bluuebunny/crossref_metadata_2025 huggingface dataset:
Defective Telomerase in Familial Myelodysplasia and Leukemia
Authors: Kirwan Michael , Vulliamy Tom , Walne Amanda J. , Beswick Richard , Hillmen Peter , Kelly Richard , Stewart Andrew , Bowen David , Schonland Stefan , Whittle Annika Maria , McVerry Tony , Gilleece Maria , Dokal Inderjeet
Published: October 2019

Myelodysplastic syndromes (MDS) comprise a diverse range of hematological conditions, all of which involve impaired or disturbed hematopoiesis, cytopenias, marrow dysplasia and the risk of progression to acute myeloid leukemia (AML). MDS and AML are believed to involve a multi-step process arising within bone marrow stem cells, but the specific defects responsible remain poorly understood. In cases of inherited, familial forms of the disease two genes have been implicated: the hematopoietic transcription factors CBFA2 (core binding factor a2) and CEBPA (CCATT box enhancer binding protein a) and these account for only a small number of the familial cases so far reported. The uncharacterized families represent an ideal resource for identifying the primary pathology of the disease. In this study we have screened 21 families where MDS/AML appears to be familial for mutations in the telomerase enzyme components TERC (telomerase RNA component) and TERT (telomerase reverse transcriptase). In two families, we have identified novel mutations in TERC and in another two we have identified mutations in TERT directly resulting in MDS/AML. Functional analysis has demonstrated that all mutations adversely impact telomerase activity in vitro and affected individuals have short telomeres (see Table 1 for summary). These results have several important implications: First, they identify a novel genetic pathway (defective telomere maintenance) that can lead to familial MDS/AML; second, they suggest that TERC and TERT act as tumour suppressors and; finally, they have implications for the biology, treatment and screening regimen for de novo cases of MDS/AML.

Family Number of affected individuals Mutation Telomerase activity compared to wild type Age-adjusted telomere length 1 3 TERC c.212C>G 1% −4.62 2 5 TERC c.309G>T 4% 0.01 3 2 TERTc.1892G>A 0% −4.45 4 2 TERTc.2354C>T 11% −3.12

Table 1 – Summary of clinical and molecular analyses of four families with MDS/AML
A Common Single Nucleotide Polymorphism in the Chromosome 7q22.3 Region, Which Is Frequently Deleted in Myeloid Malignancies, Is Associated with Mean Platelet Volume and Platelet Function in Healthy Individuals
Authors: Soranzo Nicole , Tijssen Marloes R. , Rendon Augusto , Meisinger Christine , Jones Chris I. , Gieger Christian , Watkins Nicholas A. , Menzel Stephan , Prokisch Holger , Stephens Jonathan , Doering Angela , Erber Wendy N. , Potter Simon C. , Burns Philippa , Jolley Jennifer , Falchi Mario , Garner Stephen F. , Rankin Angela , Thein Swee Lay , Wichmann H-Erich , Goodall Alison H. , Illig Thomas , Spector Tim D. , Deloukas Panos , Ouwehand Willem H.
Published: October 2019

The variation in blood cell indices between individuals is to a large extent genetically controlled. In the normal population mean platelet volume (MPV) is inversely correlated with platelet count. We undertook a genome-wide association analysis of MPV on 2.5 million imputed genotypes in 1,475 individuals from the UK Blood Services Common Controls, followed by replication in an additional 7,098 samples from four independent collections. We identified a novel locus on chromosome 7q22.3, in a region frequently deleted in myeloid malignancies, where the lead single nucleotide polymorphism (SNP) had a highly significant association with MPV (average G allele effect size 0.15 log fl, 95% CI 0.0118–0.0174, P-value = 9.5×10−24) and an opposite effect on platelet counts (G allele effect −4.51 109/l, 95% CI −6.112 - −2.900, P-value = 1.46×10−7), but the SNP did not exert an effect on red cell indices. The lead SNP with a minor allele frequency of 0.46 is intergenic between the hypothetical gene FLJ36031 and the PIK3CG gene. There are 6 genes in the 1-Mb window centred around the lead SNP and all but HBP1 are transcribed in megakaryocytes (MKs) and to a variable degree in the other seven blood cell lineages. The formation and regulation of volume of platelets is critically dependent on the interaction of MK-expressed integrins with extracellular matrix proteins like fibrinogen and collagen. This prompted us to analyze, in our platelet function cohort of 500 healthy individuals, the association between the lead SNP and binding of both fibrinogen and annexin V to platelets following activation with the collagen mimetic CRP-XL, both associations were significant, with P-values of 0.05 and 0.003, respectively. In summary, we describe a common SNP associated with differences in volume, count and function of platelets from healthy individuals. Intriguingly, our results indicate that the same 7q22.3 region which is frequently deleted in myeloid malignancies harbours important regulatory elements of several platelet phenotypic traits.
Effects of the Mcl-1/Bcl-2 Inhibitor GX015-070 (Obatoclax®) on Growth and Viability of Canine and Human Neoplastic Mast Cells
Authors: Peter Barbara , Aichberger Karl J. , Gleixner Karoline V. , Ferenc Veronika , Gruze Alexander , Pickl Winfried F. , Willmann Michael , Valent Peter
Published: October 2019

