Epidermal Growth Factor Receptor Mutant Tumors Research Articles

NEP010, a novel compound with minor structural modification from afatinib, exhibited significantly improved antitumor activity

Non-small cell lung cancer (NSCLC) is classified into many subclasses according to the specific kinase mutations. The most common mutation is epidermal growth factor receptor (EGFR) somatic mutation, which has promoted the development of several novel drugs (Tyrosine kinase inhibitors, TKIs). Although various TKIs are recommended as targeted therapy for NSCLC with EGFR mutations in NCCN guidelines, not all patients respond equally to the recommended TKIs, which lead to series of novel compound under development to satisfy actual clinical needs. Based on the structure of afatinib, a marketed drug recommended as the first-line therapy for patients with EGFR mutation, NEP010 was synthesized with structural modification. The antitumor efficacy of NEP010 on mouse tumor xenograft models with different EGFR mutations was determined. Results showed that with minor structural modifications on afatinib, the inhibitory effect of NEP010 on EGFR mutant tumors was significantly improved. Pharmacokinetics test was adopted, and compared with afatinib, the increased tissue exposure of NEP010 may be the potential factor responsible for the increased efficacy. Furthermore, tissue distribution test showed a high concentration of NEP010 in the lung which happens to be the target organ of NEP010 in clinical practice. In conclusion, data obtained suggested that NEP010 has an increased anti-tumor effect via improving pharmacokinetics, and may provide a potent therapeutic option for the patients with EGFR mutation of NSCLC in the future.

CD70 is a therapeutic target upregulated in EMT-associated EGFR tyrosine kinase inhibitor resistance.

Effective therapeutic strategies are needed for non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations that acquire resistance to EGFR tyrosine kinase inhibitors (TKIs) mediated by epithelial-to-mesenchymal transition (EMT). We investigate cell surface proteins that could be targeted by antibody-based or adoptive cell therapy approaches and identify CD70 as being highly upregulated in EMT-associated resistance. Moreover, CD70 upregulation is an early event in the evolution of resistance and occurs in drug-tolerant persister cells (DTPCs). CD70 promotes cell survival and invasiveness, and stimulation of CD70 triggers signal transduction pathways known to be re-activated with acquired TKI resistance. Anti-CD70 antibody drug conjugates (ADCs) and CD70-targeting chimeric antigen receptor (CAR) Tcell and CAR NK cells show potent activity against EGFR TKI-resistant cells and DTPCs. These results identify CD70 as a therapeutic target for EGFR mutant tumors with acquired EGFR TKI resistance that merits clinical investigation.

EGFL7 drives the evolution of resistance to EGFR inhibitors in lung cancer by activating NOTCH signaling

Accumulating evidence supports evolutionary trait of drug resistance. Like resilience in other systems, most tumor cells experience drug-tolerant state before full resistance acquired. However, the underlying mechanism is still poorly understood. Here, we identify that EGF like domain multiple 7 (EGFL7) is a responsive gene to epidermal growth factor receptor (EGFR) kinase inhibition during a period when tumors are decimated. Moreover, our data reveal that the adaptive increase of EGFL7 during this process is controlled by the depression of nonsense-mediated mRNA decay (NMD) pathway. Upregulation of EGFL7 activates NOTCH signaling in lung cancer cells, which slows down the decrease of c-Myc caused by EGFR inhibition, thereby helping the survival of cancer cells. Our data, taken together, demonstrate that EGFL7 is a driver gene for resistance to EGFR kinase inhibition, and suggest that targeting EGFL7/NOTCH signaling may improve the clinical benefits of EGFR inhibitors in patients with EGFR mutant tumors.

Abstract 6217: TP53 loss with whole genome doubling mediates heterogeneous intra-patient therapy response in EGFR-driven lung adenocarcinoma: A TRACERx study

