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Papers by Maurits de Koning

Journal of Clinical Microbiology, 2010

A large number of human papillomavirus (HPV) types, distributed over five papillomavirus genera, ... more A large number of human papillomavirus (HPV) types, distributed over five papillomavirus genera, are detectable in the skin. HPV types belonging to the alpha, gamma, and mu genera have been detected in cutaneous warts. A state-of-the-art HPV genotyping assay for these cutaneous wart-associated HPV types does not exist although warts constitute a highly prevalent skin condition, especially in children (33%) and organ transplant recipients (45%). Cutaneous warts are again the focus of attention as their clinical relevance rises with the increasing number of chronically immunosuppressed patients. The objective of this study was to develop and evaluate a DNA-based genotyping system for all known cutaneous wart-related HPV types using PCR and Luminex xMAP technology. The broad-spectrum PCR amplified DNA of all known wart-associated HPV types from the genera alpha , and nu (HPV41). The probes were evaluated using plasmid HPV DNA and a panel of 45 previously characterized cutaneous wart biopsy specimens showing high specificity. HPV was also identified in 96% of 100 swabs from nongenital cutaneous warts. HPV types 1, 2, 27, and 57 were the most prevalent HPV types detected in 89% of the swabs. In conclusion, this Luminex-based genotyping system identifies all known cutaneous wart HPV types including phylogenetically related types, is highly HPV type specific, and is suitable for large-scale epidemiological studies.

Journal of Clinical Virology, 2009

s u m m a r y Background: Based on epidemiologic studies, 18 mucosal human papillomavirus (HPV) t... more s u m m a r y Background: Based on epidemiologic studies, 18 mucosal human papillomavirus (HPV) types have been classified as (probably) high-risk (HR) (i.e., HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82). Recognition of HR HPV at the individual type level may be valuable in clinical management of HR HPV-positive women. Objectives: The goal of this study was to evaluate the performance of the novel digene HPV Genotyping RH Test (digene RH Test), which uses type-specific probes for the 18 HR HPV genotypes, in comparison to the established in-house Reverse Line Blot (RLB) genotyping assay on PCR products generated with the clinically validated GP5+/6+-PCR method. Study design: GP5+/6+ amplimers, generated from 493 digene High Risk HPV HC2 DNA Test (HC2)positive and 95 HC2-negative cervical smears, were genotyped by both the digene RH Test and the RLB assay. Results: Both genotyping assays demonstrated high concordance for overall HR HPV detection (ú = 0.886) and type-specific identification of the 18 HR types (overall ú = 0.951, individual ú range 0.777 to 1.000) in 493 HC2-positive samples. The digene RH Test revealed positivity for one or more HR HPV type(s) in 86.6% of the HC2-positive women, and negativity was confirmed in 97.9% of the HC2-negative women.

International Journal of Cancer, 2010

Human papillomaviruses from the genus beta (betaPV) are a possible cause of cutaneous squamous ce... more Human papillomaviruses from the genus beta (betaPV) are a possible cause of cutaneous squamous cell carcinoma (SCC). We compared the betaPV infections in SCC and in sets of cutaneous tissues collected from a series of individual SCC patients to determine concordance and to assess the adequacy of eyebrow hairs as noninvasive markers of betaPV infection. Biopsies of SCC tumors, perilesional tissue, normal skin from the mirror image of nonfacial SCC and plucked eyebrow hairs were collected from 21 patients with incident SCC living in Queensland, Australia. These were tested for the presence of DNA from 25 different betaPV types. Overall prevalence of betaPV was high in every sample type, ranging from 81% to 95%. The median number of types was significantly higher in the SCC tumour (6), perilesional skin (5) and eyebrow hairs (5) than in normal skin (2). Comparing SCC tissue with other sample types within patients showed 63 overlapping infections with eyebrow hairs (71%; 95% CI: 60–80); 56 with perilesional skin samples (63%; 95% CI: 52–73) and 23 with normal skin samples (26%; 95% CI: 17–36). The sensitivity of eyebrow hair testing for detection of betaPV in the tumor was 82% (95% CI: 57–96) with concordance defined as 50% of betaPV types in common and 29% (95% CI: 10–56) for 100% concordance. These findings support the concept that perilesional skin represents an area of field change involving betaPV preceding SCC development and indicate that eyebrow hairs can serve to some degree as an easily collected marker of tumor betaPV status in epidemiological studies.

Infectious Agents and Cancer, 2008

Background We investigated the role of infection with genital and cutaneous human papillomavirus ... more Background We investigated the role of infection with genital and cutaneous human papillomavirus types (HPV) in the aetiology of ocular surface squamous neoplasia (which includes both conjunctival intraepithelial neoplasia (CIN) and carcinoma) using data and biological material collected as part of a case-control study in Uganda. Results Among 81 cases, the prevalence of genital and cutaneous HPV types in tumour tissue did not differ significantly by histological grade of the lesion. The prevalence of genital HPV types did not differ significantly between cases and controls (both 38%; Odds ratio [OR] 1.0, 95% confidence interval [CI] 0.4–2.7, p = 1.0). The prevalence of cutaneous HPV types was 22% (18/81) among cases and 3% (1/29) among controls (OR 8.0, 95% CI 1.0–169, p = 0.04). Conclusion We find no evidence of an association between genital HPV types and ocular surface squamous neoplasia. The prevalence of cutaneous HPV was significantly higher among cases as compared to controls. Although consistent with results from two other case-control studies, the relatively low prevalence of cutaneous HPV types among cases (which does not differ by histological grade of tumour) indicates that there remains considerable uncertainty about a role for cutaneous HPV in the aetiology of this tumour.

