Nadarajah Vigneswaran

Epidemiologic Trends in Head and Neck Cancer and Aids in Diagnosis

Head and neck squamous cell carcinoma is the sixth most common cancer worldwide predominately associated with tobacco use. Changing cause and increased incidence in oropharyngeal carcinomas is associated with high-risk types of human papilloma virus and has an improved survival. Optical devices may augment visual oral examination; however, their lack of specificity still warrants tissue evaluation/biopsy. Histologic factors of oral carcinomas are critical for patient management and prognostic determination. Clinical biomarkers are still needed to improve early detection, predict malignant transformation, and optimize therapies.

Selective Upregulation of microRNA Expression in Peripheral Blood Leukocytes in IL-10 −/− Mice Precedes Expression in the Colon

IL-10−/− mice, an animal model of Th1-mediated inflammatory bowel disease, were screened for the expression of 600 microRNAs (miRNAs) using colonic tissues and PBLs from animals having either mild inflammation or severe intestinal inflammation. The development of colonic inflammation in IL-10−/− mice was accompanied by upregulation in the expression of 10 miRNAs (miR-19a, miR-21, miR-31, miR-101, miR-223, miR-326, miR-142-3p, miR-142-5p, miR-146a, and miR-155). Notably, the expression of all of these miRNAs plus miR-375 was elevated in PBLs of IL-10−/− mice at a time when colonic inflammation was minimal, suggesting that changes in specific miRNAs in circulating leukocytes may be harbingers of ensuing colonic pathology. In vitro exposure of colonic intraepithelial lymphocytes to IL-10 resulted in downregulation of miR-19a, miR-21, miR-31, miR-101, miR-223, and miR-155. Interestingly, unlike IL-10−/− mice, changes in miRNAs in PBL of dextran sulfate sodium-treated mice were minimal but selectively elevated in the colon after pathology was severe. We further show that miR-223 is a negative regulator of the Roquin ubiquitin ligase, Roquin curtails IL-17A synthesis, and the 3′ untranslated region of Roquin is a target for miR-223, thus defining a molecular pathway by which IL-10 modulates IL-17–mediated inflammation. To identify additional miRNAs that may be involved in the regulation of Roquin, transcriptome analysis was done using cDNAs from HeLa cells transfected with 90 miRNA mimics. Twenty-six miRNAs were identified as potential negative regulators of Roquin, thus demonstrating functional complexity in gene expression regulation by miRNAs.

Advances in fluorescence imaging techniques to detect oral cancer and its precursors

Oral cancer is a significant health problem in the USA and throughout the world. Most oral cancer patients are diagnosed at a late stage, when treatment is less successful and treatment-associated morbidity is more severe. A number of new diagnostic aids to conventional oral examination have recently been introduced to assist in the early detection of oral neoplasia. In particular, autofluorescence imaging has emerged as a promising adjunctive technique to improve early identification of oral premalignant lesions. Direct visual inspection of tissue autofluorescence has shown encouraging results in high-prevalence populations, but the technique requires subjective interpretation and depends on the visual recognition skills of the examiner. Capturing and analyzing digital fluorescence images can reduce subjectivity and potentially improve sensitivity of detection of precancerous changes. Recent studies of wide-field autofluorescence imaging in low-prevalence populations suggest that benign lesions such as inflammation may give rise to false-positive results. High-resolution fluorescence imaging is a new modality that can be used in conjunction with wide-field imaging to improve specificity by imaging subcellular detail of neoplastic tissues. The combination of wide-field and high-resolution fluorescence imaging systems with automated image analysis should be investigated to maximize overall diagnostic performance for early detection of oral neoplasia.