Abstract Introduction: Mutations in the epidermal growth factor receptor (EGFR) are often found in never-smokers who develop lung adenocarcinoma (LUAD) and resistance to receptor-targeted tyrosine kinase inhibitors (TKIs) generally occurs within five years of treatment. Mixed responses, where individual tumor lesions within the same patient respond differently to treatment, contribute to early treatment failure and indicate the involvement of multiple genomic alterations. TP53 loss of function has been associated with both tolerance to chromosomal instability (CIN) and with shortened progression free survival in EGFR-driven tumors. The aim of this study was to investigate the hypothesis that CIN, together with loss of p53 function, may lead to diverse genotypes that underlie the mixed responses observed in EGFR-driven LUAD. Experimental Procedures: We used genetically engineered mouse models (GEMMs), driven by EGFR with or without concomitant Trp53 loss and human isogenic cell lines to investigate cellular evolution and the effect of whole genome doubling on targeted therapy responses and mechanisms of resistance. Next generation sequencing and shallow whole genome single cell analyses, together with longitudinal imaging analysis from the Aura clinical trials (AURA2, AURA3 and the AURA extension cohort, Identifiers: NCT02094261, NCT02151981 and NCT01802632) was used to investigate the effect of p53 loss on tumor evolution. Results: EGFR mutant tumors with clonal Trp53 loss or TP53 pathway disruption displayed increased Weighted Genome Integrity Index (wGII) and higher cell to cell variation in both the mouse and TRACERx data sets. We found that TP53 loss of function increased the incidence of mixed responses and resistance to targeted therapy in both mouse and human tumors leading to early treatment failure and reduced survival. Whole-exome sequencing (median depth of 92x, range: 58-169x) of nine erlotinib-resistant EGFR mouse tumors identified four EGFR bypassing mutations (oncogenic KRAS mutations; Q61H, Q61R, and two G12D mutations) and one likely driver mutation in FGFR2 (C286R). We could only identify one known resistance associated mutation, EGFRT790M, in EGFR mutant tumors with concomitant loss of Trp53. In depth analysis revealed no major copy number differences in treatment naÏve vs resistant EGFR mutant tumors. In contrast, 70% of all tumors with concomitant Trp53 loss had amplified a region of mouse chromosome 7, harboring MET and BRAF. Investigating an isogenic EGFR/TP53 mutant cell model system revealed whole genome doubling as advantageous in overcoming the selection pressure induced by targeted therapy. Conclusion: We find that loss of TP53 in the context of mutated EGFR leads to an altered and plastic genomic landscape, with multiple copy number changes, which in turn facilitates therapy resistance. Citation Format: Sebastijan Hobor, Maise Al Bakir, Marcin Skrzypski, Alexander M. Frankell, Bjorn Bakker, Thomas B. Watkins, Aleksandra Markovets, Jonathan R. Dry, Andrew P. Brown, Jasper van der Aart, Dahmane Oukrif, Marco Novelli, Matthew J. Renshaw, William Hill, Hilda van den Bos, Diana C. Spierings, Juliann Chmielecki, Carl Barrett, Kevin Litchfield, Elza de Bruin, Floris Foijer, Karen H. Vousden, TRACERx consortium, Robert E. Hynds, Crispin T. Hiley, Nnennaya Kanu, Simone Zaccaria, Eva C. Gronroos, Charles Swanton. TP53 loss with whole genome doubling mediates heterogeneous intra-patient therapy response in EGFR-driven lung adenocarcinoma: A TRACERx study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6217.

Abstract 1827: CD70 is a novel therapeutic target for EGFR mutant NSCLC with acquired, EMT-associated EGFR TKI resistance

Abstract Approximately 15% of all patients with non-small cell lung cancer (NSCLC) and nearly 35% of Asian patients with NSCLC harbor activating mutations within the epidermal growth factor receptor (EGFR). Although these patients are initially highly sensitive to first or second generation EGFR tyrosine kinase inhibitors (TKIs) including erlotinib or third-generation inhibitors including osimertinib, EGFR TKI-refractory disease inevitably emerges. While therapeutic strategies to target resistant disease that emerges though secondary EGFR mutations or MET amplification have been developed, there remains a void of therapeutic options for patients where resistance occurs through EGFR-independent mechanism such as epithelial to mesenchymal transition (EMT) or transformation to small cell lung cancer (SCLC). To identify cell surface proteins that could be targeted by antibody-based or adoptive cell therapy approaches we interrogated RNAseq data from EGFR mutant NSCLC cell lines (HCC827 and HCC4006) and their associated EGFR TKI resistant variants previously shown to have developed resistance through EMT and filtered gene expression data to include only genes which transcribed proteins localized to the cell surface. We identified CD70 as a being highly upregulated in EGFR TKI resistant cells (p = 7.2e-42). Given that CD70 expression is highly restricted and only transiently expressed on immune cells, CD70 was selected as a top candidate cell surface protein for targeting studies. Western blotting and flow cytometry analysis confirmed CD70 protein levels to be highly upregulated in EGFR TKI resistant cells that had undergone EMT but not in cells harboring secondary EGFR mutations or MET amplifications. We also observed CD70 upregulation in osimertinib-treated drug tolerant persister cells, indicating that CD70 upregulation is an early event in the evolution of TKI resistance. Moreover, patient-derived models of acquired EGFR TKI resistance also exhibited CD70 positivity. Our data also indicated that in EGFR mutant NSCLC cells, CD70 could be upregulated through decreased CD70 promoter methylation as well as by the EMT regulators, transforming growth factor-β (TGF-β) and ZEB1, both of which were upregulated in TKI resistant cells. In EGFR TKI resistant cells, CD70 knockdown impaired cell viability and invasiveness, and stimulation of CD70 using the exogenous binding partner CD27 resulted in activation of AKT and MAPK, pathways known to be re-activated with acquired TKI resistance. CD70-targeting approaches including anti-CD70 antibody drug conjugates (ADCs) and CD70-targeting CAR T cell and CAR NK cells showed promising in vitro and in vivo activity against CD70 positive tumor cells and in osimertinib drug-tolerant persister cells. These results identify CD70 as a novel therapeutic target for EGFR mutant tumors with acquired EGFR TKI resistance that merits further investigation in the clinic. Citation Format: Monique B. Nilsson, Yan Yang, Sonia Patel, Simon Heeke, Xiuning Le, Thiru Aruguman, Jacqulyne Robichaux, Xiaoxing Yu, Alissa Poteete, Xiaoyang Ren, Lixia Diao, Li Shen, Qi Wang, Fahao Zhang, Leticia Campos Clemente, Luisa Solis Soto, Chunhua Shi, Hai Tran, Jason Bock, Jing Wang, Ignacio I. Wistuba, John D. Minna, John V. Heymach. CD70 is a novel therapeutic target for EGFR mutant NSCLC with acquired, EMT-associated EGFR TKI resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1827.

PD-L1 expressed from tumor cells promotes tumor growth and invasion in lung cancer via modulating TGF-β1/SMAD4 expression.

