Chronic Myelomonocytic Leukemia Research Articles

Update of a Phase II Trial of Guadecitabine in Higher-Risk Myelodysplastic Syndrome and Chronic Myelomonocytic Leukemia Under the International Working Group 2023 Criteria

Introduction: Guadecitabine is a dinucleotide of decitabine which has shown high response rates in higher-risk myelodysplastic syndrome. In this update we seek to evaluate the performance of the International Working Group (IWG) 2023 criteria in a prospective, systematically annotated clinical trial. Methods: Patients older than 18 years old with higher-risk MDS/CMML as defined by the International Prognostic Scoring System - Revised (IPSS-R), or those with more than 10% bone marrow blasts were eligible. Guadecitabine was administered at a dose of 60 mg/m2 subcutaneously daily for 5 days every 28 days. We re-classified patients by World Health Organization 2022, calculated IPSS-Molecular (IPSS-M) scores and clinical responses were reassessed under the IWG-2023 criteria and compared to IWG-2006. Results: From 2014 to 2018, 82 patients with MDS and 18 patients with CMML were enrolled and treated. Median age was 69 (IQR 62.5 - 75), 62% were male, 38.4% had complex karyotype, 86.6% were categorized as IPSS-M high or very-high risk. Additionally, 33.7% were TP53 mut, 21.4% were TP53 multi-hit. Forty percent of patients were classified as MDS-IB-2, 21.4% as MDS-biTP53, and 17.1% as MDS-IB1. Common mutations were TP53 (38.5%), ASXL1 (22%), DNMT3A (18%) in MDS. ASXL1 (61%), TET2 (61%), NRAS (28%), and SRSF2 (22 %) for CMML patients. Most common treatment-emergent adverse events were fatigue (79% G1-2, 6% G3), nausea (37% G1), injection site reaction (29% G1), and constipation (23% G1). Grade 3 events were febrile neutropenia (32%), infection (25%), and thrombocytopenia (16%). Eight-week mortality was 4%. Ninety-six patients were evaluable for response. The median number of cycles was 5 (IQR:3-8) number of cycles to best response was 3 (IQR:2-5). Median overall survival (mOS) was 16.8 months. Survival was response-dependent with mOS in months being 32, 23.3, 15.5 and 8 for complete remission (CR), mCR (marrow complete remission), HI (hematologic improvement) and NR (no response) by IWG 2006 criteria (Figure 1). By IWG-2023 overall response rate (ORR) was 52%, with a complete remission (CR) of 29%, 3% with complete remission with bilineage limited hematologic recovery (CRLbi), 11% with CRL with unilineage recovery (CRLuni), 5% complete remission with incomplete hematologic recovery (CRh), and 4% had hematologic improvement (HI). Patients classified as mCR by 2006 criteria (n=30) were reclassified as CR (6.6%), CRLbi (6.6%), CRLuni (33.3%), CRh (16.6%), HI (6.6%), NR (26.6%),and PR (3.3%). Patients in mCR who underwent allogeneic-stem cell transplant (allo-SCT) (n=9) had a mOS of 46 months, compared to 14.4 months to those who did not (log-rank, p < 0.005). Median OS in months was 32.8, 28.6, 17.5, 7.8, 16.8, and 9.2 for CR, CRLbi, CRLuni, CRh, HI, and NR, respectively (log-rank < 0.005). In a multivariate analysis TP53 multi-hitwas the only factor significant in prognosticating response (p = 0.023) and overall survival (p =0.03). Discussion: In this report we examine the performance of the IWG 2023 criteria in a prospective clinical trial. Patients classified as mCR had significantly heterogenous outcomes which were more precise when re-classified by IWG-2023. Patients in mCR may still benefit from allo-SCT. These criteria also demonstrate response-dependent overall survival outcomes. TP53 multi-hitwas predictive of response and overall survival. This analysis shows the improved accuracy of the IWG 2023 model in stratifying clinical responses and survival. These may serve as future benchmarks for drug development and trial design. Figure 1. Overall survival by A) IWG 2006 criteria and B) IWG 2023 criteria.

