Common Gene Expression Signature of B-Cells of Waldenström Macroglobulinemia (WM) and IgM Monoclonal Gammopathies of Undetermined Significance (IgM MGUS) Compared to Healthy Subjects
Authors: Trojani Alessandra , Di Camillo Barbara , Bossi Luca Emanuele , Greco Antonino , Leuzzi Livia , Tedeschi Alessandra , Frustaci Anna Maria , Deodato Marina , Zamprogna Giulia , Melluso Agostina , Panzeri Alessia , Montillo Marco , Beghini Alessandro , Cairoli Roberto 
Published: November 2021

Abstract
We performed a comparative gene expression profiling (GEP) study on B-cells and plasma cells of Waldenström Macroglobulinemia (WM), IgM monoclonal gammopathies of undetermined significance (IgMMGUS), and normal individuals (CTRLs) to identify GEP changes as reliable predictors of progression of IgMMGUS to WM.
We analyzed bone marrow B-cells and plasma cells from 36 WM patients, 13 IgMMGUS subjects, and 7 CTRLs by Affymetrix microarray, respectively (Table 1). GEP experiments were performed on the CD19+ and CD138+ cells using GeneChip-HGU133 Plus 2.0. Data were preprocessed and normalized by Robust Multi-Array Average and ComBat. Selection of the different expressed genes was performed separately for CD19+ and CD138+ cells, using Significance Analysis of Microarrays (SAM) on the 3 groups and a false discovery rate threshold of 5%, followed, for significance comparisons, by a pair-wise SAM test corrected for multiple testing.
We focused on the comparison of the CD19+ cells of WM vs. IgMMGUS vs. CTRLs which highlighted 2038 unique genes whereas the same comparison of the CD138+ cells determined 29 unique genes (Trojani et.al. Cancers 2021). Among the 2038 DEGs, 115 genes were grouped in KEGG pathways involved in Wnt-signaling, BCR-signaling, calcium signaling, hematopoietic cell antigens, cell adhesion, adherens junctions, coagulation cascade, platelet activation, cytokine receptor, and signaling pathways responsible for cell cycle, apoptosis, and survival.
Interestingly, most of the 115 DEGs in B-cells were different expressed in WM vs. IgMMGUS and CTRLs. Only 9/115 DEGs were significantly different expressed in WM vs. CTRLs and in IgMMGUS vs. CTRLs, but no significant expression changes were noted between WM and IgMMGUS (Table 2).
To further inspect the similarities and the differences among WM and IgMMGUS, we computed the Euclidean pair-wise distance between subjects and, using this distance as weight, constructed a minimum spanning tree (MST) (Figure 1).
Considerably, four probesets identified ADRB2 (transmembrane Beta adrenergic receptor) which was up regulated in WM and IgMMGUS compared to CTRLs. The over expression of ADRB2 was also demonstrated in Mantle Cell Lymphoma cell lines and in Diffuse large B-cell lymphoma (DLBCL) lymphocytes compared to normal B-cells (doi10.1016/j.cellsig.2017.08.002), and in most malignancies (doi10.1007/s11033-021-06250-y) .
As far as we know, ADAM23 (ADAM Metallopeptidase Domain23) has not been found in WM, whereas we suggest its possible role in WM patients with Sjogren's syndrome (SS). ADAM23 plays a role in the peripheral neuropathy by controlling the activity of potassium channels in SS (doi10.1007/s10067-016-3499-z). Some authors found that sensory/motor neuropathies were associated with MGUS patients (doi10.1017/s0317167100011483). We strongly believe that the down regulation of ADAM23 in WM and IgMMGUS has a good chance to be associated with clinical neuropathy in WM and IgMMGUS.
RASGRP3 (RAS Guanyl Releasing Protein3) and PIK3AP1 (Phosphoinositide 3 Kinase Adaptor Protein1) play crucial roles in BCR signaling pathway: PIK3AP1 activates the PI3K-Akt signaling while RASGRP3 stimulates MAPK signaling pathway.
The deregulation of LEF1 (Lymphoid Enhancer Binding Factor1) and genes of Wnt-pathway were previously demonstrated in B-cell disorders and multiple myeloma (doi10.1007/s00277-017-3207-3, doi10.1016/j.pathol.2019.09.009). According to these studies, we showed the under expression of LEF1 in WM and IgMMGUS compared to CTRLs.
We identified the down regulation of EZH2 (Enhancer Of Zeste2 Polycomb Repressive Complex2Subunit) in WM and IgMMGUS compared to CTRLs. EZH2 is involved in Follicular lymphoma and DLBCL (doi10.1080/2162402X.2017.1321184).
CDHR3 (Cadherin Related Family Member3), CHEK1 (Checkpoint Kinase1), and HIST1H1B (Histone-H1.5) were over expressed in CTRLs compared to IgMMGUS and WM.
In conclusion, the common gene-set in WM and IgMMGUS could suggest two-hit hypothesis. First, the gene-set could play a role in the risk of progression of IgMMGUS to WM. Until now, all the IgMMGUS subjects have not been transformed in WM or other NHL, but they have been monitored every 6 months, and their possible transformation to lymphoma could highlight new insights. The second hypothesis suggests their involvement in the biological processes of leukemogenesis in WM and IgMMGUS which will be further investigated.
Figure 1 Figure 1.

Disclosures
Tedeschi: AbbVie: Honoraria, Speakers Bureau; AstraZeneca: Honoraria, Speakers Bureau; Beigene: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau.