bluuebunny/crossref_metadata_2025 huggingface dataset:
Shigatoxin Triggers Thrombotic Thrombocytopenic Purpura in Genetically Susceptible ADAMTS13-Deficient Mice.
Authors: Motto David G. , Chauhan Anil K. , Zhu Guojing , Homeister Jonathon , Lamb Colin B. , Desch Karl C. , Tsai Han-Mou , Wagner Denisa D. , Ginsburg David
Published: April 2020

The life threatening disease TTP is associated with ultra-large von Willebrand Factor multimers (UL-VWF) in the circulation due to inherited or acquired deficiency of the ADAMTS13 metalloprotease. Here we show that ADAMTS13-deficient mice generated by gene targeting are viable and exhibit normal survival through 2 years of age. Despite the absence of VWF-cleaving protease activity (<1% of normal), wild-type and ADAMTS13-deficient plasma exhibit identical VWF multimer distributions, and Adamts13−/− mice develop spontaneous TTP at an extremely low rate (2 cases out of 358 mice). However, intravital microscopy demonstrated that VWF-mediated platelet-endothelial interactions are significantly prolonged in Adamts13−/− mice. These observations suggested that additional environmental triggers and/or genetic modifying factors may be required to bring about TTP in the setting of ADAMTS13 deficiency. To address the effect of VWF level on development of TTP, Adamts13−/− mice were crossed to mice of the CASA/Rk strain which exhibit markedly elevated plasma VWF levels. Resulting CASA/Adamts13−/− mice demonstrated plasma VWF ranging from 150% to 600% of C57BL/6 controls, and we found that 21% of these mice were thrombocytopenic at baseline (vs. 0% of controls). Introduction of the CASA/Rk genetic background also resulted in the appearance of UL-VWF in CASA/Adamts13−/− mice, further prolonged VWF-mediated platelet-endothelial cell interactions, increased the rate of spontaneous TTP, and markedly decreased survival. Challenge of CASA/Adamts13−/− mice with shigatoxin (derived from bacterial pathogens associated with the related human disease hemolytic uremic syndrome) resulted in a striking syndrome closely resembling human TTP, with thrombocytopenia, profound microangiopathic hemolytic anemia, and platelet- and VWF-thrombi seen in multiple organs. Surprisingly, we observed no correlation between plasma VWF level and severity of TTP, implying the existence of TTP-modifying genes distinct from VWF. Our laboratory is pursuing the identification of these genes which may provide insight into the pathogenesis and treatment of TTP in humans. Finally, our data also suggest that microbial-derived toxins, or possibly other sources of endothelial injury, may be among the key factors required to trigger acute TTP in the setting of ADAMTS13 deficiency.
The Prothrombotic Polymorphism Prothrombin G20210A Is an Ancestral Mutation That Arose in Caucasians Approximately 20,000 Years Ago.
Authors: Zivelin Ariella , Mor-Cohen Ronit , Kovalsky Victoria , Conard Jacqueline , Peyvandi Flora , Pabinger Ingrid , Bertina Rogier , Peyvandi Ferial , Emmerich Joseph , Seligsohn Uri
Published: April 2020

The prothrombotic prothrombin G20210A polymorphism is associated with increased plasma levels of prothrombin, it confers a 2–5 fold increase in the risk of venous thromboembolism and is prevalent in Caucasians. We previously showed that prothrombin G20210A originated in a single ancestral founder (Zivelin et al. Blood92:1119, 1998). In this study we estimated the age of the G20210A polymorphism by assessing linkage disequilibrium (LD) between A20210 and 9 microsatellites markers or 5 SNPs located upstream and downstream of the prothrombin gene (Figure). The study group included 93 unrelated A20210 homozygotes (21 Israelis, 19 French, 17 Italians, 17 Austrians, 16 Dutch and 3 Germans) and 63 unrelated healthy Caucasian homozygotes for G20210. Calculation of the mutation age was done by the DMLE+ program (Reeve and Rannala, Bioinformatics18:894, 2002), which allows Bayesian inference of mutation age based on observed LD at multiple genetic markers. Assuming 20 years as a generation, analysis of the microsatellite markers yielded an estimated age of 19,750 years (95% CI 16,200–29,500), and analysis of the SNPs yielded an estimated age of 20,500 years (95% CI of 14,700–27,950). We previously estimated by a different method (Risch et al. Nature Gen9:152, 1995) that the age of factor V Leiden, also prevalent only in Caucasians, is 21,000–34,000 years (Zivelin et al. Blood89:397, 1997). In the present study we recalculated the age of factor V Leiden by the DMLE+ program in 117 homozygous for factor V Leiden and 160 controls and obtained a value of 20,600 years (95% CI of 15,400–29,800). These data suggest that both relatively common prothrombotic polymorphisms occurred about 20,000 years before our time following the evolutionary divergence of Africans from Non-Africans and of Caucasians from Oriental subpopulations.