British Journal of Dermatology, 2009

Background Nonmelanoma skin cancer (NMSC) has been linked to cutaneous human papillomaviruses of... more Background Nonmelanoma skin cancer (NMSC) has been linked to cutaneous human papillomaviruses of the genus beta (betaPV).Objectives We sought to assess the presence of betaPV in NMSC biopsies from a group of Scottish skin cancer patients, both immunocompetent (IC) patients and immunosuppressed (IS) organ transplant recipients.Methods One hundred and twenty-one paraffin-embedded skin tumours (27 actinic keratosis, 41 intraepidermal carcinoma, 53 squamous cell carcinoma) and 11 normal skin samples were analysed for the presence of betaPV by a polymerase chain reaction–reverse hybridization assay designed to detect the presence of the 25 known betaPV genotypes.Results In IC patients, betaPV was detected in 30 of 59 (51%) tumours and two of 11 (18%) normal skin samples (P = 0·046). In IS patients, betaPV was found in 27 of 62 (44%) tumours; no normal skin samples were available for comparison. The most frequently found genotypes were HPV-24, HPV-15 and HPV-38. Of those tumours infected with betaPV, 28 of 57 (49%) were infected with more than one genotype (range 2–8). Tumours from IS patients were from a younger age group (mean age 57·4 years) than IC patients (mean age 73·8 years). Multiple infections were more common in tumours from IC patients (21 of 30; 70%) compared with those from IS patients (seven of 27; 26%) (P < 0·001). In the IC group, age did not appear to influence the distribution of single and multiple infections whereas in IS patients the proportion of multiple infections to single infections increased with age. There were no multiple infections in normal skin.Conclusions A wide spectrum of betaPV types was detected in our samples. Further characterization of betaPV in vivo is needed in order to determine the mechanisms by which the virus contributes to cutaneous carcinogenesis.

Journal of General Virology, 2010

Betapapillomavirus (bPV) DNA and seroresponses are highly prevalent in the general population and... more Betapapillomavirus (bPV) DNA and seroresponses are highly prevalent in the general population and both are frequently used as infection markers in epidemiological studies to elucidate an association with cutaneous squamous cell carcinoma (SCC). Little is known about the natural history of bPV infection and the aspects of infection that drive antibody responses. To investigate the relationship between these markers, this study assessed whether the presence or persistence of bPV DNA in eyebrow hairs and L1 antibodies of the same bPV type co-occurred more frequently than would be expected by chance in both a cross-sectional assessment and a longitudinal study. bPV DNA in plucked eyebrow hairs and L1 antibodies in serum were measured in 416 participants of the Australian community-based Nambour Skin Cancer Study in 1996. Similar data were available for a subset of 148 participants in 2003. Observed co-occurrence of bPV DNA and antibodies was compared with expected values based on prevalence. A case-wise concordance index was used to calculate the overall concordance of bPV DNA and antibodies of the same type. No significant associations were found between the presence or persistence of bPV DNA and antibody responses. The age and sex of the host did not influence the association, and nor did SCC status or a history of sunburns. It was concluded that bPV antibody responses in adults are not primarily driven by bPV infection as measured in eyebrow hairs. Other factors, such as viral load, may play a more pivotal role in the induction of detectable seroresponses.

Journal of Virology, 2009

Cutaneous human betapapillomaviruses (beta-HPVs) are widespread in the general population and hav... more Cutaneous human betapapillomaviruses (beta-HPVs) are widespread in the general population and have been associated with skin cancer. To evaluate the impact of continuous person-to-person contact within families on an individual's beta-HPV type spectrum, we collected serial skin swab samples from parents and children from 10 families. All participants were found to be beta-HPV DNA positive, with 1 to 13 types at study entry (median, 4.0 types). Initial and cumulative (2 to 16 types) HPV type multiplicities varied widely between different families but only a little between family members. The high intrafamilial correlation of HPV multiplicity is already obvious for babies aged 10 days to 10 months. Family members typically displayed similar spectra of HPV types. More than 75% of the HPV types in babies were also detected in their parents. This indicates that HPV transmission mainly results from close contact between family members. Type-specific persistence for at least 9 months was more prevalent in parents (92%) than in children (66%). Of the types detected throughout the study, 24% turned out to persist in the parents and only 11% in the children. Interestingly, about one-half of the HPV types found to persist in one of the parents occurred less frequently or even only sporadically in the spouse. Similarly, only one-third of the persisting parental types also persisted in their children. This indicates that even regular exposure to cutaneous HPV does not necessarily lead to the establishment of a persistent infection, which may point to type-specific susceptibilities of different individuals.

Journal of Clinical Virology, 2010

Journal of General Virology, 2009

Solar UV radiation is the main risk factor for cutaneous squamous cell carcinoma (SCC), but infec... more 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 b-HPV types, 40-48 % for c-HPV types), and the most frequent b-HPV and c-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 b-HPV [odds ratio (OR) 2.81, 95 % confidence interval (CI) 1.64-4.82] and c-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 b-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.

Journal of Molecular Diagnostics, 2011

A novel Chlamydia trachomatis (Ct) microsphere suspension (MS) assay was evaluated for identifica... more A novel Chlamydia trachomatis (Ct) microsphere suspension (MS) assay was evaluated for identification of the different serovars, using the same PCR primer set established for the Ct Detection and genoTyping assay. Both assays can detect and identify all 14 major serovars (A, L2/L2a, and L3) and one genovariant of serovar J. The probe specificity for the Ct-MS assay was determined using 14 Ct reference strains and 1 clinical isolate from a genovariant of serovar J. Also, the Ct-MS assay and the Ct detection and genoTyping assay were compared in 712 Ct-positive clinical samples. The Ct-MS assay showed a highly specific reaction for all probes with the amplicons of the reference strains, giving a very low background median fluorescence intensity signal (median fluorescence intensity < 10). An excellent overall agreement in the Ct detection (kappa ‫؍‬ 0.947, 95% con-fidence interval, 0.89 to 0.999; McNemar's test, P ‫؍‬ 1.000) and the Ct genotyping (kappa ‫؍‬ 0.993, 95% confidence interval, 0.977 to 1.000; McNemar's test, P ‫؍‬ 0.053) was observed between the Ct detection and genoTyping (DT) assay and the Ct-MS assay. In conclusion, the novel Ct-MS assay permits simultaneous detection and genotyping of Ct serovars, making the Ct-MS assay an excellent high throughput method. ( J Mol Diagn 2011, 13:152-159;