Mcl-1 is a Bcl-2 family-member that has been described to act anti-apoptotic in various myeloid neoplasms. We and others have recently shown that neoplastic mast cells (MC) in patients with systemic mastocytosis (SM) display Mcl-1, Bcl-2, and Bcl-xL. In the present study, we examined the effects of the Mcl-1/Bcl-2-targeting drug GX015-070 (obatoclax®; GeminX, Montréal, Quebéc, Canada) on growth and viability of primary neoplastic MC obtained from patients with SM (n=3), the human MC leukemia cell line HMC-1, and the canine mastocytoma cell line C2. Two HMC-1 subclones, one lacking KIT D816V (HMC- 1.1) and one expressing KIT D816V (HMC-1.2) were examined. As assessed by RT-PCR and immunostaining, primary neoplastic MC as well as HMC-1 cells (both subclones) were found to express Mcl-1 mRNA and the Mcl-1 protein in a constitutive manner, but did not express significant amounts of proapoptotic Bim. Transfection of HMC-1 cells with Mcl-1-specific siRNA resulted in reduced proliferation and increased apoptosis compared to cells transfected with a control siRNA. GX015-070 was found to inhibit 3H-thymidine uptake and thus proliferation in HMC-1 cells in a dose-dependent manner, with higher IC50 values obtained in HMC-1.2 cells (0.5 μM) compared to HMC-1.1 cells (0.05 μM). GX015-070 also inhibited the growth and survival in the canine mastocytoma cell line C2 (IC50: 0.5-1 μM). Moreover, GX015-070 was found to inhibit the proliferation of primary human neoplastic MC in all SM patients tested (IC50: 0.05-0.1 μM). We next attempted to combine obatoclax with a modulator of Mcl-1/Bim expression in MC, in order to enhance drug effects. Since Bim is degraded via the proteasome, we applied the proteasome inhibitor bortezomib. Whereas GX015-07 did not modulate the production/expression of Mcl-1 or Bim in HMC-1 cells, bortezomib was found to promote the expression of Bim in our Western blot experiments. In addition, bortezomib was found to suppress 3H-thymidine uptake in both HMC-1 subclones. Finally, bortezomib was found to cooperate with GX015-070 in producing apoptosis in HMC-1.1 cells, HMC-1.2 cells, and C2 cells. Together, our data show that the Mcl-1/Bcl-2-targeting drug GX015-070 is a potent inhibitor of in vitro growth and survival of canine and human neoplastic MC. Targeting of Mcl-1 in neoplastic MC alone or in combination with a Bim-regulator may be an interesting pharmacologic approach in advanced SM.
Pomalidomide (CC4047) Plus Low-Dose Dexamethasone (Pom/dex) Is Highly Effective Therapy in Relapsed Multiple Myeloma
Authors: Lacy Martha Q. , Hayman Suzanne R. , Gertz Morie A. , Allred Jacob B. , Mandrekar Sumithra J. , Dispenzieri Angela , Zeldenrust Stephen R. , Kumar Shaji , Greipp Philip R. , Lust John A. , Russell Stephen J , Buadi Francis , Kyle Robert A. , Bergsagel Peter Leif , Fonseca Rafael , Roy Vivek , Mikhael Joseph , Stewart A. Keith , Rajkumar S. Vincent
Published: October 2019

Background: Thalidomide and its analogue lenalidomide have high response rates among patients with newly diagnosed as well as previously treated myeloma. Pomalidomide (CC4047) is the newest immunomodulatory (IMiD) agent that has shown single-agent activity in phase I studies. We report on the first Phase 2 trial of pomalidomide combined with low dose dexamethasone (Pom/dex) in patients with relapsed or refractory multiple myeloma.

Methods: 37 patients (21 male and 16 female) were enrolled. Pomalidomide was given orally 2 mg daily on days 1–28 of a 28-day cycle. Dexamethasone was given orally at a dose of 40 mg daily on days 1, 8, 15 and 22 of each cycle. Response was assessed by the International Myeloma Working Group Uniform Response criteria. All patients received aspirin 325 mg daily as prophylaxis against DVT.

Results: The median age was 66 years (range, 40 – 88). All patients were evaluable for response and toxicity, and all analysis were done on intent to treat basis. All patients had received prior therapy; 38% had 3 prior regimens; 35% had 2 prior regimens and 27% had one prior regimen. 76% had previous autologous stem cell transplant (ASCT) and 24% had 2 prior ASCT. 62% had previous IMiD therapy. Toxicity was mild and consisted primarily of myelosuppression. Grade 3 neutropenia occurred in 31%; grade 3 thrombocytopenia 3%; grade 3 anemia 3 %. Other grade 3/4 toxicities seen in less than 5% pts included: diarrhea, atrial fibrillation, pneumonia, dehydration and renal insufficiency. 16 % had grade 1/2 neuropathy. No grade 3 neuropathy was seen and there have been no thromboembolic events. Thirty (81%) patients are continuing study treatment. Seven patients have discontinued treatment due to: disease progression (5), died on study (1) and the medical doctor’s discretion (1). Twenty three of 37 patients (62%) achieved an objective response to therapy; including 9 (24%) with VGPR; 14 patients (38%) with PR; 6 (16%) with stable disease. Objective responses were seen in 4 of 13 patients (29%) who were refractory to lenalidomide.

