Epstein-Barr Virus DNA Is Associated With Conjunctival Squamous Cell Carcinomas: A Case-Control Study From Zimbabwe.

Incidence of conjunctival squamous cell carcinoma (cSCC) in Zimbabwe is >30-fold higher than the global average. cSCC risk is notably higher among people with human immunodeficiency virus, implicating impaired immune response and a yet unknown infectious etiology. Formalin-fixed, paraffin-embedded blocks from Zimbabwe, comprising conjunctival precancer (n = 78), invasive cSCC cases (n = 148) and nonmalignant eye lesions (n = 119), were tested for multiple DNA viruses using Luminex bead-based technology. Epstein-Barr virus (EBV) type 1 positivity was strongly associated with cSCC diagnosis (adjusted odds ratio [aOR], 5.6 [95% confidence interval {CI}, 3.0-10.4) and marginally associated with precancer (aOR, 2.1 [95% CI, 1.0-4.5]). On analyzing EBV transcriptional activity with any of LMP1, EBNA1, and BZLF1, RNA transcripts were detected in 5 of 112 controls, 3 of 67 precancers, and 10 of 139 cases and none were associated with conjunctival case status. Our EBV DNA data suggest that EBV may play a role in cSCC. However, the low detection rate of EBV RNA supports further investigation to infer causality.

Prevalence of Alpha, Beta, and Gamma Human Papillomaviruses in Patients With Head and Neck Cancer and Noncancer Controls and Relation to Behavioral Factors.

BACKGROUND: Human papillomaviruses (HPVs) cause head and neck cancer (HNC), which is increasing in incidence in developed countries. We investigated the prevalence of alpha (α), beta (ß), and gamma (γ) HPVs among HNC cases and controls, and their relationship with sociodemographic, behavioral, and oral health factors. METHODS: We obtained oral rinse and brush samples from incident HNC cases (n = 369) and hospital-based controls (n = 439) and tumor samples for a subsample of cases (n = 121). We genotyped samples using polymerase chain reaction with PGMY09-PGMY11 primers and linear array for α-HPV and type-specific multiplex genotyping assay for ß-HPV and γ-HPV. Sociodemographic and behavioral data were obtained from interviews. RESULTS: The prevalence of α-, ß-, and γ-HPV among controls was 14%, 56%, and 24%, respectively, whereas prevalence among cases was 42%, 50%, and 33%, respectively. Prevalence of α- and γ-HPV, but not ß-HPV, increased with increase in sexual activity, smoking, and drinking habits. No HPV genus was associated with oral health. Tumor samples included HPV genotypes exclusively from the α-genus, mostly HPV-16, in 80% of cases. CONCLUSIONS: The distribution of α- and γ-HPV, but not ß-HPV, seems to vary based on sociodemographic and behavioral characteristics. We did not observe the presence of cutaneous HPV in tumor tissues.

Detection of serum biomarkers of HPV-16 driven oropharynx and oral cavity cancer in Brazil.

BACKGROUND: HPV-16 driven oropharynx/oral cavity squamous cell carcinomas prevalence varies globally. We evaluated the presence of HPV-16 ctDNA and HPV-16 E6 antibodies in samples obtained from participants treated at the Instituto do Cancer do Estado de Sao Paulo, ICESP, and from whom tumoral HPV DNA, HPV-16 E6*I mRNA, and p16INK4a status was also accessed. METHODS: HPV was genotyped by PCR-hybridization. All HPV DNA positive and ∼10 % HPV DNA negative cases underwent p16INK4a immunohistochemistry and E6*I RNA testing using a multiplex bead based protocol. HPV-16 ctDNA and anti-E6 antibodies were assessed by ddPCR (digital droplet PCR) and multiplex serology, respectively. RESULTS: The prevalence of HPV-16 in oropharynx carcinoma (OPC) cases was low (8.7 %) when considering solely HPV-16 DNA detection, and even lower (5.2 %) when taken into consideration the concomitant detection of HPV-16 E6*I RNA and/or p16INK4 (HPV-16 attributable fraction - AF). None of the oral cavity cancer (OCC) cases were detected with HPV-16 DNA. HPV-16 ctDNA was more commonly detected than HPV-16 E6 antibodies (29.8 % versus 10.6 %). Both serum biomarkers attained 100 % sensitivity of detecting HPV-16 AF OPC, however the specificity of the HPV-16 anti-E6 biomarker was higher compared to ctDNA (93.2 % versus 75.0 %). Finally, when both HPV-16 ctDNA and anti-E6 biomarkers were considered together, the sensitivity and specificity for HPV-16 OPC detection was 100 % and about 70 %, respectively, independently of analyzing HPV-16 DNA positive or HPV-16 AF tumors. CONCLUSIONS: Our findings corroborate that serum biomarkers are highly sensitive and specific biomarkers for detection of HPV-associated OPC.

