And/or Acute Myeloid Leukemia Research Articles

Blastic Plasmocytoid Dendritic Cell Neoplasm (BPDCN): Clinical Features and Histopathology with a Therapeutic Overview.

Blastic Plasmacytoid Dendritic Cell Neoplasms (BPDCNs) are a rare, highly aggressive hematological malignant neoplasm that primarily involve the skin, bone marrow, lymph nodes and even extra-nodal sites. The rarity and relative poor description of cases in the literature make it necessary to review and further studies that deeply investigate this entity not only in a histopathological but also molecular field. In August-September 2023, we searched MEDLINE, PubMed and Scopus for randomized controlled trials (RCTs), narrative and systematic reviews, meta-analyses, observational studies (either longitudinal or retrospective), and case series published in English in the last 25 years using the keywords BPDCN, PDCs, Blastic NK-cell lymphoma, agranular CD4+ NK leukemia/lymphoma, agranular CD4+ CD56+ hematodermic neoplasm/tumor. Despite the progress made in recent years in the diagnosis and biological understanding of the disease, until 2018 there was no clear consensus regarding its treatment and the main therapeutic schemes used were based on chemotherapy regimens already used in the treatment of lymphomas, acute lymphoblastic leukemia (ALL) and/or acute myeloid leukemia (AML). In this narrative review, we address the definition and epidemiological features of BPDCN, provide the different theories on the etiopathogenesis with particular attention to the presumed cell of origin, discuss the main clinical manifestations that provide a sign of its presence, summarize the main histopathological and immunophenotypic characteristics with special attention to the most important markers, and finally, we provide some of the most effective information on the therapeutic treatment modalities of BPDCN.

Targeting Nucleotide Biosynthesis Ameliorates Neutropenia in a Zebrafish Model of Dnajc21-Mutant Shwachman-Diamond Syndrome

Germline mutations in the ribosome maturation factors, SBDS, DNAJC21 and EFL1, lead to Shwachman-Diamond syndrome (SDS), a type of inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency and growth restriction. Further, SDS patients have a 36% increased cumulative risk of developing myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML) by 30 years of age. Given the rarity of SDS, large numbers of primary human samples are not readily available for mechanistic studies, warranting the use of animal models. Mouse and zebrafish models of SBDS mutations exhibit early lethality. In contrast, no in vivo models of DNAJC21 mutations have been published to date. We generated the first in vivo model of dnajc21 loss using zebrafish ( Danio rerio), given their highly conserved hematopoietic program and ease of genetic manipulation. Introduction of a homozygous deletion mutation in the zebrafish dnajc21 gene using CRISPR-Cas9 genomic editing resulted in stunted body growth and neutropenia, recapitulating hallmark SDS patient phenotypes. Whole-mount in situ hybridization for key hematopoietic lineage markers revealed reduced l-plastin +mature leukocytes and hbbe3 + mature erythrocytes in dnajc21 mutant embryos at 48 hours post-fertilization (hpf). A concomitant accumulation of myb + hematopoietic stem and progenitor cells (HSPCs) both in the dorsal aorta (equivalent to aorta-gonad-mesonephros) and the caudal hematopoietic tissue (fetal liver equivalent) was observed. Treatment of embryos with zebrafish granulocyte colony stimulating factor (gcsf) mRNA resulted in a modest expansion of neutrophils in dnajc21 mutant zebrafish compared to wildtype. Further, we observed reduced cell proliferation marked by phosphohistone-H3 (pH3) as well as low ATP:AMP levels measured by liquid chromatography/mass-spectrometry (LC/MS) in the dnajc21 mutants, suggesting a poor bioenergetic profile. At 4 months of age, flow cytometry and histopathological analysis identified reduced mature myelomonocytes and mild erythroid dysplasia in the whole kidney marrow (human bone marrow equivalent) of mutant fish. The link between TP53 somatic mutations and MDS/AML has been established in SBDS-mutant SDS, but has not been explored for DNAJC21-mutant SDS. We hence crossed our dnajc21 mutant zebrafish with a zebrafish line carrying a gain-of-function mutation (R217H) in the tp53 gene. Compound mutant fish had improved neutrophil counts but importantly, this was accompanied by an expansion of myeloid progenitors and significant erythroid dysplasia. To understand the disease mechanisms underlying the neutropenia and hypoproliferation in SDS, we performed bulk RNA sequencing of whole zebrafish larvae at 48 hpf. Biological processes such as oxidative stress response, drug catabolism and pre-mRNA spliceosome assembly were downregulated; whereas, acyl-CoA and nucleotide metabolism were upregulated in the mutants. In parallel, we performed untargeted metabolomics by LC/MS to determine the metabolic changes induced by dnajc21 loss. Upregulated pathways included urea cycle, amino acid and phenylacetate metabolism. Pyrimidine biosynthesis, including both the de novo and salvage pathways were downregulated in the mutants. Remarkably, exogenous supplementation with uridine or thymidine significantly improved neutrophil counts in the mutants. Our findings suggest that in dnajc21-mutant SDS1) neutropenia is caused by a combination of impaired granulocyte differentiation and cell proliferation; 2) the kidney marrow features myelodysplasia-like changes and 3) nucleotide biosynthesis may be a previously unrecognized therapeutic strategy in SDS.

