基于 PCR 的下一代测序方法检测和定量急性淋巴细胞白血病和多发性骨髓瘤微小残留病的验证，使用 gBlocks 作为校准品。

Validation of a PCR-Based Next-Generation Sequencing Approach for the Detection and Quantification of Minimal Residual Disease in Acute Lymphoblastic Leukemia and Multiple Myeloma Using gBlocks as Calibrators.

机构信息

Molecular Hematology Laboratory, Vrije Universiteit Brussel, Universitair Ziekenhuis Brussel, Brussels, Belgium.

Department of Hematology, Vrije Universiteit Brussel, Universitair Ziekenhuis Brussel, Brussels, Belgium.

出版信息

J Mol Diagn. 2021 May;23(5):599-611. doi: 10.1016/j.jmoldx.2021.01.009. Epub 2021 Feb 4.

DOI:10.1016/j.jmoldx.2021.01.009

PMID:33549860

Abstract

Detection of minimal residual disease (MRD) to guide therapy has been a standard practice in treatment of childhood acute lymphoblastic leukemia (ALL) for decades. In multiple myeloma (MM), a clear correlation is found between absence of MRD and longer survival. Quantitative allele-specific oligonucleotide (qASO)-PCR is the standard molecular method for MRD detection in these hematologic malignant tumors. However, this technique has some drawbacks that can be overcome by next-generation sequencing (NGS). In this study, NGS is validated as an alternative method for qASO-PCR for MRD detection in both ALL and MM. MRD results obtained by NGS and qASO-PCR were compared in 59 and 39 bone marrow samples of 33 and 14 patients with ALL and MM, respectively. Our results indicate that the use of gBlocks as calibrators makes the NGS approach a powerful tool to quantify MRD. With an input of 400 ng of DNA (corresponding to approximately 7 × 10 cells), a limit of detection of 0.01% can be achieved. The specificity of the NGS-MRD technique was 100%, and a correlation with qASO-PCR for quantifiable MRD results of 0.93 and 0.91 was found in ALL and MM, respectively. Especially for MM, the higher applicability (100%) of the NGS-MRD protocol, compared with qASO-PCR (57%), was clearly demonstrated. These results demonstrate that NGS is an even better alternative to qASO-PCR.

摘要

检测微小残留病（MRD）以指导治疗已经成为儿童急性淋巴细胞白血病（ALL）治疗的标准做法已有数十年。在多发性骨髓瘤（MM）中，MRD 阴性与更长的生存时间之间存在明确的相关性。定量等位基因特异性寡核苷酸（qASO）-PCR 是这些血液恶性肿瘤中 MRD 检测的标准分子方法。然而，这种技术存在一些缺点，可以通过下一代测序（NGS）来克服。在这项研究中，NGS 被验证为 qASO-PCR 替代方法，可用于 ALL 和 MM 中的 MRD 检测。将 NGS 和 qASO-PCR 分别在 33 名 ALL 患者和 14 名 MM 患者的 59 份和 39 份骨髓样本中进行了比较。结果表明，使用 gBlocks 作为校准物使 NGS 方法成为定量 MRD 的有力工具。用 400ng DNA（相当于大约 7×10 个细胞）作为输入，可以达到 0.01%的检测极限。NGS-MRD 技术的特异性为 100%，在 ALL 和 MM 中分别发现与 qASO-PCR 检测可定量的 MRD 结果的相关性为 0.93 和 0.91。特别是对于 MM，与 qASO-PCR（57%）相比，NGS-MRD 方案的适用性（100%）更高。这些结果表明 NGS 是 qASO-PCR 的更好替代方法。