$250K International Collaborative Grant Funds Genetic Research on Sarcomas
May 1, 2014 - The Wendy Walk and the Liddy Shriver Sarcoma Initiative are pleased to award a $250,000 International Collaborative Grant to the International Sarcoma Kindred Study (ISKS). The research project, which focuses on the genetic risks for sarcoma, is a global effort of investigators in Australia, France, India, the United Kingdom and the United States.
The new grant will be used to explore several issues within the well-established framework of the ISKS. These issues include the genetic risks for Ewing's sarcoma and liposarcoma, the genetic variants that predict response to sarcoma treatments, and the genetics of sarcoma predisposition syndromes. The study's investigators will also seek to further understand international attitudes toward the knowledge of genetic risk for sarcoma.
Studying the Genetics of Pediatric and Adult-Onset Sarcomas
Familial genetics play an important role in the development of many cancers and in their treatment. There are several large-scale studies in breast, bowel, ovary, prostate, pediatric cancer, and melanoma kindreds, and each of these has made major contributions to cancer biology and clinical practice. The ISKS is the first study to investigate genetic risks in both childhood and adult-onset sarcomas. Dr. Ajay Puir said, "This study brings together a group of international specialists who are vastly experienced in the treatment of these tumors. Pooling together their resources and expertise will help facilitate new insights to help identify patients who may be at a greater risk of developing these lesions."
Aiming for Prevention and Early Diagnosis
According to Dr. David Thomas, one of the study's principal investigators, research indicates that the risk of sarcomas is strongly genetically determined. He and the global team will be using new technologies to identify the genes associated with increased sarcoma risk. The researchers aim to use this information to offer genetic testing, counselling and risk-reduction strategies to sarcoma survivors and their family members in order to potentially prevent cancers or identify them at the earliest possible stage.
Understanding and Meeting the Needs of Patients & Families
Genetic testing is of great importance to famlies dealing with sarcoma. Preliminary ISKS data indicates that 95% of sarcoma patients and their families want genetic tests to be made available, and 78% want the test even if there is nothing that can be done to change their risk. Anticipating the development of genetic testing programs, the investigators will seek to understand what people know about the genetics of sarcomas and how they feel about the personal impact of genetic tests. The researchers are particularly interested in differences in these attitudes between populations, and will start by comparing findings in French, Australian, UK and US populations.
Focusing on Ewing's sarcoma
By Dr. Joshua Schiffman
One of the biggest challenges in Ewing sarcoma is that we still do not understand who is at genetic risk for this disease, or even what type of tissue it comes from. We will focus on examining the DNA from people with Ewing sarcoma from all across the globe. We will look to see if repeating regions of DNA called "microsatellites" in the germline (inherited) DNA of people with Ewing sarcoma are associated with an increase risk of disease development. We know that these repeating regions are necessary for the rearranged EWS-FLI1 chromosome in Ewing sarcoma to make the tumor grow. We will investigate whether these DNA microsatellites need to contain a specific length of repeating DNA regions in order to develop Ewing sarcoma in the first place. This may one day lead us to better understand who could be at risk to develop Ewing sarcoma and whether we can think about novel prevention strategies.
Despite the aggressive nature of Ewing sarcoma, we're actually quite hopeful to really make some progress. We believe that understanding the genetic risk for Ewing sarcoma is going to give us an amazing opportunity to change the outcome of this disease for children and young adults.
Focusing on Liposarcoma
By Dr. David Thomas
Liposarcoma are a group of cancers of fat cells. There are at least 3 different molecular subtypes. The first and most common is called well-differentiated liposarcoma, and it's de-differentiated form. It is characterised by amplification of MDM2 and CDK4, in the form of an accessory neochromosome. The second is myxoid liposarcoma, which is characterised by a completely different mutation, generating a fusion gene between the FUS and CHOP genes. The final rare form is pleomorphic liposarcoma, which appears similar to other genomically unstable sarcomas, and lacks a known recurrent mutation. However, we don't know what allows these very different cancers to form. We hope that we will discover heritable mutations in genes that explain the predisposition to these quite different cancers. This information will shed light on how these cancers develop and perhaps allow us to understand who is at risk so the cancers can be detected earlier and treated more effectively.
The Collaborative Model
The Liddy Shriver Sarcoma Initiative awards international collaborative grants like this one in order to increase the power of private donations and to accelerate research that has the potential to save and improve lives.
"The project aims to gather individual and familial epidemiological data, associated to blood samples to create a genetic, biological, epidemiological and clinical resource to be used at an international level."
- Isabelle Ray-Coquard, MD
"Sarcoma specific research is vital to improve our understanding of sarcomas, leading to better screening, prevention and treatment options." - Mandy Ballinger, PhD
"This project offers the opportunity to learn from individuals affected by sarcoma and their family members; their thoughts and attitudes about new genetic discoveries and technology."
- Mary-Anne Young, MHSc
"My personal motivation and drive in carrying out this research is applying my knowledge and training in genetic epidemiology to pertinent clinical questions."
- David Cox, PhD
"The Liddy Shriver Sarcoma Initiative has given us the opportunity to come together...to truly impact the care of children and adults with sarcoma."
- Joshua Schiffman, MD
By uniting nine researchers in five countries in the study of both pediatric and adult sarcomas, this grant creates an incredible opportunity for the sharing of knowledge, experience and resources. Dr. Lor Randall explains: "What's exciting about this particular initiative is that it's a global effort. This is the first of its kind, where people are coming from all countries with the same sort of fervor and passion that Dr. Schiffman and I have, to put our minds together to answers these questions. There's never been anything quite like this for these orphan diseases."
