bluuebunny/crossref_metadata_2025 huggingface dataset:
Anatomical Compartments Modulate the Response of Human Hematopoietic Cells to Mitogenic Signaling, In Vivo.
Authors: Wang Linlin , Nagasawa Yasuo , Wood Brent , Lintmaer Ingrid , Guo Wenjin , Harkey Michael , Papayannopoulou Thalia , Nourigat Cynthia
Published: October 2019

Hematopoietic cells can respond differently to a mitogenic signal depending on whether the signal is delivered in vitro or in vivo. Results presented here suggest that the compartment in which a human hematopoietic progenitor resides can influence its response to a proliferative stimulus. An ectopically expressed conditional derivative of the thrombopoietin receptor (F36VMpl) in human cord blood CD34+ cells induces, upon provision of an agonist ligand, the differentiation (but not expansion) of transduced human burst forming units erythroid (BFUe) in culture. Here we show that upon transplantation into immune deficient mice, F36VMpl-expressing human BFUe and multipotential mixed progenitors (CFU-mix) decline in the marrows of ligand-exposed mice, replaced by a 12–17 fold expansion of differentiated erythroid precursors. In contrast, F36VMpl signaling dramatically expands BFUe and CFUmix in the spleen. Clonal analysis is consistent with the interpretation that F36Vmpl signaling induces an in situ expansion of BFUe and CFUmix in the spleen. In aggregate, these findings suggest that differences in environmental signals present in the marrow versus spleen can regulate the differentiation versus expansion of BFUe and CFU-mix.
Myeloablative Single Cord Transplants (SCT) Versus Dual Cord Transplants (DCT) in Adults with Acute Leukemia.
Authors: Creer Michael H. , Regan Donna , Alonso Mario , Wooford Jonathan
Published: October 2019

Background: Combining 2 cord blood products overcomes the total nucleated cell (TNC) dose limitation and may improve clinical outcomes following allogeneic cord blood (CB) transplantation in adults. We performed a retrospective case-control study in adult patients with acute leukemia receiving myeloablative conditioning and 1 or 2 cord blood units to identify potential causes for differences in clinical outcomes following SCT and DCT and product characteristics predictive of long-term engraftment in DCT recipients.

Study Design: To evaluate 20 DCT cases, we selected 20 SCT with the largest TNC dose as controls from a cohort of 103 adult SCT patients. SCT and DCT were matched for age, sex, diagnosis, disease status, ethnicity and body weight (0.11 ≤ p ≤ 0.78) excluding patients with graft failure or previous transplants.

Results: Administered TNC dose for DCT (3.7x10e7/kg) was nearly 2-fold greater than SCT (1.9x10e7/kg, p < 0.0001) with no significant differences in post-thaw TNC recovery (p=0.13). Time to ANC > 500/uL for DCT was 17 days compared to 20 days for SCT (p<0.02). For SCT, there were 9 and 11 pts. with 1 or 2 Ag mismatch (molecular) at HLA-A,B or DRB1, respectively, (mean = 1.6 Ag mismatch). Three-way comparison of both donors and recipient for DCT also gave mean = 1.6 Ag mismatch (molecular). Overall survival (OS) at 12 months was 18% for SCT and 72% for DCT (p<0.0001) with median duration of follow-up of > 18 mos. for both groups. OS was significantly correlated with post-thaw TNC dose for SCT (p<0.04) but not for DCT (p=0.88). In the first 100 days post-transplant, infection rates for SCT (65%) and DCT (50%) were similar with no significant differences in frequency of viral, fungal or bacterial infections (p=0.74). Most importantly, infection was the primary cause of death at 100 days for SCT (9/20 patients) whereas for DCT, there were no infection-related deaths! TNC and CD34+ cell dose did not differ between engrafting and non-engrafting units, however, we found that the engrafting units had a lower absolute lymphocyte count (ALC) in 8/10 evaluable cases.

Conclusion: DCT is an effective approach to overcome the TNC dose-limiting therapeutic benefit of CB transplantation. Although 100-day post-transplant infection frequency remains high, deaths from infection are markedly reduced and OS is significantly improved in DCT recipients, however, additional studies are needed to define the mechanism. For DCT, the engrafting unit does not have a higher TNC or CD34 cell dose but paradoxically; a lower ALC dose may be predictive.
Using the NOD/SCID Mice Model of Human Erythroleukemia for Evaluating the Cytotoxicity Activity of CB-CIK/NK Cells Stimulated by K562-DCs Fusion Vaccines.
Authors: Li Yang , Huang Shaoliang , Fang Jianpei , Zhang Xuchao , Wei Jing , Huang Wenge , Wu Yanfeng
Published: October 2019

