|Year : 2018 | Volume
| Issue : 6 | Page : 1403-1406
A study of BRAF mutation in colorectal carcinoma in Indian population
Shilpi Saxena1, V Srinivas2, Prabal Deb3, Deep Kumar Raman4, Rajat Jagani2
1 Department of Pathology, Military Hospital, Roorkee, Uttarakhand, India
2 Department of Pathology, Command Hospital, Pune, Maharashtra, India
3 Department of Pathology, Army Hospital Research and Referral, New Delhi, India
4 Department of Pathology, AFMC, Pune, Maharashtra, India
|Date of Web Publication||28-Nov-2018|
Department of Pathology, Military Hospital, Roorkee, Haridwar, Uttarakhand
Source of Support: None, Conflict of Interest: None
Context: BRAF mutation has been extensively studied and associated with various tumors. Targeted therapeutic intervention against BRAF mutation is established modality against many such tumors. Various studies have estimated that the prevalence of BRAF mutation in colorectal carcinoma (CRC) is 5%–25%. Considering epidemiology differences from Western population and paucity of studies on BRAF mutation in CRC in Asian patients, the present study was done to study the BRAF mutation in CRC in Indian population.
Aims: The aim is to study the distribution of BRAF mutation and its correlation with the American Joint Committee on Cancer (AJCC) stage, grade, and other clinicopathological parameters in CRC.
Settings and Design: This was a cross-sectional study.
Subjects and Methods: Immunohistochemistry study was done using BRAFV600E monoclonal antibody (Clone VE1) for 65 consecutive cases of CRC in a tertiary care center. The results were statistically analyzed using SPSS version 2.0.
Results: This study found that BRAF mutation is not significantly present in CRC as only 4.6% of cases were positive for BRAFV600E mutation. However, there was statistically significant relation between increasing AJCC stage and BRAF mutation.
Conclusions: This study concluded that BRAF mutation is not prevalent in Indian population with CRC. However, it is significantly related with advanced AJCC stages.
Keywords: BRAF, colorectal carcinoma, immunohistochemistry, India
|How to cite this article:|
Saxena S, Srinivas V, Deb P, Raman DK, Jagani R. A study of BRAF mutation in colorectal carcinoma in Indian population. J Can Res Ther 2018;14:1403-6
| > Introduction|| |
Colorectal carcinoma (CRC) is the third most common cancer worldwide with surging incidence in India., Genetics and molecular pathways are implicated in initiation and progression of CRC, among which “Kinzler–Vogelstein model” is well recognized, which conveys step-wise accumulation of mutations of adenomatous polyposis coli gene, p53, and members of β-catenin pathway., Recent “Big-Bang model” proposes that after initial mutation, intratumoral heterogeneity is favored by the evolution of tumor cell subpopulations, bearing other oncogenic mutations. Mutations in BRAF gene in about 5%–25% of CRC patients illustrate such event.,, BRAFV600E mutation identification by immunohistochemistry (IHC) may supplement clinical and pathological staging as poor prognostic marker and may overtake multiplex polymerase chain reaction (PCR) mass., Although the studies done on Western population suggest low incidence of BRAF mutation in CRC. However, in view of epidemiological differences of CRC in Western and Indian population, an analysis of BRAF mutation in CRC in Indian patient is necessary. Very few studies are done on the subject previously.
The present study was aimed to study the distribution of BRAFV600E mutation in CRC in Indian population with IHC and its correlation with the American Joint Committee on Cancer (AJCC) stage, grade, and other clinicopathological parameters in CRC.
| > Subjects and Methods|| |
The present study included 65 consecutive cases of CRC from 2010 to 2015. Inclusion criteria were all cases of primary CRC, confirmed by histopathological examination. All nonadenocarcinomas of colon, for example, melanoma, gastrointestinal stromal tumors, and carcinoids were excluded from the study. Patients of CRC, who were already given radiotherapy or chemotherapy, were also excluded from the study. Written informed consent was obtained from all patients and the study was approved by the Institutional Ethical Committee.
The IHC for BRAF was performed on 3 μm thick sections using Mouse Antihuman BRAFV600E Monoclonal Antibody (Clone VE1), Isotype IgG2a, E 19290, and LOT 130508H (Spring Biosciences). This antibody was used in 1:50 dilution as per manufacturer's direction. The cases with positive IHC staining (defined as diffuse strong brown colored positive staining of >:75% of malignant cells) were scored as positive [Figure 1]. Malignant melanoma cases positive for BRAFV600E mutation were taken as positive control. For all the cases, same slide was used as a negative control by subtracting the primary antibody from the reaction. Interpretation of IHC result was performed by two pathologists independently, blinded to all clinical and pathological data. The statistical analysis of the result was performed using IBM SPSS (Statistical Package for the Social Sciences, IBM CORP, New Delhi, India) statistics software version 20.
|Figure 1: BRAF-positive case of colorectal carcinoma showing diffuse strong brown colored positive staining of >75% of malignant cells (IHC, ×400)|
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| > Results|| |
In the present study, 44 (67.69%) male and 21 (32.31%) female patients of CRC were included. The age of the cases ranged from 26 to 90 years, with a mean of 64 years and the highest percentage (40%) of patients were in the age group of 51–60 years. Out of total 65 CRC cases, 17 (26.15%) were Grade I (well differentiated), 33 (50.76%) cases were Grade II (moderately differentiated), and 15 (23.07%) cases comprised Grade III (poorly differentiated) CRC. Most of the cases, that is, 31 of 65 cases (47.69%) were in Stage II, whereas 23 cases (35.38%) were in Stage III. Stage I and IV were assigned to 8 (12.31%) and 3 (4.62%) cases, respectively.
This study showed that out of the total 65 cases, three cases (4.62%) were BRAF positive [Table 1].
When the positivity of BRAF mutation in CRC was related to AJCC stages, it was found that there was a significant relation between them. One of the three cases was in Stage IV and two were Stage III. The expression of BRAF was significantly higher in higher stages (P = 0.029). However, the study failed to draw any statistically significant relation between expression of BRAF and tumor grade.
| > Discussion|| |
The incidence of CRC vary worldwide, the highest estimated rates being in Australia/New Zealand and lowest rates in Western Africa There has been a waning trend in the incidence rate of CRC in the United States since mid-1980s. The acceleration in the decline in the past decade has mostly been attributed to the early detection and timely removal of precancerous polyps as a result of CRC screening. There is also a small but demonstrable impact of reduction in risk factors of CRC. The incidence rates are gradually rising in countries that were previously considered to be having lower incidence of CRC, including India and other Asian countries, Africa, and South America.
In India, the incidence rates of CRC in males and females are 4.3 and 3.4 per 100,000, respectively. Although the age-adjusted incidence rates of CRC in the Indian cancer registries are very close to the lowest rates in the world, population-based time-trend studies have revealed a rising trend in the incidence of CRC in India. A worrisome finding is that the incidence rates of CRC in Indian immigrants to the USA and UK are much higher, suggesting that the lifestyle and dietary habits play an important role in causation of the CRC. It also means that with economic transition from a low- to middle-income economy, there will be a big increase in the burden of CRC in India.
The BRAF oncogene codes for a serine/threonine kinase and the effects of BRAF are mediated by activating mitogen-activated protein kinase kinase (MAPKK), which promotes cell proliferation. BRAFV600E is an activating mutation that accounts for approximately 90% of all BRAF mutations seen in CRC. The prognostic role of the BRAF mutation in CRC is well established and is usually associated with significant poorer prognosis. BRAF status is also believed to be responsible for the 12%–15% of patients of CRC, who fail anti-epidermal growth factor receptor. Due to the increasing significance of this mutation, the National Comprehensive Cancer Network Guidelines now recommend BRAF mutation testing in patients with metastatic disease.
In this study, the age and gender distribution were comparable with a recent review of Indian patients of CRC by Mohandas, who also found increased incidence in male as compared to females. The majority of tumors were seen in the rectosigmoid region which is comparable to that seen in other studies.
In the present study, BRAF was found positive in 3 (4.62%) of the 65 cases studied. This was in comparison with a recent study of 344 CRC cases by Zhang et al., who found BRAF mutation to be present in 4.7% (16/344) and 4.1% (14/344) cases using real-time quantitative PCR-Sanger sequencing and TaqMan probe, respectively. However, in contrast to most other studies conducted in various countries, BRAF positivity in the present study was lower.,, A recent study by Javadi et al. studied BRAFV600E mutation in 100 Iranian patients of CRC with conventional PCR and DNA sequencing using Sanger method and found none of the 100 cases (0%) were positive for BRAF mutation.
The present study also did not find a statistically significant relation between expression of BRAF and tumor grade. Furthermore, none of the nine mucinous carcinomas were positive for BRAF. The results were in contrast to Li et al., who studied BRAFV600E mutation in 275 CRC cases using fluorescent single-strand conformation polymorphism for screening and DNA sequencing for confirmation and reported 8% (23/275) of cases with the presence of mutation. Their study found strong associations between BRAF mutation and mucinous appearance, poor histological grade, and tumor origin in the proximal side of the large bowel. Higher frequency of BRAF mutation observed in females was also in contrast to present study in which only one female patient was positive for BRAF mutation. Patil et al. in their recent study on Indian patients found BRAF mutation in 8% (12/150) of the cases, all of them were V600E type. In contrast to our study, Patil et al. noticed that BRAF mutation was more frequent in well-differentiated tumors.
The present study also revealed significant relation of BRAF with CRC stage (P = 0.029), similar to a recent study by Zhang et al. which showed the frequency of the BRAF mutation was higher in Stage III-IV cases as compared to Stage I-II cases. Korphaisarn et al. studied BRAF mutation in 405 CRC patients and did not find any association with any clinicopathological features such as gender, age, site of the primary tumor, UICC stage, lymphovascular or perineural invasion, histologic grade, pT, pN, and M stage.