Dr. Thomas adds, "This program is a beautiful example of how collaboration is critical to achieve progress in rare diseases. And it's not just collaboration between people of different continents and different centers internationally, but it's also collaboration between people with genetic expertise in cancer as well as those of us who are working in the lab and the clinic, and it's collaboration with people who are affected by sarcoma."
Dr. Schiffman expresses great hope that the collaborative team can make a difference: "We are a collection of oncologists, surgeons, psychologists, and genetic epidemiologists, and we are all driven by the same passion: to understand the genetic basis of sarcoma so that we can finally do something about it. The Liddy Shriver Sarcoma Initiative has given us the opportunity to come together...to truly impact the care of children and adults with sarcoma."
This grant is co-funded by the Wendy Walk and the Liddy Shriver Sarcoma Initiative. The two organizations are working together to initiate and support high-quality basic and translational research in sarcoma.
The Wendy Walk funds are contributed in memory of Philip and Rita Rosen, the parents of Wendy Landes. The Wendy Walk continues to build on its five year history to provide hope, strength, and courage to those fighting these rare cancers while funding research with the Liddy Shriver Sarcoma Initiative. The Initiative greatly appreciates this very productive partnership.
The Initiative also gratefully acknowledges the following support for this research: a generous donation from the Arlo and Susan Ellison family; donations in honor of Emily Oberst, a Ewing's sarcoma patient; donations in memory of Michael Lio, Christi Campbell, Jeremiah Weingrod, Gage Dole, Kristen Hoodak, Barret Lehman, Stephen Meier, and Lea Anne Dean, who lost their lives to Ewing's sarcoma; donations from the Peter Skelton Sarcoma Research Foundation and donations made in memory of Edward Shrier.
Together, we are making a difference!
V11N3 ESUN Copyright © 2014 Liddy Shriver Sarcoma Initiative.
The International Sarcoma Kindred Study:
A Global Multi-Site Prospective Cancer Genetics Study
The International Sarcoma Kindred Study (ISKS) is a global effort to create a genetic resource to study the origins of sarcoma recently funded by a Liddy Shriver Sarcoma Initiative International Collaborative Research Grant. Working with partners from around the world, ISKS consortium members will investigate various genetic aspects of sarcoma risk and treatment. This article will describe ISKS, including sarcoma background and ISKS’s planned investigations. The current areas of ISKS investigation include:
- Ewing sarcoma
- Sarcoma genetic testing
- Genomic modifiers of treatment response
- Sarcoma predisposition syndromes.
Importantly, patients, physicians, and researchers are all invited to participate in ISKS.
There are over 130,000 new cases of sarcoma world‐wide each year, accounting for approximately 1‐3% of all malignancies. The number of new sarcoma cases diagnosed each year continues to be increasing across the globe.1,2 Sarcomas disproportionately affect the young, representing 20% of cancers in children and 10% of cancers <30 years of age.
Sarcomas & Familial Genetics
Sarcoma has a strong genetic, or inherited, component. Early age of onset increases the likelihood that genetic factors are present. Sarcomas arise on average 20 years earlier than epithelial cancers (like lung cancer or melanoma). Sarcomas are more common in persons with recognized hereditary cancer syndromes, including retinoblastoma, Li‐Fraumeni syndrome (LFS), Gardner’s syndrome, Werner’s syndrome, neurofibromatosis type 1, and some immunodeficiency syndromes.2 Up to 33% of pediatric sarcomas are estimated to be associated with a significant family history of cancers.3,4 The risk of sarcomas in relatives of all children with sarcoma is increased over 6‐fold compared to age‐matched controls and, where a causal gene mutation is found, this risk increases to over 250‐fold.5 Although little data on familial risk are available in adult‐onset sarcoma, a Scandinavian population‐based study of over 800 patients showed that 20% of cases developed a second cancer at a median of 10 years after treatment, and that the hazard ratio for a second sarcoma was 17.6‐fold increased over the general population.6 The incidence of second sarcomas was increased almost 30‐fold in patients with a first sarcoma, and there was a 5.3‐fold increased risk of sarcomas in parents of patients with leiomyosarcoma.7 Because these data are entirely based on retrospective registries, they almost certainly underestimate actual genetic risk. One of the goals of ISKS is to better understand the actual genetic risk for sarcoma.
Current knowledge regarding genetic risk in adult‐onset sarcoma (90% of all sarcomas) is limited by the major ascertainment biases inherent in family cancer registry data. Such registries are over‐represented in common cancers with well‐defined genetic risk and where clinical interventions can modify that risk, exemplified by breast and bowel cancers.8 Existing familial sarcoma studies tend to be heavily biased towards pediatric populations,3,5,9-11 with a median age in one of the larger studies of 5 years.3 Pediatric sarcomas are biologically and pathologically different than adult‐onset sarcomas, with different genetic causes, treatments and cure rates. The median age of sporadic sarcomas is ~50 years.12 Finally, family history is an imperfect guide to genetic risk. Twenty-five to 50% of sarcoma probands (patients) with germline TP53 mutations lack a family history in prior generations,4,13 similar to early-onset breast cancer.14 These may be due to ‘de novo’ (new) mutations, gonadal mosaicism in parents (with implications for siblings), or to recessive alleles. More precise identification of those patients at genetic risk for sarcoma are required to better understand the etiology of this rare disease and most importantly, to design novel treatment and effective prevention strategies.
