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1.  Activation of the NOTCH pathway in Head and Neck Cancer 
Cancer research  2013;74(4):1091-1104.
NOTCH1 mutations have been reported to occur in 10 to 15% of head and neck squamous cell carcinomas (HNSCC). To determine the significance of these mutations, we embarked upon a comprehensive study of NOTCH signaling in a cohort of 44 HNSCC tumors and 25 normal mucosal samples through a set of expression, copy number, methylation and mutation analyses. Copy number increases were identified in NOTCH pathway genes including the NOTCH ligand JAG1. Gene set analysis defined a differential expression of the NOTCH signaling pathway in HNSCC relative to normal tissues. Analysis of individual pathway-related genes revealed overexpression of ligands JAG1 and JAG2 and receptor NOTCH3. In 32% of the HNSCC examined, activation of the downstream NOTCH effectors HES1/HEY1 was documented. Notably, exomic sequencing identified 5 novel inactivating NOTCH1 mutations in 4/37 of the tumors analyzed, with none of these tumors exhibiting HES1/HEY1 overexpression. Our results revealed a bimodal pattern of NOTCH pathway alterations in HNSCC, with a smaller subset exhibiting inactivating NOTCH1 receptors mutations but a larger subset exhibiting other NOTCH1 pathway alterations, including increases in expression or gene copy number of the receptor or ligands as well as downstream pathway activation. Our results imply that therapies that target the NOTCH pathway may be more widely suitable for HNSCC treatment than appreciated currently.
doi:10.1158/0008-5472.CAN-13-1259
PMCID: PMC3944644  PMID: 24351288
2.  Clusterin is a Gene Specific Target of MicroRNA-21 in Head and Neck Squamous Cell Carcinoma 
Purpose:
MicroRNA-21 (miRNA-21) has proto-oncogenic properties, though no miRNA-21 specific targets have been found in head and neck squamous cell carcinoma (HNSCC). Further study of miRNA-21 and its specific targets is essential to understanding HNSCC biology.
Experimental Design:
miRNA expression profiles of 10 HNSCC and 10 normal mucosa samples were investigated using a custom miRNA microarray. 13 HNSCC and 5 normal mucosa primary tissue specimens underwent mRNA expression microarray analysis. To identify miRNA-21 downstream targets, oral keratinocyte cells were subjected to microarray analysis after miRNA-21 transient transfection. miRNA and mRNA expression were validated by RT-qPCR in a separate cohort of 16 HNSCC and 15 normal mucosal samples. Microarray and bioinformatics analyses were integrated to identify potential gene targets. In vitro assays looked at the function and interaction of miRNA-21 and its specific gene targets.
Results:
miRNA-21 was upregulated in HNSCC and stimulated cell growth. Integrated analyses identified Clusterin (CLU) as a potential miRNA-21 gene target. CLU was downregulated after forced expression of miRNA-21 in normal and HNSCC cell lines. The activity of a luciferase construct containing the 3’UTR of CLU was repressed by the ectopic expression of miRNA-21. CLU was also downregulated in primary HNSCC and correlated with miRNA-21 over-expression. CLU variant 1 (CLU-1) was the predominant splice variant in HNSCC, and showed growth suppression function that was reversed by miRNA-21 over-expression.
Conclusions:
CLU is a specific, functional target of oncogenic miRNA-21 in HNSCC. CLU-1 isoform is the predominant growth suppressive variant targeted by miRNA-21.
doi:10.1158/1078-0432.CCR-13-2675
PMCID: PMC3970211  PMID: 24327270
Clusterin; microRNA-21; gene target; tumor-suppressor gene; head and neck cancer
3.  Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma 
Purpose
Myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) play a key role in the progression of head and neck squamous cell carcinoma (HNSCC). On the basis of our preclinical data demonstrating that phosphodiesterase-5 (PDE5) inhibition can modulate these cell populations, we evaluated whether the PDE5 inhibitor tadalafil can revert tumor-induced immunosuppression and promote tumor immunity in patients with HNSCC.
Experimental Design
First, we functionally and phenotypically characterized MDSCs in HNSCCs and determined, retrospectively, whether their presence at the tumor site correlates with recurrence. Then, we performed a prospective single-center, double-blinded, randomized, three-arm study in which patients with HNSCC undergoing definitive surgical resection of oral and oropharyngeal tumors were treated with tadalafil 10 μg/day, 20 μg/day, or placebo for at least 20 days preoperatively. Blood and tumor MDSC and Treg presence and CD8+ T-cell reactivity to tumor antigens were evaluated before and after treatment.
Results
MDSCs were characterized in HNSCC and their intratumoral presence significantly correlates with recurrence. Tadalafil treatment was well tolerated and significantly reduced both MDSCs and Treg concentrations in the blood and in the tumor (P < 0.05). In addition, the concentration of blood CD8+ T cells reactive to autologous tumor antigens significantly increased after treatment (P < 0.05). Tadalafil immunomodulatory activity was maximized at an intermediate dose but not at higher doses. Mechanistic analysis suggests a possible off-target effect on PDE11 at high dosages that, by increasing intracellular cAMP, may negatively affect antitumor immunity.
