Search Results (13)

Background and Objectives: p16 immunohistochemistry (IHC) is widely used as a surrogate marker for high-risk human papillomavirus (HPV)-driven carcinogenesis in oropharyngeal squamous cell carcinoma. Diagnostic reliability in laryngeal squamous cell carcinoma (LSCC) is still up for debate, particularly because HPV DNA detection, p16 overexpression, and transcriptionally active HPV infection may be discordant at this anatomical site. This systematic review aimed to assess the diagnostic performance of p16 IHC as a surrogate marker for high-risk HPV status in primary invasive LSCC. Materials and Methods: A systematic review was conducted in accordance with PRISMA principles and prospectively registered in PROSPERO. PubMed, Scopus, and Web of Science, and citation searches were used to identify studies reporting paired p16 IHC and tumour-based molecular HPV testing in invasive LSCC. Eligible HPV reference standards included HPV DNA PCR/genotyping, DNA in situ hybridization, RNA in situ hybridization, and E6/E7 mRNA detection. Data were synthesized narratively because of substantial heterogeneity in p16 thresholds, HPV assays, and study populations. Results: Fourteen studies were included. p16 positivity thresholds varied widely, ranging from ≥30% moderate/strong staining to ≥70–75% diffuse nuclear and cytoplasmic staining. HPV reference standards also differed substantially across studies. Overall, p16 IHC showed inconsistent concordance with molecular HPV testing. Studies using RNA-based reference standards showed that transcriptionally active HPV was uncommon in LSCC and that p16-positive tumours often lacked evidence of active HPV transcription. Conclusions: p16 IHC should not be used as a standalone surrogate marker for high-risk HPV-driven carcinogenesis in invasive LSCC. When HPV attribution is clinically or analytically important, p16-positive cases should be confirmed using HPV-specific molecular testing, preferably RNA-based assays. Future prospective studies using standardized p16 protocols and transcriptionally active HPV reference standards are needed. Full article

Background/Objective: Cervical intraepithelial neoplasia grade 2 (CIN2) shows heterogeneous clinical behavior, with substantial rates of spontaneous regression under active surveillance. Reliable molecular biomarkers are needed to distinguish regressive from transforming lesions and reduce overtreatment. We evaluated the prognostic role of host and viral DNA methylation, alone and combined with HPV genotyping, in predicting CIN2 regression. Methods: This subanalysis derives from a prospective, multicenter Italian cohort of women with histologically confirmed CIN2 managed conservatively. Among 319 enrolled women, 134 with single HPV infections and valid host (FAM19A4/miR124-2) and viral (HPV L1 region) methylation results were included. HPV genotyping was performed with partial stratification (HPV16/18 vs. non-16/18). Clinical outcomes at 24 months were classified as regression versus persistence/progression. Logistic regression models assessed associations between biomarkers and regression. Results: At 24 months, 50% of women showed regression. Host and viral methylation positivity rates were more frequent in non-regressive lesions (40.3% vs. 19.4%, p = 0.01, and 52.2% vs. 32.8%, p = 0.02, respectively). Negative host methylation was significantly associated with regression (Odds Ratio OR = 0.37, 95% CI 0.17–0.81, p = 0.02), as was negative viral methylation (OR = 0.47, 95% CI 0.23–0.96, p = 0.04). Conclusions: Both host and viral methylation are inversely associated with CIN2 regression. Combining methylation markers did not substantially improve predictive accuracy; however, methylation negativity emerged as a potential molecular reassurance marker. When integrated with HPV genotyping, the highest probability of regression was observed among women with non-HPV16/18 infections and negative methylation results. These results endorse DNA methylation testing as a molecular tool for the conservative management of CIN2. Full article

