat T-cellline: a model for positive 

selection in the thymus 

Positive selection involves downregulation of the 

recombinant activation genes 1 and -2 (RAG 1 and 

RAG2), responsible for the V(D)J recombination of the 

TCR genes. In addition, differentiation ofDP cells results 

in down regulation of terminal deoxytransferase (Tdn, 

and in upregulation of the activation antigen, CD69, the 

co-stimulatory molecule CD27 and IL-2 mRNA. To 

study regulation ofthis differentiation process in vitro, we 

used the Jurkat T -cell line. This cell line has gene rally 

been considered as a mature T-cellline, but by RT-PCR 

we detected expression of RAGl, RAG2, and TdT by 

flow cytometry in Jurkat cells. Mature T cells do not 

express RAGs or TdT. Expression of these antigens could 

be downregulated within two hours af ter activation of 

Jurkat with a crosslinked anti-CD3 mAb. 

Development ofthymocytes from mice deficient for the 

tyrosine phosphatase CD45, is arrested at the DP stage, 

indicating that a critical step in differentiation of DP cells 

is blocked by the absence of CD45. In order to study 

whether CD45 has a role in the downregulation of RAG, 

we examined this process in a mutant of Jurkat, which 

does not express CD45 (mutant #5). As a positive 

control we used a subclone of the CD45 Jurkat cells that 

were reverted from the CD45- phenotype and have 

become CD45 + (revertant 26). After crosslinking of the 

TCR of mutant #5 or revertant 26 with an anti-CD3 

mAb, we found th at RAG 1 was downregulated in the 

mutant as well as in the revertant. In contrast, activation 

with anti-CD3 induced IL-2 mRNA in CD45 + Jurkat 

cells but not in CD45- mutant cells. We conclude that 

downregulation of RAG 1 is independent of CD45, while 

upregulation of IL-2 is CD45 dependent. In addition it 

was found th at RAG 1 was downregulated both in the 

absence and presence of Herbimycin A (a PTK inhibitor) 

in the CD45- mutant as weil as the CD45 + revertant 

Jurkat after activation by anti-CD3. As expected 

Herbimycin A completely inhibited anti-CD3 mediated 

upregulation of IL-2 mRNA and delayed the 

upregulation of c-fos mRNA. We conclude that the 

downregulation of RAG 1 in the CD45- mutant and 

CD45 + revertant Jurkat does not require PTK activity. 

Publications 

Izon DJ et al. J Immunol 1994; 153: 2939-50. 

lzon DJ et al. Int Immunol 1994; 6: 31-9. 

Kruisbeek A, Storb U . Curr Opin Immunol 1994; 6: 

199-202. 

Notes 


2 Funding: NWO (Gebied Medische Wetenschapen), 

Project 900-509-188. 

3 Funding: Junta Nacional de Investigacao Cientifica e 

Tecnologica (JNICT), Portugal. 

4 Funding: American Cancer Society. 


Project 900-507-178. 

6 Funding: BAE, nr. 94 / 5388, Ministerio Sanidad y 

Cousuma, Spain. 


8 Funding: UKE. 

9 Oklahoma Medical Research Foundation, Oklahoma 

City, OK 83104, USA. 

10 Robert Koch Institute, BerIin, Germany.

V Division of Molecular Biology 

Head 

RHA Plasterk PhD 

Permanent academic staff 

P Borst MD PhD, APM Jongsma PhD, HV Westerhoff 

PhD (80%, until October) 

C and C Huygens-fellow, honorary staff member 

F Baas MDPhD 


IJL De Baere PhD, MB Bakker PhD, W Bitter PhD, 

PA Blundell MSc, A Broeks MSc, EWHM Eijdems 

MSc, FMI van den Ent MSc, WC van Heeswijk MSc, 

G Jansen PhD, PR Jensen PhD, R Ketting MSc, 

M Kool PhD, HC Korswagen MSc, F van Leeuwen 

MSc, HGAM van Luenen MSc, R McCulloch PhD, 

D Molenaar PhD, RA Puras Lutzke MSc, JM Rohwer 

MSc, G Rudenko PhD, AH Schinkel PhD, JJM Smit 

MSc, AJ Smith MSc, B Teusink MSc, A Vaz Gomes 

MSc, C Vink PhD, JC Vos PhD, A de Waal PhD, 

GJR Zaman PhD, R ZwaaI MSc 

Permanent technica} staff 

A Dirks-Mulder (80%, from October), L van Deemter 

(75%), F Fase-Fowler (80%, until September), 

KH van der Linden (80%), A Riethorst, 

MJ de Vroomen (until October), E Wagenaar, 

M van Workum 

Other technica} staff 

S van Dooren, M de Haas, S Hoving, R Kieft, 

ML Loman, CAAM Mol (80%), MTh Srnits, 

B Stegeman, HC Stoffers MSc (12%), 

C van der Weijden, P Wielinga 

Undergraduate students and trainee technicians 

C Bontekoe, J van Eerden, M Entius, NEppens, 

M van Haandel, E Kamst, M van Leusden, 

Y Rombout, DGPW Satijn 

Guests 

A Bobok MSc, M Cross PhD, SL Hajduk PhD, 

JRS Hofmeyr PhD, P van Iwaarden PhD, 

BNS Kholodenko PhD, J Pettitt PhD, R Rezsohazy 

PhD, J Snoep PhD, M Taylor PhD, S Youngman PhD 

Secretaries 

J Helfrich, BK van Houten, NCM Immink (62%), 

J Wijker (62%) 

53

versions thereof. Similarly, a marked Tc3 element can be 

introduced and followed, which allows the investigations 

of DNA sequences important for jumping. The inverted 

repeats ofTc3 are exceptionally long (462 bp) and contain 

two binding sites for Tc3A. Elimination of most of the 

internal sequences ofthe inverted repeats up to 80 bp does 

not affect the transposition efficiency, although it 

removes one of the binding sites of Tc3A. Further 

restriction of the length of the inverted repeat to 40 bp 

dramatically reduces the transposition frequency. 

Another aspect of the cis-requirements are the sequences 

flanking the transposon, for instance the TA di nucleotide 

which is duplicated upon Tc3 insertion. Substitution of 

the TA sequence for GC had no influence on either the 

transposition frequency or the choice of a TA as target 

integration site. Based on sequence analysis it was 

suggested that the transposases of the Tcllmariner 

family share a common motif with the retroviral 

integrases and prokaryotic IS transposases. The motif is 

characterized by three crucial amino acids with a 

conserved spacing which play an important role in the 

catalysis of the transposition reaction; referred to as the 

DDE-motif. Mutation of any of these three amino acids 

abolishes Tc3A activity in vivo. In contrast, mutation of 

two nonconserved aspartic acid residues does not abolish 

transposition. 

The Tcl transposase, TclA, and derivatives thereof 

have been partially purified from E co/i and tested for 

specific DNA-binding and endonucleolytic activity in 

vitro. The N- terminus of the transposase contains a 

bipartite DNA-binding domain which binds a single site 

in the 54 bp Tc I inverted repeat. The DNA-binding 

domain is structurally related to the paired domain found 

in Drosophila and marnmalian genes involved in 

development. Tc1A is able to introduce single stranded 

nicks at the 5' ends ofthe transposon. Furthermore, TclA 

can mediate a phosphoryl transfer reaction. A mutation 

in the DDE-motif abolishes both endonucleolytic and 

phosphoryl transfer activities, but does not affect DNAbinding. 

Current experiments aim to study the mechanism of 

transposition in vivo and in vitro, to isolate potential host 

factors involved in the transposition process and to 

determine the three-dimensional structure of Tc3A. We 

also aim to further develop Tcl and Tc3 as tools for 

genetic analysis of C e/egans. 

Publications 

Van Luenen HGAM and Plasterk RHA. NAR 1994; 

22: 262-9. 

Van Luenen HGAM et af. Cell 1994; 79: 293-301. 

Vos JC and Plasterk RHA. EMBO J 1994; 13: 6125-32. 

Notes 

, Funding: European Community. 


Research (NWO/Pionier program). 

3 Funding: European Molecular Biology Organization 

(EMBO). 


Research (NWO/SON). 

5 Funding: Special grant from the Ministry of Education 

and Sciences/Netherlands 

Mo/ecu/ar Bi%gy 55 

Organization for Scientific Research (NWO/ Pionier 

program). 

Reverse gene tics in Caenorhabditis 

elegans 

RHA Plasterk, A Broeks', G Jansen 2 , HC Korswagen 3 , 

KH van der Linden, J Pettitt 4 , MJ de Vroomen, 

S Youngman5, RR ZwaaI' 

The nematode Caenorhabditis e/egans is a model 

organism that is easily accessible for genetics. The project 

of sequencing the entire gen ome is planned to be finished 

within the next three years (approximately 5 Mb has 

already been determined) and this win reveal the sequence 

of the 15,000 to 20,000 genes that are present in the 

nematode genome. A way to determine the function of 

these genes in the development and behavior of C e/egans 

is to inactivate them and to study mutant phenotypes. 

Since homologous recombination cannot easily be used 

as a method to inactivate or alter genes in a targeted 

fashion , we developed a transposon based method to 

obtain null alleles. First a Tel transposon insertion in the 

gene is isolated. This insertion is subsequently used to 

obtain deletion derivatives, which arise as aresult ofTc1 

excision. To isolate Tcl insertion alleles we established a 

library of frozen nematode cultures with a random 

pattern of Tel insertions. This library is screened by PCR 

using gene- and Tel-specific primers to obtain mutants in 

which a gene of interest is interrupted. Tel alleles of over 

70 genes have now been isolated and the library serves as a 

resource for researchers all over the world. In line with the 

C e/egans genome project we are developing new 

technologies to obtain Tel alleles of genes. One of these 

approaches involves shotgun sequencing of Tel insertion 

sites. We have now sequenced an estimated 400 Tel 

insertion sites spread over the genome. These insertions 

can be used to isolate deletion mutants and also serve as 

genetic markers. We are using reverse genetics to study 

GTP-regulatory proteins, P glycoproteins, Pim-1 related 

kinases and cadherins in C e/egans. 

GTP-regulatory proteins in C elegans 

Heterotrimeric G proteins transduce signals from 

membrane spanning receptors to intracellular pathways 

and th us play a role in the reaction of cells on changing 

extracellular conditions. Six G protein a-subunit genes 

and one fJ-subunit gene have been c1oned. The a-subunit 

genes gsa-l, goa-l and gqa-l show very high homo10gy to 

mammalian Gas, Gao and Gaq, respectively. The three 

gpa a-subunit genes show a much lower homology to 

marnmalian G proteins. Deletion mutants for the gpa 

genes, gsa-l and the fJ-subunit gene gpb-l we re obtained. 

Null alleles of both gpb-l and gsa-l are lethal. Animals 

homozygous for the deletion arrest in larval development. 

The lethal phenotype of these mutants is rescued by 

introducing the wild type gene as a transgene. We are 

isolating weaker alleles of gsa-l to use in screens for 

extragenic suppressors. Single, double and triple mutants 

ofthe gpa genes were obtained. Animals mutant for gpa-2 

and gpa-3 seem to be partially defective in the response to

56 M o/ecu/ar Bi%gy 

the pheromone that induces an alternative developmental 

route in the absence offood (dauer larvae). The gpa genes 

will be tested for epistatic interactions with other genes 

involved in dauer induction (daf genes) to fit them into 

this pathway. goa-J mutants are hyperactive and resistant 

to serotonin (see Figure V. 2). 

goa-1 mutant phenotypes 

goa-1 

overexpression 

wild type 

90a-1 null 

mutant 

Figure V.2 

Behavioral effect of goa-J rnutants: GOA-J overproducing 

C elegans show lethargic phenotypes, whereas knock-out 

rnutants show the opposite (hyperactivity) . 

P glycoproteins in C elegans 

P glycoproteins (Pgps) were first identified in 

mammalian tumor cells as a cause ofmultidrug resistance 

(MDR) by active drug transport over the plasma 

membrane. Four genes are known to encode Pgps in C 

elegans (pgp-J to pgp-4). Using a monoclonal antibody 

th at recognizes a highly conserved epitope on almost all P 

glycoprotein isoforms, Pgp expression was analyzed in 

transgenic worms that overexpress pgp-l and pgp-3. Both 

proteins are expressed in the intestinal cell membrane. In 

addition, pgp-3 is expressed in the H-shaped excretory 

cell. These expression patterns suggest a role for these 

Pgps in protecting the animal against environmental 

toxins. Null mutants of pgp-J and pgp-3 were isolated as 

described above and tested for sensitivity to various drugs 

that are known substrates for Pgps. The pgp-3 knockout 

worms are sensltlve to the drugs chloroquine and 

colchicine. The sensitivity can be reversed by introducing 

the wildtype pgp-3 gene; pgp-J mutants are not sensitive 

to these drugs. Colchicine and chloroquine are alkaloids 

of plant origin so pgp-3 may indeed protect nematodes 

from these toxins in the wild. 

Pirn genes in C elegans 

As part of a cDNA sequencing project in .C elegans, 2 

partial cDN As were recovered encoding homologs of the 

mammalian proto-oncogene pirn-J. For both prk ('pim 

related kinase') genes, Tc1 insertion mutants have been 

obtained. Analysis of mutant phenotypes and of tissue 

specific expression of prk-J and prk-2 are in progress. 

Publication 

Greenstein D et al. Genes & Development 1994; 8: 

1935-48. 

Notes 


Research (NWO/Pionier program). 


3 Funding: Human Frontier Science Program. 


(EMBO). 

5 Funding: The Wellcome Trust. 

Integration of HIV DNA into the 

human gen ome 

C Vinkl , FMI van den Ent 2 , RA Puras Lutzke 3 , 

KH van der Linden 2 , RHA Plasterk 

Integration of HIV DNA into the human chromosome 

is essential [or replication of HIV. The integration 

reaction is, therefore, a potential target for antiviral 

therapies. One protein is known to be required for 

integration, the viral integrase (IN) protein. IN mediates 

two distinct reactions: 1) specific removal of two 

nucleotides from the 3' ends of the vi ral DNA (donor 

cleavage) and 2) integration of the viral DNA into target 

DNA. Previously, th ree regions were identified in the IN 

protein: 1) the amino terminus; this region probably 

contains a motif which binds Zn2+ ions, 2) the central 

region, which contains the single active site ofthe protein, 

and 3) the carboxyl terminus, which contains a DNAbinding 

domain. These three regions are all necessary for 

full activity of the IN protein. 

We examined the DNA-binding characteristics of 

HIV -1 IN and found that a sta bie complex of IN and the 

viral DNA ends is formed in the presence of Mn2+. The 

IN-viral DNA complex is resistant to challenge by an 

excess of competitor DNA. StabIe binding of IN to the 

viral DNA requires that the protein contains an intact 

N-terminal domain and active site, in addition to the 

C-terminal DNA-binding domain. To further define the 

boundaries of the DNA-binding domain of IN, we 

carried out an extensive deletion mapping of the carboxyl 

terminus ofthe protein. We found that the region which is 

minimally required for nonspecific DNA binding is

located between amino acids 220 and 270 of the 288residues 

IN protein. Binding of DNA by this region is 

independent of divalent cations. Arnong ten different 

point mutations we generated in the DNA-binding 

domain, we identified one mutation (lysine 264 to 

glutamic acid) which resulted in strong reduction of both 

DNA binding and catalytic activity of IN. In 

collaboration with the group of Prof. R Kaptein (Utrecht 

University) we are currently trying to determine the threedimensional 

structure of the DNA-binding domain by 

NMR spectroscopy. 

Besides donor cleavage and integration, IN can also 

mediate intermolecular disintegration. This reaction has 

been regarded as the reversal of the integration reaction. 

We found, however, that disintegration is not the exact 

reversal of integration but rather is a sequenceindependent, 

phosphoryl-transfer reaction between 

gapped DNA duplex molecules. 

As mentioned above, DNA integration is essential for 

replication of HIV. Inhibitors of IN are therefore 

potential inhibitors of the progression of AIDS in HIVinfected 

humans. In order to find peptide inhibitors ofIN, 

we screened a 'synthetic' peptide combinatorial library' 

(developed by R Houghten, San Diego). An inhibiting 

peptide was identified with an IC so of approximately 2 

I-lM. Currently, we are analyzing the mode of action of 

this peptide. We wiU also screen for IN inhibitors among 

other combinatorial libraries which are composed of 

other compounds than peptides. 

Publications 

Puras Lutzke RA et al. NAR 1994; 22: 4125-3l. 

Van den Ent FMI et al. J Vi rol 1994; 68: 7825-32. 

Vink C and Plasterk RHA. TIG 1993; 9: 433-7. 

Vink C et al. J Viro11994; 68: 1468-74. 

Vink C et al. NAR 1994; 22: 2176-7. 

Vink C et al. NAR 1994; 22: 4103-10. 

N otes 

I Funding: Glaxo Group Research Limited. 

2 Funding: AIDS foundation. 


Research (NWO). 

Gene rearrangement 

P Borst, W Bitter l , P Blundell, M Cross 2 , S Hajduk 3 , 

G Rudenko, MC Taylor4, F van Leeuwen I, F Fase 

Fowler, R Kief tI , A Dirks-Mulder, R McCulloch 4 

Antigenie variation in trypanosomes 

African trypanosomes are unicellular protozoa 

transmitted to the mammalian host by an insect vector, 

the tse-tse fly. In the bloodstream of the mammal, 

trypanosomes are covered by a dense homogeneous 

protein coat consisting of Variant-specific Surface 

Glycoprotein (VSG). A given VSG coat is encoded by a 

single gene from a repertoire ofup to a thousand different 

VSG genes. During the course of a parasitemia the host 

develops antibodies against the prevailing VSG coat, 

effectively killing these trypanosomes. However, within 

Mo/ecu/ar Bi%gy 57 

the population, individual trypanosomes are capable of 

switching to the expression of a new VSG with no surface 

epitopes in common with the preceding one. Eradication 

is therefore never complete. 

The active VSG gene is expressed in one of several 

complex expression sites, containing several other 

Expression Site Associated Genes (ESAGs) and located 

adjacent to a telomere. Antigenic variation of the surface 

coat is brought about by replacing the VSG gene in an 

active expression site or by switching to another site. VSG 

gene replacement occurs predominantly by duplicative 

transposition. How trypanosomes switch from one 

expression site to another is not known. In this project we 

try to unravel the molecular genetics of antigenic 

variation. 

Control of VSG gene expression sites 

There are some 20 VSG gene expression sites in the 

trypanosome nucleus and these have to be tightly 

regulated. In bloodstream trypanosomes only one is 

active, whereas in the insect they are all switched off and 

the VSG coat is replaced by a procyclin coat. Since these 

expression sites are so similar, it has been difficult to study 

how individual sites are switched on and off. We have 

found, however, that the promoter regions of different 

sites differ by up to 7% in sequence. We have used these 

small differences to distinguish nascent RNAs coming 

from different expression sites and to target marker genes 

into individual sites. By this approach we have found th at 

the con trol of expression sites in insect form 

trypanosomes and bloodstream form trypanosomes is 

organized in a different fashion. In insect trypanosomes 

most if not all expres sion sites are transcribed at a low rate 

and transcription terminates at about 700 bp from the 

promoter. Transcriptional repression cannot be relieved 

by duplicating the promoter or inverting it in the context 

of the expression site. However, derepression was 

observed when the promoter was transposed to the 

nontranscribed spacer of the ribosomal RNA genes. The 

repression operating on the expression site promoter is 

sequence specific, as there is no down-regulation of a 

rDNA promoter introduced in this location. 

In marked contrast with these results, the con trol of 

VSG gene expression sites in bloodstream trypanosomes 

seems to be entirely at the level oftranscription initiation. 

Marker genes inserted downstream of the promoter are 

not transcribed or are transcribed at a very low rate. 

Replacement of the expression site promoter with a 

rDNA promoter does not relieve repression, showing that 

repression is insensitive to the precise sequence of the 

promoter. These results provide the basis for a more 

detailed study of the mechanism of expression site 

repression in bloodstream trypanosomes. Several 

possible mechanisms can be envisaged. In 1994 we have 

concentrated on the possible role of DNA modification 

(see below). 

We have also attempted to reconstitute the 

transcriptional attenuation observed in insect 

trypanosomes. This was done by integrating a VSG 

expression site promoter plus the attenuation region into 

a transcriptionally silent part of the trypanosome 

gen ome, but no attenuation was observed. As genomic

58 Mo/ecular Biology 

location may be important, we are attempting to 

construct an artificial expression site at a telomere to 

study VSG attenuation. 

The expression site is most likely transcribed by the 

same RNA polymerase used for transcription of the 

rDNA, so we are also investigating the rDNA 

transcription terminator to compare it with the site of 

expression site attenuation. The 3' boundary of 

transcription in the rDNA unit was mapped by nuclear 

run-on experiments to a position some 250 bp 

downstream ofthe end ofthe 28S coding region. Different 

rDNA fragments of up to 4 kb in length, spanning the 

drop in transcription, we re tested for their ability to block 

transcription from a ribosomal promoter by both 

transient transfection and following stabIe integration 

into the rDNA spacer. No transcription termination was 

seen. It is therefore possible that the rDNA terminator of 

trypanosomes does not work like that of other 

eukaryotes. 

Telomeric DNA modification 

We have previously shown that T brucei contains an 

unusual form of DNA modification that correlates with 

antigenic variation. It is present in and around VSG genes 

near telomeres but not in chromosome-internal genes. It 

is only present in nontranscribed genes but not in the VSG 

gene in the active expression site. It is lacking in insect 

trypanosomes, which do not undergo antigen ic variation. 

In 1993 we demonstrated that this modification is due to a 

novel base, fJ-glucosyl-hydroxymethyluracil (fJ-glc 

HOMeU), or J. To determine whether J is involved in 

expression site control we have studied how and where J is 

introduced into the DNA. Since expression sites are 

invariably located near telomeres and the inactive VSG 

genes alone do not harbor all the Jin the DNA, we have 

tested whether J is present in telomeres. Partially purified 

telomeric repeats from bloodstream trypanosome DNA 

show a ten times enrichment for J. Separation of the 

complementary telomeric strands by alkaline cesium 

chloride equilibrium centrifugation indicates that J may 

be present in both strands. To study how J is introduced 

into the DNA, we have cultured insect form 

trypanosomes in the presence of hydroxymethyluridine 

(HOMedU), which we expect to be a precursor of J. 

HOMedU was incorporated randomly into the DNA and 

a small fraction of it was converted into J. This supports 

our hypothesis that HOMeU in DNA is a precursor of J. 

The group of Prof. J van Boom (Organic Chernistry, 

University of Leiden) has chemically synthesized J and 

used this to make J-containing oligonucleotides. These 

will be used to search for J-binding proteins. 

Transferrin-binding proteins are encoded by 

expression site associa ted genes 

In the bloodstream of the mammalian host, 

trypanosomes take up host transferrin by means of a 

high-affinity uptake system, presumably a transferrin 

receptor. Transferrin binding activity is exclusively 

located in an invagination of the cellular .Ilembrane, 

designated the flagellar pocket, and is absent in insect 

form trypanosomes. By transfections we have recon- 

stituted a transferrin-binding complex in insect form 

trypanosomes. This required the expression of two 

homologous genes, the expression site associated gene 

(ESAG) 6 and ESAG 7. These proteins form a complex, 

which is attached to the membrane by the GPI anchor of 

ESAG 6. A number of hybrids we re constructed between 

the highly homologous ESAG 6 and 7 genes and these 

we re expressed in insect form trypanosomes. From these 

experiments we conclude that only the hybrid which 

contains mainly ESAG 7 and the C-terminal part of 

ESAG 6, which codes for the GPI anc hor addition signal, 

is capable of forming a transferrin-binding complex with 

the complete ESAG 6 gene product. This complex with 

two GPI anchors is more stabie on the cell surface than 

the native complex. 

The ESAG 6 and 7 gen es from different expression sites 

are not identical and this variability may affect either the 

binding specificity or the surface epitopes exposed to the 

immune system. We have shown that the transferrinbinding 

complex encoded by a single expression site 

enables the trypanosome to utilize transferrins from 

several different mammais. We therefore favor the 

hypothesis that switching between expression sites may 

serve to change the surface epitopes; th is hypothesis is 

under investigation. 

Publications 

Borst P. In: The encyclopaedia of Molecular Biology 

1994. 

Borst P et al. Science 1994; 264: 1872-4. 

Gommers-Ampt JH. [Dissertation]: University of 

Amsterdam, 1994. 

Ligtenberg MJL et al. EMBO J 1994; 13: 2565-73. 

Rudenko G et al. EMBO J 1994; 13: 5470-82. 

Steverding D et al. Eur J Cell Biol 1994; 64: 78-87. 

Notes 


Research (NWO/ Stichting Scheikundig Onderzoek). 


(EMBO) Fellowship. 

3 Funding: Fogarty Fellowship, United States. 

4 Funding: The Wellcome Trust. 

Multidrug resistance (MDR) 

P Borst, APM Jongsma, F Baas I , AH SchinkeF, 

JJM Smit 2 , EWHM Eijdems 2 , GJR Zaman 3 , AJ Smith 2 , 

E Wagenaar, E van Deemter, CAAM MoF, 

M de Haas 2 , A Riethorst, M KooF 

Upon selection with a single cytostatic drug, 

mammalian cells can become resistant to a wide spectrum 

of drugs that do not share a common structure or target. 

This kind of drug resistance is known as multidrug 

resistance (MDR). The classical form of MDR is caused 

by P glycoproteins (Pgps). These are large, glycosylated 

membrane proteins that can extrude a range of 

hydrophobic drugs from the cell against a concentration 

gradient. An increase in Pgp activity can therefore result 

in lowered intracellular drug concentration and hence, 

lowered drug toxicity. To establish the normal

60 Ma/ecular Bialagy 

MDR not mediated by P glycoprotein 

Many cell lines selected for resistance to natural 

product drugs do not contain increased levels of Pgp. 

Several, but not all, of these nonPgp lines contain raised 

levels of MRP (MDR-associated Protein). To examine 

whether MRP can confer drug resistance, gene 

transfection experiments were performed. By 

hybridization screening of cDNA libraries and by reverse 

PCR we isolated a set of partial cDNA clones that 

together covered the complete predicted open reading 

frame of MRP. To analyze the expression level and the 

cellular location of MRP, we raised polyclonal antisera 

and generated monoclonal antibodies against bacterial 

fusion proteins of MRP. This was done in collaboration 

with M Flens and RJ Scheper (Pathology, Free 

University Hospital Amsterdam). 

To make sta bIe transfectants in human non-small cell 

lung cancer SW-1573 cells, the full-length MRP cDNA 

was reconstructed and cloned in an expression vector 

containing the bacterial nea gene as a selectable marker. 

Neomycin resistant clones were obtained that 

overexpressed MRP. These clones were resistant to 

various anthracyclins, vincristine, etoposide, colchicine 

and rhodamine. This proves that M RP is a drug 

resistance gene. In contrast to MDRI Pgp, MRP did not 

confer resistance to taxol or gramicidin D, indicating that 

the detailed mechanism of MRP mediated MDR is 

different from that of Pgp mediated MDR. 

On cytological preparations the anti-MRP polyclonal 

antisera and monoclonal antibodies predominantly 

stained the plasma membrane of MRP-overexpressing 

cells. Immunoelectron microscopy and confocal laser 

scan microscopy confirmed the plasma membrane 

location of MRP. Also in drug selected cells that 

overexpress MRP, such as the small cell lung cancer 

GLC4/ ADR line, MRP was predominantly located in 

the plasma membrane. 

The mechanism of MRP mediated MDR was 

investigated in collaboration with J Lankelma, H 

Broxterman and HM Pinedo (Medical Oncology, Free 

University Amsterdam). In the MRP-transfected cells the 

intracellular accumulation of drug (daunorubicin, 

vincristine, VP-16) was decreased. The efflux of drug 

(daunorubicin) was increased. The decreased 

accumulation of daunorubicin was abolished by 

permeabilization ofthe plasma membrane with digitonin, 

showing that MRP can lower the intracellular 

concentration of drug against a concentration gradient. 

We conclude th at MRP is a plasma membrane drug efflux 

pump. 

A clue as to how this pump might work came from M 

Müller, E de Vries and PLM Jansen (Gastroenterology 

and Medical Oncology, Groningen) who showed that the 

increased expression of MRP in the transfectant 

increased the A TP-dependent glutathione S-conjugate 

carrier activity in plasma membrane vesicles isolated 

from these cells. This indicated that MRP is a glutathione 

S-conjugate export carrier, or GS-X pump. The GS-X 

pump mediates the excretion of bivalent anionic 

conjugates and is present in many mammalian cells, yeast 

and even in plants. MRP mediated MDR might therefore 

be linked to resistance associated with glutathione 

conjugation. Indeed, we observed that resistance to 

doxorubicin, daunorubicin and vincristine in the MRPtransfected 

SW-1573 cells was dependent on the 

intracellular level of glutathione, as pretreatment of the 

cells with an inhibitor of glutathione synthesis 

substantially reduced resistance against these drugs. 

Doxorubicin, daunorubicin and vincristine are not 

known to be substrates for glutathione-S-transferases. 

However, it is possible that negatively charged complexes 

of these drugs are formed in the cell but that these 

complexes have escaped detection, for instance because of 

their instability. Alternatively, MRP might be able to 

transport both conjugated and unconjugated drugs. To 

examine which of these alternatives is correct, we will 

investigate (in collaboration with J Beijnen, Slotervaart 

Hospital) whether anionic metabolites of natura I product 

drugs exist and are excreted by M RP-overexpressing 

cells. Furthermore, we will exarnine whether MRP in 

plasma membrane vesicles is also able to transport 

cationic compounds (in collaboration with M Müller and 

PLM Jansen). 

In 1994 we also obtained new information about an 

interesting form of nonPgp MDR previously found in 

SW -1573 cells and characterized in collaboration with H 

Broxterman, J Lanke\ma and HM Pinedo (Medical 

Oncology, Free University, Amsterdam) and F Baas 

(Academic Medical Center, University of Amsterdam). 

This form of resistance is associated with a decreased 

uptake of drug (which explains the resistance) and a 

decrease rather than an increase in the level of MDRI 

mRNA. In initial experiments we had not observed 

significant changes in MRP mRNA, but a reinvestigation 

of this problem has now implicated MRP in resistance. 

Wh en MRP mRNA levels were analyzed in a large new 

series of resistant variants, these were found to be slightly 

increased (1.2 to 2.0-fold paren tal levels). The level of 

MRP was also slightly increased and in all resistant clones 

a novel MRP band was present that migrated slightly 

slower than the wildtype 180 kDa MRP band in SDSpolyacrylamide 

gels. Confocal scanning rnicroscopy (in 

collaboration with LOomen, Department of Biophysics) 

showed that in sensitive parental cells most of the MRP 

was located in the membranes of intracellular vesicles, 

whereas in the resistant mutants a substantial fraction 

was present in the cell membrane. These results suggest 

that MRP is responsible for the nonPgp MDR in 

SW -1573 cells after all. We have shown that the change in 

migration of MRP in the mutants is due to an alteration 

in glycosylation, but the cause of this alteration and its 

relation to resistance remain to be determined. 

Publicatians 

Burger H et al. Leukemia 1994; 8: 990-7. 

Burger H et al. Br J Haematology 1994; 88: 348-56. 

Eijdems EWHM. [Dissertation): University of 


Eijdems EWHM et al. Int J Cancer (in press). 

Eijdems EWHM et al. Br J Cancer (in press). 

Flens MJ et al. Cancer Res 1994; 54: 4557-63. 

Mauad TH et al. Am J Pathol 1994; 145: 1237-45 . 

Müller M et al. PNAS (in press). 

Oude Elferink RPJ et al. J Clin Invest (in press). 

Schinkel AH et al. Cell1994; 77: 491-502.

Smit JJM et al. Lab Invest (in press). 

Smit JJM et al. BBA (in press). 

Smith AJ et al. FEBS Letters 1994; 354: 263-6. 

Zaman GJR et al. PNAS 1994; 91: 8822-6. 

Notes 


Research (NWO/ Huijgens fellowship). 

2 Funding: Dutch Cancer Society, Projects NKI 91-18 

and NKI 92-41 . 

3 Collaborative project with University of Amsterdam. 

Regulation of eell funetion 

BN Bakker, JH Daams 1 , S van Dooren, 

AA van der Gugten 1, M Guirol, WC van Heeswijk 2 , 

S Hoving, PR Jensen 3 , APM Jongsma, 

BN Kholodenk0 4 , L Loman, D Molenaar, A Riethorst, 

JM Rohwer, J Snoep, B Stegeman, B Teusink 2 , 

CC van der Weijden, P Wielingal, M van Workum, 

HV Westerhoff 

Regulation of DNA structure and gene expression 

Both the base sequence of the DNA and its three 

dimensional structure determines gene expression. To 

study the role of DNA supercoiling in the model cell 

E co!i, the activity of DNA gyrase is modulated to induce 

changes in DNA supercoiling. The modulation is 

achieved by varying the concentration of IPTG 

(isopropyl-thio-tJ-galactoside) in strains in which the 

promoters of the chromosomal atp operon or of an 

artificial chromosomal gy r operon have been substituted 

by lac type promoters. Negative supercoiling decreased 

strongly when the cells ran out of glucose, especially at 

low concentrations of the H + -ATPase. Modulation of 

the artificial gyr operon affected the concentration of the 

gyrA sub unit, intracellular supercoiling and growth rate. 

The results suggest th at the energetics of DNA structure 

affect cell function and that DNA gyrase controls growth 

rate. 

Principles and quantitative analysis of control and 

regulation 

The functioning of the living cell, like that of a largesize 

factory, depends on sufficient activity of individual 

processes and on the proper adjustment of these processes 

relative to one another. Accordingly, the living cell avails 

itself of a hierarchy of management levels, which serve the 

purpose of administering proper contro!. Persistent 

perturbations at these management levels are found to be 

associated with tumorigenesis and oncogenic 

transformation of cell lines. In view of the complexity of 

the living cell and its management, we sollicit support 

from biomathematics in our search for the rules of cell 

management. 

It is difficult to determine proper ties of intracellular 

molecules without disrupting the cello Yet one can 

sometimes measure how the function of intact cells is 

controlled. We developed a biomathematical method to 

calculate molecular properties from the con trol of cell 

Molecular Biology 61 

function. This method may help in suggesting, on the 

basis of differences in behavior between tumor cells and 

their untransformed counterparts, which molecular 

activity has been compromised by the primary oncogenic 

event. A similar principle was developed for individual 

enzymes, where ra te limitation was related to the 

distribution of the enzyme over its states. 

We examined the differences in con trol principles 

between two types of signal transduction pathways, i. e. 

one in which a chemical group such as phosphate is 

transferred between proteins and a second in which 

proteins are merely catalyzing modification reactions of 

subsequent proteins in a cascade. We proved th at in the 

former case the total con trol strength of the proteins on 

the signal transduction flux amounts to 2. In the latter 

case it amounts to 1, the same as in traditional metabolic 

pathways, with the difference that many proteins may 

exert strong positive contro!. 

Again using E coh as the model system, growing on 

glucose, we confirmed our prediction that the Pil protein 

of the glutamine synthetase signal transduction cascade 

does not con trol the steady state activity of glutamine 

synthetase. Contrary to our expectations, the same 

protein did not con trol the time it took cells to respond to 

changes in ammonia concentration. In cells grown on 

succinate, the Pil signal transduction cascade was 

important for th at response. This shows th at the function 

of a signal transduction route may be to reduce response 

times and that the activity of transduction of one signal 

(nitrogen status) may depend strongly on other, 

apparently unrelated signals (carbon or energy status). 

For the activity of E coh's phosphotransferase signaltransduction 

system, we have demonstrated astrong 

dependence on the cellular energy state (measured as 

ATP / ADP ratios), modulating the latter in various ways. 

Our biomathematical models have been applied to 

various systems. They helped explain the energetics of 

finger flexor muscle, as measured by phosphorous NMR. 

They led to a prediction of what controls glycolysis in 

trypanosomes, which is now tested experimentally. 

Moreover, they helped us understand what controls the 

steady oscillations we observe in metabolites and heat 

produced in yeast. Of particular interest was how an 

intercellular signal molecule can control the synchrony of 

the oscillations in the individual cells. 

Control of multi drug resistance 

The effectiveness of many anti-tumor drugs is 

compromised by their extrusion from tumor cells that 

have enhanced concentrations of P glycoprotein. The 

cytotoxic drug concentration is a complex function ofthe 

kinetic properties of the P glycoprotein, the passive 

permeability, the drug target and possible detoxification 

mechanisms. By elucidating the relevant kinetic 

properties we aim at understanding what determines drug 

toxicity and how drug toxicity and drug selectivity may be 

improved. 

We approach the problem from two sides. On one side, 

we employ kinetic models to interpret experimental data, 

obtained by Lankelma and colleagues, with intact 

multidrug resistant cells. This analysis confirms that P 

glycoprotein pumping ra te is a saturabIe function of the

62 Mo/ecu/ar Biology 

concentration of daunorubicin, with a Km of a few 

micromolar. Most importantly, the pumping ra te varies 

almost quadratically with intracellular drug 

concentration at low drug concentrations. The quadratic 

cooperativity is important because it suggests that the 

effectiveness of the P glycoprotein mediated drug 

resistance decreases strongly with drug concentration. 

These results were confirmed in our second, experimental 

approach, in which plasma-membrane vesicles were 

assayed for A TP dependent drug uptake. The original 

assay system was improved by including DNA in the 

plasma membrane vesicles and adding a fluorescent label, 

allowing for on-line measurement of fluorescent drug 

uptake. 

Vesicles from cells overexpressing multidrug resistance 

associated protein (MRP) also exhibited drug up take 

wruch was stimulated by ATP but not by a 

nonhydrolyzable A TP analog. Both in the vesicles and in 

the intact cells, drug pumping was sensitive to ouabain, 

but not to vanadate, distinguishing the activity from that 

of P glycoprotein. The MRP mediated daunorubicin 

pumping was also cooperative in daunorubicin 

concentration. 

Publications 

Cortassa S et al. , Kahn D and Westerhoff HV, Stoffers 

HJ et al. 3 chapters In: BioThermoKinetics 1994. 

Daams H et al. , HellingwerfKJ et al. , Jensen PR et al. , 

Kahn D and Westerhoff HV, Kholodenko BN and 

Westerhoff HV, Meszena G and Westerhoff HV, 

Richard P et al. , Schuster S et al. , Stoffers HJ et al. , 

Van der Gugten AA and Westerhoff HV, Van 

Heeswijk WC et al. , Westerhoff HV et al. 12 chapters 

In: Modem Trends in Biothermokinetics 1994. 

Guiral M et al. FEBS Lett 1994; 346: 141-5. 

Jensen PR et al. J Biol Syst (in press). 

Kahn D, Westerhoff HV. Biotheor Acta 1993; 41 : 

85-96. 

Kholodenko BN et al. FEBS Lett 1993; 336: 381-4. 

. Kholodenko BN et al. Eur J Biochem 1994; 225: 179-86. 

Kholodenko BN et al. J Mol Cell Biochem 1994; 

133/134: 313-3l. 

Kholodenko BN et al. FEBS Lett 1994; 349: 131-4. 

Kholodenko BN, Westerhoff HV. Biochim Biophys 

Acta (in press). 

Kholodenko BN et al. In: Proceedings of the 2-nd 

ECMBM (in press). 

Meszena G , WesterhoffHV. Biophys Chem 1994; 48: 

321-36. 

Mülder HS et al. Eur J Biochem 1993; 218: 871-82. 

Richard P et al. FEBS Lett 1994; 341 : 223-6. 

Snoep JL et al. Biochem Mol Biol Inter 1994; 33: 

1023-32. 

Spoelstra EC et al. Eur J Biochem 1994; 221 : 363-73. 

Westerhoff HV et al. Biophys Chem 1994; 50: 273-83. 

Westerhoff HV et al. Biophys Chem (in press). 

Westerhoff HV et al. In: Nonlinear Analysis- 

Mathematical Modelling of Enzyme Systems (in 

press). 

Wijker JE et al. Biophys Chem (in press). 

Notes 

Funding: NWO (SLW, Pionier), NKB, EC. 

1 Division of Tumor Biology, Netherlands Cancer 

Institute. 

2 EC Slater Institute, University of Amsterdam. 

3 Danish Technical University, Lyngby, Denmark. 

4 University of Moscow, Russia. 

The basis for selective toxicity of 

membrane active peptides 

A Vaz Gomes, APM Jongsma, A Riethorst, 

HV Westerhoff 

Magainins are amphiphilic cationic peptides secreted 

by Xenopus laevis. We are examining the physicochemical 

basis for their cytotoxic activity. 

We established that with liposomes, magainin activity 

is an all-or-none affair; once a liposome is affected, its 

contents mix completely with the outside medium. 

Heterogeneity of liposomal preparations is a major 

determinant of the extent to which a given magainin 

concentration permeabilizes them. Although magainin 

action on liposomes is reversible, the peptide creates large 

holes in the membranes, as evident from permeation of 

large dextran molecules. 

PGLa and other magainins act synergistically against 

prokaryotes. We now established that they also do this 

against a human melanoma cell line and in liposomes. 

Clearly the synergism occurs both at the molecular and at 

the functional level. This may be a basis for further 

development of effective peptide-based drug mixtures. 

Publications 

Kamp F et al. Biochemistry 1993; 32: 11074-86. 

Vaz Gomes A et al. Biochemistry 1993; 32: 5365-72. 

Vaz Gomes A et al. In: Modem Trends in 

Biothermokinetics 1994. 

Vaz Gomes A. [Dissertation]: University of Amsterdam, 

1994 . 

Westerhoff HV et al. Eur J Biochem (in press). 

No te 

Funding: NWO Pionier.

VI Division of Tumor Biology 

Head 

WJ Mooi MD PhD 

PROGRESSION AND DIFFERENTIATION OF 

LUNG AND BREAST TUMORS 

Coordinator 

RJAM Michalides PhD 


J Hilkens PhD (75 %), WJ Mooi, MD PhD (10 %), 

JL Peterse, MD (10 %), M Sluyser PhD 


JL Koopman PhD, A Leyte PhD, HL Vos PhD, 

J Wesseling MSc 


MBoer, F Buys, CCJ de Goeij 


AJ van Hof, R Klompmaker, SW van der Valk 

U ndergraduate students and trainee technicians 

H Bakker, EH van Beumei, N Dantuma, E Löwik, 

AMG van der Wal 

CLINICAL APPLICATION OF MONOCLONAL 

ANTIBODIES 

Coordinator 

WJ Mooi MD PhD (10 %) 


H Daams MSc (5 %), PhC Hageman PhD (50 %), 

D Ivanyi MD PhD (50 %) 


DAtsma, F Verhofstad, E Groeneveld 


G van Doornewaard 

IN VIVO APPLICATION OF MONOCLONAL 

ANTIBODIES 

Coordinator 

J Hilkens PhD (25 %) 

63 


B Kwa (10 %), I Groenenberg, CA Hoefnagel MD PhD 

(10 %), EJT Rutgers MD PhD (10 %), AHM Verhoeven 

MSc, HL Vos, PhD 

Secretary 

JE Wijker 

DEPARTMENTOFPATHOLOGY 

Head 

WJ Mooi MD PhD (90 %) 


MPW Gallee MD PhD, D de Jong, MD PhD, B Loftus 

Coll MD, JL Peterse MD (90 %), P van Heerde MD 

PhD, LJ van 't Veer, PhD 


HA Maas PhD, MAJ van Dijk MSc 


LH Boerrigter-Barendsen (60 %), G Brink, A Floore, 

ME Verbruggen (10 %), P Wisman 


AJ Breedijk, N Kürten 

U ndergraduate students 

N Haver, Y Oei, M Vandeputte, E Vos

64 Tumor Bi%gy 

Introduction 

Within the Division of Tumor Biology basic scientific 

and clinical expertise are combined in order to contribute 

to improved diagnosis and treatment of cancer through a 

better understanding of the pathophysiology of 

malignant tumors. Some observations of clinicians and 

surgical pathologists, notably the work on neuroendocrine 

differentiation in lung cancer, on the estrogen 

receptor in breast cancer and on cell kinetics as predictors 

of treatment response, have kindled the interest of basic 

scientists in the division. Conversely, some ofthe findings 

originating in the lab have been extended to clinical 

investigations; especially, the prognostic implications of 

NCAM expression in lung cancer, the effects of episialin 

overexpression and loss of polarization in infiltrating 

adenocarcinomas, and keratin subtypes in carcinomas of 

the head and neck region. 

Episialin overexpression in invasive 

breast cancer 

HL VOSI, J Wesseling l , J Storm 2 , MBoer, 

SW van der Valkl, MCE Maas l , S van Damme 3 , 

C Patriarca 4 , JL Peterse, T ThingstadS, J Hilkens 

Episialin (also known as PEM, EMA, CA 15-3 antigen, 

etc.), encoded by the MUCI gene, is a transmembrane 

glycoprotein, the extracellular domain of which contains 

a region ofnearly identical repeats of20 amino acids. As a 

result of genetic polymorphism, the number of repeats 

varies in each individual. The repeats, together with 

adjacent degenerated repeats, are proline rich and contain 

many serines and threonines, which are attachment sites 

for O-linked glycans. This part ofthe molecule constitutes 

the mucin-like domain. As a result of the abundance of 

prolines, the protein backbone of the mucin-like domain 

forms a polyproline f3-helix, resulting in an elongated 

structure, stabilized by the attached glycans, which 

protrudes 200-500 nm above the plasma membrane. For 

comparison, the extracellular part of most membrane 

molecules, e.g. cell adhesion molecules, do not exceed a 

length of 30 nm. 

The molecule is normally expressed at the apical side of 

glandular epithelial cells. In carcinoma cells, which have 

lost their normal polarization, episialin is present across 

the entire cell surface. Moreover, in breast cancers, the 

expression level of episialin can be increased more than 

10-fold. 

We have previously shown that overexpression of th is 

elongated and relatively rigid molecule on the cell surface 

can mask other surface molecules, thus impeding cell-cell 

and cell-matrix interactions. Furthermore, a high level of 

episialin expression renders cells less susceptible to 

immune destruction by cytotoxic Iymphocytes. All of 

these phenomena may result in an increased invasive and 

metastatic potential. 

We have concentrated on the further characterization 

of the anti-adhesive effect of episialin, its effect on 

invasion in vitro, the formation of metastases, the 

identification of post-translational modifications and 

their effects on the function of episialin, and the 

regulation of expression. In addition, we have started to 

clone the gene for epiglycanin, another membrane bound 

mucin highly expressed on a mouse mammary carcinoma 

cell line with strongly reduced adhesiveness. 

The effect of episia/in on adhesion and aggregation 

We have previously shown that episialin overexpression 

can diminish cellular adhesion to extracellular 

matrix components of melanoma cells and to alesser 

extent also of SV -40 transformed normal breast epithelial 

cells (HBL-IOO). MDCK cells transfected with episialin 

cDNA also show the anti-adhesion effect of episialin in a 

standard adhesion assay. Interestingly, the cel!s also show 

an altered morphology. Normal MDCK cel!s form a 

monolayer ofwel!-polarized cells, whereas the transfected 

MDCK cells acquired a more fibroblastic morphology 

and failed to form epithelial sheets. 

Removal of the cytoplasmic tail of episialin did not 

diminish the anti-adhesion effect, showing that this 

property was not caused by competition with integrins for 

cytoskeletal proteins or by an as yet unidentified 

signalling process. Thus, the property causing the antiadhesion 

effect must be situated in the extracellular 

domain. Since the extracellular domain is heavily 

sialylated and sulphated, the resultant net negative charge 

of the molecule may be involved. However, removal of 

these residues had only a minor effect on the antiadhesion 

effect. When the number ofrepeats was reduced 

to three, the anti-adhesion effect was no longer apparent, 

indicating that the si ze of the molecule may be of major 

importance. 

Overexpression of episia/in and tumor progression 

Electron microscopy (J Calafat, Division I) showed 

that overexpression of episialin in transfected human 

melanoma cells, growing as xenografts in nude mice, 

resulting in reduced cell-cel! contacts. Sometimes the 

contact was confined to filopodia. Often relatively large 

intercellular spaces we re present. In contrast, the tumor 

cel! membranes in the episialin negative xenografts were 

closely apposed to each other. Thus, the anti-adhesion 

effect of episialin is also operating in vivo. In human 

primary breast carcinomas where the cells had lost their 

normal polarized archltecture, episialin was often present 

at cell-stroma boundaries. At these boundaries, large 

clefts were sometimes present. We are presently 

investigating whether this subgroup of tumors has a 

higher propensity to metastasize. 

Overexpression of episia/in and invasiveness 

Previously, we have shown that in transfected cells 

growing as cell clusters in a reconstituted extracellular 

matrix (Matrigel), episialin can promote invasion into the 

extracellular matrix. We have now quantified this 

phenomenon by growing the cells on Matrigel coated 

filters in a Boyden chamber. Episialin transfected MDCK 

cells and HBL-I 00 cells were able to invade the Matrigel 

and were passing the filters, whereas the revertants 

showed this property to a much more limited extent. 

These results suggest th at episialin mayalso be involved

in invasion in vivo. 

Transgenic mice expressing human episialin 

In cooperation with A Berns and E Robanus Maandag 

(Division VII), we have raised human episialin transgenic 

mice. The episialin gene was placed under the con trol of 

the MTV-LTR promoter which allows induction of the 

gene by dexamethasone. As expected, the expression 

before and after induction was mainly present in 

glandular epithelial cells, since the gene is under the 

con trol of the MTV promoter. We did not observe any 

phenotype as a result of overexpression of episialin in 

these mice. We have selected two strains for further study; 

one with a very high and one with an intermediate 

episialin expression level. We are presently inducing 

mamrnary tumors in these animals by transplanting the 

pituitary gland under the kidney capsule and in F 1 (C3H x 

episialin transgenic) mice. The metastatic capacity of the 

primary tumors and transplanted forms of these tumors 

will be assessed with and without enhancement of 

episialin expression. So far, two tumors have been 

obtained, one of which was transplantable. 

Phosphorylation of the membrane anchor of 

episialin 

Episialin is synthesized as a single polypeptide chain 

which is immediately cleaved in the ER, but both moieties 

remain npn-covalently associated. The smaller 

C-terminal domain serves as the membrane anchor for 

the larger N-terminal mucin-like domain. We have 

identified the membrane anchor as a set of glycoproteins 

of 25-30 kDa. Part of the heterogeneity is the result of 

N-glycosylation. However, we found th at the molecule is 

highly phosphorylated on serine and threonine (in 

cooperation with L Smits, Division 111), causing further 

heterogeneity. The phosphorylation was stimulated by 

TPA treatment, suggesting PKC dependence. Moreover, 

arresting the cells in the G2 / M phase by nocodazole 

treatment caused additional phosphorylation, probably 

at a different site. 

mAbs against the proteoly tic e/eavage site region 

We have generated monoclonal antibodies against the 

region encompassing the proteolytic cleavage site to study 

this biochemical event. mAbs against this region mayalso 

be useful for the quantitation of the protein, since this 

region does not contain repeats, and is not 

O-glycosylated, which can variably affect mAb binding. 

With these anitbodies, it appeared that episialin is not 

overexpressed in colon carcinoma, which is in contrast to 

the situation in breast cancer. Large differences in the 

reactivity of episialin / MUCl in various parts of the 

crypts and in adenomas versus carcinomas could be 

detected using a panel of anti-episialin mAbs. 

The e/oning ofthe epiglycanin cDNA 

Epiglycanin is a cell surface bound mucin with a 

structure similar to episialin, which is highly expressed on 

the murine mammary carcinoma cell line T A3Ha. 

Tumor Biology 65 

Expression of the protein results in loss of adhesion and in 

allotransplantability of this cellline, suggesting a masking 

of other cell-surface molecules, similar to the effect of 

episialin. We are presently attempting to clone the cDNA 

encoding the epiglycanin gene. We have made an 

expression library from mRNA of this cell line and 

screened it with various polyclonal and monoclonal 

antibodies. The mAbs had been generated by 

Kemperman and Roos from the division ofCell Biology. 

We are currently characterizing the positive clones 

obtained in the various screening procedures. 

The genomic region containing the MUCl gene 

We have previously reported on our finding of the 

thrombospondin 3 gene and glucocerebrosidase gene, 

which is defective in Gaucher's disease, upstream from 

the MUel gene. Another gene, provisionally named 

GeneX , is located between these genes, but in the opposite 

transcriptional orientation. GeneX has been further 

characterized in the laboratories of Drs Bornstein and 

Ginns. lts sequence has been determined and GeneX 

knock out mice, which carry an insertion mutation in the 

last exon of GeneX , have been obtained. These mice we re 

initially generated to study the effects of point mutations 

in the glucocerebrosidase gene. Mice lacking functional 

GeneX die early during gestation, suggesting an 

important role of the GeneX protein during development. 

However, the phenotype may be complicated by effects 

on the transcript processing of the nearby glucocerebrosidase 

gene. An extensive database search for similar 

molecules showed a distant similarity to glutathione 

S-transferases (GSTs) and related proteins. However, the 

level of similarity is so low that it seems unlikely that the 

GeneX protein will function as a GST. 

No vel monoe/onal antibodies to human and murine 

ep is ia lin 

The glycosylation of episialin is tumor and tissue 

dependent and may affect the binding of mAbs. Thus, 

many mAbs show a clear tumor or tissue preference. This 

property may allow us to select mAbs that show a 

preferential reactivity with lung carcinomas. We have 

raised several new mAbs against the repeat moiety of 

episialin, derived from pleural effusions of lung cancer 

patients, that preferentially reacted with episialin from 

lung carcinomas in solid phase assays. Subsequently, we 

selected these mAb further on tissue sections. Selected 

mAbs are presently being tested for their potential 

usefulness as serum markers. 

Publications 

Hilkens et al. In: Biochemistry of Cell Membranes (in 

press). 

Vos et al. In: Biomembranes Volume 3 (in press). 

Zhrihan-Licht et al. Eur J Biochem 1994; 224: 787-95. 

Notes 

I Funding: Dutch Cancer Society, Projects NKI 91-16 

and 93-523. 

2 Funding: Centocor. 

3 Undergraduate student.

66 Tumor Biology 

4 Guest from the Department ofPathology, Ospedale S 

Paolo, University of Milan. 

5 Guest from the Department of Phannacology, 

University of Oslo. 

Cell cycle control genes and tumor 

progression 

R Michalides, N Hoflandl, R Klompmaker 2 , P Kristei' , 

N van Veelen, E Wientjens, R Zwijsen 2 

In this project, we aim to investigate the role of 

overexpression of Cyclin Dl in the process of tumor 

progression. In a previous study we found that in breast 

cancer, amplification of Cy clin Dl as part of a very large 

amplicon on chromosome llql3 is associated with an 

unfavorable prognosis (Schuuring E et al. Cancer 

Research 1992; 52: 5229-34). In this project, we extend 

this finding to archival material and aim to provide an 

experimental basis for the observed association with 

apparently increased tumor aggressiveness. 

Retrospective clinical studies 

In order to analyze archival series of tumors for 

overexpression of Cyclin Dl, we generated a polyclonal 

rabbit antiserum against the carboxyl-terminal part ofthe 

Cyclin DI protein. After affinity purification, the 

antiserum appeared specific for CycJin DI, also on 

sections from formalin fixed / paraffin embedded tissues. 

The antiserum detects overexpression of Cyclin D I in 

tumor cells with at least a three-fold amplification of 

Cyclin Dl. With this antiserum, we performed two 

retrospective studies. 

I) In a series of 47 resected TI-2, NO-I squamous cell 

carcinomas of the head and neck region (collaboration 

with F Balm, B Loftus and G Hart), overexpression was 

found in over 50 % of tumor cells in II cases and in 

10-50% of tumor cells in 21 cases. Recurrence of disease 

was significantly associated with a high degree of 

overexpression of Cyclin Dl (p = 0.026). 

2) In a series of 248 consecutive TI-2, NO-I breast 

cancer patients treated with curative intent (collaboration 

with ] Peterse, Ph Hageman and H van Tinteren), 

overexpression of Cyclin Dl was significantly associated 

with estrogen receptor positivity (p < 0.000) and weakly 

associated with disease-free survival in the subgroup of 

estrogen receptor-negative breast cancer. The difference 

between this finding and the association between 

amplification of Cyclin Dl and poor prognosis as 

reported previously by us and by others may be due to 

selection of more advanced tumor stages or larger tumors 

in the groups of patients analyzed previously. 

Overexpression of 1 and loss of growth control 

Cyclin DI is a regulatory protein essen ti al for 

progression through the G I phase of the cell cycle in 

normal cells, as well as in cancer cells. Biochemical 

analysis of Cyclin D I levels in nocodazole-synchronized 

tumor cells revealed a characteristic cell-cycle dependent 

oscillation, with maximalievels ofthe Cyclin Dl protein 

in the G I phase. A two parameter flow cytometry analysis 

for cylin DI and counterstaining for DNA confirmed the 

predominantly G I phase-specific expression of the 

protein in Cylin Dl overexpressing cell types. Immunocytochemical 

examination of exponentially growing 

h uman breast cancer and squamous carcinoma cells 

showed a characteristic nuclear pattern of Cyclin DJ as 

reported for human non-transformed cells. 

In order to investigate the role of Cyclin DJ in the cell 

cycle regulation, we have generated transfectants of 

MCF-7 cells with Cyclin DJ constructs under the control 

of a tetracycJin sensitive transactivator. These transfectants 

showed a 4-6 fold increase in expression levels of 

Cyclin DJ. In these transfectan ts, induction of Cyclin DJ 

increased the growth fraction of the cells but it did not 

acceJerate the G I phase of the cell cycle. In the absence of 

exogenous growth factors, induction of ectopic Cyclin Dl 

was sufficient for completion of the cell cycle, a process 

requiring growth factor stimulation in con trol cells. These 

data indicate th at Cyclin Dl overexpression may reduce 

the effects of physiological restraints and could thus 

contribute to loss of growth contro!. 

Notes 



Radiolabeled DNA precursor 

incorporation into tumor cell DNA 

M Sluyser, CC] de Goeij, ] Mester' , F Gerbrandy2 

Incorporation of a radioactive precursor into DNA 

leads to non-repairable double strand breaks, causing 

mutations or, if the incorporated radioactivity is 

sufficiently high, to cell death. We are using mammary 

tumors ofGR mice as a model system to explore different 

strategies for the preferential incorporation of '25IdU into 

tumor DNA. 

Pubfications 

Sluyser M. Human Mutation (in press). 

Sluyser M. Apoptosis in nonnal development and in 

cancer (in press). 

Verhagen A et al. Nucl Med Biol 1994; 21: 941-52. 

Notes 

, Centre de Recherches Paris Saint-Antoine, Paris, 

France. 

2 Undergraduate student. 

Polysialylation of the neural cell 

adhesion molecule (NCAM) 

R Michalides, ] Koopman' , A] van het Hof', ABosma, 

B Kwa 2 , W Mooi 

We studied the possible contribution of polysialic acid 

(PSA) to the invasiveness of NCAM containing tumor 

cells in two ways: 1) by investigating whether PSA 

containing non-small cell lung tumors exhibit a more

aggressive phenotype than PSA negative non-small cell 

lung tumors; 2) by investigating the effect of 

polysialylation ofNCAM on aggregation in vitro. 

P SA as a prognostic factor in resected non-smal! 

cel!lung cancer? 

In previous studies (Kibbelaar R et al. J Path 1989; 159: 

23-8; Eur J Cancer 1991 ; 27: 431-5), we found that PSA is 

most consistently expressed in small celllung cancer, but 

that approximately 15% of non-sm all cell lung cancers 

also expresses this marker. We recently investigated 

whether expression of PSA, detected irnmunohistochemical!y 

with monoclonal anti body 735, was related to 

disease free survival in 99 weil characterized non-small 

celllung cancer patients. In contrast to our earlier study, 

no significant correlation was found. This discrepancy 

could be due to immunohistochemical technicalities (in 

this study we used formalin fixed paraffin embedded 

tumor specimens, which resuJt in far superior 

morphology but lower sensitivities compared with frozen 

sections used in the earl ier study). Possibly, the frequency 

of PSA positive tumors is lower than th at of NCAM 

positive tumors. This wil! be studied further. 

