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Abstract: Diabetic retinopathy is a posterior eye disorder in which damage occurs to the light sensitive retina due to diabetes mellitus. This disorder specifically affects people aged between 18-64 with type ІІ diabetes. This disease progresses through different pathophysiological pathways, which include oxidative stress, inflammation, stimulation of the growth factor in the eye’s vasculature, isoforms of protein kinase C, and also the activation of the hexosamine pathway. It starts as micro aneurysms and advances in complicated stage, which results in retinal detachment. Treatment of posterior eye diseases has complications due to the structural design of the eye and physiological barriers present. The current treatment approach involves the use of intravitreal anti- VEGFs, corticosteroids implants, laser and surgery; these treatment methods have drawbacks attributed to them despite their benefits. The development of a robust delivery system with minimal or no invasion to tackle the issues of diabetic retinopathy will be of considerable benefit to patients having diabetic retinopathy; the dependency on ophthalmologists for multiple injections will significantly reduce and provide a promising approach in drug delivery. In this review article, the authors provided information related to existing treatment methods available for diabetic retinopathy, the most significant among which is nanotechnology approach through which local delivery via the ocular route to posterior eye can be achieved. It also possesses the various carriers studied for the non-invasive approach for retinal delivery of medicaments. Non-invasive approach for delivery of drugs can be considered as potential for the treatment of diabetic retinopathy.

Neurodegenerative disorders occur when nerve cells in the brain or peripheral nervous system partial or complete fail in their functions and sometimes even die due to some injuries or aging. Neurodegenerative disorders such as Alzheimer's Disease (AD) and Parkinson's Disease (PD), have been majorly resulted due to degeneration of neurons and neuroinflammation progressively. There are many similarities that correlates both AD and PD on a cellular and sub-cellular level. Therefore, a hope for therapeutic advancement for simultaneous upgradation in both the diseases are directly depending on the discovery of common mechanism at molecular and cellular level. Recent and past evidences from scientific literature supporting the efficacy of plants flavonoids in treatment and protection of both AD and PD. Further, dietary flavones, specially Heptamethoxyflavone, Kaempferitrin, Vitexin and Amentoflavone gains recently much more attention for producing many health beneficiary effects including neuroprotection. Despite of these evidence a detailed updated overview of neuroprotective effects against both AD and PD by Heptamethoxyflavone, Kaempferitrin, Vitexin and Amentoflavone are still missing. In this context several published studies were assessed by using various online electronic search engines/databases to meet the objective from 1981 to 2021 (Approx. 224). Therefore, present review was designed to deliver the detailed description on these flavones including therapeutic benefits in AD, PD and other CNS complications with critical analysis on underlying mechanisms.

Targeting inhibitor of apoptosis proteins (IAP) with second mitochondria-derived activator of caspase (SMAC) mimetics may promote cancer cell death. We tested whether cIAP1 predicts poor prognosis in head and neck squamous cell carcinoma (HNSCC) and whether a novel Smac-mimetic, LCL161, could radiosensitize human papillomavirus–positive (HPV+) and -negative (HPV−) HNSCC. The association of BIRC2 (encoding cIAP1) mRNA level with HPV status in HNSCC was analyzed using The Cancer Genome Atlas (TCGA) database. cIAP1 was assessed by IHC on an HNSCC tissue microarray (TMA, n = 84) followed by correlation analysis with HPV status and patient outcomes. Human cell culture and animal models of HNSCC were used to analyze the outcome and molecular characteristics following radiotherapy in combination with LCL161. cIAP1 expression is increased in HPV− compared with HPV+HNSCC tumors in the TCGA database. In our TMA, cIAP1 was overexpressed in HNSCC compared with normal tissues (P = 0.0003) and associated with a poor overall survival (P = 0.0402). cIAP1 levels were higher in HPV− than that in HPV+HNSCC tumors (P = 0.004) and patients with cIAP1+/HPV− HNSCC had the worst survival. LCL161 effectively radiosensitized HPV− HNSCC cells, which was accompanied with enhanced apoptosis, but not HPV+ HNSCC cells. Importantly, LCL161 in combination with radiotherapy led to dramatic tumor regression of HPV− HNSCC tumor xenografts, accompanied by cIAP1 degradation and apoptosis activation. These results reveal that cIAP1 is a prognostic and a potential therapeutic biomarker for HNSCC, and targeting cIAP1 with LCL161 preferentially radiosensitizes HPV− HNSCC, providing justification for clinical testing of LCL161 in combination with radiation for patients with HPV− HNSCC.

Objective To identify predictors of overall survival (OS) and to stratify patients according to significant prognostic variables. Methods A retrospective study of 274 consecutive patients with primary Oral Cavity Squamous Cell Carcinoma. Kaplan-Meier, Cox proportional hazard models, and recursive partitioning analysis (RPA) were used for analysis of OS. These results were further validated using National Cancer Database cohort of 21 895 patients. Results Median OS was 3.65 years. T-classification and N-classification, alcoholic beverages/week, age, and adjuvant treatment were significant predictors of OS. RPA identified high-risk subpopulations: N0-1 patients with CCI ≥ 4.5 and N2-3 patients ordered by those not receiving adjuvant treatment, those with T3-4 disease despite adjuvant therapy, and those having T1-2 disease with adjuvant therapy. Conclusions This study utilized significant prognostic indicators and RPA to highlight the importance of age, N-classification, T-classification, comorbidity, and adjuvant therapy in conjunction with American Joint Committee on Cancer staging to improve preoperative counseling.

OBJECTIVES To elucidate predictive factors in the perioperative period resulting in gastrostomy tube (G-tube) dependence for patients undergoing primary surgical treatment of oropharyngeal squamous cell carcinoma (OPSCC) in the modern era. METHODS Two hundred and thirty patients with known OPSCC treated with primary surgery were screened and selected from a retrospective database spanning from 2002 to 2012 at The Ohio State University Wexner Medical Center (Columbus, Ohio), with univariable and multivariable logistic regression modeling used to determine independent predictive factors resulting in G-tube dependence (defined as tube persistence/presence 1 year after surgery). RESULTS Surgical approach, baseline characteristics, tumor (T)-nodal-metastasis stage, human papillomavirus status, extent of tissue resected, surgical complications, reconstructive technique, preoperative G-tube presence, and adjuvant treatment were recorded. Patients undergoing open surgery for OPSCC without adjuvant treatment had 42.9% G-tube dependence (44.6% with adjuvant chemoradiation [CRT]) compared to 0% for those undergoing transoral nonrobotic surgery (8.1% with adjuvant CRT) and 0% for those undergoing transoral robotic surgery (10.3% with adjuvant CRT). In multivariable analysis, greater than 25% of the oral tongue resected (odds ratio [OR] 12.29; P = 0.03), an open surgical approach (OR 5.72; P

Background Neurodegenerative disorders are among the most common challenging diseases that affect the population with extreme medical and financial burdens. Widely seen neurodegeneration affects population of all ages, as it progresses with age, affecting a large proportion of elderly population including patients, caregivers, and immensely increasing the financial load of the country. These diseases have a very complex nature that frequently results from combined genetic, environmental and pathological factors. Various challenges are faced by the researchers working on the pathogenesis and the possible treatment of neurodegenerative disorder. Objective The review has analysed for recent patent documents and treatment approaches for neurodegenerative disorders. This review does not relate to potential targets such as ( i.e. protein where modulation could be predicted to impact on pathophysiology), rather it mainly focuses on various available patented approaches for neurodegenerative disorders. Method The study design is based on updating the international and national literatures and an exhaustive patent search, compiling various patented documents for the treatment of neurodegenerative disorders (EP2282779A1, US20110229555A1) to provide information in the state of technological innovation in terms of research and development. Results In the present review, the authors described various neurodegenerative diseases, there treatment strategies and emphasized on various patented approaches for age-related neurodegenerative disorders such as novel therapeutic methods for treating Alzheimer's and associated disorders via modulated cell stress response EP2282779A1, through combined therapies that modulate angiogenesis US20120058992A1. Conclusion The review will attract the interest of academics, researchers, students and pharmaceutical companies with regard to the recent on-going activities in neurodegenerative disorders.