Endocervical adenocarcinoma is now classified within an etiologic framework based on the presence or absence of high-risk human papillomavirus (HPV) infection. Gastric-type endocervical adenocarcinoma (GAS) is the prototypical HPV-independent subtype, accounting for up to 25% of endocervical adenocarcinomas and showing a particularly high frequency in East Asia. GAS is typically diagnosed at a more advanced stage than usual-type HPV-associated endocervical adenocarcinoma (UEA); exhibits deep stromal and parametrial invasion, lymphovascular space invasion, and a strong propensity for ovarian and peritoneal metastasis; and is associated with markedly worse survival, even in stage I disease. Radiological evaluation is challenging because of diffuse infiltrative growth, prominent mucin production, and frequent underestimation of extra-cervical spread. Histologically, GAS shows gastric-type (pyloric) differentiation, ranging from minimal deviation adenocarcinoma to poorly differentiated forms, and often overlaps with precursor lesions such as atypical lobular endocervical glandular hyperplasia and gastric-type adenocarcinoma in situ. Immunophenotypically, GAS is typically p16-negative, ER/PR-negative, and frequently exhibits mutant-type p53 and expression of gastric markers including MUC6, HIK1083, and claudin 18.2. Recent next-generation sequencing and multi-omics studies have revealed recurrent alterations in TP53, CDKN2A, STK11, KRAS, ARID1A, KMT2D, and homologous recombination-related genes, together with the activation of PI3K/AKT, WNT/β-catenin, TGF-β, and EMT pathways and characteristic metabolic reprogramming. GAS is highly resistant to conventional chemotherapy and radiotherapy, and its current management follows guidelines for squamous and usual-type adenocarcinoma. Emerging data support precision-medicine approaches targeting HER2/HER3, PD-1/PD-L1, and claudin 18.2, and suggest a role for PARP inhibition and other genotype-directed therapies in selected subsets. Given its aggressive biology and rising relative incidence in the HPV-vaccination era, GAS represents a critical unmet need in gynecologic oncology. Future progress hinges on developing reliable diagnostic biomarkers, refining imaging protocols, and validating targeted therapies through international clinical trials. Full article

Cervical cancer remains the fourth most common malignancy among women worldwide. Despite well-developed prevention measures, incidence and mortality continue to rise, especially in low- and middle-income countries due to low screening coverage and unavailability of human papillomavirus (HPV) vaccination. The cervical cancer screening coverage could be improved by the implementation of a self-sampling modality for HPV testing. Multiple research pieces support the validity and reliability of a self-sampling modality as an alternative approach to clinician-collected samples for primary cervical cancer screening via HPV genotyping. Moreover, growing research evidence on the self-sampling modality reception shows high acceptance of the method among screened populations. Studies on the self-sampling approach economic efficiency also revealed a high cost-effectiveness of HPV testing through a self-sampling modality compared to other screening modalities for cervical cancer. It is specifically important for low-resource settings, which should use the self-sampling cost advantages to improve cervical cancer screening coverage by attracting underscreened populations. Overall, self-sampling modality has a higher participation rate and better patient satisfaction reported; thus, the method is highly recommended by the World Health Organization for cervical cancer screening. Full article

Background and Objectives: Human Papillomavirus (HPV) is a leading cause of cervical and other anogenital cancers, with over 200 known genotypes classified into high-risk, probable high-risk, and low-risk groups. While conventional diagnostic and classification approaches often rely on sequence alignment, phylogenetic relationships, or protein structure analyses, these methods are limited in scalability, cost efficiency, and generalizability to emerging HPV types. This study aims to develop a novel, machine learning-based framework for classifying HPV genotypes by oncogenic risk using genome-derived numerical features. A key objective is to introduce TATA-box, CAAT-box, and CpG-island-based features to HPV risk prediction for the first time. Methods: We constructed a comprehensive feature set that integrates regulatory sequence motifs (TATA-box, CAAT-box, CpG islands) with dinucleotide and trinucleotide (k-mer) composition derived from full HPV genomes. Multiple machine learning algorithms were implemented to evaluate classification performance across all risk categories. Model accuracy, precision, recall, and F1-score were calculated to assess the effectiveness and robustness of the proposed feature set. Results: The proposed method achieves an average precision of 0.95, a recall of 0.95, an F1-score of 0.95, and an accuracy of 97.47%. The experimental findings indicate that the proposed method not only attains high classification accuracy across all HPV risk groups but also surpasses existing models in generalizability by utilizing genomic data and novel biologically informed features. Conclusions: This study introduces regulatory motif-based numerical features to HPV classification for the first time and demonstrates that integrating these with k-mer descriptors yields a highly accurate and scalable machine learning model. Unlike previous studies, which often focus on specific HPV genes or a limited subset of types, our method is scalable, robust, and capable of classifying known and emerging HPV types with high reliability. This highlights its potential for real-world deployment in large-scale epidemiological screening and vaccine development programs. Full article

Background/Objectives: Human papillomavirus (HPV) infection is a well-established risk factor for oropharyngeal squamous cell carcinoma (OPSCC), where p16 immunohistochemistry serves as a surrogate marker. However, the role of HPV in sinonasal squamous cell carcinoma (SNSCC) remains less defined, and the reliability of p16 as a standalone surrogate is under debate. This study aimed to assess the concordance between p16 expression and HPV-DNA status in SNSCC and characterize clinicopathologic features in HPV-associated cases. Methods: We retrospectively analyzed 111 SNSCC cases diagnosed between 2008 and 2024 at two German centers. p16 status was determined by immunohistochemistry using site-specific antibody protocols. HPV-DNA testing and genotyping were performed via PCR and reverse hybridization. Clinical and histopathological data were collected and compared between HPV-positive and -negative tumors. Results: HPV-DNA was detected in 31/111 cases (27.9%), with HPV16 and HPV33 (Site A) and HPV 16 and HPV18 (Site B) being the most frequent subtypes. Discordance between p16 and HPV-DNA status was observed in 29.7% of cases, with site-specific discordance rates of 44.6% and 14.5%. Patients with HPV-positive tumors were younger than their HPV-negative counterparts. Conclusions: Our findings underscore the limitations of p16 as a single surrogate marker for detecting HPV-associated sinonasal cancer. Future research on the role of HPV in sinonasal cancer should integrate complementary testing methods (like p16Ink4A immunohistochemistry and HPV DNA/mRNA analysis) and aim for test standardization. Full article

