Human papillomavirus type 16 (HPV16) plays a role in the development of a subgroup of head and neck squamous cell carcinomas (HNSCC). However, uncertainty exists about the true impact of HPV in this tumor type as conflicting reports have been published with prevalence rates from 0 to 100%. We aimed to find a detection algorithm of a biologically and thus clinically meaningful infection, applicable for high-throughput screening of frozen and formalin-fixed paraffin embedded (FFPE) specimens. By considering detection of HPV E6 oncogene expression in frozen biopsies as gold standard for a meaningful HPV infection, the value of several assays was evaluated on FFPE tumor specimens and sera of 48 HNSCC patients. The following assays were evaluated on FFPE tissue samples: HPV DNA general primer (GP)5+/6+ PCR, viral load analysis, HPV16 DNA FISH detection, HPV16 E6 mRNA RT-PCR, p16 immunostaining, and on corresponding serum samples detection of antibodies against the HPV16 proteins L1, E6 and E7. Comparing single assays on FFPE tissue samples detection of E6 expression by RT-PCR was superior, but application remains at present limited to HPV16 detection. Most suitable algorithm with 100% sensitivity and specificity appeared p16 immunostaining followed by GP5+/6+ PCR on the p16-positive cases. We show that clinically meaningful viral HPV infections can be more reliably measured in FFPE HNSCC samples in a standard and high throughput manner, paving the way for prognostic and experimental vaccination studies, regarding not only HNSCC, but possibly also cancer types with HPV involvement in subgroups such as penile and anal cancer.

Oncogenic human papillomavirus (HPV) is a causative agent in a subgroup of head and neck carcinomas, particularly tonsillar squamous cell carcinomas (TSCC). This study was undertaken because controversial data exist on the physical status of HPV-DNA and the use of p16INK4A overexpression as surrogate HPV marker, and to examine the impact of HPV and tobacco consumption on the clinical course of TSCC. Tissue sections of 81 TSCC were analyzed by HPV 16-specific fluorescence in situ hybridization (FISH) Head and neck squamous cell carcinomas (HNSCC) account for 4% of all malignancies in the Western world, for up to 50% of all malignancies in Southeast Asian countries and for 6.5% of all annual cancer cases worldwide.1 HNSCC is associated with severe disease-and treatment-related morbidity and because treatment has not improved greatly in recent years, the 5-year survival rate remains 50%. HNSCC develop in various anatomical defined regions, including the oral cavity, larynx and pharynx. These organ-specific tumors each show specific clinical presentations and outcome, and are treated by different strategies. 2,3 The median age at presentation is 60 years and approximately two-third of patients are male. Well-known risk factors in the etiology of HNSCC are cigarette smoking combined with alcohol consumption in Western countries, or with betel quid chewing in Asia. A history of tobacco use is present in 90% of patients who develop oral cavity cancers. 2,3Despite these evident associations, the exact mechanisms by which these factors cause tumor initiation and progression are not fully understood. Furthermore, the fact that most tobacco and alcohol users do not develop HNSCC and that in recent years more often individuals without a history of these traditional risk factors have been witnessed, 4 underlines the complexity of HNSCC pathogenesis and a role for additional factors in the disease process.Increasing evidence suggests that human oncogenic papillomaviruses (HPVs), known to cause uterine cervical and other anogenital cancers, may also be of importance in the pathogenesis of HNSCC. 5 The strongest association has been found for oropharyngeal carcinomas, especially tonsillar carcinomas.6-11 Sero-positive patients for HPV 16 or with a history of HPV-related anogenital cancer also show increased risk rates of developing oropharyngeal cancer.12,13 The prevalence of HPV-exhibiting HNSCC, however, varies broadly amongst several studies (2-76%) due to differences in the population, combination of histological subsites, type and number of specimens analyzed, and detection methods used. 7,14 Thus, besides determining the presence of HPV DNA it has been suggested to better define the biological association of oncogenic HPV with these tumors, e.g., by means of assessing the viral copy number per cell, the viral oncoprotein E6/E7 expression levels, perturbation of pRb-dependent cell cycle control, or the physical status of the virus (episomal or integrated). 15 In this way, several reports have shown that HPV 16 is predomi...

