bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of Beta Blocker on Clinical Outcomes of Multiple Myeloma (MM) Patients
Authors: Hwa Yi Lisa , Lacy Martha Q. , Gertz Morie A , Buadi Francis , Rajkumar S. Vincent , Kumar Shaji , Kapoor Prashant , Go Ronald S. , Hayman Suzanne R , Lin Yi , Lust John A. , Dingli David , Russell Stephen J. , Gonsalves Wilson I , Dispenzieri Angela
Published: October 2019

Background: A recent preclinical study revealed an antiproliferative and apoptotic effect of propranolol on MM cell lines. This study was to investigate if propranolol and other beta blocker intake improved survival in MM patients.

Methods: Among patients seen at Mayo Clinic, Rochester from 1993 to 2010, we identified those taking propranolol. Age, year of diagnosis, International Staging System (ISS), Mayo Stratification of Myeloma and Risk-Adapted Therapy (mSMART) were used as case-match variables to match propranolol users to two control populations: no beta blocker and other beta-blocker. Cox proportional hazard modeling was utilized to analyze the effect of propranolol and other beta blockers intake on overall survival (OS) and disease-specific survival (DSS).

Results: In 3 groups: propranolol (n=52), no beta blocker (n= 136), or other beta blocker (n=84), transplant eligibility was 63.5%, 66.9%, 66.7% respectively (p=0.90). Median age was 63 years. Median follow-up was 86 months. No statistically differences were seen in age, ISS, mSMART or year of diagnosis in 3 groups (p=0.46).In univariate analysis comparing propranolol vs no beta blocker or other beta blocker cohort, 5-year OS rate was 60.2% vs 47% or 64% (p=0.039), and 5-year DSS rate was 71.7% vs 50.3% or 69.1% (p=0.0029). In multivariate analysis adjusted for age, ISS, transplant eligibility, mSMART and year of diagnosis, compared to propranolol intake, no beta blocker group had poorer OS [HR= 2.081; 95% CI (1.353- 3.200); p=0.0008], and DSS [HR=2.833 (1.709-4.697); p<0.0001], but other beta blocker users had no difference in OS [HR =1.145 (0.728-1.803);p=0.56],and DSS [HR= 1.276 (0.745-2.185); p=0.38].

Conclusion: Propranolol and other beta-blocker intake among MM patients were equally associated with a more favorable OS and DSS.

Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures

Lust: Senesco: PI Other.
HPRT As a Selectable Safe Harbor for Transgenesis
Authors: Conway Anthony , Laganière Josée , Paschon David E , Hacke Katrin , Kasahara Noriyuki , Gregory Philip D , Holmes Michael C. , Cost Gregory J
Published: October 2019

A current limitation in gene therapy is obtaining a sufficient number of modified cells to produce a therapeutic effect in vivo. In several diseases, correction of a mutant allele confers a selective growth advantage to the modified cells, thus enhancing efficacy with moderate initial modification. For most diseases, however, there is no selective advantage to the corrected cells. One potential strategy to address this limitation is in vivo selection of modified cells using pharmacological agents. It has previously been shown that 6-thioguanine (6-TG), an FDA-approved chemotherapeutic small molecule, is cytotoxic to cells expressing the enzyme HPRT, allowing for selective growth of HPRT knockout cells. Knockout of HPRT can be achieved by creating a nonsense mutation in an upstream exon, or by terminating splicing by introducing a large transgene into an intron. To allow for selectable transgenesis of only cells which have undergone targeted integration (TI), engineered zinc-finger nucleases (ZFNs) were used to insert a virally-delivered transgene into an HPRT intron. After two weeks of in vitro 6-TG selection following genome modification, a 95-fold increase in TI was observed in pooled K562 cell populations to a final level of 72% TI, whereas a 30-fold increase in transgene-expressing live cells was seen in peripheral blood-mobilized primary CD34+ cells resulting in 90% transgene-positive live cells. Furthermore, a 72-fold increase in transgene mRNA transcript was observed after two weeks of erythroid differentiation and 6-TG selection of CD34+ cells compared to unselected genome-modified controls. These results represent an important step in developing hematopoietic stem cell (HSC)-based gene therapies, as well as a platform technology for creating gene-modified HSC populations with high proportions of therapeutic transgene expression via precise, targeted integration of a transgene of interest.

Disclosures

Conway: Sangamo Biosciences: Employment. Paschon:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment. Cost:Sangamo Biosciences: Employment.
A T2* MRI Prospective Survey on Cardiac and Hepatic Iron in Non-Trasfusion-Dependent Thalassemia Intermedia Patients Treated with Desferrioxamine
Authors: Meloni Antonella , Maggio Aurelio , Cosmi Carlo , D'Ambrosio Alfonso , Facchini Elena , Chiari Roberta , Sardella Leonardo , Grassedonio Emanuele , Valeri Gianluca , Neri Maria Giovanna , Positano Vincenzo , Pepe Alessia
Published: October 2019

Background. In thalassemia intermedia (TI) patients no observational study prospectively evaluated in the real life the efficacy of the desferrioxamine (DFO) therapy in removing or preventing iron overload from the heart and the liver by T2* Magnetic Resonance Imaging (MRI). The efficacy endpoint of this study is represented by the changes in cardiac T2* and MRI LIC (liver iron concentration) values in non-transfusion dependent (NTD) TI patients after 18 months of desferrioxamine therapy.

Methods. Among the 325 TI patients enrolled in the MIOT (Myocardial Iron Overload in Thalassemia) network, we selected 129 TI patients NTD. We considered 29 patients who had been received DFO alone between the two MRI scans. Cardiac iron overload was assessed by the T2* multiecho technique. Hepatic T2* values were converted into liver iron concentration (LIC) values.

