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Purpose: Perineural invasion (PNI) in oral cavity squamous cell carcinoma (OSCC) is associated with poor survival. Because of the risk of recurrence, patients with PNI receive additional therapies after surgical resection. Mechanistic studies have shown that nerves in the tumor microenvironment promote aggressive tumor growth. Therefore, in this study, we evaluated whether nerve density (ND) influences tumor growth and patient survival. Moreover, we assessed the reliability of artificial intelligence (AI) in evaluating ND. Experimental Design: To investigate whether increased ND in OSCC influences patient outcome, we performed survival analyses. Tissue sections of OSCC from 142 patients were stained with hematoxylin and eosin and IHC stains to detect nerves and tumor. ND within the tumor bulk and in the adjacent 2 mm was quantified; normalized ND (NND; bulk ND/adjacent ND) was calculated. The impact of ND on tumor growth was evaluated in chick chorioallantoic-dorsal root ganglia (CAM-DRG) and murine surgical denervation models. Cancer cells were grafted and tumor size quantified. Automated nerve detection, applying the Halo AI platform, was compared with manual assessment. Results: Disease-specific survival decreased with higher intratumoral ND and NND in tongue SCC. Moreover, NND was associated with worst pattern-of-invasion and PNI. Increasing the number of DRG, in the CAM-DRG model, increased tumor size. Reduction of ND by denervation in a murine model decreased tumor growth. Automated and manual detection of nerves showed high concordance, with an F1 score of 0.977. Conclusions: High ND enhances tumor growth, and NND is an important prognostic factor that could influence treatment selection for aggressive OSCC.

The aim of this study is to investigate the prognostic role of body mass index (BMI) and survival from head and neck cancer (HNC). We performed a pooled analysis of studies included in the International Head and Neck Cancer Epidemiology consortium in order to investigate the prognostic role of BMI and survival from HNC. We used Cox proportional hazards models to estimate the adjusted hazard ratios (HR) for overall survival and HNC-specific survival, by cancer site. The study included 10,177 patients from 10 studies worldwide. Underweight patients had lower overall survival (HR = 1.69, 95%CI: 1.31–2.19) respect to those having normal weight with consistent results across the HNC sites. Overweight and obese patients with oropharyngeal cancers had a favourable HNC-specific survival (HR = 0.50 (95%CI: 0.33–0.75) and HR = 0.51 (95%CI: 0.36–0.72), respectively). Among ever smokers overweight and obese patients showed a favourable HNC-specific survival (HR = 0.69 (95%CI: 0.56–0.86) and HR = 0.70 (95%CI: 0.61–0.80)). Our findings show that high BMI values at cancer diagnosis predict the survival rates in smoking patients with HNC. This association may be explained by residual confounding, reverse causation, and collider stratification bias, but may also suggest that a nutritional reserve may help patients survive HNC cancer.

This figure shows that Tongue SCC patients with IND equal or larger than T-AND have poor DSS

Adjusted Cox modeling of intratumoral and normalized nerve density in tongue SCC using AJCC 8th edition

Purpose:Perineural invasion (PNI) in oral cavity squamous cell carcinoma (OSCC) is associated with poor survival. Because of the risk of recurrence, patients with PNI receive additional therapies after surgical resection. Mechanistic studies have shown that nerves in the tumor microenvironment promote aggressive tumor growth. Therefore, in this study, we evaluated whether nerve density (ND) influences tumor growth and patient survival. Moreover, we assessed the reliability of artificial intelligence (AI) in evaluating ND.Experimental Design:To investigate whether increased ND in OSCC influences patient outcome, we performed survival analyses. Tissue sections of OSCC from 142 patients were stained with hematoxylin and eosin and IHC stains to detect nerves and tumor. ND within the tumor bulk and in the adjacent 2 mm was quantified; normalized ND (NND; bulk ND/adjacent ND) was calculated. The impact of ND on tumor growth was evaluated in chick chorioallantoic-dorsal root ganglia (CAM-DRG) and murine surgical denervation models. Cancer cells were grafted and tumor size quantified. Automated nerve detection, applying the Halo AI platform, was compared with manual assessment.Results:Disease-specific survival decreased with higher intratumoral ND and NND in tongue SCC. Moreover, NND was associated with worst pattern-of-invasion and PNI. Increasing the number of DRG, in the CAM-DRG model, increased tumor size. Reduction of ND by denervation in a murine model decreased tumor growth. Automated and manual detection of nerves showed high concordance, with an F1 score of 0.977.Conclusions:High ND enhances tumor growth, and NND is an important prognostic factor that could influence treatment selection for aggressive OSCC.

Network statistics for network constructed from 89 genes with/near integration events.

Summary of Integration Transcription Analysis in HPV16-Positive Tumors. (F), (R) = Forward or Reverse viral orientation in relation to the cellular gene. Bold text= Viral/cellular fusion transcript, Italicized text=Transcript spans integration site, Grey shade= No transcript produced, a = Nonsense sequence, no alignment, b= Sequence was correct and spliced in frame, c= Sequence mapped to unexpected or rearranged sequences.

