Study validates Transgenomic's COLD-PCR technology for colorectal tumor-associated KRAS mutation detection

Transgenomic, Inc. (OTC Bulletin Board: TBIO) announced today that it has completed a preliminary study with a leading pharmaceutical company that validates the use of its licensed COLD-PCR technology to detect colorectal tumor-associated KRAS mutations that determine efficacy of recently developed therapies. These were detected in plasma samples in which the mutation levels were too low for detection by standard DNA analysis methodologies such as Sanger sequencing.

The study, which was performed by Transgenomic's Pharmacogenomics Services Laboratory in Omaha, NE and the company's Molecular Biology research team in Gaithersburg, MD, consisted of testing DNA extracted from colorectal tumor-matched plasma samples. Screening with standard Sanger sequencing of the plasma samples resulted in only 70% concordance between matched plasma and tumor due to missing KRAS codon 12 and 13 mutations.

The samples were enriched by Transgenomic's COLD-PCR mutation enrichment technology before analysis with the SURVEYOR SCAN KRAS assay. COLD-PCR preferentially amplifies genomic DNA mutations in comparison to normal, wild-type sequences. This powerful enrichment and screening method can detect matched-tumor KRAS mutations in serum mutation levels at less than 0.1% of total wild-type DNA.

This resulted in the accurate detection of all KRAS codon 12 and KRAS codon 13 mutations with full discrimination against wild-type or normal samples tested concurrently. Due to the extent of the enrichment process, all of these KRAS containing plasma samples could subsequently be verified by DNA sequencing.

It was confirmed by the study's pharmaceutical partner that there was a 100% concordance between Transgenomic's mutation results and the matched tumor KRAS genotypes.

"We believe that the developments in COLD-PCR that we are pursuing at Transgenomic will open the way for detecting tumor mutations in surrogate tissues and body fluids such as serum, plasma and urine rather than by tumor biopsy," said Craig Tuttle, CEO of Transgenomic. "This study is a very promising start of the process of validating this technology in a clinically oriented setting. We intend to participate in further studies such as this with the aim of developing COLD-PCR serum assays for key gene mutations that are biomarkers for early tumor detection, tumor therapy efficacy, emergence of drug resistance biomarkers and relapse."

COLD-PCR was discovered in the laboratory of Dr. Mike Makrigiorgos at the Dana Farber Cancer Institute. It has been exclusively licensed by Transgenomic for all Sanger Sequencing and mitochondrial DNA applications. Furthermore, it can be coupled with Transgenomic's proprietary DNA mutation detection technologies to further improve its sensitivity.

Technical Information

When mutant and reference DNA samples from the same gene are mixed and re-annealed, variations between these sequences cause double-stranded DNA heteroduplexes to form. The WAVE System employs denaturing HPLC to separate these homo- and hetero-duplexes by ion-pairing reverse-phase HPLC. This technology has been in widespread use for genomic analysis being cited in over 2000 peer-reviewed publications. As an alternative offering, Transgenomic's SURVEYOR Nuclease cleaves such heteroduplexes with high specificity at sites of base mismatch or small insertions/deletions. It has a proven track record as a robust and reliable tool in analyzing DNA variations, especially where the mutant alleles are at a very low concentration within the sample (less than 1% of the total wild type allele concentration). COLD-PCR protocols preferentially amplify heteroduplexes such that mutant alleles become enriched compared to normal alleles. The range of enrichment demonstrated to date varies from 3 to 100-fold, which will contribute to Transgenomic's target of achieving a 1/10,000 mutant to normal allele ratio detection in a routine, cost-effective and high throughput protocol. This level of detection will allow straightforward tumor analysis via surrogate tissues such as blood and urine.