Prevalence of viral DNA in high-grade serous epithelial ovarian cancer and correlation with clinical outcomes.

INTRODUCTION: Currently 11 infectious agents are classified as carcinogenic but the role of infectious agents on outcomes of epithelial ovarian cancer is largely unknown. OBJECTIVE: To explore the association between infectious agents and ovarian cancer, we investigated the prevalence of viral DNA in primary ovarian cancer tumors and its association with clinical outcomes. METHODS: Archived tumors from 98 patients diagnosed with high-grade serous epithelial ovarian cancer were collected between 1/1/1994 and 12/31/2010. After DNA extraction, Luminex technology was utilized to identify polymerase chain reaction-amplified viral DNA for 113 specific viruses. Demographic data and disease characteristics were summarized using descriptive statistics. We used logistic regression and Cox proportional hazards model to assess associations between tumor viral status and disease outcome and between tumor viral presence and overall survival (OS), respectively. RESULTS: Forty-six cases (45.9%) contained at least one virus. Six highly prevalent viruses were associated with clinical outcomes and considered viruses of interest (VOI; Epstein-Barr virus 1, Merkel cell polyomavirus, human herpes virus 6b, and human papillomaviruses 4, 16, and 23). Factors independently associated with OS were presence of VOI (HR 4.11, P = 0.0001) and platinum sensitivity (HR 0.21, P<0.0001). Median OS was significantly decreased when tumors showed VOI versus not having these viruses (22 vs 44 months, P<0.0001). Women <70 year old with VOI in tumors had significantly lower median OS versus age-matched women without VOI (20 vs 57 months, P = 0.0006); however, among women ≥70 years old, there was no difference in OS by tumor virus status. CONCLUSIONS: The presence of a VOI was significantly associated with a lower OS. These findings may have implications for clinical management of ovarian cancer but require additional studies.

Evaluation of human papillomavirus DNA in colorectal cancer and adjacent mucosal tissue samples.

BACKGROUND: Although the role of viral agents, such as human papillomavirus (e.g. HPV16, HPV18) in colorectal cancer (CRC) has been previously investigated, results remain inconclusive. METHODS: To further evaluate the involvement of oncogenic HPV types in CRC, 40 frozen neoplastic and 40 adjacent colonic tissues collected from Italian patients were analyzed by Luminex-based assays that detect a broad spectrum of HPV types, i.e. Alpha (n = 21), Beta (n = 46) and Gamma HPVs (n = 52). In addition, 125 frozen CRC samples and 70 surrounding mucosal tissues were collected from Czech patients and analyzed by broad spectrum PCR protocols: (i) FAP59/64, (ii) FAPM1 and (iii) CUT combined with Next Generation Sequencing (NGS). RESULTS: Using Luminex-basedassays, DNA from HPV16 was detected in 5% (2/40) CRC tissues from Italian patients. One HPV16 DNA-positive CRC case was subsequently confirmed positive for E6*I mRNA. Cutaneous beta HPV types were detected in 10% (4/40) adjacent tissues only, namely HPV111 (n = 3) and HPV120 (n = 1), while gamma HPV168 (n = 1) and HPV199 (n = 1) types were detected in adjacent and in tumor tissues, respectively. The NGS analysis of the CRC Czech samples identified HPV sequences from mucosal alpha-3 (HPV89), alpha-7 (HPV18, 39, 68 and 70) and alpha-10 species (HPV11), as well as cutaneous beta-1 (HPV20, 24, 93, 98, 105,124) beta-2 (HPV23), beta-3 (HPV49) and gamma-1 species (HPV205). CONCLUSIONS: Our findings indicate that HPV types belonging to the mucosal alpha, and the 'cutaneous' beta and gamma genera can be detected in the colonic mucosal samples with a low prevalence rate and a low number of HPV reads by Luminex and NGS, respectively. However, additional studies are required to corroborate these findings.

