Download Metastatic Soft Tissue Sarcoma Chemotherapy

Clinically validated predictive tests for
systemic therapy response in soft tissue
sarcoma are still being sought.
Jeffrey Hessing. Maison Ancienne. Oil on canvas, 31¾″ × 39″.
Metastatic Soft Tissue Sarcoma Chemotherapy:
An Opportunity for Personalized Medicine
Damon Reed, MD, and Soner Altiok, MD, PhD
Background: Soft tissue sarcoma (STS) includes biologically and histologically diverse mesenchymal tumors
that are relatively chemotherapy-resistant compared with other sarcoma subtypes.
Methods: The authors discuss the clinical challenges frequently encountered by medical oncologists and review
the literature for predictive strategies to systematically approach chemotherapy decision making.
Results: There are no clinically validated predictive tests for chemotherapeutic response or resistance in STS.
Clinical features including histology, stage, and patient age are currently used to guide therapy decisions in STS.
Conclusions: A method to predict response or resistance to chemotherapy, utilizing both targeted and conventional agents, would be beneficial in reducing toxicity and improving response rates for patients with STS and
also in designing clinical trials for this disease.
Introduction
Soft tissue sarcoma (STS) encompasses a range of over
40 histologic diagnoses with varying biology, from
translocation-defined entities to diseases with complex
karyotypes.1-4 Roughly 10,000 new cases are diagnosed
in the United States per year across all ages, comprising
1% of adult cancers and 7% of childhood cancers. While
localized resected disease can often be cured, controversy exists regarding optimal management of tumors
in younger patients with high-grade disease or larger
tumors. Prognosis is significantly worse when tumors
cannot be controlled by surgery and radiation therapy
or in the setting of metastatic disease, and chemotherapy
is the primary modality of treatment. Because of the
poor overall survival of 20% to 25% at 2 years, as well as
the short median survival of 12 months and the rarity
of these tumors, there is a need for novel approaches
for therapy.5 Due to the biologic diversity, it is unlikely
that a single agent or a combination of agents would be
successful across the spectrum of diagnoses.
Chemotherapy in STS
From the Departments of Sarcoma (DR) and Pathology (SA) at the
H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida.
Submitted June 17, 2010; accepted September 23, 2010.
Address correspondence to Damon Reed, MD, Moffitt Cancer Center,
Sarcoma Department, 12902 Magnolia Drive, FOB 1, Tampa, FL
33612. E-mail: [email protected]
The authors receive support from the Sarcoma Foundation of
America, V Foundation, and the Moffitt Cancer Center Sarcoma
Foundation.
No significant relationship exists between the authors and the
companies/organizations whose products or services may be referenced in this article.
188 Cancer Control
Historically, doxorubicin-based therapy has been the
standard first-line agent in the treatment of STS when
chemotherapy is indicated, typically in metastatic or unresectable disease. While combination therapy, mainly
with ifosfamide, has improved response rates, toxicities
have increased with no clear corresponding benefit in
overall survival.6-10 Response rates to front-line chemotherapy vary, ranging from 10% to 46% with single agents
and with combinations of chemotherapy.5,9-19 Confounding this even further is the spectrum of response rates
July 2011, Vol. 18, No. 3
between histologies.4,16 In addition, patient age affects
analyses; improved responses in younger patients have
been reported.20-24 Finally, specific subtypes may benefit
from agents outside the typical approach of anthracyclines and alkylators (eg, taxanes for angiosarcoma and
trabectedin for myxoid/round cell liposarcomas).25,26
Tyrosine kinase inhibitors have revolutionized the management of gastrointestinal stromal tumors (GISTs), a
subtype of STS, showing that significant clinical benefit
is possible among the diagnoses currently categorized
as STS if the correct therapy can be matched to the tumor biology.27 Interpretation between trials is difficult
in part due to the heterogeneity of diagnoses and ages
among cohorts and has contributed to conflicting results between trials. Neither a systematic method to
guide chemotherapy for an individual patient nor a clear
national standard of care has yet been developed, thus
necessarily leading to a multifactorial, patient-specific
analysis to determine whether or not to offer therapy.
