Analytical evaluation of the clonoSEQ Assay for establishing measurable (minimal) residual disease in acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma
Analytical evaluation of the clonoSEQ Assay for establishing measurable (minimal) residual disease in acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma
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DOI：
10.1186/s12885-020-07077-9
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发表时间：
2020-06-30
期刊：
影响因子：
3.8
通讯作者：
Sherwood, Anna
中科院分区：
文献类型：
--
作者：
Ching, Travers;Duncan, Megan E.;Sherwood, Anna
ClonoSEQ(R)Assay(美国西雅图自适应生物技术公司)通过对恶性B细胞中IgH、IgK和IGL重排以及IgH-BCL1/2易位的下一代测序来识别和跟踪独特的疾病相关免疫球蛋白(Ig)序列。方法通过对66例多发性骨髓瘤(MM)、急性淋巴细胞白血病(ALL)或慢性淋巴细胞白血病(CLL)患者的基因组DNA(GDNA)中检测限(LOD)、定量限(LOQ)和空白限(LOB)的定义，建立了灵敏度和特异性。以健康供体gDNA为稀释剂，制备具有特定DNA质量和恶性细胞频率的样品。使用从患者gDNA、健康供者gDNA和9个细胞系设计的一系列样本来验证精确度，以产生跨越临床相关阈值的可测量残留病(MRD)频率。使用从细胞系gDNA中提取的样本添加到健康的gDNA中，为每个DNA输入产生11个MRD频率，然后使用临床样本进行验证，来确定线性。通过(1)比较在健康单个核细胞中稀释的ALL和MM细胞系的ClonoSEQ和多参数流式细胞术(MPFC)测量，以及(2)分析稀释gDNA的ClonoSEQ MRD结果与基于原始未稀释样本的mPFC预期结果之间的偏差来评估定量准确性。通过该方法恢复多个重复的恶性克隆序列的能力、过程特征和MRD水平来评估核苷酸碱基调用的重复性。结果LOD和LOQ估计分别为1.903个细胞和2.390个恶性细胞。健康供者gDNA中LOB为零。精密度范围从较高DNA输入时的18%变异系数(CV)到LOD附近的68%变异系数。方差分量分析表明，MRD结果是稳健的，预期的实验室过程变化也起到了作用
BackgroundThe clonoSEQ (R) Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines.MethodsSensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay's ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels.ResultsLoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing