bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the Somatic Mutation Rate in Patients with Classical Myeloproliferative Neoplasms (MPN) with and without Other Neoplasms
Authors: Berardi Margherita , Rondelli Tommaso , Susini Maria Chiara , Pellecchia Annamaria , Antonioli Elisabetta , Boni Luca , Luzzatto Lucio , Vannucchi Alessandro M. , Notaro Rosario
Published: October 2019

Abstract 5055

Classical chronic myeloproliferative neoplasms (MPNs) are amongst the best-characterized neoplasms associated with one of a set of specific somatic mutations, the most common of which is in the JAK2 gene. MPN is a relatively rare condition, and it could be surmised that it develops preferentially in people who have an increased tendency to somatic mutations, i. e. an increased somatic mutation rate (μ). Since measuring μ is rather labor-intensive, in this study we have measured instead the frequency of peripheral blood granulocytes that have inactivating mutations in a reporter gene, namely the gene PIG-A, a gene whose protein product is required for numerous glycosyl-phosphatidylinositol-anchored proteins to become surface bound. Since several such proteins are displayed by normal granulocytes, mutant cells can be numbered accurately by flow cytometry; and the frequency (ƒ) of such mutant cells may be a good surrogate of μ (Peruzzi et al. Mutation Research. 2010, 705:3).

We have determined ƒ in 74 patients with MPN (including 29 with polycythemia vera (PV), 17 with essential thrombocythemia (ET), 16 with primary myelofibrosis (PMF) 12 with post-PV/post-ET (PPV/PET) MF. Overall the proportion of those with a JAK2(V617F) mutation was 90%. In a subset of 59 of these patients, who had “uncomplicated” MPNs, the median of ƒ was no different compared to 142 healthy controls (4. 74×10−6vs. 4. 87×10−6: Figure 1). However, in a subset of 15, consisting of patients who in addition to MPN had at least one primary malignancy (2 had lymphomas and 13 had one of several types of solid tumors), the median value of ƒ was 15. 22×10−6, i. e. significantly elevated (Mann Whitney test: MPNs vs. MPNs with a second neoplasm, p<0. 013; Healthy controls vs. MPNs with a second neoplasm, p<0. 005: Figure 1).

Clearly we cannot automatically extrapolate from our reporter gene, PIG-A, to the actual probability that another gene undergoes somatic mutation; besides, the rate of mutation of JAK2 is influenced by the local genomic context. With that proviso, our data suggest that JAK2 mutations are unlikely to result from an overall increased mutation rate. In keeping with this notion, we found no correlation between the value of ƒ and the allelic burden of JAK2(V617F) (R2= 0. 001): i. e., there is no evidence for genetic instability in MPNs (although, once there is a JAK2 mutation, this may produce a mutator phenotype). On the other hand, an intrinsically increased mutation rate does play a significant role in subjects with multiple neoplasms. This is not surprising: but, to the best of our knowledge, for patients who did not otherwise appear to be ‘cancer-prone’, direct evidence for this is provided here for the first time.

Disclosures:

No relevant conflicts of interest to declare.
Interleukin-15 Levels in Patients with Essential Thrombocythemia
Authors: Cacciola Emma , Francesco Ernesto Di , Cipolla Antonino , Ferlito Carmen , Pezzella Francesca , Seria Elisa , Cacciola Rossella Rosari
Published: October 2019

Abstract 5071

The essential thrombocythemia (ET) is a myeloid neoplasm characterized by a inflammatory component responsible for myeloproliferation and hypercoagulability. It has been showed that interleukin-15 (IL-15) affects myelopoiesis and coagulation. Therefore, we evaluated platelets, hematocrit (HCT), as myeloproliferative indicator, tissue factor (TF), as marker of coagulative activation and IL-15. We recruited 25 patients with ET (10 males and 15 females, mean age 58 years) who fulfilled WHO criteria. Their mean duration of disease was 8 years (range, 5–21 years). All patients were on aspirin. None of the patients had thrombotic risk factors. Platelets and HCT were measured by automated analyser. IL-15 and TF were measured by ELISA. All patients had thrombocytosis (961±235×109/L), high HCT (42±4 vs 37±1 %) (p<. 0001), increased TF (214±266 pg/ml vs 5±3 pg/ml) (p<. 0001) and elevated IL-15 (21±8 pg/ml vs 2±1 pg/ml) (p<. 0001). A correlation there was between IL-15 and HCT (p=0. 076) and a strong correlation was found between IL-15 and TF (p=0. 046). These results suggest a novel relationship between IL-15 and myeloproliferation and thrombosis that might mean worse prognosis.

Disclosures:

No relevant conflicts of interest to declare.
Long-term Use of Anagrelid in Essential Thrombocythemia Treatment of a Child Under 12 Months
Authors: Laguna Pawel , Klukowska Anna , Pawelec Katarzyna , Matysiak Michal
Published: October 2019

Abstract 5072

Essential thrombocythemia (ET) is currently classified as a chronic myeloproliferative disorder. Anagrelide is a novel platelet lowering agent that has recently been approved for use n ET.

We present the case of ET in an 8 –month- old girl, who was treated with Anagrelid for 13 months.

The girl was admitted to hospital because of elevated platelet count. She had stomach pains periodically. On admission the child's condition was good but on palpation her stomach was sore and her spleen was enlarged. Laboratory investigations showed an elevated platelet count (1. 5m) and hypercalemia (6. 3mEq/l). Thrombopoetin was normal.

Acesan was added to the treatment. A morphology performed after 1 month revealed further elevation of platelets (up to 2. 65m). On the basis of laboratory investigations, bone-marrow biopsy and trepanobiopsy, we eliminated an oncological disease and infectious diseases of connective tissues.

On bone-marrow investigation, acquired mutation of JAK2 (V617F) tyrosine kinase was diagnosed, which confirmed ET.

