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Yoshihisa Kobayashi



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    P1.13 - Targeted Therapy (Not CME Accredited Session) (ID 945)

    • Event: WCLC 2018
    • Type: Poster Viewing in the Exhibit Hall
    • Track:
    • Presentations: 2
    • Moderators:
    • Coordinates: 9/24/2018, 16:45 - 18:00, Exhibit Hall
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      P1.13-41 - In Vitro Evaluation for Optimal MET-TKI Selection in Lung Cancers with MET Mutations Including Exon 14 Skipping (ID 12825)

      16:45 - 18:00  |  Author(s): Yoshihisa Kobayashi

      • Abstract

      Background

      MET exon 14 skipping mutation present in 3-5% of adenocarcinoma of the lung is an emerging driver gene alteration. Clinical responses of these tumors to MET-TKI have been reported. However, response rates are not very satisfactory compared with EGFR/ALK/ROS1 TKI. Therefore, it is necessary to create in vitro model system to understand sensitivity/resistance mechanism for various types of MET-TKI, to establish optimal treatment strategy.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      We introduced MET exon 14 skipping mutation as well as Y1003F, D1010Y which had been also reported to be present in lung cancer, to mouse pro-B cell line, Ba/F3. Since Ba/F3 requires interleukin 3 (IL-3) for its growth, IL-3 independent growth of Ba/F3 indicates that transduced mutation is oncogenic. The growth inhibitory assays were then performed using 9 MET-TKIs that include all classes of MET-TKIs ; Type Ia (crizotinib), Type Ib (capmatinib, tepotinib, savolitinib and AMG337) , Type II (cabozantinib, merestinib and glesatinib) and Type III (tivantinib).

      4c3880bb027f159e801041b1021e88e8 Result

      Ba/F3 transfected with wild-type MET did not grow in the absence of IL-3, while all transfected with any of three mutated MET did so. In general, all type Ia/b / type II inhibitors were active for any of 3 MET mutations. Interestingly MET point mutations (Y1003F/D1010Y) were more sensitive to type Ib inhibitors except AMG337 than type II, while exon 14 skipping was likely to be more sensitive to type II inhibitors than type Ib compared with point mutations. IC50 / Cmax of cabozontinib was least for exon 14 skipping while that of capmatinib was least for Y1003F and D1010Y, suggesting most promising activity of these drugs (Table).

      table.png

      8eea62084ca7e541d918e823422bd82e Conclusion

      We found that the MET exon 14 skipping, Y1003F, and D1010Y mutations were all oncogenic in Ba/F3 system. Several type I/II inhibitors especially cabozantinib and capmatinib are expected to be active for treating lung cancer patients with MET mutations.

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      P1.13-42 - Activity of Novel HER2 Inhibitor, Poziotinib, for HER2 Exon 20 Mutations in Lung Cancer and Mechanism of Acquired Resistance (ID 12717)

      16:45 - 18:00  |  Author(s): Yoshihisa Kobayashi

      • Abstract

      Background

      Oncogenic HER2 mutations are present in 2-4% of adenocarcinoma of the lung. However, clinical trials of HER2 inhibitors such as afatinib or neratinib has been unsatisfactory. Recently, a novel HER2 inhibitor, poziotinib has been developed and clinical trial results are being expected. Here, we evaluated poziotinib in comparison with pre-existing TKIs using Ba/F3 system. We also derived resistant clones against poziotinib and investigated their resistant mechanism.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      We introduced three common HER2 mutations into Ba/F3 cells (i.e. G776delinsVC (VC), A775_G776insYVMA (YVMA) and P780_Y781insGSP (GSP)) which account for 13, 72, 9% of HER2 mutations in human lung cancer, respectively. We defined sensitivity index (SI) as an IC90divided by trough concentration of a given drug at the recommended dose for humans in the literature, as a surrogate for drug activity in humans. Poziotinib activity was compared with 8 TKIs (afatinib, osimertinib, erlotinib, neratinib, lapatinib, dacomitinib, irbinitinib, and AZ5104). In addition, we created resistant clones by exposing poziotinib in the presence of N-ethyl-N-nitrosourea (ENU) and HER2 secondary mutations were searched.

      4c3880bb027f159e801041b1021e88e8 Result

      All drugs but lapatinib showed the highest activity against VC (Table). In contrast, YVMA was most resistant in all but neratinib and poziotinib. For most common YVMA, poziotinib was the only drug that had SI of less than 10 (Table). Furthermore, poziotinib was most potent for VC and GSP except dacomitinib for GSP (Table). We established 19 poziotinib-resistant clones, all of which harbored C805S secondary mutation of the HER2 gene homologous to C797S of the EGFR gene.

      Ctrough [nM] YVMA VC GSP
      Afatinib 69 28 5.4 12
      Dacomitinib 166 20 4.4 6.6
      Erlotinib 2969 337 22 98
      Osimertinib 400 40 4.5 30
      Neratinib 100 11 11 28
      Lapatinib 516 133 117 91
      Poziotinib 20 6.0 2.7 10
      Irbinitinib 520 34 33 16
      AZ5104 50 60 8.0 48

      8eea62084ca7e541d918e823422bd82e Conclusion

      Poziotinib showed the most potent activity against HER2 exon 20 mutations. We also found that secondary C805S HER2 mutation was the common mechanism of acquired resistance, which most likely inhibit covalent binding of poziotinib with HER2.

      6f8b794f3246b0c1e1780bb4d4d5dc53

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    P2.03 - Biology (Not CME Accredited Session) (ID 952)

    • Event: WCLC 2018
    • Type: Poster Viewing in the Exhibit Hall
    • Track:
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/25/2018, 16:45 - 18:00, Exhibit Hall
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      P2.03-17 - EGFR T790M Mutation may not be Generated through Selection by EGFR-TKI from Randomly Occurring Mutations in Vitro Using ENU (ID 13205)

      16:45 - 18:00  |  Presenting Author(s): Yoshihisa Kobayashi

      • Abstract

      Background

      It is postulated that T790M resistant mutation is a result of selection by EGFR-TKI from clones that have randomly generated mutations. If so, it would be possible to detect the process of convergence from random mutations to T790M, depending on exposure time for EGFR-TKI.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      After exposure of Ba/F3 cells expressing EGFR Del19 to ENU (N-ethyl-N-nitrosourea) for 24 hours, they were cultured with various concentrations of 3 EGFR-TKIs at trough concentrations (Fig.). At 1, 6, 12, 24, and 48 hours, cell viability was evaluated and single cell cloning was performed. EGFR exons 18 to 21 were analyzed by Sanger sequencing.

      4c3880bb027f159e801041b1021e88e8 Result

      Percentages of viable cells exposed to TKI were approximately 80%, 50%, < 20%, and < 3% at 1, 6, 12, 48 hours, respectively. These ratios were almost similar among 3 TKIs. Of 50 clones that had survived TKI treatment, T790M was detected in 12 (24%) (Fig.). Notably, two were detected in cells exposed to erlotinib or afatinib only for one hour (Fig.). In addition, we found 2 G873E (c.2876G>A) mutations, known to be oncogenic, with the same TKI treatment. In contrast, 36 clones (72%) with TKI or additional 19 clones without TKIs that had been exposed to only ENU did not harbor any secondary EGFR mutations. secondary mutations of established clones.tif

      8eea62084ca7e541d918e823422bd82e Conclusion

      It is unlikely that EGFR T790M mutation is generated through selection by EGFR-TKI from randomly occurring mutations. Rather, it appears that T790 is the preferred target of mutagenesis through yet an unknown mechanism which occurs after only one-hour treatment.

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