Current and Recent Past Sarcoma Genomic Studies
Project GENESIS (Genetics of Ewing Sarcoma International Study)
PI: Joshua Schiffman, MD
Huntsman Cancer Institute, University of Utah
Study Recruiting. Project GenESis is a global study designed to identify genetic factors that contribute to the development of Ewing Sarcoma. Participants complete a medical and family history questionnaire, give a saliva sample, and receive a gift card for their participation. Anyone diagnosed with Ewing sarcoma around the world can participate. DNA is extracted from saliva samples to try to find the genetic basis of disease and new treatment targets in Ewing sarcoma. Analysis ongoing.
Molecular Analysis Of Solid Tumors (MAST) Study
PI: Sara Federico, MD
St. Jude Children's Research Hospital (SJCRH)
Study recruiting. This SJCRH research study characterizes the molecular, cellular and genetic properties of primary and metastatic neuroblastoma, osteosarcoma, retinoblastoma, Ewing sarcoma family of tumors, soft tissue sarcomas, adrenocortical tumors and liver malignancies. These cell isolates will be used for gene expression array analysis, genomic analysis by single nucleotide polymorphism chip, array comparative genomic hybridization and next generation sequencing, and transmission electron microscopy analysis. Additionally cell lines and orthotopic xenografts will be created from the obtained tumor specimens. High throughput drug screening will be done on a subset of xenografts and carry out preclinical trials with promising agents. Analysis ongoing.
The 23andMe Sarcoma Study
Study closed. Industry study to recruit 1,000 participants to combine a person's genetic information with online questionnaires about environment and sarcoma history offered from 23andMe.com (direct-to-consumer genetic testing company). Goal of study to look for novel genetic markers that increase susceptibility to sarcoma and affect response to common treatments, including risk for adverse events. Awaiting study analysis and results.
Genetics of Ewing’s Sarcoma Study
PI: Shahab Asgharzadeh, MD
Children’s Hospital Los Angeles, University of Southern California
Study currently not recruiting. Designed to look at inherited changes in the DNA to learn about the causes of Ewing Sarcoma and how it can be prevented. Blood collected from patients with Ewing sarcoma and their relatives. Analysis ongoing.
NIH-funded Project FLAG (Families Learning About GIST)
PI: Judy Garber, MD with collaborators George Demetri, MD and Suzanne George, MD
Dana-Farber Cancer Institute and Harvard Medical School
Study closed to further accrual, data analysis ongoing. Project FLAG is research study, sponsored by the National Institutes of Health (NIH), focused on learning more about GISTs (gastrointestinal stromal tumors) that may be hereditary. 800 individuals with GIST were enrolled into Project FLAG. Information about family cancer history was collected and, when possible, a sample of DNA from blood or saliva also collected. Project FLAG will identify the frequency of hereditary GIST and whether other cancers or conditions may be signs that a GIST may have occurred because of an inherited factor.
Identification of risk alleles (genetic markers) is critical for young adult sarcoma patients for several reasons. Firstly, the mutated gene can be passed onto the next generation, affecting reproductive choices. Many sarcoma patients are in their reproductive years, and effective strategies now exist for antenatal and pre‐implantation diagnosis.15 Secondly, the risk of second malignancies appears alarmingly increased in patients with hereditary cancer syndromes. If a TP53 mutation is identified, the estimated cumulative probability of second cancer occurrence is 57% at 30 years after a first cancer diagnosis,16 and the relative risk of second cancers is increased up to 83 times compared to control populations. Thirdly, the risk to other relatives, parents and siblings may vary from 50% to an unquantifiable risk, depending on penetrance, de novo mutations, recessive alleles or parental gonadal mosaicism. Finally, ionizing radiation increases cancer risks synergistically in the presence of mutations in sarcoma genes such as Rb and TP53.17-19 Diagnostic and therapeutic radiation exposure is modifiable in high‐risk individuals, provided such mutations are identified.
Although not all family members may wish to know the results of mutation testing, it is arguable that the option ought to be available due to novel early tumor surveillance strategies being offered. For example, it is now recommended that TP53 mutation carriers (LFS) undergo annual mammograms and breast MRIs starting at 20 to 25 years old (in women), colonoscopy every 2 to 5 years starting at 25 years old, and consider regular whole-body MRI, abdominal ultrasounds, and brain MRI (Familial Breast Cancer, NICE Clinical Guideline 2006, NCCN Clinical Practice in Oncology Guidelines 2013). In fact, a recent study which included patients from University of Toronto, Children’s Hospital of Los Angeles, and Huntsman Cancer Institute (HCI) at the University of Utah, demonstrated 100% overall survival in patients with LFS who underwent early cancer surveillance with MRI and biochemical testing compared to 20% survival in those patients who chose no surveillance.20
It is becoming clear that understanding of genetic risk for cancer, especially sarcoma, improves clinical outcome. Moreover, our preliminary data indicate that 95% of sarcoma patients and their families want genetic tests to be made available, and 78% want the test regardless of risk modifiability. The true incidence and penetrance of de novo/gonadal mosaic/medium penetrance germline mutations in adult‐onset sarcomas is simply not known, and this remains a pressing clinical issue. If identified, these patients can be properly counseled about risk reduction and early tumor surveillance protocols.