This is particularly complicated in second-line therapies, where many agents are available with similar low
response rates, and the short median survival of these
patients does not always allow for multiple treatment
attempts. Response rates are so low in the second-line
setting that progression-free survival (PFS) has become
the standard endpoint in clinical trials, with PFS at 3
months above 40% demonstrating activity and below
20% demonstrating inactivity.28
Ifosfamide is a standard second-line agent, with dosedependent activity ranging from 10% to 40% with particular activity in synovial cell sarcoma.12,18,19 Gemcitabine
and docetaxel combination chemotherapy has demonstrated particularly good response rates in leiomyosarcoma, especially uterine leiomyosarcoma, and has become
front-line therapy in our institution for this malignancy.
Response rates of 16% to 43% have been reported in STS
series.13,15-17 Vinorelbine has particular activity in rhabdomyosarcoma, having been studied particularly in the
pediatric population; it also has a 6% response rate in
heavily pretreated STS.11,14,29
Many agents beyond the scope of this article are being explored in STS. These include agents with a wide
range of activity, such as cytotoxic and targeted agents.
Trabectedin and ifosfamide derivatives are currently
being studied in phase III studies, and mTOR inhibitors
have been studied in maintenance, with approval pending. Insulin-like growth factor (IGF) pathway inhibitors,
proteasome inhibitors, cell cycle and growth modulators,
kinase inhibitors and others are being explored in earlier
phase trials, at times with corresponding biomarker
development. The vast numbers of available agents and
numerous histologic diagnoses complicate study design,
which can lead to overall negative conclusions and also
overlook clinical benefit in a particular subtype of STS. A
means to enrich responders and exclude nonresponders
in early-phase trials for STS is needed.
July 2011, Vol. 18, No. 3
Assay-Guided Therapy
Prospective determination of antibiotic sensitivity and
resistance has been the standard of care in infectious
diseases for many years. In contrast, due to the lack of
reproducible and predictive assays, treatment protocols
of cancer patients have been designed according to tumor histology rather than to the tumor’s sensitivity to a
given chemotherapeutic agent. Therefore, development
of novel drug sensitivity studies to predict patient response before initiation of therapy would help to design
the most efficient therapeutic regimen for individual patients while sparing others who are not likely to benefit
from the given therapy.
A formal analysis conducted by both the American
Society of Clinical Oncology and the Blue Cross and
Blue Shield Association’s Technology Evaluation Center
concluded that predictive assays were not ready for routine clinical use.30,31 However, both studies noted that
versions of these assays that meet specific parameters
might improve prediction of patient response to therapy.
Reasons for specific problems with previous assays included the low percentage of successfully completed
assays that yielded clinically useful results, rare interpretations that differed from standard of care, trial design
that did not include choices among several treatment
options, technical complexity that made application
beyond a single laboratory/institution unlikely, and the
long time needed to study completion, thus delaying
therapy.30,31 Minimum criteria that have been proposed
for a successful assay include standardizing materials, testing a range of concentrations to provide a dose response
curve, applying simple techniques with the possibility
of automation, and measuring cell survival with a clear
interpretation.32 Rather than calling for an end to predictive assays, the authors recognized that the need for such
an assay has even more appeal as new agents become
available. Making rational choices between increasing
numbers of regimens is not possible beyond matching a
particular histology to a regimen. A method of enhancing responders on a given regimen or excluding nonresponders would be of great benefit to sarcoma patients.
These studies concluded that future assays should be
studied in the context of a clinical trial, particularly for
diseases with a short median survival, when the window
for multiple, sequential therapeutic regimens is narrow
and when performance status can rapidly decline and
thus preclude additional therapies. The importance of
a predictive assay would be even more appealing when
there is a choice among multiple agents with relatively
low percentages of activity, as is the case with STS.30-32
Despite substantial progress made in recent years
for other malignant diagnoses, no clinically validated
tests are currently available to predict the efficacy of a
given agent for an individual patient with STS other than
GIST. Although there is consensus supporting the need
to develop and integrate the evaluation of predictive
Cancer Control 189
biomarkers in clinical trials, the practical application of
such an approach is still lacking.33
Assay-Guided Therapy Trials and
Retrospective Analyses
A search of PubMed literature was conducted that included the following terms and combinations thereof:
“drug screening assays, antitumor,”“predictive assay,”“ex
vivo,”“cancer prediction,”“individualized medicine,”“chemosensitive,”“chemoresistant,”“in vitro,”“biomarker,”“prediction,”“cancer,”“prognosis,”“clinical trial,” and “sarcoma.”
