HPV as well as LBC testing, has largely reduced the incidence of cervical cancer and morbidity and mortality. Some countries use HPV testing as a primary screening for cervical cancer. The biggest challenge in implementing HPV primary screening is managing the large number of women who are found to have transient HPV infection. HPV testing is considered to have a high sensitivity, but HPV testing can only detect the presence or absence of the virus and cannot distinguish between transient and persistent infections with the virus. The test identifies the virus but not the cancer precursor lesion or the cancer itself. It is well known that the main risk factor for cervical cancer is persistent HPV infection. It promotes impaired cell growth and differentiation, leading to dysplasia (cervical intraepithelial neoplasia, CIN). Positive HPV test does not mean that cervical lesions have occurred, it indicates an increased risk of future cervical cancer. LBC has low sensitivity and poor reproducibility, and poor LBC staining may lead to misdiagnosis or overdiagnosis. Cytologic findings of ASC-US/LSIL are currently a key area of focus for stratified management of cervical cancer at the screening stage to reduce unnecessary biopsies and avoid overtreatment. Cytology is the test of choice for classifying HPV-positive women because it is highly specific, but it is a subjective judgment and its accuracy depends on the cytologist's level of profession. For clinicians, the most important aspect of diagnostics for CIN is to differentiate LSIL from HSIL. For this reason, many currently ongoing studies search for new markers that may streamline the diagnostic process. Accurate screening for ≥CIN2 lesions prior to colposcopic triage is key to early screening for cervical cancer. The use of p16/Ki-67 DS to detect cervical precancerous lesions has received attention from investigators for its high sensitivity and specificity[14, 21]. The technique of CB production has been widely used in non-gynecologic pathology, which is mature, simple and easy to operate, and can maximize the enrichment of cells and also obtain morphological details similar to histology such as nests, sheets, papillae and three-dimensional structures of certain solid tissues. CB not only has histological advantages, but also provides sufficient material for subsequent molecular pathological examinations such as special staining, Immunohistochemical(IHC), PCR and sequencing. The prepared CB can be stored for a long time, while the preparation of CB provides convenience and possibility for the transportation and preservation of samples in the multicenter construction of cervical cancer screening, but its use in cervical cancer screening has been rarely reported in the literature. In this paper, we applied CB made from leftover specimens of LBC combined with p16/Ki-67 DS to compare the role of HR-HPV, DNA ploidy and p16/Ki-67 DS in early screening of cervical cancer, especially before colposcopic triage.
P16, also known as p16INK4a, is encoded by the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene located on chromosome 9p21.3. It is a cell cycle protein that regulates cell proliferation in the G1-S phase due to the reciprocal relationship with another tumor suppressor protein-Rb(20). In HPV-infected cells, interference with the pRb-E2F1 pathway by E7 through a negative feedback loop [22, 23]. Induced overexpression and accumulation of p16 in cells. Therefore, p16 is considered as an alternative marker of persistent HR-HPV infection. Cells with heterogeneous proliferation exhibit p16 overexpression, which is easily detected by immunohistochemistry. Since E7 oncogene induces cell transformation of all HR-HPV types, dysplastic cells exhibit p16 overexpression, which is easily detected by immunohistochemistry). In recent years, P16 immunohistochemical staining has been increasingly recommended to differentiate HSIL from unrelated neoplastic disease, and studies have shown that P16 immunohistochemical staining can be used as a specific biomarker to predict the progression of CIN. Ki-67 is a proliferating cell-associated antigen, also known as MIB-1, that is detected in the nuclei of proliferating cells in all phases of the cell cycle (G1, S, G2, and M), but is largely unexpressed in quiescent cells (G0). Ki-67 has been shown to play multiple roles in regulating cell cycle progression as a marker of tumor cell proliferation capacity. When CIN occurs, Ki-67 expression gradually extends and spreads from the basal layer to the mid-surface layer, consistent with the extension of CIN. Detection of Ki-67 has been widely used in the ancillary diagnosis of precancerous cervical cancer and cancer. Alshenawy et al. found that Ki-67 expression gradually increased with increasing CIN grade, and in cancerous tissues, Ki67 expression reached 100%. p16 and Ki67 act as tumor suppressors and cell proliferation markers, respectively, and overexpression of p16 and Ki67 are mutually exclusive under normal conditions and do not usually occur in the same cervical epithelial cells. Detection of p16/Ki-67 co-expression can be used as a predictor of HR-HPV cell transformation and high-grade CIN lesions. It has been shown that p16/Ki67 DS can be a more valuable method in detecting cervical lesions when comparing the combination of p16/Ki67 DS test, LBC and HPV test. Wu Y et al. repoerted that the ROC curve for p16/Ki-67 was greater than HC-II in ASCUS, LSIL (0.799 vs 0.696, 0.708 vs 0.531; all 0.05). For patients with cytologic diagnosis of ASCUS, LSIL, the p16/Ki-67 DS method can be an effective method to assist in the diagnosis of high-grade cervical lesions, and the screening efficiency is superior to that of HR-HPV. Excellent performance of p16/Ki-67 DS was observed in one study, especially in women under 30 years of age, with an area under the ROC curve of 0.762 (p<0.001). As described in the previous article， the area under the ROC curve for p16/Ki-67 DS on CB in our study was greater than that for HR-HPV and DNA ploidy (0.900 vs. 0.557,0.583), (p<0.001).