Supplemental Table 6. Summary of genomic aberrations to identified genes with integrated HPV. The Oncomine database was used to assess genomic aberrations in all publicly available HNSCC next generation sequencing data sets including (1, 2) 1. Agrawal N, Frederick MJ, Pickering CR, Bettegowda C, Chang K, Li RJ, Fakhry C, Xie TX, Zhang J, Wang J, Zhang N, El-Naggar AK, Jasser SA, Weinstein JN, Trevino L, Drummond JA, Muzny DM, Wu Y, Wood LD, Hruban RH, Westra WH, Koch WM, Califano JA, Gibbs RA, Sidransky D, Vogelstein B, Velculescu VE, Papadopoulos N, Wheeler DA, Kinzler KW, Myers JN. Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1. Science. 2011;333(6046):1154-7. doi: 10.1126/science.1206923. PubMed PMID: 21798897; PMCID: PMC3162986. 2. Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K, Sivachenko A, Kryukov GV, Lawrence MS, Sougnez C, McKenna A, Shefler E, Ramos AH, Stojanov P, Carter SL, Voet D, Cortes ML, Auclair D, Berger MF, Saksena G, Guiducci C, Onofrio RC, Parkin M, Romkes M, Weissfeld JL, Seethala RR, Wang L, Rangel-Escareno C, Fernandez-Lopez JC, Hidalgo-Miranda A, Melendez-Zajgla J, Winckler W, Ardlie K, Gabriel SB, Meyerson M, Lander ES, Getz G, Golub TR, Garraway LA, Grandis JR. The mutational landscape of head and neck squamous cell carcinoma. Science. 2011;333(6046):1157-60. doi: 10.1126/science.1208130. PubMed PMID: 21798893; PMCID: PMC3415217.

Number of viral-cellular amplicons generated and sequenced from responsive and recurrent tumors.

TaqMan quantitative RT-PCR assay primer and probe sequences.

The incidence of human papillomavirus (HPV)–related oropharynx cancer has steadily increased over the past two decades and now represents a majority of oropharyngeal cancer cases. Integration of the HPV genome into the host genome is a common event during carcinogenesis that has clinically relevant effects if the viral early genes are transcribed. Understanding the impact of HPV integration on clinical outcomes of head and neck squamous cell carcinoma (HNSCC) is critical for implementing deescalated treatment approaches for HPV+ HNSCC patients. RNA sequencing (RNA-seq) data from HNSCC tumors (n = 84) were used to identify and characterize expressed integration events, which were overrepresented near known head and neck, lung, and urogenital cancer genes. Five genes were recurrent, including CD274 (PD-L1). A significant number of genes detected to have integration events were found to interact with Tp63, ETS, and/or FOX1A. Patients with no detected integration had better survival than integration-positive and HPV− patients. Furthermore, integration-negative tumors were characterized by strongly heightened signatures for immune cells, including CD4+, CD3+, regulatory, CD8+ T cells, NK cells, and B cells, compared with integration-positive tumors. Finally, genes with elevated expression in integration-negative specimens were strongly enriched with immune-related gene ontology terms, while upregulated genes in integration-positive tumors were enriched for keratinization, RNA metabolism, and translation.Implications: These findings demonstrate the clinical relevancy of expressed HPV integration, which is characterized by a change in immune response and/or aberrant expression of the integration-harboring cancer-related genes, and suggest strong natural selection for tumor cells with expressed integration events in key carcinogenic genes. Mol Cancer Res; 16(1); 90–102. ©2017 AACR.

Supplemental Methods, Tables S5-S14, and Figures S1-S3

Supplemental Figure 1. Representative DIPS PCR and transcript analysis electrophoretic gel images. M=100 bp ladder. Panel A. Representative Responsive Tumor DIPS PCR Gels. Panel B. Representative Recurrent Tumor DIPS PCR Gels. Panel C. Gel Electrophoresis of Tumor Integration Transcript Analysis. Bold text= Viral/cellular fusion transcript, Italicized text=Transcript spans integration site

DIPS primer sequences and predicted episome-only amplicon sizes.

Purpose: Perineural invasion (PNI), a common occurrence in oral squamous cell carcinomas, is associated with poor survival. Consequently, these tumors are treated aggressively. However, diagnostic criteria of PNI vary and its role as an independent predictor of prognosis has not been established. To address these knowledge gaps, we investigated spatial and transcriptomic profiles of PNI-positive and PNI-negative nerves. Experimental Design: Tissue sections from 142 patients were stained with S100 and cytokeratin antibodies. Nerves were identified in two distinct areas: tumor bulk and margin. Nerve diameter and nerve-to-tumor distance were assessed; survival analyses were performed. Spatial transcriptomic analysis of nerves at varying distances from tumor was performed with NanoString GeoMx Digital Spatial Profiler Transcriptomic Atlas. Results: PNI is an independent predictor of poor prognosis among patients with metastasis-free lymph nodes. Patients with close nerve-tumor distance have poor outcomes even if diagnosed as PNI negative using current criteria. Patients with large nerve(s) in the tumor bulk survive poorly, suggesting that even PNI-negative nerves facilitate tumor progression. Diagnostic criteria were supported by spatial transcriptomic analyses of >18,000 genes; nerves in proximity to cancer exhibit stress and growth response changes that diminish with increasing nerve-tumor distance. These findings were validated in vitro and in human tissue. Conclusions: This is the first study in human cancer with high-throughput gene expression analysis in nerves with striking correlations between transcriptomic profile and clinical outcomes. Our work illuminates nerve-cancer interactions suggesting that cancer-induced injury modulates neuritogenesis, and supports reclassification of PNI based on nerve-tumor distance rather than current subjective criteria.