Results. Mean age was 39.69 ± 8.12 years and 14 (48.3%) patients were females. Patients started regular chelation therapy at a mean age of 21.92 ± 15.89 years. The mean administered dosage of DFO via subcutaneous route was 38.46 ± 10.27 mg/kg body weight on 3.32 ± 1.54 days/week. The percentage of patients with excellent/good levels of compliance to the chelation treatment was 82.1%.

At baseline only one patient showed cardiac iron overload (global heart T2*=15.23 ms) but he recovered at the FU (global heart T2*=26.93 ms). All patients without cardiac iron maintained the same status at the follow-up (FU).

Eighteen patients (62.1%) had hepatic iron overload (MRI LIC ≥3 mg/g/dw) at the baseline.

For this subgroup, the baseline and the FU LIC values were, respectively, 9.15 ± 7.97 mg/g/dw and 7.41 ± 6.28 mg/g/dw. The reduction in MRI LIC values was not significant (P=0.102). Out of the 11 patients with a baseline MRI LIC <3 mg/g/dw, only one (9.1%) showed hepatic iron at the FU. The Figure shows the evolution of different hepatic iron overload risk classes between the baseline and the FU.

Conclusions. In this small population of sporadically or non transfused TI patients, DFO showed 100% efficacy in maintaining a normal global heart T2* value. As regards as the hepatic iron overload, the DFO therapy did not prevent the transition to a worst class in 2 patients.

Figure 1 Figure 1.

Disclosures

Pepe: Chiesi: Speakers Bureau; ApoPharma Inc.: Speakers Bureau; Novartis: Speakers Bureau.
Lack of Cardiac Iron in SCD Patients Despite Severe Iron Overload
Authors: Ahmad Rasha I , Keyrouz Sara , Arabi Mariam , Bitar Fadi , al Jaroudi Wael , Abboud Miguel
Published: October 2019

In sickle cell disease (SCD), transfusions improve blood flow by reducing the proportion of red cells capable of forming sickle hemoglobin polymer. The major and unavoidable complication of transfusions in SCD is iron overload. Patients with significant transfusion load and iron siderosis, often have iron deposit into multi organs, including liver, pancreas and heart. However, patients with SCD may be relatively protected from iron mediated cardiac toxicity as compared to patients with thalassemia and similar transfusion load. Nonetheless while patients with SCD have less iron deposition in the heart it is presumed that severe loading will eventually lead to cardiac involvement. We report here two patients with scd with severe iron overload who had no cardiac involvement.

These are an 18 year old female and her 20 year old male sibling, known to have SCD (HbSS) with significant blood transfusions history, once a month or every two weeks for 12 years duration. Both had undergone splenectomy, and multiple hospitalizations for pain crises and acute chest syndrome. They had received intermittent iron chelation therapy for only 2 years. Examination revealed marked hepatomegaly and highly elevated ferritin levels, 11964ng/ml and 7098ng/ml respectively, suggestive of iron overload. Both patients had unremarkable electrocardiogram and echocardiogram. SGPT and SGOT are normal. Both patients are below the 5thpercentile for height, and l the 18 year old girl is pre-pubertal, has growth hormone deficiency and is on treatment.

Cardiac, liver and pancreas magnetic resonance imaging with T2* showed normal cardiac structure and function without siderosis ( heart T2*32 ms and 35 ms in the female and male siblings, respectively [normal >20 ms]). There was however considerable liver siderosis with estimated liver iron content 16.6±4.4 mg/g dry weight (female sibling) and 13.9±1.8 mg/g dry weight (male sibling). The corresponding R2* were 646±165 HZ and 541±62 Hz, respectively. Also, there was mild to moderate pancreatic siderosis in the 18 year old female (R2* 111 HZ) and mild pancreatic siderosis in her sibling (R2* 55 Hz)

These two cases while consistent with what has been reported are unusual because of the degree of iron ovreloead. It is an unexepected finding that despite such extensive iron deposits in the liver, there is no evidence of cardiac iron. It thus seems that in patients with sickle cell disease, even with extensive liver siderosis and endocrine dysfunction there is sparing of the heart. Collecting and studying such cases may shed light on the mecahins of cardiac protection from iron overload in sickle cell disease.

Disclosures

No relevant conflicts of interest to declare.
High Concentration of Immunoglobulin Allows Long-Lasting Suppression of Allo-Immune Reaction By Directly Inhibiting T-Cell Activation
Authors: Maeba Hideaki , Kuroda Rie , Fukuda Masaki , Fujiki Toshihiro , Mase Shintaro , Araki Raita , Ikawa Yasuhiro , Koizumi Shoichi , Yachie Akihiro , Nishimura Ryosei
Published: October 2019