Secondary Acute Myeloid Leukemia from a Previous Chronic Myeloproliferative Neoplasm: A Study of Grelam-Chile Concerning Chilean Patients on Behalf of AML Pethema Registry

Introduction: Secondary Acute Myeloid Leukemia (sAML) from Chronic Myeloproliferative Neoplasm (chMPN) is associated with an unfavorable prognosis and is characterized by a unique set of cytogenetic and molecular features distinct from de novo AML. The only cure for these patients is allogeneic Stem Cell Transplantation (SCT), which usually is not possible given the advanced age and comorbidities from this patient population. Methods: We present a retrospective analysis of sAML Chilean patients due to chMPN from the multicentric epidemiological registry of AML of the PETHEMA Spanish Group. Results: There are 802 AML Chilean patients incorporated in the online PETHEMA platform. 158 are sAMLand 26 are secondary to chMPN. Compared with the sAML group, patients with previous chMPN were mainly male (56% vs. 76%, respectively), had a larger white blood cell count (32,720 mm3 vs. 61,130 mm3), and had splenomegaly (P <0.05). There was no difference in age (median 62,35 years old), peripheral blood blasts (media 28,8%), fibrinogen (287 mg/dl), or other laboratory tests (p>0.05). Regardless of the type of chMPN: Chronic Myeloid Leukemia: 8 patients: Chronic myelomonocytic leukemia: 5, Primary Myelofibrosis: 5, Chronic Myeloproliferative Syndrome not specified subtype: 6, Polycythemia Vera/essential Thrombocytosis 2. 50% received previously chemotherapy, mainly Tyrosine Kinase Inhibitors (7) and hydroxycarbamide (7). Regardless sAML % had an abnormal karyotype. Treatment used, it was Anthracycline/Daunorubicin (38%), azacytidine with or without Venetoclax (19%), and palliative care (23%). In the first induction, the rate of Complete response (CR), or complete response with incomplete hematological response (iCR), was reached in only 3 patients, and these had negative MRD. From the patients with partial responses, 9 patients received a second line of treatment, mainly azacytidine. After that, three more patients reached CR. Only one patient received allogeneic SCT. The average stay in the hospital was 41,4 days, and Overall survival (OS) was 12,2 months (OS in all sAML was 14,2 months, N.S. p>0.05). The mortality rate was 84%, and the main cause was progressive disease in 60% of those cases. Conclusion: We present our local experience with this type of sAML. The outcome remains poor; it seems that allogeneic SCT is the only cure, but most patients cannot reach it. More research is needed to improve the high rates of mortality for this patient population.

Transcriptomic Signature and Functional Abnormalities of Bone Marrow Mesenchymal Stromal Cells Mediate Disease Progression of Myelodysplastic/Myeloproliferative Neoplasms

Background Mesenchymal stromal cells (MSC) are vital components of the bone marrow (BM) microenvironment niche and regulate hematopoietic stem and progenitor cell (HSPC) activity. While HSPC harbor primary disease driver mutations, MSC in myelodysplastic syndrome and myeloproliferative neoplasm (MDS/MPN) have been shown to influence the emergence and selection of leukemic clones and disease progression. However, it remains unclear if there are specific functional characteristics or genetic signals of MSC that interact with HSPC at different stages of disease progression. We examined BM samples from MDS/MPN patients with high rate of leukemic transformation in less than 2 years (HR) versus those with stable disease for 5 years (SD), to investigate the phenotypic differentiation, functional characteristics, and transcriptomic profiles of MSC and the HSPC counterparts. Methods We obtained BM samples at diagnosis and follow-up from MDS/MPN patients including chronic myelomonocytic leukemia (CMML) treated at Singapore General Hospital. We studied 5 HR and 13 SD patient samples to compare against 5 healthy donor samples. MSC were derived in MesenCult-ACF media (StemCell Technologies), and phenotypic characterization was done after 1-2 passages. HSPC were subjected to co-culture expansion with MSC in MyeloCult-H5100 for 7 days, and long-term culture-initiating cell assays (LTC-IC) using MSC as feeder cells for 6 weeks. Total RNA extracted from MSC and HSPC were subjected to bulk and single-cell RNA sequencing (scRNA-Seq) (10X Genomics platform). Principal component analysis (PCA) was used for the uniform manifold approximation and projection (UMAP). Gene set enrichment analysis (GSEA) was performed on differentially expressed genes to examine canonical signaling pathways. Cellchat algorithm was applied to predict intercellular communication between MSC and HSPC using scRNA-Seq data. Results Baseline clinical variables for the international prognostic scoring system (IPSS) including age at diagnosis, myeloblast percentage and cytogenetics were not significantly different between HR and SD patients. Decreased osteogenic and chondrogenic differentiation, but relatively preserved adipogenic differentiation capacity was observed in all MDS/MPN MSC samples. A significantly lower yield of CD34+ CD38- primitive HSPC with myeloid skewing was observed in the co-culture experiments with MDS/MPN MSC, compared to healthy MSC. Pre-treatment of SD-MSC feeder with the hypomethylating agent 5-azacytidine improved the clonogenic potential of the corresponding co-cultured HSPC, but did not restore the HSPC colony yield with HR-MSC feeder. Unbiased PCA of bulk RNA-Seq data demonstrated greater clustering of HR-MSC, while SD and healthy MSC overlapped significantly. Genes associated with mesenchymal multipotency and differentiation (PODXL20, FOXQ122, SERPINA923) were generally downregulated in MDS/MPN MSC, likely attributed to promoter hypermethylation. Significant differential upregulation of CD74 and IL-6 expression was observed in HR-MSC, which suggested a uniquely enriched inflammatory signature. GSEA results showed that pathways associated with G2M cell cycle checkpoint, DNA repair, glycolysis, and response to azacytidine were suppressed in HR-MSC. UMAP on scRNA-Seq data of longitudinal MSC samples identified distinct clustering of HR-MSC at disease progression. Cell-cell interaction analysis predicted a strong communication between collagen-encoding genes (COL1A1 and COL1A2) in HR-MSC at diagnosis and CD44, a negative regulator of HSPC proliferation, and the interaction was further enhanced at disease progression (Figure 1). Ongoing analysis of downstream gene expression changes and secretome profile of HSPC in MDS/MPN MSC-based in-vivo scaffold models will be presented. Conclusion Together, our data point towards a degree of co-development and communication between MSC and HSPC during disease progression of MDS/MPN. Further characterization of the unique functional and transcriptomic signatures in MSC may help to optimize disease prognostication and identify novel therapeutic targets in MDS/MPN patients.

