Oral human papilloma virus infection: an overview of clinical-laboratory diagnosis and treatment.

OBJECTIVE: The aim of this review is to describe the "hot points" of current clinical governance for oral HPV comprising the use of new diagnostic molecular procedures, namely, Pyrosequencing and Next Generation Sequencing. MATERIALS AND METHODS: The data on oral HPV was collected through two levels of research. First for all, we used the canonical medical search engines, PubMed, and Medline, followed by the study of current commercial tools for HPV diagnosis, particularly within commercial companies involved in the molecular procedures for HPV detecting and genotyping. RESULTS: Different medical procedures are now described and used throughout the world in HPV diagnosis and treatment. However, the laboratory methods are often validated and used for genital infections, and, in these cases, data are missing in the literature as regards the clinical approach for oral lesions. CONCLUSIONS: Dental care units are often the front line for a clinical evaluation of a possible HPV lesion in the oral cavity, which means that correct clinical governance could avoid a viral neoplastic progression of this disease with great advantages for the patient. In this case, the problem is due to the difficulty in lesion recognition but also and more especially the absence of correct laboratory diagnosis and subsequent treatment in the clinical course.

Human papillomavirus infection is not involved in esophageal verrucous carcinoma.

Verrucous carcinoma of the esophagus (VCE) is a rare variant of squamous cell cancer, with a puzzling clinical, etiological, and molecular profile. The etiological involvement of human papillomavirus (HPV) in the cancer's natural history is controversial. This study considers 9 cases of VCE, focusing on patients' clinical history before surgery, histologic phenotype, immunophenotype (epidermal growth factor receptor [EGFR], E-cadherin, cyclin D1, p16, and p53 expression), HPV infection, and TP53 gene mutational status (exons 5-8). Using 3 different molecular test methods, not one of these cases of VCE featured HPV infection. The only case with synchronous nodal metastasis was characterized by a TP53 missense point mutation in association with high EGFR and low E-cadherin expression levels. In conclusion, HPV infection is probably not involved with VCE, while TP53 gene mutation, EGFR overexpression, and E-cadherin loss might fuel the tumor's proliferation and lend it a metastatic potential.

Human Papilloma Virus-Associated Lips Verrucous Carcinoma in HIV-Infected Male.

Human papillomavirus (HPV) infection, widely known as the necessary cause of cervical cancer, has been established as a major etiologic factor for head and neck cancer (HNC). HIV-infected individuals are at higher risk of HPV-associated cancers than the general population. We describe a 45-year-old man with HIV and HPV coinfection, who presented progressively enlarging verrucous neoformations of the lips. The final diagnosis of verrucous carcinoma was delayed. Early detection of HPV lesions in oral mucosa and HPV screening activities could be important in improving the diagnostic sensitivity for the HIV-infected patients with oral cancer.

Anal verrucous carcinoma is not related to infection with human papillomaviruses and should be distinguished from giant condyloma (Buschke-Löwenstein tumour).

AIMS: Verrucous carcinoma (VC) is a variant of well-differentiated squamous cell carcinoma and in the anal region is regarded as synonymous with giant condyloma (Buschke-Löwenstein tumour) (BLT). Aetiology, diagnostic criteria and clinical behaviour of both lesions are controversial. Recent studies suggest that VC at other sites is not associated with human papillomaviruses (HPV). We hypothesized that anal VC is also not related to HPV, while BLT is a HPV-induced lesion. METHODS AND RESULTS: Ten cases of VC and four cases of BLT were included. Several techniques were used for HPV detection: in-situ hybridization for HPV6, 11, 16 and 18, six different polymerase chain reaction (PCR) protocols for detection of at least 89 HPV types from alpha-, beta-, gamma- and mu-PV genera and in-situ hybridization for high-risk HPV E6/E7 mRNA; p16 immunohistochemistry and morphometric analysis were also performed. Alpha-, gamma- and mu-PVs were not found in any case of VC, while HPV6 was detected in all cases of BLT. p16 overexpression was not present in any of the lesions. Among microscopic features, only the absence of koilocytosis and enlarged spinous cells seem to be useful to distinguish VC from BLT. CONCLUSIONS: Our results suggest that anal VC, similarly to VC at other sites, is not associated with HPV infection, and must be distinguished from BLT, which is associated with low-risk HPV. Only with well-set diagnostic criteria will it be possible to ascertain clinical behaviour and optimal treatment for both lesions.

