Aspartate ß-hydroxylase Regulates Expression of Ly6 Genes.

Background: Overexpression of aspartate ß-hydroxylase (ASPH) in human tumors contributes to their progression by stimulating cell proliferation, migration, and invasion. Several signaling pathways affected by ASPH have been identified, but the high number of potential targets of ASPH hydroxylation suggests that additional mechanisms may be involved. This study was performed to reveal new targets of ASPH signaling. Methods: The effect of ASPH on the oncogenicity of three mouse tumor cell lines was tested using proliferation assays, transwell assays, and spheroid invasion assays after inhibition of ASPH with the small molecule inhibitor MO-I-1151. ASPH was also deactivated with the CRISPR/Cas9 system. A transcriptomic analysis was then performed with bulk RNA sequencing and differential gene expression was evaluated. Expression data were verified by quantitative PCR and immunoblotting. Results: Inhibition or abrogation of ASPH reduced proliferation of the cell lines and their migration and invasiveness. Among the genes with differential expression in more than one cell line, two members of the lymphocyte antigen 6 (Ly6) family, Ly6a and Ly6c1, were found. Their downregulation was confirmed at the protein level by immunoblotting, which also showed their reduction after ASPH inhibition in other mouse cell lines. Reduced production of the Ly6D and Ly6K proteins was shown after ASPH inhibition in human tumor cell lines. Conclusions: Since increased expression of Ly6 genes is associated with the development and progression of both mouse and human tumors, these results suggest a novel mechanism of ASPH oncogenicity and support the utility of ASPH as a target for cancer therapy.

Prevalence of oral and anal human papillomavirus infection in Czech predominantly HIV-positive men having sex with men - data from a previously unreported population.

BACKGROUND: HIV-positive men who have sex with men (MSM) are more likely to experience human papillomavirus (HPV) infection. The persistent HPV infection is the major factor in the development of anal and oropharyngeal neoplasms. Data on the prevalence of anal and oral HPV in MSM are almost absent from the countries of Central and Eastern Europe. We conducted a cross-sectional study focused on the prevalence of oral and anal HPV infections and the relationship between current anal and oral HPV intrapersonal infection in a Czech population of predominantly HIV-positive MSM. METHODS: Oral gargle and anal swab samples from 205 predominantly HIV-positive MSM from the Czech Republic were analysed for HPV infection using PCR. Selected sociodemographic and clinical data were correlated with HPV detection using generalized linear models and multivariate analysis. RESULTS: HPV infection was detected in 183 (96.8%) anal and 48 (23.6%) oral samples. The most common type of HR-HPV was HPV16 in both anal (25.4%) and oral (2.5%) samples. Multiple anal HPV infections and the presence of vaccine-targeted HR-HPV types were significantly correlated with abnormal anal cytology and HIV status. CONCLUSION: The prevalence of anal HPV infection in Czech predominantly HIV-positive MSM ranks among the highest reported, while oral HPV prevalence is consistent with MSM populations. Minimal overlap of oral and anal HPV types within a patient was observed.

Prevalence of genital and oral human papillomavirus infection among psoriasis patients on biologic therapy.

