We evaluated the MYD88 L265P mutation in Waldenström's macroglobulinemia (WM) and B-cell lymphoproliferative disorders by specific polymerase chain reaction (PCR) (sensitivity ∼10(-3)). No mutation was seen in normal donors, while it was present in 101/117 (86%) WM patients, 27/31 (87%) IgM monoclonal gammapathies of uncertain significance (MGUS), 3/14 (21%) splenic marginal zone lymphomas and 9/48 (19%) non-germinal center (GC) diffuse large B-cell lymphomas (DLBCLs). The mutation was absent in all 28 GC-DLBCLs, 13 DLBCLs not subclassified, 35 hairy cell leukemias, 39 chronic lymphocytic leukemias (16 with M-component), 25 IgA or IgG-MGUS, 24 multiple myeloma (3 with an IgM isotype), 6 amyloidosis, 9 lymphoplasmacytic lymphomas and 1 IgM-related neuropathy. Among WM and IgM-MGUS, MYD88 L265P mutation was associated with some differences in clinical and biological characteristics, although usually minor; wild-type MYD88 cases had smaller M-component (1.77 vs 2.72 g/dl, P=0.022), more lymphocytosis (24 vs 5%, P=0.006), higher lactate dehydrogenase level (371 vs 265 UI/L, P=0.002), atypical immunophenotype (CD23-CD27+ +FMC7+ +), less Immunoglobulin Heavy Chain Variable gene (IGHV) somatic hypermutation (57 vs 97%, P=0.012) and less IGHV3-23 gene selection (9 vs 27%, P=0.014). These small differences did not lead to different time to first therapy, response to treatment or progression-free or overall survival.

The tumoral clone of Waldenströ m's macroglobulinemia (WM) shows a wide morphological heterogeneity, which ranges from B lymphocytes (BL) to plasma cells (PC). By means of genomewide expression profiling we have been able to identify genes exclusively deregulated in BL and PC from WM, but with a similar expression pattern in their corresponding cell counterparts from chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), as well as normal individuals. The differentially expressed genes have important functions in B-cell differentiation and oncogenesis. Thus, two of the genes downregulated in WM-BL were IL4R, which plays a relevant role in CLL B-cell survival, and BACH2, which participates in the development of class-switched PC. Interestingly, one of the upregulated genes in WM-BL was IL6. A set of four genes was able to discriminate clonal BL from WM and CLL: LEF1 (WNT/b-catenin pathway), MARCKS, ATXN1 and FMOD. We also found deregulation of genes involved in plasma cell differentiation such as PAX5, which was overexpressed in WM-PC, and IRF4 and BLIMP1, which were underexpressed. In addition, three of the target genes activated by PAX5 -CD79, BLNK and SYK -were upregulated in WM-PC. In summary, these results indicate that both PC and BL from WM are genetically different from the MM and CLL cell counterpart.

It is an open question whether in multiple myeloma (MM) bone marrow stromal cells contain genomic alterations, which may contribute to the pathogenesis of the disease. We conducted an array-based comparative genomic hybridization (array-CGH) analysis to compare the extent of unbalanced genomic alterations in mesenchymal stem cells from 21 myeloma patients (MM-MSCs) and 12 normal donors (ND-MSCs) after in vitro culture expansion. Whereas ND-MSCs were devoid of genomic imbalances, several non-recurrent chromosomal gains and losses (>1 Mb size) were detected in MM-MSCs. Using real-time reverse transcription PCR, we found correlative deregulated expression for five genes encoded in regions for which genomic imbalances were detected using array-CGH. In addition, only MM-MSCs showed a specific pattern of 'hot-spot' regions with discrete (<1 Mb) genomic alterations, some of which were confirmed using fluorescence in situ hybridization (FISH). Within MM-MSC samples, unsupervised cluster analysis did not correlate with particular clinicobiological features of MM patients. We also explored whether cytogenetic abnormalities present in myelomatous plasma cells (PCs) were shared by matching MSCs from the same patients using FISH. All MM-MSCs were cytogenetically normal for the tested genomic alterations. Therefore we cannot support a common progenitor for myeloma PCs and MSCs.

