Gene expression profiling reveals novel molecular marker candidates of laryngeal squamous cell carcinoma

Laryngeal squamous cell carcinoma is a very common in head and neck cancer, accounting for 25% of all cases, with high mortality rates and poor prognosis. In this study, we compared expression profiles of clinical samples from15 larynx tumors and 10 non-neoplastic larynx tissue using a custom-built cDNA microarray containing 332 probes for 285 genes previously identified as up- or down-regulated in head and neck tumors by the Head and Neck Annotation Consortium (Reis et al., Cancer Res 65:1693-99, 2005). Thirty-five genes showed statistically significant differences (SNR ≥|1.0|,p-value ≤ 0.001) in expression between tumor and non-tumor larynx tissue samples. Functional annotation indicated that these genes are involved in cellular processes relevant to the cancer phenotype, such as apoptosis, cell cycle, DNA repair, proteolysis, protease inhibition, signal transduction, and transcription regulation. Six of the identified transcripts map to intronic regions of protein-coding genes and may comprise unannotated exons or yet uncharacterized long ncRNAs with a regulatory role in the gene expression program of larynx tissue. Differential expression of 10 of these genes (ADCY6, AES, AL2SCR3, CRR9, CSTB, DUSP1, MAP3K5, PLAT, UBL1 and ZNF706) was independently confirmed by quantitative real-time RT-PCR. Among these, the CSTB gene product has cysteine protease inhibitor activity that has been associated to an antimetastatic function. Interestingly, CSTB showed low expression in all tumor samples analyzed (p-value < 0.0001). The set of genes identified here contribute to a better understanding of the molecular basis of larynx cancer, and at the same time provide novel candidate markers for improving diagnosis, prognosis and treatment of this carcinoma. Keywords: Gene expression profiling of larynx tumors and non-neoplastic adjacent tissue Total RNA was isolated from snap-frozen tissue samples from 13 larynx tumors and 10 non-neoplastic larynx tissue.Labeled targets for hybridizations were generated from total mRNA in reverse transcription reactions using oligo-dT primers. The Cy5 (tumor or non-tumor sample) labeled cDNAs were hybridized separately with microarrays on an Automated Slide Processor (ASP, GE Healthcare) and incubated for 16 h at 42ºC. Processed slides were scanned with a 700 V PMT setting. Background-subtracted artifact-removed median intensities of Cy5 emissions were extracted for each spot from raw images, using the ArrayVision V.7.2 software (Imaging Research Inc., Ontario, Canada). To make the experiments comparable, intensity data derived from Cy5-labeled test samples from different hybridizations were LOWESS normalized by, using as reference the intensity data from a sample with total energy comparable to the average intensity of all samples.