The impact of familial cancer genetics cannot be over‐stated, and many of the clinically important cancer genes were originally identified in kindred studies similar ISKS.21-24 Several of these large‐scale family studies in breast, bowel, ovary, prostate, pediatric cancer, and melanoma kindreds have made major contributions to cancer biology and clinical practice. Despite the clear importance of genetic factors in sarcoma, no adult‐onset sarcoma kindred studies currently exist. Moreover, ascertainment biases in age, cancer‐type, and family history continue to collectively limit the clinical relevance of findings from other familial cancer registry studies to the wider sarcoma population. A population‐based case‐control study like ISKS will generate new and important insights into the genetic basis and clinical risks associated with adult‐onset sarcoma.
Clinical Outcome and Survival
For many cancers, family history is significantly associated with individual cancer risk. There is mounting evidence that survival after cancer is also hereditary. This has been observed for breast,25,26 prostate,27,28 and colorectal cancers.29 Finally, the recent work of Greaves and Maley (2012) explores clonal evolution in cancers and the finding that tumor cells undergo successive rounds of selection (Figure 1).30 This clonal selection could be highly influenced by SNPs that influence the biology of different “ecosystems” in the body. Studies in hybrid cell lines,31 transfected mouse embryonic fibroblasts,32 and mouse models33 have shown that host-tumor interactions influence the ability of tumors to metastasize. All together, these observations open the door for further investigation in sarcoma where response rate and survival after initial treatment (surgery plus radiotherapy with or without chemotherapy) seem to be influenced by more than the traditional prognostic factors of stage, histological subtype or histological grading. It is quite likely that genomic modifiers of sarcoma risk equally influence risk of recurrence through the biological and evolutionary mechanisms briefly described above.
Given the rarity of sarcomas, studying the influence of genetics on response to treatment and progression among sarcoma patients has not been possible prior to the development of the ISKS study. Combining genetic analyses and highly detailed clinical information in the framework of this ISKS consortium provides important information that can be used in the future to improve the clinical approach to and care of sarcoma patients.
ISKS Preliminary Collection Summary
More than 1000 families are enrolled in the ISKS (March 2014) with participation still increasing. Worldwide, 1067 sarcoma probands (720 Australia, 3 New Zealand, 203 France, 120 India, 15 USA, 6 UK), 2010 family members, and 380 controls. Almost 2200 study questionnaires have been completed and 1894 blood samples have been collected. The clinical impression of significant inherited cancer risk in patients with sarcoma is being borne out by early data. Of the first 560 sarcoma index cases, 88 have >1 cancer and 23 have >2 cancers. The average age of diagnosis for sarcoma is 43.8 years comprised of 65% soft tissue subtypes and 35% bone sarcomas. In Australia, ISKS has identified 2005 cancers in family members with an average age of onset of 58.3 years, significantly younger than the general Australian population (65.6yrs). Analysis of the pedigree information shows 35% of families having no family history of cancer, 7% uninformative, and a surprisingly high number (57%) of recognized TP53 or sarcoma-related cancer predisposition classifications and 1% other cancer-related syndromes.
TP53 mutation studies
Initial germline testing on the index cases showed an unexpectedly high 3.6% (19/523) incidence of clinically significant TP53 mutation events, compared to 0.005% in the general population, and almost half of these are seen in the absence of family history (Table 1).34 An assessment of the families of the TP53 mutation positive ISKS probands reveals that 104 first degree relatives are potentially affected. Of these 104 relatives, 31 individuals have been screened thus far and 13 have tested positive. Extrapolating to the UK population where approximately 2,800 new cases of bone and soft tissue sarcoma are diagnosed each year, a 3.6% mutation rate translates into 100 new patients and 547 relatives as potential carriers of a pathogenic TP53 mutation.
Deep sequencing of high risk family
ISKS has identified families with what appears to be markedly increased risk for sarcomas. In the first of these families, the parents have not developed any cancers, but the offspring are markedly affected. Two children developed four cancers at 18 years of age and both children developed sarcomas (a Ewing sarcoma and a rhabdomyosarcoma). In addition three relatives developed cancer under 20 years of age, including one with a leiomyosarcoma. This family did not have an identified TP53 mutation. This is the type of highly informative family that can teach us about the inherited risks for different sarcomas, including Ewing sarcoma that has no currently known high penetrant disease causing alleles.35 DNA collected from this family through ISKS is now being deep sequenced to identify novel genes possibly associated with sarcomagenesis.
Research Grant Objectives
Now that the ISKS has been established and proven to be successful in Australia, the Liddy Shriver International Collaborative Research Grant will allow us to collect enough samples from sarcoma patients around the world to help answer specific sarcoma questions. Obtaining an adequate number of samples to answer these questions only can be accomplished through the cooperation and collaboration of international institutions. The five study goals to be initially tackled by ISKS include the following:
- Ewing sarcoma: To determine the association between the length of repeating DNA binding sites (microsatellites) of the EWS-FLI1 fusion protein and the risk of Ewing sarcoma
- Liposarcoma: To investigate the genetic basis for development of myxoid and well-/de-differentiated liposarcoma
- Sarcoma genetic testing: To define international cross-cultural attitudes to genetic knowledge
- Genomic modifiers: To measure germline genetic variants as predictors of response to treatment,
- Sarcoma predisposition syndromes: To identify familial cancer patterns and novel gene mutations in sarcoma predisposition syndromes.