Articles were chosen based on review of the abstract and
English language. In the following, we present a spectrum
of methods and highlight the strengths and limitations of
each method. Clinical trials are emphasized, particularly
in sarcoma, though these are limited (Table 1).34-39
Cell Viability Assays
The concept of utilizing tumor cells exposed to chemotherapeutic agents and extrapolating the effects to in vivo
disease is as old as modern chemotherapy. Growth inhibition or cell death has been used in previous iterations of
assays of sensitivity to conventional chemotherapeutic
agents. 40-44 However, due to poor tumor growth under
assay conditions, methods that are labor-intensive and
time-consuming, and the use of uncertain criteria for
defining “sensitivity” or “resistance,” these assays have not
yet gained wide clinical acceptance. Several methods
have been explored using the cell-based platform.
The human tumor cloning assay (HTCA) and capillary cloning system (CCS) utilize tumor cell proliferation,
measured as the number of colony-forming units in solid
media over a period of 2 to 4 weeks.37,45 While these
assays measure tumor cell growth, they remove the tumor
microenvironment and the three-dimensionality of an in
situ tumor. The techniques are also labor-intensive, taking
weeks to complete. They have been studied in a relatively
large number of patients and histologic subtypes. One
trial reported a 64% rate of successful assay completion.37
In this trial, a range of malignancies had successfully
completed assays, and those treated according to the
assay results had an overall response rate of 25%. Patients
placed on empiric therapy due to an unsuccessful assay
had a response rate of 14%. Of the 470 patients, 14 (3%)
had sarcoma.37
The above assays measure tumor cell growth but do
not take into account tumor cells that are alive but not
dividing. This important distinction, which can be clinically relevant, is incorporated into the differential staining
cytotoxicity (DiSC) assay. This assay uses cells cultured
from the primary tumor and then counts live cells after
incubation with agents for 4 to 6 days. Modified versions
of this test have been used in lung cancer trials in the
past with a trend toward improved response-based assay results. Multiple trial designs, including selections of
combinations of therapy, have been attempted.36,46,47 A
series that included limited-stage small cell lung cancer
patients demonstrated that the biopsy needed for the test
Table 1. — Selected Studies Evaluating Predictive Assays in Cancer Therapy
Study/Assay
Method Overview
Successful Assay
Outcome
ATP34
Multicenter study of metastatic melanoma patients
98% successful;
71% with successful
assay received therapy
Response 36.4% in chemosensitive
patients vs 16.1% in chemoresistant
patients; overall survival 14.6 mos
in chemosensitive patients vs 7.4 mos
in chemoresistant patients
SRCA35
Primary tumor cells implanted under renal capsule
90% tumor growth in
xenograft and assessable
after chemotherapeutic
therapy
11 patients received a tested combination
of chemotherapy; 6 of 7 with relapse
had corresponding nonresponder status
of xenograft
DiSC36
Dye exclusion study, cells treated at reference range,
10% of reference range and 1000% of reference
range. Sensitivity < 50% of control in individual
drug assay (7 drugs tested); 3-drug combination
starting at week 13 from among 13 combinations
proven active in small cell lung cancer
44% of assays completed
in 12-wk period
All patients had a complete or partial
response to first 12 wks of standard
radiation therapy and chemotherapy;
among group of 8 who received assayguided therapy, median survival was
38.5 vs 17.5 mos
HTCA37,38
Cells from primary tumor grown in vitro in the
presence of chemotherapeutic assay and colony
counts done
Multiple trials: 45% to 64%
successful
470 patients on single trial
Reported higher response rates but
nonrandomized
ATP39
Extremity sarcomas were minced, enzymatically
digested tumors plated in 96 well plates, and tested
against a panel of chemotherapy
100% successful
No clinical data available
ATP = adenosine 5ʹ-triphosphate, SRCA = subrenal capsule assay, DiSC = differential staining cytotoxicity, HTCA = human tumor clonogenic assay.
