K. Casper

Kv1.3 Channels Mark Functionally Competent CD8+ Tumor-Infiltrating Lymphocytes in Head and Neck Cancer

Tumor-infiltrating lymphocytes (TIL) are potent mediators of an antitumor response. However, their function is attenuated in solid tumors. CD8+ T-cell effector functions, such as cytokine and granzyme production, depend on cytoplasmic Ca2+, which is controlled by ion channels. In particular, Kv1.3 channels regulate the membrane potential and Ca2+ influx in human effector memory T (TEM) cells. In this study, we assessed the contribution of reduced Kv1.3 and Ca2+ flux on TIL effector function in head and neck cancer (HNC). We obtained tumor samples and matched peripheral blood from 14 patients with HNC. CD3+ TILs were composed of 57% CD4+ (82% TEM and 20% Tregs) and 36% CD8+ cells. Electrophysiology revealed a 70% reduction in functional Kv1.3 channels in TILs as compared with peripheral blood T cells from paired patients, which was accompanied by a decrease in Ca2+ influx. Immunofluorescence analysis showed that CD8+ TILs expressing high Kv1.3 preferentially localized in the stroma. Importantly, high expression of Kv1.3 correlated with high Ki-67 and granzyme B expression. Overall, these data indicate that defective Kv1.3 channels and Ca2+ fluxes in TILs may contribute to reduced immune surveillance in HNC. Cancer Res; 77(1); 53-61. ©2016 AACR.

Predictors of severe long-term toxicity after re-irradiation for head and neck cancer

OBJECTIVE To identify predictive factors of severe long-term toxicity after re-irradiation of recurrent/persistent or second-primary head and neck cancer. METHODS Outcomes and treatment plans of patients who underwent modern IMRT based re-irradiation to the head and neck from 2008-2015 were reviewed. Co-variables including demographic, clinical and oncologic factors, as well as interval to re-irradiation and re-irradiated planning tumor volume (PTV) were analyzed as predictors of developing severe (CTCAE grade⩾3) long-term toxicity with death as a competing risk. RESULTS A total of 66 patients who met inclusion criteria were eligible for analysis. A median re-irradiation dose of 70Gy was delivered at a median of 37.5months after initial radiotherapy. Re-irradiation followed surgical resection in 25 (38%) patients, and concurrent chemotherapy was delivered to 41 (62%) patients. Median follow-up after re-irradiation was 23months and median overall survival was 22months (predicted 2year overall survival 49%). Of the 60 patients who survived longer than 3months after re-irradiation, 16 (25%) patients experienced severe long-term toxicity, with the majority (12 of 16) being feeding tube -dependent dysphagia. In multivariable analysis, shorter intervals to re-irradiation (<20months) and larger re-irradiated PTVs (>100cm(3)) were independent predictors of developing severe long-term toxicity. Patients with longer disease-free intervals and smaller PTVs had a 94% probability of being free of severe toxicity at two years. CONCLUSION Selection of patients with longer re-irradiation intervals and requiring smaller re-irradiated PTVs can independently predict avoidance of severe long-term toxicity.

Pilot testing of a novel surgical simulator for endoscopic zenker's diverticulotomy

Restrictions on resident work hours and the increasing purview of otolaryngology reduce the efficacy of the traditional surgical training model. With limited case volumes at many institutions and the unique instrumentation of endoscopic Zenkers diverticulotomy (EZD), simulation may be useful to improve training. In this study, a novel surgical simulator for EZD is developed and validated.

Decreased plasma DEK Oncogene Levels Correlate with p16-Negative Disease and Advanced Tumor Stage in a Case–Control Study of Patients with Head and Neck Squamous Cell Carcinoma

Head and neck cancer (HNC) remains the sixth most common malignancy worldwide and survival upon recurrence and/or metastasis remains poor. HNSCC has traditionally been associated with alcohol and nicotine use, but more recently the Human Papilloma Virus (HPV) has emerged as a favorable prognostic risk factor for oropharyngeal HNSCC. However, further stratification with additional biomarkers to predict patient outcome continues to be essential. One candidate biomarker is the DEK oncogenic protein, which was previously detected in the urine of patients with bladder cancer and is known to be secreted by immune cells such as macrophages. Here, we investigated if DEK could be detected in human plasma and if DEK levels correlated with clinical and pathological variables of HNSCC. Plasma was separated from the peripheral blood of newly diagnosed, untreated HNSCC patients or age-matched normal healthy controls and analyzed for DEK protein using ELISA. Plasma concentrations of DEK protein were lower in p16-negative tumors compared to both normal controls and patients with p16-positive tumors. Patients with lower plasma concentrations of DEK were also more likely to have late stage tumors and a lower white blood cell count. Contrary to previously published work demonstrating a poor prognosis with high intratumoral DEK levels, we show for the first time that decreased concentrations of DEK in patient plasma correlates with poor prognostic factors, including HPV-negative status as determined by negative p16 expression and advanced tumor stage.

