Development Of New Combination Strategies Research Articles

The Trend of the Treatment of Advanced Hepatocellular Carcinoma: Combination of Immunotherapy and Targeted Therapy.

Hepatocellular carcinoma (HCC) is a common type of tumor worldwide. The development of systemic treatment of advanced HCC has remained stagnant for a considerable period. During the last years, a series of new treatment regimens based on the combination of immunotherapeutic drugs and targeted drugs have been gradually developed, increased the objective response rate (ORR), overall survival (OS), and progression free survival (PFS) of HCC patients. Among the different combination therapy groups, atezolizumab plus bevacizumab and sintilimab plus IBI-305 seem to have unique advantages, while head-to-head comparisons are still needed. A comprehensive understanding of the developments, the ongoing clinical trials and the mechanisms of combination of immunotherapy and targeted therapy might lead to the development of new combination strategies and solving current challenges such as the molecular biomarkers, the clinical administration order of drugs and the second-line treatments after combination therapy.

The Abscopal Effect in the Era of Cancer Immunotherapy: a Spontaneous Synergism Boosting Anti-tumor Immunity?

Radiotherapy is one of the main treatment strategies used in cancer. Aside from the local control of the disease, which is mediated by a direct cytotoxic effect on tumor cells, radiotherapy has also been shown to exert immune-mediated local and systemic effects. Radiotherapy can elicit anti-tumor responses in distant sites from the radiation field; this phenomenon is known as the abscopal effect and has been described in patients previously treated with immune checkpoint blockade (ICB). Considering that the efficacy of immunotherapy has been demonstrated only in a subset of patients-who often benefit with lasting responses-efforts are ongoing to potentiate its activity with the development of new combination strategies. Radiotherapy might represent a potential candidate for a synergistic combination with immunotherapy, by improving the immunogenicity of tumors and by enhancing local and systemic immune effects. This review aims to summarize the current pre-clinical and clinical data on the immune effects of radiotherapy and their potential implications for cancer immunotherapy.

Tumor-Intrinsic RIG-I Signaling Promotes Anti-CTLA-4 Checkpoint Inhibitor-Mediated Anticancer Immunity

Strong inter-individual variation in clinical response to checkpoint inhibitors such as anti-CTLA-4 remains a major challenge. In a retrospective analysis, expression of viral defense genes in human melanomas including the cytosolic RNA receptor RIG-I (DDx58) has been associated with clinical benefit to CTLA-4 blockade. However, possible interconnections of these two distinct pathways remain unknown. Using CRISPR/Cas9 technology to generate B16 melanoma cells lines that lack nucleic acid receptors or downstream signaling molecules (RIG-I, STING, IRF3/7) together with available genetically deficient mouse models, we addressed the importance of nucleic acid receptor signaling in both tumor and host cells for the efficacy of anti-CTLA-4 immunotherapy. We here provide experimental proof that anti-CTLA-4 immunotherapy relies on tumor cell-intrinsic RIG-I but not STING signaling. Following anti-CTLA-4 treatment, tumor-intrinsic RIG-I signaling critically impacts on cross-presentation of tumor-associated antigen by CD103+ dendritic cells, the expansion of tumor antigen-specific CD8+ T cells and ultimately the accumulation of CD8+ T cells within tumor tissue. Consistently, therapeutic targeting of RIG-I with 5'-phosphorylated-RNA in both tumor and non-malignant cells potently augmented the efficacy of CTLA-4 checkpoint blockade. These processes were additionally dependent on host STING, MAVS and type I interferon signaling and were closely linked to RIG-I-mediated tumor cell death. In summary, our study identifies both tumor- and host-intrinsic RIG-I signaling as fundamental requirements for anti-CTLA-4-mediated antitumor immunity. Our data predict that targeting RIG-I may serve as a basis for the development of new combination strategies to increase the response rate of checkpoint inhibitor-based immunotherapy of malignancy including lymphoma, particularly in individuals that do not have a sufficient spontaneous antitumor T-cell immune response. Disclosuresvan den Brink:Jazz Pharmaceuticals: Consultancy; Seres: Research Funding; PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement.

