Favorable outcome in children and adolescents with a high proportion of advanced phase disease using single/multiple autologous or matched/mismatched allogeneic stem cell transplantations: Favorable outcome in children and adolescents with a high proportion of advanced phase disease usingsingle/multiple autologous or matched/mismatchedallogeneic stem cell transplantations

Purpose: We determined the indication, outcome and risk factors of single and multiple hematopoietic stem cell transplantation(s) (HSCT) in children and adolescents mostly with advanced disease. Methods: Forty-one out of 483 patients (8.5%; median age 9 years) diagnosed at the University of Leipzig with haematological and oncological diseases required HSCT from 1999 to 2011. Results: Patients had overall survival (OS) of 63±10% and 63±16%, event-free survival (EFS) of 57±10% and 42±16%, relapse incidence (RI) of 39±10% and 44±18% and non-relapse mor-tality (NRM) of 4±4% and 13±9% at 10-years after one or more HSCT for allogeneic and autologous HSCT, respectively. One patient in complete remission (CR)1 and five with advanced disease received two HSCT. Four of the six patients maintained/achieved CR for a median of 13 months. Three died of progression and one of NRM. Two patients had a third HSCT and one survived in CR +231 days after HSCT. Risk factors for OS and EFS were disease stage at HSCT and EBMT risk-score. Center (paediatric or JACIE accredited paediatric/adult) was not a determinant for survival. Conclusion: Paediatric single and multiple HSCT are important curative approaches for high-risk malignant diseases with low NRM. Efforts to reduce high RI remain the major aim.:Bibliographic description 3 Introduction: 4 Infections 6 Veno-occlusive disease (VOD) 7 Graft rejection 7 Graft-versus Host Disease (GvHD) 8 Non-relapse mortality (NRM) 9 Relapse of the underling disease 9 Indications for HSCT 10 HSCT in Children. 10 Research questions: 12 Publication 13 Discussion 22 Future developments 25 References 26 Abbreviations 28 Summary 29 Zusammenfassung 33 Erklärung über die eigenständige Abfassung der Arbeit 38 Curriculum vitae 39 Acknowledgement 4

Plasmodium falciparum Heterochromatin Protein 1 Marks Genomic Loci Linked to Phenotypic Variation of Exported Virulence Factors

Epigenetic processes are the main conductors of phenotypic variation in eukaryotes. The malaria parasite Plasmodium falciparum employs antigenic variation of the major surface antigen PfEMP1, encoded by 60 var genes, to evade acquired immune responses. Antigenic variation of PfEMP1 occurs through in situ switches in mono-allelic var gene transcription, which is PfSIR2-dependent and associated with the presence of repressive H3K9me3 marks at silenced loci. Here, we show that P. falciparum heterochromatin protein 1 (PfHP1) binds specifically to H3K9me3 but not to other repressive histone methyl marks. Based on nuclear fractionation and detailed immuno-localization assays, PfHP1 constitutes a major component of heterochromatin in perinuclear chromosome end clusters. High-resolution genome-wide chromatin immuno-precipitation demonstrates the striking association of PfHP1 with virulence gene arrays in subtelomeric and chromosome-internal islands and a high correlation with previously mapped H3K9me3 marks. These include not only var genes, but also the majority of P. falciparum lineage-specific gene families coding for exported proteins involved in host–parasite interactions. In addition, we identified a number of PfHP1-bound genes that were not enriched in H3K9me3, many of which code for proteins expressed during invasion or at different life cycle stages. Interestingly, PfHP1 is absent from centromeric regions, implying important differences in centromere biology between P. falciparum and its human host. Over-expression of PfHP1 results in an enhancement of variegated expression and highlights the presence of well-defined heterochromatic boundaries. In summary, we identify PfHP1 as a major effector of virulence gene silencing and phenotypic variation. Our results are instrumental for our understanding of this widely used survival strategy in unicellular pathogens

Antibody response after vaccination against SARS-CoV-2 in adults with hematological malignancies: a systematic review and meta-analysis

