A "Bedside to Bench" Investigational Platform
for the Study of Myxoid Liposarcoma
Myxoid liposarcoma is one of the most common sarcomas bearing a characteristic chromosomal translocation encoding an oncogenic fusion transcription factor. In the case of myxoid liposarcoma it is the FUS-DDIT3 fusion protein. Although local control measures (surgery and radiation) are highly effective and cure about half of patients, this cancer is prone to distant metastases to sites around the body. The risk of metastasis is higher when there is round cell histology present, as well as when certain biomarkers are expressed (such as those implying activation of the IGF/Akt/mTOR axis). Conventional chemotherapy provides little, if any, survival benefit, although recent results suggest trabectedin may help some patients. Regardless, we have yet to convert these molecular insights into specific therapies targeting the FUS-DDIT3 fusion protein or its direct oncogenic effects. One major reason for slow progress has been a lack of available tissue samples from annotated cohorts and clinical trials; another has been a lack of representative model systems.
Translocations are a type of mutation that occurs when DNA from two pieces of different chromosomes gets abnormally spliced together. If the break points on each chromosome are in the middle of genes, this can result in an abnormal "fusion protein" which has a front part coded by one gene and a back part coded by a different gene originally from a different chromosome. If the two involved genes are transcription factors – master regulators of the cell’s biology – then you can get a situation where the DNA targeting element (e.g. DDIT3) is abnormally spliced to a new on/off switch (e.g. FUS). If this causes cancer, it is termed an oncogenic fusion transcription factor. Several such mutant proteins appear to be fundamental events in sarcomas (e.g. Ewing family tumors) and leukemias, and with new research methods have become increasing recognized in other cancers (e.g. prostate).
We have assembled an international team of sarcoma experts who have the ultimate goal of developing a multi-investigator driven "bed-side to bench and back" platform for advancing the study of myxoid liposarcoma.
A "bedside to bench and back" strategy starts with physicians (such as the MD researchers in our team) recognizing pressing, unsolved clinical problems and using their bedside practice connections to assemble patient data and tissue specimens. Returning to the laboratory bench as clinician-scientists and/or in collaboration with PhD scientists, a molecular biology or similar research approach is then used to scientifically develop a solution that not only advances scientific knowledge, but also suggests new diagnostic tools or treatments that might work to improve patient care. When ready, these are brought back to the bedside by inspiring new clinical trials, informing their best design, and making scientific sense of the results.
We will do so by drawing on our institutions’ large combined catchment of sarcoma specimens and our connections to major sarcoma clinical trial groups in Europe and North America, to generate annotated archival resources and high quality material from incident cases. We have extensive experience with tissue microarrays, primary tumor culture and xenograft models. Team members also bring expertise developed in related sarcoma systems to detail the function of the FUS-DDIT3 oncogene and its relation to key molecular pathways including Akt/mTOR and histone modifications. This work will be linked to an experimental therapeutics program assessing trabectedin, histone deacetylase inhibitors, mTOR inhibitors and other targeted therapies suggested by our research. If successful, we will be able to organize future targeted therapy trials for myxoid liposarcoma, and obtain long term funding from national sources.
Introduction and Background
Clinical Features and Pathology of Myxoid Liposarcoma
Myxoid liposarcomas represent about one third of liposarcomas and account for ~10% of all adult soft tissue sarcomas (recent reviews included in Romeo & de Toi1 and Singer et al).2 These tumors most commonly arise in the deep soft tissues, particularly thigh or retroperitoneum. Patients are typically younger (peak age 30-50) than for other types of liposarcoma, with no gender predilection. Standard treatment by surgical wide local excision and adjuvant radiation is often, but not always, sufficient to achieve local control. Most problematically, myxoid liposarcoma is prone to metastasis (seen in 35% of cases overall), with an unusual propensity for spread to extrapulmonary sites (such as other soft tissues throughout the body) which can be difficult to detect clinically. Conventional cytotoxic chemotherapy provides at best a small benefit to survival, and in the metastatic setting achieves at most partial responses. Five year survival figures have variously been quoted between 20-70%, depending in part on the amount of round cell histology present.
Under the microscope, myxoid liposarcoma consists of small histologically bland spindle cells, dispersed within an abundant hyaluronidase-sensitive myxoid matrix, with frequent pools of stromal mucin3 (Figure 1). This type of sarcoma displays an unusual, characteristic plexiform vasculature: a network of small capillaries, with larger arborizing vessels notably absent. Lipoblasts, cells representing an early stage of adipocytic differentiation, are a characteristic feature, as are varying amounts of mature fat. Round cells, defined as closely-packed high grade small blue cells with no evident line of differentiation and no matrix production, are present in a subset of myxoid liposarcomas. These define higher grade disease, portend increased rates of metastasis and poor outcomes,4 and can create problems for differential diagnosis on small biopsies.
Molecular Features of Myxoid Liposarcoma
Myxoid liposarcoma is one of the more common entities within the class of sarcomas bearing a fusion transcription factor oncogene (see box below for definition of transcription). In this disease, the defining molecular event is a balanced translocation, most commonly t(12;16)(q13;p11), fusing FUS with DDIT3.5 Twelve FUS-DDIT3 transcript variants (varying in the exons retained at splice sites) (Figure 2) have been reported, and EWSR1 (the Ewing sarcoma gene) may substitute for its homologue FUS in rare cases. The resulting chimeric transcript fuses 5’ amino terminal exons of FUS (transcriptional regulatory domains interacting with the RNA polymerase II complex) to the full coding sequence of DDIT3, which is a DNA-binding leucine-zipper transcription factor that plays a role in cell cycle control and adipocytic differentiation. Importantly, FUS-DDIT3 expression constructs can induce a sarcoma phenotype in cell model systems,6 although characteristic myxoid liposarcoma is not so derived, suggesting the in vivo cellular and tissue background for the translocation may be critical to modeling this disease. Relatively little is known about the mechanism of action of FUS-DDIT3 in molecular oncogenesis. Native DDIT3 dimerizes and inhibits fellow C/EBP family members, which are central effectors of growth arrest and terminal adipocyte differentiation, suggesting that the fusion oncoprotein may directly interfere with these critical functions. Evidence also exists suggesting net downstream results include activation of angiogenic factors (IL-8), early adipose differentiation (PPARγ), growth factor signaling (IGF, RET) and cell cycle progression (cyclinD-cdk4) among others.7
Transcription: This is a basic and important biological process whereby deoxyribonucleic acid (DNA) is copied into ribonucleic acid (RNA), which is a chain of nucleotides that is similar to DNA. Unlike the double-stranded DNA, RNA is single-stranded, and is much shorter and less stable. The type of nucleotides used in RNA is also slightly different from the DNA. Transcription of DNA results in messenger RNA (mRNA), a specialized type of RNA that carries the template to make proteins, which are the main workhorses of a cell.
Therapeutic Opportunities in Myxoid Liposarcoma
Myxoid liposarcoma is highly susceptible to adjuvant or neoadjuvant radiotherapy, which aids in impressive local control rates achievable by experienced surgeons.8 Metastatic disease remains incurable in the great majority of cases, although multiple chemotherapeutic approaches can elicit at least temporary responses. Both radiation and chemotherapy cause extensive hyalinization and loss of neoplastic cellularity in myxoid liposarcoma, but the reduction in tumor size is less impressive (most likely due to the large portions of tumor volume composed of myxoid stroma).
Hyalinization refers to the replacement of cellular cancer tissue with non-descript protein material, as viewed under the microscope. In such cases, the apparent size of a tumor mass on a follow-up CT scan may not change much with treatment, but the content of that mass will be dramatically different if biopsied and viewed under the pathologist’s microscope – a sign of successful treatment. This is one type of response that can be seen with successful anticancer therapies, with imatinib treatment of GIST (gastrointestinal sarcomas) being a prominent example.
More recently, the use of trabectedin (ET-743 or Yondelis) in these tumors has been associated with extensive lipid maturation and loss of characteristic myxoid liposarcoma cellularity. While overall patient response rates with this drug in advanced sarcomas are low (10%), myxoid and round cell liposarcomas appear particularly sensitive to trabectedin.9 It has been postulated that this drug directly interacts with DNA chromatin grooves, displacing the FUS-DDIT3 chimeric transcription factor. Thus it may interrupt the key molecular initiator and driver of this tumor, an ideal characteristic for a "targeted therapy," although this remains far from proven. Interestingly, high-dose adriamycin/ifosfamide has recently been noted to show identical lipid maturation features (Lazar, unpublished data) and thus the morphologic changes induced by trabectedin treatment may not be unique. The molecular underpinnings of these similar responses to very different agents is largely unexplored.
Other agents with very different mechanisms may also be effective against fusion transcription factor associated sarcomas. The emerging delineation of histone deacetylase-driven epigenetic alterations that coincide with tumorigenicity and malignant progression has provided the impetus to develop HDAC inhibitors as novel cancer therapeutics.10 These initiatives were prompted by preclinical observations of broad growth inhibitory effects in cultured cancer cells while sparing normal cells, and significant in vivo effects in various human tumor xenografts. To date, clinical trials have documented the potential efficacy of HDAC inhibitors in multiple cancers and two compounds have already received FDA approval for cutaneous T-cell lymphoma therapy.11 The study of HDACs in soft tissue sarcomas is one of the major research goals of the Lev and Nielsen laboratories. These efforts have identified in vitro and in vivo efficacy for several HDAC inhibitors against a range of soft tissue sarcomas,12 where they may even be capable of disrupting the fusion oncoprotein’s effector complex.13 These insights have supported a recently initiated phase I/II clinical study examining the effects of PCI-24781 in combination with doxorubicin in advanced sarcomas and of SB939 in translocation-associated sarcomas. To the best of our knowledge, little is known about the potential effect of HDAC inhibitors on human myxoid liposarcoma. Our previous studies did include one myxoid liposarcoma cell line (402-91) demonstrating anti-tumor effects,14 and have since extended this finding to primary myxoid and myxoid-round cell liposarcoma xenograft models (see section 2c, below). These encouraging preliminary data form the basis for some of the studies proposed in this research program.
Current Research in Myxoid Liposarcoma by the Co-Principal Investigators
Dr. Nielsen began work in this field by helping generate the world’s first gene expression profile data on myxoid liposarcomas.15 Subsequent investigations identified gene signatures characteristic of early adipocytic development and branching vascular morphogenesis. Using tissue microarrays as a validation platform, Nielsen’s group confirmed that expression of the RET, IGF1R and IGF2 proteins is linked to poor outcome in myxoid liposarcoma, supporting a role for the MAPK and Akt/mTOR pathways in aggressive cases.16 Dr. Nielsen has also been active in developing HDAC inhibitors as experimental therapies, demonstrating that translocation-associated sarcomas, including myxoid liposarcoma, are particularly sensitive to such agents.14 This work has led to the opening of a multicentre Canadian trial, IND.200 (NCT01112384), for which Nielsen is the correlative science director.
Drs. Lazar and Lev, already working closely together in the MD Anderson Sarcoma Research Laboratory, have been involved in ongoing studies into the molecular events underlying progression of myxoid to round cell liposarcoma. Using multiple techniques, including tissue microarrays to investigate phosphorylation of downstream targets, Lazar’s group confirmed activation of the Akt pathway in myxoid liposarcoma, and found a further increase in flux through the pathway upon round cell transformation. Targeted mutational analysis of PIK3CA identified activating mutations in 14% of cases, confirming previously reported prevalence;17 these mutations were linked to round cell change. Moreover, loss of PTEN, a tumor suppressor and critical inhibitor of the phosphoinositide 3-kinase (PI3K) pathway, was found in an additional 13% of cases and was mutually exclusive with PIK3CA mutations. Lazar’s studies into IGF1R expression validated Nielsen’s prior findings, and further showed that poor outcomes could be linked to preferential IGF1R expression in round cell tumors. Taken together, these findings support a role for the PI3K/Akt pathway in round cell transformation, and provide an explanation for reports of poor prognosis associated with PIK3CA mutation and high pAKT expression in myxoid liposarcoma.18 Dr. Lazar has also collaborated with investigators at Texas Children’s Hospital in developing an optimized RT-PCR assay to detect FUS-DDIT3 fusion transcripts for clinical use.19
Overall, development in the field of myxoid liposarcoma has been hampered in part by a lack of available models. Prof. Pierre Aman’s research group (Lundberg Laboratory for Cancer Research, Department of Pathology, Göteborg University, Sweden) has provided the community with a number of established myxoid liposarcoma cell lines,20,21 immortalized using viral sequences from the SV40 early region, which alters the function of a number of critical tumor suppressors including p53, PP2A, and RB family members. For this reason their relevance is limited for the study of growth and differentiation, although they are valued for isolation and study of the endogenous fusion protein. FUS-DDIT3 has been introduced into a number of cellular backgrounds including NIH3T3 and HT1080 cells, and more recently into murine22 and human23 mesenchymal progenitor cells. In mice only p53 null cells were tumorigenic, whereas in humans no tumorigenic clones were obtained. Myxoid liposarcoma therefore appears to arise in a different background in humans, possibly from more lineage-committed cells, and possibly with requirement for different contributing mutations (since human tumors are largely wild type for p53). Very recently, research by Dr. Igor Matushansky suggested that myxoid liposarcoma cells are mesenchymal cells that are committed to adipocytic differentiation, but for reasons yet unknown, are unable to complete the terminal differentiation program. Their study results were based on a p53 null mouse model; which while very elegant for research, may not provide an authentic cellular background for myxoid liposarcoma.
The conclusion which we share with many groups is that native human myxoid liposarcoma cells obtained from patient tumors are the most representative models for the biology and behavior of this disease. Such cultures are hard to obtain even in designated sarcoma centres due to the usual management strategy of core needle biopsy (yielding insufficient cells for research) followed by neoadjuvant radiation (damaging viability of cells for research in the subsequent surgical excision). Dr. Bovée’s work in the field includes not only the genetic characterization of 2 established myxoid liposarcoma cell lines originally generated by Prof. Aman,20 but also the establishment of new myxoid liposarcoma cell cultures from patient samples.24 Her research group additionally performed kinome profiling and subsequent pathway analysis in these models of myxoid liposarcoma, to identify new molecular targets for systemic treatment. Kinome profiling revealed that kinases associated with the nuclear factor-kappaB and Src pathways are active in myxoid liposarcoma. Inhibition of Src by the small molecule tyrosine kinase inhibitor dasatinib showed mild effect on cell viability, whereas inhibition of the nuclear factor-kappaB pathway, which is regulated by the FUS-DDIT3 fusion product, showed a significant decrease in myxoid liposarcoma cell viability, decreased phosphorylation of nuclear factor-kappaB pathway proteins, and caspase 3 mediated apoptosis.
Although a pathognomonic FUS-DDIT3 translocation has been identified that appears to drive the biology of myxoid liposarcoma, it is not directly targeted by any known drugs. A better understanding of the critical mechanisms of oncogenesis by FUS-DDIT3, including transcriptional effector partners and target genes, may suggest much-needed more specific and effective systemic therapies. However, the limited availability and relevance of model systems and difficulties in obtaining viable primary tumor samples have slowed research progress worldwide.
The Myxoid Liposarcoma Investigative Team
Myxoid liposarcoma is among the more common sarcoma subtypes for which a defined underlying oncogenic event has been identified. However, this has yet to lead to breakthroughs in patient care, due to a lack of critical mass in resources: tissue specimens, patient data, funding and investigators pursuing myxoid liposarcoma research. In agreement with the Liddy Shriver Sarcoma Initiative’s philosophy, we believe improved patient management and outcome can best be made by a team of dedicated investigators (Figure 3) working cohesively on this disease.
Judith Bovée, MD, PhD, a pathologist / associate professor at the department of Pathology of the Leiden University Medical Center, where she is the principal investigator in the bone and soft tissue tumor pathology group. Her main focus is on translational sarcoma research, for which she has several grants from the Netherlands Organization for Scientific research (NWO) and the KWF Dutch Cancer Society. Her group coordinated a trans-European Network of Excellence, EuroBoNeT, on pathology and genetics of bone tumours, in which Dr. Bovée was work package leader on the biology of chondrosarcoma. Since March 1st she became work package leader for "Molecular diagnosis and translational research" within the EuroSarc collaborative project "European Clinical trials in Rare Sarcomas within an integrated translational trial network." She has published close to 80 research articles related to mesenchymal tumors.
Alexander Lazar, MD, PhD is on faculty in the Sarcoma Research Center at The University of Texas MD Anderson Cancer Center (UTMDACC) where he has designated laboratory space and works collaboratively with Dr. Dina Lev. He is an Associate Professor in the Department of Pathology and oversees the Institutional Sarcoma Tumor Bank. Dr. Lazar’s particular expertise lies in tissue-based translational research, molecular diagnostics and early genetic changes in sarcoma. Based on his clinical role as an academic pathologist, he has extensive experience in the classification of soft tissue tumors and serves as a reference pathologist for the Sarcoma Alliance for Research through Collaboration. He has helped author more than 100 papers and book chapters, mostly pertaining to sarcomas.