Kdm6a Modulates Hematopoiesis and Leukemogenesis Via Demethylase-Dependent Epigenetic Programming

Hematopoietic stem and progenitor cells (HSPCs) maintain their differentiation by precisely regulating gene expression patterns at the transcriptional and epigenetic levels. KDM6A encodes an epigenetic regulator of the repressive chromatin mark histone 3 lysine 27 tri-methylation (H3K27me3). Growing evidence suggested the involvement of KDM6A in hematological malignancies. KDM6A mutations in leukemia frequently occur in males, reflecting that KDM6A escapes X chromosome inactivation. Therefore, female patients with single-allele loss-of-function KDM6A mutations will partially retain KDM6A expression, which is called haploinsufficiency. Furthermore, KDM6A haploinsufficiency was reported to be associated with refractory and/or relapsed leukemia, while the role of KDM6A haploinsufficiency in hematopoiesis remains unexplored. We established kdm6a-null zebrafish using CRISPR-Cas9 gene editing. We found that kdm6a loss impaired HSPCs formation. kdm6a-null embryos showed reduced expression of the hemogenic endothelial and HSPC markers runx1 at 28 hours post-fertilization (hpf). Using Tg(kdrl:mCherry;cmyb:en-GFP) embryos, we also found that the number of hemogenic endothelium and emerging HSPCs in kdm6a-null was significantly decreased. To study the importance of Kdm6a-mediated demethylase activity, we ectopically expressed kdm6a constructs (wild-type kdm6a, or catalytically-dead kdm6a) in kdm6a-null embryos. The result showed that, unlike restoring effect by wild-type kdm6a, the ectopic expression of catalytically-dead kdm6a constructs ( kdm6a H1134A) showed no rescue effect for decreased HSPC numbers. Then, we aimed to understand the potential role of kdm6a haploinsufficiency in hematopoiesis and leukemogenic mechanisms. We found that zebrafish mutants that are haploinsufficient in kdm6a ( kdm6a +/-) exhibited uncontrolled myeloid differentiation. The expression of myeloid-specific markers substantially increased in kdm6a +/- mutants, compared with WT siblings at 72 hpf. Confocal analysis showed that the ratio of myeloid-primed HSPCs was markedly increased in kdm6a +/- embryos compared to WT siblings. Consistently, a significant upregulation in transcripts encoding key myeloerythorid TFs including cebps, irf8 and gata1a was observed in kdm6a +/- embryonic HSPCs. Furthermore, kdm6a +/- adults exhibited the phenotype of monocytosis, which mimics chronic myelomonocytic leukemia, or exhibited the phenotype of increased progenitors, which mimics acute myeloid leukemia. We next sorted HSPCs from WT or kdm6a +/- at 72 hpf for RNA-sequencing and ATAC-sequencing. Enrichment analysis showed transcriptional dysregulation of genes involving “Purine metabolism”, “Cysteine and methionine metabolism”, “TGF-beta signaling pathway”, and “Cell cycle”. GSEA further uncovered positive enrichment of gene sets in kdm6a +/- embryonic HSPCs, including “Oxidative phosphorylation”, “mTORC1 signaling” and “DNA repair”. These dysregulated pathways in kdm6a +/- HSPCs are highly involved in cellular homeostasis and hallmarks of aged hematopoietic system. ATAC analysis also revealed robust changes in chromatin accessibility, especially including genes related to aged hematopoietic system. Integrated RNA-seq and ATAC-seq analyses showed that the chromatin accessibility at promoters and transcription of 63 genes were decreased in kdm6a +/- HSPCs. And we found that the negative regulator of Jak-Stat signaling- socs3a was on the list. Ectopic expression of socs3a in kdm6a +/- could partially rescue the uncontrolled myeloid differentiation of HSPCs. Consistently, abnormal myeloid differentiation from kdm6a +/- mutations was rescued by Jak or Stat3 inhibitors. We also tested the importance of Kdm6a-mediated demethylase activity. Unlike restoring effect by wild-type kdm6a, the ectopic expression of catalytically-dead kdm6a constructs ( kdm6a H1134A) showed no rescue effect for dysregulated expression of socs3a and key myeloerythorid genes including cebps. In conclusion, our data demonstrate the multiple roles of kdm6a in regulating HSPC homeostasis and provide new insight into the mechanism of kdm6a haploinsufficiency in uncontrolled myeloid differentiation through demethylase-dependent epigenetic programming.

