bluuebunny/crossref_metadata_2025 huggingface dataset:
A Global Role for EKLF in Definitive and Primitive Erythropoiesis.
Authors: Perkins Andrew C. , Coghill Elise , Maguire Tina , Hartmann Belinda , McDowall Alasdair , Weiss Mitchell , Grimmond Sean , Keys Janelle , Hodge Denise
Published: April 2020

Erythroid Kruppel-like factor (EKLF or Klf1) is an erythroid specific C2H2 zinc-finger transcription factor which is essential for definitive erythropoiesis and β-globin gene expression. The absence of EKLF results in fatal anaemia but correction of globin chain imbalance does result in rescue, suggesting the existence of additional EKLF target genes. The aim of this study was to search for such genes by expression profiling. We performed profiling on fetal livers from wild-type versus EKLF null litter mates, and also EKLF null erythroid cell lines containing an inducible EKLF-ERTM fusion construct. Hybridisations were performed on microarray slides printed with a 23K oligo library from Compugen. Target gene validation was performed by real-time RT-PCR, chromatin immuno-precipitation (ChIP) and promoter-reporter assays. A large number of genes were down regulated in the absence of EKLF but few were up regulated, suggesting EKLF acts primarily as a transcriptional activator in vivo. One hundred genes were EKLF dependent in both systems. These include heme synthesis enzymes, red cell surface proteins including Rh and the transferrin receptor, and erythroid transcription factors. Two interesting highly EKLF-dependent genes are α-haemoglobin stabilising protein (AHSP), a key chaperone for free a-globin chains, and dematin (band 4.9) which links the cytoskeleton to the red cell membrane. A search for EKLF binding sites within the dematin and AHSP genes demonstrated a number of phylogenetically conserved CACC sites, and ChIP demonstrated in vivo EKLF occupancy at some but not all of these. Promoter-reporter assays showed EKLF directly activates dematin gene transcription through two promoters containing these sites. Lastly, investigation of EKLF target genes in the yolk sac lead to the discovery of unexpected defects in the embryonic red cell membrane and cytoskeleton. In conclusion, EKLF regulates global erythroid gene expression which is critical for development of primitive as well as definitive red cells.
Differential GATA-1 Sensitivities of Target Loci.
Authors: Johnson Kirby D. , Pal Saumen , Grass Jeffrey A. , Bresnick Emery H.
Published: April 2020

The transcription factor GATA-1 is a key regulator of red cell differentiation, activating numerous erythroid-specific genes while downregulating genes that permit proliferation of erythroid precursors. We have demonstrated that GATA-1 only occupies a limited number of the high affinity WGATAR motifs present within target gene loci, FOG-1 is required for GATA-1 to occupy a subset of chromatin sites, and GATA-1 occupancy often coincides with GATA-2 displacement or a “GATA switch”. Once bound, GATA-1 elicits changes in the histone modification patterns and the three dimensional structure of target gene loci via recruitment of cofactors such as FOG-1 and CBP. As detailed mechanistic studies have not been conducted with most GATA-1 target genes, it is unclear whether these genes are equally sensitive to GATA-1 or if they respond differently to the rising GATA-1 levels during erythropoiesis. Using GATA-1 fusions to the estrogen receptor ligand binding domain (ER-GATA-1) in the GATA-1-null erythroid precursor cell line, G1E, we analyzed the responses of endogenous GATA-1 target genes to varied levels of GATA-1 activity. We found that transcriptional activation of Tac-2 required higher concentrations of ER-GATA-1 than is required for other GATA-1 target genes. Previously, we showed that Tac-2, which encodes the neurokinin-B precursor protein preprotachykinin B, is regulated by GATA-1 in erythroid cell lines and is induced upon ex vivo differentiation of human CD34+ peripheral blood cells. Importantly, whereas regulation of many GATA-1 target genes is only partially disrupted by removal of the N-terminal 193 amino acids from ER-GATA-1 (ER-GATA-1 ΔN), Tac-2 expression was very sensitive to this truncation. Whereas NK-B signals through G-protein-coupled receptors to modulate neuronal function, its functions beyond the nervous system are poorly understood. Although erythroid cells do not express NK-B receptors, the receptors, but not NK-B, are expressed in certain endothelial cell subtypes, and elevated levels of NK-B are implicated in the pregnancy-associated disorder pre-eclampsia. Tac-2 represents the first GATA-1 target gene that critically requires the N-terminus. Studies are underway to elucidate mechanisms underlying the exquisite sensitivity of Tac-2 to deletion of the GATA-1 N-terminus, the relationship between Tac-2 deregulation and GATA-1 N-terminal deletions in megakaryoblastic leukemia, and the function of erythroid cell-derived NK-B.
Strikingly Homologous Immunoglobulin Gene Rearrangements and Poor Outcome in VH3-21-Utilizing Chronic Lymphocytic Leukemia Independent of Geographical Origin and Mutational Status.
Authors: Murray Fiona , Thorselius Mia , Krober Alexander , Thunberg Ulf , Tobin Gerard , Buhler Andreas , Kienle Dirk , Albesiano Emilia , Dao-Ung Lan-Phuong , Wiley James , Vilpo Juhani , Laurell Anna , Roos Goran , Karlsson Karin , Chiorazzi Nicholas , Marasca Roberto , Dohner Hartmut , Stilgenbauer Stephan , Rosenquist Richard
Published: April 2020

