bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between Conditioning Intensity and Comorbidity in Patients (Pts) with Acute Myeloid Leukemia (AML) or Myelodysplasia (MDS) Receiving Allogeneic Hematopoietic Cell Transplantation (HCT).
Authors: Sorror Mohamed , Storer Barry , Sandmaier Brenda M. , Maris Michael , Baron Frederic , Maloney David , Scott Bart , Deeg H. Joachim , Storb Rainert
Published: April 2020

We developed a new HCT-specific comorbidity index (HCT-CI) as a sensitive tool to capture pretransplant comorbidties among pts given allogeneic HCT (Sorror et al, online, 2005; DOI 10.1182/blood-2005-05-2004). In the current report, we used the HCT-CI to explore differences in the tolerability of nonablative compared to ablative HCT in MDS or AML pts with pretransplant comorbidities. Consecutive pts (n=88) receiving nonablative conditioning with 2 Gy total body irradiation (TBI) alone (6%) or with 90 mg/kg of fludarabine (94%) were compared to concurrent pts (n=361) conditioned with cyclophosphamide plus busulfan (70%) or 12–14 Gy TBI (30%). All pts were classified into low (AML-first complete remission or MDS-refractory anemia) and high-risk disease (all others). Overall, 76% of nonablative pts had AML compared to 66% of ablative pts. At HCT, nonablative pts differed substantially from ablative pts with respect to age (median 60 vs 46 years), use of unrelated donor grafts (57% vs 42%), use of marrow grafts (5% vs 20%), and HCT-CI scores of ≥2 (75% vs 45%). Smaller differences existed for high-risk disease (64% vs 58%) and pt cytomegalovirus positive sero-status (59% vs 55%). Proportional hazards models were used to estimate the hazard ratio (HR) for non-relapse mortality (NRM) and survival for pts receiving nonablative compared to ablative conditioning regimens; these models were adjusted for stem cell source, pt age, donor type, pt cytomegalovirus sero-status, diagnoses (MDS vs AML), and HCT-CI scores (Table). Adjusted HRs were not statistically significantly different, except among pts with high-risk disease and HCT-CI scores of ≥2 where nonablative pts had lower HR for NRM (0.34, P=0.005) and worse survival (0.51, P=0.006, Fig.1). These results suggest that pts with high-risk MDS or AML and HCT-CI scores of ≥2 might have less NRM and better survival with nonablative than with ablative HCT. Prospective randomized trials are warranted to confirm this retrospective observation. Additional data are needed for AML and MDS pts with low comorbidities or low disease risk in order to clarify the usefulness of nonmyeloablative conditioning.

Table: Adjusted hazard ratios for pts receiving nonablative relative to ablative conditioning Nonablative/ablative NRM Survival Number HR* (95% CI) P HR* (95% CI) P *HR <1 indicates more favorable outcome for pts receiving nonablative conditioning Low-risk disease HCT-CI scores 0–1 7/86 0.0 (undefined) 0.33 0.8 (0.1–6.3) 0.83 HCT-CI scores ≥2 25/64 1.97 (0.7–5.9) 0.23 1.86 (0.8–4.1) 0.13 High-risk disease HCT-CI scores 0-1 15/112 0.60 (0.1–2.7) 0.51 0.96 (0.5–2.0) 0.92 HCT-CI scores ≥2 41/99 0.34 (0.2–0.7) 0.005 0.51 (0.3–0.8) 0.006

Figure Figure
Severe Thrombocytopenia with Impaired Megakaryocytopoiesis in a Family with von Willebrand Disease Type 2B.
Authors: Nurden Paquita , Debili Najet , Vainchenker William , Pasquet Jean-Max , Bobe Regis , Bredoux Raymonde , Nurden Alan T. , Enouf Jocelyne
Published: April 2020

Thrombocytopenia associated with type 2B von Willebrand disease (VWD) is said to result from an increased elimination of platelets due to an abnormal binding of the higher molecular weight VWF multimers. Here we report a family (brother and sister) with severe thrombocytopenia, presence of agglutinated platelets and a very severe spontaneous bleeding syndrome since childhood. Their mutation concerns the VWF A1 domain, with a R1308P substitution which corresponds to an interactive site for GPIb (French VWD network). Electron microscopy showed agglutinates composed of adjacent discoid platelets with plasma membrane domains either in very close contact or showing signs of being fusioned. By flow cytometry, increased amounts of VWF and sometimes also fibrinogen were found at the platelet surface, but not P-selectin. Binding of antibodies to the GP Ibalpha N-terminal domain was decreased whereas GPIX and GPV were normally detected. In Western Blotting (WB), GPIbalpha was normally present but there were signs of filamin degradation. Basal Ca2+ levels were elevated. Platelet proteins involved in Ca2+ regulation were analysed by quantitating mRNA or by WB. Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)-type 3b was found to be increased but not the isoforms 2b or 3a. Furthermore, plasma membrane Ca2+ ATPase (PMCA)-4b as well as Inositol triphosphate receptor (InsP3-R3) were also increased in concentration. Interestingly, this platelet profile, mimics that observed in normal immature MK before proplatelet formation. As PMCA4b is a target of caspase-3 in MK, another substrate the polyadenosine diphosphate (ADP)-ribose polymerase protein (PARP) was evaluated. For both patients, platelets contained increased PARP and a degradation product indicating an abnormal persistance of caspase-3 activity. In vitro, megakaryocyte (MK) culture from peripheral blood CD34+ cells in the presence of SCF and TPO showed impaired MK maturation and the production of self-associated proplatelets. All these results are in favour of a role for abnormal platelet production with abnormal apoptosis that can explain at least in part the marked thrombocytopenia in this family with VWD type 2B.
SP1 and NF-Y-Dependent Gene Transactivation Defines a Gain-of-Function for the PML-RARα Oncoprotein.
Authors: van Wageningen Sake , Breems-de Ridder Marleen C. , Nikoloski Gorica , Erpelinck-Verschueren Claudia A.J. , Nigten Jeannet , Löwenberg Bob , de Witte Theo , van der Reijden Bert A. , Jansen Joop H.
Published: April 2020

