Although studies of risk factor profiles have been conducted to identify biological markers to predict the natural history of cervical intraepithelial neoplasia (CIN) grade III, there is not sufficient information to support the routine clinical use of any biomarker.
Objectives
The purpose of this study was to examine aberrant promoter methylation, which is implicated in cancer development and progression, in CIN III lesions in order to identify markers associated with more aggressive biological behavior that could be used to recognize women who are at higher risk of recurrence.
Patients and methods
We used methylation-specific polymerase chain reaction to analyze promoter hypermethylation of 8 genes (p16, RARβ, GSTP1, MGMT, p14, TIMP3, E-cad and DAPk) in 33 uterine cervix cones with CIN III that were also submitted to human papillomavirus (HPV) genotyping. All 33 patients in this study had been clinically followed after conization with Papanicolaou smears, colposcopy, and biopsy when indicated, every 6 months during 5 years.
Results
Of the 33 patients, 12 (36%) underwent immediate hysterectomy after conization for having compromised cone margins, 14 (43%) have not relapsed, and 7 (21%) presented CIN relapse. The frequency of HPV infection in this group was 97% and no significant difference between the groups was observed. HPV of high oncogenic risk was present in 29 (87.9%) cases; HPV 16 was the most frequent (69.7%), while HPV 18 was found in 33.3%; however, it was associated with HPV 16 in 15.1%. Concomitant infection by HPV 6/11 was detected in 21.2% (15.1% with HPV 16 and 6.1 with HPV 18). 85.7% (6/7) of patients with recurrence had HPV 18 vs 0% (0/14) of patients without recurrence (P = 0.0001). At least 1 of the 8 genes was found hypermethylated in all samples. Concomitant hypermethylation of several genes was frequently found. However, CIN relapse was only seen in the cases with hypermethylation of 3 or more of the 8 genes studied (P = 0.0039).
Conclusion
We suggest that aberrant promoter methylation may play a role and may serve as a useful biomarker in the recurrence of CIN.
Zur HausenH., MeinhofW., ScheiberW., BornkammG.W.: Attempts to detect virus-specific DNA sequences in human tumors: I - Nucleic acid hybridizations with complementary RNA of human wart virus. Int J Cancer, 13: 650–656, 1974.
2.
Zur HausenH.: Papillomavirus infections: a major cause of human cancers. Biochim Biophys Acta, 1288: 55–78, 1996.
3.
HolowatyP., MillerA.B., RohanT., ToT.: Natural history of dysplasia of the uterine cervix. J Natl Cancer Inst, 91: 252–258, 1999.
4.
MunozN.: Human papillomavirus and cancer: the epidemiological evidence. J Clin Virol, 19: 1–5, 2000.
5.
SchiffmanM.H., BrintonL.A.: The epidemiology of cervical carcinogenesis. Cancer, 76: 1888–1901, 1995.
6.
NakagawaH., SuganoK., FujiT., KubushiroK., TsukazakiS.F.: Frequent detection of human papillomaviruses in cervical dysplasia by PCR single-strand DNA-conformational polymorphism analysis. Anticancer Res, 22: 1655–1660, 2002.
7.
JohnsonT.J., PliethA.D., CrissmanJ.D., SarkarF.H.: HPV detection by polymerase chain reaction (PCR) in verrucous lesions of the upper aerodigestive tract. Mod Pathol, 4: 461–465, 1991.
8.
SarkarF.H., CrissmanJ.D.: Detection of human papillomavirus DNA sequences by polymerase chain reaction. Biotechniques, 9: 180–185, 1990.
9.
BosJ.L.: Ras oncogenes in human cancer: a review. Cancer Res, 49: 4682–4689, 1989.
10.
MouronS.A., AbbaM.C., GuerciA., GomezM.A., DuloutF.N., GolijowC.D.: Association between activated K-ras and c-erbB-2 oncogenes with “high-risk” and “low-risk” human papillomavirus types in preinvasive cervical lesions. Mutat Res, 21: 127–134, 2000.
11.
GreenleeR.T., MurrayT., BoldenS., WingoP.A.: Cancer statistics, 2000. CA Cancer J Clin, 50: 7–33, 2000.
