Simon Jiang

Abstract 5651: In vivo generation of EGFR-TKI resistance in a patient-derived xenograft (PDX) with an activating EGFR mutation (L858R), and molecular characterisation of resistance mechanisms..

BACKGROUND: Non-small cell lung cancer (NSCLC) patients that have activating mutations in the EGFR gene are treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) e.g. Erlotinib (Tarceva®) and Gefitinib (Iressa®). Most NSCLC patients with activating EGFR mutations will respond to EGFR-TKIs; however, in about 50% of these cases a secondary mutation in EGFR (T790M) subsequently occurs which results in resistance to treatment. Other mechanisms of clinical resistance can also occur such as amplification of MET kinase and EMT conversion; however, additional routes of resistance are poorly defined. Using a novel patient-derived NSCLC xenograft model, driven by the L858R EGFR mutation, we set out to recapitulate the reported clinical routes of resistance to EGFR inhibitors and to evaluate if additional mechanism could also be identified. METHODS: LION102 is a NSCLC adenocarcinoma patient-derived xenograft (PDX) model with an activating EGFR mutation (L858R) which is maintained subcutaneously in vivo admixed with a human stromal cell component. Resistant models were generated in vivo through repeated cycling of treatment for up to 10 weeks with EGFR-TKI alone or in combination with a MET inhibitor. Resistant tumour material was characterised for further mutations in the EGFR gene by direct sequencing as well as for MET, AXL and HER2 over-expression and genomic amplification by quantitative PCR; Epithelial-to-Mesenchymal transition (EMT) was assessed by immunohistochemistry (IHC). RESULTS: Naive LION102 PDX tumours exhibited exquisite sensitivity to EGFR-TKIs (100% reduction in pre-treatment tumour volume, p CONCLUSIONS: EGFR-TKI resistant subtypes were generated in vivo from a proprietary patient-derived xenograft model (LION102) bearing an activating EGFR mutation (L858R) and characterised for their resistance mechanism. PDX models of resistance will be invaluable in assessing novel agents targeting the EGFR pathway and the development of new combination strategies which seek to prevent or overcome resistance to EGFR-TKIs. Citation Format: Andrew Mckenzie, Aaron Cranston, Phil Mallinder, Nektaria Papadopoulou, Simon Jiang, Kerry Moakes, Yinfei Yin, David Onion, Anna Grabowska, Martin Page, Rajendra Kumari. In vivo generation of EGFR-TKI resistance in a patient-derived xenograft (PDX) with an activating EGFR mutation (L858R), and molecular characterisation of resistance mechanisms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5651. doi:10.1158/1538-7445.AM2013-5651

Abstract 342: In vivo response and molecular characterisation of a NSCLC squamous cell carcinoma PDX model exhibiting reproducible sensitivity to FGFR inhibitors.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases, carries a poor prognosis and remains an area of high unmet need. Despite recent advances in treating NSCLC adenocarcinomas with targeted agents such as Erlotinib (Tarceva©) and Crizotinib (Xalkori©) few advances have been made in the treatment of squamous cell carcinoma (SCC), which accounts for 25% of all NSCLC. Although the incidence of fibroblast growth factor receptor (FGFR) mutations in SCC is modest, amplification of FGFR is well documented and there are several first generation FGFR inhibitors currently in clinical trial. It is therefore essential to develop relevant in vivo patient-derived xenograft (PDX) models for the development and characterisation of new FGFR agents and/or combination strategies which may prolong benefit and delay the emergence of resistance. METHODS: LION137 is a novel NSCLC SCC patient-derived xenograft (PDX) model which is maintained subcutaneously in vivo admixed with a human stromal cell component. Cohorts of mice were dosed with AZD4547 alone or in combination with chemotherapy (CTx; parallel or serial dosing regimens). The extent of inhibition was measured over several in vivo passages and following cryopreservation/resuscitation. Response to treatment was evaluated by tumour growth inhibition and waterfall plots. Tumour material was characterised for 50 key oncogenes and mutations by Ion Torrent sequencing and FGFR1-4 expression and genomic amplification by quantitative PCR. Immunohistochemistry (IHC) was used to confirm SCC subtype, EMT status and FGFR expression. RESULTS: LION137 PDX tumours (SCC confirmed by IHC) exhibited reproducible sensitivity to AZD4547 alone (P CONCLUSIONS: LION137 is a novel patient-derived NSCLC SCC xenograft model with reproducible FGFRi-sensitivity which is directly relevant to the clinical setting. This model could be invaluable in assessing novel agents targeting the FGFR pathway and in the development of new combination strategies which may prevent or overcome resistance. Citation Format: Andrew Mckenzie, Aaron Cranston, Phil Mallinder, Nektaria Papadopoulou, Simon Jiang, Kerry Moakes, Yinfei Yin, David Onion, Anna Grabowska, Martin Page, Rajendra Kumari. In vivo response and molecular characterisation of a NSCLC squamous cell carcinoma PDX model exhibiting reproducible sensitivity to FGFR inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 342. doi:10.1158/1538-7445.AM2013-342

