Divisions of Blood and Marrow Transplantation and Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
Proc Natl Acad Sci U S A. 2011 Dec 27;108(52):21194-9. doi: 10.1073/pnas.1118357109. Epub 2011 Dec 12.
The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention to prevent clinical relapse; however, MRD quantification remains an uncommon diagnostic test because of logistical and financial barriers to widespread use. Here we describe a method for quantifying CLL MRD using widely available consensus primers for amplification of all Ig heavy chain (IGH) genes in a mixture of peripheral blood mononuclear cells, followed by high-throughput sequencing (HTS) for disease-specific IGH sequence quantification. To achieve accurate MRD quantification, we developed a systematic bioinformatic methodology to aggregate cancer clone sequence variants arising from systematic and random artifacts occurring during IGH-HTS. We then compared the sensitivity of IGH-HTS, flow cytometry, and allele-specific oligonucleotide PCR for MRD quantification in 28 samples collected from 6 CLL patients following allogeneic HCT. Using amplimer libraries generated with consensus primers from patient blood samples, we demonstrate the sensitivity of IGH-HTS with 454 pyrosequencing to be 10(-5), with a high correlation between quantification by allele-specific oligonucleotide PCR and IGH-HTS (r = 0.85). From the same dataset used to quantify MRD, IGH-HTS also allowed us to profile IGH repertoire reconstitution after HCT-information not provided by the other MRD methods. IGH-HTS using consensus primers will broaden the availability of MRD quantification in CLL and other B cell malignancies, and this approach has potential for quantitative evaluation of immune diversification following transplant and nontransplant therapies.
同种异体造血细胞移植 (HCT) 后慢性淋巴细胞白血病 (CLL) 不良预后的主要原因是疾病复发。HCT 后微小残留病 (MRD) 增加的检测可能允许早期干预以预防临床复发; 然而，由于广泛使用的后勤和财务障碍，MRD 定量仍然是一种不常见的诊断测试。在这里，我们描述了一种使用广泛可用的共识引物来扩增混合外周血单核细胞中的所有免疫球蛋白重链 (IGH) 基因，然后进行高通量测序 (HTS) 以对疾病特异性 IGH 序列进行定量的方法来检测 CLL 的 MRD。为了实现准确的 MRD 定量，我们开发了一种系统的生物信息学方法，用于聚合在 IGH-HTS 过程中出现的系统性和随机伪影引起的癌症克隆序列变体。然后，我们比较了 IGH-HTS、流式细胞术和等位基因特异性寡核苷酸 PCR 在 6 例接受同种异体 HCT 的 CLL 患者 28 个样本中的 MRD 定量的敏感性。使用来自患者血液样本的共识引物生成的扩增子文库，我们证明 454 焦磷酸测序的 IGH-HTS 灵敏度为 10(-5)，等位基因特异性寡核苷酸 PCR 和 IGH-HTS 的定量之间具有高度相关性 (r = 0.85)。从用于定量 MRD 的同一数据集，IGH-HTS 还使我们能够在 HCT 后分析 IGH 受体库的重建信息-其他 MRD 方法无法提供的信息。使用共识引物的 IGH-HTS 将扩大 CLL 和其他 B 细胞恶性肿瘤中 MRD 定量的可用性，并且这种方法具有在移植和非移植治疗后定量评估免疫多样化的潜力。