Oral verrucous carcinoma: A retrospective analysis for clinicopathologic features.

AIM: To study the clinicopathological features of oral verrucous carcinoma (VC). METHODS: Archives of the department were retrieved for verrucous lesions. After thorough histopathologic examination, 10 cases were confirmed as VC. Age, sex, site, tobacco habit, clinical presentation, and histologically, the presence of dysplasia and koilocytic changes were studied. RESULTS: Oral VC showed a distinct male preponderance with male:female ratio of 8:2 and occurring predominantly in sixth and seventh decade. Tobacco association in the form of chewing (50%), smoking (40%) or both (10%) was found in all the cases. In chewers, the site of lesion corresponded to the site of tobacco placement that is gingivobuccal sulcus. In smokers, the posterior part of the oral cavity was affected, and the lesions were extensive. Painless, exophytic, cauliflower-like growth was the most common presentation with surrounding whitish (leukoplakic) mucosa. Fifty percentage of the cases showed human papilloma virus-induced changes in the epithelium. Dysplasia was seen in two cases. CONCLUSION: Oral VCs are invariably associated with tobacco habits. In smokers, the lesions are extensive, affecting the posterior parts of the oral cavity. Although evidence of viral infection was seen but its role as an etiological agent is still controversial. Site and depth of the biopsy along with thorough histopathological sampling is essential to avoid erroneous diagnosis.

Notch1 Activation or Loss Promotes HPV-Induced Oral Tumorigenesis.

Viral oncogene expression is insufficient for neoplastic transformation of human cells, so human papillomavirus (HPV)-associated cancers will also rely upon mutations in cellular oncogenes and tumor suppressors. However, it has been difficult so far to distinguish incidental mutations without phenotypic impact from causal mutations that drive the development of HPV-associated cancers. In this study, we addressed this issue by conducting a functional screen for genes that facilitate the formation of HPV E6/E7-induced squamous cell cancers in mice using a transposon-mediated insertional mutagenesis protocol. Overall, we identified 39 candidate driver genes, including Notch1, which unexpectedly was scored by gain- or loss-of-function mutations that were capable of promoting squamous cell carcinogenesis. Autochthonous HPV-positive oral tumors possessing an activated Notch1 allele exhibited high rates of cell proliferation and tumor growth. Conversely, Notch1 loss could accelerate the growth of invasive tumors in a manner associated with increased expression of matrix metalloproteinases and other proinvasive genes. HPV oncogenes clearly cooperated with loss of Notch1, insofar as its haploinsufficiency accelerated tumor growth only in HPV-positive tumors. In clinical specimens of various human cancers, there was a consistent pattern of NOTCH1 expression that correlated with invasive character, in support of our observations in mice. Although Notch1 acts as a tumor suppressor in mouse skin, we found that oncogenes enabling any perturbation in Notch1 expression promoted tumor growth, albeit via distinct pathways. Our findings suggest caution in interpreting the meaning of putative driver gene mutations in cancer, and therefore therapeutic efforts to target them, given the significant contextual differences in which such mutations may arise, including in virus-associated tumors.

Laser micro-dissection and qPCR for identifying specific HPV types responsible for malignancy in penile lesions.

The aim of the study was to identify specific human papillomavirus (HPV) type responsible for malignancy in penile tissue samples using laser micro-dissection and TaqMan quantitative real-time PCR (qPCR). The study was based on two pre-malignant and seven malignant penile tissue samples and laser micro-dissection was performed on all. Genotyping was performed on whole tissue sections and laser micro-dissection samples using qPCR. Two whole tissue section samples were HPV negative while seven were HPV positive. In four samples that were single HPV infections with whole tissue section PCR, identical HPV types were confirmed with laser micro-dissection PCR. Clearly confirming that the single HPV type detected is responsible for malignancy. In two samples that had multiple HPV infections with whole tissue section PCR, only one HPV type with the highest viral load was detected with laser micro-dissection PCR, suggesting that the HPV type with the highest viral load is most likely the cause of that particular lesion. HPV 11 and/or HPV 16 were the only types detected with laser micro-dissection PCR in these cases, compared to multiple HPV types (HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, and HPV 39) initially detected with whole tissue section PCR. HPV 11 was associated with verrucous lesions while HPV 16 was associated with squamous cell carcinoma and PIN 3 lesions. This study confirms that laser micro-dissection and qPCR are essential tools in identifying the HPV types responsible for malignancy in penile lesions, particularly in samples with multiple infections.

