HPV-negative Tumors Research Articles

Generation of affibody molecules specific for HPV16 E7 recognition.

Cervical cancer caused by infection with high-risk human papillomavirus remains to be the most deadly gynecologic malignancy worldwide. It is well documented that persistent expression of two oncogenes (E6/E7) plays the key roles in cervical cancer. Thus, in vivo detection of the oncoproteins is very important for the diagnosis of the cancer. Recently, affibody molecules have been demonstrated to be a powerful targeting probe for tumor–targeted imaging and diagnosis. In this study, four HPV16 E7-binding affibody molecules (ZHPV16 E7127, ZHPV16E7301, ZHPV16E7384 and ZHPV16E7745) were screened from a phage-displayed peptide library and used for molecular imaging in tumor-bearing mice. Biosensor binding analyses showed first that the four affibody molecules bound to HPV16 E7 with very high affinity and specificity. They co-localized with E7 protein only in two HPV16-positive cancer cells (SiHa and CaSki). Furthermore, affibody ZHPV16E7384 was conjugated with Dylight755 and used for in vivo tumor-imaging. Strongly high-contrast tumor retention of this affibody only occurred in HPV16-derived tumors of mice as early as 30 min post-injection, not in HPV-negative and HPV18-derived tumors. The accumulation of Dylight755-conjugated ZHPV16E7384 in tumor was achieved over a longer time period (24 h). The data here provide strong evidence that E7-specific affibody molecules have great potential used for molecular imaging and diagnosis of HPV-induced cancers.

Human papillomavirus as a favorable prognostic factor in a subset of head and neck squamous cell carcinomas: A meta-analysis.

Many epidemical and biological studies have proposed that human papillomavirus (HPV), primarily high-risk HPV16/18, is an etiological factor for a subset of head and neck (HN) cancers. On that premise, we systematically reviewed relevant articles and improved the understanding of HPV-related cancers. This article comprehensively described the characteristics of HPV-associated HN tumors according to demography, histopathology, molecular biology, and prognosis. Meta-analyses were conducted to combine the studies that reported the association between HPV status and these variables using Rev Man 5.0. The pooled results showed that HPV-positive tumors were not only poorly differentiated (OR = 2.77, 95% CI: 2.3-3.32) and smaller (OR = 2.21, 95% CI: 1.75-2.8) but were also strongly associated with oropharynx (OR = 5.8, 95% CI: 4.01-8.38) and node involvement (OR = 2.77, 95% CI: 2.3-3.32). HPV-related tumors showed significantly more p16 overexpression (OR = 34.55, 95% CI: 20.91-57.09) and less TP53 mutations (OR = 0.27, 95% CI: 0.18-0.41) than HPV-negative tumors. The patients with HPV-positive cancers had different clinical behaviors, such as a reduced risks of death (HR = 0.32, 95% CI: 0.29-0.36). This study supported the view point that HPV is a favorable indicator of prognosis and that HPV-related HN tumors are distinct from traditional tumors. This etiological relationship could impact future strategies of diagnosis, prevention, therapy, and prognosis for this subset of patients. J. Med. Virol. 89:710-725, 2017. © 2016 Wiley Periodicals, Inc.

Candidate Biomarkers for HPV-Negative Head and Neck Cancer Identified via Gene Expression Barcode Analysis.

(1) Reanalyze publicly available genomic data for HPV-negative oral cavity squamous cell carcinoma to look for candidate biomarkers. (2) Evaluate the association of the identified biomarkers with survival. Retrospective cohort study. Tertiary care. Gene expression barcode analysis was applied to an existing publicly available data set of 54 HPV-negative oral cavity squamous cell carcinoma tumor samples to identify candidate genes associated with poor prognosis. Genes identified were evaluated for their association with survival on the basis of univariable and multivariable Cox proportional hazards models. Three genes were found to be associated with poor prognosis. The most significant association was seen with spectrin expression. Subjects whose tumors expressed spectrin were 4.60 times more likely (hazard ratio; 95% confidence interval: 1.88-11.25) to die at any given time when compared with those without spectrin (P = .001). On univariable analysis, subjects with late-stage cancer were 6.34 times more likely (hazard ratio; 95% confidence interval: 1.41-28.53; P = .02) to die at any given time, but interestingly, after controlling for spectrin, this effect was attenuated (P = .07). Despite controlling for several possible confounding effects, the effect of spectrin remained hazardous throughout all multivariable models. This was true even after controlling for cancer stage and extracapsular extension (P = .004). Our analysis of public genomic data shows promise in identifying biomarkers that may allow clinicians to make more accurate survival predictions. Spectrin is a strong candidate for further biomarker testing.