Journal of Clinical Microbiology, 2010

A large number of human papillomavirus (HPV) types, distributed over five papillomavirus genera, ... more A large number of human papillomavirus (HPV) types, distributed over five papillomavirus genera, are detectable in the skin. HPV types belonging to the alpha, gamma, and mu genera have been detected in cutaneous warts. A state-of-the-art HPV genotyping assay for these cutaneous wart-associated HPV types does not exist although warts constitute a highly prevalent skin condition, especially in children (33%) and organ transplant recipients (45%). Cutaneous warts are again the focus of attention as their clinical relevance rises with the increasing number of chronically immunosuppressed patients. The objective of this study was to develop and evaluate a DNA-based genotyping system for all known cutaneous wart-related HPV types using PCR and Luminex xMAP technology. The broad-spectrum PCR amplified DNA of all known wart-associated HPV types from the genera alpha , and nu (HPV41). The probes were evaluated using plasmid HPV DNA and a panel of 45 previously characterized cutaneous wart biopsy specimens showing high specificity. HPV was also identified in 96% of 100 swabs from nongenital cutaneous warts. HPV types 1, 2, 27, and 57 were the most prevalent HPV types detected in 89% of the swabs. In conclusion, this Luminex-based genotyping system identifies all known cutaneous wart HPV types including phylogenetically related types, is highly HPV type specific, and is suitable for large-scale epidemiological studies.

Journal of Clinical Virology, 2008

Human papillomaviruses (HPV) are found in almost all squamous epithelia where they can cause hype... more 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.

Journal of Clinical Virology, 2009

Background: Epidemiologic studies have classified 18 genotypes of the human papillomavirus (HPV) ... more Background: Epidemiologic studies have classified 18 genotypes of the human papillomavirus (HPV) as (probably) high-risk (HR) based on their association with cervical cancer, i.e., HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. Given the fact that certain HR HPV types confer an increased risk of cervical (pre)cancer, type-specific identification might aid clinical management of women tested positive for HR HPV. Therefore, the development of robust, highthroughput genotyping assays is important. Objectives: An analytical comparison of the digene HPV Genotyping LQ Test (digene LQ Test), capable of identifying 18 HR types using bead-based xMAP suspension array technology, with the established Reverse Line Blot (RLB) genotyping assay was carried out on amplimers generated with the clinically validated GP5+/6+-PCR method. Study design: GP5+/6+ amplimers, generated from 434 digene High Risk HPV HC2 DNA Test (HC2)positive and 95 HC2-negative cervical smears, were genotyped by both the digene LQ Test and the RLB genotyping assay. Results: The genotyping assays revealed high agreement for overall HR HPV detection (ú = 0.884) and type-specific identification of the 18 HR HPV types (overall ú = 0.958, individual ú range 0.795 to 1.000). The digene LQ Test demonstrated a very good inter-laboratory reproducibility (ú = 0.987). Among the HC2-positive women, the digene LQ Test revealed positivity for one or more HR HPV type(s) in 85.9%, and negativity was observed in 97.9% of the HC2-negative women.

Journal of General Virology, 2007

Infections with human papillomaviruses (HPVs) belonging to the genus Betapapillomavirus have been... more Infections with human papillomaviruses (HPVs) belonging to the genus Betapapillomavirus have been linked to the development of non-melanoma skin cancer. Although persistence is expected, systematic investigation of this aspect of betapapillomavirus (b-PV) infection has not been conducted. This study investigated the prevalence and persistence of 25 known b-PV types in the skin of immunocompetent individuals. Over a 2 year period, eight consecutive plucked eyebrow hair samples taken from 23 healthy individuals were analysed for the presence of b-PV DNA. Using a recently published general b-PV PCR and genotyping method, 61 % of the individuals were b-PV DNA positive for one or more types at intake, whereas during follow-up this percentage rose to 96 %. HPV23 was the most frequently detected b-PV type. Type-specific b-PV DNA was detected over 6 months or longer in 74 % of the individuals. In 57 % of the individuals, DNA from multiple b-PV types was detected simultaneously for 6 months or longer. When the detection intervals of all b-PV type-specific infections in the study population were considered, a substantial proportion, 48 %, lasted at least half a year. The consistent b-PV patterns found over time in most individuals strongly suggested that b-PV DNA detection in plucked eyebrow hairs reveals true b-PV infection. If the minimum interval of detection was set at 6 months, persistent b-PV infections were found in the majority of the study population (74 %).

Journal of Molecular Diagnostics, 2007

Chlamydia trachomatis (Ct) comprises distinct serogroups and serovars. The present study evaluate... more Chlamydia trachomatis (Ct) comprises distinct serogroups and serovars. The present study evaluates a novel Ct amplification, detection, and genotyping method (Ct-DT assay). The Ct-DT amplification step is a multiplex broad-spectrum PCR for the cryptic plasmid and the VD2-region of ompl. The Ct-DT detection step involves a DNA enzyme immunoassay (DEIA) using probes for serogroups (group B, C, and intermediate) and the cryptic plasmid, permitting sensitive detection of 19 Ct serovars (A, and L3) without any cross-reactivity with other Chlamydia species and pathogenic bacteria or commensal organisms of the genital tract. Ct-positive samples are analyzed by a nitrocellulose-based reverse hybridization assay (RHA) containing probes for the 19 different serovars and for the cryptic plasmid. The sensitivity of the PCR-DEIA on clinical specimen is equivalent to that of the Cobas TaqMan assay [ ‫؍‬ 0.95 (95% confidence interval ‫؍‬ 0.92 to 0.99)]. Using the RHA, 98% of the Ct-DT detection step-positive samples could be typed. Analysis of cervical swabs from Uganda and The Netherlands revealed that the most common serovars in Uganda are G/Ga (45%), E (21%), K (13%), and F (8%), and in The Netherlands serovars E (38%), F (23%), G/Ga (11%), and D/Da (7%) were most common.

Journal of General Virology, 2009

Betapapillomavirus (betaPV) infections are often associated with squamous-cell carcinoma (SCC) an... more 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.

