Human papillomavirus infection in head and neck cancer: the role of the secretory leukocyte protease inhibitor.

We previously showed that secretory leukocyte protease inhibitor (SLPI) gene and protein expression is significantly lower in metastatic versus non-metastatic head and neck squamous cell carcinoma (HNSCC). However, we did not assess the human papillomavirus (HPV) status of these cases. Since SLPI plays a role in HIV and herpes simplex virus (HSV) infections, we hypothesized that SLPI may be involved in HPV-infected HNSCC. In HNSCC tissue (n=54), HPV DNA was determined and correlated with SLPI expression. Additionally, to investigate a possible role of smoking on SLPI expression in clinically normal mucosa, 19 patients treated for non­malignant diseases (non-HNSCC) were analyzed for SLPI expression and correlated with smoking habits. In HNSCC patients, SLPI expression showed a significant inverse correlation with HPV status. In patients with moderate/strong SLPI expression (n=19), 10.5% were HPV-positive. By contrast, patients with absent/weak SLPI expression (n=35), 45.7% were HPV-positive. Low SLPI expression was correlated with metastasis (P=0.003) independent of HPV status. HPV-positivity was clearly associated with lymph node status (81.3% N1-3 cases). In smoking non-HNSCC patients (n=7), 42.9% showed absent/weak and 57.1% moderate/strong SLPI staining. In non-smoking non-HNSCC patients (n=10) 83.3% showed absent/weak and 16.7% moderate/strong SLPI expression. For the first time, a correlation between SLPI downregulation and HPV infection was demonstrated, suggesting that high levels of SLPI, possibly induced by environmental factors such as tobacco smoking, correlate with protective effects against HPV infection. SLPI may be a potential biomarker identifying head and neck cancer patients not at risk of developing metastases (SLPI-positive), and those at risk to be infected by HPV (SLPI-negative) and likely to develop metastases.

HPV DNA, E6*I-mRNA expression and p16INK4A immunohistochemistry in head and neck cancer - how valid is p16INK4A as surrogate marker?

It has been proposed that p16(INK4A) qualifies as a surrogate marker for viral oncogene activity in head and neck cancer (HNSCC). By analyzing 78 HNSCC we sought to validate the accuracy of p16(INK4A) as a reliable marker of active HPV infections in HNSCC. To this end we determined HPV DNA (HPVD) and E6*I mRNA (HPVR) expression status and correlated these results with p16(INK4A) staining. In tonsillar SCC 12/20 were HPVD+ and 12/12 of these showed active HPV infections whereas in non-tonsillar SCC 10/58 were HPVD+ and 5/10 showed active HPV infections. Thus, we prove about 8% of non-tonsillar SCC to be also correlated with HPV-associated carcinogenesis. Strikingly, 3/14 (21.4%) of tonsillar and non-tonsillar HPVD+/HPVR+ cases did not show p16(INK4A) overexpression and these cases would have been missed when applying initial p16(INK4A) staining only. However, in 13 cases negative for HPV, DNA p16(INK4A) was overexpressed. In conclusion, our data confirm tonsillar SCC to be predominantly but not only associated with active HPV infections. Furthermore, our data show that p16(INK4A) overexpression is not evident in a subgroup of HNSCC with active HPV infection. Definitive HPV data should therefore be utilized in diagnostics and treatment modalities of HPV positive and HPV negative HNSCC patients, resulting in a paradigm shift regarding these obviously different tumor entities.

p16(INK4a) overexpression predicts translational active human papillomavirus infection in tonsillar cancer.

