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.     

Systemic and local human papillomavirus 16-specific T-cell immunity in patients with head and neck cancer

Squamous cell carcinomas of the head and neck (HNSCC), in particular those of the oropharynx, can be caused by human papilloma virus Type 16 (HPV16). Whereas these HPV-induced oropharyngeal carcinomas may express the HPV16 E6 and E7 oncoproteins and are associated with better survival, the nonvirally induced HNSCC are associated with overexpression of p53. In this study we assessed the presence of systemic and local T cells reactive against these oncoproteins in HNSCC. An exploratory study on the presence, type and function of HPV16- and/or p53-specific T cells in the blood, tumor and/or metastatic lymph node as measured by several immune assays was performed in an unselected group of 50 patients with HNSCC. Tumor tissue was tested for HPV DNA and the overexpression of p53 protein. Almost all HPV16+ tumors were located in the oropharynx. Circulating HPV16- and p53-specific T cells were found in 17/47 and 7/45 tested patients. T cells were isolated from tumor cultures and/or lymph nodes of 20 patients. HPV16-specific T cells were detected in six of eight HPV+ tumors, but in none of the 12 HPV-tumors. Tumor-infiltrating p53-specific T cells were not detected. In depth analysis of the HPV16-specific T-cell response revealed that this response comprised a broad repertoire of CD4+ T-helper Type 1 and 2 cells, CD4+ regulatory T cells and CD8+ T cells reactive to HPV16. The local presence of HPV16-specific T-cell immunity in HPV16-induced HNSCC implicates a role in the antitumor response and support the development of immunotherapy for HNSCC.     

High-risk human papillomavirus in oral squamous cell carcinoma of young patients

The aim of this study was to investigate a possible relation between oral squamous cell carcinoma (SCC), the presence of high-risk human papillomavirus (HR-HPV) DNA and p16 expression in young patients. Paraffin-embedded tumor blocks from 47 oral SCC of young (≤40-year old) patients were evaluated. The presence of HPV DNA in tumor specimens was analyzed by polymerase chain reaction (PCR) using GP5+/GP6+ generic primers (L1 region) followed by dot blot hybridization for HPV typing. When necessary, the HPV16 positivity was confirmed by PCR HPV16 E7-specific primers. Cases involving young patients were compared with 67 oral SCC from patients ≥50-year old (controls). Demographic and clinical data were collected to analyze patient outcomes. p16(ink4) expression was evaluated by immunostaining of tissue microarrays. HPV16 was detected in 22 (19.2%) cases; 15 (68.2%) young and 7 (31.8%) control patients, a statistically significant difference (p = 0.01). In 1 (1.7%) young group specimen, HPV DNA 16 and 18 was detected. p16 expression was observed in 11 (25.6%) cases from the young group and in 11 (19.6%) controls (p = 0.48). Association between HPV and p16 was verified, and it was statistically significant (p = 0.002). The higher prevalence of high-risk HPV types, especially HPV16, may be a contributing factor to oral carcinogenesis in younger individuals.     

Lack of Association between High-risk Human Papillomaviruses and Oral Squamous Cell Carcinoma in Young Japanese Patients

Background: Human papillomaviruses (HPV) may play an important role as one of the possible etiologiesof oral squamous cell carcinoma (OSCC). The present study aimed to investigate the association between HPVand OSCC in young Japanese patients by examining the presence of HPV DNA and surrogate markers in OSCCtissues. Materials and Methods: Forty young patients with OSCC whose surgical specimens were available wereanalyzed and compared with 40 patients randomly recruited from a pool of patients aged >40 years. HPV DNAwas detected using the polymerase chain reaction-based AMPLICOR® HPV test, and surrogate markers ofHPV infection were analyzed using immunohistochemical techniques to detect p16INK4a and p53. Results: Onlytwo (5%) young patients and one (2.5%) older patient were positive for HPV DNA. p16INK4a overexpressionwas identified in six (15%) young patients. p53 staining levels were not high in tissues of most young patients(27 patients, 67.5%). HPV DNA status did not significantly correlate with p16INK4a expression levels. Profiles ofincreased levels of p16INK4a expression with diminished levels of p53 staining were not associated with the presenceof HPV DNA. The combined p53 with p16INK4a profiles were significantly correlated with alcohol consumptionin younger patients (p=0.006). Conclusions: Results of the present study indicate that HPV is less likely to causeOSCC in young Japanese patients, and the p16INK4a expression level is not an appropriate surrogate marker forHPV infection in OSCC.     

