Singapore Med J 2018; 59(3): 139-143 Original Article https://doi.org/10.11622/smedj.2017012 Extent of field change in colorectal cancers with BRAF mutation Aaron Poh1, FRCS, Heidi Sian Ying Chang2, MRCS, Kok Yang Tan1, FRCS, Xin Xiu Sam3, BSc(Hons), Avery Khoo3, BSc, Shoa Nian Choo4, BSc, Min En Nga4, FRCPath, Wei Keat Wan5, FRCPath INTRODUCTION Sporadic colorectal cancers with BRAF mutations constitute two distinct subgroups of colorectal cancers. Recent studies have linked the presence of the BRAF mutation to a familial inheritance pattern. This was a proof- of-concept study that aimed to examine: (a) the extent of field change in sporadic colorectal cancers with BRAF mutation; and (b) the extent of resection margins required and the pattern of DNA mismatch repair protein loss in these tumours. METHODS Eight microsatellite instability-high tumours with positive BRAF mutation from an existing histopathological database were selected for BRAF mutation and mismatch repair protein analysis. RESULTS All the resection margins were negative for BRAF mutation. Three tumours had loss of MLH1 and PMS2 expressions, and five tumours had no protein loss. Six peritumoral tissues were negative and one was positive for BRAF mutation. CONCLUSION The results suggest that any early field change effect is restricted to the immediate vicinity of the tumour and is not a pan-colonic phenomenon. Current guidelines on resection margins are adequate for BRAF mutation-positive colorectal cancers. Any suggestion of a hereditary link to these tumours is likely not related to germline BRAF gene mutations. The pattern of protein loss reinforces previous findings for the two subgroups of BRAF mutation-positive colorectal cancers. Keywords: BRAF mutation, colorectal cancer INTRODUCTION partial methylation of the MLH1 gene, presence of BRAF Colorectal cancers can be divided into several groups based mutation, and stable microsatellite instability (MSS) or low levels on their molecular characteristics. These characteristics are of microsatellite instability (MSI-L). Tumours in the second group underpinned by different pathways of tumorigenesis (Table I). are also chromosomally stable and make up 8% of colorectal The earliest known pathways are the chromosomal instability cancers.(1-8) The two subgroups of colorectal cancers share some (familial adenomatous polyposis [FAP]) and DNA mismatch repair common characteristics, namely the presence of BRAF mutation, (MMR) (hereditary non-polyposis colorectal cancer [HNPCC]) high levels of CIMP methylation, an element of MLH1 gene pathways.(1-3) The serrated pathway was subsequently noted after methylation (either partial or full) and an element of microsatellite the observation that a subgroup of hyperplastic polyps behaved in instability (MSI-L or MSI-H).(1-8) a manner similar to adenomatous polyps, resulting in an analysis Microsatellite instability is also seen in a third subgroup of serrated polyps and their associated pathway.(1) of colorectal cancers, namely in HNPCC or Lynch syndrome. Further work on the classification of colorectal cancers In contrast to the above two subgroups, HNPCC tumours are focused on additional molecular characteristics for further hereditary in nature but do not have BRAF mutations, are CIMP- delineation. Jass(2) hypothesised in 2007 that there are five separate negative and always show MSI-H. HNPCC is characterised by subgroups of colorectal cancers based on multiple molecular germline mutations in the genes that encode DNA MMR proteins. characteristics. Sporadic colorectal cancers with BRAF mutations More than 90% of these mutations occur in the genes encoding constitute two separate subgroups out of the five possible for MLH1 and MutS homolog 2 (MSH2), with the remainder subgroups of colorectal cancers according to this classification. occurring in the MSH6 and PMS1 homolog 2 (PMS2) genes. Due These two subgroups were the focus of our study. to the inherent germline mutations present, there is a generalised The first subgroup is made up of colorectal cancers field change, and the entire colon and rectum are at risk for characterised by high levels of DNA methylation at CpG islands development of tumours.(1,2,9) (CIMP-high), methylation of the MutL homolog 1 (MLH1) gene, The group of BRAF mutation-positive colorectal cancers presence of BRAF mutation and high levels of microsatellite was the subject of the present study. The presence of BRAF instability (MSI-H). These are chromosomally stable tumours mutation in the presence of MSI-H status has been touted as a and make up 12% of colorectal cancers.