bluuebunny/crossref_metadata_2025 huggingface dataset:
Bone Mineral Density Changes In Patients with Paraproteinemia After Treatment with Bortezomib
Authors: Berno Tamara , Boucher Kenneth , Zhan Fenghuang , Tricot Guido J. , Mughal Benjamin , Zangari Maurizio
Published: October 2019

Abstract 4989

Background:

Bone disease is present at diagnosis in almost all patients with multiple myeloma (MM) and can impact substantially on patient morbidity and quality of life. Decreased bone mineral density is also observed not only in MM but also in patients with monoclonal gammopathy of undetermined significance (MGUS). The pathogenesis of bone disease in MM is complex. The activity of proteasome inhibitor bortezomib has been linked to increased bone formation and osteoblastic activation. Evidence from the available clinical data indicates that bortezomib has a positive impact on bone health in MM and demonstrates a bone anabolic effect.

Methods:

We analyzed retrospectively 53 patients with MM and 16 with MGUS who have completed bone density at least at diagnosis. 21 patients have completed two bone density (3 MGUS and 18 MM). The bone density was obtained in all patients at baseline and in 16 patients repeated after bortezomib treatement with a median time of bortezomib exposure of 6 months. We analyzed T-score values at lumbar spine and at femoral neck.

Results:

With a median age of 66 years, 41 male and 28 female were analyzed. At baseline the mean lumbar spine T-score of all subjects and of 16 MM treated with bortezomib was -0.50 and -0.76 respectively. At baseline the mean femoral neck T-score for all subjects and for 16 MM treated with Bortezomib was -1.56 and -1.31 respectively. The baseline mean lumbar spine T-score for MGUS and MM was -0.71 and -0.43 respectively. The baseline mean femoral neck T-score of MGUS and MM was -1.61 and -1.54 respectively. In the group of 16 patients treated with Bortezomib we observed from baseline a change in lumbar bone mineral density T-score of 0.36 and at femoral neck bone density T-score of 0.25.

Conclusion:

These data show that patients treated with proteasome inhibitor showed moderate increment in bone mineral density at lumbar spine and at femoral neck.

Disclosures:

No relevant conflicts of interest to declare.
IDH1 and IDH2 mutations are Not Frequent In Multiple Myeloma
Authors: Langer Christian , Ibañez Mariam , Liebisch Peter , Zenz Thorsten , Knop Stefan , Einsele Hermann , Paschka Peter , Doehner Hartmut , Doehner Konstanze
Published: October 2019

Abstract 4992

Multiple myeloma (MM) is characterized by frequent and complex genomic abnormalities. However, most genetic abnormalities are already present in the precursor state of MM, the monoclonal gammopathy of undetermined significance (MGUS). Therefore, it is likely that secondary genetic events might contribute to the development from MGUS to symptomatic MM. Mutations in isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) genes have recently been described as relatively frequent molecular lesions in gliomas and in acute myeloid leukemia (AML). However no larger study has so far examined the frequency of IDH1 and IDH2 gene mutations in patients (pts) with MM.

Methods:

In 188 pts with MM genomic DNA from CD138 sorted plasma cells was used for analyses. Exon 4 of both IDH1 and IDH2 were amplified by PCR and the amplicons were analyzed using a combination of denaturing high-performance liquid chromatography and DNA sequencing. All patients were also characterized by a comprehensive set of FISH probes for the presence of recurring cytogenetic abnormalities.

Results:

185 out of 188 samples were evaluable for analyses. One missense mutation in the IDH2 gene (c.G419A) was identified in the cohort of 185 MM pts (0.5%). This mutation was described as the most frequent IDH2 mutation in AML and is predicted to cause an amino acid change from arginine to glutamin at position 140 (p.R140Q). On cytogenetic analysis this patient harbored a translocation t(11;14) resulting in aberrant expression of CCND1. Additionally, in 15 pts (8%) the recently described single nucleotide polymorphism (SNP) in the IDH1 gene (rs11554137) was detected that has been reported as an adverse prognostic factor in cytogenetically normal AML.

Mutations in the IDH1/2 genes are a rare event in MM (0.5%). Further studies are warranted to address the issue if IDH1/2 mutations are restricted to distinct genetic subgroups as for example the group of MM pts with translocation t(11;14).

Disclosures:

No relevant conflicts of interest to declare.
Multi-Parameter Flow Cytometry(FC) Can Detect Proliferating Myeloma Cells After Treatment: Implications for Targeting the Myeloma Proliferative Component
Authors: Lust John , Kumar Shaji , Timm Michael , Donovan Kathleen , Greipp Philip R. , Morice William
Published: October 2019

Abstract 4995

Background:

Multiple myeloma results from an accumulation of monoclonal nonproliferating plasma cells arising from a small subpopulation of proliferating myeloma cells. In an effort to optimize detection of the human myeloma cell growth fraction and obviate the need for slide review, a novel FC strategy was developed combining elements of light chain restriction, surface antigen expression, and ploidy analysis.

Methods:

Bone marrow cells from 41 patients with plasma cell proliferative disorders were lysed with ACK and resuspended in 3% BSA. Cells were stained using a 6-color assay with anti-CD45, anti-CD38, anti-CD138, and anti-CD19. Cells were washed and 100 ul Caltag solution A was added for 15 min. Cells were washed and 100 ul of Caltag solution B, anti-kappa and anti-lambda were added for 10 min. Cells were washed and 100 ul PBS and 3 ul RNAse are added for 15 min. Cells were washed and 400 ul of a 1:1000 solution of 3uM DAPI in Tris 0.1% NP-40 was added. Cells were incubated at 4°C for 45 minutes before running on a FACSCanto instrument for ploidy determination. All patients were analyzed both by flow cytometry and the slide based plasma cell labeling index. Results: Forty-one patients were studied; 34 demonstrated a proliferative fraction and 7 had too few plasma cells for analysis after therapy. Of the 34 patients, 6 had MGUS/SMM, 5 newly diagnosed MM, 7 amyloid, and 16 were treated MM. The mean percent proliferating cells were 1.1% (range 0 – 8.6%) with PCLI and 1.4% (range 0.1 – 12.7%) by flow. The correlation between PCLI and flow gave a RSquare value of 0.54. Twelve patients with a PCLI of 0% had a flow proliferation between 0.1 – 1.4% (mean 0.49%). Treated patients received lenalidomide, dexamethasone, bortezomib, and/or autologous transplantation. All 16 treated myeloma patients with adequate plasma cells had a flow proliferation between 0.2 – 12.7% (mean 2.2%). Conclusion: Flow cytometry offers a useful way to detect the proliferative myeloma component at diagnosis and after treatment. The continued presence of proliferating myeloma cells after treatment may explain why most patients relapse and offers another important marker to monitor and cell population to target in patients with active disease.

