Risk-stratification of HPV-positive women with low-grade cytology by FAM19A4/miR124-2 methylation and HPV genotyping

Background The introduction of primary HPV screening has doubled the number of colposcopy referrals because of the direct referral of HPV-positive women with a borderline or mild dyskaryosis (BMD) cytology (ASC-US/LSIL) triage test. Further risk-stratification is warranted to improve the efficiency of HPV-based screening. Methods This study evaluated the discriminative power of FAM19A4/miR124-2 methylation, HPV16/18 genotyping and HPV16/18/31/33/45 genotyping in HPV-positive women with BMD (n = 294) in two Dutch screening trials. Absolute CIN3+ risks and colposcopy referrals within one screening round were calculated. Results Methylation analysis discriminated well, yielding a CIN3+ risk of 33.1% after a positive result and a CIN3+ risk of 9.8% after a negative result. HPV16/18 and HPV16/18/31/33/45 genotyping resulted in a 27.6% and 24.6% CIN3+ risk after a positive result, and a 13.2% and 9.1% CIN3+ risk after a negative result. Colposcopy referral percentages were 41.2%, 43.2%, and 66.3% for FAM19A4/miR124-2 methylation, HPV16/18 and HPV16/18/31/33/45 genotyping, respectively. The CIN3+ risk after a negative result could be lowered to 2.8% by combining methylation and extended genotyping, at the expense of a higher referral percentage of 75.5%. Conclusion The use of FAM19A4/miR124-2 methylation and/or HPV genotyping in HPV-positive women with BMD can lead to a substantial reduction in the number of direct colposcopy referrals.

Development of models for cervical cancer screening: construction in a cross-sectional population and validation in two screening cohorts in China

Background Current methods for cervical cancer screening result in an increased number of referrals and unnecessary diagnostic procedures. This study aimed to develop and evaluate a more accurate model for cervical cancer screening. Methods Multiple predictors including age, cytology, high-risk human papillomavirus (hrHPV) DNA/mRNA, E6 oncoprotein, HPV genotyping, and p16/Ki-67 were used for model construction in a cross-sectional population including women with normal cervix (N = 1085), cervical intraepithelial neoplasia (CIN, N = 279), and cervical cancer (N = 551) to predict CIN2+ or CIN3+. A base model using age, cytology, and hrHPV was calculated, and extended versions with additional biomarkers were considered. External validations in two screening cohorts with 3-year follow-up were further conducted (NCohort-I = 3179, NCohort-II = 3082). Results The base model increased the area under the curve (AUC, 0.91, 95% confidence interval [CI] = 0.88–0.93) and reduced colposcopy referral rates (42.76%, 95% CI = 38.67–46.92) compared to hrHPV and cytology co-testing in the cross-sectional population (AUC 0.80, 95% CI = 0.79–0.82, referrals rates 61.62, 95% CI = 59.4–63.8) to predict CIN2+. The AUC further improved when HPV genotyping and/or E6 oncoprotein were included in the base model. External validation in two screening cohorts further demonstrated that our models had better clinical performances than routine screening methods, yielded AUCs of 0.92 (95% CI = 0.91–0.93) and 0.94 (95% CI = 0.91–0.97) to predict CIN2+ and referrals rates of 17.55% (95% CI = 16.24–18.92) and 7.40% (95% CI = 6.50–8.38) in screening cohort I and II, respectively. Similar results were observed for CIN3+ prediction. Conclusions Compared to routine screening methods, our model using current cervical screening indicators can improve the clinical performance and reduce referral rates.

Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples

The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/μl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies.

Evaluation of Cervical Cancer Screening Tools; INNO-LiPA® HPV Genotyping Extra-II Assay versus E7/E6 oncoproteins, How is reliable and practical?

Background: High-Risk Human papillomavirus (HR-HPV) has been well established as the cervical cancer (CC) risk factor. In recent years, various diagnostic methods of human papillomaviruses (HPV) have been developed to promote sensitivity and specificity of CC screening which leads to a low mortality rate. This study aimed to compare diagnostic test metrics of two HPV diagnostic techniques, including Western blot and INNO-LiPA HPV Genotyping Extra II assay methods in asymptomatic or subclinical patients, among the South-Eastern Iranian women. Methods: 323 women were referred to the Pathology and Stem Cell Research Center, from February 2018 to January 2020. HPV-DNA with the INNO-LiPA HPV Genotyping Extra-II Assay kit and the western blot assays for HPV E7 and E6 assessment were employed. Results: Overall, 163 (50.4%) samples were dysplastic pap smear, the specificity of the HPV DNA test by INNO-LiPA HPV Genotyping Extra-II Assay test was significantly higher than the E7/E6 oncoproteins finding (67.3 vs. 49.9%), and the sensitivity was lower (96.6 vs. 74.8%), respectively. Conclusions: HR-HPV E7/E6 oncoproteins expression was evaluated as a possible novel biomarker for CC screening in pap smear as the preliminary test with satisfactory diagnostic values for HR-HPV types 16 and 18. The corresponding diagnostic values may be further improved by combining HPV DNA tests with the INNO-LiPA HPV Genotyping Extra-II test. Also, they may prove helpful for HR-HPV infection diagnosis in cases that the patients are asymptomatic or subclinical. Keywords: Cervical Cancer; Human Papillomavirus (HPV); Diagnostic Screening Programs; Oncogene Proteins