Dina Lev, MD is the principal investigator of the Sarcoma Research Laboratory (SRL) at UTMDACC, Houston, Texas. Integrated within one of the largest clinical sarcoma treatment centers worldwide, the SRL is provided unique access to a large number of sarcoma patient derived samples permitting establishment and development of relevant human soft tissue sarcoma experimental models. These resources are highly valuable and have already formed the basis for a comprehensive translational platform for the study of sarcomas. Dr. Lev’s research focuses on the identification of molecular markers and therapeutic targets for a range of soft tissue malignancies, and she has published over 120 peer reviewed manuscripts.
Torsten Nielsen, MD, PhD is a clinician-investigator pathologist at the University of British Columbia, Vancouver. His centre draws sarcoma patients from Western Canada and is affiliated with the National Cancer Institute of Canada-Clinical Trials Group, wherein Dr. Nielsen is a member of the sarcoma executive committee, and its director of correlative science. He runs research labs active in translating novel molecular findings into practical diagnostics and new treatments, and he holds active funding for investigations of translocation-associated sarcomas from three major Canadian national granting agencies. His work has already led to two new, widely-used diagnostic biomarkers in sarcoma and to two clinical trials that are currently underway. Dr. Nielsen has published over 100 cancer research articles in scientific journals.
Each of the above investigators has been able to establish an infrastructure of equipment, specimens, models, laboratory and supporting personnel for sarcoma research, and has demonstrated a track record of productivity. Funding from the Liddy Shriver Sarcoma Initiative will synergize with existing infrastructure to bring a large array of research resources to bear on myxoid liposarcoma. Just as importantly, by working as an international team with complementary strengths, we will also be able to synergize our skills and research directions to address those questions which are both scientifically tractable and clinically relevant.
Our core team will also be supported by several close collaborators. Raphael Pollock MD, PhD, Head of the Division of Surgery and Director for the Sarcoma Research Center at UTMDACC, has extensive experience as a leading clinician-investigator in soft tissue sarcoma and will assist the group in securing access to needed human bioresources as well as troubleshooting experiments. Chad Creighton, PhD, Associate Professor in the Department of Medicine and the Dan L. Duncan Cancer Center Division of Biostatistics at UTMDACC, will serve as the biostatistics & bioinformatics advisor. T. Michael Underhill, PhD, a musculoskeletal developmental biologist at UBC, will assist in the molecular characterization work of FUS-DDIT3 and its targets. From Leiden, Bob van de Water, PhD (Toxicology) and Hans Gelderblom, MD, PhD (Clinical Oncology) will provide support for drug screening studies and for clinical trial translational work, respectively.
Aims of the Integrated Myxoid Liposarcoma Research Program
Our ultimate goal is to develop a multi-investigator driven "bed-side to bench and back" program for the study of myxoid liposarcoma that would remain operative beyond the Liddy Shriver Sarcoma Initiative funding timeline. The long term goals identified below have been identified as critical to developing and implementing this research program and their accomplishment will be our measure of success.
- To assemble the world’s largest clinically annotated, high quality frozen and formalin-fixed human myxoid liposarcoma tissue repository, and develop and characterize urgently needed experimental research models
- To use these resources to unravel myxoid liposarcoma-associated biomarkers and molecular predictors of therapeutic response, treatment resistance and disease outcome
- To elucidate the molecular mechanisms underlying myxoid liposarcoma biology to identify pathways that can be targeted with existing or experimental therapies
- To assess the anti-myxoid liposarcoma effects of biologically justified therapeutic strategies
All four principal investigators will contribute to each of the 5 specific objectives set out below (Figure 4), but with primary responsibility assigned to one PI. The critical tissue repository (Section 1a) – comprising formalin-fixed and frozen tissues, primary cultures and xenografted tumor tissues – will be consolidated, characterized and shared among all participants as a basis for biomarker (Section 1b), molecular biology (Section 2a) and experimental therapeutics (Section 2b and 2c) studies. Biomarker and molecular biology investigations will focus on pathways relevant to therapeutic intervention, supporting our "bedside to bench and back" theme.
1. Human Tumor-Based Insights
1A. Develop Tissue Repository
Pooled resources across our institutions provide a robust initial basis for our tissue repository. In addition to the existing resources described below, each of our investigators has established mechanisms in place for accrual of incident tumor specimens.
- Formalin Fixed Tissue: Dr. Lazar and colleagues have developed a myxoid liposarcoma tissue microarray (TMA)18 with annotated clinical data, including patient outcomes. This tissue microarray includes 165 tumors from 111 patients, including 82 primaries, 27 locally recurrent tumors and 56 metastases, representing 75 myxoid tumors, 43 with round cell features and 45 with marked treatment effect. Six tumors are represented by pre- and post- therapy cores to include a total of 76 untreated tumors, 35 treated with chemotherapy, 13 by radiation alone, and 28 by combination chemoradiation. Clinical follow-up for specimens included in this TMA is available for 150 tumors, on the full spectrum of outcomes, including disease-specific mortality (n=36, median follow-up time 1.7 years) alive with disease (n=57, median follow-up 3 years), and alive with no evidence of disease (n=61, median follow-up 3 years). MDACC resources also include additional formalin-fixed paraffin-embedded (FFPE) material for 75 cases included in the TMA, as well as 32 tumors with marked treatment effect which were not included on the TMA. This resource is complemented by the UBC adipose tumor TMA constructed by Dr. Nielsen's group, which includes 32 primary myxoid liposarcomas (11 of which have >5% round cell component) with detailed clinical annotation including median 78 month follow-up data.16 Both arrays have undergone detailed expert pathology review to confirm specimen quality for histology and immunohistochemistry.