Conclusions: Pomalidomide plus dexamethasone (Pom/dex) is highly active and well tolerated for treatment of relapsed/refractory multiple myeloma with an objective responserate of 62%, including a 29% response rate among patients who are lenalidomiderefractory.
Functional Analysis of CD9 during Megakaryopoiesis
Authors: Kono Tomoko , Mukai Harumi , Kozuma Yukinori , Kojima Hiroshi , Ninomiya Haruhiko
Published: October 2019

CD9 is a four transmembrane protein belonging to a tetraspanin family and regulates cell motility and adhesion. Several reports have indicated that CD9 form complexes with integrin including platelet fibrinogen receptor integrin aIIb-b III and involve in platelet function. We have previously reported that the c-myb knock down (KD) mice exhibited anemia and thrombocytopenia, and the expression level of CD9 mRNA was markdly increased in the c-myb KD mice. Reverse correlation of c-Myb expression with the CD9 gene expression was verified and agonistic antibody of CD9 stimulated megakaryocytic colony formation. These observations suggested that the CD9 expression was downregulated by c-myb. In our current study, we investigated the role of CD9 during megakaryopoiesis and platelet function by using CD9-null mice. Numbers of megakaryocytes and platelets, CFU-Meg, and ploidy were not different between wild-type and CD9-null mice. However, proplatelet formation (PPF) was significantly impaired in CD9-null megakaryocytes, and the size of proplatelets was smaller than those generated by wild-type megakaryocytes. Furthermore, after the bone marrow suppression with 5-fluorouracil (5-Fu), the recovery phase of platelet counts were delayed in the CD9-null mice. To clarify the reason of this platelet- recover delay, the number of bone marrow megakaryocyte was investigated using anti-vWF antibody staining, but the serial measurement of megakaryocyte number in CD9-null mice was not changed compared with wild-type mice. And also megakaryocyte ploidy was not changed in CD9-null mice compared with wild-type mice. Previous reports revealed that the cytoskeleton reorganization has a key role for the PPF formation, and Rac1-PAK1 signaling is important for actin restructure and aggregation prior to the cytoskeleton reorganization. The PPF formation was suppressed by Rac1 inhibitor. Although the mRNA expression levels of Rac1 in CD9-null mice were almost same as that in wild-type mice, PAK1 activation, major target of Rac1, was delayed in CD9-null mice compared with wild-type mice using thrombin-stimulating platelets. These results suggested that the delay of PAK1 activation cause the suppression of PPF formation in CD9-null mice.

Our previous and current studies demonstrate that c-Myb suppresses the CD9 expression in a steady-state condition, while in the stress megakaryocytosis, CD9 is upregulated and acts to induce megakaryopoiesis and platelets production through Rac1-PAK1 signaling pathway. Elucidation of c-Myb-CD9 regulatory function seems to be important to understand the stress megakaryopoiesis.
Significance of Initial Lumbar Puncture on Day8 of Remission Induction Therapy in Childhood Acute Lymphoblastic Leukemia: The Results from the Tokyo Children’s Cancer Study Group L99-15 Protocol.
Authors: Hasegawa Daisuke , Manabe Atsushi , Ohara Akira , Koh Katsuyoshi , Ogawa Chitose , Hanada Ryoji , Tsuchida Masahiro
Published: October 2019

Traumatic lumbar puncture with the presence of leukemic blasts (TLP+) in the cerebrospinal fluid (CSF) has been shown to adversely affect the outcome of children with acute lymphoblastic leukemia (ALL). In the Tokyo Children’s Cancer Study Group (TCCSG), we adopted a strategy to defer the initial lumbar puncture (LP) and intrathecal (IT) chemotherapy until 1 week after prednisolone (PSL) monotherapy so that circulating blasts were substantially reduced when the initial LP/IT was performed. The result of L89-12 study was previously reported, in which the incidence of TLP+ was significantly diminished but the impact on the rate of central nervous system (CNS) relapse was not determined because over 80% of patients received cranial irradiation (CRT). We analyzed the result of L99-15 study in which CRT was employed in only 16% of patients. Seven hundred and fifty-five children with newly diagnosed ALL were enrolled onto the TCCSG L99-15 study between April 1999 and June 2003. Patients received the initial IT administration of methotrexate, hydrocortisone, and cytarabine on day 8 following PSL monotherapy. Patients with CNS2 (<5 leukocytes/microL of CSF with leukemic blasts) received additional IT therapy. Patients with CNS3 (>5 leukocytes/microL of CSF with leukemic blasts) or CNS1s (negative CSF finding, with cranial nerve palsy) received additional IT therapy and CRT. Patients with TLP+ did not require any reinforcement of CNS-directed therapy. The frequency of CNS1s, CNS2, CNS3, or TLP+ was 0.5%, 1.1%, 0.5% and 0.8%, respectively. These figures were almost equal to the results from L89-12 study and much lower than those reported by other study groups such as BFM. Of 22 patients (2.9% of all) classified as CNS positive group (CNS-1s, -2, -3 and TLP+), only one patient had a CNS relapse concurrently with bone marrow (BM) relapse, whereas 22 of 723 patients classified as CNS negative had CNS relapse (isolated 12, combined with BM 10). Overall, 4-year event-free survival rate was 78.2 +/− 1.6%, and 4-year cumulative incidence of overall CNS relapse was 3.3 +/− 0.7%. Our strategy may underestimate the frequency of CNS leukemia at diagnosis, however, unnecessary intensification of CNS treatment may be avoided while overall CNS relapse rate is quite low in spite of restricted indication for cranial irradiation.
Superiority of Rasburicase Versus Allopurinol on Serum Uric Acid Control in Adult Patients with Hematological Malignancies at Risk of Developing Tumor Lysis Syndrome : Results of a Randomized Comparative Phase III Study.
Authors: Cortes Jorge , Seiter Karen , Maziarz Richard Thomas , Wetzler Meir , Craig Michael , Matous Jeffrey , Luger Selina , Dey Bimalangshu , Schiller Gary J. , Pham Dat , Abboud Camille N. , Krishnamurthy Muthuswamy , Brown Archie , Moore Joseph O.
Published: October 2019

