Treatment of Soft Tissue Sarcomas

TARGETING TREATMENT OF
SOFT TISSUE SARCOMAS

Cancer Treatment and Research
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TARGETING TREATMENT OF
edited by
Jaap Verweij
Department of Medical Oncology
Erasmus University Medical Center
Rotterdam, The Netherlands
and
Herbert M. Pinedo
Department of Medical Oncology
Free University Medical Center
Amsterdam, The Netherlands
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Table of Contents
1. Targeted therapy: Ready for prime time? 1

C. Seynaeve and J. Verweij, Erasmus University Medical Center,
Rotterdam, The Netherlands
2. Volume-based radiotherapy targeting in soft tissue sarcoma 17
Iain Ward, Tara Haycocks, Michael Sharpe, Anthony Griffin,
Charles Catton, David Jaffray, Brian OSullivan, Princess Margaret
Hospital, University of Toronto, Toronto, Canada
3. Preoperative therapy for soft tissue sarcoma 43
Janice N. Cormier, Howard N. Langstein, Peter W. T. Pisters,
The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA
4. TNF-based isolated limb perfusion: A decade of experience with 65
antivascular therapy in the management of locally advanced
extremity soft tissue sarcomas
Dirk J Grnhagen, Flavia Brunstein, Timo L.M. ten Hagen, Albertus N.
van Geel, Johannes H.W. de Wilt, and Alexander M.M. Eggermont,
Erasmus University Medical Center, Rotterdam, The Netherlands
5. Pitfalls in pathology of soft tissue sarcomas 81
Judith V.M.G. Bove and Pancras C.W. Hogendoorn, Leiden Universtiy
Medical Center, Leiden, The Netherlands
6. Molecular biology and cytogenetics of soft tissue sarcomas: 99
Relevance for targeted therapies
Jonathan Fletcher, Brighams and Womens Hospital, Boston, MA, USA
7. KIT and PDGF as targets 117
Jaap Verweij, Erasmus University Medical Center, Rotterdam,
The Netherlands
8. Targeting mutant kinases in gastrointestinal stromal tumors: 129
A paradigm for molecular therapy of other sarcomas
Mike C. Heinrich, Christopher L. Corless, Portland, OR, USA
9. Targeting other abnormal signalling pathways in sarcoma: 151
EGFR in synovial sarcomas, in liposarcoma
Jean-Yves Blay, Isabelle Ray-Coquard, Laurent Alberti,
Dominique Ranchere, Hospital Edouard Herriot, Lyon, France
10. Angiogenesis: A potential target for therapy of soft tissue sarcomas 169
K. Hoekman and H.M. Pinedo, Free University Medical Center,
Amsterdam, The Netherlands
Index 181
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Preface
The last decade we have witnessed a major change in the

development of new techniques and agents for the treatment of cancer in
general, and for soft tissue sarcomas in particular. The important
achievements of molecular biology research have changed the landscape
markedly. Increasingly subtypes of soft tissue sarcomas are shown to be
related to changes in cellular growth factors in the cell signaling
pathways. This in theory enabled to development of agents with specific
activity against these factors. The presence of the KIT receptor at the
surface of the gastrointestinal stroma tumor cell, and the constitutive
activation by mutations, has lead to the discovery of the specific KIT
tyrosine kinase inhibitor Imatinib, an agent with impressive activity in this
disease. Before the era of Imatinib, GIST was an untreatable disease once
metastasized. Imatinib clearly is a breakthrough in the approach of soft
tissue sarcomas, and will likely serve as a role model for the development
of other agents acting towards other receptors. Likewise, soft tissue
sarcomas with their specific molecular characteristics, will likely serve as
role model diseases for targeted treatment approaches. Whether the future
lies in drugs with selective inhibition of only one receptor and one
pathway, or multiple receptors and multiple pathways is currently a matter
of debate, and again soft tissue sarcomas serve as role model.
Fully in line with the more targeted approach in drug use, the
changes in the field of radiation therapy and surgery basically also focus
on a better targeting of the disease, albeit not based on the molecular
characteristics yet.
The present volume of this series reflects all of the above
mentioned changes. World wide renowned experts have been willing to
contribute to this book, and we would like to thank all of them for their
efforts. Hopefully this book will contribute to a better understanding of
the changes in the field, and will serve our patients in helping getting a
better future.
Jaap Verweij
Herbert M. Pinedo
Editors
Chapter 1
Targeted therapy: Ready for prime time?
Caroline Seynaeve and Jaap Verweij
Dept. of Medical Oncology,

Erasmus University Medical Centre-Daniel den Hoed Cancer Centre, Rotterdam,
the Netherlands
Correspondence to:
Caroline Seynaeve, MD, PhD
Erasmus University Medical Centre-Daniel den Hoed Cancer Centre,
Groene Hilledijk 301
3075 EA Rotterdam, the Netherlands
2
1 INTRODUCTION
Soft tissue tumours are a heterogeneous group of rare neoplasms with

several histiotypes that all share a putative common mesenchymal origin, and
account for only 1% of all adult malignancies. Variation in pathological
definition has made it difficult to obtain exact numbers of patients with
sarcomas. Based on the Surveillance, Epidemiology, and End Results (SEER)
database, approximately 8,000 new cases of soft tissue sarcoma (STS) are
diagnosed each year in the United States, while 4000 (50%) are dying per annum
due to advanced or metastatic disease. This is a 10 times greater mortality that
that of testicular cancer or Hodgkins disease, diseases with a similar or lower
incidence (Stojadinovic et al, 2002). Since metastatic disease is only amenable to
curative therapy in very selected cases (Blay et al, 2003) and very few drugs are
available with meaningful activity, the search for effective systemic agents
remains extremely important in order to improve the outcome and decrease
mortality.
Progress has long been modest and slow because of the rarity of the
disease, the lack of systematic referral of adult patients to specialised centres also
being influenced by the advanced age the disease mostly occurs (> 50 years), and
the insufficient tumour selectivity of therapies. Further, research in the field of
soft tissue sarcoma (STS) has been hampered by the wide range of histological
appearances, with overlapping architectural and cytological characteristics,
within which at this moment more than 50-100 different entities have been
described (Weiss/Goldblum, 2001). Still new entities are being defined by means
of improvement in light and electron microscopical appearances,
immunohistochemical and molecular biological tools.
While it has longer been recognised that many of the subtypes are
associated with distinctive clinical and prognostic features, until recently a one-
size-fits-all approach has been pursued because of the lack of truly targeted
therapies. Being aware that this will change in the near future, results of
randomised clinical trials on the efficacy of systemic therapy incorporating
different subtypes are already difficult to interpret to day, and unfortunately
probably will become even less informative in the future.
Nevertheless, progress in diagnosis and therapy of adult STS over the

last decades of the past century has been achieved resulting from improvement in
pathological definition, imaging techniques, staging, surgical operating
procedures and advances in limb preservation, the use of radiotherapy as an
adjunct to other treatment modalities, a better delineation of the activity of the
available chemotherapeutic agents doxorubicin and ifosfamide, and the search
for new active drugs. Not to forget, advance has been obtained by the concerted
action of the different specialists involved in the care of sarcoma patients
working together in a multidisciplinary setting, while the efforts of co-operative
3
groups facilitating cross-talk with respect to the communication of trial results

and the initiation of new ideas for further studies greatly contributed.
The start of this new century is characterised by exciting developments
in all of the above as well as in genetic profiling of tumour specimens linking
some sarcoma subtypes with distinct histopathological differences to each other
and resulting in clarification of the classification of different sarcoma subtypes,
and the molecular identification of oncogenes and protein products that will
enable the development of targeted therapies. While the administered
chemotherapy in the last century seemed independent of the subtype of STS, we
are entering an era shifting towards targeted therapy for a specific subtype which
hopefully will yield more benefit for both the patient and the scientific research
group. As said, clinical trial design will also undergo change to reflect the nature
of these therapies.
However, since this strategy is not yet to-days practice, attempts to
refine the currently available therapeutic armamentarium to maximise the
therapeutic index by means of dose intensification and the identification of new
agents with certain activity also remain of paramount importance.
On the way to a new approach and further advance in systemic therapy

in STS, various issues may be of importance which we will address in the
following paragraphs: optimal use of systemic therapy at the beginning of the
century, new and pipeline agents, molecular targets and signal transduction
pathways, and changing methodology on testing new agents for activity.
2 CHEMOTHERAPY AT THE BEGINNING OF THE

CENTURY
Although over the last decades several known and new compounds have
been tested for activity in STS, only doxorubicin and ifosfamide have
meaningful activity. For both drugs a dose-response curve in STS has been
identified, with higher response rates for doxorubicin administered at a cycle
dose of and ifosfamide at a cycle dose of or more.
Reported single agent response rates vary between 16-36%. Dacarbazine, while
yielding some activity, has only shown short lasting responses of limited value
(Seynaeve/Verweij, 1999; OSullivan et al, 2002). The value of these drugs has
been studied in neo-adjuvant, adjuvant and metastatic setting.
2.1 (Neo-)Adjuvant Chemotherapy
The value of neo-adjuvant or adjuvant chemotherapy continues to be a

matter of debate. Although the only randomised study investigating the value of
neo-adjuvant therapy, by means of standard doses of adriamycin/ifosfamide,
conducted by the EORTC failed to show any benefit in disease free and overall
4
survival (Gortzak et al, 2001), the question remains actual since other groups
have suggested impressive response rates using more dose-intensive regimens
(Patel et al, 1998; Patel, 2002). The discussion has recently been stirred up by the
results of the study by Delaney et al. reporting on the activity of MAID (mesna,
adriamycin, ifosfamide and dacarbazine) interdigitated with radiotherapy and
followed by surgery and postoperative chemotherapy in a subset of STS at very
high risk of distant metastasis (Delaney et al, 2003, OSullivan/Bell, 2003). In
comparison with a cohort of historical controls, the MAID regimen resulted in a
dramatic improvement of distant disease free, disease-free and overall survival
being 75% and 44%, 70 and 42%, and 87% and 58% respectively, all being
statistically significant. As appealing these data may seem however, they have to
be interpreted with great caution since all results come from non-randomised
studies, while this approach should be investigated in randomised studies to
avoid for bias in the comparative groups before it can be incorporated as
standard of care.
A similar discussion is ongoing with respect to the role of adjuvant
therapy in STS (Verweij/Seynaeve, 1999). The most powerful evaluation of the
value of chemotherapy (doxorubicin-based, standard doses versus control)
originated from the Sarcoma Meta-Analysis Collaboration (SMAC) and showed
a statistically significant improvement in local relapse-free survival (6%), distant
metastasis-free (10%) and disease-free survival (10%) for treated patients, but
only a trend toward an increased overall survival (4%) after a median follow-up
of 9.4 years (SMAC, 1997). In 2001, the Italian Sarcoma Group reported that an
intensified chemotherapy regimen consisting of epirubicin/ifosfamide
in comparison with a control group, resulted in a significant
increase of disease-free (48 versus 16 months, p=.04) and overall survival (75
versus 46 months, p=.03) in high-risk STS after a follow up of 59 months.
Although this study was prematurely closed because of the interim results in
favour of the chemotherapy group, it has to be noticed that where fewer
metastatic events were seen at 2 years in the chemotherapy group (28% vs.
45%), identical metastatic rates were observed at the 4-year time point (Frustaci
et al, 2001). Long term follow-up data from this study are therefore crucial,
especially, where two other small studies using intensive
anthracycline/ifosfamide regimens failed to confirm a benefit (OSulllivan/Bell,
2003). Further, the data of the ongoing EORTC study investigating the value of
doxorubicin versus controls will hopefully add
relevant information on this issue
2.2 Metastatic disease
In contrast with above-mentioned settings, the aim of systemic therapy

in metastatic disease is disease control, symptom palliation and prolonged
survival. As there are only modest gains in survival with the use of known
chemotherapeutic agents, studies in the last decades of the century have
focused on schedule optimisation and/or dose intensification (Bramwell et al,
5
2000; Seynaeve/Verweij, 2002). Conclusions from these studies are that

combination chemotherapy regimens produced higher response rates and more
toxicity, but did not improve the complete response rate, the time to failure or
overall survival. Some recent studies investigating more dose-intensive
anthracyclin/ifosfamide regimens show unexpectedly high response rates, and
resulted in a higher complete response percentage that may be important aiming
at improved survival in selected individuals (Patel et al, 1998; Blay et al, 2003).
However, these regimens have not yet been tested in a randomised setting.
Therefore, the recently activated international world-wide EORTC-lead study,
realised through the global co-operation of different collaborative groups, in
which patients are randomised between doxorubicin (control) and a
dose-intensified regimen (doxorubicin plus ifosfamide with
G-CSF support) is warmly welcomed, while results are eagerly awaited and
hopefully will bring an answer to a long-lasting question.
3 SYSTEMIC THERAPY OLD DRUGS IN A NEW

JACKET / NEW DRUGS
Over the last decades of clinical research it has been recognised that
many of the STS subtypes are associated with distinctive clinical and prognostic
features. Therefore, it has already longer been questioned whether the one-size-
fits-all-approach with respect to chemotherapy as has been applied till now is
still appropriate. An analysis of the Soft Tissue and Bone Sarcoma Group Study
(STBSG) of the EORTC into prognostic factors for the outcome of
chemotherapy in advanced STS reported in univariate analysis an increased
overall survival (OS) in lipo- and synovial sarcoma (SS), a decreased survival
time in malignant fibrous histiocytoma (MFH), a lower response rate in
leiomyosarcoma (LMS), and a higher response rate in liposarcoma (p<0.05, for
all log-rank and X2 tests). In multivariate analysis, the subtype dropped out of
the logistic model as independent prognostic factor (van Glabbeke et al, 1999).
Comments that should be considered hereby are: in the multivariate analysis
liver involvement, irrespective of the subtype, was included as covariate, being
the more important with respect to the subtypes as for example synovial sarcoma
where this metastatic pattern is only infrequently observed; the EORTC database
has been set up at a moment that the histologic entity of gastrointestinal stromal
tumours (GIST) had not yet been identified, and therefore a number of so-called
leiomyosarcomas probably were GISTs which at this moment are known to be
chemotherapy-insensitive. In a more recent publication of the EORTC,
progression-free rates (PFR) at 6 months (as an alternative for response rate) in
non-pre-treated patients differed between subtypes, showing a PFR > 50% in
synovial and liposarcoma, and in LMS and MFH (see figure 1) (van
Glabbeke et al, 2002). This is in accordance with the clinical observation that SS
is more chemo-sensitive. Whether this chemosensitivity indeed especially
6
concerns high-dose ifosfamide as has been reported in small series (Rosen et al,
1994, Spillane et al, 2000), remains to be proven in larger studies. However, the
above mentioned may partly explain why some investigators report impressive
results of a certain agent in a specific STS subtype, albeit that this was never
observed in properly designed studies allowing inclusion of a variety of
subtypes.
Paxlitaxel has been studied in patients after or without prior exposure to

first line chemotherapy showing response rates between 0% and 12,5%
(Blacerzak et al, 1995; Patel et al, 1997; Casper et al, 1998). Despite the lack of
meaningful activity of paclitaxel for the whole STS group, responses in two of
these studies were seen in patients with an angiosarcoma (Blacerzak et al, 1995;
Casper et al, 1998). Based on these observations, Fata et al. reviewed their
institutional experience with paclitaxel (different schedules) in nine patients with
an angiosarcoma of the scalp/face, and found an impressive response rate of 89%
with a median duration of 5 months (Fata et al, 1999). Attempts are being
undertaken to try to investigate this further in a global study. Fewer studies have
investigated docetaxel. In an initial phase II study conducted by the EORTC
activity was seen in 18% of the patients. In a subsequent randomised study by
the same EORTC group with doxorubicin as the control agent, docetaxel did not
show any activity in 34 patients including 3 patients with an angiosarcoma, while
a response was seen in 1/3 angiosarcoma patients in the doxorubicin arm
(Verweij et al, 2000). In the EORTC analyses, LMS was reported to be less
chemo-sensitive. One has to be aware that these data certainly are influenced by
the fact that some of the LMS nowadays would be classified as GIST, known to
be chemo-resistant. Investigating the value of gemcitabine in STS in small phase
II studies some activity has been observed, with a pharmacological advantage
being suggested with a 150-minute infusion. Responses to gemcitabine were
particularly seen in uterine leiomyosarcoma whereas none were noted in
gastrointestinal LMS (Spath-Swalbe et al, 2000; Patel et al, 2001; Svancarova et
al, 2002; Okuno et al, 2002 and 2003). Interestingly, the combination of
gemcitabine and docetaxel showed an impressive response rate of 53% in a non-
randomised phase II study in chemotherapy-naive or pretreated LMS, being 55%
in uterine and 40% in other LMS, respectively. The performed pharmacokinetic
evaluation in this study demonstrated that the 90-minute infusion time resulted in
approximately 50% longer period of time above the gemcitabine concentration
threshold of which may be important for greater DNA incorporation
of gemcitabine affecting cell kill (Hensley et al, 2002). Although it is difficult to
assess from this small study whether the high response rate is caused by the
chemo-sensitivity of uterine LMS, which also has been suggested by others
(Leyvraz et al, 2001; Pautier et al, 2002), the longer infusion schedule of
gemcitabine or the synergistic activity of the chemotherapy combination, or just
chance, these results are interesting enough to warrant further investigation into
this issue.
7
ET-743 (Trabectedin), a cytotoxic tetrahydroisoquinolone alkaloid

isolated from a murine organism that binds to DNA and causes single strand
breaks resulting in cell death, has over the last years been studied as novel
antitumor agent in STS, because it had shown potent antiproliferative activity in
vitro (Delaloge et al, 2001). While objective response rates in second line
therapy for the whole group were modest, ranging between 6% and 8%, better
response rates were seen in LMS and liposarcoma (14% and 16%) and the
response duration was months (le Cesne, 2002). Even more important, in all
studies performed in Europe and the United States impressive long-lasting major
responders as well as a high number of durable stable diseases (+/- 50%) were
seen. Overall survival at 12 months was consistently between 45-55%, for a
category of patients being refractory to first line therapy and starting on the drug
at documented progression (Demetri, 2002; Brain, 2002). The observation of a
long durable response and stable disease, associated with clinical benefit in
symptomatic disease, resulted in reconsideration of the optimal way of response
assessment of novel antitumor agents in STS, as we will comment on later.
4 MOLECULAR TARGETS AND SIGNAL

TRANSDUCTION PATHWAYS IN SOFT TISSUE SARCOMA
8
The success of imatinib mesylate in treatment of GIST has led to a better

appreciation of how studies of STS can enhance the understanding of cancer
biology and development of targeted therapies. It also has taught us that
expression of a target may not be enough to build a targeted clinical trial upon,
but that it may be relevant to conduct model studies on the functional relevance
of the target for tumour growth. Once this is established there is a justification to
perform clinical studies directed towards this target. If functional relevance can
not be proven because of the lack of appropriate assays, one has to be aware that
the pragmatism of nevertheless studying the target in a clinical setting may be
useful and may yield relevant information.
The identification of new and relevant targets being involved in STS is
one of the crucial steps. Microarray analysis allowing for the determination of
gene expression profiles in sarcoma specimens may prove to be most useful
hereby. Nielsen et al. recently reported data from a set of 41 sarcomas and
described characteristic expression profiles for GIST (with kit being the
discriminator gene), monophasic synovial sarcomas involving the retinoic-acid
pathway and the epidermal-growth-factor receptor (EGF), subgroups of LMS
(calponin-positive and negative), while MFH and liposarcoma exhibited
considerable heterogeneity (Nielsen et al, 2003). Lee et al., studying gene
expression profiles in SS, LMS and MFH (n=9 in each group), also found a
distinct pattern in SS, and identified a subset of MFH, but did not distinguish two
separate LMS groups (Lee et al, 2003). Unfortunately, there was very little
overlap between the identified genes in the two SS clusters, on one hand because
other gene sets have been studied and selected, on the other hand possibly
influenced by the subtype of SS studied which is not specified in the study of
Lee et al. Further, these findings reflect that it is necessary to study large enough
sarcoma samples before coming to conclusions.
However, the observation that EGF is expressed in monophasic SS,
while erb-B2 expression has been demonstrated in the epithelial component of
biphasic SS (Nielsen et al, 2003; Borden et al, 2003) opens avenues to test the
value of EGF-inhibitors or herceptin in this disease, which is further addressed in
chapter 9. In addition, in vitro studies have shown that epithelioid sarcoma cells
overexpressing EGFR1 respond to EGFR1-antibody therapy, lending support to
test these inhibitors in epithelioid sarcoma.
In inflammatory myofibroblastic tumours (IMTs) composed of spindled
mesenchymal cells admixed with a striking inflammatory infiltrate
predominantly consisting of plasma cells and lymphocytes, the neoplastic nature
became apparent by the observation that translocations and other rearrangements
in the short arm of chromosome 2 occurred in the IMT spindle cells. These
aberrations create fusion oncogenes that encode activated forms of the ALK
receptor tyrosine kinase. ALK fusion oncoproteins are also characteristic for
many anaplastic large cell lymphomas (Tuveson/Fletcher, 2001). Specific
antagonists of these proteins may be effective in this type of diseases, but are not
yet available.
9
Most dermatofibrosarcoma protuberans tumours contain a translocation

of chromosomes 17 and 22, resulting in oncogenic juxtaposition of the COL1A1
and platelet-derived growth factor beta genes. Since imatinib mesylate
is also an inhibitor of and it is assumable that glivec may be

effective in this disease. Activity of glivec in this entity, indeed, has been
reported in two case reports, underscoring this assumption (Schuetze et al, 2002;
Labropoulos et al, 2003). Likely, most desmoplastic round cell tumours
(DRCTs) express an EWS-WT1 fusion oncogene resulting from a translocation
of chromosomes 11 and 22. The oncoprotein EWS-WT1 is a transcriptional
regulator inducing expression of which binds and activates both PDGF-
receptors and (Tuveson/Fletcher, 2001). Since DRCTs are quite
chemotherapy-resistant it certainly is worthwhile to study the activity of glivec in
this entity as well. This topic is further highlighted in chapter 9.
The use of farnesyl transferase inhibitors (FTI) to target the oncogenic
ras protein may be applicable in sarcomas overexpressing the ras protein, as is
the case in the neurofibromatosis syndrome 1, which can predispose to malignant
peripheral nerve sheath tumours (MPNST). FTIs can inhibit the trafficking of ras
protein to the cell membrane by inhibition of the farnesylation of this protein
(Scappaticci/Marina, 2001).
Liposarcomas tend to be of low grade and generally have a better
survival than many other STS subtypes. There is some laboratory evidence that
the heterodimeric complex of peroxisome proliferator-activated receptor-gamma
and the retinoid acid receptor (RAR) alpha functions as a central
regulator or adipocyte differentiation, while it has been demonstrated that human
liposarcoma cells can be induced to undergo terminal differentiation by treatment
10
by PPAR ligand pioglitazone. Demetri et al. have used troglitazone, an analogue

drug mediating differentiation, to attempt differentiation in poorly differentiated
subtypes of liposarcoma (Scappaticci/Marina, 2001). This is further addressed in
chapter 9.
Finally, angiogenesis inhibitors are of great interest in STS, on one hand
because of the important involvement of vessels in vascular sarcomas e.g.
angiosarcomas and hemangioendotheliomas, and on the other hand because it
may give us yet another mechanism to attack STS. In chapter 10 an overview is
provided.
5 CHANGING METHODOLOGY ON TESTING NEW

AGENTS FOR ACTIVITY
Although phase II studies performed in a small group of patients suffer

from biases with respect to random and prognostic variables (age, performance
status, disease free interval, histotype....) or other unknown variables, they
remain useful as screening studies to evaluate whether a new agent has biological
activity (rather than therapeutic benefit) in the intended cohort of patients. If the
results of the phase II trial are consistent with activity as expected from an active
drug, the new agent deserves further testing. If results are consistent with the
level of activity from an inactive drug, then the experimental agent is rejected
from further investigation. Sample size is computed to ensure that these 2
decision rules are mutually exclusive. The statistical design to use by preference
remains the two steps optimal and minimax design as proposed by Simon (van
Glabbeke et al, 2002)
Response to therapy, based on a measured decrease in the size of
objective lesions, is considered the most effective end-point to document
biological activity of cytotoxic agents. However, for non-cytoreductive
anticancer drugs, as for example for signal transduction inhibitors and
angiogenesis inhibitors, biological activity is frequently not expected to translate
into a diminution of lesions, but rather in slowing down or arrest the growth
acceleration. This still may result in clinical benefit (decrease of symptoms,
improved quality of life, increase of progression free survival). The benefit of
static disease (long term SD) during a certain therapy has since long been
recognised for breast cancer, showing that survival in patients with durable SD
(> 6 months) was similar to the survival in patients achieving an objective
response for both first- and second-line endocrine therapies (Robertson et al,
1997 and 1999). However, these observations have been obtained in randomised
studies, while data on stable disease rate in most phase II studies are lacking.
Nevertheless, the data on ET-743 in STS suggest that stable disease induced by a
cytotoxic agent may be worthwhile and actually underestimated in this entity.
This is further substantiated by a recent retrospective literature review on a large
number of cytotoxic agents tested in STS (Verweij/van Glabbeke, 2003).
11
Since in phase II screening studies of biological agents response rate is

not always an appropriate endpoint, an alternative endpoint may be progression
free survival/rate (PFR) or time to progression. This endpoint has properly
been defined by the RECIST Working Group and was considered a valuable
alternative to estimate the biological activity of this type of agents in phase II
studies by the EORTC and also by others (Therasse et al, 2000; Korn et al,
2001). However, the appropriate time point of assessing PFR has not been
clearly defined, partly because this may be disease- or agent-specific and
therefore impossible to define in general.
The purpose of the earlier mentioned analysis of the EORTC studying
the PFR in STS obtained by an active or an inactive agent, in the assumption that
doxorubicin, ifosfamide and dacarbazine (as generally accepted) are the only
active agents, has exactly been set up to clarify this for STS (van Glabbeke et al,
2002). In pre-treated patients (n=380), which is the relevant group for the
purpose of phase II screening studies, the 3- and 6- months progression free rates
were respectively 39% and 14% for an active drug (n=234), and 21% and 8% for
an inactive agent (n=146). For the whole cohort, the rates were 28% and 10%.
The Kaplan-Meier estimate of the PFR in patients treated with an active of an
inactive drug is shown in figure 2 (standard error approximately 5%). The
selection of an appropriate time point for PFR is known to be a compromise
between the need to avoid false-positive trials, and practical burdens coincided
with a long period of observation. If the disease is slowly progressing, absence of
objective progression at the first evaluation (generally 6-8 weeks after the start of
therapy) may not reflect any substantial drug activity. In the EORTC study there
was no major discrimination between active and inactive agents at this time
point. On the other hand, a study requiring a long treatment and follow-up period
in this setting, which possibly can extend over years (as in GIST), may
logistically be difficult to conduct and is unattractive in view of the purpose of a
phase II study, which is to screen new agents for activity. Therefore, the EORTC
suggested evaluating the progression free status at 3 and 6 months after the start
of therapy. Further, the EORTC proposed to consider a drug as active in first line
therapy of STS if the PFR at 6 months is 30-55% (depending on histology).
For second line therapy, a PFR at 3 months of 40% would suggest drug
activity, and 20% would suggest inactivity (van Glabbeke et al, 2002;
Verweij/van Glabbeke, 2003). Another way of looking at the data could be to
take together objective response and stable disease rate, resulting in the
determination of the rate of no progression. Although van Oosterom already in
1986 proposed to use progression arrest as an endpoint in phase II studies (van
Oosterom, 1986), the idea was only recently picked up by the NCIC that
developed a multinomial phase II stopping rule using response and early
progression. They showed that this was more efficient as compared to the usually
used stopping rules (Dent et al., JCO, 2001).
These data certainly would gain strength if PFR or progression arrest
rates could be assessed by means of the use of the EORTC database in
12
combination with those of other co-operative groups, possibly even resulting in

the determination of subtype specific progression arrest rates. Further, this would
provide strong suitable tools that could serve as reference for future phase II
studies aiming at more efficient screening of new and old drugs for activity in
STS.
6 CONCLUSION
The fast development and application of microscopic and new

immunohistochemical tools, molecular and cytogenetic analytical methods
results in a better identification of specific or clusters of subtypes in STS, and
provides specific molecular targets to which selected agents are being developed.
This allows for a further evaluation of the characteristics and chemo-sensitivity
of these different subtypes to known agents, and necessitates the prospective
screening of selected (old and new) drugs in subtype specific cohorts of patients.
Further, the drugs aiming at targeting the identified molecular target also have to
be tested in subtype specific studies.
In order to make this process as efficient as possible the endpoints of
phase II screening studies should be clearly selected. We propose to use the 3-
and 6- month PFR as a reference value in phase II screening studies, while
progression arrest also may be of relevance. Moreover, performing these
screening studies of molecular targeting agents in a cohort of patients expressing
the specific target in a disease already rarely occurring will not be possible
13
without a joined effort and a global co-operation of the several co-operative

groups.
We hope that the topics in this book may contribute to a better
understanding hereby, and stimulate the co-operation and participation into
initiated screening studies.
7 REFERENCES
1. Balcerzak SB, Benedetti J, Weiss GR et al. (1995) A phase II trial of paclitaxel in patients with
advanced soft tissue sarcomas. Cancer 76, 2248-2252
2. Blay J-Y, van Glabbeke M, Verweij J, et al. (2003) Advanced soft tissue sarcoma: a disease that
is potentially curable for a subset of patients treated with chemotherapy. Eur J Cancer 39: 64-69
3. Borden EC, Baker LH, Bell RS et al. (2003) Soft tissue sarcomas of adults: state of the
translational science. Clin Cancer Research 9: 1941-56
4. Brain EGC (2002) Safety and efficacy of ET-743: the French experience. Anti-cancer Drugs 13
(suppl 1): 11-14
5. Bramwell V, Anderson HC, Charette ML et al. (2000) Doxorubicin-based chemotherapy for
the palliative treatment of adult patients with locally advanced or metastatic soft tissue sarcoma: a
meta-analysis and clinical practice guidelines. Sarcoma, 4: 103-112
6. Casper ES, Waltzman RJ, Schwartz GK et al. (1998) Phase II trial f paclitaxel i patients with
soft tissue sarcoma. Cancer Invest 16: 442-446
7. Delaloge S, Yovine A, Taamma A et al. (2001) Ecteinasidin-743: a marine derived compound
in advanced, pretreated sarcoma patients preliminary evidence of activity. J Clin Oncol 19: 1248-
1255
8. Delaney TF, Spiro IJ, Suit HD et al. (2003) Neoadjuvant chemotherapy and radiotherapy for
large extremity soft tissue sarcoma. Int J Radiat Oncol Biol Phys, 56: 1117-1127
9. Demetri GD (2002) ET-743: the US experience in sarcomas of soft tissues. Anti-cancer Drugs
13 (Suppl 1): 7-9
10. Dent S, Zee B, Dancey J et al. (2001) Application of a new multinomial phase II stopping rule
using response and early progression. J Clin Oncol 19: 785-791
11. Fata F, OReilly E, Ilson D et al. (1999) Paclitaxel in the treatment of patients with
angiosarcoma of the scalp or face. Cancer 86: 2034-2037
12. Frustaci S, Gherlinzoni F, de Paoli A, et al. (2001) Adjuvant chemotherapy for adult soft tissue
sarcomas of the extremities and girdles : results of the Italian randomized cooperative trial. J Clin
Oncol, 19: 1238-1247
13. Gortzak E, Azarelli A, Buesa J et al. (2001) A randomised phase II study on neo-adjuvant
chemotherapy for high-riskadult soft tissue sarcoma. Eur J Cancer 37: 1096-1103
14. Hensley ML, Maki R, Venkatraman E et al. (2002) Gemcitabine and docetaxel with
unresectable leiomyosarcoma: results of a phase II trial. J Clin Oncol 20: 2824-2831
15. Korn EL, Arbuck SG, Pluda JM et al. (2001) Clinical trial designs for cytostatic agents: are
new approaches needed? J Clin Oncol 19: 265-272
16. Labropoulos SV, Papadopoulos S, Hadjiyiassemi L et al. (2003) Response of metastatic
dermatofibrosarcoma protuberans to imatinib mesylate. Proceedings ASCO 2003, J Clin Oncol 22:
830 (#3334)
17. Le Cesne A. (2002) Improving efficacy in soft tissue sarcoma. Satellite symposium ESMO
2002, 18 October, Nice, France.
18. Lee Y-F, John M, Edwards S et al. (2003) Molecular classification of synovial sarcomas,
leiomyosarcomas and malignant fibrous histiocytomas by gene expression profiling. Brit J Cancer
88: 510-515
14
19. Leyvraz S, Jundt G, Lissoni A et al. (2001) High-dose Ifosfamide and doxorubicin for the
treatment of gynaecological sarcomas. Proceedings ASCO 2001, J Clin Oncol 20 (part 1): 362a
(#1443)
20. Nielsen TO, West RE, Linn SC et al. (2002) Molecular expression of soft tissue tumours: a
gene expression study. Lancet 359: 1301-1307
21. Okuno S, Edmonson J, Mahoney M et al. (2002) Phase II trial of gemcitabine in advanced
sarcoma. Cancer 94: 3225-3229
22. Okuno S, Ryan LM, Edmonson J et al. (2003) Phase II trial of gemcitabine in patients with
advanced sarcomas. Cancer 97: 1969-1973
23. OSullivan B, Bell RS, Bramwell V (2002) Sarcoma of the soft tissue. In: Souhami R,
Tannock I, Hohenberger P, and Horiot JC (eds): Oxford Textbook of Oncology. Oxford
University Press, Oxford, UK, 2002, 2495-2523
24.OSullivan B, Bell RS (2003) Has MAID made it in the management of high-risk soft tissue
sarcoma? Int J Radiat Oncol Biol Phys, 56: 915-916
25. Pautier P, Genestie C, Fizazi K et al. (2002) Cisplatin-based chemotherapy regimen (DECAV)
for uterine sarcomas. Int J Gynaecol Cancer 12: 749-754
26. Patel SR, Linke KA, Burgess MA et al. (1997) Phase II study of paclitaxel in patients with soft
tissue sarcomas. Sarcoma 1, 95-97
27. Patel SR, Vadhan-Rai S, Burgess MA, et al. (1998) Results of two consecutive trials of dose-
intensives chemotherapy with doxorubicin and ifosfamide in patients with sarcoma. Am J Clin
Oncol 21: 317-321
28. Patel SR, Gandhi V, Jenkins J et al. (2001) Phase II clinical investigation of gencitaine in
advanced soft tissue sarcomas and window evaluation of dose rate on gemcitabine triphosphate
accumulation. J Clin Oncol 19: 3483-4389
29. Patel SR (2002) Systemic therapy for advanced soft tissue sarcoma. Curr Oncol Rep 4: 299-
304
30. Robertson JFR, Wilsher PC, Cheung KL et al. (1997) The clinical relevance of static disease
category for 6 months on endocrine therapy in patients with breast cancer. Eur J Cancer 33: 1774-
1779
31. Robertson JFR, Howell A, Buzdar A et al. (1999) Static disease on anastrozole provides
similar benefit as objective response in patients with advanced breast cancer. Breast Ca Res &
Treatm 58: 157-162
32. Rosen G, Forscher C, Lowenbraun S et al. (1994) Synovial sarcoma: uniform responsee of
metastases to high-dose ifosfamide. Cancer 73: 2506-2511
33. Sarcoma Meta-Analysis Collaboration (1997) Adjuvant chemotherapy for localised respectable
soft tissue sarcoma of adults: meta-analysis of individual data. Lancet 350: 1647-1654
34. Scappaticci FA, Marina N (2001) New molecular targets and biological therapies in sarcomas.
Ca Treatm Reviews 27: 317-326
35. Schuetze SM, Rubin BP, Eary JF et al. (2002) Molecular targeting of PDGF beta by imatinib
mesylate in dermatofibrosarcoma protuberans. Proceedings CTOS 2002, Sarcoma 6 (suppl 2): 71
(#25)
36. Seynaeve C, Verweij J (1999) High-dose chemotherapy in adult sarcomas: no standard yet.
Semin Oncol, 26: 119-133
37. Seynaeve C, Verweij J. (2002) High dose chemotherapy in sarcomas: science, fiction or
science fiction? In: Lorigan P, Vandenberghe E (eds): High dose chemotherapy, Principles and
Practice. Dunitz Publishers, London, UK, 2002, 167-179,
38. Spath-Schwalbe E, Genvresse I, Koschuth A et al. (2000) Phase II trial of gemcitabine in
patients with pretreated advanced soft tissue sarcoma. Anti-cancer drugs 11: 325-329
39. Spillane AJ, AHern R, Judson I et al. (2000) Synovial sarcoma: a clinicopathologic, staging,
and prognostic assessment. J Clin Oncol, 16: 1794-3803
40. Stojadinovic A, Leung DHY, Allen P et al. (2002) Primary adult soft tissue sarcoma: Time-
dependent influence of prognostic variables. J Clin Oncol 20: 4344-4352
15
41. Svancarova L, Blay J-Y, Judson I et al. (2002) Gemcitabine in advanced adult soft tissue
sarcomas. A phase II study of the EORTC soft tissue and bone sarcoma group. Eur J Cancer 38:
556-559
42. Therasse P, Arbuck GA, Eisenhauer EA et al. (2000) New guidelines to evaluate the response
to treatment in solid tumors. J Natl Cancer Inst 92: 205-216
43. Tuveson DA, Fletcher JA (2001) Signal transduction pathways in sarcoma as targets for
therapeutic intervention. Curr Opin Oncol 13: 249-255
44. Van Glabbeke M, van Oosterom AT, Oosterhuis JW et al. (1999) Prognostic factors for the
outcome of chemotherapy in advanced soft tissue sarcoma: an analysis of 2,185 patients treated
with anthracycline-containing first line regimens an EORTC soft tissue and bone sarcoma group
study. J Clin Oncol 17: 150-157
45. Van Glabbeke M, Verweij J, Judson I et al. (2002) Progression-free rate as the principal end-
point for phase II trials in soft-tissue sarcomas. Eur J Cancer 38: 543-549
46. Van Oosterom AT (1986) Phase II new drug trials in soft tissue sarcomas. In: Pinedo H and
Verweij J (eds): Clinical management of soft tissue sarcomas. Boston, MA, Martinus Nijhoff
Publishers, 131-138
47. Verweij J, Seynaeve C. (1999) The reason for confining the use of adjuvant chemotherapy in
soft tissue sarcoma to the investigational setting. Semin in Radiation Oncology, 9: 352-359
48. Verweij J, Lee SM, Ruka W et al. (2000) Randomized phase II study of docetaxel versus
doxorubicin in first- and second-line chemotherapy fr locally advanced or metastatic soft tissue
sarcoma in adults: a study of the EORTC soft tissue and bone sarcoma group. J Clin Oncol 18:
2081-2086
49. Verweij J, van Glabbeke M (2003) Translating targets into treatment: changes in trial
methodology and treatment approaches for soft tissue sarcomas. In: Educational book, ASCO
2003, 522-530
50. Weiss SW, Goldblum JR (2001) In: Weiss SW, Goldblum JR (eds): Enzinger and Weisss Soft
Tissue Tumors. Mosby Inc, St Louis, Missouri, 2001: 1-19
Chapter 2
Volume-based radiotherapy targeting in soft tissue

sarcoma
Iain Ward1 ,Tara Haycocks2, Michael Sharpe3, Anthony Griffin4, Charles

Catton1, David Jaffray3, Brian OSullivan1
Departments of Radiation Oncology1, Radiation Therapy2, Medical Physics3, and Surgical

Oncology4, Princess Margaret Hospital, University of Toronto, Toronto, Canada.
Correspondence:
Brian OSullivan
Department of Radiation Oncology
Princess Margaret Hospital
University of Toronto
610 University Avenue
Toronto, Ontario
Canada, M5G 2M9
Tel: 416 946 2123
Fax: 416 946 6556
Email: brian.osullivan@rmp.uhn.on.ca
18
1 INTRODUCTION
Although a highly effective adjuvant therapy, traditional

radiotherapy (RT) target volumes used for STS have largely been
constrained by available technology and are not ideal in some situations;
this includes inadequate dose administration because of normal tissue
constraints and/or the necessity for excessive volume coverage of normal
tissue to encompass the tumor region. The advent of very precise
treatment planning and delivery systems, including three dimensional
conformal radiotherapy (3D CRT) and intensity modulated radiotherapy
(IMRT), means it is now possible to select target volumes that more
closely approach the optimum. Consequently, these new approaches
provide great opportunity for treatment enhancement in the future. In this
chapter, the principles of RT will be discussed as they relate to current or
potential uses in the management of soft-tissue sarcoma (STS). Specific
examples of situations in STS that lend themselves to volumetric-based RT
planning approaches will be depicted to illustrate theses concepts and detailed
background to the use of such approaches will be provided.
2 DEFINING TARGETS FOR RADIOTHERAPY OF STS
2.1 Tissues at risk
The choice of RT volumes in STS is profoundly influenced by the

appreciation of the existence of a zone that may contain sub-clinical disease in
proximity to the presenting site of the primary tumour. The size and extent of
the putative risk zone depends on a number of factors, and appreciating this
will effect the target volume chosen for radiotherapy. Also, perhaps more than
most cancers, the pathway to appropriate treatment may already have been
declared by events that have taken place prior to referral. For example, the type
of biopsy that may have already been performed, or a prior inappropriate
excision may jeopardize the form and outcome of local treatment thereafter. 1
2.2 Local patterns of spread
In broad terms, soft tissue sarcomas tend to spread in a longitudinal

direction within the muscle groups of the extremity. They generally respect
barriers to tumor spread in the axial plane of the extremity such as bone,
interosseous membrane, major fascial planes, etc. Thus the margins of radiation
therapy must be wide in the cephalo-caudal direction but in the cross section
there may be much greater security in defining non-target structures. For non-
extremity lesions (e.g. head and and neck and torso lesions), the direction of
sarcoma growth is also along the involved musculature but care must be taken to
19
ensure that the fascial planes are appropriately recognized and encompassed in
the radiation target volume. 2
2.3 Regional lymphatic pathways of spread
Regional lymph node involvement in STS is unusual and for most

histological sub-types the nodal areas are not ordinarily treated electively.
Important exceptions to this generalization include epithelioid sarcoma, clear
cell sarcoma, angiosarcoma, and embryonal rhabdomyosarcoma.2 However the
presence of overt regional lymph node disease generally prompts their inclusion,
if the patient is being considered for curative management, although we
recognize that institutional preference may vary in this regard. In targeting the
lymphatic drainage areas, it is usual to treat the chain along the vascular supply
to eventually reach the terminal group of lymph nodes that are at risk. 2
2.4 Paucity of available evidence
Unfortunately, with one exception,3 no formal assessment of target

volumes in STS has been undertaken using contemporary hypothesis solving
techniques such as comparative clinical trials. Also, problems in defining the
gross tumor volume (GTV) will persist until resolution of dilemmas surrounding
imaging characteristics occurs, at least when considering pre-operative
radiotherapy where the volumes are most selectiveFor example, one obvious
problem concerns the significance of peritumoral edema evident on magnetic
resonance imaging4 and whether such areas should be considered part of the
GTV (see figure 1).
2.5 Extracompartmental sarcoma
Certain anatomic areas are potential spaces without good

definition from the standpoint of tumour containment and in such situations it is
usual to design the radiotherapy target volume to encompass the extreme limits
of the structure or region in question (generally determined by where the fascial
reflections eventually merge). Areas such as the axilla, popliteal fossa, femoral
triangle, and the entire subcutaneous compartment present problems of target
volume delineation that must be evaluated on an individual basis.2 Evaluation
of the region in terms of potential tissues involved or tissues that have already
been surgically violated is paramount in deciding the most appropriate volume
to treat. Again the principles are determined by the proximity of the most
reliable barrier to tumor invasion (hopefully an intact durable anatomical
boundary). Alternatively, an effective distance (e.g. 2-5 cm, where anatomically
feasible) is maintained from the highest risk area, most typically manifested by
the existing GTV or the pre-operative GTV; this should bear in mind the
surgical-pathologic findings at the time of the resection.
20
2.6 Margins for geometric uncertainty
An additional feature in radiotherapy target delineation concerns

allowance for uncertainties in set up and treatment delivery. A region of
additional margin must be defined to account for geometric uncertainty and is
especially important where there is respiratory movement (e.g. abdominal and
thoracic areas). The expansion of volume should be defined around the Clinical
target volume (CTV) as the Planning Target Volume (PTV) to insure the
inclusion of the areas at risk in the treated area.5, 6 A similar margin should be
used when protecting normal tissues vulnerable to radiotherapy (e.g. the spinal
cord in paraspinal, retroperitoneal or head and neck sarcomas) because
21
uncertainties in set up and treatment delivery may equally result in inadvertent

treatment of these structures. The additional zonal expansion surrounding these
structures is termed a planning risk volume (PRV).6
2.7 Influence of scheduling of modalities
Pre-operative and post-operative RT represent the two usual

approaches to external beam delivery for STS but effectively comprise two
disease scenarios from the standpoint of delineating targets. Pre-operative RT
approaches can focus on the extent of definable disease (generally using
imaging characteristics) and the choice of target is based on the anatomic
location, containment by barriers to spread, estimated distance from the GTV
that may contain microscopic disease, and allowance for geometric uncertainty.
In contrast, post-operative radiotherapy volumes must encompass all surgically
manipulated tissues and are often less specific because anatomic planes have
been disrupted and no longer provide barriers to contain tumor growth and are
consequently significantly larger.3, 7
Another consideration that is relevant to cases undergoing induction
chemotherapy is the determination of the pre-chemotherapy volume in chemo-
responsive tumors. In these situations, vigorous tumor response will likely have
manifested by the time radiotherapy is ordinarily scheduled to commence and
little if any radiologically apparent disease may be present at the time of
radiotherapy planning. The initial pre-chemotherapy volume must be the
reference for treatment planning and imaging studies must be carefully
performed and recorded to facilitate subsequent RT planning.2
3 VOLUMETRIC TREATMENT PLANNING AND

DELIVERY
3.1 Evolution of volumetric-based planning
The initial introduction of adjuvant radiotherapy more than two decades

ago took place in an era when limb conservation had only recently become
established as an alternative to amputation.8-10 The paradigm involved the use of
radiotherapy to sterilize a broad field of tissue that generally targeted an entire
muscle compartment from which the tumour had been resected and almost
exclusively involved post-operative radiotherapy.
Subsequently a desire to minimize morbidity resulted in progressive
reduction in radiation treatment volumes and evidence accumulated that smaller
radiation field margins could be used without compromising the very high rates
of local control which were being achieved. 10-12 Refinement of external beam
radiotherapy (EBRT) fields was limited by imprecise cross-sectional imaging
22
and treatment planning and delivery systems could not tailor radiation dose to
irregularly shaped volumes. Consequently, for many years conventional
radiotherapy technique for limb STS essentially comprised two parallel opposed
rectangular fields.10
Over the last decade unprecedented improvements in treatment planning
and delivery have become available. Two new approaches, three-dimensional
conformal radiotherapy (3D CRT) and intensity-modulated radiation therapy
(IMRT), have been successfully applied to the treatment of other cancers, but
their application to the management of STS is very new. The introduction of 3D
CRT and IMRT is an important opportunity to reevaluate many aspects of the
current treatment paradigm, which had evolved under the technological
constraints of the past.
3.2 Three-Dimensional Conformal Radiotherapy (3D CRT)
3.2.1 The elements of 3D CRT
Three-dimensional conformal irradiation has been described as

external-beam radiation therapy in which the prescribed dose volume
(treatment volume) is made to conform closely to the target volume. 13 The
increased conformality in comparison to two-dimensional techniques is made
possible by innovations in hardware associated with the treatment apparatus, as
well as in software which allows improved calculations of the absorbed dose
and improved presentation of the results.
3.2.2 The multi-leaf collimator (MLC)
Simple modulation of the treatment beam to improve conformality may

be achieved by physical blocks, missing-tissue compensators, physical wedges
or dynamic wedges generated by moving collimators, but the single most
important hardware innovation in the development of 3D CRT has been the
multi-leaf collimator (MLC). This device consists of multiple interlocking
finger-like blocks mounted in the treatment head of a linear accelerator, which
may be advanced from either side by individual motors under computer control.
The sides of the leaves interlock and are dove-tailed together to minimize
radiation leakage in between the leaves. In the past, conformation required the
manufacture and handling of individual physical blocks. The ability of an MLC
to rapidly change configuration under computer control, and to produce
individualised beam shaping represents a significant advance and has made the
delivery of complex multi-beam treatment plans practical and achievable.
Furthermore, the MLC facilitates modulation of beam intensity by
superimposing a smaller radiation field within a larger field (beam
segmentation).
3.2.3 3D CRT plan calculation, visualization and evaluation
23
The hallmark of three-dimensional planning is the calculation of the

absorbed radiation dose throughout the volume of interest, rather than in
selected axial cross-sections only. This allows accurate assessment of the dose
deposited by beams that are not aligned with the axial plane (non-coplanar
beams) and provides many more beam configurations, which may be utilized to
produce optimal conformality to the target volume.
Software that permits clear presentation of treatment data is an integral
part of such planning systems. The effects of a candidate treatment plan on
multiple sub-volumes may be evaluated, representing tumour and other
structures of interest including normal tissues. These include features such as
beams eye views (BEV) (i.e. visualization of any beam along its central axis
irrespective of its direction and that include the target volume and tissues to be
avoided in the view) (figure 2) and rooms eye views (that allow rapid visual
24
assessment of coverage of the target volume by a single beam and the potential
for collision of couch and gantry). Presentation of dose distributions in different
planes and in three dimensions allow assessment of RT dose perturbation within
and adjacent to the target volume, including hot spots and cold spots.
3D CRT procedures facilitate development of a plan which deposits
dose to the target volume with minimal dose to surrounding tissues. The degree
of conformality may be quantified by the conformity index (CI), which is the
ratio of volume enclosed by the prescription isodose surface to the planning
target volume (PTV). 14 This is somewhat helpful in comparing candidate plans
but in practice it is more useful to compare dose-volume histograms (DVHs) for
volumes of interest (figure 3), 15 as the CI does not fully reveal the effects on
critical structures.
3.3 Intensity modulated radiotherapy (IMRT)
3.3.1 The elements of IMRT
The impact of computer technology enhancements has provided the

opportunity for extraordinary improvement in the physical basis of radiation
therapy. Leading these advances in the contemporary era is the development of
intensity-modulated radiotherapy (IMRT). IMRT is an advanced form of three-
25
dimensional conformal radiotherapy in which radiation beams are not only

shaped at their perimeters, but also include variable intensity across the profiles
of the beams. This permits the creation of exquisite conformation of dose to
targets of irregular shape while generating high dose gradients between tumor
and normal tissues. In addition to the use of intensity modulated beams, the
other key enhancement of IMRT over conventional conformal treatments, is the
use of computer assisted iterative treatment plan optimization. In its complete
form, IMRT combines inverse treatment planning (where dose to normal tissues
as well as target regions is specified in advance), with computer controlled
dynamic beam shaping and filtration (usually with MLC).
3.3.2 The mechanism of IMRT
The IMRT concept relies on the fact that multiple beams crossing a
target from different directions do so with great redundancy that can be
harnessed to provide substantial flexibility in distributing dose. Instead of
permitting the full intensity of a beam to traverse the target, the dose in part(s)
of the field aperture is reduced in a variable way (by programming the
configuration and timing of the MLC leaf positions), according to the
requirements. This can permit sparing of a structure on the one hand or delivery
of relatively enhanced dose to the target in another part of the field aperture
thereby causing deliberate perturbation of the RT dose distribution of a beam.
At the same time, the variable dose incurred in components of the aperture of a
given beam by this process can be compensated by enhancing or reducing the
dose in components of the apertures of other beams directed at the target from
other directions. In this way a beam directed from a given direction, can be
fashioned to comprise numerous (e.g. dozens to hundreds) of mini-beams
(termed beamlets) with variable shape and intensity of radiation exposure.
The ultimate result from the combination of numerous beams of
different direction, intensity patterns, and shape provides an unprecedented three
dimensional configuration to the composite dose distribution from all beams.
Ironically, it is now reasonably certain that the capability of placing dose and
avoiding tissues is significantly more precise than our knowledge of what issues
are indeed involved and the measures required to minimize geometric
uncertainty related to normal physiologic movements in the patient (eg.
breathing, adjustment related to hollow viscous filling and emptying, etc).
3.3.3 Dose sculpting and painting
Using the IMRT approach, the delivered RT dose can be fitted to

volumes tailored to complex shape specifications. These can be both shaped
externally (i.e. convex shaping), as in traditional fields, but also the external
surface of the intended RT dose region can be excavated thereby providing
concave or indented shaping (dose sculpting)16 that permit previously
26
inaccessible target areas to be treated while avoiding adjacent vulnerable

anatomy that may be partially surrounded by the target (figure 4).
Most radiotherapy treatment plans, including 3D CRT plans and IMRT
plans that use dose sculpting strategies, accept a certain amount of dose
heterogeneity within the target volume as a by-product of efforts to conform a
given isodose to the periphery. This apparently anomalous situation can be
further enhanced with IMRT where the opportunity exists to create deliberate
variation in absorbed dose within the target. This has been termed dose
painting 16 and provides further opportunity to relatively spare normal tissues
while delivering even more intense dose accumulation into diseased tissues.
3.3.4 Inverse Planning
IMRT targeting comprises a powerful engineering accomplishment

employing concepts of radiation physics, advanced computation, and a
revolution in hardware design. To achieve this an intended dose distribution is
displayed on anatomic details obtained from thin CT scan slices upon which the
target and the anatomic areas of risk are outlined. The CT scan also provides
the radiation attenuation properties of tissues, which are necessary for the
computation of absorbed dose. This is accomplished using numerical models
that approximate the radiation transport physics in tissues. The planning
objective includes two important specifications: a description of the required
dose to each part of the target and the normal tissues in the treated region; and
the importance of achieving that dose in relative terms compared to the other
structures (target or normal tissues) as a weighting expressed in a
mathematical equation.
Altogether this process is characterized as inverse planning, in which
the requirements for the dose configuration in the patient is specified precisely
in advance in mathematical terms and computer technology is then used to
calculate the intensity pattern specifications of each beam to achieve the
intended result. Thus the clinical objectives are specified mathematically in the
form of an objective function (also called the score function or cost function)17
(see table 1).
In practice the number of beams and selection of their orientation are
chosen by the treatment planner because the computerized optimization process
outlined below that searches for the optimal plan would not be possible, in
practical terms, with our current computation tools. This process can be
facilitated by the use of libraries of plans (including number and direction of
beams) that successfully met a need in the past and the development of class
solutions that generally fit the problem at hand.
Following the specification of treatment goals, an iterative search
algorithm is used to compute the intensity patterns that optimize the dose
distribution throughout the normal tissues and the target. Many thousands of
27
28
iterative calculations are generally required to solve the mathematical task of

calculating an objective function that takes account of the desired dose, the
calculated dose (at each iteration), and the weighting (or priority) to eventually
achieve a distribution of radiation dose in the volume that comes as close as
possible to what is intended.
A variety of computational methods are available for this process, all

with their own advantages and disadvantages, which in itself compromises an
active area of research. These include different degrees of accuracy in
accounting for tissue heterogeneity corrections, geometric dose reproduction
(e.g. adjustment and synchronization of leaf position to take account of leakage
between the leaves of the MLC and passage of radiation through multileaves of
partial depth, the method of objective function calculation, and, of great
29
practical importance, speed of performing the calculations).17, 18 Unfortunately,

