Erlotinib Response Research Articles

Abstract 5829: Elucidating the significance of amphiregulin and its implications for EGFR inhibition strategies in LKB1-mutant NSCLC

Abstract LKB1-mutant non-small cell lung cancer (NSCLC) is aggressive and treatment-refractory, underscoring the need for new therapeutic strategies. Our comparative study involving mouse lung tumors driven by oncogenic KrasG12D and LKB1 loss as well as tumors driven solely by KrasG12D, revealed that LKB1 loss upregulated amphiregulin (Areg), a pro-growth EGFR ligand. Therefore, this study investigates how the inactivation of LKB1 enhances AREG expression, the significance of elevated AREG in sustaining lung cancer growth, and the responses of AREG-high lung cancer to EGFR inhibitors. We first evaluated the potential clinical significance of AREG using The Cancer Genome Atlas (TCGA) lung adenocarcinoma dataset and the Cancer Cell Line Encyclopedia (CCLE) database. We observed that LKB1-null tumors with high AREG levels were associated with shorter patient survival and exhibited transcriptional signatures similar to EGFR-mutant tumors. High AREG expression also correlated positively in NSCLC cell lines with LKB1 mutations and Western blot analysis confirmed that elevated AREG levels in lung cancer cells were associated with constitutive EGFR signaling. We next explored the impact of LKB1 loss on AREG expression in lung cancer. Our prior RNA-seq and ChIP-seq investigations demonstrated CRTC2 and CREB enrichment on the AREG promoter in LKB1-null lung cancer cells. Upon LKB1 reintroduction, the enrichment diminished, indicating that CRTC-CREB transcriptionally induces AREG in LKB1-null lung cancer. To corroborate, reintroducing LKB1 or expressing a dominant-negative construct (dnCRTC), disrupting CREB/CRTC interaction, decreased AREG levels, as confirmed by RT-qPCR in LKB1-null lung cancer cells. This supports that AREG is a direct target of CRTC-CREB activation induced by LKB1 loss. To assess the impact of elevated AREG in LKB1-mutant lung cancer growth, we used RNA interference and antibodies to inhibit AREG. Both interventions decreased cell growth and colony formation, underscoring AREG is critical for LKB1-mutant lung cancer growth. Moreover, we investigated the potential correlation between AREG and the response to the EGFR tyrosine-kinase inhibitor Erlotinib in NSCLC cell lines, utilizing AREG expression data from CCLE and Erlotinib response data from the Genomics of Drug Sensitivity in Cancer (CDSC) databases. Stratifying NSCLC cell lines into sensitive, intermediate, and resistant categories revealed a potential positive association between AREG expression and Erlotinib sensitivity. Notably, LKB1-null lung cell lines with high AREG expression demonstrated heightened sensitivity compared to those with low AREG expression. Our study revealed that LKB1 loss increases AREG, triggering aberrant EGFR signaling critical for lung cancer growth, and AREG expression might impact the Erlotinib response in LKB1-inactive cells. Citation Format: Xzaviar Kaymar Solone, Xin Zhou, Mu Yu, Jennifer W. Li, Finn Rimay, Frederic Kaye, Lizi Wu. Elucidating the significance of amphiregulin and its implications for EGFR inhibition strategies in LKB1-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5829.

Effect of macrocytosis on erlotinib response in metastatic non-small cell lung cancer

Background/Aim: Numerous studies have assessed the relationship between macrocytosis and responses to chemotherapeutic agents and TKIs such as sunitinib and imatinib. However, there is limited data in the literature regarding the prognostic or predictive value of macrocytosis in using erlotinib. If a relationship is detected, early response/resistance assessment can be performed before imaging time in the follow-up of treatments, and a more cost-effective, non-invasive method can be employed for response monitoring. This study aimed to elucidate the effect of macrocytosis on response rates in patients treated with erlotinib for non-small cell lung cancer. Methods: Seventy-five individuals diagnosed with non-small cell lung cancer (NSCLC) and admitted to our institution were enrolled in this retrospective cohort study. Baseline demographics, time of diagnosis, previous treatment, and the initiation or cessation of erlotinib were recorded. Data of patients with and without macrocytosis were analyzed. Stable disease, partial and complete response rates, and progressive disease response were evaluated separately as response rates. Progression-free survival between drug initiation and discontinuation due to progression was interpreted using Kaplan-Meier curves. Results: The distribution of the overall survival (OS) and progression-free survival (PFS) evaluations revealed that 84% (n=63) of the patients were deceased, and the progression rate was 94.7% (n=71). The median OS of the patients was 18 months, and the median PFS was 11 months. There was a statistically significant difference in overall survival in females, with a median OS of 25 months (95% CI 17–32 months) and a median OS of 13 months in males (95% CI 9–20 months) (P=0.008). PFS was 14.5 months (95% CI 11–21 months) in women and six months (95% CI 4–17 months) in men, and there was a statistically significant difference (P=0.02). A statistically significant difference was achieved between MCV values measured during diagnosis and the third month between age groups (P=0.044). Conclusion: The outcomes of this research suggest a statistically significant difference between the MCV values measured at the time of diagnosis and the third month regarding age groups. Both OS and PFS in women were statistically significantly higher than in men.