PML-RARa is the product of the chromosomal translocation t(15;17) and is the causative oncogene in more than 98% of the cases of acute promyelocytic leukemia. PML-RARa interferes with the transcription of retinoic acid receptor target genes in a dominant manner, contributing to the transformation of the cells. RARa regulates transcription via heterodimerisation with RXR. PML-RARa can bind as a homodimer to the same sequences as RARa/RXR. At supra-physiological dosis of all-trans retionoic acid (ATRA), the block in differentiation in APL can be overcome. ATRA releases co-repressors from PML-RARa and activates RARa target genes.

The ID1 gene is rapidly induced by ATRA in acute promyelocytic leukemia cells. Promoter analysis showed that the ID1 promoter was activated by PML-RARa but, unexpectedly, not by wild type RARa/RXR. Surprisingly, PML-RARa lacking the DNA binding domain could still transactivate the ID1 promoter indicating that transactivation was mediated without direct DNA binding. Promoter deletion studies showed that adjacent NF-Y and SP1 binding sites were essential for this transactivation. A direct physical interaction was shown between PML-RARa and SP1 in vitro and chromatin immunoprecepitation assays confirmed that a complex of PML-RARa/NF-Y/Sp1 is present on the ID1 promoter in vivo. In addition, we found that ectopic expression of PML-RARa induced expression of the endogenous ID1 gene in response to retinoic acid. We propose a novel, gain-of-function model for PML-RARa in which the fusion protein interferes with the transcription of SP1-NF-Y regulated genes. Interference is mediated without DNA-binding, through direct interaction with SP1. This implicates that apart from the previously described deregulation of retinoic acid receptor target genes, PML-RARa may deregulate an additional class of genes that are nomally not regulated by retinoid receptors.
Survival Prediction Acute Myeloid Leukemia (AML) Using a Combination of DNA Methylation Analysis and Gene Expression Profiling.
Authors: Ehrich Mathias , Bullinger Lars , Nelson Mattew R. , Döhner Konstanze , Döhner Hartmut , van den Boom Dirk
Published: April 2020

Acute myeloid leukemia is classified by the presence or absence of recurrent cytogenetic aberrations. In order to improve diagnosis and therapy, more recently new studies have been performed to supplement the current classification with refined molecular information based on gene expression profiling. However, it has been established that expression levels of genes are often largely controlled by the state of cytosine methylation in the adjacent promoter region. Thus we were interested to evaluate the quantitative methylation levels for a previously identified predictive set of genes (Bullinger et al. 2004) using a novel technology based on a unique combination of base specific cleavage of single stranded nucleic acids with MALDI TOF detection. We have employed this new quantitative high throughput DNA methylation analysis technology to analyze 147 promoter regions in a total of 192 individuals. The resulting quantitative methylation data was analyzed using a semi-supervised approach to evaluate the quantitative methylation data as a predictor for patient survival. We used a first set of 96 individuals to train a statistical learning algorithm and a second set of 96 samples to validate the trained algorithm. The analysis revealed quantitative methylation patterns as a reliable predictor for survival (p < 0.001). Subsequently, we combined the methylation based predictive model with the results from the expression based predictor. The combination of both models yielded a superior predictive model for patient survival, which outperformed all clinical and cytogenetic risk stratification in the given sample set.

The results of this work revealed a potential significance of DNA methylation in the pathophysiology of AML and suggest that DNA-methylation patterns might be useful molecular markers for patient survival prediction based on the fact that large-scale DNA methylation studies can now be performed with reasonable efforts in a limited amount of time. Therefore, these results lay the groundwork for future research which might ultimately enable individualized therapy based on improved molecular characterization of AML.
C-MYC Induces KIR Expression Via a Novel Control Region Upstream of the Conventional Adult KIR Promoter.
Authors: Haack Becky , McCullar Valarie , Lenvik Todd , Pitt Michelle , Anderson Stephen , Miller Jeffrey S.
Published: April 2020

