Validation of selected molecular methods for the mutations determination in codons 12 and 13 of K-RAS gene in five Polish oncological research centers

Abstract

Metastatic colorectal cancer patients will benefit from treatment with panitumumab only when they don't have mutation in K-RAS gene. Therefore, estimation of mutational status of K-RAS is necessary for the selection of patients, who should be treated with panitumumab.
The aim of this study was to evolve a standard method of estimation of K-RAS mutational status in the material isolated from paraffin blocs. The second aim was the validation of selected molecular methods of K-RAS mutation evaluation in five Polish oncological centers where mCRC patients are treated. Four methods were evaluated: SSCP, DHPLC, RFLP/PCR and direct sequencing.
We found that all groups in five selected oncological centers, who took part in the validation process, were well prepared for molecular diagnosis of K-RAS mutational status. The following recommendations for diagnostic laboratories were approved: 1. At least 70% of cancer cells should be present in a tissue for DNA isolation; 2. The method of DNA isolation should be standardized, the most appropriate is usage of DNA isolation kits; 3. In case of equivocal results two independent molecular methods should be employed, one of them should be direct sequencing.