p53 and p16ink4a As Predictive and Prognostic Biomarkers for Nodal metastasis and Survival in A Contemporary Cohort of Penile Squamous Cell Carcinoma

BackgroundHuman papilloma virus (HPV) infection is implicated in a proportion of invasive squamous cell carcinoma of the penis (PC). A subset of PC involves dysregulation of the p53 pathway. HPV in situ hybridization (ISH) and p16^ink4a positivity are surrogate markers for HPV infection, and p53 immunohistochemistry (IHC) denotes abnormality in the p53 pathway. There remains an ambiguity with regard to the contribution of both the pathways in the prognosis of PC. We sought to analyze the clinicopathologic characteristics of a cohort of Indian PC patients with respect to p16 ^ink4a and p53 expression.Patients and MethodsA cohort of 123 PC patients was studied for p16^ink4aand p53IHC and HPVISH. The results of these biomarkers were correlated with various clinicopathologic parameters.Resultsp16^ink4a and HPV ISH were positive in 47% and 53% of the tumors, respectively. The proportion of warty, basaloid, or mixed warty-basaloid tumor subtypes showed significant p16^ink4apositivity (P < .0001) compared to other subtypes. Twenty-eight patients were dual negative (p53- /p16^ink4a-), 32 were dual positive (p53+/p16^ink4a+), 38 were p53+/p16^ink4a-, and 25 were p53-/p16^ink4a +. In patients where p16^ink4a was negative, a p53-positive phenotype had a higher propensity for lymph node metastases (OR, 5.42; 95% CI, 1.75-16.80; P = .003). Similarly, p53 positivity dictates nodal involvement in the p16^ink4a-positive subset of tumors (OR, 5.00; 95% CI, 1.23-20.17; P = .024). On multivariate analyses, pathologic subtypes (warty, warty-basaloid, and basaloid) (P < .0001), p16^ink4aexpression (P < .0001), and absence of nodal metastasis (P < .0001) were significant predictors of improved overall (OS) and cancer specific survival (CSS). In Kaplan-Meier analysis, the OS was significantly longer in patients with p16^ink4a + tumors (P < .0001), as was the CSS (P < .0001). Patients with dual positive tumors had a significantly higher OS (P < .001) and CSS (P = .012), in the entire cohort. In the node positive patients, dual positivity was associated with significantly higher OS (P < .0001); however, the median CSS for p53+/p16^ink4a+tumors were not significantly different compared to p53- /p16^ink4a- tumors (P = .064), although there was a trend towards improved CSS.ConclusionsThere is a strong concordance between p16^ink4aIHC and HPV ISH results. p16^ink4a status is an independent predictor of survival (OS and CSS) in our cohort of PCs. p53 is a predictor of nodal metastasis irrespective of p16 status. Dual positive tumors have a significantly better outcome in comparison to dual negative tumors;     

HPV16 DNA status is a strong prognosticator of loco-regional control after postoperative radiochemotherapy of locally advanced oropharyngeal carcinoma: Results from a multicentre explorative study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG)

ObjectiveTo investigate the impact of HPV status in patients with locally advanced head and neck squamous cell carcinoma (HNSCC), who received surgery and cisplatin-based postoperative radiochemotherapy.Materials and methodsFor 221 patients with locally advanced squamous cell carcinoma of the hypopharynx, oropharynx or oral cavity treated at the 8 partner sites of the German Cancer Consortium, the impact of HPV DNA, p16 overexpression and p53 expression on outcome were retrospectively analysed. The primary endpoint was loco-regional tumour control; secondary endpoints were distant metastases and overall survival.ResultsIn the total patient population, univariate analyses revealed a significant impact of HPV16 DNA positivity, p16 overexpression, p53 positivity and tumour site on loco-regional tumour control. Multivariate analysis stratified for tumour site showed that positive HPV 16 DNA status correlated with loco-regional tumour control in patients with oropharyngeal carcinoma (p = 0.02) but not in the oral cavity carcinoma group. Multivariate evaluation of the secondary endpoints in the total population revealed a significant association of HPV16 DNA positivity with overall survival (p < 0.01) but not with distant metastases.ConclusionsHPV16 DNA status appears to be a strong prognosticator of loco-regional tumour control after postoperative cisplatin-based radiochemotherapy of locally advanced oropharyngeal carcinoma and is now being explored in a prospective validation trial.     

