Department of hematology laboratory, The Third Affiliated Hospital of Soochow University, Changzhou, China.
Department of Hematology, The Third Affiliated Hospital of Soochow University, Changzhou, China.
J Leukoc Biol. 2022 Dec;112(6):1633-1648. doi: 10.1002/JLB.6MA0822-720RR. Epub 2022 Aug 30.
Diffuse large B-cell lymphoma (DLBCL) is the most common invasive type of non-Hodgkin lymphoma. Cell-of-origin (COO) classification is related to patients' prognoses. Primary drug resistance in treatment for DLBCL has been observed. The specific serum biomarkers in these patients who suffer from relapsed and refractory (R/R)-DLBCL remains unclear. In the current study, using single-cell RNA sequencing (scRNA-seq) and mass cytometry (CyTOF), we determined and verified immune cell biomarkers at the mRNA and protein levels in single-cell resolution from 18 diagnostic PBMC specimens collected from patients with R/R DLBCL. As controls, 5 PBMC specimens from healthy volunteers were obtained. We identified a panel of 35 surface marker genes for the features of R/R DLBCL unique cell cluster by scRNA-seq of 8 R/R DLBCL patient samples and validated its efficiency in an external cohort consisting of 10 R/R DLBCL patients by CyTOF. The cell clustering and dimension reduction were compared among R/R DLBCL samples in CyTOF Space with COO as well as the C-MYC expression designation. Immune cells from each patient occupied unique regions in the 32-dimensional phenotypic space with no apparent clustering of samples into discrete subtypes. Significant heterogeneity observed in subgroups was mainly attributed to individual differences among samples and not to expression differences in a single, homogeneous immune cell subpopulation. The marker panel showed reliability in labeling R/R DLBCL without any influence from COO stratification and C-MYC expression designation. Furthermore, we compared all the markers between R/R DLBCL and normal samples. A total of 12 biomarkers were significantly overexpressed in R/R DLBCL relative to the normal samples. Therefore, we further optimized the diagnostic biomarker panel of R/R DLBCL comprising CD82, CD55, CD36, CD63, CD59, IKZF1, CD69, CD163, CD14, CD226, CD84, and CD31. In summary, we developed a novel set of biomarkers for the diagnoses of patients with R/R DLBCL. Detections procedures at single-cell resolution provide precise biomarkers, which may substantially overcome intertumoral and intratumoral heterogeneity among primary samples. The findings confirmed that each case was unique and may comprise multiple, genetically distinct subclones.
弥漫性大 B 细胞淋巴瘤（DLBCL）是最常见的侵袭性非霍奇金淋巴瘤。细胞起源（COO）分类与患者的预后相关。在 DLBCL 的治疗中已经观察到原发性药物耐药。在患有复发和难治性（R/R）-DLBCL 的这些患者中，具体的血清生物标志物仍然不清楚。在本研究中，我们使用单细胞 RNA 测序（scRNA-seq）和质谱流式细胞术（CyTOF），从 18 名 R/R DLBCL 患者的诊断性 PBMC 标本中以单细胞分辨率确定和验证了 mRNA 和蛋白质水平的免疫细胞生物标志物。作为对照，从 5 名健康志愿者中获得 5 个 PBMC 标本。我们通过对 8 名 R/R DLBCL 患者样本的 scRNA-seq 鉴定了一个由 35 个表面标记基因组成的面板，用于 R/R DLBCL 独特细胞簇的特征，并通过 CyTOF 验证了其在由 10 名 R/R DLBCL 患者组成的外部队列中的效率。在 CyTOF Space 中，我们比较了 R/R DLBCL 样本中的细胞聚类和降维与 COO 以及 C-MYC 表达指定之间的关系。来自每个患者的免疫细胞在 32 维表型空间中占据独特的区域，样本没有明显聚类为离散亚型。在亚组中观察到的显著异质性主要归因于样本之间的个体差异，而不是单个同质免疫细胞亚群中的表达差异。该标记面板在不进行 COO 分层和 C-MYC 表达指定的情况下可靠地标示 R/R DLBCL。此外，我们比较了 R/R DLBCL 和正常样本之间的所有标记。与正常样本相比，R/R DLBCL 中有 12 个标志物显著过表达。因此，我们进一步优化了包含 CD82、CD55、CD36、CD63、CD59、IKZF1、CD69、CD163、CD14、CD226、CD84 和 CD31 的 R/R DLBCL 诊断生物标志物的面板。总之，我们为 R/R DLBCL 患者的诊断开发了一套新的生物标志物。单细胞分辨率的检测程序提供了精确的生物标志物，这可能极大地克服了原发性样本中的肿瘤间和肿瘤内异质性。研究结果证实，每个病例都是独特的，可能包含多个遗传上不同的亚克隆。