Human papillomavirus-related verrucous carcinoma in a renal transplant patient after long-term immunosuppression: a case report.

BACKGROUND: Verrucous carcinoma is a slow-growing tumor with 3 main localizations: Oral cavity, ano-urogenital region, and plantar surface of the foot. On the sole it may rise adjacent to viral warts and very often is mistaken for the common verruca plantaris. Although both conditions-viral warts and cutaneous squamous cell carcinoma-are often diagnosed in immunosuppressed patients, in literature we have found only 3 case reports of verrucous carcinoma in organ transplant recipients. CASE REPORT: We present a case of 26-year-old man after deceased donor renal transplantation with plantar verrucous carcinoma successfully treated with excision and 5% imiquimod.

Retrospective case-control study of viral pathogen screening in proliferative verrucous leukoplakia lesions.

OBJECTIVE: This study aimed to survey the presence of known oncoviruses in oral biopsies from patients diagnosed with the aetiologically undetermined proliferative verrucous leukoplakia and compare results to those from milder oral leukoplakia (OL) cases, oral squamous cell carcinoma, a common outcome of the lesions of interest, and healthy controls. DESIGN: Blind, retrospective, case-control study. SETTING: A stomatology unit in an academic Hospital and a Public Health laboratory. PARTICIPANTS: Forty patients were divided in four groups. Ten patients had been diagnosed with proliferative verrucous leukoplakia, 10 with OL and 10 with OSCC, and 10 were healthy subjects. MAIN OUTCOME MEASURES: The presence or absence of oncovirus DNA was assayed with the amplification of viral genetic markers using PCR and subsequent gel electrophoresis confirmation. Amplified fragments were sequenced and identified bioinformatically. RESULTS: No DNA from the herpesvirus, papillomavirus or polyomavirus species was detected in the samples. CONCLUSIONS: No association between proliferative verrucous leukoplakia and target viruses was detected. A higher throughput viral metagenomic approach may prove valuable for future analyses, as it would not be restricted to a priori knowledge of potential targets.

Next-generation sequencing analysis for detecting human papillomavirus in oral verrucous carcinoma.

OBJECTIVE: The etiology of oral verrucous carcinoma is unknown, and human papillomavirus 'involvement' remains contentious. The uncertainty can be attributed to varied detection procedures and difficulties in defining 'gold-standard' histologic criteria for diagnosing 'verrucous' lesions. Their paucity also hampers investigation. We aimed to analyze oral verrucous lesions for human papillomavirus (HPV) subtype genomes. STUDY DESIGN: We used next-generation sequencing for the detection of papillomavirus sequences, identifying subtypes and computing viral loads. We identified a total of 78 oral verrucous cases (62 carcinomas and 16 hyperplasias). DNA was extracted from all and sequenced at a coverage between 2.5% and 13%. RESULTS: An HPV-16 sequence was detected in 1 carcinoma and 1 hyperplasia, and an HPV-2 sequence was detected in 1 carcinoma out of the 78 cases, with viral loads of 2.24, 8.16, and 0.33 viral genomes per cell, respectively. CONCLUSIONS: Our results indicate no conclusive human papillomavirus involvement in oral verrucous carcinoma or hyperplasia.

Verrucous carcinoma of the head and neck - not a human papillomavirus-related tumour?