- Frozen Tumor Samples: Frozen tissue from 57 myxoid liposarcoma tumors is presently retained in the UTMDACC institutional and sarcoma-specific biorepositories, including 19 tumors with predominately myxoid features, 6 round cell tumors, and 32 with variable treatment effect. Dr. Bovée adds 35 frozen cases of myxoid and round cell liposarcoma, while Dr. Nielsen at UBC contributes an additional 12 frozen specimens from the UBC orthopaedic oncology tissue bank. Both UBC and MDACC capture incident cases (approx. 15 / year total) under a procurement protocol designed to ensure high specimen quality.
- Primary cell culture: Both Bovée and Nielsen have obtained two established myxoid liposarcoma cell line models (402-91 and 1765-92) from Prof. P. Aman (Goteburg, Sweden).20 Dr. Bovée has established and characterized 8 primary cell cultures at Leiden. These cell lines and primary cultures were previously successfully used for karyotype and fusion gene characterization, drug assays and Western blot analyses,24 and cells from early passages are still available. Primary cell culture strains have been established at MDACC from 7 additional myxoid liposarcoma cases.
- Primary tumor xenograft lines: The UBC group contributes expertise with 4 subrenal implant primary tumor xenograft lines that have been passaged in up to 5 generations of mice. These xenograft lines are an effective means for successfully propagating even low grade tumors in a fashion amenable to drug assays.25 Importantly, the gross and characteristic microscopic features (including matrix, lipoblasts and vascularity) appear perfectly preserved in the subcapsular xenografts (Figure 6).
Formalin fixed tissue is the main way tumor specimens excised at surgery are stored long term. Research tests that can be used on formalin fixed tissue can be applied to the largest number of cases giving the best statistical power for scientific studies, and also are easy to take back into hospital labs as a new clinical test. Unfortunately, the formalin preservative destroys RNA and damages some DNA and proteins, limiting the types of research that can be applied. Frozen tissue has all these molecular preserved intact, but due to the expense of freezers and need to act immediately on a fresh tumor specimen, is only captured at major research centers and is a very limited resource. Cell cultures grown from fresh tissue remain alive and can be expanded, but living conditions in a dish may not resemble the real life tumor biology very well. Xenografts are tumor cells grown (with considerably greater effort and cost) in an experimental mouse, and are better models in that they grow in 3 dimensions with a blood supply – important if you are going to test new drugs, for example.
Resources to be acquired during grant period: Additional primary tumor material acquisition is ongoing as routine institutional and sarcoma-specific tumor banking at MDACC and UBC based on IRB-approved active protocols. This is enabling accumulation of additional frozen tumor tissues and primary cell cultures as well as access to FFPE materials for further studies on patient tumor specimens. We expect 25 to 30 incident cases a year at MD Anderson alone. Dr. Lazar is a reference pathologist and correlative science coordinator for the SARC clinical trial group and Dr. Nielsen serves a similar role for NCIC-CTG, facilitating access to clinical trial specimens from ongoing studies accruing myxoid liposarcomas, including studies of trabectedin and of HDAC inhibitors.
Significance: This program would create the world’s largest and best-characterized repository of human myxoid liposarcoma tissues and experimental models suitable for research, forming the basis for the rest of the proposed studies, and for future work by our group and for other dedicated researchers worldwide needing such a resource to translate their scientific insights.
1B. Unravel Biomarkers and Molecular Prognosticators of Therapeutic Response and Outcome in Myxoid Liposarcoma
Twelve fusion variants of FUS-DDIT3 have been described (Figure 2).19,24 However, data is mixed as to the clinical relevance of the different variants.4,26 We will perform RT-PCR on samples from patients included in our TMA population in order to perform correlative analysis of specific fusion variants with both patient outcome, as well as response to therapy and biomarker expression. This work will create an efficient pipeline for identification of myxoid liposarcoma fusion subtype on diagnostic materials, and the resulting resource will be able to assess the biologic significance of potential fusion variants, both in untreated tumors, as well as in response to therapy.
PIK3CA mutations have been reported in up to 18% of all myxoid liposarcomas, based on the most detailed mutational profiling data published to date.17 Work by the Lazar/Lev group has confirmed this finding in a subset of 44 cases, and demonstrated that mutational frequency is higher in round cell tumors compared to myxoid tumors. In this proposal we will isolate DNA and RNA from our frozen and paraffin-embedded resources to assess the mutation status of the IGF/PI3K/PTEN/Akt/mTOR pathway using new and efficient technologies fit for this purpose. Mutation status will be correlated with expression of related molecules in this pathway, interrogating our existing rich dataset on growth factors (e.g. IGF2), receptor tyrosine kinases (IGF1R, RET), AKT and activated pAKT, as well as downstream markers pS6K, nuclear p27, and in particular phospho-4EBP1, which in recently-published experiments18 we identified as the most analytically and clinically robust immunohistochemical biomarker of Akt pathway activation, Of note, assessing the status of the Akt/mTOR pathway in sarcomas has taken on added importance following the recently-reported positive result of the SUCCEED trial using radiforolimus, an mTOR inhibitor which significantly improves progression-free survival in metastatic sarcomas.27
Once we have assembled the annotated patient myxoid liposarcoma tissue cohorts, and enhanced their scientific value by detailing fusion oncogene and Akt pathway status, we will have the best resource in the world to interrogate other candidate biomarkers of targetable oncogenic pathways. The strength of the pathology expertise in our team will allow us to generate such results quickly and efficiently. Together with ongoing efforts to identify novel biomarkers from gene expression profile data,16 these additional investigations will include components of Akt/mTOR interrelated pathways including ERK. Dr. Nielsen has recently demonstrated high level expression of the therapeutic target histone deacetylase 2 and has reported on the adverse prognosis associated with RET expression in myxoid liposarcoma.16,28 These findings will be expanded upon and confirmed in the MDACC patient cohort using the Lev/Lazar TMA, which has increased numbers of round cell tumors as well as treated tumors. This will not only validate expression of these potential therapeutic targets, but also enable a more clear determination of these biomarkers as independent risk factors, and allow correlation with changes associated with both round cell transformation and treatment-related effects. Biostatistical support for TMA analysis is provided by experienced biostatisticians associated with the Sarcoma Research Center at UTMDACC (key collaborator: C. Creighton).
