An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering.

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1-autoinhibited for chromatin binding-that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering.

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1 - autoinhibited for chromatin binding - that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

New insights into the molecular mechanisms of glutaminase C inhibitors in cancer cells using serial room temperature crystallography.

Cancer cells frequently exhibit uncoupling of the glycolytic pathway from the TCA cycle (i.e., the "Warburg effect") and as a result, often become dependent on their ability to increase glutamine catabolism. The mitochondrial enzyme Glutaminase C (GAC) helps to satisfy this 'glutamine addiction' of cancer cells by catalyzing the hydrolysis of glutamine to glutamate, which is then converted to the TCA-cycle intermediate α-ketoglutarate. This makes GAC an intriguing drug target and spurred the molecules derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (the so-called BPTES class of allosteric GAC inhibitors), including CB-839, which is currently in clinical trials. However, none of the drugs targeting GAC are yet approved for cancer treatment and their mechanism of action is not well understood. Here, we shed new light on the underlying basis for the differential potencies exhibited by members of the BPTES/CB-839 family of compounds, which could not previously be explained with standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 or its analogs. Using an emerging technique known as serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors with significantly different potencies. We also developed a computational model to further elucidate the molecular basis of differential inhibitor potency. We then corroborated the results from our modeling efforts using recently established fluorescence assays that directly read out inhibitor binding to GAC. Together, these findings should aid in future design of more potent GAC inhibitors with better clinical outlook.

Impact of age and comorbidity on survival among patients with oral cavity squamous cell carcinoma.

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.

Fixed-target serial oscillation crystallography at room temperature.

A fixed-target approach to high-throughput room-temperature serial synchrotron crystallography with oscillation is described. Patterned silicon chips with microwells provide high crystal-loading density with an extremely high hit rate. The microfocus, undulator-fed beamline at CHESS, which has compound refractive optics and a fast-framing detector, was built and optimized for this experiment. The high-throughput oscillation method described here collects 1-5° of data per crystal at room temperature with fast (10°â€…s-1) oscillation rates and translation times, giving a crystal-data collection rate of 2.5 Hz. Partial datasets collected by the oscillation method at a storage-ring source provide more complete data per crystal than still images, dramatically lowering the total number of crystals needed for a complete dataset suitable for structure solution and refinement - up to two orders of magnitude fewer being required. Thus, this method is particularly well suited to instances where crystal quantities are low. It is demonstrated, through comparison of first and last oscillation images of two systems, that dose and the effects of radiation damage can be minimized through fast rotation and low angular sweeps for each crystal.

Predictors of gastrostomy tube dependence in surgically managed oropharyngeal squamous cell carcinoma.

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 < 0.01) and T3/T4 tumor stage (OR 2.84; P = 0.02) were independent and significant predictors of G-tube dependence. CONCLUSION: Surgical approach, advanced tumor stage, and oral tongue resection may influence the development of nutritional dependence for surgically treated patients with OPSCC. LEVEL OF EVIDENCE: 4 Laryngoscope, 129:415-421, 2019.

Effect of adjuvant radiotherapy treatment center volume on overall survival.

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.

Notch1 Overexpression Correlates to Improved Survival in Cancer of the Oropharynx.

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.

High expression of myoferlin is associated with poor outcome in oropharyngeal squamous cell carcinoma patients and is inversely associated with HPV-status.

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<0.001) and these patients had 5.5 times increased hazard of death (95% Cl 3.4-8.8). Nuclear myoferlin expression was also directly associated with tumor recurrence (p<0.001), perineural invasion (p=0.008), extracapsular spread (p=0.009), higher T-stage (p=0.0015) and distant metastasis (p<0.001). In addition, nuclear MYOF expression was directly associated with IL-6 (p<0.001) and inversely with HPV status (p=0.0014). In a subgroup survival analysis, MYOF nuclear+/IL-6+ group had worst survival (84.6% mortality), whereas MYOF nuclear-/IL-6- had the best survival. Similarly, patients with HPV-negative/MYOF-positive tumors had worse survival compared to HPV-positive/MYOF-negative. Taken together, our results demonstrate for the first time that nuclear myoferlin expression independently predicts poor clinical outcome in OPSCC patients.