The causal role of human papillomaviruses (HPV) in squamous cell carcinogenesis of tonsillar cancers (TSCC) depends on the activity of the viral oncoproteins E6 and E7, leading to inactivation of the cellular tumor suppressor p53 and the retinoblastoma gene product pRb. Because of the negative feedback mechanisms, the pRb inactivation causes an increase of the inhibitor of the cyclin-dependent kinases p16(INK4a). In 39 TSCC specimens, genotyping based on the amplification of HPV DNA was carried out using PCR by applying HPV type-specific oligonucleotides. Subsequently, amplicons were hybridised with fluorescence-labeled complementary probes using the Southern blot technology. For HPV E6/E7 mRNA expression, Northern hybridization and RT-PCR were performed, and for p16(INK4a) detection, immunohistochemistry was performed. With 21/39 (53%) HPV-positives, the detection rate is within the range that can be expected in TSCC. The E6/E7 oncogene mRNA was detectable in 11 cases, 10 of which showed positive signals after p16(INK4a) staining. Albeit the small study group was investigated, the correlation of the HPV DNA status with the p16(INK4a) expression was of statistical significance (p = 0.02). Kaplan-Meier estimations revealed better survival outcome for patients with HPV-positive tumors with detectable E6/E7 mRNA and p16(INK4a) overexpression (p = 0.02, median observation time 29 months). As mRNA expression tests are not routinely available in many clinical diagnostic laboratories, and based on the high correlation of p16(INK4a) staining with HPV E6/E7 mRNA expression, in conclusion we suggest for a deeper exploration for the use of p16(INK4a) as a surrogate marker with the potential to impact the standard of care of HPV DNA-positive head and neck carcinomas.

Human papillomavirus and p53 polymorphism in codon 72 in head and neck squamous cell carcinoma.

The impact of a polymorphism of the wild-type human tumour suppressor gene p53(wt) on carcinogenesis is subject of controversy ever since a higher susceptibility of p53 to HPV-E6 mediated degradation when encoding for Arginine at codon 72 (p53Arg) was first reported. The issue remained unclear because various studies investigating this question for different tumour entities and different geographical regions demonstrated diverging results. In the present study, the HPV status and p53 genotype frequency of 42 head and neck cancers was analysed and compared to results reported in the recent literature. Applying PCR and cycle sequencing techniques, HPV DNA was demonstrated in 12/42 (29%) of the cases and the overall distribution of the p53 allele was: 64, 31 and 5% for p53Arg, p53Arg/Pro and p53Pro, respectively. There was no statistically significant association between HPV status and p53 genotype distribution. The results of our study and of the reviewed literature do not support a relevant role of the p53 polymorphism in head and neck carcinogenesis, either taken alone or in association with the HPV status.

HPV16 DNA in histologically confirmed tumour-free neck lymph nodes of head and neck cancers.

BACKGROUND: Human papillomavirus (HPV) has been demonstrated in lymph node neck metastases (NM) of HPV-positive squamous cell carcinomas of the head and neck (HNSCC), underscoring the possible role of HPV for HNSCC progression. Reports on HPV infections in histopathologically tumour-free lymph-nodes of the SCC of the uterine cervix developing higher rates of lymph-node metastases and recurrences later in the survey of the patients was the starting point of the present study. MATERIALS AND METHODS: The presence of HPV-DNA in primary tumours (PT, n=45), NM (n=45) and histologically confirmed tumour-free neck lymph-nodes (LN, n=102) of HNSCC from 60 patients was analysed by PCR and Southern blot hybridisation. RESULTS: A highly positive correlation of simultaneous HPV-DNA detection in PT and NM was demonstrated. In the case of HPV-positivity of PT and/or NM [24/60 cases (40%)], 11/24 (45.8%) LN contained HPV-DNA, as well. Accepting HPV demonstration as a marker for the presence of micro-metastasis, HPV analysis would result in an upstaging of the N category in 4 out of these 11 patients. CONCLUSION: Considering the high agreement of HPV-DNA detection in PT and simultaneous HPV-DNA demonstration in the draining NM corroborating the monoclonal character of the tumour cells, the HPV-DNA presence in LN seems to be indicative of micro-metastasis in these lymph nodes. Thus, HPV analysis might be another powerful tool for the definition of the N-status of HPV-positive HNSCC.

Detection of human papillomavirus DNA in benign and malignant sinonasal neoplasms.

Infections with human papillomaviruses are divided basically into three different infection types: those producing specific clinically visible lesions, those remaining subclinical, and those being latent. The assumed infection type thought to be present in tissue specimens has influence on the conclusions that can be made from an analysis, i.e. whether or not the HPV infection has a causal relationship with other epidemiological or molecular investigation observations. To determine whether HPV DNA detection in different entities of the upper aerodigestive tract represents a coincidental, persistent/latent or specific infection, 20 clinically intact mucosa specimens of the upper aerodigestive tract, 20 sinonasal polyps, 26 inverted papillomas, and 20 squamous cell carcinomas of the paranasal sinuses were investigated. HPV DNA was not detectable in specimens derived from clinically intact mucosa or in nasal polyps. Yet, three out of 26 inverted papillomas were HPV-positive, each showing double infection with HPV6 and 11. Four out of 20 squamous cell carcinomas were HPV16 positive. To our knowledge, we are presenting the first study contemporaneously analyzing benign as well as malignant non-proliferative and proliferative mucosal entities whilst applying identical methodical standards. The data corroborate the hypothesis that HPV DNA demonstration in tissue specimens represents a specific infection of the mucosa of the upper aerodigestive tract. It can thus be assumed that there is a causative involvement of HPV infections in the alteration of cell proliferation and in the case of infection with high risk HPV types even on progression to malignant transformation.

