Early Hematopoietic Cells Research Articles

A Retrospective Audit of the Use of Next-Generation Sequencing in the Management of Myelofibrosis Patients in North London Hospitals

Background: Myelofibrosis (MF) is typically characterized by one of three classic driver mutations identified in the JAK2, CALR, and MPL genes. However, a small proportion of MF patients are triple negative and do not carry any of these driver mutations. In the latest WHO 2016 classification of myeloid neoplasms, the authors recommended further genetic testing in this subset of patients to confirm clonality 1. In MF, mutations in ASXL1, EZH2, IDH1, IDH2, SRSF2, U2AF predicted shorter survival and increased risk of leukemic transformation 2,3,4. The presence of one or more of these mutations defined a higher risk category termed high molecular risk (HMR) 4. In transplant eligible patients with HMR, early transplantation should be considered. The hematological malignancy diagnostic service (HMDS) at University College London Hospital (UCLH) serves 8 secondary or tertiary hospitals in North London. At our HMDS, transplant eligible patients with suspected MF are offered the TruSight Myeloid NGS (Illumina) with a panel of 54 genes. The panel is run on the MiSeq Illumina platform. All results are integrated by the oncogenomics multidisciplinary team (MDT). Aim: We evaluated the diagnostic, prognostic, and therapeutic utilization of 54 gene panel myeloid NGS in management of MF patients. Method: Eligible patients were those: (1) Aged >18 years at diagnosis of MF, with a diagnostic date between 1/1/2017 and 12/31/2019. (2) Availability of diagnostic bone marrow samples at HMDS at UCLH. Data was retrospectively collected from patients' medical records. Gene variance screening was performed as described before 5. Results: 67 patients were referred to HMDS at UCLH with suspected myelofibrosis between 1/1/2017 and 12/31/2019. 36 patients were included in this retrospective audit (94% [n= 34/36] with primary MF (PMF) and 6% [n=2/36] with secondary MF). The median age at MF diagnosis was 69 years (range 48-90 years). 55.5% patients (n=20/36) had JAK2 mutation, 13.8% (n=5/36) had CALR mutation, 5.5% (n=2/36) had MPL mutation, 27.3% (n=9/36) were negative for JAK2, CALR and MPL mutation (triple negative) by polymerase chain reaction (PCR). 24/36 patients had myeloid NGS. This includes 15/17 (88%) transplant eligible patients and 9/19 (47%) transplant ineligible patients. 12% patients (n=3/24) had no pathogenic mutations, 42% (n=10/24) had 1 pathogenic mutation, 21% (n=5/24) had 2 pathogenic mutations, 21% (n=5/24) had 3 pathogenic mutations, and 4% (n=1/24) had 4 mutations (Figure 1). The most commonly identified pathogenic mutation by NGS was JAK2 (42%), followed by ASXL1 (21%), SRSF2 (17%), TET2 (13%), CALR (13%). Other less frequent mutations were identified in U2AF1 (8%), SETBP1 (8%), MPL (4%), TP53 (4%), SF3B1 (4%), NRAS (4%), PHF6(4%), CUX1(4%), RUNX1 (4%), BCOR (4%) (Figure 2). 57% (n=4/7) triple negative patients showed evidence of clonal hematopoiesis by NGS. Compared with our standard PCR, NGS showed 88% concordance in identifying JAK2, MPL and CALR mutations. 12% mutations identified by PCR were not identified by NGS. No cryptic mutations were identified. 29% patients (n=7/24) had high molecular risk, of whom 4 were transplant eligible with a DIPSS-plus score of intermediate-2 or above. All of the 4 patients were referred for early Hematopoietic Stem Cell Transplant (HSCT). Despite early HSCT, 2 patients showed early relapse and disease progression of MF. Conclusion: Myeloid NGS showed clinical utility in diagnosis of MF through verifying clonal hematopoiesis in triple negative patients and refining clinical decisions relating to HSCT.

Zrsr2 Deficient Zebrafish Display Hematopoietic Defects and U12-Type Intron Retention in mRNA Processing Genes

