Reference gene evaluation for digital PCR; Applications for RNA biomarker testing in cervical precancer.

Background Cervical and breast cancer are the leading malignancies among females in Nepal. The World Health Organization (WHO) reported a crude cervical cancer incidence rate in Nepal of 14.2 per 100,000 women per year in 2022. Antenatal cervical screening provides an excellent opportunity to screen and create awareness for further surveillance. This study aimed to determine the prevalence of abnormal cervical cytology among pregnant Nepalese women and identify associated risk factors. MethodsAn observational cross-sectional study was conducted in Department of Obstetrics and Gynaecology at a tertiary center. Pregnant women between 20 and 28 weeks of gestation attending antenatal check-ups underwent liquid-based cytology (LBC). Cytology results were reported using the Bethesda classification system (2014). ResultsAmong 78 enrolled pregnant women who completed the study, cytology indicated abnormalities in 58 women (74.4%), while 20(25.6%) were negative for intraepithelial lesion or malignancy (NILM). Among abnormal findings, inflammatory changes were most frequent 29(37.2%), followed by bacterial vaginosis 12(15.4%), Trichomonas vaginalis 12(15.4%), and candidiasis 5(6.4%). No premalignant or malignant lesions were detected. Conclusions Antenatal cervical screening offers a valuable opportunity to detect genital infections, screen for cervical cancer precursors, and raise awareness for future screening. Liquid based cytology test is easy to perform, cost effective and does not adversely affect pregnancy, playing a crucial role in reducing preventable cervical disease burden in low-resource settings.

Objective, To explore the infection status of high-risk human papillomavirus (HR-HPV) in postmenopausal women and the value of liquid-based thin-layer cytology test (TCT) in cervical lesion screening. Methods: A retrospective analysis was conducted on the HR-HPV and TCT results of 193 postmenopausal women aged 55 and above who underwent colposcopy at Luancheng People's Hospital in Shijiazhuang from 2018 to 2023. Biopsy results were used as the gold standard to calculate and compare the screening efficacy of the two methods. Results: Among the 193 women, 162 were TCT positive, with a positive rate of 83.94%; 107 were HR-HPV positive, with a positive rate of 55.44%. The HR-HPV positive rate was significantly lower than that of TCT （χ² = 109.003, P < 0.001）. Among the 108 biopsy-positive patients, TCT detected 97 cases, with a sensitivity of 89.81%; HR-HPV detected 78 cases, with a sensitivity of 72.22%. In the TCT-negative group, 11 cases of pathological positivity were detected, with a missed diagnosis rate of 10.19%; in the HR-HPV-negative group, 30 cases of pathological positivity were detected, with a missed diagnosis rate of 27.78%. Conclusion: In cervical cancer screening for postmenopausal women, TCT has a high sensitivity and low missed diagnosis rate, while HR-HPV has a relatively low sensitivity and high missed diagnosis rate. The combined screening strategy of HR-HPV and TCT can improve the detection rate of cervical lesions and is an ideal cervical screening model for postmenopausal women.

Cervical cancer remains a major public health challenge, especially in developing countries, and is strongly associated with persistent infection by oncogenic types of Human Papillomavirus (HPV). This article addresses the main molecular mechanisms involved in cervical carcinogenesis, highlighting the action of the viral oncoproteins E6 and E7 on tumor suppressor genes and their role in the progression of squamous intraepithelial lesions to invasive carcinoma. The relevance of the cervicovaginal cytopathological examination as a central screening tool is discussed, as well as the contribution of complementary methods, such as DNA-HPV detection tests using liquid-based cytology samples, to improve diagnostic accuracy. From a clinical perspective, the differential diagnosis of lesions is of utmost importance, as treatment is guided by the type of lesion. In the context of prevention, strategies aimed at reducing the risk of cervical cancer development are discussed, including preventive measures related to HPV infection. It is emphasized that this article focused exclusively on squamous cervical lesions, which are responsible for the majority of precursor alterations of cervical cancer.