Vaccines against SARS-CoV-2 have shown remarkable efficacy and thus constitute an important preventive option against coronavirus disease 2019 (COVID-19), especially in fragile patients. We aimed to systematically analyze the outcomes of patients with hematological malignancies who received vaccination and to identify specific groups with differences in outcomes. The primary end point was antibody response after full vaccination (2 doses of mRNA or one dose of vectorbased vaccines). We identified 49 studies comprising 11,086 individuals. Overall risk of bias was low. The pooled response for hematological malignancies was 64% (95% confidence interval [CI]: 59-69; I²=93%) versus 96% (95% CI: 92-97; I²=44%) for solid cancer and 98% (95% CI: 96-99; I²=55%) for healthy controls (P<0.001). Outcome was different across hematological malignancies (P<0.001). The pooled response was 50% (95% CI: 43-57; I²=84%) for chronic lymphocytic leukemia, 76% (95% CI: 67-83; I²=92%) for multiple myeloma, 83% (95% CI: 69-91; I²=85%) for myeloproliferative neoplasms, 91% (95% CI: 82-96; I²=12%) for Hodgkin lymphoma, and 58% (95% CI: 44-70; I²=84%) for aggressive and 61% (95% CI: 48-72; I²=85%) for indolent non-Hodgkin lymphoma. The pooled response for allogeneic and autologous hematopoietic cell transplantation was 82% and 83%, respectively. Being in remission and prior COVID-19 showed significantly higher responses. Low pooled response was identified for active treatment (35%), anti-CD20 therapy ≤1 year (15%), Bruton kinase inhibition (23%), venetoclax (26%), ruxolitinib (42%), and chimeric antigen receptor T-cell therapy (42%). Studies on timing, value of boosters, and long-term efficacy are needed. This study is registered with PROSPERO (clinicaltrials gov. Identifier: CRD42021279051)

Use of busulfan in conditioning for allogeneic hematopoietic stem cell transplantation in adults : a survey by the Transplant Complications Working Party of the EBMT

A survey was carried out among EBMT centers about the use of busulfan for conditioning in allogeneic stem cell transplantation. Of 109 responding centers, 106 used busulfan for conditioning, 102 in conventional myeloablative doses, and 93 in reduced doses (RIC). The route of administration was mostly intravenous, but similar to 10% of the centers gave the drug orally. The number of doses in i.v. administration varied and was in myeloablative conditioning mostly one (50 centers) or four (43 centers) doses a day. Seventeen of the 106 centers used pharmacokinetics for dose adjustment in myeloablative conditioning, nine in RIC. The details of pharmacokinetic monitoring varied markedly. Three quarters of the centers reported adjusting the dose based on obesity in myeloablative conditioning and about 60% in RIC. The most common method for dose calculation was ideal body weight + 0.25 x (actual body weight - ideal body weight). In conclusion, the present survey showed marked heterogeneity in the current practices of busulfan administration for conditioning. The impact of the heterogeneity is not well known. Due to this and the scarcity of support from controlled clinical studies, no clear guidelines can be presented, but some prevailing policies to be recommended were identified.Peer reviewe

Mapping and functional analysis of heterochromatin protein 1 phosphorylation in the malaria parasite Plasmodium falciparum

Previous studies in model eukaryotes have demonstrated that phosphorylation of heterochromatin protein 1 (HP1) is important for dynamically regulating its various functions. However, in the malaria parasite Plasmodium falciparum both the function of HP1 phosphorylation and the identity of the protein kinases targeting HP1 are still elusive. In order to functionally analyze phosphorylation of P. falciparum HP1 (PfHP1), we first mapped PfHP1 phosphorylation sites by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of native PfHP1, which identified motifs from which potential kinases could be predicted; in particular, several phosphorylated residues were embedded in motifs rich in acidic residues, reminiscent of targets for P. falciparum casein kinase 2 (PfCK2). Secondly, we tested recombinant PfCK2 and a number of additional protein kinases for their ability to phosphorylate PfHP1 in in vitro kinase assays. These experiments validated our prediction that PfHP1 acts as a substrate for PfCK2. Furthermore, LC-MS/MS analysis showed that PfCK2 phosphorylates three clustered serine residues in an acidic motif within the central hinge region of PfHP1. To study the role of PfHP1 phosphorylation in live parasites we used CRISPR/Cas9-mediated genome editing to generate a number of conditional PfHP1 phosphomutants based on the DiCre/LoxP system. Our studies revealed that neither PfCK2-dependent phosphorylation of PfHP1, nor phosphorylation of the hinge domain in general, affect PfHP1's ability to localize to heterochromatin, and that PfHP1 phosphorylation in this region is dispensable for the proliferation of P. falciparum blood stage parasites

