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• 11515

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  P2.05 - Poster Session 2 - Preclinical Models of Therapeutics/Imaging (ID 158)

  • Event: WCLC 2013
  • Type: Poster Session
  • Track: Biology
  • Presentations: 4
  • Moderators:
  • Coordinates: 10/29/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 11519

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    P2.05-004 - Synergistic antitumor activity of bevacizumab and erlotinib in EGFR-mutated non-small cell lung cancer xenograft models (ID 310)

    09:30 - 09:30  |  Author(s): N. Harada
    • Abstract

    Background
    Erlotinib (ERL), a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, benefits patients with non-small cell lung cancer (NSCLC), especially if the cancer harbors EGFR active mutations (mtEGFR). However, those who initially respond eventually develop progressive disease through acquired resistance. Bevacizumab (BEV), a humanized anti-vascular endothelial cell growth factor monoclonal antibody, has been demonstrated to be effective in combination with standard chemotherapies for advanced NSCLC patients. We hypothesized that the combination of the two agents may be more effective because they work through different modes of action. In the present study, we examined the antitumor activity of BEV in combination with ERL in human NSCLC xenograft models harboring mtEGFR.

    Methods
    Mice (BALB-nu/nu) were subcutaneously inoculated with NSCLC cell lines harboring mtEGFR (exon19 deletion); HCC827 and B901L. BEV (5 mg/kg) was intraperitoneally administered once a week for 3 weeks, and ERL was orally given daily for 21 days at doses of 5 and 40 mg/kg for HCC827 and B901L, respectively. Antitumor activity was evaluated by tumor volume (TV; mm[3]) on day 22. In order to examine the prolonged antitumor effect of the combination of BEV with ERL, the B901L xenograft model was used. BEV (5 mg/kg) was intraperitoneally administered once a week for 13 weeks and ERL (60 mg/kg; maximum effective dose) was orally given daily for 91 days. The antitumor activity was evaluated on day 92, with an interim evaluation on day 22. The microvessel density (MVD) of tumor tissues was evaluated by CD31 immunohistochemistry of specimens obtained on day 2 from mice treated with BEV (5 mg/kg) and ERL (40 mg/kg). Statistical analysis was performed using the Wilcoxon test.

    Results
    In the HCC827 model, the TV (mean±SD) of control, BEV, ERL, and BEV+ERL was 1300±424, 686±84, 615±185, and 194±29, respectively. In the B901L model, the TV of control, BEV, ERL, and BEV+ERL was 1739±761, 819±293, 294±198, and 57±19, respectively. The antitumor activity of BEV+ERL was significantly better than that of BEV or ERL monotherapy (p≤0.05) in both models. In the prolonged treatment experiment using B901L, on day 22, BEV (1096±298) showed significantly higher (p≤0.05) tumor growth inhibition than control (2452±731), but the ERL (61±31) and BEV+ERL (53±30) showed even better (p≤0.05) tumor regression effects than BEV. However, during further treatment, tumor regrowth was observed in the ERL group, even though ERL was consecutively given, and the TV on day 92 (1105±1001) was significantly greater than that on day 22 (61±31). Tumor regrowth was also observed in the BEV+ERL group; however, it was much slower compared to the ERL group and there was no significant difference between TV on day 92 (79±70) and that on day 22 (53±30). The MVD was significantly decreased to the same level in the BEV and BEV+ERL groups.

    Conclusion
    Compared to ERL monotherapy, the combination of BEV and ERL demonstrated promising efficacy in mouse models of NSCLC harboring EGFR activating mutations. The encouraging preclinical results warrant further investigation in a clinical setting.

  • 11520

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    P2.05-005 - Antitumor activity of bevacizumab in combination with docetaxel in EGFR-TKI resistant non-small cell lung cancer xenograft models. (ID 313)

    09:30 - 09:30  |  Author(s): N. Harada
    • Abstract

    Background
    Bevacizumab (BEV), a humanized anti-vascular endothelial cell growth factor monoclonal antibody, is used in combination with chemotherapy for patients with non-small cell lung cancer (NSCLC). The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) benefit patients with NSCLC, especially when this cancer harbors EGFR mutations (mtEGFR); however, those who initially respond eventually develop resistance. To treat the patients with EGFR-TKI acquired resistance, a combination of BEV and docetaxel (DTX) may be a possible treatment modality based on different mechanisms of action. In the present study, we investigated the antitumor activity of BEV in combination with DTX in EGFR-TKI-resistant NSCLC xenograft models.