BackgroundProgrammed death ligand‐1 (PD‐L1) has a known association with the prognosis of human cancers because of its ability to alter tumor immune surveillance via its interaction with PD‐1. We questioned whether expression of PD‐L1 in tumor cells could directly promote tumor growth and invasiveness in non–small cell lung cancer (NSCLC).MethodsReal‐time reverse transcription‐polymerase chain reaction (RT‐PCR) was performed to evaluate PD‐L1 messenger RNA (mRNA) expression in lung tumors. The prognostic value of PD‐L1 mRNA was assessed by Cox regression model. Transcriptional regulation of PD‐L1 by human papillomavirus (HPV) 16/18 E6 oncoprotein or by epidermal growth factor receptor (EGFR) mutation in lung cancer cells was examined by Western blot and luciferase reporter assay. The cell growth and invasion were evaluated by colony formation, soft agar growth, and Boyden chamber assay.ResultsThe PD‐L1 mRNA levels showed a positive association with HPV 16/18 E6 oncoprotein and with EGFR mutation in 223 surgically resected NSCLC patients. The prognostic significance of PD‐L1 was more commonly observed in patients with high PD‐L1/E6 positive and high PD‐L1/EGFR mutant tumors. Mechanistically, upregulation of PD‐L1 transcription by E6 or mutant EGFR occurred largely through the ERK‐C/EBPβ‐TLR4‐NF‐κB cascade. PD‐L1 promotes the efficacy of colony formation, soft agar growth, and cell invasion. PD‐L1 upregulates BAG‐1 to reduce transforming growth factor (TGF)‐β1 expression, and the decrease in SMAD4 because of TGF‐β1 occurs through the p53/microRNA (miR)‐224 axis. The decreases in TGF‐β1 and SMAD4 are responsible for PD‐L1‐mediated cell invasiveness.ConclusionInduction of PD‐L1 by E6 oncoprotein or mutant EGFR through the ERK‐C/EBPβ‐TLR4‐NF‐κB cascade may promote tumor growth and invasiveness in NSCLC because of decreasing TGF‐β1 and SMAD4 expression.

Dysregulation of TFH-B-TRM lymphocyte cooperation is associated with unfavorable anti-PD-1 responses in EGFR-mutant lung cancer

Patients with non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations exhibit an unfavorable response to PD-1 inhibitor through unclear mechanisms. Hypothesizing that EGFR mutations alter tumor-immune interactions, we compare tumor-infiltrating lymphocytes between EGFR mutant (EGFR-MT) and wild type (EGFR-WT) tumors through single-cell transcriptomic analysis. We find that B cells, CXCL13-producing follicular helper CD4+ T (TFH)-like cells, and tissue-resident memory CD8+ T (TRM)-like cells decreased in EGFR-MT tumors. The NOTCH-RBPJ regulatory network, which is vital for persistence of TRM state, is perturbed, and the interactions between TFH and B cells through the CXCL13-CXCR5 axis disappear in EGFR-MT tumors. Notably, the proportion of TRM-like cells is predictive for anti-PD-1 response in NSCLC. Our findings suggest that the impairment of TFH-B-TRM cooperation in tertiary lymphoid structure formation, accompanied by the dysregulation of TRM homeostasis and the loss of TFH-B crosstalk, underlies unfavorable anti-PD-1 response in EGFR-MT lung tumors.

Abstract 1094: Genetic determinants of EGFR-driven lung cancer growth and therapeutic response in vivo

Abstract Oncogenic alterations in the Epidermal Growth Factor Receptor (EGFR) gene are found in ~15% of lung adenocarcinomas (LUADs). EGFR mutations predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs) and these drugs are approved for the first-line treatment of patients with EGFR mutant lung cancer. Response rates to TKIs are high, however, there is variability in the duration and depth of response between patients and acquired resistance to TKIs inevitably emerges. EGFR mutant tumors harbor numerous additional co-occurring alterations in addition to the driver mutations that are likely to contribute to the malignant phenotype of these tumors and the variability in therapeutic response. TP53 is the most common tumor suppressor gene (TSG) co-mutated with EGFR, and alterations in TP53 correlate with worse outcomes upon TKI treatment. However, the functional contribution of loss of other LUAD-associated TSGs to tumor progression and drug resistance in EGFR mutant tumors remains poorly understood. To study the role of 10 putative tumor suppressor genes–that are frequently altered in human LUADs–for EGFR mutant tumors, we developed a conditional lung cancer model based on the inducible expression of EGFRL858R and deletion of Tp53 in the lungs via lentiviral delivery of Cre recombinase. We coupled this EGFRL858R;p53flox/flox mouse model with multiplexed CRISPR-Cas9-mediated genome editing and performed an in vivo screen to quantify the effects of inactivation of these 10 TSGs on tumor growth and TKI sensitivity. We found that inactivation of Apc, Rb1, and Rbm10 each strongly promoted growth while loss of Lkb1 or Setd2 reduced tumor growth. In contrast, in a KrasG12D;p53flox/flox model inactivation of Lkb1 or Setd2 were the strongest drivers of tumor growth. To establish the relevance of the functional data from mouse models for human tumors, we examined the AACR Project Genomics Evidence Neoplasia Information Exchange (GENIE) database. Consistent with the mouse data, of the 10 TSGs screened, APC, RB1 and RBM10 were the most frequently mutated in EGFR/TP53 mutant LUADs. We also tested whether inactivation of any of the 10 TSGs was associated with a reduced response to TKIs, by treating mice infected with the lentiviral pool targeting the TSGs with the 3rd generation TKI osimertinib and found that loss of Keap1 was associated with a diminished response to therapy. Collectively, our data have: i) uncovered striking specificity of TSG function in EGFR mutant LUADs, ii) discovered that inactivation of some TSGs can have diametrically opposite effects depending on the nature of the initiating oncogene and iii) established a causal link between certain tumor genotypes and differential responses to EGFR inhibition. Ongoing studies are aimed at investigating the mechanistic underpinnings of these findings. Citation Format: Giorgia Foggetti, Chuan Li, Hongchen Cai, Wen-Yang Lin, Deborah Ayeni, Katherine Hastings, Laura Andrejka, Dylan Maghini, Robert Homer, Dmitri A. Petrov, Monte M. Winslow, Katerina Politi. Genetic determinants of EGFR-driven lung cancer growth and therapeutic response in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1094.