In vitro effect of polysialylation of NCAM 

In order to study the contribution of PSA to the 

invasive phenotype ofNCAM containing tumor cells, it is 

necessary to clone the genees) encoding the enzyme(s) 

responsible for the synthesis of PSA. In the previous 

annual report (1993), we reported on the cloning of a 

cDNA encoding a novel sialyJtransferase, STF 114, th at 

could weil be involved in the synthesis ofPSA. The cDNA 

encoding STF 114 was cloned in an eukaryotic expression 

vector and transfected into the NCAM positive/ PSA 

negative variant ofneuroblastoma cellline CHP 212. The 

transfected cells expressed high levels ofSTF 114 mRNA, 

but showed no polysialylation ofNCAM. These cells also 

demonstrated no ex-2.8 sialyltransferase activity (D vd 

Eynden, Free University, Amsterdam). At that time, two 

independent publications reported on the identification 

and characterization of an ex-2.8 sialyltransferase with an 

amino acid sequenee identical to STF 114 (Sas aki K et al. 

J Biol Chem 1994; 269: 15950-6; Nara K et al. Proc Natl 

Acad Sci USA 1994; 91: 7952-6). Both reports showed 

that this sialyJtransferase is involved in the biosynthesis of 

ganglioside GD3. Whether this GD3 -ex-2.8 sialyltransferase 

is also involved in the polysialylation of NCAM 

remains to be studied. 

As another approach to deterrnine the effect ofPSA on 

the in vivo invasiveness ofNCAM containing tumor cells, 

two variants of small cell lung cancer cellline H69 we re 

established in collaboration with R Gerardy-Schahn, 

Medizinische Hochschuhle, Hannover. One variant 

demonstrated fully polysialylated NCAM on its cel1 

surface, the other lacked polysialylation of NCAM. The 

amounts of NCAM, E-Cadherin and integrins we re 

virtually identical. In vitro aggregation experiments 

demonstrated that the PSA negative variant aggregated 

faster. Removal ofPSA by endo-N treatment ofH69 PSA 

positive cells gave the same result. To deterrnine the effect 

Tumor Biology 67 

of PSA expression on invasiveness in vivo, both variants 

wil1 be injected into nude mice. 

Publications 

Michalides R et al. Int J ofCancer 1994, suppl. 8, 34-7. 

Michalides R et al. Cell Adhesion and Communication 

(in press). 

Notes 


2 Department of Pulmonology, University of Leiden. 

Cytoskeletal antigens 

D Ivanyi, Ph Hageman, AJM Balm, E Groeneveld, 

G van Doornewaard, DAtsma, WJ Mooi 

Expression of simple epithe/ial keratins 8/ 18 in 

head and neck squamous carcinomas ( NH-SCC) 

and SCC cel!-/ines 

Keratin pair 8/ 18 is expressed in simple and glandular 

epithelia. Originally cytokeratin 18 (KI8) was 

recommended for the differential diagnosis of 

adenocarcinomas (K18 positive) and squamous 

carcinomas (SCC) (K18 negative). With the development 

of a number of monospecific monoclonal antibodies, it 

became possible to irnmunolocalize individual keratin 

proteins in a small subpopulation of cells even when 

present at low levels. Site-related variations in K8/18 

expression in human epithelia of the head and neck 

(H&N) region were reported. Using a panel of 

monospecific mAbs, we have tested expression ofvarious 

keratins in normal epithelia of this region and we have 

confirmed and completed these data. Specifically, DE 

K18, specific for KI8 (Ivanyi D et al. Am J Vet Res 1992; 

53: 304-14) did not stain stratified epithelium ofthe floor 

of the mouth, gingiva and cheek, while it did stain basal 

cells in stratified epithelium ofhypopharynx as wel1 as the 

pseudostratified epithelium of the hypopharynx and 

larynx. K14 and K19 we re present in basal parts of all 

these epithelia. 

We have deterrnined the keratin phenotype of various 

carcinomas in H&N area and found that the mutually 

quantitative relationship between basal ce 11 and 

differentiation-related keratins of normal epithelia was 

altered in SCC in favor of bas al eell keratins. Most 

carcinomas recapitulated the expression pattern of 

keratins present in the basal layer of the normal 

epithelium ofthe tumor origin. Seventeen out of 18 SCC 

of larynx and hypopharynx were Kl8 positive; in these, 

more than 50% cells, and often al1 cells, were stained by 

DE-KI8. On the other hand, in only 3 out of9 SCC ofthe 

oral cavity, were K18 positive, in single cells. These 

findings suggest a potential application for K18 typing to 

distinquish between carcinomas originating in different 

sites of the head and neck region. 

In addition, we have analyzed keratin phenotype in 

several celllines established from HN-SCC. Irrespective 

of their site of origin (floor of mouth, gingiva, 

hypopharynx, larynx, buccal mucosa) all expressed 

keratins 8 and 18. However, after transplantation into

68 Tumor Biology 

nude mice, expression of K8/18 was downregulated to 

barely detectable levels, indicating that expression of 

K8 / l8 is also modulated by various environmental 

factors. 

Clinical application of monoclonal 

antibodies 

Tissue bank 

The central bank of deep-frozen tumor tissue, which is 

available as a central facility to the entire institute, 

expanded this year to 7,025 specimens. 

Cellular proliferation parameters 

during initial breast cancer treatment as 

predictors of clinical response 

RA Maas 1, AJ Breedijk 1, JL Peterse, PF Bruning 

The aim of the project is to predict the clinical response 

of patients with advanced breast cancer, by studying 

cellular proliferation in serial fine needie aspirates 

(FNAs) early in the course of treatment. As potential 

early indicators of response, changing mRNA levels of 

proliferation-associated genes are studied. A serniquantitative 

RT-PCR assay is used to monitor these 

changes. As a model, we started to measure the effect of 

the antiestrogen Tamoxifen on the expression of 

proliferation-associated genes in Tamoxifen-sensitive 

breast carcinoma cell lines. A decrease in the mRNA 

levels of Cy clin A, histone H4, c-myc, and Cy clin Dl was 

found within 3 to 7 days. The 8 to 32-fold decrease of 

Cy clin A mRNA levels was the most pronounced. At the 

same time, an increase of the mRNA levels of W AFI and 

c-fos was observed. The latter was probably related to the 

induction of apoptosis, since fragmentation of DNA and 

the expression of TRPM2, a marker of apoptosis, 

increased during Tamoxifen treatment. 

We have measured mRNA levels of proliferation 

related genes in a series of FNAs. Mammary carcinoma 

cells were purified immunomagnetically from FNAs 

before mRNA isolation. This purification proved highly 

efficient in a series of experiments using cell lines and 

FNAs. In 11 / 13 FNAs PB GD mRNA could be amplified 

by RT-PCR. In 4 /4 FNAs, positive for PBGD, cyclin A 

and TRPM2 gene transcripts could also be amplified and 

visualized. 

A study has now been started to measure early changes 

in mRNA levels during Tamoxifen treatment in women 

with primary palpable breast cancer. In this study women 

take Tamoxifen (20mg l day) for seven days prior to 

surgery. In these patients mRNA levels are compared in 

diagnostic FNAs from primary breast carcinomas and 

FNAs taken from the surgical specimen. 

Notes 


Malignant lymphoma 

P van Heerde, D de Jong 

During 1994, the lymphoma panel of the Amsterdam 

Comprehensive Cancer Center (I KA) reviewed more 

than 500 referred cases of malignant Iymphoma. 

Discrepancies between referral and definite diagnoses 

and diagnostic pro bI ems could mostly be resolved with 

the aid of extensive immune marker typing, occasionally 

complemented by molecular biologic techniques, 

available in the Department of Pathology. 

Publications 

Van Heerde P et al. In: A Colour Atlas of Cytology and 

Histology of Malignant Lymphomas (in press). 

De Bruin Pc. [Dissertation]: Free University of 


Molecular pathology laboratory 

G Brink, LH Boerrigter-Barendsen, MPW Gallee, 

M Vandeputte, E Vos, P Wisman, NJ Kürten, SJ Oei, 

D de Jong, MAJ van Dijk, AN Floore, WJ Mooi, 

LJ van 't Veer 

Mo/ecu/ar diagnostics 

The Molecular Pathology laboratory continued to 

expand in 1994. Analysis of gene rearrangements in 

immunoglobulin, T-cell receptor and bcl-2 (tI4;18) are 

performed on a routine basis. A clonal rearrangement of 

bcl-2 is indicative for the diagnosis follicular center cell 

lymphoma and clonal rearrangement of Ig or TCR can 

distinguish a lymphoma from a benign disorder. For two 

clinical trials patients are entered on the basis of 

molecular diagnosis. K-ras mutations in non-small cell 

lung cancer makes patients eli gab Ie for an adjuvant 

chemotherapy trial (phase 11, Rodenhuis) and patients 

with melanoma highly expressing the MAGE-I antigen 

are entered in an immunotherapy protocol (ph ase I, 

Rankin). 

New developments include X-chromosome 

inactivation analysis, which can discriminate between 

mono- or polyclonality and sometimes between multiple 

'primary' tumors in women. This clonality is potentially 

an important parameter for treatment protocols and is 

currently tested for archival material of metachrone 

tumors. 

In the past year more became known about genetic 

predisposition in high-risk cancer families, e.g. for 

Hereditary Nonpolyposis Colon Carcinoma (HNPCC), 

melanoma and breast carcinoma. Recognition of highrisk 

farnily members will greatly improve predictive 

screening and will help to design accurate treatmentl 

follow-up protocols. As a first indication for high-risk 

assessment in HNPCC, we are using a PCR-based test to 

determine genomic instability (replication error +) in the 

colon tumor tissue. A functional yeast growth assay for 

the melanoma susceptibility genes MTSI and MTS2 is 

being developed, which rnight enable us to identify highrisk 

familiar melanoma family members.

Molecular pathology is positioned at the interface 

between the laboratory and the clinic, and several of these 

projects are performed in close collaboration with other 

groups within the institute (Divisions 11, IV, VI, VIII, X 

and XI). 

Functional activity of the estrogen receptor in 

human breast cancer 

The estrogen receptor (hER) is an important regulator 

of growth and differentiation in the mammary gland and 

is also implicated in the development of breast cancer. 

Elevated levels of the estrogen receptor are found in 60% 

of mammary carcinomas and its presence correlates well 

with disease-free and overall survival when patients are 

treated with antiestrogens. However, 40% of the patients 

with an ER positive tumor do not respond to the 

treatment. Recent findings suggest that variant estrogen 

receptors might be present in these tumors that are nonfunctional 

and non-responsive to antiestrogens. 

Estrogen receptors are nuclear receptors which, upon 

hormone binding, act as transcriptional activa tors of 

target genes by binding to a specific DNA sequence, the 

estrogen responsive element (ERE), in the vicinity of 

these genes. We are using a yeast growth assay to 

determine the transcriptional activity of the estrogen 

receptors present in mammary carcinoma. In th is assay, 

PCR products of the ORF of hER cDNA (2004 bp) are 

directly cloned into a yeast expression vector in the yeast 

Saccharomyces cerevisiae, in vivo. Subsequently, the 

transcriptional activity of the cloned PCR products is 

tested, using a hER activated reporter gene, whose 

product is required for growth of the yeast. Each yeast 

colony represents a single hER molecule and is tested 

individually for hER activity. This allows us to 

discriminate between constitutively active, inactive and 

estrogen inducible hER variants. This assay is also 

suitable to appreciate heterogeneity of estrogen receptors 

within one specimen. So far, we have in deed been able to 

identify different hER variants that are present in the 

mammary carcinoma celllines T47D and MCF7 on the 

basis of their different functional activity. The nature of 

these variants is subsequently determined by DNAsequencing. 

The prevalence of variant receptors in 

mammary carcinoma is currently under investigation. 

The role of bcl-2 in B-non-Hodgkin 's lymphomas 

About 15% of low-grade follicle center celllymphomas 

(FCCL) fail to express immunoglobulins. The mechanism 

of the defect may be postulated by the absence of a 

funcional immunoglobulin gene, either by mutations or 

by non-functional rearrangement to pseudo-genes or outof-frame 

rearrangements. A PCR-based assay to clone 

complete immunoglobulin variabie regions, including the 

whole VH, DH and JH-regions, was developed and 

currently 15 cases ofimmunoglobulin negative FCCL are 

being studied. The mechanism ofthe defect has important 

consequences for the role of bcl-2 in the development of 

FCCL, since in normal B-cell development there are two 

bcl-2 regulated, immunogJobulin-dependent selection 

moments; upon functional heavy-chain rearrangement in 

pre-B-ceJls and in the germinal center. In FCCL, bcl-2 is 

Tumor Bi%gy 69 

constitutively expressed. Therefore, tumor development 

may be rendered immunoglobulin-independent in either 

of these selection steps. In collaboration with D Acton 

(Division VII), the same question will be investigated in 

bcl-2 transgenic mice, which can be expected to have a 

higher number of immunoglobulin negative mature 

B-cells. 

The t(14; 18) translocation th at is characteristic of 

FCCL can also be found in healthy individuals at a very 

low frequency. Transloctions that are not Iymphoma 

related and involve the T -cel receptor genes occur at a 

somewhat higher frequency in normal individuals. Assays 

to detect both translocations with high sensitivity were 

developed to be able to study the environmental factors 

that influence translocation incidences in vivo and in vitro.

VII Division of Molecular Genetics 

Head 

AJM Berns PhD 

SUBSECTION ON GENETICS 

Coordinator 

P Demant MD PhD 


M Snoek PhD (80%) 

Other academic staff 

RJA Fijneman MSc, PC Groot PhD, CJA Moen MSc, 

AStassen PhD, J van Wezel, MSc 

Permanent technica I staff 

E Delzenne-Goette (60%), J de Moes (84%), A de Moes 

Bezemer (60%), M Treur-Mulder (40%), H van Vugt 


AMJ Sachs, C Schippers-Gillissen (50%) 

Guests 

S van den Eijnde, C Moen, N Mori, K Schmeler 


L van Dinten, J de Graaf, N Kürten, L Mulder, AMJ 

Sachs, S de Vries 

Secretary 

MH Sonne-Gooren (50%) 

SUBSECTION ON MOLECULAR GENETICS 

Coordinator 

AJM Berns PhD 


MA van der Valk MSc 

Other academic staff 

D Acton MSc, MAlkema MSc, J Allen PhD, H Jacobs 

MSc, J Jonkers MSc, E Kennedy MSc, P Krimpenfort 

PhD, T Nijar MSc, HPJ te Riele PhD, EC Robanus 

Maandag, G Scheijen MSc, T Nijjar, C Tölg MSc, 

NMT van der Lugt MSc, M van Roon PhD, M Vooijs 

MSc 


L Rijswijk (84%), H van der Gulden (80%), 

EVerhoeven 


C Brouwers, A Floore, R Regnerus 


M Bronk, J Dannenberg 

Guest 

K Akagi, H Brady, G Gil Gomez, H Jacobs, 

NMT van der Lugt, C Tö!g 

Secretary 

C van Diepen (40%) 

71

72 Molecular Genetics 

Introduction 

The Division of Molecular Genetics has two 

subsections: the subsection ofMolecular Genetics and the 

subsection of Genetics. Emphasis is on the study of genes 

involved in tumorigenesis. For th is purpose, advanced 

mouse genetics is combined with molecular biological 

analysis. The studies indude the identification, 

characterization and functional analysis of oncogenes 

and tumor suppressor gen es (subsection Molecular 

Genetics) and the genetic mapping and identification of 

genes mediating resistance or susceptibility to carcinogeninduced 

solid tumors (subsection Genetics). Recombinant 

congenic mouse strains were developed for this 

purpose. Transgenic mice bearing oncogenes are being 

used to identify collaborating oncogenes and tumor 

suppressor genes th at are involved in later stages of the 

tumorigenic process. Targeted mutagenesis in embryonic 

stem cells, to inactivate or mutate resident protooncogenes 

and tumor suppressor genes, is being applied 

to study the normal function of these genes in 

development and their role in inherited and somatically 

acquired diseases. The division also supports ongoing 

transgenic projects in other divisions at the institute. 

Ca neer Genetics 

Understanding the genetic control of cancer 

development is one of the major challenges of cancer 

research. While considerable progress has been made in 

the identification of genes responsible for familial cancer 

syndromes, the nature of genetic factors influencing the 

susceptibility to the non-familial, sporadic cancer remains 

unknown. Comparison of susceptibility of mouse inbred 

strains to various types of spontaneous and induced 

tumors indicated that th ere is a large group of tumor 

susceptibility genes (TSGs). These, largely unknown, 

genes are polymorphic and exhibit organ-specific or 

organ-restricted action. Most of these genes are different 

from the presently known oncogenes and tumor 

suppressor genes. Elucidation of their mechanisms of 

action can provide further insight into the neoplastic 

process and allow a better risk assesment in man. 

Mapping of TSGs and working towards their 

identification is being combined with the study of their 

mechanisms of action, their role in specific stages of the 

neoplastic process and their possible functional 

interactions with known oncogenes and tumor 

suppressor genes. 

One ofthe major obstades in the analysis ofthis family 

of genes has been their multiplicity, which prevented their 

genetic mapping. In order to overcome this problem we 

developed the Recombinant Congenic Strains (RCS), 

that greatly facilitate the mapping of multiple loci 

controlling the same trait. By using this genetically weil 

characterized system, we we re able to dissect the genetic 

control of colon tumors, tumors of small intestine, lung 

tumors and radiation-induced leukemias. In addition, 

these strains turned out to be instrumental in mapping 

genes involved in the control of apoptosis and of various 

Immune responses. 

After the chromosomal linkage of these unknown 

genes has been established, they can be precisely mapped 

and subsequently identified. Presently, two regions 

bearing tumor susceptibility genes are being analyzed in 

detail, the region between Hsc70t and G7 in the center of 

the Major Histocompatibility Complex (H2) on 

chromosome 17, which harbors a lung tumor 

susceptibility gene, and the region between CD44 and 

D2Mit9 on chromosome 2 which contains the colon 

tumor susceptibility gene SccJ. 

MHC class III region and 

tumorigenesis 

M Snoek, H van Vugt' , RJH Fijneman 2 , P Demant 

The Major Histocompatibility Complex has been 

found to affect many traits, including susceptibility to a 

number of diseases. Previously, we have mapped tumor 

susceptibility genes for chemically induced lung tumors 

and hormonally induced mammary tumors to the central 

part of the H2 complex, within the H2-I-D interval 

(Oomen et al. Cancer Res 1988; 48: 6634-41 ; Röpcke et al. 

Immunogenetics 1990; 31 : 347-55). Analysis of various 

recombinant haplotypes in the C4-H2D interval showed 

the presence of a recombinational hot spot in this region 

between Hsp70.1 and Bat5 (Snoek et al. Immunogenetics 

1991 ; 34: 409-12). Our structural analysis, combined with 

functional data from the literature, has shown that 

susceptibility to several diseases map to this hot spot area, 

namely the Orchl gene controlling the susceptibility to 

experimentally induced allergic orchitis, the Cpsl gene 

controlling the susceptibility to corticoid induced deft 

palate, as well as Acp, a gene regulating the vitamin A 

sensitivity of this hormonally induced cleft palate. 

Moreover, the as yet structurally indistinghuishable 

strains BI0.A(lR) and BI0.A(2R), defining the crossover 

interval of the H2 b and H2 d haplotypes, show a 

different phenotype after prenatal ENU-induced lung 

tumorigenesis, indicating the presence of a susceptibility 

element in this hot spot area. To determine the genomic 

organization of this interval, we have cloned a ± 100 kb 

long segment derived from the C4-H2D interval of the 

H2 b haplotype (Snoek et al. Genomics 1993; 15:350-6). 

Our contig of overlapping cosmid dones contains murine 

counterparts of the genes described in the homologous 

region in man, namely G9, Hsp70.1 , Hsp70.3, Hsc70t , 

G7b, G7a (Bat6) , and G7. Hsc70t, G7b and part of G7a 

were also cloned from the H-2 d haplotype. Simple 

sequence repeat length polymorphism analysis and 

extensive sequence comparisons of Hsc70t and flanking 

regions of both haplotypes led to the condusion that the 

described functionally active genes map to the Hsc70t-G7 

segment. This further reduction of the cross-over interval 

leaves G7b and G7a as candidate genes. However, 

whereas in man the G7b and G7a genes are separated by ± 

1 kb of DNA, in the mouse there is ± 17 kb of DNA at this 

position. We examined the presence of additional genes in 

this stretch of DNA by hybridizing small genomic 

fragments isolated from the cosmid insert to N orthern 

blots. Three mRNA were observed. One mRNA was 

transcribed from the G7a gene, enabling us to map the 

transcriptional start site. A second mRNA of ± 2 kb is

expressed in liver tissue; the encoding gene spans ± 9 kb of 

DNA and is located telomeric of G7b. A third message of 

approximately 3 kb is expressed in lymphoid tissue. The 

encoding gene spans ± 4 kb centromeric of G7a. Cloning 

and sequencing of the cDNAs is expected to give insight 

into the nature and function of these genes. Furthermore, 

sequence analysis of the genomic cosmid insert spanning 

this interval is in progress. This might help us in reducing 

the number of candidate genes involved in orchitis, cleft 

palate and lung tumor susceptibility. 

Publications 

Snoek M et al. Mammalian Genome 1994; 5: 174-6. 

Snoek M et al. Immunogenetics 1994; 40: 159-62. 

Notes 


2 Funding: EEC Project SCl000213. 

Genetics of lung tumor development 

RJ A Fijneman I , MA van der Valk, E Delzenne-Goette, 

M Treur-Mulder, C Schippers-Gillissen, P Demant 

Mice transplacentally treated with N-ethyl-Nnitrosourea 

(END) develop lung tumors and tumors of 

the small intestine. The 0201 A-strain is susceptible to 

lung tumors but resistant to tumors ofthe small intestine. 

The B10.020 / Dem-strain is resistant to lung tumors but 

susceptible to tumors of the small intestine. In order to 

map the responsible TSGs, the OcB / Dem RC-strains, 

containing 87.5 % of genome of the strain 0201 A and 

12.5% of genome of the BlO.020/Dem strain were 

treated with END. Neither the (020 x BlO.020)F 1 

hybrids nor any of the OcB-strains developed tumors of 

the sm all intestine after the END treatment, indicating 

that several genes of BlO.020-origin are required for 

development of these tumors. This has been confirmed in 

the second backcross generation ((020 X BlO.020) X 

BlO.020) X BlO.020, where less than 20% of the mice 

developed tumors of the sm all intestine as opposed to 

50% in BlO.020-mice. Among these mi ce the 

homozygosity at the D4Mit13 locus was more frequent 

than expected (p < 0.0001), indicating that a locus (ssicJ 

susceptibility to small intestinal cancer - I) resides in the 

vicinity of this marker. 

Analysis of lung tumors in END-treated OcB-mice 

revealed several RC-strains (including OcB-9) more 

resistant than 0201 A. Our results show that the Kras-2 

allele is not the major gene responsible for these 

differences, as was suggested in the literature. The TSGs 

controlling differences in tumor numbers and tumor size 

are presently being mapped in the F2 progenies of these 

RC-strains and 0201 A. The transgene 

Tg(Alb-l ,HRASF24)Brli46, which leads to early 

appearance of lung tumors, has been crossed into 020 

and BlO.020 genetic background. The 020-mice bearing 

this transgene appear more susceptible to lung tumor 

development than the BIO.020 transgenic mice. In a cross 

involving transgenic 020- and OcB-9-mice we are 

presently testing whether the same TSGs control 

susceptibility to carcinogen-induced as weIl as transgene- 

Molecular Genetics 73 

induced tumors. 

We previously showed that one ofthe regions involved 

in control ofsusceptibility to tumors ofthe lung and small 

intestine is the H2 complex on chromosome 17, which 

contains several lung tumor susceptibility genes. We 

showed that two congenic strains, BlO.A(lR) and 

BIO.A(2R), with recombinant H2 haplotypes differing 

only in an 50 kb segment between Hsc70t and G7, differ in 

susceptibility to alveolar but not papillary lung tumors. 

Publications 

Fijneman RJA et al. Oncogene 1994; 9: 1417-21. 

Fijneman RJA et al. Immunogenetics 1994 (in press). 

Notes 

I Funding: EEC Project SC 1000213. 

Genetic dissection of colon tumor 

susceptibility 

APM Stassen I , JT van WezeJ2, A de Moes-Bezemer, 

J de Moes, MA van der Valk, P Demant 

The BALB Ic strain is relatively resistant and the STS I 

A strain susceptible to the 1,2-dimethyl-hydrazineinduced 

colon tumors. The RC strains of the CcS IDem 

series, each of which contains a different subset of 

approximately 12.5% genes from the strain STS I A and 

87.5% genes from the strain BALBIc, exhibit large 

differences in colon tumor susceptibility. Some strains are 

highly susceptible, whereas others are resistant, indicating 

that the susceptible strains received one or more nonlinked 

susceptibility genes from the STS parent. Linkage 

studies indicated th at one of the susceptibility genes 

(SccJ ; Susceptibility to colon cancer-l), present in the 

strains CcS-19, is located on chromosome 2 (Moen et al. 

Oncogene 1992; 7: 563-6) in the vicinity of D2Ndsl . The 

study also indicated the presence of TSGs on 

chromosomes 7, 10, and 11. Recently a more precise 

mapping has been obtained for the locus on chromosome 

10. 

The tumors th at were induced in these mapping studies 

were almost exclusively benign adenomas. As colon 

tumorigenesis proceeds from the early stages, manifested 

as aberrant crypts, through benign adenomas, to 

malignant carcinomas, we tested the susceptibility to the 

induction of these three stages in the strains BALBIc, 

STS I A, CcS-19, and several other CcS strains. The data 

obtained do not show a significant correlation between 

the different end-points. Strain BALB Ic, although 

resistant to colon tumors, is highly susceptible to the 

induction of aberrant crypts, whereas strain STS IA, 

which is highly susceptible to benign adenomas, is 

relatively resistant to aberrant crypt induction. Several 

other CcS strains also exhibit marked differences between 

susceptibility to aberrant crypts and benign adenomas. 

The induction procedure used for malignal}t tumors leads 

to more tumors in the strain CcS-19 than in STS 1 A, while 

STS I A is more susceptible to benign adenomas than 

CcS-19. These data suggest that different stages of the 

tumorigenic process are controlled by different subsets of 

susceptibility genes.


Notes 



Genetic and physical map of the Sec! 

region 

PC Groot', APM Stassen 2 , JT van WezeP, N Mori, 

P Demant 

The original mapping experiments of Moen et al. (see 

above) 10cated the Scel locus in a 40 cM interval between 

He and IU on chromosome 2. A set of 40 recombinant 

haplotypes has been produced, each with an independent 

recom bination in this region. The progenies of the crosses 

of these recombinants with BALB/ c were tested for 

tumor susceptibility and the linkage of tumor 

susceptibility with each recombinant haplotype assessed. 

The results limited the location of Seel to a region of 

approximately 4 cM between D2Mit66 and D2Mit43, 

including the CD44 locus. Novel recombinants were 

produced in this shorter 4 cM region and added to the 

subset of the previous recombinants. Their progeny tests 

indicated th at this segment of chromosome 2 contains the 

Seel gene located in an approximately 1.2 cM interval 

around D2Mit66 and another, previously undetected 

susceptibility gene, located 7 cM centromerically of 

D2Mit66. 

Further tests of the recombinants in the 1.2 cM interval 

will map the Seel locus to a very short segment of DNA. 

In order to obtain the linear order of recombination sites 

of the various recombinant haplotypes, high density 

coverage of the region with microsatellite pro bes is 

required. A Y AC contig comprising more than 120 Y ACs 

covering the region has been constructed. The Y ACs will 

be subcloned, the clones bearing simple sequence repeats 

sequenced and the flanking sequences used as primers to 

detect SSLPs. 

Publieations 

Groot PC et al. In: Genetic Variants and Strains of the 

Laboratory Mouse (in press). 

Mori N et al. Genornics (in press). 

Notes 




Molecular Genetics 

Identification and characterization of 

collaborating· oncogenes 

D Acton 7 , MAlkema', J Allen 9 , H BradylO, C Brouwerss, 

A Floore 3 , G Gil Gomez'o, H van der Gulden, J Jonkers 2 , 

E Kennedy5, P Krimpenfort 6 , NMT van der Lugt 8 , 

R Regnerus 2 , M van Roon 2 , G Scheyen 4 , 

MA van der Valk, E Verhoeven 4 

Transgenic mice exp;essing (putative) oncogenes can 

provide insight into the oncogenic potentialof the genes, 

disclose their tissue-specific activity and reveal their roles 

in the transformation process. Furthermore, genetic 

crosses between transgenic mice carrying different 

oncogenes allow assessment of their cooperative activity. 

Oncornice can also be used in combination with the 

provirus tagging technique to identify new genes, who se 

gain- or loss-of-function synergizes with the transgene(s). 

This approach is particularly suited to uncover events 

associated with tumor progression. By accelerating 

Iymphomagenesis through Moloney Murine Leukemia 

Virus infection (proviral tagging) in EI1-mye and 

E{l-pim-l transgenic mice, a number of collaborating 

genes we re identified. In EI1-mye transgenic mice the 

proviral insertion sites pim-l ,pim-2, pal-I , and bmi-l were 

identified as common insertion sites, whereas in EI1-pim-I 

transgenic mice proviruses integrated with high incidence 

near c-myc, N-mye, pal-I , and tiam-I. In a significant 

fraction of the tumors proviral insertions were found in 

the vicinity of more than one proto-oncogene, indicating 

that each activation contributed to the tumorigenic 

phenotype of the cell. By transplanting the primary 

tumors to syngeneic hosts, additional genes specifically 

involved in late stages of the tumorigenic process (e.g. 

tie-I andfat-l) were marked. With this approach up to 

four distinct genet ic mutations in the tumorigenic process 

were found. The progress made with respect to the role of 

these genes in normal and aberrant growth is summarized 

below. 

Pim-l and Pim-2 

Pim-l is a serine-threonine protein kinase with a short 

half-life and primarily cytoplasmic localization. The 

pathway in which the pim-l oncogene acts is still elusive. 

Recently, a homolog of pim-l has been identified by PCR 

cloning using degenerate oligonucleotide primers 

corresponding to conserved regions of the Pim-l kinase. 

Pim-2 is also frequently activated by proviral insertional 

mutagenesis in MuLV-induced Band T celllymphomas 

and it has many biochemical characteristics in common 

with Pim-l, such as substrate specificity and cation 

requirement for optimal kinase activity. We have 

generated pim-2 knock-out mice, which showed no 

obvious defects as was the case for pim-l deficient rnice. 

Pim-lI pim-2 double knock-out mice have been 

generated. Preliminary analysis again revealed little effect 

on hematopoietic populations. We are currently 

investigating whether hematopoietic ceUs from these mice 

exhibit specific deficiencies in response to growth factors 

or other mitogenic stimuli. 

Identification of Pim-l interacting proteins 

To search for interacting ceUular proteins that might 

act as a substrate of the Pim kinases or modulate their 

activity, we have applied the yeast two hybrid system. As 

a result of the screening of a mouse embryonic library 

using p34 Pim - ' as a bait we identified three cDNA clones 

that encode Pim-l binding proteins (Pib). Pibl and pib3 

are previously unidentified genes, whereas pib2 is the 

murine homolog of Ki, a protein frequently acting as an 

autoantigen in Lupus (SLE) patients. Pib3 belongs to the

mutagenesis and thereby the probability of oncogene 

activation. Transgenic mice have been generated carrying 

different variants of the MCF glycoprotein gene under 

the control of the CD2 promoter/enhancer. We will 

assess the tumorigenic potentialof the MCF envelope 

gene. Subsequently, we will investigate whether 

tumorigenesis in these mice can be accelerated with 

replication-defective viruses. 

Further refinement of proviral tagging 

There are a number of advantages to using fluorescence 

in situ hybridization (FISH) for the identification of 

proviral integration sites in Iymphoid tumors. 1) FISH 

allows identification of common insertion regions in 

which insertions are less clustered. 2) It will permit fast 

screening for the presence of new common insertion sites. 

3) It wiU enable unequivocal assessment of the coexistence 

of two or more common insertion sites within 

one tumor cell clone. We have shown that a defective 

pro virus carrying the LacZ gene can be effectively 

detected by FISH. We are currently further improving the 

sensitivity of detection and are introducing the various 

painting methods to identify the different mouse 

chromosomes in the metaphase spreads. We expect this 

approach to become operational in the coming year. 

Publications 

Berns AJM et al. In: Cold Spring Harbor Symposia on 

Quantitative Biology, Volume LVIX (in press). 

De Wind N et al. Virology 1994; 200: 784-90. 

Habets G et al. Cell 1994; 77: 537-49. 

Klein H et al. J Biol Chem 1994; 269: 25521-8. 

Renauld J-C et al. Oncogene 1994; 9: 1327-32. 

Van der Lugt NMT et al. Genes & Development 1994; 

8: 757-69. 

Van der Lugt NMT [Dissertation]. University of 


Notes 







Research (NWO), Program 900-502-095. 


8 Funding: SBDO. 

9 Fellow of the Leukemia Society of America. 

10 Fellow ofEMBO. 

T -cell development and T -cell receptor 

genes 

D Acton 2 , J Borst 5 , H Brady4, H Jacobs 3 , P Krimpenforti 

We have further analyzed the T-cell development and 

requirements for tumor induction in TCRó{3 transgenic 

mice. These mice, which carry a T -cell receptor transgene 

in which most of the varia bie region has been deleted, 

show aberrant T -cell development with a concomitant 

Molecular Genetics 77 

increase in Iymphoma incidence. We have further 

dissected this system by a number of crossbreeding 

experiments (e. g. bcl-2, TCR{3, TCRa transgenic mice; 

RAG-l KO mice) combined with biochemical analysis of 

the surface expressed TCR complex. The data suggest 

that continued signalling of the pre-T-cell receptor in 

combination with an increased cycling of progenitor T 

cells, the latter caused by a disturbance of homeostasis 

due to apoptosis or irnpairment of T -cell differentiation, 

aUows accumulation of additional mutations with the 

resulting high tumor incidence. 

To investigate the pathways involved in the apoptosis 

ofT cells, CD2-bax transgenic mice have been generated. 

Since bax negatively regulates bcl-2, we expect these mice 

to exhibit increased apoptosis. MuLV infection wiU be 

applied to determine whether this 'death' phenotype can 

be rescued through the insertional mutation of a gene that 

acts in this pathway. 

The IL-2Ry chain (y-chain) is an essential component 

of the high and intermediate affinity IL-2 receptors. The 

y-chain gene is located on the X chromosome in both man 

and mouse. Recently, it has been demonstrated that the 

y-chain is also an essential component of the receptors of 

several other Iymphokines (IL-4, IL-7, IL-15). This 

finding might explain why y-chain deficiency has such a 

profound effect on the development of the Iymphoid 

system as observed in X-linked SCID patients. To assess 

the function of the y-chain in the development of the 

Iymphoid system we have generated mice lacking the 

y-chain by targeted disruption in ES ceUs. Mice carrying a 

disrupted y-chain gene would be affected in all the 

functions of the Iymphoid system. These mice might 

constitute a better system for (heterologous) tissue 

transplantation than the currently available mutant 

mouse strains which exhibit residuallymphoid cells. Since 

the y-chain gene is 10cated on the X chromosome and the 

parent ES cellline is of male origin, the targeted ES ceUs 

do not have a functional y-chain. We have therefore 

analyzed the contribution of the KO ES cells to the 

lymphoid compartment of the chimeric mice (using the 

Ly-9.1 antibody specific for 129 lymphoid cells). The 

results indicate that y-chain deficient cells are unable to 

colonize thymus, spleen and lymph nodes, whereas there 

might be a minor contribution to bone marrow. The 

development into myeloid and erythroid ceUs seems not 

to be affected. Nullizygous y-chain mutant mice, obtained 

in the next generation, will be studied in detail and their 

suitability as a transplantation host wiU be tested. 

Publication 

Jacobs H et al. Eur J Immunol 1994; 24: 934-9. 

Notes 




3 Funding: SBDO. 

4 FeUow of EMBO. 

5 Division of Cellular Biochemistry, The Netherlands 

Cancer Institute.


Functional analysis of tumor 

suppressor genes 

K Akagi4, EC Robanus Maandag 3 , HPl te Riele', 

M Vlaar', M Vooijs2 

Targeted disruption of the Rb gene 

Rb- i - mi ce die in utero at day 12-14 of gestation with 

defects in liver hematopoiesis, neuronal development and 

eye lens differentiation. Heterozygous Rb mice are 

normal but succumb later in life from pituitary tumors. 

Since the embryonic lethality precludes monitoring the 

effects of loss of Rb in later stages of development and 

tumorigenesis, we have extensively studied phenotypic 

alterations in chimeric mice generated from Rb double 

knock-out ES cells. Remarkably, Rb- i - ES cells can 

contribute efficiently to all tissues, with the exception of 

the retina. In the retina an increased level of apoptosis is 

observed late in gestation, whereas such cell death is not 

observed in chimeric mice generated with the paren tal 

Rb + i - ES cellline. This observation is in accordance with 

other studies in which the retina-specific sequestering of 

Rb by viral oncoproteins leads to a similar pathology. We 

cannot exclude that in other chimeric tissues, such as 

brain, a subset of cells cannot be formed from the Rb- i -ES 

cells. In pure Rb-i-embryos, this might lead to widespread 

death of cells that themselves do not need Rb for survival. 

To gain further insight into the requirement for Rb in the 

various cell types, we have generated ES celllines in which 

an IRES-IacZ gene is used to disrupt the Rb gene. Rb- i - 

cells will therefore express lacZ in an Rb-specific fashion. 

Sublines will be made that will in addition express CAT 

constitutively. The simultaneous detection of these 

markers in situ will hopefully enable us to follow the fate 

of Rb- i -ES cells that do or do not have the Rb promoter 

switched on. Analysis of these mice is expected to provide 

us with a better insight into the cell type-specific 

requirement of Rb. 

Conditional inactivation of tumor suppressor genes 

in the mouse 

Mice carrying targeted mutations in tumor suppressor 

genes are often embryonic lethal (Rb, Apc, WTl, NF2). 

To study the fuction of these genes later in development 

as weil as their role in tumorigenesis, we wish to employ 

methods that allow tissue-specific inactivation of these 

genes, using Flp/ Frt and Cre/ LoxP site-specific 

recombination systems. These systems allow, in a 

chromosomal context, specific deletion of DNA 

fragments. We are testing the feasibility of this approach 

on the Rb-locus in which a homozygous targeted 

mutation causes embryonic lethality. In addition, we will 

modify these loci in such a way that successful 

recombination, and thereby inactivation of the target 

gene, is accompanied by the switch-on ofthe lacZ marker 

gene. As a first approach, ES-cell lines have been 

generated in which recombination target sites (Frt or 

LoxP) have been introduced around an exon that is 

essential for normal Rb-function. Mice were obtained 

that transmitted the modified Rb-allele through the germ 

line and are currently being crossed with Rb + /- 

heterozygotes to ascertain that the recombination sites do 

not interfere with normal transcription and splicing of the 

Rb transcript. A panel of transgenic mice will be 

generated th at express the recombinase in a tissue-specific 

fashion. Crossing of these transgenics with rnice th at have 

one inactive Rb-allele and one Frt or LoxP modified allele 

is expected to lead to the tissue-specific deletion of Rb. In 

the first instance, the consequences of retina-specific 

inactivation of Rb will be studied. Subsequently, Rb 

inactivation will be induced in tissues in which loss-of-Rbfunction 

is correlated with the predisposition and 

progression of tumorigenesis in man (e. g. osteosarcoma, 

breast and lung cancer). Other oncogenic mutations, such 

as dominant negative p53 mutations, will be combined 

with the conditional Rb-inactivation to unleash the 

tumorigenic capacity of Rb. 

lnducible expression of tumor suppressor gene 

functions 

We have designed a stringent binary gene suppression 

and induction system allowing tissue-specific shut-off and 

induction of gene expression in vivo. This system takes 

advantage of a modified tetracycline repressor gene, 

carrying the activation domain of the herpes simplex 

VP16 protein. Several transgenes have been constructed. 

To express the hybrid transactivator (tTA) in the mouse, 

the tTA gene was fused to the elongation factor la and 

actin promoter. In addition, transgenic lines we re 

generated in which the human Rb gene or the lacZ gene 

are under control of the tPA. Crossbreeding experiments 

are in progress to generate the mice carrying the 

regulatable human Rb transgene in the Rb-i-background. 

Publications 

Berns AlM Curr Bio11994; 4: 137-9. 

Robanus Maandag EC et al. EMBO 1 1994; 13: 4260-8. 

Notes 




3 Funding: Amgen. 

4 Kumamoto University School of Medicine 

Functional analysis of other genes by 

targeted mutations and transgenesis 

K Akagi, H Brady, P Krimpenfort, EC Robanus 

Maandag, L Rijswijk, HPl te Riele, C Tölg, M van Roon, 

M Vlaar 

The Division has been involved in the generation ofKO 

mice for a variety of genes in collaboration with other 

investigators: 

The E12/ E47 Helix-Loop-Helix transcriptional 

activator has been disrupted in collaboration with C 

Murre, UCSD, San Diego. The mutant mice show a 

profound phenotype that has been analyzed in some 

detail. 

The bcl-3 gene has been disrupted and mutant mice 

lacking both allel es are currently being investigated. This

work was initiated with IM Verma ofThe Salk Institute in 

La Jolla, California. 

The ADA gene has been inactivated in cooperation 

with D Valerio (University of Leiden). Contrary to our 

expectations the loss of ADA results in early postnatal 

death of the ADA-deficient mice. Biochemical analysis 

confirms the severe defect in the nucleotide synthetic 

pathway of these mice. 

Mutant CD44 genes will be studied in collaboration 

with the laboratory ofP Herrlich from Karlsruhe. Loss of 

CD44 does not result in embryonic lethality (data 

obtained by the Laboratory of T Mak, University of 

Toronto, Canada). We intend to generate more subtie 

mutations and analyze their competitive capacity to 

colonize specific organs/tissues. 

Tap-l knock-out mice have been generated to make 

these valuable mice available to the scientific community. 

Mutant mice have been distributed to a large number of 

laboratories. 

Mutant mice of the NF-2 tumor suppressor gene are 

being produced in collaboration with G Thomas in Paris. 

The targeting of ES cells has just started. 

In collaboration with the group of W Mooienaar 

(Division lIl), one of the receptor phosphatase genes is 

being inactivated in mice. These activities started recently. 

We are participating in the generation of single and 

compound mutant mice for the mdr genes (collaboration 

with group of P Borst, Division V). 

A variety of transgenic lines have been produced for 

groups in house as weil as for outside laboratories. 

During 1994 15 different constructs were used to yield 80 

different transgenic founders. 

Publications 

Bain G et al. Cell 1994; 79: 885-892. 

Schinkel AH et al. Cell 1994; 77: 491-502. 

Mo/ecu/ar Genetics 79

VIII Division of Experimental Therapy 

Head 

AC BeggPhD 


H Bartelink MD PhD (10%), WJ Nooijen PhD (10%), 

S Rodenhuis MD PhD (20%), LA Smets PhD, 

FA Stewart PhD 


JD van den Berg MSc, JM Coco Martin PhD, 

ME Craanen MD PhD, I van Geel MSc, EC Heesbeen 

PhD, A Kuin MSc, AC Lambrechts PhD, 

JHB Masselink MSc, JG Post MSc, M Rutgers PhD 

(80%), GS Salomons, MFMA Smeets MSc, 

B van Stekelenburg MSc, A Top PhD, 

PJM van der Vaart MD, R Veenhuizen MD 


SG Klaver (80%), I Hofland, EHM Mooren (80%), 

Y Oussoren, H van Rooy, MC Ruevekamp-Helmers 

(70%), M Verwijs-Janssen 


MC Aalders, CKM Buitenhuis, MS de Lange, 

HJ Knaken (65%), HOppelaar, CPE Ottenheim, 

JAM te Poele, UH Smolders, GR Westerhof 


E Boot, M van den Brand, MEssers, ESJ Mangal, 

L 't Mannetje, LM Nijenhuis 

Secretary 

TEA Eggenhuizen 

81

82 Experimen ta! Therapy 

Introduction 

Research in the division includes studies on 

experimental chemotherapy, experimenta1 radiotherapy 

and new forms of therapy such as photodynamic therapy 

and boron neutron capture therapy. The aim of the 

division is to build and maintain good links between the 

laboratory and the clinic, both by attracting young 

clinicians to do laboratory research for a year or more, 

and by carrying out clinically based or clinically relevant 

projects. 

This year, two new Dutch Cancer Society projects were 

begun, one on MIBG, a radiopharmaceutical showing 

promise in the treatment and diagnosis of neuroadrenergic 

tumors, and one on the relevance of the bcl-2 

oncogene in drug sensitivity (Smets). Research highlights 

include the interesting data emerging on apoptosis and 

treatment sensitivity, where two groups (Smets, Smeets) 

have found a divergence between clonogenic cell killing 

and apoptosis induction frequency for a number of cell 

lines and treatments. In the predictive assay field, the 

studies on chromosome damage have thrown up two 

unexpected findings which could shed light on damage 

tolerance as a factor in radiosensitivity (Co co Martin, 

Smeets). In the cell kinetic field, 3-color flow cytometry 

with cytokeratin and IdUrd antibodies shou1d lead to 

improvements in T ot measurements (Begg), and analysis 

of ploidy and %S-phase in childhood leukemia appears to 

have strong predictive value, potentially leading to 

reduction in treatment intensity in some subgroups 

(Smets). 

Oncogenes and response prediction in 

human tumors 

S Rodenhuis, A Top', SG Klaver', AC Lambrechts', 

MS de Lange 2 , WJ Mooi, LH Boerrigter, P Wisman, 

G Brink, ME Craanen, LJ van 't Veer 

The clinical significance of ras mutations 

We have previously shown that ras mutations in 

completely resected lung adenocarcinomas are associated 

with a very poor prognosis. This finding has been 

confirmed by others in several independent studies and is 

the basis for a European clinical study (EOR TC 08922), 

in which lung cancer patients with ras mutations in their 

tumors are being randomized to receive either adjuvant 

chemotherapy or no further treatment after operation. 

This study began to recruit patients in 1993 and should 

determine whether chemotherapy is helpful in this 

prognostically unfavorable group of patients. 

A single-institution clinical study in patients with 

advanced lung cancer, which was initiated in 1991, was 

closed in the summer of 1994, after a total of 83 patients 

had been entered. This study aimed to determine whether 

ras mutations could predict the response to 

chemotherapy. This question is relevant, since the 

introduction of mutated ras oncogenes in rodent celllines 

leads to radiation and chemotherapy resistance. The 

survival and response data should reach maturity in 1995, 

but it is already clear that tumors harboring ras mutations 

are not invariably resistant to chemotherapy. 

The presence and type of ras and p53 mutations have 

been investigated in a series of 54 non small cell lung 

cancers, employing immunostaining, denaturing gradient 

gel electrophoresis and cycle sequencing. Missense 

mutations in p53 proved to be detectable by immunostaining, 

but nonsense mutations were not. Simultaneous 

occurrence of ras and p53 mutations in the same tumor 

was surprisingly uncommon. Abnormalities in p53 were 

not associated with any clinical characteristics of the 

tumor. 

p16 mutations in lung cancer 

The p16 protein is an inhibitor ofthe cell cycle, that has 

been shown to be commonly mutated or deleted in a 

variety of tumors, including melanoma, breast cancer and 

kidney cancer. The gene coding for the protein has been 

identified as the first tumor suppressor gene that is an 

intrinsic component of the cell cycle machinery. A 

suitable screening test that is able to detect abnormalities 

in p16 in sizable series ofuncultured tumors is, at present, 

not available. We have attempted to develop an assay 

based on PCR and denaturing gradient gel electrophoresis 

to achieve this. This is technically difficult 

because of the unusually high melting temperature of the 

amplified DNA fragments. Strategies to overcome these 

difficulties are currently under investigation. 

Quantification of gene expression employing 

RT-PCR 

Work on the quantification of gene expression in very 

small samples (such as fine-needle aspirations) has 

continued. The main focus of the experiments has been 

the linear relationship between the number ofPCR cycles 

and the logarithm of the signal intensity, as quantified by 

a phosphorus imager. This relationship was shown to be 

independent of the amount of input DNA, but the 

efficiency of the PCR varies between different genes and 

different samples. Fine-tuning of the reaction conditions 

has led to a general method th at allows the estimation of 

the relative amount of input RNA (as compared to an 

internal standard) with approximately 20% accuracy. 

Experiments are in progress that will show whether this 

level of accuracy is sufficient to obtain clinically useful 

information from fine-needle aspirations from human 

tumors in situ. Genes of interest include ras, neu, 

topoisomerases I, IIa and IIb, and mdr 1. 

Publications 

Kern JA et al. J Clin Invest 1994,93: 516-20. 

Rodenhuis S. In: Lung Cancer: Principles and Practice 

(in press). 

Top B et al. Int J Cancer (in press). 

Van Halteren HK et al. Int J Cancer 1994; 57: 362-4. 

Notes 

, Funding: Dutch Cancer Society, Project NKB 91-08. 

2 Funding: Bristol-Myers-Squibb.

, 84 Experimenfal Therapy 

Apoptosis and cytostatic treatment 

The role of apoptosis in mediating the death of 

hematopoietic cells from various physiological 

(glucocorticoids; growth factor withdrawal) or cytostatic 

(anti-leukemic drugs, ionizing radiation) inducers, is 

attracting much attention. The clinical relevance of 

apoptotic death has been questioned, however, for the 

lack of comparisons with clonogenic cell death, 

considered to be the gold standard for effects of cytostatic 

treatment. We have compared the induction of apoptosis 

and the loss of clonogenic capacity in clonal variants of 

S49 lymphoma cells, sensitive or resistant to 

glucocorticoids. Apoptosis-resistant cells ('deathless 

phenotypes') were cross-resistant to the effect of growth 

factor withdrawal but had unchanged sensitivity to the 

drugs cytosine arabinoside and vincristine and to ionizing 

radiation. The bcl-2 gene was not expressed in the various 

cell lines nor induced by prolonged growth delay. 

Formation of DNA ladders or of apoptotic bodies 

underestimated by 10-100 fold the effects of various 

treatments on clonogenic survival and we re even falsepositive 

for the latter parameter after low doses of 

ionizing radiation. These experiments have identified a 

novel, Bcl-2-independent, mechanism of resistance 

against apoptosis by physiological triggers which did not 

apply for the action of cytostatic drugs and ionizing 

radiation. 

Publications 

Smets LA et al. Med Ped Oncol (in press). 

Smets LA et al. Blood 1994; 84: 1613-9. 

Smets LA. Anti-cancer Drugs 1994; 5: 3-9. 

Van den Berg JD et al. J Ster Biochem Mol Biol 1994; 

51: 33-40. 

Notes 


2 Collaborations with Dutch Childhood Leukemia 

Study Group, Den Haag and Emma Children's 

Hospital/Children's Academic Medical Center, 

Amsterdam. 

Tumor pH and drug sensitivity 

LA Smets, A Kuin I , MAalders I, M Nijenhuis, MEssers 

Intratumoral pH is a recognized modulator of several 

non-surgical treatment modalities of cancer. Selective 

reduction of intratumoral pH may increase the 

therapeutic index of several conventional and 

experimental (pro)drugs. We have developed a protocol 

of glucose administration to stimulate the glycolytic flux 

in hypoxic tumor cells in vivo, combined with the 

mitochondrial inhibitor meta-iodobenzylguanidine 

(MIBG) to convert oxygenated tumor eeUs into 

functionally hypoxic cells. Combined treatment results in 

protracted, tumor-selective acidification of potential 

therapeutic interest. 

To screen for drugs that will be more active at increased 

H + -ion concentration, we have developed an in vitro 

model for the animal studies. Af ter acidification (pH 6.2) 

of the culture medium by enhanced lactate production in 

the presence of MIBG, enhancement of the cytotoxicity 

of several conventional and experimental (pro )drugs was 

tested. The cytotoxicity of the bioreductive agent E09 

and of the alkylating agents chlorambucil and melphalan 

was most potentiated at reduced pH. Potentiation with 

mitomycin C of cytotoxicity by MIBG was less than 

predicted from the potentiation observed after lowering 

the pH by addition oflactate. These observations suggest 

that MIBG can have negative effects on drug action, 

unrelated to its capacity to stimulate lactic acid 

production. Contrary to expectations, the topoisomerase 

I inhibitor Topotecan was not potentiated by conditions 

of low pH that may promote its uptake. 

Publication 

Kuin A et al. Cancer Res 1994; 54: 3785-92. 

No te 


Membrane transport of cisplatin in 

human ovarian earcinoma eells 

EC Heesbeen I , JH Schornagel 

We have investigated cisplatin (cDDP) sensitivity and 

transport in two human ovarian carcinoma cell lines, 

A2780 and COV and their cDDP resistant sub-lines 

ADDP (25-fold) and COV.Pt (9-fold) towards cDDP. In 

accumulation studies, cellular drug content increased in 

proportion to extracellular cDDP concentration in all 

four cell lines, although the cDDP-resistant ADDP and 

COV.Pt lines accumulated 80% and 65% less cDDP 

respectively compared to their parent celllines. In all cell 

bnes, accumulation of cDDP was non-saturable up to 

2mM and linear up to 240 min. A TP depletion by sodium 

fluoride and sodium azide resulted in a 50% decrease in 

cDDP accumulation in A2780, COV and COV.Pt cells, 

and a 2-fold increase in ADDP cells. In all cell lines, 

accumulation ofcDDP was reduced 2-fold in the presence 

ofCaCl 2 • 

It appeared th at the efflux ra te of cDDP was only 

minimal and not significantly different in the parent and 

resistant cell lines. Depletion of intracellular glutathione 

(GSH) with buthionine sulfoximine and 

dimethylfumarate did not change the cDDP 

accumulation pattern in any of the four cell lines. 

Moreover, cells preloaded with [S 35]-cysteine, which will 

be incorporated into GSH, did not show any efflux ofthe 

label after exposure to cDDP, except for A2780 cells 

which showed some efflux af ter one hour of cDDP 

treatment. These latter findings suggest that a role for the 

glutathione-S-conjugated efflux pump as the mechanism 

behind the cDDP transport defect in the ADDP and 

COV.Pt cells is not likely. 

Note 

I Funding: Dutch Cancer Society, Project NKI 90-19.

esponse for mTHPC-mediated PDT but not for 

Photofrin-PDT. This could be related to greater 

photochemical efficiency, and thus more rapid oxygen 

depletion, for mTHPC. 

Normal tissue response to PDT 

One of the major si de effects of Photofrin mediated 

PDT is persistent skin photosensitivity. Skin reaction 

experiments demonstrated that mTHPC 1 day before 

illumination caused slightly greater skin phototoxicity 

than Photofrin. At 3 to 7 days, ho wever, there was little 

skin phototoxicity remaining af ter mTHPC whereas the 

Photofrin reactions remained maximal. 

PDT has clinical application for the treatment of 

multiple superficial bladder tumors. However, since the 

entire organ is treated, normal tissue toxicity is dose 

limiting and must be carefully evaluated. Bladder damage 

(increased urination frequency, hematuria, uJceration) 

and recovery were compared for Photofrin, mTHPC or 

BCA mediated PDT in a mouse model. Intravesicle 

illumination with 5-1 OJ.cm- 2 at 1 day after mTHPC gave a 

similar amount of damage to Photofrin. Higher light 

doses (15-20 J.cm- 2 ) could be applied at 1 hour af ter BCA 

injection for the same amount of damage. Since 

equivalent tumor responses are achieved with much lower 

light doses in combination with mTHPC or BCA than 

with Photofrin (see above), it should be possible to 

achieve a better tumor response for less bladder damage 

using these new photosensitizers. 

Intraperitoneal P DT 

The aim of th is project is to evaluate the potentialof 

PDT for the treatment of sm all peritoneal tumors, as a 

model for minimal residual ovarian cancer. Tumors 

(CC531 colon carcinoma) we re inoculated in abdominal 

fat pads of female rats and grown to 5 mm diameter. Rats 

were then given i.V. or i.p. injections of Photofrin or 

mTHPC before iUumination of the lower abdomen at 

laparotomy. Tumor response was evaluated by 

laparoscopic caliper measurements. Light or 

photosensitizer alone did not induce tumor growth delay 

relative to untreated rats. Significant growth delay was 

achieved for light doses of 25J.cm- 2 at 1 day after 

Photofrin, or with 6J.cm- 2 after mTHPC (equitoxic with 

respect to weight loss). Intraperitoneal administration of 

photosensitizers did not improve tumor response relative 

to i.V. Drug uptake studies with 14C-labeled mTHPC did, 

however, demonstrate much higher drug levels in very 

sm all (l-2mm) peritoneal tumors after i.p. 

administration. lt seems likely that i.p. drug 

administration will only offer an advantage for 

microscopic disease and not for larger tumors with an 

established blood supply. 

MMC alone induced only smalI, non-significant tumor 

growth delay but MMC in combination with Photofrin or 

mTHPC mediated PDT gave longer growth delays than 

PDT alone, confirming resuIts with subcutaneous mouse 

tumors (see above). 

A method has been developed for minimally invasive 

laparoscopic illumination, suitable for repeated 

treatments of intraperitoneal tumors. Spherical light 

Experimental Therapy 87 

diffusers were fed into catheters with inflatable balloon 

tips. These catheters were introduced into the abdominal 

cavity via a smaU, midaxillary incision, and inflated in 

situ. Tumor bearing, photosensitized rats were 

illuminated 1 to 4 times using this set-up and resuIts for 

tumor response and toxicity are being evaluated for 

comparison with the more invasive laparatomic 

iUuminations (single treatments only). 

Detection of singlet oxygen yieldfrom new 

photosensitizers 

Many different types of new photosensitizers are 

currently being developed for clinical use. Requirements 

of new photosensitizers are that they have low dark 

toxicity, preferential uptake and retention in tumor 

tissue, strong absorption peaks in the red part of the 

spectrum (for good tissue penetration oflight) and a high 

yield of singlet oxygen on excitation. Singlet oxygen yields 

of three new photosensitizers and Photofrin were 

measured by the detection of luminal chemiluminescence 

at 445 nm in Menzel's buffer solution. The relative singlet 

oxygen yields were nPe6 (chlorin) > ATX-SIO 

(Porphyrin) > mTHPC (chlorin) > Photofrin 

(porphyrin). The new compounds all produced greater 

singlet oxygen yields than Photofrin, indicating th at they 

should be effective photosensitizers. 

Publications 

Baas P. [Dissertation]. Amsterdam: University of 


Baas P et al. Int J Cancer 1994; 56: 880-5. 

Baas P et al. Photochem Photobiol1994; 59: 448-54. 

Baas P et al. Lasers in Surgery and Medicine (in press). 

Van Geel IPJ et al. Int J Cancer 1994; 56: 224-8. 

Van Geel IPJ et al. Int J Cancer (in press). 

Veenhuizen RB et al. Int J Cancer (in press). 

Notes 

J Funding: Dutch Cancer Society, Project NKI 91-05. 

2 Outside funding. 

3 Dr Daniel den Hoed Cancer Center, Rotterdam. 

4 University of Leiden. 

5 University Hospital St Radboud, Nijmegen. 

Boron neutron capture therapy (BNCT) 

R Verrijk I, UH Smolders 1 , AC Begg, R Huiskamp2 

Studies in th is last project year concentrated on two 

boronated porphyrins, BOPP and MSK-l, which are 

candidate compounds for clinical BNCT. In vitro studies 

revealed greater uptake of BOPP in three glioma lines 

than in two melanoma lines, with ceU / medium ratios 

varying between land 8. MSK-l showed worse uptake 

than BOPP in two glioma lines (ratios < 1), but an equal 

uptake in a melanoma line. Cellular retention of BOPP 

also tended to be greater than for MSK -1. These resuIts 

indicate that BOPP is the preferred porphyrin for BNCT, 

although in vivo irradiation studies need to be done for 

confirmation. 