Abstract A140: Check point inhibitor modulation of tumor microenvironment at orthotopic and metastatic sites using bioluminescent syngeneic cell line models in immune competent mice

Introduction: Syngeneic models are widely used to model the impact of cancer immunotherapy on tumor growth and tumor invading leucocytes (TILs), and the majority of such work is typically carried out in the subcutaneous setting. However, orthotopic models are known to better model cancer in patients as they form a single focal disease area as in the patient situation, facilitate metastatic spread via intra- and extra-thoracic lymph nodes and, in the case of syngeneic models, strain-specific tumor microenvironment interactions (immune and stromal components). Bioluminescent imaging (BLI) increases the usefulness of such models, as it allows for non-invasive longitudinal monitoring of tumor burden, allowing for optimal randomisation and reduction of false positives. It also allows continuous feedback allowing one to optimise treatment regimen mid-study. Herein we describe the generation of several bioluminescent variants of syngeneic cell lines commonly used for immunotherapy studies and assess the impact of orthotopic growth on response to immune check point therapy. Experimental Procedures: Bioluminescent cell line variants of syngeneic cell lines were established by lentiviral transduction for: 4T1 (breast), B16-F10 (melanoma), MBT-2 (bladder) H22 (Hepatoma), LL/2 (lung), Pan02 (Pancreatic) and RM-1 (prostate). Following establishment of stable cultures, DNA profiling and in vitro cytotoxicity assays was carried out to ensure there was no significant changes in DNA, cell doubling time or response to SoC agents following transduction. Subcutaneous growth of wild-type and bioluminescent variants was compared to assess any impact of luciferase expression on tumor growth, inflammation and TILs. Orthotopic models were established for most cell lines, and a metastatic model for B16-F10, BLI was carried out to assess real-time tumor growth and tumor burden at end stage (Spectrum CT; PerkinElmer). Immune checkpoint therapy was also assessed and TIL infiltration by FACs analysis and IHC. Results: Stable transduced bioluminescent cell lines were established; cell doubling time, morphology and growth in vitro was found to be consistent with their wild-type counterparts. Bioluminescent 4T1 cells exhibited growth consistent to that previously reported and readily metastasised to the lungs from both the orthotopic and subcutaneous sites; B16-F10 cells readily metastasise to bone following intracardiac administration. Subcutaneous response to immunotherapy did not appear to be affected. Modulation of immune cell infiltration will be reported and correlated to response. Conclusions: The growth and response to immunotherapy does not appear to be significantly impacted in the bioluminescent cell line models tested; as such they are a useful tool for further assessing the impact of complex orthotopic, spontaneous and experimental metastasis modelling in immune competent mice. Citation Format: Andrew McKenzie, Nektaria Papadopoulou, Simon Jiang, Jane Wrigley, Kelly Jones, Russell Garland, Neil Williams, Rajendra Kumari. Check point inhibitor modulation of tumor microenvironment at orthotopic and metastatic sites using bioluminescent syngeneic cell line models in immune competent mice [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A140.

