Zavodna K, Konecny M, Krivulcik T, Spanik S, Behulova R, Vizvaryova M, Weismanova E, Galbavy S, Kausitz J
Department of Clinical Genetics, St.Elizabeth Cancer Institute, Bratislava, Slovak Republic.
Neoplasma. 2009;56(3):275-8. doi: 10.4149/neo_2009_03_275.
Colorectal carcinoma (CRC) represents a serious problem worldwide: in the Slovak republic are diagnosed about 2600 new CRC cases annually and its incidence is increasing. Colorectal cancer patients may succumb to the disease because of local recurrence or local formation of metastasis. Therefore, it is necessary to modulate therapeutic algorithm with new methods, leading to early diagnostic of CRC or changing the existing therapeutic procedures. Recent progresses have been made in understanding of EGFR pathway involved in CRC carcinogenesis, especially the role of Ras protein. Mutations in KRAS oncogene are frequently found in human cancers, particularly colorectal, pancreatic, billiary tract and lung tumors. The presence of the KRAS mutations in metastatic colorectal cancer patients correlates with lack of response to the certain epidemal growth factor receptor (EGFR) inhibitor therapies, such as Panitumumab and Cetuximab. Consequently, screening for KRAS mutations status may be used as a prognostic marker, because the CRC patients with KRAS positive tumors have a worse prognosis. The aim of our study was to establish the methods for rapid and sensitive detection of KRAS mutation status in formalin fixed paraffin embedded (FFPE) tissues DNA. We applied Real Time PCR analysis (TheraScreen KRAS Mutation Test Kit) and sequencing analysis (optimised for the analysis of FFPE tissues) to detect somatic mutations in codon 12 and 13 of KRAS gene. Both methods were used concurrently in the panel of DNA isolated from 25 colorectal FFPE tissues tumor. The positive or negative results from all 25 samples were identified by both methods independently. The KRAS mutations were presented in 8 of 25 patients (32%). Our results demonstrate that the Real Time PCR analysis can be used for detection of somatic KRAS mutations in FFPE clinical samples. However, we also recognize that the sequencing analysis of approximately 200bp amplicons may be used for mutations status screening, but with care of method sensitivity.
结直肠癌（CRC）是全球范围内的一个严重问题：在斯洛伐克共和国，每年约有2600例新的CRC病例被诊断出来，且其发病率正在上升。结直肠癌患者可能会因局部复发或局部转移的形成而死于该疾病。因此，有必要用新方法调整治疗方案，以实现CRC的早期诊断或改变现有的治疗程序。在理解参与CRC致癌过程的表皮生长因子受体（EGFR）途径方面，尤其是Ras蛋白的作用，已经取得了最新进展。KRAS癌基因的突变在人类癌症中经常被发现，特别是在结直肠癌、胰腺癌、胆管癌和肺癌中。转移性结直肠癌患者中KRAS突变的存在与对某些表皮生长因子受体（EGFR）抑制剂疗法（如帕尼单抗和西妥昔单抗）缺乏反应相关。因此，筛查KRAS突变状态可作为一种预后标志物，因为KRAS阳性肿瘤的CRC患者预后较差。我们研究的目的是建立在福尔马林固定石蜡包埋（FFPE）组织DNA中快速、灵敏地检测KRAS突变状态的方法。我们应用实时荧光定量PCR分析（TheraScreen KRAS突变检测试剂盒）和测序分析（针对FFPE组织分析进行优化）来检测KRAS基因第12和13密码子的体细胞突变。这两种方法同时用于从25个结直肠癌FFPE组织肿瘤中分离的DNA样本组。所有25个样本的阳性或阴性结果均由这两种方法独立鉴定。25例患者中有8例（32%）存在KRAS突变。我们的结果表明，实时荧光定量PCR分析可用于检测FFPE临床样本中的体细胞KRAS突变。然而，我们也认识到，对大约200bp扩增子的测序分析可用于突变状态筛查，但要注意方法的灵敏度。