CD3+CD56+ cytokine-induced killer (CIK) cells are prospective effectors for adoptive immunotherapy, CIK/NK cells incubated with K562-dendritic cells (DCs) fusion vaccines have more higher cytotoxicity activity. In this study, the efficacy and the safety of application of cord blood (CB) derived CIK/NK cells stimulated by K562-dendritic cells (DCs) fusion vaccines were evaluated in vivo by the NOD/SCID mice model of human erythroleukemia (K562 line, CD13+). DCs and CIK/NK cells were inducted by combination of cytokines from CB MNCs, DCs were fused with inactivated K562 tumor line by PEG (mw1500). 5 days before the harvest of CIK/NK cells, 1×105 K562-DCs fusion vaccines were co-cultured with 1×106 CB-CIK/NK cells to prepare for the K562-DCs fusion vaccines stimulated CIK/NK cells. NOD/SCID mice divided into six groups, eight in one group. Mice in A,B and C groups were inoculated with 1×106 K562 cells by tail vein. 24 hours later, 1×107 K562-DCs fusion vaccines stimulated CIK/NK cells and 1×107 unstimulated CIK/NK cells were transfued into the mice of group A and B, respectively. Group D and E were K562-DCs fusion vaccines stimulated CIK/NK cells and no-stimulated CIK/NK cells control, transfued by 1×107 stimulated CIK/NK cells and 1×107 no-stimulated CIK/NK cells, respectively. Group F is a normal control that no any inoculation were taken. None of the NOD/SCID mice in group C that inoculated with 1×106 K562 cells survived longer than 39 days, hepatosplenomegalic mass was seen in five mice. Death in group A and B were only one and two, respectively, at day 65 and day 56, 62. There was no tumour mass can be seen in group A and B, and the survial were more than 70 days. Both survival time of group A(69.38±1.77 days) and B(67.25±5.34 days) were longer than that of group C(30.38±4.57 days) significantly (P<0.01). The tumor marker (CD13) in periperal blood, live and lung of CIK/NK cells treated NOD/SCID mice (group A and B) significantly less than group C(P<0.01). There was no difference of CD56 positive in periperal blood of group A and B survival mice between that of the control (group D and E). These result indicated that K562-DCs fusion vaccines stimulating CB-CIK/NK cells have a potent anti-tumour activity in vivo and without any side-effect.
Reduced Toxicity Condition, Mixed Chimerism and Donor Stem Cells Infusion — A More Effective and Safely Model of Nonmyeloablative Allogeneic Stem Cell Transplantation (Seventy-Two Cases Report).
Authors: Ai Hui-Sheng , Guo Mei , Yu Chang-Lin , Wang Dan-Hong , Qiao Jian-Hui , Sun Qi-Yun , Sun Wan-Jun , Shi Zhang
Published: October 2019

Background Recently, the development of nonmyeloablative allogeneic stem cell transplantation (NST) which are associated with lower toxicity and mixed chimerism has improved the survival of old and young patients with haematology diseases. Otherwise, donor lymphocyte infusion (DLI) that is associated with high immunological activity and more graft versus leukaemia effect has also improved the complete remission rate of relapsed patients after transplant. But there are remain of some problems to have resolved which includes how to prevent from graft-versus-hostdisease (GVHD) and leukaemia relapsed after NST. We conducted A more effective and safely model of nonmyeloablative allogeneic stem cell transplantation which haved successfully been used in the seventy-two cases of haematology diseases.

Methods Seventy-two parients (year range 17–61) with HLA-march donor in acute leukaemia(n=33), chromic leukaemia (n=23) and MDS or SAA(n=16) were received reduced toxicity condition (cyclophosphamide, fludarabine, anti-thymocyte gloubin and cytarabine or busulfan) following a allogenic perpheral blood stem cell transplantation. GVHD prophylaxis consisted of cyclosporine A and Mycophenolate mofetil (MMF) for 90 days. All cases who has no aGVHD were received the donor frozen mobilization and apheresis perpheral blood stem cells(DSI) in 35 to 55 days after NST.

Results All Seventy-two patients passed smoothly the hematopoietic suppression stage and achieved engraftment of the donor cells which included 42 cases of full donor chimerism FDC), 30 cases of mixed chimerism (MC) in which 27 cases converted to FDC in 1–16 monthes and 3 cases developed graft rejection. Among the all of Seventy-two cases, the incidence of aGVHD (grade I–II=18, and grade III–IV=3) was 29.2% (n=21) in which 5 cases of aGVHD was caused by the DSI and stopped CSA and MMF. There are much higher rate aGVHD in the FDC group (n=15) than the MC group (n=6) The incidence of cGVHD was 30.6% (n=22). Among the 33 cases of acute leukaemia patients, five cases died from leukaemia relapsed (15.2%). Follow-up 3 to 75 months, survive rate of patients is 77.8% (n=56) and the 3 year estimated disease-free survival is 41.7%.

Conclusions These results shown that new protocol of NST is a much more effective and safty strategy for haematology patients, the use of conditions with CTX, Flud, ATG, Ara-C/BU reduced toxicity of transplant, the mixed chimerism help to prevent from the aGVHD, use of DSI both improved the MC converted to FDC and GVL effect but only slightly GVHD and no hypocellular which is much better than the DLI.
Efficacy and Safety of Using G-CSF on Day before Stem Cell Infusion (Day -1) in Hematopoietic Stem Cell Transplantation (HSCT) -A Single Institution Experience from India.
Authors: Sastry P.S.R.K. , Narayanan Prasad , Khadwal Alka , Biswas Ghansyam , Bhagwat Roshni , Bakshi Ashish , Parikh Purvish
Published: October 2019

Background: It is well established that recombinant human G-CSF accelerates neutrophil recovery following hematopoietic stem cell transplantation. However, the optimal timing of G-CSF following transplantation remains unknown. We have done a Phase II non randomized open label study on effects of adding an extra dose of G-CSF (filgrastim 5μgm/kg) on Day -1 of HSCT.

Patients and methods: Thirty nine consecutive patients who underwent HSCT in one year period were given G-CSF on Day -1(Day before stem cell infusion) in addition to the regular doses from Day +1 till engraftment.

Results: Among the 39, males were 31 and females 8 with 15 allograft and 24 autografts. The type of harvest was PBSC in 35, BM alone in 1and both in 3 cases. Disease wise distribution was AML-11, MM-8, NHL-6, HL-5, CML-4, MDS-2 and Aplastic Anemia, ALL and Ca nasopharynx 1 each. The median MNC and CD 34 doses infused were 5.4X108/Kg and 2.26X106/Kg respectively. Median days of Grade IV neutropenia was 11 (range 5–19) and that of ANC< 100 was 6 (range 2–19). Neutropenic fever was present for a median of 10 days (range 4–19). Neutrophil engraftment and platelet engraftment occurred at Day +11(range 8–13) and Day +21(range 11–73) respectively. Median number of packed cells and platelets (SDP) transfused were 4 and 5 respectively. Mucositis was present for a median of 9 days with Grade III in 51% (n=20) and Grade IV in 23% (n=9) patients. TPN was used in 76% (n=30) patients for a median days of 6 days. Median number of days of antibiotics use was 10 and 57% (n=22) needed 3 lines of antibiotics. Antifungals were used in 57% (n=22) and 95% of use was empirical. Infections were documented in 42% (n=16). Median days of hospitalization was 22 (range 13–38). Transplant related mortality was 10% (2 Auto and 2 Allo). After a median follow up of 113 days 79.71% (n=34) patients are alive with 71% (n=28) in complete remission. One patient with Ca nasopharynx had progressive disease on evaluation and one patient with peripheral T cell lymphoma developed a secondary leukemia (myeloid) post transplant.

