Comparison of human papillomavirus in situ hybridization and p16 immunohistochemistry in the detection of human papillomavirus‐associated head and neck cancer based on a prospective clinical experience

BACKGROUND:

Human papillomavirus (HPV) is a causative agent in a subset of head and neck squamous cell carcinomas (HNSCCs). These HPV‐related cancers have a clinicopathologic profile that diverges from HPV‐negative HNSCCs. Accordingly, HPV testing may soon become integrated into standard pathologic assessment of HNSCCs.

METHODS:

Data were prospectively collected for all patients with head and neck carcinomas who had undergone HPV testing at the Johns Hopkins Hospital as part of clinical care during a 57‐month period. HPV testing consisted of concurrent HPV16 in situ hybridization (ISH) and p16 immunohistochemistry (IHC). Wide spectrum HPV ISH was reserved for p16‐positive cases that were HPV‐16 negative.

RESULTS:

HPV analysis was performed on 256 head and neck carcinomas in an effort to predict clinical outcomes (56%), localize primary tumor origin (21%), establish tumor classification (9%), determine patient eligibility for vaccine trials (8%), or satisfy patient curiosity (5%). A total of 182 (71%) tumors were HPV positive. HPV positivity correlated with oropharyngeal site (82% vs 9%) and male sex (77% vs 48%). p16 positivity was present in all 176 HPV16‐positive cases, and in 19 of 80 (24%) cases that were HPV‐16 negative. In 6 (32%) discordant cases, p16 expression was because of the presence of another HPV type.

CONCLUSIONS

A feasible strategy that incorporates p16 IHC and HPV ISH is able to detect HPV in a high percentage of oropharyngeal carcinomas. HPV status is frequently requested by the oncologist to estimate clinical outcome, and used by pathologists to establish tumor classification and determine site of tumor origin. Cancer 2010. © 2010 American Cancer Society.     

The interplay between HPV and host immunity in head and neck squamous cell carcinoma

Persistent infection with human papillomavirus (HPV) type 16 is a major risk factor for the development of head and neck squamous cell carcinoma (HNSCC), in particular oropharyngeal squamous cell carcinoma (OPSCC). The oropharyngeal epithelium differs from the mucosal epithelium at other commonly HPV16‐infected sites (i.e., cervix and anogenital region) in that it is juxtaposed with the underlying lymphatic tissue, serving a key immunologic function in the surveillance of inhaled and ingested pathogens. Therefore, the natural history of infection and immune response to HPV at this site may differ from that at other anatomic locations. This review summarizes the literature concerning the adaptive immune response against HPV in the context of HNSCC, with a focus on the T‐cell response. Recent studies have shown that a broad repertoire of tumor‐infiltrating HPV‐specific T‐cells are found in nearly all patients with HPV‐positive tumors. A systemic response is found in only a proportion of these. Furthermore, the local response is more frequent in OPSCC patients than in cervical cancer patients and HPV‐negative OPSCC patients. Despite this, tumor persistence may be facilitated by abnormalities in antigen processing, a skewed T‐helper cell response, and an increased local prevalence of T‐regulatory cells. Nonetheless, the immunologic profile of HPV‐positive vs. HPV‐negative HNSCC is associated with a significantly better outcome, and the HPV‐specific immune response is suggested to play a role in the significantly better response to therapy of HPV‐positive patients. Immunoprofiling may prove a valuable prognostic tool, and immunotherapy trials targeting HPV are underway, providing hope for decreasing treatment‐related toxicity.     

Time trends in the prevalence of HPV in oropharyngeal squamous cell carcinomas in northern Spain (1990–2009)

Recent studies support an important role for human papillomavirus (HPV) in oropharyngeal squamous cell carcinomas (OPSCC), although the incidence varies widely depending on the geographic location and time period studied. The aim of this study was to determine the proportion of HPV in a large cohort of OPSCC in northern Spain in the years 1990–2009. Clinical records and paraffin embedded tumor specimens of 248 consecutive patients surgically treated for OPSCC (140 tonsillar and 108 base of tongue) between 1990 and 2009 were retrieved. OPSCC cases were histomorphologically evaluated, and protein expression of p16 and p53 was analyzed by immunohistochemistry. Detection of high‐risk HPV DNA was performed by GP5+/6+‐PCR and in situ hybridization (ISH). Thirty cases (12%) were positive for p16 immunostaining, of which eight (3.2% of the total series) were found positive for HPV type 16 by genotyping of GP5+6+‐PCR products. All HPV GP5+/6+‐PCR‐positive tumors were p53‐immunonegative, seven had a basaloid morphology and seven were also positive by HPV ISH. Presence of HPV correlated inversely with tobacco and alcohol consumption (p < 0.001), but not with age of onset of OPSCC. Overall survival was better in the HPV‐positive group, although not statistically significant (p = 0.175). OPSCC patients in northern Spain demonstrated a low involvement of HPV, increasing (although not significantly, p = 0.120) from 1.8% in 1990–1999 to 6.1% of cases in 2000–2009.     

Antitumour effects of metformin and curcumin in human papillomavirus positive and negative head and neck cancer cells

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has significantly increased in recent decades due to human papillomavirus (HPV)‐mediated oncogenesis. Unfortunately, a growing number of HPV‐positive (+) OPSCC survivors are living with the irreversible side effects of treatment. The novel, well‐tolerated chemotherapeutics with improved side effect profiles are, therefore, in high demand. Metformin is one such drug, widely used as a first‐line oral agent in the treatment of type 2 diabetes mellitus. Curcumin is another well‐tolerated agent quickly gaining attention for its medicinal properties. Both metformin and curcumin have been shown to display anticancer properties. This study aimed to determine the antitumor effects of these agents, individually and combined, in HPV+???? ???and HPV‐negative (?) head and neck squamous cell carcinoma (HNSCC) cell lines. This was achieved by assessing the efficacy of varying drug concentrations on the overall cell viability, proliferation, and expression of common HNSCC biomarkers. The results from protein and RNA expression data are highly variable, as expected, with multiple pathways being affected in cancer. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assays and immunofluorescence microscopy suggest that both agents are capable of slowing proliferation and inducing apoptosis. We conclude that curcumin and metformin display effective antitumor effects in both HPV+ and HPV? HNSCC cell lines. The curcumin effects appear more pronounced in the HPV? cell lines. Metformin appears to be more effective at reducing the overall cell numbers in HPV+ cell lines. Metformin and curcumin combined did not appear to have synergistic effects on the proliferation or apoptosis of the treated cell lines.     

Human papillomavirus 16 and head and neck squamous cell carcinoma

Evidence suggests that human papillomavirus (HPV)16 seropositivity reflects past HPV16 exposure and is associated with risk for head and neck squamous cell carcinoma (HNSCC). Our objectives were to test the hypothesis that HPV16 seropositivity is associated with risk for HNSCC, to correlate HPV16 seropositivity with HPV16 tumor DNA, and to correlate HPV16 seropositivity and HPV16 DNA with sexual history and patient survival. In a case-control study of approximately 1,000 individuals, we assessed serology to the HPV16 L1 protein and in cases only, assayed tumors for HPV16 DNA. HPV16 seropositivity was associated with 1.5- and 6-fold risks for tumors of the oral cavity and pharynx, respectively. There was a dose response trend for HPV16 titer and increasing risk of HNSCC (p < 0.0001) and HPV16 tumor DNA (p < 0.0001). In cases, HPV16 DNA and seropositivity were significantly associated with sexual activity; odds ratios (ORs) of 12.8 and 3.7 were observed for more than 10 oral sexual partners and ORs of 4.5 and 3.2 were associated with a high number of lifetime sexual partners, respectively. Finally, HPV16 seropositivity and HPV16 tumor DNA were associated with hazard ratios of 0.4 and 0.5, respectively, indicating better survival for HPV positive individuals. HPV16 seropositivity was associated with risk for HNSCC, with greatest risk for pharyngeal cancer. We observed dose response relationships between serology titer and both risk for HNSCC and HPV16 tumor DNA. In cases, HPV16 tumor DNA and positive serology were associated with sexual history and improved disease free survival.     

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.     

