Tumor Human Papillomavirus Research Articles

Comorbidity, human papillomavirus infection and head and neck cancer survival in an ethnically diverse population

To demonstrate the importance of comorbid conditions in head and neck squamous cell carcinoma (HNSCC), we assessed the association between comorbidity and survival in an inner-city population of HNSCC patients. Comorbid status at diagnosis was derived using medical records and the Adult Comorbidity Evaluation-27 (ACE-27) index on 288 patients with histologically confirmed HNSCC from Montefiore Medical Center in the Bronx (NY) between 2002 and 2011. The association between comorbidity, tumor human papillomavirus (HPV) status and overall and disease specific survival was assessed by Kaplan-Meier analysis and multivariable Cox regression adjusting for clinico-pathologic factors. The study population consisted of primary oropharyngeal (36%), laryngeal (33%) and oral cavity cancer patients (31%). Overall, 19% had no comorbidity, 43% mild comorbidity, 29% moderate comorbidity, and 9% severe comorbidity. The most common comorbid conditions were hypertension, diabetes mellitus, respiratory disease, other malignancies, and illicit drug use. Survival analyses revealed that increased comorbidity at diagnosis was significantly related to poorer overall survival (p=0.016), but not to cancer survival (p=0.369) or recurrence (p=0.652). Oropharyngeal cancer patients with HPV DNA positive tumors and lower levels of comorbidity had significantly better overall survival compared to patients with HPV negative tumors (hazard ratio=0.2, 95%CI: 0.04-0.8), however there was no significant difference in overall (or disease specific) survival by HPV status among patients with higher levels of comorbidity at diagnosis (hazard ratio=0.7, 95%CI: 0.2-2.8). In an inner-city predominantly minority population, comorbidity at HNSCC diagnosis is relatively common and associated with poor overall survival, but not cancer survival or recurrence. Interestingly, the relationship between HPV and improved survival appears to be specific to patients with low comorbidity at diagnosis.

Association of tumor necrosis factor-alpha promoter variants with risk of HPV-associated oral squamous cell carcinoma

BackgroundTumor necrosis factor alpha (TNF-α) plays an important role in inflammation, immunity, and defense against infection and clearance of human papillomavirus (HPV). Thus, genetic variants may modulate individual susceptibility to HPV-associated oral squamous cell carcinoma (OSCC).MethodsIn this study we genotyped four common single nucleotide polymorphisms (SNPs) in the TNF-α promoter [ −308G > A(rs1800629), -857C > T (rs1799724), -863C > A (rs1800630), and -1031T > C (rs1799964)] and determined HPV16 serology in 325 OSCC cases and 335 matched controls and tumor HPV status in 176 squamous cell carcinomas of the oropharynx (SCCOP) patients. Univariate and multivariable logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs).ResultsWe found that HPV16 seropositivity alone was associated with an increased risk of OSCC (OR, 3.1; 95% CI, 2.1–4.6), and such risk of HPV16-associated OSCC was modified by each SNP. Patients with both HPV16 seropositivity and variant genotypes for each SNP had the highest risk when using patients with HPV16 seronegativity and a wild-type genotype as a comparison group. Moreover, similar results were observed for the combined risk genotypes of four variants and all such significant associations were more pronounced in several subgroups, particularly in SCCOP patients and never smokers. Notably, the combined risk genotypes of four variants were also significantly associated with tumor HPV-positive SCCOP.ConclusionTaken together, these results suggest that TNF-α SNPs may individually or, more likely, jointly affect individual susceptibility to HPV16-associated OSCC, particularly SCCOP and never smokers. Validation of our findings is warranted.

Statistical analysis of human papillomavirus in a subset of upper aerodigestive tract tumors

Human papilloma virus (HPV) has been detected in some upper aerodigestive tract tumors, but the correlation between HPV and these tumors is not well understood. There is also some controversy regarding this correlation because a large variation in the prevalence of HPV in these tumors has been reported. To discuss the impact of HPV infection on upper aerodigestive tract tumors, this review estimated systematically the prevalence and risk of HPV in upper aerodigestive tract tumors from studies published between 1982 and 2012. In total, 418 articles were selected, which included 273 studies on the head and neck and 145 studies on esophageal squamous cell carcinoma. The overall prevalence of HPV in the 5,467 head and neck squamous cell carcinoma specimens was 36.3%. HPV was detected more frequently in the Americas (40%), than in Asia (38.6%), Europe (32%), or others regions (30.5%). The prevalence of HPV was significantly higher in oropharyngeal (48.5%) than in oral (32.5%), laryngeal (30.7%), and unselected head and neck squamous cell carcinoma (33.3%) (P < 0.001). The pooled prevalence of HPV in the 6,912 esophageal squamous cell carcinoma cases was 38.9% and it was significantly higher in China (44%) versus other regions (31.3%) (P < 0.05). Meta-analysis showed that head and neck and esophageal squamous cell carcinoma are associated with HPV infection (combined OR: 3.58, OR for head and neck and esophageal squamous cell carcinoma: 4.20). These findings suggest that HPV might be linked etiologically to the development of some upper aerodigestive tract tumors.