Natural History of Patients with Clonal Hematopoiesis Seen in Hematology Clinics

Background: The International Consensus Classification (ICC) recently recognized clonal hematopoiesis of indeterminate potential (CHIP) as a clinically defined pre-malignant state. A subgroup of patients with clonal hematopoiesis (CH), herein termed progressors, ultimately develop to a frank clinical diagnosis, while others, herein termed non-progressors, may have CH indefinitely with variable risk of progression. Although eight years have passed since the large-scale descriptions of CHIP as an age-related phenomenon, the risk factors for progression and management guidelines are still not well-defined. Moreover, while many quaternary care centers have developed CHIP databases, little is known about the epidemiology of CHIP from tertiary care centers that draw patients from the community setting. Methods: Patients with CH were identified via concurrent morphologic, cytogenetic, and molecular diagnostics of bone marrow aspirates reviewed by UMass Hematopathology between 2013 and 2022. CH was defined here as per the 2022 ICC: presence of a somatic mutation with variant allele frequency (VAF) ≥ 2% on molecular diagnostics or non-myelodysplastic syndrome (MDS)-defining clonal cytogenetic aberration in a patient without morphologic evidence of myeloid neoplasm. Patients with germline mutations were excluded. Independent review of all bone marrow aspirates was performed by at least two pathologists. This study included a pre-selected group of patients who had an indication for bone marrow evaluation; reasons for performing bone marrow evaluation included presentation with pancytopenia (41.7%), anemia (14.6%), leukocytosis (12.5%), thrombocytopenia (6.3%), leukopenia (4.2%), bicytopenia (4.2%), staging workup for a lymphoma (4.2%), erythrocytosis (4.2%), thrombocytosis (4.2%), abnormal radiographic marrow signal (2.1%), eosinophilia (2.1%). A total of 48 patients with CH were identified. Results: A total of 102 aberrations were found among 48 patients with CH. The mean, median, mode, and range for CH aberrations were 2.15, 1.5, 1, and 5, respectively. The mean age at detection of CH for non-progressors was 69.2 ± 1.8 years vs. 63.1 ± 3.5 years for progressors (p = 0.072). The most prevalent somatic variants in CH were: TET2 (29.1%), DNMT3A (14.5%), and gross structural abnormalities (7.7%) (Panel A, outer circle). Progressors had a distinct profile (Panel A, inner circle). Zero of 48 patients with CH carried TP53, IDH1, or EZH2 aberrations. The most prevalent genomic disruptions were missense mutations (45.6%), nonsense mutations (15.6%), and frameshift deletions (13.3%). We assessed the clinical trajectory of patients with CH (Panel B). Nine (18.8%) of 48 patients progressed to neoplasm (confirmed by bone marrow evaluation). Median overall survival (OS) at the time of analysis was 6.85 years, with longest follow up of 9.58 years. For non-progressors, median OS was not reached. For progressors, median OS was 6.85 years (from time of identification of CH). Median time from identification of CH to frank neoplasm was 1.05 years. For progressors, median time from diagnosis of frank neoplasm to death was 1.76 years. Of the 14 patients harboring DNMT3A, TET2, or ASXL1 (DTA) mutations alone, without mutations in other biological clusters, none (0%) progressed (Panel B,blue bars). Conclusion: In contrast to the original reports of CH, which studied unselected patients from multiple consortia, the patients in our study were pre-selected for suspicion of an underlying hematologic condition, reflecting real-world data. CH spans multiple functional clusters that are recurrently mutated in MDS and/or acute myeloid leukemia (AML), with the notable exception of TP53, IDH1, and EZH2. This may be due to more rapid progression to malignancy for those mutations, with only a transient, if any, CH state. All patients who progressed with DTA mutations did so only in the presence of additional mutations - at least a second mutation was required for DTA cases to progress. There was a trend toward significance for younger mean age at CH detection for progressors vs. non-progressors. Lead-time bias may skew OS results for patients found to have CH at an early point; and CH detected at a younger age may be more likely progress, leading to shorter OS. Systematic studies are needed to more comprehensively profile CH and develop formalized risk-adapted management strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Hyperglycemia cooperates with Tet2 heterozygosity to induce leukemia driven by proinflammatory cytokine-induced lncRNA Morrbid.

