Abstract
Background/Aim: To evaluate p16/Ki-67 dual-staining performance for detection of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) in the management of women with minor cervical abnormalities. Patients and Methods: All 759 enrolled patients were tested for cytology, high-risk human papillomavirus (HR-HPV) and dual p16/Ki-67 staining. Results: Positivity rates for HR-HPV and dual staining increased as dysplasia was worsened from non-CIN (37.6% and 0%) to CIN1 (62.5% and 1.6%) and CIN2+ (98.7% and 97.3%), respectively. HPV18 and HPV16 exhibited the highest odds ratios (53.16 and 11.31) in the CIN2+ group. Both p16/Ki-67 dual staining and HR-HPV presented similar sensitivities (97.3% and 98.7%, respectively) for CIN2+ detection. Dual staining specificity, however, was 99.3%, significantly higher compared to HR-HPV testing (52.2%). The utility of dual staining was evaluated in different screening strategies and appeared to reduce the number of colposcopies required for the detection of CIN2+ cases. Conclusion: p16/Ki-67 dual-staining cytology is a surrogate triage biomarker in cytology-based screening programs, with high performance for efficient risk stratification of women with mild cervical abnormalities.
- HPV
- cervical cancer
- p16/Ki-67 dual-stained cytology
- triage
- CIN2
- CIN3
- HSIL
- screening
- risk stratification
- Pap test
For decades, cytology-based screening has contributed to the reduction of the cervical cancer incidence globally (1, 2). However, the incidence and mortality rates remain high, as primary and secondary prevention are either lacking or not well established or accessible in many countries worldwide. In Greece, the cervical cancer screening program still remains opportunistic, involving mainly an annual cytology screening which has achieved an estimated coverage of 68.8% (3). From this point of view, efficient management of human papillomavirus (HPV)-infected women who remain at a high risk for developing cervical cancer is critical, and this necessity requires revision and optimization of existing screening programs.
The persistence of high-risk (HR)-HPV infection as a prerequisite for cervical cancer development was the fundamental concept which has led to the implementation of HPV testing in cervical cancer screening (4, 5). The increased sensitivity of HPV testing has led to the transition from cytology-based primary screening to HPV-based, either alone or as co-testing with cytology, in several countries (6, 7). The idea behind HPV-based screening strategies is that HR-HPV-positive women with mild [low-grade squamous intraepithelial lesions (LSIL)] or borderline atypical squamous cells of undetermined significance (ASCUS) cytology are at greater risk and should have a strict follow-up. However, the natural history of HPV infection has shown that the majority of infections will resolve spontaneously, especially in younger women, without consequences; however, this constitutes the main weakness of this screening strategy (8, 9). Specifically, HPV-based screening produces a high rate of colposcopy referrals and over-treatments, which greatly increases the psychological and economic burden of cervical cancer screening (10, 11).
Therefore, the introduction of effective biomarkers for direct and more accurate risk stratification is an urgent need. In various studies, positivity in p16/Ki-67 dual-staining cytology seems to be a promising triage marker for women with borderline/mild dyskaryosis or with normal cytology but HR-HPV-positive results and exhibits high sensitivity and specificity (12-15). p16/Ki-67 dual staining allows the simultaneous detection of both the tumor-suppressor protein p16INK4a and the cell proliferation marker Ki-67 within the same cell through immunocytochemical staining. Co-expression of these markers is indicative of cell-cycle deregulation and may predict cervical dysplasia. Therefore, p16/Ki-67 dual staining may serve as a marker for risk stratification (16).
The aim of our study was to evaluate the clinical efficiency of p16/Ki-67 dual-staining cytology in identifying underlying cervical intraepithelial neoplasia (CIN) 2 or worse lesions, in order to employ it as a triage biomarker for accurate and reliable stratification of women with minor cervical abnormalities in cytology or with normal cytology but positivity for HR-HPV.