Tumor lysis syndrome (TLS) is a potentially lethal metabolic complication of chemotherapy or cytolytic antibody therapy usually seen in patients with hematologic malignancies, especially those malignancies with a high proliferative rate, large cellular burden and/or sensitivity to chemotherapy. The prevention and management of TLS includes hydration and reduction of serum uric acid (SUA) levels. Although Allopurinol (ALLO) has had longstanding use for TLS prophylaxis, its efficacy in controlling SUA is limited, especially due of its lack of action on pre-existing hyperuricemia. Rasburicase (RAS), a recombinant urate oxidase, effectively reduces SUA due to conversion of UA into allantoin, a readily excretable and soluble substance. RAS has significant activity in the initial management of TLS-associated acute hyperuricemia in pediatric populations, and is currently indicated in the US for this condition in children and adolescents. A prospective, randomized, controlled phase III study was conducted in adult pts to compare the efficacy in SUA control of RAS (0.20 mg/kg/d, IV) days 1–5, versus RAS+ALLO (RAS 0.20 mg/kg/d, IV days 1–3 plus oral ALLO 300 mg/day days 3–5) versus ALLO alone (300 mg/d) days 1–5. 280 pts (275 evaluable) with hematological malignancies at high or potential risk for TLS were enrolled. 92 pts received RAS, 92 pts received RAS+ALLO, and 91 received ALLO. Treatment arms were well balanced in terms of demographics, baseline characteristics, TLS risk, and percentage of pts with baseline hyperuricemia. The SUA response rate - defined as normalization of SUA (≤ 7.5mg/dl) at days 3–7 was 87.0% in the RAS arm, 78.3% in the RAS+ALLO arm and 65.9% in the ALLO arm. RAS was superior over ALLO (p=0.0009) in the overall study population as well as in pts at high risk TLS (89.0% vs. 62.8%, p=0.0012), and in pts with baseline hyperuricemia (89.5% vs. 52.9%, p=0.0151). The time to control SUA in hyperuricemic pts was 4.1 h in the RAS arm and 27 h in the ALLO arm. The mean SUA area under the curve (AUC) results indicated that there was an 8.4-fold increase in UA exposure in the ALLO arm compared to the RAS arm. There were no significant differences in the incidence or severity of adverse events, serious adverse events or deaths. The majority of RAS and/or ALLO-related adverse events were grade 1 and 2, and most of these events were hypersensitivity-related reactions. No cases of anaphylaxis, methemoglobinemia or hemolysis were observed with RAS treatment. In conclusion, RAS is superior to ALLO in normalization of SUA, with a faster effect, in adult pts at risk for TLS. RAS alone or followed by ALLO are two valid options for this patient population.
Arsenic Trioxide Entered Cerebrospinal Fluid with the Help of Mannitol Overwhelm the Meningeal Relapse of Acute Promyelocytic Leukemia.
Authors: Zhou Jin , Meng Ran , Sui Xinhua , Feng Guosheng , Li Baoyu
Published: October 2019

Background and objective Central nervous system (CNS) relapse of acute promyeloytic leukemia (APL) is increasingly reported after treatment with all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3), the optimal therapy for these cases remains unclear. As2O3 highly effective in APL marrow relapse, but its efficacy in APL CNS relapse is undefined. Recent researches showed that little As2O3 could pass through the intact blood- brain-barrier (BBB), which limits its use in prevention and treatment of central nervous system acute promyelocytic leukaemia (CNSAPL). After discovered the different tolarance between NB4 cell line and human cortex neuron to As2O3in vitro, and primarily cleared the safe range of As2O3 level in central nervous system (CNS), we created a non-invasive method to help As2O3 enter into CNS effectively.

Methods Five volunteers with isolated meningle APL relapse received the new As2O3 regimen (mannitol helped As2O3 penetration) after accepted the conventional treatment: As2O3 0.16mg/kg/d diluted with 5%glucose 250ml common speed intravenous infusion with intensive intrathecal chemotherapy (MTX 12mg/dose + cytarabine 50mg / dose + Dexamethasone 10mg/dose) and showed chemotherapy resistance. The new regimen included 125ml of 20% mannitol bolus through medial cubital vein with the speed of 12„d`30ml / min, followed by As2O3 0.08mg/kg/d diluted with 250ml-20% mannitol intravenous infusion with the speed of 6ml /min, and then followed by As2O3 0.08mg/kg/d diluted with 5%glucose 250ml intravenous infusion with the speed of 0.5ml/min, meanwhile, associated with intensive intrathecal chemotherapy. The elemental arsenic levels in CSF and plasma during the new regimen performance were dynamic monitored with atomic fluorescence, and the differentiation and apoptosis of the blast cells in CSF were detected with flow cytometry assay.

Results The elemental arsenic level in the CSF treated with mannitol helped As2O3 penetration was fluctuated between 0.04 mg /L (0.2 micromol /L) and 0.05mg / L(0.25 micromol /L), which was much higher than that with common speed As2O3 intravenous infusion, and reached to the As2O3 effectively therapeutic level. The blast cells in CSF showed significant differentiation and apoptosis after treated with mannitol helped As2O3 penetration. Three of the five patients obtained complete remission, two of them reached partial remission.