Conclusions
Tadalafil seems to beneficially modulate the tumor micro- and macro-environment in patients with HNSCC by lowering MDSCs and Tregs and increasing tumor-specific CD8+ T cells in a dose-dependent fashion.
doi:10.1158/1078-0432.CCR-14-1711
PMCID: PMC4322895  PMID: 25320361
4.  Saliva and Plasma Quantitative Polymerase Chain Reaction–Based Detection and Surveillance of Human Papillomavirus–Related Head and Neck Cancer 
IMPORTANCE
Human papillomavirus type 16 (HPV-16) is a major causative factor in oropharyngeal squamous cell carcinoma (OPSCC). The detection of primary OPSCC is often delayed owing to the challenging anatomy of the oropharynx.
OBJECTIVE
To investigate the feasibility of HPV-16 DNA detection in pretreatment and posttreatment plasma and saliva and its potential role as a marker of prognosis.
DESIGN, SETTING, AND PARTICIPANTS
This is a retrospective analysis of a prospectively collected cohort. Among a cohort of patients with oropharyngeal and unknown primary squamous cell carcinoma with known HPV-16 tumor status from the Johns Hopkins Medical Institutions and Greater Baltimore Medical Center (from 1999 through 2010), 93 patients were identified with a complete set of pretreatment and posttreatment plasma or saliva samples, of which 81 patients had HPV-16–positive tumors and 12 patients had HPV-16–negative tumors. Real-time quantitative polymerase chain reaction was used to detect HPV-16 E6 and E7 DNA in saliva and plasma samples.
MAIN OUTCOMES AND MEASURES
Main outcomes included sensitivity, specificity, negative predictive value of combined saliva and plasma pretreatment HPV-16 DNA status for detecting tumor HPV-16 status, as well as the association of posttreatment HPV DNA status with clinical outcomes, including recurrence-free survival and overall survival.
RESULTS
The median follow-up time was 49 months (range, 0.9–181.0 months). The sensitivity, specificity, negative predictive value, and positive predictive value of combined saliva and plasma pretreatment HPV-16 DNA status for detecting tumor HPV-16 status were 76%, 100%, 42%, and 100%, respectively. The sensitivities of pretreatment saliva or plasma alone were 52.8%and 67.3%, respectively. In a multivariable analysis, positive posttreatment saliva HPV status was associated with higher risk of recurrence (hazard ratio [HR], 10.7; 95% CI, 2.36–48.50) (P = .002). Overall survival was reduced among those with posttreatment HPV-positive status in saliva (HR, 25.9; 95% CI, 3.23–208.00) (P = .002) and those with HPV-positive status in either saliva or plasma but not among patients with HPV-positive status in plasma alone. The combined saliva and plasma posttreatment HPV-16 DNA status was 90.7%specific and 69.5%sensitive in predicting recurrence within 3 years.
CONCLUSIONS AND RELEVANCE
Using a combination of pretreatment plasma and saliva can increase the sensitivity of pretreatment HPV-16 status as a tool for screening patients with HPV-16–positive OPSCC. In addition, analysis of HPV-16 DNA in saliva and plasma after primary treatment may allow for early detection of recurrence in patients with HPV-16–positive OPSCC.
doi:10.1001/jamaoto.2014.1338
PMCID: PMC4313904  PMID: 25078109
5.  Evaluation of an integrated clinical workflow for targeted next-generation sequencing of low-quality tumor DNA using a 51-gene enrichment panel 
BMC Medical Genomics  2014;7(1):62.
Background
Improvements in both performance and cost for next-generation sequencing (NGS) have spurred its rapid adoption for clinical applications. We designed and optimized a pan-cancer target-enrichment panel for 51 well-established oncogenes and tumor suppressors, in conjunction with a bioinformatic pipeline informed by in-process controls and pre- and post-analytical quality control measures.
Methods
The evaluation of this workflow consisted of sequencing mixtures of intact DNA to establish analytical sensitivity and precision, utilization of heuristics to identify systematic artifacts, titration studies of intact and FFPE samples for input optimization, and incorporation of orthogonal sequencing strategies to increase both positive predictive value and variant detection. We also used 128 FFPE samples to assess clinical accuracy and incorporated the previously described quantitative functional index (QFI) for sample qualification as part of detailing complete system performance.
Results
We observed a concordance correlation coefficient of 0.99 between the observed versus expected percent variant at 250 ng input across 4 independent sequencing runs. A subset of the systematic variants were confirmed to be barely detectable on an independent sequencing platform (Wilcox signed-rank test p-value <10−16), and the incorporation of orthogonal sequencing strategies increased the harmonic mean of sensitivity and positive predictive value of mutation detection by 41%. In one cohort of FFPE tumor samples, coverage and inter-platform concordance were positively correlated with the QFI, emphasizing the need for pre-analytical sample quality control to reduce the risk of false positives and negatives. In a separate cohort of FFPE samples, the 51-gene panel achieved 78% sensitivity (95% CI = 56.3, 92.5) with 100% PPV (95% CI = 81.5, 100.0) based on known mutations at 7.9% median abundance. By sequencing specimens using an orthogonal NGS technology, sensitivity was improved to 87.0% (95% CI = 66.4,97.2) while maintaining PPV.
Conclusions
The results highlight the value of process integration in a comprehensive targeted NGS system, enabling both discovery and diagnostic applications, particularly when sequencing low-quality cancer specimens.
Electronic supplementary material
The online version of this article (doi:10.1186/s12920-014-0062-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12920-014-0062-0
PMCID: PMC4241214  PMID: 25395014
6.  EDNRB and DCC salivary rinse hypermethylation has a similar performance as expert clinical examination in discrimination of oral cancer/dysplasia vs. benign lesions 
Purpose
Promoter hypermethylation has been recently proposed as a mean for HNSCC detection in salivary rinses. In a prospective study of a high-risk population, we showed that EDNRB promoter methylation in salivary rinses is a useful biomarker for oral cancer and premalignancy.