P8‐187: A case of lung adenocarcinoma with 4.5 years of sustained erlotinib response and reversion of EGFR mutation positiveness

P8‐187: A case of lung adenocarcinoma with 4.5 years of sustained erlotinib response and reversion of EGFR mutation positiveness

Abstract PO-052: Exploring patient derived xenografts based pharmacogenomic data for precision oncology

Abstract Preclinical cancer models play a vital role in oncology research and precision medicine. Patient-derived tumor xenografts (PDXs) are used as reliable preclinical models for studying tumor biology and for testing anti-cancer therapies that are tailored according to genomic characteristics of tumors. Several academic groups, research institutes, and commercial organizations are generating and distributing PDX models. However the distributed nature of PDX model generation and lack of central repository make it challenging to find PDX models with specific characteristics. Furthermore this also hinders meta-analysis (across datasets) of PDX pharmacogenomic data. International consortia and catalogs of PDX models such as PDXNet, EurOPDX and PDXFinder are being developed to standardize PDX associated metadata and facilitate material sharing. Recently we have developed Xenograft Visualization & Analysis (Xeva), an open-source software package in R programming language. Xeva allows PDX growth curve visualization, different response metrics computation and biomarker discovery. Extending to this we have developed XevaDB, a database of PDX drug response and genomic profiles. XevaDB is the first resource to allow concurrent visualization of drug response and associated molecular data such as mutation and copy number alterations. Furthermore XevaDB enables exploration of the tumor growth curve of a PDX model, along with corresponding control. XevaDB contains PDXs from >600 individual patients, spanning across nine different tissue types and >70 drugs. Using XevaDB, we have performed meta-analysis of PDX pharmacogenomic data and have identified 90 pathways significantly associated with response to 53 drugs (FDR < 5%). Our results show that activity of the EGFR signaling pathway is significantly associated with erlotinib response in lung cancer PDXs. We have also found that in PDXs, response to binimetinib is associated with the MAP kinase activation pathway. XevaDB provides a comprehensive resource to search and explore PDX pharmacogenomic data. By combining drug response with genomic data of PDXs, XevaDB allows researchers to quickly find the model of interest and access the data to answer their biological questions. As PDXs based pharmacogenomic datasets continue to expand, XevaDB will facilitate easy access and analysis of this valuable data by the scientific community. Citation Format: Arvind Singh Mer, Benjamin Haibe-Kains. Exploring patient derived xenografts based pharmacogenomic data for precision oncology [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-052.

Classical versus non-classical EGFR mutations: Erlotinib response and impact of renal insufficiency.

Erlotinib is an effective treatment option for EGFR-mutant non-small cell lung cancer. It is important to predict patients who will respond better to erlotinib. We designed this study to investigate the effect of renal insufficiency (RI) on erlotinib treatment outcomes. All patients receiving erlotinib were stratified into 3 groups. Group 1 consisted of non-RI subjects with classical epidermal growth factor receptor (EGFR) mutations, Group 2 consisted of those with RI (Estimated glomerular filtration rate <60 mL/min) and classical EGFR mutations, and Group 3 consisted of those with non-classical EGFR mutations. 82 patients were included in the study. Median progression-free survival (PFS) in patients with classical mutation was approximately 6 months shorter in those with RI, although not statistically significant. Median overall survival (OS) in Group 1, 2 and 3 was 34.1 months, 35.2 months, and 15 months, respectively and although not statistically significant, median OS was 20 months shorter in Group 3. Univariate and multivariate cox-regression analysis revealed shorter PFS and OS in males and those with ECOG ≥2 while PFS and OS were longer in those with recurrent lung tumors and generating rash during erlotinib treatment. There was no difference between RI and non-RI patients in terms of adverse events except for fatigue and appetite loss. This research showed OS in patients with and without RI was comparable. Although not statistically significant, PFS in patients with classical mutation was approximately 6 months shorter in those with RI patients.

