Purpose: Epidermal growth factor receptor (EGFR) antibody therapy is established in patients with wild-type KRAS colorectal carcinoma; however, up to 50% of these patients do not respond to this therapy. To identify the possible causes of this therapy failure, we searched for mutations in different EGFR-dependent signaling proteins and analyzed their distribution patterns in primary tumors and corresponding metastases.

Experimental Design: Tumor tissues, macrodissected from tumor centers, invasion fronts (n = 100), lymph nodes (n = 55), and distant metastases (n = 20), respectively, were subjected to DNA extraction and mutation analysis of KRAS, BRAF, and PIK3CA.

Results: Activating mutations were detected in 41% (KRAS), 7% (BRAF), and 21% (PIK3CA) of the primary tumors. By comparing tumor centers and invasion fronts, the intratumoral heterogeneity of KRAS, BRAF, and PIK3CA mutations was observed in 8%, 1%, and 5% of primary tumors, respectively. Heterogeneity between primary tumors and lymph node metastases was found in 31% (KRAS), 4% (BRAF), and 13% (PIK3CA) of the cases. Heterogeneity between primary tumors and distant metastases was present in two patients (10%) for KRAS and one patient for PIK3CA (5%), but not for BRAF. Discordant results between primary tumors and metastases could markedly be reduced by testing the additional tumor samples.

Conclusions: Failure of EGFR antibody therapy in patients with wild-type KRAS colorectal cancer may result from activating BRAF or PIK3CA mutations and false-negative sequencing results caused by intratumoral heterogeneity. Due to the particularly high rates of heterogeneity between primary tumors and lymph node metastases, the latter are least suitable for diagnostic mutation analysis. Clin Cancer Res; 16(3); 790–9