Many cell types release extracellular vesicles (EVs), including exosomes, microvesicles (MVs), and apoptotic bodies, which play a role in physiology and diseases. Presence and phenotype of circulating EVs in hematological malignancies (HMs) remain largely unexplored.The aim of this study was to characterize EVs in peripheral blood of HM patients compared to healthy subjects (controls). We isolated serum EVs from patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), Waldenstrom's macroglobulinemia (WM), Hodgkin's lymphoma (HL), multiple myeloma (MM), acute myeloid leukemia (AML), myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDS), and controls. EVs were isolated from serum of peripheral blood by ultracentrifuge steps and analyzed by flow cytometry to define count, size, and immunophenotype. MV levels were significantly elevated in WM, HL, MM, AML, and some MPNs and, though at a lesser degree, in CLL and NHL as compared to healthy controls. HL, MM, and MPNs generated a population of MVs characterized by lower size (below 0.3 μm) when compared to controls. MVs from patients specifically expressed tumor-related antigens, such as CD19 in B cell neoplasms, CD38 in MM, CD13 in myeloid tumors, and CD30 in HL. Both total and antigen-specific count of MVs significantly correlated with different HM clinical features such as Rai stage in CLL, International Prognostic Scoring System in WM, International Staging System in MM, and clinical stage in HL. MVs may represent a novel biomarker in HMs.

Purpose: Primary plasma cell leukemia (pPCL) is a rare and very aggressive form of plasma cell dyscrasia. To date, no information on microRNA (miRNA) expression in pPCL has been reported. This study aimed at investigating the involvement of miRNAs in pPCL and their possible relationship with higher tumor aggressiveness.Experimental design: Global miRNA expression profiles were analyzed in highly purified malignant plasma cells from 18 pPCL untreated patients included in a prospective clinical trial. MiRNA expression patterns were evaluated in comparison with a representative series of multiple myeloma patients, in relation to the most recurrent chromosomal abnormalities (as assessed by fluorescence in situ hybridization and single-nucleotide polymorphism-array analysis), and in association with clinical outcome. MiRNA expression was also integrated with gene expression profiles in pPCL and multiple myeloma samples.Results: We identified a series of deregulated miRNAs in pPCL (42 upregulated and 41 downregulated) in comparison with multiple myeloma. Some of them, on the basis of their reported functions and putative target genes computed by integrative analysis, might have a role in the pathobiology of pPCL. As regards chromosomal aberrations, the expression of some miRNAs mapped to hotspot altered regions was associated with DNA copy number of the corresponding loci. Finally, 4 miRNA (miR-497, miR-106b, miR-181a à , and miR-181b) were identified as having expression levels that correlated with treatment response, and 4 (miR-92a, miR-330-3p, miR-22, and miR-146a) with clinical outcome. Conclusions: Overall, our study provides insights into the possible contribution of miRNAs in the pathogenesis of pPCL and suggests targets for future therapeutic investigations.

Primary plasma cell leukemia (pPCL) is a rare, yet aggressive form of de novo plasma cell tumor, distinct from secondary PCL (sPCL) which represents a leukemic transformation of pre-existing multiple myeloma (MM). Herein, we performed a comprehensive molecular analysis of a prospective series of pPCLs by means of FISH, single nucleotide polymorphism (SNP) array and gene expression profiling (GEP). IGH@ translocations were identified in 87% of pPCL cases, with prevalence of t(11;14) (40%) and t(14;16) (30.5%), whereas the most frequent numerical alterations involved 1p (38%), 1q (48%), 6q (29%), 8p (42%), 13q (74%), 14q (71%), 16q (53%), and 17p (35%). We identified a minimal biallelic deletion (1.5 Mb) in 8p21.2 encompassing the PPP2R2A gene, belonging to a family of putative tumor suppressors and found to be significantly down-regulated in deleted cases. Mutations of TP53 were identified in four cases, all but one associated with a monoallelic deletion of the gene, whereas activating mutations of the BRAF oncogene occurred in one case and were absent in N-and K-RAS. To evaluate the influence of allelic imbalances in transcriptional expression we performed an integrated genomic analysis with GEP data, showing a significant dosage effect of genes involved in transcription, translation, methyltransferase activity, apoptosis as well as Wnt and NF-kB signaling pathways. Overall, we provide a compendium of genomic alterations in a prospective series of pPCLs which may contribute to improve our understanding of the pathogenesis of this aggressive form of plasma cell dyscrasia and the mechanisms of tumor progression in MM. Am. J. Hematol. 88:16-23, 2013. V