Disclosures:

Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.
Multi-Organ Involvement of Multiple Myeloma Cells In the Cases of Recent 10 Years: A Clinicopathologic Study of 81 Consecutively Autopsied Cases
Authors: Hagiwara Shotaro , Mochizuki Makoto , Endo Hisako , Hirai Risen , Tanimura Akira , Takeshita Masataka , Murakami Hirokazu , Miwa Akiyoshi
Published: October 2019

Abstract 5004

Background:

Despite recent progress in treatment, multiple myeloma is still uncurable disease. The impact of modern therapy on the causes of death and pathological feature of end-stage myeloma is not fully understood.

Methods:

We studied autopsied cases with multiple myeloma between 1979 and 2009 at National Medical Center of Global Health and Medicine, Tokyo, Japan. We compared the clinicopathological feature of the autopsied cases in recent 10 years with the cases before 2000. Statistical analysis was performed using student's t-test and chi-square test.

Results:

There were 81 autopsied cases between 1979 and 2009. 31 cases were autopsied in recent 10 years and the older 50 cases were before 2000. Mean age at death was 59.2 and 65.1 years old, and the mean duration of illness was 46.1 and 31.9 months, respectively. Stem cell transplantation was performed in 13 (12 autologous, 1 allogeneic) of recent cases and 3 (2 autologous and 1 allogeneic) of older cases. In recent cases, five patients were treated with bortezomib, 2 were with thalidomide and 2 were with both. Extramedullary infiltration of myeloma cells were observed in both groups. The frequent sites of involvement were spleen, liver, kidney, lymph nodes, lung, pancreas, adrenal gland and perioneum. The infiltration in liver and lung was significantly frequent in the recent cases than in the older cases (58.1% vs. 28.0%, p=0.007, and 38.7% vs. 18%, p=0.039). Infection as a cause of death was noted more frequently in the recent cases than in the older cases (41.9% vs. 18.0%, p=0.019). Amyloid deposition was detected in 16.1% and 22.0% (ns.), and myeloma kidney was noted in 48.4% and 60% (ns.).

Conclusion:

High dose chemotherapy with stem cell support and novel agents have been contributed to improving the survival. However, the increase in resistant bacterial and fungal infection is serious problem. Also, extramedullary relapse after autologous and allogeneic stem cell transplantation is not rare, and extramedullary progression under thalidomide has been reported. In our recent autopsied cases, the incidence of fatal infection and extramedullary involvement of myeloma cells was significantly higher than in the older cases.

Disclosures:

No relevant conflicts of interest to declare.
The Autophagy Gene ULK1 Plays a Role In AML Differentiation and Is Negatively Regulated by the Oncogenic MicroRNA 106a
Authors: Tschan Mario P. , Jost Melanie , Batliner Jasmin , Fey Martin F.
Published: October 2019

Abstract 503

Autophagy is a degradative mechanism mainly involved in the recycling and turnover of cytoplasmic constituents allowing for example survival upon a variety of stress stimuli. Although often referred to as type II programmed cell death, autophagy is primarily a pro-survival mechanism rather than an important cell death effector. During autophagy, part of the cytoplasm or entire organelles are sequestered into double-membraned vesicles, called autophagosomes, that ultimately fuse with lysosomes and degrade their contents. Regarding hematopoiesis there is accumulating evidence that autophagic mechanisms are operative in the development of different hematopoietic lineages. To identify ATG genes with a function in neutrophil differentiation, we employed an shRNA library targeting autophagy regulators in acute myeloid leukemia (AML) cells as a model for neutrophil differentiation. Among others, we found that inhibiting the key autophagy kinase ULK1 (also known as ATG1) using RNAi significantly attenuated all-trans retinoic acid (ATRA)-induced neutrophil differentiation of NB4 and HL60 AML cells. In agreement, we observed significantly elevated ULK1 mRNA and protein levels during neutrophil differentiation of these cells. Moreover, ULK1 expression was significantly reduced in primary AML patient samples as compared to mature myeloid cells from healthy donors. Given the aberrant microRNAs (miRNAs) expression in myeloid malignancies, we wondered if altered miRNA expression in AML might contribute to the low ULK1 levels seen. MiRNAs are 19–25 nucleotide, non-protein coding RNAs that regulate gene expression by directly binding to partially complementary sequences in the 3′-untranslated regions (3′-UTR) of their target mRNAs. Several target prediction software's identified a putative miR-106a binding site in the ULK1 3′-UTR. Interestingly, miR-106a is part of a highly oncogenic miRNA family. We then confirmed direct inhibition of ULK1 by miR-106a using wild-type and miR-106a mutated 3′-UTR ULK1 reporters. Moreover, a decrease of miR-106a expression was seen during ATRA-induced differentiation of AML cells paralleled by increased ULK1 mRNA levels. Lastly, stable overexpression of miR-106a in NB4 cells resulted in decreased ULK1 levels and markedly impaired neutrophil differentiation upon ATRA treatment. In summary, we show for the first time a significant involvement of the autophagy gene ULK1 in neutrophil differentiation and an association of low ULK1 levels with primary AML. Our studies indicate that ULK1 is a putative tumor suppressor in AML that is targeted by the oncogenic miR-106a.

