Ashida S, Okuda H, Chikazawa M, Tanimura M, Sugita O, Yamamoto Y, Nakamura S, Moriyama M, Shuin T
Department of Urology, Kochi Medical School, Kochi, Japan.
Clin Cancer Res. 2000 Oct;6(10):3817-22.
Mutations of the von Hippel-Lindau (VHL) tumor suppressor gene have been detected in up to 60% of sporadic clear cell renal carcinomas (RCCs). Even patients with RCCs believed to be curable with radical nephrectomy sometimes develop distant metastasis 5-10 years after surgery, suggesting hematogenous circulation of cancer cells. Useful tumor markers have not yet been established for RCC. To detect patients at high risk of metastasis after surgery, we developed a highly sensitive and specific nested reverse transcription-PCR method using VHL gene mutation to detect circulating cancer cells. We screened 29 sporadic clear cell RCCs from patients for mutations of the VHL gene by direct sequencing. We next examined blood samples from patients with the VHL gene mutation using mutation-specific nested reverse transcription-PCR. Somatic mutations were detected in 20 of 29 (69.0%) sporadic clear cell RCCs. The VHL gene mutations were detected in peripheral and/or renal venous blood from 15 of 20 (75%) patients. The mutations were detected in the peripheral blood in 2 of 17 (11.8%) patients before surgery, 6 of 16 (37.5%) patients within 24 h after surgery, 3 of 16 (18.8%) patients on day 7 after surgery, and 2 of 11 (18.2%) patients on day 30 after surgery. In seven of nine (77.8%) patients, mutations were detected in renal venous blood during surgery. These findings indicate the presence of circulating cancer cells with VHL gene mutation. Although much larger studies are needed to determine the clinical significance, our study shows that this technique is feasible for detecting circulating RCC cells.
在高达60%的散发性肾透明细胞癌（RCC）中检测到了冯·希佩尔-林道（VHL）肿瘤抑制基因突变。即使是那些被认为可通过根治性肾切除术治愈的RCC患者，有时也会在术后5至10年发生远处转移，提示癌细胞发生了血行转移。目前尚未建立用于RCC的有效肿瘤标志物。为了检测术后有高转移风险的患者，我们开发了一种高度敏感且特异的巢式逆转录-聚合酶链反应（RT-PCR）方法，利用VHL基因突变来检测循环癌细胞。我们通过直接测序对29例散发性肾透明细胞癌患者的VHL基因进行了突变筛查。接下来，我们使用突变特异性巢式逆转录-聚合酶链反应检测了VHL基因突变患者的血样。在29例（69.0%）散发性肾透明细胞癌中，有20例检测到体细胞突变。在20例（75%）患者的外周血和/或肾静脉血中检测到了VHL基因突变。术前17例患者中有2例（11.8%）在外周血中检测到突变，术后24小时内16例患者中有6例（37.5%），术后第7天16例患者中有3例（18.8%），术后第30天11例患者中有2例（18.2%）。在9例患者中的7例（77.8%）手术期间在肾静脉血中检测到突变。这些发现表明存在带有VHL基因突变的循环癌细胞。尽管需要更大规模的研究来确定其临床意义，但我们的研究表明该技术对于检测循环肾癌细胞是可行的。
