慢性髓性白血病中不同类型bcr-abl转录本的实时定量分析

Real-time quantification of different types of bcr-abl transcript in chronic myeloid leukemia.

作者信息

Amabile M, Giannini B, Testoni N, Montefusco V, Rosti G, Zardini C, Terragna C, Buonamici S, Ottaviani E, Soverini S, Fiacchini M, Bassi S, de Vivo A, Trabacchi E, Saglio G, Pane F, Baccarani M, Tura S, Martinelli G

机构信息

Institute of Hematology and Medical Oncology "Seràgnoli", University of Bologna, Italy.

出版信息

Haematologica. 2001 Mar;86(3):252-9.

PMID:11255271

Abstract

BACKGROUND AND OBJECTIVES

The most common translocation in chronic myeloid leukemia (CML) t(9;22) (q34;q22) produces the BCR/ABL fusion gene. We set up and evaluated a rapid and reliable real-time reverse-transcription-polymerase chain reaction (RT-PCR) approach using TaqMan technology for detection and quantification of bcr-abl transcripts in CML patients at diagnosis and during therapy.

DESIGN AND METHODS

A pair of primers and probe complementary to ABL exon 2 were designed, enabling detection of the most frequent bcr-abl transcripts, and also of the normal ABL-Ia transcript as an internal control. Conditions were established to amplify less than 1(-10) target molecules/reaction and detect one CML cell in 10(6) cells from healthy donors. To determine the utility of the assay, we quantified the bcr-abl/ABL-Ia ratio in 59 bone marrow samples (45 samples with evidence of different Ph+ chromosome percentages and 14 samples in complete cytogenetic remission) from 48 CML patients, 34 of them at diagnosis and 14 in clinical remission (CR). In 14 cases, this ratio was compared with results obtained by a competitive-quantitative RT-PCR/capillary electrophoresis method from contemporary specimens.

RESULTS

By real-time RT-PCR, the median value of bcr-abl/ABL-Ia ratio at diagnosis was 15.334 (range 3.3-28.81) and fell to 0.9 (range 0.003-26.1) in CR. The median value of bcr-abl/ABL-Ia ratio at cytogenetic remission was 0.7 (range 0.003-2.83). The real-time bcr-abl/ABL-Ia ratios correlated with those obtained by competitive RT-PCR (p < 0.0001) and the percentage of Ph+ metaphases (p < 0.0001). The high sensitivity and specificity of the real-time RT-PCR procedure was confirmed in all 14 patients with minimal residual disease. INTERPRETATION AND CONCLUSIONS. We conclude that this real-time RT-PCR procedure is a reliable and sensitive method of monitoring CML patients after therapy, and that the bcr-abl/ABL-Ia ratio correlates strongly with cytogenetic analysis.

摘要

背景与目的

慢性髓性白血病（CML）中最常见的易位t(9;22) (q34;q22)产生BCR/ABL融合基因。我们建立并评估了一种使用TaqMan技术的快速可靠的实时逆转录-聚合酶链反应（RT-PCR）方法，用于检测和定量CML患者诊断时及治疗期间的bcr-abl转录本。

设计与方法

设计了一对与ABL外显子2互补的引物和探针，能够检测最常见的bcr-abl转录本，同时也能检测正常的ABL-Ia转录本作为内对照。确定了扩增条件，使每个反应中少于1(-10)个靶分子，并能从健康供体的10(6)个细胞中检测出1个CML细胞。为了确定该检测方法的实用性，我们对48例CML患者的59份骨髓样本（45份有不同Ph+染色体百分比证据的样本和14份完全细胞遗传学缓解的样本）中的bcr-abl/ABL-Ia比值进行了定量，其中34例在诊断时进行检测，14例处于临床缓解期（CR）。在14例病例中，将该比值与同期样本通过竞争性定量RT-PCR/毛细管电泳方法获得的结果进行了比较。

结果

通过实时RT-PCR，诊断时bcr-abl/ABL-Ia比值的中位数为15.334（范围3.3 - 28.81），在CR时降至0.9（范围0.003 - 26.1）。细胞遗传学缓解时bcr-abl/ABL-Ia比值的中位数为0.7（范围0.003 - 2.83）。实时bcr-abl/ABL-Ia比值与竞争性RT-PCR获得的比值相关（p < 0.0001），也与Ph+中期细胞百分比相关（p < 0.0001）。在所有14例微小残留病患者中证实了实时RT-PCR方法的高敏感性和特异性。