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B. Hennessy
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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
- Moderators:T.E. Stinchcombe, N. Pavlakis
- Coordinates: 9/07/2015, 16:45 - 18:15, Mile High Ballroom 4a-4f
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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): B. Hennessy
- Abstract
- Presentation
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 NSCLCPathway/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
- Moderators:Y. Koh, R.K. Thomas
- Coordinates: 9/08/2015, 10:45 - 12:15, 205+207
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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): B. Hennessy
- Abstract
- Presentation
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) studyMutation 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
- Moderators:S.S. Ramalingam, E. Thunnissen
- Coordinates: 9/09/2015, 16:45 - 18:15, Mile High Ballroom 4a-4f
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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): B. Hennessy
- Abstract
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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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-109 - Epithelial-To-Mesenchymal Transition (EMT) and Acquired Resistance to PI3K-mTOR Inhibition in NSCLC (ID 934)
09:30 - 09:30 | Author(s): B. Hennessy
- Abstract
Background:
The PI3K-Akt-mTOR pathway regulates cell growth and proliferation and is often dysregulated in NSCLC, making it an attractive therapeutic target in this setting. GDC-0980 is a selective dual inhibitor of PI3K and mTOR, which is currently in Phase II clinical trials for solid tumours. As with all targeted therapies, acquired resistance to GDC-0980 is anticipated to be a major hurdle in the success of this drug. The aims of this project are to (i) elucidate the frequency of PIK3CA mutations in an Irish cohort of NSCLC patients and (ii) develop and characterise three cell line models of resistance to GDC-0980, each representing a different molecular subtype of NSCLC, in order to identify biomarkers of response/resistance to the drug that may dictate beneficial treatment strategies.
Methods:
DNA was extracted from 250 NSCLC patient tissue samples, and screened for 547 clinically relevant mutations in 46 genes using the Sequenom platform. H460, A549, and H1975 cells were cultured in GDC-0980 at IC50 concentrations over a period of several months, along with matched ‘parent’ cell lines. Development of resistance was assessed by monthly BrdU proliferation assays. Cell growth patterns were compared across the sensitive and resistant cell lines in real time using the xCELLigence platform. Cell lines were then interrogated for alterations in DNA (Sequenom), mRNA (SABiosciences arrays profiling expression of >150 genes), miRNA (Exiqon expression profiling of 2100 miRNAs) and protein (R&D Phospho Kinase array expression profiling of 43 kinases and 2 associated total proteins, PTMScan[®] Ubiquitin Remnant Motif (K-ε-GG) Kit from CST and Western blot analysis).
Results:
PIK3CA mutations occur in ~5% adenocarcinomas & 12% squamous cell carcinomas. H1975 cells (PIK3CA mutant and activated pAkt (Ser473/Thr308), pmTOR, pS6R) were most sensitive to GDC-0980, however they were the first to develop resistance to the drug. Results obtained from xCELLigence studies identified H1975 resistant (H1975R) cells as having the highest cell index out of all parent and resistant cell lines after 100 hours of cell growth, suggesting that these are the most aggressive cells. Initially a 33 miRNA signature was identified contrasting H1975P and H1975R. qPCR validation of miR-205 (a regulator of EMT) identified expression in H1975P cells but miR-205 was undetectable in H1975R cells. mRNA expression of Zeb1 & Zeb2 (direct targets of miR-205) were increased in H1975R cells compared to H1975P cells. 1,200 proteins were found to be differentially expressed between H1975P and H1975R cells. Increased expression of EMT proteins vimentin, desmin and filamin was detected in H1975R cells (p < 0.05, fold change >2). Vimentin overexpression in H1975R cells was confirmed by western blot analyis. Activation of EMT was identified as one potential mechanism of resistance to GDC-0980 in H1975R cells.
Conclusion:
The PI3K-mTOR pathway is frequently mutated in NSCLC, in particular squamous cell carcinoma, making it an ideal therapeutic target. Acquired resistance to GDC-0980 developed rapidly in NSCLC cell lines, (4-6 months) and correlates to the induction of EMT. Further elucidation of EMT regulation is under investigation and is crucial to the design of improved treatment protocols.