From in vivo pharmacokinetic data in mice, an index of

88 Experimental Therapy 

merit was calculated to compare compounds and 

administration routes. The index combines tumor I 

plasma ratios, absolute tumor levels and the total dose 

administered. For BOPP, the data indicated the 

superiority of muliple injections of BOPP over single 

admninistrations, whether oral or intravenous, and the 

superiority of BOPP over the present European clinical 

candidate drug BSH. 

This project has now been completed, although a 

clinically related project is still running within the 

institute (Division IX) and at the Petten nuclear reactor 

site. 

Publications 

Verrijk R et al. Int J Radiat Bio11994; 65: 241-53. 

Verrijk R et al. Br J Cancer 1994; 69: 641-7. 

Notes 


2 Energy Research Foundation ECN, Petten. 

Mechanisms of radiation sensitivity 

MFMA Smeets I , JHB Masselink, EHM Mooren, 

AC Begg 

Cel! cycle blocks, p53, and apoptosis 

The p53 tumor suppressor gene plays an important role 

in inducing a G 1 arrest and in triggering apoptosis after 

irradiation. In five human tumor cell lines with a wide 

range of radiosensitivities, the relevance of this cell cycle 

block and apoptosis for cell survival and the correlation 

with p53 status and DNA damage induced protein 

expression was investigated. Using F ASA Y (functional 

analysis of separated alleles in yeast) and cycle 

sequencing, we found only one cell line, MCF7, to 

contain wild-type p53 and to display the expected G 1 IS 

arrest. The other lines we re either heterozygous (SQ20B 

and SQD9), homozygous (SCC61) or p53 null mutants 

(SC8S) and did not arrest at the Gi l S border after 

irradiation, despite the induction of protein expression. 

Contrary to expectation, the G 1 IS block in MCF7 cells 

did not confer radioresistance, nor did the absence of the 

block in mutant p53 cens result in increased 

radiosensitivity. Apoptosis was induced in a p53 

independent way and there was a linear relationship with 

dose and time until maximum levels we re reached one day 

after radiation. No correlation was found with 

radiosensitivity, however. 

Manipulation of cel! cycle genes 

DNA damage causes cell cycle blocks at the Gi l S and 

G2/ M boundaries. The biological function of these 

blocks is thought to facilitate DNA repair before the cell 

attempts DNA replication or mitosis. Alternatively, the 

cen may undergo apoptosis if the damage is beyond 

repair. To establish a possible relationship between cell 

cycle progression and DNA damage after ionizing 

radiation, a system to manipulate cen cycle progression 

was set up. Initial experiments with microinjections 

proved to be time consuming and toxic to cens. As an 

alternative approach, transfections were chosen using a 

binary vector system with a tetracyclin-dependent 

inducible promoter. Advantages are that the effect of 

expression of a single gene can be studied in a uniform 

genetic background, while the duration and level of 

expression can be varied. The changes in cen cycle 

progression caused by ectopic gene expression win be 

correlated with changes in cell survival after cytotoxic 

treatment. 

Three cell lines selected for this approach were 

characterized by cen cycle progression, clonogenic 

capacity and apoptosis induction after irradiation. For all 

three cell lines, transfectants stably expressing a 

transactivator molecule required for the inducible 

expres sion have been generated and successfully tested 

with a luciferase reporter construct. A number of genes 

implicated in cell cycle con trol have been cloned behind 

the tetracycline dependent operator I promoter to 

generate inducible overexpression. 

We have attempted to decrease G liS arrest after 

irradiation by inducible cyclin DI overexpression in 

MCF-7 cens (collaboration with R Michalides, Division 

VI). Although changes in cell cycle distribution were 

observed by cyclin DI overexpression, the presence of the 

G liS arrest was unaffected and no significant change in 

radiosensitivity was observed. We are currently 

transfecting cell lines stably (and inducibly) 

overexpressing cyclin E and p21 wafl /cipl , in order to 

further explore the importance of the G 11 S arrest for the 

radiosensitivity of tumor cens. 

Chromosome aberrations 

In an extension of previous studies on the induction 

and stability of chromosome aberrations as predictors for 

human tumor radiosensitivity, we have investigated the 

tolerance for chromosome aberrations in MCF7 and 

SC8S carcinoma eens. Surviving SC8S cens did not 

exhibit any chromosome aberrations, as expected fr om 

previous results on radiosensitive celllines (see below). In 

contrast, survivors of the MCF7 cells showed a dose 

dependent increase in chromosome aberrations, despite 

its radiosensitive phenotype and the presence of wild type 

p53 . In the resistant SQD9 cens, aberrations in survivors 

were almost exclusively of the stabIe kind and had been 

induced in greater numbers than in sensitive SCC61 cel1s. 

Publications 

Smeets et al. Radiat Res 1994; 140: 153-60. 

Smeets et al. Radiother Oncol (in press). 

Notes 

I Outside funding. 

Prediction of radiosensitivity 

JM Coco-Martinl, MFMA Smeets 2 , E Mooren, 

CPE Ottenheim I , AC Begg 

Previous experiments designed to validate 

chromosome aberrations as a predictor of radiation-

induced cell kill in tumors showed that there was a 

moderate correlation (r =0.84) between cell survival and 

induced chromosome aberrations when assessed 24h after 

irradiation using fluorescence in situ hybridization 

(FISH). A better correlation (r = 0.94) was found using 

the difference in aberrations between those induced (1 

day) and those in survivors (14 days). 

To further investigate the lethality of different 

aberration types, the FISH method was modified by using 

a pan centromere probe together with whole chromosome 

probes. Visualization of centromeres allowed 

discrimination between 'stabie' (translocations, deletions, 

insertions) and ' unstable' (acentric fragments, dieentrics) 

aberrations. Aberrations 1 day af ter irradiation were, as 

expected, ofthe stabie as wen as unstable types. The ratio 

between unstable and sta bie aberrations was similar for 3 

of the cell lines (approximately 1). In a fourth, 

radiosensitive, line, the ratio was far greater than I. This 

could partly explain its radiosensitive phenotype. 

In the radioresistant lines, a dose related increase in 

stabie aberrations was observed in survivors (14d). In 

contrast, no cells with aberrations we re observed in 

survivors in the radiosensitive lines. In one line this was 

mainly due to few stabie aberrations being produced (see 

above), whereas in the other, even so-called stabIe 

aberrations disappeared. In all cell lines studied, 

aberrations ofthe unstable type were lost by 5-6 days af ter 

irradiation (a few divisions). For purposes of predicting 

radiosensitivity using the 'difference' parameter, scoring 

could therefore be done within a week, making it more 

attractive as a potential predictive assay. 

The observed differences between the two sensitive cell 

lines in terms of aberration types at the first postirradiation 

mitosis has still to be resolved. Since the cell 

lines have approximately the same cell cyc1e time it is 

unlikely to be a cell cyc1e effect. The differences could 

possibly be attributed to the processing of aberrations. 

Experiments have therefore been performed to determine 

the short term kinetics of chromosome aberration 

(collaboration with A Wojcik, Essen) using replication 

banding. First experiments show that induced 

aberrations decrease more rapidly in the resistant A549 

than in sensitive A1847 cells within the first cyc1e. 

Publications 

Coco Martin JM et al. Int J Radiat Biol 1994; 66: 

297-307. 

Begg AC et al. Int J Radiat Biol 1994; 65: 103-8. 

Lambin P et al. Radiat Res 1994; 138: S40-3. 

Notes 


2 Outside funding. 

Predictive assays in non-small celllung 

cancer (NSCLC) 

PJM van de Vaartl, HJ Knaken l , L Moonen, AC Begg 

The purpose of these studies is to explore possibilities 

for more individualized and effective use of combined 

radiotherapy and cisplatin (cDDP) for patients with non- 

Experimental Therapy 89 

metastatic NSCLC by prediction of sensitivity to cDDP. 

To search for more sensitive methods to detect cDDP 

DNA adducts, an immunocytochemical test (lCC) was 

compared with a quantitative PCR-based method and a 

post-labeling method. 

Antiserum NKI-A59 was used for visualizing and 

quantifying cDDP-DNA adducts in individual cells. This 

ICC method was found to have a detection limit of 

1 Ptll0 6 bp. The quantitative PCR method, based on the 

blocking of Taq polymerase by a cDDP-DNA adduct, 

was found to have a detection limit of 1 PtlI04 bp for a 2.4 

kbp fragment. Inhibition of the PCR only occurred at 

doses giving more than 90% cell kill. A 32P-postlabelling 

method developed in our institute (Division 11) was found 

to have a detection limit of 1 Ptl1û 8 bp, coneentrations 

which are expected in patient samples. This therefore 

appears to be the most sensitive technique, but doesn't 

retain the histological and geographical information of 

ICe. 

White blood cells and buccal cells we re obtained from 

28 patients treated with cDDP and lung tumor biopsies 

from 9 patients. The number of cDDP-DNA adducts will 

be measured with the most sensitive method. Patient 

accrual is continuing and a correlation of cisplatin-DNA 

adducts with treatment outcome will be made after 

sufficient follow-up. 

Publication 

Begg AC et al. Radiother Oncol 1994; 312: 129-37. 

Notes 

I Outside funding. 

Predictive assays: tumor cell kinetics 

AC Begg, I Hofland, K Haustermans l , H Awwad 2 , 

T Shouman 2 , JM Coco Martin3, D Ivanyi, A Balm, 

H Bartelink 

Cy tokeratin 

Antibodies to cytokeratin (CK) have been further 

explored as potential tumor cen markers in squamous cen 

carcinomas. The aim of these studies is to increase 

accuracy of cen kinetic measurements on human tumor 

biopsies in order to select fast growing tumors for 

shortened (accelerated) radiotherapy schedules. In a 

collaboration with University Hospital, Leuven, frozen 

sections from 20 he ad and neck carcinomas (Amsterdam) 

and 22 oesphagus carcinomas (Leuven) were first 

screened for cytokeratin positivity for 4 different CK 

antibodies using immunohistochemistry methods (lHC). 

The most positive anti body for each tumor was then used 

in flow cytometry studies. 

All tumors tested using IHC were positive for at least 

one of the antibodies, with the non-malignant stroma 

being mostly negative. The success rate in Oow cytometry 

(cytokeratin positivity, analyzable DNA histogram) was 

over 70%. Tumor cen enrichment (tested in aneuploid 

tumors) could be obtained by gating on cytokeratin 

positive eells. The percentage tumor cells increased from 

an ave rage of 50% to over 80% after CK gating. Imperfect

IX Division of Radiotherapy 

Board 

JV Lebesque MD PhD, Head 

H Bartelink MD PhD, BJ Mijnheer PhD 

Permanent academie staff (research funded) 

RW de Boer PhD (25%), IAD Bruinvis PhD (25%), 

AAM Hart MSc (30%), BJ Mijnheer PhD (50%) 

AvL fellow 

M van Herk PhD 


A Bel MSc, RB Boellaard MSc, LJ Boersma MD, 

MEssers MSc, KGA Gilhuijs MSc, MW Konijnenberg 

PhD, SLS Kwa PhD, JC de Munck PhD, 

CPJ Raaijmakers MSc, JCM Theuws MD, 

PJM van de Vaart MD 


AJ van Dalen, TJ Minderhoud (20%), LG de Mooy 

(20%), LH ten Zende 


JH Lanson (50%), A Touw, PJH van de Ven, 

FC Verster, RE Vijlbrief, AC Wagenaar (40%) 

Guests 

A Burian, PhD 

BA Fraass, PhD 

PF Kukolowicz, PhD 

T Shouman, MD 

Secretaries 

A Hooiveld (15%), EJP Hofmann (15%) 

DEPARTMENT OF RADIOTHERAPY 

Head 

H Bartelink MD PhD 

Permanent academic staff of Department of 

Radiotherapy 

RW de Boer PhD, JH Borger MD, IAD Bruinvis PhD, 

BNFM van Bunningen MD, JMV Burgers MD PhD, 

EMF Damen PhD, LGH Dewit MD PhD, AAM Hart 

MSc, RB Keus MD, JV Lebesque MD PhD, 

H Masselink MD, LMF Moonen MD, SH Muller PhD, 

BJ Mijnheer PhD, V de Ru MD PhD (from November), 

NS Russell MD, CCE Schaake-Koning MD PhD, 

FW Wittkämper PhD 

Other academie staff of Department of 

Radiotherapy 

BMP Aleman MD, JSA Beiderbos MD, AM Bruce 

MD, FCJM van Gils MD PhD, RLM Haas MD, 

EPM Jansen MD, Ph.W. Koken PhD, APG Kroes 

MSc, A Minken MSc, CRN Rasch MD, JG Salverda 

MD, Th Schnabel MD PhD, ALJ Schuster-Uiterhoeve 

MD, M Verheij MD, JCM van der Voet MD 

91

92 Radiotherapy 

Introduction 

Research in the Division of Radiotherapy includes 

studies on dosimetric and geometrical accuracy of 

external beam therapy, clinical studies of normal tissue 

tolerance, boron neutron capture therapy and combined 

modality treatments. Three new projects were started this 

year. In a three-dimensional (3D) imaging project the 

clinical applications of 3D image matching will be 

studied. In a second project, the application of the 

electronic portal imaging device for transit dosimetry will 

be investigated. Finally, a follow-up project was started 

concerning detailed measurements of radiation induced 

lung damage. 

Highlights of the research this year include the work of 

M van Herk and his colleagues who successfully applied 

3D image matching techniques to quantify organ motion 

during conformal radiation therapy for prostate cancer. J 

Lebesque and coworkers we re able to quantify the 

recovery from radiation induced local lung damage 1.5 

year after radiotherapy. For her major contribution in 

this project, L Boersma received the V ARIAN award for 

clinical research during the ESTRO meeting in Granada. 

B Mijnheer and colleagues demonstrated in the boron 

neutron capture therapy project that rapid on-site 

monitoring ofthe blood-loB concentration is feasible and 

consequently that the actual irradiation with neutrons 

can be adjusted to the individually determined boron 

elimination curve. 

Dose calculation models and computer 

programs for treatment planning of 

photon beams 

IAD Bruinvis, LM Chin I , BA Fraass 2 , S Heukelom 3 , 

JH Lanson, BJ Mijnheer 

Heterogeneity corrections for photon beams 

Perturbations of dose distributions at interfaces of 

media with different densities are complex and difficult to 

measure or calculate. At present no reliable dose 

calculation algorithm is incorporated in treatment 

planning systems to take these perturbations into 

account. In order to quantify these effects a series of 

benchmark measurements have been performed at airtissue 

and lung-tissue interfaces. The preliminary results 

indicate that only a smalliateral build-up region adjacent 

to the lung volume exists where the dose is slightly lower 

than in the region of the lung where lateral electron 

equilibrium exists. 

Dose build-up near the skin surface 

For many conformal photon beam treatments (e.g. 

parotid gland) the precise location of the high dose 

volume near the skin surface is of concern. For conformal 

fields (involving trays, blocks, wedges and varying 

source-skin-distance, SSD), the dose build-up is not so 

weIl characterized. Therefore dose distributivns in the 

build-up region for three different types of accelerators 

and beam qualities between 6 and 25 MV have been 

studied. Depth dose curves and beam profiles for open 

and wedged fields, with and without trays and blocks, 

were measured as a function of field size and SSD. For a 

large 8 MV field and 1.25 cm PMMA tray, addition ofthe 

tray to the open beam a) increases the dose at 2 mm depth 

by 55% at 80 cm SSD but only by 25% at 100 cm SSD, 

relative to the dose at 10 cm depth, and adds 1 % at 100 cm 

SSD to 3% at 80 cm SSD to the dose at the nominal depth 

of dose maximum (1.8 cm); b) decreases the depth of dose 

maximum (up to 7 mm). These differences are quite 

important for in-vivo dosimetry and monitor unit 

calculations, and are usually not taken into account by a 

treatment planning syslem. A simple phenomenological 

model is under development in order to improve the dose 

calculations in the build-up region for complex conformal 

fields. 

Output factors of megavoltage photon beams 

In order to quantitatively explain the behaviour of 

wedge factors, the he ad and phantom scatter contribution 

to the output of a treatment machine have been 

determined for open and wedged 60CO y-ray beams and 4, 

8, 16 and 25 MV X-ray beams, using an extended and a 

mini-phantom. For the wedged beams a stronger increase 

of the head scatter contribution with field size, i. e. 4-9% 

for field sizes increasing from 5 x 5 cm 2 to 20 x 20 cm 2 , has 

been observed compared with open beams. This result 

indicates that the wedge factor variation with field size is 

related to a change of the primary photon fluence. Our 

study shows that the ratio of the head and phantom 

scatter contribution for the wedged and open beams 

remains unchanged for all beams except the 4 and 25 MV 

X-ray beam. This implies th at, except for these latter 

energies, the variation of the wedge factor with phantom 

depth is determined by the wedge-induced change of the 

primary photon energy fluence. For the 4 and 25 MV 

X-ray beam it is shown that the wedge factor is also 

influenced by a change of the phantom scatter 

contribution. The wedge factor for the 25 MV X-ray 

beam is strongly influenced by the electron contamination 

for phantom depths up to 6 cm. For the 60CO and 

the 4 MV photon beams it is shown that the wedge factor 

decreases slightly with increasing source-to-skin distance 

due to a reduced contribution to the total dose from 

photons scattered in the wedge. For clinical use, an 

algorithm has been developed to calculate the wedge 

factor variation with field size and phantom depth. 

Publication 

Heukelom S et al. Radiother Oncol 1994; 32: 73-83. 

Notes 

I Joint Center for Radiation Therapy, Harvard Medical 

School, Boston, USA. 

2 University of Michigan, Ann Arbor, USA. 

3 Free University, Amsterdam. 

Experimental dosimetry of photon and 

electron beams 

RB Boellaard I, IAD Bruinvis, M Essers 2 , S Heukelom 3 ,

patients, (62 in our institution (A), 31 in Tilburg (B) and 

42 in Rotterdam (C)). 

Although the distribution of initial systematic 

deviations in the three institutions were different, the 

application of the verification procedure increased the 

setup accuracy to a similar level (Figure IX.2). This 

accuracy and the number of corrections and measurements 

necessary to obtain this accuracy could be weil 

predicted by the computer simulation. 

Clinical implementation of de cis ion rules for 

patient setup corrections 

In addition to the ongoing patient setup study 

concerning prostate treatments, the setup accuracy for 

patients treated for parotid carcinoma was studied. It 

became apparent that our method for imrnobilization was 

insufficiently robust and allowed out-of-plane rotations. 

Since the estimated deviations in the fields were 

inconsistent, the patient position could not be reconstructed 

in three dimensions from the oblique views. 

By improving the immobilization system and by 

application of a lead bullet in the homolateral ear, 

consistent measurements could be obtained . A start has 

been made with the clinical application ofthe verification 

procedure for this group of patients. 

Study of organ motion using 3D image correlation 

Knowledge of organ mobility is important for 

conformal radiotherapy. 3D image correlation has been 

applied to study organ motion. Besides the planning CT, 

three repeat CT scans were made during the course of 

treatment of 11 patients with prostate ca ncer. Contours 

were drawn ofprostate and seminal vesicles, bladder and 

rectum in all scans. The bony anatomy from the repeat 

scans was matched automatically on the planning scan. 

Femurs and pelvic bone were matched separately to 

quantify motion of the legs. By matching the contours of 

prostate and seminal vesicles, the motion of the prostate 

relative to the pelvic bone was quantified with an 

accuracy better than 1 mm translation (l SD) and I 

degree rotation (1 SD). 

Astrong correlation was found between differences in 

recta I volume and rotation ofthe prostate around the leftright 

axis. A corresponding translation was found that 

indicated th at the center of rotation was located near the 

apex of the prostate. Surprisingly, bladder filling had no 

significant influence on the position of the prostate. 

However, it was found that rotation of the femurs had a 

small influence on rotation of the pro state around the 

cranio-caudal axis. Finally, an interesting correlation was 

observed between the width of leg opening and the 

bladder volume. 

Publications 

Bel A et al. Radiother Onco11994; 31 : 176-80. 

Gilhuijs KGA et al. In: Proc XIth ICCR 1994; 228-9. 

Kooy HM et al. Int J Radiat Oncol Biol Phys 1994; 28: 

1229-34. 

Mijnheer BJ. In: Radiation Dose in Radiotherapy from 

Prescription to Delivery. IAEA, Vienna, 

1994; 269-74. 

Radiotherapy 95 

Van Herk M et al. In: Proc XIth ICCR 1994; 220-I. 

Van Herk Mand Kooy HM. Med Phys 1994; 21: 

1163-78. 

Notes 




4 Joint Center for Radiation Therapy, Harvard Medical 

School, Boston, MA, USA. 


6 Dr Bernard Verbeeten Institute, Tilburg. 

Treatment planning 

A Bel', RW de Boer, EMF Damen, BA Fraass 2 , 

H Huizenga 3 , MH Idzes 3 , RB Keus, JH Lanson, 

TJ Minderhoud, AA van 't Veld 4 , JV Lebesque, 

BJ Mijnheer, IAD Bruinvis 

A ccuracy in volume sampling 

The influence of the shape of a region of interest (ROl) 

on the uncertainty in the sampled volume of the ROl for 

computations with regular Cartesian grids was 

investigated in detail. For a number of mathematically 

defined volumes the sampling uncertainty was studied as 

a function of the compactness of the ROl and of the 

degree of grid-alignment. In a spherical ROl, gridmatching 

effects were demonstrated by means of Fourier 

transforms; the effect on the sampling uncertainty can be 

20%. For sphere-like ROIs the volume can be sampled, 

however, with 1 % uncertainty using 10 3 grid points. For a 

volume encompassed by an isodose surface of a treatment 

plan with rectangular fields the same uncertainty level 

requires 10 4 grid points. Thus care should be taken in 

grid-sampling ofROIs with a high degree oftranslational 

symmetry. 

Quality assurance of treatment planning systems 

The accuracy of dose calculations for the parotid gland 

treatments with 6 MV photon beams has been 

investigated, particularly the aspect of target coverage. 

This concerned the position of the 95% isodose line in 

three orthogonal planes (axial, sagittal, coronal) through 

the center of the target volume; the axial plane contained 

the beams' axes. The treatment setup with two oblique 

wedged beams was simulated in a water phantom. In each 

plane, 4 scans were made every 45° through the isocenter 

with a small semi-conductor detector. From these data 

the position of the 95% isodose could be reconstructed 

around the isocenter in each plane. With our threedimensional 

planning system (UM-plan), we calculated 

the isodose distribution in these three planes and 

compared the positions ofthe 95% isodose along the scan 

lines with the measurements. For the points near the 

surface, deviations up to 3.5 mrn were found; this relates 

to dose calculations in the build-up region for which an 

improved algorithm is being developed. In the sagittal 

plane deviations of similar magnitude were found on the 

scanline parallel to the surface, perpendicular to the

96 Radiotherapy 

wedge. This mainly reflects the accuracy of dose 

calculations in the penumbra region of the beams. 

Possible improvements of the calculation by adjustment 

of the octree/ edge model parameters and adaptions of the 

treatment technique are being investigated. 

Irradiation of the breast and adjacent lymph nodes 

as su mes a geometrically perfect match between the two 

tangential breast fields and the supraclavicular fields. 

Patient movement and/ or uncertainties in patient setup 

may however, cause considerable under- or overdosage 

volumes. In order to quantify these setup deviations we 

investigated the accuracy of such a technique under 

clinical circumstances. Field matching is performed by 

the radio grap hers during each session on a match line 

drawn on the patient's skin. Field edge positions we re 

assessed in the cranial match plane of tangential breast 

fields and supraclavicular-axillary fields using an 

electronic portal imaging device and match line markers 

placed on the skin of the patients. The mean gap/ overlap 

of the four fields for individual patients during each 

treatment session was + 0.5 mm, indicating that no 

systematic gap or overlap was observed. The uncertainty 

in the position of the four fields with respect to the match 

plane ranged from 3.1 to 5.1 mm (l SD) for the individual 

patients. Gaps and overlaps between fields were also 

related to an absolute match line position, found by 

comparison of simulator and portal images, showing a 

small systematic uncertainty of 2.4 mm and a standard 

deviation of 3.3 mmo It can be concluded that the use of an 

electronic portal imaging device in combination with 

match line markers is a good method to quantify the 

accuracy of field matching in vivo. The results showed 

good stability and reproducibility in the field matching 

region for this treatment technique of breast cancer 

irradiation. 

Incorporation of random setup uncertainties in 3D 

treatment planning 

The aim of this study was to determine if margins for 

random setup uncertainties and organ motions around a 

clinical target volume could be defined taking into 

account the shape ofthe target volume and the irradiation 

technique. By convolving the 3D dose distribution with 

these random translations and rotations, the actual 

delivered total dose distribution of a multifractioned 

treatment was estimated. The required margin was 

defined by the resulting displacement of the 95% isodose 

surface. 

For a 4-field box technique (field sizes 10xl0 cm 2 ) , 

random translations with a standard deviation of 0.6 cm 

in three directions, without rotations, resulted in a shift of 

the 95% isodose of about 0.6 cm in all three directions. 

Additional rotations (SD = 5 degrees around the 3 main 

axes) did not alter this result significantly. However, 

rotations had a considerable effect on a 4 field 'elongated' 

box technique (field sizes 10x5 cm 2 ) . In the direction of 

the elongated axis of the box, the shift increased from 0.6 

cm to 1 cm after adding rotations (5 degrees around the 3 

main axes). In the other two directions, rotations had only 

a small influence, yielding an increase of the shift of less 

than 0.1 cm. 

It could be concluded that, in general, the anisotropy of 

the margins increased with decreasing symmetry of 

treatment techniques and target volume. Consequently, a 

unique definition of margins is impossible and the 

convolution should be included in the planning process 

itself. 

Publications 

Holmberg 0 et al. Radiother Oncol (in press). 

Van Bree NAM et al. In: Radiation Dose in 

Radiotherapy from Prescription to Delivery. IAEA, 

Vienna, 1994; 251-7. 

Van 't Veld AA and Bruinvis IAD. In: Proc XI th ICCR 

1994; 76-7. 

Wambersie A et al. In: Radiation Dose in Radiotherapy 

from Prescription to Delivery. IAEA, Vienna, 

1994; 11-35. 

Notes 


2 University of Michigan, Ann Arbor, USA. 


4 Academic Center Groningen. 

Studies of dose-effect relationships and 

fractionation in normal tissues 

P Baas, LJ Boersma I, EMF Damen, CA Hoefnagel, 

RB Keus, M Koelman I, SLS Kwa 2 , JGJ Letschert 3 , 

SH Muller, CM Roos3, NS Russell, J Theuws 2 , 

RA Valdés Olmos, AC Wagenaar 2 , N van Zandwijk, 

RW de Boer, JV Lebesque 

Lung 

The aim of this study is to develop a method to predict 

the reduction in overall pulmonary function and the 

probability to develop radiation pneumonitis based on 

the full 3D dose distribution. Twenty-five patients treated 

for malignant lymphoma were examined prior to, 3-4 

months after and 18 months af ter irradiation. Single 

Photon Emission Computed Tomography (SPECT) 

ventilation (V) and perfusion (Q) scans were performed to 

quantify local V and Q changes and CT scans were made 

to quantify density changes and to calculate the 3D dose 

distribution. Standard pulmonary function tests (Vital 

Capacity (VC), Forced Expiratory Volume in 1 second 

(FEV I)' Alveolar Volume (V A)' transfer factor for carbon 

monoxide (TL eoJ) were performed to quantify overall 

lung function èhanges. 

Dose-effect relations for local Q, V and density changes 

we re calculated using correlated SPECT and CT data. 

Subsequently, these dose-effect relations we re fitted to a 

logistic function. At 3-4 months af ter irradiation, the 

NTD I5 (Normalized Total Dose required for a 15% 

reduction in function) was 31 Gy, 34 Gy and 41 Gy for Q, 

V and density, respectively. At 18 months af ter 

irradiation the values of NTD I5 were 40 Gy, 38 Gy, and 

45 Gy, for Q, V and density, respectively. The data 

indicate th at after both follow-up periods a dose-effect 

relation is present for all three end-points (Figure IX.3). 

In addition, after an initial reduction of local Q and V at 

3-4 months af ter irradiation, a partial recovery of local

captopril. In patients with a posltlve captopril 

renography, a selective angiography was performed to 

exclude pre-existing central renal artery stenosis and to 

assess the type and extent of the vascular changes. The 

captopril 99mTc-DTPA demonstrated a longer time until 

maximal renal activity (T max) compared with the baseline 

study in 5 out of 8 hypertensive patients. This increase in 

T max was observed in both high-dose (40 Gy 1 5.5 weeks) 

and in low-dose (12-13 Gy 1 3 weeks) irradiated kidneys. 

No increase in T max was observed in the normotensive 

patients. In the five hypertensive cases with an increased 

T max' selective angiography demonstrated severe stenotic 

and tortuous changes in the sm all intrarenal branches of 

the high-dose irradiated kidneys without stenosis of the 

main renal artery. Captopril also induced an increase in 

peripheral plasma renin level in the hypertensive group, 

but not in the normotensive patients. These data strongly 

suggest a radiation-induced renovascular hypertension, 

mediated by the renin-angiotension system, due to 

damage predominantly in small renal arteries. Since the 

increase in T max was also observed in low-dose irradiated 

kidneys (bel ow the 'classica!' threshold dose of20 Gy 12-3 

weeks), these data imply that the 'tolerance dose' of the 

human kidney may have to be reassessed. 

Publications 

Dewit L et al. Eur J Cancer 1993; 29A: 2239-43. 

Verheij M et al. Radiat Res 1994; 137: 202-7. 

Verheij M et al. Int J Rad Oncol Biol Phys 1994; 30: 

677-83. 

Notes 

J Department of Blood Coagulation, Central 

Laboratory of The Netherlands Red Cross Blood 


2 Department of Nephrology, Academic Medical 

Center, Amsterdam. 

Breast carcinoma 

JH Borger, JA van Dongen, AAM Hart, H Kemperman, 

JV Lebesque, H Bartelink 

Since our analysis of local recurrence (LR) after breast 

conserving therapy for stage 1 and 11 breast cancer 

showed a significant correlation between risk factors for 

LR and survival, we investigated the role of LR as a 

prognostic factor for survival in a muItivariate analysis. 

In a retrospective study of 1,026 patients treated with 

tumorectomy, axillary dissection and radiotherapy, 

factors associated with disease specific survival (DSS) 

were analyzed. 

Actuarial estimates for DSS are 91 % at 5 years and 86% 

at 10 years. The multivariate analysis revealed 5 factors: 

clinical stage, number of affected axillary nodes, 

histologic grade, degree of tubule formation, left-sided 

primary tumor. Controlled for these factors, LR appears 

to be significantly correlated with DSS. The hazard ra te 

of DSS is estimated to increase by a factor 8.8 (95% 

confidence interval: 4.6-16.8) upon occurrence of a LR. 

Local recurrence per se, apart from the identified 

prognostic factors, is a risk factor for DSS. The exact 

Radiotherapy 99 

mechanism by which LR has an influence on survival 

cannot be determined from these data. 

An example of some of the practical problems 

associated with the interim monitoring of accumulating 

time to event data was illustrated through an EOR TC 

clinical trial in locally advanced breast cancer which 

studied the effects of both hormon al therapy and 

chemotherapy using a 2 x 2 factorial design. The resuIts 

2.5 years after closing the trial to patient entry differed 

quite markedly from those observed during the period of 

patient accrual, thus showing the potential dangers of 

drawing conclusions based on only short term follow-up 

data. 

Publications 

Borger JH et al. J Clin Onco11994; 12: 653-60. 

Borger JH et al. Int J Rad Oncol Biol Phys 1994; 

30: 1073-81 . 

Kemperman H et al. Eur J Cancer (in press). 

Sylvester R et al. Stat Med 1994; 13: 1329-35. 

Cis-diamminedichloroplatinum 11 as a 

potential radiosensitizer 

CCE Schaake-Koning, T Benraadt', D Gonzalez 

Gonzalez 2 , LMF Moonen, L Schuster-Uitterhoeve 2 , 

PJM van de Vaart, H Bartelink 

A feasibility study was carried out to assess the acute 

and late toxicity of the combination of radiotherapy 

according to a concomitant boost technique and 6 mg/ m 2 

cisplatin (daily) in patients with inoperable non-small cell 

lung cancer. The elective field was irradiated with a dose 

of 2 Gy per fraction, irnrnediately followed by a boost 

dose of 0.75 Gy to a total dose of 55 Gy in 4 weeks. The 

length ofthe esophagus, ifencompassed, was limited to 17 

cm in the elective fields and 11 cm in the boost fields. Until 

now, 3 patients received cisplatin for 1 week, 6 patients for 

2 weeks, 4 patients for 3 weeks and 20 patients for 4 

weeks. The acute toxicity was acceptable and no 

enhancement of late toxicity has been observed. 

Consequently, there seems to be possibilities for further 

radiation do se escalation, which will be carried out in a 

stepwise fashion with and without cisplatin. When the 

definitive dose has been found, a Phase 111 study proposal 

will follow . 

In a parallel study, cisplatin-DNA adducts in buccal 

cells, leukocytes and tumor cells obtained from cisplatin 

R T treated patients are measured. Results from this study 

are reported by Division VIII. 

Publication 

Schaake-Koning CCE et al. Lung Cancer 1994; 10 

suppl.l: S263-70. 

Notes 

J Comprehensive Cancer Center, Amsterdam. 

2 Academic Medical Center, Amsterdam.

X Division of Medical Oncology 

Head 

R Somers MD (until August) 

S Rodenhuis MD PhD (from September) 


JW Baars MD PhD (25%), P Baas MD PhD (50%), 

EM Bais MD, JH Beijnen PhD, WW ten Bokkei 

Huinink MD, W Boogerd MD PhD, H Boot MD, 

JMG Bonfrer MSc, PF Bruning MD PhD (50%), 

ME Craanen (50%), CC Delprat MD, WR Gerritsen 

MD PhD (50%), CA Hoefnagel MD PhD, SP Israëls 

MD, C Mattern MD, SH Muller PhD, H Neering MD 

PhD, WJ Nooijen PhD, EM Rankin MD PhD (50%), 

JJ van der Sande MD PhD, JH Schornagel MD PhD 

(50%), R Somers MD, 0 van Tellingen PhD, BG Taal 

MD PhD, RA Valdés Olmos MD PhD, 

N van Zandwijk MD PhD (25%), APE Vielvoye 

KerkmeerMD 


AC Ogilvie, RF A ten Hoeve, W Kool MSc, IH Liem 

MD, E van der Wall, J Wanders, AM Westermann 


TC Linders 

Graduate students 

MW Dercksen, MT Huizing, LJC van Warmerdam 

Guest 

H Sakai MD 

Research nurses 

D Batchelor, AC Dubbelman, M Schot 

ABMTnurses 

M Holtkamp, GAM Linthorst (from August) 

Secretary 

CJKamp 

101

102 M edical Oncology 

Introduction 

For Division X, 1994 was a year oftransition. As part 

of a major reorganization of the Cancer Hospital, the 

Cluster of Medical Oncology was molded which will 

incorporate most, but not all of the former division. The 

Department of Nuclear Medicine and the Clinical 

Chemistry Laboratory will become members of the novel 

Cluster of Diagnostics by the end of the year. 

Even more importantly, a number of ambitious 

projects were launched in 1994 that orchestrate the main 

research themes of the Medical Oncology group. 

1) The immunotherapy group began a gene therapy trial 

in melanoma, which employs GM-CSF transduced 

autologous tumor cells as the basis for a vaccination. The 

study can be viewed as a 'proof of concept' experiment 

and it is conducted in close collaboration with workers 

from the Division of Immunology and the SOMATIX 

Corporation in Alameda, California. 

2) The high-dose therapy group has brought about the 

official start of a national adjuvant chemotherapy study 

in high-risk breast cancer, in which al! Dutch University 

Hospitals and the two Cancer Institutes collaborate. The 

protocol is based on clinical and technical experience 

gained in our single-institution study in apical nodepositive 

patients. By the end of October, over 125 patients 

had al ready been randomized. 

3) The pharmacology group has begun to focus its 

attention on the development of treatment strategies that 

employ combinations of topoisomerase land II 

inhibitors. lt has continued to develop methods to 

monitor the concentrations and metabolisms of these 

drugs in humans and has developed quantitative RNA 

detection methods to be used on small numbers of tumor 

cells. 

After completing his additional training in molecular 

biology methods, ME Craanen joined the clinical 

gastroenterology group in October 1994. In recognition 

of his outstanding accomplishments in drug formulation 

and pharmacodynamics, JH Beijnen was appointed 

professor in Pharmacy at the University of Utrecht. The 

Netherlands Cancer Institute and his laboratory at the 

neighboring Slotervaart Hospital wil!, however, continue 

to be the major locations of his work. 

Clinical Pharmacology Unit 

ww ten BokkeI Huinink, S Rodenhuis, J Schornagel, 

JH Beijnen, J Baars, E van der Wall, AM Westermann, 

AC Dubbelman, GC Roodbergen 

The work in the Clinical Pharmacology Unit 

concentrated on the clinical development of taxoids 

(paclitaxel and docetaxel) and on the application of 

topoisomerase land II inhibitors. The unit continued to 

take part in phase II studies of the Early Clinical Trials 

Group of the EOR TC. 

Taxoids 

Pa clitaxe I 

A study that investigated a potential role for escalated 

doses of paclitaxel in patients with documented 

doxorubicin resistance was completed. In this study, 

comprehensive pharmacokinetic studies were performed. 

From these, it was apparent why patients with liver 

involvement experience more severe toxicity from 

paclitaxel. At high doses (250 mg/ m 2 in 3 hours), 

disproportionately high paclitaxel AUC values we re 

observed and the excess toxicity may be caused by 

prolonged drug concentrations above a critical threshold. 

The antitumor activity was disappointing, probably as a 

result of the heavy prior treatment of most patients. 

In a dose-finding study of the combination of 

carboplatin and paclitaxel in ovarian cancer patients, the 

MTD had not yet been reached by November 1994. 

Unexpectedly, doses of carboplatin leading to an AUC of 

8-10 mg.ml-I.h and 225 mg / m 2 of paclitaxel did not 

require the addition of G-CSF to the treatment regimen. 

Satisfactory responses have been documented, even In 

patients with bulky disease. 

Docetaxel 

Studies with this European analog of paclitaxel were 

conducted in breast and lung cancer patients. lts antitumor 

activity was confirmed in both cancer types. 

Topoisomerase I inhibitors 

Topotecan 

Activity of topotecan in relapsing ovarian cancer 

patients was documented in a phase II study ofthis drug, 

which employed a daily times 5 intravenous administration 

schedule. Topotecan appears to be active in smal! 

cell lung cancer, even in patients resistant to standard 

(anthracycline containing) chemotherapy, as has been 

found in an ongoing ph ase II study of the ECTG. 

Irinotecan ( CPT 11) 

The topoisomerase-l inhibitor mnotecan was 

investigated in pancreatic cancer patients and some 

beneficial effects were noted. The drug caused toIerabIe 

and manageable side effects, mainly diarrhoea. 

T allimus t in 

Tallimustin is an intercalating agent that binds to the 

minor groove of DNA. lt was studied in breast and 

ovarian cancer patients. In a single patient with ovarian 

cancer who received the agent every 4 weeks 

intravenously, disease stabilization occurred. In none of 

the other patients (9 x breast cancer and 10 x ovarian 

cancer), could any signs of activity be documented. A 

ph ase I study of this interesting intercalating drug was 

initiated, with a daily times 3 intravenous dose escalation 

schedule. So far, no myelotoxicity or other toxicities

could be shown. Even hair loss was absent. 

Other studies 

Studies with rhizoxin did not yield responses in our 

patients. Phase II studies with the mitomycin analog E09 

were initiated. Limited sampling pharmacokinetic studies 

we re continued in patients treated with paclitaxel or 

topotecan. As a prelude for a study investigating 

sequential therapy with topotecan and etoposide, the 

bioavailability of orally administered low dose (20 mg) 

etoposide capsules was studied. Finally, a feasibility and 

pharmacokinetic study of the intraperitoneal administration 

of suramin was begun. Suramin is a potent 

inhibitor of the growth factor lysophosphatidylic acid 

(LPA), a high concentration of which was shown to be 

present in malignant ascites. 

Publications 

Franklin HR et al. Ann Oncol 1994; 5: 113-7. 

Neijens V et al. Ann Oncol (in press). 

Oza AM et al. Ann Onco11994; 5: 343-7. 

Ten Bokkei Huinink WW et al. Ann Oncol 1994; 5: 

133-9. 

Ten Bokkei Huinink WW et al. Ann Oncol 1994; 5: 

527-32. 

Ten Bokkei Huinink WW et al. Semin Oncol 1994; 21 

Suppl 2: 27-33. 

Verweij J et al. Ann Oncol 1994; 5: 375-6. 

Autologous hematopoietie stem eell 

transplantation program 

S Rodenhuis, JH Schornagel, JW Baars, E van der Wall, 

WR Gerritsen, JH Borger, MW Dercksen I, M Holtkamp, 

GAM Linthorst, GC Roodbergen, WJ Nooijen, 

lCM Slaper-Cortenbach 2 , CE van der Schoot 2 

High-dose adjuvant therapy in breast cancer 

In 1991 , a single-institution randomized ph ase II study 

was initiated to investigate the usefulness of high-dose 

chemotherapy with hematopoietic stem ceB support in 

breast cancer patients with positive apical-node biopsies 3 . 

This study, which had accrued 74 patients by September 

1994, is expected to reach its target number of patients 

early in 1995. It has served to further develop the 

techniques for peripheral stem cell mobilization and 

harvest and it has documented the feasibility and 

tolerability of the CTC regimen (cyclophosphamide 6 

g/m 2 , thiotepa 480 mg/ m 2 and carboplatin 1600 mg/m 2 ) . 

Based on the technical developments in the study 

mentioned above, a Dutch national study4 has begun to 

accrue patients with high-risk breast cancer, defined as 

Mo disease with 4 or more axillary lymph nodes. In 

October 1994, 120 of the targeted 250 patients had been 

entered. All university hospitais, the two cancer institutes 

and the 'Medisch Spectrum Twente' collaborate in this 

study, which is the second multi-center study ofthe Dutch 

Collaborative Working Group on Autotransplantation 

in Solid Tumors (NW AST). 

MedicalOncology 103 

Salvage Chemotherapy in Germ Cel! Cancer 

Pilot experience from our institute has shown that 

patients with germ cell cancer who relapse from a 

complete remission have an excellent chance ofachieving 

long-term survival with high-do se carboplatin-based 

chemotherapy. These results, 64% long-term disease-free 

survival, appear to be dramatically better than the 30% 

salvage rate after standard-dosed 'non-cross resistant' 

therapy, but the number of patients in the pilot study is 

small and the confidence interval is wide. 

To confirm the improved survival with intensive 

therapy, a Dutch national study was initiated in 1994, 

that aims to treat a series of 30 patients in a prospective, 

but non-randomized manner. Treatment consists of a 

stem cell mobilization course ofifosfamide and etoposide, 

which is foIlowed by a single cycle of carboplatin (1600 

mg/m 2 ) and etoposide (1500 mg/ m 2 ). Two high-dose 

courses ofCTC (see previous paragraph) with autologous 

stem ceIl reinfusion conclude the salvage therapy 

sequence. The centers participating in the NW AST (see 

above) coIlaborate in this study, in which 8 patients had 

been entered between January and October 1994. 

Multiple courses of high-dose alkylating therapy 

In an attempt to further increase the dose intensity in 

the autologous stem cell transplantation setting, multiple 

courses of CTC have been adrninistered in a very tight 

time frame in a series of feasibility studies. A total of 22 

second cycles of CTC we re administered, and a total of 11 

third cycles. While 2 cycles we re tolerated reasonably weB 

(which is clear from the germ ceIl cancer program), triple 

CTC is associated with major non-meduIlary toxicity, 

such as veno-occlusive disease and hemolytic uremic 

syndrome. Further studies focus on multiple CTC courses 

th at contain lower doses of the three alkylating agents to 

avoid cumulative toxicity ('tiny CTC')3.5. 

Autologous transplantation in lymphomas 

High-dose therapy with autologous stem ceB 

transplantation in lymphomas may be expected to 

become even more frequent in the next few years, now 

that its superiority to standard-dose therapy has been 

documented in the salvage setting in a prospective 

randomized multi-center study. In 1994, contributions 

were made to several mul ti-center studies in lymphomas. 

In addition, the facilities for Total Body Irradiation (TBI) 

were set up in the institute, and the first two lymphoma 

patients received their transplants after a cyclophosphamide/TBI 

regimen. 

Publications 

Rodenhuis S et al. Cancer Invest (in press). 

Van der WaIl E et al. Ann Oncol 1994; 5: 795-802. 

Van Warmerdam LJC et al. Cancer Chemother 

Pharmacol (in press). 

Notes 

I Funding: European Cancer Center Fellowship. 

2 Central Laboratory of the Netherlands Red Cross 

Blood Transfusion Service, Amsterdam.

104 MedicalOncology 

3 Supported in part by the 'Schumacher-Kramer 

Stichting' (SK Foundation). 

4 Supported by Ziekenfondsraad Ontwikkelingsgeneeskunde 

Project 09-94-051 . 

5 Supported in part by the 'Stichting Fondsenwerving 

Volksgezondheid' . 

Gastrointestinal tract cancer 

BG Taal, HBoot, M Craanen, BAleman, J Lebesque, 

E Gortzak, F van Coevorden, FAN Zoetrnulder, 

D de Jong, B Loftus, R Kröger, C Hoefnagel, 

RA Valdès Olmos. 

Esophagealcancer 

A total of 150 patients has been entered in a treatment 

project that evaluates intraluminal irradiation with an 

Iridium High-Dose-Rate source. The results are 

promising: an objective response rate of 77% (53 of 69 

patients) has been noted in locally advanced esophageal 

cancer and adequate short-term palliation of dysphagia 

was achieved in 49% of patients with metastatic disease. 

A new generation of coated self-expandable stents has 

become available and has been applied in esophagotrachea 

I fistulas and in recurrent esophageal cancer 

following irradiation. The ma in advantage over the 

plastic tygon endoprostheses, that had been developed in 

our hospitalover 10 years ago, is the easier insertion 

procedure and therefore lower risk of perforation. The 

only, but significant, disadvantage is the high cost of these 

stents. 

Non-Hodgkin 's lymphoma of the stomach 

Previous analyses have concentrated on the endoscopic 

characteristics and on treatment results, especially 

radiotherapy. In a recent analysis of prognostic factors, 

the histological grade of malignancy (using the MALT 

classification of Isaacson) and age were found to be 

significant prognostic factors. The role of H pylori in the 

etiology of gastric lymphoma is currently of great interest. 

In a pilot study, a favorable response to antibiotic therapy 

was seen. Based on this observation and on similar 

literature data, a clinical study that evaluates triple 

therapy to eradicate H pylori in early stomach lymphoma 

is in preparation. 

Percutaneous ultra-sound guided gastrostomy 

(PUG) 

Percutaneous endoscopic gastrostomy (PEG) is an 

accepted method for both nutrition and decompression. 

Using the new method of PUG with fluoroscopic and 

ultrasound guidance, endoscopy is no longer needed. This 

makes it the preferred gastrostomy procedure in patients 

with extreme stenosis of the upper gastrointestinal tract. 

PUG was shown to be a simple and safe procedure in 23 

patients. 

Carcinoid tumors 

Treatment with the radioactive drug 131-1 MIBG 

(Meta-iodo benzyl guanidine) may lead to significant 

subjective improvement ofthe characteristic symptoms of 

the carcinoid syndrome (flushes, diarrhoea) in 

approximately 60% of cases. In a dose finding study of the 

pharmaceutical preparation MIBG without the 

radioactive label ('cold MIBG') a similar subjective 

response ra te was found, but the duration of the 

responses was brief. The study is being continued. A 

combination of'cold MIBG' with radioactive MIBG may 

be advantageous (see 'Radio nuclide tumor imaging and 

therapy'). 

Chronology of p53 accumulation in gastric 

carcinogenesis 

Accumulation of p53 protein reflecting p53 gene 

mutation, has been reported in up to 60% of advanced 

gastric canceL In a study performed in cooperation with 

several hospitaIs, this accumulation proved to be absent 

in early gastric canceL This suggests th at p53 gene 

mutations are relatively late events in the gastric 

carcinogenesis. 

Partialliver resections 

In collaboration with the Department of Surgery (F 

Zoetrnulder, F van Coevorden and E Gortzak), the 

results of liver resections have been analyzed. In a recent 

series of50 consecutive resections, no peri / post-operative 

mortality was seen and major rnorbidity was at an 

acceptably low level. The survival curves compared 

favorably with the literature. 

Publications 

Taal BG et al. Ann Oncol 1994; 5: 90-2. 

Taal BG et al. Gastroint Endosc (in press). 

Taal BG et al. Gastrointest Endosc (in press). 

Vasen HFA et al. Gut 1994; 35: 1262-6. 

Zoetrnulder FAN et al. Neth J Med 1994; 45: 134-5. 

Lung cancer 

N van Zandwijk, P Baas, WW ten BokkeI Huinink, 

S Rodenhuis, G Giaccone 1 , WJ Mooi, H van Tinteren, 

AJM Balm, S de Flora 2 , U Pastorin0 3 , N de Vries 4 , 

JPA Marijnissen 5 , W Stars, L van 't Veer, A Breedijk, 

CCE Schaake-Koning, G Wigbout, FJ van Schooten 6 , 

M Meijer, M de Kwant 

Chemotherapy of SCLC 

Although combination chemotherapy has had a 

substantial impact on the management of patients with 

small cell lung cancer (SCLC), little improvement in 

overall survival has been observed since the beginning of 

the eighties. Topotecan, a new semi-synthetic analog of 

the alkaloid camptotecin, is a specific inhibitor of 

topoisomerase I. To further increase its efficacy, a phase 

11 study with an almost continuous schedule (21 days) was

initiated in 1994, in patients with recurrent SCLC. 

Chemotherapy in NSCLC 

The Netherlands Cancer Institute contributed to 

European studies (EORTC 08884 and 08875) that 

confirmed that the addition of cisplatin not only 

improved local con trol in patients receiving chest 

irradiation for locoregional disease, but was also related 

to better survival in patients with distant metastatic 

disease (confirmed in a meta-analysis). A consensus has 

now been reached on cisplatin, as an important ingredient 

of polychemotherapeutic regimens against NSCLC. 

Endobronchial treatment 7 

The technology assessment (TA) project 

(Ziekenfondsraad) investigating the additive role of 

photodynamic therapy and high dose rate (HDR) 

brachytherapy in patients with locoregional advanced 

non-small cell lung cancer was analyzed after accrual of 

78 patients. The reason for this early analysis was the 

observation th at a substantial number of serious side 

effects ocurred in the combined HDR brachytherapy / 

external radiotherapy arm. This observation was 

confirmed by an independent review board and led to a 

premature closure of the trial. However, in recent years 

photodynamic therapy, and also HDR brachytherapy, 

have gradually obtained a place in the treatment of early 

lung cancer and the palliation of advanced (locoregional) 

non-smal1 cell lung cancer respectively. The TA project 

will therefore focus also on the cost benefit analysis of 

both modalities in a non-randomized group of patients. 

Addition of mitomycin C to photodynamic therapy 

showed a supra-additive effect on skin metastases of 

breast cancer, which had been previously predicted by 

animal experiments (see also Division VIII). 

Chemoprevention 8 

Euroscan, the study which evaluates the potentialof 

N-acetylcysteïne (NAC), retinol palmitate, or the 

combination of both agents to prevent or delay the 

occurrence of second primary cancers, was closed for 

patient entry in September 1994, after inclusion of 2600 

subjects in 6 years. After an interim analysis earlier in 

1994, it was decided to delay the analysis of the efficacy of 

the potential chemopreventive agents until the majority 

of follow-up data have been obtained on all patients. 

In addition to Euroscan, in 1994 a type IIB prevention 

study with NAC was initiated in smoking volunteers, with 

PHA DNA adducts, proliferation markers and 

immunohistological signs of tumor suppressor gene 

mutations as surrogate endpoints. 

Publications 

Bunn PA et al. Eur J Cancer 1994; 30A: 710-3. 

Van Zandwijk N. Anticancer Res 1994; 14: 309-12. 

Van Zandwijk N. Anticancer Res 1994; 14: 313-6. 

Van Zandwijk N et al. Sem Onco11994; 21 (suppI6): 

66-71. 

Van Zandwijk N et al. J Cell Biochem (in press). 

Van Zandwijk et al. Chest (in press). 

Notes 


I Free University Hospital, Amsterdam. 

2 Institute of Hygiene and Preventive Medicine, Genoa. 

3 Royal Brompton Hospita), London. 

4 Lucas Hospital, Amsterdam. 

S Dr Daniel den Hoed Cancer Center, Rotterdam. 

6 University of Limburg, Maastricht. 

7 Funding: Ziekenfondsraad. 

8 Funding: Partical financial support from the Dutch 

Cancer Society, the EEC, Cancer Research Campaign 

(UK) and Zambon International and Mucos Pharma. 

Malignant lymphoma 

R Somers, JMV Burgers, JW Baars, S Rodenhuis, 

AC Ogilvie, EM Rankin 

In collaboration with the EORTC Lymphoma Group, 

studies in Hodgkin's disease and non-Hodgkin's 

lymphomas were continued. In early Hodgkin's disease, 

decrease in treatment intensity is attempted to prevent 

late side effects. In higher risk groups, the number of 

chemotherapy courses is being studied. The monitoring 

of late toxicity and of quality of life remain important 

issues in the final evaluation of the results. 

In the higher stages of Hodgkin's disease, the role of 

adjuvant radiotherapy af ter chemotherapy is still under 

investigation. High risk groups of patients, as defined on 

the basis of meta-analyses, will be selected for new trials 

of more intensive approaches. 

In the non-Hodgkin's lymphomas, the results of a 

number of pivotal studies became known th is year. 

Adjuvant treatment with interferon in low grade 

lymphomas was shown to be able to prolong remission 

duration in stage lIl-IV follicular lymphomas. In 

recurrent high grade lymphomas, high dose 

chemotherapy with ABMT could finally be demonstrated 

to improve both relapse-free survival and survival. 

In the field of second cancers after treatment of 

malignant lymphomas the thesis of F van Leeuwen on 

this subject was acclaimed as a very important 

contribution in this area. 

Publications 

Somers R et al. J Clin Oncol 1994; 12: 279-87. 

Somers R et al. Ann Onco11994; 5 (suppl s): S85-S9. 

Van Leeuwen FE et al. J Nat! Cancer Inst (in press). 

Pain management 

APE Vielvoye-Kerkmeer, C Mattern, EM Bais, 

W Boogerd, JH Beijnen, FSAM van Dam, 

M Hel1endoorn-Smit, EJTh Rutgers, GJ in 't Veld, 

JC Visser, LM Gualthérie van Weezei, R de Wit 

The task of the multidisciplinary pain management 

team is to provide advice to hospital physicians and 

family practitioners regarding cancer patients who suffer 

from severe pain. These patients are often in the final 

stages oftheir diseases. Different treatment modalities are 

used, such as analgesics (oral, transdermal, spinal), nerve

studied further. The posltlve clinical resuJts of this 

collaborative phase II trial will be published separately by 

the IDBBC group. 

The resuIts of project NKI 90-13 studying very early 

cellular proliferation parameters in relation to clinical 

response to systemic treatment (e.g. with Tamoxifen) are 

reported in the section of Division VI. 

Adjuvant breast cancer treatment and risk of 

osteoporosis 

Premature menopause due to adjuvant chemotherapy 

may cause premature osteoporosis. The bisphosphonate 

APD is very poorly resorbed when administered orally 

and may cause gastrointestinal erosions. We therefore 

studied the duration of metabolic-hormonal effects from 

a single intravenous infusion of APD in 5 women with 

premature menopause and low lumbar spine bone 

density. We found that the effects last for at least 8 weeks. 

Intermittent use of intravenously administered APD in 

trials for the prevention of osteoporosis should therefore 

probably not use schedules with shorter intervals. 

Prospective measurements of the lumbar spine bone 

density (BMD) have demonstrated th at Tamoxifen, 20 

mg daily, or oral APD, 150 mg daily, maintains 

trabecular BMD over more than 18 months in women 

with premature menopause due to adjuvant chemotherapy 

for breast canceL Women receiving 75 mg of 

APD or no treatment showed a similar loss of BMD. 

Publications 

Koenig K et aL. Cancer Epidemiol Biomarkers Prev 

1993; 2: 411-4. 

Van Tulder M et al. Ann Onco11994; 5: 153-8. 

Nole 

I Funding Dutch Cancer Society, Project NKI 90-13. 

Radionuclide tumor imaging and 

therapy 

CA Hoefnagel, RA Valdés Olmos, SH Muller, 

RKL Panday, JH Beijnen, HBoot, BP Israëls, 

BAE Kapteijn, J de Kraker 1 , IH Liem, H Maessen, 

EM Rankin, BG Taal, PA Voûte 1 , AR Wafelman 

13 I I-MIBG as first fine treatment in advanced 

neuroblastoma 

First line preoperative 131I-MIBG treatment at 

diagnosis in advanced / high-risk inoperable neuroblastoma 

was performed in 37 children. Nineteen of 27 

evaluable patients had complete or over 95% primary 

tumor resection or did not require surgery at all. Only 

mild hematological toxicity was observed. 131I-MIBG 

therapy of neuroblastoma at diagnosis is feasible and at 

least equally effective as chemotherapy. It is, however, 

considerably less toxic and allows the administration of 

postoperative chemotherapy for minimal residual 

disease. 

M edicaL OncoLogy 107 

MIBG and IJl In-pentetreotide in neuroendocrine 

tumors 

131I-MIBG and lllIn-pentetreotide are currently in use 

for the diagnosis and therapy of neural crest tumors. The 

agents interact with characteristic properties of these 

tumors, i.e. an active cell membrane uptake mechanism 

and storage system in cytoplasm neurosecretory granules 

for MIBG and cell membrane specific receptors for 

pentetreotide. The department has now performed 1,267 

131I-' 23I_MIBG total body scintigrams in 419 patients 

with neural crest tumors and 58 lllIn-pentetreotide 

studies in 54 patients. Comparative scintigraphies were 

performed in 23 patients (see Table X.l). Since its first 

application in neuroblastoma, in Janary 1984, diagnostic 

scintigraphy and radionuclide therapy of neural crest 

tumors, using radioiodinated MIBG has become a 

comprehensive clinical research program at the institute, 

in which the Departments of Nuclear Medicine and 

Pharmacy cooperate with clinicians, safety / radiation 

protection officers and the Division of Experimental 

Therapy. 

Both radiopharmaceuticals were found to be sensitive 

neural crest tumor indicators, which have a 

complementary role. Unlike MIBG, which can be used 

effectively for therapy, radiolabeled pentetreotide is not 

specific for neural crest tumors and its biodistribution 

does not allow radionuclide therapy. 

13II-MIBG af ter 'cold' MIBG in carcinoid 

Apart from 131I-MIBG therapy, unlabeled ('cold') 

MIBG has been used at increasing dose levels for the 

palliation of symptomatic, metastatic carcinoids. In 5 of 

these patients, who on the basis of an 131I-MIBG tracer 

study had been excluded from 131I-MIBG therapy, 

scintigraphies we re repeated during treatment with 'cold' 

MIBG, in order to see how the high dose of MIBG would 

affect the lower radiolabeled dose biodistribution. In all 

but one patient, this resulted in decreased specific target 

organ activity (salivary glands, myocardium, liver, 

adrenal glands). In two patients, a markedly increased 

tumor/ non-tumor ratio was observed, to such a degree 

that they qualified for 131I-MIBG therapy. One of them 

subsequently received 'cold' MIBG therapy immediately 

followed by a therapeutic dose of 131I-MIBG. This was 

associated with a satisfactory palliative response. 

