Steffi Silling

Comprehensive Analysis of HPV16 Integration in OSCC Reveals No Significant Impact of Physical Status on Viral Oncogene and Virally Disrupted Human Gene Expression

Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the development of oropharyngeal squamous cell carcinomas (OSCC). However, it is unclear whether viral integration is an essential hallmark in the carcinogenic process of OSCC and whether HPV integration correlates with the level of viral gene transcription and influences the expression of disrupted host genes. We analyzed 75 patients with OSCC. HPV16-positivity was proven by p16INK4A immunohistochemistry, PCR and FISH. Viral integration was examined using DIPS- as well as APOT-PCR. Viral E2, E6 and E7 gene expression levels were quantified by quantitative reverse transcriptase (RT-q)PCR. Expression levels of 7 human genes disrupted by the virus were extracted from mRNA expression profiling data of 32 OSCCs. Viral copy numbers were assessed by qPCR in 73 tumors. We identified 37 HPV16-human fusion products indicating viral integration in 29 (39%) OSCC. In the remaining tumors (61%) only episome-derived PCR products were detected. When comparing OSCC with or without an integration-derived fusion product, we did not find significant differences in the mean RNA expression of viral genes E2, E6 and E7 or the viral copy numbers per cell, nor did the RNA expression of the HPV-disrupted genes differ from either group of OSCC. In conclusion, our data do not support the hypothesis that integration affects the levels of viral and/or HPV-disrupted human gene transcripts. Thus constitutive, rather than a high level, of expression of oncogene transcripts appears to be required in HPV-related OSCC.

Viral load, gene expression and mapping of viral integration sites in HPV16-associated HNSCC cell lines.

HPV‐related HNSCC generally have a better prognosis than HPV‐negative HNSCC. However, a subgroup of HPV‐positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16‐positive HNSCC cell lines currently available have been carefully analyzed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD‐SCC‐2, UM‐SCC‐047, UM‐SCC‐104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT‐PCR and DIPS‐PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration‐specific staining patterns and signals indicating transcriptional activity using FISH. APOT‐ and DIPS‐PCR identified integration‐derived fusion products in six cell lines and only episomal products for UM‐SCC‐104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression and aneusomy (except UPCI:SCC154). In conclusion, all HPV16‐positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies.

Case–control study of genus-beta human papillomaviruses in plucked eyebrow hairs and cutaneous squamous cell carcinoma

Cutaneous human papillomaviruses (HPV) have been reported in cutaneous squamous cell carcinoma (SCC). We conducted a clinic‐based case–control study to investigate the association between genus‐beta HPV DNA in eyebrow hairs (EBH) and SCC. EBH from 168 SCC cases and 290 controls were genotyped for genus‐beta HPV DNA. SCC tumors from a subset of cases (n = 142) were also genotyped. Viral load was determined in a subset of specimens positive for a single HPV type. Associations with SCC were estimated by odds ratios (OR) and 95% confidence intervals (CI) adjusted for age and sex using logistic regression. Statistical tests were two‐sided. EBH DNA prevalence was greater in cases (87%) than controls (73%) (p < 0.05), and the association with SCC increased with the number of HPV types present, (≥4 types vs. HPV‐negative: OR = 2.02, 95% CI = 1.07–3.80; ptrend = 0.02). Type‐specific associations were observed between SCC and DNA in EBH for HPV23 (OR = 1.90, 95% CI = 1.10–3.30) and HPV38 (OR = 1.84, 95% CI = 1.04–3.24). Additionally, when compared with the controls, the DNA prevalence in EBH was significantly higher among cases for 11 of the 25 genus‐beta types tested, when accounting for DNA for the same HPV type in the tumor (ORs = 3.44–76.50). Compared to controls, the mean viral DNA load in EBH among the selected cases was greater for HPV5, HPV8 and HPV24, but lower for HPV38. SCC cases were more likely than controls to have HPV DNA+ EBH for single and multiple HPV types, providing additional support for the potential role of genus‐beta HPV infections in SCC development.

Human papillomavirus oncogene mRNA testing for the detection of anal dysplasia in HIV-positive men who have sex with men.

