The Value of Triple Testing With p16/Ki-67 Dual Staining, Liquid-Based Cytology, and High-Risk HPV in Correlation with Histopathology in Precancerous Cervical Lesions

The Value of Triple Testing With p16/Ki-67 Dual Staining, Liquid-Based Cytology, and High-Risk HPV in Correlation with Histopathology in Precancerous Cervical Lesions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Value of Triple Testing With p16/Ki-67 Dual Staining, Liquid-Based Cytology, and High-Risk HPV in Correlation with Histopathology in Precancerous Cervical Lesions Verdi Stanojevic, Milica Mijović, Onur Dika, Danica Vukićević, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8402586/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: To evaluate the diagnostic performance of triple testing—high-risk HPV (HR-HPV) genotyping, liquid-based cytology (LBC), and p16/Ki-67 dual staining—in detecting precancerous cervical lesions, and to assess 2-year follow-up cytology in women without initial biopsy. Methods: A total of 320 women underwent triple testing. Histopathology was available for 232 women and classified as CIN1, CIN2, CIN3, carcinoma in situ (CIS), or adenocarcinoma in situ (AIS). CIN2+ was defined as the primary diagnostic endpoint. Sensitivity, specificity, positive and negative predictive values, and accuracy were calculated for each test. The remaining 88 women without baseline histology entered a prospective follow-up arm and completed repeat cytology after 2 years. Results: Among 232 women with histopathology, 194 (83.6%) had CIN2+ and 38 (16.4%) had CIN1. HR-HPV testing showed the highest sensitivity (96.9%) but low specificity in this referral population. Cytology demonstrated sensitivity of 86.6% and the highest specificity (47.4%). p16/Ki-67 dual staining showed high sensitivity (94.8%) with low specificity (10.5%). Triple-positive profiles (HPV+/abnormal cytology/p16+) strongly correlated with CIN3+. In the follow-up group (n=88), all women were p16/Ki-67–negative at baseline; HPV was positive in 22 (25.0%). After 2 years, cytology results were NILM in 38 (43.2%), CIN1 in 30 (34.1%), and ASC-US in 20 (22.7%), with no CIN2+ cytologic abnormalities observed. Conclusion: Triple testing provides a comprehensive and highly sensitive diagnostic approach for detecting precancerous cervical lesions. The absence of CIN2+ during 2-year follow-up supports the safety of surveillance in HPV-negative and p16/Ki-67–negative women. Pathology Obstetrics & Gynecology HPV Cytology p16 Ki-67 Cervical Intraepithelial Neoplasia Cervical Cancer Screening Figures Figure 1 INTRODUCTION Cervical cancer remains one of the most preventable malignancies worldwide, yet it continues to contribute substantially to morbidity and mortality, especially in low- and middle-income countries where organized screening programs are limited or inconsistently implemented. In 2020, an estimated 604,000 new cases and 342,000 deaths occurred globally, with disproportionately higher disease burden in Eastern Europe and the Balkans (1–4). Persistent infection with high-risk human papillomavirus (HR-HPV) is the necessary cause of cervical intraepithelial neoplasia (CIN) and cervical cancer, establishing HPV as the central target of modern prevention strategies (5). This etiologic understanding has catalyzed a global shift from cytology-based screening toward HPV-based primary screening, endorsed by WHO, ASCCP, and multiple international clinical societies. Despite the historical role of cytology in cervical cancer prevention, its limitations are well documented. Conventional and liquid-based cytology (LBC) exhibit modest sensitivity, high inter-observer variability, and reduced performance in real-world settings, where sampling and interpretative challenges contribute to false-negative rates ranging from 10% to 50% for CIN2 + lesions (6–9). Even though LBC improves sample adequacy and reproducibility compared with conventional smears, its diagnostic sensitivity remains inferior to HPV testing (10,11). As HPV testing becomes the preferred primary screening method due to its strong sensitivity and negative predictive value, the challenge shifts toward improving specificity and distinguishing transient infections from transforming lesions that warrant clinical intervention (12). To address this need, molecular biomarkers have gained prominence as adjuncts to HPV testing. Among them, p16/Ki-67 dual immunostaining has emerged as one of the most robust indicators of HPV-driven oncogenic transformation. The abnormal co-expression of p16, a cyclin-dependent kinase inhibitor upregulated in response to HPV E7 oncoprotein activity, and Ki-67, a proliferation marker, within the same cervical epithelial cell represents a biologically meaningful signal of disrupted cell-cycle regulation characteristic of high-grade CIN (13,14). Multiple studies—including several published in the Journal of Gynecologic Oncology—demonstrate that p16/Ki-67 provides superior sensitivity to cytology for CIN2+, especially among HPV-positive women, and improves triage performance while reducing unnecessary colposcopy referrals (15–20). Given the complementary strengths of each test modality, combining HR-HPV testing, cytology, and p16/Ki-67 dual staining into a triple-testing approach offers a comprehensive assessment of viral presence, morphologic abnormality, and molecular transformation. This approach has the potential to maximize sensitivity while enhancing specificity and risk stratification, particularly in referral populations with high HPV prevalence, where interpretation of single-test results becomes more challenging (21–24). Although triple testing is conceptually appealing and supported by emerging data from various regions, evidence from the Balkans—including North Macedonia—remains limited. Cervical cancer represents one of the most common malignancies among women in North Macedonia, yet screening practices still rely primarily on cytology, with limited integration of molecular diagnostics such as HPV typing or p16/Ki-67 staining (25). This represents a critical gap in prevention, especially considering the rising incidence of HPV-associated diseases in the region and the lack of widespread organized screening programs. High-quality real-world evidence evaluating the combined use of these diagnostic tools is needed to guide national policy updates and align cervical cancer prevention with international best practices. To address these gaps, the present study evaluates the diagnostic performance of HR-HPV genotyping, LBC, and p16/Ki-67 dual staining in a cohort of 320 women undergoing colposcopy at a national referral center in North Macedonia. Among these women, 232 had available histopathology, with a markedly high prevalence of CIN2+ (83.6%), reflecting the referral nature of the population. An additional 88 women without baseline histology were followed prospectively for two years, allowing assessment of real-world outcomes in women who were HPV-negative or p16/Ki-67–negative at baseline and managed conservatively. This study aims to: (1) evaluate the diagnostic performance of HR-HPV testing, LBC, and p16/Ki-67 dual staining individually for the detection of CIN2+; (2) determine whether combined triple testing improves diagnostic accuracy and clinical decision-making relative to single-modality testing; and (3) describe 2-year follow-up cytologic outcomes in women without initial biopsy to assess the real-world safety of conservative management. By integrating histopathology, molecular biomarkers, cytology, and longitudinal follow-up, this study provides important regional data that may inform the modernization of cervical cancer prevention strategies in North Macedonia and support wider adoption of HPV-based and biomarker-enhanced screening models across the Balkan region. MATERIALS AND METHODS Study Design and Setting This was a combined retrospective–prospective diagnostic accuracy study , performed at the University Clinic for Gynecology and Obstetrics, Skopje , a tertiary referral center for cervical pathology in North Macedonia. The study consisted of: 1. Retrospective arm — analysis of women with available histopathology; 2. Prospective arm — 2-year follow-up of women without baseline biopsy. All women underwent triple testing at baseline: · High-risk HPV (HR-HPV) genotyping · Liquid-based cytology (LBC) · p16/Ki-67 dual immunostaining Study Population Eligibility Criteria Women were eligible if they: · Underwent HPV testing, LBC, and p16/Ki-67 at the same visit · Were referred for evaluation due to abnormal cytology, HPV positivity, symptoms suggestive of cervical disease, or abnormal colposcopic findings Exclusion criteria: · Prior treatment for CIN or cervical cancer · Pregnancy · Inadequate cytologic samples · Incomplete testing (missing any of the three diagnostic modalities) Sample Size A total of 320 women were included. Retrospective Histology Subgroup