The earliest events in BRAF ‐mutant colorectal cancer: exome sequencing of sessile serrated lesions with a tiny focus dysplasia or cancer reveals recurring mutations in two distinct progression pathways

Around 15–30% of colorectal cancers (CRC) develop from sessile serrated lesions (SSLs). After many years of indolent growth, SSLs can develop dysplasia and rapidly progress to CRC through events that are only partially understood. We studied molecular events at the very early stages of progression of SSLs via the MLH1‐proficient and deficient pathways to CRC. We collected a cohort of rare SSLs with a small focus (<10 mm) of dysplasia or cancer from the pathology archives of three hospitals. Whole‐exome sequencing was performed on DNA from nonprogressed and progressed components of each SSL. Putative somatic driver mutations were identified in known cancer genes that were differentially mutated in the progressed component. All analyses were stratified by MLH1 proficiency. Forty‐five lesions with a focus dysplasia or cancer were included, of which 22 (49%) were MLH1‐deficient. Lesions had a median diameter of 10 mm (interquartile range [IQR] 8–15), while the progressed component had a median diameter of 3.5 mm (IQR 1.75–4.75). Tumor mutational burden (TMB) was high in MLH1‐deficient lesions (23.9 mutations per MB) as compared to MLH1‐proficient lesions (6.3 mutations per MB). We identified 34 recurrently mutated genes in MLH1‐deficient lesions. Most prominently, ACVR2A and RNF43 were affected in 18/22 lesions, with mutations clustered in three hotspots. Most lesions with RNF43 mutations had concurrent mutations in ZNRF3. In MLH1‐proficient lesions APC (10/23 lesions) and TP53 (6/23 lesions) were recurrently mutated. Our results show that the mutational burden is exceptionally high even in the earliest MLH1‐deficient lesions. We demonstrate that hotspot mutations in ACVR2A and in the RNF43/ZNRF3 complex are extremely common in the early progression of SSLs along the MLH1‐deficient serrated pathway, while APC and TP53 mutations are early events in the the MLH1‐proficient pathway. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.