HPV38 impairs UV-induced transcriptional activation of the IL-18 pro-inflammatory cytokine.

IMPORTANCE: Here, we demonstrate that the direct binding of p53 on the IL-18 promoter region regulates its gene expression. However, the presence of E6 and E7 from human papillomavirus type 38 impairs this mechanism via a new inhibitory complex formed by DNA methyltransferase 1 (DNMT1)/PKR/ΔNp73α, which binds to the region formerly occupied by p53 in primary keratinocytes.

Betapapillomaviruses in p16-Negative Vulvar Intraepithelial Lesions Associated with Squamous Cell Carcinoma.

Approximately 40% of vulvar squamous cell carcinoma (vSCC) cases are etiologically associated with high-risk human papillomaviruses (HPVs) of the alpha genera (α-HPV) that cause other anogenital cancers; however, the etiology of α-HPV-negative vSCC is poorly understood. HPVs of the beta genera (ß-HPV) are risk factors for cutaneous squamous cell carcinoma (cSCC) and may be related to carcinomas originating in other cutaneous sites such as the vulva. In this study, we investigate the presence of ß-HPVs, with an emphasis on p16-negative squamous lesions adjacent to vSCC. We subjected 28 vulvar squamous intraepithelial lesions adjacent to vSCC for comprehensive HPV genotyping, p16 and p53 immunohistochemistry, and consensus morphology review. Selected cases were subjected to qPCR and RNA in situ hybridization. Clinical data were obtained from medical records. ß-HPV DNA was detected in eight of ten p16-negative lesions and three of fourteen p16-positive high-grade squamous intraepithelial lesions. The HPV DNA loads in vulvar squamous intraepithelial lesions ranged between less than 1 HPV DNA copy per cell to more than 100 HPV DNA copies per cell. This is, to the best of our knowledge, the first report of the association of p16-negative vulvar intraepithelial squamous lesions with detection of ß-HPVs. These findings expand possible etiologic mechanisms that may contribute to p16-negative lesions of the vulva.

Prevalence of novel gamma HPV types 223 and 225 in oral cavity and skin of Indian normal and neoplastic participants.

Gamma-papillomaviruses, though traditionally classified as cutaneotropic, actual tissue tropism is largely unexplored. This study aimed to evaluate the tissue-specific prevalence of two novel-HPV 223 and 225 in samples of oral mucosa and keratinized epithelium of varied skin parts from 226 female and male subjects, with or without neoplastic/dysplastic lesions in oral cavity or cervix. The gamma-human papillomavirus (gamma-HPV) 223 and 225 DNA presences were determined by polymerase chain reaction (PCR) ursing the HPV type-specific primers and confirmed by Sanger sequencing. Viral load in the HPV 223 and HPV 225 positive samples were determined by absolute real-time quantification method. Alpha-HPV DNA prevalence was also checked in oral mucosa to ascertain coinfection status. Novel HPV 223 was present in 4.4% (10/226) oral mucosal samples of the study population; interestingly all were females with no prevalence in their corresponding skin swab samples. Whereas, the prevalence of HPV 225 was found both in the skin and oral mucosa of 28.2% (N = 37/131) female and 17.9% (N = 17/95) male participants. Alongside, HPV 223 viral load was found to be significantly higher (p = 0.02 < 0.05) in the oral mucosa of diseased participants, whereas, HPV 225 viral load was higher in the oral mucosa of normal participants. Our results suggest that gamma-HPV 223 has its prevalence only in the oral mucosal epithelium, whereas, HPV 225 has its prevalence on both mucosal and keratinized skin epithelium, indicating its dual tropism nature.

