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T. Bivona



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    MO21 - Prognostic and Predictive Biomarkers V - EGFR (ID 98)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      MO21.05 - Integrated genomic analysis by whole exome and transcriptome sequencing of tumor samples from EGFR-mutant non-small-cell lung cancer (NSCLC) patients with acquired resistance to erlotinib. (ID 1426)

      10:50 - 10:55  |  Author(s): T. Bivona

      • Abstract
      • Presentation
      • Slides

      Background
      NSCLC patients with EGFR mutations initially respond to EGFR tyrosine kinase inhibitors (TKIs) but ultimately relapse. Sub-genomic molecular studies indicate that the EGFR T790M mutation and the activation of MET, PI3K, AXL, HER2 and MAPK can lead to acquired resistance to EGFR TKIs. To date, no integrated comprehensive genomic investigation of EGFR TKI resistance has been reported.

      Methods
      FFPE biopsies of erlotinib-sensitive and erlotinib-resistant tumors were obtained from 13 EGFR mutant NSCLC patients. The samples were analyzed by whole exome sequencing and whole transcriptome sequencing utilizing the Illumina HiSeq2500 platform. In addition, targeted gene sequencing was performed with the Illumina TruSeq Amplicon-Cancer Panel and run on the MiSeq system.

      Results
      Erlotinib resistant NSCLC specimens harbored known resistance drivers, including EGFR T790M mutations (9/13; 69%), MET amplification (3/13; 23%), HER2 amplification (3/13; 23%), and AXL upregulation (3/13; 23%). Differential expression analysis between resistant and pre-treatment states revealed enrichment in the pre-treatment tumors of immune signaling pathways, and in the resistant tumors upregulation of ERBB2, mTOR, PI3 kinase and ribosomal signaling pathways. PI3K/AKT pathway upregulation also occurred through somatic mutations in AKT and LKB1 in the resistant tumors. Copy number analysis demonstrated both large scale and focal amplifications and deletions in the resistant tumors, including the focal loss of EGFR and gain of c-Myc and NKX2-1. There was strong correlation between the copy number changes observed and the expression mRNA levels of the involved cancer-associated genes. Of note, each resistant tumor exhibited greater copy number similarity to the corresponding matched pre-treatment sample compared to other tumors within the resistance cohort.

      Conclusion
      We conducted the first ever comprehensive integrated genomic analysis of EGFR TKI resistant NSCLC patients, and identified both known and potentially novel drivers of EGFR TKI resistance. This study demonstrated the feasibility and utility of comprehensive genomic analysis in the clinical management of NSCLC receiving targeted therapy. Together, our data provide unprecedented insight into the molecular pathogenesis of escape from EGFR oncogene inhibition in NSCLC. We are now conducting a prospective observational study in additional NSCLC patients on targeted therapy.

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    P3.02 - Poster Session 3 - Novel Cancer Genes and Pathways (ID 149)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.02-009 - <b>ROR1 as a novel therapeutic target for EGFR-mutant non-small-cell lung cancer (NSCLC) patients with the EGFR T790M mutation</b> (ID 1395)

      09:30 - 09:30  |  Author(s): T. Bivona

      • Abstract

      Background
      Molecular cross-talk between EGFR and other signaling pathways creates alternative means of tumor cell proliferation and promotes resistance to single-agent erlotinib therapy in NSCLC driven by EGFR mutations. ROR1 knockdown inhibited the growth of NCI-H1975 cells (harboring EGFR L858R and T790M mutations). A pro-survival function for ROR1/MEK/ERK signaling in cooperation with AKT has been demonstrated. We have assessed ROR1 expression in 45 patients from the EURTAC trial (clinicaltrials.gov NCT00446225), 27 of whom harbored pretreatment concomitant EGFR T790M mutations, and correlated results with outcome.

