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M.W. Wynes



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    MO05 - Prognostic and Predictive Biomarkers II (ID 95)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      MO05.02 - Overexpression of FGFR1 mRNA and protein are more frequent than FGFR1 gene amplification in non-small cell lung cancer (NSCLC) patients (ID 2459)

      16:20 - 16:25  |  Author(s): M.W. Wynes

      • Abstract
      • Presentation
      • Slides

      Background
      Somatic mutations and gene fusions have been identified as oncogenic drivers in lung cancer, however, a number of lung cancers have no apparent molecular aberration driving oncogenesis. It appears that gene/protein overexpression may sustain these “pan-negative” cancers. Fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate cell proliferation, differentiation, migration and survival and dysregulation of this signaling pathway is observed in a proportion of lung cancers. A number of compounds targeting FGF/FGFR are in clinical development but clinically applicable biomarker assays and companion diagnostics that accurately identify patients with tumors sensitive to these agents are needed. We previously presented cell line data demonstrating that FGFR1 mRNA (ME) or protein expression (PE) better identified FGFR1 inhibitor sensitive tumors compared to gene copy number (GCN). The goal of this study was to examine FGFR1 ME, PE and GCN in a surgically treated NSCLC clinical cohort and explore possible associations with clinical features and prognosis.

      Methods
      Immunohistochemistry, brightfield in situ hybridization, and silver in situ hybridization were used to investigate ME, PE and GCN, respectively, in a cohort of 189 NSCLC surgically-treated patients. PE was scored by the H-score method (0-300) and ME on a semiquantative integer scale (0-4+), both evaluating the entire tumor specimen. GCN was scored on continuous scale by counting the individual signals in 50 cells and determining the average GCN per tumor cell.

      Results
      Amplification (GCN >=4) was present in 8% of the entire cohort and in 11% of the squamous cell carcinoma (SCC) or mixed histology subgroup. No amplifications were found in the adenocarcinomas (ADC) or tumors from never smokers. In contrast, 29% of SCC and ADC patients had high ME (= 4+). Elevated PE (>= 100) was observed in 20% of the cohort, with the highest expression observed in SCC/mixed histology, but 6% of ADCs also showed elevated PE. There was no elevated FGFR1 PE in the never smokers. There was significant correlation but incomplete overlap between biomarkers. There were no prognostic associations, either with overall or disease-free survival, for FGFR1 GCN, ME, or PE. There was excellent inter-observer agreement among the readers of all 3 biomarker assays.

      Conclusion
      Overexpression of FGFR1 mRNA and protein are more frequent than FGFR1 gene amplification in NSCLC patients. Although GCN amplification was restricted to SCC, elevated ME and PE were found in both ADC and SCC. There was no prognostic association with FGFR1 GCN, ME, or PE. These data are consistent with our previous cell line data that showed elevated PE and ME in non-amplified cells and suggests that GCN may not identify all the potential patients who could benefit from FGF/FGFR pathway inhibitors.

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    O04 - Molecular Pathology I (ID 126)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Pathology
    • Presentations: 1
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      O04.06 - An international standardization study using the ALK IHC antibody D5F3 and a sensitive detection kit demonstrates high concordance between ALK IHC and ALK FISH and between evaluators (ID 2875)

      11:25 - 11:35  |  Author(s): M.W. Wynes

      • Abstract
      • Presentation
      • Slides

      Background
      The goal of personalized medicine is treating patients with a therapy predicted to be efficacious based on the molecular characteristics of the tumor, thereby sparing the patient futile or detrimental therapy. Anaplastic lymphoma kinase (ALK) inhibitors are effective against ALK positive non-small cell lung cancer (NSCLC) tumors, but to date the only US Food and Drug Administration approved companion diagnostic is a break-apart fluorescence in situ hybridization (FISH) assay. Immunohistochemistry (IHC) is a clinically applicable cost-effective test that is sensitive and specific for ALK protein expression. The purpose of this study was to assemble an international team of expert pathologists to evaluate and standardize the interpretation of a new automated standardized ALK IHC assay.

