HPV-negative Cancers Research Articles

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<.01 for HNSCCs; Fibronectin’s p=.04 for CESCS and p<.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<.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<.01). Several genes involved in apoptotic pathways were higher in HPV+ HNSCCs and CESCs, including BIM (p<.01 for CESCs and for HNSCCs) and cleaved caspase 7 (p<.01 for CESCs and for HNSCCs). BRD4 was also higher in HPV-positive HNSCCs (p<.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<.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<.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

1121P - Nimotuzumab-cisplatin-radiation versus cisplatin-radiation in HPV-negative oropharyngeal cancer

BackgroundAddition of nimotuzumab to weekly cisplatin and radiation improves outcomes in head and neck cancer. HPV-negative oropharyngeal cancer has unsatisfactory treatment outcomes and is a candidate for escalation of treatment. Hence we wanted to determine whether the addition of nimotuzumab to cisplatin-radiation can improve outcomes in these subsets of poor-risk tumors. MethodsThis was a subgroup analysis of a phase 3 randomized study. In this study locally advanced head and neck cancer patients undergoing definitive chemoradiation were randomly allocated to weekly cisplatin (30mg/m2 IV )- radiation (66-70Gy) {CRT arm} or nimotuzumab (200mg weekly) -weekly cisplatin (30mg/m2)-radiation (66-70Gy) {NCRT arm}. The data for HPV-negative oropharyngeal cancer was extracted from the database of this study for the current analysis. HPV testing was done with p16 IHC staining and reported according to the CAP criteria. The outcomes assessed were progression-free survival (PFS), disease-free survival (DFS), locoregional control (LRC), and overall survival (OS). Interaction test was performed between the study arms and HPV status prior to doing any HPV-specific analysis for each of the studied outcomes. Kaplan Meier estimates for 2-year OS with 95% CI is provided. The hazard ratio was calculated using Cox regression analysis. ResultsWe had 187-HPV negative oropharyngeal cancers, 91-CRT arm and 96 in NCRT arm. The interaction test was significant for PFS (p=0.000), locoregional control (p=0.007) and overall survival (p=0.002) but not for DFS (p=0.072). The 2-year PFS was 31.5% (95%CI 21.5-42) in CRT arm versus 57.2% (95%CI 45.8-67.1) in NCRT arm (HR -0.54;95%CI 0.36-0.79, p=0.002). While the 2-year LRC was 41.4 % (95%CI 29.8-52.6) in CRT arm versus in 60.4% (95%CI 48.7-70.2) in NCRT arm (HR -0.61;95%CI 0.4-0.94, p=0.024). The addition of nimotuzumab also led to an improvement in OS from 39.0% (95%CI 28.4-49.6) to 57.6% (95%CI 46.3-67.4) (HR-0.63, 95%CI 0.43-0.92, p=0.018). ConclusionsThe addition of nimotuzumab to weekly cisplatin-radiation improves outcomes inclusive of OS in HPV-negative oropharyngeal cancers. Clinical trial identificationCTRI/2014/09/004980. Legal entity responsible for the studyThe authors. FundingBiocon Ltd, TRAC. DisclosureK. Prabhash: Research grant / Funding (institution): Dr. Reddy’s Laboratories Inc; Research grant / Funding (institution): Fresenius Kabi India Pvt Ltd; Research grant / Funding (institution): Alkem Laboratories; Research grant / Funding (institution): Natco Pharma Ltd; Research grant / Funding (institution): BDR Pharmaceutics Intl Pvt Ltd; Research grant / Funding (institution): Roche Holding AG. V. Noronha: Research grant / Funding (institution): Dr. Reddy’s Laboratories Inc; Research grant / Funding (institution): Amgen; Research grant / Funding (institution): Sanofi Aventis. All other authors have declared no conflicts of interest.

Prognostic implications of stromal hyaluronic acid protein expression in resected oropharyngeal and oral cavity cancers.

