Molecular triage of cervical screening samples in women 55–59 years of age: a pilot study

Abstract Background With HPV screening the specificity of screening positives has decreased, even with a cytological triage test. Increases in colposcopies and detection of benign or low-grade dysplasia are reported, not least in older women. These results highlight the necessity to find other triage tests in HPV screening strategies, so that women can be more accurately selected for colposcopy, thus minimizing the clinically irrelevant findings. Methods The study included 55- to 59-year-old women who exited the screening with normal cytology, but later in a follow-up test were positive for the HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 and had a cervical cone biopsy done. To model a screening situation with hrHPV-positive women, three different triage strategies, namely, cytology, genotyping and methylation, were performed. The study considered the effect of direct referral to colposcopy for HPV genotypes 16, 18, 31, 33, 45, 52 and 58, and methylation for FAM19A4 and hsa-mir124-2 and/or any form of abnormal cytology. Results Seven out of 49 women aged 55–59 years with hrHPV had a cone biopsy with high-grade squamous intraepithelial lesion. No triage method found all cases, and when comparing positive and negative predictive value and false negative rate, cytology showed better results than genotyping and methylation. Conclusion This study does not support a switch in triage strategies from cytology to hrHPV genotyping and methylation for women above 55 years of age yet, but demonstrates the need for more evidence on molecular triage strategies

HPV-based screening for cervical cancer among women 55-59 years of age.

AimMany cervical cancers occurs among women over 65 and prevalence of HPV genotypes in this age cohort is sparingly studied. One aim of this study was to study the prevalence and distribution of HPV genotypes in women 55-59 years, with normal cytology when exiting the screening program. Secondly, HPV clearance as well as the value of HPV genotyping and/or liquid based cytology as triage tests for identifying histological dysplasia among women with persistent HPV was studied.MethodsWomen that exited the screening program with normal cytology, between the years 2012-2014, in Örebro County, Sweden, were invited to this study. A total of 2946 samples were analyzed with a broad-spectrum assay to detect both hrHPV and lrHPV in order to investigate the distribution of genotypes. In the consent group, women with a positive hrHPV test were offered a follow-up test and a cone biopsy for histological confirmation, and a follow up sample 6 months post cone.ResultsThe overall prevalence of hrHPV was 7.4% and 59% of them remained hrHPV positive in a follow-up test after 12 months. A total of 99 women had a cone biopsy done, where 19% showed histological dysplasia. HPV 53 was the most common genotype, and among women with histology confirmed LSIL or HSIL, HPV 31 was most common. A positive hrHPV result showed a PPV of 25% for LSIL+ and 12.5%for HSIL+. Using detection of HPV 16/18 genotypes as a triage test for hrHPV positive tests, indicated FNR for histological LSIL+ and HSIL+ of 94% and 87.5% respectively, whilst triage based on cervical cytology had a FNR of 69% for LSIL+ and 37.5% for HSIL+.ConclusionThe most common hrHPV genotypes among women 55-59 years of age were non HPV16/18 genotypes, and in this population, these genotypes represented most of the histological verified HSIL lesions. This result does not support the proposition of a HPV 16/18 triaging test after a positive hrHPV test as a marker of histological HSIL+ cervical lesions in women over 55 years of age. Similarly, cytological triage after a positive hrHPV showed no additional benefit in this population. Specific triaging tests should be validated to follow post-menopausal women with a positive hrHPV test

HPV16 viral characteristics in primary, recurrent and metastatic vulvar carcinoma

Vulvar carcinoma is the fourth most common gynecological malignancy. Two separate carcinogenic pathways are suggested, where one is associated with the human papillomavirus (HPV) and HPV16 the most common genotype.The aim of this study was to evaluate HPV-markers in a set of primary tumors, metastases and recurrent lesions of vulvar squamous cell carcinomas (VSCC). Ten HPV16-positive VSCC with metastatic regional lymph nodes, distant lymphoid/hematogenous metastases or local recurrent lesions were investigated for HPV genotype, HPV16 variant, HPV16 viral load, HPV16 integration and HPV16 E2BS3 and 4 methylation.In all 10 analyzed case series, the same HPV genotype (HPV16), HPV16 variant and level of viral load were detected in all lesions within a patient case. Primary tumors with a high E2/E6 ratio were found to have fewer vulvar recurrences and/or metastases after diagnosis and treatment. Also, a significantly lower viral load was evident in regional lymph nodes compared to primary tumors.The data presented strengthens the evidence for a clonal HPV-induced pathway for vulvar carcinoma Keywords: Vulvar carcinoma, Human papillomavirus, Recurrences, Metastases, Integration, Viral loa

Distribution of the HPV16-variants present in both series.

<p>Distribution of the HPV16-variants present in both series.</p

Distribution of methylation in percentages (%) for each position in E2BS3 and 4 in the two series.

<p>In total series, 6 samples, of 54 samples analysed, showed high methylation (>50%) and 5 samples fulfilled the criteria of medium methylation.</p><p>Distribution of methylation in percentages (%) for each position in E2BS3 and 4 in the two series.</p

A. Results from FAM19A4/miR-124-2 hypermethylation analysis.

Presented in comparison between groups of differing age, screening outcome, and HPV status. The HPV groups presented were positive vs. negative genotyping test, single vs. multiple genotypes within positive samples, positive for IARC1 genotype vs. positive for other non-IARC1 genotype, single vs. multiple IARC1 genotype within IARC1 positive samples, and HPV16/18 positive vs. positive for other IARC1 genotype. B. Multivariate analysis. Binary logistic regression analysis of predictive factors for hypermethylation positivity in the FAM19A4 and hsa-miR124-2 genes.</p

Genotype-specific hypermethylation patterns in samples among controls (normal cytology, ≤LSIL cytology, LSIL histology, and normal histology) and cases (≥HSIL histology).

Genotype-specific hypermethylation patterns in samples among controls (normal cytology, ≤LSIL cytology, LSIL histology, and normal histology) and cases (≥HSIL histology).</p

Study flowchart.

The steps within the green middle box are part of the cervical cancer screening program in Örebro County, Sweden, where women 30 years and older are screened with primary HPV. All mRNA-positive results lead to reflex cytology analysis on the same sample with liquid-based cytology (LBC) method. For this study, all HPV mRNA–positive samples were subjected to full DNA genotyping (left) and methylation analysis of the host genes FAM19A4 and miR-124-2 (right).</p

History of HPV in HPV-positive elderly women

Background: The aim of this study was to examine the natural course of HPV infection in women of 60 years and older who were HPV positive at inclusion, and any association between HPV positivity in historical samples and dysplasia outcome. Methods: Eighty-nine women aged 60-82 years, who tested positive for HPV between 2012 and 2016 were included. Sampling for cytology and/or histology was also performed. HPV genotyping was carried out on archived material back to 1999. Results: Of the 89 HPV-positive women 16 had HSIL, 34 had LSIL and 39 were benign at inclusion. Of the women with HSIL, 50.0% had the same HPV type in the archive samples, 12.5% had another type, and 37.5% were HPV negative. Among the 34 women with LSIL, 47.1% had the same HPV type in archive samples, 5.8% had another type, and 47.1% were HPV negative. Of the 39 women without dysplasia at inclusion, 25.6% had the same HPV type in archive samples, 5.1% had another HPV type and 69.2% were HPV negative. Conclusion: Surprisingly few of the elderly women thus seem to have a history with the same or any HPV infection the years before being diagnosed with an HPV infection and dysplasia. The significance of an HPV infection for dysplasia development in elderly women is still not fully understood