Evans D B, Frazier M L, Charnsangavej C, Katz R L, Larry L, Abbruzzese J L
Department of Surgical Oncology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Ann Surg Oncol. 1996 May;3(3):241-6. doi: 10.1007/BF02306278.
The K-ras oncogene is activated by point mutations at codon 12 in most patients with exocrine pancreatic cancer. Mutant-enriched polymerase chain reaction (PCR) amplification can enhance the detection of mutated K-ras. This technique was applied to patients undergoing percutaneous fine-needle aspiration (FNA) biopsy of suspect pancreatic lesions.
Twenty-five patients underwent percutaneous FNA of the pancreas for cytologic and molecular analysis. After preparing cytologic smears, the 22-gauge needle and syringe used for FNA were rinsed in RPMI-1640. The specimen was centrifuged, and DNA was extracted from the supernatant and subjected to mutant-enriched PCR using appropriate mismatched primers that introduce a BstNI restriction endonuclease clevage site at codon 12 of wild-type, but not mutant, K-ras. After digestion with BstNI, the DNA was reamplified. To increase assay sensitivity, the final five PCR cycles were completed incorporating 5 microCi of (alpha-32P)dCTP. The DNA was then redigested and subjected to gel electrophoresis and autoradiography.
The median amount of DNA retrieved per specimen was 3.33 micrograms. Mutant K-ras was detected as a band of 143 bps; residual wild-type DNA was seen as a 114-bp fragment. Twenty-one of 25 specimens demonstrated mutated K-ras DNA. Two patients with nondiagnostic cytology results had mutated K-ras DNA; adenocarcinoma of pancreatic origin was confirmed in both cases after pancreatectomy.
The molecular diagnosis of pancreatic cancer through identifications of mutations in K-ras can be readily performed on specimens obtained by percutaneous FNA. As aggressive multimodality management of this disease becomes more common, pretreatment analysis of molecular determinants may have greater clinical significance.
在大多数外分泌型胰腺癌患者中，K-ras癌基因通过密码子12处的点突变被激活。富含突变体的聚合酶链反应（PCR）扩增可增强对突变型K-ras的检测。该技术应用于对可疑胰腺病变进行经皮细针穿刺（FNA）活检的患者。
25例患者接受胰腺经皮FNA以进行细胞学和分子分析。制备细胞学涂片后，用于FNA的22号针头和注射器在RPMI-1640中冲洗。标本离心后，从上清液中提取DNA，并使用适当的错配引物进行富含突变体的PCR，这些引物在野生型而非突变型K-ras的密码子12处引入BstNI限制性内切酶切割位点。用BstNI消化后，DNA重新扩增。为提高检测灵敏度，在最后五个PCR循环中加入5微居里的（α-32P）dCTP。然后对DNA再次消化并进行凝胶电泳和放射自显影。
每个标本回收的DNA中位数为3.33微克。突变型K-ras被检测为一条143 bp的条带；残留的野生型DNA表现为114 bp的片段。25个标本中有21个显示出突变型K-ras DNA。两名细胞学检查结果不明确的患者有突变型K-ras DNA；胰腺切除术后这两例均确诊为胰腺来源的腺癌。
通过鉴定K-ras突变对胰腺癌进行分子诊断可很容易地在经皮FNA获得的标本上进行。随着对该疾病积极的多模式治疗变得更加普遍，分子决定因素的术前分析可能具有更大的临床意义。
