Quantification of T790M mutation in EGFR-mutant lung adenocarcinoma with nanofluidic digital PCR arrays.

11054 Background: Patients with EGFR-mutant lung adenocarcinoma develop acquired resistance to EGFR-TKI (TKI) in 10-16 months. About 50% of patients with acquired resistance to TKI have T790M mutation (T790M). However, repeat biopsy to monitor mutation status is not practical. Digital PCR arrays limiting dilution of DNA and can detect single molecules, thus enabling extremely sensitive detection and quantification. We evaluated the usefulness of nanofluidic digital PCR arrays in EGFR-mutant lung adenocarcinoma. Methods: We enrolled 12 patients with primary lung adenocarcinoma and EGFR mutation (exon 19 deletion in 8; L858R in 4) at the pretreatment primary site who acquired resistance to gefitinib. Patients were divided into 2 groups according to T790M status after TKI resistance, as confirmed by rebiopsy. Nanofluidic digital PCR arrays (BioMark HD System, Fluidigm Japan K.K. Tokyo, Japan) were used to quantify T790M in genomic DNA from the pretreatment primary site and serum cell-free DNA (cfDNA) after TKI resistance. Numbers of mutant molecules were estimated by the number of positive chambers in the digital PCR chip and were corrected using the Poisson equation. We assessed the ratio of the number of positive T790M molecules to the number of positive exon 2 molecules. Results: The digital PCR array detected and quantified T790M (0.00-1.21%) in samples from the pretreatment primary site and serum cfDNA after TKI resistance. On digital PCR, the rebiopsy-positive T790M group (n=4) had a significantly higher quantified T790M at the pretreatment primary site than did the T790M-negative group (n=8) (0.59±0.58% vs 0.07±0.09%, p < 0.001). However, the results of analysis of serum cfDNA after TKI resistance did not significantly differ (0.09±0.14% vs 0.05±0.15%, respectively, p = 0.41). T790M at the pretreatment primary site as quantified by digital PCR significantly positively correlated with progression-free survival (PFS) after gefitinib therapy (r = 0.71, p < 0.001). Conclusions: Use of digital PCR to quantify T790M at the primary site of EGFR-mutant lung adenocarcinoma was useful for predicting T790M positivity in rebiopsies after TKI resistance and PFS after gefitinib therapy.