Oropharyngeal squamous cell carcinoma with known human papillomavirus status treated with definitive chemoradiotherapy: patterns of failure and toxicity outcomes

BackgroundTumor human papillomavirus (HPV) status has emerged as one of the most powerful prognostic factors for disease control and survival in patients with oropharyngeal squamous cell carcinoma (OPSCC). We reviewed our experience in patients with OPSCC and known tumor HPV status treated with definitive chemoradiotherapy (CRT).MethodsPatients with stage III-IVb OPSCC and known tumor HPV status treated with CRT between 2006 and 2011 were identified from an IRB approved registry for this retrospective review. Outcomes were estimated using the Kaplan-Meier method and compared between HPV-positive and negative patients using the log-rank test.ResultsOf the 121 pts (89% male, 93% Caucasian) included in this study, median age was 57 (range: 40–73) and median follow-up was 21 months (range: 6–63). Ninety-seven (80%) patients were HPV-positive and 24 (20%) were HPV-negative. Primary site was base of tongue (55%), tonsil (44%), and oropharyngeal wall (2%). Two year rates of locoregional recurrence (3% vs. 26%; p = 0.002), disease free survival (93% vs. 64%; p = 0.001) and overall survival (94% vs 73%; p = 0.002) were superior in HPV-positive patients, while rates of distant recurrence were similar (3% vs. 5%; p = 0.98). While acute toxicities were similar between both groups, patients with HPV-positive disease were more likely to resume a normal diet (90% vs. 65%; p = 0.017) at last follow up. Also, no HPV-positive patient required a feeding tube beyond 6 months after treatment, compared with 24% of HPV-negative patients.ConclusionsDefinitive CRT produces excellent rates of disease control with minimal late toxicity for patients with HPV-positive OPSCC. Studies of OPSCC should account for tumor HPV status when identifying factors prognostic for outcome.

Weekly chemotherapy with radiation versus high-dose cisplatin with radiation as organ preservation for patients with HPV-positive and HPV-negative locally advanced squamous cell carcinoma of the oropharynx

Optimal treatment for locally advanced squamous cell carcinoma of the oropharynx (SCCOP) is not well defined. Here we retrospectively compare survival and toxicities from 2 different organ preservation protocols. The matched dataset consisted of 35 patients from each trial matched for age, stage, smoking, and tumor human papillomavirus (HPV) status. Patients in the University of Michigan Cancer Center (UMCC) trial 9921 were treated with induction chemotherapy (IC) followed by high-dose cisplatin and radiation in responders or surgery in nonresponders. Patients in the UMCC trial 0221 were treated with weekly carboplatin and paclitaxel and radiation. Survival was comparable for both studies and did not differ significantly across each trial after stratifying by HPV status. Grade 3 and 4 toxicities were more frequent in UMCC 9921. At 6 months posttreatment, gastrostomy tube (G-tube) dependence was not statistically different. These data suggest that survival outcomes in patients with locally advanced SCCOP are not compromised with weekly chemotherapy and radiation therapy, and such treatment is generally more tolerable.

Cisplatin and fluorouracil with or without panitumumab in patients with recurrent or metastatic squamous-cell carcinoma of the head and neck (SPECTRUM): an open-label phase 3 randomised trial

Previous trials have shown that anti-EGFR monoclonal antibodies can improve clinical outcomes of patients with recurrent or metastatic squamous-cell carcinoma of the head and neck (SCCHN). We assessed the efficacy and safety of panitumumab combined with cisplatin and fluorouracil as first-line treatment for these patients. This open-label phase 3 randomised trial was done at 126 sites in 26 countries. Eligible patients were aged at least 18 years; had histologically or cytologically confirmed SCCHN; had distant metastatic or locoregionally recurrent disease, or both, that was deemed to be incurable by surgery or radiotherapy; had an Eastern Cooperative Oncology Group performance status of 1 or less; and had adequate haematological, renal, hepatic, and cardiac function. Patients were randomly assigned according to a computer-generated randomisation sequence (1:1; stratified by previous treatment, primary tumour site, and performance status) to one of two groups. Patients in both groups received up to six 3-week cycles of intravenous cisplatin (100 mg/m(2) on day 1 of each cycle) and fluorouracil (1000 mg/m(2) on days 1-4 of each cycle); those in the experimental group also received intravenous panitumumab (9 mg/kg on day 1 of each cycle). Patients in the experimental group could choose to continue maintenance panitumumab every 3 weeks. The primary endpoint was overall survival and was analysed by intention to treat. In a prospectively defined retrospective analysis, we assessed tumour human papillomavirus (HPV) status as a potential predictive biomarker of outcomes with a validated p16-INK4A (henceforth, p16) immunohistochemical assay. Patients and investigators were aware of group assignment; study statisticians were masked until primary analysis; and the central laboratory assessing p16 status was masked to identification of patients and treatment. This trial is registered with ClinicalTrials.gov, number NCT00460265. Between May 15, 2007, and March 10, 2009, we randomly assigned 657 patients: 327 to the panitumumab group and 330 to the control group. Median overall survival was 11·1 months (95% CI 9·8-12·2) in the panitumumab group and 9·0 months (8·1-11·2) in the control group (hazard ratio [HR] 0·873, 95% CI 0·729-1·046; p=0·1403). Median progression-free survival was 5·8 months (95% CI 5·6-6·6) in the panitumumab group and 4·6 months (4·1-5·4) in the control group (HR 0·780, 95% CI 0·659-0·922; p=0·0036). Several grade 3 or 4 adverse events were more frequent in the panitumumab group than in the control group: skin or eye toxicity (62 [19%] of 325 included in safety analyses vs six [2%] of 325), diarrhoea (15 [5%] vs four [1%]), hypomagnesaemia (40 [12%] vs 12 [4%]), hypokalaemia (33 [10%] vs 23 [7%]), and dehydration (16 [5%] vs seven [2%]). Treatment-related deaths occurred in 14 patients (4%) in the panitumumab group and eight (2%) in the control group. Five (2%) of the fatal adverse events in the panitumumab group were attributed to the experimental agent. We had appropriate samples to assess p16 status for 443 (67%) patients, of whom 99 (22%) were p16 positive. Median overall survival in patients with p16-negative tumours was longer in the panitumumab group than in the control group (11·7 months [95% CI 9·7-13·7] vs 8·6 months [6·9-11·1]; HR 0·73 [95% CI 0·58-0·93]; p=0·0115), but this difference was not shown for p16-positive patients (11·0 months [7·3-12·9] vs 12·6 months [7·7-17·4]; 1·00 [0·62-1·61]; p=0·998). In the control group, p16-positive patients had numerically, but not statistically, longer overall survival than did p16-negative patients (HR 0·70 [95% CI 0·47-1·04]). Although the addition of panitumumab to chemotherapy did not improve overall survival in an unselected population of patients with recurrent or metastatic SCCHN, it improved progression-free survival and had an acceptable toxicity profile. p16 status could be a prognostic and predictive marker in patients treated with panitumumab and chemotherapy. Prospective assessment will be necessary to validate our biomarker findings. Amgen Inc.

