Myeloid-associated Genes Research Articles

Modelling acquired resistance to DOT1L inhibition exhibits the adaptive potential of KMT2A-rearranged acute lymphoblastic leukemia

In KMT2A-rearranged acute lymphoblastic leukemia (ALL), an aggressive malignancy, oncogenic KMT2A-fusion proteins inappropriately recruit DOT1L to promote leukemogenesis, highlighting DOT1L as an attractive therapeutic target. Unfortunately, treatment with the first-in-class DOT1L inhibitor pinometostat eventually leads to non-responsiveness. To understand this we established acquired pinometostat resistance in pediatric KMT2A::AFF1+ B-ALL cells. Interestingly, these cells became mostly independent of DOT1L-mediated H3K79 methylation, but still relied on the physical presence of DOT1L, HOXA9 and the KMT2A::AFF1 fusion. Moreover, these cells selectively lost the epigenetic regulation and expression of various KMT2A-fusion target genes such as PROM1/CD133, while other KMT2A::AFF1 target genes, including HOXA9 and CDK6 remained unaffected. Concomitantly, these pinometostat-resistant cells showed upregulation of several myeloid-associated genes, including CD33 and LILRB4/CD85k. Taken together, this model comprehensively shows the adaptive potential of KMT2A-rearranged ALL cells upon losing dependency on one of its main oncogenic properties.

Uncovering transcriptomic landscape alterations of CAN-2409 in in vitro and in vivo glioma models.

CAN-2409 is a locally delivered oncolytic therapy, which results in vaccination against the injected tumor. CAN-2409 consists of a non-replicating adenovirus armed with the Herpes virus thymidine kinase, which metabolizes ganciclovir into a phosphorylated nucleotide that is incorporated into the tumor cell's genome, thereby inflicting immunogenic cancer cell death. While CAN-2409's immunological impact has been well characterized, its effects on the tumor cells transcriptome remains unknown. We compared the transcriptomic landscape after treatment of glioblastoma models with CAN-2409 in vitro and in vivo to assess how the interplay with the tumor microenvironment influences CAN-2409-mediated transcriptome alterations. We performed RNA-Seq with CAN-2409 treated patient-derived glioma stem-like cells and tumors of C57/BL6 mice and compared KEGG pathway usage and differential gene expression focusing on immune cell and cytokine profiles. T-cell -killing assays were performed to assess candidate effectors. PCA analysis showed distinct clustering of control and CAN-2409 samples under both conditions. KEGG pathway analysis revealed significant enrichment for p53 signaling and cell cycle pathway, with similar dynamics for key regulators of both pathways in vitro and in vivo, including MYC, CCNB1, PLK1 and CDC20. Selected alterations (PLK1 and CCNB1) were validated at the protein level. Cytokine expression analysis revealed upregulation of pro-inflammatory IL12a under both conditions; immune cell gene profiling showed reduction of myeloid associated genes. T-cell-killing assays showed increased killing in the presence of IL-12. CAN-2409 significantly alters the transcriptome both in vitro and in vivo. Comparison of pathway enrichment revealed mutual and differential utilization of pathways under both conditions, suggesting a modulating influence on the cell cycle in tumor cells, and of the tumor microenvironment on the transcriptome in vivo. IL-12 synthesis likely depends on interactions with the tumor microenvironment, and it facilitates CAN-2409 cell killing. This dataset provides potential to understand resistance mechanisms and identify potential biomarkers for future studies.

P985: THE ALLELIC RATIO OF DRIVER AND ASXL1 MUTATIONS IS PROGNOSTICALLY RELEVANT IN PMF

Background: Primary myelofibrosis (PMF) is a clonal disorder driven by mutations in JAK2, CALR, and MPL. Somatic mutations in myeloid-associated genes were shown to impact prognosis of PMF patients (pts). Among these, ASXL1 mutations (ASXL1mt) are by far the most frequent and prognostically relevant, in fact they are included in the category of “high molecular risk” (HMR), along with EZH2, IDH1/2, SRSF2, and U2AF1Q157 mutations. Aims: To investigate the phenotypic and prognostic implications of ASXL1mt and variant allele frequency (VAF) in PMF. Methods: After IRB approval, consecutive pts with WHO-defined PMF were included in the study. Mutational analysis by targeted NGS was performed by previously described methods (Guglielmelli P et al., JCO 2017). Results: The study enrolled a total of 384 pts, including 190 (49%) prefibrotic and 194 (51%) overt PMF. Median age was 60 (18-90) years, 236 (61%) were male. JAK2, CALR and MPL mutations were found in 255 (66%), 83 (22%), and 17 (4%) pts, respectively; 36 (9%) were triple negative (TN). Among HMR mutations, ASXL1 was mutated in 88 (23%) pts, SRSF2 in 35 (9%), EZH2 in 32 (8%), U2AF1 in 15 (5%), and IDH1/2 in 11 (3%). VAF distribution of driver and HMR mutations was as reported in Fig.1A. We did not find any significant correlation of ASXL1mt VAF with other clinical and molecular characteristics, including PMF subtype, gender, age, hemoglobin, leukocyte, platelet and blast counts, splenomegaly, BM fibrosis grade, cytogenetics, mutational status for driver and other HMR genes and their VAFs. The only exception was the association of CALR-mutated PMF with lower ASXL1 VAF (median 28% vs 40%, p=.0332). Median overall survival (OS) was 120 (102-151) months. In univariate Cox analysis, ASXL1mt VAF did not correlate with OS, nor did other single driver and HMR mutations. When considered as a whole (for the purpose of this study, TN pts were considered has having driver VAF equal to 0), the VAF of driver mutations inversely correlated with survival (HR 0.4 [0.2-0.9], p=.0219). We next investigated the allelic ratio between driver and ASXL1 mutations (d/Aratio) and its prognostic correlates. Median (range) of d/Aratio was 1.24 (0-13), and ROC analysis with death as an endpoint identified 1.31 as the optimal cut-off value. In univariate analysis, pts with a d/Aratio <1.31 had a significantly worse OS compared to those with a ratio ≥1.31 (median 45 vs 104 months, p=.0014; HR 2.4 [1.4-4.1]) (Fig.1B). When TN pts with ASXL1mt were considered apart, having a d/Aratio <1.31 retained its inferior prognostic impact compared to the d/Aratio ≥1.31 group (median OS 57 vs 104 months, p<0.0453; HR 1.8 [1-3.2]), with TN/ASXL1mt pts being associated with the worst outcome (median OS 24 months) (Fig.1C). In a multivariate Cox model including VAF-adjusted ASXL1 mutant status (ASXL1wtvs ASXL1mt with d/Aratio ≥1.3 vs ASXL1mt with d/Aratio <1.3) and other HMR mutations, the former and mutated SRSF2 were confirmed to be independent predictors of inferior OS. Both d/Aratio <1.31 and ≥1.31 remained significant compared to ASXL1wt with respective HRs of 3.4 (2.3-5.1; p<.0001) and 1.6 (1-2.6; p=.0342). Notably, d/Aratio <1.31 still retained its inferior prognostic impact compared to d/Aratio ≥1.31 (HR 2 [1.2-3.5]; p=.0095). Image:Summary/Conclusion: This study explores the implications of ASXL1mt VAF and its interplay with driver mutant burden. The adverse prognosis of pts with a d/Aratio <1.31 suggests that this disease entity is predominantly driven by ASXL1mt-clones characterized by a more aggressive biology. Further research is needed.

