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: Naïve LION102 PDX tumours exhibited exquisite sensitivity to EGFR-TKIs (100% reduction in pre-treatment tumour volume, p<0.0001). Following successive cycles of EGFR-TKI treatment in vivo several resistant subtypes were generated and characterized. 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

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<0.001) which was maintained both in continuous serial passage and following cryopreservation (at -196°C). FGFR expression and amplification and mutational analysis were assessed in naïve tumour material, following 10 weeks of continuous dosing and following outgrowth from CTX treatment and correlated with treatment response. 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

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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

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