ALBERT

Description: Inflammation represents a key feature and hallmark of tumor microenvironment playing a major role in the interaction with mesenchymal stromal cells (MSC) in cancer progression. The aim of the present study was to investigate the crosstalk between MSCs and myeloma cells (MM) in the pro-inflammatory microenvironment promoting immune evasion and tumor growth. MSC were collected from patients with diagnosis of MGUS (n = 10), smoldering myeloma (n = 7), multiple myeloma at diagnosis (n = 16), relapse (n = 5) or refractory (n = 3), and from age-matched healthy controls (HC, n = 10) and cultured with peripheral blood mononucleated cells (PBMC) from healthy volunteer donors. Similarly to MM, we showed that MSC from smoldering multiple myeloma (SMM) patients activated neutrophils and conferred an immunosuppressive and pro-angiogenic phenotype. Furthermore, co-cultures of plasma cells (PC) and HC-MSC suggested that such activation is driven by MM cells through the switching into a pro-inflammatory phenotype mediated by toll-like receptor 4 (TLR4). These results were further confirmed using a zebrafish as an immunocompetent in vivo model, showing the role of MM–MSC in supporting PCs engraftment and Th2 response. Such effect was abolished following inhibition of TLR4 signaling in MM–MSC before co-injection with PC. Moreover, the addition of a TLR4 inhibitor in the co-culture of HC-MSC with MM cells prevented the activation of the pro-tumor activity in PC-educated MSC. In conclusion, our study provides evidence that TLR4 signaling plays a key role in MSC transformation by inducing a pro-tumor phenotype associated with a permissive microenvironment allowing immune escape and tumor growth.

Description: An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Description: Background and objective: Multiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. Previous reports in murine models and our previous work in MM patients, showed that myeloid cells, both granulocyte-myeloid derived suppressor cells (G-MDSC) and neutrophils sedimenting on top of red cells after density gradients, defined as high-density neutrophils (HDN) are increased and exert an immunosuppressive activity. Moreover, we showed that myeloid precursors could be activate and acquire G-MDSC phenotype if cultures in presence of MM mesenchymal cells. Thus, we characterized functionally HDN to gauge their contribution in immunesuppression, angiogenesis and bone resorption in MM microenvironment. Methods: In HDN freshly-isolated from a series of 60 newly-diagnosed MM, 30 smoldering MM and 30 MGUS we investigated by RT-PCR, flow-cytometry, western-blot and immunofluorescence genes and proteins related to immunesuppression (Arg-1, IDO), angiogenesis (BV8, VEGF, MMP9) and bone resorption (IL1-b, MMP9, CHI3L). Results: First, MM-HDN exhibited an increased expression of immunesuppressive molecules Arg-1 and IDO compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity, immunostaining and WB, positively correlated with advanced disease. Second, MM-HDN showed increased expression of BV8 and MMP9 (p=0.01, p=0.05) but not VEGF, as confirmed by immunofluorescence. In line with this observation, by using migration assay (CIM plate) MM-HDN induced neo-vasculogenesis in vitro by forming more and aberrant tubes than healthy or MGUS-HDN, thus to disclosure a novel proangiogenic mechanism driven by BV8. Third, MM-HDN showed increased expression of IL1-b, MMP9, CHI3L, associated to increased bone resorption activity, evaluated by dentin-disk assay. The same findings could be obtained treating bone matrix with MM-HDN-conditioned media, suggesting the crucial role of molecules released by MM-HDN but no from MGUS-HDN. Conclusion: taken together, our observations disclosure the contribution of aberrantly activated HDN in MM clinical features, contributing to immunosuppression, angiogenesis and bone resorption. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Our previous work showed that in Multiple Myeloma (MM) the immune function is impaired, including immunosuppressive properties of granulocytes due to their increased amount of arginase-1 and reduced phagocytic activity (Parrinello, manuscript in preparation). It is currently unknown if granulocyte dysfunction occurs in progression from MGUS to MM. Aim Providing a gene expression profile of mature granulocytes isolated from peripheral blood at the steady-statein MGUS and MM. Methods Using oligonucleotide microarrays we first evaluated the gene expression profile of granulocytes at the steady state in 5 MM, 3 MGUS and 3 healthy subjects matched for sex and age. Then, we validated the first up-regulated gene PROK-2, obtained from preliminary findings in granulocytes from peripheral blood in 85 consecutive newly diagnosed MGUS (N=45), MM (N=40) and 15 healthy subjects, in RT-PCR (validation set). Results We found 708 genes differentially expressed (467 up- and 241 down regulated) in MGUS versus healthy granulocytes at the steady state. The set of annotated, differentially expressed genes could befunctionally organized by “gene ontology” (http://www.geneontology.org/) into the following major categories: i) receptors and signal transduction (including up-regulation of CD14, Toll-like receptor 5 (TLR-5), IL-7 Receptor (CD127), IL-11 receptor, TGF-beta receptor 2, hematopoietic cells kinase (HCK), IFNAR1); ii) negative regulation of adaptive immune response (including up regulation of CD127, STAT6, IFNAR1, OSCAR, PROK-2 and down regulation of p50, p65,NFKBIA, IL8, ELK-1, HIF-1 alpha, CEBP-beta, CEBP-zeta). In MM samples we confirmed a statistically significant up-regulation of PROK-2 (a key molecule of VEGF-independent angiogenesis), CD14 (mediator hypersensitive innate immune response to lipopolysaccharide) and HCK (the hematopoietic cell kinase, involved in neutrophil migration and degranulation). In the validation set, PROK-2 expression was two times higher in MGUS than healthy subjects (p=.02) and up to ten times higher in MM (p=.001). In MM patients, increased levels of PROK-2 were positively associated with advanced bone disease and unfavourable cytogenetics. Conclusion Granulocytic impairment is present in MGUS and worsened in MM patients due to increased expression of genes that negatively regulate adaptive immune response. PROK-2 is a key molecule involved in the granulocyte dysfunction and could be involved in the progression from MGUS to MM. Disclosures Musto: Celgene: Honoraria; Janssen: Honoraria.

Description: Background In sickle cell disease (SCD), profound anaemia and severe hemosiderosis cause functional and physiological abnormalities in various organ systems, including immune system. Infectious complications are common, constitute the second most common cause of mortality and a main cause of morbidity. During the haemolytic crisis, large amount of arginase (s-Arg-1) are released, potentially acting as immunosuppressor molecule. Despite its clinical impact, only a few is known about myeloid dysregulation in SCD. Aim Detecting immunological impairment at the steady state evaluating myeloid and lymphoid cells in peripheral blood of SCD patients. Material and Methods Between May and June 2014,peripheral blood obtained from 30 consecutive SCD patients at the steady state plus 30 healthy subjects was studied for evaluation of myeloid subpopulations and lymphoid paresis. Myeloiddys function was evaluated as percentage and absolute count of circulating myeloid suppressor cells (MDSC) in peripheral blood assessed by flow cytometry as follows: im-MDSC (CD34+/CD11b+/CD13+/CD14-/ HLA-DR-/CD45+), neutrophilic-like N-MDSC (CD11b+/CD13+/CD15+/CD14-/HLA-DR-/Lin-) and monocytic-likemo-MDSC (CD14+/HLA-DRlow/-), phagocytic activityof granulocytes using a commercially available kit (Phagotest R), amount of Arg-1 expressed in mature granulocytes by RT-PCR and circulating s-Arg-1 using a commercially available ELISA kit (Biovendor). Results The capability of phagocytosis of granulocytes wassignificantly reduced compared to healthy subjects (p=0.001). G-MDSC subset was not increased, while mo-MDSC subpopulation was increased in SCD (p=0.001) but not in thalasso-SCD. s-ARG-1 was increased in both SCD and thalasso-SCD (respectively 203±3 ng/mL and 248±6ng/mL, p=0.003) and positively correlated with the amount of HbS (r=0.7, p=0.002). Arg-1 expression in granulocytes was increased (20 times higher than healthy controls, p=0.002) Conclusion SCD and thalasso-SCD caucasian patients exhibiti mmunosuppressive myeloid markers including reduced phagocytosis, increased amount of mo-MDSC, Arg-1 expression in granulocytes and circulating s-Arg-1. Further analysis are ongoing to detect if the same myeloid impairment occurs during vase occlusive crisis and in a different genetic background, like in Afro-Americans. Disclosures No relevant conflicts of interest to declare.