Abstract 3994: Immune competent syngeneic models demonstrate additive effects of combination strategies using checkpoint immunotherapy and inducers of immunogenic cell death (ICD)

INTRODUCTION: Recent progress in the field of cancer immunotherapy have made it possible to translate several emerging immunostimulatory strategies, e.g. anti-CTLA-4, and anti-PD-1 into the clinic resulting in promising clinical benefits. In addition, a number of treatment strategies such as radiotherapy (RT) oncolytic viruses and certain chemotherapeutic agents e.g. Doxorubicin, Bortezomib and Mitoxantrone have been highlighted as potential inducers of immunogenic cell death through a mechanism resulting in the increased presentation of cell-associated antigens to CD4+ and CD8+ T lymphocytes by dendritic cells. Thus combination strategies of ICD inducers with immunotherapy (IT) could provide opportunities to harness the immune system to extend survival, even among metastatic and heavily pre-treated cancer patients, and may increase the efficacy of immunotherapy in those cancer types to be of a low immunogenic status. Here we compare the efficacy of immune checkpoint inhibitors in combination with documented ICD inducers to demonstrate an additive combination outcome in preclinical syngeneic models. EXPERIMENTAL PROCEDURES: Bioluminescent CT26 mouse colon cells, 4T1 mammary carcinoma cells or H22 hepatoma cells were implanted subcutaneously or orthotopically into BALB/c mice. Subcutaneous tumour growth was monitored by calliper measurement and bioluminescent imaging (BLI) was carried out to confirm orthotopic and/or metastatic growth. Established tumours were treated with immunotherapy in combination with chemotherapy, or hypofractionated image-guided micro-irradiation (IGMI) using the small animal research platform (SARRP; Xstrahl Ltd; body weight and clinical condition of mice were monitored daily. At termination the tumours were collected and assessed for immune cell infiltration and/or ICD markers by FACS and IHC. RESULTS: Response to treatment was evaluated by tumour growth inhibition or BLI following treatment of monotherapy or combinations of immunotherapy and ICD inducers (Oxaliplatin, Doxorubicin, and IGMI). Monotherapy with anti-CTLA4 exerted no statistically significant effect on primary or metastatic (4T1) tumour growth whereas ICD such as IR resulted in a modest tumour growth inhibition (TGI); When combined significant additive effect was observed (60% increase in TGI) on the primary tumour and reduction in tumour burden in the lungs indicating an abscopal effect. Details of modulation of immune cell infiltration and ICD markers observed in all models will be reported and correlated to response. CONCLUSIONS: Combination of immune checkpoint immunotherapy with a known ICD inducer (IGMI) resulted in an additive TGI in both CT26 and 4T1 models and effectively demonstrates their applicability for further exploring combination strategies involving immunotherapy. Citation Format: Andrew Mckenzie, Rajendra Kumari, Qian Shi, Nektaria Papadopoulou, Yinfei Yin, Simon Jiang, Jane Wrigley, Jason King, Neil Williams, Russell Garland. Immune competent syngeneic models demonstrate additive effects of combination strategies using checkpoint immunotherapy and inducers of immunogenic cell death (ICD). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3994.

