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S.M. Lim
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MINI 09 - Drug Resistance (ID 107)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:L. Villaruz, J. Minna
- Coordinates: 9/07/2015, 16:45 - 18:15, 205+207
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MINI09.07 - Activation of the MET Kinase Confers Acquired Resistance to FGFR-Targeted Therapy in FGFR-Dependent Squamous Cell Carcinoma of the Lung (ID 1212)
17:20 - 17:25 | Author(s): S.M. Lim
- Abstract
- Presentation
Background:
Fibroblast growth factor receptor (FGFR) tyrosine kinase plays a crucial role in cancer cell growth, survival, and resistance to chemotherapy. FGFR1 amplification occurs at a frequency of 10-20% and is a novel druggable target in squamous cell carcinoma of the lung (SCCL). A number of FGFR-targeted agents are currently being developed in SCCL harboring FGFR alterations. The aim of the study is to evaluate the activity of selective FGFR inhibitors (AZD4547, BAY116387) and the mechanisms of intrinsic and acquired resistance to these agents in SCCL.
Methods:
The antitumor activity of AZD4547 and BAY116387 was screened in a panel of 12 SCCL cell lines, among which 4 cell lines harbored FGFR1 amplification. To investigate mechanisms of acquired resistance, FGFR1-amplified H1581 cells which were exquisitely sensitive to FGFR inhibitors, were exposed to AZD4547 or BAY116387 to generate polyclonal resistant clones (H1581-AR, H1581-BR). Characterization of these resistant clones was performed using receptor tyrosine kinase (RTK) array, immunoblotting and microarray. Migration and invasion assays were also performed.
Results:
Among 12 SCCL cell lines, two FGFR1-amplified cells, H1581 and DMS114, were sensitive to FGFR inhibitors (IC~50~<250 nmol/L). Compared with resistant cells, sensitive cells showed increased phosphorylation of FRS2 and PLC-γ, but decreased phosphorylation of STAT3. There was no noticeable difference in FGFR1-3 protein expression level between sensitive and resistant cells. Importantly, phosphorylation of ERK1/2 was significantly suppressed upon treatment of FGFR inhibitors only in sensitive cells, suggesting phospho-ERK1/2 as a pharmacodynamic marker of downstream FGFR signaling. RTK array and immunoblots demonstrated strong overexpression and activation of MET in H1581-AR and H1581-BR, in comparison to almost nil expression in parental cells. Four different SCCL cells with intrinsic resistance to FGFR inhibitors also showed intermediate to high MET expression, suggesting that MET may be involved in both intrinsic and acquired resistance to FGFR inhibitors. Gene-set enrichment analysis against KEGG database showed that cytokine-cytokine receptor interaction pathway was significantly enriched, with MET contributing significantly to the core enrichment, in H1581-AR and H1581-BR, as compared with parental cells. Stimulation with HGF strongly activated downstream FGFR signaling or enhanced cell survival in the presence of FGFR inhibitors in both acquired and intrinsic resistant cells. Quantitative PCR on genomic DNA and fluorescent in situ hybridization revealed MET amplification in H1581-AR, but not in H1581-BR. MET amplification led to acquired resistance to AZD4547 in H1581-AR by activating ERBB3. The combination of FGFR inhibitors with ALK/MET inhibitor, crizotinib, or small interfering RNA targeting MET synergistically inhibited cell proliferation in both H1581-AR and H1581-BR, whereas it resulted in additive effects in SCCL cells with intrinsic resistance to FGFR inhibitors. Acquisition of resistance to FGFR inhibitors not only led to a morphologic change, but also promoted migration and invasion of resistant clones via inducing epithelial to mesenchymal transition phenotype, as documented by a decrease in E-cadherin and an increase in N-cadherin and vimentin.
Conclusion:
MET activation is sufficient to bypass dependency on FGFR signaling and concurrent inhibition of these two pathways may be desirable when targeting FGFR-dependent SCCL.
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ORAL 06 - Next Generation Sequencing and Testing Implications (ID 90)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:G. De Lima Lopes, V. Miller
- Coordinates: 9/07/2015, 10:45 - 12:15, Mile High Ballroom 1a-1f
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ORAL06.02 - Targeted Deep Sequencing of EGFR/KRAS/ALK-Negative Lung Adenocarcinoma Reveals Potential Therapeutic Targets (ID 622)
11:16 - 11:27 | Author(s): S.M. Lim
- Abstract
- Presentation
Background:
Identification of clinically relevant molecular drivers in patient tumors is essential in selecting appropriate targeted therapy. Using next-generation sequencing (NGS) -based clinical cancer gene test, we performed genomic profiling of lung adenocarcinoma tumors.
Methods:
We collected formalin-fixed paraffin-embedded tumors from 41 lung adenocarcinoma patients whose tumors previously tested negative for EGFR/KRAS/ALK by conventional methods in an ongoing trial (NCT01964157). We performed hybridization capture of 4,557 exons from 287 cancer-related genes and 47 introns from 19 genes frequently rearranged in cancer (FoundationOne). Illumina HiSeq2000 platform was used to sequence to high uniform depth.
Results:
Figure 1Tumors were sequenced to a median coverage of 529x. Overall, we identified a total of 170 known and 492 unknown individual genomic alterations. The number of known alterations per sample was average of 3.8 alterations (range 0-10). Cancer genomes are characterized by 45% (77/170) non-synonymous base substitutions, 17% (29/170) insertions or deletions, 2% (4/170) splice site mutations, 20% (34/170) gene amplifications, 5% (8/170) homozygous loss and 5% (8/170) gene fusions. TP53 was the most commonly mutated gene (13%, n=10/77) among non-synonymous base substitutions, followed by KRAS (10%, n=8/77) and PIK3CA (8%, 6/77). Insertions or deletions commonly occurred TP53 (17%, 5/29) and ERBB2 (14%, 4/29), and splice site mutations occurred in TP53, INPP4B, ATR, and MAP2K4 (n=1 each). Among gene amplification, MDM2 amplification was the most frequent (12%, 4/34), followed by ERBB2 (8%, 3/34) and CDK4 (8%, 3/34) amplification. All 8 cases of homozygous loss were observed with CDKN2A and CDKN2B. Fusion genes were most commonly observed with RET (50%, n=4/8). Based on NCCN guidelines, actionable genomic alterations with a targeted agent were identified in 16 patients (39%) (BRAF mutation [n=1], EGFR mutation [n=7], ERBB2 mutation [n=4], MET amplification [n=1], KIF5B-RET rearrangement [n=2], CCDC6-RET rearrangement [n=1], and CD74-ROS1 rearrangement [n=1]). Nine out of all patients (22%) showed discordance in targetable alterations when compared between NGS and conventional non-NGS methods.
Conclusion:
Thirty-nine percent of lung adenocarcinoma wild type for EGFR/KRAS/ALK may harbor a genomic alteration revealed by NGS approach. These results highlight the importance of profiling lung adenocarcinomas using NGS in the clinic.
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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-101 - Utility of Patient-Derived Cell Line Models Using Conditional Reprogramming for in Vitro Pharmacogenomics Platform (ID 963)
09:30 - 09:30 | Author(s): S.M. Lim
- Abstract
Background:
To evaluate the potential of conditional reprogrammed cells (CRCs) established from biopsy or effusion samples of advanced non-small cell lung cancer (NSCLC) for in vitro pharmacologic screen and identification of drug resistance mechanisms.
Methods:
A total of 48 tumor specimens obtained from 46 patients with NSCLC were cultured with irradiated fibroblast feeder cells and Rho kinase inhibitor (Y-27632) to induce tumor cells to proliferate indefinitely. The cell lines established from patients harboring EGFR mutation or other druggable oncogenes were subjected to genetic analyses and pharmacologic screen. Corresponding tumor cells were injected into nude mice to test for tumorigenicity and efficacy of targeted agents in vivo.
Results:
Twenty one male patients and twenty five female patients were assessed for establishment of CRC. Adenocarcinoma was the most frequent histologic type (84.7%). There were 21 patients (46%) who harbored an active EGFR mutation. There were four patients with ALK fusion and five with ROS1 fusion. Twenty-six patients experienced disease progressed while on treatment with EGFR (20), ALK (2) or ROS1 (4) tyrosine kinase inhibitors. Tumor cells came from primary or distant metastases in 48% and 52%, respectively. Thirty one (65%) samples were obtained by tumor biopsy and 17 from malignant pleural effusion. Nine CRC model were successfully established (18.7%, 9/48). The successful growth was not dependent on the clinicopathologic characteristics. Both cells from pleural effusion (4 of 17) and biopsy (5 of 31) and adenocarcinoma (8 of 41) and squamous cell carcinoma (1 of 3) were successfully cultured. For biopsy samples, the success rate of cells obtained from primary lung lesion was 21.7% (5 of 23) and cells from metastatic site outside lung was 0% (0 of 8) (P = 0.3). For effusion samples, volume of effusion required for CRC was not significant factors for establishment (success vs. failure cases: mean volume 500 ml vs. 267 ml). The genetic characteristics of patients with non-squamous cell carcinoma did not affect the success rate of CRC (EGFR mutation, 4 of 21; ALK translocation, 0 of 4; ROS1 translocation, 2 of 5; wild or unknown, 2 of 15). Two xenograft models with CRC were successfully established and passaged to maintain tumor in vivo.
