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Non-small cell lung cancer (NSCLC) tumors harboring mutations in EGFR ultimately relapse to therapy with EGFR tyrosine kinase inhibitors (EGFR TKIs). Here, we show that resistant cells without the p.T790M or other acquired mutations are sensitive to the Aurora B (AURKB) inhibitors barasertib and S49076. Phospho-histone H3 (pH3), a major product of AURKB, is increased in most resistant cells and treatment with AURKB inhibitors reduces the levels of pH3, triggering G1/S arrest and polyploidy. Senescence is subsequently induced in cells with acquired mutations while, in their absence, polyploidy is followed by cell death. Finally, in NSCLC patients, pH3 levels are increased after progression on EGFR TKIs and high pH3 baseline correlates with shorter survival. Our results reveal that AURKB activation is associated with acquired resistance to EGFR TKIs, and that AURKB constitutes a potential target in NSCLC progressing to anti-EGFR therapy and not carrying resistance mutations., Non-small cell lung cancer with EGFR mutations are known to develop resistance to EGFR tyrosine kinase inhibitors. Here, the authors show AURKB activation to be associated with resistance in EGFR mutant lung cancer cells, and that AURKB is a therapeutic target in resistant tumours that lack the p.T790M or other acquired mutations.

Background: The EGF/EGFR pathway is involved in the emergence of resistance to BRAF and MEK inhibitors and the combination of encorafenib with the anti-EGFR antibody cetuximab has been recently approved in BRAF-positive, advanced colorectal cancer (CRC). Vaccination against EGF represents an alternative to the administration of monoclonal antibodies against EGFR 1-2. In this study, we tested if antibodies generated by vaccination with IN01 (biologic fusion molecule), could improve the antitumor activity of encorafenib and sotorasib in BRAF and KRAS colorectal (CRC) and non-small cell lung cancer (NSCLC) cell lines. Methods: Cell lines with BRAF V600E (HT29, CRC) and KRAS G12C mutations (H2122 and H23, NSCLC) were used. Anti-EGF VacAbs were obtained by immunizing rabbits with recombinant human EGF. Cell lines were treated with encorafenib or sotorasib in combination with anti-EGF VacAbs and the antitumor effects compared to the combination of encorafenib/cetuximab and sotorasib/panitumumab. Cell viability was determined by MTT, changes of total and phosphorylated proteins by Western blotting and emergence of resistance by direct microscopic examination in low density cultures. Results: Anti-EGF VacAbs suppressed the EGF-induced proliferation and blocked the EGFR signaling pathways more efficiently than cetuximab and panitumumab in KRAS and BRAF cell lines. In combination, anti-EGF VacAbs significantly enhanced the antitumor activity of encorafenib (in HT29 cells) and sotorasib (in H2122 and H23 cells), further suppressing EGFR, PRAS40 and ERK 1/2 phosphorylation. Also, the antitumor effects of the anti-EGF VacAbs in combination were superior to those of cetuximab and panitumumab. Finally, the addition of anti-EGF VacAbs to the culture medium significantly delayed the emergence of resistant clones to encorafenib (in HT29) and sotorasib (in H23). Again, the effect was more pronounced than the delay observed when adding cetuximab or panitumumab. Resistant clones generated under all combinations are being analyzed by NGS and Western blotting to determine the possible mechanisms of acquired resistance. Conclusions: Anti-EGF VacAbs potentiate the antitumor effects of encorafenib and sotorasib in cancer cell lines more efficiently than cetuximab and panitumumab. Our data provide a rationale for clinical trials testing the combination of anti-EGF VacAbs with targeted inhibitors in BRAF and KRAS mutant CRC and NSCLC patients. (1)“Anti-Epidermal Growth Factor Vaccine Antibodies Enhance the Efficacy of Tyrosine Kinase Inhibitors and Delay the Emergence of Resistance in EGFR Mutant Lung Cancer Cells” García-Roman S, Molina-Vila MA, et al. J Thorac Oncol. 2018. (2) “Anti-epidermal growth factor vaccine antibodies increase the antitumor activity of kinase inhibitors in ALK and RET rearranged lung cancer cells” García-Roman S, Molina-Vila MA, et al. Transl. Oncol. 2020. Citation Format: Silvia Garcia-Roman, Miguel A. Molina-Vila, Erik d'Hondt, Rafael Rosell. Anti-EGF antibodies significantly improve the activity of BRAF and KRAS inhibitors in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 338.