The transition from conventional smear (CS) cytology to liquid-based cytology (LBC) in thyroid fine-needle aspiration (FNA) has been a subject of debate, with varying reports on diagnostic performance. This retrospective observational study evaluates this institution's 5-year experience with LBC in thyroid cytology from 2020 to 2024, comparing its advantages and results with past experience using CS combined with rapid on-site assessment (ROSE) from 2016 to 2018, and correlating cytological diagnoses with histopathological outcomes. We performed a retrospective analysis of all thyroid FNAs processed and reported at the cytopathology department of Cleveland Clinic Abu Dhabi-National Reference Laboratory (CCAD/NRL) using the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC). A total of 741 nodules were aspirated from January 2016 to May 2018 using primarily conventional smears with the routine practice of ROSE (pre-LBC), while 4993 nodules were aspirated via LBC (ThinPrep) between July 2020 and September 2024 without ROSE (post-LBC). Cases with subsequent surgical resection were reviewed and correlated with the pre-operative cytology diagnosis to assess diagnostic accuracy. Data analysis before and after transitioning to LBC was performed to compare diagnostic performance, Bethesda category distribution, and surgical outcomes. LBC was associated with improved pre-analytical standardization, with nondiagnostic rates stabilizing at 10.8% compared with 4%-7% during the pre-LBC period when ROSE was routinely used. The atypia of undetermined significance (AUS; Bethesda III) rate significantly decreased from 16% pre-LBC to 7% post-LBC, with architectural atypia alone comprising 3% post-LBC. Histologic correlation was available for 199 pre-LBC and 258 post-LBC nodules. Pre-LBC cytology demonstrated sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 80.8%, 95.1%, 93.3%, and 85.3%, respectively. Post-LBC metrics improved to 92.3% sensitivity and PPV, and 98.8% specificity and NPV. Among Bethesda II nodules, the false-negative rate decreased from 14.3% pre-LBC to 2.3% post-LBC. AUS nodules with nuclear atypia demonstrated a 92.3% malignancy rate post-LBC, compared to 40.4% in unstratified AUS cases pre-LBC. Oncocytic cell-rich indeterminate nodules were predominantly benign in both cohorts. LBC is associated with several practical advantages over CS in thyroid cytology. In addition to streamlining and standardizing diagnostic workflows, it significantly helped to improve diagnostic accuracy. We observed a significant decrease in AUS rate, reducing interpretive uncertainty, and enhanced detection of malignancy with nuclear atypia. These findings validate Bethesda's recommendations for more granular classification of indeterminate cytology. The nondiagnostic rate increased slightly in the post-LBC period, a predictable effect of ROSE withdrawal. However, this modest rise was offset by the substantial gains in workflow efficiency and overall diagnostic accuracy achieved with LBC. This long-term experience reinforces LBC as a reliable and efficient method for thyroid FNA evaluation and better risk stratification.

Human papillomavirus (HPV) testing is the preferred method for cervical cancer screening. We aimed to assess the distribution of HPV genotypes among women with screen-positive (SP) results and histopathological (HP) confirmed high-risk lesions. Women aged 25-65 years presenting to the outpatient department who underwent routine cervical cancer screening and who screened positive were enrolled in the study. Patients referred with an SP report were also enrolled. HPV testing, colposcopic evaluation, and guided biopsy were performed. Follow-up cases of genital malignancies and hysterectomized women were excluded. Out of 5687 women screened, 123 (2.2%) tested positive via cytology or visual methods, and 118 were analyzed. Most (84.7%) tested positive through liquid-based cytology (atypical squamous cells of undetermined significance and above, ASCUS+). HPV was detected in 56.8% of cases (67/118); 25% had multiple strains. HPV 16 was most prevalent (38.1%). Among women with biopsy-proven HSIL and above (bHSIL and bHSIL+) lesions, HPV 16 was most common (78%), followed by HPV 18 (14%) and 33 (12%). There was a strong correlation between HPV genotype positivity and presence of bHSIL and bHSIL+ lesions (ϕ = 0.7; P < 0.001). HPV 16, 18, 31, 33, and 45 were found in high-grade and invasive lesions. Testing for either all high-risk HPV types or a subset (16, 18, 31, 33, 45) had similar diagnostic accuracy. This study highlights the predominance of HPV 16, 18, 31, 33, and 45 in cervical lesions, emphasizing the need for targeted screening and vaccination against these high-risk genotypes to reduce HPV-related disease burden in the Indian population.