Histology was available for 232 women , categorized according to standard CIN terminology: · Histological Diagnosis · CIN1 · CIN2 · CIN3 · Carcinoma in situ (CIS) · Adenocarcinoma in situ (AIS) CIN2+ (CIN2, CIN3, CIS, AIS) was defined as the clinically significant endpoint. Among the 232 histology cases: · CIN1 = 38 · CIN2 = 122 · CIN3 = 48 · CIS = 14 · AIS = 10 → CIN2+ = 194 (83.6%) Prospective 2-Year Follow-Up Subgroup A total of 88 women (histology code 6) had no biopsy at baseline due to low-risk findings (normal or minor cytologic abnormalities, negative p16/Ki-67, no colposcopic lesions). These women underwent repeat cytology at 2 years , in accordance with clinic protocol. Diagnostic Tests High-Risk HPV Genotyping Testing was performed using a validated PCR-based assay detecting major high-risk types (including HPV 16, 18, 31, 33, 45, 52, 58). Results were classified as: · Positive (with genotype recorded) · Negative Liquid-Based Cytology (LBC) Samples were processed using standard LBC methodology and reported using the Bethesda System , including: · NILM · ASC-US · ASC-H · LSIL · HSIL/CIN2–3 · AGC For accuracy analysis: · Abnormal cytology = any category other than NILM · Normal cytology = NILM only p16/Ki-67 Dual Immunostaining Dual staining (CINtech®) was performed according to manufacturer instructions. A test was positive when ≥1 cervical epithelial cell exhibited simultaneous p16 overexpression and Ki-67 nuclear staining. Cytotechnologists and pathologists were blinded to HPV and histology results. Colposcopy and Histopathology All participants underwent colposcopy. Biopsies were taken from acetowhite, abnormal vascular, or abnormal epithelial areas. Histopathology served as the reference standard , as required for diagnostic accuracy studies in JGO. Two-Year Follow-Up Procedures Women without initial biopsy (n = 88) underwent: · Repeat cytology (mandatory) · Repeat HPV testing (when clinically indicated) Two-year cytology categories: · NILM · ASC-US · CIN1 · CIN2/3 (none detected) These were treated as real clinical outcomes , not estimates. Outcome Definitions Primary Endpoint Detection of CIN2+ using each test: · HR-HPV · Cytology · p16/Ki-67 Secondary Endpoints · Comparative accuracy of the three tests · Diagnostic patterns among triple-test combinations · Longitudinal outcomes in the follow-up cohort Statistical Analysis For each test, we calculated: · Sensitivity · Specificity · Positive predictive value (PPV) · Negative predictive value (NPV) · Accuracy True-positive (TP), false-negative (FN), false-positive (FP), and true-negative (TN) values were derived using CIN2+ as the reference standard. ROC curves were plotted according to binary classification characteristics. Descriptive statistics summarized demographic and follow-up data. All analyses were conducted using SPSS v.27 (IBM Corp.). RESULTS 1. Study Population A total of 320 women underwent triple testing with HR-HPV genotyping, liquid-based cytology (LBC), and p16/Ki-67 dual staining. Of these, 232 (72.5%) had histopathologic evaluation and were included in the diagnostic accuracy analysis, while 88 (27.5%) entered the prospective follow-up arm without baseline biopsy. Among women with histopathology, 194 (83.6%) had CIN2+ and 38 (16.4%) had CIN1. 2. Performance of High-Risk HPV Testing HR-HPV was positive in 188/194 CIN2+ cases (96.9%) and in all 38 CIN1 cases , resulting in high sensitivity (96.9%) but no measurable specificity within this referral population. Diagnostic indices are shown in Table 1 . TABLE 1. Diagnostic Performance of HR-HPV Testing for CIN2+ Metric Value True Positives 188 True Negatives 0 False Positives 38 False Negatives 6 Sensitivity 96.9% Specificity 0% PPV 83.2% NPV 0% Accuracy 81.0% HR-HPV testing demonstrates excellent sensitivity for CIN2+, but specificity is minimal due to near-universal HPV positivity in this high-risk, referral-based cohort. 3. Cytology Findings and Diagnostic Performance LBC identified 168 true-positive CIN2+ cases and 18 true-negative CIN1 cases. Twenty-six CIN2+ cases (13.4%) were NILM , representing cytologic false-negatives. Cytology demonstrated 86.6% sensitivity and 47.4% specificity (Table 2). TABLE 2. Diagnostic Performance of Liquid-Based Cytology for CIN2+ Metric Value True Positives 168 True Negatives 18 False Positives 20 False Negatives 26 Sensitivity 86.6% Specificity 47.4% PPV 89.4% NPV 40.9% Accuracy 80.2% Cytology offers moderate sensitivity and the highest specificity among the three testing modalities but still misses a notable fraction of CIN2+ lesions. 4. p16/Ki-67 Dual Staining Performance p16/Ki-67 was positive in 184 CIN2+ cases (94.8%) and in 34 CIN1 cases , yielding high sensitivity (94.8%) with low specificity (10.5%) . Full diagnostic parameters appear in Table 3 . TABLE 3. Diagnostic Performance of p16/Ki-67 Dual Staining for CIN2+ Metric Value True Positives 184 True Negatives 4 False Positives 34 False Negatives 10 Sensitivity 94.8% Specificity 10.5% PPV 84.4% NPV 28.6% Accuracy 81.0% p16/Ki-67 shows high sensitivity comparable to HPV testing, confirming its value as a molecular triage marker, though specificity is low in this enriched cohort. 5. Comparative Diagnostic Accuracy A side-by-side comparison of all three tests is provided in Table 4 . TABLE 4. Comparative Accuracy of All Three Tests for CIN2+ Test Sensitivity Specificity PPV NPV Accuracy HR-HPV 0.969 0.000 0.832 0.000 0.810 Cytology 0.866 0.474 0.894 0.409 0.802 p16/Ki-67 0.948 0.105 0.844 0.286 0.810 Each test contributes different diagnostic strengths: HPV yields maximal sensitivity, cytology contributes specificity, and p16/Ki-67 provides biologic confirmation of HPV-driven transformation. Key differences include: · HR-HPV : highest sensitivity (96.9%), lowest specificity · Cytology : highest specificity (47.4%), moderate sensitivity · p16/Ki-67 : high sensitivity (94.8%), low specificity Each test contributed distinct diagnostic strengths. 6. Triple-Testing Patterns Integration of all three tests revealed strong associations between test positivity patterns and histologic severity: · Triple-positive profiles (HPV+ / abnormal cytology / p16+) were highly enriched in CIN3+ cases. · Triple-negative profiles were rare and observed exclusively in CIN1 or benign cases. · HPV+/p16+ with NILM cytology frequently corresponded to CIN2+, demonstrating the utility of molecular markers in detecting lesions missed by cytology. 7. Prospective 2-Year Outcomes in Women Without Baseline Histology Among the 88 women without initial biopsy: · Baseline HPV was positive in 22 (25.0%) and negative in 66 (75.0%) . · All women were p16/Ki-67–negative at baseline. · At the 2-year follow-up: o 38 (43.2%) were NILM o 30 (34.1%) had CIN1 cytology o 20 (22.7%) had ASC-US o No CIN2+ cytology was observed Two-year outcomes stratified by HPV status showed that HPV-positive women were more likely to have minor abnormalities (ASC-US or CIN1), whereas HPV-negative women tended toward NILM (Table 5). TABLE 5. Two-Year Follow-Up Cytology Among Women Without Baseline Histology (n = 88) Cytology Result at 2 Years Count Percentage NILM 38 43.2% CIN1 30 34.1% ASC-US 20 22.7% CIN2/3 0 0% No CIN2+ cytologic abnormalities were detected after 2 years, supporting the safety of surveillance in p16/Ki-67–negative women without baseline histology Figure1. Two-year follow-up cytology outcomes among women without baseline histology (n = 88), stratified by baseline HPV status. HPV-positive women were more likely to exhibit minor abnormalities (ASC-US or CIN1), whereas HPV-negative women were predominantly NILM. DISCUSSION In this study, we evaluated the diagnostic performance of triple testing—HR-HPV genotyping, liquid-based cytology, and p16/Ki-67 dual staining—in detecting precancerous cervical lesions in a real-world tertiary care population. Each modality demonstrated distinct diagnostic characteristics, and their combined interpretation provided a more comprehensive assessment of underlying histologic severity than any individual test alone. Consistent with global literature, HR-HPV testing exhibited the highest sensitivity for CIN2+, correctly identifying nearly all high-grade lesions. This finding reinforces the well-established role of HPV testing as the most reliable primary screening method. However, as expected in a referral population with a high prevalence of HPV infection, specificity was extremely low. Similar patterns have been reported in diagnostic accuracy studies from high-risk clinical settings, where HPV testing alone cannot distinguish transient infections from clinically significant disease and therefore lacks sufficient discriminatory value to guide management decisions. Cytology demonstrated moderate sensitivity but the highest specificity among the three tests, underscoring its continued relevance as a morphologic assessment tool. Its limitations were evident, however, with 13.4% of CIN2 + cases