Shi Yan, Li Li-Zhen, Sun Jian-Zhi, Zhang Ti, Peng Jun, Xu Cong-Gao
Department of Hematology, Qilu Hospital, Shandong University, Jinan, 250012, People's Republic of China.
Ann Hematol. 2008 Jan;87(1):35-41. doi: 10.1007/s00277-007-0365-8. Epub 2007 Sep 11.
Currently, quantitative and semiquantitative assays for minimal residual disease detection include fluorescence in situ hybridisation, multiparameter flow cytometric immunophenotyping and real-time quantitative polymerase chain reaction (RQ-PCR). We have developed a new approach to detect hybrid breakpoint cluster region and Abelson proto-oncogene (BCR-ABL) transcripts inside suspension cells using in situ RT-PCR and light upon extension (LUX) primer, followed by rapid quantitative analysis with flow cytometry. After cellular permeabilization and fixation of single cell suspension, the neoplastic mRNA was reverse transcribed and amplified by PCR with LUX primer. The results demonstrated that a strong positive yellow-green signal was observed in 99-100% cells of K562 cell line, only the red nucleus was detected in NB4 cell line and normal controls. The technique has been utilised to study 12 patients with chronic myeloid leukemia, and the results were compared with those of BCR-ABL fusion mRNA by RT-PCR and BCR-ABL fusion gene of the interphase cells by fluorescence in situ hybridization (FISH). In the five diagnosed patients, 90-98% cells were strongly positive. Four patients, including three patients treated with interferon-alpha and hydroxyurea and one patient treated with imatinib mesylate, had 26-82.5% positive cells. Three patients treated with imatinib mesylate were negative. The in situ RT-PCR results demonstrated complete concordance with the results of I-FISH and RT-PCR. A fluorescence signal was detectable at 1/10(4) cells and became negative below this threshold with flow cytometry. The results of the present study suggest that (1) LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR; (2) the novel technique is a specific and sensitive way of detecting fusion gene with potential clinical usefulness.
目前，用于微小残留病检测的定量和半定量分析方法包括荧光原位杂交、多参数流式细胞免疫表型分析和实时定量聚合酶链反应（RQ-PCR）。我们开发了一种新方法，使用原位逆转录聚合酶链反应（RT-PCR）和延伸发光（LUX）引物来检测悬浮细胞内的杂交断点簇区域和Abelson原癌基因（BCR-ABL）转录本，随后通过流式细胞术进行快速定量分析。在单细胞悬液进行细胞通透化处理和固定后，肿瘤mRNA通过使用LUX引物的PCR进行逆转录和扩增。结果表明，在K562细胞系99% - 100%的细胞中观察到强烈的阳性黄绿信号，在NB4细胞系及正常对照中仅检测到红色细胞核。该技术已用于研究12例慢性髓性白血病患者，并将结果与通过RT-PCR检测的BCR-ABL融合mRNA以及通过荧光原位杂交（FISH）检测的间期细胞BCR-ABL融合基因的结果进行比较。在5例确诊患者中，90% - 98%的细胞呈强阳性。4例患者，包括3例接受α干扰素和羟基脲治疗的患者以及1例接受甲磺酸伊马替尼治疗的患者，有26% - 82.5%的阳性细胞。3例接受甲磺酸伊马替尼治疗的患者为阴性。原位RT-PCR结果与I-FISH和RT-PCR结果完全一致。通过流式细胞术在1/10⁴个细胞时可检测到荧光信号，低于此阈值则变为阴性。本研究结果表明：（1）LUX引物可用于通过细胞内RT-PCR有效检测BCR-ABL融合mRNA；（2）这项新技术是一种检测融合基因的特异性和灵敏方法，具有潜在临床应用价值。
