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S. Rafee



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    MINI 08 - Prognostic/Predictive Biomarkers (ID 106)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      MINI08.10 - Co-Occurrence of Driver Mutations of MAPK and PI3K Pathways in Non Small Cell Lung Cancer: A Report from Lung Cancer Genomics Ireland (LCGI) Study (ID 2627)

      17:45 - 17:50  |  Author(s): S. Rafee

      • Abstract
      • Presentation
      • Slides

      Background:
      The mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are frequently altered in human cancers. Targeting these pathways is an attractive therapeutic strategy in malignant disease. The frequency of single and dual pathway alterations varies substantially across various cancers. Co-occurrence of the MAPK and PI3K pathway aberrations is reported in 5-7% of melanomas, gastric and colorectal cancers, and is associated with a worse clinical outcome. In this report we aim to determine the co-occurrence of the MAPK and PI3K pathway mutations in a large cohort of surgically resected NSCLC tumors.

      Methods:
      We used the platform of Sequenom’s MassArray to perform genotyping for 548 somatic hotspot mutations in 49 genes including genes in the MAPK and PI3K pathways in surgically resected NSCLC tumors. MAPK pathway genes that were screened include: KRAS, HRAS, BRAF, RAF1, MAP3K1, MAP3K2, MAP3K3, MAP3K4, MAP3K5, MAP2K1, MAP2K2, MAP2K3, and PTPN11. PI3K pathway genes that were screened include: PIK3CA, PIK3R1, PIK3R2, PTEN, PDPK1, AKT1, AKT2, and MTOR. Fisher’s exact test was used to determine the statistical significance of association between the MAPK and PI3K pathway mutations. The strength of association was determined in the form of odds ratio.

      Results:
      NSCLC tumors from 356 patients (258 squamous cell, 98 adenocarcinomas) were tested using Sequenom’s MassArray. The frequency of mutations in the MAPK and PI3K pathways was 22.5% (n=80) and 22.8% (n=81) respectively. Among these patients, 38 patients had mutations in both pathways (i.e: 47.5% of patients with a MAPK pathway mutation also had a mutation in the PI3K pathway, and 46.9% of patients with a PI3K pathway mutation also had a mutation in the MAPK pathway, see table 1). Fisher’s exact test revealed that mutations in the MAPK and the PI3K pathways are mutually inclusive (p<0.0001, odds ratio=4.95, 95% CI 2.9-8.5) Table 1: The co-occurrence of MAPK and PI3K pathway mutations in NSCLC

      Pathway/no of patients PI3K WT PI3K MT
      MAPK WT 235 43
      MAPK MT 42 38


      Conclusion:
      38 (10.7%) of 356 NSCLC patients included in the LCGI study had hotspot somatic mutations in both the MAPK and PI3K pathways. Contrary to previous reports, we observed that activating mutations of the MAPK and PI3K pathways are mutually inclusive in NSCLC. These findings may have implications in designing clinical trials of targeted therapies in lung cancer.

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    MINI 13 - Genetic Alterations and Testing (ID 120)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      MINI13.08 - Targetable Genomic Aberrations in Squamous Cell Lung Cancer (SCC): A Report from the Lung Cancer Genomics Ireland (LCGI) Study (ID 766)

      11:25 - 11:30  |  Author(s): S. Rafee

      • Abstract
      • Presentation
      • Slides

      Background:
      The prognosis of lung SCC continues to be poor with no molecularly targeted agents specifically developed for its treatment. LCGI aims to identify potential targetable oncogenes in lung SCC.

      Methods:
      The LCGI study is being carried out in 500 patients with surgically resected lung SCC, treated at St James’s University Hospital and Beaumont University Hospital, Dublin. We used the platform of Sequenom’s MassArray to perform genotyping for accustomed panel of 258 somatic hotspot mutations in 49 genes including genes in the MAPK and PI3K pathways. We also evaluated FGFR1 amplification by fluorescence in situ hybridization (FISH) and MET protein expression by immunohistochemistry (IHC).

      Results:
      Lung SCCs from 258 patients have been tested by Sequenom MassArray to date. Lung SCCs from 150 patients have been evaluated for MET protein expression and 89 for FGFR1 amplification. 163 (63.2%) patients were male. The median age of the cohort was 68. The majority of patients were either current (39.5%) or former (58.1%) smokers at the time of diagnosis. 138 (53.5%) were stage I, 87 (33.7%) were stage II, and 33 (12.8%) were stage III SCCs. At least one aberrant, potentially targetable oncogene was identified in the SCC of 101 (39.1%) patients (see Table). The presence of PIK3CA or KRAS mutations, or FGFR1 amplification did not have a statistically significant impact on median overall survival or recurrence-free survival. However, the presence of two or more aberrations in driver oncogenes in a tumor (patients, n=19) was associated with a worse median overall survival compared to patients with either a single driver aberration (p=0.04) or no aberrations (p<.01). Table: Frequency of driver mutations in LCGI compared to The Cancer Genome Atlas (TCGA) study

