High-Throughput Molecular Profiling of Multiple Myeloma (MM) Clonotypic CD19+ B Cells Highlights Pathways Potentially Involved in the Disease Endurance
Authors: Martello Marina , Dico Angela Flores , Remondini Daniel , Martinelli Giovanni , Borsi Enrica , Santacroce Barbara , Pantani Lucia , Zamagni Elena , Tacchetti Paola , Brioli Annamaria , Zannetti Anna Beatrice , Mancuso Katia , Rocchi Serena , Pezzi Annalisa , Testoni Nicoletta , Marzocchi Giulia , Ameli Gaia , Cavo Michele , Terragna Carolina 
Published: October 2019

Abstract
Background Although remarkable advances have been achieved in MM therapy, mainly thanks to the introduction of novel-agent-based regimens, the disease remains incurable. Neoplastic CD138+ plasma cells are the hallmark of MM: both their expansion in the bone marrow (BM) and the production of monoclonal immunoglobulin (Ig) are responsible for the clinical manifestation of the disease. However, the existence of a Myeloma Propagating Cells (MPCs) has been proposed as a major cause of MM drug-resistance, leading to relapse. Several studies support the hypothesis that MPCs are phenotypically close to memory B cells residing in the CD138- compartment; however, very little is known concerning their molecular characteristics.
Here we present an extensive molecular characterization of clonotypic CD19+ B cells clones obtained from newly diagnosed MM patients (pts), in order to recognize biological pathways possibly explaining the malignant clone’s persistence.
Methods CD138+ and CD138- cell fractions were collected from BM and peripheral blood (PBL) of 50 newly diagnosed MM pts. CD19+ B cell and CD27+ memory B cell populations were isolated from CD138- cell fraction. Clonogenic assays were performed by plating cell fractions obtained from RPMI-8226 and NCI-H929 cell lines. The molecular characterization included: IgH gene rearrangement Sanger sequencing; analysis of the whole spectrum of genomic aberrations and gene expression profiling, by Affymetrix 6.0 SNPs array and HG-U133 Plus 2.0 microarray, respectively.
Results Clonogenic assays showed that CD138- cells, plated on conditioned media, were able to form colonies after two weeks of culture more efficiently than CD138+ cells.
By VDJ gene rearrangement sequencing, a clonal relationship between the CD138+ clone and the memory B ones was confirmed.
SNPs arrays showed that both BM and PBL CD138+ cell fractions carried exactly the same genomic macro-alterations. On the contrary, in the CD138-19+27+ cell fractions from BM and PBL any macro-alteration was detected, whereas several micro-alterations (median number per sample: 32 amplifications and 16 losses, range: 8-122 Kb, average markers per region: 50) unique of the memory B cells clone were highlighted. An enrichment analysis revealed the involvement of genes affected by losses (17 genes) in both DNA repair mechanisms and transcriptional regulation and the involvement of genes affected by gains (46 genes) in both the negative regulation of apoptosis and the angiogenesis. Interestingly, KRAS, WWOX and XIAP genes, renown to be involved in MM pathogenesis, are located in the amplified regions in the immature cells. Moreover, several LOH regions were described, which covered at least 106 tumor suppressor genes involved in MM and leukemia (including TP53, CDKN2C and RASSF1A).
Transcriptome profiles analysis of the CD19+ cell fractions highlighted pathways suggesting a possible involvement of immature cells in MM pathogenesis. The gene expression profiles of 20 MM CD19+ cells samples (12 from PBL, 8 from BM) were compared both to their normal counterpart and to the mature CD138+ cell fractions. In particular, unsupervised analysis by hierarchical clustering discriminated the differential expression of 11480 and 11360 probes in the PBL and BM CD19+ clones, respectively (<-2FC>2; FDR=0,05; p <0,05). An overall de-regulation of pathways involved in self-renewal mechanisms was highlighted, with Notch and Wnt signaling over-expressed in every analyzed cell compartment; on the contrary, Hedgehog pathway was overall down regulated.
Interestingly, the protein homeostasis deregulation possibly caused by ER stress, resulted particularly evident in the BM 19+ cells (p=7,25E-14; FDR= 2,98E-11); moreover, the down-regulation of genes related to the unfolded protein response (e.g. IRE1α and XBP1 FC=-18,0; -19,96. p<0,05) suggests the expression of a proteasome inhibitor-resistant phenotype of these cells.
Conclusions Presented data support the emerging role of the immature cell compartment in the MM disease course, where the MM CD138+ clone might resume the end of the complex process of tumorigenesis, whereas the putative CD19+ MPCs, by displaying peculiar genomic micro-alterations and a unique transcriptional profile, might be involved in the neoplastic clone supply.
Supported by ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project.

Disclosures
Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; Ariad: Consultancy. Cavo:BMS: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Honoraria; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.