Detection of human papillomavirus in cervical lymph nodes: a highly effective strategy for localizing site of tumor origin.

PURPOSE: Patients with head and neck squamous cell carcinoma (HNSCC) often come to clinical attention with a neck mass due to metastatic spread to lymph nodes. Treatment is dictated by the subsequent determination of primary tumor site and stage. However, the primary site remains elusive in some patients even after an exhaustive examination. Human papillomavirus type 16 (HPV-16) is an important etiologic agent for HNSCCs that arise within the oropharynx but less so for tumors from nonoropharyngeal sites. Detection of HPV-16 or a surrogate marker may be useful in localizing tumor origin in patients who present with metastatic HNSCC. EXPERIMENTAL DESIGN: We performed in situ hybridization (ISH) for HPV-16 on lymph node metastases from 68 patients with HNSCC. P16 immunohistochemistry was also performed because HPV-16 integration disrupts the retinoblastoma pathway and induces an overexpression of p16. RESULTS: HPV-16 was detected in 22 of the 68 (32%) cases by ISH. When stratified by site of origin, HPV-16 was detected in 22 of 31 (71%) metastases from the oropharynx, but in none of the 37 (0%) metastases from other sites (P < 0.001; Fisher's exact). P16 expression was associated with the presence of HPV-16 by ISH: 21 of 22 HPV-16 positive tumors exhibited p16 expression, whereas only 4 of the 46 HPV-16-negative tumors were p16 positive (95% versus 9%; P < 0.001; Fisher's exact). P16 expression in the node metastases also correlated with site of tumor origin: 24 of 31 oropharyngeal tumors were p16 positive, whereas only 1 of 37 nonoropharyngeal tumors was p16 positive (77% versus 3%; P < 0.001; Fisher's exact). CONCLUSIONS: For patients with metastatic HNSCC, detection of HPV-16 is a reliable way to establish origin from the oropharynx, either directly by ISH or indirectly by immunohistochemistry for p16 overexpression.     

Inverse relationship between human papillomavirus-16 infection and disruptive p53 gene mutations in squamous cell carcinoma of the head and neck.

PURPOSE: Squamous cell carcinomas of the head and neck (HNSCC) often harbor p53 mutations, but p53 protein degradation by the viral oncoprotein E6 may supercede p53 mutations in human papillomavirus 16 (HPV16)-positive tumors. The prevalence of p53 mutations in HPV-positive HNSCCs is indeed lower, but in some tumors these alterations coexist. The purpose of this study was to discern whether HNSCCs differ in the type of p53 mutations as a function of HPV16 status. EXPERIMENTAL DESIGN: The study was nested within a prospective multicenter study (ECOGE 4393/RTOG R9614) of patients with HNSCC treated surgically with curative intent. Tumors from one study center were used to construct a tissue microarray. The tumors were well characterized with respect to p53 mutational status. The tissue microarray was evaluated by HPV16 in situ hybridization. HPV16 analysis was also done on a select group of tonsillar carcinomas known to harbor disruptive p53 mutations defined as stop mutations or nonconservative mutations within the DNA binding domain. RESULTS: HPV16 was detected in 12 of 89 (13%) HNSCCs. By tumor site, HPV16 was detected in 12 of 21 (57%) tumors from the palatine/lingual tonsils, but in none of 68 tumors from nontonsillar sites (P < 0.00001). Both HPV16-positive and HPV16-negative HNSCCs harbored p53 mutations (25% versus 52%), but disruptive mutations were only encountered in HPV16-negative carcinomas. Of seven tonsillar carcinomas with disruptive p53 mutations, none were HPV16 positive, in contrast to HPV16-positive tonsillar carcinomas without disruptive p53 mutations (0% versus 57%; P = 0.008). CONCLUSIONS: Although HPV16 and mutated p53 may coexist in a subset of HNSCCs, HPV16 and disruptive p53 mutations seem to be nonoverlapping events. A less calamitous genetic profile, including the absence of disruptive p53 mutations, may underlie the emerging clinical profile of HPV16-positive HNSCC such as improved patient outcome.     