ZRSR2 is a small nuclear riboprotein necessary for assembly of the minor spliceosome and subsequent splicing of U12-type introns. It is involved in the recognition of 3' splice sites during the assembly of the spliceosome. Somatic mutations in ZRSR2 gene are present in hematopoietic malignancies, most notably myelodysplastic syndrome (MDS). These mutations are spread throughout the gene, indicating that its function is critical for hematopoiesis. In spite of these clinical associations, the role of ZRSR2 in hematopoiesis has not been well studied. Zebrafish make an excellent animal model for investigating this gene as all of the functional domains in the human protein are conserved with a high level of protein similarity in zebrafish. Additionally, hematopoietic development is well understood in this species. We used CRISPR/Cas9 to generate a zebrafish zrsr2 knockout model with an 11 base pair deletion (zrsr2Δ11) that results in frameshift with premature truncation and loss of all functional domains (p.W167fs*175). The zrsr2Δ11/Δ11 mutants began to appear morphologically different from their siblings by 4 days post fertilization (dpf), showing aberrant development in the mandible and pharyngeal arches. The zrsr2Δ11/Δ11 mutants developed mild to severe edema by 6 dpf and died by 8 dpf. To understand its role in hematopoiesis, we performed o-dianisidine staining and whole mount in situ hybridization (WISH) with lineage-specific markers during embryonic development. Our data showed that zrsr2Δ11/Δ11 embryos exhibit mild anemia by 2 dpf, suggesting that primitive hematopoiesis is defective. WISH showed that zrsr2Δ11/Δ11 mutants have normal early hematopoietic stem cell (HSC) emergence (c-myb) at 36 hours post fertilization. However, these cells are absent through 3 and 5 dpf. At 5 dpf zrsr2Δ11/Δ11 mutants had severely reduced expression of hematopoietic markers for erythroid (hbae1), lymphoid (rag1), and myeloid (mpo) lineages. This verifies that knockout of zrsr2 in zebrafish disrupted normal hematopoiesis and leads to cytopenias - emulating the symptoms of MDS. We then investigated the transcriptional and splicing changes underlying these phenotypes using RNA sequencing on zrsr2Δ11/Δ11 mutant embryos at 3dpf. We found a total of 285 intron retention events in zrsr2Δ11/Δ11 mutants as compared to the control embryos, with about 76% of those events occurring within U12-type introns. Genes with retained introns were associated with mRNA decay and destabilization. We also found that genes related to cell cycle arrest were upregulated in zrsr2Δ11/Δ11 mutants . These results indicate that aberrant mRNA splicing and cell cycle deregulation contribute to arrested hematopoiesis when zrsr2 is knocked out in zebrafish. Disclosures No relevant conflicts of interest to declare.

Haploinsufficiency of cohesin protease, Separase, promotes regeneration of hematopoietic stem cells in mice.

Cohesin recently emerged as a new regulator of hematopoiesis and leukemia. In addition to cohesin, whether proteins that regulate cohesin's function have any direct role in hematopoiesis and hematologic diseases have not been fully examined. Separase, encoded by the ESPL1 gene, is an important regulator of cohesin's function. Canonically, protease activity of Separase resolves sister chromatid cohesion by cleaving cohesin subunit-Rad21 at the onset of anaphase. Using a Separase haploinsufficient mouse model, we have uncovered a novel role of Separase in hematopoiesis. We report that partial disruption of Separase distinctly alters the functional characteristics of hematopoietic stem/progenitor cells (HSPCs). Although analyses of peripheral blood and bone marrow of Espl1+/Hyp mice broadly displayed unperturbed hematopoietic parameters during normal hematopoiesis, further probing of the composition of early hematopoietic cells in Espl1+/Hyp bone marrow revealed a mild reduction in the frequencies of the Lin- Sca1+ Kit- (LSK) or LSK CD48+ CD150- multipotent hematopoietic progenitors population without a significant change in either long-term or short-term hematopoietic stem cells (HSCs) subsets at steady state. Surprisingly, however, we found that Separase haploinsufficiency promotes regeneration activity of HSCs in serial in vivo repopulation assays. In vitro colony formation assays also revealed an enhanced serial replating capacity of hematopoietic progenitors isolated from Espl1+/Hyp mice. Microarray analysis of differentially expressed genes showed that Separase haploinsufficiency in HSCs (SP-KSL) leads to enrichment of gene signatures that are upregulated in HSCs compared to committed progenitors and mature cells. Taken together, our findings demonstrate a key role of Separase in promoting hematopoietic regeneration of HSCs.

Production of Murine Macrophages from Hoxb8-Immortalized Myeloblasts: Utility and Use in the Context of Salmonella Infection.

Salmonella enterica is a Gram-negative intracellular pathogen that causes a range of life-threatening diseases in humans and animals worldwide. In a systemic infection, the ability of Salmonella to survive/replicate in macrophages, particularly in the liver and spleen, is crucial for virulence. Transformed macrophage cell lines and primary macrophages prepared from mouse bone marrow are commonly used models for the study of Salmonella infection. However, these models raise technical or ethical issues that highlight the need for alternative methods. This chapter describes a technique for immortalizing early hematopoietic progenitor cells derived from wild-type or transgenic mice and using them to produce macrophages. It validates, through a specific example, the interest of this cellular approach for the study of Salmonella infection.

Second-Tier Next Generation Sequencing Integrated in Nationwide Newborn Screening Provides Rapid Molecular Diagnostics of Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) and other T cell lymphopenias can be detected during newborn screening (NBS) by measuring T cell receptor excision circles (TRECs) in dried blood spot (DBS) DNA. Second tier next generation sequencing (NGS) with an amplicon based targeted gene panel using the same DBS DNA was introduced as part of our prospective pilot research project in 2015. With written parental consent, 21 000 newborns were TREC-tested in the pilot. Three newborns were identified with SCID, and disease-causing variants in IL2RG, RAG2, and RMRP were confirmed by NGS on the initial DBS DNA. The molecular findings directed follow-up and therapy: the IL2RG-SCID underwent early hematopoietic stem cell transplantation (HSCT) without any complications; the leaky RAG2-SCID received prophylactic antibiotics, antifungals, and immunoglobulin infusions, and underwent HSCT at 1 year of age. The child with RMRP-SCID had complete Hirschsprung disease and died at 1 month of age. Since January 2018, all newborns in Norway have been offered NBS for SCID using 1st tier TRECs and 2nd tier gene panel NGS on DBS DNA. During the first 20 months of nationwide SCID screening an additional 88 000 newborns were TREC tested, and four new SCID cases were identified. Disease-causing variants in DCLRE1C, JAK3, NBN, and IL2RG were molecularly confirmed on day 8, 15, 8 and 6, respectively after birth, using the initial NBS blood spot. Targeted gene panel NGS integrated into the NBS algorithm rapidly delineated the specific molecular diagnoses and provided information useful for management, targeted therapy and follow-up i.e., X rays and CT scans were avoided in the radiosensitive SCID. Second tier targeted NGS on the same DBS DNA as the TREC test provided instant confirmation or exclusion of SCID, and made it possible to use a less stringent TREC cut-off value. This allowed for the detection of leaky SCIDs, and simultaneously reduced the number of control samples, recalls and false positives. Mothers were instructed to stop breastfeeding until maternal cytomegalovirus (CMV) status was determined. Our limited data suggest that shorter time-interval from birth to intervention, may prevent breast milk transmitted CMV infection in classical SCID.

