Background:Analysis of T-cell receptor (TCR) clonality is a major diagnostic tool for lymphomas, particularly for cutaneous T-cell lymphomas (CTCL) like Mycosis fungoides and Sézary syndrome. However, a fast and cost-effective workflow is needed to enable widespread use of this method.Methods: We established a procedure for TCR rearrangement analysis via Oxford Nanopore Technology (ONT) sequencing. TCR receptor rearrangements (TCR-gamma and TCR-beta chains) were analyzed in samples from 45 patients with various diagnoses: Mycosis fungoides (37/45), Sézary Syndrome (2/45), folliculotropic CTCL (1/45), and non-CTCL diagnoses as polyclonal controls (5/45). Sample types included formalin-fixed paraffin-embedded (FFPE) samples (27/45), fresh frozen samples (9/45), and CD3-isolated cells (9/45). In addition, DNA of a Jurkat cell line was used as a monoclonal control. TCR amplicons were generated employing an optimized version of the protocol from the Euro Clonality consortium. Sequencing was conducted on the ONT GridION and Illumina MiSeq platforms, followed by similar bioinformatic analysis protocols. The tumor clone frequency (TCF), a crucial prognostic factor for CTCL patients, was used for method comparison.Results:The use of an optimized amplicon protocol and adapted bioinformatic tools demonstrated a strong correlation in TCF values between both sequencing methods across all sample types (range R: 0.992–0.996; range r2: 0.984–0.991).Conclusions: In summary, ONT sequencing was able to detect TCR clonality comparable to NGS, indicating its potential as a faster and more cost-effective option for routine diagnostic use.
背景：T细胞受体（TCR）克隆性分析是淋巴瘤，特别是蕈样肉芽肿和Sézary综合征等皮肤T细胞淋巴瘤（CTCL）的重要诊断工具。然而，需要一种快速且经济高效的工作流程以实现该方法的广泛应用。 方法：我们建立了一种通过牛津纳米孔技术（ONT）测序进行TCR重排分析的操作流程。对45例不同诊断患者的样本进行了TCR受体重排（TCR-γ和TCR-β链）分析，包括：蕈样肉芽肿（37/45）、Sézary综合征（2/45）、毛囊性CTCL（1/45）以及作为多克隆对照的非CTCL诊断（5/45）。样本类型包括福尔马林固定石蜡包埋（FFPE）样本（27/45）、新鲜冷冻样本（9/45）和CD3分选细胞（9/45）。此外，使用Jurkat细胞系的DNA作为单克隆对照。采用欧洲克隆性联盟方案的优化版本生成TCR扩增子。分别在ONT GridION和Illumina MiSeq平台上进行测序，随后采用类似的生物信息学分析流程。使用CTCL患者的关键预后因子——肿瘤克隆频率（TCF）进行方法学比较。 结果：使用优化的扩增子方案和适配的生物信息学工具表明，在所有样本类型中，两种测序方法的TCF值均呈现高度相关性（R值范围：0.992–0.996；r²范围：0.984–0.991）。 结论：总之，ONT测序检测TCR克隆性的能力与NGS相当，表明其有潜力成为常规诊断中更快速、更经济高效的选择。
Nanopore Sequencing for T-Cell Receptor Rearrangement Analysis in Cutaneous T-Cell Lymphoma
肿瘤(癌症)患者之家