Abstract.Background:Forthepurposeofearlydetectionofcarcinomas,detectionofp16hypermethylationbymethylation-specificpolymerasechainreaction(MSP)intheserumofmanykindsofmalignancieshasbeenintroduced.Anattempttoincreasethesensitivityofthisassayisreportedforcolorectalcancer.MaterialsandMethods:DNAsamplesweresubjectedtolimitingdilutionbeforebeingdividedintotensmallersamples.Subsequently,hemi-nestedMSPwasperformedonthosetensamples.Thelimitingdilution-MSP(LD-MSP)provideda10-foldincreaseinsensitivityofthedetectionofmethylatedDNAcomparedwithconventionalMSP.Results:Of44colorectalcancerpatients,30(68%)exhibitedabnormalpromotermethylationofp16intheirserumDNAbyLD-MSP,while13(30%)exhibiteditbyconventionalMSP.Asacontrol,theserumDNAof50patientswithcolorectalcarcinomaswhosecorrespondingtumorDNAhadnomethylationinthep16promoterwasscreenedforaberrantmethylation.NomethylationwasfoundintheserumDNAofthiscontrolgroupbyLD-MSPorconventionalMSP.Conclusion:ThehighsensitivityofLD-MSPmakesitpossibletodetectsmalleramountsoftumorDNAintheserumthanconventionalMSP.Thistechniquealsohasgreatspecificityandnoabnormalmethylationinserumhasyetbeenobservedifthecorrespondingtumordoesnotexhibitmethylation.ThisobservationsupportstheideathatLD-MSPcouldbeappliedinclinicaluseforthedetectionoftumorDNAinserum.PreviousstudieshaveproposedthatenrichedcirculatingDNAcanbefoundintheserumofcancerpatients(1,2).Onthebasisofthesestudies,manyattemptshavebeenmadetoachievetheearlydetectionoftumor-relatedaberrantDNAintheserumofpatientswithvariousmalignancies(3,4).Ourstudieshavealsoshownthatitispossibletodetecttumor-specificDNAintheserumofvariouscancerpatientsusingamismatchligationassayforK-rasandmitochondrialDNAmutations(5-8).Recentstudieshaveindicatedthatpromotermethylationisanimportantmechanismforgenetranscriptionalinactivation.Severalgenessuchasp16(9),p14(10),HLTF(11),SOCS-1(12)andCDH13(13)havebeenfoundtoharborpromoterhypermethylationassociatedwithalossofgeneexpressionindigestivetractcarcinomas.Thepresenceofepigeneticmethylat...
