Eachkoti Rafiqa, Farooq Sanah, Syeed Sheikh Imran, Wani Hilal Ahmed, Majid Sabhiya, Pampori Mohd Rafiq
Department of Biochemistry, Karanagar, Srinagar, Jammu and Kashmir, India.
Department of Physiology, Karanagar, Srinagar, Jammu and Kashmir, India.
Mutagenesis. 2018 Sep 17;33(3):225-230. doi: 10.1093/mutage/gey008.
Molecular studies have implicated mutant B-type Raf kinase (BRAFMut) of MAP-kinase signalling pathway in the pathogenesis of several cancers including colorectal cancer. Recently, the prognostic and therapeutic relevance of the most frequent BRAFV600E mutation also has been highlighted in colorectal carcinomas (CRC). Thus, the aim of this study was to investigate the prevalence of BRAFV600E mutation and to determine the correlation between this mutation and indicators of poor prognosis and outcome in patients with CRCs from Kashmir, North India. Here, we developed a highly sensitive technique, mutation allele-specific multiplex PCR (MASMP), for detection of BRAFV600E/BRAFc.1799T>A mutation, the results of which were confirmed by sequencing the product and compared to direct DNA sequencing. In total, BRAFV600E mutation status was analyzed in 57 colorectal tumour samples and an equal number of adjacent normal tissues. A high frequency of BRAFV600E mutation 21% (12/57) was identified in tumour tissues by MASMP compared to only 5.2% by direct DNA sequencing. Statistical analysis indicated that compared to BRAF-negative colorectal tumours, patients with BRAFV600E colorectal tumours were more likely to be >50 years old (61%) (P < 0.03). These tumours were more likely to be of clinical tumour stages III and IV (63%) (P < 0.04) with lymph node metastasis (52%) (P < 0.02) and characterised by a high-grade histology (63%) (P < 0.04). Colorectal patients harbouring BRAFV600E mutation experience more relapse/recurrence (52%) (P < 0.02). We, therefore, conclude that BRAFV600E mutation can be used as an indicator of poor prognosis to predict the outcome for CRC patients from Kashmir. MASMP proved to be a simple, sensitive and reliable technique for screening patients for BRAFV600E mutation. Testing for this mutation may be useful for selecting initial therapy mode and for follow-up monitoring in CRC patients.
分子研究表明，丝裂原活化蛋白激酶（MAP）信号通路中的突变型B型 Raf 激酶（BRAFMut）与包括结直肠癌在内的多种癌症的发病机制有关。最近，最常见的BRAFV600E突变在结直肠癌（CRC）中的预后和治疗相关性也得到了强调。因此，本研究的目的是调查BRAFV600E突变的发生率，并确定该突变与印度北部克什米尔地区CRC患者预后不良指标和结局之间的相关性。在此，我们开发了一种高度敏感的技术，即突变等位基因特异性多重PCR（MASMP），用于检测BRAFV600E/BRAFc.1799T>A突变，其结果通过对产物进行测序得到证实，并与直接DNA测序进行比较。总共对57个结直肠肿瘤样本和相同数量的相邻正常组织进行了BRAFV600E突变状态分析。通过MASMP在肿瘤组织中检测到BRAFV600E突变的频率较高，为21%（12/57），而直接DNA测序仅为5.2%。统计分析表明，与BRAF阴性的结直肠肿瘤相比，BRAFV600E结直肠肿瘤患者年龄更可能大于50岁（61%）（P < 0.03）。这些肿瘤更可能处于临床肿瘤分期III期和IV期（63%）（P < 0.04），伴有淋巴结转移（52%）（P < 0.02），且具有高级别组织学特征（63%）（P < 0.04）。携带BRAFV600E突变的结直肠癌患者复发/再发的情况更多（52%）（P < 0.02）。因此，我们得出结论，BRAFV600E突变可作为预后不良的指标，用于预测克什米尔地区CRC患者的结局。MASMP被证明是一种用于筛查患者BRAFV600E突变的简单、敏感且可靠的技术。检测该突变可能有助于选择CRC患者的初始治疗模式和进行随访监测。