      Methods
      Archival NSCLC tumor specimens (n=103) previously tested for ALK rearrangement by FISH were provided by the international collaborators. These specimens were stained by IHC with the anti-ALK (D5F3) primary antibody (Ventana Medical Systems, Inc) combined with OptiView DAB IHC detection and OptiView amplification (Ventana Medical Systems, Inc). The evaluators went through an interpretation training session and scored the specimens as positive, if strong granular cytoplasmic brown staining was present in tumor cells, or negative. IHC results were compared to the FISH results and inter-evaluator agreement comparisons made.

      Results
      Overall for the 100 evaluable cases the ALK IHC assay was highly sensitive (90%), specific (95%) and accurate (93%) relative to the ALK FISH results. Similar results were observed using a majority score. For the discrepant cases IHC negativity was scored by 7/7 on 3 FISH positive cases and 6/7 evaluators on 2 additional FISH positive cases. IHC positivity was scored on 2 FISH negative cases by 7/7 readers. There was agreement among 7/7 and 6/7 readers on 88% and 96%% of the cases before a consensus review, respectively, and following a review there was agreement among 7/7 and 6/7 on 95% and 97% of the cases, respectively.

      Conclusion
      Based on expert evaluation the ALK IHC assay using the D5F3 antibody combined with Optiview Detction and Optiview amplification is sensitive, specific and accurate, relative to FISH, and a majority score of multiple readers does not improve these results over an individual reader’s score. Excellent inter-reader agreement was observed for the IHC assay. These data support the algorithmic use of ALK IHC as a screening procedure for ALK protein expression in NSCLC.

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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-004 - ROS1 Immunohistochemistry Among Major Genotypes of Non-Small Cell Lung Carcinoma (ID 739)

      09:30 - 09:30  |  Author(s): M.W. Wynes

      • Abstract

      Background
      ROS1 (c-ros oncogene 1) is a receptor tyrosine kinase that can become constitutively active and drive cellular proliferation in a variety of cancers. Approximately 1-2% of patients with non-small cell lung cancer (NSCLC) harbor activating ROS1 gene fusions and these patients may benefit from ROS1-targeted inhibitor therapy.

      Methods
      Immunohistochemistry for ROS1 expression was performed on 33 NSCLC specimens previously characterized for the presence of genetic abnormalities. These specimens were selected for ROS1 gene rearrangements (6 specimens) detected by RT-PCR and FISH, ALK gene rearrangements (5 specimens), EGFR mutations (5 specimens), KRAS mutations (5 specimens), HER2 mutations (3 specimens), RET gene rearrangements (3 specimens), and pan-negative (6 specimens). Immunohistochemistry was performed in a CLIA-certified laboratory with manual application of the ROS1 DFD6 antibody (Cell Signaling Technology, Inc) for 1 hour. ROS1 protein expression was evaluated by a pathologist with a hybrid (H)-score scale of 0 (no expression in any tumor cells) to 300 (intense expression in all tumor cells). ROS1 over-expression was defined as an H-score greater than 100.

      Results
      ROS1 protein over-expression was detected by immunohistochemistry in all 6 of the NSCLC specimens with ROS1 gene fusions detected by RT-PCR (example in figure below). None of the remaining 27 lung cancer specimens with ALK gene rearrangements, EGFR mutations, KRAS mutations, HER2 mutations, RET gene rearrangements, or pan-negative exhibited ROS1 protein over-expression. Figure 1

      Conclusion
      Detection of ROS1 over-expression by immunohistochemistry exhibited 100% concordance with results of ROS1 gene rearrangement for 33 NSCLC specimens and did not overlap with any of the other genetic alterations. Six specimens were positive for ROS1 gene rearrangement by both RT-PCR and immunohistochemistry. Tumors positive for genetic alterations associated with the ALK, EGFR, KRAS, HER2, and RET genes were all negative for ROS1 gene rearrangement and ROS1 immunohistochemistry. ROS1 immunohistochemistry is a sensitive, specific and cost-effective method for identification of a subset of patients with lung cancer that may benefit from ROS-1 targeted-therapy.

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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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      P3.05-023 - Treatment with the mTOR kinase inhibitor CC-223 overcomes resistance to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib in non-small cell lung cancer cells (ID 3429)

      09:30 - 09:30  |  Author(s): M.W. Wynes

      • Abstract

      Background
      Activation of the mTOR pathway is a common down-stream mechanism of resistance to Epidermal Growth Factor receptor (EGFR) tyrosine kinase inhibitors. CC-223 (Celgene) is an mTOR kinase inhibitor under clinical development. We evaluated CC-223 in combination with erlotinib to overcome resistance to EGFR tyrosine kinase inhibition in non-small cell lung cancer (NSCLC) cell lines and xenografts in nude mice.