Partitioning of Circulating Monocyte Subsets in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP) is an autoimmune disease characterized by accelerated platelet destruction with impaired platelet production leading to a platelet count decreased +/- bleeding manifestations, mostly caused by antiplatelet autoantibodies. The role of monocytes in ITP pathophysiology is not clearly established. Circulating monocyte subpopulations were identified phenotypically by flow cytometry and defined according to the surface expression of CD14 and of CD16 into classical (cMo), intermediate (iMo) and nonclassical (ncMo) monocytes. A new subpopulation, defined by the expression of the slan marker, has recently been described and represents a fraction of ncMo (ncMo slan +). Objectives The 1 st objective of the study was to analyze the partitioning of peripheral blood monocyte subsets by flow cytometry in ITP patients compared to a group of healthy blood donors (HD). Our second objective was to further characterize different phenotypic profiles of monocytes among ITP patients based on baseline characteristics, severity and treatment(s). Methods From April 2019 to January 2023, peripheral blood samples from ITP patients and HD were collected on EDTA and stained with the following antibodies: anti-CD45, CD2, CD56, CD24, CD14, CD16 and anti-slan. Acquisition analysis was performed either with a Navios or a DxFLEX cytometer. All files were analyzed with the previously described exclusion strategy of non-monocytes populations, using Kaluza™ analysis software, to characterize cMo, iMo, ncMo and ncMo slan + subsets. ITP patients were included regardless of their ITP status and/or phase of the disease. Results In total, 56 peripheral blood samples from 46 ITP patients and from 19 HD were analyzed. ITP patients had a median age of 50.5 years [19-91], 54.3% were females, whereas the median age of HD was 36 years [21-65] with 42.1% of females. ITP was defined as primary in 82.6% of the cases with a median disease duration of 3 years (<3 months or newly-diagnosed in 26.8% and >12 months or chronic ITP in 60.7%). Nine patients had undergone splenectomy (19.6%) at time of analysis. The platelet count was <50 x 10 9/L in 60.7% of ITP patients and absolute monocyte count ≥ 1 x 10 9/L was found in 21.4%. Regarding ITP treatment, 14 patients (25%) were receiving glucocorticoids (GC) at time of analysis whereas 9 (16.1%) had been exposed to GC within the previous month. Eleven patients (19.6%) had received intravenous immunoglobulin within a month prior to the analysis, 20 (35.7%) were on thrombopoietin receptor agonists, 9 (16.1%) had received rituximab in the previous year, 4 (7.1%) received another immunosuppressive therapy and 6 (10.7%) received another treatment. Eighteen patients (32.1%) were considered as “treatment-free” as they had not received any treatment for their ITP in the previous month or rituximab in the previous year. The latter group of “untreated” patients displayed a significant increase of the iMo relative fraction when compared to HD (4.92% vs 3.18%, p=0.018). There was no significant difference between the two groups regarding other monocytic subpopulations, including cMo, ncMo, ncMo slan+, the absolute and relative counts of monocytes. As for the treatment effect, the ongoing intake of GC therapy did not have a significant impact on monocytosis but was strongly associated with an increased proportion of cMo (94.2% vs 85.0%, p < 0.0001; Figure 1) and a decreased proportion of ncMo (2.31% vs 9.85%, p<0.0001) and ncMo slan+ (3.12% vs 7.46%, p<0.0001), mimicking the cMo accumulation characteristic of chronic myelomonocytic leukemia. Splenectomy and other treatments did not significantly interfere neither with monocyte count nor with the monocyte subpopulation partitioning. When excluding patients receiving GC, splenectomized patients had a higher monocyte count compared to non-splenectomized patients (1.0 vs 0.5G/L, p=0.008), with no difference in monocyte subset repartition. Conclusion Using multiparameter flow cytometry analysis, we demonstrated the expansion of iMo in untreated ITP patients. GC treatment led to the accumulation of cMo with reduced ncMo proportion, hence should be considered when interpreting the monocyte subset partitioning. These data suggest a potential role of monocytes in ITP pathogenesis and clinical response to GC therapy.