The Molecular Landscape of Recurrent and Metastatic Head and Neck Cancers

ImportanceRecurrent and/or metastatic head and neck cancer is usually incurable. Implementation of precision oncology for these patients has been limited by incomplete understanding of the molecular alterations underlying advanced disease. At the same time, the molecular profiles of many rare head and neck cancer types are unknown. These significant gaps in knowledge need to be addressed to rationally devise new therapies.ObjectiveTo illuminate the distinct biology of recurrent and metastatic head and neck cancers and review implementation of precision oncology for patients with advanced disease.Design, Setting, and ParticipantsAfter exclusions, 151 patients with advanced, treatment-resistant head and neck tumors, including squamous cell carcinoma (HNSCC), adenoid cystic carcinoma (ACC), and other salivary and cutaneous cancers, whose tumors were sequenced between January 2014 and July 2015 at Memorial Sloan Kettering were recruited. Next-generation sequencing of tumors as part of clinical care included high-depth (median 600×) exonic coverage of 410 cancer genes and whole-genome copy number analysis.InterventionsNext-generation sequencing of tumors and matched normal DNA.Main Outcomes and MeasuresFeasibility, the frequency of actionable molecular alterations, the effect on decision making, and identification of alterations associated with recurrent and metastatic disease.ResultsOverall, 151 patients (95 men and 56 women; mean [range] age, 61.8 [17-100] years) were included in the study. Next-generation sequencing ultimately guided therapy in 21 of 151 patients (14%) (13 of 53 [25%] of patients with HNSCC) by refining diagnoses and matching patients to specific therapies, in some cases with dramatic responses on basket studies. Molecular alterations were potentially actionable in 28 of 135 patients (21%). The genetic profiles of recurrent and metastatic tumors were often distinct from primary tumors. Compared to primary human papillomavirus (HPV)-positive tumors, many recurrent and metastatic HPV-positive tumors exhibited a molecular profile more similar to HPV-negative tumors, including enriched frequencies of TP53 mutation (3 of 20 tumors [15%]), whole genome duplication (5 of 20 tumors [25%]), and 3p deletion (11 of 20 tumors [55%]). There were high rates of TERT promoter mutation in recurrent and metastatic HPV-negative HNSCC (13 of 30 tumors [43%]), cutaneous SCC (11 of 21 tumors [52%]), basal cell carcinoma (3 of 4 tumors [75%]), and ACC (5 of 36 tumors [14%]). Activating NOTCH1 mutations were enriched in metastatic ACCs (8 of 36 tumors [22%]).Conclusions and RelevanceThese findings reveal the molecular landscape of advanced disease and rare cancer subtypes, both predominant challenges in head and neck oncology. To understand the repertoire of targetable alterations in advanced cancers, it is necessary to sequence recurrent and metastatic tumors. These data are important first steps toward implementation of precision head and neck oncology.