Experimental Design
Using that cohort, we evaluated EDNRB methylation status and 8 additional genes. Clinical risk assessment by expert clinicians was performed and compared with biomarker performance in the prediction of premalignant and malignant disease. Methylation status of 9 genes was analyzed in salivary rinses of 191 patients by Quantitative methylation-specific PCR.
Results
HOXA9, EDNRB and DCC methylation were associated (p= 0.012; p<0.0001; p=0.0005) with premalignant or malignant disease. On multivariable modeling, histological diagnosis was only independently associated with EDNRB (p=0.0003) or DCC (p=0.004) methylation. A subset of patients received clinical risk classification (CRC) by expert clinicians based on lesion examination. CRC, DCC and EDNRB were associated with diagnosis of dysplasia/cancer on univariate (p=0.008; p=0.026; p=0.046) and multivariate analysis (p=0.012; p=0.037; p=0.047). CRC identified dysplasia/cancer with56% of sensitivity and 66% of specificity with a similar AUC (0.61, 95%CI=0.60-0.81) when compared to EDNRB and DCC combined AUC (0.60, 95%CI=0.51-0.69), sensitivity of 46% and specificity of 72%. A combination of EDNRB, DCC and CRC was optimal AUC (0.67, 95%CI=0.58-0.76).
Conclusion
EDNRB and/or DCC methylation in salivary rinses compares well to examination by an expert clinician in CRC of oral lesions. These salivary biomarkers may be particularly useful in oral premalignancy and malignancy screening in clinical care settings in which expert clinicians are not available
doi:10.1158/1078-0432.CCR-12-3496
PMCID: PMC3687013  PMID: 23637120
HNSCC; hypermethylation; saliva; DCC; EDNRB
7.  The Gastrointestinal Manifestations of Telomere-Mediated Disease 
Aging cell  2013;12(2):319-323.
Summary
Defects in telomere maintenance genes cause pathological telomere shortening, and manifest in syndromes which have prominent phenotypes in tissues of high turnover: the skin and bone marrow. Because the gastrointestinal (GI) epithelium has rapid turnover, we sought to determine whether telomere syndromes cause GI disease, and to define its prevalence, spectrum and natural history. We queried subjects in the Johns Hopkins Telomere Syndrome Registry for evidence of luminal GI disease. In sixteen percent of Registry subjects (6 of 38), there was a history of significant GI pathology, and 43 additional cases were identified in the literature. Esophageal stenosis, enteropathy and enterocolitis were the recurrent findings. In the intestinal mucosa, there was striking villous atrophy, extensive apoptosis, and anaphase bridging pointing to regenerative defects in the epithelial compartment. GI disease was often the first and most severe manifestation of telomere disease in young children. These findings indicate that telomere dysfunction disrupts the epithelial integrity in the human GI tract manifesting in recognizable disease processes. A high index of suspicion should facilitate diagnosis and management.
doi:10.1111/acel.12041
PMCID: PMC3602337  PMID: 23279657
telomere; telomerase; enterocolitis; immunodeficiency
8.  Expression Microarray Analysis Reveals Alternative Splicing of LAMA3 and DST Genes in Head and Neck Squamous Cell Carcinoma 
PLoS ONE  2014;9(3):e91263.
Purpose
Prior studies have demonstrated tumor-specific alternative splicing events in various solid tumor types. The role of alternative splicing in the development and progression of head and neck squamous cell carcinoma (HNSCC) is unclear. Our study queried exon-level expression to implicate splice variants in HNSCC tumors.
Experimental Design
We performed a comparative genome-wide analysis of 44 HNSCC tumors and 25 uvulopalatopharyngoplasty (UPPP) tissue samples at an exon expression level. In our comparison we ranked genes based upon a novel score—the Maximum-Minimum Exon Score (MMES) – designed to predict the likelihood of an alternative splicing event occurring. We validated predicted alternative splicing events using quantitative RT-PCR on an independent cohort.
Results
After MMES scoring of 17,422 genes, the top 900 genes with the highest scores underwent additional manual inspection of expression patterns in a graphical analysis. The genes LAMA3, DST, VEGFC, SDHA, RASIP1, and TP63 were selected for further validation studies because of a high frequency of alternative splicing suggested in our graphical analysis, and literature review showing their biological relevance and known splicing patterns. We confirmed TP63 as having dominant expression of the short DeltaNp63 isoform in HNSCC tumor samples, consistent with prior reports. Two of the six genes (LAMA3 and DST) validated by quantitative RT-PCR for tumor-specific alternative splicing events (Student's t test, P<0.001).
Conclusion
Alternative splicing events of oncologically relevant proteins occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression.
doi:10.1371/journal.pone.0091263
PMCID: PMC3967989  PMID: 24675808
9.  Novel Insight into Mutational Landscape of Head and Neck Squamous Cell Carcinoma 
PLoS ONE  2014;9(3):e93102.
