Heterogeneous EGFR gene copy number increase is common in colorectal cancer and defines response to anti-EGFR therapy

• Published in: PloS one Vol. 9; no. 6; p. e99590
• Main Authors: Ålgars, Annika, Avoranta, Tuulia, Österlund, Pia, Lintunen, Minnamaija, Sundström, Jari, Jokilehto, Terhi, Ristimäki, Ari, Ristamäki, Raija, Carpén, Olli
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 18.06.2014; Public Library of Science (PLoS)
• Subjects: Adult; Aged; AKT protein; Antibody response; Biotechnology; Cancer; Cancer therapies; Cell Line, Tumor; Chemotherapy; Cohort Studies; Colorectal cancer; Colorectal carcinoma; Colorectal Neoplasms - drug therapy; Colorectal Neoplasms - genetics; Copy number; Cytotoxicity; Disease-Free Survival; Drug Resistance, Neoplasm; Epidermal growth factor; Epidermal growth factor receptors; Extracellular signal-regulated kinase; Female; Gastric cancer; Gene amplification; Gene Dosage; Gene Expression Regulation, Neoplastic; Genetic Heterogeneity; Heterogeneity; Histology; Humans; Immunohistochemistry; In Situ Hybridization; K-Ras protein; Kaplan-Meier Estimate; Male; Medical diagnosis; Medical prognosis; Medicine and Health Sciences; Metastases; Metastasis; Middle Aged; Mutation; NMR; Nuclear magnetic resonance; Oncology; Pathology; Patients; Phosphorylation; Protein expression; Proteins; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins p21(ras); Radiation therapy; ras Proteins - genetics; Receptor, Epidermal Growth Factor - antagonists & inhibitors; Receptor, Epidermal Growth Factor - genetics; Reproducibility of Results; Rodents; Silver; Stem cells; Studies; Therapy; Treatment Outcome; Tumors
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0099590

Anti-EGFR therapy is commonly used to treat colorectal cancer (CRC), although only a subset of patients benefit from the treatment. While KRAS mutation predicts non-responsiveness, positive predictive markers are not in clinical practice. We previously showed that immunohistochemistry (IHC)-guided EGFR gene copy number (GCN) analysis may identify CRC patients benefiting from anti-EGFR treatment. Here we tested the predictive value of such analysis in chemorefractory metastatic CRC, elucidated EGFR GCN heterogeneity within the tumors, and evaluated the association between EGFR GCN, KRAS status, and anti-EGFR antibody response in CRC cell lines. The chemorefractory patient cohort consisted of 54 KRAS wild-type (WT) metastatic CRC patients. EGFR GCN status was analyzed by silver in situ hybridization using a cut-off value of 4.0 EGFR gene copies/cell. KRAS-WT and KRAS mutant CRC cell lines with different EGFR GCN were used in in vitro studies. The chemorefractory CRC tumors with EGFR GCN increase (≥4.0) responded better to anti-EGFR therapy than EGFR GCN (<4.0) tumors (clinical benefit, P = 0.0004; PFS, HR = 0.23, 95% CI 0.12-0.46). EGFR GCN counted using EGFR IHC guidance was significantly higher than the value from randomly selected areas verifying intratumoral EGFR GCN heterogeneity. In CRC cell lines, EGFR GCN correlated with EGFR expression. Best anti-EGFR response was seen with KRAS-WT, EGFR GCN = 4 cells and poorest response with KRAS-WT, EGFR GCN = 2 cells. Anti-EGFR response was associated with AKT and ERK1/2 phosphorylation, which was effectively inhibited only in cells with KRAS-WT and increased EGFR GCN. In conclusion, IHC-guided EGFR GCN is a promising predictor of anti-EGFR treatment efficacy in chemorefractory CRC.