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Genetic mutations are being thoroughly mapped in human cancers, yet a fundamental question in cancer biology is whether such mutations are functionally required for cancer initiation, maintenance of established cancer, or both. Here, we study this question in the context of human acute myeloid leukemia (AML), where DNMT3A R882 missense mutations often arise early, in pre-leukemic clonal hematopoiesis, and corrupt the DNA methylation landscape to initiate leukemia. We developed CRISPR-based methods to directly correct DNMT3A R882 mutations in leukemic cells obtained from patients. Surprisingly, DNMT3A R882 mutations were largely dispensable for disease maintenance. Replacing DNMT3A R882 mutants with wild-type DNMT3A did not impair the ability of AML cells to engraft in vivo , and minimally altered DNA methylation. Taken together, DNMT3A R882 mutations are initially necessary for AML initiation, but are largely dispensable for disease maintenance. The notion that initiating oncogenes differ from those that maintain cancer has important implications for cancer evolution and therapy., Competing Interests: Declaration of interests: R.M. is on the Advisory Boards of Kodikaz Therapeutic Solutions, Orbital Therapeutics, Pheast Therapeutics, 858 Therapeutics, Prelude Therapeutics, Mubadala Capital, and Aculeus Therapeutics. R.M. is a co-founder and equity holder of Pheast Therapeutics, MyeloGene, and Orbital Therapeutics.

Hematopoietic multipotent progenitors (MPPs) regulate blood cell production to appropriately meet the biological demands of the human body. Human MPPs remain ill-defined whereas mouse MPPs have been well characterized with distinct immunophenotypes and lineage potencies. Using multiomic single cell analyses and complementary functional assays, we identified new human MPPs and oligopotent progenitor populations within Lin-CD34+CD38dim/lo adult bone marrow with distinct biomolecular and functional properties. These populations were prospectively isolated based on expression of CD69, CLL1, and CD2 in addition to classical markers like CD90 and CD45RA. We show that within the canonical Lin-CD34+CD38dim/loCD90CD45RA-MPP population, there is a CD69+ MPP with long-term engraftment and multilineage differentiation potential, a CLL1+ myeloid-biased MPP, and a CLL1-CD69-erythroid-biased MPP. We also show that the canonical Lin-CD34+CD38dim/loCD90-CD45RA+ LMPP population can be separated into a CD2+ LMPP with lymphoid and myeloid potential, a CD2-LMPP with high lymphoid potential, and a CLL1+ GMP with minimal lymphoid potential. We used these new HSPC profiles to study human and mouse bone marrow cells and observe limited cell type specific homology between humans and mice and cell type specific changes associated with aging. By identifying and functionally characterizing new adult MPP sub-populations, we provide an updated reference and framework for future studies in human hematopoiesis.

Mutations in additional sex combs like 1 (ASXL1) confer poor prognosis both in myeloid malignancies and in premalignant clonal hematopoiesis (CH). However, the mechanisms by which these mutations contribute to disease initiation remain unresolved, and mutation-specific targeting has remained elusive. To address this, we developed a human disease model that recapitulates the disease trajectory from ASXL1-mutant CH to lethal myeloid malignancy. We demonstrate that mutations in ASXL1 lead to the expression of a functional, truncated protein and determine that truncated ASXL1 leads to global redistribution of the repressive chromatin mark H2AK119Ub, increased transposase-accessible chromatin, and activation of both myeloid and stem cell gene-expression programs. Finally, we demonstrate that H2AK119Ub levels are tied to truncated ASXL1 expression levels and leverage this observation to demonstrate that inhibition of the PRC1 complex might be an ASXL1-mutant-specific therapeutic vulnerability in both premalignant CH and myeloid malignancy., Significance: Mutant ASXL1 is a common driver of CH and myeloid malignancy. Using primary human HSPCs, we determine that truncated ASXL1 leads to redistribution of H2AK119Ub and may affect therapeutic vulnerability to PRC1 inhibition., (©2024 The Authors; Published by the American Association for Cancer Research.)