Correction: Self-collected and clinician-collected anal swabs show modest agreement for HPV genotyping.

[This corrects the article DOI: 10.1371/journal.pone.0250426.].

The E2F4/p130 Repressor Complex Cooperates with Oncogenic ΔNp73α To Inhibit Gene Expression in Human Papillomavirus 38 E6/E7-Transformed Keratinocytes and in Cancer Cells.

Tumor suppressor p53 and its related proteins, p63 and p73, can be synthesized as multiple isoforms lacking part of the N- or C-terminal regions. Specifically, high expression of the ΔNp73α isoform is notoriously associated with various human malignancies characterized by poor prognosis. This isoform is also accumulated by oncogenic viruses, such as Epstein-Barr virus (EBV), as well as genus beta human papillomaviruses (HPV) that appear to be involved in carcinogenesis. To gain additional insight into ΔNp73α mechanisms, we have performed proteomics analyses using human keratinocytes transformed by the E6 and E7 proteins of the beta-HPV type 38 virus as an experimental model (38HK). We find that ΔNp73α associates with the E2F4/p130 repressor complex through a direct interaction with E2F4. This interaction is favored by the N-terminal truncation of p73 characteristic of ΔNp73 isoforms. Moreover, it is independent of the C-terminal splicing status, suggesting that it could represent a general feature of ΔNp73 isoforms (α, ß, γ, δ, ε, ζ, θ, η, and η1). We show that the ΔNp73α-E2F4/p130 complex inhibits the expression of specific genes, including genes encoding for negative regulators of proliferation, both in 38HK and in HPV-negative cancer-derived cell lines. Such genes are not inhibited by E2F4/p130 in primary keratinocytes lacking ΔNp73α, indicating that the interaction with ΔNp73α rewires the E2F4 transcriptional program. In conclusion, we have identified and characterized a novel transcriptional regulatory complex with potential implications in oncogenesis. IMPORTANCE The TP53 gene is mutated in about 50% of human cancers. In contrast, the TP63 and TP73 genes are rarely mutated but rather expressed as ΔNp63 and ΔNp73 isoforms in a wide range of malignancies, where they act as p53 antagonists. Accumulation of ΔNp63 and ΔNp73, which is associated with chemoresistance, can result from infection by oncogenic viruses such as EBV or HPV. Our study focuses on the highly carcinogenic ΔNp73α isoform and uses a viral model of cellular transformation. We unveil a physical interaction between ΔNp73α and the E2F4/p130 complex involved in cell cycle control, which rewires the E2F4/p130 transcriptional program. Our work shows that ΔNp73 isoforms can establish interactions with proteins that do not bind to the TAp73α tumor suppressor. This situation is analogous to the gain-of-function interactions of p53 mutants supporting cellular proliferation.

Human papilloma virus-16-specific CD8+ T-cell expansions characterize different clinical forms of lichen planus and not lichen sclerosus et atrophicus.

Lichen planus (LP) is a cutaneomucosal chronic inflammatory disease characterized by a CD8+ cytotoxic T-lymphocytes (CTL) infiltrate. In erosive oral LP, we found HPV16-specific activated CTL in lesions, supporting a pathogenic contribution of HPV16. Here, we investigated whether a similar scenario occurs in other clinical forms of LP and in lichen sclerosus et atrophicus (LSA), another chronic disease also affecting the mucosa and/or the skin. Blood CTL from LP and LSA patients expressed significant higher levels of granzyme B, perforin and CD107a proteins than healthy donors. Expansions of TCRVß3+ CTL, with presence of TCR clonotypes identical to those previously detected in erosive oral LP, were found both in blood and mucosal/skin lesions of LP, and not of LSA patients. These expansions were enriched with HPV16-specific CD8+ T-cells as shown by their recognition of the E711-20 immunodominant epitope. In LSA patients, the peripheral repertoire of CTL was oligoclonal for TCRVß6+ CTL. Finally, although patients with LP and LSA have developed antibodies against HPV16 capsid L1, antibodies against HPV16 E6 were only observed in patients with LP. Overall, our data collectively suggest an involvement of HPV16-specific CTL in different clinical forms of LP, not only in erosive oral LP, while a different scenario operates in LSA.

