JMDCMEProgramTechnicalAdvanceHighQualityAssessmentofDNAMethylationinArchivalTissuesfromColorectalCancerPatientsUsingQuantitativeHigh-ResolutionMeltingAnalysisMarijaBalic,*MartinPichler,*JasminStrutz,*EllenHeitzer,*ChristophAusch,†HellmutSamonigg,*RichardJ.Cote,‡andNadiaDandachi*FromtheDivisionofOncology,*DepartmentofInternalMedicine,MedicalUniversityofGraz,Graz,Austria;theDepartmentofSurgery,†LudwigBoltzmannResearchInstituteofSurgicalOncology,DanubeHospital,Vienna,Austria;andtheDepartmentofPathology,‡KeckSchoolofMedicine,UniversityofSouthernCalifornia,LosAngeles,CaliforniaHigh-resolutionmelting(HRM)analysisisanoveltoolforanalysisofpromotermethylation.TheaimofthepresentstudywastoestablishandvalidateHRManalysisfordetectionofpromotermethylationonarchivalformalin-fixedparaffin-embeddedtissuesfromcolorectalcancerpatients.WefirstevaluatedHRMassaysforO6-methylguanine-DNAmethyltrans-ferase(MGMT)andadenomatouspolyposiscoli(APC)promotermethylationonamethylatedDNAdilutionmatrixandDNAextractedfromeightfreshorforma-lin-fixedparaffin-embeddedhumancancercelllines.ThenweusedtheseassaysfortheanalysisofMGMTandAPCpromotermethylationinasubsetofarchivalformalin-fixedparaffin-embeddedcolorectaltumorspecimens.AllsampleswithpromotermethylationofMGMTorAPCandrandomlyselectedsampleswith-outpromotermethylationwereanalyzedtwice.AllresultsgeneratedbyHRMwerevalidatedwithMGMTandAPCMethyLightassays.APCandMGMTpromotermethylationdatawereconsistentandreproduciblethroughoutthedilutionsandallthreereplicatesinthemethylatedDNAdilutionmatrixandbetweentwoexperimentsinclinicalsamples.Therewashighcon-cordancebetweenHRMandMethyLightresults.HRMforAPCpromotermethylationrevealedconsistentresultsbetweenfreshandformalin-fixedparaffin-em-beddedhumancancercelllineDNA.ThemethylationstatusinarchivaltumorspecimensfrompatientswithcolorectalcancercanthereforebedeterminedwithhighqualitybyHRM.Theabilitytoanalyzearchivaltissuesgreatlyfacilitatesfurtherresearchanditsclin-icalimplementation.(JMolDiagn2009,11:102–108;DO...
