HPV-positive Head Research Articles

Abstract 5175: Birinapant enhances death agonist antibody against TRAILR2 anti-tumor activity in HPV-positive head and neck squamous cell carcinomas

Abstract Human papilloma virus positive (HPV+) head and neck squamous cell carcinomas (HNSCC) exhibit a better prognosis and response to therapies than HPV(-) cancers. Analysis of HNSCC TCGA dataset provides evidence for distinct alterations in expression of components of the NF-κB and cell death pathways in HPV(+/-) HNSCC. Previously, we have found that birinapant, a novel SMAC mimetic that inhibits inhibitor of apoptosis proteins (IAPs), sensitizes a subset of HPV(-) HNSCC cell lines to death agonists like TNF-α and TRAIL. In our current study, we have observed that birinapant also sensitizes several HPV(+) cell lines to TNF-α and TRAIL in vitro. The IC50 for birinapant was under 50nM with TRAIL and TNF-α for the HPV (+) UPCI-SCC-90 and UM-SCC-47 cell lines. As TRAIL is known as a selective cancer cell death inducer, we explored its potential for enhancing death signaling in HPV(+) HNSCC cells via the effects of an agonistic polyclonal TRAILR2 antibody. Flow cytometry analysis confirmed the presence of TRAILR2 expression on the cell surface of the two HPV(+) cell lines. Treatment of cells with the antibody or TRAIL alone showed little or no inhibitory effect on growth of either cell lines. However, treatment with birinapant plus antibody, and especially the triple combination of birinapant, TRAIL, and antibody showed additive or synergistic effects to inhibit cell proliferation in a dose dependent manner. The Fixed Apoptotic Necrotic test using anti-phosphatidylserine and Zombie fixable dye showed that late apoptosis was the predominant mode of cell death for all combination groups for both cell lines at 48 hours post treatment. The proportion of cells undergoing apoptosis/necrosis was highest in the triple combination group in both cell lines, followed by birinapant and TRAIL combination for UM-SCC-47, or birinapant and antibody combination for UPCI-SCC-90, which was consistent with the relative percentage of sub-G0 DNA determined by flow cytometry analysis using propidium iodide staining. Enhanced apoptosis/necrosis with the triple combination was observed earlier than with single or dual treatment (12 versus 24 hours post treatment). Specific cell death mechanism was analyzed using pan-caspase (ZVAD), caspase-8 (ZIETD), and RIPK1 (necrostatin) inhibitors. In both UPCI-SCC-90 and UM-SCC-47 cells, caspase inhibition (ZVAD and ZIETD) completely reversed the effects of the double or triple combination treatments, whereas necrostatin did not. This suggests that the TRAILR2 agonist antibody mediated cell death in combination with birinapant is predominantly caspase-8 dependent. These results indicate that the TNF-α, TRAIL, and TRAILR2 agonist antibody sensitized birinapant anti-tumor activity, and triple combination exhibited synergistic effects. Supported by NIDCD projects ZIA-DC-000016, -73, -74, and the Medical Research Scholars Program. Citation Format: Yi An, Jun W. Jeon, Lillian Sun, Adeeb Derakhshan, Jianhong Chen, Hui Cheng, Xinping Yang, Christopher Silvin, Carter Van Waes, Zhong Chen. Birinapant enhances death agonist antibody against TRAILR2 anti-tumor activity in HPV-positive head and neck squamous cell carcinomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5175.

Abstract 2114: HPV-driven cancers show distinct methylation signatures in cell-free DNA (cfDNA)