Background/AimsHyaluronic acid (HA) regulates cell adhesion, migration and proliferation in various cancers. The clinical implications of HA in resected head and neck squamous cell carcinoma have not been elucidated. We investigated the clinical significance and prognostic value of the expression of tumoral and stromal HA and its related proteins in oropharyngeal and oral cavity cancer.MethodsResected tissues from oropharyngeal or oral cavity cancer patients undergoing surgery were analysed in tissue microarrays divided into stroma and cancer panels. The expression levels of HA, HA synthases and hyaluronidases were also assessed by immunohistochemistry.ResultsA total of 160 resected oropharyngeal or oral cavity cancer tissues were analysed. Stromal HA expression was observed more frequently in human papilloma virus (HPV)-negative tumors, but other clinicopathological characteristics did not differ. In patients with HPV-negative oral cavity cancers, high stromal HA expression was associated with significantly shorter recurrence-free survival and overall survival compared with low stromal HA expression. The expression of HA in both tumors and stroma was significantly correlated with poorer outcomes than other combinations in patients with HPV-negative oral cavity cancers. However, these prognostic roles of HA were not observed in patients with HPV-negative oropharyngeal cancers. In the HPV-stratified multivariate analysis, high stromal HA expression remained an independent indicator of poor prognosis in terms of recurrence-free survival.ConclusionsHigh stromal HA and expression of HA in both tumors and stroma were correlated with poor prognosis in HPV-negative oral cavity cancer, but not in HPV-negative oropharyngeal cancers.

Oncological outcome following initiation of treatment for stage III and IV HPV negative oropharyngeal cancers with transoral robotic surgery (TORS)

ObjectiveTo report long-term oncological and functional outcome of Transoral Robotic Surgery escalated treatment including radiotherapy or chemoradiotherapy for Stage III-IV HPV negative oropharyngeal malignancies. MethodFrom March 2013 to September 2015, 153 patients with oropharyngeal carcinoma were included in the study. Patients were evaluated for disease free survival, overall survival and post-treatment functional outcomes. Results153 patients (96 males and 57 females) underwent TORS for oropharyngeal carcinoma. 142 patients on final histopathology had stage III and IV disease and received adjuvant treatment based on final histopathology. One hundred and sixteen (81.7%) patients were disease free on average follow-up of 48 months with an overall survival of 91.5% at mean follow-up of 48 months. ConclusionTORS can be used to intensify treatment of Stage III/IV oropharyngeal carcinoma and avoid early and late toxicities due to higher doses of upfront RT/CTRT and achieve better oncological outcome.

Oropharyngeal Cancers in the East and the West – Implications of Aetiopathogenesis on Prognosis and Research

There has been a surge in human papillomavirus (HPV)-positive oropharyngeal cancers (OPCs) in the West. Although the prognosis of HPV-positive OPC is good, de-escalation strategies have so far not been able to confirm comparable cancer control. We examine the strategies implemented across the globe to safely reduce toxicities in HPV-positive disease. HPV-negative OPC has a poorer prognosis and is more prevalent in Eastern countries. We outline the intensification strategies currently used in HPV-negative cancers, with an aim to better prognosis. With recent improvements in clinical trial frameworks in Eastern countries such as India, we discuss areas where joint collaborative research between Western and Eastern countries could further improve outcomes in OPC.

A phase I multicenter study to assess the safety, tolerability, and immunogenicity of mRNA-4157 alone in patients with resected solid tumors and in combination with pembrolizumab in patients with unresectable solid tumors.

2523 Background: T-cell targeting of mutation-derived epitopes (neoantigens) has been demonstrated to drive anti-tumor responses. Immunizing patients against such neoantigens in combination with a checkpoint inhibitor (CPI) may elicit greater anti-tumor responses than CPI alone. Mutations are rarely shared between patients, thus requiring a personalized approach to vaccine design. Methods: A phase I dose escalation study of mRNA-4157 as adjuvant monotherapy in patients with resected solid tumors (melanoma, bladder carcinoma, HPV negative HNSCC, NSCLC, SCLC, MSI-High, or TMB High cancers) and in combination with pembrolizumab in patients with advanced or metastatic cancer is being conducted to evaluate safety. mRNA-4157 is a lipid encapsulated personalized vaccine encoding multiple neoantigens selected using a proprietary algorithm designed to induce neoantigen specific T cells and associated anti-tumor responses. Patients may receive up to 9 cycles (Q3W) of mRNA-4157 by intramuscular injection (0.04 – 1 mg). In the combination arm, pembrolizumab (200 mg) is administered for two cycles prior to combination with mRNA-4157; patients may continue pembrolizumab after completion of 9 cycles of combination therapy. Primary end points include safety, tolerability, and recommended phase 2 dose. Results: 33 patients received mRNA-4157; 13 as monotherapy and 20 in combination with pembrolizumab. No DLTs were reported, and treatment related AEs have generally been of low grade and reversible, and no drug related SAEs or AEs ≥ grade 3 have been observed. Of the 13 patients on adjuvant monotherapy (3 melanoma, 8 NSCLC, 2 MSI-High), 12 patients remain disease free on study, median follow-up of 8 months. 20 patients have been treated in combination (1 TMB-high, 4 bladder, 2 HNSCC, 1 melanoma, 7 NSCLC, 2 SCLC, 3 MSI-high), 12 had progressed on prior CPI, 16 have been restaged and there are 1 CR (on pembrolizumab prior to vaccination), 2 PR, 5 SD for at least 5 combination cycles, 5 PD, 2 iuPD, and 1 patient is non-evaluable for response but remains on study. Neoantigen specific T cell responses have been detected by IFN-γ ELISpot from PBMCs. Conclusions: mRNA-4157 is safe and well tolerated at all dose levels tested. Clinical responses have been observed in combination with pembrolizumab and neoantigen-specific T cells have been induced, supporting the advancement of mRNA-4157 to phase 2. Clinical trial information: NCT03313778.

Identifying adverse molecular features of HPV+ head and neck cancers using patient-derived models.

6057 Background: Advancing therapy for human papilloma virus-related (HPV+) head and neck squamous cell carcinoma (HNSCC) is hindered by inadequate preclinical models and risk stratification tools. This study addresses both barriers by characterizing a panel of patient-derived xenografts (PDXs) that includes nine new models of HPV+ disease. Methods: Exome-sequenced genetic features of the PDXs were compared to their growth properties and the outcomes of their patients of origin. Genetic traits with potential prognostic utility were validated in multiple retrospective patient cohorts. Results: The HPV+ PDXs avoided known artifacts in HPV+ cell lines, including 3q amplifications and loss of PIK3CA mutants, while enriching for alterations in H3K4 methyltransferases and Notch pathway genes. A positive association emerged between PDX tumor mutational burden (TMB) and their growth efficiency both in vivo and as organoids. This finding led to identification in The Cancer Genome Atlas (TCGA) of an association between high TMB and worse survival of early-stage HPV-negative cancers but not HPV+ ones. Insight into aggressive HPV+ disease came from a PDX established from a patient before lethal relapse. The reduced levels of viral E7 and p16INK4A present in this model were also detected in early lethal HPV+ cases in TCGA as well as the recurrences in a second HPV+ HNSCC cohort (JHU). This observation suggested a diminished contribution of viral oncogenes to cell cycle dysregulation in aggressive cases. To evaluate this possibility, hierarchical clustering of both cohorts was performed based on expression of E2F target genes. This analysis discovered a distinct cell cycle-related transcriptional pattern in the clusters of cases containing the recurrences and early lethal events. Furthermore, a subset of these transcripts proved to be stage-independent predictors of survival for the HPV+ HNSCCs in both TCGA and JHU cohorts. Conclusions: Characterizing the most HPV+ patient-derived models described to date revealed novel prognostic utility for E2F target expression in HPV+ HNSCCs and TMB in HPV-negative HNSCCs. These features have potential for application to risk stratification, biomarker development, and trial design.

