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Purpose: Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3′UTR in CCND2 overexpression in multiple myeloma.Experimental Design: Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3′UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3′ rapid amplification of cDNA ends (RACE)-PCR.Results: We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3′RACE experiments revealed that changes in CCND2 3′UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3′UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Furthermore, functional analysis showed significant CCND2 mRNA shortening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggesting that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction.Conclusions: Overall, these results highlight the impact of CCND2 3′UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma. Clin Cancer Res; 22(1); 207–17. ©2015 AACR.

Supplementary Tables Supplementary Table 1. Cytogenetic characteristics of MM samples Supplementary Table 2. Sequences of primers used in the study Supplementary Table 3. CCND2 mRNA FISH probes sequences Supplementary table 4. microRNAs predicted to target CCND1 or/and CCND2 Supplementary table 5. microRNAs expression in MM cell lines. Supplementary Figures Supplementary Figure 1. CCND2 mRNA level 24h after transfection of MM1S, RPMI and H929 cell lines with indicated miRNAs Supplementary Figure 2. Expression of miR-15a and miR-196b in H929 and MM1S cell lines after transfection Supplementary Figure 3. (A) Alignment of 3'RACE PCR product sequence from MM1S cell line with CCND2 mRNA (NM_001759.3). (B) Sequence of the 3'RACE PCR product withhighlighted APA signal. Supplementary Figure 4. CCND2 mRNA expression visualized with FISH probes Supplementary Figure 5. Luciferase activity in HEK923 cells cotransfected with various miRNAs, plasmid encoding GFP and plasmid containing luciferase and 3'UTR of CCND2 short isoform Supplementary Figure 6. Regulation of cyclin D2 level by CCND1/CCND3.

(1) Background: New therapeutic strategies have improved the prognosis of multiple myeloma (MM), changing the accepted view of this disease from being incurable to treatable. (2) Methods: We studied 1001 patients with MM between 1980 and 2020, grouping patients into ten-year periods by diagnosis 1980–1990, 1991–2000, 2001–2010 and 2011–2020. (3) Results: After 65.1 months of follow-up, the median OS of the cohort was 60.3 months, and OS increased significantly over time: 22.4 months in 1980–1990, 37.4 months in 1991–2000, 61.8 months in 2001–2010 and 103.6 months in 2011–2020 (p < 0.001). Using novel agents in the front-line setting for myeloma patients yielded a significantly better OS than in those treated with conventional therapies, especially when combinations of at least two novel agents were used. The median OS of patients treated with the combination of at least two novel agents in induction was significantly prolonged compared to those treated with a single novel agent or conventional therapy in induction: 143.3 vs. 61.0 vs. 42.2 months (p < 0.001). The improvement was apparent in all patients regardless of age at diagnosis. In addition, 132 (13.2%) patients were long-term survivors (median OS ≥ 10 years). Some independent clinical predictors of long-term survival were identified: ECOG < 1, age at diagnosis ≤ 65 years, non-IgA subtype, ISS-1 and standard-risk cytogenetic. Achieving CR and undergoing ASCT were positively associated with >10 years of survival. (4) Conclusions: The combination of novel agents appears to be the main factor for the improvement in survival in MM, which is becoming a chronic and even curable disease in a subtype of patients without high-risk features.

Immunoparesis is the suppression of normal polyclonal immunoglobulins and is present in most patients with newly diagnosed multiple myeloma (MM). The association of immunoparesis at diagnosis, and particularly its recovery along with treatment, with survival in patients ineligible for autologous stem-cell transplantation (ASCT) has not been well established. This retrospective study evaluated the impact of immunoparesis in 431 patients diagnosed with MM, ineligible for ASCT, with a median overall survival of 36 months [95% confidence interval (CI): 31-40]. Immunoparesis was present in 81.2% of patients at diagnosis and was associated with a trend to a worse overall response rate (ORR: 84.8% vs. 74.9%; OR 1.88 (95% CI: 0.97-3.63), shorter progression-free survival (PFS) [22.0 vs. 18.2 months; hazard ratio (HR) 0.775; 95%CI: 0.590-1.018; p = 0.066], and overall survival (OS) (45.9 vs. 34.2 months; HR 0.746; 95% CI: 0.551-1.010; p = 0.057). Twenty-four per cent of patients who had immunoparesis at diagnosis recovered polyclonal immunoglobulins in the follow-up period. Interestingly, these patients had a better ORR (96.3% vs. 68.2%; OR 12.29 (95% CI: 3.77-40.06), PFS (HR 0.703; 95CI%: 0.526-0.941; p = 0.018) and OS (HR 0.678; 95 CI%: 0.503-0.913; p = 0.011) than patients who did not recover it. In summary, restoring a healthy immune system along with first-line treatment in patients with MM, not receiving ASCT, is associated with better outcomes.

Background and objective SARS-CoV-2 infection has bimodal distribution in Europe with a first wave in March to June 2020 and a second in September 2020 to February 2021. We compared the frequency, clinical characteristics and outcomes of adults with acute lymphoblastic leukemia (ALL) and infection in the first vs. second pandemic waves in Spain. Patients and Methods In this prospective study the characteristics of ALL and COVID-19 infection, comorbidities, treatment and outcome in the two periods were compared. The study ended when vaccination against SARS-CoV-2 was implemented in Spain. Results Twenty eight patients were collected in the first wave and 24 in the second. The median age was 46.5 years (range 20–83). Patients from the first wave had a trend to more severe ALL (higher frequency of patients under induction or submitted to transplantation or under immunosuppressive therapy). No significant differences were observed in need for oxygen support, intensive care unit (ICU) requirement, days in ICU and time to COVID-19 infection recovery. Seventeen patients (33%) died, with death attributed to COVID infection in 15 (29%), without significant differences in the 100 day overall survival (OS) probabilities in the two waves (68% ± 17% vs. 56% ± 30%). The only prognostic factor for OS identified by was the presence of comorbidities at COVID-19 infection (HR: 5.358 [95% CI: 1.875- 15.313]). Conclusion The frequency and mortality of COVID-19 infection were high in adults with ALL, without changes over time, providing evidence in favor of vaccination priority for these patients., Microabstract The characteristics and outcome of ALL in adults with COVID-19 infection in the first two waves of the pandemic in Spain were compared. The frequency and mortality of COVID-19 infection were high in adults with ALL, without changes over time. Comorbidities at COVID-19 infection was the only prognostic factor for survival.