OBJECTIVES: to examine the impact of radiotherapy center volume on overall survival in patients with oral cavity and oropharyngeal squamous cell carcinoma getting adjuvant radiation therapy after receiving surgery at a high-volume center. MATERIALS AND METHODS: a retrospective study was conducted on patients with oral cavity squamous cell carcinoma or oropharyngeal squamous cell carcinoma treated surgically at a tertiary institution from 2000 to 2012 who received adjuvant radiotherapy. The outcome variable was overall survival and the independent variable was location of adjuvant radiation therapy: high-volume center (HVC) versus low-volume center (LVC). Cox proportional hazards models were used to assess associations between predictors of death. Variables that were found to be significant at the α = 0.10 were included in a multivariable model. RESULTS: 336 patients met inclusion criteria. One-hundred thirty-nine patients received adjuvant radiation therapy at HVC and 197 patients received adjuvant radiation therapy at LVC. A univariate Cox proportional hazards model identified the variables location, age, marital status, subsite, T stage, extracapsular extension, and smoking status to include in a multivariable model. Age, subsite, T stage, and extracapsular extension were independent predictors of overall survival (p < .05). Location (p=.55), marital status (p=.29), and smoking status (p=.22) were not statistically significant predictors of survival. CONCLUSION: After surgery at a HVC, the volume of adjuvant radiation therapy center was not significantly associated with overall survival. Significant predictors of survival included age, subsite, T stage, and extracapsular extension.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive and often fatal disease. Cisplatin is the most common chemotherapeutic drug in the treatment of HNSCC, but intrinsic and acquired resistance are frequent, and severe side effects occur at high doses. The second messenger cyclic GMP (cGMP) is produced by soluble guanylate cyclase (sGC). We previously reported that activation of the cGMP signaling cascade caused apoptosis in HNSCC cells, while others found that this pathway enhances cisplatin efficacy in some cell types. Here we found that sGC stimulators reduced HNSCC cell viability synergistically with cisplatin, and enhanced apoptosis by cisplatin. Moreover, the sGC stimulators effectively reduced viability in cells with acquired cisplatin resistance, and were synergistic with cisplatin. The sGC stimulator BAY 41-2272 reduced expression of the survival proteins EGFR and β-catenin, and increased pro-apoptotic Bax, suggesting a potential mechanism for the anti-tumorigenic effects of these drugs. The sGC stimulator Riociguat is FDA-approved to treat pulmonary hypertension, and others are being studied for therapeutic use in several diseases. These drugs could provide valuable addition or alternative to cisplatin in the treatment of HNSCC.

// Eric A. Smith 1, * , Bhavna Kumar 2, 3, * , Kakajan Komurov 1 , Stephen M. Smith 4 , Nicole V. Brown 5 , Songzhu Zhao 5 , Pawan Kumar 2, 3 , Theodoros N. Teknos 2, 3 , Susanne I. Wells 1 1 Cancer and Blood Diseases Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 45229, USA 2 Department of Otolaryngology–Head and Neck Surgery, The Ohio State University, Columbus, OH, 43210, USA 3 The Ohio State University, Comprehensive Cancer Center, Columbus, OH 43210, USA 4 Department of Pathology, The Ohio State University, Columbus, OH, 43210, USA 5 Center for Biostatistics, The Ohio State University, Columbus, OH, 43210, USA * Co-first author Correspondence to: Susanne I. Wells, email: Susanne.Wells@cchmc.org Theodoros N. Teknos, email: ted.teknos@osumc.edu Keywords: DEK, oropharyngeal squamous cell carcinoma, human papillomavirus, IL6, p16 Received: July 18, 2016 Accepted: February 12, 2017 Published: February 21, 2017 ABSTRACT Oropharyngeal squamous cell carcinomas (OPSCC) are common, have poor outcomes, and comprise two biologically and clinically distinct diseases. While OPSCC that arise from human papillomavirus infections (HPV+) have better overall survival than their HPV- counterparts, the incidence of HPV+ OPSCC is increasing dramatically, affecting younger individuals which are often left with life-long co-morbidities from aggressive treatment. To identify patients which do poorly versus those who might benefit from milder regimens, risk-stratifying biomarkers are now needed within this population. One potential marker is the DEK oncoprotein, whose transcriptional upregulation in most malignancies is associated with chemotherapy resistance, advanced tumor stage, and worse outcomes. Herein, a retrospective case study was performed on DEK protein expression in therapy-naive surgical resections from 194 OPSCC patients. We found that DEK was associated with advanced tumor stage, increased hazard of death, and interleukin IL6 expression in HPV16+ disease. Surprisingly, DEK levels in HPV16- OPSCC were not associated with advanced tumor stage or increased hazard of death. Overall, these findings mark HPV16- OPSCC as an exceptional malignancy were DEK expression does not correlate with outcome, and support the potential prognostic utility of DEK to identify aggressive HPV16+ disease.

// Bhavna Kumar 1, 2 , Arti Yadav 2 , Nicole V. Brown 3 , Songzhu Zhao 3 , Michael J. Cipolla 1 , Paul E. Wakely 4 , Alessandra C. Schmitt 4, 6 , Robert A. Baiocchi 5 , Theodoros N. Teknos 1, 2 , Matthew Old 1, 2, * , Pawan Kumar 1, 2, * 1 Department of Otolaryngology-Head and Neck Surgery, The Ohio State University, Columbus, OH 43210 USA 2 The Ohio State University Comprehensive Cancer Center, Columbus, OH 43210 USA 3 Center for Biostatistics, The Ohio State University, Columbus, OH 43210 USA 4 Department of Pathology, The Ohio State University, Columbus, OH 43210 USA 5 Department of Internal Medicine, The Ohio State University, Columbus, OH 43210 USA 6 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30303 USA * These authors contributed equally to this work Correspondence to: Pawan Kumar, email: Pawan.Kumar@osumc.edu Keywords: PRMT5, OPSCC, HPV, cyclin D1, IL-6 Received: July 22, 2016 Accepted: December 27, 2016 Published: January 17, 2017 ABSTRACT Protein arginine methyltransferase-5 (PRMT5) plays an important role in cancer progression by repressing the expression of key tumor suppressor genes via the methylation of transcriptional factors and chromatin-associated proteins. However, very little is known about the expression and biological role of PRMT5 in head and neck cancer. In this study, we examined expression profile of PRMT5 at subcellular levels in oropharyngeal squamous cell carcinoma (OPSCC) and assessed its correlation with disease progression and patient outcome. Our results show that nuclear PRMT5 was associated with poor overall survival ( p 10 pack-years ( p = 0.013). In addition, nuclear PRMT5 was directly correlated with cyclin D1 ( p = 0.0101) and IL-6 expression ( p < 0.001). In a subgroup survival analysis, nuclear PRMT5-positive/IL-6-positive group had worst survival, whereas nuclear PRMT5-negative/IL-6-negative group had the best survival. Similarly, patients with p16-negative/nuclear PRMT5-positive tumors had worse survival compared to patients with p16-positive/nuclear PRMT5-negative tumors. Our mechanistic results suggest that IL-6 promotes nuclear translocation of PRMT5. Taken together, our results demonstrate for the first time that nuclear PRMT5 expression is associated with poor clinical outcome in OPSCC patients and IL-6 plays a role in the nuclear translocation of PRMT5.