Abstract 977: RNA-sequencing analysis implicates novel non-coding RNAs in human papillomavirus-associated head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease marked by frequent recurrence and metastasis and stagnant survival rates. Given the recent emergence of human papillomavirus (HPV) as a major etiological factor in head and neck cancers, there exists a pressing need to better resolve the molecular heterogeneity of HNSCCs, define more precise diagnostic and prognostic tools, and develop a wider repertoire of targeted therapies. Non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), have been increasingly viewed as promising sources of insight into cancer onset and progression, with their diverse regulatory roles, tissue specificity, and aberrant expression in various human diseases now established by a multitude of studies. Using RNA-sequencing data from 466 HNSCC patients in The Cancer Genome Atlas (TCGA), we sought to investigate the transcriptome-wide dysregulation of both classes of ncRNAs in HPV-associated HNSCCs. Differential expression analyses controlled for patient smoking status revealed 5 lncRNAs and 9 miRNAs exhibiting significantly altered regulation between HPV-negative and HPV-positive HNSCC tumors (FDR < 1 × 10-10). Among these, 8 ncRNAs were additionally correlated with HNSCC tumor stage, invasion, and anatomic site, among other clinical characteristics (Kruskal-Wallis, p < 0.05). We subsequently validated the dysregulation of several candidate HPV-associated ncRNAs in vitro after ectopically expressing HPV16 E6/E7 in a panel of normal oral epithelial and HPV-negative HNSCC cell lines. To explore the functional consequences of our identified HPV-associated ncRNAs, we further investigated lnc-SP9-1:1, an lncRNA upregulated by 73.89 fold in HPV-positive compared to HPV-negative HNSCCs (FDR < 1 × 10-10). Cell proliferation, migration, and matrigel invasion assays were performed to evaluate the effects of siRNA-mediated lnc-SP9-1:1 knockdown in E6/E7 overexpressing cell lines. While the specific roles of the vast majority of non-coding transcripts remain unknown, their consistent dysregulation in HPV-positive HNSCCs, coupled with their ongoing functional characterization, promises to unravel new layers of understanding of the molecular underpinnings of HPV-associated HNSCCs and nominate new biomarkers and therapeutic candidates for this specific disease. Citation Format: Angela E. Zou, Aswini R. Krishnan, Yinan Xuan, Maarouf A. Saad, Avinaash Korrapati, Sunil J. Advani, Jessica Wang-Rodriguez, Weg Ongkeko. RNA-sequencing analysis implicates novel non-coding RNAs in human papillomavirus-associated head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 977.

Erratum to: Variants of EVER1 and EVER2 (TMC6 and TMC8) and human papillomavirus status in patients with mucosal squamous cell carcinoma of the head and neck.

There is a growing association of human papillomavirus (HPV) with some cases of mucosal squamous cell carcinoma of the head and neck (HNSCC), particularly of the oropharynx. Persistent oral HPV infection is believed to increase the likelihood of malignancy, and it is possible that host genetic factors can determine susceptibility to persistent HPV infection. Polymorphisms in the two EV genes (EVER1 and EVER2, also known as transmembrane channel protein (TMC) 6 and 8) have been identified as strong candidate genes, since a small number of critical mutations in these genes have been shown to cause profound and florid skin HPV infections, and some of them have been linked to susceptibility to cervical cancer. We sought to determine whether there was a difference in the frequency of single nucleotide polymorphisms (SNPs) in EVER1 (rs2613516, rs12449858) and EVER2 (rs7205422, rs12452890) between HNSCC patients with HPV-positive and HPV-negative tumors, and healthy controls. We used logistic regression to analyze SNPs in 219 patients with histologically confirmed primary SCC of the oropharynx, oral cavity, hypopharynx, or larynx, and 321 healthy controls. We did not find any associations with the EVER1/EVER2 SNPs and HPV status or being a HNSCC case or a control. The present data do not provide evidence for a role of genetic variations in EVER1 or EVER2 for HPV status of mucosal HNSCC or between HNSCC patients and controls.

Human papilloma virus prevalence in HIV patients with head and neck squamous cell carcinoma.

The implication of human papilloma virus (HPV) in head and neck squamous cell carcinoma (HNSCC) is well established, especially in oropharyngeal SCC. HIV patients have a higher risk of persistent HPV infection. We investigated the role of HPV in HNSCC carcinogenesis in HIV population. Retrospective monocentric study. We studied HIV patients who presented with HNSCC between 1994 and 2014. For each patient, tumor characteristics, HIV disease, and survival information were collected. Tumor HPV testing was performed using p16 immunohistochemistry (IHC), in-situ hybridization and PCR. We assessed the percentage of HPV in this population of HIV patients with HNSCC and compared HIV disease characteristics based on HPV status. Forty-seven patients were included: 11 women/36 men, the median age was 50 years. Tumor HPV testing was performed in 40 patients. Tumors were located in oropharynx (32%), oral cavity (32%), larynx (21%), and hypopharynx (11%). At the time of diagnosis, median CD4 level was 385 cells/μl, 31% of the patients were stage (Centers for Disease Control, stage C). The percentage of HPV linked to HNSCC for all locations in HIV patients was 30% (n = 12). HPV16 accounted for 50% of all HPV genotypes. HPV positive status was associated with a CD4 nadir of less than 200 (P = 0.026), but not with CD4 level at time of diagnosis (P = 0.414). HPV-negative tumors tend to be associated with poorer 5-year overall survival (hazard ratio = 2.9, P = 0.0711). HPV plays a critical role in HNSCC development in HIV population. HIV immunodeficiency may increase HPV persistence and progression of HNSCC.

