Clinical Significance for detection of circulating renal cancer cells in the blood by RT-PCR

RT-PCR检测血液中循环肾癌细胞的临床意义

• 批准号: 12470337
• 负责人: HIAO Yoshihiko
• 金额: $ 8.38万
• 依托单位: Nara Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470337/
• 关键词: renal cell carcinoma; MN; CA9; antigen peptide; RT-PCR

Renal cell carcinoma is considered as a very poor prognostic disease due to absence of specific tumor marker and effective therapeutic modalities. Conventional treatments such as chemotherapy and radiotherapy are not useful at all. We are previously investigating to detect circulating renal cancer cells in the patient's blood and established RT-PCR assay to detect MN/CA9 positive cells in the peripheral blood. The blood-based RT-PCR resulted in detection of MN/CA9 mRNA in 76% of RCC patients. However; detection of MN/CA9 positive cells was observed in 30% of healthy volunteers (false positive). This may be because the skin expresses MN/CA9 antigen and aspiration of MN/CA9 positive cell when taking the blood via skin. Considering this situation, we try to use blood sample from renal vein during radical nephrectomy. To optimize PCR condition, we investigated 96 sets of PCR primers, and were able to set up blood-based RT-PCR with approximately 70 % sensitivity and 90 % specificity. We expected such high detection rates and no correlation with prognostic factors such as stage, grade and metastases, was observed. Based on these findings, we focused on cadherin-6 as an additional targeting molecule. Cadherin-6 is cell-cell adhesional molecule and considered as an as an important biomarker in renal cell carrinomas. We set up blood-based RT-PCR double assay targeting to MN/CA9 and Cadherin-6. Prospective study with blood samples from renal vein during radical nephrectomy revealed that significantly high detection rate of MN/CA9 and/or Cadherin-6 positive cells was observed in RCC patients with metastasis. This finding suggests that our double assay may be useful for diagnosis of RCC as well as early detection of metastasis.

由于缺乏特异的肿瘤标志物和有效的治疗方法，肾细胞癌被认为是一种预后很差的疾病。常规的治疗方法，如化疗和放射治疗根本没有用。我们之前正在调查检测患者血液中的循环肾癌细胞，并建立了RT-PCR方法来检测外周血中的MN/CA9阳性细胞。以血为基础的逆转录聚合酶链式反应检测到76%的肾癌患者MN/CA9基因表达。然而，在30%的健康志愿者中检测到MN/CA9阳性细胞(假阳性)。这可能是皮肤表达MN/CA9抗原，经皮采血时吸入MN/CA9阳性细胞所致。考虑到这种情况，我们尝试在根治性肾切除术中采用肾静脉采血。为了优化聚合酶链式反应的反应条件，我们考察了96套聚合酶链式反应引物，建立了灵敏度约为70%、特异度约为90%的血液RT-PCR方法。我们预计会有如此高的发现率，但没有观察到与分期、分级和转移等预后因素相关。基于这些发现，我们将重点放在钙粘蛋白-6作为另一种靶向分子上。钙粘蛋白-6是细胞间的黏附分子，被认为是肾细胞癌的重要生物标志物。我们建立了针对MN/CA9和Cherin-6的血基RT-PCR双重检测方法。对肾癌根治性切除术中肾静脉血标本的前瞻性研究显示，在有转移的肾癌患者中，MN/CA9和/或钙粘连蛋白-6阳性细胞的检出率显著高于非转移组。这一发现表明，我们的双重检测可能对肾癌的诊断以及转移的早期发现有用。

项目成果

植村天受: "腎細胞癌の新しいバイオマーカーMN/CA9"臨床泌尿器科. 55・5. 329-335 (2001)

Tenke Uemura：“肾细胞癌的新生物标志物 MN/CA9”《临床泌尿学》55・5（2001 年）。

植村天受: "腎細胞癌の新しいバイオマーカーMN/CA9"臨床泌尿器科. 55. 329-335 (2001)

Tenke Uemura：“肾细胞癌的新生物标志物 MN/CA9”《临床泌尿学》55. 329-335 (2001)。

Y Hirao, K Fujimoto, M Yoshii, N Tanaka, Y Hayashi, H Momose, S Samma, E Okajima, H Uemura, et al: "Non-ischemic Nephron-sparing Surgery for Small Renal Cell Carcinoma: Complete Tumor Enucleation Using a Microwave Tissue Coagulator"Jpn J Clin Oncol. 32. 9

Y Hirao、K Fujimoto、M Yoshii、N Tanaka、Y Hayashi、H Momose、S Samma、E Okajima、H Uemura 等人：“小肾细胞癌的非缺血性肾单位保留手术：使用微波完成肿瘤摘除术

仲川嘉紀, 植村天受, 清水一宏, 趙順規, 吉川元祥, 平尾佳彦 他: "泌尿器科腫瘍学における分子研究の展望:腎細胞癌の発癌・転移とMN/CA9"泌尿器科紀要. 47. 809-814 (2001)

Yoshiki Nakakawa、Tensuke Uemura、Kazuhiro Shimizu、Junki Cho、Motoyoshi Yoshikawa、Yoshihiko Hirao 等：“泌尿肿瘤学分子研究的前景：肾细胞癌和 MN/CA9 的癌变和转移”泌尿外科通报 47. 809。 -814 (2001)

M Cho, H Uemura, S-C Kim, Y Kawada, K Yoshida, Y Hirao, N Konishi, S Saga and K Yoshikawa: "Hypomethylation of the MN/CA9 promoter and upregulated MN/CA9 expression in human renal cell carcinoma"British Journal of Cancer. 85. 563-567 (2001)

M Cho、H Uemura、S-C Kim、Y Kawada、K Yoshida、Y Hirao、N Konishi、S Saga 和 K Yoshikawa：“人肾细胞癌中 MN/CA9 启动子的低甲基化和上调的 MN/CA9 表达”英国杂志