Megakaryocytic Dysplasia Research Articles

Overexpression of Histone Demethylase KDM6B in the Hematopoietic Compartment Promotes the Effect of Innate Immune Activation and Results in Myelodysplastic Syndrome (MDS)-like Phenotype in Mice

The histone demethylase KDM6B regulates transcriptional activity of genes involved in innate immune signals and controls differentiation of hematopoietic cells. Our laboratory previously reported that KDM6B is overexpressed in bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) of over 50% MDS pts (Leukemia 2013). Pharmacologic targeting of KDM6B has shown pre-clinical activity in several neoplasms such as glioma and ALL. It still remains to be elucidated whether KDM6B overexpression directly alters hematopoiesis, in order to test the hypothesis that overexpression of KDM6B contributes to aberrant epigenetic and innate immune signal in MDS and thus forms an important pathological mechanism and potential therapeutic target.Here we report an animal model of KDM6B overexpression in mouse hematopoietic compartment (Vav-KDM6B). In 20-wk old mice, KDM6B overexpression led to mild phenotype including reduced white cell count (WBC) in peripheral blood (PB) (15% p<0.05 N=47) and decreased LSK in BM (20% p<0.05 N=12). When exposed to chronic low dose Toll-like receptor ligand lipopolysaccharide (LPS) (6mg every other day, i.p. 6 wks), phenotype severity was more profound in Vav-KDM6B mice than in wildtype (WT) mice. First, hematopoietic cell numbers in both PB and BM were significantly reduced: LPS led to a 40% decrease of PB WBC (p<0.01 N=17) and a 38% decrease of BM cell (p<0.01 N=13 Fig 1A) in KDM6B mice, compared to 20% and 28% reduction respectively in WT mice treated by LPS. Second, both number and repopulating function of BM long term (LT) HSC were severely compromised: LPS led to decrease of BM LT-HSC by 52% (p<0.01 N=13) in KDM6B mice compared to a 35% decrease in WT mice. Functionally, colonies formed in methylcellulose by LSKs of LPS treated KDM6B mice decreased by 75% (p<0.01 N=13) at 2nd plating, whereas LPS led to a reduction of 60% for WT-LSK colony formation at 2nd plating. Similar effect was also observed when BM repopulating capacity was evaluated by chimerism (CD45.2:CD45.1) in competitive transplantation assay (p<0.05 N=11). Third, increase of megakaryocytic dysplasia was documented in BM of LPS treated KDM6B mice compared to WT counterparts (Fig 1B).To identify KDM6B overexpression mediated molecular signals under innate immune stimuli, we performed genome-wide transcriptome study using BM LSK of treated mice. Results reveal that over 130 genes were more profoundly activated by LPS in LSK of Vav-KDM6B than in WT LSK. Gene set enrichment assays (GSEA) indicated that major molecular pathways related to these genes include MDS-relevant signals, such as inflammatory and complement innate immune signals, MAPK pathway, as well as activated gene signature of RPS14 knockout in human BM CD34+ cell. We also performed RNA-Seq analysis in BM CD34+ HSPCs of MDS (N=26) and healthy controls (N=10). Results confirmed that a significant proportion of these genes (N>20) are overexpressed in MDS patients. Furthermore, we demonstrated in mouse BM cells that KDM6B overexpression mediates histone H3K4 and H3K27 methylation at promoters of several key innate immune genes towards transcription activation.We next evaluated effect of pharmacological KDM6B targeting using a highly specific KDM6B inhibitor (GSK-J4). At relatively low-dose (0.5mM), GSK-J4 could increase in vitro colony formation of LSKs of LPS treated KDM6B mice by 80% (p<0.05) at 2nd plating. Injection of GSK-J4 (160mg once a week, 5 weeks) resulted in 50% increase of WBM cells in LPS treated Vav-KDM6B mice (p<0.05). These results suggest that GSK-J4 can improve compromised hematopoiesis of Vav-KDM6B mice. We next treated primary BM HSPCs isolated from patients with low risk MDS (N=11) with GSK-J4. Results indicated that at 0.5mM, GSK-J4 could improve colony formation of MDS CD34+ cells (p=0.05). Flow cytometry analysis using cells from colonies indicated that GSK-J4 led to increased myeloid mark CD11b (p=0.08) and reduced apoptotic mark annexin-V (p<0.05). The same concentration of GSK-J4 did not affect colony formation of normal BM CD34+ HSPCs isolated from healthy donors (N=4).Taken together, our results indicate that overexpression of KDM6B mediates the hyperactivation of innate immune signals and has a functional impact in BM HSPCs, supporting role for KDM6B overexpression in MDS pathogenesis. Furthermore, our study also indicates that pharmacological inhibition of KDM6B may have therapeutic potential in MDS. [Display omitted] DisclosuresKhoury:Kiromics: Research Funding; Angle: Research Funding; Stemline Therapeutics: Research Funding; Pfizer: Research Funding.

Clinical and Pathologic Spectrum of DDX41-Mutated Hematolymphoid Neoplasms.

This study seeks to further characterize the clinicopathologic spectrum of DDX41-mutated hematolymphoid malignancies. We identified DDX41 mutations from a cohort of known or suspected hematologic disorders and reviewed the corresponding clinical, genetic, phenotypic, and morphologic findings. DDX41 mutations were identified in 20 (1.4%) of 1,371 cases, including 8 cases of acute myeloid leukemia (AML), 5 cases of myelodysplastic syndrome (MDS), 2 cases of therapy-related MDS/AML, 1 case of primary myelofibrosis, 1 case of chronic myeloid leukemia, 1 case of clonal cytopenia of uncertain significance (CCUS), 1 case of T-cell large granular lymphocytic leukemia (T-LGL), and 1 case of multiple myeloma. DDX41-mutated neoplasms were morphologically heterogeneous with a median cellularity of 20% (range, 10%-100%). Megakaryocyte dysplasia occurred in 7 (35%) of 20 cases and trilineage dysplasia in 1 (5%). Frequently comutated genes include a second, somatic DDX41 mutation (8/19, 42%) followed by mutations in TET2 (20%), DNMT3A (20%), ASXL1 (20%), and CUX1 (20%). Karyotypes were noncomplex in 17 (89%) of 19. This report extends the spectrum of DDX41-mutated disorders to include CCUS, T-LGL, and plasma cell disorders. The morphologic features are heterogeneous and nonspecific, highlighting the importance of DDX41 testing during routine workup of hematolymphoid neoplasms.

