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Single-cell phenotypic profiling to identify a set of immune cell protein biomarkers for relapsed and refractory
Yuan Shi1, Weidong Ding2, Weiying Gu2
1Department of hematology laboratory, The Third Affiliated Hospital of Soochow University, Changzhou, China.
Insights
Researchers identified novel immune cell biomarkers for diagnosing relapsed and refractory (R/R) Diffuse Large B-cell Lymphoma (DLBCL). This single-cell analysis overcomes heterogeneity, offering precise diagnostic tools for R/R DLBCL patients.
Area of Science:
- Immunology
- Oncology
- Biomarker Discovery
Background:
- Diffuse Large B-cell Lymphoma (DLBCL) is the most common non-Hodgkin lymphoma, with observed primary drug resistance.
- Prognoses in DLBCL are linked to cell-of-origin (COO) classification, yet specific biomarkers for relapsed and refractory (R/R)-DLBCL remain unclear.
Purpose of the Study:
- To identify and verify immune cell biomarkers at mRNA and protein levels in single-cell resolution for R/R DLBCL.
- To develop a precise diagnostic biomarker panel for R/R DLBCL, addressing intertumoral and intratumoral heterogeneity.
Main Methods:
- Employed single-cell RNA sequencing (scRNA-seq) on 8 R/R DLBCL patient samples and 5 healthy controls.
- Utilized mass cytometry (CyTOF) for validation on an external cohort of 10 R/R DLBCL patients and 5 healthy controls.
- Compared cell clustering and dimension reduction with COO and C-MYC expression, analyzing 32-dimensional phenotypic space.
Main Results:
- Identified a 35-surface marker gene panel for R/R DLBCL unique cell clusters via scRNA-seq, validated by CyTOF.
- Observed significant heterogeneity attributed to individual differences, not single subpopulations.
- Developed an optimized diagnostic biomarker panel for R/R DLBCL including CD82, CD55, CD36, CD63, CD59, IKZF1, CD69, CD163, CD14, CD226, CD84, and CD31.
Conclusions:
- Developed a novel set of biomarkers for diagnosing R/R DLBCL patients.
- Single-cell resolution detection provides precise biomarkers, overcoming sample heterogeneity.
- Confirmed individual case uniqueness and potential for multiple, genetically distinct subclones in R/R DLBCL.
Abstract:
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.