Disclosures:

No relevant conflicts of interest to declare.
A Multicenter Randomized Phase II Trial of Mapatumumab, a TRAIL-R1 Agonist Monoclonal Antibody, In Combination with Bortezomib In Patients with Relapsed/Refractory Multiple Myeloma (MM)
Authors: Belch Andrew , Sharma Atul , Spencer Andrew , Tarantolo Stefano , Bahlis Nizar J , Doval Dinesh , Gallant Gilles , Kumm Elizabeth , Klein Jerry , Chanan-Khan Asher A.
Published: October 2019

Abstract 5031

Mapatumumab, a fully human monoclonal antibody, targets and activates the TRAIL-R1 receptor. We conducted this randomized, controlled phase II trial to evaluate the efficacy and safety of mapatumumab in combination with bortezomib in subjects with relapsed/refractory MM. Response was evaluated using the Bladé criteria. Patients were required to have a measurable serum and/or urine M-protein and have failed 1 or 2 prior therapies for MM. Patients were excluded if they had received prior bortezomib. Patients were randomly assigned to Arm A, 1.3 mg/m2 bortezomib (days 1, 4, 8, 11) every 21 days; Arm B10, bortezomib + 10 mg/kg mapatumumab (day 1) every 21 days; or Arm B20, bortezomib + 20 mg/kg mapatumumab (day 1) every 21 days. Cycles were repeated every 21 days for a maximum of 17 cycles (1 year), progressive disease or unacceptable toxicity. Subjects with complete response (CR) were treated for an additional 2 cycles after documentation of CR (not to exceed 17) and then followed. A total of 104 subjects from 4 countries were randomly assigned to the treatment arms. The addition of mapatumumab to bortezomib did not increase the response rate, progression-free survival (PFS) or duration of response compared to bortezomib alone. Adverse events were generally balanced across treatment groups; there was no evidence that mapatumumab exacerbated toxicities associated with bortezomib. As expected, the most common toxicities were hematological and peripheral neuropathies. The results do not support further evaluation of mapatumumab in combination with bortezomib in patients with advanced MM. Additional trials of mapatumumab in other indications are on-going.

Arm A (n= 35) Arm B10 (n=33) Arm B20 (n=36) CR 0 0 2 PR 18 10 17 CR + PR n (%) 18 (51.4) 10 (30.3) p=0.08a 19 (52.8) p=0.91b Median PFS mo (95% CI) 8.7 (7.6, 10.0) 4.7 (2.5, 7.4) p=0.29a 5.7 (5.2, 8.9) p=0.21b Median Duration of Response mo (range) 8.5 (6.9, 14.2) 9.3 (3.9, 16.1) p=0.92a 7.6 (4.3, 8.8) p=0.41b a P value for comparison of Arm A with Arm B10 b P value for comparison of Arm A with Arm B20

Disclosures:

Gallant: Human Genome Sciences, Inc.: Employment, Equity Ownership. Kumm:Human Genome Sciences, Inc.: Employment, Equity Ownership. Klein:Human Genome Sciences, Inc.: Employment, Equity Ownership.
T Regulator Cells (Treg) In Patients with Myelofibrosis
Authors: Shakil Shams A , Chen Chi , Surahio Ali , Mirza Mariam B , Su Marisa Y , Sun Yiwu , Wang Jen-Chin
Published: October 2019

Abstract 5051

Primary myelofibrosis (PMF) and myelofibrosis post essential thrombocytosis (MF-ET) or myelofibrosis post polycythemia vera (MF-PV) have been reported to be associated with autoimmune phenomenon, such as Coomb positive anemia, lupus anticoagulant, positive ANA and the presence of circulating immune complex etc. Regulatory T cells (Treg) also have known to play important roles in modulating immune responses. Therefore we studied Treg cells in 25 patients with MF including PMF (12), MF-ET (8), MF-PV (5) and compared with other MPD including ET (7), PV (8), and normal volunteer controls (17). Mononuclear cells (MNC) were separated from peripheral blood. 106 MNCs were stained for flow cytometry analysis using Treg Detection Kit from Miltenyi Biotec Inc (Auburn, CA). The numbers of Treg was calculated as the percentage of positive CD4+ CD25+ FoxP3+ T cells (Treg) from the numbers of gated CD4+ cells. Treg function was evaluated by XTT cell proliferation assay (Invitrogen) with ratios of Treg vs. T-effector cells (CD4+ CD25-) at 1:1, 1:2, 1:4, and 0:1 in the presence of anti-CD3 CD28 microbeads (Invitrogen). The results (mean ± SE) showed numbers of Treg cells in MF were 0.73 + 0.08, other MPD were 1.37 ± 0.22, and normal controls were 0.96 ± 0.27 (p=NS). Treg function was evaluated in 12 patients with MF and 6 normal volunteer controls. Four MF patients were found to have significant lower values than controls. We concluded that in MF, quantitatively Treg were not different from other MPD or normal controls but Treg dysfunction was observed in 30–40 % of MF patients. This could explain why some patients with MF are prone to develop autoimmune phenomenon. Further studies with more patients are in progress.

Disclosures:

No relevant conflicts of interest to declare.
LY2784544, a Novel JAK2 Inhibitor, Decreases In Vitro Growth of Hematopoietic Human Progenitors From JAK2 V617F Positive Polycythemia Vera Patients
Authors: Florensa Lourdes , Bellosillo Beatriz , Arenillas Leonor , Ma Liandong , Walgren Richard , Alvarez Alberto , Gonzalez Judit , Vila Rosa M , Longarón Raquel , Besses Carles
Published: October 2019

Abstract 5054

Introduction:

The discovery of JAK2 V617F mutation in patients with myeloproliferative disorders (MPD) has opened new perspectives for the development of targeted therapies. We have studied the efficacy of a novel molecule LY2784544 with JAK2 inhibitory activity in the in vitro growth of myeloid progenitors from JAK2 V617F-positive polycythemia vera (PV) patients.

Objectives:

To investigate the efficacy of LY2784544 in the inhibition of endogenous(e)BFU-E and CFU-GM growth in PV patients.