Conclusions: Addition of G-CSF on Day -1 of HSCT is safe and provides stable engraftment and acceptable results in terms of neutropenic fever, requirement of blood products, antibiotics, TPN and duration of hospital stay. It needs to be compared in randomized trials with use of G-CSF from Day +1 onwards to prove its superiority over that schedule.
The Clinical Research of Umbilical Cord Blood Transplantation for Children with Leukemia or Whom with Beta-Thalassemia.
Authors: Fang Jianpei , Huang Shaoling , Zhou Dunhua
Published: October 2019

Objective To evaluate the clinical efficacy and complications of related (RD) umbilical cord blood transplantation (UCBT) or unrelated (UD) UCBT for children with leukemia or whom with b-thalassemia (TM).

Methods Ten patients with TM received RD-UCBT (group 1), while another 10 patients with TM had UD-UCBT (group 2).13 patients with leukemia (4 high risk ALL and 9 AML) were transplanted from UD-UCB (group3). Of 7 were HLA-identical, a patient was a loci-mismatched and two of them were half matched in group 1. Meanwhile, in the group2, three of ten patients were HLA matched, six was a loci-mismatched and one received double unites of UD-UCB that was from HLA matched to a loci-mismatched. Furthermore, ten of 13 patients were one loci-mismatched in group 3. Two were two loci-mismatched and one received double unites of UD-UCB from HLA matched to a loci-mismatched in the group. The conditioning regimen was consisted of busulfan (Bu) 14–20 mg /kg, cyclophosphages(Cy) 120–200 mg /kg and horse antithymocyte globulin (ATG)90 mg /kg or rabbit ATG 25 mg /kg for all TM patients. Of them, 6 also received melphalan (Melph) 90 mg /m2. Four patients had fludarabine (Flu) 150 mg /m2. Three combined Flu with thiotepa (TT) 6 mg /kg. Five used Flu, TT and TBI together. In the group of patients with leukemia (group 3), the regimen was divided into two branches: (1) Non-TBI regimen: the regimen mainly focused on 10 patients with leukemia in the CR phase except a patient was too young although him in the NR phase, which was consisted of Bu, Cy, ATG and Flu. A patient added high dose Ara-C. One had IDA, and 3 Melph. (2). Accompanying with TBI regimen. Only had two patients who were in the phase of NR used this regimen. The dose of TBI was 7.5 Gy and 6.5 Gy respectively. The patients received the nucleated cells 7.5 (3.4–19.4)’107/kg, CD34 cell 4.5 (0.6–11.7) ’105/kg, CFU-GM 1.1 (0.2–23) ’105/kg in three groups.

Result: The engraftment occurred 7 in 10 at the group 1. One patient developed acute GVHD grade III. Two patients rejected 2 months post RD-UCBT. The following up was from 34–69 months in the group and 4 cases remained in the EFS. However, only 3 out of 10 were engraftment in the group 2. The others all were antologous reconstitution in the failure patients. One developed acute GVHD grade II in the group 2. The following up was from 3 months to 43 months in the group. Two cases kept EFS but one died of IP at the 6 months post transplantation. 11 out of 13 patients engrafted in the group 3. Nine patients were EFS post transplantation and the time of following up was from 4 to 37 months in the group. One developed severe acute GVHD and 3 had extensive chronic GVHD.
dSLIM Immunomodulators Induce Anti-Tumor Responses Both In Vitro and In Vivo.
Authors: Schmidt Manuel , de Cristobal Javier , Sander Astrid , Brzezicha Bernadette , Koenig-Merediz Sven A. , Wittig Burghardt
Published: October 2019

Cytosine-guanine (CpG) motifs containing oligonucleotides (ODN) are commonly used for immunomodulatory purpose in cancer therapy and for the treatment of allergic diseases since they resemble bacterial DNA and serve as “danger signals”. These CpG-ODNs promote predominately a TH1-response with secretion of IL-12 and IFN-γ, In addition their broad potential includes activation of B-cell proliferation, monocyte stimulation and secretion of IgM and IL-6, and stimulation of plasmacytoid DC to produce IFN-α/-β and thus γδT-cells and NK-cells to express CD69 and secrete IFN-γ. Usually phosphorothioate (PS) modifications are to enhance the stability, but these are leading to several side-effects, like severe organ enlargements, morphological changes and immunosuppression in mice. We designed immunomodulatory molecules based on short covalently-closed dumbbell-like structures (dSLIM) to stabilize the DNA without the otherwise necessary PS-modification. To evaluate the anti-tumor effect of the dSLIM molecules we developed an in vitro anti-tumor assay. This assay uses supernatant from dSLIM-activated human PBMCs for incubation with tumor cells in vitro. We observed increased apoptosis and necrosis by the HT-29 tumor cell line after incubation with supernatant from dSLIM-treated PBMC which was significantly higher than the effect of supernatant from non-treated PBMC. In addition, supernatant from dSLIM-treated PBMC increased the expression of HLA-ABC on the tumor cells, a pre-requisite for tumor cell recognition by the immune system. These effects were confirmed with human HEK293 and murine Renca cell lines. Analyzing the effect with neutralizing antibodies to various apoptosis-related cytokines, we observed a crucial role of IFN-γ but not IFN-α or TNFα. To investigate the anti-tumor effects of dSLIM in vivo, we employed a SKH1 murine model which is prone to spontaneous development of papillomas. Using chemicals for initiation and weekly promotion of de novo papilloma development we compared groups of weekly s.c. or i.p. dSLIM injections, respectively, with the PBS control group. The number of papilloma developing mice was significantly lower in the dSLIM groups and the total number of papillomas on all mice was reduced by approximately 50%. In conclusion, we showed that dSLIM immunomodulators exhibit potent anti-tumor effects in vitro and in vivo.
A National Survey of Treatment Practices for Elderly Cancer Patients in India.
Authors: Parikh Purvish M. , Arora Brijesh , Vora Amish , Nair Reena , Gupta Sudip , Sastry P.S.R.K.
Published: October 2019