KIR determine whether NK cells will be alloreactive against targets. Although epigenetic control is thought to play a role in KIR expression, the mechanism of KIR repertoire formation is unknown. Recently, a probabilistic transcriptional mechanism has been shown to control the acquisition of mouse Ly49 receptors using a switch region upstream of each Ly49 promoter (S. Anderson). Based on this information, the DNA upstream of known KIR genes was scanned for similar motifs to test the hypothesis that similar control mechanisms are operant in human NK cells. A putative c-myc/AML binding site was found approximately 1Kb upstream of the traditional KIR promoters. Initiation of transcription in this region was observed by RNAse protection, and spliced KIR mRNAs originating in the upstream region were cloned. To look at the effects of c-myc on KIR expression, we transduced NK92 cells using retroviral murine stem cell virus vectors containing either c-myc/eGFP or eGFP alone. Cells expressing high levels of c-myc were 18% KIR positive as compared to 0.9% of control cells. To further look at the effect of c-myc on KIR from primary cells, we transduced CD34+ progenitors isolated from umbilical cord blood with c-myc/eGFP and eGFP vectors. eGFP+ cells were plated on the murine fetal liver line AFT024 and cultured with IL-15, IL-7, IL-3, Flt3 ligand, and c-kit ligand, all known to induce NK cell differentiation. Although c-myc is an oncogene, it did not promote autonomous growth of NK cells in the absence of exogenous cytokines. Proliferation was significantly increased in single cell progenitors over-expressing c-myc with 739.3x103 NK cell progeny compared to 0.56x103 from each control cell after 21 days (p<0.0001). At this early time point, 21.68 ± 3.25% of c-myc cells were KIR positive while 0.0% of control cells were KIR positive (p<0.0001) suggesting that c-myc targets KIR expression. Furthermore, bulk-transduced cells over expressing c-myc had 3.9 times the level of early non-coding transcripts originating from the upstream promoter as compared to eGFP transduced cells. A direct effect of c-myc on the upstream promoter was supported by ChIP assays demonstrating increased binding to the c-myc/AML site. The presence of these upstream transcripts directly correlates with increased conventional KIR transcripts as shown by Q-RT-PCR and an increase of KIR expression as shown by flow cytometry. These data show that c-myc play an important role in the differentiation of NK cells and in KIR acquisition. Experiments are currently in progress to look at the effects of the competing AML site on KIR expression and to investigate how non-coding RNA affects adult KIR expression. Understanding the mechanisms of KIR expression may lead to development of novel strategies to manipulate NK cells for therapeutic benefit.
Decreased Generation and Function of CD4+CD25hi T Regulatory Cells in Human STAT5b Deficiency.
Authors: Cleary Aileen , Nadeau Kari , Tu Wenwei , Hwa Vivian , Dionis Kira Y. , Bezrodnik Liliana , Teper Alejandro , Gaillard Maria , Heinrich Juan J. , Krensky Alan M. , Rosenfeld Ron G. , Lewis David B.
Published: April 2020

CD4+ CD25+ regulatory T cells (Tregs) are a well characterized population of cells that play an important role in limiting inflammation and in the maintenance of tolerance to self. Here we describe a patient with a homozygous missense mutation (A630P) in the STAT5b gene who clinically displays immune dysregulation in association with decreased numbers and function of Tregs. Freshly isolated or in vitro-derived CD4+CD25high Treg cells from this patient had low Foxp3 expression, did not suppress non Treg T-cell proliferation, and were unable to kill autologous CD4+CD25neg T cells compared to controls. CD25 expression in response to IL-2 did not increase on freshly isolated CD4 T cells and was decreased on T-cell blasts derived from the patient. The patients mother who was heterozygous for this mutation had an intermediate phenotype for all of these immune abnormalities, indicating a gene dosage effect. In contrast, IL-2 upregulated expression of the common gamma chain (γc) cytokine receptor and perforin by T cells normally. Activation-induced T-cell expression of CD40-ligand (CD154) and interferon-gamma (IFN-γ) were also normal in the patient. These results suggest that the STAT5 pathway propagates an important IL-2 mediated signal that is necessary for Treg generation and function in humans in vivo.
The TNF-a Gene Modifies Stroke Risk in Children with Sickle Cell Anemia: Results from the STOP Trial.
Authors: Hoppe Carolyn , Klitz William , D’Harlingue Katherine , Cheng Suzanne , Steiner Lori , Adams Robert , Styles Lori
Published: April 2020

Background: The etiology of stroke in sickle cell anemia (SCA) is complex and multiple genetic factors outside of the sickle gene undoubtedly contribute to stroke risk in SCA. Simultaneous analyses of multiple genetic variants may therefore be more informative than evaluation of individual candidate genes. Moreover, stroke in SCA is phenotypically heterogeneous, varying from large cortical vessel (LV) distribution infarcts to milder lacunar infarcts due to small vessel disease, suggesting distinct pathogenetic mechanisms for stroke in SCA.

We previously reported risk associations with particular TNFA, IL4R and ADRB2 polymorphisms in children enrolled in the CSSCD.

Methods: To confirm our previous findings from the CSSCD and identify other potential “susceptibility” loci that may influence the development of LV stroke, we extended our investigations to SCA children who participated in the Stroke Prevention Trial in Sickle Cell Anemia (STOP). Children with MRA evidence of obstruction or stenosis of one or more large vessels (LV disease) were compared with untransfused children with a normal MRA. Ninety-seven patients were included in this study. Of the 119 patients originally enrolled in STOP, 77 (30 MRA+, 47 MRA−) were eligible for this study and were analyzed together with a local sample of 20 MRA(+)patients. Genotyping for 110 polymorphisms among 65 candidate cardiovascular and proinflammatory genes was performed using multiplex linear array methods. Of the 110 variant sites examined, 95 were sufficiently polymorphic to permit statistical testing, using 2 x 2 exact tests.