the optimization process must be repeated several times if the initial result is not
satisfactory. This may arise if the planning objectives cannot be met and may
include excessive dosing of areas including hot spots in normal tissues that
exceed tolerance and rendering the plan vulnerable from a safety perspective, or
if parts of the target do not receive reliable dose coverage.
4 RATIONALE FOR VOLUMETRIC-BASED PLANNING
4.1 Reducing late tissue adverse events
One of the main potential advantages to conforming the treated

volume to the target volume is to reduce toxicity to surrounding tissues. Non-
randomised studies of limb STS have demonstrated an association of radiation
field size with limb oedema and tissue induration 19 and of radiation dose with
increased oedema, decreased muscle strength, decreased range of motion,
fibrosis and worse functional outcome in general 19, 20. More recently, these
findings have also been borne out in a prospective fashion in the comparison of
different outcomes following pre-operative compared to post-operative
radiotherapy. The latter generally requires higher doses and larger fields and is
associated with significantly higher rates of fibrosis and edema two years
following completion of treatment compared to the pre-operative approach.21
Because of its ability to produce high dose volumes with concave surfaces,
IMRT will usually be able to spare some subcutaneous tissue to reduce the risk
of lymphoedema.22
In addition, late (i.e. many years following treatment) irradiation
induced bone fracture is evident with doses of exceeding 60 Gy in weight-
bearing bone 23, and minimization of dose to, or even complete avoidance of,
such structures would seem desirable in reducing the risk of this debilitating
complication. Dose modeling studies have shown that the ability of IMRT to
produce high dose volumes with concave surfaces can substantially lower mean
dose to the femur and the volume of bone irradiated to 95% of the prescription
dose.24 This may reduce the incidence of late fracture, particularly in the setting
of postoperative radiotherapy where higher doses are prescribed.
4.2 Facilitate curative radiotherapy
Lesions in proximity to vital structures may need specific

volume modifications in order to encompass disease while sparing normal
tissue. This may be particularly attractive in disease sites where critical organ
tolerance may be paramount yet undertreatment of the target poses additional
risks. This may involve potentially achieving kidney protection, spinal cord or
30
lung avoidance in different situations. This differs from the discussion of late
tissue toxicity (section 4.1), because the context here concerns whether
radiotherapy can be delivered at all with curative intent, out of concern for life
threatening complications to critical anatomy. This approach may be best
exemplified by retroperitoneal sarcomas and paraspinal tumours.
Retroperitoneal sarcoma frequently recurs locally, despite optimal
surgery, and radiotherapy is often used pre-, intra- or postoperatively to improve
control.25-28 Local recurrences are still the rule, however. The need to observe
the tolerance of bowel, liver, kidneys and other organs generally requires that
lower doses be prescribed than for sarcomas at other sites and target coverage is
often compromised. 3D CRT and MRT provide the opportunity to treat large
and complex retroperitoneal tumor volumes that were previously close to
impossible to treat. These modalities are especially well suited to pre-operative
radiotherapy because the target is in situ with less risk of intra-abdominal
contamination so that the region to be treated can be readily defined. In
addition, bowel is both mobile and displaced by the tumor and treatment is well
tolerated 28 in contrast to the post-operative setting 29 where the bowel is
frequently tethered in the surgical bed making safe delivery of substantive doses
of radiotherapy problematic. In addition, a problem that is especially difficult is
presented by the large right sided retroperitoneal sarcoma where tumor is in
direct proximity to the liver and frequently hooded by this stucture. Standard
radiotherapy cannot achieve safe coverage of the target while permitting the
liver to be spared. Even 3D CRT is problematic for these lesions and IMRT
presents many advantages (figure 5).
Paraspinal tumors also pose prodigious problems for the safe delivery of
radiotherapy. The real possibility exists for the spine to be severely injured by
the treatments themselves (radiotherapy and surgery are both severely
constrained by normal tissue tolerance) or by tumor. Target volumes partially
enveloping the spinal cord pose a challenge in dose delivery not previously
achievable with standard planning but that can be overcome with IMRT (see
figure 3 and 4, and table 2).
4.3 Retreatment
Reirradiation of limb soft tissue sarcoma after local recurrence has been
shown to contribute to limb conservation, although there is a risk of inducing
radionecrosis, chronic ulceration and fracture. 30, 31 Brachytherapy has been
advocated as the optimal radiation modality for this situation 31-34 but IMRT has
the potential advantages, through inverse planning, to tailor the dose to treat
sites inaccessible to brachytherapy. This approach has been used successfully to
retreat vertebral metastases, including sarcoma,35 and it is well suited to locally
recurrent STS.
31
32
4.4 Reduction of acute radiation morbidity
Combined modality treatment of STS improves local control,36, 37 but

some structures may suffer enhanced toxicity. It is plausible that in the future
IMRT may ameliorate this problem. For instance, preoperative radiotherapy
increases the incidence of major wound complications compared with
postoperative radiotherapy 3, despite reducing late fibrosis and oedema.21 One
potential strategy to enhance wound healing might be to investigate the use
IMRT to avoid irradiating skin in the region of the planned wound.38
Highly conformal RT may also reduce adverse interactions with

chemotherapy. Ifosfamide is a drug that is being actively investigated in
combination with radiotherapy because of its high activity against sarcoma,39, 40
but the incidence of moist desquamation of skin may be increased.39, 41 One
suggestion is that IMRT may allow the radiation dose to skin to be limited to
avoid this problem.38 3D CRT has also been used to reduce radiation toxicity to
bone marrow, so that the intensity of chemotherapy is not compromised. 42
33
4.5 Radiation dose escalation
An intriguing aspect in considering the radiotherapy of many STS sites

is that the results of treatment are generally satisfactory in terms of local control.
The therapeutic strategy must therefore focus on the opportunity to either reduce
the intensity of treatment application or modify radiotherapy volumes compared
to traditional approaches. The exception may be retroperitoneal sarcoma where
the local control rates, even with adjuvant radiotherapy, have been disappointing
although the reason for this are multifactorial and with strong potential that the
tumor is being underdosed and the target area at risk is not covered due to organ
tolerance (see section 4.2). It is plausible that retroperitoneal sarcoma results
could improve if doses to tumour were escalated while conformally avoiding
organs at risk. This is probably achievable with IMRT.43
5 APPLYING VOLUMETRIC-BASED PLANNING
5.1 Multidisciplinary interactions
For several reasons, several specialties should be involved when very

selected treatment RT volumes are proposed. Predominantly this applies to
interaction between the radiation medicine disciplines and surgical oncology,
medical imaging, and pathology, but also extends to medical oncology
collaboration.
We have already noted that prior surgical interventions may influence
the choice of target tissues at risk in a striking fashion. We also discussed the
implications of absence of pretreatment appropriate imaging since initial
chemotherapy may eliminate all evidence of overt disease before radiotherapy
can be administered. Clearly both circumstances may equally result in a
situation where the use of selected conformal volumes may prove impossible.
In retroperitoneal sarcoma, following pre-operative radiotherapy
that may have treated a generous portions of involved or adjacent liver, it is very
important that the surgical team be aware of the area of liver that has been
treated so that sufficient liver is left intact in the patient if partial liver resection
is contemplated. This information can only be imparted by detailed review of
the planning and dosimetry records by the surgeons with the radiation
oncologists and treatment planners 44
As high intensity dose escalation chemo-radiotherapy techniques
evolve, there may be opportunity to spare superficial tissues using IMRT and
prevent acute and long term skin and subcutaneous morbidity that may be a
hallmark of the concurrent use of chemotherapy and radiotherapy.
In this way as treatment strategies evolve, it is likely that the evolution
to more selective radiotherapy volumes will require more rather than less
interaction among the disciplines. Hopefully, it may be possible to develop
34
protocols where anatomic areas treated by one discipline may be avoided

deliberately by the other to reduce the overall morbidity.
5.2 Imaging and image fusion
Imaging is key to the application of 3D CRT and IMRT. Targets as well

as structures to be avoided must be identified accurately with respect to
anatomical site and local extension to surrounding structures. They must then be
delineated on the images of the radiotherapy planning system. Usually the
radiation oncologist will examine the other images beside the planning CT
images, mentally coregister them and then electronically contour the structures
by drawing on the latter. This obviously has potential to introduce error. An
alternative, which is often used when adjacent critical structures require narrow
margins for uncertainty, is to fuse the two sets of images. This is routinely done
in the planning of stereotactic IMRT for skull base sarcoma. It is then relatively
simple for the oncologist to trace around the MRI image of the structure and the
resulting contour is registered on the CT image. The use of other methods of
simulation are being investigated, such as MR simulators and combination PET
/ CT simulators.
5.3 Reduction in geometric uncertainty and treatment

variability
Geographic miss is a serious error as it exposes the patient to toxicity

without the prospect of tumour control. However, as radiation treatment
becomes more conformal the risk of geographic miss increases because any
error is more likely to move the CTV out of the high-dose volume. Uncertainty
is an important component in radiotherapy. The quality assurance requirements
for IMRT present new and unique problems and involve technical issues at the
radiotherapy delivery level as well as the planning problems already discussed.
45, 46
Consistency in set up and the use of appropriate immobilisation (see section
6.1) of anatomic regions should be integral to the planning process to insure that
correct anatomic treatment and avoidance takes place. While strict
immobilisation is not necessarily an integral part of 3D CRT or IMRT, in
practice unless attention is directed towards reducing movement, much of the
benefit of highly conformal treatment will be lost and the risk of unsatisfactory
treatment increased.47
35
6 DOSE HETEROGENEITY AND DOSE DISPERSION
6.1 Dose dumping
An increase in dose heterogeneity within the target volume is

commonly encountered in IMRT plans, frequently beyond the 95% -107%
range that is commonly considered acceptable.5 This phenomenon has been
noted in the treatment of many sites, including the thigh. 24 It is enhanced by a
tendency for users to ascribe high priorities to conformality and target coverage
rather than dose homogeneity in the objective function. 48 Generally, provided
the radiotherapy is prescribed such that no part of the PTV is underdosed and
the hot spots fall within the GTV away from sensitive normal structures,
adverse consequences are unlikely.
When computer-assisted dose optimisation takes place in the IMRT
planning process, the algorithm will consider all plans that satisfy the specified
geometric and dosimetric constraints. This may result in high doses being
dispersed into unexpected sites, and represents the converse of delivering
inadequate dose to the target because unintended dose may be delivered to
normal structures. This phenomenon is called dose dumping. Examples are the
allocation of high doses to skin and subcutaneous tissues in a plan to treat limb
sarcoma or to brain in the treatment of skull base disease. Experience generally
allows prediction of when this might occur, but all IMRT plans should be
carefully examined for unanticipated hot spots. If identified, the optimisation
may need to be repeated after adjustment of contours for the relevant tissue and
allocating appropriate constraints, and the incidence of dose-dumping is
reduced with experience.
6.2 Increased integral dose
In order to achieve highly conformal irradiation of the target, IMRT

plans tend to use more beams than traditional RT approaches. Therefore more
normal tissue can fall within the irradiated volume. Furthermore, the complexity
of IMRT treatments, with many beamlets being delivered at less than 100%
intensity, results in wider dispersion of lower doses of radiation but effectively a
higher patient whole-body (or integral) dose than is the case with conventional
external beam radiotherapy. 17 Concerns have been expressed that this could
substantially enhance the rate of radiation carcinogenesis in cancer survivors49,
50
. This may be acceptable for older adults but in younger patients, particularly
children, very careful consideration should be applied to balancing as yet
unproven improvements in local control and toxicity against possible second
malignancies. In principle, the excess radiation leakage that may lead to excess
secondary cancers can be reduced by careful consideration in the design of
radiation treatment technologies of the future.
36
7 OTHER MODALITIES
External beam photon therapy is only one method of delivering

volumetric-based radiotherapy.
Postoperative brachytherapy has been shown in a prospective
randomised trial to improve local control of high grade STS over resection
alone.36 However, it requires skills and facilities that are not available in many
centers but the advantages of a short overall treatment time and irradiation of
less normal tissue than traditional EBRT are understandably attractive.34
Technological enhancements now also permit full 3D planning for
brachytherapy with dose-volume histograms and other tools employed by 3D
CRT.51 There is also no reason why brachytherapy treatment planning systems
should not be extended to include inverse planning capability, which might
allow further sparing of nearby critical structures. The low integral dose
resulting from brachytherapy makes it a useful option for recurrent tumour in
previously irradiated tissue.52
In proton beam irradiation, high-dose volume treatment volumes can be
made to conform precisely to the target by varying the depth and breadth of the
Bragg peak. 53 If the peak is positioned at the target, there is no dose deposited
in deeper tissues but also less dose in superficial tissues than if photon beams
were used. Therefore proton RT has the advantage of a lower integral dose to
normal tissues. The major drawback to the application of this technology is the
cost of constructing facilities for delivery and currently it is only available at a
handful of sites. Nevertheless, work is underway to improve access by
increasing the number of treatment units and to improve its capability by
introduction of intensity modulated proton beam (IMPT) planning and delivery.
53
Modulated electron therapy (MERT) is an investigational modality,

which shows promise in the treatment of superficial targets. Modulation of
electron beam energy, as well as intensity, allows limitation of the depth of
penetration with a reduction in the dose to tissues that lie beyond tumour.54 This
may result in a lower integral dose than photon IMRT produces. Theoretical
applications include breast tumours (with sparing of lung) and situations where
large areas of scalp need to be treated, such as for angiosarcoma (with sparing of
underlying brain).
8 FUTURE PERSPECTIVES
8.1 Biological targeting / dose-painting
In STS, it has long been appreciated that biological heterogeneity exists.

If different subvolumes have different biology, they may require different
radiation doses to eradicate malignant clones. Larger radiation doses may be
needed if clonogen density is greater, if hypoxia is present or if there is intrinsic
37
radioresistance. A number of functional imaging modalities are showing

potential to identify such regions and could permit a biological target volume
(BTV) to be conceived within the GTV on the basis of such imaging
characteristics.16 These could include the incorporation of single positron
emission tomography (PET), single photon emission computed tomography
(SPECT)55, or proton MR spectroscopy (MRS).56, 57 Other novel applications of
MRI include the identification of regions of hypoxia using blood-oxygen level
dependent (BOLD) imaging sequences.58
The ability of IMRT to design and deliver specific nonuniform dose
distributions within a target (dose painting) has provided the capability to
target different parts of the CTV. The BTV might be targeted to receive a higher
radiation dose than the surrounding gross tumour, resulting in biological
conformality as well as physical conformality.16 In this way, a paradigm of
multidimensional conformal radiotherapy potentially acknowledges biological
tumour heterogeneity in addition to the goals of physical conformality.16
8.2 Image-guided radiotherapy using on-line imaging
A highly active area of research is the use of CT imaging at the time of

radiotherapy delivery. This reduces uncertainty regarding the positions of the
target and organs at risk, as they no longer need to be inferred from images
acquired days or weeks earlier. If the CT scanner and the linear accelerator
gantry are mounted about the same axis, then the coordinates of structures of
interest can be related directly to the radiotherapy delivery system, rather than to
surface markings on the patient that are in turn related to the delivery system.
One approach, under development at our centre is flat-panel cone-beam
computed tomography where a kilovoltage photon source is mounted at 90 to
the treatment head.59 A cone-shaped beam passes through the patient and is
collected by a flat detector mounted on the opposite side during a single rotation
to construct high-resolution images that allow adjustments to the treatment plan
to be made to improve target coverage (adaptive radiotherapy). An alternative
strategy is to mount a linear accelerator onto the ring gantry of a helical CT
scanner to produce a treatment and imaging system known as helical
tomotherapy. As well as delivering rotational IMRT, the transmitted portion of
the beam is collected by a detector array on the opposite side of the gantry to
produce megavoltage CT images that can be compared to the planning images
to verify the accuracy of the delivered treatment.60
8.3 Clinical assessments
A key recommendation from the recent U.S. National Cancer Institute

State-of-the-Science meeting on adult sarcomas in June 2002 included the view
that IMRT) is a promising technique that should be actively studied in well-
controlled trials of patients with soft tissue sarcomas.61 To achieve this,
38
prospective assessment of outcome should include established and validated

methods of measurement for normal tissue toxicity, functional assessment,
quality of life and careful evaluation of acute and late complications and local
control of disease.
There is an urgent need to correlate toxicity with modern
measures of delivered dose, such as DVHs that allow assessment of the effects
of partial organ tolerance and of inhomogeneous irradiation of whole organs.
Certain structures, not previously considered avoidance targets, may be spared
toxicity provided appropriate dose limits can be defined. For example,
individual lymphatic trunks can be identified by indirect lymphography.62
Avoidance of these might allow more reliable prevention of lymphoedema
following limb irradiation. In the long term, is conceivable that every part of the
irradiated volume, including bone, joints and muscle beyond the PTV, might be
contoured and assigned dose limits to reduce late toxicity.
9 CONCLUSION
RT targeting of STS presents considerable challenges in realizing

optimum outcome in tissue and function preservation while maintaining high
local control for most anatomic sites. While a highly effective adjuvant, RT
delivered improperly may cause substantial disability by excessive volume or
dose delivery. The advent of very precise treatment planning and delivery
systems, including 3D CRT and IMRT, means it is now possible to choose to
treat ideal volumes rather than ones that are merely feasible. At the same time
precise knowledge of appropriate targets continues to evolve for the different
clinical scenarios and will likely be greatly influenced in the future by enhanced
imaging capability. Clinical trials are needed that include relevant end-points to
measure improvements in the therapeutic ratio resulting from more precise RT
targeting and without loss of local control. In addition, advancement of 3D
CRT and IMRT over the next decade will rely on the consistent reporting and
sharing of results concerning outcome of normal tissue from volumetric
treatment planning.47
10 REFERENCES
1. Mankin HJ, Mankin CJ, Simon MA. The hazards of the biopsy, revisited. Members of the
Musculoskeletal Tumor Society [see comments]. J Bone Joint Surg Am 1996;78(5):656-63.
2. OSullivan B, Wunder J, Pisters PW. Target description for radiotherapy of soft tissue
sarcoma. In: Gregoire V, Scalliet P, Ang KK, editors. Clinical target volumes in conformal
radiotherapy and intensity modulated radiotherapy. Heidelberg: Springer; 2003. p. 205-227.
3. OSullivan B, Davis AM, Turcotte R, Bell R, Catton C, Chabot P, Wunder J, Kandel R,
Goddard K, Sadura A, Pater J, Zee B. Preoperative versus postoperative radiotherapy in soft-
tissue sarcoma of the limbs: a randomised trial. Lancet 2002;359(9325):2235-41.
4. Panicek DM, Schwartz LH. Soft tissue edema around muskuloskeletal sarcomas at
magnetic resonance imaging. Sarcoma 1997; 1:189-191.
39
5. ICRU Report 50. Prescribing, Recording, and Reporting Photon Beam Therapy. Bethesda:
International Commission on Radiation Units and Measurement; 1993.
6. ICRU Repor t62. Prescribing, Recording, and Reporting Photon Beam Therapy
(Supplement to ICRU Report 50). Bethesda: International Commission on Radiation Units
and Measurement; 1999.
7. Nielsen OS, Cummings B, OSullivan B, Catton C, Bell RS, Fornasier VL. Preoperative
and postoperative irradiation of soft tissue sarcomas: effect of radiation field size. Int J Radiat
Oncol Biol Phys 1991;21(6):1595-9.
8. Enneking WF, Spanier SS, Goodman MA. Current concepts review. The surgical staging
of musculoskeletal sarcoma. J Bone Joint Surg Am 1980;62(6):1027-30.
9. Simon MA, Enneking WF. The management of soft-tissue sarcomas of the extremities. J
Bone Joint Surg Am 1976;58(3):317-27.
10. Tepper J, Rosenberg SA, Glatstein E. Radiation therapy technique in soft tissue sarcomas
of the extremity--policies of treatment at the National Cancer Institute. Int J Radiat Oncol Biol
Phys 1982;8(2):263-73.
11. Lindberg RD, Martin RG, Romsdahl MM, Barkley HT, Jr. Conservative surgery and
postoperative radiotherapy in 300 adults with soft-tissue sarcomas. Cancer 1981;47(10):2391-
7.
12. Suit HD, Spiro I. Role of radiation in the management of adult patients with sarcoma of
soft tissue. Semin Surg Oncol 1994;10(5):347-56.
13. Emami B, Graham MV, Michalski JM, Perez CA. Three-Dimensional Conformal
Radiation Therapy: Clinical Aspects. In: Perez CA, Brady LW, editors. Principles and
Practice of Radiation Oncology. 3rd ed. Philadelphia: Lippincott-Raven; 1998. p. 371-386.
14. Knoos T, Kristensen I, Nilsson P. Volumetric and dosimetric evaluation of radiation
treatment plans: radiation conformity index. Int J Radiat Oncol Biol Phys 1998;42(5):1169-
76.
15. Mohan R, Brewster LJ, Barest GD. A technique for computing dose volume histograms
for structure combinations. Med Phys 1987;14(6):1048-52.
16. Ling CC, Humm J, Larson S, Amols H, Fuks Z, Leibel S, Koutcher JA. Towards
multidimensional radiotherapy (MD-CRT): biological imaging and biological conformality.
Int J Radiat Oncol Biol Phys 2000;47(3):551-60.
17. IMRT. Intensity-modulated radiotherapy: current status and issues of interest. IMRT
Collaborative Working Group. Int J Radiat Oncol Biol Phys 2001;51(4):880-914.
18. Webb S. Advances in three-dimensional conformal radiation therapy physics with
intensity modulation. Lancet Oncol 2000;1(1):30-6.
19. Robinson MH, Spruce L, Eeles R, Fryatt I, Harmer CL, Thomas JM, Westbury G. Limb
function following conservation treatment of adult soft tissue sarcoma. Eur J Cancer
1991;27(12):1567-74.
20. Stinson SF, DeLaney TF, Greenberg J, Yang JC, Lampert MH, Hicks JE, Venzon D,
White DE, Rosenberg SA, Glatstein EJ. Acute and long-term effects on limb function of
combined modality limb sparing therapy for extremity soft tissue sarcoma. Int J Radiat Oncol
Biol Phys 1991;21(6):1493-9.
21. OSullivan B, Davis A. A Randomized phase III trial of pre-operative compared to post-
operative radiotherapy in extremity soft tissue sarcoma.[Abstract].Proc 43rd Annual Meeting,
American Society of Therapeutic Radiology and Oncology. Int J Radiation Oncology Biol
Phys 2001;51(3, supplement 1):151.
22. Millar BM, Bragg CM, Conway J, Robinson MH. Investigation of the use of intensity
modulated radiotherapy (IMRT) in comparison with conformal radiotherapy in the
management of soft tissue sarcoma.[Abstract]. Proc 43rd Annual Meeting, American Society
of Therapeutic Radiology and Oncology. International Journal of Radiation Oncology
Biology Physics 2001;51(3 (Supplement 1)):412.
23. Holt GE, Wunder JS, Griffin AM, Bell RS. Fractures following radiation therapy and
limb salvage surgery for soft tissue sarcomas: high versus low dose radiotherapy.[Abstract]
Proc Muskuloskeletal Tumor Society 2002:41.
40
24. Hong L, Alektiar K, Hunt M, Leibel S. Intensity Modulated Radiotherapy for Soft Tissue
Sarcoma of Thigh [Abstract]. Proc 44th Annual Meeting, American Society of Therapeutic
Radiology and Oncology. Int J Radiat Oncol Biol Phys 2002;54 (2S):140-1.
25. Gieschen HL, Spiro IJ, Suit HD, Ott MJ, Rattner DW, Ancukiewicz M, Willett CG.
Long-term results of intraoperative electron beam radiotherapy for primary and recurrent
retroperitoneal soft tissue sarcoma. Int J Radiat Oncol Biol Phys 2001;50(1):127-31.
26. Petersen IA, Haddock MG, Donohue JH, Nagorney DM, Grill JP, Sargent DJ, Gunderson
LL. Use of intraoperative electron beam radiotherapy in the management of retroperitoneal
soft tissue sarcomas. Int J Radiat Oncol Biol Phys 2002;52(2):469-75.
27. Stoeckle E, Coindre JM, Bonvalot S, Kantor G, Terrier P, Bonichon F, Nguyen Bui B.
Prognostic factors in retroperitoneal sarcoma: a multivariate analysis of a series of 165
patients of the French Cancer Center Federation Sarcoma Group. Cancer 2001;92(2):359-68.
28. Jones JJ, Catton CN, OSullivan B, Couture J, Heisler RL, Kandel RA, Swallow CJ.
Initial results of a trial of preoperative external-beam radiation therapy and postoperative
brachytherapy for retroperitoneal sarcoma. Ann Surg Oncol 2002;9(4):346-54.
29. Gilbeau L, Kantor G, Stoeckle E, Lagarde P, Thomas L, Kind M, Richaud P, Coindre JM,
Bonichon F, Bui BN. Surgical resection and radiotherapy for primary retroperitoneal soft
tissue sarcoma. Radiother Oncol 2002;65(3):137-43.
30. Graham JD, Robinson MH, Harmer CL. Re-irradiation of soft-tissue sarcoma. Br J Radiol
1992;65(770):157-61.
31. Nori D, Schupak K, Shiu MH, Brennan MF, Shupak K. Role of brachytherapy in
recurrent extremity sarcoma in patients treated with prior surgery and irradiation. Int J Radiat
Oncol Biol Phys 1991;20(6):1229-33.
32. Catton C, Davis A, Bell R, OSullivan B, Fornasier V, Wunder J, McLean M. Soft tissue
sarcoma of the extremity. Limb salvage after failure of combined conservative therapy.
Radiother Oncol 1996;41(3):209-14.
33. Pearlstone D, Janjan NA, Feig B, Yasko A, Hunt K, Pollock R, Lawyer A, Horton J,
Pisters P. Re-resection with brachytherapy for locally recurrent soft tissue sarcoma arising in a
previously irradiated field. Cancer J Sc Am 1999;5(1):26-33.
34. Crownover RL, Marks KE. Adjuvant brachytherapy in the treatment of soft-tissue
sarcomas. Hematol Oncol Clin North Am 1999;13(3):595-607.
35. Milker-Zabel S, Zabel A, Thilmann C, Schlegel W, Wannenmacher M, Debus J. Clinical
results of retreatment of vertebral bone metastases by stereotactic conformal radiotherapy and
intensity-modulated radiotherapy. Int J Radiat Oncol Biol Phys 2003;55(1):162-7.
36. Pisters PW, Harrison LB, Leung DH, Woodruff JM, Casper ES, Brennan MF. Long-term
results of a prospective randomized trial of adjuvant brachytherapy in soft tissue sarcoma. J
Clin Oncol 1996;14(3):859-68.
37. Yang JC, Chang AE, Baker AR, Sindelar WF, Danforth DN, Topalian SL, DeLaney T,
Glatstein E, Steinberg SM, Merino MJ, Rosenberg SA. Randomized prospective study of the
benefit of adjuvant radiation therapy in the treatment of soft tissue sarcomas of the extremity.
J Clin Oncol 1998;16(1):197-203.
38. OSullivan B, Bell R. Has MAID made it in the management of high-risk soft-tissue
sarcoma. International Journal of Radiation Oncology Biology Physics 2003;56(4):915-916.
39. Cormier JN, Patel SR, Herzog CE, Ballo MT, Burgess MA, Feig BW, Hunt KK, Raney
RB, Zagars GK, Benjamin RS, Pisters PW. Concurrent ifosfamide-based chemotherapy and
irradiation. Analysis of treatment-related toxicity in 43 patients with sarcoma. Cancer
2001;92(6):1550-5.
40. Sauer R, Schuchardt U, Hohenberger W, Wittekind C, Papadopoulos T, Grabenbauer GG,
Fietkau R. [Neoadjuvant radiochemotherapy in soft tissue sarcomas. Optimization of local
functional tumor control]. Strahlenther Onkol 1999;175(6):259-66.
41. DeLaney TF, Spiro IJ, Suit HD, Gebhardt MC, Hornicek FJ, Mankin HJ, Rosenberg AL,
Rosenthal DI, Miryousefi F, Ancukiewicz M, Harmon DC. Neoadjuvant Chemotherapy and
Radiotherapy for Large Extremity Soft Tissue Sarcomas. International Journal of Radiation
Oncology Biology Physics 2003;56(4): 1117-1127.
41
42. Coles CE, Twyman N, Earl HM, Burnet NG. Conformal radiotherapy facilitates the
delivery of concurrent chemotherapy and radiotherapy: a case of primitive neuroectodermal
tumour of the chest wall. Sarcoma 2000;4(3):129-133.
43. Haycocks T, Kelly V, Islam M, OSullivan B, Swallow CJ, Catton CN. High resolution,
intensity modulated radiation therapy (IMRT) for retroperitoneal soft tissue sarcoma (RPS)
[Abstract]. Proceedings of the 7th Annual Meeting of the Connective Tissue Oncology
Society. Sarcoma 2001;5(Supplement 1,):S24-S25.
44. OSullivan B, Wylie J, Catton C, Gutierrez E, Swallow CJ, Wunder J, Gullane P, Neligan
P, Bell R. The local management of soft tissue sarcoma. Semin Radiat Oncol 1999;9(4):328-
48.
45. Low DA. Quality assurance of intensity-modulated radiotherapy. Semin Radiat Oncol
2002;12(3):219-28.
46. Dixon P, OSullivan B. Radiotherapy quality assurance: time for everyone to take it
seriously. Eur J Cancer 2003;39(4):423-9.
47. Purdy JA. Dose-volume specification: New challenges with intensity-modulated radiation
therapy. Semin Radiat Oncol 2002;12(3):199-209.
48. Pirzkall A, Carol M, Lohr F, Hoss A, Wannenmacher M, Debus J. Comparison of
intensity-modulated radiotherapy with conventional conformal radiotherapy for complex-
shaped tumors. Int J Radiat Oncol Biol Phys 2000;48(5):1371-80.
49. Verellen D, Vanhavere F. Risk assessment of radiation-induced malignancies based on
whole-body equivalent dose estimates for IMRT treatment in the head and neck region.
Radiother Oncol 1999;53(3):199-203.
50. Hall EJ, Wuu CS. Radiation-induced second cancers: the impact of 3D-CRT and IMRT.
Int J Radiat Oncol Biol Phys 2003;56(1):83-8.
51. Kovacs G, Hebbinghaus D, Dennert P, Kohr P, Wilhelm R, Kimmig B. Conformal
treatment planning for interstitial brachytherapy. Strahlenther Onkol 1996;172(9):469-74.
52. Catton CN, Swallow CJ, OSullivan B. Approaches to local salvage of soft tissue sarcoma
after primary site failure. Semin Radiat Oncol 1999;9(4):378-88.
53. Suit H. The Gray Lecture 2001: coming technical advances in radiation oncology. Int J
Radiat Oncol Biol Phys 2002;53(4):798-809.
54. Ma CM, Pawlicki T, Lee MC, Jiang SB, Li JS, Deng J, Yi B, Mok E, Boyer AL. Energy-
and intensity-modulated electron beams for radiotherapy. Phys Med Biol 2000;45(8):2293-
311.
55. Nishizawa K, Okunieff P, Elmaleh D, McKusick KA, Strauss HW, Suit HD. Blood flow
of human soft tissue sarcomas measured by thallium-201 scanning: prediction of tumor
response to radiation. Int J Radiat Oncol Biol Phys 1991;20(3):593-7.
56. Nelson SJ. Multivoxel magnetic resonance spectroscopy of brain tumors. Mol Cancer
Ther 2003;2(5):497-507.
57. Sijens PE. Phosphorus MR spectroscopy in the treatment of human extremity sarcomas.
NMR Biomed 1998;11(7):341-53.
58. Baudelet C, Gallez B. How does blood oxygen level-dependent (BOLD) contrast
correlate with oxygen partial pressure (pO2) inside tumors? Magn Reson Med
2002;48(6):980-6.
59. Jaffray DA, Siewerdsen JH, Wong JW, Martinez AA. Flat-panel cone-beam computed
tomography for image-guided radiation therapy. Int J Radiat Oncol Biol Phys
2002;53(5):1337-49.
60. Mackie TR, Kapatoes J, Ruchala K, Lu W, Wu C, Olivera G, Forrest L, Tome W, Welsh
J, Jeraj R, Harari P, Reckwerdt P, Paliwal B, Ritter M, Keller H, Fowler J, Mehta M. Image
guidance for precise conformal radiotherapy. Int J Radiat Oncol Biol Phys 2003;56(1):89-105.
42
61. Borden EC, Baker LH, Bell RS, Bramwell V, Demetri GD, Eisenberg BL, Fletcher CD,
Fletcher JA, Ladanyi M, Meltzer P, OSullivan B, Parkinson DR, Pisters PW, Saxman S,
Singer S, Sundaram M, Van Oosterom AT, Verweij J, Waalen J, Weiss SW, Brennan MF.
Soft tissue sarcomas of adults: state of the translational science. Clin Cancer Res
2003;9(6):1941-56.
62. Partsch H, Stoberl C, Wruhs M, Wenzel-Hora BI. Indirect lymphography with iotrolan.
Fortschr Geb Rontgenstrahlen Nuklearmed Erganzungsbd 1989;128:178-81.
Chapter 3
Preoperative therapy for soft tissue sarcoma
Janice N. Cormier, MD, MPH, Howard N. Langstein, MD,

Peter W. T. Pisters, MD
The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030-4009
Correspondence to:
Peter Pisters MD
The University of Texas M. D. Anderson Cancer Center,
1515 Holcombe Boulevard
Box 444
Houston, TX 77030-4009
USA
44
1 INTRODUCTION
A number of theoretical advantages are associated with the use of

preoperative therapy for solid tumors. For patients with soft tissue sarcomas,
a survival advantage has been difficult to establish, but there is evidence that
induction therapy (e.g., chemotherapy, radiation therapy, or multimodality
regimens) may result in cytoreduction that facilitates less radical surgical
resections with improved postoperative function. This is particularly
important for patients presenting with large tumors that are initially resectable
only by means of amputation. The focus of this review is to summarize the
rationale, treatment options and ultimate impact of induction therapy for
patients with soft tissue sarcoma.
2 RATIONALE FOR INDUCTION THERAPY
2.1 Solid Tumors
The primary goal of induction therapy for solid tumors is to decrease

the tumor burden. The concept of induction therapy, consisting of either
preoperative local therapies (e.g., radiation, chemoradiation, or limb
perfusion) or systemic therapy (e.g., chemotherapy), arose because surgical
resection alone was inadequate for a number of tumors (1).
There are several theoretical reasons for using induction or

preoperative therapies for localized tumors. First, reducing the size of the
tumor may enable margin-negative (R0) resections, resection of tumors that
were initially unresectable, or less radical resections that allow preservation of
function. Second, there may be systemic benefits from the early delivery of
cytotoxic agents, such as elimination of micrometastatic disease and improved
vascular delivery of therapies to undisturbed tumors (1). Finally, induction
therapy allows rapid assessment of tumor response in situ. This is not
possible with postoperative therapy, which requires long-term follow-up for
assessment of its effectiveness (2).
There are also potential disadvantages to induction therapy, including
the associated toxicity (morbidity), possible ineffectiveness of induction
therapy and delayed local treatment of the primary tumor, cost of therapy, and
obscuring of pathologic staging information (3). When induction therapy
includes radiation, the risk of wound-healing complications after surgery is
increased. The effectiveness of induction therapy must be demonstrated prior
to its generalization because many patients may be cured by locoregional
treatment alone (1,3).
The sequence of treatments -- chemotherapy, radiation therapy, and
surgery-- may be important in determining outcomes for patients with some
tumor types. Three categories of tumors have been defined with regard to
45
induction chemotherapy: (1) tumors for which chemotherapy has been

demonstrated to be the primary therapeutic modality (e.g., embryonal
rhabdomyosarcoma, small cell lung cancer, and lymphoma), (2) tumors for
which there is evidence of downstaging with chemotherapy (e.g., osteogenic
and Ewings sarcoma, locally advanced breast cancer, anal carcinoma, and
laryngeal cancer), and (3) tumors for which the benefits of induction
chemotherapy have not been scientifically validated (e.g., esophageal cancer,
gastric and pancreas cancers, non-small cell lung cancer, prostate cancer,
cervical carcinoma, nasopharyngeal cancer, and soft tissue sarcomas) (1).
2.2 Sarcomas
Sarcomas are among the group of solid tumors for which the benefits
of induction therapy have been difficult to establish. Survival benefits have
not been definitively demonstrated because of the paucity of adequately
powered randomized clinical trials. However, single-institution reports
suggest that preoperative therapy enables tumor downstaging and organ
sparing in some patients.
Several distinct groups of sarcomas are recognized: soft tissue
sarcomas, bone sarcomas (osteosarcomas and chondrosarcomas), Ewings
sarcomas, and peripheral primitive neuroectodermal tumors. Since the late
1980s, preoperative chemotherapy has been the standard treatment for
patients with osteosarcoma based on data from randomized controlled trials
demonstrating a significant survival advantage with systemic therapy (4,5).
The histologic subtypes rhabdomyosarcoma and Ewings sarcoma have been
demonstrated to have a higher propensity for systemic metastases, and for
these histologies, the addition of chemotherapy may have survival advantages
and is considered standard care (6,7).
The use of chemotherapy (preoperative or postoperative) for other
sarcomas remains controversial. Patients with large (> 5 cm), high-grade,
deep, extremity soft tissue sarcomas (American Joint Committee on Cancer
stage III) commonly develop distant recurrence and subsequently die of
sarcoma. Consequently, pre- or postoperative anthracycline-based
chemotherapy is often considered in these patients.
46
3 INDUCTION THERAPY OPTIONS
3.1. Preoperative Radiation Therapy
3.1.1. Extremity and Trunk Sarcomas
The primary goal of pre- or postoperative radiation therapy is to

maximize local tumor control. Surgical resection and radiation therapy
(external beam or brachytherapy) are generally considered standard treatment
modalities for most patients with large, high-grade extremity soft tissue
sarcomas. This therapeutic approach is based on data from two phase III
trials demonstrating improved local control with the addition of radiation
therapy for patients with localized extremity and trunk sarcomas (8,9). In the
randomized trial from the National Cancer Institute (NCI), 91 patients with
high-grade extremity tumors were treated with limb-sparing surgery followed
by chemotherapy alone or by radiation therapy plus adjuvant chemotherapy.
A second group of 50 patients with low-grade tumors were treated with
resection alone or resection plus radiation therapy. The 10-year rate of local
control for all patients receiving radiation therapy was 98%, compared with
70% for those not receiving radiation therapy (p = 0.0001) (8). Similarly, in
the randomized trial from Memorial Sloan-Kettering Cancer Center, 164
patients with extremity or trunk sarcomas underwent observation or
brachytherapy after conservative surgery. The 5-year local control rate for
patients with high-grade tumors was 66% in the observation group and 89%
in the brachytherapy group (p = 0.003) (9).
No consensus exists on the optimal sequence of radiation therapy and
surgery. Proponents of preoperative radiation therapy cite several advantages.
First, multidisciplinary planning with radiation oncologists, medical
oncologists, and surgeons is facilitated early in the course of therapy while the
tumor is in place. Second, lower doses of preoperative radiation can more
easily be delivered to an undisturbed tumor bed, which may have improved
tissue oxygenation (10-12). Third, the size of preoperative radiation fields is
smaller and the number of joints included in those fields is fewer than in
postoperative radiation fields, and this may result in improved functional
outcome (13). And finally, preoperative radiation may induce tumor
shrinkage and thus facilitate surgical resection (11). On the other hand, critics
of preoperative radiation therapy cite as disadvantages the difficulty of
pathologic assessment of margins in irradiated specimens and the increased
rate of wound complications.
The only randomized comparison of preoperative and postoperative

radiation therapy for soft tissue sarcoma conducted to date was performed by
the National Cancer Institute of Canada Clinical Trials Group (14). This trial
was designed to compare complications and functional outcomes of sarcoma
patients treated with preoperative or postoperative external-beam radiation
47
therapy. From October 1994 to December 1997, 190 patients were

randomized to receive preoperative radiation therapy (50 Gy) or postoperative
radiation therapy (66 Gy). At a median follow-up of 3.3 years, wound
complications had occurred in 35% of patients given preoperative radiation
therapy but only 17% of patients given postoperative radiation therapy (p =
0.01). The majority of wound complications occurred in patients with lower
extremity sarcomas. Both groups achieved similarly high rates of local
control and progression-free survival at 3 years (14). Interestingly, overall
survival appeared to be improved in patients treated with preoperative
radiation. These findings suggest that preoperative external-beam radiation
therapy is effective but that patients should be informed of the increased risk
for major wound complications particularly for patients with lower
extremity soft tissue sarcoma.
3.1.2. Retroperitoneal Sarcomas
Based on the high rates of local control obtained with surgery plus
radiation therapy in patients with extremity and trunk sarcomas, there has
been interest in attempting such strategies for patients with retroperitoneal
sarcomas.
Administering preoperative radiation therapy to retroperitoneal soft
tissue sarcomas is complex. Large tumors in proximity to vital radiosensitive
anatomic structures frequently hinder safe delivery of treatment. However,
there are several advantages to administering radiation therapy preoperatively
for retroperitoneal sarcomas: the gross tumor volume is definable, which
allows accurate treatment planning; tumors often displace radiosensitive
viscera outside of the radiation field; and biologically effective radiation
doses may be lower in the preoperative setting (15).
Several groups have prospectively examined the effects of
preoperative and intraoperative radiation therapy administered to patients with
retroperitoneal sarcomas (16-19). These studies demonstrate that preoperative
radiation doses of 45 to 50.4 Gy can be delivered to the retroperitoneum with
acceptable treatment-related toxicity.
3.2 Preoperative Chemotherapy
Chemotherapy given either preoperatively or postoperatively for soft

tissue sarcomas remains controversial. Soft tissue sarcomas encompass a
diverse group of cancers that vary greatly in natural history and response to
treatment, and the results of conventional chemotherapy regimens have
generally been poor. While some histologic subtypes of sarcoma are very
48
responsive to cytotoxic chemotherapy, most subtypes are resistant to current

agents.
The most active chemotherapeutic agents for bone sarcomas are
doxorubicin, methotrexate, cisplatin, and ifosfamide. For Ewings sarcoma,
doxorubicin, vincristine, cyclophosphamide, and ifosfamide have
demonstrated response rates of up to 90% (20,21). Dactinomycin, vincristine,
and etoposide are active only against small-cell sarcomas, including Ewings
sarcoma, rhabdomyosarcoma, primitive neuroectodermal tumor, and
neuroblastoma. For other subtypes of sarcoma doxorubicin and ifosfamide
are the two most active agents, with consistently reported response rates of
20% to 40% (20,22,23). Both agents have demonstrated positive dose-
response curves (24,25). Response rates to ifosfamide at higher doses or in
combination with doxorubicin have been reported to range from 20% to 60%
in single-institution series (24,26-29).
There are several theoretical benefits associated with early systemic
treatment. In addition to early treatment of micrometastatic disease,
preoperative systemic chemotherapy may induce primary tumor shrinkage,
resulting in increased rates of limb salvage. In addition, a major deterrent to
the use of postoperative chemotherapy has been the risk of adverse toxic
effects in patients who do not respond to therapy. The ability to assess the
effects of treatment by assessing tumor response in situ argues in favor of
preoperative chemotherapy. Such an approach spares those patients who fail
to respond to therapy from the prolonged toxicities of an ineffective
treatment. Patients who are deemed to be responsive to preoperative
chemotherapy can be treated postoperatively as well with hope of improving
their outcome.
3.3 Multimodality Therapy / Chemoradiation
3.3.1 Extremity and Trunk Sarcomas
The objectives of sequential multimodality therapy or concurrent

chemoradiation therapy are similar to those of other induction regimens, to
achieve tumor reduction and provide local control while allowing the timely
administration of systemic therapy to eradicate potential micrometastatic
disease.
Treatment approaches that combine systemic chemotherapy with
radiosensitizers and concurrent external-beam radiation may improve disease-
free survival by treating microscopic disease while enhancing the treatment of
macroscopic disease. Concurrent chemoradiation with doxorubicin-based
regimens reportedly produces favorable local control rates for patients with
soft tissue sarcoma (30,31). The initial experience involved intra-arterial
doxorubicin combined with high-dose-per-fraction radiation therapy in
patients with extremity soft tissue sarcomas. Since those findings were
49
published, several groups have attempted to evaluate the optimal route of

administration (30,32-35), alternative chemotherapeutic agents (36-38), and
the toxicity of combined therapies (39).
The route of administration of doxorubicin, intra-arterial versus
intravenous, was evaluated in a small phase III trial, which demonstrated no
difference between the two routes in limb salvage, local recurrence,
complications, or pathologic response (40). Given these results and the
complexity of intra-arterial administration, intravenous administration of
chemotherapeutic agents during concurrent chemoradiation is the current
standard (31).
Doxorubicin is the most commonly studied radiosensitizer for soft
tissue sarcoma (31). A number of other agents have been evaluated, including
idoxuridine (38), razoxane (36), and ifosfamide (41). In addition, various
chemotherapy approaches have been studied including short-duration
chemotherapy with concurrent rapid-fractionation radiation therapy (31), low-
dose continuous-infusion chemotherapy with concurrent radiation therapy
(42), and sequential chemotherapy and radiation therapy (43). The toxicity
and responses for each approach appear acceptable, but additional survival
data are required to determine if one is superior.
It is difficult to make comparisons between chemotherapeutic agents
and regimens because of the diversity of the patient populations, but in
general preoperative chemoradiation combined with surgery has been
demonstrated to be feasible, to have acceptable toxicity, and to result in
favorable local control rates for patient with localized and locally advanced
soft tissue sarcomas. Theoretical advantages of concurrent treatment
notwithstanding, the concurrent use of local and systemic therapies decreases
the total treatment time for patients with high-risk sarcoma. This decrease
represents a specific advantage over current sequential multimodality
treatment approaches, for which the total time for radiation, chemotherapy,
surgery, and rehabilitation frequently exceeds 6 to 9 months. The toxicity
associated with chemoradiation depends on the particular chemotherapeutic
agent and route of administration as well as the radiation dose/fractionation
regimen. Tumor factors such as size and anatomic site may also be critical in
treatment-related toxicity. As with preoperative radiation alone, the rate of
postoperative wound complications for patients treated with preoperative
chemoradiation is high, reported as 26% in some series (30).
3.3.2 Retroperitoneal Sarcomas
Anatomic considerations favor preoperative over postoperative

radiation for patients with retroperitoneal sarcomas (31), and pilot and phase I
studies using idoxuridine-based and doxorubicin-based (44,45)
50
chemoradiation have demonstrated that these approaches are safe and feasible
for patients with retroperitoneal sarcomas.
The Radiation Therapy Oncology Group is conducting a multicenter
phase II trial of combined multimodality treatment for patients with
intermediate- or high-grade retroperitoneal sarcomas (RTOG S-0124,
www.rtog.org). Patients are given preoperative systemic therapy with
doxorubicin and ifosfamide (up to 4 cycles) followed by preoperative
external-beam radiation therapy (45 to 50 Gy) and then surgical resection with
an intraoperative or postoperative radiation boost. The objective of the trial is
to assess the feasibility, toxicity, and complications of this multimodality
treatment regimen.
4 IMPACT OF PREOPERATIVE THERAPY
4.1 Scope of Surgery
The goal of surgical therapy for soft tissue sarcoma is to achieve

grossly and microscopically negative (R0) margins of resection with the best
possible functional result. Experience has demonstrated that surgical
resection alone is generally inadequate treatment except for patients with
small, superficial, well-differentiated lesions (46-48). The translation of a
response to induction therapy (e.g., radiation therapy or chemotherapy) into a
clinically meaningful outcome reduction in the scope of surgical resection
is not well defined.
A recent retrospective analysis examined the impact of preoperative
chemotherapy on the scope of surgery. The study included a blinded
assessment of imaging studies obtained before and after induction
chemotherapy in 65 patients with stage II or III soft tissue sarcomas (49). The
impact of induction chemotherapy was classified into one of three categories
based on the perceived impact of induction chemotherapy on the planned
surgical procedure: no change in planned surgical resection, decreased scope
of resection, or increased scope of resection. The radiographic responses to
preoperative chemotherapy included partial responses in 34%, minimal
responses in 9%, stable disease in 31%, and progressive disease in 26%. Only
8 patients (12%) were believed to have downstaging sufficient to decrease the
scope of their operation, and 6 patients (9%) had disease progression
sufficient to increase the scope of their operation (49). Of interest, none of
the 9 patients who were determined to require amputation prior to
chemotherapy were able to undergo limb salvage after chemotherapy. Thus,
there was no clear evidence to support the perception that patients with locally
advanced extremity soft tissue sarcoma (resectable only by amputation) could
be downstaged to permit function-preserving, limb-sparing surgery.
51
4.2 Functional Status and Quality of Life
Treatment for sarcomas may result in significant functional disability

and reduced quality of life (50). An adequate margin-negative (R0) tumor
resection for soft tissue sarcoma includes removal of the tumor with a margin
of normal tissue confirmed to be tumor-free by microscopic evaluation. The
proximity of some tumors to vital anatomic structures often requires resection
of such structures, including major motor nerves, resulting in significant
postoperative functional impairment. In a prospective study evaluating
functional outcome in patients with soft tissue sarcomas, large tumor size,
postoperative complications, and neural sacrifice were associated with poor
functional outcome (51).
Treatment sequencing may have an impact on long-term functional
outcome. Preoperative external-beam radiation therapy allows the delivery of
lower doses of radiation to smaller volumes of tissue with potentially less
long-term morbidity from tissue damage. In a large retrospective study, the
incidence of delayed radiation-associated complications including soft tissue
necrosis, bone fractures, osteonecrosis, edema, and fibrosis was 9% for
postoperative radiation therapy and 5% for preoperative radiation therapy (p =
0.03) (52).
The impact of the timing of radiation therapy on functional outcome
was also examined in a randomized clinical trial supported by the National
Cancer Institute of Canada (50). Two years following surgery, there were no
differences in function or quality of life, with all measures returning to
pretreatment levels, between the preoperative and postoperative radiation
therapy groups. However, long-term follow-up is needed to assess the late
manifestations of preoperative and postoperative radiation therapy,
particularly with respect to physical function, limb edema, and bone fractures
(14).
4.3 Pathologic Response
An advantage of preoperative therapy is the ability to assess tumor

response in situ using radiologic imaging as well as pathologically following
surgical resection. This strategy allows patients with unresponsive tumors to
be identified and spared the toxicity and cost of additional treatment with a
regimen that is not effective. The pathologic responses identified following
induction chemotherapy have ranged from no response to complete response
with no residual viable cancer cells. In most cases, responses are partial or
incomplete. Studies have demonstrated that cells located in the well-
vascularized tumor periphery are most likely to be affected by induction
52
chemotherapy and that if left untreated, these peripheral tumor cells are likely
to be responsible for tumor recurrence (53).
Chemotherapy-induced pathologic necrosis is a predictor of survival
in patients who receive preoperative chemotherapy for osteogenic and
Ewings sarcoma (54-57). However, the incidence of treatment-induced
pathologic necrosis and its correlation with clinical outcomes are not well
defined in patients with soft tissue sarcomas (2). Small studies have reported
rates of complete pathologic tumor necrosis following doxorubicin-based
induction chemotherapy of only 5% to 9% (58-60).
In a retrospective analysis of 496 patients with intermediate- and
high-grade extremity soft tissue sarcomas who were treated with preoperative
therapy (consisting primarily of doxorubicin-based chemotherapy and
radiation), complete pathologic responses were noted in 69
patients (14%). With a mean follow-up of 10 years, the 10-year local
recurrence rate for patients with complete pathologic necrosis was 11%,
compared to 23% for patients with less than 95% pathologic necrosis.
However, the 10-year survival rate for patients with complete pathologic
responses was 71%, compared to 55% for other patients (p = 0.0001)(2).
Based on these results, the authors concluded that pathologic assessment of
necrosis can be considered a surrogate endpoint for survival outcomes in
patients with soft tissue sarcomas and, as such, can be used as a valid and
timely endpoint by which novel agents and treatment protocols are evaluated
(2).
5 SURVIVAL
5.1 Radiation Therapy
The relationship between local control and distant metastasis/disease-

specific survival has long been debated in the treatment of solid tumors. It is
biologically plausible that with high-risk disease, more complete eradication
of aggressive residual sarcoma could reduce the risk of distant progression
and sarcoma-specific death. Important issues in this debate include (1)
whether a local treatment modality can impact distant metastasis and tumor-
related mortality and (2) whether patients who have increased rates of local
failure have an increased risk of subsequent distant metastasis. However, the
prevention of local recurrence by either amputation or radiation therapy has
not translated into a survival benefit in sarcoma patients (61).
Examination of the published phase III trials of postoperative
radiation for sarcoma indicates that local tumor control does not have an
impact on disease-specific survival (62,63). However, the existing phase III
trials (8,9,62,63) of postoperative radiation included relatively small numbers
of patients at risk for recurrence 52 patients in the NCI trial (J. Yang,
personal communication) and 77 patients in the Memorial Sloan-Kettering
53
trial (M.F. Brennan, personal communication). If one accepts that the

association between local control and survival is best addressed through
studies of high-risk patients, then it is conceivable that the existing phase III
trials may not be adequate for the assessment of a potential association.
At least two large retrospective studies have examined the outcomes
associated with preoperative versus postoperative radiation therapy (52,64).
Both studies found no difference in the rates of local control and disease-
specific survival between the two treatment sequence groups.
The only randomized comparison of preoperative and postoperative
radiation therapy is the multi-institutional study performed by the National
Cancer Institute of Canada Clinical Trials Group (14). With a median follow-
up of 3.3 years, there were no differences in local recurrence, distant
recurrence, or progression-free survival but there was a slightly higher overall
survival rate in patients who received preoperative radiation therapy than in
those who were treated with postoperative radiation therapy (p = 0.0481). It
has been noted that there was a relative imbalance in the number of patients
who died of other causes between the pre- and postoperative radiation arms.
It is not clear whether this imbalance or other confounding factors could
account for the significant survival difference or whether this observation
indicates a significant clinical advantage associated with preoperative
radiation.
Interestingly, an improvement in disease-specific survival associated
with the use of radiation has been demonstrated in phase III trials in other
malignancies, including node-positive breast cancer (65-68) and some
squamous cell carcinomas of the head and neck (69). A common theme in
those reports was an examination of the relationship between local control and
survival in subgroups of patients at high risk for distant metastasis.
5.2 Chemotherapy
In a meta-analysis of patients with extremity soft tissue sarcomas, the

estimated survival benefit attributable to postoperative chemotherapy was
only 7% (70). More recently, one randomized prospective trial of 104 patients
with heterogeneous tumors reported an overall survival rate of 75 months for
patients treated with surgery and chemotherapy versus 46 months for those
treated with surgery with or without radiation (p = 0.03) (71). Two additional
smaller randomized trials of patients with primary or locally recurrent soft
tissue sarcoma also showed higher 5-year overall survival rates associated
with chemotherapy, although the differences were not statistically significant
(72,73). The interpretation of this complex literature is difficult, and there is
no consensus on the role of chemotherapy for patients with localized high-risk
soft tissue sarcoma (74,75).
54
Although radiographic response rates to chemotherapy have been 20

to 30%, there have been conflicting reports regarding whether tumor response
is associated with improved rates of disease-specific and overall survival
(76,49,58). A major goal of preoperative or induction systemic therapy is the
early eradication of micrometastatic disease. A single randomized phase II/III
study attempted to examine the survival benefits associated with preoperative
chemotherapy for soft tissue sarcoma (77). However, the study was closed
prior to initiation of the phase III arm because of slow accrual. In the 134
patients randomized for the phase II study, the 5-year disease-free survival
rate was 52% for the no-chemotherapy arm and 56% for the chemotherapy
arm (p = 0.35).
6 SURGICAL COMPLICATIONS
6.1 Radiation Therapy
There are several studies which have examined the rate of