Analysis of endothelial-to-haematopoietic transition at the single cell level identifies cell cycle regulation as a driver of differentiation

BackgroundHaematopoietic stem cells (HSCs) first arise during development in the aorta-gonad-mesonephros (AGM) region of the embryo from a population of haemogenic endothelial cells which undergo endothelial-to-haematopoietic transition (EHT). Despite the progress achieved in recent years, the molecular mechanisms driving EHT are still poorly understood, especially in human where the AGM region is not easily accessible.ResultsIn this study, we take advantage of a human pluripotent stem cell (hPSC) differentiation system and single-cell transcriptomics to recapitulate EHT in vitro and uncover mechanisms by which the haemogenic endothelium generates early haematopoietic cells. We show that most of the endothelial cells reside in a quiescent state and progress to the haematopoietic fate within a defined time window, within which they need to re-enter into the cell cycle. If cell cycle is blocked, haemogenic endothelial cells lose their EHT potential and adopt a non-haemogenic identity. Furthermore, we demonstrate that CDK4/6 and CDK1 play a key role not only in the transition but also in allowing haematopoietic progenitors to establish their full differentiation potential.ConclusionWe propose a direct link between the molecular machineries that control cell cycle progression and EHT.

Prognostic plasma biomarkers of early complications and graft-versus-host disease in patients undergoing allogeneic hematopoietic stem cell transplantation.

Early complications post hematopoietic stem cell transplantation (HSCT) such as sinusoidal obstruction syndrome (SOS) and graft versus host disease (GVHD) can be life threatening. Although several biomarkers have been identified to correlate with these complications and their response to treatment, these are yet to be used in clinical practice. Here, we evaluated circulating endothelial cells (CECs) (n = 26) and plasma biomarkers (ST2, REG3α, VCAM1, ICAM1, TIM3) (N = 210) at early time points, to determine their association with early complications post‐HSCT. Elevated CEC counts at the end of conditioning was associated with GVHD, indicating endothelial damage during HSCT. Plasma levels of REG3α, VCAM1, ICAM1, and TIM3 on day 14 (D14) and D14 ICAM1 and D28 ST2 were significantly higher in patients with SOS and aGVHD, respectively. Upon sub‐group analysis, D28 ST2, D14/D28 REG3α, and D14 ICAM1 levels were significantly higher in patients with gastrointestinal GVHD, while D28 ST2 was higher in those with skin/liver GVHD. High ST2 levels on D28 was significantly associated with non‐relapse mortality (NRM) and overall survival. Our results suggest that elevated ST2 levels on D28 could predict the likelihood of developing aGVHD and could influence NRM and OS.

Design of New Therapeutic Agents Targeting FLT3 Receptor Tyrosine Kinase Using Molecular Docking and 3D-QSAR Approach

Background: FMS-like tyrosine kinase-3 (FLT3) belongs to the class III Receptor Tyrosine Kinase (RTK) family. FLT3 is involved in normal hematopoiesis and is generally expressed in early hematopoietic progenitor cells. Mutations either with an internal tandem duplication of FMS-like tyrosine kinase-3 (FLT3-ITD) or point mutation at the activation loop leads to the Acute Myeloid Leukemia (AML), a highly heterogeneous disease. Thus, FLT3 is an important therapeutic target for AML. Method: In the present work, docking and 3D-QSAR techniques were performed on a series of diaminopyrimidine derivatives as FLT3 kinase antagonists. Results: Docking study recognized important active site residues such as Leu616, Gly617, Val624, Ala642, Phe830, Tyr693, Cys694, Cys695, Tyr696 and Gly697 that participate in the inhibition of FLT3 kinase. Receptor-based CoMFA, RF-CoMFA and CoMSIA models were developed. RFCoMFA model revealed relatively better statistical results compared to other models. Furthermore, the selected RF-CoMFA model was evaluated using various validation techniques. Contour maps of the RF-CoMFA illustrated that steric and electronegative substitutions were favored at R1 position whereas steric and electropositive substitutions were favored at R2 position to enhance the potency. Conclusion: Based on the designed strategy, we derived from the contour map analysis, 14 novel FLT3 inhibitors were designed and their activities were predicted. These designed inhibitors exhibited more potent activity than the most active compounds of the dataset.