Detection of human papillomavirus DNA and p53 codon 72 polymorphism in prostate carcinomas of patients from Argentina.

BACKGROUND: Infections with high-risk human papillomaviruses (HPVs), causatively linked to cervical cancer, might also play a role in the development of prostate cancer. Furthermore, the polymorphism at codon 72 (encoding either arginine or proline) of the p53 tumor-suppressor gene is discussed as a possible determinant for cancer risk. The HPV E6 oncoprotein induces degradation of the p53 protein. The aim of this study was to analyse prostate carcinomas and hyperplasias of patients from Argentina for the presence of HPV DNA and the p53 codon 72 polymorphism genotype. METHODS: HPV DNA detection and typing were done by consensus L1 and type-specific PCR assays, respectively, and Southern blot hybridizations. Genotyping of p53 codon 72 polymorphism was performed both by allele specific primer PCRs and PCR-RFLP (Bsh1236I). Fischer's test with Woolf's approximation was used for statistical analysis. RESULTS: HPV DNA was detected in 17 out of 41 (41.5%) carcinoma samples, whereas all 30 hyperplasia samples were HPV-negative. Differences in p53 codon 72 allelic frequencies were not observed, neither between carcinomas and hyperplasias nor between HPV-positive and HPV-negative carcinomas. CONCLUSION: These results indicate that the p53 genotype is probably not a risk factor for prostate cancer, and that HPV infections could be associated with at least a subset of prostate carcinomas.

Human papillomaviruses in lymph node neck metastases of head and neck cancers.

CONCLUSION: The results of this study corroborate earlier findings that human papillomavirus (HPV)16 is the most prevalent type of HPV in squamous cell carcinomas of the head and neck (SCCHNs) and reinforce a possible influence of HPV on SCCHN progression by showing that the majority of HPV-positive patients harbor HPV16 (or HPV33) both in their primary tumors and in lymph node neck metastases (LNNMs). OBJECTIVE: HPVs are causally associated with carcinomas of the uterine cervix and have also been linked to a subset of SCCHNs. In order to further investigate the predicted causative role of HPV in SCCHNs, we analyzed pairs of primary tumors and LNNMs or LNNMs alone for the presence of HPV DNA using polymerase chain reaction (PCR). MATERIAL AND METHODS: DNA was extracted from fresh frozen tissue samples of primary tumors and the corresponding LNNMs of 18 patients and from LNNMs alone in 17 patients. For the detection and typing of HPV, PCR was performed using both type-specific and consensus primer pairs, followed by Southern hybridization and, in selected cases, sequencing of the PCR products. RESULTS: Of the 35 patients investigated, 22 (63%) were found to have HPV DNA in their tumors: HPV16 DNA in 21 cases and HPV33 in 1. The highest HPV prevalence was detected in tumors of Waldeyer's tonsillar ring (8/9 patients; 89%). Of the 18 patients in whom primary tumors and LNNMs were analyzed, 7 (39%) were HPV-positive in both samples (HPV16, n = 6; HPV33, n = 1), in 3 (17%) the primary tumors were HPV-negative and the LNNMs HPV16-positive and in 1 (5.5%) the primary tumor contained HPV16 and the LNNM was negative. Interestingly, of the 7 patients in whom LNNMs had been detected only several months after diagnosis and treatment of the primary tumors, only 1 showed infection with HPV (HPV33).

Human papillomaviruses in head and neck cancer: 8 year-survival-analysis of 73 patients.