postoperative wound complication following preoperative radiation therapy in
patients with extremity soft tissue sarcoma (Table 1). In two large
retrospective studies of preoperative versus postoperative radiation therapy
(64,52), rates of postoperative wound complications were reported as 31% for
preoperative radiation compared to 8% for postoperative radiation. Similarly,
there was an increased rate of 18% (95% confidence interval 5% to 30%,
p<0.01) of wound complications associated with the use of preoperative
radiation therapy in the only randomized comparative study (14). Tissue
transfer techniques are being used more often in patients undergoing
preoperative radiation therapy with better postoperative outcomes (Fig
1)(79,81). Centers with experience in pedicled and free tissue flaps have
demonstrated high success rates (> 90%) in ensuring healed wounds in a
single-stage operation (81). Unlike the results with extremity sarcomas, there
have been no reports of increased rates of wound complications in patients
with retroperitoneal sarcomas who received preoperative radiation therapy
(16). Long-term toxic effects of preoperative radiation therapy have been
reported in only a small fraction of patients. In a report from the
Massachusetts General Hospital, 4 of 37 retroperitoneal sarcoma patients
treated with preoperative radiation therapy suffered from neuropathy,
hydronephrosis, fistula formation, vascular injury and/or bowel complications
(18). Similary, 4 of 41 patients treated at the Mayo Clinic were reported to
have significant late toxic effects associated with combined therapy; these
effects included duodenitis/gastric outlet obstruction, chronic pain, bowel
obstruction requiring surgery, and, in one patient, death (16).
55
56
6.2 Chemotherapy
There are few clinical data examining the relationship between

preoperative chemotherapy and surgical complications following resection of
soft tissue sarcomas. A single-institution retrospective analysis compared the
rate of postoperative complications in 105 patients who received preoperative
chemotherapy to that in 204 patients who were treated with surgical resection
alone (82). The characteristics of the two treatment groups are presented in
Table 2. The median interval between the end of chemotherapy and surgical
resection was 45 days. Postoperative complication rates were similar in the
57
two treatment groups in both those with extremity sarcomas (34% versus
41%) and those with retroperitoneal/visceral sarcomas (29% versus 34%).
The most common complications in both groups were wound infections.
7 CONCLUSIONS
Soft tissue sarcomas continue to represent a therapeutic challenge

because of their rarity and the heterogeneity of the many histologic subtypes.
The efficacy of preoperative therapy has been difficult to establish. However,
there is evidence that induction therapy may result in reduction of tumor size
that facilitates less radical surgical resections with improved postoperative
function. Clinical evidence supports continued investigation of preoperative
treatment approaches for soft tissue sarcomas.
The use of preoperative therapy reinforces the need for cooperation
among oncologic disciplines. The timing and sequence of treatment
modalities should be discussed at the time of tumor diagnosis. Individual
treatment decisions must weigh the estimated likelihood and severity of
potential short- and long-term complications associated with one treatment
sequence versus another.
8 REFERENCES
1. Wynendaele W, van Oosterom AT. Neoadjuvant/primary chemotherapy in cancer

treatment: what advantage? Forum (Geneva) 1999;9(3):212-21.
2. Eilber FC, Rosen G, Eckardt J, et al. Treatment-induced pathologic necrosis: a
predictor of local recurrence and survival in patients receiving neoadjuvant therapy
for high-grade extremity soft tissue sarcomas. J Clin Oncol 2001;19(13):3203-9.
3. Trimble EL, Ungerleider RS, Abrams JA, et al. Neoadjuvant therapy in cancer
treatment. Cancer 1993;72:3515-24.
4. Eilber F, Giuliano A, Eckardt J, Patterson K, Moseley S, Goodnight J. Adjuvant
chemotherapy for osteosarcoma: a randomized prospective trial. J Clin Oncol
1987;5(1):21-6.
5. Link MP, Goorin AM, Horowitz M, et al. Adjuvant chemotherapy of high-grade
osteosarcoma of the extremity. Updated results of the Multi-Institutional
Osteosarcoma Study. Clin Orthop 1991(270):8-14.
6. Arndt CA, Crist WM. Common musculoskeletal tumors of childhood and
adolescence. N Engl J Med 1999;341(5):342-52.
7. Maurer HM, Beltangady M, Gehan EA, et al. The Intergroup Rhabdomyosarcoma
Study-I. A final report. Cancer 1988;61(2):209-20.
8. Yang JC, Chang AE, Baker AR, et al. Randomized prospective study of the benefit
of adjuvant radiation therapy in the treatment of soft tissue sarcomas of the extremity.
J Clin Oncol 1998;16(1):197-203.
58
9. Pisters PW, Harrison LB, Leung DH, Woodruff JM, Casper ES, Brennan MF.
Long-term results of a prospective randomized trial of adjuvant brachytherapy in soft
tissue sarcoma. J Clin Oncol 1996;14(3):859-68.
10. Suit HD, Mankin HJ, Wood WC, Proppe KH. Preoperative, intraoperative, and
postoperative radiation in the treatment of primary soft tissue sarcoma. Cancer
1985;55(11):2659-67.
11. Sadoski C, Suit HD, Rosenberg A, Mankin H, Efird J. Preoperative radiation,
surgical margins, and local control of extremity sarcomas of soft tissues. J Surg Oncol
1993;52(4):223-30.
12. Wilson AN, Davis A, Bell RS, et al. Local control of soft tissue sarcoma of the
extremity: the experience of a multidisciplinary sarcoma group with definitive surgery
and radiotherapy. Eur J Cancer 1994;30A(6):746-51.
13. Nielsen OS, Cummings B, OSullivan B, Catton C, Bell RS, Fornasier VL.
Preoperative and postoperative irradiation of soft tissue sarcomas: effect of radiation
field size. Int J Radiat Oncol Biol Phys 1991 ;21(6): 1595-9.
14. OSullivan B, Davis AM, Turcotte R, et al. Preoperative versus postoperative
radiotherapy in soft-tissue sarcoma of the limbs: a randomised trial. Lancet
2002;359(9325):2235-41.
15. Pisters PW, OSullivan B. Retroperitoneal sarcomas: combined modality
treatment approaches. Curr Opin Oncol 2002;14(4):400-5.
16. Jones JJ, Catton CN, OSullivan B, et al. Initial results of a trial of preoperative
external-beam radiation therapy and postoperative brachytherapy for retroperitoneal
sarcoma. Ann Surg Oncol 2002;9(4):346-54.
17. Petersen IA, Haddock MG, Donohue JH, et al. Use of intraoperative electron
beam radiotherapy in the management of retroperitoneal soft tissue sarcomas. Int J
Radiat Oncol Biol Phys 2002;52(2):469-75.
18. Gieschen HL, Spiro IJ, Suit HD, et al. Long-term results of intraoperative
electron beam radiotherapy for primary and recurrent retroperitoneal soft tissue
sarcoma. Int J Radiat Oncol Biol Phys 2001;50(1):127-31.
19. Pisters PWT, Ballo MT, Fenstermacher MJ, et al. Phase I trial of preoperative
concurrent doxorubicin and radiation therapy, surgical resection, and intraoperative
electron-beam radiation therapy for patients with localized retroperitoneal sarcoma. J
Clin Oncol 2003 (In Press).
20. Seynaeva C, Verweij J. High-dose chemotherapy in adult sarcomas: no standard
yet. Semin Oncol 1999;26(1):119-133.
21. Antman KH. Chemotherapy of advanced sarcomas of bone and soft tissue. Semin
Oncol 1992;19(6 Suppl 12):13-20.
22. Santoro A, Tursz T, Mouridsen H, et al. Doxorubicin versus CYVADIC versus
doxorubicin plus ifosfamide in first-line treatment of advanced soft tissue sarcomas: a
randomized study of the European Organization for Research and Treatment of
Cancer Soft Tissue and Bone Sarcoma Group. J Clin Oncol 1995;13(7):1537-3.
59
23. Benjamin RS, Legha SS, Patel SR, Nicaise C. Single-agent ifosfamide studies in
sarcomas of soft tissue and bone: the M. D. Anderson experience. Cancer Chemother
Pharmacol 1993;31:S174-9.
24. Patel SR, Vadhan-Raj S, Papadopolous N, et al. High-dose ifosfamide in bone
and soft tissue sarcomas: results of phase II and pilot studies--dose-response and
schedule dependence. J Clin Oncol 1997;15(6):2378-84.
25. OBryan RM, Baker LH, Gottlieb JE, et al. Dose response evaluation of
Adriamycin in human neoplasia. Cancer 1977;39(5):1940-8.
26. Nielsen OS, Judson I, van Hoesel Q, et al. Effect of high-dose ifosfamide in
advanced soft tissue sarcomas. A multicentre phase II study of the EORTC Soft
Tissue and Bone Sarcoma Group. Eur J Cancer 2000;36(1):61-7.
27. Frustaci S, Buonadonna A, Romanini A, et al. Increasing dose of continuous
infusion ifosfamide and fixed dose of bolus epirubicin in soft tissue sarcomas. A
study of the Italian group on rare tumors. Tumori 1999;85(4):229-33.
28. Palumbo R, Palmeri S, Antimi M, et al. Phase II study of continuous-infusion
high-dose ifosfamide in advanced and/or metastatic pretreated soft tissue sarcomas.
Ann Oncol 1997;8(11):1159-62.
29. Buesa JM, Lopez-Pousa A, Martin J, et al. Phase II trial of first-line high-dose
ifosfamide in advanced soft tissue sarcomas of the adult: a study of the Spanish Group
for Research on Sarcomas (GEIS). Ann Oncol 1998;9(8):871-6.
30. Eilber FR, Giuliano AE, Huth JH, Mirra JJ, Rosen G, Morton DL. Neoadjuvant
chemotherapy, radiation, and limited surgery for high grade soft tissue sarcoma of the
extremity. In: Ryan JR, Baker LO, eds. Recent Concepts in Sarcoma Treatment.
Dordrecht, The Netherlands: Kluwer Academic Publishers, 1988: 115-22.
31. Pisters PW, Ballo MT, Patel SR. Preoperative chemoradiation treatment
strategies for localized sarcoma. Ann Surg Oncol 2002;9(6):535-42.
32. Goodnight JE, Jr., Bargar WL, Voegeli T, Blaisdell FW. Limb-sparing surgery
for extremity sarcomas after preoperative intraarterial doxorubicin and radiation
therapy. Am J Surg 1985;150(1):109-13.
33. Levine EA, Trippon M, Das Gupta TK. Preoperative multimodality treatment for
soft tissue sarcomas. Cancer 1993;71(11):3685-9.
34. Wanebo HJ, Temple WJ, Popp MB, Constable W, Aron B, Cunningham SL.
Preoperative regional therapy for extremity sarcoma. A tricenter update. Cancer
1995;75(9):2299-306.
35. Temple WJ, Temple CL, Arthur K, Schachar NS, Paterson AH, Crabtree TS.
Prospective cohort study of neoadjuvant treatment in conservative surgery of soft
tissue sarcomas. Ann Surg Oncol 1997;4(7):586-90.
36. Rhomberg W, Hassenstein EO, Gefeller D. Radiotherapy vs. radiotherapy and
razoxane in the treatment of soft tissue sarcomas: final results of a randomized study.
Int J Radiat Oncol Biol Phys 1996;36(5): 1077-84.
37. Goffman T, Tochner Z, Glatstein E. Primary treatment of large and massive adult
sarcomas with iododeoxyuridine and aggressive hyperfractionated irradiation. Cancer
1991;67(3):572-6.
60
38. Sondak VK, Robertson JM, Sussman JJ. Preopertive idoxuridine and radiation
for large soft tissue sarcomas: clinical results with five-year follow-up. Ann Surg
Oncol 1998;5:106-112.
39. Pisters PW. Chemoradiation treatment strategies for localized sarcoma:
conventional and investigational approaches. Semin Surg Oncol 1999;17(1):66-71.
40. Eilber FR, Giuliano AE, Huth JF, Weisenburger TH, Eckardt J. Intravenous (IV)
vs. intraarterial (IA) Adriamycin, 2800r radiation and surgical excision for extremity
soft tissue sarcomas: a randomized prospective trial [Abstract 309]. Proc Am Soc Clin
Oncol 1990.
41. Cormier JN, Patel SR, Herzog CE, et al. Concurrent ifosfamide-based
chemotherapy and irradiation. Analysis of treatment-related toxicity in 43 patients
with sarcoma. Cancer 2001;92(6):1550-5.
42. Toma S, Palumbo R, Vincente M. Concomitant doxorubicin (DOXO) by
continuous infusion (CI) and radiotherapy (RT) at low doses in locally advanced
and/or metastatic soft tissue sarcomas (STS): long-term results of a phase II study
[Abstract 520]. Proc Am Soc Clin Oncol 1995.
43. Kraybill WG, Spiro IJ, Harris J. Radiation Therapy Oncology Group (RTOG) 95-
14: a phase II study of neoadjuvant chemotherapy (CT) and radiation therapy (RT) in
high risk (HR), high grade, soft tissue sarcomas (STS) of the extremities and body
wall: a preliminary report [Abstract 348a]. Proc Am Soc Clin Oncol 2001.
44. Eilber F, Eckardt J, Rosen G, Forscher C, Selch M, Fu YS. Preoperative therapy
for soft tissue sarcoma. Hematol Oncol Clin North Am 1995;9(4):817-23.
45. Pisters PWT, Patel SR, Pollock RE. Phase I trial of preoperative doxorubicin-
based concurrent chemoradiation and electron-beam intraoperative radiation therapy
(IORT) for resectable retroperitoneal sarcomas [Abstract 103]. Cancer J Sci Am 1998.
46. Alektiar KM, Leung D, Zelefsky MJ, Brennan MF. Adjuvant radiation for stage
II-B soft tissue sarcoma of the extremity. J Clin Oncol 2002;20(6):1643-1650.
47. Fleming JB, Berman RS, Cheng SC, et al. Long-term outcome of patients with
American Joint Committee on Cancer stage IIB extremity soft tissue sarcomas. J Clin
Oncol 1999;17(9):2772-80.
48. Baldini EH, Goldberg J, Jenner C, et al. Long-term outcomes after function-
sparing surgery without radiotherapy for soft tissue sarcoma of the extremities and
trunk. J Clin Oncol 1999;17(10):3252-9.
49. Meric F, Hess KR, Varma DG, et al. Radiographic response to neoadjuvant
chemotherapy is a predictor of local control and survival in soft tissue sarcomas.
Cancer 2002;95(5):1120-6.
50. Davis AM, OSullivan B, Bell RS, et al. Function and health status outcomes in a
randomized trial comparing preoperative and postoperative radiotherapy in extremity
soft tissue sarcoma. J Clin Oncol 2002;20(22):4472-7.
51. Bell RS, OSullivan B, Davis A, Langer F, Cummings B, Fornasier VL.
Functional outcome in patients treated with surgery and irradiation for soft tissue
tumours. J Surg Oncol 1991;48(4):224-31.
61
52. Zagars GK, Ballo MT, Pisters PW, Pollock RE, Patel SR, Benjamin RS.
Preoperative vs. postoperative radiation therapy for soft tissue sarcoma: a
retrospective comparative evaluation of disease outcome. Int J Radiat Oncol Biol
Phys 2003;56(2):482-8.
53. Stephens FO. Induction chemotherapy: to downgrade aggressive cancers to
improve curability by surgery and/or radiotherapy. Eur J Surg Oncol 2001 ;27(7):672-
88.
54. Lindner NJ, Scarborough MT, Spanier SS, Enneking WF. Local host response in
osteosarcoma after chemotherapy referred to radiographs, CT, tumour necrosis and
patient survival. J Cancer Res Clin Oncol 1998;124(10):575-80.
55. Rosen G, Caparros B, Huvos AG, et al. Preoperative chemotherapy for
osteogenic sarcoma: selection of postoperative adjuvant chemotherapy based on the
response of the primary tumor to preoperative chemotherapy. Cancer
1982;49(6):1221-30.
56. Wunder JS, Paulian G, Huvos AG, Heller G, Meyers PA, Healey JH. The
histological response to chemotherapy as a predictor of the oncological outcome of
operative treatment of Ewing sarcoma. J Bone Joint Surg Am 1998;80(7):1020-33.
57. Picci P, Bohling T, Bacci G, et al. Chemotherapy-induced tumor necrosis as a
prognostic factor in localized Ewings sarcoma of the extremities. J Clin Oncol
1997;15(4):1553-9.
58. Ottaiano A, De Chiara A, Fazioli F, et al. Neoadjuvant chemotherapy for
intermediate/high-grade soft tissue sarcomas: five-year results with epirubicin and
ifosfamide. Anticancer Res 2002;22(6B):3555-9.
59. Henshaw RM, Priebat DA, Perry DJ, Shmookler BM, Malawer MM. Survival
after induction chemotherapy and surgical resection for high-grade soft tissue
sarcoma. Is radiation necessary? Ann Surg Oncol 2001;8(6):484-95.
60. Rahoty P, Konya A. Results of preoperative neoadjuvant chemotherapy and
surgery in the management of patients with soft tissue sarcoma. Eur J Surg Oncol
1993;19(6):641-5.
61. Brennan MF. More is less: systemic treatment for local control in soft tissue
sarcoma. Ann Surg Oncol 2001;8(6):480-1.
62. Brennan MF, Alektiar KM, Maki RG. Sarcomas of the soft tissue and bone. In:
DeVita V, Hellman S, Rosenberg SA, eds. Cancer: Principles & Practice of
Oncology. Philadelphia: Lippincott Williams & Wilkins, 2001:1841.
63. Espat NJ, Lewis JJ. The biological significance of failure at the primary site on
ultimate survival in soft tissue sarcoma. Semin Radiat Oncol 1999;9(4):369-77.
64. Cheng EY, Dusenbery KE, Winters MR, Thompson RC. Soft tissue sarcomas:
preoperative versus postoperative radiotherapy. J Surg Oncol 1996;61:90-99.
65. Overgaard M, Jensen MB, Overgaard J, et al. Postoperative radiotherapy in high-
risk postmenopausal breast-cancer patients given adjuvant tamoxifen: Danish Breast
Cancer Cooperative Group DBCG 82c randomised trial. Lancet
1999;353(9165):1641-8.
62
66. Overgaard M, Hansen PS, Overgaard J, et al. Postoperative radiotherapy in high-

risk premenopausal women with breast cancer who receive adjuvant chemotherapy.
Danish Breast Cancer Cooperative Group 82b Trial. N Engl J Med 1997;337(14):949-
55.
67. Ragaz J, Jackson SM, Le N, et al. Adjuvant radiotherapy and chemotherapy in
node-positive premenopausal women with breast cancer. N Engl J Med
1997;337(14):956-62.
68. Whelan TJ, Julian J, Wright J, Jadad AR, Levine ML. Does locoregional
radiation therapy improve survival in breast cancer? A meta-analysis. J Clin Oncol
2000;18(6):1220-1229.
69. Mishra RC, Singh DN, Mishra TK. Post-operative radiotherapy in carcinoma of
buccal mucosa, a prospective randomized trial. Eur J Surg Oncol 1996;22(5):502-4.
70. Tierney JF. Adjuvant chemotherapy for localised resectable soft-tissue sarcoma
of adults: meta-analysis of individual data. Sarcoma Meta-analysis Collaboration.
Lancet 1997;350(9092):1647-54.
71. Frustaci S, Gherlinzoni F, De Paoli A, et al. Adjuvant chemotherapy for adult soft
tissue sarcomas of the extremities and girdles: results of the Italian randomized
cooperative trial. J Clin Oncol 2001;19(5):1238-47.
72. Brodowicz T, Schwameis E, Widder J. Intensified adjuvant IFADIC
chemotherapy for adult soft tissue sarcoma: a prospective randomized feasibility
trial. Sarcoma 2000;4:151-160.
73. Petrioli R, Coratti A, Correale P, et al. Adjuvant epirubicin with or without
ifosfamide for adult soft-tissue sarcoma. Am J Clin Oncol 2002;25(5):468-73.
74. Bramwell VHC. Adjuvant chemotherapy for adult soft tissue sarcoma: is there a
standard of care? J Clin Oncol 2001;19(5):1235-1237.
75. Figueredo A, Bramwell VHC, Bell R, Davis AM, Charette ML. Adjuvant
chemotherapy following complete resection of soft tissue sarcoma in adults: a
clinical practice guideline. Sarcoma 2002;6:5-18.
76. Pisters PW, Patel SR, Varma DG, et al. Preoperative chemotherapy for stage IIIB
extremity soft tissue sarcoma: long-term results from a single institution. J Clin Oncol
1997;15(12):3481-7.
77. Gortzak E, Azzarelli A, Buesa J, et al. A randomised phase II study on neo-
adjuvant chemotherapy for high-risk adult soft-tissue sarcoma. Eur J Cancer
2001;37(9):1096-103.
78. Bujko K, Suit HD, Springfield DS, Convery K. Wound healing after preoperative
radiation for sarcoma of soft tissues. Surg Gynecol Obstet 1993;176(2):124-34.
79. Peat BG, Bell RS, Davis A, et al. Wound-healing complications after soft-tissue
sarcoma surgery. Plast Reconstr Surg 1994;93(5):980-7.
63
80. Prosnitz LR, Maguire P, Anderson JM, et al. The treatment of high-grade soft
tissue sarcomas with preoperative thermoradiotherapy. Int J Radiat Oncol Biol Phys
1999;45(4):941-9.
81. Langstein HN, Robb GL. Reconstructive approaches in soft tissue sarcoma.
Semin Surg Oncol 1999;17(1):52-65.
82. Meric F, Milas M, Hunt KK, et al. Impact of neoadjuvant chemotherapy on
postoperative morbidity in soft tissue sarcomas. J Clin Oncol 2000;18(19):3378-383.
Chapter 4
TNF-based isolated limb perfusion: A decade of

experience with antivascular therapy in the
management of locally advanced extremity soft tissue
sarcomas
Dirk J Grnhagen, Flavia Brunstein, Timo L.M. ten Hagen, Albertus N. van
Geel, Johannes H.W. de Wilt, and Alexander M.M. Eggermont
Dept. of Surgical Oncology,
Erasmus University Medical Centre-Daniel den Hoed Cancer Centre, Rotterdam,
the Netherlands
Correspondence to:
Alexander M.M. Eggermont, MD, PhD
Professor Surgical Oncology, Head of Department
Department of Surgical Oncology
Erasmus University Medical Center - Daniel den Hoed Cancer Center
301 Groene Hilledijk
3075 EA Rotterdam
The Netherlands
Tel: 31-10-439 19 11
Fax: 31-10439 10 11
E-mail: a.m.m.eggermont@erasmusmc.nl
66
1 INTRODUCTION
In the management of locally advanced extremity soft tissue sarcomas

limb salvage has become all the more important in the light of evidence that
amputations do not improve survival rates in patients with large (>5 cm) deep
seated high grade sarcomas. Several studies have shown that marginal excisions
with a high risk for local recurrence do not influence survival significantly (1-4).
Of the 7800 new cases of STS diagnosed in the USA each year about 4700 occur
in the extremities and tumors are often large at the time of diagnosis. (5) Treat-
ment options for locally advanced extremity STS may consist of an amputation
or a limb sparing extensive surgical procedure followed by radiation therapy.
This combination may mutilate and compromise limb function considerably.
Preoperative therapies to improve limb salvage rates have been propagated.
Preoperative radiotherapy alone or in combination with intraarterial or
intravenous chemotherapy has been reported to improve resectability rates of
extremity soft tissue sarcomas. (6-8) Amputation may also be avoided and local
control improved by combining a marginal resection in combination with
brachytherapy to the tumor bed (9). Isolated limb perfusion is another strategy to
deal with locally advanced soft tissue sarcomas which can be applied also in
case of multifocal primary or multiple sarcoma recurrences in limbs, thereby
expanding the patient population that can be successfully treated.
2 ISOLATED LIMB PERFUSION
The technique of isolated limb perfusion was pioneered by Creech

and Krementz at Tulane University in New Orleans (10). Regional drug
concentrations 15-25 times higher than those reached after systemic
administration can be achieved by ILP without systemic side effects (11).
Isolation of the limb is achieved by clamping and canulation of the major
artery and vein, connection to an oxygenated extracorporeal circuit, ligation
of collateral vessels and application of a tourniquet. Once isolation is secured,
drugs can be injected into the perfusion circuit. Because of its efficacy and
low regional toxicity profile melphalan (L-phenyl-alaninemustard) is the
standard drug, most commonly used at a dose of 10 mg/L (leg) - 13mg/L
(arm) perfused tissue (12). Tissue temperatures are monitored and
radiolabeled albumen or erythrocytes is injected into the perfusion circuit to
detect leakage into the systemic circulation by precordial scintillation probe
(13). Leakage monitoring is mandatory especially for high dose tumor
necrosis factor-alpha (TNF) perfusions in the treatment of soft tissue
sarcomas. After 1-1.5 hours of perfusion the limb is rinsed with an electrolyte
solution, cannulas are removed, and the vessels are repaired. Classification of
acute tissue reactions after perfusion is done according to Wieberdink et al
(14): (I) No reaction; (II) Slight erythema and/or edema; (III) Considerable
67
erythema and/or edema with some blistering, slightly disturbed motility

permissible; (IV) Extensive epidermolysis and/or obvious damage to the deep
tissues, causing definite functional disturbances; threatening or manifest
compartmental syndrome; (V) Reaction which may necessitate amputation.
3 TNF- BASED ISOLATED LIMB PERFUSION FOR STS
3.1 Inadequate results in STS with ILP with

chemotherapeutic drugs only
In contrast to the efficacy of melphalan- based ILP in patients with

multiple intransit melanoma metastases, results wih ILP with melphalan,
doxorubicin and a variety of other drugs for large soft tissue sarcoms were
diiappointing. After studies in the seventies and eighties with poor response
rates ILP for advanced SIS was largely abandoned (15-19). The reported
studies are summarized in Table 1.
3.2 Results with ILP with TNF + Melphalan trials leading to

approval of TNF
Thanks to the pioneering work of Lejeune and Lienard this situation

changed dramatically with the application of high dose TNF in the ILP setting
(20). TNF-based ILP has been established as a highly effective new method of
induction biochemotherapy in extremity soft tissue sarcomas with a 20-30%
complete remission (CR) rate and about a 50% Partial Remission (PR) rate (21-
27). On the basis of results in a multicenter program in Europe TNF was
68
69
approved and registered in Europe for the sarcoma-indication in 1998 (27). The
European TNF/ILP assessment group evaluated 246 patients with irresectable
STS enrolled in 10 years in 4 studies. All cases were reviewed by an
independent review committee and compared with conventionally treated
patients (often by amputation) of a population based Scandinavian STS
database. In short: there were 246 patients with locally very advanced disease:
Primary sarcomas in 55%, local recurrent sarcomas in 45%, multifocal
primary or multiple local recurrences in 22 %. Overt concurrent metastatic
disease in 15%. Tumors >10 cm in 46%. Grade III tumors in 66%. Previous
radiotherapy (13%), chemotherapy (15%). Patients underwent 1 ILP (222 pts)
or 2 ILPs (24 pts) of 90 minutes at 39-40 C with 2-4 mg TNF + melphalan (10-
13 mg/L limb volume). The first 56 pts also received A delayed marginal
resection of the tumor remnant was usually (76%) done 2-4 months after ILP.
Major responses were seen in 56.5 to 82.6 % of the patients after which usually
resection of the sarcoma became possible.
Limb salvage was achieved in 74%-87% in these 4 studies and in 71 %
of the 196 patients who had been classified by the independent review
committees as cases that normally could only have been managed by
amputation (87%) or by functionally debilitating resection + radiotherapy
(13%). Comparison with the survival curves based on a matched control study
with cases from the Scandinavian Soft Tissue Sarcoma Databank showed that
TNF had no negative effect on survival (p=0.96). It was concluded that the
application of TNF in combination with melphalan in the setting of isolated
limb perfusion represents a new and successful option in the management of
irresectable locally advanced extremity soft tissue sarcomas (27).
3.3 Confirmatory single center reports on TNF + melphalan
Smaller single center studies with TNF+Melphalan have been reported

recently by Lejeune (29) reporting a 17% CR and 64% PR rate in 22 STS
patients treated for limb threatening STS tumors, achieving limb salvage in
77% of the patients. A similar limb salvage rate of 84% and excellent functional
results were reported from the Berlin team regarding their experience in a series
of 55 patients (30). We reported on very good outcome of 16 perfusions in 10
patients with multiple lymphangiosarcomas (Stewart Treves Syndrome),
achieving a CR rate of 56% and a limb salvage rate of 80% (32). The
Amsterdam group reported somewhat less favorable results in their experience
in 49 patients. The limb salvage rate of 58% was felt to reflect the selection
patients with particularly unfavorable characteristics (32). The French group
reported recently on their experience in 72 patients. In a randomized phase II
trial, utilizing various doses of TNF ranging from 0.5 mg to 4 mg, they
observed a 35% CR rate and an overall limb salvage rate of 84%. No significant
differences between the various TNF dosage groups were observed. In a
particularly unfavorable group of patients with recurrent sarcomas after surgery
70
and radiotherapy, with the recurrent sarcoma in the irradiated field, we reported
on the Rotterdam experience in 24 patients. A response rate of 74% and a limb
salvage rate of 67% was reported in these patients otherwise destined for
amputation.
3.4 Safety in Elderly Patients
A very important message is given by the report on the Rotterdam

experience with 50 TNF-based ILPs in patients older than 75 years with limb
threatening tumors. Results were very favorable in the 34 perfusions for limb
threatening sarcomas, with a 38% CR and a 38% PR rate, achieving limb
salvage in 76% of the patients as well as in 16 perfusions for bulky melanoma
intransit metastases resulting in a 75%CR and 25%PR rate. The procedure was
proven safe in the elderly with the high reward of limb salvage which is of
overriding importance in this age group as amputations lead to loss of
independency in lives in the elderly (35). Moreover we reported on the absence
of toxicity in patients without leakage and the relatively easy management and
relative lack of toxicity in patients with high leakage of TNF during ILP (36,
37)
3.5 Results with TNF + Doxorubicine
Very similar results have been obtained by Italian perfusion groups

with the drug doxorubicin in combination with TNF. Interestingly similar
response and limb salvage rates are achieved while using lower doses of only 1
mg TNF instead of the usual doses of 2-4 mg used in combination with
melphalan (28). The perfusions were performed at much higher temperatures
(40-41 degrees), which leads to higher locoregional toxicity. Grade IV
locoregional toxicity was reported in 25% as opposed to only 5% in the large
TNF+Melphalan series (21,23,27). We found that with melphalan ILPs grade
IV toxicity was clearly related to tissue temperatures of above 39 degrees when
melphalan was administered (38) Therefore we have only allowed for tissue
temperatures to rise to 39 degrees after melphalan has been added to the
perfusion circuit the last 8 years and have hardly seen any cases with grade IV
toxicity since, without a drop in response rates (27,31,34,35). Most likely
therefore the higher regional toxicity in the Italian experience with doxorubicin
is primarily related to the hyperthermia although doxorubicine may be
responsible in part.
4 TNF-BASED ILP ACTIVE IN MANY HISTOLOGIES
Since the tumor vasculature in the target of TNF and of the

TNF+chemotherapy combination it can be expected that this treatment is
effective against a wide variety of tumor types as long as there is a well-
developed vascular stromal component to the tumor. This is indeed the case.
71
Apart from activity in some 20 different histological types of soft tissue

sarcoma and activity in melanoma (39-43), the efficacy of TNF + melphalan
ILP has also been demonstrated in various skin tumors (44), bony sarcomas
(45) and limb desmoid tumors (46).
5 VASCULOTOXIC MECHANISM OF TNF +

CHEMOTHERAPY
The target of TNF is the tumor-vasculature. This common denominator

in all these tumors makes the use of TNF very attractive and explains its efficacy
in combination with chemotherapy across all these different histologies. The
selective destructive effects of TNF-ILP on tumor-associated vessels have been
illustrated in previous publications by means of pre- and post perfusion
angiographies (23). Moreover in sarcoma patients magnetic resonance
spectrometry studies we have clearly shown that the metabolic shut down of the
tumor is virtually complete within 16 hours after the perfusion, confirming the
likelihood of mediating its most important effects on the vasculature of
the tumor (48). At the histopathological level we have also studied these intra-
vascular effects such as thrombocyte aggregation, erythrostasis, endothelial and
vascular destruction already in the early and late stages after ILP (48-49).
6 NEW INSIGHTS THROUGH LABORATORY MODELS
To further insight in the mechanisms underlying the positive results

obtained with ILP in humans we developed in rats extremity perfusion models
using the BN175 non immunogenic fibrosarcoma in Brown Norways rats and
the ROS-1 osteosarcoma in WAG rats. In both models we could demonstrate
that the tumor cells were resistant to TNF in vitro and that ILP in vivo with TNF
alone had no major impact on tumor growth. In both models a strong synergistic
antitumor effect leading to CRs in some 60-70 % was observed after ILP with
TNF+Melphalan (50-51). TNF alone only caused some central necrosis and no
regression of the tumor was observed as has been reported for the clinical setting
as well. Histopathologically haemorrhagic necrosis was most prominent after
ILP with both drugs. Early endothelial damage and platelet aggregation in the
tumor vessels are observed after ILP with TNF + Melphalan and this is believed
to lead to ischemic (coagulative) necrosis, which is in line with observations in
patients. Our observations confirm that has its major effect on larger
tumors, with well-developed vasculature in contrast to small tumors (diameter <
3 mm) with lack of developed capillary bed. TNF may exert its effect mainly
through the neovasculature of the tumor, which is more abundant in large
tumors. Moreover there are distinct similarities between tumor stroma generation
and wound healing and observations by us that sites other than the tumor (recent
wounds or skin overlying tumors only when invaded by tumor), which undergo
72
angiogenesis, also become necrotic after ILP with TNF+ melphalan, but not after
ILP with melphalan alone.
We have demonstrated a number of crucial elements in our rat tumor
models identifying the mechanisms for the strong synergy between TNF and
Cytostatic drugs in ILP and have identified the prerequisites for an effective ILP:
6.1 Tumor vessel destruction
The vasculo-toxic effects of the combination of TNF + melphalan

leading to haemorrhagic and anoxic coagulative necrosis as described above.
6.2 Enhanced drug uptake by the tumor
We have recently demonstrated that the addition of high dose TNF to

the perfusate results in a 4-6 fold increased uptake by the tumor of the cytostatic
drug. For Melphalan and for Doxorubicin is was demonstrated that this uptake
was tumor specific and that no increased uptake was noted in the normal tissues,
thus emphasizing the relatively selective action of TNF on the tumor-associated
vasculature (52) This increase in concentration was also observed with
doxorubicin (53). Moreover we have demonstrated that the effect correlates with
the vascularity of the tumor. The more vascular the tumor the better the
synergistic effect between TNF and the chemotherapeutic agent (54). Whether a
TNF-mediated drop in interstitial pressure (55) in the tumor plays a role in this
mechanism remains speculative.
6.3 Role of Leukocytes
We have shown that leukocytes play also an important role in the

TNF-mediated antitumor effects. In rats that underwent total body irradiation
and underwent an ILP at the time of absolute leukopenia the antitumor effect
of an ILP with TNF+melphalan was very similar to the effects of a perfusion
with melphalan alone. In the leukopenic rat the TNF-effect was lost and the
synergy between TNF and Melphalan was no longer observed (56)
73
6.4 Dose range for TNF
We demonstrated that 10 micrograms of TNF (a fivefold reduction of

the standard dose of 50 microgram was the threshold dose for activity of TNF
in our rat tumor extremity perfusion model. At 2 microgram all TNF-effects
were lost (57). This finding would suggest that also in the clinical setting dose
reduction without loss of activity could be explored as been also suggested by
the clinical results in the UK (22) and in Italy (29) as well as the recent report
from France (33).
6.5 Duration of ILP
As the pharmacokinetics of melphalan demonstrates that almost all

melphalan uptake occurs over 20-30 minutes the minimal duration for an
effective ILP should be 30 minutes. Shorter perfusion times are associated
with a drop in CR and PR rates whereas longer than 30 minutes ILPs do not
seem to further improve the results (57)
6.6 Mild Hyperthermia
Temperatures of 38-39 degrees Centigrade were shown to be essential

for obtaining a good antitumor response without damage to the normal tissues in
the limb. True hyperthermia (42-43degrees) resulted in an increase of CRs but
was associated with very sever damage to the normal tissues. All antitumor
efficacy was lost when perfusions were performed at room temperature. (57)
6.7 Hypoxia
We demonstrated that hypoxia can enhance the antitumor effects of an

ILP with either TNF alone or Melphalan alone. Hypoxia did not further enhance
the antitumor efficacy of an ILP with TNF+Melphalan as the synergy between
these two agents overrided any minor enhancement mediated by hypoxia (57).
6.8 Interferon-gamma
In spite of many reports of the synergy between IFN-gamma and TNF

both in vitro as well as in vivo in murine tumor models the role of IFN-
gamma not very strong in our rat models. We demonstrated that about a 10%
increase in CR rate and an increase of about 20% in overall response rate was
observed in our animal models (58), which resembles the situation in the
clinic (43).
74
6.9 Idiosyncratic toxicity
Interestingly unexpected interactions may lead to idiosyncratic

reactions between TNF and certain cytostatic agents. Actinomycin D is
commonly used in combination with melphalan in the clinical setting. When
investigating whether TNF would enhance the efficacy of Actinomycin D we
discovered that it did in an idiosyncratic and nondiscriminative way. The
combination was more effective against the tumor than TNF+Melphalan, but
this advantage was annulled by the toxicity of TNF+Actinomycin D to the
normal tissues, resulting in the amputation of all extremities in these animal
models. We sent out a strong warning to the clinic not to use TNF in
combination with Actinomycin D (59).
6.10 Vasoactive drugs
Various vasoactive drugs have been and are being studied in our
laboratory models. Nitric Oxide (NO) is an important molecule in the
maintenance of both vascular tone and the integrity of the vascular wall and is
highly produced in experimental and human tumors. We postulated that its
inhibition could lead to hypoxia and an enhancement of TNF early vascular
effects in the tumor. In our ILP BN 175 rat model we performed a response study
with TNF in combination with the Arginine analogues L-NAME and LNA,
which inhibit NO synthase. In rats treated with TNF combined with L-
NAME/LNA important and immediate antitumor effects were observed in all
rats and necrosis of the skin at the tumor site. These effects are normally only
observed when hypoxia or melphalan are added to TNF as described above.
Typical TNF tumor response was observed, when NO synthase was inhibited
during ILP (60).
Another vasoactive drug is histamine, which is currently being studied.
Also in this case we see a clear synergy with melphalan in our tumor models
(61) These findings show the importance of agents that can change the patho-
physiology of tumor vasculature, rheologic conditions en thereby can improve
drug uptake in tumors. These findings underline the importance of investigating
how to modulate tumor physiology and the potential that this approach has to
improve efficacy of various standard agents.
7 CONCLUSIONS
Isolated limb perfusion methodology provides us an excellent tool in

the clinic to obtain local control and avoid amputations of limbs in patients
with limb threatening tumors. This has been largely achieved by the success
of the antivascular TNF-based biochemotherapy in this setting. TNF, for the
first time, has brought us an effective treatment against large, bulky tumors.
75
Moreover Isolated limb perfusion is a albeit somewhat exotic, but

very interesting research model to develop and study agents that modify the
pathophysiology of large tumors that blocks effective penetration of cytotoxic
drugs into the tumor.
We can now manipulate and study the tumor vascular bed in ways
that will identify new drugs that can enhance the activity of old drugs.
Moreover it has proven to be a model system that may also facilitate the
development of vector-mediated gene therapy and other innovative
approaches.
Much of these developments have been initiated by the application of
TNF in this setting. TNF-based isolated limb perfusion is a very successful
treatment option to achieve limb salvage in the management of advanced,
multiple or drug resistant extremity tumors. TNF-based ILPs are now
performed in some 30 cancer centers in Europe with referral programs for
limb salvage. TNF-based antivascular therapy of cancer is here to stay and its
potential needs to be studied further (62). Other drugs will follow and we may
well learn through this model how to use them systemically more effectively
as well.
8 REFERENCES
1. Gaynor JJ, Tan CC, Casper ES, et al. Refinement of clincopathologic staging for localized soft
tissue sarcoma of the extremity: a study of 423 adults. J Clin Oncol 1992;10:1317-1327
2. Potter DA, Kinsella D, Gladstein E et al. High grade soft tissue sarcomas of the extremities.
Cancer 1986;59:190-205
3. Stotter AT, AHearn RP, Fisher C, Mott AF, Fallowfield ME, Westbury G. The influence of
local recurrence of extremity soft tissue sarcoma on metastasis and survival. Cancer
1990;65:1119-1129
4. Gustafson P, Rser B, Rydholm A. Is local recurrence of minor importance for metastases in
soft tissue sarcoma? Cancer 1991;67:2083-2086
5. Landis S, Murray T, Bolden S, Wingo P. Cancer Statistics, 1999. CA Cancer J Clin 1999;9:8-
26
6. Suit HD, Proppe KH, Mankin HJ, Wood WC. Preoperative radiation therapy for sarcoma of
soft tissue. Cancer 47:2269-2274, 1981
7. Eilber FR, Mirra JJ, Grant T, Weisenburger T, Morton DL. Is amputation necessary for
sarcoma: a 7-year experiment with limb salvage. Ann Surg 1980;192:431-437
8. Eilber FR, Morton DL, Eckhardt J, Grant T, Weisenburger T. Limb salvage for skeletal and
soft-tissue sarcomas: multidisciplinary preoperative therapy. Cancer 1984;53:2579-2584
9. Shiu MH, Hilaris BS, Harrison LB, Brennan MF. Brachytherapy and function-saving
resection of soft tissue sarcoma arising in the limb. Int J Radiat Oncol Biol Phys 1991;21:1485-
1492
10. Creech O, Krementz E, Ryan E, Winblad J. Chemotherapy of cancer: regional perfusion
utilising an extracorporeal circuit. Ann Surg 1958;148:616-632
11. Benckhuijsen C, Kroon BB, van Geel AN, et al: Regional perfusion treatment with melphalan
for melanoma in a limb: an evaluation of drug kinetics. Eur J Surg Oncol 1988;14:157-63
12. Thompson JF, Gianoutsos MP. Isolated limb perfusion for melanoma - effectiveness and
toxicity of cisplatin compared with that of melphalan and other drugs. World J Surg 1992;16:227-
233
76
13. Klaase JM, Kroon BBR, Van Geel AN, Eggermont AMM, Franklin HR. Systemic leakage
during isolated limb perfusion for melanoma. Br J Surg 1993;80:1124-1126
14. Wieberdink K, Benckhuijsen C, Braat RP, Van Slooten EA, Olthuis GAA. Dosimetry in
isolation perfusion of the limbs by assessment of perfused tissue volume and grading of toxic
tissue reactions. Eur J Cancer Clin Oncol 1982; 18:905-910
15. Krementz ET, Carter RD, Sutherland CM, Hutton I. Chemotherapy of sarcomas of the limbs
by regional perfusion. Ann Surg 1977;185(5):555-564
16. Muchmore JH, Carter RD, Krementz ET. Regional perfusion for malignant melanoma and soft
tissue sarcoma: a review. Cancer Invest. 1985;3:129-143
17. Pommier RF, Moseley HS, Cohen J et al. Pharmacokinetics, Toxicity, and Short-term results
of cisplatin hyperthermic isolated limb perfusion for soft tissue sarcoma and melanoma of the
extremities. Am J Surg 155:667-671, 1988
18. Klaase JM, Kroon BBR, Benckhuysen C, Van Geel AN, Albus-Lutter ChE, Wieberdink J.
Results of regional isolation perfusion with cytostatics in patients with soft tissue tumors of the
extremities. Cancer 64:616-621, 1989
19. Rossi CR, Vecchiato A, Foletto M, et al. Phase II study on neoadjuvant hyperthermic-
antiblastic perfusion with doxorubicin in patients with intermediate or high grade limb sarcomas.
Cancer 73:2140-2146, 1994
20. Lienard D, Ewalenko, Delmotte JJ, Renard N, Lejeune FJ. High-dose recombinant tumor
necrosis factor alpha in combination with interferon gamma and melphalan in isolation perfusion
of the limbs for melanoma and sarcoma. J Clin Oncol 1992; 10:50-62
21. Eggermont AMM, Linard D, Schraffordt Koops H, Rosenkaimer F, Lejeune FJ. Treatment
of irresectable soft tissue sarcomas of the limbs by isolation perfusion with high dose TNF-a in
combination with gamma-Interferon and melphalan. Fiers W and Buurman WA (eds), In: Tumor
Necrosis Factor: Molecular and Cellular Biology and Clinical Relevance, Basel, Karger Verlag,
1993, pp 239-243
22. Hill S, Fawcett WJ, Sheldon J, Soni N, Williams T, Thomas JM. Low dose tumor necrosis
factor-alpha and melphalan in hyperthermic isolated limb perfusion. Br J Surg 1993; 80:995-997
23. Eggermont AMM, Schraffordt Koops H, Lienard D, et al: Isolated limb perfusion with high-
dose tumor necrosis factor-alpha in combination with interferon-gamma and melphalan for
nonresectable extremity soft tissue sarcomas: a multicenter trial [see comments]. J Clin Oncol
14:2653-65, 1996a
24. Santinami M, Deraco M, Azzarelli A, Cascinelli F, Chiti A, Costagli V, Manzi R, Quagliolo V,
Rebuffoni G, Santoro N, Vaglini M. Treatment of recurrent sarcoma of the extremities by isolated
perfusion using tumor necrosis factor alpha and melphalan. Tumori 1996;82:579-84
25. Eggermont AMM, Schraffordt Koops H, Klausner JM, et al: Isolated limb perfusion with
tumor necrosis factor and melphalan for limb salvage in 186 patients with locally advanced soft
tissue extremity sarcomas. The cumulative multicenter European experience. Ann Surg 224:756-
64; discussion 764-5, 1996b
26. Gutman M, Inbar M, Lev-Shlush D, Mozes M, Chaitchik S, Meller I, Klausner JM. High dose
tumor necrosis and melphalan administered via isolated limb perfusion for advanced limb
soft tissue sarcoma results in a > 90% response rate and limb preservation. Cancer 1997;79:1129-
37
27. Eggermont AMM, Schraffordt Koops H, Klausner JM, Schlag PM, Kroon BBR, Gustafson P,
Steinmann G, Lejeune FJ. Limb Salvage by Isolation Limb Perfusion with Tumor Necrosis Factor
Alpha and melphalan for locally advanced extremity soft tissue sarcomas: results of 270 perfusions
in 246 patients. Proceed ASCO 1999;11:497(abstract)
28. Rossi CR, Foletto M, Di Filippo F, Vaglini M, Anza M, Azzarelli A, Pilati P, Mocellin S,
Lise M. Soft tissue limb sarcomas: Italian clinical trials with hyperthermic antiblastic perfusion.
Cancer, 1999;86:1742-9
29. Lejeune FJ, Pujol N, Lienard D, Mosimann F, Raffoul W, Genton A, Guillou L, Landry M,
Chassot PG, Chiolero R, Bischof-Delaloye A, Leyvraz S, Mirimanoff RO, Bejkos D, Leyvraz
PF. Limb salvage by neoadjuvant isolated perfusion with TNFalpha and melphalan for non-
resectable soft tissue sarcoma of the extremities. Eur J Surg Oncol 2000, 26:669-78
77
30. Hohenberger P, Kettelhack C, Hermann A, Schlag PM. Functional outcome after

preoperative isolated limb perfusion with rhTNFalpha/Melphalan for high-grade extremity
sarcoma. Eur J Cancer 2001;37(6):S34-35
31. Lans TE, deWilt JHW, van Geel AN, Eggermont AMM. Isolated limb perfusion with
tumor necrosis factor and melphalan for nonresectable stewart-treves lymphangiosarcoma. Ann
Surg Oncol. 2002;9:1004-9
32. Noorda EM, Vrouwenraets BC, Nieweg OE, Slooten GW, Kroon BBR. Isolated limb
perfusion with and Melphalan for Irresectable Soft Tissue Sarcoma of the Extremities.
Ann Surg Oncol 2003;10;1:S36
33. Bonvalot S, Lejeune F, Laplanche A, Stoeckle E, Le Pechoux C, Vanei D, Lumbroso J,
Terrier P, Aubert B, LeCesne A. Limb slavage by isolated limb perfusion (iILP) in patients
with locally advanced soft tissue sarcoma (ASTS): a randomized phase II study comparing 4
doses of Proc. Am Soc Clin Oncol, 2003;22:823
34. Grnhagen D, Lans TE, de Wilt JHW, van Geel AN, Eggermont AMM. Management of
Local Recurrences of Soft Tissue Sarcomas in an Irradiated Field after Prior Surgery and
Radiotherapy: the Role of TNF-based Isolated Limb Perfusions to achieve Limb Salvage. Eur J
Cancer 2003;39:in press
35. Etten van B, van Geel AN, de Wilt JHW, Eggermont AMM. Fifty Tumor Necrosis Factor-
based Isolated Limb Perfusions for limb salvage in patients older than 75 years with limb-
threatening soft tissue sarcomas and other exremity tumors. Ann Surg Oncol, 2003;27:32-37
36. Vrouwenraets BC, Kroon BBR, Ogilvie AC, Van Geel AN, Nieweg OE, Swaak AJG,
Eggermont AMM. Absence of severe systemic toxicity after laekage controlled isolated limb
perfusion with Tumor Necrosis Factor alpha and melphalan. Ann Surg Oncol, 1999;6:405-412
37. Stam, T. C., Swaak, A. J., de Vries, M. R., ten Hagen, T. L., Eggermont, A. M. Systemic
toxicity and cytokine/acute phase protein levels in patients after isolated limb perfusion with
tumor necrosis factor-alpha complicated by high leakage [In Process Citation] Ann Surg Oncol,
2000;4:268-75
38. Vrouenraets BC, Eggermont AMM, Hart AA, Klaase JM, van Geel AN, Nieweg OE,
Kroon BBR. Regional toxicity after isolated limb perfusion with melphalan and tumour
necrosis factor- alpha versus toxicity after melphalan alone. Eur J Surg Oncol. 2001;27:390-5
39. Lejeune FJ, Lienard D, Leyvraz S, Mirimanoff RO. Regional therapy of melanoma. Eur J
Cancer 1993; 29A:606-612
40. Eggermont AMM, Linard D, Schraffordt Koops H, Kroon BBR, Rosenkaimer F, Klaase JM,
Schmitz PIM, Lejeune FJ. High dose tumor necrosis factor-alpha in isolation perfusion of the
limb: highly effective treatment for melanoma in transit metastases or unresectable sarcoma. Reg
Cancer Treat, 7:32-36, 1995
41. Eggermont AMM. Treatment of melanoma intransit metastases confined to the limb. Cancer
Surveys, 26:335-349, 1996
42. Fraker DL, Alexander HR, Andrich M, Rosenberg SA. Treatment of patients with
melanoma of the extremity using hyperthermic isolated limb perfusion with melphalan, tumor
necrosis factor, and interferon gamma: results of a tumor necrosis factor dose-escalation study.
J Clin Oncol, 1996;14:479-89
43. Lienard D, Eggermont AMM, Schraffordt Koops H, Kroon BBR, Towse G, Hiemstra S,
Schmitz P, Clarke J, Steinmann G, Rosenkaimer F, Lejeune FJ. Isolated limb perfusion with
tumour necrosis factor-alpha and melphalan with or without interferon-gamma for the
treatment of in-transit melanoma metastases: a multicentre randomized phase II study.
Melanoma Res, 1999;9:491-502
44. Olieman, A.F., Lienard, D., Eggermont, A.M., Kroon, B.B., Lejeune, F.J., Hoekstra, H.J.
& Koops, H.S. Hyperthermic isolated limb perfusion with tumor necrosis factor alpha,
interferon gamma, and melphalan for locally advanced nonmelanoma skin tumors of the
extremities: a multicenter study. Arch Surg, 1999;134, 303-7
45. Bickels, J., Manusama, E.R., Gutman, M., Eggermont, AMM, Kollender, Y., Abu-Abid, S.,
Van Geel, A.N., Lev-Shlush, D., Klausner, J.M. & Meller, I. Isolated limb perfusion with tumour
78
necrosis factor-alpha and melphalan for unresectable bone sarcomas of the lower extremity [In
Process Citation]. Eur J Surg Oncol, 1999;25:509-14
46. Lev-Chelouche D, Abu-Abeid S, Nakache R, Issakov J, Kollander Y, Merimsky O, Meller
I, Klausner JM, Gutman M. Limb desmoid tumors: a possible role for isolated limb perfusion
with tumor necrosis factor-alpha and melphalan. Surgery 1999;126:963-967
47. Sijens PE, Eggermont AMM, Van Dijk P, Oudkerk M. magnetic resonance spectroscopy as
predictor for clinical response in human extremity sarcomas treated by single dose
melphalan isolated limb perfusion. NMR in Biomedicine 1995;18:215-224
48. Renard N, Linard D, Lespagnard L, Eggermont AMM, Heimann R, Lejeune FJ. Early
endothelium activation and polymorphonuclear cell invasion precede specific necrosis of human
melanoma and sarcoma treated by intravascular high-dose tumour necrosis factor alpha
Int J Cancer 1994;57:656-663
49. Nooijen PTGA, Eggermont AMM, Schalkwijk L, Henzen-Logmans S, DeWaal RMW, Ruiter
DJ. Complete response of melanoma in-transit metastasis after isolated limb perfusion with tumor
necrosis factor-alpha and melphalan without massive tumor necrosis: clinical and histopathological
study of the delayed-type reaction patterns. Cancer Res 1998;58:4880-4887
50. Manusama ER, Nooijen PTGA, Stavast J, Durante NMC, Marquet RL, Eggermont AMM.
Synergistic antitumour effect of recombinant human tumour necrosis factor with melphalan in
isolated limb perfusion in the rat. Br J Surg 1996;83:551-555
51. Manusama ER, Stavast J, Durante NMC, Marquet RL, Eggermont AMM. Isolated limb
perfusion in a rat osteosarcoma model: a new anti-tumour approach. Eur J Surg Oncol
1996;22:152-157
52. De Wilt JHW, ten Hagen TLM, de Boeck G, van Tiel ST, de Bruijn EA, Eggermont
AMM. Tumour Necrosis Factor alpha increases melphalan concentration in tumour tissue after
isolated limb perfusion. Br J Cancer 2000;82:1000-1003
53. Veen vd AH, Wilt de JHW, Eggermont AMM, van Tiel ST, ten Hagen TLM.
augments intratumoural concentration of doxorubicin in isolated limb perfusion
in rat sarcoma models and enhances antitumour effects. Br J Cancer,2000;82:973-980
54. B van Etten, M de Vries, M van IJken, T Lans, G Guetens, G Ambagtsheer, S van Tiel, G
de Boeck, E de Bruijn, AMM Eggermont AMM, TLM Ten Hagen. Degree of tumour
vascularity correlates with drug accumulation and tumour response upon TNF-based isolated
hepatic perfusion. Br J Cancer. 2003;87:314-9
55. Kristensen CA, Nozue M, Boucher Y and Jain RK. Reduction of interstitial fluid pressure
after TNF-alpha treatment of three human melanoma xenografts. Br J Cancer 1996;74:533-
536.
56. Manusama ER, Nooijen PTGA, Stavast J, de Wilt JHW, Marquet RL and Eggermont
AMM. Assessment of the role of neutrophils on the antitumor effect of in an in vivo
isolated limb perfusion model in sarcoma - bearing Brown Norway rats. J Surg Res
1998;78:169-175
57. DeWilt JHW, Manusama ER, van Tiel ST, van IJken MGA, ten Hagen TLM, Eggermont
AMM. Prerequisites for effective isolated limb perfusion using tumour necrosis factor-alpha and
melphalan in rats. Br J Cancer 1999;80:161 -166
58. Manusama ER, de Wilt JHW, ten Hagen TLM, Marquet RL, Eggermont AMM. Toxicity and
antitumor activity of interferon-gamma alone and in combinations with TNF and Melphalan in
isolated limb perfusion in the BN175 sarcoma tumor model in rats. Oncol Rep 1999;6:173-177
59. Seynhaeve ALB, de Wilt JHW, vanTiel SA, Eggermont AMM, ten Hagen TLM.
Combination of Actinomycin D with TNF-a in Isolated Limb Perfusion results in improved
tumour response in soft tissue sarcoma-bearing rats but is accompanied by severe dose limiting
local toxicity. . Br J Cancer 2002; 86:1174-1179.
60. DeWilt JHW, Manusama ER, van Etten B, van Tiel ST, Jorna AS, Seynhaeve ALB, ten
Hagen TLM, Eggermont AMM: Inhibition of Nitric Oxide Synthesis by L-NAME results in
synergistic antitumour activity with melpahlan and tumour necrosis factor-alpha- based isolated
limb. Br J Cancer, 2000:83: 1176-11
79
61. Brunstein F, Hoving S, van Tiel S, ten Hagen TLM, Eggermont AMM. Synergistic
antitumor activity of histamine in combination with chemotherapy in the regional treatment of
soft tissue sarcomas. Eur J Cancer 2003;39:in press
62. Ten Hagen TLM, FJ Lejeune, Eggermont AMM. TNF is here to stay Revisited, Trends in
Immunology (Formerly Immunology Today), 2001;22:127-129
Chapter 5
Pitfalls in pathology of soft tissue sarcomas
Judith V.M.G. Bove and Pancras C.W. Hogendoorn
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands
Correspondence to:
Pancras C.W.Hogendoorn
Dept. of Pathology,
Leiden University Medical Center,
PO Box 9600
2300 RC Leiden,
The Netherlands
82
1 INTRODUCTION
Soft tissue sarcomas are rare, constituting fewer than 1% of all

cancers. Soft tissue tumors are generally regarded difficult by surgical
pathologists, since they constitute a very heterogeneous, relatively uncommon
group of tumors comprising more than 40 entities, with considerable
morphological overlap between the diagnostic entities (1). However,
distinction is essential since these entities differ widely in treatment and
outcome. Classification of soft tissue tumors is assisted by
immunohistochemistry, confirming the line of differentiation, and in the last
decade also by molecular diagnostics, detecting tumor-specific translations.
Histological grading schemes have been developed for soft tissue sarcomas as
a group and seem to be a valuable predictor of patient survival for many, but
not all, types of soft tissue sarcomas. Accurate histological subtyping is
essential for accurate histological grading. Clinicians and pathologists should
be aware of the limitations, prognostic significance, and relationship of
histological subtyping and grading in the therapeutic management of soft
tissue sarcomas.
2 HISTOPATHOLOGICAL TYPING OF SOFT TISSUE

TUMORS
2.1 Method: Needle Biopsy Versus Open Biopsy
There has been a continuous debate over the past years whether core-
needle biopsies, or open biopsies should be used in the diagnostic process of
soft tissue tumors. Though a diagnostic accuracy has been documented as
high as 90% in bone tumors (2), this number is debated widely for soft tissue
tumors. Unfortunately due to increasing economic issues in health care and
patient expectations the work up of patients with soft tissue tumors focuses on
speed and patient friendliness instead of accuracy and a scientific basis for
treatment. As a result core needle biopsies become more and more popular
complicating accurate diagnosis and making grading virtually impossible.
This is especially worthwhile realizing the more and more popular use of pre-
operative chemotherapy and isolated limb perfusion, which if successful leave
virtual no tissue left for definite diagnosis, meaning that a substantial number
of patients will be treated with toxic therapies, while one honestly does not
know what kind and grade of tumor has been treated. Core needle biopsies are
however very useful for the differential diagnosis with metastatic carcinoma,
melanoma and to rule out lymphoma. In a specialized hospital setting it is
useful for the diagnosis of a number of tumors with consistent genetic
abnormalities which can be very accurately assessed by molecular techniques
83
on needle biopsies as well (3).
2.2 Classification of Soft Tissue Tumors
Classification of soft tissue tumors is based primarily on the line of

differentiation displayed by the tumor (the type of tissue formed by the tumor)
rather than the type of tissue from which the tumor arises. The classification
of soft tissue tumors has been changing over the past decades. An increasing
knowledge of immunohistochemical and molecular genetic characteristics has
resulted in a more accurate classification and a better understanding of the
biology of these tumors. For instance, malignant fibrous histiocytoma (MFH)
was for long considered the most common adult soft tissue sarcoma.
However, its line of differentiation could not be established and the group of
tumors seemed very heterogeneous in terms of clinical behavior and ultimate
outcome, questioning its existence as a real entity. Fletcher et al (4) showed in
a retrospective study that MFH could be subclassified, and that this
subclassification was prognostically relevant. For instance, pleomorphic MFH
could be subclassified as dedifferentiated liposarcoma (metastatic risk <25%),
high grade myxofibrosarcoma (metastatic risk 35-40%) and pleomorphic
rhabdomyosarcoma (most patients have metastases within 2 years) (4). In
general, pleomorphic sarcomas demonstrating myogenic differentiation (high
grade leiomyosarcoma, pleomorphic rhabdomyosarcoma, high grade
myogenic sarcoma NOS) were shown to have a worse prognosis (4),
underlining the need to minimize the use of the term MFH and necessitating
the development of more intense therapeutic strategies for this category of
tumors.
2.3 WHO Classification
The most recent classification is the WHO classification 2002, which

is based upon consensus of several experts in the field (1)(table 1). This
Working Group chose to divide the tumors according to their biological
potential into four categories; benign, intermediate (locally aggressive),
intermediate (rarely metastasizing) and malignant. Most benign soft tissue
tumors do not recur locally, and if they do the recurrence is non-destructive
and almost always cured by complete local excision. Soft tissue tumors in the
intermediate locally aggressive category often recur locally with an
infiltrative, locally destructive growth pattern, thus requiring wide excision
with a margin of normal tissue. They do not have an evident potential to
metastasize, in contrast to soft tissue tumors in the intermediate, rarely
metastasizing category. In addition to the locally destructive growth pattern,
the risk of distant metastases in this category is estimated at <2%, which is
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Table 1. WHO classification of soft tissue tumors ( 1 ).