Methods:

In vitro cultures in semisolid media were performed from peripheral blood mononuclear cells (PBMC) of 6 PV patients who had never received cytoreductive treatment (4 patients with homozygous JAK2 V617F and 2 patients with heterozygous JAK2 V617F). PBMC were suspended in methylcellulose (Methocult. StemCell, Vancouver, Canada) without the addition of EPO and containing 0–30.0 μM LY2784544 drug. Concurrent plates containing EPO were plated as control cultures. The medium was distributed in multidishes and they were incubated at 37° with 5% CO2 and 95% humidity. Hemoglobinized colonies and granulomonocytic colonies were counted on day 14 by standard criteria (BFU-E defined by an aggregate of >50 hemoglobinized cells or three or more erythroid subcolonies and CFU-GM was defined by an aggregate of >50 cells). Each in vitro assay was performed in duplicate. DNA was obtained from peripheral blood granulocytes from each patient to quantify the JAK2 V617F allele burden at the time of culture assay.

Results:

LY2784544, at concentrations ranging from 0.03–30.0 μM, inhibited growth of unselected peripheral blood eBFU-E and CFU-GM from PV patients carrying the JAK2 V617F mutation in a dose-dependent manner, although without achieving complete inhibition of all colonies (fig.1).

Conclusions:

In vitro studies show that LY2784544 decreases the eBFU-E and CFU-GM growth in therapy-naive JAK2 V617F positive PV patients. Our data suggest that LY2784544 may be a candidate for the treatment of MPD carrying the JAK2 V617F mutation.

Disclosures:

Ma: Eli Lilly and Company: Employment. Walgren:Eli Lilly and Company: Employment.
Chronic Myeloprolipherative Neoplasms (MPNs) and Splanchnic Venous Thrombosis(SVT): Incidence and Risk Factors
Authors: Martino Bruno , Alati Caterina , Cufari Patrizia , Vincelli Iolanda , Ronco Francesca , Oriana Vincenzo , Nobile Francesco
Published: October 2019

Abstract 5057

There is evidence in literature of the clinical and pathogenetic role of JAK2 V617F mutation in MPN, while its contribute as an additional thrombotic risk factor in MPN patients is still under discussion. The jak2 mutation has been identified as occult marker in several patients with splanchnic venous thrombosis (SVT); morover, 45% of patients with Budd-Chiari syndrome (BCS) and 34% of patients with portal vein thrombosis (PVT) have associated MPN. On the other hand, the majority of BCS patients without MPN present additional congenital or acquired thrombosis risk factors.

The aim of the present study is to evaluate the incidence of SVT in MPN patients. We also evaluated the presence of other prothrombotic risk factors in MPN patients, in addition to the JAK-2 mutation.

Out of the 460 Ph1 negative MPN patients observed in our center from January 2000 to January 2010 and retrospectively evaluated, 9 patients (7 females and 2 males; 2%) presented SVT.

Six cases had Essential Thrombocythemia (ET), 2 Primary Myelofibrosis (PMF), and 1 Polycitemia Vera (PV). Five of the 6 cases with ET were females. Among the entire population of ET, SVT incidence was 3%. All the 9 patients diagnosed of SVT were JAK2 V617F positive and they were treated with antiaggregant and anticoagulation therapy; 6 received hydrossiurea.

Seven patients had SVT before MPN diagnosis, 2 of them had splenectomy at diagnosis for surgical decision. In these patients developing SVT before MPN diagnosis no other major thrombotic event occurred during follow-up. The remaining 2 MPNs patients presented asymptomatic SVT diagnosed by imaging techniques routinely performed during MPN follow up.

Interestingly, 75% of MPNs patients with SVT demonstrated at least one prothromobtic risk factor, such as factor V Leiden, Protein C deficiency, hyperhomocystinemia and 50% had 2 or more associated defects. MPNs patients without SVT (396) had a lower prevalence of prothrombotic risk factors and developed venous thrombosis in different anatomical sites: in these cases white blood cell count, platelets values and the presence of JAK2 V617F mutation correlate with the development of the thrombotic event.

Conclusion.

Even though SVT has a low incidence in MPNs patients, according to the results of the present retrospective study we suggest the potential benefit of searching for additional prothrombotic risk factors in the whole MPN population in order to prevent and/or properly treat this complication.

Disclosures:

No relevant conflicts of interest to declare.
A Retrospective Epidemiological Analysis of 1572 Cases of Ph1- Myeloprolypherative Neoplasms (MPNs) From 9 Centers In the Latium: Preliminary Results
Authors: Montanaro Marco , Latagliata Roberto , Montefusco Enrico , Cedrone Michele , Villivà Nicoletta , Cotroneo Ettore , Rago Angela , Spirito Francesca , De Muro Marianna , Breccia Massimo , Anaclerico Barbara , Porrini Raffaele , Pessina Gloria , Felici Stefano , Trapè Giulio , Tafuri Agostino , Mazzucconi Maria Gabriella , Cimino Giuseppe , Majolino Ignazio , Avvisati Giuseppe , Lo Coco Francesco , Andriani Alessandro
Published: October 2019