Final Evaluation of a Clinical Phase III Trial Comparing Treosulfan to Busulfan-Based Conditioning Therapy Prior to Allogeneic Hematopoietic Stem Cell Transplantation of Adult Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients Ineligible to Standard Myeloablative Regimens

Background Allogeneic hematopoietic stem cell transplantation (HCT) remains a challenge in elderly and comorbid AML and MDS patients. This patient population is at increased risk for non-relapse mortality (NRM) when treated with standard myeloablative conditioning and was selected to compare a newly developed treosulfan-based with a well-established reduced intensity busulfan-based preparative regimen in a prospective randomized clinical phase III trial. Methods Adult patients with AML in remission or MDS scheduled for HCT from matched related or unrelated donors, aged ≥50 years or with a comorbidity index (HCT-CI) of >2 were enrolled by a central stratified randomization procedure. Treatment arms consisted of intravenous (IV) treosulfan (10 g/m²/day [d-4 to d-2]) or IV busulfan (3.2 mg/kg/day [d-4 to d-3]), both combined with IV fludarabine (30 mg/m²/day [d-6 to d-2]). The primary objective was to compare event-free survival (EFS) at two years with relapse/progression of disease, graft failure, or death reported as events. Secondary endpoints were safety evaluation (according to CTCAE v4.03), engraftment, chimerism, overall survival (OS), relapse/progression incidence (RI), NRM and acute or chronic GvHD. After a previously conducted confirmatory interim analysis (based on 476 patients), which resulted in early termination of patient accrual due to significant non-inferiority of treosulfan treatment with improved EFS, NRM and OS (Beelen et al., ASH 2017), results of the final analysis of all 570 randomized patients including post surveillance data are provided here. Results Median age of the 551 patients (352 AML; 199 MDS) included in the full analysis set (268 treosulfan; 283 busulfan) was 60 years (range: 31, 70). Frequencies of early adverse events (d-6 to d+28) and incidences of acute and chronic GvHD were largely comparable between the two regimens, while extensive chronic GvHD was numerically in favor of treosulfan (19.7% vs. 26.7%; p=0.0750). Primary neutrophil recovery at day +28 was comparable, while the rate of complete donor-type chimerism (day +28) was higher after treosulfan (93.2% vs. 83.3%; p Conclusions Final evaluation of this phase III trial substantiates the previous confirmatory analysis resulting in significantly improved survival after treosulfan-based conditioning. Due to the reduction of NRM a major clinical benefit of the new treosulfan conditioning regimen was demonstrated in the selected AML/MDS patient population

Addition of Rituximab in Reduced Intensity Conditioning Regimens for B-Cell Malignancies Does Not Influence Transplant Outcomes: EBMT Registry Analyses Following Allogeneic Stem Cell Transplantation for B-Cell Malignancies

Rituximab (R) is increasingly incorporated in reduced intensity conditioning (RIC) regimens for allogeneic hematopoietic cell transplantation (alloHCT) in patients with B-cell malignancies, not only to improve disease control, but also to prevent graft-versus-host disease (GVHD). There are no randomized prospective data to validate this practice, although single center data and the CIBMTR analysis have shown promising results. We aimed at validation of these findings in a large registry study. We conducted a retrospective analysis using the EBMT registry of 3,803 adult patients with B-cell malignancies undergoing alloHCT (2001–2013) with either rituximab (R-RIC-9%) or nonrituximab (RIC-91%) reduced intensity regimens respectively. Median age and median follow up were 55 years (range 19.1–77.3) and 43.2 months (range 0.3–179.8), respectively. There was no difference in transplant outcomes (R-RIC vs RIC), including 1-year overall survival (69.9% vs 70.7%), 1-year disease-free survival (64.4% vs 62.2%), 1-year non-relapse mortality (21% vs 22%), and day-100 incidence of acute GVHD 2-4° (12% vs 12%). In summary, we found that addition of rituximab in RIC regimens for B-cell malignancies had no significant impact on major transplant outcome variables. Of note, data on chronic GVHD was not available, limiting the conclusions that can be drawn from the present study

