National Program to Standardize the BCR-ABL qRT-PCR Assay for CML

CML BCR-ABL qRT-PCR 检测标准化国家计划

• 批准号: 8157693
• 负责人: John Jessup
• 金额: $ 1.84万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157693
• 关键词: Biological Assay; bcr-abl Fusion Proteins; programs

a.) Objectives and Postulates The primary objective of the second phase of the standardization project was to test the hypothesis that use of common primers and fluorescent probe in the qRT-PCR assay with a common lot of master mix and a Standard Operating Protocol (SOP) will reduce the variability in BCR-ABL transcript copy number per g RNA at MMR between labs compared to the situation of each lab using its own home brew method for clinical qRT-PCR assays. Further, it will be determined whether predicted maximum magnitude of difference between labs can be reduced to the level of less than 1 log10 for samples in the MMR range. A secondary objective of this project is to determine the source(s) of variability within the assay and their contribution to the overall variance in the assay. Although the samples will be limited to mixtures of cell lines that contain either of the two major variants of BCR-ABL in CML (b3a2 and b2a2) in a negative cell line, the three phases of the assay will be assessed using the cells, their total RNA and cDNA. This will assess the errors generated during the three phases of the assay: RNA isolation, reverse transcription, and qPCR itself. Due to budgetary constraints, the assessment of all three phases of the assay (i.e., assay of RNA and cDNA in addition to cells) will occur only for the b3a2 variant. b.) Development of Calibrators The Jessup lab prepared Standard Reference Materials (SRM) of ssRNA in control (HL-60 total RNA) to act as calibrators. The two major variants of BCR-ABL are b3a2 and b2a2 in which the exons of BCR are fused to Abl as part of the Ph1+ translocation t (9, 22) (q34, q11) e13a2 = b2a2; e14a2 = b3a2. The sources of SRM were the following cell lines: 1) for the b3a2 fusion gene the K562 cell line (CB Lozzio and BB Lozzio Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 45: 321-334, 1975; Gabert J, Beillard E, van der Velden VH, et al. Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program. Leukemia 17:2318-57, 2003) and 2) for the b2a2 fusion gene the KCL-22 cell line (I Kubonishi, Y Ohtsuki, S Yoshimoto and I Miyoshi. Heterotransplantation and maturation of a chronic myelogenous leukemia cell line (KCL-22) in vivo. Int. J. of Cell Cloning, 2, 243-253, 1984; Saussele S, Weisser A, Mller MC, Emgi M, La Rose P, Paschka P, Kuhn C, Willer A, Hehlmann R, Hochhaus A. Frequent polymorphism in BCR exon b2 identified in BCR-ABL positive and negative individuals using fluorescent hybridization probes. Leukemia. 