Abstract 5065

The Latium is a region of central Italy that counts approximatively 5.600.000 of residents. We reported herein a preliminary analysis of 1572 patients affected by MPN diagnosed in the last 20 years and treated in 9 of the Centers belonging to our group. Our centers are mainly located in Rome (7 centers) and neighboring districts (2 centers, Latina and Viterbo). The diagnosis was performed according to PVSG criteria until 2001 and then according to the WHO criteria: the majority of the patients underwent bone marrow biopsy. 218 patients were affected by primary myelofibrosis (PMF), 779 by Essential thrombocytemia (ET) and 575 by Policytemia Vera (PV). Epidemiological and clinical findings of all patients at diagnosis as well as thrombotic complications and evolution are reported in the table:ET (779 pts)PV (575 pts)PMF (218 pts)M/F288/491350/225134/84Age (yrs)59 (r: 20-93)60 (r: 19-91)67 (r: 30-86)WBC (x109/L)9192 (+/− 3483 SD)10423 (+/− SD 4307)13313 (+/−5220)Hb (g/dL)13, 9 (+/− 1,8)18,1 (+/− 2,3)11,7 (+/− 2,8)Plts (x 109/L)877 (+/− 353)445 (+/−247)457 (+/− 358)Splenomegaly (%)193579Bone marrow biopsy (performed/total))557/779 (71,5%)254/575 (44,2%)207/218 (95%)JAK-2 V617F mut (%)284/484 (59%)256/369 (69,4%)69/116 (59,5 %)Jak-2 allele burdenPerformed in 188/284 cases 26,49% (SD +/− 24)Performed in 199/256 cases: 59,4 % (SD +/−30,9)Performed in 51/69 cases 57,5% (SD +/− 29,4)Median Follow-up (years)7,157,863,89Thrombosis pre-diagnosis of MPNN. of patients (%)108/779 (13,8%)100/575 (17,4%)29/218 (13,3%)(arterial+venous)74+3476+2424+5Thrombosis post diagnosis of MPNN. of patients (%)71/779 (9,1%)66/575 (11,5%)20/218(9,17%)(arterial+venous)42+2942+2412+8EvolutionMyelofibrosis9/779 (1,1%)24/575 (4,1%)AML9/779 (1,1%)13/575 (2,3%)17/143 (11,8%)

Comments:

In our opinion, this casistic of patients with different types of MPNs reflects clinical presentation and evolution of three variants of the same disease. Many clinical findings in our unselected cohort of patients are similar to those reported in literature; in particular, thrombotic events were seen in about 13 – 17% of patients, without any correlation with Jak-2 status in both pre-diagnosis and follow-up. However, 2 main differences were noted: a lower incidence of JAK-2 V617F mutation among our PV patients and a lower rate of evolution in myelofibrosis and AML among our ET and PV patients. Both these features warrant further insights to be fully elucidated.

Disclosures:

No relevant conflicts of interest to declare.
Nuclear Area Predicts Outcome In Peripheral T-Cell Lymphoma, Not Otherwise Specified: Morphometric Image and Immunohistochemical Analyses of 78 Patients
Authors: Niino Daisuke , Kimura Yoshizo , Kiyasu Junichi , Takeuchi Masanori , Yoshino Tadashi , Nakamura Shigeo , Ohshima Koichi
Published: October 2019

Abstract 5081

Background:

Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is an uncommon tumor with unfavorable prognosis. The purpose of this retrospective study was to investigate the cytological features and treatment outcomes in patients with PTCL-NOS and to obtain new information on the therapeutic implications of PTCL-NOS.

Method:

A total of 6 institutions in Japan and Asia submitted clinical and pathologic information on PTCL-NOS diagnosed and treated at their respective centers. Among these cases, 55 (71%) were from Japan, 8 (10%) were from Hong Kong, 8 (10%) were from Thailand, and 7 (9%) were from Korea. From 1992 to 2006, 78 cases of PTCL-NOS were examined for cytological features, focusing on morphometric image analysis. The average nuclear area of lymphoma cells, as well as the median, the minimum, the maximum and the standard deviation, was estimated in 50 cells for each case. In addition, we performed an immunohistochemical study of CCR4 expression in all cases. Univariate analyses were performed using Kaplan-Meier survival estimates, and data were compared using the log-rank test. Cox proportional-hazard regression test was used for multivariate analysis. P-values of less than 0.05 were considered significant.

Results:

Clinical information was available for review in 78 cases. There was a 1.52:1 male/female ratio, and median age was 62 years (range, 3 to 87 years). Sixty-two percent of patients had advanced clinical stages. Lactic dehydrogenase (LDH) was elevated in 53% of cases, and most patients were treated with combination chemotherapy. Median survival was 693 days. On the morphometric image analysis, median nuclear area was 41.7 μ m2 (range, 22 to 119 μ m2). Thirty-three of the cases (42%) showed positive CCR4 immunostaining. Statistical analysis confirmed that nuclear area of lymphoma cells in PTCL-NOS is correlated with overall survival, whereas immunohistochemical expression of CCR4 is not correlated with overall survival or nuclear area of lymphoma cells. Multivariate analysis confirmed that the nuclear area of lymphoma cells in PTCL-NOS is an independent prognostic factor.

Conclusion:

These results indicate that nuclear area of lymphoma cells in PTCL-NOS is an independent prognostic factor that may predict overall survival. Because PTCL-NOS is a heterogeneous disease with regard to histological type and pathological state, nuclear area of lymphoma cells could be used to stratify patients with PTCL-NOS for therapies, although we are continuing to accumulate data.

Disclosures:

No relevant conflicts of interest to declare.
Absence of Mutations of the Histone Methyltransferase Gene EZH2 In Splenic B-Cell Marginal Zone Lymphoma
Authors: Martínez-Avilés Luz , Salido Marta , Bellosillo Beatriz , Adema Vera , Ferrer Ana , Salar Antonio , Garcia Mar , Espinet Blanca , Florensa Lourdes , Besses Carles , Serrano Sergi , Sole Francesc
Published: October 2019

Abstract 5086

Background

Splenic marginal zone lymphoma (SMZL) is a rare low-grade B-cell lymphoproliferative disorder with characteristic clinical, cytological, histological and immunophenotypical features. The most common cytogenetic abnormality, present in 30–40% of the patients is the 7q deletion, that extends from 7q21 to 7q36. This aberration may represent a primary pathogenic event in SMZL. Recently, mutations in the EZH2 gene, located at 7q36.1, have been described in different hematological malignancies including B-cell lymphomas. However, the role of the EZH2 gene in SMZL has to be elucidated.

Aim

To determine the prevalence of EZH2 mutations in a cohort of SMZL patients.

