Jean-Damien Combes

Role of human papillomavirus in non-oropharyngeal head and neck cancers.

Accurate estimates of the fraction of head and neck cancer (HNC) attributable to human papillomavirus (HPV) infection are essential to predict the effectiveness of interventions based on vaccination against HPV or HPV-testing. In addition, if supported by currently on-going clinical trials, attribution of a HNC to HPV may allow better and less toxic treatments. Here we focused on studies in which the prevalence of molecular and serological HPV markers was similarly assessed in oropharyngeal and non-oropharyngeal HNC. Large data on HPV DNA detection by PCR and p16 expression in HNC biopsies suggests that the probability of a cancer of the oral cavity, larynx, and hypopharynx being attributable to HPV is at least 5-fold lower than that for oropharyngeal cancer. Seropositivity for HPV16 E6 or E7 shows larger differences across sites, but findings vary between studies. Because HPV DNA and p16 detection lack specificity, and E6 and E7 antibody detection lacks sensitivity, these tests are not totally satisfactory. Limited data on in situ hybridization or HPV E6/E7 mRNA, mainly from the United States, suggests that HPV-attributable HNC is rare in the oral cavity (∼3%), larynx (∼7%), and hypopharynx (∼0%). Data on HPV in other rarer HNCs are extremely limited and essentially negative. Available data do not allow the establishment of the way HPV infection and tobacco may interact in non-oropharyngeal HNC. The exclusion of oropharynx as a site of tumor origin and the identification of robust fingerprints of HPV-driven carcinogenesis are the priorities to improve the estimate of HPV-attributable non-oropharyngeal HNC.

Plasmacytoid dendritic cells infiltrating ovarian cancer are associated with poor prognosis

Using two different and complementary approaches (flow cytometry and immunohistochemistry) on two independent cohorts of ovarian cancer patients, we found that accumulation of plasmacytoid dendritic cells (pDC) in tumors is associated with early relapse. This deleterious effect of tumor-associated pDC was evident when they are present in cancer epithelium but not in lymphoid aggregates.

Antibodies against high-risk human papillomavirus proteins as markers for invasive cervical cancer

Different human papillomavirus (HPV) genes are expressed during the various phases of the HPV life cycle and may elicit immune responses in the process towards malignancy. To evaluate their association with cervical cancer, antibodies against proteins from HPV16 (L1, E1, E2, E4, E6 and E7) and HPV18/31/33/35/45/52/58 (L1, E6 and E7) were measured in serum of 307 invasive cervical cancer cases and 327 controls from Algeria and India. Antibody response was evaluated using a glutathione S‐transferase‐based multiplex serology assay and HPV DNA detected from exfoliated cervical cells using a GP5+/6+‐mediated PCR assay. Among HPV16 DNA‐positive cases, seroprevalence of HPV16 antibodies ranged from 16% for HPV16 E1 to 50% for HPV16 E6 and all were significantly higher than controls. Seroprevalence of E6, E7 and L1 antibodies for HPV18 and for at least one of HPV31/33/35/45/52/58 were also higher in cases positive for DNA of the corresponding type (50% and 30% for E6 of HPV18 and HPV31/33/35/45/52/58 combined, respectively). E6 and E7 antibodies were rarely found in controls, but cross‐reactivity was evident among cancer cases positive for DNA of closely phylogenetically‐related HPV types. E6 or E7 antibodies against any of the eight HPV types were detected in 66.1% of all cervical cancer cases, as compared to 10.1% of controls. E6, and to a lesser extent E7, antibodies appear to be specific markers of HPV‐related malignancy. However, even among cases positive for the same type of HPV DNA, approximately one‐third of cervical cancer cases show no detectable immune response to either E6 or E7.