BACKGROUND Anal human papillomavirus (HPV) infection and anal dysplasia are frequent in HIV-positive men who have sex with men (HIV+MSM), and progression of low-grade (LSIL) to high-grade squamous intraepithelial lesions (HSIL) or anal cancer (AC) occurs faster than in HIV-negative individuals. High-risk (HR)-HPV-E6/E7 oncogene mRNA testing has a higher specificity and a higher positive predictive value (PPV) than HR-HPV-DNA testing for detecting high-grade cervical lesions. OBJECTIVE To evaluate the diagnostic accuracy of the NucliSENS-EasyQ HPV1.1 E6/E7-mRNA-assay for the detection of anal dysplasia in HIV+MSM. STUDY DESIGN 289 intraanal swabs from HIV+MSM participating in a screening program that included anal cytology, high-resolution anoscopy and histology were analyzed. HR-HPV-DNA detection was performed by PCR and hybridization using a bead-based multiplex genotyping assay. E6/E7-mRNA detection of HR-HPV-types 16, 18, 31, 33 and 45 was performed using the NucliSENS-EasyQ assay. RESULTS 269 swabs had valid results in both test formats (111 normal, 10 ASCUS, 105 LSIL, 42 HSIL, 1 AC). For the detection of LSIL+(LSIL+HSIL+cancer) sensitivity, specificity, negative predictive value (NPV) and PPV were 80.4%, 26.4%, 52.5%, and 57.2% for HR-HPV-DNA testing, respectively, compared to 75.7%, 57.9%, 66.0% and 68.7% for E6/E7-mRNA testing. The respective values for the detection of HSIL/cancer were 95.3%, 26.1%, 96.7%, 19.7% for HR-HPV-DNA and 95.3%, 46.0%, 98.1%, 25.2% for E6/E7-mRNA detection. CONCLUSION Compared to HR-HPV-DNA detection, E6/E7-mRNA testing has an increased specificity (approximately two-fold), similar sensitivity and higher NPV and PPV for the detection of low- and high-grade anal dysplasia in HIV+MSM.

Merkel Cell Polyomavirus Infection in HIV-Positive Men

OBJECTIVE To evaluate Merkel cell polyomavirus (MCPyV) DNA prevalence and load among men with human immunodeficiency virus (HIV) (hereafter referred to as HIV-positive men) and among healthy male control subjects. DESIGN Prospective study from February 4, 2009, through April 24, 2010. SETTING Dermatology department of a university hospital. PATIENTS A total of 449 male adults were prospectively recruited, including 210 HIV-positive men who have sex with men and 239 healthy controls. Cutaneous swabs were obtained once from the surface of the forehead in all participants. MAIN OUTCOME MEASURES Swabs were evaluated for the presence of MCPyV DNA using single-round and nested polymerase chain reaction. The MCPyV DNA load (the number of MCPyV DNA copies per β-globin gene copy) was determined in MCPyV-positive samples using quantitative real-time polymerase chain reaction. RESULTS Among 449 forehead swabs analyzed, MCPyV DNA was detected in 242 (53.9%). Compared with healthy controls, HIV-positive men more frequently had MCPyV DNA on nested polymerase chain reaction (49.4% vs 59.0%, P = .046) and on single-round polymerase chain reaction (15.9% vs 28.1%, P = .002). The MCPyV DNA loads in HIV-positive men were similar to those in HIV-negative men, but HIV-positive men with poorly controlled HIV infection had significantly higher MCPyV DNA loads than those who had well-controlled HIV infection (median and mean MCPyV DNA loads, 2.48 and 273.04 vs 0.48 and 11.84; P = .046). CONCLUSIONS Cutaneous MCPyV prevalence is increased among HIV-positive men who have sex with men. Furthermore, MCPyV DNA loads are significantly higher in HIV-positive men with poorly controlled HIV infection compared with those who have well-controlled HIV infection. This could explain the increased risk of MCPyV-associated Merkel cell carcinoma observed among HIV-positive individuals.

Prevalence of human polyomaviruses in common and rare types of non-Merkel cell carcinoma skin cancer.

Background  Little is known about the association of human polyomaviruses (HPyVs) other than Merkel cell polyomavirus (MCPyV) with nonmelanoma skin cancer.

Human polyomaviruses 6, 7, 9, 10 and Trichodysplasia spinulosa-associated polyomavirus in HIV-infected men

Recently, several novel human polyomaviruses (HPyVs) have been detected. HPyV6, 7, 9 and 10 are not associated with any disease so far. Trichodysplasia spinulosa (TS)-associated polyomavirus (TSPyV) can cause the rare skin disease TS. We have evaluated cutaneous DNA prevalence and viral loads of five HPyVs in HIV-infected men compared to healthy male controls. 449 forehead swabs were analysed by HPyV-specific real-time PCR. HPyV6, HPyV7, TSPyV and HPyV10 were found significantly more frequently on the skin of 210 HIV-infected compared to 239 HIV-negative men (HPyV6, 39.0 vs 27.6 %; HPyV7, 21.0 vs 13.4 %; TSPyV, 3.8 vs 0.8 %; HPyV10, 9.3 vs 3.4 %; P<0.05, respectively). HPyV9 was not detected. Multiple infections were more frequent in HIV-positive men, but HPyV-DNA loads did not differ significantly in both groups. In contrast to HPyV6, 7 and 10, TSPyV and HPyV9 do not seem to be a regular part of the human skin microbiome.

Integration of HPV6 and downregulation of AKR1C3 expression mark malignant transformation in a patient with juvenile-onset laryngeal papillomatosis.

