The Expression And Clinical Significance Of P16^INK4A, △NP63, HPV And Brn-3α In Atypical Squamous Cells Of Undetermined Significance (ASCUS) And The Correlation Between These Biomarkers

Objective: To analyze the clinical, histopathology and molecular biological characteristics of atypical squamous cells of undetermined significance(ASCUS), to examine the expression and clinical significance of P16^INK4A,ΔNP63, HPV, Brn-3αin ASCUS and the correlation between these biomarkers, and to investigate the value of these biomarkers in screening dysplastic epithelial cells in ASCUS and the molecular mechanism of ceivical dysplastic epithelial cells.Methods: 1. A total of 150 patients cytologically diagnosed with ASCUS were referred for colposcopy. Biopsy or endocervical curettage samples were sent for pathological examination. We collected clinical and histopathological data of these 150 patients.2. Streptavidin peroxidase (SP) immunocytochemical assay was used to determine the expression of P16^INK4A andΔNP63 in ThinPrep smears of the 150 patients who were cytologically diagnosed with ASCUS. 3. RT-PCR was used to detect the mRNA of HPV16 E7, HPV18 E6 and Brn-3a in these 150 patients.Results: 1. The clinical and histopathological features and the treatment of ASCUS:(1)A total of 150 patients cytologically diagnosed with ASCUS were referred for colposcopy. The histopathological analysis based on colposcopic biopsy and endocervical curettage showed that there were 92 cases (61.33%) of cervical inflammation, 20 cases (13.33%) of CINⅠ, 17 cases (11.33%) of CINⅡ, 21 cases (8%) of CINⅢ, 8 cases(5.33%)of condyloma acuminatum or condylomatoid diseases, and one case of cervical cancer. The coincidence rate between colposcopy and histopathology diagnosis was 68%.(2)The 92 cases pathologically diagnosed with cervical inflammation were reexamined every 4-6 months, among whom 2 cases were found to progress to CINI, 20 cases of CINⅠturned normal when reexamined 3 months after shallow cutting or conization of cervix, 30 cases of CINⅡ/CINⅢ/cervical cancer were referred for conization of cervix or hysterectomy, and TCT test performed 3 months later found that only one case of them had ASCUS who however became normal after another 3 months, Among the 8 cases of condyloma or condylomatoid diseases, 6 underwent shallow cutting or conization of cervix and then anti-virus treatment and they were found normal when re-examined by colposcopy or TCT 3-6 months later. Another 2 of the 8 cases were given anti-virus treatment directly, and the re-examiantion 3 months later showed 1case became normal and 1 case had ASCUS.2. The expression and clinical significance of P16^INK4A andΔNP63 in ASCUS: (1)The positive rate of P16^INK4A andΔNP63 in ASCUS was 37.33% and 39.33% respectively. The positive staining of P16^INK4A was detected on cytoplasm and part nucleus of the dysplastic epithelial cells of the cervix, whileΔNP63 was detected on nucleus. The positive staining and the positive rate would be raised with the severer histopathology diagnosis: the positive rate of P16^INK4A in cervical inflammation, CINⅠ, CINⅡ, CINⅢand condyloma/condylomatoid diseases was 5.43%, 80%, 88.24%, 100% and 87.5% respectively, and the positive rate ofΔNP63 was 6.52% , 85%, 91.12%, 100% and 87.5% respectively. Both P16^INK4A andΔNP63 were positive in the 1 case of cervical cancer. (2)P16^INK4A andΔNP63 ICC did best in screening dysplastic epithelial cells in ASCUS, showing 0.88 and 0.91 sensitivity, 0.95 and 0.93 specificity, 0.91and 0.89 positive predictive value (PPV) , 0.93 and 0.95 negative predictive value (NPV). If we detected both P16^INK4A andΔNP63, the SE, SP, PPV and NPV were improved to 0.97, 0.92, 0.89 and 0.98 respectively. The incidence of cervical cancer and cervical pre-malignant lesions in the cases with positive P16^INK4A andΔNP63 was 88.89%, while in negative cases the incidence was only 2.30%.3. The expression and clinical significance of HPV and Brn-3αin screening dysplastic epithelial cells in ASCUS:(1)The positive rate of HPV16 E7/HPV18 E6 and Brn-3αin ASCUS was 46.67% and 36% respectively, and the positive rate of these two biomarkers was raised with the severer histopathology level: the positive rate of HPV16 E7/HPV18 E6 and Brn-3αin cervical inflammation, CINⅠ, CINⅡ, CINⅢand condyloma/condylomatoid diseases was 25%, 75%, 88.25%, 100% and 50% respectively, and the positive rate of Brn-3αwas 14.13%, 60%, 70.59%, 91.67% and 62.5% respectively. In addition, the positive rate in each pathological level was significantly different. The positive rate was higher in cervical cancer and pre-malignant lesions than in inflammation.