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.     

Human papillomavirus and p16 expression in inverted papillomas of the urinary bladder

Human Papillomaviruses (HPVs) have been found in association with benign and malignant growth of epithelia. The cell cycle inhibitor p16^Ink4a has been shown to be overexpressed in HPV-positive cervical pre-malignant and malignant lesions, probably as a result of pRB targeting by the viral E7 protein. Inverted papillomas of the urinary bladder are epithelial tumors considered to be of benign nature. In this report we analyze the expression of p16^Ink4a and the presence of HPV sequences in inverted papillomas and in non-tumoral bladder controls. Our results show no association of HPV infection and inverted papillomas. Further, no correlation between p16 overexpression and HPV positivity was found. We conclude that HPV does not play an indispensable role in the development of urinary bladder inverted papillomas and that overexpression of p16^Ink4a does not correlate with HPV infection in these tumors.     

Detection of human papillomavirus-16 in fine-needle aspirates to determine tumor origin in patients with metastatic squamous cell carcinoma of the head and neck.

PURPOSE: Patients with head and neck squamous cell carcinoma (HNSCC) often clinically present with metastases to regional lymph nodes. Fine-needle aspiration of neck masses is routinely used to establish the presence of metastatic carcinoma and in turn to initiate a subsequent workup to determine the site of tumor origin. Human papillomavirus (HPV) 16 is an important etiologic agent for HNSCCs that arise from the oropharynx but less so for tumors from non-oropharyngeal sites. HPV16 detection thus provides a strategy for localizing an important subset of HNSCCs, but this approach has not been applied to fine-needle aspiration specimens. EXPERIMENTAL DESIGN: We did in situ hybridization for HPV16 on 77 consecutive aspirated neck masses diagnosed as metastatic squamous cell carcinoma. P16 immunohistochemistry was also done because p16 overexpression may serve as a surrogate marker of HPV-associated HNSCC. RESULTS: HPV16 was detected in 13 of the 77 (17%) aspirates. By site of origin, HPV16 was detected in 10 of 19 metastases from the oropharynx but in none of 46 metastases from other sites (53% versus 0%; P < 0.0001). HPV16 was not detected in 2 branchial cleft cysts misdiagnosed as metastatic squamous cell carcinoma, but it was detected in 3 of 10 metastases from occult primary tumors. P16 expression was associated with the presence of HPV16: 12 of 13 HPV16-positive metastases exhibited p16 expression, whereas only 4 of 62 HPV16-negative metastases were p16 positive (92% versus 6%; P < 0.0001). P16 expression also correlated with site of tumor origin: 13 of 19 oropharyngeal metastases were p16 positive, whereas only 1 of 46 non-oropharyngeal metastases was p16 positive (68% versus 2%; P < 0.0001). CONCLUSIONS: HPV16 status can be determined in tumor cells aspirated from the necks of patients with metastatic HNSCC. Its presence is a reliable indicator of origin from the oropharynx.     

11q13 amplification status and human papillomavirus in relation to p16 expression defines two distinct etiologies of head and neck tumours

Two distinct etiologies of head and neck squamous cell carcinoma (HNSCC) have been proposed, DNA damage owing to tobacco and alcohol exposure and human papillomavirus (HPV) oncogene-mediated transformation. Common genetic alterations in HNSCC include TP53 mutations, 11q13 amplification (amp) and CDKN2A/p16 mutations or promoter methlyation. However, in HPV+ HNSCC it is frequent to observe wild-type TP53 and expression of p16. The relationship of this unusual pattern with 11q13 amp has not been tested. In a retrospective study on 125 HNSCC patients, only 17% (five out of 30) of HPV+ vs 44% (39 out of 89) of HPV - tumours expressed 11q13 amp (adjusted odds ratio (OR)=0.2, 95% confidence interval (CI)=0.1-0.6). A subpopulation of tumours (n=69) were classified according to the three molecular markers, TP53, p16 and 11q13 amp. In addition to wild-type TP53, and p16 expression, HPV+ tumours were more likely not to be amplified at 11q13 (OR=6.5, 95% CI=1.8-23.9). As HPV+ HNSCC lack the genetic alterations which are common in other tumours, we hypothesise that HPV infection may represent an early event in the HNSCC carcinogenic process, thus suggesting a distinct molecular pathway.     