Research Plan and Experimental Design:
Establishing a global resource for studying genetic basis for sarcoma
As described above, the goal of ISKS is to understand the hereditary genetic basis and clinical risks associated with adult-onset sarcoma. ISKS Australia began recruitment in July 2009 and since then additional, international study sites have become active in India, France, New Zealand, USA, Canada, and the UK (Figure 2). Working together, each local ISKS center contributes data and samples from ISKS participants to the "Global Study Centre" located in Australia (Figure 3). These samples, with corresponding data, are then freely available for participating ISKS members in order to achieve the objectives of each specific aim which we will describe below. Each aim covers a different aspect of sarcoma risk and clinical outcome, and will best be answered through the continued enrollment of patients through ISKS.
AIM 1. Ewing Sarcoma
To determine the association between the length of repeating DNA binding sites (microsatellites) of the EWS-FLI1 fusion protein and the risk of Ewing sarcoma.
Ewing sarcoma (ES) is the second most common bone malignancy in adolescents and young adults. However it remains a relatively rare disease with less than 500 cases diagnosed per year in North America and no known genetic causes or clues to its etiology (35). The outcome for patients with ES remains poor with only 60-70% survival, and less than 20% survival for those patients with metastatic or relapsed disease. ES is unusual for its recurring cytogenetic alteration in that nearly every tumor contains a translocation of the EWSR1 gene encoding the EWS transcript.
Dr. Stephen Lessnick (Huntsman Cancer Institute, University of Utah) discovered a significant over-representation of highly repetitive GGAA-containing elements (microsatellites) in the regions upstream of genes targeted by EWS-FLI1.36 Microsatellites are simple nucleotide sequence (DNA) repeats with a repeat unit length between 2 and 6 base pairs, and typically contain between 10 and 100 repeats. Microsatellites comprise approximately 3% of the human genome, and are often considered “junk” DNA with no known function.37 Dr. Lessnick reported that the EWS-FLI1 translocation uses GGAA microsatellite repeats to regulate the expression of target genes in ES, and that the ability to do so depends on the number of consecutive GGAA motifs in the promoter regions of target genes.36,38 Dr. Lessnick’s laboratory has discovered a direct correlation between the number of consecutive GGAA repeats, EWS-FLI1 binding, and transcriptional activation mediated by EWS-FLI1. A very specific number of microsatellite repeats promote more active binding leading to increased tumorigenesis.39Other groups have since reported similar findings.40 It has been speculated that microsatellite length polymorphisms may contribute to differences in individual and population susceptibility to ES.38 Dr. Lessnick's group recently combined transcriptional analysis, whole genome localization data, and RNA interference knockdown to identify glutathione S-transferase M4 (GSTM4) as a critical EWS-FLI target gene in ES.41 They found that the recurrent EWS-FLI1 fusion protein directly binds and regulates GSTM4 expression through the same repeat GGAA-microsatellite described above. Higher GSTM4 expression correlated with worse clinical outcome. Microsatellite sizes differ between individuals, and so in addition to possible genetic contribution to ES susceptibility, microsatellite repeats may lead to inherited differences in ES therapeutic responses. ES studies analyzing microsatellite repeat length in germline and somatic (tumor) DNA are now required to support their contribution to disease susceptibility and outcome.42
Microsatellite repeat lengths are often polymorphic between individuals.43 As described above, the ability of EWS-FLI1 to bind to GGAA-microsatellites in target genes depends highly on the number of consecutive GGAA repeats.36,38,41 Thus, GGAA-repeat polymorphisms have a significant potential to modulate EWS-translocation-dependent tumorigenesis and susceptibility to develop ES.39 The presence of the EWS-translocation may be necessary, but not sufficient for ES to occur. Therefore, a minimum length of GGAA-microsatellite repeats may be the determining factor of whether an acquired EWS-translocation will lead to ES. ISKS will test this hypothesis using genomic PCR and sequencing of the GGAA-microsatellite repeats at key genes from germline and paired tumor DNA from patients with ES enrolled through ISKS (total expected number = 100 over 2 years). Our analysis will examine if patients with ES have specific microsatellite lengths compared to people without ES. In addition, we will explore if there is a connection between microsatellite length and the new ES-related genes (e.g., TARDBP and EGR2) that recently have been described in the literature.
Clinical Implications of Aim 1
If GGAA-polymorphisms (microsatellites) are associated with differences in disease susceptibility, then these may offer a genetic clue to who may be more or less likely to develop ES. Also, at the completion of this ES study aim, we will have created a valuable resource for future ES microsatellite analyses for the research community. We, and others, will be able to use this information in the future to correlate the size of GGAA-repeats with clinical features including outcome. We also will begin to explore whether microsatellite changes in length will occur during sarcomagenesis by comparing microsatellite length between paired germline and tumor DNA (preliminary data indicate there is no change). These types of studies may have potential therapeutic implications and highlight the importance of studies like ISKS to inform novel treatment approaches.
AIM 2. Liposarcoma
To investigate the genetic basis for development of myxoid and well-/de-differentiated liposarcoma.