P1649: GFI1B AND LSD1 REPRESS A MYELOID DIFFERENTIATION PROGRAM DURING INDUCED PLURIPOTENT STEM CELL DERIVED MEGAKARYOCYTE DIFFERENTIATION

Background: Megakaryocytic development is driven by the transcription factor Growth Factor Independence 1B (GFI1B) and its interaction with the chromatin remodeler Lysine Specific Demethylase 1 (LSD1/KDM1A). GFI1B aberrations are linked to inherited bleeding disorders, resulting in macro-thrombocytopenia, gray platelets and increased expression of CD34. One such mutation, which was previously show to be the disease-causing mutation in a family with gray-platelet syndrome, introduces a premature stop codon (Q287*) in the fifth zinc finger. Aims: Here, we study the effect of the Q287* mutation on gene expression regulation, as well as GFI1B interaction with LSD1, during induced pluripotent stem cells (IPSC) derived megakaryocytic development. Methods: We differentiated IPSCs that contain wildtype GFI1B or GFI1BQ287* into megakaryocytes. During differentiation, megakaryocytes derived from GFI1B wildtype IPSCs were also treated with an LSD1 inhibitor that disrupts GFI1B-LSD1 interaction for 24 hours. The transcriptomic landscape of differentiated IPSCs was explored using single-cell RNA sequencing and downstream computational analysis. We applied a novel filtering strategy, removing cells based on doublet annotation, mitochondrial gene count and exclusive differential expression of ribosomal or mitochondrial genes. Results: After filtering and data integration we retained 14,848 cells. Within this population we were able to annotate six different cell types using single-cell GSEA and publicly available reference datasets. Megakaryocytes and megakaryocyte progenitors made up roughly 75% of all cells differentiated from IPSCs containing wildtype GFI1B. They are characterized by approximately 220 differentially expressed genes (p-value < 0.05, log-fold change > 0.69), including common megakaryocyte markers such as CD41 and CD42b. Strikingly, we also observed a population of myeloid and myeloid-progenitor cells after differentiation towards megakaryocytes. This population specifically expressed common myeloid markers, such as VIM, CD81, and SPI1 as well as approximately 200 other genes (p-value < 0.05, log-fold change > 0.69) and made up around 10% of the entire GFI1B wildtype cell population. This population was expanded two-fold in cells differentiated from GFI1B-wildtype IPSCs treated with the LSD1 inhibitor and three-fold in GFI1BQ287* IPSCs. Notably, in the GFI1BQ287* cell population megakaryocytes were reduced by 2-fold. Pseudotime analysis showed a differentiation trajectory starting at megakaryocytes and going towards myeloid cells. Megakaryocyte and platelet associated genes, such as CD41, CD42b and PF4, are gradually downregulated along this trajectory while myeloid associated genes exhibited a mirrored expression pattern. Thus, LSD1i treated and GFI1BQ287* IPSC-derived cells showed an increase in myeloid marker gene expression after differentiation and downregulation of coagulation-associated genes, hinting at a decreased potential to differentiate into megakaryocytes. Summary/Conclusion: Our data show that GFI1B and LSD1 function to repress a myeloid cell fate during IPSC-induced megakaryocytic development. Disruption of the interaction between GFI1B and LSD1 and a dominant-negative GFI1B mutation results in myeloid differentiation at the expense of megakaryocyte development.

518 Bavituximab Effectively Targets Suppressive Myeloid Cells in Patients with Glioblastoma

INTRODUCTION: Glioblastoma and tumor endothelial cells express phosphatidylserine, an immunosuppressive membrane phospholipid. Bavituximab – a chimeric monoclonal antibody – competitively binds to the β2-glycoprotein 1-phosphatidylserine complex, resulting in anti-tumor immune activation and anti-angiogenesis. Bavituximab may act in the tumor immune microenvironment (TIME) through myeloid cells. METHODS: 33 adults with newly diagnosed glioblastoma were enrolled in this phase II trial (NCT03139916). Bavituximab was given weekly, starting week 1 of Stupp. The primary objective was % overall survival at 12 months. We collected peripheral blood mononuclear cells (PBMCs) at enrollment and at regular intervals thereafter. Tumor tissue was banked and analyzed retrospectively. The nCounter Myeloid Innate Immunity Panel, which includes 770 myeloid-associated genes, was used to profile the myeloid transcriptome in both PBMCs and tumor tissue. Multispectral immunofluorescence was used to characterize the intratumoral immune environment. RESULTS: In the TIME, bavituximab-treated patients saw a reduction in the number of MDSCs. While the pretreatment RNA profile in PBMCs was not associated with outcome, above-median progression-free and overall survival were associated with significantly enriched expression of myeloid-associated genes in pretreatment tumor tissue. CONCLUSION: Bavituximab reduces MDSCs in the glioblastoma microenvironment. Elevated tumor expression of myeloid-associated genes may be a predictive biomarker for response to bavituximab.

Analysis of Single Cell Transcriptomics in Paired Pediatric T-ALL Samples Collected at Diagnosis and Following End of Induction Therapy Reveals an MRD-Associated Stem Cell Signature

IntroductionDespite recent improvement in outcomes for de novo disease, pediatric T-cell acute lymphoblastic leukemia (T-ALL) remains challenging to treat at relapse. Investigation into genomic markers of treatment response and therapy resistance offers an opportunity to further enhance outcomes for these patients. We previously identified a T-ALL blast-associated gene signature at diagnosis (Dx) and characterized the immune microenvironment in Dx T-ALL marrow samples using single cell transcriptome analysis (Bhasin et al. Blood 2020(ASH)). This approach allowed us to generate a granular expression map of both the T-ALL landscape and the Dx bone marrow (BM) immune microenvironment. Here we expand this work by evaluating samples collected from the same patients Dx and End of Induction (EOI) BM samples from pediatric T-ALL patients. The use of paired samples provides insight into treatment-induced changes in the microenvironment. Further, the inclusion of both minimal residual disease (MRD) positive and MRD negative samples allowed us to compare differences between these groups.MethodsUsing the 10X genomics platform, we profiled the single cell transcriptome of ~18,000 BM and immune microenvironment cells from viably frozen samples collected from T-ALL patients at Dx or EOI. Five paired Dx and EOI samples and one EOI sample from a patient with relapsed T-ALL were evaluated, for a total of 11 samples. Three paired samples were MRD positive at EOI and two were MRD negative; the relapsed sample was MRD negative. Cell clustering was performed using the Seurat package and differential expression analysis was performed using R/Bioconductor packages (Hao et al. Cell 2021). Cell communication analysis was conducted using the CellChat R tool (v 1.0.0) to infer cell-cell communication within the EOI MRD positive and MRD negative subsets and compare their communication networks (Jin et al. Nature Comm 2021).ResultsUsing our previously described blast-associated gene signature (Bhasin et al. ASH 2020) we were able to identify residual blast populations at EOI in MRD-positive samples. Comparative analysis of gene profiles at Dx and EOI showed significant changes in the microenvironment cell populations with highest increase in erythroid cell populations after induction therapy. The gene expression profiles were significantly different for immune cells at Dx and EOI and the relapsed sample had greater similarity to the Dx samples indicating a persistent immunosuppressive environment. Clustering analysis of the EOI samples (3 MRD positive and 2 MRD negative) demonstrated the presence of patient specific blast cells in MRD positive samples that retained patient-specific transcriptomeheterogeneity at EOI (Fig.1A). Analysis of communication networks between different cell types based on receptor and ligand expression levels between different cell types identified a CD34 + cluster of stem cells that had different interactions with other immune populations in the MRD positive and negative subsets. Differential expression analysis between the MRD positive and MRD negative cells in this CD34 + stem cell cluster identified higher expression of myeloid associated genes such as CEBPB, CEBPD, AZU1 in the MRD negative group relative to the MRD positive cells, which showed higher expression of B-cell related genes such as IGHM, VPREB1, CD79A/ B along with upregulation of P13K signaling in B-lymphocytes, B-cell receptor signaling and autophagy pathways. Analysis of upstream regulators based on the differential gene signature between the MRD positive and MRD negative group demonstrated upregulation of MYC and TCF3 activity and inhibition of TGFB1, CSF3 and CEBPA in MRD positive compared to MRD negative samples (Fig.1B).Conclusions: Leukemic blasts exhibit patient-specific gene expression signatures that are present at EOI in MRD positive samples. Exploration of the impact of minimal residual disease at EOI revealed differential gene expression patterns in stem cells from MRD positive samples, characterized by activation of B cell related signaling pathways and regulators such as MYC and TCF3. In contrast, a more myeloid-like expression signature was observed in stem cells from MRD negative samples. These findings open the avenues for exploration of therapeutic targets of T-ALL progression. [Display omitted] DisclosuresDeRyckere: Meryx: Other: Equity ownership. Graham: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