Impact of HPV vaccination on HPV-related oral infections.

BACKGROUND: Human papillomavirus (HPV) is one of the most common sexually transmitted infections worldwide. Although the efficacy of the HPV vaccine in preventing the development of cervical pre-malignant lesions has been well demonstrated, the efficacy of the HPV vaccine in preventing HPV infection in the upper respiratory tract has been poorly studied. METHODS: In the context of the IARC cohort study of two versus three doses of HPV vaccine in India, we compared the HPV type prevalence in the oral cavity of women vaccinated with three doses, two doses, or a single dose of quadrivalent HPV vaccine with that of unvaccinated women. A total of 997 oral samples, from 818 vaccinated women and 179 unvaccinated women, were collected at three study sites. All the participants were sexually active at the time of sample collection. RESULTS: The age-standardized proportion (ASP) of HPV16/18 infections was 2.0 % (95 % CI, 1.0-3.0 %) in vaccinated women and 4.2 % (95 % CI, 1.2-7.2 %) in unvaccinated women. HPV16 was detected in 3.5 % of single-dose recipients, 1.2 % of two-dose recipients (days 1 and 180), and 1.5 % of three-dose recipients (days 1, 60, and 180), whereas 3.3 % of the unvaccinated women tested positive for HPV16. The same trend was observed for HPV18. DISCUSSION: Our findings agree with those of previous studies on the efficacy of HPV vaccination in reducing oral HPV infections and provide indications that a single vaccine dose may be less efficient than two or three doses in preventing oral HPV infection.

Evaluation of human papillomavirus (HPV) knowledge among healthcare professionals: A study of conference attendees in Angola.

Human papillomavirus (HPV) plays an essential role in cervical cancer development. Angola has a high cervical cancer incidence rate (36.1 per 100,000); therefore, knowledge of HPV among clinicians is essential for the prevention of cervical cancer and educating at-risk individuals. This study aimed to evaluate knowledge of HPV among healthcare professionals in Angola. A 44-item questionnaire was distributed to 65 healthcare professionals who attended a conference on the cancer burden in Angola. Non-parametric and multiple regression analyses were conducted. Of the participants (55.6%) were male, and 33.3% were aged 31-40 years, 56.3% had an undergraduate degree, and 35.9% had a postgraduate degree. All participants worked in healthcare in academic and/or clinical settings, with 36.7% in joint contracts. Most (62.2%) knew that early sexual debut increases the risk of contracting HPV, and HPV vaccines are most effective if administered before sexual debut. However, there was limited knowledge regarding the virus transmission. The mean HPV knowledge score was 11.08 ± 8.76 and knowledge was higher among older healthcare workers (>50 years) with a postgraduate degree and working in a clinical role. Overall, HPV knowledge was limited among the respondents. Policymakers should consider promoting knowledge and targeted public health initiatives among healthcare professionals in Angola.

Bariatric surgery-induced weight loss and associated genome-wide DNA-methylation alterations in obese individuals.