Intravenous immunoglobulin (IVIg) has been used after allogeneic stem cell transplants (HSCT) mainly to prevent infections. In addition, some studies have also demonstrated that IVIg reduced GVHD and GVHD-related mortality, but others failed to show these effects. To answer whether IVIg could reduce GVHD and/or has preventive effects, as a first step, we compared the mixed lymphocyte reactions (MLRs) between a variety of responder and stimulator combinations (Balb/c and C57BL/6) with/without immunoglobulin (Ig). We showed that the MLR was strongly inhibited when high concentration of Ig was added to the cocluture (Stimulator (S): bone marrow-derived dendritic cells (BM-DCs) obtained from C57BL/6 using GM-CSF, Responder (R): splenocytes obtained from Balb/c). Interestingly, dose dependent inhibition was not observed only when high concentration of Ig could suppresses allo-reaction. The next concern was whether Ig could suppresse MLR via modulation of DCs or T-cell directly, or both. To test this, splenocytes were stimulated with phytohemagglutinin (PHA) in the presence of various Ig concentrations. We observed that Ig suppressed PHA-induced T-cell proliferation in a dose dependent manner. To eliminate the possibility that Ig might have direct cytotoxicity against lymphocytes and consequently allo-immune reaction seemed to be inhibited, we performed dead cell analysis by flowcytometry using 7-AAD and annexin V on T-cells in the various concentrations of Ig. Even in high concentration of Ig, the frequency of apoptotic or dead cells were observed as same levels as in low concentration or absence of Ig, suggesting that Ig inhibited T-lymphocyte activation directly. Next concern was when Ig allowed tolerogenic function to T-cells and/or DCs. That is, only pretreatment of Ig before MLR was enough to suppress allo-reaction or persistent high concentration of Ig exposure was needed during MLR. To test this, splenocytes and BM-DCs were cultured with Ig for 24 hours before MLRs, and then washed twice and we compared the MLRs. Ig pretreatment to either splenocytes or BM-DCs or both did not show any suppression of MLRs as shown in the figure below, indicating that persistent high levels of Ig during interaction were needed to induce maximum inhibiting effects. In conclusion, we have clearly demonstrated that Ig has a strong capacity to suppress allo-immune reaction. Repetitive high dose IVIg treatment would be a promising approach to reduce or prevent sever acute GVHD, only if serum Ig concentration could be kept in high level. In addition, to apply this in clinical settings, disadvantage of high dose IVIg should be further investigated.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Gorham's Disease: A Case of Vanishing Ribs and Its Effective Treatment with Sirolimus
Authors: Hall Nathan Scott , Carpenter Shannon L.
Published: October 2019

Introduction: Gorham’s disease, also known as Gorham-Stout syndrome or the vanishing bone disease, is a rare clinical entity characterized by a non-neoplastic proliferation of vascular and/or lymphatic vasculature with resultant osteolysis of adjacent bone1.With just over 200 reported cases, the etiology of Gorham’s disease is no better understood than when originally described by Dr. Jackson in 18382. Due to the rarity of Gorham’s disease, no standard treatment algorithm exists. Many therapeutic options have been proposed, however, the efficacy and success of these options is limited due to the lack of randomized controlled trials.

Case Presentation: Our patient is a 10 year old Caucasian female with past medical history significant for abnormal CXR (8months prior) showing absence of right sided 9th rib, who presented with right lateral abdominal pain, swelling and erythema. She denied fever, shortness of breath, malaise, decreased appetite, weight loss, recent travel, TB exposure or recent trauma. Laboratory work showed a normal CBC, BMP, LFTs, lipase, PT/PTT, and urinalysis. CT scan of the abdomen showed a right side pleural effusion as well as edema and fluid in the right anterior abdominal wall and subcutaneous fat. A chest tube was placed and 1.5 liters of fluid drained. An infectious and oncologic workup was initiated. Chest tube output was shown to be chylous and rib radiographs demonstrated absence of the complete right ninth rib, partial absence of the right eighth and tenth ribs. A rib biopsy was performed and showed of lymphatic proliferation with erosion/effacement of surrounding bone with no increased or abnormal osteoclastic activity, and no evidence of infectious or malignant etiology. This confirmed the previously presumed diagnosis of Gorham’s Disease.

Management and Outcome: Our patient was initially treated with bisphosphonate and interferon alpha 2b with minimal success. It was not until initiation of treatment and achieving a steady state of sirolimus that her disease was able to be controlled. She is now over two year in remission and continues on sirolimus therapy.

Disccussion:The rare diagnosis of Gorham disease unveils the difficulty in treating vascular and lymphatic malformations that medical providers encounter. The successful use of sirolimus in our patient adds another potentially useful medication in the treatment of vascular malformations. There are clinical trials currently being conducted to evaluate the safety and efficacy of sirolimus use in complex vascular malformations. Franchi, A, Bertoni, F, Bacchini, P, Mourmouras, V, & Miracco, C. CD105/endoglin expression in Gorham disease of bone. J Clin Pathol. 2009;62:163–167.Jackson JBS. A boneless arm. Boston Med Surg J. 1838;18:368-369.

Disclosures

No relevant conflicts of interest to declare.
the Diagnostic Value of Plasma Level, Activity, Ratios between von Willebrand Factor and ADAMTS13 in Patients with Cerebral Infarction
Authors: Qu Le , Ruan Changgeng , Zhao Yiming
Published: October 2019

Introduction: Large population studies have revealed that higher levels of von Willebrand Factor (VWF) and lower levels of ADAMTS13 activity (ADAMTS13) are associated with a risk of thrombosis. In this study, we aimed to investigate the relationship among the plasma levels of VWF, ADAMTS13 and the occurrence of cerebral infarction in patients.

Methods: In a case-control study of 94 cerebral infarction patients (CIP) and 103 age-matched healthy controls (Control), the plasma levels of VWF antigen (VWF:Ag ), VWF ristocetin cofactor activity (VWF:Rcof), and VWF collagen binding activity (VWF:CB) were measured using ELISA. ADAMTS13 activity (ADAMTS13) was measured with FREST-VWF73. The study was approved by the institutional Ethics Committee at the First Affiliated Hospital of Soochow University, China. All participants were given write informed consents.