Successful Treatment of Concurrently Diagnosed Multiple Myeloma and Myelodysplastic Syndrome with Isolated Del(5q) with Lenalidomide, Bortezomib, and Dexamethasone

Lenalidomide is known to be an effective therapy for multiple myeloma (MM) and for myelodysplastic syndrome with isolated del(5q). However, there have been very few reports of treatment of both conditions using lenalidomide when they are diagnosed concurrently. A review of the literature revealed two reports of MM and del(5q) MDS treated with lenalidomide. We report the case of a patient simultaneously diagnosed with multiple myeloma and myelodysplastic syndrome with isolated del(5q) who was treated successfully with lenalidomide. The patient is a 74 year old female who was referred to hematology for worsening chronic macrocytic anemia with a hemoglobin of 9.4 g/dL. A serum protein electrophoresis (SPEP) was obtained during her workup and demonstrated an IgG kappa monoclonal spike of 4.7 g/dL. Free light chain analysis demonstrated a kappa/lambda ratio of 36.7. The patient was mildly hypercalcemic at 10.6 g/dL but had no renal insufficiency. Platelet and white blood cell counts were normal. There were no osteolytic lesions on skeletal survey and a whole body PET scan identified no bony disease or plasmacytomas. A β-2 microglobulin level was 3.7 mg/L and albumin was 3.3 g/dL. Bone marrow biopsy revealed 60% plasma cells in a 70% cellular marrow. Granulocytic and megakaryocytic dysplasia was identified. Fluorescence in situ hybridization returned showing a 4:14 translocation in 72% of analyzed nuclei and monosomy 13 in 61% of nuclei analyzed consistent with an unfavorable risk profile. Chromosome analysis also revealed a 5q deletion in 15 of 20 analyzed cells. Bone marrow blasts were measured at 1%. Therefore, the patient concurrently met diagnostic criteria for stage II IgG kappa multiple myeloma per the International Staging System and low risk myelodysplastic syndrome with isolated del(5q) per the 2016 WHO classification of MDS with a Revised International Prognostic Scoring System Score (IPSS-R) of 2. She was started on lenalidomide 25 mg daily, bortezomib 1.3 mg/m2 on days 1, 4, 8, and 11 and dexamethasone 20 mg on days 1, 8, and 15 of a 21 day cycle. After 3 cycles of therapy, serum immunofixation electrophoresis showed an unquantifiably low IgG kappa monoclonal spike and the patient's kappa/gamma light chain ratio had normalized to 1.1. Hemoglobin and calcium returned to normal. On repeat bone marrow biopsy, there was normocellular marrow with 4% polytypic plasma cells by kappa/lambda immunohistochemistry. No dysplasia was identified and bone marrow blasts were 1.5%. Therefore, the patient achieved a very good partial response (VGPR) to therapy for multiple myeloma according to International Myeloma Working Group criteria within 3 months. She met National Comprehensive Cancer Network criteria for response of her MDS to lenalidomide by normalization of hemoglobin. The patient's case demonstrates successful treatment of concurrently diagnosed multiple myeloma and MDS with isolated del(5q) using lenalidomide. Among the two other similar cases we discovered in the literature, one patient was treated with low-dose lenalidomide and dexamethasone [Nolte, et al. Eur J Haematol. 2017 Mar;98(3):302-310.], and the other patient was treated with high-dose lenalidomide and dexamethasone, achieving a partial response [Ortega, et al. Leuk Res. 2013 Oct;37(10):1248-50.]. Neither patient received a proteasome inhibitor. In our case, the patient was treated with higher intensity induction therapy for multiple myeloma and achieved a VGPR. She did not have worsening cytopenias during therapy, and in fact experienced normalization of her blood counts. Therefore, it is reasonable to treat patients simultaneously diagnosed with MM and MDS with isolated del(5q) with standard three-drug induction therapy for multiple myeloma. While our approach makes sense in the abstract, hematology/oncologists should be aware that it works in practice. Disclosures No relevant conflicts of interest to declare.

Optimizing Response to Enasidenib with Dose Escalation: Case Series

Background: Isocitrate dehydrogenase enzymes catalyze the conversion of isocitrate to alpha-ketoglutarate. Mutated IDH enzymes generate the "oncometabolite" 2-hydroxyglutarate (2-HG), which inhibits TET2 function. IDH2 mutations have been reported in 9% to 19% of acute myeloid leukemia (AML) cases. Inhibition of the mutant IDH2 enzyme led to decreased 2-HG levels and induced myeloid differentiation in IDH2 mutant AML. Enasidenib, a small-molecule inhibitor of mutant IDH2, was approved in 2017, after a successful phase I/II trial showing an overall response rate (ORR) of 40.3% in relapsed/refractory (R/R) disease, with 19.3% of patients achieving complete remission (CR). A Phase III trial is currently ongoing. It targets the mutant IDH2 variants R140Q, R172S, and R172K; at an approved dose of 100 mg oral daily dose. At higher doses the drug was less tolerated, however, few subjects received dose modification at the 650 mg daily dose group; these events did not qualify as a dose-limiting toxicity (DLT). In this descriptive study, we are reporting a case series of R/R AML, with an IDH2 mutation, that are treated with escalated dose enasidenib. Method: This case series is based on retrospective observations of patients with R/R IDH2 mutant AML since January 2017, who are treated with enasidenib. We are reporting two cases treated with an escalated dose of 200 mg daily. We included patients with intermediate to poor-risk AML, who received at least one prior line of therapy, started on standard dose enasidenib (100 mg daily) for a minimum of 6 months and followed until disease relapse or death. We excluded patients who are primarily resistant to enasidenib (AML risk stratification and response evaluation by ELN-Leukemia NET. IDH2 mutations were identified by a local diagnostic laboratory that is regulated under CLIA through PCR which is validated to detect 10% or more mutant allele frequency. To quantify IDH2 variant frequency, confirmatory testing was performed via ARUP next-generation sequencing panel (NGS). Results: Here we report the descriptive outcomes of 2 cases from a single institution, who were treated with escalated dose enasidenib (200 mg oral daily) for R/R AML with an IDH2 mutation. The first case describes a 65-year-old female diagnosed with poor-risk AML in the setting of pancytopenia with bone marrow evaluation consistent with the background of erythroid and granulocytic dysplasia, with normal karyotype analysis. The patient has treated with induction chemotherapy and 2 cycles consolidation that was complicated with bacterial infections, prolonged hospitalization, and profound deconditioning led to stopping treatment. After 18 months of surveillance, repeat bone marrow evaluation for new-onset pancytopenia revealed relapsed AML with evidence of IDH2 mutation at codon 140, with a variant frequency of 36.9% by NGS. The case was started on enasidenib at 100 mg oral daily dose. After 6 months of therapy, the patient continued to be cytopenic, repeated bone marrow evaluation showed residual AML with IDH2 variant frequency of 27.6%. The decision was made to increase the enasidenib dose to 200 mg daily. With follow up for another 5 months on the escalated dose, the patient achieved hematologic complete response (hCR) with normalization of peripheral platelet count and ANC. [Figures 1-2] The patient maintained hCR for another 5 months on 200 mg enasidenib daily until she died from COVID-19 infection. The second case describes a 59-year-old female with poor-risk AML in the setting of leukocytosis, with bone marrow evaluation consistent with the background of megakaryocytic dysplasia and karyotype analysis positive for monosomy 7. The patient was treated with induction chemotherapy and failed to respond. Repeat bone marrow evaluation showed primary refractory disease with positive IDH2 mutation at codon 140 with a variant frequency of 40.7%. Treatment with 100 mg enasidenib was started. The patient maintained a partial response for 6 months, then peripheral blasts counts started to gradually increase. The decision was made to increase enasidenib dose to 200 mg daily, which improved peripheral blasts within one month of escalated dose therapy. [Figure 3] The patient maintained a response to therapy for another 3 months before she relapsed. Conclusion: Our limited data showed that initial response to standard dose enasidenib could potentially be optimized by dose escalation to 200 mg daily. Disclosures No relevant conflicts of interest to declare.

Evolution of histomorphologic, cytogenetic, and genetic abnormalities in an untreated patient with MIRAGE syndrome

Gain of function variants in SAMD9 cause MIRAGE syndrome, a rare Mendelian disorder that results in myeloid dysplastic syndrome (MDS), poor immune response, restricted growth, adrenal insufficiency, ambiguous genitalia, feeding difficulties and most often significantly reduced lifespan. In this study, we describe histomorphologic and genetic changes occurring in serial bone marrow measurements in a patient with MIRAGE syndrome and untreated MDS of 9 years. Histomorphological analysis during childhood showed progressive hypocellularity with erythroid and megakaryocytic dysplasia and cytogenetic testing demonstrated monosomy 7. Serial leukemia gene panel testing performed over a seven year period revealed multiple pre-leukemic clones arising at age 7 years followed by sequential mutational events in ETV6 and RUNX1 driving acute myeloid leukemia (AML) at age 9. Comprehensive genotype-phenotype analysis with 28 previously reported patients found the presence of MDS did not impact overall survival, but in silico variant pathogenicity prediction scores for SAMD9 distinguished patients with poor prognosis. Overall, our analysis shows progression of MDS to AML can be monitored by following mutation evolution in leukemia related genes in patients with MIRAGE syndrome, and specific SAMD9 mutations likely influence disease severity and overall survival.

Hippo kinase loss contributes to del(20q) hematologic malignancies through chronic innate immune activation

AbstractHeterozygous deletions within chromosome 20q, or del(20q), are frequent cytogenetic abnormalities detected in hematologic malignancies. To date, identification of genes in the del(20q) common deleted region that contribute to disease development have remained elusive. Through assessment of patient gene expression, we have identified STK4 (encoding Hippo kinase MST1) as a 20q gene that is downregulated below haploinsufficient amounts in myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN). Hematopoietic-specific gene inactivation in mice revealed Hippo kinase loss to induce splenomegaly, thrombocytopenia, megakaryocytic dysplasia, and a propensity for chronic granulocytosis; phenotypes that closely resemble those observed in patients harboring del(20q). In a JAK2-V617F model, heterozygous Hippo kinase inactivation led to accelerated development of lethal myelofibrosis, recapitulating adverse MPN disease progression and revealing a novel genetic interaction between these 2 molecular events. Quantitative serum protein profiling showed that myelofibrotic transformation in mice was associated with cooperative effects of JAK2-V617F and Hippo kinase inactivation on innate immune-associated proinflammatory cytokine production, including IL-1β and IL-6. Mechanistically, MST1 interacted with IRAK1, and shRNA-mediated knockdown was sufficient to increase IRAK1-dependent innate immune activation of NF-κB in human myeloid cells. Consistent with this, treatment with a small molecule IRAK1/4 inhibitor rescued the aberrantly elevated IL-1β production in the JAK2-V617F MPN model. This study identified Hippo kinase MST1 (STK4) as having a central role in the biology of del(20q)-associated hematologic malignancies and revealed a novel molecular basis of adverse MPN progression that may be therapeutically exploitable via IRAK1 inhibition.

How Morphologic Features Are Shaped By Underlying Somatic Genotype in MDS

Morphology has been the pillars of the MDS diagnosis. In the genetic era, the subjectivity of pathologic evaluation in the absence of integration with mechanistic correlations is a clear limitation of current diagnostic schemes. Well-known genotype/phenotype associations, including e.g., linking of SF3B1 mutations to RARS or JAK2/SF3B1 mutations to RARS-T, suggest that somatic events can shape morphologic features and vice versa. However, the complexity of morphologic and genetic changes precludes identification of many consequential genotype/phenotype associations. As objective image analytic tools are being developed, this project aims at determining the most mutually predictive relationships between combinations of morphologic features and genomic changes. We have analyzed 1,079 MDS patients for somatic CNVs and mutations in a targeted panel of 33 genes frequently mutated in myeloid cancers. Our stringent bioanalytic pipeline removed artifacts, SNPs and errors. We applied this pipeline to discovery (2/3) and validation (1/3) cohorts. Bone marrow morphological alterations were mapped to binary features called by an independent pathologist in a blinded fashion based on uniformly defined criteria occurring in &gt;10% of cells. A total of 10 such features were investigated. For instance, myeloid, erythroid and megakaryocytic dysplasia occurred in 54%, 70% and 72% of patients, respectively. 89% had at least one cytopenia, 57% multiple cytopenias, and 50% had at least one myeloproliferative feature (e.g., monocytosis). In addition, all cases were partitioned in accordance with two risk groups defined by IPSS-R (lower risk &lt;3.5 and higher risk &gt;3.5). NGS analysis identified 1,929 somatic mutations, but for proper correlation with morphologic definitions only mutations with a clonal burden &gt;10% were used. Our initial univariate analysis yielded 52 significant associations (q&lt;.1). For instance, myeloid dysplasia was more associated with ASXL1, NRAS,SRSF2, STAG2, and TET2 mutations. Univariate associative analyses failed to yield signatures due to the extensive presence of the heterogeneity in genotype profiles. Noting patterns of interdependence exhibited by the 24 individual morphologic features, an unsupervised cluster analysis based on the consensus clustering method was used to identify intrinsic patterns of co-occurrence and morphologic subtypes of MDS. A semi-supervised machine learning (ML) technique based on Bayesian partial exchangeability investigated the extent to which morphologic subtypes of MDS can be discriminated on the basis of patterns of mutation incidence and co-occurrence. To augment existing clinical models of prognosis, ML was applied to low risk patient MDS subtypes. A single model, selected and subsequently validated using our independent test set, yielded genetic signatures demonstrating morphologic orientation. An unsupervised cluster analysis revealed 5 discrete morphological MDS groups on the basis of 24 features (Fig.1a). High-risk subtypes were clustered in group 1 (G1), whereas low risk subtypes clustered in the other 4 groups, each of which had unique prominent morphological features (Fig.1b). G2 had pancytopenia, G3 had monocytosis, G4 had elevated megakaryocytes, and G5 had erythroid dysplasia. Exhibiting prognostic utility, overall survival differed significantly among these groups (Fig.1c). Mutational frequencies also differed between these groups, e.g., G1 frequently had mutations of RUNX1, TP53, and STAG2. 8 genetic signatures characterize the morphologic phenotypes of low risk MDS patients (S1-8; Fig.1d). Signature-A (SA) was enriched for TET2 mutations, SB had the co-occurrence of TET2 and SRSF2 mutations, whereas SG was enriched in SF3B1 mutations. Deviation in survival (p&lt;0.026) demonstrated the prognostic value of these genetic signatures among low risk patients. The genetic signatures were predictive of specific morphological features (Fig.1e). SB dominated G3 (73%), and SC were frequent for G4 (57%). Among 18 morphologic/genetic associations identified in the discovery set, 9 were confirmed in the validation cohort. For instance, we validated that SB was enriched in monocytosis and TET2 mutations; while SD in anemia and SF3B1 mutations. This is the first comprehensive analysis that links somatic molecular lesions to morphological patterns to identify distinct phenotype/genotype associations. Disclosures Nazha: Jazz Pharmacutical: Research Funding; Abbvie: Consultancy; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Daiichi Sankyo: Consultancy; Tolero, Karyopharma: Honoraria; Incyte: Speakers Bureau. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Hobbs:Amgen: Research Funding; SimulStat Inc.: Consultancy. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

Phf6-null hematopoietic stem cells have enhanced self-renewal capacity and oncogenic potentials

AbstractPlant homeodomain finger gene 6 (PHF6) encodes a 365-amino-acid protein containing 2 plant homology domain fingers. Germline mutations of human PHF6 cause Börjeson-Forssman-Lehmann syndrome, a congenital neurodevelopmental disorder. Loss-of-function mutations of PHF6 are detected in patients with acute leukemia, mainly of T-cell lineage and in a small proportion of myeloid lineage. The functions of PHF6 in physiological hematopoiesis and leukemogenesis remain incompletely defined. To address this question, we generated a conditional Phf6 knockout mouse model and investigated the impact of Phf6 loss on the hematopoietic system. We found that Phf6 knockout mice at 8 weeks of age had reduced numbers of CD4+ and CD8+ T cells in the peripheral blood compared with the wild-type littermates. There were decreased granulocyte-monocytic progenitors but increased Lin–c-Kit+Sca-1+ cells in the marrow of young Phf6 knockout mice. Functional studies, including competitive repopulation unit and serial transplantation assays, revealed an enhanced reconstitution and self-renewal capacity in Phf6 knockout hematopoietic stem cells (HSCs). Aged Phf6 knockout mice had myelodysplasia-like presentations, including decreased platelet counts, megakaryocyte dysplasia, and enlarged spleen related to extramedullary hematopoiesis. Moreover, we found that Phf6 loss lowered the threshold of NOTCH1-induced leukemic transformation at least partially through increased leukemia-initiating cells. Transcriptome analysis on the restrictive rare HSC subpopulations revealed upregulated cell cycling and oncogenic functions, with alteration of key gene expression in those pathways. In summary, our studies show the in vivo crucial roles of Phf6 in physiological and malignant hematopoiesis.

PS1022 PREFERENTIAL TRANSCRIPTION OF THE MUTATED ALLELE IN NPM1 MUTATED ACUTE MYELOID LEUKAEMIA

Background: Early studies in the 1980 s concentrated on defining AMLs poorly responding/resistant to SICT as cases of AMLs with trilineage (TLD) or multilineage dysplasia (MLD). These AMLs with MLD were supplemented with AMLs exhibiting cytogenetic findings typical for myelodysplastic syndromes (MDS) and with AMLs arising from previous MDS into the category of AML with MDS-related changes (AML-MRC) in the WHO 2016 classification. AML with MLD have to exhibit erythroblastic and/or megakaryocytic dysplasia (EMD) and originate from myeloid pluripotent progenitors. However, AML M0-M5 with granulocyte-monocyte blasts exhibiting a single line dysplastic features of EMD (not MLD by definition but two lines involved) belong to EMD-AML and not to single-line GM-AMLs which responded well to SICT (Lemez et al., Leuk Res 2000;24:207). Aims: The aim of this study was to review our published and unpublished de novo AML patients examined for dysplastic features and treated with SICT as well as similar de novo AML cases from the literature and their response to SICT. Methods: We have analyzed our cases examined for dysplastic bone marrow features treated with SICT or high-dose cytarabine with daunorubicin cycle 10/2. A literature review of reported AML cases examined for dysplasia and treated with SICT was also analyzed. Results: Our single-institution study on consecutive adult de novo AML cases <65 years of age showed that those with GM-AML reached complete remission (CR) in 78% after SICT but cases with EMD-AML in 29% (p = 0.02). However, 7/10 EMD-AML cases reached CR after the cycle with high-dose cytarabine and daunorubicin 10/2 (Lemez et al., 2000). Coasguen et al. (Leukemia 1992;6:520) reported 74.4% CR after SICT in 156 cases without dysplastic features (GM-AML), but 56% CR in 39 cases with TLD AMLs. Brito-Babapulle et al. (Br J Haematol 1987;66:445), Estienne et al. (Clin Lab Haematol 1990;12:57), and Kuriyama et al. (Br J Haematol 1994;86:767) reported post SICT 86%, 79%, and 81% CR in 136, 118, and 190 selected non-TLD cases, but CR 50%, 43%, and 63% in TLD AML cases, respectively. Patients’ selection of cases suitable for SICT with median age below 50 years probably increased the CR rates in non-TLD and TLD AML categories. Papers with high or intermediate doses of cytarabine during induction were not included in this literature review. Our 6 of 12 consecutive cases with de novo AML over 80 years of age opted for SICT administration. Three cases with GM-AML reached CR after one cycle of SICT and survived 16.5–28 months on maintenance therapy while none of the three octogenarians with EMD-AML reached CR and all died within 0.4–2.7 months. A 79-year old man with GM-AML reached CR post 2 cycles of low-dose cytarabine (LDAC) and survived on 53 repeated cycles LDAC 84 months. Another 67-year old man with GM-AML and near-tetraploid karyotype reached CR post SICT and after two further same cycles he survived in CR 51 months and with further treatment altogether 80 months. Summary/Conclusion: Our results and the literature review showed that GM-AML cases achieve CR after SICT in 74–85% of patients <60/65 years of age. Five older patients > 65 years including three > 80 years of age reached CR after SICT in spite of poor performance status 3 in two and had survival 16.5–28 months on maintenance therapy. We estimate that de novo GM-AML cases constitute about 20% of all cases 80+ years old with newly diagnosed AML. GM-AML cases > 65 years exhibit a good sensitivity to SICT. Further reports and studies are needed to enlarge the number of older GM-AML cases treated with SICT.

PS1021 GOOD SENSITIVITY OF ACUTE MYELOID LEUKEMIAS INVOLVING ONLY GRANULOCYTE‐MONOCYTE LINE (GM‐AML) TO STANDARD‐DOSE INDUCTION THERAPY (SICT) IS NOT INFLUENCED BY AGE

Background:Early studies in the 1980 s concentrated on defining AMLs poorly responding/resistant to SICT as cases of AMLs with trilineage (TLD) or multilineage dysplasia (MLD). These AMLs with MLD were supplemented with AMLs exhibiting cytogenetic findings typical for myelodysplastic syndromes (MDS) and with AMLs arising from previous MDS into the category of AML with MDS‐related changes (AML‐MRC) in the WHO 2016 classification. AML with MLD have to exhibit erythroblastic and/or megakaryocytic dysplasia (EMD) and originate from myeloid pluripotent progenitors. However, AML M0‐M5 with granulocyte‐monocyte blasts exhibiting a single line dysplastic features of EMD (not MLD by definition but two lines involved) belong to EMD‐AML and not to single‐line GM‐AMLs which responded well to SICT (Lemez et al., Leuk Res 2000;24:207).Aims:The aim of this study was to review our published and unpublished de novo AML patients examined for dysplastic features and treated with SICT as well as similar de novo AML cases from the literature and their response to SICT.Methods:We have analyzed our cases examined for dysplastic bone marrow features treated with SICT or high‐dose cytarabine with daunorubicin cycle 10/2. A literature review of reported AML cases examined for dysplasia and treated with SICT was also analyzed.Results:Our single‐institution study on consecutive adult de novo AML cases &lt;65 years of age showed that those with GM‐AML reached complete remission (CR) in 78% after SICT but cases with EMD‐AML in 29% (p = 0.02). However, 7/10 EMD‐AML cases reached CR after the cycle with high‐dose cytarabine and daunorubicin 10/2 (Lemez et al., 2000). Coasguen et al. (Leukemia 1992;6:520) reported 74.4% CR after SICT in 156 cases without dysplastic features (GM‐AML), but 56% CR in 39 cases with TLD AMLs. Brito‐Babapulle et al. (Br J Haematol 1987;66:445), Estienne et al. (Clin Lab Haematol 1990;12:57), and Kuriyama et al. (Br J Haematol 1994;86:767) reported post SICT 86%, 79%, and 81% CR in 136, 118, and 190 selected non‐TLD cases, but CR 50%, 43%, and 63% in TLD AML cases, respectively. Patients’ selection of cases suitable for SICT with median age below 50 years probably increased the CR rates in non‐TLD and TLD AML categories. Papers with high or intermediate doses of cytarabine during induction were not included in this literature review. Our 6 of 12 consecutive cases with de novo AML over 80 years of age opted for SICT administration. Three cases with GM‐AML reached CR after one cycle of SICT and survived 16.5–28 months on maintenance therapy while none of the three octogenarians with EMD‐AML reached CR and all died within 0.4–2.7 months. A 79‐year old man with GM‐AML reached CR post 2 cycles of low‐dose cytarabine (LDAC) and survived on 53 repeated cycles LDAC 84 months. Another 67‐year old man with GM‐AML and near‐tetraploid karyotype reached CR post SICT and after two further same cycles he survived in CR 51 months and with further treatment altogether 80 months.Summary/Conclusion:Our results and the literature review showed that GM‐AML cases achieve CR after SICT in 74–85% of patients &lt;60/65 years of age. Five older patients &gt; 65 years including three &gt; 80 years of age reached CR after SICT in spite of poor performance status 3 in two and had survival 16.5–28 months on maintenance therapy. We estimate that de novo GM‐AML cases constitute about 20% of all cases 80+ years old with newly diagnosed AML. GM‐AML cases &gt; 65 years exhibit a good sensitivity to SICT. Further reports and studies are needed to enlarge the number of older GM‐AML cases treated with SICT.

Bone Marrow Morphology Associated With Germline RUNX1 Mutations in Patients With Familial Platelet Disorder With Associated Myeloid Malignancy.

Germline mutations in RUNX1 result in autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM). To characterize the hematopathologic features associated with a germline RUNX1 mutation, we reviewed a total of 42 bone marrow aspirates from 14 FPDMM patients, including 24 cases with no cytogenetic clonal abnormalities, and 18 with clonal karyotypes or leukemia. We found that all aspirate smears had ≥10% atypical megakaryocytes, predominantly characterized by small forms with hypolobated and eccentric nuclei, and forms with high nuclear-to-cytoplasmic ratios. Core biopsies showed variable cellularity and variable numbers of megakaryocytes with similar features to those in the aspirates. Granulocytic and/or erythroid dysplasia (≥10% cells per lineage) were present infrequently. Megakaryocytes with separate nuclear lobes were increased in patients with myelodysplastic syndrome (MDS) and acute leukemia. Comparison to an immune thrombocytopenic purpura cohort confirms increased megakaryocytes with hypolobated eccentric nuclei in FPDMM patients. As such, patients with FPDMM often have atypical megakaryocytes with small hypolobated and eccentric nuclei even in the absence of clonal cytogenetic abnormalities; these findings are related to the underlying RUNX1 germline mutation and not diagnostic of MDS. Isolated megakaryocytic dysplasia in patients with unexplained thrombocytopenia should raise the possibility of an underlying germline RUNX1 mutation.