Description: Background: Survival of patients (pts) with multiple myeloma (MM) decreases with increased age (Pulte, Oncologist, 2011). In addition, MM pts with advanced disease who have exhausted treatment (Tx) with novel agents have a poor prognosis (Kumar, Leukemia, 2012). Pomalidomide (POM) is a new oral agent with antimyeloma, stromal cell inhibitory, and immune modulatory effects (Quach, Leukemia, 2010; Mark, Leuk Res, 2014). In the pivotal MM-003 trial, POM in combination with low-dose dexamethasone (LoDEX) demonstrated significant progression-free survival (PFS) and overall survival (OS) benefits vs. high-dose dexamethasone, with a tolerable safety profile in refractory or relapsed and refractory MM (RRMM; Dimopoulos, Blood, 2013). In MM-003, significant PFS and OS benefits with POM + LoDEX Tx were seen in different age groups; tolerability and dose intensity were not affected by age (Weisel, Blood, 2013). STRATUS is a multicenter, single-arm, open-label, European, phase 3b trial to further evaluate safety and efficacy of POM + LoDEX in a large pt population. This analysis examines outcomes by age (≤ 65 vs. 〉 65 yrs, and ≤ 70 vs. 〉 70 yrs). Methods: Eligible pts had refractory or relapsed and refractory disease (progressive disease [PD] during or within 60 days of last line of Tx), having previous BORT and LEN failure and adequate prior alkylator therapy as defined in study protocol. Pts must have been refractory to the last prior Tx line. Pts with Eastern Cooperative Oncology Group performance status 〉 2 were excluded. Pts received 28-day cycles of POM 4 mg D1-21 + DEX 40 mg (20 mg for pts aged 〉 75 yrs) once weekly. All pts received thromboprophylaxis with low-dose aspirin, low-molecular-weight heparin, or equivalent based on clinical recommendations. Tx continued until PD or unacceptable toxicity. The primary endpoint was safety; the secondary endpoints included POM exposure, overall response rate (ORR; ≥ partial response), duration of response (DOR), PFS, and OS. Outcomes were analyzed by pt age at baseline (≤ 65 vs. 〉 65 yrs, and ≤ 70 vs. 〉 70 yrs). Results: As of March 17th, 2014, a total of 456 pts have been enrolled, and 452 had received POM + LoDEX; 48% were aged ≤ 65 yrs, 52% were 〉 65 yrs, 71% were ≤ 70 yrs, and 29% were 〉 70 yrs. Pts were heavily pretreated (median 4-5 prior Tx depending on age subgroup). Median follow-up was 6.8 mos. Younger pts (≤ 65 yrs) were more likely to have better renal function (creatinine clearance ≥ 60 mL/min; 80%) than those aged 〉 65 (49%). Median relative dose intensity was similar independent of age (range, 0.95-0.97 mg/day). The most common grade (Gr) 3-4 treatment-emergent adverse events (TEAEs) across age groups were neutropenia, anemia, thrombocytopenia, and pneumonia (Table). Gr 3-4 deep vein thrombosis (DVT) with prophylaxis was 1% in each subgroup. Discontinuations of POM due to TEAEs were low in pts aged ≤ 65 (0.9%), 〉 65 (3.0%), ≤ 70 (1.3%), and 〉 70 (3.8%) yrs. Outcomes by age are summarized in the Table. ORR was consistent for pts aged ≤ 65 (38%), 〉 65 (32%), ≤ 70 (35%), and 〉 70 (34%) yrs; DORs were 5.1, 6.8, and 5.8 mos and not estimable (NE) in these pt populations, respectively. Median PFS and median OS were similar across all age groups (PFS range, 4.0-4.9 mos, OS range, 10.6-11.5 mos). Conclusions: The data reported here further demonstrate the tolerability and efficacy of POM + LoDEX in pts with RRMM in the age subgroups analyzed (≤ 65 vs. 〉 65 yrs and ≤ 70 vs. 〉 70 yrs). Safety profiles were consistent, while dose intensity was similar across age groups. PFS, OS, and response rates were comparable with those previously reported in trials of POM + LoDEX in pts with RRMM and reinforce 4 mg POM as an appropriate starting dose irrespective of age. These data support POM + LoDEX as a standard Tx option for pts with refractory or RRMM regardless of age. Table 1. Age ≤ 65 yrs (n = 215) Age 〉 65 yrs (n = 237) Age ≤ 70 yrs (n = 319) Age 〉 70 yrs (n = 133) Grade 3-4 TEAEs, % Neutropenia Anemia Thrombocytopenia Pneumonia 38 30 22 13 41 24 16 10 38 29 21 11 44 23 15 11 Grade 3-4 EOI, % DVT/PE PN 1