Figure Figure
A Platelet Shedding Defect in Mice Lacking the Basic Helix-Loop-Helix Transcription Factor Scl.
Authors: McCormack Matthew P. , Hall Mark A. , Schoenwaelder Simone M. , Jackson Shaun P. , Ellis Sarah , Jane Stephen M. , Curtis David J.
Published: April 2020

Transcriptional regulation of megakaryocyte maturation including platelet shedding is poorly characterised. One potential regulator is Scl, a basic helix-loop-helix transcription factor essential for hematopoietic specification in embryonic development. Scl is expressed in megakaryocytes and erythroid cells, forming DNA binding complexes with other transcription factors including GATA-1. To overcome embryonic lethality of Scl knockout mice, we have generated conditional Scl knockout mice to allow deletion of Scl in cell lineages including megakaryocytes. Scl-null adult mice have mild thrombocytopenia (730±90x109/L compared with 1400±220x109/L in control mice) despite normal numbers of megakaryocytes. Megakaryocyte ploidy was increased in the absence of Scl (modal ploidy 32n compared with 16n in control mice) indicating that Scl is not required for megakaryocyte development and endomitosis. Platelet survival studies demonstrated a normal platelet half-life of Scl-null platelets, suggesting that thrombocytopenia was due to impaired platelet production. This defect was more pronounced in the setting of pharmacologic administration of thrombopoietin (Tpo) or 5-fluorouracil, with the platelet count in Scl-null mice remaining static despite appropriate increases in megakaryocyte number and ploidy. Ultrastructural studies of Scl-null megakaryocytes were performed to examine the function of Scl in later stages of megakaryocyte maturation. Transmission electron microscopy demonstrated markedly disordered platelet demarcation membranes and a lack of platelet granules, defects reminiscent of GATA-1-null megakaryocytes. Gene expression analyses of FACS-sorted Scl-null megakaryocytes demonstrated a 5-fold reduction in β-1 tubulin, a proposed target gene of GATA-1 and NF-E2 in megakaryocytes. Consistent with the role of this gene in maintaining platelet discoid shape, Scl-null platelets were abnormally round. The similarity of defects of GATA-1- and Scl-null mice implies that these genes act together to regulate platelet shedding and structure.
CTCF and Nucleophosmin Mediate Long Distance Cyclin D1 Deregulation in t(11;14) B Cell Malignancies.
Authors: Epner Elliot M. , Wang Jin , Liu Hui , Clurman Bruce
Published: April 2020

Translocations involving the immunoglobulin heavy chain gene locus (IgH) are common in B cell malignancies. One common target gene is cyclin D1, which is deregulated in most patients with mantle cell lymphoma (MCL) and 20–30% of patients with multiple myeloma (MM). Cyclin D1 is known not to be expressed in normal lymphocytes, yet is deregulated by IgH translocations and insertions. The molecular mechanisms of long distance cyclin D1 activation by IgH regulatory elements in B cell malignancies have been obscure. Chromatin immunoprecipitation (ChIP) assays demonstrated the presence of the CCCTC-binding factor (CTCF) at both the cyclin D1 promoter and 3′ IgH regulatory regions only in cyclin D1 expressing malignant B cell lines containing IgH translocations or insertions. The nucleolar protein nucleophosmin (B23; NPM), a newly identified CTCF interaction partner, exhibited a similar distribution in ChIP assays. Combined FISH/immunofluorescence labelling studies have shown the presence of the cyclin D1 loci colocalized with CTCF and nucleophosmin at the nucleolus in MCL cell lines and patient samples, “Knockdown” of NPM by shRNA shows a specific growth arrest in MCL and MM cell lines containing the t(11;14) and cell context dependent apoptosis. This growth arrest is not mediated by changes in cyclin D1 RNA or protein levels. Tethering of the cyclin D1 promoter to the 3′Cα IgH regulatory regions at the nucleolar membrane by CTCF/nucleophosmin allows spatial juxtaposition of the cyclin D1 promoter and 3′IgH regulatory elements and provides a mechanism for long distance cyclin D1 deregulation. Nucleophosmin is also identified as a target for novel therapies in t(11;14) B cell malignancies.
PLAGL2 Cooperates in Leukemia Development by Direcly Inducing c-Mpl Expression.
Authors: Landrette Sean F. , Castilla Lucio H.
Published: April 2020

Inversion of chromosome 16 is found in 12% of human acute myeloid leukemia (AML). This inversion creates the CBFΒ-SMMHC fusion protein, which hinders myeloid differentiation and triggers AML in collaboration with additional mutations. We have previously shown that the zinc finger transcription factor PLAGL2 efficiently cooperates with CbfΒ-SMMHC in murine AML, and that PLAGL2 transcript levels are significantly increased in inversion 16 human AML. The mechanism of PLAGL2 action in AML, however, is not understood.

In order to identify candidate PLAGL2 target genes in AML, we performed gene expression profiling of primary hematopoietic progenitors expressing CbfΒ-SMMHC and PLAGL2, as well as in AML samples induced by CbfΒ-SMMHC and PLAGL2. The expression of c-Mpl transcript was increased 8 to 32 fold in progenitors and AML samples when compared to controls not expressing PLAGL2. This increase was confirmed by quantitative PCR analysis and correlated with increased c-Mpl receptor in membrane by flow cytometry. Analysis of the c-Mpl proximal promoter identified three conserved Plag binding sites. PLAGL2 binding to these sites was confirmed by chromatin immunoprecipitation and luciferase reporter assays. Furthermore, PLAGL2/CbfΒ-SMMHC AML cells show a significant increase in sensitivity to the c-Mpl-ligand thrombopoietin when compared to non-PLAGL2 CbfΒ-SMMHC-AML samples as measured by Jak2 phosphorylation.

To test whether PLAGL2 induces AML by increasing c-Mpl levels, AML development was analyzed in bone-marrow transplantation assays using CbfΒ-SMMHC-expressing donor cells transduced with c-Mpl expressing retrovirus. All transplanted mice (8/8) succumbed to leukemia with a median latency of 8 weeks, while control groups remained unaffected for 16 weeks. The pathology of Mpl/CbfΒ-SMMHC AML was immuno-phenotypically and morphologically identical to that of PLAGL2/CbfΒ-SMMHC AML.