Melanoma targeting with specific 

radiopharmaceuticals 

Af ter an initial period in which various radiolabeled 

F(ab')2 fragments and 67Ga-citrate were tested for their 

ability to detect melanoma, subsequent efforts have 

turned to the use of specific metabolic tracers. 131I-MIBG 

was positive in 1/14 patients (7%), 123I-IBZM in 8/16 

patients (50%) and the somatostatin analogue lllIn 

pentetreotide in 14/17 patients (82%). The somatostatin 

receptor status of these tumors is being investigated. A 

new metabolic melanoma-specific tracer is being 

developed in cooperation with Technical University in 

Eindhoven.

108 M edica/ Oncology 

TableX.l 

Clinical diagnosis 

Imaging 

neural crest tumors 

1-123 vs 1-l31 MIBG 

MIBG at the NKI/ AvL, a comprehensive program 

Clinical therapy 

1-l31 MIBG 

neuralcrest tumors 

1-125 MIBG 

neuroblastoma 

MIBG vs pentetreotide Preop. therapy inoperable 

neuro blastoma 

MTC multitracer study 

1-123 MIBG i.V. vs i.a. 

1-123 MIBG in an thracycline 

cardiomyopathy 

Intra-arterial MIBG 

Cold MIBG therapy 

carcinoid 

1-131 MIBG after 

'cold' MIBG 

SPECT QC I pharmaco kinetics I 

metabolites I dosimetry 

radiation safety 

Pub lica tions 

Gelfand M] et al. Pediatric Nuc1ear Imaging 1994; 

309-21. 

Hoefnagel CA. ] Nuc1 Biol Med (in press). 

Hoefnagel CA et al. 2 chapters. In: Nuc1ear Medicine in 

Clinical Diagnosis and Treatment 1994. 

Hoefnagel CA. Eur] Nuc1 Med 1994; 21: 561-81. 

Hoefnagel CA et al. Nuc1 Med Commun 1994; 15: 

712-7. 

Hoefnagel CA et al. Horm Met Res (in press). 

Hoefnagel CA et al. ] Nuc1 Biol Med (in press). 

Hoefnagel CA et al. Eur] Nuc1 Med 1994; 21: 587-8. 

Wafelman AR et al. Appl Radiat Isot 1994; 45: 183-9. 

Wafelman AR et al. Eur] Nuc1 Med 1994; 21: 545-59. 

Wafelman AR [Dissertation]. Utrecht: Rijksuniversiteit 

Utrecht, 1994. 

Notes 

lEmma Kinderziekenhuis / Children's Academic 

Medical Center. 

Radionuclide assessment of organ 

injury in ca neer therapy 

RA Valdés Olmos, CA Hoefnagel, SH Muller, AF] Balm, 

L] Boersma I, WW ten BokkeI Huinink, PF Bruning, 

L Dewit, RB Keus,] de Kraker, JV Lebesque, IH Liem 2 , 

H van Tinteren, N van Zandwijk 

Radiation pneumonitis 

1llln-pentetreotide scintigraphy was used for the early 

recognition of radiation pneumonitis. 1llln-pentetreotide 

lung uptake, which may reflect peptide activation in 

Research 

eells: 

uptake mechanisml 

pharmacokinetics 

effects of 'cold' MIBG 

up take in platelets 

and megakaryocytes 

Animal modeis: 

PC-12 pheochromocytoma 

SK-N-SH neuroblastoma 

dosimetry, 

specific activities 

1-125 vs 1-l31 MIBG, 

drug interventions, 

influence 'coId' MIBG 

injured areas, was strongly positive in 5/7 symptomatic 

patients 2-5 months after irradiation. In 2 patients, IiI Inpentetreotide 

was affected by steroid therapy. A 

prospective study is ongoing to assess the value of 1111n_ 

pentetreotide in steroid treatment monitoring. 

Cardiac injury 

1231_MIBG heart scintigraphy has now been used to 

study late cardiac effects of anthracyc1ines or thoracic 

irradiation. Abnormal parameters indicating myocardial 

adrenergic injury we re found in 8/9 asymptomatic 

patients. The time interval between therapy and 123 1_ 

MIBG examination was 6-60 months. This study will be 

extended to a larger number of patients. 

SPECT data we re analyzed in relation to planar images 

in patients investigated with 1llln-antimyosin for the 

detection of anthracyc1ine-associated myocyte injury. 

SPECT confirmed myocardial damage in 14/18 patients. 

The quality of cardiac SPECT images depended strongly 

on the degree of myocardial uptake. 

Radiation-induced salivary gland injury 

The prospective scintigraphic study of salivary gland 

function after radiotherapy for head and neck 

malignancies was continued. Twenty-eight patients have 

been entered; 24 we re examined one month after 

radiotherapy, 17 after 6 months and 12 af ter one year. A 

short-term effect evaluation shows abnormal excretory 

function of the irradiated glands in 87% of the patients.

lfosfamide renal dysfunction 

99mTc-DMSA kidney uptake, evaluated in 11 children 

with cancer during ifosfamide chemotherapy, decreased 

proportionally to the cumulative ifosfamide dose in 

patients independent of clinical toxicity. 99mTc-DMSA 

uptake, a proximal tubular function indicator, was more 

consistent than beta-2-microglobulin urine values, 

quantitative hyperaminoaciduria and phosphate tubular 

resorption. On follow-up (mean 22 months), 4 patients 

showed persistently reduced uptake; in one of them, a 

sudden onset of the Fanconi syndrome was observed 

upon treatment with carboplatin. 

Publications 

Anninga JK et al. Eur J Nucl Med 1994; 21: 658-62. 

Boersma LJ et al. Radiother Oncol (in press). 

Damen E et al. J Nucl Med 1994; 35: 784-92. 

Dewit L et al. Eur J Cancer 1993; 29A: 2239-43. 

Taal BG et al. Gastrointest Endosc (in press). 

Valdés Olmos RA. In: Nuclear Medicine in Clinical 

Diagnosis and Treatment 1994. 

Valdés Olmos RA [Dissertation]. Amsterdam: 

University of Amsterdam, 1994. 

Valdés Olmos RA et al. Ann Oncoll994; 5: 617-22. 

Valdés Olmos RA et al. Cancer 1994; 73: 2886-93. 

Valdés Olmos RA et al. Eur J Cancer (in press). 

Verheij M et al. Int J Radiat Oncol Biol Phys 1994; 30: 

677-83. 

Notes 


2 Supported in part by an ADAC Clinical Research 

Program Grant. 

Clinical chemistry 

WJ Nooijen, JMG Bonfrer, 0 van Tellingen, JH Beijnen, 

CM Korse, TC Linders, E Jansen, JGW Hilkens, 

KN Gaarenstroom', CFM Molthoff 2, J van Asperen, 

MJX Hillebrand, A Sparreboom, HR van der Woude 

Pharmacokinetics of paclitaxel and metabolites in 

mice 

Three metabolites of the cytotoxic drug paclitaxel 

(Taxol; Tx) have been isolated and purified from the feces 

of cancer patients receiving the drug. Based on UV and 

mass-spectral data, the metabolites were identified as 

6a,3' -p-dihydroxypaclitaxel (I), 3' -p-hydroxypaclitaxel 

(11) and 6a-hydroxypaclitaxel. They have lost cytotoxic 

activity as demonstrated by clonogenic assays and also 

displayed a reduced myelotoxic effect in an hemopoietic 

progenitor toxicity assay. A sensitive and selective 

HPLC-procedure has been developed for the quantitative 

determination ofTx and its metabolites. It has been used 

in a pharmacokinetic study ofTx and metabolites in mice. 

Tx was rapidly distributed over most tissues. Metabolites 

have been observed in the liver and the bile and, at higher 

dose levels, in colon and gut. We observed non-linear 

pharmacokinetic behavior in plasma, whereas the 

concentrations in tissues increased linearly with the dose 

administered. 


Pharmacokinetics of anti-cancer drugs in 

transgenie mice with inactivated P-glycoprotein 

genes 

Agents that reverse multidrug resistance may affect 

drug uptake in both tumor and normal cells. 

Consequently, effects on normal tissues may limit their 

application. In collaboration with the group of P Borst 

(Division V) pharmacokinetic studies with cytotoxic 

drugs are being performed in transgenic mice with 

inactivated mdr P-gp genes. The use of this knock-out 

(K/ O) mouse model may provide a better understanding 

of the role of P-gp in the protection of norm al tissues by 

P-gp. Studies with vinblastine (VBL) in mdrla P-gp K / O 

mice are in progress. Inactivation of this gene leads to a 

diminished excretion of the parent drug, which is partly 

compensated by an increased metabolic breakdown to 

more polar products. We observed a 22-fold higher VBL 

level in the brain of mdr 1 a( -/ -) mice 4 hours af ter the 

administration of 1 mg/ kg of VBL, whereas VBL levels 

we re 6.7, 3.4 and 2.9-fold higher in the muscle, heart and 

the gut, respectively. Although the brain still contained a 

12-fold higher drug level 4 hours after the administration 

of 6 mg/ kg VBL, the differences in all other organs were 

minimal. This is probably due to a saturation of P-gp 

caused by the high drug levels present during the first 

hours. However, at 24 hours drug levels have declined and 

a marked difference in drug retention between transgenic 

and wild-type mice is evident. 

Pharmacokinetics miscellaneous 

We are investigating the bio-availability ofVP-16 from 

a new oral formulation which has been developed in the 

Pharmacy Department and which will be used for a 

clinical trial using long-term oral adrninistration. We 

have further developed an analytical method for the antiemetic 

drug ondansetron to support a comparative 

double-blind clinical study of the bio-availability of this 

drug. Analyses of samples collected in several European 

institutes as part of a phase I clinical trial with carzelesin 

we re conducted. Finally, a standard operation procedure 

for 'Biomedical Method Validation' has been developed, 

which meets international criteria. 

Tumor markers 

Recently, a new assay has been developed to measure 

the S-100 protein, specifically the S-100 fJ chain. S-100 is 

an acidic calcium binding protein found in the nervous 

system. The S-100 fJfJ is present in glial cells and Schwann 

cells, the S-100 afJ in glial cells, but not in Schwann cells. 

S-100 has also been detected in melanocytes. We have 

measured the serum S-100 concentration in 265 patients 

with malignant melanoma and 17 with benign conditions. 

Although the tests may be insufficiently sensitive for early 

detection, the S-100 concentration significantly 

correlated with stage. Further investigations must clarify 

the applicability of S-1 00 in clinical practice. 

The Cyfra 21.1 assay measures the presence of 

fragments of cytokeratin 19 (CK 19) released in blood.

110 Medica! Onco!ogy 

CK 19 is a protein, expressed in several simple epithelia 

including the bronchial epithelium. We have investigated 

the possible usefulness of this marker in sera from 25 

patients with malignant lung disease, excluding small cell 

lung cancer. In adenocarcinomas of the lung, CEA is still 

the marker of choice during follow up of treatment. In 

mesotheliomas, Cyfra 21 .1 is a marker for progression of 

disease. Measurement of this marker may predict 

progression of disease or failure of treatment in non small 

cell lung cancer. 

Publications 

Bonfrer JMG et al. Eur J Clin Chem Clin Biochem 

1994; 32: 201-7. 

Bonfrer JMG et al. Tumor Bio11994; 15: 210-22. 

Bonfrer JMG et al. Tijdschr NVKC 1994; 19: 22-5. 

Bonfrer JMG et al. Gynecol Oncol (in press). 

Schinkel AH et al. Cell1994; 77: 491-502. 

Si ps JHM et al. Cancer Chemother Pharmacol (in 

press). 

Sparreboom A et al. Cancer Chemother Pharmacol (in 

press). 

Sparreboom A et al. J Chromatogr (in press). 

Van Tellingen 0 et al. Cancer Chemother Pharmacol 

1993; 33: 425-34. 

Van Tellingen 0 et al. J Chromatogr 1994; 652: 51-8. 

Notes 

1 University of Leiden, Department of Obstetrics and 

Gynecology. 

2 Free University Hospital Amsterdam, Department of 

Obstetrics and Gynecology. 

Pharmacy 

JH Beijnen, ACA Paalman l , JD Jonkman-de Vries l , 

AR Wafelman 1 , DM Burger 1 , R van Gijn 1 , MT H uizing2 , 

CHW Koks I , S van Oene I, C Kroon I, H Rosing 1 , 

A Sparreboom I, J van Asperen, MJX Hillebrand, 

LJC van Warmerdam I , 0 van Tellingen 

The research programs of the Pharmacy Department 

include the formulation, chemical stability and drug level 

monitoring of (investigational) anticancer drugs in both 

animal toxicology and early clinical studies. The research 

is conducted in the setting of the foundation Netherlands 

Laboratory for Anticancer Drug Formulation 

(NLADF), founded in 1990, which is a collaboration -on 

a 'fifty-fifty' basis- between the Netherlands Cancer 

Institute and Slotervaart Hospital. We have close 

relationships with the Department of Pharmaceutical 

Analysis and Toxicology (A Bult and RAA Maes) of the 

Faculty of Pharmacy of Utrecht University and the 

EORTC-New Drug Development Office (NDDO). 

Formulation 

At the end of 1994, a large project was completed that 

involved the production of 5,000 vials with the 

aziridinylquinone E09 which are used in a broad phase 11 

program in many European hospitals including the 

Netherlands Cancer Institute. The chemical stability of 

the cyclopropapyrroloindole derivative carzelesin 

(U-80,244) appeared to be very much dependent upon the 

quality of the polyethylene glycol 400 used in its 

formulation. We found traces of acid or alkali to be the 

cause of the inter-batch differences. 

A new, stabIe formulation was developed for the 

benzoylurea derivative c1anfenur. The EORTC-NDDO 

now contemplates the further development of this 

compound. 

Chemical stability studies 

Stability studies of 1311-metaiodobenzylguanidine (' 31 1_ 

MIBG) in concentrates (3.7 GBq in 7.5 mL) for iv 

in fusion revea1ed th at the percentage of free [ 131 l]iodide 

increased from 1.9 % ± 0.34 % to 4.4% ± 0.67% during a 

3-day storage period at -78 oe. With these experiments we 

have completed our stability studies with this 

radiopharmaceutical for all relevant conditions from the 

production site to the bed side. 

Drug level monitoring 

Most studies mentioned in this section are performed in 

close collaboration with the Clinical Chemistry 

Department and are within the setting of the Bio-analysis 

and Early Clinical Trials Broup (BECTG) jointly chaired 

by WW ten BokkeI Huinink and JH Beijnen. 

The analysis of carboplatin by Zeeman atomie 

absorption spectrometry has been adapted and 

thoroughly va1idated for different biological matrices 

(plasma, plasma ultrafiltrate, urine, saliva). The method 

is now used in different clinical studies. Carboplatin 

pharmacokinetics was studied in the high-dose CTC 

(carboplatin-thiotepa-cyclophosphamide) regimen using 

a validated limited sampling model. Ototoxicity was 

strongly correlated with the cumulative AUC. Platinum 

pharmacokinetics was also investigated as part of the 

carboplatin-paclitaxel combination trial in non-small cell 

lung cancer. We have investigated the usefulness of the 

serum creatinine level as a measure for the glomerular 

filtration ra te in the Calvert formula. By using a factor of 

0.9 the serum creatinine concentration was appropriate 

for this purpose. 

A large clinical research program has just been started 

with the topoisomerase I inhibitor topotecan also in 

combination regimens. Pharmacokinetic - pharmacodynamic 

relationships constitute a major part of these 

studies. Topotecan pharmacokinetics is also investigated 

as part of the EOR TC phase 11 evaluation of the drug 

using the limited sampling model developed by us. The 

HPLC assay has been further optimized and is now 

capable of detecting levels down to 50 pg/ mL. 

Metabo1ites of paclitaxel (Taxol®) have been isolated 

in sufficient quantities from human feces for 

chromatographic purification and identification with 

spectrometric methods. Paclitaxel calibration curves can 

be utilized for the quantitation of the metabolites. 

Quantification of metabolites was performed in samples 

from breast cancer patients refractory to anthracyclines 

and treated with the drug in relatively high dosages (250 

mg/ m 2 in 3 hours). Low serum albumin levels and liver 

dysfunction were strongly corre1ated with a decreased

clearance of paclitaxel and hydroxylated metabolites. 

Preliminary results of paclitaxel kinetics in the 

combination and sequence study with carboplatin 

indicate that it is only marginally influenced by 

carboplatin. The limited sampling models developed by 

us for paclitaxel were succesfully implemented in the 

ongoing pharmacokinetic studies. An HPLC assay for 

paclitaxel analysis in urine was developed . 

HPLC assays have also been designed for the 

staurosporine derivative and protein kinase C inhibitor 

CGP 41 251 and th ree potential hydroxylated 

metabolites. The method is based on reversed phase 

HPLC with fluorescence detection and liquid-liquid 

extraction with di-isopropyl ether as sample pretreatment. 

The detection limit is around 0.5 ng/ m!. We 

await the start of the phase I study which appears to be 

delayed due to stability problems ofthe oral formulation. 

Other ongoing studies include the pharmacokinetics of 

paclitaxel and docetaxel in laboratory animaIs, 

pharmacokinetics of cytotoxic agents in MDR knock-out 

mice and the clinical pharmacokinetics of etoposide and 

ondansetron (see Clinical Chemistry section) and clinical 

pharmacokinetics of anti-retroviral and anti-fungal 

drugs. 

Publications 

Beijnen JH et al. Cancer Chemother Pharmacol 1994; 

33: 523-6. 

Beijnen JH et al. Semin Oncol 1994; 21(Suppl 8): 53-62. 

Burger DM et al. Ann Pharmacother 1994; 28: 327-30. 

Burger DM et al. J Drug Dev 1994; 6: 187-94. 

Burger DM et al. Drug Invest 1994; 7: 282-7. 

Burger DM et al. Neth J Med 1994; 44: 161-5. 

De Vries et al. Cancer Chemother Pharmacol 1994; 34: 

416-22. 

Van Warmerdam LJC et al. Ann Onco11994; 5: 259-64. 

Van Warmerdam LJC et al. J Cancer Res Clin Oncol 

1994; 120: 427-33. 

Wafelman AR et al. Eur J Nucl Med 1994; 21: 545-59. 

Wafelman AR et al. Appl Rad Isot 1994; 45: 183-9. 

Notes 

I Slotervaart Hospita!. 

2 European Cancer Centre. 

MedicalOncology III

XI Division of Surgical Oncology 

Head 

JA van Dongen MD PhD 

Secretary 

AJM Balm MD PhD 

Manager 

SW van Bergen ' 


General surgery 

JA van Dongen MD PhD, Head (until april 1994) 

BBR Kroon MD PhD, Head (from april 1994) 

F van Coevorden MD PhD, E Gortzak MD, 

OE Nieweg MD PhD, EJTh Rutgers MD PhD 

FAN Zoetrnulder MD PhD 

Head and neck surgery 

FJM Hilgers MD PhD, Head 

AJM Balm MD PhD, RT Gregor MB BCh FRCS 

FACS PhD, S Gonggrijp DDS, AP Timmers DDS 

Orall maxillofacial surgery 

FHM Kroon DDS PhD 

Gynecology 

ThJM Helrnerhorst MD PhD, Head 

EJ Aartsen MD, P Kenemans MD PhD (10%), 

Urology 

S Horenbias MD PhD, Head 

W Meinhardt MD 

Reconstructive surgery 

KE Bos MD PhD, JB de Boer MD 

Other academie staff involved in research activities 

AH Ackerstaff MSc, RKL Baidjnath Panday MD, 

LMF Beenen MD, CL Berendsen MD, 

SC Donkervoort MD, MM Duisters MD, MD Dijkstra 

MD, HWW de Gier MD, JAL Groenenberg MD PhD, 

AJM Janse MD, R Kaas MD, JM Klaase MD PhD, 

R Klicks MD, BAE Kapteijn MD, IH Liem MD, 

S Mak-Kregar DDS PhD, ChR Leemans MD PhD, 

FA Peccatori, BEC Plaat MD, TA Roeleveld MD, 

R Ritz MD, NMJ van Veelen MD, RB Veenhuizen 

MD, HP Verschuur MD PhD, BC Vrouenraets MD, 

PAC van Vuuren MD, J Wedman MD 


J Wijnia, N Bijker, PE Wolffs, JPH Leyer, M Klop, 

W Deenink, M Heijdendaal, E, Voogd, L. Bouwma 

Secretary 

Evan Damme 

Guest 

MMZOsman MD 

113

11 4 Surgical Oncology 

Introduction 

Members of Division XI were active in many 

multidisciplinary working parties. Hence many aspects of 

their research activities are also mentioned in other parts 

of this annual report. In the following survey we have 

restricted the reports to the more specific surgically 

oriented research. 

This year our research again focused on the 

development oftechniques leading to better quality oflife 

without impairing cure rate. The successful start of the 

project to reconstruct a continent perineal stoma after 

removing the anus and rectum was important. The 

expertise with the reconstruction of a continent urinary 

stoma (artificial urinary bladder) was also extended. The 

studies centered around methods to improve quality of 

life after laryngectomy we re reported in a thesis which will 

be defended in January 1995 (AH Ackerstaff). 

The full time appointment ofa plastic surgeon from the 

University of Amsterdam will further increase the 

possibilities for research in reconstructive techniques. The 

expertise with the use rTNF-alpha in limb perfusion was 

enlarged, confirming the impressive effect on melanoma 

and soft tissue sarcoma. 

A major step forward this year, was the introduction of 

the sentinel node biopsy technique which probably will 

solve the dilemma of elective removal of clinically tumor 

negative lymph node areas. 

Head and neck oncology 

AJM Balm, RT Gregor, FJM Hilgers, NK Aaronson, 

AH Ackerstaff, JB de Boert, KE Bost , H Ceha 2 , 

S Gonggrijp, AAM Hart, RB Keus, JD Kerrebijn 3 , 

BBR Kroon, FHM Kroon 4 , ChR Leemans 5 , 

BEC Plaat, S Mak-Kregar 6 , RP Takes, AP Timmers, 

HP Verschuur 5 , PAC van Vuuren 

Analysis of head and neck cancer treatment results 

Following our institutional oropharynx carcinoma 

studies, a similar nationwide study was addressed by the 

Dutch Head and Neck Oncology Cooperative Group 

(NWHHT), coordinated by the NKII AvL-AMC head 

and neck working party. 640 patients, treated between 

1986 and 1990 and referred for treatment of their 

squamous cell carcinoma (628; 98%) or undifferentiated 

carcinoma (12; 2%) were analyzed. The 5-year overall 

survival and 5-year disease-specific survival were, 

respectively, 28% and 41 % in males, 51 % in females 

(p = 0.003). On multivariate analysis, lower stage and 

female sex emerged as significant beneficial prognostic 

indicators. On the basis of this study, the current 

UICC'87 / AJCC'88 staging system was re-evaluated with 

respect to patient distribution and prognostic value. 

Staging was first evaluated in a proportional hazard 

regression analysis controlled for the known 

prognosticators. Next, all possible combinations of T, N 

and M were tested in a stepwise backward elimination 

model until all remaining indicator variables had a 

P-value of < 0.05 . The revised stage revealed two 

advantages compared with the UICC'87 / AJCC'88 

version: a more balanced distribution of patients (31 % in 

stage I, 31 % in lI, 18% in lIl, 14% in IV and 5% unknown 

in the revised staging versus 7%, 17%, 24%, 50% and 2%, 

respectively, in UICC'87 / AJCC'88 staging) and an 

improved prognostic discrimination for the diseasespecific 

survival (5 year results in the revised staging were 

67%, 42%, 28% and 11% for stages I, lI, III and IV, 

respective1y, versus 68%, 64%, 44% and 27% in UICC'87 / 

AJCC'88). The second tumor site studied, concerned 

carcinoma of the pyriform sinus, where 101 cases treated 

in the institute from 1970 to 1989 were retrospectively 

analyzed. The 5-year overall survival was 27%, whereas 

the 5 year disease specific survival was 37%. Stage 

according to the UI CC '87 criteria emerged as an 

important prognostic variabie (p = 0.0026). Furthermore, 

significantly less locoregional recurrences and a better 

disease free survival were seen in the combined surgery 

and radiotherapy group than in the radiotherapy alone 

group (p < 0.0001). It could be concluded that, with 

proper selection, a patient group can be .dentified with a 

favourable prognosis after a planned combined surgical 

and radiotherapeutic treatment. The third major site 

concerned supraglottic larynx carcinoma, for which 

treatment planning is still controversial on the issue of 

radiotherapy (RT) and horizontal partial laryngectomy 

(HPL). Recent publications have suggested th at bilateral 

neck dissections should also be carried out electively in all 

surgical procedures in supraglottic disease. In a group of 

94 patients treated in our institute from 1980 to 1989, the 

5 year overall survival was 51 % and the disease free 

survival was 73%. No significant differences were found 

between the applied treatment modalities. When 

comparing the HPL and primary R T groups, the HPL 

group had more advanced nodal metastases. For the No 

patients the locoregional control was equivalent, but for 

the N + patients, HPL combined with neck dissection 

were far superior in locoregional control (p < 0.0024). 

Bilateral neck dissection, with or without R T did not 

demonstrate an increase in morbidity, however our data 

do not support routine elective neck dissection. An 

analysis of the neck nodes suggests that patients with 

bilateral or centrally located primary lesions and with 

ipsilateral nodal disease (> 3 cm or multiple) should be 

considered for bilateral neck dissection. When applying 

multivariate analysis, age « 65» (p = 0.0005) and 

staging (p = 0.0001) emerged as strongly significant 

prognostic indicators. 

Diagnosis and treatment of rare head and neck 

tumors 

For many years the Netherlands Cancer Institute has 

been a major referral centre for rare he ad and neck 

tumors. Retrospective analysis of treatment results of 

these neoplasms remains important for identification of 

the tumor characteristics. Merkel cell carcinoma is a rare 

cutaneous tumour that typically arises in the head and 

neck, following an aggressive course with frequent local 

recurrences and (distant) metastases. We have identified 

five patients with such malignancy since 1984. Four were 

cured by a combination of surgery and radiotherapy. 

Based on the experience presented in the literature and 

our own experience we formulated the following

guidelines for treatment: wide excision of the prirnary 

tumor followed by radiotherapy in case of narrow 

excision margins. Neck dissection, preferably followed by 

radiotherapy, is indicated in the presence of (occult) 

Iymph node metastases. Carcinoma arising in the 

thyroglossal remnants is extremely rare and the diagnosis 

is seldom made preoperatively. From the charts we could 

identify three patients with this disease. Adding our 

experience to that of 153 cases described in literature, we 

conclude that surgical excision according to Sistrunk's 

procedure is still the treatment of choice. All three 

patients surgically treated in this manner we re cured. 

Fibromatosis is a locally infiltrative fibrous tissue 

proliferation with a tendency to local recurrence af ter 

inadequate therapy. From 1977 to 1994 we treated nine 

adult patients with this disease. Five of these lesions we re 

localized in the posterior triangle of the neck. The 

majority of patients were treated by surgery in 

combination with radiotherapy. None ofthe patients died 

of disease. Alf'JO based on literature data, we advise wide 

surgical excision as the first therapy of choice. In case of 

positive surgical margins, surgery may be followed by 

radiotherapy if an expected recurrence is in opera bIe i. e. 

might occur near vital structures. Otherwise close followup 

with careful observation including CT / MRI scans is 

adequate. If a recurrence occurs, repeated surgery 

followed by radiotherapy is indicated. 

Quality of life assessment of head an neck cancer 

patients 

Laryngectomy not only leads to the loss of the normal 

voice but also to extensive respiratory symptoms. These 

symptoms can be positively influenced by the regular use 

of a stomafiJter with heat and moisture exchanging 

capacities (HME), which also has a positive effect on the 

physical and psychosocial problems following 

laryngectomy. The decline of several respiratory 

symptoms, due to the HME use, resulted in a decrease of 

fatigue, sleeping problems and feelings of anxiety and 

depression. Moreover, an improvement of the perceived 

quality of voice was observed. In a multicenter 

prospective study 59 laryngectomy patients were 

provided with HME's either immediately post-surgery or, 

in case of post-surgical radiotherapy, upon its 

completion. Patients reported the same range of 

respiratory symptoms 6 months post-Iaryngectomy as 

was previously observed in patients with longer followup. 

However, the frequency and severity of these 

symptoms were stillless pronounced. For the total sample 

statistically significant improvements over time (between 

3 and 6 months) could be found in forced expectoration 

(P < .05), in the perceived voice quality (P < .001), social 

anxiety (P < .001), social interactions (P < .001) and in 

feelings of anxiety and depression (P < .05). A clear trend 

was observed in respiratory symptoms over time, with 

regular HME users (n = 29) reporting a decrease, as 

compared with non-regular HME users (n = 30), who 

reported a slight increase in these symptoms. Repeated 

measures analysis of variance indicated statistically 

significant group differences over time in forced 

expectoration and stoma cleaning (P < .05). The results of 

this study are promising for the potential use of an HME 

Surgical Oncology 115 

in preventing and / or resolving respiratory problems 

during the first 6 months following totallaryngectomy. 

Publications 

Ackerstaff AH et al. Ann ORL 1993; 102: 878-83. 

Ackerstaff AH et al. Laryngoscope 1993; 103: 1391-4. 

Ackerstaff AH et al. Clin Otolaryngol1994; 19: 

295-300. 

Balm AJM et al. Facial Plastic Surgery (in press). 

Elias MM et al. Clin Otolaryngol (in press). 

Gregor RT et al. Proceedings 2nd World Congress on 

Laryngeal Cancer (in press). 

Mak-Kregar S et al. Eur J Cancer 1993; 29B: 119-25. 

Mak-Kregar S et al. Clin Oto1aryngol1994; 19: 22-7. 

Mak-Kregar S et al. Eur Arch ORL (in press). 

Plaat BEC et al. Clin Otolaryngol (in press). 

Takes RP et al. Clin Otolaryngol1994; 19: 222-9. 

Van Vuuren PAC et al. Clin Otolaryngol (in press). 

Notes 

, Department of Plastic, Reconstructive and Hand 

Surgery, Academic Medical Center, Amsterdam. 

2 Department of Radiotherapy, Academic Medical 

Center, Amsterdam. 

3 Department of Otolaryngology / Head&Neck Surgery, 

Dijkzigt Hospital, Rotterdam. 

4 Department of Oral/Maxillofacial Surgery, Academic 

Medical Center, Amsterdam. 

5 NKB/ KWF-fellowship head and neck oncology. 

6 NWHHT (Dutch Working Party for Head and Neck 

Tumors)/ IKA (Comprehensive Cancer Center 

Amsterdam). 

Breast Cancer 

EJTh Rutgers, JA van Dongen, F van Coevorden, 

E Gortzak, BBR Kroon, FAN Zoetrnulder, P Besnard, 

JH Beijnen, JB de Boer, J Borger, N Bijker, T van Dijk, 

B van Geel', M Heijdendaal, J Hilkens, CA Hoefnagel, 

R Kaas, BAE Kapteijn, B Kwa, FE van Leeuwen, 

JK Lafleur, IH Liem, SH Muller, R Baidjnath Panday, 

JL Peterse, J Pruyn 2 , C van Roosmalen, E Voogd, 

G Went, T Wiggers' 

Prognostic factors for local recurrence af ter radical 

mastectomy 

In a group of 691 patients treated consecutively at the 

NKI with a modified radical mastectomy from 

1971-1988, clinicopathologic (age, hormonal status, 

tumor size, lymph node status, pathology) and treatment 

(type of mastectomy, adjuvant radiotherapy, chemo- or 

hormonal therapy) factors we re analyzed. More than 

90% ofthe patients presented with a clinical tumor size of 

more than 2 cm; 44% was node positive. In 27 patients 

thoracic wall recurrence was seen (4% at 5 years, 5% at 10 

years) in 18 as first event. Univariate analysis revealed 9 

factors related to 10ca1 recurrence. The presence of an 

extensive in situ component, a major risk factor for 

recurrence after BCT proved to be no risk factor for local 

recurrence after mastectomy. In a multivariate analysis 

only the nodal status and the histological tumor si ze we re

116 Surgical Oncology 

risk factors for recurrence. 

Prognostic factors for survival af ter breast 

conserving therapy: the role of 10 cal recurrence 

Prognostic factors for disease specific survival (DSS) 

and the relation between local recurrence (LR) and DSS 

were studied in a multivariate analysis of the data of 1,026 

patients treated for early breast cancer by breast 

conservation at NKV AvL from 1978-1988. The risk 

factors for LR, young age, vascular invasion and 

incomplete excision, were also connected with survival. 

Local and distant failure therefore cannot be regarded as 

independent events. Step-wise regression analysis 

revealed as significant factors for DSS: high clinical stage, 

high number of involved Iymph nodes, high histologic 

grade and left sided primary tumor. Controlled for these 

factors, local recurrence appears significantly correlated 

with DSS. 

Out-patient substitution of hospital care and 

continuity of information in patients with primary 

breast cancer 

This study was started in October 1993 in cooperation 

with the Daniel den Hoed Cancer Center. The aim is 

evaluation of short versus long hospital stay in patients 

who underwent axillary dissection. The parameters are: 

postoperative complications, the physical and 

psychosocial wellbeing and satisfaction after short versus 

long stay and financial consequences. Tools are a daily 

diary for the first four post-operative weeks, a weekly 

diary for three months thereafter, one preoperative 

interview and two postoperative interviews at four weeks 

and four months. At the same intervals, shoulder and arm 

functions are measured by a physiotherapist. The 

interviews include the Rotterdam Symptom checklist. 

Until October 1994, 154 women we re asked to 

participate, 100 women consented: 53 in short stay and 47 

in long stay. 29 women refused consent, 15 women 

refused after surgery and 10 women had inoperable breast 

cancer because of tumor positive apex biopsy. Accrual is 

ongoing. 

Radio-immunoscintigraphy (RIS) for detection of 

breast cancer 3 

In a total of 31 patients RIS was performed 24, 48 and 

72 hours after administering three different monoclonal 

antibodies (mAbs). RIS was only considered true positive 

when increased activity was observed at all known tumor 

10calizations. With every scintigram, blood samples were 

taken to calculate the kinetics of the mAbs. At 72 hours 

post-injection all patients underwent surgery, during 

which tissue samples were taken from the tumor, fat, 

muscIe, skin tissue and if present, suspicious Iymph nodes. 

Percentages of the injected dose per gram tissue were 

calculated. Immunohistochemistry was performed using 

the corresponding mAb on tumor tissue. A summary of 

the RIS results is shown in Table XLI. As far as tumor 

imaging is concerned, the results of RIS with these mAbs 

were disappointing. In order to improve the tumor 

targeting, in the next study the antibody dosage will be 

increased by administering a large dose of cold antibody 

before the labelled mAb. 

Table XI.1 

Moab n TP TPI FN %TP 

FN 

SF25 11 2 8 18 

C95 (anti 17-lA) 16 3 12 19 

l75F4 (anti MAM-7) 4 4 0 

TP: true positive FN: false negative 

FP: false positive n: number of patients 

The value of intensified follow-up in women with 

increased risk for developing breast cancer 

Women with astrong family history for breast cancer 

or with a so called 'marker lesion' (lobular carcinoma in 

situ, atypical ductal hyperplasia) discovered by breast 

biopsy, are at increased risk for developing breast cancer 

and are usually offered in intensified follow-up schedule 

including bi-annual physical examination and annual 

mammography. The value of such an intensified followup 

is still not established: does it really provide the ability 

to detect breast cancer at an early stage with an increased 

chance for cure in the majority of women? 

We studied retrospectively 134 patients who developed 

breast cancer while they were followed for 'benign' 

reasons according an intensified con trol schedule. 

Reasons for benign follow-up were: family history of 

breast cancer in 51 patients (38%), prior biopsy with 

marker lesion in 26 patients (19%), on patients demand 

(usually patients after benign breast biopsy) 57 patients 

(43%). The histological diagnosis was: ductal carcinoma 

in situ 11 (8%), tumors < 2 cm without node metastases 

51 (38%), larger tumors and / or axillary node metastases 

56 (42%), unknown 16 (12%). The first sign ofmalignancy 

was heralded by 88 patients theirselves (66%), in 13 (10%) 

by routine physical examination of the attending 

physician, in 29 (22%) by routine mammography and in 

2% by cytology or breast biopsy. Despite this intensive 

follow-up, the majority ofpatients discovered their breast 

cancer themselves and over 1 13 already had more 

advanced disease. Further analysis will ex amine the 

relation between tumor stage (and consequently the 

outcome of patients) and factors such as follow-up 

control interval, mammography-quality and interval, 

age, histology of prior breast biopsy and family history to 

pro vide more information on the value of intensified 

follow-up for women at high risk for developing breast 

cancer. 

T-Staging in breast cancer and what is the correct 

size? 

T -staging is crucial for therapy choice and therapy 

evaluation. Standardization is essential. Pathology 

measurement is considered 'gold standard', but usually is 

only a macroscopic measurement of the tumor af ter 

cutting the specimen. The microscopic tumor extensions 

are usually not measured. We compared the clinical, 

roentgenographic and pathology size interpretation in a

etrospective analysis of data in patients files (N = 180) 

with size measurements in a prospective series ofpatients 

rigidly applying standardized measuring criteria (N = 35). 

A good concordance of the measurements was found in 

the prospective study. However, in the retrospective series 

this was poor: the clinical and mammographical si ze often 

considerably exceeded the pathological size. 

Notes 

, Dr Daniel den Hoed Kliniek, Rotterdam. 

2 Instituut voor Gezondheids en 

Omgevingsvraagstukken, Willemstad. 

3 Funding: STIPT / Centocor. 

Gynecologic oncology 

ThJM Helmerhorst, EJ Aartsen, 

WW ten BokkeI Huinink, BNFM van Bunningen, 

JMG Bonfrer, PW Gallee, EH Hopman, D Ivanyi, 

P Kenemans, H van Tinteren, RB Veenhuizen, 

AC Ansink', PFJ van BommeF, FV Cromme 3 , 

KN Gaarenstroom4, APM Heintz 5 , CJLM Meijer3, 

MJ Stukart 3 , FJ Voorhorst 3 , JMM Walboomers 3 

HPV genotype as a prognosticfactor for 

p rogression to cervical carcinoma 

In a retrospective study of 227 patients presenting with 

abnormal cervical cytology, all patients we re biopsied 

before studyentry. The patients were followed 

cytologically and colposcopically for a mean of 19 

months (range 6-42 months). Progression of acervical 

lesion was defined as progressions to a higher 

premalignant (= CIN) grade, confirmed by histological 

examination. HPV DNA detection and genotyping was 

done in cervical smears by general primer and type 

specific primer mediated PCR technique. It was 

demonstrated th at the presence of HPV DNA increased 

with CIN grade (p

11 8 Surgical Oncology 

homo logo us insemination. Delivery was by cesarean at 

31.5 weeks of a 2,000 gr male infant. This is the first report 

of a patient treated by radium needIes for endodermal 

sinus tumor of the vagina and the only reported patient 

with a subsequent pregnancy. 

Publications 

Aartsen EJ et al. Obstet Gynecol 1993; 81: 893-5. 

Ansink AC et al. Gynecol Oncol 1994; 52: 184-4. 

Bommel van PFJ et al. Cancer 1994; 74: 2314-20. 

Cromme FV. [Dissertation]. Free University 


Cromme FV et al. Br J Cancer 1994; 69: 1176-8l. 

Gaarenstroom KN et al. Int J Gynecol Cancer 1994; 4: 

73-8. 

Kenemans P. Eur J Obstet Gynecol Reprod Biol 1994; 

55: 24-5. 

Notes 

I KWF / NKB fellow, England. 

2 Ignatius Hospital, Breda. 

3 Free University Hospital, Amsterdam. 

4 Academic Hospital, Leiden. 

5 Academic Hospital, Utrecht. 

GI tract cancer 

FAN Zoetrnulder, F van Coevorden, E Gortzak, 

L Beenen, C Blackburn, HBoot, M van Eekeren, 

S van Ravensbergen, BG Taal, EJ Vos 

Dynamic gracilo-pLasty af ter abdominaL perineaL 

resection 1 

Abdornino-perineal resection remains the treatment of 

choice in low rectal cancer. As a consequence these 

patients need to have a permanent abdominal colostomy. 

The construction of a perineal colostomy was until 

recently not feasible because of the lack of an active 

sphincter mechanism. Experience with the use of a 

dynamic gracilo-plasty in fecal incontinence has shown 

that the gracilis muscIe stimulated by a pace-maker can 

transform into a functional anal sphincter. In close 

cooperation with C Baeten, of the University of 

Maastricht and the Bakker Research Center Medtronic, 

Maastricht, this technology has now been adapted for the 

use in reconstruction of the ano-rectum after abdominoperineal 

resection. The operation and reconstruction is 

performed as a three step procedure. Step one includes a 

standard abdomino-perineal resection, followed by the 

construction of a perineal colostomy. Around the distal 

colon the gracilis muscles from both upper legs are circled 

to form a new sphincter. The reconstruction is protected 

bya temporary ileostomy. Six weeks later electrodes are 

implanted in the gracilis muscles and the leads are 

connected to a subcutaneously implanted pace maker. 

This pace-maker can be externally programmed. After a 

training period the pace-maker can be continuouslyon, 

resulting in a tonic contraction of the gracilis muscIe and 

effective closure of the new rectum. The patient can 

switch off the pace-maker himself, using am external 

magnet, enabling him to defecate (see Figure XLI). When 

reconstruction has shown function on manometry and 

defecography, the ileostomy is closed. This full schedule 

has now been completed in two patients with very 

encouraging results. 

Perioperative morbidity in 50 consecutive hepatic 

resections 

Hepatic resection for metastatic and primary cancer 

has become a feasible procedure, performed with 

increasing frequency. Pringle's clamping of the 

hepatoduodenal ligamental structures and the 

introduction of new techniques, such as the Cavitron 

Ultrasonor Surgical Aspirator (CUSA), have decreased 

the perioperative mortality and morbidity. 

Our experience with 50 consecutive hepatic resections 

in 49 patients (26 male, 23 female; age 27 - 77 years, 

median 56 years) in the period J anuary 1989 - A pril 1994 

was analyzed. The indication for surgery was metastatic 

cancer in most cases, the majority of colorectal origin 

(38 / 47 cases). The disease free interval between primary 

tumour treatment and metastatic disease ranged from 0 - 

144 months (mean 22.2 months, median 8); 24 patients 

had solitary metastasis, 23 multiple. Diaphragm 

involvement was seen in 9 patients. The extent of surgery 

was at least three segments in 28 patients. Operation time 

ranged from 80 - 330 minutes (median 175 minutes). 

Pringle's procedure was performed in 45 hepatic 

resections. The total clamping time ranged from 25 - 120 

minutes. In almost all cases CUSA parenchymal 

dissection technique was used. 

Total blood loss during surgery was less than 1,500 ml 

in 24, 1,500-3,000 mI in 20 and more than 3,000 mI in 6 

patients and was not related to the extent ofthe resection. 

No blood transfusion was required in 27 patients, only 

two patient needed more than 6 units of blood. 

Abdominal drains were used routineIy. Drains were 

removed within 8 days in 38 patients, and had to stay in 

for more than 2 weeks in 4 patients. In 39 resections the 

postoperative course was uneventful in 39 resections. 

Postoperative bile leakage occurred in 9 patients. In 11 

patients 13 complications were seen: subhepatic fluid 

collection (4x), subphrenic abscess (3x), jaundice (3x) and 

other (3x). Four surgical reinterventions were needed. 

There was no peri- or postoperative mortality. 

Hospital discharge within 14 days was possible for 25 

patients. Only 8 patients stayed in the hospital for more 

than 21 days. We concluded that hepatic surgery for 

metastatic cancer can be performed with acceptable low 

perioperative morbidity and minimal mortality. 

No te 

I Funding: Stichting Fondsenwervingsacties 

Volksgezondheid, St. Mundo Crastini Meliori, 

Maurits en Anna de Kock Stichting.


HPV detection in partners of patients with 

squamous cell carcinoma of the penis 

HPV is a weil known risk factor for the developing of 

penile cancer as weil as cervical cancer. In a prospective 

way all the partners of patients presenting with penile 

cancer are being screened on the presence or absence of 

HPV in acervical scrape. The study aims at increasing the 

insight of the sexually transmittabIe character of HPV in 

a very specific patient population. The PCR for detecting 

HPV is done at the labo ra tory of histopathology at the 

Free University. So far, no HPV possitive cervical smear 

was detected in more than 15 partners of patients with 

penile cancer. 

Note 

I Free University Hospital, Amsterdam. 

Soft tissue sarcoma 

F van Coevorden, E Gortzak, BBR Kroon, OE Nieweg, 

EJTh Rutgers, FAN Zoetrnulder, RC van Doorn, 

MPW Gallee, AAM Hart, AJ Janse, RB Keus, 

BM Loftus-Call, R Somers, T van Vreeland, 

JD Blankensteyn I, EDM Bruggink 2 , AN van Geel I , 

HJ Hoekstra 3 , S Meyer4, B Schuurman4, CW TaatS 

Treatment of retroperitoneal soft tissue sarcomas 

Retroperitoneal soft tissue sarcomas are rare. lts 

prognosis seems to be particularly influenced by local 

control of the tumor. In a retrospective study, our 

experience between 1973 - 1990 was reviewed. In 34 

patients initially treated in the NKI AvL, complete 

(extended or marginal) resection was achieved in 29 

patients. The 5 year survival in this group was 35 % and 

the local recurrence ra te 63%. Extended surgery, which 

was performed in eight patients, resulted in a 50 % local 

recurrence rate. Adjuvant high dose radiotherapy was 

found to have a significantly favorable effect on the 

recurrence free interval (P < 0.01). Adequate preoperative 

planning and specific technical features during 

surgery and radiotherapy are important to improve the 

results. 

Surgical treatment of the retroperitoneum requires 

adequate exposure of the retroperitoneal space. A 

continuous abdominolumbar incision offers such an 

exposure, but was never decribed as such in the literature. 

lts theoretical and practical advantages now have been 

described. 

Lymphangiosarcoma in the edematous extremity 

Lymphangiosarcoma is a rare tumour, seen usually as a 

late complication of chronic Iymphedema. Between 1958 

and 1992 eight cases of lymphangiosarcoma were seen in 

the NKII AvL. In six, the Iymphangiosarcoma followed 

mastectomy with or without radiotherapy of the chest 

wall, in two cases a benign cause for the lymphedema was 

seen. All patients showed quite typical clinical features 

and all but one a typical rapid progression of the disease 

with fatal outcome. One patient, however, after bilateral 

mastectomy and radiotherapy showed a predominance of 

lesions on the chest wall. Without any therapy, an 

uncommon slow progression was seen in this patient. 

Treatment in general is hampered by the multifocality of 

the lesions. Ablative surgery is often dismissed as a 

possibility because hematogenic metastases are of ten 

already present. Radiotherapy sometimes plays a 

palliative role, as does chemotherapy. 

Pulmonary metastasectomy 

The role of pulmonary metastasectomy for soft tissue 

sarcomas has been evaluated in two Dutch studies in 

collaboration with the Dr Daniel den Hoed Cancer 

Clinic. Af ter proper slection of patients a 5 and 10 year 

survival of 40% and 30%, respectively, was seen. Grade 

111 malignancy was the only significant prognostic factor 

for poor outcome. In a second study the value ofrepeated 

resection of recurrent pulmonary metastatic soft tissue 

sarcoma was studied. In selected cases, where 

extrapulmonary disease is absent and all lesions seem 

resectable, repeated resection is feasible, with minimal 

morbidity, and worthwile in terms of disease free and 

overall survival. 

Publications 

Van Doorn RC et al. Cancer 1994; 73: 637-42. 

Van Geel AN et al. J Surg Oncol 1994; 56: 172-7. 

Van Geel AN et al. Eur J Surg Oncol 1994; 20: 436-40. 

Van Vreeland TC et al. J Am CoB Surg (in press). 

Notes 

I Dr Daniel den Hoed Cancer Center, Rotterdam. 

2 University Hospital, Nijmegen. 

3 University Hospital, Groningen. 

4 Free University Hospital, Amsterdam. 

S Academic Medical Center, Amsterdam. 

Melanoma 

BBR Kroon, OE Nieweg, JA van Dongen, AJM Balm, 

D Batchelor, JF Bierhorst, JMF Bonfrer, 

WR Gerritsen, AAM Hart, SP Israels, JM Klaase, 

WJ Mooi, H Neering, EM Rankin, EJTh Rutgers, 

CCE Schaake-Koning, HJC Sombroek 

Lymph node metastases in the neck and parotid 

gland from unknown primary melanoma 

Out of a total of 300 patients with head and neck 

melanoma, Iymph node metastasis in the neck or parotid 

region from an unknown primary melanoma was found 

in 17 (5.7%) patients. The most common site for 

metastatic Iymph nodes (18 nodes in 17 patients) was level 

V (n = 7), followed by the parotid region (n = 4), level 11 

(n =4), level 111 (n =2), and level IV (n = 1). Two patients 

had local excision ofthe neck node metastasis only, while 

the remaining 15 patients underwent more extensive 

surgical treatment. The 5-year disease-specific survival 

rate in this group was 48%, with a median survival of 36 

months, which is similar to the prognosis of stage 11 

melanoma of the head and neck with a known, surgically

treated primary tumor. No relation was found between 

disease-free interval and sex, the number of positive 

lymph nodes or the duration of symptoms. 

Publication 

Balm AJM et al. Clin Otolaryngol1994; 19: 161-5. 

Regional isolated perfusion for 

malignant tumors ofthe limbs 

BBR Kroon, OE N ieweg, JA van Dongen, 

GW van Slooten, JH Beijnen, W Buurman I , 

AMM Eggermont 2 , HR Franklin, AN van GeeP, 

C Hack 3 , AAM Hart, HR Hoekstra4, BAE Kapteijn, 

JM Klaase, M Klop, LB Kristensen, FJ Lejeune 5 , 

D Liénard 5 , SA van de Merwe 6 , AC Ogilvie, GA Pool, 

EM Rankin, H Schraffordt KOOpS4, BC Vrouenraets, 

J van der Zee 2 

High do se rTNF-rJ.., interferon-y and melphalan in 

irresectable melanoma in-transit metastases of the 

ex tremities 

Isolated perfusion of the limbs allows the delivery of 

high dose rTNF -a in a closed system with acceptable sideeffects. 

A protocol with a triple-drug regimen combined 

with mild hyperthermia (tissue temperatures between 

39-40°C) is based on the reported synergism of rTNF-a 

with chemotherapy (melphalan), interferon-y and 

hyperthermia. In melanoma in - transit metastasis (stage 

lIlA and 111AB) a complete remission rate of about 80% 

can be obtained with this immuno-chemotherapeutic 

approach (data on > 100 patients treated in the perfusion 

centers of Lausanne, Rotterdam , Groningen and 

Amsterdam). In single perfusions using melphalan alone 

remission rates of about 50% are achieved. The limb 

disease control ra te of triple-drug perfusions, however, 

does not seem to be improved compared with perfusions 

applying melphalan alone. The role of interferon-y is not 

yet weIl established and more data are awaited from a 

prospective phase 11 trial, randomizing immunochemotherapeutic 

perfusions with and without 

interferon-y. High dose rTNF-a can be administered 

safely in perfusion without systemic side-effects, provided 

systemic leakage is weIl controlled. So far, in about 50 

rTNF-a perfusions, performed in our institute, no 

substantial systemic toxicity was encountered. 

High dose rTNF-rJ.., interferon-y and melphalan in 

advanced soft tissue sarcomas of the ex tremities 

Application of the immuno-chemotherapeutic tripledrug 

regimen of rTNF-a, interferon-y and melphalan, 

combined with mild hyperthermia has promising 

antitumor activity in patients with soft tissue sarcomas. In 

54 patients treated in the perfusion centers of Lausanne, 

Rotterdam, Groningen and Amsterdam, a complete 

remission ra te of 38% and a partial remission rate of 52% 

was achieved. Remission rates are based upon 

compilation of clinical and histopathological data. A 

100% necrosis on histological examination was observed 

SurgicalOncology 121 

in 15 patients. Acute softening of the tumor and total 

disappearance of the tumor vascular bed on angiography 

are striking phenomena. Most tumors we re resected 

between 2-5 months after perfusion. In 5 patients 

amputation of the extremity was unavoidable. The 

combination of high dose rTNF-a, interferon-y and 

melphalan seems to be the first regimen in isolated 

perfusion that has substantial antitumor activity in 

patients with soft tissue sarcomas of the extremities. 

Results of perfusionfor locally inoperable 

melanoma ofthe limbs 

Forty-nine patients with locally inoperable melanoma 

we re treated with perfusion according to four different 

schedules. Perfusion resulted in a complete remission in 

28 patients (57%), with a median duration of 10 (1-55 +) 

months, and a partial remission in 10 patients (21 %), with 

a median duration of 3 (1-9) months. In patients treated 

with a double (normotherrnic or sequential hyperthermie) 

perfusion schedule the complete remission rate was 

higher. Regional lymph node involvement reduced the 

chance of achieving complete remission. Twelve patients 

with complete remission (43%) showed a relapse in the 

perfused area. The corresponding 3-year limb recurrencefree 

interval was 46%. This interval was mainly influenced 

by the number of lesions at the moment of perfusion. 

Three of the patients who failed to respond eventually 

required amputation. The median follow-up of the 

surviving patients was 23 (5-142) months. At the time of 

analysis 23 patients we re still alive, 12 of whom had no 

evidence of disease. Perfusion is an important modality in 

the management of patients with locally inoperable 

melanoma and provides a val ua bie alternative to 

amputation. 

Unexpected severe acute regional toxicity af ter 

routine perfusion with melphalan for melanoma of 

the extremities 

Although regional isolated perfusion is generally a safe 

procedure, even with modern perfusion techniques, 

unexpected severe acute regional toxicity may occur. A 

better understanding of this phenomenon could lead to 

further improvements of the perfusion technique. The 

charts of 181 patients who underwent a single 

normotherrnic or 'mild' hyperthermie perfusion were 

studied. Thirty (16.5%) of them did encounter 

unexpectedly severe acute regional toxicity. Apart from 

proximal isolation level, well-known other prognostic 

factors, such as acute regional toxicity, temperature, 

deviation of pH, deviation of PC0 2 and melphalan peak 

concentration, could not be identified in this study group. 

Although not statistically significant, it is remarkable that 

all but 4 patients were women (26 / 30; 87%). Probably a 

combination of known and as yet unknown risk factors 

may have caused the unexpected severe acute toxicity. 

Incomplete wash out from the limb accounts for 

systemic toxicity af ter perfusion with rTNF-rJ.. 

Five patients who underwent perfusion with rTNF-a 

we re compared with 5 patients who were perfused


without this cytokine. Systemic leakage (99mTc-HSA) was 

< 0.1 %. Blood pressure remained normal in both groups. 

In the rTNF-a group there was fever (peak 38.6°C at 5.5 

hours af ter the start of rTNF-a administration), 

tachycardia (peak 103 / minutes at 5.5 hours) and an 

increase in cardiac index (peak 4.5 l/min/m 2 at 3.5 

hours), with minimal decrease in systemic vascular 

resistance index (1277 dynes. sec/cm 3 /m 2 at 3.5 hours). 

Plasma samples were analyzed for the following 

parameters: rTNF-a (N

CA Hoefnagel, A Kapteijn, B Kwa, IH Liem, WJ Mooi, 

SH Muller, R Baidjnath Panday, JL Peterse 

In vivo tumor imaging with monoc/onal antibodies 

Monoclonal antibody C-95 (a 17-AI antibody) was 

tested in 23 patients. Results were disappointing with only 

3 true positive and 20 false negative scans, irrespective of 

the type of tumor (breast, colon, melanoma, lung) or the 

antibody form (Fab or F(ab)2). 

In order to determine why C-95 appears to have a low 

tumor seeking property in the clinical situation, C-95 is 

compared to the anti body C-I , a weIl known tumor 

seeking antibody. For this purpose C-95 is labeled with 

131 1 and C-l with 1231. This combination was applied in 

6 patients; 2 with breast cancer, 3 with colorectal cancer 

and 1 with colorectal adenoma. One of the patients with 

breast cancer and one with colorectal cancer showed a 

true positive scan. The patient with the colorectal 

adenoma showed a false positive scan. The others were 

false negative; both true positive findings and the false 

positive were se en on the 131I-C95 scans. The tumor/ 

normal tissue ratios were promising (2.2-46.0) for 

eventual therapeutic use and the study will continue. In 

order to improve the tumor uptake of the monoclonal 

antibody, the dosage will be increased from I mg to 10 mg 

First 9 mg of cold anti body will be administered, followed 

by 1 mg of radiolabeled antibody. 

The N-CAM monoclonal antibody 123C3 was tested in 

5 patients with small celllung cancer (SCLC) using 1 mg 

123C3 labeled with 13 11. One patient with extensive liver 

metastasis showed a true positive image, the four patients 

with primary tumor all had false negative scans. The 

study was extended by using 5 mg 123C3 in the next 3 

patients. These 3 patients all showed false negative scans. 

As final step three more patients will be entered in this 

study using an even higher dose of 123C3, to try to 

decrease the amount of aspecific uptake in the liver and 

the spleen; 24 mg of cold anti body will be injected, 

followed by 1 mg of 13 I I-123C3. 

F(ab)2 fragments of Mab 175F4 (anti-episialine) 

labeled with 131 1 were applied in 11 patients, 6 with 

primary breast cancer and 5 with primary head and neck 

cancer. Two true positive scans were seen in patients with 

head and neck cancer. All others we re false negative. The 

tumor/ non tumor ratio was > 1 in all cases (1.2-5.1). The 

immunohistochemistry varied from 0-100%. The study 

will not be continued. 

Radioimmunoguided surgery in melanoma J 

Overall, radioimmunoguided surgery was applied in 10 

patients with clinically apparent regional lymph node 

metastases in axilla or groin. The first 3 patients received 

i.v. 1 mg Fab 99Tc 225.28S (Sorin Tecnemab), after which 

tumor deposits could not be discerned intraoperatively by 

the gamma-pro be because of high blood pool 

backgrounds. In the next 7 patients, 1 mg 125 1 labelled 

whole Ig 225.28S was administered i. v. and operated after 

a median of 9 days (3-15). In 4 out of 7 patients clinically 

overt tumor had 1.5 fold higher than background activity, 

however most probably due to a tumor volume effect. Ex 

vivo, no difference in tumor non-tumor ratio could be 

SurgicalOncology 123 

seen as measured in the coulter counter. Surgery was not 

altered in any patient by probe findings. In the future, the 

occurance of clinically overt regional metastases in 

melanoma should be a rare event, as a result of the 

sentinel node directed immediate selective lymph node 

treatment. The lack of clinical effectiveness, at least with 

this monoclonal anti body, therefore means that the RIGS 

idea in regional melonama has to be viewed critically. 

Radioimmunoguided surgery in colon carcinoma' 

Radioimmunoguided surgery was performed in 4 

patients with operabie colon carcinoma; 3 patients with 

liver metastases and 1 patient with primary tumor. All 

patients received 1 mg of whole antibody IgG C95 

labelled with 1251. Surgery was performed 7 to 15 days 

after administration. In all patients with liver metastases 

activity was no higher than background. In the patient 

with the primary tumor, a two-fold increase in activity 

was found compared to normal colon tissue. No other 

tumor deposits were detected, but those were also not 

found on rnicroscopy. Surgery was not altered by probe 

findings. 

Biopsy of the first-echelon lymph node in melanon.a 

The sentinel node biopsy was added to the melanoma 

protocols in December 1993 and has led to the referral of 

increasing numbers of patients with clinically localized 

melanoma. The concept ofthe procedure is to remove the 

lymph node that is on a direct drainage pathway from the 

primary tumor. This node can be identified with the aid of 

lymphoscintigraphy using 99mTc-colloid (Figure X1.2), 

intraoperative use of a hand held gamma ray detector and 

administration of patent blue dye at the melanoma site. 

