Quantitative Multiplex Methylation-Specific PCR Assay for the Detection of Promoter Hypermethylation in Multiple Genes in Breast Cancer

Abstract

If detected early, breast cancer is eminently curable. To detect breast cancer in samples with little cellularity, a high level of sensitivity is needed. Tumor-specific promoter hypermethylation has provided such a valuable tool for detection of cancer cells in biological samples. To accurately assess promoter hypermethylation for many genes simultaneously in small samples, we developed a novel method, quantitative multiplex-methylationspecific PCR (QM-MSP). QM-MSP is highly sensitive (1 in 10 4 -10 5 copies of DNA) and linear over 5 orders of magnitude. For RASSF1A, TWIST, Cyclin D2, and HIN1, we observed significant differences in both the degree (P < 0.003) and incidence (P < 0.02) of hypermethylation between normal and malignant breast tissues. Evaluation of the cumulative hypermethylation of the four genes within each sample revealed a high level of sensitivity (84%) and specificity (89%) of detection of methylation. We demonstrate the application of this technique for detecting hypermethylated RASSF1A, TWIST, Cyclin D2, HIN1, and RARB in 50 -1000 epithelial cells collected from breast ducts during endoscopy or by lavage. Such an approach could be used in a variety of small samples derived from different tissues, with these or different biomarkers to enhance detection of malignancy.

Quantitative promoter methylation analysis of hepatocellular carcinoma, cirrhotic and normal liver

“…[13][14][15]17,28,29 Therefore, taking NLT for threshold calculation in real-time MSP may lead to false negative results for promoter methylation. 26,30 In our study, lower APC methylation in CT compared to HCC and NLT (97.5 in HCC vs. 6.6 in CT, both p < 0.001) could be explained by the relative lower amount of hepatocytes compared to connective tissue in cirrhosis. Similarly, the higher methylation level of DAP-K in CT when compared to HCC and NLT could be explained by aberrant promoter methylation in fibroblasts and Ito-cells (stromal cells) in connective tissue as described for GSTP1 in prostate cancer.…”

Single CpG hypermethylation, allele methylation errors, and decreased expression of multiple tumor suppressor genes in normal body cells of mutation‐negative early‐onset and high‐risk breast cancer patients

“…In addition to allele methylation errors, we found single CpG hypermethylation in multiple TS genes in normal body cells of approximately one third of EO BC patients. Previous studies45, 46, 47 observed cumulative hypermethylation of TS genes in tumor tissue and/or serum (probably due to cell‐free tumor DNA). The epigenetic abnormalities of TS genes in whole blood DNA in our study are not derived from tumor cells.…”

Profiling CpG island field methylation in both morphologically normal and neoplastic human colonic mucosa

“…However, QMSP measures CGI methylation, which probably occurs in an allelespecific manner (Supplementary Figure S2; Fackler et al, 2004;Belshaw et al, 2005). Because each colonic crypt is populated by clonal expansion of stem cells, aberrant CGI methylation is more likely to occur discretely in individual crypts.…”