| Objective1.To explore the deficiencies of the cervical lesions screening schemes currently available by evaluating the performance of Thinprep Cytology Test(TCT),HR-HPVDNA genotyping test,TCT combined HR-HPVDNA genotyping test screening schemes.2.To offer underlying new methods for cervical lesions screening in different regions and populations by exploring the values of assaying HPVE6/E7,signal transcription and activation factor3(STAT3),Survivin protein and m RNA in cervical canal secretions and exfoliated cells.Methods? 192 women with abnormal TCT results and/or HR-HPV infection were screened to indentify possible cervical lesions in cases from October 2015 to September 2016 in the gynecological clinic of Changhai Hospital of Shanghai and Changshu Second People's Hospital.Dignoses were confirmed by histopathology.Pathological diagnosis with a HSILs+ or greater was positive,low grade squamous intraepithelial lesions(LSILs+)or lower was negative.Analysed the situation of HR-HPV infection and the prevalence of HSILs+.Evaluated performance of TCT,HR-HPVDNA genotyping test,TCT combined HR-HPVDNA genotyping test schemes for cervical lesions screening and the performance of HR-HPVDNA genotyping test for ASCUS triage.? Detect and qualify HPVE6/E7,STAT3 and Survivin proteins and m RNAs by Enzyme-linked immunosorbent assay(ELISA)and Fluorescence in situ hybridization(FISH)methods in cervical canal secretions and exfoliated cells.Compare the performance of separate and combined screening schemes in these two methods with that of TCT,HR-HPVDNA genotyping test,TCT combined HR-HPVDNA genotyping test schemes respectively.To explore the schemes with the better performance to detect HSILs+,and are in line with the requirements of health economics,and are applicable to a diverse group of people in different regions.Results?(1)The overall HR-HPV infection rate was 85.71%(132/154).The three most common HR-HPV subtypes were HPV16(39.39%),HPV58(18.18%),HPV52 /33(13.64%).For women suffered HSILs +,the HR-HPV infection rate was 97.37%(74/76)and the top three HR-HPV subtypes were HPV16(60.53%),HPV58/68(13.16%),HPV33(10.53%).For 16 women suffered cervical squamous cell carcinoma(SCC),HPV16 single infection was 6,HPV58 / 68 and HPV16 /68 mixed infection was 4 and 2 respectively.HPV68 positive women were a total of 10,they were all suffered HSILs +,of which 6 suffered SCC.Women under 30 had higher HR-HPV infection rate than that of elder 30(100.00%/84.85%).Whereas,the latter had higher abnormal TCT(78.75%/61.54%)and HSILs+ rates(52.41%/38.46%).For HR-HPV multiple infection women,whose HSILs+ confirmation and HPVE6/E7 m RNA positive rate(61.90%/ 60.00%)were higher than that of single infection(55.55%/37.50%).(2)The higher sensitivity(Se)(91.75%/97.37%),the lower specificity(Sp)(40.45%/25.64%)and positive predictive value(PPV)(62.68%/56.06%),the higher the false positive rate(FPR)in TCT and HR-HPVDNA genotyping test screening alone when ASCUS and any HR-HPV positive were taken as cutoff value.On the contrary,the higher Sp(67.42%/80.00%)and PPV(72.64%/84.62%),the lower Se(79.38%/63.16%),the higher false negative rate(FNR)when LSIL and HPV16/18 positive were taken as cutoff value.(3)The Sp(64.86%)and PPV(72.92%)of TCT combined HR-HPVDNA genotyping test improved than TCT and HPVDNA genotyping test screening alone(40.45%/25.64%,62.68%/56.06%).Compared to TCT and HPVDNA genotyping test,the FPR and colposcopy referral rate declined 24.31%/10.06% and 39.22%/16.86% respectively.?