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Background: Oral squamous cell carcinoma (SCC) is associated with oral microbial dysbiosis. In this unique study, we compared pre- to post-treatment salivary microbiome in patients with SCC by 16S rRNA gene sequencing and examined how microbiome changes correlated with the expression of an anti-microbial protein., Results: Treatment of SCC was associated with a reduction in overall bacterial richness and diversity. There were significant changes in the microbial community structure, including a decrease in the abundance of Porphyromonaceae and Prevotellaceae and an increase in Lactobacillaceae. There were also significant changes in the microbial community structure before and after treatment with chemoradiotherapy, but not with surgery alone. In patients treated with chemoradiotherapy alone, several bacterial populations were differentially abundant between responders and non-responders before and after therapy. Microbiome changes were associated with a change in the expression of DMBT1, an anti-microbial protein in human saliva. Additionally, we found that salivary DMBT1, which increases after treatment, could serve as a post-treatment salivary biomarker that links to microbial changes. Specifically, post-treatment increases in human salivary DMBT1 correlated with increased abundance of Gemella spp., Pasteurellaceae spp., Lactobacillus spp., and Oribacterium spp. This is the first longitudinal study to investigate treatment-associated changes (chemoradiotherapy and surgery) in the oral microbiome in patients with SCC along with changes in expression of an anti-microbial protein in saliva., Conclusions: The composition of the oral microbiota may predict treatment responses; salivary DMBT1 may have a role in modulating the oral microbiome in patients with SCC. After completion of treatment, 6 months after diagnosis, patients had a less diverse and less rich oral microbiome. Leptotrichia was a highly prevalent bacteria genus associated with disease. Expression of DMBT1 was higher after treatment and associated with microbiome changes, the most prominent genus being Gemella Video Abstract., (© 2023. The Author(s).)

Objective: To evaluate whether nodal yields and ratios based on level serves as prognostic indicators in patients with oral cavity squamous cell carcinoma undergoing neck dissection., Materials and Methods: A retrospective analysis of 342 patients with oral cavity squamous cell carcinoma treated surgically between 1998 and 2017 were included.Demographics and clinicopathologic data were collated. Disease specific survival and overall survival were analyzed via Kaplan-Meier method and log-rank test as well as univariable and multivariable Cox models., Results: Total nodal yield is associated with improved overall and disease specific survival (p < 0.01). Total positive nodal yield (p < 0.01), positive nodal ratio per level (p < 0.001), and identification of <4 lymph nodes/level (p < 0.001) are associated with worse disease specific survival and overall survival. A ratio of at least 4 lymph nodes/level dissected yields the maximal hazard ratio on for both disease specific and overall survival optimizes the Kaplan-Meier split between survival groups. After controlling for sex, age, margin status, disease stage, extranodal extension, perineural invasion, and lymphovascular invasion as fixed covariates in the Cox models, a nodal level ratio of 4 lymph nodes/level provides hazard ratio (95% CI) of 3.59 (1.69, 7.60); p < 0.0006) for disease free survival and 2.90 (1.54, 5.46; p < 0.001) for overall survival., Conclusion: Nodal level ratio of < 4 lymph nodes/level is associated with worse disease specific and overall survival in oral cavity squamous cell carcinoma. This level-specific metric may prove useful qualitatively and in predicting survival in oral cavity cancer with broader utility to address variations in levels of neck dissection performed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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. See related commentary by Hondermarck and Jiang, p. 2342., (©2023 American Association for Cancer Research.)

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., (©2022 American Association for Cancer Research.)

Purpose: Galanin receptor 2 (GALR2) plays a significant role in the progression of head and neck squamous cell carcinomas (HNSCC). Since there is virtually no information on immunomodulation mediated by its ligand in the tumor microenvironment, we assessed the effects of galanin on peripheral blood mononuclear cells (PBMCs)., Methods: After verification of GALR2 expression and it activity in PBMCs we evaluated the effect of galanin and conditioned media from HNSCC cell lines silenced for galanin or antibody-depleted, on proliferation, apoptosis, cytokine expression and activation/differentiation of immune cells., Results: We found that galanin alone and as a component of the HNSCC secretome decreased HNSCC cell proliferation and expression of pro-inflammatory cytokines (IFNγ, IL-12, IL-17A, IL-1α, IL-6 and TNF-α), whilst increasing apoptosis and expression of pro-tumoral cytokines/growth factors (IL-10, IL-4, PDGF and GM-CSF). T cell activation (using CD69 as activation marker) and anti-tumoral phenotypes in CD4 + T cells (Th1 and Th17) were found to be suppressed. In vivo, tumor growth was found to be increased in the presence of galanin-stimulated PBMCs. Data from The Cancer Genome Atlas (TCGA) revealed that high expression of galanin was associated with a reduced overall survival of patients with HNSCC., Conclusion: Our data indicate that galanin secreted by HNSCC cells exhibits immune-suppressive and pro-tumoral effects., (© 2022. The Author(s).)

Recurrent and new tumors, attributed in part to lateral invasion, are frequent in squamous cell carcinomas and lead to poor survival. We identified a mechanism by which cancer subverts adjacent histologically normal epithelium to enable small clusters of cancer cells to burrow undetected under adjacent histologically normal epithelium. We show that suppression of DMBT1 within cancer promotes aggressive invasion and metastasis in vivo and is associated with metastasis in patients. Cancer cells via TGFβ1 and TNFα also suppress DMBT1 in adjacent histologically normal epithelium, thereby subverting it to promote invasion of a small population of tumor cells. The sufficiency of DMBT1 in this process is demonstrated by significantly higher satellite tumor nests in Dmbt1-/- compared with wild-type mice. Moreover, in patients, invasion of small tumor nests under adjacent histologically normal epithelium is associated with increased risk for recurrence and shorter disease-free survival. This study demonstrates a crucial role of adjacent histologically normal epithelium in invasion and its important role in the tumor microenvironment and opens new possibilities for therapeutic strategies that reduce tumor recurrence., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Singh et al.)

Background: The purpose of this study was to evaluate the AJCC eighth edition clinical staging system for human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma and to further understand how clinical stage and smoking history affect oncologic outcomes. The purpose of this study was to present the understanding of how clinical stage and smoking history affect oncologic outcomes in human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (SCC) is critical for selecting patients for treatment deintensification., Methods: Kaplan-Meier and Cox regression were used to evaluate overall survival (OS), locoregional recurrence-free survival (LRFS), and distant recurrence-free survival (DRFS). Concordance statistics (C-indices) were used to compare discriminating ability., Results: The OS and DRFS but not LRFS were significantly distributed using the American Joint Committee on Cancer (AJCC) seventh and eighth editions criteria. The C-indices for OS, LRFS, and DRFS were 0.57, 0.54, and 0.60, respectively, using the AJCC seventh edition, and 0.63, 0.53, and 0.65, respectively, using the AJCC eighth edition. On multivariate analysis, 1 + pack-year smoking history correlated with OS (hazard ratio [HR] 1.96; 95% confidence interval [CI] 1.2-3.1; P < .01) but not LRFS or DRFS., Conclusion: These results support implementation of the AJCC eighth edition for HPV-associated oropharyngeal SCC. Clinical stage may be more important than smoking history in selection for deintensification., (© 2018 Wiley Periodicals, Inc.)

