Introduction
Double-hit or Triple-hit lymphoma (DHL/THL) is an uncommon subset of B cell non-Hodgkin lymphoma with recurrent translocations involving
MYC/8q24 and
BCL2/18q21 and/or
BCL6/3q27, according to the 2016 updated WHO classification of lymphoid neoplasias [
1]. DHL displays a highly heterogeneous profile with complex genomic alterations, which results in aggressive clinical manifestations and inferior prognosis upon current therapeutic approaches, regardless of targeted therapy or intensive chemotherapy [
2,
3].
Despite the increasing clinical interest in DHL and the corresponding precise treatment, few deep explorations of pathogenesis at cellular and molecular level were carried out, and the previous DHL research had mainly focused on genetics and flow cytometric analysis with a limited number of markers measured per single cell [
4‐
6]. On the other hand, the immune system has many important regulatory roles in cancer development and progression, while the immune cell population is of great importance for the function and status of the immune system. Therefore, evaluating immune cell populations of DHL could be a perspective to dig the information behind the clinical phenotype and be possible to uncover immunological mechanisms and identify biomarkers that might aid in precision therapies [
7,
8]. Most of the published immune cell profiling of cancers was based on tumor tissue for biomarker identification and analysis [
9‐
11], this is reasonable since the immune cell composition of the tumor site closely reflects the interactions between the cancer cells and immune system. However, acquiring tissue is quite challenging since this approach it is an invasive procedure. Instead, analyses of peripheral blood offer a noninvasive and simpler way to monitor immune cells (peripheral blood mononuclear cells - PBMCs) that ultimately can infiltrate the TME, and therefore may provide valuable information about the status of immune system surrounding the tumor cells [
12]. As regards to the approach to characterizing the immune cell of peripheral blood, mass cytometry or CyTOF (cytometry by time of flight) has emerged as a revolutionary technology in single-cell proteomics, enabling a comprehensive understanding of cell phenotype [
13]. CyTOF displays tremendous advantages over the conventional flow cytometer, especially the feasibility to measure more than 50 markers per cell in limited samples.
In the present study, we characterized the immune cell profile of a cohort of peripheral blood samples from DHL patients, with blood samples from DLBCL patients and healthy people as the control, and CyTOF was employed with a range of surface markers. This study aimed to understand the discrepancy of peripheral immune cell composition of DHL compared to DLBCL and healthy people and thus identify potential biomarkers for pharmaceutical and clinical utility.
Materials and methods
Patient selection
This study was conducted in accordance with the guidelines of the Institutional Review Board of Zhejiang Cancer Hospital. 11 cases of B-cell lymphoma associated with
MYC and
BCL2 (4 cases),
MYC and
BCL6 (5 cases), or
MYC,
BCL2 and
BCL6 rearrangements (2 cases) confirmed by fluorescence in situ hybridization (FISH) studies were identified (Supplementary Table 2). Morphologic, immunophenotypic, and cytogenetic data were reviewed to further confirm the diagnosis and classification according to the 2016 World Health Organization. More clinical information of DHL patients was listed in Table
1. Besides, 4 cases of DLBCL diagnosed at the same institute were identified; pathologic and cytogenetic data were reviewed for all cases to confirm the diagnosis and the lack of rearrangements of
MYC,
BCL2, and
BCL6. In addition, PBMC sample of 7 cases of healthy people were purchased from commercial source (Sailybio).
Table 1
Clinical characteristics of DHL patients
age < 60 diagnosis | 6 |
age > 60 diagnosis | 5 |
Sex (n = 11)
|
male | 6 |
female | 5 |
Disease stage
|
I/II | 4 |
III/IV | 7 |
B symptoms
|
No | 8 |
Yes | 3 |
Abdominal lymph nodes
|
No | 1 |
Yes | 10 |
Marrow involved
|
No | 10 |
Yes | 1 |
GCB or NGCB
|
GCB | 5 |
NGCB | 6 |
IPI
| |
0–1 | 3 |
2 | 3 |
3 | 2 |
4–5 | 3 |
CNSIPI
| |
0–1 | 3 |
2 | 4 |
4–5 | 4 |
TP53
mutation
|
No | 7 |
Yes | 3 |
Undefined | 1 |
PBMC sample preparation
Dilute the fresh blood samples with PBS buffer and slowly add the diluted blood sample to the upper layer of the Lymphocyte Separation Medium (Beijing Solarbio), centrifuge at 800 g for 20 min, suck the middle layer of PBMC cells and filter them with a membrane filter (100 µM), then wash twice with PBS buffer.
Antibody
All the antibodies were purchased from Fluidigm, some of them are pre-labeled (with Lanthanide metal) antibodies, and the others are purified antibodies and manually conjugated with metals by using MCP9 or X8 single metal labeling kits (Fluidigm) according to the manufacturer’s instructions. The detailed antibody panel was listed in the Supplementary Table 1.