(1)The ROC area under the curve of STAT3 and Survivin proteins(0.870/0.758)confirmed using ELISA assay was higher than that of TCT and HR-HPV(0.641/0.565).STAT3,Survivin+STAT3,STAT3+HR-HPVDNA,STAT3+TCT+HR-HPVDNA schemes were the best separate and combined screening schemes after comparing screening performance.(2)STAT3 assay had perfect Sp(73.00%/75.00%)and PPV(75.89%/62.50%)for HSILs+ screening and ASCUS triage.The FPR and colposcopy referral rate decreased 32.55%/47.36% and 13.21%/19.83% respectively for screening compared with TCT and HR-HPV.The FPR and colposcopy referral rate declined 33.33% and 16.35% for ASCUS triage compared with HR-HPV.(3)STAT3+HR-HPVDNA and STAT3+TCT+HR-HPVDNA schemes had better Sp(73.75%/ 76.71%/ 64.86%)and PPV(77.17%/80.68%/72.92%),the FPR and colposcopy referral rate decreased 8.89%/11.85% and 4.25%/7.76% respectively compared with TCT+HR-HPV.Whereas,the Se(95.95%/94.61%)and Sp(73.75%/76.71%)of the two fomer schemes closed to the STAT3(92.40%/73.00%).Beyond that,which increased the cost of HR-HPVDNA genotyping test and TCT+HRHPVDNA genotyping test,did not meet the requirements of hygienic economics,could not be used as favourable combined projects for cervical lesions screening.(4)In contrast to TCT+HR-HPV,Survivin+STAT3 scheme had perfect Sp(83.76%)and PPV(84%),the FPR and colposcopy referral rate dropped 18.81% and 11.08% respectively,and had the advantages of simplicity,convenience,easier popularization and promotion,which could be used as the best combined project of ELISA method.But lower Se(66.67%),higher FNR(33.33%)made it could not be used as a perfect project for ASCUS triage.(5)No difference was observed in the Se and Sp of STAT3 and Survivin+STAT3 cases(?2=0.51,3.21,P>0.05).But the combined scheme had perfect PPV(84.00%)and Sp(83.76%),lower FPR and colposcopy referral rate,could be proper for cervical lesions diagnosis,the separate one had favourable Se(92.40%)and NPV(91.25%),lower FNR,could be propitious for cervical lesions screening.(6)After repeated experiments,HPVE6 and E7 protein were not confirmed in cervical secretions.?(1)The ROC area under the curve(0.909/0.868/0.803)confirmed using FISH assay of HPVE6/E7,STAT3 and Survivin m RNAs for screening HSILs+ was significantly higher than TCT(0.641)and HR-HPV(0.565).HPVE6/E7 m RNA,STAT3 m RNA,HPVE6/E7 m RNA+STAT3m RNA,STAT3 m RNA+HR-HPVDNA and Survivinm RNA+ STAT3 m RNA+HPVE6/E7 m RNA assaies were the best separate and combined assaies by comparing screening performance.(2)HPVE6/E7 m RNA and STAT3 m RNA and STAT3 m RNA+HR-HPVDNA cases had perfect Sp(81.63%/81.63%/87.50%/79.17%/ 82.05%/83.3%)and PPV(82.69%/82.35%/84.09%/78.57%/68.75%/71.43%)for HSILs+ screening and ASCUS triage.The FPR reduced 41.48%/55.99%/17.19% and the colposcopy referral rate decreased about 20.00%/more than 25.00%/11.17% compared with TCT and HR-HPV and TCT+HR-HPV schemes for screening.The FPR and colposcopy referral rate reduced 45.83%/37.50%/41.66% and 32.42%/22.60%/25.28% respectively compared with HR-HPV For ASCUS triage.No difference was observed in the Se and Sp of the three projects for detecting HSILs+ both in cervical lesions screening and ASCUS triage(?2=4.03,1.05/0.07,0.06,P>0.05 respectively),but the combined project had better Se(97.37%)and NPV(96.97%),lower FNR,could provide more security screening interval for women with negative results.