Maurits N. C. de Koning

Multicenter Study of the Association between Betapapillomavirus Infection and Cutaneous Squamous Cell Carcinoma

Human papillomaviruses (betaPV) from the beta genus cannot be classified according to their oncogenicity due to a paucity of information. This study evaluates the association between betaPV infection and cutaneous squamous cell carcinoma in conjunction with measures of UV exposure and susceptibility. We performed case-control studies in the Netherlands, Italy, and Australia, countries with profoundly different UV exposures. The presence of 25 betaPV types in eyebrow hair follicles was determined using a highly sensitive HPV DNA genotyping assay, and antibodies for the 15 most prevalent betaPV types in a total of 689 squamous cell carcinoma cases and 845 controls were detected using multiplex serology. Multivariate logistic regression models were used for case-control comparisons and interaction analyses. BetaPV DNA was detected in eyebrow hairs of more than 90% of all participants. The presence of betaPV DNA was associated with an increased risk of squamous cell carcinoma in the Netherlands (OR = 2.8; 95% CI 1.3-5.8) and Italy (OR = 1.7; 95% CI 0.79-3.6), but not in Australia (OR = 0.91; 95% CI 0.53-1.6). Seropositivity for betaPV in controls ranged between 52% and 67%. A positive antibody response against 4 or more betaPV types was associated with squamous cell carcinoma in Australia (OR = 2.2; 95% CI 1.4-3.3), the Netherlands (OR = 2.0; 95% CI 1.2-3.4) and fair-skinned Italians (OR = 1.6, 95% CI 0.94- 2.7). The association between UV susceptibility and squamous cell carcinoma was stronger in betaPV-seropositive people. These combined data support the hypothesis that betaPV may play a role in the development of cutaneous squamous cell carcinoma.

Prevalence and associated factors of betapapillomavirus infections in individuals without cutaneous squamous cell carcinoma.

Betapapillomavirus (betaPV) infections are often associated with squamous-cell carcinoma (SCC) and the prevalence of betaPV infections in (immunosuppressed) SCC patients is known to be high. The distribution and possible associated factors of betaPV infections in the general population, however, are largely unknown. To address this issue, betaPV infection was studied in 1405 SCC-free immunocompetent (n=845) and immunosuppressed (n=560) individuals from six countries of different latitudes. A standard study protocol was used to obtain information about age, sex, UV-irradiation and skin type, and from all participants eyebrow hairs were collected for detection and genotyping of 25 established betaPV types using the PM-PCR reverse hybridization assay (RHA) method. The frequency of betaPV-positive participants ranged from 84 to 91% in the immunocompetent population with HPV23 as the most prevalent type, and from 81 to 98% in the immunosuppressed population with HPV23 as the most or the second most prevalent type. The median number of infecting betaPV types ranged from four to six in the immunocompetent and from three to six in the immunosuppressed population. Increasing age in the immunocompetent participants and (duration of) immunosuppression in the immunosuppressed patients were associated with betaPV infection. In both groups, sex, skin phototype, sunburns and sun-exposure were not consistently associated with betaPV infection. This study demonstrates that betaPV infections are also highly prevalent in SCC-free individuals, with similar HPV types prevailing in both immunocompetent and immunosuppressed persons. Age and (duration of) immunosuppression were identified as betaPV infection-associated factors, whereas characteristics related to sun exposure and skin type were not.

Evaluation of a Novel Highly Sensitive, Broad-Spectrum PCR-Reverse Hybridization Assay for Detection and Identification of Beta-Papillomavirus DNA

ABSTRACT Human papillomavirus can be detected by amplification of viral DNA. A novel one-step PCR (PM-PCR) was evaluated for amplification of a 117-bp fragment from the E1 region. It permitted ultrasensitive detection of all 25 known human papillomavirus genotypes from the beta-papillomavirus genus. The intra- and intertypic sequence variations of the 77-bp interprimer region were studied. Genotype-specific probes as well as general probes were selected for the 25 established beta-papillomavirus types, and a reverse hybridization assay (RHA) was developed (PM-PCR RHA method). The analytical sensitivity of the PM-PCR RHA method was 10 to 100 viral genomes. The one-step PM-PCR turned out to be more sensitive than the previously described nested MaHa-PCR for beta-papillomavirus detection. The PM-PCR RHA method was able to detect and identify beta-papillomavirus types in frozen patient material as well as in poorly amplifiable material such as formalin-fixed, paraffin-embedded skin biopsy specimens. Inter- and intralaboratory variability experiments showed that the reproducibility of the assay was very high. In conclusion, the one-step PM-PCR together with the RHA allows extremely sensitive, specific, and reproducible detection of beta-papillomavirus DNA as well as reliable identification of beta-papillomavirus genotypes in both fresh and paraffin-embedded patient material.