Enriched GO terms for genes differentially expressed between HPV(+) and HPV(-) HNC tumors in the UM cohort.

This file includes Supplementary Tables S2-S4 and captions for all supplementary tables.

Purpose:Perineural invasion (PNI), a common occurrence in oral squamous cell carcinomas, is associated with poor survival. Consequently, these tumors are treated aggressively. However, diagnostic criteria of PNI vary and its role as an independent predictor of prognosis has not been established. To address these knowledge gaps, we investigated spatial and transcriptomic profiles of PNI-positive and PNI-negative nerves.Experimental Design:Tissue sections from 142 patients were stained with S100 and cytokeratin antibodies. Nerves were identified in two distinct areas: tumor bulk and margin. Nerve diameter and nerve-to-tumor distance were assessed; survival analyses were performed. Spatial transcriptomic analysis of nerves at varying distances from tumor was performed with NanoString GeoMx Digital Spatial Profiler Transcriptomic Atlas.Results:PNI is an independent predictor of poor prognosis among patients with metastasis-free lymph nodes. Patients with close nerve-tumor distance have poor outcomes even if diagnosed as PNI negative using current criteria. Patients with large nerve(s) in the tumor bulk survive poorly, suggesting that even PNI-negative nerves facilitate tumor progression. Diagnostic criteria were supported by spatial transcriptomic analyses of >18,000 genes; nerves in proximity to cancer exhibit stress and growth response changes that diminish with increasing nerve-tumor distance. These findings were validated in vitro and in human tissue.Conclusions:This is the first study in human cancer with high-throughput gene expression analysis in nerves with striking correlations between transcriptomic profile and clinical outcomes. Our work illuminates nerve-cancer interactions suggesting that cancer-induced injury modulates neuritogenesis, and supports reclassification of PNI based on nerve-tumor distance rather than current subjective criteria.

Figure S1. Generation of HLA-A*02-restricted EGFR-specific CD8+ cytotoxic T lymphocytes; Figure S2. SOX2 inhibits IFN-I signaling by promoting autophagy-mediated degradation of STING; Figure S3. Validation of C57BL/6-syngeneic mouse squamous cell carcinoma cells; Figure S4. Characterization of MOC2-E6/E7 tumors; Figure S5: Clinical and immunological correlations of SOX2 expression in human HNSCC; Figure S6. SatVax (Q19D; Q15L) effectively controls tumor growth in larger established tumors and improves survival; Figure S7. Treatment schedule and individual tumor growth curves of Sox2-positive tumors

Purpose: The response rates of Head and Neck Squamous Cell Carcinoma (HNSCC) to checkpoint blockade are below 20%. We aim to develop a mechanism-based vaccine to prevent HNSCC immune escape.Experimental Design: We performed RNA-Seq of sensitive and resistant HNSCC cells to discover central pathways promoting resistance to immune killing. Using biochemistry, animal models, HNSCC microarray, and immune cell deconvolution, we assessed the role of SOX2 in inhibiting STING-type I interferon (IFN-I) signaling-mediated antitumor immunity. To bypass SOX2-potentiated STING suppression, we engineered a novel tumor antigen–targeted nanosatellite vehicle to enhance the efficacy of STING agonist and sensitize SOX2-expressing HNSCC to checkpoint blockade.Results: The DNA-sensing defense response is the most suppressed pathway in immune-resistant HNSCC cells. We identified SOX2 as a novel inhibitor of STING. SOX2 facilitates autophagy-dependent degradation of STING and inhibits IFN-I signaling. SOX2 potentiates an immunosuppressive microenvironment and promotes HNSCC growth in vivo in an IFN-I-dependent fashion. Our unique nanosatellite vehicle significantly enhances the efficacy of STING agonist. We show that the E6/E7–targeted nanosatellite vaccine expands the tumor-specific CD8+ T cells by over 12-fold in the tumor microenvironment and reduces tumor burden. A combination of nanosatellite vaccine with anti-PD-L1 significantly expands tumor-specific CTLs and limits the populations expressing markers for exhaustion, resulting in more effective tumor control and improved survival.Conclusions: SOX2 dampens the immunogenicity of HNSCC by targeting the STING pathway for degradation. The nanosatellite vaccine offers a novel and effective approach to enhance the adjuvant potential of STING agonist and break cancer tolerance to immunotherapy. Clin Cancer Res; 24(17); 4242–55. ©2018 AACR.