Comparative genomic hybridization analysis of tonsillar cancer reveals a different pattern of genomic imbalances in human papillomavirus-positive and -negative tumors

Our aim was to map and compare genomic imbalances in human papillomavirus (HPV)-positive and -negative squamous cell carcinomas of the tonsil. Twenty-five primary carcinomas were analyzed by comparative genomic hybridization. Fifteen (60%) were found to be HPV-positive by PCR, and the majority were HPV-16. There were statistically significant differences in the distribution of DNA gains and losses between the HPV-positive and -negative samples. Eleven of 15 HPV-positive samples (73%) showed gain on chromosome 3q24-qter, while only 4/10 (40%) HPV-negative samples had the same gain (p = 0.049). Furthermore, 4/10 (40%) HPV-negative samples but no HPV-positive samples had gain on chromosome 7q11.2-q22 (p = 0.017). As expected, and similar to previous studies, patients with an HPV-positive tumor had a statistically significantly better disease-specific survival than patients with an HPV-negative tumor (p = 0.002). The most common changes, e.g., gain on 3q or 8q, loss on 11q or 13 and loss on chromosome 7q in HPV-negative tumors, did not have any influence on prognosis. However the number of cases in each subgroup was limited.     

Prevalence of Human Papillomavirus and Co-Infection with Epstein-Barr Virus in Oral and Oropharyngeal Squamous Cell Carcinomas

Background: Head and neck squamous cell carcinoma is one of the most important malignancies, worldwide. Oncogenic viruses, such as human papilloma virus (HPV) and Epstein-Barr virus (EBV), are linked to these cancers and studies suggest a possible interaction between HPV and EBV during co-infections to promote oncogenesis. Nonetheless, these reports are controversial and demand more investigations in this regard. The present work to assessed the prevalence of HPV and co-infection with EBV in oral and oropharyngeal squamous cell carcinomas. Methods: Formalin-fixed paraffin-embedded tissues were collected from 166 archived oral and oropharyngeal squamous cell carcinoma samples from Ahvaz Imam Khomeini hospital, Ahvaz, Iran, from March 2013 and December 2019. Nested-PCR was used to detect the viruses and type-specific PCR/nested-PCR and sequencing were performed for virus genotyping. Results: Out of the 166 specimens, 84.33% and 16.42% were from oral cavity and oropharynx, respectively; of which, 32 cases (19.3%) were HPV-positive (16.42% of oral cavity and 34.6% of oropharynx). HPV was detected in 36.36%, 25%, and 16.42% of base of tongue, tonsil, and oral tongue tumors, respectively. HPV was more associated with well differentiated tumors (24;18.04%) in compared to moderately and poorly differentiated ones. Regarding HPV-16 genotyping, 7 (21.8%) out of the 32 samples were found to be HPV-16 (4/26 (15.38%) for oropharynx and 3/140 (2.14%) for oral cavity). Moreover, 90 samples were evaluated for EBV infection and co-infection; of which, 4 (4.4%) subjects tested positive for EBV, including two cases with HPV co-infection. All the positive cases were EBV type B, from oral cavity, and histologically well differentiated. Conclusions: HPV was more associated with oropharyngeal cancer. This association has been linked to various factors such as repeated oral and oropharyngeal exposure to HPV due to change in patterns of sexual behaviors; a phenomenon that may demand routine HPV vaccination.     

A subset of head and neck squamous cell carcinomas exhibits integration of HPV 16/18 DNA and overexpression of p16INK4A and p53 in the absence of mutations in p53 exons 5-8