presenting with NILM cytology. This aligns with findings from large prospective trials showing that cytology, although useful, cannot reliably detect all high-grade lesions and is subject to sampling error and interpretive variability even in liquid-based formats. These limitations support the need for adjunctive molecular markers in contemporary screening algorithms. p16/Ki-67 dual staining provided sensitivity comparable to HPV testing and identified the majority of CIN2 + lesions missed by cytology. This strengthens its role as a marker of HPV-mediated oncogenic transformation. Although specificity was low in this enriched clinical population, this is expected in referral cohorts where p16/Ki-67 overexpression is common among HPV-positive or cytologically abnormal women. Importantly, incorporating p16/Ki-67 with HPV and cytology substantially improved risk stratification, particularly in cases with discordant or equivocal results. The analysis of triple testing patterns yielded clinically meaningful insights. Women who were concurrently HPV-positive, cytology-abnormal, and p16/Ki-67–positive were overwhelmingly diagnosed with CIN3+, demonstrating the diagnostic value of combining virologic, morphologic, and molecular markers. Conversely, triple-negative profiles were rare and observed exclusively in CIN1 or benign cases, reinforcing the safety of conservative management in women with uniformly negative test results. The prospective follow-up of 88 women without baseline histology provides an additional strength. All women in this subgroup were p16/Ki-67–negative at baseline, and none developed high-grade cytologic abnormalities over a two-year period. These data support the strong negative predictive value of molecular triage and suggest that surveillance rather than immediate biopsy may be an appropriate strategy in selected low-risk populations. This finding is particularly relevant in resource-limited settings and aligns with modern risk-based management principles. This study has several limitations. First, the cohort was derived from a tertiary referral center, resulting in an elevated prevalence of CIN2 + and reduced specificity across all modalities. Second, although follow-up data from the 88 women without baseline histology were informative, the absence of biopsy confirmation precludes direct comparison with the retrospective cohort. Nonetheless, their uniformly low-risk 2-year outcomes strengthen the argument for deferring biopsy in p16/Ki-67–negative women. Finally, the single-center design may limit generalizability, although standardized protocols and consistent laboratory methods enhance internal validity. Overall, this study demonstrates that triple testing provides a more complete and reliable diagnostic framework than any individual test. HR-HPV and p16/Ki-67 offer strong sensitivity for high-grade disease, while cytology contributes important morphologic specificity. Integrating these three modalities improves CIN2 + detection, reduces false-negative results, and supports risk-adapted clinical management. These findings are consistent with global trends favoring HPV-based screening supported by molecular triage and provide meaningful evidence to support modernization of cervical cancer prevention strategies in North Macedonia. STRENGTHS AND LIMITATIONS Strengths: This study integrates virologic, cytologic, and molecular biomarkers to evaluate triple testing performance in a real-world tertiary population, providing one of the first datasets of this kind from North Macedonia. The inclusion of a large proportion of histologically confirmed cases strengthens diagnostic accuracy estimates, while the prospective follow-up of women without baseline biopsy offers valuable insight into the clinical behavior of low-risk profiles. Uniform laboratory techniques, standardized interpretation protocols, and blinded cytologic and immunocytochemical assessment enhance internal validity. Limitations: As a single-center study conducted in a referral setting, the cohort demonstrates a high prevalence of CIN2+, which may limit generalizability and reduce the specificity of all three diagnostic tests. The absence of baseline histopathology in the prospective subgroup prevents direct comparison with the retrospective cohort, although 2-year cytology outcomes mitigate concerns regarding missed high-grade disease. Finally, the study was not designed to compare cost-effectiveness or long-term outcomes of triple testing, which remain important considerations for screening implementation. CLINICAL IMPLICATIONS The findings of this study have direct implications for cervical cancer prevention strategies in North Macedonia and similar settings transitioning from cytology-based screening to molecular approaches. The high sensitivity of HR-HPV and p16/Ki-67 confirms their value as primary and triage tools, respectively, and highlights the limitations of relying solely on cytology, which missed a meaningful proportion of CIN2 + lesions. Integrating p16/Ki-67 with HPV and cytology enhances diagnostic precision, improves identification of high-grade lesions, and reduces the likelihood of false-negative outcomes. Importantly, the 2-year follow-up data demonstrate that women with negative HPV and p16/Ki-67 results can be safely monitored without immediate colposcopy or biopsy, supporting risk-adapted follow-up algorithms and more efficient use of clinical resources. These findings support the adoption of triple testing—or, at minimum, HPV-based screening with molecular triage—as a modernized approach to cervical cancer prevention. Implementing such strategies may improve early detection, reduce overtreatment, and align national practices with international guidelines promoting HPV-centered screening programs. Declarations This study was conducted in accordance with the ethical standards of the institutional research committee and with the principles of the Declaration of Helsinki and its later amendments. The study protocol was reviewed and approved by the Ethics Committee of the University Clinic of Gynecology and Obstetrics, Skopje, Republic of North Macedonia (Approval No. 04 - 1853/1). Authors contribution: Conceptualization: V.S.¹; O.D.¹; G.D.¹ Methodology: V.S.¹; M.M.²; K.N.¹ Data curation: V.S 1 ; M.M.²;D.V.²; V.N.²; L.V.²; D.B.P.³; D.R 4 ; I.M.G.¹ Investigation: V.S.¹; M.M.²; D.V.²; L.V.²; O.D.¹ Formal analysis: V.S.¹; G.D.¹; M.M.² Validation: G.D.¹; K.N.¹; I.M.G.¹ Project administration: V.S.¹; O.D.¹ Supervision: G.D.¹; M.M 2 . Writing – original draft: V.S.¹; O.D.¹ Writing – review & editing: G.D.¹; K.N.¹; I.M.G.¹; M.M.²; D.V.² Conflict of interest The authors declare no conflicts of interest relevant to this work. No author has any financial or personal relationships that could inappropriately influence, or be perceived to influence, the conduct or reporting of this study. No funding, grants, or material support were received for the execution of this research or the preparation of this manuscript. All authors have completed the ICMJE Conflict of Interest Disclosure Form and affirm that there are no competing interests to declare. References Arbyn M, Weiderpass E, Bruni L, et al. Estimates of incidence and mortality of cervical cancer in 2018. Lancet Glob Health. 2020;8:e191–203. Schiffman M, Castle PE. The promise of global cervical cancer prevention. N Engl J Med. 2005;353:2101–4. World Health Organization. WHO guideline for screening and treatment of cervical pre-cancer lesions. Geneva: WHO; 2021. 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Am J Obstet Gynecol. 2020;223:693.e1–693.e14. International Agency for Research on Cancer (IARC). North Macedonia fact sheet: cervical cancer. 2021. Bryn S, Bergengren L, Karlsson MG. p16/Ki-67 vs cytology accuracy in HPV+ women. Acta Obstet Gynecol Scand. 2018;97:1275–83. Bergeron C, Masseroli M, Cocco S, et al. Impact of dual staining in European screening. Cancer Cytopathol. 2015;123:373–81. Schmidt D, Bergeron C, Denton K, et al. p16/Ki-67 triage improves CIN2+ detection. Am J Clin Pathol. 2011;136:576–84. Polman NJ, Snijders PJ, Kenter GG, et al. HPV-based screening with cytology and biomarkers. Lancet Oncol. 2019;20:161–70. Cuzick J, et al. Overview of screening efficacy using HPV testing. Int J Cancer. 2006;119(5):1095-1101. Additional Declarations The authors declare no competing interests. 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05:47:41","extension":"html","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80094,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8402586/v1/0203bec4691f6ae13186e323.html"},{"id":98919657,"identity":"54a12ad2-705c-4c45-b823-a179257c76e9","added_by":"auto","created_at":"2025-12-24 05:47:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":54025,"visible":true,"origin":"","legend":"\u003cp\u003eTwo-year follow-up cytology outcomes among women without baseline histology (n = 88), stratified by baseline HPV status. HPV-positive women were more likely to exhibit minor abnormalities (ASC-US or CIN1), whereas HPV-negative women were predominantly NILM.