Diabetes mellitus (DM) is a risk factor for cancer. The role of DM-induced hyperglycemic (HG) stress in blood cancer is poorly understood. Epidemiologic studies show that individuals with DM are more likely to have a higher rate of mutations in genes found in pre-leukemic hematopoietic stem and progenitor cells (pre-LHSPCs) including TET2. TET2-mutant pre-LHSPCs require additional hits to evolve into full-blown leukemia and/or an aggressive myeloproliferative neoplasm (MPN). Intrinsic mutations have been shown to cooperate with Tet2 to promote leukemic transformation. However, the extrinsic factors are poorly understood. Using a mouse model carrying Tet2 haploinsufficiency to mimic the human pre-LHSPC condition and HG stress, in the form of an Ins2Akita/+ mutation, which induces hyperglycemia and type 1 DM, we show that the compound mutant mice developed a lethal form of MPN and/or acute myeloid leukemia (AML). RNA-Seq revealed that this was due in part to upregulation of proinflammatory pathways, thereby generating a feed-forward loop, including expression of the antiapoptotic, long noncoding RNA (lncRNA) Morrbid. Loss of Morrbid in the compound mutants rescued the lethality and mitigated MPN/AML. We describe a mouse model for age-dependent MPN/AML and suggest that hyperglycemia acts as an environmental driver for myeloid neoplasms, which could be prevented by reducing expression levels of the inflammation-related lncRNA Morrbid.

Hyperglycemia Cooperates with Tet2 Heterozygosity to Induce Leukemia Driven By Pro-Inflammatory Cytokine Induced Lncrna Morrbid

Diabetes mellitus (DM) is a risk factor for cancer development. However, the role of DM induced hyperglycemic stress (HG) in the development of blood cancer is poorly understood, largely due to lack of appropriate animal models. Epidemiologic studies show that individuals with DM are more likely to possess higher rate of mutations in genes found in pre-leukemic stem and progenitor cells (pre-LHSC/Ps) including in the epigenetic regulator TET2. TET2-mutant pre-LHSC/Ps require additional hits to evolve into a full-blown leukemia and/or aggressive myeloproliferative neoplasm (MPN). Cell intrinsic mutations have been shown to cooperate with Tet2 to promote leukemic transformation. However, the role of extrinsic factors is poorly understood. Utilizing a novel mouse model bearing haploinsufficiency of Tet2, to mimic the human pre-LHSC/P condition and HG stress, in the form of an Ins2Akita/+mutation, which induces HG and Type-1 DM, we show that the compound mutant mice develop a lethal form of MPN and/or acute myeloid leukemia (AML). RNA-seq revealed that this is in part due to upregulation of pro-inflammatory pathways, thereby generating a feed-forward loop, including the expression of an anti-apoptotic lncRNA Morrbid. Loss of Morrbid in the compound mutants rescues the lethality and mitigates the development of MPN/AML. Our results describe a novel mouse model for age-dependent AML/MPN and suggest that HG stress acts as an environmental driver for myeloid neoplasm, which could be effectively prevented by reducing the expression of inflammation-related lncRNA Morrbid. Disclosures No relevant conflicts of interest to declare.

Oral and dental management of people with myelodysplastic syndromes and acute myeloid leukemia: A systematic search and evidence-based clinical guidance.

To systematically search all studies that discussed dental procedures in patients diagnosed with myelodysplastic syndromes (MDS) and/or acute myeloid leukemia (AML) and to provide an evidence-based clinical guidance on oral and dental management of people with MDS and/or AML. The systematic search followed the Preferred Reporting Item for Systematic Review and Meta-analyses Protocols (PRISMA-P) guideline. Two databases systems were used (MEDLINE and EMBASE). PROSPERO was searched for ongoing or recently completed systematic reviews. The International Clinical Trials Registry Platform Search was searched for ongoing or recently completed trials. Level of evidence was evaluated based on the Oxford Level of Evidence. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach was used to summarize the evidence. Only 18 articles were selected and included in data synthesis and analysis. The level of evidence and recommendation ranged from 1 to 5 and A to D, respectively. All the included studies in the data synthesis (n=18) showed no specific guidelines were followed; however, all reflected the importance of liaison with the patient's hematoncologist at all stages of MDS and/or AML therapy. Oral and dental assessment is crucial prior to MDS therapy to help reduce anticipated complications. Dental treatment prior to hematopoietic stem cell transplantation and/or active stage of MDS therapy is tricky and always required liaison with the hematoncologist. MDS can progress to AML; hence, dental care providers are in a good position to spot any changes and refer early to the hematoncologist for further assessment.