190 Cancer Control
July 2011, Vol. 18, No. 3
did not delay initial chemotherapy and could be used to
guide the second 12 weeks of therapy. In addition, in
44% of patients, the median survival was 38.5 months vs
17.5 months in the standard care cohort. While the assay
tested seven different agents as monotherapy, the patients
were treated with a three-drug combination of assay-active
agents that had proven efficacy in this disease type.36
While the above-described assays may eventually
be automated, this technology does not currently exist. Higher-volume analyses using MTT and adenosine
5ʹ-triphosphate (ATP)-based chemiluminescence assays
have utilized microplate readers to measure chemical
surrogates of cell survival or proliferation. These have
been investigated clinically in a variety of tumor types
and are readily automated.48 Whether they accurately
correlate to cell survival or number is a matter of ongoing debate. Nevertheless, a multicenter melanoma study
demonstrated an overall survival advantage in chemosensitive patients of 14.6 months compared to 7.4 months
in chemoresistant patients.34 In this study, combination
cytotoxic chemotherapy was analyzed in the ATP-based
assay. The authors concluded that further optimization
regarding in vitro drug dosages and combinations tested
may need to be explored, though this strategy is currently being tested in the phase III setting through the
Dermatologic Cooperative Oncology Group.34 Importantly, this trial used combinations not routinely used in
melanoma and found higher response rates than those
reported in historical data. Feasibility of chemosensitivity
testing with an ATP-based assay in an exclusive sarcoma
population was performed showing that results can be
obtained. Unfortunately, no clinical response data was
included in the published study.39
Genome-Wide Microarray Assays
Genome-wide microarray assays allow for a snapshot of
the transcribed genes and level of transcript in a given
tumor. These assays have been used to explore aberrant
pathways and to define signatures of tumor subtypes
typically within a given histology. Genome-wide microarray assays have been investigated retrospectively
due to the cost and time necessary to analyze the large
volume of data. Recent reports in bone sarcomas have
demonstrated that enriched subsets of genes produce
reliable predictive signatures compared to clinical history.
This was validated prospectively in 1 case, compared to
histologic tumor necrosis in 8 of 8 patients.49-51 Ongoing
studies in sarcoma appear to be underway comparing
gene signatures to outcome based on observed abstracts
at meetings; however, publications remain limited to biology without clinical outcomes data.52 With large data sets
and numbers of genes upregulated and downregulated
in a given experiment, it is often difficult to sort out the
relative contributions of the individual genes. Furthermore, the data is not necessarily correlated with protein
expression and is unable to identify protein modifications
July 2011, Vol. 18, No. 3
that may be important in the pathogenesis of a given
tumor. Inherent biases built into the methodologies also
complicate the interpretation of results.53
Protein Assays
Putative markers of chemotherapy resistance including
efflux pumps, enhanced DNA repair, mutated target proteins, and variation of metabolizing enzymes have been
thoroughly investigated with varying results. Examples
of single proteins with prognostic and therapeutic importance can be readily found in cancer literature, with
a notable example being hormone receptors in breast
cancer. However, markers that affect therapeutic decision making have yet to be developed in non-GIST STS.
Examples of clinical trials in sarcoma include investigating the efflux pump P-glycoprotein (P-gp) and O6methylguanine-DNA-methyltransferase (MGMT). P-gp
levels were assessed by immunohistochemistry in an
osteosarcoma clinical trial that included P-gp substrates
doxorubicin and methotrexate in the treatment of all
patients. There was no difference in overall or event-free
survival in this study based on P-gp expression.54 MGMT,
which repairs DNA damage due to methylation and is
inhibited by O6-benzylguanine, varied considerably in
the blood of patients with STS, and targeted inhibition
of MGMT did not produce any objective responses.55
Xenograft Assays
Patient-derived mouse xenograft models allow for recapitulation of three-dimensional tumor architecture and
incorporate aspects of tumor stroma. Two major limitations of animal studies are cost and labor intensity. While
sarcoma xenografts have been generated, a comparison
of xenograft response to clinical outcome data has not
yet been published. One group reported establishment
of a series of xenografts that were treated with conventional cytotoxic agents and analyzed for biologic markers of resistance including MDR1, topoisomerase IIα,
and glutathione S-transferase. This panel showed low
responses to conventional agents and did not have a
strong correlation between selected gene markers and
chemotherapeutic effect.56
Xenografts have been used recently in non–small
cell lung cancer (NSCLC) to explore the chemosensitivity of selected agents or combinations. The subrenal
capsule assay uses subcapsular renal transplants of human tumors into murine xenografts treated with various
chemotherapies in this disease, with a recently published
report establishing a series of xenografts and treating
them with three chemotherapeutic combinations.35 A
90% engraftment rate was achieved, and about half of the
tumors were tested against all three chemotherapeutic
regimens. The xenografts resembled the primary tumor
histologically. Almost a third of the tumors were not
sensitive to any of the tested combinations. Perhaps
most importantly, a series of 11 patients received one
Cancer Control 191
of the tested combinations (vinorelbine and cisplatin).
