摘要目的:比较高通量测序（NGS）与Sanger测序在评估免疫球蛋白重链可变区（IGHV）基因体细胞高突变（SHM）状态中的应用差异，并着重探讨导致两种技术结果不一致的关键因素（尤其在复杂克隆背景下）。方法:回顾性分析2016-2021年于北京大学人民医院收集的Sanger测序结果显示为非单克隆（43例）和单克隆（10例）的淋系肿瘤样本，并采用NGS对其进行IGHV SHM检测。将两种方法的结果进行系统性比对，并从克隆丰度量化、引物设计差异及解读标准等角度，对结果不一致的病例进行分析。结果:53例同时进行Sanger测序及NGS检测的患者中，男36例，女17例，中位年龄64（33~88）岁。慢性淋巴细胞白血病35例（66.0%）、弥漫大B细胞淋巴瘤9例（17.0%）、滤泡性淋巴瘤3例（5.7%）、套细胞淋巴瘤3例（5.7%）、其他3例（5.7%）。在43例Sanger测序结果为非单克隆背景的样本中，NGS检测结果显示23例为双克隆或多克隆，17例为单克隆，3例未检出克隆性。Sanger与NGS结果的主要差异体现在克隆性评估、IGHV基因重排类型以及突变率方面。在10例Sanger测序结果为单克隆的病例中，NGS检测出2例双克隆，另有4例的IGHV重排类型与Sanger结果差异明显。尽管两种方法在SHM比例检测上存在细微差异，但对突变状态的整体判断无实质影响。结论:与Sanger测序相比，NGS在IGHV SHM的复杂克隆背景中更具优势，尤其在识别亚克隆成分和精确量化克隆比例方面更具敏感性和准确性，可为慢性淋巴细胞白血病等淋系肿瘤的精准诊断及预后评估提供更为精准的分子依据。

abstractsObjective:To compare the performance of next-generation sequencing (NGS) and Sanger sequencing in investigating somatic hypermutation (SHM) status of immunoglobulin heavy chain variable region (IGHV) genes. It specifically focuses on identifying key factors contributing to discrepancies between the two methods, particularly under complex clonal backgrounds, to inform optimized strategies for clinical application.Methods:This retrospective analysis included 53 samples, comprising 43 identified as non-monoclonal and 10 as monoclonal using Sanger sequencing. All samples were further analyzed using NGS to assess IGHV SHM. The two methods were used for systematic comparison. For discordant cases, in-depth attribution analysis was conducted, considering factors, including clonal abundance quantification, differences in primer design, and interpretation criteria.Results:Among the 53 patients who underwent both Sanger and NGS testing, 36 were male and 17 were female, with a median age of 64 years (range: 33–88). Diagnoses included chronic lymphocytic leukemia (CLL) in 35 (66.0% ), diffuse large B-cell lymphoma in 9 (17.0% ), follicular lymphoma in 3 (5.7% ), mantle cell lymphoma in 3 (5.7% ), and other types in 3 (5.7% ) cases. In the 43 cases with non-monoclonal profiles using Sanger sequencing, NGS revealed 23 cases as biclonal or polyclonal, 17 as monoclonal, and 3 with no detectable clonality. The primary discrepancies between the two methods involved variations in clonality assessment, IGHV gene rearrangement types, and mutation rates. Among the 10 cases identified as monoclonal using Sanger sequencing, NGS detected biclonality and markedly different IGHV rearrangement types in 2 and 4 cases, respectively. Minor differences were observed in SHM percentage between the two methods; however, these did not substantially affect the overall determination of mutational status.Conclusion:Compared with Sanger sequencing, NGS exhibits superior performance in assessing IGHV SHM status under complex clonal conditions. It provides greater sensitivity and accuracy in detecting subclonal components and quantifying clonal proportions, thereby providing a more precise molecular basis for diagnosing and prognostically assessing lymphoid malignancies, including CLL.