A secondary epidermal growth factor receptor (EGFR) mutation, the substitution of threonine 790 with methionine (T790M), leads to acquired resistance to reversible EGFR-tyrosine kinase inhibitors (EGFR-TKIs). A non-invasive method for detecting T790M mutation would be desirable to direct patient treatment strategy. Plasma DNA samples were obtained after discontinuation of gefitinib or erlotinib in 75 patients with non-small cell lung cancer (NSCLC). T790M mutation was amplified using the SABER (single allele base extension reaction) technique and analyzed using the Sequenom MassARRAY platform. We examined the T790M mutation status in plasma samples obtained after treatment with an EGFR-TKI. The SABER assay sensitivity using mixed oligonucleotides was determined to be 0.3%. The T790M mutation was detected in 21 of the 75 plasma samples (28%). The presence of the T790M mutation was confirmed by subcloning into sequencing vectors and sequencing in 14 of the 21 samples (66.6%). In this cohort of 75 patients, the median progression-free survival (PFS) of the patients with the T790M mutation (n = 21) was not statistically different from that of the patients without the mutation (n = 54, P = 0.94). When patients under 65 years of age who had a partial response were grouped according to their plasma T790M mutation status, the PFS of the T790M-positive patients (n = 11) was significantly shorter than that of the T790M-negative patients (n = 29, P = 0.03). The SABER method is a feasible means of determining the plasma T790M mutation status and could potentially be used to monitor EGFR-TKI therapy. (Cancer Sci 2013; 104: 1198-1204

Background:Germline alterations in the proapoptotic protein Bcl-2–like 11 (BIM) can have a crucial role in tumor response to treatment. To determine the clinical utility of detecting BIM deletion polymorphism in non–small-cell lung cancer positive for epidermal growth factor receptor (EGFR) mutation, we examined outcomes of patients with and without BIM alterations.Methods:We studied 70 patients with EGFR mutation-positive non–small-cell lung cancer who were treated with an EGFR tyrosine kinase inhibitor between January 2008 and January 2013. BIM deletion was analyzed by polymerase chain reaction in 58 samples of peripheral blood and 24 formalin-fixed paraffin-embedded slides of surgical specimens (20 of lung tissue and four of brain tissue); both blood and tissue specimens were available for 12 patients. We retrospectively analyzed clinical characteristics, response rate, toxicity, and outcomes among patients with and without BIM deletion.Results:BIM deletion was present in 13 of 70 patients (18.6%). There were no significant differences between patients with and without BIM deletion in clinical characteristics, rate of response to EGFR tyrosine kinase inhibitor, or incidence of adverse events. Patients with BIM deletion had significantly shorter progression-free survival (PFS) than those without BIM deletion (median, 227 versus 533 days; p < 0.001). Multivariate Cox regression analysis showed that BIM deletion was an independent indicator of shorter PFS (hazard ratio, 3.99; 95% confidence interval, 1.864–8.547; p < 0.001).Conclusions:Polymerase chain reaction successfully detected BIM deletion in samples of peripheral blood and formalin-fixed paraffin-embedded slides of surgical specimens. BIM deletion was the most important independent prognostic factor in shorter PFS.

Long non-coding RNAs (lncRNAs) have been reported to be involved in the physiological and pathological processes of tumor pathogenesis, including epithelial-mesenchymal transition (EMT). However, epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) resistance is a major challenge in the treatment of advanced and recurrent EGFR-mutant lung adenocarcinoma. An increased understanding of the underlying mechanisms would aid in the development of effective therapeutic strategies against EGFR-TKI resistance, strategies which are urgently required for clinical therapy. In this study, long non-coding RNA LINC00460 was identified as a novel marker of a poor response to EGFR-TKI and prognosis. In lung cancer cells, LINC00460 promoted EGFR-TKI resistance as a competitive decoy for miR-149-5p, thereby facilitating interleukin (IL)-6 expression and inducing EMT-like phenotypes. The knockdown or knockout of LINC00460 in gefitinib-resistant non-small cell lung cancer cells restored the response to EGFR-TKI. In addition, as compared with patients with a low LINC00460 expression in tumors, those with a high LINC00460 expression had a significantly shorter progression-free survival following gefitinib therapy, and a shorter overall survival. Therefore, LINC00460 may be a predictor of and potential therapeutic target for EGFR-TKI resistance.

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11052 Background: Germ line alterations in the proapoptotic protein BCL2-like 11 (BIM) can have a crucial role in how a tumor responds to treatment. To clarify the clinical usefulness of detecting BIM deletion polymorphism in non-small cell lung cancer (NSCLC) with positive epidermal growth factor receptor (EGFR) mutation we examined the prognosis in patients with or without the BIM changes. Methods: Seventy NSCLC patients with positive EGFR mutation treated with EGFR-tyrosine kinase inhibitor (EGFR-TKI) between January 2008 and January 2013 were enrolled in this study. BIM deletion polymorphism was analyzed by PCR in 58 peripheral blood samples, 24 formalin-fixed paraffin-embedded (FFPE) slides of surgical specimens (lung tissue in 20, brain tissue in 4), and 12 samples that included both blood and tissue specimens. We performed retrospective analyses on clinical characteristics, response rate and toxicity of EGFR-TKI, and estimated the prognosis in patients with or without BIM deletion polymorphism. Results: BIM deletion polymorphism was present in 13 of 70 patients (18.6%) with homozygous deletion in 1 and heterozygous deletion in 12. There was no discordance between the two types of samples among the 12 patients. There were no significant differences between patients with or without BIM deletion polymorphism on clinical characteristics, response rate to EGFR-TKI and incidence of EGFR-TKI toxicity. Patients with BIM deletion polymorphism showed significantly shorter PFS that in patients without BIM deletion polymorphism (median: 216 days vs. 430 days, p < 0.001). There was no significant difference in OS. In multivariate analyses using the Cox regression model, BIM deletion polymorphism (hazard ratio = 4.2, p< 0.001, 95% CI = 2.026-8.777) was identified as an independent factor for shorter PFS. Conclusions: BIM deletion polymorphism could be detected by PCR on peripheral blood samples and FFPE slides of surgical specimens. BIM deletion polymorphism has emerged as an independent prognostic factor for shorter PFS. Therefore, new treatment strategies should be established for patients with BIM deletion polymorphism.