P-0194 A Friendly, Cheap and Very Sensitive Method for K-Ras Point Mutations Detection Based on PNA-Directed Pcr-Clamping in Metastatic Colorectal Carcinoma

ABSTRACT Introduction K-Ras mutations, occurring prevalently within codons 12/13, are detected in different cancer types including colorectal carcinoma (CRC) and might be responsible for resistance to anti-EGFR monoclonal antibodies (Cetuximab®) treatment. A sensitive method for K-Ras mutations detection is therefore crucial issue for choosing the most appropriate therapy. Currently, Sanger sequencing (SS) is accepted as gold standard method for mutational analysis but it is time-consuming and cost-ineffective. In addition, since in tumor analysis mutated clone may be present at different percentage, the SS sensitivity limit of 15-20% is crucial. Pyrosequencing (PS) has 5% detection limit, but still cost-ineffective, not largely available and shown a grey area (5-10%) in which genotype remains undeterminable. Therefore, a more sensitive method for detecting k-Ras mutations represents still a challenge. In this study we developed a novel, sensitive, friendly to use and very cheap assay for K-Ras mutations in CRC, based on PNA-directed PCR-clamping. Methods PNAs are synthetic molecules acting as potent DNA mimic in terms of sequence specific hybridization. As consequence, PNA/DNA heteroduplex is thermodynamically more stable than DNA/DNA counterpart but PNA cannot be extended by polymerase to function as a primer. Based on this, we designed a PNA molecule and one of the PCR primers perfectly matching the WT K-Ras sequence (encompassing codons 12/13), thus leading to a direct competition towards complementary template genomic DNA. In WT samples, perfect matching occurs because PNA-template hybridization is favoured and DNA amplification suppressed. By contrast, in case of mutated K-ras a single base pair mismatch will strongly impair the PNA-template stability favouring PCR amplification. Sixty formalin-fixed paraffin-embedded specimens from metastatic CRC were analyzed for K-Ras mutations by using both SS and PS and results and compared with PNA-directed PCR clamping method. Sensitivity was tested performing serial dilutions with plasmids carrying either wt or mutated sequences. Results PNA-methods displayed higher sensitivity compared to both SS and PS, allowing mutations detection up to 98.5%. The results showed a concordance of 63.3% among the three methods, while the concordance between PS and PNA-method resulted significantly increased. 47 samples were found to be mutated by PNA-PCR clamping, while 30 and 26 by PS and SS, respectively. Major discrepancy (13 out of 14) regards the detection of mutations by PNA-methods, otherwise unidentified either by SS or PS. Four samples, previously fallen into grey area, were classified as mutated by PNA-method. PNA-method failed to detect mutations just in 1 out of 30 cases, thus displaying a specificity of 96.66%. Correlation between clinical outcome after Cetuximab treatment and genotyping by PNA-method, which will confirm the clinical usefulness of the new method, is ongoing. Conclusion We developed an innovative method, able to detect K-Ras mutations in tumor paraffin-embedded specimens with very high sensitivity (98.5%). This is a cheap, easy to apply, quick and sensitive technique that could represent a novel tool, useful in clinical practice to drive therapeutic decisions.