Surgical management of oropharyngeal squamous cell carcinoma: Survival and functional outcomes.

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.

Suppression of Proinflammatory and Prosurvival Biomarkers in Oral Cancer Patients Consuming a Black Raspberry Phytochemical-Rich Troche.

Black raspberries (BRB) demonstrate potent inhibition of aerodigestive tract carcinogenesis in animal models. However, translational clinical trials evaluating the ability of BRB phytochemicals to impact molecular biomarkers in the oral mucosa remain limited. The present phase 0 study addresses a fundamental question for oral cancer food-based prevention: Do BRB phytochemicals successfully reach the targeted oral tissues and reduce proinflammatory and antiapoptotic gene expression profiles? Patients with biopsy-confirmed oral squamous cell carcinomas (OSCC) administered oral troches containing freeze-dried BRB powder from the time of enrollment to the date of curative intent surgery (13.9 ± 1.27 days). Transcriptional biomarkers were evaluated in patient-matched OSCCs and noninvolved high at-risk mucosa (HARM) for BRB-associated changes. Significant expression differences between baseline OSCC and HARM tissues were confirmed using a panel of genes commonly deregulated during oral carcinogenesis. Following BRB troche administration, the expression of prosurvival genes (AURKA, BIRC5, EGFR) and proinflammatory genes (NFKB1, PTGS2) were significantly reduced. There were no BRB-associated grade 3-4 toxicities or adverse events, and 79.2% (N = 30) of patients successfully completed the study with high levels of compliance (97.2%). The BRB phytochemicals cyanidin-3-rutinoside and cyanidin-3-xylosylrutinoside were detected in all OSCC tissues analyzed, demonstrating that bioactive components were successfully reaching targeted OSCC tissues. We confirmed that hallmark antiapoptotic and proinflammatory molecular biomarkers were overexpressed in OSCCs and that their gene expression was significantly reduced following BRB troche administration. As these molecular biomarkers are fundamental to oral carcinogenesis and are modifiable, they may represent emerging biomarkers of molecular efficacy for BRB-mediated oral cancer chemoprevention.

Head and neck cancers, Version 2.2014. Clinical practice guidelines in oncology.

This selection from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Head and Neck Cancers focuses on glottic laryngeal cancer, which is the most common type of laryngeal cancer and has an excellent cure rate. The lymphatic drainage of the glottis is sparse, and early stage primaries rarely spread to regional nodes. Because hoarseness is an early symptom, most glottic laryngeal cancer is early stage at diagnosis. Updates to these guidelines for 2014 include revisions to "Principles of Radiation Therapy" for each site and "Principles of Surgery," and the addition of a new section on "Principles of Dental Evaluation and Management."

Does body mass index matter while selecting the flap type for pharyngeal reconstructions?

The aim of our study was to investigate the effect of patient-related factors, such as the body mass index (BMI) and tumor size, in selecting the flap type for the reconstruction of pharyngeal defects. This retrospective review included 182 patients with pharyngeal defect reconstructions with free and pedicled flaps at the Ohio State University from January 2005 to December 2008. We conducted a retrospective comparison of variety of different flap reconstruction techniques. We compared different flap reconstruction with BMI and tumor size without functional outcome such as swallowing and speech data. Although there was no statistically significant correlation (P > 0.05) when comparing the free flaps with pedicled flaps according to the BMI and tumor size, there was an obvious tendency to prefer radial forearm free flap over anterolateral thigh free flap in patients who are overweight and those with obesity with a ratio of 32:3. In the same group of patients, a similar tendency was observed to prefer fibular free flap over iliac crest free flap with a ratio of 14:5, whereas the ratio was becoming 3:5 in favor of iliac crest free flap over fibular free flap in patients with BMI of 24 or lower. Despite the fact that surgeons' experience with a certain flap type is one of the most important factors while determining which flap to reconstruct, BMI might have a significant impact while selecting the free flap types for the reconstruction of pharyngeal defects.

Elevated intrinsic cancer stem cell population in human papillomavirus-associated head and neck squamous cell carcinoma.