Human papillomavirus (HPV) is the most prevalent sexually transmitted infection worldwide and a leading cause of cervical cancer, accounting for over 300,000 deaths annually, primarily due to high-risk genotypes HPV-16 and HPV-18. Conventional molecular diagnostic methods, such as polymerase chain reaction (PCR), require expensive instrumentation and well-equipped laboratories, which limits their applicability in low-resource or decentralized settings. To address this challenge, the aim of this study was to develop a prototype point-of-care (POC) diagnostic platform based on helicase-dependent amplification (HDA) integrated into a microfluidic device for the specific detection of HPV-16 and HPV-18. The proposed POC platform comprises a disposable poly (methyl methacrylate) (PMMA) microfluidic device, a portable warming mat for isothermal amplification at 65 °C, and a compact electrophoresis chamber for fluorescence-based visualization using SYBR Safe dye, with an approximate total cost of $320 USD. Platform validation was performed on 33 samples, demonstrating amplification of target sequences in less than 60 min with only 20 µL of reaction volume, a limit of detection (LOD) of 15 copies (cp) per reaction, a sensitivity of 95.52%, and a specificity of 100%. This portable and scalable platform constitutes a cost-effective and reliable tool for the detection of HPV, supporting global health initiatives, including those driven by the World Health Organization (WHO), aimed at eliminating cervical cancer as a public health threat, as it can be implemented in decentralized or resource-limited settings. Full article

Cervical cancer screening plays a crucial role in preventing invasive disease through early detection of high-grade lesions. However, traditional cytology and histology often fail to reliably differentiate between transient HPV infections and those likely to progress. This study investigates the feasibility of integrating molecular HPV testing into histopathological workflows using FFPE tissue samples to improve diagnostic precision. A retrospective analysis was conducted on 55 FFPE cervical specimens from patients undergoing colposcopy with biopsy or conization. The workflow included automated DNA extraction and real-time PCR-based HPV genotyping with the Seegene Anyplex II HPV28 assay. HPV DNA was detected in 56.4% of samples, with 21 genotypes, including multiple high-risk types. High viral loads correlated with high-grade lesions, supporting the clinical value of HPV quantification. Compared to histology, molecular analysis reduced potential overdiagnosis by confirming HPV absence in morphologically suspicious but HPV-negative lesions. Integrating viral load and genotyping improved risk stratification, optimizing colposcopy referrals and reducing unnecessary follow-ups. This study introduces a novel, fully automated molecular workflow applicable to FFPE samples, enhancing cervical cancer screening beyond traditional methods. Although based on a limited sample, the findings support the method’s potential for broader implementation and further validation in multicenter settings. Full article

In postmenopausal women with high-grade cervical intraepithelial neoplasia (CIN3), hysterectomy is frequently performed after loop electrosurgical excision procedure (LEEP) due to the concern for residual disease or occult carcinoma. However, the decision to proceed with hysterectomy is often made without validated predictive criteria, increasing the risk of overtreatment or underdiagnosis. The aim of this study is to identify independent predictors of residual CIN2+ (CIN2, CIN3, adenocarcinoma in situ, invasive carcinoma) or invasive disease in hysterectomy specimens following LEEP in this high-risk population. Methods: We conducted a multicenter retrospective study including 154 postmenopausal women (aged 50–75) who underwent total hysterectomy within 12 months after LEEP for histologically confirmed CIN3. Data collected included human papillomavirus (HPV) genotyping (pre- and post-LEEP), endocervical curettage (ECC), cone margin status, transformation zone type, and histopathological outcomes of the hysterectomy specimen. Logistic regression and ROC curve analysis were used to assess predictive factors. Results: Residual disease (CIN2+, AIS, or carcinoma) was found in 38 patients (24.7%), including 7 cases (4.5%) of occult carcinoma. Persistent high-risk HPV post-LEEP was the strongest independent predictor (adjusted OR for HPV 16/18: 74.0; p < 0.001), followed by positive ECC (OR: 3.64; p = 0.028). Cone margin status was not independently associated. The multivariate model showed good discriminative performance (AUC = 0.860; sensitivity 67.2%, specificity 72.8%). Conclusions: Our findings suggest that persistent high-risk HPV infection and positive ECC are reliable predictors of residual or occult disease. These markers should be integrated into post-LEEP follow-up protocols to better identify candidates for hysterectomy and minimize unnecessary surgeries. Full article