Patients and Methods
Study design and population. This was a prospective population-based study, including women with mild cervical cytology findings who were referred for colposcopy. The study was conducted at the First Department of Obstetrics and Gynecology at Alexandra University Hospital in Athens, Greece. Women attended the colposcopy clinic for their annual cytology-based examination from March 2018 through April 2021. They provided written informed consent and completed a brief questionnaire with demographic and clinical characteristics. The study was approved by the Ethics Committee of the Alexandra University Hospital.
Patients were eligible for participation in the study if they met the following criteria: a) sexually active women aged 20 to 60 years, b) not pregnant, c) with cytology of ASCUS, LSIL or negative for intraepithelial lesion or malignancy (NILM), either HR-HPV-positive or HR-HPV-negative, and d) with no history of any treatment for CIN or hysterectomy.
Prior to colposcopy/biopsy, liquid-based cytology samples were collected from 1,292 patients. After applying inclusion criteria, the final cohort comprised of 759 cases as shown in Figure 1. Specifically, we recruited 382 patients with LSIL cytology, 118 with ASCUS, both irrespective of HPV type, and 259 with NILM cytology, comprising 99 HR-HPV-positive cases and a random subset of 160 HR-HPV-negative women.
Flow diagram of the study population. ASCUS: Atypical squamous cells of undetermined significance; HR-HPV: high-risk human papillomavirus; LBC: liquid based cytology; LSIL: low-grade squamous intraepithelial lesion; NILM: negative for intraepithelial lesion or malignancy.
Cytology. Liquid-based cytology samples were collected from all subjects in Thinprep® PreservCyt solutions (Thinprep; Hologic, Inc., Marlborough, MA, USA) and a first slide was prepared for cytological evaluation. Cytology interpretation was carried out by experienced cytopathologists and classified according to the Bethesda 2014 system (17).
HPV DNA testing. From the residual liquid-based cytology material, HPV DNA testing was performed using a Cobas® 4800 HPV test (Roche Molecular Systems, Inc., Branchburg, NJ, USA) which identifies HPV16 and HPV18 genotypes and detects another 12 HR-HPV types as a pooled result. Subsequently, a second HPV DNA assay was conducted, namely CLART® HPV3 genotyping (Genomica S.A.U, Madrid, Spain) in order to genotype the 12 pooled HR-HPV types (types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68).
p16/Ki-67 dual-staining cytology. A second slide from the residual Thinprep® specimens was stained by CINtec® PLUS Cytology kit (Roche mtm Laboratories AG, Mannheim, Germany). The assay was performed on an automated Benchmark ULTRA instrument according to the manufacturer’s instructions. Samples synchronously stained for p16INK4a and Ki-67 were considered as positive regardless of the cells’ morphological appearance (Figure 2). We emphasize the assumption that the presence of a single dual-stained cell in the slide is a sufficient indicator of a positive result. Slides with no dual-stained cells were considered negative, whereas those that did not meet the minimum squamous cellularity criteria (<5,000 squamous cells) according to the Bethesda 2014 system were marked as invalid. All slides were evaluated and reviewed by two experienced, trained cytopathologists unaware of the HPV test results and colposcopy findings.
Dual p16INK4a (p16)/Ki-67-stained cells (×40). A: Positive dual-stained cell. B: Cluster with dual-stained cells. Brown cytoplasmic stain: p16; and red nuclear stain: Ki-67.
Colposcopy and histology. All women underwent colposcopy and directed biopsies by experienced gynecologists. Histological evaluation was based on CIN classification: No CIN (non-neoplasia, inflammation and chronic cervicitis), CIN1, and CIN2+ (CIN2, CIN3, adenocarcinoma or cervical cancer). The clinical endpoint of the study was biopsy-confirmed CIN2+.
Statistical analysis. Data are expressed as mean±standard deviation for continuous variables and as percentages for categorical data. The Kolmogorov–Smirnov test was utilized for normality analysis of the parameters. The risk of CIN2+ according to HPV genotype was expressed as odds ratio (OR) with 95% confidence intervals (CI) and evaluated using the chi-square test. The diagnostic ability of the HR-HPV test and p16/Ki-67 for the detection of CIN2+ was evaluated using sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). Comparison between them was performed using the paired samples t-test. All tests were two-sided, statistical significance was set at p<0.05. All analyses were carried out using the statistical package SPSS Statistics 21.0 (IBM Corporation, Somers, NY, USA).