Overexpression of p16(INK4a) in urothelial carcinoma in situ is a marker for MAPK-mediated epithelial-mesenchymal transition but is not related to human papillomavirus infection.

BackgroundThe role of human papillomavirus (HPV) in bladder carcinogenesis remains controversial. Overexpression of p16INK4a, a surrogate marker for infection with oncogenic HPV in other tumours, has been described for urothelial carcinoma in situ (UCIS). Our goal was therefore to evaluate whether overexpression of p16INK4a is associated with HPV infection and to identify mechanisms of p16INK4a upregulation in UCIS.Materials and MethodsIn 60 tissue specimens from a total of 45 patients (UCIS and controls), we performed p16INK4a immunohistochemistry followed by detection and subclassification of HPV DNA. In a subset of samples, we tested for gene amplification of p16INK4a applying fluorescence in situ hybridization (FISH). RAS/MAPK signalling and epithelial-mesenchymal transition (EMT) was assessed using immunohistochemistry. Finally, we transfected urothelial carcinoma cells with KRAS and examined the expression of p16INK4a as well as markers of EMT.ResultsWe found overexpression of p16INK4a in 92.6% of UCIS and in all cervical intraepithelial neoplasia (CIN) controls. In contrast, we detected high-risk HPV DNA in 80% of CIN, but none in UCIS. There was no gene amplification of p16INK4a. High levels of phosphorylated kinases and urokinase plasminogen activator (uPA) and loss of membraneous E-cadherin were detected in UCIS. KRAS transfection of urothelial carcinoma cells led to upregulation of p16INK4a and uPA accompanied by loss of E-cadherin that could be inhibited by application of the kinase-inhibitor Sorafenib.ConclusionsOur results show that overexpression of p16INK4a in UCIS is neither associated with HPV infection nor p16INK4a gene amplification but is a consequence of enhanced RAS/MAPK signalling that promotes EMT, possibly due to Sorafenib-sensitive paracrine secretion of the EMT activator uPA. These findings might open a novel therapeutic option for localized but aggressive urothelial cancer.

OP028: A modified prognostic model for oropharyngeal squamous cell carcinoma based on presence of human papillomavirus, nodal stage and comorbidity

Purpose To determine the prevalence and clinical significance of human papillomavirus (HPV) in a large, consecutive cohort of patients with oropharyngeal squamous cell carcinoma (OPSCC) and to develop a prognostic model for patients with OPSCC. Patients and methods All patients diagnosed with OPSCC between 2000 and 2006 in two main university hospitals in the Netherlands were included ( n = 841). The presence of an oncogenic HPV infection was determined by immunostaining for p16 INK 4 A , followed by a high-risk HPV GP5+/6+ DNA PCR on the p16 INK 4 A immuno-positive cases. Cox regression analysis was performed to compare survival rates between HPV-positive and HPV-negative patients and a prognostic model was generated by recursive partitioning. Results In total, 163 of 841 (19.4%) patients were p16 and HPV DNA-positive. HPV-positive patients had a more favorable overall survival (73.5% vs. 40.9% after 5 years; p Conclusions Tumor HPV status is a strong and independent prognostic factor for survival among patients with OPSCC. The existing prognostic model (1) for OPSCC patients was validated. An additional model was proposed, based on our large, unselected cohort of OPSCC patients. In this model, HPV-status, comorbidity and nodal stage were the important prognostic factors.

