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Biomarkers of invasiveness in racially disparate populations using an isogenic & progressive model of human DCIS
Breast Cancer Disparities
Based on a novel breast tissue engineering system developed by the Latimer Laboratory, we have been able to derive several sets of isogenically matched Ductal Carcinoma In Situ cell lines as well as 55 additional cell lines from more advanced stage, but chemotherapy naïve tumors at a success rate for the creating tumor cell lines (explants over passage 13) of over 60%. From these unprecedented cell lines we have now developed a set of biomarkers from microarray analysis that correlate well with invasiveness potential in these isogenic sets of DCIS and associate contralateral cell lines. These lines are expressed differentially between the contralateral (non-diseased breast) and the matching DCIS cell line. Validation of these biomarkers has been obtained using additional stage I and II cell lines we have also established (in the early and mid 1990s), with known clinical recurrence patterns in the patients. We hypothesize that the cell lines with the more transformed (malignant) cytogenetic profiles will correlate with DCIS that has a higher potential to invade and become stage I disease. The same markers that correlate with recurrent stage I and II tumors will be the markers that will be associated with invasiveness, allowing for the prognostication of DCIS that is not possible now. We further hypothesize that African American subjects with DCIS will manifest a more invasive phenotype because of the increased odds ratio shown for invasive recurrence in these women versus Asian and white subjects.
Our first objective will be to further validate our biomarkers using additional cell lines with known recurrence potential and microarray using Affymetrix U133A chips. We will then perform validation in a functional way by determining the invasiveness of these cell lines by placing all DCIS and isogenically matched contralateral and non-tumor cell lines into transwell chambers and immuno-compromised mice. Any invasive (metastatic population) cells will be harvested from the transwells and from metastatic sites in the mice for subsequent expression microarray analysis and direct comparison to the parental line already analyzed with microarray analysis. Our third and concurrent objective is to establish similar matched sets of DCIS cell lines from African American women. These women will be available to us from the contacts we have established with the Center for Minority Health. We will compare the markers we have validated in aims 1 and 2 with what we find in the AA samples to determine if the same markers apply in terms of DCIS in African Americans vs white and Asian cell lines.
We have developed a novel tissue engineering system for Human Mammary Epithelial Cells (HMEC), both normal and malignant. This system allows for the long-term (>3 months) establishment of normal primary cultures that begin as 3-dimensional mammospheres. These mammospheres differentiate into complex branching ducts and lobules. The ability to form ductal structures from human breast tissue is absolutely unique to our laboratory. Tumor cells lack the ability to form these epithelial architectures. From primary cultures we have generated 2 unprecedented DCIS cell lines and a contralateral line from the same Asian patient’s breast without the use of transforming agents. Recently we have also created a similar set of lines from a caucasian woman. The DCIS lines show chromosomal abnormalities consistent with malignancy, although not as abnormal as MCF7. We have now developed a set of biomarkers from microarray analysis that correlate well with invasiveness in these isogenic sets of cell lines. Validation of these biomarkers has been obtained using a learning set of stage I and II cell lines with known clinical recurrence/outcomes patterns in the patients. We hypothesize that the cell lines with the more transformed (malignant) cytogenetic profiles will correlate with DCIS that has a higher potential to invade and become stage I disease. We will perform further microarray validation of these markers on 15 additional early stage cell lines with known recurrence patterns. We will functionally evaluate the invasiveness of these cell lines by placing DCIS and isogenically matched contralateral and non-tumor cell lines into transwell chambers and immuno-compromised mice. Subcultures of the most motile cells will be analyzed in each case with microarray to confirm the markers most associated with a motile and invasive phenotype. Concurrently, we will establish similar matched sets of DCIS cell lines from African American (AA) women. Black women have a relative risk of mortality from DCIS of 1.35 compared to white women, while Asian women had a reduced RR. The results obtained on the white and Asian DCIS cell lines will be compared with those of the AA women to assess whether DCIS, is associated with a higher risk of invasiveness in AA women. We hypothesize that AA subjects with DCIS will manifest a more invasive phenotype because of the increased odds ratio shown for recurrence in these women compared to other populations. DCIS is one of the most commonly diagnosed forms of BC and one of the least understood forms of BC prognostically. From our identified biomarkers associated with the early stages of breast oncogenesis using our unique model systems, we will now develop a population specific set of invasiveness markers for DCIS to assist in the tailoring of treatments for DCIS in AA women.