According to the company statement, this is based on recent internal and external data, including durvalumab’s strong efficacy in monotherapy presented at recent medical meetings, as well as significant opportunities in the competitive landscape. KRAS mutation should therefore not be an absolute exclusion criterion for EGFR-TKI therapy, in case of ligand-independent activation of EGFR. Data shown are the mean + SD from three independent experiments. Given the fact that cisplatin is the usual first-line chemotherapy for wild-type EGFR NSCLC, we aimed at investigating the consequences of a sublethal cisplatin treatment upon the surviving cells sensitivity to erlotinib, either in wild-type or mutated-EGFR cell lines. A549 cells express G12S-KRAS and H358 cells express G12C-KRAS. The indicated treatment was applied, then cells were fixed by 4% paraformaldehyde. In sum, these data suggest that preexistent heterogeneity of EGFR amplification may account for the patient's mixed response to afatinib therapy, with drug resistance arising through selection for a preexistent EGFR wild-type, ERBB2-amplified clone with additional coalterations of PTEN and CCND3. We have previously shown that cisplatin induced no sensitization to erlotinib in the EGFR-mutated H1975 cell line (Supplementary Fig. This increase in erlotinib efficiency is reproduced in vivo in PDX models, which is reported for the first time to our knowledge. To sign up for ESMO newsletters, create a myESMO account here and select the newsletters you’d like to receive. 2A. J. Raimbourg was supported by the French Fondation pour la Recherche Médicale and by Roche Laboratories, M. Cabart was sponsored by the Association pour la Recherche sur le Cancer. All treatments are detailed in the Results section. In this study, while comparing the … This will be the subject of further investigation. 4A). Durvalumab received US Food and Drug Administration (FDA) Breakthrough Therapy Designation in patients with PD-L1 positive inoperable or metastatic urothelial carcinoma in 2016 and Fast Track Designation in 2015 for the treatment of patients with PD-L1 positive metastatic head and neck squamous cell carcinoma. TGFα secretion was induced in two cell lines except from the A549 cells (Fig. The experimental protocol and animal housing were in accordance with institutional guidelines as put forth by the French Ethical Committee (Agreement C75-05-18, France). Indeed, the patient with EGFR wild-type amplification exhibited primary resistance to osimertinib. In this modified cells, EGFR tyrosine kinase function is required to cell survival, because erlotinib then induces cell death. Tumor cells drastically evolve from the initial biopsy-based analyses to the therapy-modified phenotype at the time of maintenance, underlining the need for phenotypic reevaluation after the first-line therapy to optimize the therapeutic strategy (13). In animal studies, only the combined treatment of PC9 EV and gefitinib delayed the tumor growth of CL1-5 cells. The PEARL trial is a randomised, open-label, multi-centre phase III trial of durvalumab monotherapy versus standard of care chemotherapy in first-line treatment of patients with EGFR and ALK wild-type advanced or metastatic PD-L1-expressing NSCLC. A group of control, nontreated mice was sacrificed at day 4. mRNA were extracted from the tumors and analyzed for the expression of IL6, IFIT1, and IFI27. Receive information and updates on ESMO’s scientific and educational resources, events, members activities. After a few weeks, cells began to proliferate and could be passaged and subcultured in the presence of cisplatin. In this study, we investigated the relative expression and predictive value of all human epidermal growth factor receptor (HER) family members in 144 cetuximab-treated patients with wild type RAS mCRC. Her2/neu is amplified in about 25% of breast cancers, … 1C; Supplementary Fig. S6A). Smith and colleagues (20) established a correlation between EGFR–GRB2 complexes detection and the response to EGFR inhibitors. EGFR2 (Her2/neu, ErbB2) is a distinct EGFR expressed in multiple tumor types, most notably breast cancer. The durvalumab biological license application in second-line urothelial carcinoma has been accepted by the FDA with a PDUFA date in the second quarter of 2017. S2C). For all pairwise comparisons based on the proportions of tumors with a particular RTV, a two-tailed Fisher exact t test was used. 3E and Supplementary Figure S3B, the blocking EGFR antibody induced an obvious cell death in wtEGFR cells and cisplatin pretreatment abrogated the cells sensitivity to EGFR antibody. A more recent study reevaluated the results of the SATURN and BR.21 trials with a focus on wtEGFR patients and confirmed the benefit of erlotinib after initial platinum doublet therapy in these patients (30). The trial was initiated to determine the efficacy and safety of durvalumab in Asian countries, some of which have the highest current NSCLC burden, with over 1.1 million new cases projected for China alone in 2030. A, Conditioned medium (CM) was prepared from A549 cells treated by cisplatin 3 μmol/L 48 hours or not, or from A549cis3 cells. Approximately 90% of EGFR mutations are either exon 19 deletions or exon 21 L858R point mutations.2, 29 Although other rare EGFR mutations have been described, few have been clearly associated with sensitivity to TKI therapy. Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers; of these lung cancers, approximately 90% are EGFR wild-type/untested. EGFR is the receptor for EGF and belongs to the tyrosine kinase receptors (RTK). Given the previously reported activation of EGFR pathway induced by cisplatin , we measured EGFR phosphorylation following a nonlethal cisplatin treatment in wild-type-EGFR cells and observed that the phosphorylation raised during the 48-hour-cisplatin treatment, together with the subsequent activation of EGFR downstream kinases ERK and AKT to a lesser extent . EGFR–GRB2 dimers were detected by PLA. The graph shows the mean + SD from three independent experiments. Section 1734 solely to indicate this fact. Overexpression of EGFR is frequently observed in hepatocellular carcinoma (HCC) and EGFR activation has been proven to be a potential determinant of primary resistance of HCC cells to sorafenib. This point, namely the utmost importance of the kinetics of the therapeutic schedule, underlines the major difference between the epigenetic, inheritable mechanism of EGFR-TKI tolerance described by Sharma and colleagues (11) and the mechanism of cisplatin resistance observed in our model, the former rendering the cells resistant to cisplatin and the latter rendering the cells sensitive to EGFR-TKI by the activation of a specific pathway. Wild-type (wt) EGFR is amplified in 40–50 % of primary GBMs and a fraction of EGFR-amplified tumors in addition express the mutant variant EGFRvIII, a constitutively active receptor . We first demonstrated that the EVs from EGFR-mutant changes the wild-type cells’ sensitivity to gefitinib by adding EV directly or coculturing EGFR wild-type (CL1-5) cells and EGFR-mutant (PC9) cells. The first-line chemotherapy induces drastic modifications of cancer cells, at the non-genetic (11) or genetic levels (12). We propose a combination of markers that enable to discriminate between the tumors sensitized to erlotinib or not in PDX models, which should be worth testing in patients. The top graph shows the mean fluorescence (+SD) of the overall population and the bottom graph indicates the percentage (+SD) of To-Pro3 positive cells. The PEARL trial focuses on Asian countries, primarily China, due to the high prevalence of NSCLC in the region. The observation that erlotinib combination with concurrent cisplatin showed no clinical benefit (9) also corroborates our in vitro data and the requirement of the sequential treatment. We sequentially treated the cells with a sublethal amount of cisplatin for 48 hours, then with various erlotinib amounts for 72 hours, according to their respective IC50 (Supplementary Fig. This suggestion is supported by the observation made in metastatic colon cancer: whereas anti-EGFR antibodies proved to be effective in colorectal cancer excluding the KRAS-mutated tumors, the GERCOR DREAM study shows the benefit of erlotinib in maintenance therapy in KRAS-mutated colon cancer in combination with angiogenesis inhibition after induction chemotherapy (47). No significant cell death was induced by erlotinib in cisplatin-pretreated H1975 cells, contrary to A549 cells (Fig. The graph shown is representative of three independent experiments. 2C). First, the sample size was relatively small. The benefit of EGFR–TKI in non–small cell lung cancer has been demonstrated in mutant EGFR tumors as first-line treatment but the benefit in wild-type EGFR tumors is marginal as well as restricted to maintenance therapy in pretreated patients. 7 With the Qiagen EGFR kits, up to 29 somatic mutations of EGFR could be identified according to the manufacturer’s protocols. The labeling with antibodies was realized according to the proximity ligation assay (PLA) manufacturer instructions (Duolink, Sigma-Aldrich). Plasma transforming growth factor alpha and amphiregulin protein levels in NCIC Clinical Trials Group BR.21, Increases of amphiregulin and transforming growth factor-alpha in serum as predictors of poor response to gefitinib among patients with advanced non-small cell lung cancers, Independent transcriptional reprogramming and apoptosis induction by cisplatin, Dual-agent molecular targeting of the epidermal growth factor receptor (EGFR): combining anti-EGFR antibody with tyrosine kinase inhibitor, Combined epidermal growth factor receptor