Clonal Dynamics of JAK2V617F and Non-Driver Mutations in Polycythemia Vera and Essential Thrombocythemia Patients Receiving Hydroxyurea Therapy

▪Introduction:Hydroxyurea (HU) is the most widely used cytoreductive treatment for patients with essential thrombocythemia (ET) and polycythemia vera (PV) at high risk of thrombosis. It remains unknown whether long-term HU therapy modulates or promotes the acquisition of mutations in non-driver (ND) genes, especially, when assessing hematological (HR) and molecular (MR) response. The objective of the study was to analyze the clonal dynamics of ND genes in HR and MR with HU in a cohort of JAK2V617F-mutated PV and ET patients.Methods:The study included 144 JAK2V617F positive patients (PV n = 73, TE n = 71) receiving HU as first-line cytoreductive treatment. The baseline sample (before HU treatment) and at the timepoint of best molecular response to JAK2V617F were analyzed. The allelic burden of J AK2V617F was assessed by allele-specific PCR and the mutational profile of ND genes was analyzed by next generation sequencing with a custom panel including 27 myeloid-associated genes. HR was defined according to the criteria of the European LeukemiaNet 2009 and MR of JAK2V617F was defined as complete, major, partial and no response (Table I).Results:Median molecular follow-up was 54.1 months for PV and 55.5 months for ET. Patients with PV were more likely to be males (p<0.001), and displayed higher leukocyte count (p<0.001) compared to those with ET. The respective numbers of deaths, leukemic transformations and fibrotic progressions were: 22 (30%), 4 (5%), 6 (8%) for PV cases, and 19 (27%), 1 (1%), 0 (0%) for ET patients.At baseline, a total of 62 somatic mutations in ND genes were detected in 42/73 (57%) PV patients while 58 were detected in 36/71 (51%) ET patients.Complete HR was observed in 102 patients: 44 (60%) PV and 58 (81%) ET. Partial MR in 67 cases: 35 (48%) PV and 32 (45%) ET and major or complete MR in 21 cases: 8 (11%) PV and 13 (18%) ET. The median duration of HU treatment was 45.8 months (range: 17.5-189.5) for PV and 45.6 months (range: 14.6-168.6) for ET.The most frequently mutated genes detected at pre-therapy samples were TET2 (34%), ASXL1 (12%), SF3B1 (7%) and EZH2 (5%) in PV patients, and TET2 (34%), ASXL1 (13%), DNMT3A (13 %) and SRSF2 (5%) in ET patients. No significant differences were observed in the MR (p=0.358) or HR (p=0.917) according to the presence or absence of mutations in ND genes at baseline.Clonal dynamics of DNMT3A, ASXL1, and TET2 (DAT) genes were not modulated by HU therapy to the same extent as JAK2V617F. Disappearance and emergence of additional mutations in DAT genes were observed independently of the molecular response achieved by the JAK2V617F clone. These findings suggest the existence of clones with mutations in ND genes independent from the pathogenic driver clone, and the lack of modulation by HU treatment.Finally, an increase of allelic burden or the appearance of mutations in TP53, a gene related to progression, and in other DNA repair genes (PPM1D and CHEK2) was observed in 14 (19.1%) PV patients and 9 (12.6%) ET cases during HU treatment. However, no increased risk of myelofibrotic transformation or progression to acute myeloid leukemia was observed in these patients.Conclusions: 1.Pre-treatment ND mutations are not associated with HR and MR to HU in JAK2V617F-mutated patients. 2. The clonal dynamics of ND mutations (decrease, increase, appearance, disappearance) are not related to the evolutionary dynamics of JAK2V617F. 3. An increase or appearance of progression-related mutations in TP53 and/or other genes of the DNA repair pathway such as CHEK2 and PPM1D is observed during HU treatment.Acknowledgments: Instituto de Salud Carlos III-FEDER, PI16/0153, PI19/0005, 2017SGR205, PT20/00023 and XBTC. [Display omitted] DisclosuresSalar: Janssen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Gilead: Research Funding; Celgene: Consultancy, Speakers Bureau. Besses: Gilead: Research Funding. Bellosillo: Thermofisher Scientific: Consultancy, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau; Roche: Research Funding, Speakers Bureau.

Mutation Landscape and Prognostic Correlates of ASXL1 Variants in Primary and Secondary Myelofibrosis

▪Introduction: Myelofibrosis (MF), whether primary (PMF) or secondary (SMF) to polycythemia vera or essential thrombocytemia, is characterized by a complex and partially undeciphered molecular architecture. Besides mutations in driver genes (JAK2, CALR, MPL), somatic mutations in selected myeloid-associated genes have been shown to impact prognosis of MF patients (pts). Among these, ASXL1 mutations (ASXL1MTs) are associated with poor outcomes in myeloid malignancies including PMF, where they are included in the category of “high molecular risk” (HMR) mutations along with EZH2MTs, IDH1/2MTs, and SRSF2MTs (Vannucchi AM, Leukemia 2013). However, a recent study (Luque Paz D, Blood Adv 2021) questioned the value of ASXL1MTs in MF. The current study aimed at further characterizing the prognostic role of ASXL1MTs in MF.Methods: After IRB approval, pts with WHO-defined MF were included in the study. Mutational analysis by targeted NGS was performed as previously described (Guglielmelli P, JCO 2017). All deposited variants were manually curated to assess pathogenicity. In this study, we also used the molecular model proposed by Luque Paz et al. that identifies 4 genetic groups: TP53MT; High-risk (≥1 mutation in EZH2, CBL, U2AF1, SRSF2, IDH1/2); ASXL1MT-only; and “Others”.Results: A total of 525 pts were included in the study, including 331 (63%) PMF and 194 (37%) SMF. Median age at diagnosis was 89 (18-90) years, 314 (60%) were male. The median follow-up time was 80 (98% CI, 68-90) months. Overall, 324 (62%) pts were JAK2MT, 126 (24%) CALRMT, 24 (5%) MPLMT, 40 (8%) triple negative (TN), and 11 (2%) double mutated. Among non-driver genes, ASXL1MTs were found in 158 (30%) pts, EZH2MTs in 45 (9%), SRSF2MTs in 37 (7%), NRASMTs in 30 (6%) U2AF1MTs in 27 (5%), TP53MTs and CBLMTs in 25 (5%) each, IDH1/2MTs in 18 (3%), and KRASMTs in 15 (3%). Pts in the HMR category were 125 (38%) in PMF and 63 (32%) in SMF.According to the above model, distribution of pts was as follows: TP53MT n=25 (5%), High-risk n=137 (26%), ASXL1MT-only n=64 (12%), and Others n=299 (57%). Pts in the TP53MT and ASXL1MT-only groups were more likely to be diagnosed with SMF compared to pts in the High-risk and Others groups (44% and 48% vs 28% and 38%, respectively). In addition, the High-risk group was enriched in TN pts (16%), while CALRMTs were more common in the ASXL1MT-only and Others compared to the TP53MT and High-risk groups (25% and 27% vs 12% and 18%, respectively).In univariate analysis, the TP53MT and High-risk groups were associated with the worst overall survival (OS), with median values of 38 (14-110) and 55 (45-85) months (P=.0039), respectively (Fig 1A). Albeit remarkably better, the OS of pts in the ASXL1MT-only group was inferior compared to pts in the Others group (median 124 [91-156] vs 193 [142-NR] months; P=.0118) (Fig 1A).We then analyzed separately PMF and SMF cohorts. In the former, the TP53MT and High-risk groups remained associated with the worst OS (median 58 [20-126] vs 55 [36-85] months), although with no significant difference, likely due to the low frequency (4%) of TP53MTs mutations in PMF (Fig 1B). Concurrently, the negative prognostic impact of the ASXL1MT-only group was confirmed in comparison to the Others group (median 103 [78-NR] vs 320 [178-NR] months; P=.0170). In pts with SMF, while the TP53MT group (6%) had by far the worst OS (median 13 [6-NR] months), the OS of the ASXL1MT-only group (median 141 [56-171] months) was comparable to that of the Others group (median 131 [106-NR] months; P=.5188) and not different from the High-risk group (median 58 [45-174] months; P=.3606) (Fig 1C). In a further analysis including only pts in the High-risk group, ASXL1MTs were found in 62% and 63% of patients with PMF and SMF, respectively. In survival analysis, the presence of ASXL1MTs was associated with an increased risk of death only in PMF (median OS 47 [31-73] vs 102 [34-317] months; P=.0240), unlike in SMF (median OS 90 [47-174] vs 25 [16-338] months; P=.3296) (Fig 1D-E).Conclusion: In the current study, we critically re-addressed the prognostic impact of ASXL1MTs by applying a genetic model recently developed by Luque Paz et al. to our cohort of molecularly annotated, WHO-defined MF pts. Overall, our results confirm that ASXL1MTs -even in the absence of other co-occurring high-risk mutations- harbor a negative prognostic impact mainly in PMF. These findings also reinforce the idea that PMF and SMF represent two different biological entities. [Display omitted] DisclosuresVannucchi: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Non-Invasive Genetic Profiling and Monitoring in Myelodysplastic Syndromes

