Searching for molecular markers in head and neck squamous cell carcinomas (HNSCC) by statistical and bioinformatic analysis of larynx-derived SAGE libraries

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies in humans. The average 5-year survival rate is one of the lowest among aggressive cancers, showing no significant improvement in recent years. When detected early, HNSCC has a good prognosis, but most patients present metastatic disease at the time of diagnosis, which significantly reduces survival rate. Despite extensive research, no molecular markers are currently available for diagnostic or prognostic purposes. Methods: Aiming to identify differentially-expressed genes involved in laryngeal squamous cell carcinoma (LSCC) development and progression, we generated individual Serial Analysis of Gene Expression (SAGE) libraries from a metastatic and non-metastatic larynx carcinoma, as well as from a normal larynx mucosa sample. Approximately 54,000 unique tags were sequenced in three libraries. Results: Statistical data analysis identified a subset of 1,216 differentially expressed tags between tumor and normal libraries, and 894 differentially expressed tags between metastatic and non-metastatic carcinomas. Three genes displaying differential regulation, one down-regulated (KRT31) and two up-regulated (BST2, MFAP2), as well as one with a non-significant differential expression pattern (GNA15) in our SAGE data were selected for real-time polymerase chain reaction (PCR) in a set of HNSCC samples. Consistent with our statistical analysis, quantitative PCR confirmed the upregulation of BST2 and MFAP2 and the downregulation of KRT31 when samples of HNSCC were compared to tumor-free surgical margins. As expected, GNA15 presented a non-significant differential expression pattern when tumor samples were compared to normal tissues. Conclusion: To the best of our knowledge, this is the first study reporting SAGE data in head and neck squamous cell tumors. Statistical analysis was effective in identifying differentially expressed genes reportedly involved in cancer development. The differential expression of a subset of genes was confirmed in additional larynx carcinoma samples and in carcinomas from a distinct head and neck subsite. This result suggests the existence of potential common biomarkers for prognosis and targeted-therapy development in this heterogeneous type of tumor.Fundação de Amparo a Pesquisa do Estado de São Paulo/FAPESP [05/51467-0]; [04/12054-9]; [07/50894-7]Ludwig Institute for Cancer ResearchConselho Nacional de Pesquisas/CNPqCoordenacao de Aperfeicoamento do Pessoal do Ensino Superior/CAPE

A Population Proportion approach for ranking differentially expressed genes

<p>Abstract</p> <p>Background</p> <p>DNA microarrays are used to investigate differences in gene expression between two or more classes of samples. Most currently used approaches compare mean expression levels between classes and are not geared to find genes whose expression is significantly different in only a subset of samples in a class. However, biological variability can lead to situations where key genes are differentially expressed in only a subset of samples. To facilitate the identification of such genes, a new method is reported.</p> <p>Methods</p> <p>The key difference between the Population Proportion Ranking Method (PPRM) presented here and almost all other methods currently used is in the quantification of variability. PPRM quantifies variability in terms of inter-sample ratios and can be used to calculate the relative merit of differentially expressed genes with a specified difference in expression level between at least some samples in the two classes, which at the same time have lower than a specified variability within each class.</p> <p>Results</p> <p>PPRM is tested on simulated data and on three publicly available cancer data sets. It is compared to the t test, PPST, COPA, OS, ORT and MOST using the simulated data. Under the conditions tested, it performs as well or better than the other methods tested under low intra-class variability and better than t test, PPST, COPA and OS when a gene is differentially expressed in only a subset of samples. It performs better than ORT and MOST in recognizing non differentially expressed genes with high variability in expression levels across all samples. For biological data, the success of predictor genes identified in appropriately classifying an independent sample is reported.</p

Clinical relevance of nine transcriptional molecular markers for the diagnosis of head and neck squamous cell carcinoma in tissue and saliva rinse

<p>Abstract</p> <p>Background</p> <p>Analysis of 23 published transcriptome studies allowed us to identify nine genes displaying frequent alterations in HNSCC (<it>FN1, MMP1, PLAU, SPARC</it>, <it>IL1RN, KRT4, KRT13, MAL</it>, and <it>TGM3</it>). We aimed to independently confirm these dysregulations and to identify potential relationships with clinical data for diagnostic, staging and prognostic purposes either at the tissue level or in saliva rinse.</p> <p>Methods</p> <p>For a period of two years, we systematically collected tumor tissue, normal matched mucosa and saliva of patients diagnosed with primary untreated HNSCC. Expression levels of the nine genes of interest were measured by RT-qPCR in tumor and healthy matched mucosa from 46 patients. <it>MMP1 </it>expression level was measured by RT-qPCR in the salivary rinse of 51 HNSCC patients and 18 control cases.</p> <p>Results</p> <p>Dysregulation of the nine genes was confirmed by the Wilcoxon test. <it>IL1RN, MAL </it>and <it>MMP1 </it>were the most efficient diagnostic markers of HNSCC, with ROC AUC > 0.95 and both sensitivity and specificity above 91%. No clinically relevant correlation was found between gene expression level in tumor and T stage, N stage, tumor grade, global survival or disease-free survival. Our preliminary results suggests that with 100% specificity, <it>MMP1 </it>detection in saliva rinse is potentially useful for non invasive diagnosis of HNSCC of the oral cavity or oropharynx, but technical improvement is needed since sensitivity was only 20%.</p> <p>Conclusion</p> <p><it>IL1RN, MAL </it>and <it>MMP1 </it>are prospective tumor diagnostic markers for HNSCC. <it>MMP1 </it>overexpression is the most promising marker, and its detection could help identify tumor cells in tissue or saliva.</p