ADIPOCYTIC TUMORS
Benign Intermediate (locally Malignant
aggressive)
Lipoma, lipomatosis, Atypical lipomatous tumor / Myxoid / round
lipomatosis of Nerve well differentiated cell liposarcoma
liposarcoma
Lipoblastoma/lipoblasto- Dedifferentiated
matosis liposarcoma
Angiolipoma Pleomorphic
liposarcoma
Myolipoma Mixed-type
liposarcoma
Chondroid lipoma Liposarcoma, not
otherwise
specified
Extrarenal angiomyolipoma
Extra-adrenal myelolipoma
Spindle cell / pleomorphic
lipoma
Hibernoma
FIBROBLASTIC / MYOFIBROBLASTIC TUMORS
aggressive)
Nodular / proliferative Superficial fibromatosis Adult
fasciitis (palmar/plantar) fibrosarcoma
Proliferative myositis, Desmoid-type fibromatosis Myxofibrosarcoma
myositis Ossificans
Ischemic fasciitis Lipofibromatosis Low grade
fibromyxoid
sarcoma /
hyalinizing
spindle cell tumor
Elastofibroma Sclerosing
epithelioid
fibrosarcoma
Fibrous hamartoma of Intermediate (rarely
infancy metastasizing)
Myofibroma / Solitary fibrous tumor and
myofibromatosis haemangiopericytoma
Fibromatosis colli Inflammatory
myofibroblastic tumor
Juvenile hyaline fibromatosis Low grade myofibroblastic
sarcoma
Inclusion body fibromatosis Myxoinflammatory
fibroblastic sarcoma
Fibroma of tendon sheath Infantile fibrosarcoma
Desmoplastic fibroblastoma
Mammary-type myofibroblastoma
85
Calcifying aponeurotic
fibroma
Angiomyofibroblastoma
Cellular angiofibroma
Nuchal type fibroma
Gardner fibroma
Calcifying fibrous tumor
Giant cell angiofibroma
SO CALLED FIBROHISTIOCYTIC TUMORS
Benign Intermediate (rarely Malignant
metastasizing)
Giant cell tumor of tendon Plexiform fibrohistiocytic Pleomorphic
sheath tumor MFH /
undifferentiated
pleomorphic
sarcoma
Diffuse type giant cell tumor Giant cell tumor of soft Giant cell MFH /
tissue undifferentiated
pleomorphic
sarcoma with
giant cells
Deep benign fibrous Inflammatory
histiocytoma MFH /
undifferentiated
pleomorphic
sarcoma with
prominent
inflammation
SMOOTH MUSCLE TUMORS
Benign Malignant
Angioleiomyoma Leiomyosarcoma (excl skin)
Deep leiomyoma
Genital leiomyoma
PERICYTIC (PERIVASCULAR) TUMORS
Glomus tumor (and variants), malignant glomus tumor
Myopericytoma
SKELETAL MUSCLE TUMORS
Benign Malignant
Rhabdomyoma (adult, fetal, Embryonal rhabdomyosarcoma (incl. spindle cell,
genital type) botryoid, anaplastic)
Pleomorphic rhabdomyosarcoma
Alveolar rhabdomyosarcoma (incl. solid,
anaplastic)
86
VASCULAR TUMORS
aggressive)
Haemangioma (incl.capillary, Kaposiform Epithelioid
cavernous, arteriovenous, haemangioendothelioma haemangioendo-
venous, intramuscular, thelioma
synovial, subcutis /
soft tissue)
Epithelioid haemangioma Intermediate (rarely Angiosarcoma of
metastasizing) soft tissue
Angiomatosis Retiform
haemangioendothelioma
Lymphangioma Papillary intralymphatic
angioendothelioma
Composite
haemangioendothelioma
Kaposi sarcoma
CHONDRO-OSSEOUS TUMORS
Benign Malignant
Soft tissue chondroma Mesenchymal chondrosarcoma
Extraskeletal osteosarcoma
TUMORS OF UNCERTAIN DIFFERENTIATION
Benign Intermediate (rarely Malignant
metastasizing)
Intramuscular myxoma Angiomatoid fibrous Synovial sarcoma
(incl. cellular variant) histiocytoma
Juxta-articular myxoma Ossifying fibromyxoid Epithelioid
tumor (incl. atypical / sarcoma
malignant)
Deep (aggressive) Mixed tumor / myoepi- Alveolar soft part
angiomyxoma thelioma / parachordoma sarcoma
Pleomorphic hyalinizing Clear cell sarcoma
angiectatic tumor of soft tissue
Ectopic hamartomatous Extraskeletal
thymoma myxoid
chondrosarcoma
PNET/
extraskeletal
Ewing tumor
Desmoplastic
small round
cell tumor
Extra-renal
rhabdoid tumor
Malignant
mesenchymoma
87
PEComa /
neoplasms with
perivascular
epithelioid cell
differentiation
Intimal sarcoma
TUMOURS OF PERIPHERAL NERVES (5)
Benign Malignant
Schwannoma (incl. cellular, plexiform, Malignant peripheral nerve sheath
Melanotic) tumor (MPNST)
(incl. epithelioid, with divergent
mesenchymal and / or
epithelial differentiation, melanotic,
melanotic psammomatous)
Neurofibroma (incl. plexiform)
Perineurioma (incl. intraneural, soft tissue)
not predictable on the basis of histology. Malignant soft tissue tumors, called
soft tissue sarcomas, have a significant risk of distant metastases, ranging
from 20-100% depending on histological type and grade (1). Some low grade
sarcomas have initially a lower metastatic risk but they may advance in grade
upon local recurrence, increasing the risk of distant spread. It is important to
note that the intermediate categories of biological potential as defined in the
WHO classification do not correspond to the histologically defined
intermediate grade of malignancy (see histological grading) (1).
3 HISTOPATHOLOGICAL GRADING
Malignant soft tissue tumors (sarcomas) represent a heterogeneous

group of neoplasms differing widely in their clinico-biological behavior,
ranging from lesions that only very rarely metastasize to lesions that behave
in a highly aggressive manner with metastases in most of the cases which may
happen very rapidly or may take many years. Part of this behavior is histotype
specific (e.g. well differentiated liposarcoma), underscoring the importance of
adequate histopathological classification. Some lesions show however a broad
spectrum of behavior not predictable from histological typing alone (e.g.
leiomyosarcoma, gastrointestinal stromal tumor). Therefore, histopathological
grading systems were developed in an attempt to identify histotype-
independent histological parameters that may be of help to predict prognosis
(6). Thus, in addition to the histological type of a soft tissue sarcoma,
histological grading also gives information about the degree of malignancy
and the probability of distant metastases and survival. Histological grading is
therefore a feature of the major tumor staging systems and constitutes
important information for therapeutic decisions. Grading is of poor value for
predicting local recurrence, which is mainly related to the quality of surgical
margins.
88
3.1 Grading Systems
Histological grading schemes have been proposed for soft tissue

sarcomas as a group, since the relative rarity of soft tissue sarcomas severely
hampers optimal and significant histotype specific clinicopathological studies.
Grading aims at identifying and separating tumors with a favorable prognosis
from those with a poor prognosis (6). Grading is based on histological
parameters only and is very subjective. There is no ideal grading system and
several different grading systems have been published so far and have been
shown to correlate with prognosis. The two most widely used grading systems
are the NCI (United States National Cancer Institute) system (7) and the
FNCLCC (French Fdration Nationale des Centres de Lutte Contre le
Cancer) system originally described by Trojani et al in 1984 (8), later
modified by Guillou et al (9). The NCI system uses a combination of
histological typing and histological parameters (cellularity, pleomorphism,
and mitotic rate) for identification of grade 1 tumors. All the other types of
sarcomas are classified as either grade 2 or 3, with 15% necrosis as the
threshold for separating grade 2 from grade 3 tumors (1;7). The FNCLCC
system is based on the evaluation of three parameters: tumor differentiation,
mitotic rate and amount of tumor necrosis (table 2). There is however
considerable interobserver variation in the assessment of these histological
parameters and evaluation of the reproducibility of the FNCLCC system
revealed a 75% agreement for tumor grade, while the agreement for the
Table 2. FNCLCC grading system for (adult) soft tissue sarcoma (8;9)
TUMOR DIFFERENTIATION (see table 3)

Score 1 Sarcomas resembling normal adult mesenchymal tissue
Score 2 Sarcomas for which histological typing is certain
Score 3 Embryonal and undifferentiated sarcomas, sarcomas of
doubtful type, synovial sarcomas, osteosarcomas, PNET
MITOTIC COUNT
Score 1 0-9 mitoses per 10 HPF*
Score 2 10-19 mitoses per 10 HPF
Score 3 20 mitoses per 10 HPF
TUMOR NECROSIS
Score 0 No necrosis
Score 1 <50% tumor necrosis
Score 2 50% tumor necrosis
HISTOLOGICAL GRADE
Grade 1 Total score 2,3
Grade 2 Total score 4,5
Grade 3 Total score 6,7,8
* HPF (high power field) defined as
89
histological typing was only 61% (10). When both grading systems are
compared using the same set of 410 tumors concordance rates are only around
65% (9). The FNCLCC system is favored because it correlates better with
overall and metastasis free survival, and it allocates less patients in the
intermediate grade category and is more reproducible than the NCI system
(9). The establishment of the tumor differentiation score in the FNCLCC
system can however be problematic. A listing of the differentiation scores for
the most common tumors has been reported (table 3)(9), but the rationale for
some of these scores is not clear (11).
Table 3. tumor differentiation score according to histological type in the updated

version of the FNCLCC system (modified from Guillou et al (9) and the WHO classification
(1))
TUMOR
HISTOLOGIC TYPE DIFFERENTIATION
SCORE
Well differentiated liposarcoma 1
Myxoid liposarcoma 2
Round cell liposarcoma 3
Pleomorphic liposarcoma 3
Dedifferentiated liposarcoma 3
Fibrosarcoma
Well differentiated 1
Conventional 2
Poorly differentiated 3
Malignant peripheral nerve sheath tumor (MPNST)
Conventional 2
Poorly differentiated 3
Epithelioid 3
Malignant Triton tumor 3
Myxofibrosarcoma (myxoid MFH) 2
Pleomorphic sarcoma (pleomorphic MFH)
With storiform pattern 2
Patternless pleomorphic sarcoma 3
With giant cells (giant cell MFH) 3
With prominent inflammation (inflammatory MFH) 3
90
Leiomyosarcoma
Conventional 2
Poorly differentiated / pleomorphic / epithelioid 3
Biphasic / monophasic synovial sarcoma 3
Embryonal / alveolar / pleomorphic rhabdomyosarcoma 3
Myxoid chondrosarcoma 2
Mesenchymal chondrosarcoma 3
Conventional angiosarcoma 2
Poorly differentiated / epithelioid angiosarcoma 3
Extraskeletal osteosarcoma 3
Ewing sarcoma / PNET 3
Alveolar soft part sarcoma 3
Epithelioid sarcoma 3
Malignant rhabdoid tumor 3
Clear cell sarcoma 3
Undifferentiated sarcoma 3
3.2 Limitations and drawbacks of histological grading
Grading soft tissue sarcomas as a group, due to their rarity, has

several major disadvantages. One should realize that grading is not a
substitute for the histological diagnosis, which should alsways be assessed
first and may even be considered the most important predictor of outcome,
illustrated by the fact that both th eNCI as well as the FNCLCC grading
schemes are based on adequate histotyping. Some tumor types appear to be
characterized by a biological behavior that can be predicted by
histopathological classification alone (Table 4). Therefore, not all soft tissue
sarcomas have to be graded since for some histological subtypes grade is of
no prognostic value. Studying 1240 cases revealed that the histological grade
(FNCLCC) is an independent predictor of metastasis development for the
main histological types of adult soft tissue sarcomas, with the exception of
MPNST and rhabdomyosarcoma (13). The WHO classification does not
recommend histological grading for MPNST, angiosarcoma, extraskeletal
myxoid chondrosarcoma, alveolar soft part sarcoma, clear cell sarcoma and
epithelioid sarcoma (1). The latter three tumors will often metastasize within
10-20 years of follow-up (14). Histological grading is however useful in
91
Table 4. Soft tissue sarcomas for which the histotype determines the biological
behavior / grade (adapted from (6) and (14)).
HIGH GRADE BEHAVIOR

Angiosarcoma
Desmoplastic small round cell tumor
Extrarenal rhabdoid tumor
Extraskeletal Ewing sarcoma / PNET
Extraskeletal osteosarcoma
Myxoid / round cell liposarcoma with >5% round cell component
Mesenchymal chondrosarcoma
Pleomorphic liposarcoma
Rhabdomyosarcoma (except spindle cell variant)
LOW GRADE BEHAVIOR

Atypical lipomatous tumor / well differentiated liposarcoma
Congenital fibrosarcoma
Dermatofibrosarcoma protuberans
Myxoid / round cell liposarcoma with <5% round cell component
tumors with varying behavior (e.g. leiomyosarcoma, myxofibrosarcoma,

fibrosarcoma). One should also realize that grading does not differentiate
benign from malignant lesions (1). Ideally, the clinicopathological study of
sarcomas, including histological grading, should be histotype specific since
evaluation of histological parameters in 1116 tumors revealed that there is no
single universal histological prognostic parameter that is valid for all types of
soft tissue sarcomas (15). However, due to the rarity of soft tissue sarcomas
comparison of larger groups of individual tumor types is problematic, often
hampering the identification of histotype specific prognostic histological
parameters. Eventually, especially with targeted treatment becoming more
and more applied, grading systems will be devised for an increasing number
of individual sarcoma types. For instance, the biological behavior of myxoid /
round cell liposarcoma can be predicted by the significance of the round cell
component; if this component constitutes >5% and there is necrosis, the tumor
will behave as a high grade sarcoma (1), although the mitotic rate can be low.
Another example concerns gastrointestinal stromal tumors (GISTs).
Stromal/mesenchymal tumors of the gastrointestinal tract have long been a
source of confusion and controversy with regard to classification, line of
differentiation and prognostication (16). With the discovery of targeted
treatment using STI571 / Glivec, GISTs have become the center of attention.
Since an accurate and reproducible diagnosis is essential to ensure appropriate
treatment for GIST patients, a consensus approach to diagnosis and
morphological prognostication has led to a scheme for estimating metastatic
risk in these lesions, based on tumor size and mitotic count (Table 5) (16).
Finally, one should be aware that only untreated primary soft tissue sarcomas
should be graded, since radiation treatment and chemotherapy prior to
92
surgical excision make grading of the resected specimen unreliable, for

instance due to treatment induced necrosis, fibrosis and pleomorphism.
Histological grading should only be performed on representative and well
processed material. The mitotic rate for instance is greatly influenced by
sampling error, tissue fixation, the presence of necrosis and section thickness
(17).
Table 5. Risk assessment for GIST (gastrointestinal stromal tumor) (16).
Size* Mitotic rate

Very low risk <2cm <5 / 50 HPF#
Low risk 2-5 cm <5 / 50 HPF
Intermediate risk <5 cm 6-10 / 50 HPF
5-10 cm <5 / 50 HPF
High risk >5 cm >5 / 50 HPF
>10cm Any mitotic rate
Any size >10 / 50 HPF
*size represents the single largest dimension. # HPF (high power field) not defined
The distinction of mitoses from apoptosis and karyorrhexis can be

difficult. The number of high power fields counted need to be standardized,
taking into account the exact microscopic field size which may show marked
variation between various types of microscopes. It is as yet unknown whether
necrosis is best assessed microscopically or macroscopically (9;17). Since
necrotic areas will be avoided when selecting blocks, microscopic assessment
of necrosis will be an underestimate. Gross assessment of necrosis can be
made only on the resected specimen, precluding retrospective (re)evaluation.
Furthermore, naturally occurring intralesional heterogeneity also
automatically implies the risk of sampling error. Thus, enough blocks should
be sampled. The association of directors of anatomic and surgical pathology
(ADASP) recommends sampling with an overall block number of
approximately 1 per cm of the tumors greatest dimension (14). The
consequence of these limitations is that reliable grading can rarely be
performed on a needle biopsy or small incisional biopsy, unless it shows an
obvious high grade tumor.
93
4 ADDITIONAL TECHNIQUES
4.1 Immunohistochemistry
Immunohistochemistry is especially helpful in the classification of

soft tissue sarcomas, since classification can be difficult due to their rarity,
and their broad and partly overlapping morphological spectrum.
Immunohistochemistry is a valuable diagnostic tool in determining the line of
differentiation. In addition, in the past decade targeted treatment of soft tissue
tumors has rapidly made its introduction as an adjuvant therapy, making
immunohistochemistry a tool to investigate whether this treatment can be
useful.
4.1.1 Immunohistochemistry for Classification Purposes
Immunohistochemistry is widely used as an adjunct to light

microscopy aiding the classification of tumors. After the establishment of the
differential diagnosis on conventional haematoxylin and eosin stained slides,
the line of differentiation can be determined or confirmed using
immunohistochemistry. In soft tissue tumors, skeletal or smooth muscle
differentiation, nerve sheath differentiation, melanocytic, fibrohistiocytic, or
endothelial differentiation can be distinguished (Table 6). A growing list of
antibodies is available, each with its own sensitivity and specificity. Since no
antibody is 100% specific for a certain tumor type, a combination of
antibodies should be used. For instance, in daily practice a standard panel of
antibodies is used in case of monomorphic spindle cell tumors or
undifferentiated round cell tumors, two of the most difficult groups of soft
tissue tumors in terms of differential diagnosis in which the application of
immunohistochemistry is essential to come to an accurate diagnosis (Tables 7
&8). Several studies have been performed to detect proliferation markers,
overexpression of oncogenes and loss of expression of tumor suppressor
genes by immunohistochemistry in soft tissue sarcomas to gain further insight
into their pathogenesis, and to investigate whether clinical behavior could be
predicted. However, results so far have been disappointing and
immunohistochemistry is not yet used as a prognostic tool in routine daily
practice.
94
Table 6. Immunohistochemistry as a tool to determine the line of differentiation.
Muscle differentiation
Desmin, smooth muscle actin, muscle specific actin (HHF35),
Myogenic transcription factors (MyoD1, Myf4 (myogenin)),
myoglobin, heavy caldesmon, calponin
Nerve sheath differentiation
S100, CD57
Melanocytic differentiation
HMB-45, Melan-A (MART-1), tyrosinase, microphtalmia
transcription factor
Endothelial differentiation
Von Willebrand Factor (Factor VIII-related antigen), CD34 (human
haematopoetic progenitor cell antigen), CD31 (platelet endothelial
cell adhesion molecule-1), Ulex Europaeus Lectin
Fibro-histiocytic differentiation
CD68, Factor 13A, vimentin
Epithelial differentiation
EMA (epithelial membrane antigen), cytokeratin
95
4.1.2 Immunohistochemistry for Drug Targeting
A number of molecular events at the DNA level, leading to altered

protein expression can be identified by immunohistochemistry. A number of
them have been implied as potential targets for drugs such as Her-2Neu,
PDGF and c-kit. The most exciting results are presented by studies in
gastrointestinal stromal tumors (GISTs) targeting c-kit. Overexpression as a
result of an activating mutation can be identified by clear
immunohistochemical positivity using antibodies directed against c-kit
(CD117), which is of paramount importance in its differential diagnosis as
well (18). Mutations in c-kit leading to constitutive activation of the tyrosine
kinase are believed to play a role in the majority of GISTs. STI571 has been
shown to be a selective inhibitor of the tyrosine kinase activity of kit, and was
shown to be clinically effective in patients with GISTs (19-21). Eligibility for
clinical trials is dependent on the expression of c-kit, as determined by
immunhistochemical staining.
4.2 Limitations and Pitfalls of Immunohistochemistry
Since the introduction of (adjuvant) treatment of GIST by STI571,

different tumor types have been investigated for c-kit overexpression using
immunohistochemical staining, to investigate whether other tumor types
might also benefit from STI571 treatment. These investigations demonstrated
some important limitations. Variations in immunohistochemical techniques
96
used in the different laboratories can lead to major discrepancies in positive

staining. The type of antibody used can vary since several antibodies may be
(commercially) available for the same antigen. In addition, the staining
protocols used in different laboratories vary somewhat with regard to antigen
retrieval. High background staining, as can be seen in some polyclonal
antibodies or can be due to insufficient dilution of the antibody, may lead to
false positive results. In general, it is of crucial importance to evaluate, if
possible, positive internal controls, such as vessels walls (CD34, CD31, factor
VIII, smooth muscle actin, muscle specific actin / HHF35, desmin), mast cells
(c-kit/CD117) and nerve fibers (S100) to avoid false negative results.
4.3 Molecular Diagnostics
In the past decade, molecular diagnostics have played an increasing

role aiding the pathologist in the differential diagnosis. Especially the
detection of translocations, specific for certain tumor types (see next chapter)
by reverse transcription PCR or by fluorescence in situ hybridization has
found its way in routine daily practice in sarcoma specialized centers.
Moreover, studies are beginning to indicate that the type of translocation
found may be of prognostic value, and future clinicopathological studies and
clinical trials will reveal whether these histotype specific prognostic
parameters are superior to histological grading in making therapeutic
decisions.
5 REFERENCES
1. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours

of Soft Tissue and Bone. Lyon: IARC Press, 2002.
2. Van der Bijl AE, Taminiau AHM, Beerman H, Hogendoorn PCW. Accuracy of the Jamhidi
trocar biopsy in the diagnosis of bone tumors. Clin Orthop 1997; 334:233-243.
3. Graadt van Roggen JF, Bovee JVMG, Morreau J, Hogendoorn PCW. Diagnostic and
prognostic implications of the unfolding molecular biology of bone and soft tissue tumours. J
Clin Pathol 1999; 52:481-489.
4. Fletcher CD, Gustafson P, Rydholm A, Willen H, Akerman M. Clinicopathologic re-
evaluation of 100 malignant fibrous histiocytomas: prognostic relevance of subclassification. J
Clin Oncol 2001; 19(12):3045-3050.
5. World Health Organization Classification of Tumours. Pathology ans Genetics of Tumours
of the Nervous System. Lyon: IARC Press, 2003.
6. Graadt van Roggen JF. The histopathological grading of soft tissue tumours: current
concepts. Curr Diagn Pathol 2001; 7(1):1-7.
97
7. Costa J, Wesley RA, Glatstein E, Rosenberg SA. The grading of soft tissue sarcomas.
Results of a clinicohistopathologic correlation in a series of 163 cases. Cancer 1984; 53:530-
541.
8. Trojani M, Contesso G, Coindre JM, Rouesse J, Bui NB, De Mascarel A et al. Soft-tissue
sarcomas of adults; study of pathological prognostic variables and definition of a
histopathological grading system. Int J Cancer 1984; 33(1):37-42.
9. Guillou L, Coindre JM, Bonichon F, Nguyen BB, Terrier P, Collin F et al. Comparative
study of the National Cancer Institute and French Federation of Cancer Centers Sarcoma Group
grading systems in a population of 410 adult patients with soft tissue sarcoma. J Clin Oncol
1997; 15(1):350-362.
10. Coindre JM, Trojani M, Contesso G, David M, Rouesse J, Binh Bui N et al.
Reproducibility of a histopathologic grading system for adult soft tissue sarcoma. Cancer 1986;
58:306-309.
11. Weiss SJ, Goldblum JR. Soft Tissue Tumors. 4 ed. St.Louis: the C.V. Mosby Company,
2001.
12. Brown FM, Fletcher CD. Problems in grading soft tissue sarcomas. Am J Clin Pathol 2000;
114 Suppl:S82-S89.
13. Coindre JM, Terrier P, Guillou L, Le D, V, Collin F, Ranchere D et al. Predictive value of
grade for metastasis development in the main histologic types of adult soft tissue sarcomas: a
study of 1240 patients from the French Federation of Cancer Centers Sarcoma Group. Cancer
2001; 91(10):1914-1926.
14. Recommendations for the reporting of soft tissue sarcomas. Association of Directors of
Anatomic and Surgical Pathology. Hum Pathol 1999; 30(1):3-7.
15. Hashimoto H, Daimaru Y, Takeshita S, Tsuneyoshi M, Enjoji M. Prognostic significance
of histologic parameters of soft tissue sarcomas. Cancer 1992; 70:2816-2822.
16. Fletcher CD, Berman JJ, Corless C, Gorstein F, Lasota J, Longley BJ et al. Diagnosis of
gastrointestinal stromal tumors: A consensus approach. Hum Pathol 2002; 33(5):459-465.
17. Oliveira AM, Nascimento AG. Grading in soft tissue tumors: principles and problems.
Skeletal Radiol 2001; 30(10):543-559.
18. Graadt van Roggen JF, Van Velthuysen MLF, Hogendoorn PCW. The histopathological
differential diagnosis of gastrointestinal stromal tumours. J Clin Pathol 2001;(54):96-103.
19. Demetri GD, von Mehren M, Blanke CD, Van den Abbeele AD, Eisenberg B, Roberts PJ
et al. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N
Engl J Med 2002; 347(7):472-480.
20. Joensuu H, Roberts PJ, Sarlomo-Rikala M, Andersson LC, Tervahartiala P, Tuveson D et
al. Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal
stromal tumor. N Engl J Med 2001; 344(14):1052-1056.
21. Van Oosterom AT, Judson 1, Verweij J, Stroobants S, Donato dP, Dimitrijevic S et al.
Safety and efficacy of imatinib (STI571) in metastatic gastrointestinal stromal tumours: a phase
I study. Lancet 2001; 358(9291):1421-1423.
Chapter 6
Molecular biology and cytogenetics of soft tissue

sarcomas: Relevance for targeted therapies
Jonathan A. Fletcher, M.D.
Department of Pathology; Brigham and Womens Hospital

Boston, USA
Correspondence to :
Jonathan A Fletcher, MD
Department of Pathology;
Brigham and Womens Hospital;
75 Francis Street
Boston, MA 02115
USA
100
1 INTRODUCTION
Various genomic mechanisms can result in activation of therapeutic

targets in soft tissue sarcomas (Table 1). Some mechanisms involve
rearrangements of sizeable chromosomal regions, and given that they can be
demonstrated by conventional cytogenetic methods, they are often referred to
as cytogenetic aberrations. A well known example is the rearrangement of
chromosomes 17 and 22 (often in a circular ring form) which activates the
platelet derived growth factor receptor beta (PDGFRB) pathway in
dermatofibrosarcoma protuberans (1,2). Other genomic mechanisms cannot
be shown by conventional cytogenetic methods, because they involve small
deletions or point mutations of DNA material which are below the resolution
of karyotyping and fluorescence in situ hybridization (FISH) methods. Such
mutations can be referred to as molecular aberrations. A well known
example in this category is the gain-of-function KIT gene point mutation
found in most gastrointestinal stromal tumors (3).
Most soft tissue sarcomas contain clonal cytogenetic and molecular

aberrations, some of which are diagnostic for particular tumor types (4-8).
Demonstration of characteristic chromosome abnormalities has been useful
diagnostically, especially in undifferentiated small round cell or spindle cell
soft tissue tumors (5). However, only recently have these aberrations become
useful as therapeutic targets. The advantage of such targets is that their
relevance to the sarcoma is clear, given that they are activated by specific
genomic mutations, and that they are selected for during the clinical
progression of the sarcoma. For example, the fusion oncogenes that result
from chromosomal translocations in sarcomas are invariably retained by the
neoplastic cells even when the sarcomas undergo dedifferentiation, or
progress to a more advanced histological grade. These observations suggest
that the oncogenes are essential in maintaining the transformed state of the
sarcoma cells, and the proteins encoded by these oncogenes therefore
represent compelling therapeutic targets.
In general, the cytogenetic and molecular mutations of greatest

relevance to targeted therapies are those which activate an oncoprotein, and
which can be countered directly with a therapeutic inhibitor of the
oncoprotein. Examples include the various tyrosine kinase proteins that are
activated by cytogenetic or molecular aberrations in different types of
sarcomas. Transcription factor proteins, which regulate gene expression, are
also activated by many of the cytogenetic aberrations in sarcomas.
Therapeutic inactivation of the transcription factor oncoproteins appears to be
more challenging compared to inhibition of kinase proteins, but nonetheless,
this family of proteins will doubtless provide many targets for future
treatment strategies. Tumor suppressor mechanisms are also relatively
challenging as therapeutic targets, in that the conceptual approach involves
101
restoring the lost tumor suppressor function to the neoplastic cells. It has
been difficult to devise methods in which such restoration of function is
accomplished with high efficiency and at physiological levels across a
heterogeneous neoplastic cell population. On the other hand, tumor suppressor
mutations result inevitably in activation of downstream proteins whose
function would normally be inhibited by the tumor suppressor. These
downstream proteins might be more tractable to targeted therapies than the
tumor suppressor proteins themselves.
2 METHODOLOGICAL CONSIDERATIONS
The classic cytogenetic approach requires fresh, viable, tumor

specimens which should be processed rapidly and transported to the
cytogenetics laboratory in sterile tissue culture media or in a physiologic
buffer such as Hanks Buffered Salt Solution. It is important that the
cytogenetic sample be removed from the overall tumor specimen with sterile
scalpel blades or scissors. Otherwise, bacterial or fungal contamination may
lead to microbial overgrowth in the subsequent tissue cultures. Because
viable tumor cells are essential in establishing the tissue cultures, it is also
important that the specimen be selected carefully so as to contain a minimum
of necrotic tissue. Also, it is crucial to minimize nonneoplastic components,
particularly fibroblasts, lest these cells overwhelm the tumor population after
the cultures are established. The success of the cytogenetic analysis depends
largely on the quality of the tumor specimen, whereas the amount of tumor is
less important: thus, percutaneous needle biopsies of small round cell tumors,
and other cellular sarcomas, can be karyotyped (5). At least 80% of all soft
tissue sarcomas can be cultured successfully if the specimens are carefully
selected so as to minimize necrotic and nonneoplastic components.
Fluorescence in situ hybridization (FISH), PCR and sequencing

methods, unlike conventional karyotyping, can be performed in archival
sarcoma specimens. These methods are most successful in fresh or frozen
tumor specimens, but can also be performed in paraffin materials. FISH and
molecular methods will likely play an increasing role in identifying
therapeutic targets in sarcomas, and it is likely that one or another form of
these analyses will be used routinely as an adjunct to histological appraisal
in the near future.
102
Several molecular cytogenetic methods have expanded the

capabilities of FISH by permitting genome-wide evaluation of
chromosomal aberrations. Examples include comparative genomic
hybridization and spectral karyotyping. However, these methods are
labor-intensive, and better suited to research applications than in routine
clinical evaluation of sarcoma patients.
3 CYTOGENETIC MECHANISMS
As mentioned above, recurring chromosome rearrangements,

particularly those that serve as diagnostic and/or prognostic markers, are
critical events in sarcoma tumorigenesis (Table 1).
The specific nature of each chromosome rearrangement -
whether translocation, deletion, or amplification can identify the
mechanisms by which specific genes are altered during tumorigenesis.
Certain sarcoma translocation breakpoints interrupt genes directly,
resulting in novel fusion oncogenes. Other translocation breakpoints
are adjacent to a particular gene, and result in deregulated expression
(typically overexpression) of the gene. Deletions of whole chromosomes,
or chromosomal regions, generally signify loss of one or more tumor
suppressor genes. Amplification events, whether extrachromosomal
(double minute chromosomes) or intrachromosomal (homogeneously
staining regions) signify increased copies - and associated overexpression
- of one or more oncogenes.
4 MOLECULAR AND CYTOGENETIC

ABERRATIONS IN SARCOMAS
4.1 Adipose Tumors Diverse Genetic Mechanisms
Adipose differentiation is regulated by (peroxisome

proliferator-activated receptor gamma), which is a nuclear receptor and
transcription factor (9). Notably, ligands have shown promise as
therapeutic adjuncts in liposarcoma, being most effective in
myxoid/round-cell liposarcoma (10). The selectivity of targeted
therapy is in keeping with molecular and cytogenetic evidence showing
that liposarcomas have distinctive genetic and biological profiles.
Indeed, most adipose tumors, whether benign or malignant, contain
distinctive chromosome aberrations (Table 1). Useful diagnostic markers
include 12q rearrangement in lipoma, ring chromosomes in well-
differentiated and dedifferentiated liposarcoma, t(12;16) translocations in
mxyoid/round cell liposarcoma, and cytogenetic complexity in
pleomorphic liposarcomas.
103
104
The most diagnostically useful aberration, in malignant adipose

tumors, is a translocation of chromosomes 12 and 16, t(12;16)(q13;p11).
This translocation is found in myxoid liposarcomas (11-13), and is
retained in those cases that acquire round cell features (14,15). The
t(12;16) translocation results in fusion of the CHOP gene on
chromosome 12 with the TLS gene on chromosome 16 (16,17), and the
resultant fusion oncoprotein is an activated transcription factor. The
t(12;16) translocation appears to be diagnostic for myxoid liposarcoma
and has not been found in other subtypes of liposarcoma or in other types
of myxoid soft tissue tumors (18,19). Detection can be accomplished by
cytogenetics or reverse transcriptase PCR.
Well-differentiated liposarcomas (atypical lipomas) contain large

giant marker chromosomes or ring chromosomes. These chromosomes
are generally comprised of chromosome 12 material, often admixed with
components of several other chromosomes (13,20). The ring and giant
marker chromosomes contain various amplified genes, but the essential
gene amplification targets have not been pinpointed. Notably, these
amplifications are retained when well-differentiated liposarcomas
progress to clinically more aggressive, dedifferentatiated, liposarcomas.
Therefore one can hope that eventual identification of the gene
amplification targets will reveal therapeutic pathways that might be
particularly useful in dedifferentiated liposarcoma. Pleomorphic
liposarcomas differ cytogenetically from other liposarcomas in that they
have exceedingly complex karyotypes with multiple clonal chromosome
aberrations (12). The genomic complexity in pleomorphic liposarcomas
has hampered attempts to define consistent chromosomal aberrations that
might be of diagnostic and therapeutic utility.
4.2 Clear Cell Sarcoma (malignant melanoma of soft

parts)
Clear cell sarcomas of soft tissues, also referred to as

melanomas of the soft parts, share many phenotypic features with
cutaneous malignant melanomas. Hence, it can be difficult to distinguish
between clear cell sarcoma and cutaneous melanoma histologically.
Despite the histologic similarities between clear cell sarcoma and
cutaneous melanoma, these two tumors are quite different clinically.
Clear cell sarcomas usually present as isolated masses located in deep
soft tissues without apparent origin from skin. It is notable, therefore,
105
that more than 75% of clear cell sarcomas contain a chromosome

translocation, t(12;22)(q13;q12), that has never been reported in
cutaneous melanoma. The t(12;22) translocation fuses the ATF1 gene on
chromosome 12 with the EWS gene on chromosome 22 (21,22). ATF1
encodes a transcription factor, and the biological implications of the
translocation are probably similar to those - as discussed below - in
Ewings sarcoma translocations. The clear cell sarcoma t(12;22)
translocation can be detected by karyotyping or in situ hybridization,
whereas the associated ATF1-EWS fusion can be detected conveniently
by reverse transcriptase PCR.
4.3 Desmoplastic Small Round Cell Tumor Is PDGFRA

a Target?
Desmoplastic small round cell tumors are aggressive and

chemotherapy-resistant neoplasms that arise predominantly, but not
exclusively, from intraabdominal soft tissues (23). These tumors are
composed of undifferentiated malignant small round cells within a florid
desmoplastic reaction (23), and virtually all cases express an EWS-WT1
fusion oncogene (24,25). The EWS-WT1 fusion oncogene results from
translocation between the chromosome 11 short arm and the chromosome
22 long arm, juxtaposing the WT1 (Wilms tumor) and EWS (Ewings)
genes, respectively (26,27). Notably, the EWS-WT1 oncoprotein,
expressed in the neoplastic small round cells, is a transcriptional regulator
that induces expression of platelet derived growth factor alpha (PDGFA)
(28). PDGFA binds and activates both PDGFRbeta and PDGFRalpha,
serving to activate potent mitogenic signaling pathways in fibroblasts
(29). Therefore, it is likely that oncogenically induced PDGFA
expression contributes to the prominent desmoplastic reaction in these
tumors. However, it is unclear whether PDGFA also serves an autocrine
or paracrine function in directly stimulating growth of the neoplastic
cells. Irrespective, these observations have fueled hopes that therapeutic
inhibition of PDGFRA (e.g. with imatinib) might benefit patients with
this highly lethal disease.
4.4 Ewings Sarcoma
Most Ewings sarcomas and peripheral primitive neuroectodermal

tumors contain chromosome translocations involving the Ewings
sarcoma gene, EWS. More than 80% of Ewing tumors contain a
cytogenetic translocation in which material is exchanged between the
long arms of chromosomes 11 and 22, resulting in oncogenic fusion of
106
the chromosome 11 FLI1 gene with the chromosome 22 EWS gene (30-
33). FLI1 encodes a transcription factor belonging to the ETS family of
transcription factors, and the oncogenic EWS-FLI1 fusion gene encodes
an activated version of this transcription factor. A smaller subset of
Ewing tumors, perhaps 5-15% of the total, have variant translocations in
which the EWS gene is fused with other ETS family transcription factor
genes (Table 1) (34-38). The Ewings gene translocations are considered
to be essential genetic aberrations because they are found in virtually all
cases and are assumed to be the critical genetic aberration in these
tumors. However, targeted therapies for the EWS oncoproteins are still
developmental, with no substantial clinical responses in early studies of
vaccine therapies to EWS (39). Alternate targets might be found amongst
the kinase protein family, but thus far have not shown great promise in
preclinical studies (40-42).
4.5 Dermatofibrosarcoma protuberans Activated

PDGFRB
The typical cytogenetic abnormality in dermatofibrosarcoma

protuberans (DFSP) is a ring chromosome composed of sequences from
chromosomes 17 and 22 (43,44). The DFSP ring chromosomes contain
multiple copies of a fusion gene, COL1A1-PDGFB, in which COL1A1 (a
collagen gene) is contributed by chromosome 17 and PDGFB (platelet
derived growth factor beta gene) by chromosome 22 (2,45). The
diagnostic COL1A1-PDGFB oncogene fusion can be demonstrated by
fluorescence in situ hybridization or reverse transcriptase PCR.
Occasional dermatofibrosarcoma protuberans have balanced t(17;22)
translocations - associated with the COL1A1 -PDGFB fusion gene - rather
than the usual ring chromosomes. Irrespective of the cytogenetic
mechanism, the COL1A1-PDGFB oncogene results in overexpression of
PDGFB, which is a growth factor that activates platelet derived growth
factor receptor beta (PDGFRB) and platelet derived growth factor
receptor alpha (PDGFRA). This observation suggested the possibility
that patients with inoperable DFSP might benefit from treatment with
PDGFR inhibitors, e.g. imatinib, and this hypothesis has been confirmed
by striking clinical responses in several patients (46,47).
4.6 Desmoid tumors APC and beta-catenin
Deep fibromatoses (desmoid tumors) can contain various

cytogenetic or molecular aberrations, including APC (adenomatous
polyposis coli) and beta-catenin mutations (48,49), and trisomies for
chromosomes 8 or 20 (50). Cytogenetic deletions of the chromosome 5
107
long arm are seen in occasional desmoids, resulting in loss of the APC
tumor suppressor gene, and the remaining APC allele in these cases is
typically inactivated by a point mutation (51,52). The most common
known mutations in sporadic desmoid tumors are activating beta-catenin
mutations, which were demonstrated in 22 of 42 desmoids by Alman et
al. (48,49). These mutations result in stabilization, and resultant
overexpression, of the beta-catenin protein. Therefore, it is likely that
targeted therapies of the Wnt-APC-beta-catenin pathway, will be useful
in treatment of patients with advanced desmoid tumors. Notably,
PDGFRB activation plays a highly mitogenic role in myofibroblasts, and
this knowledge has led to therapeutic evaluation of PDGFRB inhibition
(by imatinib) with promising preliminary results (53). It is unkown
presently whether PDGFRB activation in desmoids results from Wnt-
APC-beta-catenin pathway perturbations, or whether this is an unrelated
biological mechanism.
4.7 Fibrosarcoma (Infantile/Congenital) and Mesoblastic

Nephroma Activated NRTK3/TRKC
Trisomies of chromosomes 8, 11, 17 and 20 are characteristic

aberrations in infantile fibrosarcomas (54). It is interesting that one or
more of this same group of trisomies is also found in the cellular variant
of mesoblastic nephroma, which is an infantile renal tumor having
substantial histological overlap with infantile fibrosarcoma (55). In
addition, most infantile fibrosarcomas and cellular mesoblastic
nephromas contain the same diagnostic chromosome translocation,
t(12;15)(p13;q26) (56-58). This translocation results in fusion of the
chromosome 12 ETV6 (also known as TEL) gene with the chromosome
15 NTRK3 (also known as TRKC) gene. The t(12;15) translocation is
difficult to detect by conventional cytogenetic banding approaches, but is
demonstrated readily by FISH or reverse transcriptase PCR (56-58).
From a clinical standpoint, infantile fibrosarcomas and cellular
mesoblastic nephromas are undifferentiated and mitotically active tumors
that nonetheless have an excellent prognosis after excisional biopsy
(59,60). Therefore, these tumors appear to be closely related at the
pathogenetic, morphologic, and clinical levels, perhaps representing a
single neoplastic entity, arising in either renal or soft tissue locations.
Most infantile fibrosarcomas and cellular mesoblastic nephromas are
cured by surgical excision, and those that are inoperable respond well to
cytotoxic therapies. A small minority of cases progress, and can be lethal,
on conventional therapies, and development of NTRK3 kinase inhibitors
would doubtless be highly useful for this subgroup.
108
4.8 Inflammatory Myofibroblastic Tumor Activated