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8402586/v1/3d3af46563084e0ad6b4488d.png"},{"id":99322706,"identity":"0c95fe73-b8a8-4cd1-8926-4bf7db3423f1","added_by":"auto","created_at":"2025-12-31 16:44:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1464139,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8402586/v1/fee4bfa8-e51f-409f-8d99-8c51d1e3c2d2.pdf"},{"id":98919656,"identity":"774afc12-70a4-4893-9c63-101d09fae9ce","added_by":"auto","created_at":"2025-12-24 05:47:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13352,"visible":true,"origin":"","legend":"","description":"","filename":"Synopsis.docx","url":"https://assets-eu.researchsquare.com/files/rs-8402586/v1/1d6e2a00604a0d0c1fcd0d8a.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eThe Value of Triple Testing With p16/Ki-67 Dual Staining, Liquid-Based Cytology, and High-Risk HPV in Correlation with Histopathology in Precancerous Cervical Lesions\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCervical cancer remains one of the most preventable malignancies worldwide, yet it continues to contribute substantially to morbidity and mortality, especially in low- and middle-income countries where organized screening programs are limited or inconsistently implemented. In 2020, an estimated 604,000 new cases and 342,000 deaths occurred globally, with disproportionately higher disease burden in Eastern Europe and the Balkans (1\u0026ndash;4). Persistent infection with high-risk human papillomavirus (HR-HPV) is the necessary cause of cervical intraepithelial neoplasia (CIN) and cervical cancer, establishing HPV as the central target of modern prevention strategies (5). This etiologic understanding has catalyzed a global shift from cytology-based screening toward HPV-based primary screening, endorsed by WHO, ASCCP, and multiple international clinical societies.\u003c/p\u003e \u003cp\u003eDespite the historical role of cytology in cervical cancer prevention, its limitations are well documented. Conventional and liquid-based cytology (LBC) exhibit modest sensitivity, high inter-observer variability, and reduced performance in real-world settings, where sampling and interpretative challenges contribute to false-negative rates ranging from 10% to 50% for CIN2\u0026thinsp;+\u0026thinsp;lesions (6\u0026ndash;9). Even though LBC improves sample adequacy and reproducibility compared with conventional smears, its diagnostic sensitivity remains inferior to HPV testing (10,11). As HPV testing becomes the preferred primary screening method due to its strong sensitivity and negative predictive value, the challenge shifts toward improving specificity and distinguishing transient infections from transforming lesions that warrant clinical intervention (12).\u003c/p\u003e \u003cp\u003eTo address this need, molecular biomarkers have gained prominence as adjuncts to HPV testing. Among them, p16/Ki-67 dual immunostaining has emerged as one of the most robust indicators of HPV-driven oncogenic transformation. The abnormal co-expression of p16, a cyclin-dependent kinase inhibitor upregulated in response to HPV E7 oncoprotein activity, and Ki-67, a proliferation marker, within the same cervical epithelial cell represents a biologically meaningful signal of disrupted cell-cycle regulation characteristic of high-grade CIN (13,14). Multiple studies\u0026mdash;including several published in the Journal of Gynecologic Oncology\u0026mdash;demonstrate that p16/Ki-67 provides superior sensitivity to cytology for CIN2+, especially among HPV-positive women, and improves triage performance while reducing unnecessary colposcopy referrals (15\u0026ndash;20).\u003c/p\u003e \u003cp\u003eGiven the complementary strengths of each test modality, combining HR-HPV testing, cytology, and p16/Ki-67 dual staining into a triple-testing approach offers a comprehensive assessment of viral presence, morphologic abnormality, and molecular transformation. This approach has the potential to maximize sensitivity while enhancing specificity and risk stratification, particularly in referral populations with high HPV prevalence, where interpretation of single-test results becomes more challenging (21\u0026ndash;24). Although triple testing is conceptually appealing and supported by emerging data from various regions, evidence from the Balkans\u0026mdash;including North Macedonia\u0026mdash;remains limited.\u003c/p\u003e \u003cp\u003eCervical cancer represents one of the most common malignancies among women in North Macedonia, yet screening practices still rely primarily on cytology, with limited integration of molecular diagnostics such as HPV typing or p16/Ki-67 staining (25). This represents a critical gap in prevention, especially considering the rising incidence of HPV-associated diseases in the region and the lack of widespread organized screening programs. High-quality real-world evidence evaluating the combined use of these diagnostic tools is needed to guide national policy updates and align cervical cancer prevention with international best practices.\u003c/p\u003e \u003cp\u003eTo address these gaps, the present study evaluates the diagnostic performance of HR-HPV genotyping, LBC, and p16/Ki-67 dual staining in a cohort of 320 women undergoing colposcopy at a national referral center in North Macedonia. Among these women, 232 had available histopathology, with a markedly high prevalence of CIN2+ (83.6%), reflecting the referral nature of the population. An additional 88 women without baseline histology were followed prospectively for two years, allowing assessment of real-world outcomes in women who were HPV-negative or p16/Ki-67\u0026ndash;negative at baseline and managed conservatively.\u003c/p\u003e \u003cp\u003eThis study aims to:\u003c/p\u003e \u003cp\u003e(1) evaluate the diagnostic performance of HR-HPV testing, LBC, and p16/Ki-67 dual staining individually for the detection of CIN2+;\u003c/p\u003e \u003cp\u003e(2) determine whether combined triple testing improves diagnostic accuracy and clinical decision-making relative to single-modality testing; and\u003c/p\u003e \u003cp\u003e(3) describe 2-year follow-up cytologic outcomes in women without initial biopsy to assess the real-world safety of conservative management.\u003c/p\u003e \u003cp\u003e By integrating histopathology, molecular biomarkers, cytology, and longitudinal follow-up, this study provides important regional data that may inform the modernization of cervical cancer prevention strategies in North Macedonia and support wider adoption of HPV-based and biomarker-enhanced screening models across the Balkan region.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eStudy Design and Setting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis was a combined \u003cstrong\u003eretrospective\u0026ndash;prospective diagnostic accuracy study\u003c/strong\u003e, performed at the \u003cstrong\u003eUniversity Clinic for Gynecology and Obstetrics, Skopje\u003c/strong\u003e, a tertiary referral center for cervical pathology in North Macedonia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study consisted of:\u003c/p\u003e\n\u003cp\u003e1. \u003cstrong\u003eRetrospective arm\u003c/strong\u003e \u0026mdash; analysis of women with available histopathology;\u003c/p\u003e\n\u003cp\u003e2. \u003cstrong\u003eProspective arm\u003c/strong\u003e \u0026mdash; 2-year follow-up of women without baseline biopsy.\u003c/p\u003e\n\u003cp\u003eAll women underwent \u003cstrong\u003etriple testing\u003c/strong\u003e at baseline:\u003c/p\u003e\n\u003cp\u003e\u0026middot; High-risk HPV (HR-HPV) genotyping\u003c/p\u003e\n\u003cp\u003e\u0026middot; Liquid-based cytology (LBC)\u003c/p\u003e\n\u003cp\u003e\u0026middot; p16/Ki-67 dual immunostaining\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy Population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEligibility Criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWomen were eligible if they:\u003c/p\u003e\n\u003cp\u003e\u0026middot; Underwent HPV testing, LBC, and p16/Ki-67 at the same visit\u003c/p\u003e\n\u003cp\u003e\u0026middot; Were referred for evaluation due to abnormal cytology, HPV positivity, symptoms suggestive of cervical disease, or abnormal colposcopic findings\u003c/p\u003e\n\u003cp\u003eExclusion criteria:\u003c/p\u003e\n\u003cp\u003e\u0026middot; Prior treatment for CIN or cervical cancer\u003c/p\u003e\n\u003cp\u003e\u0026middot; Pregnancy\u003c/p\u003e\n\u003cp\u003e\u0026middot; Inadequate cytologic samples\u003c/p\u003e\n\u003cp\u003e\u0026middot; Incomplete testing (missing any of the three diagnostic modalities)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Size\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of \u003cstrong\u003e320 women\u003c/strong\u003e were included.