Introduction. Myelodysplastic syndromes (MDS) are clonal heterogeneous disorders in which molecular studies and cytogenetics are essential for diagnosis, classification and prognosis. Cell-free DNA (cfDNA) analysis has been reported as a reliable non-invasive approach for detecting molecular abnormalities in MDS. However, there is limited information about cytogenetic alterations and monitoring in cfDNA from MDS patients.Objective. To assess and monitor the molecular and cytogenetic abnormalities by next generation sequencing (NGS) using cfDNA from patients with MDS.Patients and methods. Bone marrow (BM) aspirates and peripheral blood (PB) samples were collected from 70 newly diagnosed or treatment-naïve patients with MDS (Table). PB samples from 21 healthy controls were also studied. Molecular characterization was performed in paired samples of BM DNA and cfDNA by NGS in all patients. Libraries were prepared using a custom panel including 48 myeloid-associated genes and genomic regions localized at the most frequently altered chromosomes in MDS (QIAseq Custom DNA Panels, Qiagen) and sequenced using Illumina. 20/70 (28.6%) showed cytogenetic/FISH alterations at diagnosis, 2 of them with infrequent alterations in MDS, not covered by the panel (+14, del(9q)). 6 cases presented chromosome Y loss as a single alteration, not covered by the NGS panel. Copy number variant (CNV) analysis was performed by NGS to detect cytogenetic alterations in both cfDNA and BM, and confirmed by chromosomal microarrays (CMA) in BM DNA (CytoScan/OncoScan, ThermoFisher).Results. We obtained a median amount of cfDNA of 58.4 ng/ml in MDS patients that was significantly higher than that obtained in healthy controls (median 32.4 ng/ml, P=0.023). Sequencing of BM DNA and cfDNA showed a comparable mutational profile (187/201 mutations, 93.0% concordance). The most frequently mutated genes were TET2 (44.3%), SF3B1 (37.1%), ASXL1 (20.0%), DNMT3A (20.0%), SRSF2 (15.7%), ZRSR2 (12.9%) and U2AF1 (12.9%). A strong correlation was observed between the VAFs of BM DNA and cfDNA (r s=0.797, P<0.001). VAFs of SF3B1 mutations detected in cfDNA were significantly higher than VAFs in BM DNA (P=0.016).The analysis of cytogenetic alterations by NGS showed abnormalities in 10/70 MDS patients in both BM DNA and cfDNA. Interestingly, in a patient without analyzable metaphases in karyotype, del(20q) was found by NGS and confirmed by CMA. Overall, CMA and NGS were highly concordant to detect chromosomal aberrations although they do not reach the sensitivity achieved by karyotype/FISH (Figure 1). However, all cytogenetic aberrations detected by NGS in BM DNA were also detected in cfDNA.Molecular and cytogenetic alterations were monitored in sequential samples from 7 cases (median follow up: 13 months, range 10-30). We observed an excellent correlation between the VAF of mutations in BM DNA and ctDNA across multiple matched time points. A decrease in the VAF was detected in patients responding to therapy (either hypomethylating agents or chemotherapy), but not in non-responding patients (Figure 2). Of note, cfDNA analysis also showed cytogenetic evolution in 2 cases not responding to azacitidine (del(12p) and +21). From the two untreated patients, one acquired a subclonal del(7q) not detected by NGS and observed only in few metaphases, and the second patient showed a clonal expansion of the NF1 mutation at the time of AML transformation.Conclusions. Analysis of cfDNA allows the characterization and monitoring of molecular abnormalities in patients with MDS. Cytogenetic alterations were detectable in most cases by NGS in both BM DNA and cfDNA although with a lower detection rate than karyotype/FISH.Acknowledgements. ISCIII-FEDER, PI16/0153, PI19/0005, 2017SGR205, PT20/00023 and XBTC.Figure/Table legends:Table. MDS patients included, classified by disease subtype. (SLD: single lineage dysplasia; MLD: multilineage dysplasia; RS: ring sideroblasts; EB: excess blasts; MDS-U: MDS-unclassifiable)Figure 1. Detection of cytogenetic alterations by conventional karyotype, FISH, CMA and NGS.Figure 2. Monitoring of molecular and cytogenetic alterations in 7 patients with MDS. 5 patients receiving treatment (3 azacitidine, 1 FLAG-IDA + HCT, 1 inhibitor of hypoxia-inducible factor (HIF)) and 2 untreated cases were included. BM VAF dynamics are shown with a dotted line and cfDNA dynamics are shown with a solid line. [Display omitted] DisclosuresBesses: Gilead: Research Funding. Salar: Janssen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Gilead: Research Funding; Celgene: Consultancy, Speakers Bureau. Bellosillo: Thermofisher Scientific: Consultancy, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau.

Multiple TET2 Mutations As a New Biological Clue for Differentiating Oligomonocytic Chronic Myelomonocytic Leukemia from Myelodysplastic Syndromes