Molecular landscape and prognostic impact of FLT3 -ITD insertion site in acute myeloid leukemia : RATIFY study results

In acute myeloid leukemia (AML) internal tandem duplications of the FLT3 gene (FLT3- ITD) are associated with poor prognosis. Retrospectively, we investigated the prognostic and predictive impact of FLT3 -ITD insertion site (IS) in 452 patients randomized within the RATIFY trial, which evaluated midostaurin additionally to intensive chemotherapy. Next-generation sequencing identified 908 ITDs, with 643 IS in the juxtamembrane domain (JMD) and 265 IS in the tyrosine kinase domain-1 (TKD1). According to IS, patients were categorized as JMDsole (n = 251, 55%), JMD and TKD1 (JMD/TKD1; n = 117, 26%), and TKD1sole (n = 84, 19%). While clinical variables did not differ among the 3 groups, NPM1 mutation was correlated with JMDsole (P = 0.028). Overall survival (OS) differed significantly, with estimated 4-year OS probabilities of 0.44, 0.50, and 0.30 for JMDsole, JMD/TKD1, and TKD1sole, respectively (P = 0.032). Multivariate (cause-specific) Cox models for OS and cumulative incidence of relapse using allogeneic hematopoietic cell transplantation (HCT) in first complete remission as a time-dependent variable identified TKD1sole as unfavorable and HCT as favorable factors. In addition, Midostaurin exerted a significant benefit only for JMDsole. Our results confirm the distinct molecular heterogeneity of FLT3 -ITD and the negative prognostic impact of TKD1 IS in AML that was not overcome by midostaurin

A Major Role for the Plasmodium falciparum ApiAP2 Protein PfSIP2 in Chromosome End Biology

The heterochromatic environment and physical clustering of chromosome ends at the nuclear periphery provide a functional and structural framework for antigenic variation and evolution of subtelomeric virulence gene families in the malaria parasite Plasmodium falciparum. While recent studies assigned important roles for reversible histone modifications, silent information regulator 2 and heterochromatin protein 1 (PfHP1) in epigenetic control of variegated expression, factors involved in the recruitment and organization of subtelomeric heterochromatin remain unknown. Here, we describe the purification and characterization of PfSIP2, a member of the ApiAP2 family of putative transcription factors, as the unknown nuclear factor interacting specifically with cis-acting SPE2 motif arrays in subtelomeric domains. Interestingly, SPE2 is not bound by the full-length protein but rather by a 60kDa N-terminal domain, PfSIP2-N, which is released during schizogony. Our experimental re-definition of the SPE2/PfSIP2-N interaction highlights the strict requirement of both adjacent AP2 domains and a conserved bipartite SPE2 consensus motif for high-affinity binding. Genome-wide in silico mapping identified 777 putative binding sites, 94% of which cluster in heterochromatic domains upstream of subtelomeric var genes and in telomere-associated repeat elements. Immunofluorescence and chromatin immunoprecipitation (ChIP) assays revealed co-localization of PfSIP2-N with PfHP1 at chromosome ends. Genome-wide ChIP demonstrated the exclusive binding of PfSIP2-N to subtelomeric SPE2 landmarks in vivo but not to single chromosome-internal sites. Consistent with this specialized distribution pattern, PfSIP2-N over-expression has no effect on global gene transcription. Hence, contrary to the previously proposed role for this factor in gene activation, our results provide strong evidence for the first time for the involvement of an ApiAP2 factor in heterochromatin formation and genome integrity. These findings are highly relevant for our understanding of chromosome end biology and variegated expression in P. falciparum and other eukaryotes, and for the future analysis of the role of ApiAP2-DNA interactions in parasite biology