ALK
Inflammatory myofibroblastic tumor (also known as
inflammatory pseudotumor) is composed of fascicles of bland
myofibroblastic cells admixed with a prominent inflammatory infiltrate.
The inflammatory component of these tumors is reactive (and therefore
normal cytogenetically), whereas the myofibroblastic cells contain clonal
chromosome aberrations (61-63). A subset of inflammatory
myofibroblastic tumors have cytogenetic rearrangements that activate the
ALK receptor tyrosine kinase gene on chromosome 2 (64). This is the
same gene activated, typically by fusion with the NPM gene on
chromosome 5, in many anaplastic large cell lymphomas. Inflammatory
myofibroblastic tumors with ALK gene rearrangements express the ALK
protein strongly, and can be identified by immunohistochemical staining
for ALK in tumor paraffin sections, or by FISH to demonstrate the ALK
gene rearrangement. Therapeutic ALK inhibition would be a useful
therapeutic adjunct in patients with oncogenic ALK activation,
particularly those with extensive disease, where local control is
occasionally difficult.
4.9 Gastrointestinal Stromal Tumors Activated KIT

and PDGFRA
The oncogenic molecular mutations in KIT and PDGFRA are

discussed extensively in other chapters, and will not be detailed here. In
brief, most gastrointestinal stromal tumors display strong
immunostaining for the KIT receptor tyrosine kinase protein, and contain
activating mutations of the KIT or PDGFRA oncogenes (3,65,66). These
mutations have been targeted with spectacular success using the KIT
inhibitor, imatinib (Gleevec) (67,68). In addition, germline (inherited)
KIT mutations are responsible for rare syndromes of familial, multifocal,
gastrointestinal stromal tumors (69). Both germline and somatic KIT
aberrations are point mutations that are inevident at the cytogenetic level
of resolution. However, gastrointestinal stromal tumors have distinctive
karyotypes that generally include deletion of chromosomes 14 and 22
(66,70). Less often, gastrointestinal stromal tumors contain deletions of
the chromosome 1, 9 and 11 short arms (Table 1). KIT or PDGFRA
activation result from early and even initiating mutational events in
gastrointestinal stromal tumors, and these kinase activations likely
stimulate proliferation of the neoplastic progenitor cell. Subsequently,
the various cytogenetic aberrations are acquired, which presumably drive
the progression of the tumor from benign to malignant GIST (71).
109
4.10 Malignant Peripheral Nerve Sheath Tumors Ras

pathway
Benign and malignant peripheral nerve sheath tumors are seen

with greatly increased frequency in patients with the hereditary
neurofibromatosis syndromes. These are the most common tumor
predisposition syndromes, affecting 1 in 3500 individuals worldwide.
Neurofibromas and malignant peripheral nerve sheath tumors are
common in individuals with neurofibromatosis type 1, whereas benign
schwannomas are associated with neurofibromatosis type 2 (central
neurofibromatosis). Characterization of the neurofibromatosis syndrome
genes has shed substantial light on the pathogenesis of peripheral nerve
sheath tumors. The neurofibromatosis type 1 (NF1) and type 2 (NF2)
genes are located on chromosomes 17 and 22, respectively, and both of
these genes encode tumor suppressor proteins that normally constrain cell
proliferation (72-77). Malignant peripheral nerve sheath tumors
(MPNST) often have deletions of the NF1 gene, which can be
demonstrated by FISH assays. The NF1 gene aberrations are
accompanied by a generally complex karyotype, suggesting that genetic
instability plays a prominent role in the development of MPNST (Figure).
Notably, NF1 gene deletions can also be shown in the Schwann cell
component of neurofibromas (78). This observation supports the view
that neurofibromas are clonal schwannian neoplasms, whereas the other
admixed cell lineages - including fibroblasts, mast cells, and perineural
cells - are reactive.
The NF1 gene encodes a large protein which is related to the

p120 Ras GTPase Activating Protein (Ras-GAP) (79,80). Ras-GAP
proteins diminish signaling through the Ras pathway by stimulating the
hydrolysis of GTP to GDP, and resulting in conversion of the active Ras-
GTP complex to the inactive Ras-GDP complex. The tumorigenic role of
this function is highlighted by the finding that a subset of familial NF1
mutations are point mutations resulting in loss of Ras binding or Ras-
GAP activity. The roles of NF1 in suppressing Ras activation, and the
loss of this normal role in MPNST, suggest that inhibition of the Ras
pathway might be beneficial in patients with MPNST, and in other NF1-
associated cancers. One potential site of intervention is Ras itself, which
can be targeted with farnesyl protein transferase inhibitors (FTIs). FTIs
are peptidomimetics which inhibit the farnesylation of ras and thus
prevent the proper trafficking of Ras to the plasma membrane (81).
Signaling pathways downstream of ras offer alternative targets for
intervention, including members of the RAF, MEK and MAPK kinase
families (82).
110
4.11 Rhabdoid Tumor
Most malignant rhabdoid tumors - whether arising in soft tissues,

kidney, or the central nervous system - are characterized by deletions of
the chromosome 22 long arm. The chromosome 22 deletions target a
tumor suppressor gene, INI1 (also known as SNF5, or SMARCB1, which
encodes a protein involved in chromatin remodeling (83,84). The
rhabdoid tumor karyotypic profile is quite characteristic inasmuch as the
chromosome 22 deletion is often the only detectable cytogenetic
aberration, suggesting that INI1 inactivation is a relatively early event in
rhabdoid tumorigenesis. Additional evidence of an essential tumorigenic
role includes the finding of germline INI1 mutations in some individuals
with rhabdoid tumors (84,85), and the predictable development of
rhabdoid tumors in mice with inactivating INI1 mutations (86). These
observations suggest that novel therapies targeting the INI1 pathway
might be very effective in patients with rhabdoid tumors .
4.12 Rhabdomyosarcoma
Cytogenetic analyses have been useful in reaffirming the distinct

nature of embryonal and alveolar forms of rhabdomyosarcomas.
Alveolar rhabdomyosarcomas are characterized by reciprocal
chromosome translocations involving the FKHR (Forkhead transcription
factor) gene on chromosome 13. In most alveolar rhabdomyosarcomas,
the FKHR gene is fused with the PAX3 gene on chromosome 2 (87-89),
but a minority of cases contain fusions of FKHR with the PAX7 gene on
chromosome 1 (90). FKHR, PAX3, and PAX7 encode transcription
factors, and the PAX3-FKHR and PAX7-FKHR fusion oncogenes encode
activated forms of those transcription factors (91,92). By contrast,
embryonal rhabdomyosarcomas typically lack FKHR translocations, but
in experimental models can result from activation of various receptor
tyrosine kinase proteins, including MET and ERBB2 (93,94), and can
respond to inhibition of other receptor tyrosine kinase proteins,
particularly IGF1R (95,96). Therefore, it is possible that therapeutic
inhibition of these tyrosine kinase proteins might be useful clinically in
patients with rhabdomyosarcoma.
4.13 Synovial sarcoma
Most synovial sarcomas contain a reciprocal translocation of

chromosomes X and 18, t(X;18)(p11;q11), resulting in oncogenic fusion
between one of several SSX family genes on chromosome X, and the
SYT (SS18) gene on chromosome 18 (97). The t(X; 18) translocation is
found in more than 90% of synovial sarcomas, but is not found in
potential histologic mimics such as hemangiopericytoma, mesothelioma,
leiomyosarcoma, or malignant peripheral nerve sheath tumor. There are
no therapies as of yet which reproducibly inhibit the t(X;18) oncogenic
mechanism, or the associated SYT-SSX fusion oncoproteins. In a
111
preliminary report, Matsuzaki et al. described temporary stabilization of

synovial sarcoma pulmonary metastases following immunotherapy with
autologous dendritic cells that had been exposed to the SYT-SSX2
junctional region (98). Microarray expression profiling has shown higher
ERBB2 (her2/neu) expression in synovial sarcoma compared to other soft
tissue sarcomas, and the ERBB2 expression is found predominantly in the
glandular components of the synovial sarcomas (99). Based on these
findings, Allander et al. have suggested that ERBB2 might provide a
therapeutic target in synovial sarcoma (99).
5 REFERENCES
1. Greco, A., Roccato, E., Miranda, C., Cleris, L., Formelli, F., and Pierotti, M. A.
Growth-inhibitory effect of STI571 on cells transformed by the COL1A1/PDGFB
rearrangement. Int. J. Cancer, 92: 354-360, 2001.
2. OBrien, K. P., Seroussi, E., Dal Cin, P., Sciot, R., Mandahl, N., Fletcher, J. A., Turc-
Carel, C., and Dumanski, J. P. Various regions within the alpha-helical domain of the
COL1A1 gene are fused to the second exon of the PDGFB gene in dermatofibrosarcomas
and giant-cell fibroblastomas. Genes Chromosomes. Cancer, 23: 187-193, 1998.
3. Hirota, S., Isozaki, K., Moriyama, Y., Hashimoto, K., Nishida, T., Ishiguro, S.,
Kawano, K., Hanada, M., Kurata, A., Takeda, M., Muhammad Tunio, G., Matsuzawa, Y.,
Kanakura, Y., Shinomura, Y., and Kitamura, Y. Gain-of-function mutations of c-kit in
human gastrointestinal stromal tumors. Science, 279: 577-580, 1998.
4. Sandberg, A. A., Turc-Carel, C., and Gemmill, R. M. Chromosomes in solid tumors
and beyond. Cancer Res., 48: 1049-1059, 1988.
5. Fletcher, J. A., Kozakewich, H. P., Hoffer, F. A., Lage, J. M., Weidner, N., Tepper, R.,
Pinkus, G. S., Morton, C. C., and Corson, J. M. Diagnostic relevance of clonal
cytogenetic aberrations in malignant soft-tissue tumors. N. Engl. J. Med., 324: 436-442,
1991.
6. Sandberg, A. A. and Bridge, J. A. The Cytogenetics of Bone and Soft Tissue
Tumors.Austin: R.G. Landes Company, 1995.
7. Heim, S. and Mitelman, F. Cancer Cytogenetics, Second ed.New York: Wiley-Liss,
1995.
8. Sreekantaiah, C., Ladanyi, M., Rodriguez, E., and Chaganti, R. S. Chromosomal
aberrations in soft tissue tumors. Relevance to diagnosis, classification, and molecular
mechanisms. Am. J. Pathol., 144: 1121-1134, 1994.
9. Tontonoz, P., Singer, S., Forman, B. M., Sarraf, P., Fletcher, J. A., Fletcher, C. D.,
Brun, R. P., Mueller, E., Altiok, S., Oppenheim, H., Evans, R. M., and Spiegelman, B. M.
Terminal differentiation of human liposarcoma cells induced by ligands for peroxisome
proliferator-activated receptor gamma and the retinoid X receptor. Proc. Natl. Acad. Sci.
U. S. A., 94: 237-241, 1997.
10. Demetri, G. D., Fletcher, C. D., Mueller, E., Sarraf, P., Naujoks, R., Campbell, N.,
Spiegelman, B. M., and Singer, S. Induction of solid tumor differentiation by the
peroxisome proliferator-activated receptor-gamma ligand troglitazone in patients with
liposarcoma. Proc. Natl. Acad. Sci. U. S. A, 96: 3951-3956, 1999.
11. Turc-Carel, C., Limon, J., Dal Cin, P., Rao, U., Karakousis, C., and Sandberg, A. A.
Cytogenetic studies of adipose tissue tumors. II. Recurrent reciprocal translocation
t(12;16)(q13;p11) in myxoid liposarcomas. Cancer Genet. Cytogenet., 23: 291-299, 1986.
12. Sreekantaiah, C., Karakousis, C. P., Leong, S. P., and Sandberg, A. A. Cytogenetic
findings in liposarcoma correlate with histopathologic subtypes. Cancer, 69: 2484-2495,
1992.
13. Fletcher, C. D., Akerman, M., Dal Cin, P., De Wever, I., Mandahl, N., Mertens, F.,
Mitelman, F., Rosai, J., Rydholm, A., Sciot, R., Tallini, G., Van Den Berghe, H., Van de
Ven, W., Vanni, R., and Willen, H. Correlation between clinicopathological features and
karyotype in lipomatous tumors. A report of 178 cases from the Chromosomes and
Morphology (CHAMP) Collaborative Study Group. Am. J. Pathol., 148: 623-630, 1996.
112
14. Hisaoka, M., Tsuji, S., Morimitsu, Y., Hashimoto, H., Shimajiri, S., Komiya, S., and
Ushijima, M. Detection of TLS/FUS-CHOP fusion transcripts in myxoid and round cell
liposarcomas by nested reverse transcription-polymerase chain reaction using archival
paraffin-embedded tissues. Diagn. Mol. Pathol., 7: 96-101, 1998.
15. Kuroda, M., Ishida, T., Horiuchi, H., Kida, N., Uozaki, H., Takeuchi, H., Tsuji, K.,
Imamura, T., Mori, S., and Machinami, R. Chimeric TLS/FUS-CHOP gene expression
and the heterogeneity of its junction in human myxoid and round cell liposarcoma. Am. J.
Pathol., 147: 1221-1227, 1995.
16. Aman, P., Ron, D., Mandahl, N., Fioretos, T., Heim, S., Arheden, K., Willen, H.,
Rydholm, A., and Mitelman, F. Rearrangement of the transcription factor gene CHOP in
myxoid liposarcomas with t(12;16)(q13;p11). Genes Chromosom. Cancer, 5: 278-285,
1992.
17. Crozat, A., Aman, P., Mandahl, N., and Ron, D. Fusion of CHOP to a novel RNA-
binding protein in human myxoid liposarcoma. Nature, 363: 640-644, 1993.
18. Mandahl, N., Heim, S., Arheden, K., Rydholm, A., Willen, H., and Mitelman, F.
Rings, dicentrics, and telomeric association in histiocytomas. Cancer Genet. Cytogenet.,
30: 23-33, 1988.
19. Turc-Carel, C., Dal Cin, P., Rao, U., Karakousis, C., and Sandberg, A. A. Recurrent
breakpoints at 9q31 and 22q12.2 in extraskeletal myxoid chondrosarcoma. Cancer Genet.
Cytogenet., 30: 145-150, 1988.
20. Pedeutour, F., Forus, A., Coindre, J. M., Berner, J. M., Nicolo, G., Michiels, J. F.,
Terrier, P., Ranchere-Vince, D., Collin, F., Myklebost, O., and Turc-Carel, C. Structure of
the supernumerary ring and giant rod chromosomes in adipose tissue tumors. Genes
Chromosomes. Cancer, 24: 30-41, 1999.
21. Zucman, J., Delattre, O., Desmaze, C., Epstein, A. L., Stenman, G., Speleman, F.,
Fletchers, C. D., Aurias, A., and Thomas, G. EWS and ATF-1 gene fusion induced by
t(12;22) translocation in malignant melanoma of soft parts. Nat. Genet., 4: 341-345, 1993.
22. Brown, A. D., Lopez-Terrada, D., Denny, C., and Lee, K. A. Promoters containing
ATF-binding sites are de-regulated in cells that express the EWS/ATF1 oncogene.
Oncogene, 10: 1749-1756, 1995.
23. Gerald, W. L., Miller, H. K., Battifora, H., Miettinen, M., Silva, E. G., and Rosai, J.
Intra-abdominal desmoplastic small round-cell tumor. Report of 19 cases of a distinctive
type of high-grade polyphenotypic malignancy affecting young individuals. Am. J. Surg.
Pathol., 15: 499-513, 1991.
24. Ladanyi, M. and Gerald, W. Fusion of the EWS and WT1 genes in the desmoplastic
small round cell tumor. Cancer Res., 54: 2837-2840, 1994.
25. Gerald, W. L., Ladanyi, M., de Alava, E., Cuatrecasas, M., Kushner, B. H.,
LaQuaglia, M. P., and Rosai, J. Clinical, pathologic, and molecular spectrum of tumors
associated with t(11;22)(p13;q12): desmoplastic small round-cell tumor and its variants. J.
Clin. Oncol., 16: 3028-3036, 1998.
26. Rodriguez, E., Sreekantaiah, C., Gerald, W., Reuter, V. E., Motzer, R. J., and
Chaganti, R. S. A recurring translocation, t(11;22)(p13;q11.2), characterizes intra-
abdominal desmoplastic small round-cell tumors. Cancer Genet. Cytogenet., 69: 17-21,
1993.
27. Biegel, J. A., Conard, K., and Brooks, J. J. Translocation (11;22)(p13;q12): primary
change in intraabdominal desmoplastic small round cell tumor. Genes Chromosom.
Cancer, 7: 119-121, 1993.
28. Lee, S. B., Kolquist, K. A., Nichols, K., Englert, C., Maheswaran, S., Ladanyi, M.,
Gerald, W. L., and Haber, D. A. The EWS-WT1 translocation product induces PDGFA in
desmoplastic small round-cell tumour. Nat. Genet., 17: 309-313, 1997.
29. Kelly, J. D., Haldeman, B. A., Grant, F. J., Murray, M. J., Seifert, R. A., Bowen-Pope,
D. F., Cooper, J. A., and Kazlauskas, A. Platelet-derived growth factor (PDGF) stimulates
PDGF receptor subunit dimerization and intersubunit trans-phosphorylation. J. Biol.
Chem., 266: 8987-8992, 1991.
30. Delattre, O., Zucman, J., Plougastel, B., Desmaze, C., Melot, T., Peter, M., Kovar, H.,
Joubert, I., de Jong, P., Rouleau, G., and et al Gene fusion with an ETS DNA-binding
domain caused by chromosome translocation in human tumours. Nature, 359: 162-165,
1992.
113
31. Turc-Carel, C., Philip, I., Berger, M. P., Philip, T., and Lenoir, G. M. Chromosome
study of Ewings sarcoma (ES) cell lines. Consistency of a reciprocal translocation
t(11;22)(q24;q12). Cancer Genet. Cytogenet., 12: 1-19, 1984.
32. Turc-Carel, C., Aurias, A., Mugneret, F., Lizard, S., Sidaner, I., Volk, C., Thiery, J. P.,
Olschwang, S., Philip, I., Berger, M. P., and et al Chromosomes in Ewings sarcoma. I. An
evaluation of 85 cases of remarkable consistency of t(11;22)(q24;q12). Cancer Genet.
Cytogenet., 32: 229-238, 1988.
33. Ewen, M. E., Ludlow, J. W., Marsilio, E., DeCaprio, J. A., Millikan, R. C., Cheng, S.
H., Paucha, E., and Livingston, D. M. An N-terminal transformation-governing sequence
of SV40 large T antigen contributes to the binding of both p110Rb and a second cellular
protein, p120. Cell, 58: 257-267, 1989.
34. Buckler, A. J., Chang, D. D., Graw, S. L., Brook, J. D., Haber, D. A., Sharp, P. A.,
and Housman, D. E. Exon amplification: a strategy to isolate mammalian genes based on
RNA splicing. Proc. Natl. Acad. Sci. U. S. A., 88: 4005-4009, 1991.
35. Jeon, I. S., Davis, J. N., Braun, B. S., Sublett, J. E., Roussel, M. F., Denny, C. T., and
Shapiro, D. N. A variant Ewings sarcoma translocation (7;22) fuses the EWS gene to the
ETS gene ETV1. Oncogene, 10: 1229-1234, 1995.
36. Peter, M., Couturier, J., Pacquement, H., Michon, J., Thomas, G., Magdelenat, H., and
Delattre, O. A new member of the ETS family fused to EWS in Ewing tumors. Oncogene,
14: 1159-1164, 1997.
37. Kaneko, Y., Yoshida, K., Handa, M., Toyoda, Y., Nishihira, H., Tanaka, Y., Sasaki,
Y., Ishida, S., Higashino, F., and Fujinaga, K. Fusion of an ETS-family gene, EIAF, to
EWS by t(17;22)(q12;q12) chromosome translocation in an undifferentiated sarcoma of
infancy. Genes Chromosomes. Cancer, 15: 115-121, 1996.
38. Ishida, S., Yoshida, K., Kaneko, Y., Tanaka, Y., Sasaki, Y., Urano, F., Umezawa, A.,
Hata, J., and Fujinaga, K. The genomic breakpoint and chimeric transcripts in the
EWSR1- ETV4/E1AF gene fusion in Ewing sarcoma. Cytogenet. Cell Genet., 82: 278-
283, 1998.
39. Dagher, R., Long, L. M., Read, E. J., Leitman, S. F., Carter, C. S., Tsokos, M., Goletz,
T. J., Avila, N., Berzofsky, J. A., Helman, L. J., and Mackall, C. L. Pilot trial of tumor-
specific peptide vaccination and continuous infusion interleukin-2 in patients with
recurrent Ewing sarcoma and alveolar rhabdomyosarcoma: an inter-institute NIH study.
Med. Pediatr. Oncol., 38: 158-164, 2002.
40. Hotfilder, M., Lanvers, C., Jurgens, H., Boos, J., and Vormoor, J. c-KIT-expressing
Ewing tumour cells are insensitive to imatinib mesylate (STI571). Cancer Chemother.
Pharmacol., 50: 167-169, 2002.
41. Scotlandi, K., Manara, M. C., Strammiello, R., Landuzzi, L., Benini, S., Perdichizzi,
S., Serra, M., Astolfi, A., Nicoletti, G., Lollini, P. L., Bertoni, F., Nanni, P., and Picci, P.
C-kit receptor expression in Ewings sarcoma: lack of prognostic value but therapeutic
targeting opportunities in appropriate conditions. J. Clin. Oncol., 21: 1952-1960, 2003.
42. Ye, D., Maitra, A., Timmons, C. F., Leavey, P. J., Ashfaq, R., and Ilaria Jr, R. L. The
Epidermal Growth Factor Receptor HER2 Is Not a Major Therapeutic Target in Ewing
Sarcoma. J. Pediatr. Hematol. Oncol., 25: 459-466, 2003.
43. Pedeutour, F., Simon, M. P., Minoletti, F., Sozzi, G., Pierotti, M. A., Hecht, F., and
Turc-Carel, C. Ring 22 chromosomes in dermatofibrosarcoma protuberans are low- level
amplifiers of chromosome 17 and 22 sequences. Cancer Res., 55: 2400-2403, 1995.
44. Naeem, R., Lux, M. L., Huang, S. F., Naber, S. P., Corson, J. M., and Fletcher, J. A.
Ring chromosomes in dermatofibrosarcoma protuberans are composed of interspersed
sequences from chromosomes 17 and 22. Am. J. Pathol., 147: 1553-1558, 1995.
45. Simon, M. P., Pedeutour, F., Sirvent, N., Grosgeorge, J., Minoletti, F., Coindre, J. M.,
Terrier-Lacombe, M. J., Mandahl, N., Craver, R. D., Blin, N., Sozzi, G., Turc-Carel, C.,
OBrien, K. P., Kedra, D., Fransson, I., Guilbaud, C., and Dumanski, J. P. Deregulation of
the platelet-derived growth factor B-chain gene via fusion with collagen gene COL1A1 in
dermatofibrosarcoma protuberans and giant-cell fibroblastoma. Nat. Genet., 15: 95-98,
1997.
46. Maki, R. G., Awan, R. A., Dixon, R. H., Jhanwar, S., and Antonescu, C. R.
Differential sensitivity to imatinib of 2 patients with metastatic sarcoma arising from
dermatofibrosarcoma protuberans. Int. J. Cancer, 100: 623-626, 2002.
114
47. Rubin, B. P., Schuetze, S. M., Eary, J. F., Norwood, T. H., Mirza, S., Conrad, E. U.,
and Bruckner, J. D. Molecular targeting of platelet-derived growth factor B by imatinib
mesylate in a patient with metastatic dermatofibrosarcoma protuberans. J. Clin. Oncol.,
20: 3586-3591, 2002.
48. Alman, B. A., Li, C., Pajerski, M. E., Diaz-Cano, S., and Wolfe, H. J. Increased beta-
catenin protein and somatic APC mutations in sporadic aggressive fibromatoses (desmoid
tumors). Am. J. Pathol, 151: 329-334, 1997.
49. Tejpar, S., Nollet, F., Li, C., Wunder, J. S., Michils, G., Dal Cin, P., Van Cutsem, E.,
Bapat, B., van Roy, F., Cassiman, J. J., and Alman, B. A. Predominance of beta-catenin
mutations and beta-catenin dysregulation in sporadic aggressive fibromatosis (desmoid
tumor). Oncogene, 18: 6615-6620, 1999.
50. Fletcher, J. A., Naeem, R., Xiao, S., and Corson, J. M. Chromosome aberrations in
desmoid tumors. Trisomy 8 may be a predictor of recurrence. Cancer Genet. Cytogenet.,
79: 139-143, 1995.
51. Miyaki, M., Konishi, M., Kikuchi-Yanoshita, R., Enomoto, M., Tanaka, K.,
Takahashi, H., Muraoka, M., Mori, T., Konishi, F., and Iwama, T. Coexistence of somatic
and germ-line mutations of APC gene in desmoid tumors from patients with familial
adenomatous polyposis. Cancer Res., 53: 5079-5082, 1993.
52. Sen-Gupta, S., Van der Luijt, R. B., Bowles, L. V., Meera Khan, P., and Delhanty, J.
D. Somatic mutation of APC gene in desmoid tumour in familial adenomatous polyposis.
Lancet, 342: 552-553, 1993.
53. Mace, J., Sybil, B. J., Sondak, V., McGinn, C., Hayes, C., Thomas, D., and Baker, L.
Response of extraabdominal desmoid tumors to therapy with imatinib mesylate. Cancer,
95: 2373-2379, 2002.
54. Schofield, D. E., Fletcher, J. A., Grier, H. E., and Yunis, E. J. Fibrosarcoma in infants
and children. Application of new techniques. Am. J. Surg. Pathol., 18: 14-24, 1994.
55. Schofield, D. E., Yunis, E. J., and Fletcher, J. A. Chromosome aberrations in
mesoblastic nephroma. Am. J. Pathol., 143: 714-724, 1993.
56. Knezevich, S. R., McFadden, D. E., Tao, W., Lim, J. F., and Sorensen, P. H. A novel
ETV6-NTRK3 gene fusion in congenital fibrosarcoma. Nat. Genet., 18: 184-187, 1998.
57. Rubin, B. P., Chen, C. J., Morgan, T. W., Xiao, S., Grier, H. E., Kozakewich, H. P.,
Perez-Atayde, A. R., and Fletcher, J. A. Congenital mesoblastic nephroma t(12;15) is
associated with ETV6- NTRK3 gene fusion: cytogenetic and molecular relationship to
congenital (infantile) fibrosarcoma. Am. J. Pathol., 153: 1451-1458, 1998.
58. Knezevich, S. R., Garnett, M. J., Pysher, T. J., Beckwith, J. B., Grundy, P. E., and
Sorensen, P. H. ETV6-NTRK3 gene fusions and trisomy 11 establish a histogenetic link
between mesoblastic nephroma and congenital fibrosarcoma. Cancer Res., 58: 5046-5048,
1998.
59. Bolande, R. P. Congenital mesoblastic nephroma of infancy. Perspect. Pediatr.
Pathol., 1: 227-250, 1973.
60. Chung, E. B. and Enzinger, F. M. Infantile fibrosarcoma. Cancer, 38: 729-739, 1976.
61. Treissman, S. P., Gillis, D. A., Lee, C. L., Giacomantonio, M., and Resch, L.
Omental-mesenteric inflammatory pseudotumor. Cytogenetic demonstration of genetic
changes and monoclonality in one tumor. Cancer, 73: 1433-1437, 1994.
62. Snyder, C. S., DellAquila, M., Haghighi, P., Baergen, R. N., Suh, Y. K., and Yi, E. S.
Clonal changes in inflammatory pseudotumor of the lung: a case report. Cancer, 76: 1545-
1549, 1995.
63. Su, L. D., Atayde-Perez, A., Sheldon, S., Fletcher, J. A., and Weiss, S. W.
Inflammatory myofibroblastic tumor: cytogenetic evidence supporting clonal origin. Mod.
Pathol., 11: 364-368, 1998.
64. Lawrence, B., Perez-Atayde, A., Hibbard, M. K., Rubin, B. P., Dal Cin, P., Pinkus, J.
L., Pinkus, G. S., Xiao, S., Yi, E. S., Fletcher, C. D., and Fletcher, J. A. TPM3-ALK and
TPM4-ALK oncogenes in inflammatory myofibroblastic tumors. Am. J. Pathol, 157: 377-
384, 2000.
65. Rubin, B. P., Singer, S., Tsao, C., Duensing, A., Lux, M. L., Ruiz, R., Hibbard, M. K.,
Chen, C. J., Xiao, S., Tuveson, D. A., Demetri, G. D., Fletcher, C. D., and Fletcher, J. A.
KIT Activation Is a Ubiquitous Feature of Gastrointestinal Stromal Tumors. Cancer Res.,
61: 8118-8121, 2001.
66. Heinrich, M. C., Corless, C. L., Duensing, A., McGreevey, L., Chen, C. J., Joseph, N.,
Singer, S., Griffith, D. J., Haley, A., Town, A., Demetri, G. D., Fletcher, C. D., and
115
Fletcher, J. A. PDGFRA activating mutations in gastrointestinal stromal tumors. Science,

299: 708-710, 2003.
67. Demetri, G. D., von Mehren, M., Blanke, C. D., Van den Abbeele, A. D., Eisenberg,
B., Roberts, P. J., Heinrich, M. C., Tuveson, D. A., Singer, S., Janicek, M., Fletcher, J. A.,
Silverman, S. G., Silberman, S. L., Capdeville, R., Kiese, B., Peng, B., Dimitrijevic, S.,
Druker, B. J., Corless, C., Fletcher, C. D., and Joensuu, H. Efficacy and safety of imatinib
mesylate in advanced gastrointestinal stromal tumors. N. Engl. J. Med., 347: 472-480,
2002.
68. van Oosterom, A. T., Judson, I., Verweij, J., Stroobants, S., Donato, d. P.,
Dimitrijevic, S., Martens, M., Webb, A., Sciot, R., Van Glabbeke, M., Silberman, S., and
Nielsen, O. S. Safety and efficacy of imatinib (STI571) in metastatic gastrointestinal
stromal tumours: a phase I study. Lancet, 358: 1421-1423, 2001.
69. Nishida, T., Hirota, S., Taniguchi, M., Hashimoto, K., Isozaki, K., Nakamura, H.,
Kanakura, Y., Tanaka, T., Takabayashi, A., Matsuda, H., and Kitamura, Y. Familial
gastrointestinal stromal tumours with germline mutation of the KIT gene. Nat. Genet., 19:
323-324, 1998.
70. Sarlomo-Rikala, M., el-Rifai, W., Lahtinen, T., Andersson, L. C., Miettinen, M., and
Knuutila, S. Different patterns of DNA copy number changes in gastrointestinal stromal
tumors, leiomyomas, and schwannomas. Hum. Pathol., 29: 476-481, 1998.
71. Heinrich, M. C., Rubin, B. P., Longley, B. J., and Fletcher, J. A. Biology and genetic
aspects of gastrointestinal stromal tumors: KIT activation and cytogenetic alterations.
Hum. Pathol., 33: 484-495, 2002.
72. Legius, E., Marchuk, D. A., Collins, F. S., and Glover, T. W. Somatic deletion of the
neurofibromatosis type 1 gene in a neurofibrosarcoma supports a tumour suppressor gene
hypothesis. Nat. Genet., 3: 122-126, 1993.
73. Basu, T. N., Gutmann, D. H., Fletcher, J. A., Glover, T. W., Collins, F. S., and
Downward, J. Aberrant regulation of ras proteins in malignant tumour cells from type 1
neurofibromatosis patients. Nature, 356: 713-715, 1992.
74. DeClue, J. E., Papageorge, A. G., Fletcher, J. A., Diehl, S. R., Ratner, N., Vass, W. C.,
and Lowy, D. R. Abnormal regulation of mammalian p21ras contributes to malignant
tumor growth in von Recklinghausen (type 1) neurofibromatosis. Cell, 69: 265-273, 1992.
75. Rouleau, G. A., Merel, P., Lutchman, M., Sanson, M., Zucman, J., Marineau, C.,
Hoang-Xuan, K., Demczuk, S., Desmaze, C., Plougastel, B., and et al Alteration in a new
gene encoding a putative membrane-organizing protein causes neuro-fibromatosis type 2.
Nature, 363: 515-521, 1993.
76. Twist, E. C., Ruttledge, M. H., Rousseau, M., Sanson, M., Papi, L., Merel, P.,
Delattre, O., Thomas, G., and Rouleau, G. A. The neurofibromatosis type 2 gene is
inactivated in schwannomas. Hum. Mol. Genet., 3: 147-151, 1994.
77. Lutchman, M. and Rouleau, G. A. The neurofibromatosis type 2 gene product,
schwannomin, suppresses growth of NIH 3T3 cells. Cancer Res., 55: 2270-2274, 1995.
78. Kluwe, L., Friedrich, R., and Mautner, V.-F. Loss of NF1 allele in Schwann cells but
not in fibroblasts dervied from an NF1-associated neurofibroma. Genes Chromosomes
Cancer, 24: 283-285, 1999.
79. Xu, G. F., Lin, B., Tanaka, K., Dunn, D., Wood, D., Gesteland, R., White, R., Weiss,
R., and Tamanoi, F. The catalytic domain of the neurofibromatosis type 1 gene product
stimulates ras GTPase and complements ira mutants of S. cerevisiae. Cell, 63: 835-841,
1990.
80. Xu, G. F., OConnell, P., Viskochil, D., Cawthon, R., Robertson, M., Culver, M.,
Dunn, D., Stevens, J., Gesteland, R., White, R., and et al The neurofibromatosis type 1
gene encodes a protein related to GAP. Cell, 62: 599-608, 1990.
81. Cox, A. D. and Der, C. J. Farnesyltransferase inhibitors and cancer treatment:
targeting simply Ras? Biochim. Biophys. Acta, 1333: F51-F71, 1997.
82. Cox, A. D. and Der, C. J. Ras family signaling: therapeutic targeting. Cancer Biol.
Ther., 1: 599-606, 2002.
83. Versteege, I., Sevenet, N., Lange, J., Rousseau-Merck, M. F., Ambros, P.,
Handgretinger, R., Aurias, A., and Delattre, O. Truncating mutations of hSNF5/INI1 in
aggressive paediatric cancer. Nature, 394: 203-206, 1998.
116
84. Biegel, J. A., Zhou, J. Y., Rorke, L. B., Stenstrom, C., Wainwright, L. M., and
Fogelgren, B. Germ-line and acquired mutations of INI1 in atypical teratoid and rhabdoid
tumors. Cancer Res., 59: 74-79, 1999.
85. Sevenet, N., Sheridan, E., Amram, D., Schneider, P., Handgretinger, R., and Delattre,
O. Constitutional mutations of the hSNF5/INI1 gene predispose to a variety of cancers.
Am. J. Hum. Genet., 65: 1342-1348, 1999.
86. Roberts, C. W., Galusha, S. A., McMenamin, M. E., Fletcher, C. D., and Orkin, S. H.
Haploinsufficiency of Snf5 (integrase interactor 1) predisposes to malignant rhabdoid
tumors in mice. Proc. Natl. Acad. Sci. U. S. A, 97: 13796-13800, 2000.
87. Galili, N., Davis, R. J., Fredericks, W. J., Mukhopadhyay, S., Rauscher, F. J. 3.,
Emanuel, B. S., Rovera, G., Barr, F. G., and Rauscher, F. J. Fusion of a fork head domain
gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nat. Genet., 5: 230-235,
1993.
88. Barr, F. G., Galili, N., Holick, J., Biegel, J. A., Rovera, G., and Emanuel, B. S.
Rearrangement of the PAX3 paired box gene in the paediatric solid tumour alveolar
rhabdomyosarcoma. Nat. Genet., 3: 113-117, 1993.
89. Shapiro, D. N., Sublett, J. E., Li, B., Downing, J. R., and Naeve, C. W. Fusion of
PAX3 to a member of the forkhead family of transcription factors in human alveolar
rhabdomyosarcoma. Cancer Res., 53: 5108-5112, 1993.
90. Davis, R. J., DCruz, C. M., Lovell, M. A., Biegel, J. A., and Barr, F. G. Fusion of
PAX7 to FKHR by the variant t(1;13)(p36;q14) translocation in alveolar
rhabdomyosarcoma. Cancer Res., 54: 2869-2872, 1994.
91. Fredericks, W. J., Galili, N., Mukhopadhyay, S., Rovera, G., Bennicelli, J., Barr, F.
G., Rauscher, F. J. 3., and Rauscher, F. J. r. The PAX3-FKHR fusion protein created by
the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional
activator than PAX3. Mol. Cell Biol., 15: 1522-1535, 1995.
92. Bennicelli, J. L., Fredericks, W. J., Wilson, R. B., Rauscher, F. J. 3., and Barr, F. G.
Wild type PAX3 protein and the PAX3-FKHR fusion protein of alveolar
rhabdomyosarcoma contain potent, structurally distinct transcriptional activation domains.
Oncogene, 11: 119-130, 1995.
93. Sharp, R., Recio, J. A., Jhappan, C., Otsuka, T., Liu, S., Yu, Y., Liu, W., Anver, M.,
Navid, F., Helman, L. J., DePinho, R. A., and Merlino, G. Synergism between
INK4a/ARF inactivation and aberrant HGF/SF signaling in rhabdomyosarcomagenesis.
Nat. Med., 8: 1276-1280, 2002.
94. Nanni, P., Nicoletti, G., De Giovanni, C., Croci, S., Astolfi, A., Landuzzi, L., Di
Carlo, E., Iezzi, M., Musiani, P., and Lollini, P. L. Development of Rhabdomyosarcoma
in HER-2/neu Transgenic p53 Mutant Mice. Cancer Res., 63: 2728-2732, 2003.
95. Kalebic, T., Tsokos, M., and Helman, L. J. In vivo treatment with antibody against
IGF-1 receptor suppresses growth of human rhabdomyosarcoma and down-regulates
p34cdc2. Cancer Res., 54: 5531-5534, 1994.
96. Kalebic, T., Blakesley, V., Slade, C., Plasschaert, S., Leroith, D., and Helman, L. J.
Expression of a kinase-deficient IGF-I-R suppresses tumorigenicity of
rhabdomyosarcoma cells constitutively expressing a wild type IGF-I-R. Int. J. Cancer, 76:
223-227, 1998.
97. Clark, J., Rocques, P. J., Crew, A. J., Gill, S., Shipley, J., Chan, A. M., Gusterson, B.
A., and Cooper, C. S. Identification of novel genes, SYT and SSX, involved in the
t(X;18)(p11.2;q11.2) translocation found in human synovial sarcoma. Nat. Genet., 7: 502-
508, 1994.
98. Matsuzaki, A., Suminoe, A., Hattori, H., Hoshina, T., and Hara, T. Immunotherapy
with autologous dendritic cells and tumor-specific synthetic peptides for synovial
sarcoma. J. Pediatr. Hematol. Oncol., 24: 220-223, 2002.
99. Allander, S. V., Illei, P. B., Chen, Y., Antonescu, C. R., Bittner, M., Ladanyi, M., and
Meltzer, P. S. Expression profiling of synovial sarcoma by cDNA microarrays:
association of ERBB2, IGFBP2, and ELF3 with epithelial differentiation. Am. J. Pathol.,
161: 1587-1595, 2002.
Chapter 7
KIT and PDGF as targets
Jaap Verweij
Dept. of Medical Oncology, Erasmus University Medical Centre-Daniel den Hoed Cancer
Centre, Rotterdam, the Netherlands
Correspondence to:
Jaap Verweij, MD, PhD
Erasmus University Medical Centre-Daniel den Hoed Cancer Centre,
Groene Hilledijk 301
3075 EA Rotterdam, the Netherlands
118
1 INTRODUCTION
The development of one particular drug (STI571, Glivec,

Gleevec) paired with one specific discovery of a gain of function
receptor in Gastrointestinal stromal tumors has paved the way for a
completely new approach in the treatment of soft tissue sarcomas.
Gastrointestinal stromal tumors (GISTs) are rare diseases but
they constitute the most frequent sarcomas of the gastrointestinal tract. At
presentation, metastatic disease is found in up to half of patients [1].
Metastases are predominantly found in the liver and in the peritoneal
cavity. The annual incidence of this rare disease[2] is estimated at 10-20
per 1.000.000 [3, 4], yielding 5000-10000 new cases each year in both
the US A and Europe [5].
In 1983, Mazur and Clark introduced the term GIST to
describe non-epithelial neoplasms of the gastrointestinal tract that stained
positive for S100 protein, and showed neither smooth muscle nor
Schwann cell features [6]. Diagnostic criteria have recently been more
clearly outlined by a pathology consensus [4]. In this consensus, GISTs
have been defined as spindle-cell epitheloid, or occasionally
pleiomorphic mesenchymal tumors of the gastrointestinal tract that
express the KIT receptor kinase, except for rare cases [2, 4]. Of crucial
importance for our insight in molecular etiology, cellular origin,
classification and treatment approach of GIST was the discovery of the
gain-of-function mutations of the mentioned KIT receptor by Hirota et al
[7]. The KIT proto-oncogene contains 21 exons and is located on
chromosome segment 4q12 [8]. The frequency of gain-of-function
mutations in KIT is high [9, 10]. These mutations result in the permanent
activation of KIT signaling in the absence of binding of the stem cell
factor (SCF), which leads to uncontrolled cell proliferation and resistance
to apoptosis. The expression of the CD117 antigen, an epitope of the
transmembrane KIT protein, has emerged as the most important defining
feature for diagnosing GIST [11]. Staining positive for CD117 by
immunochemistry suggests a histiogenesis from the interstitial cells of
Cajal, the gastrointestinal pacemaker cells which control gut motility [12,
13].
Surgery is the standard of care for non-metastatic GIST.
Five-year survival after surgical resection ranges from 35-65% [14].
Once metastasized, prognosis becomes poor. Independent risk factors for
aggressive behavior following resection are: localization other than
stomach, tumor size > 5 cm), and a high mitotic count [15]. Metastatic
tumors are considered insensitive to conventional chemotherapy [15, 16]
and median survival for metastatic or inoperable cases ranges from 9-12
months [17, 18].
119
2 IMATINIB
As said, the development of a single drug, has been the backbone

for considerable changes and improvement in the therapeutic approach in
GIST.
Imatinib Mesylate (imatinib; STI571; Glivec , Gleevec) [19]
is a 2-phenylaminopyrimidine compound (Figure 1). Its molecular weight
is 589.7 g/mol. Imatinib is highly soluble in water (50 mg/l at pH 7.4)
[20]. About 90% of patients with metastatic or unresectable GIST, treated
with imatinib in a phase I study showed clinical benefit [21]. The
impressive response results obtained with imatinib have drastically
changed the management of patients with unresectable or advanced
GIST.
Imatinib is the first rationally designed competitive
inhibitor of several specific protein tyrosine kinases involved in signal
transduction pathways [22, 23]. Imatinib competes with ATP for its
specific binding site in the intracellular kinase domain, which inhibits the
ability of the kinase to transfer phosphate groups from ATP to tyrosine
residues on substrate proteins. By inhibiting substrate protein
phosphorylation, imatinib prevents transduction of signals. Thus, the
downstream signaling from the KIT-kinase that is activated by SCF or is
abnormally activated in GIST, is inactivated by imatinib, which switches
the fragile cell balance into apoptosis [24].
Imatinib inhibits is relatively selective and potently inhibits the

tyrosine kinase activity of the intracellular Abelson (ABL) protein, the
BCR-ABL fusion protein, the KIT protein, and the platelet-derived
growth factor receptor (PDGF-R) [21, 25-27]. BCR-ABL is a fusion
product of the Philadelphia chromosome which results from the
120
translocation t(9;22), and plays a critical role in the development of

chronic myelogenous leukemia (CML) [28], the disease for which
Imatinib was initially developed [29]. In a GIST-cell line (GIST882) with
a KIT-gene gain-of-function mutation, yielding constitutive activation of
KIT, imatinib was found to switch off KIT-activity at [30]. In
SCF dependent cell lines, imatinib inhibits KIT at an (inhibitory
concentration of 50% of the maximal effect) of
depending on the assay [25, 31, 32]. In view of the clinical activity in
CML [29, 33] confirming proof of concept in that disease, the
information that imatinib inhibits KIT [25] as well as proliferation of
GIST in vitro [30], and a proof of concept of the activity of imatinib in a
single GIST patient [34], imatinib was also developed in GIST [35].
3 PHARMACOKINETICS AND METABOLISM
When given orally, imatinib has a very good mean absolute

bioavailability of 98% [36]. Food does not affect uptake [37].
Pharmacokinetic profiles in GIST and CML patients are reported to be
similar [36, 38].
After a single 400 mg dose, the mean area under the plasma-
concentration curve approzimates 24.75 mg.h/mL at steady
state [38]. After one month, GIST patients receiving a 600 mg daily dose
showed at steady state a 1.2 fold higher AUC compared to patients
receiving 400 mg daily [39]. Mean imatinib AUC increases
proportionally with increasing doses up to 1000 mg. The estimated
elimination half-life of imatinib is 18-20 hours [36, 38, 39], indicating
that from this perspective once-daily oral dosing would be sufficient.
On repeated dosing clearance tended to be somewhat lower [40],
which may be related to improvement in albumin levels and higher
creatinine levels. After long-term treatment (3-12 months), imatinib
clearance seems to increase above the first day level [40], although the
available data are yet not conclusive. Liver impairment does not require
dose adaptation [41] while renal impairment does [43]. Importantly
patients homozygous for one of three determined single nucleotide
polymorphisms had a higher plasma AUC at steady state concentration
[44].
4 KIT-DRIVEN GIST
The development of Imatinib in KIT-driven GIST initially

involved parallel phase I and II studies. The European Organization for
Research and Treatment of Cancer (EORTC) Soft Tissue and Bone
Sarcoma Group performed a formal phase I study in which efficacy was a
secondary objective[21, 35]. Eligible patients had to show histological
evidence of soft tissue sarcoma, and GIST patients had to be positive for
KIT, as indicated by expression of CD117 on immunohistochemical
staining. This dose-escalation study included a total of 40 sarcoma
121
patients, 35 of whom had GIST. The other 5 sarcomas did not express
KIT. By common toxicity criteria of the National Cancer Institute (NCI-
CTC version 2.0) [45], at the 500 mg twice daily dose-level 5 of 8
patients showed a dose-limiting toxicity (DLT), including nausea,
vomiting, edema, and skin rash. A maximum tolerated dose of 800 mg
per day, 400 mg bid, was advised for future studies. At a follow-up of 9-
12 months, 82% of the included GIST patients still benefited from
imatinib therapy (Table 1).
At the same time an initial phase II study was performed by
investigators in the USA and Finland to formally assess activity and
safety in metastatic or unresectable GIST patients [39]. In this study, 147
patients were included. In 135 patients KIT positive GIST was confirmed
by expression of CD117. In 10 cases no material was available to confirm
this diagnosis. Patients were randomized to receive either a daily dose
of 400 mg or 600 mg imatinib. Side effects were frequent at both

dose levels and comparable to those observed in CML, with the exception
of a lower frequency of myelotoxicity and a higher frequency of serious
gastrointestinal bleeding, which is probably disease related. Both doses
were well tolerated. Because of lack of statistical power, this study was
not primarily intended to compare efficacy between both doses. On
neither dose, patients reached complete remission. Taking both doses
together, 82% of patients showed remarkable benefit from therapy. The
1-year survival was estimated to be 88%. No statistically significant
differences in outcome between both groups were found. In both studies
in 5-10% of patients imatinib was totally inactive. Three of nine patients
in the USA-Finland study, achieved a partial response or stable disease
after dose escalation to 600 mg daily after experiencing progressive
disease at 400 mg, indicating that there might be a dose-response
relationship [39]. Finally the activity data were further confirmed by an
EORTC phase II study [46] (See Table 1). The latter included 27 GIST
122
patients treated with 400 mg imatinib bid. GIST patients again showed a
high response rate, even one complete remission [46].
In view of remaining questions on dose-response relationships,
subsequently 2 randomized phase III studies have been performed in
pretreated patients with CD117-positive GIST receiving an oral dose of
either 400 mg or 800 mg per day. Primary study objectives differ slightly.
The study coordinated by the EORTC, in cooperation with the Italian
Sarcoma Group and the Australian Gastrointestinal Tumors Group, has
included 946 patients and had time to progression of disease as primary
endpoint [47, 48], whereas the study coordinated by the South West
Oncology Group (SWOG) accruing 746 patients has overall survival as
primary endpoint [49, 50]. With a median follow-up of 8.4 months, at the
time of the latest interim analysis, the EORTC study does show a better
progression free survival for the higher dose group [48] (progression free
survival at 6 and 12 months 78% and 69% versus 73% and 64%).
Objective response rates by RECIST criteria [51] are reported to be
identical in both dose groups (43%), with 2% to 3% complete responses.
The SWOG study does not show significant differences between
both dose groups at their most recent interim analysis as well [50].
Objective response rates by RECIST criteria [51] are reported to be
comparable as well (Table 1). Data from cross-over from 400 to 800 mg
are currently not yet available.
Little is yet known about the potential of imatinib as neo-
adjuvant and/or adjuvant treatment. One study presents data of only 5
patients, who received adjuvant imatinib therapy after radical surgery.
With a follow up of 7 to 13 months, none of these patients has developed
recurrent disease yet [52], but clearly publishing such data is strange
based upon flawed methodology, and surely these data to not justify
routine use of the approach.
The data on neo-adjuvant treatment are similarly scantly and
incoclusive. A small study involved only 18 GIST patients that were
operated after a period of imatinib treatment [53]. Radical resection was
achieved in seven out of eight patients, who underwent the surgery
because of residual tumor mass and had a partial response, and in two out
of ten patients, who were operated because of progressive disease.
Currently, various phase II and phase III studies are being performed
investigating the potential role of imatinib in the treatment of resectable
GISTs. Clearly only phase III studies can yield undebatable results in this
setting.
Most gain-of-function KIT mutations in GISTs are within the
juxtamembrane region, encoded by exon 11 [54]. Mutations are also
found in exon 9, and to a much lesser extent in exons 13 and 17, encoding
for the extracellular region, the first part of the split kinase domain, and
for the catalytic activation loop in the second part of the kinase domain,
respectively [54, 55]. From in vitro studies, it was suggested that GISTs
with a mutation of KIT in its regulatory part would respond better on
imatinib as those with a KIT mutation in its enzymatic part [56].
Recently, it was reported that GIST patients with a KIT mutation in exon
11 have a significant higher partial response rate than patients with a
123
mutation in exon 9 (72% versus 32%; p=0.033) as well as a longer time

to progression of (23 months versus 6 months) [55, 57]. If no mutations
were found, only 12% showed a partial response and the median time to
treatment failure was approximately 3 months. More recent and extended
data are discussed in chapter 8.
Presented studies show that response to imatinib appears to be
long lasting [21, 39, 48, 50]. However, resistance to imatinib, like in
CML [59], after initial response or stable disease does occur. In GIST
heterogeneous mechanisms may be responsible. Reported elucidating
factors for acquired resistance in GIST are the overexpression of KIT due
to genomic amplification of KIT, the acquisition of new mutations (e.g.
in the enzymatic part of KIT), the loss of KIT expression, accompanied
by gain-of-function mutations in other receptor tyrosine kinases, and not
otherwise characterized functional resistance [60-62].
At doses up to 800 mg imatinib daily, side effects are generally
mild to moderate and relatively easily manageable [21, 39, 46, 47, 49],
and particularly include granulocytopenia, anemia, edema, fatigue, rash,
and nausea. Higher dosing is associated with a higher frequency of some
side effects, i.e. edema, fatigue, rash, and nausea [63]. Although most
patients (90% to 100%) experience some toxicity, grade 3 and 4 adverse
events are seen in 20% [39, 49],. Side effects seem to decrease in
severity with ongoing treatment [21, 40, 46].
5 PDGF DRIVEN SARCOMAS
In view of the success of the inhibition of KIT in GIST by

treatment with imatinib, and the fact that the agent in models also actively
inhibited Platelet derived growth factor (PDGF), the latter target has also
been assessed. Almost all sarcomas are reported to show some expression
of PDGF although it is not known if this is truly the reflection of a tumor
growth factor. activating mutations have meanwhile been
described in GIST as a factor of responsiveness beside KIT mutations
[64]. As indicated the EORTC phase I study [21, 35] already included 5
patients with other sarcomas than GIST, but none of them responded. In
their phase II study the EORTC also included a stratum of 24 non-GIST
sarcomas, not expressing KIT but assumed to be expressing PDGF.
Importantly none of these patients had an objective response and the
stable diseases obtained were so few and so short that it is unlikely that
further studies will show any activity in such sarcomas [46]. The results
of a phase II study with KIT expressing other sarcomas have yet not been
published.
An exception to the inactivity of imatinib in PDGF expressing
tumors may be the activity of the agent in dermatofibrosarcoma
protruberans (DFSP) and desmoid tumors.
DFSP is an extremely rare disease that tends to remain local, but
has a high potential for infiltration. Most DFSPs possess either ring
chromosomes or translocations that result in fusion of 17q22 and 22q13,
the location of the gene. Since imatinib was known to inhibit the
124
latter it was given to a patient with DFSP, yielding such a dramatic

reduction in tumor size that resection became possible [65]. Other case
reports suggest the same.
Similarly, two cases of PDGF (and KIT) expressing
extraabdominal desmoid tumors have been reported [66], that both
showed long lasting benefit. Further studies to assess the activity in these
diseases are clearly warranted.
Meanwhile several other inhibitors of KIT and PDGF have
entered development. Although it is too early to assess activity without a
reasonable level of doubt, early data are encouraging.
6 CONCLUSION
The development of Imatinib has clearly created a new paradigm

for the treatment of soft tissue sarcomas. Although long term follow-up
data are yet lacking it is already quite evident the drug is nothing less
than a break-through in the treatment of GIST. It will be of crucial
importance in the next coming years to establish a functional relationship
between presence of a target for treatment and its involvement in actual
tumor growth. For KIT this was evident in GIST and this is likely one of
the reason for the success. And other development of signal transduction
inhibitors may have failed due to the lack of such information.
7 REFERENCES
1. DeMatteo RP, Lewis JJ, Leung D, Mudan SS, Woodruff JM, Brennan MF. Two hundred
Gastrointestinal Stromal Tumors: Recurrence patterns and prognostic factors for survival. Ann
Surg. 231(1), 51-58 (2000).
2. Miettinen M, Lasota J. Gastrointestinal stromal tumors--definition, clinical, histological,
immunohistochemical, and molecular genetic features and differential diagnosis. Virchows Arch.
438(1), 1-12 (2001).
3. Emory TS, Sobin LH, Lukes L, Lee DH, OLeary TJ. Prognosis of gastrointestinal smooth-
muscle (stromal) tumors: Dependence on anatomic site. Am J Surg Pathol. 23(1), 82-87 (1999).
4. Fletcher CD, Berman JJ, Corless C et al. Diagnosis of Gastrointestinal Stromal Tumors: A
consensus approach. Hum Pathol. 33(5), 459-465 (2002).
5. Blanke CD, Eisenberg BL, Heinrich MC. Gastrointestinal Stromal Tumors. Curr Treat Options
Oncol. 2(6), 485-491 (2001).
6. Mazur MT, Clark HB. Gastric Stromal Tumors. Reappraisal of histogenesis. Am J Surg Pathol.
7(6), 507-519 (1983).
7. Hirota S, Isozaki K, Moriyama Y et al. Gain-of-function mutations of c-KIT in human
Gastrointestinal Stromal Tumors. Science. 279(5350), 577-580 (1998).
8. Giebel LB, Strunk KM, Holmes SA, Spritz RA. Organization and nucleotide sequence of the
human KIT (mast/stem cell growth factor receptor) proto-oncogene. Oncogene. 7(11), 2207-2217
(1992).
9. Lux ML, Rubin BP, Biase TL et al. KIT extracellular and kinase domain mutations in
Gastrointestinal Stromal Tumors. Am J Pathol. 156(3), 791-795 (2000).
10. Rubin BP, Singer S, Tsao C et al. Kit activation is a ubiquitous feature of Gastrointestinal
Stromal Tumors. Cancer Res. 61(22), 8118-8121 (2001).
125
11. Sarlomo-Rikala M, El-Rifai W, Lahtinen T, Andersson LC, Miettinen M, Knuutila S.

