Expression Of HPV16/18 E6, P16 And Ki-67 In Cervical Lesions And Their Clinical Diversion Value Of CIN2

Background and ObjectiveCervical cancer is a common and important public health problem for adult women in developing countries.High-risk human papillomavirus(HPV16 and HPV18)is the most important risk factor for cervical cancer.Histology is considered the gold standard for the pathological progression of cervical cancer development.Accurate colposcopy and histological evaluation of cervical precursor lesions are critical to determining clinical management.Due to the high conversion rate of cervical intraepithelial neoplasia grade 2(CIN2),it is unclear which lesion will eventually develop into invasive cancer,and there are differences in the management of CIN2 about its diagnosis and treatment.Because the pathologist's diagnosis is subjective and based on personal experience,the use of H&E staining alone has limitations that make it impossible to visually judge the extent of cervical lesions.For the clinical pathology of this part of the tissue,immunohistochemical staining(IHC)or biomarker classification system can improve the accuracy and reproducibility of CIN classification,thereby making the diagnosis more standardized.The cell proliferating nuclear antigen Ki-67 and the multi-tumor suppressor gene p16 are widely used to guide pathologists in classifying CIN.However,the specificity of the markers p16 and Ki-67 is low enough to assist in providing a clear pathological diagnosis.Etiology studies suggest that high-risk HPV E6 protein is associated with epithelial transformation,which plays an important role in the development of the virus from a transient infection to a persistent infection,which is a necessary factor in the development of cervical precancerous lesions and cancer.The detection of persistent infections with transformative potential from transient infections makes the detection of high-risk HPV E6 proteins extremely meaningful.However,the use of HPV16/18 E6 as a reference for CIN2 shunting requires further comparative analysis of its expression characteristics in cervical lesions.In this study,CIN2+ was used as the end point of the disease to elucidate the stage of CIN.HPV16/18 E6?p16 and Ki-67 were performed on formalin-fixed paraffin-embedded cervical tissue sections using the avidin-biotin peroxidase method.Observe their expression characteristics in the process of cervical lesions,and explore their clinical diversion value of CIN2.Materials and Methods1.MaterialsThis retrospective study selected patients from the Second Affiliated Hospital of Zhengzhou University from September 1,2016 to August 31,2018,who was with HPV 16 and/or HPV 18 positive(no other types of HPV infection)through HPV-DNA typing text in cervical exfoliated cells,and further be diagnosed as cervicitis,cervical intraepithelial neoplasia or cervical squamous cell carcinoma by cervical biopsy under colposcopy.There were 190 cases.Patients with the following criteria were excluded from the study:(i)age <20 years;(ii)negative for HPV 16/18;(iii)no pathological diagnosis of cervical tissue;(iv)uterine or cervical resection;(v)The case date is incomplete.This study has been reviewed by the Ethics Committee of the Second Affiliated Hospital of Zhengzhou University.2.MethodsAll cervical lesion tissue wax blocks were serially sliced and distributed onto charged slides in the order of cutting.The first and last sections were stained with hematoxylin and eosin(H&E)to ensure that the pathological specimens taken in the middle were of the same grade of cervical lesions,and the middle part were subjected to immunohistochemical staining with the anti-HPV16/18 E6 antibody,the Ki-67 antigen(DAKO,clone MIB-1)and the mouse monoclonal antibody of p16 antigen.High-risk HPV-DNA in the tissue was detected by PCR to identify the type of HPV virus infected in the tissue.The following studies were performed separately:(1)H&E staining;(2)PCR;(3)HPV16/18 E6,p16,Ki-67 IHC;(4)HPV DNA ISH;(5)negative control;(6)H&E staining.The results of staining results were: HPV E6,p16 IHC positive results interpretation: brown nucleus staining or cytoplasmic staining positive,and vice versa.According to the literature,the percentage of positive cells and the immunohistochemical scores(IHS)were: IHS = a × b.a represents the percentage of positive cells: a = 0,no positive cells;a = 1,positive cells are 1% to 10%;a = 2,positive cells are 11% to 50%;a = 3,positive cells are 51% ~ 80%;a = 4,more than 81% of positive cells.b represents the positive staining of positive cells: b = 0,negative;b = 1,weakly positive;b = 2,moderately positive;b = 3,strong positive.IHS 0 is divided into(-);IHS 1 ~ 4 is divided into(+);IHS 5 ~ 8 is divided into(+ +);IHS 9 ~ 12 is divided into(+ + +).Each slice was rated by IHS.Ki-67 IHC positive results were interpreted as positive for brown particles in the nucleus.Divided into 4 groups: 0(all cells negative),+(6-25% positive staining in cells),+ +(26-50% positive staining in cells)or + + +(positive staining more than 50% of cells).The results of HPV-DNA in situ hybridization were positive: the brown granules were positive in the nucleus.Results1.PCR detection of HPV types of infection in cervical lesions were 16 and/or 18 infections,the same as HPV infection in cervical exfoliated cells.2.Expression characteristics of HPV16/18 E6,p16 and Ki-67 proteins in cervical precancerous lesions:2.1 The expression of E6 protein in cervicitis and CIN1 was localized in the nucleus and cytoplasm of squamous epithelial cells,mainly weakly positive(+)expression,with positive rates