Both myxoid and well-/de-differentiated liposarcoma are characterized by specific molecular defects. In the case of myxoid liposarcoma (mLPS), a fusion gene is almost always observed between FUS and CHOP, while in the case of well-/de-differentiated liposarcoma (WDLPS), a neochromosome is always found. Both of these defects are highly specific and invariant features of these sarcomas. Our group hypothesizes that a defect in DNA repair is required to permit the formation of these two pathologic events. However, the defect in mLPS and WDLPS is almost certainly different than the gross chromosomal instability characteristic of other sarcomas — such as in leiomyosarcoma, undifferentiated sarcoma, or osteosarcoma. The reason for this belief is that: 1) the remainder of the genome outside of the mLPS and WDLPS-specific mutations is relatively diploid, unlike the striking aneuploidy seen with many sarcoma,44,unpublished data and 2) unlike other sarcomas, mLPS and WDLPS have an absence of mutations in key genes known to play critical roles in genome integrity, such as TP53.45 Certain DNA repair processes are known to be mutated in specific cancer types. For example, there are heritable mutations in the mismatch repair deficient colorectal cancers; mutations in homologous recombination genes in breast and ovarian cancers; and mutations in the helicase genes in osteosarcoma. Understanding which mutations are linked to specific cancer subtypes may shed light on the nature of the DNA repair defect that are permissive for development of the mutations seen in mLPS and WDLPS.
AIM 2A: Inherited mutations in mLPS and WDLPS
We plan to undertake a massively parallel sequencing screen for mutations in key heritable cancer genes in the cohort of patients within ISKS who have mLPS and WDLPS, and compare the allele frequencies observed with:
- Control group of pooled cases of undifferentiated pleomorphic sarcoma and leiomyosarcoma.
- Allele frequencies in a control population derived from the Exome Variant Server and dbSNP.
In some cases, dominant alleles will be identified which should conform to rare familial patterns of cancer development. In most cases, we anticipate more common, less penetrant alleles will account for predisposition (genetic risk for tumor development). For the dominant alleles, we anticipate that identification of pathogenic mutations will be relatively obvious using standard criteria (frameshift, premature stop, previously reported missense, exon or gene deletion). For the less penetrant alleles, statistical analyses will determine assignment of putative pathogenicity. We anticipate that pathogenic germline variants will be enriched in the disease groups, allowing identification of enrichment for defects in specific pathways.
Currently in the ISKS, we have data and biospecimens on patients and families from: mLPS probands (N=35), WDLPS (N=56), leiomyosarcoma (N=82), undifferentiated pleomorphic sarcoma (N=90), controls (N=380). We anticipate more patients to be rapidly accrued internationally through ISKS now that the enrollment pipeline has been established and local institutional IRBs have been approved. The candidate gene list to be studied includes 85 genes already identified as critical to predisposition to cancers in general, and sarcomas in particular. It encompasses all known DNA repair genes in the homologous recombination pathways, genes implicated in telomere maintenance, mismatch repair, and helicases.
AIM 2B: Whole exome sequencing on familial lipomatoses.
We also intend to conduct whole exome sequencing on familial lipomatoses. This relatively rare familial condition is characterized by an autosomal dominant pattern of inheritance, and the development of multiple lipomas. There are a shared set of molecular events observed in lipomas and WDLPS, including the rearrangement of HMGA2. We anticipate that mapping the genetic basis of this familial disease will potentially shed light on less penetrant alleles observed in the ISKS cohort.
AIM 2C: Somatic tumor analysis
Heritable defects in the above pathways identified in the germline DNA of ISKS participants will likely affect a subset of cases of each sarcoma. We hypothesize that a larger fraction of patients will sustain somatic mutations in key genes within the tumor. Accordingly, the subaims described above will be complemented by a parallel study of somatically acquired mutations in the same set of genes in an interrogation of primary tumors. To date we have conducted a screen for somatic mutations in 23 WDLPS, 9 mLPS and 24 leiomyosarcoma/undifferentiated pleomorphic sarcoma, with a high frequency of TP53 gene mutations in leiomyosarcomas and pleomorphic sarcomas. We would like to extend this analysis to include more examples of these tumors, which would be made possible through funding of the ISKS and its collaborating study centers.
Clinical Implications of Aim 2
This information will not only shed light on the molecular basis for these diseases, but may also provide insights into therapeutic opportunities based on DNA repair defects. An example of this has been the development of PARP inhibitors for cancers with defects in BRCA1, or even more generally in homologous recombination. It appears that such tumors demonstrate synthetic lethality in response to inhibition of the enzyme PARP, something that is not seen in normal cells. Through the sequencing efforts of ISKS on an unprecedented number of sarcoma germline and tumor DNA samples, we may find additional defects in other DNA repair pathways that can be incorporated into novel therapeutic approaches for patients diagnosed with sarcoma.
AIM 3. Sarcoma genetic testing
To define international cross-cultural attitudes to genetic knowledge.
The use of new genomic technologies in large scale international population-based studies, such as ISKS, will result in the identification of incidental genetic information (i.e. genetic information which is of clinical or personal interest to study participants and their family). It is generally accepted that researchers have an ethical duty to inform research participants of research findings that may significantly affect their health and/or for which therapeutic or preventative measures are available.46,47 Researchers, health professionals and lawyers, and bioethicists continue to explore the myriad of dilemmas associated with the medical, ethical, psychosocial and social implications of incidental information generated as a result of genomic testing in both clinical practice and research.48-51
It is important that the contribution and opinions from research participants and the general public be included in this discussion. There is considerable evidence in some countries e.g. Australia, the United Kingdom and the United States of America, to show that research participants and the general public are interested in receiving incidental information.52-56 It is important to hear from other groups, particularly those where cultural differences or knowledge may influence attitudes, as individual's background, knowledge and attitudes has been shown to influence response to genetic information.57,58
Cultural differences have previously been considered in many areas of clinical genetics including the uptake of risk reducing strategies for BRCA1/2 mutation carriers at risk for breast cancer. Differences have been observed between countries with much lower uptake of risk reducing mastectomy in Israel and southern Europe compared with Northern Europe, Holland and America.59,60 These differences may relate to population characteristics, recommendations regarding risk reducing strategies, fundamental beliefs concerning body image, feminity and risk reducing surgery as well as differences in funding of health care systems.60 Many of these variables can be considered as cultural i.e. the characteristics of a particular group of people, defined by everything from language, religion, cuisine, social habits, music and arts. Cultural differences have not been specifically examined in the arena of genomic research and ancillary information, especially in patients with sarcoma. The ISKS study cohort provides a unique opportunity to explore this important question.
