NF-κB and stat3 transcription factor signatures differentiate HPV-positive and HPV-negative head and neck squamous cell carcinoma.

Using high-throughput analyses and the TRANSFAC database, we characterized TF signatures of head and neck squamous cell carcinoma (HNSCC) subgroups by inferential analysis of target gene expression, correcting for the effects of DNA methylation and copy number. Using this discovery pipeline, we determined that human papillomavirus-related (HPV+) and HPV- HNSCC differed significantly based on the activity levels of key TFs including AP1, STATs, NF-κB and p53. Immunohistochemical analysis confirmed that HPV- HNSCC is characterized by co-activated STAT3 and NF-κB pathways and functional studies demonstrate that this phenotype can be effectively targeted with combined anti-NF-κB and anti-STAT therapies. These discoveries correlate strongly with previous findings connecting STATs, NF-κB and AP1 in HNSCC. We identified five top-scoring pair biomarkers from STATs, NF-κB and AP1 pathways that distinguish HPV+ from HPV- HNSCC based on TF activity and validated these biomarkers on TCGA and on independent validation cohorts. We conclude that a novel approach to TF pathway analysis can provide insight into therapeutic targeting of patient subgroup for heterogeneous disease such as HNSCC.

Integrative computational analysis of transcriptional and epigenetic alterations implicates DTX1 as a putative tumor suppressor gene in HNSCC.

Over a half million new cases of Head and Neck Squamous Cell Carcinoma (HNSCC) are diagnosed annually worldwide, however, 5 year overall survival is only 50% for HNSCC patients. Recently, high throughput technologies have accelerated the genome-wide characterization of HNSCC. However, comprehensive pipelines with statistical algorithms that account for HNSCC biology and perform independent confirmatory and functional validation of candidates are needed to identify the most biologically relevant genes. We applied outlier statistics to high throughput gene expression data, and identified 76 top-scoring candidates with significant differential expression in tumors compared to normal tissues. We identified 15 epigenetically regulated candidates by focusing on a subset of the genes with a negative correlation between gene expression and promoter methylation. Differential expression and methylation of 3 selected candidates (BANK1, BIN2, and DTX1) were confirmed in an independent HNSCC cohorts from Johns Hopkins and TCGA (The Cancer Genome Atlas). We further performed functional evaluation of NOTCH regulator, DTX1, which was downregulated by promoter hypermethylation in tumors, and demonstrated that decreased expression of DTX1 in HNSCC tumors maybe associated with NOTCH pathway activation and increased migration potential.

Serum biomarkers for detection of head and neck squamous cell carcinoma.

BACKGROUND: Detection of hypermethylated circulating tumor DNA has the potential to be a minimally invasive, low cost, and reproducible method for cancer detection. METHODS: We evaluated serum from 100 patients with known head and neck squamous cell carcinoma (HNSCC) and 50 healthy control patients for 3 previously described methylation targets, endothelin receptor type B (EDNRB), cyclin-dependent kinase inhibitor 2A (CDKN2A or p16), and deleted in colorectal carcinoma (DCC), using quantitative methylation specific polymerase chain reaction (qMSPCR). RESULTS: EDNRB hypermethylation was identified in the serum of 10% of the patients with HNSCC but in none of the control patients. DCC hypermethylation was detected in 2 serum samples from patients with cancer that also amplified EDNRB and one of these samples also had p16 hypermethylation. EDNRB hypermethylation was statistically significant by Fisher's exact test (p = .03) when comparing HNSCC to controls. CONCLUSIONS: Serum EDNRB hypermethylation is a highly specific but not sensitive serum biomarker for HNSCC.

Outlier Analysis Defines Zinc Finger Gene Family DNA Methylation in Tumors and Saliva of Head and Neck Cancer Patients.

Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas). Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC.

Chapter 9: Benign sinonasal neoplasms.

Benign sinonasal neoplasms are a heterogeneous group of tumors that present with similar symptoms including nasal obstruction, anosmia, rhinorrhea, and epistaxis. The proper workup and accurate diagnosis is essential for these tumors so that the appropriate treatment plan can be established. In this article of benign sinonasal neoplasms, we discuss their typical clinical presentation, histological and radiographic findings, and treatment options.

Chapter 9: Benign sinonasal neoplasms.