ASXL1 Truncating Mutations Drive Leukemic Resistance to T Cell Attack

Restoration of graft-versus leukemia activity underlies the efficacy of donor lymphocyte infusion (DLI), a curative immunotherapy for leukemic relapse following allogeneic stem cell transplant (SCT). We found that while specific exhausted T cell subsets defined DLI response, no pattern of T cell dysfunction appeared in nonresponders (NRs) [PMID: 34758319]. We hypothesized that leukemic molecular pathways define DLI resistance (DLIr). We performed whole exome sequencing on bone marrow samples collected before and after DLI from 11 patients (pts) with relapsed chronic myeloid leukemia (CML) at Dana-Farber Cancer Institute. We used MuTect2 to discover somatic alterations using genomic DNA from recipient-derived mesenchymal stromal cells and donor peripheral blood cells as germline sources. The burden of nonsynonymous mutations and mutational spectra was similar between responders (Rs) and non-responders (NRs). Known non-Philadelphia (Ph) chromosome cancer drivers were found in 0/7 Rs and 4/4 NRs (RUNX1 (1/4), IKZF1 (2/4), and truncating exon 12 ASXL1 mutations (ASXL1 m) (3/4)). We profiled 6 more DLI-treated pts with relapsed CML from the MD Anderson Cancer Center with a targeted mutation panel that identified ASXL1 m in 4/5 NRs. Altogether, we found ASXL1 m in 78% (7/9) of NRs and 0% (0/10) of Rs (p=7e-4, two-sided Fisher's test), suggesting ASXL1 m as a driver of DLIr. To discover molecular pathways of DLIr, we interrogated unpublished non T-cell single cell (sc)RNA-seq data in our DLI cohort (8 R, 6 NR). Leukemic cells were identified for patients (n=8) with sex-mismatched SCT by male sex-defining genes, which correlated with Ph chromosomal positivity (Pearson correlation = 0.85). These cells were primarily confined to the myeloid population, so we performed a scRNA myeloid reclustering (138,152 cells) with Harmony batch correction and identified 5 populations enriched in R vs NR; 4 clusters in NR, and 1 monocyte cluster in R (p=0.039, 0.005, 0.038, 0.043, and 0.025, respectively). These NR clusters were enriched in progenitor cell identities, notably the LSC17 [PMID: 27926740] signature (p<2e-16). Interrogation of immune pathways in the LSC cluster showed downregulation of HLA-I and multiple antigen processing/presentation (APP) genes in NRs (p<2e-16), while HLA-II remained unchanged. Thus, ASXL1 m may mediate immune escape through suppression of HLA-I expression and antigen presentation. To directly test this hypothesis, we CRISPR corrected the endogenous ASXL1 m (Y591*) in a BCR-ABL-driven leukemic cell line (K562). This wildtype reversion (ASXL1 c) increased HLA-I expression at the RNA (RNA-seq; HLA-B/C, log2foldchange= -4.77, q=7e-42) and protein level (flow cytometry; 49.0% vs 1.62%, q=2e-4); here too, HLA-II expression remained unaffected by ASXL1 mutation status. Gene-set enrichment analysis also showed increased APP expression in ASXL1 c K562 (NES=-1.75, q=0.01). We then sought to identify whether correction of ASXL1m acted through known regulators of HLA-I. Treatment with IFNɣ or EPZ011989, an inhibitor of enhancer of zeste-homolog 2 (EZH2), resulted in greater HLA-I in K562 ASXL1 c (85.0% vs 50.2% and 89.5% vs 39.3%, respectively, q<0.001) suggesting that ASXL1 cacts orthogonally to IFNɣ or EZH2. ASXL1 c also mediated increased T-cell cytotoxicity towards K562 ASXL1 c vs ASXL1 m without (43% vs 16%, q=2e-4) and with IFNɣ pre-treatment (71% vs 34%, q=1e-6) at 10:1 target to effector ratios. Finally, to study other disease contexts, we examined published sc chromatin accessibility and scRNA-seq data from a chronic myelomonocytic leukemia cohort [PMID: 35301312] and an acute myeloid leukemia cohort [PMID:32649887], respectively. We found both chromatin and transcriptional states enriched in ASXL1m samples to have decreased accessibility and expression, respectively, of HLA-I and APP genes (p<2E-16). In summary, we integrated molecular analyses with immuno-functional studies to define a novel oncogenetic pathway of immune evasion. Specifically, we link DLIr to ASXL1 m that suppresses transcription of HLA-I and antigen presentation machinery in myeloid diseases; moreover, its correction restores anti-leukemic CD8+ T-cell cytotoxicity. Ongoing studies will examine additional clinical contexts and the epigenetic mechanisms by which ASXL1m regulates HLA-I expression.