ISKS will define international cross-cultural attitudes to genetics, genomic research and ancillary information as a result of participation in genetic research within several parts of the world. We will test the hypothesis that ISKS participants will respond differently depending on their country of origin to the following:
- Beliefs about the genetic versus environmental component of aspects to health and well being
- Feelings towards new genetic discoveries
- Attitudes towards genetic testing for inherited conditions
- Attitudes towards predictive testing for sarcoma
- Attitudes towards the possibility of “incidental findings” as a result of genetic investigations
A discrete set of questions has been developed to include in the larger baseline questionnaire administered to all ISKS participants. Study specific questions have been designed to examine attitudes to incidental information arising from participation in research. The questions about new genetic discoveries cover six items on a seven point Likert scale (bored/excited, valuable/worthless, uninterested/interested, indifferent/passionate, don’t care/care and important/unimportant). The questions about genetic testing for inherited conditions cover five domains on a seven point Likert scale (favourable/unfavourable, calm/anxious, trusting/sceptical, good idea/bad idea and acceptable/unacceptable).
Clinical Implications of Aim 3
Through this aim, ISKS will accomplish 3 major clinical goals.
- Generate knowledge about the offering of genetic testing to apply to cancer screening programs adopted by future countries,
- Identify major critical points for patients and their family to inform the future culturally-sensitive feedback of genetic research results, and
- Identify differences by country to adapt or homogenize the informed consent process for future genetic/genomic research for patients in all countries.
AIM 4. Genomic modifiers.
To measure germline genetic variants as predictors of response to treatment.
The post-genome era has shed considerable light on the genetic component of risk for a number of diseases. However, the vast majority of this work has been carried out in the context of prospective cohorts and large case-control series of common diseases. While these studies are very well designed to evaluate disease risk, they often do not collect sufficient clinical information. This is particularly the case with respect to treatment and time to progression, to properly evaluate genetic variants as predictors of response to treatment. ISKS is particularly conducive to studying genetic variants as predictors of response to treatment. Recruiting patients through clinical centers provides access to data regarding treatments and response. Furthermore, federating large cohorts of patients increases statistical power of the study. Sufficient statistical power is paramount in identifying predictors of response to treatment. Highly powered studies will provide markers that segregate patients that respond to treatments from those that do not. Such markers can then be used in clinical trials to tailor treatments to patients based on their potential to respond. ISKS’s global collaboration will allow for enough patients to study these questions related to genomic modifiers in sarcoma.
We will conduct a genome-wide association study (GWAS) to examine the influence of genetic polymorphisms on response to treatment and survival among sarcoma patients. Primary response to treatment will be measured through time to failure, defined as appearance of second primary tumor(s) and/or progression to metastases. Secondary response to treatment will be defined through time to clinical progression of metastases (as defined by imaging) or death. Analyses will be controlled for age of the patient at the time of primary diagnosis, sex, type and histology of the primary tumor, and treatment strategy. For analyses of response to treatment in the metastatic phase, we will additionally control for age at diagnosis of metastasis, location of the metastasis, and treatment in the metastatic phase.
Clinical implications of Aim 4
This ISKS genomic modifier project is a “bench-to-bed” study, aimed at the mechanistic understanding disease progression in the context of sarcomas. We will identify genetic polymorphisms associated with response to treatment, and use these variants to generate a progression risk score. This risk score can then be used in clinical trials to stratify patients to more or less aggressive treatment regimens as opposed to standard care. The risk score developed by ISKS could be then validated in prospective clinical trials.
AIM 5. Sarcoma predisposition syndromes.
To identify familial cancer patterns and novel gene mutations in sarcoma predisposition syndromes.
There are some families that carry multiple sarcoma cases, not explained by known mutations. We propose whole exome analysis of all the known human genes to identify new cancer risk alleles (cancer-causing genes). Our initial studies using the strategy described below have yielded important insights into the biological basis and clinical course in several families. To date, ISKS has enrolled at least 40 families with multiple sarcoma cases not explained by mutations in TP53 and the current Liddy Shriver Sarcoma Initiative International Collaborative Research Grant will allow ISKS to rapidly expand the number of families to enroll. For this last ISKS aim, families will be selected on the basis that they carry two or more sarcoma cases occurring within 1st or second degree relatives, along with another early-onset (less than 40 years) cancer or another sarcoma occurring in a 3rd or greater degree relation in a pattern-consistent Mendelian dominant (inherited) genetic transmission. Through the ISKS enrollment infrastructure, we will make certain that tumor DNA is available from at least one affected individuals. Whole exome sequencing of germline (normal) DNA and somatic (tumor) DNA will be analyzed by the ISKS-developed pipeline to look for genes that are mutated or altered that may be causing sarcoma development.
Clinical implications of Aim 5
Identifying the novel genetic basis for highly penetrant, or inherited, sarcoma predisposition in families will: 1) Assist in genetic counseling, testing, and early tumor surveillance, and 2) Reveal new biological pathways relevant to sarcoma development, that may in the future have diagnostic, prognostic, and therapeutic implications. The discovery of new sarcoma-causing genes through ISKS is very exciting due to its strong clinical impact.
