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

一个)。标准化项目第二阶段的主要目标是验证这样一个假设，即与每个实验室使用自己的自制方法进行临床qRT-PCR分析相比，在使用共同的主混合物和标准操作方案（SOP）的qRT-PCR分析中使用共同的引物和荧光探针，可以减少实验室之间在MMR中每g RNA BCR-ABL转录本拷贝数的差异。此外，还将确定在MMR范围内的样品，实验室之间预测的最大差异幅度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的来源及其对测定总体变异的贡献。虽然样品将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）中的任一种的细胞系的混合物，但检测的三个阶段将使用细胞及其总RNA和cDNA进行评估。这将评估在测定的三个阶段产生的错误：RNA分离，逆转录和qPCR本身。由于预算限制，检测的所有三个阶段（即除了细胞外的RNA和cDNA检测）的评估将仅针对b3a2变体进行。b。)Jessup实验室制备了ssRNA对照（HL-60总RNA）标准参比物（SRM）作为校准器。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的来源为：1)b3a2融合基因的K562细胞系（CB Lozzio和BB Lozzio）费城染色体阳性的人慢性髓性白血病细胞系。中国科学进展（英文版）；1975；张建军，张建军，张建军，等。标准化和质量控制研究的“实时”定量逆转录酶聚合酶链反应融合基因转录物残留疾病检测白血病-欧洲抗癌计划。I Kubonishi, Y Ohtsuki， S Yoshimoto和I Miyoshi。b2a2融合基因在KCL-22细胞系中的应用。慢性粒细胞白血病细胞系（KCL-22）的体内异源移植和成熟。Int。中国生物医学工程学报（英文版），2004；Saussele S, Weisser A, Mller MC, Emgi M, La Rose P, Paschka P, Kuhn C, Willer A, Hehlmann R, Hochhaus A. BCR- abl阳性和阴性个体BCR外显子b2多态性的荧光杂交检测。白血病。14:2006-10,2000)。这些基因被克隆并测序。在确认身份后，将T7启动子添加到引物中，并创建ssRNA。RT-PCR分析结果表明，两种校正器的大小正确，并且在缺乏BCR-ABL融合基因的HL-60细胞中均缺失融合基因。校准器的测试样本被发送到七个参与联盟的实验室，这些实验室确认它们有能力在测试校准器中检测到BCR-ABL变体。杰瑟普实验室随后在标准量的HL-60 RNA中创建了440,000个变体拷贝的对数稀释液，用作创建检测标准曲线的校准样品。c。)七个实验室将评估来自细胞混合物、RNA和cDNA混合物样品的BCR-ABL转录水平。细胞混合物将包含不同比例的细胞系HL-60 （BCR-ABL融合转录本阴性）和K562 （b3a2融合变体阳性）或KCL-22 （b2a2融合变体阳性）的混合物。RNA和cDNA混合物也含有b3a2或b2a2变体的混合物，以模拟细胞混合物中的浓度的比例添加到HL-60的总RNA或cDNA中。一家商业试剂盒制造商已根据批准的UBMTA为其qRT-PCR BCR-ABL临床检测提供了一套引物和探针，该制造商将保持匿名。然而，作为减少实验室间差异的一种手段，它们是在分析中要测试的标准引物的来源。该供应商的另一个方面是使用标准操作协议（SOP），并使用一套标准试剂，根据SOP进行分析。所有这些都提供给了财团成员。此外，每个实验室将使用自己的基于clia的实验室分析以及涉及标准引物的分析，以便可以在每个实验室的自酿分析和标准引物分析之间进行比较。为了确定误差的来源，评估从细胞混合物中分离的RNA的质量将是重要的。该质量评估将包括260/280比率和提取或制备的总RNA在用于一步法和/或实验室qRT-PCR检测之前的RIN数。此外，将准备SRM ssRNA校准器，以建立两种变体的标准曲线。这些校准器将由标准量的基本正常RNA组成，不含BCR-ABL转录本，其中添加了不同量的b3a2或b2a2 ssRNA。这将允许在家庭酿造协议或通用协议下产生的分析结果被校准到标准单位（每个杯子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，然后每种校准剂的高浓度和低浓度将包括在每次检测中，以确保每次运行在校准曲线的可接受范围内。最后，当数据返回时，将对数据的准确性进行审计，然后NCI生物计量研究部的Lisa McShane博士将进行适当的统计分析，以评估实验室间的可比性是否得到了改善，以及主要的误差来源在哪里。这将告知是否需要进一步继续这个标准化项目。b。)进展和未来方向：数据已经返回，并证明使用共同的引物和试剂协调分析，因此转录本编号在所有实验室的一个日志内。使用校准器与家庭酿造化验只略微提高化验性能。不幸的是，由于统计学家还没有参与到项目中，我们已经花了半年的时间才将这些数据提交出版。

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.