We recently reported that Swedish VH3-21-utilizing chronic lymphocytic leukemia (CLL) patients showed restricted immunoglobulin gene features and poor prognosis despite VH mutation status. To investigate whether VH3-21+ CLLs have similar characteristics in different parts of the world, we analyzed the VH and VL gene rearrangements in 90 patients from Sweden, Germany, Italy, USA, Finland and Australia and correlated these data with survival. Sixty-three percent of cases exhibited mutated VH genes and 37% had unmutated VH genes. Fifty patients (56%) displayed a short and homologous heavy-chain CDR3, many of these with the amino acid motif, DANGMDV. Also, a highly biased Vλ2-14 usage was evident in 73% of patients with a restricted light-chain CDR3, QVWDS(S/G)SDHPWV. Combined restricted heavy- and light-chain CDR3s were found in patients from all included countries. Although VH3-21+ CLLs have a remarkably predominant λ-expression, analyses of kappa deleting element showed a conserved rearrangement order of the light-chain loci. The overall survival was poor in the VH3-21+ cohort (median survival 88 months) with no significant difference in relation to mutation status or homologous/non-homologous CDR3. In summary, highly restricted B-cell receptors and worse outcome characterize VH3-21+ CLLs independent of geographical origin and mutation status. VH3-21 usage should now be included in prognostic stratification of CLL when assessing mutation status.
Increased Risk for Treatment-Related Mortality of Bone Marrow Transplantation in GSTM1-Positive Recipients.
Authors: Terakura Seitaro , Murata Makoto , Nishida Tetsuya , Emi Nobuhiko , Akatsuka Yoshiki , Riddell Stanley R. , Morishima Yasuo , Kodera Yoshihisa , Naoe Tomoki
Published: April 2020

Treatment-related mortality (TRM) is a major obstacle to successful allogeneic hematopoietic stem cell transplantation (HSCT). A variety of drugs are used in allogeneic HSCT, such as chemotherapeutic agents, immunosuppressants, antibiotics, antifungal and antiviral drugs, thus, a genetic polymorphism in metabolic enzymes would affect the metabolism of drugs and subsequent TRM after HSCT. We recently reported a metabolic enzyme UDP glycosyltransferase 2 family, polypeptide B17 (UGT2B17) has a null phenotype, and the use of a UGT2B17-positive donor is an independent risk factor for higher TRM and lower survival after HSCT (ASH annual meeting 2004, abstract# 1837). Here, we focused attention on glutathione S-transferase (GST) M1 and GSTT1 enzymes. These enzymes mainly metabolize chemotherapeutic agents, chemical carcinogens and by-products of oxidative stress and are absent from more than 50% of some populations. To assess the significance of homozygous GSTM1 and GSTT1 gene deletion in allogeneic HSCT, we analyzed DNA from 373 patients with a hematological disease and their HLA-A, B, C, DRB1-identical unrelated bone marrow donors using PCR method. All patients received an intensive myeloablative pretransplant conditioning regimen, an unmanipulated marrow graft, and cyclosporine A or tacrolimus as GVHD prophylaxis. Homozygous GSTM1 and GSTT1 gene deletion were observed in 56% and 45% of patients with a hematological disease, respectively, and 57% and 46% of healthy donors, respectively. There was no difference in the frequencies between disease group and donor group. We next applied Cox proportional hazard model to multivariate analysis for TRM which was defined as any death that occurred while the patient was in remission, acute GVHD, chronic GVHD, relapse, disease-free survival (DFS) and overall survival (OS). The analysis showed no significant association between GSTT1 polymorphism and any outcome. However, GSTM1-positive recipient was significantly associated with higher TRM and lower survival: TRM; Odds ratio (OR), 1.49; 95% confidence interval (CI), 1.03–2.16; P=0.036: DFS; OR, 1.50; 95% CI, 1.08–1.97; P=0.013: OS; OR, 1.41; 95% CI, 1.04–1.92; P=0.030. TRM was analyzed in relation to GSTM1 genotype in the patient by the Kaplan-Meier method (Fig 1). TRM in the GSTM1-positive patients (42.9%, n=166) was significantly higher than that in the GSTM1-negative patients (29.3%, n=201) (P=0.04). These data suggest the presence of the GSTM1 null genotype in recipient provide better protection against TRM after allogeneic HSCT. Some reactive intermediates or toxic metabolites generated by GSTM1 may initiate or promote the development of TRM.

Figure Figure
Multidrug Resistance-1 Gene Polymorphism Associated with the Outcomes after Allogeneic HLA-Identical Stem Cell Transplantation.
Authors: Kim Dong Hwan , Jeon Seok Bong , Baek Jin Ho , Lee Nan Young , Kim Jong Gwang , Sohn Sang Kyun , Suh Jang Soo , Do Young Rok , Kwon Ki Kyung
Published: April 2020

Background: The pharmacokinetic impact of multidrug resistance-1 (MDR1) gene single nucleotide polymorphisms (SNPs) has been already investigated in solid organ transplantation field, however, data is still lacking in an allogeneic stem cell transplantation (SCT) setting.

Methods: A total of 82 patients receiving an allogeneic HLA-identical sibling (n=70) or unrelated SCT (n=12) with graft-versus-host disease (GVHD) prophylaxis of cyclosporine-A (CSA) plus methotrexate (MTX) were included in the current study. Two SNPs of MDR1 gene (C3435T and G2677T/A) were analyzed using PCR/RFLP assay.

Results: As regards G2677T/A SNP, GG genotype showed a higher incidence of NRM compared to non-GG genotype (67% vs. 32%, p=0.0073), yet not C3435T (p=0.2026) or MDR1 haplotype (p=0.2238). Accordingly, overall survival (OS) was significantly correlated with G2677T/A genotype (p=0.0048), yet not with C3435T (p=0.5041) or MDR1 haplotype (p=0.4086). However, no difference in the relapse incidence was noted according to G2677T/A, C3435T genotype or MDR1 haplotype. In a multivariate analysis, those patients without GG genotype at G2677T/A were found to have favorable prognosis in terms of OS (p=0.003) or NRM (p=0.031) along with occurrence of chronic GVHD (p<0.001 for OS, p=0.001 for NRM), standard disease risk (p=0.045 for OS) or acute grade 0,1 GVHD (p=0.019 for NRM). However, no correlation was found between the blood concentrations of CSA and MDR1 genotype and CSA neurotoxicity and MDR1 genotype.