This study demonstrates that PLAGL2 directly induces c-Mpl receptor expression, and that such increase cooperates with CbfΒ-SMMHC in AML development. These results suggest that AML can result from the alteration of upstream regulators of Mpl/Jak2 signaling.
Trends and Socio-Economic Inequalities in Cancer Survival of Patients with Hodgkin’s Lymphoma Diagnosed in England and Wales between 1986 and 1999.
Authors: Bohlius Julia , Rachet Bernard , Nolte Ellen , Engert Andreas , Coleman Michel
Published: April 2020

Background: Similar to other cancers in England and Wales, survival from Hodgkin’s lymphoma is influenced socially patterned, with those in the most deprived groups showing worse survival rates. Survival from Hodgkin’s lymphoma increased during the 1980s, however, so did the gap in survival between the most affluent and most deprived groups. It is not known whether the suggested widening of this gap continued into the 1990s.

Objective: To examine trends in relative survival (RS) from Hodgkin’s lymphoma in England and Wales diagnosed between 1986 and 1999. It explores how survival varies by socio-economic background and how these differences change over time.

Methods: Anonymized data of 14,831 patients with Hodgkin’s lymphoma diagnosed in England and Wales between 1986 and 1999 and followed up until the end of 2001 were analyzed. We estimated 5-year relative survival (ratio of observed survival of cancer patients and survival that would have been expected if the patients had had the same age- and sex-specific mortality in each time period as the general population). We examined the periods 1986-1990, 1991–1995 and 1996–2001. Patients were assigned to one of five deprivation categories based on the area of residence, from ‘affluent’ to ‘deprived’ using both the Carstairs score (Carstairs 1995) and the ward income domain score, a subcomponent of the indices of multiple deprivation. We estimated differences in survival between the deprivation categories using linear regression, weighted by the variance of the relative survival estimate. Men and women were analyzed separately.

Results: Among men 5-year relative survival improved consistently and statistically significant from 76% in the late 1980s to 83% in the early 1990s. The 5-year relative survival improved on average by 4.23% (95% CI 1.22 to 7.25, p=0.007) every five years. For women there was no evidence that RS improved over time (RS 0.89, 95% CI −3.04; 4.82, p-value = 0.66). For both men and women there was evidence for a deprivation gap in survival. Relative survival at 5 years was on average 5% percent less in men from poor (‘deprived’) areas compared to men from wealthy (‘affluent’) areas (RS −4.9; 95% CI −9.5; −0.39, p=0.036). In women the RS at 5 years was 6% less in women from poor areas compared to women from wealthy areas (RS −6.12; 95% CI −12. to −0.24, p=0.04). There is no conclusive evidence whether the survival gap by deprivation changed over time.

Conclusion: Relative survival of patients diagnosed with Hodgkin’s lymphoma in England and Wales improved only for men, but not for women. The existing deprivation gap in relative survival for both men and women confirms that cancer survival depends on socio-economic background and is inequitable. More research is required to better understand how socio-economic factors affect survival from Hodgkin’s lymphoma.
Emp Null Mice Are Non-Viable and Exhibit Erythroid Differentiation Defect.
Authors: Soni Shivani , Bala Shashi , Gwynn Babette , Sahr Kenneth E. , Peters Luanne L. , Hanspal Manjit
Published: April 2020

Emp, erythroblast macrophage protein, was originally detected in erythroblasts and macrophages, which form erythroblastic islands during erythropoiesis in the human bone marrow. The physical contact between erythroblasts and macrophages was suggested to promote the terminal maturation of erythroblasts, leading to their enucleation in vitro. To evaluate the function of Emp in vivo, we employed gene targeting studies to develop an Emp(−/−) mouse model. Mouse embryonic stem cells containing a gene-trap insertion in Emp were obtained from BayGenomics. Insertion of the gene-trap vector into Emp was verified by direct sequencing of cDNA obtained by 5′RACE. Chimeric mice generated by blastocyst microinjection were intercrossed, and the offspring were genotyped by PCR and Southern hybridization. The Emp (+/−) mice were healthy and fertile. However, no live Emp (−/−) mice were found among the progeny of the Emp (+/−) intercrosses. Analysis of timed pregnancies revealed that Emp (−/−) embryos were present at a frequency roughly consistent with Mendelian inheritance throughout the embryonal stages. Homozygous Emp (−/−) embryos were small and pale compared to their littermates, and they survived embryonic development but died at birth. To determine the effect, if any, of Emp gene deletion on definitive hematopoiesis, livers of +/+, +/−, and −/− embryos at E15.5 were examined after H&E and Giemsa staining of paraffin-embedded serial sections, and cytospins. We found few mature erythroid cells in the sinusoids of homozygotes, in contrast to those of either wild-type or heterozygotes, where abundant enucleated red blood cells were observed. Although nucleated erythrocytes were found in both wild-type and mutant embryos, their relative proportions were very different: the less mature forms (proerythroblasts) predominated in the −/− embryos whereas the more mature forms (polychromatophilic/orthochromatic and enucleated erythrocytes) were most common in +/+ and +/− embryos. Furthermore, erythroblastic islands consisting of a central macrophage surrounded by developing erythroblasts were seen in the cytospin preparations of wild-type and heterozygote livers but not in those of homozygous null livers. Since fetal liver macrophages (FLMs) are indispensable for definitive erythropoiesis, we investigated the effect that Emp’s absence might have on development of FLMs. The E15.5 fetal liver sections were stained with the macrophage-specific F4/80 antigen. Numerous F4/80-positive macrophages were present throughout the liver of normal embryos whereas, the number was substantially reduced in Emp (−/−) liver. In summary, in the absence of Emp, FLMs are significantly reduced and terminal maturation of erythroid cells is negatively affected. Thus, the availability of Emp(−/−) embryos will provide a unique experimental model to study the function of macrophages in definitive erythropoiesis.
Recent Interim Analysis of the HD11 Trial of the GHSG: Intensification of Chemotherapy and Reduction of Radiation Dose in Early Unfavorable Stage Hodgkin’s Lymphoma.
Authors: Diehl Volker , Brillant Corinne , Engert Andreas , Mueller Ralf-Peter , Eich Hans-Theodor , Mueller-Hermelink Konrad , Herrmann Richard , Markova Jana , Ho Anthony , Hiddemann Wolfgang , Doerken Bernd , Greil Richard , Josting Andreas , Pfistner Beate
Published: April 2020

Background: Combined modality treatment consisting of chemotherapy (CT) followed by involved field radiotherapy (IF-RT) is the standard treatment for early unfavourable Hodgkin’s lymphoma (HL). Despite high complete remission (CR) rates, failures are common. We thus compared the baseline-dose BEACOPP regimen with ABVD and 20 with 30 Gy IF-RT in a prospectively randomized trial (HD11) in an attempt to improve outcome in this group of patients.

