Integration of human papillomavirus (HPV) DNA into the host cell genome is a frequent event in cervical carcinogenesis, even though this phenomenon does not seem to be mandatory for cervical cancer development. Our objective was to describe the load and physical state of HPV type 16 (HPV16) DNA in a series of cervical samples representative of the natural history of cervical cancer. We used a combination of three real-time PCR assays targeting E6, E2, and albumin genes to calculate HPV16 load (E6 and albumin) and the E2/E6 ratio as a surrogate of integration. This method was applied to 173 HPV16-positive cervical samples. Results show that viral load increases with the lesion grade (from 102 HPV16 DNA copies per 10 ³ cells in normal samples up to 56,354 copies per 10 ³ cells in cancers), while E2/E6 ratio decreases (from 1 in normal samples down to 0.36 in cancers). We propose that, according to this technique, an HPV16 viral load of higher than 22,000 copies/10 ³ cells or an E2/E6 ratio of lower than 0.50 allows the identification of women with prevalent high-grade lesions or worse with a high specificity. In conclusion, both viral load and E2/E6 ratio, used in combination with an appropriate cutoff value, are suitable to screen women with prevalent cervical intraepithelial neoplasia grade 2 or 3 or cancer. Therefore, these assays would be useful in addition to routine HPV testing to more accurately identify women with (pre)cancerous lesions.

Extracellular vesicles (EVs), including exosomes (Exos), microvesicles (MVs) and apoptotic bodies (ABs) are released in biofluids by virtually all living cells. Tumor-derived Exos and MVs are garnering increasing attention because of their ability to participate in cellular communication or transfer of bioactive molecules (mRNAs, microRNAs, DNA and proteins) between neighboring cancerous or normal cells, and to contribute to human cancer progression. Malignant traits can also be transferred from apoptotic cancer cells to phagocytizing cells, either professional or non-professional. In this review, we focus on Exos and ABs and their relationship with human papillomavirus (HPV)-associated tumor development. The potential implication of EVs as theranostic biomarkers is also addressed.

Human Papillomavirus (HPV) infection is known to be associated with a number of conditions including cervical, vaginal, vulvar, penile, anal neoplasias and cancers, oropharynx cancers and genitals warts (GW). Two prophylactic vaccines are currently available: a bivalent vaccine designed to prevent HPV type 16 and 18 infection and a quadrivalent vaccine targeting HPV 6, 11, 16, and 18. In France, HPV vaccination is recommended in 11-14 year-old girls with a catch-up for girls aged 15-19. The objective of this study was to assess the potential impact of an HPV 6/11/16/18/31/33/45/52/58 nonavalent vaccine on anogenital and oropharyngeal HPV-related diseases in France. HPV genotype distributions from 6 multicentric retrospective studies (EDiTH I to VI) were analyzed including 516 cases of invasive cervical cancers (ICC), 493 high-grade cervical neoplasias (CIN2/3), 397 low-grade squamous intraepithelial lesions (LSIL), 423 GW, 366 anal cancer and 314 oropharyngeal carcinomas. Low and high estimates of HPV vaccine impact were calculated as follows: low estimate: prevalence of HPV 6/11/16/18/31/33/45/52/58 genotypes alone or in association but excluding presence of another HPV type; high estimate: prevalence of HPV 6/11/16/18/31/33/45/52/58 genotypes alone or in association, possibly in presence of another HPV type. Estimates of potential impact varied from 85% (low estimate) to 92% (high estimate) for ICC, 77% to 90% for CIN2/3, 26% to 56% for LSIL, 69% to 90% for GW, 81% to 93% for anal cancer, and 41% to 44% for oropharyngeal carcinomas. Compared to the quadrivalent vaccine, the proportion of additional cases potentially prevented by the nonavalent vaccine was 9.9%-15.3% for ICC, 24.7%-33.3% for CIN2/3, 12.3%-22.7% for LSIL, 2.1%-5.4% for GW, 8.5%-10.4% for anal cancer, and 0.0%-1.6% for oropharyngeal carcinoma. The nonavalent HPV vaccine showed significant increased potential impact compared to the HPV 6/11/16/18 quadrivalent vaccine for ICC, CIN2/3 and LSIL. Considering a 100% vaccine efficacy and high vaccine coverage, about 90% of ICC, CIN2/3, GW or anal cancer cases could be prevented by a nonavalent HPV vaccine in France.

High-risk (HR) human papillomavirus (HPV)-associated carcinogenesis is driven mainly by the overexpression of E7 and E6 oncoproteins following viral DNA integration and the concomitant loss of the E2 open reading frame (ORF). However, the integration of HR-HPV DNA is not systematically observed in cervical cancers. The E2 protein acts as a transcription factor that governs viral oncogene expression. The methylation of CpGs in the E2-binding sites (E2BSs) in the viral long control region abrogates E2 binding, thus impairing the E2-mediated regulation of E7/E6 transcription. Here, high-resolution melting (HRM)–PCR was developed to quantitatively analyze the methylation statuses of E2BS1, E2BS2, and the specificity protein 1 (Sp1)-binding site in 119 HPV16-positive cervical smears. This is a rapid assay that is suitable for the analysis of cervical samples. The proportion of cancer samples with methylated E2BS1, E2BS2, and Sp1-binding site CpGs was 47%, whereas the vast majority of samples diagnosed as being within normal limits, low-grade squamous intraepithelial lesions (LSIL), or high-grade squamous intraepithelial lesions (HSIL) harbored unmethylated CpGs. Methylation levels varied widely, since some cancer samples harbored up to 60% of methylated HPV16 genomes. A pyrosequencing approach was used as a confirmation test and highlighted that quantitative measurement of methylation can be achieved by HRM-PCR. Its prognostic value deserves to be investigated alone or in association with other biomarkers. The reliability of this single-tube assay offers great opportunities for the investigation of HPV16 methylation in other HPV-related cancers, such as head and neck cancers, which are a major public health burden.