Background: Elderly cancer patients are underrepresented in cancer services utilization and clinical research in India. National data on providers’ knowledge, attitude and practices with regard to elderly cancer patients is sparse and is urgently required to address needs of this vulnerable and growing population.

Methods: A self administered questionnaire was mailed to nationally representative sample of practicing oncologists all over India. 74 Oncologists (Medical Oncologists-58%; Radiation Oncologists-25%; Surgical Oncologists-12%; allied fields-6%) responded out of > 250 mailed Questionnaires (response rate-29%).

Results: Majority (52%) of respondents considered patients with chronological age of >60 years as elderly. Although 75%(median) of elderly patients receive some therapy, only 50% of early stage-potentially curable patients and a similar percentage of advanced stage-potentially incurable patients receive curative and palliative therapies respectively. Also, half of treated patients require modification in their treatment approach and only two-third of treated patients complete therapy. The existent barriers to treatment included poor performance status (51%), advanced stage (18%), co-morbidities (14%) and financial constraints (8%). Only 50% Oncologists always discussed and 31% always enrolled elderly patients in clinical trials. Inadequacies with respect to care and evidence based recommendations for elderly patients as compared to young patients were felt by 47% and 92% of respondents respectively. The need of separate trials for elderly and a separate discipline of Geriatric Oncology was voiced by 92% and 87% of participants respectively. Major differences in treatment practice between Hemato-oncologists and non Hemato-oncologists are highlighted in table-1.

Conclusions: Treatment practices and clinical trial accrual of elderly cancer patients in India is far from optimal. Formation of a National Geriatric Oncology society with a focus on research and treatment in this vulnerable group may enhance the understanding and clinical care of this growing population.

Table-1 : Differences in the treatment practice between Hemato-oncologists and non Hemato-oncologists Treatment Practice Hemato-oncologists(%) Non Hemato-oncologists(%) *Patients given therapy as a percentage of those eligible Age cut-off for elderly ≥60 years 59 39 Palliative therapy * 60 30 Curative therapy * 50 75 Discuss trials with patients 42 64 Enroll patients in trials 36 21 Felt inadequacy in care 52 39 Felt inadequacy in evidence based recommendations 96 85 Felt need for seperate trials 96 85
Evidence for a Novel Blood Lineage Commitment Pathway from Lympho-Myeloid Hematopoietic Stem Cells.
Authors: Jacobsen Sten Eirik W. , Mansson Robert , Hultquist Anne , Sigvardsson Mikael , Buza-Vidas Natalija , Thoren Lina , Liuba Karina , Luc Sidinh , Yang Liping
Published: October 2019

We recently identified a novel Lin−Sca-1+c-kithiCD34+Flt3hi (LSKCD34+Flt3hi) lymphoid-primed multipotent progenitor (LMPP) in adult mouse bone marrow which, although possessing a combined lymphoid (B and T cell) and myeloid (granulocyte-monocyte; GM) differentiation potential, have little or no ability to adopt erythroid (E) and megakaryocyte (MK) lineage fates (Adolfsson et al, Cell121:295, 2005). The identification of this lineage restricted lymphomyeloid progenitor implicates the existence of alternative roadmaps for lineage commitment of pluripotent hematopoietic stem cells (HSCs), distinct from the classical model suggesting that the first HSC commitment step results in a strict separation into common lymphoid and myeloid progenitors. Herein we provide further, genetic evidence for such a model. Affymetrix global gene profiling, quantitative PCR, and multiplex single cell PCR analysis of LSKCD34−Flt3− long-term (LT)-HSCs, LSKCD34+Flt3− short-term (ST)-HSCs and LSKCD34+Flt3hi LMPPs, demonstrate that LMPPs in contrast to LT-HSCs and ST-HSCs down-regulate or turn off a number of genes critically involved in MkE lineage development, including GATA-1 and the receptors for erythropoietin and thrombopoietin. In contrast, a number of genes specific for early lymphoid development, including Rag-1, sterile Ig and IL-7 receptor are upregulated in LMPPs but absent in LT-HSCs and ST-HSCs. Importantly, within the LMPP, these lymphoid genes are typically co-expressed with a number of GM associated genes such as G-CSF receptor and MPO, but virtually never co-expressed with MkE associated genes. Investigating fetal liver day 14.5 we also provide evidence for existence of the LSKCD34+Flt3hi LMPPs at this early stage of development, and using a single cell clonal assay promoting combined B, T and myeloid lineage development, we demonstrate that a large fraction of fetal LMPPs lacking MkE potential possess a combined GM, B and T cell potential. Thus, evaluation at the single cell level of combined lineage potentials and multilineage gene expression provide compelling evidence for lymphoid-priming within the HSC compartment being preceeded by a loss of MkE potential, but occurring prior to loss of GM potential.
Self-Managed Long-Term Low-Molecular-Weight Heparin Compared with Usual-Care Anticoagulation for Patients with Proximal Venous Thrombosis: Harm Associated with Bleeding!.
Authors: Hull Russell D. , Pineo Graham F. , Brant Rollin F. ,   for the LITE Trial Investigators
Published: October 2019

Context: A substantial clinical need exists for an alternate to vitamin-K-antagonists for treating deep-vein thrombosis in many patients. Long-term low-molecular-weight heparin, body-weight adjusted, avoids anticoagulant monitoring and may be associated with less harm due to bleeding.