Prevalence of Human Papillomavirus in Cancer of the Oropharynx by Gender

Oropharyngeal cancer (OPC) is more frequent in men than women mainly due to the heavier and longer duration of smoking in men. Human papillomavirus (HPV) has a role in the rising incidence of OPC in the United States and other high-income countries. To determine whether there is a difference in the proportion of HPV-attributable OPC between men and women, we systematically retrieved HPV prevalence data from 63 studies reporting separately on OPC by gender. The male/female (M/F) ratios of HPV prevalence in OPC across different countries and the corresponding M/F ratios of cumulative lung cancer risk (a proxy for smoking) were compared. The United States had the highest M/F ratios of HPV prevalence in OPC (1.5). The lowest M/F ratios (≤0.7) were found in Asia and some European countries (e.g., France). The countries in which the M/F ratio of HPV prevalence in OPC was ≥1.0 had the most similar lung cancer risks for men and women. When HPV prevalence data were applied to age-standardized OPC incidence rates in the United States, Australia, the United Kingdom, and France, the M/F ratio for the HPV-positive OPC incidence rates was rather stable (around 4) in all countries. In contrast, the M/F ratio for the HPV-negative OPC incidence rates reached 10.2 in France versus <3 elsewhere. We showed that HPV prevalence in OPC differs by gender and country mainly as a consequence of the vast international variation in male smoking habits. Nevertheless, HPV-positive OPC may affect men more heavily than women in different populations for reasons that are unclear. Cancer Epidemiol Biomarkers Prev; 23(12); 2954–8. ©2014 AACR.

Isolation and characterization of a novel putative human polyomavirus.

The small double-stranded DNA polyomaviruses (PyVs) form a family of 73 species, whose natural hosts are primarily mammals and birds. So far, 13 PyVs have been isolated in humans, and some of them have clearly been associated with several diseases, including cancer. In this study, we describe the isolation of a novel PyV in human skin using a sensitive degenerate PCR protocol combined with next-generation sequencing. The new virus, named Lyon IARC PyV (LIPyV), has a circular genome of 5269 nucleotides. Phylogenetic analyses showed that LIPyV is related to the raccoon PyV identified in neuroglial tumours in free-ranging raccoons. Analysis of human specimens from cancer-free individuals showed that 9 skin swabs (9/445; 2.0%), 3 oral gargles (3/140; 2.1%), and one eyebrow hair sample (1/439; 0.2%) tested positive for LIPyV. Future biological and epidemiological studies are needed to confirm the human tropism and provide insights into its biological properties.

Human papillomavirus type 16 E6 variants in France and risk of viral persistence.

BackgroundOnly a small portion of HPV 16 infections persist and can lead to cervical intraepithelial lesions and cancer. Factors that favour HPV persistence versus clearance are still poorly understood, but several studies have suggested that HPV intra-type variants may influence persistence and clinical outcome. The aim of this study was to assess the possible association between HPV 16 variants and the risk for viral persistence in the general population of France.MethodsOne hundred and forty two women infected with HPV 16 with normal cytology, without previous treatment for cervical lesions, and with a valid second follow-up visit 4 to 16 months later, were selected from patients participating in routine cervical cancer screening in the Reims HPV Primary Screening Cohort Study. HPV intra-type variants were determined by sequencing the HPV 16 E6 open reading frame, and were compared for viral persistence at the second visit using odds ratios (OR) to estimate relative risk.ResultsAlthough no statistically significant differences in risk for persistence were observed by the HPV 16 variant lineage, European variants containing the polymorphism 350 T (EUR-350 T) appeared to persist more often than those containing 350 G (EUR-350 G) (OR = 1.6, 95% CI = 0.8-3.4).ConclusionsNo strong differences were observed in the risk of viral persistence for the HPV 16 variants that predominate in France.

Deep brush‐based cytology in tonsils resected for benign diseases

A fraction of oropharyngeal cancer (OPC), especially in the tonsil, is caused by human papillomavirus (HPV), mainly HPV16. Noninvasive diagnostic methods to detect precancerous lesions in the tonsil would be useful, e.g., liquid‐based cytology (LBC). However, ill‐characterized precancerous lesions may be hidden in the depth of the tonsillar crypts. We therefore conducted a study on HPV and tonsillar precancerous lesions to evaluate, among other things, the utility of LBC obtained by deep brushing of the resected tonsils. Two hundred non‐paediatric patients (mean age: 30.3 years) who underwent tonsillectomy for infection‐related conditions (69%) or other conditions (mainly obstructive sleep apnoea, 31%) were included. An ultra‐sensitive Luminex bead‐based platform was used to test for the DNA of 21 mucosal HPV types; 56% of slides were unsatisfactory due to low number of squamous epithelial cells or the masking effect of a large number of lymphocytes. Three patients (1.5%; 95% CI: 0.5–4.3) showed suspicious cytological findings (atypical squamous cells‐cannot exclude high‐grade squamous intraepithelial lesion, ASC‐H) while 3 others were HPV‐positive (2 for HPV16 and 1 for HPV39). None of the ASC‐H patients and HPV‐positive patients showed dysplasia at histological examination. The rarity of HPV infection in the tonsil conflicts with the relatively frequent detection of the virus in the mouth. In conclusion, aggressive deep brushing of tonsils, while hardly applicable in vivo, is unlikely to be a reliable method to detect precancerous lesions. The absence of OPC screening modalities places the priority on multi‐purpose primary prevention strategies, i.e., HPV vaccination and reduction of smoking and drinking.