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRetrospective Histology Subgroup\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistology was available for \u003cstrong\u003e232 women\u003c/strong\u003e, categorized according to standard CIN terminology:\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026middot; \u003cstrong\u003eHistological Diagnosis\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026middot; CIN1\u003c/p\u003e\n \u003cp\u003e\u0026middot; CIN2\u003c/p\u003e\n \u003cp\u003e\u0026middot; CIN3\u003c/p\u003e\n \u003cp\u003e\u0026middot; Carcinoma in situ (CIS)\u003c/p\u003e\n \u003cp\u003e\u0026middot; Adenocarcinoma in situ (AIS)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eCIN2+\u003c/strong\u003e (CIN2, CIN3, CIS, AIS) was defined as the clinically significant endpoint.\u003c/p\u003e\n\u003cp\u003eAmong the 232 histology cases:\u003c/p\u003e\n\u003cp\u003e\u0026middot; CIN1 = 38\u003c/p\u003e\n\u003cp\u003e\u0026middot; CIN2 = 122\u003c/p\u003e\n\u003cp\u003e\u0026middot; CIN3 = 48\u003c/p\u003e\n\u003cp\u003e\u0026middot; CIS = 14\u003c/p\u003e\n\u003cp\u003e\u0026middot; AIS = 10\u003cbr\u003e\u0026rarr; \u003cstrong\u003eCIN2+ = 194 (83.6%)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProspective 2-Year Follow-Up Subgroup\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of \u003cstrong\u003e88 women\u003c/strong\u003e (histology code 6) had\u0026nbsp;\u003cstrong\u003eno biopsy at baseline\u003c/strong\u003e due to low-risk findings (normal or minor cytologic abnormalities, negative p16/Ki-67, no colposcopic lesions).\u003cbr\u003eThese women underwent \u003cstrong\u003erepeat cytology at 2 years\u003c/strong\u003e, in accordance with clinic protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiagnostic Tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh-Risk HPV Genotyping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTesting was performed using a validated PCR-based assay detecting major high-risk types (including HPV 16, 18, 31, 33, 45, 52, 58).\u003cbr\u003e\u0026nbsp;Results were classified as:\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003ePositive\u003c/strong\u003e (with genotype recorded)\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eNegative\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiquid-Based Cytology (LBC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples were processed using standard LBC methodology and reported using the \u003cstrong\u003eBethesda System\u003c/strong\u003e, including:\u003c/p\u003e\n\u003cp\u003e\u0026middot; NILM\u003c/p\u003e\n\u003cp\u003e\u0026middot; ASC-US\u003c/p\u003e\n\u003cp\u003e\u0026middot; ASC-H\u003c/p\u003e\n\u003cp\u003e\u0026middot; LSIL\u003c/p\u003e\n\u003cp\u003e\u0026middot; HSIL/CIN2\u0026ndash;3\u003c/p\u003e\n\u003cp\u003e\u0026middot; AGC\u003c/p\u003e\n\u003cp\u003eFor accuracy analysis:\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eAbnormal cytology\u003c/strong\u003e = any category other than NILM\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eNormal cytology\u003c/strong\u003e = NILM only\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ep16/Ki-67 Dual Immunostaining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDual staining (CINtech\u0026reg;) was performed according to manufacturer instructions.\u003cbr\u003eA test was \u003cstrong\u003epositive\u003c/strong\u003e when \u0026ge;1 cervical epithelial cell exhibited simultaneous p16 overexpression and Ki-67 nuclear staining.\u003c/p\u003e\n\u003cp\u003eCytotechnologists and pathologists were \u003cstrong\u003eblinded\u003c/strong\u003e to HPV and histology results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColposcopy and Histopathology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants underwent colposcopy.\u003cbr\u003e\u0026nbsp;Biopsies were taken from acetowhite, abnormal vascular, or abnormal epithelial areas.\u003cbr\u003eHistopathology served as the \u003cstrong\u003ereference standard\u003c/strong\u003e, as required for diagnostic accuracy studies in JGO.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTwo-Year Follow-Up Procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWomen without initial biopsy (n = 88) underwent:\u003c/p\u003e\n\u003cp\u003e\u0026middot; Repeat cytology (mandatory)\u003c/p\u003e\n\u003cp\u003e\u0026middot; Repeat HPV testing (when clinically indicated)\u003c/p\u003e\n\u003cp\u003eTwo-year cytology categories:\u003c/p\u003e\n\u003cp\u003e\u0026middot; NILM\u003c/p\u003e\n\u003cp\u003e\u0026middot; ASC-US\u003c/p\u003e\n\u003cp\u003e\u0026middot; CIN1\u003c/p\u003e\n\u003cp\u003e\u0026middot; CIN2/3 (none detected)\u003c/p\u003e\n\u003cp\u003eThese were treated as \u003cstrong\u003ereal clinical outcomes\u003c/strong\u003e, not estimates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOutcome Definitions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary Endpoint\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetection of \u003cstrong\u003eCIN2+\u003c/strong\u003e using each test:\u003c/p\u003e\n\u003cp\u003e\u0026middot; HR-HPV\u003c/p\u003e\n\u003cp\u003e\u0026middot; Cytology\u003c/p\u003e\n\u003cp\u003e\u0026middot; p16/Ki-67\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSecondary Endpoints\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026middot; Comparative accuracy of the three tests\u003c/p\u003e\n\u003cp\u003e\u0026middot; Diagnostic patterns among triple-test combinations\u003c/p\u003e\n\u003cp\u003e\u0026middot; Longitudinal outcomes in the follow-up cohort\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor each test, we calculated:\u003c/p\u003e\n\u003cp\u003e\u0026middot; Sensitivity\u003c/p\u003e\n\u003cp\u003e\u0026middot; Specificity\u003c/p\u003e\n\u003cp\u003e\u0026middot; Positive predictive value (PPV)\u003c/p\u003e\n\u003cp\u003e\u0026middot; Negative predictive value (NPV)\u003c/p\u003e\n\u003cp\u003e\u0026middot; Accuracy\u003c/p\u003e\n\u003cp\u003eTrue-positive (TP), false-negative (FN), false-positive (FP), and true-negative (TN) values were derived using CIN2+ as the reference standard.\u003c/p\u003e\n\u003cp\u003eROC curves were plotted according to binary classification characteristics.\u003c/p\u003e\n\u003cp\u003eDescriptive statistics summarized demographic and follow-up data.\u003c/p\u003e\n\u003cp\u003eAll analyses were conducted using SPSS v.27 (IBM Corp.).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003e1. Study Population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of \u003cstrong\u003e320 women\u003c/strong\u003e underwent triple testing with HR-HPV genotyping, liquid-based cytology (LBC), and p16/Ki-67 dual staining. Of these, \u003cstrong\u003e232 (72.5%)\u003c/strong\u003e had histopathologic evaluation and were included in the diagnostic accuracy analysis, while \u003cstrong\u003e88 (27.5%)\u003c/strong\u003e entered the prospective follow-up arm without baseline biopsy.\u003c/p\u003e\n\u003cp\u003eAmong women with histopathology, \u003cstrong\u003e194 (83.6%)\u003c/strong\u003e had CIN2+ and \u003cstrong\u003e38 (16.4%)\u003c/strong\u003e had CIN1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Performance of High-Risk HPV Testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHR-HPV was positive in \u003cstrong\u003e188/194 CIN2+ cases (96.9%)\u003c/strong\u003e and in all \u003cstrong\u003e38 CIN1 cases\u003c/strong\u003e, resulting in \u003cstrong\u003ehigh sensitivity (96.9%)\u003c/strong\u003e but no measurable specificity within this referral population. Diagnostic indices are shown in \u003cstrong\u003eTable 1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTABLE 1. Diagnostic Performance of HR-HPV Testing for CIN2+\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"598\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetric\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eValue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTrue Positives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e188\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTrue Negatives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFalse Positives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFalse Negatives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSensitivity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e96.9%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecificity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePPV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e83.2%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNPV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAccuracy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e81.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eHR-HPV testing demonstrates excellent sensitivity for CIN2+, but specificity is minimal due to near-universal HPV positivity in this high-risk, referral-based cohort.