Current knowledge about human papillomavirus (HPV) infection in psoriasis patients treated with biologics is limited. In this study we evaluated the prevalence of oral and genital HPV infection in psoriasis patients treated with biologics or topical therapy for at least 6 months. The presence of HPV DNA in oral rinse and genital smears was evaluated. In total, 267 patients who met the inclusion criteria and agreed to participate were enrolled: 110 (41.2%) on topical therapy, 84 (31.5%) on anti-TNF-alpha therapy, 31 (11.6%) on anti-IL-12/23 therapy and 42 (15.7%) on anti-IL-17 therapy. The presence of genital HPV infection was detected in 34.6% of men receiving anti-TNF-α treatment, in 25.0% of patients on anti-IL-12/23 and 18.8% of patients on anti-IL-17 therapy. The difference in prevalence was not statistically different from men on topical treatment (26.3%). Prevalence of oral HPV infection was higher across all of the biologic groups (11.9% for anti-TNF-α, 12.9% for anti-IL-12/23 and 19.0% for anti-IL-17) compared to patients on topical therapy (7.3%), but statistically significant only for anti-IL-17 (p < 0.05). The presence of oral HPV infection in patients treated with biologics was significantly higher (44.0%) in patients on long-term biologic treatment (>8 years) compared to patients taking biologics for a shorter period (9.1%; p < 0.01). Our results suggest that patients on biologics for psoriasis have a higher prevalence of oral HPV infection compared to patients on topical treatment. Long-term treatment with biologics seems to be associated with a higher prevalence of oral HPV infection, independent of previous conventional immunosuppressive therapy.

Outcomes After Human Papillomavirus Vaccination in Patients With Recurrent Respiratory Papillomatosis: A Nonrandomized Clinical Trial.

Importance: Recurrent respiratory papillomatosis (RRP) is a rare benign chronic disease of the larynx etiologically linked with the infection of low-risk human papillomavirus (HPV). Combination of surgical and immunomodulatory therapy has limited success. Possible use of prophylactic HPV vaccine that includes HPV-6 and HPV-11 antigens has been studied. Objective: To evaluate if the HPV vaccination is associated with a lower number of recurrences requiring surgical intervention in patients with new and recurrent RRP. Design, Setting, and Participants: This was a non-placebo-controlled intervention study. Enrollment data were collected from October 2011 to August 2013. The patients were followed up at 1 month, 12 months, and 5 years after the third dose of the vaccine and clinically monitored until December 31, 2018. Data were analyzed from 2019 to 2021. Altogether, 50 adults with active RRP were enrolled and followed up in referral centers. For the final outcome, follow-up data for 42 patients were available. Eight patients who did not fulfill the protocol were excluded. Interventions: All patients received HPV vaccine as an adjuvant treatment and were clinically followed up. When RRP progression or a significant recurrent lesion was detected, surgical removal via direct laryngoscopy was indicated. No adjuvant therapy with antiviral or biological agents was used. Main Outcomes and Measures: This study compared the prevaccination and postvaccination positivity for HPV-specific antibodies. The main outcome was the difference in the frequency of RRP recurrences in the prevaccination and postvaccination period. Results: A total of 50 patients with RRP were enrolled (median [SD] age, 41.5 [12.3] years [range, 21-73 years]; 39 [78%] men and 11 [22%] women). After HPV vaccination, patients with previously no HPV-specific antibodies showed seroconversion, and all patients developed 100-fold higher levels of HPV vaccine type-specific antibodies compared with the prevaccination period. In patients with recurrent RRP, decreased frequency of recurrences requiring surgical treatment was present after vaccination (from 0.85 to 0.36 recurrences/y). No difference in postvaccination recurrences was found between patients with newly diagnosed and recurrent RRP. Conclusions and Relevance: In this nonrandomized clinical trial, the frequency of RRP recurrences was significantly lower after HPV vaccination, and patients with RRP thus had a reduced burden of disease. Because no difference was detected in the frequency of recurrent postvaccination lesions in patients with new and recurrent disease, it appears that both groups showed equal benefit following HPV vaccination. These findings suggest that the earlier that patients with RRP receive HPV vaccine, the sooner they may show reduced burden of disease. Trial Registration: EudraCT Identifier: 2011-002667-14; ClinicalTrials.gov Identifier: NCT01375868.

Prevalence of high-risk human papillomavirus DNA and mRNA and its association with abnormal anal cytology in the Czech male anal cancer screening cohort.