Variants of EVER1 and EVER2 (TMC6 and TMC8) and human papillomavirus status in patients with mucosal squamous cell carcinoma of the head and neck.

There is a growing association of human papillomavirus (HPV) with some cases of mucosal squamous cell carcinoma of the head and neck (HNSCC), particularly of the oropharynx. Persistent oral HPV infection is believed to increase the likelihood of malignancy, and it is possible that host genetic factors can determine susceptibility to persistent HPV infection. Polymorphisms in the two EV genes (EVER1 and EVER2, also known as transmembrane channel protein (TMC) 6 and 8) have been identified as strong candidate genes, since a small number of critical mutations in these genes have been shown to cause profound and florid skin HPV infections, and some of them have been linked to susceptibility to cervical cancer. We sought to determine whether there was a difference in the frequency of single nucleotide polymorphisms (SNPs) in EVER1 (rs2613516, rs12449858) and EVER2 (rs7205422, rs12452890) between HNSCC patients with HPV-positive and HPV-negative tumors, and healthy controls. We used logistic regression to analyze SNPs in 219 patients with histologically confirmed primary SCC of the oropharynx, oral cavity, hypopharynx, or larynx, and 321 healthy controls. We did not find any associations with the EVER1/EVER2 SNPs and HPV status or being a HNSCC case or a control. The present data do not provide evidence for a role of genetic variations in EVER1 or EVER2 for HPV status of mucosal HNSCC or between HNSCC patients and controls.

Cell-cycle gene expression analysis using real time PCR in locally advanced squamous-cell head and neck cancer

PurposeThe analysis of gene expression, especially those involved in cell cycle control, can help to discover mechanisms determining the outcome of radiation treatment. The main purpose of this study was to examine the expression level of genes responsible for cell cycle regulation in samples of the head and neck cancer, obtained during surgery. MethodsPostsurgical samples of SCC of head and neck region were collected. Over 80 genes were analysed using cell cycle quantitative real-time RT-PCR Array method. Presence of 14 high-risk HPV types DNA in frozen or paraffin-embedded tumour pathological samples was also assessed. To correlate gene expression with selected pathological features and clinical outcome we used different hierarchical clustering method. ResultsHierarchical clustering demonstrated the association between gene expression within certain clusters and gender, tumour site, T stage, N stage, grade, pathological subtype or tumour recurrence. ConclusionsDespite some limitations we were able to identify gene clusters that allowed to classify patients according to selected clinical features and occurrence of tumour recurrence. The results of the analysis also confirm that the incidence of HPV infection among the patients from Upper Silesia is relatively low, whereas HPV negative tumours, likely associated with smoking, appeared dominant.

Integrative Genomics and Transcriptomics Analysis Reveals Potential Mechanisms for Favorable Prognosis of Patients with HPV-Positive Head and Neck Carcinomas.

Patients with HPV-positive head neck squamous cell carcinomas (HNSCC) usually have a better prognosis than the HPV-negative cases while the underlying mechanism remains far from being well understood. We investigated this issue by an integrative analysis of clinically-annotated multi-omics HNSCC data released by the Cancer Genome Atlas. As confirmatory results, we found: (1) Co-occurrence of mutant TP53 and HPV infection was rare; (2) Regardless of HPV status, HNSCCs of wild-type TP53 implied a good survival chance for patients and had fewer genome-wide somatic mutations than those with a mutation burden on the gene. Our analysis further led to some novel observations. They included: (1) The genes involved in “DNA mismatch repair” pathway were up-regulated in HPV-positive tumors compared to normal tissue samples and HPV-negative cases, and thus constituted a strong predictive signature for the identification of HPV infection; (2) HPV infection could disrupt some regulatory miRNA-mRNA correlations operational in the HPV-negative tumors. In light of these results, we proposed a hypothesis for the favorable clinical outcomes of HPV-positive HNSCC patients. That is, the replication of HPV genome and/or its invasion into the genomes of cancer cells may enhance DNA repair mechanisms, which in turn limit the accumulation of lethal somatic mutations.