Besides well-known risk factors such as tobacco use and alcohol consumption, oncogenic human papillomavirus (HPV) infection also has recently been suggested to promote head and neck tumorigenesis. HPV is known to cause cancer by inactivation of cell cycle regulators p53 and pRb via expression of viral oncoproteins E6 and E7. This indicates that p53 mutations are not a prerequisite in HPV-induced tumor development. However, discrepancy exists with respect to the frequency of head and neck squamous cell carcinomas (HNSCC) harboring DNA of oncogenic HPV and the fraction of these tumors showing p53 mutations. In our study, we examined the frequency of HNSCC demonstrating HPV 16/18 integration as identified by fluorescence in situ hybridization (FISH) and investigated their p53 (mutation) status by immunohistochemistry and single-strand conformation polymorphism (SSCP) analysis of exons 5-8. Paraffin-embedded, archival biopsy material from 27 premalignant mucosal lesions and 47 cases of HNSCC were analyzed. Ten of the 47 (21%) HNSCC unequivocally exhibited HPV 16 integration, including 8 of 12 (67%) tonsillar carcinomas. This is supported by the immunohistochemical detection of p16(INK4A) overexpression in all 10 HPV-positive tumors. Although FISH is considered to be less sensitive than PCR-based methods for HPV detection, our data clearly demonstrate clonal association of HPV with these tumors, as illustrated by the presence of integrated HPV 16 in both the primary tumor and their metastases in 2 patients. In contrast, HPV 16/18 DNA could not be detected in the premalignant lesions. In 30 of 47 (64%), HNSCC accumulation of p53 was observed, including 8 of the 10 HPV-positive carcinomas. However, in none of the latter cases could mutations in exons 5-8 be identified, except for a polymorphism in codon 213 of exon 6 in one patient. Evaluation of clinical data revealed a significant inverse relation between tobacco use with or without alcohol consumption, and HPV positivity of the tumors.     

HPV检测原发灶不明颈淋巴结转移癌的诊断作用初探(附6例报告)

目的 初步探讨HPV检测在诊断原发灶不明的颈淋巴结转移性鳞癌中的价值。方法 收集复旦大学附属肿瘤医院6例首诊为原发灶不明的颈淋巴结转移性鳞癌,最终确诊为HPV相关口咽鳞癌,分析确诊过程。结果 首诊时6例患者均被确诊为颈淋巴结转移性鳞癌,且p16表达阳性和HPV-16亚型阳性,而EBER表达阴性。经全面检查均未发现明显的原发病灶。随后对其中4例予同侧扁桃体盲检(2例)和切除(2例),病理均证实为扁桃体鳞癌。另2例分别在随访第149天、545天MRI检查发现同侧口咽侧壁和舌根增厚伴强化,经活检分别证实为扁桃体鳞癌、舌根鳞癌。结论 对于原发灶不明的HPV阳性颈淋巴结转移性鳞癌,应高度怀疑原发病灶来源于口咽部位的可能性,特别是扁桃体和舌根部位。     

Real-time quantitative PCR demonstrates low prevalence of human papillomavirus type 16 in premalignant and malignant lesions of the oral cavity.

PURPOSE: Human papillomavirus (HPV) type-16 has been associated with invasive squamous cell carcinoma of the head and neck. This study examines the role of HPV-16 in the progression of oral head and neck cancer by determining the quantity of HPV-16 DNA in premalignant and malignant lesions, using real-time quantitative PCR, to more accurately determine the role of HPV-16 in oral head and neck squamous cell carcinogenesis. EXPERIMENTAL DESIGN: We examined 102 microdissected premalignant head and neck lesions (85 from the oral cavity), 34 invasive oral cavity squamous cell carcinomas, as well as 18 invasive tumors known to be HPV positive by traditional molecular technology for the presence of HPV-16 DNA using real-time quantitative PCR. RESULTS: HPV DNA was detected in 1 of 102 premalignant lesions (0.98%), 1 of 34 (2.9%) invasive oral cavity carcinomas, and 14 of 18 (78%) known HPV-positive tumors. CONCLUSIONS: HPV-16 infection and integration is seldom found in oral premalignant lesions and invasive carcinoma, and therefore rarely contributes to malignant progression in the oral cavity. Furthermore, quantitative PCR is a useful technique that reliably excludes contaminated samples and those with minimal HPV DNA content that is unlikely to be significant in carcinogenesis.     