    Methods
    Mice (BALB-nu/nu) were subcutaneously inoculated with NSCLC cell lines; NCI-H1993 (MET amplification) and NCI-H1975 (mtEGFR/T790M). The treatment was started (day 1) after tumor growth was confirmed. BEV (5 mg/kg) was administered intraperitoneally on days 1, 8, 15 and DTX was administered intravenously on day 1 at a dose of 60 or 20 mg/kg as a maximum effective dose in NCI-H1993 or NCI-H1975 xenografts, respectively. The antitumor activity was evaluated by tumor volume (TV; mm[3]) on day 46 in the NCI-H1993 model and on day 50 (with an interim evaluation on day 19) in the NCI-H1975 model. Tumor cells in the proliferation phase were immunohistochemically evaluated using Ki-67 as an index (count/1000 tumor cells) on day 8 in both tumors. Statistical analysis was performed using the Wilcoxon test.

    Results
    In the NCI-H1993 model, the TV (mean±SD) of control, BEV, DTX, BEV+DTX on day 46 was 1630±492, 670±116, 204±85, 49±34, respectively. The antitumor activity of BEV+DTX was significantly better than that of DTX monotherapy (p≤0.05). In the NCI-H1975 model, TV of control, BEV, DTX, BEV+DTX was 4631±2384, 1581±572, 18±19, 16±10, respectively, on day 19. The BEV+DTX combination and DTX monotherapy showed similar effects on tumor regression at this point. However, after that, tumor regrowth was observed in the DTX group, and increased beyond the initial TV in 5 out of 6 mice, whereas no tumor regrowth was observed in BEV+DTX group at day 50. The TV of DTX and BEV+DTX groups on day 50 was 2631±2391 and <10 respectively. The number of Ki-67 positive cells (mean±SD) in tumor tissue taken from control, BEV, DTX, BEV+DTX on day 8 was, respectively, as follows: NCI-H1993: 672±50, 638±25, 559±38, 459±37; NCI-H1975: 533±28, 401±57, 381±49, 290±34. The number of Ki-67 positive cells in tumors treated with BEV+DTX was significantly lower than in those treated with DTX (p≤0.05) in both models, although there were no significant differences in TV between the two groups on day 8.

    Conclusion
    Combining BEV with DTX can decrease the tumor cells in a proliferation phase more effectively than DTX alone in the early stage of the therapy. This may provoke a stronger antitumor effect later on and delay tumor regrowth. These data are encouraging, and the combination of BEV+DTX may be a promising treatment modality for patients with NSCLC after acquiring a resistance to EGFR-TKIs.

  • 11521

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    P2.05-006 - Antitumor activity of bevacizumab in combination with pemetrexed as maintenance therapy for non-small cell lung cancer in a xenograft model: comparison with pemetrexed monotherapy and analysis of the combination mechanism (ID 655)

    09:30 - 09:30  |  Author(s): N. Harada
    • Abstract

    Background
    Bevacizumab (BEV) is a humanized monoclonal antibody against vascular endothelial cell growth factor (VEGF) that inhibits VEGF-mediated angiogenesis in many types of tumors. OS and PFS were significantly prolonged in patients with advanced non-small cell lung cancer (NSCLC) who received BEV maintenance after induction with BEV+chemotherapy (ECOG4599 study). The maintenance study AVAPERL demonstrated that, after induction with pemetrexed (PEM)+BEV+cisplatin therapy, BEV+PEM combination significantly prolonged PFS compared to BEV monotherapy. However, the comparative efficacy of BEV+PEM to PEM monotherapy is not clear. In the present study, we investigated the antitumor effect of the BEV+PEM combination compared to BEV or PEM monotherapy as continuation maintenance after BEV+PEM treatment in a human NSCLC xenograft model.

