A prognostic gene expression signature for oropharyngeal squamous cell carcinoma

BACKGROUND: Robust prognostic stratification of patients with oropharyngeal squamous cell carcinoma (OPSCC) is important for developing individualized treatment plans. This study was conducted to develop and validate a clinically feasible prognostic classifier based on transcriptome-wide gene expression profiles. METHODS: Tumor tissues were collected from 208 OPSCC patients treated at Washington University in St. Louis and 130 OPSCC patients treated at Vanderbilt University, used for model training and validation, respectively. OPSCC patients (n = 70) from the TCGA cohort were also included for independent validation. Based on RNA-seq profiling data, Cox proportional hazards regression analysis was performed to identify genes associated with disease outcomes. Then, Lasso-penalized multivariate survival models were constructed to identify biomarker genes for developing a prognostic gene signature. FINDINGS: A 60-gene signature was identified by RNA-seq profiling analysis. Computed risk score of the gene signature was significantly predictive of 5-year overall survival of the training cohort (Hazard ratio (HR) 28·32, P = 4·3E-41). Subgroup analysis stratified by HPV status revealed that the signature was prognostic in HPV-positive OPSCC patients (HR 30·55, P = 7·0E-37) and was independent of clinical features. Importantly, the gene signature was validated in two independent patient cohorts, including the TCGA cohort (HR 3·94, P = 0·0018) and the Vanderbilt cohort (HR 8·50, P = 5·7E-09) for overall survival. INTERPRETATION: The prognostic gene signature is a robust tool for risk stratification of OPSCC patients. The signature remains prognostic among HPV-positive OPSCC patients. FUNDING: National Institutes of Health

APOBEC Mutagenesis Is Concordant between Tumor and Viral Genomes in HPV-Positive Head and Neck Squamous Cell Carcinoma

APOBEC is a mutagenic source in human papillomavirus (HPV)-mediated malignancies, including HPV+ oropharyngeal squamous cell carcinoma (HPV + OPSCC), and in HPV genomes. It is unknown why APOBEC mutations predominate in HPV + OPSCC, or if the APOBEC-induced mutations observed in both human cancers and HPV genomes are directly linked. We performed sequencing of host somatic exomes, transcriptomes, and HPV16 genomes from 79 HPV + OPSCC samples, quantifying APOBEC mutational burden and activity in both host and virus. APOBEC was the dominant mutational signature in somatic exomes. In viral genomes, there was a mean of five (range 0–29) mutations per genome. The mean of APOBEC mutations in viral genomes was one (range 0–5). Viral APOBEC mutations, compared to non-APOBEC mutations, were more likely to be low-variant allele fraction mutations, suggesting that APOBEC mutagenesis actively occurrs in viral genomes during infection. HPV16 APOBEC-induced mutation patterns in OPSCC were similar to those previously observed in cervical samples. Paired host and viral analyses revealed that APOBEC-enriched tumor samples had higher viral APOBEC mutation rates (p = 0.028), and APOBEC-associated RNA editing (p = 0.008), supporting the concept that APOBEC mutagenesis in host and viral genomes is directly linked and occurrs during infection. Using paired sequencing of host somatic exomes, transcriptomes, and viral genomes, we demonstrated for the first-time definitive evidence of concordance between tumor and viral APOBEC mutagenesis. This finding provides a missing link connecting APOBEC mutagenesis in host and virus and supports a common mechanism driving APOBEC dysregulation

Summary from an international cancer seminar focused on human papillomavirus (HPV)-positive oropharynx cancer, convened by scientists at IARC and NCI

Cancer of the oropharynx has attracted considerable attention in recent years given: (1) an increasing incidence in selected populations over the past three decades; (2) the discovery of human papillomavirus (HPV) infection as the driver of the increase, as opposed to the traditional risk factors such as tobacco (smoking and chewing) and alcohol; and (3) the promise of new prevention and treatment strategies. As a result of such developments, the International Agency for Research on Cancer (IARC) and the US National Cancer Institute (NCI), convened the fourth Cancer Seminar meeting in November 2018 to focus on this topic. This report summarizes the proceedings: a review of recent science on the descriptive epidemiology, etiology, biology, genetics, early detection, pathology and treatment of HPV-positive oropharyngeal cancer, and the formulation of key research questions to be addressed

Human Papillomavirus Antibodies And Future Risk Of Anogenital Cancer: A Nested Case-control Study In The European Prospective Investigation Into Cancer And Nutrition Study

Purpose: Human papillomavirus (HPV) type 16 (HPV16) causes cancer at several anatomic sites. In the European Prospective Investigation Into Cancer and Nutrition study, HPV16 E6 seropositivity was present more than 10 years before oropharyngeal cancer diagnosis and was nearly absent in controls. The current study sought to evaluate the extent to which HPV16 E6 antibodies are present before diagnosis of anogenital cancers within the same cohort. Methods: Four hundred incident anogenital cancers (273 cervical, 24 anal, 67 vulvar, 12 vaginal, and 24 penile cancers) with prediagnostic blood samples (collected on average 3 and 8 years before diagnosis for cervix and noncervix cancers, respectively) and 718 matched controls were included. Plasma was analyzed for antibodies against HPV16 E6 and multiple other HPV proteins and genotypes and evaluated in relation to risk using unconditional logistic regression. Results: HPV16 E6 seropositivity was present in 29.2% of individuals (seven of 24 individuals) who later developed anal cancer compared with 0.6% of controls (four of 718 controls) who remained cancer free (odds ratio [OR], 75.9; 95% CI, 17.9 to 321). HPV16 E6 seropositivity was less common for cancers of the cervix (3.3%), vagina (8.3%), vulva (1.5%), and penis (8.3%). No associations were seen for non-type 16 HPV E6 antibodies, apart from anti-HPV58 E6 and anal cancer (OR, 6.8; 95% CI, 1.4 to 33.1). HPV16 E6 seropositivity tended to increase in blood samples drawn closer in time to cancer diagnosis. Conclusion: HPV16 E6 seropositivity is relatively common before diagnosis of anal cancer but rare for other HPV-related anogenital cancers