Results
Study population. The study cohort included 759 patients, aged 20 to 60 years. The demographic and clinical characteristics are presented in Table I. The mean age was 39.6±10.5 years, while 21.7% of the women were under 30 years old. Overall, 17.7% of them had had one lifetime sexual partner and approximately half of the population (47.8%) was nulliparous. Most of the women (88.4%) had participated in a cytology-based screening program in the previous 3 years, and a significant percentage (63.1%) were non-smokers, 13.6% were HPV-vaccinated and almost one-third of the population reported frequent vaginal infections.
Descriptive characteristics of participants (N=759).
Test results according to histopathology. As shown in Table II, 149 histological reports were CIN2 or worse (CIN2+). HR-HPV and HPV16/18 positivity rates increased with lesion severity, representing 37.6% (136/362) and 5.2% (19/362) in the no-CIN group, 62.5% (155/248) and 11.7% (29/248) in CIN1, 98.7% (147/149) and 70.5% (105/149) in CIN2+ lesions, respectively. An ascending trend was also observed for p16/Ki-67 dual-stain positivity rates, which increased from 0% (0/362) in no CIN, to 1.6% (4/248) in CIN1, through 97.3% (145/149) in CIN2+ lesions.
High-risk human papillomavirus (HR-HPV) test and p16/Ki-67 dual-staining results according to histology.
Among the NILM group, the two cases of CIN2+ were also both positive for HPV16/18 and p16/Ki-67. Interestingly, in the same group, none of the mild/benign lesion (no neoplasia/CIN1) cases were positive by dual staining. In the ASCUS population, HR-HPV, HPV16/18 and p16/Ki-67 positivity all increased as the pathology worsened (from 38.7%, 12.2% and 0% in the no-CIN group to 68%, 20% and 2% in the CIN1 group and 100%, 73.7% and 94.7% in CIN2+ group, respectively). A similar trend was observed in the LSIL group, where CIN2+ subgroup presented the highest rates of HR-HPV, HPV16/18 and p16/Ki-67 positivity (98.4%, 69.5% and 97.7%, respectively) in contrast with the no-CIN and CIN1 groups, which demonstrated lower HR-HPV/HPV16/18 positivity rates (48.2%/4.7% and 59.2%/10.1%) and lower p16/Ki67 positivity rates (0% and 1.8%).
Risk of CIN2+ lesions associated with HR-HPV genotype. The ORs for the incidence of CIN2+ were calculated and correlated with each of the 14 HR-HPV genotypes (Table III). For HPV18 positivity, the odds of CIN2+ development was calculated to be 53.16 (95% CI=16.0-176.70) and was the highest among all HPV subtypes. HPV16 positivity was the second most risky factor for CIN2+ (OR=11.31, 95% CI=7.39-17.32), followed by HPV33 (OR=2.96, 95% CI=1.34-6.52) and HPV31 (OR=2.59, 95% CI=1.56-4.32). The remaining HR-HPV types (types 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) presented lower ORs with no significant correlation to CIN2+ lesions.
Risk of cervical intraepithelial neoplasia 2 or worse (CIN 2+) histology correlated with positivity for high-risk human papillomavirus (HR-HPV) genotype.
Performance of HR-HPV test and p16/Ki-67 dual-staining regarding CIN2+ detection. There were no statistical differences concerning sensitivity for CIN2+ between HR-HPV testing (98.7%, 95% CI=95-100%) and p16/Ki-67 dual-staining cytology (97.3%, 95% CI=93-100%). NPVs were also comparable, with 99.3% (95% CI=98-100%) for p16/Ki-67 staining and 99.3% (95% CI=97-100%) for HR-HPV testing. On the other hand, specificity and PPV of p16/Ki-67 dual staining were significantly higher, at 99.3% (95% CI=98-100%) and 97.3% (95% CI=93-100%), compared to 52.2% (95% CI=48-56%) and 33.6% (95% CI=30-38%) for the HR-HPV test.