Different patterns of DNA copy number changes in Gastrointestinal Stromal Tumors,
Leiomyomas, and Schwannomas. Hum Pathol. 29(5), 476-481 (1998).
12. Sircar K, Hewlett BR, Huizinga JD, Chorneyko K, Berezin I, Riddell RH. Interstitial cells of
cajal as precursors of Gastrointestinal Stromal Tumors. Am J Surg Pathol. 23(4), 377-389 (1999).
13. Kindblom LG, Remotti HE, Aldenborg F, Meis-Kindblom JM. Gastrointestinal Pacemaker
Cell Tumor (GIPACT): Gastrointestinal stromal tumors show phenotypic characteristics of the
interstitial cells of Cajal. Am J Pathol. 152(5), 1259-1269 (1998).
14. Roberts PJ, Eisenberg B. Clinical presentation of Gastrointestinal Stromal Tumors and
treatment of operable disease. Eur J Cancer. 38 Suppl 5, S37-38 (2002).
15. Joensuu H, Fletcher C, Dimitrijevic S, Silberman S, Roberts P, Demetri G. Management of
malignant Gastrointestinal Stromal Tumours. Lancet Oncol. 3(11), 655-664 (2002).
16. Plaat BE, Hollema H, Molenaar WM et al. Soft Tissue Leiomyosarcomas and malignant
Gastrointestinal Stromal Tumors: Differences in clinical outcome and expression of multidrug
resistance proteins. J Clin Oncol. 18(18), 3211-3220 (2000).
17. Mudan SS, Conlon KC, Woodruff JM, Lewis JJ, Brennan MF. Salvage surgery for patients
with recurrent Gastrointestinal Sarcoma: Prognostic factors to guide patient selection. Cancer.
88(1), 66-74 (2000).
18. Van Glabbeke M, van Oosterom AT, Oosterhuis JW et al. Prognostic factors for the outcome
of chemotherapy in advanced soft tissue sarcoma: An analysis of 2,185 patients treated with
anthracycline-containing first-line regimens - a European Organization for Research and
Treatment of Cancer Soft Tissue and Bone Sarcoma Group Study. J Clin Oncol. 17(1), 150-157
(1999).
19. Dagher R, Cohen M, Williams G et al. Approval summary: Imatinib Mesylate in the
treatment of metastatic and/or unresectable malignant Gastrointestinal Stromal Tumors. Clin
Cancer Res. 8(10), 3034-3038 (2002).
20. Manley PW, Cowan-Jacob SW, Buchdunger E et al. Imatinib: A selective tyrosine kinase
inhibitor. Eur J Cancer. 38 Suppl 5, S19-27 (2002).
21. van Oosterom AT, Judson IR, Verweij J et al. Update of phase I study of Imatinib (STI571) in
advanced soft tissue sarcomas and Gastrointestinal Stromal Tumors: A report of the EORTC Soft
Tissue and Bone Sarcoma Group. Eur J Cancer. 38 Suppl 5, S83-87 (2002).
22. Kolibaba KS, Druker BJ. Protein tyrosine kinases and cancer. Biochim Biophys Acta. 1333(3),
F217-248 (1997).
23. Druker BJ, Lydon NB. Lessons learned from the development of an abl tyrosine kinase
inhibitor for Chronic Myelogenous Leukemia. J Clin Invest. 105(1), 3-7 (2000).
24. Taylor ML, Metcalfe DD. KIT signal transduction. Hematol Oncol Clin North Am. 14(3),
517-535 (2000).
25. Buchdunger E, Cioffi CL, Law N et al. Abl protein-tyrosine kinase inhibitor STI571 inhibits
in vitro signal transduction mediated by c-KIT and platelet-derived growth factor receptors. J
Pharmacol Exp Ther. 295(1), 139-145 (2000).
26. Druker BJ, Tamura S, Buchdunger E et al. Effects of a selective inhibitor of the abl tyrosine
kinase on the growth of bcr-abl positive cells. Nat Med. 2(5), 561-566 (1996).
27. Verweij J, Judson I, van Oosterom A. Sti571: A magic bullet? Eur J Cancer. 37(15), 1816-
1819 (2001).
28. Faderl S, Talpaz M, Estrov Z, OBrien S, Kurzrock R, Kantarjian HM. The biology of chronic
myeloid leukemia. N Engl J Med. 341(3), 164-172 (1999).
29. Druker BJ, Talpaz M, Resta DJ et al. Efficacy and safety of a specific inhibitor of the bcr-abl
tyrosine kinase in Chronic Myeloid Leukemia. N Engl J Med. 344(14), 1031-1037 (2001).
30. Tuveson DA, Willis NA, Jacks T et al. Sti571 inactivation of the gastrointestinal stromal
tumor c-KIT oncoprotein: Biological and clinical implications. Oncogene. 20(36), 5054-5058
(2001).
31. Heinrich MC, Griffith DJ, Druker BJ, Wait CL, Ott KA, Zigler AJ. Inhibition of c-KIT
receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor. Blood. 96(3),
925-932 (2000).
32. Krystal GW, Honsawek S, Litz J, Buchdunger E. The selective tyrosine kinase inhibitor
STI571 inhibits Small Cell Lung Cancer growth. Clin Cancer Res. 6(8), 3319-3326 (2000).
33. Druker BJ, Sawyers CL, Kantarjian H et al. Activity of a specific inhibitor of the bcr-abl
tyrosine kinase in the blast crisis of Chronic Myeloid Leukemia and Acute Lymphoblastic
Leukemia with the Philadelphia chromosome. N Engl J Med. 344(14), 1038-1042 (2001).
126
34. Joensuu H, Roberts PJ, Sarlomo-Rikala M et al. Effect of the tyrosine kinase inhibitor STI571
in a patient with a metastatic Gastrointestinal Stromal Tumor. N Engl J Med. 344(14), 1052-1056
(2001).
35. van Oosterom AT, Judson I, Verweij J et al. Safety and efficacy of Imatinib (STI571) in
metastatic Gastrointestinal Stromal Tumours: A phase I study. Lancet. 358(9291), 1421-1423
(2001).
36. (imatinib mesylate) tablets prescribing information. 2003, East Hanover, New
Jersey, USA: Novartis Pharmaceuticals Corporation.
37. Reckmann AH, Fischer T, Peng B et al. Effect of food on STI571 Glivec pharmacokinetics
and bioavailability. Proc. Am. Soc. Clin. Oncol. 20, abstract 1223 (2001).38. Glivec. Gist clinical
monograph. 2002, Basel, Switserland: Novartis Pharma AG.
39. Demetri GD, von Mehren M, Blanke CD et al. Efficacy and safety of Imatinib Mesylate in
advanced Gastrointestinal Stromal Tumors. N Engl J Med. 347(7), 472-480 (2002).
40. Judson IR, Donato Di Paola E, Verweij J et al. Population pharmacokinetic (pk) analysis and
pk-pharmacodynamic (pd) correlations in phase I / II trial of Imatinib in Gastrointestinal Stromal
Tumours (GIST) conducted by the European Organisation for Research and Treatment of Cancer
Soft Tissue and Bone Sarcoma Group. Proc. Am. Soc. Clin. Oncol. 22, abstract 3287 (2003).
41. Ramanathan RK, Remick SC, Mulkerin D et al. P-5331: A phase I pharmacokinetic (pk)
study of STI571 in patients (pts) with advanced malignancies and varying degrees of liver
dysfunction (ld). Proc. Am. Soc. Clin. Oncol. 22, abstract 502 (2003).
42. OBrien SG, Peng B, Dutrix C et al. A pharmacokinetic interaction of Glivec and Simvastatin,
a cytochrome 3a4 substrate, in a patients with Chronic Myeloid Leukemia. Proc. Am. Soc.
Hematology 98, 141a, abstract 593 (2001).
43. Remick SC, Ramanathan RK, Mulkerin D et al. P-5340: A phase I pharmacokinetic study of
STI-571 in patients (pts) with advanced malignancies and varying degrees of renal dysfunction.
Proc. Am. Soc. Clin. Oncol. 22, abstract 503 (2003).
44. Gurney H, Wong M, Rivory L et al. Imatinib elimination: Characterisation by in vivo testing
of phenotype and genotype. Proc. Am. Soc. Clin. Oncol. 22, abstract 775 (2003).
45. Cancer therapy evaluation program. Common toxicity criteria, version 2.0. 1998, Bethesda,
USA: National Cancer Institute.
46. Verweij J, van Oosterom A, Blay JY et al. Imatinib Mesylate (STI-571 Glivec, Gleevec) is
an active agent for Gastrointestinal Stromal Tumours, but does not yield responses in other soft-
tissue sarcomas that are unselected for a molecular target. Results from an EORTC Soft Tissue
and Bone Sarcoma Group phase II study. Eur J Cancer. 39(14), 2006-2011 (2003).
47. Casali PG, Verweij J, Zalcberg J et al. Imatinib (Glivec) 400 vs 800 mg daily in patients with
Gastrointestinal Stromal Tumors (GIST): A randomized phase III trial from the EORTC Soft
Tissue and Bone Sarcoma Group, the Italian Sarcoma Group (ISG), and the Australasian Gastro-
Intestinal Trials Group (AGITG). A toxicity report. Proc. Am. Soc. Clin. Oncol. 21, abstract 1650
(2002).
48. Verweij J, Casali PG, Zalcberg J et al. Early efficacy comparison of two doses of Imatinib for
the treatment of advanced Gastro-Intestinal Stromal Tumors (GIST): Interim results of a
randomized phase III trial from the EORTC-STBSG, ISG and AGITG. Proc. Am. Soc. Clin.
Oncol. 22, abstract 3272 (2003).
49. Demetri GD, Rankin C, Fletcher C et al. Phase III dose-randomized study of Imatinib
Mesylate (Gleevec, STI571) for GIST: Intergroup s0033 early results. Proc. Am. Soc. Clin. Oncol.
21, abstract 1651 (2002).
50. Benjamin RS, Rankin C, Fletcher C et al. Phase III dose-randomized study of Imatinib
Mesylate (STI571) for GIST: Intergroup s0033 early results. Proc. Am. Soc. Clin. Oncol. 22,
abstract 3271 (2003).
51. Therasse P, Arbuck SG, Eisenhauer EA et al. New guidelines to evaluate the response to
treatment in solid tumors. European Organization for Research and Treatment of Cancer, National
Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst.
92(3), 205-216 (2000).
52. Bumming P, Andersson J, Meis-Kindblom JM et al. Neoadjuvant, adjuvant and palliative
treatment of Gastrointestinal Stromal Tumours (GIST) with imatinib: A centre-based study of 17
patients. Br J Cancer. 89(3), 460-464 (2003).
53. Hohenberger P, Bauer S, Schneider U et al. Tumor resection following imatinib pretreatment
in GI Stromal Tumors. Proc. Am. Soc. Clin. Oncol. 22, abstract 3288 (2003).
127
54. Fletcher JA, Fletcher CD, Rubin BP, Ashman LK, Corless CL, Heinrich MC. KIT gene
mutations in Gastrointestinal Stromal Tumors: More complex than previously recognized? Am J
Pathol. 161(2), 737-738; author reply 738-739 (2002).
55. Heinrich MC, Corless CL, Blanke C et al. KIT mutational status predicts clinical response to
STI571 in patients with metastatic Gastrointestinal Stromal Tumors (GISTs). Proc. Am. Soc. Clin.
Oncol. 21, abstract 6 (2002).
56. Heinrich MC, Rubin BP, Longley BJ, Fletcher JA. Biology and genetic aspects of
Gastrointestinal Stromal Tumors: KIT activation and cytogenetic alterations. Hum Pathol. 33(5),
484-495 (2002)
57. Heinrich MC, Corless CL, Von Mehren M et al. PDGFRA and KIT mutations correlate with
the clinical responses to Imatinib Mesylate in patients with advanced Gastrointestinal Stromal
Tumors (GIST). Proc. Am. Soc. Clin. Oncol. 22, abstract 3274 (2003).
58. Heinrich MC, Corless CL, Duensing A et al. Pdgfra activating mutations in Gastrointestinal
Stromal Tumors. Science. 299(5607), 708-710 (2003).
59. Deininger MW, Druker BJ. Specific targeted therapy of Chronic Myelogenous Leukemia with
imatinib. Pharmacol Rev. 55(3), 401-423 (2003).
60. Fletcher JA, Corless CL, Dimitrijevic S et al. Mechanisms of resistance to Imatinib Mesylate
(IM) in advanced Gastrointestinal Stromal Tumor (GIST). 22, abstract 3275 (2003).
61. Andersson J, Sjogren H, Meis-Kindblom JM, Stenman G, Aman P, Kindblom LG. The
complexity of KIT gene mutations and chromosome rearrangements and their clinical correlation
in Gastrointestinal Stromal (pacemaker cell) Tumors. Am J Pathol. 160(1), 15-22 (2002).
62. Eisenberg BL, von Mehren M. Pharmacotherapy of Gastrointestinal Stromal Tumours. Expert
Opin Pharmacother. 4(6), 869-874 (2003).
63. Van Glabbeke MM, Verweij J, Casali PG et al. Prognostic factors of toxicity and efficacy in
patients with Gastro-Intestinal Stromal Tumors (GIST) treated with imatinib: A study of the
EORTC-STBSG, ISG and AGITG. Proc. Am. Soc. Clin. Oncol. 22, abstract 3286 (2003).
64. Heinrich MC, Corless CL, Duensing A, et al. PDGFRA activating mutations in
gastrointestinal Stromal tumors. Science 229, 708-710 (2003).
65. Rubin BP, Schuetze SM, Eary JF, et al. Molecular targeting of platelet-derived growth factor
B by Imatinib mesylate in a patient with metastatic dermatofibrosarcoma protruberans.
J.Clin.Oncol. 20:3586-3591 (2002)
66..Mace J, Biermann JS, Sondak V, et al. Response of extraabdominal desmoid tumors to therapy
with imatinib mesylate. Cancer 95:2372-2370 (2002)
Chapter 8
Targeting mutant kinases in Gastrointestinal

Stromal Tumors: A paradigm for molecular
therapy of other sarcomas
Michael C. Heinrich and Christopher L. Corless
Departments of Medicine and Pathology, Oregon Health Science University Cancer

Institute and Portland VA Medical Center, Portland, USA
Correspondence to:
Michael C.Heinrich, MD
Departments of Medicine and Pathology,
Oregon Health Science University Cancer Institute and Portland VA Medical Center,
3710 SW US Veterans Hospital Road
Portland, OR 97201
USA
130
1 INTRODUCTION
Gastrointestinal stromal tumors (GISTs) are the most common

mesenchymal neoplasm of the gastrointestinal tract. The classification of
these tumors has long been a subject of controversy in the pathology
literature. Likewise, the medical management of metastatic or
unresectable tumors has been problematic as these tumors are uniformly
resistant to standard cytotoxic chemotherapy agents. Fortunately, recent
advances in the understanding of the molecular biology and pathogenesis
of these tumors has lead to advances in both diagnosis and medical
therapy. In particular, the use of the tyrosine kinase inhibitor imatinib
(formerly STI571; Gleevec in the US and Glivec in Europe, Novartis
Pharma, Basel, Switzerland), has lead to a revolution in the therapeutic
approach to metastatic GIST. In this chapter, we will review the
development of targeted therapy for GISTs and will further suggest how
this paradigm might be applied to other soft tissue sarcomas.
2 ONCOGENIC MUTATIONS OF KIT ARE

COMMON IN GISTS
KIT is a 145 kD transmembrane glycoprotein that serves as the

receptor for stem cell factor (SCF) and has tyrosine kinase activity.1,2 A
member of the subclass III family of receptor tyrosine kinases, KIT is
closely related to the receptors for PDGF, M-CSF and FLT3 ligand.3,4
Based on studies of naturally occurring loss-of-function mutations in
mice, signaling through KIT is critical to the development of the
interstitial cells of Cajal, as well as hematopoietic progenitor cells, mast
cells and germ cells.5-9 Binding of SCF to KIT results in receptor
homodimerization and activation of the tyrosine kinase activity, which
leads to the phosphorylation of a variety of signaling intermediates.10,11 In
many cases, these substrates are themselves kinases and serve to amplify
intracellular signal transduction.12
2.1 Mutations of the Juxtamembrane Domain (Exon 11)
The juxtamembrane region of KIT (exon 11) functions to

inhibit receptor dimerization in the absence of SCF. From in vitro
mutagenesis studies it has been established that small, in-frame deletions,
insertions or single amino acid substitutions in this domain disrupt its
function, allowing spontaneous, ligand-independent receptor
dimerization.13-16 In 1998, Dr. Seichi Hirota and colleagues discovered
that GISTs not only express KIT protein, but that mutations in the
juxtamembrane domain of the KIT gene can be found in these tumors.17
As predicted, the mutant KIT isoforms had constitutive kinase activity
when expressed in vitro, that is, their kinase domains were active even in
the absence of SCF. The oncogenicity of the KIT mutants was confirmed
in a nude mouse tumorigenesis assay with transfected BA/F3 cells.
131
supporting the view that activation of KIT plays an important role in the
growth and survival of GISTs.
The reported frequency of exon 11 mutations in GISTs
has varied over a wide range (20% to 92%), but the highest frequencies
have come from studies based on cDNA prepared from frozen tumor
samples. Hirota and colleagues observed exon 11 mutations in 5 of 6
tumors (83%), while Rubin et al. uncovered 34 exon 11 mutations in 48
tumors (71%).17,18 In most studies that have used genomic DNA extracted
out of paraffin-embedded tumor tissue the frequency has been lower
(20% to 57%).19-24 This likely reflects technical issues related to the lower
quality of DNA obtainable from such samples. In addition, the PCR
primers used to amplify exon 11 in some paraffin-based studies did not
permit analysis of the entire exon. Screening methodologies are another
factor. Many investigators have relied on single strand conformation
polymorphism (SSCP) to look for the presence of a deletion or point
mutation, but this technique is not as sensitive as capillary gel
electrophoresis, which in a recent study by Emile et al. yielded exon 11
mutations in 67.5% of 40 GIST samples.25
Denaturing HPLC (D-HPLC) is another highly sensitive
screening methodology that has yielded high frequencies of
deletions/insertions and point mutations in KIT exon 11. In a series of 322
paraffin-embedded GISTs for KIT gene mutations screened by this
approach in our laboratory the frequency of sequence-confirmed exon 11
mutations was 66.1%. This series includes 127 malignant GISTs and 13
very low-risk GISTs that have previously been published.26,27 The
spectrum of mutations, was similar to that reported by other groups.
Deletions and insertions tend to affect the first part of the exon,
particularly codons 557-559. Point mutations are limited to just four of
the codons within the exon: 557, 559, 560 and 576. Internal tandem
duplications are observed near the end of the exon. A subset of the
tumors (17.8%) were either hemizygous or homozygous for the observed
mutation, suggesting that there is negative selective pressure on
expression of the wild-type KIT allele in exon 11-mutant tumors. In vitro
experiments demonstrating that a peptide corresponding to the wild-type
juxtamembrane domain is inhibitory to activated isoforms of KIT support
this view.16
2.2 Mutations in the Extracellular Domain (Exon 9)
Exon 9, which encodes the extracellular domain of KIT,

is the second most common site of mutation in GISTs. An insertion-type
mutation that results in duplication of Ala501 and Tyr502, first reported by
Lux and colleagues, was confirmed by Hirota et al. to encode a KIT
isoform that has a constitutively activated kinase.28,29 Other groups have
also observed this mutation and noted its preferential association with
small intestinal origin.30-32 Among the 322 GISTs that we have analyzed,
42 (13%) had exon 9 mutations, only 1 of which was hemi/homozygous.
All were the AY501-502 duplication/insertion, with the exception of a
132
single FAF505-508 duplication/insertion.26 Based on our data and other

published examples, 95% of exon 9-mutant GISTs arise in the small
bowel, suggesting that the biology of this class of mutations differs from
the exon-11 mutant tumors that most commonly occur in the stomach.
2.3 Mutations in the Kinase I Domain (Exon 13)
Lux and colleagues were the first to identify the K642E

point mutation in KIT exon 13.28 Though this mutation is rare, it has since
been observed by several other investigators in frequencies ranging from
0.8 to 4.1%.28,30,33 Our series of 322 GISTs yielded just 4 of these exon 13
mutations (1.2%). There is evidence that this mutation results in ligand-
independent activation of the receptor, although it is unclear whether
spontaneous receptor homodimerization is the mechanism.34,35
2.4 Mutations in the Activation Loop (Exon 17)
Mutations involving the activation loop of KIT are

uncommon in GISTs. Rubin et al. reported an N822K and an N822H
mutation in one case each.18 Two additional N822K mutants were
detected in our series of 322 GISTs (0.6%), but no such mutations were
observed by Kinoshita and colleagues amongst 124 GISTs.33 As is
discussed further below, a germline D820Y substitution has been related
to familial GIST;36 however, this mutation has not been reported in
sporadic tumors. The N822K and D820Y mutations cause constitutive
activation of the kinase domain, although the mechanisms remain
unclear.26,36 It is interesting that a nearby codon in exon 17 (Asp816) is
commonly mutated in other human malignancies, including mast cell
disease, seminoma/dysgerminoma, acute myelogenous leukemia, and
sinonasal natural killer/T-cell lymphoma.37-42 While mutations of this
codon are highly activating, they have not been observed in more than
700 GISTs published to date. Conversely, activating mutations of KIT
exon 11 have been found in only 5 cases of human mastocytosis. The
implication of these observations is that the stem cells that give rise to
GISTs have different transforming requirements than those that give rise
to mastocytosis, and these requirements may be reflected in alternative
signaling initiated by the various KIT mutations.
3 KIT MUTATIONS OCCUR EARLY IN GIST

DEVELOPMENT
Several lines of evidence support the hypothesis that activating

mutations of KIT are the initiating event in most adult GISTs: 1) KIT
mutations are common in small, incidentally discovered GISTs; 2) KIT
mutation status does not correlate pathologic grade; 3) germline
activating KIT mutations are associated with a heritable susceptibility to
133
GIST; 4) expression of mutant KIT in mice results in GISTs; 5) KIT

mutations antedate cytogenetic abnormalities.
Early reports on KIT mutations in GISTs emphasized an apparent

association with aggressive clinical behavior.19-21,23,24 These studies,
however, were based on SSCP and/or direct sequence analyses of exon
11 amplimers prepared from paraffin-extracted DNA. Using D-HPLC,
we screened 13 incidentally discovered, morphologically benign GISTs
that were 1 cm or less in size. Ten of these sub-clinical tumors harbored
exon 11 mutations (77%), and one had an exon 9 insertion.27 We have
since analyzed 275 GISTs for which information on grade and stage was
available. The exon 11 mutation frequency among tumors of low
malignant potential (<5 cm and <5 mitoses/50 hpf) was 87.1%.26 Reports
from Rubin and colleagues and by Wardelmann et al. show similar high
frequencies of exon 11 mutations in low grade GISTs.18,43 Clearly, these
mutations occur early in GIST oncogenesis and are therefore unlikely to
influence malignant potential.
In contrast to exon 11 mutations, the frequency of exon 9
mutations in our series of 275 GISTs was higher among malignant tumors
(17.3%) than among high-risk (3.0%) and low-risk tumors (2.5%). These
mutations appear to support altered intracellular signaling compared with
exon 11-mutant tumors.44 Thus, the biology of exon 9-mutant tumors is
inherently different and perhaps more aggressive than that of other
GISTs.
In theory, the progression of GISTs might be related to
the accumulation of secondary mutations in KIT. For example, if a tumor
harboring an exon 11 point mutation subsequently acquired an exon 9
deletion, it might have an additional growth advantage. This hypothetical
phenomenon has not been observed in our series of GISTs. Among 127
tumors studied in detail for mutations in KIT exons 9, 11, 13 and 17, none
had more than a single mutation.45
Germline mutations in the KIT gene have been reported in a
number of kindreds, as summarized in Table 1. Individuals who inherit an
activating mutation in exon 11 (V559A or deletion 559) are predisposed
to skin hyperpigmentation, skin mastocytosis (urticaria pigmentosa), and
the development of multiple GI stromal tumors. Interestingly, there is
diffuse ICC hyperplasia in the GI tract in affected patients, consistent
with continuous growth signaling from the mutant KIT isoform. GISTs
that arise from this background hyperplasia are not usually clinically
evident until early adulthood, suggesting that other mutations are
necessary to allow full neoplastic progression.
Other germline KIT mutations have been reported in exons 13
and 17 (Table 1). These mutations also lead to ICC hyperplasia and a
predisposition to multifocal GIST formation, but they are not associated
with skin pigmentation or mast cell proliferations. Thus, signaling from
these particular tyrosine kinase domain mutations does not appear to
influence melanocytes and mast cells in the same manner as the exon 11
mutations.
134
From the above studies, it is clear that germline inheritance of an

activating KIT mutation results in a greatly increased risk of developing
one or more GISTs, strongly supporting the notion that KIT mutation is
an initiating event in GIST pathogenesis. As GISTs in affected patients
usually develop after the second decade of life, it is inferred that
additional molecular events are required for development of a clinically
advanced lesion. This inference is supported by studies of lesional
clonality in two different kindreds with familial GIST (one kindred with
an exon 11 point mutation, the other with an exon 17 point mutation).52 In
the studied patients, the diffuse ICC hyperplasia present within the
muscularis propria of the GI tract was found to be polyclonal in nature. In
contrast, discrete mass lesions diagnosed as GIST were monoclonal, and
different GIST lesions from the same patient were derived from
independent clones.
Recently, Sommer et al. have generated a murine model of GIST
using a knock-in strategy to introduce a mutation found in human familial
GIST (del V559 human = del V558 mouse) into the mouse genome.53
Heterozygous mutant mice developed patchy hyperplasia of
KIT-positive cells of the myenteric plexus throughout the GI tract. The
morphological, immunohistochemical, and ultrastructural features of
these hyperplastic cells were consistent with ICC. In addition, nearly all
mice developed stromal tumors of the cecum, with morphologic and
immunophenotypic features similar to those seen in human GIST
specimens. Relative to control mice, the heterozygous mutant mice had
shorted survival due to bowel obstruction. None of the mice developed
metastatic disease. It is interesting to note that GISTs occur
spontaneously in dogs and juxtamembrane mutations in KIT are present
in these tumors.54 Thus, the relationship between KIT signaling and GIST
development is constant across several mammalian species.
The majority of GISTs have one or more cytogenetic
abnormalities, most commonly chromosomal deletions, and have a
cytogenetic profile that is distinct from histologic mimics such as
leiomyoma and leiomyosarcoma. These cytogenetic abnormalities are
typically found in only a subset of GIST cells and thus are likely to be
secondary events that occur after the initiating oncogenic KIT mutation.
Indeed, some GISTs have normal karyotypes but have an activating KIT
mutation. To summarize, the molecular, clinical, animal models and
cytogenetic studies suggest a consistent pathogenic process for GIST
progression. A simplified version of this progession is as follows: KIT
135
activating mutation 14 q deletion 22q deletion 1p deletion

8q gain 11p deletion 9p deletion 17q gain.55 This schema is a
generalization and is not meant to suggest that all GISTs acquire each of
these cytogenetic abnormalities or that the abnormalities are necessarily
acquired in this order. This model is analogous to the biology of chronic
myelogenous leukemia where expression of BCR-ABL oncoprotein is the
initiating event and additional cytogenetic abnormalities are acquired
during progression to accelerated phase and blast crisis.56
4 PDGFRA IS AN ALTERNATIVE ONCOGENE IN

GISTS
Some GISTs are negative for mutations in the KIT gene, despite
best efforts to find these mutations, including complete cDNA
sequencing. Heinrich, Fletcher and colleagues recently applied a novel
methodology to search for other activated kinases in tumors wild-type for
KIT (KIT-WT).57 A cocktail of antibodies to epitopes shared by a wide
range of receptor tyrosine kinases was used to immunoprecipitate kinases
from extracts of these tumors, and western blotting with a phospho-
tyrosine specific antibody revealed a novel band that was identified as
PDGF receptor alpha (PDGFRA). Phosphorylated PDGFRA was
detectable in a subset of KIT-WT tumors, but was not present in extracts
of tumors with known KIT mutations. Conversely, extracts of KIT-mutant
tumors had phosphorylated KIT, but were negative for phosphorylated
PDGFRA. These results suggested that PDGFRA is the active kinase in
some KIT-WT tumors.
Examination of genomic DNA from KIT-WT tumors yielded a
variety of mutations in the juxtamembrane (exon 12) and activation loop
(exon 18) domains of the PDGFRA gene.57 When cloned and transfected
into CHO cells, the PDGFRA mutant isoforms were found to be
constitutively phosphorylated in the absence of PDGF-AA ligand,
consistent with oncogenic activation. The frequency of PDGFRA
mutations among KIT-WT GISTs is 34% (23/67 cases), and among all
GISTs is 7.1% (23/322). PDGFRA exon 12 mutations appear to be less
common than exon 18 mutations (1.5% versus 5.6%, respectively).
PDGFRA -mutant tumors tend to have an epithelioid morphology,
but are not histologically distinguishable from KIT-mutant tumors.
Likewise, the signal transduction profiles for the two types of tumors are
similar.57 No PDGFRA mutations have been found in 146 KIT-mutant
tumors, so the activation of the two genes appears to be mutually
exclusive. Although not as extensively studied as KIT mutations in
GISTs, it seems likely that PDGFRA mutations might be an initiating
event in some GISTs lacking KIT mutations. The impact of PDGFRA
mutations on the diagnosis and treatment of GISTs is considered in later
sections.
136
5 OTHER GIST VARIANTS
As discussed above, we favor a model in which activating

mutations of KIT or PDGFRA serve as the initiating event in the vast
majority of these tumors. There are, however, a number of variant
clinical syndromes in which GISTs appear to arise independent of KIT
and PDGFRA mutations. The association of paraganglioma, pulmonary
chondromas and gastric leiomyosarcoma, known as Carneys triad, is a
rare, sporadic syndrome that presents almost exclusively in young
women.58 While the genetic basis for the syndrome is not known,
virtually all reported patients have had one or more gastric tumors that
were morphologically and immunophenotypically consistent with
GIST.59 In most instances, the tumors are diagnosed before the patient
reaches age 30. Preliminary studies of Carney triad-associated GISTs
suggest that they do not harbor KIT or PDGFRA mutations (authors
unpublished results).
Gastric GISTs are occasionally diagnosed in pediatric patients
outside of Carneys triad.60 A malignant GIST of the stomach from one
patient was recently screened for mutations in KIT exons 9, 11 and 13
and found to be negative.61 Preliminary studies of additional non-
syndromic pediatric GISTs indicate that KIT and PDGFRA mutations are
much less common than in adult GISTs (authors unpublished results).
Patients with neurofibromatosis type I (NF1, von
Recklinghausens neurofibromatosis) often develop neurofibromas in the
GI tract, but in addition, a subset of these patients suffer from one or
more gastric, intestinal or colonic GISTs. Described as autonomic nerve
tumors, stromal tumors with skeinoid fibers or leiomyomatosis in
the older literature,62-66 the presence of strong KIT positivity in these
tumors supports their designation as GISTs.32,67 Based on a Swedish
study of 70 NF1 patients, the incidence of GISTs in this population is
approximately 7%.68 Why the tumors arise in only a minority of these
patients and yet may be multifocal remains an interesting mystery.
6 TARGETING ONCOGENIC KINASES IN GISTS

USING IMATINIB
The accepted standard treatment for localized GISTs is complete

surgical resection.69 Recurrence after complete resection is common,
occurring in up to 90% of patients with larger tumors,70 and survival
following recurrence is short. Patients with advanced GIST have
historically done poorly, with a 30% 6-month progression-free survival
following initial chemotherapy.71 The sensitivity of GISTs to standard
chemotherapy agents has been difficult to define because most published
series have not adequately distinguished GISTs from leiomyosarcomas
and neurogenic tumors. However, the published data suggest that the
137
response rate of GISTs to single- or multi-agent chemotherapy regimens

is less than 5%.72
The concept that patients with GISTs might benefit from
treatment with imatinib was based in part on two experimental
observations: 1) treatment of GIST cell lines with imatinib inhibited
proliferation and induced apoptosis; 2) several GIST-associated mutant
KIT isoforms were potently inhibited by imatinib in vitro at
concentrations similar to wild-type KIT.73,74 Based on the identification
of mutated KIT as a therapeutic target in GIST and the lack of an
effective conventional medical therapy, a patient with chemotherapy-
resistant gastric GIST metastatic to omentum and liver was started on
imatinib at 400 mg po daily in March 2000.75 The tumor in this patient
expressed an exon 11 mutant KIT isoform and the patient responded
dramatically to imatinib therapy.
The strong pre-clinical rationale, coupled with the
clinical success in this solitary case, led to the development of two proof-
of-principle GIST trials utilizing imatinib. An EORTC dose-escalation
study of imatinib included 40 patients, 36 of whom had advanced GIST.
The imatinib dose ranged from 400 milligrams daily to 500 mg BID. At
the highest dose, five of eight patients had grade 3 toxicity
(nausea/vomiting in 3, edema in 1, and dyspnea in 1) and 400 mg BID
was considered the maximally tolerated dose. Nineteen patients (53%)
had objective partial responses, while thirteen (36%) had stable disease
and only four (11%) experienced frank progression. Two patients who
progressed on 400 mg subsequently responded to 800 mg. With a
minimum follow-up of 11 months, 29/36 remained on treatment. None of
the four patients in the trial with a non-GIST sarcoma responded to
imatinib.76,77
The GIST Working Group (a consortium of the Oregon Health &
Science University Cancer Institute, the Fox Chase Cancer Center, the
University of Turku, the University of Helsinki, and the Dana-Farber
Cancer Institute) performed a phase II randomized trial of 400 mg daily
versus 600 mg.78 One-hundred forty-seven patients with incurable GIST
(51% pre-treated with systemic therapy) were accrued, with a median age
of 54 and median performance status of 1. Ninety-four percent of the
patients had undergone previous surgery, often involving the stomach or
other areas in the GI tract, and therefore drug absorption was a concern in
this trial. Nevertheless, pharmacokinetic assessments showed that drug
levels were actually higher than that seen in leukemia patients given
imatinib.
Imatinib was highly effective in this advanced disease
population. Sixty-two percent of patients on 400 mg daily, and 65% on
600 mg achieved a partial response.78,79 Stable disease was seen in 19 and
20% respectively, and 16 and 8% had primary resistance, manifested as
initial disease progression. Twenty-eight percent of patients failing the
lower dose responded when crossed over to the higher dose. With a
median follow-up of 15 months, 73 percent of patients remained on study
drug, with an overall median time-to-treatment failure of 72 weeks.
138
7 USE OF KINASE MUTATIONS IN THE

DIAGNOSIS OF GIST
The use of KIT as an immunohistochemical marker for

GISTs has helped to solidify an otherwise untidy field by engendering
greater uniformity in both the diagnosis and the comparative study of
these tumors. No immunohistochemical marker is perfect, however, and
the heavy reliance on KIT staining has created some problems. The first
is that there are several commercially available KIT antibodies and these
antibodies are applied using different protocols in different laboratories.
The result is disagreement as to the specificity of this marker for GISTs
relative to other mesenchymal tumors in the abdomen, including
fibromatosis (desmoid tumor), synovial sarcoma and leiomyosarcoma.80-
87
From our experience in a referral center for GIST patients, it is
apparent that over-staining with inappropriately titered KIT antibodies is
a problem in some laboratories. Educational efforts are underway in the
U.S. to help pathology laboratories validate their KIT staining protocols,
and improved commercial packages for KIT staining may soon be
available. Thus, some of the confusion generated by improper
immunohistochemical staining for this marker may abate in coming
years.
Perhaps a larger challenge created by the emphasis on
KIT staining in GISTs is that approximately 5% to 10% of these tumors
are either weak or negative for KIT expression. In our experience, KIT-
negative GISTs often have activating mutations of PDGFRA.57 In
addition, KIT-negative GISTs are sometimes associated with activating
mutations of KIT.88 In the latter situation it appears that KIT expression is
either too low for detection with routine immunohistochemistry or is
artificially absent due to problems with tumor fixation.
We propose that genotyping for KIT and PDGFRA
kinase mutations may be useful as a confirmatory test in most GISTs,
particularly the subset of GISTs that lack KIT expression as assessed
using a routine immunohistochemical assay. As discussed below,
genotyping studies are useful not only providing molecular confirmation
of the diagnosis, but in determining the prognosis of patients treated with
imatinib.
8 KINASE MUTATIONS ARE PREDICTIVE OF

IMATINIB RESPONSE
The weight of existing evidence favors a model in which

mutations of KIT and PDGFRA are the initiating event in the vast
majority of GISTs. An additional prediction from this model is that
imatinib treatment would be most effective in GISTs with sensitive
kinase mutations. This prediction has been confirmed in correlative
studies of patient specimens from the GIST working group phase II trial.
139
Specifically, pre-treatment tumor specimens from 127 of the 147 treated

patients were genotyped for KIT and PDGFRA mutations and the
patients clinical response to imatinib, event-free and overall survival
were correlated with tumor genotype. Additionally, the imatinib
sensitivity of representative KIT and PDGFRA oncoproteins was
determined in vitro.26
Activating mutations of KIT or PDGFRA were found in
112 (88.2%) and 6 (4.7%) GISTs, respectively. Most KIT mutations
involved exon 9 (N = 23) or exon 11 (N = 85). The in vitro sensitivity of
representative KIT exon 9 (reduplication of AY502-503), 11 (V560G,
deletion WK557-557, deletion L579), 13 (K642E) and 17 (N822H,
N822K) oncoproteins to imatinib was similar to that of ligand-activated
wild-type KIT approximately 100-200 nM). In contrast, only some
of the PDGFRA oncoproteins were sensitive to imatinib in vitro
approximately 100-200 nM). Specifically, the D842V PDGFRA
mutation seen in 3 patients was 10-20 fold more resistant to inhibition by
imatinib approximately
In patients with GISTs harboring exon 11 KIT mutations, the
partial response rate (PR) was 83.5 percent, while patients with tumors
containing an exon 9 KIT mutation or no detectable mutation of KIT or
PDGFRA had PR rates of 47.8 percent (P=0.0006) and 0.0 percent
(P<0.0001), respectively. Patients whose tumors contained exon 11 KIT
mutations had a longer event free and overall survival than those whose
tumors expressed either exon 9 KIT mutations or had no detectable kinase
mutation.
The difference in partial response rate and event free survival
between the groups of patients whose GISTs had KIT exon 9 versus exon
11 mutations is particularly noteworthy, as the KIT oncoproteins encoded
by exon 9 and exon 11 mutations were equally sensitive to imatinib in
vitro. Preliminary studies suggest differences in downstream signaling in
exon 9 versus exon 11 KIT-mutant GISTs,44 and such biological
differences might influence the susceptibility of the tumor cells to
apoptosis in response to kinase suppression by imatinib. Alternatively,
the activation mechanisms for KIT exon 9 mutants might vary between
the in vitro and in vivo settings.
As only six genotyped patients in the trial had a PDGFRA
mutation, it is difficult to draw firm conclusions regarding the response of
such patients to imatinib. Nevertheless, it is noteworthy that 2 of 3
patients with an imatinib-sensitive PDGFRA mutation had a clinical
partial response whereas all 3 patients whose GISTs expressed the
imatinib-resistant D842V PDGFRA oncoprotein had progressive disease
after initiation of imatinib therapy.
The group of patients whose GISTs lacked a detectable KIT or
PDGFRA mutant oncoprotein responded poorly to imatinib therapy. Of
the nine patients whose GIST expressed wild-type KIT and PDGFRA
kinases, none of the patients obtained a partial response and only three
patients had stable disease as the best clinical response to imatinib
therapy. Additionally, the event free and overall patient survival were
140
significantly shorter in these patients. These results suggest that GISTs

lacking a KIT or PDGFRA mutation are biologically distinct and are
apparently less dependent upon these kinases that GISTs expressing
mutant kinases.
These data strongly support the notion that the responsiveness of
GIST to imatinib is mechanistically linked to the expression of imatinib-
sensitive mutant kinases. Notably, the partial response rate of patients
whose GIST expressed an imatinib sensitive KIT or PDGFRA mutant
oncoprotein was 76%, whereas the partial response of GISTs lacking a
kinase mutation or PDGFRA D842V was 0%. Overall, 100% (87/87) of
the genotyped patients that achieved a partial response during imatinib
therapy had GISTs expressing an imatinib sensitive mutant KIT or
PDGFRA oncoprotein.
Currently we are genotyping GIST specimens from patients
enrolled in the US-Canadian Phase III trial of imatinib for metastatic or
unresectable GIST. To date, GISTs from approximately 300 of the total
745 patients have been genotyped. Correlation with response data is not
yet available, but the overall frequency and spectrum of mutations is
similar to that observed in the GIST working group phase II trial
(authors unpublished observations). The larger patient numbers in the
phase III trial should be more than adequate for confirmation and
extension of the phase II genotyping results. The larger sample size of
this trial will be particularly important for analyzing the less common
mutations, such as PDGFRA D842V, and the group of GISTs without
KIT or PDGFRA mutations.
Routine genotyping of GISTs may be clinically useful in
determining the likelihood of response to imatinib therapy. Such
knowledge could prove useful in determination of prognosis and
individualizing care plans. For example, patients whose GISTs express
KIT exon 9, PDGFRA D842V, or wild-type KIT and PDGFRA proteins
have a much higher rate of treatment failure than patients whose GISTs
express KIT exon 11 mutation. Conceivably, these high-risk patients
should be monitored more frequently than the good-risk patients,
particularly during the first six months of therapy. PET imaging has
proven to be a more sensitive and earlier predictor of imatinib response
than conventional imaging. The optimal role of PET imaging in the
management of GIST has not been defined, but it might be particularly
useful in early assessment (days 21-40) of patients at moderate to high
risk of treatment failure.77,89 As additional treatment modalities
(alternative kinase inhibitors and/or combination therapy) are defined, the
choice of upfront treatment regimen could be optimized using tumor
genotype.
Another area where genotyping may help guide therapy is in the
use of adjuvant or neoadjuvant imatinib. Logically, the addition of
imatinib to surgical resection should be most efficacious in situations
where there is a high risk of failure with surgery alone and the tumor has
an imatinib-sensitive genotype. For example, the use of neoadjuvant
imatinib to treat a GIST lacking a kinase mutation seems unlikely to
result in tumor shrinkage; there would be an approximately 70% chance
141
of tumor progression and this might compromise surgical resectability of

the tumor(s). Conversely, the 84% chance of a partial response of a GIST
expressing an exon 11 KIT mutation to imatinib suggests a greater
likelihood of benefit to combination therapy. Ongoing neoadjuvant and
adjuvant trials of imatinib will define the overall benefit of such
approaches. These ongoing trials are incorporating correlative
genotyping studies that may define subsets of patients who will derive
benefit from neoadjuvant and/or adjuvant therapy.
9 MOLECULAR CLASSIFICATION OF GISTS
Based on the studies presented in this chapter, it is clear

that GISTs should be regarded as a group of closely related tumors rather
than as a single, uniform entity. While it has been only a few years since
KIT was identified as a highly sensitive marker for GISTs and imatinib
was introduced as a new therapy, it is now apparent that there are
clinically important differences among the oncogenic mutations that
occur in KIT and PDGFRA, and that wild-type GISTs constitute yet
another distinct subset. For this reason, we propose that GISTs be
classified according to the scheme outlined in Table 2, which emphasizes
the molecular context of the tumor and provides a quick reference for
other syndromes with which it may be associated.
142
To the extent that this classification is useful in identifying

patients who are likely to fail initial imatinib therapy, or in identifying
kindreds with possible germline KIT mutations, it is obvious that there
will be an increasing role for mutation screening in newly diagnosed
GISTs. Further progress in defining the oncogenic pathway(s) in wild-
type GISTs, and in the development of new therapeutics, will certainly
bring revisions to this classification, but in the meantime it may be
helpful in interpreting the results from ongoing and future clinical trials
of new targeted therapies for GIST.
10 EXTRAPOLATING THE GIST MODEL TO

OTHER SOFT TISSUE SARCOMAS
To date, the most effective use of small molecule kinase

inhibitors has been in the treatment of hematological malignancies
(Chronic Myelogenous Leukemia [CML], Chronic Myelomonocytic
Leukemia, Hypereosinophilic syndrome) and sarcomas (GIST,
Dermatofibrosarcoma Protuberans).77,78,90-97 A logical question is what is
the biological relationship between these entities? As reviewed
elsewhere, a striking common feature of hematological malignancies and
sarcomas is 1) the association of these cancers with recurrent
chromosomal translocations targeting transcription factors (e.g. AML1-
ETO in Acute Myelogenous Luekemia (AML) and PAX3-FKHR in
alveolar rhabdomyosarcoma); and 2) constitutive activation of tyrosine
kinases by translocation or intragenic mutation (e.g. KIT mutations in
systemic mastocytosis and GIST) (Table 3).98-102 Accordingly, molecular
analysis has become central to confirming the diagnosis of many
hematological and mesenchymal malignancies (e.g. BCR-ABL to
distinguish CML from other myeloproliferative diseases, PDGFRA
mutations in KIT-negative GISTs).
Abundant clinical and molecular evidence supports the
hypothesis that chromosomal translocations targeting transcription factors
serve to block hematopoietic stem cell differentiation. In contrast, kinase
mutations serve to increase cellular proliferation (e.g. BCR-ABL, FLT3).
The order of acquisition of these two types of genomic abnormalities
determines the clinical phenotype. For example, if a kinase mutation
develops first, the clinical phenotype is that of a myeloproliferative
syndrome with a massive increase in the number of well-differentiated
cells. Subsequent development of a differentiation block abnormality
results in accumulation of immature precursors and transition to an acute
leukemia/lymphoma syndrome (e.g. CML blast crisis).103 Conversely, a
differentiation block may develop first resulting in a bone
marrow/myelodysplastic syndrome with subsequent evolution to an acute
leukemia/lymphoma syndrome after acquisition of an activating kinase
mutation (e.g. AML1-ETO translocation with subsequent activating
FLT3 internal tandem duplication mutation).101,102,104
143
Based on the above model and reported trials of kinase

inhibitors, the existing evidence suggests that kinase inhibitors will be
most effective as monotherapy for those diseases in which the kinase
mutation is the initiating event, as exemplified by the clinical response of
chronic phase CML and GIST. Targeting the activity of fusion/mutant
transcription factors might serve to reverse the differentiation block in
sarcomas such as synovial sarcoma and rhabdomyosarcoma. Such a
possibility is suggested by activity of all-trans-retinoic acid in targeting
the fusion oncoprotein in some variants of acute progranulocytic
leukemia.104,105 Even more speculatively, combined therapy targeting
differentation blocks and genomically activated kinases might be
particularly effective for those sarcomas associated with both
abnormalities.106
To test the above model, we urgently need to define additional
genomic mechanisms of kinase activation and differentiation blocks in
sarcomas. It is likely that ongoing genomic and proteomic efforts will
identify such abnormalities and will set the stage for new targeted
approaches to sarcomas.107 In addition, and as discussed above in
relationship to GISTs, identification of additional molecular mechanisms
that initiate or enforce clinical progression of sarcomas will likely lead to
additional subclassification and/or reclassification of various soft tissue
neoplasms. Hopefully, refinements in diagnosis and new treatment
approaches will lead to better treatment outcomes for patients with
sarcoma.
144
11 REFERENCES
1. Besmer, P., Murphy, J. E., George, P. C., Qiu, F., Bergold, P. J., Lederman, L.,
Snyder, H. W., Jr., Brodeur, D., Zuckerman, E. E., and Hardy, W. D. A new acute
transforming feline retrovirus and relationship of its oncogene v-kit with the protein
kinase gene family. Nature., 320: 415-419, 1986.
2. Yarden, Y., Kuang, W.-J., Yang-Feng, T., Coussens, L., Munemitsu, S., Dull,
T. J., Chen, E., Schlessinger, J., Francke, U., and Ullrich, A. Human proto-oncogene c-kit:
a new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J., 11: 3341-
3351, 1987.
3. Rousset, D., Agnes, F., Lachaume, P., Andre, C., and Galibert, F. Molecular
evolution of the genes encoding receptor tyrosine kinase with immunoglobulinlike
domains. Journal of Molecular Evolution, 41: 421-429, 1995.
4. Small, D., Levenstein, M., Kim, E., Carow, C., Amin, S., Rockwell, P., Witte,
L., Burrow, C., Ratajczak, M. Z., Gewirtz, A. M., and et al STK-1, the human homolog of
Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved
in the proliferation of early progenitor/stem cells. Proc.Natl.Acad.Sci.U.S.A., 91: 459-
463, 1994.
5. Ishikawa, K., Komuro, T., Hirota, S., and Kitamura, Y. Ultrastructural
identification of the c-kit-expressing interstitial cells in the rat stomach: a comparison of
control and Ws/Ws mutant rats. Cell & Tissue Research, 289: 137-143, 1997.
6. Natali, P. G., Nicotra, M. R., Sures, I., Santoro, E., Bigotti, A., and Ullrich, A.
Expression of c-kit receptor in normal and transformed human nonlymphoid tissues.
Cancer Res., 52: 6139-6143, 1992.
7. Nocka, K., Majumder, S., Chabot, B., Ray, P., Cervone, M., Bernstein, A.,
Besmer, and P. Expression of c-kit gene products in known cellular targets of W
mutations in normal and W mutant mice-evidence for an impaired c- kit kinase in mutant
mice. Genes Dev., 3: 816-826, 1989.
8. Russell, E. S. Hereditary anemias of the mouse: A review for geneticists.
Adv.Genet., 20: 357-359, 1979.
9. Turner, A. M., Zsebo, K. M., Martin, F., Jacobsen, F. W., Bennett, L. G., and
Broudy, V. C. Nonhematopoietic tumor cell lines express stem cell factor and display c-
kit receptors. Blood, 80: 374-381, 1992.
10. Blume-Jensen, P., Claesson-Welsh, L., Siegbahn, A., Zsebo, K. M.,
Westermark, B., and Heldin, C. H. Activation of the human c-kit product by ligand-
induced dimerization mediates circular actin reorganization and chemotaxis. EMBO J.,
10: 4121-4128, 1991.
11. Huang, E., Nocka, K., Beier, D. R., Chu, T. Y., Buck, J., Lahm, H. W., Wellner,
D., Leder, P., and Besmer, P. The hematopoietic growth factor KL is encoded by the Sl
locus and is the ligand of the c-kit receptor, the gene product of the W locus. Cell, 63:
225-233, 1990.
12. Blume-Jensen, P. and Hunter, T, Oncogenic kinase signalling. Nature., 411:
355-365, 2001.
13. Irusta, P. M. and DiMaio, D. A single amino acid substitution in a WW-like
domain of diverse members of the PDGF receptor subfamily of tyrosine kinases causes
constitutive receptor activation. EMBO J., 17: 6912-6923, 1998.
14. Kitayama, H., Kanakura, Y., Furitsu, T., Tsujimura, T., Oritani, K., Ikeda, H.,
Sugahara, H., Mitsui, H., Kanayama, Y., Kitamura, Y., and et al. Constitutively activating
mutations of c-kit receptor tyrosine kinase confer factor-independent growth and
tumorigenicity of factor-dependent hematopoietic cell lines. Blood, 85: 790-798, 1995.
15. Ma, Y., Cunningham, M. E., Wang, X., Ghosh, I., Regan, L., and Longley, B. J.
Inhibition of spontaneous receptor phosphorylation by residues in a putative alpha-helix
in the KIT intracellular juxtamembrane region. J.Biol.Chem., 274: 13399-13402, 1999.
16. Chan, P. M., Ilangumaran, S., La Rose, J., Chakrabartty, A., and Rottapel, R.
Autoinhibition of the Kit Receptor Tyrosine Kinase by the Cytosolic Juxtamembrane
Region. Mol.Cell Biol., 23: 3067-3078, 2003.
17. Hirota, S., Isozaki, K., Moriyama, Y., Hashimoto, K., Nishida, T., Ishiguro, S.,
Kawano, K., Hanada, M., Kurata, A., Takeda, M., Muhammad Tunio, G., Matsuzawa, Y.,
Kanakura, Y., Shinomura, Y., and Kitamura, Y. Gain-of-function mutations of c-kit in
human gastrointestinal stromal tumors. Science, 279: 577-580, 1998.
145
18. Rubin, B. P., Singer, S., Tsao, C., Duensing, A., Lux, M. L., Ruiz, R., Hibbard,
M. K., Chen, C. J., Xiao, S., Tuveson, D. A., Demetri, G. D., Fletcher, C. D. M., and
Fletcher, J. A. KIT Activation Is a Ubiquitous Feature of Gastrointestinal Stromal
Tumors. Cancer Res, 61: 8118-8121, 2001.
19. Ernst, S. I., Hubbs, A. E., Przygodzki, R. M., Emory, T. S., Sobin, L. H., and
OLeary, T. J. KIT mutation portends poor prognosis in gastrointestinal stromal/smooth
muscle tumors. Laboratory Investigation, 78: 1633-1636, 1998.
20. Lasota, J., Jasinski, M., Sarlomo-Rikala, M., and Miettinen, M. Mutations in
exon 11 of c-Kit occur preferentially in malignant versus benign gastrointestinal stromal
tumors and do not occur in leiomyomas or leiomyosarcomas. Am.J.Pathol., 154: 53-60,
1999.
21. Li, S. Q., OLeary, T. J., Sobin, L. H., Erozan, Y. S., Rosenthal, D. L., and
Przygodzki, R. M. Analysis of KIT mutation and protein expression in fine needle
aspirates of gastrointestinal stromal/smooth muscle tumors. Acta Cytologica, 44: 981-986,
2000.
22. Moskaluk, C. A., Tian, Q., Marshall, C. R., Rumpel, C. A., Franquemont, D.
W., Frierson, and Jr, H. F. Mutations of c-kit JM domain are found in a minority of
human gastrointestinal stromal tumors. Oncogene, 18: 1897-1902, 1999.
23. Singer, S., Rubin, B. P., Lux, M. L., Chen, C. J., Demetri, G. D., Fletcher, C.
D., and Fletcher, J. A. Prognostic Value of KIT Mutation Type, Mitotic Activity, and
Histologic Subtype in Gastrointestinal Stromal Tumors. J Clin.Oncol., 20: 3898-3905,
2002.
24. Taniguchi, M., Nishida, T., Hirota, S., Isozaki, K., Ito, T., Nomura, T., Matsuda,
H., and Kitamura, Y. Effect of c-kit mutation on prognosis of gastrointestinal stromal
tumors. Cancer Res, 59: 4297-4300, 1999.
25. Emile, J. F., Lemoine, A., Bienfait, N., Terrier, P., Azoulay, D., and Debuire, B.
Length analysis of polymerase chain reaction products: a sensitive and reliable technique
for the detection of mutations in KIT exon 11 in gastrointestinal stromal tumors.
Diagn.Mol.Pathol., 11: 107-112, 2002.
26. Heinrich, M. C., Corless, C. L., Demetri, G. D., Blanke, C. D., von Mehren, M.,
Joensuu, H., McGreevey, L. S., Chen, C. J., Van den Abbeele, A. D., Druker, B. J., Kiese,
B., Eisenberg, B., Roberts, P. J., Singer, S., Fletcher, C. D. M., Silberman, S.,
Dimitrijevic, S., and Fletcher, J. A. Kinase mutations and imatinib mesylate response in
patients with metastatic gastrointestinal stromal tumor. Journal of Clinical Oncology (in
press), 2003.
27. Corless, C. L., McGreevey, L., Haley, A., Town, A., and Heinrich, M. C. KIT
mutations are common in incidental gastrointestinal stromal tumors one centimeter or less
in size. Am.J.Pathol., 160: 1567-1572, 2002.
28. Lux, M. L., Rubin, B. P., Biase, T. L., Chen, C. J., Maclure, T., Demetri, G.,
Xiao, S., Singer, S., Fletcher, C. D. M., and Fletcher, J. A. KIT extracellular and kinase
domain mutations in gastrointestinal stromal tumors. Am.J.Pathol., 156: 791-795, 2000.
29. Hirota, S., Nishida, T., Isozaki, K., Taniguchi, M., Nakamura, J., Okazaki, T.,
and Kitamura, Y. Gain-of-function mutation at the extracellular domain of KIT in
gastrointestinal stromal tumours. Journal of Pathology, 193: 505-510, 2001.
30. Lasota, J., Wozniak, A., Sarlomo-Rikala, M., Rys, J., Kordek, R., Nassar, A.,
Sobin, L. H., and Miettinen, M. Mutations in exons 9 and 13 of KIT gene are rare events
in gastrointestinal stromal tumors : A study of 200 cases. Am.J Pathol., 157: 1091-1095,
2000.
31. Sakurai, S., Oguni, S., Hironaka, M., Fukayama, M., Morinaga, S., and Saito,
K. Mutations in c-kit gene exons 9 and 13 in gastrointestinal stromal tumors among
Japanese. Jpn.J.Cancer Res., 92: 494-498, 2001.
32. Miettinen, M., Kopczynski, J., Makhlouf, H. R., Sarlomo-Rikala, M., Gyorffy,
H., Burke, A., Sobin, L. H., and Lasota, J. Gastrointestinal stromal tumors, intramural
leiomyomas, and leiomyosarcomas in the duodenum: a clinicopathologic,
immunohistochemical, and molecular genetic study of 167 cases. Am.J Surg.Pathol., 27:
625-641, 2003.
33. Kinoshita, K., Isozaki, K., Hirota, S., Nishida, T., Chen, H., Nakahara, M.,
Nagasawa, Y., Ohashi, A., Shinomura, Y., Kitamura, Y., and Matsuzawa, Y. c-kit Gene
146
mutation at exon 17 or 13 is very rare in sporadic gastrointestinal stromal tumors. J