Conclusion: The G2677T/A genotype seemed to be associated with the transplantation outcomes, especially NRM. Further study is warranted to clarify its mechanism of MDR1 SNPs other than pharmacokinetic aspects.

Figure. Overall survival (A) and cumulative incidence of non-relapse mortality (B) according to G2677T/A MDR1 genotype Figure. Overall survival (A) and cumulative incidence of non-relapse mortality (B) according to G2677T/A MDR1 genotype
A Bioinformatic Approach to CLL IGHV Sequence Analysis Identifies Extensive Ig Sequence Archetypes among BothMutated and Unmutated Sequences.
Authors: Messmer Bradley T. , Raphael Benjamin J. , Aerni Sarah J. , Widhopf George F. , Rassenti Laura Z. , Toy Traci L. , Kipps Thomas J.
Published: April 2020

Several recent studies have pointed towards a number of sets of CLL patients with highly similar IgV genes, both in terms of V, D, J segment and light chain use and also heavy chain third complementarity determining region (CDR3) architecture and amino acid composition. These findings support the notion that antigen may drive development or perpetuation of the leukemic cells. To effectively mine the available Ig sequence databases, we developed a novel sequence similarity and clustering algorithm that incorporates the mechanistic knowledge of IgV gene recombination. CDR3 sequences were initially aligned using the conventional blosum62 based scoring matrix, then rescored based on the junctional annotations. In this way, the method gives greater weight to sequence similarity created as a consequence of the VDJ recombination process, rather than simple germline homology. This modified scoring matrix allows the creation of more robust sequence “clusters” and is not restricted to sequences composed of the same germline genes. Preliminary analysis with this method on a collection of more than 1000 CLL IgV gene sequences has identified over 40 clusters, including all that have been previously described. Interestingly, a species abundance estimator indicates additional clusters of IgV genes in CLL remain to be discovered. Additionally, we performed a focused evaluation of over 100 VH4-34 containing sequences expressed by CLL cells of unrelated patients. The VH4-34 gene is among the most commonly used IVH genes expressed in CLL, has a curious bi-phasic distribution of mutational frequencies among different CLL cases, and can encode antibodies with specificity for the linear polylactosamine carbohydrate antigen “i” found on neonatal red blood cells. Prior studies by the Chiorazzi group identified a subgroup of patients with CLL cells that expressed remarkably similar mutated VH4-34 genes encoding isotype-switched Ig. Using the method described above, seven additional clusters of CLL cases that expressed highly related VH4-34-encoded Ig were identified, including three clusters containing sequences with a mutated VH4-34. The mutations seen within these clusters have a spectra that is distinct from other VH4-34 sequences from CLL, normal plasma cells, or marginal zone B cells. Furthermore, within each cluster there were conserved replacement mutations not commonly found in the other VH4-34 sequences. This clearly indicates specific selective pressures subsequent to IgV gene rearrangement and implies that the expressed Ig were selected for binding to several distinct antigens or epitopes. However, redundant antigen specificities encoded by different sequence clusters can not be excluded and the number of driving antigens may still be quite restricted. Comparative analysis of normal sequence collections is ongoing.
Thrombotic Microangiopathy™ after Allogeneic Peripheral Blood Stem Cell Transplantation with Nonmyeloablative Conditioning (NMSCT) Compared with Myeloablative Conditioning (MSCT).
Authors: Willems Evelyne , Baron Frédéric , Fillet Georges , Beguin Yves
Published: April 2020

Background: Thrombotic microangiopathy (TM) is a severe complication of allogeneic transplantation. The use of CsA for GvHD prophylaxis and unrelated donor appear to be the most significant risk factors for TM after transplantation with conventional myeloablative conditioning (MSCT). Allogeneic transplantation with reduced-intensity conditioning (RIC) is associated with a similar risk of TM, but TM has never been studied after NMSCT.

Purpose: To determine the risk factors, incidence and outcome of TM in NMSCT compared to MSCT.

Patients: Consecutive patients undergoing NMSCT (group 1, n=103) or MSCT (group 2, n=136) in our center between 1995 and 2004 were retrospectively analysed for TM. Median age was 55 vs 34 yrs in groups 1 and 2 respectively (p<0.0001). Donor was unrelated for 52% of the patients in group 1 and for 38% in group 2 (p=0.0287).

Results: TM was diagnosed in 18 (20%) and in 30 (26%) patients in groups 1 and 2, respectively (NS). Most cases were grade 2 TM (18/18 in group 1 and 24/30 in group 1, NS). Neurological signs were present in 50% of the patients in group 1 and 57% in group 2 (NS). In both groups, TM was significantly associated with unrelated donor (p=0.0476 in group 1, p=0.0005 in group 2) and with CMV disease (p=0.0332 in group 1, p=0.0346 in group 2) but not CMV infection. AGvHD of all grades was correlated with TM in group 1 (p=0.0004) but only severe (grade 3–4) AGvHD in group 2 (p<0.0001). Median time of onset of TM was 39 (range 18–157) days in group 1 and 38 (range 0–428) in group 2. First line treatment consisted in switching GvHD prophylaxis to alternative treatment (including tacrolimus) in 54% of the patients and plasmaphereses (36±10 in group 1 and 21±4 in group 2) in 63%. Vincristine (in 11% and 13% of the patients in groups 1 and 2, respectively) and other treatments were rarely used. Forty-four % of the patients died without achieving resolution of their TM in group 1, and 50% in group 2 (NS). For patients who responded to therapy, median duration of TM was 57 (range 5–165) days in group 1 and 46 (range 5–163) days in group 2 (NS). In group 1, the 100-d and 1-yr survival rates were 64% and 28% for patients who developed TM, and 84% and 69% for those without TM, respectively (p=0.0002). In group 2, survival was also significantly reduced in the case of TM (48% vs 70% at day 100 and 23% vs 53% at 1 yr, respectively, p=0.0126).

