Abstract 1521: High-throughput molecular profiling of Multiple Myeloma clonotypic CD19+ B cells highlights pathways potentially involved in the disease endurance
Authors: Martello Marina , Dico Angela Flores , Remondini Daniel , Santacroce Barbara , Borsi Enrica , Procacci Mauro , Pantani Lucia , Zamagni Elena , Tacchetti Paola , Brioli Anna Maria , Rocchi Serena , Marzocchi Giulia , Martinelli Giovanni , Cavo Michele , Terragna Carolina 
Published: August 2015

Abstract
The bulk of current knowledge on Multiple myeloma (MM) disease is mainly based on the molecular characterization of 138+ plasma cells, but we become aware that MM heterogeneity has more deepen roots. Recent studies support that Myeloma Propagating Cells (MPCs) might be responsible of relapses and they residing in the 138- compartment.
In order to molecularly characterize mature and precursor cell fractions, we collected the 138+ and 138-19+27+ cell fractions from bone marrow (BM) and peripheral blood (PBL) of 50 newly diagnosed MM patients (pts). Clonogenic assays were performed by plating cell fractions of MM cell lines. The molecular characterization included: IgH rearrangement sequencing, analysis of genomic aberrations and gene expression profiling by Affymetrix 6.0 SNPs array and HG-U133 Plus 2.0 microarray.
Clonogenic assays showed that 138- cells were able to form colonies more efficiently than 138+ cells. By VDJ gene rearrangement sequencing, a clonal relationship between the 138+ clone and precursor ones was confirmed. SNPs arrays showed that both BM and PBL 138+ cell fractions carried the same genomic macro-alterations. On the contrary, in the 138-19+27+ cell fractions from BM and PBL any macro-alteration was detected, whereas several micro-alterations unique of the memory B cells clone were highlighted. An enrichment analysis revealed that genes are involved in DNA repair mechanisms, transcriptional regulation, negative regulation of apoptosis and angiogenesis. Interestingly, KRAS, WWOX and XIAP genes are located in the amplified regions of immature cells. Moreover, we observed several LOH regions that covered important tumor suppressor genes, including TP53, CDKN2C and RASSF1A.
The involvement of immature cells in MM pathogenesis was confirmed by the analysis of the 19+ cells’ transcriptome profiles. 20 MM 19+ cells samples (12 from PBL, 8 from BM) were compared both to their normal counterpart and to the mature 138+ cell fractions. In particular, unsupervised analysis by hierarchical clustering discriminated the differential expression of 11480 and 11360 probes in the PBL and BM 19+ clones, respectively (2; FDR = 0,05; p &lt;0,05). An overall de-regulation of pathways involved in self-renewal mechanisms was highlighted, with Notch and Wnt signaling over-expressed and Hedgehog pathway 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&lt;0,05) suggests the expression of a proteasome inhibitor-resistant phenotype of these cells.
Presented data support the emerging role of the immature cell compartment in the MM disease course, where the putative 19+ MPCs might be involved in the neoplastic clone supply.
Citation Format: Marina Martello, Angela Flores Dico, Daniel Remondini, Barbara Santacroce, Enrica Borsi, Mauro Procacci, Lucia Pantani, Elena Zamagni, Paola Tacchetti, Anna Maria Brioli, Serena Rocchi, Giulia Marzocchi, Giovanni Martinelli, Michele Cavo, Carolina Terragna. High-throughput molecular profiling of Multiple Myeloma clonotypic CD19+ B cells highlights pathways potentially involved in the disease endurance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1521. doi:10.1158/1538-7445.AM2015-1521