Supplementary methods and figures. from Subtypes of HPV-Positive Head and Neck Cancers Are Associated with HPV Characteristics, Copy Number Alterations, PIK3CA Mutation, and Pathway Signatures

Purpose: There is substantial heterogeneity within human papillomavirus (HPV)-associated head and neck cancer (HNC) tumors that predispose them to different outcomes; however, the molecular heterogeneity in this subgroup is poorly characterized due to various historical reasons.Experimental Design: We performed unsupervised gene expression clustering on deeply annotated (transcriptome and genome) HPV+ HNC samples from two cohorts (84 total primary tumors), including 18 HPV− HNC samples, to discover subtypes and characterize the differences between subgroups in terms of their HPV characteristics, pathway activity, whole-genome somatic copy number alterations, and mutation frequencies.Results: We identified two distinct HPV+ subtypes (namely HPV-KRT and HPV-IMU). HPV-KRT is characterized by elevated expression of genes in keratinocyte differentiation and oxidation–reduction process, whereas HPV-IMU has strong immune response and mesenchymal differentiation. The differences in expression are likely connected to the differences in HPV characteristics and genomic changes. HPV-KRT has more genic viral integration, lower E2/E4/E5 expression levels, and higher ratio of spliced to full-length HPV oncogene E6 than HPV-IMU; the subgroups also show differences in copy number alterations and mutations, in particular the loss of chr16q in HPV-IMU and gain of chr3q and PIK3CA mutation in HPV-KRT.Conclusions: Our characterization of two subtypes of HPV+ HNC tumors provides valuable molecular level information that point to two main carcinogenic paths. Together, these results shed light on stratifications of the HPV+ HNCs and will help to guide personalized care for HPV+ HNC patients. Clin Cancer Res; 22(18); 4735–45. ©2016 AACR.

Supplementary Data from Spatial and Transcriptomic Analysis of Perineural Invasion in Oral Cancer

List of deferentially expressed genes in HNSCC cancer cells resistant to immune killing

Tumor-infiltrating lymphocytes (TILs) and cytokines are associated with prognosis among patients with head and neck squamous cell carcinoma (HNSCC). Statins (cholesterol-lowering drugs) may improve HNSCC prognosis, particularly in human papillomavirus (HPV)-positive cases, but the mechanism remains unclear.Statin use was collected from medical records for HNSCC cases (2008-2014). TILs were counted in tumor tissue, and a total weighted score (TILws) was created. Cytokines were measured in blood. The associations between statins and biomarkers were estimated using logistic (biomarker categories:median, ≥median) and linear regression models (log-transformed continuous biomarkers) adjusted for age, smoking, and comorbidities.We observed a positive association between statins and TILs among HPV-positive patients (TILws odds ratio [OR] = 2.80; 95% CI = 1.03-7.61), but no association among HPV-negative patients. We observed no association between statins and cytokines.Statins may influence TILs in HPV-positive patients. This may be the mechanism through which they improve prognosis in HPV-positive HNSCC patients.

Radiation therapy, a mainstay of treatment for head and neck cancer, is not always curative due to the development of treatment resistance; additionally, multi-institutional trials have questioned the efficacy of concurrent radiation with cetuximab, the epidermal growth factor receptor (EGFR) inhibitor. We unraveled a mechanism for radiation resistance; that is, radiation induces EGFR, which phosphorylates TRIP13 (thyroid hormone receptor interactor 13) on tyrosine 56. Phosphorylated (phospho-)TRIP13 promotes non-homologous end joining (NHEJ) repair to induce radiation resistance. NHEJ is the main repair pathway for radiation-induced DNA damage. Tumors expressing high TRIP13 do not respond to radiation but are sensitive to cetuximab or cetuximab combined with radiation. Suppression of phosphorylation of TRIP13 at Y56 abrogates these effects. These findings show that EGFR-mediated phosphorylation of TRIP13 at Y56 is a vital mechanism of radiation resistance. Notably, TRIP13-pY56 could be used to predict the response to radiation or cetuximab and could be explored as an actionable target.