Large Cell Neuroendocrine Carcinoma of the Head and Neck: A Clinicopathologic Series of 10 Cases With an Emphasis on HPV Status.

Large cell neuroendocrine carcinoma (LCNEC) is a high-grade neuroendocrine neoplasm first described in the lung and subsequently well documented in many other anatomic sites. It has only recently been recognized that LCNEC can also occasionally arise in the head and neck. The role of human papillomavirus (HPV), which is associated with some small cell carcinomas of the head and neck, has not been investigated for LCNEC. We sought to further characterize the histologic, immunophenotypic, and clinical features of LCNEC and also investigate the role of HPV in this newly described group of tumors. The surgical pathology archives of 2 large academic institutions were searched for cases of LCNEC arising in the head and neck. p16 immunohistochemistry and HPV in situ hybridization were performed, and clinical information was obtained from electronic medical records. Ten cases of head and neck LCNEC were identified. The tumors arose in 6 men and 4 women ranging in age from 14 to 70 years (median, 63.5 y). The primary tumor sites were the oropharynx (n=4), the sinonasal tract (n=3), and the larynx (n=3). The LCNECs consisted of nests and trabeculae of medium-large cells with abundant cytoplasm, coarse chromatin, and prominent nucleoli with very high mitotic rates. The tumor nests were often associated with necrosis, peripheral palisading, and rosette formations. The LCNECs were positive for pan-cytokeratin and at least 1 neuroendocrine marker (most often synaptophysin) and were largely negative for p63 (focal staining in 2/10) and CK5/6 (staining in 1/10). The LCNECs demonstrated aggressive clinical behavior: 8 of 10 presented with advanced disease, 5 of 10 died, with 4 more living but with persistent tumor. Three of 10 LCNECs were HPV-related (HPV-LCNEC); they arose in the oropharynx (n=2) and sinonasal tract (n=1). The HPV-LCNECs did not differ from the HPV-negative tumors in histologic appearance or behavior: 2 patients with HPV-LCNEC have died because of their disease and 1 remains alive but with widespread metastases. LCNEC is a rare but distinct form of head and neck carcinoma that exhibits aggressive clinical behavior. A subset of oropharyngeal and sinonasal LCNEC is HPV related, but the presence of HPV may not impart a more favorable prognosis. Because of its aggressive behavior, LCNEC should be distinguished from moderately differentiated neuroendocrine carcinoma and squamous cell carcinoma. The morphology of LCNEC overlaps considerably with the nonkeratinizing appearance of HPV-related squamous cell carcinoma, and as a result a high index of suspicion is needed to identify LCNEC. Immunohistochemical studies for synaptophysin and p63 are helpful tools for making this distinction.

Biological Features of Human Papillomavirus-related Head and Neck Cancers Contributing to Improved Response

Head and neck squamous cell carcinomas (HNSCC) are the sixth most common malignancy globally, and an increasing proportion of oropharyngeal HNSCCs are associated with the human papillomavirus (HPV). Patients with HPV-associated tumours have markedly improved overall and disease-specific survival compared with their HPV-negative counterparts when treated with chemoradiation. Although the difference in outcomes between these two groups is clearly established, the mechanism underlying these differences remains an area of investigation. Data from preclinical, clinical and genomics studies have started to suggest that an increase in radio-sensitivity of HPV-positive HNSCC may be responsible for improved outcomes, the putative mechanisms of which we will review here. The Cancer Genome Atlas and others have recently documented a multitude of molecular differences between HPV-positive and HPV-negative tumours. Preclinical investigations by multiple groups have explored possible mechanisms of increased sensitivity to therapy, including examining differences in DNA repair, hypoxia and the immune response. In addition to differences in the response to therapy, some groups have started to investigate phenotypic differences between the two diseases, such as tumour invasiveness. Finally, we will conclude with a brief review of ongoing clinical trials that are attempting to de-escalate treatment to minimise long-term toxicity while maintaining cure rates. New insights from preclinical and genomic studies may eventually lead to personalised treatment paradigms for HPV-positive patients.

Poor prognosis in human papillomavirus-positive oropharyngeal squamous cell carcinomas that overexpress hypoxia inducible factor-1α.

