Thestudyof p16 and p15 genemethylationinheadandneck squamouscellcarcinomaandtheirquantitativeevaluationin plasmabyreal-timePCR T.-S.Wong a ,M.W.-L.Man a ,A.K.-Y.Lam b ,W.I.Wei a , Y.-L.Kwong c ,A.P.-W.Yuen a, * a Department of Surgery, The University of Hong Kong, Hong Kong, PR China b Department of Pathology, The University of Hong Kong, Hong Kong, PR China c Department of Medicine, The University of Hong Kong, Hong Kong, PR China Received4December2002;receivedinrevisedform24February2003;accepted6May2003 Abstract Epigeneticsilencingofthe p16 and p15 genesbypromotermethylationarecommonlyobservedinhumanepithelialmalignancies, including head and neck squamous cell carcinomas (HNSCC). In this study, a methylation-specific polymerase chain reaction (MSP)wasusedtoevaluatethemethylationstatusofthe p16 and p15 genesin73HNSCCsurgicalspecimens. p16 and p15 gene methylationwasalsoexaminedin29pairedmetastaticlymphnodesand29pairedhistologically,normalresectionmarginmucosae. Thequantityofcell-freemethylated p16 and p15 DNAintheplasmasamplesof20HNSCCpatientsand24healthycontrolswas alsoexaminedusingafluorescence-basedreal-timePCRassay.Thefrequenciesof p16 and p15 methylationintheprimarytumour were 49% and 60%, respectively. Concordant methylation of p16 and p15 in tumour samples and metastatic lymph nodes was foundin59and38%ofcases,respectively.Asignificantlyhigherprevalenceof p15 methylationwasfoundinhistologically-normal surgical margin epithelia of HNSCC patients with chronic smoking and drinking habits compared with non-smokers and non- drinkers. In addition, methylated p16 and p15 DNAlevelsweresignificantlyhigherintheplasmaofHNSCCpatients(mean56 copies/mlplasmaand65copies/mlplasma,respectively)comparedwithnormalcontrols(mean6copies/mlplasmaand16copies/ mlplasma,respectively).Inconclusion,promotermethylationofthe p16 and p15 genesisinvolvedinthepathogenesisofHNSCC andmayberelatedtochronicsmokinganddrinking.Thedifferentiallevelsofmethylated p16 and p15 DNAinplasmamightbe potentialusefulmarkersinscreeninghigh-riskpopulationsforearlyHNSCCandmonitoringtheirtreatmentresponse. # 2003ElsevierLtd.Allrightsreserved. Keywords: p15; p16;Tumoursuppressorgenes;HNSCC 1. Introduction Boththep16andp15proteinsareinhibitorsofcyclin- dependent kinases that prevent the cell going through theG1/Sphasetransaction.Inactivationofp16andp15 are important steps in cancer development [1]. Down- regulation of p16 and p15 expression are found in manycancers [2,3].Wehavepreviouslyreportedahigh frequency (48%) of p16 downregulation in a study of 225 head and neck squamous cell carcinomas [4]. Transcriptional silencing of these inhibitors is fre- quently associated with methylation of 5 0 CpG islands [5,6].Methylationof p16 hasbeenfoundinprecancerous oraldysplastictissues,adjacentnon-neoplasticareasof gastriccarcinomaandulcerativecolitis [7–10].Therole ofp15islesswelldocumented. Methylated p16 is present in bronchial epithelia before the clinical evidence of lung cancer in chronic smokers [11].Itthereforesuggeststhepotentialofusing methylatedDNAasatumourmarkerincancerscreen- ing, monitoring chemoprevention and the treatment of cancer. Apart from examining cells, the other possible source of detection of these tumour markers is in the absorbed tumour DNA in peripheral blood. Elevation 0959-8049/03/$-seefrontmatter # 2003ElsevierLtd.Allrightsreserved. doi:10.1016/S0959-8049(03)00428-3 EuropeanJournalofCancer39(2003)1881–1887 www.ejconline.com * Corresponding author. Tel.: +852-2855-4584; fax: +852-2855- 3464. E-mail address: pwyuen@hkucc.hku.hk(A.P.-W.Yuen).