Results: When comparing MRA(+) patients with MRA(−) patients, 9 of the polymorphic loci revealed nominally significant differences (P<0.10): TNF(−308), IL4(−589), ADRB2 16, ICAM1 56, APOC3(−482), LPL 93, AGT 235, and MMP3(−1171). For TNF G(−308)A, the frequency of the G allele was 80%, consistent with allele frequency data in African-Americans. In the current study, there was a relative excess of children with the GG genotype in the MRA(+) group compared to the MRA(−) group (OR= 2.48; p = 0.037). These results, showing the TNF(−308) locus to be predictive of LV vasculopathy, are virtually identical to our previous findings in the CSSCD population and confirm that the TNF gene plays a significant role in stroke risk. The TNF gene lies within the HLA region, making this finding particularly intriguing in light of our previous data indicating HLA associations with stroke risk. Detailed studies of this region are now necessary to determine if it is indeed the TNF locus or other genes in linkage disequilibrium that are responsible for this association.
Direct Evidence for Ex Vivo Expansion of Human Hematopoietic Stem Cells.
Authors: Ando Kiyoshi , Yahata Takashi , Sato Tadayuki , Miyatake Hiroko , Matsuzawa Hideyuki , Oki Masayuki , Kato Shunichi , Hotta Tomomitsu
Published: April 2020

Ex vivo expansion of hematopoietic stem cells (HSC) is a major challenge for clinical and experimental transplantation protocols. However, no significant clinical benefit has been demonstrated to date. Clonal kinetics of ex vivo-expanded HSCs is one of the basic transplantation biology questions to be addressed before we can optimize ex vivo expansion approaches. To characterize human HSC, xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSC, LDA is a statistical method and does not directly establish that a single HSC has self renewed in vitro. By using lentiviral gene-marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro.

To detect multiplied clones, 5x104 gene-marked CD34+ cells were cultured for 4 days in our ex vivo expansion culture system (Exp Hematol, 27:904–915, 1999), and then divided into 10 lots, each of which was transplanted directly into the bone marrow of a NOD/SCID mouse. We used linear amplification-mediated (LAM)-PCR to detect unique genomic-proviral junctions as clonal markers. Detection of the same clones in different mice would provide direct evidence of ex vivo multiplication of a SRC clone. We identified 20 clone-specific genomic-proviral junction sequences by LAM-PCR on 10 mice. Although 14 clones were detected in only one mouse, six clones were detected in more than 2 mice. In the next experiment, purified CD19+EGFP+ and CD33+EGFP+ cells from each mouse were analyzed for each clone to detect multi-lineage differentiation of amplified SRCs. We identified 15 clonal markers from 6 mice. While 12 clones were present in only one mouse, 3 clones were present in 2 independent mice and reconstituted both CD19+and CD33+cells. Finally, we designed a secondary transplantation experiment to confirm the self-renewal ability of each clone. We identified 39 clonal markers from 10 primary and 10 secondary transplanted mice, 11 of which were detected in multiple mice with secondary transplantable ability. Together, of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than two mice after serum-free and stroma-dependent culture. Some of them were secondary transplantable. Furthermore, we identified new class of stem cells based not on repopulation, or cell surface markers, but on response to cytokine stimulation in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.
Deterministic Trigger of Self-Renewal in Expanded HSCs Expressing Hoxb4 + Antisense Pbx1.
Authors: Cellot Sonia , Krosl Jana , Humphries Keith , Sauvageau Guy
Published: April 2020

We previously reported the generation of pluripotent and ultracompetitive HSCs through modulation of Hoxb4 and Pbx1 levels. These Hoxb4hiPbx1lo HSCs display a tremendous regenerative potential, yet they are still fully responsive to in vivo regulatory signals that control stem cell pool size (20 000 HSCmouse) and differentiation pathways. Further work in our laboratory attempted to circumvent these physiological constraints by expanding Hoxb4hiPbx1lo transduced HSCs in vitro, and hence revealing their intrinsic expansion potential. Independent experiments were performed where primary mouse BM cells were co-infected with retroviruses encoding antisense Pbx1 cDNA plus YFP, and Hoxb4 plus GFP (double gene transfer ranged between 20–50%). Hoxb4hiPbx1lo HSCs measured using the CRU assay expanded by 105-fold during a 12 day in vitro culture. Following serial transplantations, these cells displayed an additional 4–5 log expansion in vivo. Total stem cell content per animal remained within normal limits. Southern blot analyses of proviral integrations showed that the expansion was polyclonal, and analyses of individually expanded clones provided a molecular proof of in vitro self-renewal (SR). This unprecedented level of HSC expansion in such a short time course (105-fold in 12 days) implies an absolute HSC doubling time of approximately 17 hours in our culture, raising the possibility that virtually all dividing HSCs undergo self-renewal. This analysis prompted us to dissect the impact of Hoxb4 on cell proliferation versus cell fate (SR?).

When analyzed during the period of maximal HSC expansion, the cell cycle distribution of Sca+ or Sca+Lin− cells were comparable between the cultures initiated with neo control versus Hoxb4 BM cells (CTL vs Hoxb4: G0/G1: 66% vs 83%; S: 15% vs 9%; G2/M: 18% vs 7%). Correspondingly, CFSE tracking studies confirmed the identical, or even lower, number of cellular divisions in Sca+ cells isolated from cultures initiated with Hoxb4 versus neo transduced cells. Annexin V studies precluded protection from apoptosis as the major mechanism to increase HSC numbers since similar results (3–10% positive cells) were observed in the Hoxb4 versus neo-transduced cells.

