PAXgene系统用于稳定慢性髓性白血病伊马替尼耐药患者骨髓RNA的适用性。

Suitability of the PAXgene system to stabilize bone marrow RNA in imatinib-resistant patients with chronic myeloid leukemia.

作者信息

Ernst Thomas, Hoffmann Jana, Erben Philipp, Hehlmann Rüdiger, Hochhaus Andreas, Müller Martin C

机构信息

III. Medizinische Klinik, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany.

出版信息

Clin Chem Lab Med. 2008;46(3):318-22. doi: 10.1515/CCLM.2008.086.

DOI:10.1515/CCLM.2008.086

PMID:18303987

Abstract

BACKGROUND

Optimum sample quality is a crucial requirement for molecular monitoring of patients with chronic myeloid leukemia (CML) on therapy. Bedside RNA stabilization systems (e.g., PAXgene) have been developed to inhibit RNA degradation during shipment of samples from the clinical site to the specialized laboratory. In CML, blood but not bone marrow samples have been examined using RNA stabilization in previous studies. Therefore, we sought to investigate the applicability of the PAXgene system for bone marrow samples in CML.

METHODS

Simultaneously stabilized blood and bone marrow samples were obtained from 55 imatinib-resistant CML patients to compare RNA yield and purity, expression of two housekeeping genes (total ABL and beta-glucuronidase; GUS) by quantitative reverse-transcriptase polymerase chain reaction, BCR-ABL expression (ratios BCR-ABL/ABL and BCR-ABL/GUS), and BCR-ABL kinase domain mutations analyzed by denaturing high-performance liquid chromatography and direct sequencing.

RESULTS

RNA extraction revealed high-quality RNA derived from both stabilized blood and bone marrow samples. RNA yield was significantly higher in bone marrow (median 9.9 microg RNA/mL bone marrow) than in blood (median 4.3 microg RNA/mL blood) (p=0.0005). The number of housekeeping gene transcripts was comparable in blood and bone marrow (median ABL copies/2 microL cDNA 13,260 vs. 25,590; median GUS copies/2 microL cDNA 35,490 vs. 60,200; n.s.). Further, ratios BCR-ABL/ABL (blood vs. bone marrow, median 47% vs. 57%) and ratios BCR-ABL/GUS (blood vs. bone marrow, median 26% vs. 21%) were not significantly different. Results of mutation analysis corresponded in 51 out of 55 patients (93%), whereas moderate differences were observed in four patients.

CONCLUSIONS

We conclude that bone marrow can be effectively stabilized using the PAXgene system and shows concordance with blood in terms of BCR-ABL mRNA quantification and mutation analysis in imatinib-resistant CML patients.

摘要

背景

对于接受治疗的慢性髓性白血病（CML）患者进行分子监测而言，最佳样本质量是一项关键要求。已开发出床边RNA稳定系统（如PAXgene），以抑制样本从临床位点运送至专业实验室过程中的RNA降解。在CML研究中，既往研究仅使用RNA稳定技术检测血液样本，而未检测骨髓样本。因此，我们试图探究PAXgene系统在CML骨髓样本中的适用性。

方法

从55例对伊马替尼耐药的CML患者中同时获取经稳定处理的血液和骨髓样本，以比较RNA产量和纯度、通过定量逆转录聚合酶链反应检测两个管家基因（总ABL和β-葡萄糖醛酸酶；GUS）的表达、BCR-ABL表达（BCR-ABL/ABL和BCR-ABL/GUS比值），以及通过变性高效液相色谱和直接测序分析BCR-ABL激酶结构域突变。

结果

RNA提取显示，经稳定处理的血液和骨髓样本均能获得高质量RNA。骨髓中的RNA产量（中位数9.9μg RNA/mL骨髓）显著高于血液（中位数4.3μg RNA/mL血液）（p = 0.0005）。血液和骨髓中管家基因转录本数量相当（ABL拷贝数/2μL cDNA中位数分别为13,260和25,590；GUS拷贝数/2μL cDNA中位数分别为35,490和60,200；无显著差异）。此外，BCR-ABL/ABL比值（血液与骨髓，中位数分别为47%和57%）和BCR-ABL/GUS比值（血液与骨髓，中位数分别为26%和21%）无显著差异。55例患者中有51例（93%）的突变分析结果一致，4例患者观察到中度差异。