Human papillomavirus in lung carcinomas among three Latin American countries

The presence of human papillomavirus (HPV) genome in lung carcinomas has been reported worldwide but its frequency varies from country to country. We examined HPV genome in 36 lung carcinomas, consisting of 14 squamous cell carcinomas, 13 adenocarcinomas, and 9 small cell carcinomas, collected from Colombia, Mexico and Peru. PCR analysis using GP5+/GP6+ primers, combined with Southern blot hybridization, found the presence of HPV genome in 10 (28%) of 36 cases. This percentage is similar to the value of 22% reported by Syrj?nen, who conducted a meta-analysis of nearly 2500 lung carcinomas examined to date. Genotype analysis revealed that the most predominant genotype was HPV-16 (7 cases), followed by HPV-18 (2 cases) and HPV-33 (1 case). HPV-16 was more frequently found among female than male cases (P=0.008) but was not detected in any adenocarcinoma cases. On the other hand, HPV-18 and HPV-33 were detected only among male cases. These HPV genotypes were detected only in adenocarcinomas, and all the HPV genotypes detected in this histological type were HPV-18 or HPV-33. The frequency of HPV-16 positive cases among all the HPV positive cases differed in the sexes (P=0.033) and differed in the three histological types (P=0.017). The presence of HPV tended to be more frequent in well-differentiated tumors when squamous cell carcinomas and adenocarcinomas were combined. However, it was not statistically significant (P=0.093). Neither p16 nor p53 expression in carcinoma cells was related to the proportion of HPV-positive cases. In conclusion, high-risk HPV DNA was detected in 28% of lung carcinomas. The predisposition of HPV-16 to female cases and to non-adenomatous carcinomas warrants further investigation.     

HPV integration begins in the tonsillar crypt and leads to the alteration of p16, EGFR and c-myc during tumor formation

The prevalence of human papillomavirus (HPV) infection is high in the oropharyngeal mucosal regions, of which the tonsil is the most commonly affected. There may be a link between HPV and the pathogenesis of tonsillar cancer (TC), because of common anatomical characteristics between cervical and tonsillar cancer. We aimed to clarify whether HPV directly affects the oncogenesis and biologic behavior of TC by making a comparison between infection prevalence, physical status and viral loading numbers, and clinicopathologic prognostic factors. To compare HPV-related molecules between TC and tonsillitis (CFT), p16, survivin, HIF-1alpha, skp-1, cyclin A, cyclin B1, c-myc and EGFR were investigated. We observed a significant difference in HPV prevalence between 52 TCs and 69 CFTs (73.1% vs. 11.6%), and most of the HPVs were type 16 (87.2%) and nonepisomal (94.1%). Most TCs associated with HPV arose from the tonsillar crypts, and tended to be inverted and poorly differentiated. Compared with HPV-negative TC, HPV-positive TC showed a strong association with p16 overexpression (p<0.0001), and an inverse association with EGFR amplification (p=0.0478). HPV-16 integration status was strongly associated with c-myc amplification (p=0.034) and HIF-1alpha overexpression (p=0.022). HPV-16 integration could be directly related to tonsillar carcinogenesis initially in tonsillar crypts, followed by cell cycle aberration such as p16 overexpression related to the G1-S phase.     

High-risk human papillomavirus E7 oncoprotein detection in cervical squamous cell carcinoma.

PURPOSE: Persistent infections by high-risk human papillomavirus (HPV) types are the main etiologic factor for cervical cancer. The objective of this study was to evaluate whether high-risk E7 oncoprotein is adequate as a marker for the detection of cervical cancer. EXPERIMENTAL DESIGN: HPV typing was done in biopsies from 58 cervical carcinoma and 22 normal cervical squamous epithelia. The HPV-16 E7, HPV-18 E7, and HPV-45 E7 oncoprotein levels were monitored by immunohistochemistry and compared with those of p16(INK4a) and Ki67. RESULTS: Fifty-five (94.8%) tumors were high-risk HPV-DNA-positive (46 HPV-16, 2 HPV-16 and HPV-18, 4 HPV-18, 1 HPV-33, and 2 HPV-45). HPV-DNA could not be detected in three tumors (5.2%). High HPV E7 oncoprotein levels were shown in 57 cervical cancers (98.3%), without correlation between expression levels and tumor stages. CONCLUSION: This is the first study which systematically analyzes the levels of the major HPV oncoproteins in cervical carcinomas demonstrating that the high-risk HPV E7 proteins are regularly expressed in these cancers. This suggests that high-risk E7 oncoproteins are necessary for cervical cancers and apparently essential as tumor marker.     