Acquired Aplastic Anemia as a Clonal Disorder of Hematopoietic Stem Cells

Aplastic anemia is rare disorder presenting with bone marrow failure syndrome due to autoimmune destruction of early hematopoietic stem cells (HSCs) and stem cell progenitors. Recent advances in newer genomic sequencing and other molecular techniques have contributed to a better understanding of the pathogenesis of aplastic anemia with respect to the inflammaging, somatic mutations, cytogenetic abnormalities and defective telomerase functions of HSCs. These have been summarized in this review and may be helpful in differentiating aplastic anemia from hypocellular myelodysplastic syndrome. Furthermore, responses to immunosuppressive therapy and outcomes may be determined by molecular pathogenesis of HSCs autoimmune destruction, as well as treatment personalization in the future.

A comparison of clinical outcomes and transplant complications/morbidity with early (

A comparison of clinical outcomes and transplant complications/morbidity with early (<4 months of age) versus late (~ 1 yr of age) hematopoietic stem cell transplant in sibling pairs with a diagnosis of MPS I (Hurler syndrome)

Deciphering Common Gene Fusions and Mutations in Acute Leukemia of Ambiguous Lineage

Background The acute leukemia of ambiguous lineage (ALAL) is high risk and uncommon in acute leukemia, including acute undifferentiated leukemia (AUL) and mixed phenotype acute leukemia (MPAL). On account of the rareness and age-influenced genome landscape, the genetic characterization was always limited and showed discrepancy among different cooperative groups. Methods By integrating the immunophenotype, cytogenetics, and molecular biology of ALAL patients, we found that the molecular profile played an important role in the diagnosis, treatment, and prognosis of ALAL patients. Through an in-depth analysis of the molecular features, we found the genetic differences between different subtypes of ALAL, and studied the unique gene mutation patterns and synergy of genomic abnormalities in different subtypes. Results Most patients in the cohort were children and young adults, and among B/myeloid and T/myeloid MPAL, while rare in AUL. Here we showed that ALAL patients show both genetic commonality and discrepancy among subtypes. Except for the BCR-ABL1 and KMT2A-r, which were mainly in B/myeloid MPAL, we reported that SET-NUP214 was recurrent in T/myeloid MPAL and AUL patients. The PHF6 were also frequently mutated in T/myeloid MPAL with most loss of function and significantly associated with SET-NUP214. Otherwise, there was a big difference between B/myeloid and T/myeloid MPAL in genetic profile. The most mutated genes in B/myeloid MPAL were CEBPA, RUNX1, NRAS, TET2, WT1, PTPN11, and FLT3, while NOTCH1, WT1, RUNX1, FLT3, PHF6, ETV6, and EZH2 were frequently in T/myeloid MPAL. Moreover, the alterations in the genes encoding transcriptional regulation were both recurrently mutated in B/myeloid and T/myeloid MPAL, especially WT1 and RUNX1, which were the most mutated genes and mutually exclusive with each other. Alterations in the genes involved in JAK_STAT signaling, RAS signaling, and epigenetic regulation were also recurrently mutated in B/myeloid and T/myeloid MPAL, despite some differences in incidence rate. However, the deletion of IKZF1 exclusively occurred in B/myeloid MPAL, while genes involved in NOTCH signaling were mainly altered in T/myeloid MPAL. When analyzing the synergy among genetic abnormalities, we found that SET-NUP214 and PHF6 (p = 0.00002), FLT3 and RUNX1 (p = 0.009), BCR-ABL1 and RUNX1 (p = 0.02) were the most significantly co-occurred genes (Figure 1-A, B). Although the association between NRAS and WT1 were not significantly (p = 0.11), they co-occurred in five patients (Figure 1-C). Since this was the first time to report that SET-NUP214 was recurrent in ALAL patients, we retrospectively review the therapy of patients with the aberration. According to the treatment of these ALAL patients, we confirmed that even with transplantation therapy the prognosis of SET-NUP214-positive ALAL patients was still very poor with a high risk of relapse. Discussion We charted the genomic landscape of ALAL to comprehensively reveal the commonality and discrepancy among subtypes of ALAL patients. The founding lesion of SET-NUP214 in early primitive hematopoietic cells was showed to be involved in the block of differentiation. Together with the loss of function in PHF6, which was showed a contribution to the lineage promiscuity and enhance the proliferation of hematopoietic cells, the combination event could make leukemia with ambiguous lineage. After deciphering the molecular characterization of ALAL patients, we proposed several candidate updates to WHO 2016 (Table 1), as these molecular features may help the diagnosis, therapy, and prognosis of the ALAL patients. Disclosures No relevant conflicts of interest to declare.