Objective: Considerable evidence suggests that reporting of harms-related data* from randomized clinical trials needs improvement. In accordance with CONSORT* we present a harm analysis regarding hemorrhagic complications.

Design: Randomized clinical trial using objective outcome measures.

Setting: 30 centres across Canada.

Participants: Acute symptomatic proximal-vein thrombosis patients.

Intervention: Therapeutic tinzaparin subcutaneously once-daily, or usual-care intravenous unfractionated heparin/vitamin-K-antagonists for 3 months.

Main Outcome Measures: Benefit was assessed by assessing the frequency of symptomatic objectively documented recurrent venous thromboembolism and harm by objectively documented hemorrhagic complications.

Results: A broad-spectrum of patients was randomized. Of 737 patients, 18 of 369 receiving tinzaparin (4.9 percent) suffered recurrent venous thromboembolism at three months compared with 21 of 368 (5.7 percent) receiving usual-care; (absolute difference, −0.9 percent, 95 percent confidence interval −4.1 to 2.4). Hemorrhagic complications occurred less frequently in the low-molecular-weight heparin group; 48 of 369 patients, (13.0 percent) versus usual-care 73 of 368, (19.8 percent), (absolute difference −6.8 percent; p=0.011; risk ratio = 0.66). The prevalence of major bleeding according to the duration of study treatment favored low-molecular-weight heparin therapy. Major bleeding persisted throughout study treatment for patients in the usual-care group receiving warfarin but ceased early by day 23 in the low-molecular-weight heparin group (p=0.034). Twenty-five patients (6.8 percent) in the low-molecular-weight heparin group and 24 patients (6.5 percent) receiving usual-care died (absolute difference 0.3 percent, 95 percent confidence interval −3.4 to 3.9).

Conclusion: Our findings are clinically relevant and important, offering the clinician an alternate therapy to vitamin-K-antagonist therapy which does not require anticoagulant monitoring in a wide range of patients with proximal venous thrombosis. Self-management with once-daily subcutaneous tinzaparin was at least as effective as usual-care and vitamin-K-antagonist therapy and offers a safety advantage with less harm due to bleeding.
Intensive Chemotherapy (LMB-86 Regimen) for AIDS-Related Burkitt’s Lymphoma/Leukemia.
Authors: Galicier Lionel , Fieschi Claire , Gerard Laurence , Chevrey Sylvie , Boulanger Emmanuelle , Daniel Marie-Therese , Meignin Veronique , Oksenhendler Eric
Published: October 2019

Early studies report extremely poor outcome in AIDS-related Burkitt’s lymphoma treated with standard dose chemotherapy. This poor prognosis is not improved by Highly Active Antiretroviral Therapy (HAART). 53 patients with HIV infection and stage IV Burkitt lymphoma/leukemia (BL) participated a unicentric and prospective clinical trial to evaluate the safety and efficacy of the intensive LMB-86 chemotherapy regimen. The median CD4 cell count was 236 x 106/l. 32 tumors were of the Burkitt type and 21 were Burkitt-like. All patients had stage IV disease with bone marrow involvement in 40 and central nervous system disease in 37. The LMB-86 regimen combined: (1) cytoreductive phase, with low dose cyclophosphamide, vincristine and steroids; (2) induction phase, with 2 consecutive courses of COPADM with high dose (8 g/m2) methotrexate (MTX); (3) consolidation phase with two courses of CYVE with high dose cytarabine and etoposide; (4) maintenance phase, with four courses combining previous drugs with lower dosage.

Thirty eight patients (71.7 %) achieved complete remission after the induction phase. With a median follow-up of 74.1 months (range, 8–153.1), 11 have progressed on therapy or relapsed. The median survival was 14.2 months and 32 patients have died. Most of deaths were due to BL (n=17, 53.1%), 5 (15.6%) to treatment toxicity, 7 (21.9%) to AIDS and 3 (9.4%) to other causes. The 1-year and 2-year estimate survival were 51% (95% CI, 44.1%–57.9%) and 47% (95% CI, 40.1%–53.9%), respectively (figure). Median disease free survival (DFS) was 41.7 months (range, 11.5-not reached) with a 2-year estimate DFS of 59.3%. Using the univariate analysis, four baseline covariates were associated with shorter survival: CD4<200 x 106/L (HR 3.3; 95% IC, 1.6–6.6), CD8 <1500 x 106/L (HR 2.5; 95% IC, 1.1–10), "B" symptoms (HR 3.6; 95% IC, 1.1–11.9), and ECOG score > 2 (HR 2.9, 95% IC, 1.3–6.4). No other HIV covariates, lymphoma baseline data or demographic data were associated with outcome. Using multivariate analysis only association with low CD4 count was significant (p=0.03).

Intensive chemotherapy regimen (LMB-86) containing high dose MTX and cytarabine is highly efficient in stage IV AIDS-related Burkitt’s lymphoma. Toxicity is high but acceptable with regard to the best overall survival reported to date.

Figure Figure
A Severe Case of Delayed Haemolytic Transfusion Reaction in a Sickle Cell Disease Patient: Immuno-Haematological Study, Cytokine Transcripts and Lymphocyte Subsets Analysis.
Authors: Noizat-Pirenne France , Ansart-Pirenne Helene , Plonquet Anne , Chadebech Philippe , Habibi Anoosha , Bachir Dora , Galacteros Frederic , Bierling Philippe
Published: October 2019

Background: Delayed haemolytic transfusion reaction (DHTR) may be a life-threatening complication in sickle cell disease (SCD) patients. DHTR is characterized by a marked drop in hemoglobin (Hb) and is frequently accompanied with intensification of the disease symptoms. Pathogenesis of DHTR is not completely understood as autologous RBCs are probably destroyed and allo-antibodies against transfused RBCs in the course of the accident are not always detected. Here we describe a case of serious DHTR and analyse lymphocyte subsets and cytokine transcripts.

