Molecular detection of circulating renal cell carcinoma cells by RT-PCR

一种针对人肾细胞癌（RCC）的小鼠单克隆抗体G250 (MAbG25O) （Oosterwijk, et al, 1986）已知能与很大比例的RCC起反应，但对正常肾组织不起作用。MAbG25O识别的抗原存在于质膜中，并在几种类型的恶性肿瘤中表达。最近，G250抗原基因被分离出来（Oosterwijk et al. 1995），数据库分析显示G250抗原与原来源于Hela细胞的MN/CA IX相同（Pastorek et al. 1994）。为了确定G250抗原（MN/CA IX/G250）是否可以作为潜在的治疗靶点和肿瘤标志物，我们对147例RCC进行了免疫组织化学和逆转录聚合酶链反应（RT-PCR）分析。此外，从患者外周血样本中提取总rna，分析MN/CA9/G250 mRNA信号。免疫组化结果显示，在128/147（87.1%）的RCC中表达强烈，而在正常组织中则不表达。对冷冻标本进行RT-PCR分析，137/147（93.2%）中明确检测到MN/CA9/G25OmRNA信号，尽管存在细微差异，但结果与免疫组织化学几乎相同。虽然高分级和分期肿瘤的表达明显低于低分级和分期肿瘤，但有很大比例的肿瘤表达MN/CA9 IX/G250蛋白和mRNA。患者血液样本的RT-PCR分析显示存在循环MN/CA9/G25O表达细胞。这些发现表明，该抗原可能是一种潜在的治疗靶点，也是rcc的诊断标志物。

A murine monoclonal antibody G250 (MAbG25O) raised against human renal cell carcinoma (RCC)(Oosterwijk, et al, 1986) has been known to react with a large proportion of RCC, but not with the norma1 kidney tissue. The antigen recognized by MAbG25O is present in the plasma membrane and expresses in several types of malignancies. Recently, G250 antigen gene has been isolated (Oosterwijk et al. 1995), and database analysis revealed that G250 antigen is identical to MN/CA IX originally deribed from Hela cell (Pastorek et al, 1994). To determine whether G250 antigen (MN/CA IX/G250) could be a potential therapeutic target and a tumor marker, a total of 147 cases of RCC were investigated immunohistochemically as well as, by reverse transcriptase polymerase chain reaction (RT-PCR) anilysis. In addition, total RNAs extracted from patients' peripheral blood samples were analyzed for MN/CA9/G250 mRNA signals. Immunohistochenistry resulted in strong expression in 128/147 (87.1%) of RCC, in contrast to the lack of expression observed in normal tissues. RT-PCR analyses of frozen specimens resulted in the clear detection of MN/CA9/G25OmRNA signals in 137/147 (93.2 %), and despite of the subtle differences, the results were almost identical to those for immunohistochemistry. Although high grade and stage tumors exhibited significaitly lower expression than low grade and stage tumors, a large proportion of tumors expressed MN/CA9 IX/G250 protein as well as mRNA.RT-PCR analysis of patients' blood samples revealed the presence of circulating MN/CA9/G25O expressing cells. These findings suggest that this antigen may be a potential therapeutic target as well as diagnostic marker for RCCs.