Rare preleukemic hematopoietic stem cells (pHSC) harboring only the initiating mutations can be detected at the time of acute myeloid leukemia (AML) diagnosis. pHSCs are the origin of leukemia and a potential reservoir for relapse. Using primary human samples and gene editing to model isocitrate dehydrogenase 1 (IDH1) mutant pHSCs, we show epigenetic, transcriptional, and metabolic differences between pHSCs and healthy hematopoietic stem cells (HSC). We confirm that IDH1-driven clonal hematopoiesis is associated with cytopenia, suggesting an inherent defect to fully reconstitute hematopoiesis. Despite giving rise to multilineage engraftment, IDH1-mutant pHSCs exhibited reduced proliferation, blocked differentiation, downregulation of MHC class II genes, and reprogramming of oxidative phosphorylation metabolism. Critically, inhibition of oxidative phosphorylation resulted in the complete eradication of IDH1-mutant pHSCs but not IDH2-mutant pHSCs or wild-type HSCs. Our results indicate that IDH1-mutant preleukemic clones can be targeted with complex I inhibitors, offering a potential strategy to prevent the development and relapse of leukemia., Significance: A high burden of pHSCs is associated with worse overall survival in AML. Using single-cell sequencing, metabolic assessment, and gene-edited human models, we find human pHSCs with IDH1 mutations to be metabolically vulnerable and sensitive to eradication by complex I inhibition. See related commentary by Steensma., (©2023 American Association for Cancer Research.)

Relapse of acute myeloid leukemia (AML) is highly aggressive and often treatment refractory. We analyzed previously published AML relapse cohorts and found that 40% of relapses occur without changes in driver mutations, suggesting that non-genetic mechanisms drive relapse in a large proportion of cases. We therefore characterized epigenetic patterns of AML relapse using 26 matched diagnosis-relapse samples with ATAC-seq. This analysis identified a relapse-specific chromatin accessibility signature for mutationally stable AML, suggesting that AML undergoes epigenetic evolution at relapse independent of mutational changes. Analysis of leukemia stem cell (LSC) chromatin changes at relapse indicated that this leukemic compartment underwent significantly less epigenetic evolution than non-LSCs, while epigenetic changes in non-LSCs reflected overall evolution of the bulk leukemia. Finally, we used single-cell ATAC-seq paired with mitochondrial sequencing (mtscATAC) to map clones from diagnosis into relapse along with their epigenetic features. We found that distinct mitochondrially-defined clones exhibit more similar chromatin accessibility at relapse relative to diagnosis, demonstrating convergent epigenetic evolution in relapsed AML. These results demonstrate that epigenetic evolution is a feature of relapsed AML and that convergent epigenetic evolution can occur following treatment with induction chemotherapy., Competing Interests: Declaration of Interests R.M. is on the Advisory Boards of Kodikaz Therapeutic Solutions, Orbital Therapeutics, and 858 Therapeutics and is an inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences. R.M. is a co-founder and equity holder of Pheast Therapeutics, MyeloGene, and Orbital Therapeutics. A.T.S. is a founder of Immunai and Cartography Biosciences and receives research funding from Allogene Therapeutics and Merck Research Laboratories. C.A.L. is a consultant for Cartography Biosciences.

Disease-initiating mutations in the transcription factor RUNX1 occur as germline and somatic events that cause leukemias with particularly poor prognosis. However, the role of RUNX1 in leukemogenesis is not fully understood, and effective therapies for RUNX1-mutant leukemias remain elusive. Here, we used primary patient samples and a RUNX1-KO model in primary human hematopoietic cells to investigate how RUNX1 loss contributes to leukemic progression and to identify targetable vulnerabilities. Surprisingly, we found that RUNX1 loss decreased proliferative capacity and stem cell function. However, RUNX1-deficient cells selectively upregulated the IL-3 receptor. Exposure to IL-3, but not other JAK/STAT cytokines, rescued RUNX1-KO proliferative and competitive defects. Further, we demonstrated that RUNX1 loss repressed JAK/STAT signaling and rendered RUNX1-deficient cells sensitive to JAK inhibitors. Our study identifies a dependency of RUNX1-mutant leukemias on IL-3/JAK/STAT signaling, which may enable targeting of these aggressive blood cancers with existing agents.

The conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is a key step in DNA demethylation that is mediated by ten-eleven translocation (TET) enzymes, which require ascorbate/vitamin C. Here, we report the 5hmC landscape of normal hematopoiesis and identify cell type-specific 5hmC profiles associated with active transcription and chromatin accessibility of key hematopoietic regulators. We utilized CRISPR/Cas9 to model TET2 loss-of-function mutations in primary human hematopoietic stem and progenitor cells (HSPC). Disrupted cells exhibited increased colonies in serial replating, defective erythroid/megakaryocytic differentiation, and in vivo competitive advantage and myeloid skewing coupled with reduction of 5hmC at erythroid-associated gene loci. Azacitidine and ascorbate restored 5hmC abundance and slowed or reverted the expansion of TET2-mutant clones in vivo. These results demonstrate the key role of 5hmC in normal hematopoiesis and TET2-mutant phenotypes and raise the possibility of utilizing these agents to further our understanding of preleukemia and clonal hematopoiesis., Significance: We show that 5-hydroxymethylation profiles are cell type-specific and associated with transcriptional abundance and chromatin accessibility across human hematopoiesis. TET2 loss caused aberrant growth and differentiation phenotypes and disrupted 5hmC and transcriptional landscapes. Treatment of TET2 KO HSPCs with ascorbate or azacitidine reverted 5hmC profiles and restored aberrant phenotypes. This article is highlighted in the In This Issue feature, p. 265., (©2022 American Association for Cancer Research.)

Background: Immunotherapy of acute myeloid leukemia has experienced considerable advances, however novel target antigens continue to be sought after. To this end, unbiased approaches for surface protein detection are limited and integration with other data types, such as gene expression and somatic mutational burden, are poorly utilized. The Cell Surface Capture technology provides an unbiased, discovery-driven approach to map the surface proteins on cells of interest. Yet, direct utilization of primary patient samples has been limited by the considerable number of viable cells needed., Methods: Here, we optimized the Cell Surface Capture protocol to enable direct interrogation of primary patient samples and applied our optimized protocol to a set of samples from patients with acute myeloid leukemia (AML) to generate the AML surfaceome. We then further curated this AML surfaceome to exclude antigens expressed on healthy tissues and integrated mutational burden data from hematologic cancers to further enrich for targets which are likely to be essential to leukemia biology. Finally, we validated our findings in a separate cohort of AML patient samples., Results: Our protocol modifications allowed us to double the yield in identified proteins and increased the specificity from 54 to 80.4% compared to previous approaches. Using primary AML patient samples, we were able to identify a total of 621 surface proteins comprising the AML surfaceome. We integrated this data with gene expression and mutational burden data to curate a set of robust putative target antigens. Seventy-six proteins were selected as potential candidates for further investigation of which we validated the most promising novel candidate markers, and identified CD148, ITGA4 and Integrin beta-7 as promising targets in AML. Integrin beta-7 showed the most promising combination of expression in patient AML samples, and low or absent expression on healthy hematopoietic tissue., Conclusion: Taken together, we demonstrate the feasibility of a highly optimized surfaceome detection method to interrogate the entire AML surfaceome directly from primary patient samples and integrate this data with gene expression and mutational burden data to achieve a robust, multiomic target identification platform. This approach has the potential to accelerate the unbiased target identification for immunotherapy of AML., (© 2022. The Author(s).)

Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

Cancer-related anemia is present in more than 60% of newly diagnosed cancer patients and is associated with substantial morbidity and high medical costs. Drugs that enhance erythropoiesis are urgently required to decrease transfusion rates and improve quality of life. Clinical studies have observed an unexpected improvement in hemoglobin and RBC transfusion-independence in patients with acute myeloid leukemia (AML) treated with the isocitrate dehydrogenase 2 (IDH2) mutant-specific inhibitor enasidenib, leading to improved quality of life without a reduction in AML disease burden. Here, we demonstrate that enasidenib enhanced human erythroid differentiation of hematopoietic progenitors. The phenomenon was not observed with other IDH1/2 inhibitors and occurred in IDH2-deficient CRISPR-engineered progenitors independently of D-2-hydroxyglutarate. The effect of enasidenib on hematopoietic progenitors was mediated by protoporphyrin accumulation, driving heme production and erythroid differentiation in committed CD71+ progenitors rather than hematopoietic stem cells. Our results position enasidenib as a promising therapeutic agent for improvement of anemia and provide the basis for a clinical trial using enasidenib to decrease transfusion dependence in a wide array of clinical contexts.