Circulating miR-30b and miR-30c predict erlotinib response in EGFR-mutated non-small cell lung cancer patients

ObjectivesMiR-30b, miR-30c, miR-221 and miR-222 are known to induce gefitinib resistance in lung cancer cell lines with activation of mutations in the epidermal growth factor receptor (EGFR). However, the role of these four microRNAs in tyrosine kinase inhibitor (TKI)-resistance in non-small cell lung cancer (NSCLC) patients is unknown. Thus, the aim of this study was to investigate the predictive value of miR-30b, miR-30c, miR-221 and miR-222 in plasma from EGFR-mutated lung cancer patients receiving erlotinib. Materials and methodsThe cohort consisted of 29 EGFR-mutated lung cancer patients receiving erlotinib. Plasma levels of miR-30b, miR-30c, miR-221 and miR-222 were analyzed by qPCR from blood samples collected before treatment start. Plasma concentration of each microRNA was correlated to clinical outcome. ResultsPlasma concentrations of miR-30b and miR-30c could be determined in all 29 patients. Low plasma concentrations of miR-30b and miR-30c showed significant correlation with superior progression-free survival (PFS) (miR-30b: HR = 0.303 [0.123–0.747], p < 0.05; miR-30c: HR = 0.264 [0.103–0.674], p < 0.05). Low plasma concentrations of miR-30c were also significantly correlated with superior overall survival (OS) (HR = 0.30 [0.094–0.954], p < 0.041). ConclusionHigh plasma concentrations of miR-30b and miR-30c predicted shorter PFS and OS. This implies that miR-30b and miR-30c could have clinical potential as biomarkers in EGFR-mutated lung cancer patients.

Abstract 3378: Systematic pharmacogenomic analysis of large patient derived xenografts data

Abstract One of the key challenges in cancer precision medicine is finding robust biomarkers of drug response. Patient-derived tumor xenografts (PDXs) have emerged as reliable preclinical models since they better recapitulate tumor response to chemo- and targeted therapies. However, the lack of standard tools poses a challenge in the analysis of PDXs with molecular and pharmacological profiles. Efficient storage, access and analysis is key to the realization of the full potential of PDX pharmacogenomic data. To address this, we have developed Xeva (XEnograft Visualization &amp; Analysis), an open-source software package for processing, visualization and integrative analysis of a compendium of in vivo pharmacogenomic datasets. The Xeva package follows the PDX minimum information (PDX-MI) standards and can handle both replicate-based and 1x1x1 experimental designs. We used Xeva to characterize the variability of gene expression and pathway activity across passages. We found that only a few genes and pathways have passage specific alterations (median intraclass correlation of 0.53 for genes and positive enrichment score for 92.5% pathways). For example, activity of the mRNA 3'-end processing and elongation arrest and recovery pathways were strongly affected by model passaging (gene set enrichment analysis false discovery rate [FDR] &amp;lt;5%). We then leveraged our platform to link the drug response and the pathways whose activity is consistent across passages by mining the Novartis PDX Encyclopedia (PDXE) data containing 1,075 PDXs spanning 5 tissue types and 62 anticancer drugs. We identified 87 pathways significantly associated with response to 51 drugs (FDR &amp;lt; 5%), including associations such as erlotinib response and signaling by EGFR in cancer pathways and MAP kinase activation in TLR cascade and binimetinib response. Among the significant pathway-drug associations, we found novel biomarkers based on gene expressions, Copy Number Aberrations (CNAs) and mutations predictive of drug response (concordance index &amp;gt; 0.60; FDR &amp;lt; 0.05). Xeva provides a flexible platform for integrative analysis of preclinical in vivo pharmacogenomics data to identify biomarkers predictive of drug response, a major step toward precision oncology. Citation Format: Arvind Singh Mer, Wail Ba-alawi, Petr Smirnov, Yi Xiao Wang, Ben Brew, Ming-Sound Tsao, David Cescon, Anna Goldenberg, Benjamin Haibe-Kains. Systematic pharmacogenomic analysis of large patient derived xenografts data [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 3378.

Abstract 3578: Comprehensive pharmacogenomic analysis establishes lncRNAs as protein-coding independent biomarker of drug response in human cancers

Abstract Somatic alterations in the cancer genome influence a patient’s response to anti-cancer therapeutics. Thus, identifying context-specific alterations associated with differential drug sensitivity can improve patient response prediction and therapeutic selection. The discovery of pervasive dysregulation of long non-coding RNAs (lncRNA) in human cancers and their perceived role in gene regulation has led to the speculation that lncRNAs may also play a role in determining cancer drug response. However, it is unclear whether lncRNAs can augment the existing, predominantly protein coding, repertoire of pharmacogenomic biomarkers of anti-cancer agents. We performed comprehensive analysis of whole genome and transcriptome data from over 1000 cancer cell lines and drug screening data for over 500 anti-cancer agents to systematically analyze over 5 million lncRNA-drug associations. We developed statistical and machine-learning frameworks to study these associations while controlling for the effects of confounding PCGs. Sparse regression and network-based methods demonstrated the predictive ability and biological relevance of literature-supported and novel lncRNAs biomarkers. Using regression analysis of lncRNA expression controlling for neighboring PCGs against drug response, we established that a large proportion of the lncRNAs candidates are PCG-independent biomarkers and potent predictors of cancer drug response. We further identified response-associated somatic alterations specifically in lncRNA genome that do not overlap PCGs. In addition, we demonstrated that collinear lncRNAs might be biologically relevant determinants of drug response. As an example, we identified EGFR-AS1 and MIR205HG as novel predictors of anti-EGFR therapy, which explain a significantly larger proportion of variability in erlotinib and gefitinib response as compared to EGFR mutations and copy-number alterations in drug screens and in patient data. We validated our findings in 16 non-small cell lung cancer and erlotinib-resistant cell lines. Our knockdown experiments revealed mechanisms of erlotinib sensitivity mediated by the two lncRNAs without influencing EGFR expression. In conclusion, our comprehensive analysis generated unprecedented insights into the role of lncRNAs in cancer drug response that reaches beyond PCGs. This study will serve as a foundation for future cancer pharmacogenomic studies and will be an invaluable resource for investigators seeking insights into mechanisms of drug response. Citation Format: Aritro Nath, Eunice Y. Lau, Adam M. Lee, Paul Geeleher, William C. Cho, R. Stephanie Huang. Comprehensive pharmacogenomic analysis establishes lncRNAs as protein-coding independent biomarker of drug response in human cancers [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 3578.

