Clonal Disease Research Articles

IL-18 and IL-18 Binding Protein Are Related to Disease Severity of Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) was defined as a clonal hematopoietic disorder disease, although current stratified therapy for MDS has greatly improved overall survival, there is still a subset of them who suffered from progression to AML with a poor prognosis. Immune factors play essential roles in the pathogenesis and evolution of MDS. It is known that the level of plasma IL-18 which has anti-tumor and immunomodulatory effects increased in MDS patients, recent research found that the presence of IL-18BP depresses its effect. In this study, we intended to discuss the importance of IL-18 and IL-18BP on the severity of MDS and the potential mechanisms affecting the efficacy and explore new targets for MDS further therapy. Methods: 43 MDS patients, 14 AML patients, and 16 healthy volunteers were enrolled in our study, the levels of IL-18, IL-18BP of bone marrow supernatant were tested by ELISA and clinical information was collected; the expression of perforin, granzyme B, and IFN-γ, as well as PD-1 and TIM-3 expression on the surface CD8+ T and NK cells, was tested in MDS patients by flow cytometry, correlations among all these data were analyzed by SPSS. Results: We found that the levels of IL-18, IL-18BP, and fIL-18 in the bone marrow supernatants of both MDS and AML were higher than those of healthy controls. fIL-18 were negatively correlated with the severity of MDS. CD8+ T cells in MDS were hypofunctional, with a lower secretion of perforin, granzyme B, and IFN-γ than in healthy controls. The level of fIL-18 was positively correlated with perforin and IFN-γ; the expression of IL-18Rα on the surface of CD8+ T cells was low in MDS patients. The expression of IL-18Rα was negatively correlated with perforin, granzyme B, and IFN-γ. In addition, elevated levels of PD-1 and TIM-3 on the surface of CD8+ T cells in MDS seemed no significant correlation with fIL-18 and IL-18BP. Conclusion: MDS is in a state of immunosuppression as evidenced by the decreased number and depressed function of CD8+ T cells and NK cells. Increased expression of fIL-18 in MDS patients was shown, compared to healthy controls. Due to the presence of IL-18BP, fIL-18is relatively insufficient. Thus antagonizing IL-18BP is a promising new target for MDS.

Interferon-alpha2 Favorably Impacts Deregulated Atherosclerosis Genes in Polycythemia Vera and Related Neoplasms

Introduction: Chronic inflammation has been suggested to be the driving force for clonal evolution and disease progression in the Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) but also potentially having an impact upon the development of accelerated (premature) atherosclerosis, which is recorded in several other chronic inflammatory diseases. Using whole blood gene expression profiling, we have previously shown massive deregulation of genes involved in oxidative stress. Importantly, we have also reported these genes to be favorably regulated during treatment with interferon-alpha2 (IFN). Herein, we report the favorable regulation of deregulated atherosclerosis genes by IFN as well. Objectives: By whole blood gene expression profiling to investigate the regulation of atherosclerosis-promoting genes during treatment with IFN. Methods: Global gene expression profiling was performed at baseline and after 3 months of treatment with IFN in 8 patients with ET, 21 patients with PV, and 4 patients with PMF. Hydroxyurea (n = 18) or anagrelide (n = 6) were discontinued 1 week prior to enrollment. The Affymetrix HG-U133 2.0 Plus microarray was used to generate gene expression profiles. Total RNA was purified from whole blood, amplified to biotin-labeled aRNA, and hybridized to microarray chips. Data preprocessing and statistical analysis of microarray data were done in R. Results: Single gene analysis was performed on 84 genes represented on the Qiagen Human Atherosclerosis gene panel. In response to treatment with IFN in all three patient categories, 10 genes became upregulated in ET including ABCA1, BCL2, CCL2, CCR1, KLF2, LIF, SELL, TGFB1, RXRA, and VEGFA, and 8 genes became downregulated including APOE, BCL2L1, CD44, IL1A, IL1R1, IL1R2, LPA and SPP1. In PV patients, ABCA1, ADFP, BAX, BCL2, CCL1, CCR2, CCR2, CSF1, HBEGF, LIF, NR1H3, RXRA, SELL, TGFB1, TNFAIP3, TNF, and VEGFA were among the 17 upregulated genes, and CD44, CFLAR, IL1A, IL1R1, IL1R2, ITGAX, MMP1, and SPP1 among the 8 downregulated genes. In PMF, 5 genes became upregulated including ABCA1, BAX, CCL2, CCR1, and TNFAIP3, and 5 genes became downregulated including BCL2L1, IL1R1, IL1R2, ITGAX, and SPP1 (all P<0.05) (Table 1 and 2). In a previous microarray study comparing another cohort of patients with ET, PV, or PMF with healthy controls, we have shown significant downregulation of the atherosclerosis genes BCL2, CCR2, CFLAR, FAS, IL1A, ITGA5, KLF2, NFKB1, PPARA, and SELPLG, and significant upregulation of BCL2L1, COL3A1, ELN, FGA, FGF2, IL4, IL5, KDR, LIF, MMP1, PDGFA, PDGFB, PDGFRB, PTGS1, SERPINE1, TGFB2, and THBS4 in all three disease entities (FDR<0.05). Comparing 3 months IFN treatment with baseline in ET, PV and PMF, COL3A1, ELN, FAS, FGA, FGF2, IL4, IL5, ITGA5, KDR, NFKB1, PDGFA, PDGFB, PDGFRB, PPARA, PTSG1, SELPLG, SERPINE1, TGFB2 and THBS4 were not significantly deregulated in any of the disease entities. Furthermore, IFN upregulated downregulated atherosclerosis genes including BCL2 (ET, PV), CCR2 (PV), and KLF2 (ET) and downregulates upregulated atherosclerosis genes including BCL2L1 (ET, PMF) and MMP1 (PV). Discussion and conclusions: Atherosclerosis is a chronic inflammatory disease, which involves several circulating blood cells, including both leukocytes and platelets, which all are variably elevated in MPNs and normalized during IFN-treatment. Therefore, we speculated, if IFN might favorably impact deregulated atherosclerosis genes in patients with MPNs. Indeed, for the first time, we herein report that IFN impacts several atherosclerosis-promoting genes, including both significant upregulation and downregulation of key genes of importance during atherosclerosis development, plaque formation and rupture. Our results add novel important information in regard to the beneficial impact of IFN in regulating deregulated atherosclerosis-promoting genes of potential importance for the development of premature atherosclerosis and accordingly the heavy cardiovascular disease burden in MPNs. The significance of our observations in MPNs should be further explored and confirmed in other MPN-cohorts, including comparative studies of atherosclerosis gene signatures in atherosclerotic plaques from patients with and without MPNs being operated for occlusive vascular diseases and aneurysms. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular Characterization of Clinical Response and Relapse in Patients with IDH1m ND-AML Treated with Ivo+AZA in the AGILE Study