Introduction. Oligomonocytic chronic myelomonocytic leukemia (OM-CMML) is defined as those myelodysplastic syndromes (MDS) or MDS/MPN unclassifiable cases with relative monocytosis (≥ 10% monocytes) and a total monocyte count of 0.5-<1x10 9/L. These patients show similar clinical, genomic and immunophenotypic features than overt CMML, although the majority of them are currently classified as MDS following WHO 2017 classification. TET2 is the most prevalent mutated gene in CMML and it is frequent to find multiple TET2 mutations in the same patient.Objective. We investigated TET2 mutations in 42 OM-CMML, their diagnostic value and their correlation with immunophenotypic pattern, and compared them with that observed in 54 CMML and 86 MDS.Patients and methods. Samples were collected from 182 patients: 42 OM-CMML, 54 overt CMML, and 86 MDS. Molecular characterization was performed by next-generation sequencing (NGS) using a custom panel (QIAseq Custom DNA Panels, Qiagen) including the whole codifying region of 25 myeloid-associated genes and sequenced using Illumina technology. Multiparametric flow cytometry (FC) analysis of monocyte subsets was performed on whole PB collected on EDTA. Cell surface staining of 2 × 10 6 cells was performed and at least 500 000 total events were acquired per tube (FACS Canto II; BD Biosciences). The FC strategy analysis, the 5-tube experimental panel, and the NGS custom panel are described in Calvo & Garcia-Gisbert, Blood Adv 2020.Results. 1.Patients with multiple TET2 (multi-TET2) mutations (2 or more) were identified with similar percentages in OM-CMML (17/42, 40.5%) and CMML (29/54, 53.7%) while they were infrequent in MDS (5/86, 5.8%) (P<0.001). In MDS patients, 4/5 with multi-TET2 showed a percentage of monocytes >10% though they were not considered OM-CMML since the monocyte count was below 500/microL. CMML and OM-CMML patients with multi-TET2 showed predominantly 2 TET2 mutations (CMML: 20/29, 69%; OM-CMML: 14/17, 82%;). The distribution of patients with >2 TET2 mutations was similar in CMML and OM-CMML. We did not observe differences among the frequency of nonsense, frameshift or missense TET2 mutations, nor between early or late-truncating (early: aa 1-1128, late: 1128-1936) TET2 mutations between OM-CMML and CMML.2.TET2 variant allele frequency (VAF) was significantly lower in MDS (median 11.83%) than in OM-CMML and CMML (P<0.001 in both comparisons). OM-CMML and CMML showed similar TET2 VAF (medians 41.4% and 40.3%). In those patients with TET2 VAF >55% or a sum of muti-TET2 mutations VAF above 55% (17 OM, 19 CMML, 2 MDS), we could infer a biallelic alteration in the same clone (Coltro, Leukemia 2020). We did not observe differences in the frequency of OM-CMML showing biallelic TET2 mutations when compared to CMML.3.The main clinical characteristics of CMML patients (overt and OM-CMML), grouped by TET2 mutational status (unmutated: TET2 WT; 1 TET mutation: TET2 single; ≥2 mutations: multi-TET2) are depicted in Table 1. As shown, we did not observed significant differences when comparing TET2 single to TET2 WT or multi-TET2 Of note, multi-TET2 patients showed a higher percentage of PB and BM monocytes, BM promonocytes and more dysgranulopoiesis than TET2 WT patients.4.Patients with TET2 WT presented a lower number of mutated genes than TET single and multi-TET2 patients (P=0.019 and P=0.029). In line with previous studies, IDH mutations and TET2 mutations where mutually exclusive (P=0.001). Co-mutation of SRSF2 and TET2, the well-accepted gene signature of CMML, was found in 25/96 patients (26.0%, 13 OM-CMML, 12 CMML) in 10 cases co-mutated with TET2 single and in 15 cases with multi-TET2.5.We assessed the proportion of OM-CMML patients with MO1>94% since this has been shown as very sensitive and specific feature of CMML. In TET2 mutated OM-CMML, we observed a higher proportion of patients with MO1>94% (90% mut. vs 40% WT; P=0.001) and CD56 expression on monocytes (70% mut. vs 30% WT; P=0.025). We observed a trend (non-significant) difference in the proportion of patients showing these features when comparing TET2 single vs multi-TET2 OM-CMML (single, CD56: 57.1%, MO1>94%: 85.7%; multi-TET2, CD56: 81.3%, MO1>94%: 93.8%).Conclusion. The high occurrence of multiple TET2 mutations in overt and OM-CMML and its infrequency in MDS is a new biological clue for supporting the consideration of OM-CMML in the continuum of CMML. [Display omitted] DisclosuresSalar: Celgene: Consultancy, Speakers Bureau; Gilead: Research Funding; Roche: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau. Bellosillo: Roche: Research Funding, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau; Thermofisher Scientific: Consultancy, Speakers Bureau.

MKP-1 is required to limit myeloid-cell mediated oral squamous cell carcinoma progression and regional extension

Mitogen-activated protein kinases (MAPKs) require MAPK phosphatases (MKPs) for deactivation of MAPK intracellular signaling. MKP-1 (encoded by Dusp1) is a key negative regulator of MAPKs and prior reports have indicated that MKP-1 regulates oral cancer-associated inflammation and leukocyte infiltration. ObjectiveTo determine the significance of myeloid-based expression of MKP-1 in oral cancer. MethodsThe Cancer Genome Atlas (TCGA) was used to address DUSP1 expression in oral squamous cell carcinoma (OSCC). Syngeneic and carcinogen-induced mouse models using global and myeloid-specific Dusp-1 deficient mice with immunophenotypic, histologic, and transcriptomic analyses and in vitro migration assays. ResultsData from TCGA indicates the DUSP1 expression is inversely related to oral cancer burden and nodal involvement. Using murine models of OSCC, the role of MKP-1 signaling in tumor associated macrophages (TAMs) was assessed. Dusp1-deficient mice had increased tumor burden and TAM infiltrate with increased M2 macrophage polarization. Transcriptomic signatures of TAMs from Dusp1-deficent mice indicated a pro-metastatic phenotype as well as concomitant differences in myeloid-associated genes, cytokine/chemokine signaling, and Notch signaling consistent with tumor progression. In vitro and in vivo assays revealed mouse OSCC cells had a higher migration rate using TAM cell-free supernatant from Dusp1 deficiency mice compared to controls with enhanced regional cervical lymph node metastasis, respectively. To validate TAM studies using implantable mouse models, an OSCC progression model with conditional myeloid-specific Dusp-1 deficient mice demonstrated enhanced OSCC disease progression, characterized by advanced onset, histological stage, and tumor burden. ConclusionMyeloid-based Dusp1-deficiency increases OSCC burden and metastasis through alteration in TAM recruitment, gene profile, and polarity suggesting that MKP-1 could be a viable target to reprogram TAM to limit local/regional OSCC extension.

Checkpoint Expression By Acute Myeloid Leukemia (AML) and the Immune Microenvironment Suppresses Adaptive Immunity

Abstract Introduction AML suppresses immune responses directly by expressing cytokines and immune checkpoint ligands as well as indirectly, by recruiting suppressive immune cells like regulatory T cells (Tregs). Prior studies have looked at a small number of immune checkpoint receptors and ligands on limited subsets of T cells and AML blasts, respectively. The frequency of circulating Tregs has been associated with induction failure in AML (Szczepanski et al CCR 2009) and the coexpression of PD1 and TIM3 on CD8 T cells has been associated with immune exhaustion (Zhou et al Blood 2011) and relapse following allogeneic stem cell transplant (Kong et al Blood Can J). Recent clinical trials have demonstrated encouraging response rates and durable responses in patients with relapsed AML treated with PD-1 based therapies (Daver et al EHA 2017) and following allogeneic stem cell transplantation in relapsed AML with CTLA-4 based therapies (Davids et al NEJM 2016). Better defining the immune checkpoint landscape of human AML will facilitate the rational selection of immune checkpoint targets and combination approaches for future clinical trials. Methods Multi-parameter flow-cytometry assay were performed by Immunotherapy Platform on bone marrow (BM) aspirates from 107 patients with AML treated at the MD Anderson Cancer Center. We evaluated the expression of inhibitory (PD1, CTLA4, LAG3, TIM3) and stimulatory receptors (GITR, OX40, 41BB, ICOS) on T cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3-, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells and the expression of their ligands (41BBL, B7-1, B7-2, ICOSL, PDL1, PDL2 and OX40L) on AML blasts. Eight healthy donor BMs were used as controls. We correlated the expression of these markers on T cell subsets and blasts, with age, karyotype and a baseline next generation sequencing for 28 myeloid-associated genes including P53, DNA methylation proteins (DNMT3a, IDH1, IDH2, TET2) and FLT3. Results BM aspirates from AML patients demonstrated significantly higher frequency of CD45 gated CD3+ total T cell infiltrate compared to healthy donors. The proportion of CD8+ T cells expressing PD1 was higher in patients with relapsed (32.8%) and with newly diagnosed AML (26.4%) compared to healthy donors (18.5%) (P Compared with healthy donors, patients with relapsed and newly diagnosed AML had significantly higher percentage of CD8+ T cells co-expressing the inhibitory markers PD1 with TIM3 (P=0.02) and PD1 with LAG3 (P We attempted to identify potential relationships between molecular characteristics and immune checkpoint expression and found that BM blasts in patients with adverse karyotype more frequently expressed PD-L1 as compared to patients with non-adverse karyotype (17.5% vs 8.8%, P=0.03) (Figure 1D). Conclusions Increased Treg and PD1+ CD8 T cell infiltration in AML and the increased PD1+TIM3+ and PD1+LAG3+ CD8 T cells in the BMs of patients with AML, point to worsening exhaustion of an adaptive immune response. The correlation between increased Treg infiltration, increased PD1+CD8 cells and increased PD-L1 expression on blasts in the BM indicates immune suppression at multiple levels. The association between adverse cytogenetics and higher PD-L1 expression on BM blasts further suggests links between AML biology and immune suppression. The dual contributions by T cells and blasts to immune suppression indicate that these pathways may play an important role in AML survival and therefore patients may benefit from checkpoint antibody therapy. Download : Download high-res image (123KB) Download : Download full-size image Figure 1 . Disclosures Cortes: ImmunoGen: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; ARIAD: Consultancy, Research Funding; Sun Pharma: Research Funding; Teva: Research Funding. Allison: Merck: Patents & Royalties: Owns patent licensed to Merck; BMS: Consultancy, Patents & Royalties; Jounce: Consultancy, Patents & Royalties: Owns patent licensed to Jounce; Jounce: Consultancy, Other: Owns stock; Kite Pharma: Consultancy, Other: stock; Evelo: Consultancy, Other: stock; Neon: Consultancy, Other: stock; Constellation: Consultancy, Other: stock; GSK: Consultancy; Astra Zeneca: Consultancy; Amgen: Consultancy; EMD Serono: Consultancy; Astellas: Consultancy. Kantarjian: Delta-Fly Pharma: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding; Amgen: Research Funding; Bristol-Meyers Squibb: Research Funding; Novartis: Research Funding. Sharma: Jounce: Consultancy, Other: stock, Patents & Royalties: Patent licensed to Jounce; Astellas: Consultancy; EMD Serono: Consultancy; Amgen: Consultancy; Astra Zeneca: Consultancy; GSK: Consultancy; Consetellation: Other: stock; Evelo: Consultancy, Other: stock; Neon: Consultancy, Other: stock; Kite Pharma: Consultancy, Other: stock; BMS: Consultancy. Daver: Karyopharm: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Immunogen: Research Funding; Incyte Corporation: Honoraria, Research Funding; Pfizer Inc.: Consultancy, Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Jazz: Consultancy; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Kiromic: Research Funding; Daiichi-Sankyo: Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding.