(3-8) The second subgroup negative predictor for germline MMR mutations and a possible is made up of colorectal cancers characterised by CIMP-high, exclusion criterion for germline testing. However, some recent 1Department of General Surgery, Khoo Teck Puat Hospital, 2University Surgical Cluster, National University Hospital, 3Histopathology Translational Research Group, Department of Pathology, Singapore General Hospital, 4Department of Pathology, National University Hospital, 5Department of Pathology, Singapore General Hospital, Singapore Correspondence: Dr Chang Sian Ying Heidi, Resident, University Surgical Cluster, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074. [email protected] 139 Original Article Table I. Classification of colorectal cancers based on molecular characteristics. Characteristic Chromosomal instability (FAP) Mismatch repair (HNPCC) Serrated pathway Hybrid pathway pathway pathway Heredity Sporadic and hereditary (FAP Hereditary only (HNPCC Sporadic and hereditary Sporadic only syndrome) syndrome) MSI status MSS MSI-H MSI-H MSI-L MSS or MSI-L Chromosomal Present Absent Absent Absent Present instability BRAF mutation No No Yes Yes No KRAS mutation Yes Yes/No No No Yes CIMP status Negative Negative High High Low MLH1 status Normal Mutated Methylated Partial methylation Normal CIMP: CpG island methylator phenotype; FAP: familial adenomatous polyposis; HNPCC: hereditary non-polyposis colorectal cancer; KRAS: V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; MLH1: MutL homolog 1; MSI: microsatellite instability; MSI-H: microsatellite instability-high; MSI-L: microsatellite instability-low; MSS: microsatellite stable population-based studies have suggested a familial tendency METHODS for colorectal cancers with BRAF mutation or CIMP-high status, This was a retrospective study. Colorectal tumours from an suggesting a possible new cancer family syndrome.(10-12) There is existing histopathological database were filtered and eight MSI-H no direct evidence of the extent of the field change around this tumours with positive BRAF mutation were selected for further group of tumours that can provide support for either theory, and testing. Paraffin-fixed slides from the tumour and the proximal hence, the adequacy of current resection margins is questionable. and distal margins were extracted from the database and used Current resection margin standards of a 5-cm margin proximal for testing. and distal to the tumour are derived from research on a separate Tumour blocks, adjacent non-tumour blocks and margin group of tumours arising from the chromosomal instability blocks were selected for analysis. Histological review was pathway (i.e. the FAP pathway). There is a need to exclude the performed to ensure the absence of histological evidence of a presence of a generalised field change and, if it is excluded, to tumour. Adjacent normal mucosa was defined as histologically determine the extent of the local field change in sporadic BRAF normal mucosa at a distance of at least 5 mm from the tumour. mutation-positive tumours. Each of the aforementioned formalin-fixed paraffin-embedded Mutation of the BRAF gene occurs early in the tumorigenesis (FFPE) blocks were divided into ten sections of 5 μm in thickness pathway leading to these two subgroups of colorectal cancers.(1-4,13) and deparaffinisation using standard xylene with the automated Using this knowledge as a foundation for this study, BRAF Ventana system was performed. DNA extraction was performed mutations can be used as a surrogate marker for detection of early using the Qiagen FFPE Kit and DNA concentration was assessed field change in apparently normal mucosa that can potentially lead using the Nanodrop analyser. Minimum DNA amounts of 300 ng to tumour formation in the future. The concept of field change, were required for BRAF analysis. BRAF mutational analysis was also known as field cancerisation, was first mooted in a study by performed using the Entrogen KRAS/BRAF Mutation Analysis Kit Slaughter et al in 1953.(14) Field change is described as a ‘process (v1.2). This kit uses allele-specific real-time polymerase chain whereby cells in a particular tissue or organ are transformed such reaction (PCR) and detects the V600E mutation of exon 15 of that genetically altered but histologically normal-appearing cells the BRAF gene. It may also detect some BRAF V600K mutations precede the development of neoplasia or coexist with malignant as V600E mutations. The BRAF V600E mutation accounts for a cells, irrespective of clonality’.(15) The essence of field change is the majority of BRAF mutations. The assay demonstrates a limit of existence of normal-looking cells or tissue that harbours genetic detection of up to 1% sensitivity. alterations predisposing them to overt malignant transformation. The presence of four MMR proteins, namely MLH1, MSH2, Multiple mechanisms of field change have been proposed,(16)