Conclusion:
The CRC models derived from NSCLC patients provide useful in vitro platforms of preclinical studies evaluating novel targeted therapies and uncovering the drug resistance mechanisms.
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P3.01 - Poster Session/ Treatment of Advanced Diseases – NSCLC (ID 208)
- Event: WCLC 2015
- Type: Poster
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.01-077 - A Randomized, Phase II Study of Nimotuzumab Plus Gefitinib vs Gefitinib in Advanced Non-Small Cell Lung Cancer After Platinum- Based Chemotherapy (ID 1176)
09:30 - 09:30 | Author(s): S.M. Lim
- Abstract
Background:
Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) monoclonal antibody. We aim to evaluate the efficacy of dual inhibition of EGFR with nimotuzumab plus gefitinib in advanced non-small cell lung cancer (NSCLC) previously treated with platinum-based chemotherapy.
Methods:
An open label, randomized, phase II trial was conducted in 6 centers; 160 patients were randomized (1:1) to either nimotuzumab (200mg, IV weekly) plus gefitinib (250mg p.o. daily) or gefitinib alone until disease progression or intolerable toxicities. The primary endpoint was progression free survival (PFS) rate at 3 months. Secondary endpoints included PFS, overall survival (OS), overall response rate (ORR) and safety.
Results:
A total of 155 patients (78 in nimotuzumab plus gefitinib, 77 in gefitinib) were evaluable for efficacy and toxicity. Patient characteristics were well balanced in both groups. Majority of patients had adenocarcinoma histology (65.2%) and ECOG performance status 0 to 1 (83.5%). Among 102 patients with EGFR mutation results available, activating EGFR mutation was documented in 27 patients (12/50 in nimotuzumab plus gefitinib, 15/52 in gefitinib). With a median follow-up of 12.1 months, PFS rate at 3 months was 37.2% in nimotuzumab plus gefitinib and 48.1% in gefitinib [HR 1.03; 95% CI, 0.71–1.40; P=0.98]. Median PFS and OS were 2.0 months and 14.0 months in nimotuzumab plus gefitinib and 2.8 months and 13.2 months in gefitinib [HR 1.03, 95% CI 0.71-1.41, P=0.98 for PFS; HR 0.86, 95% CI 0.57–1.30, P=0.47 for OS]. The ORRs were 14.1% in nimotuzumab plus gefitinib and 22.1% in gefitinib, which was not statistically significant (P=0.76). As expected, patients with EGFR mutation showed significantly longer survival than those with wild-type EGFR or unknown EGFR mutation status (10.3 vs. 1.2 vs. 2.7 months, P < 0.001 for PFS; 23.5 vs. 13.5 vs. 10.5 months, P= 0.001 for OS). Combined treatment of nimotuzumab plus gefitinib did not show superior PFS compared to gefitinib alone in patients with EGFR mutation (13.5 vs. 10.2 months in gefitinib alone, P=0.30) and patients with wild-type EGFR (0.9 vs. 2.0 months in gefitinib alone, P=0.90). The median PFS was not significantly different between two treatment arms according to histology (2.8 vs. 2.9 months in gefitinib alone for adenocarcinoma, P=0.64; 1.2 vs. 2.8 months in gefitinib alone for non-adenocarcinoma, P=0.35). Adverse events (AEs) in both treatment arms were mostly grade 1 to 2 and easily manageable. Importantly, combined EGFR inhibition with nimotuzumab and gefitinib did not increase EGFR inhibition-related AEs, such as acneiform rash (32.4 vs. 30.3% in gefitinib alone, P=0.38), diarrhea (30.7 vs. 35.7% in gefitinib alone, P=0.32), and stomatitis (11.5 vs. 13.4% in gefitinib alone, P=0.19). There was no treatment-related death.
Conclusion:
The dual inhibition of EGFR with nimotuzumab plus gefitinib did not show superiority over gefitinib alone for second-line treatment of advanced NSCLC (NCT01498562).