In summary, our studies support the emerging concept that distinct molecular pathways regulate cell proliferation and self-renewal, suggesting that Hoxb4 + antisense Pbx1 predominantly triggers self-renewal over HSC proliferation.
Investigation of Hematopoietic Stem Cell and Progenitor Populations: Implication for Cell Fate Determination and Lineage Commitment.
Authors: Passegue Emmanuelle , Forsberg Camilla , Serwold Thomas , Kogan Scott , Weissman Irving L.
Published: April 2020

A thorough understanding of the lineage potential of each subset of hematopoietic stem cells (HSC) and progenitor populations is critical to establish an accurate map of cell fate determination during hematopoietic development. A controversy exists whether multipotentiality is conserved until a mutually exclusive segregation of myeloid and lymphoid potentials or whether early progenitor populations sequentially lose lineage potential as they differentiate from the long-term self-renewing HSC (LT-HSC), starting with loss of megakaryocyte/erythrocyte (MegE) potential.

Hematopoietic cells at different developmental stages can be prospectively isolated based on a combination of cell surface phenotypes and functional assays in vitro and in vivo. However, assessment of lineage potential of cells other than LT-HSC is complicated by the progressive loss of self-renewal activity in progenitor populations and the lack of congenic surface markers on mature cells of the MegE lineage. Using sensitive in vitro and in vivo approaches, we quantitatively and kinetically assessed the MegE potential of Lineage−/c-Kit+/Sca-1+ (KLS) subsets of mouse bone marrow, including LT-HSC (Thy1.1int/Flk-2−), sort-term HSC (ST-HSCF: Thy1.1int/Flk-2+) and multipotent progenitor population (MPPF: Thy1.1−/Flk-2+), and compared it with the MegE potential of downstream myeloid progenitors (CMP, GMP and MEP) and with their ability to give rise to mature myelomonocytic and lymphoid cells. In contrast to previous reports, we demonstrate that Flk2-positive ST-HSCF and MPPF populations have readily detectable but transient MegE potential in vivo that is more robust than committed myeloid progenitors CMP and MEP. We also show that these cells make clonal colonies in vitro and in vivo in the spleen that contained megakaryocytes and erythrocytes. Moreover, we established the kinetics of mature cell production from each stem and progenitor population, hence providing the timing of these early differentiation events in vivo that is of critical importance when investigating lineage potential.

Our results demonstrate that multipotentiality is retained in the KLS “stem cell” fraction of the bone marrow and support a model of hematopoietic development with mutually exclusive segregation of myeloid and lymphoid lineage potential. Taken together with previous findings, they indicate that transition from LT-HSC to ST-HSCF and then to MPPF, is accompanied by progressive lose of self-renewal ability, increased proliferation and change in gene expression programs to prepare multipotent cells to leave the stem cell niche and undergo lineage differentiation. This model is by definition a simplification of a complex biological process but accounts for most, if not all, differentiation events, tolerates plasticity in lineage segregation at early steps of commitment and it accommodates intrinsic lineage preferences during ontogeny and aging.
Adducin Forms a Bridge between the Spectrin-Actin Junctional Complex and Band 3.
Authors: Anong William A. , Mohandas Narla , An Xuli , Bodine David M. , Markham Nicholas O. , Gallagher Patrick G. , Low Philip S.
Published: April 2020

The classical model of the human erythrocyte membrane (RBCM) shows two bridges connecting the lipid bilayer to the membrane skeleton: 1) a bridge attaching the cytoplasmic domain of band 3 (CDB3) to ankyrin, which in turn binds β-spectrin, and 2) a bridge linking the cytoplasmic domain of glycophorin C to protein 4.1, which in turn binds the spectrin-actin junctional complex. Recent data, however, suggest while disruption of the band 3-ankyrin- β-spectrin interaction profoundly alters membrane stability, disruption of the glycophorin C-protein 4.1R linkage has no effect on membrane mechanical properties. In a search for additional bridges between the junctional complex and the bilayer, we discovered that adducin, a component of the spectrin-actin junctional complex, binds to band 3. Evidence for this interaction derives from a number of different experimental strategies. We have been able to show that: i) photoactivation of sulfo-SBED-labeled adducin reconstituted onto the RBCM leads to label transfer to band 3, ii) adducin binds to KI-stripped IOVs with a Kd of 280 nM and this binding is inhibited by an antibody to CDB3 (as well as by unlabeled adducin), iii) IOVs derived from normal erythrocytes retain adducin, whereas similar IOVs prepared from erythrocytes deficient in band 3 retain very little adducin, iv) the tail domain (but not the headpiece domain) of β-adducin binds to KI-stripped IOVs and this binding is competed by both anti-CDB3 and intact adducin, v) GST-labeled β-adducin tail domain can pull down band 3 in co-pelleting studies and this co-precipitation is blocked by anti-CDB3, and vi) the tail domain of β-adducin directly binds CDB3. Because adducin is an important structural component of the junctional complex, these data suggest that the junctional complex is linked to the RBCM via CDB3 and that part of the band 3 population must be located adjacent to the junctional complex. This putative new bridge between the RBCM and the spectrin-actin skeleton may also help explain why β-adducin knockout mice have unstable erythrocyte membranes.
The C-Terminus of Alpha Spectrin Binds Protein 4.2 and Is Necessary for Optimal Spectrin-Actin Binding.
Authors: Korsgren Catherine , Birkenmeier Connie S. , Barker Jane E. , Peters Luanne L. , Lux Samuel E.
Published: April 2020