Methylation Status of P16Ink4a in Human Papillomavirus-Associated Cancer of Oral Cavity and Oropharynx in Northeastern Thailand

Background: Over-expression of p16INK4a protein is a biomarker for human papillomavirus (HPV)-associated cervical cancer. However, absence of p16INK4a protein expression in HPV-associated cancer of the oral cavity and oropharynx has been reported. Among a number of possible reasons for this is methylation, which is frequently noted in the promoter region of p16INK4a and is associated with silencing of the gene and disease severity. Methods: We investigated the relationships between p16INK4a protein expression, HPV infection and methylation status of the p16INK4a promoter in cancers of the oral cavity and oropharynx. Fifty-three formalin-fixed paraffin-embedded (FFPE) cancer tissue samples from the oral cavity (49 cases) and oropharynx (4 cases) were studied. P16INK4a protein expression was determined using immunohistochemical staining (IHC). Additional oral tissues lacking squamous intraepithelial lesions (SILs), and cervical tissues with high-level SILs, were used as negative and positive controls, respectively. High-risk HPV infection was detected using HPV E6/E7 mRNA in situ hybridization. Methylation status of the p16INK4a promoter was investigated using sodium bisulfite treatment and methylation-specific PCR (MS-PCR).Results: HPV infection was found in 40.8% (20/49) and 50.0% (2/4) of oral cavity and oropharynx cancers, respectively. Promoter methylation of p16INK4a occurred in 73.6 % of all cases and differed significantly in frequency between HPV-positive (90.9%, 20/22) and HPV-negative (61.3%, 19/31) samples. Expression of p16INK4a was found in 35.8% (19/53) and commonly detected in samples with p16INK4a unmethylation (79.5%). Interestingly, the silencing of p16INK4a (64.2%, 34/53) was significantly associated with methylation status (91.2%, 31/34), especially in HPV-infected samples in which the p16INK4a promoter was methylated (52.9%, 18/34). Conclusions: This result demonstrated high frequency of p16INK4a promoter methylation status in HPV-associated HNSCC subsets that could influence the silent p16INK4a expression and might promote disease severity.     

人乳头瘤病毒１６型感染与非小细胞肺癌的相关性研究

目的　了解人乳头瘤病毒１６型（ＨＰＶ-１６）感染与非小细胞肺癌（ＮＳＣＬＣ）发生、发展及预后的相关性。方法应用免疫组织化学技术（ＳＡＢＣ法）检测ＨＰＶ １６在４３例ＮＳＣＬＣ组织和２７例肺良性病变组织中的表达情况,结合临床病理资料和随访资料进行回顾性研究。结果　ＨＰＶ-１６在ＮＳＣＬＣ组织中阳性表达率为３９.５％（１７／４３）,高于肺良性病变的７.４％（Ｐ＜０.０１）。ＮＳＣＬＣ组织ＨＰＶ-１６表达与患者的性别、年龄、肿瘤部位、肿瘤大小、病理类型、淋巴结转移、ＴＮＭ分期、吸烟与否无关（Ｐ＞００５）。ＨＰＶ-１６阳性和阴性表达的ＮＳＣＬＣ患者的术后３年、５年生存率分别为３９.３％、３４.３％和５９.９％、４４.７％（Ｐ＞０.０５）。结论　ＨＰＶ-１６感染对ＮＳＣＬＣ的发生可能起到一定的作用。     

Prevalence and physical status of human papillomavirus in squamous cell carcinomas of the head and neck