Benign sinonasal neoplasms are a heterogeneous group of tumors that present with similar symptoms including nasal obstruction, anosmia, rhinorrhea, and epistaxis. The proper workup and accurate diagnosis is essential for these tumors so that the appropriate treatment plan can be established. In this article of benign sinonasal neoplasms, we discuss their typical clinical presentation, histological and radiographic findings, and treatment options.

Is there a "July effect" for head and neck cancer surgery?

OBJECTIVES/HYPOTHESIS: A "July effect" of increased complications when new trainees begin residency has been reported widely by the media. We sought to determine the effect of admission month on in-hospital mortality, complications, length of hospitalization, and costs for patients undergoing head and neck cancer (HNCA) surgery. STUDY DESIGN: Retrospective cross-sectional study. METHODS: Discharge data from the Nationwide Inpatient Sample for 48,263 patients who underwent an ablative procedure for a malignant oral cavity, laryngeal, hypopharyngeal, or oropharyngeal neoplasm in 2005 to 2008 were analyzed using cross-tabulations and multivariate regression modeling. RESULTS: There were 3,812 cases admitted in July (8%). July admission was significantly associated with Medicaid (RRR 1.40, P = 0.011) or self-pay payor status (RRR 1.40, P = 0.022), medium hospital bed size (RRR 1.63, P = 0.033) and large hospital bed size (RRR 1.73, P = 0.013). There was no association between July admission and other patient or hospital demographic characteristics. Major procedures and comorbidity were significantly associated with in-hospital death, surgical and medical complications, length of hospitalization, and costs, but no association was found for July admission, July through September discharge, or teaching hospital status and short-term morbidity or mortality. Teaching hospitals and large hospital bed size were predictors of increased length of hospitalization and costs; and private, for profit hospitals were additionally associated with increased costs. No interaction between July admission and teaching hospitals was found for any of the outcome variables studied. CONCLUSIONS: These data do not support evidence of a "July effect" or an increase in morbidity or mortality at teaching hospitals providing HNCA surgical care.

Serum microRNA biomarkers for detection of non-small cell lung cancer.

Non small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality world-wide and the majority of cases are diagnosed at late stages of disease. There is currently no cost-effective screening test for NSCLC, and the development of such a test is a public health imperative. Recent studies have suggested that chest computed tomography screening of patients at high risk of lung cancer can increase survival from disease, however, the cost effectiveness of such screening has not been established. In this Phase I/II biomarker study we examined the feasibility of using serum miRNA as biomarkers of NSCLC using RT-qPCR to examine the expression of 180 miRNAs in sera from 30 treatment naive NSCLC patients and 20 healthy controls. Receiver operating characteristic curves (ROC) and area under the curve were used to identify differentially expressed miRNA pairs that could distinguish NSCLC from healthy controls. Selected miRNA candidates were further validated in sera from an additional 55 NSCLC patients and 75 healthy controls. Examination of miRNA expression levels in serum from a multi-institutional cohort of 50 subjects (30 NSCLC patients and 20 healthy controls) identified differentially expressed miRNAs. A combination of two differentially expressed miRNAs miR-15b and miR-27b, was able to discriminate NSCLC from healthy controls with sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 100% in the training set. Upon further testing on additional 130 subjects (55 NSCLC and 75 healthy controls), this miRNA pair predicted NSCLC with a specificity of 84% (95% CI 0.73-0.91), sensitivity of 100% (95% CI; 0.93-1.0), NPV of 100%, and PPV of 82%. These data provide evidence that serum miRNAs have the potential to be sensitive, cost-effective biomarkers for the early detection of NSCLC. Further testing in a Phase III biomarker study in is necessary for validation of these results.

Promoter methylation in head and neck squamous cell carcinoma cell lines is significantly different than methylation in primary tumors and xenografts.

Studies designed to identify novel methylation events related to cancer often employ cancer cell lines in the discovery phase of the experiments and have a relatively low rate of discovery of cancer-related methylation events. An alternative algorithm for discovery of novel methylation in cancer uses primary tumor-derived xenografts instead of cell lines as the primary source of nucleic acid for evaluation. We evaluated DNA extracted from primary head and neck squamous cell carcinomas (HNSCC), xenografts grown from these primary tumors in nude mice, HNSCC-derived cell lines, normal oral mucosal samples, and minimally transformed oral keratinocyte-derived cell lines using Illumina Infinum Humanmethylation 27 genome-wide methylation microarrays. We found >2,200 statistically significant methylation differences between cancer cell lines and primary tumors and when comparing normal oral mucosa to keratinocyte cell lines. We found no statistically significant promoter methylation differences between primary tumor xenografts and primary tumors. This study demonstrates that tumor-derived xenografts are highly accurate representations of promoter methylation in primary tumors and that cancer derived cell lines have significant drawbacks for discovery of promoter methylation alterations in primary tumors. These findings also support use of primary tumor xenografts for the study of methylation in cancer, drug discovery, and the development of personalized cancer treatments.