OP041: Impact of human papillomavirus (HPV) and p16 status on survival and response with cisplatin plus 5-FU and cetuximab in recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN): Analysis of the phase III extreme trial

Purpose Retrospective analysis to assess the role of tumor HPV/p16 status in patients (pts) with R/M SCCHN receiving cisplatin+5-FU chemotherapy (CT)+/−cetuximab. Material and methods Cervista HPV 16/18 and HPV high-risk assays and IHC detection of p16INK4A were used to determine tumor HPV/p16 status. Results Of 442 pts in the trial, p16 status was available for 381 and HPV status for 321. For survival, hazard ratios in favor of CT+cetuximab were seen in HPV−, HPV+, p16− and p16+ subgroups ( Table 1 ). Interaction tests confirmed the treatment effect of CT+cetuximab vs CT was independent of tumor HPV or p16 status. Similar findings were made for PFS (interaction test p = 0.98 for HPV and p = 0.43 for p16: no significant interaction was noted). CT+cetuximab also improved the chances of achieving a response across all subgroups ( Table 1 ). Within the CT+cetuximab and CT arms, HPV+/p16+ status was associated with a more favorable outcome than HPV−/p16− status. Conclusion Although the number of HPV+/p16+ pts was low, the results suggest that the survival benefits of CT+cetuximab vs CT are independent of tumor HPV/p16 status but that HPV+/p16+ status seems associated with a more favorable outcome than HPV−/p16− status.

Abstract 4776: Expression of Human Papillomavirus E6 and E7 genes in colorectal cancer.

Abstract Purpose: The role of human papillomavirus (HPV) has been well established in the pathogenesis on cervical cancers and anogenital. However, HPV-DNA has also been detected in tumor tissues of other malignancies such as head and neck cancer, esophageal cancer, and some skin cancers. Viral genes expression and multiplication occur exclusively in the nuclei of the infected cells and are tightly linked to the state of differentiation of the cells. Integration of viral DNA into host genome is essential for carcinogenesis since it promotes disruption of HPV E2 gene, leading to abnormal expression of E6 and E7 oncoproteins. Both E6 and E7 can form specific complexes with tumor suppressor gene p53 and pRB respectively. However, the role of HPV in the pathogenesis of colorectal cancer is still undetermined. Our objectives were (1) to evaluate the presence of HPV infection in patients with CRC; (2) to type HPV positive cases using primer specific for HPV-16, and (3) to determine the mRNA expression levels of the viral oncogenes HPV-16 E6 and HPV-16 E7. Experimental Designs: To determine the association of HPV and CRC, we conducted an age-and-gender matched case-control study using tumor and tumor-adjacent colorectal tissues from patients with CRC (cases) and without CRC (controls). Presence of L1, E2, and E6 genes was analyzed to determine whether a full-length HPV genome existed in tumor and normal tissue samples. Human HPV-16 DNA detection was performed by a nested PCR strategy using the PGMY09/PGMY11 as outer primer, and GP5+/GP6+ as inner primers. We detected the expression of E6 and E7 mRNA in the HPV-16 (+) cases. Hypothesis testing was performed using Pearson Chi-square analysis of proportions and conditional logistic regression using SPSS 17. Results: A total of 54 (ß-actin+) CRC patients (mean age 61 ±11 years, 27 males) and 36 controls (mean age was 60 ± 9 years, 18 males) were evaluated. HPV DNA was identified in 25 of 54 (46.3%) patients with CRC and in 1 of 36 (2.8%) controls (OR = 25.58; 95% CI 3.22 to 203.49, p &amp;lt; 0.001). HPV-16 was identified in 18 of 25 (72.0%) HPV-positive CRC cases, and in one (2.8%) control was HPV (+). All 18 HPV-16 CRC cases showed expressed E6 mRNA. Six of 18 (33.3%) HPV-16 positive cases expressed E7 mRNA. Among the 12 CRC cases where both tumor and adjacent tissue was available, 2 of 9 (16.7%) exhibited HPV-16 DNA in both tumor and tumor-adjacent tissues. Conclusion: These results support that colorectal HPV infection is common among patients with CRC. The high presence of HPV in tumors located proximal to the rectum suggests that this infection might not result from direct spread from anogenital sites. Moreover, our finding of highly frequent active expression of E6 and E7 viral oncogenes in the host strongly suggests that HPV oncoproteins may play a role in the pathogenesis of colorectal carcinogenesis. Citation Format: Wesley Villavicencio-Torres, Mercedes Y. Lacourt-Ventura, Sharon C. Fonseca-Williams, Maria Del Mar Gonzalez- Pons, Yaritza Diaz-Algori, Carlos Torres-Ramos, Raul D. Bernabe-Dones, Marcia R. Cruz-Correa. Expression of Human Papillomavirus E6 and E7 genes in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4776. doi:10.1158/1538-7445.AM2013-4776

1018O - Safety and Efficacy of Cisplatin Plus 5-Fu and Cetuximab in HPV-Positive and HPV-Negative Recurrent and/or Metastatic Squamous Cell Carcinoma of the Head and Neck (R/M SCCHN): Analysis of the Phase III Extreme Trial