Abstract 5066: Investigating the role of IL-6 to modulate the immunosuppressive tumor microenvironment of EGFR mutant NSCLC

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death with a less than 15% five-year survival. Approximately 15% of NSCLC cases in the US harbor epidermal growth factor receptor (EGFR) activating mutations and are initially highly sensitive to EGFR tyrosine kinase inhibitors (TKI). Unfortunately, the vast majority of tumors acquire EGFR TKI resistance, and treating resistant disease remains a clinical challenge. Additional therapeutic modalities, such as immunotherapy, are needed to improve clinical outcome. While immunotherapy has shown clinical activity in multiple cancer types including NSCLC, a lack of efficacy has been observed in EGFR mutant NSCLC patients. The biological mechanisms driving immunotherapy resistance in EGFR mutant NSCLC are not well understood, and additional EGFR mutant NSCLC patients. We previously reported that IL-6 is upregulated in NSCLC cells with acquired resistance to EGFR TKIs. Given the role of IL-6 as an immunomodulatory cytokine, we evaluated the effects of IL-6 signaling on immunosuppression in genetically engineered mouse models (GEMMs) of EGFR mutant NSCLC. IL-6 knockout (EGFRL858R/IL-6KO) resulted in decreased PD-L1 expression on the surface of EGFR mutant tumor cells compared to tumors in IL-6 proficient mice. Additionally, we observed a modest increase in PD-1 expression on CD8+ T cells in EGFRL858R/IL-6KO mice compared to EGFRL858R mice, suggesting that IL-6 acts on both tumor and immune populations to facilitate a “cold” immunosuppressive tumor microenvironment. We next analyzed immune populations in EGFR mutant tumors from mice with or without knockout of IL-6 by single cell RNA sequencing and validated these findings by immunohistochemistry. Tumors in EGFRL858R/IL-6KO mice displayed decreased B-cell (CD20) populations, increased macrophages (F480), and increased natural killer cells (NKp46) compared to tumors from IL-6 expressing EGFRL858R mice. Our data suggests that the increased expression of IL-6 in EGFR TKI resistant NSCLC may alter immune cell populations within the tumor microenvironment and impact immunosuppression, and that therapeutic approach targeting IL-6 merit further investigation for EGFR-mutant NSCLC. Citation Format: Sonia A. Patel, Monique Nilsson, Sungnam Cho, Xiuning Le, John Heymach. Investigating the role of IL-6 to modulate the immunosuppressive tumor microenvironment of EGFR mutant NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5066.

Abstract 1675: Enhanced antitumor effect of dual EGFR and VEGFR2 inhibition in EGFR-mutated non-small cell lung cancer (NSCLC) patient-derived tumor xenograft (PDX) models

Abstract Background: NSCLC accounts for nearly 85% of all lung cancers. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are effective for EGFR mutated NSCLCs, but these drugs often stop working after EGFR acquires additional mutations. Preclinical and clinical data suggest a stronger antitumor effect with dual blockade of the EGFR and vascular endothelial growth factor (VEGF) pathways in EGFR-mutated NSCLC. RELAY, a randomized, double-blind, phase 3 trial, demonstrated superior progression-free survival (PFS) with ramucirumab (RAM) + erlotinib compared with placebo + erlotinib in patients with untreated EGFR-mutated advanced NSCLC. Additionally, RAM was tested in a Phase 1 study with the third-generation irreversible EGFR TKI osimertinib (OSI); AstraZeneca) in NSCLC patients with advanced T790M-positive EGFR mutant tumors after progression on first-line EGFR TKI therapy. OSI can be used with L858R, exon 19 del and T790M EGFR mutant tumors. We investigated the combined use of OSI+RAM in EGFR mutated NSCLC PDX mouse models. Materials and Methods: The antimurine VEGFR2 antibody DC101 (40 mg/kg, BIW) and the EGFR TKI OSI (10 mg/kg, QD) were used in this study. Twenty-seven PDX NSCLC mouse models carrying EGFR activating and/or T790M resistance mutations, commonly detected in patients with advanced NSCLC, were evaluated at 3 independent sites. Mice were divided into 4 treatment arms: vehicle (PBS), DC101, OSI and combination (DC101+OSI). Each study arm included two transplanted mice per model. Sensitivity to DC101 and OSI monotherapies, enhancement of anti-tumor activity with the combination therapy and durability of the response were evaluated. Desirability score, a weighted composite score of six features of tumor growth curves including best median % response, number of days in best response category, time to doubling, re-growth rate (last 5 observations) relative to vehicle growth rate, last observed tumor volume and last observed day, was calculated for each treatment arm. Analysis of variance was used to compare summary scores per animal across treatments. Results: OSI alone and OSI+DC101 combination therapy had stronger antitumor effects than DC101 monotherapy in significantly more models 11 vs 1 and 12 vs 0, respectively. A small but consistent shift in favor of OSI+DC101 vs OSI was observed across most of the models. Paired t-test analyses showed a moderate but significant benefit with combination OSI+DC101 vs OSI. Overall desirability score was statistically improved with OSI+DC101 compared to either single agent. Conclusions: Dual inhibition of EGFR and VEGFR2 with OSI+DC101 had a stronger antitumor effect than those of the respective monotherapies in EGFR mutated NSCLCs in vivo. Future studies will investigate the molecular mechanisms underlying the enhanced antitumor effect of OSI+DC101 combination therapy. Citation Format: Sudhakar Chintharlapalli, Anthony Fischl, Chun Ping Yu, Philip Iversen. Enhanced antitumor effect of dual EGFR and VEGFR2 inhibition in EGFR-mutated non-small cell lung cancer (NSCLC) patient-derived tumor xenograft (PDX) models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1675.