Recent studies have shown that IL-6 signaling plays an important role in the aggressive and metastatic phenotype of head and neck squamous cell carcinoma (HNSCC). Therefore, we hypothesized that targeting of IL-6 signaling in HNSCC could enhance the therapeutic efficacy of standard chemoradiation treatment. We used both in vitro and in vivo models to test the efficacy of Bazedoxifene (BZA), a drug that was originally developed as a newer-generation selective estrogen receptor modulator (SERM) for the treatment of postmenopausal osteoporosis. Recently, BZA was also shown to exhibit potent anti-cancer effects that were both estrogen receptor (ER)-dependent and ER-independent. Our results suggest that BZA inhibits IL-6 signaling by disrupting IL-6R/gp130 protein-protein interactions. BZA treatment of CAL27-IL-6 (IL-6 overexpressing cells) or UM-SCC-74A (naturally expressing high levels of IL-6) significantly inhibited cell proliferation, migration and colony formation ability in a dose-dependent manner. In addition, BZA significantly decreased IL-6-mediated tumorsphere formation by markedly reducing nanog expression. BZA treatment also markedly reduced chemo and radioresistance in head and neck cancer cells by downregulating ERCC-1, XRCC-1 and survivin expression. In a SCID mouse xenograft model, BZA significantly enhanced the anti-tumor effects of cisplatin and radiation treatment with no added systemic toxicity. Furthermore, combination treatments significantly decreased tumor metastasis, pSTAT3 expression and nanog expression, in vivo. Taken together, our results suggest that targeting IL-6 signaling with bazedoxifene could be an effective treatment strategy for the treatment of HNSCC patients.

// Bhavna Kumar 1, 2 , Nicole V. Brown 3 , Benjamin J. Swanson 4 , Alessandra C. Schmitt 4, 5 , Matthew Old 1, 2 , Enver Ozer 1, 2 , Amit Agrawal 1, 2 , David E. Schuller 1, 2 , Theodoros N. Teknos 1, 2 , Pawan Kumar 1, 2 1 Department of Otolaryngology-Head and Neck Surgery, The Ohio State University, Columbus, OH 43210, USA 2 Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA 3 Center for Biostatistics, The Ohio State University, Columbus, OH 43210, USA 4 Department of Pathology, The Ohio State University, Columbus, OH 43210, USA 5 Current affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30303, USA Correspondence to: Pawan Kumar, e-mail: Pawan.Kumar@osumc.edu Keywords: myoferlin, OPSCC, HPV, IL-6, nanog Received: December 22, 2015 Accepted: February 11, 2016 Published: February 23, 2016 ABSTRACT Myoferlin (MYOF) is a member of ferlin family of membrane proteins that was originally discovered as a muscle specific protein. Recent studies have shown that myoferlin is also expressed in other cell types including endothelial cells and cancer cells. However, very little is known about the expression and biological role of myoferlin in head and neck cancer. In this study, we examined expression profile of myoferlin in oropharyngeal squamous cell carcinoma (OPSCC) and assessed its correlation with disease progression and patient outcome. In univariate analyses, nuclear MYOF was associated with poor overall survival (p

Background The purpose of this study was to further define the impact of primary surgery in the management of oropharyngeal squamous cell carcinoma (SCC). Methods Two hundred ninety-six patients with oropharyngeal SCC treated with primary surgery were included. Multivariable analysis and recursive partitioning analysis (RPA) identified predictors of survival and gastrostomy tube presence. Results Multivariable analysis identified that HPV negativity (p = .0002), presence of extranodal extension (p = .0025), and advanced T classification (p = .0081) were independent predictors of survival. For HPV-positive patients, surgical approach (p = .0111) and margin status (p = .0287) were significant predictors of survival. For HPV-negative patients, extranodal extension (p = .0021) and advanced T classification (p = .0342) were significant predictors of survival. Smoking status and advanced neck disease did not impact survival, and the addition of adjuvant chemotherapy did not confer survival benefit in HPV-positive or HPV-negative subgroups. Conclusion Independent predictors of survival are unique in patients with oropharyngeal SCC treated with primary surgery. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1794–E1802, 2016

Acquisition of chemoresistance and metastatic phenotype are the major causes of treatment failure and mortality in head and neck squamous cell carcinoma (HNSCC) patients. Histone deacetylases (HDACs) have been shown to be overexpressed in many tumor types and directly linked to poor prognosis. In this study, we demonstrate that HDACs are markedly elevated in HNSCC. HDACs expression was further increase in cisplatin resistant cell lines (CisR). In addition, cisplatin-resistant cells showed enhanced stem cell properties and tumor metastasis. Depletion of HDAC1 and 2 in CisR cell lines significantly reversed cisplatin resistance and tumorsphere formation. Next, we tested the efficacy of Suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, by using both in vitro and in vivo models. SAHA significantly inhibited cell proliferation and synergistically enhanced the anti-proliferative effects of cisplatin. In addition, SAHA significantly decreased tumorsphere formation by markedly reducing nanog expression. In a SCID mouse xenograft model, SAHA significantly enhanced the anti-tumor effects of cisplatin treatment with no added systemic toxicity. Furthermore, SAHA and cisplatin combination treatment significantly decreased tumor metastasis and nanog expression, in vivo. Taken together, our results suggest that targeting HDACs with SAHA could be an effective treatment strategy for the treatment of HNSCC patients.

Objectives The Notch1 signaling pathway has recently been shown to be highly dysregulated in head and neck squamous cell carcinoma, but the value of Notch1 as a predictive biomarker is yet to be elucidated in oropharyngeal squamous cell carcinoma (OPSCC). The objective of this study is to evaluate Notch1 expression in surgical OPSCC specimens and determine clinicopathologic correlates. Study Design Case series with planned data collection. Setting Tertiary academic medical center. Subjects and Methods Surgical specimens from 181 patients with OPSCC were collected to create a tissue microarray (TMA). Human papillomavirus (HPV) status and Notch1 expression were determined and correlated with clinicopathologic characteristics. Results In univariate analysis, Notch1 expression correlated with improved survival as a categorical variable (hazard ratio [HR], 0.346; P < .0001) and correlated with HPV/p16 positivity as a continuous variable ( P < .0001). In multivariate analysis, Notch1 expression retained significance in HPV-positive (HR, 0.303; P = .033) and HPV-negative (HR, 0.416; P = .0055) subgroups. Similarly, Notch1 expression positively correlated with survival in p16-positive (HR, 0.469; P = .031) and p16-negative subgroups (HR, 0.310; P = .014). Conclusions In the largest OPSCC cohort to date, we found that Notch1 receptor expression positively correlates with overall survival, regardless of HPV or p16 status. Furthermore, we found higher Notch1 receptor expression in HPV/p16-positive tumors than their HPV/p16-negative counterparts.

