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A. Giménez-Capitán



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    P1.01 - Advanced NSCLC (ID 757)

    • Event: WCLC 2017
    • Type: Poster Session with Presenters Present
    • Track: Advanced NSCLC
    • Presentations: 1
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      P1.01-075 - Simultaneous Multiplex Profiling of Gene Fusions, METe14 Mutations and Immune Genes in Advanced NSCLC by NCounter Technology (ID 9481)

      09:30 - 16:00  |  Author(s): A. Giménez-Capitán

      • Abstract
      • Slides

      Background:
      Assessment of several immune-genes and tumor drivers is critical for individualized treatment of non-small cell lung cancer (NSCLC). We have previously demonstrated the ability of the transcript-based nCounter Technology for the detection of ALK, ROS1 and RET gene fusions, using a customized codeset (Reguart et al. Clinical Chemistry 2017). Here, we present the first results of the validation in advanced NSCLC samples of a new CodeSet designed to simultaneously characterize clinically relevant gene fusions, MET alterations and the expression of immune genes.

      Method:
      We have designed an in-house custom set to detect driver fusions involving 4 genes (ALK, ROS, RET, NTRK), MET exon 14 skipping mutation, MET overexpression and immune genes (PD1, PDL-1, CD4, CD8, FOXP3, GZMM, IFNG). A panel of ALK-ROS-RET-NTRK positive cell lines (H2228, H3122, SU-DHL-1, HCC78, BaF3 pBABE, LC2/ad and a NTRK-positive cell line), Hs746T (METex14), EBC-1 (overexpressing MET) and a negative cell line (PC9) were used for the initial validation of the panel. Subsequently, 58 FFPE samples from advanced NSCLC patients, previously characterized by FISH, RT-PCR and IHC, have been analyzed. Total amount of 100-150 ng RNA was used for detection. Workflow includes RNA isolation, hybridization and digital counting with for a total turnaround of 3 days. Raw counts were normalized using positive controls, negative controls and house-keeping genes.

      Result:
      .Results obtained with the cell lines positive for ALK, ROS1, RET and NTRK1 fusion genes were exactly coincident with their genotypes, with fusion transcripts successfully detected in all cases by 3’/5’ imbalance and direct fusion probes. In addition, METex14 splicing transcripts were detected in the Hs746T cells at significant levels, higher than those of wt MET mRNA. In contrast, METex14 mRNA counts were almost undetectable in the rest of cell lines. Regarding FFPE samples from advanced patients, 46 could be successfully analyzed by nCounter. Among 13 patients positive for ALK and ROS1 fusions, 12 were confirmed by nCounter. Regarding the METex14 splicing variant, 5 out of 6 patients previously detected by RT-PCR were also positive by nCounter.

      Conclusion:
      Preliminary data suggest that multiplex detection of clinically relevant drivers can be successfully achieved using nCounter Technology. The assay is simple, requires short hands-on-time, needs low input RNA and is highly efficient in detecting gene rearrangements and METex14 splicing variants. Results will be prospectively validated in a larger cohort of advanced NSCLC patients and we will determine if clusters of different inmune-phenotypes exist among oncogenic-driven NSCLC tumors.

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    P1.07 - Immunology and Immunotherapy (ID 693)

    • Event: WCLC 2017
    • Type: Poster Session with Presenters Present
    • Track: Immunology and Immunotherapy
    • Presentations: 1
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      P1.07-015 - Interferon-Gamma (INFG) as a Biomarker to Guide Immune Checkpoint Blockade (ICB) in Cancer Therapy (ID 8939)

      09:30 - 16:00  |  Author(s): A. Giménez-Capitán

      • Abstract
      • Slides

      Background:
      PD-L1 is induced by oncogenic signals or via INFG. STAT3, through DNMT1, epigenetically silences STAT1 and RIG-I and opposes INFG signaling. TET1 is a DNA demethylase. I kappa B kinase epsilon (IKBKE), a noncanonical I-kappa-B kinase, is essential for INFG induction, but can also promote NFATc1 phosphorylation and T cell response inhibition. Eomesodermin (Eomes) regulates T cell exhaustion. CCL5 (or Rantes), dependent on STAT3, causes myeloid-derived suppressor cell (MDSC) recruitment. YAP1 can also drive MDSC recruitment via CXCL5 signaling. We have explored whether the expression of genes related to INFG signaling, T cell exhaustion and MDSC recruitment is associated with response to ICB.