Hypoxia induces stabilization of the transcription factor HIF-1alpha (HIF-1α), associated with (chemo-)radiotherapy resistance in oropharyngeal squamous cell carcinoma (SCC). We investigated the effect of HIF-1α expression on survival in relation to human papillomavirus (HPV) status in oropharyngeal SCC. We conducted an immunohistochemical analysis of HIF-1α protein expression and downstream targets carbonic anhydrase-IX (CA-IX) and glucose transporter-1 (GLUT-1) in 274 patients with oropharyngeal SCC. Overall survival (OS) was analyzed in total and stratified for HPV status and treatment. In HPV-positive tumors (n = 44), HIF-1α overexpression predicted worse OS (hazard ratio [HR] = 6.23; p = .012), whereas TNM classification or treatment modality did not. In HPV-negative tumors (n = 218), advanced T and N classification and HIF-1α overexpression all independently predicted worse OS. However, the effect of HIF-1α overexpression on OS was lower in HPV-negative (HR = 1.50; p = .024) than in HPV-positive tumors. HIF-1α overexpression is associated with worse OS and characterized a subgroup of patients with HPV-positive oropharyngeal SCC with poor prognosis. Possibly, patients with HIF-1α overexpressing HPV-positive tumors should not be eligible for treatment dose deescalation. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1338-1346, 2016.

Squamous cell carcinoma of the oral cavity often overexpresses p16 but is rarely driven by human papillomavirus

Human papillomavirus (HPV) is a causal and prognostic factor for oropharyngeal cancer, but its role in squamous cell carcinoma of the oral cavity (SCCOC) is unclear. We sought to clarify HPV's role in SCCOC. Patients with newly diagnosed SCCOC (N=460) were prospectively recruited, treated, and followed at one institution. p16/HPV status was determined by p16 immunohistochemistry (IHC) (N=210), PCR-based HPV 16/18 E6/7 DNA testing (N=403), and/or HPV in situ hybridization (ISH) (N=178). Kaplan-Meier curves and log-rank tests were used to compare survival by p16/HPV status. p16 expression was detected in 30% of tumors (62/210) and HPV 16/18 E6/7 DNA in 28% (114/403), although correlation between these two assays was poor (r=-0.01). Patients with p16-positive tumors were more likely to be younger and have primary tumors of the oral tongue. Only 4% of tumors (7/171) were positive for HPV by ISH. Comparisons of patients with p16-positive and p16-negative tumors, patients with HPV-positive and HPV-negative tumors by PCR, and patients with HPV-positive and HPV-negative tumors by ISH showed no significant differences in disease-specific or disease-free survival by p16/HPV status. When we applied a more stringent definition of HPV positivity based on a combination of assay results, only 10 of 166 tumors were HPV positive, and there were no significant differences in demographic, exposure, clinical, or survival characteristics between these patients and the 156 HPV-negative patients. Very few patients with SCCOC have HPV-driven tumors. SCCOC that overexpresses p16 may be a unique subset deserving of further study.

Oropharyngeal squamous cell carcinomas differentially express granzyme inhibitors.

ObjectivesPatients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinomas (OPSCCs) have an improved prognosis compared to HPV-negative OPSCCs. Several theories have been proposed to explain this relatively good prognosis. One hypothesis is a difference in immune response. In this study, we compared tumor-infiltrating CD3+, CD4+, CD8+ T-cells, and granzyme inhibitors (SERPINB1, SERPINB4, and SERPINB9) between HPV-positive and HPV-negative tumors and the relation with survival.MethodsProtein expression of tumor-infiltrating lymphocytes (TILs) (CD3, CD4, and CD8) and granzyme inhibitors was analyzed in 262 OPSCCs by immunohistochemistry (IHC). Most patients (67 %) received primary radiotherapy with or without chemotherapy. Cox regression analysis was carried out to compare overall survival (OS) of patients with low and high TIL infiltration and expression of granzyme inhibitors.ResultsHPV-positive OPSCCs were significantly more heavily infiltrated by TILs (p < 0.001) compared to HPV-negative OPSCCs. A high level of CD3+ TILs was correlated with a favorable outcome in the total cohort and in HPV-positive OPSCCs, while it reached no significance in HPV-negative OPSCCs. There was expression of all three granzyme inhibitors in OPSCCs. No differences in expression were found between HPV-positive and HPV-negative OPSCCs. Within the group of HPV-positive tumors, a high expression of SERPINB1 was associated with a significantly worse overall survival.ConclusionHPV-positive OPSCCs with a low count of CD3+ TILs or high expression of SERPINB1 have a worse OS, comparable with HPV-negative OPSCCs. This suggests that the immune system plays an important role in the carcinogenesis of the virally induced oropharynx tumors.