Background: This study aims to evaluate the association between area deprivation and survival of head and neck squamous cell carcinoma (HNSCC) in the context of social and behavioral factors. Previously, area deprivation indices (ADIs) corresponding to decreasing neighborhood affluence have been associated with poorer survival of ovarian and lung cancer (Hufnagel et. al., 2021; Fairfield et. al., 2020). Further, neighborhood deprivation indices have been shown to be related to the survival of oropharynx cancer (Reitzel et. al., 2012). However, the relationship between area deprivation and the survival of multiple head and neck cancer sites has yet to be examined while considering the full extent of possible mediating or interacting factors, including the following: smoking, alcohol, human papillomavirus (HPV), urbanicity, and other dietary factors. Methods: We used 759 patients collected as a part of the University of Michigan Head and Neck Specialized Program of Research Excellence (UM HN-SPORE) prospective cohort study for whom we were able to assign an Area Deprivation Index (ADI) score. We calculated the ADI based on 17 census variables to describe neighborhood socioeconomics. Census block group level data was downloaded from the American Community Survey. The neighborhoods were categorized into ADI tertiles, least (Q1) to most deprived (Q3) neighborhoods. The role of ADI on disease-specific and overall survival was evaluated using Cox proportional hazards models adjusting for factors such as gender, age, body mass index (BMI), stage, site, smoking status, and, HPV status. Effect modification by comorbidity indices was also evaluated. Analyses used R studio (r-project.org).Results: Initial findings show that the hazard of head and neck cancer mortality is 1.74 (95% CI = 1.27, 2.39) times higher among the highest tertile of ADI scores relative to the lowest tertile, indicating that those in more highly disadvantaged areas had a lower survival proportion. However, after adjusting for age, stage at diagnosis, site, HPV status, BMI, and smoking status, and comorbidities the hazard ratio for was reduced to 1.12 (95% CI = 0.80, 1.58). Conclusion: Higher ADI indicative of increased area deprivation is associated with poorer survival of HNSCC. However, much of this association is accounted for by the presence of comorbidities, smoking and BMI in our cohort. Citation Format: Sarah Soppe, Mackenzie Gilson, A. A. Chamila Perera, Madhav KC, Edward S. Peters, Emily Belile, Steven Chinn, Gregory T. Wolf, Jeremy M.G. Taylor, Laura Rozek. Examination of the role of area deprivation indices on head and neck cancer survival adjusting for clinical and behavioral factors [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A015.

BACKGROUND: The associations between specific types of fat and head and neck squamous cell carcinoma (HNSCC) recurrence and mortality rates have not yet been examined. OBJECTIVES: The purpose of this study was to determine how intakes of various fat subtypes before cancer treatment are associated with recurrence and mortality in adults diagnosed with HNSCC. METHODS: This was a secondary analysis longitudinal cohort study of data collected from 476 newly diagnosed patients with HNSCC. Patients completed baseline FFQs and epidemiologic health surveys. Recurrence and mortality events were collected annually. Fat intakes examined included long-chain fatty acids (LCFAs), unsaturated fatty acids (FAs), PUFAs, ω-3 (n–3) PUFAs, ω-6 (n–6) PUFAs, MUFAs, animal fats, vegetable fats, saturated FAs, and trans fats. Associations between fat intake (categorized into tertiles) and time to event were tested using multivariable Cox proportional hazards models, adjusting for age, sex, smoking status, human papillomavirus status, tumor site, cancer stage, and total caloric intake. Intake of fats was compared with the lowest tertile. RESULTS: During the study period, there were 115 recurrent and 211 death events. High LCFA intake was associated with a reduced all-cause mortality risk (HR: 0.55; 95% CI: 0.34, 0.91; P-trend = 0.02). High unsaturated FA intake was associated with a reduced all-cause mortality risk (HR: 0.62; 95% CI: 0.40, 0.97; P-trend = 0.04) and HNSCC-specific mortality risk (HR: 0.51; 95% CI: 0.29, 0.90; P-trend = 0.02). High intakes of ω-3 PUFAs (HR: 0.56; 95% CI: 0.35, 0.91; P-trend = 0.02) and ω-6 PUFAs (HR: 0.57; 95% CI: 0.34, 0.94; P-trend = 0.02) were significantly associated with a reduced all-cause mortality risk. There were no significant associations between other fat types and recurrence or mortality risk. CONCLUSIONS: In this prospective survival cohort of 476 newly diagnosed patients with HNSCC, our data suggest that HNSCC prognosis may vary depending on the fat types consumed before cancer treatment. Clinical intervention trials should test these associations.

Background Human papillomavirus (HPV) oncogenes E6, E7, and shorter isoforms of E6 (E6*) are known carcinogenic factors in head and neck squamous cell carcinoma (HNSCC). Little is known regarding E6* functions. Methods We analyzed RNA-seq data from 68 HNSCC HPV type 16-positive tumors to determine host genes and pathways associated with E6+E7 expression (E6E7) or the percent of full-length E6 (E6%FL). Influence scores of E6E7 and E6%FL were used to test for associations with clinical variables. Results For E6E7, we recapitulated all major known affected pathways and revealed additional pathways. E6%FL was found to affect mitochondrial processes, and E6%FL influence score was significantly associated with overall survival and tumor size. Conclusions HPV E6E7 and E6* result in extensive, dose-dependent compensatory effects and dysregulation of key cancer pathways. The switch from E6 to E6* promotes oxidative phosphorylation, larger tumor size, and worse prognosis, potentially serving as a prognostic factor for HPV-positive HNSCC.

