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Analytical evaluation of the clonoSEQ Assay for establishing measurable (minimal) residual disease in acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma.
BMC Cancer ( IF 3.8 ) Pub Date : 2020-06-30 , DOI: 10.1186/s12885-020-07077-9
Travers Ching 1 , Megan E Duncan 2 , Tera Newman-Eerkes 3 , Mollie M E McWhorter 4 , Jeffrey M Tracy 3 , Michelle S Steen 5 , Ryan P Brown 3 , Srivatsa Venkatasubbarao 5 , Nicholas K Akers 5 , Marissa Vignali 1 , Martin E Moorhead 3 , Drew Watson 6 , Ryan O Emerson 7 , Tobias P Mann 8 , B Melina Cimler 2 , Pamela L Swatkowski 2 , Ilan R Kirsch 9 , Charles Sang 10 , Harlan S Robins 11 , Bryan Howie 1 , Anna Sherwood 3
Affiliation  

The clonoSEQ® 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. Sensitivity 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. LoD 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 ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low. These studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.

中文翻译:

在急性淋巴细胞白血病,慢性淋巴细胞性白血病和多发性骨髓瘤中建立可测量(最小)残留疾病的clonoSEQ分析的分析评估。

ClonoSEQ®Assay(美国西雅图,自适应生物技术公司)通过下一代测序对恶性B细胞中IgH,IgK和IgL重排以及IgH-BCL1 / 2易位进行鉴定和跟踪与疾病相关的独特免疫球蛋白(Ig)序列。在这里,我们描述了一些研究,以验证使用患者样品和细胞系进行分析的分析性能。通过定义66例多发性骨髓瘤(MM),急性淋巴细胞白血病(ALL)患者的基因组DNA(gDNA)中的检出限(LoD),定量限(LoQ)和空白限(LoB),建立了敏感性和特异性。或慢性淋巴细胞性白血病(CLL)和三种细胞系。健康的供体gDNA用作稀释剂,用于配制具有特定DNA质量和恶性细胞频率的样品。使用从患者gDNA,健康供体gDNA和9个细胞系中提取的一系列样品来验证其准确性,以产生可跨越临床相关阈值的可测量残留疾病(MRD)频率。使用从掺入健康gDNA的细胞系gDNA中制成的样品确定线性,以便为每个DNA输入生成11个MRD频率,然后使用临床样品进行确认。通过(1)比较在健康单核细胞中稀释的ALL和MM细胞系的clonoSEQ和多参数流式细胞术(mpFC)测量,以及(2)分析精密研究数据中克隆gSEQ的MRD结果与gDNA稀释之间的偏差,来评估定量准确性。来自mpFC的原始未稀释样品。核苷酸碱基检出的可重复性是通过该测定方法在多个重复,过程特征和MRD水平上恢复恶性克隆型序列的能力来评估的。LoD和LoQ分别估计为1.903个细胞和2.390个恶性细胞。健康供体gDNA中的LoB为零。精度范围从较高DNA输入的18%CV(变异系数)到LoD附近的68%CV。方差成分分析表明,MRD结果很可靠,预期的实验室工艺变化对CV的贡献≤3%。证明了每种疾病在克隆频率数量级上的线性和准确性。核苷酸序列错误率极低。这些研究验证了clonoSEQ分析的分析性能,并证明了其作为针对所选淋巴恶性肿瘤的高度敏感的诊断工具的潜力。
更新日期:2020-06-30
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