Endometrial cancer represents a major global health concern, with rising incidence particularly in developed countries despite declining mortality rates. This comprehensive review examines the evolution of endometrial cancer screening techniques, encompassing traditional methods, emerging technologies, and integrated approaches. Traditional screening methods including transvaginal ultrasound and hysteroscopy demonstrate varying diagnostic capabilities. Hysteroscopy combined with endometrial biopsy exhibits superior diagnostic accuracy with sensitivity ranging from 62.5 to 100% and specificity from 67.8 to 98.9%. Recent advances in molecular diagnostics show promising potential for non-invasive screening, with DNA methylation analysis achieving sensitivity of 91.8% and specificity of 95.5% when utilizing specific gene panels. Cell-based screening methods including liquid-based cytology and immunocytochemistry provide additional diagnostic pathways, demonstrating sensitivity of 84% and specificity of 98%. Digital image analysis integrated with deep learning technologies enables molecular subtype prediction from histopathological images without requiring expensive sequencing. Combination screening approaches, particularly integrating molecular diagnostics with traditional imaging, demonstrate enhanced diagnostic performance while potentially reducing costs through decreased unnecessary procedures. Molecular testing carries broader implications beyond individual patients, as detection of germline mutations such as Lynch syndrome enables cascade genetic screening benefiting family members through early detection and surveillance programs. While universal screening remains cost-prohibitive for asymptomatic populations, evidence strongly supports personalized screening strategies based on individual risk factors including obesity, diabetes, and hereditary cancer syndromes. Future developments will likely emphasize integrated approaches combining molecular diagnostics with traditional methods to optimize diagnostic accuracy, accessibility, and cost-effectiveness.

Cervical cancer (CC) can be prevented through continuous screening and the timely detection of cervical intraepithelial neoplasia (CIN) using immunohistochemistry techniques to identify biomarker expressions. In a previous study, we proposed nuclear REST loss as a biomarker in precancerous lesions and CC; however, no validated antibodies are available for detecting REST in cytology or cervical tissues. Although we have developed an IgM-type anti-REST monoclonal antibody capable of detecting REST in liquid-based cytology cells, it was not useful for the detection of REST in cervical tissues by immunohistochemistry. The main objective of this study is to generate single-chain variable fragments (scFvs) for the clinical evaluation of REST in cervical tissues from women with CIN and CC. Using RNA from an IgM-producing hybridoma anti-REST, we conducted RT-PCR and PCR to obtain the coding sequences for the variable regions of the heavy and light chains. These sequences were joined with a linker to create a single-chain antibody. The scFv was then cloned into the pSyn1 vector, expressed in E. coli TG1, and purified through chromatography. Subsequently, it was characterized using immunological methods to assess its biological activity and employed to evaluate REST expression in cytological samples and cervical tissues. The anti-REST scFv represents an innovative detection tool that retains the antigen recognition of the parental IgM while overcoming its size limitation, enabling tissue penetration and detection of REST in cervical samples. Its application facilitates the identification of REST in cervical samples, reinforcing REST’s potential as a diagnostic biomarker for CC and CIN.

Molecular testing is increasingly used to improve preoperative risk assessment of thyroid nodules, especially those with indeterminate cytology. This study evaluated the performance of the Myriapod next-generation sequencing (NGS) DNA-only cancer panel in fine-needle aspiration cytology thyroid samples, correlating findings with postsurgical diagnoses. A retrospective analysis was performed on fine-needle aspiration cytology specimens from 74 thyroid nodules in the TIR3A, TIR3B, TIR4, and TIR5 categories according to the Italian Consensus for the Classification and Reporting of Thyroid Cytology. DNA from these samples, obtained from residual liquid-based cytology material, was analyzed with the Myriapod NGS panel, targeting 16 genes implicated in thyroid cancer. All patients underwent surgery, allowing for histopathologic correlation. The residual liquid-based cytology material yielded adequate DNA for molecular testing in 89.2% of the nodules. All TIR3A (low-risk intermediate) nodules were histologically benign, whereas 50% of TIR3B (high-risk intermediate) nodules were malignant; mutations were identified only in the malignant nodules. In the TIR4 category (suspicious for malignancy), BRAF V600E was the most frequent mutation in malignant nodules. Both TIR5 (malignant) nodules were papillary thyroid carcinomas with a BRAF V600E mutation. The molecular test demonstrated 100% sensitivity, 95.5% specificity, 91.7% positive predictive value, and 100% negative predictive value for samples that were adequate for molecular testing. An intention-to-diagnose analysis that included samples inadequate for molecular testing was also performed, yielding 84.6% sensitivity, 87.5% specificity, 91.7% positive predictive value, and 84% negative predictive value. The Myriapod NGS panel aids in the preoperative assessment of thyroid nodules. Its high negative predictive value may help avoid unnecessary surgery, whereas the detection of specific mutations strongly correlates with malignancy, thus informing surgical planning.

One of the biggest causes of cancer-related fatalities among women is still Cervical cancer, especially in low and middle-income nations where access to broad screening and early detection may be limited. Cervical cancer is curable if detected in its early stages, but asymptomatic progression frequently results in late diagnosis, which makes treatment more difficult and lowers survival chances. Even though they work well, current screening methods including liquid-based cytology and Pap smears have drawbacks in terms of consistency, sensitivity, and specificity. Recent developments in Deep Learning and Artificial Intelligence have shown promise for greatly improving Cervical cancer detection and diagnosis. In this work, we have introduced CervixCan-Net, a novel Deep Learning based model created for the precise classification of Cervical cancer from histopathology images. Our approach offers a solid and dependable classification solution by addressing common problems like overfitting and computational inefficiency. CervixCan-Net performs better than many state-of-the-art models according to a comparison investigation. CervixCan-Net, with an impressive test accuracy of 99.83%, provides a scalable, automated Cervical cancer classification solution that has great promise for improving patient outcomes and diagnostic accuracy.

High-grade cervical intraepithelial lesions (CIN 2/3) are critical targets for early detection in cervical cancer prevention. MicroRNAs are stable, tissue-specific molecules involved in cancer-related pathways and can be detected in minimally invasive samples, making them suitable for use in the context of liquid biopsies. This exploratory study aimed to identify differentially expressed microRNAs in paired liquid-based cytology (LBC) and plasma samples from women with CIN 2/3, and to evaluate miR-339-3p as a potential biomarker. Samples from 70 women (35 cases with histologically confirmed high-grade lesions and 35 age-matched controls with normal cytology and negative HPV tests) were analyzed using a commercial microRNA panel on the NanoString platform. We identified 57 dysregulated microRNAs in LBC samples and 33 in plasma, with four shared between both matrix types. Among these, miR-339-3p was the only one consistently upregulated and significantly associated with case status. In LBC samples, the area under the curve was 0.65; in plasma, 0.64. Pathway analysis suggested its involvement in apoptosis-related pathways, including PI3K-Akt and p53. Moreover, this study highlights the value of integrating microRNA profiling across local and systemic samples to advance biomarker discovery in cervical cancer.

Background/Objectives: Atypical cytological findings in cervical screening, such as ASC-US, ASC-H, and AGC, present a clinical challenge due to their variable risk of underlying high-grade lesions. The precise stratification of this risk is crucial for effective patient management. This study aimed to correlate Bethesda cytology categories with HPV genotyping, including viral load, and histological follow-up to improve risk prediction for cervical intraepithelial neoplasia grade 2 or worse (CIN2+). Materials and Methods: In this retrospective single-center study, we analyzed 407 patients with cytological reports of ASC-US, ASC-H, or AGC. All patients underwent HPV DNA testing with genotyping for 21 types, with viral load quantification for HPV16/18, and subsequent histological verification. Statistical analyses included non-parametric tests, correlation analysis, and multivariate logistic regression to identify independent predictors of CIN2+. Results: The prevalence of CIN2+ differed significantly among the cytological categories: 23.2% in ASC-US, 47.3% in ASC-H, and 19.5% in AGC. ASC-H and a high HPV16 viral load were identified as independent predictors of CIN2+ in the multivariate analysis. An ASC-H result increased the probability of CIN2+ by 2.5 times (aOR = 2.51; 95% CI: 1.28–4.94). For each 1 log10 increase in HPV16 viral load, the risk of CIN2+ increased by 30% (aOR = 1.30; 95% CI: 1.16–1.46). Stratification of ASC-US cases by HPV16 status revealed a dramatically higher positive predictive value (PPV) for CIN2+ in HPV16-positive patients (66%) compared to HPV16-negative patients (12.6%). The AGC category showed the strongest association with glandular pathology, including adenocarcinoma in situ. Conclusions: The combination of cytological findings and HPV16 viral load provides a powerful model for risk stratification. An ASC-H result is a strong independent risk marker, while the clinical significance of ASC-US is fundamentally determined by HPV16 status. These findings advocate for a risk-based management algorithm that integrates liquid-based cytology with extended HPV genotyping and viral load assessments to optimize patient triage and follow-up.

High risk human papillomaviruses (hrHPVs) cause most cervical cancers. Cervical screening programmes aim to identify precancerous disease using detection of hrHPV nucleic acid using clinician-taken liquid-based cytology (LBC) samples, or increasingly, self-taken samples (STSs). However, STSs are incompatible with traditional cytology triage. Therefore, development of novel triage tests is essential. Quantification of cellular and viral mRNA biomarkers is one option, but little is known about mRNA quality in STSs. We extracted RNA from two sets of STSs (reflecting separate sampling devices) from the Scottish HPV Archive (SHA) and the Swedish Cervical Cytology Biobank (SCCB). We investigated whether the ACTB, GAPDH and p16 RNAs could be amplified by reverse transcription quantitative PCR (RT-qPCR). RNA was degraded in both sets of samples, but samples from the Scottish HPV Archive were generally suitable for RT-qPCR analysis, while samples from the SCCB were mostly unsuitable. Genomic DNA contamination was detected in 11.6% of samples. The quantity and quality of RNA derived from STSs was unaffected by storage of the original sample at -70°C for a period of 1 year. These data suggest that feasibility of utilising STSs for mRNA expression work is device-dependent and that optimisation of collection and storage systems is warranted.

Artificial intelligence-assisted diagnostic systems in cervical liquid-based cytology: A multi-centered study

The aim of this study was to evaluate the clinical performance of 2 DNA-based assays (DNA-xGT, DNA-lGT) and 1 RNA-based assay (mRNA), using specimens from women with cervical carcinoma or high-grade cervical lesions (≥CIN3). Liquid-based cytology samples collected from 154 patients with ≥CIN3 histology results were tested with DNA-xGT, DNA-lGT, and mRNA assays. The clinical sensitivities of the HPV testing for disease detection were evaluated. Genotype-specific agreement was also assessed between the assays. The clinical sensitivity of the DNA-xGT, DNA-lGT, and mRNA in detecting ≥CIN3 were 96.8%, 96.1%, and 91.6%, respectively. The overall high-risk HPV positive percent agreement ranged from 92.6% to 99.3% across different assay pairs analyzed. Composite comparator analysis demonstrated no significant difference between the DNA assays; however, a significant difference was observed between the RNA assay and the DNA assays. While no statistically significant difference was observed between DNA-xGT and DNA-lGT assays in disease detection rate, the mRNA test showed a significantly lower detection rate when compared to the DNA-xGT assay. Most of the missed infections (8/13) were identified as HPV16 or HPV18.

Liquid biopsy offers a minimally invasive approach for cancer diagnosis by detecting circulating biomarkers, such as microRNAs (miRNAs), in different types of specimens. The present study aimed to identify the most effective specimen for miRNA detection in cervical cancer precursor lesions by comparing plasma, urine and liquid-based cytology (LBC). A total of 798 miRNAs were analyzed using NanoString technology in 24 women, equally divided between those with high-grade cervical intraepithelial neoplasia (2/3) and controls with negative Pap and human papillomavirus tests. Comparative analyses revealed that LBC exhibited the highest detection efficiency, with only 1.75% of miRNAs demonstrating low counts, compared with 20.68% in plasma and 15.79% in urine. Additionally, LBC exhibited 21.3% of miRNAs above the 9th decile of expression, compared with 5.89% in plasma and 2.88% in urine. LBC revealed the most consistent detection performance across samples, establishing its potential as the most effective specimen for detecting cervical cancer precursor lesions biomarkers. Plasma also showed promise as a detection medium, whilst urine exhibited higher variability and lower detection consistency. In conclusion, LBC demonstrated the highest efficacy for miRNA detection among the specimen types tested. Plasma remains a viable alternative, whilst urine presents challenges due to its inherent variability. These results underscore the importance of specimen selection for optimizing diagnostic sensitivity in cervical cancer precursor lesions screening.

IntroductionCervical cancer has become one of the most malignant tumors that threatens women's health worldwide. Liquid-based cytology (LBC) examination has become the most common screening method for detecting cervical cancer early and preventing it. Currently, nuclear segmentation technology for cervical clinical LBC images based on convolutional neural networks has become a vital means of assisting in the diagnosis of cervical cancer. However, the existing nuclear segmentation techniques fail to segment the nuclei of severely overlapping nuclei in highly aggregated cell clusters, which will inevitably lead to the misdiagnosis of cervical cancer pathology.MethodsTherefore, a novel bending loss and dual-task decoding network (Bloss-DDNet) is proposed for overlapping cell nucleus segmentation of cervical clinical LBC images. First, the network architecture search method is introduced to search and optimize the architecture of the decoding module in the dual-task branch, determining the mask and boundary decoding modules (dual-task decoding modules) of the Bloss-DDNet. Second, two feature maps, separately generated from dual-task decoding branches composed of a shared encoder module and dual-task decoder modules, are fused to enhance the sensitivity to cell nucleus boundaries. Third, a bending loss is introduced to the loss function to focus on the curvature variation characteristics of the intersection of overlapping cell nucleus boundaries, thereby constraining the training process of the dual-task decoding branch and increasing the constraint on the cell nucleus boundary.ResultsThe results show that all evaluation metrics of the proposed Bloss-DDNet achieved the best performance on public datasets.DiscussionTherefore, the proposed Bloss-DDNet can effectively address the segmentation problem of overlapping cell clusters and nuclei in clinical LBC images, providing strong support for subsequent clinical auxiliary diagnosis of cervical cancer.

Introduction: Oral squamous cell carcinoma (OSCC) is the most prevalent oral malignant neoplasm. Cytopathology may represent an important tool in the screening of OSCC, and liquid-based oral brush cytology (LBOBC) has been widely studied because of its clearer cell sample results. These cytopathological analyses could be more efficient with the aid of artificial intelligence. The objective of this study was to analyze the effectiveness of two AI models (Papanicolaou and AgNOR Slide Image Examiners) in LBOBC analyses. Methods: Two human evaluators and the AI models performed cell maturation pattern analysis and mean nucleolar organizer region (NOR) per nucleus count in Papanicolaou and silver-stained nucleolar organizer region (AgNOR) oral cytopathological samples of 20 individuals, respectively. Inter-evaluator agreement was evaluated by kappa and intraclass correlation coefficient. Chi-square and Wilcoxon matched-pairs/Friedman tests analyzed differences between the conventional and LBOBC methods and among evaluators. Results: Kappa between the Papanicolaou AI model and each human researcher was substantial (k = 0.69) for the conventional method and moderate for the LBOBC (k = 0.55–0.53. There were statistical differences in the cellular type analysis between cytology methods and among evaluators (p < 0.001). The automated AgNOR model showed an excellent/highly good agreement with human evaluators for NOR count in both cytology methods, with and without bounding boxes. There was no statistical difference in the NOR count between methods (p > 0.05). In the conventional method, there were differences among evaluators (p < 0.05); in the LBOBC, there were not (p > 0.05). Conclusion: The AgNOR automated model is reliable when assessing NOR count in oral samples processed by different cytological methods, when compared to the human analysis. The Papanicolaou model still needs more training with LBOBC samples.