PURPOSE: The incidence of oropharyngeal squamous cell carcinoma continues to rise with the majority of patients receiving definitive or adjunctive radiation. For patients with locoregional recurrence after radiation, optimal treatment involves salvage surgery. The aim of this study is to identify factors that predict survival in order to ultimately improve patient selection for salvage surgery. METHODS: Retrospective cohort study at an NCI-designated cancer center. We analyzed patients with a history of head and neck radiation who presented with persistent/recurrent or second primary disease requiring salvage oropharyngeal resection from 1998–2017 (n=120). Patients were stratified into three classes based on time to recurrence and presence of laryngopharyngeal dysfunction. Primary outcomes were five-year overall survival (OS) and disease specific survival (DSS). RESULTS: Median OS was 27 months (median follow-up 20 months). Five-year OS was 47% for class I (recurrence > 2 years), 26% for class II (recurrence ≤ 2 years), and 0% for class III (recurrence ≤ 2 years and laryngopharyngeal dysfunction), (p

Objective IRX-2 is a homologous cell-derived multi-cytokine biologic with multifaceted immune modulatory effects that has been shown to induce increased lymphocyte infiltration into primary tumors in oral cavity carcinoma. Our objective was to characterize tumor immune gene expression and epigenomic changes after neoadjuvant IRX-2 immunotherapy in patients with squamous cell carcinoma of the oral cavity. Methods A randomized phase II trial was conducted of the IRX regimen 3 weeks prior to surgery for previously untreated patients with Stage II-IV oral cavity carcinoma. The treatment regimen consisted of low dose (300 mg/m2) cyclophosphamide (day 1) followed by 10 days of regional perilymphatic IRX-2 cytokine injections and daily oral indomethacin, zinc and omeprazole (Regimen 1) compared to the identical regimen without the IRX-2 cytokines (Regimen 2). The NanoString immune panel (730 genes) and Infinium MethylationEPIC BeadChip were performed to assess the gene expression and DNA methylation signatures, respectively, in pre- and post-immunotherapy tumor samples. Results A total of 51 and 79 immune-related genes were found upregulated and downregulated, respectively, in the samples from Regimen 1 patients after treatment, while 51 and 56 were found upregulated and downregulated in the samples for Regimen 2. When comparing the changes between the two regimens, we identified 9 genes significantly different, including DMBT1, a potential tumor suppressor, functioning in tumor invasion of head and neck cancer. The exploration of DNA methylation showed slight overall hypermethylation after treatment in both regimens, especially for Regimen 1 immune responders, and methylation-based cell type deconvolution demonstrated high concordance with tumor infiltrating T lymphocyte cell counts. Conclusion While a consistent patient response after treatment was observed, most changes were similar between regimens, indicating a subtle, targeted, or patient-specific effect of IRX-2 cytokines. Change in DMBT1 expression was a unique finding that will require further study to better understand its significance.

No studies, to date, have scrutinized the role of

Despite major progress in elucidating the pathobiology of head and neck squamous cell carcinoma (HNSCC), the high frequency of disease relapse correlates with unacceptably deficient patient survival. We previously showed that cancer stem-like cells (CSCs) drive tumorigenesis and progression of HNSCC. Although CSCs constitute only 2–5% of total tumor cells, CSCs contribute to tumor progression by virtue of their high tumorigenic potential and their resistance to chemo-, radio-, and immunotherapy. Not only are CSCs resistant to therapy, but cytotoxic agents actually enhance cancer stemness by activating transcription of pluripotency factors and by inducing expression of Bmi-1, a master regulator of stem cell self-renewal. We hypothesized therapeutic inhibition of interleukin-6 receptor (IL-6R) suppresses Bmi-1 to overcome intrinsic chemoresistance of CSCs. We observed that high Bmi-1 expression correlates with decreased (p = 0.04) recurrence-free survival time in HNSCC patients (n = 216). Blockade of IL-6R by lentiviral knockdown or pharmacologic inhibition with a humanized monoclonal antibody (Tocilizumab) is sufficient to inhibit Bmi-1 expression, secondary sphere formation, and to decrease the CSC fraction even in Cisplatin-resistant HNSCC cells. IL-6R inhibition with Tocilizumab abrogates Cisplatin-mediated increase in CSC fraction and induction of Bmi-1 in patient-derived xenograft (PDX) models of HNSCC. Notably, Tocilizumab inhibits Bmi-1 and suppresses growth of xenograft tumors generated with Cisplatin-resistant HNSCC cells. Altogether, these studies demonstrate that therapeutic blockade of IL-6R suppresses Bmi-1 function and inhibits cancer stemness. These results suggest therapeutic inhibition of IL-6R might be a viable strategy to overcome the CSC-mediated chemoresistance typically observed in HNSCC patients.