Advanced NSCLC patients harboring EML4-ALK and CCDC6-RET rearrangements derive benefit from treatment with ALK and RET TKIs but not immune checkpoint inhibitors. New immunotherapeutic approaches, such as immunization against growth factors, can be of particular interest for combination treatment in these patients. Here, we investigated the effects of anti-EGF antibodies generated by vaccination (anti-EGF VacAbs), TKIs and combinations in EML4-ALK and CCDC6-RET NSCLC cell lines. We found that EGF and tumor growth factor alpha (TGFα) significantly decreased the antiproliferative activity of the RET inhibitor BLU-667 in CCDC6-RET cells and brigatinib, alectinib and crizotinib in EML4-ALK translocated cells. The addition of anti-EGF VacAbs reversed the effects of EGF and TGFα, potentiated the antitumor effects of the kinase inhibitors and delayed the appearance in vitro of resistant clones. Western blotting demonstrated that the combination of anti-EGF VacAbs with ALK or RET TKIs effectively suppressed EGFR downstream pathways in EML4-ALK translocated and CCDC6-RET cells, respectively. In conclusion, anti-EGF VacAbs significantly increased the antitumor activity of TKIs in ALK and RET-positive cell lines. Clinical trials of an EGF vaccine in combination with ALK and RET TKIs are warranted.

Introduction: Stage IIIB-IV non-small cell lung cancer patients with mutations in the EGF receptor gene (EGFR) usually derive clinical benefit from to tyrosine kinase inhibitors (EGFR TKIs) but ultimately relapse. In preclinical studies, we have showed that anti-EGF antibodies generated by vaccination significantly increased the antitumor activity of TKIs in EGFR-mut cell lines, blocking EGFR, Erk1/2, Akt and STAT3 activation and delaying emergence of resistance. Based on these findings, the EPICAL trial was initiated (ClinicalTrials.gov number, NCT03623750). Methods: The EPICAL was a single arm, phase 1b, single arm study to evaluate the safety and efficacy of first line anti-EGF vaccination combined with afatinib. The trial enrolled advanced NSCLC patients with sensitizing EGFR mutations confirmed in a central laboratory. Patients received 40 mg/day of afatinib and five intramuscular anti-EGF vaccinations every 14 days and then every three months until progression. Four medical centers in Spain participated, with a target enrollment of 30 patients. However, the COVID-19 outbreak forced an early termination of the study in March 2020 with only 23 patients included. Serial blood samples were collected and used to evaluate the levels of selected growth factors by ELISA and biological activity by addition of sera to in vitro cultures of EGFR-mut cells followed by Western blotting. Results: Of the 23 patients enrolled in the trial, nine (39%) had exon 19 in-frame deletions, twelve (52%) exon 21 substitutions and two (9%) exon 18 missense mutations. Combination treatment was well tolerated and no SAES related to anti-EGF vaccination were reported. Objective response and disease control rates were 78.3% (95%CI=53.6-92.5) and 95.7% (95%CI=78.1-99.9), respectively. At data cut-off, with a median follow-up of 11.4 months (95%CI=8.1-15.2), the median progression-free survival was 17.4 months (95% CI=13.22-NA) and median survival not reached (95% CI=15.21-NA). Median PFS for patients with exon 19 deletions and exon 21 point mutations were 13.9 months (95%CI=8.7-NR) and 17.4 months (95%CI=13.2-NR), respectively. Three months after initiation of treatment, high titers of anti-EGF antibodies were detected in all patients and serum EGF and TGFα levels were found to be significantly lower compared to baseline levels. Finally, treatment with post-vaccination patient’s sera inhibited EGFR, AKT and ERK1/2 phosphorylation in EGFR-mut cells growing in vitro. Conclusion: The combination of an anti–EGF vaccine with afatinib is well tolerated and induces a sustained immunogenic effect. Vaccination against EGF might enhance the clinical efficacy of EGFR TKIs. Keywords: anti-EGF vaccination, EGFR-mutant non-small cell lung cancer, EGFR inhibitors