Abstract 2773: A prospective observational study evaluating c-MET expression and EGFR gene mutation correlation with erlotinib response in second line treatment for advanced/metastatic non-small cell lung cancer patients

Abstract Background: The c-MET is associated with tumor progression and acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). The aim of this study was to evaluate the prevalence and predictive role of c-MET and EGFR mutation in non-small cell lung cancer (NSCLC). Methods: This was a multicenter prospective observational study for patients with stage IV or recurrent NSCLC who had progressive disease after 1st line chemotherapy and was followed by erlotinib for 2nd line treatment from September 2011 to September 2014. The primary outcome was to measure the rates of c-MET expression/amplification and EGFR mutation in tumor tissue. MET expression was evaluated by immunohistochemistry (IHC) and gene copy number was assessed by silver in situ hybridization (SISH). EGFR mutations were analyzed by Cobas™ version 1. Results: A total of 196 patients were enrolled and major histologic types were adenocarcinoma (67%). c-MET was overexpressed (MET IHC-positive) in 44.4% of the patients and dominant in female and non-squamous histology. c-MET amplification and high polysomy (MET SISH-positive) were observed in 1.5% and 8.7%, but there was no specific clinical characteristics. The detection rate of EGFR mutation was 10.2% (19 deletion: 11, L858R: 7, exon 20 insertion: 2 cases). The response rate of erlotinib was significantly higher in EGFR sensitive mutant group (55% vs. 4%, p&amp;lt;0.001) and the median progression-free survival (PFS) was significantly longer than wild type (173 vs 58 days, p&amp;lt;0.001). c-MET positivity (IHC- or SISH-positive) did not show significant correlation with response or PFS. Patients with SISH-positive for c-MET tended to have shorter overall survival (OS) than patients with SISH-negative (127 vs 303 days, p=0.051). Conclusion: In this observational study with 2nd line erlotinib treatment, we reconfirmed EGFR mutation is strong predictive marker, and EGFR-TKI use in EGFR wild-type patients should be considered carefully and may be harmful. c-MET overexpression was related to some clinical characteristics such as female and non-squamous histology. Although c-MET positivity was not associated with response or PFS, c-MET SISH may be predictive marker for OS. Citation Format: Cheol-Kyu Park, Yoo-Duk Choi, In-Jae Oh, Jung-Eun Lee, Shin Yup Lee, Tae-Won Jang, Jeong-Seon Ryu, Young-Chul Kim. A prospective observational study evaluating c-MET expression and EGFR gene mutation correlation with erlotinib response in second line treatment for advanced/metastatic non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2773. doi:10.1158/1538-7445.AM2017-2773

Abstract 5712: Potential clinical significance of downregulation of MAPK pathway components mRNA expression in head and neck squamous cell carcinoma (HNSCC)

Abstract We previously showed the MAPK pathway can be mutationally activated, and may implicate drug sensitivity in HNSCC [1]. Here, we report that RNA expression of several MAPK pathway components may be associated with HNSCC patients' outcome based on the US-TCGA Provisional data. We found that loss of DUSP4, a negative regulator of MAPK pathway, was correlated with poor overall survival (OS; P=0.0286) and disease free survival (DFS) in HNSCC (0.00150; median progression time = 38.11 vs. 71.22 months in DUSP4-unaltered patients) which can potentially be explained by activation of oncogenic MAPK pathway upon DUSP4 underexpression. Interestingly, we identified a group of HNSCC patients with homozygous loss and mRNA downregulation of MAPK pathway scaffold protein components (GRB2, SHC2 and SHC3) with significantly poorer DFS (P=0.000871). RPPA analysis showed a trend of decreased protein expression level of phospho-RAFs, downstream of scaffold proteins, decreased MEKs and MAPKs, supportive of an overall decreased MAPK pathway signaling in this subset of patients. Unexpectedly, downregulation (homozygous deletion/ RNA expression less than 2 SD) of multiple MAPK pathway components which normally support activation of the pathway: RAF1 (9%), MAPK1 (3%) and RPS6KA1 (1.9%) was found to be significantly associated with poorer DFS (P=6.643x10^-5) with median time to progression of 18.17 months vs. 71.22 months in the unaltered group. Subsequent proteomic analysis of the respective patient tumors from the TCGA cohort (N=357 with RPPA data) showed that these patient tumors had elevated levels of E2F1 protein expression (P=0.0146), along with increase FOXM1 expression (P=8.499x10^-4), which is known to drive cell cycle progression [2]. As E2F1 is involved in cell survival upon DNA damage [3], it is likely that upregulation of E2F1 protein expression may enable cancer cells to survive after DNA insults by radiotherapy or chemotherapy, and contributes to disease relapse. Acknowledgements: VWYL is funded by the Research Grant Council, Hong Kong (#17114814, General Research Fund), Theme-based Research Scheme (T12-401/13-R), and the Start-up Fund from the School of Biomedical Sciences, Faculty of Medicine, the Chinese University of Hong Kong. Reference: 1. Van Allen, E.M., et al., Genomic Correlate of Exceptional Erlotinib Response in Head and Neck Squamous Cell Carcinoma. JAMA Oncol, 2015. 1(2): p. 238-44. 2. Barger, C.J., et al., Genetic determinants of FOXM1 overexpression in epithelial ovarian cancer and functional contribution to cell cycle progression. Oncotarget, 2015. 6(29): p. 27613-27. 3. Berton, T.R., et al., Regulation of epidermal apoptosis and DNA repair by E2F1 in response to ultraviolet B radiation. Oncogene, 2005. 24(15): p. 2449-60. Citation Format: Hoi Lam Ngan, Vivian W.Y. Lui. Potential clinical significance of downregulation of MAPK pathway components mRNA expression in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5712. doi:10.1158/1538-7445.AM2017-5712

Abstract 3142: Aberrantly expressed microRNAs drive the development of acquired Erlotinib resistance in non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer-related deaths in the world. Non-small cell lung cancer (NSCLC) accounts for ~85% of the cases. NSCLC patients frequently harbor causal gene mutations. Epidermal Growth Factor Receptor (EGFR), an NSCLC causal gene, is mutated in 10-35% of NSCLC patients. Patients with EGFR activating mutation are treated with a tyrosine-kinase inhibitor, Erlotinib, specifically targeting EGFR. However, most patients develop Erlotinib resistance within a year. Although multiple mechanisms are involved in the development of Erlotinib resistance, the role of microRNAs in mediating Erlotinib resistance is largely unexplored. MicroRNAs (miRNAs) are small non-coding RNAs that regulate normal cellular physiology. In cancers, miRNAs are severely dysregulated, contributing to multiple cancer processes, including drug response. Despite the involvement of miRNAs in cancer, their direct role as drivers of drug resistance remain understudied, therefore, there is a critical need to understand the role of miRNAs in inducing Erlotinib resistance. To this end, we propose an unbiased two-prong approach to identify the miRNAs that drive the development of Erlotinib resistance. Therefore, miRNAs are either being (i) overexpressed, or (ii) silenced in Erlotinib sensitive cells with the hypothesis that perturbed miRNA levels will drive Erlotinib resistance. Erlotinib sensitive NSCLC cell lines, EKVX and H322M were identified from the NCI-60 Developmental Therapeutics Project and their Erlotinib dose response curves were established. To perform the miRNA overexpression screen, EKVX and H322M cells stably expressing renilla and firefly luciferase genes were generated, which will be used to monitor cell number and transfection efficiency, respectively. The luciferase-expressing cells will be transfected with a library of 2,019 individually arrayed human miRNAs and cell growth in the presence of Erlotinib will be monitored. The second prong of the study will identify miRNAs, that when lost, confer Erlotinib resistance. 400-fold coverage of small guide RNA (sgRNA) library of the CRISPR-Cas9 system was transduced in Cas9 overexpressing EKVX cells, to knockout ~21,000 human encoded genes (~1800 miRNA genes). Cells are being cultured in the presence of 75% or 90% growth inhibitory concentrations (GI75 or GI90) of Erlotinib so that only cells with sgRNAs against genes critical for Erlotinib response, survive and grow due to acquired resistance. DNA from these cells will be harvested, the sgRNAs sequenced, and compared to the sgRNAs present in Erlotinib-untreated cells. Successful completion of this project will identify miRNAs that drive Erlotinib resistance and may contribute towards development of miRNA therapeutics to enhance Erlotinib sensitivity of NSCLC tumors. Citation Format: Arpita S. Pal, Alejandra Agredo, Andrea L. Kasinski. Aberrantly expressed microRNAs drive the development of acquired Erlotinib resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3142. doi:10.1158/1538-7445.AM2017-3142

