Colorectal Cancer in Focus: The BRAF Mutation in MLH1-Deficient Colorectal Cancer

Sameer A. Mahesh, MD

Clinical Advances in Hematology & Oncology

April 2013, Volume 11, Issue 4


Sameer A. Mahesh, MD

Assistant Professor, Northeast Ohio Medical University, Rootstown, Ohio, Medical Oncologist, Summa Health System, Akron, Ohio

H&O  How common is the mismatch repair protein deficiency (MMR-d) in colorectal cancer (CRC)?

SM  Approximately 15% of CRC cases are attributable to MMR-d.1 The deficient proteins are MLH1, MSH2, MSH6, and PMS2. The most common cause of MMR-d is hypermethylation of MLH1.2 Hence, the etiology of MLH1-deficient CRC is more often sporadic than genetic.

H&O  Does MMR-d affect prognosis or treatment?

SM  Patients with MMR-d CRC exhibit better prognosis than those with MMR-proficient (MMR-p) tumors.3 In patients with MMR-d tumors, single-agent fluoropyrimidine (5-FU)-based therapy is not beneficial and may even be detrimental.4 In stage II colon cancer, testing for MMR-d is one of the risk assessment modalities used to make decisions regarding adjuvant use of single-agent, 5-FU–based treatment.5

H&O  What is the current status of BRAF mutation in CRC?

SM  BRAF is a member of the Raf kinase family of serine/threonine-specific protein kinases. These proteins play a role in regulating the signaling pathway of mitogen-activated protein (MAP) kinases/extracellular signal-regulated kinases (ERKs), which affects cell division, differentiation, and secretion. The BRAF V600E mutation is found in 5–10% of patients with metastatic colon cancer and is an adverse prognostic factor, with a median survival of 9–14 months.6,7 In early-stage CRC, the situation is less clear. Hutchins and colleagues evaluated BRAF status in 1,584 stage II CRC patients from the QUASAR (Quick and Simple and Reliable) trial and found a BRAF V600E mutation rate of 8%.8 Risk of recurrence did not differ between BRAF-mutated and wild-type tumors (relative risk [RR], 0.84). However, 53% of BRAF-mutated tumors were MMR-d, and when the confounding effect of MMR-d was eliminated, the trend was reversed (RR, 1.32). A combined translational analysis of the PETACC (Pan-European Trials in Alimentary Tract Cancer) 3, EORTC (European Organisation for Research and Treatment) 40993, and SAKK (Swiss Group for Clinical Cancer Research) 60-00 trials showed that the BRAF mutation was present in 8% of patients and was negatively prognostic for overall survival in only the microsatellite-low (MSI-L) and microsatellite-stable (MSI-S) tumors. MSI-L and MSI-S are synonymous with MMR-p tumors.9

H&O  What was the design of your study of the BRAF V600E mutation in colorectal cancer patients with MMR-d due to loss of MLH1?

SM  We sought to investigate the contribution of the BRAF V600E mutation in MLH1-deficient CRC to identify any specific genotype/phenotype relationship.10 The study population included 128 patients newly diagnosed with CRC at Akron City Hospital from March 2010 to February 2011. The patients underwent mismatch repair (MMR) protein testing by immunohistochemistry (IHC). Their baseline characteristics are listed in Table 1. Deficiency of MLH1 prompted BRAF V600E testing. If the BRAF V600E mutation was detected, further testing was stopped since it was unlikely that the cancer was due to Lynch syndrome. If BRAF was wild in MLH1-deficient patients, sequencing of MLH1 was performed. If PMS2 deficiency coexisted with MLH1, then sequencing of PMS2 was performed as well if MLH1 sequencing was normal.

IHC for the MMR proteins (MLH1, PMS2, MSH2, and MSH6) was performed on 4 µm–sections of formalin-fixed tissue. Staining protocols from the manufacturer (Cell Marque) were followed. Adequately stained positive and negative controls were included in each patient sample. The slides were interpreted by a single pathologist. Nuclear staining in greater than 50% of tumor nuclei was considered positive (ie, an indication that the protein was not deficient). Normal and abnormal MLH1 stains are shown in Figure 1.

BRAF V600E mutation testing was performed by Clarient. Briefly, the procedure included the selection and microdissection of the tumor with lysis and extraction of DNA. Single-primer, real-time polymerase chain reaction was used to amplify the region containing the BRAF mutation. Two fluorogenic probes detected BRAF wild-type and V600E-mutant sequences.

H&O  What were the study findings?

SM  Fourteen percent of the patients (n=18) were MMR-d. Among these 18 patients, 16 were MLH1 deficient (15 were MLH1/PMS2 deficient, and 1 was solely MLH1 deficient). The BRAF V600E mutation was found in 81% of patients with MLH1 deficiency (13 of 16 patients). The remaining 3 patients were sequenced for MLH1 and PMS2, and no mutations in either gene were found.