Principal Investigators and Associated Institutes
The success of ISKS depends directly on the strength and dedication of the global collaborators and its principal investigators (PIs). The ISKS PIs supervise the assembly and operations of the local research teams. Specifically, the PIs oversee the local clinical database and biospecimen repository, including data entry and management, quality control, biospecimen processing, storage and shipping to the global study centre in Australia (where ISKS originated). Every PI, with the support of two co-PIs, will take the “lead” on a specific ISKS sarcoma aim. This collaborative, interdisciplinary nature of ISKS will contribute directly to its success.
ISKS Global Participants
ISKS Global Principal Investigator
Prof David Thomas
The Kinghorn Cancer Centre in Sydney, Australia
Peter MacCallum Cancer Centre in Melbourne, Australia
ISKS Global Steering Committee
Dr Isabelle Ray-Coquard, Centre Leon Berard in Lyon, France
A/Prof Josh Schiffman, Huntsman Cancer Institute in Salt Lake City, Utah, USA
Prof Ajay Puri, Tata Memorial Hospital in Mumbai, India
Prof Ian Judson, Royal Marsden Hospital in London, United Kingdom
Dr Beatrice Seddon, University College London Hospital in London, United Kingdom
Dr Paul Clarkson, British Columbia Cancer Agency in Vancouver, Canada
Prof Robert Maki, Mt Sinai Hospital in New York, New York, USA
ISKS Global Coordinator
Dr Mandy Ballinger, Peter MacCallum Cancer Centre in Melbourne, Australia
ISKS Data Manager
Ms Eveline Niedermayr, Peter MacCallum Cancer Centre in Melbourne, Australia
Additional Site Investigators
Dr Gillian Mitchell, British Columbia Cancer Agency in Vancouver, Canada
Ms Heather Thorne, Peter MacCallum Cancer Centre in Melbourne, Australia
Dr Kathy Tucker, Prince of Wales Hospital in Sydney, Australia
Dr Craig Lewis, Prince of Wales Hospital in Sydney, Australia
Prof Martin Tattersall, Royal Prince Alfred Hospital in Sydney, Australia
Dr Michael Gattas, Queensland Health in Brisbane, Australia
A/Prof Susan Neuhaus, Royal Adelaide Hospital in Adelaide, Australia
Dr Richard Carey-Smith, Hollywood Private Hospital in Perth, Australia
Prof David Wood, Hollywood Private Hospital in Perth, Australia
Ms Mary-Anne Young, Peter MacCallum Cancer Centre in Melbourne, Australia
Dr Gillian Dite, University of Melbourne in Melbourne, Australia
Dr Paul James, Peter MacCallum Cancer Centre in Melbourne, Australia
Dr Sue Shanley, Peter MacCallum Cancer Centre in Melbourne, Australia
Dr Iain Ward, Christchurch Hospital in Christchurch City, New Zealand
Dr Angel Cioffi, Mt Sinai Hospital in New York, New York, USA
Dr Charlotte Benson, Royal Marsden Hospital in London, United Kingdom
Prof Lor Randall, Huntsman Cancer Institute in Salt Lake City, Utah, USA
Prof Rajiv Sarin, Tata Memorial Hospital in Mumbai, India
The ISKS, funded by the newly awarded Liddy Shriver Sarcoma Initiative International Collaborative Research Grant, will establish an international collaboration focused on a neglected and critical area of cancer research: the genetic basis for development of sarcomas. With recent astonishing advances in genomics and computational biology, the creation of suitably annotated cohorts with sufficient statistical power will be rate-limiting to our ability to exploit these tools to benefit patients – and the ISKS overcomes this challenge. Already, ISKS has demonstrated features consistent with previously unrecognized and untreated inherited cancer risk, which does not correspond to known risk stratification criteria. An immediate outcome of ISKS will be increased engagement between familial cancer centers and sarcoma clinics. Furthermore, identifying variables that are associated with response to treatment will lead to improved therapeutic strategies for sarcoma patients. ISKS creates the necessary resources to identify genetic risk in patients and families with sarcoma and this knowledge will benefit patients, physicians, and sarcoma research.
As the era of genomics spreads across different cancer types, there will be increasing recognition of genetic predictors of cancer risk beyond breast and bowel cancers. Analogous to those diseases, programs will be developed for recognizing and addressing differential risk, on the basis that early detection and cure is better than treating advanced disease. To reach that goal, cohorts such as the ISKS bring together major centres globally to achieve statistically meaningful numbers and to unite experts in complementary disciplines (oncologists, surgeons, clinical geneticists, basic scientists, and molecular epidemiologists). ISKS is one of the first and only sarcoma-specific efforts to undertake a global initiative of this kind.
By Joshua Schiffman, MD at Huntsman Cancer Institute in the USA
Isabelle Ray-Coquard, MD at Centre Léon Bérard in France
Ajay Puir, MD at Tata Memorial Hospital in India
Ian Judson, MD at The Royal Marsden in the United Kingdom
David Cox, PhD at Centre Léon Bérard in France
R. Lor Randall, MD at Huntsman Cancer Institute in the USA
Mary-Anne Young, MHSc at Peter MacCallum Cancer Centre in Australia
Mandy Ballinger, PhD at Peter MacCallum Cancer Centre in Australia
and at Peter MacCallum Cancer Centre in Australia
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