Methods: Between May 1998 and January 2003, 1570 patients (pts) aged 16–75 with untreated intermediate stage HL (CS I, IIA with risk factors or IIB with elevated ESR and/or ≥3 nodal areas only) were randomized according to a factorial design between 4 cycles of ABVD followed by 30 Gy IF-RT (arm A - standard treatment), 4 ABVD + 20 Gy IF-RT (arm B), 4 baseline-dose BEACOPP + 30 Gy IF-RT (arm C) and 4 baseline-dose BEACOPP + 20 Gy IF-RT (arm D).

Results: In the fifth preplaned interim analysis, 1293 pts were evaluable for the chemotherapy comparison and 1274 for the radiotherapy comparison. Patient characteristics were well balanced between the treatment arms. 95% of patients treated reached CR, 2% had pogressive disease, 8% relapsed and the total mortality rate was 4% with no significant differences between treatment arms for either endpoint. The most frequent haematological toxicities during chemotherapy were leucopenia observed in 32% of pts (ABVD: 25%, BEACOPP: 39%) and anemia in 4% of pts (ABVD <1%, BEACOPP 7%). Infection rate was 5% (ABVD 3%, BEACOPP 7%). The most frequent toxicity during radiotherapy was dysphagia in 5%. 14 secondary neoplasias were observed: 2 AML, 4 NHL, 8 solid tumors with no significant differences between treatment arms.

After a median observation time of three years, freedom from treatment failure (FFTF) was 87% (95%-CI 85–89) and overall survival (OS) was 96% (95%-CI 95–97). Both for FFTF and OS, there was no sequential significant difference either between ABVD (FFTF 87%, OS 97%) and BEACOPP (FFTF 88%, OS 96%) nor 30 Gy (FFTF 90%, OS 97%) and 20 Gy IF-RT (FFTF 87%, OS 97%).

Conclusions: At three years of median observation time, no sequential significant differences in treatment outcome were detected, neither between chemotherapy regimens nor between the different doses of radiotherapy, despite more relapses in 20 Gy radiotherapy arms.
Nuclear Dynamics and Gene Activation during Erythroid Maturation.
Authors: Bender M. A. , Ragoczy Tobias , Byron Rachel , Telling Agnes , Groudine Mark
Published: April 2020

We have investigated the relationships among nuclear positioning, association with RNA polymerase II (PolII) and expression of the murine β-globin locus during erythroid differentiation, as well as the role of the locus control region (LCR) in these processes. Fetal liver cells from wildtype and LCR-deletion mouse strains were stained with a panel of antibodies, and flow cytometry was used to define and isolate cells from four stages of erythropoiesis spanning pro-erythroblasts (stage 1) to orthochromatic normoblasts and nucleated RBC (stage 4). DNA FISH analyses reveal that with increasing erythroid maturation the β-globin locus is less likely to be located in the nuclear periphery. Immuno-FISH demonstrates that PolII speckles initially are spread diffusely throughout the nucleus, with the exception of extreme periphery. With erythroid maturation the number of speckles decline and become more centrally located. Combined DNA FISH /PolII immuno-staining reveals an increase in co-localization of PolII and β-globin alleles during erythroid maturation. These results are consistent with a model in which the locus is more likely to be in the nuclear periphery and away from PolII foci prior to activation, and with maturation, the locus re-locates more centrally and associates with PolII speckles, leading to β-globin activation. To investigate this model, α and β-globin primary transcript FISH were performed and revealed that while β activation lags behind α, both start at stage 2 and increase with maturation. Notably, activation occurs prior to the increased localization of the locus away from the periphery during maturation, suggesting that localization of the locus away from the periphery may be secondary to the redistribution of PolII during erythroid maturation. Analysis of sorted erythroid cells from mice with a targeted deletion of the LCR reveals less co-localization of the β-globin locus and PolII speckles and loss of re-localization away from the periphery during maturation. Moreover, primary transcript FISH analysis of sorted cells from mice lacking the LCR and those carrying a combined deletion of HS 2 and 3 suggests that the LCR affects the likelihood that an allele is expressed, as well as the amount of transcript generated during periods of expression. Taken together our results suggest that the LCR plays a role in locating to, or stabilization of, interactions between the locus and PolII speckles, increasing the probability of β-globin expression, after which the LCR also affects the rate of transcription.
Identification of Switching-Related Transcription Factors from a Comparison of Cord and Adult Human Blood Reticulocytes Transcriptomes.
Authors: Goh Sung-Ho , Joselyn Matthew , Lee Y. Terry , Reed Christopher , Miller Jeffery L.
Published: April 2020