Post-Radiation Immunosuppression is Associated with Poor Clinical Outcomes in HPV-negative Oropharyngeal Cancers

An elevated neutrophil to lymphocyte ratio (NLR) in the peripheral blood has been associated with poor disease outcomes in a number of solid tumors sites. However, the prognostic value of NLR in human papilloma virus (HPV)-negative oropharyngeal cancers (OPCs) remains controversial. We hypothesized that post-radiation lymphopenia and NLR would be associated with patient outcomes. HPV-negative oropharyngeal cancer patients treated with radiation therapy at an academic center from 1997-2013 were queried from a prospectively maintained database. Absolute lymphocyte counts (ALC) and absolute neutrophil counts (ANC) were evaluated before treatment, at the lymphocyte count nadir during treatment, 3 months, 6 months, and 12 months after radiation (RT) start. Logistic regression was used to determine clinical and treatment-related predictors of lymphopenia. Kaplan-Meier and Cox analyses were done for loco-regional control (LRC), progression free survival (PFS), and overall survival (OS). We identified 108 patients with HPV-negative oropharyngeal cancers, of whom 57 received surgery followed by post-operative radiation (PORT) and 51 received definitive RT. Median radiation dose was 70Gy for definitive RT and 66Gy for PORT. Median follow up was 37 months (range 2-197). During treatment, 85% of patients developed grade ≥ 3 lymphopenia and 22% with grade 4 lymphopenia, with no difference in lymphopenia rates between patients receiving PORT and those receiving definitive RT. Stage T3-4 tumor vs. T1-2 (OR = 5.3, 95% CI 1.4-20, p = 0.015) was the only clinical factor associated with developing grade 4 lymphopenia. Chemotherapy and surgery did not significantly associate with grade ≥ 3 lymphopenia at any timepoint. Pre-treatment and treatment nadir blood counts were not associated with OS. Three months after RT start, 19 (29%) out of 65 patients with blood counts had grade ≥ 3 lymphopenia. The estimated 3-year LRC, PFS, and OS in patients with and without grade ≥ 3 lymphopenia at 3 months after RT was 73% vs. 82% (p = 0.334), 36% vs. 63% (p = 0.014), and 34% vs. 64% (p = 0.016), respectively. In multivariable analysis, definitive radiation (HR = 3.2, 95% CI 1.5-6.7) and grade ≥ 3 lymphopenia (HR =2.5, 95% CI 1.3-5.0) at 3 months after RT were associated with worse OS. Lymphopenia and NLR as early as 3 months post-radiation associate with poor clinical outcomes in HPV-negative oropharyngeal cancer patients. Validation in a prospective clinical trial is warranted.

Impact of Primary Payer on Survival in Patients

Prior studies have demonstrated better outcomes for cancer patients with health insurance coverage compared to those with none, but less has been reported regarding the effect of insurance type. The U.S. health insurance landscape is highly variable in patients <65 years old, given they do not have access to uniform coverage such as Medicare. We sought to determine whether the type of primary payer (private versus government-sponsored insurances [GSI]) impacted survival in insured patients younger than 65 with localized head and neck cancer undergoing definitive radiotherapy (RT). The National Cancer Data Base was queried for patients age <65 with stage I-IVB squamous cell carcinoma of the oropharynx, nasopharynx, hypopharynx, or larynx diagnosed from 2004 to 2014 and treated with definitive RT (total dose: 6600 – 8160 cGy) with or without chemotherapy. Patients with no insurance, unknown insurance status, or oropharynx cancer (OPC) with unknown HPV status were excluded. Multivariate Cox regression and propensity score matching were used to adjust for imbalances in covariates. A Bonferroni correction was applied to p-values from subgroup analyses to account for multiple hypothesis testing. The analysis included 27,292 HNSCC patients with median follow-up of 52.1 months. In propensity score-matched cohorts, 3-year overall survival (OS) was 75.2% versus 63.5% for privately insured versus government-insured patients, respectively (p<0.001). In multivariate analysis, private health insurance was associated with significantly improved OS (HR 0.61, 95% CI 0.57-0.65, p<0.001) in comparison to GSI after accounting for other covariates. In subgroup analysis, private insurance was associated with improved OS across all subgroups. However, the greatest impact of private insurance was observed among patients with HPV-positive OPC (interaction p<0.001), stage T1-2 tumors (interaction p<0.001), age ≤50 (interaction p=0.001), no comorbidity (interaction p=0.001), and those receiving chemotherapy (interaction p=0.001) when performing tests of interaction in multivariable models corrected for multiple hypothesis testing. For example, private insurance was associated with a 54% (95% CI 46%-62%) relative decrease in the risk of death for HPV-positive OPC, in comparison to a 37% (95% CI 34%-40%) relative decrease in mortality for HPV-negative cancers. Patients under 65 with HNSCC with private health insurance have improved OS relative to patients with GSI. Although this effect was observed in all subgroups, the relative survival difference was largest in young, healthy patients with HPV-positive OPC and those receiving chemotherapy. This illustrates a significant disparity in our complex healthcare-delivery system which may be mitigated with future policy changes.

Expression of PD-L1 on Immune Cells Shows Better Prognosis in Laryngeal, Oropharygeal, and Hypopharyngeal Cancer.

Despite great enthusiasm towards immunotherapy, reliable biomarkers are still lacking. The importance of subsets based on human papillomavirus (HPV) status is supported by a growing body of evidence. However, role of other possible subgroups such as anatomic localization of primary tumor remains controversial. Our objective was to investigate immune cell infiltrate and checkpoint inhibitor proteins in above-mentioned head and neck cancer subsets. Archival tumor samples of 106 laryngeal, oropharyngeal, and hypopharyngeal cancer patients were stained with PD-L1, PD-L2, PD-1, and CTLA-4 antibodies. Proportion of tumor-infiltrating lymphocytes was assessed as well. In HPV-negative tumors, PD-L1 immune cell positivity was associated with better disease-specific survival. PD-L1 expression on immune cells correlated with improved disease-specific survival in laryngeal tumors. Furthermore, PD-L1 immune cell positivity correlated with CTLA-4 expression on immune cells and it was accompanied by high tumor-infiltrating lymphocyte rate. PD-L1 expression on tumor cells and PD-1 status showed strong correlation in all patients and in oropharyngeal and laryngeal localization, but not in hypopharynx. HPV-negative oropharyngeal cancers showed negative PD-L1 status on tumor cells. CTLA-4 positivity was observed in 49.5% and 20.6% on immune cells and on tumor cells, respectively. We concluded that PD-L1 expression on immune cells indicates better prognosis in laryngeal squamous cell carcinoma and in HPV-negative head and neck squamous cell carcinoma. We have not found any essential differences between anatomic subgroups. A possibly distinct role of hypopharyngeal localization regarding immune activity requires further clarification.

Evaluation of pemetrexed and etoposide as therapeutic regimens for human papillomavirus-positive oral and oropharyngeal cancer.

Although human papillomavirus (HPV) positive oral and oropharyngeal cancers have distinct epidemiologic and molecular characteristics compared to HPV-negative cancers, all patients with oral and oropharyngeal cancers received same standard regimen regardless of HPV status. For these reasons, specific regimens for patients with HPV-positive oral and oropharyngeal cancer are needed. Differentially expressed genes (DEG) between HPV-positive and HPV-negative oropharyngeal cancers were re-analyzed and categorized from public database. Then, druggable targets to HPV-positive oral and oropharyngeal cancer were identified and were validated with E6/E7, which is oncogene of HPV, transfected oral and oropharyngeal cancer cell lines and HPV infected cell lines. In DEG analysis, HPV-positive oral and oropharyngeal cancer showed distinct disease entity from HPV-negative cancers. Unlike HPV-negative oral and oropharyngeal cancer, thymidylate synthase (TS) and topoisomerase II (Topo II) were overexpressed in HPV-positive cancers. Transfection of Lenti-virus containing E6/ E7 to HPV-negative oral and oropharyngeal cancer cells induced upregulation of TS and Topo II in those cells. Although cisplatin, which is standard regimen in head and neck cancers, showed more effectiveness in HPV-negative cells, 5-FU and pemetrexed, which are TS inhibitors, or etoposide, which is Topo II inhibitors, worked more effectively in HPV-positive cells. In addition, cisplatin/etoposide and cisplatin/pemetrexed combination regimens showed synergic effects in HPV-positive cells. Pemetrexed or etoposide alone, or in combination with other chemotherapeutic agents such as cisplatin, can be used as novel substitutes in a regimen of concurrent chemoradiotherapy or a palliative regimen for HPV-positive oral and oropharyngeal cancer patients. However, a well-designed clinical trial is needed.