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Mini-BEAM and ESHAP are two non-cross-resistant salvage regimens that have been used separately in patients with lymphoma. The aim of the present study was to investigate the efficacy of the combination of these two regimens, administered in alternating cycles, as salvage therapy for refractory non-Hodgkin's lymphoma (NHL) patients. A total of 28 patients were included in the study: 14 patients were primary refractory, seven were partial responders, and seven were in relapse. The alternating cycles of mini-BEAM and ESHAP were given until there was maximum response or progression. The overall response rate to mini-BEAM/ESHAP was 39%; 25% of patients achieved a complete response and 14% a partial response. Nevertheless, it should be noted that none of the primary refractory patients responded to this protocol. Nine of the 11 patients who responded to mini-BEAM/ESHAP were consolidated with autologous transplantation using BEAM as a conditioning regimen. The survival at 3 years in this group of 11 patients who responded to the salvage regimen is 64%, with a disease-free survival of 67% at 2 years. No major toxic effects were observed with mini-BEAM/ESHAP. Myelosuppression was the most frequent complication, especially with the mini-BEAM cycles. Other toxicities were infrequent and no treatment-related deaths were observed. These results suggest that alternating mini-BEAM/ESHAP chemotherapy is a safe regimen that is effective in partial responders or relapsing patients with NHL who have sensitive disease, but not in primary refractory patients. Moreover, although this therapy has a potential advantage, combining as it does two non-cross-resistant regimens, it does not seem superior to ESHAP alone.

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The complex interplay between bone marrow-derived mesenchymal stromal cells (BM-MSC) and neoplastic hematopoietic cells is involved in the progression of myeloproliferative neoplastic (MPN) diseases. Extracellular vesicles (EV) have emerged as a complex cell-to-cell communication system within the neoplastic microenvironment. EV are able to reprogram recipient cells by transferring proteins, mRNA and microRNA from their cell of origin. We aimed to analyze the microRNA content of EV obtained from MPN BM-MSC, as well as the changes induced when these EV are incorporated into hematopoietic progenitor cells (HPC). EV were isolated from BM-MSC of MPN patients (n=22) and healthy donors (HD) (n=19) by ultracentrifugation. Characterization of EV by transmission electron microscopy (TEM), immunoblot, multiparametric flow cytometry (MFC) and NanoSight analysis revealed vesicles with a typical bilayer-membrane characteristic morphology with a size inferior to 500 nm, which were positive for various EV markers as CD63 and CD81, and for MSC markers as CD73, CD90 and CD44 (Figure 1). MicroRNA profiling by 384-well microfluidic cards (TaqMan® MicroRNA Array A) showed an overall increase in the microRNA expression in the MPN-MSC-derived EV, when compared to EV from donor MSC. Using RT-PCR, we observed that miR-155 was selectively enriched in EV released by MPN-MSC. An overexpression of this microRNA was observed in EV (p=0.032), while a downregulation was observed in BM-MSC (p=0.0078) (Figure 2). EV incorporation was demonstrated by fluorescence microscopy and MFC, where HPC (CD34+ cells obtained by immunomagnetic selection) were co-cultured with Vybrant Dil-labeled EV. For functional studies apoptosis and clonogenic assays (CFU-GM) were performed. We observed an increase in CD34+ cell viability after incorporating EV from BM-MSC (HD and MPN). Moreover, an increase (p=0.04) in miR-155 expression was observed when HD HPC incorporated EV from MPN-MSC. When neoplastic CD34+ cells incorporated the EV derived from MPN-MSC an increase of CFU-GM number was also observed. We suggest that EV released from MPN-MSC represent a mechanism of intercellular communication between malignant stromal and hematopoietic cells, through the transfer of genetic information that may be relevant in the pathophysiology of these diseases. Funding: GRS 1034/A/14 (C. Sanidad, JCYL) and FCT (SFRH/BD/86451/2012) Figure 1 EV characterization by TEM (A), Immunobloting - CD63 (B) and MFC (C). Scale bar: 200 and 500 nm. Figure 1. EV characterization by TEM (A), Immunobloting - CD63 (B) and MFC (C). Scale bar: 200 and 500 nm. Figure 2 Expression of miR-155. RT-PCR from EV released from HD and MPN-MSC (A), and the expression of miR-155 in BM-MSC (B). Figure 2. Expression of miR-155. RT-PCR from EV released from HD and MPN-MSC (A), and the expression of miR-155 in BM-MSC (B). Disclosures Sánchez-Guijo: Bristol-Myers-Squib: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Del Cañizo:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.