Abstract 769: In vivo response and molecular characterization of a Caucasian NSCLC squamous cell carcinoma PDX sensitive to FGFR inhibitors

BACKGROUND: The fibroblast growth factor receptor (FGFR) family are reported to be involved in key cellular processes such as proliferation, differentiation, migration & survival with the deregulation of signalling through genetic modification or amplification being observed in cancer. FGFR1 has been reported to be amplified in squamous cell non-small cell lung cancer (NSCLC) as well as other indications (e.g. breast) and there are several FGFR inhibitors currently in clinical trial. It is therefore essential to develop relevant in vivo models for the development and characterisation of new FGFR agents and/or combination strategies which may prolong benefit and delay the emergence of resistance. METHODS: Patient derived xenograft (PDX) models were maintained subcutaneously in vivo in serial passage and their growth monitored by calliper measurements three times weekly; mice were recruited to treatment groups when the mean tumour volume was approximately 200mm3. Response to treatment was evaluated by tumour growth inhibition in response to FGFR inhibitors such as AZD4547 and chemotherapy. Resistance to both chemotherapy (Paclitaxel/Carboplatin) and AZD4547 in the squamous cell carcinoma (SCC) PDX model, LU6429, was generated in vivo through repeated dosing and the resulting models characterised for their treatment response. Molecular characterisation was carried out by RNA sequencing. RESULTS: A number of novel PDX models have been developed which harbour FGFR amplification and mutations including LU6429, a Caucasian NSCLC SCC PDX model which has modest FGFR1 and FGFR2 amplification and responds to FGFR inhibitors. LU6429 PDX tumours (SCC confirmed by IHC) exhibited reproducible sensitivity to AZD4547 (p CONCLUSIONS: Pre-clinical models such as LU6429, a novel patient-derived NSCLC SCC xenograft model with moderate FGFR amplification which showed significant sensitivity to the FGFR inhibitor AZD4547, are invaluable in assessing novel agents targeting the FGFR pathway and in the development of new combination strategies which may prevent or overcome resistance. Citation Format: Andrew McKenzie, Nektaria Papadopoulou, Yinfei Yin, Simon Jiang, Jane Wrigley, Jie Cai, Martin Page, Henry Li, Rajendra Kumari. In vivo response and molecular characterization of a Caucasian NSCLC squamous cell carcinoma PDX sensitive to FGFR inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 769. doi:10.1158/1538-7445.AM2015-769

Abstract 765: Overcoming acquired EGFRi resistance in NSCLC with targeted beam irradiation in combination with targeted agents

BACKGROUND: Non-small cell lung cancer (NSCLC) patients undergo primary, adjuvant or neoadjuvant radiotherapy treatment with image-guided radiotherapy (IGRT) being widely used to provide more accurate treatment plans and reduced side effects. Patients who have activating mutations in the epidermal growth factor receptor (EGFR) gene are treated with EGFR tyrosine kinase inhibitors (EGFRi). However, resistance to EGFRi occurs in the majority of patients with the main clinical drivers being secondary gatekeeper mutations (T790M) or amplification of genes such as c-MET and Her2. The small animal radiation research platform (SARRP), which allows the treatment of rodent models of cancer more accurately with planned protocols similar to those utilised in the clinic, was used in combination with EGFRi, to assess the efficacy of combination treatment strategies of irradiation (IR) in EGFRi resistant NSCLC xenografts. METHODS: Resistance to EGFRi in the NSCLC cell line HCC827, which harbours an activating EGFR mutation, was generated through repeated exposure to Gefitinib or Erlotinib. Resistant models were characterised for further mutations in the EGFR gene by direct sequencing and for c-MET, Axl and Her2 over-expression/genomic amplification by quantitative PCR. EGFRi resistant or parental HCC827 cells were implanted subcutaneously into nude mice, xenograft growth was monitored by calliper measurements and treatment initiated when the mean tumour volume reached ∼200mm3. An image guided IR dose of 3Gy/mouse/day (30Gy total) was targeted directly to the xenografts whilst sparing the surrounding normal tissue. Mice were treated with IR either alone or in combination with Erlotinib or Crizotinib; response to treatment was evaluated by tumour volume measurement. RESULTS: EGFR-TKI resistant HCC827 cell lines were generated following successive cycles of treatment. Amplification of c-MET was identified in a number of clones, but no Her2 amplification or gatekeeper mutations; therefore Crizotinib, a c-MET inhibitor, was selected for evaluation in these models. When assessed in vivo, treatment of parental HCC827 xenografts with targeted IR or Erlotinib treatment resulted in tumour regression. However, in the c-MET driven resistant model, both the IR and Erlotinib response was significantly attenuated, resulting in tumour re-growth. Combination testing with Crizotinib restored the efficacy of both IR and Erlotinib to naive treatment levels confirming the role of c-MET in resistance. CONCLUSIONS: Resistance to EGFRi and IR in a HCC827 model is driven by c-MET amplification. Treatment with Crizotinib restored sensitivity to both EGFRi and IR demonstrating that pre-clinical models of resistance are invaluable in assessing novel agents targeting the EGFR pathway and the development of new combination strategies which seek to prevent/overcome resistance to EGFRi. Citation Format: Andrew McKenzie, Nektaria Papadopoulou, Lucy Page, Simon Jiang, Jane Wrigley, Yinfei Yin, Qian Shi, Martin Page, Ian Wilson, Rajendra Kumari. Overcoming acquired EGFRi resistance in NSCLC with targeted beam irradiation in combination with targeted agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 765. doi:10.1158/1538-7445.AM2015-765

