Quantitative multiplex methylation-specific PCR assay for the detection of promoter hypermethylation in multiple genes in breast cancer.

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-methylation-specific 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.

Very high frequency of hypermethylated genes in breast cancer metastasis to the bone, brain, and lung.

PURPOSE: Most often it is not the primary tumor, but metastasis to distant organs that results in the death of breast cancer patients. To characterize molecular alterations in breast cancer metastasis, we investigated the frequency of hypermethylation of five genes (Cyclin D2, RAR-beta, Twist, RASSF1A, and HIN-1) in metastasis to four common sites: lymph node, bone, brain, and lung. EXPERIMENTAL DESIGN: Methylation-specific PCR for the five genes was performed on DNA extracted from archival paraffin-embedded specimens of paired primary breast cancer and its lymph nodes (LN) metastasis (n = 25 each); in independent samples of metastasis to the bone (n = 12), brain (n = 8), and lung (n = 10); and in normal bone, brain, and lung (n = 22). RESULTS: No hypermethylation was detected in the five genes in the normal host tissues. In paired samples, LN metastasis had a trend of higher prevalence of methylation compared with the primary breast carcinoma for all five genes with significance for HIN-1 (P = 0.04). Compared with the primary breast carcinomas, all five genes had higher methylation frequencies in the bone, brain, and lung metastasis, with HIN-1 and RAR-beta methylation being significantly higher (P < 0.01) in each group. Loss of expression of all five genes correlated, with a few exceptions, to hypermethylation of their promoter sequences in metastatic carcinoma cells microdissected from LNs. CONCLUSION: The frequent presence of hypermethylated genes in locoregional and distant metastasis could render them particularly susceptible to therapy targeted toward gene reactivation combining demethylating agents, histone deacetylase inhibitors, and/or differentiating agents.

Estrogen receptor/progesterone receptor-negative breast cancers of young African-American women have a higher frequency of methylation of multiple genes than those of Caucasian women.

PURPOSE: To provide a molecular rationale for negative prognostic factors more prevalent in African-American (AA) than Caucasian (Cau) women, we investigated the frequency of promoter hypermethylation in invasive ductal breast cancers in the two races. EXPERIMENTAL DESIGN: HIN-1, Twist, Cyclin D2, RAR-beta, and RASSF1A were analyzed in DNA from 67 AA and 44 Cau invasive ductal breast cancers, stratified by age and estrogen receptor/progesterone receptor (ER/PR) status, by methylation-specific PCR. Hierarchical multiple logistic regression analysis was applied to determine estimated probabilities of methylation. Expression of HIN-1 mRNA was analyzed by in situ hybridization and quantitative reverse transcribed PCR. RESULTS: Significant differences between races were observed in the ER-/PR-, age < 50 subgroup; AA tumors had higher frequency of methylation (P < 0.001) in four of five genes as compared with Cau and also a higher prevalence (80 versus 0%; P < 0.005) of three or more methylated genes per tumor. No differences in gene methylation patterns were observed across the two races for ER+/PR+ tumors in all ages and ER-/PR- tumors in age > 50. ER+/PR+ status was associated with higher frequency of methylation in Cau tumors of all ages but only with the age > 50 subgroup in AA. Frequent Cyclin D2 methylation was significantly associated (P = 0.01) with shorter survival time. CONCLUSION: ER-/PR-, age < 50 tumors in AA women, have a significantly higher frequency of hypermethylation than in those of Cau women. Comparative studies, such as these, could provide a molecular basis for differences in tumor progression and pathology seen in the two races.

DNA methylation of RASSF1A, HIN-1, RAR-beta, Cyclin D2 and Twist in in situ and invasive lobular breast carcinoma.

Little is known about epigenetic silencing of genes by promoter hypermethylation in lobular breast cancers. The promoter methylation status of 5 cancer-related genes (RASSF1A, HIN-1, RAR-beta, Cyclin D2 and Twist) was evaluated in 2 types of lobular cancers, in situ (LCIS) and invasive lobular carcinomas (ILC) (n = 32), and compared to ductal in situ (DCIS) and invasive (IDC) breast cancers (n = 71). By using methylation-specific PCR (MSP), 100% of ILC and 69% of LCIS cases were found to have 1 or more hypermethylated genes among the panel of 5 genes (compared to 100% IDC and 95% of DCIS). Two or more hypermethylated genes were detected per tumor in 79% of invasive and 61% of in situ lobular carcinomas compared to 81% of IDC and 77% of DCIS. By contrast, DNA from nearly all normal reduction mammoplasty tissues (n = 8) was unmethylated for the 5 genes. The methylation profiles of lobular vs. ductal carcinomas with respect to RASSF1A, Cyclin D2, RARbeta, and Hin-1 genes were similar, suggesting that gene silencing by promoter hypermethylation is likely to be important in both groups of diseases. Distinctly different, Twist was hyper- methylated less often in ILC (16%, 3/19 cases) than in IDC (56%, 15/27 cases) (p = 0.01). These results suggest that these 2 types of tumors share many common methylation patterns and some molecular differences. Additional studies might lend further understanding into the etiology and clinical behavior of this tumor type.