Characteristics and long-term outcome in a large series of chronic myelomonocytic leukaemia patients including 104 formerly referred to as oligomonocytic.

Recently modified diagnostic criteria for chronic myelomonocytic leukaemia (CMML) have lowered the cut-off for absolute monocytosis. In the largest series to date, we have analysed 313 CMML patients, including 104 with oligomonocytic (OM)-CMML. Five-year survival was longer for OM-CMML than for other patients (p < 0.001). Multivariate analysis identified OM-CMML as a favourable prognostic factor (HR 0.58; p = 0.002). The 5-year cumulative incidence of progression to classical CMML was 47%. Older age and transfusion dependence were adverse prognostic factors for OM-CMML. Our results support the inclusion of OM-CMML in the CMML category as a subtype with superior outcomes.

Leukemia Cutis-The Current View on Pathogenesis, Diagnosis, and Treatment.

Leukemia cutis (LC) is defined as the leukemic infiltration of the epidermis, the dermis, and the subcutaneous tissue. Leukemia cutis may follow or occur simultaneously with the diagnosis of systemic leukemia. However, cutaneous lesions are occasionally diagnosed as the primary manifestation of leukemia. Leukemic skin infiltrations demonstrate considerable variation regarding a number of changes, distribution, and morphology. The highest incidence of LC is observed in chronic lymphocytic leukemia, monocytic and myelomonocytic acute myeloid leukemia, and T-cell lineage leukemia. Although the pathogenic mechanism of the invasion of leukemic cells into the skin is not well understood, chemokine receptors and adhesion molecules as well as the genetic characteristics of leukemia are thought to play a role. Leukemic skin lesions may be localized or disseminated and may occur alone or in combination on any site of the skin, most frequently in the trunk and extremities. The most common clinical presentations of leukemia cutis are papules, nodules, macules, plaques, and ulcers. In most patients, the complete or partial resolution of cutaneous infiltrations occurs simultaneously with hematologic remission. However, in patients with resistant disease or recurrent skin infiltration, local radiotherapy can be used. This review presents recent data on the pathogenesis, diagnosis, and treatment of leukemic skin involvement in different types of leukemia.

Venetoclax (VEN) Improves Response Rates but Not Survival in Patients with Chronic Myelomonocytic Leukemia (CMML) Treated with Hypomethylating Agents (HMA): A Multicenter, Propensity Score Analysis