On account of the growing number of platelets, which was life - threatening, we decided to administer anagrelid, starting with a dose of 0. 25 mg per day. The number of platelets decreased to 1. 4m, so the dose was increased to 0. 25 mg twice daily after 3 weeks.

At present, after 37 months of anagrelide treatment, the number of platelets in the child ranges between 400 and 650.

The patient visits our, department for monthly check-up (morphology, biochemical tests, ECG and echocardiography).

In international literature there is no information on the use of Anagrelid in ET treatment of children under 12 months old. However, on the basis of our observations and initial results of treatment, it seems that the above protocol for Anagrelid administration is safe for infants of this age.

Disclosures:

No relevant conflicts of interest to declare.
Association of Renal Cell Carcinoma and B-Cell Hematological Malignancy
Authors: Chintapatla Rangaswamy , Varella Leticia , Wiernik Peter , Rusciano Valerie , Dutcher Janice P.
Published: October 2019

Abstract 5086

Purpose of the study:

We observed an increased frequency of hematologic malignancy (HM) in patients and family members of patients with renal cell cancer (RCC) and sought to characterize the association further in terms of frequency and characteristics of HM, and the importance of such an association.

Methods:

We performed a chart review of our data base of approximately 700 RCC patients seen by us from 2004 to the present in an effort to determine the frequency of HM in patients and in the families of patients diagnosed with RCC.

Results:

Of the 700 charts reviewed, both HM and RCC occurred in 19 individuals. [11 males and 8 females]. HM diagnosis included acute myeloid leukemia in 1 patient, Hodgkin's lymphoma (HL) in 4 pts, non-Hodgkin lymphoma (NHL) in 7 pts, (3 small cell and 4 large B-cell lymphoma), chronic lymphocytic leukemia (CLL) in 2 pts and hairy cell leukemia (HCL), monoclonal gammopathy of undetermined significance (MGUS) myelodysplasia (MDS) in one patient each. A family history of HM was found in 71 relatives involving 56 families of patients with RCC. Of these, 48/71 cases of HM were in first degree relatives and 18/71 were in second degree relatives. The most common HMs were lymphoma and leukemia: 24 NHL, 9 HL, 6 lymphoma not further specified (NOS), 11 CLL, 1 acute myeloid leukemia, 5 acute leukemia NOS and 6 leukemia NOS. Other HM observed once were multiple myeloma, Waldenström's macroglobulinemia, chronic myeloid leukemia, myelofibrosis and polycythemia vera. In addition, 2 family members had blood cancers that were NOS. Thus, of 77 patients/family members with known HM diagnosis 94% were B-cell malignancies. Clear cell histology was the most common subtype of RCC, and all subtypes of RCC occurred in the study population with expected frequency. RCC and HM occurred in the same patient in this study more frequently at 2. 7% than would be expected from a SEER database. In that database the observed to expected (O/E) ratio of NHL and RCC was 1. 86 to 2. 07% [Kunthur et al. Am J Hematol 2006; 81:271–80].

Conclusions:

Increased incidence of B-cell malignancy has been reported in individuals with RCC [Dutcher et al. Proc Am Fed Clin Res, Eastern Division, April 2011]. Wiernik et al. [Cancer J 2000] reported a similar increase was noted between adenocarcinoma of the breast and B cell malignancies in same individual and mouse mammary tumor virus was proposed a potential causative agent. There is a preponderance of B-cell malignancy in both the individuals and in the families of the patients with RCC noted in this study. The etiology of this association between RCC and HM is unclear and suggests a common etiopathogenesis for RCC and B-cell tumors, or a familial immunologic defect that facilitates both malignancies. We plan to further explore the relationship of HM to RCC.

Disclosures:

No relevant conflicts of interest to declare.
A Mendelian Predisposition to B Cell Lymphoma Caused by IL-10R2 Deficiency
Authors: Neven Benedicte , Mamessier Emilie , Bruneau Julie , Kaltenbach Sophie , Suarez Felipe , Doz Francois , Orbach Daniel , Minard Veronique , Frange Pierre , Canioni Danielle , Radford-Weiss Isabelle , Asnafi Vahid , Casanova jean-Laurent , Blanche Stephane , Picard Capucine , Rieux-Laucat Frederic , Davi Frederic , Brousse Nicole , Nadel Bertrand , Ruemmele Frank , Fischer Alain
Published: October 2019

Abstract 5092

Background

The fact that monogenic interleukin (IL)-10 and IL-10 receptor deficiencies cause very early-onset, severe inflammatory bowel disease demonstrates the IL-10 pathway's crucial role in preventing microbiota-driven gut inflammation. However, no other severe complications of IL-10/IL-10R deficiencies have been reported to date.

Methods

A cohort of six patients with IL-10/IL-10R deficiency was retrospectively surveyed for phenotypic expression. The identification of a number of cases of lymphoma prompted an in-depth characterization of available biopsy material, with immunohistochemical staining, cytogenetic studies and gene expression profiling.

Results

Four patients (all with a complete IL-10R2 deficiency) had developed B cell non-Hodgkin's lymphoma between the ages of 5 and 6 years. Cytogenetic and IgH clonality analyses suggested that one of the patients developed at least 3 distinct lymphomas. The patients' tumors had the characteristics of diffuse large B cell lymphomas and contained monoclonal, Epstein-Barr-virus-negative germinal center B cells. Nuclear expression of the NF-κB factor c-REL was detected in all of the six lymphomas tested.

Conclusions

IL-10R deficiency is associated with a high risk of developing a B cell lymphoma with an unusual phenotype. Our results highlight an unexpected role of the IL-10R pathway in the control of lymphomagenesis.