Conclusions The new regimen of mannitol helped As2O3 penetration promoted the entrance of elemental arsenic to CNS, which might be benefit to prevent and cure CNS APL, especially to chemotherapy resistant CNS APL.
One Antigen HLA-Mismatched Related and 8/8 Allele Matched Unrelated Donors Are Associated with Similar Survival after Hematopoietic Cell Transplantation for Acute Leukemia.
Authors: Valcarcel David , Kan Fangyu , Wang Tao , Lee Stephanie J. , Spellman Stephen R , Hale Gregory A. , Marino Susana , Marks David , McCarthy Philip L. , Oudshoorn Machteld , Petersdorf Effie W , Ringdén Olle , Setterholm Michelle , Sierra Jorge
Published: October 2019

Patients in need of an allogenetic hematopoietic cell transplant but who lack an HLA genotypically identical sibling donor, are faced with the decision to consider a single HLA antigen mismatched related donor, or a search for a suitable 8/8 matched unrelated donor. We compared the outcomes of adult patients (≥18 years old) receiving a transplant for the treatment of AML or ALL in first or second remission from either a one-antigen mismatched related donor (MMRD group, N=89) reported to the CIBMTR or an 8/8 HLA-A, B, C and DRB1 allele matched unrelated donor (UD group, N=700) facilitated by the NMDP between 1995–2005. MMRD group was typed by serological or DNA-based methods for HLA-A, -B and –DR with all results verified by lab report review. The UD group was retrospectively typed for HLA-A, B, C and DRB1 by high resolution typing methods. Most received myeloablative conditioning regimens (77%), calcineurin inhibitor-based GVHD prophylaxis (100%) and T cell replete grafts (100%). 13% received ATG with the conditioning regimen. Median follow-up was 54 and 38 months in the MMRD and UD groups, respectively. The MMRD group was younger (median age 35 vs 43, p=0.002), had more ALL patients with low-risk cytogenetics (43% vs 18%, p=0.005), had older donors (median age: 38 vs 34, p=0.047), were more likely to receive methotrexate for GVHD prophylaxis (89% vs 77%, p=0.014) and were more likely to be transplanted prior to 2001 (62% vs 24%; p<0.001). There were no differences in patient or donor gender, diagnosis, disease-status, cytogenetic-risk of AML, time from diagnosis to transplant, stem cell source, conditioning regimen, use of ATG and Karnofsky index. Univariate comparisons (MMRD vs. UD) showed: 3-year OS (42% vs 44%, p=0.647), 3-year DFS (41% vs 41%, p=0.931), 3-year TRM (39% vs 31%, p=0.136), 3-year incidence of relapse (20% vs 28%, p=0.094), grade III–IV acute GVHD by 100 days (22% vs. 15%, p=0.147), chronic GVHD by 1 year (35% vs 47%, p=0.029). All multivariate analyses were adjusted for patient and transplant characteristics and are shown in the table below.

In summary, transplants utilizing one-antigen mismatched related and 8/8 allele-matched unrelated donors did not significantly differ in overall survival or disease free survival, but chronic GVHD was more frequent after UD transplantation.

Outcome RR (MMRD vs. UD) 95% CI p-value Survival 0.99 0.73–1.34 0.94 Disease-free survival 1.06 0.80–1.41 0.69 Treatment related mortality 1.14 0.77–1.69 0.52 Relapse 0.81 0.50–1.30 0.38 Acute GVHD III–IV 1.53 0.91–2.57 0.11 Chronic GVHD 0.58 0.39–0.85 0.006
Risks and Benefits of Unrelated Donor Peripheral Blood Progenitor Cells (PBPC) in Children and Adolescents with Acute Leukemia.
Authors: Eapen Mary , Ringden Olle , Locatelli Franco , Frangoul Haydar , Remberger Mats , Laughlin Mary J. , Rocha Vanderson
Published: October 2019

Although PBPC is an acceptable alternative to bone marrow (BM) for transplanting children with leukemia, there are no published studies describing outcomes after unrelated donor PBPC transplants. We compared the results of 385 unrelated donor BM transplants that were allele-matched (n=186) or mismatched (n=199) at HLA A, B, C, DRB1 and 110 PBPC transplants that were matched (n=60) or mismatched (n=50) at HLA A, B, C, DRB1 in patients younger than 18 years of age. All patients had acute leukemia and were transplanted in 2000–2006. Median follow up of surviving patients was 2 years in both treatment groups. There were no significant differences in patient and disease characteristics, transplant conditioning regimen, graft-versus-host disease (GVHD) prophylaxis and donor-recipient HLA disparity by graft type. Though the early probability of neutrophil recovery (³500/ul) was faster after transplantation of PBPC (31% vs. 10% at day-14, p<0.001) the probability of recovery by day-28 was similar after PBPC and BM transplants (94% and 91%, p=0.391). In contrast, platelet recovery (³20,000/ul) was better after PBPC transplants (86% vs. 76% at day-60, p=0.022). Risks of grade 2–4 (hazard ratio [HR] 1.24, p=0.147) and grade 3–4 (HR 1.07, p=0.785) acute GVHD were similar after PBPC and BM transplants. The risk of developing chronic GVHD was significantly greater after PBPC transplants compared to BM transplants (HR 2.36, p<0.001). After adjusting for disease status, donor-recipient HLA disparity and age, the of transplant-related mortality (TRM) relapse, treatment failure (relapse or death from any cause; inverse of leukemia-free survival and overall survival were similar after PBPC and BM transplants. The Table below shows the day-100 probability of grade 2–4 acute GVHD and the 3-year probabilities of chronic GVHD, TRM, relapse, leukemiafree survival and overall survival by graft type. These results differ from transplantation of PBPC from HLA-matched siblings where higher chronic GVHD translated into higher TRM and lower LFS. It remains to be seen whether the observed higher chronic GVHD after PBPC transplants will eventually result in the long term in higher mortality or fewer leukemia recurrence.