Differential etiopathogenic features of vulvar squamous cell carcinomas in sub-Saharan Africa and Europe

Two pathways have been described for vulvar squamous cell carcinomas (VSCC), one associated with human papillomavirus (HPV), and the other HPV-independent. We compared the etiopathogenic features of a series of VSCC from Mozambique, a sub-Saharan country with high prevalence of HPV and HIV, with those of Spain, a European country with low prevalence of HPV and HIV. All VSCC diagnosed at the two institutions from January 2018 to December 2020 were included (n = 35 and n = 41, respectively). HPV DNA detection and genotyping, and immunohistochemistry for p16 and p53 were performed. Tumors showing p16 positive staining and/or HPV DNA positivity were considered HPV-associated. 34/35 tumors (97%) from Mozambique and 8/41 (19%) from Spain were HPV-associated (P < .001). Mean age of the patients from Mozambique and Spain was 45 ± 12 and 72 ± 14, respectively (P < .001). No differences were found in terms of HPV genotypes or multiple HPV infection rates. 1/35 tumors (3%) from Mozambique and 29/41 (70%) from Spain showed abnormal p53 immunostaining (P < .001). In contrast with the predominance of HPV-independent VSCC affecting old women in Europe, most VSCC in sub-Saharan Africa are HPV-associated and arise in young women. This data may have important consequences for primary prevention of VSCC worldwide.     

HPV DNA,E6I-mRNA expression and p16 immunohistochemistry in head and neck cancer – How valid is p16 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.     

Human papillomavirus and p53 mutations in head and neck squamous cell carcinoma among Japanese population

We aimed to reveal the prevalence and pattern of human papillomavirus (HPV) infection and p53 mutations among Japanese head and neck squamous cell carcinoma (HNSCC) patients in relation to clinicopathological parameters. Human papillomavirus DNA and p53 mutations were examined in 493 HNSCCs and its subset of 283 HNSCCs. Oropharyngeal carcinoma was more frequently HPV‐positive than non‐oropharyngeal carcinoma (34.4% vs 3.6%, P < 0.001), and HPV16 accounted for 91.1% of HPV‐positive tumors. In oropharyngeal carcinoma, which showed an increasing trend of HPV prevalence over time (P < 0.001), HPV infection was inversely correlated with tobacco smoking, alcohol drinking, p53 mutations, and a disruptive mutation (P = 0.003, <0.001, <0.001, and <0.001, respectively). The prevalence of p53 mutations differed significantly between virus‐unrelated HNSCC and virus‐related HNSCC consisting of nasopharyngeal and HPV‐positive oropharyngeal carcinomas (48.3% vs 7.1%, P < 0.001). Although p53 mutations were associated with tobacco smoking and alcohol drinking, this association disappeared in virus‐unrelated HNSCC. A disruptive mutation was never found in virus‐related HNSCC, whereas it was independently associated with primary site, such as the oropharynx and hypopharynx (P = 0.01 and 0.03, respectively), in virus‐unrelated HNSCC. Moreover, in virus‐unrelated HNSCC, G:C to T:A transversions were more frequent in ever‐smokers than in never‐smokers (P = 0.04), whereas G:C to A:T transitions at CpG sites were less frequent in ever‐smokers than in never‐smokers (P = 0.04). In conclusion, HNSCC is etiologically classified into virus‐related and virus‐unrelated subgroups. In virus‐related HNSCC, p53 mutations are uncommon with the absence of a disruptive mutation, whereas in virus‐unrelated HNSCC, p53 mutations are common, and disruptive mutagenesis of p53 is related with oropharyngeal and hypopharyngeal carcinoma.     

