Abstract 2089: Fully automated SNP detection from whole blood and mutation detection from plasma-free DNA

Purpose Gene polymorphism and genetic mutation are related to the therapeutic effect of many drugs, and a simple genetic test method is preferred. To meet this demand, we have developed a platform for genetic testing called i-densy, which can complete fully automated gene polymorphism analysis from whole blood in approximately 90 minutes, and compared it with the existing test method. Also, recent reports indicate that DNA derived from cancer cells are liberated in blood (it is called “circulating cell-free DNA”). Using the i-densy, we here tested EGFR gene T790M mutation in circulating cell-free DNA from a sample derived from advanced lung cancer patient with EGFR-TKI aquired resistance, and then compared with the existing test method. Methods The i-densy newly developed can perform fully automated gene polymorphism testing from 70μL of whole blood and allows typing of 3 polymorphisms or mutations in a single reaction. Four samples can be tested at once with this instrument, and testing is also possible using oral mucosa or purified nucleic acid instead of whole blood. In addition to the i-densy, circulating cell-free DNA was also tested using Mutation Biased PCR (MBP) method that can preferentially amplify mutation sequence. Results As a result of using the i-densy with whole blood for gene testing on 1236C>T, 3435C>T and 2677 G>(T/A) polymorphism of ABCB1 gene related to toxicity and pharmacokinetics of EGFR-TKI erlotinib, results of 1236C>T and 3435C>T matched with that of Taqman assay method and 2677 G>(T/A) matched with Direct sequencing method for all 20 samples. We also extracted free DNA from peripheral blood plasma of a patient, and tested EGFR T790M mutation using Mutation Biased PCR (MBP) method, PNA-LNA PCR clamp method and Cycleave method. Among 19 patients with acquired resistance to EGFR-TKI, number of patients tested positive for T790M were 10 (53%) using MBP method, 3 (16%) using PNA-LNA PCR method and 5 (26%) using Cycleave method. Also, when using MBP method, there was not a single case with T790M mutation detected among cases with continued therapeutic effect or which EGFR-TKI is not used. Conclusion The i-densy newly developed has been identified to be simple and fast that allows accurate gene polymorphism from whole blood, and also confirmed to be useful in clinical practice. As a result of EGFR gene T790M mutation testing from circulating cell-free DNA using the i-densy and MBP method, appearance ratios of acquired resistance mutati