Different screening strategies with respect to the number of colposcopies needed for CIN2+ detection. The American Society for Colposcopy and Cervical Pathology (ASCCP) guidelines advise that women with HPV16/18+ and women positive for the other 12 HR-HPV types plus ASCUS or worse cytology should be referred for colposcopy (18). Based on that, concerning our population, 352 colposcopies should have been recommended to detect 147 out of 149 actual CIN2+ cases (2.4 colposcopies/CIN2+ case). Thus, we studied a number of hypothetical scenarios to find the optimal combination of HPV types and cytology which can detect CIN2+ with minimum referrals for colposcopy. As shown in Table IV, the combination of a positive result for the 14 HR-HPV test led to a ratio of 3.0 colposcopies/CIN2+, for HPV16/18-positive to 1.5 colposcopies/CIN2+ and for HPV16/18/31-positive and HPV16/18/31/33-positive to 1.8 and 1.9 colposcopies/per CIN2+, respectively.
Different screening strategies for cervical intraepithelial neoplasia 2 or worse (CIN 2+), based on the ratio of colposcopies (Cpx) to CIN2+ cases.
Our next step was to add positivity for p16/Ki-67 dual-staining to all the different strategies mentioned above. We observed that the addition of dual-staining positivity to the algorithm (positivity for HPV16/18;12 HR-HPV ASCUS+) reduced the number of cases for colposcopy from 352 to 147 (1.0 colposcopy/CIN2+). A similar reduction in the number of colposcopies was shown in all strategies with dual-staining implementation, with the best combination being that combined with positivity for HPV16/18. The population of our study might also have benefited from the use of positivity for p16/Ki-67 as a stand-alone assay, requiring 149 colposcopies and detecting 145 CIN2+ cases, thus resulting in a ratio of 1.0 colposcopy needed per CIN2+ case detected.
Discussion
The clinical management of women with repetitive equivocal or mildly abnormal cytology of ASCUS or LSIL, as well as those positive for HR-HPV with normal cytology, represents a major burden in cervical cancer screening. The optimization of risk stratification will benefit these women and provide financial profit in the healthcare system. In this study, we compared the effectiveness of p16/Ki-67 dual-staining cytology versus HR-HPV test under the scope of a triage model in a population which was referred for colposcopy.
We observed that the more severe lesions were associated with higher p16/Ki-67 positivity rates, ranging from 0% in no CIN, to 1.6% in CIN1 lesions through 97.3% in CIN2 or worse in accordance with other studies (19, 20). An ascending trend was also observed in HR-HPV-positive and HPV16/18-positive cases, which similarly correlated with lesion severity corresponding to 37.6% and 5.2% in no CIN, 62.5% and 11.7% in CIN1 and 98.7% and 70.5% in CIN2+ lesions, respectively.
To evaluate extended genotyping and its association with cancer progression, we further calculated the risk of CIN2+ according to HR-HPV genotype. In our population, we noted that HPV18-positive cases exhibited the highest OR (53.16) for CIN2+, followed by HPV16 (11.31) and HPV31/33 (2.59/2.96) respectively, as we have reported previously and in accordance with the literature (21-23). Slight differences in ORs for CIN2+ detection and their association with HR-HPV genotypes between our study and others may be attributed to different screening populations or various epidemiological factors.
Our findings, similar to other published studies, illustrated that p16/Ki-67 dual-staining cytology achieved similarly high sensitivity (97.3%) to HR-HPV testing (98.7%), showing no statistical significance, while conferring significantly higher specificity of 99.3% (p<0.005) versus 52.2% of HR-HPV testing (13, 15, 24-26). A small number of studies also examined its utility in triaging HR-HPV-positive patients with normal cytology results, reporting good performance of p16/Ki-67 dual-staining for CIN2+ detection, with sensitivity being 91.9% and 68.8%, and specificity 82.1% and 72.8%, respectively (14, 27).