Depending on the primary tumour's anatomical location, squamous cell carcinoma of the head and neck (HNSCC) shows HPV prevalences between 20 and 30% for oro-, hypopharyngeal as well as laryngeal SCC and up to over 50% for SCC of the Waldeyer's tonsillar ring. There is persistent controversy on the role of HPV infection in HNSCC-progression, and on the influence of these infections on the final clinical outcome. To evaluate the possible relevance of HPV infection on survival and prognosis, 73 patients with HNSCC were investigated statistically with a median follow-up time of 28 (0.3-94) months. The statistical analysis revealed no differences in the overall survival of HPV-positive and HPV-negative cancer patients. A correlation between decreased survival and increased lymph node status was expected. Patients with carcinomas of the Waldeyer's tonsillar ring with a high HPV prevalence rate as compared to tumours of other anatomical locations revealed a better survival. Moreover, an association between HPV positivity and higher lymph node status at time of first diagnosis, and a better survival of HPV-positive patients compared to HPV-negative patients given the same initial nodal status (N0 vs. N1-N2b vs. N2c-N3) could be demonstrated. The influence of HPV on the patient's survival can only be observed statistically in combination with other prognostic factors, as the lymph nodal status of the patients. The better prognosis of survival of HPV-positive vs. the HPV-negative patients with lymph node neck metastasis is attributable to a better response of the HPV-positive group to therapy, especially radiotherapy.

Human papillomavirus type 16 E6 and E7 genotypes in head-and-neck carcinomas.

Human papillomavirus type 16 (HPV16) is associated with squamous cell carcinomas of the head and neck (HNSCC) particularly from the Waldeyer's tonsillar ring. A causal role of HPV16 in carcinogenesis is linked to the activity of the viral oncoproteins E6 and E7 which inactivate the cellular tumor suppressors p53 and pRB, respectively. Lack of E6 expression in HPV16-positive HNSCC has been reported, in some cases caused by disruption of the E6 gene. We have examined the status of the HPV16 E6-E7 gene region in tumor and metastasis samples of 24 HNSCC patients employing genomic PCR. No cases with a disrupted E6-E7 region could be identified. Sequence analysis of the E6-E7 segments revealed three different HPV16 E6-E7 genotypes: the HPV16 prototype (6 of 21 cases), the E6 variant T350G (8 of 21 cases), and the E6-E7 variant A131G/C712A (7 of 21 cases). The E6 variants T350G and A131G have been associated with increased oncogenic potential in cervical cancer patients depending on host genetic factors. Their high prevalence in the HNSCC samples studied indicates that they may be important also in HNSCC development.

Refined physical map of the human PAX2/HOX11/NFKB2 cancer gene region at 10q24 and relocalization of the HPV6AI1 viral integration site to 14q13.3-q21.1.

BACKGROUND: Chromosome band 10q24 is a gene-rich domain and host to a number of cancer, developmental, and neurological genes. Recurring translocations, deletions and mutations involving this chromosome band have been observed in different human cancers and other disease conditions, but the precise identification of breakpoint sites, and detailed characterization of the genetic basis and mechanisms which underlie many of these rearrangements has yet to be resolved. Towards this end it is vital to establish a definitive genetic map of this region, which to date has shown considerable volatility through time in published works of scientific journals, within different builds of the same international genomic database, and across the differently constructed databases. RESULTS: Using a combination of chromosome and interphase fluorescent in situ hybridization (FISH), BAC end-sequencing and genomic database analysis we present a physical map showing that the order and chromosomal orientation of selected genes within 10q24 is CEN-CYP2C9-PAX2-HOX11-NFKB2-TEL. Our analysis has resolved the orientation of an otherwise dynamically evolving assembly of larger contigs upstream of this region, and in so doing verifies the order and orientation of a further 9 cancer-related genes and GOT1. This study further shows that the previously reported human papillomavirus type 6a DNA integration site HPV6AI1 does not map to 10q24, but that it maps at the interface of chromosome bands 14q13.3-q21.1. CONCLUSIONS: This revised map will allow more precise localization of chromosome rearrangements involving chromosome band 10q24, and will serve as a useful baseline to better understand the molecular aetiology of chromosomal instability in this region. In particular, the relocation of HPV6AI1 is important to report because this HPV6a integration site, originally isolated from a tonsillar carcinoma, was shown to be rearranged in other HPV6a-related malignancies, including 2 of 25 genital condylomas, and 2 of 7 head and neck tumors tested. Our finding shifts the focus of this genomic interest from 10q24 to the chromosome 14 site.