Abstract IA10: Modeling sensitivity and resistance to systemic therapies in lung cancer

Abstract Targeted therapies have transformed the landscape for the treatment of metastatic lung cancer. Despite their success, targeted therapies are not curative and acquired resistance is a major impediment to cures for patients treated with these therapies. A paradigm for the success of targeted therapies in lung cancer comes from epidermal growth factor receptor (EGFR) mutant lung cancer where mutations in exons encoding the tyrosine kinase domain of EGFR confer sensitivity to tyrosine kinase inhibitors (TKIs), and several TKIs are currently approved for the first-line treatment of EGFR mutant lung cancer. Moreover, there is heterogeneity in the durability and depth of responses between patients and at different metastatic sites within individual patients. Therefore, there is a need to understand the biology of EGFR mutant tumors and identify factors that affect TKI sensitivity. We developed transgenic mouse models of EGFR mutant lung cancer and have used them to study tumor progression and TKI resistance. When we modeled acquired resistance to the TKI osimertinib as a first-line therapy in transgenic mouse models of EGFRL858R-induced lung adenocarcinoma, we found that it is mediated largely through secondary mutations in EGFR—either C797S or L718V/Q or Kras mutations. We also identified a therapeutic approach to prevent the emergence of secondary mutations in EGFR. To determine whether the presence of co-occurring genetic alterations contributes to a more complex spectrum of resistance mechanisms in vivo, we have developed new mouse models to specifically evaluate the consequences of co-occurring tumor suppressor gene alterations on the progression and TKI sensitivity of EGFR mutant tumors. Collectively, our findings highlight how genetically engineered mouse models of lung cancer, including those with complex genotypes, can be leveraged to study tumor progression and drug resistance in vivo. Citation Format: Katerina A. Politi. Modeling sensitivity and resistance to systemic therapies in lung cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr IA10.

IA31 Genetic Contributors to Tumor Progression and Drug Resistance in EGFR Mutant Lung Cancer

Targeted therapies have transformed the landscape for the diagnosis and treatment of metastatic lung cancer. These tumors are now routinely tested for the presence of mutations or rearrangements in specific oncogenic drivers that, if present, predict sensitivity to targeted therapies directed to the genomic alterations present. Genotype-directed therapies have improved outcomes in specific subsets of patients with metastatic lung cancer. Despite this success, targeted therapies are not curative and acquired resistance is a major impediment to cures for patients treated with these therapies. Moreover, there is heterogeneity in the durability and depth of responses between patients. A paradigm for the success of targeted therapies in lung cancer comes from Epidermal Growth Factor Receptor (EGFR) mutant lung cancer. Mutations in exons encoding the tyrosine kinase domain of EGFR confer sensitivity to tyrosine kinase inhibitors (TKIs), and several are currently approved for the first-line treatment of EGFR mutant lung cancer. Most recently, the third-generation TKI osimertinib was approved and is increasingly being used in the first line. However, we have very limited knowledge of the mechanisms of resistance to osimertinib given its recent adoption in the clinic. Without knowledge about resistance mechanisms, optimal post-osimertinib treatment strategies remain to be defined. We modeled acquired resistance to first-line osimertinib treatment in transgenic mouse models of EGFRL858R-induced lung adenocarcinoma and found that it is mediated largely through secondary mutations in EGFR and identified therapeutic strategies to treat these tumors and prevent their emergence. Moreover, since EGFR mutant tumors in patients harbor additional genetic alterations beyond EGFR, many of them in tumor suppressor genes, we tested how the presence of co-occurring genetic alterations in tumor suppressor genes contributes to the progression and osimertinib sensitivity of the tumors in the mouse models of EGFR mutant lung cancer. Collectively, our findings highlight how genetically engineered mouse models of lung cancer, including those with complex genotypes, can be leveraged to study tumor progression and drug resistance in vivo.

Angiogenesis and epidermal growth factor receptor inhibitors in non-small cell lung cancer.