Development of head and neck squamous cell carcinoma (HNSCC) is characterized by accumulation of mutations in several oncogenes and tumor suppressor genes. We have formerly described the mutation pattern of HNSCC and described NOTCH signaling pathway alterations. Given the complexity of the HNSCC, here we extend the previous study to understand the overall HNSCC mutation context and to discover additional genetic alterations. We performed high depth targeted exon sequencing of 51 highly actionable cancer-related genes with a high frequency of mutation across many cancer types, including head and neck. DNA from primary tumor tissues and matched normal tissues was analyzed for 37 HNSCC patients. We identified 26 non-synonymous or stop-gained mutations targeting 11 of 51 selected genes. These genes were mutated in 17 out of 37 (46%) studied HNSCC patients. Smokers harbored 3.2-fold more mutations than non-smokers. Importantly, TP53 was mutated in 30%, NOTCH1 in 8% and FGFR3 in 5% of HNSCC. HPV negative patients harbored 4-fold more TP53 mutations than HPV positive patients. These data confirm prior reports of the HNSCC mutational profile. Additionally, we detected mutations in two new genes, CEBPA and FES, which have not been previously reported in HNSCC. These data extend the spectrum of HNSCC mutations and define novel mutation targets in HNSCC carcinogenesis, especially for smokers and HNSCC without HPV infection.
doi:10.1371/journal.pone.0093102
PMCID: PMC3965530  PMID: 24667986
10.  Transcervical Ultrasonography Is Feasible to Visualize and Evaluate Base of Tongue Cancers 
PLoS ONE  2014;9(1):e87565.
Background
Base of tongue (BOT) is a difficult subsite to examine clinically and radiographically. Yet, anatomic delineation of the primary tumor site, its extension to adjacent sites or across midline, and endophytic vs. exophytic extent are important characteristics for staging and treatment planning. We hypothesized that ultrasound could be used to visualize and describe BOT tumors.
Methods
Transcervical ultrasound was performed using a standardized protocol in cases and controls. Cases had suspected or confirmed BOT malignancy. Controls were healthy individuals without known malignancy.
Results
100% of BOT tumors were visualized. On ultrasound BOT tumors were hypoechoic (90.9%) with irregular margins (95.5%). Ultrasound could be used to characterize adjacent site involvement, midline extent, and endophytic extent, and visualize the lingual artery. No tumors were suspected for controls.
Conclusions
Ultrasonography can be used to transcervically visualize BOT tumors and provides clinically relevant characteristics that may not otherwise be appreciable.
doi:10.1371/journal.pone.0087565
PMCID: PMC3907536  PMID: 24498138
11.  Validation of nucleolar protein 4 as a novel methylated tumor suppressor gene in head and neck cancer 
Oncology Reports  2013;31(2):1014-1020.
Methylation of CpG islands in the promoter region of genes acts as a significant mechanism of epigenetic gene silencing in head and neck cancer. In the present study, we assessed the association of epigenetic alterations of a panel of 12 genes [nucleolar protein 4 (NOL4), iroquois homeobox 1 (IRX1), SLC5A8, LRRC3B, FUSSEL18, EBF3, GBX2, HMX2, SEPT9, ALX3, SOCS3 and LHX6] with head and neck squamous cell carcinoma (HNSCC) via a candidate gene approach. After the initial screening of methylated CpG islands on the promoter regions by bisulfite sequencing using salivary rinse samples, only two genes had methylated CpG dinucleotides on their promoter regions in tumor samples and absence of methylated CpGs were found in normal salivary rinse samples after bisulfite modification and bisulfite sequencing. We then performed real-time quantitative methylation-specific PCR (QMSP) on 16 salivary rinse and 14 normal mucosal samples from healthy subjects and 33 HNSCC tumor samples for the two genes selected. After validation with QMSP, one gene, NOL4, was highly methylated (91%) in tumor samples and unmethylated in normal salivary rinses and minimally methylated in normal mucosal samples demonstrating cancer-specific methylation in HNSCC tissues. Although the IRX1 gene was observed as methylated in normal mucosal and salivary rinse samples, the methylation values of these normal samples were very low (<10%). In conclusion, we identified NOL4 as a highly specific promoter methylated gene associated with HNSCC. IRX1 may have potential as a biomarker for HNSCC and should be assessed in a larger cohort.
doi:10.3892/or.2013.2927
PMCID: PMC3896520  PMID: 24337411
nucleolar protein 4; methylation; head and neck cancer; candidate gene approach
12.  Preferential Activation of the Hedgehog Pathway by Epigenetic Modulations in HPV Negative HNSCC Identified with Meta-Pathway Analysis 
PLoS ONE  2013;8(11):e78127.
Head and neck squamous cell carcinoma (HNSCC) is largely divided into two groups based on their etiology, human papillomavirus (HPV)-positive and –negative. Global DNA methylation changes are known to drive oncogene and tumor suppressor expression in primary HNSCC of both types. However, significant heterogeneity in DNA methylation within the groups results in different transcriptional profiles and clinical outcomes. We applied a meta-pathway analysis to link gene expression changes to DNA methylation in distinguishing HNSCC subtypes. This approach isolated specific epigenetic changes controlling expression in HPV− HNSCC that distinguish it from HPV+ HNSCC. Analysis of genes identified Hedgehog pathway activation specific to HPV− HNSCC. We confirmed that GLI1, the primary Hedgehog target, showed higher expression in tumors compared to normal samples with HPV− tumors having the highest GLI1 expression, suggesting that increased expression of GLI1 is a potential driver in HPV− HNSCC. Our algorithm for integration of DNA methylation and gene expression can infer biologically significant molecular pathways that may be exploited as therapeutics targets. Our results suggest that therapeutics targeting the Hedgehog pathway may be of benefit in HPV− HNSCC. Similar integrative analysis of high-throughput coupled DNA methylation and expression datasets may yield novel insights into deregulated pathways in other cancers.
doi:10.1371/journal.pone.0078127
PMCID: PMC3817178  PMID: 24223768
13.  Tissue Imprint for Molecular Mapping of Deep Surgical Margins in Patients with Head and Neck Squamous Cell Carcinoma 
Head & neck  2012;34(11):1529-1536.
