Uttam K. Sinha

Salivary Transcriptome Diagnostics for Oral Cancer Detection

Purpose: Oral fluid (saliva) meets the demand for noninvasive, accessible, and highly efficient diagnostic medium. Recent discovery that a large panel of human RNA can be reliably detected in saliva gives rise to a novel clinical approach, salivary transcriptome diagnostics. The purpose of this study is to evaluate the diagnostic value of this new approach by using oral squamous cell carcinoma (OSCC) as the proof-of-principle disease. Experimental Design: Unstimulated saliva was collected from patients (n = 32) with primary T1/T2 OSCC and normal subjects (n = 32) with matched age, gender, and smoking history. RNA isolation was done from the saliva supernatant, followed by two-round linear amplification with T7 RNA polymerase. Human Genome U133A microarrays were applied for profiling human salivary transcriptome. The different gene expression patterns were analyzed by combining a t test comparison and a fold-change analysis on 10 matched cancer patients and controls. Quantitative polymerase chain reaction (qPCR) was used to validate the selected genes that showed significant difference (P < 0.01) by microarray. The predictive power of these salivary mRNA biomarkers was analyzed by receiver operating characteristic curve and classification models. Results: Microarray analysis showed there are 1,679 genes exhibited significantly different expression level in saliva between cancer patients and controls (P < 0.05). Seven cancer-related mRNA biomarkers that exhibited at least a 3.5-fold elevation in OSCC saliva (P < 0.01) were consistently validated by qPCR on saliva samples from OSCC patients (n = 32) and controls (n = 32). These potential salivary RNA biomarkers are transcripts of IL8, IL1B, DUSP1, HA3, OAZ1, S100P, and SAT. The combinations of these biomarkers yielded sensitivity (91%) and specificity (91%) in distinguishing OSCC from the controls. Conclusions: The utility of salivary transcriptome diagnostics is successfully demonstrated in this study for oral cancer detection. This novel clinical approach could be exploited to a robust, high-throughput, and reproducible tool for early cancer detection. Salivary transcriptome profiling can be applied to evaluate its usefulness for other major disease applications as well as for normal health surveillance.

Effects of steel scalpel, ultrasonic scalpel, CO2 laser, and monopolar and bipolar electrosurgery on wound healing in guinea pig oral mucosa.

Objective The studys objective was to compare instrument performance and tissue healing when steel scalpel, ultrasonic scalpel, monopolar or bipolar electrosurgical instruments, or CO2 laser was used in an animal oral surgery model.

Serum Circulating Human mRNA Profiling and Its Utility for Oral Cancer Detection

PURPOSE The purpose of this study is to explore the presence of informative RNA biomarkers from human serum transcriptome, and evaluate the serum transcriptome diagnostics for disease detection. Oral squamous cell carcinoma (OSCC) was selected as the proof-of-concept disease. PATIENTS AND METHODS Blood samples were collected from patients (n = 32) with primary T1/T2 OSCC and matched healthy patients (n = 35). Circulating RNA was isolated from serum and linearly amplified using T7 polymerase. Microarrays were applied for profiling transcriptome in serum from 10 cancer patients and controls. The differential gene expression was analyzed by combining the present calls, t tests, and fold-change statistics. Quantitative polymerase chain reaction (PCR) was used to validate the selected candidate RNA markers identified by microarray. Receiver operating characteristic curve and classification models were exploited to evaluate the diagnostic power of these markers for OSCC. RESULTS Human serum circulating mRNAs were presented by reverse transcriptase PCR. Microarray identified 2,623 +/- 868 probes assigned present calls in OSCC (n = 10) versus 1,792 +/- 165 in healthy patients (n = 10), indicating a higher complexity of serum transciptome in OSCC patients (P = .002, Wilcoxon test). Three hundred thirty-five serum RNAs exhibited significantly differential expression level between the two groups (P < .05, t test; fold > or = 2). Five cancer-related gene transcripts were consistently validated by quantitative PCR on serum from OSCC patients (n = 32) and controls (n = 35). The best combination of biomarkers yielded a receiver operating characteristic curve value of 88%, sensitivity (91%), and specificity (71%) in distinguishing OSCC. CONCLUSION The utility of serum transcriptome diagnostics is successfully demonstrated for OSCC detection. This novel concept could be developed as an adjunctive tool for disease diagnosis.

Auditory system degeneration in Alzheimer's disease

We observed a highly topographically specific and consistent pattern of degeneration in the auditory system of Alzheimers disease (AD) patients. Senile plaques (SP) and neurofibrillary tangles (NFT) were distributed throughout the ventral nucleus of the medial geniculate body (MGB) and the central nucleus of the inferior colliculus (IC) in nine of nine AD patients. Adjacent nuclei within the MGB and IC were consistently spared. NFT and SP were also present in the primary auditory and the auditory association cortices. In all control tissues, there were neither SP nor NFT in any of the above sites. The cochlear nuclei were normal in tissues from both AD and control patients. The ventral nucleus of the MGB is the major thalamic relay station for auditory function and receives fibers from neurons of the central nucleus of the IC, with projections arranged tonotopically in a laminar pattern corresponding to a gradient of high-to-low frequency ranges. The degenerative changes distributed throughout these nuclei suggest that neuronal loss may include all frequency ranges in AD. In contrast, the clinical features of presbycusis in elderly patients include only high-frequency loss due to lesions peripherally in the cochlea or auditory nerves, rather than in the central auditory nuclei. These histologic changes in the brains of AD patients may provide an additional basis for altered cognitive function due to primary sensory deafferentation.