BACKGROUND: Obesity is a multifactorial and chronic condition of growing universal concern. It has recently been reported that bariatric surgery is a more successful treatment for severe obesity than other noninvasive interventions, resulting in rapid significant weight loss and associated chronic disease remission. The identification of distinct epigenetic patterns in patients who are obese or have metabolic imbalances has suggested a potential role for epigenetic alterations in causal or mediating pathways in the development of obesity-related pathologies. Specific changes in the epigenome (DNA methylome), associated with metabolic disorders, can be detected in the blood. We investigated whether such epigenetic changes are reversible after weight loss using genome-wide DNA methylome analysis of blood samples from individuals with severe obesity (mean BMI ~ 45) undergoing bariatric surgery. RESULTS: Our analysis revealed 41 significant (Bonferroni p < 0.05) and 1169 (false discovery rate p < 0.05) suggestive differentially methylated positions (DMPs) associated with weight loss due to bariatric surgery. Among the 41 significant DMPs, 5 CpGs were replicated in an independent cohort of BMI-discordant monozygotic twins (the heavier twin underwent diet-induced weight loss). The effect sizes of these 5 CpGs were consistent across discovery and replication sets (p < 0.05). We also identified 192 differentially methylated regions (DMRs) among which SMAD6 and PFKFB3 genes were the top hypermethylated and hypomethylated regions, respectively. Pathway enrichment analysis of the DMR-associated genes showed that functional pathways related to immune function and type 1 diabetes were significant. Weight loss due to bariatric surgery also significantly decelerated epigenetic age 12 months after the intervention (mean = - 4.29; p = 0.02). CONCLUSIONS: We identified weight loss-associated DNA-methylation alterations targeting immune and inflammatory gene pathways in blood samples from bariatric-surgery patients. The top hits were replicated in samples from an independent cohort of BMI-discordant monozygotic twins following a hypocaloric diet. Energy restriction and bariatric surgery thus share CpGs that may represent early indicators of response to the metabolic effects of weight loss. The analysis of bariatric surgery-associated DMRs suggests that epigenetic regulation of genes involved in endothelial and adipose tissue function is key in the pathophysiology of obesity.

Prevalence of 13 polyomaviruses in actinic keratosis and matched healthy skin samples of immunocompetent individuals.

BACKGROUND: Actinic keratosis (AK) is a precursor of cutaneous squamous cell carcinoma (cSCC). UV radiation is the major risk factor for AK, but certain human papillomaviruses (HPVs) of the beta genus are also involved in its development. Differently, the role of polyomaviruses (PyVs) in skin carcinogenesis is still debated. Fiftheen PyVs have been isolated from human tissues so far, including Merkel cell polyomavirus (MCPyV), the aetiological agent of Merkel cell carcinoma. METHODS: The presence of 13 PyVs was assessed in skin samples from AK patients (n = 342). Matched fresh-frozen scrapings from healthy skin (HS) and AK lesions from 242 patients, and formalin-fixed paraffin-embedded AK biopsies from a different cohort of 100 patients were analyzed by multiplex PyVs genotyping assay. RESULTS: The most frequent lesion site was the scalp in men (27.3%), and the cheek area in women (29.0%). Differences between men and women were significant for the scalp, the cheek area and the lips. Almost all the scrapings were PyV-positive (HS: 89.7%, AK: 94.6%; p = 0.04). The three most frequent PyVs were MCPyV, HPyV6 and JCPyV (HS: 87.2%, 58.7%, 6.6%, respectively; AK: 88.8%, 51.2%, 9.9%, respectively). HPyV9, TSPyV, BKPyV, HPyV7, LIPyV and SV40 were detected in < 2% of the scrapings. In most cases, matched HS and AK scrapings were both positive (MCPyV: 78.1%, HPyV6: 41.7%), or both negative for the individual genotypes (for the remaining PyVs). PyV prevalence in AK biopsies was 22.0%. Only MCPyV (21.0%) and HPyV6 (3.0%) were detected in these samples. CONCLUSIONS: PyV prevalence in HS and AK scrapings was high, but detection of PyVs exclusively in AK scrapings was rare. PyV positivity rate in AK biopsies was modest. Further research is need to reach firm conclusions regarding the role of these viruses in AK development.

The Role of HPV in Determining Treatment, Survival, and Prognosis of Head and Neck Squamous Cell Carcinoma.