Background: Fusions involving the tyrosine kinase receptor genes ALK, ROS1, RET, NTRK or MET exon 14 skipping variant (METex14) are present in a significant percentage of advanced solid tumors and their accurate identification is critical to guide targeted therapies. While FISH has traditionally been considered the gold standard for fusion analysis, it is costly and shows biases. GeneReader Next Generation Sequencing (NGS) (Qiagen) and nCounter (Nanostring) are two technologies allowing simultaneous detection of fusion transcripts and splicing variants. In this work we compared the performance of both platforms for fusion and splicing variant detection in advanced solid tumor patients. Methods: RNAs from 40 selected solid tumors were purified using High Pure FFPET RNA Isolation Kit (Hoffman-La Roche) and prospectively analyzed by GeneReader and nCounter. The custom nCounter codeset used targets fusions involving ALK, ROS1, RET, NTRK1-3, NRG1 and MET exon 14 skipping variant based on a dual strategy: detection of specific fusion transcripts and imbalances between the 3' and 5' mRNA regions, enabling the recognition of even those fusions not identified with the specific primers. Reporter counts from nCounter were collected with the nSolver Analysis software (Nanostring) and analyzed using an algorithm developed in the laboratory. The design of QIAact Lung Fusion Custom GeneReader panel contains specific junction probes for the detection of fusions in ALK, ROS1, RET, FGFR1- 3, NRG1, NTRK1- 3, EGFR, BRAF, and MET exon 14 skipping variant. GeneReader analysis and interpretation were performed with the QCI-Analyze and QCI-Interpret software's (Qiagen). Results: Valid results were obtained for 40/40 (100%) of samples tested. Paired analysis showed a 97.5% concordance (39/40 cases) between the results obtained by nCounter and GeneReader NGS, corresponding to a Cohen's kappa of 0.935 [CI=0.809-1.0]. Overall, 8 samples tested positive for fusion transcripts, namely EML4-ALK (n=4), CCDC6-RET (n=1), KIF5B-RET (n=1), EZR-ROS1 (n=1) and ETV6-NTRK3 (n=1). In addition, MET exon 14 skipping variant was detected in two samples. The discordant case between nCounter and NGS corresponded to a rare RET fusion only detected by 3'-5' imbalance using nCounter, while the remaining 29 patients were pan-negative. In one of them, an uncommon HLA-DRB1-MET fusion not included in the nCounter codeset, was found by the GeneReader custom panel. Conclusions: RNA-based NGS and nCounter show excellent concordance for detection of gene fusions and MET splicing variant in advanced solid tumors. Citation Format: Mónica Garzón Ibañez, Ana Gimenez-Capitán, Marta Vives Usano, Ruth Román Lladó, Sonia Rodriguez, Erika Aldeguer, Beatriz García Pelaez, Nuria Jordana Ariza, Cristina Aguado, Santiago Viteri, Andrés Aguilar, Irene Moya, Carlos Cabrera, Maria Jose Catalán, Maria Gonzalez cao, Silvia Garcia-Roman, Jordi Bertran Alamillo, Florencia Garcia Casabal, Rafael Rosell, Miguel Angel Molina-Vila, Clara De La Caridad Mayo De Las Casas. Comparison of clinically relevant fusions detection using two multiplexing RNA based platforms: nCounter and GeneReader [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 290.