Modern Pathology, 2008

Two independent pathways of vulvar carcinogenesis have currently been identified, one related to ... more Two independent pathways of vulvar carcinogenesis have currently been identified, one related to infection with mucosal human papillomaviruses (HPVs) and a second related to chronic inflammatory or autoimmune processes. The goal of the study was to examine a possible role of cutaneous HPVs from the beta genus in vulvar carcinogenesis and to evaluate the distribution of intratypic variants of HPV 16 in HPV 16-positive vulvar cancer. Consecutive cases of vulvar carcinoma were retrieved from the files and included the following histologic subtypes: keratinizing (n ¼ 21), basaloid (n ¼ 7), warty (n ¼ 1), mixed basaloid-warty (n ¼ 4), verrucous (n ¼ 4), keratoacanthoma (n ¼ 1), basal cell carcinoma (n ¼ 1). All tumors were microdissected and tested for 25 beta HPV types and 25 mucosal HPV types. Cases identified as positive for HPV 16 were further tested for intratypic variants. All cases were immunostained for p16 INK4a . Beta HPVs were not detected in any of the tumor cases. Mucosal HPVs were detected in all but one basaloid/warty carcinomas; of these, nine cases (82%) were positive for HPV 16, including five European subtypes, one African subtype, one North American subtype and two indeterminate subtypes. Two of four verrucous carcinomas were positive for HPV 6. Mucosal HPVs were not detected in keratinizing carcinomas, keratoacanthoma and basal cell carcinoma. All cases of basaloid/warty carcinomas, but none of the remaining tumors, overexpressed p16 INK4a protein. Our data do not support a role of beta HPVs in the pathogenesis of vulvar carcinoma. The study reaffirms the role of mucosal HPVs, in particular that of HPV 16, in the pathogenesis of basaloid and warty tumor subtypes. The HPV 16 intratypic variation showed correlation with patients' ethnic background. P16 INK4a immunostaining seems to be a sensitive and specific marker of vulvar carcinomas positive for oncogenic mucosal HPVs.

Journal of Cellular and Molecular Medicine, 2009

Studies in cervical dysplasia have reported overexpression of the tumour suppressors p14 and p16 ... more Studies in cervical dysplasia have reported overexpression of the tumour suppressors p14 and p16 – and absence of p53 – in high-risk human papilloma virus (HPV)- associated lesions. In skin carcinogenesis, the relation between these tumour suppressors and HPV remain unclear. We evaluated the expression of the tumour suppressors p14, p16 and p53 in pre-malignant and malignant squamous skin tumours, and its relation with risk factors for skin carcinogenesis (HPV, immune status and sun exposure). We performed immunohistochemical stainings for p14, p16 and p53 on paraffin embedded material of 71 pre-malignant squamous skin lesions and 34 squamous cell carcinomas, from 52 renal transplant recipients (RTRs) and 53 immunocompetent individuals. PCR-based assays were used for detection and genotyping of β-papilloma virus (β-PV) types and mucosal HPV types. P14 expression was independent of the expression of p16 and p53, irrespective of immune status and skin site. In 49 of 105 specimens (46.6%), one or more β-PV types were detected. We found no significant association between p14, p16 or p53 protein expression and overall presence of β-PV, irrespective of immune status. There was a significant association between presence of β-PV and lesions from sun-exposed skin sites in the RTRs (P= 0.002). We conclude that in skin carcinogenesis, relations between the herein studied tumour suppressors and HPV are different from what one would expect based on findings in cervical neoplasia. P14, p16 and p53 expressions are independent of immune status. Our data indicate that in immunosuppressed patients, β-PV together with ultraviolet radiation act synergetic in promoting carcinogenesis.

Cancer Research, 2009

Human papillomaviruses from the β genus (βPV) are a possible cause of cutaneous squamous cell car... more Human papillomaviruses from the β genus (βPV) are a possible cause of cutaneous squamous cell carcinoma (SCC). We assessed the extent to which βPV infections persisted longterm in a subtropical Australian community and whether βPV persistence is positively associated with actinic keratoses, precursor for SCC. Eyebrow hairs were collected from 171 participants of the community-based Nambour Skin Cancer Study in 1996 and 2003. Hair samples were tested for the presence of DNA from 25 different βPV types and assessed in relation to actinic keratosis presence in 2007. In 1996, a total of 413 βPV infections were found in 73% of participants, increasing to 490 infections among 85% in 2003. Of the total βPV infections detected, 211 (30%) were found to persist. Age was significantly associated with βPV persistence: those ages >60 years had 1.5fold (95% confidence interval, 1.1-1.9) increased risk of typespecific viral persistence than those ages <40 years. After accounting for actinic keratoses at baseline, persistence of βPV DNA resulted in a 1.4-fold (95% confidence interval, 1.0-1.9) increase in risk of having actinic keratoses on the face in 2007. In conclusion, persistent βPV infections in this population were associated with an increased occurrence of actinic keratosis. Additional studies are needed to determine the possible association of βPV persistence with SCC. [Cancer Res 2009;69(23):8926-31] 8926

International Journal of Cancer, 2009

Genetic alterations in metastatic cutaneous squamous cell carcinoma (CSCC) which might serve as p... more Genetic alterations in metastatic cutaneous squamous cell carcinoma (CSCC) which might serve as prognostic biomarkers are not well investigated. We investigated the mutation status and protein expression of the CDKN2A (INK4a-ARF) and TP53 genes in metastatic CSCCs and correlated this with clinicopathological variables, HPV presence, and survival data. Sequence analysis was performed on formalin-fixed and paraffin-embedded tissue of 35 metastases and their primary tumors, and was correlated with immunohistochemical stainings for p53, p16 and p14. Beta-PV and alpha-PV DNA was detected using PCR-based assays. Kaplan–Meier and Cox regression methods were used for survival assessment. CDKN2A was mutated in 31% of the metastases and their primary tumors, while the TP53 gene was mutated in 51% of the metastases. P53 protein expression was significantly associated with missense type of mutations (p = 0.002). No persistent HPV types were detected. CDKN2A mutations were significantly associated with disease-specific death (p = 0.001). A significant difference was observed in disease-specific survival between patients with or without a CDKN2A mutation (p = 0.010), while this was not the case for TP53. At univariate Cox's regression analysis tumor size (p = 0.010), invasion depth (p = 0.030) and CDKN2A mutations (p = 0.040) were significantly related to shorter disease-specific survival. At multivariate Cox's regression only tumor size had an adverse effect on survival (p = 0.002). In conclusion, our study indicates that the CDKN2A mutation status might be of prognostic value in metastatic CSCCs. In most cases, CDKN2A and TP53 mutations are early genetic events in CSCC tumorigenesis. The possible role of HPV in metastatic CSCC needs further exploration.