BACKGROUND: Anal cancer (AC) screening is justified in high-risk populations, particularly HIV-positive men having sex with men (MSM). HR-HPV testing could improve the efficiency of cytologically based screening of AC, as in the screening of biologically analogical cervical cancer. The specificity of HR-HPV testing is influenced by the prevalence of HR-HPV infection in the screened population. Reported anal HR-HPV DNA prevalence in MSM is high, but HR-HPV mRNA reflects rather long-term infections and is more specific for high-grade lesions. However, no data were published about HR-HPV DNA and mRNA prevalence in the Czech AC screening population. METHOD: Results of liquid-based anal cytology of 203 predominantly HIV-positive MSM from the Czech AC screening cohort were correlated with results of DNA and E6/E7 mRNA testing of 14 HR-HPV types, and HPV16 genotyping. Eighty-one MSM underwent a standard anoscopy. RESULTS: A total of 109 (53.7%) samples had abnormal cytology, with 12 (5.9%) ASC-H/HSIL, 67 (33.0%) samples cytologically negative, and 27 (13.3%) unsatisfactory. HR-HPV DNA was detected in 134 (66.0%) and HR-HPV RNA in 72 (35.5%) anal smears. HR-HPV mRNA and HPV16 mRNA positivity were associated with abnormal cytology (p = .0037, p = .0021). No significant association was found between HR-HPV DNA or HPV16 DNA positivity and abnormal cytology. No high-grade lesions were revealed by anoscopy. CONCLUSION: Prevalence of anal HR-HPV DNA among Czech MSM is high, however, the prevalence of HR-HPV mRNA is half and associated with abnormal cytology. Our results indicate an increased efficiency of cytological screening when combined with HR-HPV mRNA testing.

Distinct Responsiveness of Tumor-Associated Macrophages to Immunotherapy of Tumors with Different Mechanisms of Major Histocompatibility Complex Class I Downregulation.

Tumor-associated macrophages (TAMs) plentifully infiltrate the tumor microenvironment (TME), but their role in anti-tumor immunity is controversial. Depending on the acquired polarization, they can either support tumor growth or participate in the elimination of neoplastic cells. In this study, we analyzed the TME by RNA-seq and flow cytometry and examined TAMs after ex vivo activation. Tumors with normal and either reversibly or irreversibly decreased expression of major histocompatibility complex class I (MHC-I) molecules were induced with TC-1, TC-1/A9, and TC-1/dB2m cells, respectively. We found that combined immunotherapy (IT), composed of DNA immunization and the CpG oligodeoxynucleotide (ODN) ODN1826, evoked immune reactions in the TME of TC-1- and TC-1/A9-induced tumors, while the TME of TC-1/dB2m tumors was mostly immunologically unresponsive. TAMs infiltrated both tumor types with MHC-I downregulation, but only TAMs from TC-1/A9 tumors acquired the M1 phenotype upon IT and were cytotoxic in in vitro assay. The anti-tumor effect of combined IT was markedly enhanced by a blockade of the colony-stimulating factor-1 receptor (CSF-1R), but only against TC-1/A9 tumors. Overall, TAMs from tumors with irreversible MHC-I downregulation were resistant to the stimulation of cytotoxic activity. These data suggest the dissimilarity of TAMs from different tumor types, which should be considered when utilizing TAMs in cancer IT.

Lack of Conserved miRNA Deregulation in HPV-Induced Squamous Cell Carcinomas.

Squamous cell carcinomas (SCCs) in the anogenital and head and neck regions are associated with high-risk types of human papillomaviruses (HR-HPV). Deregulation of miRNA expression is an important contributor to carcinogenesis. This study aimed to pinpoint commonly and uniquely deregulated miRNAs in cervical, anal, vulvar, and tonsillar tumors of viral or non-viral etiology, searching for a common set of deregulated miRNAs linked to HPV-induced carcinogenesis. RNA was extracted from tumors and nonmalignant tissues from the same locations. The miRNA expression level was determined by next-generation sequencing. Differential expression of miRNAs was calculated, and the patterns of miRNA deregulation were compared between tumors. The total of deregulated miRNAs varied between tumors of different locations by two orders of magnitude, ranging from 1 to 282. The deregulated miRNA pool was largely tumor-specific. In tumors of the same location, a low proportion of miRNAs were exclusively deregulated and no deregulated miRNA was shared by all four types of HPV-positive tumors. The most significant overlap of deregulated miRNAs was found between tumors which differed in location and HPV status (HPV-positive cervical tumors vs. HPV-negative vulvar tumors). Our results imply that HPV infection does not elicit a conserved miRNA deregulation in SCCs.