Abstract 4875: In vivo and in vitro generation and characterisation of EGFR-TKI resistance in patient-derived xenograft (PDX) and cell line-derived xenograft (CDX) models of NSCLC with activating EGFR mutations

Background: Non-small cell lung cancer (NSCLC) patients that have activating mutations in the epidermal growth factor receptor (EGFR) gene are treated with EGFR tyrosine kinase inhibitors (TKIs) e.g. Erlotinib (Tarceva®) and Gefitinib (Iressa®). Most NSCLC patients with activating EGFR mutations will respond to EGFR-TKIs; however, in about 50% of these cases a secondary mutation in EGFR (T790M) subsequently occurs which results in resistance to treatment. Other mechanisms of clinical resistance can also occur such as amplification of c-MET kinase, Her-2 and EMT conversion; however, additional routes of resistance are poorly defined. Using a novel NSCLC patient-derived xenograft (PDX) model, driven by the L858R EGFR mutation, we set out to recapitulate the reported clinical routes of resistance to EGFR inhibitors and to evaluate if additional mechanism could also be identified. Methods: LU6422 is a Caucasian NSCLC adenocarcinoma PDX model with an activating EGFR mutation (L858R) which is maintained subcutaneously in vivo admixed with a human stromal cell component. HCC827 is NSCLC adenocarcinoma cell line with an activating EGFR mutation (exon 19 del E746-A750). Resistant models of LU6422 and HCC827 were generated in vivo and in vitro (respectively) through repeated dosing or exposure to EGFR-TKIs. Resistant tumour material was characterised for further mutations in the EGFR gene by direct sequencing and for c-MET, Axl and Her2 over-expression and genomic amplification by quantitative PCR. Combination efficacy treatment (in vitro and in vivo) was carried out to verify documented resistance mechanisms. Results: Naive LU6422 and HCC827 tissue exhibited exquisite sensitivity to EGFR-TKIs (100% reduction in pre-treatment tumour volume, p Conclusions: EGFR-TKI resistant subtypes were generated in vivo and in vitro from a proprietary patient-derived xenograft model (LU6422) and the HCC827 cell line and were characterised for their resistance mechanisms. Pre-clinical modes of acquired resistance will be invaluable in assessing novel agents targeting the EGFR pathway and the development of new combination strategies which seek to prevent or overcome resistance to EGFR-TKIs. Citation Format: Andrew McKenzie, Nektaria Papadopoulou, Simon Jiang, Martin Page, Henry Li, Rajendra Kumari, Jane Wrigley. In vivo and in vitro generation and characterisation of EGFR-TKI resistance in patient-derived xenograft (PDX) and cell line-derived xenograft (CDX) models of NSCLC with activating EGFR mutations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4875. doi:10.1158/1538-7445.AM2014-4875

Abstract A142: In vivo response and molecular characterization of a Caucasian NSCLC squamous cell carcinoma PDX sensitive to FGFR inhibitor.