Fresh-frozen biopsies were obtained from 61 patients at diagnosis of squamous cell carcinoma of the head and neck (HNSCC) for study of the prevalence and physical status of human papillomavirus (HPV) DNA. The frequency of HPV DNA and genotypes were determined by SPF10 PCR screening with a general probe hybridization and INNO-LiPA HPV genotyping assay. In addition, a single-phase PCR with primers FAP 59/64 and a nested PCR with primers CP 65/70 and CP 66/69 served to detect particularly cutaneous HPV types. By the sensitive SPF10 PCR and INNO-LiPA assay, 37 of 61 (61%) samples were positive for HPV. HPV-16 was the most frequently detected type (31 of 37, 84%). Multiple infections were found in 8 of 37 (22%) of the HPV-positive samples, and co-infection by HPV-16 and HPV-33 was predominant. No cutaneous HPV types were detected. Patients with HPV-positive tumors had similar prognosis as those with HPV-negative ones. Real-time PCR analysis of the HPV-16 positive samples indicated the presence of integrated (11 of 23, 48%), episomal (8 of 23, 35%) and mixed forms (4 of 23, 17%) of HPV DNA. The viral load of HPV DNA exhibited large variation. The median copy numbers of E6 DNA in tonsillar specimens were approximately 80,000 times higher than that in nontonsillar HNSCC ones. Patients with episomal viral DNA were more frequently found to have large (T3-T4) tumors at diagnosis than were those with integrated or mixed forms.     

Decreased p21 Expression in HPV-18 Positive Cervical Carcinomas

The aim of this study was to investigate the relationship between different human papillomaviruse (HPV) genotypes and the expression of p53, p21 and p27 in cervical carcinomas. A total of 103 cases of cervical carcinomas were assayed for expression of p53, p21 and p27 by immunohistochemistry. HPV typing was carried out by two polymerase chain reaction-based methods. Overall, HPV prevalence was 97.1% among the cervical carcinomas in this study. HPV-16 was detected in 66% of the tumors, HPV-18 in 7.8%, HPV-16/18 in 7.8% and other HPV types in 15.5%. The expression of p53 and p27 was not related to HPV genotype. However, in the HPV-18 positive cervical carcinomas, expression of p21 was significantly decreased or completely absent (P = 0.019). Our results indicated that down-regulation of p21 was strongly associated with HPV-18 positive cervical carcinomas. The significantly lower expression of p21 protein in HPV-18 positive samples compared to HPV-18 negative cervical carcinomas supports the hypothesis that inactivation and degradation of p21 proteins by HPV-18 E7 may play an important role in the carcinogenesis of HPV-18 positive cervical neoplasia.     

Differential Biomarker Expression in Head and Neck Cancer Correlates with Anatomical Localization

We tested the expression of known (p16^ink4, Ki67, p53, EGFR) and a new immunohistochemical (collagen XVII/BP180) biomarker in head and neck squamous cell carcinomas (SCC) of diverse anatomical localization. Tissue microarrays (TMA) of 124 SCC were created, immunostained, and analyzed following whole slide digitalization using the Pannoramic Scan and the TMA Module software (3DHISTECH Kft, Budapest, Hungary). Statistical analysis of scoring results was carried out using Pearson’s chi-square test. We observed the significant elevation of p16^ink4 and Ki67 expression in supraglottic, tonsillar and tonsillo-lingual SCCs compared to those affecting the oral cavity, oropharynx without tonsils, larynx without supraglottis and the hypopharynx. This differential antigen expression may reflect the diverse route of embryologic differentiation followed by the affected regions except those of the tonsils and the supraglottis which show similar antigenic pattern but diverse developmental path. All the other biomarkers tested including p53, collagen XVII and EGFR were detected in the majority of cancers including high grade cases, but did not reveal any significant regional difference. Based on our results oropharyngeal squamous cell carcinomas may not be regarded as one entity. Concerning the oral cavity and the oropharynx, cancers affecting the tonsils (palatine and lingual) show significantly elevated p16^ink4 and Ki67 expression; so as the cancers of the supraglottis compared to the rest of larynx. Consequently, tonsillar and supraglottic cancers show similar biomarker profiles. Correlation of differential biomarker expression with diverse biological behavior in head and neck cancers need further investigations.     

Does pretreatment seropositivity to human papillomavirus have prognostic significance for head and neck cancers?