Background: We have recently demonstrated that antibodies generated by vaccination (anti-EGFVacAbs) potentiate the effects of EGFR TKIs in epidermal growth factor receptor mutant (EGFR-mut) non-small cell lung cancer (NSCLC) cells growing in vitro and prevent the emergence of resistance to EGFRTKIs (1). Based on these preclinical results we have started the Epical Phase Ib clinical trial(NCT03623750), testing anti-EGF vaccination in combination with EGFR TKI in advanced EGFR-mut NSCLC patients. Methods: The EPICAL trial is a Multicenter, Open-Label, Exploratory Phase Ib Clinical Study in which 23 patients have been enrolled. Anti-EGF VacAbs is a vaccine for intramuscular administration that is designed to be used in conjunction with standard of care treatment which, in this study, is afatinib. Blood samples have been collected at baseline and every three months and sent to a central laboratory. The presence of EGFR mutations has been determined on purified DNA by 5′-nuclease real-time PCR (Taqman®) assay in presence of PNA. All the serum samples have been analyzed by ELISA to determine the levels of EGF, TGFα, HGF and GAS6, together with anti-EGF antibody titers. Sera of patients were added to EGFR-mutant cell lines growing in vitro and Western blot was used to analyze EGFR, AKT andERK activation. Results: Median EGF levels in the sera of enrolled patients was 179.5 pg/mL at baseline and no anti-EGFantibodies were detected. Three months after first immunization, median serum EGF levels decreased to30.8 pg/mL (p=0.0008) while the anti-EGF vaccination titers increased to 1:6000 (p=0.0012). Anti-EGF titers remained high and EGF levels low in the patient's sera for the duration of the trial. Regarding serum TGFα and HGF levels baseline were 19.4 pg/mL and 861.2 pg/mL, respectively, and after three months dropped to 7.9 pg/mL (p=0.0037) and 341.3 pg/mL (p=0.0259). In EGFR-mutant cell lines in culture, the activation of the EGFR, AKT or ERK was abrogated by the sera of immunized patients. Conclusions: Anti-EGF vaccination induces high anti-EGF antibody titers and effectively reducing the levels of EGF and other growth factors in the blood of advanced NSCLC patients. 1J Thorac Oncol. 2018 Sep;13(9):1324-1337. Citation Format: Silvia Garcia-Roman, Jordi Codony, Miguel Angel Molina-Vila, Nuria Jordana-Ariza, Beatriz Garcia, Monica Garzon, Clara Mayo de las Casas, Maria Jose Catalan, Andres Aguilar, Santiago Viteri, Andres F. Cardona, Delvys Rodríguez, Manuel Cobo, Noemi Reguart, Erik d'Hondt, Rafael Rosell. Evolution of antibody titers, growth factor levels and in vitro activity in the sera of patients enrolled in the EPICAL trial of afatinib combined with anti-EGF vaccination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 951.

Background: Somatic copy number gains (CNGs; i.e. amplifications) have been related to tumorogenesis and some of them are designated targets for therapies, such as HER2 amplification in breast cancer. In the case of NSCLC, MET alterations are receiving increasing attention as targets in precision medicine and several clinical trials of anti-MET agents are ongoing. Routine testing for CNGs on formalin-fixed paraffin embedded (FFPE) samples is usually carried out by in-situ hybridization (FISH) covering a single gene. Although this methodology is still the gold standard of CNG detection, it presents several drawbacks and cannot be used in liquid biopsies. Here we aimed to determine the potential of next generation sequencing (NGS) to simultaneously determine CNGs across many genes in FFPE and liquid biopsy samples in NSCLC patients. Methods: FFPE biopsies (n=137) and blood samples (n=24) at presentation from NSCLC patients (p) of our institutions were prospective tested. In addition, 40 tumor and 19 blood samples at relapse were also analyzed, most of them from p progressing to tyrosine kinase inhibitors. DNA was purified and submitted to NGS using the 16-gene QIAact Lung Panel (Genereader®, Qiagen). For each sample, CNGs were determined using an algorithm developed in house. Results: Validation analyses in 8 cell lines showed 100% concordance between FISH and NGS for detection of EGFR, MET and HER2 amplifications. Among the 137 NSCLC biopsies at presentation, MET was the gene showing a higher frequency of CNGs (11%), followed by PIK3CA, KRAS and EGFR (7% each). Regarding tumor tissues at progression, the frequency of MET amplification raised to 15%, and HER2 was the second most common gene with CNGs (13%). CNGs were associated with sensitizing mutations only in the case of EGFR, with 6 samples showing both alterations concomitantly. Of the 24 blood samples at presentation, CNGs were detected only in one case, corresponding to an EGFR-mut patient with EGFR amplification. In contrast, MET and EGFR amplification were the most frequent CNGs (16% each) of liquid biopsies at progression to EGFR TKIs. Additionally, ALK amplification was detected in a 11% of cases progressed to ALK therapies. Paired tumor-blood samples were available for 9 patients and concordance in CNG detection was 70 %. Conclusions: CNGs in clinically relevant genes are present in a significant percentage of advanced NSCLC patients. However, with the only exception of EGFR, they are not concomitant with driver mutations in the same gene. NGS can be used to determine CNGs in multiple genes in both tumor tissue and liquid biopsy samples and further research is warranted to determine the clinical implications of these findings. Citation Format: Núria Jordana- Ariza, Mónica Garzón, Beatriz García-Peláez, Ruth Román, Jordi Bertrán- Alamillo, Sonia Rodriguez, Erika Aldeguer, Silvia García-Román, Santiago Viteri, Andrés Aguilar, María González Cao, Alejandro Martínez- Bueno, Miguel Angel Molina Vila, Rafael Rosell Costa, Clara Mayo de las Casas. Copy number gains of clinically relevant genes in advanced non small cell lung cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3175.

