Transcriptomics,Genomics

Dataset Information

36

Hypoxia related splice variants in HNSCC


ABSTRACT: The identifcation of alternatively spliced transcript variants specific to particular biological processes in tumours should increase our understanding of cancer. Hypoxia is an important factor in cancer biology and associated splice variants may present new markers to help with planning treatment. A method was developed to analyse alternative splicing in exon array data, using probeset multiplicity to identify genes with changes in expression across their loci, and a combination of the splicing index and a new metric based on the variation of reliability weighted fold changes to detect changes in the splicing patterns. The approach was validated on a cancer/normal sample dataset in which alternative splicing events had been confirmed using RT-PCR. We then analysed ten head and neck squamous cell carcinomas using exon arrays and identified differentially expressed splice variants in five samples with high versus five with low levels of hypoxia-associated genes (Winter et al, 2007; Cancer Res 67:3441-9). The analysis identified a splice variant of LAMA3 (Laminin 3), LAMA3-A, known to be involved in tumour cell invasion and progression. The full-length transcript of the gene (LAMA3-B) did not appear to be hypoxia-associated. The results were confirmed using qualitative real time PCR. In a series of 59 prospectively-collected head and neck tumours (Winter et al, 2007; Cancer Res 67:3441-9), expression of LAMA3-A had prognostic significance whereas LAMA3-B did not. This work illustrates the potential for alternatively spliced transcripts to act as biomarkers of disease prognosis with improved specificity for particular tissues or conditions over assays which do not discriminate between splice variants. Overall design: In (Winter et al, 2007; Cancer Res 67:3441-9) 59 Head and Neck Squamous Cell Carcinoma (HNSCCs) samples were processed onto Affiymetrix HG-U133 Plus2 3'IVT arrays, prior to any treatment at the time of primary surgery and a set of 99 genes up-regulated in hypoxia was obtained by analysis of genes whose in vivo expression clustered with the expression of 10 well-known hypoxia-regulated genes (e.g. CA9, GLUT1, and VEGF). A Hypoxia Score (HS) was defined as the median value of expression for these 99 genes. High HS values indicate higher hypoxia relative to lower values and were an adverse prognostic factor in an independent microarray dataset. Here, we first eliminated samples from the study with a high percentage of absent calls by removing the top 10-th quantile of the samples ordered by number of absent calls, and then selected the 5 least and 5 most hypoxic samples as defined by the HS values. Confirmation of hypoxia status was carried out by investigating CAIX protein expression in histological sections. There was a statistically significant increased CAIX expression in the samples with high HS values (p=0.024). These 10 samples were then processed onto Affiymetrix Human Exon 1.0ST arrays using manufacturers' standard protocols. Following hybridization, we investigated the similarity in expression profiles among the 10 exon arrays. Multidimensional scaling and hierarchical clustering of the samples based on a reduced set of probesets (exonic probesets flagged present; DABG p-value< 0:01 in at least half of all the samples; N=172,204) confirmed that the samples are partitioned by high and low HS values, as expected.

INSTRUMENT(S): [HuEx-1_0-st] Affymetrix Human Exon 1.0 ST Array [probe set (exon) version]

SUBMITTER: Carla Sofia Moller-Levet  

PROVIDER: GSE18300 | GEO | 2009-10-07

SECONDARY ACCESSION(S): PRJNA118029

REPOSITORIES: GEO

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Publications

Exon array analysis of head and neck cancers identifies a hypoxia related splice variant of LAMA3 associated with a poor prognosis.

Moller-Levet Carla S CS   Betts Guy N J GN   Harris Adrian L AL   Homer Jarrod J JJ   West Catharine M L CM   Miller Crispin J CJ  

PLoS computational biology 20091120 11


The identification of alternatively spliced transcript variants specific to particular biological processes in tumours should increase our understanding of cancer. Hypoxia is an important factor in cancer biology, and associated splice variants may present new markers to help with planning treatment. A method was developed to analyse alternative splicing in exon array data, using probeset multiplicity to identify genes with changes in expression across their loci, and a combination of the splici  ...[more]

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