Background: Multiplex PCR based on consensus primers followed by capillary electrophoresis and Sanger sequencing are considered as the gold standard method for the evaluation of clonality and somatic hypermutation in lymphoid malignancies. As an alternative, the next-generation sequencing (NGS) of immune receptor genes has recently been proposed as a solution, due to being highly effective and sensitive. Here, we designed a phase III diagnostic accuracy study intended to compare the current gold standard methods versus the first commercially available NGS approaches for testing immunoglobulin heavy chain gene rearrangements. Methods: We assessed IGH rearrangements in 68 samples by means of both the NGS approach (LymphoTrack®IGH assay, and LymphoTrack®IGH somatic hypermutation assay, run on Illumina MiSeq) and capillary electrophoresis/Sanger sequencing to assess clonality and somatic hypermutations (SHM). Results: In comparison to the routine capillary-based analysis, the NGS clonality assay had an overall diagnostic accuracy of 96% (63/66 cases). Other studied criteria included sensitivity (95%), specificity (100%), positive predictive value (100%) and negative predictive value (75%). In discrepant cases, the NGS results were confirmed by a different set of primers that provided coverage of the IGH leader sequence. Furthermore, there was excellent agreement of the SHM determination with both the LymphoTrack®FR1 and leader assays when compared to the Sanger sequencing analysis (84%), with NGS able to assess the SHM rate even in cases where the conventional approach failed. Conclusion: Overall, conventional Sanger sequencing and next-generation-sequencing-based clonality and somatic hypermutation analyses gave comparable results. For future use in a routine diagnostic workflow, NGS-based approaches should be evaluated prospectively and an analysis of cost-effectiveness should be performed.
背景：基于通用引物的多重PCR结合毛细管电泳与Sanger测序，是评估淋巴系统恶性肿瘤克隆性及体细胞超突变的金标准方法。新一代测序技术因其高效性与高灵敏度，近年来被提议作为检测免疫受体基因的替代方案。本研究设计了一项III期诊断准确性试验，旨在比较现行金标准方法与首个商用化NGS方案在免疫球蛋白重链基因重排检测中的效能。 方法：我们采用NGS方案（LymphoTrack®IGH检测及LymphoTrack®IGH体细胞超突变检测，于Illumina MiSeq平台运行）与毛细管电泳/Sanger测序两种方法，对68例样本的IGH重排进行克隆性及体细胞超突变评估。 结果：相较于常规毛细管电泳分析，NGS克隆性检测总体诊断准确率达96%（63/66例）。其他研究指标包括灵敏度（95%）、特异度（100%）、阳性预测值（100%）及阴性预测值（75%）。在结果不一致的病例中，NGS结果通过另一组覆盖IGH前导序列的引物得到验证。此外，与Sanger测序分析相比，LymphoTrack®FR1与前导序列检测在体细胞超突变判定方面具有高度一致性（84%），且NGS能在传统方法失效的病例中成功评估超突变率。 结论：传统Sanger测序与基于新一代测序的克隆性及体细胞超突变分析总体结果具有可比性。为推进NGS技术在常规诊断流程中的应用，需开展前瞻性评估并进行成本效益分析。
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