Response to Anti-EGFR Therapy in Patients with BRAF non-V600-Mutant Metastatic Colorectal Cancer

Abstract

Purpose: While mutations in BRAF in metastatic colorectal cancer (mCRC) most commonly occur at the V600 amino acid, with the advent of next-generation sequencing, non-V600 BRAF mutations are increasingly identified in clinical practice. It is unclear whether these mutants, like BRAF V600E, confer resistance to anti-EGFR therapy.

Experimental design: We conducted a multicenter pooled analysis of consecutive patients with non-V600 BRAF-mutated mCRCs identified between 2010 and 2017. Non-V600 BRAF mutations were divided into functional classes based on signaling mechanism and kinase activity: activating and RAS-independent (class 2) or kinase-impaired and RAS-dependent (class 3).

Results: Forty patients with oncogenic non-V600 BRAF-mutant mCRC received anti-EGFR antibody treatment [n = 12 (30%) class 2 and n = 28 (70%) class 3]. No significant differences in clinical characteristics were observed by mutation class. In contrast, while only 1 of 12 patients with class 2 BRAF mCRC responded, 14 of 28 patients with class 3 BRAF responded to anti-EGFR therapy (response rate, 8% and 50%, respectively, P = 0.02). Specifically, in first- or second-line, 1 of 6 (17%) patients with class 2 and 7 of 9 (78%) patients with class 3 BRAF mutants responded (P = 0.04). In third- or later-line, none of 6 patients with class 2 and 7 of 19 (37%) patients with class 3 BRAF mutants responded (P = 0.14).

Conclusions: Response to EGFR antibody treatment in mCRCs with class 2 BRAF mutants is rare, while a large portion of CRCs with class 3 BRAF mutants respond. Patients with colorectal cancer with class 3 BRAF mutations should be considered for anti-EGFR antibody treatment.See related commentary by Fontana and Valeri, p. 6896.

Fig. 1.. Response to EGFR inhibitors in preclinical patient-derived models.

PDXs established from class 2 (T599dup, K601E) or class 3 (D594N, G466V) mutant BRAF were treated with either vehicle or cetuximab. Each treatment group consisted of 5 mice. Error bars indicate standard deviation.

Fig. 2.. Classification of BRAF mutants.

(A) Mouse embryonic fibroblasts (MEFs) were transfected to express the V5-tagged, indicated BRAF proteins or the same BRAF proteins together with R509H mutation. Cells were collected to assay expression of activated pathway intermediates. (B) Expression of the indicated BRAF proteins was induced in the conditional “RAS-less” MEFs after pre-treatment with 4-hydroxytamoxifen (4-OHT) to knock out the last RAS allele. Cells were collected to assay expression of activated pathway intermediates.

Fig. 3.. Survival of patients with non-V600 BRAF alterations.

(A) PFS in a total of 25 patients treated with anti-EGFR antibody at the third- or later-line, or (B) stratified by BRAF mutation class. (C) Measurements of cell tumor DNA before and during treatment with panitumumab and irinotecan. Numbers for highest allelic fraction correspond to BRAF D594G mutant allelic fraction detected in blood.

Fig. 4.. Class 2 mutations can cause secondary resistance to EGFR inhibitors.

(A) Timeline of patient’s treatment history, (B) Representative images from PET/CT showing biopsy-proven, hypermetabolic metastatic thoracic lymphadenopathy at the start of FOLFIRI/cetuximab treatment and after one year of treatment, (C) Representative MRI image showing new cerebellar metastasis, (D) Schema of the MKRN1-BRAF fusion identified on sequencing the cerebellar metastasis, (E) Growth curve of mice bearing the PDX with MKRN1-BRAF fusion treated with vehicle or cetuximab. Five mice were treated in each group and standard deviations are indicated with error bars.