Single-cell phenotypic profiling to identify a set of immune cell protein biomarkers for relapsed and refractory diffuse large B cell lymphoma: A single-center study

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.