ABSTRACT Background Tumor human papillomavirus (HPV) status is a strong predictor of survival and response to treatment in patients (pts) with early and locally advanced oropharyngeal cancer, but its effects in R/M SCCHN remain to be clarified. This retrospective analysis of data from the EXTREME trial assessed the role of tumor HPV status in pts with R/M SCCHN receiving cisplatin + 5-FU chemotherapy (CT) alone or in combination with cetuximab. Material and methods: Immunohistochemical detection of p16INK4A was used to determine HPV status: p16-positive and p16-negative disease is referred to as HPV-positive (HPV+) and HPV-negative (HPV-) disease, respectively. Results 196/222 (88%) pts in the CT + cetuximab and 185/220 (84%) pts in the CT arm had tissue evaluable for p16. Of these, 91% and 88%, respectively, had HPV- tumors. Among HPV- pts, CT + cetuximab (n = 178) improved overall survival (OS), progression-free survival (PFS) and overall response rate (ORR), compared with CT (n = 162) (Table). A similar pattern was observed among HPV+ pts, between CT + cetuximab (n = 18) and CT (n = 23) for OS, PFS and ORR. Interaction tests between treatment and HPV status for OS and PFS confirmed that the treatment effect of CT + cetuximab vs CT was independent of tumor HPV status (p = 0.482 and p = 0.430, respectively: no significant interaction was noted). Within the CT + cetuximab and CT arms, HPV (p16) expression is associated with a more favorable outcome than HPV- (Table). In the CT + cetuximab arm, the overall incidence of grade 3/4 adverse events was slightly higher among HPV+ than HPV- pts. Conclusions Although the number of HPV+ pts in this analysis was low, the results suggest that in R/M SCCHN pts in the EXTREME trial, the survival benefits ofCT + cetuximab vs CT are independent of tumor HPV status and HPV (p16) expression is associated with a more favorable outcome than HPV-. Table: 1018O: Tumor HPV status: effects on prognosis and efficacy of CT + cetuximab and CT in R/M SCCHN. Population Comparison OS PFS ORR CT + cetuximab vs CT HPV- (n = 340) Median (months)/rate (%) 9.7 7.3 5.7 3.1 37 17 HR/odds ratio 95% CI p 0.822 (0.647–1.043) 0.1064 0.486 (0.376–0.628) 2.753 (1.655–4.579) HPV+ (n = 41) Median (months)/rate (%) 12.6 9.6 5.6 3.6 50 22 HR/odds ratio 95% CI p 0.628 (0.295–1.338) 0.2241 0.730 (0.363–1.469) 0.3761 3.600 (0.929–13.953) 0.0614 HPV+ vs HPV- CT + cetuximab (n = 196) Median (months)/rate (%) 12.6 9.7 5.6 5.7 50 37 HR/odds ratio 95% CI p 0.592 (0.319–1.098) 0.0925 1.172 (0.685–2.005) 0.5624 1.738 (0.657–4.600) 0.2620 CT (n = 185) Median (months)/rate (%) 9.6 7.3 3.6 3.1 22 17 HR/odds ratio 95% CI p 0.827 (0.504–1.355) 0.4492 0.871 (0.529–1.434) 0.5870 1.329 (0.455–3.880) 0.6025 Data are medians and hazard ratios (HR) for PFS and OS and rates and odds ratios for ORR. p: log-rank test for OS/PFS; Cochran-Mantel-Haenszel test for ORR. Disclosure B. De Blas: The author is an employee of Merck KGaA. I. Celik: The author is an employee of Merck KGaA J.B. Vermorken: I have participated in advisory boards of Merck-Serono, Amgen, Boehringer-Ingelheim, Genentech and Sanofi-aventis and have lectured (compensated) for Merck-Serono, Amgen, Bristol-Myers Squibb and Sanofi-aventis All other authors have declared no conflicts of interest.

Improved survival of Japanese patients with human papillomavirus-positive oropharyngeal squamous cell carcinoma

The prevalence of oropharyngeal carcinoma is rising in western Europe and the United States, where there appears to be a strong association between human papillomavirus (HPV) and oropharyngeal squamous cell carcinoma (OPSCC). However, such a correlation has not yet been fully evaluated in Japan. We performed a retrospective analysis of the association between tumour HPV status and the demographic and clinicopathological parameters of 71 patients with OPSCC at Hokkaido University Hospital, Japan, between 1998 and 2009. The parameters included age, gender, survival, tumour subsite, tumour-node-metastasis (TNM) stage, smoking history, second primary tumour status, recurrence/residual disease at the primary site, and overall survival. HPV status was established by multiplex polymerase chain reaction (PCR) analysis. Of the 71 oropharyngeal cancers, 20 were positive for HPV-16, two for HPV-18, and one for HPV-58. Kaplan-Meier survival analysis showed improved overall survival rates in patients with HPV-positive tumours (p = 0.0038) compared with HPV-negative tumours. Of the 45 patients who received chemoradiotherapy, HPV-positive patients experienced better overall survival than HPV-negative patients (p = 0.0032). In a multivariate analysis, the survival benefit of HPV-positive patients was independent of age and T and N classification. HPV status is a significantly favourable prognostic factor in oropharyngeal cancer and could be used as a marker to optimize the treatment of patients with this type of cancer in Japan.