Can Predictive Modeling Identify Head and Neck Oncology Patients at Risk for Readmission

Objective Unplanned readmission within 30 days is a contributor to health care costs in the United States. The use of predictive modeling during hospitalization to identify patients at risk for readmission offers a novel approach to quality improvement and cost reduction. Study Design Two-phase study including retrospective analysis of prospectively collected data followed by prospective longitudinal study. Setting Tertiary academic medical center. Subjects and Methods Prospectively collected data for patients undergoing surgical treatment for head and neck cancer from January 2013 to January 2015 were used to build predictive models for readmission within 30 days of discharge using logistic regression, classification and regression tree (CART) analysis, and random forests. One model (logistic regression) was then placed prospectively into the discharge workflow from March 2016 to May 2016 to determine the model’s ability to predict which patients would be readmitted within 30 days. Results In total, 174 admissions had descriptive data. Thirty-two were excluded due to incomplete data. Logistic regression, CART, and random forest predictive models were constructed using the remaining 142 admissions. When applied to 106 consecutive prospective head and neck oncology patients at the time of discharge, the logistic regression model predicted readmissions with a specificity of 94%, a sensitivity of 47%, a negative predictive value of 90%, and a positive predictive value of 62% (odds ratio, 14.9; 95% confidence interval, 4.02-55.45). Conclusion Prospectively collected head and neck cancer databases can be used to develop predictive models that can accurately predict which patients will be readmitted. This offers valuable support for quality improvement initiatives and readmission-related cost reduction in head and neck cancer care.

The Spider Limb Positioner in subscapular system free flaps

BACKGROUND The subscapular system is a versatile vascular network that can provide multiple flaps for reconstruction of the head and neck. A significant drawback of using the subscapular system is that patient positioning can preclude the use of simultaneous two-team ablative and reconstructive surgery. Herein, we describe a novel use of an upper extremity limb positioner (Spider Limb Positioner) used primarily in orthopedic surgery to facilitate concurrent two-team technique in head and neck surgery. METHODS Using a bean bag and the Spider Limb Positioner for the upper extremity, a semidecubitus position was used for subscapular donor site dissection. Ablative and reconstructive teams worked concurrently in all cases. RESULTS This technique was utilized 78 times on 73 patients, with chimeric flaps used in 38% of cases. The average operative time was 466 min. Only one patient required repositioning intraoperatively due to a change in the subscapular donor site side. There were no nerve compression injuries or positioning related complications. CONCLUSION Scapular and parascapular free flaps are useful tools for reconstruction of the head and neck. In a two-team approach, the use of a semidecubitus position in conjunction with the Spider Limb Positioner facilitates exposure for the reconstructive team without compromising access for the ablative team.

Differential expression and prognostic value of long non-coding RNA in HPV-negative head and neck squamous cell carcinoma

BACKGROUND Long non-coding RNA (lncRNA) has emerged as a new avenue of interest due to its various biological functions in cancer. Abnormal expression of lncRNA has been reported in other malignancies but has been understudied in head and neck squamous cell carcinoma (HNSCC). METHODS The lncRNA expression was interrogated via quantitative real-time polymerase chain reaction (qRT-PCR) array for 19 human papillomavirus (HPV)-negative HNSCC tumor-normal pairs. The Cancer Genome Atlas (TCGA) was used to validate these results. The association between differentially expressed lncRNA and survival outcomes was analyzed. RESULTS Differential expression was validated for 5 lncRNA (SPRY4-IT1, HEIH, LUCAT1, LINC00152, and HAND2-AS1). There was also an inverse association between MEG3 expression (not significantly differentially expressed in TCGA tumors but highly variable expression) and 3-year recurrence-free survival (RFS). CONCLUSION We identified and validated differential expression of 5 lncRNA in HPV-negative HNSCC. Low MEG3 expression was associated with favorable 3-year RFS, although the significance of this finding remains unclear.

Individualized survival prediction for patients with oropharyngeal cancer in the human papillomavirus era: Assessment of OPSCC Survival Calculators

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.

Abstract 26: Exosomal microRNA as salivary biomarkers of head and neck squamous cell carcinoma