Abstract Accurate tissue of origin (TOO) prediction is crucial for effective clinical follow-up in early cancer detection from blood. In the second substudy of the Circulating Cell-free Genome Atlas (CCGA; NCT02889978), we trained logistic regression classifiers under cross-validation to detect and localize cancer. Input features were methylation states from a targeted cfDNA assay of 2023 participants. TOO classification accuracy was 89% across 20 pre-specified prediction classes. We subsequently sought to understand the causes of the remaining TOO errors. 45% of the errors fell into clusters reflecting similarities in developmental biology, histology, or oncological drivers. Here, we analyzed tissues that may be affected by HPV-driven cancers; these accounted for 21% of TOO errors. The original classifier, which used only human epigenetic states as input, demonstrated cross-scoring between likely HPV-driven cancers of the anus (N = 14) and cervix (N = 11), as well as confirmed HPV-positive head & neck (H&N) cancers (37/62). We also observed HPV-associated vulva (N = 9) and penis (N = 1) cancers, which were not directly trained as TOO classes, were assigned high H&N scores. To test the hypothesis of HPV-driven TOO confusion, we assessed HPV cfDNA and HPV-driven methylation in human peripheral blood cfDNA, and used a specialist classifier restricted to HPV-associated cancers to resolve errors. We corroborated putative HPV-positive participants using targeted sequencing of HPV16 and HPV18 cfDNA fragments, and showed that the number of unique HPV-derived fragments in a sample matched with expected cancer localizations, HPV subtypes, and HPV status. Consistent with the literature, we found little evidence of HPV viremia in non-cancer participants despite the high prevalence of transient HPV infections in the US population. At 99.8% specificity, a cross-validated cutoff on the number of HPV cfDNA fragments in a sample achieved sensitivities of 78.6% (11/14), 36.3% (4/11), 66.6% (6/9), 100% (1/1), and 81.0% (30/37), for anus, cervix, vulva, penis, and confirmed HPV-positive H&N cancers, respectively. These sensitivities were similar to those achieved by the epigenetic classifier. Finally, we trained a cross-validated specialist classifier using the same features as the TOO classifier, but restricted to HPV-driven cancers. This improved TOO accuracy for detected anal cancers from 11% (1/9) to 100% (9/9), with little effect on other classes. These data support that HPV presence may explain observed cross-scoring patterns between H&N, cervix, and anus TOO prediction classes, which were driven by recurrent epigenomic changes in participants with HPV-positive cancers as detected by this assay. This suggests that modelling axes of shared biology across cancer types can be useful for accurate cfDNA TOO classification, which is critical to direct diagnostic work-up of diverse cancer types in a multi-cancer early detection test. Citation Format: Robert Calef, Oliver Venn, M. Cyrus Maher, John F. Beausang, Earl Hubbell, Aman Patel, Alexander P. Fields, Joerg Bredno, Arash Jamshidi, Alexander M. Aravanis. HPV-driven cancers show distinct methylation signatures in cell-free DNA (cfDNA) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2114.

BCCIPβ facilitates p53 ubiquitination via binding with E6 protein in high-risk HPV positive head and neck squamous cell carcinoma

BRCA2 And CDKN1A Interacting Protein (BCCIP) is initially identified as a tumor suppressor. Some recent studies confirmed its p53 binding capability. In this study, we explored the regulatory effect of BCCIPβ on p53 stability in HPV-positive and HPV-negative HNSCC cells. RNA-seq data from TCGA-HNSC were extracted for transcript isoform analysis in HPV-positive and HPV-negative tumors. HPV16-positive UM-SCC-47 (SCC47) and UM-SCC-104 (SCC104) and HPV-negative SCC-9 (SCC9) and UM-SCC-1 (SCC1) cell lines were used as in vitro cell models. Results showed that BCCIPβ was the dominant transcript in both HPV-positive and HPV-negative HNSCC cases. Knockdown of BCCIPβ decreased p53 protein concentration in the two HPV-negative cell lines but increased p53 concentration in the two HPV-positive cell lines. BCCIPβ inhibition increased proliferation and G1/S transition of SCC9 and SCC1 cells. In comparison, BCCIPβ inhibition slowed proliferation and increased G1 arrest of SCC104 and SCC47 cells. BCCIPβ inhibition prolonged the half-life of p53 protein and reduced p53 ubiquitination in the two HPV16-positive cell lines. Co-IP assay confirmed interactions among BCCIPβ, HPV E6, and p53 in both SCC104 and SCC47 cells. In comparison, only the interaction between BCCIPα and p53 was confirmed in these two cell lines. Either E6 or BCCIPβ inhibition reduced p53 ubiquitination and increased p53 concentration. However, inhibiting E6 and BCCIPβ at the same did not generate synergistic effects. On the contrary, p53 ubiquitination level was even higher in the combination group, with lower p53 concentration compared to the shE6 group. BCCIPβ overexpression in SCC47 cells with HPV E6 depletion significantly reduced p53 ubiquitination. In conclusion, this study found a novel interaction between HPV E6 and BCCIPβ in HPV16-positive HNSCC cells. The presence of HPV E6 turned BCCIPβ from a p53 stabilizer to a ubiquitination facilitator. This mechanism helps explain why BCCIPβ acted as a tumor suppressor in HPV-negative HNSCC but exerted oncogenic function in HPV16-positive HNSCC.

Endogenous APOBEC3B overexpression characterizes HPV-positive and HPV-negative oral epithelial dysplasias and head and neck cancers.

