The medical records of patients with CINs detected by cervical biopsy or conization from April 2018 to April 2019 in Beijing Chao-Yang Hospital were retrospectively reviewed. All outpatients within 9-65 years old were routinely inquired whether they recently received CC screening examination. If not, cytology (HOLOGIC, USA) and HPV detection (Fluorometric Real-time PCR, Shanghai ZJ Bio-Tech, China) were performed under oral informed consent. The results for the cervical cytology were reported using the 2014 Bethesda (TBS) system . According to the 2012 American Society for Colposcopy and Cervical Pathology (ASCCP) revised guidelines for the abnormal treatment of CC screening results , patients with abnormal cytology (worse than atypical squamous cells of undetermined significance, >ASC-US or continuous ASC-US), HR-HPV16/18 positive testing, persistent infection (more than one year) of the other subtypes of HR-HPV, ASC-US and HR-HPV positive testing, and/or visible suspicious lesions were scheduled for colposcopy. A biopsy was performed, when necessary. Most of the patients who were pathologically diagnosed with HSIL underwent physical therapy or loop electrosurgical excision procedure (LEEP). For young women with desire for fertility, patients with CIN2 were followed up when there was a satisfactory colposcopy .
Patients with CIN1, CIN2 and CIN3 were compiled for further evaluation. Patients with concurrent invasive CC, which is a simultaneous or subsequent primary malignant tumor in other parts of the body, and incomplete medical records were excluded. Furthermore, patients with insufficient tissues for the ISH or IHC examination were also excluded.
The patient clinical data, which included age at diagnosis, smoking history, drinking history, age at first intercourse, sex partners, education, fertility status, menopausal status, body mass index (BMI), monthly income, cytology, HR-HPV detection, surgical method, pathological diagnosis and screening history, was collected and evaluated. The BMI was calculated by dividing the weight in kilograms by the height in meters squared. Subjects who smoked at least one cigarette a day and continuously for more than six months were considered positive for smoking history. Drinking at least once a month, including social engagements, was considered positive for drinking history.
The H&E slides of these patients were reviewed by two independent gynecologic pathologists, who were blind to the results. Any disagreements between these two reviewers were discussed and resolved. If a disagreement could not be resolved, a third review would be conducted until a consensus diagnosis (at least a two-way agreement) is reached. The histological classification criteria of CINs were referred to the World Health Organization (WHO) classification criteria for cervical intraepithelial lesions in 2014 . For CIN1, the undifferentiated cells extended no more than one-third of the way up the epithelium. Nuclear hyperchromasia nuclear membrane irregularities and few mitotic features are usually present. Furthermore, koilocytosis can be observed. For CIN2, the undifferentiated cells were confined in the lower 2/3 of the epithelium, but exceeding the criteria for LSIL. Furthermore, these tended to more obviously present with nuclear atypia and mitotic features. For CIN3, differentiation and stratification may be totally absent, or only present in the superficial quarter of the epithelium. Nuclear abnormalities could be observed in the full-thickness epithelium. However, the lesion did not break through the basement membrane.
Five 4-µm-thick sections were cut from the FFPE tissue. Hematoxylin and eosin (H&E), RISH and IHC staining were respectively performed.
The RNAscope HR 18 HPV assay is designed to detect the E6/E7 RNA for 18 HR HPV genotypes (HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82). The RNAscope® 2.5HD probes and detection kit were purchased from Advanced Cell Diagnostics (ACD, Hayward, USA). The assay was performed according to supplier’s instructions (ACD). Pretreatment: After being de-paraffinized and dehydrated, these sections were serially treated with the Pre-Treatment 1 solution and Pre-Treatment. Overnight, Pre-Treatment 3 was performed. Hybridization: The sections were hybridized in tge HR 18 HPV hybridization solution without a cover slip in a HybEZ Oven (ACD, Hayward, USA). Signal amplification: The hybridized probe was performed through the serial application of Amp 1-6. Visualization: Diaminobenzidine (DAB) was used to demonstrate the amplified signal. The sections were counterstained with H&E, dehydrated with graded ethanol and xylene, and mounted with Cytoseal.
The staining data was recorded with the presence of dark brown, nuclear+ and dot-like cytoplasmic+/−. The positivity pattern was recorded according to thickness of the epithelial staining, the presence and amount of diffuse, and/or the punctate nuclear staining and cytoplasmic staining, as well as the signal intensity. Morphologically, normal epithelia in the FFPE block were used as the internal negative control. A positive control was added to ensure that the dyeing process of the slides was successful. If the positive control did not show a positive staining, the batch of slides were considered as invalid.
The RISH slides were scanned using an automatic digital pathological slide scanner at 20× magnification. The expression intensity of the HR-HPV E6/E7 mRNA was analyzed using the StrataQuest software. The nuclei were identified and screened by H&E staining channels. The effective nucleus was used as the core to identify the areas with DAB staining signals within the cell range. Then, the E6/E7mRNA expression of a single cell was quantified using the software, according to the DAB staining intensity and number of cells in the lesion area.
IHC was performed to detected the expression level of P16 and Ki67
IHC was performed using the antibodies of P16 (clone: G175-405, ZSGB-BIO, China) and Ki67 (clone: MIB1, ZSGB-BIO, China), respectively, according to manufacturer’s instructions. Then, the sections were de-paraffinized and dehydrated. The antigen retrieval was performed by boiling the slides with the ethylene diamine tetraacetic acid (EDTA) Antigen Retrieval solution (pH 9.0) in a pressure cooker for 15 minutes. After blocking the endogenous peroxidase with H2O2, the sections were incubated with the antibodies. Then, the secondary antibody reagent was performed. The reaction was detected by DAB and counterstained with H&E. A positive control slide was used to ensure the validity of the staining procedure.
Definition of P16 positivity: According to the LAST deﬁnition of p16 positivity , which calls “block positivity” continuous staining in the nucleus and/or cytoplasm, this was extended up from the basal layer through at least one-third of the epithelium. Definition of Ki67 positivity: Nuclear staining only and continuous staining, and this extended up from the basal layer through the lower third of the epithelium.
SPSS (21.0) was used for the statistical analysis. The rate and percentage of expression was used to describe the general situation of the study subject. HSIL was used as the clinical endpoint of the disease to evaluate the diagnostic efficiencies of HPV E6/E7 mRNA ISH, P16 and P16/Ki67 on CINs, and calculate the sensitivity and specificity of these various detection methods. Chi-square was used to test for differences in expression rate, sensitivity and specificity. Binary logistic regression was used to calculate the OR and 95% confidence interval (CI), which describes the risk factors associated with CINs. A P-value of <0.05 was considered statistically significant.