Formal lymph node dissection is reserved for patients 

with a sentinel node that is found to contain metastatic 

tumor. 

Figure X/.2 

Lymphoscintigraphy in a patient with melanoma of the 

right lower limbo Lymphatic drainage to an inguinal 

sentinel node is visualized. 

Thirty-nine patients with clinically localized melanoma 

have been included in the study. Forty-six drainage basins 

have been explored and 84 sentinel nodes have been 

removed. In 8 patients, the sentinel node has been found

XII Division of Psychosocial Research and 

Epidemiology 

Head 

NK Aaronson PhD (80%) 

SUBSECTION ON PSYCHOSOCIAL RESEARCH 

Coordinator 

NK Aaronson PhD (80%) 


FSAM van Dam PhD (60%) 


AH Ackerstaff MSc (50%), JB de Boer MSc (20%), 

H Huisman (50%), MEJ de Rond MSc, KCA Sneeuw 

MSc, MAG Sprangers PhD, A te Velde MSc (80%), 

RdeWitMSc 


MJ Muller MSc 


EM Abbink (50%), AC Buitelaar (80%), JF van Buuren 

(80%), BThM van Camp en (80%), SB Detmar MSc 

(80%), YM den Hartog (80%), CAM van der Heijden 

(80%), R Klievink (80%), GHMW Leenhouts (60%), 

LDV Wever, LC Zand belt (80%) 


J Broersen, E Golderberger, MJG Hanneman, 

HA Lammers, MJM Litjens, LN Lodder, TJ Neve, 

MM Schouten, HHM Steenbergen, Y Welegan 

Secretary 

M van den Hoorn 

SUBSECTION ON EPIDEMIOLOGY 

Coordinator 

FE van Leeuwen PhD 


TA van Barneveld MSc, AMJ Chorus MSc, R Noyon 

MD (50%), MA Rookus PhD (70%), 

GAC van der Sanden MSc (80%) 


WJ Klokman MD MSc 

125 

Other technica} staff 

AW van den Belt-Dusebout, JK Bos (80%), PAM Braas 

(60%), AC Buitelaar (80%), CSM Bijen (80%), 

DF Jansen MSc (80%), TM Mooy (50%), 

DMP Portocarero, EG Wits (80%), 

MMPGM Woordes-van BaaIen (60%) 


N Bakker, B van Benthem, SM van Gastel, 

M Louwman, TM Mooy, M van Wely, 

Guest 

K van der Kooy 

Secretary 

M van den Hoorn

126 Psychosocial Research and Epidemiology 

Introduction 

In 1994, the Psychosocial Research Group continued 

its work on the development of valid, reliable and 

practical methods for assessing the quality of life of 

patients with cancer, AIDS and other chronic conditions. 

Via a long-term grant from the Dutch Cancer Society, 

methodologic studies are being pursued to validate both 

generic and cancer-specific quality of life questionnaires, 

to develop and test diagnosis-specific questionnaire 

modules, and to investigate the feasibility of employing 

proxy ratings of patients' quality of life in selective 

situations. Close international collaboration continues 

with the European Organization for Research and 

Treatment of Cancer (EORTC) and the International 

Quality of Life Assessment Project. 

In the area of symptom management, the psychosocial 

research group is conducting severallarge scale studies of 

the role of (district) nurses in facilitating effective pain 

management in cancer patients, and pilot research on the 

cognitive functioning of cancer patients receiving 

intensive chemotherapy. 

The cancer epidemiology group is currently 

concentrating on two principal research lines: the etiology 

of breast cancer, and the long-term health consequences 

ofthe treatment of cancer, particularly in terms ofthe risk 

of developing a second canceL In 1994, analyses were 

completed of a population-based case-control study of 

oral contraceptives (OCs) and breast cancer risk. A twofold 

increased risk of developing breast cancer before age 

36 was found for 4 or more years of OC use compared to 

shorter use. The use of OC, also for long durations or at 

an early age, was not associated with the risk of 

developing breast cancer between age 36 and 45. Recent 

use of OC was associated with two-fold risk in the oldest 

age group (46-54 years). It was concluded that OC use in 

both the early and late fertile years is associated with 

increased risk of breast canceL 

A case-con trol study was conducted to evaluate the 

effect ofTamoxifen on the risk of developing endometrial 

cancer subsequent to breast canceL The results of this 

study show that Tamoxifen use, regardless of dose 

intensity, is associated with increased risk of endometrial 

canceL In 1994, we also examined risk factors for lung 

cancer following Hodgkin's disease (HD). The risk of 

lung cancer was found to increase significantly with 

increasing radiation dose. In patients who smoked after 

diagnosis of HD, the increase of lung cancer risk with 

radiation dose was significantly greater than among 

patients who refrained from smoking. Chemotherapy did 

not affect lung cancer risk. 

Psychosocial Research 

Methodologie issues in health-related 

quality of life assessment 

KCA Sneeuwl, A te Velde 2 , MJ Muller, H Huisman 2 , 

SB Detmar l , AC Buitelaar 2 , LDV Wever l , 

EM Abbink 2 , JH Schornagel, FSAM van Dam, 

MAG Sprangers 3 , NK Aaronson 

Quality of life assessment in clinical oncology 

research: development and testing of questionnaires 

and administration procedures 

The primary objectives ofthis study are: 1) to compare 

the psychometric characteristics of 3 self-report 

questionnaires (the EOR TC QLQ-C30, the CARES-SF 

and the MOS SF-36) in assessing the quality of life of 

cancer patients; and 2) to develop and test supplementary 

questionnaire modules for patients with breast, colorectal 

and lung cancer (in collaboration with the EOR TC 

Quality of Life Study Group). 

Patient accrual began in September, 1992 and was 

completed in September, 1994. Of the 617 patients 

approached to participate in the study, 487 (79%) 

completed at least the baseline questionnaires (n = 171 

with breast cancer, 151 with lung cancer, 117 with 

colorectal cancer, and 48 with other tumor sites; stratified 

by stage of disease). Complete quality-of-life data (i.e. 

baseline plus 2 follow-up assessments) wer e obtained for 

71 % of patients. A subsample of patients completed the 

questionnaires a fourth time for purposes of assessing 

test-retest reliability. Patient attrition was due primarily 

to severe illness or death. 

Preliminary psychometric analyses of the baseline data 

have largely confirmed the hypothesized scale structure of 

the 3 questionnaires. More extensive evaluation of 

instrument reliability (internal consistency and testretest) 

and validity (construct, criterion-based and 

clinical) is currently underway. 

The role of health care providers and significant 

others in evaluating the quality of life of patients 

with cancer 

This study is examining the viability of employing 

health care providers and significant others (e.g. spouses, 

relatives, friends) as either complementary or alternative 

sources of information on cancer patients' quality of life 

(QL). The study sample will be composed of 200 

consenting triads (outpatients under active treatment, 

their significant others, and their treating physician) and 

120 consenting quartets (inpatients, significant others, 

physicians, and nurses). Patients and significant others 

are asked to complete 2 QL questionnaires (the COOP/ 

WONCA-charts and the EOR TC QLQ-C30). Physicians 

and nurses complete the COOP/WONCA-charts only. 

Additionally, ratings of the quality of the patient-proxy 

relationship and of psychological distress will be 

obtained. 

Patient accrual began in November 1993. To date, 140 

outpatients and 39 inpatients have been accrued 

(response ra te is 84%). The percentage of complete triads 

and quartets is 92%. Patient attrition during the follow-up 

period (3 months following the initial assessment) is 30%, 

primarily due to progressive disease and death. Patient 

accrual will continue through November, 1995. 

The international quality of life assessment 

(IQOLA) project 

The IQOLA project is an international collaborative 

effort (currently involving 15 countries) to translate,

128 Psychosocial Research and Epidemiology 

diarrhea was found to have a major impact on patients' 

self-reported social functioning and overall quality oflife. 

A worrisome finding was that half of these patients we re 

incontinent, resulting in increased anxiety levels, and 

restricted movement outside of the home. 

Publication 

De Boer JB et al. Psychol Health 1994; 9: 65-77. 

Notes 

I Funding: Ministry of Welfare, Public Health and 

Culture, Project 88-52. 

2 Academic Medical Center, University of Amsterdam. 

3 Department of Clinical Psychology, University of 

Amsterdam. 

4 Funding: Dutch Cancer Society, Project NKI 90A. 

Symptom perception and symptom 

management in cancer patients 

R de Wit l ,2, MJ Muller, N Moerman 3 , JF van Buuren l , 

CAM van der Heijden l , GHMW Leenhoutsl, 

LC Zandbeltl, MEJ de Rond 2 , BThM van Campen 2 , 

YM den Hartog 2 , R Klievink 2 , MG Nieweg 2 ,3, 

J Noort 2 ,4, MJ Wagenaar 2 ,5, C Mattern, APE Vielvoye 

Kerkrneer, H Huijer Abu-Saad 6 , E van der Wall, 

MMJ Holtkamp, GC Roodbergen, 

LM Gualthérie van Weezei, S Rodenhuis, H Oosting 3 , 

FSAM van Dam 

The (district) nurse and the cancer patient in pain: 

an intervention study 

The primary objectives of this study are: 1) to assess 

longitudinally the nature and treatment of pain among 

cancer patients; and 2) to evaluate a nursing intervention 

designed to enhance the assessment and management of 

pain among cancer patients in the home setting. The 

nursing intervention consists of three components: 1) 

informing patients about pain and pain management; 2) 

stimulating patients to record their pain on a daily basis 

on an I I-point numeric scale; and 3) promoting patients' 

help-seeking behavior. Patient accrual (N = 314) was 

completed in November, 1994. Data analysis is currently 

underway. 

Inpatients' pain assessment: a nursing intervention 

study 

Based on the results of an earlier study conducted in the 

Netherlands Cancer Institute, a simple method for nurses 

to monitor cancer patients' pain was introduced into 3 

hospitals in the Netherlands. A quasi-experimental 

research design was employed to assess the impact of the 

nursing pain assessment on the quality of the pain 

management. Patient accrual has recently been 

completed, with 720 patients enrolled into the study. Data 

analysis is currently underway. 

Cognitive functioning in patients receiving intensive 

chemotherapy 

This pilot study was undertaken to gain initial 

experience in assessing the potential cognitive problems 

that may be associated with the use of intensive 

chemotherapy. Three months after the end of 

chemotherapy, nine patients completed a standard 

neuropsychological test battery. Six of these patients 

performed significantly below age- and educationadjusted 

norms on a test ofverbal memory, and 5 patients 

performed below adjusted norms on tests of attention and 

concentration. Seven patients reported having problems 

with memory and concentration. For 5 of these patients, 

the subjective reports of cognitive problems we re 

confirmed by standard tests. The magnitude of cognitive 

problems observed in this pilot study is sufficient to 

justify further study. A large investigation is currently 

being planned. 

Anx iety and need for information in the ' 

preoperative ph ase 

A short, 6-item questionnaire was developed to assess 

patients' preoperative anxiety levels and information 

requirements. The questionnaire was administered to 320 

patients attending an anesthesiology outpatient department 

for preoperative screening. The results 

indicated th at: 1) women we re more anxious than men; 2) 

patients with a high information requirement also had a 

high level of anxiety; and 3) patients who had never 

undergone surgery expressed a greater need for 

information than patients with prior surgical experience. 

Notes 


2 Funding: Dutch Ministry of Welfare, Public Health 

and Culture, Project NKI 15884. 

3 Academic Medical Center, University of Amsterdam. 

4 Ziekenhuis Gooi-Noord, Blaricum. 

5 St. Ziekenhuis De Heel, Zaandam. 

6 Department of Nursing Science, Limburg State 

University. 

Cancer Epidemiology 

Risk factors for breast cancer 

MA Rookusl, PAM Braas l , AW van den Belt 

Dusebout l , MMPGM Woordes-van Baalen l , 

AC Buitelaar I , AMJ Chorus 2 , K van der Kooy3, 

JA van Dongen, CCE Schaake-Koning, EEngelsman, 

AAM Hart, JL Peterse, JHH Thijssen 4 , 

FE Van Leeuwen 

In 1994 analyses we re completed of a large, populationbased 

case-control study of oral contraceptive use (OCs) 

and breast cancer. Breast cancer cases, aged 20-54 at 

diagnosis (N = 918), we re pair-matched on age with 

controls randomly selected from municipal registries. 

Information on OC use was obtained from both the 

women and their prescribers. The two sources of OC

130 Psychosocial Research and Epidem iology 

th at Tamoxifen use is associated with increased risk of 

endometrial cancer. Contrary to what has been assumed, 

our results suggest that Tamoxifen's estrogenic effect on 

the endometrium is not related to the dose intensity of the 

drug. Physicians should be alert to the elevated risk of 

endometrial cancer in Tamoxifen users and regular 

gynecologic examinations might be considered for longterm 

users. 

Several studies have shown that survivors of HD have 

increased risk of lung cancer, but factors responsible for 

this excess risk are not well-known. We conducted a casecontrol 

study of lung cancer risk in a cohort of 1,939 

patients treated for HD between 1966 and 1986 in the 

Netherlands. Detailed treatment information from the 

medical records was collected for 30 cases of lung cancer 

and 82 matched controls in whom lung cancer had not 

developed. Nearly complete smoking histories were 

obtained from multiple sources. For each case-con trol 

set, the radiation dose to the area of the lung where the 

case's tumor had developed was estimated on the basis of 

radiotherapy charts and experimental simulations of 

treatments. The risk of lung cancer increased significantly 

with increasing radiation dose (p trend = 0.01), with a 

relative risk (RR) of 9.6 (95% Cl: 0.93-98) for patients 

who received 9 Gy or more, compared to those who 

received less than 1 Gy. Patients who smoked more than 

10 pack-years after HD diagnosis had a six-fold elevated 

risk oflung cancer compared to patients who smoked less 

than one pack-year (p = 0.03). Positive interaction on a 

multiplicative scale was present between the carcinogenic 

effects of smoking and radiation. In patients who smoked 

af ter diagnosis of HD, the increase of lung cancer risk 

with radiation dose was significantly greater than among 

patients who refrained from smoking (p = 0.04). There 

was no increase in lung cancer risk with the number of 

cycles of chemotherapy, or with the cumulative doses of 

the drugs mechlorethamine and procarbazine. It was 

concluded that treating physicians should make a 

particular effort to dissuade patients irradiated for HD 

fr om smoking. 

We participated in an international, NCI-coordinated, 

study ofleukemia risk following NHL. Within a cohort of 

11 ,386 two-year survivors of NHL, 35 cases of secondary 

ANLL were identified and matched to 140 controls with 

NHL who did not develop ANLL. Significant excesses of 

ANLL followed therapy with either prednimustine (RR 

13.4; P trend for do se

Biometrics Department 

Head 

OB Dalesio MSc 

Coordinator Trial Bureau 

I Mandjes BSc 

Coordinator Tumor Registry 

GWent 

Coordinator Computer Unit 

W Dirkx BSc 

Coordinator Euroscan Office 

JS Dijkstra 

Academic staff 

H van Tinteren MSc, JL te Velde MSc, B Schaefer MSc 

(from November) 


F Bierhorst, H Franklin (until July), M Broug 

(deceased, November), K Hogerna, I Jansen, 

C Noordhout, H van der Pol BSc, W van Waardenberg, 

A Wals, E Willemse 


R Bakx, M Buitenhuis, E Deen-Ruiter, E van der Donk, 

J Fletcher, A Hiemstra, J Kruit, T Kuipers, M Meyer, 

S Neelakandan, P van Rhee, M Schaefers, J Schipper, 

H Sombroek, L Valkenet MD, S Visser, 

L van Warmerdam 

Secretary 

JS Dijkstra 

131

Study on chemotherapy with vitamin A 

andjor N-acetylcysteine (Euroscan) 

OB Dalesio, JS Dijkstra, E Deen-Ruiter, J Kruit, 

JL te Velde, H van Tinteren, N van Zandwijk 

Euroscan is an ambitious EORTC study to assess the 

value of medical treatment on second tumors in high risk 

patients with lung or head and neck cancer. The Euroscan 

Coordinating Center was created in the department to 

provide efficient coordinating facilities for distribution of 

drugs, protocols and forms, contact with the 

pharmaceutical companies and partlclpants, the 

organization of the 2 weekly meeting of the Euroscan 

Steering Committee, the publication and dissemination of 

the Euroscan Newsletter and the organization of the 

Annual Euroscan Congress. A Data Monitoring 

Committee, comprising investigators not involved in the 

study, was formed to review the data and to ensure careful 

and independent control of the study. 

In June the chairman of the Data Monitoring 

Committee, S Piantadosi from Johns Hopkins in 

Baltimore, visited the department to review the data and 

to assess a preliminary report of results including data on 

compliance, toxicity and activity. Based on the 

recomrnendations ofthe Data Monitoring Comrnittee the 

number of patients to be entered in the study was 

established at 2,600. Accrual was completed in 1994. 

These were accrued by 67 participating institutes in 14 

European countries. Drug distribution will continue for 2 

more years and follow-up for at least 5 years. Statistical 

analysis is performed by the department. Data files in 

SAS format are sent through network from the EOR TC 

Data Centre in Brussels, which is responsible for data 

management. 

Ancillary studies are organized around the Euroscan. 

A fellowship program has been organized with EOR TC 

and IARe. The forms regarding patients smoking habits 

will be analyzed within this program. A follow-up 

chemoprevention study (EUROSCAN 11) is already 

being planned. 

Note 

Funding: Dutch Cancer Society / EEC DG V / EORTC 

fellowship. 

Overviews in Oncology 

OB Dalesio, H van Tinteren, JL te Velde 

Biometrics Department 135 

The department is involved in carrying out two 

overviews (meta-analysis), one in prostate cancer and one 

in non-small cell lung cancer. In prostate cancer the 

question of interest concerned the evaluation of the 

possible benefit of the addition of antiandrogens to 

medicalor surgical castration, in terms of survival. 

Twenty-five studies were identified and individual data 

on more than 5,500 patients entered in those studies were 

collected and processed by specially written computer 

software. When performing an overview it is essential to 

take all possible measures to avoid bias, therefore efforts 

were made to include all relevant studies. In spite of this, 

there are still 2 studies for which no mortality data are 

available and which cannot yet be included. Current 

results of the overview are being discussed with the 

investigators for publication. To assess the possibility and 

importance of extending the overview to include all 

therapeutic questions which are addressed in randomized 

clinical trials of prostate cancer, a literature study was 

performed. Consequently a registry was created 

consisting of references to trials and trial groups. 

Currently this register contains over 125 randomized 

trials, including more than 26,000 patients. The register is 

a potentially useful source not only in conducting the 

overview, but also for planning of new studies and the 

promotion of comrnunication and collaboration among 

investigators throughout the world. 

An overview on the role of cisplatin in the treatment of 

non-small cell lung cancer (NSCLC) is also being 

performed in collaboration with A Ardizzoni and P 

Bruzzi from the University of Genoa, Italy. The 

department is currently in the process of data collection 

and verification. Through exhaustive literature searches 

and contacts with experts in the field, 15 trials have been 

identified. In 11 of the trials the benefit of the addition of 

cisplatin to other chemotherapy regimens is being studied 

and in 4 other trials cisplatin is added to radiotherapy. 

Most ofthe trial coordinators have been located and have 

given their consent for collaboration. Computer 

programs have been adapted from the prostate cancer 

overview software to check the newly received data. 

Queries about inconsistencies, outliers and missing va lues 

are sent back to the local data processing units for 

correction or verification. There are, however, difficulties 

in obtaining data for some of the studies, specially those 

performed a long time ago.

Biophysics Department 

Head 

GJF Blommestijn PhD 


LCJM Oomen PhD, HJ Stoffers MSc (80%) 


CJP Slee BSc, IV van de Pavert 


LJ Korbee BSc (80%), RO van Drimmelen BSc 


B Guvenc, Z Imak, G de Leeuw, M Lolkus, 

AJ van Meelen, BJ Olofsen 

Secretary 

AJ Linden (20%) 

137

138 Biophysics Department 

Introduction 

The Biophysics Department is responsible for a 

number of central facilities and activities for the research 

divisions of the institute: the central computer and 

network infrastructure, electronics maintenance and 

development, digital microscopical image acquisition, 

image analysis, and participation in projects with 

biocomputing and biophysical aspects. 

We added a microscope-CCD camera system to our 

image acquisition equipment, thereby enhancing the 

functionality of the confocallaser scanning system. 

In image processing and analysis, our main focus was 

the standardization of the present SCIL-Image 

applications and the setting up of a library of image 

analysis routines to be used by the present and future 

SCIL-Image programs. 

Computers and N etwork 

HJ Stoffers, LJ Korbee, RO van Drimmelen, B Guvenc, 

Z Imak, CJP Slee, GJF Blommestijn 

The migration to NovelI PC-file servers was completed 

in the first half of 1994. In the process, the primary PC file 

server 'OZ' was upgraded to accommodate more than 50 

simultaneous users. This already became a necessity wh en 

the migration was 70% completed, because the users 

evidently liked the new servers a lot more than the old one 

and usage increased. The support for MS-Windows 

applications and the fact that much of the institute's 

library catalogues and documentation can now be 

consulted on a desktop computer, via the local area 

network, seem to be appreciated. 

User friendly public domain implementations of 

'Gopher' and 'Mosaic' clients (Internet general 

information browsing software) that run under MS 

Windows have been configured to run on every PC that 

can start MS-Windows from the primary PC file server. 

To exploit the possibilities of our Internet connection 

further, we also made arrangements with NCBI (National 

Center for Biotechnology Information) to the effect th at 

any computer with an lP address in the NKI.NL domain 

can access their remote 'Entrez' databases. The Entrez 

software is especially weU suited to quickly gather sets of 

related sequences (protein and/ or nucleotide) together 

with abstracts of the associated relevant scientific 

literature. NCBI has pre-processed their databases to 

optimize th is type of 'inter-database' queries. We have 

configured the Entrez application to run on every PC th at 

can run MS-Windows from our PC primary file server 

'OZ'. 

Unlike the old system, the new PC servers have no hard 

copy user guide. However, considerable effort has been 

spent to make the system more self evident for first time 

users, by providing menus for DOS users, and online 

documentation and application help for both DOS and 

MS-Windows users. A news-Ietter, to appear three or 

four times a year, has been started to supplement the 

online documentation with important tips and 

announcements of new applications and facilities, or 

important changes in existing ones. 

During the year we have experimented with several 

options to give non PC users, who cannot login to a PC 

file server, some form of indirect access to files stored on 

such a server. Users of Apple Macintosh computers 

particularly requested a possibility to transfer files to and 

from a PC file server. Since august 1994 the central PC file 

servers for the NKI research community, 'OZ' and 'Lion', 

can be reached by FTP (File Transfer Protocol). This also 

allows NKI researchers abroad to easily and efficiently 

transfer files from anywhere on Internet to and from their 

home directories on our local PC file servers (provided 

they remember their account and associated password of 

course). 

Two trainee technicians designed, implemented, and 

tested a database application especially tailored to the 

needs of the subsection Genetics of the Division of 

Molecular Genetics, in about 6 months. The system can 

be used on PCs and enables the geneticists to store 

genOlnic and phenotypical data of mice, and implements 

certain analytic tools that are important for the study of 

multigenically controUed traits. 

By the end of October the first components of a new 

U nix system that will replace our current central mail and 

GCG (Genetics Computer Group)-application server, the 

microV AX, arrived. We started migration to the new 

system immediately. Like the migration to the NoveU PC 

file servers that was completed 6 month ago, this wiU be a 

piecemeal process of conversion, since day to day 

activities ofusers must be hampered at little as possible by 

the migration process. Final retirement ofthe V AX is due 

in July 1995. 

Digital Microscopical Image 

Acquisition 

LCJM Oomen, IV van de Pavert, GJF Blommestijn 

This year the microscopic imaging facility at the 

Department of Biophysics, has been extended with a 

microscope-CCD camera system. It consists of a Zeiss 

inverted microscope and a cooled Photometrics CCD 

camera which is operated from a Macintosh computer. 

The key features of the system are its high sensitivity with 

near perfect linearity and a large dynamic range. This 

implies th at the system is weil suited for quantitative 

measurements, also of dim signals like those obtained in 

fluorescence in situ hybridization (FISH) experiments. 

The system can be used to detect both UV and visible light 

excitable fluorescent dyes and, of course, transmission 

imaging is also possible. The basic configuration of the 

system is such th at it can be extended to a 

(semi-)automatic imaging device and an ion-imaging 

system for studies of living cells. 

As in preceding years, the confocal laser scanning 

micro scope (CLSM), with its ability to obtain high 

resolution images at weil defined positions in space, has 

provided detailed information on the (sub)cellular 

localization of proteins and other cell components as 

studied in a great number of different research projects. 

Studies included the functions of cell surface and 

cytoskeletal proteins important in tumor growth and 

metastasis (Divisions I and VI), the interplay between

signal transduction and changes in the cytoskeleton 

(Division 111), localization of tumor specific cell surface 

markers in relation to tumor diagnosis (Division VI) and 

protein distribution in subcellu1ar organelles and / or 

structures (Divisions 111, V, and VI). Consequently, 

CLSM-imaging work has again found its way into several 

publications from researchers in our institute. Apart from 

studies on fixed specimens, studies on dynamic events in 

living cells (monitoring of changes in membrane 

potential, Ca2+ concentration, protein 10calization and 

adhesion plaque formation (Division 111)) have been 

initiated. To be able to perform these experiments at 

physiologically relevant temperatures, a tightly 

controlled temperature regulation assembly coupled to a 

culture chamber has been deve10ped and critically tested. 

The confocal microscope and the CCD system can be 

regarded as complementary units: the CLSM offers high 

resolution and 3D imaging properties (restricted to the 

visible light range), while the CCD is better suited for 

quantitative measurements with high sensitivity (also into 

the UV). With both systems a large range of digital 

microscopical imaging techniques is now available. 

Publication 

Bonfrer JMG et al. Tumor Bio11994; 15: 210-22. 

Biophysics Department 139

Laboratory Animal Department 

Head 

RGM ten Berg DVM PhD 

Permanent Academic staff 

MA van der Valk MSc (20%) 

Permanent technical staff: 

AC Bender, M Beumkens, RC Bobeldijk, PE Boot, 

NH Bosnie, Nl Bovenkerk, H Grimminck, 

Pl Handgraaf, E Herwerden (50%), TL Hetem 

Maidment, 11 lanssen, 1 Kleinsma, FC Krekko, 

Al Linden (30%), FT Nicolaas, lH Pater, 

N Roosmaelen, H Scherpenzeel (50%), Al Schrauwers, 

K Snoep, C Spaans, HP Starrevelt, EH Tanger, 

MAM Timpico, AlM Tolkamp, R van den Berg, 

D van der Pijl, W Wolff, A Zwerver (80%) 

Other technial staff 

Hl Besseis, D Grund, BC Schul te, FH van der Ahe 

141

142 Anima! Deparlmenl 

Introduction 

A large part of oncological research in the Netherlands 

Cancer Institute is carried out using inbred mouse strains. 

The tasks of the Laboratory Animal Department are: 

production of mice and rats, maintenance of all animais, 

rendering of biotechnical assistance, sanitation of strains 

and production of gnotobiotic animaIs, microbiological 

surveillance of the animals and process con trol. 

Training programs for the animal technicians were 

completed this year. This program has, so far, not lead to 

an increased involvement of the technicians in the 

execution of experiments, because of shortage of manpower 

due to occupational diseases. 

The Animal Experiments Advisory Board has reviewed 

all experiments planned for 1994 (total of 23 new 

protocols). According to the Laboratory Animal Law, all 

animal experiments have to be reviewed by this board 

before experiments can be carried out. 

The action for improvement ofthe working-conditions 

for the technicians in the Laboratory Animal Department 

was continued. The breeding-unit on the fifth floor was 

renovated. A new type of isolator, which meets 

ergonomic requirements, was developed on the advice of 

the Safety Health and Welfare Team. Transport logistic 

advices we re implemented. An approach to prevention of 

allergy, which causes substantial sick-leave among 

laboratory animal personnel, is still difficult to formulate. 

Production, sanitation 

A list of the strains of mice and rats produced at the 

Netherlands Cancer Institute is available on request. In 

1993 the breeding production was 78,498 mice and 1,896 

rats. Seventy percent of the mice were used for 

experiments or breeding. The average number of animals 

housed was 21,000 mice and 500 rats (weekly fI uctuation 

of 10%). The average number of animals housed in 

extemal facilities was 70 rabbits. Experiments with cats 

were terminated. A total of2,418 mice and 122 rats were 

delivered to non-resident investigators at production 

price. For sanitation of l3 strains, 53 hysterectomy-foster 

nursing procedures have been performed. 

Microbiological screening 

Microbiological screening of animals has continued in 

the same thorough way as in preceding years. 

Microbiological screening of personnel was terminated. 

Virological screening was carried out by the Department 

of Virology of the University of Nijmegen (ICLAS 

Reference Center for Rodent Viruses). In 1994 the 

animals remained free of Adeno viruses, LCM, MHV, 

Sendai, Ectromelia, REO-virus type 3, K Virus, Po1yoma, 

Theiler, EMC, PVM, MVM, MCMV and Hantaan. 

Serological tests were also carried for Tyzzer and 

Mycoplasma pulmonis. In 1991 a contamination with 

Rota virus was diagnosed. All units were contaminated 

with Rota, except the breeding unit (G3 north, RCS 

lines). In 1993 and 1994 all tests of all units were negative 

for Rota. Rota is a self-limiting disease but it is unusual 

that, in a large breeding-unit, this contamination fades 

out. Screening for pathogens other than viruses was done 

by our own Microbiological Diagnostics Laboratory. 

This showed the animals to be free ofthe main pathogenic 

bacteria. Contaminations have been found with potential 

pathogens such as Pasteurella pneumotropica, Pseudomonas 

aeruginosa, Staphylococcus aureus, Pneumocystis 

camIOn, Syphacia obvelata, Spironucleus muris, 

Tritrichomonas spp and Myocoptes musculinus. The 

microbiological condition of the colony remains stabIe in 

comparison with previous reports. 

Histological screening 

Histo-pathological examinations are routinely being 

carried out by the laboratory animal pathologist of the 

Research Division of Molecular Genetics (MA van der 

Valk). Histological investigations are executed if specific 

clinical symptoms are manifest, or if routine post mortem 

examination warrants this. In 1994 the Sick Animal 

Program (SAP) was fully operative. SAP-reports are 

delivered during the monthly Laboratory Animal 

Committee meetings. Enteropathy is the most prominent 

finding. It is evident that the endoparasite Spironucleus 

muris is an important factor (see Annual Report 1993). 

Treatment of several sections with antiflagelates and/ or 

anthelmintics were carried out on a fixed schedule, and 

proved to be effective. 

Genetic screening 

A screening program using PCR-analyzed microsatellits 

is performed by staff members of the Division of 

Molecular Genetics (P Demant). In 1994, tests on 17 

strains were carried out. 

Cryo-preservation of murine embryos 

Two methods of cryopreservation have been tested: 

freezing of 8-cell stage mice embryos in 1.5 M propylene 

glycol and vitrification of 8-cell stage embryos in 6.5 M 

glycerol and 6% BSA both by use of plastic insemination 

straws. Both methods have been shown to be successful 

and are fully operational. 

Tumors 

The greatest risk of virus contaminations of a mouse 

colony comes from the introduction of various kinds of 

biological entities (tumors, transplants, isolated cells, 

homogenate, etc.). We are therefore testing all new 

biological entities for contamination(s) by means of the 

Mouse Antibody Production test and bacteriological 

cultivation methods. In total, 4 tests have been performed 

in order to screen 4 entities before allowing introduction 

into the animal house.

Cancer Hospital 

Introduction 

The year 1994 was an eventful one for the hospital and 

a considerable change of the organization was realized. 

We started building a new outpatient clinic and treatment 

center, as well as an extension of the operating theatre 

complex. Our official request for building a new hospital 

and renovating the existing one was completed in October 

and sent to the government for official approval. 

Realization ofthis plan is very important for the future of 

our institute. 

In order to improve the organization and make it more 

flexible and ready for the future, a new organization was 

designed and partly implemented this year. 

Organizational divisions ('Clusters') and the delegation 

of operational responsibilities and authorities to lower 

levels in the organization are the most important aspects 

of this new structure. After the completion of this 

operation the clinical center of our institute consists of 

three clusters: 'Surgical Oncology', 'Medical Oncology' 

and 'Radiotherapy'. Next to this the clinical diagnostic 

support will be concentrated in the 'Diagnostic Cluster', 

whereas the service facilities will be brought together in a 

'Service Cluster'. 

In our 1993 annual report we mentioned the 

government approval for building new outpatient 

facilities. Building is now underway and, assuming th at 

everything goes according to schedule, we intend to start 

using this new facility early 1995. This expansion of 

capacity is absolutely necessary in order to attend an 

increasing number of patients, reduce waiting times and 

solve logistic bottle-necks. Almost simultaneously we 

Figure 1 Building of the new outpatient facility, to be completed early 1995. 

143 

started the expansion of our operating capacity. 

Our organization is frequently confronted with the 

limitations of the present building. Rebuildings that are 

necessary for logistic and infrastructual improvement are 

often impossible or only partially possible. Our recent 

plans are focused on building a new hospital next to our 

present building and renovation ofthe present building to 

accommodate part of our research activities. 

Outpatient department 

In January, preparations were started for building the 

new outpatient clinic and treatment center which finally 

resulted in laying the first stone at the end of March. 

Meanwhile, the future users we re consulted about the layout, 

furnishings and equipment for the medical 

examination rooms. The new outpatient clinic and 

treatment center occupies about 3000 m 2 , which is almost 

double the present capacity. The present outpatient 

center was built in the seventies and was intended for a 

capacity of about 35,000 patients. Currently the 

outpatient clinic receives about 70,000 patients a year. 

The expectation is that after moving into the new 

accommodation (March 1995) the number ofpatients will 

further increase, as will, the quality of care. 

This year a number of patients with lung and 

esophageal cancer was treated with expandable wallstents. 

This treatment of obstructions is less burdensome 

for these patients than other procedures. The purchase of 

aspecific gastro-duodenoscope makes ER CP 

(Endoscopic Retrograde Cholangio Pancreaticography)

144 Cancer Hospita! 

examination possible within our institute instead of 

sending these patients elsewhere. Gynecology started 

using a CO 2 laser treatments for pre-malignant lesions. 

The cytostatic-outpatient facility, rebuilt in 1993, 

experienced the first complete year at the new location, 

were it became possible to treat blood transfusion 

patients outside the clinic. Blood sampling through the 

'port-a-cath' and Hickman catheters, and their 

maintenance, mainly occurred in the cytostatic outpatient 

clinic. A number of new cytostatic regimes and studies 

were also introduced. 

Surgical departments 

This year E Aartsen, gynecologist, left after a long and 

fruitfuI clinical period of 28 years at the institute. In 

December a symposium and a liber amicorum we re 

dedicated to him personally and to his professional I i fe. E 

Tasseron also left after serving the institute for 20 years as 

voluntary part-time gynecologist. 

The decentralization of the organizational structure of 

the hospital management ('Clustering') was further 

implemented, and the close cooperation between the 

surgical departments was intensified. BBR Kroon took 

over from JA van Dongen as head of the surgical 

department and was appointed as chairman of the 

surgical oncology cluster, further consisting of the 

Departments of Otolaryngology/ Head and Neck 

surgery, Gynecology, Urology, and Anesthesia. SW van 

Bergen was appointed as manager for Patient Care and 

Economics of the Surgical Cluster and head of the 

Surgical Nursing Staff. OE Nieweg was appointed as a 

full time surgeon ofthe permanent staffand W Meinhardt 

as half time urologist of the permanent staff. This last 

post is combined with a half time appointment in the 

Academic Hospitalof the Free University. Cooperation 

with the Academic Medical Center in Amsterdam (AMC) 

was strengthened in the form of participation in the 

training program for surgical residents. The cooperation 

with the Department of Plastic & Reconstructive surgery 

of the AMC Amsterdam was intensified, resuIting in the 

appointment of a resident, while an application 

procedure for a plastic surgeon is in progress. ChR 

Leemans (4 months) and HP Verschuur (6 months) were 

clinical KWF-fellows for advanced training in head and 

neck oncologic surgery. The Gynecology Department 

hosted FA Peccatori as an AGOG/ ECC-fellow, working 

on the ECC project 'Immunomodulation therapy in 

cancer of the cervix u teri'. 

The continuous pressure on the operating theater 

facilities resulted in approval for extension of the 

operating theater complex. Construction activities for the 

fourth operating theatre are ongoing. 

Highlights in surgical oncology this year were the 

development of dynamic gracilo-plasty af ter abdominoperineal 

resection for recta I cancer to restore continence 

and the program on sentinel node biopsy in patients with 

melanoma, breast cancer, penile cancer and vulvar 

cancer. Gracilo-plasty is developed in cooperation with 

the University Hospital Maastricht. The program on 

sentinel node biopsy will be incorporated in an 

international study, headed by DL Morton from the John 

Wayne Cancer Institute, Santa Monica, California, to 

assess this procedure with regard to survival bene fit. The 

application of minimally invasive surgery was further 

explored, both for staging procedures, such as esophageal 

cancer, and for therapeutic options such as Iymph node 

dissection in urology, ovariectomy and lung 

metastasectomy. Activities in the field of reconstruction, 

making use of free flap transpositions we re intensified. 

Urinary tract reconstruction with bladder substitution, 

aiming at continence after cystectomy, has become a well 

established procedure. The same is true for perineal 

prostatectomy. The first line phase 111 study of Taxol and 

Carboplatin in advanced ovarian cancer led to the referral 

of an increasing number of patients with primary ovarian 

cancer. Further experience was gained in the field of 

isolated limb perfusion in patients with advanced 

sarcoma and melanoma, applying immunochemotherapy 

(rTNF-a, interferon-y, melphalan), in cooperation with 

the Center Pluridisciplinaire d'Oncologie Lausanne, Dr 

Daniel den Hoed Cancer Center Rotterdam, and the 

University Hospital Groningen. 

The surgical group participated in many prospective 

studies initiated by the multidisciplinary tumor groups of 

NKI/ AvL, the Comprehensive Cancer Center 

Amsterdam (I KA) and of many EORTC, WHO, and 

other international groups. The surgical group hasjoined 

the Department of Surgical Oncology of the University 

Hospital Groningen and the PET Center of the 

University of Groningen in the investigation of the value 

of positron emission tomography for tumor detection, 

staging and evaluation of treatment. Several fellows from 

the Netherlands and other countries stayed for varying 

periods in the different surgical departments. The annual 

post graduate course for general surgeons attracted 

several participants from abroad. New developments in 

surgical oncology we re discussed in lectures and round 

table sessions. The guests also participated in the 

operating theatre sessions and in follow-up clinics. Two 

successful teaching sessions for family doctors we re 

organized with topics of prostatic cancer and lung cancer. 

The Department of Otolaryngology / Head and Neck 

surgery, organized several successful courses; a course on 

soft tissue sarcoma in the head and neck region together 

with the general surgeons and the Soft Tissue Sarcoma 

Working Party and 6 bimonthly training courses for 

prosthetic voice rehabilitation after total laryngectomy. 

These latter courses were fully booked with the maximum 

of 8 participants, the vast majority coming from abroad 

(10 different countries). The surgical group contributed 

substantially to the Breast Cancer Working Conference 

of the EOR TC, held this year in Amsterdam, while the 

HPV W orking Party (departmen t of Gynecology) served 

as alocal scientific committee of the 13th International 

Papillomavirus Conference, also organized in 

Amsterdam. R T Gregor became a fellow of the American 

Laryngological Association by invitation. 

Members of the general surgical department acted as 

consultants in meetings of oncology groups in 21 

hospitals in the region covered by the lKA, including 

several hospitals in eastern parts of the Netherlands.

Radiotherapy department 

The Radiotherapy Department showed a steady 

increase in the number of patients treated and in research 

activities in 1994. This year 3,100 new patients were 

treated and 50,000 treatment sessions we re applied with 

extern al irradiation. This increased number of patients 

was reached despite a major renovation ofthe ventilation 

system in the departnent, which took place to remove the 

asbestos, with the accompanying inconvenience for all 

concerned. 

The department welcomed two distinguished visitors 

from the United States this year. Professor S Hellman, 

former dean of the University of Chicago, and D Fraass, 

he ad of the Physics Department of the University of 

Michigan, spent one and six months respectively. 

Th Schnabel, JG Salverda, VJ de Ru, BMP Aleman and 

JSA Beiderbos were appointed as radiation oncologists in 

the department. 

The clinical activities focused on conformal therapy, 

and treatment of breast, prostate and anal cancer. 

Conformal therapy was applied in more patients with 

tumors of the brain, head and neck, prostate and soft 

tissues. A major step forward was the clinical 

introduction of matching of images made on MRI- and 

CT -scan to define the target volume for irradiation. This 

matching process, developed in the research group, 

allows the use of images made before tumor removal, 

even when the images were obtained in other hospitais. 

Breast cancer remained a major focus of attention. 

Many members of the department were involved in the 

organization of the biannual EOR TC Breast Cancer 

Working Conference, which attracted about 900 

participants. The results of several studies were presented 

at this meeting by the department members and by Prof. S 

Hellman in his Karnofski Lecture at the conference of the 

American Society of Clinical Oncology. These results 

we re also published as leading articles accompanied by 

editorials in two major cancer journals in the United 

States. 

The treatment results in patients with anal cancer 

became available this year, revealing a high local control 

ra te with extern al irradiation and iridium 192 implants, 

resulting in a normal anal function in nearly all patients. 

F or patients with locally advanced disease, the results of a 

randornized study proved that the concomitant use of 

radiotherapy and chemotherapy contributed to an 

improved colostomy free survival. 

The continuous efforts of members of the department 

were weIl recognized, i.e. an award given by the European 

Society for Therapeutic Radiology and Oncology 

(ESTRO) to L Boersma for her study on prediction of 

lung function changes after irradiation. In addition a very 

positive report was received from the Dutch Academy of 

Science (KNA W) on the integration of fundamental 

research in physics and biology with the clinical activities 

of the department. 

Medical oncology 

As part of a general reorganization of the hospital, a 

'Cluster' ofmedical specialist groups was formed in 1994, 


that includes the Departments of Medical Oncology, 

Gastro-enterology, Pulmonary Medicine, Neurology and 

the Pain Management group. The Cluster is not limited to 

the Medical Staff, but will also incorporate the Nursing 

Staff ofthe two Medical wards, the Chemotherapy Clinic 

and the Research Nurses. S Rodenhuis was appointed 

Chief of Medicine from September 1994, and P Terwijn 

joined the cluster as Manager for Patient Care and 

Economics in December. 

One ofthe objectives ofthe reorganization is to be able 

to more effectively deal with some of the pressing 

problems in health care in the Netherlands. As a result of 

the restrictive hospital budgeting system, the rapidly 

rising cost of quality care is not matched by 

reimbursement through government agencies or by the 

health care insurers. For example, 1994 has seen the 

introduction ofa number ofnovel and effective drugs that 

may be expected to improve both the curative and 

palliative treatment of cancer patients. These drugs, 

however, are extremely expensive and any use of these 

agents necessarily leads to an unacceptable strain on the 

hospital budget. Other technical innovations have similar 

effects. For example, coated expandable stents became 

available in 1994, which are ideal for use in palliation of 

patients with esophageal cancer who are no longer able to 

swallow. These stents replace the older tygon 

endoprostheses, the insertion of which was technically 

demanding and was associated with an appreciable (and 

often lethal) perforation rate. Thus, the novel stents are 

more convenient, much more acceptable to the patient 

and safer. They do, however, cost three times as much as 

their predecessors, while no proportional reimbursement 

from the health insurers is available. It is hoped that the 

new cluster structure will facilitate the making of the 

painful choices that, sadly, have now become 

unavoidable. 

On a more cheerful note, 1994 witnessed the returns of 

a number of investments in top-clinical care and clinical 

research of the previous few years. The autologous blood 

stem cell transplantation program for solid tumors 

flourished, and some 80 transplantation procedures 

should be completed by the end of the year. The clinical 

and technical developments resulting from the NKV AvL 

program in high-risk breast cancer have led to a national 

trial in which all Dutch University Hospitals and both 

Cancer Institutions participate and th at began to register 

patients in May 1994. A second national study with the 

same study partners, in relapsing germ cells cancers, (also 

based on NKV AvL work) began patient recruitment 

only a few moths later. 

The establishment of a Clinical Immunology Unit in 

1993 and the building of an associated 'Good 

Manufacturing Practice' laboratory have allowed the 

start of an ambitious gene therapy study in melanoma. 

Autologous tumor cells are transduced in vitro with the 

GM-CSF gene, irradiated and subsequently re-injected 

into patients, as a vaccination designed to elicit T-cell 

anti-melanoma responses. The study is performed in 

collaboration with the SOMATIX corporation of 

Alameda, California. 

There were no major personnel changes in 1994. Dr 

ME Craanen, a young gastro-enterologist with a keen 

interest in basic science, finished a year of additional

146 Cancer Hospital 

training in molecular biology and was we1comed to the 

clinical gastro-enterology group in September. He will 

share his time between the Academic Medical Center in 

Amsterdam and the NKV AvL, thereby contributing to 

the intensive collaboration between the two centers. 

Pathology Department 

In 1994, the histologic diagnostic workload of the 

department continued to ri se to a total of about 11 ,900 

investigations (1993: 10,600), whilst the number of 

cytologic investigations stabilized at ab out 6,200. An ever 

growing part of the total workload consists of diagnostic 

problem cases submitted for expert opinion. 

The 22nd European Congress of Cytology was held 

Amsterdam' on 25-28 September 1994, attracting 400 

participants. The congress was presided by P van Heerde, 

who played a major part in its organization. All other 

staff members were also actively involved in the 

organization ofvarious congresses, symposia, workshops 

and teaching courses, covering a wide spectrum of topics 

concerning cancer diagnosis. This part of our work is 

always somewhat under threat, since it involves neither 

diagnostic work directly related to patient care, nor 

innovative research. The molecular pathology group, 

headed by LJ van 't Veer, tlourished in 1994. It initiated a 

number of new research lines and set up a panel of new 

molecular diagnostic tests, outlined in the reports of 

Divisions VI and X. 

Pharmacy department 

The Pharmacy Department of the Slotervaart Hospital 

also functions as the pharmacy of the Antoni van 

Leeuwenhoek Huis and provides it with all drugs. 

Current pharmacy services include the delivery of 

registered and non-registered drugs, special preparations 

according to the needs of the patient, drug level 

monitoring, pharmaceutical quality con trol of radiopharmaceuticals, 

providing information on drugs, 

education, support in clinical pharmacologic research, 

etc. Discussions are ongoing regarding the extension of 

our service for the preparation of cytotoxic drugs to the 

week-ends. In 1993 we subjected the Central Preparation 

Room (CPR) (Slotervaart Hospital) for cytotoxic drugs 

to an internal review with respect to the guidelines 

currently in force for safe handling of cytotoxic agents. As 

the premises did not meet the requirements, mainly from 

an architectural nature, we rebuilt the CPR in 1994 and 

have adapted working procedures. In 1995 we will 

probably combine the CPR and preparation room on the 

outpatient clinic into a new unit in the outpatient clinic 

now under construction. The workload at the CPR and 

the preparation room in the outpatient clinic has been 

stabie for some years; ab out 110 preparations per week 

for the clinic and around 150 per week for the outpatient 

clinic. 

The Pharmacy department holds responsibility for the 

quality for all registered and non-registered drugs. We 

we re involved in setting up the quality systems and hold 

responsibility for the ongoing clinical study in melanoma 

patients with gene transfected systems. The following 

investigational drugs we re delivered in 1994: 

2-chlorodeoxyadenosine, coumarin, CPT -11 , docetaxel, 

E09, tludarabine phosphate, miltefosine, suramin, 

tallimustine, topotecan. 

N uclear Medicine department 

Since delivery of two dual head gamma cameras 

(ADAC Dual Genesys and Vertex) incorporated in a 

computer network, late 1993, the department has been 

functioning as a totally digital department. 

A total of 3,730 in vivo procedures was performed, 652 

in in-patients, 2,178 in out-patients and 900 for other 

hospitaIs. A total of 112 radionuclide therapies was 

carried out. 

Despite an incidental reduction of the department's 

budget it has been possible to stay within the budget by 

increasing the efficiency of the supply of materials and by 

a critical evaluation of requests for relatively expensive 

procedures. 

Apart from routine nuclear medicine the department is 

actively involved in many research projects in 

cooperation with other departments, outlined in the 

report of Division X. 

Throughout the year the department has received 

many visitors and short term residents, both because ofits 

role as a reference site for the equipment used and because 

of its experience in 131I-MIBG therapy. 

Staff members we re actively involved in the European 

and World Congresses of Nuclear Medicine, in the 

organization of an international workshop and 

symposium on radionuclide therapy. This year the 

department brought forward 32 publications and 20 

abstracts on nuclear medicine, as well as two 

dissertations. On June 29, RA Valdés Olmos, nuclear 

medicine physician, defended his thesis on 'The role of 

nuclear medicine in the detection of organ injury and 

adverse effects of cancer therapy' at the University of 

Amsterdam (promotor Prof. JB van der Schoot, copromotor 

Dr CA Hoefnagel). On October 3, AR 

Wafelman, pharmacist, defended his thesis 'Pharmaceutical 

characteristics and pharmacokinetics in cancer 

patients of iodine-131 Iabelled meta-iodobenzylguanidine 

and unlabelled meta-iodobenzylguanidine' at the 

University of Utrecht (promoters Prof. RAA Maes and 

Prof. JH Beijnen, co-promotor Dr CA Hoefnagel). 

Medical Ethics Committee 

Members ofthe committee in 1994 we re RB Keus, MD, 

chairman, radiotherapist, M Bos, RN, secretary, RT 

Gregor, MD, ENT surgeon, M Piek-den Hartog, MD, 

general practitioner, JS Reinders, theologian, JH 

Schornagel, MD, medical oncologist (until October), EB 

van Veen, lawyer. 

The Medical Ethics Committee (MEC) is installed by 

the medica I staff and the board of directors of the NKI/ 

AvL. lts main tasks are the ethical review of clinical 

research protocols involving patients and / or healthy 

persons and advising on medical ethical aspects of patient

care. This year Schornagel left the committee because of 

his appointment as chairman of the Medical Staff. His 

contribution to the work of the committee is greatly 

appreciated. A procedure has been started to recruit a 

new medical oncologist for his replacement. 

In 1994 the frequency of meetings was bimonthly. 

During this period 45 protocols were reviewed. 31 

Research protocols were accepted (mostly after 

amendments suggested by the MEC), 8 were sustained, 3 

we re rejected, 1 was delayed and 2 protocols returned to 

the study coordinator for answering questions. 

Other topics in 1994 included the preparation of 

guidelines for the monitoring of clinical trials with 

optimal protection of privacy and patient rights. A new 

set of guidelines for patient information and a standard 

informed consent form we re also prepared and accepted. 

The implementation of the new Dutch law on medical 

practice (WGBO) has been discussed by the committee. 

Moreover, the moral aspects of limited prescription of 

expensive drugs has been discussed by the committee and 

will be the subject of further consideration. 

Sodal Medical Service 

The primary goal of the Social Medical Service (SMD) 

is to improve the quality oflife of cancer patients and their 

families at all stages of the illness trajectory. To optimize 

this service, the Social Medical Service has developed a 

systematic registration form for in-and out patients. 

In 1994, 631 psychosocial care consultations (308 

patients) have been registered during a three month trial 

period. Breast cancer patients were the largest group of 

patients demanding psychosocial care, followed by 

gynecologic and lung cancer patients. The number of 

outpatients asking for psychosocial support also 

increased and the care for clinical patients was intensified. 

Activities of 1994 included: multidisciplinary groups 

we re established to develop a genetic counseling program 

and to address the issues of sexual rehabilition; in 

collaboration with the National Society for General 

Practitioners (LHG), we are developing a trainingprogram 

for 'counseling cancer patients'; participation in 

national and international conferences on the subject of 

'farnily and cancer'; training in communication skills for 

personnel working on the radiation ward and outpatient 

department; together with the Amsterdam Comprehensive 

Cancer Center, we prepared a nationwide 

conference on rehabilitation issues in cancer care for 

social workers specialized in oncology; development of a 

database of all community resources available in the 

Netherlands.- 

Furthermore, the Social Medical Service continues to 

develope programs to better address the needs of cancer 

patients and identify high risk patients for long-term 

psychosocial morbidity. 

Tumor documentation 

A computerized Tumor Registry is held by the 

Biometrics Department of the Netherlands Cancer 

Institute. A total of 4,458 new tumors was seen in the 


hospital from July 1993 to June 30 1994: 758 nonmalignancies 

and 3,700 malignancies (see Table 1). From 

the 3,552 malignancies, 527 were seen at the hospital for 

advice only and 3,173 were treated in the hospita!. Figure 

2 shows the distribution of treated malignancies by tumor 

site. 

Table 1 

Malignant disease localization or diagnosis seenfor: 

lCD Code Treatment Advice only 

140 Lip 7 

141 Tongue 15 

142 Salivary glands 10 

143 Gingiva 

144 Floor of mouth 11 

145 Other parts of the mouth 9 

146 Oropharynx 21 5 

147 N asopharynx 9 

148 Hypopharynx 18 

150 Esophagus 69 13 

151 Stomach 36 20 

152 Small intestine 

153 Large intestine 65 35 

154 Rectum 128 23 

155 Liver 3 3 

156 Gall bladder 4 3 

157 Pancreas 9 10 

158 Peritoneum 1 1 

160 Nose 7 1 

161 Larynx 44 4 

162 Lung 484 66 

163 Pleura 22 10 

164 Thymus, heart, 5 1 

mediastinum 

170 Bone 9 1 

171 Connective tissue 73 10 

172 Melanoma 153 34 

173 Skin 129 5 

174 Breast (female) 983 150 

175 Breast (male) 1 

180 Cervix uteri 64 6 

181 Placenta 1 

182 Corpus uteri 61 1 

183 Ovary 72 19 

184 Vagina, vulva 10 1 

185 Prostate 153 17 

186 Testis 49 4 

187 Penis 18 1 

188 Bladder 84 5 

189 Kidney 37 10 

191 Brain 14 11 

192 Nervous system 7 

193 Thyroid gland 21 

194 Endocrine glands 2 

199 Unknown primary site 71 34 

200 Non-Hodgkin lymphoma 113 12 

201 Morbus Hodgkin 29 6 

203 Multiple myeloma 33 1 

204 Lymphocytic leukemia 5 1 

205 Myeloid leukemia 3 2 

Total 3173 527

148 Cancer Hospital 

Documentation of childhood 

malignancies 

Registration of all tumors reported by the Regional 

Pediatrie Oneology Working Party is also performed by 

the Tumor Registry of the Biometries Department. A 

total of 147 new tumors diagnosed from Ju1y 1993 

through June 30, 1994 were entered into the database; 22 

of these tumors we re non-malignaneies. Table 2 shows the 

tumor site of the 125 malignaneies. 

Table 2 

ICDO code/ Primary site 

147 Nasopharynx 

155 Liver 

170 Bone 

171 Conneetive tissue 

183 Ovary 

186 Testis 

189 Kidney 

191 Brain 

192 Nervous system 

194 Endoerine glands 

200 Non Hodgkin lymphoma 

201 Morbus Hodgkin 

204 Lymphoeytie leukemia 

205 Myeloid leukemia 

206 Leukemia NOS 

Total 

2 

2 

6 

18 

3 

3 

9 

13 

10 

11 

8 

8 

29 

2 

1 

---us 

G:U. 

Figure 2 

Distribution of 3,173 patients treated in the hospital by 

tumor site in 1993.

Clinical W orking Parties 

Introduction 

The mu1tidisciplinary character of the Netherlands 

Cancer Institute/ Antoni van Leeuwenhoek Huis (NKV 

AvL) and the collaboration between basic research, 

clinical research and patient care is expressed by the way 

the tumor oriented working parties perform their 

research activities. 

The working parties meet at regular intervals to discuss 

clinical cases and exchange scientific information and 

clinical experience. The aims of the parties are also to 

develop, conduct and stimulate new clinical studies for 

the evaluation of cancer treatments. Furthermore, the 

parties develop and update patient management 

guidelines. The parties have long-standing and close 

collaboration with national and international clinical 

cooperative cancer groups. 

Antiemetics W orking Group 

Chairman: BG Taal 

The novel HT3-blocking antiemetics have been 

incorporated successfully in clinical practice as a routine 

prophylactic measure. Pharmacological research has 

been planned for a new application: the suppository in 

comparison to the oral preparation. 

Bioanalysis and Early Clinical Trials 

Group (BECTG) 

Chairman: ww ten Bokkei Huinink 

For details, see Division X, Pharmacy text. 

Breast Cancer W orking Group 

Chairman: EJTh Rutgers 

This year more than 200 breast cancer patients were 

entered in ongoing trials concerning primary treatment (8 

trials) or advanced disease (8 trials). Studies of dose 

intensive adjuvant chemotherapy in high risk primary 

patients and of Taxanes in advanced breast cancer we re 

started. The nation wide 'N4 + ' -trial, comparing 

standard adjuvant chemotherapy (5 x FEC) and the 

addition ofhigh dose CTC chemotherapy with peripheral 

stem cell re-infusion (co-initiated by the NKV AvL 

149 

group). has already recruited over 120 patients. 

A pilot study on the use of the sentine1lymph node 

procedure in breast cancer has been started. In a modified 

radical mastectomy procedure the tumor-draining lymph 

nodes are identified: the main initial goal is to determine 

whether these lymph nodes can be found and whether 

their tumor status predicts the status of the remaining 

axillary nodes. 

The patient-care protocol for patients undergoing 

axillary dissection for primary breast cancer, examining 

the short and long term effects of early hospital discharge 

on psychological and physical well-being, morbidity, arm 

function, micro- and macro-economics, has been weIl 

received by patients and nursing staff: over 100 patients 

have entered within a one year period, equally divided 

between short and long stay. 

The increasing possibilities to analyze hereditary 

factors for cancer risk and the concomitant increasing 

awareness of patients and relatives has unified clinicians, 

epidemiologists, psychologists, pathologists and 

molecular biologists in a 'Hereditary Tumor Working 

Group'. Breast cancer will be a major issue for this 

working group. A hereditary tumor clinic, in close 

cooperation with clinical genetic counsellors and 

molecular pathologists, will be effective early 1995. 

The epidemiology department published the first 

results of their large-scale case-con trol study of life stylerelated 

breast cancer risk factors. Long-term oral 

contraceptive use starting at a young age appeared to be 

related to an increased risk of developing breast cancer at 

a young age ( < 36 years). They also published a study on 

the risk of endometrial cancer following breast cancer in 

which it was found that use of Tamoxifen for 2 or more 

years doubles the risk of endometrial cancer (see Division 

XII). 

The intensified cooperation with the reconstructive 

surgeons resulted in a rapidly increasing use of plastic 

reconstructive techniques in primary breast cancer 

management, mainly by the immediate or delayed use of 

modified TRAM-flap techniques (circumventing the use 

of silicon prosthesis). 

Chaired and organized by Prof. JA van Dongen, the 

EORTC Breast Cancer Working Group held its third 

workshop on ductal carcinoma in situ in Venice in 

February. A major spin-off is the conception by 

European pathologists of a new simplified classification 

of DCIS, which is increasingly gaining acceptance 

throughout the world. 

Under the chairmanship of Prof. GMM Barte1ink the 

group organized the 6th EOR TC Breast Cancer Working 

Conference fr om 6-9 September in Amsterdam; over 800

150 Clinical Working Parties 

participants from 40 different countries attended this weil 

appreciated and successful meeting. 