Positive Effect of Surgery Regardless of Stage on Oropharynx Subsites Base of Tongue and Tonsillar-Fossa: A SEER Analysis

(OPSCC) undergoing definitive concurrent chemoradiation (CCRT) remains unclear. The purpose of this review was to examine these effects on survival outcomes among a single institute population. Materials/Methods: This retrospective review of OPSCC patients treated with CCRT between 2008 and 2015 was conducted. All tumors were examined for HPV 16/18 status (+/). Smoking status and other clinical characteristics were abstracted from the electronic medical record. Former smokers are patients who quit within a month of diagnosis or treatment. Descriptive summaries, overall survival (OS), and multivariate cox proportional hazard ratios (HR) were completed. Results: Out of 134 patients, 94 patients (71%) had HPV-positive (HPV+) tumors. Age, gender, and overall stage were not statistically different between patients with HPV+ or HPV-negative (HPV-) tumors. HPV+ patients had higher tumor grade (P<.01). Patients with HPV+ tumors had a higher percentage of never smokers than patients with HPVtumors (10.3% vs 26.6%). Median pack-years were 18 for the HPV+ group versus 30 for the HPVgroup. OS did not differ for HPV+ versus HPVpatients. Within HPV+ patients, current and former smokers had significantly worse OS than never smokers (P<.01). The same was true for HPVpatients, but the log-rank test for this group did not reach statistical significance (PZ.06). The 3-year survival rate for former smokers in each group was similar (HPV+: 0.77 (0.60, 0.88) and HPV-: 0.75 (0.52, 0.88). Hazard ratios for OS among current smokers compared to never/former smokers in each patient group were statistically significantly higher. Conclusion: Current smoking is associated with poor prognosis, independent of HPV status in OPSCC patients treated with CCRT. Former smokers have similar outcomes irrespective of HPV tumor status. The amount of tobacco a patient is exposed to before diagnosis cannot be altered, but every effort should be made to get patients to quit smoking as soon as possible before CCRT regardless of HPV status. Author Disclosure: A. Platek: None. V. Jakaprakash: None. M. Platek: None. V. Gupta: None. D. Cohan: None. W. Hicks: None. T. Winslow: None. A. Groman: None. S.S. Dibaj: None. H. Arshad: None. M.A. Kuriakose: None. G.W. Warren: None. A.K. Singh: None.

A beneficial tumor microenvironment in oropharyngeal squamous cell carcinoma is characterized by a high T cell and low IL-17(+) cell frequency.

Patients with HPV-positive oropharyngeal squamous cell carcinomas (OPSCCs) have a better prognosis than patients with non-HPV-induced OPSCC. The role of the immune response in this phenomenon is yet unclear. We studied the number of T cells, regulatory T cells (Tregs), T helper 17 (Th17) cells and IL-17+ non-T cells (mainly granulocytes) in matched HPV-positive and HPV-negative OPSCC cases (n = 162). Furthermore, the production of IFN-γ and IL-17 by tumor-infiltrating T cells was analyzed. The number of tumor-infiltrating T cells and Tregs was higher in HPV-positive than HPV-negative OPSCC (p < 0.0001). In contrast, HPV-negative OPSCC contained significantly higher numbers of IL-17+ non-T cells (p < 0.0001). Although a high number of intra-tumoral T cells showed a trend toward improved survival of all OPSCC patients, their prognostic effect in patients with a low number of intra-tumoral IL-17+ non-T cells was significant with regard to disease-specific (p = 0.033) and disease-free survival (p = 0.012). This suggests that a high frequency of IL-17+ non-T cells was related to a poor immune response, which was further supported by the observation that a high number of T cells was correlated with improved disease-free survival in the HPV-positive OPSCC (p = 0.008). In addition, we detected a minor Th17 cell population. However, T cells obtained from HPV-positive OPSCC produced significantly more IL-17 than those from HPV-negative tumors (p = 0.006). The improved prognosis of HPV-positive OPSCC is thus correlated with higher numbers of tumor-infiltrating T cells, more active Th17 cells and lower numbers of IL-17+ non-T cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-016-1805-x) contains supplementary material, which is available to authorized users.