Introduction HMAs are the only approved therapy for CMML. In order to improve response rates and survival, the addition of VEN has been utilized in both CMML and after blast transformation (CMML-BT) with conflicting results in small single center studies. To clarify the role of upfront HMA+VEN in CMML and CMML-BT, we performed a large multicenter analysis. Methods We included patients with HMA-naïve CMML and CMML-BT treated with combination HMA and VEN from 4 academic centers. The primary outcome was overall survival (OS) with secondary outcomes including overall response rate (ORR) by 2015 MDS/MPN IWG criteria for CMML and 2022 ELN criteria for CMML-BT. To better appreciate the benefit of adding VEN to HMA treatment, we also performed 1:1 propensity score matched (PSM) analysis of patients treated with HMA alone as upfront therapy for CMML or CMML-BT. Matching was based on age, sex, ECOG performance status, CPSS-Mol, blood counts, peripheral and bone marrow blasts. Results Eighty-nine patients were treated with frontline HMA+VEN, 51 CMML and 38 CMML-BT (5 had received HMA alone for prior CMML). The median age was 71 years, 70% were male, and 83% had ECOG 0-1. Among CMML patients, 55% had MP-CMML subtype, 65% had CMML-2 and 12%, 30%, 58% were CPSS-mol intermediate-1, intermediate-2 and high risk, respectively. In the entire cohort, ASXL1 (63%), TET2 (47%), SRSF2 (39%) and RUNX1 (27%) were the most commonly mutated genes and 55% of patients harbored a RAS mutation ( KRAS [20%], NRAS [18%], CBL [13%], NF1 [11%], PTPN11 [9%]). For HMA combination partner, 46 (52%) were treated with azacitidine, 35 (39%) with decitabine, and 8 (9%) with both during their VEN course. During a median follow-up time of 15.8 months (mos) 52 patients (58%) died: 23 (45%) with CMML and 29 (76%) with CMML-BT. Among CMML patients, the ORR was 90%, including complete remission (CR) in 22%, complete cytogenic remission (CCR) in 4%, optimal marrow response (CMR) in 50%, and partial marrow response (PMR) in 6%. The OS of CMML patients was 25.1 mo (95% CI 15.7 - NR). A PSM population of HMA patients matched to HMA+VEN treated patients showing similar baseline characteristics. ORR was significantly higher with HMA+VEN (95%) as compared those treated with HMA alone (46%), p &amp;lt; 0.001. However, there was no significant OS difference between HMA+VEN vs HMA alone (19.1 mos vs 19.1 mos, p = 0.85, Figure 1). For CMML-BT patients, the ORR of HMA+VEN was 81% including CR in 17%, CR with partial hematologic recover (CRh) in 3%, CR with incomplete hematologic recovery (CRi) in 28%, morphologic leukemia-free state (MLFS) in 22% and partial response (PR) in 11%. The OS of CMML-BT patients was 8 mos (95% CI 4.6 - 26.2). A PSM population of CMML-BT patients treated with HMA alone showed a significantly increased ORR with HMA+VEN (82% vs 40%, p = 0.039). However, there was no difference in OS (7.49 mos vs 9.26 mos, p = 0.83, Figure 2). Among HMA-VEN treated patients, achievement of a CR was associated with an improved OS in the CMML (HR 0.31, 95% CI 0.13 - 0.75) and CMML-BT (HR 0.37, 95% CI 0.19 - 0.74). No predictors of response were identified, including the presence of a RAS mutation. In terms of safety, 7 patients (7.9%) had a grade 3/4 (G3/4) neutropenic infection and 2 patients (2.2%) had a G3/4 hemorrhagic event while being treated with HMA+VEN. A total of 29 patients went on to receive allogeneic transplant (alloSCT): 22 (43%) with CMML and 7 (18%) with CMML-BT. CMML patients who underwent alloSCT after HMA+VEN were younger as compared to non-transplanted patients (64 vs 74 years, p &amp;lt; 0.0001), but had otherwise similar baseline characteristics including stage, CPSS-mol category and prevalence of RAS mutations. Similarly, CMML-BT patients bridged to alloSCT were younger (66 vs 74 years, p &amp;lt; 0.002) and had a better ECOG performance status (p = 0.047). Of patients who received alloSCT, 10 patients (34%) died, 5 with CMML and 5 with CMML-BT. The median OS after alloSCT was 26.7 mo. Conclusions Although the use of VEN with HMA in the upfront treatment of CMML and AML secondary to CMML results in higher response rates compared to HMA alone, this combination does not improve survival based on this multi-center PSM analysis. The current sample size of CMML-BT may limit detection of OS differences between treatment groups. The role of VEN to reduce blasts and serve as a bridge to alloSCT, particularly after HMA failure, requires further investigation in larger studies in both the CMML and CMML-BT populations.

Sting-Mediated Interferon Signaling Exerts Potent Antileukemic Effects in High-Risk Myeloid and Monocytic Malignancies

