Transmembrane Receptors, Cytoskeleton and Cell Cycle Genes Were Progressively Deregulated in the Bone Marrow CD19+ and CD138+ Cells of Patientswwith Waldenstrom's Macroglolubinemia (WM) Vs. Subjects with IgM Monoclonal Gammopathy of Undetermined Significance Igmmgus Vs. Healthy Subjects
Authors: Trojani Alessandra , Tedeschi Alessandra , Frustaci Annamaria Maria , Deodato Marina , Bossi Luca Emanuele , Di Camillo Barbara , Montillo Marco , Morra Enrica , Cairoli Roberto 
Published: November 2019

In this study, we investigated the transcriptome differences of selected bone marrow (BM) CD19+ and BM CD138+ cells of Waldenström's Macroglobulinemia (WM) patients vs. IgM Monoclonal Gammopathy of Undetermined Significance (IgMMGUS) subjects vs. healthy donors (CTLRs). We performed the gene expression profiling (GEP) considering all the different transcript isoforms of genes, coding and non-coding transcripts. Microarrays were performed on BM CD19+ cells (n=36) and BM CD138+ (n=32) in WM, BM CD19+ cells (n=10) and BM CD138+ (n=10) cells in IgMMGUS, BM CD19+ cells (n=7) and BM CD138+ (n=7) cells in healthy donors (CTRLs), respectively. Data were preprocessed and normalized using RMA and ComBat. We performed a functional annotation clustering and enrichment analysis on each pattern using DAVID 6.7 (https://david.ncifcrf.gov/). Selection of differentially expressed genes (DEg) was performed separately for CD19+ and CD138+ cells using Statistical Analysis for Microarrays (SAM) on 3 groups (WM, IgMMGUS and CTRLs). A false discovery rate threshold of 5% was used followed, for significance comparisons, by a pair-wise SAM test.
We identified 3095 unique probe-sets corresponding to 2559 unique genes and 494 not annotated probe-sets from the comparison of WM vs. IgMMGUS vs. CRTLs in CD19+ cells.
The comparison of WM, IgMMGUS and CRTLs in CD138+ cells demonstrated 38 unique probe-sets corresponding to 32 unique genes and 2 not annotated probe-sets. There were no genes in common between the CD19+ and the CD138+ DEg lists.
To better characterize the genes selected as differentially expressed we considered, for all genes that resulted significantly DE in at least one of the below listed comparisons, the following patterns of differential expression: 1) WM>IgMGUS, IgMGUS>CTRLs, and WM>CTRLs; 2) WM<IgMGUS, IgMGUS<CTRLs, and WM<CTRLs.
We identified 265 unique genes in the CD19+ cells pattern 1 whereas the CD19+ cells pattern 2 contained 403 unique genes.
Enrichment analyses showed that transmembrane, adhesion and flavo proteins were progressively significantly under expressed in the WM>IgMGUS>CTRL CD19+ pattern.
IL2RA (CD25), ATP1B1 and ADRB2 (cGMP-PKG signaling pathway), GPER (activating PI3K signaling pathway) and IGHM were up regulated in WM. We found in the hematopoietic lineage that CD1D (positive in WM Natural Killer T cells), CD44 (marker in WM stromal cells), IL6 and IGHM were progressively under expressed in WM vs. IgMMGUS vs. CTRLs. CEACAM1 and ANG, both inducing new blood vessels formation, were progressively down regulated in WM vs. IgMMGUS vs. CTRLs. Notably, ANG was over expressed in WM vs. IgMMGUS vs. CTRLs with the fold change (FC) of 1.54, 2.13 and 3.27, respectively.
The WM<IgMGUS<CTRL CD19+ pattern showed that 50 genes involved in cytoskeleton and 35 genes belonging to the cell cycle, were progressively over expressed in WM vs. IgMMGUS vs. CTRLs.
ADARB1 (alternative splicing) and LEF1 (transcription regulation) were progressively over expressed in WM vs. IgMMGUS vs. CTRLs with the following FC: -2.19, -2.16, -4.72 and -1.50, -2.58, -3.86, respectively. In addition, IL4R (plasma membrane protein) which has been demonstrated to be down regulated in WM B cells, was progressively up regulated in WM vs. IgMMGUS vs. CTRLs with the FC of -1.83, -2.18, and -3.99, respectively.
GEP data determined few genes in the CD138+ cells patterns: no genes were differently expressed in the WM>IgMGUS>CTRL CD138+ pattern 1 whereas 26 genes emerged from the WM<IgMMGUS< CTRLs CD138+ pattern 2. Enrichment analyses demonstrated that cell junctions genes were progressively significantly over expressed in the WM<IgMMGUS< CTRLs CD138+ pattern 2.
TJP1 (apoptosis) was progressively over expressed in WM vs. IgMMGUS vs. CTRLs with the FC -2.65, -2.33 and -6.18, respectively. In previous studies it has been demonstrated that the TJP1 lower expression induced resistance to proteasome inhibitors in myeloma. TUBB2B, SEPT10, TTLL,SYNM, PARD3, ARHGAP32, involved in the cytoskeleton, as well as PERP (TP53 apoptosis effector), ESPR1 (RNA splicing), and CADPS2 (protein transport) were progressively down regulated in CTRLs vs. IgMMGUS vs. WM.
In conclusion, we demonstrated that transmembrane receptors, cell cycle, and cytoskeleton proteins in BM CD19+ cells as well as transmembrane/cell junctions molecules in BM CD138+ cells were differently and progressively deregulated in WM vs. IgMMGUS vs. CTRLs, respectively.

Disclosures
Tedeschi: Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; BeiGene: Honoraria; SUNESIS: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen spa: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy. Montillo:AbbVie: Consultancy, Honoraria, Speakers Bureau; Versatem: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Speakers Bureau; Acerta: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau.