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N. Pham



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    MA17 - Genetic Drivers (ID 409)

    • Event: WCLC 2016
    • Type: Mini Oral Session
    • Track: Biology/Pathology
    • Presentations: 1
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      MA17.06 - Landscape of Somatic Mutations Involving Lung Cancer Associated Genes in Non-Small Cell Lung Cancer (NSCLC) Patient-Derived Xenografts (ID 6084)

      14:20 - 15:50  |  Author(s): N. Pham

      • Abstract
      • Presentation
      • Slides

      Background:
      Patient-derived tumor xenografts (PDXs) have high fidelity to their histological origins, and maintain the molecular heterogeneity and genetic aberrations of the donor patient tumors more faithfully than established in non-small cell lung cancer (NSCLC) cell lines. This study evaluated whether our panel of PDX models recapitulate known cancer-related gene mutations.

      Methods:
      Whole-exome sequencing was completed on 103 NSCLC PDX models, 47 adenocarcinoma (AdC) and 56 squamous (SqCC), with a mean coverage of 84x. After filtering for contaminating mouse reads, the exome data were aligned using the Burrows-Wheeler Aligner, processed using the standard GATK pipeline, and mutations were identified using MuTect. Additional filtering using dbSNP, ExAC and ESP was performed for cases without corresponding normal adjacent lung exome data (n = 80). The identified mutations were compared to 1260 frequently mutated cancer-related genes, which were compiled from a panel of cancer-related mutated genes (555) and a panel of lung cancer-specific mutated genes (1082).

      Results:
      High rates of somatic mutations were observed in both AdC (mean of 12.4 mutations/megabase) and SqCC (mean of 11.7 mutations/megabase) PDX models. Compared to the rates observed in primary lung cancers in The Cancer Genome Atlas studies (mean of 8.9 mutations/megabase in AdC; 8.1 mutations/megabase in SqCC), these values appear higher, but may be inflated due to the lack of data from corresponding normal tissues. AdC models had a total of 953 mutated genes (median: 57 genes/model; range: 5-307), while SqCC models were characterized by 1007 mutated genes (median: 55 genes/model; range: 21-354). Specific mutation frequencies were compared to those determined in a recent study involving genomic alterations in human primary lung AdC and SqCC (Nature Genetics 2016; 48; 607–616). This comparison, based on mutated genes common in both studies, demonstrated significant correlation of the frequencies in 791 genes in AdC (ρ=0.78; p<2.2×10[-16]), as well as in 799 genes in SqCC (ρ=0.73; p<2.2×10[-16]). Three genes that were reported as significantly mutated in both AdC and SqCC primaries, and had higher mutation frequencies in SqCC, were also observed to be higher in our SqCC PDX models (TP53: 48.9% in AdC vs. 55.4% in SqCC; CDKN2A: 4.3% vs. 7.1% and PIK3CA: 2.1% vs. 23.2%); however, the statistical significance of these differences needs to be tested.

      Conclusion:
      Mutation landscapes in cancer genes are recapitulated in AdC and SqCC PDX models. The fidelity of these landscapes in matched patient primary tumour samples is being investigated.

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    P1.02 - Poster Session with Presenters Present (ID 454)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Biology/Pathology
    • Presentations: 1
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      P1.02-036 - An EGFR Tyrosine Kinase Inhibitor Sensitive Patient-Derived Lung Cancer Xenograft Model without Classical Sensitizing Mutations (ID 5398)

      14:30 - 15:45  |  Author(s): N. Pham

      • Abstract

      Background:
      Mutations in the tyrosine kinase (TK) domain of EGFR are oncogenic driver in 10-20% of lung adenocarcinoma (AdC) patients in Western countries. Approximately 90% of EGFR-TK inhibitor (TKI) sensitizing mutations occur as small in-frame deletions in exon 19 or L858R point mutations in exon 21. Recently, novel driver mutations in EGFR with oncogenic and TKI sensitizing activity have been reported. We present here an AdC patient-derived xenograft (PDX) model (PDX12) that is highly sensitive to EGFR-TKI, yet failed to demonstrate classical TKI sensitizing mechanisms.

      Methods:
      Comprehensive genomics profiling was used to characterize the genotype of PDX12, which was established from a resected stage IIIA AdC patient grafted in NGS mouse. The primary human lung cancer cell line (PHLC12) was extracted from its PDX model (PDX12). Aberrant EGFR cell lines used were H3255 (L858R), H2935 (exon 19 deletion), H1975 (L858R and T790M), and H1944 (wild type). Cell viability was assessed after erlotinib treatment at 1nM - 2μM for 72 hours using MTS assay. Levels of EGFR activation in both pre- and post-treatment by Western blot analysis.

      Results:
      PDX12 model had no known oncogenic mutations (EGFR wild type) on exons 18-21 by next-generation sequencing, RT-qPCR, and SISH, but was highly sensitive to EGFR-TKI. The IC50 to erlotinib treatment at 72 hr was 67.13 ± 7.63 nM for PHLC12, compared to 9.70 ± 2.64 nM for H3255, 64.88 ± 8.49 nM for HCC2935, > 2 μM for H1975, and > 2 μM for H1944 EGFR mutant or wild type cells, respectively. Western blot analysis demonstrated a relatively higher molecular weight band for EGFR protein with high expression level in PHLC12 when compared to other lung cancer cell lines. Using RT-qPCR, relative expression level of each EGFR domain (extracellular, tyrosine kinase, and c-terminal domain) in PHLC12 showed no difference compared to EGFR wild type. Phosphorylation status of EGFR in PHLC12 was similar in activity as compared to erlotinib sensitive cell lines.

      Conclusion:
      PHLC12 represents an enigmatic EGFR TKI sensitive lung PDX model without classical TKI sensitizing aberrations. Additional potential mechanisms of EGFR dependency including exon duplication, or post-translational modification of EGFR protein are being investigated.

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    P2.03b - Poster Session with Presenters Present (ID 465)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Advanced NSCLC
    • Presentations: 3
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      P2.03b-070 - Establishment of Organoid Cell Lines from Lung Squamous Cell Carcinoma (ID 5362)

      14:30 - 15:45  |  Author(s): N. Pham

      • Abstract

      Background:
      The limited number of available lung squamous cell carcinoma (LUSC) cell lines poses significant challenge for biological, experimental therapeutic and biomarker research in LUSC. Novel approaches to establish new preclinical models are urgently needed. We have previously established patient-derived xenografts (PDX) from resected tumours of LUSC patients and characterized them on the genomic, transcriptomic, and proteomic levels. We have used these PDX models to develop a method for establishment of 3D organoid cultures and cell lines as new in vitro preclinical models of LUSC.

      Methods:
      PDX models were established and propagated from resected primary non-small cell lung cancer (NSCLC) in NOD/SCID mice; they were molecularly profiled by exome sequencing, SNP array for copy number analysis, and immunohistochemistry (IHC). PDX tissue harvested from mice was dissociated into single cells and plated in 100% matrigel dome, with overlaying media on top. Organoids were characterized by H&E, and IHC of p63, CK5/6, TTF-1, and CK7. Organoid growth rate and drug screening were assessed using Celltiter glo cell viability assay.

      Results:
      A total of 17 LUSC PDX models have been used for this study. All organoids were able to initiate in culture at passage 1, and the organoid establishment rate (beyond passage 4) is 50% (6/12). 4/12 (33%) LUSC organoids were able to be propagated beyond 10 passages for over 60 days with an average doubling rate of 2-3 days. Organoid tumour cells recapitulated the histological features of LUSC and were positive for p63 and CK5/6, and negative for TTF-1 and CK7 by IHC. Molecular characterization of LUSC PDX models revealed PIK3CA mutations, amplifications, and PTEN loss. Over 40% (4/9) of PI3K altered LUSC organoids were sensitive to PI3K inhibitor BKM120.

      Conclusion:
      LUSC organoids can be established for long term culture and recapitulate the phenotypic features of the PDX. The culture protocol is currently being tested on primary patient LUSC tumours. Organoid cultures and cell lines may be useful as additional preclinical models for functional validation of novel therapeutic targets in LUSC.