Disclosures:

No relevant conflicts of interest to declare.
The Mir-17∼92 Cluster Activates mTORC1 Signaling in MCL by Targeting Multiple Regulators of the LKB1/AMPK/mTOR Pathway
Authors: Bi Chengfeng , Jiang Chunsun , Jiang Xiaoxing , Huang Xin , Fernandez Mario R , Iqbal Javeed , Lewis Robert E , Chan Wing Chung , McKeithan Timothy , Fu Kai
Published: October 2019

Abstract 5118

Mantle cell lymphoma (MCL) is an aggressive hematological malignancy with a median survival ranging between 3 and 5 years. Novel therapeutic strategies are urgently needed to improve the outcome. Mammalian target of rapamycin (mTOR) pathway which plays a central role in controlling cell growth, proliferation and metabolism has been shown to be deregulated in MCL. mTOR inhibitors, such as rapamycin and its analogues, have been approved for treatment of relapsed/refractory MCL. However, the molecular mechanism of mTOR activation in MCL has yet to be defined. MiRNA (miR)-17∼92 is a cluster of six miRNAs which are frequently overexpressed in MCL cases and overexpression of the miR-17∼92 cluster in MCL predicts poor prognosis. Our previous study demonstrated that miR-17∼92 activated the PI3K/AKT pathway by directly targeting PTEN and PHLPP2; and knock-down of miR-17∼92 expression inhibited MCL tumor growth in a xenograft/SCID MCL mouse model. In the present study, we further demonstrated that knock-down miR-17∼92 decreased the cell size, similar effect as seen in cells treated with mTORC1 inhibitors. Knockdown of miR-17∼92 expression also decreased the glycolysis, protein synthesis and glucose uptake in MCL cells. We found that knockdown miR-17∼92 activated AMPK signaling as demonstrated by increased phosphorylation of AMPK at Thr172, especially under low glucose condition. Activated AMPK further phosphorylated TSC2 and Raptor at S1387 and S792, respectively, thereby inhibiting mTORC1 signaling as evidenced by decreased phosphorylation of RPS6 and 4E-BP1. Using TargetScan and other prediction algorithms, we found that several factors in the LKB1/AMPK/mTOR pathway, such as LKB1, CAB39, PRKAA1 and TSC1, are predicted the targets of miR-17∼92. In this study, we validated these factors as direct targets of the miR-17∼92 by 3'UTR luciferase assays using reporter plasmids containing the 3'UTR of the targets or the 3'UTR with mutations in the predicted miRNA binding sites. The protein levels of these targets decreased in MCL cells with the miR-17∼92 overexpression. Conversely, the levels of these factors were increased upon knockdown of miR-17∼92 cluster. Our results indicate that overexpression of miR-17∼92 in MCL plays important role in mTORC1 activation by inactivating the LKB1/AMPK signaling, in addition to its effect on the PI3K/AKT pathway activation.

Disclosures:

No relevant conflicts of interest to declare.
Calmodulin Inhibition Rescues the Effects of Ribosomal Protein Deficiency by Modulating p53 Activity in Models of Diamond Blackfan Anemia
Authors: Taylor Alison M. , Raiser David M. , Humphries Jessica M. , Ebert Benjamin L. , Zon Leonard I.
Published: October 2019

Abstract 512

Ribosomal protein mutations are common in patients with Diamond Blackfan anemia (DBA), who have red cell aplasia and craniofacial abnormalities, and rps14 deficiency has been linked to 5q- myelodysplastic syndrome. We have characterized a zebrafish mutant in rps29, a ribosomal protein in the small subunit. Rps29−/− embryos have hematopoietic and endothelial defects, including decreased hematopoietic stem cells, defects in hemoglobinization, and decreased staining of vessel markers. Heads of mutant embryos are also morphologically affected, with increased apoptosis. Consistent with other models of DBA, knockdown of p53 completely rescues the rps29 hematopoietic and apoptotic phenotypes. We wanted to identify chemicals that could rescue the rps29 mutant phenotype. Using 600 compounds from known bioactive libraries, we performed an in vivo chemical screen for rescue of the rps29 apoptotic and endothelial defects. Treatment with one compound, A-3, improved the morphology of the apoptotic embryo head. A-3 is an inhibitor of calmodulin, which interacts with a host of calmodulin-dependent enzymes and can bind to other proteins in the cell, including p21. W-7, another calmodulin inhibitor related to A-3, can rescue the endothelial defect in the rps29 mutant. Calmodulin inhibition in rps29 mutant embryos rescues the hemoglobin defect. Furthermore, structurally unrelated chemicals that inhibit calmodulin rescue the endothelial defect. We hypothesized that calmodulin inhibition was affecting some part of the p53 pathway. We tested whether A-3 had an effect on the phenotype in vitro in a cancer cell line where shRNA knockdown of RPS19 has been shown to stabilize p53 and induce p21. When rps19 is knocked down in A549 cells, treatment with calmodulin inhibitors resulted in a decrease of p21 protein, as measured by flow cytometry, although p53 protein levels were stable. This effect was also observed in primary human cord blood-derived CD34+ hematopoietic stem and progenitor cells. In situ immunofluorescence in A549s further revealed that nuclear localization of p53 and p21 upon RPS19 knockdown was disrupted by chemical treatment. Calmodulin inhibitors also rescue ribosomal protein deficiency-mediated erythroid defects both in vitro and in vivo. When RPS19 is knocked down in CD34+ cells, there is a defect in erythroid differentiation. Treatment with calmodulin inhibitors rescued production of CD71+ cells upon differentiation. In conclusion, we have demonstrated the use of calmodulin inhibitors as a novel approach for mediating p53 activation upon ribosomal protein knockdown, thereby rescuing the erythroid defects of ribosomal deficiency. Our data suggests that small molecules that inhibit calmodulin may be effective therapies for patients with ribosomal deficiency.