Journal of Clinical Microbiology, 2010

A large number of human papillomavirus (HPV) types, distributed over five papillomavirus genera, ... more A large number of human papillomavirus (HPV) types, distributed over five papillomavirus genera, are detectable in the skin. HPV types belonging to the alpha, gamma, and mu genera have been detected in cutaneous warts. A state-of-the-art HPV genotyping assay for these cutaneous wart-associated HPV types does not exist although warts constitute a highly prevalent skin condition, especially in children (33%) and organ transplant recipients (45%). Cutaneous warts are again the focus of attention as their clinical relevance rises with the increasing number of chronically immunosuppressed patients. The objective of this study was to develop and evaluate a DNA-based genotyping system for all known cutaneous wart-related HPV types using PCR and Luminex xMAP technology. The broad-spectrum PCR amplified DNA of all known wart-associated HPV types from the genera alpha , and nu (HPV41). The probes were evaluated using plasmid HPV DNA and a panel of 45 previously characterized cutaneous wart biopsy specimens showing high specificity. HPV was also identified in 96% of 100 swabs from nongenital cutaneous warts. HPV types 1, 2, 27, and 57 were the most prevalent HPV types detected in 89% of the swabs. In conclusion, this Luminex-based genotyping system identifies all known cutaneous wart HPV types including phylogenetically related types, is highly HPV type specific, and is suitable for large-scale epidemiological studies.

Journal of Clinical Virology, 2009

s u m m a r y Background: Based on epidemiologic studies, 18 mucosal human papillomavirus (HPV) t... more s u m m a r y Background: Based on epidemiologic studies, 18 mucosal human papillomavirus (HPV) types have been classified as (probably) high-risk (HR) (i.e., HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82). Recognition of HR HPV at the individual type level may be valuable in clinical management of HR HPV-positive women. Objectives: The goal of this study was to evaluate the performance of the novel digene HPV Genotyping RH Test (digene RH Test), which uses type-specific probes for the 18 HR HPV genotypes, in comparison to the established in-house Reverse Line Blot (RLB) genotyping assay on PCR products generated with the clinically validated GP5+/6+-PCR method. Study design: GP5+/6+ amplimers, generated from 493 digene High Risk HPV HC2 DNA Test (HC2)positive and 95 HC2-negative cervical smears, were genotyped by both the digene RH Test and the RLB assay. Results: Both genotyping assays demonstrated high concordance for overall HR HPV detection (ú = 0.886) and type-specific identification of the 18 HR types (overall ú = 0.951, individual ú range 0.777 to 1.000) in 493 HC2-positive samples. The digene RH Test revealed positivity for one or more HR HPV type(s) in 86.6% of the HC2-positive women, and negativity was confirmed in 97.9% of the HC2-negative women.

International Journal of Cancer, 2010

Human papillomaviruses from the genus beta (betaPV) are a possible cause of cutaneous squamous ce... more Human papillomaviruses from the genus beta (betaPV) are a possible cause of cutaneous squamous cell carcinoma (SCC). We compared the betaPV infections in SCC and in sets of cutaneous tissues collected from a series of individual SCC patients to determine concordance and to assess the adequacy of eyebrow hairs as noninvasive markers of betaPV infection. Biopsies of SCC tumors, perilesional tissue, normal skin from the mirror image of nonfacial SCC and plucked eyebrow hairs were collected from 21 patients with incident SCC living in Queensland, Australia. These were tested for the presence of DNA from 25 different betaPV types. Overall prevalence of betaPV was high in every sample type, ranging from 81% to 95%. The median number of types was significantly higher in the SCC tumour (6), perilesional skin (5) and eyebrow hairs (5) than in normal skin (2). Comparing SCC tissue with other sample types within patients showed 63 overlapping infections with eyebrow hairs (71%; 95% CI: 60–80); 56 with perilesional skin samples (63%; 95% CI: 52–73) and 23 with normal skin samples (26%; 95% CI: 17–36). The sensitivity of eyebrow hair testing for detection of betaPV in the tumor was 82% (95% CI: 57–96) with concordance defined as 50% of betaPV types in common and 29% (95% CI: 10–56) for 100% concordance. These findings support the concept that perilesional skin represents an area of field change involving betaPV preceding SCC development and indicate that eyebrow hairs can serve to some degree as an easily collected marker of tumor betaPV status in epidemiological studies.

Infectious Agents and Cancer, 2008

Background We investigated the role of infection with genital and cutaneous human papillomavirus ... more Background We investigated the role of infection with genital and cutaneous human papillomavirus types (HPV) in the aetiology of ocular surface squamous neoplasia (which includes both conjunctival intraepithelial neoplasia (CIN) and carcinoma) using data and biological material collected as part of a case-control study in Uganda. Results Among 81 cases, the prevalence of genital and cutaneous HPV types in tumour tissue did not differ significantly by histological grade of the lesion. The prevalence of genital HPV types did not differ significantly between cases and controls (both 38%; Odds ratio [OR] 1.0, 95% confidence interval [CI] 0.4–2.7, p = 1.0). The prevalence of cutaneous HPV types was 22% (18/81) among cases and 3% (1/29) among controls (OR 8.0, 95% CI 1.0–169, p = 0.04). Conclusion We find no evidence of an association between genital HPV types and ocular surface squamous neoplasia. The prevalence of cutaneous HPV was significantly higher among cases as compared to controls. Although consistent with results from two other case-control studies, the relatively low prevalence of cutaneous HPV types among cases (which does not differ by histological grade of tumour) indicates that there remains considerable uncertainty about a role for cutaneous HPV in the aetiology of this tumour.

