经典霍奇金淋巴瘤 (cHL) 的恶性霍奇金细胞和里德斯滕伯格 (HRS) 细胞在受影响的淋巴结中很少，这给检测驱动体细胞突变带来了挑战。作为细胞纯化技术的替代方案，我们假设超深度外显子组测序将允许对 HRS 细胞进行基因组研究，从而简化分析并避免技术陷阱。为了测试这一点，对 31 个 cHL 肿瘤/正常对进行了约 1000 倍中位覆盖深度的外显子组测序。正交纠错测序方法验证了超过 95% 的已发现突变。我们鉴定了 cHL 的新基因突变，包括：CDH5 和 PCDH7、IL4R 中新的停止增益突变，以及调节 Hippo 信号传导途径中新的复发突变模式。作为外显子组测序的进一步应用，我们试图在我们队列中的一名患者生成的单核 RNA-seq (snRNA-seq) 数据中识别表达的体细胞单核苷酸变异 (SNV)。我们的 snRNA 分析发现了一个清晰的细胞簇，其中包含我们的深层外显子组数据中发现的体细胞 SNV。该簇具有差异表达的基因，与已知在 HRS 细胞中失调的基因（例如 PIM1 和 PIM3）一致。该簇还包含具有扩展的 B 细胞克隆型的细胞，进一步支持恶性表型。这项研究提供了原理证明，即超深度外显子组测序可用于识别 HRS 细胞中的复发性突变，并证明了 snRNA-seq 在 cHL 背景下的可行性。这些研究为进一步分析大量 cHL 患者的基因组变异奠定了基础。

The malignant Hodgkin and Reed Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) are scarce in affected lymph nodes, creating a challenge to detect driver somatic mutations. As an alternative to cell purification techniques, we hypothesized that ultra-deep exome sequencing would allow genomic study of HRS cells, thereby streamlining analysis and avoiding technical pitfalls. To test this, 31 cHL tumor/normal pairs were exome sequenced to ~1000x median depth of coverage. An orthogonal error-corrected sequencing approach verified >95% of the discovered mutations. We identified mutations in genes novel to cHL including: CDH5 and PCDH7, novel stop gain mutations in IL4R, and a novel pattern of recurrent mutations in pathways regulating Hippo signaling. As a further application of our exome sequencing, we attempted to identify expressed somatic single nucleotide variants (SNVs) in single nuclei RNA-seq (snRNA-seq) data generated from a patient in our cohort. Our snRNA analysis identified a clear cluster of cells containing a somatic SNV identified in our deep exome data. This cluster has differentially expressed genes that are consistent with genes known to be dysregulated in HRS cells (e.g., PIM1 and PIM3). The cluster also contains cells with an expanded B-cell clonotype further supporting a malignant phenotype. This study provides proof-of-principle that ultra-deep exome sequencing can be utilized to identify recurrent mutations in HRS cells and demonstrates the feasibility of snRNA-seq in the context of cHL. These studies provide the foundation for the further analysis of genomic variants in large cohorts of cHL patients.