Gastroenterol.Hepatol., 18: 147-151, 2003.
34. Heinrich, M. C., Corless, C. L., Blanke, C. D., Demetri, G., Joensuu, H., von
Mehren, M., McGreevey, L., Wait, C.L., Griffith, D., Chen, C. J., Haley, A., Kiese, B.,
Druker, B., Roberts, P. J., Eisenberg, B. L., Singer, S., Silberman, S., Dimitrijevic, S.,
Fletcher, C. D., and Fletcher, J. A. KIT mutational status predicts clinical response to
STI571 in patients with metastatic gastrointestinal stromal tumors (GISTs). Proc Am Soc
Clin Oncol 21, 2a. 2002.
35. Chen, H., Isozaki, K., Kinoshita, K., Ohashi, A., Shinomura, Y., Matsuzawa,
Y., Kitamura, Y., and Hirota, S. Imatinib inhibits various types of activating mutant kit
found in gastrointestinal stromal tumors. Int.J.Cancer, 105: 130-135, 2003.
36. Hirota, S., Nishida, T., Isozaki, K., Taniguchi, M., Nishikawa, K., Ohashi, A.,
Takabayashi, A., Obayashi, T., Okuno, T., Kinoshita, K., Chen, H., Shinomura, Y., and
Kitamura, Y. Familial gastrointestinal stromal tumors associated with dysphagia and
novel type germline mutation of KIT gene. Gastroenterology, 122: 1493-1499, 2002.
37. Buttner, C., Henz, B. M., Welker, P., Sepp, N. T., and Grabbe, J. Identification
of activating c-kit mutations in adult-, but not in childhood-onset indolent mastocytosis: a
possible explanation for divergent clinical behavior. J.Invest.Dermatol., 111: 1227-1231,
1998.
38. Gari, M., Goodeve, A., Wilson, G., Winship, P., Langabeer, S., Linch, D.,
Vandenberghe, E., Peake, I., and Reilly, J. c-kit proto-oncogene exon 8 in-frame deletion
plus insertion mutations in acute myeloid leukaemia. Br.J.Haematol., 105: 894-900, 1999.
39. Hongyo, T., Li, T., Syaifudin, M., Baskar, R., Ikeda, H., Kanakura, Y., Aozasa,
and Nomura, T. Specific c-kit mutations in sinonasal natural killer/T-cell lymphoma in
China and Japan. Cancer Res, 60: 2345-2347, 2000.
40. Longley, B. J., Tyrrell, L., Lu, S. Z., Ma, Y. S., Langley, K., Ding, T. G., Duffy,
T., Jacobs, P., Tang, L. H., and Modlin, I. Somatic c-KIT activating mutation in urticaria
pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell
neoplasm. Nature Genetics, 12: 312-314, 1996.
41. Nagata, H., Worobec, A. S., Oh, C. K., Chowdhury, B. A., Tannenbaum, S.,
Suzuki, Y., and Metcalfe, D. D. Identification of a point mutation in the catalytic domain
of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have
mastocytosis with an associated hematologic disorder. Proc.Natl.Acad.Sci.U.S.A., 92:
10560-10564, 1995.
42. Tian, Q., Frierson, H. F. J., Krystal, G. W., and Moskaluk, C. A. Activating c-
kit gene mutations in human germ cell tumors. Am.J.Pathol., 154: 1643-16470, 1999.
43. Wardelmann, E., Neidt, I., Bierhoff, E., Speidel, N., Manegold, C., Fischer, H.
P., Pfeifer, U., and Pietsch, T. c-kit mutations in gastrointestinal stromal tumors occur
preferentially in the spindle rather than in the epithelioid cell variant. Mod.Pathol., 15:
125-136, 2002.
44. Heinrich, M. C., Rubin, B. P., Longley, B. J., and Fletcher, J. A. Biology and
genetic aspects of gastrointestinal stromal tumors: KIT activation and cytogenetic
alterations. Hum Pathol, 33: 486-495, 2002.
45. Corless, C. L., Fletcher, J. A., and Heinrich, M. C. Biology of gastrointestinal
stromal tumors. Journal of Clinical Oncology (submitted), 2003.
46. Beghini, A., Tibiletti, M. G., Roversi, G., Chiaravalli, A. M., Serio, G., Capella,
C., and Larizza, L. Germline mutation in the juxtamembrane domain of the kit gene in a
family with gastrointestinal stromal tumors and urticaria pigmentosa. Cancer, 92: 657-
662, 2001.
47. Maeyama, H., Hidaka, E., Ota, H., Minami, S., Kajiyama, M., Kuraishi, A.,
Mori, H., Matsuda, Y., Wada, S., Sodeyama, H., Nakata, S., Kawamura, N., Hata, S.,
Watanabe, M., Iijima, Y., and Katsuyama, T. Familial Gastrointestinal Stromal Tumor
With Hyperpigmentation: Association With a Germline Mutation of the c-kit Gene.
Gastroenterology, 120: 210-215, 2001.
48. OBrien, P., Kapusta, L., Dardick, I., Axler, J., and Gnidec, A. Multiple familial
gastrointestinal autonomic nerve tumors and small intestinal neuronal dysplasia.
Am.J.Surg.Pathol., 23: 198-204, 1999.
49. Hirota, S., Okazaki, T., Kitamura, Y., OBrien, P., Kapusta, L., and Dardick, I.
Cause of familial and multiple gastrointestinal autonomic nerve tumors with hyperplasia
147
of interstitial cells of Cajal is germline mutation of the c-kit gene [letter]. American
Journal of Surgical Pathology, 24: 326-327, 2000.
50. Nishida, T., Hirota, S., Taniguchi, M., Hashimoto, K., Isozaki, K., Nakamura,
H., Kanakura, Y., Tanaka, T., Takabayashi, A., Matsuda, H., and Kitamura, Y. Familial
gastrointestinal stromal tumours with germline mutation of the KIT gene. Nature
Genetics, 19: 323-324, 1998.
51. Isozaki, K., Terris, B., Belghiti, J., Schiffman, S., Hirota, S., and Vanderwinden,
J.-M, Germline-activating mutation in the kinase domain of KIT gene in familial
gastrointestinal stromal tumors. Am.J Pathol., 157: 1581-1585, 2000.
52. Chen, H., Hirota, S., Isozaki, K., Sun, H., Ohashi, A., Kinoshita, K., OBrien, P.,
Kapusta, L., Dardick, I., Obayashi, T., Okazaki, T., Shinomura, Y., Matsuzawa, Y., and
Kitamura, Y. Polyclonal nature of diffuse proliferation of interstitial cells of Cajal in
patients with familial and multiple gastrointestinal stromal tumours. Gut, 51: 793-796,
2002.
53. Sommer, G., Agosti, V., Ehlers, I., Rossi, F., Corbacioglu, S., Farkas, J., Moore,
M., Manova, K., Antonescu, C. R., and Besmer, P. Gastrointestinal stromal tumors in a
mouse model by targeted mutation of the Kit receptor tyrosine kinase. Proc
Natl.Acad.Sci.U.S.A, 100: 6706-6711, 2003.
54. Frost, D., Lasota, J., and Miettinen, M. Gastrointestinal stromal tumors and
leiomyomas in the dog: a histopathologic, immunohistochemical, and molecular genetic
study of 50 cases. Vet.Pathol, 40: 42-54, 2003.
55. Heinrich, M. C., Rubin, B. P., Longley, B. J., and Fletcher, J. A. Biology and
genetic aspects of gastrointestinal stromal tumors: KIT activation and cytogenetic
alterations. Hum.Pathol, 33: 484-495, 2002.
56. Faderl, S., Talpaz, M., Estrov, Z., OBrien, S., Kurzrock, R., and Kantarjian, H.
M. The biology of chronic myeloid leukemia. N.Engl.J Med., 341: 164-172, 1999.
57. Heinrich, M. C., Corless, C. L., Duensing, A., McGreevey, L., Chen, C. J.,
Joseph, N., Singer, S., Griffith, D. J., Haley, A., Town, A., Demetri, G. D., Fletcher, C.
D., and Fletcher, J. A. PDGFRA Activating Mutations in Gastrointestinal Stromal
Tumors. Science, 299: 708-710, 2003.
58. Carney, J. A., Sheps, S. G., Go, V. L., and Gordon, H. The triad of gastric
leiomyosarcoma, functioning extra-adrenal paraganglioma and pulmonary chondroma.
N.Engl.J.Med., 296: 1517-1518, 1977.
59. Carney, J. A. Gastric stromal sarcoma, pulmonary chondroma, and extra-
adrenal paraganglioma (Carney Triad): natural history, adrenocortical component, and
possible familial occurrence. Mayo Clin.Proc., 74: 543-552, 1999.
60. Kerr, J. Z., Hicks, M. J., Nuchtern, J. G., Saldivar, V., Heim-Hall, J., Shah, S.,
Kelly, D. R., Cain, W. S., and Chintagumpala, M. M. Gastrointestinal autonomic nerve
tumors in the pediatric population: a report of four cases and a review of the literature.
Cancer, 85: 220-230, 1999.
61. Li, P., Wei, J., West, A. B., Perle, M., Greco, M. A., and Yang, G. C.
Epithelioid gastrointestinal stromal tumor of the stomach with liver metastases in a 12-
year-old girl: aspiration cytology and molecular study. Pediatr.Dev.Pathol., 5: 386-394,
2002.
62. Hamanaka, S., Hamanaka, Y., Yamashita, Y., and Otsuka, F. Leiomyoblastoma
and leiomyomatosis of the small intestine in a case of von Recklinghausens disease.
J.Dermatol., 24: 117-119, 1997.
63. Ishida, T., Wada, I., Horiuchi, H., Oka, T., and Machinami, R. Multiple small
intestinal stromal tumors with skeinoid fibers in association with neurofibromatosis 1
(von Recklinghausens disease). Pathol.Int., 46: 689-695, 1996.
64. Min, K. W. and Balaton, A. J. Small intestinal stromal tumors with skeinoid
fibers in neurofibromatosis: report of four cases with ultrastructural study of skeinoid
fibers from paraffin blocks. Ultrastruct.Pathol., 17: 307-314, 1993.
65. Schaldenbrand, J. D. and Appelman, H. D. Solitary solid stromal
gastrointestinal tumors in von Recklinghausens disease with minimal smooth muscle
differentiation. Hum Pathol, 15: 229-232, 1984.
66. Walsh, N. M. and Bodurtha, A. Auerbachs myenteric plexus. A possible site of
origin for gastrointestinal stromal tumors in von Recklinghausens neurofibromatosis.
Arch.Pathol.Lab Med., 114: 522-525, 1990.
148
67. Boldorini, R., Tosoni, A., Leutner, M., Ribaldone, R., Surico, N., Comello, E.,
and Min, K. W. Multiple small intestinal stromal tumours in a patient with previously
unrecognised neurofibromatosis type 1: immunohistochemical and ultrastructural
evaluation. Pathology, 33: 390-395, 2001.
68. Zoller, M. E., Rembeck, B., Oden, A., Samuelsson, M., and Angervall, L.
Malignant and benign tumors in patients with neurofibromatosis type 1 in a defined
Swedish population. Cancer, 79: 2125-2131, 1997.
69. Blanke, C. D., Eisenberg, B. L., and Heinrich, M. C. Gastrointestinal stromal
tumors. Current Treatment Options in Oncology, 2: 485-491, 2001.
70. Ng, E. H., Pollock, R. E., and Romsdahl, M. M. Prognostic implications of
patterns of failure for gastrointestinal leiomyosarcomas. Cancer, 69: 1334-1341, 1992.
71. van Glabbeke, M., Hogendoorn, P. C., Mouridsen, H., Radford, J. A., Verweij,
J., Rodenhuis, S., le Cesne, A., Buesa, J., Keizer, H. J., Van Oosterom, A., and Nielsen,
O. Progression free fate as the principal end-point for phase II trial on soft tissue sarcoma:
what should be the target? Proc Am Soc Clin Oncol 20, 345a. 2001.
72. Edmonson, J., Marks, R., Buckner, J., and Mahoney, M. Contrast of response to
D-MAP + Sargramostim between patients with advance malignant gastrointestinal
stromal tumors and patients with other advanced leiomyosarcomas. Proc Am Soc Clin
Oncol 18, 541a. 1999.
73. Tuveson, D. A., Willis, N. A., Jacks, T., Griffin, J. D., Singer, S., Fletcher, C.
D., Fletcher, J. A., and Demetri, G. D. STI571 inactivation of the gastrointestinal stromal
tumor c-KIT oncoprotein: biological and clinical implications. Oncogene, 20: 5054-5058,
2001.
74. Heinrich, M. C., Griffith, D. J., Druker, B. J., Wait, C. L., Ott, K. A., and Zigler,
A. J. Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine
kinase inhibitor. Blood, 96: 925-932, 2000.
75. Joensuu, H., Roberts, P. J., Sarlomo-Rikala, M., Andersson, L. C.,
Tervahartiala, P., Tuveson, D., Silberman, S. L., Capdeville, R., Dimitrijevic, S., Druker,
B. J., and Demetri, G. D. Effect of the tyrosine kinase inhibitor STI571 in a patient with a
metastatic gastrointestinal stromal tumor. N Engl J Med, 1052: 1052-1056, 2001.
76. Van Oosterom, A. T., Judson, I., Verweij, J., Di Paola, E., Dimitrijevic, S.,
Dumez, H., Scurr, M., Sciot, R., Silberman, S., van Glabbeke, M., and Nielsen, O. S.
Update of the Imatinib (STI571, Glivec) phase I study in gastro intestinal stromal tumours
(GISTs). Proc Am Soc Clin Oncol 21, 82a. 2002.
77. Van Oosterom, A. T., Judson, I., Verweij, J., Stroobants, S., Donato, d. P.,
Dimitrijevic, S., Martens, M., Webb, A., Sciot, R., van Glabbeke, M., Silberman, S., and
Nielsen, O. S. Safety and efficacy of imatinib (STI571) in metastatic gastrointestinal
stromal tumours: a phase I study. Lancet, 358: 1421-1423, 2001.
78. Demetri, G. D., von Mehren, M., Blanke, C. D., Van den Abbeele, A. D.,
Eisenberg, B., Roberts, P. J., Heinrich, M. C., Tuveson, D. A., Singer, S., Janicek, M.,
Fletcher, J. A., Silverman, S. G., Silberman, S. L., Capdeville, R., Kiese, B., Peng, B.,
Dimitrijevic, S., Druker, B. J., Corless, C., Fletcher, C. D., and Joensuu, H. Efficacy and
safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N.Engl.J Med.,
347: 472-480, 2002.
79. von Mehren, M., Blanke, C. D., Joensuu, H., Heinrich, M. C., Roberts, P. J.,
Eisenberg, B. L., Silberman, S., Dimitrijevic, S., Kiese, B., Fletcher, Jonathan A.,
Fletcher, Christopher D. M., and Demetri, G. D. High incidence of durable responses
induced by imatinib mesylate (Gleevec) in patients with unresectable and metastatic
gastrointestinal stromal tumors (GISTs). Proc Am Soc Clin Oncol 21, 403a. 2002.
80. Fletcher, C. D. and Fletcher, J. A. Testing for KIT (CD117) in gastrointestinal
stromal tumors: another HercepTest? Am J Clin.Pathol, 118: 163-164, 2002.
81. Hornick, J. L. and Fletcher, C. D. Immunohistochemical staining for KIT
(CD117) in soft tissue sarcomas is very limited in distribution. Am J Clin.Pathol, 117:
188-193, 2002.
82. Mace, J., Sybil, B. J., Sondak, V., McGinn, C., Hayes, C., Thomas, D., and
Baker, L. Response of extraabdominal desmoid tumors to therapy with imatinib mesylate.
Cancer, 95: 2373-2379, 2002.
83. Miettinen, M., Sobin, L. H., and Sarlomo-Rikala, M. Immunohistochemical
spectrum of GISTs at different sites and their differential diagnosis with a reference to
CD117 (KIT). Mod.Pathol., 13: 1134-1142, 2000.
149
84. Miettinen, M. Are desmoid tumors KIT positive? Am.J.Surg.Pathol., 25: 549-
550, 2001.
85. Montgomery, E., Torbenson, M. S., Kaushal, M., Fisher, C., and Abraham, S.
C. Beta-catenin immunohistochemistry separates mesenteric fibromatosis from
gastrointestinal stromal tumor and sclerosing mesenteritis. Am.J.Surg.Pathol., 26: 1296-
1301, 2002.
86. Sarlomo-Rikala, M., Kovatich, A. J., Barusevicius, A., and Miettinen, M.
CD117: a sensitive marker for gastrointestinal stromal tumors that is more specific than
CD34. Mod.Pathol., 11: 728-734, 1998.
87. Sabah, M., Leader, M., and Kay, E. The Problem With KIT: Clinical
Implications and Practical Difficulties With CD117 Immunostaining.
Appl.Immunohistochem.Mol.Morphol., 11: 56-61, 2003.
88. Bauer, S., Corless, C. L., Heinrich, M. O., Dirsch, O., Antoch, G., Kanja, J.,
Seeber, S., and Schutte, J. Response to imatinib mesylate of a gastrointestinal stromal
tumor with very low expression of KIT. Cancer Chemother.Pharmacol., 51: 261-265,
2003.
89. Van den Abbeele, A. D., Badawi, R. D., Cliche, J., Janicek, M. J., Tetrault, R.,
Spangler, T., Potter, A., Merriam, P., Silberman, S., Dimitrijevic, S., and Demetri , G.
18F-FDG-PET predicts response to imatinib mesylate (Gleevec) in patients with advanced
gastrointestinal stromal tumors (GIST). Proc Am Soc Clin Oncol 21, 403a. 2002.
90. OBrien, S. G., Guilhot, F., Larson, R. A., Gathmann, I., Baccarani, M.,
Cervantes, F., Cornelissen, J. J., Fischer, T., Hochhaus, A., Hughes, T., Lechner, K.,
Nielsen, J. L., Rousselot, P., Reiffers, J., Saglio, G., Shepherd, J., Simonsson, B.,
Gratwohl, A., Goldman, J. M., Kantarjian, H., Taylor, K., Verhoef, G., Bolton, A. E.,
Capdeville, R., and Druker, B. J. Imatinib compared with interferon and low-dose
cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N.Engl.J Med.,
348: 994-1004, 2003.
91. Druker, B. J., Talpaz, M., Resta, D. J., Peng, B., Buchdunger, E., Ford, J. M.,
Lydon, N. B., Kantarjian, H., Capdeville, R., Ohno-Jones, S., and Sawyers, C. L. Efficacy
and safety of a specific inhibitor of the bcr-abl tyrosine kinase in chronic myeloid
leukemia. N Engl J Med, 344: 1031-1037, 2001.
92. Druker, B. J., Sawyers, C. L., Kantarjian, H., Resta, D. J., Reese, S. F., Ford, J.
M., Capdeville, R., and Talpaz, M. Activity of a specific inhibitor of the BCR-ABL
tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic
leukemia with the Philadelphia chromosome. N Engl.J Med., 344: 1038-1042, 2001.
93. Apperley, J. F., Gardembas, M., Melo, J. V., Russell-Jones, R., Bain, B. J.,
Baxter, E. J., Chase, A., Chessells, J. M., Colombat, M., Dearden, C. E., Dimitrijevic, S.,
Mahon, F. X., Marin, D., Nikolova, Z., Olavarria, E., Silberman, S., Schultheis, B., Cross,
N. C. P., and Goldman, J. M. Response to Imatinib Mesylate in Patients with Chronic
Myeloproliferative Diseases with Rearrangements of the Platelet-Derived Growth Factor
Receptor Beta. The New England Journal of Medicine, 347: 481-487, 2002.
94. Cools, J., DeAngelo, D. J., Gotlib, J., Stover, E. H., Legare, R. D., Cortes, J.,
Kutok, J., Clark, J., Galinsky, I., Griffin, J. D., Cross, N. C., Tefferi, A., Malone, J., Alam,
R., Schrier, S. L., Schmid, J., Rose, M., Vandenberghe, P., Verhoef, G., Boogaerts, M.,
Wlodarska, I., Kantarjian, H., Marynen, P., Coutre, S. E., Stone, R., and Gilliland, D. G.
A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic
target of imatinib in idiopathic hypereosinophilic syndrome. N.Engl.J.Med., 348: 1201-
1214, 2003.
95. McArthur, G, Demetri, G., Heinrich, M., Van Oosterom, A., Fletcher, C.,
Corless, C., Fletcher, J. A., Dimitrijevic, S., and Nikolova, Z. Molecular and clinical
analysis of response to imatinib for locally advanced dermatofibrosarcoma protuberans.
Proceedings of ASCO 2003, 195. 2003.
96. Maki, R. G., Awan, R. A., Dixon, R. H., Jhanwar, S., and Antonescu, C. R.
Differential sensitivity to imatinib of 2 patients with metastatic sarcoma arising from
dermatofibrosarcoma protuberans. Int.J Cancer, 100: 623-626, 2002.
97. Rubin, B. P., Schuetze, S. M., Eary, J. F., Norwood, T. H., Mirza, S., Conrad, E.
U., and Bruckner, J. D. Molecular targeting of platelet-derived growth factor B by
imatinib mesylate in a patient with metastatic dermatofibrosarcoma protuberans. J
Clin.Oncol., 20: 3586-3591, 2002.
150
98. Sandberg, A. A. Cytogenetics and molecular genetics of bone and soft-tissue

tumors. Am J Med.Genet, 115: 189-193, 2002.
99. Mackall, C. L., Meltzer, P. S., and Helman, L. J. Focus on sarcomas. Cancer
Cell, 2: 175-178, 2002.
100. Bennicelli, J. L. and Barr, F. G. Chromosomal translocations and sarcomas.
Curr.Opin.Oncol, 14: 412-419, 2002.
101. Deguchi, K. and Gilliland, D. G. Cooperativity between mutations in tyrosine
kinases and in hematopoietic transcription factors in AML. Leukemia, 16: 740-744, 2002.
102. Kelly, L. M. and Gilliland, D. G. Genetics of Myeloid Leukemias. Annu Rev
Genomics Hum Genet, 3: 179-198, 2002.
103. Dash, A. B., Williams, I. R., Kutok, J. L., Tomasson, M. H., Anastasiadou, E.,
Lindahl, K., Li, S., Van Etten, R. A., Borrow, J., Housman, D., Druker, B., and Gilliland,
D. G. A murine model of CML blast crisis induced by cooperation between BCR/ABL
and NUP98/HOXA9. Proc.Natl.Acad.Sci.U.S.A, 99: 7622-7627, 2002.
104. Kelly, L. M., Kutok, J. L., Williams, I. R., Boulton, C. L., Amaral, S. M.,
Curley, D. P., Ley, T. J., and Gilliland, D. G. PML/RARalpha and FLT3-ITD induce an
APL-like disease in a mouse model. Proc Natl.Acad.Sci.U.S.A, 99: 8283-8288, 2002.
105. Fenaux, P., Chastang, C., Chevret, S., Sanz, M., Dombret, H., Archimbaud, E.,
Fey, M., Rayon, C., Huguet, F., Sotto, J. J., Gardin, C., Makhoul, P. C., Travade, P.,
Solary, E., Fegueux, N., Bordessoule, D., Miguel, J. S., Link, H., Desablens, B.,
Stamatoullas, A., Deconinck, E., Maloisel, F., Castaigne, S., Preudhomme, C., and Degos,
L. A randomized comparison of all transretinoic acid (ATRA) followed by chemotherapy
and ATRA plus chemotherapy and the role of maintenance therapy in newly diagnosed
acute promyelocytic leukemia. The European APL Group. Blood, 94: 1192-1200, 1999.
106. Sohal, J., Phan, V. T., Chan, P. V., Davis, E. M., Patel, B., Kelly, L. M.,
Abrams, T. J., OFarrell, A. M., Gilliland, D. G., Le Beau, M. M., and Kogan, S. C. A
model of APL with FLT3 mutation is responsive to retinoic acid and a receptor tyrosine
kinase inhibitor, SU11657. Blood, 101: 3188-3197, 2003.
107. Nielsen, T. O., West, R. B., Linn, S. C., Alter, O., Knowling, M. A., OConnell,
J. X., Zhu, S., Fero, M., Sherlock, G., Pollack, J. R., Brown, P. O., Botstein, D., and Van
de Rijn, M. Molecular characterisation of soft tissue tumours: a gene expression study.
Lancet, 359: 1301-1307, 2002.
Chapter 9
Targeting other abnormal signaling pathways in

sarcoma: EGFR in synovial sarcomas, in
liposarcomas
Jean-Yves Blay 1,2, Isabelle Ray-Coquard3, Laurent Alberti2, Dominique
Ranchre4
1. Unit dOncologie Mdicale

Hpital Edouard Herriot
Place dArsonval, 69003 Lyon
2. Equipe Cytokine et Cancer, Unit INSERM 590

Centre Lon Brard
28, rue Lannec, 69008 Lyon
3. Dpartement de Mdecine
Centre Lon Brard, 28, rue Lannec
69008 Lyon
4. Departement of Pathology
Centre Lon Brard, 28, rue Lannec
69008 Lyon
Correspondance to:
JY Blay
Equipe Cytokine et Cancer, Unit INSERM 590
Centre Lon Brard
28, rue Lannec 69008 Lyon
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1 INTRODUCTION
Soft tissue sarcomas (STS) are a family of rare malignant

diseases originating from mesenchymal cells whose incidence is around
2-3/100000 per year and causing to 2% of the total cancer-related
mortality. According to the EUROCARE data (1) the 5-year survival in
Europe of adult STS (excluding visceral ones) averages 60%. Further
improvements in the treatment of these rare tumours are therefore needed.
2 SOFT TISSUE SARCOMAS: UNIFORM

TREATMENTS FOR HETEROGENOUS DISEASES?
There are multiple histological subtypes of STS and histological

classifications have actually evolved constantly in the past years (2) as a
consequence of the refinements of immunohistochemical, and molecular
investigations. New molecular techniques, such as conventional
cytogenetics, molecular cytogenetics, comparative genomic
hybridization, or sequence of an overexpressed gene have enabled to
identify specific molecular alterations associated with discrete
histological subtypes (2,3). These specific molecular alterations are now
used for diagnosis purposes: a diagnosis of Ewing family of tumors is
now based on the detection of one of the specific translocations involving
the EWS gene on chromosome 22. Other examples are detailed in
different chapters of this book. These tools have enabled to profoundly
modify the classifications of these tumors, with the identification of
previously unrecognized subtypes of sarcoma, eg. gastrointestinal stromal
tumors (GIST), or the splitting of malignant fibrous histiocytoma
(MFH), into various new of other histological subtypes.
Despite of these major improvement in the understanding of the
pathogenesis of these sarcomas, until recently, most soft tissue sarcomas
subtypes were grouped for the purpose of treatment. This remains the
case regarding local treatment : standard treatment in localized tumors is
generally a wide surgical excision combined with radiotherapy,
whenever feasible, or in some cases radical surgery, i.e. compartmental
resection or amputation of the extremity. Pre or post-operative
radiotherapy reduces the rate of local recurrence significantly (4).
Although the effect adjuvant chemotherapy has been studied in several
studies, a recent international meta-analysis indicated an effect on
progression free survival but no significant effect on overall survival (5).
Chemotherapy is in contrast widely used in the treatment of
advanced disease, basically with a palliative intent. Until recently, similar
chemotherapy regimens were given to all types of sarcomas: doxorubicin
and ifosfamide, yielding response rates in the range of 10-25% in
monotherapy, of 20-35% in combination therapy are the most frequently
prescribed agents (6). The systemic treatment of locally advanced or
metastatic soft tissue sarcoma (STS) remains however unsatisfactory,
with few long lasting response and only a small proportion of patients
achieving long term survival (7,8). However, it has become clear that
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some subtypes have a distinct behavior with regards to response to

chemotherapy. For instance, and as described elsewhere, gastrointestinal
stromal tumors are resistant to all known cytotoxic agents, but exhibit
specific sensitivity to the c-kit tyrosine kinase inhibitor imatinib (9,10).
Synovial sarcomas, but not leiomyosarcomas, are characterized by high
response rates to ifosfamide (6). We are therefore now moving from an
undifferenciated therapy for all sarcoma subtypes to specific
chemotherapy regimens for distinct sarcoma subtypes.
One of the key questions regarding clinical sarcoma research in
the next years is whether the knowledge gained in the understanding of
the neoplastic transformation of various sarcoma subtype will be useful in
routine clinical practice to select appropriate targets for the treatment of
these patients. This has already been the case for tumors of the Ewing
family, in which molecular diagnosis leads to specific therapeutic
protocol. This has also been the case for the targeting of KIT and PDGFR
in GIST and few other sarcoma subtypes (9-11). The purpose of this
chapter is to discuss whether the recent identification of molecular
alterations in specific sarcoma subtypes could lead to specific targeted
treatment in these subtypes. Two histological subtypes, synovial
sarcomas and liposarcomas, will be presented here and analyzed for
possible new therapeutic strategies based on recent knowledge on the
specific bioloy to these subtypes.
3 SYNOVIAL SARCOMA
Synovial sarcomas represent 5 to 10% of all sarcoma subtypes,

and is the fourth most common type of sarcoma. Synovial sarcoma occur
at all ages but predominantly in adolescent and young adults, between 15
and 40 years mostly with a slight male predominance (12-14). Typically,
this tumor occurs in the lower (60%) or upper limbs (20%), in para-
articular areas of the tendon sheaths and joints, but can be encountered in
regions with no relation to synovial structure, such as the head and neck
(8%), thoracic and abdominal wall (8%) or even lungs and heart. Despite
its name, there is no evidence that tumor cells come from synovial tissues
and actually, some authors have suggested to rename them
carcinosarcomas of the soft part (12). Synovial sarcomas comprises
actually different histological subtypes: the biphasic type (Figure 1A),
with distinct spindle cell and epithelioid cell components, the
monophasic fibrous type, with no detectable epithelial cell component,
and the rare monophasic epithelial subtype. Calcifying and poorly
differentiated synovial sarcomas are also distinguished. Synovial
sarcomas are grade 2 or 3 tumors according to the FNCLCC grading (12-
14).
Local treatment follows the general rules of STS treatment with
wide excision macroscopically and microscopically complete excisions
followed by adjuvant radiotherapy chemotherapy , with or without
adjuvant chemotherapy. Reccurence occur in 30 to 70% of patients in
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most series, mostly in the lung, lymph nodes and bones. 5 year survival
rates range between 50 and 80% are reported in most series (12-14).
3.1 Specific translocations of synovial sarcoma
Specific t(X, 18)(p11.2;q11.2) translocations have been reported

to be associated with synovial sarcomas. These translocations represent a
hallmark of the tumor and is probably now a gold standard for the
establishment of a diagnosis of synovial sarcoma in 2003 (12,15,16). It
has for instance been observed in 100% and 96% of biphasic and
monophasic synovial sarcomas in a series of 221 cases using RT PCR
(17). This translocation involves the SYT gene on chromosome 18p11,
and 3 of the 6 members of the SSX gene family on chromosome Xq11,
namely SSX1 or SSX2 genes, and less frequently the SSX4 gene (16,17).
These translocations can be detected using RT PCR or FISH techniques,
on fresh or paraffin embedded tissues or even on FNA product with a
high level of specificity and sensitivity. The nature of the SSX partner in
the t(X, 18) translocation has been found to be correlated with distinct
clinical presentation (smaller size for SSX2, female gender for SSX2)
The biphasic subtype may more frequent harbor the translocation with the
SSX1 partner, although this has not consistently been found (20).
Synovial sarcomas with translocations involving SSX2 have also been
155
reported to be associated with an improved outcome in particular in

patients with localized disease (18-21).
Other genetic changes can be detected in synovial sarcomas,
including complex losses and gains of chemosomes, some of which
(gains of 8p) being correlated with poor risk factors. Important genes
reported to be altered in structure or expression levels in synovial
sarcomas include the RB, p53 and p21 genes, overexpression of the
antiapoptotic bcl-2 protein, overexpression of HER2/neu and the MET
tyrosine kinase in the epithelial cell component compartment, a
phenomenon which may contribute to epithelial mesenchymal transition
with is a hallmark of these tumors (16). It is interesting to note in this
sarcoma with an epithelial component that E-cadherin expression is
frequently lost while translocation of the catenin in the nucleus is also
frequently observed (16). These alterations are not consistently found in
all tumors and are felt to represent late genetic alterations correlated with
tumor progression. It is also likely that some of these phenotypic or
genetic characteristics observed in synovial sarcomas are controled by the
protein product of the t(X, 18) translocation. Indeed, the product of the
genes rearrangements, SYT-SSX1 and SYT-SSX2, are transcription
factors which also activates a discrete set of genes (16).
3.2 Protein products of fusion genes regulate gene

expression
The identification of specific translocations associated with

sarcoma subtypes has enabled to improve the understanding of the
deregulation of proliferation and apoptotic pathways in specific sarcomas
subtypes. Usually, the translocations observed in soft tissue sarcomas
fuse a gene with a wide pattern of expression to a gene encoding for a
protein with a more restricted pattern of expression, leading to the
creation of a new transcriptional activator with abnormal transcription
function. For instance, it has been shown that the EWS-Fli1 fusion gene
product induces the transcription of specific set of genes in the Ewing
family of tumors, including PDGF C, myc, and MFNG, a soluble factor
of the Fringe family with transforming properties (22). Protein product of
fusion genes are therefore capable to stimulate the transcription of genes,
which may play an important role in tumor proliferation.
Similarly, the t(X,18) translocations results in a gene encoding
for a protein in which the addition of the C terminal domain of ssx to the
syt protein redirects the syt activation motif to different new genes,
presumably with important transforming properties. The SYT gene is
expressed in all embryonic and adult tissue, while the SSX genes have a
much more discrete pattern of expression, in the testis and thyroid, and
actually are members of the Cancer Testis Antigen family. The SYT-SSX
translocation products contain both activation (in the syt part of the fusion
protein) and repression motifs (in the ssx part) for the transcription of
target genes. The gene product is detectable in the nucleus and inteferes
with the SNF2/Brahma (brm) protein, a phenomenon which may be
156
essential for transformation in synovial sarcoma cells (16). Specific

target genes for the syt-ssx fusion protein have however not been
precisely identified. The SYT-SSX gene product appears to act as a
transcriptional inhibitor for some genes, in particular DCC, a tumor
suppressor gene involved in cell adhesion. There is no direct evidence
however that it triggers EGFR transcription. The specific pattern of gene
expressed in synovial sarcoma has actually been investigated recently
using expression microarray strategy.
3.3 Gene expression patterns across histological subtypes

of sarcomas
Four recent studies have investigated gene expression patterns in

synovial sarcomas subtypes using microarrays (23-25). A summary of
genes found overexpressed in these four papers is presented in Table 1
Nielsen et al (23) used a 42K gene array and identified a specific
set of genes associated with the histological subtype of synovial sarcoma.
Reference RNA was isolated from a pool of 11 cell lines. 13 genes were
found overexpressed in this specific subtypes including the relevant
SSX3 and SSX4 genes, previously identified in translocation variants of
synovial sarcomas, cytokines BMP 2 and BMP7), and
molecules involved in signal transduction such as and EGFR. The
mechanisms through which these genes are overexpressed specifically in
synovial sarcomas remain unclear.
Other members of the EGFR family of genes may be
overexpressed in synovial sarcomas: the HER2/neu gene was found to be
overexpressed in a series of 37 synovial sarcoma tumor samples tested
with expression microarrays containing 6548 sequence-verified human
cDNAs in the study reported by Allander et al (24). In this study
reference RNA was obtained from an osteosarcoma cell line.
In the paper by Nagayama et al (25), using a 23K micrarray, 26
genes were found specifically overexpressed in synovial sarcomas, not
including EGFR. Of note in this report, gene expression pattern of
synovial sarcomas was found to be close to that of MPNST, including a
large number of neural crest specific genes such as ephrins, N-myc or
neurofilaments. In a fourth recent paper by Lee et al (25), 48 genes, for
most of them different to those described in the three previously
mentionned works, enabled to distinguish synovial sarcomas from other
sarcoma subtypes.
In these 4 studies, overexpressed gene were found in the cytokine
and cytokine receptor genes, gene involved in transduction signal and
regultation of expression, adhesion molecules, matrix components and
cytoskeleton. Actually, only few genes were found overexpressed in
synovial sarcomas in more than one study: these were the SSX4 gene
(possibly because of its homology with the overexpressed SYT-SSX), the
CRABP-1, PRAME- a melanoma Ag, IGF2, and a neuronspecific protein
(23-26) (Table 1). The differences in the gene expression pattern of
synovial sarcoma in these different works (single cell line, a panel of
different tumor cell lines) may be related to various technical issues,
157
including the nature of reference cDNA used, the microarrays used in

these works. It is however likely that the gene described in these studies
contribute to tumor progression in synovial sarcoma. Whether they are
3.4 EGFR protein expression in synovial sarcomas
EGFR is actually the first member of a large family of growth

factor receptor tyrosine kinases that share a common structure composed
of an extracellular ligand binding domain, a short transmembrane
domain, and an intracellular domain that has tyrosine kinase activity.
Binding of the cognate ligand, for example, EGF or transforming growth
factor to the extracellular domain of EGFR initiates a signal
transduction cascade that can influence many aspects of tumor cell
biology including growth, survival, metastasis, and angiogenesis, as well
as tumor cell sensitivity to chemo- and radio-therapeutic drugs. Tyrosine
phosphorylation provides docking sites on the EGFR for recruitment of
proteins that are either direct substrates for EGFR-mediated
phosphorylation, or adaptor proteins that link the receptor to a cascade of
downstream biochemical reactions, for example the ras-raf-MAP-fos
pathway, which drives tumor cell proliferation (27,28).
Whether EGFR is indeed present at the surface of synovial
sarcoma cells needed anyway confirmation using immunohistochemistry
To our knowledge, there is no reported study investigating the
expression of EGFR in fresh tumor samples of synovial sarcomas. In our
hands, all 15 molecularly confirmed synovial sarcomas tested were found
to have detectable expression of EGFR at the cell surface. Expression
levels however vary considerably between each tumors, but also between
the primary tumor and their metastasis. Figure 1 presents the expression
of EGF using the Dako Ab in a patients with translocation positive
biphasic synovial sarcoma. There is no demonstration to our knowledge
that EGFR is expressed in its phosphorylated form in synovial sarcomas.
However, the presence of this protein suggest that it may be an interesting
target for a specific tyrosine kinase inhibitor in this tumor.
We selected the HER1 protein to evaluate microarray-oriented

targeted therapy in synovial sarcomas because 1) this gene is a well
known protoncogene for a wide range of tumors, and therefore may
contribute to transformation process and progression of synovial
sarcomas. 2) EGFR tyrosine kinase inhibitors were available in clinical
practice.
158
Table 1 : Examples of enes preferentially expressed in synovial sarcomas as compared to

other sarcomas in four different studies
Family Function Ref
Cytokine and receptors
Axl Receptor tyrosine kinase 24

BMP-2 Cytokine 23
BMP-4 Cytokine 26
BMP-7 Cytokine 23
Endothelin 3 Cytokine 25
EPHA4 Ephrin A4 25
EPHB1 Ephrin B1 26
EPHB3 Ephrin B3 25
FGF9 Fibroblast growth factor 26
FGF18 Fibroblast growth factor 25
FGFF3 FGF receptor 24
Frizzled homolog 10 Receptor 25
GFRA1 GDNF receptor 26
HER1/EGFR Receptor TK for EGF 23
HER2 Receptor TK 24
IGF2 Cytokine 24,26
IGFBP2 IGF binding protein 24
TGFB2 Cytokine 23
WNT inhibitory factor Ligand inhibitor 25
Transcription factors and related
CRABP-1 Transport of retinoic acid 23,24,25

MYB Transcription facor 26
N-MYC Transcription factor 25
NCOA3 Nuclear receptor co-activator 26
PGK1 Transcription regulator 24
Nuclear receptor 26
Nuclear receptor 23
SSX3 RNA binding protein 23
SSX4 RNA binding protein 23,26
SOX9 Transcription factor 24
TLE2 Nuclear protein 24
Transduction signal machinery
CDK10 Cdc2-related kinase 24

CNL1 GTP binding protein 24
MAPK10 MAP kinase 26
NFAT4 Nuclear factor 24
PDK2 Kinase 24
PTK7 Tyrosine kinase 24
PTPN2 Nuclear Phosphatase 24
Matrix proteins
COLIXa3 Collagen IX 25
COLXVIII A1 Collagen XVIII 24
THSB3 Thrombospondin 3 24
159
Table 1 : Genes preferentially expressed in synovial sarcomas as compared to

other sarcomas in four different studies (contd)
Adhesion molecules
CDH1 E Cadherin 26
PCAD P Cadherin 25
GYPB Glycophorin B 26
ICAM1/CD54 Ig gene superfamily 26
ITGAM Integrin 26
SELL L selectin 26
Cytosketon
ACTA2 Alpha actin 24
ARPC1B Actin polym. regulator 24
BAT8 Actin associated protein 24
CTNNA Alpha catenin 24
Ectodermal-neural cortex 1 Actin associated protein 23
MSN Moesin 24
Neurofilament heavy polypeptide, - 25
Neuron specific proteins Hs 79404 - 23,25
Spectrin Actin associated protein 26
Other neuroectodermic specific proteins

CNTNAP l Contactin associated prot. 24
CST3 Cystatin 3 24
Dachshund Neural development 23
DRPLA Neural developemet 24
DYT1 Dystonia 26
Granin-like neuroendocrin peptide - 25
IRX5 homeobox protein 5 23
MSX2 homeobox cont gene 23
OLFM1 Olfactomedin 24
FRAME Melanoma Ag 24,25
Serin protease inhibitor Kunitz type, 2 Serin protease inhibitor 25
SERPIN B6 Serin protease inhibitor 24
3.5 Different types of targets for targeted therapy of

cancer
Until recently, targeted therapy of solid tumors could be

classified according in three different categories, according the the type
of target:
1) Early targets displaying activating mutation(s) which play a
causal role in the oncogenic process, acting presumably at an early step
on oncogenesis;
2) Late Targets whose mutations occurs occuring at a later step
of tumor progression;
3) Uncertain targets which are expressed in tumor cells, but
whose role in the oncogenic process is uncertain in the pathogenesis of
the tumor.
160
1) The first example of early target is the efficacy of imatinib

for the treatment of GIST As mentioned in a different chapter, GIST cells
almost consistently contain activating mutations of the KIT gene. These
mutations play an early and essential role in the oncogenesis of this
tumor. This observation led investigators to investigate a specific
inhibitor of the kit gene for the treatment of this tumor. The high response
rate to a KIT tyrosine kinase inhibitor in 3 consecutive studies (9,10), has
provided the first demonstration of the relevance of specific molecular
approaches in solid tumors.
2) Treatment of breast carcinoma with Herceptin is an example
of a late target. Only 15-25% of breast cancer are susceptible to this
treatment which gives a relatively low response rate as single agent, but
prolongs progression free and overall survival in combination with
chemotherapy.
3) Regarding uncertain targets, PDGFR, an other target of
imatinib, is almost consistently expressed at the surface of sarcoma cells,
but its exact role is unknown in most sarcomas. The treatment of non
GIST sarcomas with imatinib, with the hope of targeting this receptor
whose role in the oncogenic process is uncertain, gives an example of the
third type of target. In this situation, only occasional signs of antitumor
activity were reported (29).
3.6 Targeting EGFR pathway in synovial sarcomas?
In view of the different tumor models studied so far in clinical

practice, targeted therapy is therefore much more efficient when the
target plays an early role in the oncogenic process. The observation of a
consistent overexpression of the EGFR gene in synovial sarcoma in the
study by Nielsen et al suggest that this protein may play an important role
in the process of progression, possibly through an overexpression process
driven by the product of the fusion gene. In this situation , however, the
target is presumably a secondary event, whose over expression is the
consequence of the initial genetic alteration caused by the translocation.
To test this hypothesis, the Soft Tissue and Bone Sarcoma Group
of EORTC is currently investigating the antitumor activity of a HER1
tyrosine kinase inhibitor, ZD1839, in synovialosarcoma overexpressing
the HER1 receptor.
3.6.1 ZD1839 in human cancer
ZD1839 is a signal transduction inhibitor of the epidermal growth

factor receptor (EGFR) tyrosine kinase, and has been developed as an
oral anti-tumor agent (30). ZD1839 has been found capable to delay
growth and, at higher doses, cause regression in a wide range of other
tumor xenografts. NSCLC express detectable levels of EGFR in 60-80%
of the cases using immunohistochemistry, and, although the exact
oncological role of EGFR in NSCLC remains unclear, ZD1839 was
investigated in this model (31,32). In NSCLC clinical trials, clinically
significant objective responses have been seen for both the 250 mg dose
161
and 500 mg dose, in patients pretreated with platinum compounds and/or

taxanes. Objective responses were seen in 10% of patients with large or
bulky tumor and generally occurred by Day 28, although responses
continued to occur for up to 4 months. Clinically significant
improvements in disease-related symptom rates were also seen at both
doses. Rate of disease-related symptom improvement was substantial
with slightly more than 40% of the patients experiencing improvement
for at least one month. The median time to symptom improvement
occurred within 8 to 10 days. Approximately 80% of patients who had
symptom improvement were still showing an improvement at the time of
data cutoff. In addition to the efficacy findings, quality of life (QoL)
improvement was seen in a significant proportion of patients and was
consistent with disease-related symptom improvement, which reflects the
lack of significant therapy-related toxicity observed. Tumor response and
stable disease were associated with disease-related symptom
improvement in the majority of patients; while association of better
survival with objective response, disease control and disease-related
symptom control was seen.
3.6.2 ZD1839 in EGFR+ synovial sarcomas
EGFR is one of the most overexpressed gene in synovial

sarcomas in the study by Nielsen et al, and its protein product is
detectable at the surface of most synovial sarcomas tested. Although the
exact role of EGFR in the transformation process of synovial sarcomas,
and the link between its expression and the specific genetic alterations
associated with SS are unclear, EGFR may be a secondary target playing
a role in tumor progression. The EORTC STBSG has initiated a clinical
trial investigating ZD1839 for the treatment of synovial sarcomas
expressing detectable EGFR at their cell surface. Translational research
with investigation of the phosphorylation status of EGFR, as weel as gene
expression profiles in SS prior and after ZD1839 treatment in scheduled.
The trial is ongoing. This is to our knowledge the first example of
conception of a targeted therapy trial based on results of expression
microarrays demonstrating the presence of the target.
4 LIPOSARCOMA
The second example of such strategy is liposarcoma.

Liposarcoma is one of the most common histological subtype of
malignant soft tissue tumor occuring during adult life. 10 to 18% of
malignant soft tissue tumors are liposarcomas, and this tumor may occur
at virtually all ages (33). Its includes a wide variety of different
histological subtypes with considerable heterogeneity regarding
histological grade, molecular abnormalities, and clinical behavior.
Liposarcoma may be classified in 3 major histological subtypes:

162
1) The first subgroup comprises lipoma-like, well differentiated

(WDLPS) and dedifferentiated liposarcomas (DDLPS), which represent
more than one half of all LPS (Figure 2). These occur preferentially in the
limbs, and in the retroperitoneum, less frequently in other parts of the
body. 6 to 30% of these tumors exhibit a dedifferenciated phenotype,
which increases the risk of metastatic relapse. In contrast, WDLPS are
associated maily with a risk of local relapse. The risk of relapse and
strongly correlates to the topography of the tumor. While limb LPS are
most frequently efficiently treated, retroperitoneal LPS tend to reccur
almost constantly when followed-up is sufficiently long, with a resulting
risk of death which is considerably increased as compared to limb
locations. Of note the number of liposarcomas belonging to this category
may be higher than previously thought.
2) Myxoid and round cell liposarcoma, although they were given

separated designation in the WHO classification of soft tissue tumors,
share a similar set of molecular alterations and are distinguished
essentially by the percentage of small round cell within the tumor mass.
M/RLPS have a much more agressive behavior, with an increased risk of
metastasis as compared to the remaining subtypes. In a large series of
patients treated in a single institution and including 95 patients, the risk of
metastasis ranged from 23% to 58% according to the percentage of round
cell population in the tumor. Metastasis sites are somewhat different that
163
those reported in other liposarcomas and soft tissue sarcomas: M/RLPS

tend to relapse with metastasis in the soft part, in the bone and the lung.
3) Pleomorphic liposarcoma represent the third group of

liposarcoma, including 10 to 15% of all liposarcomas. This is a less
frequent tumor, less well characterized. These tumors may occur both in
the limbs and retroperitoneum, later in life, and are associated with a high
risk of relapse and death.
4.1 Heterogeneity of molecular alterations in synovial

sarcomas
The two largest subgroups of liposarcoma are characterized by

very different genetic alterations.
M/RLPS exhibit a typic reciprocal translocation
t(12,16)(q13,p11) resulting in the fusion of the CHOP gene on
chromosome 12, with the TLS gene on chromosome 16 (33,34). Less
frequently, alternative translocations t(12, 22) with a different partner for
CHOP are observed. CHOP is a DNA binding protein , while TLS/FUS
is an RNA binding protein capable to interact with several nuclear
receptors for thyroid , steroids and retinoid receptors, a combination
reminding the t(11,22) translocation associated with Ewing sarcomas.
These translocations yield different transcripts, in particular a type II
transcript capable to inhibit the adipocytic maturation of preadipocytes
when transfered into adipocytes and to promote a transformed pheotype
in these cells (35).
In contrast, well differentiated and dedifferentiated liposarcomas
exhibit typical genetic alterations characterized by giant marker and ring
chromosomes. These giant markers or ring chromosomes consistently
contain amplified portion of the 12q13-q15 portion of chromosome 12.
This region contains a number of gene playing a potential role in the
oncogenesis of these tumors in particular the mdm2 gene, CDK4, GLI1,
HMGI-C, SAS).The amplification of this region may be useful to
distinguish benign adipose tissue from lipoma like, well differentiated
liposarcoma
4.2 The PPAR gamma pathway in adipogenesis and

liposarcoma
Interestingly, despite a distinct set of genetic abnormalities, all

liposarcoma subtypes still express an important nuclear receptor involved
in adipocyte differentiation.
The nuclear receptor peroxysome proliferator activated receptor
gamma is an essential signaling molecule for the process of
adipocyte differentiation. This nuclear receptor binds several natural or
synthetic ligands, such as prostaglandins, and the thiazolidindione family
of antidiabetic drugs including troglitazone, rosiglitazone. It interacts
with to form a DNA binding complex which is capable to promote
164
adipocyte differentiation (36,37). Indeed, this complex triggers the

transcription of specific adipocytic gene, and its transfection into
fibroblasts is capable to stimulate the ectopic expression of adipocytic
genes and to promote the acquisition of an adipocytic phenotype (37).
The combination of agonists of the and pathways exert a
synergistic activity for adipocytic differentiation.
4.3 Targeting PPAR-gamma pathway in liposarcoma to

force differentiation
has been reported to be expressed in each of the major

histological subtypes of liposarcoma. It is a physiological component of
the molecular apparatus for adipocytic differentiation. Synthetic ligands
of this receptor are capable to promote the terminal differentiation of
liposarcoma cell lines, with an intracellular accumulation of lipids, and
cell cycle arrest (39). A synergy with agonist was observed in
vitro. This observation strongly suggested that these ligands could enable
to promote the differentiation of liposarcoma cell in vivo, in the clinical
setting (36). This induction of differentiation associated with cell cycle
arrest could be of therapeutic benefit, since 1) WDLPS have a much
slower progression rate and indolent course than DDLPS, 2) tumor
stabilization in soft tissue sarcoma has similar impact on overall survival
than partial response (39). The objective is therefore not to block a kinase
will possible growth promoting properties, but to force the tumor cell
towards their physiological differentiation pathway, on which they are
blocked because of neoplastic transformation.
In vivo, Demetri and colleagues (39) fist investigated the
capacity of a agonist, troglitazone, to modulate the
differentiation liposarcoma cells in vivo in 3 patients. Troglitazone was
found capable to induce LPS diffenciation in vivo, as evaluated by
histological reassessment showing an important intracellular lipid
accumulation,, NMR analysis of triglyceride content in the tumor,
reduction of the Ki67 positivity, induction of the expression of fat
specific genes, such as aP2 and adipsin (39). On MRI imaging of the
tumor, troglitazone induced subtle changes in tumor signal, compatible
with intratumoral lipid accumulation. Still no responses were observed,
but tumors exhibited a less aggressive phenotype with a slow growth or
stabilization, consistent with the effects observed in tumor cells in vitro.
Another trials on 49 patients from the same group was reported in an
abstract form and confirmed these biological observations (40). Median
progression free survival for WDLPS, DDLPS, MRLPS and pleomorphic
liposarcoma were 408, 81, 92 and 89 weeks respectively. There results
are encouraging and further investigation of this family of compounds in
liposarcoma is therefore mandatory.
165
5 CONCLUSIONS: IDENTIFY CAUSAL

MOLECULAR ALTERATION IN SARCOMAS
The observation that imatinib can induce a dramatic response and

growth arrest rate in GIST associated with KIT mutation provided the
first demonstation of the efficacy of a targeted therapy directed towards
the initial molecular event causing tumor transformation, soon followed
by the demonstration of its efficacy in dermatofiborsarcoma protuberans
with a t(17,22) translocation yielding high levels of autocrine PDGF
production (41). However, only a limited number of solid tumors have
such a characteristic simple and targetable activating molecular
alteration,and many are sarcomas. Indeed, specific molecular alterations
associated with specific sarcoma subtypes have been characterized in a
growing number of histological subtypes of sarcomas in the last years:
these include specific reciprocal translocation resulting in an oncogene
with translation activating properties, or more complex massive losses
and gains of chromosomes in other histological subtypes.
It is therefore essential to identify the target genes of the
abnormal transcriptional activators encoded by most protein products of
sarcoma translocations. Among these, it is necessary to identify which
genes play an essential role in the transformation process of these
sarcomas. With the identification of these genes, specific treatment
directed towards their protein products may be proposed
In the present paper, an example of this strategy is presented,
with the clinical trial of the EGFR1 inhibitor ZD1839 in synovial
sarcoma. Although the direct link between the malignant transformation
and EGFR overexpression has not been formally demonstrated, this trial
will enable to establish whether this inhibitor exerts significant growth
inhibitory properties in this tumor. Presumably, it will be necessary to
combine different inhibitors in such strategy. Other potential targets may
be the ras family of proteins, using farnesyl transferase inhibitors in
particular when this pathway is constitutionally activated through NF1
mutation. The inhibition of cdk4 in liposarcomas harbouring the 12p13-
15 amplification is another potential target.
Another stragy is to try to circumvent the differentiation blockade
observed in most cancer cells by triggering pharmacologically the
physiological pathways of differenciation when they remain intact in the
tumor cell. The example of agonist in liposarcomas provided
here is the first piece of evidence showing that this strategy is relevant in
vivo in cancer patients. Although no responses were observed, growth
arrest could be feasible using this strategy. The identification and
investigation of new and more active activators in liposarcoma
remains a relevant strategy for the control of these tumors.
166
6 REFERENCES
1. Storm H.H. Survival of adult patients with cancer of soft tissues or bone in Europe.
Eur J Cancer 1998; 34:2212-2217.
2. Pathology and Genetics. Tumors of Soft Tissue and Bone.Christopher
DM Fletcher, K. Krishnan Unni, Fredrick Mertens eds. Lyon: IARC press
3. Fletcher, Jonathan. Cytogenetic analysis of soft tissue tumors. in Enzinger and
Weisss Soft Tissue Tumors 4th. edition , Sharon Weiss, John R. Goldblum eds. St-
Louis, MO: Mosby, 2001.
4. Leibel S.A., Tranbaugh R.F., Wara W.M. et al.: Soft tissue sarcomas of the
extremities. Survival and patterns of failure with conservative surgery and postoperative
irradiation compared to surgery alone. Cancer 50: 1076-1083, 1982.
5. Adjuvant chemotherapy for localised resectable soft-tissue sarcoma of adults: meta-
analysis of individual data. Sarcoma Meta-analysis Collaboration. Lancet 350: 1647-54
6. Nielsen OS, Blay JY, Judson IR, van Glabekke M, Verweij J, van Oosterom A,.
Metastatic Soft Tissue Sarcoma in adults. Prognosis and treatment options. Am. J. Cancer
2003:2:1-12.
7. Van Glabbeke M., van Oosterom AT., Oosterhuis JW et al. Pronostic factors in
advanced soft tissue sarcoma (STS): analysis of 2185 patients treated with doxorubicin
containing first line regimens- A European Organization for Research and Treatment of
Cancer Soft Tissue and Bone Sarcoma Group study. J. Clin. Oncol. 1999: 17:150-157
8. Blay JY, van Glabbeke M, Verweij J, et al. Advanced soft-tissue sarcoma: a disease
that is potentially curable for a subset of patients treated with chemotherapy. Eur J
Cancer. 2003; 39: 64-9.
9. Van Oosterom A, Judson I, Verwej J, et al. Safety and efficacy of Imatimib (STI-571)
in metastatic gastrointestinal stromal tumors: a phase I study. Lancet 2001, 358: 1421-3
10. Demetri GD, von Mehren M, Blanke CD, et al. Efficacy and safety of imatinib
mesylate in advanced gastrointestinal stromal tumors. N Engl J Med 2002; 347: 472-80.
11. Maki RG, Awan RA, Dixon RH, Jhanwar S, Antonescu CR. Differential sensitivity to
imatinib of 2 patients with metastatic sarcoma arising from dermatofibrosarcoma
protuberans. Int J Cancer 2002; 100: 623-6.
12. Weiss SW, Goldblum JR. Malignant soft tissue tumors of uncertain type. in
Enzinger and Weisss Soft Tissue Tumors 4th. edition, Sharon Weiss, John R. Goldblum
eds. St-Louis, MO: Mosby, 2001.
13. Trassard M, Le Doussal V, Hacene K, et al. Prognostic factors in localized primary
synovial sarcoma: a multicenter study of 128 adult patients. J Clin Oncol 2001; 19:525-
34.
14. Okcu MF, Munsell M, Treuner J, et al. Synovial sarcoma of childhood and
adolescence: a multicenter, multivariate analysis of outcome. J Clin Oncol. 2003; 21:
1602-11.
15. Ladanyi M. Fusions of the SYT and SSX genes in synovial sarcoma. Oncogene. 2001;
20: 5755-62.
16. Sandberg AA, Bridge JA.Updates on the cytogenetics and molecular genetics of bone
and soft tissue tumors. Synovial sarcoma. Cancer Genet Cytogenet 2002;133:1-23
17. Guillou L, Coindre J, Gallagher G, et al. Detection of the synovial sarcoma
translocation t(X;18) (SYT;SSX) in paraffin-embedded tissues using reverse
transcriptase-polymerase chain reaction: a reliable and powerful diagnostic tool for
pathologists. A molecular analysis of 221 mesenchymal tumors fixed in different
fixatives. Hum Pathol. 2001; 32:105-12.
18. Kawai A, Woodruff J, Healey JH, Brennan MF, Antonescu CR, Ladanyi M. SYT-
SSX gene fusion as a determinant of morphology and prognosis in synovial sarcoma. N
Engl J Med 1998 ; 338:153-60
19. Nilsson G, Skytting B, Xie Y, et al. The SYT-SSX1 variant of synovial sarcoma is
associated with a high rate of tumor cell proliferation and poor clinical outcome. Cancer
Res. 1999; 59:3180-4.
20. Mezzelani A, Mariani L, Tamborini E, et al. SYT-SSX fusion genes and prognosis in
synovial sarcoma. Br J Cancer 2001; 85: 1535-9
21. Ladanyi M, Antonescu CR, Leung DH, et al. Impact of SYT-SSX fusion type on the
clinical behavior of synovial sarcoma: a multi-institutional retrospective study of 243
patients. Cancer Res. 2002 Jan 1;62(1):135-40.
167
22. De Alava E., Gerald W. Molecular diagnosis of the Ewing family of tumors. J Clin
Oncol 2000;18:204-213.
23. Nielsen TO, West RB, Linn SC, et al. Molecular characterisation of soft tissue
tumours: a gene expression study. Lancet 002;359: 1301-7
24. Allander SV, Illei PB, Chen Y, et al. Expression profiling of synovial sarcoma by
cDNA microarrays: association of ERBB2, IGFBP2, and ELF3 with epithelial
differentiation. Am J Pathol. 2002;161:1587-95.
25. Nagayama S, Katagiri T, Tsunoda T, et al. Genome-wide analysis of gene expression
in synovial sarcomas using a cDNA microarray.
Cancer Res. 2002; 62: 5859-66.
26. Lee YF, John M, Edwards S, et al. Molecular classification of synovial sarcomas,
leiomyosarcomas and malignant fibrous histiocytomas by gene expression profiling. Br J
Cancer. 2003; 88: 510-5.
27. Schlessinger J. Ligand-induced, receptor-mediated dimerization and activation of EGF
receptor. Cell 2002; 110: 669-72
28. Olayioye MA, Neve RM, Lane HA, Hynes NE The ErbB signaling network: receptor
heterodimerization in development and cancer. EMBO J 2000; 19:3159-67.
29. Judson I, Verweij J, Van Oosterom A et al. Imatimib (Imatinib) an active agent for
gastrointestinal stromal tumors but not for other soft tissue sarcoma subtypes not
characterized for KIT and PDGR-R expression, results of EORTC phase II studies Proc
Am Soc Clin Onc 2002, 21 (abstract 1609).
30. Culy CR, Faulds D. Gefitinib. Drugs 2002; 62:2237-50
31. Bunn PA Jr, Franklin W. Epidermal growth factor receptor expression, signal
pathway, and inhibitors in non-small cell lung cancer. Semin Oncol 2002; 29(5 Suppl
14):38-44
32. Herbst RS, Kies MS. ZD1839 (Iressa) in non-small cell lung cancer. Oncologist
2002;7 Suppl 4:9-15
33. Weiss SW, Goldblum JR. Liposarcoma in Enzinger and Weisss Soft Tissue Tumors
4th. edition, Sharon Weiss, John R. Goldblum eds. St-Louis, MO: Mosby, 2001.
34. Knight JC, Renwick PJ, Cin PD, Van den Berghe H, Fletcher CD.Translocation
t(12;16)(q13;p11) in myxoid liposarcoma and round cell liposarcoma: molecular and
cytogenetic analysis. Cancer Res 1995; 55:24-7
35. Kuroda M, Ishida T, Takanashi M, et al. Oncogenic transformation and inhibition of
adipocytic conversion of preadipocytes by TLS/FUS-CHOP type II chimeric protein. Am
J Pathol. 1997; 151: 735-44
36. Tontonoz P, Singer S, Forman BM, et al. Terminal differentiation of human
liposarcoma cells induced by ligands for peroxisome proliferator-activated receptor
gamma and the retinoid X receptor. Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):237-41.
37. Tontonoz, P., Hu, E., Graves, R. A., Budavari, A. I. & Spiegelman, B. M. mPPAR
gamma 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev. 1994; 8, 1224-
1234
38. Van Glabbeke M, Verweij J, Judson I, Nielsen OS; EORTC Soft Tissue and Bone
Sarcoma Group. Progression-free rate as the principal end-point for phase II trials in soft-
tissue sarcomas. Eur J Cancer. 2002; 38:543-9.
39. Demetri GD, Fletcher CD, Mueller E, et al. Induction of solid tumor differentiation
by the peroxisome proliferator-activated receptor-gamma ligand troglitazone in patients
with liposarcoma. Proc Natl Acad Sci USA. 1999; 96:3951-6
40. Demetri GD, Speigelman B, Fletcher CD, et al. Differenciation of liposarcomas in
patients treated with the PPAR-g ligand troglitazone: documentation of biological activity
in myxoid round cell and pleomorphic subtypes. Proc Am Soc Clin Oncol. 1999; 18: 535
a (abstract 2064).
41. Heinrich MC, Corless CL, Duensing A, et al. PDGFRA activating mutations in
gastrointestinal stromal tumors. Science. 2003; 299:708-10.
Chapter 10
Angiogenesis: a potential target for therapy of

soft tissue sarcomas
K. Hoekman and H.M. Pinedo
Department of Medical Oncology, Free University Medical Center,