British Journal of Dermatology, 2009

Background Nonmelanoma skin cancer (NMSC) has been linked to cutaneous human papillomaviruses of... more Background Nonmelanoma skin cancer (NMSC) has been linked to cutaneous human papillomaviruses of the genus beta (betaPV).Objectives We sought to assess the presence of betaPV in NMSC biopsies from a group of Scottish skin cancer patients, both immunocompetent (IC) patients and immunosuppressed (IS) organ transplant recipients.Methods One hundred and twenty-one paraffin-embedded skin tumours (27 actinic keratosis, 41 intraepidermal carcinoma, 53 squamous cell carcinoma) and 11 normal skin samples were analysed for the presence of betaPV by a polymerase chain reaction–reverse hybridization assay designed to detect the presence of the 25 known betaPV genotypes.Results In IC patients, betaPV was detected in 30 of 59 (51%) tumours and two of 11 (18%) normal skin samples (P = 0·046). In IS patients, betaPV was found in 27 of 62 (44%) tumours; no normal skin samples were available for comparison. The most frequently found genotypes were HPV-24, HPV-15 and HPV-38. Of those tumours infected with betaPV, 28 of 57 (49%) were infected with more than one genotype (range 2–8). Tumours from IS patients were from a younger age group (mean age 57·4 years) than IC patients (mean age 73·8 years). Multiple infections were more common in tumours from IC patients (21 of 30; 70%) compared with those from IS patients (seven of 27; 26%) (P < 0·001). In the IC group, age did not appear to influence the distribution of single and multiple infections whereas in IS patients the proportion of multiple infections to single infections increased with age. There were no multiple infections in normal skin.Conclusions A wide spectrum of betaPV types was detected in our samples. Further characterization of betaPV in vivo is needed in order to determine the mechanisms by which the virus contributes to cutaneous carcinogenesis.

Journal of General Virology, 2010

Betapapillomavirus (bPV) DNA and seroresponses are highly prevalent in the general population and... more Betapapillomavirus (bPV) DNA and seroresponses are highly prevalent in the general population and both are frequently used as infection markers in epidemiological studies to elucidate an association with cutaneous squamous cell carcinoma (SCC). Little is known about the natural history of bPV infection and the aspects of infection that drive antibody responses. To investigate the relationship between these markers, this study assessed whether the presence or persistence of bPV DNA in eyebrow hairs and L1 antibodies of the same bPV type co-occurred more frequently than would be expected by chance in both a cross-sectional assessment and a longitudinal study. bPV DNA in plucked eyebrow hairs and L1 antibodies in serum were measured in 416 participants of the Australian community-based Nambour Skin Cancer Study in 1996. Similar data were available for a subset of 148 participants in 2003. Observed co-occurrence of bPV DNA and antibodies was compared with expected values based on prevalence. A case-wise concordance index was used to calculate the overall concordance of bPV DNA and antibodies of the same type. No significant associations were found between the presence or persistence of bPV DNA and antibody responses. The age and sex of the host did not influence the association, and nor did SCC status or a history of sunburns. It was concluded that bPV antibody responses in adults are not primarily driven by bPV infection as measured in eyebrow hairs. Other factors, such as viral load, may play a more pivotal role in the induction of detectable seroresponses.

Journal of Virology, 2009

Cutaneous human betapapillomaviruses (beta-HPVs) are widespread in the general population and hav... more Cutaneous human betapapillomaviruses (beta-HPVs) are widespread in the general population and have been associated with skin cancer. To evaluate the impact of continuous person-to-person contact within families on an individual's beta-HPV type spectrum, we collected serial skin swab samples from parents and children from 10 families. All participants were found to be beta-HPV DNA positive, with 1 to 13 types at study entry (median, 4.0 types). Initial and cumulative (2 to 16 types) HPV type multiplicities varied widely between different families but only a little between family members. The high intrafamilial correlation of HPV multiplicity is already obvious for babies aged 10 days to 10 months. Family members typically displayed similar spectra of HPV types. More than 75% of the HPV types in babies were also detected in their parents. This indicates that HPV transmission mainly results from close contact between family members. Type-specific persistence for at least 9 months was more prevalent in parents (92%) than in children (66%). Of the types detected throughout the study, 24% turned out to persist in the parents and only 11% in the children. Interestingly, about one-half of the HPV types found to persist in one of the parents occurred less frequently or even only sporadically in the spouse. Similarly, only one-third of the persisting parental types also persisted in their children. This indicates that even regular exposure to cutaneous HPV does not necessarily lead to the establishment of a persistent infection, which may point to type-specific susceptibilities of different individuals.

Journal of Clinical Virology, 2010

Journal of General Virology, 2009

Solar UV radiation is the main risk factor for cutaneous squamous cell carcinoma (SCC), but infec... more 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 b-HPV types, 40-48 % for c-HPV types), and the most frequent b-HPV and c-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 b-HPV [odds ratio (OR) 2.81, 95 % confidence interval (CI) 1.64-4.82] and c-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 b-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.

Journal of Molecular Diagnostics, 2011

A novel Chlamydia trachomatis (Ct) microsphere suspension (MS) assay was evaluated for identifica... more A novel Chlamydia trachomatis (Ct) microsphere suspension (MS) assay was evaluated for identification of the different serovars, using the same PCR primer set established for the Ct Detection and genoTyping assay. Both assays can detect and identify all 14 major serovars (A, L2/L2a, and L3) and one genovariant of serovar J. The probe specificity for the Ct-MS assay was determined using 14 Ct reference strains and 1 clinical isolate from a genovariant of serovar J. Also, the Ct-MS assay and the Ct detection and genoTyping assay were compared in 712 Ct-positive clinical samples. The Ct-MS assay showed a highly specific reaction for all probes with the amplicons of the reference strains, giving a very low background median fluorescence intensity signal (median fluorescence intensity < 10). An excellent overall agreement in the Ct detection (kappa ‫؍‬ 0.947, 95% con-fidence interval, 0.89 to 0.999; McNemar's test, P ‫؍‬ 1.000) and the Ct genotyping (kappa ‫؍‬ 0.993, 95% confidence interval, 0.977 to 1.000; McNemar's test, P ‫؍‬ 0.053) was observed between the Ct detection and genoTyping (DT) assay and the Ct-MS assay. In conclusion, the novel Ct-MS assay permits simultaneous detection and genotyping of Ct serovars, making the Ct-MS assay an excellent high throughput method. ( J Mol Diagn 2011, 13:152-159;