Conclusion: TM remains a frequent and severe complication of allografting. Its incidence and severity in NMSCT is not decreased compared to MSCT and the same factors appear to contribute to its development.
Risk Factor Analysis for Thrombotic Microangiopathy after Reduced Intensity or Myeloablative Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Nakamae Hirohisa , Yamane Takahisa , Kiyoyuki Hagihara , Takeoka Yasunobu , Sakamoto Erina , Nakane Takahiko , Nakamae Mika , Koh Ki-Ryang , Ohta Kensuke , Hino Masayuki
Published: April 2020

Thrombotic microangiopathy (TMA) impairs long-term survival after allogeneic hematopoietic stem cell transplantation (HSCT). As the allogeneic HSCT procedure has developed, addressing risk factors for TMA has become more complicating. The aim of this study was to investigate the impact of transplant-associated factors on TMA incidence of in patients who underwent HSCT in various settings. One hundred and twenty-three consecutive allogeneic HSCT patients (15 to 69 years old) with hematologic diseases receiving myeloablative and reduced intensity conditioning were evaluated retrospectively. Of 123 HSCTs, 22 (17.9%) patients were diagnosed with TMA after HSCT. The median time to the diagnosis of TMA from HSCT was 33.5 days (range, 1–126). In 21of 22 patients, TMA occurred within 120 days after HSCT. Ten of 22 patients with TMA received some treatments for TMA. Any treatment for TMA including plasma exchange, tapering immunosuppressive agents, avoiding platelet transfusion, and administration with fresh frozen plasma and/or anti-thrombin III preparation, did not significantly improve overall survival in patients with TMA compared with those not treated for TMA. Using univariate Cox proportional hazard model analysis, age, HLA mismatch, GVHD grade II–IV, the use of FK506 for GVHD prophylaxis and the use of high-dose Bu were significantly associated with an increased incidence of TMA after HSCT. Furthermore, multivariate analysis showed the significance of GVHD grade II–IV, the use of FK506 and the use of high-dose Bu (16 mg/kg) persisted. On the other hand, the use of half-dose Bu (8 mg/kg) for conditioning was not significantly associated with increased TMA. The hazard ratios of the use of FK506, the use of high-dose Bu (16 mg/kg) and GVHD grade II–IV for TMA were 8.7 (95% CI 2.0–37), 5.7 (95% CI 1.5–21) and 3.4 (95% CI 1.3–9.1), respectively. In the present study, reduced intensity conditioning did not have an advantage over myeloablative conditioning for decreasing the incidence of TMA after HSCT. Our results also showed that high-dose Bu (16 mg/kg) for a conditioning and FK506 for a prophylaxis of GVHD might contributes more to TMA onset after HSCT than other agents.
First Molecular Characterization of a Family with Combined FV and FVII Deficiency.
Authors: Traynis Ilana , Jones Carol D. , Gibb Connie B. , Acharya Suchitra , Zehnder James L.
Published: April 2020

Combined factor V and VII deficiency is a rare bleeding disorder, with 3 cases reported in the literature. None of these cases were characterized at a molecular level. We now report a 4th case of factor V and VII deficiency, and for the first time characterize the factor V and VII mutations/polymorphisms in the proband and parents.

Case history: The proband is a 5-year-old female who presented with microscopic hematuria and mild mucosal bleeding. She has 47% FV activity and 38% FVII plasma activity. Her father has a history of delayed wound healing and mild mucosal bleeding and her mother has history of mild epistaxis, menorrhagia and gingival bleeding.

Results: The proband was heterozygous for a novel FV mutation, a one base deletion in exon 4 (524delG) introducing a frameshift and resulting in premature truncation of translation 33 amino acids later. In addition, she was heterozygous for 4 previously described factor VII polymorphisms: a 10-bp insertion [CCTATATCCT] at -323 in the 5′ region of the promoter, a G to A substitution in intron 1a (73 g>a), and the R353Q polymorphism in exon 8 of the F7 gene. These 3 polymorphisms have been found to be in strong allelic association in the Italian population, with mild FVII deficiency (Peyvandi et al, 1999). In addition, a R315W mutation was found in exon 8. This mutation has been reported to decrease both FVII coagulant function and factor X activation (Furlan Freguia et al, 2004). The mother was also heterozygous for FV and FVII deficiency, with FV 524 delG and FVII R315W, while as expected the father was heterozygous for the factor VII allele with the 3 associated FVII polymorphisms (Table 1). Thus the proband is heterozygous for FV deficiency and a compound heterozygote for Factor VII deficiency, inheriting FVII mutations from both parents. This is the first molecular characterization of a family with combined factor V and VII deficiency, and also the first family described with 2 different forms of combined FV and FVII deficiency within the same family. These results are consistent with this syndrome being due to chance co-inheritance of FV and FVII mutations rather than a post-translational defect as seen in combined FV and FVIII deficiency.

Table 1 F5 524delG F7 R315W F7 -323 F7 IVS1 73 g>a F7 R353Q Summary of mutations/polymorphisms in a family with combined factor V and factor VII deficiency. X denotes presence of a given mutation/polymorphism in an individual. Proband X X X X X Mother X X Father X X X
Genetic and Phenotypic Analysis of Families with Inherited Hypo- and Dys-Fibrinogenemia. Fibrinogen Bratislava.
Authors: Batorova Angelika , Horvathova Daniela , Mistrik Martin , de Moerloose Philippe , Neerman-Arbez Marguerite
Published: April 2020

Inherited hypofibrinogenemia (hypo-FBG) and dysfibrinogenemia (dys-FBG) are rare blood coagulation disorders caused by quantitative or qualitative defects of fibrinogen. We reviewed the clinical course in 47 individuals with dys-FBG and 16 patients with hypo-FBG with median fibrinogen coagulant/antigen levels of 0.76/2.8 and 1.1/1.2 g/L, respectively. Genetic analysis was performed in 5 families (16 individuals) with dys-FBG and 4 families (8 individuals) with hypo-FBG.

In the dysfibrinogenemia group 21 (45%) individuals were asymptomatic, 24(51%) patients suffered from bleeding and/or prolonged wound healing (only four had serious bleeding) and 2(4%) patients had a history of thrombosis. A total of 96 surgeries were performed without preoperative fibrinogen replacement in 31 dys-FBG patients, only 9 (9%) procedures were complicated with bleeding, two requiring fibrinogen substitution. Genetic analysis in dysfibrinogenemia has revealed heterozygous mutation in FGA exon 2 (Aα Arg16 His) known to cause delayed fibrinopeptide A cleavage by thrombin in 6 individuals (3 families). Five patients from two other families were heterozygous for a novel mutation in FGA exon 2 (Aα Gly13Glu). Fibrinogen coag/Ag median levels were comparable for both mutant genotypes: 0.5/2.9 g/L and 0.56/2.8 g/L, respectively.

In the hypofibrinogenemia group 6/14 (43%) patients had mild spontaneous bleeding, 8/36 (22%) surgeries performed in 12 patients w/o replacement were complicated with bleeding. A novel mutation in FGG exon 1 (Trp3 Stop) was identified in heterozygosity for 3 patients (3 families) with FBG coag/Ag median level of 1.1/1.2 g/L. An additional unrelated patient with a fibrinogen level of 0.7 g/L and serious postpartum bleeding was heterozygous for a novel mutation in FGG exon 7 (Trp253Cys) - Fibrinogen Bratislava.
Micro-Array and Protein Analyses Reveal a Preferential Autocrine VEGF Survival Loop in CD38+ Sub-Clones When Compared with CD38− Sub-Clones Derived from the Same CLL Patient.
Authors: Pepper Chris , Ward Rachel , Starczynski Jane , Rowntree Clare , Pratt Guy , Mills Ken , Fegan Chris
Published: April 2020

CD38 expression is a marker of poor prognosis in chronic lymphocytic leukemia (CLL) but to-date no clear rationale for this has been elucidated. Using high speed cell sorting and subsequent gene expression profiling, we investigated the transcriptional characteristics of purified CD38+ and CD38− sub-clones derived from the same patient (n = 12). Our analysis revealed a distinct, differential gene expression profile between CD38+ and CD38− subsets with 35 genes found to be consistently over expressed in the CD38+ cells (of which CD38 was one; 9.1 fold). Of particular interest were four genes, CXCL-2 (9.4 fold), vascular endothelial growth factor (VEGF) (5.8 fold), MIP1α /CCL3 (5.4 fold) and Interleukin 1β (IL-1β ) (4.1 fold). In a number of different tumor models IL-1β has been shown to induce the expression of CXCL-2, VEGF, and MIP1α resulting in angiogenesis, survival signaling and/or tumor proliferation. Protein analysis revealed 2–3 fold elevated levels of IL-1β and VEGF in CD38+ cells confirming that the elevated transcription was translated. Furthermore, the differential transcription of these genes corroborates our previous findings; CD38+ cells have a higher proliferative index and higher levels of the anti-apoptotic protein Mcl-1. Our data strongly suggests that the CD38+ clones preferentially utilise an autocrine VEGF loop and given that the CD38− clones also express VEGF receptor the potential for bystander paracrine effects of secreted VEGF seem likely in bimodal patients. Therefore, the relative over expression of IL-1β and VEGF provide a rationale for the disease progression and poor clinical prognosis of CD38+ CLL patients.
A Randomized, Double Blind Trial, of Hydroxychloroquine for the Prevention of Graft-Versus-Host Disease after Allogeneic Peripheral Blood Stem Cell Transplantation.
Authors: Fong Tom , Trinkaus Kim , Adkins Douglas R. , Vij Ravi , Devine Steven , Tomasson Michael , Goodnough Lawrence T. , Graubert Timothy , Shenoy Shalini , DiPersio John F. , Khoury Hanna J.
Published: April 2020

Hydroxychloroquine (HCQ) is an immunosuppressive lysosomotropic amine that has activity against graft-versus-host disease (GVHD). We previously reported low incidences of acute GVHD in unrelated donor transplant recipients who received prophylactic HCQ in addition to standard GVHD prophylaxis (BBMT2003; 9: 714–721). We herein report results of a single-institution phase III trial, in which 95 recipients of matched sibling allogeneic peripheral blood stem cell transplantation were randomized to receive, in a double-blind fashion, and in addition to prophylactic cyclosporine A (CSA), HCQ or placebo starting 21 days pre-transplant and continued until d+365. HCQ was very well tolerated and not associated with side effects. The addition of HCQ had no effects on lymphocyte subsets both pre- and post-transplant. Overall, the incidence of acute GVHD was 59% in both arms, and severe acute GVHD occurred in 11% (HCQ) and 14% (placebo) (p=0.76). Sixty-one and 46% of patients developed chronic GVHD in the placebo and the HCQ arms, respectively (p = 0.15). With a median follow-up of 18 months, relapse-free and overall survivals were comparable in both groups. In summary, in this randomized trial, the addition of HCQ to single agent CSA was not associated with a reduction of either acute or chronic GVHD; additionally, no significant effects on relapses or survival were observed.
Clinical Outcomes and the Impact of Anti-Thymocyte Globulin (ATG) and Chronic Graft Versus Host Disease (cGVHD) Following Pediatric Allogeneic Stem Cell Transplantation: A Comparison between Peripheral Blood and Bone Marrow as a Source of Stem Cells.
Authors: Cheema Parneet , Desai Sunil , Anderson Ronald , Coppes Max , Lewis Victor A.
Published: April 2020

INTRODUCTION: The use of peripheral blood as a source of stem cells for pediatric allogeneic stem cell transplantation remains controversial. We present a retrospective analysis evaluating the safety and efficacy of allogeneic peripheral blood stem cell transplantation (PBSCT) compared to allogeneic bone marrow transplantation (BMT) for the treatment of acute leukemia at our centre. The impact of ATG added to the treatment regimen was also evaluated in this analysis.