      Mutation LCGI (n=258) TCGA (n=178)
      FGFR1 amp (n=89) 13 % 16.8 %
      PIK3CA 15.1 % 10.1 %
      KRAS 6.5 % 0.6 %
      PTPN11 3.5 % 1.7 %
      STK11 3.1 % 1.7 %
      MYC 1.9 % 0.0 %
      NRAS 1.6 % 0.0 %
      BRAF 1.2 % 3.9 %
      HRAS 1.6 % 1.7 %
      CTNNB1 1.5 % 1.7 %
      FBXW7 1.5 % 3.4 %
      MET Overexpression (n=150) 1.3 % NA
      EGFR 0.9 % 2.8 %
      AKT1 0.4 % 0.6 %
      CDK4 0.4 % 0.0 %
      GNA11 0.4 % 0.6 %
      MAP2K1 0.4 % 0.6 %
      DDR2 0 % 1.1 %


      Conclusion:
      39.1% of lung SCC patients have an aberrant, potentially targetable driver oncogene in their tumor. The presence of two or more aberrant oncogenes is a poor prognostic factor in lung SCC. These findings can be used to guide clinical trials in lung SCC.

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    ORAL 37 - Novel Targets (ID 146)

    • Event: WCLC 2015
    • Type: Oral Session
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      ORAL37.02 - Protein Tyrosine Phosphatase Non Receptor 11 PTPN11/Shp2 as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer NSCLC (ID 1590)

      16:56 - 17:07  |  Author(s): S. Rafee

      • Abstract
      • Slides

      Background:
      PTPN11/Shp2 somatic mutations occur in 25% of Juvenile myelomonocytic leukemias (JMML) and less commonly in adult solid tumors. PTPN11/Shp2 activates the mitogen-activated protein kinase (MAPK) and the phosphatidylinositide 3-kinase (PI3K) pathways. Accordingly, PTPN11/Shp2 mutations were shown to sensitize leukemia cells to MEK and PI3K inhibitors.

      Methods:
      We applied mass-spectrometry based genotyping (Sequenom Inc., Germany) to DNA extracted from tumor and matched normal tissue of 356 NSCLC patients (98 adenocarcinomas and 258 squamous cell (SCC)). PTPN11/Shp2 constructs with mutations (E76A, A72D) were generated and stably expressed in IL-3 dependent BaF3 cells and NSCLC cell lines (H1703, H157). The acquisition of MAPK and PI3K pathways activation was evaluated using western blotting and reverse phase protein array (RPPA). PTPN11/Shp2 phosphatase activity was measured in whole cell protein lysates using Shp2 assay kit (R&D Systems).

      Results:
      Somatic PTPN11/Shp2 hotspot mutations occurred in 3 (3.1%) and 9 (3.4%) of adenocarcinomas and SCCs, respectively. Mutant PTPN11/Shp2, compared to PTPN11/Shp2 wild type, promoted ten-fold IL-3 independent BaF3 cell survival. BaF3, H1703, and H157 cells expressing mutant PTPN11/Shp2 exhibited increased PTPN11/Shp2 phosphatase activity, phospho-ERK1/2, and phospho-AKT levels. Sequencing of NSCLC cell lines revealed that NSCLC H661 cell line has a PTPN11/Shp2 activating mutation (N58S). H661 had significantly higher PTPN11/Shp2 phosphatase activity when compared to PTPN11 wild-type H1703 and Calu3 NSCLC cells. Since the biological functions of PTPN11/Shp2 are mediated through its phosphatase domain, we stably expressed the inactivating PTPN11/Shp2 phosphatase domain mutation (C459S) in H661, H1703 and H157 cells resulting in catalytically inactive PTPN11/Shp2. This led to decreased phospho-ERK1/2 levels in all three cell lines. Importantly, the inactivation of PTPN11/Shp2 resulted in decreased phospho-AKT levels in H661 cells (PTPN11-mutated) and had no effect on phospho-AKT levels in the PTPN11/Shp2-wild type H1703 and H157 cells. Taken together, this data suggests that PTPN11/Shp2 activating mutations are oncogenic in NSCLC cells. Moreover, these findings reveal that PTPN11/Shp2 mutations may selectively activate the PI3K pathway in NSCLC cells. Parental H661 (PTPN11-mutated, KRAS and PIK3CA-wild type), parental H1703 (PTPN11, KRAS and PIK3CA-wild type) and parental H157 (KRAS-mutated, PTPN11 and PIK3CA-wild type) cells were treated with the novel MEK (BAY86-9766) and PI3K (BAY80-6946) inhibitors. IC50 values (table 1) suggest that PTPN11-mutated NSCLC cells have modest sensitivity to MEK inhibitors and profound sensitivity to PI3K inhibitors.

      Table 1 IC 50 valuse
      Cell Line BAY86-9766 (nM) BAY80-6946 (nM)
      H661 2880 ± 600 13 ± 4.7
      H157 1450 ± 520 < 50% inhibition @ 200
      H1704 < 50% inhibition @ 10000


      Conclusion:
      PTPN11/Shp2 demonstrates the in vitro features of a driver oncogene, and potentially represents a new target in NSCLC.

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