Nano-bio-chip sensor platform for examination of oral exfoliative cytology

Oral cancer is a deadly and disfiguring disease that could greatly benefit from new diagnostic approaches enabling early detection. In this pilot study, we describe a nano-bio-chip (NBC) sensor technique for analysis of oral cancer biomarkers in exfoliative cytology specimens, targeting both biochemical and morphologic changes associated with early oral tumorigenesis. Here, oral lesions from 41 dental patients, along with normal epithelium from 11 healthy volunteers, were sampled using a noninvasive brush biopsy technique. Specimens were enriched, immunolabeled, and imaged in the NBC sensor according to previously established assays for the epidermal growth factor receptor (EGFR) biomarker and cytomorphometry. A total of 51 measurement parameters were extracted using custom image analysis macros, including EGFR labeling intensity, cell and nuclear size, and the nuclear-to-cytoplasmic ratio. Four key parameters were significantly elevated in both dysplastic and malignant lesions relative to healthy oral epithelium, including the nuclear area and diameter (P < 0.0001), the nuclear-to-cytoplasmic ratio (P < 0.0001), and EGFR biomarker expression (P < 0.03). Further examination using logistic regression and receiver operating characteristic curve analyses identified morphologic features as the best predictors of disease (area under the curve ≤0.93) individually, whereas a combination of all features further enhanced discrimination of oral cancer and precancerous conditions (area under the curve, 0.94) with high sensitivity and specificity. Further clinical trials are necessary to validate the regression model and evaluate other potential biomarkers, but this pilot study supports the NBC sensor technique as a promising new diagnostic tool for early detection of oral cancer, which could enhance patient care and survival. Cancer Prev Res; 3(4); 518–28. ©2010 AACR.

Cathepsin B mediates TRAIL-induced apoptosis in oral cancer cells.

Purpose: The death ligand TRAIL (tumor necrosis factor-related apoptosis inducing ligand) triggers apoptosis in a variety of cancer cells, which implies the potential for therapeutic applications. The purpose of this study was to investigate the role of the lysosomal protease cathepsin B (CB) in mediating TRAIL-induced cell death in oral squamous cell carcinoma (OSCC) cells. Methods: OSCC cell lines from primary tumor and lymph node metastasis were examined for expression of apoptosis markers by Western blots, enzyme activity assays, nuclear fragmentation assays, and FACS analysis. Gene-specific ribozymes or chemical inhibitors were used to inhibit CB or caspases in target cells. Results: TRAIL-induced activation of caspase-3, cleavage of Bid and poly–ADP-ribose polymerase, release of cytochrome c, and DNA fragmentation were blocked either by a pan-caspase inhibitor (zVAD-fmk) or a CB inhibitor (CA074Me), consistent with the involvement of TRAIL as well as CB in cell death. The primary tumor cells were more susceptible to apoptosis than their corresponding lymph node metastatic cells. Stable transfection of a ribozyme which inhibited CB expression also decreased the apoptotic process. Conclusions: We conclude that TRAIL-induced apoptotic cell death in OSCC cells is mediated through CB or through caspase activation. Our data point to a new tumor-suppressive role for CB in OSCC which is opposed to the invasion- and metastasis-promoting functions of lysosomal proteases.

Erlotinib and the Risk of Oral Cancer: The Erlotinib Prevention of Oral Cancer (EPOC) Randomized Clinical Trial