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

Abstract 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<0.001) which was maintained in continuous serial passage and following cryogenic storage. FGFR expression and mutational analysis was assessed in naïve tumours, tumours following continuous AZD4547 treatment and in the acquired chemo-resistance setting. Other models show differential response to FGFR inhibitors. 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

Abstract 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 naïve 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

Abstract 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: Naïve LU6422 and HCC827 tissue exhibited exquisite sensitivity to EGFR-TKIs (100% reduction in pre-treatment tumour volume, p<0.0001). Following successive cycles of EGFR-TKI treatment several resistant subtypes were generated, cloned and characterized. Combination treatment of resistant HCC827 variants restored efficacy to naïve treatment levels. 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.

Abstract 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 naïve 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.

Abstract 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: Naïve 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.

Preclinical modeling of EGFR-specific antibody resistance: oncogenic and immune-associated escape mechanisms

To define the molecular basis of secondary resistance to epidermal growth factor receptor (EGFR)-specific antibodies is crucial to increase clinical benefit in patients. The limited access to posttreatment tumor samples constitutes the major barrier to conduct these studies, representing preclinical experimentation as a useful alternative. Anti-EGFR antibody-based therapy has been reported to mediate tumor regression by interrupting oncogenic signals and, more recently, by inducing antitumor immunological responses. However, resistance models have been focused only on tumor escape associated with EGFR blockade, whereas studies describing immune-associated escape mechanisms have not been reported thus far. To address this idea, we modeled resistance induction in D122 metastasis-bearing C57BL/6 mice treated with 7A7 (an anti-murine EGFR antibody). Similarly to patients receiving EGFR-specific antibodies, 7A7 resistance promotion represents an important drawback to successful therapy. Characterization of primary cultures derived from metastasis in 7A7-treated mice revealed a high frequency of tumor variants resistant to in vivo and in vitro antibody treatment. We showed, for the first time, the convergence of alterations in oncogenic and immunological pathways in 7A7-resistant variants. To identify key molecules behind resistance, seven 7A7-resistant variants were screened. HER3 overexpression and PTEN deficiency leading to hyperactivation of protumoral downstream signaling were found in these variants as a consequence of 7A7-mediated EGFR inhibition. Concomitantly, we found a high percentage of resistant variants carrying abnormalities in the constitutive and/or interferon gamma (IFN-γ)-inducible major histocompatibility complex I (MHC-I) expression. A significant decrease in mRNA levels for MHC-I heavy chains, β2-microglogulin and antigen processing machinery genes as well as transcriptional alterations in IFN-γ pathway components were identified as the main mechanisms underlying MHC-I expression defects in 7A7-resistant variants. Notably, these defects have not been previously associated with EGFR-specific antibody resistance, providing novel immunological escape mechanisms. This study has strong implications for the development of new combination strategies to overcome anti-EGFR antibodies refractoriness.

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

Abstract 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

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..

Abstract 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

Age, outcomes, and treatment effects of fibrinolytic and antithrombotic combinations: Findings from Assessment of the Safety and Efficacy of a New Thrombolytic (ASSENT)-3 and ASSENT-3 PLUS

Elderly patients with acute myocardial infarction are at particularly high risk for death and bleeding complications. The efficacy and safety of antithrombotic strategies in these patients remain unclear. To provide more insight into the risk and benefit of antithrombotic strategies in the elderly, we examined patients from the ASSENT-3 and ASSENT-3 PLUS trials with STEMI who were treated with tenecteplase (TNK) and unfractionated heparin (UFH) or enoxaparin, or half-dose TNK with abciximab and reduced-dose UFH. Older patients had a higher risk profile, and lower use of concomitant therapies and revascularization procedures. We found an interaction between age and treatment effect for the efficacy end point (P = .0007) and the efficacy plus safety end point (P < .0001). Younger patients (<65 years) had a lower risk of the composite efficacy plus safety end point with enoxaparin (relative risk [RR] 0.84, 95% CI 0.74-0.94) or abciximab (RR 0.79, 95% CI 0.69-0.90) compared with UFH. In patients >65 years of age, the benefit of enoxaparin appeared to be offset by an increased risk of bleeding complications. The risk of the efficacy plus safety end point tended to be higher in elderly patients receiving abciximab and half-dose TNK (RR 1.18, 95% CI 0.91-1.51 for 76-85 years of age and RR 1.48, 95% CI 0.88-2.49 for >85 years of age). Although TNK with either enoxaparin or abciximab appeared to be more effective than with standard UHF in younger patients, these combinations tended to be less effective and even may be unsafe in the elderly. Development of new combination strategies and dosing schemes of fibrinolytics and antithrombotics with improved efficacy and safety in the elderly remains a high priority.