BACKGROUND Human papillomavirus 16 (HPV16) is a major risk factor for the development of head and neck squamous cell carcinoma (HNSCC), particularly the development of oropharyngeal squamous cell carcinoma (OPSCC). Cancer stem cells (CSCs) are resistant to conventional therapies, and it is postulated that they are responsible for disease recurrence and/or progression. Because the prognoses of patients with HPV16-positive and HPV-negative HNSCC are distinct, the authors sought to determine whether differences in the number of CSCs could account for this clinical observation. METHODS CSC populations in HPV16-positive and HPV-negative HNSCC were assessed using a proprietary assay based on expression of the enzyme aldehyde dehydrogenase (ALDH), an in vitro tumorsphere formation assay, and an in vivo limiting cell dilution in nonobese diabetic/severe combined immunodeficiency mice. A high-density tissue microarray was stained with ALDH1, a CSC marker, to determine the association between CSCs and HPV16-positive/HPV-negative OPSCC. RESULTS HPV16-positive HNSCC had a greater intrinsic CSC pool than HPV-negative HNSCC. Inactivation of p53 has been identified as a major mechanism for the elevated CSC population in HPV16-positive HNSCC. In vivo limiting cell dilution experiments using tumors from patients with HPV16-positive and HPV-negative OPSCC indicated that the CSC frequency was 62.5-fold greater in an HPV16-positive OPSCC tumor than in an HPV-negative OPSCC tumor. Primary tumors from patients with HPV16-positive OPSCC were associated with elevated tumor ALDH1 staining, further extending the association between HPV16 and CSCs. CONCLUSIONS The current data and the clinical observation that patients with HPV16-positive HNSCC respond more favorably to current treatment paradigms than patients with HPV-negative HNSCC support the suggestion that CSC phenotype is not homogeneous. Therefore, the reliance on the CSC number may be insufficient to accurately assess the potential of a particular tumor for disease recurrence and/or progression. Cancer 2014;120:992–1001. © 2014 American Cancer Society.

To determine if tumor biomarkers were predictive of outcome in a prospective cohort of patients with advanced larynx cancer treated in a phase II clinical trial.Prospectively collected biopsy specimens from 58 patients entered into a Phase II trial of organ preservation in advanced laryngeal cancer were evaluated for expression of a large panel of biomarkers, and correlations with outcome were determined.Tissue microarrays were constructed from pretreatment biopsies and stained for cyclin D1, CD24, EGFR, MDM2, PCNA, p53, survivin, Bcl-xL, Bcl-2, BAK, rhoC, and NFκB. Pattern of invasion and p53 mutations were assessed. Correlations with overall survival (OS), disease-specific survival (DSS), time free from indication of surgery, induction chemotherapy response, and chemoradiation response were determined. Cox models were used to assess combinations of these biomarkers.Low expression of BAK was associated with response to induction chemotherapy. Low expression of BAK and cytoplasmic NFκB was associated with chemoradiation response. Aggressive histologic growth pattern was associated with response induction chemotherapy. Expression of cyclin D1 was predictive of overall and disease-specific survival. Overexpression of EGFR was also associated with an increased risk of death from disease. Bcl-xL expression increased significantly in persistent/recurrent tumors specimens when compared to pretreatment specimens derived from the same patient (P = 0.0003).Evaluation of biomarker expression in pretreatment biopsy specimens can lend important predictive and prognostic information for patients with advanced larynx cancer.

Background Optimal treatment for locally advanced SCCOP is not well defined. Here we retrospectively compare survival and toxicities from two different organ preservation protocols.

Background The purpose of this study was to describe the relationship of p16 and epidermal growth factor receptor (EGFR) expression with survival in surgically treated patients who had oropharyngeal or oral cavity squamous cell carcinoma (SCC). Methods Tissue from 36 patients with oropharyngeal SCC and 49 patients with oral cavity SCC treated between 1997 and 2001 was imbedded and immunostained using a tissue microarray. Results The p16 was positive in 57% and 13% of patients with oropharyngeal SCC and oral cavity SCC, respectively. EGFR was positive in 60% and 63% of patients with oropharyngeal SCC and oral cavity SCC, respectively. In patients with oropharyngeal SCC, p16 expression was associated with improved disease-specific survival (DSS), overall survival (OS), and time to recurrence (TTR) (p < .01, < .01, and

Objectives/Hypothesis: Human papillomavirus-16 (HPV-16)–associated carcinoma of the oropharynx has a favorable prognosis. Such patients have elevated CD8+ T-lymphocyte levels that correlate with response to chemotherapy and survival. Tumor-infiltrating lymphocyte (TIL) subpopulations were assessed in pretreatment biopsies from a prospective patient cohort to determine if TIL subsets differed by HPV status, clinical factors, or patient outcome or correlated with peripheral blood T-cell levels. Study Design: Retrospective immunological correlative study of patients entered in a prospective Phase 2 clinical trial. Methods: Measured were CD8, CD4, CD68, and Treg (FoxP3) lymphocytes by immunohistochemistry in a tissue microarray created from patients (n = 46) with advanced oropharyngeal cancer. Correlations with peripheral blood levels, HPV status, expression of epidermal growth factor receptor (EGFR), clinical tumor, and patient characteristics and outcome were determined. Median follow-up was 6.6 years. Results: HPV-16–positive patients had improved survival (P = .016). Degree of T-cell infiltration did not differ by HPV status but was significantly related to disease-specific survival (DSS) and overall survival (OS). Even after adjusting for HPV status, we found that CD8, FoxP3, and total T cells were significantly associated with DSS (P = .0236, P = .0040, and P = .0197, respectively) and OS (P = .0137, P = .0158, and P = .0115, respectively). Less T-cell infiltration (P = .0130) and CD4 cells in particular (P = .0792) were associated with higher EGFR expression. Conclusions: Improved outcomes are associated with increased TILs independent of HPV status and suggest the local immune response may be more related to factors such as tumor size, EGFR expression, or performance status than HPV status. Further study of larger numbers of patients and infiltrates combined with functional analysis of individual subsets may be necessary to detect significant differences in local immunity in HPV-16–related cancers.Laryngoscope, 122:121–127, 2012

Purpose: The goal of this study was to examine the effect of tobacco use on disease recurrence (local/regional recurrence, distant metastasis, or second primary) among patients with human papillomavirus (HPV)–positive squamous cell carcinoma of the oropharynx (SCCOP) following a complete response to chemoradiation therapy. Experimental Design: Between 1999 and 2007, 124 patients with advanced SCCOP (86% with stage IV) and adequate tumor tissue for HPV analysis who were enrolled in one of two consecutive University of Michigan treatment protocols were prospectively included in this study. Patients were categorized as never-, former, or current tobacco users. The primary end points were risk of disease recurrence and time to recurrence; secondary end points were disease-specific survival and overall survival. Results: One hundred and two patients (82.3%) had HPV-positive tumors. Over two thirds (68%) of patients with HPV-positive tumors were tobacco users. Among HPV-positive patients, current tobacco users were at significantly higher risk of disease recurrence than never-tobacco users (hazard ratio, 5.2; confidence interval, 1.1-24.4; P = 0.038). Thirty-five percent of HPV-positive ever tobacco users recurred compared with only 6% of HPV-positive never users and 50% of HPV-negative patients. All HPV-negative patients were tobacco users and had significantly shorter times to recurrence (P = 0.002), and had reduced disease-specific survival (P = 0.004) and overall survival (P < 0.001) compared with HPV-positive patients. Compared with HPV-positive never-tobacco users, those with a tobacco history showed a trend for reduced disease-specific survival (P = 0.064) but not overall survival (P = 0.221). Conclusions: Current tobacco users with advanced, HPV-positive SCCOP are at higher risk of disease recurrence compared with never-tobacco users. Clin Cancer Res; 16(4); 1226–35