Disclosures:

Ebert: Celgene: Consultancy; Genoptix: Consultancy. Zon:Fate Therapeutics: Founder Other.
Detection of SNPs of Dck and Cda Genes in Five Hematological Malignant Tumor Cell Lines
Authors: Xiaoping Zhang , Jian Ziying , Chen Bao-An , Xu Peipei , Peng Miaoxin , Xia Guohua , Cheng Lu , Lu Zuhong
Published: October 2019

Abstract 5129

Background & Aim:

This study detected single nucleotide polymorphisms (SNPs) of deoxycytidine kinase(DCK) gene, cytidine deaminase(CDA) gene in five hematological malignant tumor cell lines, to find connections between sNPs and Cytarabine dose selection in the treatment of some hematopathy.

Materials & Methods:

Cell line K562, Ka, HL-60, U937, and Raji were cultured. The genomic DNA was isolated by QIAamp DNA Blood Mini kit. Designing primes were amplified by PCR to get the related DNA fragments. DCK gene A674G(rs111454937) C1644T(rs72552079), CDA gene A79C(rs2072671) G208A(rs60369023) was genotyped by means of matrix assisted laser desorption ionisation-time of flight mass spectrometry method (MALDI-TOF MS).

Results:

The genotype of locus A674G(rs111454937) on DCK gene in all five hematology system cell lines was AA; The genotype of locus C1644T(rs72552079) on DCK gene in all five hematology system cell lines was CC. The genotype of locus G208A(rs60369023) on CDA gene in all five hematology system cell lines was GG; For HL-60, U937, and Raji cell lines, the genotype of locus A79C(rs2072671) on CDA gene was AA, while for K562 and Ka cell lines, that was C/A.

Conclusions:

The genotype of locus A79C(rs2072671) on CDA gene was not all the same in the detected hematological malignant tumor cell lines, while the genotypes of the other 3 loci were same in the 5 cell lines.

Disclosures:

No relevant conflicts of interest to declare.
Tissue Factor and Hematological Neoplasias
Authors: Cesarman-Maus Gabriela N. , Braggio Esteban , Lome Carmen , Morales-Leyte Ana Lilia , Fonseca Rafael
Published: October 2019

Abstract 5132

There is a well- recognized correlation between cancer and aberrant hemostasis. Venous thromboembolism (VTE) in individuals with solid epithelial tumors has been associated with a poor prognosis, with more than three-fold higher risk of early death as compared to cancer patients without thrombosis. The expression of tissue factor (TF), a cell-surface membrane glycoprotein that triggers the activation of coagulation by cancer cells, is one of the main underlying mechanisms linking thrombosis and aggressive tumor behavior. TF is expressed in a variety of solid tumors in association with genetic events affecting oncogenes and tumor suppressor genes. However, the mechanisms of thrombosis in individuals with hematological malignancies may differ from those with solid tumors. We have previously shown that despite the high rate of thrombosis in multiple myeloma, malignant plasma cells only rarely express TF. We sought to determine TF gene (F3) and protein expression in hematological neoplasias. F3 expression profiling was studied on a variety of cell lines established from lymphoid and myeloid neoplasias available at Glaxo Smith Kline (GSK) Cancer Cell Line Genomic Profiling Dataset (https://array.nci.nih.gov/caarray/project/woost-00041). Interestingly, F3 expression was absent in all lymphoid neoplasias studied, in sharp contrast to acute myeloid leukemias (AML) and solid tumors, of which 30 and 90% expressed F3, respectively. Immunohistochemistry (IHC) confirmed the absence of TF protein expression in all indolent and high-grade B-cell lymphomas (99 patients, including germinal center and activated B-cell phenotype diffuse large B-cell lymphomas) and in all T-cell lymphomas/leukemias (20 patients) studied. IHC for TF was also negative in AML (11 patients) but positive in representative solid tumors (breast, pancreas, prostate), except for renal cell carcinoma which has been previously shown to lack TF. We propose that the pathogenesis of VTE associated with hematological neoplasias differs from that of solid tumors. Though TF from non-neoplastic cell sources may still be important for the prothrombotic state often seen in these patients, we show there is no evidence for a role of tumor-derived TF in the development of DVT, nor in neoplastic behavior. Thus, treatments directed against TF may not impact on prognosis in lymphoid, and non TF-expressing myeloid neoplasias.

Disclosures:

No relevant conflicts of interest to declare.
Hemostatic Factors and Shortened Sleep
Authors: Koktas Zehra , Vida Loyda , De Chavez Peter John D , Carnethon Mercedes , Green David
Published: October 2019

Abstract 5137

Introduction:

Enhanced coagulation has been reported in persons with sleep disturbances and sleep apnea. However, it is unclear whether shortened sleep duration in the absence of disordered breathing is associated with higher levels of hemostatic factors.

Methods:

In this cross-sectional study, 582 community-dwelling adults were randomly sampled. They underwent polysomnography to determine the apnea hypopnea index (AHI), a measure of sleep disordered breathing, and wrist actigraphy for 7 nights to assess sleep duration. A morning blood sample was collected for measurements of factor VIII (FVIII), von Willebrand Factor (vWF), thrombin antithrombin complexes (TAT), and plasminogen activator inhibitor-1 (PAI-1). Logistic regression analyses were used to model the odds of being in the upper quintile of each hemostatic factor by AHI score or sleep duration.

Results:

Participant ages ranged from 35 to 65 (mean 48), with 57% women and 30% Blacks. Hypertension was present in 18% and diabetes in 6%. The mean (SD) sleep duration was 7 (1. 07) hours/day, and 34% had an AHI ≥5. Mean values (SD) for hemostatic factors were FVIII (U/ml), 1. 06 (0. 41); vWF (U/ml), 1. 21 (0. 51); TAT (ng/ml), 2. 66 (2. 25); and PAI-1(ng/ml), 23. 4 (21. 4). vWF increased with age and FVIII, vWF, and TAT were higher in Blacks vs Whites. Associations (odds ratio, 95% confidence interval) were observed for FVIII with diabetes (3. 16, 1. 36–7. 32) and PAI-1 with hypertension (1. 9, 1. 06–3. 41) and diabetes (2. 58, 1. 15–5. 76) after adjustment for demographic and cardiovascular covariates.