The Relationship between Transplant Status and Patient-Reported Outcomes in Patients with FLT3-Mutated Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML): Results from the Phase 3 Admiral Study

The Relationship between Transplant Status and Patient-Reported Outcomes in Patients with FLT3-Mutated Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML): Results from the Phase 3 Admiral Study

Single-Cell RNA Sequencing Reveals Distinct Hematopoietic Stem Cell Hierarchies in MDS

Myelodysplastic Syndromes (MDS) are a group of heterogeneous stem cell disorders that result in inefficient hematopoiesis. Although the genetic and cytogenetic landscapes of MDS have been well characterized (Papaemmanuil 2013, Sperling 2017), little is known about the differentiation abnormalities that underlie the MDS phenotype. Gaining insights on how different hematopoietic stem and progenitor cell (HSPC) types contribute to MDS is essential for the design of new targeted therapies to supplement the currently limited effective therapeutic options. To understand the contribution of different cell types to the pathogenesis of MDS, we analyzed the expression profile of the Lin-CD34+ HSPC compartment at the single-cell level. Single-cell RNA-sequencing (scRNA-seq) analysis of HSPCs isolated from 2 MDS patients and 2 age-matched healthy donor samples revealed distinct cell clusters driven by the sample type and the differentiation potential of the cells. To annotate the specific subsets of HSPCs in each cluster, we scored them on the basis of previously reported population-specific gene signatures (Laurenti 2013, Psaila 2016, Van Galen 2019). Whereas CD34+ cells from the 2 healthy donor bone marrow (BM) samples largely overlapped with each other and displayed 2 distinct erythroid/megakaryocytic (Er/Mk; cluster 3) and lympho/myeloid (clusters 2, 5) differentiation trajectories in line with the current view of hematopoiesis, CD34+ cells from the 2 MDS BM samples clustered separately and showed predominantly myeloid differentiation routes (Fig a). Importantly, differential expression analysis of the HSPCs from the 2 MDS samples (Fig b) showed that cells residing atop of the HSPC hierarchy retained the transcriptional profile of immature HSCs in one of the samples (clusters 2, 4), while they were characterized by the expression of genes involved in the differentiation of myelo/lympho multipotent progenitor cells (clusters 0, 1) in the other. However, pseudotime analysis of the HSPCs' transcriptional dynamics showed that, despite the distinct differentiation state of the early hematopoietic cells in each group, the differentiation trajectories of those cells converged at the late myeloid progenitor state (clusters 3, 5, 6). These results suggest that, although the earlier HSC architecture is heterogeneous across MDS patients, the more differentiated myeloid progenitor compartment is similarly compromised and is responsible for the clinical phenotypes of MDS. To confirm differential cell-type contributions to the MDS hierarchy, we immunophenotyped BM samples from 123 untreated patients using multicolor flow cytometry. We applied principal component analysis and logistic regression to group samples based on their cellular compositions. Our mathematical classifier stratified patients in 2 groups, which had markedly different cellular repertoires consistent with our scRNA-seq results (Fig c). Patients with different MDS stem cell hierarchies did not present with significantly different clinical characteristics at diagnosis. These data confirm that different abnormal hematopoietic trajectories converge in the myeloid bias typically observed in MDS hematopoiesis. Next, we exome-sequenced mononuclear cells and T-cells from 45 untreated MDS patients and identified high-confidence somatic mutations in known oncogenes and/or leukemia driver genes. The median number of mutations (n=3) was not significantly different between MDS groups 1 and 2. We identified 4 genes that were differentially mutated in the 2 MDS architectures (Fig d), which suggested that certain mutations may predispose for a specific HSPC phenotype. However, mutation specificity could not fully account for the origin of the 2 differentiation architectures, which were independent on the genetic background in most patients. In conclusion, we demonstrated that MDS are sustained by distinct and recurrent abnormal HSPC differentiation hierarchies. Diverse cellular compositions suggest that different cell-type specific signaling pathways maintain the disease in each group of patients. Our work shows that the characterization of the cellular diversity in the hematopoietic compartment can be used as a biomarker to stratify MDS patients, and warrants further studies to predict the intrinsic vulnerabilities of the cells involved in the pathogenesis and maintenance of MDS in a patient-specific manner. Figure Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Colla:IONIS: Other: Intellectual property and research material IONIS); Amgen: Research Funding; Abbvie: Research Funding.

2017 - MICROBIAL SIGNAL INSTRUCTS EARLY HEMATOPOIESIS UPON INTESTINAL TISSUE DAMAGE

Hematopoietic stem cells (HSCs) slowly self-renew and differentiate in adult bone marrow (BM) to sustain lifelong hematopoiesis, and can be activated to self-renew or differentiate when hematopoietic need increases. However, it remains unclear how HSC and progenitor cells (HSPCs) integrate the peripheral organ-derived demand signal to hematopoietic production and what the biological consequence of HSPCs activation is on their cell fate decision. We have previously shown that systemic challenge of gram negative bacteria directly activates dormant HSCs to proliferation and impairs their competitive fitness via Toll like receptor (TLR)-4 signaling (Cell Stem Cell 2017). These findings let us hypothesize that commensal bacteria, often referred to as microbiota, might also impact on early hematopoiesis upon intestinal barrier damage. Here, we employed inflammatory bowel disease (IBD) model to induce gut inflammation. Acute IBD induced expansion of HSPCs such as multipotent progenitors (MPPs) in primary BM followed by their localization in mesenteric lymph node (MLN), a local inflammatory site. Myeloid compartment, especially neutrophils and monocytes dominantly increased and suppressed IBD-induced colitis as shown by antibody-based cell depletion study, indicating possible contribution of MPP in tissue damage and repair. Genetic and pharmacological studies revealed that the HSPC expansion and their directed migration depend on a TLR/IL-1 receptor signaling and specific type of microbiota, suggesting that microbial signal generated in the distal organs regulates early hematopoietic cell proliferation and localization. Uncovering the underlying mechanism for gut associated inflammation will help to understand inflammatory feedback signals through cross-organ communications that orchestrate hematopoiesis, and might be relevant to ageing-associated chronic disorders.