© 2021 by the authors., B-cell regeneration during therapy has been considered as a strong prognostic factor in multiple myeloma (MM). However, the effects of therapy and hemodilution in bone marrow (BM) B-cell recovery have not been systematically evaluated during follow-up. MM (n = 177) and adult (≥50y) healthy donor (HD; n = 14) BM samples were studied by next-generation flow (NGF) to simultaneously assess measurable residual disease (MRD) and residual normal B-cell populations. BM hemodilution was detected in 41 out of 177 (23%) patient samples, leading to lower total B-cell, B-cell precursor (BCP) and normal plasma cell (nPC) counts. Among MM BM, decreased percentages (vs. HD) of BCP, transitional/naïve B-cell (TBC/NBC) and nPC populations were observed at diagnosis. BM BCP increased after induction therapy, whereas TBC/NBC counts remained abnormally low. At day+100 postautologous stem cell transplantation, a greater increase in BCP with recovered TBC/NBC cell numbers but persistently low memory B-cell and nPC counts were found. At the end of therapy, complete response (CR) BM samples showed higher CD19− nPC counts vs. non-CR specimens. MRD positivity was associated with higher BCP and nPC percentages. Hemodilution showed a negative impact on BM B-cell distribution. Different BM B-cell regeneration profiles are present in MM at diagnosis and after therapy with no significant association with patient outcome., This work has been supported by the International Myeloma Foundation-Black Swan Research Initiative, the EuroFlow Consortium (grant LSHB-CT-2006-018708); Centro de Investigación Biomédica en Red de Cáncer (CIBER-ONC; Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain and FONDOS FEDER), numbers: CB16/12/00400, CB16/12/00233, CB16/12/00369, CB16/12/00489 and CB16/12/00480; grant from Bilateral Cooperation Program between Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES (Brasília/Brazil) and Dirección General de Políticas Universitárias (DGPU)-Ministério de Educación, Cultura y Deportes (Madrid/Spain) number DGPU 311/15; Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro of Brazil (FAPERJ) numbers: E26/110.105/2014 and E26/102.191/2013; grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico of Brazil (CNPQ), number: 400194/2014-7. R.M.d.P. was supported by a grant from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/DGPU), number: 000281/2016-06 and CAPES/PROEX 641/2018, Brazil; Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro of Brazil (FAPERJ) number: E01/200/537/2018.

Recommended genetic categorization of acute myeloid leukaemias (AML) includes a favourable‐risk category, but not all these patients have good prognosis. Here, we used next‐generation sequencing to evaluate the mutational profile of 166 low‐risk AML patients: 30 core‐binding factor (CBF)‐AMLs, 33 nucleophosmin (NPM1)‐AMLs, 4 biCEBPα‐AMLs and 101 acute promyelocytic leukaemias (APLs). Functional categories of mutated genes differed among subgroups. NPM1‐AMLs showed frequent variations in DNA‐methylation genes (DNMT3A, TET2, IDH1/2) (79%), although without prognostic impact. Within this group, splicing‐gene mutations were an independent factor for relapse‐free (RFS) and overall survival (OS). In CBF‐AML, poor independent factors for RFS and OS were mutations in RAS pathway and cohesin genes, respectively. In APL, the mutational profile differed according to the risk groups. High‐risk APLs showed a high mutation rate in cell‐signalling genes (P = 0·002), highlighting an increased incidence of FLT3 internal tandem duplication (ITD) (65%, P < 0·0001). Remarkably, in low‐risk APLs (n = 28), NRAS mutations were strongly correlated with a shorter five‐year RFS (25% vs. 100%, P < 0·0001). Overall, a high number of mutations (≥3) was the worst prognostic factor RFS (HR = 2·6, P = 0·003). These results suggest that gene mutations may identify conventional low‐risk AML patients with poor prognosis and might be useful for better risk stratification and treatment decisions., This work was partially supported by the ‘Instituto de Salud Carlos III’ (ISCIII), Spanish Ministry of Economy and Competitiveness PI15/01706, PI16/00517, PI18/01946, CIBERONC‐CB16/12/00233 and ‘Una manera de hacer Europa’ (Innocampus; CEI‐2010‐1‐0010). MIPC and MGA are supported by the Fundación Española de Hematología y Hemoterapia (FEHH). MCC has been supported by the Spanish Association Against Cancer Scientific Foundation, (AECC). MES is supported by Miguel Servet programme (CP13/00080) from the ISCIII, Ministerio de Ciencia e Innovación, Madrid, Spain. CJ is supported by the ‘Beca de investigación FEHH‐CRIS 2018’.