Background: We have recently shown that antibodies generated by vaccination (anti-EGF VacAbs) potentiate the effects of tyrosine kinase inhibitors (TKIs) in epidermal growth factor receptor mutant (EGFR-mut) cell lines (1) and a Phase I/II clinical trial of an anti-EGF vaccine in combination with afatinib has been initiated. In this study we aimed to determine the efficacy of anti-EGF VacAbs to improve the antitumor activity of RET, BRAF, MEK and PI3K inhibitors in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cell lines. Methods: Cell lines with RET translocations (LC-2/ad) and BRAF, KRAS and PIK3CA mutations (HT29, DLD1, LS174T and H508) were used. Anti-EGF VacAbs were obtained by immunizing rabbits with recombinant human EGF. Cell lines were treated with anti-EGF VacAbs alone and in combination with RET, BRAF, MEK and PI3K inhibitors. Cell viability was determined by MTT, cell cycle was analyzed by flow cytometry, changes of total and phosphorylated proteins by Western blot and emergence of resistance by direct microscopic examination in low density cultures. Results: Anti-EGF VacAbs suppressed EGF-induced cell proliferation and blocked EGFR transduction signaling pathway in all cell lines tested. In combination, the anti-EGF VacAbs significantly enhanced the antitumor activity of BLU667 in LC-2/ad, trametinib and encorafenib in HT29, trametinib in DLD1 and LS174T and trametinib, taselisib, alpelisib and copanlisib in H508 cells. In these cell lines, anti-EGF VacAbs in combination with kinase inhibitors suppressed EGFR, Akt and Erk 1/2 phosphorylation. Cell cycle experiments revealed that anti-EGF VacAbs significantly increased the antiproliferative effects of the kinase inhibitors, measured as the percentage of cells in G2+M. Finally, the addition of the anti-EGF VacAbs to the culture medium significantly delayed the emergence resistant clones to trametinib in DLD1 cells. Conclusions: Anti-EGF VacAbs potentiate the antitumor effects of RET, MEK, BRAF and PI3K inhibitors in tumor cell lines. Our data provide a rationale for clinical trials testing the combination of anti-EGF VacAbs with kinase inhibitors in RET-translocated, KRAS, BRAF and PIK3CA mutant NSCLC and CRC patients.(1)“Anti-Epidermal Growth Factor Vaccine Antibodies Enhance the Efficacy of Tyrosine Kinase Inhibitors and Delay the Emergence of Resistance in EGFR Mutant Lung Cancer Cells” Codony-Servat J, García-Roman S, Molina-Vila MA, et al. J Thorac Oncol. 2018. Citation Format: Silvia Garcia Roman, Jordi Codony Servat, Miguel Angel Molina Vila, Jordi Bertran Alamillo, Monica Garzon, Alejandro Martínez Bueno, Santiago Viteri, María González Cao, Andrés Aguilar, Juan José García, Erik d'Hondt, Rafael Rosell. Anti-EGF antibodies significantly improve the activity of RET, BRAF, MEK and PI3K kinase inhibitors in preclinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6645.

Significant advances have been made in the analysis of the human genome in the first decades of the 21st century and understanding of tumor biology has matured greatly. The identification of tumor-associated mutations and the pathways involved has led to the development of targeted anticancer therapies. However, the challenge now in using chemotherapy to treat nonsmall-cell lung cancer is to identify more molecular markers predictive of drug sensitivity and determine the optimal drug sequences in order to tailor treatment to each patient. This approach could permit selection of patients who could benefit most from a specific type of chemotherapy by matching their tumor and individual genetic profile. Nevertheless, this potential has been limited so far by reliance on the single biomarker approach, though this is now on the way to being overcome through whole genome studies.