Results: The levels of VWF:Ag and VWF:Rcof in cerebral infraction patients (mean±SEM, 223±15% and 256±20%) were significantly higher compared with controls (117±8% and 128±9%). Levels of ADAMTS13 and ratios of VWF:CB/VWF:Ag, ADAMTS13/VWF:Ag and ADAMTS13/VWF:Rcof in patients (107±4%, 0.73±0.08, 0.76±0.07 and 0.86±0.10, respectively) were significantly lower compared with controls (125±3%, 1.37±0.08, 1.69±0.14 and 1.45±0.10, P<0.001, respectively). However, there was no difference between cerebral infarction patients and controls in the levels of VWF:CB, VWF:Rcof/VWF:Ag and ADAMTS13/VWF:CB (P>0.05). Unconditional logistic regression analysis demonstrated the highest quartile of VWF:Ag (odds ratio [OR] = 5.11, 95% confidence interval [CI], 1.49-17.50), VWF:Rcof (OR = 5.04, 95% CI, 1.62-15.66), and the lowest quartile of VWF:CB/VWF:Ag (OR = 5.91, 95% CI, 1.95-17.93), ADAMTS13/VWF:Ag (OR = 9.11, 95% CI, 2.49-33.33), ADAMTS13/VWF:Rcof (OR = 3.73, 95% CI, 1.39-10.03) were associated with cerebral infarction (P<0.01). The receiver operating characteristic (ROC) curves analysis demonstrated the cutoff values of VWF:Ag (137.88%), VWF:Rcof (135.88%) and VWF:CB/VWF:Ag (0.538), ADAMTS13/VWF:Ag (0.974), ADAMTS13/VWF:Rcof (0.946) showed a better diagnostic value of cerebral infarction (AUC > 0.7).

Conclusions: High levels of VWF:Ag, VWF:Rcof and lower levels of VWF:CB/VWF:Ag, ADAMTS13/VWF:Ag, ADAMTS13/VWF:Rcof are relative risk factors. The threshold of VWF:Ag ( > 137.88%), VWF:Rcof ( > 135.88%), VWF:CB/VWF:Ag ( ≤ 0.538), ADAMTS13/VWF:Ag ( ≤ 0.974), ADAMTS13/VWF:Rcof ( ≤ 0.946) can be diagnostic indicators of cerebral infarction.

Research funding: This work was supported by grants from National Scientific Foundation of China (NSFC) (81270593), Jiangsu Provincial Special Program of Medical Science (BL2012005) and Jiangsu Province’s Key Medical Center (ZX201102), China

Disclosures

No relevant conflicts of interest to declare.
Recombinant Human Thrombopoietin: a Novel Therapeutic Option for Patients with Immune Thrombocytopenia in Pregnancy
Authors: Kong Zhangyuan , Qin Ping , Wang Xin , Hou Ming
Published: October 2019

BACKGROUND:

Thrombopoietin (TPO), the major physiological regulator of platelet formation, binds to and activates TPO receptor on megakaryocytes, thereby promoting platelet production. Recombinant human thrombopoietin (rhTPO) is a novel therapeutic option for patients with immune thrombocytopenia (ITP) in China. However, the safety and efficacy of rhTPO for ITP patients in pregnancy is unclear. We report two ITP patients in pregnancy successfully treated with rhTPO.

METHODS:

Patient one, who was diagnosed as ITP according to the ASH guidelines two years ago, was a 28-year-old G2P1 pregnant woman, with a platelet count of 10×109/L and gingival bleeding. Before rhTPO therapy her TPO level was 231pg/mL. Patient two, who was diagnosed as ITP one year ago, was a 23-year-old primigravid woman with gross hematuria at 22-week gestation. Her platelet count was 19×109/L and the level of TPO in her plasma was 107pg/mL before rhTPO admission. Both patients failed to respond to prednisone (1mg/kg, 14d) and intravenous immunoglobulin (IVIG, 0.4g/kg, 5d). Due to persistent hemorrhage, they received rhTPO at a daily dose of 300U/kg daily (3SBIO Pharmaceutical Co., LTD, Shenyang, China). When their platelet counts rose above 50×109/L, maintenance therapy (rhTPO 300U/kg, every other day) started. This treatment protocol was approved by the Medical Ethical Committee of Qilu Hospital, Shandong University.

RESULTS:

The platelet count of patient one rose to 165×109/L after 10 days of rhTPO therapy. Then rhTPO maintenance therapy was applied. On the 39th week, she vaginally delivered a female infant with normal platelet count. The TPO level of the newborn was 127pg/mL. In the second case, after seven days of rhTPO therapy, her platelet count rose to 58×109/L, then we started rhTPO maintenance therapy. On the 40th week, she delivered a female infant without any signs of neonatal ITP. The level of TPO in the infant's plasma was 174pg/mL. Maintenance therapy continued after delivery with a dose of 300U/kg per week. The platelet counts of patients during treatment were illustrated in Figure 1. At a 1-month follow-up visit, no adverse events were observed in both infants.

CONCLUSION:

RhTPO may be an effective and fast-onset treatment for ITP in pregnancy. Future studies are necessary to investigate the safety and efficacy of rhTPO in this clinical setting.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Analysis of the Quality of Life in Children and Their Parents with Immune Thrombocytopenia in China
Authors: Zhou Min , Li Xiao-jing , Shu Hui-ying , Meng Fan-yi , Yang Mo
Published: October 2019