Based on the ASCCP screening guidelines, women testing positive for HPV16/18 or 12 HR-HPVs plus positive cytology (ASCUS or higher) should be referred for colposcopy (18). By applying this guideline to our population, it led to 2.4 colposcopies for the diagnosis of one CIN2+ case. The addition of positivity for HR-HPV types other than HPV16/18 according to their estimated risk for CIN2+ lesions (HPV16/18/31 and HPV16/18/31/33) did not significantly reduce the number of colposcopies for CIN2+ detection, which underscored the necessity of a reliable triage biomarker.
However, the combination of p16/Ki-67 dual-staining cytology with all of the different screening scenarios minimized the number of colposcopies for CIN2+ detection to just 1.0 in all strategies. Additionally, the application of p16/Ki-67 dual-staining cytology as a stand-alone assay to triage our study population resulted in 149 referrals for colposcopy for the identification of 145 CIN2+ cases, with a colposcopy ratio of only 1.0. Our findings are comparable with other studies which also assessed p16/Ki-67 dual-stain implementation in different strategies in HPV-positive referral populations for CIN2+ or CIN3+ identification (28-30).
Therefore, we suggest that p16/Ki-67 dual-staining immunocytochemical reaction offers both high sensitivity and specificity, and provides a better risk stratification for women who are at a higher risk of CIN2 or worse with the advantage of reducing the number of colposcopy referrals. Interestingly, the utility of p16/Ki-67 in NILM/HR-HPV-negative cases was proven to be beneficial, as no positive dual staining case was observed in this subset, leading to a proposed safe return to routine screening for all these women. However, further evaluation of NILM/HR-HPV-positive individuals is also critical, as in our screening, two cases were found to be dual-staining-positive with CIN2+ on histology, which could have been missed according to the ASCCP algorithm.
One of the strengths of the study is the absence of similar research, evaluating the diagnostic accuracy of p16/Ki-67 dual-stain, in an opportunistic cytology-based screening setting in Greece. Furthermore, all diagnostic tests and histological evaluations were performed prospectively within 3 to 4 months of specimen collection. With regards to the limitations, our study included a colposcopy referral population rather than a general screening population and employed CIN2+ histology outcomes as a clinical endpoint, as we did not have a large group of patients with cervical intraepithelial grade 3 or worse as in other studies.
Conclusion
Our findings strongly support the use of p16/Ki-67 dual-staining cytology as a stand-alone assay, which displays significantly high sensitivity and specificity, suggesting that it may be a suitable approach in the management of women with mild cervical lesions for the detection of CIN2+. Moreover, the addition of p16/Ki-67 staining to existing cervical cancer screening strategies might improve the triaging system and minimize colposcopy referrals. These points are important, especially in countries with mainly opportunistic, annual cytology-based screening mode or with no organized screening settings. In these cases, implementation of p16/Ki-67 dual-staining cytology will provide accuracy and financial benefits for healthcare systems.
Acknowledgements
This research was co-financed by Greece and the European Union (European Social Fund) through the Operational Program ‘Human Resources Development, Education and Lifelong Learning’ in the context of the Project Strengthening Human Resources Research Potential via Doctorate Research (MIS-5000432) and implemented by the State Scholarships Foundation (ΙΚΥ). Roche Diagnostics (Hellas) S.A partially supported the statistical analysis of the current study (no. 16742). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.
Footnotes
Authors’ Contributions
MM: Conceptualization, investigation, methodology, visualization, writing the original draft, writing-reviewing and editing; PM, EM and AP: project administration and resources; NV, EK, GD, ED, NT and AR: validation and visualization; NA: visualization, writing-reviewing and editing; KP: supervision, conceptualization, methodology, project administration, visualization, writing the original draft, writing-reviewing and editing. All Authors read and approved the final version of the article.
Conflicts of Interest
The Authors have no conflicts of interest to declare.
- Received March 1, 2022.
- Revision received March 24, 2022.
- Accepted March 25, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