Although most patients with acute myeloid leukemia (AML) achieve clinical remission with induction chemotherapy, relapse rates remain high. Next-generation sequencing enables minimal/measurable residual disease (MRD) detection; however, clinical significance is limited due to difficulty differentiating between pre-leukemic clonal hematopoiesis and frankly malignant clones. Here, we investigated AML MRD using targeted single-cell sequencing (SCS) at diagnosis, remission, and relapse (n = 10 relapsed, n = 4 nonrelapsed), with a total of 310 737 single cells sequenced. Sequence variants were identified in 80% and 75% of remission samples for patients with and without relapse, respectively. Pre-leukemic clonal hematopoiesis clones were detected in both cohorts, and clones with multiple cooccurring mutations were observed in 50% and 0% of samples. Similar clonal richness was observed at diagnosis in both cohorts; however, decreasing clonal diversity at remission was significantly associated with longer relapse-free survival. These results show the power of SCS in investigating AML MRD and clonal evolution., (© 2020 by The American Society of Hematology.)

Objectives: Innovative post-remission therapies are needed to eliminate residual AML cells. DC vaccination is a promising strategy to induce anti-leukaemic immune responses., Methods: We conducted a first-in-human phase I study using TLR7/8-matured DCs transfected with RNA encoding the two AML-associated antigens WT1 and PRAME as well as CMVpp65. AML patients in CR at high risk of relapse were vaccinated 10× over 26 weeks., Results: Despite heavy pretreatment, DCs of sufficient number and quality were generated from a single leukapheresis in 11/12 cases, and 10 patients were vaccinated. Administration was safe and resulted in local inflammatory responses with dense T-cell infiltration. In peripheral blood, increased antigen-specific CD8 + T cells were seen for WT1 (2/10), PRAME (4/10) and CMVpp65 (9/10). For CMVpp65, increased CD4 + T cells were detected in 4/7 patients, and an antibody response was induced in 3/7 initially seronegative patients. Median OS was not reached after 1057 days; median RFS was 1084 days. A positive correlation was observed between clinical benefit and younger age as well as mounting of antigen-specific immune responses., Conclusions: Administration of TLR7/8-matured DCs to AML patients in CR at high risk of relapse was feasible and safe and resulted in induction of antigen-specific immune responses. Clinical benefit appeared to occur more likely in patients <65 and in patients mounting an immune response. Our observations need to be validated in a larger patient cohort. We hypothesise that TLR7/8 DC vaccination strategies should be combined with hypomethylating agents or checkpoint inhibition to augment immune responses., Trial Registration: The study was registered at https://clinicaltrials.gov on 17 October 2012 (NCT01734304) and at https://www.clinicaltrialsregister.eu (EudraCT-Number 2010-022446-24) on 10 October 2013., Competing Interests: DJS is employed by Medigene Immunotherapies GmbH, and holds patents and receives royalties for DC vaccines. All other authors declare that they have no conflict of interest., (© 2020 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology Inc.)

Cotton production is reaching a global limit, leading to a growing demand for bio-based textile fibers produced by other means. Textile fibers based on regenerated cellulose from wood holds great potential, but in order to produce fibers, the components need to be dissolved in suitable solvents. Furthermore, the dissolution process of cellulose is not yet fully understood. In this study, we investigated the dissolution state of microcrystalline cellulose in aqueous NaOH by using primarily scattering methods. Contrary to previous findings, this study indicated that cellulose concentrations of up to 2 wt % are completely molecularly dissolved in 8 wt % NaOH. Scattering data furthermore revealed the presence of semi-flexible cylinders with stiff segments. In order to improve the dissolution capability of NaOH, the effects of different additives have been of interest. In this study, scattering data indicated that the addition of ZnO decreased the formation of aggregates, while the addition of PEG did not improve the dissolution properties significantly, although preliminary NMR data did suggest a weak attraction between PEG and cellulose. Overall, this study sheds further light on the dissolution of cellulose in NaOH and highlights the use of scattering methods to assess solvent quality.

In spite of therapeutic improvements in the treatment of different hematologic malignancies, the prognosis of acute myeloid leukemia (AML) treated solely with conventional induction and consolidation chemotherapy remains poor, especially in association with high risk chromosomal or molecular aberrations. Recent discoveries describe the complex interaction of immune effector cells, as well as the role of the bone marrow microenvironment in the development, maintenance and progression of AML. Lipids, and in particular omega-3 as well as omega-6 polyunsaturated fatty acids (PUFAs) have been shown to play a vital role as signaling molecules of immune processes in numerous benign and malignant conditions. While the majority of research in cancer has been focused on the role of lipid mediators in solid tumors, some data are showing their involvement also in hematologic malignancies. There is a considerable amount of evidence that AML cells are targetable by innate and adaptive immune mechanisms, paving the way for immune therapy approaches in AML. In this article we review the current data showing the lipid mediator and lipidome patterns in AML and their potential links to immune mechanisms.