(2)The sensitivity, specificity, PPV and NPV of HPV16 E7/HPV18 in screening dysplastic epithelial cells were 0.81, 0.75, 0.67, 0.86 and those of Brn-3αwere 0.71, 0.86, 0.76 and 0.82, and those of both HPV16 E7/HPV18 E6 and Brn-3αwere 0.86, 0.74, 0.68 and 0.89 respectively. The incidence of cervical cancer and cervical pre-malignant lesions was 67.14% and 75.93% in ASCUS patients with positive HPV16 E7/HPV18 E6 or positive Brn-3α, while in negative cases it was only 13.75% and 17.71%4. Correlation between expression of P16^INK4A,ΔNP63, HPV16 E7/HPV18 E6 and Brn-3αin ASCUS: over-expression of P16^INK4A,ΔNP63, HPV16 E7/HPV18 E6 and Brn-3αwas detected in cervical cancer or cervical pre-malignant lesions. Expression of P16^INK4A in ASCUS was highly consistent with that ofΔNP63, and similar consistency was detected between P16^INK4A and HPV16 E7/HPV18 E6, and between HPV16 E7/HPV18 E6 and Brn-3α.5. Relationship between"the expression of P16^INK4A,ΔNP63, HPV16 E7/HPV18 E6 and Brn-3α"and"the outcome of ASCUS":Patients with ASCUS were followed up for 3-9 months after their treatment according to histopathology diagnosis. In the 92 cases with cervical inflammation, 2 patients progressed to CIN, one of whom had positive P16^INK4A,ΔNP63, HPV16 E7/HPV18 E6 and Brn-3α, and the other patient were found positive in P16^INK4A, HPV16 E7/HPV18 E6 and Brn-3α. 49 cases with CIN were given cervical conizaion or hysterectomy treatment and then followed up, and one of them who had positive expression of P16^INK4A,ΔNP63, HPV16 E7/HPV18 E6 and Brn-3αwas cytologically diagnosed with ASCUS 3 months after treatment. 8 cases of condyloma/condylomatoid diseases were re-examined 3 months after treatment, and among them, one case with positive P16^INK4A,ΔNP63, HPV and Brn-3αwas diagnosed with ASCUS, while the others became normal .Conclusions: 1. ASCUS has a high potential of high grade cervical lesions , therefore, to search the underlying cervical neoplastic lesions in ASCUS and give them normalization treatment plays a significant role in decreasing the incidence of cervical lesions.2. P16^INK4A,ΔNP63, HPV16 E7/HPV18 E6 and Brn-3αover-expressed in ASCUS patients who were histopathologically diagnosed as having cervical cancer or cervical pre-malignant lesions. These biomarkers showed high sensitivity (SE), specificity (SP), positive predictive value (PPV) and negative predictive value (NPV) in detecting dysplastic epithelial cells in ASCUS, indicating that P16^INK4A,△NP63, HPV16 E7/HPV18 E6 and Brn-3αcan increase the significance of screening program for ASCUS patients, and become specific biomarkers to identify cervical cancer and cervical pre-malignant lesions,.3. P16^INK4A,ΔNP63, HPV16E7/HPV18E6, Brn-3αand histopathological diagnosis were positively correlated in cervical cancer or cervical pre-malignant lesions. The high consistencies between P16^INK4A andΔNP63, between P16^INK4A and HPV16E7/HPV18E6, and between HPV16E7/HPV18E6 and Brn-3α, demonstrate that these biomarkers and their reciprocities were involved in the genesis and development of cervical dysplastic epithelial cells and in the development of cervical cancer or cervical pre-malignant lesions.5. HPV16E7/HPV18E6 could directly show the activation of HPV's oncogene, and its consistency with the expression of P16^INK4A and Brn-3αin ASCUS, demonstrates that P16^INK4A and Brn-3αcan indirectly reflect levels of HPV oncogene's transcription and activation. Therefore, the three biomarkers, HPV16E7/HPV18E6, P16^INK4A and Brn-3αcould be used to evaluate HPV's oncogenicity.