Single Cell Phenotypic Profiling of 27 DLBCL Cases Reveals Marked Intertumoral and Intratumoral Heterogeneity

Abstract

Diffuse large B‐cell lymphoma (DLBCL) is the most common histologic subtype of non‐Hodgkin lymphoma and is notorious for its clinical heterogeneity. Patient outcomes can be predicted by cell‐of‐origin (COO) classification, demonstrating that the underlying transcriptional signature of malignant B‐cells informs biological behavior in the context of standard combination chemotherapy regimens. In the current study, we used mass cytometry (CyTOF) to examine tumor phenotypes at the protein level with single cell resolution in a collection of 27 diagnostic DLBCL biopsy specimens from treatment naïve patients. We found that malignant B‐cells from each patient occupied unique regions in 37‐dimensional phenotypic space with no apparent clustering of samples into discrete subtypes. Interestingly, variable MHC class II expression was found to be the greatest contributor to phenotypic diversity. Within individual tumors, a subset of cases showed multiple phenotypic subpopulations, and in one case, we were able to demonstrate direct correspondence between protein‐level phenotypic subsets and DNA mutation‐defined subclones. In summary, CyTOF analysis can resolve both intertumoral and intratumoral heterogeneity among primary samples and reveals that each case of DLBCL is unique and may be comprised of multiple, genetically distinct subclones. © 2019 International Society for Advancement of Cytometry

Mass cytometry defines distinct immune profile in germinal center B-cell lymphomas

“…To our knowledge, this is the first immune profiling of the myeloid compartment in lymphoma tumors by mass cytometry. Prior work has characterized T cells and B cells in FL, DLBCL, and HL using mass cytometry [38,40,41] and characterized myeloid cells in lung and breast cancer [11,13]. The resolution of the complex, heterogeneous lymphoma microenvironment by mass cytometry was striking when compared to prior low-dimensional studies measuring fewer than eight markers.…”

TBL1XR1 Mutations Drive Extranodal Lymphoma by Inducing a Pro-tumorigenic Memory Fate

“…To further support the notion that C5/MCD reflects a MB COO, we conducted CyTOF on patients from an independent DLBCL cohort (Table S6) (Nissen et al, 2019). Cells from reactive LN specimens were used to define B cell subpopulations.…”

“…Clinic-pathological characteristics of these cases are detailed in the original publication. For CyTOF immunoprofiling, diagnostic pre-treatment LN biopsies from patients with DLBCL, or non-malignant ''reactive'' LN (rLN) were acquired from the lymphoma tumor bank at the BC Cancer Agency (as described in Nissen et al, 2019), and were selected based on sufficient numbers of viable cells for CyTOF analysis (2 million per sample). Clinic-pathological characteristics of these cases are detailed in the original publication.…”

“…Viable cells were gated in FlowJo based on negative staining for cisplatin, and all subsequent analysis was performed using R. Malignant tumor cells were identified as previously described (Nissen et al, 2019). Isolated malignant cells and manually-defined germinal center B cells and memory B cells were then pooled together for principal component analysis.…”

ISSUE HIGHLIGHTS ‐ March 2020

“…A very special barcoding technique using palladium isotopes has been applied in the paper by GK Behbehani (Behbehani et al, ), where an extensive study of myelodysplastic syndromes (MDS) by 34‐parameter mass cytometry is illustrated. Mass cytometry is a mature analytical technique that can be applied today outside its traditional research boundaries, as shown in many recent valuable papers on leukemia, myelodysplasia (Zeng et al, ; Das Gupta et al, ; Nissen et al, ; Wierz et al, ; Bandyopadhyay et al, ), immunological studies (Leipold et al, ) and even erythroid development (Thomson‐Luque et al, ). Mass cytometry allows for the analysis of cell heterogeneity on a very large scale, theoretically measuring up to 100 different cell features simultaneously with metal isotope‐tagged antibodies without any spectral overlap (Olsen et al, ).…”