A microRNA signature of response to erlotinib is descriptive of TGF\u03b2 behaviour in NSCLC

Our previous work identified a 13-gene miRNA signature predictive of response to the epidermal growth factor receptor (EGFR) inhibitor, erlotinib, in Non-Small Cell Lung Cancer cell lines. Bioinformatic analysis of the signature showed a functional convergence on TGFβ canonical signalling. We hypothesized that TGFβ signalling controls expression of the miRNA genes comprising an erlotinib response signature in NSCLC. Western analysis revealed that TGFβ signalling via Smad2/3/4 occurred differently between erlotinib-resistant A549 and erlotinib- sensitive PC9 cells. We showed that TGFβ induced an interaction between Smad4 and putative Smad Binding Elements in PC9. However, qRT-PCR analysis showed that endogenous miR-140/141/200c expression changes resulted from time in treatments, not the treatments themselves. Moreover, flow cytometry indicated that cells exited the cell cycle in the same manner. Taken together these data indicated that the miRNA comprising the signature are likely regulated by the cell cycle rather than by TGFβ. Importantly, this work revealed that TGFβ did not induce EMT in PC9 cells, but rather TGFβ-inhibition induced an EMT-intermediate. These data also show that growth/proliferation signals by constitutively-activated EGFR may rely on TGFβ and a possible relationship between TGFβ and EGFR signalling may prevent EMT progression in this context rather than promote it.

Early Change in FDG-PET Signal and Plasma Cell-Free DNA Level Predicts Erlotinib Response in EGFR Wild-Type NSCLC Patients

INTRODUCTION: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are a treatment option in the second- or third-line palliative setting in EGFR wild-type (wt) non–small cell lung cancer (NSCLC) patients. However, response rates are low, and only approximately 25% will achieve disease control. Early prediction of treatment resistance could accelerate discontinuation of ineffective treatment and reduce unnecessary toxicity. In this study, we evaluated early changes on 18F-fluoro-D-glucose (F-18-FDG) positron emission tomography/computed tomography (PET/CT) and in total plasma cell-free DNA (cfDNA) as markers of erlotinib response in EGFR-wt patients. METHODS: F-18-FDG-PET/CT scans and blood samples were obtained prior to erlotinib initiation and were repeated after 1 week (PET/CT) and 1 to 4 weeks (blood sample) of treatment. Level of cfDNA was measured by droplet digital polymerase chain reaction. Percentage change (%∆) in SULpeak and total lesion glycolysis (TLG) on FDG-PET/CT and in plasma cfDNA was correlated to radiological response, progression-free survival (PFS), and overall survival (OS). RESULTS: Fifty patients were prospectively enrolled. A significant correlation was found between CT response and %∆TLG (P=.003). All patients with early metabolic progression showed radiological progression. Increased %∆TLG and %∆cfDNA were significantly correlated with shorter PFS (P=.002 and P=.004, respectively) and OS (P=.009 and P=.009, respectively). Multivariate analysis indicated %∆cfDNA to be the strongest predictor of OS. CONCLUSION: Early increase in TLG on F-18-FDG-PET/CT correlates with radiological progression, and shorter PFS and OS. Early increase in cfDNA predicts shorter PFS and OS. Both assessments are promising tools for early detection of nonresponders and reduced OS in TKI-treated EGFR-wt NSCLC patients.

MiR-17-5p down-regulation contributes to erlotinib resistance in non-small cell lung cancer cells

Non-small cell lung cancer (NSCLC) is the major cause of lung cancer-related deaths. Erlotinib is an effective drug for NSCLC patients, but its effect in advanced NSCLC is compromised because of the drug resistance. The mechanism of erlotinib resistance in NSCLC is largely unknown. In the current study, we found that erlotinib treatment down-regulated miR-17-5p level in A549 cells and miR-17-5p was lower in acquired erlotinib-resistant A549 cells (A549-ER) compared with erlotinib-sensitive A549 cells. Consistently, miR-17-5p was down-regulated in the tumor tissues and the plasma of erlotinib-resistant NSCLC patients in comparison to those who are sensitive to erlotinib. Moreover, miR-17-5p mimic increased the sensitivity of A549 and A549-ER to erlotinib. In contrast, miR-17-5p inhibitor decreased the cell death of A549 and A549-ER induced by erlotinib. In addition, we also demonstrated that miR-17-5p could inhibit the mRNA and protein levels of enhancer of zeste homolog (EZH) 1, a member of the EZH family that contributes to drug resistance in several types of cancer. The luciferase assay of 3′-untranslated region (UTR) demonstrates that EZH1 is a direct target of miR-17-5p and EZH1 RNAi recapitulates the effect of miR-17-5p overexpression on erlotinib resistance. Further, mutation in seed sequence of miR-17-5p could totally abolish the sensitization of A549-ER to erlotinib induced by miR-17-5p overexpression. Our results indicate that miR-17-5p down-regulation contributes to erlotinib resistance of NSCLC by modulating its target genes such as EZH1 and plasma miR-17-5p might be a potential biomarker of erlotinib response in NSCLC patients.