Significance: The overall exchange of biomarker data and confirmation of each contributor's finding on an external TMA increases the overall power and level of evidence of our studies, to validate and prioritize investigations of novel therapeutic strategies.
2. Myxoid Liposarcoma Biology and Experimental Therapeutics
2A. Fusion Gene Protein Binding Partners
Recent work in the Nielsen lab, in collaboration with Underhill, has elucidated the nature of the abnormal transcriptional complex in synovial sarcoma,13 revealing druggable targets currently under evaluation. We will apply a similar strategy in myxoid liposarcoma, using the primary cell models generated by Dr. Bovée which endogenously express FUS-DDIT3 in an appropriate cellular context. The aim of this study will be to identify the specific proteins that bind to and interact with the FUS-DDIT3 fusion protein, especially those that may be targeted by existing drugs.
To identify novel interacting proteins of FUS-DDIT3, we are using a global proteomic approach by obtaining proteins from the primary cultures and performing immunoprecipitation with an anti-DDIT3 antibody. The isolated protein complex, now comprised of FUS-DDIT3 (wild-type DDIT3 is absent under normal culture conditions30) and its interacting proteins, will then be separated by SDS-PAGE and the individual proteins sequenced and identified by mass spectrometry. The identities of the interacting proteins will be vigorously validated using multiple approaches including co-immunoprecipitation, glycerol gradient co-elution studies and chromatin immunoprecipitation.
Gene knockdown of identified binding partners will then be employed to interrogate dysregulatory effects on target gene transcription and consequent effects on oncogenic capacity. For both novel fusion gene binding partners and their relevant target genes, Dr. Lazar will perform validation on larger numbers of primary specimens and determine clinical relevance using the tissue microarray series described in Section 1a.
Significance: As of yet, we do not have available therapies targeting the FUS-DDIT3 oncoprotein directly. Identification of the immediate partners may reveal targetable components (e.g. HDAC1, EZH2 in synovial sarcoma) or abnormal protein interactions that can be disrupted using emerging strategies.31 Such investigations would form a natural basis for a subsequent grant application by our consortium.
2B. Trabectedin Related Studies
Trabectedin (Yondelis, ET 743) is a novel chemotherapeutic agent derived from the marine tunicate Ecteinascidia turbinata. By binding to the DNA minor groove, it disrupts repair mechanisms and causes inhibition of cell proliferation.32 Phase I and II clinical trial studies showed promising results in myxoid liposarcoma patients with advanced disease, though recent studies reported an increasing number of side effects.33,34 The exact mechanism by which trabectedin inhibits myxoid liposarcoma proliferation is currently unknown, although it seems to induce terminal adipogenic differentiation.20 Therefore, we will aim to identify the pathways that are driving tumor proliferation in order to establish targets for treatment, while concurrently investigating whether trabectedin affects these pathways.
The current proposed project will be performed in collaboration with Prof. Hans (AJ) Gelderblom, medical oncologist at Leiden. The LUMC is the leading sarcoma center in the Netherlands and Prof. Gelderblom is head of the systemic treatment committee of the EORTC Soft Tissue and Bone Sarcoma group and EORTC board member, which guarantees clinical translational overview of the project. Prof. Gelderblom currently participates in 2 trabectedin studies: the EORTC TRUSTS study and (as PI) the Dutch observational phase IV Trabectedin trial.
Dr. Bovée has access to an XCelligence system, which will allow us to assess proliferation, apoptosis and migration. In collaboration with the lab of Prof. van de Water, Department of Toxicology, Leiden University, with whom Dr. Bovée established a successful collaboration,35 we will have access to high throughput screening and live cell imaging. As an additional read-out, we can assess adipogenic differentiation, reportedly induced by trabectedin,20 using Oil-red-O staining in combination with live cell imaging.
- Subproject 1: Test the response to trabectedin in myxoid liposarcoma cell lines and primary cultures to test a possible relation to fusion type. Our panel of cell lines and cultures reflects the heterogeneity found with regards to fusion type in this disease. We will assess response to trabectedin within this panel of cell lines, evaluating proliferation, apoptosis and migration using the Xcelligence system as we reported previously for chondrosarcoma, and adipogenic differentiation using oil-red-o staining. Results will be correlated to fusion type and sensitive models will be assessed for treatment-induced changes in the FUS-DDIT3 complex and its targets identified by Nielsen and Lev. Doxorubicin will be used as a comparative positive control, a drug used world-wide with known activity in this disease.
- Subproject 2: Focused synthetic lethal kinase screen to identify genes or pathways that modify the response to trabectedin. In this subproject, we will identify genes and pathways that modify sensitivity to trabectedin in myxoid liposarcoma. This project is performed in collaboration with Prof. PCW Hogendoorn, Prof. B. van de Water and Dr. E. Danen. We will use an siRNA screen for kinase genes combined with trabectedin to identify the most important genes involved in the response to trabectedin.