The red blood cell (RBC) membrane skeleton is composed principally of short F-actin filaments crosslinked by α2β2-spectrin tetramers with the assistance of protein 4.1R. Actin and 4.1R bind to the actin-binding domain (βABD) at the N-terminus of the spectrin β-chain. The adjacent, C-terminal end of α-spectrin, contains a calmodulin-like domain (αCML, aa 2262–2418) that is also called the EF hand domain and is thought to be inert or vestigial. However, the sph1J/sph1J mouse, which has severe hereditary spherocytosis and unstable RBC membranes, makes a mutant α-spectrin that lacks the last 13 amino acids (αCMLΔC13), showing that the domain has some important function. To investigate this function we “fished” for interacting proteins using glutathione-S-transferase (GST)-fused to the CML domain—either the wildtype (αGST-CML) or sph1J (αGST-CMLΔC13). αGST-CML retrieved protein 4.2 from a 2M Tris HCl extract of spectrin-actin depleted human RBC membranes. Protein 4.2 bound αGST-CML with high affinity (Kd = 2.7 x 10−7M) but did not bind αGST-CMLΔC13. Binding was abolished by 1 mM Ca2+, which converts the CML domain to the liganded conformation. The binding site on protein 4.2 localized, at least partly, to amino acids 411–492. Because red cells lacking protein 4.2 are not as severely affected as sph1J/sph1J RBCs, we also tested the effect of the αCMLΔC13 mutation on spectrin-actin binding. A minispectrin was prepared containing the actin-binding domain plus the first four spectrin repeats of the β-chain, combined with the CML domain (±ΔC13) and the last four repeats of the α-chain. The normal and mutant minispectrins were incubated with protein 4.1R, F-actin, or both proteins. The results were striking. The minispectrin containing the normal CML domain bound actin in the presence of protein 4.1R, but the minispectrin containing the mutant CML domain did not. Similarly, the mutant minispectrin was defective in its ability to bind 125I-4.1R in the presence of a constant amount of F-actin. However, the mutation did not affect binding of the minispectrin to protein 4.1R in the absence of actin. We have not yet tested whether protein 4.2 or Ca2+ modulate the effects of the CML domain on spectrin-actin binding. In summary, these experiments clearly show that the calmodulin-like (EF hand) domain of α-spectrin, which was previously considered inert, binds protein 4.2 and also contributes to spectrin-actin binding in the presence of protein 4.1R. Further experiments will be needed to determine whether the CML domain binds actin directly or strengthens the binding of the adjacent actin-binding domain.
Relapse Patterns and Subsequent Outcomes of Patients Treated on the NCIC CTG HD.6 (ECOG JHD06) Randomized Trial Evaluating ABVD Alone in Patients with Limited Stage Hodgkin Lymphoma (HL).
Authors: Macdonald David A. , Gospodarowicz Mary K. , Wells Woodrow A. , Pearcey Robert G. , Connors Joseph M. , Winter Jane N. , Horning Sandra J. , Djurfeldt Marina S. , Ding Keyue , Shepherd Lois E. , Meyer Ralph M.
Published: April 2020

Background: We completed a randomized trial comparing ABVD alone with treatment that included extended field (EF) radiation therapy (RT) in 399 patients with limited stage HL. Patients allocated to EF RT received this as a single modality if categorized as favourable risk or preceded by 2 cycles of ABVD if unfavourable risk. The 5-year FFP was superior in patients receiving RT but no difference in overall survival was detected as the difference in FFP has been offset by deaths due to other causes (Meyer, J Clin Oncol2005; 23: 4634 42). Overall, 34 patients had progressive disease (PD) and 15 patients died (4 from HL, 2 from treatment-related toxicity and 9 from other causes). To better understand the implications of treatment with ABVD alone, we compared the patterns of PD and outcomes with secondary therapy of the two randomized groups. We were particularly interested in assessing how frequently sites of PD observed in patients assigned to ABVD alone occurred within the EF of RT that was prescribed in the HD.6 trial, or within a hypothetical involved field (IF) of RT that constitutes an accepted current practice.

Methods: Two expert reviewers, blinded to treatment allocation, were provided a list of disease sites at diagnosis and at PD. Sites of PD were categorized as ’within field only’, ’out of field only’ and ’in + out of field’. Two analyses were performed by considering both EF and IF RT fields. A third blinded reviewer adjudicated disagreements.

Results: Inter-observer agreement was excellent for both the EF (kappa 0.87) and IF (kappa = 1.0) analyses. More cases of PD were observed in patients allocated to ABVD alone (24 vs. 10). More within field only PD was observed in patients allocated to ABVD alone when assessed according to both the EF (88% vs 30%; P = 0.002) and IF (71% vs. 20%; P = 0.01) analyses. Individual investigators determined the actual choice of therapy for PD; among patients allocated to ABVD alone, secondary therapy included chemotherapy alone (2), RT alone (8), combined modality therapy (6) and autologous stem cell transplantation (8). No difference in the proportion of patients alive without second progression was detected between patients allocated to ABVD alone (18 /24; 75%) or treatment that included RT (8 /10; 80%) (P = 1.0).