To understand neutrophil impairment in the progression from MGUS through active MM, we investigated the function of mature, high-density neutrophils (HDNs), isolated from peripheral blood. In 7 MM, 3 MGUS and 3 healthy subjects by gene expression profile, we identified a total of 551 upregulated and 343 downregulated genes in MM-HDN, involved in chemokine signaling pathway and FC-gamma receptor mediated phagocytosis conveying in the activation of STAT proteins. In a series of 60 newly diagnosed MM and 30 MGUS patients, by flow-cytometry we found that HDN from MM, and to a lesser extend MGUS, had an up-regulation of the inducible FcγRI (also known as CD64) and a down-regulation of the constitutive FcγRIIIa (also known as CD16) together with a reduced phagocytic activity and oxidative burst, associated to increased immune-suppression that could be reverted by arginase inhibitors in co-culture with lymphocytes. In 43 consecutive newly-diagnosed MM patients, who received first-line treatment based on bortezomib, thalidomide and dexamethasone, high CD64 could identify at diagnosis patients with inferior median overall survival (39.5 versus 86.7 months, p = 0.04). Thus, HDNs are significantly different among healthy, MGUS and MM subjects. In both MGUS and MM neutrophils may play a role in supporting both the increased susceptibility to infection and the immunological dysfunction that leads to tumor progression.

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Purpose: Plasma cell leukemia (PCL) is a rare form of plasma cell dyscrasia that presents either as a progression of previously diagnosed multiple myeloma, namely secondary PCL, or as initial manifestation of disease, namely primary PCL (pPCL). Although the presenting signs and symptoms include those seen in multiple myeloma, pPCL is characterized by several aspects that define a more aggressive course. Here, we have investigated the transcriptome of pPCLs and correlated differential expression profiles with outcome to provide insights into the biology of the disease. Experimental Design: The expression profiles of 21 newly diagnosed pPCLs included in a multicenter prospective clinical trial were generated using high-density microarray, then evaluated in comparison with a representative series of patients with multiple myeloma and in association with clinical outcome. Results: All but one of the pPCLs had one of the main immunoglobulin heavy-chain locus translocations, whose associated transcriptional signatures resembled those observed in multiple myeloma. A 503-gene signature distinguished pPCL from multiple myeloma, from which emerged 26 genes whose expression trend was associated with progressive stages of plasma cells dyscrasia in a large dataset from multiple institutions, including samples from normal donors throughout PCL. Finally, 3 genes were identified as having expression levels that correlated with response to the first-line treatment with lenalidomide/dexamethasone, whereas a 27-gene signature was associated with overall survival independently of molecular alterations, hematologic parameters, and renal function. Conclusions: Overall, our data contribute to a fine dissection of pPCL and may provide novel insights into the molecular definition of patients with poorer prognosis. Clin Cancer Res; 19(12); 3247–58. ©2013 AACR.

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Regulatory T-cells (Tregs) are increased in chronic lymphocytic leukemia(CLL) and correlates with clinical and biological features of active/progressive disease. However, little is known about their ability to predict the time to first treatment (TFT). We evaluated 75 patients with Rai stage 0 CLL, in whom the absolute number of Tregs was determined at diagnosis, and correlated to main clinical and biological features, as well as to the need of receiving any specific therapy during the course of the disease. After a median follow-up of 30 months, 12 patients(16%) required therapy at some time from the diagnosis. Treated patients showed a significant higher number of peripheral white blood cells and B-lymphocytes, platelet count, cases with unmutated immunoglobulin heavy chain status, and high-risk cytogenetic abnormalities,as well as lower hemoglobin values, than patients who did not need therapy. A greater number of circulating Tregs was detected in treated patients (P < 0.001). Multivariate analysis confirmed that the absolute number of Tregs was an independent predictor of TFT in these patients, the best predictive cut-off being 41/mL. These data show that the absolute Tregs cell number is able to identify Rai stage 0 CLL patients at higher risk of requiring therapy.