Staining of surface proteins and nuclear proteins
Cells in PBMC samples were stained with 0.5 µM cisplatin in PBS without Ca2+ and Mg2+ at room temperature to distinguish dead from living cells. The cisplatin-stained cells were blocked with FcR Blocking Solution (BioLegend), followed by incubation with a prepared mixture of metal-conjugated antibodies at 4 °C for 30 min. Cells were washed and incubated again for 30 min at 4 °C with the prepared mixture of metal-conjugated nucleoprotein antibodies. After the second round of washing, the cells were fixed with 1.6% formaldehyde solution for 10 min. Then, the cells were incubated with Intercalator-Ir with a final concentration of 125 nM at 4 °C overnight.
Data acquisition
The stained cells were washed and resuspended with Cell Acquisition Solution (CAS) with the cell concentration at around 1.1 × 106/mL, 10% EQ Beads (EQ beads: CAS = 1:9) were then added to the cell suspension. Adherent cell clumps were filtered using a membrane filter (35 µM), followed by data acquisition on a Helios (Fluidigm) at 300 event/s.
Data preprocessing
The data were gated to identify cell events and the exclusion of dead cells by using Cytobank (premium.cytobank.cn). The live cells were then gated to separate the CD45+ cell for subsequent clustering and high dimensional analyses.
Dimensionality reduction and clustering
All the FCS files obtained from the last step were exported from cytobank and the data were input as R using the flowCore R language package. Preprocessed data were down-sampled to a maximum of 10,000 cells per sample and merged into a single dataset, followed by arcsine transformation to normalize the data.
For dimensionality reduction and clustering of the data, we performed principal component analysis using variable genes, followed by dimensionality reduction of the data by t-stochastic neighbor embedding (t-SNE) analysis to obtain a visual 2D image. Next, the dataset is clustered by the module optimization algorithm, and each cluster is manually annotated according to the biomarker expression.
TP53 gene mutation
Peripheral blood samples sent to Tianjin Hematology Research Institute, then p53 (17p13) probe kit produced by Anbiping Pharmaceutical Technolog (Guangzhou, China) was used to detect TP53 gene mutation by fluorescence in situ hybridization (FISH).
Statistical analysis
Significant differences were analyzed using paired Student’s t-test or analysis of variance, and p < 0.05 was considered significant.
Discussion
CyTOF is a powerful high-dimensional single-cell immune system analysis platform that can simultaneously analyze more than 50 markers at the single-cell level. It integrates the advantage of high-speed analysis of traditional flow cytometers and the high-resolution capability of mass spectrometry detection. To date, no study has been done to investigate the comprehensive landscape of peripheral circulating immune cells in DHL patients using CyTOF technology. In this study, we evaluated the PBMC cells extracted from the peripheral blood of DHL patients for high-throughput analysis by CyTOF, and used PBMC samples from healthy people and DLBCL patients as controls. The subsets of peripheral circulating immune cells in patients were determined, and the causes and effects of differences in their abundance were analyzed.
The 5th edition of the World Health Organization Classification of Lymphoid Neoplasms was published in 2022, and only the high-grade B cell lymphoma involving
MYC and
BCL2 rearrangements was retained in this entity (DHL), while lymphoid neoplasms with dual
MYC and
BCL6 rearrangements are now classified either as a subtype of DLBCL, NOS or HGBL, NOS according to their cytomorphological features [
20]. Nonetheless, all the peripheral blood samples in this study were acquired before 2022, and we still considered the DLBCL with
MYC and
BCL2 and/or
BCL6 rearrangement as DHL/THL according to the 2016 World Health Organization classification of lymphoma.
In the analysis of T cells, we found the absolute number of TEM cells and TEMRA cells were elevated in patients, and increased expression of PD-1 was observed in several TEM subsets. CD8
+ TEMRA cells are associated with immunosenescence and have been found at a high level in age-related chronic inflammatory diseases [
14], whilst PD-1 is often regarded as a biomarker of T cell exhaustion, all of which suggests that T cell population of the patients was ongoing with cellular senescence and depletion. We also noticed that the portion of double-negative T cells (DNT) significantly declined in patients. DNTs is a kind of natural suppressor (NS) cells which was characterized as abnormal regulatory T cells with αβ-TCR and CD25 expression but no CD4 or CD8 expression [
21,
22]. Despite the small portion of DNTs in peripheral lymphocytes, it had been demonstrated that DNTs have strong suppressive ability towards CD8
+ T cells, CD4
+ T cells, B cells and NK cells in vitro and in vivo, which leads to xenograft or allograft transplantation tolerance and powerful prevention of GVHD (graft-versus-host disease) [
23,
24]. DNTs was also proved to play a role of tumor suppressor with the ability to suppress the tumor growth of lung cancer and inhibit cell proliferation of acute myeloid leukemia [
25‐
27]. In addition, Fang et al. demonstrated that the growth of non-small cell lung cancer could be inhibited in vitro by using a combination of PD-1 blockade and DNT cell therapy [
28]. Considering T cell depletion occurred in both DHL and DLBCL patients, as well as the tumor suppressor role of DNT cells, the level of DNT cells could be an indication of the prognosis of DHL, and DNT cells and their derivatives can be novel therapeutic agents as monotherapy or combination with PD-1 blockade for the treatment of DHL. Furthermore, significant enrichment of DPT cell subsets and Treg cell subsets were detected in the DHL sample compared to the DLBCL group and healthy group, while DPT had been found in various cancers, including T-cell lymphomas, with abnormally high abundance [
29‐
31].