In unadjusted analyses, participants with AHI ≥5 had elevated levels of FVIII and vWF, but this association was attenuated after adjustment for demographic and cardiovascular covariates (see Table). However, AHI ≥5 remained significantly associated with elevated PAI-1 in fully adjusted models. The majority of participants (90%) had a low likelihood of apnea (AHI <15), and their sleep duration showed no associations with hemostatic factors in any model.

Conclusion:

In a random population sample, we confirmed that mild apnea is associated with elevated PAI-1. However, among those persons free of apnea, there were no associations of sleep duration with FVIII, vWF, TAT or PAI-1.

Disclosures:

No relevant conflicts of interest to declare.
Elevated Plasma Levels of Soluble Platelet Glycoprotein VI (GPVI) in Patients with Thrombotic Patients
Authors: Wada Hideo , Nobori Tsutomu , Naitoh Katsuki
Published: October 2019

Abstract 5145

Introduction

Thrombotic microangiopathy (TMA) is caused by various conditions, such as decreased a ADAMTS13 level, activated or injured vascular endothelial cells or activated platelets and it must be diagnosed at the early stage. This study, examined the activation of platelets by measuring of soluble platelet glycoprotein VI (sGPVI) level in patients with TMA.

Materials and Methods

The plasma levels of sGPVI, ADAMTS13 activity, von Willebrand factor (VWF) and VWF propeptide (VWFpp) were measured in 40 healthy volunteers, 46 patients without thrombosis (TH), 15 postoperative patients, 13 with disseminated intravascular coagulation (DIC) and 70 with TMA. The plasma levels of GPVI and VWFpp were measured by ELISA and ADAMTS13 was measured by the FRETS assay. There were 27 TMA with markedly decreased ADAMTS13 levels (TMA-ADAMTS13) and 43 TMA without (TMA-Other).

Results

The plasma levels of sGPVI (median; 25. 0–75. 0%tile) were significantly higher in patients without TH (16. 2 ng/mL 12. 6–22. 5 ng/mL), postoperative patients (31. 6 ng/mL; 28. 3–35. 1 ng/mL), DIC (44. 5 ng/mL; 36. 6– 60. 8 ng/mL), TMA (40. 8 ng/mL; 32. 9–56. 7 ng/mL) than in healthy volunteers (11. 4 ng/mL; 9. 1–14. 8 ng/mL). In addition, the plasma levels of sGPVI post in postoperative patients were significantly higher than in patients without TH (p< 0. 001), and these levels were also significantly higher in those with TMA and DIC than in without TH (p< 0. 001, respectively). The plasma levels of sGPVI were significantly higher in patients with TTP-ADAMTS13 (36. 8 ng/mL; 30. 5–49. 0 ng/mL) and TMA-Other (51. 9 ng/mL; 37. 4–66. 3 ng/mL) than in patients without TH (p< 0. 001, respectively).

Conclusion

The measurement of sGPVI is therefore considered to be important for the diagnosis of TMA.

Disclosures:

Wada: Mochida pharmaceutical CO., LTD: Suporting GPVI assay Other.
Arsenic Trioxide Induces Platelet Apoptosis
Authors: Wu Yicun , Dai Jin , Zhang Weilin , Yan Rong , Ruan Changgeng , Dai Kesheng
Published: October 2019

Abstract 5149

Objective:

Arsenic trioxide, a component of Traditional Chinese Medicine, is known as an effective anticancer drug especially in the treatment of acute promyelocytic leukaemia (APL). APL has emerged as the most curable subtype of acute myeloid leukaemia since the widely use of arsenic trioxide-based chemotherapy. However, recent researches show that thrombocytopenia occurred in 79% of the relapsed or refractory APL patients treated with arsenic trioxide, and part of the APL patients had to be stopped treatment because of catastrophic bleeding, such as intracranial and pulmonary haemorrhage. Thrombocytopenia also occurred in 43% of the myelodysplastic syndrome patients treated with arsenic trioxide. Recently, arsenic trioxide has been proved to have a pro-apoptotic effect on various kinds of nucleated tumour cells or non-tumour cells. The effect of arsenic trioxide on enucleated platelet, however, still remains unclear. The aim of current study is to investigate whether arsenic trioxide induces platelet apoptosis.

Methods:

Washed platelets (3 × 108/ml) were incubated with different concentrations of arsenic trioxide or vehicle at 37°C for 4 hours. Then, mitochondrial inner transmembrane potential (ΔΨm) and phosphatidylserine (PS) exposure were tested by flow cytometry. In the mean time, the treated platelets were analyzed by western blot for the expression levels of pro-apoptotic protein (Bax), and anti-apoptotic proteins (Bcl-2 and Bcl-XL). Activation of caspase-3 was also examined by western blot using an anti-caspase-3 antibody.

Results:

ΔΨm depolarization and PS exposure were dose-dependently induced in platelets incubated with different concentrations (2 uM, 4 uM, 8 uM, 16 uM) of arsenic trioxide as detected by flow cytometry, and the lowest concentration of arsenic trioxide incurring ΔΨm depolarization and PS exposure was 4 umol/L. Simultaneously, arsenic trioxide induced up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL in a dose-dependent manner. Furthermore, 17 kD cleaved caspase-3 fragments were dose-dependently induced in platelets treated with different concentrations of arsenic trioxide indicating that caspase-3 was activated by arsenic trioxide.

Conclusions:

Taken together, the data indicate that arsenic trioxide induces platelet apoptosis in vitro, which might suggest a novel pathogenic mechanism of thrombocytopenia in the patients who treated with arsenic trioxide.

