TY - JOUR
T1 - MethyQESD, a robust and fast method for quantitative methylation analyses in HNPCC diagnostics using formalin-fixed and paraffin-embedded tissue samples
AU - Bettstetter, Marcus
AU - Dechant, Stefan
AU - Ruemmele, Petra
AU - Vogel, Corinna
AU - Kurz, Katrin
AU - Morak, Monika
AU - Keller, Gisela
AU - Holinski-Feder, Elke
AU - Hofstaedter, Ferdinand
AU - Dietmaier, Wolfgang
PY - 2008/12
Y1 - 2008/12
N2 - Promoter hypermethylation occurs in various tumors and leads to silencing of tumor-relevant genes. Thus, promoter methylation analysis (MA) has been established as an important tool in cancer research and diagnostics. Here we present MethyQESD (methylation-quantification of endonuclease-resistant DNA) as a fast, easy, precise and reliable method for quantitative MA without the need of bisulfite-treatment or fluorescent probes. Though MethyQESD principally works with any gene promoter we established MethyQESD for the mismatch repair gene MLH1 and tested its utility to differentiate between sporadic microsatellite unstable (MSI-H) colorectal cancer and hereditary nonpolyposis colorectal cancer (HNPCC) by quantitative MLH1 MA. We investigated formalin-fixed and paraffin-embedded tissue samples from a previously published, well-characterized tumor collective comprising 25 HNPCC, 14 sporadic MSI-H CRC and 16 sporadic microsatellite stable (MSS) CRC. We found a high accuracy of MethyQESD by spiking experiments with dilution series of methylated (SW48 cancer cell line) and unmethylated (blood) DNA (Pearson's r=0.9997 (proximal MLH1 promoter region), r=0.9976 (distal MLH1 promoter region)). MethyQESD and conventional quantitative MA using of 96 formalin-fixed and paraffin-embedded CRC showed a high degree of concordance of both methods (Pearson's r=0.885). HNPCC tumors showed either null MLH1 methylation or a significantly lower degree of MLH1 methylation than sporadic MSI-H CRC (P<0.001). MLH1 methylation was negative in all MSS tumors. Receiver operating characteristic (ROC) curve analyses defined a cutoff value of 16.5% MLH1 methylation for specific and sensitive identification of sporadic MSI-H CRC (area under ROC curve: 1.000; asymptotic significance: P<0.001). Thus, quantitative MLH1 MA by MethyQESD provides a simple, fast and valuable tool to identify HNPCC candidates. Furthermore, MethyQESD works reliably with formalin-fixed paraffin-embedded tissue and simplifies DNA MA both for research and diagnostic purposes.
AB - Promoter hypermethylation occurs in various tumors and leads to silencing of tumor-relevant genes. Thus, promoter methylation analysis (MA) has been established as an important tool in cancer research and diagnostics. Here we present MethyQESD (methylation-quantification of endonuclease-resistant DNA) as a fast, easy, precise and reliable method for quantitative MA without the need of bisulfite-treatment or fluorescent probes. Though MethyQESD principally works with any gene promoter we established MethyQESD for the mismatch repair gene MLH1 and tested its utility to differentiate between sporadic microsatellite unstable (MSI-H) colorectal cancer and hereditary nonpolyposis colorectal cancer (HNPCC) by quantitative MLH1 MA. We investigated formalin-fixed and paraffin-embedded tissue samples from a previously published, well-characterized tumor collective comprising 25 HNPCC, 14 sporadic MSI-H CRC and 16 sporadic microsatellite stable (MSS) CRC. We found a high accuracy of MethyQESD by spiking experiments with dilution series of methylated (SW48 cancer cell line) and unmethylated (blood) DNA (Pearson's r=0.9997 (proximal MLH1 promoter region), r=0.9976 (distal MLH1 promoter region)). MethyQESD and conventional quantitative MA using of 96 formalin-fixed and paraffin-embedded CRC showed a high degree of concordance of both methods (Pearson's r=0.885). HNPCC tumors showed either null MLH1 methylation or a significantly lower degree of MLH1 methylation than sporadic MSI-H CRC (P<0.001). MLH1 methylation was negative in all MSS tumors. Receiver operating characteristic (ROC) curve analyses defined a cutoff value of 16.5% MLH1 methylation for specific and sensitive identification of sporadic MSI-H CRC (area under ROC curve: 1.000; asymptotic significance: P<0.001). Thus, quantitative MLH1 MA by MethyQESD provides a simple, fast and valuable tool to identify HNPCC candidates. Furthermore, MethyQESD works reliably with formalin-fixed paraffin-embedded tissue and simplifies DNA MA both for research and diagnostic purposes.
KW - BRAF mutation
KW - HNPCC
KW - MLH1
KW - MSI
KW - Microsatellite instability
KW - Quantitative methylation analysis
UR - http://www.scopus.com/inward/record.url?scp=56649097479&partnerID=8YFLogxK
U2 - 10.1038/labinvest.2008.100
DO - 10.1038/labinvest.2008.100
M3 - Article
C2 - 18936738
AN - SCOPUS:56649097479
SN - 0023-6837
VL - 88
SP - 1367
EP - 1375
JO - Laboratory Investigation
JF - Laboratory Investigation
IS - 12
ER -