In this study, we assessed the health-related quality of life (QoL) in children with immune thrombocytopenia (ITP) and their parents in China using a disease-specific QoL measure, the Kids' ITP Tools (KIT). Forty-three Children less than 18 years of age with newly diagnosed ITP and their parents were recruited to complete the KIT study. The children’s version of the KIT consisted of 26 items which divided into 5 domains: treatment side effects, intervention-related, disease-related, activity-related, and family-related concerns. Parental version of KIT consisted of 26 items which divided into 6 domains: diagnosis-related, monitoring-related, child’s restricted activity-related, daily life-related, disease outcome, and emotional impact. Each item was rated on a 6-point Likert scale from 1 (not at all) to 6 (a great deal). A high score represented a high concern level. Scores for the individual domains were summed to yield a total KIT score. The reliability of KIT was found to be high in assessing QoL of children with ITP and their parents in China (children’s version: Cronbach’s a=0.933, parents’ version: Cronbach’s a=0.905). Parent KIT scores was significantly higher than child KIT scores, (31.46±14.58）vs（79.05±14.99）Z=7.625，P=0.000. Which suggested that QoL of parents was significantly lower than children’s. Among the children KIT, the highest mean score was noted in the “intervention-related” (1.92±1.30) and “activity related (1.82±1.47)”. Among the parents KIT, the highest mean score was noted in the “emotional impact（4.88±0.97)” and “disease outcome (4.78±1.01)”; (3) The KIT scores for different age groups of children with ITP were significantly different which showed that the older the children were, they cared for more and worried more about the disease. But the KIT scores for parents were no different in different age groups, which suggested that whatever their children are old or young, Chinese parents worried about ITP disease in the same degree. The QoL of children with ITP and their parents were remarkably lower in Chinese, especially for parents. The cross-culturally translated KIT was a valid and reliable disease-specific measure of health-related quality of life for children with ITP. KIT could be used as an tool in clinical trials and management of childhood ITP.

Disclosures

No relevant conflicts of interest to declare.
Complications of Central Venous Therapy in Children with Hemophilia in Poland
Authors: Laguna Pawel , Klukowska Anna , Pawelec Katarzyna , Matysiak Michal
Published: October 2019

Introduction

Central venous catheters (CVC) are essential in the management of children with hemophilia who need frequent infusions. Peripheral access is usually sufficient but the use of a central venous line is necessary in many cases.

Methods

CVCs have been used in our department since 1997. During this time 75 CVCs (port-a-cath) have been inserted in 54 patients aged 3 months to 18 years. 52 patients had hemophilia A and 2 had hemophilia B.

21 out of 52 had hemophilia A complicated by high-titre inhibitors. CVCs were inserted 3 times in 2 patients and twice in 11 patients. In the remaining patients 47 they were inserted once. All the ports were inserted on the left side of the body and replacements were inserted on the right side. CVCs were used for ITI in 10 patients.

Results

The period of vascuport use ranged from 2 to 103 months CVL injections were observed 17 times in 13 children. All infected ports were successfully treated with antibiotics. Trombotic complications occurred in 8 boys 11 times. In 2 cases the ends of the CVCs got torn and moved to the heart, while in 2 other cases the reservoir stated leaking.

Conclusion

Our investigations show that CVLs are convenient in use but complications can occur.

Disclosures

No relevant conflicts of interest to declare.
The Effect of Tanshinone-IIa on IL-1β Induced-Thrombocytosis in an Immune Vasculitis Model
Authors: Zhou Min , Zhou Li-xia , Shu Hui-ying , Li Xiao-jing , Lian Qi-zhou , Meng Fan-yi , Yang Mo
Published: October 2019

Inflammation cytokines may be involved in the pathogenesis of thrombocytosis with vasculitis. Our previous study showed that inflammation cytokine IL-1β plays an important role on in-vitro megakaryopoiesis (Yang M et al, Br J Haematol 2000). The role of IL-1β and Tanshinone IIA (TIIA) (Isolated from Danshen, Radix Salviae Miltiorrhiza Bge) on platelets and megakaryocytes (MKs) in immune vasculitis model was investigated in this study. Rabbit model with immune vasculitis was established by injection (iv) of BSA. After treatment with BSA for 7 days, the platelet count, platelet aggregation and the expression of AnnexinⅤ were significantly increased in this vasculitis model group compared with normal control group (n=7). IL-1β levels was also significantly higher in vasculitis model. There were positive correlations between platelet count and IL-1β levels (R=0.65), platelet aggregation and IL-1β levels (R=0.60). Treatment with TIIA (5 mg/kg/day, iv) and aspirin significantly decreased all these parameters. MKs and CFU-MK number were also significantly increased in vasculitis group as compared to normal group. Treatment with TIIA and aspirin significantly reduced the number of MKs and CFU-MK in this model. Study further demonstrated that IL-1β alone or in combination with TPO induced in-vitro CFU-MK formation. The mRNA of of IL-1 type I and type II receptors (IL-1 RI and RII) were detected in cultured MK (CD61+ CD41+) cells. The expression of IL-1 RI and RII was also confirmed by immunofluorescence staining in bone marrow MKs. Moreover, the IL-1R bloker can reduced IL-1β induced megakaryopoiesis. TIIA on in-vitro megakaryopoiesis was also investigated. TIIA at 10-30 ug/ml significantly inhibited CFU-MK formation. TIIA also induced the apoptosis of MKs in a dose dependent manner by Anexin V assay. Caspase 3 assay showed the activation of Caspase 3 increased from 5% to 16%. Using JC-1 assay found that the depolarized cells increased from 9% to 17% suggesting the involvement of intrinsic apoptotic pathway. IL-1β may play an important role in the thrombocytosis of immune vasculitis by inducing megakaryopoiesis. TIIA has anti-platelet effect in this model which may be mediated via inhibiting IL-1β induced-megakaryopoiesis.

Disclosures

No relevant conflicts of interest to declare.
Improving the Appropriate Use of Intravenous Immunoglobulin in the Saskatoon Health Region
Authors: Manna Mita , Karim Safiya , Dallas Karen L , Brose Kelsey
Published: October 2019

Intravenous Immunogloblulin (IVIG) is a plasma protein used in the treatment of various diseases. The cost of IVIG is approximately $50-$80 per gram. In 2005, Saskatchewan's usage of IVIG was above the national average. There are several published guidelines that outline recommended uses of IVIG. These are largely based on case studies and expert opinion. Despite these guidelines, IVIG is often used in multiple settings where there is no supporting evidence.

