Abstract

   Oral Tongue Squamous cell carcinoma (OTSCC), the most frequently
   affected oral cancer sub-site, is associated with a poor therapeutic
   outcome and survival despite aggressive multi- modality management.
   Till date, there are no established biomarkers to indicate prognosis
   and outcome in patients presenting with tongue cancer. There is an
   urgent need for reliable molecular prognostic factors to enable
   identification of patients with high risk of recurrence and treatment
   failure in OTSCC management. In the current study, we present the
   meta-analysis of OTSCC microarray based gene expression profiles,
   deriving a comprehensive molecular portrait of tongue cancer biology,
   showing the relevant genes and pathways which can be pursued further to
   derive novel, tailored therapeutics as well as for prognostication. We
   have studied 5 gene expression profiling data sets available on
   exclusively oral tongue subsite comprising of sample size; n = 190,
   consisting of 111 tumors and 79 normals. The meta- analysis results
   showed 2405 genes differentially regulated comparing OTSCC tumor and
   normal. The top up regulated genes were found to be involved in
   Extracellular matrix degradation (ECM) and Epithelial to mesenchymal
   transition (EMT) pathways. The top down regulated genes were found to
   be involved in detoxication pathways. We validated the results in
   clinical samples (n = 206), comprising of histologically normals (n =
   10), prospective (n = 29) and retrospective (n = 167) OTSCC by
   evaluating MMP9 and E-cadherin gene expression by qPCR and
   immunohistochemistry. Consistent with meta-analysis results, MMP9 mRNA
   expression was significantly up regulated in OTSCC primary tumors
   compared to normals. MMP9 protein over expression was found to be a
   significant predictor of poor prognosis, disease recurrence and poor
   Disease Free Survival (DFS) in OTSCC patients. Analysis by univariate
   and multivariate Cox proportional hazard model showed patients with
   loss of E-cadherin expression in OTSCC tumors having a poorer DFS (HR =
   1.566; P value = 0.045) and poorer Overall Survival (OS) (HR = 1.224; P
   value = 0.003) respectively. Combined over-expression of MMP9 and loss
   of E-cadherin membrane positivity in the invasive tumor front (ITF) of
   OTSCC had a significant association with poorer DFS (Log Rank = 16.040;
   P value = 0.001). These results suggest that along with known clinical
   indicators of prognosis like occult node positivity, assessment of MMP9
   and E-cadherin expression at ITF can be useful to identify patients at
   high risk and requiring a more intensive treatment strategy for OTSCC.
   Meta-analysis study of gene expression profiles indicates that OTSCC is
   a disease of ECM degradation leading to activated EMT processes
   implying the aggressive nature of the disease. The triggers for these
   processes should be studied further. Newer clinical application with
   agents that can inhibit the mediators of ECM degradation may be a key
   to achieving clinical control of invasion and metastasis of OTSCC.

Introduction

   Oral Tongue Squamous Cell Carcinoma (OTSCC) is regarded as a
   biologically unique entity compared to cancers occurring in the other
   oral sub-sites. The trend in epidemiology of oral cancer in Asia in the
   past decade (2000–2012) shows OTSCC as the most frequently affected
   oral sub-site. [[36]1] Earlier studies also report a higher incidence
   of OTSCC in India compared to other countries. [[37]2, [38]3, [39]4,
   [40]5] According to population based cancer registry (PBCR), the age
   adjusted incidence rate (AAR) for OTSCC in Chennai is showing an
   increasing trend from 3.6 to 5.7 per 100,000 persons above 25 years.
   Though there are poor prognostic indicators for OTSCC like occult node
   positivity, tumor depth, lymphovascular invasion and perineural
   invasion, there is still a need for molecular prognostic biomarkers
   that are reliable and robust to identify patients who are likely to
   have an adverse outcome.

   Microarrays, a tool for genomic scale profiling of gene expression, is
   a well known potentially valuable means of understanding the complex
   interactions and networks in development of several diseases including
   cancer. [[41]6, [42]7] These high throughput studies have offered the
   advantage of understanding the biology of a cancer through an
   exhaustive analysis. The launching of public microarray data archives
   like Gene Expression Omnibus and the advent of advanced computational
   informatics tools have made it possible to compare and converge gene
   expression studies done independently across different platforms.
   However, the hallmark of scientific progress is reproducibility of
   published outcomes which has been difficult in the case of several
   microarray studies with major sources of discordance because of
   variation caused by random noise, biological and experimental
   differences, and differences in technical methods. [[43]8] Most often
   we have findings that are not reproducible across studies due to data
   perturbations of individual studies, improper validations, and
   insufficient control of false positives. Despite these obstacles,
   several groups have successfully gleaned important insights from the
   focused comparison of disparate microarray results. [[44]9, [45]10]
   Many of the limitations can be mitigated by the use of standard
   reporting methods, together with careful application of large-scale
   meta-analysis techniques.

   Current study presents meta-analysis of OTSCCs as an exclusive sub-site
   for the first time as our primary objective. It was attempted to
   overcome the limitations of the individual expression profiling
   studies, resolving inconsistencies and reducing the likelihood of
   random errors, thus laying a foundation for uncovering the molecular
   aspects of OTSCC. We present the differentially expressed genes (DEG)
   comparing the OTSCC and normal expression profiles along with the
   involved signaling pathways. We have validated two biomarkers, MMP9 and
   E-cadherin found in meta–analysis in prospective and retrospective
   clinical samples as our second objective.

Materials and Methods

Identification of eligible OTSCC gene expression data sets

   OTSCC expression profiling studies were identified by searching the
   PubMed database. The following keywords and their combinations were
   used: “Oral tongue cancer gene expression microarray”. The Gene
   Expression Omnibus database ([46]http://www.ncbi.nlm.gov/geo) was also
   searched for terms “Oral Tongue Cancer”, “Oral Tongue Squamous cell
   carcinoma”, “mobile tongue cancer”.

Inclusion Criteria

   Gene expression data sets from exclusively anterior 2/3 (mobile tongue
   cancer) were taken for the study. The original experimental studies
   comprising of gene expression values for tongue tumor and normal
   tissues were taken. The expression data sets obtained from only
   standard microarray platforms were undertaken for the current study.

Exclusion Criteria

   Studies on head and neck cancer with a few samples on tongue cancer
   were excluded. Studies from datasets with base of tongue samples,
   tongue cancer cell lines, non human tissues were excluded. Studies
   without inclusion of normal samples were excluded.

Individual Study Analysis

   GEO accession number, sample type, platform, number of cases and
   controls, references and gene expression data were extracted from each