Patients and Methods

Twenty-nine patients with SMZL were screened for mutations in the EZH2 gene. From the whole cohort, 11 patients presented 7q deletion (three of them as a single anomaly), 11 had a normal karyotype and 7 had other cytogenetic aberrations. The mutational analysis of the EZH2 gene was performed by direct sequencing using primers covering the whole exome of the gene. DNA was extracted from CD19 isolated B-cells from peripheral blood or from total lymphocytes if the percentage of pathologic B-cell was higher than 50%.

Results

From the whole cohort of 29 SMZL patients, no pathogenic mutations (frameshift or nonsense mutations) were detected in the EZH2 gene in any of the patients analyzed. Five patients harboured the missense mutation D185H in exon 6, that has been previously described as a single nucleotide polymorphism (SNP).

Conclusions

In conclusion, the EZH2 gene is not mutated in our series of SMZL patients suggesting that this gene is not involved in the pathogeny of this entity.

Acknowledgments:

Fellowship FI2008 (AGAUR) to LMA, This work was supported (in part) by grants from Instituto de Salud Carlos III FEDER; Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER): RD06/0020/0031 and RD07/0020/2004; Ministerio de Sanidad y Consumo (Spain): PI07/0586.

Disclosures:

No relevant conflicts of interest to declare.
Activation of Raf-1 through the Depletion of RKIP by Bcr-Abl Promotes CML Cell Proliferation
Authors: Nakamura Satoki , Takemura Tomonari , Nagata Yasuyuki , Yokota Daisuke , Hirano Isao , Ono Takaaki , Shigeno Kazuyuki , Fujisawa Shinya , Ohnishi Kazunori
Published: October 2019

Abstract 5105

[Background and Aims]

RKIP (Raf kinase inhibitor protein) regulates cell proliferation, apoptosis, and cell cycle through the inhibition of Raf-1, and it has been reported that the suppression of the RKIP expression promotes the proliferation of tumor cells. However, its function is little known in CML. We found that the expression of RKIP gene and protein was suppressed in CML cells. In this study, we have investigated the expression and the function of the RKIP in CML cell proliferation. [Methods] The cells used in this study were human CML cell lines, K562, Meg01, and SHG3 cells. Primary CML cells (ALDHhicells) were obtained from the bone marrow of CML (CP) patients (n=12). Human normal ALDHhi cells were isolated from bone marrow of healthy volunteers after obtaining informed consents. For analysis of RKIP mRNA expression, quantitative RT-PCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For proliferation analysis and the levels of Erk1/2 phosphorylation in CML cells, MTT assays, western blot and cell cycle analysis were performed in all cell lines transfected with Bcr-Abl, RKIP siRNA, or RKIP cDNA respectively. For colony analysis, the colonies of CFU-GEMM, CFU-GM, and BFU-E were counted in CML stem/progenitor cells transfected with RKIP siRNA or treated with Abl kinase inhibitors. [Results] In CML cell lines treated with Abl kinase inhibitors or transfection with Bcr-Abl siRNA, the expressions of RKIP mRNA and protein were significantly increased more than untreated cells. Moreover, in CML cells transfected with the RKIP siRNA, the cell proliferation inhibition by treatment of the Abl kinase inhibitors was weakened compared to RKIP siRNA-untransfected cells, and the phosphorylation levels of Erk1/2 and Raf-1 were markedly increased compared to theRKIP siRNA-untransfected cells. On the other hands, the overexpression of RKIP induced G1 cell cycle arrest through p27 and p21 accumulation, decreased the phosphorylation levels of ERK1/2 and Raf-1, and inhibited the proliferation in CML cells. In CML stem/progenitor cells obtained from patients with CML, the expression of RKIP mRNA was suppressed in 10/10 (100 %) of CML. The transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of RKIP mRNA and protein, and the overexpression of RKIP decreased the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusion] Our results demonstrated that the Bcr-Abl suppressed the expression of RKIP, the depletion of RKIP induced Raf-1 activation, and induced the proliferation of CML cells through ERK1/2 phosphorylation. Moreover, in CML, the induction of RKIP expression inhibited the proliferation of CML stem/progenitor cells.

Disclosures:

No relevant conflicts of interest to declare.
CDP7657, and Other Anti-CD40L Antibody Formats Lacking a Functional Fc Region, Do Not Cause Platelet Activation In Vitro
Authors: Amirkhosravi Ali , Meyer Todd , Harari Olivier , Burkly Linda , Su Lihe , Hsu Yen-Ming , Bourne Timothy , Wakefield Ian , Peters Christopher , Shock Anthony , Francis John
Published: October 2019

Abstract 5128

Objectives:

Hu5c8, a whole IgG1 anti-CD40L antibody, is known to cause thrombotic side effects in patients with Systemic Lupus Erythematosus (SLE). Anti-CD40L-mediated activation of platelets in vitro was shown to be FcγRIIa-dependent1, suggesting that the adverse effects of hu5c8 could be due to this mechanism, and that anti-CD40L antibodies lacking a functional Fc region would not activate platelets. CDP7657 is a monovalent Fab' PEG anti-CD40L antibody being developed for the treatment of SLE. The aim of the present work was to study a range of anti-CD40L antibody formats in isolated platelet assays and to evaluate the role of the Fc region and of valency.

Methods:

Isolated human platelets from healthy donors were used in the platelet aggregometry and serotonin release assay systems. Antibodies were added either alone, or pre-complexed with soluble CD40L trimer (sCD40L) at optimal stoichiometry. Antibody IV.3 was used to block FcγRIIa. The antibodies and antibody fragments tested included hu5c8 in IgG1, aglycosyl IgG, Fab′, & (Fab′)2 formats, CDP7657, and the CDP7657 variable regions in di-Fab' PEG, tri-Fab' PEG and aglycosyl formats.

Results:

Hu5c8 in complex with sCD40L caused strong aggregation (in 2–3 minutes) and >85% serotonin release, both alone and in complex with sCD40L. This activation was completely abrogated by blocking FcγRIIa. In contrast, platelet activation did not occur with CDP7657 or other antibody formats, in any assay, regardless of the valency or presence of sCD40L.