Journal of Clinical Microbiology, 2010

A large number of human papillomavirus (HPV) types, distributed over five papillomavirus genera, ... more A large number of human papillomavirus (HPV) types, distributed over five papillomavirus genera, are detectable in the skin. HPV types belonging to the alpha, gamma, and mu genera have been detected in cutaneous warts. A state-of-the-art HPV genotyping assay for these cutaneous wart-associated HPV types does not exist although warts constitute a highly prevalent skin condition, especially in children (33%) and organ transplant recipients (45%). Cutaneous warts are again the focus of attention as their clinical relevance rises with the increasing number of chronically immunosuppressed patients. The objective of this study was to develop and evaluate a DNA-based genotyping system for all known cutaneous wart-related HPV types using PCR and Luminex xMAP technology. The broad-spectrum PCR amplified DNA of all known wart-associated HPV types from the genera alpha , and nu (HPV41). The probes were evaluated using plasmid HPV DNA and a panel of 45 previously characterized cutaneous wart biopsy specimens showing high specificity. HPV was also identified in 96% of 100 swabs from nongenital cutaneous warts. HPV types 1, 2, 27, and 57 were the most prevalent HPV types detected in 89% of the swabs. In conclusion, this Luminex-based genotyping system identifies all known cutaneous wart HPV types including phylogenetically related types, is highly HPV type specific, and is suitable for large-scale epidemiological studies.

Journal of Clinical Virology, 2008

Human papillomaviruses (HPV) are found in almost all squamous epithelia where they can cause hype... more 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.

Journal of Clinical Virology, 2009

Background: Epidemiologic studies have classified 18 genotypes of the human papillomavirus (HPV) ... more Background: Epidemiologic studies have classified 18 genotypes of the human papillomavirus (HPV) as (probably) high-risk (HR) based on their association with cervical cancer, i.e., HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. Given the fact that certain HR HPV types confer an increased risk of cervical (pre)cancer, type-specific identification might aid clinical management of women tested positive for HR HPV. Therefore, the development of robust, highthroughput genotyping assays is important. Objectives: An analytical comparison of the digene HPV Genotyping LQ Test (digene LQ Test), capable of identifying 18 HR types using bead-based xMAP suspension array technology, with the established Reverse Line Blot (RLB) genotyping assay was carried out on amplimers generated with the clinically validated GP5+/6+-PCR method. Study design: GP5+/6+ amplimers, generated from 434 digene High Risk HPV HC2 DNA Test (HC2)positive and 95 HC2-negative cervical smears, were genotyped by both the digene LQ Test and the RLB genotyping assay. Results: The genotyping assays revealed high agreement for overall HR HPV detection (ú = 0.884) and type-specific identification of the 18 HR HPV types (overall ú = 0.958, individual ú range 0.795 to 1.000). The digene LQ Test demonstrated a very good inter-laboratory reproducibility (ú = 0.987). Among the HC2-positive women, the digene LQ Test revealed positivity for one or more HR HPV type(s) in 85.9%, and negativity was observed in 97.9% of the HC2-negative women.

Journal of General Virology, 2007

Infections with human papillomaviruses (HPVs) belonging to the genus Betapapillomavirus have been... more Infections with human papillomaviruses (HPVs) belonging to the genus Betapapillomavirus have been linked to the development of non-melanoma skin cancer. Although persistence is expected, systematic investigation of this aspect of betapapillomavirus (b-PV) infection has not been conducted. This study investigated the prevalence and persistence of 25 known b-PV types in the skin of immunocompetent individuals. Over a 2 year period, eight consecutive plucked eyebrow hair samples taken from 23 healthy individuals were analysed for the presence of b-PV DNA. Using a recently published general b-PV PCR and genotyping method, 61 % of the individuals were b-PV DNA positive for one or more types at intake, whereas during follow-up this percentage rose to 96 %. HPV23 was the most frequently detected b-PV type. Type-specific b-PV DNA was detected over 6 months or longer in 74 % of the individuals. In 57 % of the individuals, DNA from multiple b-PV types was detected simultaneously for 6 months or longer. When the detection intervals of all b-PV type-specific infections in the study population were considered, a substantial proportion, 48 %, lasted at least half a year. The consistent b-PV patterns found over time in most individuals strongly suggested that b-PV DNA detection in plucked eyebrow hairs reveals true b-PV infection. If the minimum interval of detection was set at 6 months, persistent b-PV infections were found in the majority of the study population (74 %).

Journal of Molecular Diagnostics, 2007

Chlamydia trachomatis (Ct) comprises distinct serogroups and serovars. The present study evaluate... more Chlamydia trachomatis (Ct) comprises distinct serogroups and serovars. The present study evaluates a novel Ct amplification, detection, and genotyping method (Ct-DT assay). The Ct-DT amplification step is a multiplex broad-spectrum PCR for the cryptic plasmid and the VD2-region of ompl. The Ct-DT detection step involves a DNA enzyme immunoassay (DEIA) using probes for serogroups (group B, C, and intermediate) and the cryptic plasmid, permitting sensitive detection of 19 Ct serovars (A, and L3) without any cross-reactivity with other Chlamydia species and pathogenic bacteria or commensal organisms of the genital tract. Ct-positive samples are analyzed by a nitrocellulose-based reverse hybridization assay (RHA) containing probes for the 19 different serovars and for the cryptic plasmid. The sensitivity of the PCR-DEIA on clinical specimen is equivalent to that of the Cobas TaqMan assay [ ‫؍‬ 0.95 (95% confidence interval ‫؍‬ 0.92 to 0.99)]. Using the RHA, 98% of the Ct-DT detection step-positive samples could be typed. Analysis of cervical swabs from Uganda and The Netherlands revealed that the most common serovars in Uganda are G/Ga (45%), E (21%), K (13%), and F (8%), and in The Netherlands serovars E (38%), F (23%), G/Ga (11%), and D/Da (7%) were most common.

Journal of General Virology, 2009

Betapapillomavirus (betaPV) infections are often associated with squamous-cell carcinoma (SCC) an... more 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.