Introduction: Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases, carries a poor prognosis and remains an area of high unmet need. Despite recent advances in treating defined subsets of NSCLC adenocarcinomas with targeted agents such as erlotinib and crizotinib few advances have been made in the treatment of squamous cell carcinoma (SCC), which accounts for 25% of all NSCLC. Although the incidence of fibroblast growth factor receptor (FGFR) mutations in SCC is modest, amplification of FGFR is well documented and there are several first generation FGFR tyrosine kinase inhibitors (TKI) currently in clinical trials. It is therefore essential to develop relevant in vivo patient-derived xenograft (PDX) models for the development and characterisation of new FGFR agents and/or combination strategies which may prolong benefit and delay the emergence of resistance. Methods: LU6429 is a novel Caucasian NSCLC SCC PDX model which is maintained subcutaneously in vivo admixed with a human stromal cell component. Cohorts of mice were dosed with AZD4547, which is an FGFR TKI undergoing evaluation in the clinic, alone or in combination with chemotherapy (CTx; parallel or serial dosing regimens). The extent of inhibition was measured over several in vivo passages and following cryopreservation/resuscitation. CTx and FGFR TKI resistant models of LU6429 were generated in vivo through repeated dosing and the resulting models characterized for their treatment response. Tumor material was characterized for 50 key oncogenes and mutations by Ion Torrent sequencing, for FGFR1-3 expression and genomic amplification by quantitative PCR and FGFR1-2 by FISH analysis. Immunohistochemistry (IHC) was used to confirm NSCLC subtype and EMT status. Results: LU6429 PDX tumours (SCC confirmed by IHC) exhibited reproducible sensitivity to AZD4547 alone (P<0.001) which was maintained both in continuous serial passage and following cryopreservation (at -196°C). FGFR expression, amplification and mutational analysis were assessed in naive tumour material, following 10 weeks of continuous dosing and following outgrowth from CTx treatment and correlated with treatment response. Conclusions: LU6429 is a novel patient-derived NSCLC SCC xenograft model with reproducible FGFR TKI sensitivity, which is directly relevant to the clinical setting. This model could be invaluable in assessing novel agents targeting the FGFR pathway and in the development of new combination strategies which may prevent or overcome resistance. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A142. Citation Format: Andrew J. McKenzie, Aaron N. Cranston, Nektaria Papadopoulou, Simon Jiang, Jane Wrigley, Yinfei Yin, Henry Li, Martin Page, Rajendra Kumari. In vivo response and molecular characterization of a Caucasian NSCLC squamous cell carcinoma PDX sensitive to FGFR inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A142.

Abstract A145: In vivo and in vitro generation and characterization of EGFR TKI resistance in a patient-derived and a cell line-derived xenograft model of NSCLC with activating EGFR mutations.