Conclusion: For patients with limited stage HL, treatment with ABVD alone is associated with a greater risk of PD at disease sites that would be included in an IF of RT. Secondary therapy is often successful and further testing is required to determine the optimum choice of therapy for these patients.
Dendritic Cell Deficiencies in Pediatric Acute Lymphoblastic Leukemia Patients.
Authors: Maecker Britta , Mougiakakos Dimitrios , Zimmermann Martin , Behrens Mirja , Hollander Sophia , Schrauder Andre , Schrappe Martin , Welte Karl , Klein Christoph
Published: April 2020

Acute lymphoblastic leukemia (ALL) cells are particularly poor at generating anti-leukemia immunity, despite residing in lymphoid organs. The mechanisms accounting for this phenomenon of immunoevasion are poorly understood. To assess a potential role of dendritic cells (DC) in poor anti-leukemia immunity, we analyzed peripheral blood DC in 55 pediatric ALL patients at the time of initial diagnosis and 44 age-matched healthy controls. DC were identified by their expression of HLA-DR, lack of B-, T-, NK-, and monocyte-markers, and expression of CD11c (myeloid DC) or BDCA-2 (plasmacytoid DC) using flow cytometry. T-lineage ALL patients showed normal plasmacytoid DC and significantly elevated myeloid DC (p=0.003) levels with normal expression of HLA-DR and co-stimulatory molecules. Preliminary data suggest that these DC may be less capable of stimulating allogeneic T cell responses than DC from healthy volunteers. In contrast, we found that in children with B-lineage ALL, numbers of both myeloid and plasmacytoid DC were significantly reduced (p=0.0001) as compared to healthy controls. A decrease in DC could not be explained by impaired bone marrow function, since we could not demonstrate a correlation of DC numbers with granulocyte or monocyte counts, hemoglobin levels, or platelet counts in patients with B-lineage ALL. Interestingly, in univariate analysis there was a significant correlation of lack of myeloid DC with aberrant expression of myeloid surface marker CD13 on B-lineage ALL blasts. This unexpected finding raises the hypothesis that in some cases of B-lineage ALL the physiological differentiation of a common myeloid progenitor cell might be blocked and differentiation might be skewed towards leukemia-development. No correlation of DC numbers with subtype of ALL (proB, common, preB ALL) could be demonstrated. Furthermore, in a subgroup of B-lineage ALL patients, a complete lack of plasmacytoid DCs correlated with prednisone poor response, a surrogate marker of poor prognosis. It remains currently unclear, whether lack of myeloid or plasmacytoid DC directly correlates with long-term survival. Thus, depletion of DC in B-lineage ALL patients may contribute to poor anti-leukemia immune responses in pediatric patients with ALL. Further investigations will prove whether these deficiencies may impact on patient survival and may support the concept of immunosurveillance in lymphatic malignancies.
Induction of CD33 Expression in B-Precursor ALL by E2A-HLF Fusion Transcription Factor Derived from t(17;19).
Authors: Akahane Koshi , Inukai Takeshi , Inaba Toshiya , Kurosawa Hidemitsu , Endo Mikiya , Goto Hiroaki , Kiyokawa Nobutaka , Look A. Thomas , Goi Kumiko , Sugita Kanji , Nakazawa Shinpei
Published: April 2020

CD33 (Siglec-3), one of myeloid-specific antigens, belongs to a family of sialic acid-binding, immunoglobulin-like lectins (Siglecs), which is adhesion receptor for sialic-acid bearing ligands and regulates a signal transduction through its cytoplasmic tyrosine residues designated immunoreceptor tyrosine-based inhibitory motif (ITIM) as representatively well-characterized in CD22 (Siglec-2). CD33 is ectopically expressed in 5–20% of B-precursor ALL, and has been reported to be potentially associated with poor prognosis. This myeloid-specific antigen expression in B-precursor ALL is considered as lineage promiscuity or lineage infidelity, but underlying mechanism remains totally unknown. We have recently found that B-precursor childhood ALL with t(17;19), which has extremely poor prognosis, is principally CD33 positive, suggesting that CD33 expression might be regulated by E2A-HLF fusion transcription factor generated by t(17;19). To test this hypothesis, using zinc inducible expression vector, we have introduced E2A-HLF into Reh cells, which is a CD33-negative B-precursor ALL cell line. In flow cytometric analysis, by the addition of zinc, CD33 expression was gradually induced in Reh cells transfected with E2A-HLF but not in control cells, and returned to baseline after deprivation of zinc. Of note, mutant of E2A-HLF defective in its DNA-binding domain did not induce CD33 expression. Induction of CD33 expression was not due to “myeloid differentiation”, since upregulation of other myeloid markers including CD11b, CD13, CD14, CD15 and cytoplasmic myeloperoxidase was not observed. In real-time PCR analysis, despite the immediate expression of E2A-HLF protein after addition of zinc, the CD33 mRNA was slowly induced and progressively upregulated to the level approximately 20 times higher than baseline at 5 days after addition of zinc. In promoter region of the CD33 gene, there was no authentic HLF-binding consensus sequence. These observations suggest an indirect induction of CD33 transcription by E2A-HLF. As reported in myeloid cells, when CD33-positive t(17;19)-ALL cell lines were treated with a phosphatase inhibitor pervanadate and analyzed with immunoprecipitaions and Western blotting assay using anti-CD33 antibody, CD33 was confirmed to be tyrosine-phospholyrated and recruit both the protein-tyrosine phosphatases, SHP-1 and SHP-2. This is a first report indicating that an oncogenic fusion transcription factor is evidenced to be associated with ectopic expression of CD33 in B-precursor ALL and that CD33 might play some roles in signal transduction in B-precursor ALL cells.
Upregulation of FLIPs upon CD40 Stimulation - A Novel Inhibititory Mechanism of CD95-Induced Apoptosis in Precursor B-ALL Blasts in Children.
Authors: Troeger Anja , Schmitz Ingo , Glouchkova Ludmila , Siepermann Meinolf , Janka-Schaub Gritta , Schulze-Osthoff Klaus , Dilloo Dagmar
Published: April 2020

