Clinical Value Of HPV-DNA Testing Combined With TCT In Cervical Cancer Screening

PartⅠClinical Application of HC2-HPV Combined with TCT in Cervical Cancer ScreeningObjectiveTo investigate clinical significance of human papillomavirus DNA testing and Thinprep liquid-based cytology in diagnosis and post-treatment follow-up of early cervical cancer and precancer lesion.Methods1. Cervical exfoliated cellular specimens of 740 patients were archived from cervical disease diagnosis and treatment center of the Department of Gynecology at the First Affiliated Hospital of Anhui Medical University from April 2008 to February 2009. All cytological specimens exfoliated from cervix (collected by clinician) were prepared using Thinprep liquid-based cytology test (TCT) system. The Hybrid Capture II assay detected 13 types of high-risk HPV DNA including HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. Colposcopically directed biopsy or endocervical curettage (ECC) were performed in the patients who had TCT≥ASCUS (atypical squamous cells of undetetemined significance) with positive results of high-risk HPV DNA or HPV-negative with TCT≥low grade squamous intraepithelial lesion (LSIL), then all biopsy specimen were examined by pathology. Patients were divided into normal control group, CIN1, CIN2, CIN3 and cervical carcinoma according to the final pathological results. Correlation of HPV infection and cervical cancer screening and cervical disease progression were investigated by the comparative analysis between HPV infection rates and HPV load of the different degree of cervical disease.2. Loop electrosurgical excision procedure (LEEP) was performed in 72 cases of positive HR-HPV result with different degree of CIN. HPV DNA testing was taken pre-treatment and post-treatment respectively, then to compare the variation of HPV load before and after treatment.Result1. The pathology results were used as gold standards, the sensitivity,specificity,positive predictive value,negative predictive value of TCT alone for≥CIN2 were 76.3%(408/536),62.8%(128/204),84.3%(408/484),50.0%(128/256), respectively; 89.3%(266/298),50.7%(224/442),54.9%(266/484),87.5%(224/256) for the HC2 test alone; 97.4%,81.6%,90.6%,94.5% for HC2-TCT combination。2. HR-HPV positive rates in normal cytology,ASC/AGC,ASC-H,LSIL and HSIL of 740 cases were 37.3%﹙76/204﹚,68.0%﹙174/256﹚,92.3%﹙24/26﹚,73.8%﹙90/122﹚and 90.9%﹙120/132﹚, respectively. HR-HPV positive rates were increased gradually along with the increase of cervical lesion. To compare two groups randomly, the difference of HR-HPV positive rate between normal cytology and ASC/AGC,ASC-H,LSIL,HSIL was statistically significant﹙χ2=21.583, P<0.001;χ2=14.221, P<0.001;χ2=20.366, P<0.001;χ2=47.461, P<0.001﹚. The difference of HR-HPV positive rate between ASC/AGC and LSIL was not as statistically significant as that between ASC/AGC and ASC-H﹙χ2=0.660, P>0.05;χ2=3.343, P>0.05﹚, The difference of HR-HPV positive rate between ASC/AGC and HSIL was statistically significant﹙χ2=12.484, P<0.001﹚. The difference of HR-HPV positive rate between LSIL and ASC-H was not statistically significant﹙χ2=2.081, P>0.05﹚, The difference of HR-HPV positive rate between LSIL and HSIL was statistically significant﹙χ2=6.502, P<0.05﹚. The difference of HR-HPV positive rate between HSIL and ASC-H was not statistically significant﹙χ2=0.026, P>0.05﹚.3. 740 patients were divided into normal control group, CIN1, CIN2, CIN3( cervical carcinoma in situ) and invasive carcinoma of cervix according to the final pathological results, then to study correlation between them and HR-HPV infection rate. HR-HPV positive rate in chronic cervicitis, CIN1, CIN2, CIN3 and invasive carcinoma of cervix was 37.7%﹙116/308﹚,76.1%﹙102/134﹚,85.9%﹙110/128﹚,91.8%﹙134/146﹚,91.7%﹙22/24﹚, respectively. HR-HPV positive rates were increased gradually along with the increase of cervical lesion. To compare two groups randomly, the difference of HR-HPV positive rate between chronic cervicitis and CIN1, CIN2, CIN3 and cervical cancer was statistically significant﹙χ2=27.625, P<0.001;χ2=42.202, P<0.001;χ2=58.621, P<0.001;χ2=13.367, P<0.001﹚. The difference of HR-HPV positive rate between CIN1 and CIN2 was as statistically significant as that between CIN1 and CIN3﹙χ2=4.056, P<0.001;χ2=8.320, P<0.001﹚, the difference of HR-HPV positive rate between CIN1 and cervical cancer was not statistically significant﹙χ2=2.147, P> 0.05﹚. The difference of HR-HPV positive rate between CIN2 and CIN3 was not as statistically significant as that between CIN2 and cervical cancer﹙χ2=0.687, P>0.05;χ2=0.350, P>0.05﹚. The difference of HR-HPV positive rate between CIN3 and cervical cancer was not statistically