In 2013, we conducted a retrospective study on IVIG usage and found that the Saskatoon Health Region (SHR) adhered poorly to published guidelines. Following this, we aimed to determine IVIG usage in the Hematology and Neurology departments after implementing education sessions and order forms. We conducted six education sessions from December 2012 to January 2014 in the SHR. We also circulated IVIG order forms outlining appropriate indications for IVIG on the neurology and heme-oncology ward from January 1, 2014 to March 31, 2014. We then conducted a retrospective chart review of patients who received IVIG from January 1, 2014 to March 31, 2014 and compared the usage to patients who received IVIG in 2011 and 2012. Our primary endpoint was any improvement in the appropriate usage. Our secondary endpoint was adverse reactions seen in patients receiving IVIG. Statistical analysis was conducted with the use of two sample z-test for proportions.

Our results (Figure 1) showed that for hematological conditions, the proportion of patients treated appropriately increased from 0.61 in 2011 to 0.85 in the first three months of 2014 (P value = 0.05). For neurological conditions, the proportion of patients treated appropriately declined from 0.85 in the first three months of 2012 to 0.72 in the first three months of 2014 (P value = 0.52). No adverse reactions were reported.

In conclusion, our results show that after our interventions, there was significant improvement in the appropriate use of IVIG for hematologicalconditions compared to 2011. However, there was a decrease in appropriate use of IVIG for neurological conditions. Further analysis and larger sample size is required to determine the overall significance of our interventions.

Figure 1. IVIG use in Hematology and Neurology after interventions implemented Figure 1. IVIG use in Hematology and Neurology after interventions implemented

Disclosures

No relevant conflicts of interest to declare.
Age-Related Alterations in Human Hematopoietic Stem and Progenitor Cells
Authors: Rundberg Nilsson Alexandra , Bryder David , Pronk Cornelis J.H.
Published: October 2019

Aging of the human hematopoietic system is typically associated with various disease states and functional changes, such as decreased bone marrow (BM) cellularity, reduced adaptive immune responses and increased incidence of anemia. Similar observations are made in mice and have been shown to, at least in part, have their origin already at the hematopoietic stem cell (HSC) level. Current ideas on age-related alterations of the hematopoietic system are to a large extent based on studies of the murine system, as studies on the human system are few and to some extent contradictory. We further detailed human hematopoietic aging by comparing adult BM samples from young and old individuals, as well as samples obtained from cord blood (CB). We observed similar changes to those observed in mice, including a functional decline in the proliferative and cloning potential of HSCs and impaired lymphoid cell production with age. We revealed alterations in the frequencies of immunophenotypically defined HSCs and progenitor cells of the megakaryocytic/erythroid, granulocyte/macrophage and lymphoid lineages within the CD34+ population, consistent with the observed HSC impairment and decreased lymphopoiesis. Transcriptome profiling demonstrated a myelo-meg-erythroid bias and downregulation of genes associated with lymphoid specification in the adult aged HSC compartment compared to the young. Further, single cell gene expression analysis of 45 individual genes, including lineage affiliated genes and a panel of transcription factors, in a large number of HSCs isolated from young and old BM and CB revealed heterogeneity within the HSC compartment as well as a pattern of transcription fluctuation for a set of genes across age boundaries. Collectively, we demonstrate age-related alterations in the human hematopoietic system to have, in least in part, their origin already in the immature HSC level and find a number of genes differentially expressed within the HSCs compartment and with age.

Disclosures

No relevant conflicts of interest to declare.
From the Bone Marrow to the Thymic Niche
Authors: Susini Sandrine , Mouraud Séverine , Elkaim Elodie , Roullier Julien , Luce Sonia , Pellé Olivier , Bruneau Julie , Cavazzana Marina , Andre-Schmutz Isabelle
Published: October 2019

To generate T cells throughout adult life, the thymus must import hematopoietic progenitor cells from the bone marrow via the blood. The cellular and molecular mechanisms governing the circulation of thymus-seeding progenitor cells are well characterized in mice but not in humans. The aim of the present study was to characterize the molecular mechanisms and cellular components involved in thymus colonization by lymphoid progenitors (CD34+/CD10+/CD7-/CD24-) and the early steps of thymopoiesis under physiological conditions in humans. Our results demonstrate that circulating lymphoid progenitor cells express CCR9 and CXCR4 chemokine receptors, VLA-4, VLA-5 and VLA-6 integrins and PSGL-1 and CD44 adhesion molecules. We used in vitro migration and adhesion assays to validate the functional status of these markers.

As in the mouse, human circulating progenitor cells enter the thymus at the corticomedullary junction (CMJ). Once in the thymus, crosstalk with thymic epithelial cells causes the circulating progenitors to commit to the T-cell differentiation pathway. In order to characterize thymic niches and interactions between circulating progenitors and the thymic stroma, we undertook a chemokine/chemokine-receptor-focused gene expression analysis of sorted lymphoid progenitor cells and CMJ epithelial cells (based on the expression of EpCAM and Delta-like-4). We observed an unexpected gene expression profile for chemokines and chemokine regulators in thymus-seeding CD34+/CD10+/CD7-/CD24- cells and epithelial cells at the CMJ. The present results should help us to highlight candidate genes involved in the early steps of human thymopoiesis.