Background: Recurrent laryngeal squamous cell carcinomas (LSCCs) are associated with poor outcomes, without reliable biomarkers to identify patients who may benefit from adjuvant therapies. Given the emergence of tumor-infiltrating lymphocytes (TIL) as a biomarker in head and neck squamous cell carcinoma, we generated predictive models to understand the utility of CD4 + , CD8 + and/or CD103 + TIL status in patients with advanced LSCC., Methods: Tissue microarrays were constructed from salvage laryngectomy specimens of 183 patients with recurrent/persistent LSCC and independently stained for CD4 + , CD8 + , and CD103 + TIL content. Cox proportional hazards regression analysis was employed to assess combinations of CD4 + , CD8 + , and CD103 + TIL levels for prediction of overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS) in patients with recurrent/persistent LSCC., Results: High tumor CD103 + TIL content was associated with significantly improved OS, DSS, and DFS and was a stronger predictor of survival in recurrent/persistent LSCC than either high CD8 + or CD4 + TIL content. On multivariate analysis, an "immune-rich" phenotype, in which tumors were enriched for both CD103 + and CD4 + TILs, conferred a survival benefit (OS hazard ratio: 0.28, p = 0.0014; DSS hazard ratio: 0.09, p = 0.0015; DFS hazard ratio: 0.18, p = 0.0018) in recurrent/persistent LSCC., Conclusions: An immune profile driven by CD103 + TIL content, alone and in combination with CD4 + TIL content, is a prognostic biomarker of survival in patients with recurrent/persistent LSCC. Predictive models described herein may thus prove valuable in prognostic stratification and lead to personalized treatment paradigms for this patient population.

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., (© 2018 American Cancer Society.)

A diagnosis of perineural invasion (PNI), defined as cancer within or surrounding at least 33% of the nerve, leads to selection of aggressive treatment in squamous cell carcinoma (SCC). Recent mechanistic studies show that cancer and nerves interact prior to physical contact. The purpose of this study was to explore cancer-nerve interactions relative to clinical outcome. Biopsy specimens from 71 patients with oral cavity SCC were stained with hematoxylin and eosin and immunohistochemical (IHC; cytokeratin, S100, GAP43, Tuj1) stains. Using current criteria, PNI detection was increased with IHC. Overall survival (OS) tended to be poor for patients with PNI (P = .098). OS was significantly lower for patients with minimum tumor-nerve distance smaller than 5 μm (P = .011). The estimated relative death rate decreased as the nerve-tumor distance increased; there was a gradual drop off in death rate from distance equal to zero that stabilized around 500 μm. In PNI-negative patients, nerve diameter was significantly related to OS (HR 2.88, 95%CI[1.11,7.49]). Among PNI-negative nerves, larger nerve-tumor distance and smaller nerve diameter were significantly related to better OS, even when adjusting for T-stage and age (HR 0.82, 95% CI[0.72,0.92]; HR 1.27, 95% CI[1.00,1.62], respectively). GAP43, a marker for neuronal outgrowth, stained less than Tuj1 in nerves at greater distances from tumor (OR 0.76, 95% CI[0.73,0.79]); more GAP43 staining was associated with PNI. Findings from a small group of patients suggest that nerve parameters other than presence of PNI can influence outcome and that current criteria of PNI need to be re-evaluated to integrate recent biological discoveries., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Patients undergoing salvage laryngectomy are predisposed to radiation-induced hypothyroidism and impaired wound healing secondary to the tissue effects of prior treatment. The impact of hypothyroidism on postoperative wound healing is not established., Methods: A single-institution retrospective case series was performed. The inclusion criteria specified preoperatively euthyroid adults who underwent salvage laryngectomy with concurrent neck dissection between 1997 and 2015 for persistent or recurrent laryngeal squamous cell carcinoma after radiation or chemoradiation therapy (n = 182). The principal explanatory variable was postoperative hypothyroidism, defined as thyroid-stimulating hormone (TSH) higher than 5.5 mIU/L. The primary end points of the study were pharyngocutaneous fistulas and wounds requiring reoperation. Multivariate analysis was performed., Results: The fistula rate was 47% among hypothyroid patients versus 23% among euthyroid patients. In the multivariate analysis, the patients who experienced hypothyroidism in the postoperative period had a 3.6-fold greater risk of fistula [95% confidence interval (CI) 1.8-7.1; p = 0.0002]. The hypothyroid patients had an 11.4-fold greater risk for a required reoperation (24.4 vs 5.4%) than the euthyroid patients (95% CI 2.6-49.9; p = 0.001). The risk for fistula (p = 0.003) and reoperation (p = 0.001) increased with increasing TSH. This corresponds to an approximate 12.5% incremental increase in the absolute risk for fistula and a 10% increase in the absolute risk for reoperation with each doubling of the TSH., Conclusion: Postoperative hypothyroidism independently predicts postoperative wound-healing complications. The association of hypothyroidism with fistula formation may yield opportunities to modulate wound healing with thyroid supplementation or to provide a biomarker of wound progression.

Due to the high incidence of recurrent squamous cell carcinoma of the head and neck and the toxicity profile of current salvage regimens, there is a need for tolerable and effective treatment options. We performed a retrospective matched case series to report our experience with recurrent high-risk patients who received capecitabine (CAP) therapy in the adjuvant setting after salvage therapy. The 5-year recurrence-free survival rates for the CAP and control cohorts were 54% (95% CI, 0.27%-0.75%) and 27% (95% CI, 0.09%-0.50%), respectively. Multivariable Cox modeling showed a significant improvement in recurrence-free survival in the CAP cohort (hazard ratio, 0.19; 95% CI, 0.04-0.92; P = .0392). While this was a respective analysis that could not control for all variables, these exploratory findings offer insights that may inform a prospective study to determine CAP efficacy.

Background: We examined the prognostic value of a panel of biomarkers in patients with squamous cell carcinoma of the head and neck (SCCHN) who were human immunodeficiency virus (HIV) positive (HIV-positive head and neck cancer) and HIV negative (HIV-negative head and neck cancer)., Methods: Tissue microarrays (TMAs) were constructed using tumors from 41 disease site-matched and age-matched HIV-positive head and neck cancer cases and 44 HIV-negative head and neck cancer controls. Expression of tumor biomarkers was assessed by immunohistochemistry (IHC) and correlations examined with clinical variables., Results: Expression levels of the studied oncogenic and inflammatory tumor biomarkers were not differentially regulated by HIV status. Among patients with HIV-positive head and neck cancer, laryngeal disease site (P = .003) and Clavien-Dindo classification IV (CD4) counts <200 cells/μL (P = .01) were associated with poor prognosis. Multivariate analysis showed that p16 positivity was associated with improved overall survival (OS; P < .001) whereas increased expression of transforming growth factor-beta (TGF-β) was associated with poor clinical outcome (P = .001)., Conclusion: Disease site has significant effect on the expression of biomarkers. Expression of tumor TGF-β could be a valuable addition to the conventional risk stratification equation for improving head and neck cancer disease management strategies., (© 2017 Wiley Periodicals, Inc.)