ARG1 mRNA Level Is a Promising Prognostic Marker in Head and Neck Squamous Cell Carcinomas.

Head and neck squamous cell carcinomas (HNSCC) can be induced by smoking or alcohol consumption, but a growing part of cases relate to a persistent high-risk papillomavirus (HPV) infection. Viral etiology has a beneficial impact on the prognosis, which may be explained by a specific immune response. Tumor associated macrophages (TAMs) represent the main immune population of the tumor microenvironment with a controversial influence on the prognosis. In this study, the level, phenotype, and spatial distribution of TAMs were evaluated, and the expression of TAM-associated markers was compared in HPV positive (HPV+) and HPV negative (HPV-) tumors. Seventy-three formalin and embedded in paraffin (FFPE) tumor specimens were examined using multispectral immunohistochemistry for the detection of TAM subpopulations in the tumor parenchyma and stroma. Moreover, the mRNA expression of TAM markers was evaluated using RT-qPCR. Results were compared with respect to tumor etiology, and the prognostic significance was evaluated. In HPV- tumors, we observed more pro-tumorigenic M2 in the stroma and a non-macrophage arginase 1 (ARG1)-expressing population in both compartments. Moreover, higher mRNA expression of M2 markers-cluster of differentiation 163 (CD163), ARG1, and prostaglandin-endoperoxide synthase 2 (PTGS2)-was detected in HPV- patients, and of M1 marker nitric oxide synthase 2 (NOS2) in HPV+ group. The expression of ARG1 mRNA was revealed as a negative prognostic factor for overall survival of HNSCC patients.

Aspartate ß-hydroxylase as a target for cancer therapy.

As metastasis is a major cause of death in cancer patients, new anti-metastatic strategies are needed to improve cancer therapy outcomes. Numerous pathways have been shown to contribute to migration and invasion of malignant tumors. Aspartate ß-hydroxylase (ASPH) is a key player in the malignant transformation of solid tumors by enhancing cell proliferation, migration, and invasion. ASPH also promotes tumor growth by stimulation of angiogenesis and immunosuppression. These effects are mainly achieved via the activation of Notch and SRC signaling pathways. ASPH expression is upregulated by growth factors and hypoxia in different human tumors and its inactivation may have broad clinical impact. Therefore, small molecule inhibitors of ASPH enzymatic activity have been developed and their anti-metastatic effect confirmed in preclinical mouse models. ASPH can also be targeted by monoclonal antibodies and has also been used as a tumor-associated antigen to induce both cluster of differentiation (CD) 8+ and CD4+ T cells in mice. The PAN-301-1 vaccine against ASPH has already been tested in a phase 1 clinical trial in patients with prostate cancer. In summary, ASPH is a promising target for anti-tumor and anti-metastatic therapy based on inactivation of catalytic activity and/or immunotherapy.

Prevalence and Risk Factors for Oral HPV in Healthy Population, in Central Europe.