Disclosures

No relevant conflicts of interest to declare.
HDAC1 Can Deacetylate GATA-1 and Regulates Its Activity through a FOG-1 Independent Manner
Authors: Yan Bowen , Yang Tao , Huang Suming , Qiu Yi
Published: October 2019

The transcription factor GATA-1 is essential for erythroid and megakaryocytic cell differentiation and maturation. It has been well documented that GATA-1 can indirectly interact with histone deacetylase 1 (HDAC1) containing NuRD corepressor complexes through an association with FOG-1. Our previous work also shows that HDAC1 acetylation modulates the activity of FOG-1 associated NuRD complexes. Earlier studies show GATA-1 can be acetylated by p300/CBP, and the acetylation modulates GATA-1 binding activity to chromatin. However, it is not clear whether the acetylation can be reversed by a deacetylase. In this study, we found that GATA-1 can directly interact with HDAC1 in a FOG-1 independent manner. The interaction results in the deacetylation of GATA-1. We have identified two arginine sites within GATA-1 that are important for its interaction with HDAC1. The arginine to alanine mutation on these sites (2RA) can largely decrease the interaction of these two proteins, but doesn't affect its interaction with FOG-1, indicating that the direct interactions with HDAC1 and FOG-1 dependent association of NuRD complexes are separate events. The mutations also do not affect GATA-1 DNA binding activity in vitro. To further investigate the function of this interaction in erythropoiesis, we introduced wild type or the 2RA mutant of GATA-1 fused with estrogen receptor ligand binding domains into G1E cells, a GATA-1-null erythroid progenitor cell line. Upon estradiol induction, GATA-1 2RA inhibits the differentiation in G1E cells. We performed RNA sequencing to study the effect of GATA-1 2RA in gene expression in comparison with wild type GATA-1 and GATA-1-V205M (a mutation abolished binding with FOG-1) on a genome wild scale. GATA-1 2RA affects GATA-1 function in both GATA-1 activated and repressed genes. Although there is some overlap between 2RA and V205M mutations in both activated and repressed genes, more genes affected by these two mutations are different. Thus, this study unveils a novel regulation for GATA-1 by its direct interaction with HDAC1 during hematopoiesis.

Disclosures

No relevant conflicts of interest to declare.
Exosomes Secreted By Multiple Myeloma Cell Lines (MMCLs) and Bone Marrow Stromal Cells (BMSCs) Reciprocally Modulate Cell Adhesion and Impact on MM Cell Migration and Bortezomib-Induced Apoptosis
Authors: Udi Josefina , Voll Reinhard , Ureta Daniela , López Margarita , Wäsch Ralph , Álvarez Élida , Engelhardt Monika
Published: October 2019

Introduction: In MM pathogenesis, the interaction of plasma cells and the bone marrow microenvironment plays a crucial role. Exosomes are secreted by nearly all cell types, including cancer cells, and have been implicated in intercellular communication, drug resistance and tumor progression. We therefore purified, characterized and studied the influence of exosomes secreted by MMCLs and BMSCs on cell adhesion, bortezomib-induced apoptosis and cell migration.

Methods: Exosomes were purified from the supernatants of MMCLs (IM-9, MM.1S, U266, RPMI8226) and M2-10B4 BMSCs after 72h of culture with RPMI1640+10% exosome-depleted FBS by differential centrifugation. Purity and quality of exosomes was confirmed by electron microscopy and western blot for CD63. Exosomal protein concentration per 5x106 cells was quantified by Bradford. CD44 and ICAM1 were evaluated by flow cytometry. Apoptosis was assessed via annexin V/7-AAD-staining. Chemotaxis to M2-10B4 exosomes (100-500μg/ml) was studied using 24-well chemotaxis chambers. Migrated cells were counted by flow cytometry.

Results: Exosomes from IM-9 cells were identified by electron microscopy, thereby showing a characteristic saucer-like morphology, ranging in diameter between 30 and 120nm. Both MM cell and M2-10B4 exosomes expressed CD63. Mean exosomal protein concentration was 1.4μg/μl for M2-10B4 and varied for MMCLs between 1.2 and 1.7μg/μl. M2-10B4 cells expressed higher levels of both adhesion molecules CD44 and ICAM-1 when incubated with exosomes from MMCLs compared to basal conditions, however, not reaching statistical significance. Conversely, M2-10B4 exosomes induced a significant increase in CD44 expression on IM-9 cells (p=0.03), reflecting an increased adhesion to components of the extracellular matrix that might contribute to cell-adhesion mediated drug resistance. IM-9 apoptotic cells after 24h with 10nM bortezomib decreased from 72% to 61% in the presence of M2-10B4 exosomes and from 74% to 62% with IM-9 exosomes, suggesting a lower sensitivity to bortezomib. Migration of IM-9 cells slightly increased with 200μg/ml M2-10B4 exosomes and decreased with higher exosome concentrations.

Conclusions: Our findings underline the role of M2-10B4 exosomes on adhesion and migration of one representative MMCL (IM-9), and the impact of M2-10B4 and IM-9 exosomes on bortezomib cytotoxicity in this MMCL, suggesting their involvement in a paracrine/autocrine loop modulating treatment response and MM cell survival.

Disclosures

No relevant conflicts of interest to declare.
Preliminary Study on the Abnormal DNA Methylation in T Cells from the Patients with Immune Related Pancytopenia
Authors: Shao Zonghong , Ren Yue , Fu Rong
Published: October 2019

Objective To explore the global DNA methylation and the expression of regulatory genes for methylation in CD4 + T cells of the patients with immune related pancytopenia (IRP) and explore the role of methylation in pathogenesis of IRP.

Methods Thirty IRP patients (untreated, n=15; remission, n=15) and 15 healthy donors as controls were enrolled from December 2012 to December 2013. CD4+ T cells were sorted by immunomagnetic separation. The global DNA methylation was tested with enzyme-linked immunosorbent assay (ELISA). The mRNA levels of DNA methylation-related regulating genes, DNA methyltransferases (DNMTs) and methylated CpG binding proteins (MBDs), were measured by real-time quantitative polymerase chain reaction (RT-PCR).

