Comparative analysis of somatic K-RAS gene mutation frequency in the clinical NSCLC specimens

Accurate and reliable oncological diagnosis is an important tool that facilitates selection of the most efficient treatment available. Based on the somatic and autosomal genetic makeup of a particular cancer patient, treatment selection can be customised to provide maximum potential benefit to the patient. The identification of somatic mutations in the K-RAS gene can provide very useful information regarding the responsiveness of cancer patients (especially smokers) to selected drugs. However, screening of cancerous tissue specimens collected during biopsies and surgeries is not yet routinely performed due to the lack of a cost-efficient and well-standardized detection method, sufficiently reliable for the detection of somatic mutations in heterogeneous oncological specimens. Several alternative K-RAS mutation detection sequencing and non-sequencing methods have been described in the literature. We have selected three methods and have compared their reliability in detecting K-RAS mutation frequency in clinical specimens. Analysis of seventy one NSCLC (NonSmall Cell Lung Cancer) biopsy samples employing the nested-PCR method of DNA amplification failed to detect any K-RAS mutations after direct dideoxy sequencing (BigDye Terminator). The subsequent the same cohort of patients’ specimens, using direct genomic DNA as a template for the amplification of a PCR fragment for sequencing, resulted in a finding that 3% of the specimens contained somatic mutations in either codon 12 or codon 13 of the K-RAS gene. This comparison clearly demonstrates the inferiority of a nested-PCR method compared to single PCR amplification of genomic DNA template. A BigDye-termination method following regular PCR amplification of the genomic DNA template represents a reliable and reproducible method that allows finding K-RAS gene mutation(s) but the extracted genomic DNA template should contains at least 30% of mutated at the K-RAS locus DNA. Therefore, in some cases when less than 30% of genomic DNA comes from non-mutated cells, this analysis may not detect an existent mutation. Furthermore, these results suggest that the nested PCR, which by definition re-amplifies the targeted DNA fragment, should be avoided when using genomic DNA isolated from specimens that are likely to contain both nonmutated normal tissue, and cancer cells that may contain somatic mutations in the K-RAS gene. The observed difference in frequency of detection of KRAS mutations between the two methods most likely results from preferential amplification of non-mutated DNA. This bias could lead to a distortion of the results when the PCR amplification step is repeated. The ratio between the non-mutated and mutated genomic DNA can be reliably assessed by using a pyrosequencing method. The pyrosequencing method is designed not to be influenced by differences in amplification efficiency. Even when less than 10% of the DNA present in the genomic specimens is mutated, it can still be reliably quantified and sequenced using this method. Therefore, we intend to employ the Biotage PyroMark pyrosequencing method to analyse the same genomic DNA samples that were previously analyzed by the two methods described above. We anticipate that using the pyrosequencing method will allow us to establish the ratio between genomic DNA that is mutated or nonmutated at the K-RAS locus, and will improve our ability to detect K-RAS mutations in our cohort.
The project was supported in parts from grants MSM6198959216 and MPO 1H-PK/45.