Background/Aim: The incidence of oropharyngeal tumours induced by human papillomaviruses (HPV) is ever increasing. Information about oral HPV prevalence and its risk factors are very important for future screening and early diagnosis of the disease. The present study aimed to assess oral HPV prevalence in healthy population and risk factors for HPV infection, since this data is scarce or even missing in Central Europe. Patients and Methods: HPV prevalence in oral rinse and HPV-specific antibodies in peripheral blood were investigated in two groups of healthy participants. Group I consisted of 294 students who reached sexual maturity after the HPV vaccine had been licensed with mean age 23.2 years, and Group II of 215 unvaccinated participants with the mean age 55.7 years. Additionally, the risk factors were evaluated. Results: In Group I, 2% of participants were positive for oral HPV DNA. A statistically significantly higher rate (8.8%) was found in Group II. The seropositivity rates for anamnestic HPV antibodies were comparable in both groups. None of the analysed risk factors was significantly associated with oral HPV positivity. Conclusion: The lower prevalence of oral HPV DNA in younger participants suggests the positive influence of vaccination against oral HPV.

Detailed Characteristics of Tonsillar Tumors with Extrachromosomal or Integrated Form of Human Papillomavirus.

The human papillomavirus (HPV) integration, the critical step in viral carcinogenesis, most frequently occurs in the E2 gene, which results in its inactivation and in an increase of E6/E7 transcription. However, in a substantial number of tumors, the virus is present in an extrachromosomal form. For those tumors, the transformation mechanisms are not fully elucidated. Here we evaluated the possible mechanism of inactivating the E2 without interruption of the gene, methylation or mutation of the E2 binding sites (E2BSs) in HPV16-positive tonsillar tumors by next-generation and Sanger sequencing. Viral genome status was analyzed by the amplification of papillomavirus oncogene transcripts assay (APOT) and mRNA mapping, and expression of viral oncogenes was performed by qPCR. The methylation of E2BSs was significantly higher in tumors with an integrated, in comparison to extrachromosomal, form of the viral genome. No mutations were detected in the E2BSs. The viral oncogenes were equally expressed in samples with an integrated and extrachromosomal form of the virus. Only the nucleotide variants were identified in the E2 gene. No proposed mechanism of E2 inactivation was confirmed in tonsillar tumors with an extrachromosomal form of the HPV genome. The expression of E6/E7 genes seems to be sufficient to initiate and maintain the carcinogenic process.

Experimental Combined Immunotherapy of Tumours with Major Histocompatibility Complex Class I Downregulation.

Combined immunotherapy constitutes a novel, advanced strategy in cancer treatment. In this study, we investigated immunotherapy in the mouse TC-1/A9 model of human papillomavirus type 16 (HPV16)-associated tumors characterized by major histocompatibility complex class I (MHC-I) downregulation. We found that the induction of a significant anti-tumor response required a combination of DNA vaccination with the administration of an adjuvant, either the synthetic oligodeoxynucleotide ODN1826, carrying immunostimulatory CpG motifs, or α-galactosylceramide (α-GalCer). The most profound anti-tumor effect was achieved when these adjuvants were applied in a mix with a one-week delay relative to DNA immunization. Combined immunotherapy induced tumor infiltration with various subsets of immune cells contributing to tumor regression, of which cluster of differentiation (CD) 8⁺ T cells were the predominant subpopulation. In contrast, the numbers of tumor-associated macrophages (TAMs) were not markedly increased after immunotherapy but in vivo and in vitro results showed that they could be repolarized to an anti-tumor M1 phenotype. A blockade of T cell immunoglobulin and mucin-domain containing-3 (Tim-3) immune checkpoint had a negligible effect on anti-tumor immunity and TAMs repolarization. Our results demonstrate a benefit of combined immunotherapy comprising the activation of both adaptive and innate immunity in the treatment of tumors with reduced MHC-I expression.

Concordance of HPV-DNA in cervical dysplasia or genital warts in women and their monogamous long-term male partners.