The median age of patients with MLH1-deficient/BRAF V600E–mutated CRC was 80 years, which was significantly older than the overall population (70 years) as well as the population that was MLH1-deficient/BRAF V600E wild-type (67 years). Of note, among the 13 patients with MLH1-deficient/BRAF V600E–mutated CRC, 12 were women with right-sided tumors who had deficient MLH1 and PMS2, and 1 was a man with a left-sided tumor who had only MLH1 deficiency. Three had stage I disease, 5 had stage II, 4 had stage III, and 1 had stage IV. The patient with stage IV was the man with left-sided cancer. These results are summarized in Table 2. Among the 13 patients with MLH1-deficient/BRAF V600E–mutated tumors, 3 had mucinous and/or signet cell features.

H&O  Does your study have implications for the management of CRC patients?

SM  We identified a specific genotype/phenotype in CRC: MLH1/PMS2-deficient BRAF-mutated right-sided CRC in elderly women. The numbers in this study are too small to provide meaningful data on prognosis, and follow-up is not mature. However, it does seem that even within MMR-d BRAF tumors, there might be different subgroups based on the type of MMR-deficient protein, which could have prognostic implications. This suggestion must be confirmed in larger trials. MMR testing is relatively inexpensive, and BRAF mutation analysis has been performed in various large, adjuvant study samples.

Based on the reported data, it appears that early-stage CRC patients with BRAF mutations have 2 different prognostic subsets: MMR-p and MMR-d. The MMR-p subsets do worse when compared to their MMR-d counterparts as well as their BRAF wild-type counterparts. In metastatic CRC, MMR-p BRAF-mutated tumors might account for a higher percentage than MMR-d, leading to worse outcomes. Thus, it would be important to know the MMR status from the CRYSTAL (Cetuximab Combined With Irinotecan in First-Line Therapy for Metastatic Colorectal Cancer) and AGITG MAX (Australasian Gastro-Intestinal Trials Group Mitomycin C, Avastin and Xeloda) trials.4,5

It seems like we have reached a ceiling in the one-size-fits-all approach to the adjuvant treatment of colon cancer, and it is necessary to identify poor prognostic factors that require further efforts to improve outcomes. Patients with MMR-p/BRAF V600E are one such subset, and might benefit from intensification of chemotherapy efforts and use of targeted agents.


1. Jenkins MA, Hayashi S, O’Shea AM, et al. Pathology features in Bethesda guidelines predict colorectal cancer microsatellite instability: a population-based study. Gastroenterology. 2007;133:48-56.

2. Poynter JN, Siegmund KD, Weisenberger DJ, et al. Molecular characterization of MSI-H colorectal cancer by MLHI promoter methylation, immunohistochemistry, and mismatch repair germline mutation screening. Cancer Epidemiol Biomarkers Prev. 2008;17:3208-3215.

3. Popat S, Hubner R, Houlston RS. Systematic review of microsatellite instability and colorectal cancer prognosis. J Clin Oncol. 2005;23:609-618.

4. Sargent DJ, Marsoni S, Monges G, et al. Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancer. J Clin Oncol. 2010;28:3219-3226.

5. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) Colon Cancer. Version 3.2013. Accessed March 4, 2013.

6. Van Cutsem E, Köhne CH, Láng I, et al. Cetuximab plus irinotecan, fluorouracil, and leucovorin as first-line treatment for metastatic colorectal cancer: updated analysis of overall survival according to tumor KRAS and BRAF mutation status. J Clin Oncol. 2011;29:2011-2019.

7. Price TJ, Hardingham JE, Lee CK, et al. Impact of KRAS and BRAF gene mutation status on outcomes from the phase III AGITG MAX trial of capecitabine alone or in combination with bevacizumab and mitomycin in advanced colorectal cancer. J Clin Oncol. 2011;29:2675-2682.

8. Hutchins G, Southward K, Handley K, et al. Value of mismatch repair, KRAS, and BRAF mutations in predicting recurrence and benefits from chemotherapy in colorectal cancer. J Clin Oncol. 2011;29:1261-1270.

9. Roth AD, Tejpar S, Delorenzi M, et al. Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial. J Clin Oncol. 2010;28:466-474.

10. Mahesh SA, Hanna E, Khan, MS, Ravichandran P, Slezak F. Incidence and characteristics of BRAF V600E mutation in colorectal cancer (CRC) with mismatch repair (MMR) protein defect due to loss of MLH1: a prospective evaluation of 104 consecutive patients. J Clin Oncol (Proceedings from the 2011 American Society of Clinical Oncology Annual Meeting). 2011;29(suppl): Abstract 3539.