We report data from a prospective, observational study (ZAGAL) evaluating miglustat 100mg three times daily orally. in treatment-naïve patients and patients with type 1 Gaucher Disease (GD1) switched from previous enzyme replacement therapy (ERT). Clinical evolution, changes in organ size, blood counts, disease biomarkers, bone marrow infiltration (S-MRI), bone mineral density by broadband ultrasound densitometry (BMD), safety and tolerability annual reports were analysed. Between May 2004 and April 2016, 63 patients received miglustat therapy; 20 (32%) untreated and 43 (68%) switched. At the time of this report 39 patients (14 [36%] treatment-naïve; 25 [64%] switch) remain on miglustat. With over 12-year follow-up, hematologic counts, liver and spleen volumes remained stable. In total, 80% of patients achieved current GD1 therapeutic goals. Plasma chitotriosidase activity and CCL-18/PARC concentration showed a trend towards a slight increase. Reductions on S-MRI (p=0.042) with an increase in BMD (p

Although information about the molecular pathogenesis of Waldenström macroglobulinemia (WM) has significantly advanced, the precise cell of origin and the mechanisms behind WM transformation from immunoglobulin-M (IgM) monoclonal gammopathy of undetermined significance (MGUS) remain undetermined. Here, we undertook an integrative phenotypic, molecular, and genomic approach to study clonal B cells from newly diagnosed patients with IgM MGUS (n = 22), smoldering (n = 16), and symptomatic WM (n = 11). Through principal component analysis of multidimensional flow cytometry data, we demonstrated highly overlapping phenotypic profiles for clonal B cells from IgM MGUS, smoldering, and symptomatic WM patients. Similarly, virtually no genes were significantly deregulated between fluorescence-activated cell sorter-sorted clonal B cells from the 3 disease groups. Interestingly, the transcriptome of the Waldenström B-cell clone was highly different than that of normal CD25-CD22+ B cells, whereas significantly less genes were differentially expressed and specific WM pathways normalized once the transcriptome of the Waldenström B-cell clone was compared with its normal phenotypic (CD25+CD22+low) B-cell counterpart. The frequency of specific copy number abnormalities [+4, del(6q23.3-6q25.3), +12, and +18q11-18q23] progressively increased from IgM MGUS and smoldering WM vs symptomatic WM (18% vs 20% and 73%, respectively; P = .008), suggesting a multistep transformation of clonal B cells that, albeit benign (ie, IgM MGUS and smoldering WM), already harbor the phenotypic and molecular signatures of the malignant Waldenström clone., This study was supported by Cooperative Research Thematic Network grants RD12/0036/0058 and RD12/0036/0048 of the Red de Cancer (Cancer Network of Excellence), Consejería de Sanidad, Junta de Castilla y Leon, Valladolid, Spain (557/A/10).

Immunoglobulin M (IgM) monoclonal gammopathies show considerable variability, involving three different stages of presentation: IgM monoclonal gammopathy of undetermined significance (IgM-MGUS), asymptomatic Waldenstrom's macroglobulinemia (AWM), and symptomatic WM (SWM). Despite recent findings about the genomic and transcriptomic characteristics of such disorders, we know little about the causes of this clinical heterogeneity or the mechanisms involved in the progression from indolent to symptomatic forms. To clarify these matters, we have performed a gene expression and mutational study in a well-characterized cohort of 69 patients, distinguishing between the three disease presentations in an attempt to establish the relationship with the clinical and biological features of the patients. Results showed that the frequency of genetic alterations progressively increased from IgM-MGUS to AWM and SWM. This means that, in contrast to MYD88 p.L265P and CXCR4 WHIM mutations, present from the beginning of the pathogenesis, most of them would be acquired during the course of the disease. Moreover, the expression study revealed a higher level of expression of genes belonging to the Toll-like receptor (TLR) signaling pathway in symptomatic versus indolent forms, which was also reflected in the disease presentation and prognosis. In conclusion, our findings showed that IgM monoclonal gammopathies present higher mutational burden as the disease progresses, in parallel to the upregulation of relevant pathogenic pathways. This study provides a translational view of the genomic basis of WM pathogenesis.

Bortezomib-melphalan-prednisone combination is one of the standards of care for nontransplant eligible patients with newly diagnosed multiple myeloma. However, bortezomib intravenous (twice weekly for 4 cycles then weekly for 5 cycles) results in ~13% of patients with grade 3-4 peripheral neuropathy. Bortezomib subcutaneous (SQ) and weekly delivery, improves tolerability without impairment of efficacy. The aim of this study was to evaluate the safety and effectiveness of SQ bortezomib-based combinations in nontransplant eligible patients with newly diagnosed myeloma in a real-world setting. A total of 135 patients (median age [range] = 76 [58-89], International Staging System-III = 54%, median follow-up = 14.8 months [1-40], Intensive group [twice weekly bortezomib] = 65%, Optimized group [weekly bortezomib] = 35%) were included and evaluable for safety, whereas 121 were evaluable for effectiveness. Overall response rate (95% CI) was 61% (53%, 71%) (complete response = 27%, very good partial response = 13%, and partial response = 21%) and median progression-free survival was 22.2 months (95% CI: 16.1-not reached). The 3-year overall survival was 75%. The most frequent grade 3-4 adverse events were thrombocytopenia (18%), neutropenia (17%), and anemia (11%). Peripheral neuropathy of any grade was observed in 44% of patients (2% with grade 3). Comparison between regimens (Intensive vs Optimized) showed similar overall response rate (57% vs 70%) and PFS (25 vs 19 months). A similar safety profile was observed between regimens. Thus, SQ bortezomib showed similar effectiveness and better tolerability as compared with results from intravenous bortezomib studies, and showing no differences either in effectiveness or safety in different bortezomib-based combinations.

Eltrombopag is safe and effective in primary chronic ITP. However, lack of clinical trials avoids a clear demonstration of its utility in newly diagnosed and persistent ITP. Our aim here is to report Spanish results for this type of patients. We retrospectively evaluated 220 adult primary ITP patients. According to standard definition, patients were allocated to newly diagnosed (n = 30), persistent (n = 30), and chronic (n = 160) ITP. Groups were homogenous regarding most relevant parameters. 180 (90%) of 220 patients achieved a platelet response (R) with 167 (75.9%) complete responses (CR) after a 15-month follow-up. No statistical significant differences among groups but a trend towards a greater efficacy in newly diagnosed ITP were observed (93.3% of responses with 86.7% of CR). Efficacy in persistent ITP (83.3% of responses with 80.0% of CR) and chronic ITP (79.4% of responses with 73.1% of CR) was similar. 70 patients (31.8%) experienced adverse events. 15 of them were grade 3–4. Most common adverse effects were headache and hepatobiliary laboratory abnormalities (HBLAs). One persistent ITP had a venous thrombosis and one chronic ITP had grade II myelofibrosis. We consider Eltrombopag use for the early stage ITP as effective and safe as it is in chronic ITP.