Prevalence of human papillomavirus in tonsil brushings and gargles in cancer-free patients: The SPLIT study

OBJECTIVE To evaluate human papillomavirus (HPV) prevalence in the tonsil using extensive ex vivo brushing and gargling in a large age-stratified sample of cancer-free patients. MATERIALS AND METHODS From 2012 to 2016, consecutive patients undergoing tonsillectomy for benign indications in 19 French University Hospitals were invited to participate in the SPLIT study. Immediately after resection, half-tonsils were extensively brushed at the pathology laboratories on the surface epithelium and in tonsil crypts to collect exfoliated cells. In 11 centers, patients aged 15 and over (adults) were also asked to provide gargle samples before surgery. HPV-DNA detection used a very sensitive Luminex technology to evaluate 21 HPV types. RESULTS Tonsil brushings from 692 patients aged 1-70 years and gargles from 268 adults were tested for HPV. Among adults, overall HPV prevalence was 3.6% in tonsil brushings and 13.1% in gargles and HPV16 prevalence was 2.2% and 4.1%, respectively. Among 139 children, tonsil brushings were positive in two girls (1.4%). Percent agreement in HPV detection in paired tonsil brushings and gargles in adults was 85.8% and positive agreement 9.5%. HPV prevalence in gargles significantly varied by sex (prevalence ratio in men vs women=2.1; 95% confidence interval; 1.1-4.1) and tonsillectomy indication (non-infectious vs. infectious=4.9; 1.4-17.0). CONCLUSION HPV infection is infrequent in tonsil brushings of cancer-free children and adults. In contrast, HPV infection in gargles in adults is rather common. Low agreement in paired tonsil brushings and gargles suggests that gargle is not representative of HPV prevalence in the tonsil.

Human papillomavirus antibody response following HAART initiation among MSM.

Objective:To describe effects of HAART on high-risk human papillomavirus (HPV) antibody response in HIV-positive MSM and the meaning of this response for subsequent HPV-related cancer risk. Design:Prospective seroepidemiological study of 281 HIV-positive MSM initiating HAART between 1995 and 2004 in the Swiss HIV Cohort Study. Methods:For each individual, two serum samples, one at HAART initiation (pre-HAART) and another 24 months later (post-HAART), were tested for L1 antibodies to HPV6, 11, 16, 18, 31, 33, 35, 45, 52 and 58, as well as HPV16-E6 antibodies, using a multiplex serology assay. Identification of HPV-related cancer included data linkage with Swiss cancer registries. Results:Pre-HAART, 45.2% were seropositive for any high-risk HPV-L1 and 32.4% for HPV16-L1. Sexual intercourse during the last 6 months was the only evaluated factor associated with L1 seropositivity pre-HAART. Seropositivity increased post-HAART to 60.5% for any high-risk HPV-L1 [prevalence ratio versus pre-HAART = 1.34, 95% confidence interval (CI) 1.14–1.57] and 48.0% for HPV16-L1 (prevalence ratio versus pre-HAART = 1.48, 95% CI 1.20–1.83), and seroconversion was significantly associated with both lower CD4+ cell count and CD4+/CD8+ ratio (P < 0.01). Only one individual was HPV16-E6-seropositive pre-HAART, but two more seroconverted post-HAART. Anal cancer incidence among the three HPV16-E6-positives post-HAART was significantly increased compared with HPV16-E6-negatives (incidence rate ratio = 63.1, 95% CI 1.1–1211). Conclusion:HAART-related immune reconstitution increases HPV-specific antibody responses, which may discriminate future anal cancer risk in this high-risk population.