Study of bone marrow abnormalities in patients with HIV infection-a prospective study in tertiary care center of Nalgonda district

Context: Bone marrow abnormalities reported in various stages of HIV infection have not attracted much attention; this is partly because the clinical picture of HIV infection is still dominated by opportunistic infections and malignancies. Aims: 1. To study the bone marrow abnormalities in patients with HIV / AIDS and to find their association with peripheral hematological abnormalities. 2. To draw conclusions and compare with previous studies.Settings and Design: The present Prospective study was conducted at the Department of pathology, Kamineni Institute of Medical Sciences, Narketpalli over a period of 2 years starting from January 2017 to Dec 2018. Methods and Material: The study population included 54 newly detected HIV positive symptomatic or asymptomatic patients. Patients were classified into two clinical groups according to NACO criteria 1) AIDS and 2) Non-AIDS. Bone marrow analysis was done for HIV patients who presented with anemia, leucopenia, Thrombocytopenia and Bi-cytopenia. Results: Patients were classified into two clinical groups: AIDS (55.5%) and NON-AIDS (44.5%). Most common age group involved was 21-40 years and Male to Female ratio is 4:1 in AIDS Group and 1.6:1 in Non-AIDS group. Most common haematological abnormality was Anaemia present in 73.3% of AIDS group and 79.1% of Non-AIDS group. Bone marrow was predominantly normocellular (AIDS: 50%, Non-AIDS: 62.5%) with normoblastic maturation. Myelodysplasia was predominantly seen in Granulocytic series (AIDS: 26.6%, Non-AIDS: 12.5%). Erythroid dysplasia was reported in 23.3% of cases of AIDS group and 8.3% in Non-AIDS group. Megakaryocytic dysplasia was seen in 2 cases of AIDS group. Myelodysplasia was common in patients with CD4 counts < 200/µL. Patients with myelodysplasia presented with anaemia (52.9%), Leukopenia (41.1%) and Thrombocytopenia (11.7%).Conclusions: Bone marrow examination is usually performed in HIV patients to evaluate peripheral cytopenia. Myelodysplasia commonly observed in HIV patients predominantly involves Granulocytic series. Erythroid dysplasia is prominent in AIDS Patients. Myelodysplasia associated with anemia carries poor prognosis and marks progression to advanced disease. Bone marrow abnormalities evolve and increase in frequency during progression of HIV disease and thorough schematic evaluation carries prognostic significance.

Genotype-Resultant Morphology of Myelodysplastic Syndromes (MDS)

Morphology has dominated the diagnosis and classification of MDS for decades. With the advent of NGS, morphology is used as a gold standard to assess associations of specific disease sub-entities with individual mutations or their combinations. An example is the association between SF3B1 mutations and ringed sideroblasts, or with disease-defining lesions, such as t(8:21), 11q23 translocations, and EVI1 mutations. Beyond these discrete examples, assignments of diverse morphologic features to combinations of specific genotypes has been more challenging.To identify potentially novel paradigms of associations between molecular hits and morphologies we examined 835 patients with MDS. Common somatic mutations, chromosomal abnormalities and morphologic reviews were subjected to blinded, objective standard diagnostic criteria. Abnormalities included individual lineage dysplasia and cytopenias, monocytosis, fibrosis, and increased megakaryocytes, all classified using standard criteria. To correlate the presence of mutations to morphologic criteria, only mutations with a clonal burden >10% were used. In total, 794 out of 835 patients (95%) harbored at least one lineage dysplastic feature. Myeloid, erythroid and megakaryocytic dysplasia occurred in 56% (470/835), 71% (590/835) and, 70% (587/835) of patients, respectively. Bi- and tri-lineage dysplasia were also identified in 36% (297/835) and 33% (278/835). In 90% (753/835) of patients there was cytopenia: 48% (402/835) had neutropenia, 61% (510/835) anemia, and 61% (510/835) thrombocytopenia. More than 50% of patients had multiple-cytopenias; 23% (194/835) had pancytopenia. Monocytosis was present in 20% (168/835) of patients and 31% (256/835) had elevated numbers of megakaryocytes. For analysis all possible combinations (25 pairs in total) were queried and those present in >80 patients were evaluated. Based on NGS among 36 genes, TET2 (27%), SF3B1 (23%), ASXL1 (19%), del(5q) (16%), SRSF2 (14%), DNMT3A (11%) and del(7q) (10%), were frequently mutated genes/CNAs, each present in >10% of patients. A number of relationships between phenotypic features and molecular context were uncovered. For example, patients with thrombocytopenia also commonly had tri-lineage dysplasia [38% (198/510) vs. 25%, (80/325), p<.001, q<.001] or more myeloid dysplasia [63% (321/510) vs. 46% (149/325), p<.0001, q<.0001] but no megakaryocyte dysplasia [72% (366/510) vs. 68% (221/325), p=.027]. Moreover lineage specific dysplasia was not associated with lineage specific cytopenias. In fact, myeloid or erythroid dysplasia had no significant association with neutropenia or anemia (p=.14 or p=.1, respectively).When we performed statistical analyses that sought correlations between clinical features and genetic alterations, 78 significant associations were identified. For instance, patients with erythroid dysplasia more frequently had SF3B1 mutations [in 15% of erythroid dysplasia patients, 90/590] than patients without it [6%, 15/245] (p=.0002, q=.001). In contrast, these patients were less likely to have EZH2 mutations [4.2% (25/590) vs. 11.4% (28/245), p<.001, q=.002]. Compared to patients with no myeloid dysplasia, patients with myeloid dysplasia were more likely to have TET2, STAG2, and SRSF2 mutations, and less likely to have SF3B1 mutations (p<.02). Compared to patients without megakaryocytic dysplasia, those with it more commonly had ASXL1, mutation, but less commonly SF3B1 and BCORL1 mutations. Patients with STAG2 mutations [8%, 66/835] were more likely to have neutropenia [62% (41/66) vs. 47% (361/769), p=.02, q=.08]. TP53 and ASXL1 mutations occurred frequently in patients with thrombocytopenia. Monocytosis co-existed more with 5 genetic mutations, including SRSF2 and RAS pathway genes.Using various conventional and more advanced strategies, we present the first comprehensive genotype-phenotype association study that linked somatic molecular events to morphologic and clinical features. We demonstrate that the complexities of how molecular context governs morphologic changes can be deciphered. Certain rigorously defined morphologic configurations show specific associations with individual mutations or their combinations. DisclosuresNazha:MEI: Consultancy. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.