Juvenile-onset recurrent respiratory papillomatosis (RRP) is associated with low risk human papillomavirus (HPV) types 6 and 11. Malignant transformation has been reported solely for HPV11-associated RRP in 2–4% of all RRP-cases, but not for HPV6. The molecular mechanisms in the carcinogenesis of low risk HPV-associated cancers are to date unknown. We report of a female patient, who presented with a laryngeal carcinoma at the age of 24 years. She had a history of juvenile-onset RRP with an onset at the age of three and subsequently several hundred surgical interventions due to multiple recurrences of RRP. Polymerase chain reaction (PCR) or bead-based hybridization followed by direct sequencing identified HPV6 in tissue sections of previous papilloma and the carcinoma. P16INK4A, p53 and pRb immunostainings were negative in all lesions. HPV6 specific fluorescence in situ hybridization (FISH) revealed nuclear staining suggesting episomal virus in the papilloma and a single integration site in the carcinoma. Integration-specific amplification of papillomavirus oncogene transcripts PCR (APOT-PCR) showed integration in the aldo-keto reductase 1C3 gene (AKR1C3) on chromosome 10p15.1. ArrayCGH detected loss of the other gene copy as part of a deletion at 10p14-p15.2. Western blot analysis and immunohistochemistry of the protein AKR1C3 showed a marked reduction of its expression in the carcinoma. In conclusion, we identified a novel molecular mechanism underlying a first case of HPV6-associated laryngeal carcinoma in juvenile-onset RRP, i.e. that HPV6 integration in the AKR1C3 gene resulted in loss of its expression. Alterations of AKR1C gene expression have previously been implicated in the tumorigenesis of other (HPV-related) malignancies.

No evidence for a causal role of Merkel cell polyomavirus in keratoacanthoma

BACKGROUND Merkel cell polyomavirus (MCPyV) is a recently discovered virus that is monoclonally integrated into the genome of approximately 80% of all Merkel cell carcinomas (MCCs). While some evidence exists that MCPyV does not play a pathogenic role in other nonmelanoma skin cancers, such as basal cell carcinoma and squamous cell carcinoma (SCC), little is known about the presence of MCPyV in keratoacanthoma (KA). OBJECTIVES To evaluate the prevalence, viral DNA-load, and large T(umor)-antigen expression of MCPyV in KA of immunocompetent patients and to compare the results with those found in SCC and MCC. METHODS Paraffin-embedded tissue samples were analyzed for the presence of MCPyV-DNA by polymerase chain reaction (PCR). MCPyV-DNA load (MCPyV-DNA copies per beta-globin gene copy) was determined by using quantitative real-time PCR. Immunohistochemical analysis of the MCPyV large T-antigen was performed with the monoclonal antibody CM2B4. RESULTS A total of 137 samples (42 KA, 52 SCC, and 43 MCC) were analyzed. MCPyV-DNA was found significantly more frequently in MCC (37/43, 86.0%) compared with KA (12/42, 28.6%) and SCC (14/52, 26.9%). Moreover, MCPyV-DNA loads were more than two orders of magnitude lower in KA and SCC compared with MCC (median/mean loads 0.005/0.015 [KA] vs 0.023/0.059 [SCC] vs 2.613/56.840 [MCC] MCPyV-DNA copies per beta-globin gene copy). All MCC analyzed (n = 3) expressed MCPyV large T-antigen, whereas 8 KA and 7 SCC were negative in immunohistochemistry. LIMITATIONS The relatively small number of samples is a limitation. CONCLUSIONS Our findings argue against a pathogenic role of MCPyV in KA and SCC.

Differential Regulation of Human Papillomavirus Type 8 by Interferon Regulatory Factors 3 and 7

ABSTRACT The genus β human papillomavirus (HPV) type 8 is associated with nonmelanoma skin cancer in patients with epidermodysplasia verruciformis, and evidence for its protumorigenic potential in the general population increases. To date, strategies to suppress genus β HPV infections are limited. Interferon regulatory factors IRF-3 and IRF-7 play key roles in the activation of the innate immune response to viral infections. In this study, we show for the first time that both IRF-3 and IRF-7 regulate transcription of a papillomavirus, but with opposing effects. IRF-7, expressed in the suprabasal layers of human epidermis, increased HPV8 late promoter activity via direct binding to viral DNA. UV-B light-induced activation of the HPV8 promoter involved IRF-7 as a downstream effector. In contrast, IRF-3, expressed in all layers of human epidermis, induced strong HPV8 suppression in primary keratinocytes. IRF-3-mediated suppression prevailed over IRF-7-induced HPV8 transcription. Unlike the E6 oncoprotein of the mucosal high-risk HPV16, the HPV8 E6 protein did not bind to IRF-3 and only weakly antagonized its activity. Strong antiviral activity was also observed, when keratinocytes were treated with potent IRF-3 activators, poly(I:C) or RNA bearing 5′ phosphates. In conclusion, we show that IRF-3 activation induces a state of cell-autonomous immunity against HPV in primary human keratinocytes. Our study suggests that local application of IRF-3-activating compounds might constitute an attractive novel therapeutic strategy against HPV8-associated diseases, particularly in epidermodysplasia verruciformis patients.