of 4.5% and 13.6%,respectively.As the lesion progressed,the positive rate and expression intensity of E6 protein increased significantly,the positive rate of CIN2 was 53.0%,which was mainly positive(+ +)expression.The positive rate of CIN3 was 72.0%,mainly showing moderate-strong positive(+ + ~ + + +)expression;the positive rate in cervical cancer tissues was 86.7%,mainly strongly positive(+++).The positive rate of HPV16/18 E6 protein in the five groups of cervical lesions was statistically significant(P<0.001).Pairwise comparison results showed that the positive rate of E6 protein in cervicitis and CIN1 was significantly lower than that in CIN2,CIN3 and cervical cancer tissues(P<0.05).There was no significant difference in the positive rate of E6 protein between CIN2 and CIN3(P>0.05).2.2 The expression of p16 protein in cervicitis and CIN1 was localized in the cytoplasm of squamous epithelial basal cells,and the expression was weak.As the lesion progresses to the surface,the whole epithelial layer of CIN3 showed strong positive expression.Almost all cancer cells showed p16 positive staining.The positive rate of cervicitis was 4.55%;the positive rate of CIN1 was 40.9%;the positive rate of CIN2 was 71.2%,mainly positive,transition to strong positive;the expression of p16 protein in CIN3 was the strongest in cytoplasm and nucleus.The positive rate was 92.0%,mainly showing moderate-strong positive expression;the positive rate of cervical cancer reached 100.0%.There was a statistically significant difference in the positive rate of p16 between the five groups of cervical lesions(P<0.001).Pairwise comparison results showed that the positive rate of p16 in cervicitis was significantly lower than that in CIN and cervical cancer(P<0.05).2.3 Ki-67 expression in CIN1 was localized in the nucleus of basal and parabasal layers,and it was distributed in granular form.As the lesion progressed,it gradually promoted to the surface layer.CIN3 and cervical cancer showed strong positive expression in the nucleus of epithelial cells.The positive rate of cervicitis was 9.1%;the positive rate of CIN1 was 90.9%,which was mainly weakly positive;the positive rates of CIN2,CIN3 and cervical cancer were 100%,mainly showing strong positive expression.The positive rate of Ki-67 in the five groups of cervical lesions was statistically significant(P < 0.001).Pairwise comparison results showed that the positive rate of Ki-67 protein in cervicitis was significantly lower than that in CIN and cervical cancer(P<0.05).3.Correlation analysis between HPV16/18 E6,p16 and Ki-67 expression intensity: There was no significant correlation between E6 and p16 expression intensity in CIN1(P>0.05);In CIN2,with the increasing of expression intensity,the correlation between E6 and p16 protein was gradually enhanced,and the correlation coefficient was the highest when the expression was strongly positive,0.489(P<0.05).The correlation coefficient between the expression of E6 and p16 in CIN3 was lower than that of CIN2 in the positive expression and strong positive expression,which were 0.342 and 0.327,respectively(P < 0.05).The correlation between the expression intensity of E6 and p16 in CIN2 was the strongest,the strong positive expression being the most significant.There was no correlation between the expression levels of Ki-67 and E6 in cervical lesions.4.Sensitivity and specificity of E6,p16 and Ki-67 for the diagnosis of CIN1 and CIN2/3: When E6 protein is positive(1+~3+),the sensitivity of diagnosis of high-grade cervical lesions(CIN2+)is 0.2969,the specific was 0.9595 and the likelihood ratio was 7.323,which was statistically significant(P < 0.0001).When p16 was positive(1+~3+),the sensitivity of diagnosis of high-grade cervical lesions(CIN2+)was 0.3611,the specificity was 0.9118,the likelihood ratio was 4.093(P=0.003),and p16 was moderately strong.When it was moderate-strong positive expression,the sensitivity was 0.2537,the specificity was 0.9231(2+~3+),and the likelihood ratio was 3.299(P=0.005).When p16 was strongly positive(3+),the diagnosis of high grade cervical lesions(CIN2+)was no statistical significance.(P = 0.4077).When Ki-67 was expressed in medium-strong positive(2+~3+),the sensitivity of diagnosis of high-grade cervical lesions(CIN2+)was 0.7907,the specificity was 0.9145,and the likelihood ratio was 9.251,which was statistically significant(P<0.0001).).5.Expression of HPV16/18 E6 protein in lymphocytes of cervical lesions: HPV16/18 E6 oncoprotein expression in lymphocytes in adjacent normal tissues of cervical lesions.This result seems to suggest that HPV16/18 may be transmitted through the blood circulation to other organs.6.Distribution of HPV-DNA in cervical lesions: HPV-DNA in cervical lesions detected by in situ hybridization showed that it was mainly distributed in surface hollow cells of cervical epithelial tissue,not in basal and subbasal HPV-DNA was detected at the bottom layer.Conclusions1.The expressions of HPV16/18 E6,p16 and Ki-67 are related to the extent of cervical lesions.As the lesion progresses,the expression levels of the three are gradually increased.2.The correlation between the expression intensity of E6 and p16 in CIN2 was the strongest,the strong positive expression being the most significant,suggesting that there may be some intrinsic association between the two.3.Compared with p16 and Ki-67,HPV16/18 E6 has low sensitivity and high specificity for the diagnosis of high-grade cervical lesions(CIN2+).We can consider HPV E6,p16,Ki-67 as three indicators in clinical diagnosis of high-grade cervical lesions to improve diagnostic performance.