Mühlbauer M, Langenbach N, Stolz W, Hein R, Landthaler M, Buettner R, Bosserhoff A K
Department of Dermatology, University of Regensburg Medical School, Germany.
Clin Cancer Res. 1999 May;5(5):1099-105.
The detection of tumor-specific mRNA transcripts in the blood of patients by reverse transcription (RT)-PCR has been used as a very sensitive technique for determining systemically disseminated tumor cells. On the basis of previous expression studies, we aimed to trace melanoma cells in the blood of melanoma patients by RT-PCR of melanoma-inhibitory activity (MIA) mRNA. To detect sensitively MIA transcripts in total RNA isolated from peripheral blood mononuclear cells (PBMCs), we established a sensitive PCR-ELISA system. With this assay, we detected one melanoma cell in 2 ml of blood by a single round of 32 PCR cycles. A total of 295 PBMC samples isolated from 166 patients with melanocytic tumors were tested with the MIA RT-PCR-ELISA: (a) 58 patients (99 samples) with malignant melanomas in stage I; (b) 49 patients (65 samples) with malignant melanomas in stage II; and (c) 47 patients (116 samples) with metastasized melanomas (stages III and IV), with an additional 12 patients (15 samples) with benign melanocytic nevi. Forty-four (26.8%) of 164 samples isolated from patients with melanomas in stages I and II were positive for MIA mRNA; in stages III/IV, 33 (28.4%) of 116 samples of patients, irrespective of clinically evident disease, were positive. Eleven (84.6%) of 13 PBMC samples from patients with metastasized melanoma and clinically evident disease without treatment were MIA mRNA-positive in contrast to only 19 (25.7%) of 74 samples isolated from patients in stage IV with metastasis during chemotherapy. Furthermore, none of the 16 PBMC samples of patients in stage IV without clinically detectable metastases at that time point during chemotherapy was MIA mRNA-positive. Interestingly, of the 44 positive samples (26.8%) isolated from patients with melanomas in stages I and II, 20 were still positive when retested after complete excision of the tumor. Our results reveal that amplification of MIA mRNA from the PBMCs of patients with malignant melanomas by PCR-ELISA provides a useful means to detect tumor cells in the systemic blood circulation. A correlation between positive blood samples and tumor burden in stages III and IV was detected, and, in addition, a significant effect of chemotherapy with respect to the reduction of the number of systemically spread tumor cells was observed. However, MIA amplification seems to be of little value as a surrogate marker for clinical staging or the detection of metastatic disease.
通过逆转录（RT）-PCR检测患者血液中的肿瘤特异性mRNA转录本，已被用作一种非常灵敏的技术来确定全身播散的肿瘤细胞。基于先前的表达研究，我们旨在通过对黑色素瘤抑制活性（MIA）mRNA进行RT-PCR来追踪黑色素瘤患者血液中的黑色素瘤细胞。为了灵敏地检测从外周血单个核细胞（PBMC）中分离的总RNA中的MIA转录本，我们建立了一种灵敏的PCR-ELISA系统。通过该检测方法，经过一轮32次PCR循环，我们在2ml血液中检测到一个黑色素瘤细胞。用MIA RT-PCR-ELISA检测了从166例黑素细胞肿瘤患者中分离的295份PBMC样本：（a）58例（99份样本）I期恶性黑色素瘤患者；（b）49例（65份样本）II期恶性黑色素瘤患者；（c）47例（116份样本）转移性黑色素瘤（III期和IV期）患者，另外还有12例（15份样本）良性黑素细胞痣患者。从I期和II期黑色素瘤患者中分离的164份样本中有44份（26.8%）MIA mRNA呈阳性；在III/IV期，116份患者样本中有33份（28.4%）呈阳性，无论临床疾病是否明显。13份来自转移性黑色素瘤且有临床明显疾病但未接受治疗的患者的PBMC样本中有11份（84.6%）MIA mRNA呈阳性，相比之下，从IV期化疗期间有转移的患者中分离的74份样本中只有19份（25.7%）呈阳性。此外，在化疗期间该时间点临床未检测到转移的IV期患者的16份PBMC样本中，没有一份MIA mRNA呈阳性。有趣的是，从I期和II期黑色素瘤患者中分离的44份阳性样本（26.8%）中，有20份在肿瘤完全切除后重新检测时仍为阳性。我们的结果表明，通过PCR-ELISA从恶性黑色素瘤患者的PBMC中扩增MIA mRNA，为检测全身血液循环中的肿瘤细胞提供了一种有用的方法。检测到III期和IV期血液样本阳性与肿瘤负荷之间存在相关性，此外，观察到化疗对减少全身播散肿瘤细胞数量有显著效果。然而，MIA扩增作为临床分期或检测转移性疾病的替代标志物似乎价值不大。