Seven of these patients recurred, 6 with corresponding
nonresponsive xenografts. One xenograft was sensitive
to the combination and recurred. Results from this assay
took 6 to 8 weeks. The authors concluded that there is
a correlation between the assay results and clinical data
in the recurrence group and that xenograft-based testing
may help identify new active combinations in NSCLC.35
Summary of Predictive Assay Approaches
Individual gene tests, translocation analysis, and protein
tests are currently widely used in breast cancer, leukemia,
and other malignancies to tailor therapy. For example,
FLT3 testing modifies prognosis for acute myelogenous
leukemia patients, and therapy with a targeted tyrosine
kinase inhibitor molecule or a lower threshold for allogeneic transplantation is being explored in clinical trials
at this time. Similarly, the BCR-ABL translocation is used
to guide therapy, also with a tyrosine kinase inhibitor
in chronic myelogenous leukemia and childhood acute
lymphoblastic leukemia. However, no cell-based assays
or genome-wide analyses are currently used for the selection of chemotherapy as the accepted standard of care.
In non-GIST STS, histology and clinical features are often
used to guide therapy, but predictive assays are currently
not routinely used, if at all. [18F]fluorodeoxy-D-glucose
positron emission tomography (FDG-PET) scans are being evaluated in sarcomas to determine the extent of disease and residual disease. One study of 46 patients with
STS showed that baseline PET intensity and metabolic
response were predictors of relapse-free survival, and a
combination of baseline signal intensity and metabolic
response was a robust predictor of overall survival.57
A recent study of high-grade bone sarcomas showed
good negative correlation of PET avidity changes after
one cycle of chemotherapy with tumor necrosis, with
a threshold signal reduction of 60% after one cycle of
chemotherapy.58 However, a sarcoma cohort including
intermediate- and high-grade tumors was more complex,
with lower sensitivity and specificity.59 A recent task
force report has no recommendation on the routine use
of a PET scan during therapy to follow response or to
predict response in sarcomas.60
Many STS clinical trials have compared cytotoxic
agents alone or in combination with a spectrum of response rates. While clinical data have led to some insight in matching tumor types that tend to have better
response rates, a mechanism-based analysis of tumor
tissue treated with chemotherapy is lacking. Doxorubicin has remained the standard of care for 35 years.6 It
is not always clear how cytotoxic agents, which damage
DNA or inhibit critical pathways in cells, induce a response. While mechanisms of action of these agents are
postulated, biomarkers of efficacy or resistance remain
largely unknown. Markers of DNA damage, apoptosis,
autophagy, and cell cycle arrest have been identified,
though these pathways are complex, interconnected,
and often redundant (Table 2).
Traditional approaches to the preclinical investigation
of novel cancer therapies rely on the use of established
human cancer cell lines. These cell lines are maintained
in vitro in serum-based growth media, and their responses
to experimental cancer therapeutic agents are assessed
by studying growth and apoptosis in vitro or in vivo.