Abstract 2063: SAMD9/SAMD9L mutations in familial monosomy 7

Abstract The purpose of this study was to define the mutations causing familial monosomy 7 with myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). We have previously reported frequent germline mutations in the genes SAMD9 and SAMD9L in pediatric MDS. We obtained samples on 32 individuals in 7 families with multiple siblings affected with MDS/AML and monosomy 7. We conducted targeted sequencing of genes frequently mutated in MDS/AML and amplicon sequencing of SAMD9 and SAMD9L. We performed cell cycle analysis assays on SAMD9/9L mutations to study their effects on cell proliferation. Our data show that 5 of the 7 families have germline pathologic mutations in SAMD9 (n=1) and SAMD9L (n=4), with significant clinical variability in the phenotypes displayed, ranging from transient monosomy 7 to overt AML with monosomy 7. Consistent with previous data, the mutant SAMD9 or SAMD9L allele was underrepresented in the bone marrow. This decrease in the mutant allele paralleled the extent of monosomy 7, suggesting that only the wild-type allele was retained in cells with monosomy 7. Functional analysis revealed that all germline SAMD9/9L mutations displayed a gain-of-function ability to suppress cell cycle progression. The patients with more aggressive disease were found to harbor somatic mutations in SETBP1, ETV6 or RUNX1. In many individuals we found additional somatic revertant mutations in cis with the germline mutation, which overcame the growth-suppressive effects of the germline mutation. Our data show that germline mutations in SAMD9/9L represent a new class of predisposition genes for familial monosomy 7 with MDS/AML and should be screened for in all children with MDS. Citation Format: Victoria Bryant, Jasmine Wong, Jason Schwartz, Tamara Lamprecht, Jing Ma, Charles Mullighan, Mignon Loh, Kevin Shannon, Jeffery Klco. SAMD9/SAMD9L mutations in familial monosomy 7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2063.

Acute myeloid leukemia and myelodysplastic syndrome after adjuvant chemotherapy: A population-based study among older breast cancer patients.

Chemotherapy for early breast cancer is associated with a small risk of developing myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). The aim of this study was to determine the risk of developing AML or MDS after modern adjuvant chemotherapy in older breast cancer patients and to further define the risk of individual chemotherapy regimens. Patients diagnosed with stage I to III breast cancer from 2003 to 2009 were identified in the Surveillance, Epidemiology, and End Results-Medicare and Texas Cancer Registry-Medicare linked databases. The development of AML/MDS, chemotherapy use, and comorbidities were identified with International Classification of Diseases, Ninth Revision and Healthcare Common Procedure Coding System codes. Analyses included descriptive statistics, cumulative incidences, and Cox proportional hazards models to estimate the hazard of AML/MDS after adjustments for clinically relevant covariates. In all, 92,110 patients were included; after a median follow-up of 85 months, the overall rates per 1000 person-years were 0.65 for AML and 1.56 for MDS. Patients who received an anthracycline (A) or anthracycline and taxane (A+T) regimen were more likely to develop AML (hazard ratio [HR] for A, 1.70; 95% confidence interval [CI], 1.16-2.50; HR for A+T, 1.68; 95% CI, 1.22-2.30) or MDS (HR for A, 2.18; 95% CI, 1.70-2.80; HR for A+T, 1.62; 95% CI, 1.29-2.03) than patients who did not receive chemotherapy. Patients using docetaxel and cyclophosphamide (TC) were not at increased risk for AML or MDS. Adjuvant chemotherapy is associated with a small but significant increase in the risk of AML and MDS, especially with regimens that include A. Longer follow-up is needed to confirm that risk is not increased with the recently adopted TC regimen. Cancer 2018;124:899-906. © 2017 American Cancer Society.

Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.

Monosomy 7/del (7q) in inherited bone marrow failure syndromes: A systematic review.

Inherited bone marrow failure syndromes (IBMFS) are rare cancer predisposition syndromes with an especially high risk of transformation to myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). We performed a retrospective systematic review of reported MDS/AML arising in the eight most common IBMFS to determine the frequency and outcome of chromosome 7 abnormalities. We identified 738 MDS/AML cases of 4,293 individuals. Monosomy 7 or del (7q) occurred in ∼17%. Greater understanding of the roles played by sequential acquisition of genetic and cytogenetic changes will provide insights into myeloid leukemogenesis and improve the surveillance and hopefully outcomes for individuals with IBMFS.