Background: Mutations in isocitrate dehydrogenase 1 (mIDH1) occur in approximately 6-10% of patients (pts) with AML. Ivosidenib (IVO), a potent, oral, targeted inhibitor of mIDH1, is FDA approved as monotherapy for the treatment of relapsed/refractory AML and newly-diagnosed AML (ND-AML) in adults >75 years of age or with comorbidities that preclude the use of intensive chemotherapy. In addition, the combination of IVO with azacitidine (AZA) was recently FDA approved in older or unfit AML pts with mIDH1 as a result of the global Phase 3 AGILE trial (NCT03173248). Compared to placebo (PBO) + AZA, the combination of IVO + AZA extended event-free survival (EFS; hazard ratio 0.33, 95% CI [0.16 - 0.69]; P = 0.002), overall survival (OS; median 24.0 months vs. 7.9 months; hazard ratio 0.44; 95% CI [0.27 - 0.73]; P = 0.001), and increased the rates of complete remission or complete remission with partial hematologic recovery (CR/CRh; 52.8% vs. 17.6%). To date, biomarker analysis has shown that IVO+AZA is associated with deep and durable clearance of IDH1m clones with improved clinical responses and EFS/OS in several pt subgroups, including those with mutations in receptor tyrosine kinase (RTK) pathway genes, a subgroup refractory to IVO monotherapy. Aim: To characterize clonal evolution and relapse mechanisms in pts with mIDH1 ND-AML treated with IVO+AZA. Methods: Patients received oral IVO + AZA per study protocol. Next-generation DNA sequencing (DNA-seq) on baseline and longitudinal samples was performed on bone marrow aspirates (BMA), bone marrow mononuclear cells (BMMCs), or peripheral blood mononuclear cells (PBMCs) depending on sample availability using the ACE Extended Cancer Panel (Personalis, Inc); limit of detection (LOD) 2% variant allele frequency (VAF). This analysis was focused on variants in 95 AML-associated genes with known or likely oncogenic potential. Results: Among 72 pts enrolled on the IVO+AZA arm, 46 had both baseline and longitudinal DNA-seq data available, with a median follow-up of 366 days on study (range 16 - 1210). The median number of baseline mutations was 3 (range 1 - 10). Suppression of all baseline non-DTA (DNMT3A / TET2 / ASXL1) mutations to below the LOD ("mutation clearance") was observed in 28 patients, including 22/34 (65%) pts who achieved CR/CRh [Table 1]. To identify potential mechanisms of acquired resistance, on-treatment samples were analyzed for the emergence of mutations not detected at baseline. Overall, emerging mutations were identified in 22/46 (48%) pts (median 2 emerging mutations; range 1-6). 2/46 (4%) pts had emerging mutations in IDH2, but discontinued treatment due to adverse events and stable disease, respectively. No second-site IDH1 mutations were observed across any response category. Longitudinal samples were available for 8/10 (80%) pts who discontinued treatment due to relapse (RL) following an objective response (4 CR, 2 CRh, 1 CRi, 1 MLFS). Emerging mutations were detected in 6/8 (75%) of RL pts [Table 2]. Genes with emerging mutations recurrently associated with RL included ASXL1, FLT3, RUNX1, and TET2 (2 pts each). The IDH1 mutation was not detected at RL in 6/8 (75%) pts. Among the two RLs that occurred in the absence of emerging mutations, one was associated with expansion of a baseline FLT3-ITD subclone, and one was not associated with any new emerging or expanding mutations. To further evaluate clonal evolution and disease progression of these pts, scDNA-seq is being performed and will be presented. Conclusion: IVO+AZA combination therapy leads to deep and durable remissions associated with clearance of mIDH1 and baseline co-mutations. To date, no second-site IDH1 mutations have been observed, but mutations in other genes, including IDH2, can emerge and may provide an opportunity for relapse. Relapses were observed in a subset of responders and were generally associated with expansion or emergence of high-risk mutations occurring independently of mIDH1. Further study is needed to better characterize mechanisms of RL to IVO+AZA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TIF1β Enhances Self-Renewal Capacity of BCR-ABL Leukemic Stem Cells and Inhibits the Myeloid Differentiation

Chronic myeloid leukemia (CML), characterized by expression of p210-BCR-ABL, is a clonal myeloproliferative disease initiated by malignant transformation of hematopoietic stem cells (HSC). Blastic crisis phase (BC) of CML that shows expansion of therapy-refractory and differentiation-arrested blasts, remains a therapeutic challenge. Transcription intermediary factor TIF1β/TRIM28/KAP1 modulates chromatin structure to repress or activate transcription of target genes. Tif1β is essential for the proliferation and differentiation of normal hematopoietic stem cells, but the role of Tif1β in myeloid leukemia is unclear. Recently, high expression of TIF1β was correlated with poor prognosis in patients with various types of cancer. By analyzing datasets of gene expression in bone marrow (BM) cells in CML patients, we found that TIF1β expression levels were robustly elevated in CML cells at the BC stage. Thus, we hypothesized that TIF1β functions as an oncogene for the development of BCR-ABL leukemia. To determine whether TIF1β promoted the development of BCR-ABL-induced myeloid leukemia in vivo, we generated BCR-ABL;Tif1βf/f;Cre-ERT2 compound mice by crossing Rosa26 locus BCR-ABL conditional knock-in mice and Tif1βf/f;Cre-ERT2 conditional KO mice. We transplanted BM cells isolated from Cre-ERT2 (WT), Tif1βf/f;Cre-ERT2 (Tif1β KO), BCR-ABL;Cre-ERT2 (BCR-ABL), and BCR-ABL;Tif1βf/f;Cre-ERT2 (BCR-ABL;Tif1β KO) mice into lethally irradiated CD45.1+ WT recipient mice, and injected tamoxifen 4 weeks after BM transplantation. While BCR-ABL KI mice showed rapid expansion of leukemic blast cells and died in a short period of time, the deletion of Tif1β gene resulted in a significantly longer survival with reduced tumor burden in BM, liver and spleen in BCR-ABL KI mice. BCR-ABL KI mice mostly developed myeloid leukemia, in contrast, BCR-ABL;Tif1β KO mice dominantly showed B-cell leukemia or bone marrow failure diseases. A subset of BCR-ABL;Tif1β KO mice survived without development of lethal leukemia in the primary and the secondary transplantation settings, indicating that TIF1β maintained the self-renewal capacity of leukemic stem cells (LSCs) but also controlled the myeloid/lymphoid lineage commitment. To elucidate the mechanisms underlying the impaired development of leukemia in BCR-ABL;Tif1βKO mice, we performed RNA sequencing analyses of HSCs isolated from WT, Tif1β KO, BCR-ABL, BCR-ABL;Tif1βKO mice. Gene set enrichment analysis revealed that the loss of Tif1β repressed BCR-ABL-induced activation of proliferative stem cell signatures, but increased expression of myeloid differentiation genes including PU.1 gene in HSCs. We found that BCR-ABL;Tif1βKO HSCs reduced expression levels of Myc and Myc-target genes including Fra1 transcription factor (TF), which was known to promote tumor cell proliferation. Notably, human CML-CP and CML-BC CD34+ cells markedly increased expression levels of MYC and FRA1 genes. To clarify how the Tif1β protein controlled self-renewal of LSCs, we performed ATAC-sequencing in murine HSCs and TIF1β-ChIP-sequencing using BCR-ABL-expressing human leukemia cells. ATAC-seq revealed that BCR-ABL KI HSCs showed enrichments of binding motif of Fra1 in open chromatin and that of PU.1 in closed chromatin, compared to WT HSCs, while BCR-ABL;Tif1βKO HSCs cancelled those enrichments of binding motifs of TFs. TIF1β-ChIP-seq revealed that the TIF1β protein directly bound to a promoter region of the FRA1 gene. TIF1β binding sites were enriched with canonical PU.1- and JUN/FRA1-binding motifs. We also found that treatment with imatinib decreased enrichment of FRA1-binding motif among TIFβ-binding regions in BCR-ABL+ cells, indicating that TIFβ opened chromatin accessibility to activate expression of and transcriptional function of the Fra1 gene in a BCR-ABL dependent manner, but repressed the accessibility of myeloid differentiation regulator TFs. Furthermore, knockdown of Fra1 significantly impaired the proliferation of BCR-ABL stem cells in an in vitro condition. In conclusion, we demonstrate that the Tif1β gene was required for the propagation of BCR-ABL-induced myeloid leukemia stem cell in mice. Tif1β enhanced chromatin accessibility of Fra1 TF to promote transcription of LSC signature genes, but also reduced chromatin accessibility at the PU.1-binding motif, resulting in the enhanced proliferation and limited differentiation of LSCs.