Three mutation-specific targeted therapies have recently been approved by the FDA for the treatment of acute myeloid leukemia (AML): midostaurin for FLT3 mutations, enasidenib for relapsed or refractory cases with IDH2 mutations, and ivosidenib for cases with an IDH1 mutation. Together, these agents offer a mutation-directed treatment approach for up to 45% of de novo adult AML cases, a welcome deluge after a prolonged drought. At the same time, a number of computational tools have recently been developed that promise to further accelerate progress in mutation-specific therapy for AML and other cancers. Technical advances together with comprehensively annotated AML tissue banks have resulted in the availability of large and complex data sets for exploration by the end-user, including (i) microarray gene expression, (ii) exome sequencing, (iii) deep sequencing data of sub-clone heterogeneity, (iv) RNA sequencing of gene expression (bulk and single cell), (v) DNA methylation and chromatin, (vi) and germline quantitative trait loci. Yet few clinicians or experimental hematologists have the time or the training to access or analyze these repositories. This review summarizes the data sets and bioinformatic tools currently available to further the discovery of mutation-specific targets with an emphasis on web-based applications that are open, accessible, user-friendly, and do not require coding experience to navigate. We show examples of how available data can be mined to identify potential targets using synthetic lethality, drug repurposing, epigenetic sub-grouping, and proteomic networks while also highlighting strengths and limitations and the need for superior models for validation.

Some patients with acute myeloid leukemia (AML) who are in complete remission after induction chemotherapy harbor persisting pre-leukemic clones, carrying a subset of leukemia-associated somatic mutations. There is conflicting evidence on the prognostic relevance of these clones for AML relapse. Here, we characterized paired pre-treatment and remission samples from 126 AML patients for mutations in 68 leukemia-associated genes. Fifty patients (40%) retained ≥1 mutation during remission at a VAF of ≥2%. Mutation persistence was most frequent in DNMT3A (65% of patients with mutations at diagnosis), SRSF2 (64%), TET2 (55%), and ASXL1 (46%), and significantly associated with older age (p < 0.0001) and, in multivariate analyses adjusting for age, genetic risk, and allogeneic transplantation, with inferior relapse-free survival (hazard ratio (HR), 2.34; p = 0.0039) and overall survival (HR, 2.14; p = 0.036). Patients with persisting mutations had a higher cumulative incidence of relapse before, but not after allogeneic stem cell transplantation. Our work underlines the relevance of mutation persistence during first remission as a novel risk factor in AML. Persistence of pre-leukemic clones may contribute to the inferior outcome of elderly AML patients. Allogeneic transplantation abrogated the increased relapse risk associated with persisting pre-leukemic clones, suggesting that mutation persistence may guide post-remission treatment.

Certain ionic liquids are powerful cellulose solvents, but tend to be less effective when small-molecule hydrogen bond donors are present. This is generally attributed to competition with cellulose for hydrogen bonding opportunities to the anion of the ionic liquid. We show that the solubility of cellulose in dimethyl sulfoxide solutions of tetrabutylammonium acetate is less strongly affected by water than by ethanol on a molar basis, contrary to what can be expected based on hydrogen bond stoichiometry. Molecular dynamics simulations indicate that the higher tolerance to water is due to water-cellulose interactions that improves solvation of cellulose and, thereby, marginally favors dissolution. Through Kirkwood-Buff theory we show that water, but not ethanol, improves the solvent quality of DMSO and partly compensates for the loss of acetate-cellulose hydrogen bonds.

The advent of new immunotherapeutic agents in clinical practice has revolutionized cancer treatment in the past decade, both in oncology and hematology. The transfer of the immunotherapeutic concepts to the treatment of acute myeloid leukemia (AML) is hampered by various characteristics of the disease, including non-leukemia-restricted target antigen expression profile, low endogenous immune responses, and intrinsic resistance mechanisms of the leukemic blasts against immune responses. However, considerable progress has been made in this field in the past few years.Within this manuscript, we review the recent developments and the current status of the five currently most prominent immunotherapeutic concepts: (1) antibody-drug conjugates, (2) T cell-recruiting antibody constructs, (3) chimeric antigen receptor (CAR) T cells, (4) checkpoint inhibitors, and (5) dendritic cell vaccination. We focus on the clinical data that has been published so far, both for newly diagnosed and refractory/relapsed AML, but omitting immunotherapeutic concepts in conjunction with hematopoietic stem cell transplantation. Besides, we have included important clinical trials that are currently running or have recently been completed but are still lacking full publication of their results.While each of the concepts has its particular merits and inherent problems, the field of immunotherapy of AML seems to have taken some significant steps forward. Results of currently running trials will reveal the direction of further development including approaches combining two or more of these concepts.