Several preclinical studies suggested a potential benefit from combined treatment with inhibitors of epidermal growth factor receptor (EGFR) and angiogenesis, both effective in patients with advanced non-small-cell lung cancer (NSCLC). In pretreated patients with advanced EGFR wild type NSCLC, bevacizumab plus erlotinib improved progression-free survival as second-line therapy in the BeTa study and as maintenance therapy in the ATLAS trial, although the benefit was modest and did not translate into an advantage in overall survival. Disappointing results were reported with oral VEGF inhibitors plus erlotinib in pretreated patients with EGFR wild type NSCLC. On the contrary, erlotinib plus bevacizumab or ramucirumab showed a clinically relevant improvement of progression-free survival in naïve patients with EGFR mutations, leading to the approval of these two regimens as first-line treatment of NSCLC patients with EGFR mutant tumors. Several clinical studies are evaluating the feasibility and activity of osimertinib plus bevacizumab or ramucirumab. However, limits that could affect its use in clinical practice are the need of an intravenous infusion for angiogenesis inhibitors, the increased incidence of treatment associated adverse events, the exclusion of patients with tumors located in central position or at risk of hemorrhage. The identification of predictive biomarkers is an important goal of research to optimize the combined use of these agents.

Abstract 4744: A prospective biospecimen collection study from patients with EGFR mutant tumors

Abstract Background: Somatic mutations in the epidermal growth factor receptor (EGFR) are detected in nearly 15% of patients with lung adenocarcinoma. These mutations are critical for tumor development and maintenance; some but not all are sensitive to EGFR tyrosine kinase inhibitors (TKI). EGFR mutations fall into 3 major categories: 1) Exon 19 deletions and L858R which are detected in 85% of patients; 2) Atypical EGFR mutations including G719X, L861Q which are detected in 5 to 8% of patients and 3) Exon 20 insertion mutations which are also detected in 5 to 8% of patients. These different subtypes exhibit varying responses to TKI treatment, with osimertinib being the standard of care for patients with exon 19 deletion or L858R mutations. However, no currently-approved TKI is available for patients with Exon 20/EGFR mutant lung cancer. Method: Patient advocacy groups representing oncogene-driver mutations in lung cancer (ALK, EGFR, Exon 20, RET, ROS1) have assembled with the objectives of providing patient education/support and driving research into lung cancer’s causes and treatments. The EGFR Resisters represent over 775 patients from 26 countries. In an effort to understand mechanisms of resistance and develop new treatments, EGFR Resisters embarked on a collaboration with Dr. Pasi Jänne, the Addario Lung Cancer Medical Institute, the Bonnie J. Addario Lung Cancer Foundation, and Champions Oncology to develop patient-derived xenograft (PDX) models for EGFR mutant lung cancer. Result: In mid-2018 the EFGR Resisters assembled a coalition of key leaders in the lung cancer space to advance development of research models for EGFR mutant lung cancer. The IRB-approved study, “A Prospective Biospecimen Collection Study from Patients with EGFR mutant Tumors” was launched in November 2018 with a goal of developing at least ten EGFR mutant PDX models with full characterization including whole exome sequencing (WES) and RNA sequencing. Model development will be divided among three cohorts: 1) EGFR T790M patients who have progressed on osimertinib or other third-generation TKIs; 2) EGFR exon 19 del or L858R patients who have progressed on first-line osimertinib; 3) patients with Exon 20 insertion mutations (includes EGFR Exon 20 and HER2 Exon 20). The models will be made available to the scientific community. Conclusion: Understanding mechanisms of resistance in EGFR mutant lung cancer is high priority as most patients will ultimately develop resistance to currently-approved TKIs. Additionally, there is an urgent need to develop next-generation TKIs since some EGFR mutation subtypes do not respond to current therapies. By leveraging the skills and expertise of diverse partners, the EGFR Resisters have advanced an initiative to create a unique cohort of PDX models that will provide a rich resource for clinical and translational research to understand mechanisms of resistance and develop new therapies for patients with EGFR mutant lung cancer. Citation Format: Ivy B. Elkins, Jill Feldman, Anita Figueras, Teri Kennedy, Allen Lee, Ildiko Medve, Colleen Sturdivant, Pasi Janne, Tony Addario, Alicia Sable-Hunt, Bonnie Addario, David LeDuc, Amy Moore, Jennifer Jaskowiak, Maria Mancini. A prospective biospecimen collection study from patients with EGFR mutant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4744.

Abstract 1055: Evaluation of immune checkpoint inhibitor efficacy in EGFR mutant tumors

Abstract Tyrosine Kinase Inhibitor (TKI) of Epidermal Growth Factor Receptor (EGFR) is a first-line therapy for non-small cell carcinoma lung cancer patients with EGFR mutations, but patients often develop resistance to such a target therapy. Recently several PD-1 and PD-L1 inhibitors were approved for treating NSCLC, however, it remains unclear whether EGFR mutant NSCLC would benefit from PD-1/PD-L1 treatment. PD-1 response in cancer patients is closely associated with oncogenic mutation status, tumor microenvironment, PD-L1 expression and tumor mutation burden (TMB); however, the lack of pre-clinical models limited the investigation of the checkpoint inhibitors in EGFR mutant tumors. Here we use human PBMC reconstitution system for immune-humanization, and established a series of IO-Xenograft models (IO-CDX and IO-PDX) with cancer cell lines (IO-CDX) or patient-derived tissues (IO-PDX) that contain various EGFR mutations, including EGFR 19 del, L858R, T790M, and EGFR over-expression, amplification and fusion. We treated these models with either EGFR TKI (erlotinib, afatinib or osimertinib) or nivolumab, a PD-1 inhibitor. The predictive biomarkers including PD-L1 expression and TMB were analyzed by whole exom sequencing (WES) and FACS for tumor infiltrating lymphocytes (TILs), respectively. We found that 3 TKI-resistant PDX models bearing EGFR T790M mutation exhibited lower TILs than those bearing EGFR wild type or exon 19del mutation. Of the 8 IO-CDX or IO-PDX humanized models we have examined, those exhibiting higher TILs achieved better responses to nivolumab treatment. The 3 most sensitive models are EGFR amplification (wt), EGFR over-expression (wt) and EGFR exon 19del. In contrast, the EGFR T790M model from H1975 cell line was observed with hyper-progressive disease after nivolumab treatment. We will also discuss the contribution of PD-L1 expression and TMB to nivolumab in other models. In summary, these well-established IO-CDX and IO-PDX models can provide a human-resembling immune system for of immune therapeutics and combination with targeted therapies, and help to facilitate the understanding of relationship among tumor microenvironment, driver oncogenic mutations and drug response. Citation Format: Xuzhen Tang, Li Yang, Hui Qi, Xianzhi Zhai, Fuyang Wang, Xiangnan Qiang, Jie Xu, Xiaoran Qin, Qingyang Gu, Shaoyu Yan, Qunsheng Ji. Evaluation of immune checkpoint inhibitor efficacy in EGFR mutant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1055.