The DNA cytosine deaminase APOBEC3B (A3B) is a newly recognized endogenous source of mutations in a range of human tumors, including head/neck cancer. A3B inflicts C-to-T and C-to-G base substitutions in 5'-TCA/T trinucleotide motifs, contributes to accelerated rates of tumor development, and affects clinical outcomes in a variety of cancer types. High-risk human papillomavirus (HPV) infection causes A3B overexpression, and HPV-positive cervical and head/neck cancers are among tumor types with the highest degree of APOBEC signature mutations. A3B overexpression in HPV-positive tumor types is caused by the viral E6/E7 oncoproteins and may be an early off-to-on switch in tumorigenesis. In comparison, less is known about the molecular mechanisms responsible for A3B overexpression in HPV-negative head/neck cancers. Here, we utilize an immunohistochemical approach to determine whether A3B is turned from off-to-on or if it undergoes a more gradual transition to overexpression in HPV-negative head/neck cancers. As positive controls, almost all HPV-positive oral epithelial dysplasias and oropharyngeal cancers showed high levels of nuclear A3B staining regardless of diagnosis. As negative controls, A3B levels were low in phenotypically normal epithelium adjacent to cancer and oral epithelial hyperplasias. Interestingly, HPV-negative and low-grade oral epithelial dysplasias showed intermediate A3B levels, while high-grade oral dysplasias showed high A3B levels similar to oral squamous cell carcinomas. A3B levels were highest in grade 2 and grade 3 oral squamous cell carcinomas. In addition, a strong positive association was found between nuclear A3B and Ki67 scores suggesting a linkage to the cell cycle. Overall, these results support a model in which gradual activation of A3B expression occurs during HPV-negative tumor development and suggest that A3B overexpression may provide a marker for advanced grade oral dysplasia and cancer.

Galectin-3 Expression in High-Risk HPV-Positive and Negative Head & Neck Squamous Cell Carcinomas and Regional Lymph Node Metastases.

Despite higher stages at presentation, patients with high-risk (HR) HPV-related (HPV+) head and neck squamous cell carcinomas (HNSCCs) have better survival rates compared to those with non-HPV-related (HPV-) disease. However, significant comorbidity and the number of patients who suffer failed treatment, recurrent disease, late metastasis, and deathare increasing along with the incidence of HPV+ HNSCC. A cytotoxic T-cell-dependent immune response is required to clear these antigenic cancers. This provides a unique opportunity to employ immune modulators in therapy. Galectin-3 (Gal-3) is a lectin and glycoprotein involved in numerous immunosuppressive functions. Inhibitors are currently under clinical investigation for various diseases. Gal-3 expression was evaluated in HR-HPV+ and HPV- HNSCCs and regional lymph node metastases by tissue microarray. HR-HPV+ cases were more likely to be Gal-3-positive (Gal+) [50% (14/28)] than HPV- cases [18% (9/50), p = 0.004]. No difference in the number of Gal+ cases was identified between primary [30% (16/53)] and metastatic [28% (7/25)] cancers (p = 1); 53% (9/17) of primary HPV+ cancers were Gal+ and 45% (5/11) of metastatic HPV+ cancers were Gal+ (p = 1). Nineteen percent (7/36) of primary HPV- cancers were Gal+ and 14% (2/14) of metastatic HPV- cancers were Gal+ (p = 1). Gal-3 positivity was observed in a subset of HNSCC, suggesting a potential role for therapeutic inhibition in this tumor type. The significantly higher rates of expression seen in HR-HPV+ versus HPV- HNSCC suggest particularpromise in the setting of HPV infection. The relatively consistent Gal-3 expression rates observed between metastatic and primary tumors argues against progressive Gal-3 expressionin metastasis.

Targeting DNA Double-Strand Break Repair Enhances Radiosensitivity of HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinoma to Photons and Protons.

The response of head and neck squamous cell carcinoma (HNSCC) to radiotherapy depends on human papillomavirus type 16 (HPV) status, and where improved outcome and survival is observed in HPV-positive disease. However, strategies to further radiosensitise the tumours, particularly relatively radioresistant HPV-negative HNSCC, are actively being sought. The impact of targeting the major protein kinases involved in the signaling of DNA double-strand break (DSB) repair, namely ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and the catalytic subunit of DNA-dependent protein kinase (DNA-Pkcs), on the radiosensitisation of HNSCC cells was examined. The response to both conventional photon radiotherapy, but also proton beam therapy, was analysed by clonogenic assays and 3D spheroid growth. We observed that inhibition of ATM, ATR, and particularly DNA-Pkcs, caused a significant reduction in HNSCC cell survival post-irradiation with both photons and protons, with less of an impact on the most radiosensitive HPV-positive cell line. The inhibition of DNA-Pkcs and, to a lesser extent ATM, in combination with radiation was also more effective at inhibiting the growth of 3D spheroids derived from relatively radioresistant HPV-negative HNSCC. Similar effects of the inhibitors were observed comparing photon and proton irradiation, demonstrating the potential for targeting DSB repair as an effective combination treatment for HNSCC.

