Clinical Performance Of HPV Assays Targeting On E6/E7 Region And Extended HPV Genotyping On Precise Cervical Cancer Screening And Triage

ObjectivesThis study aimed to explore the correlation among HPV E6/E7 DNA,mRNA and E6 oncoprotein,moreover,to evaluate their clinical performance on cervical cancer screening and triage;to explore superior screening and triage strategies using extended HPV genotyping and p16/Ki-67,and provide scientific evidence of their implementation in precise cervical screening and triage in China.Materials and MethodsWe launched a multicenter study by case-enriched study design and recruited 1967 women who participated in cervical cancer screening or attended gynecologic clinic,from April 2014 to March 2015.The cervical samples were obtained using a cytobrush and kept in PreservCyt solution(Hologic Inc.,Bedford,MA),stored at 4℃ until transported to central lab for HPV DNA test(cobas4800 HPV test),Liquid-based cytology(LBC)detection,OncoE6 test and p16/Ki-67 dual stain detection.Women from screening site with positive results for any tests were called back for colposcopy and biopsy.Cervical samples of women who attended gynecologic clinic were collected before treatment.The residue samples were stored at ultralow temperature refrigerator(-78 ℃)in central lab of NCC(National Cancer Center/Cancer Hospital,Chinese Academy of Medical Sciences)until tested for extended HPV genotyping(BD Onclarity HPV assay)and HPV mRNA assays(APTIMA and APTMA-GT).Masked testing with HPV DNA and HPV mRNA was performed by technicians in NCC,from July 2016 to September 2017.Results1.The clinical performance of HPV DNA,mRNA and E6 oncoprotein in cervical cancer screening:HPV DNA test and the HPV mRNA test were in high agreement.The agreements between HPV nucleic acid tests and E6 oncoprotein were poor,but they were improved with the pathological grade increased.There was a positive correlation between E6 oncoprotein expression and HPV DNA viral load increasing(P=0.000).And no correlation was observed between E6 oncoprotein expression and the strength of mRNA(P=0.940).Pathological grade increased as the improvement of HPV DNA viral load and E6 oncoprotein expression.For detection of CIN2/3+,HPV DNA assay and HPV mRNA assay provied higher sensitivity,and E6 oncoprotein test provided higher specificity.However,for detection of cervical cancer,E6 oncoprotein provided similar sensitivity and better specificity in comparison with HPV DNA/mRNA assays.2.Clinical performance of extended HPV genotyping combined with p16/Ki-67 for detecting cervical cancer and precancer in HPV positive women:HPV genotypes were divided into 4 strata according to the association between each HPV type channel and CIN3+.Dual staining positive rates increased with increasing risk of HPV strata from 64.4%in HPV51/39/68/35-positive women to 87.8%in HPV16/18-positive women(Ptrend=0.000).Increasing trend of pl6/Ki-67 positivity with the raising grade of histology was observed,and the corresponding positive rate of p16/Ki-67 was 18.6%(normal),53.2%(CIN1),69.8%(CIN2),87.6%(CIN3)and 86.4%(cancer)for each histology category,respectively(Ptrend=0.00).For 8-type HPV-positive women(HPV31/33/58/52/45/59/56/66),pl6/Ki-67 was an effective triage tool.Triage by p16/Ki-67 for 8-type positive-women combined with HPV16/18 genotyping proved to be better than by pl6/Ki-67 for HPV positive-women,furthermore,the former presented fewer referral(P<0.0167 for all)and comparable clinical performance to p16/Ki-67 triage of 12-type HPV with HPV16/18 genotyping.3.Evaluation of extended HPV testing for detecting cervical cancer and precancer in ASC-US and LSIL women:Comparing with ASC-US and HPV negative patients,women with ASC-US and HPV 16、45、18、52、33/58、31、39/68/35 positive gained higher risk for CIN2+.The similar results were observed in LSIL HPV 31、16、39/68/35、45、33/58、51、18 positive women.Triage of ASC-US women with HPV 16/45/18/52/33/58/31/39/68/35 positive provided better clinical performance for detecting CIN2+.Triage of LSIL women with HPV 16/31/39/68/35/45/33/58/51 positive provided better clinical performance for detecting CIN2+.Conclusions1.HPV DNA and mRNA testing,with similar clinical performace,both can service as primary screening tools.E6 oncoprotein testing was a potential useful triage method for primary HPV positive women,and it also could accuratly identify cervical cancer women.2.p16/Ki-67 dual stain cytology was effective to stratify risk of CIN2/3+ among HPV31/33/58/52-and HPV45/59/56/66-positive women.Moreover,the combination of HPV16/18 genotyping with p16/Ki-67 triage of 8-type HPV(HPV31/33/58/52/45/59/56/66)could provide reliable clinical performance,with decreasing referral rate,for primary HPV-positive women.3.Cytological ASCUS linked HPV 16/18/31/33/58/45/52/35/39/68 positive women should immediately refer to colposcopy,which can provide better clinical performance.Cytological LSIL women with HPV16/31/39/68/35/33/58/51/18 positive require immediate colposcopy.