This was a retrospective study. From January 2014 to December 2014, patients who were negative for the HR-HPV subtype were recruited in this study to evaluate the value of p16INK4a immunostaining for detecting HSIL and above (HSIL+). These patients had undergone colposcopy and colposcopically-directed multiple punch cervical biopsy at our hospital. In our study, even cases with HPV neg/cytology neg underwent colposcopy because these patients had several clinical symptoms. All biopsy tissues underwent pathological examination. This study was conducted in accordance with the Declaration of Helsinki and approved by the Human Ethics Committee of Peking University First Hospital. All methods were carried out per cervical cancer screening guidelines. All eligible participants provided written, informed consent to be included in this study.
1.2 Inclusion and exclusion criteria
Inclusion criteria: (1) patients who were negative for the HR-HPV subtype; (2) older than 18 years of age; (3) patients who have signed informed consent. Exclusion criteria: (1) patients who were pregnant or nursing women; (2) patients whose data were incomplete.
1.3 Cytological detection
A liquid-based, thin-layer cytologic preparation was used, and the 2001 Bethesda System (TBS) was used for diagnosis. Tissues were evaluated as within normal limits (WNL), atypical squamous cells of undetermined significance (ASC-US), atypical squamous cells – cannot exclude high-grade squamous intraepithelial lesion (ASC-H), a low-grade squamous intraepithelial lesion (LSIL), HSIL, squamous cell carcinoma (SCC), and atypical glandular cells (AGC).
1.4 HPV detection
HPV was tested using the digene Hybrid Capture 2 (HC2) High-Risk HPV DNA Test (QIAGEN, Gaithersburg, MD, USA) with the Rapid Capture System (QIAGEN), which is based on signal amplification using RNA probes to target the entire HR-HPV genome.8 All steps were performed according to the manufacturer’s protocols. Briefly, cervical brush samples collected in preserve cytological solution underwent denaturation, hybridisation, capture, and amplification of chemiluminescent signal detection. We also used the HybriMax HPV blot, which captures 21 HPV genotypes: namely six low-risk types (HPV 6, 11, 42, 43, 44, and CP8304) and 15 HR types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68) that are common in the Chinese population.9 The HC2 test was used for 43 patients, and the HPV blot was used for 74 patients.
Patients with cytological LSIL+ and all patients with AGC underwent colposcopy. Patients with no cytological abnormality or with HPV-negative ASC-US, but with suspected clinical symptoms ( such as contact bleeding, irregular vaginal bleeding, increased vaginal discharge, etc. ) of cervical cancer, also underwent colposcopy. Colposcopy was carried out per standard procedures. We carried out colposcopically-directed multiple punch cervical biopsy for the most abnormal part of the suspected lesion. Cervical four-quadrant randomised biopsy and endocervical curettage were used when the colposcopy was unsatisfactory.
1.6 Pathological diagnosis of cervical biopsy samples
We used a three-level classification method for the pathological H&E-stained sections of CIN1, CIN2, and CIN3. CIN1 was considered LSIL; CIN2 and CIN3 were classified as HSIL.
1.7 Detection of p16INK4a protein and evaluation of positive immunostaining results The immunohistochemical method was adopted for detecting p16INK4a. Paraffin sections of cervical tissue were stained according to the reagent kit instructions (Ventana Medical System，Inc，Arizona USA). We used 1:100 dilution of the primary antibody (mouse anti-human p16INK4a monoclonal antibody; clone number E6H4, USA). The primary antibody was replaced with phosphate buffer solution (PBS) to construct the negative control; known p16INK4a-positive pancreas sections were used as the positive control.
Cells with positive p16INK4a immunostaining had brownish-yellow nuclei and cytoplasm. We determined the staining grade according to the percentage of p16INK4a-positive cells: positive, epithelial diffuse layer staining; focal positive, focal, discontinuous positive staining; negative, no obvious staining.
1.8 Cervical loop electrosurgical excision procedure (LEEP)
For patients with H&E pathological diagnosis of CIN2 and above (CIN2+), cervical LEEP was carried out in the next menstrual cycle, and the obtained sample was once again pathologically diagnosed.
1.9 Statistical analysis
We used the software program SPSS 13.0 (SPSS, Chicago, IL, USA) to conduct the statistical analysis. The continuous variables of normal distribution were expressed as mean ± standard deviation, the continuous variables of non-normal distribution were expressed as a median (interquartile range[IQR]), and the categorical variables were expressed as a frequency (percentage[%]). For two comparisons, each value was compared by t-test. For multiple comparisons, each value was compared by one way ANOVA following Dunnett test when each datum conformed to a normal distribution, while the non-normally distributed continuous data were compared using non-parametric tests. The chi-square test tested the counting data. A value of P<0.05 was considered statistically significant.