Hemoglobin switching patterns during human ontogeny are highly correlated with transcription of the alpha and beta globin genes on chromosomes 16 and 11, but the molecular and genetic mechanisms responsible for the switching phenomenon are not yet defined. For a better understanding of post-natal globin gene switching and silencing, the expression profiles from purified reticulocyte mRNA (28 separate clinical samples; 14 cord, 14 adult) were studied using Affymetrix HG-U133 arrays (44,229 probe sets). To validate the transcriptome expression levels derived from the microarrays and to discriminate the differences between cord blood and adult blood reticulocytes, we performed quatitative real-time PCR. Among 21 tested control genes, the ratios of adult-to-cord blood (AB/CB) array hybridization intensities versus PCR copy numbers demonstrated a correlation coefficient of 0.978. Hence, the AB/CB array hybridization intensities provide a reliable description of erythroid transcriptome patterns associated with post-natal hemoglobin switching. Stringent screening criteria were then used to identify genes that may be involved in fetal-to-adult hemoglobin switching. All 44,229 probe sets were filtered to identify genes that demonstrated greater than a five fold difference in AB and CB mean signal intensities as well as consistent differences among the 14 samples from each group. In addition, the screening criteria required high-level signals in either AB or CB (>1000 FU), and t-test p-values between AB and CB <0.0001. 107 switching-related probe sets from 94 genes met these criteria, and over 90% of them were detected at higher levels in CB. When this inventory of probe sets was categorized using Gene Ontology, less than half were assigned a known molecular function. Among those, five transcription factors were identified including LIM domain only 2 (LMO2) and Kruppel like factor 3 (KLF3). Those two transcription factors play known roles in hematopoiesis, and LMO2 is required for embryonic erythropoiesis. Two other genes encode zinc finger proteins, but no description of their expression in hematopoietic tissues has been reported. Finally, a novel gene encoding chromatin remodeling and PHD-finger domains was discovered. Postnatal hemoglobin switching is associated with significant changes in the reticulocyte transcriptome. Among the transcripts demonstrating highly significant shifts in their levels, known and novel transcription factors were identified.
Vaccination with the Heptavalent Pneumococcal Conjugate Vaccine Prevenar™ Provides Early Protective Antibody Responses in Children after Allogeneic Stem Cell Transplantation.
Authors: Meisel Roland , Dirksen Uta , Schubert Ralf , Gruhn Bernd , Strauss Gabriele , Duffner Ulrich , Groll Andreas , Beutel Karin , Bluetters-Sawatzki Renate , Holter Wolfgang , Feuchtinger Tobias , Gruettner Hans-Peter , Zielen Stefan , Laws Hans-Juergen , Dilloo Dagmar
Published: April 2020

Children undergoing allogeneic hematopoietic stem cell transplantation (HSCT) are at significant risk for life-threatening infections with Streptococcus pneumoniae especially in the first two years after transplantation. Due to immaturity of the immune system, immunization with conventional pneumococcal polysaccharide vaccine fails to elicit protective immunity in the majority of pediatric HSCT recipients. The heptavalent pneumococcal conjugate vaccine Prevenar™ (PCV7; Wyeth Pharma) provides effective protection from invasive disease in infants, who like aHSCT recipients are unable to respond to polysaccharide vaccines due to immunologic immaturity. Currently there is a dearth of data on the safety and efficacy of PCV7 vaccination in pediatric transplant recipients. Here we report tolerability and profound immunogenicity of PCV7 in children after HSCT and show for the first time that PCV7 provides early protective anti-pneumococcal antibody responses in a homogeneously treated cohort of pediatric HSCT recipients recruited into a prospective, multicenter vaccination trial. 53 children (median age 8,3 (1,416,9) years), who underwent HSCT from related (n=24) or unrelated (n=29) donors were immunized with three doses of PCV7 in monthly intervals starting at 6–9 months after HSCT. PCV7 was administered in combination with a hexavalent vaccine against Tetanus, Diphtheria, Pertussis, Poliovirus, Haemophilus influenzae type B and Hepatitis B (Infanrix hexa™; GlaxoSmithKline Pharma). Serum was obtained at baseline and 4–6 weeks after the 2nd and 3rd vaccine and local and systemic adverse events were recorded for 28 days after each vaccination. Responses, defined as seroconversion to protective serotype-specific antibody concentrations (Ab conc.) of 0,5 mg/ml or >2-fold increase were detected in 41.9% – 86.0% of patients (pts) after the 2nd vaccine and in 58.1% – 90.7% of pts after the 3rd vaccine (n=43). As a result of a highly significant increase in mean geometric Ab conc. (GMCs) (p<0.001) protection to all 7 vaccine serotypes was achieved in 56% and 74% of pts after the 2nd and 3rd application of PCV7 at a median of 10 and 11 months after transplantation, respectively. Even in a small subgroup of pts suffering from chronic GvHD 5/7 pts attained protective Ab conc. to 6/7 serotypes (n=7). Local and systemic tolerability was very good and no vaccine-related severe adverse events were observed. In conclusion, our data show that vaccination with PCV7 elicits highly protective anti-pneumococcal antibody responses in pediatric HSCT recipients within the 1st year following aHSCT. Therefore, PCV7 should be included in routine revaccination programs for pediatric HSCT recipients.
Methylation Status and Polymorphism of Asparagine Synthetase Gene in Acute Lymphoblastic Leukemia Cells.
Authors: Akagi Tadayuki , Yin Dong , Kawamata Norihiko , Bartram Claus R. , Miller Carl W. , Koeffler Phillip H.
Published: April 2020