significant﹙χ2=0.032,P>0.05﹚.4. HPV-DNA levels (mean±SD) were 156.87±30.97 in the chronic cervicitis, 262.45±56.72 in the CIN1, 422.40±75.43 in the CIN2, 390.65±60.02 in the CIN3, 389.98±44.75 in the cervical carcinoma. HR-HPV DNA load levels, measured by RLU ratio detected by the Hybrid Capture II, correlated significantly with the severity of CIN although the HPV DNA load was lower in the CIN3 group than in the CIN2 groups. To compare two groups randomly, the difference of HR-HPV DNA levels between CIN2～3 and chronic cervicitis was as statistically significant as that cervical carcinoma and chronic cervicitis(P<0.05﹚. The difference of HR-HPV DNA levels between CIN1 and chronic cervicitis was not statistically significant( P>0.05). The difference of HR-HPV DNA levels between CIN and cervical carcinoma was not statistically significant( P>0.05).5. We analysised the 72 patients with CIN who underwent the treatment of LEEP, including 16 cases of CIN1, 28 cases of CIN2, 28 cases of CIN3. Using commercially available Hybrid CaptureⅡ( HC2) system , we detected the loads of human papillomavirus among all the specimens pre-treatment and post-treatment respectively. The negative ratio of HPV after LEEP were 87.5%(14/16),85.7%(24/28),64.29%(18/28), respectively. The total negative ratio of HPV after LEEP were 77.8%(56/72). The mean virus load of HPV before and after treatment was 314.56±298.68 and 34.19±117.56, the mean virus load of HPV of CIN1 before and after treatment was 165.91±133.72 and 19.41±54.02, the mean virus load of HPV of CIN2 before and after treatment was 255.91±267.07 and 10.82±38.77, the mean virus load of HPV of CIN3 before and after treatment was 314.56±298.68 and 65.99±179.78. There was remarkable variation of HPV mean load between pre-treatment and post-treatment for the patients with CIN (P < 0.01).Conclusion1. The sensitivity,specificity,positive predictive value and negative predictive value were significantly increased when HC2-HPV combined with TCT.2. HR-HPV infection rates were increased with the increase of cervical disease severity.3. HPV-DNA load is gradually increased with the severity of the cervical disease, it can be as an indicator of severity and progression of cervical disease.4. HPV-DNA load of the patients with CIN decreased significantly post-treatment, HPV-DNA load variation of patients with CIN before and after the treatment can be as an indicator of therapeutic efficacy and follow-up. PartⅡHPV Genotyping in Cervical Cancer ScreeningObjectiveTo investigate the clinical value of one new HPV test method-gene chips technology in cervical cancer screening and comprehend the status and infection of HPV genotypes and provide evidence for the diagnosis and treatment of cervical lesion.MethodsResidual cell suspensions of ThinPrep samples of 43 patients with normal cytology and 72 patients with abnormal cytology were analyzed. HPV genotype analysis was performed through gene chip technique, which could detect 23 genotypes of HPV. The genotyping result was compared with HC2.Result1. HPV positive rates of normal cytology, ASC/AGC, LSIL and HSIL of residual cell suspensions of 115 ThinPrep samples were 19.05%(8/42),75%(45/60),77.78%(7/9),100%(4/4), respectively. According to pathology, the HPV inflection rates of normal cervical histology,CIN1,CIN2,CIN3 and cervical cancer were 40.74%(33/81),82.35%(14/17),100%(9/9),100%(3/3),100%(3/3), respectively.2. The total infection rate of HPV was 55.65%(64/115)in residual cell suspensions of 115 ThinPrep specimens. 38.26%(44/115)of cervical cases were detected with single HPV genotype infection, 9.57%(11/115)of cervical cases were detected with double HPV genotype infection, 7.05%(9/115)of cervical cases were detected with muitiple HPV genotype infection. The most common HPV genotype found in 115 cases of cervical specimen was HPV16 (43.8%), followed by HPV18 (18.8%), 58 (17.2%), 31 (14.1%), 52 (10.9%).3. Of 115 samples, 53 samples were positive with both tests, 46 samples were negative with both tests,the overall agreement is 86.1%(Kappa=0.721). For the discordant samples, 5 samples including 3 cases of ASCUS, 2 cases of LSIL were positive by HC2 and negative by genotyping, final pathological result shows 3 cases of chronic cevicitis and 2 cases of CIN1; 11 samples including 6 cases of normal cytology, 3 cases of ASCUS, 2 cases of LSIL were negative by HC2 and positive by genotyping, final pathological result were chronic cevicitis totally.Conclusion1. The date from HPV-DNA genotyping and HC2 were consistent.2. HPV-DNA genotyping could be used as a reliable screening tool in a clinical setting, to understand HPV type, multiple type infection and repeated infection.