Direct and Indirect Costs of Patients with Myeloproliferative Neoplasm Diseases

Background: Myeloproliferative neoplasms (MPNs) are clonal blood diseases characterized by an overproduction of blood cells and include myelofibrosis (MF), polycythemia vera (PV), and essential thrombocythemia (ET). Over the course of illness, these disorders can impose substantial economic burden, in both direct and indirect healthcare costs, due to lost work productivity and medical leave. The objective of this study was to measure work loss (short-term and long-term disability [STD/LTD]) and direct and indirect costs for patients with MPNs among US employees. Methods: In this retrospective cohort study, MarketScan administrative claims databases were used to identify employed patients (aged 18-64 years) with ≥1 diagnosis of an MPN (cohorts for MF, PV, and ET identified separately) from 1/1/2009 to 12/31/2019. The latest date meeting all eligibility criteria in the selection window was set as the index. Patients were required to have 6 months of continuous database enrollment (with medical, pharmacy, and work loss data available) before (pre-index period) and 12 months after (follow-up period) the index date. Patients with evidence of pregnancy during follow-up, a diagnosis of acute myeloid leukemia (AML; all cohorts), myelodysplastic syndrome (MDS; PV and ET cohort only), or MF (PV and ET cohort only) at any point during the study were excluded. A control cohort of patients without evidence of MPN, AML, or MDS during the entire study period and meeting the same enrollment criteria as the MPN cohorts was also selected (index date randomly assigned). Direct matching on age and sex followed by propensity score matching (1:3 ratio) methods were used to adjust for differences in other patient demographics and comorbidities before measuring outcomes. The proportion of patients with STD or LTD, number of days lost from work, and associated indirect and direct healthcare costs (overall and by service category) were reported for the MF, PV, and ET cohorts and compared with matched controls during the 12-month follow-up period; subsets of each MF, PV, and ET cohort who experienced a thrombotic event (TE) were also analyzed. Results: The number of patients with MF, PV, and ET included in the analysis were 173, 4,477, and 6,061, respectively. After matching, patients with an MPN were well balanced with controls (defined as standard mean difference of <0.1) on all demographic and clinical characteristics. Median age was 54, 51, and 48 years for MF, PV, and ET patients, respectively. Total direct healthcare costs and those of each of the components (ie, inpatient, outpatient, and pharmacy) were significantly higher among all 3 MPN cohorts compared with controls (Figure). The largest mean cost difference was observed in the MF cohort ($67,456 higher), followed by the ET cohort ($22,279 higher) and the PV cohort ($10,970 higher). The MPN cohorts also had a significantly larger proportion of patients with an STD claim, a larger number of days lost from work due to STD, and higher associated indirect costs related to STD compared with controls. Patients in the MF cohort had an 8.1% higher chance of filing a STD claim, followed by the ET cohort (7.5% higher) and the PV cohort (3.3% higher). Results were consistent but less pronounced for LTD due to the small proportion of patients with LTD during the 12-month follow-up period, which may not be long enough to observe the diseases' impact (Table). In the subsets of patients with ET and PV with a TE, the mean total direct healthcare costs were substantially larger than those observed for the full ET and PV cohorts (ET: $74,279 higher and PV: $45,219 higher), and the difference in the percentage of patients filing a STD claim was also substantially larger (ET: 20.5% higher and PV: 11.6% higher). Conclusions: This real-world analysis found high economic burden among employed patients with MPNs. Patients with MF, PV, and ET were significantly more likely to take disability leave and had higher direct and indirect costs compared with matched controls. The presence of TEs more than tripled direct costs and nearly tripled use of disability leave for patients with PV or ET compared with the full PV or ET cohorts. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inpatient Burden, Characteristics and Outcomes Associated with Venous Thrombotic Complications Among Patients with Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, clonal hematopoietic stem cell disease caused by mutations in an X-linked gene called phosphatidylinositol glycan class A (PIG-A). Venous thromboembolism (VTE) is known to be a leading cause of death in patients with PNH and is considered to be a clinical emergency, warranting treatment with eculizumab and therapeutic anticoagulation. Although several studies have analyzed the overall disease burden and predictors of outcomes associated with VTE in patients with PNH, data on inpatient burden and outcomes are scarce. We sought to investigate the inpatient burden, characteristics and overall outcomes associated with VTE-related complications among patients with PNH using a large nation-wide database. Methods: The current nation-wide retrospective analysis was conducted using the National Inpatient Sample (NIS), which includes administrative and demographic data from a 20% sample of inpatient hospitalizations in the United States. We interrogated the NIS for admissions among patients with PNH from 2016-2019 using appropriate ICD-10 diagnosis codes. Admissions with known hypercoagulable conditions such as anti-phospholipid antibody syndrome, factor V Leiden mutation, prothrombin gene mutation, protein C and S deficiency, etc. were excluded. Hospitalizations with PNH were further assessed for VTE-related complications using appropriate ICD-10 codes and data on baseline characteristics and inpatient outcomes were collected and analyzed. Chi-square test was used for categorical data and Student's t-test was used for continuous variables. A p-value < 0.05 was considered statistically significant. SAS version 9.4 (SAS Institute Inc.) was used for statistical analysis. Results: A total of 3,600 hospitalizations were included for analysis, out of which, 345 (9.6%) were associated with VTE. Lower extremity deep vein thrombosis (DVT) was noted to be the most common (100/345, 29%) followed by hepatic vein thrombosis/ Budd Chiari syndrome (80/345, 23.2%), portal vein thrombosis (70/345, 20.3%), pulmonary embolism (60/345, 17.4%), upper extremity DVT (60/345, 17.4%), non-specific VTE (15/345, 4.3%) and renal vein thrombosis (5/345, 1.5%). Table 1 shows baseline characteristics and co-morbidities in hospitalizations associated with and without VTE. Patients in the VTE group were significantly younger (47.3±17.4 vs 50.4±22.1, p=0.01) and had a higher prevalence of co-morbid obesity (27.5% vs 10.1%, <0.001) and chronic liver disease (23.2% vs 8.3%, p<0.001). The prevalence of myelodysplastic syndrome (5.8% vs 11.4%, p=0.002) and aplastic anemia (20.3% vs 27.2%, p=0.006) was noted to be significantly lower in the VTE group, while no difference was noted with iron deficiency anemia (4.3% vs 4.1%, p=0.85) and primary myelofibrosis (2.9% vs 1.5%, p=0.06). No patients with acute myeloid leukemia could be captured in our cohort in either of the groups. Overall, there was no difference in all-cause inpatient mortality between groups with and without VTE (1.4% vs 2.5%, p=0.24), however, hospitalizations with VTE-related complications were associated with longer average length of stay (10 days vs 6.3 days, p<0.001), higher average cost of hospitalizations ($178,143 vs $100,547, p<0.001) and lower rate of discharge to home (66.7% vs 72.5%, p=0.02). Conclusions: Overall, 10% of the hospitalizations in our analysis were associated with VTE, out of which, lower extremity DVT, hepatic and portal vein thrombosis were the most common sites (approximately 75% of the VTE burden). Patients with VTE were younger and had a higher prevalence of co-morbid obesity and chronic liver disease. Prevalence of myelodysplastic syndrome and aplastic anemia was noted to be significantly lower in the VTE group, however, further prospective clinical data are needed to analyze the impact of these diagnoses on overall risk of VTE in patients with PNH. Although VTE did not appear to increase all-cause inpatient mortality; associated increase in average length of stay, average cost of hospitalization and decreased likelihood of discharge to home were noted. Concerted efforts to strengthen and enhance care for patients with PNH in the ambulatory setting are needed to prevent inpatient hospitalizations with VTE and reduce associated healthcare burden and costs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TP53 Gene Deletion or Mutation in Newly Diagnosed Multiple Myeloma Patients By FISH with Cytoscan Arrays and Targeted Panel Sequencing

Background Multiple myeloma (MM) is a plasma cell clonal malignant disease. In the past 20 years, the progression free survival rate and overall survival rate of patients have been significantly improved, however, due to the potential complex cytogenetic abnormalities, the clinical progresses are heterogeneous and the clinical benefits of patients are not consistent, especially for high-risk patients. In Chinese clinical practice, 17p-, t(4;14), t(14;16) are considered high-risk cytological abnormalities for newly diagnosed multiple myeloma (NDMM) patients according to R-ISS stage and 2022 Chinese guidelines for diagnose and treatment for MM. As a genome guardian, TP53 plays an important role in regulating cell cycle, apoptosis, aging, DNA repair and maintaining gene stability. But the threshold of deletion positive proportion with clinical significance by CD138-sorted FISH was unclear. TP53 mutation is also not regularly examined in Chinese patients. For that aim, our center has taken TP53 seriously as a heterogeneity problem for clinical discussion. Methods The clinical data of 248 NDMM patients in the First Affiliated Hospital of Soochow University of a VRD clinical study, with VRD in combination with autologous stem cell transplantation or VRD treatment for 8 cycles, from September 1, 2018 to August 31, 2021, were analyzed retrospectively. The samples for CD138-sorted FISH, CytoScan and targeted panel sequencing are obtained from the Multiple Myeloma Specialized Disease Bank of the National Center for Clinical Medical Research of Hematological Diseases. Results In this study, the deletion rate of TP53 was 13.0% (32/248), the mutation rate was 2.0% (5/248), the incidence of any TP53 event was 14.5% (36/248), and the incidence of double allele event was 0.8% (2/248). The detection rate of TP53 gene mutation was low, which was related to the short clinical application time of targeted panel sequencing with CD138-sorted samples. All patients with TP53 deletion or mutation were DS-III stage, with anemia (72.2%), hypoalbuminemia (58.3%) and renal insufficiency (38.9%). The most common accompanied abnormalities were 13q14- (63.9%), followed by t (4;14), 1q21+, t (11;14). Abnormal karyotypes were detected by FISH and CytoScan, indicating gene complexity and instability, and the clonal evolution, which is the root of disease recurrence and progression. The positive rate of TP53 mutation of one case study increased from 7.87% at the first recurrence to 53.44% at the second recurrence with 3rd line therapy (Figure 1a), indicating that TP53 gene lead to disease progression and the formation of multidrug resistance. In terms of induction effects, patients with TP53 gene abnormalities had ≥VGPR with 77.8%, lower than that of negative group (84.8%, P=0.0627)(Figure 1b). The median follow-up time is 20 months (1-42 months). Compared with negative ones, TP53 positive subgroup had an adverse PFS result (33 months vs not reached, P=0.0403), while the OS difference hasn't been clinical significant (both not reached, P=0.0541) (Figure 1c). The result showed that TP53 deletion with more than 50% was an independent adverse factor associated with poor prognosis of NDMM patients (PFS: HR=2.2441, 95%CI 1.117-5.335, P=0.0025; OS: HR=2.771, 95%CI 1.006-7.629, P=0.049). The prognosis of patients with TP53 deletion more than 50% was significantly worse than that of the negative group (PFS 25months vs not reached, P=0.0032, OS not reached P=0.0198), but TP53 deletion less than 50% had no difference compared with negative group. (Figure 1d).Conclusions Patients with TP53 deletion or mutation have obvious clinical symptoms and are prone to cytogenetic abnormalities such as 1q+, 1p-, -13, IgH translocation, chromosome abnormality and so on. TP53 deletion with more than 50% was an independent adverse factor associated with poor prognosis of NDMM patients, as a result, TP53 deletion more than 50% is the clinical significant threshold for clinical use. This study comprehensively describes the detection of genetic abnormalities and prognostic survival analysis of NDMM patients by using FISH, CytoScan and targeted panel sequencing. Identifying the real high-risk patients, we can earlier take targeted and more active measures, such as intensive induction treatment, tandem transplantation or CART treatment, to better deepen and consolidate the therapeutic effect for long-term survival benefits of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Using a Humanized Animal Model to Determine the Minimum JAK2V617F Mutant Allele Burden Required to Generate MPN Pathologies