Non-coding RNAs profiling in head and neck cancers.

The majority of studies on human cancers published to date focus on coding genes. More recently, however, non-coding RNAs (ncRNAs) are gaining growing recognition as important regulatory components. Here we characterise the ncRNA landscape in 442 head and neck squamous cell carcinomas (HNSCs) from the cancer genome atlas (TCGA). HNSCs represent an intriguing case to study the potential role of ncRNA as a function of viral presence, especially as HPV is potentially oncogenic. Thus, we identify HPV16-positive (HPV16+) and HPV-negative (HPV−) tumours and study the expression of ncRNAs on both groups. Overall, the ncRNAs comprise 36% of all differentially expressed genes, with antisense RNAs being the most represented ncRNA type (12.6%). Protein-coding genes appear to be more frequently downregulated in tumours compared with controls, whereas ncRNAs show significant upregulation in tumours, especially in HPV16+ tumours. Overall, expression of pseudogenes, antisense and short RNAs is elevated in HPV16+ tumours, while the remaining long non-coding RNA types are more active in all HNSC tumours independent of HPV status. In addition, we identify putative regulatory targets of differentially expressed ncRNAs. Among these ‘targets’ we find several well-established oncogenes, tumour suppressors, cytokines, growth factors and cell differentiation genes, which indicates the potential involvement of ncRNA in the control of these key regulators as a direct consequence of HPV oncogenic activity. In conclusion, our findings establish the ncRNAs as crucial transcriptional components in HNSCs. Our results display the great potential for the study of ncRNAs and the role they have in human cancers.

Characterization of gene methylation in human papillomavirus associated-head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) has become one of the most common forms of cancer worldwide. Hypermethylation-induced silencing of tumor-associated gene has been proposed as an important cofactor in cancer pathology. This paper aimed to characterize the gene methylation patterns in human papillomavirus (HPV) associated-HNSCC. TIMP3 and APC methylation status in neoplastic (N = 92) and non-neoplastic tissues (N = 92) of HNSCC as well as their association with HPV infection were investigated via methylation-specific PCR assays. Results indicated that methylation level of TIMP3 was markedly higher in HPV-positive tumors as compared with HPV-negative tumors. Both TIMP3 and APC methylation were associated with lymph node metastasis and higher clinical stage of tumors. Patients with methylation at TIMP3 or APC had worse prognoses as compared to those without these alterations. This is the first study that shows a possible linkage between HPV infection and APC methylation. Methylation patterns of tumor-related genes may contribute to different disease prognosis in HNSCC according to the HPV infection status.

Human papillomavirus genotype and oropharynx cancer survival in the United States of America

BackgroundThe presence of human papillomavirus (HPV) DNA in oropharyngeal squamous cell cancer (OPSCC) tissue appears to be a strong predictor of improved prognosis, but this observation has not been explored in a population-based sample with generalisable findings. MethodsFollow-up data from a large sample of OPSCC patients identified through six population-based cancer registries in the United States of America (USA) were used to characterise the association of tumour HPV status with survival. ResultsHPV DNA was detected in tumour tissue from 71% (378 in 529) of the OPSCC patients. A total of 65% of patients with HPV16-associated tumours survived 5 years compared to 46% of patients with other HPV types and 28% of patients with HPV-negative tumours (p log-rank test <0.0001). The OPSCC patients with detectable HPV16 DNA had a 62% reduced hazard of death at 5 years, and patients with other HPV types had a 42% reduced hazard of death at 5 years compared to HPV-negative patients. Compared to non-Hispanic Whites, Blacks with OPSCC had a 2.6-fold greater risk of death at 5 years after adjustment for HPV status and other prognostic variables. Both surgery and radiation therapy were associated with a reduced 5-year risk of death, but no evidence was found for an interaction between HPV status and radiotherapy or surgery on survival time. ConclusionsData from this US study suggest that HPV16-positive OPSCC patients survive longer than HPV-negative patients regardless of treatment, highlighting the prognostic importance of HPV status for this malignancy. Optimal treatment regimens for OPSCC could be tailored to each patient’s HPV status and prognostic profile.