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; 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。）目标并假设标准化项目的第二阶段的主要目标是测试以下假设：在QRT-PCR测定中使用常见的引物和荧光探针，并具有常见的主要组合和标准操作方案（SOP）将降低每种实验室之间使用MMR之间的BCR-ABL转录副本数量的变异性，将其与每个实验室之间的情况相比。此外，将确定是否可以将实验室之间的最大差异降低到MMR范围内样品的小于1 log10的水平。该项目的次要目标是确定测定中的可变性源及其对测定总体差异的贡献。尽管样品将仅限于在CML（B3A2和B2A2）负细胞系中包含BCR-ABL两个主要变体中的细胞系的混合物，但测定的三个阶段将使用细胞，其总RNA和CDNA进行评估。这将评估分析的三个阶段中产生的误差：RNA隔离，逆转录和QPCR本身。由于预算限制，对测定的所有三个阶段的评估（即除了细胞外的RNA和cDNA测定）仅对B3A2变体进行。 b。）开发校准器Jessup Lab在对照中制备了SSRNA的标准参考材料（SRM）（HL-60总RNA），以充当校准器。 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T（9，22）（Q34，Q11）E13A2 = B2A2融合到ABL中； 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人类慢性骨髓性白血病细胞线，带有阳性的费城染色体。血液45：321-334，1975年，1975年； Gabert J，Beillard J，Beillard e，van der'vh''vh'vh'vh'vh'vh'vh'vh'vh'vh'vh'vh'vh'vh'vh'vh'vh'vh'vh'vh。融合基因转录的定量逆转录酶聚合酶链链反应在白血病中进行残留疾病检测 - 欧洲针对癌症计划的17：2318-57，2003）和2）b2a2融合基因Kcl-22细胞系（I Kubonishi，Y ohtsuki，y ohtsuki and yoshimosh and yoshimosh and yoshimososo and yoshimososo yoshimosh and yoshimosh and yoshimososo and yoshimosh and yoshimososo。慢性粒细胞性白血病细胞系（KCl-22）在体内。使用荧光杂交探针的负个体。白血病。 14：2006-10，2000）。将基因克隆并测序。在确认身份后，将T7启动子添加到引物和ssRNA中。对RT-PCR产物的分析表明，两个校准器是正确的大小，并且缺乏BCR-ABL融合基因的HL-60细胞中没有融合基因。将校准器的测试样本发送到七个参与联盟实验室中的每个实验室中的每个实验室，他们确认了他们在测试校准器中检测两个BCR-ABL变体的能力。然后，JESSUP实验室继续创建来自4 40,000个变体副本的日志稀释液，该副本以标准量的HL-60 RNA用作校准器样品，以创建标准曲线的测定法。 c。）研究设计七个实验室将评估来自细胞混合物，RNA和cDNA混合物样品样品的BCR-ABL转录水平。细胞混合物将包含各种比例的混合物HL-60（BCR-ABL融合转录物为阴性）和K562（B3A2融合变体的阳性）或KCl-22（B2A2融合变体阳性）。 RNA和cDNA混合物还包含HL-60中总RNA或cDNA的B3A2或B2A2变体的混合物，比例模仿细胞混合物中的浓度。商业套件的制造商为其QRT-PCR BCR-ABL临床测定法提供了一系列引物和探针，并将保持匿名。但是，它们是要在测定中测试的标准引物的来源，以减少实验室间差异。该提供商的另一个方面是使用标准操作协议（SOP）以及使用SOP下执行测定的标准试剂集。所有这些都向财团成员提供。此外，每个实验室将使用自己的基于CLIA的实验室测定法以及涉及标准引物的测定法，以便可以在每个实验室家庭酿造测定法与标准引物测定法之间进行比较。为了定义误差源，评估从细胞混合物中分离出的RNA的质量将很重要。该质量评估将包括260/280的比率和提取或准备的​​总RNA上的RIN编号，然后再在1步中使用，在1步中和/或实验室QRT-PCR分析中使用。此外，SRM SSRNA校准器将准备为两个变体建立标准曲线。这些校准器将由标准量的基本正常RNA组成，该RNA不包含带有不同量的B3A2或B2A2 SSRNA的BCR-ABL转录本。这将允许根据家用啤酒协议或通用协议产生的测定结果校准为标准单元（＃融合成绩单副本，每杯RNA），以进行整个实验室的比较。将为每个BCR-ABL融合基因变体创建标准曲线，然后将每个校准器的高浓度和低浓度都包含在每个测定中，以确保每次运行在校准曲线的可接受范围内。最后，当返回数据时，数据将进行准确性审核，然后进行生物识别研究分支的Lisa McShane博士，NCI将执行适当的统计分析，以评估是否已改善了实验室间可比性，并且在何处存在主要误差源。这将告知是否需要进一步继续该标准化项目。 b。）进度和未来方向：数据已返回，并证明使用常见引物和试剂可以协调该测定法，以使成绩单编号在所有实验室中彼此对数范围内。使用校准器与家庭酿造测定法仅略有改进的测定性能。 不幸的是，由于统计学家尚未进入该项目，因此我们已经在提交这些数据出版的情况下被困了半年。