      Method:
      Total RNA from pre-treatment tissue samples of 17 NSCLC and 21 melanoma patients treated with nivolumab and pembrolizumab respectively, was analyzed by qRT-PCR. INFG, STAT3, IKBKE, STAT1, RIG-I and PD-L1 mRNA were examined. CCL5, YAP1, CXCL5, NFATC1, EOMES and TET1 expression was additionally assessed. Gene expression was categorized with respect to tertiles and patients were divided into two risk groups (low and intermediate/high). CD8[+ ]tumor-infiltrating lymphocytes (TILs) and PD-L1 protein expression in tumor and CD8[+ ]TILs were examined by immunohistochemistry (SP57 and SP142 assay, respectively). Progression free survival (PFS), overall survival (OS) and Disease Control Rate (DCR) were estimated.

      Result:
      Seventeen NSCLC patients, previously treated with one or more prior systemic therapies, received nivolumab. IKBKE was positively correlated with INFG (r=0.65, p=0.0124) and PD-L1 (r=0.58, p=0.0225) expression. RIG-I was loosely anticorrelated with NFATc1 (r=-0.55, p=0.0518). Among all biomarkers explored, only INFG was associated with PFS, OS and DCR. Specifically, PFS was significantly longer for nivolumab-treated patients with intermediate/high versus low INFG expression (5.1 versus 2.0 months, p=0.0124). OS was longer (though not statistically significant) for patients with intermediate/high versus low INFG expression (10.2 versus 4.9 months, p=0.0687). DCR to nivolumab was 71.43% for patients with intermediate/high INFG versus 0% for patients with low INFG expression. Neither PD-L1 immunohistochemistry expression nor CD8[+ ]TILs were related to nivolumab outcome. The same results were observed for 21 melanoma patients treated with pembrolizumab.

      Conclusion:
      IFNG production by T-cells plays critical roles in anti-cancer immune responses by augmentation of MHC Class I expression, growth arrest, post-proteasomal trimming of antigen epitopes, recruitment of effector cells, induction of T-regs fragility and PD-L1 expression. Further research is warranted in order to validate whether INFG is more accurate than PD-L1.

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    P2.02 - Biology/Pathology (ID 616)

    • Event: WCLC 2017
    • Type: Poster Session with Presenters Present
    • Track: Biology/Pathology
    • Presentations: 2
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      P2.02-014 - Simultaneous Gene Profiling of Advanced NSCLC: Single-Molecule Quantification of DNA and RNA by nCounter3D™ Technology (ID 9808)

      09:30 - 16:00  |  Author(s): A. Giménez-Capitán

      • Abstract
      • Slides

      Background:
      Currently, assessment of several tumor drivers is critical for individualized treatment of non-small cell lung cancer (NSCLC). Tools for molecular profiling are based on DNA, RNA and protein (PCR, NGS, FISH, IHC). However, these tests have several disadvantages including hands-on-time and tissue mass requirements. Nanostring digital barcoding technology enables simultaneous assay of different analytes, DNA and RNA from a single sample with 3D Biology Technology.