METHODS: A chart review was conducted on all pediatric patients who underwent an allogeneic stem cell transplant for acute leukemia between April 1991 and August 2003 at the Alberta Children’s Hospital. All patients who received stem cells from HLA matched or one-antigen mismatched donors were included in this analysis. Data were analyzed using SPSS.

RESULTS: 85 children (ages 9 months to 17 years) met study criteria. 24 patients underwent a PBSCT while 61 patients, a BMT for the treatment of either ALL (n=54) or AML/MDS (n=31). Median follow up time was 5.9 years. Six of 24 patients in the PBSCT group, and 30 of the 61 patients in the BMT group received ATG (p=.042). There was a trend towards increased overall survival (OS) at 5 years following PBSCT (p=.078). Disease free survival (DFS) at 5 years following PBSCT and BMT was similar (p=.297). Subgroup analysis found no difference in OS or DFS between PBSCT and BMT in patients receiving (p=.221, p=.820) or not receiving ATG as part of the conditioning regimen (p=.448, p=.820). Chronic GVHD was increased following PBSCT (45.8%) compared to BMT (19.7%) (p=.015). The rate of severe cGVHD, however, was not significantly different (25% vs. 11.5% for PBSCT and BMT, p=.119) between the two groups. No patients receiving ATG prior to PBSCT developed cGVHD (p=.009). Addition of ATG to the transplant regimen had no impact on the rate of cGVHD following BMT (p=.221). There was no increase in mortality due to cGVHD following PBSCT (p=.243) or BMT (p=.865). Although, there was a trend to improved DFS in patients who developed cGVHD following PBSCT, this was not statistically significant (p=.057). Despite a statistically significant increase in TRM found in all patients with cGVHD at one year post-transplantation (p=.048), there was no difference in TRM between the PBSCT (4.2%) and BMT (14.8%) groups (p=.173).

CONCLUSION: Allogeneic PBSCT is a safe and effective alternative to allogeneic BMT for the treatment of acute leukemia in the pediatric population. Although there is an increased rate of cGVHD following PBSCT this does not appear to influence OS, DFS or TRM in our analysis. Addition of ATG into the transplant regimen decreases the occurrence of cGVHD after PBSCT. Studies to further assess the role of ATG in PBSCT are warranted.
Chronic Cutaneous GVHD Does Not Correlate with Frequency and FoxP3 Gene Expression of Regulatory T Cells.
Authors: Haeusermann Peter , Tabellini Laura , Flowers Mary E. , Martin Paul J. , Carpenter Paul A. , Hansen John A.
Published: April 2020

OBJECTIVE: The pathogenesis of chronic GVHD and particularly of its skin involvement is incompletely understood. Regulatory CD4+CD25+ T-cells have been shown to influence acute and chronic GVHD disease and so far heterogeneous results considering the frequency of these cells in chronic GVHD have been published. Additionally, reports indicate that chronic GVHD is associated with reduced expression of FoxP3 of these specific T-cells potentially related to posttransplant thymic insufficiency. These investigations have so far not been conducted in the particular setting of isolated chronic cutaneous GVHD. Our objective therefore was to investigate number, immunophenotype and gene expression of T-cells and particularly of regulatory T-cells in whole blood, CD4-enriched T-cells and regulatory T-cells in these patients. Moreover, as immunosuppressive drugs (calcineurin inhibitors and Rapamycine) have been shown to influence the frequency of regulatory T-cells in vitro, our focus was to specifically study highly selected patients without immunosuppressive medications including cyclosporine, FK-506, Rapamycine, Glucocorticosteroids and MTX and with or without active chronic cutaneous GVHD.

METHODS: Since June 2005, 14 patients more than 365 days posttransplant because of hematologic malignancy and 4 normal controls have been investigated in this still ongoing study clinically and by T-cell immunophenotyping (whole blood, CD4+ T cells and regulatory T-cells) and gene expression (FoxP3, Il–10 and TGF-beta) with real time PCR.

RESULTS: Of all patients included so far, preliminary results indicate, that patients without GVHD at all and without immunosuppressive drugs as well as patients with isolated chronic cutaneous GVHD and off immunosuppressive drugs have a comparable amount of regulatory T-cells and level of gene expression for FoxP3. Patients within these two highly selected subgroups do not have clinical and/or laboratory evidence for gastrointestinal or liver affection.

CONCLUSION: With respect to our preliminary results, active chronic cutaneous GVHD in posttransplant patients after day 365 is not related to a decreased number of regulatory T-cells as well as to a reduced level of FoxP3 expression.
Foamy Viral Vectors Efficiently Transduce Quiescent Hematopoietic Stem/Progenitor Cells (HSC) and Restore the Long Term Repopulating Activity of Fancc −/− Stem Cells.
Authors: Clapp D. Wade
Published: April 2020