      Methods
      ROR1 mRNA expression was examined by quantitative RT-PCR and categorized by terciles; patients were classified as having low/intermediate or high ROR1 expression. The T790M mutation was determined by Taqman with a PNA to inhibit amplification of the wild-type (wt) allele. Tumor samples were run in octuplicates; this method can detect 1 mutated allele among 10,000 wt alleles.

      Results
      Median age 65; 68.9% female; 57.8% never-smokers; 95.6% ECOG PS <2; 91.1% adenocarcinoma; 68.9% exon 19 deletion. No differences in baseline characteristics were observed according to ROR1 expression levels. 24 patients (53.3%) were treated with erlotinib and 21 (46.7%) with chemotherapy. 10 (41.7%) erlotinib-treated patients and 6 (28.6%) chemotherapy-treated patients had high ROR1 mRNA levels. Among erlotinib-treated patients, response rate (RR) was 40% for the 10 patients with high ROR1 levels vs 71.4% for the 14 with low/intermediate levels (P=0.058). Among chemotherapy-treated patients, RR for the 15 patients with low/intermediate ROR1 levels was 6.7%; the 6 patients with high ROR1 levels did not respond. Progression-free survival (PFS) was 11.8 months (m) for erlotinib-treated patients with low/intermediate ROR1 levels vs 5.8 m for those with high levels. PFS for chemotherapy-treated patients was 5.6 and 9 m, respectively (P=0.0165). 15 erlotinib-treated patients harbored concomitant T790M mutations; for these patients, PFS was10.8 m for those with low/intermediate ROR1 levels vs 2.7 m for those with high levels (P=0.0138).

      Conclusion
      ROR1 expression has a differential effect on outcome to erlotinib and chemotherapy in EGFR-mutant NSCLC patients. High ROR1 expression significantly limits PFS in erlotinib-treated patients with T790M mutations and ROR1-directed therapies can enhance the efficacy of treatment. In contrast, high ROR1 expression confers longer PFS to chemotherapy in the same group of patients. The role of chemotherapy and erlotinib in EGFR-mutant NSCLC patients with high ROR1 expression warrants further investigation.

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    P3.05 - Poster Session 3 - Preclinical Models of Therapeutics/Imaging (ID 159)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.05-019 - Bim expression in EGFR-mutant lung cancer 3D spheroids predicts responsiveness to tyrosine kinase inhibitors (ID 3173)

      09:30 - 09:30  |  Author(s): T. Bivona

      • Abstract

      Background
      The pro-apoptosic Bcl-2 family member BIM is known to be a critical mediator of targeted therapy-induced apoptosis in lung cancer. Also, pretreatment level of BIM strongly predicted the capacity of EGFR inhibitors to induced apoptosis in EGFR-mutant cancers. But prevous studies were mostly done in two-dimensional (2D) cell cultures (monolayers).

      Methods
      So we used three-dimensional(3D) spheroid culture model that were proved to be a valuable platform to study acquired multicellular apoptotic resistance and effectively recapitulate some of the complexity of solid tumors such as mesothelioma. 3D spheroids with EGFR-mutant cell lines (H3255, HCC827, H1650, 11-18, H4006) were generated in polyHEMA-coated plates and TKI treatments were done both in 2D and 3D. The degree of apoptosis and expressions of BIM by immunoblotting before and after treatment were compared.

      Results
      Contrary to other solid tumors, spheroids showed more prominent apoptosis to TKI than monolayer. Spheroids expressed more BIM than did monolayers except H4006. BIM was not expressed in H4006 (2D, 3D), but BIM expression was found to be elevated after TKI treatment only in spheroids. Higher level of BIM in spheroids had the tendency of higher apoptotic response to TKI.

      Conclusion
      3D spheroids cultures are useful in studying apoptotic mechanism in EGFR-mutant lung cancer. BIM expression before and after treatment of TKI might be useful predictor in EGFR-mutant spheroids model. So therapies that upregulated BIM expression can improve the efficacy of TKI therapy.