Epigenetic silencing of the endothelin-B receptor gene in non-small cell lung cancer.

Endothelin-1 is overexpressed in several tumor types. Activation of the endothelin-A (ETA) receptor may promote cell growth, angiogenesis and invasion, and inhibits the apoptotic process, while activation of the endothelin-B (ETB) receptor may induce cell death by apoptosis and inhibit tumor progression. Hypermethylation and subsequent silencing of the ETB receptor gene promoter has been reported in some cancer types. As the endothelin pathway is subject to research for pharmacological cancer treatment, we investigated the extent of epigenetic deregulation of the ETB receptor gene in non-small cell lung cancer (NSCLC). We scanned 64 NSCLC paired tumor/normal surgical specimens for the ETB receptor promoter for methylation by developing four pyrosequencing assays that covered 24 CpGs. The ETB receptor promoter was significantly hypermethylated in 31 (48%) of tumor samples, presenting considerably higher methylation in 22/24 CpG sites compared with the normal counterpart tissues. ETB receptor mRNA levels were reduced in all lung tumors compared with normal adjacent lung tissue, indicating the potentially important involvement of this gene in lung cancer development. Furthermore, tumor samples with ETB receptor gene methylation tended to have lower receptor mRNA levels compared with unmethylated tumor specimens, suggesting a primary epigenetic role in ETB receptor silencing. Our results point to a significant involvement of ETB receptor epigenetic deregulation in the pathogenesis of lung cancer making the gene a promising candidate biomarker for response to regimens modulating the endothelin axis.

The use of real-time PCR methods in DNA sequence variation analysis.

BACKGROUND: Real-time (RT) PCR methods for discovering and genotyping single nucleotide polymorphisms (SNPs) are becoming increasingly important in various fields of biological sciences. SNP genotyping is widely used to perform genetic association studies aimed at characterising the genetic factors underlying inherited traits. The detection and quantification of somatic mutations is an important tool for investigating the genetic causes of tumorigenesis. In infectious disease diagnostics there is an increasing emphasis placed on genotyping variation within the genomes of pathogenic organisms in order to distinguish between strains. METHODS: There are several platforms and methods available to the researcher wishing to undertake SNP analysis using real-time PCR methods. These use fluorescent technologies for discriminating between the alternate alleles of a polymorphism. There are several real-time PCR platforms currently on the market. Two of the key technical challenges are allele discrimination and allele quantification. CONCLUSIONS: Applications of this technology include SNP genotyping, the sensitive detection of somatic mutations and infectious disease subtyping.

RNA-Seq Differentiates Tumour and Host mRNA Expression Changes Induced by Treatment of Human Tumour Xenografts with the VEGFR Tyrosine Kinase Inhibitor Cediranib.

Pre-clinical models of tumour biology often rely on propagating human tumour cells in a mouse. In order to gain insight into the alignment of these models to human disease segments or investigate the effects of different therapeutics, approaches such as PCR or array based expression profiling are often employed despite suffering from biased transcript coverage, and a requirement for specialist experimental protocols to separate tumour and host signals. Here, we describe a computational strategy to profile transcript expression in both the tumour and host compartments of pre-clinical xenograft models from the same RNA sample using RNA-Seq. Key to this strategy is a species-specific mapping approach that removes the need for manipulation of the RNA population, customised sequencing protocols, or prior knowledge of the species component ratio. The method demonstrates comparable performance to species-specific RT-qPCR and a standard microarray platform, and allowed us to quantify gene expression changes in both the tumour and host tissue following treatment with cediranib, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor, including the reduction of multiple murine transcripts associated with endothelium or vessels, and an increase in genes associated with the inflammatory response in response to cediranib. In the human compartment, we observed a robust induction of hypoxia genes and a reduction in cell cycle associated transcripts. In conclusion, the study establishes that RNA-Seq can be applied to pre-clinical models to gain deeper understanding of model characteristics and compound mechanism of action, and to identify both tumour and host biomarkers.

Application of oligonucleotide arrays to high-content genetic analysis.

The scope of single nucleotide polymorphism genotyping for genetic association studies has expanded recently from the use of relatively small numbers of candidate genes and markers, to include hypothesis-free, whole-genome approaches using hundreds of thousands of polymorphisms. The ability to perform such large-scale association studies has been dependant on the development of highly parallel and cost-effective genotyping platforms, of which those based on oligonucleotide arrays have proved to be the most scalable and widely adopted. It is to be expected that the new array-based genotyping methods will not only greatly expand the scope of genetic studies, but, as further content is added to arrays, will also form part of an integrated set of DNA, RNA and proteomic analyses enabling the detailed, multilayered study of complex disease-linked phenotypes.

Development of a facile fluorescent assay for the detection of 80 mutations within the p53 gene.

BACKGROUND: Alterations in the p53 tumor suppressor gene constitute one of the most frequent genetic events associated with the development of human cancers. Determination of an individual's p53 status may be of value in early diagnosis, prediction of response to treatment, and for the detection of minimal residual cancer. Recent studies have also revealed that specific mutations affecting the p53 gene are associated with a poor outcome. The majority of tumor biopsies that are sent for study in the laboratory contain neoplastic cells intermingled with stroma, such that the detection of alterations in the p53 gene requires a tumor enrichment technique and/or highly sensitive mutation detection technologies. Thus, it is desirable that a clinically useful assay for detecting point mutations in the p53 gene function in the presence of significant quantities of wild-type sequence and identify the critical sequence aberrations. MATERIALS AND METHODS: We utilized molecular beacons in a real-time allele-specific PCR format to obtain reference data on samples of quantitatively known p53 mutation status. These data have been statistically analyzed and the results used to detect p53 mutations, indicating the presence of occult tumor. RESULTS: We describe validation of a simple, rapid, sensitive, and quantitative ARMS assay for identifying the levels of 80 point mutations within the p53 gene that, when mutated, constitute at least 1% of the total p53 sequences. CONCLUSIONS: The assay successfully identifies rare p53 gene mutations in clinical samples and overcomes many of the limitations of current technologies.