Fanconi anemia (FA) is characterized by bone marrow aplasia and myeloid leukemia. The identification of FA genes raises the potential of using gene transfer technology to introduce cDNAs into autologous HSCs. Current strategies using Moloney retroviruses require a 2–4 day ex vivo culture of HSC to facilitate stable integration of the transgene. However, ex vivo culture results in a time-dependent increase in apoptosis of Fancc−/− primitive HSC and mice reconstituted with the surviving cells have an increased risk of acquiring myeloid malignancies. Therefore we examined the potential of a recombinant foamy virus construct (MD9-FANCC-EGFP) to transduce murine Fancc −/− HSC in the absence of prestimulation. Forty-80% of progenitors that were in G0 – G1 at the time of transduction were transduced following a single 10–14 hr transduction. Aliquots of MD9-FANCC-EGFP transduced BM cells or cells encoding the EGFP transgene only were transplanted into irradiated recipient mice or recipients treated with IFN-g only. Four-six months following transplantation, recipient BM cells were isolated and clonogenic assays were established in a range of mitomycin c (MMC) concentrations. Fancc−/− progenitors encoding recombinant FANCC were found to have a similar resistance to MMC as wildtype (WT) controls while Fancc−/− progenitors encoding the reporter construct only retained a high sensitivity to MMC. To assess the potential of MD9-FANCC-EGFP to correct stem cell repopulating ability, we next utilized the competitive repopulating assay. The repopulating activity of MD9-FANCC-EGFP-transduced Fancc−/− stem cells was comparable to WT controls 18–24 months following transplantation in primary and secondary recipients. Additionally, while mice reconstituted with Fancc−/− cells transduced with the reporter construct had reduced repopulating ability as compared to the other groups, none of these recipients acquired myeloid malignancies. Collectively, these data provide in vivo evidence that an abbreviated transduction protocol utilizing a foamy-viral based vector allows efficient transduction of Fancc−/− HSC, and diminishes the selection pressure that occurs during ex vivo culture of Fancc−/− HSCs.
Secondary or Concomitant Neoplasms among Adults Diagnosed with Acute Lymphoblastic Leukemia (ALL) and Treated According to the LALA-87 and LALA-94 Trials.
Authors: Tavernier Emmanuelle , de Botton Stephane , Dhedin Nathalie , Bulabois Claude-Eric , Reman Oumedaly , Vey Norbert , Garban Frederic , Boiron Jean-Michel , Gardin Claude , Fegueux Nathalie , Witz Francis , Lioure Bruno , Gratecos Nicole , Isnard Francoise , Delannoy Andre , Vernant Jean-Paul , Dombret Herve , Lheritier Veronique , Fiere Denis , Thomas Xavier
Published: April 2020

Second malignant neoplasms are a serious complication after successful treatment of childhood ALL. Although treatment intensity and outcome were not comparable, with improvements in survival, it is important to evaluate the rate and the type of second neoplasms in adults with ALL. We analyzed the data from the GET-LALA group. A cohort of 1493 patients, aged 15 to 60 years and enrolled on two successive multicenter protocols between 1987 and 2002, was observed to determine the incidence of second neoplasms and associated risk factors. The median follow-up time from diagnosis was 6 years. By February 2005, secondary or concomitant neoplasms were documented in 23 patients (median age: 36 years, range:18–57) including 9 acute myeloid leukemias, 4 non Hodgkin lymphomas, 5 skin tumors, and 5 other solid tumors (1 lung cancer, 1 tongue carcinoma, 1 thymoma, 1 condrosarcoma, 1 histiocytosis). Neoplasms developed 0.5 to 13.8 years (median, 4.5 years) after the diagnosis of ALL. 22 patients were in first remission, one was in second remission. The overall cumulative risk of secondary neoplasms was 2.1% at 5 years, 4.9% at 10 years, 9.4% at 15 years. The cumulative risk of developing a second hematologic malignancy was 1.8% at 5 years, 2.2% at 10 years, 3.3% at 18 years; that of developing a solid tumor was 0.2% at 5 years, 2.8% at 10 years, 6.2% at 15 years. The development of secondary neoplasm was not associated with the use of any specific cytotoxic agent. However, risk of skin tumor increased with radiation dose and transplantation (p = 0.01). Overall survival after the diagnosis of a second malignant neoplasm was 55% at 10 years. However, the median overall survival in patients developing acute myeloid leukemia was of 5.7 months. Considering the low-survival rate of this large unselected adult ALL cohort (32% at 10 years), considering the poorer results comparing to childhood ALL treatment, the risk of secondary or concomitant neoplasm remains probably under estimated. Larger series with long-term follow-up are, however, mandatory.

Figure Figure
SNX5, a Novel Gene Linked to Fanconi Anemia Causes Hematopoietic Failure When Knocked down in Zebrafish.
Authors: Flynn Catherine M. , Mendenhall Eric M. , Eckfeldt Craig E. , Wagner John E. , Verfaillie Catherine M.
Published: April 2020

Sorting nexin 5 (SNX 5) is a member of the sorting nexin family, a diverse group of cellular trafficking proteins unified by the presence of a phospholipid binding domain (PHOX) which is involved in protein-protein interactions. While the function of SNX5 is unknown, there is evidence from yeast two hybrid studies that SNX5 binds to FANCA, and SNX5 is one of the 20 genes located on a region on mouse chromosome 2 associated with accelerated stem cell aging. We have recently shown that SNX5 is significantly higher expressed in CD34+CD33-CD38-Rho+ (Rhohi) cells from umbilical cord blood (UCB) and bone marrow (BM), depleted of SCID repopulating cells (SRCs) than in CD34+CD33-CD38-cKIT+ Rho- cells (Rholo), containing all SRCs. A large scale high throughput functional analysis was carried out in zebrafish and genes differentially expressed between Rholo and Rhohi cells were knocked down using morpholino (MO) antisense oligonucleotides. Morpholinos are a useful tool which can specifically inhibit the translation of target mRNA. 16 out of 70 genes knocked down, including snx5, demonstrated decreased circulating blood in the zebrafish injected. Further analysis has shown that MO knock down of snx5 in zebrafish (n=152) results in hematopoietic failure with normal vasculature, with normal expression of scl and gata-1 by in situ hybridization, indicating a defect at or beyond the hematopoietic stem cell stage. Quantitative RT-PCR was used to further confirm the phenotype. Levels of hemoglobin, l-plastin and myeloperoxidase mRNA in snx5 morphants compared to uninjected controls, were significantly lower, consistent with a multi-lineage hematopoietic differentiation defect. The decrease in circulating blood could be partially rescued by overexpressing human SNX5 cDNA, confirming specificity of the morphant phenotype. To determine whether SNX5 might be linked to Fanconi anemia (FA), we measured levels of SNX5 mRNA and protein in EBV transformed cell lines from patients with Fanconi-complementation group A (FANCA), -complementation group C (FANCC) or unknown complementation group (FANC-NX). We have found no significant difference between SNX5 mRNA expression in 11 cell lines compared to Jurkat controls. However, levels of SNX5 protein appeared lower in FANCA cells suggesting a possible role of SNX5 in FANCA, which is being evaluated further. Ongoing studies evaluating the role of SNX5 in normal human hematopoiesis will also be presented.
Update of the Modified Hyper-CVAD Regimen with or without Rituximab in Newly Diagnosed Adult Acute Lymphocytic Leukemia (ALL).
Authors: Thomas Deborah A. , Cortes Jorge , O’Brien Susan , Faderl Stefan , Ravandi Farhad , Garcia-Manero Gullermo , Giles Francis , Beran Miloslav , Koller Charles , Wierda William , Verstovsek Srdan , Ferrajoli Alessandra , Kornblau Steven , Keating Michael , Andreeff Michael , Kantarjian Hagop
Published: April 2020