Phase IB/II Study of Lirilumab with Azacytidine (AZA) in Relapsed AML

Phase IB/II Study of Lirilumab with Azacytidine (AZA) in Relapsed AML

Genomic Analysis of Congenital Myeloid Sarcoma Identifies Significant Bone Marrow Involvement in the Absence of Morphologic Blasts

Myeloid sarcoma is a tumor mass consisting of blasts at extramedullary sites, most frequently the skin and soft tissues. Skin involvement, also termed leukemia cutis, is a well-described entity in congenital leukemia and is present in approximately 50% of patients diagnosed within the first 4 weeks of life. Most cases of congenital leukemia cutis harbor KMT2A rearrangements, as do up to 80% of cases of infantile leukemia, and are typically accompanied by bone marrow involvement. Here we describe a rare CIC-rearrangement as the driver mutation for a case of congenital cutaneous myeloid sarcoma in a newborn female who was noted at birth to have a violaceous, nodular rash. Skin biopsy of the nodular rash shortly after birth with immunohistochemical staining was consistent with myeloid sarcoma. Bilateral bone marrow aspirates and biopsies were also performed, but failed to reveal an aberrant blast population by morphology and flow cytometry. Cytogenetics of the cutaneous myeloid sarcoma was significant for t(10;19)(q23;q13.2) and fluorescence in situ hybridization performed on the tumor confirmed a CIC-rearrangement. CIC-rearranged sarcomas are a new entity of undifferentiated small round cell sarcoma characterized by fusion events involving the CIC gene but are not a known to be driver mutations in myeloid neoplasms. Despite the absence of a blast population in the bone marrow, 75% of analyzed metaphases carried the t(10;19)(q23;q13.2). To further explore the underlying genomic events, whole genome, exome, and transcriptome sequencing was performed on both sarcoma and bone marrow specimens. Sequencing revealed an in-frame CIC-NUTM2A fusion gene present in both the skin and the bone marrow which has been previously described in a single case of undifferentiated soft tissue sarcoma. There were no large-scale chromosomal losses or gains and no copy neutral loss of heterozygosity events. Two exonic single nucleotide variations (SNVs) were detected, both of which were limited to the skin sarcoma and not predicted to be pathogenic. In vitro and in vivo modeling demonstrated that the CIC-NUTM2A fusion protein was highly leukemogenic. Colony forming assays performed using transduced murine bone marrow revealed that the CIC-NUTM2A fusion conferred self-renewal in contrast to CIC, NUTM2A, and the reciprocal NUTM2A-CIC product which failed to serially replate (p&amp;lt;0.001). Transplantation of CIC-NUTM2A modified murine bone marrow cells resulted in a fully penetrant myeloid leukemia with a median survival of 21 days in primary transplants and 16 days in secondary transplants. Transcriptome analysis of these tumors revealed a distinct gene expression profile when compared to several classic myeloid associated fusion genes including AML1-ETO and MLL-AF6. To further understand the acquisition of a malignant phenotype by morphology between the bone marrow and the sarcoma, targeted deep sequencing was performed for all tier 1, 2 and 3 mutations identified by whole genome and exome sequencing. A shared ancestral clone was identified in both specimens along with three subclones specific to the sarcoma. Four SNVs acquired in the sarcoma were found to be present in regulatory regions of genes that were also differentially expressed between the sarcoma and the bone marrow including C6orf120, SMURF1, TJAP1, and PID1. PID1 was found to be downregulated in the malignant sarcoma specimen and has been previously shown to be a regulator of the AKT/PI3K pathway. Low PID1 expression has been associated with poor outcomes in other malignancies, including pediatric glioblastoma. A genome wide CRISPR screen of our CIC-NUTM2A positive murine leukemia cells revealed an enrichment for PID1 deficient cells as well as PTEN confirming the cooperativity between CIC-NUTM2A and the AKT/PI3K pathway (p=0.009 and p=0.005 respectively). In conclusion, we describe a rare fusion gene, CIC-NUTM2A, which leads to an aggressive myeloid malignancy in both humans and mice. Targeted deep sequencing demonstrated the clonal evolution from the bone marrow and acquisition of a cooperating mutation targeting the AKT/PI3K pathway in a subset of extramedullary cells that led to morphologic transformation. A thorough interrogation of the bone marrow in patients with myeloid sarcoma is warranted even in the absence of morphologic and immunophenotypic blasts by flow cytometry to optimally track residual disease during treatment. Disclosures No relevant conflicts of interest to declare.

HMGA1 Is Upregulated in Myelodysplastic Syndromes with Mutation in Pre-mRNA Splicing Genes and Inhibits Leukemia Cell Differentiation

In myelodysplastic syndrome (MDS), genes associated with pre-mRNA splicing such as SRSF2 (Serine and Arginine Rich Splicing Factor 2) and U2AF1 (U2 small nuclear RNA auxiliary factor 1) are frequently mutated. While several reports demonstrated that MDS patients with these mutations had worse prognosis, few treatments specific to these mutations are available, partly due to the dearth of material on functions of these mutated genes. To assess the role of pre-mRNA splicing gene mutations in the pathogenesis of MDS, we performed RNA-seq analysis using CD34-postitive fractions of bone marrow samples of MDS patients. Patients with mutations in either SRSF2 or U2AF1 tended to have shorter 1-year leukemia free survival compared to patients without these mutations (66.7% vs 73.7%, respectively; p=0.067). Gene set enrichment analysis revealed that MYC target genes were enriched in patients with mutations in either SRSF2 or U2AF1 compared to patients (n=4) without these mutations (n=3). Among them, we found that HMGA1 (High Mobility Group A1) was distinctly upregulated in these patients. Expression levels of HMGA1 are known to be higher in hematopoietic stem cells. HMGA1 is involved in various cellular processes such as transcriptional regulation, DNA repair, cell differentiation and regulated cell death. We confirmed that expression levels of HMGA1 in murine bone marrow cells were higher in Lineage marker-negative, Sca1-positive, and c-Kit-positive (LSK) fractions than other more differentiated fractions. To determine whether upregulation of HMGA1 contributed to pathogenesis of MDS, we retrovirally transduced HMGA1 to murine hematopoietic progenitor cell line 32D-cl3. We found that proportions of Gr-1 positive cells after treatment with G-CSF were lower in HMGA1-transduced 32D-cl3 cells. Further, expression levels of myeloid-associated genes were also suppressed in these 32D-cl3 cells. These results suggested that 32D-cl3 with extrinsic expression of HMGA1 were more resistant to G-CSF induced myeloid differentiation. Similarly, primary murine hematopoietic progenitor cells retrovirally transduced with HMGA1 showed increased colony-forming capacities. Considering that differentiation block plays a major role in the onset of MDS, these results supported our hypothesis that upregulation of HMGA1 would contribute to pathogenesis of MDS through blocking normal myeloid differentiation. To investigate whether inhibition of HMGA1 could affect the pathogenesis of myeloid malignancies, we silenced expression of HMGA1 via short-hairpin RNA (shRNA) in various cell lines of acute myeloid leukemia (AML). Human acute monocytic leukemia cell line THP-1 became more easily induced to differentiate upon phorbol12-myristate13-acetate treatment when HMGA1 was silenced. Similarly, we silenced expression of HMGA1 in HL60, which is a human acute promyelocytic leukemia (APL) cell line. We treated HL60 cells with all-trance retinoic acid (ATRA) to induce myeloid differentiation. As expected, we found that expression levels of CEBP-β and Gr-1 in ATRA-treated HL60 cells were significantly higher when HMGA1 was silenced, which meant that HL60 cells became more sensitive to ATRA-induced myeloid differentiation by inhibition of HMGA1. Considering that differentiation induction therapy has improved clinical outcome not only in APL but also in AML, these results suggested that HMGA1 would be a potential therapeutic target for myeloid malignancies. In summary, our experiments demonstrated that expression levels of HMGA1 were higher in patients with splicing mutations. Considering its inhibitory effects on differentiation demonstrated by our experiments, upregulation of HMGA1 could be involved in the pathogenesis of MDS. Further, therapeutic intervention for HMGA1 had a potential to improve clinical outcome of myeloid malignancies. Disclosures Kurokawa: Nippon Shinyaku: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Teijin: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bioverativ Japan: Consultancy; Shire Plc: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Eisai: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Pfizer: Research Funding; Sanwa-Kagaku: Consultancy; Chugai: Consultancy, Research Funding, Speakers Bureau; MSD: Consultancy, Research Funding, Speakers Bureau.

Molecular Characterization and Clinical Outcomes of Young Adult Patients (≤45 years old) with Philadelphia-Negative Myeloproliferative Neoplasms

Background: Polycythemia vera (PV), Essential Thrombocytosis (ET) and Primary Myelofibrosis (PMF) are Myeloproliferative Neoplasms (MPNs) with median age at diagnosis of ~56-70 years old. However, around 10%-15% of cases are diagnosed during young adulthood and there are scanty data about their molecular profile and its implications in clinical outcomes. Objective: To analyze the clinical and molecular characteristics of young adult patients (≤45y.o.) with MPNs (Y-MPN) and to correlate them with clinical features and outcomes. Material and Methods: This is a retrospective single-center study including MPN patients diagnosed below the age of 45 years. Molecular characterization was performed using DNA from granulocytes at diagnosis or before the start of cytoreductive therapy. JAK2V617F was assessed by quantitative allele-specific PCR and CALR mutations by fragment analysis of exon 9. Further molecular profiling was performed by next generation sequencing (NGS) with a custom panel of 25 myeloid-associated genes (ASXL1, CALR, CBL, CSF3R, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NRAS, PRPF8, RUNX1, SETPB1, SF3B1, S2HB3, SRSF2, STAG2, TET2, TP53, U2AF1 and ZRSR2) using Illumina technology. Pathogenic mutations in genes previously related with poor outcomes (ASXL1, EZH2, IDH1, IDH2, SRSF2 and U2AF1) were named as Mutations of Adverse Significance (MAS). Molecular alterations were correlated with diagnosis, progression to PV post ET (PVpET), progression to MF post PV/ET (MFpPV/ET), start of cytoreduction and major thrombotic events (MTE). Time to progression (TTP) and overall survival (OS) were calculated from diagnosis to progression (ELN criteria) and to last visit. Results: From 646 MPN followed in our clinic, 109 (17%) cases were Y-MPN; females 72 (66.1%). At diagnosis the median age was 35 y.o. (9-45). 23 patients (21%) were PV, 91% carried JAK2V617F, 4% (1) carried an exon-12 JAK2 mutation and 1 was JAK2V617F and exon-12 negative. 84 cases (77.1%) were ET, 53.5% (45) JAK2V617F, 25% (21) CALR, (52% type-1 mutation, see table) and 21.4% (18) triple negative (TN). There was 1 PMF with CALR type-1 mutation. No MPL canonical mutations were found. ET was predominantly diagnosed in females (M/F: 26/58). Regarding clinical variables, we found a high proportion of ET-JAK2V617F with high LDH values, higher platelet count for CALR-ET and ET-TN patients (967 and 978 vs 728 for ET-JAK2V617F, p=0.03) and higher frequency of MTE at or before diagnosis for JAKV617F cases (p=0.001). The mean follow-up was 152 months (SD +/-10.4); 16 progressions were registered (PFS 305 months); 8 patients to MFpPV/ET and 8 ET-JAK2V617F to PVpET. An increase in the VAF of JAK2V617F was observed at the time of progression either to PVpET or to MFpET/PV (see table). Seven MTE were registered during this time, 3 in JAK2V617F, 2 in CALR type-1, 1 in exon-12 and 1 in a TN case. 38 (34.8%) cases started cytoreduction, with median time to cytoreductive therapy of 251 (172-330) months; JAK2V617F cases started cytoreduction more often (p=0.04) than patients with other genotypes. No progression to AML nor deaths were recorded. The NGS panel was performed in 102 (93.5%) cases. Pathogenic mutations in non-driver genes were found in 41.2% (42) of cases, being TET2 (7%), ASXL1 (6%) and DNMT3A (5%) the most frequently mutated genes. Also, in 28.4% (29) variants of unknown significance (VUS) were found, involving TET2 (6%), SETBP1 (4%), SH2B3 (5%), and JAK2 (4%) among others. The mutations in SH2B3 (1 pathogenic, 5 VUS) were more frequent in JAK2V617F patients and those in DNMT3A were more common in PV patients. The presence of mutations in non-driver genes (pathogenic or VUS) did not correlate with MTE before or after diagnosis, the start of cytoreduction nor clonal progression. Regarding the 19 TN cases, in 7 (36.8%) one or more non-canonical pathogenic variants implicating MPL, JAK2 and TET2 genes were found. Finally, 8 patients (7.8%) harbored a MAS, of which 3 progressed to MF (2 CALR to MF and 1 ET-JAK2V617F to PVpET); TTP was similar to the rest of the cohort. Conclusions: Our data show that 41% of Y-MPN patients harbor pathogenic mutations in non-driver genes. There was no correlation between their presence and clonal progression, major thrombotic events or overall survival. Mutations of adverse significance did not predict major clinical outcomes. Monitoring of JAK2V617F allele-burden can help to predict progression to MFpPV/ET or PVpET. Disclosures Andrade-Campos: Sanofi-Genzyme: Consultancy, Speakers Bureau; Takeda-Shire: Speakers Bureau; Celgene-BMS: Consultancy. Fernández:Roche: Consultancy, Speakers Bureau. Salar:Janssen: Speakers Bureau; Roche: Speakers Bureau; Celgene: Speakers Bureau. Bellosillo:Qiagen: Consultancy, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau.

Molecular Characterization of Ph-Negative Myeloproliferative Neoplasms in Tumor Circulating Nucleic Acids