Flow cytometry has become a highly valuable method to monitor minimal residual disease (MRD) and evaluate the depth of complete response (CR) in bone marrow (BM) of multiple myeloma (MM) after therapy. However, current flow-MRD has lower sensitivity than molecular methods and lacks standardization. Here we report on a novel next generation flow (NGF) approach for highly sensitive and standardized MRD detection in MM. An optimized 2-tube 8-color antibody panel was constructed in five cycles of design-evaluation-redesign. In addition, a bulk-lysis procedure was established for acquisition of ⩾107 cells/sample, and novel software tools were constructed for automatic plasma cell gating. Multicenter evaluation of 110 follow-up BM from MM patients in very good partial response (VGPR) or CR showed a higher sensitivity for NGF-MRD vs conventional 8-color flow-MRD -MRD-positive rate of 47 vs 34% (P=0.003)-. Thus, 25% of patients classified as MRD-negative by conventional 8-color flow were MRD-positive by NGF, translating into a significantly longer progression-free survival for MRD-negative vs MRD-positive CR patients by NGF (75% progression-free survival not reached vs 7 months; P=0.02). This study establishes EuroFlow-based NGF as a highly sensitive, fully standardized approach for MRD detection in MM which overcomes the major limitations of conventional flow-MRD methods and is ready for implementation in routine diagnostics., This work has been supported by the International Myeloma Foundation-Black Swan Research Initiative, the Red Temática de Investigación Cooperativa en Cáncer (RTICC); grant SA079U14 from the Consejería de Educación, Junta de Castilla y León, Valladolid, Spain and; grant DTS15/00119 from Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain.

Hematocrit control below 45% is associated with a lower rate of thrombosis in polycythemia vera. In patients receiving hydroxyurea, this target can be achieved with hydroxyurea alone or with the combination of hydroxyurea plus phlebotomies. However, the clinical implications of phlebotomy requirement under hydroxyurea therapy are unknown. The aim of this study was to evaluate the need for additional phlebotomies during the first five years of hydroxyurea therapy in 533 patients with polycythemia vera. Patients requiring 3 or more phlebotomies per year (n=85, 16%) showed a worse hematocrit control than those requiring 2 or less phlebotomies per year (n=448, 84%). There were no significant differences between the two study groups regarding leukocyte and platelet counts. Patients requiring 3 or more phlebotomies per year received significantly higher doses of hydroxyurea than the remaining patients. A significant higher rate of thrombosis was found in patients treated with hydroxyurea plus 3 or more phlebotomies per year compared to hydroxyurea with 0-2 phlebotomies per year (20.5% vs. 5.3% at 3 years; P

Prognostic factors in Hodgkin lymphoma (HL) still fail to accurately identify high-risk patients. Tumor microenvironment in HL is a current focus of research for risk definition but few studies have focused on infiltrating lymphocytes. Here, we analyzed the number of tumor infiltrating lymphocytes by flow cytometry in diagnostic biopsies from 96 HL homogeneously treated patients with ABVD with or without radiotherapy. Most lymph node cells were lymphocytes (90 ± 17), with a median T/B/NK distribution of 74%/26%/0.7%, and CD4 T-cell predominance. The amount of CD19 B cells, and NK cells did not show association with disease features. However, high numbers of CD8 and CD4 cells were associated with better and poorer outcomes, respectively. Patients with ≥15% cytotoxic CD8 cells among the total cell population had a longer 10-year freedom from treatment failure (FFTF) (93% vs. 73%, p=.04). In turn, cases with ≥75% of CD4 infiltrating cells showed a significantly decreased FFTF (73% vs. 96%, p=.021). Consequently, CD4/CD8 ratio ≥5 associated with a poorer 10-year FFTF (69.5% vs. 94%, p=.02). This deleterious effect was particularly prominent in advanced disease (n = 58, p=.01). In multivariate analysis, a CD4/CD8 ratio ≥5 was the only independent variable to predict for treatment failure (HR = 4.5, 95% confidence interval, 1.2–16.8). In conclusion, our study shows that high CD4 and low CD8 T-cells infiltrates of tumor specimens associate with poor prognosis in HL patients, and CD4/CD8 ratio might be potentially useful for tailoring therapy.

BackgroundEltrombopag is effective and safe in chronic immune thrombocytopenia (ITP). However, clinical trials may not accurately reflect what happens in clinical practice. We evaluated the efficacy and safety of eltrombopag in primary chronic ITP in a real-world setting. MethodsA total of 164 primary patients with chronic ITP from 40 Spanish centers, who had been treated with eltrombopag, were retrospectively evaluated. ResultsThe median age of our cohort (72% women) was 63 yr (interquartile range, IQR, 45-75 yr). The median time with ITP diagnosis was 81 months (IQR, 30-192 months). The median number of therapies prior to eltrombopag was 3 (IQR, 2-4). At the time of eltrombopag start, 45 patients (30%) were receiving concomitant treatment for ITP. Forty-six patients (30%) had bleeding signs/symptoms the month before the treatment started. The median platelet count at eltrombopag initiation was 22 x 10(9)/L (IQR, 8-39 x 10(9)/L). A total of 135 patients (88.8%) achieved a platelet response. The median time to platelet response was 12 d (95% CI, 9-13 d). Maintained platelet response rate during the 15-month period under examination was 75.2%. Twenty-eight patients (18.4%) experienced adverse events, mainly grades 1-2. ConclusionEltrombopag is highly effective and well tolerated in unselected patients with primary chronic ITP.