De-Escalation After DE-ESCALATE and RTOG 1016: A Head and Neck Cancer InterGroup Framework for Future De-Escalation Studies.

Human papillomavirus (HPV)-positive oropharyngeal cancer (OPC) is increasing rapidly. The younger age, significantly improved prognosis, and relative morbidity of the standard-of-care cisplatin and radiotherapy in this population have led to the popularization of the concept of treatment de-escalation. The recent results of the first 3 randomized de-escalation trials, however, have shown a clear detriment in survival when cisplatin is omitted or substituted. In view of these results, the Head and Neck Cancer International Group identified the need to issue guidance regarding future de-escalation studies for patients with HPV-positive head and neck cancer to avoid the possibility of patients being harmed. We review the current state of the literature regarding HPV de-escalation trials and present a framework and guidance on future and existing clinical trials for treatment de-escalation of HPV-positive OPC. De-escalation paradigms of HPV-positive OPC should be evaluated in phase II studies, and results should be awaited before proceeding to phase III studies. Implementation into clinical practice before high-level evidence is available should not be undertaken in this context. Finally, harm-minimization techniques should also be evaluated as an alternative to de-escalation of treatment in these patient groups.

Cannabinoids Promote Progression of HPV-Positive Head and Neck Squamous Cell Carcinoma via p38 MAPK Activation.

Human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is associated with daily marijuana use and is also increasing in parallel with increased marijuana use in the United States. Our study is designed to define the interaction between cannabinoids and HPV-positive HNSCC. The expression of cannabinoid receptors CNR1 and CNR2 was analyzed using The Cancer Genome Atlas (TCGA) HNSCC data. We used agonists, antagonists, siRNAs, or shRNA-based models to explore the roles of CNR1 and CNR2 in HPV-positive HNSCC cell lines and animal models. Cannabinoid downstream pathways involved were determined by Western blotting and analyzed in a primary HPV HNSCC cohort with single-sample gene set enrichment analysis (ssGSEA) and the OncoGenome Positioning System (Onco-GPS). In TCGA cohort, the expression of CNR1 and CNR2 was elevated in HPV-positive HNSCC compared with HPV-negative HNSCC, and knockdown of CNR1/CNR2 expression inhibited proliferation in HPV-positive HNSCC cell lines. Specific CNR1 and CNR2 activation as well as nonselective cannabinoid receptor activation in cell lines and animal models promoted cell growth, migration, and inhibited apoptosis through p38 MAPK pathway activation. CNR1/CNR2 antagonists suppressed cell proliferation and migration and induced apoptosis. Using whole-genome expression analysis in a primary HPV HNSCC cohort, we identified specific p38 MAPK pathway activation signature in tumors from HPV HNSCC patients with objective measurement of concurrent cannabinoid exposure. Cannabinoids can promote progression of HPV-positive HNSCC through p38 MAPK pathway activation.

DNMT1 Enhances the Radiosensitivity of HPV-Positive Head and Neck Squamous Cell Carcinomas via Downregulating SMG1.

IntroductionHead and neck squamous cell carcinoma (HNSCC), which rank the 7th malignant tumors worldwide, is closely related to methylation and HPV infection. Ionizing radiation therapy is the main strategy for HNSCC patients in advanced stage. Previously, HPV-positive HNSCC predict better prognosis than HPV-negative HNSCCs under radiotherapy, however its molecular mechanism is unresolved. SMG1 serves as a potential tumor suppressor in various cancers, including HNSCC.MethodsThe mRNAs and proteins expression of HPV E6/E7, p16, p53, DNMT1, SMG1 were detected after different treatments by qPCR and Western blot. The clone formation ability was measured in radiation dose after different treatments.ResultsIn our study, the expression of HPV16 E6, DNA Methyltransferase 1(DNMT1) and SMG1 in head and neck carcinomas cell lines was detected by RT-qPCR and Western blot. Forced E6 level in HPV-negative cells by overexpression plasmid promoted the expression of DNMT1, which resulted in decreased SMG1 expression. Silenced SMG1 in HPV-negative HNSCC cells elicited increased radiation sensitivity, suggesting that SMG1 may be an effective switch to regulate the effect of radiotherapy in HNSCC.ConclusionOur study indicated that DNMT1 enhances the radiosensitivity of HPV-positive head and neck squamous cell carcinomas via downregulating SMG1.