High Mobility Group A1/2 Chromatin Remodeling Proteins Associate with Polycythemia Vera Transformation to Acute Leukemia in Humans and a JAK2 V617F Transgenic Mouse Model

Introduction: The MPN are clonal hematopoietic stem cell (HSC) disorders characterized by an overproduction of blood cells and an increased risk of transformation to an aggressive phase with myelofibrosis (MF) and/or acute myeloid leukemia (AML). Polycythemia vera (PV) is the most common clinical subtype, and while PV starts as an indolent process, nearly 25% of patients will progress to MF and/or AML. PV is caused by acquired mutations of JAK2, yet JAK2 mutations alone do not account for MF or AML transformation. Mutations in genes encoding epigenetic regulators are associated with MPN transformation, but the mechanism of action is not understood. HMGA1/2 chromatin binding proteins are potent oncogenes that drive tumor progression by activating oncogenic and stem cell transcriptional networks. Both HMGA1/2 are overexpressed in acute leukemia and have been shown to be drivers of clonal expansion in myeloid disease in humans and in murine myeloproliferative disease models. We hypothesized that HMGA proteins could be critical drivers of transformation in PV and therefore tested the association of HMGA1/2 expression to transformation in human and murine PV.Methods: We examined the HSC genomic context and clonal evolution in 49 JAK2V617F-positive PV patients using standard and SNP-array karyotyping and a targeted resequencing panel of 163 genes associated with myeloid cancers. We examined HSC clonal burden by examining JAK2V617F HSC genotypes on a single cell basis. We measured HMGA1 and HMGA2 expression in a JAK2V617F positive human cell line, in isolated CD34+ HSCs from PV patients during chronic and transformation phases, in JAK2V617F transgenic murine models of PV (tgJAK2V617F) and PV-AML (tgJAK2V617F/MPLSV; Blood 2015;126:484) using a real-time quantitative RT-PCR (qRT-PCR) assay.Results: Both HMGA1 and HMGA2 mRNA were up-regulated in all JAK2V617F-positive contexts. In primary human PV CD34+ HSCs, HMGA1 and HMGA2 were found to be increased by 7 and 100 fold, respectively, compared to controls. Moreover, there was a dramatic up-regulation in both HMGA1/2 in patients who transformed from PV to MF or AML compared to chronic phase PV, whether analyzed cross-sectionally (Figure) or prospectively in selected patients. In addition to disease phase, over-expression of HMGA1/2 correlated with clonal dominance of JAK2V617F-homozygous stem cells, and additional mutations of epigenetic regulators including EZH2 and SETBP1. Similarly, when assessed in unfractionated bone marrow or in tumor samples in the two transgenic mouse models for PV and PV-AML, Hmga1/2 were overexpressed compared to wild-type littermates, with highest levels in the PV-AML transgenic mouse model.Conclusion:HMGA1 and HMGA2 are overexpressed in PV, and higher levels associate with disease progression to MF and AML, both in human PV and in transgenic murine models of PV. These data suggest HMGA proteins are critical drivers of PV transformation and that the mechanism of HMGA1/2 overexpression is a consequence of aberrant JAK/STAT signaling and epigenetic dysregulation. Our findings indicate that HMGA1/2 overexpression may function as a necessary molecular switch for PV leukemic transformation. Therefore, HMGA proteins and their transcriptional pathways offer novel therapeutic targets aimed at the prevention of PV progression to MF and AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gene Disruption of Zebrafish Sbds Phenocopies Human Shwachman-Diamond Syndrome but Suggests More Global and Lineage Defects