      Methods
      A panel of 18 NSCLC cell lines was used to evaluate the effect of erlotinib and CC-223 treatment on cell growth using an MTT assay. Intermediately (IC50 >1 and <10 mM) and highly (IC50 >10 mM) resistant cell lines to erlotinib were used in analyses of additive/synergistic effects for the combination treatment with CC-223.

      Results
      CC-223 demonstrated anti-proliferative activity in NSCLC cell lines with various degrees of sensitivity as reflected in different IC50 values, ranging from 0.15 to 12 mM. In combination with erlotinib, CC-223 showed synergistic anti-proliferative effects in NSCLC cells resistant to erlotinib with combination indices as low as 0.1-0.2. In vivo studies in mice xenografts demonstrated a strong synergistic effect of the combination treatment of erlotinib and CC-223 with a 90% decrease of tumor volume compared to untreated and 88% compared to erlotinib treated for A549 cells. IHC analyses of apoptosis and proliferation in the tumors are ongoing; mature data will be presented at the meeting.

      Conclusion
      The mTOR kinase inhibitor CC-223 demonstrated anti-proliferative activity in a panel of NSCLC cell lines. In NSCLC cells resistant to the EGFR tyrosine kinase inhibitor erlotinib, combining erlotinib with CC-223 demonstrated a synergistic anti-proliferative effect. In vivo studies of tumors in xenografted mice also demonstrated synergistic anti-tumor effects with the combination treatment even in erlotinib resistant tumors. The present data suggest that mTOR inhibition using the mTOR kinase inhibitor CC-223 may be a therapeutic strategy to overcome resistance to EGFR tyrosine kinase inhibitors in NSCLC.

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    P3.18 - Poster Session 3 - Pathology (ID 177)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pathology
    • Presentations: 1
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      P3.18-012 - The hidden 50% of the ALK positive NSCLC patients (ID 2469)

      09:30 - 09:30  |  Author(s): M.W. Wynes

      • Abstract

      Background
      Patients with lung adenocarcinoma carrying the ALK gene rearrangement show dramatic response to ALK TKIs (e.g. crizotinib). The currently approved method for detection of ALK gene rearrangements is fluorescence in situ hybridization (FISH), however gene sequencing can also be used for detecting ALK rearrangement. Aberrant ALK protein expression, as ALK is not normally expressed in the lung, can be detected with immunohistochemistry (IHC). Our experience has shown a high rate of false negative ALK FISH patients, therefore we retrospectively investigated 53 cases with IHC and Next Generation Sequence (NGS).

      Methods
      53 patients with NSCLC adenocarcinoma were tested for ALK rearrangement by FISH (Oncotest-TEVA Ltd, Israel). Retrospective IHC (D5F3 antibody, Cell Signaling) was done at the University of Colorado Cancer Center. Discordance cases were sequenced by "FoundationOne” (Foundation Medicine Inc).

      Results
      Out of the 53 cases, 4 (7.5%) were FISH positive, and 8 (15%) were IHC positive with 3 cases both FISH+ and IHC+ (Figure 1 , Table 1). One specimen was FISH+ and IHC-. NGS was performed on the 5 IHC+/FISH- mismatched samples and found 4 out of the 5 cases positive for ALK rearrangement. The true positive incidence of ALK rearrangement in our cohort increased by 100%, from 7.5% to 15%. Interestingly, two patients were found to harbor a unique ALK rearrangement at intron 19. One of them showed a dramatically improvement to crizotinib (PFS=18 months).

      FISH IHC Patients NGS ALK rearrangement +
      + + 3 3
      - - 44 0*
      + - 1(1.8%) 0
      - + 5(9.4%) 4 4
      Total 53 4 7
      Table 1 - FISH and IHC summary Figure 1

      Conclusion
      The current FISH based approach to detect ALK gene rearrangement misses numerous (50%) patients who might benefit from the most efficient lung cancer therapy for their disease. Screening for ALK protein expression by IHC may identify ALK positive patients that would otherwise be missed. Borderline IHC staining should be further sequenced by NGS to cover other abnormalities such as intron19 or other uncommon rearrangements.