Transmission of human papillomavirus (HPV) is a premise for development of cervical dysplasia and genital warts (GWs). This cross-sectional study assesses concordance of HPV types present in GWs or cervical dysplasia in women and genital infection of their monogamous male partners in conjunction with seroprevalence of HPV-6, -11, -16, and -18 antibodies. Blood was taken from both women and men, as well a smear of the urogenital area of men. HPV DNA detection in women was done in fixed paraffin embedded tissues under histological control. Of 143 couples who agreed to participate in the study, 68 met inclusion criteria. Type-specific concordance was observed in 32.5% (13/40) of couples in which women had genital warts and in 32.1% (9/28) of couples in which women had cervical dysplasia. In multivariate analysis only smoking in women was associated with concordance (P < 0.05). Prevalence of HPV-specific antibodies was high in male partners, but was not associated with presence of the same HPV type on their genitals. The same type-specific HPV antibodies were detected in 81.8% of men in couples with HPV-6 concordant genital warts, but only in 14.3% of men in couples with HPV-16 concordant cervical dysplasia (P < 0.01). These results suggest that type-specific HPV concordance in genital warts and cervical dysplasia lesions of women and genital infection of their male partners is common and similar. Higher seroconversion in couples with HPV-6 concordant genital warts compared with couples with HPV-16 concordant cervical dysplasia may be explained by viral load exposure.

The prevalence of HPV infections in HPV-vaccinated women from the general population.

Currently, three prophylactic HPV vaccines are commercially available to prevent HPV 16/18 infection and associated lesions. The aim of the study was to assess markers of HPV infection in women/girls before vaccination and to ascertain the prevalence and spectrum of post-vaccination HPV types. Three hundred and thirty subjects of which 75 were virgins were enrolled. Before the first dose of the HPV vaccine and 1, 3 and 5 years after the completion of HPV vaccination, the samples for cytology, HPV detection and anti-HPV antibody response were taken. At enrolment, HPV DNA was detected in 38% of sexually active girls/women. At the first, second and third follow-up, HPV DNA was found in 40, 45, and 39% of them. The seroprevalence rates to HPV 6, 11, 16 and 18 in these subjects were 31, 21, 18 and 10%. On the follow-up significantly higher levels of antibodies to HPV 16/18 were found after application of divalent vaccine. Results of the study demonstrate high prevalence of HPV infection in young women. In a substantial number of women, HPV-specific antibodies as well as high-risk HPV types were detected. HPV-specific antibodies were also frequently found in non-sexually active girls. The acquisition of HPV after the onset of sexual life was very fast.

High prevalence of genital HPV infection among long-term monogamous partners of women with cervical dysplasia or genital warts-Another reason for HPV vaccination of boys.

We conducted a cross-sectional study on the occurrence of a specific type of genital human papillomavirus (HPV) among long-term monogamous male partners of women with cervical dysplasia and genital warts. The purpose of the study was to improve knowledge with regards to the management of these couples. The presence of genital HPV-DNA was detected by PCR with broad spectrum primers followed by hybridization. 82 males met the study criteria, 41 in each group. Genital HPV-DNA prevalence was 67.5% in the genital warts group and 72.2% in the cervical dysplasia group. The prevalence of high risk HPVs was higher in the cervical dysplasia group, while low risk HPVs were more prevalent in the genital warts group (p < .05). The prevalence of HPV in males was independent of the duration of the relationship (73.5% for 6-24 months and 66.7% for longer relationships). In conclusion, our results suggest that the prevalence of the genital HPV infection in both groups of male partners is comparable and very high, but the spectrum of HPV types varies significantly. The presence of the genital HPV infection in male sexual partners seems to be independent of the duration of the relationship. Applying the HPV vaccination to boys may prevent this phenomenon.

Expression of genes encoding centrosomal proteins and the humoral response against these proteins in chronic myeloid leukemia.