Prevalidation of Salivary Biomarkers for Oral Cancer Detection

Background: Oral cancer is the sixth most common cancer with a 5-year survival rate of approximately 60%. Presently, there are no scientifically credible early detection techniques beyond conventional clinical oral examination. The goal of this study is to validate whether the seven mRNAs and three proteins previously reported as biomarkers are capable of discriminating patients with oral squamous cell carcinomas (OSCC) from healthy subjects in independent cohorts and by a National Cancer Institute (NCI)-Early Detection Research Network (EDRN)-Biomarker Reference Laboratory (BRL). Methods: Three hundred and ninety-five subjects from five independent cohorts based on case controlled design were investigated by two independent laboratories, University of California, Los Angeles (Los Angeles, CA) discovery laboratory and NCI-EDRN-BRL. Results: Expression of all seven mRNA and three protein markers was increased in OSCC versus controls in all five cohorts. With respect to individual marker performance across the five cohorts, the increase in interleukin (IL)-8 and subcutaneous adipose tissue (SAT) was statistically significant and they remained top performers across different cohorts in terms of sensitivity and specificity. A previously identified multiple marker model showed an area under the receiver operating characteristic (ROC) curve for prediction of OSCC status ranging from 0.74 to 0.86 across the cohorts. Conclusions: The validation of these biomarkers showed their feasibility in the discrimination of OSCCs from healthy controls. Established assay technologies are robust enough to perform independently. Individual cutoff values for each of these markers and for the combined predictive model need to be further defined in large clinical studies. Impact: Salivary proteomic and transcriptomic biomarkers can discriminate oral cancer from control subjects. Cancer Epidemiol Biomarkers Prev; 21(4); 664–72. ©2012 AACR.

Use of AlloDerm for coverage of radial forearm free flap donor site

Objectives To evaluate and discuss the role of acellular human dermal matrix (AlloDerm, LifeCell Corp., Branchburg, NJ) graft for coverage of radial forearm free flap donor site.

Safety of thyroidectomy in residency: A review of 186 consecutive cases

A retrospective review was performed to assess the surgical complications of thyroidectomy performed by otolaryngology residents under close supervision by faculty. One hundred eighty‐six consecutive cases of thyroidectomy performed by the residents in the Department of Otolaryngology—Head and Neck Surgery at the University of Southern California were reviewed. Sixty‐nine percent of thyroidectomies were performed for malignant or possible malignant diseases, and 31% were performed for benign conditions. Transient (less than 2 weeks in duration) hypocalcemia occurred in 26% of the patients; the vast majority of cases resolved within the first week. Five percent of the patients developed persistent hypocalcemia requiring prolonged replacement therapy. The incidence of unexpected postoperative permanent vocal cord paralysis was 0.9%. These complication rates are comparable to those in previously published large series on results of thyroidectomy. Thyroidectomy performed in an otolaryngology residency training program is a safe procedure when closely supervised by experienced faculty.

EphB4 provides survival advantage to squamous cell carcinoma of the head and neck

The receptor tyrosine kinase EphB4 and its ligand EphrinB2 play critical roles in blood vessel maturation, and are frequently overexpressed in a wide variety of cancers. We studied the aberrant expression and biological role of EphB4 in head and neck squamous cell carcinoma (HNSCC). We tested the effect of EphB4‐specific siRNA and antisense oligonucleotides (AS‐ODN) on cell growth, migration and invasion, and the effect of EphB4 AS‐ODN on tumor growth in vivo. All HNSCC tumor samples express EphB4 and levels of expression correlate directly with higher stage and lymph node metastasis. Six of 7 (86%) HNSCC cell lines express EphB4, which is induced either by EGFR activation or by EPHB4 gene amplification. EphrinB2 was expressed in 65% tumors and 5 of 7 (71%) cell lines. EphB4 provides survival advantage to tumor cells in that EphB4 siRNA and AS‐ODN significantly inhibit tumor cell viability, induce apoptosis, activate caspase‐8, and sensitize cells to TRAIL‐induced cell death. Furthermore, EphB4‐specific AS‐ODN significantly inhibits the growth of HNSCC tumor xenografts in vivo. Expression of EphB4 in HNSCC tumor cells confers survival and invasive properties, and thereby provides a strong rationale for targeting EphB4 as novel therapy for HNSCC.

Sensory recovery in noninnervated free flaps for head and neck reconstruction.

Recent studies have reported sensory recovery in innervated (“sensate”) microvascular free flaps used for oromandibular reconstruction. To evaluate the efficacy of sensate free flaps used for head and neck reconstruction, the natural outcome of noninnervated flaps must be known. Data on the natural recovery of sensation in noninnervated head and neck free flaps are lacking in the literature. This study evaluates the degree of spontaneous sensory reinnervation in noninnervated microvascular free flaps used for reconstruction of a variety of head and neck defects. Eighteen flaps were evaluated–9 fibula osseocutaneous and 9 radial forearm. The fibula flapswere used to reconstruct composite defects of the mandible and oral cavity mucosa. The radial forearm flaps were used to reconstruct defects resulting from floor of mouth resection (3), total glossectomy (2), pharyngectomy (1), full‐thickness cheek (1), and facial skin (2). Sensation to pinprick, light touch, and temperature discrimination were tested over the skin paddle at time intervals ranging from 6 to 24 months. The pattern of sensory reinnervation in these noninnervated flaps over time and by location is discussed.

Increased radiation sensitivity of head and neck squamous cell carcinoma with sphingosine kinase 1 inhibition

Sphingosine kinase 1 (SphK1) is an important regulator of apoptosis, survival, and proliferation in cancer cells. SphK1 expression in head and neck squamous cell cancer (HNSCC) cell lines and tumor tissue was assessed, and the efficacy of SphK1 knockdown in increasing tumor radiosensitivity was evaluated in vitro and in vivo.