Purpose To test induction chemotherapy (IC) followed by concurrent chemoradiotherapy (CRT) or surgery/radiotherapy (RT) for advanced oropharyngeal cancer and to assess the effect of human papilloma virus (HPV) on response and outcome. Patients and Methods Sixty-six patients (51 male; 15 female) with stage III to IV squamous cell carcinoma of the oropharynx (SCCOP) were treated with one cycle of cisplatin (100 mg/m2) or carboplatin (AUC 6) and with fluorouracil (1,000 mg/m2/d for 5 days) to select candidates for CRT. Those achieving a greater than 50% response at the primary tumor received CRT (70 Gy; 35 fractions with concurrent cisplatin 100 mg/m2 or carboplatin (AUC 6) every 21 days for three cycles). Adjuvant paclitaxel was given to patients who were complete histologic responders. Patients with a response of 50% or less underwent definitive surgery and postoperative radiation. Pretreatment biopsies from 42 patients were tested for high-risk HPV. Results Fifty-four of 66 patients (81%) had a greater than 50% response after IC. Of these, 53 (98%) received CRT, and 49 (92%) obtained complete histologic response with a 73.4% (47 of 64) rate of organ preservation. The 4-year overall survival (OS) was 70.4%, and the disease-specific survival (DSS) was 75.8% (median follow-up, 64.1 months). HPV16, found in 27 of 42 (64.3%) biopsies, was associated with younger age (median, 55 v 63 years; P = .016), sex (22 of 30 males [73.3%] and five of 12 females [41.7%]; P = .08), and nonsmoking status (P = .037). HPV titer was significantly associated with IC response (P = .001), CRT response (P = .005), OS (P = .007), and DSS (P = .008). Conclusion Although the numbers in this study are small, IC followed by CRT is an effective treatment for SCCOP, especially in patients with HPV-positive tumors; however, for patients who do not respond to treatment, alternative treatments must be developed.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer worldwide, and the 5-year survival rates are among the worst of the major cancers. Oncolytic herpes simplex viruses (oHSV) have the potential to make a significant impact in the targeted treatment of these patients. Here, we tested antitumor efficacy of RAMBO, an oHSV armed with the antiangiogenic Vstat120, alone and in conjunction with ATN-224, a copper chelator against HNSCC in vitro and in vivo animal models. We found that all tested HNSCC cells responded well to virus treatment and were sensitive to RAMBO-mediated oncolytic destruction. In vivo, RAMBO had a significant antiangiogenic and antitumorigenic effect. Physiologic levels of copper inhibited viral replication and HNSCC cell killing. Chelation of copper using ATN-224 treatment significantly improved serum stability of RAMBO and permitted systemic delivery in HNSCC tumor xenografts models. Furthermore, our results show that the combination of ATN-224 and RAMBO strongly inhibits lung metastases in a mouse model of HNSCC. These findings suggest that combining ATN-224 with RAMBO has potential for clinical trials in both early and advanced HNSCC patients.

Background and purpose: Evasion of apoptosis contributes to radioresistance of head and neck squamous cell carcinoma (HNSCC), calling for novel strategies to overcome apoptotic resistance. Second mitochondria-derived activator of caspase (SMAC) - mimetics are a class of targeted drugs that antagonize selected inhibitor of apoptosis protein (IAP) to block pro-survial signalling, and eventually induce cell apoptosis. The present study was designed to investigate the radiosensitizing effect of one SMAC mimetic, LCL161, in HNSCC and the underlying mechanisms for radiosensitization. Material and methods: We analyzed the correlation between apoptotic molecules and HPV status in HNSCC via mRNA expression using The Cancer Genome Atlas (TCGA) database, as well as protein expression in 6 HNSCC cell lines by immunoblotting. We also examined cIAP1 expression on a tissue microarray (TMA) of HNSCC tumors, and assessed its correlation with HPV status and patient outcome. Clonogenic survival and WST-1 assays were carried out to explore the potential of LCL161 as a radiosensitizer in HNSCC cell lines. Cell cycle analysis, Annexin-V assays and immunoblotting were performed to investigate effects of LCL161 on radiation induced cell apoptosis. Human tumor xenografts were generated to explore the radiosensitization effect of LCL161 on HPV[-] tumors in vivo. Results: TCGA database analysis and immunoblotting from HNSCC cells revealed that expression of cIAP1, Survivin, DIABLO, p65, IK-b, TNF-α, BCL-XL, Caspase-7, and Caspase-8 were elevated in HPV[-] compared to HPV[+] HNSCC tumors. IHC staining showed cIAP1 expression was associated with HPV[-] status (p=0.0239). In addition, cIAP1 expression was significantly higher in HNSCC compared with adjacent normal tissue (p=0.0003). In univariate analysis, cIAP1 was significantly associated with poor overall survival (p=0.0359). LCL161 degraded cIAP1 at nanomolar concentrations but has minimal single-agent cytotoxicity with IC50 range of 32 μM – 95 μM. Interestingly, we found that LCL161 could induce radiosensitization only in HPV[-], but not in HPV[+] HNSCC cells. Mechanistic studies showed LCL161 mediated radiosensitisation was associated with increased cell apoptosis, with enhanced activation of caspases-3, -7, -8, -9, and PARP. Finally, in vivo data showed that LCL161 degraded cIAP1, activated caspases, and profoundly radiosensitized two human HPV[-] HNSCC xenograft models. Conclusion: Taken together, our results demonstrate for the first time that cIAP1 is a potential prognostic and therapeutic biomarker for HNSCC patients. Through targeting cIAP1 and other IAP family members, LCL161, radiosensitizes HNSCC tumors, particularly HPV [-] tumors, via caspase activation and apoptosis induction. Taken together, LCL161 holds promise for future clinical development as a novel radiosensitizer in the treatment of HPV[-] HNSCC tumors. Citation Format: Linlin Yang, Bhavna Kumar, Tianyun Li, Thenodoros N. Teknos, Arnab Chakravarti, Terence M. Williams. Targeting inhibitors of apoptosis in combination with radiation for the treatment of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5185. doi:10.1158/1538-7445.AM2017-5185

Chemotherapy constitutes the standard modality of treatment for localized head and neck squamous cell carcinomas (HNSCC). However, many patients fail to respond and relapse after this treatments due to the acquisition of chemo-resistance. Therefore, there is an urgent need to develop novel drugs that could reverse the resistant phenotype. Curcumin, the constituent of the spice turmeric has been shown to have anti-inflammatory, anti-oxidant and anti-proliferative properties in several tumor types. However, use of curcumin has been limited due to its poor bio-absorption. Recently, a novel class of curcumin analogs, based on diarylidenylpiperidones (DAP), has been developed by incorporating a piperidone link to the beta-diketone structure and fluoro substitutions on the phenyl groups. In this study, we evaluated the effectiveness of H-4073, a parafluorinated variant of DAP, using both in vitro and in vivo head and neck cancer models. Our results demonstrate that H-4073 is a potent anti-tumor agent and it significantly inhibited cell proliferation in all the HNSCC cell lines tested in a dose-dependent manner. In addition, pretreatment of cisplatin-resistant HNSCC cell lines with H-4073 significantly reversed the chemo-resistance as observed by cell viability assay (MTT), apoptosis assay (Annexin V binding) and cleaved caspase-3 (Western blot). H-4073 mediated its anti-tumor effects by inhibiting JAK/STAT3, FAK, Akt and VEGF signaling pathways that play important roles in cell proliferation, migration, survival and angiogenesis. In the SCID mouse xenograft model, H-4073 significantly enhanced the anti-tumor and anti-angiogenesis effects of cisplatin, with no added systemic toxicity. Interestingly, H-4073 inhibited tumor angiogenesis by blocking VEGF production by tumor cells as well as directly inhibiting endothelial cell function. Taken together, our results suggest that H-4073 is a potent anti-tumor agent and it can be used to overcome chemotherapy resistance in HNSCC.