12.
KimY.I., GiulianoA., HatchK.D., SchneiderA., NourM.A., DallalG.E., SelhubJ., MasonJ.B.: Global DNA hypomethylation increases progressively in cervical dysplasia and carcinoma. Cancer, 74: 893–899, 1994.
13.
ChengP., SchmutteC., CoferK.F., FelixJ.C., YuM.C., DubeauL.: Alterations in DNA methylation are early, but not initial, events in ovarian tumorigenesis. Br J Cancer, 75: 396–402, 1997.
14.
FergusonA.T., LapidusR.G., BaylinS.B., DavidsonN.E.: Demethylation of the estrogen receptor gene in estrogen receptor-negative breast cancer cells can reactivate estrogen receptor gene expression. Cancer Res, 55: 2279–2283, 1995.
15.
NuovoG.J., PlaiaT.W., BelinskyS.A., BaylinS.B., HermanJ.G.: In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis. Proc Natl Acad Sci USA, 96: 12754–12759, 1999.
16.
KangS., KimJ., KimH.B., ShimJ.W., NamE., KimS.H., AhnH.J., ChoiY.P., DingB., SongK., ChoN.H.: Methylation of p16INK4a is a non-rare event in cervical intraepithelial neoplasia. Diagn Mol Pathol, 15: 74–82, 2006.
17.
VirmaniA.K., MullerC., RathiA., Zoechbauer-MullerS., MathisM., GazdarA.F., AdiF.G.: Aberrant methylation during cervical carcinogenesis. Clin Cancer Res, 7: 584–589, 2001.
18.
FengQ., BalasubramanianA., HawesS.E., ToureP., SowP.S., DemA., DembeleB., CritchlowC.W., XiL., LuH., McIntoshM.W., YoungA.M., KiviatN.B.: Detection of hypermethylated genes in women with and without cervical neoplasia. J Natl Cancer Inst, 97: 273–282, 2005.
FongK.M., ZimmermanP.V., SmithP.J.: Correlation of loss of heterozygosity at 11p with tumour progression and survival in non-small cell lung cancer. Genes Chromosomes Cancer, 10: 183–189, 1994.
21.
MatlashewskiG.: The cell biology of human papillomavirus transformed cells. Anticancer Res, 9: 1447–1456, 1989.
22.
BarbosaM., SchlegelR.: The E6 and E7 genes of HPV-18 are sufficient for inducing two-stage in vitro transformation of human keratinocytes. Oncogene, 4: 1529–1532, 1989.
23.
de Roda HusmanA.M., WalboomersJ.M., van de BruleA.J., MeijerC.J., SnijdersP.J.: The use of general primers GP5 and GP6 elongated at their 3'ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J Gen Virol, 76: 1057–1062, 1995.
24.
SanguinettiC.J., Dias NetoE., SimpsonA.J.G.: Rapid silver staining and recovery of PCR products separated on polyacrylamide gels. Biotechniques, 17: 914–921, 1994.
25.
HermanJ.G., GraffJ.R., MyohanenS., NelkinB.D., BaylinS.B.: Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA, 93: 9821–9826, 1996.
26.
RemminkA.J., WalboomersJ.M., HelmerhorstT.J., VoorhorstF.J., RozendaalL., RisseE.K., MeijerC.J., KenemansP.: The presence of persistent high-risk HPV genotypes in dysplastic cervical lesions is associated with progressive disease: natural history up to 36 months. Int J Cancer, 61: 306–311, 1995.
27.
NoronhaV., MelloW., VillaL., BritoA., MacedoR., BisiF., MotaR., SassamotoK., MonteiroT., LinharesA.: Papilomavírus humano associado a lesões de cérvix. Rev Soc Bras Med Trop, 32: 235–240, 1999.
28.
KonnoR., PaezC., SatoS., YajimaA., FukaoA.: HPV histologic grade and age risk factors for the progression of cervical intraepithelial neoplasia. J Repr Med, 43: 561–566, 1998.
29.