Dietary intake is understood to contribute to nutrition impact symptoms (NIS) in patients with head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to evaluate the performance of four a priori-defined diet quality indices on the presence of NIS 1 year following diagnosis using data on 323 participants from the University of Michigan Head and Neck Specialized Program of Research Excellence (UM-SPORE). Pretreatment dietary intake was measured before treatment initiation using a food frequency questionnaire. NIS were measured along seven subdomains. Multivariable binary logistic regression models were constructed to evaluate relationships between pretreatment scores on a priori-defined diet quality indices (AHEI-2010, aMED, DASH, and a low-carbohydrate score) and the presence of individual symptoms in addition to a composite “symptom summary score” 1-year postdiagnosis. There were several significant associations between different indices and individual NIS. For the symptom summary score, there were significant inverse associations observed for aMED (ORQ5-Q1: 0.36, 95% CI: 0.14–0.88, ptrend = 0.04) and DASH (ORQ5-Q1: 0.38, 95% CI: 0.15–0.91, ptrend = 0.02) and the presence of NIS 1-year postdiagnosis. Higher adherence to the aMED and DASH diet quality indices before treatment may reduce NIS burden at 1-year postdiagnosis.

Key Points Question Can a model be developed for individualized survival, locoregional recurrence, and distant metastasis prognostication for patients newly diagnosed with oropharyngeal cancer, incorporating clinical, oncologic, and imaging data? Findings In this prognostic study, model predictions for 5-year overall survival demonstrated excellent discrimination in cohort study training data for models with and without imaging variables. This model appeared to possess good calibration and well stratified patients in terms of likely outcomes among many competing events. Meaning This prognostic model and web-based application may serve as a useful tool to provide clinicians, researchers, and patients with oropharyngeal cancer robust, individualized prognostic estimations., Importance Recent insights into the biologic characteristics and treatment of oropharyngeal cancer may help inform improvements in prognostic modeling. A bayesian multistate model incorporates sophisticated statistical techniques to provide individualized predictions of survival and recurrence outcomes for patients with newly diagnosed oropharyngeal cancer. Objective To develop a model for individualized survival, locoregional recurrence, and distant metastasis prognostication for patients with newly diagnosed oropharyngeal cancer, incorporating clinical, oncologic, and imaging data. Design, Setting, and Participants In this prognostic study, a data set was used comprising 840 patients with newly diagnosed oropharyngeal cancer treated at a National Cancer Institute–designated center between January 2003 and August 2016; analysis was performed between January 2019 and June 2020. Using these data, a bayesian multistate model was developed that can be used to obtain individualized predictions. The prognostic performance of the model was validated using data from 447 patients treated for oropharyngeal cancer at Erasmus Medical Center in the Netherlands. Exposures Clinical/oncologic factors and imaging biomarkers collected at or before initiation of first-line therapy. Main Outcomes and Measures Overall survival, locoregional recurrence, and distant metastasis after first-line cancer treatment. Results Of the 840 patients included in the National Cancer Institute–designated center, 715 (85.1%) were men and 268 (31.9%) were current smokers. The Erasmus Medical Center cohort comprised 300 (67.1%) men, with 350 (78.3%) current smokers. Model predictions for 5-year overall survival demonstrated good discrimination, with area under the curve values of 0.81 for the model with and 0.78 for the model without imaging variables. Application of the model without imaging data in the independent Dutch validation cohort resulted in an area under the curve of 0.75. This model possesses good calibration and stratifies patients well in terms of likely outcomes among many competing events. Conclusions and Relevance In this prognostic study, a multistate model of oropharyngeal cancer incorporating imaging biomarkers appeared to estimate and discriminate locoregional recurrence from distant metastases. Providing personalized predictions of multiple outcomes increases the information available for patients and clinicians. The web-based application designed in this study may serve as a useful tool for generating predictions and visualizing likely outcomes for a specific patient., This prognostic study reports on the development of a model including factors used in establishing an individualized prognosis for locoregional recurrence, distant metastasis, and survival in patients with oropharyngeal cancer.

Background: The updated American Joint Committee on Cancer (AJCC) 8th Edition staging manual restructured nodal classification and staging by placing less prognostic emphasis on nodal metastases for human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC). However, there was no change for HPV-negative OPSCC. The purpose of our study is to examine the impact of nodal metastases on survival in HPV-negative OPSCC. Methods: HPV-negative OPSCC was queried from the National Cancer Database (NCDB) and Surveillance, Epidemiology and End Results program (SEER) databases. Univariable and multivariable models were utilized to determine the impact of nodal status on overall survival. These patients were reclassified according to AJCC 8 HPV-positive criteria (TNM8+) and risk stratification was quantified with C-statistic. Results: There were 11,147 cases of HPV-negative OPSCC in the NCDB and 3,613 cases in SEER that were included in the nodal classification analysis. Unlike nonoropharyngeal malignancies, increased nodal stage is not clearly associated with survival for patients with OPSCC independent of HPV status. When the TNM8+ was applied to HPV-negative patients, there was improved concordance in the NCDB cohort, 0.561 (plus minus) 0.004 to 0.624 (plus minus) 0.004 (difference +0.063) and the SEER cohort, 0.561 (plus minus) 0.008 to 0.625 (plus minus) 0.008 (difference +0.065). Conclusions: We demonstrated a reduced impact of nodal metastasis on OPSCC survival, independent of HPV status and specific to OPSCC. Impact: We demonstrate, for the first time that when nodal staging is deemphasized as a part of overall staging, we see improved concordance and risk stratification for HPV-negative OPSCC. The exact mechanism of this differential impact remains unknown but offers a novel area of study.