Immunotherapy improves efficacy and safety of patients with HPV positive and negative head and neck cancer: A systematic review and meta-analysis.

Despite multiple modalities used to management of head and neck squamous cell carcinoma (HNSCC), disease control remains unsatisfactory. Immunotherapy is emerging as a novel therapeutic approach. This systematic review assesses clinical data regarding immunotherapy efficacy and safety. Data from 11 clinical trials testing immunotherapy in HNSCC were assessed. We performed the meta-analysis to correlate the overall survival (OS), response rate (RR), adverse effects, HPV status, and PD-L1 expression. Immunotherapy extended OS (hazard ratio = 0.77, p < 0.0001) and RR significantly (risk ratio = 1.41, p = 0.02). Patients with HPV-positive HNSCC exhibited a better RR (risk ratio = 1.29, p = 0.24) and OS (11.5 vs. 6.3 months). PD-L1 positive tumors showed a higher OS (9.9 vs. 6.5 months). Moreover, immunotherapy caused less adverse effects than standard therapy. Our results indicate the benefit of immunotherapy for improving RR and OS of HNSCC patients. The benefit is higher in patients with HPV and PD-L1 positive tumors.

Abstract A36: CXCL14-mediated antigen-specific CD8+ T-cell responses suppress HPV-positive head and neck cancer

Abstract Human papillomaviruses (HPVs) cause over 5% of all human cancer incidences, including a subset of head and neck squamous cell carcinoma (HNSCC), resulting in about half a million deaths every year. Persistent infection with high-risk HPV is necessary for development of HPV-associated malignancies. To establish persistence in the host, HPV must evade host antiviral defenses, including the innate and adaptive immune responses. As antiviral and antitumor immune responses share similar mechanisms, it is likely that HPV-induced immune suppression leads to immune evasion and survival of HPV-infected cancer cells. We have previously shown that expression of the chemokine CXCL14 is significantly downregulated by promoter methylation mediated by the HPV oncoprotein E7 during cancer progression. Restoration of CXCL14 expression in HPV-positive HNSCC cells dramatically suppresses tumor growth through an immune-dependent mechanism in mice. While CXCL14 recruits NK and CD8+ T cells to the tumor microenvironment, the roles of NK and T cells to enact the CXCL14-mediated tumor suppression remained undefined. To determine the roles of NK and CD8+ T cells, tumor growth was assessed in wild-type C57BL/6 mice depleted of NK or CD8+ T cells and CD8-knockout mice injected with the HPV-positive HNSCC cells re-expressing CXCL14. The results show that that CD8+ T cells are required for CXCL14-mediated tumor suppression. The antitumor CD8+ T-cell responses require antigen specificity as a transgenic model of mice with a restricted CD8+ T-cell receptor failed to control tumor growth. Counteracting the HPV-mediated downregulation of major histocompatibility complex class I (MHC-I), CXCL14 expression restores MHC-I expression on HPV-positive HNSCC cells. Furthermore, knockdown of MHC-I expression in CXCL14 expressing HNSCC cells results in loss of tumor suppression, defining a critical role for antigen presentation. These results suggest that CXCL14 expression drives antigen-specific CD8+ T-cell responses and suppresses tumor growth through the restoration of MHC-I expression in HPV-positive HNSCC. Our findings provide useful insight into the CXCL14-mediated antitumor mechanism that increases CD8+ T-cell recognition of tumor cells and could boost the efficacy of current immunotherapies. Citation Format: Joseph A. Westrich, Daniel W. Vermeer, Stephanie Bonney, Alexa Silva, Jennifer N. Berger, Marianna Madeo, Louis Cicchini, John H. Lee, William C. Spanos, Dohun Pyeon. CXCL14-mediated antigen-specific CD8+ T-cell responses suppress HPV-positive head and neck cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A36.

Analysis of HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinomas and Paired Normal Mucosae Reveals Cyclin D1 Deregulation and Compensatory Effect of Cyclin D2.