Introduction: Mutations in EGFR correlate with impaired response to immune checkpoint inhibitors and the development of novel immunotherapeutic approaches for EGFR mutant NSCLC is of particular interest. Immunization against epidermal growth factor (EGF) has shown efficacy in a phase III trial including unselected NSCLC patients, but little was known about the mechanisms involved in the effects of the anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) or their activity in tumor cells with EGFR mutations. Methods: The EGFR-mutant, NSCLC cell lines H1975, and PC9, together with several gefitinib and osimertinib-resistant cells derived from PC9, were treated with anti-EGF VacAbs and/or EGFR tyrosine kinase inhibitors (TKIs). Cell viability was analyzed by proliferation assays, cell cycle by fluorescence-activated cell sorting analysis, and levels of RNA and proteins by quantitative retro-transcription polymerase chain reaction and Western blotting. Results: Anti-EGF VacAbs generated in rabbits suppressed EGF-induced cell proliferation and cycle progression and inhibited downstream EGFR signaling in EGFR-mutant cells. Sera from patients immunized with an EGF vaccine were also able to block activation of EGFR effectors. In combination, the anti-EGF VacAbs significantly enhanced the antitumor activity of all TKIs tested, suppressed Erk1/2 phosphorylation, blocked the activation of signal transducer and activator of transcription 3 (STAT3) and downregulated the expression of AXL receptor tyrosine kinase (AXL). Finally, anti-EGF VacAbs significantly delayed the emergence in vitro of EGFR TKI resistant clones. Conclusions: EGFR-mutant patients can derive benefit from immunization against EGF, particularly if combined with EGFR TKIs. A phase I trial of an EGF vaccine in combination with afatinib has been initiated. (C) 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Background: We have previously described that EGF blocks the activity of EGFR tyrosine kinase inhibitors (EGFR-TKIs) in EGFR mutant NSCLC cells, an effect that is reversed by anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) (1). In this study we aimed to determine the effect of EGF and anti-EGF Vac Abs in the activity of different kinase inhibitors in tumor cell lines with different genetic alterations. Methods: Anti-EGF VacAbs were obtained by immunizing rabbits with recombinant EGF. The cell lines used in this study were H2228 and H3122 (NSCLC, EML4-ALK positive), H23 (NSCLC, KRAS-G12C), DLD1 (colorectal carcinoma, KRAS-G13D), PC9 and PC9-GR4 (NSCLC, EGFR-mut). Tumor cell lines were treated with EGF, anti-EGF VacAbs and combinations with the kinase inhibitors alectinib, crizotinib, brigatinib (ALK inhibitors), trametinib (MEK inhibitor), dacomitinib and osimertinib (EGFR inhibitors). Cell viability was analyzed by MTT, changes of total and phosphorylated proteins were determined by Western blot and emergence of resistance by direct microscopic examination in low density cultures. Results: EGF significantly decreased the antitumor activity of alectinib, crizotinib and brigatinib in ALK translocated cells (H2228 and H3122), trametinib in KRAS mutant cells (H23 and DLD1) and osimertinib and dacomitinib in EGFR mutant cells (PC9). In combination with these TKIs, the anti-EGF VacAbs reversed the effects of EGF and significantly potentiated the antitumor activity of all the kinase inhibitors, blocking the activation of EGFR, Akt and Erk. Finally, the addition of the anti-EGF VacAbs to the culture medium delayed the appearance of resistant clones to kinase inhibitors. Conclusions: Anti-EGF VacAbs potentiate the antitumor effects of ALK, MEK and EGFR kinase inhibitors in tumor cell lines and delay the emergence of resistance in vitro. A clinical trial is currently testing anti-EGF vaccination in combination with afatinib in EGFR-mut advanced NSCLC patients.(1)Anti-Epidermal Growth Factor Vaccine Antibodies Enhance the Efficacy of Tyrosine Kinase Inhibitors and Delay the Emergence of Resistance in EGFR Mutant Lung Cancer Cells” Codony-Servat J, García-Roman S, Molina-Vila MÁ, et al. J Thorac Oncol. 2018. Citation Format: Jordi Codony-Servat, Silvia Garcia-Roman, Miguel Ángel Molina-Vila, Jordi Bertran-Alamillo, Ana Giménez-Capitán, Santiago Viteri, Andrés F. Cardona, Delvys Rodríguez, Manuel Cobo, Noemi Reguart, Niki Karachaliou, Erik d'Hondt, Rafael Rosell. Anti-EGF antibodies generated by vaccination significantly improve the activity of kinase inhibitors in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1450.

