The molecular and clinicopathological study related to detection of K-ras point mutation in the blood of pancreatic cancer cases

胰腺癌患者血液中K-ras点突变检测相关的分子及临床病理学研究

• 批准号: 08671480
• 负责人: NAGAI Hideo
• 金额: $ 1.47万
• 依托单位: JICHI MEDICAL SCHOOL
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671480/
• 关键词: pancreatic cancer; peripheral blood; K-ras point mutation; PCR-RFLP; magnetic cell sorting system; allele specific PCR

Purpose : We have attempted to detect K-ras mutation from blood specimens in order to assist in the diagnosis of pancreatic cancer.Materials : The blood (perioperative peripheral blood and intraoperative portal blood) and tissues of the primary lesion in the pancreas were taken from 16 patients with pancreatic cancer (n=14) and benign diseases (n=2 ; chronic pancreatitis and insulinoma).Methods and Results :1.Detection of K-ras mutation in the primary lesionAfter microdissection we extracted DNA whose K-ras gene was amplified by PCR system. Its sequence was identified by digoxigenin dot blotting. The incidence of mutation at codon 12 was 85%.2.Detection of K-ras mutation from blood specimensBy the magnetic cell sorting system (MCSS) using anticytokeratine antibody and magnetic microspheres, we concentrated epithelial cells from the blood. Following amplification of the K-ras gene we enriched the mutated gene by the PCR-RFLP method. The sensitivity of PCR-RFLP without MCSS was only 10^< … More -4>, whereas that of combination of PCR-RFLP and MCSS reached 10^<-6> -10^<-7> With this combined method we could detect K-ras mutation in all samples of the portal blood and postoperative peripheral blood when the patients had K-ras mutation in the primary tumor. Furthermore, the mutation was detected in 25% even in the preope-rative peripheral blood. These cases rapidly deteriorated after surgery and died 3-6 months postoperatively.Discussion and ConclusionThe current study showed that tumor cells existed in the peripheral blood of pancreatic cancer patients at the rate of one tumor cell per 105 to 107 normal nucleated cells. We conjectured that the number of circulating cancer cells in the preoperative blood might be several thousand per body, and that the operative injury seemed to increase their number ten times. It was suggested that prognosis would be extremely poor if patients had tens of thousand of circulating cancer cells in the peripheral blood before surgey. We think that even the combination of PCR-RFLP and MCSS is not appropriate for early detection of pancreatic cancer. However, this system appears to be useful in excluding contraindicated operative cases from surgery. Less

目的：我们尝试从血液标本中检测K-ras基因突变，以协助胰腺癌的诊断。收集16例胰腺癌患者（n=14）和16例胰腺良性疾病患者的胰腺原发灶组织及围手术期外周血和术中门静脉血方法和结果：1.原发灶K-ras基因突变检测显微切割后提取DNA，PCR扩增K-ras基因。地高辛斑点杂交法鉴定其序列。第12位密码子突变发生率为85%。2.血细胞K-ras基因突变的检测应用抗细胞角蛋白抗体和磁性微球的磁性细胞分选系统（MCSS）从血细胞中富集上皮细胞。扩增K-ras基因后，通过PCR-RFLP方法富集突变基因。不加MCSS的PCR-RFLP法的敏感性仅为10^< ...更多信息 PCR-RFLP与MCSS联合检测K-ras基因突变的敏感性达10 ~ <-6>10 ~ 10%。<-7>当原发灶有K-ras基因突变时，PCR-RFLP与MCSS联合检测K-ras基因突变的敏感性达10 ~ 10%。此外，在术前外周血中也检测到25%的突变。这些情况下迅速恶化手术后死亡3-6个月postoperatives.Discussion和ConclusionThe目前的研究表明，肿瘤细胞存在于胰腺癌患者的外周血中的肿瘤细胞每105至107正常有核细胞的比率。我们证实，术前血液中循环的癌细胞数量可能是每个人几千个，而手术损伤似乎使其数量增加了十倍。提示术前外周血中循环癌细胞数达数万个者预后极差。我们认为，即使PCR-RFLP和MCSS相结合，也不适合胰腺癌的早期检测。然而，这个系统似乎是有用的，从手术中排除禁忌手术病例。少

项目成果

永井秀雄 他: "No-touch isolatioin techniqueによる膵頭十二指腸切除-自治医大方式-" 胆と膵. 19(12). 1109-1114 (1998)

Hideo Nagai 等：“使用非接触隔离技术的胰十二指肠切除术 - 自治医科大学方法”Bile and Pancreas 19(12) 1109-1114 (1998)。

Nagai H et al.: "Pancreaticogastrostomy : a mucosa-to-mucosa anastomosis for non-dilated pancreatic duct." Shujutsu (Operation). 51 (7). 1487-1497 (1997)

Nagai H 等人：“胰腺胃造口术：用于非扩张胰管的粘膜间吻合术。”

Nagai H et al.: "Pancreatogastrostomy" Shujutsu (Operation). 51 (11). 1741-1746 (1997)

Nagai H等人：“胰胃造口术”Shujutsu（手术）。

永井秀雄 他: "膵(管)胃吻合" 手術. 51巻11号. 1741-1746 (1997)

Hideo Nagai 等人：“胰腺（导管）胃吻合术”，第 51 卷，第 11 期。1741-1746 (1997)

Nagai H et al.: "Gastroduodenal artery-preserving PPPD with special reference to operative indication for so-called mucin-producing pancreatic tumor" Geka (Surgery). 60 (10). 1174-1179 (1998)

Nagai H 等人：“保留胃十二指肠动脉的 PPPD，特别提到所谓的产生粘蛋白的胰腺肿瘤的手术指征”Geka（外科）。