Aberrant regulation of the cell cycle is a typical feature of all forms of cancer. In head and neck squamous cell carcinoma (HNSCC), it is often associated with the overexpression of cyclin D1 (CCND1). However, it remains unclear how CCND1 expression changes between tumor and normal tissues and whether human papillomavirus (HPV) affects differential CCND1 expression. Here, we evaluated the expression of D-type cyclins in a cohort of 94 HNSCC patients of which 82 were subjected to whole genome expression profiling of primary tumors and paired normal mucosa. Comparative analysis of paired samples showed that CCND1 was upregulated in 18% of HNSCC tumors. Counterintuitively, CCND1 was downregulated in 23% of carcinomas, more frequently in HPV-positive samples. There was no correlation between the change in D-type cyclin expression and patient survival. Intriguingly, among the tumors with downregulated CCND1, one-third showed an increase in cyclin D2 (CCND2) expression. On the other hand, one-third of tumors with upregulated CCND1 showed a decrease in CCND2. Collectively, we have shown that CCND1 was frequently downregulated in HNSCC tumors. Furthermore, regardless of the HPV status, our data suggested that a change in CCND1 expression was alleviated by a compensatory change in CCND2 expression.

Analysis of spatial relationships between CD8 and FoxP3 cells using digital imaging in head and neck squamous cell carcinoma

T cell-mediated anti-tumor immune responses are gated in part by the relative abundance of cytotoxic T cells (Teffs) and regulatory (Tregs) in the tumor microenvironment (TME) whereby Treg may impair the efficacy of Teffs. Tregs can affect Teff function by direct cell-to-cell contact or by secretion of soluble mediators, consistent with the hypothesis that proximity of Tregs to Teff within tumor tissues may have prognostic significance. Here we used a novel chromogenic multiplex assay to investigate the spatial relationship of these functionally diverse T cell subsets in HPV positive and HPV negative head and neck squamous cell carcinomas (HNSCCs).

Risk Factors for Human Papillomavirus-Positive Nonoropharyngeal Squamous Cell Carcinoma

BACKGROUND Human papillomavirus (HPV)-positive oropharyngeal cancer (HPV-OPC) is distinct from HPV-unassociated head and neck cancer. However, whether risk factors for HPV-positive oropharyngeal and nonoropharyngeal squamous cell cancer are the same is unclear. METHODS Incident cases of HPV-positive head and neck cell cancer and matched non-cancer controls were enrolled in a multi-institutional, prospective study examining risk factors, biomarkers, and survival. RESULTS HPV-nonOPC (n = 20) were more likely to be ever smokers than controls (n = 80, OR 3.49, 95%CI 1.11-10.9) and HPV-OPC (n = 185, OR 3.28, 95%CI 1.10-10.2). Compared with HPV-OPC, HPV-nonOPC were less likely to have had over 3 oral sexual partners (OR 0.29, 95%CI 0.06-0.9), more likely to have multimorbidity (OR 3.30, 95%CI 1.04-10.5), and less likely to have antibodies to HPV16 E6 (90% vs 28%, OR 0.05, 95%CI 0.02-0.2). HPV-nonOPC had worse 4-year OS (77% vs 96%, P = .001) and RFS (69% vs 94%, P < .001) than HPV-OPC. CONCLUSIONS HPV-positive nonoropharyngeal are distinct from HPV-positive oropharyngeal cancers.

Risk factors for human papillomavirus-positive nonoropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV)-positive oropharyngeal cancer (HPV-OPC) is distinct from HPV-unassociated head and neck cancer. However, whether risk factors for HPV-positive oropharyngeal and nonoropharyngeal squamous cell cancer are the same is unclear. Incident cases of HPV-positive head and neck cell cancer and matched non-cancer controls were enrolled in a multi-institutional, prospective study examining risk factors, biomarkers, and survival. HPV-nonOPC (n = 20) were more likely to be ever smokers than controls (n = 80, OR 3.49, 95%CI 1.11-10.9) and HPV-OPC (n = 185, OR 3.28, 95%CI 1.10-10.2). Compared with HPV-OPC, HPV-nonOPC were less likely to have had over 3 oral sexual partners (OR 0.29, 95%CI 0.06-0.9), more likely to have multimorbidity (OR 3.30, 95%CI 1.04-10.5), and less likely to have antibodies to HPV16 E6 (90% vs 28%, OR 0.05, 95%CI 0.02-0.2). HPV-nonOPC had worse 4-year OS (77% vs 96%, P = .001) and RFS (69% vs 94%, P < .001) than HPV-OPC. HPV-positive nonoropharyngeal are distinct from HPV-positive oropharyngeal cancers.

Survival-Associated Metabolic Genes in Human Papillomavirus-Positive Head and Neck Cancers.

