Purpose:The challenges of cytology for accurate diagnosis of breast cancer are well recognized.We previously showed that normal and tumor tissue can be distinguished using a technique called quantitative multiplex methylation-specific PCR (QM-MSP). We hypothesized that quantitative analysis of methylated genes will provide enhanced detection of cancer cells present in cytologic specimens. Experimental Design: QM-MSP was done on ductal lavage cells from a set of 37 ductal lavage samples from women undergoing mastectomy (27 with cancer and 3 without). Duct histology information was available for each lavaged duct. QM-MSP data was assessed by measuring cumulative methylation index and by receiver operating characteristic threshold analysis.To determine the baseline level of methylation for each gene in this population, cells from 60 ducts of women at high risk of developing breast cancer were analyzed. Results: QM-MSP findings on a panel of nine genes were correlated to duct histology and ductal lavage cytology. Cytology detected cancer in 33% (7 of 21 ducts) with a specificity of 99% (92 of 93). QM-MSP detected cancer as calculated by cumulative methylation index with a sensitivity of 62% (13 of 21) and specificity of 82% (62 of 76) and by receiver operating characteristic threshold analysis with a sensitivity of 71% (15 of 21) and specificity of 83% (63 of 76). Conclusions: Compared with cytology, QM-MSP doubled the sensitivity of detection of cancer. This study provides proof of principle by showing the advantages of using methylation analyses to query cytologic specimens and indicates its potential use in diagnosis and in stratifying risk.Detection of breast cancer is dependent on sensitive screening methods. Mammography is widely used but is falsely negative in 15% of women due to factors, such as breast density. This has led to a search for improved methods of imaging and sampling of breast tissue for cytologic examination (1 -4). Cytology is the current gold standard for the identification of abnormalities typical of cellular transformation. However, recent findings have cast doubt on its effectiveness as a single discriminator of cancer cells. For instance, a recent study documented lack of reliability of cytology in cells collected by ductal lavage in women with biopsy-proven breast cancer (5).Here, similar to previous reports on cells derived through ductoscopy (6) or fine needle aspiration (7), the sensitivity of cytology to detect cancer in ductal lavage was determined to be as low as 43% (5). Additional methods to identify tumor cells, such as those detecting molecular alterations, are clearly needed (8, 9).Multigene methylation of CpG islands is common in early breast cancer and leads to silencing of genes responsible for tumor suppression (10 -13). Recently, we developed a highly sensitive method called quantitative multiplex methylationspecific PCR (QM-MSP) to quantitate cumulative gene promoter hypermethylation in multiple genes in samples where DNA is limiting, such as ductal lavage, nipp...
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