Description: INTRODUCTION: Gaucher disease (GD) is a rare, autosomal recessive genetic disorder. It is due to a deficiency of the lysosomal enzyme, glucocerebrosidase, which leads to an accumulation of its substrate, glucosylceramide, in tissue macrophage with damage to hematological, visceral, and skeletal organ systems. Although GD has a continuous spectrum of severity, it is traditionally classified into three forms: type 1 (chronic; lacking early onset neuronopathy), type 2 (acute; with early onset neuronopathy),and type 3 (chronic; with early onsetneuronopathy). Type 1GD accounts for more than 90% all GD patients. Its prevalence world-wide is 1 in 50,000-100,000 but it is as high as ~1 in 850 in individuals of Ashkenazi heritage. Type 1 GD is frequently associated to monoclonal gammopathies; despite the emergence of theories advanced to explain these observations, the cause remains unknown. OBJECTIVE: Aim of the ongoing observational study is to determine the prevalence of unrecognized type I GD in a selected Italian population with MGUS. MATERIALS AND METHODS: From January 2018, dried blood spots (DBS) sample from patients with laboratory evidence of MGUS coming from five hematology units of Sicily and Calabria were collected and tested for the acid β-glucosidase enzyme activity. The study was approved by the local institutional review board. All patients provided informed consent for the prospective collection of their data. In case of DBS positive result, a confirmatory test was carried over and, if GD was confirmed, the patient was referred to one of the Regional Reference Centers for Metabolic Disease, as for current clinical practice in Italy. RESULTS: To date, 308 patients with MGUS were enrolled; acid β-glucosidase enzyme activity was low in 22 patients (7%). Sequence analysis of GBA gene was performed in these selected patients, but we have found only 4 patients with heterozygous mutation in the GBA1 gene, 1 homozygous(c.1226A〉G -N370S) and 1 compound heterozygous (c.1226A〉G -N370S and c.1448T〉C -L444P); the last 2 patients had signs of GD (hepato-splenomegaly and mild thrombocytopenia). CONCLUSIONS: Type 1 GD remains a rare lysosomal storage disorder but preliminary results of our observational study show that it should be considered in the diagnostic framework of patients with MGUS, particularly when other GD symptoms are present. Disclosures Conticello: Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Di Raimondo:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Amgen: Consultancy, Honoraria, Research Funding.

Description: Background: Treatment (Tx) with novel agents, including lenalidomide (LEN) and bortezomib (BORT), has extended survival of patients (pts) with multiple myeloma (MM); however, pts who have relapsed on or become refractory to Tx have significantly shortened overall survival (OS) and poorer outcomes (Kumar, Leukemia, 2012). Renal impairment (RI) occurs frequently, in approximately 20% to 40% of MM pts (Kastritis, Haematologica, 2007), and is a leading cause of death in this pt population (Korbet, J Am Soc Nephrol, 2006). In the phase 3 MM-003 trial, pomalidomide plus low-dose dexamethasone (POM + LoDEX) significantly extended progression-free survival (PFS) and OS vs. high-dose dexamethasone in pts in whom BORT and LEN Tx failed, including those with moderate RI (creatinine clearance [CrCl] 〈 60 mL/min; Weisel, Blood, 2013). STRATUS is a multicenter, single-arm, open-label, European, phase 3b trial to further evaluate safety and efficacy of POM + LoDEX in pts with refractory or relapsed and refractory MM (N = 456), including those with moderate RI. Methods: Pts must have had refractory or relapsed and refractory disease (progressive disease [PD] during or within 60 days of last line of Tx), at least 2 prior therapies, BORT and LEN failure after ≥ 2 cycles of each (alone or in combination), and adequate prior alkylator therapy as defined in study protocol. Pts must have been refractory to their last line of Tx; pts with CrCl 〈 45 mL/min were excluded. POM was administered 4 mg D1-21/28-day cycle and LoDEX 40 mg/day (20 mg for pts aged 〉 75 yrs) on D1, 8, 15, and 22 until PD or unacceptable toxicity. All pts received thromboprophylaxis with low-dose aspirin, low-molecular-weight heparin, or equivalent based on clinical recommendations. The primary endpoint was safety, and secondary endpoints included POM exposure, overall response rate (ORR; ≥ partial response), duration of response (DOR), PFS, and OS. For this analysis, pts were retrospectively grouped by baseline CrCl (〈 60 mL/min vs. ≥ 60 mL/min). Results: A total of 456 pts have been enrolled, of whom 452 received POM + LoDEX and 165 (36%) had moderate RI (CrCl 〈 60 mL/min). After a median follow-up of 6.8 mos, the most frequently reported grade (Gr) 3-4 treatment-emergent adverse events (TEAEs) in pts with moderate RI vs. without moderate RI (CrCl ≥ 60 mL/min) were neutropenia (39% vs. 40%), anemia (33% vs. 24%), and thrombocytopenia (19% vs. 19%); the most common Gr 3-4 nonhematologic TEAEs were pneumonia (12% vs. 11%), fatigue (6% vs. 4%), and hypercalcemia (4% vs. 4%), respectively. Only 2% of pts in each respective renal subgroup discontinued POM due to Tx-related TEAEs. Gr 3-4 renal toxicities were similar in both subgroups: acute renal failure occurred in 3% of pts with moderate RI and 2% of pts without moderate RI; blood creatinine increased in 2% and 1% of pts, respectively. Overall, Gr 3-4 deep vein thrombosis (DVT), pulmonary embolism (PE), and peripheral neuropathy (PN) were infrequent independent of renal status (Table). Median relative POM dose intensity was similar between subgroups (0.95 in pts with moderate RI vs. 0.96 in pts without moderate RI). In pts with moderate RI vs. without moderate RI, ORR was 35% vs. 34%, median DOR was 5.8 mos vs. 6.5 mos, median PFS was 3.7 vs. 4.6 mos, and median OS was 9.3 vs. not reached, respectively (Table). The 1-yr OS was 33% for pts with moderate RI vs. 53% for pts without moderate RI. Conclusions: POM + LoDEX has acceptable safety and efficacy profiles comparable to those observed in the pivotal MM-003 trial. Tolerability was consistent across renal function subgroups, with few discontinuations due to adverse events. In addition, responses to POM + LoDEX were similar between pts, irrespective of renal function. POM is currently being prospectively evaluated in pts with severe RI in the MM-008 (US) and MM-013 (EU) trials. Table.With Moderate RICrCl 〈 60 mL/min (n = 162)Without Moderate RICrCl ≥ 60 mL/min(n = 290)Grade 3-4 TEAEs, %Neutropenia Anemia Thrombocytopenia Pneumonia39 33 19 1240 24 19 11Grade 3-4 EOI, %DVT/PE PN2 20 1Efficacy(n = 165)(n = 291)ORR (≥ PR), % (95% CI) Median DOR (95% CI), mos Median PFS (95% CI), mos Median OS (95% CI), mos35 (27.9-43.0) 5.8 (3.7-NE) 3.7 (2.9-5.2) 9.3 (6.3-11.5)34 (28.9-40.1) 6.5 (4.7-7.9) 4.6 (3.7-5.6) NR (10.9-NR) EOI, events of interest; NE, not estimable, NR, not reached. Disclosures Weisel: Celgene Corporation: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Onyx: Consultancy, Honoraria; BMS: Consultancy; Noxxon: Consultancy. Dimopoulos:Celgene: Consultancy, Honoraria. Cavo:Celgene: Consultancy, Honoraria, Speakers Bureau. Ocio:Celgene Corporation: Honoraria, Research Funding. Palumbo:Bristol-Myers Squibb: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Array BioPharma: Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria. Delforge:Celgene Corp: Honoraria; Janssen: Honoraria. Oriol:Celgene Corporation: Consultancy. Goldschmidt:Celgene Corp: Consultancy, Honoraria, Speakers Bureau. Miller:Celgene Corporation: Employment, Equity Ownership. Peluso:Celgene: Employment. Sternas:Celgene Corp: Employment, Equity Ownership. Zaki:Celgene : Employment, Equity Ownership. Moreau:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 3386 It has been demonstrated that long term treatment of CML patients with Imatinib (IM) is associated with altered bone and mineral metabolism. The mechanisms that are responsible for this effect are not fully understood but an inhibition of the PDGF-Rβ (Platelet Derived Growth Factor- Receptor beta) axis has been documented. In order to evaluate if stimulation of osteoblastogenesis is a common feature of other tyrosine kinase inhibitors (TKIs) approved for the treatment of patients with CML, we evaluated the osteoblatic differentiation of Mesenchymal Stem Cells derived from adult bone marrow donors (BM-MSCs) after in vitro treatment with Dasatinib (DA), Nilotinib (NI) or Bosutinib (BO). BM-MSCs were induced to differentiate in osteoblastic cells by treatment with osteogenic medium (OM) with or without DA, NI or BO. BM-MSCs was induced to differentiate in osteoblastic cells by treatmet with osteogenic medium (0.2 mM ascorbic acid, 0.1 μ m dexamethasone and 10 mM β-glycerophosphate, OM) with or without DA 2nM, BO 5nM or NI 100nM. After 21 days of treatment, in the cultures treated with DA e NI for 21 days we have observed a significant increase of extracellular mineralization and of osteogenic markers such as bone morphogenetic protein (BMP2) (p