Background: Total laryngectomy remains the treatment of choice for recurrent/persistent laryngeal squamous cell carcinoma (SCC) after radiotherapy (RT) or chemoradiotherapy (CRT). However, despite attempts at aggressive surgical salvage, survival in this cohort remains suboptimal., Methods: A prospectively maintained single-institution database was queried for patients undergoing total laryngectomy for recurrent/persistent laryngeal SCC after initial RT/CRT between 1998 and 2015(n = 244). Demographic, clinical, and survival data were abstracted. The Kaplan-Meier survival curves and hazard ratios (HRs) were calculated., Results: Five-year overall survival (OS) was 49%. Five-year disease-free survival (DFS) was 58%. Independent predictors of OS included severe comorbidity (Adult Comorbidity Evaluation-27 [ACE-27] scale; HR 3.76; 95% confidence interval [CI] 1.56-9.06), and positive recurrent clinical nodes (HR 2.91; 95% CI 1.74-4.88)., Conclusion: Severe comorbidity status is the strongest predictor of OS, suggesting that increased attention to mitigating competing risks to health is critical. These data may inform a risk prediction model to allow for focused shared decision making, preoperative health optimization, and patient selection for adjuvant therapies., (© 2017 Wiley Periodicals, Inc.)

Objective Emerging data have demonstrated suboptimal outcomes among patients with stage II larynx cancer. Our objective is to report survival outcomes for T2N0M0 larynx cancer and to determine the cause-specific survival. Study Design Case series with planned data collection. Setting Tertiary academic center. Subjects Adults with T2N0M0 squamous cell carcinoma of the larynx treated with curative intent. Methods A head and neck cancer epidemiology database was queried for eligible subjects from 2003 to 2014. Data were extracted from the electronic medical record and research database, and survival analyses were performed. Results Thirty-four patients with previously untreated stage II larynx cancer were identified (median follow-up 48 months). Patients included 27 males and 7 females with a mean age of 59 years. The majority of tumors arose from the glottis (59%). Of the cohort, 12% were treated with surgery, 65% radiation therapy, and 24% chemoradiation therapy. The estimated 2-year overall survival was 81%, (95% confidence interval [CI], 59%-92%), disease-specific survival was 91% (95% CI, 69%-98%), and recurrence-free survival was 84% (95% CI, 65%-93%). Four of 5 patients with persistent or recurrent disease posttreatment were successfully salvaged with total laryngectomy with 100% locoregional control. There were 11 mortalities (2 disease related, 2 due to metachronous primaries, 3 treatment related, and 4 from other/unknown causes). Conclusion Stage II laryngeal cancer has suboptimal survival outcomes. This appears to be a reflection of medical comorbidities, propensity for metachronous primaries, and the sequelae of late treatment effects rather than poor locoregional control.

Objectives It is unknown if preoperative tracheostomy for persistent/recurrent laryngeal squamous cell carcinoma (LSCC) plays a role in unrecognized local disease spread and disease recurrence after salvage laryngectomy. The goals of this study were to determine the effect of preoperative tracheostomy on disease-free survival (DFS) in patients with recurrent/persistent LSCC undergoing salvage laryngectomy. Study Design Retrospective case series derived from prospectively maintained database. Setting Tertiary care academic center. Subjects Patients with recurrent/persistent LSCC after radiation/chemoradiation (RT/CRT) who underwent salvage laryngectomy at the University of Michigan from 1997 to 2015. Methods Demographic, clinical, pathologic, and survival data were collected. Kaplan-Meier survival estimates were performed. Results DFS was worse for patients with tracheostomy prior to laryngectomy than patients without a tracheostomy (5 year: 39% vs 67%; P < .001). Patients with tracheostomy prior to RT/CRT compared to patients with tracheostomy after RT/CRT or patients without a tracheostomy had worse DFS (5-year: 25%, 49%, and 67%, respectively; P < .001). In bivariable analyses controlling for T classification, N classification, or overall stage, preoperative tracheostomy was associated with worse DFS. In multivariable analysis, presence of a preoperative tracheostomy had a worse DFS (hazard ratio, 1.63; 95% confidence interval, 1.00-2.67; P = .048). Conclusion Preoperative tracheostomy is associated with disease recurrence in patients with persistent/recurrent LSCC undergoing salvage laryngectomy, particularly in patients who had tracheostomy prior to completion of initial RT/CRT. Notably, preoperative tracheostomy as a causal factor vs marker for disease recurrence is difficult to ascertain. Nevertheless, clinicians should be aware of the increased risk of locoregional recurrence in patients with preoperative tracheostomy when counseling on surgical salvage and when considering the role of additional therapy.

Objective: Patients with a family history of cancer may be genetically predisposed to carcinogenesis. This could affect risk of recurrence, second primary tumors, and overall outcomes after treatment of a primary cancer. We evaluated the association between family history of cancer and disease-specific survival in a cohort of patients with primary head and neck squamous carcinoma (HNSCC)., Study Design: Six hundred and forty-three incident HNSCC patients recruited through the University of Michigan Specialized Program of Research Excellence were followed for up to 5 years for survival. Participants were interviewed about personal and family cancer history, demographic information, and behavioral habits., Methods: Cox proportional hazards models were used to estimate the association between family history of cancer in a first-degree relative and disease-specific survival., Results: After multivariable adjustment, we found a nonsignificant inverse association between family history and HNSCC mortality (hazard ratio [HR] = 0.88, 95% confidence interval [CI] 0.57-1.35). This association was stronger and statistically significant among patients who currently both drank alcohol and smoked cigarettes at diagnosis (HR = 0.46, 95% CI = 0.22-0.97); no association was observed among participants who did not both drink and smoke at the time of diagnosis (HR = 1.14, 95% CI = 0.68-1.91; p-interaction = 0.046)., Conclusion: Results from this study suggest that having a family history of cancer may be associated with improved disease-specific survival in patients who use tobacco and alcohol. Additional large studies, particularly in populations including nonwhites and women, are needed to confirm or refute the association and to elucidate the genetic factors that may underlie this potential association., Level of Evidence: 2b. Laryngoscope, 127:1816-1820, 2017., (© 2017 The American Laryngological, Rhinological and Otological Society, Inc.)