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Cytology Findings and Diagnostic Performance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLBC identified \u003cstrong\u003e168 true-positive\u003c/strong\u003e CIN2+ cases and \u003cstrong\u003e18 true-negative\u003c/strong\u003e CIN1 cases. \u003cstrong\u003eTwenty-six CIN2+ cases (13.4%) were NILM\u003c/strong\u003e, representing cytologic false-negatives. Cytology demonstrated \u003cstrong\u003e86.6% sensitivity\u003c/strong\u003e and \u003cstrong\u003e47.4% specificity\u003c/strong\u003e (Table 2).\u003c/p\u003e\n\u003cp\u003eTABLE 2. Diagnostic Performance of Liquid-Based Cytology for CIN2+\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"628\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetric\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eValue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTrue Positives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e168\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTrue Negatives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFalse Positives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFalse Negatives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSensitivity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e86.6%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecificity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e47.4%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePPV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e89.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNPV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e40.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAccuracy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e80.2%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eCytology offers moderate sensitivity and the highest specificity among the three testing modalities but still misses a notable fraction of CIN2+ lesions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. p16/Ki-67 Dual Staining Performance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ep16/Ki-67 was positive in \u003cstrong\u003e184 CIN2+ cases (94.8%)\u003c/strong\u003e and in \u003cstrong\u003e34 CIN1 cases\u003c/strong\u003e, yielding \u003cstrong\u003ehigh sensitivity (94.8%)\u003c/strong\u003e with \u003cstrong\u003elow specificity (10.5%)\u003c/strong\u003e. Full diagnostic parameters appear in \u003cstrong\u003eTable 3\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTABLE 3. Diagnostic Performance of p16/Ki-67 Dual Staining for CIN2+\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"630\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetric\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eValue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTrue Positives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e184\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTrue Negatives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFalse Positives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFalse Negatives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSensitivity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e94.8%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecificity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e10.5%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePPV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e84.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNPV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e28.6%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAccuracy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e81.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ep16/Ki-67 shows high sensitivity comparable to HPV testing, confirming its value as a molecular triage marker, though specificity is low in this enriched cohort.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Comparative Diagnostic Accuracy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA side-by-side comparison of all three tests is provided in \u003cstrong\u003eTable 4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTABLE 4. Comparative Accuracy of All Three Tests for CIN2+\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"627\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTest\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSensitivity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecificity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePPV\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNPV\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAccuracy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHR-HPV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.969\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.832\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.810\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCytology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.866\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.474\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.894\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.409\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.802\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ep16/Ki-67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.948\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.844\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.810\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eEach test contributes different diagnostic strengths: HPV yields maximal sensitivity, cytology contributes specificity, and p16/Ki-67 provides biologic confirmation of HPV-driven transformation.\u003c/p\u003e\n\u003cp\u003eKey differences include:\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eHR-HPV\u003c/strong\u003e: highest sensitivity (96.9%), lowest specificity\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eCytology\u003c/strong\u003e: highest specificity (47.4%), moderate sensitivity\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003ep16/Ki-67\u003c/strong\u003e: high sensitivity (94.8%), low specificity\u003c/p\u003e\n\u003cp\u003eEach test contributed distinct diagnostic strengths.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Triple-Testing Patterns\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntegration of all three tests revealed strong associations between test positivity patterns and histologic severity:\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eTriple-positive profiles (HPV+ / abnormal cytology / p16+)\u003c/strong\u003e were highly enriched in CIN3+ cases.\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eTriple-negative profiles\u003c/strong\u003e were rare and observed exclusively in CIN1 or benign cases.\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eHPV+/p16+ with NILM cytology\u003c/strong\u003e frequently corresponded to CIN2+, demonstrating the utility of molecular markers in detecting lesions missed by cytology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Prospective 2-Year Outcomes in Women Without Baseline Histology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the \u003cstrong\u003e88 women\u003c/strong\u003e without initial biopsy:\u003c/p\u003e\n\u003cp\u003e\u0026middot; Baseline HPV was \u003cstrong\u003epositive in 22 (25.0%)\u003c/strong\u003e and \u003cstrong\u003enegative in 66 (75.0%)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026middot; All women were \u003cstrong\u003ep16/Ki-67\u0026ndash;negative\u003c/strong\u003e at baseline.\u003c/p\u003e\n\u003cp\u003e\u0026middot; At the 2-year follow-up:\u003c/p\u003e\n\u003cp\u003eo \u003cstrong\u003e38 (43.2%)\u003c/strong\u003e were \u003cstrong\u003eNILM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eo \u003cstrong\u003e30 (34.1%)\u003c/strong\u003e had \u003cstrong\u003eCIN1 cytology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eo \u003cstrong\u003e20 (22.7%)\u003c/strong\u003e had \u003cstrong\u003eASC-US\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eo \u003cstrong\u003eNo CIN2+ cytology\u003c/strong\u003e was observed\u003c/p\u003e\n\u003cp\u003eTwo-year outcomes stratified by HPV status showed that HPV-positive women were more likely to have minor abnormalities (ASC-US or CIN1), whereas HPV-negative women tended toward NILM (Table 5).\u003c/p\u003e\n\u003cp\u003eTABLE 5. Two-Year Follow-Up Cytology Among Women Without Baseline Histology (n = 88)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"632\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCytology Result at 2 Years\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCount\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNILM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e43.2%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCIN1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e34.1%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eASC-US\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e22.