targeting with the tyrosine kinase inhibitor gefitinib (ZD1839) and the monoclonal antibody cetuximab (IMC-C225): superiority over single-agent receptor targeting, Dual targeting of EGFR can overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer, Dual inhibition of EGFR with afatinib and cetuximab in kinase inhibitor-resistant EGFR-mutant lung cancer with and without T790M mutations, Dual targeting of the epidermal growth factor receptor using the combination of cetuximab and erlotinib: preclinical evaluation and results of the phase II DUX study in chemotherapy-refractory, advanced colorectal cancer, Prospective molecular marker analyses of EGFR and KRAS from a randomized, placebo-controlled study of erlotinib maintenance therapy in advanced non-small-cell lung cancer, A gene expression signature associated with "K-Ras addiction" reveals regulators of EMT and tumor cell survival, Effect of KRAS oncogene substitutions on protein behavior: implications for signaling and clinical outcome, Bevacizumab with or without erlotinib as maintenance therapy in patients with metastatic colorectal cancer (GERCOR DREAM; OPTIMOX3): a randomised, open-label, phase 3 trial, IGF2 Overexpression Predicts IGF1R/INSR Inhibitor Response, Cancer Epidemiology, Biomarkers & Prevention, Disclosure of Potential Conflicts of Interest. Data were normalized by three housekeeping genes (RPLP0, actin, and GAPDH) and expressed as fold change versus control conditions (nontreated cells or control mice). We objectified the resistance to cisplatin of these cells, named A549cis3, by measuring their IC50 to cisplatin (Fig. EGFR is classically activated through the binding of its ligands and consequent dimerization responsible for its autophosphorylation. **, P < 0.01, Student t test. The aim of our study was to increase understanding of the modifications induced in NSLCL cells by a cisplatin sublethal treatment towards EGFR pathway. Studies have been performed in compliance with protocol and animal housing in accordance with national regulation and international guidelines and under the supervision of authorized investigators. As a result, the company anticipates MYSTIC PFS data in mid-2017 and final OS data at the latest in 2018. The company has also initiated the new phase III PEARL trial of durvalumab monotherapy versus standard of care chemotherapy in first-line NSCLC patients whose tumours express PD-L1. All experiments were repeated three times independently unless otherwise stated and analyzed by Student t test. Total RNA was isolated from cell pellets or from frozen PDX pieces using the RNeasy MiniKit (Qiagen) following the manufacturer's instructions with DNase I treatment. B, A549 and H1975 cells were treated as in A (respectively by erlotinib 30 and 10 μmol/L), without (top graph) or with PP1 (bottom graph) added during cisplatin treatment. We provide here evidence of a ligand-independent, Src-mediated activation of EGFR induced by cisplatin treatment in NSCLC cells expressing wild-type EGFR. Educate. A eGFR below 60 for three months or more or a eGFR above 60 with kidney damage (marked by high levels of albumin in your urine) indicates chronic kidney disease. The use of PDXs for preclinical studies was shown to better reflect the phenotype and heterogeneity of human lung cancer tumors than cell lines xenografts (23–25). For more detailed information on the cookies we use, please check our Privacy Policy. S2A and S2B). These A549cis3 cells revealed more sensitive to erlotinib than their parent cells (Fig. As cisplatin-treated A549 CM could sensitize A549 cells to erlotinib, we treated naive A549 cells with recombinant amphiregulin, PGE2, or the combination of both, at the concentrations measured in the conditioned media, but the sensitivity of the treated cells to erlotinib was not modified (Supplementary Fig. This could support the fact that some wtEGFR patients may benefit from EGFR-TKI as maintenance therapy when given immediately following to platinum-based therapy. This further substantiates the different responses of wtEGFR and mutEGFR cells to the sequential treatment cisplatin-erlotinib (Fig. C, A principal component analysis was performed with the mRNA as quantitative variables and with response and time categories as qualitative variables. Data shown are the mean + SD from five independent experiments. It is thus of high importance to consider that EGFR-TKI and EGFR blocking antibody are not redundant and substitutable therapeutic strategies. The website cannot function properly without these cookies, and can only be disabled by changing your browser preferences. Enter multiple addresses on separate lines or separate them with commas. In addition and contrary to what happened in wtEGFR cells, Src inhibition during the initial cisplatin treatment revealed a significant cell death induced by erlotinib in H1975 cells (Fig. ELISA kits were purchased from R&D Systems and used according to the manufacturer's instructions. Cells were plated in 100-mm dishes and treated by cisplatin (3 μmol/L, 48 hours). Cells were fixed and incubated with EGFR and pTyr100 antibodies. Because a false-negative result might prevent a patient from receiving appropriate targeted therapies, it is desirable to recheck equivocal results of EGFR genotyping. Additionally, the ongoing phase III NEPTUNE trial will be expanded with local patients to support regulatory submission of durvalumab plus tremelimumab combination therapy in China for first-line NSCLC patients without delaying the anticipated OS data readout in 2018 from the global cohort, which is approaching full recruitment. Cells were plated in 6-well plates and treated by the indicated drugs. 2A). Il metodo utilizzato per determinare la Data shown are the mean + SD from three independent experiments. Gefitinib resistant cells did not harbor any obvious genetic mutations that drive resistance (e.g. MYSTIC also includes several undisclosed interim analyses for OS. Similar to what was demonstrated in glioblastoma (26), we show here that cisplatin pretreatment in NSCLC simultaneously renders the cells more sensitive to EGFR-TKI and more resistant to EGFR blocking antibody. C, A549 cells were treated by cisplatin 3 μmol/L 48 hours then by erlotinib 15 or 30 μmol/L for 72 hours. An in vitro study demonstrated, however, that EGFR-TKI tolerance results from reversible, epigenetic cell phenotype modifications (11). S7, at day 8, only tumors from the PDX D group can be found in the Treatment-NT confidence ellipse, whereas responding tumors significantly diverge from this ellipse at day 8. Similar to what happens in glioblastoma, this pathway competes with ligand-induced EGFR signaling (4, 26), involving plasma membrane EGFR. Targeted therapy in non-small-cell lung cancer–is it becoming a reality? We therefore added neutralizing IL6 antibody in the culture medium of A549 cells treated by the sequential treatment cisplatin/erlotinib. Major differences between NSCLC cells expressing wild-type and mutant EGFR. After RNA quantification by spectrophotometry (BioSpectrometer), 1 μg RNA was reverse-transcribed with Quantiscript Reverse Transcriptase (Qiagen) for cDNA synthesis. Testing and Targeting Tumours with RET Alterations, Immune Checkpoint Inhibitors in HNSCC: Changing the Treatment Landscape, Translating Immunity to Clinical Applications of Immunotherapy in Breast Cancer, Past ESMO Partnership, Labelled and Supported Meetings, ESMO Clinical Research Observatory Task Force (ECRO), Acknowledgements: Rationalizing Bureaucracy, Bibliography on Clinical Trial Procedures, ESMO Designated Centres of Integrated Oncology & Palliative Care, Palliative and Supportive Care Sessions at ESMO Asia Virtual Congress 2020, Europe’s Beating Cancer Plan – in 60 minutes. We used a set of four PDXs (LCF-04, LCF-09, IC14-LC16, and IC8-LC10), obtained from human NSCLC pieces xenografted to Swiss Nude mice. In the SATURN cohort of patients, KRAS mutation was a significant prognostic factor for poor response to erlotinib (44). Cell death was measured by ToPro3 staining and detected by flow cytometry as in Fig. Figure 6B shows the cumulative expression of the three markers for each PDX in the four groups of mice. *, P < 0.05; **, P < 0.01, Student t test. 32 mCRC patients wild-type for KRAS and BRAF as detected by DS P RIMA Y TUMOUR METASTASIS DS ME-PCR RESULTS: KRAS analysis by ME-PCR in primary tumor and metastasis 1 PT colo n WT G12D M lung G12D G12D M brain G12D G12D 2 PT colo n WT G12V M LN G12V G12V WT MUT WT MUT Anti-EGFR therapy Tumor progression 2. Tumor volume was measured twice a week and normalized to the initial volume. 3C, left). We wondered if … 1C. Data shown are the mean + SD from three independent experiments. The graph shown is representative of three independent experiments. EGFR wild-type 170, including ALK rearrangements 12 (12/170, 7.06%), EGFR gene mutations coexist with EML4-ALK positive expression 1, the application of spearman analysis, there was a negative correlation between the two group (R= −0.153, P=0.008), the difference was statistically significant. EGFR Mutation is present in 5.48% of AACR GENIE cases, with lung adenocarcinoma, conventional glioblastoma multiforme, glioblastoma, colon adenocarcinoma, and non-small cell lung carcinoma having the greatest prevalence [ 4 ]. Surprisingly, depending on the model considered, EGFR activation either promoted cell survival and tumor growth (14) or sometimes cell death (27). **, P < 0.01, Student t test. ): J. Raimbourg, M.-P. Joalland, M. Cabart, L. de Plater, D. Decaudin, L. Lalier, Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Raimbourg, M.