Validation of Methods for Oropharyngeal Cancer HPV Status Determination in US Cooperative Group Trials

Tumor human papillomavirus (HPV) status is a prognostic factor for oropharyngeal cancer, but classification methods are not standardized. Here we validate the HPV classification methods used in US cooperative group trials. Tumor DNA and RNA purified from 240 paraffin-embedded oropharyngeal cancers diagnosed from 2000 to 2009 were scored as evaluable if positive for DNA and mRNA controls by quantitative polymerase chain reaction (PCR). Eighteen high-risk (HR) HPV types were detected in tumors by consensus PCR, followed by HR-HPV E6/7 oncogene expression analysis by quantitative reverse transcriptase PCR. The sensitivity (S), specificity (SP), and positive (PPV) and negative predictive values (NPV) of p16 expression detected by immunohistochemistry (IHC) and HPV16 detected by in situ hybridization (ISH) were evaluated in comparison with HR-HPV E6/7 oncogene expression. Interrater agreement among 3 pathologists was evaluated by κ statistics. Of 235 evaluable tumors, 158 (67%; 95% confidence interval, 61.2-73.3) were positive for HR-HPV E6/7 oncogene expression [HPV type 16 (92%), 18 (3%), 33 (3%), 35 (1%), or 58 (1%)]. p16 IHC had high sensitivity (S 96.8%, SP 83.8%, PPV 92.7%, and NPV 92.5%), whereas HPV16 ISH had high specificity (S 88.0%, SP 94.7%, PPV 97.2%, and NPV 78.9%) for HR-HPV oncogene expression. Interrater agreement was excellent for p16 (κ=0.95 to 0.98) and HPV16 ISH (κ=0.83 to 0.91). Receiver operating curve analysis determined the cross-product of p16 intensity score and percentage of tumor staining to optimally discriminate HR-HPV E6/7-positive and HR-HPV E6/7-negative tumors. p16 IHC and HPV16 ISH assays show excellent performance, with high sensitivity and specificity, respectively. A new validated H-score for p16 IHC assessment is proposed. Appropriate assay choice depends on clinical implications of a false-positive or false-negative test.

Tumor stage, human papillomavirus and smoking status affect the survival of patients with oropharyngeal cancer: an Italian validation study

Tumor human papillomavirus (HPV) status strongly affects overall survival (OS) of oropharyngeal cancer (OPC) patients. Recently, three groups with different outcomes were identified based on HPV status, smoking history and tumor stage. Our objective was to validate this model using a single-institutional retrospective database. Patients (n=120) diagnosed with OPC at our institution, treated with concomitant cisplatin plus radiotherapy (RT) (n=64), induction chemotherapy followed by concomitant chemoradiation (n=39) or RT alone (n=17), were stratified in three groups with respect to the risk of death (low 26, intermediate 46 and high 49 patients) according to tumor p16 expression as surrogate of HPV status, pack-years of tobacco smoking and nodal/tumor stage. Group-stratified Kaplan-Meier OS curves were estimated and compared using the log-rank test. The 2-year OS estimates were 100%, 86% and 70%, respectively. The difference between the survival curves was statistically significant (P=0.009). The Harrell's concordance index was 0.70. The calibration plot showed a good concordance between our results and those observed in the original study. This study validates the risk grouping previously identified. Risk-driven clinical decision making and trial designs will help in better defining the most appropriate treatment in OPC patients.

Local Inflammation and Human Papillomavirus Status of Head and Neck Cancers

To determine whether periodontitis is associated with human papillomavirus (HPV) status of head and neck squamous cell carcinoma (HNSCC). Hospital-based case-control study in a comprehensive cancer center. Evaluation included all patients diagnosed with incident primary squamous cell carcinoma of the oral cavity, oropharynx, and larynx between 1999 and 2007 for whom tissue samples and dental records were available (N = 124). Patients younger than 21 years and those with a history of cancer were excluded. Periodontitis history was assessed by alveolar bone loss in millimeters from panoramic radiographs by one examiner blinded to cancer status. The presence of HPV-16 DNA in paraffin-embedded tumor samples was identified by polymerase chain reaction. The prevalence of HPV-positive HNSCC was 50 of 124 patients (40.3%). A higher proportion of oropharyngeal cancers were HPV-positive (32 of 49 [65.3%]) compared with oral cavity (9 of 31 [29.0%]) and laryngeal (9 of 44 [20.5%]) cancers. Each millimeter of alveolar bone loss was associated with 2.6 times increased odds (odds ratio [OR], 2.61; 95% CI, 1.58-4.30) of HPV-positive tumor status after adjustment for age at diagnosis, sex, and smoking status. The strength of the association was greater among patients with oropharyngeal SCC (OR, 11.70; 95% CI, 2.09-65.53) compared with those with oral cavity SCC (OR, 2.32; 95% CI, 0.65-8.27) and laryngeal SCC (OR, 3.89; 95% CI, 0.95-15.99). A history of chronic inflammatory disease in the oral cavity may be associated with tumor HPV status in patients with HNSCC. This association seems to be stronger among patients with oropharyngeal cancer compared with those who have oral cavity or laryngeal SCC.