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.

Intensive care unit versus non-intensive care unit postoperative management of head and neck free flaps: comparative effectiveness and cost comparisons.

BACKGROUND: Despite its widespread use, there is no consensus on the postoperative management in patients undergoing free flap reconstructions. We report the largest study comparing flap outcomes, morbidity, and cost in patients with head and neck cancer free flaps who recovered in the intensive care unit (ICU) versus a "specialty floor" setting. METHODS: This was a retrospective review of patients undergoing free flap surgery for head and neck defects over a 4-year period. Patients before a certain date went to the ICU for immediate postoperative care and after to a non-ICU setting. Postoperative medical and surgical complications and hospital charges were analyzed. RESULTS: Patients in the ICU group had a longer length of stay (LOS) and incurred greater hospital costs than the patients in the non-ICU setting. There was no difference in the flap failure rate between the 2 groups. CONCLUSION: Consideration should be given to a floor-based postoperative management regimen for this patient population.

Head and neck cancers, version 2.2013. Featured updates to the NCCN guidelines.

These NCCN Guidelines Insights focus on nutrition and supportive care for patients with head and neck cancers. This topic was a recent addition to the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Head and Neck Cancers. The NCCN Guidelines Insights focus on major updates to the NCCN Guidelines and discuss the new updates in greater detail. The complete version of the NCCN Guidelines for Head and Neck Cancers is available on the NCCN Web site (NCCN.org).

Highly aggressive human papillomavirus-related oropharyngeal cancer: clinical, radiologic, and pathologic characteristics.

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.

Transoral resection of pharyngeal cancer: summary of a National Cancer Institute Head and Neck Cancer Steering Committee Clinical Trials Planning Meeting, November 6-7, 2011, Arlington, Virginia.

Recent advances now permit resection of many pharyngeal tumors through the open mouth, an approach that can greatly reduce the morbidity of surgical exposure. These transoral techniques are being rapidly adopted by the surgical community and hold considerable promise. On November 6-7, 2011, the National Cancer Institute sponsored a Clinical Trials Planning Meeting to address how to further investigate the use of transoral surgery, both in the good prognosis human papillomavirus (HPV)-initiated oropharyngeal cancers, and in those with HPV-unrelated disease. The proceedings of this meeting are summarized.

Multiparameter analysis, including EMT markers, on negatively enriched blood samples from patients with squamous cell carcinoma of the head and neck.

Epithelial to mesenchymal transition (EMT) has been hypothesized as a mechanism by which cells change phenotype during carcinogenesis, as well as tumor metastasis. Whether EMT is involved in cancer metastasis has a specific, practical impact on the field of circulating tumor cells (CTCs). Since the generally accepted definition of a CTC includes the expression of epithelial surface markers, such as EpCAM, if a cancer cell loses its epithelial surface markers (which is suggested in EMT), it will not be separated and/or identified as a CTC. We have developed, and previously reported on the use of, a purely negative enrichment technology enriching for CTCs in the blood of squamous cell carcinoma of the head and neck (SCCHN). This methodology does not depend on the expression of surface epithelial markers. Using this technology, our initial data on SCCHN patient blood indicates that the presence of CTCs correlates with worse disease-free survival. Since our enrichment is not dependent on epithelial markers, we have initiated investigation of the presence of mesenchymal markers in these CTC cells to include analysis of: vimentin, epidermal growth factor receptor, N-cadherin, and CD44. With the aid of confocal microscopy, we have demonstrated not only presumed CTCs that express and/or contain: a nucleus, cytokeratins, vimentin, and either EGFR, CD44, or N-cadherin, but also cells that contain all of the aforementioned proteins except cytokeratins, suggesting that the cells have undergone the EMT process. We suggest that our negative depletion enrichment methodology provides a more objective approach in identifying and evaluating CTCs, as opposed to positive selection approaches, as it is not subjective to a selection bias and can be tailored to accommodate a variety of cytoplasmic and surface markers which can be evaluated to identify a multitude of phenotypic patterns within CTCs from individual patients, including so-called EMT as presented here.