Modern Pathology, 2008

Two independent pathways of vulvar carcinogenesis have currently been identified, one related to ... more Two independent pathways of vulvar carcinogenesis have currently been identified, one related to infection with mucosal human papillomaviruses (HPVs) and a second related to chronic inflammatory or autoimmune processes. The goal of the study was to examine a possible role of cutaneous HPVs from the beta genus in vulvar carcinogenesis and to evaluate the distribution of intratypic variants of HPV 16 in HPV 16-positive vulvar cancer. Consecutive cases of vulvar carcinoma were retrieved from the files and included the following histologic subtypes: keratinizing (n ¼ 21), basaloid (n ¼ 7), warty (n ¼ 1), mixed basaloid-warty (n ¼ 4), verrucous (n ¼ 4), keratoacanthoma (n ¼ 1), basal cell carcinoma (n ¼ 1). All tumors were microdissected and tested for 25 beta HPV types and 25 mucosal HPV types. Cases identified as positive for HPV 16 were further tested for intratypic variants. All cases were immunostained for p16 INK4a . Beta HPVs were not detected in any of the tumor cases. Mucosal HPVs were detected in all but one basaloid/warty carcinomas; of these, nine cases (82%) were positive for HPV 16, including five European subtypes, one African subtype, one North American subtype and two indeterminate subtypes. Two of four verrucous carcinomas were positive for HPV 6. Mucosal HPVs were not detected in keratinizing carcinomas, keratoacanthoma and basal cell carcinoma. All cases of basaloid/warty carcinomas, but none of the remaining tumors, overexpressed p16 INK4a protein. Our data do not support a role of beta HPVs in the pathogenesis of vulvar carcinoma. The study reaffirms the role of mucosal HPVs, in particular that of HPV 16, in the pathogenesis of basaloid and warty tumor subtypes. The HPV 16 intratypic variation showed correlation with patients' ethnic background. P16 INK4a immunostaining seems to be a sensitive and specific marker of vulvar carcinomas positive for oncogenic mucosal HPVs.

Journal of Cellular and Molecular Medicine, 2009

Studies in cervical dysplasia have reported overexpression of the tumour suppressors p14 and p16 ... more Studies in cervical dysplasia have reported overexpression of the tumour suppressors p14 and p16 – and absence of p53 – in high-risk human papilloma virus (HPV)- associated lesions. In skin carcinogenesis, the relation between these tumour suppressors and HPV remain unclear. We evaluated the expression of the tumour suppressors p14, p16 and p53 in pre-malignant and malignant squamous skin tumours, and its relation with risk factors for skin carcinogenesis (HPV, immune status and sun exposure). We performed immunohistochemical stainings for p14, p16 and p53 on paraffin embedded material of 71 pre-malignant squamous skin lesions and 34 squamous cell carcinomas, from 52 renal transplant recipients (RTRs) and 53 immunocompetent individuals. PCR-based assays were used for detection and genotyping of β-papilloma virus (β-PV) types and mucosal HPV types. P14 expression was independent of the expression of p16 and p53, irrespective of immune status and skin site. In 49 of 105 specimens (46.6%), one or more β-PV types were detected. We found no significant association between p14, p16 or p53 protein expression and overall presence of β-PV, irrespective of immune status. There was a significant association between presence of β-PV and lesions from sun-exposed skin sites in the RTRs (P= 0.002). We conclude that in skin carcinogenesis, relations between the herein studied tumour suppressors and HPV are different from what one would expect based on findings in cervical neoplasia. P14, p16 and p53 expressions are independent of immune status. Our data indicate that in immunosuppressed patients, β-PV together with ultraviolet radiation act synergetic in promoting carcinogenesis.

Cancer Research, 2009

Human papillomaviruses from the β genus (βPV) are a possible cause of cutaneous squamous cell car... more Human papillomaviruses from the β genus (βPV) are a possible cause of cutaneous squamous cell carcinoma (SCC). We assessed the extent to which βPV infections persisted longterm in a subtropical Australian community and whether βPV persistence is positively associated with actinic keratoses, precursor for SCC. Eyebrow hairs were collected from 171 participants of the community-based Nambour Skin Cancer Study in 1996 and 2003. Hair samples were tested for the presence of DNA from 25 different βPV types and assessed in relation to actinic keratosis presence in 2007. In 1996, a total of 413 βPV infections were found in 73% of participants, increasing to 490 infections among 85% in 2003. Of the total βPV infections detected, 211 (30%) were found to persist. Age was significantly associated with βPV persistence: those ages >60 years had 1.5fold (95% confidence interval, 1.1-1.9) increased risk of typespecific viral persistence than those ages <40 years. After accounting for actinic keratoses at baseline, persistence of βPV DNA resulted in a 1.4-fold (95% confidence interval, 1.0-1.9) increase in risk of having actinic keratoses on the face in 2007. In conclusion, persistent βPV infections in this population were associated with an increased occurrence of actinic keratosis. Additional studies are needed to determine the possible association of βPV persistence with SCC. [Cancer Res 2009;69(23):8926-31] 8926

International Journal of Cancer, 2009

Genetic alterations in metastatic cutaneous squamous cell carcinoma (CSCC) which might serve as p... more Genetic alterations in metastatic cutaneous squamous cell carcinoma (CSCC) which might serve as prognostic biomarkers are not well investigated. We investigated the mutation status and protein expression of the CDKN2A (INK4a-ARF) and TP53 genes in metastatic CSCCs and correlated this with clinicopathological variables, HPV presence, and survival data. Sequence analysis was performed on formalin-fixed and paraffin-embedded tissue of 35 metastases and their primary tumors, and was correlated with immunohistochemical stainings for p53, p16 and p14. Beta-PV and alpha-PV DNA was detected using PCR-based assays. Kaplan–Meier and Cox regression methods were used for survival assessment. CDKN2A was mutated in 31% of the metastases and their primary tumors, while the TP53 gene was mutated in 51% of the metastases. P53 protein expression was significantly associated with missense type of mutations (p = 0.002). No persistent HPV types were detected. CDKN2A mutations were significantly associated with disease-specific death (p = 0.001). A significant difference was observed in disease-specific survival between patients with or without a CDKN2A mutation (p = 0.010), while this was not the case for TP53. At univariate Cox's regression analysis tumor size (p = 0.010), invasion depth (p = 0.030) and CDKN2A mutations (p = 0.040) were significantly related to shorter disease-specific survival. At multivariate Cox's regression only tumor size had an adverse effect on survival (p = 0.002). In conclusion, our study indicates that the CDKN2A mutation status might be of prognostic value in metastatic CSCCs. In most cases, CDKN2A and TP53 mutations are early genetic events in CSCC tumorigenesis. The possible role of HPV in metastatic CSCC needs further exploration.