Human papillomavirus (HPV) causes an increasing number of head and neck squamous cell carcinomas (HNSCCs). Altered metabolism contributes to patient prognosis, but the impact of HPV status on HNSCC metabolism remains relatively uncharacterized. We hypothesize that metabolism-related gene expression differences unique to HPV-positive HNSCC influences patient survival. The Cancer Genome Atlas RNA-seq data from primary HNSCC patient samples were categorized as 73 HPV-positive, 442 HPV-negative, and 43 normal-adjacent control tissues. We analyzed 229 metabolic genes and identified numerous differentially expressed genes between HPV-positive and negative HNSCC patients. HPV-positive carcinomas exhibited lower expression levels of genes involved in glycolysis and higher levels of genes involved in the tricarboxylic acid cycle, oxidative phosphorylation, and β-oxidation than the HPV-negative carcinomas. Importantly, reduced expression of the metabolism-related genes SDHC, COX7A1, COX16, COX17, ELOVL6, GOT2, and SLC16A2 were correlated with improved patient survival only in the HPV-positive group. This work suggests that specific transcriptional alterations in metabolic genes may serve as predictive biomarkers of patient outcome and identifies potential targets for novel therapeutic intervention in HPV-positive head and neck cancers.

The radiobiology of HPV-positive and HPV-negative head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with reported incidences of ~800 000 cases each year. One of the critical determinants in patient response to radiotherapy, particularly for oropharyngeal cancers, is human papillomavirus (HPV) status where HPV-positive patients display improved survival rates and outcomes particularly because of increased responsiveness to radiotherapy. The increased radiosensitivity of HPV-positive HNSCC has been largely linked with defects in the signalling and repair of DNA double-strand breaks. Therefore, strategies to further radiosensitise HPV-positive HNSCC, but also radioresistant HPV-negative HNSCC, have focussed on targeting key DNA repair proteins including PARP, DNA-Pk, ATM and ATR. However, inhibitors against CHK1 and WEE1 involved in cell-cycle checkpoint activation have also been investigated as targets for radiosensitisation in HNSCC. These studies, largely conducted using established HNSCC cell lines in vitro, have demonstrated variability in the response dependent on the specific inhibitors and cell models utilised. However, promising results are evident targeting specifically PARP, DNA-Pk, ATR and CHK1 in synergising with radiation in HNSCC cell killing. Nevertheless, these preclinical studies require further expansion and investigation for translational opportunities for the effective treatment of HNSCC in combination with radiotherapy.

Unique Patterns of Distant Metastases in HPV-Positive Head and Neck Cancer

Background: HPV-positive head and neck squamous cell carcinoma (HPV+ HNSCC) demonstrates favorable outcomes compared to HPV-negative SCC, but distant metastases (DM) still occur. The pattern of DM in HPV+ HNSCC is unclear. Methods: 1,494 HNSCC patients were treated from 2006 to 2012. Recurrence time and metastatic sites in HPV+ HNSCC (Group 1) were compared to patients with HPV-negative/unknown cancers arising in the hypopharynx, larynx, or glottis (Group 2) as well as to patients with HPV-negative/unknown cancers in theoral cavity, oropharynx, hard palate, or tonsil (Group 3). Results: 7/109 (6.4%) patients with HPV+ HNSCC developed DM. The median time to metastases was 11 months. At a median follow-up of 18–25 months, there was no difference in the overall rate of DM for the HPV+ HNSCC group compared to Group 2 (HPV–/unknown) (p = 0.21) and Group 3 (HPV–/unknown) (p = 0.13). There was a significant difference in the rate of DM to the lung in the HPV+ HNSCC group compared to Group 2 (HPV–/unknown) (p = 0.012) and Group 3 (HPV–/unknown) (p = 0.002). Conclusions: There was no observed difference in the time to development of DM between the HPV–/unknown and HPV+ HNSCC groups. However, the HPV+ HNSCC group showed a higher rate of DM to the lung compared to the HPV–/unknown ­HNSCC group (p = 0.002).