Betapapillomaviruses: Innocent bystanders or causes of skin cancer

Human papillomaviruses (HPV) are found in almost all squamous epithelia where they can cause hyperproliferative disease of mucosa and skin. Mucosal HPV types, such as HPV6 and HPV16, are known to cause anogenital warts and dysplasia or neoplasia, respectively. These HPV types have been studied extensively, and for some of them recently preventive vaccines have become available. Although HPV that populate the skin were the first identified HPV types, knowledge of the pathogenicity of HPV in the cornified epithelia stayed behind. What the majority of cutaneous HPV types do, for instance those belonging to the beta genus (betaPV), is largely unknown. As the number of reports that describe epidemiological associations between markers of betaPV infection and skin cancer gradually increases, the need for basic knowledge about these viruses grows as well. This review aims to picture what is currently known about betaPV with respect to infection, transmission and transformation, in order to envisage their potential role in cutaneous carcinogenesis.

Development of a sensitive and specific multiplex PCR method combined with DNA microarray primer extension to detect beta-papillomavirus types

ABSTRACT Emerging lines of evidence indicate that the cutaneous human papillomavirus (HPV) types that belong to the genus Betapapillomavirus (beta HPV) are involved in the development of nonmelanoma skin cancer. Unlike the situation for mucosal HPV types, highly sensitive and reliable methods to identify characterized cutaneous HPV types in a single assay are limited. Here, we describe a novel one-shot method for the detection of all characterized beta HPV types, namely, HPV type 5 (HPV5), 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, and 96. This assay combines two different techniques: multiplex PCR using HPV type-specific primers for amplification of each E7 gene and array primer extension (APEX) for typing. This method has been validated using clinical samples which were analyzed simultaneously for the presence of cutaneous HPV types by two additional methods, i.e., the FAP59/64 PCR protocol and a commercially available PCR-reverse hybridization assay (PM-PCR RHA). Our data show good agreement between the results obtained with the multiplex PCR/APEX assay and the PM-PCR RHA method (overall HPV positivity of 92.2% for multiplex PCR/APEX assay versus 90.6% with the PM-PCR RHA) (kappa value, 50; 95% confidence interval, 13 to 88). In addition, the multiplex PCR/APEX assay showed higher sensitivity than the PM-PCR RHA did. This favorable feature and the high-throughput potential make this assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of cutaneous types in skin cancer.

Human Beta-papillomavirus infection and keratinocyte carcinomas.

Although the role of oncogenic human Alpha‐papillomaviruses (HPVs) in the development of mucosal carcinomas at different body sites (eg cervix, anus, oropharynx) is fully recognized, a role for HPV in keratinocyte carcinomas (KCs; basal and squamous cell carcinomas) of the skin is not yet clear. KCs are the most common cancers in Caucasians, with the major risk factor being ultraviolet (UV) light exposure. A possible role for Beta‐HPV types (BetaPV) in the development of KC was suggested several decades ago, supported by a number of epidemiological studies. Our current review summarizes the recent molecular and histopathological evidence in support of a causal association between BetaPV and the development of KC, and outlines the suspected synergistic effect of viral gene expression with UV radiation and immune suppression. Further insights into the molecular pathways and protein interactions used by BetaPV and the host cell is likely to extend our understanding of the role of BetaPV in KC. Copyright

Human Papillomavirus Load in Eyebrow Hair Follicles and Risk of Cutaneous Squamous Cell Carcinoma

Background: Beta-human papillomavirus (betaPV) may play a role in the development of cutaneous squamous cell carcinoma (SCC). However betaPV is highly prevalent, and it may only be people with a higher viral load who have increased risk of SCCs. We therefore examined the association between betaPV load and SCCs. Methods: We recruited 448 immunocompetent cases with SCCs and 464 controls from Italy and Australia and 497 immunosuppressed organ transplant recipients (OTR; 179 cases and 318 controls) from Europe. We used reverse hybridization to genotype 25 betaPV types in eyebrow hair follicles and determined the viral load for eight selected types using quantitative PCR. We used logistic regression to assess associations between type-specific and cumulative viral load and SCCs. Results: Australian and OTR participants in the highest cumulative load tertile were at significantly higher risk of SCCs than those in the lowest tertile. Those with more than four betaPV types in the high load tertile were at approximately three-fold increased risk of SCCs. In Australia, HPV23 and 36 loads were significantly associated with SCCs, with borderline associations for HPV5 and 38. In OTR, HPV8 and 38 loads were significantly associated and HPV20 and 36 were borderline. We found little evidence for an association between load and SCCs in Italy. Conclusions: High viral load may be associated with risk of cutaneous SCCs, with total load seemingly more important than the load of any specific type. Impact: Our findings lend weight to the hypothesis that HPV plays a role in skin carcinogenesis. Cancer Epidemiol Biomarkers Prev; 22(4); 719–27. ©2013 AACR.