IMPORTANCE Standard molecularly based strategies to predict and/or prevent oral cancer development in patients with oral premalignant lesions (OPLs) are lacking. OBJECTIVE To test if the epidermal growth factor receptor inhibitor erlotinib would reduce oral cancer development in patients with high-risk OPLs defined by specific loss of heterozygosity (LOH) profiles. Secondary objectives included prospective determination of LOH as a prognostic marker in OPLs. DESIGN The Erlotinib Prevention of Oral Cancer (EPOC) study was a randomized, placebo-controlled, double-bind trial. Accrual occurred from November 2006 through July 2012, with a median follow-up time of 35 months in an ambulatory care setting in 5 US academic referral institutions. Patients with OPLs were enrolled in the protocol, and each underwent LOH profiling (N = 379); they were classified as high-risk (LOH-positive) or low-risk (LOH-negative) patients based on their LOH profiles and oral cancer history. The randomized sample consisted of 150 LOH-positive patients. INTERVENTIONS Oral erlotinib treatment (150 mg/d) or placebo for 12 months. MAIN OUTCOMES AND MEASURES Oral cancer-free survival (CFS). RESULTS A total of 395 participants were classified with LOH profiles, and 254 were classified LOH positive. Of these, 150 (59%) were randomized, 75 each to the placebo and erlotinib groups. The 3-year CFS rates in placebo- and erlotinib-treated patients were 74% and 70%, respectively (hazard ratio [HR], 1.27; 95% CI, 0.68-2.38; P = .45). The 3-year CFS was significantly lower for LOH-positive compared with LOH-negative groups (74% vs 87%, HR, 2.19; 95% CI, 1.25-3.83; P = .01). Increased EGFR gene copy number correlated with LOH-positive status (P < .001) and lower CFS (P = .01). The EGFR gene copy number was not predictive of erlotinib efficacy. Erlotinib-induced skin rash was associated with improved CFS (P = .01). CONCLUSIONS AND RELEVANCE In this trial, LOH was validated as a marker of oral cancer risk and found to be associated with increased EGFR copy number (the target of the intervention). Erlotinib did not, however, improve CFS in high-risk patients with LOH-positive or high-EGFR-gene-copy-number OPLs. These results support incorporation of LOH testing as a prognostic tool in routine clinical practice but do not support erlotinib use in this setting. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00402779.

Hypoxia inhibits TRAIL-induced tumor cell apoptosis: Involvement of lysosomal cathepsins

Tumor hypoxia interferes with the efficacy of chemotherapy, radiotherapy, and tumor necrosis factor-α. TRAIL (tumor necrosis factor-related apoptosis inducing ligand) is a potent apoptosis inducer that limits tumor growth without damaging normal cells and tissues in vivo. We present evidence for a central role of lysosomal cathepsins in hypoxia and/or TRAIL-induced cell death in oral squamous cell carcinoma (OSCC) cells. Hypoxia or TRAIL-induced activation of cathepsins (B, D and L), caspases (-3 and -9), Bid cleavage, release of Bax and cytochrome c, and DNA fragmentation were blocked independently by zVAD-fmk, CA074Me or pepstatin A, consistent with the involvement of lysosomal cathepsin B and D in cell death. Lysosome stability and mitochondrial membrane potential were reduced in hypoxia and TRAIL-induced apoptosis. However, TRAIL treatment under hypoxic condition resulted in diminished apoptosis rates compared to treatment under normoxia. This inhibitory effect of hypoxia on TRAIL-induced apoptosis may be based on preventing Bax activation and thus protecting mitochondria stability. Our data show that TRAIL or hypoxia independently triggered activation of cathepsin B and D leading to apoptosis through Bid and Bax, and suggest that hypoxic tissue regions provide a selective environment for highly apoptosis-resistant clonal cells. Molecular therapy approaches based on cathepsin inhibitors need to address this novel tumor-preventing function of cathepsins in OSCC.

Hypoxia-induced autophagic response is associated with aggressive phenotype and elevated incidence of metastasis in orthotopic immunocompetent murine models of head and neck squamous cell carcinomas (HNSCC)

Hypoxia confers resistance to chemoradiation therapy and promotes metastasis in head and neck squamous cell carcinomas (HNSCC). We investigated the effects of hypoxia in tumor phenotype using immunocompetent murine HNSCC models. Balb/c mice were injected intraorally with murine squamous cell carcinoma cells LY-2 and B4B8. Intratumoral hypoxia fraction was evaluated by the immunohistochemical detection of hypoxic probe pimonidazole and carbonic anhydrase IX (CAIX). Tumor cell apoptosis and autophagy in hypoxic areas of these tumors were examined immunohistochemically. Hypoxia-induced apoptotic and autophagic responses in vitro were examined by treating LY2 cells with CoCl(2). B4B8 tumors exhibited a non-aggressive phenotype characterized by its slow growth rate and the lack of metastatic spread. LY2 tumors demonstrated an aggressive phenotype characterized by rapid growth rate with regional and distant metastasis. Intratumoral hypoxia fraction in B4B8 tumors was significantly lower than in LY2 tumors. The hypoxic areas in B4B8 tumors exhibited increased apoptosis rate than that of LY2 tumors. In contrast, the hypoxic areas in LY2 tumors revealed autophagy. The induction of hypoxia in vitro elicited autophagy and not apoptosis in LY2 cells. The induction of autophagy coupled with blockage of apoptosis in hypoxic areas promotes tumor cell survival and confers aggressive phenotype in immunocompetent murine HNSCC models.

Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression

BackgroundTRAIL plays an important role in host immunosurveillance against tumor progression, as it induces apoptosis of tumor cells but not normal cells, and thus has great therapeutic potential for cancer treatment. TRAIL binds to two cell-death-inducing (DR4 and DR5) and two decoy (DcR1, and DcR2) receptors. Here, we compare the expression levels of TRAIL and its receptors in normal oral mucosa (NOM), oral premalignancies (OPM), and primary and metastatic oral squamous cell carcinomas (OSCC) in order to characterize the changes in their expression patterns during OSCC initiation and progression.MethodsDNA microarray, immunoblotting and immunohistochemical analyses were used to examine the expression levels of TRAIL and its receptors in oral epithelial cell lines and in archival tissues of NOM, OPM, primary and metastatic OSCC. Apoptotic rates of tumor cells and tumor-infiltrating lymphocytes (TIL) in OSCC specimens were determined by cleaved caspase 3 immunohistochemistry.ResultsNormal oral epithelia constitutively expressed TRAIL, but expression was progressively lost in OPM and OSCC. Reduction in DcR2 expression levels was noted frequently in OPM and OSCC compared to respective patient-matched uninvolved oral mucosa. OSCC frequently expressed DR4, DR5 and DcR1 but less frequently DcR2. Expression levels of DR4, DR5 and DcR1 receptors were not significantly altered in OPM, primary OSCC and metastatic OSCC compared to patient-matched normal oral mucosa. Expression of proapoptotic TRAIL-receptors DR4 and DR5 in OSCC seemed to depend, at least in part, on whether or not these receptors were expressed in their parental oral epithelia. High DR5 expression in primary OSCC correlated significantly with larger tumor size. There was no significant association between TRAIL-R expression and OSSC histology grade, nodal status or apoptosis rates of tumor cells and TIL.ConclusionLoss of TRAIL expression is an early event during oral carcinogenesis and may be involved in dysregulation of apoptosis and contribute to the molecular carcinogenesis of OSCC. Differential expressions of TRAIL receptors in OSCC do not appear to play a crucial role in their apoptotic rate or metastatic progression.

ICOS promotes IL‐17 synthesis in colonic intraepithelial lymphocytes in IL‐10−/− mice

In the absence of IL‐10, colonic inflammation ensues, which is characterized by high levels of IL‐17. Here, we demonstrate a direct correlation between ICOS expression and IL‐17 production in cIELs. IL‐10−/− mice had increased numbers of cIELs and greater colon weight. Although the CD69 early activation antigen was expressed on cIELs from normal and IL‐10−/− mice, ICOS was expressed only on cIELs from IL‐10−/− mice. IL‐17‐producing cells in IL‐10−/− mice consisted of CD4+ and CD8+ cIELs; however, CD4+ cells were the predominant IL‐17‐producing cell population. Culture of cIELs from IL‐10−/− mice with IL‐23 resulted in an increase in ICOS and IL‐17 expression, whereas IL‐10 suppressed expression of ICOS and IL‐17. This occurred in primary cultures and recall stimulation experiments. The ICOS ligand B7RP‐1 was up‐regulated on colonic epithelial cells and on a population of large granular leukocytes during inflammation. Culture of cIELs with B7RP‐1+ DCs enhanced IL‐17A production from normal cIELs but failed to do so using cIELs from ICOS−/− mice. In vivo treatment of IL‐10−/− mice with antibody to ICOS resulted in a significant reduction in colonic pathology. These findings implicate ICOS as an activational signal of Th17 cells during chronic intestinal inflammation, and they suggest that under some conditions, control of ICOS expression may help to suppress chronic intestinal inflammation.