Human papillomavirus (HPV) infection has been identified as a significant etiological agent in the development of head and neck squamous cell carcinoma (HNSCC). HPV's involvement has alluded to better survival and prognosis in patients and suggests that different treatment strategies may be appropriate for them. Only some data on the epidemiology of HPV infection in the oropharyngeal, oral cavity, and laryngeal SCC exists in Europe. Thus, this study was carried out to investigate HPV's impact on HNSCC patient outcomes in the Irish population, one of the largest studies of its kind using consistent HPV testing techniques. A total of 861 primary oropharyngeal, oral cavity, and laryngeal SCC (OPSCC, OSCC, LSCC) cases diagnosed between 1994 and 2013, identified through the National Cancer Registry of Ireland (NCRI), were obtained from hospitals across Ireland and tested for HPV DNA using Multiplex PCR Luminex technology based in and sanctioned by the International Agency for Research on Cancer (IARC). Both overall and cancer-specific survival were significantly improved amongst all HPV-positive patients together, though HPV status was only a significant predictor of survival in the oropharynx. Amongst HPV-positive patients in the oropharynx, surgery alone was associated with prolonged survival, alluding to the potential for de-escalation of treatment in HPV-related OPSCC in particular. Cumulatively, these findings highlight the need for continued investigation into treatment pathways for HPV-related OPSCC, the relevance of introducing boys into national HPV vaccination programs, and the relevance of the nona-valent Gardasil-9 vaccine to HNSCC prevention.

Alteration of Na/H exchange regulatory factor-1 protein levels in anogenital lesions positive for mucosal high-risk human papillomavirus type 16.

Mucosal high-risk (HR) human papillomaviruses (HPV) are associated with anogenital carcinogenesis. The products of two early genes, E6 and E7, act as major viral oncoproteins. Functional studies in experimental models showed that HPV16 E6 induces degradation of the PDZ protein, the Na+/H+ exchanger regulatory factor-1 (NHERF-1). Here, we determined NHERF-1 protein levels by immunohistochemistry (IHC) in (i) benign anogenital warts (n = 8) (ii) premalignant lesions (L-SIL and H-SIL) (n = 43) and (iii) invasive cervical squamous cell carcinomas (SCC) (n = 17). A decrease of NHERF-1 protein level was not observed in genital warts in comparison to healthy epithelium. Conversely, a clearly decrease in NHERF-1 protein levels was observed in HPV16-positive pre-malignant and malignant lesions, while the phenomenon was much attenuated in lesions induced by other HR HPV types. In conclusion, these findings show that mucosal HPV types differently impact on NHERF-1 protein level in benign and malignant anogenital lesions.

Concordance of p16INK4a and E6*I mRNA among HPV-DNA-Positive Oropharyngeal, Laryngeal, and Oral Cavity Carcinomas from the ICO International Study.

Background: Tests or test algorithms for diagnosing HPV-driven oral cavity and laryngeal head and neck carcinomas (HNC) have not been yet validated, and the differences among oral cavity and laryngeal sites have not been comprehensively evaluated. We aimed to assess the utility of a diagnostic algorithm for the detection of HPV-driven oral cavity (OCC), oropharyngeal (OPC) and laryngeal (LC) carcinomas using HPV-DNA testing followed by p16INK4a immunohistochemistry, taking E6*I mRNA detection as the reference standard. Methods: Formalin-fixed paraffin-embedded OCC, OPC, and LC carcinomas were collected from pathology archives in 29 countries. All samples were subjected to histopathological evaluation, DNA quality control, and HPV-DNA detection. All HPV-DNA-positive samples (including 78 OCC, 257 OPC, and 51 LC out of 3680 HNC with valid HPV-DNA results) were also tested for p16INK4a immunohistochemistry and E6*I mRNA. Three different cutoffs of nuclear and cytoplasmic staining were evaluated for p16INK4a: (a) >25%, (b) >50%, and (c) ≥70%. The concordance of p16INK4a and E6*I mRNA among HPV-DNA-positive OCC, OPC, and LC cases was assessed. Results: A total of 78 OCC, 257 OPC, and 51 LC were HPV-DNA-positive and further tested for p16INK4a and E6*I mRNA. The percentage of concordance between p16INK4a (cutoff ≥ 70%) and E6*I mRNA among HPV-DNA-positive OCC, OPC, and LC cases was 79.5% (95% CI 69.9−89.1%), 82.1% (95% CI 77.2−87.0%), and 56.9% (95% CI 42.3−71.4%), respectively. A p16INK4a cutoff of >50% improved the concordance although the improvement was not statistically significant. For most anatomical locations and p16INK4a cutoffs, the percentage of discordant cases was higher for HPV16- than HPV-non16-positive cases. Conclusions: The diagnostic algorithm of HPV-DNA testing followed by p16INK4a immunohistochemistry might be helpful in the diagnosis of HPV-driven OCC and OPC, but not LC. A different p16INK4a expression pattern was observed in those cases HPV-DNA-positive for types other than HPV16, as compared to HPV16-positive cases. Our study provides new insights into the use HPV-DNA, p16INK4a, and HPV-E6*I mRNA for diagnosing an HPV-driven HNC, including the optimal HPV test or p16INK4a cutoffs to be used. More studies are warranted to clarify the role of p16INK4a and HPV status in both OPC and non-OPC HNC.