BACKGROUND: Human papillomavirus (HPV) is a risk factor for head and neck cancers (HNC), yet HPV-associated tumors have better prognosis than HPV-negative tumors. METHODS: We evaluated whether pretreatment presence of antibodies to HPV capsids [virus-like particles (VLP)] or to HPV-16 oncoproteins E6 and E7 was a predictor of HPV-positive HNC and clinical outcomes. Sera from 156 HNC patients were tested for antibodies to HPV-16-derived antigens using ELISA. HPV-16 in tumors was evaluated by PCR and DNA sequencing. RESULTS: HPV-16 antibodies were found in 33% with HPV-16 VLP, 21% with HPV-16 E6, and 21% with E7. HPV-16 was detected in 26% of tumors. There was a strong correlation between detection of HPV-16 tumor DNA and antibodies to HPV-16 E6 or E7 (kappa = 0.7) but not to HPV-16 VLP (kappa = 0.4). Multivariate analyses showed significantly better disease-specific survival in seropositive HPV-16 VLP [hazard ratio (HR), 0.4; 95% confidence interval (95% CI), 0.1-0.9], HPV-16 E6 (HR, 0.1; 95% CI, 0.02-0.5), and HPV-16 E7 (HR, 0.3; 95% CI, 0.1-0.9) cases. Less disease recurrence occurred among those with antibodies to both E6 and E7 compared with those negative to both (P = 0.003). There was better disease-specific survival in patients who were E6 positive at baseline and remained positive at follow-up compared with individuals who were E6 negative at both time points (P = 0.03; kappa = 0.9). CONCLUSIONS: The presence of antibodies to HPV-16 E6 and E7 is associated with HPV in tumor cells and with better clinical outcomes. These findings suggest that the presence of E6/E7 antibodies before treatment is predictive of better clinical outcomes and that they may serve as biomarkers for selecting targeted therapeutic modalities developed for HPV-associated tumors.     

Human papillomavirus type 16 is episomal and a high viral load may be correlated to better prognosis in tonsillar cancer

The aim of our study was to investigate the physical state and the viral load of HPV-16 in tonsillar cancer and to correlate these findings with clinical outcome. To distinguish between integrated and episomal forms of HPV, 22 fresh-frozen tonsillar cancer samples were analysed by a method based on restriction enzyme cleavage, ligation and PCR (rliPCR). HPV-16 was detected in 11/22 and HPV-33 in 1/22 of the cancers, hence 12/22 (55%) of the tumours were HPV positive. Only extrachromosomal forms of HPV-16 were observed. Full-length episomal HPV was detected exclusively in 7/11 of the cancers, whereas both full-length and deleted forms of episomal HPV-16 were found in parallel in 2 other tumours. In 1 tumour only a deleted episomal form of HPV-16 was present. In the remaining HPV-16 positive tumour both full-length episomal as well as an 11 kbp PCR product were detected and if the 11 kbp product contained integrated HPV, or was off-size linearised episomal could not be determined. In 2 cervical cancer controls, HPV-16 was integrated and could be chromosome located. HPV-16 was quantified by real-time PCR and most tonsillar cancers contained between 10 to a few hundred copies of HPV per beta-actin. The 6 patients with tumour sections with > or =190 HPV-16 copies/beta-actin remained tumour free (p = 0.026) and had a better survival rate (p = 0.039) when compared to the 5 patients with tumours sections with < or =60 HPV-16 copies/beta-actin. In conclusion, HPV-16 is mainly episomal in tonsillar cancer. The viral load showed a wide distribution and the clinical outcome in our study was better when the HPV load was higher.     

Human papillomavirus type 16 and squamous cell carcinoma of the head and neck.