Tobacco Smoking and Increased Risk of Death and Progression for Patients With p16-Positive and p16-Negative Oropharyngeal Cancer

Tobacco smoking is associated with oropharynx cancer survival, but to what extent cancer progression or death increases with increasing tobacco exposure is unknown. Patients with oropharynx cancer enrolled onto a phase III trial of radiotherapy from 1991 to 1997 (Radiation Therapy Oncology Group [RTOG] 9003) or of chemoradiotherapy from 2002 to 2005 (RTOG 0129) were evaluated for tumor human papillomavirus status by a surrogate, p16 immunohistochemistry, and for tobacco exposure by a standardized questionnaire. Associations between tobacco exposure and overall survival (OS) and progression-free survival (PFS) were estimated by Cox proportional hazards models. Prevalence of p16-positive cancer was 39.5% among patients in RTOG 9003 and 68.0% in RTOG 0129. Median pack-years of tobacco smoking were lower among p16-positive than p16-negative patients in both trials (RTOG 9003: 29 v 45.9 pack-years; P = .02; RTOG 0129: 10 v 40 pack-years; P < .001). After adjustment for p16 and other factors, risk of progression (PFS) or death (OS) increased by 1% per pack-year (for both, hazard ratio [HR], 1.01; 95% CI, 1.00 to 1.01; P = .002) or 2% per year of smoking (for both, HR, 1.02; 95% CI, 1.01 to 1.03; P < .001) in both trials. In RTOG 9003, risk of death doubled (HR, 2.19; 95% CI, 1.46 to 3.28) among those who smoked during radiotherapy after accounting for pack-years and other factors, and risk of second primary tumors increased by 1.5% per pack-year (HR, 1.015; 95% CI, 1.005 to 1.026). Risk of oropharyngeal cancer progression and death increases directly as a function of tobacco exposure at diagnosis and during therapy and is independent of tumor p16 status and treatment.

Abstract 552: Characterization of HPV infection in Hispanic colorectal cancer patients

Abstract Purpose: The association between Human Papillomavirus (HPV) infection and the development of cervical cancer and anogenital cancer has been established. Moreover, molecular studies have detected HPV DNA in tumor tissues of head and neck cancer, esophageal cancer, and some skin cancers. Viral genes expression and multiplication occur exclusively in the nuclei of the infected cells and are tightly linked to the state of differentiation of the cells. Integration of viral DNA into host genome is essential for carcinogenesis since it promotes disruption of HPV E2 gene, leading to abnormal expression of E6 and E7 oncoproteins. Both E6 and E7 can form specific complexes with tumor suppressor gene p53 and pRB respectively. However, the role of HPV infection in colorectal cancer (CRC) and its mechanisms of carcinogenesis have not been properly elucidated. Our objectives were to evaluate the presence of HPV infection in patients with CRC; to probe the viral integrations status of HPV 16 in CRC and its adjacent normal colonic mucosa, and to determine the mRNA expression levels of the viral oncogenes HPV-16 E6 and HPV-16 E7. Experimental Designs: To determine the association of HPV and CRC, we conducted an age-and-gender matched case-control study using tumor and tumor-adjacent colorectal tissues from patients with CRC (cases) and without CRC (controls). Presence of L1, E2, and E6 genes was analyzed to determine whether a full-length HPV genome existed in tumor and normal tissue samples. Head-to-tail junctions of HPV-16 genomes were analyzed to determine the integration the HPV-16 genome in the HPV-16 (+) cases. Moreover, we determined the mRNA expression levels of E6 and E7 in the HPV-16 (+) cases. Hypothesis testing was performed using Pearson Chi-square analysis of proportions using SPSS 17. Results: A total of 45 (ß-actin+) CRC patients (mean age 61 ±11 years, 24 males) and 47 controls (mean age was 60 ± 9 years, 18 males) were evaluated. Tumors were located in proximal colon (n=7) and distal colon (n=38). HPV DNA was identified in 19 of 45 (42.2%) patients with CRC and in 2 of 47 (4.3%) controls (OR = 16.4; 95% CI 3.5-76.3, p &amp;lt; 0.001). HPV-16 was identified in 12 of 19 (63.2%) HPV (+) CRC patients and in none (0%) HPV(+) controls. The twelve HPV-16 CRC cases showed integration of HPV-16. Three of 12 (25%) HPV16 (+) cases expressed E6 and E7 mRNA. Among the 12 CRC cases where both tumor and adjacent tissue was available, 2 of 9 (16.7%) exhibited HPV-16 DNA in both tumor and tumor-adjacent tissues.Conclusion: These results suggest that colorectal HPV infection is common among patients with CRC. The presence of HPV in tumors located proximal to the rectum suggests that this infection might not result from direct spread from anogenital sites. Moreover, our finding of highly frequent integration of viral DNA and the active expression of E6 and E7 viral oncogenes in the host strongly suggests that HPV oncoproteins may play a role in the pathogenesis of colorectal carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 552. doi:1538-7445.AM2012-552