Gastro-Intestinal Tumors W orking 

Group 

Chairman: H Boot 

The tradition of clinical research in gastric NHL has 

continued and several new aspects have been initiated: 

investigations ofthe role ofH pylori and the development 

of a prognostic model using a belief network, in 

cooperation with MWM Jaspers of the Department of 

Medical Informatics of the Academic Medical Center, 

Amsterdam. Our HDR (high dose ra te) irradiation 

program was successful both in patient accrual and 

treatment results: a response ra te of 77%. A definitive 

statistical analysis ofprognostic factors will be carried out 

in close cooperation with the Department of Medical 

Informatics of the Academic Medical Center, 

Amsterdam. The new generation of self-expandable, 

coated stents for esophageal obstruction or fistula has 

been applied in over 30 cases and in several patients 

another stent was introduced in the trachea leading to 

immediate relief of dyspnoea and dysphagia. Another 

novelty was the introduction of the PUG (percutaneous 

ultra-sound guided gastrostomy). An analysis of the 

results of 50 partialliver resections for metastatic disease 

showed an acceptably low morbidity and mortality of th is 

procedure. Non-radioactive MIBG (Meta-iodo benzyl 

guanidine) appeared to be effective in the palliation of the 

carcinoid syndrome. An intensified interest in hereditary 

tumors resulted in a multidisciplinary group to stimulate 

both clinical detection and basic research. The entry in the 

NACCP trial to study the role of adjuvant treatment in 

colorectal cancer is still adequate. 

Gynecologic Tumors W orking Group 

Chairman: ThJM Helmerhorst 

The impressive results of the European-Canadian 

study of Taxol in 2nd line treatment in patients with 

advanced ovarian cancer we re accepted for publication. 

The group has taken a major part in the patient accrual of 

this study. The group also participated in a large 

international multicenter ph ase 11 study in ovarian cancer 

patients using Topotecan. Activity was demonstrated 

even in patients considered resistant for platinum 

containing drugs. A subsequent study was designed to 

compare Topotecan and Taxol. Analysis of the 

international study, which was coordinated by one ofus, 

evaluating the roie of rh-erythropoietin (EPO) in the 

prevention of anemia in platinum treated patients, 

revealed significant differences in the number of patients 

th at needed transfusion. 

During the XIV FIGO world congress at Montreal in 

September 1994, the group provided 6 presentations, 

concerning HPV clinical studies, colposcopy, vulvar 

melanoma and surgical technique. 

Head and Neck Tumors Working 

Group 

Chairman: AlM Balm 

The Provo x voice rehabilitation system received FDA 

acceptance and this resulted in intensifying the 

international teaching courses, organized on behalf ofthe 

head and neck working group. In combination with a 

newly developed valved heat and moisture exchanger 

(HME) rehabilitation of laryngectomy patients will 

further improve. 

Collaboration was further strengthened with the 

Department of Oral/Maxillofacial Surgery of the 

Academic Medical Center, Amsterdam, which broadens 

the scope of the treatment facilities for head and neck 

patients particularly in relation to the reconstructive 

procedures. An additional post for a full-time plastic 

surgeon will further improve our reconstructive 

capability. 

ChR Leemans and HP Verschuur participated as Head 

and Neck oncology fellows of the Dutch Cancer Society 

in our multidisciplinary clinical and research activities. 

Cooperative efforts with Division VI resulted in an 

immunohistochemical study demonstrating the potential 

value of cyclin Dl overexpression as a prognostic 

indicator in he ad and neck squamous cell carcinoma. 

The group organized six international clinical training 

courses on voice restoration after totallaryngectomy and 

a symposium on the diagnosis and treatment of adult soft 

tissue sarcomas. These meetings were weil subscribed to 

from various European countries. 

Lung Cancer Working Group 

Chairman: N van Zandwijk 

In 1994 the lung cancer group continued its supervisory 

activities in Euroscan, the European-wide chemoprevention 

(EOR TC) study that was closed for patient 

entry in August (Division X and Biostatistics). 

In addition, a phase 11 study with N-acetylcysteïne was 

started under the auspices of the Netherlands Prevention 

Fund, in collaboration with the University of Limburg 

and Institute of Hygiene and Preventive Medicine, Genoa 

(Division X and VI). 

To obtain better images of small celllung cancer, a dose 

intensification was judged necessary for radiolabeled 

123C3 directed against NCAM (Division VI). The pilot 

study of cisplatin added to fractioned radiotherapy in 

inoperable locoregional non-small ceIl lung cancer, in 

coIlaboration with the Department of Radiotherapy of 

the Academic Medical Center, was completed and 

confirmed the feasibility of this new radiochemotherapeutic 

approach (Division IX). The intensification 

ofradiotherapy by the addition ofphotodynamic therapy 

or HDR brachytherapy in locoregional advanced 

NSCLC was found to cause extensive normal tissue 

damage. This project was therefore modified to focus 

primarily on technology assessment of both 

intrabronchial modalities (Division X and IX). 

The postoperative chemotherapy trial in K-ras positive

non-small cell lung cancer patients was negatively 

influenced by the complicated patient entrance procedure 

and consequently a simpier design of the Italian ALPItrial 

was adopted to answer questions on this important 

prognostic factor (Division X). 

Unfortunately, the radiation protection study (ECC) 

with N-acety1cysteïne had to be closed as a consequence 

of an unexpected reduction in pharmaceutical funding 

(Divisions X and IX). 

The Lymphoma Working Group 

Chairman: JW Baars 

The lymphoma working group is actively participating 

in peripheral stem cell/autologous bone marrow 

transplantation protocols of the EORTC and / or 

HOVON working groups. From November 1993 till 

November 1994, 3 patients with morbus Hodgkin and 10 

patients with non-Hodgkin's lymphoma have been 

treated with an ablative regimen followed by a peripheral 

stem cell transplantation. It became possible this year to 

use total body irradiation as part of the conditioning 

scheme and in 1994 2 patients were successfully treated 

(JH Borger, L Dewit, Department of Radiotherapy). 

Furthermore, we are actively participating in the 

EORTC 20932 study (role ofRT in patients with NHL of 

intermediate and high grade malignancy achieving CR 

after chemotherapy), the randomized study comparing 

the efficacy of fludarabin and CVP in low grade NHL 

stage 111 or IV (EORTC 20921) and the EORTC 20931 

study, in order to achieve the best treatment strategy for 

patients with morbus Hodgkin stage 1 or 11 above the 

diaphragm stratified according to prognostic parameters. 

D de Jong, from the Department of Pathology, will 

coordinate studies to define the significance of 

chromosomal aberrations in the several types of 

malignant lymphomas. The lymphoma group is also 

active in the development of the best treatment strategies 

for gastro-intestinal lymphomas, together with the 

Department of Gastro-enterology. 

The development of second malignancies after 

treatment for malignant lymphomas is still a subject of 

investigation in collaboration with the Department of 

Epidemiology. 

The Melanoma W orking Group 

Chairman: BBR Kroon 

This year a start was made with the phase I study (in 

collaboration with Somatix corporation in the USA), 

exploring the effects ofvaccination with autologous GM 

CSF transfected and irradiated tumor cells in melanoma 

patients with disseminated disease. The first patients 

enrolled in this study have shown immunologie reactions 

consisting of edema and erythema at the site of 

vaccination. (Division IV and X). 

The method of lymphatic mapping and sentinel node 

biopsy in patients with intermediate and thick melanoma 

was added to the melanoma protocols in December 1993 

Clinical Working Parties 151 

and has led to the referral of increasing numbers of 

patients with clinically localized melanoma. The goal of 

the procedure is to remove the lymph node that has been 

on a direct drainage pathway from the primary tumor. 

This node can be identified with the aid of 

lymphoscintigraphy using 99mTc-colloid, intraoperative 

use of a hand-held gamma ray detector and 

administration of patent blue dye at the melanoma site. 

Formal lymph node dissection is reserved for patients 

with a sentinel node th at is found to contain metastatic 

tumor. Thirty-nine patients with clinically localized 

melanoma have been included in the study. It was shown 

th at preoperative lymphoscintigraphy and intraoperative 

lymph node detection can be done with a single 

dose of 99mTc-colloid. This approach saves patients a 

second exposure to radioactivity and is now included in 

our treatment routine. The identification of sentinel 

nodes, using dye, proved to be successful in 75% of the 

cases, by tracing the remaining 99mTc-colloid the success 

rate was 100%. The mean sentinel node to background 

ratio intra-operatively was high (27, range 7-53), a 

phenomenon which makes intra-operative lymph node 

detection by 99mTc-colloid a highly sensitive procedure. 

These data indicate that we are able to identify the 

sentinel node in (currently) 100% of the patients. The 

theoretical concept of the procedure appears to be valid. 

Participation in a randomized trial to investigate a 

potential survival benefit is being considered. 

At the Nuclear Medicine Department a variety of 

specific radiotracers for the detection of disseminated 

melanoma are under investigation, e.g. with lllln_ 

pentetreotide. Intraoperative tumor detection by the use 

of the gamma probe af ter i.v. administration of 125 1 

labelled 225.28S IgG anti-melanoma antibody was 

performed in 10 patients with stage 11 (lymph node 

metastases) melanoma. Disappointingly, this method 

appeared to be ineffective in the identification of 

subclinical and even clinically overt tumor deposits. 

However, this method becomes superfluous now th at the 

sentinel node procedure in primary melanoma has proven 

to be effective. 

The occurrence and prognosis of lymph node 

metastases in the neck and parotid gland from an 

unknown primary melanoma was studied. Prognosis in 

these patients is similar to stage 11 melanoma of the head 

and neck with a known primary tumor. 

A special issue of Diagnostic Oncology was devoted to 

12 articles in which the presentations of the symposium 

'Diagnosis and Treatment of Cutaneous Head and Neck 

Melanoma', held in October 1993 in our institute, we re 

published. 

Isolated Perfusion W orking Group 

Chairman: BBR Kroon 

Further experience was gained with isolated perfusion 

of the limbs combining chemotherapy (melphalan), 

immunotherapy (rTNF-a, Interferon-I') and mild 

hyperthermia (tissue temperatures between 39-40 °C). In 

patients with irresectable melanoma complete remis sion 

rates are about 80%. The limb disease control rate,

152 Clinical Working Parties 

however, does not seem to be improved compared with 

single drug (melphalan) administration. In patients with 

sarcoma, the chemo-immunotherapeutic schedule seems 

to be the first regimen in isolated perfusion, that has 

substantial anti-tumor activity resulting in a complete 

remission rate in irresectable patients of about 30-40% 

and a partial remission rate of 50%. Both in patients with 

irresectable melanoma and irresectable sarcoma, 

perfusion provides a valuable alternative to amputation. 

The role of interferon-y in the triple drug regimen is not 

weil established and the results of a prospective phase II 

trial, randomizing between perfusions with and without 

interferon-y, conducted in the perfusion centers of 

Lausanne, Rotterdam, Groningen and Amsterdam, are 

awaited. 

It was established that, in our hands, high dose 

r-TNF-a can be administered safely in perfusion without 

systernic side effects. This is the result of areliabie surgical 

isolation technique with no or negligible systemic leakage 

and a sophisticated monitoring system using isotopes. A 

fruitful meeting was organized by the Department of 

Nuclear Medicine on the topic of optimal monitoring 

during isolated perfusion. The meeting was attended by 

representatives of all the perfusion centers in the 

Netherlands. 

Several pharmacokinetic studies, both in the isolated 

circuit and systemically, were performed aiming to clarify 

the working mechanism ofrTNF-a and ofinterferon-y. It 

was found that during perfusion, rTNF-a activity does 

not occur systemically and that this cytokine is only 

released from the circuit after wash out and restoration of 

the vessels. 

N euro-Oncology W orking Group 

Chairrnan: W Boogerd 

In 1994 a multicenter study was completed in 

cooperation with the Departments of Neurology and 

Radiotherapy of the Academic Medical Center, 

Amsterdam and of the Free University, Amsterdam on 

the efficacy of radiotherapy of brain metastases from 

solid tumors. The neurotoxicity and influence of fraction 

size and total do se of radiation therapy on the brachial 

plexus in breast cancer patients was investigated. A study 

was started on the relevance of the mode of treatment, of 

prognostic factors, and of cerebrospinal fluid tumor 

markers in patients with meningeal involvement of non 

Hodgkin's lymphoma. We studied the value of clinical 

data and of radiologic findings as parameters and 

prognostic factors in the course of the disease of patients 

with recurrent epidural metastases. In collaboration with 

the Or Daniel den Hoed Cancer Center, Rotterdam, 

prospective comparative studies are being designed on the 

efficacy of systernic therapy versus radiation therapy in 

brain metastases from breast cancer. 

Soft Tissue Sarcoma W orking Group 

Chairman: F van Coevorden 

The availability of Magnetic Resonance Imaging 

(MRI) in our institute has contributed greatly to adequate 

imaging of most primary soft tissue sarcomas. The 

introduction of PET scanning is a new valuable tooI, 

especially in the evaluation of recurrence of soft tissue 

sarcomas. 

The neo-adjuvant study for operabie soft tissue 

sarcomas, comparing preoperative chemotherapy 

followed by surgery + radiotherapy with surgery + 

radiotherapy is still ongoing. 

The study comparing Adriamycin/lfosfarnide with 

higher dose and GM-CSF in advanced disease is about to 

be closed. New phase II studies are to be started. Our 

experience with regional perfusion of extrernity sarcomas 

with Tumor Necrosis Factor (TNF) is expanding, 

showing miscellaneous results, which will be evaluated in 

an international collaborative prospective study. 

Retrospective AvL-studies on head and neck sarcomas 

and lymphangiosarcoma are concluded and accepted for 

publication. Studies on the value of pulmonary 

metastasectomy as single and repeated procedures, in 

collaboration with the Dr Daniel den Hoed Cancer 

Center, Rotterdam, have been concluded and published. 

This experience has been expanded into an international 

study, which will be the basis for a new EOR TC study for 

patients with pulmonary metastases. A retrospective 

study on PNET tumors and extraossal Ewings sarcomas 

is ongoing. These tumors will be the subject of a new 

international study in collaboration with the EORTC. On 

November 11 , a symposium was organized, in 

collaboration with the Amsterdam Comprehensive 

Cancer Center, on sarcomas in the he ad and neck region. 

U rological Tumors W orking Group 

Chairman: S Horenbias 

The analysis of the results on the treatment of 

squamous cell carcinoma of the penis was finalized and 

resulted in a thesis describing a treatment protocol that 

was introduced more or less nation wide. Further 

attention on the etiology of squamous cell carcinoma of 

the penis has resulted in a study focusing on the role of 

HPV in penile cancer and quality oflife issues in long term 

survivors. Both studies have a multidisciplinary character 

involving the Department of Gynecology, Molecular 

Pathology and Psychosocial Research and Epidemiology. 

The open procedure for interstitial radiation therapy of 

localized prostatic carcinoma has been replaced by an 

uItrasound guided method on the basis of the poor 

results. The analysis was finalized and will be published 

this year. The first analysis of interstitial radiation 

therapy of muscle infiltrating in bladder cancer was 

published and showed excellent results in terrns of local 

con trol and overall survival. Within the framework ofthe 

EORTC and a phase II clinical study, the work on neoadjuvant 

chemotherapy in advanced bladder cancer is 

rapidly progressing. Increasing experience has been

gained with urinary reconstruction af ter total cystectomy. 

These rewarding reconstructions have become the first 

choice after cystectomy instead of a urinary diversion 

with an extern al drainage bag. The European Study on 

neo-adjuvant hormonal treatment in locally confined 

prostate cancer is progressing well with a good accrual 

from our institute. Further analysis of the results of 

treatment of various stages of testicular cancer is in 

progress. 

Clinical Working Parties 153

Ongoing Trials 

in the Netherlands Cancer Institute 

AvL 

EORTC 

WHO 

IKA 

NJOSG 

AZVU 

NCI 

IKMN 

IKO 

IKN 

EORTCPAM 

LM 

HOVON 

ECTG 

NDDO 

CKVO 

: Antoni van Leeuwenhoek Ziekenhuis (Hospital ofthe Netherlands Cancer Institute) 

: European Organization for Research and Treatment of Cancer 

: World Health Organization 

: Integraal Kankercentrum Amsterdam 

: Netherlands Joint Ovarian Cancer Study Group 

: Academisch Ziekenhuis Vrije Universiteit 

: National Cancer Institute (Bethesda) 

: Integraal Kankercentrum Midden Nederland 

: Integraal Kankercentrum Oost Nederland 

: Integraal Kankercentrum Noord Nederland 

: Pharmacokinetics and Metabolism Group 

: Limited Multi-center study 

: Hemato-Oncology Foundation for Adults in the Netherlands 

: Early Clinical Trials Group (EOR TC) 

: New Drug Development Office 

: Commissie Klinisch Vergelijkend Onderzoek 

155

TYPE OF TITLE STUDYCOORDINATOR PHASE ACTIVATED NO PTS.IN COMMENTS VI 

CANCER IN AvL (in ita/ics: (CLOSED) AvL 

STUDY international coordinator) per 1. 9 .1994 

BREAST CANCER 

MTAMOX Adjuvant therapy with Tamoxifen in JMV Burgers 111 06/1983 266 Study closed with 1662 

postmenopausal women with breast (11 / 1993) patients. Interim analysis 

ca neer stage 1-111 (ASCO 1994) showed no difference 

as yet between 1 or 3 

years of Tamoxifen and no 

survival benefit. Node positive 

and ER positive patients 

have a significantly higher 

DFS. 

EORTC 10853 Excision vs excision + R T in in situ ductal JA van Dongen 111 05 / 1986 127 Cooperation with similar 

carcinoma studies; 1988, 1991 and 

1994 DCIS Consensus 

Meetings where these studies 

have been discussed. 

EORTC 10861 Aminoglutethimide with or without PF Bruning 111 09/1986 85 Continues with two arms 

Hydrocortisone vs Hydrocortisone alone comparing aminoglutein 

patients with advanced breast cancer thimide with and without 

(post-menopausal) hydrocortisone. 

EORTC 10873 Breast conserving therapy for Paget's EJTh Rutgers 11 07/1988 15 Includes registration of 

disease of the nipple all ineligible patients 

M Paget of the nipple. 

EORTC Role of booster dose R T with breast H Bartelink 111 06/1989 339 

10882/22881 conserving therapy JH Borger 

EORTC 10901 Postoperative adjuvant chemotherapy PF Bruning 111 04 / 1991 54 Prospective study of bone 

followed by adjuvant Tamoxifen vs ni1 mineral density. 

for patients with operabie breast cancer 

-0\

EORTC 10920 An EOR TC randomized controlled 

phase In trial of short term intensive 

chemotherapy after radicallocal 

treatment of isolated locoregional 

relapse in breast cancer patients 

EORTC 10921 A multicenter randomized trial of dose 

intensive chemotherapy a primary treatment 

in a multimodality approach for 

locally advanced inflammatory breast ca 

EORTC 10923 Taxol vs Doxorubicin as first line 

chemotherapy in advanced breast 

cancer; a randomized phase IJ study 

with crossover 

M88BMB lnduction chemotherapy followed by 

intensification with hematopoietic stem 

cel! support as the initial treatment in 

subclavian node positive, stage T J _ 3 + 

Mo breast cancer 

M90MCB Treatment of meningeal carcinomatosis in 

breast cancer: the relevance of intraventricular 

chemotherapy 

M9lPRE Adjuvant chemotherapy with CMF vs 

observation in premenopausal patients 

with lymph node negative but morphometrically 

unfavourable breast cancer; 

a randomised phase III study ('PREMIS') 

M92TRA Taxol efficacy in patients with advanced 

breast cancer, refractory to anthracycline 

therapy 

EJTh Rutgers III 

PF Bruning 

PF Bruning IJ 

S Rodenhuis IJ 

W Boogerd In 

J Schornagel III 

WW ten Bokkel Huinink IJ 

09/1993 

06/1993 10 

09/1993 27 

06/1991 72 

04/1991 6 

08/1991 4 

07/1992 30 

(11/1993) 

Detailed studies of Quality 

of Life and cost are included. 

Comprises Quality of Life 

study. 

Surprising low response 

rate, in contrast with 

other literature. 

... 

VI 

-....l

TYPE OF TITLE STUDYCOORDINATOR PHASE ACTIVA TED NO PTS.IN COMMENTS 0\ 

0 

CANCER IN AvL (in ita/ics: (CLOSED) AvL 

STUDY international coordinator) per 1.9.1994 

AvLN93MIC Selective lymfadenectomy for detection OE Nieweg I/II 02/1994 29 Fast accrual, promising 

of micro metastases in melanoma patients results. 

LYMPHOMAS 

EORTC20872 Randomization between 2 schedules of SP Israëls 111 04/1989 9 Total 120 patients included. 

combination chemotherapy (Tirelli) for (04/1994) Introduction of GCSF is 

elderly patients in adapted dosage considered. 

EORTC 20884 Stage lIl-IV Hodgkin's disease: role of JMV Burgers 111 09/1989 16 389 registered by October 

iceberg R T in CR R Somers 1994, trial wiU continue 

for ± 2 years to collect 

500-600 patients with a 

CR of 60%. 300 patients 

will be randomized. 

EORTC20901 Intensive consolidation therapy with R Somers 111 04/1991 9 119 patients included, 32 

autologous bone marrow rescue after randomized in ABMT arm. 

standard chemotherapy for intermediate Peripheral stem cell support 

grade and high grade non-Hodgkin's allowed. 

lymphoma 

EORTC 20921 Fludarabine vs conventional combination R Somers 111 02/1993 6 

chemotherapy (CVP) in patients with 

stages 111 and IV low grade malignant 

non-Hodgkin's lymphoma; a prospective 

randomized controlled phase 111 trial 

EORTC 20931 Protocol H8 for a prospective controlled JMV Burgers 111 10/1993 6 Within 14 months 322 

trial in clinical stage 1-11 supradia- patients are registered. 

phragmatic Hodgkin's disease; evaluation The goal of 1200 patients 

of treatment efficacy and (long term) is achievable. 

toxicity in 3 different prognostic 

subgroups

EORTC 20932 Randomized investigation of the value JMVBurgers 111 09/1994 Admission procedures 

of radiotherapy in complete remis sion in collaborating centers are 

after chemotherapy (CR) for patients with still ongoing. 11 patients 

stage 11, lIl, IV non-Hodgkin's lymphoma regis tered. 

of intermediate or high grade histology 

M87PAR DHAP ± ABMT in relapsed NHL R Somers 111 08 / 1986 10 195 patients included, 94 

after CR (12 / 1993) randomized.55% response 

ra te after DAP. No 

significant difference 

between two arms. 

M92CAM A multicenter phase 11 study of E Rankin 11 05/1993 2 Effective therapy with 

Campath 1 H given three times a (12 / 1993) considerable toxici ty. 

week to patients with non-Hodgkin's 

lymphoma (including chronic 

lymphocytic leukemia) 

M94CUP A randomized trial comparing J Baars 111 08/1994 

the efficacy of chemotherapy with 

purged or unpurged autologous bone 

marrow transplantation in adults 

with po or risk relapsed follicular NHL 

LMM88CBE High dose chemotherapy ± ABMT for R Somers 11 08/1988 7 

= Hovon-8 early relapse M Hodgkin 

GYNECOLOGICAL TUMORS 

EORTC 55852 CAP for advanced tuba cancer WW ten Bokkel Huinink 11 10/ 1987 3 Slow accrual; rare tumor. 

EORTC 55863 BEMP vs CDDP for metastatic WW ten Bokkei Huinink 11 03 / 1987 12 Still open for accrual. 

cervix cancer 

EORTC 55874 Adjuvant R T for uterine sarcomas B van Bunningen 111 02 / 1989 3 Slow accrual; rare tumor. 

LMM90HPV Prognostic value of HPV in ahnormal T Helrnerhorst cohort 06 / 1990 351 Fast accrual, long lasting 

cervix cytology study, collaborative study 

with Free University. 

..... 

0'1

TYPE OF TITLE STUDYCOORDINATOR PHASE ACTIV ATED NO PTS.IN COMMENTS 0\ 

N 

CANCER IN A v L (in italics: (CLOSED) AvL 

STUDY international coordinator) per 1. 9 .1994 

M90END The value of postoperative radio- B van Bunningen 111 09 / 1990 6 Slow accrual in our hospital, 

therapy in PTI endometrial carcinoma elsewhere better. 

M9IEPO Controlled multicenter study of the WW ten BokkeI Huinink 111 10/ 1991 8 Erythropoietin proved to be 

influence of subcutaneous rh-EPO therapy (10/1993) highly active for prevention 

on the development of anemia and trans- of anemia and reduction of 

fusion dependency in patients with ovarian transfusion dependency. 

cancer treated with platinum based 

combination chemotherapy 

M92CTG Dose finding study of the combination WW ten BokkeI Huinink 01/ 1993 27 Surprisingly high doses of 

of (high dose) Carboplatin and Taxol Carboplatin and Taxol can 

in escalated doses, with G-CSF to protect be combined. Satisfactory 

against myelosuppression, in patients anti-tumor activity. 

suffering from ovarian cancer, stage TIl 

(Bulky disease) IV (figo) 

M93HYP Combined weekly systematic Cisplatin T Helmerhorst 11 06/1993 3 ECC study. 

and local deep hyperthermia for patients 

with a recurrence of cervical carcinoma 

a phase 11 study 

M93TAO A phase 11 of Tallimustine with advanced WW ten Bokkel Huinink 11 10 / 1993 9 Still open for late recurrent 

epithelial cancer of the ovary, persistent or patients. 

recurrent after platinum based chemotherapy 

M94039 An open label multicenter randomized WW ten Bokkel Huinink 11 08 / 1994 2 Follow-up study of 

phase 111 study of Topotecan as single N93TOP. Topotecan is 

agent second line therapy (intravenously, expected to be as active 

as 5 daily doses every 21 days) vs Taxol as Taxol. 

(administered as a three hour infusion 

every 21 days) in women with advanced 

epithelial ovarian cancer 

.......

AvL N93TOP A phase II study of intravenous Topotecan WW ten Bokkei Huinink 

given as five daily doses every 21 days in 

advanced epithelial ovarian cancer 

URO-GENITAL TRACT TUMORS 

EORTC 22923 Accelerated radiotherapy with Carbogen L Moonen 

and Nicotinamide, a phase IIII study 

EORTC 30895 BOP/VIP vs BEP/EP ± G-CSF in poor JH Schornagel 

prognosis advanced testicular cancer 

and extragonadal cancer 

EORTC 30924 A randomized phase II trial in advanced JH Schornagel 

urothelial tract tumors of high dose 

intensity M-VAC chemotherapy + GCSF 

vs classic M-VAC 

LMM89SUR Suramine for advanced pro state cancer S Horenbias 

M93LAK A phase III study of Interleukine II EM Rankin 

(IL2) + alpha Interferon (ex IFN) + 

autologous IL2 activated lymphocytes 

(LAK) vs IL2 + ex IFN in patients with 

metastatic renal cell carcinoma 

M94SAL Phase II study of a salvage regimen S Rodenhuis 

incorporating repeated ablative chemotherapy 

with autologous peripheral 

stem cell reinfusion in relapsing germ 

cell cancer 

AvL N91LPC Dose finding study for large prostatic J Lebesque 

cancer, utilizing the simultaneous boost 

technique 

II 08/1993 

(03/1994) 

IIII 12/1993 

III 06/1990 

(06/1994) 

II 11/1993 

II 06/1990 

(04/1994) 

III 01/1994 

II 09/1994 

09/1992 

11 

10 

6 

12 

8 

6 

48 

Straightforward phase II 

study. Responses 

documented 

In collaboration with Free 

University. 

Collaborative, national study. 

Dutch national study 

based on NCI pilot study. 

....... 

0\ 

l;J

M91RBL Feasibility study of high dose radio- C Schaake-Koning 

(08912) therapy with concomitant boost technique P Baas 

combined with daily Cisplatin in non-smal1 

celllung cancer 

M92TCL Dose finding and sequence study of Taxol WW ten BokkeI Huinink 

and Carboplatin in patients with NSCLC PBaas 

clinical and pharmacologic study ECC, N van Zandwijk 

Amsterdam 

M94DOC A multicenter, open study ofDocetaxel WW ten Bokkei Huinink 

in the treatment of patients with 

advanced or metastatic breast or nonsmall 

cel1 lung cancer 

M94RBL Feasibility study of high dose radio- C Schaake-Koning 

therapy with concomitant boost technique 

combined with daily Cisplatin in non-small 

celllung cancer 

AvLN89MOL MICE chemotherapy for NSCLC S Rodenhuis 


AvLN90ILC Immunoscintigraphy with monoclonal BKwa 

antibodies in patients with SCLC N van Zandwijk 

SOFT TISSUE/ OSTEOSARCOMA 

EORTC 62874 Neoadjuvant chemotherapy for operabie E Gortzak 

soft tissue sarcoma with high risk factor 

EORTC 62901 Randomized phase 11 study comparing R Somers 

Adriamycine vs two schedules of high 

dose Epirubicin in advanced soft tissue 

sarcoma 

pilot 12/1991 

(08/1994) 

I 0111993 

(05 / 1994) 

11 08/1994 

pilot 10/1994 

11 03/1990 

(06/1994) 

04/1992 

(06/1994) 

11/ 111 07/1988 

11 06/1991 

30 

10 

4 

86 

7 

20 

4 

Moderate anti tumor activity 

documented. Highest dose 

level reached. 

Compassionate use more 

than study. 

Combined treatment is 

feasible with acceptable 

toxicity. A stepwise dose 

escalation ± Cisplatin wil1 

be carried out, then ph ase 

111 proposal will follow. 

Ras mutations do not 

predict drug resistance. 

Presently second dose level. 

...... 

0\ 

VI

TYPE OF 

CANCER 

TITLE STUDYCOORDINATOR 

IN AvL (in ita/ics: 

PHASE ACTIV ATED NO PTS.IN 

(CLOSED) AvL 

COMMENTS 0\ 

0\ 

STUDY international coordinator) per 1.9.1994 

EORTC 62903 Randomlzed study comparing conventional R Somers 111 06/1992 19 Good accrual, no undue 

do se Doxorubicin plus IfosfamÏde vs high toxicity in GMCSF arm. 

dose Docorubicin plus Ifosfamide plus 

GM-CSFin the treatment of advanced soft 

tissue sarcomas in adults 

EORTC 62931 RandomÏzed trial of adjuvant chemotherapy FC van Coevorden 111 04/1994 

with high dose Doxorubicin, IfosfamÏde and (05 / 1994) 

GM-CSF in high grade soft tissue sarcoma 

EORTC 62932 Phase 11 study of daily oral Etoposide 11 03 / 1994 3 

(VP16-213) as second line treatment in 

advanced soft tissue sarcomas 

M93TGM Isolated perfusion of the limb for limb B Kroon 11 03/1993 4 TNF seems to be the first 

salva ge with TNFa, Gamma Interferon (11/93) drug active against sarcoma 

and Melphalan for irresectable soft tissue in isolated perfusion. 

sarcoma 

M93ESB A randomÏzed study for the treatment of R Somers 111 10/ 1993 

(CESS92) Ewings Sarcoma of the bone 

M941LP Isolated perfusion of the limb for limb B Kroon 11 05 / 1994 4 TNF seems to be the first 

salvage with TNFa and Melphalan for drug active against sarcoma 

irresectable soft tissue in isolated perfusion. 

sarcoma 

GASTRO INTESTINAL CANCER 

EORTC22861 R T + 5FU + Mitomycin C for T 3-4 H Bartelink 11 10/ 1986 6 

N0-3' or T 1-2' N 1-3 anal cancer 

LGHDewit 

LMM89SGC Extended lymph node dissection in EJTh Rutgers 111 09/1989 5 A large national trial to 

gastric cancer (02/1994) compare standard R-1 

gastric resection vs the 

extended R-2 resection. 

--

M90LEV 5FU / Levamisole for 12 months vs no FAN Zoetmulder 

treatment following curative locoregional 

therapy in colon + rectal 

carcinoma, Dukes Band C 

M921CI Open phase 11 trial oflCI D1694 JH Schornagel 

in patients with advanced colorectal 

cancer 

M93POC Preoperative chemotherapy in opera bIe BGTaal 

gastric cancer F van Coevorden 

M93TOM An open randomized multicenter trial JH Schornagel 

ofTomudex vs 5-FU and Leucovorin 

in patients with advanced colorectal 

cancer 

M94LAN Evaluation of the efficacy of Lansoprazole BGTaal 

for prophylaxis of oesophageal ulceration 

during radical radiotherapy for oesophageal 

cancer: a double blind randomized trial 

AvLN90LUM Intraluminal irradiation for oesophageal BGTaal 

cancer 

AvLN90IBG MIBG in carcinoid syndrome BGTaal 

AvLN94DGT Dynamic graciloplasty technique F Zoetmulder 

functional reconstruction to 

achieve anal continenee after 

abdominoperineal resection (APRE) 

for rectal cancer 

111 06/1990 

11 01/1993 

(10/1993) 

111 05/1994 

111 02 / 1994 

(06 / 1994) 

111 08/1994 

11 05 / 1990 

I 05/1990 

05 / 1994 

22 

12 

7 

152 

18 

0 

National trial with slow 

patient accrual. 

A national multicentre trial. 

The combination of HDR 

and EBR T is effective in 

80% leading to 

improvement of 

dysphagia. 

Preliminary results: 

objective response in 60% 

with mild - moderate 

side effects. 

..... 

0\ 

-.....l

TYPE OF TITLE STUDYCOORDINATOR PHASE ACTIV A TED NO PTS.IN COMMENTS 0\ 

00 

CANCER IN A v L (in ita/ics: (CLOSED) AvL 

STUDY international coordinator) per l.9.1994 

HEAD AND NECK TUMORS 

EORTC 22851 Accelerated fractionation in the RKeus 111 04 / 1985 10 

radiotherapy of advanced he ad and FJM Hilgers 

neck carcinoma 

EORTC 24871 Intensive screening and/ or chemo- N van Zandwijk 111 07/1988 118 More than 2600 patients 

Euroscan prevention with Vitamin A and/ or N- (08 / 1994) have been included. 

acetyl-cysteine of second primary cancer 

patients, curatively treated for carcinomas 

of the larynx, oral cavity and lung 

EORTC24881 Epirubicin and CDDP in advanced naso- PF Bruning 11 04/1989 Rare indication. 

pharynxcarcinoma 

EORTC 24911 A phase VII dose escalation study of JH Schornagel VII 09/1992 

Cisplatin with or without (Amifostine) 

in patients with head and neck cancer 

M9ICRT Accelerated fractionation in post- RKeus 111 11 /1991 6 

operative irradiation of head and 

neck squamous cell carcinoma 

M94CTN An open study to determine if a FBalm 11/1994 

combined oxygen and temperature tissue 

11 sensor is a reliable indicator of 

myocutaneous free lap viability postoperatively 

AvLN87RTG Low dose vs high dose R T for early glottic RKeus 111 07/1988 139 

CKVO 87-11 cancer H Bartelink

AvLN90HME The effect of a Heat and Moisture FJM Hijgers 11 06 / 1990 60 In collaboration with the 

Exchanger (HME) on physical and (10/1994) Daniel den Hoed Clinic, 

psychological consequenses of a Rotterdam and Dijkzigt 

totallarynqectomy Hospital Rotterdam. 

AvLN90IMA Immunotargeting with monoclonal anti- FBalm 11/1990 5 Slow accrual because of 

body 175F4 in head and neck squamous cell (10 / 1994) absence of investigator 

carcinoma for few months. 

AvLN91RSM Radionuclide serial monitoring of salivary RKeus 08/1991 28 

function after radiotherapy in head and 

neck cancer 

AvLN91AFL Radiotherapy for T 2B and T 3 laryngeal RKeus 10/ 1991 2 

carcinoma: accelerated fractionation (10 / 1990) 

ECTG Trials 

16912 B Phase 11 trial with Rhizoxin in patients WW ten Bokkei Huinink 11 03 / 1993 5 Rhizoxin is a new spindie 

with advanced breast cancer poison with higher potency 

than Vincristin. 

16912 H Phase 11 trial with Rhizoxin in patients WW ten Bokkei Huinink 11 03 / 1993 

with squamous cell carcinoma of the head (0111994) 

and neck 

16912 M Phase 11 trial with Rhizoxin in patients WW ten Bokkei Huinink 11 03 / 1993 7 No activity observed. 

with malignant melanoma (11 /1993) 

16912 N Phase 11 trial with Rhizoxin in patients WW ten Bokkei Huinink 11 03/1993 Activity found elsewhere. 

with non-smalllung cancer (10/1993) 

169210 Ph ase 11 trial with Taxotere in WW ten Bokkei Huinink 11 06/1992 23 Activity documented, 

patients with advanced ovarian cancer (07 / 1994) probably equal or better 

than Taxol. 

16922 S Phase 11 trial with Topotecan in N van Zand wijk 11 02/1993 4 

patients with small celllung cancer 

16923 P Phase 11 trial with CPT -11 in patients WW ten Bokkei Huinink 11 07/1992 2 Moderate activity 

with adenocarcinoma of the pancreas (12 / 1993) documented. 

16931 B Phase 11 trial with E09 in patients with WW ten Bokkei Huinink 11 08 / 1994 Already temporarily closed. 

breast cancer 

-0\ 

'-0

AvL N90C95 Project on immunotargeting with EJTh Rutgers pilot 06 / 1990 23 Testing program of 8 

(N90IMM) monocIonal antibodies in carcinoma (08 / 1994) different monoclonal 

patients antibodies for their 

biolocalizational properties 

in patients with operabIe 

breast and colon cancer. 

AvLN92ITP Protocol for testing C186 IgG (175F4) EJTh Rutgers 02 / 1993 6 

radiolabeled monoclonal antibody in 

breast and lung cancer patients 

AvLN92APH Feasibility study of a triple auto- S Rodenhuis pilot 02/1993 13 Attempt to develop a 

transplant regimen employing autologous (0111994) potentially curative 

peripheral hematopoietic stem eell and regimen for advanced 

CTC chemotherapy breast cancer, ovarian 

cancer and germ tumors. 

AvLN92IgG Protocol for testing C113 IgG (AF20) EJTh Rutgers 02/ 1993 6 

radiolabeled monocIonal antibody in 

lung and colon cancer patients 

AvLN92FEN Transdermal Fentanyl for the treatment APE Vielvoye-Kerkmeer 04/1994 24 

of chronic cancer pain in patients C Mattern (12 / 94) 

not yet using strong narcotics analytics 

AvLN93APD The duration of metabolic effect of PF Bruning pilot 09/1993 5 Study closed: metabolic 

intravenous APD on the breakdown of (04 / 1994) effects of single i.v. 

bone matrix in postmenopausal women: dose of 60mg APD last 

a pilot study for at least 8 weeks. 

AvL N94C95 Protocol for testing C95IgG radio- EJTh Rutgers pilot 08/1994 

labeled monoclonal antibodies in 

breast and colon cancer patients 

A phase I study of intraperitoneally A Westerman 10/1994 

AvL N94IAS 

administered Surarnin in patients with WW ten Bokkei Huinink 

cancer localized in the abdorninal cavity 

-....l 

I-'

Ongoing Trials In Children's Hospital 'Emma 

Kinderziekenhuis/het Kinder AMC' 

SlOP 

: Société Internationale d'Oncologie Pédiatrique 

ENSG : European Neuroblastoma Study Group 

GPO 

: Gesellschaft für Pädiatrische Onkologie 

SNWLK : Stichting Nederlandse Werkgroep Leukemie bij Kinderen 

EORTC : European Organization for Research and Treatment of Cancer 

SFOP : Société Française d'Oncologie Pédiatrique 

EKZ 

: Emma Kinderziekenhuis (Emma Children's Hospital) 

EKZ/ AM : Emma Kinderziekenhuis / het Kinder AMC 

(Emma Children's Hospital / Children's Academic Medical Centre) 

CLB 

: Centraal Laboratorium van de Bloedtransfusiedienst 

CESS : Cooperative Ewing Sarcoma Study 

IRGPNCL : International Research Group Psychosocial and Neurological functioning in Childhood Leukemia 

173

TYPE OF 

CANCER 

TITLE STUDY COORDINATOR PRASE 

IN EKZ (in ita/ics: inter- 

ACTIVATED NO PTS. IN COMMENTS 

(CLOSED) EKZ/TOTAL 

-...l 

+:- 

STUDY national coordinator) per 1.10 .1994 

NEUROBLASTOMA 

ENSG-IV Retinoic acid in disseminated J de Kraker 111 11986 10/109 Randomized ENSG-study. 

neuroblastoma patients in CR 

LNESG94.01 Phase 11 trial of surgery as J de Kraker 11 12/94 No data available. 

the only treatment for IMSS 

stage 2 neuroblastoma 

EKZIAMC 1311-MIBG 'de Novo' in advanced J de Kraker 11 04/1989 31/34 Ongoing study with response 

neuroblastoma PA Voûte in preoperative phase as main 

object. 

EKZIAMC HB0 2 and l3l1-MIBG in recurrent PA Voûte 11 01/1989 unknown No data available. 

neuroblast oma J de Kraker 

AJ van der Kley 

EWING SAR COMA 

EICESS 92 Randomized study with chemotherapy PA Voûte 111 07/1992 10 No data available. 

first treatment in Ewing Sarcoma J de Kraker 

NON-HODGKIN'S LYMPHOMA 

SFOP-LMB-86/89 Non-randomized study in children H Behrendt 11 05/1986 34 No data available. 

with Burkitt-type lymphoma and CNSinvolvement 

and children with B-ALL 

EKZIAMC Treatment protocol for children H Behrendt 11 07/1989 unknown No data available. 

with T-cell ALL/NNHL 

LEUKEMIA 

SNWLK-ANLL 87 BFM like protocol without maintenance RS Weening 111 01/1989 25 Preliminary data: 

therapy 10/1992 no difference in EFS. 

-

SNWLK-ALL-VIII Randomized study in children with RS Weening 

high-risk ALL 

SNWLK-ALL- Non-randomized study by fust RS Weening 

Recidief-90 recurrence in children with ALL 

SNWLK-ANLL-92 Non-randomized study in children with RS Weening 

(pilot) ANLL with bone marrow transplantation 

SNWLK-ANLL-94 Non-randomized study in children with RS Weening 

ANLL with bone marrow transplantation 

SNWLK-NHL-94 Non-randomized study in children and RS Weening 

adolescents with Non-Hodgkinlymphoma 

NEPHROBLASTOMA 

SlOP 93-01 A therapeutic trial and prospective J de Kraker 

study PA Voûte 

FOLLOW-UP TRIALS 

NEPHROBLASTOMA 

SlOP 9 A therapeutic trial and prospective PA Voûte 

study J de Kraker 

RHABDOMYOSARCOMA 

MMT89 A prospective study on RMS and other PA Voûte 

malignant mesenchymal tumors 

MEDULLOBLASTOMEN 

PNET 111 Randomized study with chemotherapy PA Voûte 

for the treatment of central nervous J de Kraker 

system primitive neuroectodermal tumor 

in childhood. 

111 10/ 1991 

111 01/1992 

111 10/1992 

111 10/1994 

111 02/1994 

111 01/1993 

111 12/ 1987 

06/1993 

111 03/1989 

111 01/1992 

36 

10 

2 

0 

unknown 

11/162 

52/ 812 

62 

17 

No data available. 





Data collecting in progress. 

No difference in stage 

distributon between 4 and 8 

weeks of pre-operative 

chemotherapy. 

Intensity of therapy balanced 

against prognosis and quality 

of life. 


-..l 

VI

TYPE OF 

CANCER 

TITLE STUDY COORDINATOR PHASE 

IN EKZ (in ita/ics: inter- 

ACTIV A TED NO PTS. IN COMMENTS 

(CLOSED) EKZ1 TOTAL 

...... 

-.J 

0\ 

STUDY national coordinator) per 1.10.1994 

BLASTOMA/ SARCOMA 

EKZ/ AMC GM-CSF in pediatrie oneology patients RS Weening 111 11/1991 11 No data yet. 

HEPATOCELLULAR CARCINOMA/ HEPATOBLASTOMA 

SIOPEL 1 A prospeetive pediatrie liver tumor J de Kraker 11 01 / 1990 6 The value of the combination 

study (01/1994) Adrial eisplatinum for the 

liver tumor at the juvenile age. 

NEWDRUGS 

EKZ/ AMC A phase 11 trial with Rhizoxin in J de Kraker 11 01/1993 10 / 10 Data eolleetion in progress. 

ehildren with malignant neoplasma N Langeveld (01 / 1994) 

PSYCHOSOCIAL ONCOLOGY 

lKA 91-30 The effect of attention and memory AMH van Veldhuizen 111 06/1991 60 / 60 Data eolleetion in progress. 

training in ehildren with ca neer BF Last 

lKA 91-31 Double protection as a con trol BF Last 111 06/1991 463 Data evaluation in progress. 

strategy in ehildren with eaneer AMH van Veldhuizen (03/1994) 

AMC 94-880 A school and soeial reintegration BF Last 111 0111994 10 

program for ehildren and adoleseents 

with newly diagnosed eaneer

Education in Oncology 

Education in fundamental and clinical research and 

training in patient care are regular activities of the 

institute. Seven senior staff members have joint 

appointments and lecturing responsibilities as professors 

at several Dutch Universities. The research departments 

attract many undergraduate students from Dutch 

universities and fr om abroad. Graduate students, visiting 

guests and undergraduate students participate in the 

scientific projects. Post-doctoral teaching and training in 

clinical oncology and patient care is the major 

educational task ofthe cancer hospital. The collaboration 

with the medical faculties and hospitals of the two local 

universities increases continuously by shared activities of 

their graduate schools and joint projects in clinical and 

fundamental research. 


The undergraduate program in experimental oncology 

attracts students of all national universities in partial 

fulfillment of the obligations of subphase-3 of their 

curriculum. Most students have a background in Medical 

Biology or Health Sciences. The undergraduate program 

offers combined practical and theoretical training in 

various aspects of experimental oncology. Practical 

training includes participation in ongoing research 

projects for a minimum period of 4 months, after which 

the students deliver oral and written accounts of the 

results obtained. The core-element oftheoretical training 

is the lecture course in experiment al oncology, given twice 

yearly by staffmembers ofthe institute. These courses are 

attended by over 100 students, many of them being 

auditors from Amsterdam universities, and is much 

appreciated for giving an accurate account of recent 

developments in basic oncology. The trend of a 

decreasing number of undergraduate students, noticed in 

previous years, has not continued. In 1994 about 50 

students were associated with the research departments 

for a median 6 month period, approaching the maximum 

capacity of many departments to accomodate them. 

All teaching activities are supervised by a Teaching 

Committee. Specialized tasks are assigned to individual 

members of the scientific staff. These tasks include the 

Table 1 

February: 

September: 

November: 

177 

organization of lecture courses, guidance of scientific 

visits and the distribution of information regarding 

undergraduate studies at the NKI. 

Information undergraduate school: Teaching Committee 

NKI; secretariat H6, phone 020-5122035. Dean: Prof. L 

Smets. 

Graduate students 

About 60 PhD students are member of our Graduate 

School. Of these, the majority are scientists-in-training, 

who receive complementary theoretical and practical 

training covered by a 4-year contract. At the end of this 

period, the student can acquire the PhD degree from any 

national university. The tutorship for these graduate 

students is covered by the graduate school, headed by 

Prof. R Plasterk. The graduate school has been formerly 

integrated into the Oncology Graduate School 

Amsterdam, a joint activity with the medical schools of 

the Free University and the University of Amsterdam, for 

which a Faculty has been created. This was celebrated in 

May during a symposium held in the Tropenmuseum. An 

increasing number of graduate students from the 

Amsterdam Universities participated in the yearly 

meeting of all NKI graduate students, a 3-days retreat 

during which they present the highlights of their ongoing 

investigations. In 1994, 3 combined practical and 

theoretical courses we re organized. Scientists of 

international reputation in the respective are as we re 

invited to teach at these courses. 

Information graduate school: Prof. R Plasterk, Dean. 

Phone 020-5122081. 

Postdoctoral training 

Course at the NKI Graduate School- 1994 

Signal Transduction 

Model Organisms in Cancer Research 

Transcription Con trol 

The NKI research department continually hosts a 

number of postdoc fellows and trainees from the 

Netherlands, the European Cancer Center, Amsterdam 

or abroad. Staffmembers lecture on various postdoctoral 

training courses such as the Elementary Course in 

Oncology of the Dutch Oncology Society and various 

Division of Cellular Biochemistry 

Division of Molecular Biology 

Division of Molecular Carcinogenesis

178 Education in Oncology 

courses and seminars organized (tabIe 1) by the European 

School of Oncology. Moreover, the NKI is co-organizer 

of national, postdoctoral 'Oncology Days' for clinical 

oncologists . . 

Teaching for medical students and doctors in 

training 

Staff members of the institute lecture on regular 

undergraduate courses in fundamental and clinical 

oncology at the medical schools of the two Amsterdam 

Universities. Several departments of the University 

Hospitals have residents staged for periods of six months 

at the Cancer Institute, as part of their training in 

medicine, surgery or radiotherapy. Weekly courses in 

oncology are given to the residents at the institute. Special 

seminars in clinical oncology are given on Wednesday 

afternoons. 

Cancer Nursing Education 

Twice a year the NKI organizes a core curriculum postbasic 

course in cancer nursing for internal and external 

candidates. The course educates nurses in the 

development of cognitive, psycho-motorical, interactive 

and reactive skilIs in order to practice their profession 

with confidence and professionalism. The course contains 

theoretical and practical components. The theoretical 

tuition consists of 140 hours and is concluded with a 

written nursing thesis and an oral exam. The practical 

'on-site' training consists of 11 months during which the 

student encounters all aspects of oncology nursing, 

including diagnostic, curative, palliative and terminal 

care and the care of patients who participate in Clinical 

Research Trials. At present the co re curriculum is being 

revised. 

The Integral Cancer Center Amsterdam, in 

cooperation with the NKI, organized various postgraduate 

refresher courses in the treatment and care of 

cancer patients. 

Information post-basic course in cancer nursing: T van 

Rees, head education department. Phone 020-5122457. 

Post-graduate courses 

The Department of Radiotherapy has a fun training 

program for radiotherapists and radiographers. Foreign 

radiotherapists are regular guests at the department, as 

wen as residents from other hospitaIs. 

The Departments of Medicine and Surgery offer 6-12 

months elective residencies for internists and surgeons in 

training in cooperation with various universities. 

The Department of Pathology has a 3 month training 

program for residents in diagnostic cytopathology, 

including fine-needle aspiration cytology, in cooperation 

with the Universities of Amsterdam, Utrecht and 

Rotterdam. In addition, postdoctoral courses in 

diagnostic cytology and histopathology are regularly 

organized by this department. 

Every year a one-week course is held for a limited 

number of surgeons. The program involves 

demonstration of surgical techniques, demonstration of 

patients and discussion of new modalities in diagnosis 

and treatment. Regular half day courses on new 

developments in chemotherapy were also held for 

internists. 

Information clinical training: Prof. W Mooi, Dean. 

Phone: 020-5122751.

Clinical meetings 

Every Wednesday, meetings are held by the clinical staff 

to exchange information. Af ter a discussion of patients, 

a seminar is given by one of the staff members or by an 

invited speaker on a particular clinical topic. Subjects 

discussed in 1994 were: 

Baas P, Boot H 

Intraluminal therapy. 

Bartelink H 

Radiotherapy alone or in combination with 

chemotherapy in treatment of anal carcinoma, the 

results of an EORTC trial. 

BeggAC 

Predictive assays in radiotherapy. 

BosKE 

Reconstructive surgery, a profession with perspective. 

Burgers JMV 

The results of several EOR TC-trials for Morbus 

Hodgkin. 

De JongD 

Prognostic factors in large cell B-cell non-Hodgkin's 

lymphomas. 

Dercksen W 

Characterization of stem cells and its relation to 

peripheral stem cell transplantation. 

Gerritsen WR, Rankin EM 

Gene-therapy and the Somatix protocol. 

Kruisbeek AM 

Introduction to Division of Immunology. 

Meinhardt W 

Impotence 

Mooi WJ 

Introduction to Division of Tumor Biology. 

Mooienaar WH 

Introduction to Division of Cellular Biochernistry. 


Niël Ch 

Conformation therapy for head, neck and brain tumors. 

NiewegOE 

Sentinal node biopsy as staging method. 

Ogilvie AC, Rankin EM 

Effect of Interleukin 1 on coagulation and fibrinolysis; 

cytokine profiles. 

Peterse JL 

Ductal carcinoma in situ of the breast. A new 

classification; clinical consequences. 

Plasterk R 

Introduction to Division Molecular Biology. 

Rodenhuis S 

ASCO 1994 report back. 

Rookus MA, Van Leeuwen FE 

Oral contraceptives and breast cancer risk. 

RoosE 

Introduction to Division of Cell Biology. 

Schipper WJ, Snelder I 

The insurance of liability risk and the new legislation. 

Te Riele H 

Introduction to Division of Molecular Genetics. 

Van 't Veer LJ 

A simple yeast assay to screen large series of tumor 

biopsies for (onco-)gene activity. 

Verheij M 

Radiation-induced effects in human endothelial cells: 

biological and clinical aspects. 

Vielvoye-Kerkmeer APE, Mattern C 

Fentanyl-transdermal administration: mechanism; 

indications; prelirninary study results. 

Zoetrnulder FAN, Vos EJ 

First experiences with dynamic gracilioplastictechnique 

in restoring the continence aft er abdominoperineal 

resection for rectal carcinoma.


Invited speakers 

Barlow DP (Austria) 

Dissecting the role of gametic imprinting in mammalian 

development. 

Berns A (USA) 

Gene therapy as an approach to treating cancer. 

Bohmann D (Germany) 

Biochemistry and genetics of jun. 

BosJL 

Signal transduction by p21 ras. 

Brahme A (Sweden) 

Radiobiology based treatment plan optirnization. 

Braunhut SJ (USA) 

The effects of vitamin A on the differentiation of 

endothelial cells and its response on irradiation. 

Courtneidge S (Germany) 

Src farnily tyrosine kinases: their regulation and role in 

signal transduction. 

Damsky CH (USA) 

Expression of integrins during murine development. 

Dawid I (USA) 

The function of the Xlim-1 gene in mesoderm induction 

and axis formation in Xenopus. 

Ekblom P (Sweden) 

Role of laminin-1 in epithelial development. 

Featherston D 

Exploring the gen ome database (lecture and 

demonstration). 

Flügel R (Germany) 

Gene expres sion of the human spumaretrovirus and 

characterization of the foamy viral integrase. 

Freeman JL (Canada) 

Surgery of tumors of the skull base. 

Goitein M (USA) 

Incorporation of uncertainties in radiation therapy. 

Graham MV (USA) 

3D therapy for lung cancer: the Mallinckrodt 

experience. 

Haber D (USA) 

Functional properties of the Wilms tumor suppressor 

gene WTI. 

Harnilton J (Australia) 

Colony-stimulating factors and monocytes / 

macrophages. 

Hanahan D (USA) 

The switch to the hyperproliferative stage during 

tumorigenesis: IGF-U and the balance between cell 

proliferation and apoptosis. 

Heldin C-H (Sweden) 

Signalling through receptors for platelet-derived growth 

factor and transforming growth factor (J. 

Hellman S (USA) 

Thomas Hodgkin and Hodgkin's disease: learning fr om 

the patient then and now. 

Helms JB (USA) 

Regulation of intracellular protein transport by GTPbinding 

proteins. 

Henikoff S (USA) 

Heterochromatin and somatic chromosome pairing in 

Drosophila. 

Hochstenbach F (USA) 

Cell polarity in the fission yeast Schizosaccharomyces 

pombe. 

Jansen J (France) 

The t(15;17) translocation in acute promyelocytic 

leukemia; functional analysis ofthe PML-RARa hybrid 

protein. 

Johnson P (UK) 

Apoptosis and follicular lymphoma: does bcl-2 matter? 

Kanaar R (USA) 

Mechanism ofpre-mRNA splicing in Drosophila. 

Kutcher GT (USA) 

Conformal therapy at Memorial Sloan-Kettering: status 

and future. 

Laman J (USA) 

Role of CD 40 ligand during in vivo immune responses. 

Lapetina EG (USA) 

Signal transduction in platelets: role of small GTPbinding 

proteins. 

MacArthur J (USA) 

The role of CD-28 in the co-stimulation of murine 

helper T -cells. 

Marinus M (USA) 

DNA methylation and mismatch rep air in E coU. 

Marshak DR (USA) 

Casein kinase 11 and cdc2 in cellular growth con trol. 

Martens G 

A molecular chaperone in the regulated secretory 

pathway. 

Mebius R (USA) 

A developmental switch in the expression of homing

eceptors on lymphocytes and their ligands on high 

endothelial cells. 

Merel P (France) 

Cloning and characterization of the NF2 gene. 

Meyerson M (USA) 

Cyclin-dependent kinase 6 and cell cycle regulation. 

Mandi R (USA) 

Mammary carcinogenesis: a unified hypothesis. 

Osoba D (Canada) 

Conducting clinical trial-based quality-of-life studies: 

strategies for success developed by the National Cancer 

Institute Canada (NCIC). 

Peters P 

Arf affects plasma membrane recycling and coat 

assembly onto endosomes. 

Piantadosi S (USA) 

The history of clinical trials. 

Pignon JP (France), Stewart L (UK) 

A meta-analysis of chemotherapy in non-small celllung 

cancer using updated individual patient data. 

Pommier Y (USA) 

Inhibitors of HIV integrase. 

Pouysségur J (France) 

The role of MAP kinases in mitogenic signal 

transduction. 

Roncarolo M -G (USA) 

Mechanisms of tolerance in SCID patients and in SCID 

mice transplanted with human petal stem eells. 

Saito T (Japan) 

Molecular architecture and signaling of the T -cell 

receptor complex. 

Sarkadi B (Hungary) 

Functional studies on the human multidrug transporter. 

Schalken J 

Mechanisms of decreased E-cadherin function. 

Schnegelsberg P (USA) 

The role of myoD and Myf-5 during murine 

myogenesis. 

Sherouse G (USA) 

Understanding dosimetry in three dimensions. 

Shimizu T (Japan) 

MAP kinase activation through G-protein-linked 

receptors (PAF and somatostatin). 


Stuiver MH (USA) 

Proteins influencing specificity and binding of 6HLHproteins 

to the immunoglobulin enhancer. 

Treisman R (UK) 

Transcriptional regulation by growth factors. 

Trowsdale J (UK) 

A cluster of processing and presentation genes in the 

MHC. 

Van Aelst L (USA) 

The RAS signal transduction pathway in yeast and 

mammalian cells. 

Van de Rijn M (USA) 

EBV associated lymphomas. 

Van der Bliek A (USA) 

The role of dynamin in early endocytosis. 

Van Dijk M 

Pbx and Hox proteins, partners in development and 

carcinogenesis? 

Van Dijk M (USA) 

Drosophila extradenticle and the human Pbx proteins: a 

family of homeobox proteins that modulate the target 

specificity of homeotic gene products. 

Van Kooten C (France) 

Molecular interactions of T - and B-cells; the role of 

CD40-CD40L. 

Van Leeuwen F (USA) 

In vitro model for wingless signal transduction. 

Varlet I (France) 

DNA mismatch repair in Xenopus laevis egg extracts. 

Vasen HFA 

Screening for hereditary cancers. 

WeedaG 

Genetic analysis and generation of mouse models for 

human DNA-repair and Transcription Syndromes. 

Wong JW (USA) 

An integrated approach to accurate conformal radiation 

therapy. 

Wyman C (USA) 

Scanning force microscopy of nuc1eoprotein complexes. 

Ylänne J (Finland) 

Functions of the cytoplasmic domains of integrin c/'IIb/33.


Postgraduate courses 

Organizing committee: E Gortzak, F van Coevorden 

Once a year a one-week postgraduate course for surgeons 

is given at the Netherlands Cancer Institute, this year on 

March 7-11. In addition to live demonstrations ofvarious 

surgical procedures and outpatient demonstrations the 

following lectures on cancer treatment were given: 

Baas P, Rutgers EJTh 

Lung carcinoma. 

BalmAJM 

Oral and oropharyngeal cancer. 