PURPOSE: Human papillomavirus (HPV) has previously been reported to be associated with squamous cell carcinoma of the head and neck. Our objective was to investigate the presence and type of HPV infection in head and neck tumors and determine whether infection was associated with individual tumor characteristics, patients' pattern of tobacco and alcohol exposure, or with clinical outcome. Experimental Design: Using a case series design, fresh tumor samples were obtained from a series of 89 head and neck squamous cell carcinoma patients, including 64 men and 25 women. The majority of tumors were located in the oral cavity, followed by the oropharynx. A PCR-based technique with restriction fragment length polymorphism analysis was used to detect and type HPV. RESULTS: Of the 89 patients, 18 (20%) had detectable HPV 16 in their tumor samples. HPV 16 was detected in 64% of tonsil tumors, 52% oropharyngeal tumors, and 5% oral cavity tumors. The mean age of subjects with HPV 16-positive tumors was younger than the patients with HPV-negative tumors. Also, this group consumed less alcohol on a weekly basis and had a better clinical outcome compared with the HPV-negative group. Smoking, clinical stage, tumor grade, and tumor-node-metastasis status were not associated with HPV 16 presence. CONCLUSIONS: Our study supports the previous reports that suggest HPV 16 is associated with squamous cell cancers located in the oropharynx and oral cavity. The fact that HPV-positive tumors were observed in younger, lighter alcohol-consuming individuals with a better overall and disease-specific survival suggests a distinct disease process in these patients.     

Human papillomavirus detected in female breast carcinomas in Japan

To investigate the aetiological role of human papillomavirus (HPV) in breast cancer, we examined the presence, genotype, viral load, and physical status of HPV in 124 Japanese female patients with breast carcinoma. Human papillomavirus presence was examined by PCR using SPF10 primers, and primer sets targeting the E6 region of HPV-16, -18, and -33. The INNO-LiPA HPV genotyping kit was used to determine genotype. Human papillomavirus DNA was detected in 26 (21%) breast carcinomas. The most frequently detected HPV genotype was HPV-16 (92%), followed by HPV-6 (46%), HPV-18 (12%), and HPV-33 (4%). In 11 normal epithelium specimens adjacent to 11 HPV-16-positive carcinomas, 7 were HPV-16-positive. However, none of the normal breast tissue specimens adjacent to HPV-negative breast carcinomas were HPV-positive. The real-time PCR analysis suggested the presence of integrated form of viral DNA in all HPV-16-positive samples, and estimated viral load was low with a geometric mean of 5.4 copies per 10(4) cells. In conclusion, although HPV DNA was detected in 26 (21%) breast carcinomas and, in all HPV-16-positive cases, the HPV genome was considered integrated into the host genome, their low viral loads suggest it is unlikely that integrated HPV is aetiologically involved in the development of Japanese breast carcinomas that we examined.     

Age, sexual behavior and human papillomavirus infection in oral cavity and oropharyngeal cancers

There are few well-established patient risk factors associated with human papillomavirus (HPV) infection in cancers of the oral cavity and oropharynx. The purpose of this study was to determine if there were significant different risk factors and tumor characteristics between HPV-positive and HPV-negative cancer cases. HPV was evaluated in cancer tissue and exfoliated oral cells of 193 oral cavity/oropharynx cancer patients using PCR and direct DNA sequencing. A patient questionnaire collected information about risk factors, sexual practices and medical history. The prevalence of HPV high-risk (HR) types was 20% in cancer cases. Three types were identified: HPV-16 (87%), HPV-18 (3%) and HPV-33 (11%). Risk factors for HPV-HR included younger age (< or = 55 years vs. > 55 years; adjusted OR = 3.4; 95% CI = 1.6-7.3) and younger-age cases who had more lifetime sex partners (adjusted OR = 3.8; 95% CI = 1.4-10.1), practiced oral-genital sex (adjusted OR = 4.3; 95% CI = 1.8-10.4) or oral-anal sex (adjusted OR = 19.5; 95% CI = 3.4-113). Compared to HPV-negative cancers, HPV-HR cancers were more likely to have a positive HPV-HR exfoliated oral cytology test (adjusted OR = 7.8; 95% CI = 3.4-18.4), later stage (adjusted OR = 3.0), nodal involvement (adjusted OR = 4.1) and advanced grade (adjusted OR = 3.0). This study shows new evidence that the prevalence of oncogenic mucosal HPV is higher in younger-age oral cavity/oropharynx cancer cases whose sexual practices are typically associated with sexual transmission of the virus. HPV detection also appears to be an indicator of advanced disease characteristics that may require different clinical treatment for this subset of patients. An exfoliated oral cytology test for HPV was a significant predictor of HR types in the cancers, suggesting that an oral rinse may provide an early biomarker of infected tumors.