Department of Pathology, Ziauddin Medical University, Karachi, 75000, Pakistan.
Research Department, Ziauddin Medical University Karachi, Karachi, 75000, Pakistan.
BMC Cancer. 2024 Sep 19;24(1):1162. doi: 10.1186/s12885-024-12925-z.
Despite a high incidence of colorectal carcinoma, data regarding genetic aberrations in colorectal carcinoma (CRC) patients in Pakistan is scarce. This study aimed to determine the frequency of BRAFV600E mutations in colorectal carcinoma tissue in the Pakistani population and to associate BRAFV600E expression with CD133, a marker of colorectal stem cells, and CDX2 marker of differentiation.
Sanger Sequencing of exon 15 (426 bp) including the hotspot V600E was performed on formalin-fixed-paraffin-embedded (FFPE) CRC tissue samples of 115 patients. The samples were subjected to immunohistochemistry (IHC) to assess the expression of BRAFV600E, CDX2, and CD133. Additionally, homology modelling and docking were performed to investigate novel deletions revealed in sequencing.
Twenty-four (20.8%) BRAF variants were identified in the coding region, with V600E mutations detected in 14 (12.2% )cases (GenBank: PP003258.1; Pop Set: 2678087296). Moreover, a wide spectrum of novel non-V600E mutations (8.6%) were identified, including deletions and missense variations. In-silico analysis revealed that due to large deletions in the coding region of three samples, the affinity of the anti-BRAF drugs (Encorafenib and Vemurafenib) for the active site decreased in comparison to the wild type. The IHC analysis showed that BRAFV600E expression was significantly associated with CD133 expression (χ = 26.351; p = < 0.001) and with CDX2 expression (χ = 14.88; p = 0.001). Multivariate analysis using binary logistic regression revealed association of BRAFV600E mutations with age (OR = 1.123; CI = 1.024-1.232; p = 0.014), gender (OR = 0.071; CI = 0.006-0.831; p = 0.035), grade (0.007; CI = 0-0.644) and CD133 expression (OR = 65.649; CI = 2.153-2001.556; p = 0.016).
The present study demonstrates a notably high V600E frequency (12.2%) in comparison to global reported data, which ranges from 0.4 to 18%. This finding reflects the importance of upfront BRAF testing of the genetically distinct population of Pakistan. Previously unreported mutations identified in the sample may be of clinical significance and warrant further investigation. The concomitant high expression and significant association between CD133 and BRAFV600E represent vital actionable genes that may be targeted together to improve CRC patient management.
尽管结直肠癌的发病率很高，但关于巴基斯坦结直肠癌（CRC）患者遗传异常的数据却很少。本研究旨在确定巴基斯坦人群CRC 组织中 BRAFV600E 突变的频率，并将 BRAFV600E 表达与结直肠干细胞标志物 CD133 和分化标志物 CDX2 相关联。
对 115 例 CRC 组织样本的福尔马林固定石蜡包埋（FFPE）进行外显子 15（426bp）的 Sanger 测序，包括热点 V600E。对样本进行免疫组织化学（IHC）分析，以评估 BRAFV600E、CDX2 和 CD133 的表达。此外，还进行了同源建模和对接，以研究测序中发现的新缺失。
在编码区鉴定出 24 个（20.8%） BRAF 变体，其中 14 个（12.2%）病例检测到 V600E 突变（GenBank：PP003258.1；Pop Set：2678087296）。此外，还鉴定出一系列新的非 V600E 突变（8.6%），包括缺失和错义变异。计算机分析显示，由于三个样本的编码区发生了大片段缺失，与野生型相比，抗 BRAF 药物（Encorafenib 和 Vemurafenib）与活性位点的亲和力降低。IHC 分析表明，BRAFV600E 表达与 CD133 表达（χ²=26.351，p<0.001）和 CDX2 表达（χ²=14.88，p=0.001）显著相关。使用二元逻辑回归的多变量分析显示，BRAFV600E 突变与年龄（OR=1.123；CI=1.024-1.232；p=0.014）、性别（OR=0.071；CI=0.006-0.831；p=0.035）、分级（0.007；CI=0-0.644）和 CD133 表达（OR=65.649；CI=2.153-2001.556；p=0.016）相关。
与全球报道的数据（0.4%至 18%）相比，本研究显示出明显较高的 V600E 频率（12.2%），这反映了对具有遗传特征的巴基斯坦人群进行 BRAF 检测的重要性。在样本中发现的以前未报告的突变可能具有临床意义，需要进一步研究。CD133 和 BRAFV600E 之间同时高表达和显著关联代表了重要的可操作基因，可能一起靶向以改善 CRC 患者的管理。