Comprehensive analysis of Human Papillomavirus and Chlamydia trachomatis in in-situ and invasive cervical adenocarcinoma

OBJECTIVE Chlamydia trachomatis (Ct) has been implicated as a co-factor in cervical carcinogenesis. The goal of the current study was to investigate if Ct may play a role in pathogenesis of cervical adenocarcinoma and, specifically, if there is a co-infection between Ct and Human Papillomavirus (HPV) in cervical adenocarcinomas. The second goal of the study was to determine the distribution of HPV genotypes in most recent cases of in-situ and invasive cervical adenocarcinomas. METHODS Biopsies of 71 cervical adenocarcinomas (31 in-situ and 40 invasive) were tested for the presence of Ct using two novel PCR assays. In addition, all cases were tested for HPV using SPF10-PCR assay and genotyped using LIPA(25) test. RESULTS None of the cases was found to be positive for Ct using two independent PCR assays. All lesions, however, were positive for HPV with the exception of a case of minimal deviation adenocarcinoma. Overall, 94.2% of cases were positive for either HPV16 (n=44, 62.8%) or HPV18 (n=20, 28.5%), or both (n=2, 2.8%). Other single HPV types included HPV45 (n=3, 4.2%) and HPV35 (n=1, 1.4%). CONCLUSION The study demonstrated lack of co-infection between Human Papillomavirus and C. trachomatis in in-situ and invasive adenocarcinoma of the uterine cervix. The role of Ct as a carcinogenetic co-factor may be restricted to cervical squamous cell carcinomas. Accounting for type cross-protection, currently available HPV vaccines are likely to prevent close to 100% of HPV-positive cervical adenocarcinomas.

Antibody responses to 26 skin human papillomavirus types in the Netherlands, Italy and Australia

Solar UV radiation is the main risk factor for cutaneous squamous cell carcinoma (SCC), but infections with skin human papillomavirus (HPV) types have also been linked to the development of SCC. Little is known about the natural history of these infections and whether the seroprevalence of skin HPV types is affected by ambient or individual levels of sun exposure. This study investigated this by analysing sera for antibodies to 26 skin HPV types from five phylogenetic genera obtained from 807 healthy individuals from the Netherlands, Italy and Australia, countries with strong differences in sunlight intensity. Overall HPV seroprevalence was similar across the three countries (50-57 % for beta-HPV types, 40-48 % for gamma-HPV types), and the most frequent beta-HPV and gamma-HPV types were the same in all countries. The highest seroprevalences for 24 of the 26 skin HPV types were observed in Italy (14 types) and Australia (ten types). Seroprevalence among men was generally higher than among women, and the male sex was significantly associated with both beta-HPV [odds ratio (OR) 2.81, 95 % confidence interval (CI) 1.64-4.82] and gamma-HPV (OR 2.42, 95 % CI 1.40-4.18) antibodies in Australia. The only measure of sun sensitivity or UV exposure significantly associated with skin HPV seroprevalence was found for weekend sun exposure in Australia and beta-HPV antibodies. It was concluded that type spectra and HPV seroprevalence are similar in countries with different sunlight intensity, and that levels of UV exposure do not play a strong role in the development of skin HPV antibodies in this study population.

HPV genotyping and HPV16 variant analysis in glandular and squamous neoplastic lesions of the uterine cervix

OBJECTIVE The objective of the study was to compare the distribution of HPV genotypes and HPV16 variants in glandular and squamous cervical neoplasia. METHODS Cases of endocervical adenocarcinoma in-situ (AIS, n=33) invasive adenocarcinoma (ADCA, n=55), cervical intraepithelial neoplasia-3 (CIN3, n=130) and squamous cell carcinoma (SCC, n=60) were collected at the New York Hospital and tested for HPV using SPF(10)PCR-LIPA(25) (version 1) assays and for HPV16 variants using a multiplex PCR and reverse hybridization assay. RESULTS There was a difference between the spectrum of HPV genotypes detected in glandular and squamous neoplasia: 13 different HPV genotypes were detected in CIN3 as single infections and 11 in SCC, while only 4 single genotypes were detected in AIS and 3 in ADCA. The most common single HPV types in CIN3 were HPV16, 31, and 52 (56.9%, 10%, 8.4%, respectively). In SCC the most common were HPV16, 18 and 31 (70%, 6.5%, 4.9%). In AIS, HPV16, 18, 45 and 35 accounted for 69.7%, 27.2%, 3%, 3% of cases. The three single types in ADCA were HPV16 (43.6%), HPV18 (41.8%) and HPV45 (10.9%). European variants of HPV16 were the most common in CIN3 (83.8%), SCC (71.4%) and AIS (73.9%). In ADCA the Asian American (AA) variant was the most common (41.7%) followed by European variants (33.3%). AA variant was also detected in 17.4%, 4.1%, and 2.4% of HPV16 positive AIS, CIN3 and SCC, respectively. CONCLUSION Asian American variant of HPV16, HPV18 and HPV45 are preferentially associated with cervical adenocarcinoma as compared to squamous cell carcinoma.