- Subproject 3: Test whether the combination of trabectedin acts synergistically with inhibition of other pathways implicated in myxoid and round cell liposarcoma, such as AKT/PI3K and NF-kappaB. In parallel with the siRNA screen we will perform a focused compound screen using commercially available inhibitors that target the pathways known to be important in myxoid liposarcoma, with a focus on agents targeting pathways enhancing response to trabectedin emerging from subproject 2.
Significance: Existing systemic therapies are minimally effective in myxoid liposarcoma, with trabectedin recently coming into use as a new standard in Europe. We will take this bedside result back to the bench to better understand its mechanism and identify possible improved strategies to overcome resistance or enhance efficacy. This agent can act as a benchmark for molecularly-targeted experimental therapies used alone or in rational combinations, criticial information to justify their prioritization for assessment in future clinical trials.
2C. Histone Deacetylases (HDACs) and HDAC Inhibitor Related Studies
HDAC inhibitors have also shown promise and preclinical efficacy in translocation-associated sarcomas. We recently extended on our published findings by demonstrating that SB939 (the HDAC inhibitor being evaluated in NCIC-CTG IND.200) inhibits the growth of mxyoid liposarcoma xenografts (Figure 8). Herein we propose to undertake detailed preclinical translational investigations of the effects of HDAC inhibitors on myxoid liposarcoma.
Utilizing the experimental models described in Section 1a (cell lines and xenografts) and the broad array of assays we have already developed,12,14,37 we will evaluate the impact of HDAC inhibitors on the tumorigenic properties of myxoid liposarcoma in vitro and in vivo and aim to identify potential mechanisms of sensitivity/resistance. In synovial sarcoma, the Nielsen lab has recently shown that HDAC inhibitors disrupt the critical fusion oncogene transcriptional complex,13 and we will build on the results of the studies proposed above to see if this is also the case in myxoid liposarcoma.
The importance of our experimental plan is enhanced because the NCIC-Clinical Trials Group is currently undertaking a phase II clinical study to determine the anti-tumor effects of HDAC inhibition in patients afflicted by fusion gene sarcomas. Dr. Nielsen helped design this trial, and serves as its study pathologist and director of correlative science. Recruited patients already include four with myxoid liposarcoma, from whom FFPE blocks have been provided.
It is worth mentioning that HDACs are a family of isoforms divided into four classes.38 HDAC inhibitors currently in the clinic inhibit multiple HDAC isoforms, mainly of class I.39 Despite their promise, there is a clear need to improve the therapeutic index given their toxicities.40 Targeting the most relevant HDAC isoform in a particular cancer may enhance therapeutic efficacy by attenuating the toxicities associated with inhibition of multiple isoforms. As part of the current proposal, we will determine the expression of class I isoforms in myxoid liposarcoma, and using an siRNA knockout approach in our models we will assess the impact of individual isoforms on myxoid liposarcoma growth and survival. New isoform-specific HDAC inhibitor drugs are in active development (HDAC8-specific inhibitors are already in use in the Lev lab), and those with narrow spectrums incorporating the key isoforms identified by siRNA studies will be prioritized for further assessment in this disease.
Significance: Bench research by our group members has already contributed to bedside interventions (HDAC inhibitor clinical trials). Fitting our theme, we are positioning ourselves to bring the results of this knowledge back to the lab bench, to better understand the mechanism of action and most effective agents. In this way, we are likely to have a better understanding of negative and positive trial results, fitting our overall goal of conducting translational research that will inform design of followup clinical trials, to benefit patients with this disease.
The group has already met in Vancouver in March 2012 (in conjunction with the USCAP meeting), and future meetings are planned in conjunction with CTOS meetings at the participating institutions. We will also seek to arrange a joint meeting with the existing Liddy Shriver Sarcoma Initiative-funded well differentiated / dedifferentiated liposarcoma consortium.
The proposed work will create what we believe will be the world’s largest collection of primary tumor samples and cell models of myxoid liposarcoma, a resource we would not only use ourselves but also make available to qualified researchers worldwide in future collaborations. The frozen tissue would be positioned for use in sequencing-based discovery projects currently undergoing rapid technological advancement; the fixed tissue will provide a critical validation resource linked to detailed patient and outcome data allowing clinical interpretation; and the model systems can underpin functional and preclinical experiments central to translational research strategies. We will comprehensively evaluate existing biomarkers with therapeutic implications, and both existing drugs and novel targeted therapeutics for their capacity to help patients afflicted with this dangerous disease. Our links to clinical trials groups in Europe and North America will accelerate translation of our findings, allowing us to spearhead development of new trials rationally targeted to the molecular underpinnings of myxoid liposarcoma. Funding from the Liddy Shriver Sarcoma Initiative will create a collaborative group that can function as more than the sum of its parts, and commence an international program of research to translate existing and novel scientific knowledge into the best possible care.
The investigators of this study would like to acknowledge the important contributions of our trainees Jamie Lim MSc (UBC) and Elizabeth Demicco MD/PhD (MDACC) to the planning and presentation of our research plan.
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Study Plan Figure 1. Microscopic appearance of myxoid liposarcoma. The pale pink background is the water-filled "myxoid" matrix, which is criss-crossed by small vessels (plexiform vasculature). These features are often lost in the high grade "round cell" variant (inset), with its closely-packed small blue cancer cells. In both images, many of the tumor cells contain white circular vacuoles of fat indenting the sarcoma cell nuclei; these are termed "lipoblasts."