Introduction. Genetic studies in patients with Ph-negative myeloproliferative neoplasms (MPNs) are essential to establish a correct diagnosis and to optimize their management. Recently, it has been demonstrated that it is possible to detect molecular alterations present in solid tumors and hematologic neoplasms by the analysis of circulating tumor DNA in plasma samples, which is known as liquid biopsy. It has been reported that most of the circulating cell-free DNA (cfDNA) has its origin in immature hematopoietic and bone marrow cells; however, there is limited information about liquid biopsy applications in MPNs. Objective. To analyze the molecular profile of circulating tumor DNA in patients with MPNs. Patients and methods. Peripheral blood samples from 75 patients with MPNs were collected at the time of diagnosis: 21 polycythemia vera (PV), 42 essential thrombocythemia (ET), 10 primary myelofibrosis (PMF) and two non-classifiable MPNs. Cellular DNA was extracted from the granulocytic fraction isolated by density gradient centrifugation and cfDNA was obtained from 1-3ml of plasma (MagMAX Cell-Free DNA Isolation Kit, Thermo Fisher Scientific). cfDNA purity was ascertained by capillary electrophoresis (4200 TapeStation system, Agilent). Molecular characterization was performed in paired samples of granulocytes DNA and cfDNA by next generation sequencing (NGS). Libraries were prepared using a custom panel that covered the whole codifying region of 25 myeloid-associated genes (QIAseq Custom DNA Panels, Qiagen) and sequenced using Illumina technology (Miseq, Nextseq) with a 3000x minimum coverage. Results. The amount of total cfDNA/mL in plasma was significantly higher in PMF (mean 97 ng/ml) than in PV and ET (mean 18 and 23g/ml, respectively) (p = 0.003, Kruskal-Wallis). Overall, 144 mutations in driver (JAK2, CALR, MPL) and non-driver genes were detected in the granulocytic fraction with similar frequencies to what has been described for PV, ET and PMF. The most frequently mutated non-driver genes where ASXL1 (18.7%), TET2 (17.3%), DNMT3A (6.7%), SRSF2 (6.7%) and IDH2 (5.3%). Sequencing of cfDNA showed a total of 146 mutations. All mutations detected in the granulocytic fraction were also detected in the paired cfDNA sample (100% concordance); two additional mutations in MPL and ASXL1 were detected in plasma in one case. The median variant allele frequency (VAF) present in cfDNA was 29% (range 0.86 - 91.73%), which is far superior to what has been described in solid neoplasms or lymphomas (median 0.41%, range 0.03% - 97.6%). A strong correlation was observed between the VAFs of granulocytic DNA and cfDNA (r = 0.875, p &lt; 0.001, Spearman) (Figure 1). The mutation VAFs detected in cfDNA were significantly higher than VAFs detected in granulocytes (p &lt; 0.001, Wilcoxon). In particular, MPL mutations presented 2.5 higher VAF in cfDNA than in granulocytes (p = 0.018, Wilcoxon). This finding was confirmed and quantified by digital PCR. Interestingly, in one PMF patient the p.W515L MPL driver mutation was originally only detectable by NGS in cfDNA, but not in granulocytes. This mutation was identified by ultra-sensitive digital PCR in both cfDNA (VAF 2.30%) and granulocytes (VAF 0.16%). Conclusions. The analysis of circulating tumor DNA allows the characterization of the molecular abnormalities of patients with Ph negative myeloproliferative neoplasms. The sensitivity for mutation detection in driver and non-driver genes was equal or even superior to that obtained when studying the isolated granulocytic population. Disclosures Salar: Roche: Research Funding, Speakers Bureau; Janssen Pharmaceuticals: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy. Besses:Gilead: Research Funding. Bellosillo:TermoFisher Scientific: Consultancy, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau.

Replicative senescence of hematopoietic cells in patients with idiopathic cytopenia of undetermined significance

We hypothesized that a subset of idiopathic cytopenia of undetermined significance (ICUS) is associated with an increased autonomous proliferation with exhaustion of hematopoiesis. The aim of this study was to investigate the cell turnover rate and replicative history of the bone marrow cells of ICUS patients. To this end, we examined telomere length (TL), proliferation, and apoptosis of the bone marrow cells of ICUS patients and healthy controls (HCs) using telomere quantitative fluorescence in situ hybridization and immunohistochemical staining for Ki-67 and cleaved caspase-3. We also performed targeted sequencing of 88 myeloid-associated genes. A total of 37 patients with ICUS were enrolled in this study, with a median age of 66 years (range: 31–83). TLs were significantly shorter in patients with ICUS than in the HCs (8.8, interquartile range [IQR] 6.8–12.1 vs 18.4, IQR 14.4–22.0, p < 0.0001). Proliferation (Ki-67-positive) and apoptosis (cleaved caspase-3-positive) were significantly increased in patients with ICUS compared to HCs (median = 20.0% vs 5.0%, p = 0.0003; 45.0% vs 22.5%, p = 0.0005, respectively). The shortening of TL and the increased proliferation and apoptotic activity was also prominent in patients with ICUS without mutation and dysplasia than in HCs (p < 0.0001, p < 0.0001, and p = 0.0093, respectively). TL was not associated with mutational profile and clinical characteristics as well in patients with ICUS. To our knowledge, this is the first study to show that ICUS is associated with premature replicative senescence with increased proliferation and apoptosis of bone marrow cells. Further study is needed to address the cause of replicative exhaustion in ICUS patients.

The distribution of T‐cell subsets and the expression of immune checkpoint receptors and ligands in patients with newly diagnosed and relapsed acute myeloid leukemia

BackgroundPhenotypic characterization of immune cells in the bone marrow (BM) of patients with acute myeloid leukemia (AML) is lacking.MethodsT‐cell infiltration was quantified on BM biopsies from 13 patients with AML, and flow cytometry was performed on BM aspirates (BMAs) from 107 patients with AML who received treatment at The University of Texas MD Anderson Cancer Center. The authors evaluated the expression of inhibitory receptors (programmed cell death protein 1 [PD1], cytotoxic T‐lymphocyte antigen 4 [CTLA4], lymphocyte‐activation gene 3 [LAG3], T‐cell immunoglobulin and mucin‐domain containing‐3 [TIM3]) and stimulatory receptors (glucocorticoid‐induced tumor necrosis factor receptor‐related protein [GITR], OX40, 41BB [a type 2 transmembrane glycoprotein receptor], inducible T‐cell costimulatory [ICOS]) on T‐cell subsets and the expression of their ligands (41BBL, B7‐1, B7‐2, ICOSL, PD‐L1, PD‐L2, and OX40L) on AML blasts. Expression of these markers was correlated with patient age, karyotype, baseline next‐generation sequencing for 28 myeloid‐associated genes (including P53), and DNA methylation proteins (DNA methyltransferase 3α, isocitrate dehydrogenase 1[IDH1], IDH2, Tet methylcytosine dioxygenase 2 [TET2], and Fms‐related tyrosine kinase 3 [FLT3]).ResultsOn histochemistry evaluation, the T‐cell population in BM appeared to be preserved in patients who had AML compared with healthy donors. The proportion of T‐regulatory cells (Tregs) in BMAs was higher in patients with AML than in healthy donors. PD1‐positive/OX40‐positive T cells were more frequent in AML BMAs, and a higher frequency of PD1‐positive/cluster of differentiation 8 (CD8)‐positive T cells coexpressed TIM3 or LAG3. PD1‐positive/CD8‐positive T cells were more frequent in BMAs from patients who had multiply relapsed AML than in BMAs from those who had first relapsed or newly diagnosed AML. Blasts in BMAs from patients who had TP53‐mutated AML were more frequently positive for PD‐L1.ConclusionsThe preserved T‐cell population, the increased frequency of regulatory T cells, and the expression of targetable immune receptors in AML BMAs suggest a role for T‐cell–harnessing therapies in AML.

Germline polymorphisms in myeloid-associated genes are not associated with survival in glioma patients.

Immune cells of myeloid origin, including microglia, macrophages, and myeloid-derived suppressor cells adopt immunosuppressive phenotypes that support gliomagenesis. Here, we tested an a priori hypothesis that single nucleotide polymorphisms (SNPs) in genes related to glioma-associated myeloid cell regulation and function are also associated with patient survival after glioma diagnosis. Subjects for this study were 992 glioma patients treated at The University of Texas MD Anderson Cancer Center in Houston, Texas between 1992 and 2008. Haplotype-tagging SNPs in 91 myeloid-associated genes were analyzed for association with survival by Cox regression. Individual SNP- and gene-based tests were performed separately in glioblastoma (WHO grade IV, n = 511) and lower-grade glioma (WHO grade II-III, n = 481) groups. After adjustment for multiple testing, no myeloid-associated gene variants were significantly associated with survival in glioblastoma. Two SNPs, rs147960238 in CD163 (p = 2.2 × 10-5) and rs17138945 in MET (p = 5.6 × 10-5) were significantly associated with survival of patients with lower-grade glioma. However, these associations were not confirmed in an independent analysis of 563 lower-grade glioma cases from the University of California at San Francisco Adult Glioma Study (p = 0.65 and p = 0.41, respectively). The results of this study do not support a role for inherited polymorphisms in myeloid-associated genes in affecting survival of patients diagnosed with glioblastoma or lower-grade glioma.