Case report: A 33-year-old man with SCD was scheduled for hip replacement. He was poly-immunized (anti-RH2, -RH23, -RH30, -FY1, -FY3,-MNS3, -YT2) and had history of 2 DHTR. He received 7 units of crossmatch-compatible blood at day 0 of surgery. On days 5 and 8, sera were still compatible with samples of units received at day 0. Direct antiglobulin test (DAT) and eluate were negative. Hb remained stable at 6 g.dl−1. On day 14, the patient presented pain, fever and signs of hemolysis including a drop in Hb to 3.5 g.dl−1, LDH at 12460 U per L, bilirubin at 111 μmol/L and renal failure. Serological and eluate evaluation revealed the presence of antibodies against all RBCs tested (including units previously transfused at day 0), DAT positive with anti-C3 and anti-IgG. Hb dropped to 2.5 g.dl−1, the patient presenting neurologic symptoms. Because of the life-threatening anemia, he received 4 units that were compatible only with the known antibodies, associated with corticoids and cyclophosphamide. An anti-MNS5 was finally detected explaining partly the positive reactions against all RBCs tested. Then, additional U-negative units were transfused. The patient gradually improved symptomatically and was discharged from the high dependency unit on day 30. Hb level reached 6 g.dl−1. As compared to day 0, vigorous expansion (ten times) of Natural Killer subset (CD56+, CD16+) was observed on day 14 during hemolysis. This expansion was correlated to an increase of IL-10 transcripts whereas IL-2 and IFNγ transcripts were not detected. After treatment (day 32) lymphocyte subsets returned to the day 0 level and IL10 transcripts disappeared.

Conclusion : This case report confirms that DHTR can be induced by transfusion of crossmatch-compatible units. In this poly-immunized SCD patient, transfusion has elicited production of auto-antibodies. The observed expansion of Natural Killer cells during the hemolysis suggests that Natural Killer cells could participate, through an ADCC mechanism, to the destruction of both transfused and autologous RBCs. The involvement of IL-10 on Natural Killer FcγRIIIa receptor expression and Natural Killer cytotoxicity will be discussed.
ENMD-981693 Is an Orally-Active Kinase Inhibitor with Activity towards Human Hematologic Cancers In Vitro and In Vivo.
Authors: Bray Mark R. , Fletcher Graham C. , Denny Trisha A. , Xu John , Chen Xiaoru , Burke Patricia A. , Franklin Wanda , LaVallee Theresa M. , Minden Mark D. , Sidor Carolyn F.
Published: October 2019

The clinical success of Gleevec (imatinib) for chronic myelogenous leukemia has demonstrated that small-molecule inhibitors of specific kinases can be developed into effective oncology therapies. However, the rapid development of resistance in leukemic cells to drugs such as Gleevec and the limited therapeutic indications addressed with these molecules suggests that considerable opportunity exists for new inhibitors with a distinctive spectrum of activities against multiple kinase targets. ENMD-981693 is a novel, orally-active molecule that was discovered through a screening effort directed towards Aurora kinases, a family of serine/threonine kinases that are essential for mitotic progression. ENMD-981693 is selective for the Aurora A isoform, with an IC50 value of 25 nM, compared to an IC50 value of ~700 nM for Aurora B. The activity of ENMD-981693 was evaluated against a panel of 100 recombinant kinases, and the compound was shown to inhibit a broad range of tyrosine kinase targets including Flt3, CSF1R, Lck, JAK2, and c-Kit. ENMD-981693 inhibited the in vitro growth of human hematopoietic cancer lines including MV4;11, K562, THP-1, Jurkat, TF-1, U937, and HL-60 with IC50 values ranging from 0.04 – 21 μM. ENMD-981693 was shown to induce G2/M cell cycle arrest followed by apoptosis in U937 cells, without induction of the endo-reduplication phenotype (≥4N DNA content) associated with Aurora B-acting inhibitors such as MK-0457 (VX-680) and AZD1152. Primary cells derived from AML patients were sensitive to treatment with ENMD-981693 in vitro, resulting in IC50 values from 0.2 – 6.0 μM. Sensitivity of primary CML samples was more variable in in vitro cytotoxicity assays, with IC50s in the range of 0.1 – 40 μM. ENMD-981693 shows significant antitumor activity and is well tolerated in xenograft studies, with no weight loss or morbidity observed in administration schedules of up to 100 mg/kg bid or 200 mg/kg qd, given continuously for more than 30 days. Results from in vivo efficacy studies with ENMD-981693 using the MV4;11 xenograft model will be described. In conclusion, ENMD-981693 is a novel kinase inhibitor with potent activity towards a number of targets important in hematologic cancers.
Notch Regulation by the Fbw7/hcdc4/Sel-10 Ubiquitin Ligase.
Authors: Grim Jonathan E. , Sala Olga , Chung Nack Gyun , Radich Jerald , Varnum-Finney Barbara J. , Bernstein Irwin D. , Clurman Bruce
Published: October 2019

