New Test for KRAS Mutations: Faster, Cheaper, Better

Zosia Chustecka

July 03, 2012

July 3, 2012 (Barcelona, Spain) — A simpler, cheaper, more accurate test for KRAS mutations has been developed by a group of Italian researchers.

Routine testing for KRAS mutations in the treatment of colorectal cancer is now recommended by many expert bodies. Testing identifies patients who carry this mutation and are unlikely to respond to treatment with the anti–epidermal growth factor receptor (EGFR) monoclonal antibodies cetuximab (Erbitux, Bristol-Myers Squibb, Lillly) and panitumumab (Vectibix, Amgen). Targeted therapies are indicated only for use in patients with KRAS wild-type disease.

Details on the new test were presented in a poster exhibited here at the 14th World Congress on Gastrointestinal Cancer, which was organized in partnership with the European Society for Medical Oncology.

"This new test is cheaper, quicker, and more sensitive, so the genotyping of patient[s] is more accurate," senior author Christiana Lo Nigro, PhD, from the Laboratory for Cancer Genetics and Translational Oncology at the S. Croce General Hospital, in Cuneo, Italy, told Medscape Medical News.

Currently, the gold standard for KRAS mutation testing is Sanger sequencing of polymerase chain reaction (PCR) products. "Although this detects and identifies all mutations in an amplified sequence, it is time-consuming, cost-ineffective, and has a maximum sensitivity of around 15 to 20%," Dr. Lo Nigro stated.

Another method — high-throughput pyrosequencing (PS) — is faster but still takes about a day. "It too is cost-ineffective," she said, because the equipment is expensive and is available in only a few centers. In addition, although PS has a detection limit of around 5%, about 5% to 10% of samples fall into a "gray area," in which the results are undeterminable.

The new method developed by the Italian group is based on primer exclusion peptide nucleic acid (PNA)-directed PCR clamping (PNA assay). "There is PCR competition between a standard primer and a newly synthesized PNA molecule," Dr. Lo Nigro explained. "Most laboratories already have PCR facilities and only need to synthesize the new 15mer PNA [NH2-GGAGCTGGTGGCGTA-CONH2] to act as the comparator in order to perform the test," she commented.

The new test takes only 1 hour and is more sensitive than any other currently available strategy, the researchers report. "While the sensitivity limit is around 20% for Sanger sequencing and around 10% for pyrosequencing, for the new test it is only 2%," Dr. Lo Nigro explained.

"With this new test," she pointed out, "there is only a 2% chance of patients with the mutation being incorrectly identified as KRAS wild type [not having KRAS mutations]. This is important," she affirmed, "as identifying patients incorrectly could lead them to receive a drug from which they won't benefit but which may cause side effects."

Study Compares Different Approaches

In the poster, the researchers reported results from a comparison of the 3 methods when used for blind testing for KRAS mutations in tumor samples taken from 68 patients with colorectal cancer. Sanger sequencing found KRAS mutations in 29 of 68 samples (42.6%), pyrosequencing found them in 32 of 68 samples (47.1%), and the new PNA-mediated clamping PCR method found them in 53 of 68 samples (77.9%).

Another study, which used 3 independent experiments to determine the sensitivity of the PNA-mediated clamping PCR for the detection of KRAS mutations, found that the test reached a sensitivity as high as 1.4% for the mutated versus the wild-type allele (P < .005).

"We have provided evidence that the PNA-mediated clamping PCR is more sensitive," the researchers conclude. This method also has shown fast run time, low cost, and wide applicability, requiring just a single-step modified PCR and electrophoresis equipment.

"This technology is suitable for routine analysis to support clinical decisions about the administration of EGFR-directed therapy," they conclude.

14th World Congress on Gastrointestinal Cancer (WCGC): Abstract P-0194. Presented June 29, 2012.

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