This study aimed to assess the diagnostic‐support and triage value of PAX1/JAM3 methylation testing for identifying high‐grade cervical lesions among postmenopausal women referred for colposcopy. A total of 216 women aged ≥50 years who underwent colposcopy due to positive high‐risk human papillomavirus (hrHPV)/cytology detection and/or abnormal clinical symptoms were included, and 212 women aged <50 years matched 1:1 by hrHPV and cytology results as the control group. The effectiveness of PAX1/JAM3 methylation in detecting high‐grade cervical intraepithelial neoplasia (CIN) was compared to traditional screening methods. PAX1/JAM3 methylation showed a sensitivity of 93.2%[85.7%–100%] for detecting CIN2+ lesions (97.2%[91.9%–100%] for CIN3+), with a specificity of 93.6%[89.9%–97.3%] for CIN1‐lesions, outperforming liquid‐based cytology (LBC) (CIN2+/CIN3+ sensitivity: 75%[60.9%–89.1%]; specificity: 52.3%[44.9%–59.8%]) and the combination of LBC and hrHPV tests according to current guidelines (CIN2+/CIN3+ sensitivity: 81.8%[70.4%–93.2%]/83.3%[71.2%–95.5%]; specificity: 45.3%[37.9%–52.8%]). Methylation detection successfully identified two adenocarcinoma cases with negative hrHPV and LBC, as well as 7 patients with non‐16/18 hrHPV infection that were missed by LBC. The methylation levels of PAX1 and JAM3 were elevated in elderly women with CIN2+ compared to younger women, and showed no association with HPV types. In conclusion, PAX1/JAM3 methylation testing showed promising diagnostic‐support performance among postmenopausal women referred for colposcopy, suggesting potential utility as a supplementary detection method to improve diagnostic accuracy in older women.

Background/Objectives: Cervical cancer is one of the most frequent malignancies among women in Kazakhstan, where human papillomavirus (HPV) vaccination was initiated in 2024. Despite the implementation of vaccination and cytology-based screening programs, diagnostic limitations remain, and local evidence linking HPV infection to clinical outcomes is scarce. This study aimed to evaluate the correlation between HPV status, cervical cytology results, colposcopic impression, and biopsy results in a non-vaccinated female population. Methods: A cross-sectional study was conducted at the University Medical Center, Astana, between November 2024 and March 2025. A total of 396 women of reproductive age were enrolled. Cervical samples underwent liquid-based cytology and high-risk HPV testing with the RealBest assay. Colposcopy was performed following abnormal cervical cytology results, and colposcopy-guided biopsies were obtained where indicated. Sociodemographic characteristics were assessed, and associations between HPV genotype and clinical outcomes were analyzed using descriptive and inferential statistics. Results: HPV infection was detected in 140 women (35.4%). HPV-16 was the most common genotype (11.4%), followed by HPV-52 (6.6%) and HPV-33 (5.3%). Among 198 women evaluated by colposcopy, abnormal findings were observed in 72.2%, with HPV-16 showing a significant association with higher-grade abnormalities (p < 0.001). Biopsies were available for 40 participants: 12 had CIN I, 12 had CIN II, 10 had CIN III, and 4 had carcinoma in situ. HPV-16 was the only genotype significantly linked to CIN II/III lesions. Conclusions: HPV-16 was strongly associated with abnormal colposcopic findings and high-grade histology, underscoring its oncogenic importance. The prevalence of HPV-52 and HPV-33 further supports the need for HPV nonavalent vaccination. These findings highlight the importance of HPV-based screening, genotype-specific triage, and expanded vaccination to reduce cervical cancer incidence in Kazakhstan.