Marked differences in survival rate between smokers and nonsmokers with HPV 16-associated tonsillar carcinomas

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 p16(INK4A) 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) and p16(INK4A)-specific immunohistochemistry. Results were correlated with clinical and demographic data. HPV 16 integration was detected by FISH as punctate signals in 33 out of 81 (41%) TSCC, 32 of which showed p16(INK4A) accumulation. Only 5 out of 48 HPV-negative tumors showed p16(INK4A) immunostaining (p < 0.0001). The presence of HPV furthermore correlates significantly with low tobacco (p = 0.002) and alcohol intake (p = 0.011), poor differentiation grade (p = 0.019), small tumor size (p = 0.024), presence of a local metastasis (p = 0.001) and a decreased (loco)regional recurrence rate (p = 0.039). Statistical analysis revealed that smoking significantly increases the risk of cancer death from TSCC and that non-smoking patients with HPV-containing TSCC show a remarkably better disease-specific survival rate. HPV 16 is integrated in 41% of TSCC and strongly correlates with p16(INK4A) overexpression, implicating the latter to be a reliable HPV biomarker. Patients with HPV-positive tumors show a favorable prognosis as compared to those with HPV-negative tumors, but tobacco use is the strongest prognostic indicator. These findings indicate that oncogenic processes in the tonsils of non-smokers differ from those occurring in smokers, the former being related to HPV 16 infection.     

Is the improved prognosis of p16 positive oropharyngeal squamous cell carcinoma dependent of the treatment modality?

The incidence of human papilloma virus (HPV) induced oropharyngeal squamous cell carcinoma (OPSCC) increases in the western countries. These OPSCC show distinct molecular characteristics and are characterized by an overexpression of p16, considered a surrogate marker for HPV infection. When compared to patients with p16 negative OPSCC, patients with HPV induced p16 positive OPSCC show a significantly better prognosis, which is reported to be caused by increased radiosensitivity. The objective of the present study was to analyze the impact of p16 expression status on the prognosis of OPSCC treated by either radiotherapy (RT) or primary surgery. Results are based upon a tissue microarray (TMA) of 365 head neck squamous cell carcinomas (HNSCC) including 85 OPSCC with clinico‐pathological and follow‐up data. p16 positivity correlated significantly with oropharyngeal tumor localization (p < 0.001). Patients with p16 positive OPSCC exhibited a significantly better overall survival than those with p16 negative tumors (p = 0.007). In a multivariate analysis, survival benefit of patients with p16 positive OPSCC was independent of clinico‐pathological parameters such as cT and cN classification and treatment modality. The improved prognosis of p16 positive OPSCC is found after RT as well as after surgery.     

Human papillomavirus infection and p53 nuclear overexpression in anal canal carcinoma.

The product of HPV E6 and E7 genes is able to inactivate both the p53 and pRb proteins. The aim of this study was to evaluate the correlation among anal HPV infection and nuclear p53 overexpression. The Authors evaluated HPV DNA by PCR and p53 nuclear expression by immunohistochemistry in 12 cloacogenic and 6 squamocellular carcinoma. HPV DNA was detected in 71.4% of the squamocellular tumors and in 57.1% of the cloacogenic tumors. In squamocellular tumors HPV types 31-33 and 16 were found; in cloacogenic tumors type 16 alone was detected. Nuclear accumulation of p53 was found to be associated with the presence of HPV. There was no significant difference in parietal infiltration, lymph nodes involvement and prognosis between HPV+p53+ patients and HPV-p53- patients. Tumor aggressiveness is likely to be enhanced by factors other than HPV infection and p53 overexpression.     

Human papillomavirus positive squamous cell carcinoma of the oropharynx: a radiosensitive subgroup of head and neck carcinoma

BACKGROUND: Epidemiologic evidence points to a connection between viral infection by the human papillomavirus (HPV) and a subgroup of squamous cell carcinoma of the oropharynx. To assess the impact of HPV infection on the response of these tumors toward radiotherapy, the authors retrospectively determined the presence of the virus and the integrity of the viral E2 gene in tumors of patients who have undergone curative irradiation. METHODS: Paraffin embedded biopsies from 99 patients were analyzed for HPV infection and E2 gene integrity by multiplex PCR. The experimental findings were correlated with clinical characteristics, known risk factors, and treatment outcome. RESULTS: Fourteen of 99 tumors were HPV positive (11 HPV16, 1 HPV33, 1 HPV35, and 1 HPV45). Human papillomavirus positivity was closely linked to female gender (odds ratio [OR], 5.75; P = 0.004), age older than 56 years (OR, 7.42; P = 0.012), nonsmokers (OR, 21.33; P = 0.00001), and alcohol abstainers (OR, 5.35; P = 0.012). There was an inverse association with p53 nuclear immunoreactivity (OR, 0.06; P = 0.008). The Kaplan-Meier survival estimates showed a better local control (P = 0.050, log-rank) and a better overall survival (P = 0.046, log-rank) for patients with HPV positive tumors. In the multivariate analysis, HPV positivity remained to be associated with a lower risk of local failure (risk ratio [RR], 0.31; P = 0.048). Four of 11 HPV16 positive tumors had a disrupted E2 gene. Only tumors with a disrupted E2 gene manifested local treatment failure. CONCLUSIONS: Human papillomavirus positivity designates a specific subgroup of oropharyngeal squamous cell carcinomas of the oropharynx that arise preferentially among individuals with no consumption of tobacco and alcohol and that have a favorable outcome attributable to an increased sensitivity toward radiotherapy.     