MCLA-117, a CLEC12AxCD3 bispecific antibody targeting a leukaemic stem cell antigen, induces T cell-mediated AML blast lysis

ABSTRACTObjective: We report the characterization of MCLA-117, a novel T cell-redirecting antibody for acute myeloid leukaemia (AML) treatment targeting CD3 on T cells and CLEC12A on leukaemic cells. In AML, CLEC12A is expressed on blasts and leukaemic stem cells.Methods: The functional capacity of MCLA-117 to redirect resting T cells to eradicate CLEC12APOS tumor cells was studied using human samples, including primary AML samples.Results: Within the normal hematopoietic compartment, MCLA-117 binds to cells expressing CD3 and CLEC12A but not to early myeloid progenitors or hematopoietic stem cells. MCLA-117 induces T cell activation (EC50 = 44 ng/mL), T cell proliferation, mild pro-inflammatory cytokine release, and redirects T cells to lyse CLEC12APOS target cells (EC50 = 68 ng/mL). MCLA-117-induced targeting of normal CD34POS cells co-cultured with T cells spares erythrocyte and megakaryocyte differentiation as well as preserves mono-myelocytic lineage development. In primary AML patient samples with autologous T cells, MCLA-117 robustly induced AML blast killing (23–98%) at low effector-to-target ratios (1:3–1:97).Conclusion: These findings demonstrate that MCLA-117 efficiently redirects T cells to kill tumour cells while sparing the potential of the bone marrow to develop the full hematological compartment and support further clinical evaluation as a potentially potent treatment option for AML.

Putative Role of Nuclear Factor-Kappa B But Not Hypoxia-Inducible Factor-1α in Hypoxia-Dependent Regulation of Oxidative Stress in Hematopoietic Stem and Progenitor Cells.

Aims: Adaptation to low oxygen of hematopoietic stem cells (HSCs) in the bone marrow has been demonstrated to depend on the activation of hypoxia-inducible factor (HIF)-1α as well as the limited production of reactive oxygen species (ROS). In this study, we aimed at determining whether HIF-1α is involved in protecting HSCs from ROS.Results: Oxidative stress was induced by DL-buthionine-(S,R)-sulfoximine (BSO)-treatment, which increases the mitochondrial ROS level. Hypoxia rescued Lineage-Sca-1+c-kit+ (LSK) cells from BSO-induced apoptosis, whereas cells succumbed to apoptosis in normoxia. Apoptosis in normoxia was inhibited with the antioxidant N-acetyl-L-cysteine or by overexpression of anti-apoptotic BCL-2. Moreover, stabilized expression of oxygen-insensitive HIFs could not protect LSK cells from oxidative stress-induced apoptosis at normoxia, neither could short hairpin RNA to Hif-1α inhibit the protective effects by hypoxia in LSK cells. Likewise, BSO treatment of LSK cells from Hif-1α knockout mice did not suppress the effects seen in hypoxia. Microarray analysis identified the nuclear factor-kappa B (NF-κB) pathway as a pathway induced by hypoxia. By using NF-κB lentiviral construct and DNA-binding assay, we found increased NF-κB activity in cells cultured in hypoxia compared with normoxia. Using an inhibitor against NF-κB activation, we could confirm the involvement of NF-κB signaling as BSO-mediated cell death was significantly increased in hypoxia after adding the inhibitor.Innovation: HIF-1α is not involved in protecting HSCs and progenitors to elevated levels of ROS on glutathione depletion during hypoxic conditions.Conclusion: The study proposes a putative role of NF-κB signaling as a hypoxia-induced regulator in early hematopoietic cells.

PF677 DNA HYPERMETHYLATION EMERGES AS THE STRONGEST PREDICTOR FOR POOR OUTCOME AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION IN JUVENILE MYELOMONOCYTIC LEUKEMIA (JMML)