Disclosures:

No relevant conflicts of interest to declare.
Red Cell Microparticles (RMP) and Other Cell-Derived Microparticles (C-MP) in Hemolytic Anemias
Authors: Johansen Max E , Jy Wenche , Dudkiewicz Pamela B , Ahn Yeon S
Published: October 2019

Abstract 5158

Introduction:

The lifespan of red cells (RBC) are shortened by several mechanisms in hemolytic anemias (HA): Ab-mediated hemolysis in autoimmune hemolytic anemia (AIHA), complement-mediated lysis in paroxysmal nocturnal hemoglobinuria (PNH), microangiopathy in thrombotic thrombocytopenic purpura (TTP) and hemoglobinopathy in sickle cell anemia (SS) and thalassemia (Thal). During hemolysis, RMP are released along with MP from platelets (PMP), endothelia (EMP) and leukocytes (LMP). The role of MP in HA is unknown but may be involved in complications of HA.

Methods:

We investigated RMP and other MP profiles in the following hemolytic anemias in active phase: 14 patients with AIHA, 10 with TTP, 4 with PNH, 8 with SS or Thalassemia (SS - Thal), along with 60 healthy controls. Whole, citrated blood was centrifuged at 1600xgfor 10 minutes to yield platelet-poor plasma (PPP). Microparticles in the PPP were measured by FITC- or PE-conjugated mAb specific to the aforementioned cell types. Control plasma was prepared in an identical fashion from healthy volunteers. We compared RMP profiles and other MP in HA during active phase and in remission. Associations with clinical features were calculated using the R statistical software.

Results:

Means and standard deviations of C-MP in the different types of HA are shown in the TABLE. Mean RMP and PMP were highest in SS-Thal, followed by AIHA. Mean Annexin V binding procoagluant MP were higher in all subgroups of HA and highest in SS-Thal, followed by TTP. EMP in all subgroups was similar or lower than controls.

Regarding clinical associations, high RMP levels were associated with low hemoglobin (p=0. 01), high reticulocytes (p=0. 003), and high LDH (p=0. 01) across all groups. High reticuloctye counts (>3. 0 %) were also associated with significantly elevated PMP (p=0. 04) and total MP counts by Ulex (p=0. 003). HAs in remission had marginally lower RMP levels than controls (p=0. 05). None of the subtypes had significantly higher EMP, LMP, or PMP compared to each other or controls.

Conclusion:

The small population yielded large standard deviations in the MP counts and did not allow statistical study among subgroups of patients with HA. However prothrombotic MPs such as Annexin V, PMP tend to be higher in HA. A larger scale future study will clarify this issue. The constellation of elevated LDH, reticulocytes, and decreased hemoglobin were associated with increased RMP. Therefore we suggest that elevated RMP may be viewed as an additional, new diagnostic marker of active hemolysis.

Disclosures:

No relevant conflicts of interest to declare.
Diagnosis of Auto Immune Hemolytic Anemia by Detection of RBC-Bound IgG Antibodies, Using Flow-Cytometry
Authors: Khungar Jitendra Mohan , Pati Hara Prasad Prasad , Mahapatra Manoranjan
Published: October 2019

Abstract 5159

Introduction:

Auto Immune Hemolytic Anemia (AIHA) is one of the most common types of acquired hemolytic anaemias. Its main cause is auto antibody mediated rapid destruction of RBCs. Detection of these autoantibodies to erythrocytes is of fundamental importance for diagnosis. A number of methodologies have been tried for detection & evaluation of these autoantibodies. Demonstration of a positive Direct Antiglobulin Test (DAT) against these autoantibodies is an important serological assay in the diagnosis of auto immune hemolytic anemia (AIHA). This test is also considered as pathognomonic of immune-mediated hemolysis. This routinely used direct antiglobulin test (DAT) has the disadvantage of low sensitivity and does not detect low levels of red cell auto antibodies leading sometimes to false negative results. Flow cytometry can effectively diagnose such patients of auto immune hemolytic anemia with low levels of autoantibodies. Role of flow cytometry in the diagnosis of several non-malignant haematological disorders is being explored & present study has been conducted with the same objective.

Aims & Objectives:

This study was conducted with the following aims and objectives:

•To assess the utility of flow-cytometry (FCM) in the diagnosis of suspected AIHA patients.

•Compare the sensitivity of flow-cytometry (FCM) with Direct Antiglobulin Test (DAT) by Gel-card Test (GT).

•To assess the positivity in DAT negative cases by flow-cytometry in suspected AIHA cases.

Material & Methods:

This was a prospective study, carried out in Haematology Deptt of All India Institute of Medical Sciences, where patients with suspected auto immune hemolytic anemia (AIHA) were studied during two years period. Blood samples of suspected patients of AIHA were tested by Gel Card Test as well as by Flow cytometry for detection of RBC bound IgG.

Results:

A total of 50 patients with suspected diagnosis of auto immune hemolytic anemia (AIHA) were studied by flow-cytometry as well as by Gel card test (GT) for detection of RBC bound IgG. Out of these 50 cases, 41 cases have turned out to be positive and 9 were negative by flow-cytometry. The quantification of positivity by flow-cytometry was obtained by calculating percentage fluorescence. The same 50 cases were also tested by Gel card test (GT). By Gel card test, out of 50 cases, 34 were positive & 16 were negative. Therefore, there were 7 cases which were negative for RBC bound IgG by Gel card test and these were positive by flow-cytometry.

The flow cytometry figures of these cases will be shown & discussed in the presentation.

Conclusions:

Flow-cytometry is a reliable and sensitive method of detecting RBC-bound IgG antibodies for the diagnosis of auto immune hemolytic anemia. This can be used as a new routine diagnostic technique for auto immune hemolytic anemia.

Disclosures:

No relevant conflicts of interest to declare.
Efficacy and Safety of Monoclonal Anti-CD20 Antibody Rituximab Combined Cyclophosphamide in Treatment of Refractory and Recurrent Autoimmune Hemolytic Anemia
Authors: Shao Zonghong , Liu Hong , Xing Limin , Wu Yuhong , Qu Wen , Liang Yong , Guan Jing , Wang Guojin , Wang Xiaoming , Song Jia , Li Lijuan , Wang Huaquan
Published: October 2019

Abstract 5161

Objectives:

To better assess the efficacy and safety of monoclonal anti-CD20 antibody rituximab in the treatment of refractory and recurrent autoimmune hemolytic anemia.