Abstract 4674: Vitamin D enhances erlotinib response in EGFR-mutant non-small cell lung cancer

Abstract Background. Despite remarkable initial therapeutic responses, most patients develop resistance to the first generation of EGFR tyrosine kinase inhibitors (TKIs), erlotinib and gefitinib, within one year of treatment initiation. Epithelial-mesenchymal transition (EMT) is one of the key mechanisms of resistance to EGFR TKIs. We recently demonstrated that vitamin D promotes epithelial phenotype in NSCLC cells. We hypothesized that we could prevent/overcome EMT-driven resistance by combining vitamin D with EGFR TKIs. Methods. EGFR-mutant, erlotinib sensitive HCC827 cells were treated with: vehicle control, 100nM 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 1ng/ml TGF-β, or 1,25(OH)2D3+TGF-β for 14 days. Expression of EMT markers was determined by q-RT PCR and immunoblot. Sensitivity to erlotinib was analyzed by MTT assay. For the in-vivo study, HCC 827 tumor xenografts were established in vitamin-D deficient mice. Mice were stratified based on tumor volume to receive diets containing 25 IU vitamin D3/kg (deficient) or 10,000 IU vitamin D3/kg (supplemented). Erlotinib treatment (12.5 mg/kg) was initiated two weeks after the diet switch and administered 5 days per week for the duration of the study. Tumors were collected two weeks after the diet switch (baseline), 48 hours after the erlotinib initiation (acute response) and upon treatment failure (outgrowth). Results. Vitamin D attenuated expression of mesenchymal markers in the presence of TGF-β in-vitro and opposed TGF-β driven resistance to erlotinib. Erlotinib EC50 values were 9.85 nM, 122 nM and 3.83 nM for vehicle, TGF-β, and 1,25(OH)2D3+TGF-β treated cells, respectively. In-vivo, vitamin D supplementation enhanced acute response to erlotinib. We documented a greater decrease in tumor volume after 48h of erlotinib initiation (1.1% of baseline ±19.3% in vitamin D deficient diet and 25.9% ±29.5% in supplemented diet). Greater efficacy was accompanied by increased suppression of STAT3 phosphorylation and decreased vimentin expression in the vitamin D supplemented group. Although we observed a trend towards a longer progression-free survival in animals maintained on vitamin D supplemented diet (8 vs 5.5 weeks), statistical significance was not achieved. The outgrowth tumors in both groups maintained their epithelial phenotype but exhibited a significant increase in the expression of AXL tyrosine kinase. However, outgrowth tumors from the vitamin D supplemented group had lower AXL expression than those from the vitamin D deficient group. Conclusion. Vitamin D supports epithelial phenotype in-vitro and enhances acute response to erlotinib in-vivo, possibly, by suppressing STAT3 signaling. Vitamin D supplementation also attenuates overexpression of AXL tyrosine kinase in erlotinib resistant tumors. The clinical implication of our work is that patients diagnosed with EGFR mutant NSCLC who receive erlotinib may benefit from vitamin D supplementation. Citation Format: Tatiana Shaurova, Suzanne F. Shoemaker, Pamela A. Hershberger. Vitamin D enhances erlotinib response in EGFR-mutant non-small cell lung cancer. [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 4674.

Erlotinib Response in a Non-Small Cell Lung Cancer Patient with EGFR Exon 20 Mutation.

Erlotinib Response in a Non-Small Cell Lung Cancer Patient with EGFR Exon 20 Mutation.

Tyr1068-phosphorylated epidermal growth factor receptor (EGFR) predicts cancer stem cell targeting by erlotinib in preclinical models of wild-type EGFR lung cancer.