Human papillomavirus status in young patients with head and neck squamous cell carcinoma

The role of human papillomavirus (HPV) in head and neck squamous cell carcinoma (HNSCC) development has been recognized only in the last decade. Although younger patients develop HNSCC associated with HPV, the incidence in young patients has not been studied. Forty-five young HNSCC patients (<40 years) were tested for HPV and the expression of p16(ink4a) and p53 in tumor biopsies. The presence of HPV was correlated with the absence and presence of alcohol and tobacco exposure. Paraffin-embedded, archival biopsy materials from HNSCC of 45 patients younger than 40 years were analyzed. HPV subtypes were identified by PCR followed by genotyping. Expression of p16(ink4a) and p53 were determined by immunohistochemistry. Fourteen (31%) of the HNSCC specimens from 45 patients unequivocally exhibited HPV16 positivity. Sixty percentage of the oropharyngeal tumors and 5% of the oral cavity tumors were HPV16 positive. P16(ink4a) overexpression was detected in 93% of the HPV16-positive tumors. None of the HPV16 tumors showed p53 overexpression. There was no association of HPV positivity with (lack of) exposure to alcohol and smoking. HPV association was not exclusively detected in nonsmoking, nondrinking young HNSCC patients. The presence of p16(ink4a) accumulation and the absence of p53 overexpression are good surrogate markers for HPV-associated HNSCC.     

Etiologic role of human papillomavirus infection in bladder carcinoma

BACKGROUND:

The authors elucidated an etiologic role of human papillomavirus (HPV) infection in carcinoma of the bladder.

METHODS:

One hundred seventeen of 224 patients with bladder carcinoma who were treated between 1997 and 2009 were enrolled in this study. The presence of HPV DNA was tested on frozen carcinoma tissues that were obtained by transurethral resection using a polymerases chain reaction‐based method. Localization of HPV was observed on archival tissue specimens by in situ hybridization (ISH) for high‐risk HPV DNA. Cyclin‐dependent kinase (CDK) inhibitor 2A (inhibits CDK4) (p16‐INK4a) and minichromosome maintenance protein‐7 (mcm‐7)—surrogate markers for high‐risk HPV‐E7 oncoprotein—and HPV‐L1 (capsid) protein expression were evaluated by immunohistochemistry.

RESULTS:

HPV types 16, 18, 31, 33, 52, and 58, and an unknown HPV type were detected in 18 of 117 samples (15%) from patients with bladder carcinoma. HPV16 was identified in 6 samples, HPV18 was identified in 4 samples, and HPV33 was identified in 3 samples. All were single HPV type infections. HPV was detected in 38% (12 of 28) of histologic grade 1 bladder carcinomas, 8.5% (6 of 71) of grade 2 bladder carcinomas, and in 0% (0 of 18) of grade 3 bladder carcinomas. Multivariate analysis indicated that younger age (<60 years; odds ratio [OR], 10.9; 95% confidence interval [CI], 2.6‐45.3) and grade 1 tumors (OR, 4.5; 95% CI, 1.2‐17.0) were associated with HPV infection. ISH analysis indicated that high‐risk HPV DNA was localized in the nuclei of tumor cells of all HPV‐positive samples. p16‐INK4a and mcm‐7 were expressed in 94% and 89% of HPV‐positive carcinoma cells, respectively. HPV‐L1 protein expression, which suggested reproductive HPV infection, was not observed in any carcinoma.

CONCLUSIONS:

The current results indicated that high‐risk HPV is likely to be a causative agent of some low‐grade bladder carcinomas that develop in younger patients. Cancer 2011. © 2010 American Cancer Society.     

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.     

The potential impact of prophylactic human papillomavirus vaccination on oropharyngeal cancer

The incidence of oropharyngeal cancer (OPC) is significantly increasing in the United States. Given that these epidemiologic trends are driven by human papillomavirus (HPV), the potential impact of prophylactic HPV vaccines on the prevention of OPC is of interest. The primary evidence supporting the approval of current prophylactic HPV vaccines is from large phase 3 clinical trials focused on the prevention of genital disease (cervical and anal cancer, as well as genital warts). These trials reported vaccine efficacy rates of 89% to 98% for the prevention of both premalignant lesions and persistent genital infections. However, these trials were designed before the etiologic relationship between HPV and OPC was established. There are differences in the epidemiology of oral and genital HPV infection, such as differences in age and sex distributions, which suggest that the vaccine efficacy observed in genital cancers may not be directly translatable to the cancers of the oropharynx. Evaluation of vaccine efficacy is challenging in the oropharynx because no premalignant lesion analogous to cervical intraepithelial neoplasia in cervical cancer has yet been identified. To truly investigate the efficacy of these vaccines in the oropharynx, additional clinical trials with feasible endpoints are needed. Cancer 2016;122:2313–2323 . © 2016 American Cancer Society.