EGFR amplification and outcome in a randomised phase III trial of chemotherapy alone or chemotherapy plus panitumumab for advanced gastro-oesophageal cancers

Abstract

Objective: Epidermal growth factor receptor (EGFR) inhibition may be effective in biomarker-selected populations of advanced gastro-oesophageal adenocarcinoma (aGEA) patients. Here, we tested the association between outcome and EGFR copy number (CN) in pretreatment tissue and plasma cell-free DNA (cfDNA) of patients enrolled in a randomised first-line phase III clinical trial of chemotherapy or chemotherapy plus the anti-EGFR monoclonal antibody panitumumab in aGEA (NCT00824785).

Design: EGFR CN by either fluorescence in situ hybridisation (n=114) or digital-droplet PCR in tissues (n=250) and plasma cfDNAs (n=354) was available for 474 (86%) patients in the intention-to-treat (ITT) population. Tissue and plasma low-pass whole-genome sequencing was used to screen for coamplifications in receptor tyrosine kinases. Interaction between chemotherapy and EGFR inhibitors was modelled in patient-derived organoids (PDOs) from aGEA patients.

Results: EGFR amplification in cfDNA correlated with poor survival in the ITT population and similar trends were observed when the analysis was conducted in tissue and plasma by treatment arm. EGFR inhibition in combination with chemotherapy did not correlate with improved survival, even in patients with significant EGFR CN gains. Addition of anti-EGFR inhibitors to the chemotherapy agent epirubicin in PDOs, resulted in a paradoxical increase in viability and accelerated progression through the cell cycle, associated with p21 and cyclin B1 downregulation and cyclin E1 upregulation, selectively in organoids from EGFR-amplified aGEA.

Conclusion: EGFR CN can be accurately measured in tissue and liquid biopsies and may be used for the selection of aGEA patients. EGFR inhibitors may antagonise the antitumour effect of anthracyclines with important implications for the design of future combinatorial trials.

Keywords: gastric adenocarcinoma; gastrointestinal cancer; molecular oncology; oesophageal cancer.

Conflict of interest statement

Competing interests: ES declares honoraria for advisory role from Astellas, BMS, Celgene, Five Prime, Gritstone Oncology, and Servier. DW has received honoraria from Amgen. NS received research funding from AstraZeneca, Pfizer, BMS and honoraria from Servier, MSD, Merck Serono and AstraZeneca. JW received honoraria from Eisai, AstraZeneca, Sanofi-Genzyme, Ipsen, Novartis, Bayer, Celgene and Advanced Accelerator Applications. IC has had advisory roles with Merck Serono, Roche, Sanofi Oncology, Bristol Myers Squibb, Eli-Lilly, Novartis, Gilead Science. He has received research funding from Merck-Serono, Novartis, Roche and Sanofi Oncology, and honoraria from Roche, Sanofi-Oncology, Eli-Lilly, Taiho, Bayer and Prime Therapeutics. IV has received honoraria from AbbVie. He has received researched funding from Genmab and Basilea. DC received research funding from 4SC, AstraZeneca, Bayer, Celgene, Clovis, Eli Lilly, Janssen, Medimmune, Merck, Merrimack, Amgen, Sanofi. NV received honoraria from Merck Serono, Pfizer, Bayer and Eli-Lilly. All other authors declare no conflict of interest.

© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.

Figures

Figure 1

Tissue and liquid biopsy analysis in the REAL3 trial. (A) Diagram shows the number of patients for whom FFPE tissues and plasma cfDNA were available for EGFR testing. (B) Venn diagram shows the number of patients tested for EGFR amplification based on source of material (FFPE tissues vs cfDNA) and method used (ddPCR vs FISH). cfDNA, cell free DNA; ddPCR, digital-droplet PCR; EGFR, epidermal growth factor receptor; FFPE, formalin-fixed paraffin embedded; FISH, fluorescent in situ hybridisation; ITT, intention to treat.

Figure 2

Overall survival (OS) based on EGFR-amplification in the REAL3 trial. EGFR CN was determined using ddPCR on pretreatment cfDNA. Kaplan-Meier curves show the OS of patients based on the presence/absence of EGFR CN. cfDNA=cell free DNA; CN, copy number; ddPCR, digital-droplet PCR; EGFR, epidermal growth factor receptor;

Figure 3

Clinical outcome by treatment arm in EGFR-amplified cases enrolled in the REAL3 trial. (A) Bars show OS and PFS (median±SE) in EGFR-amplified patients treated with chemotherapy alone (EOX) or chemotherapy plus panitumumab (EOX-P). Similar trends are observed when a cut-off of two or five EGFR copies is used. (B) Blue bars indicate PFS, orange bars indicate EGFR copies determined by ddPCR in plasma cfDNA (graph on the top) or tissue (bottom). (C) Plots show copy number changes in EGFR (blue) and other receptor tyrosine kinase genes (red) in patients with PFS greater than 6 months on treatment with EOX-P. Values outside of the y-axis limits are plotted at the limit. cfDNA, cell-free DNA; CN, copy number; ddPCR, digital-droplet PCR; EGFR, epidermal growth factor receptor; EOX, epirubicin+oxaliplatin+capecitabine; OS, overall survival; P, panitumumab; PFS, progression-free survival.

Figure 4

Effect of epirubicin or epirubicin plus EGFR inhibitors in EGFR-amplified and non-amplified patient-derived organoids. (A) EGFR FISH images, demonstrating gain of copies and diploid status in the F-014 BL and DD191 human GEA PDO lines, respectively. (B) Concentration-dependent effect of epirubicin as a monotherapy or in combination with a stable dose of two different anti-EGFR agents (cetuximab, 20 µg/mL; gefitinib, 200 nM) in the EGFR-amplified F-014 BL and EGFR-diploid DD191 human GEA PDO lines. The combination of low epirubicin concentrations with anti-EGFR treatments results in a paradoxical increase in viability selectively in the EGFR-amplified F-014 BL GEA PDO line. Viability data shown are means±SEM of indicated independent experiments. (C) Pathway analysis of RNAseq data from the EGFR-amplified F-014 BL GEA PDO line treated with a low concentration of epirubicin alone or in combination with cetuximab for 24 hours revealed a significant reduction in the expression of cell cycle-related genes associated with the epirubicin and cetuximab combination. RNA from three independent biological replicates were sequenced per condition. (D) Protein analysis of the EGFR-amplified F-014 BL and EGFR-diploid DD191 human GEA PDO lines treated with two low concentrations of epirubicin alone or in combination with cetuximab for 24 hours. In line with RNAseq data, a reduction in p21 and cyclin B1 protein levels was observed when epirubicin was combined with inhibition of EGFR and downstream MAPK and AKT signalling specifically in the EGFR-amplified organoid line. (E) EdU DNA incorporation assay following treatment of the EGFR-amplified F-014 BL and EGFR-diploid DD191 human GEA PDO lines with two low concentrations of epirubicin alone or in combination with cetuximab for 24 hours. The addition of cetuximab antagonises the antiproliferative effect of epirubicin and accelerates DNA synthesis specifically in the EGFR-amplified organoid line. (F) Proposed model of antagonism between epirubicin and anti-EGFR treatments in EGFR-amplified GEA. EGFR, epidermal growth factor receptor; FISH, fluorescent in situ hybridisation; FSC-A; forward scatter-area; GEA, gastro-oesophageal adenocarcinoma; KEGG, Kyoto Encyclopedia of Genes and Genomes; PDO, patient-derived organoid.