Conclusions:

The data support the hypothesis that platelet activation by anti-CD40L antibodies is dependent on the presence of an active Fc moiety, via interactions with FcγRIIa, and indicate that CDP7657 has the potential to deliver the therapeutic effect of anti-CD40L blockade in SLE without thrombotic toxicity.

References:(1) Langer F et al. 2005; Thromb Haemost 93: 1137.

Disclosures:

Amirkhosravi: UCB: Research Funding.
Inadequately Regulated Adrenal Steroids through HPA Axis In Chronic Idiopathic Thrombocytopenic Purpura
Authors: Oh Doyeun , Ha Young Wan , Chong So Young , Bang Soo-Mee , Choi Man Ho , Chung Bong Chul
Published: October 2019

Abstract 5130

Idiopathic thrombocytopenic purpura (ITP) is an immune disorder along with the condition of having a low platelet count of multiple etiologies. Endogenous steroids such as corticosteroids have been known to have potent immunomodulatory effect, and also altered expression of steroid metabolism have been reported on the immune related diseases. The level of endogenous steroids are closely related with the level of cytokines (IL-6, IL-1, TNF-α) through HPA axis. Therefore, the eluciadaton of the relationship between steroid hormones and immune cytokines through hypothalamic-pituitary-adrenal (HPA) axis may help to identify biochemical roles of steroid hormones in ITP. The metabolites of steroid hormones were assayed in the plasma obtained from 54 ITP patients and 44 controls using the validated GC-MS based signatures of 70 endogenous steroids including 22 androgens, 18 estrogens, 15 corticoids, 13 progestin, 2 sterols. We also measured the plasma concentration of three HPA axis-stimulating cytokines (IL-6, IL-1, TNF-α) and adrenocorticotropic hormone (ACTH) in chronic ITP patients and healthy subjects using Luminex xMAP technology. Patients were classified to active and remission state depending on the platelet count (active, less than 50,000/mm3; remission, over 100,000/mm3). The active ITP patients showed significant lower plasma levels of adrenal steroids such as pregnenolone (1.49 ± 0.71 ng/mL vs 3.07 ± 1.30 ng/mL, p = 2.3E–08), dehydroepiandrosterone (2.98 ± 1.57 ng/mL vs 4.82 ± 1.67 ng/mL, p = 7.1E-05) and cortisol (73.8 ± 11.3 ng/mL vs 112.7 ± 30.1 ng/mL, p = 2.6E–10) compared with healthy subjects, while the levels of adrenal steroids in remission ITP patients (pregnenolone, 1.87 ± 0.76 ng/mL; dehydroepiandrosterone, 3.70 ± 1.95 ng/mL, cortisol, 83.2 ± 16.2 ng/mL) were not recovered to normal levels. The cortisol/ACTH (ng/mL vs pg/mL) ratio representing pituitary-adrenal axis was not significantly different between patients (4.63 ± 0.72) and healthy subjects (5.17 ± 0.67). However, there was a positive correlation between ACTH and IL-6 in healthy subjects (R = 0.672, p = 0.0024), while no correlation in ITP patients. In conclusion, the low levels of endogenous adrenal steroids resulted from dysregulated HPA axis may have a role in the pathogenesis of ITP.

Disclosures:

No relevant conflicts of interest to declare.
Effect of Genetic Polymorphisms Related to Folate and Homocysteine Metabolism In the Etiology of Recurrent Miscarriages
Authors: Guerra-Shinohara Elvira Maria , da Silva Giusti Kelma Cordeiro , Monteiro Nathalia Sierra , Lazaro Robson José , Seino Fernanda Midori , Bueno Carolina Tosin , Carvalho Mário Henrique Burlacchini , Filho Antônio Amorim , Chiattone Carlos , Zugaib Marcelo
Published: October 2019

Abstract 5136

Introduction:

Folic acid deficiency has been associated with obstetric complications such as preeclampsia, placental abruption and recurrent miscarriages (RM). Folic acid is a methyl group donating in various reactions. Low concentrations of 5,10-methylenetetrahydrofolate, the cofactor of enzyme thymidylate synthase, decrease the synthesis of thymidylate, which results in increased ratio deoxyuridylate monophosphate/deoxytimidylate monophosphate (dUMP/dTMP) and increased incorporation of deoxyuridylate triphosphate (dUTP) to DNA. The removal of dUTP by DNA-glycosylase can cause permanent damage to DNA, which may lead to apoptosis or increase the risk of developing cancer. Polymorphisms in genes of enzymes (MTHFR - methylene tetrahydrofolate reductase, MTR - methionine synthase and MTRR - methionine synthase reductase) and also in the gene of the reduced folate carrier (RFC1) were related to reduced folate and increased total homocysteine concentrations and have been associated as risk factors for RM.

Material and Methods:

171 women with a history of three or more recurrent miscarriages and 95 healthy women with no history of abortion and having two or more normal babies were included. Weight and height of women were obtained and the body mass index (BMI) was calculated. The presence of antibodies (ANA and anti-DNA) was evaluated using immunofluorescence kits. The genotypes of the polymorphisms MTHFR c. 677C>T, MTR c. 2756A>G and RFC1 c.80G>A were obtained by PCR-RFLP, while genotyping for polymorphisms MTRR c. 66A>G and MTHFR c. 1298A>C was made by real time PCR. Multivariate logistic regression model (forward conditional) was used to obtain the odds ratio and its 95% confidence intervals of having MR (dependent variable). The independent variables were: quartiles of BMI, age range, positive ANA (titer of 1/40), positive anti-DNA (titre 1/10), genotypes for the MTHFR c. 677C>T, MTHFR c.1298A>C, MTR c. 2756A>G, MTRR c. 66A>G and RFC1 c. 80G>A.

Results:

No differences between the groups were observed for serum total homocysteine or allele frequencies for MTHFR c. 677C>T, MTHFR c. 1298A>C, MTR c. 2756A>G and RFC1 c.80G>A polymorphisms. In a conditional logistic regression analysis the risk of RM was significantly associated with BMI OR [95% CI] = 1.40 [1.02, 1.93] per quartile increase in BMI), positive anti-DNA OR [95% CI] = 7.24 [0.92, 57.25], positive ANA OR [95% CI] = 2.48 [1.21, 5.08], and AA genotype for MTRR c. 66A>G polymorphism (OR [95% CI] = 2.19 [1.16, 4.12].