Objective Although the majority of human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinomas have a favorable prognosis, we search for markers of poor prognosis by carefully examining a subset of highly aggressive cases. Study design Seven patients with HPV-positive oropharyngeal cancer who presented with non-pulmonary distant metastasis or developed distant metastasis posttreatment were identified. Eight control cases were chosen which responded well to treatment. Pathologic and radiologic studies were reviewed and compared. Results Two cases displayed a small cell carcinoma (SmCC) component upon pathologic review. Biomarker analysis revealed lower expression of NOTCH1 in the aggressive cohort in comparison to controls ( P = .04). Cases showed a predominance of clustering of lymph nodes, extracapsular spread, and central tumor necrosis. Conclusion Although most HPV-related oropharyngeal cancers display a positive prognosis, it is evident that there is a subset, which behaves more aggressively. This early investigation identifies pathologic and radiologic features that may help to predict this behavior.

BACKGROUND AND PURPOSE: Head and neck squamous cell carcinoma tumors positive for laboratory biomarkers hrHPV and p16 and negative for EGFR often respond better to nonsurgical organ-preservation therapy than hrHPV-negative, p16-negative, and EGFR overexpressing tumors. CTP has been shown to distinguish which locally advanced head and neck squamous cell carcinomas will respond to induction chemotherapy or chemoradiation. Our purpose was to determine whether a relationship exists between CTP measures and the expression of these laboratory biomarkers, because both appear to separate head and neck squamous cell carcinoma tumors into similar groups. MATERIALS AND METHODS: We conducted an institutional review board–approved, Health Insurance Portability and Accountability Act–compliant retrospective review of head and neck CTP in 25 patients with locally advanced head and neck squamous cell carcinoma who had signed informed consent. Eight women and 17 men, 41–80 years of age, constituted a pretreatment group of 18 patients and a palliative group of 7 patients. Tumor biopsy samples were analyzed for overexpression of hrHPV, p16, and EGFR. The hrHPV, p16, and EGFR status of the tumors was correlated with CTP parameters (MTT, BV, BF, CP) by using the Wilcoxon evaluation and Fischer exact test. RESULTS: There were significantly lower CP values in pretreatment tumors overexpressing EGFR ( P = .04). CP values ≤17.23 were significantly correlated with EGFR overexpression ( P = .015). A trend toward higher CP values was present in hrHPV-positive and p16-overexpressing pretreatment tumors ( P = .14). CONCLUSIONS: A significant correlation exists between CTP measures and EGFR overexpression in head and neck squamous cell carcinomas, suggesting an association between certain imaging findings and molecular biomarkers. These results may be related to a tumor cell survival mechanism linking perfusion and biomarker expression.

6036Background: Vorinostat (Zolinza) is a potent HDAC inhibitor that sensitizes head and neck squamous cell carcinoma (HNSCC) to cytotoxic therapy while sparing normal epithelium The primary object...

Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide. Greater than 90% of SCCHN of the oropharynx overexpress the epidermal growth factor receptor (EGFR or HER1). Cetuximab (Erbitux-TM) is a humanized anti-HER1 monoclonal antibody (mAb) that binds to HER1 overexpressing tumor cells. Cetuximab has a direct effect on HER1-positive cancer cells, but it also can activate immune cells that bear receptors for the Fc (constant portion) of IgG such as natural killer (NK) cells. NK cells have an activating Fc receptor for IgG (FcγRIIIa), which mediates Ab dependent cellular cytotoxicity (ADCC) and enhances production of interferon-γ (IFN-γ) in response to Ab-coated targets. Interleukin-12 (IL-12) is a cytokine produced by antigen-presenting cells that stimulates IFN-γ production from NK cells. We hypothesized that IL-12 would enhance the anti-tumor activity of cetuximab by activating the FcR effector mechanisms of NK cells.Expression of HER1 was measured on human papilloma virus (HPV)-positive (UD-SCC2, UM-SCC47) and HPV-negative (Cal27, UM-SCC74B) SCCHN cell lines by immunoblot analysis and flow cytometry. NK cells from normal donors were treated overnight with IL-2 (100 U), IL-12, IL-15, or IL-21 (all 10 ng/mL) and tested for ADCC versus cetuximab-coated cancer cells in a 4 hr (51)Cr assay. Release of cytokines by NK cells in response to cetuximab-coated cells was measured by ELISA. Phosphorylation of the ERK transcription factor in NK cells was measured by flow cytometry. The efficacy of combination therapy with cetuximab plus IL-12 was evaluated in a murine tumor model of head and neck cancer.All cell lines showed99% expression of HER1 by flow cytometry and immunoblot analysis except UM-SCC74B (73%). Normal NK cells mediated 49.4% lysis of cetuximab-coated SCCHN cell lines as compared to 7.6% lysis of cells treated with control IgG (P = .0002). NK cell lysis of cetuximab-coated SCCHN cells was markedly enhanced by 12 hr pre-treatment of NK cells with IL-12 (71.6% lysis, P = .005 vs cetuximab alone). As a control, IL-12-activated NK cells were tested against IgG-treated cells. ADCC under these conditions was just 21.7%. Similar levels of lysis were noted for both HPV-positive and HPV-negative and cell lines. Other NK cell activating factors such as IL-2, IL-15, and IL-21 were also able to enhance NK cell ADCC. The stimulus of IL-12 and cetuximab-coated tumor cells induced the synergistic production of nanogram levels of IFN-γ (6-fold increase over controls) (P.001). A similar effect was seen for NK cell production of the chemokines RANTES, MIP-1α, and IL-8. Phosphorylation of ERK (which is critical for FcR-mediated ADCC and cytokine production) was enhanced in NK cells exposed to IL-12 and IgG as compared to control conditions. The combination of cetuximab plus IL-12 resulted in a reduction in tumor burden when compared to either agent alone in a murine xenograft model of SCCHN.Cytokine stimulation of NK cells in the presence of cetuximab-coated head and neck cancer cells leads to enhanced NK cell mediated ADCC and cytokine secretion independent of tumor cell HPV-status. Cytokine administration could be a useful adjuvant in the cetuximab treatment of HER1-positive head and neck cancer.

Background Human papillomavirus (HPV) has been detected in keratinizing nasopharyngeal carcinomas (NPCs); however, the relationship between HPV and Epstein–Barr virus (EBV) among whites with nonkeratinizing NPCs remains unclear. The HPV, p16, and EBV status was examined in current University of Michigan patients with NPC. Methods From 2003 to 2007, 89 patients, 84 with oropharyngeal cancer (OPC) and 5 with NPC, were enrolled in an organ-sparing trial. Biopsy tissues from all 89 patients were evaluated for HPV and p16 expression. A separate HPV analysis of the 84 OPC patients is in progress. Among the patients with NPC, tumor tissue was also analyzed for EBV-encoded RNA (EBER). Results Five of 89 patients (5.6%) had NPC, all with nonkeratinizing histology. The 4 white patients with NPC were HPV(+) (subtype-16, subtype-18 [2 patients], and subtype-59)/p16(+)/EBER(-). One Asian patient with NPC had an HPV(-)/p16(-)/EBER(+) NPC tumor that developed distant metastases. Conclusion We postulate that HPV may be the etiologic factor in some EBV-negative, nonkeratinizing NPCs among whites. © 2009 Wiley Periodicals, Inc. Head Neck, 2010

We established multiple University of Michigan Squamous Cell Carcinoma (UM-SCC) cell lines. With time, these have been distributed to other labs all over the world. Recent scientific discussions have noted the need to confirm the origin and identity of cell lines in grant proposals and journal articles. We genotyped the UM-SCC cell lines in our collection to confirm their unique identity.Early-passage UM-SCC cell lines were genotyped and photographed.Thus far, 73 unique head and neck UM-SCC cell lines (from 65 donors, including 21 lines from 17 females) were genotyped. In 7 cases, separate cell lines were established from the same donor.These results will be posted on the UM Head and Neck SPORE Tissue Core website for other investigators to confirm that the UM-SCC cells used in their laboratories have the correct features. Publications using UM-SCC cell lines should confirm the genotype.