Hyper-CVAD is effective therapy for adult ALL [Kantarjian et al, JCO18:547, 2000; Kantarjian et al, Cancer101:2788, 2004]. Intensive chemotherapy with hyper-CVAD (fractionated cyclophosphamide, vincristine [VCR], doxorubicin and dexamethasone) was alternated with high dose methotrexate and cytarabine every 21 days for 8 courses with G-CSF and prophylactic antibiotics, followed by maintenance with POMP (6-MP, methotrexate, VCR, prednisone). Complete response (CR) rate was 92% with 3-year disease-free survival (DFS) rates 38% overall. A modified hyper-CVAD regimen was developed to address the following: (1) higher induction mortality in patients (pts) aged 60 or older (17% versus 3%); (2) longer DFS reported with early anthracycline intensification; (3) worse survival with CD20 expression (excluding Burkitt’s [BL] and lymphoblastic lymphoma [LL] subtypes); (4) CNS relapse rate of 6% and 1% in low and high risk pts, respectively and (5) late relapses after completion of therapy.

Modifications to Hyper-CVAD Parameter Hyper-CVAD Modified Hyper-CVAD Laminar air flow rooms No For age ≥ 60 yrs or poor PS Dose-intensive anthracycline No C2 Liposomal DNR & cytarabine Rituximab No For CD20 ≥ 20 Intrathecal treatments 4–16 6–8 Maintenance (POMP) 2 years 3 years Intensifications (MTX, asparaginase) Months 7 & 11 Months 6,7 & 18,19 with hyper-CVAD

Newly diagnosed or primary refractory (1 course only) pts were eligible. BL and Ph+ ALL pts were excluded. From May 2000 to December 2001, 77 pts were treated with the modified regimen detailed above (9 courses of intensive chemotherapy). The program was then modified further with elimination of course 2 anthracycline intensification. An additional 80 pts were treated with hyper-CVAD with or without rituximab (8 courses of intensive chemotherapy). The median age for these 157 pts was 40 yrs (range, 15–83) with 20% aged ≥ 60 yrs; 57% were males. Overall response rate was 94% in the evaluable pts (8 too early) with no difference by CD20 expression (49% were CD20+). No induction deaths were observed in the elderly subgroup (2 younger pts with induction deaths). Outcome with anthracycline intensification appeared worse, particularly in the CD20 negative group. The addition of rituximab appeared to improve DFS in CD20 positive group (with or without anthracycline intensification) compared with hyper-CVAD alone (2 yr DFS 90% versus 65%, p=.03); however, overall survival in the CD20 positive group was influenced by deaths in CR (10%) in elderly patients related to GNR sepsis or pneumonia during the intensive phase. Additional accrual and follow-up is needed to further define the role of rituximab in non-Burkitt’s adult ALL.
The Role of CD4+ T Cells in a Murine Model of Acute Lymphoblastic Leukaemia.
Authors: Hegazy Ahmed N. , Wolenski Mathias , Welte Karl , Klein Christoph
Published: April 2020

To assess CD4-mediated anti-tumor immunity in a murine acute lymphoblastic leukaemia model system, we have generated a series of BCR-ABL positive pre-B cell lines expressing the surrogate tumor antigen ovalbumine. Upon intravenous injection, PKH-26-labeled leukaemia cells were taken up by splenic CD8+ but not by CD8− dendritic cells (DC). In comparison to PBS-injected DCs, CD8+ DCs also showed increased expression of CD40, CD80, and CD86. Purified DCs from leukemic mice stimulated transgenic DO11.10 T cells recognizing OVA323–339 in the context of I-Ad, suggesting efficient presentation of the surrogate tumor antigen. Next, we utilized adoptive transfer of DO11.10 T cells to measure tumor-specific T cell responses in vivo. OVA-expressing BM185 cells were unable to directly stimulate DO11.10 T cells, as shown by 3H-thymidine incorporation. In contrast, DO11.10 T cells were activated in vivo in spleen and lymph nodes, as shown by upregulation of CD44 and CFSE staining, suggesting that DC effectively present tumor antigens to DO11.10 T cells in vivo. However, despite of detectable T cell activation and proliferative T cell responses, all animals succumbed to progressive leukemia. Furthermore, adoptive transfer of naïve DO11.10 cells did not induce protective anti-leukemia immunity. Interestingly, in vivo primed DO11.10 T cells did not express interferon-γ. We therefore hypothesized that inefficient in vivo priming of TH1 cells may contribute to immune evasion of ALL cells. To address this question, we primed DO11.10 T cells in vitro prior to adoptive transfer. In this setting, DO11.10 T cells expressed interferon-γ and induced regression of preestablished leukaemia. This effect was dependent on CD8 cells, as shown by in vivo depletion experiments. Our experimental system supports the concept of CD4-dependent antitumor immunity and provides a platform to assess immunological mechanisms of novel strategies to therapeutically enhance antileukaemic immune responses.