Introduction: Shwachman-Diamond Syndrome (SDS) is a complex disorder resulting in short stature, neutropenia, bony dysplasia, and pancreatic malabsorption. Individuals with SDS are also at increased risk for developing myelodysplastic syndrome and/or acute myeloid leukemia (AML). Its estimated incidence of 1 in 77,000 births makes SDS difficult to study and develop newer therapies. SDS results from mutations in the SBDS gene. Genetic ablation of Sbds results in early embryonic lethality (ED 7.5) in mice. Zebrafish provide an attractive, alternative model organism to study human diseases. Genome analysis of zebrafish (Danio rerio) revealed the presence of a single sbds gene, encoding a protein 90% identical to the human orthologue.Methods: We used CRISPR/Cas9 genome editing technique to generate indel mutations in sbds andestablished multiple zebrafish lines. We used zebrafish sbds mutants to analyze the function of this gene during vertebrate development and in the pathophysiology of SDS.Results: Western blotting showed progressive decrease of Sbds expression with no detectable protein at 8 days post fertilization (dpf). These results suggest that the presence of Sbds during early stages of the development is due to maternal deposition, which was confirmed by qPCR and immunoblot demonstration of sbds transcript and proteins in in all stages of the development including the 1 cell-stage. Unlike Sbds-/- mice, homozygous mutant sbdsnu132live up to 6 weeks. They display marked growth retardation. We also observed a significantly low number of neutrophils at 5 dpf and 15 dpf. No differences were found in macrophages and hemoglobinization. Interestingly, multiple organs, especially the pancreas, liver, digestive tract and eye showed histologic evidence of atrophy at 21 dpf. Differences were also observed in fat deposition and bone formation. These defects were not seen at 15 dpf, suggesting a critical stage in development dependent on functional sbds.Conclusions: To our knowledge, we established the first zebrafish model of SDS, making it a highly relevant model to investigate its pathophysiology and develop new small molecule therapies. Comparison of null states between mice and zebrafish suggest that loss of Sbds results in two phenotypes: an early embryonic stage required for viability and development and a post-embryonic (larval) stage critical for granulocyte production and normal size. Data will be presented on bone formation, neutrophil chemotaxis, metabolomics, and RNAseq in the zebrafish model.This work was supported by grants from the NIH R21CA15920, Shwachman-Diamond Syndrome Foundation and the Alex Turnquist Memorial Research fund of Shwachman-Diamond America. DisclosuresNo relevant conflicts of interest to declare.

Hereditary Predispositions to Myelodysplastic Syndrome.

Myelodysplastic syndromes (MDS) are heterogeneous clonal hematopoietic disorders characterized by ineffective hematopoiesis, bone marrow dysplasia, and peripheral cytopenias. Familial forms of MDS have traditionally been considered rare, especially in adults; however, the increasing availability of somatic and germline genetic analyses has identified multiple susceptibility loci. Bone marrow failure syndromes have been well-described in the pediatric setting, e.g., Fanconi anemia (FA), dyskeratosis congenita (DC), Diamond–Blackfan anemia (DBA), and Shwachman–Diamond syndrome (SBS), hallmarked by clinically-recognizable phenotypes (e.g., radial ray anomalies in FA) and significantly increased risks for MDS and/or acute myeloid leukemia (AML) in the setting of bone marrow failure. However, additional families with multiple cases of MDS or AML have long been reported in the medical literature with little known regarding potential hereditary etiologies. Over the last decade, genomic investigation of such families has revealed multiple genes conferring inherited risks for MDS and/or AML as the primary malignancy, including RUNX1, ANKRD26, DDX41, ETV6, GATA2, and SRP72. As these syndromes are increasingly appreciated in even apparently de novo presentations of MDS, it is important for hematologists/oncologists to become familiar with these newly-described syndromes. Herein, we provide a review of familial MDS syndromes and practical aspects of management in patients with predisposition syndromes.

Novel germ line DDX41 mutations define families with a lower age of MDS/AML onset and lymphoid malignancies

AbstractRecently our group and others have identified DDX41 mutations both as germ line and acquired somatic mutations in families with multiple cases of late onset myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML), suggesting that DDX41 acts as a tumor suppressor. To determine whether novel DDX41 mutations could be identified in families with additional types of hematologic malignancies, our group screened two cohorts of families with a diverse range of hematologic malignancy subtypes. Among 289 families, we identified nine (3%) with DDX41 mutations. As previously observed, MDS and AML were the most common malignancies, often of the erythroblastic subtype, and 1 family displayed early-onset follicular lymphoma. Five novel mutations were identified, including missense mutations within important functional domains and start-loss and splicing mutations predicted to result in truncated proteins. We also show that most asymptomatic mutation carriers have normal blood counts until malignancy develops. This study expands both the mutation and phenotypic spectra observed in families with germ line DDX41 mutations. With an increasing number of both inherited and acquired mutations in this gene being identified, further study of how DDX41 disruption leads to hematologic malignancies is critical.