Asparagine synthetase (ASY) is an enzyme expressed ubiquitously in mammalian cells. Expression of the gene, however, is repressed in acute lymphoblastic leukemia (ALL), and the cells are unable to synthesize sufficient asparagine. Depletion of asparagine by L-asparaginase leads to cell death in ALL; therefore, asparaginase has been used as treatment of ALL. Here, we show the methylation status of ASY gene in ALL clinical samples, and report a novel insertional polymorphism in the gene. The ASY gene has a CpG island located from −313 to +336 including 49 CpG sites. We analyzed the methylation status in the region by sodium bisulfite conversion of genomic DNA followed by PCR analysis. A total of 18 of 22 (82%) bone marrow samples of ALL at diagnosis were methylated in the CpG island. After chemotherapic induced-remission, all the bone marrow samples were unmethylated including the 18 patients whose ALL samples were methylated. Experiments by others using an ALL cell line showed that methylation of the ASY gene silenced the gene. In addition, we discovered either a 14 bp or 28 bp inserted sequence between +83 and +84 located in the 1st intron of the gene. Methylation status of the gene was independent of the insertion. To calculate the frequency of the insertion, 92 ALL samples were analyzed by PCR and sequencing. 69 samples (75%) did not contain the insertion in either allele, 22 samples (24%) contained the insertion in one allele, and only 1 sample (1%) contained the insertion in both alleles. To examine whether the insertion was specific for leukemic cells, we analyzed 71 non-ALL samples. 48 samples (68%) did not contain the insertion in either allele, 12 samples (27%) contained the insertion in one allele, and 4 samples (6%) contained the insertion in both allele. These results showed that the insertion is a germline polymorphism; we are exploring if this insertion changes expression of the gene.

In summary, we found a novel insertional polymorphism in the ASY gene. Also, our data for the first time suggests that ALL cells cannot express ASY because the gene’s CpG island is methylated, thus explaining why ALL cells are sensitive to L-asparaginase. Notably, 18% of ALL samples were not methylated, and these patients might be expected to be resistant to L-asparaginase therapy suggesting that a simple test could be of therapeutic value.
Maternal and Birth Characteristics and Risk of Leukemia in Children with Down Syndrome.
Authors: Roesler Michelle A. , Blair Cindy K. , Spector Logan G. , Robison Leslie L. , Olshan Andrew F. , Ross Julie A.
Published: April 2020

High birth weight (> 4000 g) is a risk factor for childhood leukemia, especially for younger children. Advanced maternal age has also been associated with an increased risk, though less consistently than birth weight. Because children with Down syndrome (DS) are at a 20-fold increased risk of developing acute leukemia than the general population, we evaluated maternal and child birth characteristics that may be associated with leukemia in children with DS. A case-control study of DS and acute leukemia was conducted through the Children’s Oncology Group in North America. From 1997 to 2002, 158 children ages birth-18 years with DS and acute leukemia (including 61 acute myeloid leukemia (AML) and 97 acute lymphoblastic leukemia (ALL)) were enrolled. Children with DS alone (n= 173) were identified through the cases’ pediatric clinics and frequency matched to cases on age. Mothers of cases and controls completed a telephone interview that included questions regarding maternal and birth characteristics. Here, we present data related to singleton pregnancies (165 controls and 151 cases (96 ALL/55AML)). Advanced maternal age (≥ 35 vs. <35) was associated with an elevated risk of leukemia (Odds Ratio (OR)=1.79, 95% Confidence Interval (CI))=1.09–2.94), which was apparent for both ALL and AML. There was also a suggestion of an increased risk for children in the highest tertile (>3260 g) of birth weight (OR=1.56, 95% CI=0.87–2.78) compared to children in the reference group (2850–3260 g). Birth length ≥ 21 inches showed an increased risk for AML cases only (OR=3.06; CI=1.25–7.50). In contrast, increasing birth order was associated with a decreased risk, particularly for AML (OR=0.36; 95% CI 0.14–0.97). This is the first study to evaluate maternal and child birth characteristics in relation to leukemia risk in children with DS. Our results add to the growiA. ng body of evidence that suggests that newborn body size may be related to the underlying etiology of childhood leukemia.
Acute Lymphoblastic Leukaemia Has a Poor Outcome in Children with Down Syndrome Due to Infective Death in Remission (Results of UK MRC ALL 97 Trial).
Authors: de la Fuente Josu , Richards Sue , Webb David K. , Hann Ian M. , Mitchell Christopher D. , Vora Ajay J. , Kinsey Sally E. , Eden Osborn B. , Ancliff Phil ,   On Behalf of the UK Childhood Leukaemia Working Party
Published: April 2020

Acute lymphoblastic leukaemia (ALL) has a poorer outcome in children with Down syndrome (DS) which has been attributed in the past to a higher incidence of infective death in remission and late relapses. We report on the outcome of children with DS enrolled on the UK Medical Research Council trial for childhood ALL, MRC ALL 97, between January 1997 and June 2002. Thirty seven children had DS (2%), of whom three were treated on the high risk protocol (HR1). Thirty-three (89%) achieved complete remission at the end of induction, 3 died during induction (8%), and one died later without remission. The median follow up was 4.9 years (2.4–7.8). The 5-year event free survival (EFS: 48.0%, SD 8.9) was not an improvement on the previous MRC UKALL XI trial (57.9%, SD 8.0, p=0.2) and was significantly worse than for children without DS (p<0.00005). At the time of follow up, 46% of the children had died (n=17). Five patients suffered relapse, and the relapse rate was not significantly different from those without DS. One patient known to have cardiac disease died during maintenance due to arrhythmia and 8 died of infection, resulting in a significantly higher rate of death in remission (28%) than in children without DS (3%, p<0.00005). Infective deaths were associated with intensification therapy, except in one child who died during interim maintenance. It was possible to isolate a microorganism in 50% of the cases (two cases of Pseudomonas, one Staphylococcus aureus, one Staphylococcus epidermidis and yeast) plus Rhinovirus was found in a nasopharyngeal aspirate of a fifth case with clinical evidence of bacterial sepsis. For the randomised comparisons (prednisolone versus dexamethasone n=30, mercaptopurine versus thioguanine n=23), results within the DS patients were not significantly different from those in all patients, with benefit for dexamethasone. The increase in remission deaths with DS was greater with prednisolone, and with mercaptopurine (p for interaction = 0.0002, <0.00005, respectively). The revision of the trial in 1999 which adopted the template of CCG 1952 which improved EFS, did so for DS patients also, with no change in the DS remission death rate. In conclusion, children with DS may benefit from increased treatment intensity but still have an unacceptably high rate of infective death in remission.
Thrombotic and Hemorrhagic Events for Patients on Pediatric Oncology Group (POG) Protocols 9201, 9605, 9406.
Authors: Rice Megan A. , Bell Beverly A. , Chauvenet Allen , Kurtzberg Joanne , Abish Sharon B. , Pullen Jeanette , Devidas Meenakshi , Camitta Bruce M.
Published: April 2020

Thromboembolism (TE) and hemorrhage are known complications following L-asparaginase (L-asp) therapy, but the incidence and epidemiology of these events in children are not clearly defined. POG protocols 9201, 9605, and 9406 were phase III studies for low, standard, and higher risk patients with acute lymphoblastic leukemia (ALL). Protocol 9201 enrolled 649 patients, 9605: 1087, and 9406: 911. All 3 studies used a common 4 week induction protocol (POG 9400) of native L-asp (6000 IU/m2 IM x 6 doses), weekly vincristine, and daily prednisone. Doxorubicin was added for patients meeting higher risk criteria based on age, initial white blood cell count, and CNS/testicular status. Patients on 9201 and 9605 received no L-asp after induction. 9406 patients received 3 doses of PEG L-asp (1000 or 2500 IU/m2 IM) during consolidation. The incidence of TE and hemorrhage on 9201 was 2.2% (14/649), on 9605 1.9% (21/1087) and on 9406 5.3% (48/911). In all three protocols, TE was more common than hemorrhage. Hemorrhages were more frequent in POG 9406.

# Patients # Thromboses # Hemorrhages # Total Events # in Induction 9201 649 13 1 14 6 9605 1087 17 4 21 10 9406 911 34 14 48 27 Total 2647 64 19 83 43

The largest proportion of events occurred during the month of induction therapy. 70% of induction events (30/43) occurred in patients who received 4 drug therapy. Of the 43 induction events, 21/26 patients with reported coagulation studies had abnormal fibrinogen levels <100mg/dL, 7 of those were <50mg/dL. Antithrombin levels were not reported. 28 of 64 TE were likely central venous line (CVL) related [6 tip of catheter, 16 axillary/subclavian vein, 2 superior vena cava, 2 atrium, 1 internal jugular vein, 1 basilic vein]. Locations of remaining TE included: 13 CNS [4 venous sinus, 9 cerebral]; 10 lower extremity; 4 upper extremity; 1 pulmonary embolism; 8 site not specified. Locations of hemorrhages included: 8 CNS [7 intracranial, 1 subdural]; 2 GI [1 gastric, 1 esophageal perforation]; 2 acute epistaxis; 2 site not specified; and 1 of each of the following: CVL site, pulmonary, splenic, vitreous eye, and hemothorax. Statistical analyses correlating number of L-asp doses to induction events and correlates with gender, age, race, and congenital abnormality are being evaluated.

Figure Figure
Forodesine (FodosineTM), a PNP Inhibitor Active in Relapsed or Refractory T-Cell Leukemia Patients (Phase II Study).
Authors: Furman Richard R. , Iosava Genadi , Isola Luis , Ravandi Farhad , Zodelava Mamia , Bennett J. Claude , Kilpatrick J. Michael , Bantia Shanta ,   Fodosine Development Group
Published: April 2020

Inherited deficiency of purine nucleoside phosphorylase (PNP) is associated with a profound loss of T-cells. This rare clinical condition provides a model for developing specific inhibitors of PNP as therapy for T-cell malignancies. Forodesine is a specific PNP inhibitor that raises plasma 2′-deoxyguanosine (dGuo) and intracellular dGTP levels, leading to T-cell apoptosis. The objective of this study was to determine the efficacy and safety of forodesine in patients with relapsed or refractory T-cell leukemia. Patients received IV forodesine 40 mg/m2 5 days each week (1 cycle) for a total of 6 cycles (8 cycles if dose escalation needed after cycle 2). To date, 21 patients have been treated, 19 of which are T-ALL. Results are based on available data from 13 T-ALL patients. Median number of prior regimens was 4 (range: 1–6). Overall response rate was 38%, including 3 patients with complete response (CR) (23%) and 2 patients with partial response (PR) (15%). Three patients had stable disease and 5 patients had disease progression. Time to progression (TTP) for the 3 CR patients was 80, 119, and 160+ days. One CR patient continues to receive treatment. TTP for the 2 PR patients was 89 and 40 days. Plasma dGuo was elevated in all patients (range: 3.4 to 88.5 μM; predose levels ≤0.004 μM). Treatment was well tolerated. One serious adverse event possibly drug related was CMV pneumonitis in 1 patient. The most common adverse events possibly drug related were nausea (33%), headache (26%), anemia (20%), thrombocytopenia (20%), and weakness (20%). In 1 patient, after 2 doses of forodesine, WBC count dropped dramatically, requiring interruption of treatment for 2 weeks because of tumor lysis syndrome (Figure). After 2 cycles of treatment, the patient achieved a CR. By week 3, platelets and neutrophils recovered, indicating a specificity of forodesine for leukemic cell populations. These study results show that forodesine is active, with minimal toxicity, as a single agent in relapsed or refractory T-cell leukemia. Additional clinical efficacy and safety data will be presented.