Purpose: There is a paucity of biomarkers to predict failure in human papillomavirus-positive (HPV + ) oropharyngeal squamous cell carcinoma (OPSCC) following curative therapy. E6/E7 viral oncoproteins are constitutively expressed in HPV + tumors and highly immunogenic, resulting in readily detected serum antibodies. The purpose of this study is to determine whether serum E6 and E7 antibody levels can potentially serve as a biomarker of recurrence in patients with HPV+OPSCC. Experimental Design: We evaluated E6/E7 antibody levels in patients with previously untreated, advanced stage (III, IVa-b), HPV+OPSCC receiving definitive chemoradiation under a uniform protocol from 2003 to 2010. Baseline and longitudinal serum samples were obtained from our archived repository. E6/E7 serum levels were measured using a glutathione- S -transferase capture ELISA and quantified by approximating the area under the dilution curve, and were analyzed using ANOVA and linear mixed model for longitudinal analysis. Results: We compared 22 HPV+OPSCC patients who developed recurrence with 30 patients who remained disease-free. There were no differences in T classification, N classification, disease subsite, or smoking status between the groups. In a longitudinal analysis, recurrent patients had significantly higher E6 and E7 serum antibody levels than the nonrecurrent patients over the follow-up period ( P = 0.02 and P = 0.002, respectively). Patients who recurred had a lower clearance of E7 antibody than patients who remained disease-free ( P = 0.0016). Conclusions: Patients with HPV+OPSCC whose disease recurs have a lower clearance of E6 and E7 antibodies than patients who do not have recurrence. The ratio of E7 antibody at disease recurrence compared with baseline is potentially a clinically significant measurement of disease status in HPV+OPSCC. Clin Cancer Res; 23(11); 2723-9. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Importance: Use of chemoradiotherapy for advanced laryngeal cancer led to a major shift in treatment as an alternative to laryngectomy. Despite widespread adoption of chemoradiotherapy, survival rates have not improved and the original premise of matching neoadjuvant chemotherapy tumor response to determine subsequent treatment has not been followed., Objective: To determine whether improved survival could be achieved by incorporating a single cycle of neoadjuvant chemotherapy to select patients with advanced disease for either laryngectomy or concurrent chemoradiotherapy., Design, Setting, and Participants: An unselected cohort of 247 patients with laryngeal cancer in an academic institution between 2002 and 2012 was evaluated. Patients with limited disease (stages I and II) underwent endoscopic resection, radiotherapy, or chemoradiotherapy for deeply invasive T2 lesions. For patients with advanced disease (stages III and IV), neoadjuvant chemotherapy, concurrent chemoradiotherapy, or primary surgery was recommended. Overall survival (OS) and disease-specific survival (DSS) were analyzed. Median follow-up was 48 months. The study was conducted from January 1, 2002, to December 31, 2012; data analysis was completed December 1, 2015., Interventions: Endoscopic resection, radiotherapy, chemoradiotherapy, neoadjuvant chemotherapy, concurrent chemoradiotherapy, and primary surgery., Main Outcomes and Measures: Overall survival and DSS., Results: Of the 247 patients, 191 (77.3%) were male; mean (SD) age was 59.6 (10.4) years. Of 94 patients with limited disease, 33 (35.1%) underwent endoscopic resection; 50 (53.2%), radiotherapy alone; and 11 (11.7%), chemoradiotherapy for deeply invasive T2 lesions. Of 153 patients with advanced disease, 71 (46.4%) received neoadjuvant chemotherapy; 50 (32.7%), concurrent chemoradiotherapy; and 32 (20.9%), surgery. Five-year OS and DSS was 75% (95% CI, 68%-81%) and 83% (95% CI, 77%-88%), respectively, for the entire cohort. The DSS was 92% (95% CI, 83%-97%) for patients with stage I or II and 78% (95% CI, 69%-84%) for patients with stage III or IV disease. For patients with advanced disease, 5-year OS and DSS ranged from 78% (95% CI, 55%-90%) and 91% (95% CI, 67%-98%), respectively, for surgery; to 76% (95% CI, 63%-85%) and 79% (95% CI, 67%-88%), respectively, for neoadjuvant bioselection; and to 61% (95% CI, 44%-75%) and 66% (95% CI, 48%-79%), respectively, for primary chemoradiotherapy. Propensity-adjusted, multivariable controlling for known prognostic factors DSS was significantly improved in the neoadjuvant group compared with the chemoradiotherapy group (hazard ratio [HR], 0.48; 95% CI, 0.29-0.80)., Conclusions and Relevance: Superior survival rates were achieved with a bioselective treatment approach using a single cycle of neoadjuvant chemotherapy. Good survival rates were also achieved in patients selected for primary surgery, and both neoadjuvant chemotherapy and primary surgery were better than survival rates with concurrent chemoradiotherapy, suggesting that the optimal individualized treatment approach for patients with advanced laryngeal cancer has not yet been defined.

In this study, high-risk HPV (hrHPV) incidence, prognostic biomarkers, and outcome were assessed in HIV-positive (case) and HIV-negative (control) patients with head and neck squamous cell cancer (HNSCC). HIV-positive cases were matched to controls by tumor site, sex, and age at cancer diagnosis. A tissue microarray (TMA) was constructed and DNA isolated from tumor tissue. MultiPlex-PCR MassArray, L1-PCR, and in situ hybridization were used to assess hrHPV. TMA sections were stained for p16ink4a, TP53, RB, CCND1, EGFR, and scored for intensity and proportion of positive tumor cells. The HNSCC cohort included 41 HIV-positive cases and 41 HIV-negative controls. Tumors from 11 of 40 (28%) cases, and 10 of 41 (24%) controls contained hrHPV. p16 expression, indicative of E7 oncogene activity, was present in 10 of 11 HPV-positive cases and 7 of 10 HPV-positive controls. Low p16 and high TP53 expression in some HPV-positive tumors suggested HPV-independent tumorigenesis. Survival did not differ in cases and controls. RB expression was significantly associated with poor survival (P = 0.01). High TP53 expression exhibited a trend for poorer survival (P = 0.12), but among cases, association with poor survival reached statistical significance (P = 0.04). The proportion of HPV-positive tumors was similar, but the heterogeneity of HPV types was higher in the HIV-positive cases than in HIV-negative controls. High RB expression predicted poor survival, and high TP53 expression was associated with poorer survival in the HIV-positive cases but not HIV-negative controls., Implications: HIV infection did not increase risk of death from HNSCC, and HPV-positive tumors continued to be associated with a significantly improved survival, independent of HIV status. Mol Cancer Res; 15(2); 179-88. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: To better understand the associations between a history of tobacco use and survival outcomes, cigarette use was prospectively surveyed in 687 previously untreated patients with cancer of the oral cavity (n = 271), oropharynx (n = 257), larynx (n = 135), or hypopharynx (n = 24)., Methods: Kaplan-Meier and Cox models explored the associations of tobacco use intensity (packs/day), duration (years of use), and timing before diagnosis with overall survival (OS), disease-specific survival (DSS), and recurrence-free survival (RFS)., Results: Cigarette use duration, timing, and intensity were significant predictors for all outcomes in univariate analysis. Never smoking and pack-years were not significantly associated with outcomes after adjustment for prognostic factors, such as stage, comorbidities, and human papillomavirus (HPV) status, which were strongly associated with clinical outcomes., Conclusion: The findings confirm the association between smoking history and survival and the importance of clinical variables in evaluating smoking as a prognostic factor. Timing, intensity, and duration of cigarette use should be considered with other prognostic factors when considering risk stratification for treatment planning. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1810-1820, 2016., (© 2016 Wiley Periodicals, Inc.)

Objective: Approximately one-third of advanced head and neck squamous cell carcinomas (HNSCCs) recur within 2 years of treatment. Due to ease of collection, saliva is of interest to monitor changes that correlate with treatment. Previously this was a challenge due to xerostomia after conventional radiation. The emergence of gland-sparing radiation has made it possible to collect saliva post-treatment. This study investigated changes in cytokines in saliva pre- and post-treatment to provide foundational knowledge for future studies exploring the use of saliva to monitor treatment response., Study Design: Pre- and post-treatment saliva was evaluated for 8 cytokines by multiplex assay and enzyme-linked immunosorbent assay., Results: In oropharyngeal HNSCC, secretion of epidermal growth factor (EGF), GROα (Growth-regulated protein alpha), interleukin (IL)-1α, IL-β, IL-6, IL-8, tumor necrosis factor-α, and vascular endothelial growth factor increased significantly post-treatment. In additional patients, significant increases of GR-α and IL-6 were validated, but EGF showed no change., Conclusions: The uniqueness of this study is its comparison of salivary cytokines from HNSCC patients pre- and post-treatment., Competing Interests: The authors have no financial disclosures or conflicts of interest., (Copyright © 2016 Elsevier Inc. All rights reserved.)