14:2006-10, 2000). The genes were cloned and sequenced. After confirmation of identity T7 promoters were added to the primers and ssRNA created. Analysis of products of RT-PCR indicated that the the two calibrators were the correct size and that the fusion genes were absent from HL-60 cells that lack either BCR-ABL fusion gene. Test samples of the calibrators were sent to each of the seven participating consortium labs who confirmed their ability to detect both BCR-ABL variants in the test calibrators. The Jessup lab then proceeded with creating log dilutions from 4 40,000 variant copies in a standard amount of HL-60 RNA to be used as the calibrator samples for creation of standard curves for the assays. c.) Study design Seven laboratories will assess the BCR-ABL transcript level of samples from cell mixtures, samples of RNA and cDNA mixtures. The cell mixtures will contain mixtures, in various proportions, of the cell lines HL-60 (negative for BCR-ABL fusion transcripts) and K562 (positive for b3a2 fusion variant) or KCL-22 (positive for the b2a2 fusion variant). The RNA and cDNA mixtures also contain mixtures of either the b3a2 or b2a2 variant added to total RNA or cDNA from HL-60 in proportions that mimic the concentrations in the cell mixtures. A manufacturer of a commercial kit has provided the set of primers and probes for their qRT-PCR BCR-ABL clinical assay under an approved UBMTA and will remain anonymous. However, they are the source of the standard primers to be tested in the assay as a means of reducing interlaboratory variance. One other aspect of this provider is the use of a Standard Operating Protocol (SOP) and the use of a standard set of reagents with which to perform the assay under the SOP. All of these were provided to the consortium members. Additionally, each laboratory will use its own CLIA-based laboratory assay as well as the assay involving the standard primers so that a comparison can be made between each laboratorys Home Brew assay and the Standard Primers assay. In order to define the sources of error, it will be important to assess the quality of RNA isolated from the cell mixtures. This quality assessment will include both 260/280 ratio and the RIN number on the extracted or prepared total RNA before it is used in the 1-step and/or the labs qRT-PCR assay. In addition, SRM ssRNA calibrators will be prepared to establish standard curves for both variants. These calibrators will consist of a standard amount of essentially normal RNA that does not contain BCR-ABL transcripts spiked with varying amounts of either b3a2 or b2a2 ssRNA. This will allow for assay results produced under either the home brew protocols or the common protocol to be calibrated to standard units (# fusion transcript copies per mug RNA) for comparison across labs. The standard curve will be created for each BCR-ABL fusion gene variant and then high and low concentrations of each calibrator will be included in each assay run to assure that each run is within acceptable limits for the calibration curve. Finally, when the data are returned, the data will be subjected to audits for accuracy and then Dr. Lisa McShane of the Biometric Research Branch, NCI will perform appropriate statistical analysis to assess whether interlaboratory comparability