Molecular Features in the Diagnosis of Myeloid Neoplasms in Down Syndrome Patients Less Than Four Years of Age: Experience from a Single Institution

Introduction:Patients with Down syndrome (DS) have an increased risk of hematological disorders, including transient abnormal myelopoiesis (TAM), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Twenty percent of patients with TAM subsequently develop myeloid neoplasm in the first 4 years of life. MDS represents a clonal aberration thought to be a pre-leukemic condition characterized clinically by cytopenias and erythroid, myeloid and/or megakaryocytic dysplasia in the bone marrow with or without increase in blasts and harbors a concordant, clone-specific mutation of GATA1. WHO 2016 classification of hematopoietic neoplasms does not distinguish between MDS and AML, as their overall prognosis appears to be similar. However, due to the rarity of this disorder, limited clinical and laboratory data is available, contributing to difficulties in establishing the diagnosis. Here we describe our center's recent experience with the diagnosis and molecular findings of myeloid neoplasm associated with Down syndrome (MN-DS).Design/Method:Retrospective review of the patient's electronic medical record and review of the literature was conducted. Routine karyotype, fluorescent in-situ hybridization (FISH) and next generation sequencing (NGS) studies were reviewed where available.Results:Six patients with DS diagnosed with AML or MDS were identified over a 3-year period. Mean age of the cohort was 18.5 (range 12-24) months with a slight female predominance. Three patients had a history of TAM, all of which resolved without intervention. Three patients had asymptomatic thrombocytopenia after birth without blasts or GATA1 mutation confirmation. One of the three patients with a history of TAM presented with overt AML, while in the others diagnosis was challenging. By WHO 2008 classification of myeloid neoplasms, four patients had refractory anemia with excess blasts, one had refractory cytopenia with multilineage dysplasia, and one had AML. For two patients, in whom myeloid directed next generation sequencing was obtained, mutations were found in GATA1, EZH2, and NRAS. One of the patients in our series presented with AML with gain of MECOM, RPN1 loss and D5S23 deletion by FISH and succumbed to relapsed disease. All patients were treated per Children's Oncology Group AAML1531 arm A protocol that included 3 induction cycles and 2 intensification cycles, except for a single patient that received one cycle per AAML0431 and completed therapy per AAML1531 arm B high risk due to persistent disease following initial induction cycle. Two patients are currently receiving treatment, three have no evidence of disease recurrence on follow up ranging from 2 to 18 months, and one of the patients has died due to relapsed/refractory disease.Conclusions:We present six cases of MN-DS in patients less than four years of age. Our cohort is representative of the diversity encountered in this rare disease including patients with 1) isolated cytopenia in the absence of overt morphological findings, 2) myelodysplasia, and 3) AML. In our patient with overt AML there were karyotypic features such as gain of MECOM, which with specific translocation partners has previously been described to portend a poor prognosis. This and other cytogenetic features perhaps warrant further investigation given our patient's refractory disease. In the patient with refractory cytopenia without blasts, there was a subpopulation of cells identified by NGS panel showing mutations in GATA1, EZH2, and NRAS that led to a diagnosis of MDS/MN-DS. Four of the patients had aberrant myeloid populations and dysplasia fitting diagnostic criteria for MDS. Establishing the clonal nature of the disease either by karyotype/FISH or NGS may help with the identification, treatment and prognostication of this unique patient population, and may aid in the diagnosis of MN-DS, which may be challenging in patients with DS once they have recovered from TAM. DisclosuresNo relevant conflicts of interest to declare.

Marked Thrombocytosis and Megakaryocytic Dysplasia in NPM1 Mutated De-Novo Acute Myeloid Leukemia.

Marked Thrombocytosis and Megakaryocytic Dysplasia in NPM1 Mutated De-Novo Acute Myeloid Leukemia.

Overexpression of Short Variant Form of New Kelch Family Protein Leads to Erythroid and Megakaryocyte Dysplasia by Targeting Megakaryocyte-Erythroid Progenitors.

Nd1-S is the nuclear-localizing short variant form of Nd1 (Ivns1abp) encoding a Kelch family transcription factor. While the function of Nd1 has been investigated in the context of metastasis and doxorubicin-induced cardiotoxicity, little is known about its role in hematopoiesis. In this study, we investigated the function of Nd1-S in hematopoiesis by transplanting the Nd1-S-overexpressing murine hematopoietic stem and progenitor cells (HSPCs) into recipient mice (Nd1-S mice). Enforced expression of Nd1-S led to erythroid and megakaryocyte dysplasia, demonstrated by dramatically decreased red blood cells and platelets, and megakaryocytes in the peripheral blood and bone marrow of the Nd1-S mice. Moreover, phenotypic megakaryocyte-erythroid progenitors (MEPs) accumulated in these Nd1-S mice with aberrant morphology and defective colony-forming capability. Furthermore, these phenotypic MEPs showed impaired pathways regulating erythroid differentiation and megakaryocyte development. Therefore, our study provides de novo evidence that overexpression of Nd1-S in HSPCs leads to erythroid and megakaryocyte dysplasia in vivo by targeting MEPs.

An uncommon t(9;11)(p24;q22) with monoallelic loss of ATM and KMT2A genes in a child with myelodysplastic syndrome/acute myeloid leukemia who evolved from Fanconi anemia

BackgroundMyelodysplastic syndrome (MDS) is rare in the pediatric age group and it may be associated with inheritable bone marrow failure (BMF) such as Fanconi anemia (FA). FA is a rare multi-system genetic disorder, characterized by congenital malformations and progressive BMF. Patients with FA usually present chromosomal aberrations when evolving to MDS or acute myeloid leukemia (AML). Thus, the cytogenetic studies in the bone marrow (BM) of these patients have an important role in the therapeutic decision, mainly in the indication for hematopoietic stem cell transplantation (HSCT). The most frequent chromosomal alterations in the BM of FA patients are gains of the chromosomal regions 1q and 3q, and partial or complete loss of chromosome 7. However, the significance and the predictive value of such clonal alterations, with respect to malignant progress, are not fully understood and data from molecular cytogenetic studies are very limited.Case presentationA five-year-old boy presented recurrent infections and persistent anemia. The BM biopsy revealed hypocellularity. G-banding was performed on BM cells and showed a normal karyotype. The physical examination showed to be characteristic of FA, being the diagnosis confirmed by DEB test. Five years later, even with supportive treatment, the patient presented severe hypocellularity and BM evolution revealing megakaryocyte dysplasia, intense dyserythropoiesis, and 11% myeloblasts. G-banded analysis showed an abnormal karyotype involving a der(9)t(9;11)(p24;q?22). The FISH analysis showed the monoallelic loss of ATM and KMT2A genes. At this moment the diagnosis was MDS, refractory anemia with excess of blasts (RAEB). Allogeneic HSCT was indicated early in the diagnosis, but no donor was found. Decitabine treatment was initiated and well tolerated, although progression to AML occurred 3 months later. Chemotherapy induction was initiated, but there was no response. The patient died due to disease progression and infection complications.ConclusionsMolecular cytogenetic analysis showed a yet unreported der(9)t(9;11)(p24;q?22),der(11)t(9;11)(p24;q?22) during the evolution from FA to MDS/AML. The FISH technique was important allowing the identification at the molecular level of the monoallelic deletion involving the KMT2A and ATM genes. Our results suggest that this chromosomal alteration conferred a poor prognosis, being associated with a rapid leukemic transformation and a poor treatment response.