Background
Tissue imprinting can generate molecular marker maps of tumor cells at deep surgical margins. This study evaluates the feasibility of this method for detection of residual head and neck squamous cell carcinoma (HNSCC).
Methods
Paired fresh tissue and nitrocellulose membrane imprints of tumor and deep margins were collected from 17 HNSCC resections. DNA was amplified using quantitative methylation-specific PCR (qMSP) for p16, DCC, KIF1A, and EDNRB. Levels of methylation in tumors and deep margins were compared.
Results
DNA from imprints was adequate for qMSP. Hypermethylation of target genes was present in 12/17 tumors and in 8 deep margins. Methylation level was better from margin imprints than tissue. During follow-up (median 13 months), local or regional recurrences occurred in six cases of which five had molecularly positive margins.
Conclusion
Tissue imprinting is feasible for molecular detection of residual tumor at deep surgical margins and may correlate with locoregional recurrence.
doi:10.1002/hed.21982
PMCID: PMC3377010  PMID: 22223471
Tissue imprint; head and neck squamous cell carcinoma; deep surgical margin; molecular diagnosis; quantitative methylation-specific PCR; Imprint; Deep margin; Surgery; Recurrence
14.  Comparison of Acute Toxicities in Two Primary Chemoradiation Regimens in the Treatment of Advanced Head and Neck Squamous Cell Carcinoma 
Annals of surgical oncology  2012;19(6):1980-1987.
Purpose
The optimal dosage and frequency of platinum-based chemoradiotherapy (CRT) regimen for treating advanced head and neck squamous cell carcinoma remains unresolved. This study aims to compare the toxicity and efficacy of weekly versus more dose-intensive cisplatin-based CRTs.
Methods
We reviewed 155 stage III/IV head and neck squamous cell carcinoma patients with no evidence of distant metastasis treated with one of two CRT regimens from 2000 to 2010 at Greater Baltimore Medical Center. Twice-daily radiation was provided as a split course over a 45-day period. Regimen A consisted of concomitant cisplatin (30 mg/m2/1 h) weekly for 6 cycles; regimen B consisted of concomitant cisplatin (12 mg/m2/1 h) and 5-fluorouracil (600 mg/m2/20 h) on days 1 through 5 and days 29 through 33. Main outcome measures included acute toxicities (myelosuppression, neurotoxicity, nephrotoxicity, gastrointestinal dysfunction), unplanned hospitalizations, and disease control at 12 months.
Results
Patients on regimen A were much less likely to experience ototoxicity due to their treatment (0% vs. 9.8%, P = 0.04). They were more likely to experience thrombocytopenia acutely (46% vs. 26%, P = 0.02), but the toxicity was not limiting (grade 1–2). No significant differences exist in the incidence of other toxicities or unplanned hospitalizations. At 1 year, 97% of patients on A vs. 86% of patients on regimen B were free of disease (P = 0.11).
Conclusions
With concurrent radiotherapy, low-dose, single-agent, weekly cisplatin is less likely than higher-dose daily cisplatin plus 5-fluorouracil provided at the beginning and end of treatment to be associated with ototoxicity. The preliminary data suggest at least equivalent efficacy, but longer follow-up is required.
doi:10.1245/s10434-012-2219-4
PMCID: PMC3715048  PMID: 22290566
15.  Neck Dissection Through a Facelift Incision 
The Laryngoscope  2012;122(12):2700-2706.
Obectives/Hypothesis
To determine the feasibility and safety of neck dissection through a facelift incision.
Study Design
Prospective case series.
Methods
Cadavers and live subjects underwent neck dissection using a facelift incision with and without endoscopic assistance. In the live facelift neck dissection (FLND), the preoperative surgical indications, staging, adjuvant therapy, intraoperative technical procedure, pathology reports on lymph nodes, and short-term outcomes were reviewed.
Results
FLND was successfully performed in four cadavers and four live subjects, including selective (less than five neck levels removed) and comprehensive (levels I–V removed) neck dissections. All levels were accessible through this approach, with additional retraction required for levels I and IV. Endoscopic assistance was required in one neck dissection for adequate visualization. Short-term complications and number of excised lymph nodes were comparable to those from traditional neck dissection approaches.
Conclusions
Open neck dissection through a facelift incision is feasible and offers an alternate approach to traditional incisions. This can be performed without requiring robotic assistance and with endoscopic assistance only in certain cases. Endoscopic assistance can offer enhanced visualization of the surgical field and complement open direct approaches in neck dissection. Although FLND offers improved cosmetic outcomes when compared to those of traditional neck incisions, further study is required to determine its efficacy and indications.
doi:10.1002/lary.23386
PMCID: PMC3715054  PMID: 23023877
Facelift; neck dissection; cosmetic; radical; modified; selective; comprehensive; approach; rhytidectomy; minimally invasive
16.  Age-Related Intimal Stiffening Enhances Endothelial Permeability and Leukocyte Transmigration 
Science translational medicine  2011;3(112):112ra122.
Age is the most significant risk factor for atherosclerosis; however, the link between age and atherosclerosis is poorly understood. During both aging and atherosclerosis progression, the blood vessel wall stiffens owing to alterations in the extracellular matrix. Using in vitro and ex vivo models of vessel-wall stiffness and aging, we show that stiffening of extracellular matrix within the intima promotes endothelial cell permeability—a hallmark of atherogenesis. When cultured on hydrogels fabricated to match the elasticity of young and aging intima, endothelial monolayers exhibit increased permeability and disrupted cell-cell junctions on stiffer matrices. In parallel experiments, we showed a corresponding increase in cell-cell junction width with age in ex vivo aortas from young (10 weeks) and old (21 to 25 months) healthy mice. To investigate the mechanism by which matrix stiffening alters monolayer integrity, we found that cell contractility increases with increased matrix stiffness, mechanically destabilizing cell-cell junctions. This increase in endothelial permeability results in increased leukocyte extravasation, which is a critical step in atherosclerotic plaque formation. Mild inhibition of Rho-dependent cell contractility using Y-27632, an inhibitor of Rho-associated kinase, or siRNA restored monolayer integrity in vitro and in vivo. Our results suggest that extracellular matrix stiffening alone, which occurs during aging, can lead to endothelial monolayer disruption and atherosclerosis pathogenesis. Because previous therapeutics designed to decrease vascular stiffness have been met with limited success, our findings could be the basis for the design of therapeutics that target the Rho-dependent cellular contractile response to matrix stiffening, rather than stiffness itself, to more effectively prevent atherosclerosis progression.
doi:10.1126/scitranslmed.3002761
PMCID: PMC3693751  PMID: 22158860
17.  Detection of TIMP3 promoter hypermethylation in salivary rinse as an independent predictor of local recurrence-free survival in head and neck cancer 
Clinical Cancer Research  2012;18(4):1082-1091.
Purpose
To validate a panel of methylation-based salivary rinse biomarkers (P16, CCNA1, DCC, TIMP3, MGMT, DAPK, and MINT31) previously shown to be independently associated with poor overall survival and local recurrence in a larger, separate cohort of patients with head and neck squamous cell carcinoma (HNSCC).
Experimental Design
One hundred ninety-seven patients were included. All pre-treatment saliva DNA samples were evaluated for the methylation status of the gene promoters by quantitative methylation-specific PCR. The main outcome measures were overall survival, local recurrence-free survival and disease-free survival.
Results
In univariate analyses, the detection of hypermethylation of CCNA1, MGMT, and MINT31 was significantly associated with poor overall survival; the detection of hypermethylation of TIMP3 was significantly associated with local recurrence-free survival; and the detection of hypermethylation of MINT31 was significantly associated with poor disease-free survival. In multivariate analyses, detection of hypermethylation at any single marker was not predictive of overall survival in patients with HNSCC; detection of hypermethylation of TIMP3 in salivary rinse had an independent, significant association with local recurrence-free survival (Hazard Ratio, 2.51, 95% CI, 1.10 to 5.68); and none of the studied markers was significantly associated with disease-free survival.
Conclusion
The detection of promoter hypermethylation of the seven genes in salivary rinse as an independent prognostic indicator of overall survival in patients with HNSCC was not validated. Detection of promoter hypermethylation of TIMP3 in pretreatment salivary rinse is independently associated with local recurrence-free survival in patients with HNSCC and may be a valuable salivary rinse biomarker for HNSCC recurrence.
doi:10.1158/1078-0432.CCR-11-2392
PMCID: PMC3288549  PMID: 22228635
18.  Identification of guanine nucleotide-binding protein γ-7 as an epigenetically silenced gene in head and neck cancer by gene expression profiling 
International Journal of Oncology  2013;42(4):1427-1436.
Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. Aberrant hypermethylation in the promoter region of some known or putative tumor suppressor genes occurs frequently during the development of various types of cancer including head and neck squamous cell carcinoma (HNSCC). In this study we used an expanded mRNA expression profiling approach followed by microarray expression analysis to identify epigenetically inactivated genes in HNSCC. Two HNSCC cell lines were treated with 5-aza-2′-deoxycytidine followed by microarray analysis to identify epigenetically silenced genes in HNSCC. We found 1,960, 614 and 427 genes were upregulated in the HNSCC cell lines JHU-012, JHU-011 and the combination of both cell lines, respectively. HNSCC tumor and normal mucosal samples were used for gene profiling by a 47K mRNA gene expression array and we found 7,140 genes were downregulated in HNSCC tumors compared to normal mucosa, as determined by microarray analysis, and were integrated with cell line data. Integrative analysis defined 126 candidate genes, of which only seven genes showed differential methylation in tumors and no methylation in normal mucosa after bisulfite sequencing. Following validation by QMSP, one gene, guanine nucleotide-binding protein γ-7 (GNG7), was confirmed to be highly methylated in tumors and unmethylated in normal mucosal and salivary rinse samples demonstrating cancer-specific methylation in HNSCC tissues. TXNIP and TUSC2 were partially methylated in tumors and normal salivary rinses but unmethylated in normal mucosa. We concluded that GNG7 is a highly specific promoter methylated gene associated with HNSCC. In addition, TXNIP and TUSC2 are also potential biomarkers for HNSCC.
doi:10.3892/ijo.2013.1808
PMCID: PMC3981008  PMID: 23403885
guanine nucleotide-binding protein γ-7; gene expression; silencing; head and neck squamous cell carcinoma; epigenetics
19.  Suprabasin Is Hypomethylated and Associated with Metastasis in Salivary Adenoid Cystic Carcinoma 
PLoS ONE  2012;7(11):e48582.
Background
Salivary gland adenoid cystic carcinoma (ACC) is a rare cancer, accounting for only 1% of all head and neck malignancies. ACC is well known for perineural invasion and distant metastasis, but its underlying molecular mechanisms of carcinogenesis are still unclear.