has been improved and where the major sources of error exist. This will inform whether there is a need to continue this standardization project further. b.) Progress and Future Directions: The data have been returned and demonstrate that use of common primers and reagents harmonize the assay so that the transcript numbers were within a log of each other across all the labs. Use of the calibrator with the home brew assay only marginally improved assay performance. Unfortunately, we have been stymed for half a year in getting these data submitted for publication because the statistician has not gotten to the project.

a.) 目标和假设 标准化项目第二阶段的主要目标是测试以下假设：在 qRT-PCR 测定中使用通用引物和荧光探针，并使用通用批次的预混液和标准操作方案 (SOP) ）与每个实验室使用自己的自制方法进行临床 qRT-PCR 检测的情况相比，将减少实验室之间 MMR 下每 g RNA 的 BCR-ABL 转录本拷贝数的变异性。此外，还将确定对于 MMR 范围内的样本，实验室之间的预测最大差异是否可以降低到小于 1 log10 的水平。该项目的第二个目标是确定测定中变异性的来源及其对测定中总体方差的贡献。尽管样品仅限于阴性细胞系中含有 CML 中 BCR-ABL 的两种主要变体（b3a2 和 b2a2）之一的细胞系混合物，但将使用细胞、其总 RNA 和 cDNA。这将评估检测三个阶段中产生的错误：RNA 分离、逆转录和 qPCR 本身。由于预算限制，仅对 b3a2 变体进行所有三个检测阶段的评估（即除细胞外还对 RNA 和 cDNA 进行检测）。 b.) 校准品的开发 Jessup 实验室制备了对照ssRNA（HL-60 总RNA）的标准参考材料（SRM）作为校准品。 BCR-ABL 的两个主要变体是 b3a2 和 b2a2，其中 BCR 的外显子作为 Ph1+ 易位的一部分与 Abl 融合 t (9, 22) (q34, q11) e13a2 = b2a2； e14a2 = b3a2。 SRM的来源是以下细胞系：1)对于b3a2融合基因，K562细胞系(CB Lozzio和BB Lozzio人慢性髓性白血病细胞系，具有阳性费城染色体。Blood 45：321-334，1975；Gabert J 、Beillard E、van der Velden VH 等人“实时”定量逆转录酶聚合酶的标准化和质量控制研究。用于白血病残留疾病检测的融合基因转录物的链式反应 - 欧洲抗癌计划 17:2318-57, 2003) 和 2) 用于 KCL-22 细胞系的 b2a2 融合基因 (I Kubonishi, Y Ohtsuki, S)。 Yoshimoto 和 I Miyoshi。慢性粒细胞白血病细胞系 (KCL-22) 的体内异种移植和成熟。 Int. J. of Cell Cloning，2, 243-253, 1984；Saussele S、Weisser A、Mller MC、Emgi M、La Rose P、Paschka P、Kuhn C、Willer A、Hehlmann R、Hochhaus A。使用荧光杂交探针在 BCR-ABL 阳性和阴性个体中鉴定 BCR 外显子 b2。白血病。 14:2006-10, 2000）。基因被克隆并测序。确认身份后，将 T7 启动子添加到引物中并创建 ssRNA。 RT-PCR产物分析表明，两种校准品的大小正确，并且缺乏BCR-ABL融合基因的HL-60细胞中不存在融合基因。校准器的测试样品被发送到七个参与联盟实验室中的每一个，这些实验室确认了他们有能力检测测试校准器中的两种 BCR-ABL 变体。然后，Jessup 实验室用标准量的 HL-60 RNA 中的 4 个 40,000 个变异拷贝进行对数稀释，用作校准样品，用于创建测定的标准曲线。 c.) 研究设计 七个实验室将评估细胞混合物样品、RNA 和 cDNA 混合物样品的 BCR-ABL 转录水平。细胞混合物将含有不同比例的细胞系 HL-60（BCR-ABL 融合转录物阴性）和 K562（b3a2 融合变体阳性）或 KCL-22（b2a2 融合变体阳性）细胞系的混合物。 RNA 和 cDNA 混合物还含有 b3a2 或 b2a2 变体的混合物，以模拟细胞混合物中浓度的比例添加到来自 HL-60 的总 RNA 或 cDNA 中。商业试剂盒的制造商已根据经批准的 UBMTA 提供了用于 qRT-PCR BCR-ABL 临床测定的引物和探针组，并且将保持匿名。然而，它们是测定中待测试的标准引物的来源，作为减少实验室间差异的手段。该提供商的另一个方面是使用标准操作协议 (SOP) 以及使用一组标准试剂来根据 SOP 进行测定。所有这些都提供给财团成员。此外，每个实验室将使用自己的基于 CLIA 的实验室测定以及涉及标准引物的测定，以便可以在每个实验室的 Home Brew 测定和标准引物测定之间进行比较。为了确定误差来源，评估从细胞混合物中分离的 RNA 的质量非常重要。该质量评估将包括提取或制备的总 RNA 在用于 1 步和/或实验室 qRT-PCR 测定之前的 260/280 比率和 RIN 编号。此外，还将准备 SRM ssRNA 校准品来为两种变体建立标准曲线。这些校准品将由标准量的基本正常 RNA 组成，不含 BCR-ABL 转录本，并掺有不同量的 b3a2 或 b2a2 ssRNA。这将允许将在家庭酿造方案或通用方案下产生的测定结果校准到标准单位（每杯 RNA 的融合转录本拷贝数），以便在实验室之间进行比较。将为每个 BCR-ABL 融合基因变体创建标准曲线，然后在每次测定运行中包含每种校准物的高浓度和低浓度，以确保每次运行都在校准曲线的可接受限度内。最后，当数据返回后，数据将接受准确性审核，然后 NCI 生物识别研究分部的 Lisa McShane 博士将进行适当的统计分析，以评估实验室间可比性是否得到改善以及主要误差来源在哪里存在。这将告知是否需要进一步继续该标准化项目。 b.) 进展和未来方向：数据已返回，并证明使用通用引物和试剂可以协调测定，从而使所有实验室的转录本数量在彼此的对数范围内。使用校准器进行自制测定仅略微提高了测定性能。 不幸的是，由于统计学家还没有参与该项目，我们在提交这些数据以供发布方面已经受阻了半年。