TY - JOUR
T1 - Quantitative multiplex methylation-specific PCR assay for the detection of promoter hypermethylation in multiple genes in breast cancer
AU - Fackler, Mary Jo
AU - McVeigh, Megan
AU - Mehrotra, Jyoti
AU - Blum, Marissa A.
AU - Lange, Julie
AU - Lapides, Amanda
AU - Garrett, Elizabeth
AU - Argani, Pedram
AU - Sukamar, Saraswati
PY - 2004/7/1
Y1 - 2004/7/1
N2 - 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 104-105 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.
AB - 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 104-105 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.
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U2 - 10.1158/0008-5472.CAN-03-3341
DO - 10.1158/0008-5472.CAN-03-3341
M3 - Article
C2 - 15231653
AN - SCOPUS:3042825295
SN - 0008-5472
VL - 64
SP - 4442
EP - 4452
JO - Cancer Research
JF - Cancer Research
IS - 13
ER -