    Methods
    SCID mice were subcutaneously inoculated with human NSCLC cell line NCI-H2228. As an induction treatment, BEV (5 mg/kg; maximum effective dose) and PEM (400 mg/kg; maximum effective dose) were administered intraperitoneally on days 1, 8, and 15 after randomization by tumor volume (TV). On day 22, the mice treated with BEV+PEM were re-randomized and BEV and/or PEM were administered intraperitoneally weekly until day 85. The antitumor activity was evaluated by TV on day 85. Microvessel density (MVD) and thymidylate synthase (TS) expression in tumor tissues were evaluated using specimens taken on day 85. MVD was analyzed by CD31 immunohistochemistry and evaluated in terms of the ratio of stained area to observed area. TS expression was evaluated by Western blot analysis. Statistical analysis was performed using the Wilcoxon test.

    Results
    TV (mm[3]; mean±SD) of BEV+PEM group on days 1 and 22 was 335±72 and 329±59, respectively, whereas TV of control group on day 22 was 671±67, indicating that BEV+PEM completely inhibited tumor growth during induction treatment. TV of control, BEV, PEM, and BEV+PEM groups on day 85 was 2748±1334, 586±320, 883±271, and 348±48, respectively. Each treatment group showed a significantly higher antitumor activity (p < 0.001) compared to control. Specifically, BEV+PEM completely inhibited tumor growth throughout the experiment, and showed a significantly higher antitumor activity than BEV (p < 0.05) or PEM (p < 0.001) monotherapy. MVD (%) was significantly lower (p < 0.01) in BEV (1.40±0.36) and BEV+PEM (1.24±0.49) groups than in control (6.63±1.89) and PEM (4.62±0.71) groups, indicating that the decrease in MVD caused by BEV treatment was not affected by the combination with PEM. TS expression level, which is inversely correlated with the effect of PEM, was lower in BEV+PEM group than in PEM group.

    Conclusion
    Compared to PEM monotherapy, the combination of BEV with PEM showed better antitumor effect as a maintenance therapy in a xenograft model. Decreases in MVD and TS expression may contribute to the effect of combination therapy.

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    P2.05-007 - Significance of the recommended dose erlotinib for avoiding acquired resistance in NSCLC cells with exon19 deletion and L858R mutation of EGFR (ID 738)

    09:30 - 09:30  |  Author(s): N. Harada
    • Abstract

    Background
    Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, shows notable effects against non-small cell lung cancers (NSCLCs) harboring EGFR-activating mutations such as exon 19 deletion or L858R mutation. However, almost all patients eventually acquire resistance to erlotinib. In this study, we investigated whether the level of exposure to erlotinib affects incidence of acquired resistance in EGFR-mutated NSCLC cell lines and examined their resistant mechanisms.

    Methods
    Human NSCLC cells B901L and PC-9 harboring exon 19 deletion and II-18 cells with L858R mutation were continuously exposed to 0.5 or 5 µM of erlotinib in 96-well plates for several months. The upper dose was chosen because the recommended dose of erlotinib (150 mg) has been reported to achieve maximum plasma concentration (Cmax) of about 5 µM. Cell growth inhibition was determined by a crystal violet assay. EGFR mutation status was determined by melting curve analysis. MET copy number was determined by real-time PCR analysis. Phosphorylations of EGFR, HER2 and ERK were measured by Western blot analysis.

    Results
    Resistant B901L, PC-9 and II-18 cells were emerged in 24, 8 and 7 wells by exposing to 0.5 µM erlotinib, and in 13, 2 and 0 wells by exposing to 5 µM erlotinib, respectively. No alterations in EGFR mutation status including T790M mutation nor acquisition of MET amplification were detected in any resistant cells. In parent cells, treatment with erlotinib markedly inhibited phosphorylation of EGFR, HER2 and ERK. In the parent cells, treatment with erlotinib markedly inhibited EGFR phosphorylation but not HER2 and ERK phosphorylation, compared with the parent cells.

    Conclusion
    In present in vitro analysis, it was suggested that exposure to the recommended dose of erlotinib would avoid acquired resistance in NSCLC with exon19 deletion or L858R mutation of EGFR. Pathways activated by phosphorylated HER2 may affect acquired resistance to erlotinib.