Patients and methods:

7 cases with autoimmune hemolytic anemia (including 1 case of Evans syndrome) were enrolled into this study, they were treated with rituximab (375 mg/m2, once per week, 2–6 times) and Cyclophosphamide(1g/10days, 2–6 times) combined with intravenous immunoglobulin (IVIG) (10g/week, given 1 day after rituximab treatment).

Results:

All 7 patients showed good responses. 6 achieved complete remission (CR) and 1 achieved partial remission (PR). Responses were seen at 1th to 10th month after the first dose of rituximab and the mean response time was approximately 2. 5 months. Average follow-up time for them isfor the patients was 27 months. All patients remained in remission at the 12-month follow-up. At the time of 24-month follow up, 3 patients showed elevated indirect bilirubin and increased reticulocyte counts. One of the 3 patients achieved CR after additional rituximab therapy, and the other 2 PR after additional cyclophosphamide therapy. At the time of 36-month follow up, 1 patient relapsed and was retreated with 3 cycles of rituximab and eventually reached PR. All patients tolerated the treatment well with only mild side effects.

Conclusion:

rituximab is highly effective and relatively safe in patients with refractory and recurrent autoimmune hemolytic anemia. Maybe Additional treatment can be given in patients with relapse after 1–2 years.

Disclosures:

No relevant conflicts of interest to declare.
Alfa Thalassemia Heterozygous: How the New Information Provided by the Routine Hematology Analysers May Help in Its Differential Diagnosis or Flagging
Authors: Barella Susanna , Simon-Lopez Ramon , Gaetano Nicola Di , Galanello Renzo
Published: October 2019

Abstract 5183

Carriers of α°-thalassemia (α-thalassemia trait) show microcytosis, hypochromia, and normal percentages of HbA2 and HbF. Carriers of α + -thalassemia (α-thalassemia silent carrier) have either a silent hematologic phenotype or present with a moderate thalassemia-like hematologic picture. Homozygosity for α + -thalassemia results in an α°-thalassemia (α-thalassemia trait) hematologic phenotype.

Diagnostic of Alfa Thalassemia Heterozygous:

Classic testing for α-thalassemia includes: hematologic testing of red blood cell indices, peripheral blood smear, supravital stain to detect RBC inclusion bodies, and qualitative and quantitative hemoglobin analysis. HBA1, the gene encoding α1-globin, and HBA2, the gene encoding α2-globin, are the two genes most commonly associated with α-thalassemia. Molecular genetic testing of HBA1 and HBA2 detects deletions in about 90% and point mutations in about 10% of affected individuals.

Objective:

Recently have been developed new parameters and information in the new automated hematology analyzer called DxH8008™ from Beckman Coulter as @MSCV, @RSF, @MAF, @LHD% and many morphological parameters for RBC and Reticulocytes calles Cell Population Data. All this parameters may be used to create flagging for laboratory use only (LUO) or Research use only (RUO). The purpose of this study is to investigate the possible use or utility of this new information for the screening/flagging of Alfa Thalassemia Heterozygous.

Patient and Methods:

We have collected 48 patients with Alfa Thalassemia Heterozygous All of them were confirmed by red cell morphology, Hgb Electroforesis, cromatography in liquid phase in human whole blood for the determination of Hemoglobin A2, F, A1c, and identification of abnormal hemoglobins and DNA analysis (DNA Analysis by GAP-PCR). We have compared these patients with a control group (184 individuals) and with other anemias (see Table 1).

Results:

Using ROC analysis, the best parameters differentiating the Alfa Thalassemia Heterozygous from the normals were: MCV (AUC 0. 963), @RSF (AUC 0. 936), @MSCV (AUC 0. 912), RDW (AUC 0. 848), @MAF(AUC 0. 845), @LHD(AUC 0. 829).

Using ROC analysis, the best parameters differentiating the Alfa Thalassemia Heterozygous from other anemias (excluding normals) were: RDW (AUC 0. 885), @MAF(AUC 0. 857), @MCNRET (AUC 0. 832).

Disclosures:

Simon-Lopez: Beckman Coulter: @LHD, @MAF, @RSF, @LHD, @MAF, @RSF Patents & Royalties, Employment. Di Gaetano:Instrumentation Laboratory spa: Work for a distributor of Beckman Coulter Instruments in Italy Other. Galanello:Ferrokin: Research Funding; Apopharma: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau.
Transfer of Monocrotaline-Induced Pulmonary Hypertension to Healthy Mice Via Microparticles
Authors: Aliotta Jason M. , Pereira Mandy , Amaral Ashley , Sorokina Arina , Rounds Sharon I , Quesenberry Peter J. , Klinger James R.
Published: October 2019

Abstract 5190

Rationale:

Circulating endothelial cell-derived microparticles (MP) are altered in pulmonary arterial hypertension (PAH) but whether they are biomarkers of cellular injury or participants in disease pathogenesis is unknown. We have shown that lung-derived MP induce marrow cells to express lung-specific mRNA, protein.

Objectives:

Determine if lung, plasma-derived MP (LMP, PMP) alter pulmonary vascular endothelial or marrow progenitor cell phenotype to induce pulmonary vascular remodeling in PAH.