Conclusion:

The etiology of RM is multifactorial and it is associated with increasing of BMI, presence of autoantibodies and AA genotype for MTRR c. 66A>G polymorphism.

Disclosures:

No relevant conflicts of interest to declare.
Study On Immune Mechanism of Human Marrow Mesenchymal Stem Cells Adjusting Dendritic Cells for Treatment of Aplastic Anemia
Authors: Xiao Yang , Zhang Leqin
Published: October 2019

Abstract 5137

Objective

(1)To explore whether MSC has inhibiting effect on the proliferation ofAA patients' Tcell;(2)To discuss whether MSC affects T cell's proliferation via adjusting the growth of DCs.

Materials and Methods

(1) MSC were separated and cultured in vitro. Cell morphology was observed and the cell surface antigen was determined by flow cytometry.(2) Peripheral blood mononuclear cells were extracted from 20 patients suffered AA and then T lymphocytes were separated by nylon fiber column. Flow cytometry was applied to determine the surface antigen and subpopulation of T cell.(3)mononuclear cells were separated from normal human peripheral blood. DCs were prepared under the culture condition of recombination human granulocyte-macrophage colony stimulating factor (GM-CSF) and recombinant human interleukin-4 (IL-4). After acquiring the mature DCs induced by LPS, the phenotype analysis of DCs before and after culture was examined by flow cytometry, respectively.

Results

(1) After co-culture of MSC and T lymphocytes from AA peripheral blood, flow cytometry showed that the ratio of D8+ in T cells reduced significantly from 38.7% to 29.7 % (p < 0.05), whereas the CD4+ ratio increased from 24.9% to 34.9% significantly (p < 0.05). Meanwhile, ELISA analysis indicated that the concentration of IL-2 and IFN-γ were significantly decreased from 38.9 and 38.5 ng/L to 6.8 and 6.6 ng/L, respectively (p < 0.05). However, IL-4 and IL-10 increased from 2.8 and 2.9 to 5.3 and 8.3 ng/L, respectively (p < 0.05).(2) After the induction of immature DCs by LPS, flow cytometry showed that the expression of CD1a increased from 2.4% to 68.4% in the treatment without MSC, while that of CD14+ decreased from 83.6% to 3.5% (p < 0.05).(3) After the co-culture of mature DCs and MSC, the expression of CD14+ increased from 5.8% to 62.8% when the expression of CD1a, CD83 and CD80 decreased from 48.6%, 60.8% and 50.2% to 30.7%,40.9% and 20.3%, respectively.

Conclusions

Our study shows that (1)MSC inhibits the proliferation of T lymphocytes from AA p-atients by regulating CD8+ and CD4+; (2)futher study indicates that MSC can inhibit the growth of DCs and reverse the status of DCs from matureness to immatureness; (3)thus suggests the possible mechanism of MSC's inhibiting effect as follows: MSC decreases the liveness by controlling the growth of DCs and further inhibits its proliferation.

Disclosures:

No relevant conflicts of interest to declare.
Activation Markers of Thrombogenesis and Endothelial Dysfunction In End-Stage Renal Disease
Authors: Sharain Korosh , Davis Rachael , Hoppensteadt Debra , Cunanan Josephine , Litinas Evangelos , Bansal Vinod , Fareed Jawed
Published: October 2019

Abstract 5183

Introduction:

Over 500,000 patients currently have ESRD and it is estimated that another 200,000 will develop ESRD in the next 5 years. Hemostatic activation resulting in cardiovascular disease accounts for over 50% of the mortality seen in this patient population. Common sequelas seen in ESRD are DVT, PE, and ACS. Derangement of biochemical markers associated with these disease states have been observed in ESRD patients. In this study we profiled several markers of thrombogenesis and endothelial dysfunction to determine their levels in the ESRD population.

Materials/Methods:

117 ESRD patients on maintenance dialysis for at least 3 months over the age of 18 were included in this study. Additionally, 50 age matched control samples were obtained from healthy volunteers with no known kidney function. The markers measured include C-Reactive Protein (CRP), D-Dimer (DDMER), and Thrombomodulin (TM) which were detected by biochip array using the Randox Evidence Investigator (Antrim, United Kingdom). Additionally, Plasminogen Activator Inhibitor -1 (PAI-1) and Asymmetric Dimethylarginine (ADMA) were measured using commercially available kits by Diagnostica Stago (Paris, France) and DLD Diagnostika GmbH (Hamburg, Germany), respectively.

Result:

The mean, standard deviation, and p-value for each marker is tabulated below. Additionally, the fold increase of the ESRD samples compared to control samples were calculated and included in the table below. Compared to controls, all markers measured were increased in the ESRD samples, each of which demonstrated statistically significant p-values between the two groups. TM expressed a 5.9 fold increase in ESRD compared to controls, this was the highest fold increase amongst the markers tested. CRP, DDMER, PAI-1, and ADMA were increased at 4.4, 3.0, 1.4, and 1.6 times their control counterparts, respectively.

Conclusion:

The results in the table demonstrate that ESRD patients have a significant upregulation in markers of thrombogenesis and endothelial dysfunction. The upregulation of CRP, TM, D-DDMER, PAI-1, and ADMA is consistent with the increased thrombotic events detected in ESRD. The upregulation of the nitric oxide inhibitor ADMA points to disturbances in vascular lumen size regulation while CRP and TM suggest significant endothelial damage. Additionally, the upregulation of PAI-1 and DDimer indicates an activation of coagulation in ESRD. The combined disruption of endothelial regulation and endothelial damage along with the upregulation of thrombogenic markers helps to explain the significant prevalence of thrombosis and cardiovascular disease in ESRD. These markers should also be evaluated for their roles in kidney disease risk stratification and prognosis.

Disclosures:

No relevant conflicts of interest to declare.