Prolonged culture of human cancer cells in serum and on
tissue culture plastic results in cell lines that may not be
Table 2. — Potential Molecular Biomarkers of Chemotherapeutic Effect or Resistance
Protein
Function
Pathway
PARP
Posttranscriptional modification of cellular proteins through poly(ADP-ribose)polymerase; this protein is
cleaved by caspase-3 during apoptosis
Apoptosis
RB
Tumor suppressor that interacts with E2F family of proteins and halts G1/S transition when hypophosphorylated
Cell cycle
Caspase-3
Member of a family of proenzymes that signal apoptosis though cleavage of other family members
Apoptosis
Gamma-H2AX
Specific phosphorylation of a histone in response to double-stranded DNA breaks
DNA damage
Chk1 and Chk2
Key signal transducer kinases that are activated in response to diverse genotoxic insults to transmit the
checkpoint signals from the proximal checkpoint kinases
Cell cycle
Cdc2
A cell division control protein, also known as cyclin-dependent kinase 1 (Cdk1) or p34/cdk1, plays a key role in
the G2 to M phase transition
Cell cycle
Cdc25
Family of dual-specificity phosphatases that positively regulate the cell division cycle by activating
cyclin-dependent protein kinases
Cell cycle
PCNA
Proliferating cell nuclear antigen (PCNA) involved in DNA synthesis and postreplication repair
Cell proliferation
LC3
Microtubule associated protein that interaction between microtubules and the cytoskeleton activated
during autophagy
Autophagy
192 Cancer Control
July 2011, Vol. 18, No. 3
representative of the parent tumor. Such differences are
of concern in the study of basic cancer biology and are
fundamental to our approach in studying this disease. In
particular, culture selection in cell lines may disturb the
in vitro relationship between the cancer stem cell and
its progeny, and it also removes tumor-stromal interactions that are essential to the three-dimensional biology of
solid tumors in vivo. To investigate novel therapeutic and
diagnostic strategies with greater accuracy, new preclinical strategies such as biomarker assessments are needed
to assess anticancer therapies in order to determine if
patients are likely to benefit from a given therapy.
We propose that future STS studies should involve
evaluations of molecular pathways in primary sarcoma
tissue. There must be a focus on the biology of the tumor
in addition to the histology. Treatment with clinically relevant agents in short-term ex vivo assays and biomarker
analysis with corresponding in vivo xenograft or clinical
outcomes data would help establish patterns or proteins
that may provide information regarding tumor sensitivity
or resistance to a given treatment (Figure). Predictive
markers can serve as the basis for inclusion or exclusion
for clinical trials that can incorporate targeted agents
in addition to a cytotoxic therapy. This may eventually
lead to combinations of medications that can improve
response rates by exploiting signaling or biologic abnormalities in malignant cells.
Conclusions
The goals of predictive assays or biomarkers are to match
efficacious chemotherapy to an individual patient and to
exclude chemotherapy that would cause toxicity without
benefit. Diseases with a poor prognosis, genetic heterogeneity, many therapeutic options, and a poor response
rate to traditional chemotherapy provide the rationale for
predictive studies. A quick, reproducible, patient-specific
assay that could test many single agents and combinations based on the mechanisms of the therapeutic agents
would be ideal. In the setting of conventional cytotoxic
agents, biomarkers of biological efficacy are not always
known, though molecular markers of DNA damage, cell
cycle arrest, apoptosis, autophagy, and necrosis may provide predictive data.
Markers of tumor response to cytotoxic chemotherapy have been explored in breast and colon cancer.
While the results for colon cancer are controversial and
mixed, markers in breast cancer such as topoisomerase
IIα and erbB2 show good correlation to response, and
ABCB1 (P-gp) and BCL-2 overexpression show good
correlation to resistance.45,61 A clinical trial similar to
that shown in the Figure could be conducted to assess
the positive and negative predictive value and match
patients to chemotherapy to determine if this can improve response rates, progression-free survival, and overall survival.
First-Line Therapy
SD, PR, CR
PD
First-Line Therapy
SD, PR, CR
Regimen A
PD
Regimen B
Regimen C
Regimen D
Regimen E
PD
Ex vivo Assay Results
Second-Best Option
Sarcoma biopsied
and treated with
front-line and
regimen A–E
ex vivo for
24 hours and
molecular markers
analyzed
Ex vivo Assay Results
Best Option
Continue
Figure. — Ex vivo assay schema. This theoretical trial design incorporates an ex vivo assay strategy for metastatic or unresectable STS patients and would
allow for prospective assessment of molecular markers, calculation of positive and negative predictive value to first-line therapy, and match chemotherapy
in the second line based on ex vivo assay response. While first-line therapy would be anthracycline-based, second-line therapies could consist of cytotoxic
agents or combinations with established efficacy or with potential, utilizing the assay to match the patient to therapy. SD = stable disease, PR = partial
response, CR = complete response, PD = progressive disease.
July 2011, Vol. 18, No. 3
Cancer Control 193
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