Antibody Responses to Cancer Antigens Identify Patients with a Poor Prognosis among HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinoma Patients

The identification of high-risk patients within human papillomavirus (HPV)-positive and -negative head and neck squamous cell carcinoma (HNSCC) is needed for improved treatment and surveillance strategies. In this study, we set out to discover antibody responses (AR) with prognostic impact in HNSCC stratified by HPV status. A fluorescent bead-based multiplex serology assay on 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens, and 8 oncogenes) and 29 HPV antigens was performed in samples of 362 patients with HNSCC from five independent cohorts (153 HPV positive, 209 HPV negative). A multivariable Cox proportional hazard model with bootstrapping (M = 1000) was used for validation of prognostic antibody responses. Antibody response to any of the cancer antigens was found in 257 of 362 patients (71%). In HPV-negative patients, antibody responses to c-myc, MAGE-A1, -A4, and Rhodopsin E2 (combined as ARhigh risk) were significantly associated with shorter overall survival. In HPV-positive patients, antibody responses to IMP-1 were discovered as a negative prognostic factor. ARhigh risk (HR = 1.76) and antibody responses to IMP-1 (HR = 3.28) were confirmed as independent markers for a poor prognosis in a multivariable Cox proportional hazard model with bootstrapping (M = 1000). We identified antibody responses to cancer antigens that associate with a dismal prognosis in patients with HNSCC beyond HPV-positive status. ARhigh risk may be used to detect HPV-negative patients with an extraordinarily bad prognosis. Most importantly, antibody response to IMP-1 may serve as a marker for a subgroup of HPV-positive patients who present with a poor prognosis similar to that in HPV-negative patients.

Abstract B032: Proteomic analysis reveals alterations in apoptotic signaling machinery in HPV-associated cancers and identifies BRD4 and BCL2 as potential therapeutic targets in HPV-positive HNSCCs

Abstract Human papillomaviruses (HPV) are among the most common viral causes of cancer; infections account for a majority of cervical squamous cell carcinomas and endocervical adenocarcinomas (CESCs) and oropharyngeal squamous cell carcinomas. HPV-positive cancers demonstrate well-established differences in histopathology and clinical outcome compared to HPV-negative cancers, yet the biological mechanisms underlying these differences are not fully characterized. In this analysis, we highlight key pathways and candidate targets differentially expressed in HPV-positive head and neck squamous cell carcinomas (HNSCCs) and CESCs and investigate potential contributors to these differences, including microRNA (miRNA). Methods: We conducted an analysis of mRNA and protein expression in HPV-positive and HPV-negative HNSCCs and CESCs from the Cancer Genome Atlas (TCGA) to investigate the potential pathways that are differentially expressed between HPV-positive and HPV-negative cancers. mRNA levels were measured by RNA Seq and protein levels by reverse phase protein arrays (RPPA). We also analyzed miRNA levels for both HNSCCs and CESCs, then used TargetScan to identify potential targets for miRNA that were differentially expressed. Results: Proteins involved in extracellular matrix (ECM) adherence including Paxillin and Fibronectin were higher in HPV-negative HNSCCs and CESCs (Paxillin’s p-value= 0.04 for CESCs and p-value&amp;lt;.01 for HNSCCs; Fibronectin’s p=.04 for CESCS and p&amp;lt;.01 for HNSCCs). In addition, Rb, a cell-cycle protein which is known to be degraded by HPV’s E7 protein, was lower in HPV-positive HNSCCs and CESCs (p=.015 for CESCs and p&amp;lt;.01 for HNSCCs). Consistent with its role as a marker for HPV-positive cancers, cell cycle regulator P16INK4A was also notably elevated in HPV+ HNSCCs and CESCs (p&amp;lt;.01). Several genes involved in apoptotic pathways were higher in HPV+ HNSCCs and CESCs, including BIM (p&amp;lt;.01 for CESCs and for HNSCCs) and cleaved caspase 7 (p&amp;lt;.01 for CESCs and for HNSCCs). BRD4 was also higher in HPV-positive HNSCCs (p&amp;lt;.01). Multiple miRNA predicted to target BIM were also significantly lower in HPV+ HNSCCs and/or CESCs . For instance, Mir-891a is lower in HPV+ CESCs (p&amp;lt;.01, fold change=3.11) and potentially targets BIM (total context score=-.61). mir-3200, which potentially targets Lck, is lower in HPV+ CESCs (p&amp;lt;.01). Conclusions: Analyses of protein expression in HPV+ HNSCCs and CESCs reveal differences in HPV-induced and non-HPV-induced cancers, including differences in pathways related to apoptosis, extracellular matrix adherence, cell-cycle regulation, and immune markers. This includes increased expression of potentially druggable targets such as BIM and BRD4. miRNA may contribute to some of the differential expression of these pathways. Citation Format: Claudia Heymach, Robert Cardnell, Lixia Diao, Jing Wang, Kwok-Shing Ng, Lauren Byers. Proteomic analysis reveals alterations in apoptotic signaling machinery in HPV-associated cancers and identifies BRD4 and BCL2 as potential therapeutic targets in HPV-positive HNSCCs [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B032. doi:10.1158/1535-7163.TARG-19-B032