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The mechanisms of action of gemcitabine (GEM) and paclitaxel (PTX) have been well investigated, and shown to be the inhibition of DNA polymerase and polymerization of tubulin, respectively. Meanwhile, genomic research has revealed that mutations in the K-RAS oncogene occur in over 90% of pancreatic cancer. Oncogenic alteration rewires alternative metabolic pathways to satisfy the demands of growth. The K-RAS oncogene also has been shown to upregulate glycolysis and glutaminolysis. However, it is still unclear whether K-RAS independently plays a central role in controlling tumor metabolism. Here, we conducted a metabolomic analysis of a simple oncogenic K-RAS cell line model constructed using human telomerase catalytic subunit-immortalized human pancreatic epithelial nestin-expressing cell lines with and without K-RAS G12D . We also investigated the effect of GEM and PTX on these cells. As a result, it was shown in the cell with K-RAS G12D that the level of lactate was increased and glutamic acid, glutamine, and aspartic acid levels were decreased. In the nucleotide metabolism, GEM-treated cells showed metabolic changes, whereas these phenomena were not observed in PTX-treated cells. In conclusion, it was suggested that K-RAS G12D independently modified tumor metabolism and the difference between GEM and PTX in the nucleotide metabolism was revealed.Pancreatic cancer is the fourth-leading cause of cancer-related death in Japan and the USA (25). Surgical resection is the only potentially curative treatment, but the majority of patients have unresectable disease at the time of diagnosis due to distant metastasis or vascular involvement. Gemcitabine tetrahydrofuran-2-yl]-1H-pyrimidin-2-one) monotherapy became a standard treatment for unresectable pancreatic cancer after it demonstrated superior clinical benefit over 5-fluorouracil (3). Median overall survival for patients who received GEM monotherapy for advanced pancreatic cancer in key trials has ranged from 5.7-6.2 months (3,22). Numerous phase III trials of other treatments have failed to meaningfully improve overall survival over GEM monotherapy (18,14,28). Although GEM plus erlo-

Background: The third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), osimertinib, is effective against patients with non-small-cell lung cancer (NSCLC) showing EGFR T790M-induced resistance to first generation EGFR-TKIs. Moreover, the potential of osimertinib as an effective initial therapy for treatment-naïve patients with NSCLC harboring EGFR-activating mutations has been reported. Unfortunately, cases of acquired osimertinib resistance exist, and the involvement of genetic alterations such as EGFR C797S mutation and MET amplification has been reported, although the resistance mechanisms are not fully understood. We have previously demonstrated that first-generation EGFR-TKI-resistant NSCLC cells possess altered metabolic pathways, which might be targeted for therapy to overcome this resistance. Therefore, we aimed to identify the metabolic profile relevant to osimertinib-resistance and develop a targeted therapy for the altered metabolic signature. Methods: Osimertinib-resistant cell lines were generated by exposing HCC827 cells harboring the EGFR-activating mutation to increasing osimertinib concentrations (OSR1, OSR2). Moreover, we selected erlotinib-resistant cell lines that also showed osimertinib-resistance (EOSR1, EOSR2) from previously generated erlotinib-resistant cells obtained by prolonged exposure of HCC827 to erlotinib. Whole-exome sequencing, gene expression profiling, and metabolic profiling were performed to identify the genetic, gene expression, and metabolic signatures associated with osimertinib resistance, respectively. The growth-inhibitory effect of the inhibitors was evaluated by the WST assay. Results: Four osimertinib-resistant cell lines showed resistance to whole-generation EGFR-TKIs, namely erlotinib, afatinib and osimertinib. EGFR T790M or C797S mutations were not detected, while MET amplification was commonly observed in these resistant cell lines. These cells also contained higher concentrations of fructose 1, 6-bisphosphate and lactic acid than HCC827, possibly because of a significant increase in hexokinase expression. Activation of the pentose phosphate pathway and subsequent nucleotide metabolism were also observed in the OSR cells, but not in the EOSR cells. A combination of a competitive glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and osimertinib restored osimertinib sensitivity in the EOSR cells showing glycolysis dependence, but not in the OSR cells. Conclusions: Four resistant cell lines showed the same genetic alteration, namely, MET gene amplification; however, the metabolic signature and response to the 2-DG-osimertinib combination differed between the EOSR and OSR cells. Stratification based on tumor metabolic signature may contribute to the selection of promising therapeutic strategies for overcoming osimertinib resistance. Citation Format: Masakuni Serizawa, Rina Umehara, Keiichi Ohshima, Kenichi Urakami, Takeshi Nagashima, Ken Yamaguchi, Kyoichi Kaira, Toshiaki Takahashi, Masatoshi Kusuhara. Development of metabolic-targeting therapy for overcoming resistance to osimertinib in non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5838.

