Functional genomics of cancer

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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. 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 cancers, adult sarcomas, lymphoma, melanoma, gastrointestinal tumors, breast cancers, and hematologic disease. 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. Very recently in collaboration with Illumina, we have obtained excellent data using small FFPE samples for gene copy number (CGH), SNPs, and methylation. Expression can also be studied in FFPE, but primarily with sub-genomic samples of candidate genes. 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. Our proof-of-principle study compared follicular lymphoma to follicular hyperplasia, and identified dozens of markers which robustly distinguish these two entities. 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. In melanoma, we are primarily focused on profiling its progenitor cell, the melanocyte in a mouse model. In collaboration with Glenn Merlino (NCI/CCR) and Ed DeFabo (George Washington University) we are investigating the gene expression program of normal melanocytes in murine development using and system which specifically tags melanocytes and allows them to be purified from mouse skin by flow cytometry. This system allows us to investigate the effect of the major melanoma carcinogen, ultraviolet (UV) light on melanocyte development. Through the use of sensitive microarray technologies, we have been able for the first time to observe the in vivo effect of UV radiation on melanocytes. These results are providing unprecedented insight into the melanocyte development and promise to advance our understanding of UV carcinogenesis. In breast cancer, we have been particularly interested in the problem of ductal carcinoma in situ (DCIS). DCIS is readily diagnosed by mammography, and may represent disease which carries very little long term risk to the patient or the early presentation of an aggressive cancer. However, pathologists have a great deal of difficulty grading DCIS, and as a result, clinicians have difficulty stratifying these patients to appropriately intense therapy. In collaboration with Rosemary Balleine (Westmead Hospital, Sydney), we have profiled microdissected DCIS lesions from specimens in which invasive cancer was associated with the DCIS. Using the grade of the invasive cancer to index the samples, we have been able to develop a gene expression classifier which can cleanly separate low grad from high grade DCIS. We are currently developing a multiplex gene expression assay which can be applied to FFPE DCIS samples. After validation and refinement, this assay has the potential to be applied to prospective samples and may have a substantial impact on the diagnosis and management of DCIS. Also in the breast cancer field, we have developed high resolution gene expression and gene copy number data which has led to the emergence of several candidate genes under investigation in the laboratory. One gene which we identified by expression profiling is GATA-3, a transcription factor which is associated with estrogen receptor positive breast cancer. We have suspected that GATA-3 is an important developmental regulator in this system, and recent studies by others in mouse models have confirmed this hypothesis. To determine how GATA-3 regulates gene expression, we have carried out whole genome transcription factor localization studies using chromatin immunoprecipitation in breast cancer cells. The results establish a functional inter-relation between estrogen receptor and GATA-3 and provide insight into the interplay between mammary epithelial development and cancer. To follow up on candidate genes identified by our profiling studies, we are using RNA interference to silence panels of candidate genes coupled with phenotypic endpoints which identify genes which are responsible for tumor growth. Some of these studies overlap with technology development aspects of our work. For example, in collaboration with Agilent Technologies, we have pushed the expression and copy number analysis of a region, chromosome 8p, which is frequently amplified in breast cancer to the highest possible resolution using tiling path arrays. These studies have allowed us to map this region in breast cancer definitively and to identify conclusively the 8p genes which are active in breast cancers. We have used similar technology to map regions of gene amplification on other chromosomes, to clone the points of rearrangement and to identify fusion genes. We are also using similar technology to investigate the pattern of expression of microRNAs and their progenitors