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    Identification of Metastasis Associated Breast Cancer Genes Using a High-Resolution Whole Genome Approach

    Scientific Abstract:
    Identification of Metastasis Associated Breast Cancer Genes Using a High-Resolution Whole Genome Approach. Background: Metastasis is the single most important determinant of survival and treatment options for breast cancer patients, yet there is a paucity of knowledge of the genetic changes that determine whether a cancer will metastasize. Axillary lymph node (LN) status remains the single most important prognostic variable for breast cancer survival and is used to stratify patients into adjuvant treatment groups. It is estimated that more than 70% of patients receive adjuvant therapy needlessly because conventional pathologic and clinical parameters do not discriminate sufficiently. Molecular studies should offer a more exact prediction of women requiring these life-altering and costly treatments. Technical problems make it doubtful that research studies that have employed gene expression and conventional comparative genomic hybridization (CGH) technology to stratify these patients will develop into assays that can be used in clinical laboratories. To overcome these technical limitations, our Microarray and Genomics Group developed an array based CGH approach (aCGH) that screens for copy number aberrations (CNAs) across the entire genome at an unparalleled resolution, thus allowing for easy identification of candidate cancer genes. Objective/Hypothesis: High-resolution aCGH, a relatively new methodology used to identify gains and losses of genetic material in tumors, can be used to discover metastasis suppressing and promoting genes in small breast cancers, which are particularly heterogeneous with regard to clinical behavior. We propose that the variable metastatic potential of a phenotypically homogeneous group of breast cancers is based on specific genetic alterations. Furthermore, we speculate that metastases derived from their parental tumors contain additional genomic abnormalities. The overall goal of this study is the identification and characterization of hitherto unknown breast cancer metastasis genes to allow us to answer the important question: Which early stage breast cancers can be cured by local therapy alone, and which tumors require adjuvant systemic treatment? Specific Aims: 1. Probe for differences in DNA amplifications and deletions between non-metastatic T1, Grade III, ER+ invasive ductal carcinomas (IDC) and matched tumors associated with LN metastases by high-resolution aCGH analysis. 2. Probe for differences in DNA amplifications and deletions between LN metastases and their parental primary tumors by aCGH. 3. Confirm the abnormal copy number and expression level of candidate genes that may determine the metastatic potential of a breast carcinoma. Study Design: We will implement aCGH as our primary means of tumor analysis. Our bacterial artificial chromosome (BAC) arrays have the highest average resolution (0.5Mb) available anywhere. To leave LN status as the only major variable, we will study only high grade, ER+ IDCs smaller than 2.0 cm (T1). It is within this group that the identification of low-risk patients is most pressing. Specimens are selected from the pathology files from the 1993-1998 time period so that the minimal follow-up is 5-10 years. 30 LN+ and 30 LN- cases will be analyzed. From each case, one representative paraffin block of the primary invasive tumor will be selected and, if applicable, one LN metastasis. Genomic DNA will be isolated from the 90 specimens using a commercially available DNA extraction kit (Qiagen), and fluorescently labeled and hybridized to the arrays for subsequent scanning and bioinformatic analysis of their respective CNAs. We will generate one list of CNAs that will differentiate primary tumors without metastasis from primary tumors that are capable of metastasis and a second list that will distinguish metastatic from the parental tumors. As an initial step in the characterization of metastasis associated genes within the amplified or deleted loci, we will confirm their abnormal copy number by interphase fluorescent in situ hybridization and their altered expression level by immunohistochemistry. Potential Outcomes/Benefits of the Research: Our unrivalled high-resolution screening technique will identify unknown metastasis suppressing and promoting genes and provide new clinical diagnostic and prognostic markers as well as biomarkers for metastasis prevention studies and novel therapeutic targets. The expected results will provide justification for extended studies that utilize aCGH as a clinical assay to help eliminate overtreatment of low-stage breast cancers, which would benefit thousands of women.

    Lay Abstract:
    Identification of Metastasis Associated Breast Cancer Genes Using a High-Resolution Whole Genome Approach When breast cancer is diagnosed and found to be confined to the breast itself, the prospects of long-term survival are good. However, once tumor cells have spread and formed secondary tumors at distant sites throughout the body (a biological process called metastasis), breast cancer is a grave disease accompanied by a significant drop in cure rates and a traumatic impact on quality of life for the affected patients. Metastasis is the most important factor that determines survival and treatment options for breast cancer patients. Currently, there is a significant lack of knowledge about what changes take place at the level of the gene (DNA) that determines whether a cancer will spread (metastasize) or, importantly, will not metastasize. Genes ultimately drive cells to make proteins that can contribute to the metastasis process when they are abnormally expressed. At present, the metastatic potential of a breast cancer is estimated, to a large extent, by its pathologic features including spread to axillary lymph nodes. The lymph node status is the main factor used to stratify women into treatment regimens beyond that of surgical excision of their tumor (adjuvant therapy). Adjuvant therapy consists of using pharmaceutical agents to reach any cancer cells that may have escaped from the primary tumor. It is costly and may cause highly toxic side effects. Up to 70% of patients who would have been cured by surgery alone receive this treatment needlessly ¡°just in case¡± there is undetected tumor spread. Studies that can detect predictive changes at the level of the gene should allow more exact identification of women requiring these life-altering treatments. Our Institution pioneered a cutting edge technology called array based comparative genomic hybridization (aCGH). aCGH can be used to examine DNA extracted from tumor cells, and it screens for gains and losses of genetic material across the entire genome compared to DNA extracted from normal cells. Preliminary studies by our researchers and others have determined that this technology can indeed be used to better define the aggressiveness of a tumor. We are in the unique position of having access to the arrays with the highest resolution available anywhere. This gives us unparalleled power to identify genes of interest. We hypothesize that aCGH technology can be employed to discover metastasis suppressing and promoting genes in small breast cancers. These genes can then be used in the more accurate stratification of breast cancer patients into the appropriate adjuvant treatment groups. This stratification process would ideally eliminate the application of adjuvant therapy to patients who do not require it. Utilizing samples from our pathology files, we will probe for gene amplifications and deletions in a subset of small breast cancers that express the estrogen receptor. It is within this group of tumors that identification of low-metastasis risk patients is most pressing. Pathologically similar tumor samples that present with and without metastases (30 cases from each group) will be selected. We will extract DNA from these tumors, apply our aCGH technology to examine the DNA, and use statistical methods to group abnormalities that have metastasis predictive power. We will generate a set of metastasis associated abnormalities (genetic defects) that can then be confirmed at the DNA and protein level by techniques called fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. These techniques allow us to probe for and verify specific gene abnormalities. Once we confirm that these gene changes discovered by aCGH are real biologic events, we will have completed the groundwork needed to employ aCGH technology in large breast cancer clinical trials for further testing. Ultimately, this tool should help clinicians to eliminate overtreatment of small breast cancers, which would benefit tens of thousands of women. It will also help us to discover new prognostic and predictive markers, in addition to novel therapeutic targets. Finally, it will further our understanding of the events leading to the spread of breast cancer cells.