6th ESACP Congress, Heidelberg, April 7-11, 1999

A154
LASER MICRODISSECTION OF BREAST CANCER FOR DETERMINATION OF MICROSATELLITE INSTABILITY AND TUMOR HETEROGENEITY
Wild P, Hofstädter F, Dietmaier W, Hartmann A

University of Regensburg Medical School, Department of Pathology and Molecular Diagnostic Unit, Franz-Josef-Strauß-Allee 11, 93042 Regensburg

Most breast cancers are known to have considerable amounts of stromal cells as fibroblasts (desmoplasia) and mononuclear cells. Analysis of tumor- specific genetic alterations can be compromised by the presence of these normal cells, requiring microdissection of pure tumor cell areas for reliable detection of loss of heterozygosity (LOH) and microsatellite instability (MSI). Normal breast tissue and primary breast tumor samples of 40 patients were analyzed for MSI and LOH by polymerase chain reaction followed by polyacrylamide gel electrophoresis and silver staining using 16 microsatellite markers on ten chromosomes. Pure tumor cell populations were obtained using either manual or laser capture microdissection (PALM) of methylenblue stained paraffin-embedded tissue as well as archived hematoxylin/eosin stained slides and the results of both techniques were compared. Tumor cells of histopathologically heterogeneous areas in each tumor (i.e. scirrhous and intraductal components) were processed separately. Regions of special interest were captured with a digital camera device before microdissection, allowing an accurate assignment of genetic alterations to the histologic phenotype. The following results were obtained: 1) accurate microdissection of tumor cells revealed a higher frequency of MSI-positive breast-cancers as previously reported in literature 2) laser microdissected samples showed a significantly higher frequency of MSI-and LOH-positive tumors than the manually ones due to reduction of contamination by desmoplasia 3) MSI-positive tumors showed a high degree of intratumoral heterogeneity. Laser based microdissection is a valuable tool in genetic analysis of desmoplastic tumors and allows an accurate determination of genetic alterations in histologically different tumor regions.