Judging the carcinogenicity of rare human papillomavirus types

Dear Sir, Persistent infection with human papillomavirus (HPV) types is accepted as the necessary cause of invasive cervical cancer (ICC). However, for the development of HPV screening tests and vaccines, a large number of mucosotropic HPV types need to be judged separately. Although some types are powerful carcinogens and can be found in a sizeable proportion of ICC, the classification of rarer types is more challenging: not only is their prevalence in ICC very low, but as all HPV types share a common mode of sexual transmission, confounding from the concurrent presence of other carcinogenic HPV types is difficult to rule out. In 2009, an International Agency for Research on Cancer (IARC) Working Group (IARC, 100B) classified HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 as carcinogenic (Group 1) to humans, and HPV68 as probably carcinogenic (Group 2A), according to an algorithm that included comparison of HPV type-specific prevalence in ICC with that among women with normal cytology (as a proxy for the background population prevalence of infection). These 13 Group 1/2A types cluster together in an evolutionary clade that includes Alphapapillomavirus species groups 5, 6, 7, and 9. Other types in this clade were classified as possibly carcinogens (Group 2B) based solely on phylogenetic relatedness. With the aim to provide an epidemiological basis for judging the carcinogenicity of rarer HPV types, we expanded our previous meta-analysis published in this journal that reported the prevalence of the Group 1/2A HPV types across the full spectrum of cervical diagnoses from normal cytology to ICC, to include 29 additional HPV types. As described previously, eligible studies met the following criteria: (i) use of well-validated broad-spectrum consensus polymerase chain reaction (PCR) assays and (ii) reporting of overall and typespecific HPV prevalence by strata of cytopathological and/or histopathological cervical diagnoses. The ratio of the prevalence of individual HPV types in ICC to that in women with normal cytology (ICC:normal prevalence ratio) was calculated as a proxy of carcinogenicity, with HPV6 considered as an archetypal noncarcinogenic type (IARC Group 3) for comparison purposes. Figure 1 describes ICC:normal prevalence ratios of individual HPV types in a total of 40,706 ICC cases and 263,971 women with normal cytology, from all world regions. Of note, type-specific prevalence was estimated only among studies that tested and reported data for the given HPV type, so denominators vary by type. All Group 1/2A types (in red in Fig. 1) showed ICC:normal prevalence ratios greater than 1.0, but with a strong variation from 21.1 (95%CI 21.5–22.7) for HPV16 down to 1.2 (95%CI 1.1–1.4) for HPV51. Furthermore, for none of Group 1/2A types did the 95% confidence interval of the ICC:normal prevalence ratio overlap with those for HPV6 (0.4, 95%CI 0.3–0.5). Among Group 2B types, HPV26, 30, 67, 69, 73, and 82 each showed ICC:normal prevalence ratios above 1.0 that resembled those of Group 1/2A types and which were significantly higher than that of HPV6. Indeed, other lines of evidence also support the carcinogenicity of HPV26, 30, 67, 69, 73, and 82. These types have been reported as single infections in cervical cancer, with a ratio of single to multiple infections in meta-analytical data that resembles that of Group 1/2A types. In addition, HPV82 is able to immortalize human keratinocytes and HPV26, 73, and 82 have been reported as the only actively transcribed type in ICC and/ or periungual cancer. The 13 Group 1/2A types have been already included in all U.S. Food and Drug Administration (FDA)-approved HPV screening tests. Types HPV26, 30, 67, 69, 73, and 82 collectively account for approximately 1.6% of ICC, and may also be considered for inclusion in future HPV screening tests. Importantly, these types are reported so rarely in normal cytology (0.2–0.5% each), that they do not raise great concerns about over-referral of women at low risk for cervical cancer in screening programs. In contrast, some other types in the 2B group, for example, HPV53, 66, and 70, are indistinguishable from HPV6 and other noncarcinogenic types in terms of their ICC:normal prevalence ratio (Fig. 1), and relatively common in women with normal cytology (1.1, 0.6, and 0.8%, respectively). Many of the currently FDAapproved HPV screening tests already include HPV66, and Hybrid Capture 2 is also known to cross-react with HPV53, 66, and 70, phenomena which are expected to unfavorably impact test specificity and positive predictive value. Of note, some HPV types that were not classified as carcinogenic showed even lower ICC:normal prevalence ratios than HPV6. Some of these types may have a stronger tropism for the vagina than the cervix, and hence be more likely to contaminate cervicovaginal swabs from women with normal cytology than to be found in cervical-directed biopsies of ICC. Some limitations of cross-sectional meta-analyses of HPV type distribution have been described in our previous article in this journal including lack of sufficient sample size to explore the potential heterogeneity in the Le tt er to th e E di to r