A number of technologies are applied in parallel to determine the molecular profile of a given biospecimen. The majority of these technologies currently use microarray based methods, but they are rapidly being supplemented by evolving next generation DNA sequencing technologies. Several varieties of microarray are used for various purposes, but the predominant current technical approaches use synthetic oligonucleotides bound to a solid support and interrogated with labeled nucleic acids prepared from the biospecimen of interest. The power of this approach in the current embodiment of this technology is based largely on the direct connection between known genome sequence and the design of microarrays completely controlled by computational means. This allows the investigator to construct arrays of arbitrary design tailored specifically to the desired analysis and to adjust the resolution of the arrays to a remarkably fine level. Thus, for example, it is now possible to determine the expression of mRNAs exon by exon and to observe changes in gene copy number (amplification or deletion) at better than single gene resolution. Fluorescent probes prepared from any cell or tissue source of interest are then hybridized to these arrays providing a large scale high resolution view of the genome. 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 1500 CpGs in parallel on hundreds of samples with results which match those obtained from frozen specimens. This opens vast existing archives of FFPE samples to investigation. We now routinely obtain excellent copy number data from FFPE samples as well. 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, 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.A number of technologies are applied in parallel to determine the molecular profile of a given biospecimen. The majority of these technologies currently use microarray based methods, but they are rapidly being supplemented by evolving next generation DNA sequencing technologies. Several varieties of microarray are used for various purposes, but the predominant current technical approaches use synthetic oligonucleotides bound to a solid support and interrogated with labeled nucleic acids prepared from the biospecimen of interest. The power of this approach in the current embodiment of this technology is based largely on the direct connection between known genome sequence and the design of microarrays completely controlled by computational means. This allows the investigator to construct arrays of arbitrary design tailored specifically to the desired analysis and to adjust the resolution of the arrays to a remarkably fine level. Thus, for example, it is now possible to determine the expression of mRNAs exon by exon and to observe changes in gene copy number (amplification or deletion) at better than single gene resolution. Fluorescent probes prepared from any cell or tissue source of interest are then hybridized to these arrays providing a large scale high resolution view of the genome. 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 1500 CpGs in parallel on hundreds of samples with results which match those obtained from frozen specimens. This opens vast existing archives of FFPE samples to investigation. We now routinely obtain excellent copy number data from FFPE samples as well. 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, 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 t [summary truncated at 7800 characters]

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010759-04
Application #
7965615
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2009
Total Cost
$2,322,606
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Morrow, James J; Bayles, Ian; Funnell, Alister P W et al. (2018) Positively selected enhancer elements endow osteosarcoma cells with metastatic competence. Nat Med 24:176-185
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
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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
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
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Sun, Wenyue; Chatterjee, Bishwanath; Wang, Yonghong et al. (2015) Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma. Mod Pathol 28:1214-24

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