Association between verrucous carcinoma (VC) of the head and neck and human papillomaviruses (HPV) is highly controversial. Previous prevalence studies focused mostly on α-PV, while little is known about other PV genera. Our aim was to investigate the prevalence of a broad spectrum of HPV in VC of the head and neck using sensitive and specific molecular assays. Formalin-fixed, paraffin-embedded samples of 30 VC and 30 location-matched normal tissue samples were analysed, by using six different polymerase chain reaction-based methods targeting DNA of at least 87 HPV types from α-PV, ß-PV, γ-PV and µ-PV genera, and immunohistochemistry against p16 protein. α-PV, γ-PV and µ-PV were not detected. ß-PV DNA was detected in 5/30 VC (16.7%) and in 18/30 normal tissue samples (60.0%): HPV-19, -24 and -36 were identified in VC, and HPV-5, -9, -12, -23, -24, -38, -47, -49 and -96 in normal tissue, whereas HPV type was not determined in 2/5 cases of VC and in 6/18 normal tissue samples. p16 expression was detected in a subset of samples and was higher in VC than in normal tissue. However, the reaction was predominantly cytoplasmic and only occasionally nuclear, and the extent of staining did not exceed 75%. Our results indicate that α-PV, γ-PV and µ-PV are not associated with aetiopathogenesis of VC of the head and neck. ß-PV DNA in a subset of VC and normal tissue might reflect incidental colonization, but its potential biological significance needs further investigation.

No human virus sequences detected by next-generation sequencing in benign verrucous skin tumors occurring in BRAF-inhibitor-treated patients.

Patients treated with BRAF inhibitors (e.g. vemurafenib), a novel targeted therapy for advanced melanoma harbouring certain BRAF mutations, develop numerous adverse cutaneous side effects, including skin tumors such as squamous cell carcinoma or non-malignant verruciform keratinocyte proliferations, termed 'BRAF-inhibitor-associated verrucous keratosis (BAVK) lesions'. These keratinocyte proliferations are believed to be caused by paradoxical hyperactivation of the MAPK pathway in cells with wild-type BRAF, but mutated RAS. However, due to the clinical and histological verruca-like appearance of these lesions, additional aetiologic cofactors, such as infectious agents (i.e. oncogenic viruses), might be suspected. Therefore, we performed 454 high-throughput sequencing of BAVK lesions from vemurafenib-treated patients on the transcript level to identify actively transcribed viral sequences of known [e.g. human papilloma viruses (HPV)] or even yet-unknown viruses. Next-generation sequencing did not identify transcripts of any human viruses out of 1 595 161 reads obtained from BAVK lesions of four patients. Nevertheless, all controls were recognized correctly, and the detection of sequences derived from the cutaneous microbiome (e.g. skin commensals and bacterial phages) confirmed the validity and sensitivity of the sequencing data. Our results are consistent with preliminary histological and immunohistochemical findings recently reported by others, who also failed to detect the expression of HPV proteins in BAVK. Although the patient number is limited and we cannot exclude the possibility of having missed a viral transcript of very low abundance, our study argues against a viral aetiology of BRAF-inhibitor-associated verruciform keratoses occurring under vemurafenib.

Verrucous carcinomas of the head and neck, including those with associated squamous cell carcinoma, lack transcriptionally active high-risk human papillomavirus.

Most oropharyngeal squamous cell carcinomas (SCC) and histologic variants harbor transcriptionally active human papillomavirus (HPV). While HPV DNA can be found in many non-oropharyngeal head and neck carcinomas, transcriptionally active HPV is rare. Verrucous carcinoma is a variant with bland cytology, warty appearance, locally destructive growth, and lack of metastasis when lacking a frankly invasive carcinoma component. Studies have shown variable rates of HPV DNA and p16 protein expression in such tumors but still have not clearly addressed if the virus has biological activity or clinical relevance in the positive cases. Department files were searched for verrucous neoplasms, including pure verrucous carcinoma, verrucous carcinoma with dysplasia or minimal invasion, and SCC arising in verrucous carcinoma (ie, having a major component of frankly invasive carcinoma). p16 immunohistochemistry, HPV DNA polymerase chain reaction (PCR) and E6/E7 mRNA reverse transcription PCR for high-risk HPV types were performed. Of the 49 cases, 6 (12.2%) showed strong (>50%) staining for p16. HPV DNA was detected in 7/49 (14.3%) cases, but only one case was positive for both p16, and HPV DNA. A total of 36 cases yielded sufficient RNA for RT-PCR (18 verrucous carcinomas, 13 atypical verrucous carcinomas, and 5 SCC arising in verrucous carcinoma). All 36 were negative, including the four p16-positive and three HPV DNA-positive tumors tested. Although a minority of verrucous carcinoma lesions are p16 and HPV DNA positive, transcriptionally active high-risk HPV is uniformly absent. These findings argue that verrucous carcinoma and its related squamous cell carcinomas are not HPV-driven tumors.