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCIN2/3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNo CIN2+ cytologic abnormalities were detected after 2 years, supporting the safety of surveillance in p16/Ki-67\u0026ndash;negative women without baseline histology\u003c/p\u003e\n\u003cp\u003eFigure1. Two-year follow-up cytology outcomes among women without baseline histology (n = 88), stratified by baseline HPV status. HPV-positive women were more likely to exhibit minor abnormalities (ASC-US or CIN1), whereas HPV-negative women were predominantly NILM.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e In this study, we evaluated the diagnostic performance of triple testing\u0026mdash;HR-HPV genotyping, liquid-based cytology, and p16/Ki-67 dual staining\u0026mdash;in detecting precancerous cervical lesions in a real-world tertiary care population. Each modality demonstrated distinct diagnostic characteristics, and their combined interpretation provided a more comprehensive assessment of underlying histologic severity than any individual test alone.\u003c/p\u003e \u003cp\u003eConsistent with global literature, HR-HPV testing exhibited the highest sensitivity for CIN2+, correctly identifying nearly all high-grade lesions. This finding reinforces the well-established role of HPV testing as the most reliable primary screening method. However, as expected in a referral population with a high prevalence of HPV infection, specificity was extremely low. Similar patterns have been reported in diagnostic accuracy studies from high-risk clinical settings, where HPV testing alone cannot distinguish transient infections from clinically significant disease and therefore lacks sufficient discriminatory value to guide management decisions.\u003c/p\u003e \u003cp\u003eCytology demonstrated moderate sensitivity but the highest specificity among the three tests, underscoring its continued relevance as a morphologic assessment tool. Its limitations were evident, however, with 13.4% of CIN2\u0026thinsp;+\u0026thinsp;cases presenting with NILM cytology. This aligns with findings from large prospective trials showing that cytology, although useful, cannot reliably detect all high-grade lesions and is subject to sampling error and interpretive variability even in liquid-based formats. These limitations support the need for adjunctive molecular markers in contemporary screening algorithms.\u003c/p\u003e \u003cp\u003ep16/Ki-67 dual staining provided sensitivity comparable to HPV testing and identified the majority of CIN2\u0026thinsp;+\u0026thinsp;lesions missed by cytology. This strengthens its role as a marker of HPV-mediated oncogenic transformation. Although specificity was low in this enriched clinical population, this is expected in referral cohorts where p16/Ki-67 overexpression is common among HPV-positive or cytologically abnormal women. Importantly, incorporating p16/Ki-67 with HPV and cytology substantially improved risk stratification, particularly in cases with discordant or equivocal results.\u003c/p\u003e \u003cp\u003eThe analysis of triple testing patterns yielded clinically meaningful insights. Women who were concurrently HPV-positive, cytology-abnormal, and p16/Ki-67\u0026ndash;positive were overwhelmingly diagnosed with CIN3+, demonstrating the diagnostic value of combining virologic, morphologic, and molecular markers. Conversely, triple-negative profiles were rare and observed exclusively in CIN1 or benign cases, reinforcing the safety of conservative management in women with uniformly negative test results.\u003c/p\u003e \u003cp\u003eThe prospective follow-up of 88 women without baseline histology provides an additional strength. All women in this subgroup were p16/Ki-67\u0026ndash;negative at baseline, and none developed high-grade cytologic abnormalities over a two-year period. These data support the strong negative predictive value of molecular triage and suggest that surveillance rather than immediate biopsy may be an appropriate strategy in selected low-risk populations. This finding is particularly relevant in resource-limited settings and aligns with modern risk-based management principles.\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, the cohort was derived from a tertiary referral center, resulting in an elevated prevalence of CIN2\u0026thinsp;+\u0026thinsp;and reduced specificity across all modalities. Second, although follow-up data from the 88 women without baseline histology were informative, the absence of biopsy confirmation precludes direct comparison with the retrospective cohort. Nonetheless, their uniformly low-risk 2-year outcomes strengthen the argument for deferring biopsy in p16/Ki-67\u0026ndash;negative women. Finally, the single-center design may limit generalizability, although standardized protocols and consistent laboratory methods enhance internal validity.\u003c/p\u003e \u003cp\u003eOverall, this study demonstrates that triple testing provides a more complete and reliable diagnostic framework than any individual test. HR-HPV and p16/Ki-67 offer strong sensitivity for high-grade disease, while cytology contributes important morphologic specificity. Integrating these three modalities improves CIN2\u0026thinsp;+\u0026thinsp;detection, reduces false-negative results, and supports risk-adapted clinical management. These findings are consistent with global trends favoring HPV-based screening supported by molecular triage and provide meaningful evidence to support modernization of cervical cancer prevention strategies in North Macedonia.\u003c/p\u003e\n\u003ch3\u003eSTRENGTHS AND LIMITATIONS\u003c/h3\u003e\n\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e \u003ch2\u003eStrengths:\u003c/h2\u003e \u003cp\u003eThis study integrates virologic, cytologic, and molecular biomarkers to evaluate triple testing performance in a real-world tertiary population, providing one of the first datasets of this kind from North Macedonia. The inclusion of a large proportion of histologically confirmed cases strengthens diagnostic accuracy estimates, while the prospective follow-up of women without baseline biopsy offers valuable insight into the clinical behavior of low-risk profiles. Uniform laboratory techniques, standardized interpretation protocols, and blinded cytologic and immunocytochemical assessment enhance internal validity.\u003c/p\u003e \u003cdiv id=\"Sec35\" class=\"Section3\"\u003e \u003ch2\u003eLimitations:\u003c/h2\u003e \u003cp\u003eAs a single-center study conducted in a referral setting, the cohort demonstrates a high prevalence of CIN2+, which may limit generalizability and reduce the specificity of all three diagnostic tests. The absence of baseline histopathology in the prospective subgroup prevents direct comparison with the retrospective cohort, although 2-year cytology outcomes mitigate concerns regarding missed high-grade disease. Finally, the study was not designed to compare cost-effectiveness or long-term outcomes of triple testing, which remain important considerations for screening implementation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec36\" class=\"Section2\"\u003e \u003ch2\u003eCLINICAL IMPLICATIONS\u003c/h2\u003e \u003cp\u003eThe findings of this study have direct implications for cervical cancer prevention strategies in North Macedonia and similar settings transitioning from cytology-based screening to molecular approaches. The high sensitivity of HR-HPV and p16/Ki-67 confirms their value as primary and triage tools, respectively, and highlights the limitations of relying solely on cytology, which missed a meaningful proportion of CIN2\u0026thinsp;+\u0026thinsp;lesions. Integrating p16/Ki-67 with HPV and cytology enhances diagnostic precision, improves identification of high-grade lesions, and reduces the likelihood of false-negative outcomes.\u003c/p\u003e \u003cp\u003eImportantly, the 2-year follow-up data demonstrate that women with negative HPV and p16/Ki-67 results can be safely monitored without immediate colposcopy or biopsy, supporting risk-adapted follow-up algorithms and more efficient use of clinical resources. These findings support the adoption of triple testing\u0026mdash;or, at minimum, HPV-based screening with molecular triage\u0026mdash;as a modernized approach to cervical cancer prevention. Implementing such strategies may improve early detection, reduce overtreatment, and align national practices with international guidelines promoting HPV-centered screening programs.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eThis study was conducted in accordance with the ethical standards of the institutional research committee and with the principles of the Declaration of Helsinki and its later amendments. The study protocol was reviewed and approved by the Ethics Committee of the University Clinic of Gynecology and Obstetrics, Skopje, Republic of North Macedonia (Approval No. 04 - 1853/1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u003c/strong\u003e V.S.