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      P2.03b-071 - Therapeutic Targeting of the Phosphatidylinositol-3 Kinase Pathway in Lung Squamous Cell Carcinoma (ID 5369)

      14:30 - 15:45  |  Author(s): N. Pham

      • Abstract

      Background:
      The phosphatidylinositol-3 kinase (PI3K) belongs to a family of lipid kinases involved in the regulation of cell proliferation and survival and is often dysregulated in cancer. Comprehensive molecular profiling by The Cancer Genome Atlas (TCGA) has identified PIK3CA mutations, amplifications, and the tumor suppressor PTEN loss in 30-40% of lung squamous cell carcinoma (LUSC) patients. Inhibitors of PI3K such as BKM120 have been initiated in BASALT-1 trial (NCT01820325) of PI3K activated LUSC, however with modest response rate (40% of patients with stable disease and 3.3% with partial response). We aim to assess the efficacy of PI3K inhibition in LUSC patient-derived xenografts (PDX) harboring different PI3K pathway alterations to identify potential mechanisms of innate resistance.

      Methods:
      PDX models were established from early stage LUSC patients and molecularly characterized via exome sequencing, SNP array for copy number variation (CNV) and gene expression analysis. PIK3CA mutations were validated by direct sequencing, amplifications by fluorescence in situ hybridization (FISH), and PTEN loss by immunohistochemistry (IHC). For in vivo drug screening, each PDX model was implanted in two mice; one treated with BKM120 (50mg/kg) and the other with vehicle control by daily oral gavage. Tumors were monitored twice weekly with caliper measurement. A responder is a tumor that regresses completely, shrinks more than 30%, or remains a stable size according to the RECIST criteria.

      Results:
      Of the 75 LUSC PDX models that our laboratory has established, 11 (14%) harbored PIK3CA E545K and E542K mutations, 36 (47%) harbored PIK3CA amplifications, and 23 (30%) showed loss of PTEN protein expression. Using the RECIST criteria, BKM120 screening in selected PDX models revealed stable disease and progressive disease in 4/9 (46%) and 5/9 (54%) of the PDX models, respectively, after 21 days of treatment. Of the 9 PDX models tested, 3/5 PIK3CA mutant models were responsive to BKM120, whereas none of the other 4 PIK3CA amplified and/or PTEN deleted models were responsive to BKM120. Additionally, downregulation of pErk1/2 and pS6 in a responder model and no change in phosphorylated proteins in non-responding models were observed. Pharmacodynamics studies, validation of responders with more mouse replicates, and testing on the remaining models are ongoing and the results will be reported.

      Conclusion:
      60% of LUSC PDXs with PIK3CA mutation demonstrate high sensitivity to pan-PI3K inhibitor. Understanding innate resistance mechanisms of PI3K inhibition may provide important insights on tractable targets and therapeutic strategy for LUSC patients with aberrant PI3K pathway.

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      P2.03b-077 - EGFR/ALK+ Patient-Derived Xenografts from Advanced NSCLC for TKI Drug Selection & Resistance Development: The REAL-PDX Study (ID 6081)

      14:30 - 15:45  |  Author(s): N. Pham

      • Abstract

      Background:
      Lung cancer patient-derived xenografts (PDX) have shown to be representative models for individual patient tumors. Theoretically, such models could inform the choice of subsequent lines of therapy, since PDX development, TKI resistance induction, and subsequent drug-screening can be completed before TKI resistance develops in the patient. The goal of Resistance modeling in EGFR and ALK Lung cancer (REAL)-PDX is to develop PDX models for real-time treatment selection of subsequent lines of therapy in advanced-stage NSCLC patients.

      Methods:
      Since August 2015, Princess Margaret Cancer Centre patients with EGFR/ALK+, as well as lifetime never-smoking lung cancer patients with unknown mutation status, were consented to have additional tumor sampling for PDX development during routine- or trial-related biopsies. Tumor sufficiency was confirmed prior to implantation into non-obese severe combined immunodeficient (NOD-SCID) mice, with successful engraftment defined as propagation beyond first passage; unsuccessful implantations had no palpable tumor after 6 months.

      Results:
      72/82 (88%) approached patients consented; 49/72 (68%) had adequate tumor tissue for implantation (71% stage III/IV): 46 adenocarcinomas, 2 squamous cell carcinoma, 1 LCNEC. 36/49 (73%) were lifetime never smokers. Patients received adjuvant chemotherapy (3), TKI therapy (15), both (5), or no treatment (26) prior to sampling. Tumor samples were taken from surgically resected lung (18), metastatic adrenal (1) and brain (2), CT-guided lung biopsies (5), endoscopic ultrasound-guided (EBUS) biopsies (6), and thoracentesis pleural fluid (17) specimens. Twenty-eight implanted tumors were EGFR+ (12 exon19 deletions, 2 exon19 deletion/T790M, 1 exon19 del/exon18 mutation, 12 L858R, and 1 L858R/T790M); 7 had ALK-rearrangements, and 1 had ROS1-rearrangement. Engraftment rates of 31 assessable implanted tumors were as follows: lung resections 12/12 (100%), metastatic resections 2/3 (67%), CT- or EBUS-guided biopsies 1/5 (20%), and pleural fluid 2/11 (18%); Engraftment rate was associated with no prior treatment (14/17 no treatment vs 3/14 any treatment, p=0.001). Of 17 assessable tumors with EGFR activating mutations, 9 engrafted (53%). Of 3 assessable tumors with ALK-rearrangement, 1 was successful (33%).

      Conclusion:
      PDX development of EGFR/ALK+ models for testing with novel therapeutics from various tumor biopsy sites is feasible and will provide valuable real-time information for subsequent treatment decisions in advanced NSCLC patients. Updated engraftment and drug screening data will be presented.

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    P3.02b - Poster Session with Presenters Present (ID 494)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Advanced NSCLC
    • Presentations: 1
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      P3.02b-028 - Characterizing Residual Erlotinib-Tolerant Population Using EGFR-Mutated NSCLC Primary Derived Xenografts: The Last Holdouts (ID 5455)

      14:30 - 15:45  |  Author(s): N. Pham

      • Abstract

      Background:
      Three generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have led to multi-fold improvements in progression free survival of advanced stage non-small cell lung cancer (NSCLC) patients carrying EGFR kinase domain mutations. However, cure is not yet achievable with any EGFR TKI monotherapy, as patients will eventually progress due to acquired resistance. In vitro evidence suggests that minor populations of epigenetically modified drug tolerant cells (DTCs) may be one important mechanism for tumor cells surviving the TKI. We hypothesize that characterizing the genomic and epigenomic alterations observed in DTCs in vivo and comparing them to the bulk tumour will delineate a number of mechanisms of tolerance exhibited by DTCs.

      Methods:
      DTCs were induced via chronic erlotinib treatment of a lung adenocarcinoma primary derived xenograft (PDX) harbouring an erlotinib sensitive exon 19 deletion. Molecular profiles of DTCs are compared to untreated controls via immunohistochemistry (IHC) and gene expression array. We are now undertaking exome-sequencing, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), methylated DNA immunoprecipitation and sequencing (MeDIP-seq).

      Results:
      When compared to untreated tumours, DTCs exhibit decreased apoptosis (CC3 IHC) and proliferation (Ki67 IHC). DTCs maintained strong signaling via the EGFR pathway (pERK, pAKT, pS6). DTCs exhibited 2437 significantly differentially expressed genes (DEGs; >1.5-fold change and adjusted p-value <0.05) including multiple cancer stem cell markers (ALDH1A1, ALDH1A3, CD44). DEGs also were involved in vesicle-mediated transport (including lysosomes, exosomes and endosomes), autophagy, stress/unfolded protein response, cytoskeleton organization, chromatin organization, ion pumps and transporters, cell adhesion, WNT, NOTCH, PI3K and MAPK pathways. DTCs remained resistant to three cycles of cisplatin/vinorelbine either alone or when combined with erlotinib. Genomic and epigenomic profiling are on-going and results will be presented.

      Conclusion:
      DTCs may be a major impediment to cure by single-agent EGFR targeted therapies. Understanding the mechanisms and developing strategies to overcome DTCs may give insights on therapeutic strategy to further improve the survival of EGFR-mutated NSCLC patients.