Low rate of detection of mucosal high-risk-type human papillomavirus in Korean patients with extragenital Bowen's disease and squamous cell carcinoma, especially in digital cases.

Human papillomavirus (HPV) infection has been demonstrated in some of the nonmelanoma skin cancers as well as in precancerous lesions. Multiple infections of mucosal high-risk HPV may contribute to the onset of digital Bowen's disease through, if any, digital-genital transmission. We screened for the presence of the mucosal HPV DNA in patients with extragenital Bowen's disease (n = 30), squamous cell carcinoma (n = 11), bowenoid papulosis (n = 9), verrucous carcinoma (n = 1), actinic keratosis (n = 5), and basal cell carcinoma (n = 5). We used a PANArray HPV Genotyping Chip for high-risk and low-risk mucosal types. Genotyping data was confirmed using a conventional direct DNA sequencing method. Two cases of extragenital Bowen's disease were positive for types 16 and 33 of mucosal HPV, respectively. None of the squamous cell carcinoma cases were positive. Neither patients with digital Bowen's disease (n = 5) nor those with squamous cell carcinoma (n = 3) showed any mucosal high-risk HPV. Mucosal high-risk HPV DNA was confirmed in 5 (55.6%) of the 9 patients with bowenoid papulosis. HPV 16 was most prevalent (n = 3), while the DNA of HPVs 35 and 67 was detected in one sample for each of the two types. Our study demonstrated that two (6.7%) of the patients with 30 extragenital Bowen's disease were positive for types 16 and 33 of mucosal HPV, respectively. HPVs belonging to the mucosal high-risk group may participate in the development of extragenital Bowen's disease. However, we could not find any relationship between the mucosal high-risk HPV and Bowen's disease or squamous cell carcinoma in the fingers.

Comprehensive analysis of human papillomavirus prevalence and the potential role of low-risk types in verrucous carcinoma.

The role of human papillomavirus (HPV) infections in the development of verrucous carcinoma, a well-differentiated variant of squamous cell carcinoma with difficult differential diagnosis, is controversial in the literature. In this study, we analysed verrucous carcinoma from different origins for the presence and activity of a broad spectrum of HPV types, and carefully reviewed the histopathological features. A random series of 27 formalin-fixed, paraffin-embedded specimens of verrucous carcinoma was taken, representing the head and neck region (n=6), anogenital area (n=16) and extragenital skin region (n=5). After review of the histological slides, all samples were subjected to different polymerase chain reaction-based HPV detection techniques, together detecting a total of 83 HPV types, including both mucosal and cutaneous types. Histological revision was carefully performed. Lesions with keratinised papillae, blunt stromal invaginations and minimal cytological atypia were considered verrucous carcinoma. Condylomatous lesions with viral changes were defined as giant condyloma. Verrucous lesions that did not meet those criteria were classified as verrucous hyperplasia. Tumours with stromal infiltration were considered as invasive squamous cell carcinoma. Histological revision revealed that 13 out of 27 cases were verrucous carcinoma (one showing a double infection with HPV 35 and 45), 5 invasive squamous cell carcinomas, 5 verrucous hyperplasia (one with a double infection with HPV 4 and 8), 1 pseudoepitheliomatous hyperplasia and 3 giant condylomas. All three giant condylomas were low-risk HPV positive (HPV 6 and 11) and showed active mRNA transcription. None of the HPV-positive samples tested positive for diffuse p16(INK4A) staining. In conclusion, our results do not support a causal role of HPV in the development of verrucous carcinoma. Testing for LR-HPV, particularly HPV 6 and 11, may help in the differential diagnosis of lesions suspicious of verrucous carcinoma as those testing positive for LR-HPV most likely represent giant condylomas.