Dieterle Christoph P, Conzelmann Michael, Linnemann Ulrich, Berger Martin R
Unit of Toxicology and Chemotherapy, German Cancer Research Center, Heidelberg, Germany.
Clin Cancer Res. 2004 Jan 15;10(2):641-50. doi: 10.1158/1078-0432.ccr-1355-02.
The aim of this study was to identify K-ras mutations as marker for isolated tumor cells in liver, lymph node, and bone marrow specimens of colorectal cancer patients. To detect these, a PCR-RFLP assay was used with a sensitivity exceeding that of routine histopathology by at least 1 order of magnitude. In addition, the ratio of mutated versus wild-type alleles was determined by an internal standard. Of 199 patients, 74 (37.5%) were found to bear a K-ras-positive tumor. Of these, 60 (81%) were mutated in codon 12 and 14 (19%) in codon 13 (P < 0.001). In addition, 14 organs were found K-ras positive, 13 of which were from 12 patients with a K-ras-positive tumor (16%) and 1 from a patient with a K-ras-negative tumor (0.8%). Eight patients exhibited liver involvement and 6 showed lymph node involvement. Remarkably, no bone marrow specimen was found K-ras positive (P < 0.017 versus liver involvement). Sequence analysis of tumor DNA revealed that GGT (Gly) was replaced by GAT (Asp; 35%), GTT (Val; 32%), AGT (Ser; 13%), GCT (Ala; 10%), TGT (Cys; 8%), and CGT (Arg; 2%) for codon 12, and by GAC (Asp) as the only type of mutation for codon 13. In colorectal carcinomas the ratio of K-ras mutated versus wild-type alleles ranged over 4 orders of magnitude (10(0)-10(-4), median: 10(-2)) and was correlated with both, residual tumor load (R1/2; P = 0.028) and distant metastasis (M1; P = 0.057). These results show that detection of K-ras mutated alleles by PCR-RFLP in patients with colorectal carcinoma may aid in the identification of isolated tumor cells. High ratios of K-ras alleles were correlated with certain negative prognostic parameters (R,M). In accord with its function as a primary filter for colorectal carcinoma cells, the liver was more often contaminated with K-ras-positive cells than bone marrow.
本研究的目的是将K-ras突变鉴定为结直肠癌患者肝脏、淋巴结和骨髓标本中孤立肿瘤细胞的标志物。为检测这些突变，采用了一种聚合酶链反应-限制性片段长度多态性（PCR-RFLP）检测方法，其灵敏度比常规组织病理学至少高1个数量级。此外，通过内标确定突变型与野生型等位基因的比例。在199例患者中，发现74例（37.5%）携带K-ras阳性肿瘤。其中，60例（81%）在密码子12处发生突变，14例（19%）在密码子13处发生突变（P<0.001）。此外，发现14个器官K-ras呈阳性，其中13个来自12例K-ras阳性肿瘤患者（16%），1个来自1例K-ras阴性肿瘤患者（0.8%）。8例患者出现肝脏受累，6例出现淋巴结受累。值得注意的是，未发现骨髓标本K-ras呈阳性（与肝脏受累相比，P<0.017）。肿瘤DNA的序列分析显示，密码子12处GGT（甘氨酸）被GAT（天冬氨酸；35%）、GTT（缬氨酸；32%）、AGT（丝氨酸；13%）、GCT（丙氨酸；10%）、TGT（半胱氨酸；8%）和CGT（精氨酸；2%）取代，密码子13处仅GAC（天冬氨酸）为唯一的突变类型。在结直肠癌中，K-ras突变型与野生型等位基因的比例范围超过4个数量级（10(0)-10(-4)，中位数：10(-2)），并且与残余肿瘤负荷（R1/2；P=0.028）和远处转移（M1；P=0.057）均相关。这些结果表明，通过PCR-RFLP检测结直肠癌患者K-ras突变等位基因可能有助于识别孤立肿瘤细胞。K-ras等位基因的高比例与某些不良预后参数（R、M）相关。与其作为结直肠癌细胞的主要滤器的功能一致，肝脏比骨髓更常被K-ras阳性细胞污染。
