Abstract B180: A novel mutant-enriched liquidchip technology (MEL) for detecting circulating EGFR somatic mutations in non small cell lung cancer

Abstract

Abstract Epidermal growth factor receptor (EGFR) is a key target for developing targeted therapeutics to treat cancer. The US FDA has approved some EGFR targeted drugs that include monoclonal antibody drugs Erbitux, Panitumumab, as well as the EGFR tyrosine kinase inhibitors Iressa and Tarceva. However, clinical data indicated that only a small percentage of patients were responsive to these targeted drugs. Further studies have revealed that up to 20% of non small cell lung cancer (NSCLC) tumors that harbor the EGFR Exon 19 or 21 somatic mutations are more likely responsive, while patients without these mutations are not responsive. Therefore, physicians have a responsibility to test whether or not the patient's EGFR is mutated before prescribing these targeted drugs. In general, a very small amount of free DNA fragments can be found in the blood, especially in patients with malignant tumors. However, it is technically very difficult to detect a very small amount of DNA somatic mutations that are usually masked under a huge amount of the wild-type (WT) sequence. To cater to the clinical need, we have successfully developed a novel technology that creatively combines a mutant-enriched method to a liquidchip platform. The principle of MEL is to create a unique restriction enzyme site at the WT gene sequence in order to remove the WT gene by the restriction enzyme digestion before the mutated gene sequence could be selectively amplified by the PCR. The mutated PCR product will then be hybridized to a specific probe on polystyrene microspheres and analyzed by the Luminex. This technology is extremely sensitive and therefore allows us to use the serum or plasma sample to detect somatic gene mutations. It is also high-throughput; which allows us to assay up to 96 samples at once and read up to 100 different mutations from one sample. MEL technology has been successfully used in the clinic for detecting other somatic mutations from serum and plasma samples as well, such as KRAS, BRAF, PIK3CA, EGFR exon 20, etc. The detection sensitivity is one mutant DNA in the presence of 1×103 wild-type copies. As low as 10 copies of mutant DNA in a sample could be detected. To evaluate the MEL, the EGFR mutation status of NSCLC was tested on tissue samples using both sequencing and MEL methods. The positive samples of EGFR mutation by sequencing method were in agreement with the results by the MEL. However, 31.8% (7/22) of the negative samples by sequencing were positive by MEL. To date, our results have shown that 32.1% (146/455) serum samples of NSCLC patients showed EGFR mutant positive, from which 9.2% (42/455) were exon 19 deletion mutations; 19.8% (90/455), exon 21 single nucleotide polymorphism mutation; and 3.1% (14/455), both. Our study also showed that EGFR mutations were detected more frequently in patients with partial response following the targeted therapy than those with stable disease or progressive disease (p=0.027). In conclusion, MEL is a valuable method for detecting circulating EGFR mutations in serum or plasma of NSCLC patients for pharmacogenomic diagnostics. This method could be used on tissue samples as well. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B180.