[Purpose]: Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3′UTR in CCND2 overexpression in multiple myeloma. [Experimental Design]: Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3′UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3′ rapid amplification of cDNA ends (RACE)-PCR. [Results]: We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3′RACE experiments revealed that changes in CCND2 3′UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3′UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Furthermore, functional analysis showed significant CCND2 mRNA shortening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggesting that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction. [Conclusions]: Overall, these results highlight the impact of CCND2 3′UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma., This study was partially supported by Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias: PI08/0568 and PI13/00111), Asociacion Española Contra el Cancer (AECC, GCB120981SAN), >Gerencia Regional de Salud, Junta de Castilla y Leon> (GRS 702/A/11, BIO/SA57/13 and BIO/SA35/14) grants, the Spanish Myeloma Network Program (RD12/0036/0058) and the INNOCAMPUS Program (CEI10-1-0010). I. Misiewicz-Krzeminska was supported by Instituto de Salud Carlos III (PS09/01897). M.E. Sarasquete was supported by Contrato Miguel Servet (CP13/00080). Research in the I. Sanchez-García group was supported by FEDER and MINECO (SAF2012-32810, and Red de Excelencia Consolider OncoBIO SAF2014-57791-REDC), Instituto de Salud Carlos III (PIE14/00066), NIH (R01 CA109335-04A1), >Junta de Castilla y Leon> (BIO/SA32/14 and CSI001U14) grants, the Fundacion Inocente Inocente, the German Carreras Foundation (DJCLS R13/26) and by the ARIMMORA project (European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 282891). Research in the C.V. Duenas group was supported by >Miguel Servet> Grant (CP14/00082 AES 2013-2016) from the Instituto de Salud Carlos III (Ministerio de Economía y Competitividad). I. Sanchez-García is an API lab of the EuroSyStem project and a partner within the Marie Curie Initial Training Network DECIDE.

The clinical value of multiparameter flow cytometry (MFC) immunophenotyping in primary or light chain amyloidosis (AL) remains unknown. We studied 44 consecutive bone marrow samples from newly diagnosed patients with amyloidosis; 35 patients with AL and 9 with other forms of amyloidosis. Monoclonal plasma cells (PCs) were identifiable by MFC immunophenotyping in 34 of 35 (97%) patients with AL, whereas it was absent from all but 1 of the 9 (11%) patients with other forms of amyloidosis. Quantification of bone marrow plasma cells (BMPCs) by MFC immunophenotyping was a significant prognostic factor for overall survival (OS) (≤ 1% vs > 1% BMPC cutoff; 2-year OS rates of 90% vs 44%, P = .02). Moreover, detecting persistent normal PCs at diagnosis identifies a subgroup of patients withAL with prolonged OS (> 5% vs ≤ 5% normal PC within all BMPC cutoff, 2-year rates of 88% vs 37%, P = .01). MFC immunophenotyping could be clinically useful for the demonstration of PC clonality in AL and for the prognostication of patients with AL. © 2011 by The American Society of Hematology., This work was supported by the Cooperative Research Thematic Cancer Network (RTIC) grants RD06/0020/0006 and G03/136, MM Jevitt, SL firm, Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS) grants PI060339, 02/0905, 01/0089/01-02, PS09/01897, and Consejería de Sanidad, Junta de Castilla y León, Valladolid, Spain grant 557/A/10.

Type 1 Gaucher disease (GD1) is caused by the deficiency in the lysosomal enzyme glucocerebrosidase and causes the accumulation of glucocerebroside mainly in macrophages, inducing deterioration of the organs in which it is deposited.The new substrate inhibitor Eliglustat (ELI), approved by EMA in 2015 and available in Spain since January 2017, has proved to be an effective therapy that selectively inhibits the enzyme glucosylceramide synthase, decreasing the accumulation of substrate. It is indicated in GD1 extensive, intermediate or poor metabolizers of the cytochrome CYP2D6. The phase 2 and 3 clinical trials have demonstrated improvement and stabilization of the parameters in the naïve patients and in switched from enzyme replacement therapy. In this work we present the design of a prospective follow-up post-authorization study (GEE-ELI-2017-01) to asure the traceability of eliglustat therapy in Spanish GD1patients (TRAZELGA) is exposed. MethodsThe national multi-center study TRAZELGA, designed as a tool to uniformly evaluate the response to ELI therapy during one year, analyzing clinical parameters, biomarkers, changes in medullary infiltration quantified by MRI and DEXA, compliance and side effects. In addition, a quality of life sub-study is included as well as an exploratory study to analyze immune system activation markers (cytokine profile, ferritin, lipocalin, gamma globulins, oxidative stress markers). All the current recommendations previous ELI therapy have been reinforced, including cardiac, hepatic and renal function assessment and suitability according concomitant medications. An electronic tool has been designed to help physicians to easily registry and update the patient information. All the current rules for data protection were taken in account. Results: 40 GD1 patients have started oral treatment with Eliglustat. In this presentation we provide preliminary results of 32 patients whom fulfilled 6 months of follow-up (median age: 43.8 years (23-75), 47% males), genotype c.1226A>G in homozygosity (29.4%), c.1226A>G/c.1448T>C (41.2% ), other double heterozygotes with c.1226A>G (29.4%); metabolism of CYP2D6 (12% poor metabolizers, 64.5% intermediate and 33.5% extensive), no patient received the treatment in first line and its baseline characteristics (table1), are from patients stabilized with ERT (23 cases) or miglustat (9).Two splenectomized patients, 3 patients with palpable splenomegaly at the time of inclusion, 6 patients with multimorbidities and polymedications and 6 patients complained of fatigue as the main symptom before inclusion in the study. The median follow-up is 6 (6-14) months. Conclusions: The acceptance of the project in the medical community has been excellent with a good inclusion page. At the end of the one year follow-up period we will be available to analyze the influence of Eliglustat on biomarkers, markers of inflammation, bone mineral density also to provide information about adherence and adverse events in every day clinical practice. This work has been partially granted by FEETEG Disclosures No relevant conflicts of interest to declare.