Head and neck squamous cell carcinomas (HNSCC) account for 6.5% of annual cancer cases worldwide. During the last decades, the incidence of HNSCC has increased in Western Europe. For example, the incidence of cancer of the mouth and pharynx increased from 26.8 to 33.8 per 100,000 from 1985 to 1990. 1,2 Recent data suggest that this increase in incidence is especially high in patients younger than 40 years of age. 3,4 Median age at presentation of HNSCC is 60 years, and 66.7% of the patients are men.Tobacco smoking, alcohol drinking and betel quid chewing are well-known risk factors in the etiology of HNSCC, responsible for 90% of the cases. 5,6 The age at smoking initiation appears to be inversely associated with a higher relative risk of developing a carcinoma. 5,7 Tobacco and alcohol use are independent risk factors, but when combined a synergistic effect is observed. 5 In addition, epidemiologic and molecular data suggest that human oncogenic papillomaviruses (HPVs), known to cause cervical and other anogenital cancers, may also promote head and neck carcinogenesis. 8 -10 Discrepancy exists with respect to the percentage of HNSCC harboring HPV, as the reported frequencies in head and neck lesions vary over a wide range (2-76%). 8,11 These differences may be due to the detection method used (Southern blot hybridization, polymerase chain reaction (PCR) or in situ hybridization), the anatomic location of tumors, the type of HPV detected and/or the number of tissue samples analyzed in the various studies. 8,11 Most studies have so far concentrated on the role of HPV in the etiology of uterine cervical cancer. 12 Particularly the high-risk oncogenic HPVs, such as HPV type 16 and 18, induce preneoplastic lesions with an increased risk of progression to cancer. The transition from dysplasia to invasive cancer appears to be associated with integration of the viral DNA into the host genome, most probably at fragile sites in chromosomes. [13][14][15] Molecular studies have shown that HPV integration results in upregulation of the viral oncoproteins E6 and E7. 8,12 The E6 protein contains zincbinding motifs and can complex with the host cell p53, thereby inducing p53 degradation through the ubiquitin-mediated pathway and thus preventing a cell cycle block and induction of apoptosis in DNA-damaged cells. The E7 protein forms complexes with hypophosphorylated forms of the retinoblastoma tumor suppressor protein (pRb), resulting in a decrease of the cellular pRb level and a release of E2F, a transcription factor involved in cell cycle progression. 16,17 Since HPV inactivates both p53 and pRb, it is expected that inactivating mutations in these genes do not play an important role in HPV-infected HNSCC. This is not only the case in cervical cancers 10,12 but also in half of the HNSCC. 6 Some studies on HNSCC, however, have reported the concomitant presence of HPV DNA and p53 overexpression and/or mutation. 9,18 -21 This gives rise to the question as to whether HPV is causally related to the development of a subgroup of HN...

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 p16INK4A 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.

Purpose: Pulmonary carcinoids are well-differentiated neuroendocrine tumors showing usually a favorable prognosis. However, there is a risk for late recurrence and/or distant metastasis. Because histologic classification in typical and atypical carcinoids is difficult and its reliability to predict disease outcome varies, we evaluated three genes as potential prognostic markers, that is, orthopedia homeobox (OTP), CD44, and rearranged during transfection (RET).Experimental Design: These genes were analyzed in 56 frozen carcinoids by quantitative real-time PCR (qRT-PCR). RET was further studied by methylation and mutation analysis. Immunohistochemistry for CD44 and OTP protein expression was conducted on 292 carcinoids.Results: Low mRNA expression levels of CD44 (P ¼ 1.8e À5) and OTP (P ¼ 0.00054), and high levels of RET (P ¼ 0.025), were strongly associated with a low 20-year survival of carcinoid patients. High RET expression was not related to promoter hypomethylation or gene mutations. A direct link between gene expression and protein levels was confirmed for CD44 and OTP but not for RET. Within all carcinoids as well as atypical carcinoids, absence of CD44 protein was significantly associated with low 20-year survival (P ¼ 0.00014 and 0.00013, respectively). The absence of nuclear OTP followed by complete loss of expression was also significantly associated with unfavorable disease outcome in all carcinoids (P ¼ 5. 2 À6 ). Multivariate analyses revealed that age at diagnosis, histopathology, stage, and cytoplasmic OTP immunoreactivity were independent predictors of prognosis.Conclusions: Our study indicates that CD44 and OTP are strong indicators of poor outcome. We therefore argue for implementation of these markers in routine diagnostics in addition to histopathology to improve subclassification of pulmonary carcinoids into prognostically relevant categories.

No proof was found for the presence of HPV in tumour-free tonsil tissue, despite increased p16(INK4A) expression in a quarter of tonsil cases. Other mechanisms than HPV infection are therefore implicated in p16(INK4A) up-regulation.

Human papillomavirus (HPV) is a risk factor for the development of benign and malignant mucosal head and neck lesions. P16 INK4A is often used as a surrogate marker for HPV-infection, although there is still controversy with respect its reliability. Our aim was to determine if p16 INK4A overexpression can accurately predict both high-risk and low-risk-HPV-presence in (pre)-malignant and benign head and neck lesions. P16 INK4A immunohistochemistry was performed on paraffin-embedded tissue sections of 162 oropharyngeal squamous cell carcinomas (OPSCC), 14 tonsillar and 23 laryngeal dysplasias, and 20 tonsillar and 27 laryngeal papillomas. PCR, enzyme-immunoassay and FISH analysis were used to assess HPV-presence and type. Of the 162 OPSCC and 14 tonsillar dysplasias, 51 (31%) and 10 (71%) were HPV16-positive, respectively. All tonsillar papillomas were HPV-negative and four laryngeal dysplasias and 26 laryngeal papillomas were positive for HPV6 or 211. P16 INK4A immunohistochemistry revealed a strong nuclear and cytoplasmic staining in 50 out of 51 HPV16-positive and 5 out of 111 HPVnegative OPSCC (p < 0.0001) and in all HPV16-positive tonsillar dysplasias, whereas highly variable staining patterns were detected in the papillomas and laryngeal dysplasias, irrespective of the HPV-status. In addition, the latter lesions generally showed a higher nuclear than cytoplasmic p16 INK4A immunostaining intensity. In conclusion, our data show that strong nuclear and cytoplasmic p16 INK4A overexpression is a reliable surrogate indicator for HPV16 in OPSCC and (adjacent) dysplasias. For HPV6 or 211-positive and HPV-negative benign and premalignant lesions of the tonsil and larynx, however, p16 INK4A immunostaining is highly variable and cannot be recommended to predict HPV-presence.

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