Myeloproliferative neoplasms (MPNs) are clonal hematological diseases driven by somatic mutations acquired in hematopoietic stem cells (HSCs) and characterized by the deviant, unregulated proliferation of one or more myeloid lineages. Chronic MPN can present as polycythemia vera (PV; excess erythrocytes), essential thrombocythemia (ET; excess platelets), or myelofibrosis (MF; bone marrow fibrosis). More than 300,000 patients are currently living with an MPN diagnosis in the United States. The JAK2V617F variant is an acquired somatic mutation recurrently found in MPN patients and is used as a diagnostic marker along with increased hematocrit, increased hemoglobin, and adverse thrombotic events. In MPN patients, the frequency of JAK2V617F mutant cells varies widely but is known to induce an MPN phenotype with both high (>50%) and very low (<5%) variant allelic frequency (VAF). The reason for this is currently unknown. Most JAK2-mutant animal models model a VAF which is much higher than the lower end of detectable VAFs found in MPN patients. This results in a very limited understanding of what the minimal mutant cell burden required for disease manifestation is. The understanding of genotype/phenotype relationships in patients with low JAK2V617F VAF chronic MPN is paramount to our understanding of this disease and its hematological progression. To this end, we have designed a clinically relevant, humanized mouse model wherein we employ CRISPR and homology directed repair (HDR) via a single stranded oligo donor nucleotide sequence (ssODN) to knock-in the JAK2V617Fmutation at the endogenous locus of human cells at low VAF. In addition, a synonymous JAK2V617V mutation was introduced via CRISPR/ssODN in parallel cells to serve as a control tracked by VAF alongside the VAF of JAK2V617F in their corresponding cohorts. The respective ssODNs were nucleofected into either umbilical cord blood (UCB) derived CD34+ cells or healthy adult bone marrow (BM) derived CD34+ cells to investigate the possibility that developmental age is a variable in low JAK2V617F VAF inducing a clinical MPN phenotype. After nucleofection, 20,000 CD34+ cells were intra-tibially transplanted into sub-lethally irradiated (250 rads) NSGS mice. The low cell input was utilized to limit engraftment and initial disease burden of mutant cells. Effect was measured by mutant allele burden by droplet-digital PCR (ddPCR) analysis, flow cytometry at selected timepoints to track human cell engraftment, spleen weight, and complete blood count (CBC) analysis including hematocrit, hemoglobin, white blood cells, and platelets. At 4 months post-transplant, peripheral blood hCD45+ engraftment across all cohorts averaged less than 3%. Average VAF of circulating hCD45+ cells increased in both JAK2V617F cohorts 5 months post-transplant compared to day 0 post nucleofection. Notably, overall JAK2V617F VAF in each sample remained <5% when adjusted to include both human and mouse derived peripheral blood cells. Remarkably, both hematocrit (p= .037 in UCB cohorts and p=.0096 in BM cohorts; Figure 1A) and hemoglobin (p=.046 in UCB cohorts and p=.0026 in BM cohorts; Figure 1B) were significantly elevated in both JAK2V617F cohorts compared to their respective JAK2V617V controls, while white blood cell and platelet counts remained unchanged within normal ranges. In addition, spleen weights were significantly increased in both JAK2V617F cohorts compared to their respective controls. These data demonstrate a successful clinical model of MPN which induces a classical MPN phenotype where a low VAF of JAK2V617F corresponds with a significant increase of hematocrit, hemoglobin, and spleen weight in a humanized mouse model compared to relevant controls. In addition, we demonstrate that while engraftment due to low hCD34+ cell input remains low after 4 months, the VAF of JAK2V617F within the hCD45+ sub- population increases. This successfully enables the overall VAF contributing to the MPN phenotype to remain at clinically relevant lower levels compared to other models with much higher VAF contributions. We believe this model to be of high clinical relevance into the investigation of how low JAK2V617F burden relates to disease development and progression in MPN patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Reactive Oxygen Species-Mediated c-Jun Activation Domain-Binding Protein-1 (Jab1) Promotes Immune Escape in Hyperleukocytic Acute Myeloid Leukemia

Background: Hyperleukocytic acute myeloid leukemia (HL-AML) is a group of clonal hematopoietic stem cell diseases. Our previous studies have confirmed that Jab1 mediated by reactive oxygen species (ROS) plays a role in the pathogenesis of AML as a transcription coactivator of AP-1/JUN. Here, we reveal the specific regulatory mechanism of immune escape in HL-AML by Jab1, which serves as a new therapeutic target for HL-AML. Methods: The relationship between Jab1 expression and immune cell subtypes in clinical bone marrow samples were analyzed by RT-qPCR. Lentiviral knockout of Jab1 was combined with transcriptomics and chromatin co-precipitation to evaluate the transcriptional regulatory mechanisms involved, and bioinformatics to identify molecular interactions. The changes of each immunophenotype were analyzed by flow cytometry. Results: Compared with healthy controls and AML, Jab1 was highly expressed in HL-AML and was associated with a poor prognosis. However, in HL-AML patients, the proportion of CD8+ T cells was significantly decreased, while the proportion of regulatory T cells was slightly increased, and the secretion of cytokines IL-2, IL-4, IFN-γ, and IFN-α decreased. In H2O2-induced oxidative stress, ROS levels promote the entry of c-Jun into the nucleus, while Jab1 knockdown blocks c-Jun nuclear expression and transcriptional activity. Mechanically, Jab1 regulates leukemic immunosuppressive target LILRB4 rather than PDL1/PDL2. As a coactivator of transcription factor c-Jun, Jab1 cannot directly regulate the transcription of LILRB4 but enhances the stability of LILRB4 mRNA. By high-throughput screening, it was found that N6-methyladenosine modification directly recognized the LILRB4 mRNA site. Finally, it was concluded that Jab1/IGF2BPs/LILRB4 signal axis was involved in disease progression in HL-AML. Conclusions: We provided compelling evidence to demonstrate that Jab1 under oxidative stress is associated with tumorigenesis and poor prognosis of AML, which can serve as a target to develop therapeutics for cancer treatment.

Prolonged Use of Eltrombopag in Patients with Severe Aplastic Anemia without Complete Response at 6 Months after Immunosuppressive Therapy

Background Eltrombopag (EPAG) could improve the efficacy of immunosuppressive therapy (IST) consisted by antithymocyte immunoglobulin (ATG) and cyclosporin (CsA) in untreated severe aplastic anemia (SAA) patients. This study explored whether patients with SAA could benefit from continuous use of EPAG beyond 6 months. Methods From February 2018 to October 2021, 92 Chinese patients who were 2 years of age or older with a new diagnosis of acquired SAA and were not eligible for front-line hematopoietic stem-cell transplantation were collected in the China Eastern Cooperation Group for Anemia (CECGA). Patients were treated with rabbit ATG (r-ATG) based IST consisting of CsA. Patients over 12 years old and aging 6-11 years old took EPAG orally at dose of 75mg and 37.5mg once daily, respectively; EPAG was administered with 1.25 mg per kilogram per day for patients with age distribution between 2 and 5 years. Generally, all patients were treated with EPAG at least 6 months. The lack of response at 6 months, relapse, HSCT, development of a clonal hematologic disease including myelodysplastic syndrome, and acute myelogenous leukemia, or disease- or treatment-related death are regarded as events in event free survival (EFS). Results The median age was 38 (2-78) years, with 18 patients (20%) under 18 years old and 21 patients (43%) over 60 years old. In the whole cohort, 61 patients (66%) were diagnosed as SAA and 31 patients (34%) with very severe aplastic anemia (vSAA). At 3, 6 and 12 months, the actuarial overall response rates (ORR) were 55% (51 of 92), 73% (67 of 92) and 81% (60 of 74) and complete response (CR) rates were 11% (10 of 92), 20% (18 of 92) and 34% (25 of 74), respectively. 18 patients (20%) who achieved CR within 6 months were under steady state. In 49 patients with PR at 6 months, 14 (35%) of 40 patients who were continuously exposed to EPAG improved to CR within 6.5 (3 ~ 10) months of median time after 6 months. Among 25 patients who failed at 6 months, 11 patients continued to use EPAG, and 5 patients (45%) improved responses with extended median time of 3 (1 ~ 6) months after 6 months (Figure 1). The cumulative effect curve showed that 93% and 58% of all 12 months remission and CR occurred within 6 months. In patients with PR and NR at 6 months, the better 2-year event free survival (EFS) was found in whom continued to use EPAG (75% vs. 26%, P=0.001) (Figure 2). Discussion EPAG plus IST were used to treat previously untreated SAA patients in prospective studies. The ORR and CR rate at 6 months were 68% ~ 94% and 26% ~ 58%, respectively [1, 2]. We found that 93% of all 12 months remission occurred within 6 months for patients treated with IST and EPAG. It is suggested that EPAG should be used in sufficient dosage at least 6 months. It has been reported that patients with CR and PR were present with better OS of 100% and 92%, while OS of non-responders was 47% (P=0.0016), effect was demonstrated as a predictor for 4-year OS [3]. For patients with PR and NR at 6 months in our study, it was found that not only the response rates, but also the 2-year EFS were improved by continuous usage of EPAG, compared to them discontinued EPAG. In conclusion, additional EPAG to IST mainly take effect within 6 months. Continuous administration of EPAG could improve the hematologic response and EFS in patients without achieving CR at 6 months. REFERENCE [1]. Townsley DM, Scheinberg P, Winkler T, et al., Eltrombopag Added to Standard Immunosuppression for Aplastic Anemia. N Engl J Med, 2017. 376(16): p. 1540-1550. [2]. Peffault de Latour R, Kulasekararaj A, Iacobelli S, et al., Eltrombopag Added to Immunosuppression in Severe Aplastic Anemia. N Engl J Med, 2022. 386(1):11-23. [3]. Assi R, Garcia-Manero G, Ravandi F, et al., Addition of Eltrombopag to Immunosuppressive Therapy in Patients with Newly Diagnosed Aplastic Anemia. Cancer, 2018. 124(21):4192-4201. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prospective Follow-up of Patients with Myelofibrosis (MF) and Treated with Ruxolitinib in France: The Rumycup Study

Introduction MF is a clonal blood disease affecting 1.5 per 100,000 people worldwide. MF is characterized by megakaryocytic proliferation and atypia, usually accompanied by reticulin and/or collagen fibrosis, splenomegaly, anemia, and constitutional symptoms. Current treatments for MF are palliative, aiming to alleviate symptoms and restore quality of life. Ruxolitinib (Jakavi®) is an oral JAK1/JAK2 inhibitor, first in its class, and currently approved in >90 countries for the treatment of splenomegaly or disease-related symptoms in patients with MF. This study aimed to evaluate the use and outcome of ruxolitinib therapy during the first year after initiation in clinical practice. Methods RUMYCUP was a French, multicenter, non-interventional, prospective study. Adult patients with MF primary or secondary to polycythemia vera (PV) or essential thrombocythemia (ET) and for whom a decision was taken to initiate treatment with ruxolitinib, were included in the study. Primary data were collected at inclusion and at 3, 6, 9 and 12 months. The primary objective of this study was to assess ruxolitinib exposure during 11.5 months after treatment initiation. The secondary objectives were to describe patients' characteristics and treatment with ruxolitinib. Results Between Oct-2018 and Sep-2021, 152 patients were enrolled in the study in 49 French centers. Of them, 146 were included in the analysis population and 150 were included in the safety population. Overall, 50.7% of patients (95% CI: 42.3; 59.0) received ruxolitinib for at least 11.5 months, with a higher proportion in patients with splenomegaly at initiation than in those with no or unknown splenomegaly (53.8% vs 37.9%). At inclusion, 58.9% of patients were male, and the mean age was 70.9 years. Half of the patients were diagnosed with primary MF, 33.6% had a post-ET and 15.8% had a post-PV MF. The driver mutations were JAK2V617F in 69.2% of patients, CALR in 11.6%, and MPL in 4.8%. Among patients with available prognostic scores, 30.9% had a high-risk disease, 36.8% and 26.5% had an intermediate 2 or 1 risk disease, respectively, and 5.9% had a low risk disease, based on the International Prognosis Scoring System. At inclusion, 27.4% of patients had hepatomegaly, 32.9% had night sweats, 21.2% had weight loss, and 5.5% had fever. Within the 3 months prior to ruxolitinib initiation, 72.3% of patients did not receive any transfusion of Red Blood Cell (RBC) concentrates, 17.0% had >2 and 10.6% had 1 or 2 RBC transfusions. The median length of the spleen measured by palpation was 8.0 cm below costal margin and the median decrease was of 2.5 cm at Month 12. At inclusion, 58.2% of patients had had at least one previous treatment line and 19.9% two or more lines. As first line, 55.8% of patients received hydroxyurea. During follow-up, <43% of patients received RBC units and <10% of patients received platelet transfusions. Ruxolitinib was initiated at a median dose of 25 mg/day, and the main reasons for starting treatment were splenomegaly (78.1% of patients) and symptoms (58.9%). During follow-up, the dose was increased in 26.7% of patients (mostly for optimization of efficacy), decreased in 14.4% (mostly due to the occurrence of an adverse event [AE], including lack of efficacy) and both increased and decreased in 21.2% of patients. The treatment was temporarily stopped for 20% of patients, for a median 0.5 months. For 27.6% of them, the treatment interruption lasted >21 days. A total of 513 AEs were described in 89.3% of patients. AEs suspected to be related to ruxolitinib were reported in 57.3% of patients, and 58.0% reported 251 serious AEs (SAEs). Overall, 42 SAEs reported in 24 (16.0%) patients led to death. One SAE suspected to be related to ruxolitinib treatment (hemorrhage), led to the death of one patient. The 23 other deaths were not suspected to be related to ruxolitinib treatment and the most frequent SAEs leading to death, were disease progression (5/150 patients) or myelofibrosis and malignant neoplasm progression (3/150), COVID-19 (3/150) and other infections (5/150). Conclusion This 1-year follow-up conducted in France gives a first overview on the exposure to treatment and disease characteristics in patients with MF treated with ruxolitinib in clinical practice. The evaluation in real life conditions showed that half of the patients included in this study maintained ruxolitinib treatment for at least 11.5 months after initiation.

Bone Marrow Microenvironment Changes in Myelodysplastic Neoplasms and Its Relationship with Clonal Hematopoiesis and Disease Progression