PBPC BM Grade 2–4 acute GVHD 53% 49% Chronic GVHD 58% 33% TRM 20% 24% Relapse 34% 28% Leukemia-free survival 46% 48% Overall survival 49% 49%
Bone Marrow in Aging: Changes? Yes; Clinical Malfunction? Not So Clear.
Authors: Longo Dan L.
Published: October 2019

Like every organ system, the bone marrow undergoes changes with age. The most readily apparent change is a decline in marrow cellularity. The percentage of marrow space occupied by hematopoietic tissue goes from 40–60% in young adults to 20–40% in older people, with the remaining space being taken up by fat. It is unclear whether the expansion of marrow fat with age is a cause or an effect of aging. A similar change happens in the thymus much earlier in adulthood. At about age 30, thymic fibroblasts become fat-laden and the expansion of fatty tissue essentially replaces the cellular elements of thymopoiesis. In late life, this thymic inactivity is manifested by a reduction in naïve T cells and a compromise in cellular immunity. It is unclear whether the processes in the marrow and the thymus are qualitatively similar. Bone remodeling and osteoporosis can also lead to a decrease in trabecular bone in the marrow. Several age-related changes have been noted in hematopoietic cells, including shortening of telomeres, accumulation of mitochondrial DNA mutations, skewed X-inactivation, and others. However, in the absence of disease, sufficient stem cell proliferation and maturation is preserved so that people are capable of living their entire life span, up to 120 years, without running out of blood cells. Indeed, even when a person is given stem cells from another person, presumably in numbers far less than were present in the host before myeloablation, the transferred stem cells appear to be capable of maintaining normal hematopoiesis throughout the lifespan of their adoptive host. Normal blood counts are maintained throughout life. Physiologic compensations may be necessary to achieve this end. A longitudinal study of older people with normal hemoglobin levels over a period of 30 years showed that people increase serum levels of erythropoietin over time to maintain normalcy. Older people also seem to increase their neutrophil count to a lower extent and somewhat more slowly in response to pharmacological G-CSF than younger people, though such an intervention is generally done in people with underlying disease. Qualitative neutrophil defects in older people may include impaired phagocytosis and respiratory burst response to soluble signals. Enhanced in vitro platelet aggregation in older people does not have a clear in vivo functional correlate.
Signal Transduction Inhibitors in Lymphoproliferative Diseases (LPD).
Authors: Grupp Stephan A.
Published: October 2019

There are a wide variety of diseases characterized by abnormal proliferation of lymphocytes, with differing clinical manifestations and in a spectrum from benign to malignant. Underlying all of these disorders, however, are subversions of normal lymphocyte signaling pathways that contribute to the pathologic process, can be characterized, and can potentially be targeted. Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood and an example of malignant LPD, while patients with nonmalignant LPD, such as autoimmune lymphoproliferative syndrome (ALPS), can be difficult to diagnose, suffer from chronic infections that can be life-threatening, have a decreased life expectancy, and often require long-term treatment with immunosuppressive agents, including steroids, which can result in long-term debilitating consequences. In order to improve outcome and potentially reduce side effects and long-term sequelae from current cytotoxic therapy, the development of novel, biologically relevant, molecular, and cellular targeted therapies is critical. Potential targeted therapies can be identified and tested using in vitro assays, in vitro bone marrow stromal cell culture systems, murine models of ALL and LPD, and xenografts of primary human ALL. These pre-clinical assays have indentified promising and clinically relevant targets in the mTOR, IL-7, and Notch pathways, some which are now being tested in clinical trials. These include

rapamycin, being tested in both ALL and ALPS; temsirolimus, a second generation rapamycin analogue that shows promise in combination with cytotoxic chemotherapy in ALL; inhibitors of IL-7 signaling; and DAPT, a gamma-secretase inhibitor that has shown pre-clinical activity against both lymphoproliferation and autoimmune manifestations including nephritis in ALPS.
Importance of FLT3 Mutations and Other Genetic Abnormalities in Normal Karyotype AML.
Authors: Bloomfield Clara D. , Marcucci Guido
Published: October 2019

Cytogenetic findings at diagnosis are currently considered the most important prognostic factor in AML. By far, the most common cytogenetic finding, found in approximately 40–50% of patients, is a normal karyotype (defined as detection of only normal chromosomes in 20 or more marrow cells). Recent molecular analyses of leukemic blasts in cytogenetically normal AML (CN-AML) have revealed a striking heterogeneity with regard to the presence of acquired gene mutations and changes in gene expression. Multiple submicroscopic genetic alterations with prognostic significance have been discovered, including mutations of the FLT3 gene, both internal tandem duplications (ITDs) and point mutations (TKDs), partial tandem duplication (PTD) of the MLL gene, mutations in the NPM1, CEBPA, and WT1 genes, and high expression of the BAALC, ERG, and MN1 genes. Most of these abnormalities, except for mutations in NPM1 and CEBPA, have been associated with an adverse prognosis. CN-AML patients with FLT3 ITD, NPM1 wild type, or both have worse outcome in the range of 30% long-term survival, unless they also harbor CEBPA mutations. A high expression of the FLT3 ITD allele compared to the FLT3 wild type allele confers an especially poor prognosis. In contrast, CN-AML patients without a FLT3 ITD and with NPM1 mutations have a particularly good outcome in the range of 60% long-term survival, unless they present with high ERG expression. The prognostic significance of FLT3 TKD mutations is controversial, and, recently, the outcome of patients with the MLL PTD has improved when treated with induction therapy, including etoposide and post induction therapy with autologous transplantation. As a result, the 2008 World Health Organization classification of AML recommends evaluation of FLT3, NPM1, and CEBPA mutations in CN-AML. Application of gene expression profiling has also identified signatures that appear to separate CN-AML patients into prognostic subgroups, and, more recently, a microRNA expression signature was found to be associated with outcome in molecular high-risk CN-AML patients (i.e., those with FLT3 ITD, NPM1 wild type, or both). These and similar future findings are likely to have a major impact on the clinical management of CN-AML not only in prognostication, but also in selection of appropriate treatment, since many of the identified genetic alterations already constitute (e.g., FLT3 mutations for tyrosine kinase inhibitors) or will potentially become targets for specific therapeutic interventions.
GATA Factor Mechanisms and Globin Gene Regulation.
Authors: Bresnick Emery H. , Kim Shin-Il , Bultman Scott J. , Lee Sherry , Boyer Meghan E. , Fujiwara Tohru , Wozniak Ryan J.
Published: October 2019