Background: Immunization against Epidermal Growth Factor (EGF) has demonstrated clinical efficacy in a phase III trial including unselected NSCLC patients. We are currently analyzing if anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) showed antitumor activity in EGFR-mutant, Kras-mutant (mut) and Anaplastic Lymphoma Kinase (ALK) translocated non-small cell lung cancer cells (NSCLC), alone or in combination with tyrosine kinase inhibitors (TKI). In an EGFR-mut and in an ALK translocated cell lines we are also studying if the anti-EGF VacABs can delay the emergence of resistance to TKIs. Methods: Anti-EGF VacAbs were obtained by immunizing rabbits with recombinant EGF. Cell lines were treated with anti-EGF VacABs alone and in combination with TKIs in EGFR (PC9, H1975) and ALK translocated (H3122, H2228) cell lines. In KRAS-mut cells (A549, H23) the combination was with chemotherapy agents. Cell viability was analyzed by MTT and apoptosis and cell cycle by fluorescence-activates cell sorting analysis (FACS). Changes of total and phosphorylated proteins were determined by Western blot. Sera from advanced NSCLC patients immunized with anti-EGF vaccine were also tested Results: Anti-EGF VacAbs suppressed EGF-induced cell proliferation and inhibited downstream EGFR signaling in all cell lines tested. In combination, the anti-EGF VacAbs significantly enhanced the antitumor activity of gefitinib, erlotinib, osimertinib and afatinib in EGFR-mut cells, potentiated Erk ½ phosphorylation, arrested cell cycle progression and increased apoptosis. In addition, anti-EGF VacAbs blocked the activation of STAT3, downregulated the expression of proteins related to EGFR resistance, such as AXL, and significantly delayed the emergence in vitro of clones resistant to EGFR TKI. Sera from patients vaccinated against EGF also suppressed Erk ½ phosphorylation. Results for the combination of ALK TKIs and chemotherapy agents in ALK translocated and KRAS-mut cell lines will be presented at the meeting. Conclusions: Anti-EGF VacAbs decreased cell proliferation and inhibited the activation of EGFR-pathway downstream proteins in EGFR-mut, KRAS-mut and ALK translocated cell lines. Also, they potentiate the effects of TKIs and prevent the emergence of resistance in EGFR-mut NSCLC cells. Two clinical trials are currently testing anti-EGF vaccination in EGFR-wt and EGFR-mut advanced NSCLC patients. Citation Format: Silvia Garcia-Roman, Jordi Codony-Servat, Miguel Angel Molina-Vila, Jordi Bertran-Alamillo, Ana Gimenez-Capitán, Niki Karachaliou, Erik d'Hondt, Rafael Rosell. Antitumor effects of anti-EGF antibodies generated by vaccination in NSCLC tumor 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 837.