Evaluation of MYB promoter methylation in salivary adenoid cystic carcinoma.

The transcription factor MYB was recently proposed to be a promising oncogene candidate in salivary gland adenoid cystic carcinoma (ACC). However, the up-regulation of MYB in ACC could not be explained solely by deletion of its 3' end. It is widely accepted that the promoter methylation status can regulate the transcription of genes, especially in human cancers. Therefore, it is important to know whether MYB promoter demethylation could explain the over-expression of MYB in ACC. By using the Methprimer program, we identified nine CpG islands in the promoter of MYB. All of these CpG islands were located within the -864 to +2082 nt region relative to the transcription start site of MYB. We then used bisulfite genomic sequencing to evaluate the methylation levels of the CpG islands of MYB in 18 primary ACC tumors, 13 normal salivary gland tissues and nine cancer cell lines. Using cell lines, we also determined the relative MYB expression levels and correlated these with the methylation levels. With bisulfite genomic sequencing, we found no detectable methylation in the CpG islands of MYB in either ACC or normal salivary gland tissues. There was a variable degree of MYB expression in the cell lines tested, but none of these cell lines demonstrated promoter methylation. Promoter hypomethylation does not appear to explain the differential expression of MYB in ACC. An alternative mechanism needs to be proposed for the transcriptional control of MYB in ACC.

Advances and Perspectives in the Molecular Diagnosis of Head and Neck Cancer.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a debilitating and lethal disease. Despite significant advances in radiotherapy and surgical management, the 5-year survival rate for head and neck cancer has remained a dismal 50%. Advances in early detection have been made, but to improve patient outcomes better biomarkers and targeted therapeutic agents are needed. Novel biomarkers can improve early detection and provide data to optimize therapeutic strategy and patient survival, and could lead to potentially effective targeted therapies. OBJECTIVE: Report the advances in the discovery of novel biomarkers for HNSCC, and review the potential utility of biomarkers in the molecular diagnosis of HNSCC. METHODS: A review of the English literature (PubMed) from 1980 to 2009. RESULTS/CONCLUSION: Currently the most widely accepted biomarker for HNSCC is high risk HPV status. EGFR is another promising biomarker, however, further research is necessary to determine its prognostic benefit. A large number of promising biomarker candidates are currently being evaluated including epigenetic, expression, and genomic based markers. Studies to validate the sensitivity and specificity of these biomarkers in clinical samples from adequately powered prospective cohorts are needed for successful translation of these findings into potential molecular diagnostic, prognostic, and therapeutic biomarkers for HNSCC.

A Caenorhabditis elegans model of insulin resistance: altered macronutrient storage and dauer formation in an OGT-1 knockout.

O-linked N-acetylglucosamine (O-GlcNAc) is an evolutionarily conserved modification of nuclear pore proteins, signaling kinases, and transcription factors. The O-GlcNAc transferase (OGT) catalyzing O-GlcNAc addition is essential in mammals and mediates the last step in a nutrient-sensing "hexosamine-signaling pathway." This pathway may be deregulated in diabetes and neurodegenerative disease. To examine the function of O-GlcNAc in a genetically amenable organism, we describe a putative null allele of OGT in Caenorhabditis elegans that is viable and fertile. We demonstrate that, whereas nuclear pore proteins of the homozygous deletion strain are devoid of O-GlcNAc, nuclear transport of transcription factors appears normal. However, the OGT mutant exhibits striking metabolic changes manifested in a approximately 3-fold elevation in trehalose levels and glycogen stores with a concomitant approximately 3-fold decrease in triglycerides levels. In nematodes, a highly conserved insulin-like signaling cascade regulates macronutrient storage, longevity, and dauer formation. The OGT knockout suppresses dauer larvae formation induced by a temperature-sensitive allele of the insulin-like receptor gene daf-2. Our findings demonstrate that OGT modulates macronutrient storage and dauer formation in C. elegans, providing a unique genetic model for examining the role of O-GlcNAc in cellular signaling and insulin resistance.