T-cell neoplasms frequently sustain mutations in the Notch1 gene, leading to the expression of constitutively active Notch proteins. Such mutations often target the C-terminal PEST domain, which is known to be involved in protein stability. The ubiquitin ligase Fbw7/hdcd4/Sel-10 is a tumor suppressor that negatively regulates Notch function by targeting the Notch protein for ubiquitination and proteasomal degradation. Although the PEST domain is known to be important for Fbw7/Notch interactions, the specific residues that regulate binding of Notch to Fbw7 have not been defined. Based on the structural motifs (known as phosphodegrons) common to known substrates of Fbw7, we have identified two candidate peptide sequences within the Notch protein and have generated a series of mutants in these regions. Using co-immunoprecipitation assays, we show that one potential phosphodegron that is outside of the PEST domain does not appear to influence Notch binding to Fbw7. However, a second potential phosphodegron is present within the PEST domain and contains a conserved threonine residue (T2512) which is central to binding of Fbw7 to Notch. A mutant in which this residue is replaced by alanine (T2512A) shows a prolonged half life when compared to wild type Notch ICD, supporting its role in Notch stability. To evaluate the role of Fbw7 mediated Notch degradation in vitro and in vivo, we used lentiviral vectors to transfect hematopoietic cells with shRNA targeting Fbw7. These studies demonstrate that Fbw7 knockdown leads to phenotypes consistent with increased Notch activity. Because Notch is commonly mutated in human leukemias, we hypothesized that Fbw7 may also sustain mutations that lead to loss of Notch regulation. We evaluated primary human T cell leukemias for mutations in Fbw7 and found that 1 of 23 samples contains a heterozygous mutation in the Fbw7 common region (R505C). We show that this mutant is deficient in binding to Notch, suggesting that Fbw7 mutation may contribute to the deregulation of Notch that is commonly seen in T-cell neoplasms. Together, this work shows that Fbw7 is an important regulator of Notch function whose mutation may be an important step in leukemogenesis.
FLT3-ITD Cooperates with MOZ-TIF2 in Leukemogenesis in a Murine Bone Marrow Transplantation Model.
Authors: Tam Winnie F. , Rocnik Jennifer , Wojiski Sarah , Deguchi Kenji , Okabe Rachel , Lee Ben , Gilliland D. Gary
Published: October 2019

MOZ-TIF2 is expressed as a consequence of the chromosomal inversion inv(8)(p11q13) and is associated with AML FAB subtypes M4 and M5. Mice transplanted with MOZ-TIF2 succumb to monoclonal or oligoclonal leukemias with a long median disease latency, indicating that cooperating mutations are required for MOZ-TIF2 mediated leukemogenesis. The presence of a FLT3-ITD mutation in a patient with inv(8)(p11q13) has previously been reported, suggesting FLT3-ITD as a candidate cooperating mutation. Here we report that FLT3-ITD functionally cooperates with MOZ-TIF2 in co-transduction experiments in both a serial replating and murine bone marrow transplantation assay to induce AML that is transplantable to secondary recipients. At limit dilution, both MOZ-TIF2 and FLT3-ITD retroviruses are present, demonstrating cooperative effect. Moreover, tyrosine to phenylalanine mutations of FLT3-ITD residues 589 and 591, which we have previously reported to abrogate Stat5 signaling, also abolish the ability of FLT3-ITD to cooperate with MOZ-TIF2 both in vivo and in vitro. Furthermore, a constitutively active Stat5 mutant supports factor independent serial replating activity of MOZ-TIF2 in vitro. These data suggest a multi-step transformation model in which constitutive downstream Stat5 signaling by FLT3-ITD cooperates with MOZ-TIF2 in AML induction, and indicate that FLT3-ITD potentiates the properties of self-renewal in hematopoietic progenitors through activation of STAT5.
Association of Complement with Microvascular Endothelial Cell Injury in Thrombotic Microangiopathy In Vivo and in In Vitro Model Systems.
Authors: Di Carlo Brian A. , Stefanescu Radu , Lee Kelvin , Yin Wei , Modaressi Rozbeh , Peerschke Ellinor I.B. , Laurence Jeffrey
Published: October 2019

We have previously shown that plasma from patients with idiopathic as well as HIV and thienopyridine-linked TTP induces apoptosis of microvascular endothelial cells (MVEC) in vitro, with specific sparing of large vessel and pulmonary MVEC, consistent with the clinical syndrome. This correlates with in vivo measures of apoptosis in splenic tissue of TTP patients. We found that osteoprotegrin (OPG), an inhibitor of multiple inducers of MVEC apoptosis, could block TTP plasma-mediated MVEC injury. Utilizing OPG affinity-binding columns and MS-TOFI analysis of column eluates, we subsequently identified complement (C) components and C-regulatory factors as potentially involved in TTP plasma-mediated EC injury. Building on the observation that familial TTP syndromes have been linked to genetic deficiencies of the C-regulatory factors Factor H and MCP, and three cases of sporadic HUS in children have been accompanied by autoantibodies to factor H, we hypothesized that the C-system plays an important role in TTP-induced MVEC injury, facilitated by autoantibody-mediated suppression of C-regulatory proteins. To evaluate the role of the complement system we looked at:

IFA staining of splenic tissue from TTP patients versus controls for C-components in conjunction with CD34+ indentification of MVEC. ELISA-based assays of complement activation/deposition on MVEC exposed to TTP vs. control plasma. Ability of anti-C agents to block TTP plasma-mediated MVEC apoptosis. DNA microarray analysis of TTP plasma MVEC exposed to TTP vs. control plasma. ELISA-based search for autoantibodies against C-regulatory proteins in TTP plasma.

IFA staining demonstrated increased deposition of C-components on MVEC in vivo on splenic tissue of TTP vs. control patients (4/5 TTP, 0/3 control). Increased C-activation was demonstrated on MVEC exposed to plasmas of TTP patients in vitro as evidenced by decreased C3b (p=0.002) and C4d (p=0.0002) deposition. Microarray data revealed increased expression of C-regulatory factors in resistant vs. susceptible MVEC exposed to TTP plasma. C-inhibtion resulted in prevention of TTP-plasma induced MVEC apoptosis by annexin-V staining (P<0.02). The search for autoantibodies against five C-regulatory components: CD46 (MCP), CD55 (DAF), CD59, Factor H, and Factor I by ELISA assay is currently ongoing. In conclusion, the clinical expression of thrombotic microangiopathy likely requires a double-hit of endothelial cell injury and autoantibodies against proteins vital to hemostasis and/or MVEC survival. In some patients this may involve autoantibodies to ADAMTS13; in others, C-system activation and inhibition of C-regulation by C-regulatory protein autoantibodies may be involved. Inhibition of the C-system may be a feasible clinical approach in some patients with thrombotic microangiopathies.
Risk Factors for Umbilical Venous Catheter-Associated Thrombosis in Very Low Birth Weight Infants.
Authors: Narang Shalu , Roy Jason , Stevens Timothy P. , Butler-O’Hara Meggan , Mullen Craig A. , D’Angio Carl T.
Published: October 2019

Background: Thrombosis in neonates is a rare but serious occurrence that is usually associated with central catheterization. Among acquired risk factors, thrombocytosis has often been thought to play a role in neonatal thrombosis, but little evidence exists to support this impression.