Antibody-based immunotherapy represents a promising strategy to eliminate chemorefractory leukemic cells in acute myeloid leukemia (AML). In this study, we evaluated a novel Fc-engineered antibody against CD157 (MEN1112) for its suitability as immunotherapy in AML. CD157 was expressed in 97% of primary AML patient samples. A significant, albeit lower expression level of CD157 was observed within the compartment of leukemia-initiating cells, which are supposed to be the major source of relapse. In healthy donor bone marrow, CD157 was expressed on CD34+ cells. In ex vivo assays, MEN1112 triggered natural killer (NK) cell-mediated cytotoxicity against AML cell lines and primary AML cells. Compared to its parental analogue, the Fc-engineered antibody exhibited higher antibody dependent cellular cytotoxicity responses. Using NK cells from AML patients, we observed heterogeneous MEN1112-mediated cytotoxicity against AML cells, most likely due to well-documented defects in AML-NK cells and corresponding inter-patient variations in NK cell function. Cytotoxicity could not be correlated to the time after completion of chemotherapy. In summary, we could demonstrate that CD157 is strongly expressed in AML. MEN1112 is a promising antibody construct that showed high cytotoxicity against AML cells and warrants further clinical testing. Due to variability in NK-cell function of AML patients, the time of application during the course of the disease as well as combinatorial strategies might influence treatment results.

The bispecific T cell engager blinatumomab has shown encouraging clinical activity in B-precursor acute lymphoblastic leukemia (ALL). However, about half of relapsed/refractory patients do not respond to therapy. Here, we present the case of a 32-year-old male patient with refractory B-precursor ALL who was resistant to treatment with blinatumomab. Bone marrow immunohistochemistry revealed T cell infiltrates and an increase in programmed death-ligand 1 (PD-L1)-positive ALL cells as a potential immune escape mechanism. We were able to recapitulate the clinical observation in vitro by showing that blinatumomab was not able to mediate cytotoxicity of CD19-positive ALL cells using autologous T cells. In contrast, the addition of healthy donor T cells led to lysis of ALL cells.These results strongly encourage further systematic evaluation of checkpoint molecules in cases of blinatumomab treatment failure and might highlight a possible mechanism to overcome resistance to this otherwise highly effective treatment.

We retrospectively compared the incidence of virus infections and outcome in the context of immune reconstitution in two different HLA-haploidentical transplantation (haplo-HSCT) settings. The first was a combined T-cell-replete and T-cell-deplete approach using antithymocyte globulin (ATG) prior to transplantation in patients with hematological diseases (cTCR/TCD group, 28 patients; median age 31 years). The second was a T-cell-replete (TCR) approach using high-dose posttransplantation cyclophosphamide (TCR/PTCY group, 27 patients; median age 43 years). The incidence of herpesvirus infection was markedly lower in the TCR/PTCY (22 %) than in the cTCR/TCD group (93 %). Recovery of CD4+ T cells on day +100 was faster in the TCR/PTCY group. CMV reactivation was 30 % in the TCR/PTCY compared to 57 % in the cTCR/TCD group, and control with antiviral treatment was superior after TCR/PTCY transplantation (100 vs 50 % cTCR/TCD). Twenty-five percent of the patients in the cTCR/TCD group but no patient in the TCR/PTCY group developed PTLD. While 1-year OS was not different (TCR/PTCY 59 % vs cTCR/TCD 39 %; p = 0.28), virus infection-related mortality (VIRM) was significantly lower after TCR/PTCY transplantation (1-year VIRM, 0 % TCR/PTCY vs 29 % cTCR/TCD; p = 0.009). On day +100, predictors of better OS were lymphocytes >300/μl, CD3+ T cells >200/μl, and CD4+ T cells >150/μl, whereas the application of steroids >1 mg/kg was correlated with worse outcome. Our results suggest that by presumably preserving antiviral immunity and allowing fast immune recovery of CD4+ T cells, the TCR approach using posttransplantation cyclophosphamide is well suited to handle the important issue of herpesvirus infection after haplo-HSCT.