Development of novel Hepatitis C Virus DNA vaccines

Hepatitis C Virus (HCV) is a global pandemic affecting over 170 million individuals worldwide. Currently there is no vaccine for HCV however it is believed that an effective vaccine strategy must be able to induce strong intrahepatic HCV-specific T cell-based immunity. In this thesis, we have taken a multi-step approach to develop four distinct HCV DNA vaccines encoding consensus genotype 1 immunogens of HCV proteins, NS3/NS4A, NS4B, NS5A and NS5B. We show that these constructs are expressed in vitro and are able to induce potent peripheral HCV-specific IFN-γ+ T cell responses in C57BL/6 mice. In order to test the ability of immunization to induce functional HCV-specific T cell-based immunity in the liver, a small animal model of HCV infection was developed. In this model, we show that we are able to induce liver selective expression of HCV viral proteins in C57BL/6 mice and that liver expression of HCV NS3 is able to mimic the immunological affects of HCV infection as evidenced by liver inflammation, upregulation of liver chemokine expression, splenomegaly and recruitment of T cells to the liver. Using this model, we have shown the all four constructs are able to induce potent HCV-specific T cell-based immunity within the liver both in the presence and absence of infection as shown by upregulation of IFN-γ and clearance of HCV antigen expressing hepatocytes. While highly immunogenic in mice, given the both the immunological differences between mice and humans, as well as, the reported decrease in immunogenicity of DNA vaccines when moving into larger animal models, one HCV DNA vaccine construct was further tested in the non-human primate, rhesus macaque model. In this model, immunization was shown to induce HCV-specific T cell-based immunity strikingly similar to that seen in patients with resolving HCV infection and included strong proliferative responses, polyfunctional T cells and broadly reactive T cell responses able to recognize multiple epitopes spanning the length of the NS3 protein. Taken together our findings suggest that this type of immunization strategy merits further study in the context of future prophylactic HCV T cell-based vaccines

Induction of Intrahepatic HCV NS4B, NS5A and NS5B-Specific Cellular Immune Responses following Peripheral Immunization

<div><p>Numerous studies have suggested that an effective Hepatitis C Virus (HCV) vaccine must induce strong cytotoxic and IFN-γ+ T cell responses targeting the non-structural region of the virus. Most importantly, these responses must be able to migrate into and remain functional within the liver, an organ known to cause T cell tolerance. Using three novel HCV DNA vaccines encoding non-structural proteins NS4B, NS5A and NS5B, we assessed the ability of peripheral immunization to induce functional intrahepatic immunity both in the presence and absence of cognate HCV antigen expression within the liver. We have shown that these constructs induced potent HCV-specific CD4+ and CD8+ T cell responses in the spleen of C57BL/6 mice and that these responses were detected within the liver following peripheral immunization. Additionally, using a transfection method to express HCV antigen within the liver, we showed that intrahepatic HCV-specific T cells remained highly functional within the liver and retained the ability to become highly activated as evidenced by upregulation of IFN-γ and clearance of HCV protein expressing hepatocytes. Taken together, these findings suggest that peripheral immunization can induce potent HCV-specific T cell responses able to traffic to and function within the tolerant environment of the liver.</p> </div

Expression and immunogenicity of pConNS4B, pConNS5A and pConNS5B.

<p><b>A</b>) Human RD muscle cells were transiently transfected with each construct. Expression of each gene product was detected using an anti-HA monoclonal antibody and confocal microscopy (250×). <b>B</b>) IFN-γ ELISpot dose response. Animals (n = 5 per group) were immunized with 5 µg, 12.5 µg or 25 µg of pConNS4B, pConNS5A and pConNS5B. Animals received a total of two intramuscular immunizations, two weeks apart followed by electroporation. Animals were sacrificed one week following the last immunization after which splenocytes were isolated and analyzed. The response of each animal to each dose was determined with IFN-γ ELISpot assays. Splenocytes were isolated and individually analyzed for NS4B-, NS5A- or NS5B-specific T cell responses. <b>C, D, E</b>) Splenocytes were intracellularly stained for IFN-γ and analyzed with flow cytometry. <b>C</b>) Representative animal from each group. The values shown are the averaged response of five individual animals from each group. <b>D</b>) and <b>E</b>) graphical representation of percent HCV-specific IFN-γ+ T cell responses from isolated splenocytes. Values are reported as the average percent ± SE of the <b>D</b>) CD4+ IFN-γ+ or <b>E</b>) CD8+ IFN-γ+ T cell responses of each animal (n = 5) from each group. Significance was determined by Student's <i>t</i> test (*p<0.05, **p<0.005 and ***p<0.0005).</p

Flow cytometric analysis of the percentage of HCV-specific IFN-γ+ T cell responses from isolated liver lymphocytes.

<p>Values are reported as the average percent ± SE of HCV-specific <b>A</b>) CD4+ IFN-γ+ or <b>B</b>) CD8+ IFN-γ+ T cell responses of each animal (n = 5) from each group. Significance was determined by Student's <i>t</i> test (*p<0.05, **p<0.005 and ***p<0.0005).</p