(3)Both HPVE6/E7 m RNA+STAT3m RNA and Survivinm RNA+STAT3m RNA+HPVE6/E7 m RNA projects had perfect Sp(95.92%/ 94.00%)and PPV(95.40%/93.26%)for screening HSILs+,the FPR and colposcopy referral rate decreased 31.06%/29.14% and 22.48%/20.34% respectively compared with TCT+HR-HPV project,which could be suitable for perfect combined project for cervical lesions diagnosis.But no difference was observed in the Se and Sp of this two combined project for HSILs+ diagnosis(?2=0.18,0.09,P>0.50).In addition,they had equal YI(0.84)and concordance rate(92.19%),the PPV of double combined project was slightly higher than the triple one(95.40%/93.26%).Nevertheless,the triple project had the disadvantages of complex operation,having many intermediate links and factors and high economic costs,which would not be used as a perfect diagnostic project.?(1)No difference was observed in the Se and Sp of ELISA method STAT3 scheme as compared to that of FISH method HPVE6/E7 m RNA,STAT3 m RNA,STAT3 m RNA+ HR-HPVDNA schemes for HSILs+ screening(?2=4.03,3.44,P>0.10)and ASCUS triage(?2=0.76,1.37,P>0.25).But the Sp of FISH method screening schemes was slightly higher than that of ELISA method STAT3 scheme.ELISA method STAT3 scheme had moderate consistency with FISH method three screening schemes(?=0.52,0.53,0.46).(2)Although no significant difference was observed in the Se of ELISA method Survivin+STAT3 scheme as compared to that of FISH method HPVE6/E7 m RNA+ STAT3 m RNA scheme(?2=0.21,P>0.50),but the Sp was significantly lower(?2=8.02,P<0.005)for HSILs+ diagnosis.They had moderate consistency for cervical lesions diagnosis(?=0.59).ConclusionsTo sum up,we draw the conclusions as following.?(1)The occurrence of HSILs and SCC was closely related to HPV16,58 infection,as well as might be closely related to HPV68 infection.Women under 30 had higher HPV infection rate,lower HSILs+ incidence.In contrast,Women elder 30 had lower HPV infection rate,higher HSILs+ occurence.HR-HPV multiple infection might increase the carcinogenicity of HPV.(2)It is difficult to balance the FNR and FPR of TCT and HRHPVDNA genotyping test.The efficiency of TCT combined HR-HPVDNA genotyping test improved than TCT and HR-HPVDNA genotyping test alone for screening HSILs+.?(1)ELISA method had the advantages of fast,sensitive,simple,cheap and so on,which would be popularized in the primary hospitals of underdeveloped regions because of the experimental technicians without special training.The expressin of STAT3 and Survivin protein confirmed using ELISA assay is expected to be a new method and molecular markers for cervical lesions screening in underdeveloped regions.(2)STAT3 assay could be new approcah for cervical lesions screening and ASCUS triage in underdeveloped regions.(3)Survivin+STAT3 assay could be new approach for cervical lesions diagnosis in underdeveloped regions.?(1)FISH technology had the advantages of gene-specific fluorescent probe stable,high location accuracy,short experimental period,good sensitivity and specificity and so on.But higher test technicians and equipment requirements and higher cost made it was suitable for popularization in developed regions.The expression of HPVE6/E7 and STAT3 m RNA confirmed using FISH assay is expected to be a new method and molecular markers for cervical lesions screening in developed regions.(2)HPVE6/E7 m RNA,STAT3 m RNA and STAT3 m RNA+HR-HPVDNA assaies could be new approaches for cervical cancer screening and ASCUS triage in developed regions,and the performance of combined screening program was better than that of a separate one.(3)HPVE6/E7 m RNA +STAT3m RNA regimen is expected to be a diagnostic strategy for cervical lesions in developed regions. |
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