¹; O.D.¹; G.D.¹\u003cbr\u003e\u003cstrong\u003eMethodology:\u003c/strong\u003e V.S.¹; M.M.²; K.N.¹\u003cbr\u003e\u003cstrong\u003eData curation:\u003c/strong\u003e V.S\u003csup\u003e1\u003c/sup\u003e; M.M.²;D.V.²; V.N.²; L.V.²; D.B.P.³; D.R\u003csup\u003e4\u003c/sup\u003e; I.M.G.¹\u003cbr\u003e\u003cstrong\u003eInvestigation:\u003c/strong\u003e V.S.¹; M.M.²; D.V.²; L.V.²; O.D.¹\u003cbr\u003e\u003cstrong\u003eFormal analysis:\u003c/strong\u003e V.S.¹; G.D.¹; M.M.²\u003cbr\u003e\u003cstrong\u003eValidation:\u003c/strong\u003e G.D.¹; K.N.¹; I.M.G.¹\u003cbr\u003e\u003cstrong\u003eProject administration:\u003c/strong\u003e V.S.¹; O.D.¹\u003cbr\u003e\u003cstrong\u003eSupervision:\u003c/strong\u003e G.D.¹; M.M\u003csup\u003e2\u003c/sup\u003e.\u003cbr\u003e\u003cstrong\u003eWriting – original draft:\u003c/strong\u003e V.S.¹; O.D.¹\u003cbr\u003e\u003cstrong\u003eWriting – review \u0026amp; editing:\u003c/strong\u003e G.D.¹; K.N.¹; I.M.G.¹; M.M.²; D.V.²\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest relevant to this work.\u003c/p\u003e\n\u003cp\u003eNo author has any financial or personal relationships that could inappropriately influence, or be perceived to influence, the conduct or reporting of this study.\u003c/p\u003e\n\u003cp\u003eNo funding, grants, or material support were received for the execution of this research or the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors have completed the ICMJE Conflict of Interest Disclosure Form and affirm that there are no competing interests to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArbyn M, Weiderpass E, Bruni L, et al. Estimates of incidence and mortality of cervical cancer in 2018. Lancet Glob Health. 2020;8:e191\u0026ndash;203.\u003c/li\u003e\n\u003cli\u003eSchiffman M, Castle PE. The promise of global cervical cancer prevention. N Engl J Med. 2005;353:2101\u0026ndash;4.\u003c/li\u003e\n\u003cli\u003eWorld Health Organization. WHO guideline for screening and treatment of cervical pre-cancer lesions. Geneva: WHO; 2021.\u003c/li\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020. CA Cancer J Clin. 2021;71:209\u0026ndash;49.\u003c/li\u003e\n\u003cli\u003eNayar R, Wilbur D. The Bethesda System for Reporting Cervical Cytology. 3rd ed. Springer; 2015.\u003c/li\u003e\n\u003cli\u003eSasieni P, Adams J. Effectiveness of cervical screening with age. Int J Cancer. 1999;86:415\u0026ndash;20.\u003c/li\u003e\n\u003cli\u003eKitchener HC, Castle PE, Cox JT. HPV detection in cervical screening. BMJ. 2006;332:15\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eRonco G, Cuzick J, Carozzi F, et al. HPV-based vs cytology-based screening. Lancet. 2014;383:524\u0026ndash;32.\u003c/li\u003e\n\u003cli\u003eCarozzi F, Gillio-Tos A, Confortini M, et al. Sensitivity of LBC vs HPV testing. Br J Cancer. 2013;109:2444\u0026ndash;51.\u003c/li\u003e\n\u003cli\u003eASCCP Risk-Based Management Consensus Guidelines. J Low Genit Tract Dis. 2020;24:102\u0026ndash;31.\u003c/li\u003e\n\u003cli\u003eUSPSTF. Screening for cervical cancer. JAMA. 2018;320:674\u0026ndash;86.\u003c/li\u003e\n\u003cli\u003eBonde J, Ejegod D, Cuschieri K, et al. HPV testing and triage strategies. J Clin Virol. 2020;128:104411.\u003c/li\u003e\n\u003cli\u003eWentzensen N, Clarke MA, Bremer R, et al. Triage of HPV-positive women. J Natl Cancer Inst. 2022;114:528\u0026ndash;37.\u003c/li\u003e\n\u003cli\u003eKlaes R, Benner A, Friedrich T, et al. p16INK4a as a biomarker for CIN. Am J Surg Pathol. 2002;26:1389\u0026ndash;98.\u003c/li\u003e\n\u003cli\u003ePetry KU, Schmidt D, Scherbring S, et al. p16/Ki-67 for triage of HPV-positive women. Gynecol Oncol. 2011;121:505\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eWentzensen N, Schwartz L, Zuna RE, et al. p16/Ki-67 dual stain performance. J Natl Cancer Inst. 2015;107:djv257.\u003c/li\u003e\n\u003cli\u003eJeong NH, Lee JK, Lee NW, et al. Clinical utility of p16/Ki-67 in ASC-US triage. J Gynecol Oncol. 2016;27:e49.\u003c/li\u003e\n\u003cli\u003eCho HW, Ouh YT, Song JS, et al. Biomarker triage strategies for HPV-positive women. J Gynecol Oncol. 2019;30:e95.\u003c/li\u003e\n\u003cli\u003eKim HS, Park JS, Park NH. Role of dual staining in cervical screening. J Gynecol Oncol. 2021;32:e37.\u003c/li\u003e\n\u003cli\u003eCuzick J, Cadman L, Mesher D, et al. Performance of HPV testing with LBC and biomarkers. Int J Cancer. 2016;138:292\u0026ndash;302.\u003c/li\u003e\n\u003cli\u003eClarke MA, Cheung LC, Castle PE, et al. HPV screening with molecular triage. J Clin Oncol. 2018;36:1182\u0026ndash;90.\u003c/li\u003e\n\u003cli\u003ePerkins RB, Guido R, Castle PE, et al. Aptima HPV triage and adjunct testing. Am J Obstet Gynecol. 2020;223:693.e1\u0026ndash;693.e14.\u003c/li\u003e\n\u003cli\u003eInternational Agency for Research on Cancer (IARC). North Macedonia fact sheet: cervical cancer. 2021.\u003c/li\u003e\n\u003cli\u003eBryn S, Bergengren L, Karlsson MG. p16/Ki-67 vs cytology accuracy in HPV+ women. Acta Obstet Gynecol Scand. 2018;97:1275\u0026ndash;83.\u003c/li\u003e\n\u003cli\u003eBergeron C, Masseroli M, Cocco S, et al. Impact of dual staining in European screening. Cancer Cytopathol. 2015;123:373\u0026ndash;81.\u003c/li\u003e\n\u003cli\u003eSchmidt D, Bergeron C, Denton K, et al. p16/Ki-67 triage improves CIN2+ detection. Am J Clin Pathol. 2011;136:576\u0026ndash;84.\u003c/li\u003e\n\u003cli\u003ePolman NJ, Snijders PJ, Kenter GG, et al. HPV-based screening with cytology and biomarkers. Lancet Oncol. 2019;20:161\u0026ndash;70.\u003c/li\u003e\n\u003cli\u003eCuzick J, et al. Overview of screening efficacy using HPV testing. Int J Cancer. 2006;119(5):1095-1101.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Saints Cyril and Methodius University of Skopje","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HPV, Cytology, p16, Ki-67, Cervical Intraepithelial Neoplasia, Cervical Cancer Screening","lastPublishedDoi":"10.21203/rs.3.rs-8402586/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8402586/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e\u003cbr\u003e\nTo evaluate the diagnostic performance of triple testing—high-risk HPV (HR-HPV) genotyping, liquid-based cytology (LBC), and p16/Ki-67 dual staining—in detecting precancerous cervical lesions, and to assess 2-year follow-up cytology in women without initial biopsy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003cbr\u003e\nA total of 320 women underwent triple testing. Histopathology was available for 232 women and classified as CIN1, CIN2, CIN3, carcinoma in situ (CIS), or adenocarcinoma in situ (AIS). CIN2+ was defined as the primary diagnostic endpoint. Sensitivity, specificity, positive and negative predictive values, and accuracy were calculated for each test. The remaining 88 women without baseline histology entered a prospective follow-up arm and completed repeat cytology after 2 years.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003cbr\u003e\nAmong 232 women with histopathology, 194 (83.6%) had CIN2+ and 38 (16.4%) had CIN1. HR-HPV testing showed the highest sensitivity (96.9%) but low specificity in this referral population. Cytology demonstrated sensitivity of 86.6% and the highest specificity (47.4%). p16/Ki-67 dual staining showed high sensitivity (94.8%) with low specificity (10.5%). Triple-positive profiles (HPV+/abnormal cytology/p16+) strongly correlated with CIN3+.\u003cbr\u003e\nIn the follow-up group (n=88), all women were p16/Ki-67–negative at baseline; HPV was positive in 22 (25.0%). After 2 years, cytology results were NILM in 38 (43.2%), CIN1 in 30 (34.1%), and ASC-US in 20 (22.7%), with no CIN2+ cytologic abnormalities observed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003cbr\u003e\nTriple testing provides a comprehensive and highly sensitive diagnostic approach for detecting precancerous cervical lesions. The absence of CIN2+ during 2-year follow-up supports the safety of surveillance in HPV-negative and p16/Ki-67–negative women.\u003c/p\u003e","manuscriptTitle":"The Value of Triple Testing With p16/Ki-67 Dual Staining, Liquid-Based Cytology, and High-Risk HPV in Correlation with Histopathology in Precancerous Cervical Lesions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-24 05:47:32","doi":"10.21203/rs.3.rs-8402586/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0cfc7ad6-a15a-4d20-bf36-5d96b730c253","owner":[],"postedDate":"December 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59936341,"name":"Pathology"},{"id":59936342,"name":"Obstetrics \u0026 Gynecology"}],"tags":[],"updatedAt":"2025-12-24T05:47:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-24 05:47:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8402586","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8402586","identity":"rs-8402586","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}