Head and neck cancer is the 10th most common cancer overall and 5th most common among men in the United States, the vast majority of which are squamous cell carcinoma (HNSCC). Despite approximately two-thirds of cases being diagnosed at an advanced stage, the only routine screening approach currently in widespread use is visual inspection and palpation, which is provided by dentists and clinicians on an opportunistic basis, lacks sensitivity, and varies according to the skill of the clinician. This underscores the urgent and compelling need for establishment of novel and effective biomarkers to facilitate early detection. Exosomes may offer a new avenue for discovery and development of novel biomarkers of HNSCC. These nanosized (40-150 nm), membrane-encapsulated vesicles are released by both normal and malignant cells into the extracellular space and function as intercellular signaling vectors through the horizontal transfer of biologic molecules, including microRNA (miRNA), which can affect the phenotype of recipient cells. Importantly, exosomes are present in essentially all extracellular biofluids, including saliva, and thus can readily be collected by noninvasive means. Through previous cell culture work, we observed striking differences in exosomal miRNA secreted by HNSCC cells relative to healthy oral epithelial cells, with a high degree of overlap in exosomal miRNA profiles across HNSCC cell lines. Although salivary exosomes have been theorized to be a potentially powerful biomarker source for HNSCC, there has been an extreme paucity of work conducted in this arena. To begin to address this question, we conducted a small pilot study in which we collected 2-mL whole-saliva samples from 5 HNSCC cases (collected prior to initiation of treatment) and 5 cancer-free controls. Exosomes were isolated from each saliva sample via differential ultracentrifugation, and total RNA was extracted for miRNA-sequencing (miRNA-seq), which was performed by the University of Cincinnati Genomics, Epigenomics & Sequencing Core (GESC). The number of reads for each sample during miRNA-seq ranged from 1.6 million to 27.4 million reads (median = 12.4 million). After aligning the reads to all human miRNA sequences catalogued in miRBase, there were a total of 1,334 mature miRNA transcripts detected across samples, with 307 transcripts (12%) detected solely in salivary exosomes from cases. Moreover, despite the small sample size of the pilot study, we observed significant differential exosomal secretion of miR-10b-5p (p = 0.006), with transcripts present at relatively high levels in salivary exosomes from 3 of the 5 cases but none detected in those from the 5 controls. We also preliminarily assessed the translational potential of 8 miRNA transcripts that were solely and universally expressed by our HNSCC cell lines (i.e. not secreted by healthy oral epithelial cells) in our previous cell culture work using the salivary exosome miRNA-seq data for the 5 cases and 5 controls. All 8 transcripts were detected in salivary exosomes obtained from the 5 HNSCC cases. In particular, miR-486-5p and miR-486-3p showed considerable promise, with 40% of cases expressing drastically higher levels of these transcripts relative to controls. When either miR-486-5p or miR-486-3p was combined with miR-10b-5p, the substantial separation of these markers could clearly distinguish 80% (4/5) of the HNSCC cases from controls. These findings demonstrate the potential of salivary miRNA as biomarkers of HNSCC and highlight the necessity of further workup and validation in full-scale studies involving human saliva. Citation Format: Scott M. Langevin, Damaris Kuhnell, Xiang Zhang, Trisha M. Wise-Draper, Keith A. Casper. Exosomal microRNA as salivary biomarkers of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 26.

Abstract 50: Plasma concentrations of the DEK oncogene correlate with pathological variables in a case-control study of patients with HNSCC

Background: Head and neck squamous cell carcinoma (HNSCC) has traditionally been associated with alcohol and nicotine use, but more recently the Human Papilloma Virus (HPV) has emerged as a favorable prognostic risk factor for oropharyngeal HNSCC. However, further stratification with additional biomarkers to predict patient outcome continues to be essential. One candidate biomarker is the chromatin remodeling DEK protein, which is both an auto-antigen in autoimmune diseases and an oncogene in epithelial tissues. DEK is secreted by stimulated macrophages and neutrophils and was previously detected in the urine of patients with bladder cancer. Previously, we have reported that DEK mRNA and protein is upregulated in HNC tumor tissue and higher DEK levels are associated with poor prognoses in many types of solid tumors. We hypothesized that DEK could be detected in the plasma of HNC patients, either due to secretion from the tumor or as part of the antitumor immune response, and therefore may be a biomarker for disease status. Methods: We recruited 38 newly diagnosed HNSCC patients and 37 age-matched normal healthy controls into the study. Plasma isolated from peripheral blood was subjected to DEK specific ELISA and DEK concentration levels were compared to levels found in normal controls, and to clinical and pathological variables. Results: We show for the first time that DEK can be detected in human plasma. We did not find an association between DEK plasma concentrations and variables including sex, age, race, or drinking/smoking status. However, we detected decreased concentrations of DEK in HNC patients with p16-negative disease and in patients with larger tumor sizes, indicating an association between DEK levels and known prognostic markers. In addition, HNC patients with lower DEK concentrations had a decreased white blood cell count, largely due to differences in lymphocyte and eosinophil counts. This direct association between plasma DEK levels and white blood cell count was independent of p16 status. Conclusions: Together, the data suggest that lower levels of DEK in HNC patient plasma may be predictive of poor outcome. This is in direct contrast to what is observed with intratumoral levels of DEK protein, in which higher levels of DEK expression are an independent factor predicting poor prognosis. Future studies will investigate the role that secreted DEK, such as that found in the plasma, may have in the antitumor immune response. Citation Format: Trisha Wise-Draper, Arun Sendilnathan, Sarah Palackdharry, Nicholas Pease, Julianne Qualtieri, Randy Butler, Nooshin Hashemi Sadraei, John C. Morris, Yash Patil, Keith Wilson, Jonathan Mark, Keith Casper, Vinita Takiar, Adam Lane, Lisa M. Privette Vinnedge. Plasma concentrations of the DEK oncogene correlate with pathological variables in a case-control study of patients with HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 50.