      Method:
      The nCounter Vantage 3D SNV:Fusions Lung Assay was used to analyze a total of 36 formalin-fixed paraffin embedded (FFPE) samples from advanced NSCLC patients. Samples were known to harbor mutations (EGFR, KRAS, NRAS, PIK3CA, BRAF, P53) or gene-fusion rearrangements (ALK, ROS1, RET, NTRK1) as verified by sequencing (Ion Torrent, Gene Reader), nCounter Elements, IHC and/or FISH. Probes were designed to target 25 genes for SNVs (104 different point and InDel mutations) as well as four genes for fusion transcripts (ALK, ROS1, RET, NTRK1) including 33 specific variants. The 3D workflow requires pre-amplification of gDNA, whereas RNA does not require any enzymatic treatment. After hybridization, the analytes (DNA/RNA) are pooled for simultaneous, single-lane, digital counting in total turnaround of 3 days. A total amount of 5ng DNA and 150ng RNA from two4-micron FFPE-sections was used for the assay without microdissection.

      Result:
      A total of 72 analyses (DNA/RNA) were performed with an evaluation pass of 97.2% (70/72 analyses yielded results) and 89% concordant results (64/72). Sensitivity of the technique was 92.1%. Among 41 SNVs interrogated in this study 34 were successfully detected (two not evaluable). Five new mutations were found involving NRAS, FBXW7, GNA11, FGFR2 and KRAS genes. Of those, only two were considered false positives as they were not confirmed by alternative sequencing and/or PCR. The remaining three were not assessable for test confirmation. For gene fusion analysis, 13 known positive samples were tested. All fusion transcripts were detected for ALK (n=5) RET (n=2) and NTRK1 (n=1). For ROS1 (n=5) there were 2 false negatives only detected by nCounter Elements target-specific assay.

      Conclusion:
      We have shown that the SNV detection chemistry can be successfully combined with fusion gene expression analysis by using the nCounter 3D™ single-workflow. The Nanostring nCounter Vantage 3D SNV:Fusions Lung Assay is highly efficient in detecting hotspot mutations as well as gene rearrangements. The assay is simple, features a brief hands-on time and requires low amounts of genomic material, supporting minimal use of precious samples.

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      P2.02-034 - PD-L1 Expression Can Be a Prognostic Marker in EGFR Mutant NSCLC Patients Treated with Erlotinib (ID 8933)

      09:30 - 16:00  |  Author(s): A. Giménez-Capitán

      • Abstract
      • Slides

      Background:
      INF-gamma secreted by CD8+ lymphocytes upregulates PD-L1 expression in cancer cells. We recently identified STAT3 and YAP1 as compensatory mechanisms of resistance to EGFR tyrosine kinase inhibition in EGFR mutant cells. STAT3 and YAP1 up-regulate CCL5 (Rantes) and CXCL5, respectively, with both chemokines attracting the myeloid-derived suppressor cell. STAT3 stimulates DNMT1 by repressing STAT1 and retinoic acid-inducible gene-I (RIG-I) expression. STAT1 and RIG-I are key mediators in INF-gamma signaling. We assume that alterations in the INF-gamma signaling pathway could be present in EGFR mutant NSCLC.

      Method:
      Total RNA from 53 EGFR mutant NSCLC patients was reversed transcribed and analyzed by qRT-PCR. STAT3, YAP1, RIG-I, STAT1, PD-L1, DNMT1 and CXCL5 mRNA were examined with specific primers/probes in triplicates. Progression-free survival (PFS) and overall survival (OS) were estimated.

      Result:
      Fifty-three EGFR mutant NSCLC patients treated with erlotinib were analyzed, 72% were female, 62% never-smoked, 70% had exon 19 deletion and 36% brain metastases. A positive correlation was found between RIG-1 and STAT1 (r=0.42, p=0.003). An anti-correlation trend was noted between STAT3 and PD-L1, YAP1 and PD-L1 and DNMT1 and STAT1. Median PFS was 22, 12.9 and 8.6 months for patients with high, intermediate and low PD-L1 mRNA, respectively (P=0.04). Median PFS was numerically longer for patients with low levels of DNMT1, RIG1 STAT1 and CXCL5, although the differences were not statistically significant. A similar trend was observed for OS.