Gaucher disease (GD), has a pan-ethnic distribution with incidence in non-Ashkenazi Jewish population about 1/70-140 thousand inhabitants. The deficiency in lysosomal acid beta-glucosidase enzyme secondary to variants in the GBA gene with autosomal recessive inheritance gives rise to a variable clinical picture. It appears at any age with symptoms that include anemia, thrombocytopenia, pain and vascular bone lesions, visceral enlargement, persistent fatigue. It was the first EDL to have enzymatic replacement and substrate reduction therapy that impact in the awareness of the disease. In 1993, the Spanish Registry of Gaucher Disease was created (REsEG), within the Spanish Foundation for the Study and Therapeutics of EG (FEETEG), with the aim of providing support to all those involved in the management of patients with EG and summarize experience at the national level. This work review the characteristics at diagnosis of type 1 adult GD (GD1) patients, diagnosed outside active early diagnosis programs, along of twenty five years of REsEG existence focus on leading diagnostic symptoms in order to answer the current challenges in patients suspicions. Aim: To analyze the evolving profile of GD1 patients diagnosed before and after 2000 based on the principal manifestations including visceral, hematological involvement, bone pain, bleeding in order to define the current suspicious characteristics in our population. Methods: Patients 18+ years old included in the REsEG were selected. Demographic, genetic, clinical (hematological, visceral, bone, neurological) data, severity index, biomarkers and other relevant information were included in a database created for this purpose. Platelets Disclosures No relevant conflicts of interest to declare.

The PI3/AKT pathway is up-regulated in acute myeloid leukemia (AML), but its prognostic relevance in cytogenetically normal AML (CN-AML) is unclear. We evaluated RNA levels of AKT and two downstream substrates (FOXO3a-p27) in 110 de novo CN-AML, included in the Spanish PETHEMA therapeutic protocols. Patients with high FOXO3a gene expression displayed shorter OS (p = 0.015) and RFS (p = 0.048) than low FOXO3a expressers. Features selected in the multivariate analysis as having an independent prognostic value for a shorter survival were WBC > 50 × 109/L, age >65 years and high FOXO3a expression. We concluded that FOXO3a assessment could contribute to improve the molecular-based risk stratification in CN-AML. © 2009 Elsevier Ltd. All rights reserved., This work has been partially supported with the grants PI061351 and 00/0023-00 from the Spanish “Fondo de Investigaciones Sanitarias de la Seguridad Social”, 89/A/06 from “Gerencia Regional de Salud, Junta Castilla y León”, CR-USA Foundation-Spanish National Research Council (CSIC) Cooperative Agreement and CIC, IBMCC (USAL-CSIC), Spain.

There are few published data from real-world clinical experience with miglustat (Zavesca®), an oral inhibitor of glucosylceramide synthase, in type 1 Gaucher disease. We report data from a prospective, open-label investigational study that evaluated substrate reduction therapy with miglustat 100 mg t.i.d. as a maintenance therapy in patients with Type 1 Gaucher disease who had been switched from previous enzyme replacement therapy. Long-term data on changes in organ size, blood counts, disease severity bio-markers, bone marrow infiltration, overall clinical status and safety/tolerability were analyzed from 28 patients with Type 1 Gaucher disease who were attending routine clinic visits. Assessments were performed at six, 12, 24, 36 and 48 months of therapy. Disease severity biomarkers improved up to 48 months after initiation of miglustat, while other disease parameters remained stable. Miglustat showed an acceptable profile of safety and tolerability throughout treatment. In conclusion, miglustat is an effective therapy for the long-term maintenance of patients with Type 1 Gaucher disease previously stabilized with enzyme replacement therapy.

The clinical significance of resistance/intolerance to hydroxycarbamide (HC) was assessed in a series of 890 patients with polycythaemia vera (PV). Resistance/intolerance to HC was recorded in 137 patients (15·4%), consisting of: need for phlebotomies (3·3%), uncontrolled myeloproliferation (1·6%), failure to reduce massive splenomegaly (0·8%), development of cytopenia at the lowest dose of HC to achieve a response (1·7%) and extra-haematological toxicity (9%). With a median follow-up of 4·6 years, 99 patients died, resulting in a median survival of 19 years. Fulfilling any of the resistance/intolerance criteria had no impact on survival but when the different criteria were individually assessed, an increased risk of death was observed in patients developing cytopenia [Hazard ratio (HR): 3·5, 95% confidence interval (CI): 1·5-8·3, P = 0·003]. Resistance/intolerance had no impact in the rate of thrombosis or bleeding. Risk of myelofibrotic transformation was significantly higher in those patients developing cytopenia (HR: 5·1, 95% CI: 1·9-13·7, P = 0·001) and massive splenomegaly (HR: 9·1, 95% CI: 2·3-35·9, P = 0·002). Cytopenia at the lowest dose required to achieve a response was also an independent risk factor for transformation to acute leukaemia (HR: 20·3, 95% CI: 5·4-76·5, P < 0·001). In conclusion, the unified definition of resistance/intolerance to HC delineates a heterogeneous group of PV patients, with those developing cytopenia being associated with an adverse outcome.

Despite remarkable therapeutic improvement in multiple myeloma (MM), prognosis is very poor once patients become refractory to or ineligible for proteasome inhibitors and immunomodulatory drugs. Accordingly, new drugs are clearly needed for this patient population, and extensive efforts are leading to a new breed of antimyeloma drugs.