Background: Immune dysregulation and somatic gene mutations are known prognostic factors in myelodysplastic neoplasms (MDS). During the evolution of the disease, a decrease in the immune surveillance is observed, with impaired cytotoxicity and a decrease in mature natural killer cells (NK), among other changes. Also, over expression of inflammatory cytokines, plays a role in the immune environment. The interaction between immune populations and the malignant clone is not entirely understood, especially in the bone marrow. The aims of this study were to characterize bone marrow (BM) immune populations, especially NK cells, and its correlation with the malignant clone. Methods: We prospectively collected 70 BM samples. Fifty MDS without RS or 5q deletion, 11 cytopenia of unknown significance (CUS) and 9 healthy donors (HD). The aims were to analyze NK cells, defined as (CD45+CD3-CD19-) and differentiating an immature population (CD56brigthCD16−NKG2A/CD94+KIR+/−) from a mature one (CD56dim CD16+NKG2A/CD94−KIR+). Their different receptors: activators (NKp46, NKp30, NKG2C, NKG2D, NKp44, DNAM) and inhibitors (TIGIT, NKG2A, Irp60, and PD1), as well as clonal cells expression of their ligands (HLA-ABC, MICA-B, CD155, PD-L1). Finally, KIR haplotype was analyzed, by NGS. Other populations included were myeloid-derived suppressor cells (MDSC), differentiating granulocytic (Gr-MDSC: CD11b+CD33+HLA-DR-CD15+CD14-) and monocytic (Mo-MDSC:CD11b+CD33+ HLA-DRlow/CD15+ CD14+). The analysis was performed, by flow cytometry, on BM fresh samples, following a membrane staining protocol with 10-colors/3 lasers. Molecular analysis was performed by NGS using the Oncomine Myeloid Research Assay included 40 genes. Finally, cytokines concentrations were stablished with the commercial kit ProcartaPlexTM Multiplex immunoassay. Results: A total of 70 subjects (median age of 77 years old and 45% female) were included (Figure 1). Although, total NK was similar among the three groups [MDS 0.17x109/L ICUS 0.07x109/L, HD 0.20x109/L, p= 0.1204], we observed differences between NK subpopulations. An increase of mature NK cells CD56dimCD16+ in CUS (76.16%) compared with MDS (11.58%) and HD (13.67%) [p= 0.0005] was observed. Regarding NK activating receptors, DNAM receptor was highly expressed on MDS (96.05%) and HD (90.98%), compared to CUS (67.79%) [p= 0.007], and the expression of KIR2DS4 was decreased in MDS (28.47%) compared to HD (79.4%) [p= 0.005], as well as NKp44 on MDS (0.19%) compared with HD (0.34%) [p= 0.03]. Interestingly, no expression of KIR2DS4 or NKp44 were observed on CUS. Regarding inhibitor receptors, Irp60 was increased on MDS (95.52%) compared to CUS (85.07%) and HD (80.2%) [p= 0.036] (Figure 2). Inhibitory KIR haplotype AA was observed on 13/47 MDS (27.56%). Finally, no differences were observed between Gr-MDSC [MDS 0.51%, ICUS 0.13%, HD 0.33%, p=0.35] or Mo-MDSC [MDS 1.03%, ICUS 0.25%, HD 0.7%, p=0.28] expression, among groups. Eighty-four percent (42/50) of MDS presented mutations at diagnosis, with a median number of 3. Interestingly, 63.6% (7/11) of CUS presented mutations, 2 DMT3A (VAF < 5%), 1 TET2 (VAF 40%), 1 IDH1 and 1 SF3B1 (VAF 33%), among others. Within subjects presenting mutations, we observed differences on the expression of NKG2A, showing an increase on those with ≥3 mutations (37.23%) vs <3 mutations (35.3%) [p=0.02]. Moreover, differences were found on KIR2DL1 [≥3 mutations 6.66% vs <3 mutations 15.61%, p=0.05]. On the 7 clonal cytopenia of unknown significance (CCUS), no differences were found on the expression of NK receptors compared to idiopathic cytopenia of unknown significance (ICUS). Cytokine analysis on BM of 39 MDS, showed increased levels of IL-10 in HR compared to LR [2 pg/ml vs. 1 pg/ml, p=0.04], including 16 (53%) subjects with very high concentrations (> 40 pg/ml). In addition, we analyzed NK cells of 14 MDS subjects who had a second BM sample, one year after diagnosis. We documented an increase in CD244 NK population [15.23% follow-up vs 3.61% diagnosis, p=0.05]. Conclusions: Our study suggests the presence of immunosuppressive profile of NK cells on MDS, with gain of expression of NKG2A and higher expression of IL10 on high risk MDS. Interestingly, 63.6% CUS subjects were CCUS and showed increase cytotoxic profile. The increase of CD244 expression, at one year control, may be related to an exhausted environment leading to immunosuppression and progression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Metabolic Biomarkers Affecting Cell Proliferation and Prognosis in Polycythemia Vera

Background Polycythemia vera (PV) is a malignant clonal hematological disease of hematopoietic stem cells characterized by proliferation of peripheral blood cells, and JAK2 mutation is one of the main causes of PV peripheral blood cell proliferation. Abnormal cell metabolism is a new feature of malignant proliferation of tumor cells, but the role of metabolism in the pathogenesis and prognosis of PV remains unclear. Methods Metabolic profiling of peripheral blood sera from 32 patients with PV and 20 HCs was performed using liquid chromatography-mass spectrometry (LC-MS). Partial least-squares with latent structure discriminant analysis (PLS-DA) was used for data analysis. Results To assess whether there were significant changes in the metabolic profiles of PV patients and healthy controls, we performed a multifactorial analysis of the metabolic profiles of the two groups. Figure 1 shows the score plots of the PLS-DA in positive and negative modes. Both data from positive and negative modes suggested that the serum metabolites of PV patients were significantly changed. Subsequently, we identified endogenous metabolites between PV patients and HCs. These potential biomarkers were eventually identified as fatty acids, acylcarnitine, sphingolipids, amino acids, etc. To clarify the abnormal metabolic pathways in PV, we performed enrichment analysis of the metabolic pathways of the biomarkers. The results showed there were 9 important metabolic pathways associated with PV, namely: D-glutamine and D-glutamate metabolism, sphingolipid metabolism, pyruvate metabolism, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, tryptophan metabolism, lysine degradation, arginine biosynthesis and glycolysis/gluconeogenesis (Figure 2). Therefore, fatty acid metabolism, glucose metabolism, sphingolipid metabolism and amino acid metabolism were significantly altered in PV. To explore the effect of JAK2-associated metabolic abnormalities on cell proliferation in PV patients, we screened for endogenous metabolites associated with JAK2, and found 7 metabolites that differed significantly between the JAK2 mutated and unmutated groups. The levels of Cer(d18:2/22:6-2OH(7S, 17S)), SM(d18:0/PGF1α), CerP(d18:1/16:0), glutamic acid, lactic acid, melibiose and xylose were significantly higher in the JAK2-mutated group than in the JAK2-unmutated group. Among them, Cer(d18:2/22:6-2OH(7S, 17S)) (r=0.817; P<0.001) and SM(d18:0/PGF1α) (r=0.463; P=0.021) levels were positively correlated with JAK2 mutational burden. Then we evaluated the relationship between JAK2 mutation and blood cell count in PV patients. PLT and HCT levels were significantly higher in JAK2 mutated PV patients than in JAK2 unmutated PV patients (P=0.006; P=0.044) and found a significant positive correlation between JAK2 mutational burden and WBC and PLT levels (r=0.62, P=0.001; r=0.55, P=0.007). Finally, we found that Cer(d18:2/22:6-2OH(7S, 17S)) levels were positively correlated with WBC (r=0.650, P<0.001), and SM(d18:0/PGF1α) levels were positively correlated with WBC and PLT (r=0.444, P=0.011 and r=0.623; P<0.001, respectively). Therefore, we speculate that Cer(d18:2/22:6-2OH(7S, 17S)) and SM(d18:0/PGF1α) are closely associated with JAK2 mutations and may contribute to the proliferation of peripheral blood cells in PV patients. According to the prognostic point system, PV patients can be divided into low-risk group (n=11) and intermediate/high-risk group (n=21). Compared to the low-risk group, the levels of 8 endogenous metabolites in the intermediate/high-risk group were significantly elevated. We next used binary logistic regression to analyze the combined biomarkers and then determine the ROC curve. A set of potential biomarkers containing four endogenous metabolites with an AUC > 0.77 was found to provide an AUC of 0.952, with a sensitivity of 90.905% and specificity of 90.909% at the optimal cutoff point. Therefore, elevated levels of Cer(d18:2/22:6-2OH(7S, 17S)), SM(d18:0/PGF1α), CerP(d18:1/16:0) and octadec-13-enoylcarnitine could be used as potential metabolic biomarkers of poor prognosis in patients with PV. Conclusion Metabonomics provides an important tool for the study of the pathogenesis of PV and the relationship between JAK2 gene mutation. Furthermore, the potential biomarkers of this study may provide a reference for the prognosis of PV. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Real-World Single-Institution Experience with Hypomethylating Agents Plus BCL-2 Inhibitor for the Treatment of Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is a heterogeneous clonal disease of myeloid progenitor cells resulting from alterations in cell differentiation, proliferation, and renewal. Treatment for young patients and those with favorable-to-intermediate risk remains a combination of cytarabine and anthracycline, while the optimal regimen for the elderly has yet to be established. First-line and salvage combination hypomethylating agents azacitidine or decitabine with BCL-2 inhibitor venetoclax (HMA+Ven) has shown promise for these patients and are becoming standard therapy. We present a cohort from our institution treated with HMA+Ven vs traditional therapy. We hypothesize HMA+Ven leads to improved overall survival (OS) and complete remission with or without count recovery (CR/CRi) compared to traditional therapy in the elderly and those with unfavorable risk as defined by the European LeukemiaNet (ELN-2017) risk stratification. Methods: 232 AML patients from Baylor St. Lukes Medical Center, and Michael E DeBakey VA Medical Center from 8/26/1997 to 7/31/2021 were reviewed. These patients were stratified by HMA+Ven vs. other standard treatment. Differential survival among groups was measured by Kaplan-Meier method. The independent effect of HMA+Ven against multiple intervening variables was evaluated by Cox proportional regression analysis. Chi-square and t-test detected potential intervening variables with impact on outcome. SAS software was used for analysis. Results: Those treated with HMA+Ven comprised 28 out of 227 patients (12.3%). Average age of patients was 65 (26-80) in the HMA+Ven group and 61.3 (20-93) in the other treatment group (p=0.05). Age, ELN-2017 risk stratification, and HMA+Ven treatment were predictors for survival (Table 1). OS in our cohort favors HMA+Ven vs other treatment (Fig. 1, 348 days vs 261 days, p=0.0004). Overall survival in the elderly population of interest favors HMA+Ven (270 days vs 135 days, p<0.0001). CR/CRi was superior for HMA+Ven in those age >60 (55% vs 24.7%, p=0.006) as well as those age >70 (55% vs 15.2% p=0.007). OS for those with adverse ELN-2017 stratification favors HMA+Ven (352 days vs 164 days, p=.002). Interestingly, overall survival trends for those with active smoking history favors HMA+Ven (p<0.0001). Lastly we found CR/CRi favors HMA+Ven for those with RUNX1 gene mutation (75% vs 25%, p=0.01). Conclusion: HMA+Ven demonstrated superior response in our elderly patients and those with unfavorable risk through OS as well as CR/CRi, compared to those treated with traditional therapy. This held true across all ELN-2017 risk categories when examined by cox regression models (Table 1) and was most notable in the adverse risk subgroup. The mechanism by which smoking may enhance HMA+Ven survival is uncharacterized and warrants investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Spatial Organization of Hematopoietic Clones in the Bone Marrow of a Patient with Polycythemia Vera