Interpretation of Endocervical Cells With Gastric-Type Mucin on Pap Smears.

Early detection of endocervical adenocarcinoma is especially important for cancers that are human papillomavirus (HPV) negative. We investigated the clinicopathologic significance of yellow gastric-type mucin observed on Papanicolaou smears. We described "atypical endocervical cells with gastric-type mucin" (AEC-GAM) when yellow mucin was observed in endocervical cells. We retrieved AEC-GAM samples from 58,752 cervical smears performed at Yamanashi University Hospital during our study period and reviewed clinical, cytologic, and pathologic features. We detected AEC-GAM in 172 (0.29 %) smears from 65 patients, and 41 of these 65 patients were histologically diagnosed with lobular endocervical glandular hyperplasia (LEGH) (43%) or pyloric gland metaplasia (20%). The prevalence of adenocarcinoma was 25% (7/28) in LEGH cases and 11% (7/65) in AEC-GAM smears. Yellow mucin is a diagnostic clue for endocervical glandular lesions with gastric differentiation. We recommend describing AEC-GAM on cytologic reports to improve cytologic screening for HPV-negative cervical cancers.

OC-0177: Mid-treatment response assessment is predictive in HPV negative oropharyngeal cancers undergoing RT

OC-0177: Mid-treatment response assessment is predictive in HPV negative oropharyngeal cancers undergoing RT

Human papillomavirus and penile cancer : Thinking about measures for prevention

Two major pathways of penile carcinogenesis are known: human papillomavirus (HPV)-induced penile cancer and HPV-negative cancers associated with chronic dermatoses. Therefore, modern measures for prevention of penile cancer may for example include prophylactic HPV vaccination. The resulting B‑cell-mediated immunity to HPV capsid proteins is effective protection against future HPV infections. Contrarily when treating existing HPV infections or HPV-associated cancers an antigen-specific T‑cell immunity is necessary. To date, screening and treatment of precancerous lesions to prevent penile cancer are not established in the German health care program and the highly expected therapeutic HPV vaccines are still on the horizon. In this article, we focus on possible strategies to prevent HPV-related penile cancer on different levels of carcinogenesis.

Head and Neck Carcinoma Immunotherapy: Facts and Hopes.

Cancer of the head and neck (HNC) is a heterogeneous disease of the upper aerodigestive tract, encompassing distinct histologic types, different anatomic sites, and human papillomavirus (HPV)-positive as well as HPV-negative cancers. Advanced/recurrent HNCs have poor prognosis with low survival rates. Tumor-mediated inhibition of antitumor immune responses and a high mutational burden are common features of HNCs. Both are responsible for the successful escape of these tumors from the host immune system. HNCs evolve numerous mechanisms of evasion from immune destruction. These mechanisms are linked to genetic aberrations, so that HNCs with a high mutational load are also highly immunosuppressive. The tumor microenvironment of these cancers is populated by immune cells that are dysfunctional, inhibitory cytokines, and exosomes carrying suppressive ligands. Dysfunctional immune cells in patients with recurrent/metastatic HNC can be made effective by the delivery of immunotherapies in combination with conventional treatments. With many promising immune-based strategies available, the future of immune therapies in HNC is encouraging, especially as methods for genetic profiling and mapping the immune landscape of the tumor are being integrated into a personalized approach. Efficiency of immune therapies is expected to rapidly improve with the possibility for patients' selection based on personal immunogenomic profiles. Noninvasive biomarkers of response to therapy will be emerging as a better understanding of the various molecular signals co-opted by the tumors is gained. The emerging role of immunotherapy as a potentially beneficial addition to standard treatments for recurrent/metastatic HNC offers hope to the patients for whom no other therapeutic options exist. Clin Cancer Res; 24(1); 6-13. ©2017 AACR.