Objectives:To investigate the effects of platelet count on catheter-related thrombosis in neonates.To investigate the effects of being small for gestational age (SGA) on catheter-related thrombosis in neonates.

We hypothesized that neonates with catheter-related thrombosis would have relative thrombocytosis and would be SGA.

Methods: The present retrospective study was performed using data from a randomized trial of duration of umbilical venous catheters (UVC) placement among infants <1250 g birth weight (Butler-O’Hara, Pediatrics2006;118:e25–e35). In this study, all subjects had UVC that were left in place for up to 28 days. All subjects were screened biweekly for thrombi with echocardiograms. Twenty-two cases of UVC-associated thrombosis were identified in this sample. The remaining study sample (n=188) served as controls. Data on thrombosis, platelets, gestational age, birth weight, hematocrit, serum sodium (as a measure of dehydration), duration of catheter placement, study group assignment and demographic factors were collected using database and record review.

Results: Among the total subjects (n=210), 112 (53%) were males and 126 (60%) were Caucasians, with mean gestational age of 27.7 ± 2.1 wks (SD) and mean birth weight of 923 ± 195g. Bivariate analysis revealed significant association of thrombosis with hematocrit >55% in the first wk (OR, 5.4; 95% CI, 2.0–14.6; p=0.0003), being small for gestational age (OR, 2.9; 95% CI, 1.2–7.4; p=0.02), lower platelet counts in the first wk (193 ± 57 x 103/uL in infants with thrombus vs. 238 ± 70 x 103/uL in infants without thrombus, p=0.005) and gestational age (27.8 ± 2.5 wks in infants with thrombus vs. 27.6 ± 2.0 wks in infants without thrombus, p=0.02). In multivariate logistic regression analysis, only higher hematocrit was independently associated with thrombus (OR, 3.9; 95% CI 1.3–12.6; p=0.02). There was a trend towards an independent negative association between platelets and thrombosis (OR, 0.93 per 10 x 103/uL platelet rise; 95% CI, 0.85–1.02; p=0.12).

Conclusion: This study demonstrates a significant, independent association of elevated hematocrit and development of UVC-associated thrombosis. We did not observe an increased risk of thrombosis with increased platelet count. Careful monitoring for catheter-associated thrombosis is suggested for neonates with hematocrit >55% in the first wk of life.
Argatroban Anticoagulation for Heparin-Induced Thrombocytopenia in Obese, Versus Non-Obese, Patients.
Authors: Rice Lawrence , Hursting Marcie J. , McCollum David A.
Published: October 2019

Argatroban, a parenteral direct thrombin inhibitor used for prophylaxis or treatment of thrombosis in heparin-induced thrombocytopenia (HIT), is routinely administered in accordance with patient body weight and activated partial thromboplastin time (aPTT) response. The effect of obesity (BMI >30) on argatroban therapy in HIT however is not well described. In this retrospective analysis of a 118-patient, multicenter HIT registry database, we evaluated argatroban doses, aPTTs, and clinical outcomes in obese, vs. non-obese, argatroban-treated adults with clinically diagnosed HIT or history of HIT. Patients lacking sufficient data to calculate BMI, administered argatroban at doses required during angioplasty, or with a baseline (pre-argatroban) platelet count >150x109/L were excluded. Argatroban was infused and adjusted to achieve aPTTs 1.5–3 times baseline, at the treating physician’s discretion. We identified 41 patients for analysis: 21 non-obese (BMI 17.3–28.9, median 22.9; 52% female) and 20 obese (BMI 30.1–50.8, median 34.8; 55% female). Two patients (1 per group) had history of HIT requiring acute anticoagulation; 39 patients had HIT (median baseline platelet count of 85 and 92x109/L, respectively, in non-obese and obese patients), including 9 (5 obese) with baseline HIT-related thrombosis. Indications for heparin therapy, typically cardiac- or surgery-related, were similar between the non-obese and obese groups. Median argatroban doses were 1.0 μg/kg/min initially in each group, and 1.0 and 1.2 μg/kg/min in the non-obese and obese groups, respectively, during therapy (median 5.4 and 6.3 days, respectively). Therapeutic aPTTs occurred within a median 5.6 and 5.0 h, respectively, with median aPTTs during therapy of 60.5 and 58.7 s. By regression analysis, BMI did not significantly affect argatroban dose. Platelet counts recovered (>100x109/L or 1.5 times baseline) within 4.3 and 2.4 days for the non-obese and obese HIT patients, respectively. Within 37 days of argatroban initiation, death occurred in 7 (33%) non-obese and 5 (25%) obese patients (1 in each group while on argatroban); amputation in 0 (0%) non-obese and 1 (5%) obese patient (off argatroban); new thrombosis in 1 (5%) non-obese and 3 (15%) obese patients (2 while on argatroban); and major bleeding in 1 (5%) non-obese (off argatroban) and 0 (0%) obese patient. Cox regression analysis failed to identify any significant risk factors (BMI, obesity, gender, argatroban dose, baseline platelet count, aPTT) for new thrombosis. We conclude that argatroban dose requirements, aPTT responses, and clinical outcomes are generally comparable between obese vs. non-obese patients with HIT or history of HIT. These findings support the use of body weight-adjusted (and aPTT-adjusted) argatroban dosing for safe, effective anticoagulation, irrespective of patient BMI.