A number of technologies are applied in parallel to determine the molecular profile of a given biospecimen. The majority of these technologies historically have used microarray based methods, but they are rapidly being supplemented, and in some respects, supplanted by evolving next generation DNA sequencing technologies. The power of these approaches is based largely on the direct connection between known genome sequence, genome annotations, experimental design and computational analysis. It is now possible to characterize cancer genomes in arbitrarily fine detail and in multiple dimensions (e.g. mRNA,miRNA, lincRNA expression, DNA sequence, DNA copy number, DNA structure, DNA methylation, chromatin structure, chromatin modification, and transcription factor binding). Our recent efforts have applied this technology to pediatric and adult sarcomas. Currently we are focused on transitioning as many assays as possible to minute samples (such as may typically be collected in the course of routine clinical care) and formalin fixed paraffin embedded (FFPE) specimens. The ability to work with FFPE samples is particularly important when one considers the potential to transition discoveries made in the course of this work to clinical care where FFPE based methods are the standard method of stabilizing biospecimens in the clinical laboratory. Of importance we have demonstrated that it is possible to determine the methylation status of more than 400,000 CpGs in parallel on hundreds of samples. This opens vast existing archives of FFPE samples to investigation. We now routinely obtain excellent copy number data from FFPE samples as well. Over the last year, we have increasingly utilized the power of next generation sequencing to improve the precision and throughput of these analyses. Our laboratory has had a long standing interesting sarcoma biology, and we have been most recently applying these technologies to the pediatric bone tumor, osteosarcoma. We have successfully identified the high resolution gene expression, miRNA expression, gene copy number, and SNP profile of osteosarcoma. This work has demonstrated a pattern of recurring copy number changes which are apparent despite the highly chaotic nature of the osteosarcoma genome. In addition, it has been possible to demonstrate that copy number has a profound impact on gene expression in osteosarcoma. This pattern suggests a number of candidate genes for further investigation. To gain a comparative genomics perspective on this disease, we have also investigated the gene expression pattern of canine osteosarcoma, and plan to take advantage of the similarities between human and canine disease to refine our understanding of this tumor. We are also investigating the molecular consequences of specific mutations which occur in sarcoma, particularly the common chromosome translocations which produce the fusion gene transcription factors characteristic of several pediatric sarcomas. Using chromatin immunoprecipitation and DNA sequencing technology, we are identifying the binding sites of oncogenic transcription factors and integrating this information with the known expression profiles of these diseases. In Ewings sarcoma, we have used RNA interference technology to target the oncogenic transcription factor EWS-FLI1 to study the genes which are regulated by this protein. We have also completed a chromatin immunoprecipitations study which assigns the location of the EWS-FLI1 transcription factor across the genome. The bioinformatics analysis reveals a striking co-location of EWS-FLI1 with the cell cycle transcription factor E2F3. Our data suggests that the transforming activity of EWS-FLI1 may arise in part from its effect on cell cycle targets also regulated by E2F factors. In alveolar rhabdomyosarcoma we have used chromatin immunoprecipitation combined with next generation sequencing to identify the genes which are targeted by the oncogenic fusion protein PAX3-FKHR. This will provide the definitive framework for building the network of dysregulated genes downstream of these critical oncogenic events. In gastrointestinal stromal tumor, we have focused on the group of tumors which lack mutations in signal transducing kinases. We found that this group of tumors, uniformly characterized by lack of succinate dehydrogenase, demonstrates striking global abnormalities in DNA methylation. This provides a new insight into the mechanism of disease and provides a robust diagnostic classification for these cases.

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National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Kovar, Heinrich; Amatruda, James; Brunet, Erika et al. (2016) The second European interdisciplinary Ewing sarcoma research summit - A joint effort to deconstructing the multiple layers of a complex disease. Oncotarget 7:8613-24
Saxena, Neetu; Maio, Nunziata; Crooks, Daniel R et al. (2016) SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery. J Natl Cancer Inst 108:
Hingorani, Pooja; Janeway, Katherine; Crompton, Brian D et al. (2016) Current state of pediatric sarcoma biology and opportunities for future discovery: A report from the sarcoma translational research workshop. Cancer Genet 209:182-94
Waterfall, Joshua J; Meltzer, Paul S (2015) Avalanching mutations in biallelic mismatch repair deficiency syndrome. Nat Genet 47:194-6
Ren, Ling; Mendoza, Arnulfo; Zhu, Jack et al. (2015) Characterization of the metastatic phenotype of a panel of established osteosarcoma cells. Oncotarget 6:29469-81
Mirabello, Lisa; Koster, Roelof; Moriarity, Branden S et al. (2015) A Genome-Wide Scan Identifies Variants in NFIB Associated with Metastasis in Patients with Osteosarcoma. Cancer Discov 5:920-31
Gindin, Yevgeniy; Jiang, Yuan; Francis, Princy et al. (2015) miR-23a impairs bone differentiation in osteosarcoma via down-regulation of GJA1. Front Genet 6:233
Schwentner, Raphaela; Papamarkou, Theodore; Kauer, Maximilian O et al. (2015) EWS-FLI1 employs an E2F switch to drive target gene expression. Nucleic Acids Res 43:2780-9
Sun, Wenyue; Chatterjee, Bishwanath; Wang, Yonghong et al. (2015) Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma. Mod Pathol 28:1214-24
Isakoff, Michael S; Bielack, Stefan S; Meltzer, Paul et al. (2015) Osteosarcoma: Current Treatment and a Collaborative Pathway to Success. J Clin Oncol 33:3029-35

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