Effect of Forodesine on Blood Components in One T-All Patient Effect of Forodesine on Blood Components in One T-All Patient
Long-Term Results of the GIMEMA LAP AIDA 0493 Amended Protocol in Elderly Patients with Acute Promyelocytic Leukemia (APL).
Authors: Latagliata R. , Breccia M. , Fazi P. , Vignetti M. , Cupri A. , Sborgia M. , Vincelli D. , Candoni A. , Salvi F. , Rupoli S. , Martinelli G. , Kropp M.G. , Di Rienzo N. , Zampaglione V. , Venditti A. , Melillo L. , Cimino G. , Petti M.C. , Avvisati G. , Coco F. Lo , Mandelli F.
Published: April 2020

In order to reduce toxicity in elderly patients with newly diagnosed APL, since 3/1997 the Italian cooperative group GIMEMA evaluated an amended AIDA protocol for patients aged > 60 years, consisting of the same induction with ATRA and Idarubicin but only 1st consolidation course (Idarubicin + Cytarabine), followed by 2 years maintenance with ATRA alone. Up to now, 56 patients (25 males and 31 females, median age 66.2 years, 46 with PS 0-1 and 10 with PS 2) are fully evaluable. At onset, according to GIMEMA-PETHEMA risk score, 18 were low-risk(32.5%), 31 intermediate risk (55%) and 7 high risk (12.5%). After induction treatment, 54 patients (96.4%) achieved CR and 2 (3.6%) died from haemorrhage (1) and infection (1). ATRA syndrome was documented in 5 patients (9.3%): 13/56 patients (23.2%) showed during induction other toxicities (WHO 3 – 4) not related to ATRA. After CR achievement, 2 patients died in CR from haemorrhage (1) and infection (1) and 52 received the consolidation course: on the whole, during consolidation 4 patients (7.6%) had a toxicity WHO 3 – 4 and 2 of them (3.8%) died from haemorrhage (1) and infection (1). The remaining 50 patients started maintenance treatment: up to now, 12 patients (22.2%) relapsed, after a median time from morphological CR of 19 months (range 7 – 86). Overall survival (OS) was 76.1% and 73.3% at 3 and 5 years, respectively. Disease free survival (DFS) was 64.5% and 61.3% at 3 and 5 years, respectively. At the univariate analysis, PS =2 (p=0.0019), WBC count > 3 x 109 /l (p=0.018) and male gender (p=0.03) had a bad prognostic impact on DFS, while only PS=2 (p=0.05) did it on OS. Age, PLTS count, WBC count > 10 x 109 /l, and risk score did not affect both OS and DFS. At the multivariate analysis on DFS, only PS =2 retained prognostic significance (HR = 3.8). In conclusion, the amended GIMEMA protocol has shown to be effective and safe in elderly APL patients, as the rate of death in CR was reduced when compared with previous results in not amended GIMEMA LAP AIDA 0493: however, to face with a relapse rate > 20%, future strategies might be designed which exploit the use of more targeted approaches including combinations of ATRA, arsenic trioxide, and anti-CD33 monoclonal antibodies.
Influence of Perceived Risk Factors for Bleeding on Venous Thromboembolism Prophylaxis Practices in Acutely Ill Medical Patients: Findings from IMPROVE.
Authors: Monreal Manuel , Zotz Rainer B. , Decousus Hervé , Chong Beng H. , Merli Geno , Pavanello Ricardo , Pini Mario , Turpie Alexander G.G. , Huang Wei , Anderson Frederick A. ,   for the IMPROVE Investigators
Published: April 2020

Background Acutely ill medical patients at risk for venous thromboembolism (VTE) should receive VTE prophylaxis. However, factors perceived by physicians to increase patients’ risk of bleeding may influence VTE prophylaxis practices. In this analysis from The International Medical Prevention Registry on Venous Thromboembolism (IMPROVE), we examined whether perceived risk factors for bleeding had a significant influence on physicians’ prescribing of in-hospital prophylaxis in acutely ill medical patients.

Methods Patients aged ≥18 years and hospitalized ≥3 days with an acute medical illness have been enrolled consecutively since July 2002. Risk factors considered were: severe renal failure, known bleeding disorder, hemorrhagic stroke, thrombocytopenia, bacterial endocarditis, active gastroduodenal ulcer, NSAID use, hepatic failure, age, immobility and alcoholism. Factors associated with different prescription rates of prophylaxis compared with rates in patients without these factors were identified and included in a multiple logistic regression model (significance at p<0.05).

Results Up to 31 March 2005, 6946 patients were enrolled in 49 hospitals in 12 countries. Pharmacologic prophylaxis was received by 42%, 25%, 16%, and 14% of patients with a platelet count at admission >100, 50–100, 20–50 and <20x109/L, respectively (p<0.0001), and 43%, 39%, 30% and 32% of patients with none, 1, 2 and 3 risk factors for bleeding (p<0.0001). Factors independently associated with a lower/higher prescription rate of heparin-based prophylaxis compared with the risk in patients without these factors are shown in the Table.

Conclusions The likelihood that hospitalized acutely ill medical patients receive in-hospital pharmacologic prophylaxis decreases as their platelet count at admission decreases, or their cumulative number of perceived risk factors for bleeding increases. Further studies are needed to determine whether the changes in prophylaxis practices observed in this study are justified.

Table. Factors Independently Associated with a Higher/lower Rate of Heparin-based VTE Prophylaxis Factor Odds Ratio 95% Confidence Interval Age (per 10-year increase) 1.19 1.66–1.22 Immobility (per 10-day increase) 1.03 1.02–1.05 Alcoholism 0.62 0.46–0.83 Thrombocytopenia 0.60 0.48–0.74 Active duodenal ulcer 0.36 0.26–0.52 Hepatic failure 0.34 0.21–0.54