Melphalan & Prednisone (MP) is considered as the standard therapy for Multiple Myeloma (MM) patients not eligible for high-dose therapy. Here, we report the results of a phase I-II study to evaluate the feasibility and efficacy of the association of PLD to the conventional MP regimen during the first six cycles of the front-line therapy for untreated MM patients older than 70. Thirty patients were included in the study with a median age of 77 years (71-84) and a M/F ratio of 17/13. The phase I of the study demonstrated that the maximum tolerable dose of PLD in this setting was 30 mg/m(2), so it was the final dose evaluated in the study. Twenty-nine patients were valuable for response, which was: complete in 4 (14%) partial in 15 (52%) minor/no changes in 7 (24%) and progressive in 3 (10%). The median progression free survival (PFS) was 24 months. The median overall survival (OS) has not been reached yet, with a 3-year probability for OS and PFS of 52 and 37%, respectively. Haematological toxicity was frequent but usually weak/moderate (grades 1 & 2 of the WHO scale) and it was resolved only with dose delays. Infection was a relatively frequent event (30% of patients), but only in 4 cases it was of grade 3. No cases of palmar-plantar erythrodysesthesia were observed. In conclusion, pegylated liposomal doxorubicin can be safely added to the other chemotherapeutic drugs in the treatment of elderly MM patients, which can be very useful for patients in whom novel agents are not tolerated or inefficient.

The aim of the present randomized trial was to compare high-dose therapy (HDT) with continued conventional chemotherapy in patients with multiple myeloma (MM) who responded to the initial treatment. From May 1994 to October 1999, 216 patients (122 men/94 women; stage II or III; Eastern Cooperative Oncology Group [ECOG] score less than 3) entered the study. Initial chemotherapy consisted of 4 cycles of alternating vincristine, BCNU, melphalan, cyclophosphamide, prednisone/vincristine, BCNU, Adriamycin, dexamethasone (VBMCP/VBAD). Responding patients were randomly assigned to receive 8 additional cycles of VBMCP/VBAD, intensification with melphalan 200 mg/m2, or melphalan 140 mg/m2 plus 12 Gy fractionated total body irradiation (TBI). One-hundred sixty-four patients were randomly assigned, 83 to continued chemotherapy and 81 to HDT. The complete remission (CR) rate was significantly higher with HDT (30% vs 11%; P = .002). However, progression-free survival (PFS) was not significantly different between HDT and conventional therapy (median, 42 vs 33 months; P = not significant [NS]), and overall survival (OS) was similar in both groups (median, 61 vs 66 months). Finally, survival after relapse was identical in the 2 arms (15.9 vs 16.4 months). In conclusion, these results show that HDT intensification, when given to myeloma patients who have responded to the initial chemotherapy, significantly increases the CR rate but has no significant impact on PFS or OS.

Although multiple myeloma (MM) is predominantly a disease of the elderly, few studies have focused on the identification of prognostic factors in this group of patients. Four hundred twenty five MM patients >65 years were uniformly treated with chemotherapy (MP or VCMP/VBAD). Multivariate analysis identified 4 factors with independent unfavorable prognostic influence: high percentage of S-phase bone marrow plasma cells (>2.5%); elevated beta(2) microglobulin (B2M) (>4 mg/L); age >80 years old; and LDH serum levels (above normal limit). The S-phase value was the most powerful independent prognostic factor to discriminate subgroups of patients with different prognosis. Thus, 3 main risk categories could be identified according to S-phase values: 3%, with median survivals of 34, 22 and 12 months, respectively (p < 0.0001). Our study also proved the value for elderly patients of the recently developed International Score System (ISS) based on B2M and albumin. Furthermore, the number of S-phase cells helped to subdivide the ISS III Group identifying a subset of patients with very poor prognosis defined by an additional high S-phase, who displayed a median survival of only 8 months. These results demonstrate that elderly patients can be accurately classified according to prognosis, which may be particularly valuable when comparing the efficacy of new treatment strategies. Moreover, our results underline the high prognostic value of proliferative activity of PC, a parameter that should be considered in routine laboratory investigations of MM.

Immunophenotyping has become an essential tool for diagnosis of hematological malignancies. By contrast, for diagnosis of Waldenstrom's macroglobulinemia (WM) immunophenotyping is used only occasionally. From 150 patients with a IgM monoclonal gammopathy we have selected 60 cases with (1) morphological lymphoplasmocytoid bone marrow (BM) infiltration (>20%); (2) IgM paraprotein (>10g/L); and (3) absence of features of other lymphoma types. Immunophenotypic analysis was based on the use of the triple or quadruple monoclonal antibody (MoAb) combinations. To increase the sensitivity of the analysis of antigen expression, selected CD19(+)CD20(+) B cells were targeted. We have also explored the antigenic characteristics of both the plasma cell (PC) and mast cell (MC) compartments present in the BM from 15 WM patients. Clonal WM lymphocytes were characterized by the constant expression of pan-B markers (CD19, CD20, CD22, CD24) together with sIg, predominantly kappa (5:1, kappa:lambda ratio). A high proportion of cases (75%) were positive for FMC7 and CD25, but in contrast to hairy cell leukemia (HCL), these lymphocytes were always negative for CD103 and CD11c. CD10 antigen was also absent in all WM patients and less than one fifth of patients were positive for CD5 and CD23, while CD27, CD45RA, and BCL-2 were present in most malignant cells. In two cases, the coexistence of two different clones of B lymphocytes was identified, and in eight additional cases, intraclonal phenotypic heterogeneity was observed. As far as PCs are concerned, in most patients (85%) the number of PCs was within the normal range (median, 0.36%). The antigenic profile of these PCs differed from that observed in normal and myelomatous PC (CD38(++)CD19(++/-)CD56(-)CD45(++)CD20(+)). In three cases, PCs showed aberrant expression for CD5, CD22, or FMC7. Finally, the number of mast cells was significantly higher (0.058 +/- 0.13) as compared to normal BM (0.019 +/- 0.02) (P

