Analysis Of Clonality And HPV16 E6 Gene Integration And P16 Expression In The Precursors Of Cervical Squamous Cell Carcinoma

Backgound Cervical cancer is the second most common cancer among women worldwide.The incidence of cervical carcinoma has been rising in China and the occurrence age is getting younger than ever.Carcinogenesis of uterine cervix is a multistep process with multigene alteration.A well-defined precursor of the carcinogenesis is called cervical intraepithelial neoplasia which represent a spectrum of histological abnormalities ranging from CINâ… ,CINâ…¡and CINâ…¢(including carcinoma in situ).However,there are still some uncertain factors that impact on CIN developmemt.(1)The clinical outcome of CIN is various.Twenty percent of CINâ… could progress to CINâ…¡/â…¢,while about fifty to sixty percent of CINâ… could regress spontaneously.But about forty to fifty percent of CINâ…¡/â…¢could progress to invasive cancer.In cytological and histological examinations,some cases of spontaneously regression might not reliably be distinguished from the few developing to invasive carcinoma.(2)Most women might have been infected with HPV,high-risk types of which were related to cancer development,but only a few would progress to invasive disease.Therefore,there is a continuing need for more robust markers of disease progression than those provided by morphological examination,or testing for the presence of high-risk HPV types.The identification of these factors are still needed further analyszing for more complete understanding the risk factor for CIN occurrence and development so as to rank CINs to meet their biological behavior.It is significant to do this research for choosing a better therapeutic plan and increase life quality for CIN patients,avoiding overtreated and insufficient treatment.Ojective To analyze the clonality of cervical intraepithelial neoplasia and the integration status of HPV16 E6 gene in the same cases and their relationship with P16 expression,and to investigate their correlation with CIN grade and the clinical pathological significance.Method 150 samples of cervical leisions by biopsy or surgical dissection were randomly selected from the department of pathology of Shanghai Dongfang Hospital, including 33 cases of CINâ… ,38 cases of CINâ…¡,34 cases of CINâ…¢and 45 cases of squamous cell carcinoma.Another 20 case of cervical tissue from non-cervical-lesions were also selected as controller.4%neutral formalin-fixed,paraffin-embeded samples were utilized for microdissection,procuring pure matched normal and lesional cells from each case.Genomic DNA was extracted by routine methods.All the DNA samples were divided into three parts for further study.(1)Following by nested PCR for human AR gene(HUMARA)located on X chromosome,PAGE was used for clonality analysis.(2) HPV infection was studied by PCR using HPV consensus primer GP5+/GP6+.Meanwhile, HPV16 E2 and E6 gene were also amplified by multiplex PCR to detect HPV 16 infection and its physical status.ShineTech electrophoresis image analysis software was used to record the E2/E6 ratio so as to evaluate the episomal and integrated status of PV16 infection:in episomal form,both targets should be equivalent,and in integrated form,E2 gene would be absent,while in mixed form of episomal/integrated mixed form, the copy number of E2 would be less than that of E6,and in integrated form,only E6 can be amplified.(3)P16 expression was examined by immunohistochemstry(Envision)and the expressive patterns were also evaluated.(4)SAS 8.0 software was used for statistical analysis.Results 139 cases were successfully amplified and 12 cases of them showed homogenerous,and the heterogenerous rate is 91.4%.(1)Monoclonal leisons were identified in 26.9%CINâ… (7/26),51.5%(23/33)CINâ…¡,82.1%(17/28)CINâ…¢and 85.0% (34/40)cervical SCC by analyzing one single site.Monoclonal cases increased prominently from CINâ… to CINâ…¡/â…¢(x~2=16.492,P=0.0003).(2)HPV16 was identified positive in 8cases of CINâ… ,13 cases of CINâ…¡,14 case of CINâ…¢and 29cases of SCC. HPV16 E6 integration were detected in CINâ… ,CINâ…¡,CINâ…¢and cervical SCC in the rate of 25%(2/8),30.8%(5/13),74.3%(9/14)and 79.3%(23/29).This is correlated with CIN grade(x~2=6.747,P=0.0343).HPV16 E6 integration also correlated with CIN monoclonality(x~2=7.768,P=0.0053,r=0.36).(3)P16 was positive in 69.2%(18/26) CINâ… ,87.9%(29/33)CINâ…¡,92.9%(26/28)CINâ…¢and 97.5%(39/40)SCC.P16 positive cells were distributed focally in 50%C1Nâ… and parebasally in 42.5%CINâ…¡and diffusedly in 67.9%CINâ…¢.Among the four cases of HPV16 positive CINâ… P16 is focally positive with episomal virus DNA.Six cases of HPV16 positive CINâ…¡showed P16 expressed parabasal positive with episomal HPV16 DNA.P16 expression pattern is associated with HPV16 E6 integration(x~2=4.36,P=0.037,r=0.37). Conclusion(1)Some CINâ… cases and most of the CINâ…¡/â…¢cases were monoclonal with neoplastic nature.Proportion of Monoclonal cases is increased from CINâ… to CINâ…¢.The monoclonal feature of CIN is correlated with CIN grade.(2)HPV16 E6 integration was correlated with CIN grading,suggesting that HPV16 maybe activate CIN progression and cervical carcinogenesis.HPV16 integration maybe an early marker to indicate cervical carcinogenesis.(3)P16 is overexpressed in some CINs and cervical SCC, and P16 expression pattern correlated with CIN grade,which also can clue on the HPV16 physical status.Maybe they both have some role on the tumor progression,while P16 is an effective biomarker for CIN grading biologically and evaluation of carcinogenesis.