Background: JMML is a childhood hematopoietic malignancy characterized by hyperactive RAS signaling. For most patients early allogeneic hematopoietic stem cell transplantation (HSCT) is the therapy of choice. Risk factors for early death and relapse following HSCT are represented by age ≥ 2 yrs, low platelet count and HbF elevated for age. Most recently, genome-wide DNA methylation profiles (Lipka, Nat Comm 2017) identified distinct methylation signatures predictive for outcome. Aims: This study evaluates the clinical significance of these epigenetic changes in a large cohort of uniformly transplanted children registered to the European Working Group of MDS in Childhood (EWOG-MDS). Methods: We analyzed the outcome of 153 children with JMML (96 males, 57 females; median age at HSCT 2.7 yrs [0.3–11.5]) given a HSCT after a preparative regimen consisting of busulfan, cyclophosphamide, and melphalan. Results: Thirty-seven patients (pts) were transplanted from an HLA-identical sibling donor (MSD) and 116 from a matched unrelated donor (UD). Stem cell source was bone marrow (n = 122) or peripheral blood (n = 31). GVHD prophylaxis was CSA only in 25 (68%) MSD procedures, CSA/MTX/ATG was administered in 88 (76%) of UD-HSCT recipients. All but 9 pts engrafted. The cumulative incidence of grades II-IV acute GVHD was 44% (37–52). Thirty-three of the 133 pts at risk developed chronic GVHD, which was limited in 20 and extensive in 13 pts. With a median follow-up of 3.7 years (0.1–17.5), the 5-year leukemia-free survival (LFS) was 61% (53–69), while the cumulative incidence of non-relapse mortality (NRM) and relapse (CIR) were 13% (8–20) and 26% (20–35), respectively. There was no difference in outcome between MSD and UD-HSCT recipients. Of the 153 pts, 76 had a somatic PTPN11 mutation, 21 had neurofibromatosis type 1, 22 belonged to the NRAS- and 18 to the KRAS-mutated subgroup, while RAS pathway alterations were absent in 16 pts (“all negative”). 5-year CIR was higher in PTPN11-mutated JMML (35%) compared to that of the NF1 (24%), NRAS (23%), KRAS (11%) or all negative subtypes (14%). Methylation status was high (HM), intermediate (IM) or low (LM) in 61, 52 or 40 pts, respectively. 5-year LFS and CIR of pts of the LM class was similar to that noted in the IM class with 76% vs 69%, and 8% vs 13%, respectively. In contrast, children of the HM class had a significantly inferior LFS (44%, 31–57, p < 0.01) due to a high CIR (50%, 38–65, p < 0.01). NRM in the 3 methylation groups (LM 16%, IM 19%, HM 6%) did not differ. There was a clear correlation between methylation class and molecular genetic subtype: PTPN11-mutated JMML was less common in the LM class and the proportion of KRAS-mutated pts was highest in the LM class. Also, patients in the HM class had a higher median age at HSCT (3.8 yrs) than pts in the IM (2.1 yrs) or LM class (1.1 yrs). Multivariate analysis identified methylation class (HM vs IM: RR 5.3 [1.8–15.3], HM vs LM RR 7.6 [1.6 – 37.3]) as the only independent variable predicting relapse when analyzed with age at HSCT, platelet count and molecular genetic type (PTPN11-mutated vs others). Summary/Conclusion: Patient outcome in genome-wide methylation classes nicely recapitulated earlier data applying a DNA methylation score of candidate gene regions (Olk-Batz, Blood 2011). Thus, DNA methylation classifiers are exceptionally consistent allowing prospective assignment of DNA methylation categories for molecularly-driven risk stratification. Future research needs to be directed at better understanding the mechanistic link of epigenetic dysregulation and resistance to treatment.

PB1702 EXPRESSION OF ANGIOTENSIN‐CONVERTING ENZYME IN PATIENTS WITH ACUTE LEUKEMIAS

Background:Blast cells are characterized by aberrant expression normal markers of early hematopoietic progenitor cells (HPC). Angiotensin‐converting enzyme (ACE/CD143), a key regulator of renin‐angiotensin system, was shown to be expressed in blood‐forming tissues of the human embryo and primitive human HPC. Recent studies indicated that CD143 is over‐expressed in leukemic myeloid blast cells.Aims:To evaluate the surface expression of CD143 in blast cells in patients with newly diagnosed acute leukemia (AL).Methods:The expression of CD143 was estimated in patients with newly diagnosed untreated acute leukemia, using eight‐color flow cytometry. The blast gate was defined on the basis of CD45dim expression and side‐scatter characteristics. Anti‐CD143 monoclonal antibody (clone BB9, BD) conjugated with phycoerythrin was used for analysis of CD143 expression. The percentage of positive cells (PPC) and the mean fluorescence intensity (MFI) was applied to describe expression. The expression of CD143 was devided into three groups according range of PPC ≤1%, 1 – 20 %, and ≥20 %.Results:A total of untreated 68 patients with newly diagnosed acute leukemia were included in the study group. They were diagnosed with non‐promyelocytic acute myeloid leukemia (non‐APL AML) – 35 (51.5 %), of them 3 (8.5 %) patients were related to favorable genetic group, 9 (25.7 %) – intermediate, 23 (65.8 %) – adverse; promyelocytic leukemia (APL) – 10 (14.7 %); B acute lymphoblastic leukemia/lymphoma (B‐ALL) – 13 (19.1 %), including 2 patients with Ph‐positive B‐ALL; T acute lymphoblastic leukemia/lymphoma (T‐ALL) – 10 (14.7 %), including 2 cases of early T‐precursor ALL (ETP). Median age of patients was 35.5 years, 29 male, 39 female.In total group with CD143 expression with PPC≥20% was observed in 7% (5/68) of all cases AL. CD143 expression in non‐APL AML with a range of PPC 1 – 20 % was detected in 12/35 cases (34.2 %), PPC ≥20 % was observed in 2/35 cases with complex karyotype (5.7 %). In group with APL 2/10 (20 %) patients had 1 – 20%, and 2/10 (20 %) patients had &gt;20 %. Among T‐ALL only 1/10 (10 %) had a CD143 expression. ACE expression in B‐ALL was absent (defined as ≤1 %). Median MFI of ACE expression was higher in patients with APL – 477 (range 159 – 4796) than in non‐APL AML ‐ 175 (range 43 – 3253). CD143 expression with PPC≥1% was significantly higher in patients with AML (non‐APL AML and APL), than in ALL (B‐ and T‐ALL), 40 % (18/45) vs. 4.3 % (1/23) (p = 0.0016).Summary/Conclusion:Thus, CD143 expression with PPC≥20% was observed only in 7% (5/68) of all cases AL, 2 was observed in AML with PML‐RARA, 2 ‐ AML with complex karyotype and 1 in ETP‐ALL. No expression of CD143 was detected in B‐ALL. CD143 expression was significantly higher in myeloid blast cells than lymphoid. No correlation was found between the expression of ACE and age, sex, neuroleukemia, leucocytes count, bone marrow blasts, lactic acid dehydrogenase activity and cytogenetic abnormalities in different subgroups. The significance of CD143 expression in AL is not completely clear and has to be studied.