Nonuniform Distribution of High-risk Human Papillomavirus in Squamous Cell Carcinomas of the Oropharynx

The oral cavity and oropharynx have historically been viewed as a single anatomic compartment of the head and neck. The practice of combining the oral cavity and oropharynx has recently been revised, largely owing to the observation that human papillomavirus (HPV)-related carcinogenesis has a strong predilection for the oropharynx but not the oral cavity. The purpose of this study was to determine whether HPV is evenly distributed across squamous cell carcinomas of the oropharynx including those sites that do not harbor tonsillar tissues such as the soft palate. A search of the medical records of the Johns Hopkins Hospital identified 32 primary squamous cell carcinomas of the soft palate (n=31) and posterior pharyngeal wall (n=1). All were evaluated with p16 immunohistochemistry and high-risk HPV in situ hybridization (ISH) (29 by RNA ISH and 3 by DNA ISH). For comparison, we also reviewed the medical records to obtain the HPV status of patients who had undergone HPV testing of primary tonsillar carcinomas over the same time interval as part of their clinical care. High-risk HPV as detected by ISH was present in just 1 (3.1%) of the 32 oropharyngeal squamous cell carcinomas, including 1 of 2 p16-positive carcinomas. The difference in HPV detection rates between tonsillar and nontonsillar sites was significant (1/32, 3.1% vs. 917/997, 92%; P<0.0001). HPV is not frequently detected in squamous cell carcinomas arising from nontonsillar regions of the oropharynx. Indeed, squamous cell carcinomas of the soft palate more closely resemble those arising in the oral cavity than those arising in areas of the oropharynx harboring tonsillar tissue. This finding not only further sharpens our understanding of site-specific targeting by HPV, but may have practical implications regarding HPV testing and even the way the oral vault is oncologically compartmentalized to partition HPV-positive from HPV-negative cancers.

Surgical staging identified false HPV-negative cases in a large series of invasive cervical cancers

ObjectiveWe examined a large series of biopsy-proven invasive cervical cancers with surgical staging and HPV re-testing to estimate the relevance of HPV-negative cervical cancers in a Caucasian population. MethodsWe prospectively collected smears from 371 patients with a biopsy-proven diagnosis of cervical cancer for HC2 testing of high-risk HPV (HR-HPV). In HC2-negative cases, smears and paraffin embedded tissue blocks underwent additional HPV genotyping. ResultsHC2 tests showed 31/371 cases (8.8%) had negative findings. Surgical staging showed that 21/31 HC2-negative cases (68%) were not cervical cancer. Overall, 340/350 cases of primary cervical cancer confirmed by surgical staging tested HC2 positive (97.2%). Non-high-risk HPV subtypes were detected in five cases (one HPV-53, one HPV-70, and three HPV-73) and high-risk subtypes in four patients with HC2-negative cervical cancer (two HPV 16 and two HPV-18). The remaining case, a primary undifferentiated carcinoma of the uterine cervix, tested negative for HPV-DNA with all tests. ConclusionsThe main explanation for HPV-negative cervical cancer was a false diagnosis, followed by cancers associated with non-HR-HPV types, and false-negative HR-HPV results. Truly HPV negative seem to be very rare in Caucasian populations. Retrospective analyses without surgical staging may overestimate the proportion of HPV negative cervical cancers.

Genomic characterization of human papillomavirus-positive and -negative human squamous cell cancer cell lines.