Methods and Main Results:

LMP, PMP isolated from monocrotaline (MCT)- or vehicle-treated mice were injected into healthy mice. Right ventricular (RV) hypertrophy and pulmonary vascular remodeling were assessed by RV-to-body weight (RV/BW), blood vessel wall thickness-to-diameter (WT/D) ratios. RV/BW, WT/D ratios were elevated in MCT vs. vehicle-injected mice (1. 99+0. 09vs. 1. 04+0. 09mg/g; 0. 159+0. 002vs. 0. 062+0. 009%). MP from MCT and vehicle-treated mice were quantitatively similar; however, MCT-LMP had higher endothelial cell mRNA expression and higher expression of mRNAs of proteins known to be abnormally expressed in PAH vs. vehicle-LMP. RV/BW, WT/D ratios were higher in mice injected with MCT-MP vs. mice injected with vehicle-MP (1. 63+0. 09vs. 1. 08+0. 09mg/g; 0. 113+0. 02vs. 0. 056+0. 01%). Lineage-depleted bone marrow cells co-cultured with MCT-MP and bone marrow cells isolated from MCT-MP infused mice had higher endothelial progenitor cell gene expression vs. cells co-cultured with vehicle-MP or cells isolated from vehicle MP infused mice.

Conclusions:

MP from MCT-injured mice have increased expression of growth factors implicated in PAH pathogenesis and induce RV hypertrophy, pulmonary vascular remodeling in healthy mice. Circulating MP may contribute to the development of MCT-induced pulmonary hypertension by inducing a pathologic phenotype in pulmonary vascular endothelial and endothelial progenitor cells.

Disclosures:

No relevant conflicts of interest to declare.
Investigation of ETV2/Etsrp Role in Lymphangiogenesis: A Potential Therapeutic Target in Lymphatic Disease
Authors: Williams Jennifer D. , Desai Sharina Palencia , Sumanas Saulius
Published: October 2019

Abstract 614

Lymphangiogenesis, defined as novel growth of lymphatic vessels to constitute a functional vascular network, captures great clinical interest because of its essential role such pathologic settings as inflammation, vascular malformations, and tumor metastasis. Previous observational hurdles are now overcome by use of the optically clear and genetically modifiable zebrafish embryo, which provides a vertebrate model for understanding lymphangiogenesis and for revealing putative therapeutic targets for metastatic disease and vascular/lymphatic malformations that currently have no cure.

The zebrafish ets1-related protein, Etsrp, encodes a novel ETS domain transcription factor and is an early marker for vascular endothelial progenitors. Its function is necessary and sufficient to initiate vasculogenesis and is conserved during vertebrate development. Human and murine Er71/Etv2 proteins are functional orthologs of Etsrp. However, its role in lymphatic development is unknown. Our data show that both vascular and lymphatic vessels express etsrp driven GFP transgene. To analyze Etsrp role in lymphatic development, we performed functional Etsrp knockdown by photoactivatable morpholino oligonucleotide (MO) injection into fli1:GFP; flk1:mCherry transgenic reporter zebrafish embryos whose fluorescent background allowed distinction of lymphatic from vascular vessels.

Inhibition of etsrp function at early stages resulted in the absence of both lymphatic and vascular endothelial development. Inhibition of etsrp function at 1 day-post-fertilization, after the major axial vessels have already formed, specifically affected lymphangiogenesis without affecting vascular development. The major lymphatic vessel, the thoracic duct, was absent or discontinuous in Etsrp-depleted embryos, while the major axial and secondary intersegmental blood vessels were not affected. Further analysis demonstrated that the lymphatic progenitors, parachordal lymphangioblasts, failed to migrate from the posterior cardinal vein in the absence of Etsrp function. Control morpholino-injected but not photoactivated embryos did not show significant defects in the lymphatic development, demonstrating the specificity of the observed defects. Because expression of the major VegfC receptor, VegfR3 was strongly down regulated in Etsrp MO-injected embryos, our results suggest that Etsrp initiates lymphangiogenesis by regulating VegfR3 expression. In the absence of Etsrp function, lymphangioblasts would fail to respond to VegfC signaling.

We report for the first time the requirement of Etsrp for normal lymphangioblast migration and thoracic duct development in zebrafish. Additional studies are in process to determine the mechanism of Etsrp role in lymphangiogenesis. Our results suggest that Etsrp may represent a potential novel target for chemical modulation in multiple human lymphatic disorders.

Disclosures:

No relevant conflicts of interest to declare.
Normal Hematopoiesis and Neurofibromin-Deficient Myeloproliferative Disease Require Erk
Authors: Staser Karl , Zeng Yi , Park Su-jung , Rhodes Steven D. , He Yongzheng , Shew Matthew A. , Gehlhausen Jeff , Cerabona Donna , Chen Shi , Sun Zejin , Nalepa Grzegorz , Yang Feng-chun , Clapp D. Wade
Published: October 2019

Abstract 704

Neurofibromatosis type 1 predisposes individuals to the development of juvenile myelomonocytic leukemia, a fatal myeloproliferative disease (MPD). In genetically engineered murine models, nullizygosity of Nf1, a tumor suppressor gene which encodes a Ras GTPase activating protein, results in hyperactivity of Raf-Mek-Erk in hematopoietic stem and progenitor cells (HSPCs). Activated Erk1/2 phosphorylate kinases and transcription factors with plethoric mitogenic roles in diverse cell types. However, genetic studies examining Erk1/2's differential and/or combined control of normal and Nf1-deficient myelopoiesis are lacking. Moreover, prior studies relying on chemical Mek-Erk inhibitors have reached conflicting conclusions in normal and Nf1-deficient mice. Here, we show that while single Erk1 or Erk2 disruption does not grossly compromise myelopoiesis, dual Erk1/2 ablation rapidly ablates granulocyte and monocyte production, diminishes progenitor cell number, and prevents HSPC proliferation. Intriguingly, genetic disruption of Erk1/2 in the context of Nf1 nullizygosity (Mx1Cre+Nf1flox/floxErk1−/−Erk2flox/flox) fully protects against the genesis of MPD. Collectively, we identify a fundamental requirement for Erk1/2 signaling in normal and Nf1-deficient hematopoiesis, elucidating a critical hematopoietic function for Erk1/2 while genetically validating highly-selective Mek-Erk inhibitors for the attenuation of a leukemia that is otherwise resistant to traditional therapy.

Disclosures:

No relevant conflicts of interest to declare.