High-risk HPV (hrHPV) is the leading etiologic factor in oropharyngeal cancer. HPV-positive oropharyngeal tumors generally respond well to therapy, with complete recovery in approximately 80% of patients. However, it remains unclear why some patients are nonresponsive to treatment, with 20% of patients recurring within 5 years. In this study, viral factors were examined for possible clues to differences in tumor behavior. Oropharynx tumors that responded well to therapy were compared with those that persisted and recurred. Viral oncogene alternate transcripts were assessed, and cellular sites of viral integration were mapped and sequenced. Effects of integration on gene expression were assessed by transcript analysis at the integration sites. All of the tumors demonstrated active viral oncogenesis, indicated by expression of HPV E6 and E7 oncogenes and alternate E6 splicing. In the responsive tumors, HPV integration occurred exclusively in intergenic chromosome regions, except for one tumor with viral integration into TP63. Each recurrent tumor exhibited complex HPV integration patterns into cancer-associated genes, including TNFRSF13B, SCN2A, SH2B1, UBE2V2, SMOC1, NFIA, and SEMA6D Disrupted cellular transcripts were identified in the region of integration in four of the seven affected genes., Implications: Integration of transcriptionally active hrHPV into cellular intergenic regions associates with tumor behavior by altering gene expression. Mol Cancer Res; 14(10); 941-52. ©2016 AACR., Competing Interests: The authors disclose no potential conflicts of interest., (©2016 American Association for Cancer Research.)

Background: AT-101 is a BCL-2 Homolog domain 3 mimetic previously demonstrated to have tumoricidal effects in advanced solid organ malignancies. Given the evidence of activity in xenograft models, treatment with AT-101 in combination with docetaxel is a therapeutic doublet of interest in metastatic head and neck squamous cell carcinoma., Patients and Methods: Patients included in this trial had unresectable, recurrent, or distantly metastatic head and neck squamous cell carcinoma (R/M HNSCC) not amenable to curative radiation or surgery. This was an open label randomized, phase II trial in which patients were administered AT-101 in addition to docetaxel. The three treatment arms were docetaxel, docetaxel plus pulse dose AT-101, and docetaxel plus metronomic dose AT-101. The primary endpoint of this trial was overall response rate., Results: Thirty-five patients were registered and 32 were evaluable for treatment response. Doublet therapy with AT-101 and docetaxel was well tolerated with only 2 patients discontinuing therapy due to treatment related toxicities. The overall response rate was 11 % (4 partial responses) with a clinical benefit rate of 74 %. Median progression free survival was 4.3 months (range: 0.7-13.7) and overall survival was 5.5 months (range: 0.4-24). No significant differences were noted between dosing strategies., Conclusion: Although met with a favorable toxicity profile, the addition of AT-101 to docetaxel in R/M HNSCC does not appear to demonstrate evidence of efficacy., Competing Interests: The authors declare that they have no conflict of interest.

Background: Because immune responses within the tumor microenvironment are important predictors of tumor biology, correlations of types of tumor infiltrating lymphocytes (TILs) with clinical outcomes were determined in 278 patients with head and neck squamous cell carcinoma (HNSCC)., Methods: Infiltrating levels of CD4 (helper T cells), CD8 (cytotoxic/suppressor T cells), FoxP3 (regulatory T cells), CD68 (myeloid-derived suppressor cells,) and CD1a (Langerhans) cells were measured in tissue microarrays (TMAs). Cox models tested associations with patient outcomes after adjusting for all known prognostic factors. Median follow-up was 36.6 months., Results: Higher CD4 and CD8 TIL levels were associated with improved overall survival (OS; hazard ratio [HR] = 0.77; 95% confidence interval [CI]  = 0.65-0.93; p = .005 and HR = 0.77; 95% CI = 0.64-0.94; p = .008, respectively), and relapse-free survival (RFS; p = .03 and .05, respectively). After controlling for prognostic factors, higher CD4 levels predicted improved OS and disease-specific survival (DSS; p = .003 and p = .004, respectively)., Conclusion: The findings suggest that TILs are a significant independent prognostic factor for HNSCC that differ by treatment. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1074-1084, 2016., (© 2016 Wiley Periodicals, Inc.)

Objective: To determine biomarkers of recurrence and survival in patients with spindle cell variant squamous cell carcinoma (SpSCC) of the head and neck., Study Design: Retrospective case control study., Setting: Tertiary academic center., Subjects and Methods: Thirty-two SpSCC patients (mean age, 68.8) between 1987 and 2009 were identified and reviewed. A tissue microarray (TMA) was constructed from tumor specimens. Tumor biomarkers under study included p16, epidermal growth factor receptor (EGFR), p53, EZH2, cyclin D1, CD104, HGFa, p21, and cMET. An additional TMA was constructed from patients with non-SpSCC oral cavity squamous cell carcinoma for comparative purposes. The main outcomes were overall survival (OS), disease-specific survival (DSS), and recurrence-free survival (RFS)., Results: In the SpSCC cohort, tumors positive for cMet had worse OS (P < .001). Patients positive for cMet (P = .007), cyclin D1 (P = .019), and p16 (P = .004) had worse DSS. Recurrence-free survival was also worse in patients with tumors positive for cMet (P = .037), cyclin D1 (P = .012), and p16 (P < .001). Compared with the oral cavity cohort, there was a significantly larger proportion of patients in the SpSCC group with tumors staining positive for cMet and a lower proportion of tumors positive for cyclin D1., Conclusion: cMet, cyclin D1, and p16 are predictive tumor biomarkers for risk of recurrence and worse DSS in patients with SpSCC., (© American Academy of Otolaryngology—Head and Neck Surgery Foundation 2016.)

Background: Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with poor overall survival. New therapeutic strategies that target specific molecular lesions driving advanced disease are needed. Herein we demonstrate the utility of the chicken chorioallantoic membrane (CAM) assay for in vivo human HNSCC tumor growth and metastasis and the tumor suppressive effects of a new chemotherapeutic agent., Methods: We tested anti-metastatic effects of a WNT pathway inhibitor, WNT974 (also known as LGK974), which targets porcupine (PORCN) the palmityl-transferase that is essential for secretion of Wnt proteins. CAM assays were performed with 8 HNSCC cell lines: UM-SCC-1, UM-SCC-10A, UM-SCC-10B, UM-SCC-11A, UM-SCC-14A UM-SCC-17A, UM-SCC-17B, UM-SCC-25, and UM-SCC-34., Results: UM-SCC-1 (University of Michigan Squamous Cell Carcinoma cell line) CAM xenografts contain CD44+ and ALDH+ cancer stem cell (CSC) proportions similar to UM-SCC-1 mouse xenografts supporting the applicability of the CAM assay for study of CSCs. Inhibition of WNT signaling by the PORCN inhibitor WNT974 reduced metastatic spread of UM-SCC cells, especially in UM-SCCs with Notch1 deficiency., Conclusions: Our data demonstrate decreased tumor growth and metastases in tumors from cell lines that showed in vitro responses to WNT974, providing evidence that this agent may have a role in future HNSCC therapy.