Among the B cell subsets, the proportion of plasma cell subsets in patients was significantly higher than that in healthy people, indicating that a severe humoral immune response occurred in patients. We also detected a cohort of B cells with negative expression of CD38 that was significantly different in enrichment between DHL patients and the other two groups. Of particular note, CD38 is highly expressed in two naïve B cell subsets that are only enriched in DHL patients. As a type II transmembrane glycoprotein, the highly expressed CD38 is an important factor in the poor prognosis of B cell lymphoma [
32,
33]. This suggests that CD38 could be a biomarker for diagnosis (distinguishing from other DLBCL subtypes) or prognosis of DHL. The cancerous B cells of DHL may originally be derived from these naïve B cells and display high expression of CD38, therefore, CD38 could be a potential therapeutic target of DHL [
34], but need further validation by proper models, such as PDX model.
As regards to monocytes, research has shown that classic monocytes are important for the initial inflammatory response, while non-classical monocytes are widely believed to have anti-inflammatory effects [
35,
36]. In this analysis, we found a non-classical monocyte subset inconsistent with existing reports [
15], namely the ncMO2 cell subset, which was abnormally and significantly elevated in patient samples. For this subpopulation, we delved into the expression of its surface antigens, and most of the antibody markers we used (including CD45RA and CD45RO) showed higher levels than that in the other subpopulation (ncMO1). Coincidentally, we also found that cDC2 and cDC3 were remarkably abundant in patients but negligible in the healthy group, and were also accompanied by higher expression of CD45RA and CD45RO. The origin and functional mechanism of ncMO2, cDC2 and cDC3, which are DHL-related monocytes subsets, need to be further studied. Furthermore, the NK cell ratio was significantly elevated in DHL and DLBCL groups, which is different from the previous report that NK cells are subjected to exhaustion and at the low level in newly diagnosed DLBCL patients [
37], further investigation should be done in the next stage with dynamic blood samples.
DHL is generally considered as highly heterogeneous cancer in terms of genetic alteration and protein expression profile, and it is still true when it comes to immune cells landscape. We demonstrated the heterogeneity of DHL by analyzing the discrepancy of immune cell population within the DHL cohorts based on the clinical characteristics. Among them,
TP53 is a critical factor contributing to the heterogeneity. Loss of p53 function in lymphoma could results in profound alteration in the transcription of some vital genes and secretion of chemokine/cytokine, leading to great influence on the immune cells population and function [
38]. Therefore, it is reasonable that the DHL patients harboring
TP53 mutation associated with reduced population of CD4
+ TEM and CD4
+ TCM, since p53 plays a key role in regulating the polarization and differentiation of CD4
+ T cells [
39]. The reduced exhausted CD8
+ T cell in the
TP53 mutation cohort may due to the declined expression of PD-1, whereas PD-1 is a key co-inhibitory receptor in the process of T cell exhaustion and the activation of PD-1 is mediated by p53 [
40,
41]. Surprisingly, the Treg cells level was decreased in
TP53 mutation cohort, while
TP53 loss were usually associated with expansion and filtration of Treg in cancers, and facilitated the tumor progression [
42,
43]. In addition, the memory B cells were decreased in the DHL cohort with IPI 3 ~ 4, indicating the deficiency of immune function in these high-risk patients.
Although comprehensive and deep analysis were performed with PBMC sample by CyTOF, and some valuable clues had been unearthed, whether these information from PMBC sample truly reflect the profile of the tumor microenvironment of DHL still need to be further confirmed with tissue specimens. In addition, extending the horizontal and longitudinal research scales with large sample size (both of DHL and r/r DLBCL cohort) and dynamic samples could provide more accurate understanding of DHL.
Conclusion
We analyzed the peripheral blood samples from eleven DHL patients by CyTOF to characterize the immune profile and expect to discover biomarkers for pharmaceutical and clinical application. Despite the small size of samples, the data of this study are promising as statistically significant differences were observed among DHL cohorts, DLBCL cohorts and the healthy control. The results obtained suggested that DHL patients suffered immune dysfunction according to the changing of immune cell population, and some of the biomarkers, such as CD38, could be useful in diagnosis of DHL, or even be potential therapeutic agents or targets. Nevertheless, further studies with larger sample size, dynamic samples or in vivo models are required to confirm these results. In addition, we also demonstrated the heterogeneity of immune cells in DHL patients, and the status of TP53 is one of the biggest influencing factors. Overall, this study could provide primary guidance for the diagnosis and treatment of DHL patients.
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