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BACKGROUND

MOLECULAR ECOLOGY, MOLECULAR GENETICSAND NATURAL HISTORY OF ANTERIOR TONGUE CANCER IN YOUNG ADULTS WITHOUT (OR INSIGNIFICANT)TOBACCO EXPOSURE: CAN A DISTINCT CLINICAL ENTITY BE DEFINED?. BACKGROUND.

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BACKGROUND

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  1. MOLECULAR ECOLOGY, MOLECULAR GENETICSAND NATURAL HISTORY OF ANTERIOR TONGUE CANCER IN YOUNG ADULTS WITHOUT (OR INSIGNIFICANT)TOBACCO EXPOSURE: CAN A DISTINCT CLINICAL ENTITY BE DEFINED?

  2. BACKGROUND • Head and neck squamous cell carcinomas (HNSCCs) comprise epithelial cancers of the oral cavity, pharynx, larynx, sinuses, para nasal sinuses and the salivary glands. • The major risk factors are: • tobacco use, • alcohol consumption and • infection with high risk human papillomaviruses • Tongue Cancer is one of the most common malignant cancers of the oral cavity. • Squamous cell carcinoma of the tongue is significantly more aggressive than other forms of HNOSCCs, with a propensity for rapid local invasion and spread. It affects human health world wide due to its disappointing survival rates. • It has been suggested that oral carcinomas with no history of smoking and alcohol use represents a distinct clinical entity. Importantly, such non-habits associated oral cancers often present increased clinical aggressiveness as compared to their habits associated counterparts. • This warrants a systematic investigation of molecular distinctions between the two etiologically diverse oral tumors. • Here, we used high-throughput gene expression profiling in a systems biology framework to comparatively characterize non-habits associated tongue carcinomas.

  3. ABSTRACT: A small, albeit significant, number of head and neck squamous cell carcinoma (HNSCC) patients has no history of tobacco and alcohol use. There have been suggestions that such non-habits associated HNSCCs represent a distinct clinical entity and exhibit increased aggressiveness, especially in younger patients. To understand differences in molecular etiology of habits- and non-habits associated tongue carcinomas, high throughput gene expression profiling of 22 tumor samples was carried out. 18 genes were identified that correlate strongly with the habits- non-habits distinction. Among the genes significantly over expressed in the non- habits group are CCND1, a key cell- cycle regulator, DACT3, a modulator of the Wnt/ beta- catenin pathway, and three genes associated with the Notch signaling pathway.CCND1and DACT3overexpression in non- habits associated tongue carcinomas were subsequently validated by quantitative real-time PCR in an independent cohort (n = 18) of patient samples. Gene expression data was integrated with publicly available protein interaction data to build a small protein interaction network containing 5 out of 18 differentially expressed genes. This suggested that a functional ‘network module’ can be implicated in the subgroup distinction. All the tumors analyzed here were human papillomavirus (HPV) negative samples. An association between CCND1over-expression in oral tumors and poor prognosis has previously been reported. Thus, CCND1over-expression in non- habits associated anterior tongue carcinomas may contribute to their increased clinical aggressiveness. Further, these results suggest that non- habits associated anterior tongue cancer may represent a distinct molecular entity paralleling its reported distinct clinical behavior.

  4. OBJECTIVES • To understand differences in molecular etiology of habits and non-habits associated tongue carcinomas using high throughput gene expression profiling. • Integrate the gene expression data with available molecular interaction data in a systems biology framework to identify a molecular interaction network module that more comprehensively captures the habits Vs non- habits distinction. • Quantitative Real-time PCR (qRT- PCR) validation in a set of clinical samples. • To investigate for the presence of human papillomavirus sub-types in the study groups.