Importance: Understanding the drainage patterns to the retropharyngeal lymph nodes is an important consideration in oropharyngeal squamous cell carcinoma (OPSCC) because treatment of these nodes is related to increased morbidity. Prediction of these drainage patterns could not only help minimize treatment morbidity but also prevent failures in at-risk patients as deintensification trials are under way for this disease., Objective: To evaluate the prevalence of pathologic retropharyngeal adenopathy (RPA) in OPSCC relative to involvement of the oropharyngeal subsite, number of metastatic neck nodes, T classification, and N classification., Design, Setting, and Participants: We performed a retrospective review from January 1, 2003, through December 31, 2010, at an academic referral center of 205 previously untreated patients with pathologically confirmed, advanced-stage (III, IV) OPSCC. Data analysis was performed from January 1, 2013, through June 30, 2015., Exposure: Concurrent chemoradiotherapy., Main Outcomes and Measures: Radiologic evidence of pathologic RPA was tabulated and related to involvement of the oropharyngeal subsite, number of metastatic neck nodes, T classification, and N classification., Results: Of the 205 previously untreated patients (183 men; mean age, 56.1 years), pathologic RPA was identified in 37 (18.0%) of the 205 patients. Pathologic retropharyngeal lymph nodes were found in 12 (13.5%) of 89 patients with base of tongue cancers, 24 (22.0%) of 109 patients with tonsil cancers, and 1 (14.3%) of 7 patients with other oropharyngeal subsite cancers. Increasing prevalence of RPA was positively correlated with closer proximity to the posterior tonsillar pillar. A multivariable logistic regression model using the oropharyngeal subsite, involvement of the posterior tonsillar pillar, number of metastatic neck nodes, T classification, and N classification revealed that the number of metastatic neck nodes was statistically significant (odds ratio, 1.44; 95% CI, 1.20-1.71; P < .001)., Conclusions and Relevance: The prevalence of pathologic RPA in this cohort was 18.0%, and patients with multiple nodes had the highest risk of pathologic RPA, followed by involvement of the posterior tonsillar pillar. However, these data suggest that there is no clear algorithm that can be used for deintensification to exclude the retropharyngeal site from the treatment volume using extent of disease gathered from pretreatment imaging for patients with advanced-stage OPSCC.

Background: We recently described the imaging characteristics of multiple confluent regional metastases (matted nodes) and found that this characteristic was associated with distant metastasis in patients with oropharyngeal squamous cell carcinoma (SCC). The purpose of this study was to determine if matted nodes are a predictive marker for distant metastasis., Methods: Radiologic lymph node characteristics on 205 patients with untreated stage III/IV with oropharyngeal SCC of whom 192 had known human papillomavirus (HPV) status underwent weekly carboplatin and paclitaxel with concomitant intensity-modulated radiation therapy (IMRT) between 2003 and 2010 with a minimum of 2-year of follow-up., Results: The 3-year disease-specific survival (DSS) for patients with matted nodes was 58% versus 97% with nonmatted nodes (p = .0001). The prevalence of matted nodes in the population was 20%. The positive predictive value of matted nodes for distant metastasis was 66%, and the negative predictive value was 99%., Conclusion: Matted nodes are a predictive marker for distant disease and can be used for planning new clinical interventions., (© 2014 Wiley Periodicals, Inc.)

Background: HPV-associated HNSCCs have a distinct etiologic mechanism and better prognosis than those with non-HPV associated HNSCCs. However, even within the each group, there is heterogeneity in survival time. Here, we test the hypothesis that specific candidate gene methylation markers (CCNA1, NDN, CD1A, DCC, p16, GADD45A) are associated with tumor recurrence and survival, in a well-characterized, prospective, cohort of 346 HNSCC patients., Methods: Kaplan-Meier curves were used to estimate survival time distributions. Multivariable Cox Proportional Hazards models were used to test associations between each methylation marker and OST/RPFT after adjusting for known or identified prognostic factors. Stratified Cox models included an interaction term between HPV and methylation marker to test for differences in the associations of the biomarker with OST or RPFT across HPV status., Results: Methylation markers were differentially associated with patient characteristics. DNA hypermethylation of NDN and CD1A was found to be significantly associated with overall survival time (OST) in all HNSCC patients (NDN hazard ratio (HR): 2.35, 95% CI: 1.40-3.94; CD1A HR: 1.31, 95% CI: 1.01-1.71). Stratification by HPV status revealed hypermethylation of CD1A was associated with better OST and recurrence/persistence-free time (RPFT) (OST HR: 3.34, 95% CI: 1.88-5.93; RPFT HR: 2.06, 95% CI: 1.21-3.49), while hypomethylation of CCNA1 was associated with increased RPFT in HPV (+) patients only (HR: 0.31, 95% CI: 0.13-0.74)., Conclusions: This study is the first to describe novel epigenetic alterations associated with survival in an unselected, prospectively collected, consecutive cohort of patients with HNSCC. DNA hypermethylation of NDN and CD1A was found to be significantly associated with increased overall survival time in all HNSCC patients. However, stratification by the important prognostic factor of HPV status revealed the immune marker, CD1A, and the cell cycle regulator, CCNA1 to be associated with prognosis in HPV (+) patients, specifically. Here, we identified novel methylation markers and specific, epigenetic molecular differences associated with HPV status, which warrant further investigation.

Background: Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the 2 most common cutaneous carcinomas. Molecular profiles predicting metastasis of these cancers have not been identified., Methods: Epigenetic profiles of 37 primary cases of cutaneous SCC and BCC were quantified via the Illumina Goldengate Cancer Panel. Differential protein expression by metastatic potential was analyzed in 110 total cases by immunohistochemical (IHC) staining., Results: Unsupervised hierarchical clustering analysis revealed that metastatic BCCs had a methylation profile resembling cutaneous SCCs. Metastatic cutaneous SCCs were found to be hypermethylated at FRZB (median methylation: 46.7% vs 4.7%; p = 4 × 10(-5) ). Metastatic BCCs were found to be hypomethylated at MYCL2 (median methylation: 3.8% vs 83.4%; p = 1.9 × 10(-6) ). Immunohistochemical staining revealed few differences between metastatic and nonmetastatic cancers., Conclusion: Metastatic primary BCCs and cutaneous SCCs had distinct epigenetic profiles when compared to their nonmetastatic counterparts. Epigenetic profiling may prove useful in future diagnosis and prevention of advanced nonmelanoma skin cancers., (© 2014 Wiley Periodicals, Inc.)

Background: Cancer stem cells (CSCs) represent a subpopulation of cells responsible for tumor growth. Their role in head and neck squamous cell carcinoma (HNSCC) tumorigenesis and metastasis remains uncertain., Methods: Wound healing and an orthotopic animal model were used to study cells expressing the CSC phenotype (CD44(high) and aldehyde dehydrogenase [ALDH](+)) and assess mobility, tumorigenesis, and metastasis. A prospective collection of 40 patient-derived primary HNSCC specimens were analyzed for CSC-proportion compared to clinical variables., Results: CSCs exhibited significantly faster wound closure and greater tumorigenesis and regional metastasis in vivo than non-CSCs. In primary patient tumors, size and advanced stage were correlated with elevated proportion of CSCs, however, not with survival., Conclusion: HNSCC stem cells mediate tumorigenesis and regional metastasis in vivo. In primary patient tumors, CSC-proportion was associated with tumor size and stage, but not with metastatic spread or survival. CSC burden alone may only represent a minor variable in understanding CSCs and metastasis., (© 2014 Wiley Periodicals, Inc.)