Genetic Ablation of Sbds in Zebrafish Results in Neutropenia, Diminished Growth, and Reduced Viability, Indicating Its Roles in Embryonic and Post-Embryonic Development

Shwachman-Diamond syndrome (SDS) is a multisystem disorder characterized by neutropenia, exocrine pancreatic insufficiency, and skeletal abnormalities. Individuals with SDS are also at increased risk for developing myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). SDS results from mutations in the SBDS gene, which encodes a protein involved in 80S ribosome maturation. Genetic ablation of Sbds results in early embryonic lethality (ED 7.5) in mice. Zebrafish provide an attractive model organism with demonstrated relevance to mammalian hematopoiesis. Genome analysis of zebrafish (Danio rerio) revealed the presence of a single sbds gene, encoding a protein 90% identical to the human orthologue. qPCR demonstrated presence of sbds transcript in in all stages of the development including unfertilized egg. We used CRISPR/Cas9 genome editing technique to generate indel mutations in sbds. We established multiple zebrafish lines with mutations in the sbds gene. We have now characterized two alleles: 1) sbdsnu132, a 7-base deletion, that results in a frameshift at the amino acid G63 and a premature stop codon and 2) sbdsnu167, a 21-base insertion, that might encode a defective protein. The introduced indels correspond to the frequently mutated site, K62X, in SDS. Western blotting during the first days post fertilization (dpf) in sbdsnu132/nu132 mutants showed a decrease of Sbds with time, until 8 dpf when the protein was absent. These results suggest that the presence of Sbds during early stages of the development is due to maternal deposition. Loss of Sbds activity results in reduced viability. Unlike Sbds-/- mice, homozygous mutant sbdsnu132 live up to 6 weeks, but display a marked growth retardation phenotype (Figure 1). Interestingly, growth retardation was not observed until 15 dpf. Unlike morpholino-mediated knockdown of sbds, as reported by Provost et al., Development, 2012, pancreatic development appeared normal as determined by fluorescence microscopy using the transgenic line Tg(ptf1a:eGFP)jh1. To determine whether loss of sbds affects the number of neutrophils and macrophages, we used the transgenic lines Tg(mpx:Dendra)uwm4 and Tg(mpeg:Dendra)uwm12. We observed a significantly lower number of neutrophils in sbdsnu132/nu132 mutants compared to the wild-type fish at 5 and 15 dpf (p<0.05 and p<0.01 by t-test, respectively). However, no statistically significant difference in macrophage number was found. To our knowledge, we established, the first zebrafish model of SDS, making it a highly relevant model to understand its pathophysiology. Comparison of null states between mice and zebrafish suggest that loss of sbds results in two phenotypes: an early embryonic stage required for viability and development and a post-embryonic (larval) stage critical for granulocyte production and normal size. Ongoing studies are evaluating bone formation, pancreatic acinar function, and ectopic expression of patient-derived SBDS mutations in the context of a null background. Their phenotypes will be presented. (This work was supported by grants from the NIH and the Alex Turnquist Memorial Research fund of Shwachman-Diamond America). [Display omitted] DisclosuresCorey:Jazz Pharmaceuticals: Speakers Bureau.

Outcomes of mismatched and unrelated donor hematopoietic stem cell transplantation in Fanconi anemia conditioned with chemotherapy only.

Fanconi anemia (FA) is a genomic instability syndrome associated with bone marrow failure, myelodysplastic syndrome (MDS), and/or acute myeloid leukemia (AML) requiring hematopoietic stem cell transplantation (HSCT) to restore normal hematopoiesis. Although low-intensity fludarabine-based preparative regimens without radiation confer excellent outcomes in FA HSCTs with HLA-matched sibling donors, outcomes for FA patients with alternative donors are less encouraging, albeit improving. We present our experience with 17 FA patients who completed mismatched related or unrelated donor HSCT using a non-radiation fludarabine-based preparative regimen at Charité University Medicine Berlin. All patients engrafted; however, one patient had unstable chimerism in the setting of multi-viral infections that necessitated a stem cell boost to revert to full donor chimerism. Forty-seven percent of patients developed grade I acute graft-verus-host disease (aGVHD). No grade II-IV aGVHD or chronic graft-versus-host disease of any severity occurred. At a median follow-up of 30 months, 88 % of patients are alive with normal hematopoiesis. Two patients died of infections 4 months post-transplantation. These results demonstrate that short-term outcomes for FA patients with mismatched and unrelated donor HSCTs can be excellent using chemotherapy only conditioning. Viral reactivation, however, was a major treatment-related complication.

