> Research & Grants
> Grants Program
> Research Grants
> Research Grants Awarded
Research Grants Awarded
Phenotypic Differences in Sub-types of DCIS: Aggressiveness and Race
Detection, Diagnosis and Prognosis
Ductal adenocarcinoma of the breast is hypothesized to develop from the pre-invasive neoplastic lesion, high grade (HG) ductal carcinoma in situ (DCIS). A problem with acquiring information on DCIS is that DCIS is usually not available for study in the fresh/frozen state because current standards for the practice of pathology require DCIS lesions to be submitted entirely in order to exclude focal invasion. Thus, methods to study DCIS in paraffin embedded material are needed to increase our understanding of its development and progression. Previously, we have observed that there are several subgroups of HG-DCIS as to the molecular phenotypes of these lesions. We hypothesize that molecular markers may be used to identify aggressive subsets of DCIS because the acquisition of molecular features of aggressiveness are retained as pre-invasive lesions progress. We also hypothesize that there are racial differences in the phenotypes of HG-DCIS and that such differences are important in aggressive subsets of HG-DCIS. We recently have adapted an approach of real time quantitative PCR (RT-Q-PCR) to permit PCR analysis of mRNA in paraffin embedded material. For most genes, we get the same results for paraffin embedded tissues as we do for matching frozen tissues. We also have successfully used a multiplex form (MA) of RT-Q-PCR which permits the simultaneous analysis of up to 382 genes and 2 housekeeping genes on one tissue or 47 genes and housekeeping genes on 8 tissues, provided 1ng of RNA is available per gene. We will combine this novel approach with laser capture microdissection to study DCIS. We also will use more standard immunohistochemistry (bright field and fluorescent) to confirm at the protein level our observations at the mRNA level. This technology will be used to analyze molecular biomarkers which have been reported to be associated with primarily the prognosis of breast cancer (e.g., p185 erbB-2 ). Also, because MA-RT-Q-PCR and immunohistochemistry permit simultaneous evaluation of multiple genes and proteins in one tissue sample, we can evaluate the expression of a signal transduction pathway. For example, we can evaluate changes in EGF-R, TGF-alpha and ERK1/2 at the gene level and pEGF-R and pERK1/2 at the protein level. This observational approach is to generate novel molecular hypotheses which can be tested in order to further our understanding of DCIS.
By microscopic observation, an association has been made between microscopic changes called high grade ductal carcinoma in situ (HG-DCIS) and invasive ductal adenocarcinoma (DCa) of the breast. Thus, HG-DCIS is postulated to be a pre-invasive form of breast neoplasia. Pure DCIS is difficult to study in frozen and/or fresh tissues of the breast because all tissues containing pure DCIS are used completely in diagnosis in order to exclude the presence of invasive cancer. In diagnosis based on microscopic examination of tissues, the tissues are embedded in wax. Thus, most cases containing only DCIS end up as diagnostic tissue which is embedded in wax. We have been interested in methods to evaluate DCIS as well as DCa using methods which can be applied to the tissues in wax and have demonstrated an approach to measuring the expansion of multiple specific genes. This approach is called real time quantitative PCR (RT-Q-PCR). We have demonstrated that such analysis can be performed on paraffin tissues and that the analysis yields results that are equivalent to those based on analysis of matching frozen tissue. Also, we have demonstrated the usefulness of a multiplex form of RT-Q-PCR which can measure over 382 separate genes on one specimen of cancer or 47 separate genes on 8 specimens of cancer. We propose to microdissect DCIS lesions from paraffin slides and analyze the expression of a group of selected genes which are likely to be important in the development and progression of DCIS to invasive cancer. These results, at the mRNA level, will be compared with results at the protein level using bright field and fluorescent immunohistochemistry. Our goal is to determine a molecular phenotype of DCIS associated with progression to invasive cancer. The selection of genes to be analyzed will be based upon the literature as to genes important in primarily prognosis, but also diagnosis. Of special importance will be genes reported to vary between Caucasians and African-Americans. Using methods which permit the simultaneous analysis of multiple genes and proteins permit an understanding of the state of regulation of multiple related genes which cooperate in a biological signal within cells. HG-DCIS in cases which have progressed to invasive cancer will be compared with HG-DCIS not associated with DCa. Also, differences between DCIS in African-Americans will be compared with similar grades of DCIS in Caucasians to determine potential racial differences.