CavalcantiS.M., FrugulhettiI.C., PassosM.R., FonsecaM.E., OliveiraL.H.: Prevalence of human papillomavirus DNA in female cervical lesions from Rio de Janeiro, Brazil. Mem Inst Oswaldo Cruz, 89: 575–580, 1994.
30.
ZehbeI., RylanderE., EdlundK., WadellG., WilanderE.: Detection of human papillomavirus in cervical intra-epithelial neoplasia, using in situ hybridization and various polymerase chain reaction techniques. Virchows Arch: 428: 151–157, 1996.
31.
KrulE.J., Van De VijverM.J., SchuuringE., Van KantenR.W., PetersA.A., FleurenG.J.: Human papillomavirus in malignant cervical lesions in Surinam, a high-risk country, compared to the Netherlands, a low-risk country. Int J Gynecol Cancer, 9: 206–211, 1999.
32.
KurmanR.J., SchiffmanM.H., LancasterW.D., ReidR., JensonA.B., TempleG.F., LorinczA.T.: Analysis of individual human papillomavirus types in cervical neoplasia: a possible role for type 18 in rapid progression. Am J Obstet Gynecol, 159: 293–296, 1988.
33.
LoK.W., WongY.F., ChanM.K., LiJ.C., PoonJ.S., WangV.W., ZhuS.N., ZhangT.M., HeZ.G., WuQ.I., LiG.D., TamJ.S., KahnT., LamP., CheungT.H., ChungT.K.: Prevalence of human papillomavirus in cervical cancer: a multicenter study in China. Int J Cancer, 100: 327–331, 2002.
34.
WidschwendterA., GattringerC., IvarssonL., FieglH., SchneitterA., RamoniA., MüllerH.M., WiedemairA., JerabekS., Müller-HolznerE., GoebelG., MarthC., WidschwendterM.: Analysis of aberrant DNA methylation and human papillomavirus DNA in cervicovaginal specimens to detect invasive cervical cancer and its precursors. Clin Cancer Res, 10: 3396–3400, 2004.
35.
BaylinS.B., HermanJ.G.: DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet, 16: 168–174, 2000.
36.
LinZ., GaoM., ZhangX., KimY.S., LeeE.S., KimH.K., KimI.: The hypermethylation and protein expression of p16 INK4A and DNA repair gene O6 methylguanine-DNA methyltransferase in various uterine cervical lesions. J Cancer Res Clin Oncol, 131: 364–370, 2005.
37.
NarayanG., Arias-PulidoH., KoulS., VargasH., ZhangF.F., VillellaJ., SchneiderA., TerryM.B., MansukhaniM., MurtyV.V.: Frequent promotor methylation of CDH1, DAPK, RAR B, and HIC1 genes in carcinoma of cervix uteri: its relationship to clinical outcome. Mol Cancer, 2: 24, 2003.
38.
CheungT.H., LoK.W., YimS.F., ChanL.K., HeungM.S., ChanC.S., CheungA.Y., ChungT.K., WongY.F.: Epigenetic and genetic alternation of PTEN in cervical neoplasm. Gynecol Oncol, 93: 621–627, 2004.
39.
MurtaE.F.C., ResendeA.V., SouzaM.A.H., AdadS.J., SalumR.: Importance of surgical margins in conization for cervical intraepithelial neoplasia grade III. Arch Gynecol Obstet, 263: 42–44, 1999.
40.
MalufP.J., AdadS.J., MurtaE.F.C.: Outcome after conization for cervical intraepithelial neoplasia grade III: relation with surgical margins, extension to the crypts and mitoses. Tumori, 90: 473–477, 2004.
41.
MurtaE.F.C., ContiR., RodovalhoJ., BarcelosA.C.M., AdadS.J., de SouzaM.A.H.: Outcome after treatment of high-grade squamous intraepithelial lesions: relation between colposcopically directed biopsy, conization and cervical loop excision. Eur J Gynaecol Oncol, 25: 587–590, 2004.
42.
MurtaE.F.C., Neves JúniorM.A., SempionatoL.R., CostaM.C., MalufP.J.: Vaginal intraepithelial neoplasia: clinical-therapeutic analysis of 33 cases. Arch Gynec Obstet, 272: 261–264, 2005.