Abstract 4930: PAI1, a β catenin transcriptional target, serves as a surrogate biomarker to predict EGFR TKI mediated drug persistence in EGFR mutant NSCLC

Abstract Lung cancer management has been transformed by the discovery and effective targeting of driver mutations such as epidermal growth factor receptor (EGFR). Tyrosine Kinase Inhibitors (TKIs) that block EGFR kinase activity such as erlotinib and osimertinib cause dramatic tumor shrinkage and prolonged responses in patients whose tumors have activating EGFR mutations. However, resistance universally develops. Drug persister cells with properties of cancer stem cell-like cells play a major role in drug resistance. In tumors without pre-existing resistant clones, some tumor clonogens survive, despite expressing the driver mutation, to acquire target reactivation or bypass resistance mechanisms. Identification of biomarkers that are mechanistically linked to EGFR TKI resistant cell populations would have clinical utility in early determination of disease recurrence in EGFR mutant NSCLC patients. Previously, we have reported that EGFR-TKI treatment activates Notch3, and here we have identified that Notch3 activates β-catenin transcriptional target, PAI1, which could play a role in drug persistence and could be used for early identification of disease recurrence. Our in vitro ELISA studies have identified an increase in secreted PAI1 levels after treatment with the EGFR TKIs erlotinib and osimertinib demonstrate that measuring PAI1 levels could serve as an indicator of EGFR TKI persistence. To further validate PAI1 as a surrogate biomarker for EGFR TKI treatment, we performed a retrospective study in NSCLC patients (n = 42) with EGFR mutant tumors. Using ELISA to detect PAI1 protein in pre- and post-treatment serum samples as a mechanism-based indicator for β-catenin activation, we observed a broad range in pre-treatment PAI1 levels, spanning from 0.8 ng/ml to 134 ng/ml, suggesting a variable baseline β-catenin activation. Doing survival analysis after dichotomizing patients into low and high baseline PAI1 level groups, we found patients with low basal PAI1 levels had a significantly shorter PFS with EGFR TKI treatment compared to patients with high basal levels. Comparing patients with a ≥ 2-fold induction to those with less than 2-fold, there was a similar trend for worsened survival in those who showed significant induction of PAI1. Overall, we demonstrate that PAI1 expression is a candidate marker for β-catenin activation and the development of drug persisters; thus, it is a potential surrogate marker for poor outcome in patients receiving EGFR TKI therapy and a potential biomarker for selecting patients for β-catenin-targeted therapy. Citation Format: Rajeswara Rao Arasada, Walter Wang, Shankar Suman, David Paul Carbone. PAI1, a β catenin transcriptional target, serves as a surrogate biomarker to predict EGFR TKI mediated drug persistence in EGFR mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4930.

P1.16-60 A High Risk of Recurrence After the Resection of Lower Lobe Adenocarcinoma

According to previous reports, non-small cell lung cancers arising in the lower lobe are associated with a worse prognosis than those arising in the upper lobe; however, the reason remains to be elucidated. Thus, we attempted to identify high-risk population in patients with lower lobe disease, that may substantially cause poor prognosis of lower lobe disease. We retrospectively reviewed a consecutive series of 400 patients with completely resected lung adenocarcinoma who were treated between January 2006 and December 2015. All patients underwent major lung resection and lymph node dissection. The clinicopathological factors that were investigated included the solid component size, the pathological subtype (pre-invasive, minimally invasive/invasive), the epidermal growth factor receptor (EGFR) mutation status, and some other characteristics (TNM 8th edition). The proportion of never-smokers among patients with lower lobe disease was significantly higher than that among those with upper lobe disease. According to a multivariate proportional hazards analysis, primary site (upper/lower) was an independent predictor of early recurrence, along with the solid component size, pleural lavage cytology results, pathological pleural invasion, and pathological lymph node metastasis. However, although lower lobe disease was associated with early recurrence in never smokers (Fig 1A), it was not in smokers (Fig 1B). Furthermore, although lower lobe disease was associated with early recurrence in never smokers with EGFR wild-type tumors (Fig 1C), it was not in never smokers with EGFR mutant tumors (Fig 1D). EGFR wild-type tumors were more frequently detected in patients with lower lobe disease than in those with upper lobe disease when the analysis was restricted to never smokers. Patients with lower lobe cancer, particularly those without a smoking history or those with EGFR wild-type tumors, showed a higher risk of recurrence than their counterparts with upper lobe disease. Further studies that include the molecular profiling of such aggressive lesions, are warranted.