Principal Findings
Here, we show that a novel oncogenic candidate, suprabasin (SBSN), plays important roles in maintaining the anchorage-independent and anchorage-dependent cell proliferation in ACC by using SBSN shRNA stably transfected ACC cell line clones. SBSN is also important in maintaining the invasive/metastatic capability in ACC by Matrigel invasion assay. More interestingly, SBSN transcription is significantly upregulated by DNA demethylation induced by 5-aza-2′-deoxycytidine plus trichostatin A treatment and the DNA methylation levels of the SBSN CpG island located in the second intron were validated to be significantly hypomethylated in primary ACC samples versus normal salivary gland tissues.
Conclusions/Significance
Taken together, these results support SBSN as novel oncogene candidate in ACC, and the methylation changes could be a promising biomarker for ACC.
doi:10.1371/journal.pone.0048582
PMCID: PMC3492451  PMID: 23144906
20.  Multidisciplinary Service Utilization Pattern by Advanced Head and Neck Cancer Patients: A Single Institution Study 
Purpose. To analyze the patterns and associations of adjunctive service visits by head and neck cancer patients receiving primary, concurrent chemoradiation therapy. Methods. Retrospective chart review of patients receiving adjunctive support during a uniform chemoradiation regimen for stages III-IV head and neck squamous cell carcinoma. Univariate and multivariate models for each outcome were obtained from simple and multivariate linear regression analyses. Results. Fifty-two consecutive patients were assessed. Female gender, single marital status, and nonprivate insurance were factors associated with an increased number of social work visits. In a multivariate analysis, female gender and marital status were related to increased social work services. Female gender and stage IV disease were significant for increased nursing visits. In a multivariate analysis for nursing visits, living greater than 20 miles between home and hospital was a negative predictive factor. Conclusion. Treatment of advanced stage head and neck cancer with concurrent chemoradiation warrants a multidisciplinary approach. Female gender, single marital status, and stage IV disease were correlated with increased utilization of social work and nursing services. Distance over 20 miles from the center was a negative factor. This information may help guide the treatment team to allocate resources for the comprehensive care of patients.
doi:10.1155/2012/628578
PMCID: PMC3483826  PMID: 23118755
21.  MAGEB2 is Activated by Promoter Demethylation in Head and Neck Squamous Cell Carcinoma 
PLoS ONE  2012;7(9):e45534.
Purpose
Although promoter hypermethylation has been an accepted means of tumor suppressor gene inactivation, activation of otherwise normally repressed proto-oncogenes by promoter demethylation has been infrequently documented.
Experimental Design
In this study we performed an integrative, whole-genome analysis for discovery of epigenetically activated proto-oncogenes in head and neck cancer tumors. We used the 47K GeneChip U133 Plus 2.0 Affymetrix expression microarray platform to obtain re-expression data from 5-aza treated normal cell line and expression data from primary head and neck squamous cell carcinoma (HNSCC) tumor tissues and normal mucosa tissues. We then investigated candidate genes by screening promoter regions for CpG islands and bisulfite sequencing followed by QUMSP and RT PCR for the best candidate genes. Finally, functional studies were performed on the top candidate gene.
Results
From the top 178 screened candidates 96 had CpG islands in their promoter region. Seven candidate genes showed promoter region methylation in normal mucosa samples and promoter demethylation in a small cohort of primary HNSCC tissues. We then studied the demethylation of the top 3 candidate genes in an expanded cohort of 76 HNSCC tissue samples and 17 normal mucosa samples. We identified MAGEB2 as having significant promoter demethylation in primary head and neck squamous cell carcinoma tissues. We then found significantly higher expression of MAGEB2 in tumors in a separate cohort of 73 primary HNSCC tissues and 31 normal tissues. Finally, we found that MAGEB2 has growth promoting effects on minimally transformed oral keratinocyte cell lines but not a definite effect on HNSCC cell lines.
Conclusion
In conclusion, we identified MAGEB2 as activated by promoter demethylation in HNSCCand demonstrates growth promoting effects in a minimally transformed oral keratinocyte cell line. More studies are needed to evaluate MAGBE2's exact role in HNSCC.
doi:10.1371/journal.pone.0045534
PMCID: PMC3454438  PMID: 23029077
22.  Use of integrative epigenetic and cytogenetic analyses to identify novel tumor suppressor genes in malignant melanoma 
Melanoma research  2011;21(4):298-307.
Purpose
Identify novel tumor suppressor genes in melanoma utilizing an integrative genomic approach
Methods
Data from: 1) prior reports of DNA loss and gain in malignant melanoma accompanied by CGH high-definition array data of the entire human genome, 2) Microarray expression data from melanoma derived cell lines identifying genes with significantly increased expression due to methylation using a pharmacologic demethylating strategy, and 3) publicly available RNA expression microarray data of primary tumors and benign nevi was integrated utilizing statistical tools in order to define a population of candidate tumor suppressor genes.
Results
27 genes were identified in areas of deletion that demonstrated diminished expression in primary melanomas relative to benign nevi and were significantly increased in expression by ‘5-Aza treatment. Seven genes of these genes demonstrated methylation and deletion in a validation cohort of 14 separate primary tumors. These were: CHRDL1, SFRP1, TMEM47, LPL, RARRES1, PLCXD1, and KOX15. All of these genes demonstrated growth suppressive properties with transfection into melanoma derived cell lines.