Induction chemotherapy and concurrent chemoradiation for responders or immediate surgery for non-responders is an effective treatment strategy head and neck squamous cell carcinoma (HNSCC) of the larynx and oropharynx. Biomarkers that predict outcome would be valuable in selecting patients for therapy. In this study, the presence and titer of high risk human papilloma virus (HPV) and expression of epidermal growth factor receptor (EGFR) in pre-treatment biopsies, as well as smoking and gender were examined in oropharynx cancer patients enrolled in an organ sparing trial. HPV16 copy number was positively associated with response to therapy and with overall and disease specific survival, whereas EGFR expression, current or former smoking behavior, and female gender (in this cohort) were associated with poor response and poor survival in multivariate analysis. Smoking cessation and strategies to target EGFR may be useful adjuncts for therapy to improve outcome in the cases with the poorest biomarker profile.

Purpose: Cisplatin resistance remains a barrier to organ-sparing and survival of patients with advanced head and neck squamous cell carcinoma (HNSCC). Targeted therapies to overcome cisplatin-resistant HNSCC are being developed. Methods and Materials: Cisplatin-sensitive parental HNSCC cell lines and cisplatin-resistant progeny were studied. Pretreatment HNSCC biopsies were used to construct tissue microarrays which were stained for p53 and Bcl-xL. Results: HNSCC cell lines selected for cisplatin resistance had wild-type p53 and high levels of Bcl-xL. Expression of wild-type p53 in cell lines with low Bcl-xL enhanced cisplatin sensitivity. Expression of both Bcl-xL and wild-type p53 caused tumor cells to become cisplatin resistant. Patients whose tumors expressed low levels of p53 and Bcl-xL enjoyed the best organ preservation and disease-free survival whereas patients whose tumors expressed low levels of p53 and high levels of Bcl-xL had the worst outcome. Novel agents that inhibit Bcl-xL or activate p53 function may target cisplatin-resistant HNSCC. Conclusion: Cisplatin resistance in HNSCC is mediated, at least in part, by high Bcl-xL and functional p53.

Objective: Head and neck squamous cell carcinoma (HNSCC) remains a major health care problem worldwide, comprising almost 50% of all malignancies in some developing nations. Even with extensive surgery and full course chemoradiation treatment the survival rates of HNSCC patients have not significantly improved over the last two decades. Acquisition of chemoresistance, locoregional recurrence and metastatic phenotype are the major causes of treatment failure and mortality in these patients. It is therefore imperative that we gain a better understanding of the molecular mechanisms that contribute to the aggressive tumor phenotype in order to develop novel and effective strategies for the treatment of head and neck cancer patients. Recent studies have highlighted the role of histone deacetylases (HDACs) in regulating a number of genes that are involved in cell cycle control, proliferation, survival, DNA repair and differentiation. In this study, we examined if treatment with a HDAC inhibitor, Vorinostat could reverse chemoresistance in head and neck cancer cells and enhance the therapeutic efficacy of cisplatin treatment. Methodology: HDACs expression in tumor samples from HNSCC patients was examined by real time PCR. Cell growth was examined by MTT assay and colony formation assay. Western blot analysis was used to measure changes in protein levels. Cancer stem cell phenotype was assessed by tumorsphere formation. A SCID mouse xenograft model was used to access the in vivo efficacy of Vorinostat. Results: Our results show that HDAC1, 2, and 6 levels are markedly increased in the head and neck cancer cell lines as compared to normal human oral keratinocytes. We took two cisplatin sensitive cell lines and grew them in increasing concentrations of cisplatin to mimic acquired resistance in patients. Both of these cisplatin resistant cell lines (cisR) showed further increase in HDAC1 and 2 expressions as compared to the cispaltin sensitive parental cells. Depletion of HDAC1 and 2 in these cisR cell lines by siRNA significantly enhanced cisplatin sensitivity. Next, we used an HDAC inhibitor, Vorinostat on these cell lines and observed a dose dependent inhibition of cell proliferation (MTT). In addition, Vorinostat treatment significantly decreased tumorsphere formation and the expression of stem cell marker, Nanog. Vorinostat in combination with cisplatin showed a synergistic effect on the inhibition of cell proliferation and tumorsphere formation. We then assessed the efficacy of Vorinostat treatment as a single agent or in combination with cispaltin in SCID mouse xenograft model. Similar to the in vitro results, combination treatment was most effective in reducing the tumor burden in the mice. Conclusions: Taken together, our results suggest that targeting of HDACs with Vorinostat could be an effective treatment strategy for the treatment of HNSCC patients that do not respond to currently used treatment regimens. Citation Format: Bhavna Kumar, Arti Yadav, Theodoros N. Teknos, Pawan Kumar. Vorinostat enhances anti-tumor effects of cisplatin in head and neck cancer cells by targeting cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 353. doi:10.1158/1538-7445.AM2015-353

Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer worldwide and five-year survival rates ( Citation Format: Arti Yadav, Bhavna Kumar, Jun-Ge Yu, Matthew Old, Theodoros N. Teknos, Pawan Kumar. Tumor-associated endothelial cells promote tumor metastasis by chaperoning circulating tumor cells and protecting them from anoikis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3934. doi:10.1158/1538-7445.AM2013-3934

Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer worldwide and five-year survival rate ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5604. doi:1538-7445.AM2012-5604

5546 Background: Optimal treatment for HPV- and HPV+ LASCCOP is not well defined. Here we retrospectively compare survival and toxicity outcomes from two different organ preservation protocols in p...

Distant metastases in head and neck cancer patients almost invariably herald a poor prognosis. Five year survival rates for early stage localized head and neck cancers are over 80% but this drop to 40% where disease has spread to neck nodes, and to below 20% for patients with distant metastatic disease. Uncontrolled cell proliferation and angiogenesis are key characteristics of initiation and early growth of epithelial origin cancers. However, it is the subsequent acquisition of motility and invasiveness that leads to metastatic dissemination of these tumor cells. A key process in this metastatic cascade that converts an adherent epithelial cell into a migratory cell which can invade through the extracellular matrix is known as epithelial-mesenchymal transition (EMT). EMT plays a critical role in epithelial tumor cell metastasis and is commonly observed in tumor samples from head and neck cancer patients. However, the precise molecular events that initiate this complex process of EMT in head and neck cancers are poorly understood. There is increasing evidence that suggest an important role of tumor microenvironment in promoting EMT changes in tumor cells. We have previously shown that head and neck tumors exhibit significantly higher levels of Bcl-2 expression in tumor associated endothelial cells and overexpression of Bcl-2 alone in tumor-associated endothelial cells was sufficient to enhance tumor metastasis of oral squamous cell carcinoma in a SCID mouse model. In this study, we show that endothelial cells expressing Bcl-2 (EC-Bcl-2) when co-cultured with head and neck tumor cells significantly enhance EMT-related changes in tumor cells and this EC-Bcl-2-induced EMT in tumor cells is mediated predominantly by IL-6 secretion. Treatment with recombinant IL-6 or stable IL-6 overexpression in CAL27 cells or normal oral epithelial cells (NOE) significantly induced the expression of vimentin while repressing E-cadherin expression via the JAK/STAT3/Snail signaling pathway. This phenotype was further associated with enhanced tumor and NOE cell scattering and motility. STAT3 knock-down significantly reversed IL-6-mediated tumor and NOE cell motility by inhibiting FAK activation. Furthermore, tumor cells overexpressing IL-6 showed marked increase in lymph node and lung metastasis, in vivo. These results suggest a novel function for IL-6 in mediating head and neck tumor EMT and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3362. doi:10.1158/1538-7445.AM2011-3362

