Landon Pastushok, S. Sarda, K. Mochoruk, Wayne Hill, Loni Pickle, Michelle A. Toro, Carolina Gonzalez, Stephanie Ostresh, T. Looney, Chenchen Yang, Julie Stakiw, Mark J. Bosch, Hadi Goubran, C. Geyer, G. Lowman, J. Decoteau
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We evaluated the Oncomine BCR Pan-Clonality Assay, a novel single-tube PCR reaction that simultaneously amplifies all BCR loci for next-generation DNA sequencing, using neoplastic B-cell lines and clinical research samples from multiple myeloma (MM) patients, a plasma cell neoplasm associated with high SHM levels. The assay showed a linear detection range down to 1 ng of clonal DNA input, sensitivity to 10−6 in a polyclonal background, and high reproducibility. Clonotype lineages were identified in 42/45 (93%) MM samples. Ion Reporter software packaged with the assay permitted straightforward identification of MM subgroups. As expected, SHM was identified in 94% of MM cases, but several unexpected subgroups were identified including biased IGHV3-11 or IGHV4-34 usage in 20% of MM samples, and two cases with very low levels of SHM. Evidence of intraclonal diversity/ongoing SHM was identified in 18% of samples, suggesting a possible germinal center origin for some MM cases. 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引用次数: 0
摘要
B 细胞肿瘤具有克隆 B 细胞受体重排(BCR 克隆型系),可通过对 B 细胞重排进行测序来识别,用于诊断、风险分层和高灵敏度监测。BCR体细胞高突变(SHM)会导致PCR引物结合区的点突变造成克隆检测失败,通常需要将样本分成多个反应,这增加了检测成本、周转时间和样本要求。我们使用来自多发性骨髓瘤(MM)患者的肿瘤性 B 细胞系和临床研究样本对 Oncomine BCR 泛克隆性检测进行了评估,这是一种新型的单管 PCR 反应,可同时扩增所有 BCR 基因座以进行下一代 DNA 测序。该检测方法的线性检测范围可低至 1 纳克克隆 DNA 输入量,在多克隆背景下灵敏度可达 10-6,重现性高。在 42/45 个(93%)MM 样本中确定了克隆型系。与该检测法配套的 Ion Reporter 软件可直接鉴定 MM 亚群。不出所料,94% 的 MM 病例中都鉴定出了 SHM,但也鉴定出了几个意想不到的亚组,包括 20% 的 MM 样本中偏向使用 IGHV3-11 或 IGHV4-34,以及两个 SHM 水平很低的病例。在18%的样本中发现了克隆内多样性/持续SHM的证据,这表明一些MM病例可能起源于生殖中心。单管 Oncomine BCR 泛克隆性测定能有效检测 BCR 克隆型系,其检测率与现有的多反应测定相当,并能为细胞起源研究和淋巴瘤分类确定其特征。
A Novel Single-Tube Next Generation Sequencing Assay for B-Cell Receptor Clonality Testing
B-cell neoplasms possess clonal B-cell receptor rearrangements (BCR clonotype lineages) that can be identified by sequencing the B-cell repertoire for use in diagnostics, risk stratification, and high-sensitivity monitoring. BCR somatic hypermutation (SHM) can result in clonality detection failure from point mutations in PCR primer binding regions, often necessitating splitting samples into multiple reactions which increases test costs, turnaround times, and sample requirements. We evaluated the Oncomine BCR Pan-Clonality Assay, a novel single-tube PCR reaction that simultaneously amplifies all BCR loci for next-generation DNA sequencing, using neoplastic B-cell lines and clinical research samples from multiple myeloma (MM) patients, a plasma cell neoplasm associated with high SHM levels. The assay showed a linear detection range down to 1 ng of clonal DNA input, sensitivity to 10−6 in a polyclonal background, and high reproducibility. Clonotype lineages were identified in 42/45 (93%) MM samples. Ion Reporter software packaged with the assay permitted straightforward identification of MM subgroups. As expected, SHM was identified in 94% of MM cases, but several unexpected subgroups were identified including biased IGHV3-11 or IGHV4-34 usage in 20% of MM samples, and two cases with very low levels of SHM. Evidence of intraclonal diversity/ongoing SHM was identified in 18% of samples, suggesting a possible germinal center origin for some MM cases. The single-tube Oncomine BCR Pan-Clonality assay efficiently detects BCR clonotype lineages at rates comparable to existing multiple reaction assays and permits their characterization for cell of origin studies and lymphoma classification.