OBJECTIVE: Bioselection to assess tumor response after induction chemotherapy has been introduced as an alternative treatment strategy to total laryngectomy for patients with advanced larynx squamous cell carcinoma (LSCC). Tumor infiltrating lymphocytes (TILs) have proven to serve as prognostic biomarkers in head and neck cancer, but have not been evaluated as a way to select patients for treatment paradigms. The aim of this study is to evaluate the role of pretreatment TILs in patients with advanced LSCC undergoing the bioselection paradigm. STUDY DESIGN: Retrospective study SETTING: Tertiary care hospital METHODS: Patients with advanced LSCC treated with bioselection and with available tissue were included (n=76). Patients were stratified into CD8 low or CD8 high cohorts using the median TIL count. Kaplan-Meier survival analysis and multivariate cox regression was performed using SPSS(v. 26). RESULTS: After controlling for tobacco use, tumor site and stage, high CD8 TIL count was an independent predictor of improved five-year disease specific survival (HR 0.17, 95% CI 0.03-0.84, p=0.03). CD8 TIL counts did not predict response to induction chemotherapy, however subgroup analysis of patients treated with CRT revealed that CD8 TIL count was significantly associated with degree of response (p=0.012). CONCLUSION: These findings support prior data published by our group which shows TILS are predictive of disease specific survival in patients with head and neck cancer. CD8 TIL counts were significantly associated with degree of clinical response after induction chemotherapy. These results suggest that pretreatment assessment of tumor infiltrating CD8 cells could be useful in selecting patients.

The presence of tumor-infiltrating lymphocytes (TIL) in the tumor microenvironment has been demonstrated to be of prognostic value in various cancers. In this systematic review and meta-analysis, we investigated the prognostic value of TIL in laryngeal squamous cell carcinoma (LSCC). We performed a systematic search in PubMed for publications that investigated the prognostic value of TIL in LSCC. A meta-analysis was performed including all studies assessing the association between TIL counts in hematoxylin-eosin (HE)-stained sections, for CD8+ and/or CD3+/CD4+ TIL and overall survival (OS) or disease-free survival (DFS). The pooled meta-analysis showed a favorable prognostic role for stromal TIL in HE sections for OS (HR 0.57, 95% CI 0.36–0.91, p = 0.02), and for DFS (HR 0.56, 95% CI 0.34–0.94, p = 0.03). High CD8+ TIL were associated with a prolonged OS (HR 0.62, 95% CI 0.4–0.97, p = 0.04) and DFS (HR 0.73, 95% CI 0.34–0.94, p = 0.002). High CD3+/CD4+ TIL demonstrated improved OS (HR 0.32, 95% CI 0.16–0.9, p = 0.03) and DFS (HR 0.23, 95% CI 0.10–0.53, p = 0.0005). This meta-analysis confirmed the favorable prognostic significance of TIL in LSCC. High stromal TIL evaluated in HE sections and intra-tumoral and stromal CD3+, CD4+ and/or CD8+ TIL might predict a better clinical outcome.

Background Accurate, individualized prognostication in patients with oropharyngeal squamous cell carcinoma (OPSCC) is vital for patient counseling and treatment decision making. With the emergence of human papillomavirus (HPV) as an important biomarker in OPSCC, calculators incorporating this variable have been developed. However, it is critical to characterize their accuracy prior to implementation. Methods Four OPSCC calculators were identified that integrate HPV into their estimation of 5-year overall survival. Treatment outcomes for 856 patients with OPSCC who were evaluated at a single institution from 2003 through 2016 were analyzed. Predicted survival probabilities were generated for each patient using each calculator. Calculator performance was assessed and compared using Kaplan-Meier plots, receiver operating characteristic curves, concordance statistics, and calibration plots. Results Correlation between pairs of calculators varied, with coefficients ranging from 0.63 to 0.90. Only 3 of 6 pairs of calculators yielded predictions within 10% of each other for at least 50% of patients. Kaplan-Meier curves of calculator-defined risk groups demonstrated reasonable stratification. Areas under the receiver operating characteristic curve ranged from 0.74 to 0.80, and concordance statistics ranged from 0.71 to 0.78. Each calculator demonstrated superior discriminatory ability compared with clinical staging according to the seventh and eighth editions of the American Joint Committee on Cancer staging manual. Among models, the Denmark calculator was found to be best calibrated to observed outcomes. Conclusions Existing calculators exhibited reasonable estimation of survival in patients with OPSCC, but there was considerable variability in predictions for individual patients, which limits the clinical usefulness of these calculators. Given the increasing role of personalized treatment in patients with OPSCC, further work is needed to improve accuracy and precision, possibly through the identification and incorporation of additional biomarkers.