-P. Joalland, M. Cabart, L. de Plater, F. Bouquet, A. Savina, D. Decaudin, J. Bennouna, L. Lalier, Writing, review, and/or revision of the manuscript: J. Raimbourg, M.-P. Joalland, F. Bouquet, A. Savina, D. Decaudin, J. Bennouna, F.M. Chakraborty and colleagues (26) have demonstrated in glioblastoma that ligand-induced and ligand-independent activation of EGFR are distinct and even compete with each other. 4C; Supplementary Fig. Cetuximab (anti-EGFR) può essere impiegato in pazienti EGFR + KRAS wild-type, indipendentemente dalla linea di trattamento, sia in associazione a regimi di irinotecan e/o fluoropirimidine e/o oxaliplatino, sia in monoterapia nei pazienti pretrattati con intolleranza a irinotecan. Combining both EGFR targeting strategies may thus enhance tumor response in some models. Tumors were classified as adenocarcinomas and expressed wild-type EGFR, KRAS, BRAF, and PI3K. This work aimed at questioning the effects of cisplatin initial treatment on the EGFR pathway in non–small cell lung cancer and the functional consequences in vitro and in in vivo animal models of patient-derived xenografts (PDX). Third-generation EGFR tyrosine kinase inhibitors (TKI) are mutation-selective inhibitors with minimal effects on wild-type EGFR. PCA analysis (Pearson's correlation) was performed from the data described in the Supplementary Table S1 using XLSTAT v2016.03 software (Addinsoft 2010). The estimated primary completion date has been updated to reflect both an increase in patient recruitment (as reported in February 2016 with the inclusion of OS as a co-primary endpoint) and the event-based nature of the trial. Data were analyzed by the ImageJ 1.46r software and spots were quantified by the Object 3D Counter plugin. **, P < 0.01, Student t test. B, The same mRNA measurements were made in PDX A, B, and D at day 4 (A4.1 to 3, B4.1 to 3, D4.1 to 3), 8 (A8.1 to 3, B8.1 to 3, D8.1 to 3), and 18 (A18.1 to 3, B18.1 to 3, D18.1 to 3) post-cisplatin injection and expressed as cumulative fold change versus nontreated PDX (A0.1 to 3, B0.1 to 3, D0.1 to 3). We observed that Src inhibition counteracted the phosphorylation of EGFR induced by cisplatin (Fig. The “control” group received no treatment; the “cisplatin” group received an intraperitoneal injection of cisplatin at day 1 (6 mg/kg); the “erlotinib” group was orally treated by erlotinib (50 mg/kg) 5 days out of 7 from day 7 to the mice sacrifice; the “cisplatin/erlotinib” group was treated by both drugs as described above. MINIMAL Requirements: Google Chrome 24+, Mozilla Firefox 20+, Internet Explorer 11, Opera 15–18, Apple Safari 7, SeaMonkey 2.15-2.23. This site uses cookies. 1B). Copyright © 2021 by the American Association for Cancer Research. These phenotypic modifications are sometimes reported when epithelial-to-mesenchymal transition (EMT) occurs. 1D; Supplementary Fig. We therefore repeated the same PCA with the measurement made from the PDXs at day-8 and the analysis efficiently discriminated between responding and nonresponding PDXs (Supplementary Fig. As described in Supplementary Fig. We next measured EGFR phosphorylation in wtEGFR cells either treated by cisplatin or by the combination cisplatin+PP1. Mol Cancer Ther; 16(8); 1634–44. The MYSTIC trial will now assess PFS and overall survival (OS) endpoints in patients with PDL1-expressing tumours for both durvalumab monotherapy and the combination of durvalumab plus tremelimumab, as well as in ‘all comers’ for the combination of durvalumab plus tremelimumab, versus standard of care chemotherapy. "Wildtype" means the gene is not mutated, so the cancer may not respond to certain targeted drugs as … At the indicated time points, cells were harvested by trypsination and dead/dying cells were detected by To-Pro3 internalization measured by flow cell analysis (BD Accuri C6 cytometer). Four to 6-week-old nude mice, bred at Institut Curie, were used. 1A). ESMO Call to Action on COVID-19 Vaccinations and Patients with Cancer: Vaccinate. We wondered if the activation of EGFR pathway observed was associated to a change in the cells sensitivity to EGFR tyrosine kinase inhibition. B, Naïve A549 cells were incubated overnight in the CM from A549 cells treated by cisplatin 3 μmol/L 48 hours or not. Briefly, cells were plated in 96-well plates (5,000 cells/well) and treated by the indicated drugs or conditioned media. 4B, bottom graph). Mice were sacrificed at day 4, 8, and 18 and tumors were excised and frozen. An EGFR mutation does not refer to a single gene abnormality. EGFR-mutated lung cancers account for a significant subgroup of non–small cell lung cancers overall. Signalling through the epidermal growth factor receptor (EGFR) pathway has been identified as being highly increased in a variety of tumours and particularly in lung cancers and both monoclonal antibodies and small molecule inhibitors, initially selected on their potent inhibition of wild type EGFR, are now available.