The influence of the microenvironment on the activation status and behaviour of ALL blasts is critical for interactions with the immune system in vivo. The capacity of B cells to respond to CD40-ligand (CD40L) stimulation is critical for their sensitisation to immunological control mechanisms and susceptibility to apoptotic signals. Primary precursor B-ALL blasts (BCP-ALL; n=32) lack CD95-expression (mean±SE; 4.2±0.6% positive cells) and are resistant to apoptosis while significant up-regulation of CD95 is apparent upon CD40-stimulation in BCP-ALL blasts that reaches a plateau after 72 h. Yet, in spite of equivalent CD95-upregulation in ALL blasts (58.3±6.5%; n=17) and normal B cells (59.3±13.1%) specific apoptosis is markedly lower in ALL compared to mature B cells (19.1±3% vs 36.7±5.5%). Resistance to apoptosis in ALL blasts and its reversibility after cycloheximid treatment suggest that anti-apoptotic mechanisms prevent induction of cell death via CD95 ligation in CD40 activated blasts. In accordance, in CD40-activated ALL blasts caspase 8 and 3 activity is not enhanced upon CD95 ligation in contrast to an 1.8±0.3 and 1.7±0.3 fold increase in caspase activity in stimulated normal B cells (n=7), suggesting a block of the apoptotic cascade in BCP-ALLrelatively close to the receptor level. CD40L-activated ALL blasts and normal B cells were submitted to western blot analysis with respect to the molecules associated to the death-inducing signalling complex (DISC). FADD and the zymogen form of caspase-8 are constitutively expressed in both malignant and non malignant B cells with no modulation following CD40 ligation. In contrast, the anti-apoptotic short isoform of the c-FLICE inhibitory protein FLIPS is weakly expressed in naïve blasts and B cells, but strongly up-regulated upon 72h CD40-ligation in ALL with only barely detectable levels in CD40-activated normal B cells. We therefore propose, that prolonged induction of the FLIPS expression inhibits the onset of apoptosis despite high CD95 surface expression levels in BCP-ALL blasts. As an additional anti-apoptotic mechanism inhibiting the downstream effector caspases we demonstrated significant upregulation of the inhibitor of apoptosis protein (IAP) survivin in CD40-activated BCP-ALL (n=6) compared to the unstimulated control (632pg/ml±200pg/ml vs 180pg/ml±52pg/ml). Thus, we identified FLIPS as a CD40-regulated upstream anti-apoptotic element and concomitant downstream upregulation of survivin protein expression as critical mechanisms contributing to blast cell resistance to apoptosis.
Acute Lymphoblastic Leukemia in Children with Associated Genetic Conditions Other Than Down Syndrome. The AIEOP Experience.
Authors: Ziino Ottavio , Rondelli Roberto , Porta Fulvio , Mura Rossella , Micalizzi Concetta , Conter Valentino , Boccalatte Marie France Pinta , Messina Chiara , Luciani Matteo , Barisone Elena , Locatelli Franco , Pession Andrea , Basso Giuseppe , Biondi Andrea , Masera Giuseppe , Aricò Maurizio
Published: April 2020

Background and Objectives. Constitutional molecular defects are known to play a role in oncogenesis, as shown by the increased incidence of embryonic cancers in children with Beckwith-Wiedemann syndrome or of leukemia in children with Down syndrome. The association between acute lymphoblastic leukemia (ALL) and genetic diseases other than Down syndrome has been less widely investigated. The aim of this study was to address this issue in a large series of childhood ALL patients prospectively treated in the AIEOP-ALL studies.

Design and Methods. We retrospectively reviewed the data-bases of AIEOP studies ALL-79, 82, 87, 88, 91, 95, and 2000 to identify all patients in whom an associated genetic disease, other than DS, had been reported.

Results. Out of the total of 6.695 patients, 42 (0.62%) were reported to have an associated genetic condition, including b-thalassemia (n=10), ataxia-telangiectasia (n=5), neurofibromatosis (n=3), Sotos syndrome (n=2) and a number of other individual conditions. Complete remission was achieved after induction therapy in 37 patients (1 died, 2 were resistant, 2 were not evaluable), of whom 10 had an adverse event, either death during remission (n=1), leukemia relapse (n=8), or went off-study (n=1). In the two largest groups, while patients with b-thalassemia had an outcome comparable to that of the general population, those with ataxia-telangiectasia, all with T-cell ALL, had a higher frequency of adverse events.

Interpretations and Conclusions. In the AIEOP experience, 0.62% of the patients enrolled in the childhood ALL studies have an associated genetic disorder; due to the reasonable risk of underestimation, this should be taken as a minimal rate. Although such patients should have access to contemporary ALL-directed therapy, those with ataxia-telangiectasia are more likely to deserve treatment modifications. Childhood leukemia specialists should pay special attention for screening of clinical signs or symptoms, as well as personal and family history, which may be relevant for identification of a possible associated condition. Study of such associations may provide additional clues for studying leukemogenesis in humans. Given the high probability of cure, for most patients leukemia will be only one part of their medical history. Thus, a correct diagnosis of the underlying genetic condition may be particularly useful for the patients and their families in order to tailor the future medical follow-up also during adulthood.