Human papillomavirus 16 (HPV-16) has been implicated as a causative agent in a subset of head and neck squamous cell carcinomas (HNSCC). This study was undertaken to discern the distribution and timing of HPV viral integration during tumorigenesis of the upper respiratory tract.
A tissue array was assembled from a consecutive group of 176 patients with HNSCCs. The array was evaluated by HPV-16 in situ hybridization and p16 immunohistochemistry. Patients with HPV-positive tonsillar cancers who had undergone bilateral tonsillectomies were selected for more complete mapping of viral integration.
HPV-16 was detected in 38 of the 176 (22%) cases by in situ hybridization. When stratified by site of origin, HPV-16 was detected in 37 of 45 cancers arising from the oropharynx but in only 1 of 131 tumors arising from nonoropharyngeal sites (82% versus 0.8%, P < 0.00001). P16 expression was associated with the presence of HPV-16: 31 of 38 HPV-positive tumors exhibited p16 expression, whereas only 9 of the 138 HPV-negative tumors were p16-positive (82% versus 6%, P < 0.00001). In the bilateral tonsil sections, hybridization signals were strictly limited to the invasive cancers and associated dysplasias. P16 staining was widely distributed throughout the nonneoplastic crypt epithelium of individuals with and without tonsillar cancer.
HPV-16 is strongly associated with carcinomas arising from the oropharynx, and integration is tightly coupled to the neoplastic process. Viral integration does not occur as a field alteration throughout normal tonsillar epithelium. P16 expression localizes to HPV-positive cancers, and is intrinsic to the specialized epithelium of the tonsillar crypts. For risk assessment, early cancer detection and disease surveillance, evidence of HPV-16 integration may represent a meaningful finding, whereas high p16 expression, by itself, may not.
"HPV16 DNA is frequently detected by in situ hybridization in tonsillar squamous cell carcinoma, and expression of p16, the most commonly used marker of biologically active E6/E7, is generally confined to the crypt epithelium (Begum et al., 2005; Kim et al., 2007). However, the actual cellular targets of HPV infection in the human tonsil have not been defined (Begum et al., 2005; El-Mofty and Patil, 2006). Here we provide initial proof-of-principle evidence that the E6/E7 oncogenes encoded by HPV16 are sufficient to induce changes in the growth and differentiation control of the primitive progenitor-enriched population of CD44 + NGFR + epithelial cells present in normal human tonsillar crypts. "
[Show abstract][Hide abstract] ABSTRACT: Human palatine tonsils are oropharyngeal lymphoid tissues containing multiple invaginations (crypts) in which the continuity of the outer surface epithelium is disrupted and the isolated epithelial cells intermingle with other cell types. We now show that primitive epithelial cells detectable in vitro in 2D colony assays and in a 3D culture system are CD44 + NGFR + and present in both surface and crypt regions. Transcriptome analysis indicated a high similarity between CD44 + NGFR + cells in both regions, although those isolated from the crypt contained a higher proportion of the most primitive (holo)clonogenic cells. Lentiviral transduction of CD44 + NGFR + cells from both regions with human papillomavirus 16-encoded E6/E7 prolonged their growth in 2D cultures and caused aberrant differentiation in 3D cultures. Our findings therefore reveal a shared, site-independent, hierarchical organization, differentiation potential, and transcrip-tional profile of normal human tonsillar epithelial progenitor cells. They also introduce a new model for investigating the mechanisms of their transformation.
"This group of carcinomas shows clinicopathological and molecular characteristics that differ from alcohol- and tobacco-induced carcinomas –. Studies that have assessed the prevalence of HPV-induced OSCC report frequencies ranging from 20% to up to 90% ,–. "
[Show abstract][Hide abstract] ABSTRACT: 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 p16(INK4A) 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.
PLoS ONE 02/2014; 9(2):e88718. DOI:10.1371/journal.pone.0088718 · 3.23 Impact Factor
"Begum et al.  undertook p16 IHC and HPV DNA ISH on the contra-lateral uninvolved tonsil in eight patients with primary HPV-related tonsil carcinomas and identified p16 overexpression and ISH positivity in one of eight cases. Interestingly, and in agreement with our findings, p16 over-expression in combination with high-risk HPV DNA by ISH was associated with morphological evidence of epithelial dysplasia . These data suggest that pathologist review of pharyngeal endoscopic biopsies by conventional microscopy is likely to be sufficient to screen for multifocal HPV infection, which can then be confirmed by conventional laboratory testing (p16 IHC and high risk HPV ISH/HPV PCR). "
[Show abstract][Hide abstract] ABSTRACT: Patients with human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) have a reduced risk of developing second primary upper aerodigestive tract (UADT) tumours compared to patients with HPV-negative primary tumours at the same site. To determine whether this finding might be explained by a lack of viral-induced field cancerisation or multifocal infection, we investigated whether there was epithelial dysplasia and/or evidence of HPV infection at other pharyngeal mucosal sites in patients presenting with the disease.
Sixty-three patients with primary tonsil SCC and 108 pharyngeal endoscopic biopsies, representing at least one pharyngeal subsite from each patient, were included in this study. Tissue samples were tested using HPV PCR (GP5+/6+), p16 immunohistochemistry (IHC) and high risk HPV DNA in situ hybridisation (ISH).
There were 46 patients with HPV-related SCC and 17 patients with HPV-negative disease. PCR detected HPV DNA in a fifth of pharyngeal endoscopic biopsies and was equally likely to be from a patient with HPV-related SCC as from a patient with HPV negative disease. All PCR positive cases were tested using p16 IHC and high risk HPV ISH and only three biopsies were positive. Significantly, these three biopsies all showed evidence of epithelial dysplasia and were from patients with an HPV positive index tumour.
Our data suggest that virus-induced field cancerisation and/or multifocal oncogenic HPV infection of the pharynx is uncommon in OPSCC and supports the concept that these patients have a lower risk of developing second primary tumours of the UADT.
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