Results The level of global DNA methylation in peripheral blood CD4+ T cells of untreated IRP patients (3.525%±2.046%)and remission patients (4.790%±1.471%) were significantly lower than that of normal controls (10.101%±3.449%) respectively (both P<0.05). DNMT3b mRNA level of untreated IRP patients (1.332±0.785) was significantly lower than that of normal controls (2.077±1.059) in CD4+T cells (P<0.05). The mRNA expression of MBD2 was significantly higher in CD4+ T cells from untreated and remission IRP patients (2.999±1.601, 2.055±1.576) than that in controls (0.581±0.247) (both P<0.05). The MBD4 mRNA level was significantly higher in CD4+ T cells from untreated IRP patients (2.736±2.719) compared to that in normal controls (1.167±1.006) (p<0.05). DNMT3b mRNA expression and CD4+ T cell DNA methylation to be positive correlated within IRP patients (r=0.569, p<0.01). The MBD2 mRNA expression negatively correlated with CD4+ T cell DNA methylation and the ratio of Th1/Th2 (r=-0.763, p<0.001; r = -0.652, p<0.05). The global methylation of CD4+ T cells negatively related to the ratio of CD5+ B cells (r= -0.439, p<0.05).

Conclusions The globe DNA hypomethylation and abnormal expression of DNA methylation-related enzymes in peripheral blood CD4+ T cells may be related with the pathogenesis of IRP.

Disclosures

No relevant conflicts of interest to declare.
Should We Hold the Point of allogeneic Hematopoietic Stem Cell Transplantation for Paroxysmal Nocturnal Haemoglobinuria in Eculizumab Era?
Authors: Chen Feng , Depei Wu , Sun Aining , Tang XiaoWen , Fu Chengcheng , Ma Xiao , Xue Shengli , Jin Zhengming
Published: October 2019

PNH is a rare haematological disorder due to the clonal expansion of abnormal haemotopoietic stem cells ,carrying a mutation in the PIG-A gene. Eculizumab ,as a humanised anti-complement component (C5), has proven highly effective in controlling intravascular haemolysis, and is expected to result in a remarkable improvement of survival. Should Eculizumab thus be offered as first treatment to all PNH patients? Allogeneic hematopoietic stem cell transplantation is still the only curative treatment for PNH by eradicating the abnormal PNH clone, leading to definitive survival improvement and QOL benefits.

Methods We assessed the long-term clinical and hematologic results in 13 PNH patients who received Allogeneic hematopoietic stem cell transplantation in our centre between Jan 2010 and Apr 2014. The patients were aged 13 to 54 (median 24 years). Eight donors were HLA -haploidentical and 5 were HLA-identical (3 siblings and 2 unrelated donors).

Results Eleven patients received a myeloablative conditioning consisting of busulfan and cyclophosphamide and 2 were given a reduced intensity conditioning consisting of fludarabine, busulfan and ALG (2 HLA-identical sibling donor). All patients achieved sustained engraftment with a median time of 11 days (range 11-26) to reach 0.5 ×109/L neutrophils and 15 days (range 12-126) to reach 20 ×109/L platelets. The overall cumulative incidence of grade II-IV acute GvHD was 15.4% and 2 patients showed limited chronic GvHD in evaluable 12 patients (cumulative incidence 16.7%).Transplant-related mortality for all patients was 15.4% (1 due to TMA and 1 due to severe pulmonary infection). As of Aug 1,2014, 11 patients are alive with complete hematologic recovery and no evidence of PNH following a median follow-up of 13 months. The 3-year Kaplan-Meier probability of disease-free survival was 80.2±12.8%. No thromboembolic event nor recurrence of the disease were reported following transplant.

Conclusions The findings of this study confirm that most patients with PNH may be definitively cured with allogeneic hematopoietic stem cell transplantation.

Disclosures

No relevant conflicts of interest to declare.
Defining the Cell Surface Landscape in B-Cell Malignancies: Comprehensive Profiling of Surface Antigens for Biomarker Discovery and Patient Stratification
Authors: O'Donnell Erika A , Alvendia Erica L , Wehrman Tom S , Krutzik Peter O
Published: October 2019

Flow cytometric analysis of surface marker expression is a fundamental component of diagnosis, classification, and monitoring for many hematologic malignancies. However, most clinical implementations of surface immunophenotyping focus on only a handful of markers. A comprehensive survey of CD markers and cell-surface receptors could provide a higher resolution and more accurate characterization of cancer lineages, as well as enabling new insights into the cellular changes resulting from disease progression or therapeutic intervention. To this end, we collated and evaluated antibodies to over 400 known markers and receptors. To establish the baseline and variability of each marker in major B cell subsets within normal human blood, a protocol was established to co-stain each sample for CD19, CD20, CD27, IgD, CD38, and CD24 simultaneously with each of the 400 markers. Antigen abundance and expression frequency were evaluated to establish a surface marker profile for each B-cell subset and to establish the variation inherent in each marker across the normal human population. To validate the application of this panel for cancer immunophenotyping, 20 malignant B-cell lines were selected for comprehensive surface characterization. Prior to staining, each cell line was barcoded with a combination of fluorescent dyes to generate a unique fluorescent signature. This approach allowed multiple cell lines to be combined into a single well for staining and analysis, enabling the rapid evaluation of hundreds of surface antigens using only 1-2 million cells from each line. Hierarchical clustering of data from cancer lines and normal B cell subsets allowed the identification of markers useful for discriminating between B-cell cancer subtypes, as well as markers that differed between normal and malignant B cells. These studies have resulted in the optimization of a protocol and antibody panel for use in profiling surface protein expression on human blood cells and cell lines. Application of this procedure to clinical samples may provide information useful for biomarker discovery, improved stratification of hematologic malignancies, and the identification of new drug targets.

Disclosures

O'Donnell: Primity Bio: Employment. Alvendia:Primity Bio: Employment. Wehrman:Primity Bio: Employment. Krutzik:Primity Bio: Employment.