As the extent of centrosome abnormalities in chronic myeloid leukemia (CML) correlates with disease stage and karyotype alterations, abnormal expression of genes encoding centrosomal proteins may be an early prognostic marker of disease progression. In the present study, we showed that in comparison with healthy controls, the expression of four centrosomal genes (AURKA, HMMR, PLK1 and ESPL1) in the peripheral blood of CML patients was significantly enhanced at diagnosis and decreased to the basal level in most patients treated with imatinib mesylate for three months. In the remaining patients (17%), this decrease was delayed and was associated with worse overall survival. The detection of antibodies in sera showed that patients with higher overall antibody production had superior outcomes in terms of achieving major molecular response and failure-free survival. These data suggest that the dynamics of the response of centrosomal genes should be considered as a risk factor and immunity against centrosomal proteins may contribute to treatment response.

Beta-HPV types in patients with head and neck pathology and in healthy subjects.

BACKGROUND: Human papillomaviruses (HPV) are a heterogeneous group of viruses classified into five genera. The beta-HPV type (beta-PV) infection is very common but mostly asymptomatic in immunocompetent individuals. However, beta-PVs play a role in Epidermodysplasia verruciformis and possibly in non-melanoma skin cancer. Head and neck cancer (HNC) is a common cancer type worldwide and high-risk alpha-PV involvement in HNC has been extensively studied but beta-PV types have rarely been the focus of such studies. OBJECTIVES: To evaluate the prevalence of beta-PV types in HNC, subjects with non-malignant or potentially pre-malignant oral lesions, and healthy controls. STUDY DESIGN: The frequency of different beta-PVs in samples from oral (n=35) and oropharyngeal (n=35) cancer patients, gender- and age-matched healthy controls (n=70), and subjects with various non-malignant or potentially pre-malignant oral lesions (n=102) was assessed by a highly sensitive, bead-based, multiplex genotyping assay. RESULTS: Overall, 54.8% of all tested samples contained at least one beta-PV type. Even though the correlation between types found in lavage and tissue specimens from cancer patients was low, there was a large statistically significant difference between oropharyngeal cancer patients and matched controls for HPV5 (P=0.003; OR=15.58) and between both oral (P=0.026; OR=5.7) and oropharyngeal cancer patients (P=0.002; OR=25.5) and controls for HPV122. In addition, there was no correlation between the prevalence of alpha and beta-PVs in the study patients. CONCLUSION: The study provides new data on the prevalence of beta-PVs in HNC. HPV5 was found significantly associated with HNC as already observed by other studies. Additionally, the significant association of HPV122 with HNC might warrant further study as this type has not been extensively studied so far.

Comparison of the miRNA profiles in HPV-positive and HPV-negative tonsillar tumors and a model system of human keratinocyte clones.

BACKGROUND: Better insights into the molecular changes involved in virus-associated and -independent head and neck cancer may advance our knowledge of HNC carcinogenesis and identify critical disease biomarkers. Here we aimed to characterize the expression profiles in a matched set of well-characterized HPV-dependent and HPV-independent tonsillar tumors and equivalent immortalized keratinocyte clones to define potential and clinically relevant biomarkers of HNC of different etiology. METHODS: Fresh frozen tonsillar cancer tissues were analyzed together with non-malignant tonsillar tissues and compared with cervical tumors and normal cervical tissues. Furthermore, relative miRNAs abundance levels of primary and immortalized human keratinocyte clones were evaluated. The global quantitation of miRNA gene abundance was performed using a TaqMan Low Density Array system. The confirmation of differentially expressed miRNAs was performed on a set of formalin-fixed paraffin-embedded tumor samples enriched for the tumor cell fraction by macrodissection. RESULTS: We defined 46 upregulated and 31 downregulated miRNAs characteristic for the HPV-positive tonsillar tumors and 42 upregulated miRNAs and 42 downregulated miRNAs characteristic for HPV-independent tumors. In comparison with the expression profiles in cervical tumors, we defined miR-141-3p, miR-15b-5p, miR-200a-3p, miR-302c-3p, and miR-9-5p as specific for HPV induced malignancies. MiR-335-5p, miR-579-3p, and miR-126-5p were shared by the expression profiles of HPV-positive tonsillar tumors and of the HPV immortalized keratinocyte clones, whereas miR-328-3p, miR-34c-3p, and miR-885-5p were shared by the miRNA profiles of HPV-negative tonsillar tumors and the HPV-negative keratinocytes. CONCLUSIONS: We identified the miRNAs characteristic for HPV-induced tumors and tonsillar tumors of different etiology, and the results were compared with those of the model system. Our report presents the basis for further investigations leading to the identification of clinically relevant diagnostic and/or therapeutic biomarkers for tumors of viral and non-viral etiology.