Objectives: Tumor infiltrating lymphocytes (TILs) in the microenvironment reflect may tumor biology and predict outcome. We previously demonstrated that infiltrates of CD4, CD8, and FoxP3 positive lymphocytes were associated with HPV-status and survival in oropharyngeal cancers. To determine if TILs were of prognostic importance in oral cancer, TIL levels were evaluated retrospectively in 52 oral cancer patients treated with surgery and correlations with outcome determined., Methods: Complete TIL and clinical data were available for 39 patients. Levels of CD4, CD8, FoxP3 (Treg), CD68 and NK cells were assessed by immunohistochemistry in tumor cores on a tissue microarray. Associations with clinical variables, tobacco and alcohol use and histologic features were assessed using Spearman correlation coefficient and the non-parametric Kruskal-Wallis testing. Time-to-event outcomes were determined using univariate and multivariate Cox models. Median follow up was 60 months., Results: The ratio of CD4/CD8 (p=.01) and CD8 infiltrates (p=.05) were associated with tumor recurrence but not overall survival. Lower CD4 infiltrates were associated with alcohol use (p=.005) and poor tumor differentiation (p=.02). Interestingly, higher levels of CD68+ macrophages were found associated with positive nodes (p=.06) and poorer overall survival (p=.07). Overall and DSS survival were significantly shorter for patients with positive nodes, extracapsular spread, or perineural invasion., Conclusion: Infiltrating immune cell levels in oral cavity cancer appear influenced by health behaviors and tumor characteristics. In contrast to oropharynx cancer, infiltrates of CD68 positive tumor associated macrophages may contribute to metastatic behavior and outcome in advanced oral cavity carcinoma., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

It has been postulated that gastroesophageal reflux plays a role in the etiology of head and neck squamous cell carcinomas (HNSCC) and contributes to complications after surgery or during radiotherapy. Antacid medications are commonly used in patients with HNSCC for the management of acid reflux; however, their relationship with outcomes has not been well studied. Associations between histamine receptor-2 antagonists (H2RA) and proton pump inhibitors (PPI) use and treatment outcomes were determined in 596 patients with previously untreated HNSCC enrolled in our SPORE epidemiology program from 2003 to 2008 (median follow-up 55 months). Comprehensive clinical information was entered prospectively in our database. Risk strata were created on the basis of possible confounding prognostic variables (age, demographics, socioeconomics, tumor stage, primary site, smoking status, HPV16 status, and treatment modality); correlations within risk strata were analyzed in a multivariable model. Patients taking antacid medications had significantly better overall survival (OS; PPI alone: P < 0.001; H2RA alone, P = 0.0479; both PPI + H2RA, P = 0.0133). Using multivariable Cox models and adjusting for significant prognostic covariates, both PPIs and H2RAs used were significant prognostic factors for OS, but only H2RAs use for recurrence-free survival in HPV16-positive oropharyngeal patients. We found significant associations between the use of H2RAs and PPIs, alone or in combination, and various clinical characteristics. The findings in this large cohort study indicate that routine use of antacid medications may have significant therapeutic benefit in patients with HNSCC. The reasons for this association remain an active area of investigation and could lead to identification of new treatment and prevention approaches with agents that have minimal toxicities., (©2014 American Association for Cancer Research.)

Head and neck squamous cell carcinomas (HNSCC) contain a small subpopulation of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self-renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)-6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellular source of Interleukin-6 (IL-6), we observed a direct correlation between IL-6 levels in tumor-associated endothelial cells and the tumorigenicity of CSC. In vitro, endothelial cell-IL-6 enhanced orosphere formation, p-STAT3 activation, survival, and self-renewal of human CSC. Notably, a humanized anti-IL-6R antibody (tocilizumab) inhibited primary human CSC-mediated tumor initiation. Collectively, these data demonstrate that endothelial cell-secreted IL-6 defines the tumorigenic potential of CSC, and suggest that HNSCC patients might benefit from therapeutic inhibition of IL-6/IL-6R signaling., (© 2014 AlphaMed Press.)

Background: Proinflammatory cytokine levels may be associated with cancer stage, recurrence, and survival. The objective of this study was to determine whether cytokine levels were associated with dietary patterns and fat-soluble micronutrients in patients with previously untreated head and neck squamous cell carcinoma (HNSCC)., Methods: This was a cross-sectional study of 160 patients with newly diagnosed HNSCC who completed pretreatment food frequency questionnaires (FFQs) and health surveys. Dietary patterns were derived from FFQs using principal component analysis. Pretreatment serum levels of the proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) were measured using an enzyme-linked immunosorbent assay, and serum carotenoid and tocopherol levels were measured by high-performance liquid chromatography. Multivariable ordinal logistic regression models examined associations between cytokines and quartiles of reported and serum dietary variables., Results: Three dietary patterns emerged: whole foods, Western, and convenience foods. In multivariable analyses, higher whole foods pattern scores were significantly associated with lower levels of IL-6, TNF-α, and IFN-γ (P ≤ .001, P = .008, and P = .03, respectively). Significant inverse associations were reported between IL-6, TNF-α, and IFN-γ levels and quartiles of total reported carotenoid intake (P = .006, P = .04, and P = .04, respectively). There was an inverse association between IFN-γ levels and serum α-tocopherol levels (P = .03)., Conclusions: Consuming a pretreatment diet rich in vegetables, fruit, fish, poultry, and whole grains may be associated with lower proinflammatory cytokine levels in patients with HNSCC., (© 2014 American Cancer Society.)

Background: The current American Joint Committee on Cancer (AJCC) staging system may not accurately reflect survival in patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (SCC). The purpose of this study was to develop a system that more precisely predicts survival., Methods: CT scans from 156 patients who underwent chemoradiation for advanced-stage oropharyngeal SCC with >2 years follow-up were reviewed. We modeled patterns of nodal metastasis associated with different survival rates. We defined HPV+ N1 as a single node <6 cm, ipsilaterally, contralaterally, or bilaterally. HPV+ N2 was defined as a single node ≥6 cm or ≥2 nodes ipsilaterally/contralaterally or ≥3 nodes bilaterally. HPV+ N3 was defined as matted nodes., Results: There was no significant difference in disease-specific survival (DSS; p = .14) or overall survival (OS; p = .16) by AJCC classification. In patients grouped by HPV+ N1, HPV+ N2, and HPV+ N3 nodal classification, significant differences in DSS (100%, 92%, and 55%, respectively; p = .0001) and OS (100%, 96%, and 55%, respectively; p = .0001) were found., Conclusion: A staging system with reclassification of size, bilaterality, and matted nodes more accurately reflects survival differences in this cohort of patients. Review of the AJCC staging system with these criteria should be considered for HPV-positive oropharyngeal SCC., (Copyright © 2014 Wiley Periodicals, Inc.)