Low-density DNA microarrays are crucial in molecular diagnostics due to their cost-effectiveness and high sensitivity. However, reliable spot localization remains challenging due to positional variations and image artifacts. Traditional intensity-based methods often struggle with weak fluorescence signals. To address this, we propose a rapid spot localization method that combines template matching with point pattern matching, enhanced through vectorized programming and square (box) templates. Vectorized programming accelerated the most time-consuming calculation by 82 times on a PC and was 6000 times faster on a Raspberry Pi compared to a for-loop implementation. While this improvement applies to the vectorized square calculation alone, substantial performance gains were still achieved in the overall process. Additionally, replacing circular templates with square templates resulted in a fourfold reduction in processing time without compromising detection performance. The proposed method effectively reduces computational overhead, making it suitable for high-throughput and resource-constrained applications. The method was validated using HPV genotyping images from commercial DNA microarrays, demonstrating its practical applicability and robust performance in clinical settings. Full article

Background: Reliable Human Papillomavirus (HPV) testing and genotyping are essential for quality assurance in HPV-based primary screening, disease management and for monitoring the impact of HPV vaccination. The clinical validation of HPV molecular diagnostic assays has significantly contributed to these objectives; however, little emphasis has been placed on assuring sample quality. This study aimed to evaluate the accuracy of sample cellularity assessment using the C-C Motif Chemokine Receptor 5 (CCR5) gene target as a marker of sample adequacy in molecular diagnostics. Methods: Jurkat cell line samples were counted using both a Thoma cell-counting chamber and Fluorescence-Activated Cell Sorting (FACS). Jurkat cell line samples at three different concentrations were subsequently evaluated using the OncoPredict HPV Quality Control (QC) real-time PCR assay, employing CCR5 for molecular cellularity quantification. Results: The cellularity values obtained were comparable across the three different methods for all dilutions of the cell line tested. Conclusions: The results obtained from this study show that CCR5 represents a promising molecular marker for the accurate quantification of sample cellularity, confirming its use as a reliable sample adequacy control, thus reducing the risk of “false-negative” results. Full article

Human Papilloma Virus (HPV) can play a causative role in the development of sinonasal tract malignancies. In fact, HPV may be the most significant causative agent implicated in sinonasal tumorigenesis and is implicated in as many as 21% of sinonasal carcinomas. To date, there are no definitive, reliable and cost-effective, diagnostic tests approved by the FDA for the unequivocal determination of HPV status in head and neck cancers. We followed an exhaustive algorithm to correctly test HPV infection, including a sequential approach with p16INK4a IHC, viral DNA genotyping and in situ hybridization for E6/E7 mRNA. Here, we report a case of sinonasal carcinoma with discordant results using HPV test assays. The tumor we describe showed an irregular immunoreactivity for p16INK4a, and it tested positive for HPV DNA; nevertheless, it was negative for HR-HPV mRNA. We discuss the possible meaning of this discrepancy. It would be advisable to test HPV transcriptional status of sinonasal carcinoma on a diagnostic routine basis, not only by p16INK4a IHC assay, but also by HPV DNA genotyping and HR-HPV mRNA assessment. Full article

The newly gained knowledge of the viral etiology in cervical carcinogenesis has prompted industrial interests in developing virology-based tools for cervical cancer prevention. Due to the long incubation period from viral infection to developing an invasive cancer, a process whose outcome is influenced by numerous life-style and genetic factors, the true efficacy of the genotype-specific human papillomavirus (HPV) vaccines in cervical cancer prevention cannot be determined for another 30 years. Most HPV DNA test kits designed to replace the traditional Papanicolaou (Pap) smears for precancer detection lack the analytical sensitivity and specificity to comprehensively detect all potentially carcinogenic HPVs and to perform reliable genotyping. The authors implemented the classic nested PCR and Sanger DNA-sequencing technology for routine HPV testing. The results showed a true negative HPV PCR invariably indicates the absence of precancerous cells in the cytology samples. However, 80.5% of single positive HPV-16 tests and 97.3% of single positive HPV-18 tests were associated with a negative or a largely self-reversible Pap cytology. Routine sensitive and reliable HPV type-specific or perhaps even variant-specific methods are needed to address the issues of persistence of HPV infection if a virology-based primary cervical screen is used to replace the Pap cytology screening paradigm. Full article