HPV and head and neck cancers: Towards early diagnosis and prevention.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with an increasing trend of its incidence. Alcohol consumption, smoking, and viral infections, such as the mucosal high-risk (HR) human papillomaviruses (HPVs) are major risk factors for HNSCC development. In particular, HR HPVs are mainly associated with a subset of oropharyngeal squamous cell carcinoma (OPSCC), while other head and neck sites are marginally affected by HPV infection. HPV16 is the most frequently HR HPV type associated with HNSCC. In contrast to the cervix, no screening programs or identifiable pre-malignant lesions have been characterized for HPV-related HNSCC. Therefore, identification of general diagnostic algorithms and HPV biomarkers that could facilitate the early diagnosis, disease evolution and recurrence for HPV-driven HNSCCs are urgently needed. We herein review the role of HPV in HNSCC with a focus on epidemiology, biology, applied diagnostic algorithms and available biomarkers in body fluids as early diagnostic tools in HPV-driven HNSCCs.

Accuracy of high-risk HPV DNA PCR, p16(INK4a) immunohistochemistry or the combination of both to diagnose HPV-driven oropharyngeal cancer.

BACKGROUND: The incidence of high-risk human papillomavirus (hrHPV)-driven head and neck squamous cell carcinoma, in particular oropharyngeal cancers (OPC), is increasing in high-resource countries. Patients with HPV-induced cancer respond better to treatment and consequently have lower case-fatality rates than patients with HPV-unrelated OPC. These considerations highlight the importance of reliable and accurate markers to diagnose truly HPV-induced OPC. METHODS: The accuracy of three possible test strategies, i.e. (a) hrHPV DNA PCR (DNA), (b) p16(INK4a) immunohistochemistry (IHC) (p16), and (c) the combination of both tests (considering joint DNA and p16 positivity as positivity criterion), was analysed in tissue samples from 99 Belgian OPC patients enrolled in the HPV-AHEAD study. Presence of HPV E6*I mRNA (mRNA) was considered as the reference, indicating HPV etiology. RESULTS: Ninety-nine OPC patients were included, for which the positivity rates were 36.4%, 34.0% and 28.9% for DNA, p16 and mRNA, respectively. Ninety-five OPC patients had valid test results for all three tests (DNA, p16 and mRNA). Using mRNA status as the reference, DNA testing showed 100% (28/28) sensitivity, and 92.5% (62/67) specificity for the detection of HPV-driven cancer. p16 was 96.4% (27/28) sensitive and equally specific (92.5%; 62/67). The sensitivity and specificity of combined p16 + DNA testing was 96.4% (27/28) and 97.0% (65/67), respectively. In this series, p16 alone and combined p16 + DNA missed 1 in 28 HPV driven cancers, but p16 alone misclassified 5 in 67 non-HPV driven as positive, whereas combined testing would misclassify only 2 in 67. CONCLUSIONS: Single hrHPV DNA PCR and p16(INK4a) IHC are highly sensitive but less specific than using combined testing to diagnose HPV-driven OPC patients. Disease prognostication can be encouraged based on this combined test result.