S129 TP53 SOMATIC MUTATIONS AS PRE‐LEUKEMIC EVENTS IN ACUTE LYMPHOBLASTIC LEUKEMIA

Background:Alterations of the tumor suppressor gene TP53 are observed in 15–20% of patients with acute lymphoid leukemia (ALL) and are associated with resistance to standard treatment regimens and inferior survival. TP53 mutations (TP53mt) have been reported as an early leukemogenic event in patients with acute myeloid leukemia. In ALL however, only limited data are available.Aims:Study the pre‐leukemic mutation pattern of TP53 gene in ALL patients. Therefore, we analyzed the kinetics of the TP53mt in bone marrow and peripheral blood in the group of 43 adult ALL patients harboring TP53mt at the diagnosis (Dx) and compared it to the kinetics of standard EuroMRD IG/TR minimal residual disease (MRD). Moreover, TP53mt were assessed in the distinct hematopoietic populations.Methods:Patients received induction/consolidation treatment according to the GMALL 07/03 and GMALL register protocol. For each patient, TP53‐mutations were analyzed in at least one Dx/MRD‐positive (n = 54) and one MRD‐negative sample, if available (n = 41). The presence of TP53mt was validated by Sanger sequencing and quantified using UMI‐based next generation amplicon sequencing (sensitivity: 5x10−3). In 5 patients, selected based on TP53mt‐status and on the availability of viable cells, distinct hematopoietic populations were sorted using FACS Aria sorter in an MRD‐negative follow‐up sample and analyzed for the presence of the TP53mt using allele‐specific digital droplet PCR (ddPCR, sensitivity: 1,5x10−3‐1x10−2). MRD was monitored using real‐time quantitative PCR of clonal immune gene rearrangements (sensitivity 1x10−5) and multiparameter flow cytometry.Results:In our series of 43 TP53mt ALL patients (B‐ALL 90.7%, T‐ALL 9.3%) MRD analysis revealed quantifiable IG/TR MRD persistence in 7 patients (16.3%), complete MRD remission in 30 patients (69.8%), and MRD persistence at not quantifiable levels in 6 patients (14.0%). In 9 of the MRD‐negative patients (20.9%) the TP53‐mutation was detected at levels between 2.0 and 17.4% (median 8.1%), pointing to the presence of TP53mt in a pre‐leukemic compartment. In 5 of 7 MRD‐persisters also TP53 mutations were detected. In the 2 remaining patients MRD‐level was below the TP53‐assay sensitivity. ddPCR analysis of the sorted, progenitor and mature hematopoietic populations revealed the presence of TP53mt in early hematopoietic cells including multipotent progenitors (MPP: CD19‐34+38−), common myeloid/lymphoid progenitors (CD19‐34+38+13/33−) and early myeloid progenitors (CD34+19‐38+13/33+) but also in differentiated cells as granulocytes (SSChighCD13/33dim) and monocytes (CD19‐34‐3‐13/33high). TP53mt was generally absent in the mature T cells but present in the B‐cell precursors defined as CD19+10+20‐34+ cells (Figure 1); altogether this data point to the pre‐leukemic origin of TP53mt. Interestingly, one patient who relapsed after two years with a fully unrelated IG/TR rearrangement still harbored the TP53mt from Dx sample, pointing to the involvement of the TP53mt in the ALL reoccurrence. The detailed analysis of other relapse‐associated mutations is currently ongoing. The correlation analysis of clinical outcome with the observed TP53 mutation pattern will also be performed.Summary/Conclusion:Hereby, we have shown that in a considerable number of adult ALL patients a TP53 mutation is present in pre‐leukemic cells, causing clonal expansions in different hematopoietic compartments, and thus persisting in MRD negative follow‐up samples. Considering TP53 somatic mutations as an MRD‐marker requires careful interpretation.image

A quantitative method for detection of circulating fms related tyrosine kinase 3 (FLT-3) in acute myeloid leukemia (AML) patients

FMS related tyrosine kinase 3 (FLT-3) is a tyrosine kinase expressed in early hematopoietic precursor cells and has roles in survival, proliferation, and differentiation. Bone marrow expression and mutagenic analysis of FLT-3 in Acute Myeloid Leukemia (AML) patients is well-characterized. However, the levels of circulating FLT-3 in serum have not been previously described. In this study we describe a quantitative electrochemiluminescent immunoassay that detects FLT-3 in human serum. Using this method we find that AML patients have elevated levels of circulating FLT-3 and these levels correlated to the percent blast counts in the bone marrow (BM).