  5. METHODOLOGY • Surgically excised tongue tumor biopsies were taken from newly diagnosed and histopathologically confirmed anterior tongue carcinoma patients in “RNA later”. • Total RNA and Genomic DNA was isolated from these tissues. • High throughput gene expression profiling was done using Illumina Human Ref- 8 v3.0 expression bead chip comprising of 24, 526 transcript probes for the assays. • Micro array data analysis and identification of genes that correlate strongly with the habits Vs. non-habits distinction. • Quantitative Real-time PCR (qRT- PCR) validation was performed using TaqMan® Gene Expression Assays to measure expression level of 4 genes (CCND1, DACT3,NOTCH1, NOTCH2,) in a set of clinical samples. • The HPV status of these samples was determined by Linear array HPV genotyping test (Roche). • The prevalence of HPV in tongue tumor tissues was also determined by Luminex Assay.

  6. RESULTS

  7. Illumina BeadChip platform ~18,000 genes profiled, bead based assay ~100,000 probes per gene, replicated 30 times on platform, thus, high feature redundancy compared to many existing platform including Affy, single dye- eliminate dye bias

  8. Bio analyzer quality check results for the 22 RNA isolated Bio analyzer quality check results for cDNA Scanning results of the Micro array using Illumina platform

  9. Microarray Analysis Flowchart Microarray data -signal extraction & normalization DEG identification K-1 cross validation Pathway enrichment analysis Integration with existing interactome data Clustering Network analysis

  10. Figure-1:Identified a set of 18 genes that most accurately discriminates habit associated oral tongue from non- habits samples

  11. Figure-2:. A protein- protein interaction map for the DEGs as calculated by STRING. Gene products are represented as pale colored spheres alongside gene symbols. Presence of “edges” connecting the spheres indicates a known physical interaction between the proteins. The thicker an edge is, the stronger the evidence for the interaction.

  12. Plots of log10 fold changes in expression, determined by relative quantification method (RQ), of CCND1 (S3), DACT3 (S4), NOTCH2 (S5) and NOTCH1 (S6) between 9 pairs of non- habits- habits anterior tongue tumor samples.‘VH’ represents habits associated tumors and ‘VNH’ marks non- habits associated samples.

  13. Figure-3: Results of HPV genotyping using Linear array for the 22 samples used for gene expression profiling.

  14. Figure-4: Results of HPV genotyping on tongue tumor samples (N=250)

  15. DISCUSSION AND CONCLUSION • In the present study, we compared habits and non-habits associated tongue carcinomas by global gene expression profiling. • We used human oral tongue carcinoma tissue for the study, because of two reasons: • [i] oral carcinomas account for 70-90% of all HNSCC. • [ii] using a single tumor site (oral tongue) reduces sub-site based variability in gene expression. • A systems biology approach, identified an 18 gene signature that separated the two groups accurately. • Our pathway based analyses of these genes identified high expression levels of three of the NOTCH signaling pathway components in non-habits associated samples. • Among the other genes identified as significantly over- expressed in the non-habits associated group is CCND1, a promoter of cell cycle. • Since we intended to investigate differences between habits associated and non-habits associated oral cancers, we also analyzed the tissues for presence of HPV, a confounding variable. We did not detect HPV in any of the tissues used for the microarray. • The prevalence of HPV in tongue squamous cell carcinoma was only 6% and the most predominant subtype prevalent was HPV 16. • We thus suggest that anterior tongue cancer may represent a distinct molecular and clinical entity, characterized by a distinguishing gene expression signature. • Further, we propose that aberrant Notch signaling together with CCND1 overexpression may contribute to the increased clinical aggressiveness of the non-habits associated tumors .

  16. DISCUSSION AND CONCLUSION • One of the top ranking genes identified as over-expressed in non- habits associated oral carcinomas by both beadchip based profiling and by qRT- PCR (6 out of 9 samples in a separate set) is CCND1, the cyclin D1 gene, which is a key cell cycle promoter. CCND1 aberrations including gene amplification and over-expression have been causally associated with several different tumors Importantly, CCND1over-expression in early stage oral squamous carcinomas has been correlated with poor prognosis .We propose that increased CCND1 expression, which has previously been linked to poor prognosis in OSCCs, may contribute to the poor prognosis seen in non- habits associated tumors of the anterior tongue. • DACT3 a recently discovered epigenetic regulator of the Wnt/beta- catenin pathway, identified as over-expressed in the non- habits group, was also found to be up-regulated in an independent set of anterior oral tumor samples (5 out of 9 samples).

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