Study Plan Figure 2. The 12 FUS-DDIT3 fusion transcripts. Wild-type FUS and DDIT3 comprise of 15 and 4 exons, respectively. The fusion results in a chimeric transcript containing various 5’ amino terminal exons of FUS and the full protein coding sequence of DDIT3 (as its translational start codon is found on exon 3). Each colored rectangle represents an exon, and a half-colored rectangle indicates missing sequences in the particular exon.
Study Plan Figure 3. The myxoid liposarcoma investigative team. From left: Dr. Alexander Lazar, Dr. Judith Bovée, Dr. Torsten Nielsen, Dr. Dina Lev. Photo taken March, 2012 (Vancouver, Canada)
Study Plan Figure 4. Outline of proposed collaborative program. The structure of our program will be divided between studies based on clinical observations and samples (the bedside – Section 1: Human tumor based insights) and laboratory studies (the bench – Section 2: Biology & experimental therapeutics). Insights from the "bedside" will guide our research at the "bench", with the aim of eventually applying our "bench" findings back to the "bedside" in terms of therapeutic intervention.
Study Plan Figure 5. COBRA-FISH karyotype of primary cultures. (A) Primary culture cell line LUMC2187 showing translocation t(12;16) on the background of an otherwise near-normal diploid genome. (B) Primary culture cell line LUMC3403 showing translocation t(12;16) on the background of monosomy 15, trisomy 19 and some additional genomic events in chromosomes 2, 3, 5, 11 and 13. [Courtesy of K. Szuhai]
Study Plan Figure 6. Renal subcapsular implant of myxoid liposarcoma at passage 3. Gross image (on the left) of myxoid tumor growing from murine kidney (dark red in the centre, in between both tumors in bright red), and microscopic image showing myxoid matrix, plexiform vasculature and fatty differentiation including lipoblasts. Immunohistochemistry using species-specific antibodies and FUS-DDIT3 fluorescence in situ hybridization (FISH) studies confirm that the vessels are murine whereas the ovoid tumor cells and lipoblasts are human.
Study Plan Figure 7. Illustration showing how the FUS-DDIT3 fusion protein may result in new interactions between proteins. Colored circles A to C and D to E represent proteins that bind to FUS and DDIT3, respectively. Following the precedent in synovial sarcoma,13 this may create new interactions not seen in normal cells.
Study Plan Figure 8. The HDAC inhibitor SB939 inhibits the growth of myxoid liposarcoma xenografts. Primary tumor xenograft lines UBC616 and UBC1118 were grown in mice that were treated with the HDAC inhibitor SB939 (125mg/kg/day) or with PBS vehicle control (200ul/mouse/day) for 3 weeks. (A) Photograph of excised tumors from mice in control and treatment groups. (B) SB939 significantly shrinks myxoid liposarcoma tumor volume in a xenograft model, with similar relative and larger absolute effects in the higher grade form of the disease as represented by UBC1118. Bars represent median ± 95% confidence interval. N = 6.
V9N2 ESUN Copyright © 2012 Liddy Shriver Sarcoma Initiative.
Walking with Hope, Strength, and Courage
to Find a Cure for Liposarcoma
We are proud to announce in this issue of ESUN a $250,000 International Collaborative Grant, "A 'Bedside to Bench' Investigational Platform for the Study of Myxoid Liposarcoma." The grant brings together researchers from Canada, the Netherlands and the United States. This grant complements the $250,000 International Collaborative Grant we awarded a year and a half ago, "Translational Research in Well-Differentiated and De-Differentiated Liposarcoma," which involves researchers from Australia, Norway, and the United States.
In both grants, each investigator has an established infrastructure of equipment, specimens, models, laboratory and supporting personnel for sarcoma research. In both grants, each member has demonstrated a track record of productivity and accomplishment in studying liposarcoma. These two Liddy Shriver Sarcoma Initiative grants help enable these existing infrastructures to be collaboratively employed to bring a larger array of research personnel and resources to bear on studying liposarcoma. Importantly, each team can focus their skills and research directions to address questions that are both scientifically tractable and clinically relevant. Joint meetings between the two teams are planned so they can share insights and results. We are truly hopeful about the possible outcomes of such cross-pollinating meetings for the liposarcoma community.
These grants were made possible by generous donations raised by the dedicated and inspirational family behind the Wendy Walk. Wendy Landes’ three children, Ali, Matt and Jackie, created the Wendy Walk in 2010 after their mother was diagnosed with multi-focal dedifferentiated liposarcoma. They were inspired by their mother’s strength, courage, faith, and unwavering positive attitude, and they wanted to increase liposarcoma awareness and raise funds to support liposarcoma research.
Wendy, Robert, Matt, Ali and Jackie Landes
Wendy has already undergone three surgeries lasting over 12 hours each, and she has undergone six different chemotherapy protocols. Each surgery has placed in her in ICU! Throughout this journey the Wendy Walk has been the shining light that has encouraged her and her family to remain strong and committed to finding a cure. Wendy’s journey is an example of the battle that thousands of other patients are fighting around the world. Wendy and the Landes family are an inspiration to us and to those who learn about them. We are grateful that they are partnering with us to advance liposarcoma research, and we believe that the Landes’ legacy will be in the impact that this research makes in the lives of countless families around the world.
The International Collaborative Grants program is an integral part of the Liddy Shriver Sarcoma Initiative’s philosophy: Improved outcome for sarcoma patients is best achieved by teams of dedicated investigators working collaboratively and cohesively towards a common goal. Notably, there are few funding sources for sarcoma research that cross national boundaries. We are committed to providing a unique source of support for truly global initiatives in basic and translational sarcoma research. The approach we are taking with our International Collaborative Grants program is catalytic in bringing quality researchers and clinicians together to help find cures for these rare cancers.
V9N2 ESUN Copyright © 2012 Liddy Shriver Sarcoma Initiative