Objective To determine whether the favorable outcome associated with human papillomavirus (HPV) 16–positive oropharyngeal cancer is related to a patient's adaptive immunity. Setting Academic medical center. Patients Forty-seven of 66 previously untreated patients (6 of 20 patients with stage III and 41 of 46 with stage IV cancer) in a prospective clinical trial of chemoradiotherapy. Intervention All patients were treated with a single course of neoadjuvant chemotherapy followed by either surgery (for nonresponders) or chemoradiotherapy. Main Outcome Measures Pretreatment levels (percentages and absolute counts) of CD3, CD4, CD8, natural killer, and B cells and overall white blood cell counts were measured by flow cytometry. Correlations of subsets with HPV-16 status, tumor subsite, cancer stage, T class, N class, smoking status, performance status, sex, response to chemoradiotherapy, p53 mutation type, epidermal growth factor receptor expression, and disease-specific and overall survival were determined. Results After a median follow-up of 6.6 years, improved survival was associated with an elevated percentage of CD8 cells ( P = .04), a low CD4:CD8 ratio ( P = .01), low epidermal growth factor receptor expression ( P = .002), and HPV status ( P = .02). The percentage of CD8 cells was significantly higher ( P = .04) and the CD4:CD8 ratio was significantly lower ( P = .02) in HPV-16–positive patients. A higher percentage of CD8 cells was associated with response to induction chemotherapy ( P = .02) and complete tumor response after chemoradiotherapy ( P = .045). Conclusion These findings confirm previous correlations of outcome with circulating CD8 cell levels and support the conjecture that improved adaptive immunity may play a role in the favorable prognosis of patients with HPV-16–positive cancers.

Objectives of the study: Chemotherapy and radiotherapy constitute the standard modality of treatment for localized head and neck squamous cell carcinomas (HNSCC). However, many patients fail to respond and relapse after such treatments due to the acquisition of chemo- and radio-resistance. Therefore, there is an urgent need to develop novel drugs that could reverse the resistant phenotype. Curcumin, the constituent of the spice turmeric has been shown to have anti-inflammatory, anti-oxidant and anti-proliferative properties in several tumor types. However, use of curcumin has been limited due to its poor absorption. Recently, a novel class of curcumin analogs, diarylidenylpiperidones (DAPs), has been developed by incorporating a piperidone link to the beta-diketone structure and fluoro substitutions on the phenyl groups. We have observed that these DAP compounds, in general are more effective than curcumin in inhibiting the proliferation of a variety of cancer cell lines and xenograft tumors. The objective of this study was to evaluate the effectiveness of HO-4073, a DAP compound, in reversing chemo-and radio-resistance in head and neck cancer cell lines. HO-4073 is a parafluorinated variant of DAPs. Methodology: The study was performed using two established human head and neck cancer cell lines, Cell-viablity assay was performed by MTT. Apoptosis was quantified by Annexin V staining. Cleaved caspase-3 and JAK/STAT3 signaling proteins were measured by Western blot. Results: Our results showed that HO-4073 is a potent anti-tumor agent and it significantly inhibited cell proliferation in all the head and neck cancer cell lines tested in a dose- and time-dependent manner, irrespective of p53 mutation status or human papillomavirus positivity. In addition, pretreatment of a highly chemo- and radio-resistant HNSCC cell line (UM-SCC-74A) with HO-4073 (2.5 M) significantly reversed the chemo resistance as observed by cell viability assay (MTT), apoptosis assay (Annexin V binding) and cleaved caspase-3 (Western blot). HO-4073 induced chemo- and radio-sensitivity via the inhibition of JAK/STAT3 pathway. To further examine the effect of HO-4073 on chemo-resistance, we established a cisplatin-resistant cell line (CAL 27-PT) in the laboratory by growing a tongue cancer cell line CAL 27 in increasing concentrations of cisplatin. CAL 27-PT is highly resistant to cisplatin (IC50 20 M) as compared to the parental cell line (IC50 2 M). Treatment with HO-4073 resulted in a 4-fold increase in apoptosis in the cisplatin resistant CAL 27-PT as compared to untreated cells. Conclusion: Taken together, our results suggest that HO-4073 is a potent anti-tumor agent and it can be used to overcome chemo- and radio-resistance in head and neck cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-282.

Objective To assess tumor markers in advanced laryngeal cancer. Design Marker expression and clinical outcome. Patients Pretreatment tumor biopsy specimens were analyzed from patients enrolled in the Department of Veterans Affairs Laryngeal Cancer Study. Main Outcome Measures Expression of p53 (OMIMTP53) and Bcl-xL (OMIM600039) in pretreatment biopsy specimens was assessed for correlation with chemotherapy response, laryngeal preservation, and survival. Results Higher rates of larynx preservation were observed in patients whose tumors expressed p53 vs those that did not (80% [36 of 45 patients] vs 59% [24 of 41 patients], P =.03). Higher rates of larynx preservation were also observed in patients whose tumors expressed low levels of Bcl-xL vs high levels of Bcl-xL (90% [18 of 20 patients] vs 60% [30 of 50 patients], P =.02). Patients were categorized into 3 risk groups (low, intermediate, and high) based on their tumor p53 and Bcl-xL expression status. Patients whose tumors had the high-risk biomarker profile (low p53 expression and high Bcl-xL expression) were less likely to preserve their larynx than patients whose tumors had the intermediate-risk biomarker profile (high p53 expression and low or high Bcl-xL expression) or the low-risk biomarker profile (low p53 expression and low Bcl-xL expression). The larynx preservation rates were 100% (10 of 10 patients), 77% (26 of 34 patients), and 54% (7 of 13 patients) for the low-risk, intermediate-risk, and high-risk groups, respectively ( P =.04, Fisher exact test). Conclusion Tumor expression of p53 and Bcl-xL is a strong predictor of successful larynx preservation in patients treated with induction chemotherapy and followed by radiation therapy in responding tumors.

The measurement of body fluid levels of biochemical markers in joint tissues has begun to provide clinically useful information. Synovial fluid (SF) plays an important role in articular joint lubrication, nutrition, and metabolism of cartilage and other connective tissues within the joint. The purpose of our study was to identify and characterize osteogenic protein 1 (OP-1) in SF from patients with rheumatoid arthritis (RA) or with osteoarthritis (OA) and to correlate levels of OP-1 with those of hyaluronan (HA) and antigenic keratan sulfate (AgKS). SF was aspirated from the knees of patients with either RA or OA and from the knees of asymptomatic organ donors with no documented history of joint disease. The presence of detectable OP-1 in SF was demonstrated by western blots with specific anti-pro-OP-1 and anti-mature OP-1 antibodies. Measurement of levels of OP-1, HA and AgKS was performed using ELISAs. OP-1 was identified in human SF in two forms, pro-OP-1 and active (mature) OP-1 – mature OP-1 being detected only in SF from OA patients and RA patients. Levels of OP-1 and HA were higher in RA patients than in OA patients and asymptomatic donors, while the level of AgKS was highest in SF from asymptomatic donors. Statistically significant differences were found between SF levels of OP-1 in RA and OA patients and between SF levels of AgKS among the three groups tested. The SF content of OP-1 tended to correlate positively with HA levels, but negatively with AgKS concentrations. In conclusion, the results of this study suggest that measurement of OP-1 in joint fluid may have value in the clinical evaluation of joint disease processes.