All humans become genetic mosaics over time as our stem cells acquire somatic mutations throughout life. In the hematopoietic compartment, this phenomenon is termed clonal hematopoiesis (Steensma et al. 2015). Clonal hematopoiesis (CH) increases in prevalence with age and is associated with an increased risk for hematologic malignancy (Genovese et al. 2014; Jaiswal et al. 2014; Xie et al. 2014). CH harboring JAK2 mutations are frequently observed in healthy individuals; and yet JAK2 mutations are also the most frequent drivers of myeloproliferative neoplasms (MPNs) such as polycythemia vera (PV). MPNs are clonal diseases marked by dysregulated hematopoiesis and can progress to a secondary myelodysplastic syndrome or acute leukemia. There is increasing evidence that the clonal mutations driving MPNs arise early in life-potentially in utero-often years before the development of symptoms (Williams et al. 2022). While the intrinsic factors driving clonal expansion are increasingly known (Watson et al. 2020), the extrinsic factors driving the expansion of clones harboring these MPN driver mutations are not well characterized. Prior work has shown that clonal mutations are geographically constrained in solid organs such as skin, colon and esophagus (Lee-Six et al. 2018; Martincorena et al. 2015, 2018). Strikingly, in the esophagus non-malignant NOTCH1 mutant clones can even outcompete nascent malignant clones (Colom et al. 2021). In the hematopoietic compartment, the spatial organization of clones harboring these mutations and their interaction with the niche and each other is largely unknown. We hypothesized that clonal mutations are geographically constrained in the bone marrow niche as is seen in solid organs. To answer these questions, we developed methods for spatially-aware clonal mutation detection. As proof of principle, we characterized the hematopoietic clonal organization within a femur head explant from a 50 year-old man with PV undergoing hip arthroplasty. Briefly, the femur head was sectioned into 2mm slices, which were then further partitioned into approximately 300 bone marrow fragments (2mm3). Approximately 20% of the fragments were snap frozen and pulverized for DNA extraction. The remainder were formalin fixed and decalcified for correlative imaging. Targeted error-corrected sequencing was performed using a myeloid-specific panel (Duncavage et al. 2018) on 24 bone marrow fragments, and bulk peripheral blood and bone marrow aspirate. In the peripheral blood, JAK2 V617F (0.24 VAF) and TET2 L1081* (0.02 VAF) mutations were identified. In the bone marrow aspirate, the same JAK2 (0.62 VAF) and TET2 (0.05 VAF) mutations were identified in addition to a DNMT3A R882C mutation (0.02 VAF). Interestingly, peripheral blood sequencing two years prior identified the JAK2 and DNMT3A mutations, but not the TET2 mutation, suggesting recent clonal expansion of TET2 mutant clone. From the 24 sequenced bone marrow fragments, the geographical organization of clonal mutations in the bone marrow was determined (Figure 1). The JAK2 V617F clone was identified in all bone marrow fragments sequenced (0.12-0.69 VAF). The TET2 L1081* clone was identified in 11/24 bone marrow fragments with some regional constraint (0.03-0.05 VAF). The DNMT3A R882C mutation was detected in only a single bone marrow fragment (0.02 VAF) and co-localized with the JAK2 V617F and TET2 L1081* mutations. A previously undetected clonal SETBP1 E616V mutation was identified in two adjacent bone marrow fragments from sequential slices of the femur head (0.05 VAF and 0.10 VAF). Together this provides the first characterization of spatial organization of clonal mutations in the bone marrow niche for an MPN patient. Currently, we are utilizing digital droplet PCR targeting these detected mutations in ~60 additional bone marrow fragments to accurately quantify clone size across the tissue. We plan to employ multiplex fluorescent imaging of bone marrow fragments adjacent to detected clusters of mutations to identify the surrounding bone marrow niche elements in those regions. These findings suggest that bone marrow biopsy from a small section of bone marrow may not accurately describe disease state and clonal mutation burden in myeloid neoplasms. Future applications of these tools will help describe the extrinsic factors and local microenvironment that drive clonal evolution over time in the bone marrow. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High Expression of Plasma IL-1β Levels and Transition of Regulatory T-Cell Subsets Correlate with Disease Progression in Myelodysplastic Syndrome

Objective: Myelodysplastic syndrome (MDS) is a group of heterogeneous hematopoietic clonal diseases. Recent studies have confirmed that there are essential immune differences in the progression of MDS. However, the detailed mechanisms remain unclear. In order to explore the changes of immune disorders during the occurrence and progression of MDS, and identify critical differentially expressed factors as well as prognostic relevance. What's more, to probe the possible immunological mechanism in the progression of MDS. Methods: The study had a prospective design. A total of 86 patients with newly diagnosed MDS were enrolled in this study, where patients were classified as low risk group and high risk group based on the revised international prognostic scoring system (IPSS-R) score. The expression profiles of multiple cytokines in patients’ plasma were determined using Luminex microarray assay, and flow cytometry were used to measure lymphocyte subsets in peripheral blood, especially T regulatory cell (Treg) and its subsets: induced regulatory Treg (iTreg: CD45RO+CD3+CD4+CD25+CD127low+) and naïve regulated Treg (nTreg: CD45RA+CD3+CD4+CD25+CD127low+), and the percentage of Monocytic-myeloid derived suppressor cells (M-MDSCs) in bone marrow. The mRNA expression of NLRP3 inflammasome related molecules was detected by real-time quantitative PCR. P <0 .05 was considered statistically significant. Results: In the high risk group, the plasma levels of some cytokines or chemokines with promoting or amplifying inflammation signals in directly or indirectly way, including IL-9, MIP-1β,GRO-α and TNF-β, and two growth factors influencing the immune microenvironment by regulating the growth and differentiation of corresponding cells, including β-NGF, PDGF-bb, were significantly lower (P<0.05).The expression level of IL-1β was significantly higher in the high risk group (P<0.05) and its expression was significantly correlated with the percentage of M-MDSCs (P<0.0001). What's more, the percentage of bone marrow M-MDSCs facilitated immune tolerance was significantly higher. Although there is no significant difference of Treg between the two groups, both the percentages and absolute counts of nTreg/iTreg was decreased significantly in high-risk group of MDS patients (P<0.05) (Figure). After adjusting the Cox model, nTreg/iTreg was an independent protective factor for OS (P=0.038, HR=0.081, 95% CI: 0.008-0.869), and elevated IL-1β level was an independent prognostic factor for PFS (P=0.018, HR=2.554, 95% CI: 1.175-5.552). Then, we found that the mRNA expression of NLRP3, ASC, Caspase 1 and IL-1β were significantly increased in the high risk group(P<0.05). Conclusion: All above results demonstrated that the expression of pro-inflammatory cytokines and chemokines was increased in low risk MDS, while the amplification of bone marrow M-MDSCs and the transformation of nTreg to iTreg may be the key immune mechanisms leading to disease progression. The activation of NLRP3/IL-1β pathway may play an important role in the progression of disease. The serum levels of IL-1β and the percentage of nTreg /iTreg ratio may be an effective indicator to predict the prognosis of MDS. It provided a basis for clinical prediction and early intervention to improve the prognosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Heme Oxygenase-1 Inhibition the Cytotoxicity of NK Cells to AML By Downregulating HLA-C