De Boelelaan 1117, 1007 MB Amsterdam.
Correspondence to :
K. Hoekman, MD, PhD
Department of Medical Oncology,
Vrije Universiteit Medical Centre,
De Boelelaan 1117, 1007 MB Amsterdam, the Netherlands.
Telephone : 020 4444319
Fax : 020 4444355
E-mail : k.hoekman@vumc.nl
170
1 INTRODUCTION
Tumors depend on neo-vascularisation (angiogenesis) during

development (Folkman, 2002). The use of curative therapy in the treatment
of soft tissue sarcomas (STS) is generally only possible in the initial stage of
the disease, where surgery plus or minus adjuvant radiotherapy is used, and
in a minority of cases where metastasectomy is possible. In advanced
metastatic disease, treatment with the chemotherapeutic agents, adriamycine
and ifosfamide, is associated with low response rates (20-35% when given as
single therapy and up to 45% when given in combination) and with short
progression free intervals. This means that there is a high need for additional
chemotherapeutic compounds or effective alternatives. Tumor
vascularisation might be a new target for anti-tumor therapy. The questions
to be asked are therefore :
what evidence is there that STSs are dependent on angiogenesis,
and which factors are involved in STS-associated angiogenesis?
which agents are available for anti-angiogenesis therapy, and
what is the optimal strategy for using these compounds in patients with STS?
2 ANGIOGENESIS
Angiogenesis, the proliferation, migration and tube formation of

endothelial cells, is a process regulated by many stimulating and inhibiting
factors. In the tumor situation these factors are produced or generated by
tumor cells and by activated stromal cells, such as fibroblasts and immune
cells. The balance between pro and contra angiogenic factors determines the
presence and activity of the angiogenesis process. While these factors may be
found in the circulation, it is the local presence in tumor tissue that is of
major relevance.
Different families of proteins are involved in angiogenesis which can
be classified into three main groups. The first group comprises several
stimulating growth factors (vascular endothelial growth factor (VEGF), basic
fibroblast growth factor (bFGF), angiopoietins, e.g., and cytokines
(interleukin-6, e.g.) and several inhibiting factors such as thrombospondin,
IL-12 and fragments of collagen and circulating proteins. VEGF, for
instance, is an outstanding angiogenesis factor that is produced by all sorts of
cells, with a unique characteristic, namely, the targeting of cancer endothelial
cells. This growth factor induces the proliferation and migration of
endothelial cells, and, at the same time, it is a survival factor and a
permeability inducing factor for those cells (Ferrara, 2002). The second
group comprises proteolytic enzymes belonging to the family of
metalloproteases (MMPs) or plasminogen activators (uPA and tPA). These
proteases are responsible for the degradation of the basal membranes and
proteins of the extracellular matrix (ECM) during the migration of
endothelial cells. The third group comprises adhesion molecules, e.g.
integrins and cadherins. These molecules are a critical factor in the
communication between cells, and in the communication between cells and
constituents of the ECM, and determine largely the outcome of endothelial
171
cell migration and tubular assembly. All these different regulators of

angiogenesis are possible targets for anti-angiogenesis therapy.
There are various reasons why tumor cells produce these angiogenic
factors or induce the receptors for these factors on endothelial cells. Genetic
abnormalities found in oncogenes, such as ras and c-myc, and suppressor
genes, such as p53, are associated with on the one hand an ongoing
production of pro-angiogenic proteins and on the other with an ongoing
inhibition of the production of inhibitory factors. A mutated p53, for
example, is associated with overexpression of VEGF and bFGF-binding
protein, activation of and downregulation of thrombospondin-1, all
of which producing a pro-angiogenic effect (Broxterman et al., 2003). In
addition, hypoxia and stress are responsible for a conditional, physiologic
angiogenic response. The ongoing, genetically determined production of
angiogenic factors is responsible for tumors being wounds that never heal,
while stress conditions further stimulate the angiogenic impulse. In addition,
activated stromal cells contribute substantially to this physiologic angiogenic
response in tumor tissues. Hypoxia is a common finding in tumor tissues
because the proliferation of tumor cells, in general, outgrows the vascular
supply.
In the final stage of angiogenesis, the nascent vessel needs support
from smooth muscle cells (pericytes) for further stabilisation. The
recruitment of these cells is regulated by other growth factors like PDGF,
angiopoietin-1 and A final characteristic of tumor-associated
neovascularisation is the chaotic, immature aspect. This is caused by the
unbalanced and ubiquitous presence of multiple angiogenic factors, and the
chaotic gradients of angiogenic factors laid down by the variety of cells
involved in the process of tumor-associated angiogenesis. While tumor cells
generally are present as groups or bundles, stromal cells are found to be more
dispersed in tumor tissue. The above is more even more evident in the
periphery of tumors and might explain the chaotic appearance of tumor
neovasculature locally.
Tumor-associated endothelial cells differ from quiescent endothelial
cells in several aspects: they have a higher rate of proliferation, they have a
different phenotype under the influence of the mixture of angiogenic factors
and tumor stress conditions, and they are less mature, often missing the
support of pericytes. This means that the turnover of such vessels is high and
that the tumor endothelial cells are more vulnerable to therapeutic
interventions.
In summary, the target of angiogenic therapy may be not only the
factors involved in the process of angiogenesis (anti-angiogenesis) but also
the tumor endothelial cells themselves (anti-vascular therapy). The
implication of the above is that any therapy which reduces the tumor cell
burden will also reduce the tumoral production of angiogenic factors, and
any therapy which is toxic for endothelial cells will be anti-angiogenic, as
well.
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3 THE ROLE OF ANGIOGENESIS IN THE BIOLOGY

OF SARCOMAS
Microvessel density (MVD) is often found to be a prognostic factor
in the response to therapy and survival of cancer patients, although numerous
articles on this topic are ambiguous in their conclusions. This may be due to
technical problems regarding the way in which the tumor fields of interest
are selected and/or the manner in which vessels are counted. In four
independent studies examining MVD in soft tissue sarcomas (Yudoh et al.,
2001; Kawauchi, 1999;Tomlinson et al., 1999; Saenz et al., 1998), MVD was
not associated with histological type, grading, metastatic behavior or
survival, but MVD was correlated with survival in a fifth study (Comandone
et al., 2003). Tomlinson et al, describe a different pattern of angiogenesis in
STS versus breast carcinoma. In breast cancer the capillaries were clustered
in bursts within the stroma of the tumor, while the sarcoma capillaries were
homogeneously distributed in the tumor stroma. They credit this difference to
the greater number of activated fibroblasts in carcinomas, with their own
gradients of angiogenic factors in the tissues. This aspect has been studied in
carcinosarcoma, which contains both tumor types. In accordance with the
findings of Tomlinson a study by Yoshida et al. describes a significant
higher MVD in the carcinoma areas of the tumor than in the sarcoma parts
(Yoshida et al., 2000). However, the consequences of the above for anti-
angiogenesis therapy of STS are not clear.
Further evidence pertaining to the relevance of angiogenesis in STS
biology comes from different studies. An animal study (Mori et al., 1999),
shows that transfection of fibrosarcoma cells with VEGF resulted in massive
angiogenesis around the tumor and in increased metastatic behavior, which
findings confirm the importance of VEGF to the initiation of angiogenesis,
but also to the most detrimental aspect of cancer, namely, the metastatic
potential.
The in vitro production of VEGF by different STS cell lines is, in
general, abundant (Hu et al., 2000; own observations), which is in line with
the high VEGF production also found in the conditioned medium of many
carcinoma cell lines. The involvement of bFGF in the biology of STS was
indicated in a fibrosarcoma development model. The authors showed that the
ability to release bFGF was critical for malignant characteristics and was
associated with the induction of neovascularization (Kandel, 1991).
There have also been studies of the levels of angiogenic factors in
the blood of STS patients. Serum VEGF was shown to be elevated in patients
with STS in multiple studies (Graeven et al., 1999; Linder, 1998; Rutkowski,
2002;Heits, 1997), and correlated significantly with tumor stage and grading.
Also plasma MMP-9 activity was shown to be enhanced in STS patients
(Pallotta et al., 2000). A remarkable finding in the serum of STS patients was
the elevation of endostatin, the COOH-terminal proteolytic fragment of the
basement membrane component collagen XVIII and a potent angiogenesis
inhibitor (Feldman, 2001). The findings of the study associated an increased
serum endostatin level with an increased risk of tumor recurrence after
resection.
Investigation of STS tissue showed that the concentration of VEGF
in STS tissue was enhanced and positively correlated with grading (Yudoh et
173
al., 2001; Chao, 2001). STS tissue levels of VEGF appeared to be a

prognostic factor in the study of Yudoh. VEGF overexpression might be
expected in STS due to the fact that it is inter alia regulated by the
p53/MDM-2 pathway, which shows aberrations in the majority of STSs
(Zietz et al., 1998). The introduction of wild type p53 in sarcoma cells
containing mutant p53 significantly reduced the expression of VEGF (Zhang,
2000), and induced angiogenesis-restricted dormancy in a mouse
fibrosarcoma model (Holmgren, 1998). Overexpression of VEGF might also
be associated with hypoxia in STS and consequently with regulated
stimulation of VEGF expression (Nordsmark et al., 2001). Overexpression of
the protease uPA, an essential enzyme for the migration of cells, was also
associated with increasing grade, local recurrence and metastases in STS
(Choong et al., 1996). Thymidine phosphorylase, another angiogenesis factor
which is often coexpressed in tumor tissue, was overexpressed in uterine
leiomyosarcomas and correlated with increased MVD (Hata, 1997).
Expression of the receptors for angiogenic factors has been
described. Angiosarcoma cells and Kaposi cells are large producers of VEGF
and have VEGF receptors. It has been demonstrated that in these tumor types
VEGF is active as an autocrine growth factor (Fujimoto et al., 1998;
Hashimoto et al., 1995; Masood, 1997). The same suggestion has been
advanced concerning bFGF in some leiomyosarcomas (Tamiya, 1998).
The AIDS-induced Kaposi sarcoma (KS) is a tumor of vascular
origin. KS cells produce and respond to VEGF and bFGF. The trans-activator
HIV protein Tat activates VEGF and bFGF and the VEGF receptor-2. In
addition, Tat and bFGF synergistically activate MT-MMP-1 and TIMP-1 and
consequently induce the secretion of MMP-2 (Toschi et al., 2001). High
serum FGF-2 levels were associated with a higher risk of death in infected
patients (Ascher et al., 2001).
A more or less unique finding in STSs is the presence of large cysts
in advanced tumors. These cysts may develop due to hypoxia and,
consequently, necrosis in the center of the tumor. Indeed, STSs harbour areas
with very low oxygen pressures (Brizel et al., 1996; Nordsmark et al., 2001).
Another or additional explanation may be sought in the presence of extreme
local concentrations of VEGF-induced partly by hypoxia- which is a
powerful permeability factor for endothelial cells (Ferrara, 2002). The cystic
fluid may be regarded as the in vivo conditioned medium of the tumor, and is
therefore greatly informative for the investigation of tumor biology. The
VEGF concentration in intratumoral fluid collections was found to be
extremely high in our own STS studies, many times higher than in the
plasma of the same patients even reaching levels 450 times higher than found
in normal plasma (Verheul, 2000). A possible explanation for this finding
may be the fact that platelets contain substantial amounts of VEGF (Verheul
et al., 1997), whereas activated endothelial cells promote the adhesion and
activation of platelets (Verheul, 2000), which may result in a strongly
enhanced local release of VEGF. The concentration of basic FGF was not
enhanced in these STS fluids, except in a patient with a gastro-intestinal
stromal tumor (GIST). The activity of uPA and MMP-9 was also enhanced in
STS cyst fluid, confirming the role of these proteases in tumor-induced
angiogenesis. Finally, we found high concentrations of a number of key
174
coagulation factors in the fluid. Together with evidence of intratumoral

platelet activation, the above findings confirm the hypothesis of a putative
role of platelets and coagulation in tumor angiogenesis (Pinedo, 1998). In the
cystic fluid of a patient with GANT we found, in addition to high VEGF,
enhanced levels of endostatin, tPA and plasmin (Hansma et al., 2003). The
demonstration of the generation of endostatin inside a tumor was a unique
finding; together with the activation of tPA and consequent generation of
plasmin in the fluid it is a likely mechanism of action of endostatin
(Reijerkerk et al., 2003).
4 ANTI-ANGIOGENIC THERAPEUTIC OPTIONS FOR

Preclinical research has brought to light many clues about anti-

angiogenic therapy of patients with STS. Antibodies directed at VEGF, or
VEGF-receptor tyrosine kinase inhibitors (SU5416) decreased the growth of
human sarcoma explants in immunocompromised rodents (Angelov et al.,
1999; Wang et al., 1998). This growth inhibition was attributed to having the
effect on tumor angiogenesis of leading to a reduction of tumor cell
proliferation and increased apoptosis. Complete inhibition and regression of
a rhabomyosarcoma explant could only be induced by blocking both tumor
(human) and host (animal) VEGF (Gerber, 2000). Histological analysis of
residual tumor tissue revealed an almost complete absence of host-derived
vasculature and massive tumor necrosis. Combination of an antibody to the
VEGF type 2 receptor with continuous low dose doxorubicin resulted in an
enhanced growth inhibition of 2 different sarcoma xenografts associated with
an increased apoptosis of endothelial cells in the tumors (Zhang, 2002).
Together, these studies confirm the importance of VEGF in the initiation of
sarcoma-induced angiogenesis and further encourage anti-VEGF based trials
in STS patients.
An internal 4 kringle containing fragment of plasminogen
(angiostatin) and the 5 kringle fragment of plasminogen, the latter induced
via the uPA-plasmin pathway, are both potent inhibitors of angiogenesis.
Both compounds inhibited the growth of a murine fibrosarcoma in mice
(Cao, 1998). Stable gene transfer of angiostatin cDNA in Kaposi sarcoma
cells delayed tumor growth in nude mice, which was associated with reduced
vascularization (Indraccolo et al., 2001). Endostatin, the fragment of collagen
XVIII, is generated during breakdown of the basal membrane and ECM, and
is also a natural angiogenesis inhibitor. Gene therapy with endostatin, which
ensures a sustained endostatin overproduction, significantly inhibited tumor
formation in the lung after intravenous injection of fibrosarcoma cells
(Nakashima et al., 2003). Intermittent treatment of fibrosarcoma explants in
mice with endostatin showed regression followed by regrowth of the tumor
xenographs, but also showed no recurrence of the tumor after 4 cycles of
endostatin treatment, which would suggest that the therapy does not induce
acquired drug resistance (Boehm, 1997). These studies warrant a clinical
follow-up.
175
Anti-vascular therapy is another strategy in downgrading the

vascular support of tumors. The tumor-associated endothelium has been
described as being the target of metronomic therapy with low dose
cyclophosphamide (Man et al., 2002). It proved to be a succesful approach in
rats challenged with a sarcoma (Rozados, 2003). Low-dose cyclo-
phosphamide eradicated a high percentage of sarcomas without evident
toxicity. In studies with other tumor types, cyclophosphamide was combined
with anti-angiogenic agents, which resulted in long-term remissions. This
strategy should be further investigated in future sarcoma studies. Another
compound is ZD6126, a vascular targeting drug that disrupts the tubulin
cytoskeleton of proliferating endothelial cells, resulting in selective
destruction and congestion of tumor blood vessels. ZD6126 induced
extensive hemorrhagic necrosis in a rat fibrosarcoma model (Robinson et al.,
2003). Combination of ZD6126 with cisplatin or radiotherapy enhanced
significantly the anti-tumor effect in a mouse model with human sarcoma
xenographs (Siemann, 2002). Further investigation of ZD6126, in
combination with chemotherapeutic agents with proven efficacy for
sarcomas, is warranted.
Clinical experience with anti-angiogenesis agents for advanced STS
is scarce. The NCI list of ongoing trials in STS records a total of 35 trials;
only one of which, however, is an anti-angiogenesis based study, combining
thalidomide with in the adjuvant setting. There was also a phase
I study of IL-12 on the list, which included a number of STS patients.
An important study was performed by Kuenen et al, who treated 31
STS patients with SU5416, which is a tyrosine kinase inhibitor of the VEGF
receptors type 1 and 2 (Kuenen et al., 2003). The results include one minor
response and 5 stable diseases (less than 25% increase during 3 months of
treatment) of limited duration. In one patient a remarkable decrease of fluid
collection within the tumor could be observed together with progression of
tumor volume, which at least confirmed activity of this agent against the
permeability effects of VEGF. Different reasons were given for the rather
disappointing outcome. There was uncertainty about the bioavailability of
SU5416, the complexity and redundancy of cytokines/growth factors
involved in angiogenesis and also the fact that the vascular support of
established tumors is mostly performed by mature vessels, which are less
dependent on VEGF (Eberhard, 2000). The above suggests that modulation
of this very compound, combination with other anti-angiogenesis agents or
the use of other drugs which have an effect on established tumor vessels
might deliver better results in STS. Tumstatin, a fragment of collagen IV
(Hamano et al., 2003), and VEGF-trap, a soluble VEGF decoy receptor
(Huang et al., 2003), have recently been described as agents that have the
potency to abolish mature tumor vessels, followed by marked tumor
regression, including regression of lung metastases. New studies, using these
agents, eventually in combination or combined with chemo-therapeutics
and/or other biological agents are warranted.
There are a number of ways of achieving selective treatment of
tumors: by targeting either the tumor cells or tumor-associated endothelial
cells, by administering drugs in the tumor itself, or by administering drugs in
the feeding arterial system of the tumor. The first-mentioned approach has
176
been carried out extensively in animal studies using doxorubicin a drug

used for STS - conjugated with different kinds of peptides, which results in
higher uptake in the tumor, less toxicity to the host, and less acquired drug
resistance (van Hensbergen, 2002; Mazel et al., 2001). It is still a promising
strategy, also for therapy of patients with STS. The latter strategy has been
used in isolated limb perfusion using
(Lejeune et al., 2000) for patients with melanoma or STS located in the
extremities. TNF (plus interferon) induced apoptosis of tumor endothelium
by reduced activation of leading to detachment of endothelial cells
(Ruegg, 1998), while enhancing the selective uptake of tumor-toxic
melphalan in the sarcoma. This therapy induced about 30% complete
responses and offered the majority of patients the possibility for limb-sparing
resection of the tumor. This experimental therapy affirms the principle that
the systematic combination of a chemotherapeutic agent- even one that is not
very effective- with an angiogenic intervention can be successful for patients
with cancer. The importance of the way a drug is presented to tumor tissue or
the way it inhibits functionally a target was underlined by the negative
results of a trial with vitaxin, a humanized monoclonal antibody against the
integrin (Patel et al., 2001), given intravenously to patients with a
leiomyosarcoma.
Reduced penetration of cytotoxic drugs in tumor tissue is an
important aspect of drug resistance (Jain 2002). In two recent papers, it was
shown that tumor drug uptake could be stimulated by low-dose given
systematically (ten Hagen et al.; 2000), or by administration of STI571
(Pietras et al., 2002). STI571 (Glivec) is a tyrosine kinase inhibitor of
signaling via the BCR-ABL fusion protein, but also via c-Kit and the PDGF
receptors and Inhibition of the latter receptors, present on endothelial
cell supporting cells, is thought to be responsible for a reduction in tumor
interstitial pressure and, consequently, for stimulation of drug uptake. These
findings should also be further elaborated in clinical studies.
A different type of tumor is the Kaposi sarcoma, being of vascular
origin. It was shown that overexpression of multiple angiogenic factors, such
as VEGF, bFGF, IL-8, MMP-2 and -9 was present in KS tissue. Thus, it is
not surprising that agents such as SU5416 (Arasleh, 2000), the anti-
angiogenic agent TNP-470 (Arbiser et al., 1999; Dezube et al,. 1998) and the
MMP-inhibitor COL-3 (Cianfrocc et al., 2002) were successful in treating
this tumor type, with an overall response rate of 44% in respect of the latter
drug. In addition, the effect of the commonly used HIV protease inhibitors on
Kaposi sarcoma are partly due to the fact that a decrease is brought about in
the activity of MMP-2 (Sgadari et al., 2003).
In conclusion, STS-associated angiogenesis and STS-vessels as such
offer multiple specific avenues for further research and treatment strategies
of patients with STS (see also Heymach, 2001 and Scappaticci, 2001). It is
questionable if attacking one pivotal factor or phenotypic address of STS-
associated vessels will result in substantial anti-STS responses. An important
problem in animal studies lies in the difficulty of predicting results of the
activity of these types of agents in patients with cancer. This may be
explained by the fact that animal models only represent the initiation of
angiogenesis in recently installed tumor explants. It will be a challenge to
177
improve the design of animal studies and to look for theoretically optimal
combinations of biologicals and chemotherapeutic agents for the therapy of
patients with STS. In some STS patients the presence of large tumors offers
the opportunity to get tumor tissue samples before and after therapy.
Investigation of these tissues with microarrays and proteomic techniques will
deliver a better understanding of STS biology and the effects of therapeutic
interventions on these tumors (Borden et al., 2003); all of which will
ultimately lead to better therapies for patients with soft tissue sarcomas.
5 REFERENCES
1. Angelov L, Salhia B, Roncari L, et al: Inhibition of angiogenesis by blocking activation
of the vascular endothelial growth factor receptor 2 leads to decreased growth of
neurogenic sarcomas. Cancer Res 1999, 59:5536-41
2. Arasleh K, Hannah A. The role of vascular endothelial growth factor (VEGF) in AIDS-
related Kaposis sarcoma. Oncologist 2000, 5(suppl 1):28-31
3. Arbiser JL, Panigrathy D, Klauber N, et al: The antiangiogenic agents TNP-470 and 2-
methoxyestradiol inhibit the growth of angiosarcoma in mice J. Am Acad Dermatol
1999; 40:925-9
4. Ascher G, Sgadari C, Bugarini R, et al. Serum concentrations of fibroblast growth factor
2 are increased in HIV type 1-infected patients and inversely correlated to survival
probability. AIDS Res Hum Retrovirusses 2001; 17:1035-9
5. Boehm T, Folkman J, Browder T, OReilly MS. Antiangiogenic therapy of experimental
cancer does not induce acquired drug resistence. Nature 1997; 390:404-7
6. Borden EC, Baker LH, Bell RS, et al. Soft tissue sarcomas of adults: state of the
translational science. Clin Cancer Res 2003; 9:1941-56
7. Brizel DM, Scully SP, Harrelson J, et al. Tumor oxygenation predicts for the likelihood
of distant metastases in human soft tissue sarcoma. Cancer Res 1996; 56:941-3
8. Broxterman HJ, Lankelma J, Hoekman K. Resistance to cytotoxic and anti-angiogenic
anticancer agents: similarities and differences. Drug Resistance Updates 2003; 6:111-27
9. Cao Y, OReilly MS, Marshall B, Flynn E, Ji R-W, and Folkman J. Expression of
angiostatin cDNA in a murine fibrosarcoma suppresses primary tumor growth and
produces long-term dormancy of metastases. J Clin Investigation 1998; 101:1055-63
10. Chao C, Al-Saleem T, Brooks JJ, Rogatko A, Kraybill WG, Eisenberg B. Vascular
endothelial growth factor and soft tissue sarcomas: tumor expression correlates with
grade. Ann Surg Oncol 2001; 8:260-67
11. Choong PF, Ferno M, Akerman M, et al. Urokinase-plasminogen-activator levels and
prognosis in 69 soft-tissue sarcomas. Int J Cancer 1996; 69:268-72
12. Cianfrocca M, Cooley TP, Lee JY, et al. Matrix metalloprotease inhibitor COL-3 in the
treatment of AIDS-related Kaposis sarcoma: a phase I AIDS malignancy consortium
study. J Clin Oncol 2002; 20:153-9
13. Comandone A, Boglione E, Berardengo A, et al. Microvessel density (MVD) as a marker
of neoangiogenesis: prognostic significance in correlation with grading and stage in adult
soft tissue sarcomas (STS) of the extremities. A perspective study. ASCO 2003; abstract
3303
14. Dezube BJ, Von Roenn JH, Holden-Wiltse J, et al. Fumagillin analog in the treatment of
Kaposis sarcoma: a phase AIDS clinical trial group study, AIDS clinical trial group no
215 team. J Clin Oncol 1998; 16:1444-9
15. Eberhard A, Kahlert S, Goede V, Hemmerlein B, Plate KH, Augustin HG. Heterogeneity
of angiogenesis and blood vessel maturation in human tumors: implications for
antiangiogenic tumor therapies. Cancer Research 2000; 60:1388-93
16. Feldman AL. Serum endostatin levels are elevated in patients with soft tissue sarcoma.
Cancer 2001; 91:1525-9
17. Ferrara N. Role of vascular endothelial growth factor in physiologic and pathologic
angiogenesis: therapeutic implications. Semin Oncol 2002; 29:10-4
178
18. Folkman J. Role of angiogenesis in tumor growth and metastases. Semin Oncol 2002;
29:15-8
19. Fujimoto M, Kiyosawa T, Murata S, et al. Vascular endothelial growth factor in
angiosarcoma. Anticancer Res 1998; 18:3725-30
20. Gerber HP, Kowalski J, Sherman D, Eberhard DA, Ferrara N. Complete inhibition of
rhabdomyosarcoma xenograft growth and neovascularization requires blockade of both
tumor and host vascular endothelial growth factor. Cancer Res 2000; 60:6553-8
21. Graeven U, Andre N, Achilles E, et al: Serum levels of vascular endothelial growth factor
and basic fibroblast growth factor in patients with soft-tissue sarcoma, J Cancer Res Clin
Oncol 1999; 125:577-81
22. ten Hagen TL, van der Veen AH, Nooijen PT, van Tiel ST, Seynagen AL, Eggermont
AM. Low-dose tumor necrosis factor-alpha augments antitumor activity of stealth
liposomal doxorubicin (DOXIL) in soft tissue sarcoma-bearing rats. Int J Cancer 2000;
87:829-37
23. Hamano Y, Zeisberg M, Sugimoto H, et al. Pysiological levels of tumstatin, a fragment
of collagen IV alpha3 chain, are generated by MMP-9 proteolysis and suppress
angiogenesis via alphaVbeta3 integrin. Cancer Cell 2003; 3:589-601
24. Hansma AHG, van Hensbergen Y, Kuenen BC, et al. A patient with a VEGF and
endostatin producing gastrointestinal autonomic nerve tumor (GANT). Submitted.
25. Hashimoto M, Ohsawa M, Ohnishi A, et al. Expression of vascular endothelial growth
factor and its receptor mRNA in angiosarcoma. Lab Invest 1995; 73:859-63
26. Hata K, Hata T, and Miyazaki K. Expression of thymidine phosphorylase in uterine
sarcoma and uterine leiomyoma: association with microvessel density and Doppler blood
flow analysis. Ultrasound Obstet Gynecol 1997; 10:54-8
27. Heits F, Katschinski DM, Wiedeman GJ, Weiss C, Jelkman W. Serum vascular
endothelial growth factor (VEGF), a prognostic factor in sarcoma and carcinoma patients.
Int J Oncol 1997; 10:333-7
28. van Hensbergen Y, Broxterman HJ, Elderkamp YW, et al. A doxorubicin-CNGRC-
peptide conjugate with prodrug properties. Biochem Pharmacol 2002; 63:897-08
29. van Hensbergen Y, Broxterman HJ, Hanemaaijer R, Jorna AS, Verheul HM, Pinedo HM,
Hoekman K. Soluble aminopeptidase N/CD13 in malignant and nonmalignant effusions
and intratumoral fluid. Clin Cancer Res 2002; 8:3747-54
30. Heymach JV. Angiogenesis and antiangiogenesis approaches to sarcomas. Current
Opinion in Oncol 2001; 13:261-9
31. Holmgren L, Jackson G, Arbiser J. P53 induces angiogenesis-restricted dormancy in a
mouse fibrosarcoma. Oncogene 1998; 17:819-24
32. Hornick JL, Fletcher CDM. Immunohistochemical staining for KIT (CD117) in soft
tissue sarcomas is very limited in distribution. Am J Clin Pathol 117:188-93
33. Hu M, Nicolson GL, Trent JC, et al. Characterization of 11 human sarcoma cell strains:
evaluation of cytogenetics, tumorogenicity, metastasis, and production of angiogenic
factors. Cancer 2000; 95:1569-76
34. Huang J, Frischer JS, Serur A, et al. Regression of established tumors and metastases by
potent vascular endothelial growth factor blockade. PNAS 2003; 100:7785-90
35. Indraccolo S, Morini M, Gola E, et al. Effects of angiostatin gene transfer on functional
properties of in vivo growth of Kaposis sarcoma cells. Cancer Res 2001; 61:5441-6
36. Jain RK. Understanding barriers to drug delivery: high resolution in vivo imaging is key.
Clin Cancer Res 1999; 5:1605-6
37. Kandel J, Bossy-Wetzel E, Radvanyi F, Klagsbrun M, Folkman J, and Hanahan D.
Neovascularization is associated with a switch to the export of bFGF in the multislep
development of fibrosarcoma. Cell 1991; 66:1095-1104
38. Kawauchi S, Fukuda T, Tsuneyoshi M. Angiogenesis does not correlate with prognosis
or expression of vascular endothelial growth factor in synovial sarcomas. Oncol Rep
1999; 6:959-64
39. Kuenen BC, Taberno J, Baselga J, et al. Efficacy and toxicity of the angiogenesis
inhibitor SU5416 as a single agent in patients with advanced renal cell carcinoma,
melanoma, and soft tissue sarcoma. Clin Cancer Res 2003; 9:1648-55
40. Lejeune FJ, Pujol N, Lienard D, et al. Limb salvage by neoadjuvant isolated perfusion
with TNFa and melphalan for non-resectable soft tissue sarcoma of the extremeties. Eur J
Surg Oncol 2000; 26:669-78
179
41. Linder C, Linder S, Munck-Wikland E, and Strander H. Independent expression of serum

vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in
patients with carcinoma and sarcoma. Anticancer Res 1998; 18:2063-8
42. Man S, Bocci G, Francia G, et al. Antitumor effects in mice of low-dose (metronomic)
cyclophosphamide administered continuously through the drinking water. Cancer Res
2002; 62:2731-5
43. Masood R, Cai J, Zheng T, Smith DL, Naidu Y, and Gill PS. Vascular endothelial growth
factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi
sarcoma. Proc Natl Acad Sci USA. 1997; 94:979-84
44. Mazel M, Clair P, Rousselle C, et al. Doxorubicin-conjugates overcome multidrug
resistance. Anticancer Drugs 2001; 12:107-16
45. Mori A, Arii S, Furutani M, et al. Vascular endothelial growth factor-induced tumor
antigen and tumorigenicity in relation to metastasis in a HT1080 human fibrosarcoma
model. Int J Cancer 1999; 80:738-43
46. Nakashima Y, Yano M, Kobayashi Y, et al. Endostatin gene therapy on murine lung
metastases model utilizing cationic vector-mediated intravenous gene delivery. Gene
Ther 2003; 10:123-30
47. Nordsmark M, Alsner J, Keller J, et al. Hypoxia in human soft tissue sarcomas: adverse
impact on survival and no association with p53 mutations. Br J Cancer 2001; 84:1070-5
48. Patel S, Jenkins J, Papadopoulos N, et al. A pilot study of vitaxin -an angiogenesis
inhibitor- in patients with advanced leiomyosarcoma. Cancer 2001; 92:1347-8
49. Pallotta M, Lastiri J, Varela M, et al: Plasma MMP-9 activity in soft tissue sarcomas,
Proc Am Soc Clin Oncol 2000; 19:2201
50. Pietras K, Rubin K, Sjoblom T, et al. Inhibition of PDGF receptor signaling in tumor
stroma enhances antitumor effect of chemotherapy. Cancer Res 2002; 62:5476-84
51. Pinedo HM, Verheul HMW, DAmato RJ, Folkman J. Involvement of platelets in tumour
angiogenesis? Lancet 1998; 352:1775-7
52. Reijerkerk A, Mosnier LO, Kranenburg O, et al. Amyloid endostatin induces endothelial
cell detachment by stimulation of the plasminogen activation system. Mol Cancer Res
2003; 1:561-8
53. Robinson SP, McIntyre DJ, Checkley D, et al. Tumour dose response to the antivascular
agent ZD6126 assessed by magnetic resonance imaging. Br J Cancer 2003; 88:1592-7
54. Rozados VR, Sanchez AM, Berra HH, Matar P, Gervasoni SI, Scharovsky OG.
Metronomic therapy with cyclophosphamide induces rat lymphoma and sarcoma
regression and is devoid of toxicity. Am Assoc Cancer Res 2003; 94the Ann Meeting,
abstract 1620
55. Ruegg C, Yilmaz A, Bieler C, Bamat J, Chaubert P, Lejeune FJ. Evidence for the
involvement of endothelial cell integrin alpha Vbeta3 in the disruption of the tumor
vasculature induced by TNF and IFN-gamma. Nat Med 1998; 4:408-14
56. Rutkowski P, Kaminska JU, Kowalska M, Ruka W, Steffen J. Cytokine serum levels in
soft tissue sarcoma patients: correlations with clinico-pathological features and prognosis.
Int J Cancer 2002; 100:463-71
57. Saenz NC, Heslin MJ, Adsay V, et al. Neovascularity and clinical outcome in high-grade
extremity soft tissue sarcomas. Ann Surg Oncol 1998; 5:48-53
58. Scappaticci FA, Marina N. New molecular targets and biological therapies in sarcomas.
Cancer Treatment Rev 2001; 27:317-26
59. Sgadari C, Barillari G, Toschi E, et al. HIV protease inhibitors are potent anti-angiogenic
molecules and promote regression of Kaposi sarcoma. Nat Med 2002; 8:225-32
60. Siemann DW, Rojiani AM. Antitumor efficay of conventional anticancer drugs is
enhanced by the vascular targeting agent ZD6126. Int J Radiat Biol Phys 2002; 54:1512-
7
61. Siemann DW, Rojiani AM. Enhancement of radiotherapy by the novel targeting agent
ZD6126. Int J Radiat Biol Phys 2002; 53:164-71
62. Tamiya S, Ueki T, Tsuneyoshi M. Expressions of basic fibroblast growth factor and
fibroblast growth factor receptor mRNA in soft tissue tumors by in situ hybridization.
Mod Pathol 1998; 11:533-6
63. Tomlinson J, Barsky SH, Nelson S, el al: Different patterns of angiogenesis in sarcoma
and carcinomas. Clin Cancer Res 1999; 5:3516-22
64. Toschi E, Barillari G, Sgadari C, et al. Activalion of matrix-metalloprotease-2 and
180
membrane-type matrix-metalloprotease in endothelial cells and induction of vascular

permeability in vivo by human immunodeficiency virus-1 Tat protein and basic fibroblast
growth factor. Mol Biol Cell 2001; 12:2934-46
65. Verheul HMW, Hoekman K, Luykx-de Bakker S, et al. Platelet: transporter of vascular
endothelial growth factor. Clin Cancer Res 1997; 3:2187-90
66. Verheul HMW, Jorna AS, Hoekman K, Broxterman HJ, Gebbink MFBG, Pinedo HM.
Vascular endothelial growth factor-stimulated endothelial cells promote adhesion and
activation of platelets. Blood 2000; 96:4216-21
67. Verheul HMW, Hoekman K, Lupu F, Broxterman HJ, van der Valk P, Kakkar AK,
Pinedo HM. Platelet and coagulation activation with vascular endothelial growth factor
generation in soft tissue sarcomas. Clin Cancer Res 2000; 6:166-71
68. Wang G, Dong Z, Xu G, et al. The effect of antibody against vascular endothelial growth
factor on tumor growth and metastasis. J Cancer Res Clin Oncol 1998; 124:615-20
69. Yoshida Y, Kurokawa T, Fukuno N, et al. Markers of apoptosis and angiogenesis
indicate that carcinomatous components play an important role in the malignant behavior
of uterine carcinosarcoma. Hum Pathol 2000; 31: 1448-54
70. Yudoh K, Kanamori M, Ohmori K, Yasuda T, Aoki M, Kimura T. Concentration of
vascular endothelial growth factor in the tumour tissue as a prognostic factor of soft
tissue sarcomas. Br J Cancer 2001; 84:1610-5
71. Zhang L, Yu D, Hu M, et al: Wild-type p53 suppresses angiogenesis in human
leiomyosarcoma and synovial sarcoma by transcriptional suppression of vascular
endothelial growth factor expression. Cancer Res 2000; 60:3655-61
72. Zietz C, Rossle M, Haas C, et al: MDM-2 oncoprotein overexpression, p53 gene
mutation, and VEGF up-regulation in angiosarcomas. Am J Pathol 1998; 153:1425-33
73. Zhang L, Yu D, Hicklin DJ, Hannay JA, Ellis LM, Pollock RE. Combined anti-fetal liver
kinase 1 monoclonal antibody and continuous low-dose doxorubicin inhibits
angiogenesis and growth of soft tissue sarcoma xenografts by induction of endothelial
cell apoptosis. Cancer Res 2002; 62:2034-42
INDEX
Actinomycin D, 74 Collagen, 170

Activation loop Computerized tomography, 31f
mutations in, 132 Conformal fields
Acute myelogenous luekemia (AML), 142 parallel opposed pairs, 31
Acute radiation morbidity, 33 CT scans, 26
Acute tissue reactions, 6667 Cyclophosphamide, 48, 175
Adipogenesis Cytogenetic aberrations, 100
PPAR gamma pathway in, 163164 in adipose tumors, 102104
Adipose tumors Clear cell sarcoma, 104105
cytogenetic aberrations in, 102104 Cytogenetic approach, 101
molecular aberrations in, 102104 Cytogenetic mechanisms, 102
ALK Cytokines, 170
activated, 108 Cytotoxic agents, 10
ALK receptor tyrosine kinase, 8
Angiogenesis Dacarbazine, 11
anti, 174177 Dermatofibrosarcoma protuberans, 106
defining, 170 desmoid tumors and, 123124
Angiopoietins, 170 Desmoid tumors, 106107
Angiosarcoma, 173 dermatofibrosarcoma protuberans and,
treatment of, 6 123124
Angiostatin, 174 Desmoplastic small round cell tumors, 105
APC, 106107 Docetaxel, 6
Apoptosis, 92 Dose dispersion, 35
Dose heterogeneity, 35
Basic fibroblast growth factor (bFGF), 170 Dose painting, 2526
Beams eye view (BEV) biological targeting and, 3637
of retroperitoneal liposarcoma, 23f Dose sculpting, 2526
Beta-catenin, 106107 Dose-volume histograms (DVH), 24
Biological target volume, 37 of POP plans, 32f
Blood-oxygen level dependent (BOLD) image Doxorubicin, 2, 11, 48, 152
sequences, 37 dose-response curve of, 3
Brachytherapy, 30 TNF and, 70
postoperative, 36 Drug targeting
immunohistochemistry for, 95
CD117 antigen, 118 Drug uptake, 72
Chemoradiation
for extremity/trunk sarcomas, 4849 Early targets, 159
for retroperitoneal sarcomas, 4950 Edema, 20f
Chemotherapy EGF
isolated limb perfusion and, 67 expression of, 8
neo-adjuvant, 34 EGFR1-antibody therapy, 8
postoperative, 4748, 56t EGFR pathway
surgical complications in, 5657 targeting, 160
survival in, 5354 EGFR protein
vasculotoxic mechanisms of, 71 expression of, in synovial sarcomas, 157
Chronic myelogenous leukemia (CML), ZD1839 and, 161
142143 Elderly patients
Cisplatin, 48 safety in, 70
Clear cell sarcoma Endothelial differentiation, 94
cytogenetic/molecular aberrations of, Enes
104105 preferential expression of, 158t159t
Clinical target volume (CTV), 20 Epithelial differentiation, 94
182 Index
ET-743 (Trabectedin), 7, 10
Ewings sarcoma, 105106, 152 Idiosyncratic toxicity, 74
Exon 9 mutations, 133 Ifosfamide, 2, 11, 48, 152
Exon 11 mutations, 133 dose-response curve of, 3
External beam radiotherapy (EBRT), 2122, 36 Imatinib
Extracellular domain clinical efficacy of, 121
mutations in, 131132 dose-escalation of, 137
Extracompartmental sarcoma, 19 mesylate, 119f
Extremity sarcomas metabolism, 120
chemoradiation for, 4849 oncogene kinase targeting with, 136137
multimodal therapy for, 4849 pharmocokinetics of, 120
preoperative radiation therapy for, 4647 phase II studies of, 121
response, 138141
False-positive trials, 11 Immunohistochemistry, 93
Farnesyl transferase inhibitors (FTI), 109 for classification, 9395
ras protein targeting by, 9 for drug targeting, 95
Fibro-histiocytic differentiation, 94 limitations of, 9596
Fibrosarcoma, 107108 for monomorphic spindle cell tumors, 94t
Fluorescence in situ hybridization (FISH), 101 for undifferentiated round cell tumors, 95t
Fusion genes Induction therapy
protein products of, 155156 postoperative chemotherapy, 4748
preoperative radiation therapy, 4647
Gastrointestinal stromal tumors (GIST), 5, 91, rationale for, 4445
95, 108 for sarcomas, 45
diagnosis of, 138 for solid tumors, 4445
gastric, 136 Integral doses, 35
genotyping of, 140 Intensity modulated radiotherapy (IMRT), 18,
imatinib treatment of, 121 30
KIT-driven, 120123 dose constraints for, 28t
KIT mutations in, 130, 132136 elements of, 2425
molecular classifications of, 141t, 142 five field, 31f
neo-adjuvant treatment of, 122 inverse planning, 2629
other sarcomas and, 142143 mechanisms of, 25
PDGFRA and, 135 Interferon-gamma, 73
risk assessment for, 92t Isodose lines
variants of, 136 distribution of, 27f
Gene expression Isolated limb perfusion, 6667
histological subtypes of sarcomas and, chemotherapy and, 67
156157 with cytostatic drugs, 67t
in synovial sarcoma, 155 duration of, 73
Geometric uncertainty in histologies, 7071
margins for, 2021 TNF-based, 6769
reduction of, 34
Gross tumor volume (GTV), 19, 24f Juxtamembrane domain
mutations of, 130131
Hematological disorders
kinases in, 143t Kaplan-Meier estimates, 11
HER1 protein, 157 of progression-free rates, 12f
Histamine, 74 Kaposi sarcoma, 173, 176
Histologies Karyorrhexis, 92
TNF-based ILP in, 7071 Kinase 1 domain
Histopathological grading, 8792 mutations in, 132
FNCLCC system for, 8890 Kinase mutations
limitations of, 9092 imatinib response and, 138141
NCI system for, 8890 use of, 138
systems for, 8890 Kinases
Histopathological typing, 6267 in hematological/mesenchymal disorders,
HPLC 143t
denaturing, 131 inhibitors, 143
Hyperthemia KIT
mild, 73 activated, 108
Hypoxia, 73, 171 activation loop of, 132
Index 183
extracellular domain of, 131 for retroperitoneal sarcomas, 4950

gain-of-function mutations, 122 Muscle differentiation, 94
germline mutations in, 134t Myofibroblastic tumors, 108
inhibition of, 123 Myxofibrosarcoma, 83
juxtamembrane region of, 130131
kinase 1 domain of, 132 Needle biopsy
mutations of, 132136 versus open biopsy, 82
oncogenic mutations of, 130 Nerve sheath differentiation, 94
KIT receptor, 118 Neurofibromatosis type 1 (NF1), 136
NF1 gene, 109
L-NAME, 74 Nitric oxide (NO), 74
Laboratory models, 7174 NTRK3
Late targets, 159 activated, 107
Late tissue adverse events, 29
Leiomyosarcoma (LMS), 5 Oncogenic kinases
Leukocytes in GISTS, 136137
role of, in TNF-mediated antitumor effects, Oncogenic mutations
72 of KIT, 130
Liposarcoma, 910, 104 Open biopsy
classifying, 161162 versus needle biopsy, 82
dedifferentiated, 162
myxoid, 162163 Paraganglioma, 136
occurence of, 161 Parallel opposed pairs (POP)
overall survival in, 5 of conformal fields, 31
PPAR gamma pathway in, 163164 Paraspinal tumors, 30
retroperitoneal, 23f Pathologic responses, 5152
round cell, 162 Paxlitaxel, 6
well differentiated, 162
LNA, 74 expression of, 9
Local patterns Pericytes, 171
of spread, 19 Peroxisome proliferator-activated
receptor-gamma 9
MAID regimen, 4 Phase II studies, 10
Malignant fibrous histiocytoma (MFH), 5, 55f, of imatinib, 121
83, 152 Phase III trials, 5253
Malignant peripheral nerve sheath tumor, 109 Planning target volume (PTV), 20, 24f
T2-weighted magnetic resonance image of, Plasminogen activators, 170
20f Plasminogen (angiostatin), 174
Melanocyte differentiation, 94 Platelet derived growth factor (PDGF)
Melphalan sarcomas driven by, 123124
TNF and, 6770 Platelet derived growth factor receptor alpha
Mesenchymal disorders (PDGFRA), 105, 176
kinases in, 143t activated, 108
Mesoblastic nephroma, 107 as alternative oncogene, 135
Metalloproteases, 170 D842V, 139140
Metastatic disease Platelet derived growth factor receptor beta
systemic therapy in, 45 (PDGFRB), 100, 176
Methotrexate, 48 activated, 106
Mitoses, 92 Pleomorphic rhabdomyosarcoma, 83
Modalities Pleomorphic sarcomas, 83
scheduling, 21 Positron emission tomography (PET), 37
Modulated electron therapy (MERT), 36 Postoperative therapy
Molecular aberrations, 100 functional status and, 51
in adipose tumors, 102104 impact of, 5052
Clear cell sarcoma, 104105 pathologic response and, 5152
Molecular diagnostics, 96 scope of surgery and, 50
Molecular targets, 710 PPAR gamma pathway
Monomorphic spindle cell tumors in liposarcoma, 163164
immunohistochemistry for, 94t Preoperative radiation therapy
Multi-leaf collimator (MLC), 22 for extremity/trunk sarcomas, 4647
Multimodality therapy for retroperitoneal sarcomas, 47
for extremity/trunk sarcomas, 4849 Progression-free rates, 5, 7, 11
184 Index
Kaplan-Meier estimates of, 12f enes expression in, 158t159t

Proton beam irradiation, 36 gene expression in, 155156
Pulmonary chondromas, 136 heterogeneity of molecular alterations in, 163
local treatment of, 153154
Quality of life, 51 occurence of, 153
overall survival in, 5
Radiation doses translocations of, 154155
escalating, 33 Systemic therapy
Radiation target volume, 27f drugs and, 57
Radiation therapy for metastatic disesase, 45
surgical complications of, 5456
survival in, 5253 Three-dimensional conformal radiotherapy (3D
Radiotherapy (RT), 18 CRT), 30
conformal, 32 elements of, 22
curative, 2930 multi-leaf collimator, 22
external beam, 2122 plan calculation, 2223
image fusion in, 34 Thrombospondin, 170
image-guided, 37 Toxicity, 44
intensity modulated, 18 Transcription factors
retreatment, 3031 targeting, 142
spread patterns and, 1819 Transforming growth factor 157
targets for, 1821 TRKC
three-dimensional conformal, 18 activated, 107
tissues at risk in, 18 Trunk sarcomas
Ras pathway, 109 chemoradiation for, 4849
Ras protein multimodal therapy for, 4849
FTI targeting, 9 preoperative radiation therapy for, 4647
Regional lymphatic pathways of spread, 19 Tumor necrosis factor-alpha (TNF), 66
Retinoid acid receptor (RAR), 9 dose ranges for, 73
Retroperitoneal sarcoma doxorubicine and, 70
chemoradiation for, 4950 in histologies, 7071
multimodality therapy for, 4950 melphalan and, 6770
preoperative radiation therapy for, 47 vasculotoxic mechanisms of, 71
RT plans for, 31f Tumor vessels
Rhabdoid tumors, 110 destruction of, 72
Rhabdomyosarcoma, 110 Tyrosine kinase inhibitors, 176
Signal transduction pathways, 710 Uncertain targets, 159
Single photon emission computed tomography Undifferentiated round cell tumors
(SPECT), 37 immunohistochemistry for, 95t
Soft tissue sarcoma (STS)
angiogenesis in, 172174 Vascular endothelial growth factor (VEGF),
classification of, 83 170
diagnosis, 23 receptor tyrosine kinase inhibitors, 174
histological subtypes of, 152153 serum, 172
identifying causal molecular alteration in, Vasculotoxic mechanisms
165 of chemotherapy/TNF, 71
progression-free rates for, 7f Vasoactive drugs, 74
therapuetic targets in, 9t Vincristine, 48
Solid tumors Volumetric-based planning
induction therapy for, 44 applying, 3334
Static disease, 10 multidisciplinary interactions of, 33
SU5416, 175 rationale for, 2933
Surgical complications Volumetric treatment, 2129
of radiation therapy, 5456 evolution of, 2122
Survival, 5254
Suveillance, Epidemiology, and End Results WHO classification, 8387
(SEER), 2
Synovial sarcoma, 110111, 154f ZD1839, 160161
EGFR protein expression in, 157 EGFR protein and, 161

Treatment of Soft Tissue Sarcomas

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Treatment of Soft Tissue Sarcomas

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TARGETING TREATMENT OF

SOFT TISSUE SARCOMAS

Klastersky, J. (ed): Infectious Complications of Cancer. 1995. ISBN 0-7923-3598-8.

KLUWER ACADEMIC PUBLISHERS

2004 Kluwer Academic Publishers

Print 2004 Kluwer Academic Publishers

All rights reserved

Created in the United States of America

Visit Kluwer Online at: http://kluweronline.com

1. Targeted therapy: Ready for prime time? 1

The last decade we have witnessed a major change in the

Targeted therapy: Ready for prime time?

Caroline Seynaeve and Jaap Verweij

Dept. of Medical Oncology,

Soft tissue tumours are a heterogeneous group of rare neoplasms with

Nevertheless, progress in diagnosis and therapy of adult STS over the

groups facilitating cross-talk with respect to the communication of trial results

On the way to a new approach and further advance in systemic therapy

2 CHEMOTHERAPY AT THE BEGINNING OF THE

2.1 (Neo-)Adjuvant Chemotherapy

The value of neo-adjuvant or adjuvant chemotherapy continues to be a

2.2 Metastatic disease

In contrast with above-mentioned settings, the aim of systemic therapy

2000; Seynaeve/Verweij, 2002). Conclusions from these studies are that

3 SYSTEMIC THERAPY OLD DRUGS IN A NEW

Paxlitaxel has been studied in patients after or without prior exposure to

ET-743 (Trabectedin), a cytotoxic tetrahydroisoquinolone alkaloid

4 MOLECULAR TARGETS AND SIGNAL

The success of imatinib mesylate in treatment of GIST has led to a better

Most dermatofibrosarcoma protuberans tumours contain a translocation

is also an inhibitor of and it is assumable that glivec may be

by PPAR ligand pioglitazone. Demetri et al. have used troglitazone, an analogue

5 CHANGING METHODOLOGY ON TESTING NEW

Although phase II studies performed in a small group of patients suffer

Since in phase II screening studies of biological agents response rate is

combination with those of other co-operative groups, possibly even resulting in

The fast development and application of microscopic and new

without a joined effort and a global co-operation of the several co-operative

Volume-based radiotherapy targeting in soft tissue

Iain Ward1 ,Tara Haycocks2, Michael Sharpe3, Anthony Griffin4, Charles

Departments of Radiation Oncology1, Radiation Therapy2, Medical Physics3, and Surgical

Although a highly effective adjuvant therapy, traditional

2 DEFINING TARGETS FOR RADIOTHERAPY OF STS

2.1 Tissues at risk

The choice of RT volumes in STS is profoundly influenced by the

2.2 Local patterns of spread

In broad terms, soft tissue sarcomas tend to spread in a longitudinal

2.3 Regional lymphatic pathways of spread

Regional lymph node involvement in STS is unusual and for most

2.4 Paucity of available evidence

Unfortunately, with one exception,3 no formal assessment of target

2.5 Extracompartmental sarcoma

Certain anatomic areas are potential spaces without good

2.6 Margins for geometric uncertainty

An additional feature in radiotherapy target delineation concerns

uncertainties in set up and treatment delivery may equally result in inadvertent

2.7 Influence of scheduling of modalities

Pre-operative and post-operative RT represent the two usual

3 VOLUMETRIC TREATMENT PLANNING AND

3.1 Evolution of volumetric-based planning

The initial introduction of adjuvant radiotherapy more than two decades

3.2 Three-Dimensional Conformal Radiotherapy (3D CRT)

3.2.1 The elements of 3D CRT

Three-dimensional conformal irradiation has been described as