Genistein Protects Hematopoietic Stem Cells against G-CSF–Induced DNA Damage

Granulocyte colony-stimulating factor (G-CSF) has been used to treat neutropenia in various clinical settings. Although clearly beneficial, there are concerns that the chronic use of G-CSF in certain conditions increases the risk of myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). The most striking example is in severe congenital neutropenia (SCN). Patients with SCN develop MDS/AML at a high rate that is directly correlated to the cumulative lifetime dosage of G-CSF. Myelodysplastic syndrome and AML that arise in these settings are commonly associated with chromosomal deletions. We have demonstrated in this study that chronic G-CSF treatment in mice results in expansion of the hematopoietic stem cell (HSC) population. In addition, primitive hematopoietic progenitors from G-CSF-treated mice show evidence of DNA damage as demonstrated by an increase in double-strand breaks and recurrent chromosomal deletions. Concurrent treatment with genistein, a natural soy isoflavone, limits DNA damage in this population. The protective effect of genistein seems to be related to its preferential inhibition of G-CSF-induced proliferation of HSCs. Importantly, genistein does not impair G-CSF-induced proliferation of committed hematopoietic progenitors, nor diminishes neutrophil production. The protective effect of genistein was accomplished with plasma levels that are attainable through dietary supplementation.

Neurofibromatosis‐1 gene deletions and mutations in de novo adult acute myeloid leukemia

Germline heterozygous alterations of the tumor-suppressor gene neurofibromatosis-1 (NF1) lead to neurofibromatosis type 1, a genetic disorder characterized by a higher risk to develop juvenile myelomonocytic leukemia and/or acute myeloid leukemia (AML). More recently, somatic 17q11 deletions encompassing NF1 have been described in many adult myeloid malignancies. In this context, we aimed to define NF1 involvement in AML. We screened a total of 488 previously untreated de novo AML patients for the NF1 deletion using either array comparative genomic hybridization (aCGH) or real-time quantitative PCR/fluorescence in situ hybridization approaches. We also applied massively parallel sequencing for in depth mutation analysis of NF1 in 20 patients including five NF1-deleted patients. We defined a small ∼0.3 Mb minimal deleted region involving NF1 by aCGH and an overall frequency of NF1 deletion of 3.5% (17/485). NF1 deletion is significantly associated with unfavorable cytogenetics and with monosomal karyotype notably. We discovered six NF1 variants of unknown significance in 7/20 patients of which only one out of four disappeared in corresponding complete remission sample. In addition, only one out of five NF1-deleted patients has an acquired coding mutation in the remaining allele. In conclusion, direct NF1 inactivation is infrequent in de novo AML and may be a secondary event probably involved in leukemic progression.

Fanconi Anemia

Fanconi anemia (FA) is the most frequent inherited cause of BM failure (BMF). Fifteen FANC genes have been identified to date, the most prevalent being FANCA, FANCC, FANCG, and FANCD2. In addition to classical presentations with progressive BMF during childhood and a positive chromosome breakage test in the blood, atypical clinical and/or biological situations can be seen in which a FA diagnosis has to be confirmed or eliminated. For this, a range of biological tools have been developed, including analysis of skin fibroblasts. FA patients experience a strong selective pressure in the BM that predisposes to clonal evolution and to the emergence in their teens or young adulthood of myelodysplasia syndrome (MDS) and/or acute myeloid leukemia (AML) with a specific pattern of somatic chromosomal lesions. The cellular mechanisms underlying (1) the hematopoietic defect which leads to progressive BMF and (2) somatic clonal evolutions in this background, are still largely elusive. Elucidation of these mechanisms at the molecular and cellular levels should be useful to understand the physiopathology of the disease and to adapt the follow-up and treatment of FA patients. This may also ultimately benefit older, non-FA patients with aplastic anemia, MDS/AML for whom FA represents a model genetic condition.

Five new pedigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy

AbstractFamilial platelet disorder with propensity to myeloid malignancy (FPD/AML) is an autosomal dominant syndrome characterized by platelet abnormalities and a predisposition to myelodysplasia (MDS) and/or acute myeloid leukemia (AML). The disorder, caused by inherited mutations in RUNX1, is uncommon with only 14 pedigrees reported. We screened 10 families with a history of more than one first degree relative with MDS/AML for inherited mutations in RUNX1. Germ- line RUNX1 mutations were identified in 5 pedigrees with a 3:2 predominance of N-terminal mutations. Several affected members had normal platelet counts or platelet function, features not previously reported in FPD/AML. The median incidence of MDS/AML among carriers of RUNX1 mutation was 35%. Individual treatments varied but included hematopoietic stem cell transplantation from siblings before recognition of the inherited leukemogenic mutation. Transplantation was associated with a high incidence of complications including early relapse, failure of engraftment, and posttransplantation lymphoproliferative disorder. Given the small size of modern families and the clinical heterogeneity of this syndrome, the diagnosis of FPD/AML could be easily overlooked and may be more prevalent than previously recognized. Therefore, it would appear prudent to screen young patients with MDS/AML for RUNX1 mutation, before consideration of sibling hematopoietic stem cell transplantation.