Squamous cell carcinoma of the head and neck (SCCHN) is a common, aggressive, treatment-resistant cancer with a high recurrence rate and mortality, but the mechanism of treatment resistance remains unclear. Here we describe a mechanism where the AAA-ATPase TRIP13 promotes treatment resistance. Overexpression of TRIP13 in non-malignant cells results in malignant transformation. High expression of TRIP13 in SCCHN leads to aggressive, treatment-resistant tumors and enhanced repair of DNA damage. Using mass spectrometry, we identify DNA-PKcs complex proteins that mediate nonhomologous end joining (NHEJ), as TRIP13-binding partners. Using repair-deficient reporter systems, we show that TRIP13 promotes NHEJ, even when homologous recombination is intact. Importantly, overexpression of TRIP13 sensitizes SCCHN to an inhibitor of DNA-PKcs. Thus, this study defines a new mechanism of treatment resistance in SCCHN and underscores the importance of targeting NHEJ to overcome treatment failure in SCCHN and potentially in other cancers that overexpress TRIP13.

Background: Optimal treatment for locally advanced squamous cell carcinoma of the oropharynx (SCCOP) is not well defined. Here we retrospectively compare survival and toxicities from 2 different organ preservation protocols., Methods: The matched dataset consisted of 35 patients from each trial matched for age, stage, smoking, and tumor human papillomavirus (HPV) status. Patients in the University of Michigan Cancer Center (UMCC) trial 9921 were treated with induction chemotherapy (IC) followed by high-dose cisplatin and radiation in responders or surgery in nonresponders. Patients in the UMCC trial 0221 were treated with weekly carboplatin and paclitaxel and radiation., Results: Survival was comparable for both studies and did not differ significantly across each trial after stratifying by HPV status. Grade 3 and 4 toxicities were more frequent in UMCC 9921. At 6 months posttreatment, gastrostomy tube (G-tube) dependence was not statistically different., Conclusion: These data suggest that survival outcomes in patients with locally advanced SCCOP are not compromised with weekly chemotherapy and radiation therapy, and such treatment is generally more tolerable., (Copyright © 2013 Wiley Periodicals, Inc.)

Importance: The University of Michigan has investigated the use of induction selection (IS) with chemoradiotherapy (CRT) for patients who respond to CRT and found this approach effective in the management of advanced laryngeal cancer. The IS approach was extended to oral cavity squamous cell carcinoma (OCSCC) to help understand whether organ preservation or survival benefit resulted., Objective: To evaluate the efficacy of an IS protocol vs primary surgical extirpation and selective postoperative radiotherapy for advanced OCSCC., Design and Setting: Retrospective matched cohort study at a tertiary care hospital., Participants: Nineteen patients with resectable stages III and IV OCSCC were enrolled into a phase 2 IS trial. Patients with a response of at least 50% underwent concurrent CRT; those with a response of less than 50% underwent surgical treatment and radiotherapy. A comparison cohort of patients treated with primary surgical extirpation during a similar time period was frequency matched for inclusion criteria and patient characteristics to those patients included from the phase 2 IS trial. No difference was noted in age, sex, pretreatment American Joint Committee on Cancer stage, T and N classifications, smoking status, alcohol consumption, or tumor subsite between the IS and surgical cohorts. Median follow-up was 9.4 years in the IS cohort and 7.1 years in the surgical cohort., Interventions: Induction selection and CRT vs primary surgical extirpation with or without postoperative radiotherapy., Main Outcomes and Measures: Overall and disease-specific survival and locoregional control., Results: The Kaplan-Meier estimate for overall survival at 5 years was 32% in the IS cohort and 65% in the surgical cohort. The Kaplan-Meier estimate for disease-specific survival at 5 years was 46% in the IS cohort and 75% in the surgical cohort. The Kaplan-Meier estimate for locoregional control at 5 years was 26% in the IS cohort and 72% in the surgical cohort. Multivariable analysis demonstrated significantly better overall and disease-specific survival and locoregional control outcomes (P = .03, P = .001, and P < .001, respectively) in the surgical cohort., Conclusions and Relevance: Primary surgical treatment showed significantly better survival and locoregional control compared with IS in this matched patient cohort. Despite success of organ preservation IS protocols in the larynx, comparative survival analysis of an IS protocol vs primary surgical extirpation for OCSCC demonstrates significantly better outcomes in the surgical cohort. These findings support surgery as the principal treatment for OCSCC.

Objective: Patients with oral cavity squamous cell carcinoma (OCSCC) undergo adjuvant radiation for pathologically high-risk features including positive nodal disease and extracapsular spread (ECS). In the absence of these high-risk features, our objective was to determine if perineural invasion (PNI) is an independent risk factor and if adjuvant radiation (XRT) improves disease control rates., Study Design: Historical cohort analysis., Setting: Tertiary university hospital., Methods: Eighty-eight OCSCC patients (46 males, 42 females; mean age = 56.7 years; median follow-up = 4.6 years) treated surgically with pathologically N0 (pN0) necks were studied. Overall, 23% (20/88) were pN0/PNI+ and of those with PNI, 70% (14/20) underwent XRT. Survival analysis using Kaplan-Meier followed by multivariable Cox models was performed., Results: Multivariate analysis verified PNI to be associated with worse disease-free interval (DFI) (P = .012) and local-regional control (LRC) (P = .005) and perivascular invasion (PVI) associated with worse DFI (P = .05). Among pN0/PNI+ patients, those who received XRT demonstrated significantly improved DFI (mean = 6.5 years vs 1.7 years; P = .014) and LRC (mean 6.7 years vs 1.9 years; P = .047). There was no improvement in overall survival (P = .68) or disease-specific survival (P = .8) in those receiving XRT., Conclusions: PNI is an independent adverse risk factor in the absence of nodal metastasis and extracapsular spread. We observed a statistically significantly longer DFI and LRC when patients were treated with adjuvant radiation.

Head and neck squamous cell carcinoma (HNSCC) is the eighth most commonly diagnosed cancer in the United States. The risk of developing HNSCC increases with exposure to tobacco, alcohol and infection with human papilloma virus (HPV). HPV-associated HNSCCs have a distinct risk profile and improved prognosis compared to cancers associated with tobacco and alcohol exposure. Epigenetic changes are an important mechanism in carcinogenic progression, but how these changes differ between viral- and chemical-induced cancers remains unknown. CpG methylation at 1505 CpG sites across 807 genes in 68 well-annotated HNSCC tumor samples from the University of Michigan Head and Neck SPORE patient population were quantified using the Illumina Goldengate Methylation Cancer Panel. Unsupervised hierarchical clustering based on methylation identified 6 distinct tumor clusters, which significantly differed by age, HPV status, and three year survival. Weighted linear modeling was used to identify differentially methylated genes based on epidemiological characteristics. Consistent with previous in vitro findings by our group, methylation of sites in the CCNA1 promoter was found to be higher in HPV(+) tumors, which was validated in an additional sample set of 128 tumors. After adjusting for cancer site, stage, age, gender, alcohol consumption, and smoking status, HPV status was found to be a significant predictor for DNA methylation at an additional 11 genes, including CASP8 and SYBL1. These findings provide insight into the epigenetic regulation of viral vs. chemical carcinogenesis and could provide novel targets for development of individualized therapeutic and prevention regimens based on environmental exposures.