Background: Aberrant activity of the MET, FGFR1 and AXL receptors has been associated with the development of resistance to first, second and third generation EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutated non-small cell lung cancer (NSCLC) patients. Methods: We obtained 6 resistant lines by treating EGFR-mutated (exon 19), TKI sensitive PC9 cells with increasing concentrations of gefitinib or erlotinib. The p.T790M resistance mutation emerged in two cell lines (GR1, GR4), which remained sensitive to osimertinib, a third generation EGFR TKI. Six new cell lines to resistant to “second line” osimertinib were generated from GR1 and GR4 by exposure to increasing concentrations of the inhibitor. Finally, six more cell lines resistant to “first line” osimertinib were derived from the PC9 parental cells. All resistant cell lines were genotyped for selected genes (including EGFR) and characterized for AXL, MET and FGFR1 expression and activation by Q-RT-PCR, immunohistochemistry and Western blotting. The effects of AXL (BGB324), MET (crizotinib, capmatinib) and FGFR1 (nindetanib) inhibitors on the parental and the 18 resistant cell lines were analyzed by MTT and, in some cases, by colony formation. AXL was stably silenced in some of the resistant cell lines. Results: All cell lines resistant to “first line” gefitinib, erlotinib and osimertinib maintained the exon 19 EGFR sensitizing mutation. In contrast, three of the resistant cell lines to “second line” osimertinib lost the exon 19 and the p.T790M mutations. In two more, the p.T790M dropped to low allelic fractions (1% and 0.03%). Regardless of the EGFR status, AXL overexpression was the most common event related to EGFR TKI resistance in our panel of 18 cell lines, with FGFR1 and MET overexpression or activation as less frequent events. In proliferation assays, the IC50 of the EGFR TKI resistant cell lines for BGB324 (AXL inhibitor) was indistinguishable from the IC50 of the parental, EGFR TKI sensitive cell line. Similar results were obtained in the case of capmatinib, crizotinib (MET inhibitors) and nintedanib (FGFR inhibitor). Stable silencing of AXL on some of the AXL-overexpressing resistant cell lines had no effects in terms of doubling times, morphology of cells or sensitivity to EGFR TKIs. In combination experiments, the effect of BGB and MET inhibitors was found to be additive. Conclusions: In tumor cell line models of acquired resistance to EGFR TKIs, overexpression or activation of AXL, MET and FGFR1 was not associated to sensitivity to single-agent treatment with AXL, MET or FGFR inhibitors. Multitargeted approaches might be more effective in this setting. Citation Format: Jordi Bertran-Alamillo, Miguel Angel Molina-VIla, Cristina Teixidó, Jordi Codony-Servat, Ana Giménez-Capitán, Carles Codony-Servat, Silvia García-Román, Erika Aldeguer, Sonia Rodríguez, Rafael Rosell. Tumor cells with acquired resistance to EGFR inhibitors and overexpression or activation of AXL, MET and FGFR1 are insensitive to single-agent treatment targeting AXL, MET or FGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3077. doi:10.1158/1538-7445.AM2017-3077

Introduction: Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase (RTK) activated by collagen I and identified as an oncogenic driver in squamous cell carcinoma of the lung (SCC) (1). DDR2 mutations have been reported in 4% of SCC patients and at lower frequencies in lung adenocarcinoma, gastric, breast and brain tumors (2). In addition, DDR2 is expressed during epithelial to mesenchymal transition (EMT) (3) and acts as an upstream activator of SHP2. SHP2 in turn is a key signaling effector which leads to activation of multiple signaling pathways (4). DDR2 has recently emerged as a potential therapeutic target in DDR2-mutated tumors and drugs such as dasatinib are being tested in this setting. Results and discussion: PB1 is a small organic molecule with a simple synthesis pathway that showed strong inhibitory activity against wild type (wt) and mutated purified DDR2 protein. The compound was subsequently tested in a panel of tumor cell lines from different origins and histologies. PB1 showed good antiproliferative activity: IC50 Conclusion: PB1 shows significant antitumor activity in preclinical models of mutated and wt DDR2 in SCC of the lung and some KRAS mutated adenocarcinoma cell lines. PB1 is also active against tumor cells with secondary resistance to dasatinib. References: 1) Hammerman et al. Cancer Discovery. 2011 June; 1: 78-89. 2) Terai et al. ACS Chem Biol. 2015 Sep; Epub ahead of print. 3) Walsh et al. Matrix Biol. 2011 May; 30(4): 243-247. 4) Iwai et al. Biochem. J. (2013) 454, 501-513. Citation Format: Silvia Garcia-Roman, Miguel Angel Molina, José Ignacio Borrell, Raimon Puig de la Bellacasa, Daniela Morales, Jordi Bertran, Ana Gimenez, Niki Karachaliou, Rafael Rosell. PB1, a DDR2 inhibitor with antitumor activity in preclinical models of squamous cell carcinoma and KRAS-mutated adenocarcinoma of the lung. [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 4802.

11581Background: Targeted therapy has improved outcomes in EGFR mutated and ALK translocated NSCLC but options for squamous cell carcinoma and KRAS-mutated tumors remain scarce. Genome re-replicati...

e13516 Background: Targeted lung cancer therapy has undoubtedly made a difference to the treatment of EGFR mutation and ALK translocation carriers. However, targeted therapies for other subgroups l...