Introduction: Most non-small cell lung cancer (NSCLC) patients that have activating mutations in the EGFR gene will respond to treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) e.g. erlotinib and gefitinib. However, in about 50% of these cases a secondary mutation in EGFR (T790M) subsequently occurs, which results in resistance to treatment. Other mechanisms of clinical resistance can also occur such as amplification of c-MET kinase, HER2 and epithelial-mesenchymal transition (EMT) conversion; however, additional routes of resistance are poorly defined. Using a novel Caucasian NSCLC patient-derived xenograft (PDX) model and a cell line derived model, driven by the L858R EGFR mutation, we set out to recapitulate the reported clinical routes of resistance to EGFR inhibitors and to evaluate if additional mechanism could also be identified. Methods: LU6422 is a Caucasian NSCLC adenocarcinoma PDX model with an activating EGFR mutation (L858R) which is maintained subcutaneously in vivo admixed with a human stromal cell component. HCC827 is NSCLC adenocarcinoma cell line with an activating EGFR mutation (del E746-A750). Resistant models of LU6422 and HCC827 were generated in vivo and in vitro (respectively) through repeated dosing or exposure to EGFR TKIs. Resistant tumour material was characterised for further mutations in the EGFR gene by direct sequencing as well as for c-MET, AXL and HER2 over-expression and genomic amplification by quantitative PCR. Results: Naive LU6422 and HCC827 tissue exhibited exquisite sensitivity to EGFR TKIs (100% & 30% reduction respectively in pre-treatment tumor volume, p<0.0001). Following successive cycles of EGFR-TKI treatment several resistant subtypes were generated, cloned and characterized. Conclusions: EGFR TKI resistant subtypes were generated in vivo and in vitro from a proprietary Caucasian NSCLC PDX model (LU6422) and the HCC827 NSCLC cell line and were characterized for their resistance mechanisms. Models of resistance will be invaluable in assessing novel agents targeting the EGFR pathway and the development of new combination strategies which seek to prevent or overcome resistance to EGFR TKIs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A145. Citation Format: Andrew J. McKenzie, Aaron N. Cranston, Nektaria Papadopoulou, Simon Jiang, Jane Wrigley, Yinfei Yin, Henry Li, Martin Page, Rajendra Kumari. In vivo and in vitro generation and characterization of EGFR TKI resistance in a patient-derived and a cell line-derived xenograft model of NSCLC with activating EGFR mutations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A145.

Abstract 2796: Patient-derived xenograft models reveal a subset of clinically relevant squamous non-small cell lung cancers that respond to targeted EGFR inhibition.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases, carries a poor prognosis and remains an area of high unmet need. Despite recent advances in treating NSCLC adenocarcinomas with targeted epidermal growth factor receptor (EGFR) kinase inhibitors, few advances have been made in the treatment of squamous cell carcinoma (SCC), which accounts for 25% of NSCLC. Although the incidence of EGFR mutations in SCC is low, a retrospective clinical study of erlotinib in SCC suggested that a proportion of SCC patients would benefit from treatment with an EGFR inhibitor (Tseng et al., 2012). METHODS: NSCLC samples obtained from untreated patients undergoing surgery were collected with ethical consent, disaggregated and implanted subcutaneously in MF-1 nude mice (Harlan UK) admixed with a human stromal cell component to generate patient-derived xenograft (PDX) models (LION PDX models). Tumour material was diagnosed on the basis of operative histology and immunohistochemistry (IHC) used to confirm histological subtype. IHC was also used to evaluate EGFR expression and tumours characterised for mutations in p53, LKB1, EGFR and K-RAS by direct DNA sequencing. Patient-derived xenografts were maintained in vivo and study cohorts of mice dosed when tumours reached 100-200mm3 with either erlotinib or gefitinib or vehicle placebo. Response to treatment was evaluated by tumour growth inhibition and waterfall plots. RESULTS: We have identified a subset of tumours with squamous histology in our LION panel of patient-derived NSCLC xenografts that respond to treatment with a targeted EGFR inhibitor. Four out of 11 SCC models tested (36%) responded to treatment with either erlotinib or gefitinib with greater than 50% growth inhibition and two SCC models responded with ∼30% growth inhibition. Responses correlated well with EGFR expression. Work to characterise a further 8 LION SCC PDX models is also underway. CONCLUSIONS: These data support the proposition that a significant subset of NSCLC patients with squamous histology may benefit from treatment with targeted EGFR inhibitors. Our LION SCC models could be valuable for biomarker studies to identify the basis of the EGFR inhibitor responder subset and for evaluating new agents in a more clinically relevant setting. Citation Format: Aaron Cranston, Andrew Mckenzie, Phil Mallinder, Simon Jiang, Kerry Moakes, Yinfei Yin, Alex Reece-Smith, Anna Grabowska, Martin Page, Rajendra Kumari. Patient-derived xenograft models reveal a subset of clinically relevant squamous non-small cell lung cancers that respond to targeted EGFR inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2796. doi:10.1158/1538-7445.AM2013-2796