Summary. In order to gain further insights into the role of the p16 gene in cell cycle regulation and the prognostic implications of its inactivation, we investigated the methylation status of the p16 gene in 98 untreated patients using a polymerase chain reaction assay based on the inability of some restriction enzymes to digest methylated sequences. Forty-one patients showed a p16 methylated gene (42%). The percentage of S-phase plasma cells (PC) in these patients was almost three times higher than in those with an unmethylated p16 gene (4·16% ± 3·37%vs 1·5% ± 1·41%, P

Eltrombopag is effective and safe in chronic immune thrombocytopenia (ITP) patients not responding to previous therapy. However, when eltrombopag is discontinued, platelet counts usually return to baseline within 2 weeks. Here, we describe the clinical characteristics of the, to the best of our knowledge, largest case series of patients with ITP, who presented sustained responses after discontinuing eltrombopag (n = 12). The median time from diagnosis to eltrombopag initiation was 24 months (range, 1-480). The median number of prior therapies was 5 (range, 1-7), and the median duration of eltrombopag treatment was 5 months (range, 1-13). Three patients received eltrombopag for only 1 month. The treatment was well-tolerated. The median (range) follow-up of this case series was of 7 months (6-20), during which all patients maintained a safe platelet count without the need for anti-ITP treatment. The communication of such cases may support the conduction of new studies to investigate which predictive factors could identify ITP patients with sustained responses after discontinuing eltrombopag without additional therapy. The need of long-term use of eltrombopag should be re-examined.

Introduction: Patients with type1 Gaucher's disease (GD1) have an increased risk of gammopathy (RR,33 Taddei TH 2009), multiple myeloma (RR,25.), other haematological malignancies (RR,3.45) and overall cancer risk (RR, 1.80). The Spanish Registry of Gaucher Disease (SpRGD) was established in 1993 in response to the need to group individual experiences in the diagnosis and management of this disease, increasing knowledge related to general characteristics and to know the real incidence and prevalence in the Spanish population. Registration is open to all physicians involved in the management of patients with GD and offers free enzymatic analysis, biomarkers and molecular analysis for the diagnosis and monitoring of patients (www.feeteg.org). Aim: to analyses the incidence of malignancies in adults GD patients. Patients and methods: A review of the SpRGD to obtain data form patients over 20 years of age at May, 2016 was performed. Physicians on charge fulfilled a survey in which they inform about the incidence of malignancies and follow-up information. Ethical approval was obtained from the institutional board and all patients has signed an inform consent before to be included into the SpRGD. Results: Of the 281 adult patients (³20 years) included, 279 were GD1 and 2 GD3. The average age of the entire cohort was 52.3 (23-90), of which 140 men, 141 women. Of these, 27 (9.6%) patients with GD1, 5 homozygous for N370S and 22 heterozygous for N370S had the presence of a malignancy and / or monoclonal gammopathy (MGUS), two of them had more than one neoplasia. Male / female: 11/16, mean age 60.2 (25-90), median follow-up of 16.5 years (4-23). Six have died by the tumor complications. All MGUS (N=12) were identified at GD diagnosis, they were 6 males and 6 females mean age 55.5 y (10-82) of them 50% under 60 years of age. Sixteen patients developed seventeen different neoplasms, with a female predominant (11, 68.7%). Only eight patients were under therapy at the time of neoplasia diagnosis (table1). Mean time on therapy 7.4 years (1.2-13-6). Neoplasms were registered (M/ F): B cell malignancies: Hodgkin lymphoma 1 (M), chronic lymphocytic leukemia 1 (M), multiple myeloma 1 (M), myeloid neoplasms: chronic myeloid leukemia: 1 (F), myelodysplastic syndrome: 1 (F), solid tumors: melanoma: 1 (F), meningioma: 2 (F), uterine cancer: 3 (F), gastric carcinoma 1 (F), cancer colon 2 (F), breast cancer 1 (F), prostate adenocarcinoma: 1(M), lung cancer 1 (M), liver carcinoma 1 (M), thyroid cancer 1 (F). Conclusions: It has been widely reported the highest incidence of haematological malignancies among patients with GD. Nevertheless in this cohort of Spanish patients, the incidence of solid tumors is similar to haematological neoplasms in general and higher than B cell lymphoid. Probably the incidence of malignancy in this population and during this monitoring period is similar to the expected in Spanish general population found in 0.21% / year, however females showed two times risk increase for malignancies and this aspect warranty further studies. This work has been carried out with aid for research FIS PS15/00616 and FEETEG Disclosures No relevant conflicts of interest to declare.

Although multiparameter flow cytometry (MFC) has demonstrated clinical relevance in monoclonal gammopathy of undetermined significance (MGUS)/myeloma, immunophenotypic studies on the full spectrum of Waldenström's Macroglobulinemia (WM) remain scanty. Herein, a comprehensive MFC analysis on bone marrow samples from 244 newly diagnosed patients with an immunoglobulin M (IgM) monoclonal protein was performed, including 67 IgM-MGUS, 77 smoldering and 100 symptomatic WM. Our results show a progressive increase on the number and light-chain-isotype-positive B-cells from IgM-MGUS to smoldering and symptomatic WM (P10% B cells or 100% light-chain-isotype-positive B-cells (P, This work was supported by the Cooperative Research Thematic Network (RTICC) RD06/0020/0006, Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS: PI060339; 06/1354; 02/0905; 01/0089/01-02; PS09/01897/01370) and Consejería de Sanidad, Junta de Castilla y León, Valladolid, Spain (557/A/10).