Boot H, Kroger R, Zoetmulder FAN 

Liver tumors; diagnosis and treatment. 

Borger JH 

Breast conserving therapy. 

Bos KE, De Boer JB 

Reconstructive surgery. 

Dalesio 0 

Clinical trials: data-management. 

Delprat CC, Gortzak E 

Thyroid carcinomas. 

DewitLGH 

Radiotherapy, general principles. 

Gortzak E 

Soft tissue sarcomas. 

Gregor RT 

Cervical node metastases. 

Helmerhorst ThJ 

Gynecologicaloncology. 

Hilgers FJM 

Parotid gland tumors. 

Horenbias S 

Urologicaloncology. 

KroonBBR 

Regional chemotherapy. 

Kroon BBR, Rankin EM 

Malignant melanoma. 

NiewegOE 

Positron Emission Tomography. 

NiewegOE 

Sentinal node biopsy. 

Rodenhuis S 

Hormonal and cytotoxic treatment of breast cancer. 

Rutgers EJTh 

Tumor targeting with monoc1onal antibodies. 

Schornagel JH 

Chemotherapy, general principles. 

Schornagel JH 

Testicular cancer. 

Taal BG, Van Coevorden F 

Esophageal and stomach cancer. 

TimmersAP 

Prosthetic rehabilitation in maxillofacial surgery. 

Van Coevorden F 

Pulmonary metastases. 

Van der Eyken JW 

Oncological orthopedics. 

Van der Heijden-Schoon G 

Technical aspects in radiotherapy. 

Van Dongen JA 

Surgical oncology, general principles. 

Van Dongen JA 

Breast cancer; indication and surgical technique. 

Vielvoye-Kerkmeer APE 

Pain control in oncology. 

Zoetrnulder FAN 

Colorectal cancer. 

A course on oncology for general physicians was also 

organized (F van Coevorden). In 1994 prostatic cancer 

and lung cancer were the subjects of discussion. Mter 

introduction of the subject (by a general physician and 

specalists oncologists) a lively debate followed. 

Prostatic cancer (June 2) 

Gualthérie van Weezei LM 

Horenblas S 

Meinhardt WJ 

Moonen LMF 

Van Coevorden RS 

Lung cancer (December 8) 

BaasP 

Van Coevorden RS 

Van Zandwijk N 

Vogelenzang R

Research staff meetings 

Organizing committee: R Bemards, AM Kruisbeek, 

WH MooIenaar 

Once a month meetings are held at the Netherlands 

Cancer Institute at which staff members of each research 

division present results of their scientific work. The 

meetings have continued to be successful and have, as in 

previous years, enjoyed the attendence of numerous 

scientists from both inside and outside the institute. The 

meetings are also effective in improving contacts between 

members of the Research Laboratory and the Cancer 

Hospital. The schedule for 1994 was as follows: 

Division I (January 17) 

Kemperman H, Wijnands YM, Roos E 

The role of rx6fJ4 and rx5fJl integrins in adhesion of 

TA3 carcinoma cells to hepatocytes. Effect of the 

mucin epiglycanin. 

Delwel GO, De Melker AA, Niessen CM, Fles D, 

Kuikman I, Sonnenberg A 

Ligand specificities of the 11.3- and rx6-containing 

integrins. 

Habets GGM, Zuydgeest DATM, Scholtes E, 

Van der Kammen R, Collard JG 

Tiam-l, an invasion-inducing gene with homology to 

GDP-GTP exchangers for Rho-like and Ras-like 

proteins. 

Stam JC, Van der Kammen R, Oomen LCJM, 

Collard JG 

Characterization of Tiam-l-transfected NIH3T3 cells. 

Division 111 (February 21) 

Hordijk PL, Jalink K, Verlaan I, Van Corven E, 

MooIenaar WH 

New signaling pathways in the action of LPA, a 

platelet-derived rnitogenic lipid. 

Alblas J, Van Etten I, Khanum A, MooIenaar WH 

Cell transformation by a truncated G protein-coupled 

receptor. 

Gebbink MFBG, Zondag GCM, Wubbolts R, 

MooIenaar WH 

Characterization of RPTPI1, a receptor pro te in 

tyrosine phosphatase that functions in cell-cell 

interaction. 

Neefjes JJ, Momburg FI, Hämmerling GJI, Roelse J 

Early events in antigen presentation by MHC class I 

molecules. 

Division IV (September 12) 

Hooijberg E, Van den Berk P, Sein J, De Boer R, 

MeliefCJM2, Hekman A 

Preclinical studies on immunotherapy for B-cell 

lymphoma. 


Izon DJ, Eynon EE3, Jones LA4, Hamel ME, 

Kruisbeek AM 

Regulation of T -cell development: Contributions by 

costimulatory molecules on thymic stromal eells. 

Rinke de Wit TFS, Izon DJ, Platen burg pps, 

Bakker AQ, Van Ewijk WS, Kruisbeek AM 

Regulation of T -cell development: Expression of 

lymphoid tyrosine kinase genes in thyrnic stromal 

cells. 

Nieland JD, Haks MC, Schrauwers AJ6, Offringa R2, 

Rinke de Wit TFs, Kruisbeek AM 

T -lymphomas provide costimulation to naive T -cells 

through non-B7 related pathways. 

Staal F, Res P, Bakker A, Berns A7, Weijer K, Spits H 

New approaches to study human T-cell development. 

Division V (May 16) 

Pur as Lutzke R, Weber p8, Houghten R 8 , Plasterk R 

Identification of a hexapeptide fr om a synthetic 

peptide combinatoriallibrary which inhibits HIV 

integrase activity. 

Vaz Gomes A, Jongsma A, Riethorst B, De Waal T, 

ZasloffM9, Rosner 0°, Hendier Dll, Juretié Dil, 

Westerhoff H 

Synergism of magainin peptides: from 

physicochemical studies to biological action. 

Zaman G, Flens M12, Eijdems L, Van Leusden M, 

De Haas M, Mülder Sl3, Lankelma J13, Pinedo HM13, 

Scheper R12, Baas F, Broxterman H13, Borst P 

Mechanism of action and cellular location of MRP, a 

novel transporter protein associated with multidrug 

resistance of human tumor cells. 

Schinkel A, Smit J, Van Tellingen 0 14 , Beijnen J1S, 

Wagenaar E, Van Deemter L, Mol C, Van der Valk 

M16, Robanus-Maandag E16, Te Riele H16, Berns A16, 

Borst P 

The phenotype ofmdrla (P-glycoprotein) knockout 

mice. 

Division VI (October 10) 

Maas RA, Breedijk AJ, Top B, Peterse JL, Bruning PF 

Detection of proliferation-related mRNA levels in 

breast cancer cells as potential indicators of treatment 

response. 

Vos HL, Van Damme S, Bajramovic J, Storm J, 

Maas AJ, Atsma D, Van der Valk S, Boer M, 

Van Doomewaard G, Wesseling J, Patriarca C, 

Hageman Ph, Hilkens J 

Characteristics of episialin / MUCI expression in 

vivo. 

Balm AJM, Hilgers FJM, Gregor RT 

General aspects of head and neck cancer.


Michalides RJAM, Van Veelen N, Hart AAM, 

Loftus-Coll BM, Wientjens E, Balm AJM 

Cyc1in Dl overexpression as prognostic marker in 

squamous cell carcinomas of the he ad and neck. 

Ivanyi D, Balm AJM, Hageman PC, Van 

Doornewaard G, Groeneveld E, Atsma D, Mooi WJ 

Cytokeratin-18 expression in squamous cell 

carcinoma of the head and neck. 

Division VII (March 21) 

Acton D, Domen J, Berns A 

Prelymphomatous expansion of various B-cell 

populations in bone marrow from bcl-2-Ig transgenic 

mice. 

Jonkers J, Korswagen H, Breuer M, Berns A 

Fat-I, a gene involved in progression ofmurine 

lymphomas. 

Van der Lugt N, Domen J, Linders K, Van Roon M, 

Robanus-Maandag E, Te Riele H, Van der Valk M, 

Deschamps J, Sofroniew M, van Lohuizen M, Berns A 

The phenotype of mice lacking bmi-I. 

Alkerna M, Domen J, Van Lohuizen M, Berns A 

Overexpression of bmi-l in transgenic mice 

predisposes to lymphomagenesis and induces anterior 

transformation of the axial skeleton. 

Division VIII (November 14) 

Top B, Mooi WJ, Klaver S, Boerrigter L, Wisman P, 

EIbers HRJ, Visser S, Rodenhuis S 

PS3 alterations in non-small celllung cancer. 

Coco Martin JM, Ottenheim CPE, Bartelink H, 

BeggAC 

Prediction of human tumor radiosensitivity by 

chromosome damage using fluorescence in situ 

hybridization with whole chromosome pro bes. 

Van Geel I, Oppelaar H, Oussoren Y, Stewart FA 

Optimization of photodynamic therapy. 

Begg AC, Hofland I, Haustermans K1 7, Balm AJM, 

Shouman T1 8, Awwad HI 8 

Cell kinetics of human head and neck and 

oesophagus tumors: recent progess. 

Division IX (April 18) 

Fraass BAI9 

Three-dimensional treatment planning for conformal 

therapy. 

Essers M 

In vivo dosimetry during conformal therapy. 

Gilhuys K 

Patient set-up analysis in two and three dimensions. 

Borger J 

Dose and volume effects on fibrosis after breast 

conserving therapy. 

Division XI (December 19) 

Bos KE, De Boer JB, Balm AJM, Gregor RT, 

Hilgers FJM 

New developments in head and neck reconstructive 

surgery. 

De Boer JB, Bos KE, Rutgers EJTh, Van Dongen JA 

Mammary reconstruction: state of the art. 

Zoetrnulder FAN, Baas P 

Bronchopleural fistula af ter pneumectomy: treatment 

and prevention using de-epithelialized latissimus dorsi 

myocutaneous island flap. 

Nieweg OE, Kroon BBR, Baidjnath Panday RKL, 

Balm AJM, Rutgers EJTh, Muller S, Liem IH, 

Hoefnagel CA, Mooi WJ, Kapteijn BAE 

Lymphoscintigraphy, lymphatic mapping and 

sentinal node biopsy for the detection of 

micrometastases of melanoma. 

Division XII (June 6) 

Sneeuw KCA, Van Wouwe MCC, Sergeant JA20, 

Buunen I, Pastoor M, Braak S, Stokkermans M, 

Braas PAM, Van Tinteren H, Van Dongen JA, 

Bartelink H, Aaronson NK 

Screening for psychiatric disorder in patients with 

early stage breast cancer. 

De Boer JB, Hart AAM, Sprangers MAG, Cleijne W, 

Frissen PHJ21, Reiss p 22, De Jong M 22, Lange JMA22, 

Van Dam FSAM 

A longitudinal study of the quality of life of 

symptomatic HIV -infected patients (CDC IV) in a 

double blind trial of high versus low dose DDI. 

Noyon R, Van den Belt-Dusebout AW, Klokman WJ, 

Van Kerkhoff EHM, Peter se JL, Bontenbal M23, 

Somers R, Van Leeuwen FE 

Second cancer risk following breast cancer: a followup 

study of 6294 patients. 

Van der Kooy K, Rookus MA, Peterse JL, 

Van Leeuwen FE 

P53 overexpression in relation to risk factors for 

breast cancer. 

Notes 

I DKFZ, Heidelberg, Germany. 

2 Department of Immunohaematology and Bloodbank, 

University Hospital Leiden. 

3 Fellow American Cancer Society. 

4 Presently at Targeted Genetics, Seattle, WA, USA. 

5 Department of Immunology, Erasmus University, 

Rotterdam. 

6 Animal Department, Netherlands Cancer Institute. 

7 Division of Molecular Genetics, Netherlands Cancer

Institute. 

8 Torrey Pines Institute for Molecular Studies, San 

Diego, USA. 

9 Childrens Hospital of Philadelphia, USA. 

10 National Institute of Diabetes and Digestive and 

Kidney Diseases, NIH, Bethesda, USA. 

11 National Heart, Lung and Blood Institute, NIH, 

Bethesda, USA. 

12 Department ofPathology, Free University Hospital, 

Amsterdam. 

13 Department of Medical Oncology, Free University 

Hospital, Amsterdam. 

14 Department of Clinical Chemistry, Netherlands 

Cancer Institute, Amsterdam. 

15 Department of Pharmacy, Slotervaart Hospital, 

Amsterdam. 


16 Department of Molecular Genetics, Netherlands 

Cancer Institute, Amsterdam. 

17 University Hospital, St. Raphael, Leuven, Belgium. 

18 National Cancer Institute, Cairo, Egypt. 

19 Associate Professor and Director of Radiation 

Physics, Department of Radiation Oncology, The 

University of Michigan Medical School. 

20 Department of Clinical Psychology, The University of 

Amsterdam. 

21 Department of Internal Medicinel AIDS Unit, 

Academic Medical Center, University of Amsterdam. 

22 National AIDS Therapy Evaluation Center, 

Academic Medical Center, University of Amsterdam. 

23 Department of Internal Medicine, Dr Daniel den 

Hoed Cancer Center, Rotterdam.

Projects Financed Externally 

Projects supported by the Dutch Cancer Society 

Projects 187 

Number of Title Div Principal Starting Ended 

project investigator date 

NK190-3 Dosimetry, treatment planning and clinical IX B Mijnheer May '90 May '94 

feasibility studies on boron neutron capture RMoss 

therapy using a high intensity epithermal L Dewit 

neutron beam. 

NK190-7 Adhesion-involved cytoskeletal structures in ERoos May'90 Dec. '94 

normal and tumor cens. C Feltkamp 

NK190-8 A comparative study of bladder damage in mice VIII F Stewart July'90 June '94 

af ter photodynamic therapy or intravesical 

chemotherapy and their influence on irradiation 

tolerance. 

NK190-10 Therapeutic gain by boron neutron capture VIII A Begg Oct '90 Oct '94 

therapy (BNCT): an experimental study. R Huiskamp 

NK190-11 Analysis of pim-1 proto-oncogene VII A Berns May'90 May '94 

function: Biochemical characterization and 

mutational analysis in transgenic mice. 

NKI90-12 Cloning and characterization of onco- VII A Berns Febr '90 Febr '94 

genes involved in progression of lymphoid 

tumors. 

NK190-13 Cellular proliferation parameters during initial X I P Bruning June'90 Jan '94 

breast cancer treatment as prediction of clinical VI J Peterse 

response. 

NK190-16 Human DNA alkylation and tumor risk resulting 11 Lden June '90 June '94 

from therapeutic or environmental exposure to Engelse 

alkylating agents. 

NK190-17 Carboplatin-DNA adducts and tumor response in 11 Lden May'90 May '94 

patients with ovarian or cervical cancer. Engelse 

NK190-18 Radiation damage of lung and small bowel as a IX J Lebesque May'90 May '94 

function of dose and volume. R de Boer 

J Meerwaldt 

NK190-19 Optimalization of intraperitoneal chemotherapy VIII H Pinedo Apr '90 Nov '94 

with regional hyperthermia. 

NK190-20 The role of diacylglycerol kinase in cell 111 Wvan Febr '90 

signal transduction. Blitterswijk 

NK190-22 Predisposition to carcinogenesis by loss VII H te Riele Febr '90 Febr '94 

of function of the retinoblastoma A Berns 

susceptibility gene in mice. 

NKI90A Quality of life assessment in clinical oncology XII N Aaronson June'90 

research. F van Dam

188 Projects 

NK191-01 High-precision radiation therapy with on-line IX J Lebesque May '91 

treatment set-up verification. H Bartelink 

NK191-02 a-2,8 polysialylation of N-CAM and decreased VI R Michalides Jan '91 

intercellular adhesion in small celllung WMooi 

cancer. 

NKI 91-03 LFA-l and other adhesion molecules involved ERoos Jan '91 

in lymphoma metastasis. 

NK191-04 Proteins involved in invasion and metastasis ERoos Jan '91 

ofT-celllymphomas. 

NKI 91-05 Optimization of photodynarnic therapy: VIIV F Stewart June '91 

preclinical and clinical studies. X N van Zandwijk 

NK191-07 Oxidative base damage induced in DNA by 11 G West ra Apr '91 Nov '94 

radiation, carcinogens and cytostatics. 

NK191-08 Monitoring of gene expression in cancer of the VIII S Rodenhuis Febr '91 

lung and breast. WMooi 

NK191-13 Growth factor-like action of phosphatidic acids. 111 WMoolenaar Nov '91 

W van Blitterswijk 

NK191-14 Modification of tumor pH and cytostatic drug VIII L Smets Aug '91 

action. 

NK191-15 Identification of invasion-inducing genes by I J Collard Aug '91 

retroviral insertional mutagenesis. 

NK191-16 Involvement of episialin in progression of VI J Hilkens June '91 

carcinomas. 

NK191-17 Genetic factors in tumorigenesis: a new VII P Demant Febr '91 

system for identification and functional 

analysis. 

NKI 91-18 The mechanism of non P-glycoprotein mediated V P Borst May '91 

multidrug re si stance in a human squamous lung 

cancer cellline. 

NK191-19 A case-control study of risk factors for XII Fvan Dec '91 

contralateral breast cancer. Leeuwen 

M Rookus 

NKI 91-30 Quality of life assessment in cancer clinical XII M Sprangers Dec '91 

research: development and testing of N Aaronson 

questionnaires and administration procedures. 

NK191-260 The role of the 0:6/31 and 0:6/34 integrins in A Sonnenberg March'91 

tumor development and tumor progression. 

NKI 92-37 Gamma detection probe assisted surgery of in XI E Rutgers May '92 

vivo labeled colorectal carcinoma and melanoma 

with monoclonal antibodies. 

NKI 92-39 Receptor-like protein tyrosine phosphatases: 111 W Mooienaar Dec '92 

role in signal transduction and growth con trol. 

NK192-40 Three-dimensional dose distribution in patients IX B Mijnheer June '92 

irradiated with high-dose high-precision 

techniques.

NKI92-41 Analysis of P-glycoprotein function in mice with V P Borst Apr '92 

modified P-glycoprotein gene complement. 

NKI92-42 Lipid metabolism associated with growth factor- 111 Wvan May'92 

stimulated receptor protein-tyrosine kinases. Blitterswijk 

NKI92-43 Molecular events in receptor-mediated transport X J Schornagel Apr '92 

of folates and antifolates via a membrane- G Strous 

associated folate binding protein. 

NKI92-47 Prediction of radiosensitivity and repopulation VIII! A Begg Apr '92 

rates in human tumors. IX H Bartelink 

NKI92-48 Characterization of pal-I, a new oncogene VII A Berns Febr '92 

involved in lymphomagenesis. 

NKI92-51 Characterization and clinical evaluation of VI R Michalides March'92 

U21 B31, a gene associated with breast metastasis. J Peterse 

NKI92-52 Identification of genes that con trol J Collard July'92 

induction of invasiveness. 

NKI92-55 Adhesion molecules of human colon carcinoma ERoos Aug '92 

cells involved in liver metastasis. 

NKI92-57 The role of cytokines in the pathogenesis IV W Gerritsen July'92 

and treatment of myelodysplatic syndromes. 

NKI 92-469 The (district) nurse and the cancer patient in XII R de Wit Feb '92 

pain: a nursing intervention study. Fvan Dam 

NKI 93-139 The role of health care providers and significant XII M Sprangers Jan '93 

others in evaluating the quality of life of N Aaronson 

patients with canceL 

NK193-523 Episialin in cancer and nonnal tissues. VI J Hilkens June'93 

NK193-525 Presentation of melanoma-specific antigens by 111 J Neefjes Aug '93 

MHC Class 11 Molecules. 

NK193-527 Induction and breakdown of immunological IV A Kruisbeek March'93 

tolerance for tumor viruses by synthetic 

peptides. 

NK193-528 Human thyroid peroxidase as a model target for X I P Bruning Sept '93 

auto immune cytotoxicity in cancer therapy. IV A Kruisbeek 

NK193-536 On-line two-dimensional and three-dimensional IX M van Herk Jan'93 

patient setup error analysis in radiotherapy by 

portal imaging. 

NK193-596 Genetic and functional dissection of colon VII PDemant Febr '93 

tumorigenesis. 

NKI 94-771 Identification of oncogenes and tumor suppressor VII A Berns Febr '94 

genes collaborating in tumor progression. 

NKI94-775 Genes involved in non-P-glycoprotein mediated V P Borst Oct '94 

drug resistance in human lung cancer celllines. F Baas 

Projects 189

190 Projects 

NKI94-777 Development and clinical application of three- IX M Van Herk July'94 

dimensional image correlation algorithms with 

emphasis on applications in radiotherapy. 

NKI94-778 Optimization of patient treatment during boron IX B Mijnheer July'94 

neutron capture therapy. RMoss 

F Stecher- 

Rasmussen 

NKI94-779 Isolation and characterization of cellular genes II R Bemards Dec '94 

whose proteins interact with adenovirus EIA 

oncoproteins. 

NKI 94-795 Aberrant Cyclin Dl expression and anchorage- VI R Michalides May '94 

independent growth in breast cancer. 

NKI94-804 Targeted radiotherapy of neural-crest tumors VIII L Smets Jan '94 

with radio-iodinated MIBG. C Hoefnagel 

P Voûte 

NKI94-808 BCL-2-mediated resistance to antileukemic drugs. VIII L Smets May'94 

NKI94-809 The proto-oncogene pim-l; investigation of its V R Plasterk Oct '94 

role in signal transduction in the experiment al 

animal C elegans. 

NKI 94-819 Clinical application of SPECT lung scans in th ree- IX J Lebesque July'94 

dimensional radiotherapy treatment planning. SMuller 

P Baas 

NKI94-825 Effect of immunization on the cytotoxic T -cell lVI E Rankin Oct '94 

response against tumors expressing the MAGE-l X A Hekman 

gene. H Spits 

NKI94-826 Immune responses against tumor antigens IV A Kruisbeek Nov '94 

through induction of B7 and ICAM-l interactions. 

NKI94-878 The rol of TIAM 1 in tumor progression and J Collard Sept '94 

metastasis in human cancer. WMooi

Major projects supported by other organizations 

Granting agency Title Div Principal 

project number investigator 

Dutch Kidney Foundation Adhesion and signal transduction of A Sonnenberg 

C91.1179 (1.3/31 and (1.6/31 integrins in the kidney. J Weening 

NWO The role of the integrin (1.6/34 in the A Sonnenberg 

900-511-043 the formation of hemidesmosomes. 

EEC Molecular mechanisms and regulation A Sonnenberg 

ERBCHRXCT930246 of cell matrix interactions. 

Projects 191 

NHS The role of tyrosine phosphorylation for I A von dem Borne 

92-369 the function of P selectin, the receptor A Sonnenberg 

of platelets and endothelial cells. 

Yamamouchi Mechanisms of integrin activa ti on and A Sonnenberg 

Res. lnstitute inactivation. 

NWO Tiaml, a novel invasion and metastasis- I J Collard 

900501146 inducing gene putatively involved in signal 

transduction. 

EEG Molecular dosimetry of chemica 1 mutagens. II L den Engelse 

NWO (Gebied Studies on neuroblastoma tumor progression. II R Bernards 

Medische Wetenschappen) 

900-792-137 

HF SP Mechanism of transcriptional regulation by Myc II KSimmen 

oncoproteins studied in vitro. 

NWO (Gebied Secretion of fibroblast growth factor by a novel III J Pieters 

Medische Wetenschappen) secretory mechanism. W Mooienaar 

900-568-119 

NWO (Gebied Interactions between MHC molecules and peptides III J Neefjes 

Medische Wetenschappen) derived from products of transformed genes and 

900-509-155 oncogenes. 

NWO (Gebied Substrate specificity of the MHC-coded peptide pump III J Neefjes 

Medische Wetenschappen) and the genera ti on of MHC c1ass-l presentation. 

901-09-027 

NWO (Gebied Elucidation of the molecular composition of the III J Borst 

Medische Wetenschappen) antigen receptor complex on human B lymphocytes. 

900-509-154 

NWO Role of the mlgM -associated components in III J Borst 

900-507-170 trans-membrane signalling in B lymphocytes. 

NWO Interaction between the biologically active phospholipid III WMoolenaar 

810-405-102 LP A and the plasma membrane. 

NWO The neuron al LP A receptor: morphological effects III W Mooienaar 

900-553-039 and signaltransduction.

192 Projects 

NWO (Gebied Use of transgenic mice in assessing the role of 111 J Borst 

Medische Wetenschappen) the gamma T -cell receptor in T -cell function 

900-509-127 and differentiation. 

NWO (Gebied Intracellular transport of the MHC class 11 associated IV J Pieters 

Medische Wetenschappen) invariant chain. A Kruisbeek 

900-509-186 

NWO (Gebied Regulation of T -cell repertoire selection. IV A Kruisbeek 


900-505-273 

NWO (Gebied Molecular regulation of early T -cell development. IV A Kruisbeek 


900-507-178 

NWO (Gebied Role of cytokines in human T-cell development. IV H Spits 


900-509-188 

UKE Regulation of expression of recombinase IV H Spits 

activating genes (RAG) in the human T-celllineage. 

ECC The physiological function and pharmacological V P Borst 

significance of P-glycoproteins assessed in mice 

with disrupted P-glycoprotein genes. 

EEG Molecular characterization of P-glycoprotein V P Borst 

multidrug transporters. 

SON 331-26 Gene transpositions and gene expression in V P Borst 

trypanosomes. 

NWO/ PIONIER DNA transposition in animal cells. V R Plasterk 

HFSPO G-protein function in C elegans. V R Plasterk 

NWO (Gebied Integration of HIV in the gen ome of host ceUs. V R Plasterk 


900-502-140 

GLAXO Inhibitors of HIV integrase. V R Plasterk 

RGO/ AIDS fonds Integrase of HIV. V R Plasterk 

SON 335-210 DNA-transposition in animal cells. V R Plasterk 

JNICT Magainin-like peptides and permeation of biological V H Westerhoff 

BD276/ 90-IF membranes. 

EEC Computer aided simulation of the coupling between V H Westerhoff 

S/ BIOT-91301O mitochondrial metabolism and NADH oscillation in 

yeast. 

NWO/ PIONIER The quantitative principles relating cell physiology V H Westerhoff 

81-428 to molecular biochemistry. 

Foundation Biofysica From photon to ATP. V H Westerhoff 

810-405-14405

EECBRIDGE The role of DNA conformation in control ofmetabolic V H Westerhoff 

K2125.5.2290 processes. 

Foundation Biofysica Quantitative analyses of the con trol of cell V H Westerhoff 

NWO 810-409-12 physiology: cascade regulation of cell physiology. 

STIPT I Centocor Cancer irnmunotargeting and imaging. VII J Hilkens 

XII E Rutgers 

X C Hoefnagel 

Centocor Monoclonal antibodies against tumor and tissue VI J Hilkens 

preferent epitopes on episialin. 

EEC Molecular analysis of proteins involved in interactions VI D Ivanyi 

CIPA-CT93-0131 between cytoskeletal filament systems in non-neural cells. VViklicky 

EEC Science Program Structural and functional analysis VII PDemant 

SC 1000213 of mouse genome. 

NWO Genetic modification of the mouse germ VII A Berns 

900-502-095 line: a new generation of mutant rnice to 

study (tumor suppressor) gene function. 

EEC Studies on the functional role of the VII A Berns 

ERBCHRXCT metastasis-associated molecule CD44 by 

940662 transgenic and gene targeting strategies. 

EEC Study of the lymphoid potentialof early VII A Berns 

ERBCHRXCT embryonic hematopoietic stem cells from 

940584 transgenic mice and ES cells. 

UKE Studies on prevention and/ or circumvention of tumor VIII I J Schornagel 

cell resistance to antifolates. X 

Scotia Pharmaceuticals Photodynamic therapy of tumors restricted to the VIIII F Stewart 

peritoneal cavity. XII T Helrnerhorst 

Schumacher-Kramer Cisplatin-DNA adducts and cell kinetic measurements in lXI LMoonen 

Stichting (SK-Foundation)I inoperable non sm all celllung cancer treated with H Bartelink 

Stichting Fondsenwervings- radiotherapy combined with daily cisplatinurn. VIII A Begg 

acties Volksgezondheid 

STW Application of an electronic portal imaging device IX B Mijnheer 

for dosimetry in radiotherapy. M Van Herk 

Projects 193 

Ziekenfondsraad What is the value of photodynarnic therapy and X N van Zandwijk 

Ontwikkelings- endobronchial radiotherapy added to external 

geneeskunde radiotherapy for patients with inoperable non-small 

92-086 celllung canceL (Closed to new patients). 

Ziekenfondsraad Prospective study on the influence of high dose X S Rodenhuis 

Ontwikkelings- chemotherapy with peripheral stem cell transplantation 

geneeskunde on the disease free survival of patients with breast cancer 

09-94-051 and four or more lymph node metastases. 

Prevention Fund An experimental study on early indicators of X I N van Zandwijk

194 Projects 

28-2397 a chemoprotective effect of N -acetylcysteine against III L den Engelse 

cancer in smokers. XII A Balm 

XII F van Leeuwen 

Schumacher-Kramer Autologous bone marrow transplantation in breast X S Rodenhuis 

Stichting cancer. 

(SK-Foundation)I 

Stichting Fondsenwervingsacties 

Volksgezondheid 

ADAC Laboratories Scintigraphic assessment of salivary gland function X I R Valdes Olmos 

in irradiated head and neck cancer patients. XII A Balm 

IX RKeus 

ADAC Laboratories Prospective evaluation of major salivary gland XII FBaim 

Europe BV function after radiotherapy using 99mTc-pertechnetate X I R Valdes Olmes 

and automated quantification. IX RKeus 

Prevention Fund Does the presence of HPV in epithelial cells of the XI C Meijer 

28-1502 uterine cervix predict for the development of J Walboomers 

cervical cancer? P Kenemans 

T Helrnerhorst 

ECC Immunomodulation in cervical carcinoma. XI Nvd Vange 

R Verheijen 

F Peccatori 

R Scheper 

T Helmerhorst 

Stichting Mundo Crastino Double dynamic graciloplasty technique: functional XI F Zoetrnulder 

Meliori reconstruction to achieve anal continence after EVos 

Stichting Fondsenwervings- abdominoperineal resection for rectal cancer. 

acties Volksgezondheid 

IARC/ Directorate International study of cancer risk in biology XII F van Leeuwen 

General of the Ministry research laboratory workers. 

of Social Affairs 

Ministry of Welfare, The quality of life of patients treated for ARC or XII S Danner 

Public Health and AIDS (category IV). FvanDam 

Culture 88-52 

STG Early warning system of cancer XII F van Leeuwen 

('signaleringssysteem kanker'). o Dalesio 

Prevention Fund Risk of breast cancer to relatives of breast cancer XII F van Leeuwen 

28-1814-2 patients. MRookus 

Ministry of Welfare, Inpatients' pain assessments: a nursing intervention XII R de Wit 

Public Health and study. FvanDam 

Culture NKI 15884 

Ank van Vlissingen Fonds The relation of the tumor ideotype of gastric low-grade VI D de Jong 

B-celllymphomas to Heliobacter pylori antigens.

Publications 

Full papers 

2 

3 

4 

5 

6 

7 

8 

Aaronson NK, CulI A, Kaasa S, Sprangers MAG. 

The European Organization for Research and 

Treatment of Cancer (EOR TC) modular approach 

to quality of life assessment in oncology. Int J 

Mental Health 1994; 23: 75-96. 

Aartsen EJ, Albus-Lutter CE. Vulvar sarcoma: 

clinical implications. Eur J Obstet Gynecol Reprod 

Bio11994; 56: 181-9. 

Aartsen EJ, Snethlage RAl, Van Geel AN, GalIee 

MPW. Squamous cell carcinoma of the vagina/ 

vulva in a male pseudohermaphrodite with 

5a-reductase deficiency. Int J Gynecol Cancer 

1994; 4: 283-7. 

Ackerstaff AH, Hilgers FJM, Aaronson NK, Balm 

AJM. Communication, functional disorders and 

lifestyle changes after total laryngectomy. Clin 

Otolaryngol1994; 19: 295-300. 

Ackerstaff AH, Hilgers FJM, Aaronson NK, Balm 

AJM, Van Zandwijk N. Improvements in 

respiratory and psychosocial functioning following 

total laryngectomy by the use of a heat and 

moisture exchanger. Ann Otol Rhinol Laryngol 

1993; 102: 878-83. 

Ackerstaff AH, Souren T, Van Zandwijk N, Balm 

AJM, Hilgers FJM. Improvements in the 

assessment of pulmonary function in laryngectomized 

patients. Laryngoscope 1993; 103: 

1391-4. 

Adema GJ, De Boer AJ, Van 't HulIenaar R, 

Denijn M, Ruiter DJ, Vogel AM, Figdor CG. 

Melanocyte lineage-specific antigens recognized by 

monoclonal antibodies NKI-beteb, HMB-50, and 

HMB-45 are encoded by a single cDNA. Am J 

Patho11993; 143: 1579-85. 

Allen DG, Baak J, Belpomme D, Berek JS, 

Bertelsen K, Ten BokkeI Huinink WW, Van der 

Burg MEL, Calvert AH, Conte PF, Dauplat J, 

Eisenhauer EA, Favalli G, Hacker NF, Hamilton 

TC, Hansen HH, Hansen M, Van Houwe1ingen 

HC, Kaye SB, Levin L, Lund B, Neijt JP, Ozols 

RF, Piccart MJ, Rustin GJS, Sessa C, Soutter WP, 

Thigpen JT, Tropé C, Vermorken JB, De Vries 

195 

EGE, (consensus group in alphabetical order). 

Advanced epithelial ovarian cancer: 1993 

consensus statements. Ann Oncol 1993; 4: S83-8. 

9 Anderson RT, Aaronson NK, Wil kin D. Critical 

review of the international assessment of healthrelated 

quality of life. Quality Life Res 1993; 2: 

369-95. 

10 Anninga JK, Valdés Olmos RA, De Kraker J, Van 

Tinteren H, Hoefnagel CA, Van Royen EA. 

Technetium-99m dimercaptosuccinic acid and 

ifosfamide tubular dysfunction in children with 

cancer. Eur J Nucl Med 1994; 21: 658-62. 

11 Ansink AC, Krul MR, De Weger RA, Kleyne JA, 

Pijpers H, Van Tinteren H, De Kraker EW, 

Helrnerhorst TJM, Heintz APM. Human 

papillomavirus, lichen sclerosus, and squamous cell 

carcinoma of the vulva: detection and prognostic 

significance. Gynecol Onco11994; 52: 180-4. 

12 Baas P, Michielsen C, Oppelaar H, Van Zandwijk 

N, Stewart FA. Enhancement of interstitial 

photodynamic therapy by mitomycin C and E09 in 

a mouse tumour model. Int J Cancer 1994; 56: 

880-5. 

13 Baas P, Oppelaar H, Van der Valk M, Van 

Zandwijk N, Stewart FA. Partial protection of 

photodynamic induced skin reactions in mice by 

N-acety1cysteine: a preclinical study. Photochem 

Photobiol 1994; 59: 448-54. 

14 Bain G, Robanus Maandag EC, Izon DJ, Amsen 

D, Kruisbeek AM, Weintraub BC, Krop I, 

Schlissel MS, Feeney AJ, Van Roon M, Van der 

Valk M, Te Riele H, Berns A, Murre C. E2A 

proteins are required for proper B cell development 

and initiation of immunoglobulin gene 

rearrangements. Cell 1994; 79: 885-92. 

15 Bakker ABH, Schreurs MWJ, De Boer AJ, 

Kawakami Y, Rosenberg SA, Adema GJ, Figdor 

CG. Melanocyte lineage-specific antigen gplOO is 

recognized by melanoma-derived tumor-infiltrating 

lymphocytes. J Exp Med 1994; 179: 1005-9.

74 De Mulder PHM, Cappelaere P, Cognetti F, 

Verweij J, Schornagel JH, Vermorken JB, 

Kirkpatrick A, Lefebvre JL. A phase 11 study of 

pirarubicin in patients with advanced recurrent 

head and neck squamous cel1 carcinoma. Cancer 

Chemother Pharmacol1994; 33: 438-40. 

75 De Rode Husman A-M, Walboomers JMM, 

Meijer CJLM, Risse EKJ, Schipper MEI, 

Helrnerhorst TJM, Bleker OP, Delius H, Van den 

Brule AJC, Snijders PJF. Analysis of 

cytomorphological1y abnormal cervical scrapes for 

the presence of 27 mucosotropic human 

papillomavirus genotypes, using polymerase chain 

reaction. Int J Cancer 1994; 56: 802-6. 

76 De Vries JD, Talsma H, Henrar REC, Kettenesvan 

den Bosch JJ, Bult A, Beijnen JH. 

Pharmaceutical development of aparenteral 

lyophilized formulation ofthe novel indoloquinone 

antitumour agent E09. Cancer Chemother 

Pharmacol1994; 34: 416-22. 

77 De Vries JD, Winkelhorst J, Underberg WJM, 

Henrar REC, Beijnen JH. A systematic study on 

the chemical stability of the novel indoloquinone 

antitumour agent E09. Int J Pharm 199:t 100: 

181-8. 

78 De Waal Malefijt R, Figdor CG, De Vries JE. 

Effects of interleukin-4 on monocyte functions: 

comparison to interleukin-13 [review]. Res 

Urrununol 1993; 144: 629-33. 

79 De Waal Malefijt R, Figdor CG, Huijbens R, 

Mohan-Peterson S, Bennett B, Culpepper J, Dang 

W, Zurawski G, De Vries JE. Effects of IL-13 on 

phenotype, cytokine production, and cytotoxic 

function of human monocytes: comparison with 

IL-4 and modulation by IFN--r or IL-lO. J 

Immunol1994; 151: 6370-81. 

80 De Weers M, Brouns GS, Hinshelwood S, Kinnon 

C, Schuurman RKB, Hendriks RW, Borst J. B-cell 

antigen receptor stimulation activates the human 

Bruton's tyrosine kinase, which is deficient in 

X-linked agammaglobulinemia. J Biol Chem 1994; 

269: 23857-60. 

81 De Wind N, Peeters BPH, Zijderveld A, Gielkens 

ALJ, Berns WJM, Kimman TG. Mutagenesis and 

characterization of a 41 kilo base pair region of the 

pseudorabies virus genome: transcription map, 

search for virulence genes, and comparison with 

homologs of herpes simplex virus type 1. Virology 

1994; 200: 784-90. 

82 Delwel GO, De Melker AA, Hogervorst F, Jaspars 

LH, Fles DLA, Kuikman I, Lindblom A, Paulsson 

M, Timpl R, Sonnenberg A. Distinct and 

overlapping ligand specificities of the 1X3Af31 and 

1X6Af31 integrins: recognition of laminin isoforms. 

Mol Biol Ce111994; 5: 203-15. 

Publications - Juli papers 199 

83 Dewit L, Verheij M, Valdés Olmos RA, Arisz L. 

Compensatory renal response af ter unilateral 

partial and whole volume high-dose irradiation of 

the human kidney. Eur J Cancer 1993; 29A: 

2239-43. 

84 Durban EM, Barreto PD, Hilgers J, Sonnenberg A. 

Cel1 phenotypes and differentiative transitions in 

mouse submandibular salivary gland defined with 

monoclonal antibodies to mammary epithelial 

cel1s. J Histochem Cytochem 1994; 42: 185-96. 

85 Essers M, Keus R, Lanson JH, Mijnheer BJ. 

Dosimetrie control of conformal treatment of 

parotid gland tumours. Radiother Oncol 1994; 32: 

154-62. 

86 Fijneman RJ, Ophoff RA, Hart AAM, Demant P. 

Kras-2 al1eles, mutations, and lung tumor 

susceptibility in the mouse: an evaluation. 

Oncogene 1994; 9: 1417-21. 

87 Flens MJ, Izquierdo MA, Scheffer GL, Fritz JM, 

Meijer CJLM, Scheper RJ, Zaman GJR. 

Immunochemical detection of MRP in human 

multidrug-resistant tumor cells by monoclonal 

antibodies. Cancer Res 1994; 54: 4557-63. 

88 Franklin HR, Simonetti GPC, Dubbelman AC, 

Ten Bokkei Huinink WW, Taal BG, Wigbout G, 

Mandjes IA, Dalesio OB, Aaronson NK. Toxicity 

grading systems: a comparison between the WHO 

scoring system and the Common Toxicity Criteria 

when used for nausea and vomiting. Ann Oncol 

1994; 5: 113-7. 

89 Gaarenstroom KN, Bonfrer JMG, Kenter GG, 

Korse CM, Helmerhorst TJM, Trimbos JB. 

Prognostic significanee of antibodies to HPV-16 

E7 /E4 proteins and fragments of cytokeratin 19 in 

invasive cervical carcinoma. In: Stanley MA, 

editor. Immunology of human papillomavirus. 

New York: Plenum, 1994: 119-23. 

90 Gaarenstroom KN, Kenter GG, Bonfrer JMG, 

Korse CM, Gal1ee MPW, Hart AAM, Müller M, 

Trimbos JB, Helrnerhorst TJM. Prognostic 

significanee of serum antibodies to human 

papillomavirus-16 E4 and E7 peptides in cervical 

cancer. Cancer 1994; 74: 2307-13. 

91 Garavaglia G, Johansson K-A, Leunens G, 

Mijnheer B. The role of in vivo dosimetry. 

Radiother Oncoll993; 29: 281-2. 

92 Gebbink MF, Verheijen MH, Zondag GC, Van 

Etten I, Mooienaar WH. Purification and 

characterization of the cytoplasmic domain of 

human receptor-like protein tyrosine phosphatase 

RPTPj.t. Biochemistry 1993; 32: 13516-22.

113 Hintzen R, Lens SMA, Koopman G, PaIs ST, Spits 

H, Van Lier RAW. CD70 represents the human 

ligand for CD27. Int J Immunol1994; 6: 477-80. 

114 Hoefnagel CA. I -131 MIBG use in neuroblastoma. 

In: Shapiro B, Hoefnagel CA, editors. MIBG 

clinical atlas. CIS-US Inc. 1994: 33-57. 

115 Hoefnagel CA. Metaiodobenzylguanidine and 

somatostatin in oncology: role in the management 

ofneural crest tumours. Eur J Nuc1 Med 1994; 21 : 

561-81. 

116 Hoefnagel CA, De Kraker J. Childhood neoplasia. 

In: Murray IPC, ElI PJ, editors. Nuclear medicine 

in clinical diagnosis and treatment. London: 

Churchill Livingstone, 1994: 765-77. 

117 Hoefnagel CA, De Kraker J, Valdés Olmos RA, 

Voûte PA. 1-131 MIBG as a first line treatment in 

high risk neuroblastoma patients. Nucl Med 

Commun 1994; 15: 712-7. 

118 Hoefnagel CA, Kapucu 0 , De Kraker J, Van 

Dongen A, Voûte PA. Radioimmunoscintigraphy 

using [IIIIn]antimyosin Fab fragments for the 

diagnosis and follow-up of rhabdomyosarcoma. 

Eur J Cancer 1993; 29A: 2096-100. 

119 Hoefnagel CA, Lewington VJ. MIBG therapy. In: 

Murray IPC, ElI PJ, editors. Nuclear medicine in 

clinical diagnosis and treatment. London: 

Churchill Livingstone, 1994: 851-64. 

120 Hoefnagel CA, Rankin EM, Valdés Olmos RA, 

Israëls SP, Pavel S, Janssen AGM. Sensitivity 

versus specificity in melanoma imaging using 

iodine-123 iodobenzamide and indium-1l1 

pentetreotide [letter]. Eur J Nucl Med 1994; 21: 

587-8. 

121 Holdener EE, Clavel M, Sessa C, Ten Bokkei 

Huinink WW, Siegenthaler P, Ludwig C, Klepp 0 , 

Renard G, Decoster G, Pinedo HM. Phase II trial 

of anaxirone (TGU) in advanced colorectal cancer: 

an EOR TC Early Clinical Trials Group (ECTG) 

study. Eur J Cancer 1994; 30A: 394-5. 

122 Holland R, Peterse JL, Millis RR, Eusebi V, 

Faverly D, Van de Vijer M, Zafrani B. Ductal 

carcinoma in situ: a proposal for a new 

classification. Sernin Diagn Pathol 1994; 11: 

167-80. 

123 Hordijk PL, Verlaan I, Jalink K, Van Corven EJ, 

MooIenaar WH. cAMP abrogates the p21rasrnitogen-activated 

protein kinase pathway in 

fibroblasts. J Biol Chem 1994; 269: 3534-8. 


124 Hordijk PL, Verlaan I, Van Corven EJ, Mooienaar 

WH. Protein tyrosine phosphorylation induced by 

lysophosphatidic acid in Rat-l fibroblasts: 

evidence that phosphorylation of map kinase is 

mediated by the Gi-p21ras pathway. J Biol Chem 

1994; 269: 645-51. 

125 Horenbias S. Squamous cell carcinoma of the 

penis: update on diagnosis, staging and 

management. Update Series European Board of 

Urology 1994; 3: 58-63. 

126 Horenbias S, Kröger R, Gallee MP, Newling DW, 

Van Tinteren H. Ultrasound in squamous cell 

carcinoma of the penis: a useful addition to clinical 

staging? A comparison of ultrasound with 

histopathology. Urology 1994; 43: 702-7. 

127 Horenbias S, Newling DWW. Local recurrent 

tumour af ter penis-conserving therapy: a plea for 

long-term follow-up. Br J Urol 1993; 72: 976. 

128 HorenbIas S, Van Tinteren H. Squamous cell 

carcinoma of the penis: IV. Prognostic factors of 

survival: analysis of tumor, nodes and metastasis 

classification system [see comments]. J Urol 1994; 

151: 1239-43. 

129 Horiot JC, Bernier J, Johansson K-A, Van der 

Schueren E, Bartelink H. Minimum requirements 

for quality assurence in radiotherapy. Radiother 

Onco11993; 29: 103-4. 

130 Huizing MT, Keung AC, Rosing H, Van der Kuij 

V, Ten Bokkei Huinink WW, Mandjes IM, 

Dubbelman AC, Pinedo HM, Beijnen JH. 

Pharmacokinetics of paclitaxel and metabolites in a 

randomized comparative study in platinumpretreated 

ovarian cancer patients. J Clin Oncol 

1993; 11: 2127-35. 

131 Israëls SP. Is there a role for chemotherapy in head 

and neck melanoma? A critical review of recent 

trials with chemotherapy. Diagn Oncol 1993; 3: 

274-9. 

132 Ivanyi D, Groeneveld E, Calafat J, Minke JM, Van 

Doornewaard G. Modulation of mammary 

carcinoma cell phenotype and keratin expression 

patterns by retinoic acid. Cancer Lett 1993; 73: 

191-205. 

133 Izon DJ, Jones LA, Eynon EE, Kruisbeek AM. A 

molecule expressed on accessory cells, activated T 

cells and thymic epithelium is a marker and 

promoter ofT cell activation. J Immuno11994; 153: 

2939-50. 

134 Izon DJ, Nieland JD, Godfrey DI, Boyd RL, 

Kruisbeek AM. Flow cytometric analysis reveals 

unexpected shared antigens between histologically 

defined populations of thymic stromal cells. Int 

Immuno11994; 6: 31-9.

157 Kholodenko BN, Sauro HM, Westerhoff HV. 

Control by enzymes, coenzymes and conserved 

moieties: a generalisation of the connectivity 

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422 Neering H. Congresverslag: Het 5e wereldcongres 

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423 Nieweg OE, Woldring MG, Vaal burg W. Wat 

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424 Renckens CNM, Van Dam FSAM. Gezondheidsraad 

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425 Sprangers MAG, Aaronson NK, Van Dam 

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426 Steggerda MJ, Borger JH, Damen E, Meertens H. 

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427 Takes RP, Valdés Olmos RA, Hoefnagel CA. 

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428 Thomas CMG, Wob bes T, Bonfrer JMG, Peter se 

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429 Valdés Olmos RA. The role of nuclear medicine in 

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430 Van 't Veer L. Molecular aspects of colon cancer. 

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216 Loca! papers / Conference papers / Theses 

431 Van Balen P, Tijssen S, Dijkstra L. Cytostatica: 

naar een risicobenadering. TvZ Tijdschr 

Verpleegkd 1994; 104: 186-8. 

432 Van Barneveld TA, Jansen DF, Van Leeuwen FE. 

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433 Van Barneveld TA, Van Leeuwen FE. 

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van het Directoraat-Generaal van de Arbeid van 

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434 Van de Pol M, Twijnstra A, Aaronson NK. 

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435 Van de Pol M, Twijnstra A, Aaronson NK. 

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436 Van der Sanden GAC, Van Barneveld TA, Dalesio 

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in opdracht van de Stuurgroep Toekomstscenario's 

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437 Van der Zouwe N, Van Dam FSAM, Aaronson 

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438 Van Dongen JA. Mammacarcinoom, beleid in 

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439 Van Dun RECS, Derix MMA, Van Dam FSAM, 

Portegies P. Cognitieve functies, overlevingsduur 

en stemming bij patiënten met AIDS en ARC. 

Gedrag & Gezondheid 1994; 22: 16-25. 

440 Van Egmond MHF, Neuteboom GHG, Vrooland 

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441 Vasen HFA, Taal BG, Griffioen G, Nagengast 

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onderzoek van families met heriditair nonpolyposis 

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442 Vielvoye-Kerkmeer APE. De multidisciplinaire 

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J, redactie. Pijn informatorium. Houten: Bohn, 

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443 Vielvoye-Kerkmeer APE, Mattern C, Hilgers 

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444 Vielvoye-Kerkmeer APE, Roe1fsema F. Kan een 

injectie met corticosteroïden leiden tot 

menstruatiestoornissen? Vademecum 1994; 27: 1. 

445 Woerdenbag HJ, Beijnen JH. Farmacie-onderwijs 

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446 Beijnen JH, Soepenberg 0 , Ten BokkeI Huinink 

WW. Chemical and pharmacologic interaction 

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447 Damen EMF, Boersma LJ, De Boer RW, M uUer 

SH, Lebesque N. Predicting overalllung function 

loss based on clinically determined local dose-effect 

relations and the fuU 3-dimensional dose 

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international conference on the use of computers in 

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448 De Boer RW, Meertens H. Collection of patient 

treatment data and technical data fr om philips sllinacs 

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of the XIth international conference on the use of 

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48-9. 

449 Gilhuijs KGA, Drukker K, Van Dam PJH, Van 

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450 Hoefnagel CA. Radionuclide targeting of tumours 

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452 Horiot JC, Van der Schueren E, Johansson KA, 

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453 Mijnheer BJ. Different types of protal imaging 

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454 Mijnheer BJ. Possibilities and lirnitations of in-vivo 

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455 Nieweg OE. Lieskliertoilet. In: Symposium 

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456 Recht A, Van Dongen JA, Peterse JL. Ductal 

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457 Valdés Olmos RA, Delprat CC, Hoefnagel CA. 

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editor. Ponencias 11 Jornada de divulgación de la 

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458 Van 't Veld AA, Bruinvis IAD. Accuracy in gridbased 

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459 Van Bree NAM, Van Battum LJ, Huizenga H, 

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460 Van Dam FSAM. Die Zukunft der psychosozialen 

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461 Van Dongen JA, Borger JH, Nieweg OE, Peterse 

JL, Rutgers EJT, Van Tienhoven G, Van der Wall 

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1994 Oct 4; Vienna, Austria. 1994. 

462 Van Herk M, Kooy HM. Optirnisation of 

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463 Van Leeuwen FE, Stiggelbout AM, Delemarre 

JFM, Somers R. Second cancer risk following 

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464 Wambersie A, Van Dam J, Hanks G, Mijnheer BJ, 

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465 Baas P. Photodynarnic therapy in mice and men 

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466 Bijlmakers MJE. Early aspects of the assembly of 

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467 Burger DM. Bioanalysis and clinical pharmacokinetics 

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468 Eijdems EWHM. Mechanisms of low level drug 

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469 Gebbink MFBG. Protein tyrosine phosphatases 



470 Gommers-Ampt JH. Beta-D-glucosyl-hydroxymethyluracil: 

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471 Sijts EJAM. Cytotoxic T lymphocyte recognition 

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218 Local papers / Conference papers / Theses 

472 Tan MCAA. Glycoprotein transport and modification 

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Amsterdam: Free University, 1994. 

473 Valdés Olmos RA. The role ofnuclear medicine in 

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cancer therapy [dissertation]. Amsterdam: University 

of Amsterdam, 1994. 

474 Van der Lugt NMT. Functional analysis ofPim-1 

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475 Van Leeuwen FE. Second malignancy as a sequel 

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476 Vaz Gomes AlCQ. Magainins: functional aspects 

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477 Vielvoye-Kerkmeer APE. Gevangen in (voor)oordelen: 

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478 Wafelman AR. Pharmaceutical characteristics and 

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479 Aamdal S, Bruntsch U, Kerger J, Verweij J, Ten 

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480 Aaronson NK, Cull A, Kaasa S, Sprangers MAG. 

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481 Anderson R T, Aaronson NK, Wil kin D. Qualtiy of 

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482 Baars JW, Wolbrink GJ, Hart MHL, Hack CE, 

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483 Balm AJM, Ackerstaff AH, Hilgers FJM, Gregor 

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484 Bárcena A, Münch MO, Roncarolo M-G, Spits H. 

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485 Begg AC. Prediction of radiation response. In: 

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486 Beijersbergen RL, Kerkhoven RM, Zhu L, Carlée 

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487 Bergman B, Aaronson NK. Quality oflife and costeffectiveness. 

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488 Berns A, Van der Lugt N , Alkerna M, Van 

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489 Bonfrer JMG, Gaarenstroom KN, Kenter GG, 

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490 Boogerd W. Neurological complications of 

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491 Borger J, Kemperman H, Sillevis Smitt H, Hart 

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492 Borst P, Pinedo HM. Drug resistance. In: Peckham 

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493 Brouns G, De Vries E, Borst J. Assembly and 

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494 Bullinger M, Power M, Anderson R, Cella D, 

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495 Burger DM, Meenhorst PL, Beijnen JH. Clinical 

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496 Burger DM, Meenhorst PL, Mulder JW, Koks 

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497 Burger DM, Meenhorst PL, Mulder JW, 

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Therapeutic drug monitoring of phenytoin in 

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498 Burger DM, Meenhorst PL, Ten Napel CHH, 

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JH. Pharmacokinetic variability of zidovudine in 

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499 Burger DM, Rosing H, Ten Napel CHH, Duyts T, 

Meenhorst PL, Mulder JW, Koks CHW, Bult A, 

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500 De Haas M, Kerst JM, Van der Schoot CE, Calafat 

J, Hack CE, Nuijens JH, Roos D, Van Oers RHJ, 

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501 De Vries ID, De Graaff-Teulen MJA, Henrar 

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502 De Wit R, Stoter G, Sleyfer DT, Kaye SB, De 

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De Pauw M, Sylvester R. Four cyc1es ofBEP versus 

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503 Delwel GO, Sonnenberg A. Larninin isoforms and 

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504 Dercksen MW, Muller EJ, Gerritsen WR, 

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Van der Schoot CE. Expression of adhesion 

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505 Dijkstra MD, Balm AJM, Gregor RT, Hilgers 

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506 Eggermont AMM, Lienard D, Schraffordt Koops 

H, Kroon BBR, Rosenkaimer F, Lejeune FJ. Limb 

salva ge by isolated limb perfusion of the limbs with 

high dose TNF-alpha, gamma interferon and 

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507 Eijdems EWHM, De Haas M, Coco Martin JM, 

Ottenheim CPE, Zaman GJR, Dauwerse JG, 

Breuning MH, Twentyman PR, Borst P, Baas F. 

Mechanisms of MRP overexpression in four 

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508 Eijdems EWHM, De Haas M, Timmerman AJ, 

Van der Schans GP, Kamst E, De Nooy N, Astaldi 

Ricotti GCB, Borst P, Baas F. Reduced topoisomerase 

11 activity in multidrug resistant human 

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509 Elias MM, Hilgers FJM, Baris G, Gregor RT, 

Balm AJM. Carcinoma of the pyriform sinus: a 

retrospective analysis of treatment results over a 

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510 Fijneman RJA, Oomen LCJM, Snoek M, Demant 

P. A susceptibility gene for alveolar lung tumors in 

the mouse, maps between Hsp70.3 and G7 within 

the H2 complex. Immunogenetics 1994 (in press). 

511 Gaarenstroom KN, Bonfrer JMG, Kenter GG, 

Korse CM, Hart AAM, Trimbos JB, Helmerhorst 

TJM. Clinical value of pretreatment serum Cyfra 

21-1 , TPA, and SCC level in cervical cancer. 

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512 Gimond C, De Melker A, Aumailley M, 

Sonnenberg A. The cytoplasmic domain of a6A 

integrin subunit is an in vitro substrate for protein 

kinase C. Exp Cell Res 1994 (in press). 

513 Gravestein LA, Nieland JD, Kruisbeek AM, Borst 

J. Novel monoclonal antibodies reveal potent 

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220 Papers in press 

514 Gregor A, Drings P, Rinaldi M, Schuster L, 

Burghouts J, Postrnus PE, Dalesio 0 , Kirkpatrick 

A, Hoctin Boes G, Van Zandwijk N. Randomised 

phase II trial of alternating or sequential chemol 

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protocol 08877 of the EOR TC Lung Cancer 

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515 Groot PC, Moen CJA, Hart AAM, Snoek M, 

Demant P. Recombinant congenic strains: genetic 

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516 Habets GGM, Van der Kammen RA, Jenkins NA, 

Gilbert DJ, Copeland NG, Hagemeijer A, Collard 

JG. The invasion-inducing Tiaml gene maps to 

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517 Habets GGM, Van der Kammen RA, Stam JC, 

Michiels F, Collard JG. Sequence of the human 

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518 Hahn DEE, Bergman L, Van Dam FSAM, 

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519 Haustermans K, Hofland I, Pottie G, Ramaekers 

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520 Heimans H , Vermorken JB, Wolbers J, Meijer 0 , 

Taphoorn MJB, Beijnen JH. Taxol concentrations 

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521 Hilkens J, Wesseling J, Vos HL, Litvinov SL, Boer 

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de Wiel-van Kemenade E, Figdor C. Cell surface 

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522 Hoefnagel CA. 1311-MIBG therapy of neural crest 

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523 Hoefnagel CA. Specific diagnosis of neural crest 

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524 Hoefnagel CA. MIBG and radiolabelled octreotide 

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Voûte PA. 1311-MIBG as a first line treatment in 

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527 Holmberg 0, Huizenga H, Idzes MHM, Lebesque 

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528 Hooiberg E, Van den Berk PCM, Sein H , Wijdenes 

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529 Huizing MT, Keung AC, Rosing H, Koopman F, 

Pinedo HM, Beijnen JH. High performance liquid 

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a comparison between a liquid-liquid extraction 

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J Chromatogr B 1994 (in press). 

530 Jalink K, Hordijk PL, MooIenaar WH. Growth 

factor-like effects oflysophosphatidic acid, a novel 

lipid mediator. Biochim Biophys Acta (CR) 1994 

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531 Janse AJ, Van Coevorden F, Peterse H, Keus RB, 

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532 Jassem J, Begg AC, Stewart FA, Bartelink H. 

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533 Jensen PR, Van der Gugten AA, Bier M, Van 

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534 Jonkman MF, De Jong MCJM, Heeres K, Pas HH, 

Van der Meer JB, Owaribe K, Martinez de Velasco 

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bullous pemphigoid antigen (BPI80) is deficient in 

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535 Kemperman H, Borger J, Hart AAM, Peterse H, 

Bartelink H, Van Dongen J. Prognostic factors for 

survival after breast conserving therapy for stage 1 

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536 Kemperman H, Driessens MHE, La Rivière G, 

Meijne AML, Roos E. The role of integrins and 

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Invas Metast 1994 (in press). 

537 Kemperman H, Driessens MHE, La Rivière G, 

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Author index 

Aamdal S 248, 479 

Aarnoutse R 381,382 

Aaronson NK 1,4,5,9,29,44,58,69,88,264,333, 

361,368,383,425,434,435,437,480,481,487,494, 

518,580,616,621,625,626 

Aartsen EJ 2,3,215 

Abdel-Wahab AHA 249 

Ackerstaff AH 4-6,483,625,626 

Acquadro C 368 

Acton D 251,488 

Adami J 287 

Adams G 173 

Adema GJ 7, 15 

Ademi J 286 

Ahmedzai S 29 

Aisner J 45 

AlakI M 282 

Alberto P 45 

Albus-Lutter CE 2, 154 

Aleman BMP 178 

Alexander 111 E 169 

Alexandrov K 232 

Alkerna M 488 

Allen DG 8 

Allen J 488 

Alonso J 368 

AmsenD 14 

Andachi Y 98 

Anderson R 9, 44, 481, 494 

Anninga JK 10 

Ansink AC 11 

Aon MA 62,64 

Apolone G 368 

Arisz L 83, 345, 600 

Armand JP 572 

Arriagada R 45 

Astaldi Ricotti GCB 508 

AtemaA 173 

Aumailley M 512 

Autier P 179 

Baak JPA 8, 42, 43 

Baan AW 33 

Baars JW 213,482 

Baas F 39,376,507,508 

Baas P 12, 13,379,465,614 

Bain G 14 

Bais EM 26 

Bakke 0 209 

Bakker ABH 15 

Bakker BM 227, 533, 612 

227 

Bakker DJ 329 

Balm AJM 4-6, 16-19,99, 110, 111, 140, 153, 170, 184, 

185,278,292,317,329,384,483,505,509,561,596, 

617,625,626,628,629,630 

Banks PM 287 

Banks M 351 

Bárcena A 20,21,262,340,484 

Baris G 278,292,509,613,628,630 

Barnes PD 169 

Barreto PD 84 

Barry-Walsh C 176 

Bartelink H 23,28, 35,40,60, 129, 178,200,201,236, 

249,275,302,452,491,532,535 

Bartsch H 232 

BaruchA 377 

Batchelor DM 385 

Battermann JJ 464 

Bayssas M 282 

Bech P 368 

Beck-Sickinger AG 267 

Beex LV AM 428 

Begg AC 22,23,60, 175,214,235,245,249,347,348, 

485,519,532,574 

Beijersbergen RL 24,486 

Beijnen JH 25-27,46-51, 76, 77,96, 130, 141, 146, 160, 

167,218,220,226,242,273,274,276,277,283,307, 

308,315,316,331,332,334,335,356,363-367,374, 

381,382,386-392,408,412,413,414,440,445,446, 

495-499,501,520,529,543,573,578,579,590,597, 

598,602,607 

Bekers 0 308, 590 

Bekos EJ 374 

Bel A 28 

Belpomme D 8, 346 

Bennett B 79 

BenraadtJ 323,615 

Bentzen S 268 

Berden JA 342 

Berek JS 8 

Bergman B 29,487 

Bergman L 518 

Bergman W 244 

Berkel R 542 

Bernards R 24,486 

Bernier J 129,452 

BernsA 14,30,104,135,166,225,229,242,309,488 

Berns WJM 81 

Bertelsen K 8 

BethelI R 351 

Bey P 138 

Bier M 533

Danen EHJ 68 

DangW 79 

Dauplat J 8 

Dauwerse JG 507 

Davies B 598 

De Beer ALJ 97 

De Bock R 259 

De Boer AJ 7, 15 

De Boer Dennert M 602 

De Boer JB 17, 69, 399 

De Boer RW 32, 66, 447, 448, 528 

De Bruin PC 70, 71 , 212 

De Gast GC 370 

De Goeij CCJ 343 

De Graaff-Teulen MJ 315,501 

De Graeff A 572 

De Haas M 209, 376, 500, 507, 508 

De Haes JCJM 616 

De Jong APJM 280, 583, 584 

De Jong D 72, 305 

De Jong MCJM 534 

De Koning HJ 73 

De Kraker EW 11 

De Kraker J 10, 116-118,451 , 525, 526 

De Melker AA 82, 205, 512 

De Mulder PHM 59, 74, 502, 572 

DeNooyN 508 

De Pauw BE 258 

De Pauw M 502 

De Rode Husman A-M 75, 608 

De Vries E 135, 493 

De Vries EGE 8, 554 

De Vries JD 76, 77, 501 

De Vries JE 20, 78, 79 

De Vries N 216 

De Vries-Smits AMM 252 

De Vries-van der Zwan A 320 

DeWaalA 342, 611 , 612 

De Waal LP 320 

De Waal Malefijt R 78, 79 

De Weers M 80 

De Weger RA 11 

De Widt J 304 

De Wind N 81 

De Wit R 502 

De Wolf-Peeters C 259 

Debruyne FMJ 375 

Debusscher L 55 

Decoster G 121 

Dekker H 263 

Delaere P 16 

Delemarre JFM 138, 326, 463 

Delius H 75 

Dellemijn TA 362, 603 

Delmer A 259 

Delprat CC 457, 596 

Delwel GO 82, 205, 503 

Demant P 86, 217, 510, 515, 553 

DeminOV 540 

Den Engelse L 31 , 181 , 238, 295 

Den Hollander W 43 

Denijn M 7 

Dercksen MW 281 , 504 

Derix MMA 439 

Deschamps J 309 

Deurloo MJM 23 

Devilee P 61 

Dewit L 83, 344, 345, 563, 601 

Diderich J 227, 612 

Diepenhorst FW 323, 615 

Dijkstra L 431 

Dijkstra MD 505 

Dingemans KP 186 

Dirix LY 266 

Dittrich C 45 

Dizdaroglu M 97 

Dobberstein B 288 

Doisma WV 285 

Domen J 309, 488 

Drexhage HA 153 

Drexler H 349 

Driessens MHE 174, 189, 536, 537 

Drings P 514 

Drukker K 449 

Author index 229 

Dubbelman AC/ R 88, 130, 215, 276, 277, 283, 316, 

357, 595 

Duez N 259 

Dunbar SF 169 

Duncan GF 374 

Dupouy N 55, 211 

DurbanEM 84 

Dusseljee S 54 

Duyts T 499 

Eerenberg AJM 213, 482 

Eggermont AM 148, 161-164, 177, 179, 411 , 506, 542, 

546, 605, 606 

Eghbali H 211 , 258 

Eichholtz T 199 

Eijdems EWHM 39, 468, 507, 508 

Eisenhauer EA 8, 346 

EIbers JRJ 585, 586 

Elias MM 509, 630 

Eliel MR 326 

Elroy-Stein 0 377 

EORTC Early Clinical Trials Group 350 

EORTC Lung Cancer Cooperative Group 339 

EOR TC Study Group on Quality of Life 29 

Eppelbaum R 248 

Esaki K 553 

Essers M 85 

Euroscan Study Group 216 

Eusebi V 67, 122 

Evers LM 330 

Eynon EE 133 

Falke TM 42 

Fargeot P 55 

Farmen RH 374 

Favalli G 8, 346 

Faverly D 122 

Feeney AJ 14 

Feller A 225 

Feitkamp CA 188, 189,229 

Feitkamp MCW 558 

Fentiman IS 632

230 Author index 

Feron VJ 295 

Ferraresi S 204 

Ferreira MR 583 

Festen J 239 

Figdor CG 7, 15, 68, 78, 79, 112, 320,321, 362, 521 

Fijneman RJ 86, 510 

Finney M 98 

Flens MJ 87, 376 

Fles DLA 82 

Fleuren GJ 305 

Franklin HR 88, 161-164,248, 266, 282, 350, 479, 542 

Fraumeni Jr JF 286 

Frijhoff A 362 

Fritz JM 87 

Fukuhara S 368 

Gaarenstroom KN 89,90, 400, 489, 511 

Gallee MPW 3,63, 90, 126,297, 311 , 489 

Galy A 20, 262 

Gandek B 368 

Garavaglia G 91 

Gatzemeier U 45 

Gaulard P 70 

GaverRC 374 

Gavin PR 348 

Gebbink MF 92, 469 

Geiger B 551 

Gelfand MJ 93 

George M 102 

Gérain J 177, 546 

Gerritsen WR 94, 95, 370, 504 

Geuze HJ 571 

Giaccone G 27, 96, 220 

Gielkens ALJ 81 

Gilbert DJ 516 

Gilhuijs KGA 449 

Gimbrère CHF 323, 615 

Gimond C 512 

Ginsberg R 45 

Glimelius B 286, 287 

GoKG 343 

Godfraind C 225 

Godfrey DI 134 

Golding RP 42 

Gommers-Ampt JH 37,97,280, 470 

Gordijn R 71 

Gortzak E 154, 311 

Gospodarowicz M 231, 286, 287 

Gravestein LA 355, 513 

Greco FA 45 

Greenstein D 98 

Gregor A 514 

Gregor RT 18,99-101,278,483, 505,509,561, 596, 

617,627,628,630 

Griffioen G 341, 441 

GroenAK 560 

Groen BK 186 

Groeneveld E 34, 132 

Groeneveld P 257 

GromméM 230 

Groot PC 256, 515 

Guastalla JP 102 

Guiral M 103 

Habets GGM 104, 516, 517 

Habrand JL 138 

Hack CE 213 , 482, 500 

HackerNF 8 

Haga S 553 

Hagemeijer A 516 

HagenbeekA 55, 258,287, 324, 325 

Hahn DEE 518 

Hamers JP 40, 178 

Hamilton JA 147 

Hamilton TC 8 

Hämmerling G 194-196, 203, 230,555 

Hanewald GJFP 437, 621 

Hanks G 464 

Hansen H 8, 45, 602 

Hansen M 8 

Haritz D 563 

Harlan JM 172 

Harper P 45 

Hart AAM 31 , 35, 86, 90, 140, 154, 161 , 163, 164, 235, 

311, 378, 489, 491 , 511 , 515, 528, 535, 542, 548, 553, 

575, 630 

HartMHL 482 

Haustermans K 519 

Hayat M 55 

Heeres K 534 

Heimans 11 520 

Heintz APM 11 

Heitbrink MA 42 

Hekman A 356, 357, 528, 603 

Hellingwerf KJ 105 

Helmerhorst TJM 11 , 43, 63, 75, 89, 90, 297, 378, 400, 

401 , 489, 511 , 549, 599, 608 

Hemker M 612 

HendIer RW 142, 611 

Hendriks RW 80 

Hengeveld T 136 

Henrar REC 76, 77, 141 , 331, 501 

Henriksson R 45 

Henry K 57 

Henry-Amar M 55, 211 , 258 

Hensen A 240 

Hermans RCA 106 

Heukelom S 107 -109 

Heus HA 584 

Hickey D 57 

Hijmans EM 24 

Hilgers FJM 4-6, 16, 18, 19, 99, 110, 111 , 140, 170, 

184,185, 278, 279, 285, 292, 443, 483, 505, 509, 548, 

561 , 596, 617, 625, 626, 628, 629, 630 

Hilgers J 84 

Hilkens J 112, 192, 377, 521 

Hilkmann H 304 

Hil1ebrand MJX 232 

Hilton AM 357 

Hinshelwood S 80 

Hintzen R 113, 330 

Hird S 98 

Ho GH 154 

Hoctin Boes G 514 

Hoefnagel CA 10, 93, 114-120,272, 279, 292-294, 

363-366, 402-405, 427, 450, 451 , 457, 522-526, 582, 

588,589,607,631

Hoekstra HJ 207,312,313 

Hofland I 60,245, 519 

Hofmeyr JHS 372 

Hogervorst F 82, 205 

Holdener EE 121 

Holdrinet A 55 

Holland R 122,632 

Holmberg 0 527 

Holoway E 286, 287 

Holtkamp MJ 310 

Holupka EJ 169 

Hoogendoorn EH 566 

Hoogenhout J 239,285 

Hooiberg E 528 

Hopman EH 401 

Hordijk GJ 285 

Hordijk PL 123, 124, 199,530 

Horenbias S 125-128,201, 357, 375,406 

Hori T 262 

Horiot JC 40, 129, 178, 452 

Hortobagyi GN 208 

Howard JC 196 

Huber H 45 

Huijbens R 79 

Huiskamp R 347,348,563 

Huizenga H 298,459,527 

Huizing MT 27,96, 130,529,578 

HuIsman EH 205 

Hutchison KA 300 

Idzes MHM 298, 527 

Imai S 553 

Inskip P 286 

Inskip PD 287 

Isaacson P 231 

Israels SP 17, 120, 131 

IvanyiD 34,132,297 

Izon DJ 14, 133, 134 

Izquierdo MA 87 

Jacobs H 135 

Jacquemijns M 106 

JagieIIo C 94 

Jagt HHT 294 

Jahde E 173 

Jalink K 123, 136, 199,530 

Janse AJ 531 

Jansen DF 407,432 

Jansen E 33, 393 

Jansen G 139 

Jansen J 396, 397 

Jansen KF J 68 

Jansen PLM 554 

Janssen AGM 120 

Janssen H 54, 172, 180, 209 

Janssen M 301 

Jarrett 0 369 

Jaspars LH 82,205 

Jassem J 532 

Jeneson JAL 610 

Jenkins NA 516 

Jensen PR 137,257,373,533,609,612 

Jereb B 138 

Jiwa M 70,71 

Johansson KA 91, 129,452 

Joiner MC 175 

J olivet J 139 

Jones LA 133 

J ongsma APM 577, 611 

Jonk A 17, 19, 140 

Jonkers J 488 

Jonkman MF 534 

Jonkman-de Vries JD 141,408 

Juretic D 142,611 


Kaasa SI, 29, 368, 480 

Kahn D 143-145,246,260,318,373,609 

Kaijser GP 146 

Kamp F 142, 147 

KampsWA 575 

Kamst E 508 

Kapteijn BAE 148 

Kapteijn CAC 349 

Kapucu 0 118 

Kast WM 355, 558 

Kaufman M 247 

Kavanagh T 172 

Kawakami Y 15 

Kaye SB 8, 266, 282, 346, 350, 479, 502 

Keijer HL 409 

Kelly A 571 

Kemperman H 35, 149,491,535-539 

Kenemans P 33,42,43,63, 150, 151,247,297,401, 

489, 549, 599 

Kenter GG 89,90,305,400,489,511 

Kerger J 479 

Kerkhoven RM 486 

Kern JA 152 

Kerrebijn JD 153 

Kerst JM 500 

Kettenes-van den Bosch 11 76, 141,332,501 

Keung AC 130,529 

Keuning 11 259 

Keus RB 85, 154, 184,292,311,531,561,628 

Keydar I 377 

Kholodenko BN 155-159,257,372,373,533, 540, 541, 

609,612 

Kibbelaar R 420 

Kieft R 37, 180,234 

Kiemeney LALM 

KimEE 208 

Kimman TG 81 

323,615 

Kinnon C 80 

Kirkpatrick A 59, 74, 239, 514, 572 

Klaase JM 148, 160-165, 177,410,411, 542,605,606 

Klaver SG 585, 586 

Kleibeuker JH 341,441 

Kleijmeer MJ 571 

Klein Gebbinck JWTM 174 

Klein JC 166,584 

KIener P 45 

Klepp 0 121 

Kleyne JA 11 

Klokman WJ 325 

KluinPM 72 

Kluin-Nelemans HC 55


Knaken H 235 

Knegt PP 153, 285 

Knoester PD 167 

Koenig K 168 

Kohara Y 98 

Koks CHW 46-50,273,381,382,391,392,412-414, 

440,496-499, 543 

Koler I 602 

Konijnenberg MW 563,564 

Konings MCP 365, 366 

KooIen M 96 

Koomen GCM 601 

Koopman F 51, 529 

Koopman G 113, 330 

Koopman J 192 

Koot M 355 

Kooy HM 169, 462 

Kooy M 319 

KopW 23 

Korse CM 34,89,90,489,511 

Kortes V 31 

Koster EHM 226 

Kowalak JA 97 

Kraaijeveld CL 46, 497 

Krediet R T 600 

Krelenberg R 247 

Kriek E 166, 232 

Kristen P 247 

Kroger R 126,595 

Kroon BBR 17-19,56, 140, 148, 160-165, 170, 177, 

179,243,384,410,411,415,416,417,506,542,546, 

605,606,618-620 

Kroon FP 48 

Krop I 14 

Kropman RF 544,550 

Kruisbeek AM 14,133,134,171,513 

Krul MR 11 

Kuijpers KC 209 

Kuijpers TW 172 

Kuijs S 316 

Kuikman I 82, 205 

KuinA 173 

Kummermehr J 245 

KwaB 192 

La Bail N 266 

La Rivière G 174, 189, 536, 537 

Lacave AJ 557 

Lager D 152 

Laird PW 488 

Lambin P 175 

LangeJMA 69 

Langeveld A 559 

Lanigan D 176 

Lankelma J 103, 202, 263, 376 

Lanson JH 85, 107-109 

Le Fur R 55 

Leader M 57, 176 

Lebesque JV 28,32,66, 178,235,447,491,527 

LeChevalier T 45 

Leeman WR 295 

Leer JW 178, 285, 545 

Lefebvre JL 74, 572 

LegalJD 175 

Lejeune FJ 177, 179,243, 506,542, 546 

Lemerle J 138 

Lens SMA 113 

Lentfer D 33 

Leplège A 368 

Letschert JGJ 178 

Leunens G 91 

Levendag PC 548 

Levin L 8 

Lewensohn R 45 

Lewington VJ 119 

Leyer JPH 378 

Li C 287 

Liénard D 177, 179,506,542,546 

Ligtenberg MJL 37, 180,267 

Limatola C 547 

Lincke CR 577 

Lindblom A 82 

Linders K 309 

Lips CJM 557 

Lister TA 231 

Litvinov SL 112, 521 

Loeffler JS 169 

Loftus BM 57,99, 100, 176,278,505,561,596 

Looijenga LHJ 559 

Los G 181, 182,214 

Lubbers SCM 590 

Lubsen H 548 

Ludwig C 121 

Lund B 8,602 

Lundbeck F 183 

Luurtsema G 343 

Lycklama à Nijeholt AAB 544, 550 

Lynch CF 286, 287 

Maas A 173 

Mackie R 56 

Maes RAA 334,335,363-367,597,598,607 

Majoor D 577 

Mak-Kregar S 184, 185,548 

Malaise EP 175 

Mandard AM 211 

Mandjes I 88, 130,215 

Maniion G 40 

Manni PC 548 

Marchisio PC 204 

Margison GP 166 

Marijnissen JPA 599 

MarkhamD 41 

Martinez de Velasco AM 534 

Mason DY 72, 349 

Mattern C 443 

Mauad TH 186 

McVie JG 65,215,283 

Mead SCW 187 

Meenhorst PL 46-51,381,382,495-499,543 

Meera Khan P 61,341,441 

Meertens H 426, 448 

Meerwaldt JH 55 

Meeuwis CA 153,548 

Meijer CJLM 63, 70, 71, 75, 87, 212, 254,401,549, 

554,593,608

Meijer 0 520 

Meijne AML 149, 188, 189, 536, 537 

Meinhardt W 418, 419, 544, 550 

Melief CJM 355-357, 359, 528, 558, 603 

Mellink WAM 211 

Meloen RH 369 

Menko FH 341 , 441 

Meszena G 190, 191 

Metz H 225 

Meyer S 312 

Michael C 31 

Michalides R 192, 420, 421 , 551 

Michelsen 0 137 

Michiels F 517 

Michiels JJ 55 

Michielsen C 12 

Miettinen N 67 

Mijnheer BJ 85, 91 , 107-109, 265, 298, 453, 454, 459, 

464, 527,564 

Milano GA 219 

Millis RR 122 

Milner CM 255 

Minke JM 132 

Möbus V 247 

Modderman PW 193 

Moen CJA 515 

Mohan-Peterson S 79, 340 

Mol CAAM 242, 576, 577 

Molenaar D 257, 533, 540, 612 

Molthoff CJM 43 

Momburg F 194-196, 203, 230, 555 

Monconduit M 55, 211 , 258 

Mooi WJ 17, 19, 140, 185, 192, 197, 317, 420, 421 , 

577, 585, 586, 599 

MooIenaar K 420 

Mooienaar WH 92, 123, 124, 136, 198, 199, 530, 547, 

552 

Moonen L 200, 201 

Moore PF 284 

Mooren E 60, 249, 574 

Morabito A 56 

Morii N 136, 553 

Muggia FM 31 

Mülder HS 202, 376 

Mulder JW 48, 49, 51 , 381 , 382, 496-499 

Mulder MP 559 

Mulder NH 554 

Mulkern RV 169 

Muller EJ 504 

Müller M 90, 554 

Muller SH 32, 66, 447 

Münch M 20, 21 , 484 

MurrayN 45 

Murre C 14 

MuselIa R 55 

Naeije M 395 

Nagengast FM 341 , 441 

Nederlandse Melanoom Werkgroep 417 

Nap M 428 

Narumiya S 136 

NeefC 498 

Neefjes JJ 54, 194-196, 203, 209, 210, 230, 555, 558, 

571 

Neering H 17, 422, 619 

Neijens V 556 

Neijt JP 8, 557 

Neisig A 558 

Neuteboom GHG 440 

Newling DW 126, 127 

Niederle N 45 

Nieland JD 134, 513 

Nielsen AL 350 

Niessen CM 204, 205, 534, 539 


Nieweg OE 170, 206-208, 343, 423, 455, 461,620 

Nijenhuis M 54,209,210 

Ninnin D 101 

Nishizawa Y 290 

Nooij M 102 

Nooijen WJ 310, 331 , 332, 573, 578, 579 

Noordijk EM 55, 211 

Noorduyn LA 70, 212, 593 

Nooter K 52, 53 

Nordeng TW 209 

Nortier YLM 367 

Norton A 231 

Notenboom RGE 186 

Noyon R 324-326 

Nuijens JH 500 

Nuyten MJC 201 

O'Brien J 229 

O'Reilly RJ 95 

Oei SS 628 

Offerhaus GJA 186, 341 , 441 

Ogilvie AC 213 

Okumoto M 553 

Olavesen MG 255 

Oldenburg J 214 

Oldenburg N 137, 373 

OldhoffJ 207, 542 

Olie RA 559 

Oomen LCJM 34, 510 

Oosterhof GON 375 

Oosterhuis JW 559 

Oostrum RG 53 

OphoffRA 86 

Oppelaar H 12, 13, 314, 592 

Opstellers 436 

Oskam W 341 , 441 

Ossendorp F 558 

Ossenkoppele GJ 71 , 212 

Osterhaus AD 369 

Osterlind A 17 

Ottenheim C 60, 507 

Ottenhof R 560 

Otter R 323, 615 

Oude Elferink RPJ 186, 560 

Oudejans JJ 70 

Oussoren YG 183, 222, 268, 314, 562, 592 

Overath P 267 

Owaribe K 290, 534 

OzaAM 215 

Ozols RF 8 

Paans A 173, 207


Padbury GE 331 

PaIs ST 113,330 

Parmentier C 175 

Pas HH 534 

Pasternack BS 168 

Pastorino U 45,216 

Patriarca C 521 

Paulsson M 82 

Pavel S 120 

Pavlidis N 266, 350 

Pavy JJ 40 

Peeters BPH 81 

Pelissier EP 40 

Pels EM 331 

Pers on PL 217 

Peters GJ 139,218-220 

Peterse JL/H 35,61,67, 122,236,421,428,456,461, 

531,535,581,622,632 

Petra B 137 

Pfauth A 369 

Philipp K 348, 563 

Phillips JH 262 

Piccart M 8, 282, 350 

Pieters J 288, 289 

Pijpers H 11 

Pinard MF 139 

Pinedo HM 27,41,96, 121, 130, 181, 182,213,214, 

220,263,316,324,376,482,492,504,529 

Pizao PE 220 

Plaat BEC 561 

Planting A 602 

Plasterk RHA 98,221,223,303,327,328,351-354, 

604 

Ploegh HL 288 

Poggensee M 60 

Portegies P 439 

Post JG 222, 562 

Postmus P 45,96,514 

Pottie G 519 

Power M 494 

Priario J 41 

Prins F 72 

ProveAM 282 

Pruim J 207 

Punnonen J 20 

Puras Lutzke RA 223, 353 

Raaijmakers CPJ 563, 564 

Raat NJH 270 

Rafferty JA 166 

Rajewsky MF 173 

Raleigh M 172 

Ramaekers M 519 

Rampen FHJ 17 

Rankin EM 17,120,224,356-359,565,566,603 

Rastogi R 479 

Razavi D 368 

Recht A 456, 632 

Reed JC 72 

Regnier R 55 

Remmink A 401, 549, 608 

Renard G 121 

Renard J 102 

Renauld JC 225 

Renckens CNM 424 

Reubsaet JLE 226 

Richard P 227,228,271,533,609 

Richel DJ 310,569 

Riethorst A 611 

Rigal-Huguet F 55 

Rinaldi M 514 

Risse EKJ 75 

Robanus Maandag EC 14,229,242,309 

Robinson R 152 

Rodenhuis S 152,310, 317, 360, 504, 559, 567-569, 

573,585,586,597 

RoeIen HCPF 166 

Roelfsema F 444 

Roelofsen B 253 

Roelse J 195, 196,230,555,558 

Rohatiner A 231 

Rohwer J 533,612 

Rojas M 232 

Roncarolo M -G 20, 21, 340, 484 

Roodenburg JLN 548 

Rookus MA 233 

RoosCM 32 

Roos D 172, 500 

Roos E 149, 174, 188, 189,536-539 

Roos JC 43 

Ros JJW 419 

Rosell R 45 

Rosenberg SA 15 

Rosenkaimer F 177,179,506,546 

Rosing H 51, 130,316,335,366,367,499,529,543, 

598,602 

Rosner JL 611 

Roth S 55 

Rouesse J 41 

Royston D 176 

Rubens R 275 

Rudenko G 37,38,234 

Ruevekamp M 214,599 

Ruiter DJ 7,68,417 

Ruka W 41 

Russell NS 235 

Rustin GJS 8 

Rutgers EJT 154,236,311,360,379,461,596,614 

Rutgers M 237, 570, 587 

Rutten EHJM 55 

RuvkunG 98 

Sabourin J-C 70 

Sabt B 57 

Sagiv D 377 

Sahmoud T 557, 572 

Salem P 231 

Sanchez M -J 262 

Sanderson F 571 

Sanson-Fisher R 368 

Santoro A 41 

Saris CP 238 

Sauro HM 157 

Scagliotti G 45 

Schaaf LJ 331 

Schaafsma HE 375

Schaake-Koning C 17,239,240 

Schaap D 241,296,547 

Scheffer GL 87 

Scheil D 267 

Schellens JHM 335, 598 

Scheper RJ 87,376,554 

Scherer E 295 

SchinkelJ\H 39,186,242,253,560,576,577 

Schipper J 544 

Schipper MEI 75 

Schlissel MS 14 

Schmidt-Rhode P 247 

Schmitz P 177, 546 

Schoenmakers HJ 355 

Schols D 20, 340 

Scholtes EHM 104 

SchornagelJH 31,59,74,139,214,219,310,569,572 

Schouten LJ 323,615 

Schouwenburg P 285 

Schraffordt Koops H 41, 177, 179,207,243,506,542, 

546 

Schreurs MW J 15 

Schrier PI 244 

Schultz-Hector S 245 

Schuster L 514 

Schuster S 246, 373 

Schutter EMJ 42, 247 

Schuuring E 421 

Schuurman B 313 

Schuurman RKB 80 

Schwartz DJ\ 152 

Scotto V 102 

Sculier JP 45 

Sein JJ 356, 528, 603 

Sessa C 8, 121,248 

Shipp M 231 

Shore RE 168 

Shumaker SJ\ 58 

Siefert J\ 563 

Siegen thaler P 121 

Sijts EJJ\M 471, 558 

Siilevis Smitt H 491 

Simonetti GPC 88 

Singletary SE 208 

SipS JHM 573 

Slaper-Cortenbach lCM 310 

Slater R 575 

Slebos RJC 152 

Sleyfer DT 502 

Sluyser M 343 

Smeets MFMJ\ 60,249,574 

Smets LJ\ 173,237,250,251,300,301,570,575,587 

Smit JJM 39, 186,242,253,560,576,577 

Smit L 252 

Smith J\J 39, 253 

Smitskamp-Wilms E 220 

Smolders IJ 348 

Smyth JF 248,266 

Snethlage RJ\I 3 

Snijders PJF 75, 254, 549, 608 

Snoek M 217,255,256,510,515 

Snoep JL 257 

Snow GB 59,302 

Soepenberg 0 215,446 

Sofroniew M 309 

Sohn C 247 


Somers R 16,55,211,231,240,258,259,324-326,463, 

600 

Sonnenberg J\ 82,84, 193,204,205,290,503,512, 

534, 539 

Sonneveld P 52, 53 

Sonneveldt J\L 332 

Sonnhammer ELL 260, 270 

Souren T 6 

SoutterWP 8 

Spaander P 502 

Sparreboom J\ 578, 579 

Spits H 20,21, 113,261,262,340,484 

Splinter T J\ W 45, 557 

Spoelstra EC 263 

Sprangers MJ\G 1,69,264,395,399,425,480,580 

Springall D 192 

Stahel R 45 

Stam JC 104, 517 

Stavenuiter JFC 106, 280 

Steenbergen RDM 254 

Steggerda MJ 265, 426 

Stern PL 63 

Sternberg CN 266 

Steverding D 180, 267 

Steward W 41,282 

Stewart FJ\ 12, 13, 183,222,268,269, 314,532,562, 

592, 599 

StierhofY-D 267 

Stiggelbout J\M 326, 463 

Stoffers HJ 270, 271 

StokkelMPM 272 

Storb U 171 

Stoter G 52, 53, 502 

Stovall M 287 

Studeny M 343 

Stuijt CCM 273 

Stukart MJ 63 

Sulkes J\ 248 

Sullivan M 29,368 

Sund M 245 

Sweeb RK 274 

Sylvester R 275, 502 

Symann M 45 

TaalBG 88,264,276,277,341,366,380,396,397, 

441,557,580-582 

Taat CW 313 

Takes RP 278, 279, 292, 427 

Talsma H 76, 141 

TanMCM 472 

Tanguy J\ 55 

Taphoorn MJB 520 

Tarayre M 258 

Tarbell NJ 169 

Tay LK 374 

Taylor MC 37,234 

Te Poele JJ\ 222, 562 

Te Riele H 14, _,229,242,309 

Te Velde J\ 264,580 

Teeuwsen J 226,308


Teixeira AJR 106, 280, 583, 584 

Teller FGM 276, 277 

Ten Bokkei Huinink WW 8,26,27,88,96, 102, 121, 

130,215,248,266,276,277,281,282,283,293,316, 

334,346,350,446,479,502,556,569,573,588,595, 

602 

Ten Hoeve R 293, 556, 588 

Ten Napel CHH 498, 499 

Terhaard CHJ 285 

Terheggen PM 31 

Teske E 284 

Teuscher C 217,255 

Teusink B 227,228,533,612 

Thatcher N 45 

Theunissen EBM 313 

Thigpen JT 8 

Thijs LG 213 

Thomas CMG 428 

Thomas D 41 

Thomas J 55,211,258,259 

Thyss A 55 

Tiel-van Buul MMC 294 

Tijssen S 431 

Timmerman AJ 508 

Timmermans-Hereijgers JLPM 253 

TimplR 82 

Tirelli U 211, 259 

Tjho-Heslinga RE 285 

Tolkamp L 135 

Tomee C 369 

Tonato M 45 

Tonioio P 168 

TopB 152,251,254,317,559,585,586 

Tournade MF 138 

Travis LB 286,287 

Trimbos JB 89,90,400,511 

Tropé C 8 

Trowsdale J 571 

Tucker MA 286, 287 

Tueni EA 59 

Tulp A 54,288,289, 571 

Turrisi A 45 

Tursz T 282 

Twentyman PR 507 

Twijnstra A 434, 435 

Tytgat GAM 587 

Tytgat GN 581 

Uematsu J 290 

Underberg WJM 50, 77, 146, 167,226,307, 308, 367, 

381,382,590 

Vaalburg W 207,343,423 

Valdés Olmos RA 10,32,83, 117, 120,272,279, 

291-293,345,427,429,451,457,473,525,526,556, 

582, 588 

Van 't Hullenaar R 7 

Van 't Veer LJ 244,430 

Van 't Veld AA 458 

Van Apeldoorn L 589 

Van Ark -Otte J 220 

Van Balen P 431 

Van Barneveld TA 407,432,433,436 

Van Battum LJ 459 

Van Beek EJR 294 

Van Benthem J 295 

Van Blitterswijk WJ 199,241,253,296,304,547 

Van Bommel PFJ 63,297 

Van Boom JH 166 

Van Bree NAM 298, 459 

Van Coevorden F 311-313,378,531 

Van Corven EJ 123, 124, 136, 199 

Van DaleA 34 

Van Dam FSAM 69, 187,299,424,425,437,439,460, 

518, 621 

Van Dam J 464 

Van Dam K 227,228,373 

Van Dam PJH 449 

Van de Merwe SA 165 

Van de Pol M 434,435 

Van de Sandt MM 212 

Van de Vijver MJ 61, 122 

Van de Werken G 106,280,583,584 

Van de Wiel-van Kemenade E 68,112,521 

Van Deemter L 39,242,576 

Van den Belt-Dusebout A W 323-325,615 

Van den Berg J 251,300,301 

Van den Bergh Weerman MA 186 

Van den Berk PCM 528 

Van den Bogaert W 239 

Van den Boom FM 187 

Van den Brekel MWM 302 

Van den Brug M 60 

Van den Brule AJC 75,549,608 

Van den Elshout MM 300 

Van den EIst H 166 

Van den Ende AM 238 

Van den Ent FMI 303 

Van der Beek JMH 548 

Van der Belt-Dusebout AW 326 

Van der Bend R 199,296,304 

Van der Bijl AE 305 

Van der Burg MEL 8 

Van der Does-van den Berg A 575 

Van der Elshout MM 301 

Van der Gugten AA 306,373,533,540,612 

Van der Heijde JF 50 

Van der Houwen OAGJ 307,308,590 

Van der Kammen RA 104, 516, 517 

Van der Kogel AJ 269 

Van der Kuij V 130 

Van der Linden KH 354 

Van der Lugt N 225,229,309,474,488 

Van der Maas PJ 73 

Van der Marel GA 166 

Van der Meer JB 534 

Van der Mey AGL 548 

Van der Ploeg E 343 

Van der Sanden GAC 436 

Van der Schans GP 508 

Van der Schoot CE 310,330,500,504 

Van der Schueren E 129,452 

Van der Valk MA 13, 14,229,242,309,553,592 

Van der Valk P 70, 71, 593 

Van der Valk S 112,521 

Van der Voet JCM 201


Vos HL 112,377,521 

Vos JC 604 

Voûte PA 117, 118, 138,237,451, 525, 526, 587 

Vroege JA 361 

Vrooland JL 440 

Vrouenraets BC 605,606 

Vyth-Dreese FA 362,603 

Wacholder S 286,287 

Wafelman AR 363-367,478,607 

Wagenaar E 39, 242, 576 

WagenerT 41 

Wagner A 368 

Wagstaff JW 213, 482 

Walboomers JMM 63, 71, 75, 254, 401, 549,608 

Wambersie A 464 

WandersJ 248,282,350,479 

Wanders S 266 

Wanebo HJ 17 

WangB 98 

WareJE 368 

Warnier G 225 

Waterval JCM 226 

Watlcins PR 347 

Weijer K 369 

Weimar IS 370 

Weiner D 152 

Weintraub BC 14 

Welvaart K 409 

Wesseling J 112, 521, 539 

WesterhoffHV 62,64,103, 105, 137, 142-145, 147, 

155-159,190,191,202,227,228,246,257,263,270, 

306,318,342,371-373,533,540,541,609-612 

Westermann R 247 

Westra JG 106, 238, 280, 583 

Wheeler F J 347 

WhelanA 57 

WHO Melanoma Pro gramme 56 

Wielinga P 612 

WigboutG 88 

Wijdenes J 528 

Wijker JE 612 

Wijnands Y 149,538,539 

Wiklund I 58 

Wildiers J 59,572 

Wilkin D 9,481 

Willemze R 70 

Willey TA 374 

Wilmer JWGM 295 

Wils JA 102 

Winkelhorst J 77 

Wirtz KW A 253 

Wisman P 284, 585, 586 

Witjes WPJ 375 

Wobbes T 428 

Woerdenbag HJ 445 

Wolbers J 520 

Wolbrink GJ 482 

Woldring MG 423 

Wolffl 248 

Wolfs PE 378 

WongWH 208 

Wood-Dauphinee S 368 

Woodward SR 217 

Wreschner DH 377 

Wubbolts R 54 

Zafrani B 122 

Zaman GJR 39,52,53,87,376,507,554 

Zasloff M 142, 611 

Zhrihan-Licht S 377 

Zhu L 24,486 

Zijderveld A 81 

Zijlstra M 355 

Zittoun R 258 

Zoetmulder FAN 311,378-380,613,614 

Zomer G 106 

Zondag GC 92 

Zurawski G 79 

Zuydgeest D 104 

Zwartendijk J 550 

Zwinderman AH 544

Personnel-Project Index 

Aalders M p 84 

Aaronson NK p 114, 126, 127 

Aartsen EJ p 117, 118, 122 

Abbink EM P 126 

Ackerstaff AH p 114 

Acton D p 74, 77 

Akagi Kp 78 

Alblas J p 34 

Aleman B p 104 

Alkerna Mp 74 

Allen J p 74 

Arnsen D p 49 

Ansink AC p 117 

Arisz Lp 98 

Atsma D p 67 

AwwadHp89 

Baars JW p 102, 103, 105 

Baas F p 58 

Baas P p 86, 96, 104 

Baidjnath Panday R p 107, 115, 123 

Bais EM 105 

Bakker AQ p 49 

Bakker BN p 61 

Bakker Mp 49 

Bakx R p 132 

Balm AFJ p 108 

Balm AJM p 67, 89, 103, 114, 120, 122 

Bartelink H p 89, 93, 98, 99 

Batchelor D p 106, 120 

Beenen L p 118 

Begg AC p 87, 88, 89 

Beijersbergen RL p 28 

Beijnen JH p 102, 105, 107, 109, 110, 115, 121 , 122 

Bel A p 93, 95 

Benraadt J p 129 

Benraadt T p 99 

Bernards R p 28 

Berns A p 38, 106 

Besnard P pilS 

Bierhorst JF p 120, 132, 133 

Bijen CSM p 130 

Bijker N pIlS 

Bitter W p 57 

Blackburn C p 118 

Blankensteyn JD p 120 

BlomAp44 

BlomB p 49 

Blommaert FA P 32 

Blommestijn GJF p 138 

Blundell P p 57 

Boellaard RB P 92 

Boer M p 64 

Boerrigter-Barendsen LH p 68, 82 

Boersma LJ p 96, 108 

Boice JD p 129 

Bonfrer JM G p 106, 109, 117, 120 

Bontenbal Mp 129 

Boogerd W p 105 

Boot E P 83 

Boot H P 104, 107, 118 

Borger JH p 93,99, 103, 115 

Borst J p 38, 77 

Borst P p 57, 58 

Bos JK p 130 

Bos KE p 114 

BosmaA p 66 

Braas PAM p 128 

Brady Hp 74, 77, 78 

Breedijk AJ p 68, 103, 106 

Brink G p 68, 82 

Broeks A p 54, 55 

Broug M p 132 

Brouns GS p 38 

Brouwers C p 74 

BruceAM p93 

Bruggink EDM p 120 

Bruinvis IAD p 92, 95 

Bruning PF p 44,68, 106, 108 

Buitelaar AC p 126, 128 

Buitenhuis CKM p 85 

Buitenhuis M p 134 

Burger DM pilO 

Burgers JMV p 105, 129 

Buurman W p 121 

Cal afat J p 26, 40 

CehaH p 114 

ChinLMp92 

Chorus AMJ p 128 

Coco-Martin JM p 88, 89 

Collard JGNM p 22 

Colloms SD p 54 

Craanen ME p 82, 104 

Cromme FV p 117 

Cross Mp 57 

DaamsJH p 61 

Dalesio OB p 132, 133, 134, 135 

Damen EMF p 95, 96 

Damen I p 22 

De Baere IJL p 54 

Personnel-Project Index 239

240 Personnel-Project Index 

De Boer JB p 114, 115, 127 

De Boer RW p 95, 96 

De Flora S p 104 

De Gast GC p 44 

De Goeij CCJ p 66 

De Haas M p 58 

De Jong D p 68,104 

De Kraker J p 107, 108 

De Kwant M p 104 

De Lange C p 22 

De Lange MS p 82 

De Melker AA p 24 

De Moes J p 73 

De Moes-Bezemer A p 73 

De Munck JC p 93 

De Rond MEJ p 128 

De Vries Ep 38 

De Vries N p 104 

De Vroomen MJ p 55 

De Weers M p 38 

De Widt JMM p 37 

De Wind Np 29 

De Wit R p 105, 128 

Deen-Ruiter E p 135 

Dekker-Vlaar HMJ p 29 

Dellemijn TAM P 44 

Delwel GO p 24 

Delzenne-Goette E p 73 

Demant P p 72, 73, 74 

Den Hartog YM p 128 

Dercksen MW p 103 

Dercksen WM p 44 

Detmar SB p 126 

Dewit L p 98, 108 

Dijkstra JS p 135 

Dirks-Mulder A p 57 

Driessens MHE p 20 

Dubbelman AC p 102 

Dubbelman RD 

Dusseljee S p 40 

Ebbers RGE p 20 

Eggermont AMM p 121 

Eijdems EWHM p 58 

Engelsman E p 128 

Essers M p 84 

Essers M p 92 

Eynon EE p 48, 49 

Fase-Fowler F p 57 

Feiken E p 36 

FeItkamp CA p 20 

Fichtinger-Schepman AM p 32 

Fijneman RJA p 72, 73 

Fles DLAp 24 

Fletcher J p 134 

Floore AN p 68, 74 

Fraass BA p 92, 95 

Franke B p 22 

Franklin HR p 121 , 134 

Gaarenstroom KN p 109, 117 

Gallee MPW p 68, 106, 117, 120 

Gebbink MFGB P 36 

Gennissen AMC p 28 

Gerbrandy F p 66 

Gerritsen WR p 44, 103, 106, 120 

Giaccone G p 104 

Gil Gomez G p 74 

Gilhuijs KGA p 93 

Gimond CML p 24 

Gonggrijp S p 114 

Gonzalez Gonzalez D p 99 

Gortzak E p 104, 115, 118, 120 

GrafD p 49 

Gravestein LA p 38 

Gregor RT p 114 

Groeneveld E p 67 

GromméMp40 

Groot PC p 74 

Gualthérie van WeezeI LM p 105,128 

Guirol M p 61 

Guvenc B p 138 

Habets GGM p 22 

Hack C p 121 

Hageman Ph p 67 

Hagenbeek A p 129 

Hajduk S p 57 

Haks MC p 48, 49 

Hamel M p 48, 49 

Hämmerling GJ p 40 

Hanewald GJFP p 127 

Hart AAM p 99, 106, 114, 120, 121 , 128 

Hassink G p 22 

Hateboer G p 28 

Haustermans K p 89 

Heemskerk M p 44 

Heesbeen EC p 84 

Heijdendaal M pIlS 

Heintz APM p 117 

Hekman A p 44, 106 

Hellendoorn-Smit M p 105 

Helmerhorst ThJM p 86, 117, 119 

Hengeveld GM p 34 

Heukelom S p 92 

Hiemstra A p 132, 133 

Hijmans EM p 28 

Hijgers FJM p 114 

HiJkens JGW p 64, 109, 115, 122 

Hilkmann HAM p 37 

Hillebrand MJX p 109, 110 

Hoefnagel CA p 85, 92, 98, 104, 107, 108, 115, 123 

Hoekstra H p 120, 121 

Hofland I p 89 

Hofland N p 66 

Hogema K p 132 

Holtkamp MMJ p 103, 128 

Hooijberg E p 44 

Hopman EH p 117 

Hordijk PL p 34 

HorenbIas Sp 119, 122, 124 

Houssa B p 37 

Hoving Sp 61 

Huber 0 p 48 

Huijer Abu-Saad H p 128

Huiskamp R p 87 

Huisman H p 126 

Huizenga H p 95 

Huizing MT pilO 

HuIsman EHM p 24 

Idzes MH p 93, 95 

Imak Z p 138 

Imamura F p 34 

In 't Veld GJ p 105 

Israëls BP p 107 

Israëls SP p 106, 120 

Ivanyi D p 67, 89, 117 

Izon DJ p 49 

Jacobs H p 38, 77 

Jaleco AC p 49 

Jalink K p 34 

Janse AJ p 120 

Jansen DF p 130 

Jansen Ep 109 

Jansen G p 55, 85 

Jansen Hp 40 

Jansen I p 132 

Janssen JWRM p 26 

Jenkin L p 134 

Jensen PR p 61 

Jongsma APM p 58, 61 , 62 

Jonkers J p 74 

Jonkman-de Vries JD pIlO 

Kaas R p 115 

KainHp22 

Kapteijn BAE p 107, 115, 121 , 123 

Kemperman H p 20, 99 

Kenemans P p 117 

Kennedy E p 74 

Kerkhoven RM p 28 

Kerrebijn JD p 114 

Ketting RF p 54 

Keus RB p 93, 95, 96, 108, 114, 120 

KhanumAp 34 

Kho1odenko BN p 61 

Kieft R p 57 

Kincade PW p 49 

Klaase JM p 120, 121 

Klaver E p 134 

Klaver SG p 82 

Klein JC p 31 

Kleine Budde I p 20 

Klievink R p 128 

Klokman W p 129 

Klompmaker R p 66 

Klop M p 121 

Knaken HJ p 89 

Koelman M p 96 

Koks CHW pIlO 

Konijnenberg MW p 98 

Koningstein G p 36 

Kool M p 58 

Koopman J p 66 

Kooy Hp 93 

Korbee LJ p 138 

Korse CM p 109 

Korse T p 106 

Korswagen HC p 55 

Kriek E p 31 

Krimpenfort P p 74, 77, 78 

Kristel P p 66 

Kristensen LB p 121 

Kroczek RA p 49 

Kroes APG p 93 

Kröger R p 104 

Personnel-Project Index 241 

Kroon BBR p 106, 114, 115, 120, 121 , 122, 124 

Kroon C p 110 

Kroon FHM p 114 

Kruisbeek AM p 44, 47, 48, 49, 106 

Kruit J p 135 

Kuikman I p 24 

KuinAp 84 

Kürten NJ p 68 

Kwa B p 66, 115, 123 

Kwa SLS p 96 

La Rivière AG p 20 

Lafleur JK p 31 , 115 

LafleurMVM 

Lambrechts AC p 82 

Lanson JH p 92, 93, 95 

Lebesque JV p 93 , 95, 96, 99, 103, 108 

Leemans ChR p 114 

Leenhouts GHMW p 128 

Lejeune FJ p 121 

Letsehert JGJ p 96 

Leupers CJM p 48 

Liefkens K p 47 

Liem IH p 107, 108, 115, 123 

Liénard D p 121 

Limatola C p 37 

Linders D p 106 

Linders TC p 109 

Linthorst GAM p 103 

Loftus-Call BM p 104, 120 

Loman L p 61 

Maas MCE P 64 

Maas RA p 68 

Maas RR p 106 

Maessen H p 107 

Mak-Kregar S p 114 

Mandjes I p 132 

Mangal Ep 86 

Marijnissen JPA p 86, 104 

Martinez de Velasco AM p 24 

Mártinez-Cáceres E p 49 

Masselink JHB p 88 

Mattern Cp 105, 128 

McCulloch R p 57 

Meijer CJLM p 117 

Meijer M p 104 

Meijer S p 124 

Meijne AML p 20 

Meinhardt W p 119 

Melief CJM p 40, 44 

Mester J p 66 

Meyer M p 132


Meyer S p 120 

Michael C p 32 

Michalides RJAM p 66 

Michiels GAM p 22 

Mijnheer BJ p 92, 93, 95, 98 

Minderhoud TJ p 95 

Modderman PW p 24 

Moerman N p 128 

MolCAAMp 58 

Molenaar D p 61 

Molthoff CFM p 109 

Momburg F p 40 

Monnee M p 44, 47 

Mooi WJ p 66, 67, 68, 82, 104, 120, 123 

Mooij TM p 130 

Mooienaar WH p 34, 36, 37 

Moonen LMF p 89, 99 

Mooren EHM p 88 

MoriNp74 

Moss RL p 98 

Muller EJ p 44 

Muller MJ p 126, 128 

Muller SH p 96, 107, 108, 115, 123 

Muriana FJG p 37 

Neefjes JJ p 40 

Neelakanden S p 134 

Neering H p 120 

Neisig A p 40 

Nieland JD p 48 

Niessen CM p 24 

Nieweg MG p 128 

Nieweg OE p 120, 121 , 122 

Nijenhuis M p 84 

Nooijen WJ p 98, 103, 106, 109 

Noordhout C p 132 

Noort J p 128 

Noyon R p 129 

Oei SJ p 68 

Offringa R p 48 

Ogilvie AC p 105, 121 

Oomen LCJM p 138 

Oosterwegel M p 49 

Oosting H p 128 

Oppelaar H p 86 

Orsini D p 44 

Ottenheim CPE p 88 

Oussoren Y p 86, 90 

Paalman ACA pIlO 

Pastoors EB p 34 

Pastorino U p 104 

Patriarca C p 64 

Peterse JL p 64,68, 106, 115, 123, 128, 129 

Pettitt J p 55 

PfauthA p 49 

Pieters J p 47 

Plaat BEC p 114 

Plasterk RHA p 54, 55, 56 

Pool GA p 121 

Post JG p 90, 119 

Postma F p 34 

Pruyn J p 115 

Puras Lutzke RA p 56 

Raaijmakers CPJ p 98 

Ramakers GJA p 34 

Rankin EM p 44, 105, 106, 107, 120, 121 

Regnerus R p 74 

Reiss P p 127 

ResPCM p49 

Retel J p 31 

Rezsohazy R p 54 

Riethorst A p 58, 61, 62 

Rijken P p 86 

Rijswijk L p 78 

Rinke de Wit T p 48 

Robanus Maandag EC p 78 

Rodenhuis S p 82, 102, 103, 104, 105, 128 

Rodriguez Erena NF p 20 

Rohwer JM p 61 

Roodbergen GC p 102, 103, 128 

Rookus MA p 128 

Roos CM p 96 

Roos E p 20 

Rosing H pIlO 

Rots E p 24 

Rudenko G p 57 

Ruevekamp MC p 86 

Ruggiero G p 48 

Ruiter G p 22 

Russell NS p 96 

Rutgers EJTh p 105, 115, 120, 118, 120, 122, 124 

Rutgers M p 85 

Ruuls-Van Stalle EMF p 20 

Sakai H p 86 

Salomons GS p 83 

Sánchez-Aparicio MP p 24 

Sanderson F p 40 

Saris CP p 31,32 

Sasco AJ p 130 

Schaake-Koning CCE p 99, 104, 120, 128 

Schaap D p 37 

Schaefer B p 132, 134 

Schaefers M p 132 

Schaesbergen W p 44 

Scherer E p 30 

Scheyen G p 74 

Schinkel AH p 58 

Schippers-Gillissen C p 29, 73 

Schlax C p 47 

Schornagel JH p 32,84,85, 102, 103, 126 

Schraffordt Koops H p 121 

Schuitmaker JJ p 86 

Schuster-Uitterhoeve Lp 99 

Schuurman B p 120 

Sein JJ p 44 

Shouman T p 89, 93 

Simmen KA p 28 

Slaper-Cortenbach lCM p 103 

Slee CJP p 138 

Sluyser M p 66 

Smeets MFMA p 88 

Smets LA p 32, 83, 84, 85

Smit JJM p 58 

Smit L p 38 

Smith AJ p 58 

Smith L p 38 

Smith-S Drensen B p 28 

Smolders UH p 87 

Sneeuw KCA p 126 

Snijders F p 127 

Snoek M p 72 

Snoep J p 61 

Sombroek G p 132 

Sombroek HJC p 120 

Somers R p 105, 120, 129 

Sonnenberg A p 24 

Sparreboom A p 109, 110 

Spits H p 44, 48, 49, 106 

Sprangers MAG p 126, 127 

Staal FJT p 49 

StamJC p 22 

Star W p 104 

Stassen APM p 73, 74 

Stecher-Rasmussen F p 98 

Stegeman B p 61 

Steggerda M p 93 

Sterk L p 24 

Stewart FA p 86, 90 

Stoffers HJ p 138 

StormJ p 64 

Stovall M p 129 

Stro eken PJM p 20 

Stroom J p 93 

Stukart MJ p 117 

Taal BG P 104, 107, 118 

Taat CW p 120 

Takes RP p 114 

Taylor MC p 57 

Te Poele JAM p 90 

Te Riele HPJ p 29, 78 

Te Velde A p 126 

Te Velde JL p 132, 133, 134, 135 

Teixeira A p 31 

Ten BokkeI Huinink WW p 102, 104, 108, 117, 133 

Teusink B p 61 

Theuws J p 96 

Thijssen JHH p 128 

Thingstad T p 64 

Tirnmers AP p 114 

't Mannetje LWC p 86 

Tol 0 p 40 

Tölg C p 78 

TopAp82 

TouwAp93 

Travis LB p 129 

Treur-Mulder M p 73 

Trowsdale J p 40 

Tulp A p 40, 41 

Valdés Olmos RA p 96, 98, 104, 107, 108 

Valkenet L p 132, 133 

Van 't Veer LJ p 68,82, 104 

Van 't Veld AA p 95 

Van Asperen J p 109, 110 

Van Barneveld TA p 130 

Van Bleek GM p 44, 47 

Van Blitterswijk WJ p 37 

Van Bommel PFJ p 117 

Personnel-Project Index 243 

Van Bunningen BNFM p 117 

Van Buuren JF p 128 

Van Camp en BThM p 128 

Van Coevorden F p 104, 115, 118, 120, 124 

Van Dalen AJ p 93 

Van Dam FSAM p 105, 126, 127, 128 

Van Damme S p 64 

Van de Kastee1e W p 44 

Van de Merwe SA p 121 

Van de Pavert IV p 138 

Van de Vaart PJM p 89, 99 

Van de Ven PJH p 93 

Van de Werken G p 31 

Van Deemter E p 58 

Van den Belt-Dusebout AW p 128, 129 

Van den Berg JD p 83 

Van den Berk P p 44 

Van den Brand M p 83 

Van den Ende AMC p 31 

Van den Ent FMI p 56 

Van der Broek R p 134 

Van der Donk E p 134 

Van der Flier A p 24 

Van der Gugten AA p 61 

Van der Gulden H p 74 

Van der Heijden CAM p 128 

Van der Horst G p 38 

Van der Kammen RA p 22 

Van der Kooy K p 128 

Van der Linden KH p 55, 56 

Van der Lugt NMT p 74 

Van der Neut R p 24 

Van der Schoot E p 44, 103 

Van der Valk MA p 73, 74 

Van der Valk R p 41 

Van der Valk SW p 64 

Van der Wal J p 37 

Van der Wall Ep 102, 103, 128 

Van der Weijden CC p 61 

Van der Woude HR p 109 

Van der Zee J p 121 

Van Dijk MAJ p 68 

Van Dijk MCM p 37 

Van Dijk T p 115 

Van Dijk WJ p 31 

Van Dongen JAp 99,114,120, 121 , 128 

Van Dooren S p 61 

Van Doorn RC p 120 

Van Doornewaard G p 67 

Van Drimmelen RO p 138 

Van Eekeren M p 118 

Van Etten 1 p 34 

Van Geel AN p 115, 120, 121 

Van Geel IPJ p 86 

Van Gijn R p 110 

Van Heerde P p 66 

Van Heeswijk WC p 61 

Van Herk M p 93 

Van het Hof AJ p 66


Van Leeuwen FE p 115, 128, 129, 130 

Van Leeuwen F p 57 

Van Leeuwen FN p 22 

Van Luenen HGAM p 54 

Van Mourik JA p 98 

Van Oene S p 110 

Van Ravensbergen Sp 118 

Van Rhee P p 132 

Van Rijthoven EAM p 20 

Van Rooij Hp 83 

Van Roon M p 74, 78 

Van Roosmalen C p 115 

Van Run PEM p 20 

Van Schooten FJ p 104 

Van Slooten GW p 120, 121 

Van Tellingen 0 p 109, 110 

Van Tinteren Hp 104, 108, 117, 132, 133, 134, 135 

Van Veelen N p 66 

Van Vreeland T p 120 

Van Vugt H p 72 

Van Vuuren PAC p 114 

Van Waarden berg W p 132 

Van Warmerdam LJC p 110, 132 

Van Wezel JT p 73, 74 

Van Workum M p 61 

Van Zandwijk N p 86, 96,104, 108, 135 

Vandeputte M p 68 

Vaz Gomes A p 62 

Veen huizen RB p 86, 117 

Verbakel SE p 22 

Verheij Mp 98 

Verhoeven Ep 74 

Verlaan I p 34 

Vernie LN p 41 

Verrijk R p 87 

Verschuur HP p 114 

Verster FC p 93 

Verwijs-Janssen M p 83 

Verwoerd D p 40, 41 

Vie1voye-Kerkmeer APE p 105, 128 

Vijlbrief RE p 93 

VinkC p 56 

Visser AG p 93 

Visser JC p 105 

Visser S p 134 

Vlaar M p 78 

Von dem Borne AEGKr p 44 

Voogd E p 115 

Vooijs M p 78 

Voordouw AC p 49 

Voorhoeve PM p 28 

Voorhorst FJ p 117 

Vos E p 68 

Vos EJ p 118 

Vos HLp 64 

Vos JC P 54 

Vos PH P 93 

Voûte PA p 85, 107 

V rouenraets BC p 121 

Vyth-Dreese FA p 44 

Wafelman AR p 107,110 

Wagenaar AC p 96 

Wagenaar E P 58 

Wagenaar MJ p 128 

Walboomers JMM p 117, 119 

Wals A p 132 

Walworth NC p 28 

Weder P p 44 

Weijer K p 44,49 

Weimar lp 44 

Went G p 115, 132 

Wesseling J p 64 

WesterhofGR p 85 

WesterhoffHV p 61,62 

Westermann AM p 102 

Westra JG p 31 

Wever LDV p 126 

Wielinga P p 61 

Wientjens E p 66 

Wigbout G p 104 

Wiggers T p 115 

Wijnands YM p 20 

Willemse E p 132 

Winterwerp HHK p 30 

Wisman P p 68, 82 

Wits EG pBO 

Wojcik-Jacobs E p 40 

Woordes-van BaaIen MMPGM p 128 

Wubbo1ts R p 40 

Y oungman S p 55 

Zaman GJR p 58 

Zandbelt LC p 128 

ZangXS p 40 

Zoetmu1der FAN p 104, 115, 118, 120, 124 

Zondag GCM p 36 

Zuurbier AEM p 20 

Zwaai RR p 55 

Zwijsen R p 66

Colofon 

Chief editor 

FA Stewart 

Editors 

AC Begg 

IS Benne 

WJ van Blitterswijk 

MPWGallee 

JV Lebesque 

FA Vyth-Dreese 

EJVos 

Coordination 

THE Eggenhuizen 

MEGde Kwant 

JM Lomecky 

Photographs 

Audiovisual Service 

Photograph HM The Queen Beatrix 

Voor inlichtingen / toestemming: 

Rijksvoorlichtingsdienst, 

afd. Pers en Publiciteit/FOTO 

Postbus 20006 

2500 EA 's-Gravenhage 

Foto: Vincent MentzeI 

Copyright © RVD 

Production 

Audiovisual Service 

The Netherlands Cancer Institute 

Antoni van Leeuwenhoek Huis 

PIesmanlaan 121 

1066 CX Amsterdam 

The Netherlands 

Typesetting 

Euroset bv, Amsterdam

Research and Hospital Divisions - NKI / AvL

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