Oral tongue cancer gene expression profiling: Identification of novel potential prognosticators by oligonucleotide microarray analysis

<p>Abstract</p> <p>Background</p> <p>The present study is aimed at identifying potential candidate genes as prognostic markers in human oral tongue squamous cell carcinoma (SCC) by large scale gene expression profiling.</p> <p>Methods</p> <p>The gene expression profile of patients (n=37) with oral tongue SCC were analyzed using Affymetrix HG_U95Av2 high-density oligonucleotide arrays. Patients (n=20) from which there were available tumor and matched normal mucosa were grouped into stage (early vs. late) and nodal disease (node positive vs. node negative) subgroups and genes differentially expressed in tumor vs. normal and between the subgroups were identified. Three genes, <it>GLUT3</it>, <it>HSAL2</it>, and <it>PACE4</it>, were selected for their potential biological significance in a larger cohort of 49 patients via quantitative real-time RT-PCR.</p> <p>Results</p> <p>Hierarchical clustering analyses failed to show significant segregation of patients. In patients (n=20) with available tumor and matched normal mucosa, 77 genes were found to be differentially expressed (P< 0.05) in the tongue tumor samples compared to their matched normal controls. Among the 45 over-expressed genes, <it>MMP-1</it> encoding interstitial collagenase showed the highest level of increase (average: 34.18 folds). Using the criterion of two-fold or greater as overexpression, 30.6%, 24.5% and 26.5% of patients showed high levels of <it>GLUT3</it>, <it>HSAL2</it> and <it>PACE4</it>, respectively. Univariate analyses demonstrated that <it>GLUT3</it> over-expression correlated with depth of invasion (P<0.0001), tumor size (P=0.024), pathological stage (P=0.009) and recurrence (P=0.038). <it>HSAL2</it> was positively associated with depth of invasion (P=0.015) and advanced T stage (P=0.047). In survival studies, only <it>GLUT3</it> showed a prognostic value with disease-free (P=0.049), relapse-free (P=0.002) and overall survival (P=0.003). <it>PACE4</it> mRNA expression failed to show correlation with any of the relevant parameters. </p> <p>Conclusion</p> <p>The characterization of genes identified to be significant predictors of prognosis by oligonucleotide microarray and further validation by real-time RT-PCR offers a powerful strategy for identification of novel targets for prognostication and treatment of oral tongue carcinoma.</p

A glimpse of the ERM proteins

In all eukaryotes, the plasma membrane is critically important as it maintains the architectural integrity of the cell. Proper anchorage and interaction between the plasma membrane and the cytoskeleton is critical for normal cellular processes. The ERM (ezrin-radixin-moesin) proteins are a class of highly homologous proteins involved in linking the plasma membrane to the cortical actin cytoskeleton. This review takes a succinct look at the biology of the ERM proteins including their structure and function. Current reports on their regulation that leads to activation and deactivation was examined before taking a look at the different interacting partners. Finally, emerging roles of each of the ERM family members in cancer was highlighted

Tumor Transcriptome Sequencing Reveals Allelic Expression Imbalances Associated with Copy Number Alterations

Due to growing throughput and shrinking cost, massively parallel sequencing is rapidly becoming an attractive alternative to microarrays for the genome-wide study of gene expression and copy number alterations in primary tumors. The sequencing of transcripts (RNA-Seq) should offer several advantages over microarray-based methods, including the ability to detect somatic mutations and accurately measure allele-specific expression. To investigate these advantages we have applied a novel, strand-specific RNA-Seq method to tumors and matched normal tissue from three patients with oral squamous cell carcinomas. Additionally, to better understand the genomic determinants of the gene expression changes observed, we have sequenced the tumor and normal genomes of one of these patients. We demonstrate here that our RNA-Seq method accurately measures allelic imbalance and that measurement on the genome-wide scale yields novel insights into cancer etiology. As expected, the set of genes differentially expressed in the tumors is enriched for cell adhesion and differentiation functions, but, unexpectedly, the set of allelically imbalanced genes is also enriched for these same cancer-related functions. By comparing the transcriptomic perturbations observed in one patient to his underlying normal and tumor genomes, we find that allelic imbalance in the tumor is associated with copy number mutations and that copy number mutations are, in turn, strongly associated with changes in transcript abundance. These results support a model in which allele-specific deletions and duplications drive allele-specific changes in gene expression in the developing tumor

Current potential and limitations of molecular diagnostic methods in head and neck cancer

Item does not contain fulltextTraditional diagnostic methods such as clinical assessment, histopathological examination and imaging techniques are limited in their capacity to provide information on prognosis and treatment choice of head and neck cancer. In recent years, molecular techniques have been developed that enabled us to get more insight into the molecular biological cellular pathways underlying tumor progression and metastasis. Correlation of these molecular changes with clinical events has been explored. However, consistently useful markers have not been identified yet, although many promising developments are in progress. It may be expected that in the near future, molecular markers will be useful for clinical purposes. In this paper, an overview will be given of the several molecular techniques that may have potential to be introduced in clinical practice in the management of head and neck squamous cell carcinoma.1 juni 201