Combined P16 and human papillomavirus testing predicts head and neck cancer survival

While its prognostic significance remains unclear, p16^INK4a protein expression is increasingly being used as a surrogate marker for oncogenic human papillomavirus (HPV) infection in head and neck squamous cell carcinomas (HNSCC). To evaluate the prognostic utility of p16 expression in HNSCC, we prospectively collected 163 primary tumor specimens from histologically confirmed HNSCC patients who were followed for up to 9.4 years. Formalin fixed tumor specimens were tested for p16 protein expression by immunohistochemistry (IHC). HPV type‐16 DNA and RNA was detected by MY09/11‐PCR and E6/E7 RT‐PCR on matched frozen tissue, respectively. P16 protein expression was detected more often in oropharyngeal tumors (53%) as compared with laryngeal (24%), hypopharyngeal (8%) or oral cavity tumors (4%; p < 0.0001). With respect to prognosis, p16‐positive oropharyngeal tumors exhibited significantly better overall survival than p16‐negative tumors (log‐rank test p = 0.04), whereas no survival benefit was observed for nonoropharyngeal tumors. However, when both p16 and HPV DNA test results were considered, concordantly positive nonoropharyngeal tumors had significantly better disease‐specific survival than concordantly negative nonoropharyngeal tumors after controlling for sex, nodal stage, tumor size, tumor subsite, primary tumor site number, smoking and drinking [adjusted hazard ratio (HR) = 0.04, 0.01–0.54]. Compared with concordantly negative nonoropharyngeal HNSCC, p16(+)/HPV16(?) nonoropharyngeal HNSCC (n = 13, 7%) demonstrated no significant improvement in disease‐specific survival when HPV16 was detected by RNA (adjusted HR = 0.83, 0.22–3.17). Our findings show that p16 IHC alone has potential as a prognostic test for oropharyngeal cancer survival, but combined p16/HPV testing is necessary to identify HPV‐associated nonoropharyngeal HNSCC with better prognosis.     

High frequency of microsatellite instability in young patients with head-and-neck squamous-cell carcinoma: lack of involvement of the mismatch repair genes hMLH1 AND hMSH2.

The most prevalent risk factors in the development of head-and-neck squamous-cell carcinoma (HNSCC) are excessive tobacco and alcohol consumption. In young patients with HNSCC, these risk factors are often absent. Our purpose was to investigate the risk factors, microsatellite instability (MSI) changes and status of the mismatch repair genes hMLH1 and hMSH2 in a cohort of young patients with HNSCC. Fifty-seven HNSCC tumors were examined for the presence of MSI at 16 microsatellite sites using PCR. In the young patient group (24 cases, < or = 44 years old), 100% of tumors had MSI at 1 site at least and 88% had MSI at 2 or more loci. In older patients (33 cases, > or = 45 years), MSI at 1 or more sites was found in 61% of tumors (young vs. old, p = 0.0003) and instability at 2 or more sites was found in 36% of tumors (young vs. old, p = 0.0001). The involvement of the mismatch repair genes was investigated by examining promoter methylation, exon mutation and gene expression of hMLH1 and hMSH2. All results were negative, indicating that inactivation of these 2 genes does not play a role in the development of MSI in tumors from this patient group. Furthermore, the young patient group had a significantly lower incidence of smoking (46% young, 88% old; p = 0.001) and alcohol consumption (33% young, 67% old; p = 0.0169), emphasizing the probable importance of other environmental and/or genetic factors in the development of their disease.