Backgroud and objective: Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem cells with abnormal differentiation of myeloid primordial naive cells, which has the highest incidence in adult Acute leukemia with a low cure rate . Clinically, less than one-third of adult AML patients can achieve sustained remission after cytarabine combined with anthracycline chemotherapy, and over 50% relapse after remission. Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is still the only way to cure leukemia clinically. However, due to age limitation, infection, graft-versus-host disease, and other complications, it is not suitable for all AML patients In recent years, numerous novel drugs have provided more options for the treatment of relapsed and refractory AML while the overall response rate is unsatisfactory and there is no significant advantage in improving overall survival Exploring the mechanism affecting AML relapse and refractory treatment, formulating effective coping strategies are the key to overcome the treatment bottleneck of AML Methods: Bone marrow nucleated cells were obtained from 15 newly diagnosed AML patients, 15 recurrent AML patients and 15 normal controls. The expressions of HO-1 and HLA-C were detected by quantitative reverse transcription PCR (RT-PCR) and Western blot. In vitro cell experiments, we constructed SiHO-1 lentiviral vector to transfect AML cell lines, cocultured NK cells with it, measured the change of CD107a expression on NK cell surface, and silenced HLA-C expression with small interfering RNA. The silenced AML cell lines were co-cultured with NK cells, and CD107a secretion on NK cells was measured by flow cytometry. Results: HO-1 expression in AML patients was higher than that in healthy donors (P<0.05), while HLA-C expression was lower than that in healthy donors (P<0.01). Additionally, the plasma HO-1 levels gradually increased, while the HLA-C levels gradually decreased from healthy donors to patients with relapsed AML. In addition, Pearman correlation analysis indicated that HLA-C expression was significantly correlated with HO-1 expression (r=0.635, P<0.001). In AML cell lines, HO-1 silencing enhanced the cytotoxicity of NK cells to AML cells (P＜0.01), while HO-1 overexpression weakened the cytotoxicity of NK cells to AML cells (P＜0.05). The expression of CD107a in NK cells after HLA-C silencing was lower than the control group in U937 and THP-1 cell line. (P＜0.05). Conclusion: decreased HLA-C expression is associated with increased HO-1 expression during the progression of AML. Besides, the inhibition of HO-1 promotes HLA-C expression, which can therefore promote cytotoxicity of NK cells to AML cells.

Congenital Erythrocytosis Due to Novel CMTR1K769L Mutation Has Augmented Hypoxia Signaling and Features of Both Primary and Secondary Erythrocytosis

Primary erythrocytoses (PE) include germline gain of function mutations of erythropoietin (EPO) receptor (EPOR) gene and polycythemia vera (PV), a clonal disease due to acquired somatic JAK2 mutations. Both these PEs have intrinsically abnormal early erythroid progenitors (BFU-E) characterized by their in vitro EPO hypersensitive growth (EPORmutations), or even EPO independent growth (the hallmark of PV); both have low plasma EPO (Chapter 58, Williams Hematology, 10e). Secondary erythrocytoses (SE) have intrinsically normal BFU-Es but are caused by increased circulating erythropoiesis stimulators, chiefly EPO. In contrast, congenital Chuvash erythrocytosis is characterized by augmentation of hypoxia inducible factors (HIFs) causing high EPO and hypersensitive BFU-E's EPO responses; thus, it has features of both PE and SE. We report a 7-year-old normoxic girl with history of symptomatic erythrocytosis since birth of unknown etiology, with hemoglobin of 22 g/dL at birth, consistently elevated EPO at 50-75 mU/mL, and normal hemoglobin oxygen affinity. Her BFU-E grew without EPO yet no somatic PV defining mutations were found. A targeted analysis of previously described erythrocytoses genes was negative. Whole genome sequencing, performed under the auspices of Undiagnosed Disease Network, identified a heterozygous missense variant (p. Lys769Leu) of CMTR1(Cap Methyltransferase 1) gene, not listed in the Genome Aggregation Database (gnomAD). Cap methylation on mRNA protects mRNA from being degraded and promotes RNA translation by recruiting translation initiation factors (PMID:30312682). The variant was present in the proband but not in her asymptomatic mother (father was not available for the analysis). This variant was found in leukocytes and in nail DNA, indicating germline origin. Transcriptional X-chromosome inactivation assay (PMID:18641369) revealed polyclonal granulocytes and platelets, confirming that the erythrocytosis is caused by germline mutation, not by somatic mutation-driven clonal hematopoiesis. Because of her high EPO, we hypothesized that CMTR1K769L variant might cause high HIF activity. HIF-mediated gene regulation is tissue- and stage of differentiation-specific. Expression of HIF target genes SLC29A1, VEGFA, EDN1, and LDHA was upregulated in granulocytes while in platelets all target genes except SLC29A1 were upregulated. To test if CMTR1 regulates HIF transcriptional activity, 1) we correlated CMTR1 transcript levels with HIF target gene expression levels in granulocytes and platelets. CMTR1 transcript levels were positively correlated with expression levels of HIF target genes. 2) we tested if CMTR1 overexpression and knockdown alters HIF target gene expression. In erythroid cell lines (K562 and HEL), overexpressed CMTR1 increased SLC2A1 and VEGFA (HIF target genes transcripts) while knockdown of CMTR1 downregulated their expression. This demonstrates that CMTR1 positively regulates HIF transcriptional activity, suggesting that CMTR1K769L accounts for increased plasma EPO and for EPO mediated feature of SE. As erythroid progenitors express endogenous erythropoietin (PMID 9573013) which contributes to their differentiation and proliferation (PMID 10903342), we evaluated if CMTR1K769L also promotes endogenous erythroid EPO accounting for EPO independent growth of CMTR1K769L mutated BFU-Es and thus provides potential mechanism as a feature of PEfor the erythrocytosis. The proband's endogenous EPO transcript levels in in vitro expanded erythroid cells at day 4, when most of cells are erythroid progenitors, were 10 times higher than the mother's see Figure. We present a new cause of erythrocytosis associated with the novel germline mutation of principal epigenetic modifier of RNA, CMTR1 (PMID:33922187), associated with high EPO (SE). Her BFU-E grew without extrinsic EPO (PE) likely due to previously unobserved stimulation of EPO production in erythroid progenitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 12981: Non-Conventional Causes of ST-Elevation Myocardial Infarction: Essential Thrombocytosis

Case: A 61-year-old female with no significant past medical history, except hospitalization for COVID-19 pneumonia complicated by myocarditis ten months prior, presented to the emergency department with worsening chest pain over the last two weeks. The admission electrocardiogram (ECG) was without ischemic changes. The initial impression was that the patient's symptoms were attributed to her prior myocarditis. However, a repeat ECG obtained six hours later, due to worsening chest pain, demonstrated ST-segment elevations in leads V2, I and AVL. Emergent coronary angiography was performed, showing a 90% occlusion of the proximal LAD, which was managed with a drug-eluting stent. Admission platelet count was significantly elevated at 1,049,000mm 3 , which resulted in a hematology consultation. Her further work up included a positive JAK2-V617F mutation and negative BCR-ABL1 translocation, which confirmed the diagnosis of essential thrombocythemia. During the prior hospitalization for her COVID pneumonia, she underwent invasive coronary angiogram for suspected myocardial infarction in the setting of elevated troponin, which showed a mild, non-obstructive lesion in the proximal LAD. She underwent a cardiac MRI that demonstrated both sub-epicardial and mid-wall late gadolinium enhancement consistent with myocarditis. Discussion: Essential thrombocythemia is a clonal myeloproliferative disease caused by a pathological expansion of megakaryocytes, which results in excess platelet production. Essential thrombocythemia leads to a hematologic hyper-viscosity and subsequent increased risk of vasomotor, thrombotic and hemorrhagic manifestations. Myocardial infarction as the primary clinical presentation of essential thrombocythemia is rare but an important risk factor that should be considered in patients presenting with acute coronary syndromes and elevated platelet counts, both with and without traditional cardiovascular risk factors.

Prognostic Impact of Unsupervised Early Assessment of Bulk and Leukemic Stem Cell Measurable Residual Disease in Acute Myeloid Leukemia.

Acute myeloid leukemias (AML) are clonal diseases that develop from leukemic stem cells (LSC) that carry an independent prognostic impact on the initial response to induction chemotherapy, demonstrating the clinical relevance of LSC abundance in AML. In 2018, the European LeukemiaNet published recommendations for the detection of measurable residual disease (Bulk MRD) and suggested the exploration of LSC MRD and the use of multiparametric displays. We evaluated the performance of unsupervised clustering for the post-induction assessment of bulk and LSC MRD in 155 patients with AML who received intensive conventional chemotherapy treatment. The median overall survival (OS) for Bulk+ MRD patients was 16.7 months and was not reached for negative patients (HR, 3.82; P < 0.0001). The median OS of LSC+ MRD patients was 25.0 months and not reached for negative patients (HR, 2.84; P = 0.001). Interestingly, 1-year (y) and 3-y OS were 60% and 39% in Bulk+, 91% and 52% in Bulk-LSC+ and 92% and 88% in Bulk-LSC-. In this study, we confirm the prognostic impact of post-induction multiparametric flow cytometry Bulk MRD in patients with AML. Focusing on LSCs, we identified a group of patients with negative Bulk MRD but positive LSC MRD (25.8% of our cohort) with an intermediate prognosis, demonstrating the interest of MRD analysis focusing on leukemic chemoresistant subpopulations.