Human cancer cell lines are the most frequently used preclinical models in the study of cancer biology and the development of therapeutics. Although anatomically diverse, human papillomavirus (HPV)-driven cancers have a common etiology and similar mutations that overlap with but are distinct from those found in HPV-negative cancers. Building on prior studies that have characterized subsets of head and neck squamous cell carcinoma (HNSCC) and cervical squamous cell carcinoma (CESC) cell lines separately, we performed genomic, viral gene expression, and viral integration analyses on 74 cell lines that include all readily-available HPV-positive (9 HNSCC, 8 CESC) and CESC (8 HPV-positive, 2 HPV-negative) cell lines and 55 HPV-negative HNSCC cell lines. We used over 700 human tumors for comparison. Mutation patterns in the cell lines were similar to those of human tumors. We confirmed HPV viral protein and mRNA expression in the HPV-positive cell lines. We found HPV types in three CESC cell lines that are distinct from those previously reported. We found that cell lines and tumors had similar patterns of viral gene expression; there were few sites of recurrent HPV integration. As seen in tumors, HPV integration did appear to alter host gene expression in cell lines. The HPV-positive cell lines had higher levels of p16 and lower levels of Rb protein expression than did the HPV-negative lines. Although the number of HPV-positive cell lines is limited, our results suggest that these cell lines represent suitable models for studying HNSCC and CESC, both of which are common and lethal.

An oral keratinocyte life cycle model identifies novel host genome regulation by human papillomavirus 16 relevant to HPV positive head and neck cancer.

Many aspects of the HPV life cycle have been characterized in cervical cell lines (W12, CIN612) and in HPV immortalized primary foreskin keratinocytes. There is now an epidemic of HPV positive oropharyngeal cancers (HPV16 is responsible for 80-90% of these); therefore increased understanding of the HPV16 life cycle in oral keratinocytes is a priority. To date there have been limited reports characterizing the HPV16 life cycle in oral keratinocytes. Using TERT immortalized “normal” oral keratinocytes (NOKs) we generated clonal cell lines maintaining the HPV16 genome as an episome, NOKs+HPV16. Organotypic raft cultures demonstrated appropriate expression of differentiation markers, E1^E4 and E2 expression along with amplification of the viral genome in the upper layers of the epithelium. Using this unique system RNA-seq analysis revealed extensive gene regulation of the host genome by HPV16; many of the changes have not been observed for HPV16 before. The RNA-seq data was validated on a key set of anti-viral innate immune response genes repressed by HPV16 in NOKs+HPV16. We show that the behavior of these NOKs+HPV16 lines is identical to HPV16 immortalized human tonsil keratinocytes with regards innate gene regulation. Finally, using The Cancer Genome Atlas (TCGA) data we examined gene expression patterns from HPV positive and negative head and neck cancers and demonstrate this innate immune gene signature set is also downregulated in HPV positive cancers versus negative. Our system provides a model for understanding HPV16 transcriptional regulation of oral keratinocytes that is directly relevant to HPV positive head and neck cancer.

Prognostic implication of ERCC1 protein expression in resected oropharynx and oral cavity cancer

BackgroundExcision repair cross complement group 1 (ERCC1) expression is a predictive biomarker for platinum-containing treatment in squamous cell carcinoma of head and neck (SCCHN). However, the prognostic significance after surgical resection is not well understood. MethodsOropharynx (n=143) or oral cavity (n=61) SCCHN patients undergoing surgery were included. ERCC1 protein expression and HPV status were assessed by ERCC1 and p16 immunohistochemistry. ResultsThe ERCC1, over-expressed in 66.7% of patients, was associated with oral cavity cancer (P<0.001), well differentiation (P=0.036), and HPV negativity (P<0.001). In TCGA database, ERCC1 mRNA upregulation was enriched in HPV-negative and oral cavity cancers, and associated with HRAS mutation (P<0.001). The prognostic role of ERCC1 was not different according to HPV status. High ERCC1 expression showed a trend toward poor prognosis in patients with an advanced stage (P=0.079) with marginal significance. ConclusionsThe ERCC1 expression was not prognostic in surgically resected oropharynx/oral cavity SCCHN, irrespective of HPV status. However, it could provide additional prognostic information for advanced stage patients.