Relation of EGFR Mutation Status to Metabolic Activity in Localized Lung Adenocarcinoma and Its Influence on the Use of FDG PET/CT Parameters in Prognosis.

The purposes of this study were to assess the relation between epidermal growth factor receptor (EGFR) mutation status and FDG PET/CT findings and to evaluate the influence of this relation on the use of FDG PET/CT parameters to establish a prognosis in cases of localized lung adenocarcinoma. Patients with stage I and II lung adenocarcinomas were retrospectively enrolled. At the initial FDG PET/CT examination, maximum and peak standardized uptake, tumor-to-background ratio, and volumetric parameters of metabolic tumor volume and total lesion glycolysis were measured. The values of all the metabolic and volumetric FDG PET/CT parameters were significantly lower in EGFR mutant than in EGFR wild-type lung adenocarcinomas. All parameters were statistically significant for predicting recurrence-free survival. In multivariate analyses, peak standardized uptake and total lesion glycolysis were more significant prognostic factors than was TNM stage (p < 0.001). Optimal cutoff values of parameters for predicting recurrence-free survival were slightly different between the two groups. EGFR mutation is related to low metabolic activity of localized lung adenocarcinoma at FDG PET/CT. Because of differences in the metabolic activity of EGFR mutant and wild-type tumors, EGFR mutation status must be considered when FDG PET/CT parameters are used for prognosis.

Immune marker profiling and PD-L1, PD-L2 expression mechanisms across non-small cell lung cancer mutations.

9076 Background: PD-1/PD-L1 axis inhibitors have been proven effective, especially in patients expressing Programmed Death Ligand 1 (PD-L1). Their clinical efficacy in patients with epidermal growth factor receptor (EGFR) activating mutations is still unclear, while KRAS mutations seem to be associated with high response rates. In this study we investigated the expression of PD-L1, PD-L2 and Tumor Infiltrating Lymphocyte (TIL) status as a function of mutation status in Non-Small Cell Lung Cancer (NSCLC). Methods: We used the AQUA method of quantitative fluorescence (QIF) to compare PD-L1 and PD-L2 expression and to characterize TILs populations and their activation status in over 150 NSCLC patient tumors with known mutation status. EGFR activation was assessed in situ using the proximity ligation assay (PLA) for EGFR and GRB2 and T cell activation was assessed using a novel multiplexed QIF assay including CD3, Granzyme B and Ki67. Results: PD-L1 tumor and stroma expression was significantly lower in EGFR mutant compared to KRAS mutant (p = 0.009) and EGFR/KRAS Wild Type (p &lt; 0.0001) tumors, while they had a higher frequency of PD-L2 expression. Conversely, KRAS mutants had significantly lower PD-L2 tumor and stroma expression but they were also more inflamed with higher CD4+, CD8+ and CD20+ TILs. Subgroup analysis of patients by their TILs activation status revealed that EGFR mutants had a very high frequency of inactive TILs even though lymphocytes were present in the tumor microenvironment. In contrast, in KRAS mutants, when TILs were present, they were almost always active. Finally, we find that PLA-defined activated EGFR correlated with increased PD-L1 expression in EGFR mutants but not in EGFR WT, while TIL activation was associated with higher PD-L1 in EGFR/KRAS WT. Conclusions: Our findings are consistent with the unique biology of EGFR mutant tumors. The high frequency of inactive TILs could explain the low immune therapy response rates in this patient group. Similarly, in this group, the reason PD-L1 expression fails to predict response may be due to expression as a result of constitutive oncogenic signaling rather than immune signaling, which would be associated with high PD-L1 levels and TILs activation.

Osimertinib: a breakthrough for the treatment of epidermal growth factor receptor mutant lung adenocarcinoma

At the time of acquired resistance to the first generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), gefitinib, erlotinib or afatinib (1), 50–60% of EGFR mutant non-small cell lung cancer (NSCLC) patients have developed the secondary gatekeeper T790M point mutation on exon 20 of the EGFR gene (2,3). Osimertinib (AZD9291) is a third-generation EGFR TKI that targets EGFR mutant (including T790M positive) tumors (4). It was initially evaluated in EGFR mutant NSCLC patients who had disease progression after previous treatment with an EGFR TKI, in the phase I part of the phase I/II AURA study (5). A response rate of 51% was achieved for all patients treated across all dose levels. Among the 222 patients of the expansion cohorts, the response rate to osimertinib was 61% for EGFR T790M-positive patients while the EGFR T790M-negative patients were not able to achieve a response rate greater than 21% ( Table 1 ). No dose limiting toxicities were observed at any dose level and, based on tumor growth inhibition, the dose of 80 mg once daily was selected for being further evaluated for the treatment of EGFR T790M-positive NSCLC patients (5). On November 13 th 2015, and on February 3 rd 2016, FDA and EMA approved osimertinib 80 mg once daily for the treatment of EGFR T790M-positive NSCLC patients, respectively, based on the data from two phase II studies (AURA extension and AURA2) and the AURA phase I expansion study. Sixty-three EGFR T790M-positive, dose expansion cohort patients receiving 80 mg of osimertinib once daily in the phase I part of the AURA study demonstrated an objective response rate (ORR) of 71% [95% confidence interval (CI), 57–82] and a median progression-free survival (PFS) of 9.7 months (95% CI, 8.3–13.6). In a pre-planned pooled analysis of the AURA extension phase II study and the AURA2 phase II study with a total of 411 EGFR T790M-positive patients, ORR was 66% and median PFS was 11.0 months (95% CI, 9.6–12.4) (6).