Conclusions
7 putative tumor suppressor genes in malignant melanoma were identified utilizing a novel integrative technique.
doi:10.1097/CMR.0b013e328344a003
PMCID: PMC3131489  PMID: 21606880
DNA hypermethylation; epigenetics; malignant melanoma; tumor suppressor
23.  Detection of Promoter Hypermethylation in Salivary Rinses as a Biomarker for Head and Neck Squamous Cell Carcinoma Surveillance 
Purpose
Hypermethylation of tumor suppressor gene promoters has been found in head and neck squamous carcinoma (HNSCC) and other solid tumors. We evaluated these alterations in pretreatment salivary rinses from HNSCC patients by using real-time quantitative methylation-specific PCR (Q-MSP).
Experimental Design
Pretreatment saliva DNA samples from HNSCC patients were evaluated for patterns of hypermethylation by using Q-MSP. Target tumor suppressor gene promoter regions were selected based on a previous study describing a screening panel for HNSCC in a high-risk population subjects. The selected genes were: DAPK, DCC, MINT-31, TIMP-3, p16, MGMT, CCNA1.
Results
We analyzed the panel in a cohort of 61 HNSCC patients. Thirty-three of the analyzed patients (54.1%) showed methylation of at least one of the selected genes in the saliva DNA. Pretreatment methylated saliva DNA was not significantly associated with tumor site (P = 0.209) nor clinical stage (P = 0.299). However, local disease control and overall survival were significantly lower in patients presenting hypermethylation in saliva rinses (P = 0.010 and P = 0.015, respectively). Multivariate analysis confirmed that this hypermethylation pattern remained as an independent prognostic factor for local recurrence (HR = 12.2; 95% CI = 1.8–80.6; P = 0.010) and overall survival (HR = 2.8; 95% CI = 1.2–6.5; P = 0.016).
Conclusions
We were able to confirm an elevated rate of promoter hypermethylation in HNSCC saliva of patients by using a panel of gene promoters previously described as methylated specifically in HNSCC. Detection of hypermethylation in pretreatment saliva DNA seems to be predictive of local recurrence and overall survival. This finding has potential to influence treatment and surveillance of HNSCC patients.
doi:10.1158/1078-0432.CCR-11-0324
PMCID: PMC3215270  PMID: 21628494
24.  Dose-Dependent Activation of Putative Oncogene SBSN by BORIS 
PLoS ONE  2012;7(7):e40389.
Testis-specific transcription factor BORIS (Brother of the Regulator of Imprinted Sites), a paralog and proposed functional antagonist of the widely expressed CTCF, is abnormally expressed in multiple tumor types and has been implicated in the epigenetic activation of cancer-testis antigens (CTAs). We have reported previously that suprabasin (SBSN), whose expression is restricted to the epidermis, is epigenetically derepressed in lung cancer. In this work, we establish that SBSN is a novel non-CTA target of BORIS epigenetic regulation. With the use of a doxycycline-inducible BORIS expressing vector, we demonstrate that relative BORIS dosage is critical for SBSN activation. At lower concentrations, BORIS induces demethylation of the SBSN CpG island and disruption and activation of chromatin around the SBSN transcription start site (TSS), resulting in a 35-fold increase in SBSN expression in the H358 human lung cancer cell line. Interestingly, increasing BORIS concentrations leads to a subsequent reduction in SBSN expression via chromatin repression. In a similar manner, increase in BORIS concentrations leads to eventual decrease of cell growth and colony formation. This is the first report demonstrating that different amount of BORIS defines its varied effects on the expression of a target gene via chromatin structure reorganization.
doi:10.1371/journal.pone.0040389
PMCID: PMC3390376  PMID: 22792300
25.  Enforced Expression of miR-101 Inhibits Prostate Cancer Cell Growth by Modulating the COX-2 Pathway In Vivo 
It is commonly agreed that there is an association of chronic inflammation with tumorigenesis. COX-2, a key regulator of inflammation-producing prostaglandins, promotes cell proliferation and growth; thus, overexpression of COX-2 is often found in tumor tissues. Therefore, a better understanding of the regulatory mechanism(s) of COX-2 could lead to novel targeted cancer therapies. In this study, we investigated the mechanism of microRNA-101 (miR-101)-regulated COX-2 expression and the therapeutic potential of exogenous miR-101 for COX-2-associated cancer. A stably expressing exogenous miR-101 prostate cancer cell line (BPH1CmiR101) was generated by using lentiviral transduction as a tool for in vitro and in vivo studies. We found that miR-101 inhibited COX-2 posttranscriptional expression by directly binding to the 3′-untranslated region (3′-UTR) of COX-2 mRNA. The regulatory function of miR-101 was also confirmed by using antisense DNA. As a result, exogenous miR-101 is able to effectively suppress the growth of cultured prostate cancer cells and prostate tumor xenografts. The average tumor weight was significantly lower in the BPH1CmiR101 group (0.22 g) than the BPH1Cvec group (0.46 g). Expression levels of the cell growth regulators, such as cyclin proteins, PCNA (proliferating cell nuclear antigen), EGFR (epidermal growth factor receptor), were also studied. In conclusion, COX-2 is a direct target in miR-101 regulation of posttranscription. Exogenous miR-101 suppresses the proliferation and growth of prostate cancer cells in vitro and in vivo. These data suggest that exogenous miR-101 may provide a new cancer therapy by directly inhibiting COX-2 expression.
doi:10.1158/1940-6207.CAPR-10-0333
PMCID: PMC3305792  PMID: 21430074

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