      Conclusion:
      PD-L1 mRNA could be a prognostic marker in EGFR mutant NSCLC patients. Down-modulation of PD-L1 indicates alterations in pattern-recognition receptors (PRRs), like RIG-1 or downstream interferon signaling factors. The dysregualtion of the pathway is multifactorial, and the role of STAT3 and YAP1 hyperactivation merits further research. DNMT1 overexpression ablates STAT1. Since the cyclin-dependent kinase 4 (CDK4) interacts with DNMT1, therapies with CDK4 inhibitors can directly neutralize the main defects in the INF-gamma signaling pathway.

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    P3.01 - Advanced NSCLC (ID 621)

    • Event: WCLC 2017
    • Type: Poster Session with Presenters Present
    • Track: Advanced NSCLC
    • Presentations: 1
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      P3.01-073 - TPX-0005 with an EGFR Tyrosine Kinase Inhibitor (TKI) Overcomes Innate Resistance in EGFR Mutant NSCLC (ID 8956)

      09:30 - 16:00  |  Author(s): A. Giménez-Capitán

      • Abstract
      • Slides

      Background:
      Overexpression of several receptor tyrosine kinases (RTKs) substitutes EGFR signaling in EGFR-mutant NSCLC. The MET ligand hepatocyte growth factor (HGF) provides an alternative signaling mechanism for EGFR by inducing inter-receptor cross talk with EphA2, CUB domain-containing protein-1 (CDCP1) or AXL. SHP2, a non-receptor protein tyrosine phosphatase is central in signal transduction downstream of RTK signaling and in Src activation. We previously demonstrated that STAT3 and Src-YAP1 signaling limits EGFR TKI efficacy in EGFR-mutant NSCLC. We are now exploring the possibility of multiple RTK activation through a Src-YAP1-mediated transcriptional program. We are evaluating whether combined EGFR inhibition with TPX-0005, a novel orally available multikinase inhibitor and potent Src/FAK and JAK2 inhibitor, can be more efficient than EGFR inhibition alone in EGFR-mutant NSCLC cells.

      Method:
      We studied the mRNA expression levels of stromal HGF and tumor RTKs, AXL, CDCP1, MET, and EphA2, as well as SHP2, and clinical outcome in baseline samples of 64 EGFR-mutant NSCLC patients treated with first-line EGFR TKI. We combined in vitro approaches to explore whether gefitinib or osimertinib combined with TPX-0005 can abolish STAT3 and Src-YAP1 and downregulate the expression of RTKs.

      Result:
      High levels of AXL, CDCP1 and SHP2 mRNA expression were associated with worse outcome to EGFR TKI in 64 EGFR-mutant NSCLC patients. Median progression-free survival (PFS) was 14.5 and 23.4 months for patients with high and low AXL mRNA, respectively (p=0.0359). Median PFS was 9.1 and 20.2 months for patients with high and low CDCP1 mRNA, respectively (p=0.0179). Tumoral EPHA2 and MET or stromal HGF levels did not affect PFS. Median PFS was 11.4 and 24.1 months for patients with high and low SHP2 mRNA, respectively (p=0.0094). The combination of gefitinib/osimertinib with TPX-0005 resulted in highly synergistic suppression of cell viability and reduced colony formation in two EGFR-mutant cell lines. The combination abolished the EGFR inhibition-induced STAT3 and YAP1 phosphorylation, as confirmed by western blotting and immunofluorescence. The results of TaqMan quantitative-PCR assay revealed that gefitinib/osimertinib plus TPX-0005 reduced the mRNA levels of AXL, CDCP1 and MET, an effect that could not be obtained with EGFR inhibition alone. In vivo experiments are ongoing.

      Conclusion:
      AXL and CDCP1 are adverse predictive markers of PFS in EGFR-mutant NSCLC patients. STAT3 and Src-YAP1 signaling limits the efficacy EGFR TKI. Combined EGFR inhibition with TPX-0005 (currently in phase I clinical testing) is a particularly attractive strategy

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