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Background: EGFR mutation testing is important in the treatment decision for advanced non-small cell lung cancer (NSCLC). Because T790M mutation in exon 20 is associated with approximately 50% of cases resistant to treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), the importance of re-biopsy for detecting T790M mutation during EGFR-TKI therapy is increasing. Circulating tumor DNA (ctDNA) is recognized as a promising target of minimally invasive diagnosis. Digital polymerase chain reaction (dPCR) is a new PCR technology for accurate quantification of the copy number of target molecules from low-input DNA. This study aimed to develop a blood-based EGFR mutation analysis in patients with advanced NSCLC by using a chip-based dPCR system that enables easy handling at a moderate costs. Methods: From October 2013 to March 2014, 49 patients with NSCLC harboring EGFR-activating mutations detected with the Scorpion ARMS method were enrolled in this study. ctDNA samples were extracted from plasma samples of 21 and 28 patients before treatment and after progression with EGFR-TKI, respectively. dPCR was performed on the QuantStudio 3D (QS3D) Digital PCR System (Life Technologies) by using 20 ng ctDNA, TaqMan probes for three EGFR mutations (exon 19 deletion, L858R in exon 21, and T790M in exon 20), and QS3D Digital PCR 20K Chips (2 chips per assay), according to the manufacturer's instructions. The performance of the dPCR assay was assessed by using HDx Reference Standards (Horizon) containing each mutant sequence serially diluted with that containing wild-type EGFR sequence. Results: Mutation of 0.1% was successfully detected in the dPCR assay for the three EGFR mutations. The median copy numbers of the EGFR mutation-positive samples were 2.9 copies (range, 0.3-84.2) for exon 19 deletion, 5.1 copies (0.6-742.1) for L858R in exon 21, and 1.4 copies (0.6-36.9) for T790M in exon 20. The sensitivity and specificity of each dPCR assay calculated by comparison with corresponding tumor samples were 61.8% (21/34) and 93.3% (14/15) for exon 19 deletion, and 66.7% (10/15) and 100% (34/34) for L858R, respectively. The T790M mutations were detected in 43% (12/28) of the ctDNA samples after progression with EGFR-TKIs therapy but in none of the samples before treatment with EGFR-TKIs. Among the 12 patients with NSCLC harboring T790M mutation in ctDNA, T790M mutations were detected in 5 of the 11 patients who underwent re-biopsy. Conclusions: This system showed similar performance to other dPCR assays used in previous studies (sensitivity: 66.6-92.0% and specificity: 95.7-100%). These results indicate the possibility that EGFR mutation testing with ctDNA using the chip-based dPCR for easy handling at a moderate costs is useful as a minimally invasive monitoring method in clinical settings. Citation Format: Norimitsu Kasahara, Hirotsugu Kenmotsu, Masakuni Serizawa, Rina Umehara, Akira Ono, Kazushige Wakuda, Shota Omori, Kazuhisa Nakashima, Tetsuhiko Taira, Tateaki Naito, Haruyasu Murakami, Noriaki Sunaga, Yasuhiro Koh, Keita Mori, Masahiro Endo, Takashi Nakajima, Masanobu Yamada, Masatoshi Kusuhara, Toshiaki Takahashi. Plasma epidermal growth factor receptor mutation (EGFR) testing in advanced non-small-cell lung cancer patients harboring EGFR mutations by chip-based digital PCR system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2248.