The variations in results of different studies are likely due to several factors, apart from different methods used for analyses of BRAF mutation, for example, the use of fresh frozen biopsies in some studies, selection bias for patient criteria, and racial and environmental factors. The differences could also be because of demographic variation in the population studied. Thus, comparison of data with different population may not be appropriate. It has been argued that these discrepancies could also be related to lack of uniformity in the selection and preparation of tissue, influencing preanalytical variables, especially cold ischemic time and formalin fixation process.
The emerging resistance to BRAF inhibitors is a subject of intense investigation. For a long time, BRAF-mutant CRC patients have been considered as one subtype with high clinical resemblances. However, the many mechanisms for resistance against BRAF inhibitors suggest that tumors can have different overall gene expression profiles., An effort to further classify the BRAF-mutant population based on gene expression is necessary to improve personalized therapy. The limitation of this study was small sample size and inclusion of retrospective cases. A multicentric, prospective study with larger sample size may be necessary for firm and conclusive evidences of BRAF mutation in CRC.
| > Conclusions|| |
The BRAF mutation was found to be uncommon in Indian patients and hence routine screening for BRAF mutation in CRC is not justifiable. However, significant relation with AJCC stage and BRAF mutation was found in the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al.
Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359-86.
Yeole BB. Trends in cancer incidence in esophagus, stomach, colon, rectum and liver in males in India. Asian Pac J Cancer Prev 2008;9:97-100.
Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759-67.
Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell 1996;87:159-70.
Sottoriva A, Kang H, Ma Z, Graham TA, Salomon MP, Zhao J, et al.
A big bang model of human colorectal tumor growth. Nat Genet 2015;47:209-16.
Baldus SE, Schaefer KL, Engers R, Hartleb D, Stoecklein NH, Gabbert HE. Prevalence and heterogeneity of KRAS, BRAF, and PIK3CA mutations in primary colorectal adenocarcinomas and their corresponding metastases. Clin Cancer Res 2010;16:790-9.
Lee SH, Ahn BK, Baek SU, Chang HK. BRAF mutation in multiple primary cancer with colorectal cancer and stomach cancer. Gastroenterol Rep (Oxf) 2013;1:70-4.
Chen D, Huang JF, Liu K, Zhang LQ, Yang Z, Chuai ZR, et al.
BRAFV600E mutation and its association with clinicopathological features of colorectal cancer: A systematic review and meta-analysis. PLoS One 2014;9:e90607.
Toon CW, Walsh MD, Chou A, Capper D, Clarkson A, Sioson L, et al.
BRAFV600E immunohistochemistry facilitates universal screening of colorectal cancers for Lynch syndrome. Am J Surg Pathol 2013;37:1592-602.
Edwards BK, Ward E, Kohler BA, Eheman C, Zauber AG, Anderson RN, et al.
Annual report to the nation on the status of cancer, 1975-2006, featuring colorectal cancer trends and impact of interventions (risk factors, screening, and treatment) to reduce future rates. Cancer 2010;116:544-73.
Boyle P, Levin B. World Cancer Report 2008. Lyon: International Agency for Research on Cancer; 2008.
Mohandas KM. Colorectal cancer in India: Controversies, enigmas and primary prevention. Indian J Gastroenterol 2011;30:3-6.
Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002;418:934.
Yuan ZX, Wang XY, Qin QY, Chen DF, Zhong QH, Wang L, et al.
The prognostic role of BRAF mutation in metastatic colorectal cancer receiving anti-EGFR monoclonal antibodies: A meta-analysis. PLoS One 2013;8:e65995.
Tie J, Gibbs P, Lipton L, Christie M, Jorissen RN, Burgess AW, et al.
Optimizing targeted therapeutic development: Analysis of a colorectal cancer patient population with the BRAF(V600E) mutation. Int J Cancer 2011;128:2075-84.
Zhang X, Wang Y, Gao N, Wang J. Comparative analysis of real-time quantitative PCR-Sanger sequencing method and TaqMan probe method for detection of KRAS/BRAF mutation in colorectal carcinomas. Zhonghua Bing Li Xue Za Zhi 2014;43:77-82.
Javadi F, Geramizadeh B, Mirzai M. BRAF gene mutation analysis in colorectal cancer in South of Iran. Ann Colorectal Res 2014;2:e19917.
Li WQ, Kawakami K, Ruszkiewicz A, Bennett G, Moore J, Iacopetta B. BRAF mutations are associated with distinctive clinical, pathological and molecular features of colorectal cancer independently of microsatellite instability status. Mol Cancer 2006;5:2.
Patil HA, Barrow C, Kanwar RK, Kanwar JR, Kapat A. Clinicopathological correlation with mutation profiling of colorectal cancer for KRAS, BRAF, NRAS and PIK3CA genes in Indian patient cohort. Ann Oncol 2015;26 Suppl 9:42-70.
Korphaisarn K, Pongpaibul A, Limwongse C, Roothumnong E, Klaisuban W, Nimmannit A, et al.
Deficient DNA mismatch repair is associated with favorable prognosis in Thai patients with sporadic colorectal cancer. World J Gastroenterol 2015;21:926-34.
Barras D. BRAF mutation in colorectal cancer: An update. Biomark Cancer 2015;7 Suppl 1:9-12.