Human Papillomavirus Immunogen That Provides Protective Tumor Immunity and Induces Tumor Regression

Human papillomavirus (HPV) is associated with premalignant lesions such as high-grade cervical intraepithelial neoplasia (CIN-III) with potential progression to cervical carcinoma. There are now preventive vaccines against HPV. However, no effective therapeutic vaccine or immunological treatment exists for individuals already infected or for the 470,000 women that develop high-grade dysplasia, carcinoma in situ, and cervical cancer each year. More than half of these women die from cervical cancer. Relative non-immunogenicity of HPV infection is one of the main reasons for the difficulty in designing a comprehensive therapeutic vaccine against HPV-induced premalignant lesions and cervical carcinoma. HPV E6 and E7 proteins, the major HPV oncogenes, are highly immunogenic but fail to induce cross-reactive and protective immune responses against heterologous strains. We designed and synthesized a therapeutic peptide vaccine comprised of multivalent peptide mixtures called hypervariable epitope constructs (HECs) that represent the major epitope variants of the oncogenic E7 structural protein, and assessed their immunogenicity and in vivo efficacy in mice. Our results show that this peptide vaccine can induce strong, HPV-specific, T-helper cell and CTL responses. More significantly, we have demonstrated that the vaccine is efficacious as a therapeutic agent in a mouse HPV tumor model. Therefore, the HPV HEC vaccine approach described herein can potentially prevent progression of HPV-associated premalignant lesions, and may also be therapeutic against tumors associated with HPV.

VEGF expression and microvascular density in relation to high-risk-HPV infection in cervical carcinoma – An immunohistochemical study

BackgroundThe poor outlook of cervical carcinoma, a human papillomavirus (HPV)-related cancer mandates the search for new treatment modalities. Therapeutic targeting of tumor vasculature is a promising strategy. The aim was to study angiogenesis in cervical carcinoma in terms of VEGF expression and assessment of microvascular density (MVD) in relation to HPV antigen expression. MethodsThirty paraffin blocks of cervical carcinoma were studied for the immune expression of VEGF and MVD utilizing CD34 monoclonal antibody. Statistical analysis of these immunophenotypes in relation to tumor type, grade and HPV antigen expression was performed. ResultsThis retrospective study comprised of 17 squamous cell carcinomas, 11 adenocarcinomas and two adenosquamous carcinomas. Eleven cases were low grade and 19 were high-grade cases. VEGF expression was detected in 100% of cases. The relation between carcinoma grade and VEGF expression and MVD was statistically significant. There was no relation between VEGF intensity and tumor type although more intense VEGF staining tended to occur in cervical adenocarcinomas. VEGF density was significantly lower in squamous cell carcinomas compared to adenocarcinomas. Mean MVD was 50.37±20.0. The relation between MVD and VEGF expression was statistically significant. HPV immune expression was detected in 93.33% of cases. The relation between HPV antigen expression and each of tumor histotype and grade was not statistically significant. There was a statistically significant relation between HPV antigen expression and each of MVD and VEGF intensity. Multivariate statistical analysis showed MVD as an independent predictor of carcinoma grade. ConclusionVEGF was expressed in 100% of studied cervical carcinoma. There was a statistically significant relation between VEGF expression and MVD. Since HPV antigen expression was significantly correlated with MVD and VEGF staining intensity, we provide evidence that HPV infection may augment tumor angiogenesis in cervical carcinoma. MVD emerged as an independent predictor of cervical carcinoma grade and hence of progressive behavior.

Small Biopsy Specimens Reliably Indicate p16 Expression Status of Oropharyngeal Squamous Cell Carcinoma

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC) is associated with favorable patient survival. Tumor HPV status at primary diagnosis is critical for proper management, and p16 immunohistochemistry (IHC) has emerged as a reliable, single, surrogate marker. It is not known, however, if small biopsy specimens are completely adequate for p16 evaluation. From a database of oropharyngeal SCC for which p16 IHC and histologic typing were already performed, all patients (32) who had available in-house primary tumor biopsy specimens and also subsequent surgical resections were analyzed. p16 IHC was performed along with histologic typing into: Type 1 keratinizing SCC, Type 2 nonkeratinizing SCC with maturation, and Type 3 nonkeratinizing SCC. Staining was graded on both biopsies and resections as follows: 0=negative; 1+ =1-25% of tumor cells positive; 2+ =26-50%; 3+ =51-75%; 4+ =76-100%. Strictly considering p16 score, perfect biopsy-resection correlation was present in 28 of 32 cases (85%), including 6/9 (67%) Type 1, 6/7 (86%) Type 2, and 16/16 (100%) Type 3 cases. Considering p16 expression binarily as 51% tumor cell staining or more (3+ or 4+) being positive and lesser amounts (0, 1+, or 2+) as being negative, there was perfect biopsy-resection correlation for all 32 cases. With p16 expression in resection specimens considered the gold standard, p16 IHC in biopsies was both 100% sensitive and specific. Our results demonstrate that p16 staining in diagnostic biopsies reliably reflects whole tumor staining results, and suggest that biopsies do not suffer from false negatives or positives.