Global Genomic Diversity of Human Papillomavirus 11 Based on 433 Isolates and 78 Complete Genome Sequences.

UNLABELLED: Human papillomavirus 11 (HPV11) is an etiological agent of anogenital warts and laryngeal papillomas and is included in the 4-valent and 9-valent prophylactic HPV vaccines. We established the largest collection of globally circulating HPV11 isolates to date and examined the genomic diversity of 433 isolates and 78 complete genomes (CGs) from six continents. The genomic variation within the 2,800-bp E5a-E5b-L1-upstream regulatory region was initially studied in 181/207 (87.4%) HPV11 isolates collected for this study. Of these, the CGs of 30 HPV11 variants containing unique single nucleotide polymorphisms (SNPs), indels (insertions or deletions), or amino acid changes were fully sequenced. A maximum likelihood tree based on the global alignment of 78 HPV11 CGs (30 CGs from our study and 48 CGs from GenBank) revealed two HPV11 lineages (lineages A and B) and four sublineages (sublineages A1, A2, A3, and A4). HPV11 (sub)lineage-specific SNPs within the CG were identified, as well as the 208-bp representative region for CG-based phylogenetic clustering within the partial E2 open reading frame and noncoding region 2. Globally, sublineage A2 was the most prevalent, followed by sublineages A1, A3, and A4 and lineage B. IMPORTANCE: This collaborative international study defined the global heterogeneity of HPV11 and established the largest collection of globally circulating HPV11 genomic variants to date. Thirty novel complete HPV11 genomes were determined and submitted to the available sequence repositories. Global phylogenetic analysis revealed two HPV11 variant lineages and four sublineages. The HPV11 (sub)lineage-specific SNPs and the representative region identified within the partial genomic region E2/noncoding region 2 (NCR2) will enable the simpler identification and comparison of HPV11 variants worldwide. This study provides an important knowledge base for HPV11 for future studies in HPV epidemiology, evolution, pathogenicity, prevention, and molecular assay development.

Analysis of the integration of human papillomaviruses in head and neck tumours in relation to patients' prognosis.

Integration, which leads to the disruption of the circular HPV genome, is considered as a critical, albeit not obligatory, step in carcinogenic progression. Although cervical carcinomas with extrachromosomal HPV plasmid genomes have been described, the virus is integrated in 70% of HPV16-positive cervical tumours. Limited information is available about HPV integration in head and neck tumours (HNC). In this study, we have characterised the physical status of HPV in a set of tonsillar tumour samples using different methods--the mapping of E2 integration breakpoint at the mRNA level, the 3' RACE based Amplification of Papillomavirus Oncogene Transcripts (APOT) assay and Southern blot. Furthermore, the impact of HPV integration on patients' prognosis has been evaluated in a larger set of 186 patients with head and neck cancer. Based on the analysis of E2 mRNA, HPV was integrated in the host genome in 43% of the HPV-positive samples. Extrachromosomal or mixed form was present in 57%. In fresh frozen samples, the APOT and E2 mapping results were in agreement. The results were confirmed using Southern blotting. Furthermore, the type and exact site of integration were determined. The survival analysis of 186 patients revealed HPV positivity, tumour size and lymph node positivity as factors that influence disease specific survival. However, no statistically significant difference was found in disease specific survival between patients with HPV-positive integrated vs. extrachromosomal/mixed forms of the virus.