Denaturing high performance liquid chromatography for the detection of microsatellite instability using bethesda and pentaplex marker panels.
Microsatellite instability (MSI) is a characteristic molecular phenotype of tumors from the hereditary nonpolyposis colorectal cancer (Lynch) syndrome. Routine MSI screening of tumors in younger patients is an efficient prescreening tool for the population-based detection of Lynch syndrome in the absence of family cancer history. We describe here the optimization of a denaturing high performance liquid chromatography (DHPLC) assay for MSI analysis with the "Bethesda" panel of markers recommended by the National Cancer Institute and with a more recently proposed "pentaplex" panel of 5 mononucleotide repeat markers. By using various polymerase chain reaction primers and tumor DNA samples with known MSI status, each of the 3 standard DHPLC formats tested could correctly identify the MSI status without the "stutter peaks" inherent in the capillary electrophoresis (CE) methods that are currently in use. Dilution experiments showed that the detection limit for MSI using DHPLC was at least 1:100, thus avoiding the need for tumor enrichment by microdissection before analysis. Concordance between CE and DHPLC for the detection of instability in the Bethesda panel markers was 95%. Optimal DHPLC running conditions for the pentaplex mononucleotide panel are also described. In conclusion, DHPLC provides a sensitive and specific alternative for routine MSI analysis that is free of the stutter peaks observed with CE and which can be used with either the Bethesda or pentaplex mononucleotide marker panels.
Soong
Richie
R
Oncology Research Institute, National University of Singapore, 117456 Singapore. nmirs@nus.edu.sg
Anuar
Diyanah
D
Liu
Yanqun
Y
Eu
Kong W
KW
Han
Hwan C
HC
Salto-Tellez
Manuel
M
Iacopetta
Barry
B
D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D006801:Humans; D053842:Microsatellite Instability; D016133:Polymerase Chain Reaction
D014408:Biomarkers, Tumor