Tyrosine kinase inhibitors (TKIs) have shown strong activity against non-small-cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations. However, a fraction of EGFR wild-type (WT) patients may have an improvement in terms of response rate and progression-free survival when treated with erlotinib, suggesting that factors other than EGFR mutation may lead to TKI sensitivity. However, at present, no sufficiently robust clinical or biological parameters have been defined to identify WT-EGFR patients with greater chances of response. Therapeutics validation has necessarily to focus on lung cancer stem cells (LCSCs) as they are more difficult to eradicate and represent the tumor-maintaining cell population. Here, we investigated erlotinib response of lung CSCs with WT-EGFR and identified EGFR phosphorylation at tyrosine1068 (EGFRtyr1068) as a powerful biomarker associated with erlotinib sensitivity both in vitro and in preclinical CSC-generated xenografts. In contrast to the preferential cytotoxicity of chemotherapy against the more differentiated cells, in EGFRtyr1068 cells, erlotinib was even more active against the LCSCs compared with their differentiated counterpart, acquiring potential value as CSC-directed therapeutics in the context of WT-EGFR lung cancer. Although tumor growth was inhibited to a similar extent during erlotinib or chemotherapy administration to responsive tumors, erlotinib proved superior to chemotherapy in terms of higher tolerability and reduced tumor aggressiveness after treatment suspension, substantiating the possibility of preferential LCSC targeting, both in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) tumors. We conclude that EGFRtyr1068 may represent a potential candidate biomarker predicting erlotinib response at CSC-level in EGFR-WT lung cancer patients. Finally, besides its invariable association with erlotinib sensitivity in EGFR-WT lung CSCs, EGFRtyr1068 was associated with EGFR-sensitizing mutations in cell lines and patient tumors, with relevant diagnostic, clinical and therapeutic implications.

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

A microRNA signature of response to erlotinib is impacted by the EMT‐ inducing cytokine TGFβ1

PurposeWe endeavored to understand the contribution of canonical TGFβ signaling on expression of a 13‐gene microRNA (miR) signature of erlotinib response.BackgroundWe identified a 13‐gene miRNA signature predictive of response to the epidermal growth factor receptor (EGFR) inhibitor, erlotinib, in Non‐Small Cell Lung Cancer (NSCLC) cell lines. Bioinformatic analysis of the signature showed a functional convergence on TGFβ canonical signaling. We hypothesize that TGFβ signaling controls expression of the miR genes comprising an erlotinib response signature in NSCLC.MethodsTwo signature miR genes (miR‐140‐3p and miR‐141) were chosen for study. Promoters regions of both genes harbor putative Smad Binding Elements (SBEs), DNA binding domains for TGFβ‐responsive Smad transcription factors. TGFβ signaling impact on miR‐140 and ‐141 expression was assessed in erlotinib‐resistant (A549) and –sensitive (PC9) NSCLC cells by quantitative real‐time PCR (qRT‐PCR), luciferase reporter assays, and chromatin immunoprecipitation (ChIP).Results(1) qRT‐PCR showed endogenous miR 140/141 expression decreased after short term TGFβ1 treatment in both cell lines. After 7 days of treatment, the trend continued. (2) The promoter region of miR‐141, containing 6 SBEs, was cloned into a luciferase reporter vector. The miR‐141 luciferase construct is responsive to TGFβ stimulation like that seen in qRT‐PCR. (3) ChIP experiments will assess specific Smad complexes binding SBE sites.ConclusionWe expected that canonical TGFβ signaling molecules, Smads, bind to and control miR genes with SBEs in A549 cells. In PC9 cells, some miR genes likely control Smad protein expression while a non‐canonical TGFβ response may regulate signature miR expressionCollege funds were used.

Predicting erlotinib resistance in EGFR wild type NSCLC patients.

e22065 Background: Cancer patients with wild type EGFR respond to treatment with erlotinib at a lower rate than patients with EGFR mutations. It would be relevant to predict which EGFR wild type patients benefit from erlotinib. We have developed a response predictor based on NCI60 cancer cell lines measurements of erlotinib effect and gene expression measurements. Clinical relevance was improved by filtering the model through expression profiles from more than 3,000 clinical samples.The erlotinib response predictor consists of 94 sensitivity genes and 10 resistance genes and is completely defined before independent validation on patients treated with erlotinib. Methods: Gene expression data from pre-treatment core biopsies from 25 patients with refractory NSCLC (clinicaltrials.gov: NCT00409968) later treated with erlotinib (The BATTLE trial; Kim, ES et al. Cancer Discov. 2011; 1:44-53) were downloaded from Gene Expression Omnibus with accession number GSE33072. PFS after erlotinib treatment was recorded. The erlotinib response predictor was applied retrospectively to the gene expression measurements.Patients with a predicted sensitivity above the 0.15 quantile were categorized as sensitive, patients with a predicted sensitivity below were categorized as resistant.The median was also tested as a cutoff, and correction for multiple testing was taken into consideration. Results: In a Kaplan-Meier analysis of PFS comparing predicted sensitive and predicted resistant patients, the predicted sensitive patients survived longer (PFS 2.3 months (1.9-2.7)) than predicted resistant patients (PFS 1.2 months (0.5-1.8)). A log-rank test for PFS found the difference significant (p=0.005). Hazard ratio 4.6 (1.4-15.2). Conclusions: The small clinical data set validated the cell line derived erlotinib response predictor. It identified a subset of 16% of patients with no apparent benefit from erlotinib. We have performed similar validations for 24 other response predictors in 24 clinical trials. In 20 out of the 24 clinical trials, the agreement between prediction and clinical outcome was statistically significant. Thus, our cell line based method represents a general method for predicting clinical response to cytotoxic or cytostatic agents.