The morphology of ASC-US cells is characterized by an enlarged nucleus that is 2.5 to 3 times larger than the normal mid-layer nucleus, a mildly increased nucleoplasmic ratio, a mildly deepened nuclear chromatin, and an abnormal nucleus or a strong orangish change in the cytoplasm, which is a risk indication for the presence of lesions, but not a definitive diagnosis of abnormal cells. The diagnostic cytologic presentation of ASC-US is unclear, i.e., it may suggest the occurrence of HPV infection or other malignant lesions in the cervix, or it may represent only benign reactive changes such as inflammation, becoming a gray area for cytologic diagnosis due to its low specificity and poor reproducibility, as well as a currently recognized diagnostic difficulty. Although ASC-US is considered a low-risk cervical cytologic abnormality, about 5-10% of women with primary ASC-US actually have an underlying high-grade cervical lesion (CIN2+ and CIN3+) on histologic diagnosis and need to be followed up. If ASC-US is not effectively triaged, the best time for treatment is missed and the lesion may progress to a higher grade or squamous cell carcinoma. ZHU et al. found that CIN2 + accounted for only 18.0% and CIN 3+ for only 8.3% of ASCUS, and that colposcopy would overtreat ASC-US if all ASC-US patients underwent colposcopy. the total positive rate of HPV-DNA testing in 300 ASC-US cases was 85.3%, and the positive rate of p16/Ki67 immunocytochemistry testing was 32.0%. The overall positive rate of HPV-DNA testing was 85.3% and the positive rate of p16/Ki67 immunocytochemistry was 32.0% in 300 ASC-US cases, indicating that staging ASC-US with p16/Ki67 DS instead of HPV-DNA testing reduced the referral rate for colposcopy by 53.3%.
According to the National Comprehensive Cancer Network (NCCN) guidelines for cervical cancer screening, the treatment of LSIL cases is based on clinical observation. The progression rate of p16/Ki67 positive cases and negative progression rate of 30.8% and 4.3%, respectively, in the study of Vrdoljak-Mozetic et al. It were significantly higher than the progression rate of negative cases, confirming the predictive role of the p16/Ki67 DS assay for LSIL case regression. A European study including 27,349 women. Similarly, p16/Ki67 DS showed a high positive predictive value for detecting HSIL cases from LSIL cases. Most of the LSIL can regress or heal on their own, and only a small proportion is at risk of progressing to higher grade lesions. Since clinical obstetricians and gynecologists in different hospitals interpret these patients differently their clinical management and follow-up are somewhat divergent therefore, how to manage patients with ASC-US/LSIL cervical cytology more effectively is becoming more and more important. Clinical intervention and treatment of cases with a cytologic diagnosis of ASC-US/LSIL remain in limbo. It is important to implement effective triage methods for ASC-US and LSIL cytology findings to identify women at highest risk for potential precancerous lesions and those requiring immediate further diagnostic follow-up. In our study, it was demonstrated that the area under the AUC curve, NRI index, and IDI index were significantly increased for p16/Ki-67 DS on CB compared to HR-HPV and DNA ploidy, verifying that p16/Ki-67 DS on CB significantly improved the correct classification of patients with ≥CIN2 and was a good method for triaging ASC-US/LSIL patients.
The diagnostic challenge in cervical cancer screening is cervical glandular lesions, and it is difficult to distinguish between hyperplasia and neoplasia. One study showed that p16/Ki-67 DS was positive in 92.5% of 40 cases of cervical adenocarcinoma and only 1 of 16 cases of cervical tissue without glandular lesion was positive for DS, suggesting that p16/Ki-67 DS is a potential tool to diagnose cervical glandular lesions. In this study, DS was also positive for adenocarcinoma and squamous cell carcinoma, but due to the small number of cases, further analysis will be made after the expansion of cases in the future.
The clinical application of p16/Ki-67 DS in combination with cervical liquid-based CB technology maximizes the benefits of cervical cancer screening while reducing and avoiding unnecessary waste of resources and potential harm caused by misdiagnosis and overdiagnosis, and helps to achieve cervical lesion interpretation, prediction, and improve the diagnostic efficacy of ≥CIN2. In practice, it has very good efficacy for stratified management of ASC-US/LSIL patients, detection rate and avoidance of unnecessary biopsies and overtreatment, providing an effective test in cervical cancer screening triage management. This will benefit women by reducing the psychosocial impact of having repeated abnormal smears and undergoing colposcopy, while also reducing the cost of testing associated with colposcopy. It also has the advantages of being easy to repeat, objective and efficient. There is good agreement with histopathology, which can assist in pathologic diagnosis and determination of clinical outcome. The p16/Ki-67 DS on CB is suggested to be a good tool to triage ASCUS, but more research is needed to support this view.
Limitations of the study
In this experiment, there are also limitations, which are summarized as follows: 1. The sampling should be done as standardized as possible to ensure a certain pressure on the site taken to obtain a sufficient amount of cells. 2. 8 of the 58 patients in our study with histologically confirmed ≥CIN2 were negative for p16/Ki-67 DS, and repeat review revealed histologically small focal CIN2 involving glands. 3. For deep focal lesions in the cervical canal, the superficial location of the sampling leads to missing deep lesions, and Endocervical curettage (ECC) can resolve such false negatives. 4. The clinician should avoid menstruation and try to remove mucus and blood before sampling to obtain high quality CB. 5. Routine sampling does not take the vaginal wall, resulting in easy to miss lesions in the vaginal wall, and it is debatable whether to do additional sampling of the vaginal area. The CB were leftover LBC specimens with relatively reduced cell volume, which may also cause false-negative results.