Association of mutations with morphological dysplasia in de novo acute myeloid leukemia without 2016 WHO Classification-defined cytogenetic abnormalities.

Despite improvements in our understanding of the molecular basis of acute myeloid leukemia (AML), the association between genetic mutations with morphological dysplasia remains unclear. In this study, we evaluated and scored dysplasia in bone marrow (BM) specimens from 168 patients with de novo AML; none of these patients had cytogenetic abnormalities according to the 2016 World Health Organization Classification. We then performed targeted sequencing of diagnostic BM aspirates for recurrent mutations associated with myeloid malignancies. We found that cohesin pathway mutations [q (FDR-adjusted P)=0.046] were associated with a higher degree of megakaryocytic dysplasia and STAG2 mutations were marginally associated with greater myeloid lineage dysplasia (q=0.052). Frequent megakaryocytes with separated nuclear lobes were more commonly seen among cases with cohesin pathway mutations (q=0.010) and specifically in those with STAG2 mutations (q=0.010), as well as NPM1 mutations (q=0.022 when considering the presence of any vs. no megakaryocytes with separated nuclear lobes). RAS pathway mutations (q=0.006) and FLT3-ITD (q=0.006) were significantly more frequent in cases without evaluable erythroid cells. In univariate analysis of the 153 patients treated with induction chemotherapy, NPM1 mutations were associated with longer event-free survival (EFS) (P=0.042), while RUNX1 (P=0.042), NF1 (P=0.040), frequent micromegakaryocytes (P=0.018) and presence of a subclone (P=0.002) were associated with shorter EFS. In multivariable modeling, NPM1 was associated with longer EFS, while presence of a subclone and frequent micromegakaryocytes remained significantly associated with shorter EFS.

Deficiency of Cux1, Encoded on Human Chromosome 7q, Causes Aberrant Hematopoietic Stem Cell Function and Spontaneous Myeloproliferative Disease in Mice

Loss of all or part of chromosome 7 [-7/del(7q)] is a common, high-risk cytogenetic abnormality identified in a variety myeloid malignancies, including myelodysplastic syndrome (MDS), acute myeloid leukemia, therapy-related myeloid neoplasms, juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML). CUX1 is a non-clustered, homeodomain-containing transcription factor encoded on the commonly deleted region of 7q22. CUX1 inactivating mutations occur across solid tumors and myeloid neoplasms, and carry a poor prognosis in myeloid malignancies independently of 7q status. Previously, we reported that CUX1 is a conserved, haploinsufficient, myeloid tumor suppressor. To determine the mechanism by which CUX1 regulates leukemogenesis, we generated an inducible, shRNA-transgenic, Cux1 -knockdown mouse model. Insufficient Cux1 levels caused early mortality in mice, due to a spontaneous myeloproliferative disorder with features of CMML/JMML and MDS. Cux1 deficient mice had a significant increase in myelomonocytic cell numbers in the peripheral blood, bone marrow, and spleen. Cux1 deficiency led to extramedullary hematopoiesis, splenomegaly, and an increase in megakaryocyte numbers and megakaryocyte dysplasia. In addition, Cux1 is required for normal red cell development, as Cux1 knockdown led to a normocytic anemia due to an erythroblast maturation arrest. To identify CUX1 transcriptional targets, we performed gene expression profiling after CUX1 knockdown in CD34+ human hematopoietic stem and progenitor cells (HSPCs). Differentially expressed genes were enriched for genes deregulated in JMML and -7/del(7q) MDS, in addition to altered expression of quiescent, proliferative, and myeloid differentiation pathways. Consistent with these gene signatures, Cux1 maintains hematopoietic stem cell quiescence and represses HSPC proliferation, self-renewal, and granulocyte/monocyte differentiation. Thus Cux1 has pleotropic roles in multiple myeloid lineages. Deletion of CUX1, in the context of -7/del(7q), may facilitate myeloid transformation through aberrant transcriptional regulation of HSPC differentiation and proliferation. DisclosuresNo relevant conflicts of interest to declare.

Impending relapse of myelodysplastic syndrome after allogeneic transplant is difficult to diagnose and requires a multi-modal approach

BackgroundThe only potentially curative therapy for myelodysplastic syndrome is allogeneic hematopoietic cell transplant; unfortunately, there is a high relapse rate. The objective of this study was to perform a detailed clinicopathologic study of patients with relapsed myeloid neoplasm following allogeneic hematopoietic cell transplant for myelodysplastic syndrome.MethodsPre-transplant, post-transplant, and relapse bone marrow and peripheral blood morphologic features (including dysplasia) were retrospectively evaluated by study authors. Clinical features and results of cytogenetic analysis and engraftment/chimerism studies were obtained from the medical record.ResultsOur study describes 21 patients with a median time to relapse of 6 months (range 2–82). Ten of the patients relapsed with higher grade disease, including six with overt acute myeloid leukemia. Pre-transplant megakaryocyte dysplasia was associated with dysplastic megakaryocytes in the relapse specimen; however, neither erythroid dysplasia nor granulocytic dysplasia were associated with their counterpart in the relapse specimen. Relapse specimens had a lower marrow cellularity and higher blast percentage than pre-transplant disease. Cytogenetic comparisons before and after transplant showed variety, including clonal evolution (22%), the same abnormal clone (33%), or a different abnormal clone (22%).ConclusionsOur detailed review of post-transplant marrow biopsies prior to relapse highlights the difficulty in diagnosing relapse and particularly impending relapse.