Monitoring minimal residual disease is an important way to identify patients with acute myeloid leukemia at high risk of relapse. In this study we investigated the prognostic potential of minimal residual disease monitoring by quantitative real-time polymerase chain reaction analysis of NPM1 mutations in patients treated in the AMLCG 1999, 2004 and 2008 trials. Minimal residual disease was monitored - in aplasia, after induction therapy, after consolidation therapy, and during follow-up - in 588 samples from 158 patients positive for NPM1 mutations A, B and D (with a sensitivity of 10(-6)). One hundred and twenty-seven patients (80.4%) achieved complete remission after induction therapy and, of these, 56 patients (44.1%) relapsed. At each checkpoint, minimal residual disease cut-offs were calculated. After induction therapy a cut-off NPM1 mutation ratio of 0.01 was associated with a high hazard ratio of 4.26 and the highest sensitivity of 76% for the prediction of relapse. This was reflected in a cumulative incidence of relapse after 2 years of 77.8% for patients with ratios above the cut-off versus 26.4% for those with ratios below the cut-off. In the favorable subgroup according to European LeukemiaNet, the cut-off after induction therapy also separated the cohort into two prognostic groups with a cumulative incidence of relapse of 76% versus 6% after 2 years. Our data demonstrate that in addition to pre-therapeutic factors, the course of minimal residual disease in an individual is an important prognostic factor and could be included in clinical trials for the guidance of post-remission therapy. The trials from which data were obtained were registered at www.clinicaltrials.gov (#NCT01382147, #NCT00266136) and at the European Leukemia Trial Registry (#LN&#95;AMLINT2004&#95;230)., (Copyright© Ferrata Storti Foundation.)

Antibody-based immunotherapy represents a promising strategy to target and eliminate chemoresistant leukemic cells. Here, we evaluated the CD33/CD3-bispecific T cell engaging (BiTE) antibody (AMG 330) for its suitability as a therapeutic agent in acute myeloid leukemia (AML). We first assessed CD33 expression levels by flow cytometry and found expression in >99% of patient samples (n = 621). CD33 was highest expressed in AMLs with NPM1 mutations (P < .001) and lower in AMLs with complex karyotypes and t(8;21) translocations (P < .001). Furthermore, leukemic stem cells within the CD34(+)/CD38(-) compartment displayed CD33 at higher levels than healthy donor stem cells (P = .047). In MS-5 feeder cell-based long-term cultures that supported the growth of primary AML blasts for up to 36 days, AMG 330 efficiently recruited and expanded residual CD3(+)/CD45RA(-)/CCR7(+) memory T cells within the patient sample. Even at low effector to target ratios, the recruited T cells lysed autologous blasts completely in the majority of samples and substantially in the remaining samples in a time-dependent manner. This study provides the first correlation of CD33 expression levels with AML genotype in a comprehensive analysis of adult patients. Targeting CD33 ex vivo using AMG 330 in primary AML samples led to T cell recruitment and expansion and remarkable antibody-mediated cytotoxicity, suggesting efficient therapeutic potential in vivo.

The role of non-steroidal anti-inflammatory drugs in inflammatory bowel disease is controversial, as they have been implicated in disease aggravation. Different from other cyclooxygenase inhibitors, acetylsalicylic acid (ASA) enhances the formation of anti-inflammatory and proresolution lipoxins derived from arachidonic acid as well as resolvins from omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA). In this study, we examined the effect of ASA on murine dextran sodium sulfate colitis. A mouse magnetic resonance imaging (MRI) protocol and post mortem assessment were used to assess disease severity, and lipid metabolites were measured using liquid chromatography-coupled tandem mass spectrometry. Decreased colitis activity was demonstrated by phenotype and MRI assessment in mice treated with ASA, and confirmed in postmortem analysis. Analysis of lipid mediators showed sustained formation of lipoxin A4 and an increase of DHA-derived 17-hydroxydocosahexaenoic acid (17-HDHA) after treatment with ASA. Furthermore, in vitro experiments in RAW264.7 murine macrophages demonstrated significantly increased phagocytosis activity after incubation with 17-HDHA, supporting its proresolution effect. These results show a protective effect of ASA in a murine colitis model and could give a rationale for a careful reassessment of ASA therapy in patients with inflammatory bowel disease and particularly ulcerative colitis, possibly combined with DHA supplementation.