Key steps in hematopoiesis and the expression of genes encoding hemoglobin subunits are critically dependent upon specific members of the GATA factor family of transcription factors. Our recent efforts have focused on elucidating how GATA factors select functional sites in chromatin and how they function combinatorially with additional regulatory factors. GATA motifs are often arranged in close proximity to E-boxes, and such composite elements commonly mediate GATA factor- and Scl/TAL1-dependent transcriptional responses. Only a small fraction of these composite elements in chromatin are occupied by GATA factors and Scl/TAL1, and a specific epigenetic signature distinguishes occupied versus unoccupied elements genome-wide. In the context of hemoglobin synthesis, we are using genetic and molecular approaches to dissect the multistep mechanism underlying the control of β-globin transcription. GATA-1-containing complexes assemble at the β-globin Locus Control Region (LCR) prior to the murine adult βmajor promoter. Though the LCR physically interacts with the βmajor promoter, this interaction is not required for the binding of several trans-acting factors to the LCR or the promoter. A hypomorphic mutation of the chromatin remodeler BRG1 limits the extent to which RNA Polymerase II (Pol II) is recruited to the promoter and also abrogates the LCR-promoter interaction. Whereas looping is not required for assembly of the full complement of promoter complex components, looping is linked to the establishment of maximal levels of Pol II at the promoter. Collectively, these results provide insights into the relationship between, and importance of, individual steps in the multi-step activation mechanism. I will discuss progress on unraveling mechanisms underlying GATA-1-mediated activation of the adult β-like globin genes as well as fundamental aspects of GATA factor function, which have broad relevance in diverse systems. promoter. Though the LCR physically interacts with the β promoter, this interaction is not required for the binding of several -acting factors to the LCR or the promoter. A hypomorphic mutation of the chromatin remodeler BRG1 limits the extent to which RNA Polymerase II (Pol II) is recruited to the promoter and also abrogates the LCR-promoter interaction. Whereas looping is not required for assembly of the full complement of promoter complex components, looping is linked to the establishment of maximal levels of Pol II at the promoter. Collectively, these results provide insights into the relationship between, and importance of, individual steps in the multi-step activation mechanism. I will discuss progress on unraveling mechanisms underlying GATA-1-mediated activation of the adult β-like globin genes as well as fundamental aspects of GATA factor function, which have broad relevance in diverse systems.
DNA Methylation and Globin Gene Expression.
Authors: Saunthararajah Yogenthiran , Lavelle Donald
Published: October 2019

Understanding how the human γ-globin gene is regulated has important clinical implications because increased levels of fetal hemoglobin (HbF) are beneficial to patients with sickle cell disease and β-thalassemia. DNA methylation is strongly implicated in developmental silencing of the γ-globin gene based on:

an inverse correlation between DNA methylation of the γ-globin gene and its expression, the acquisition of CpG residues within the γ-globin 5’ region during evolution as the γ-globin gene was recruited to fetal stage expression, and the ability of pharmacological inhibitors of DNA methyltransferase (5-azacytidine; decitabine) to reactivate high-level expression of the γ-globin gene in experimental primates that led to clinical trials demonstrating that decitabine increased HbF to therapeutic levels in patients with sickle cell disease.

Decitabine treatment in vivo decreases DNA methylation of the γ-globin gene and increases association of RNA polymerase II, acetyl Histone H3 and H4, and Histone H3 (lys4) trimethyl with the γ-globin gene, strongly suggesting that decitabine increases γ-globin gene transcription. These results are consistent with the hypothesis that γ-globin expression in adults is repressed by the binding of methyl DNA binding proteins to the methylated γ-globin promoter with subsequent recruitment of co-repressor complexes that actively repress γ-globin transcription. The reduction of γ-globin gene DNA methylation induced pharmacologically in adults by decitabine is linked to high level γ-globin expression, as is the complete loss of γ-globin gene methylation attained physiologically during erythroid differentiation of fetal liver hematopoietic progenitor cells. The mechanism of action of the drug has not been definitively established, however, and the role of DNA methylation in regulation of γ-globin gene expression remains an active area of investigation. High level γ-globin expression in baboon erythroid progenitor cell cultures without a reduction of γ-globin gene DNA methylation suggests the existence of alternative mechanisms of activation. In addition to reducing DNA methylation, decitabine activates the p38 MAP kinase pathway, increases p21WAF, and accelerates terminal erythroid differentiation. The role of these effects remains to be investigated. Increased understanding of the role of DNA methylation in γ-globin gene regulation is likely to impact the design of future therapies to increase HbF levels.
Dysregulation of Hematopoietic Stem Cells with Age.
Authors: Goodell Margaret A.
Published: October 2019

Aging is an inexorable process marked by an accumulation of traits that ultimately limit the normal function of the organism. Stem cells have long been of interest to the study of aging due to their potential to rejuvenate somatic tissues. However, several groups have now shown that stem cells themselves are subject to the effects of aging. In the hematopoietic system, stem cells markedly decline in function with time, and display characteristic behaviors such as skewing of differentiation toward myeloid development. Current studies focus on both the broad effects of aging on stem cells, and also on the role of specific genes in the aging process. Our laboratory has used gene expression profiling to examine the global gene expression changes that occur in purified hematopoietic stem cells in a time course of aging in mice. Of around 14,000 genes profiled, we identified ~1500 that were age-induced and ~1600 that were age-repressed. Genes associated with the stress response, inflammation, and protein aggregation dominated the upregulated expression profile, while the downregulated profile was marked by genes involved in the preservation of genomic integrity and chromatin remodeling. Regional analysis suggested dysregulation of many gene clusters rather than alterations of a small number of aging-specific genes. These studies suggest that hematopoietic stem cells are subject to extensive epigenetic changes over time. This epigenetic dysregulation lays a fertile ground for secondary events that may enhance the impact of genetic changes, thus driving both functional attenuation and the increased propensity for neoplastic transformation with age. The influence of the aging environment on stem cells is only now being explored. The composition of the bone marrow changes with age, and this can clearly exert effects on the types and proportions of progenitors. Furthermore, environmental conditions that typify the aging state, such as chronic inflammation, can directly impact the stem cells. A broad view of aging bone marrow environment and stem cells, as well as current molecular data addressing these issues, will be presented.
Selectins, Heparins, and Cancer: Rationale for Clinical Trials.
Authors: Varki Ajit P. , Varki Nissi M. , Borsig Lubor
Published: October 2019

Heparin has been in use as an anticoagulant for many decades, based on its ability to enhance antithrombin activity. However, heparin is a natural product, comprising a complex polydisperse mixture of highly sulfated glycosaminoglycan chains, only a fraction of which bind antithrombin. We reported that heparin has an unrelated biological effect in inhibiting P- and L-selectin binding to their natural ligands. Meanwhile, animal studies and analyses of human clinical data suggest that heparin therapy increases survival in various cancers. In contrast, clinical trials using Vitamin K antagonists showed no major effect on survival in most studies. Thus, the heparin effect on cancer may not be due primarily to its anticoagulant function, but rather to inhibition of P- and L-selectin. Indeed, we have demonstrated that the selectins play critical roles in animal models of the hematogeneous phase of carcinoma metastasis and of the platelet-rich microthromboses typical of classical Trousseau’s syndrome. This involves interactions of selectins with pathological ligands produced by carcinoma cells, particularly sialylated, fucosylated, and sulfated mucins. The critical selectin-mediated interactions blocked by heparin occur early in the metastatic cascade. Also, inhibition occurs at concentrations in the clinically acceptable range. Furthermore, there are many other salutary actions of heparin in cancer, including adsorption of cytokines and growth factors, inhibition of thrombin generation, and inhibition of heparanases. We, therefore, suggest that heparin therapy for metastasis prevention in humans be revisited, with these new paradigms in mind. Most of the above studies used unfractionated heparin. We found that selectin-inhibiting effects of low molecular weight heparins (LMWH) are varied and that the synthetic heparinoid pentasaccharide that specifically binds antithrombin does not block at clinically relevant levels. Optimal dosing to sustain clinically acceptable levels of all these drugs in mice gave congruent results. These data suggest a rational clinical trial comparing an optimal LMWH with the pentasaccharide in the pre-/peri- and immediate post-operative periods of newly diagnosed carcinomas. Notably, heparin can also attenuate inflammatory pathologies in animal models by blocking L- and P-selectin.
Iron Does Not “Jiggle Free” in Mitochondria: Is Mitoferrin the Only Answer?.
Authors: Paw Barry H.
Published: October 2019

The developing erythron requires tremendous amounts of iron (Fe) for the synthesis of heme for hemoproteins, such as hemoglobin, and iron-sulfur (Fe/S) clusters of proteins, which are required to catalyze redox reactions and regulate Fe uptake and storage. The uptake of Fe from transferrin (Tf) involves the binding of Tf to its cognate receptor (TfR), followed by the endocytosis of the Tf-TfR complex.1,2 In the late endosome, the release of Fe3+ from TfR is achieved by acidification of the vesicle by the v-ATPase H+-pump. Steap3 reduces the liberated Fe3+ prior to its transport out of the endosome by the DMT1 transporter. In contrast to previous conventional models for a cytosolic intermediate state, new data have emerged showing the direct interorganellar transfer of Fe from the endosome to the mitochondria.3 Although it is assumed that the exported Fe is targeted to the mitochondria for eventual incorporation into heme and Fe/S clusters, our understanding of the precise mechanism of how Fe traverses the outer and inner mitochondrial membranes remains poorly understood. Work in yeast mutants have implicated the role of solute carriers, Mrs3/4p, in mitochondrial iron homeostasis and revealed that it is the reduced form of iron, Fe2+, that is imported into the mitochondria. Subsequent studies of the zebrafish mutant, frascati, led to the discovery of mitoferrin 1 (Mfrn1, slc25a37), the vertebrate ortholog of Mrs3/4, as the major iron importer across the inner mitochondrial membrane in developing erythroblasts.4 A structurally related paralog, mitoferrin 2 (Mfrn2, slc25a28), plays the analogous role of Fe importer in non-erythroid cells. Loss-of-function studies of Mfrn1 in the mouse have confirmed its requirement in mammalian primitive and definitive erythropoiesis and its essential role in heme and Fe/S biosynthesis. Several questions remain unanswered in mitochondrial Fe metabolism:

Do the two Mfrn importers account for all Fe imported into the mitochondria? How does Fe get across the outer mitochondrial membrane to reach the Mfrn importers? How does the translocated Fe in the matrix ultimately reach ferrochelatase to form heme? How is Fe translocated across the inner mitochondrial membrane?