Clinical outcomes of patients (pts) with high-risk (HR) myeloid neoplasms remain dismal despite available therapies. Interferons (IFN) regulate hematopoietic stem-progenitor cell (HSPC) differentiation, proliferation and self-renewal capabilities and preferentially impair clonal HSPC fitness. To evaluate if myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) HSPCs activate IFN responses we performed RNA sequencing in bone marrow (BM) CD34 + cells obtained from pts with MDS (n=58), CMML (n=27) and age-matched healthy donors (HDs, n=17). This analysis revealed significant deregulation of IFN pathway genes in HSPCs from pts with CMML and to a lesser extent in those of pts with MDS. Activation of the stimulator of interferon genes (STING) can inhibit stem-cell function and induce myeloid and monocytic cell death via IRF3-dependent and independent mechanisms. Therefore, we hypothesized that STING pathway activation might have therapeutic potential in high risk (HR) myeloid and monocytic (MyMo) neoplasms. In vitro treatment of monocytic MOLM13 and THP1 cell lines with the novel synthetic STING agonist dazostingag resulted in induction of IRF3 phosphorylation, caspase 3 cleavage and apoptosis. Dazostingag was also able to induce enhanced cell death in multiple myeloid cell line cultures. Given the ability of dazostingag to induce apoptosis of monocytic cell lines, we sought to evaluate the activity of monocyte-directed STING activation using TAK-500, an IgG1 anti-CCR2 antibody linked to dazostingag as a STING agonist payload. To do so, we first analyzed the expression of the monocyte-specific cysteine-cysteine chemokine receptor 2 (CCR2) in primary pts samples and cell lines. Both CCR2 RNA and protein expression levels were higher in CMML compared to HDs or MDS. In accordance with this finding, monocytic cell lines (MOLM13, THP1) had significantly higher CCR2 expression compared to non-monocytic cell lines (SKM1, HL60, U937, KG1). Treatment of MOLM13 and THP1 cell lines with TAK-500 induced strong IRF3 phosphorylation, caspase 3 cleavage and apoptosis. In vitro treatment of BM mononuclear cells (MNCs) from pts with MDS (n=10), CMML (n=11) or AML (n=8) in co-culture with mesenchymal stromal cells with TAK-500 (0.5 mM) or dazostingag (1 mM) resulted in increased cell death of MNCs from pts with CMML and AML. TAK-500 (0.5 mM) significantly depleted CCR2 + BM MNCs from all disease groups but did not affect CCR2 + cells from HDs. Next, to evaluate whether STING activation impairs repopulation potential of neoplastic HSPCs we performed colony-formation studies in CD34 + BM cells from pts with CMML (n=8), MDS (n=3) and AML (n=4). Treatment with TAK-500 (0.5mM) and dazostingag (1mM) resulted in significantly impaired myeloid colony-formation in all disease groups without significantly impairing erythroid colony-formation in MDS or CMML samples. Taken together, these data suggests that STING activation might induce aberrant myelomonocytic cell death and deplete aberrant HSPCs in HR-MyMo neoplasms. To evaluate the in vivo efficacy of TAK-500 and dazostingag, we established xenograft models by inoculating NOG-IL15 transgenic mice with MOLM13 cells. Treatment with TAK-500 and dazostingag resulted in reduction of tumor burden evaluated by bioluminescence with no observed toxicities. Administration of weekly doses of TAK-500 induced enhanced and sustained tumor burden suppression. To determine the in vivo effect of STING activation in an immune competent model of CMML, we then evaluated the effects of TAK-500 and dazostingag in Vav-KDM6B/Tet2 f/f/Vav-Cre mice, a previously reported (Wei et al Leukemia 2022) animal model with CMML phenotype. Treatment with TAK-500 led to enhanced apoptosis of Ccr2 + BM cells in these mice. Moreover, the number of colonies formed by lineage negative/Sca1 positive/cKit positive BM progenitor (LSK) cells isolated from TAK-500 treated mice was significantly lower than in vehicle treated control mice, suggesting a negative regulation of repopulating capacity of Tet2 deficient/ KDM6B overexpressing BM HSPCs by TAK-500. Evaluation of the efficacy of TAK-500 and dazostingag in patient-derived xenograft models of primary CMML samples is planned. Taken together, our data suggests that CCR2-directed STING activation might have therapeutic potential in HR-MyMo neoplasms by depleting CCR2 + cells and impairing clonal HSPC repopulation potential.

Phase 1/2 Study of Pacritinib in Combination with Azacitidine in Chronic Myelomonocytic Leukemia

Introduction and preclinical rationale Treatment options for chronic myelomonocytic leukemia (CMML) are limited. Hypomethylating agents (HMAs) are the only approved therapies but are associated with unsatisfactory response rates and do not impact disease evolution (Merlevede Nat Comm 2016). JAK inhibition can reduce GM-CSF signaling, which contributes to CMML pathobiology; ruxolitinib has been clinically evaluated in CMML demonstrating promising, but insufficient clinical activity. Pacritinib is a JAK inhibitor that has a unique profile which suggests it may be uniquely well suited for the treatment of CMML, including targeting of CSF1R and IRAK1, both of which are upregulated in CMML and related diseases (Obba Autophagy 2015, Rhyasen Cancer Cell 2013). Additionally, pacritinib was recently shown to be a potent inhibitor of ACVR1 (Oh AJH 2023), potentially allowing for increased iron availability and improvement in anemia. Pacritinib has been evaluated preclinically in CMML primary samples and mouse models where it demonstrated significant, albeit heterogenous activity (Yoshimi Blood 2017). To enhance the activity of pacritinib in CMML, it was also evaluated in combination with azacitidine in GM-CSF hypersensitive cell lines and primary CMML cells where profound synergy was noted (Ma ASH 2015). Based on encouraging preclinical data, we hypothesize that the combination of pacritinib with azacitidine will be an effective treatment strategy in CMML patients. Methods In this phase 1/2 trial, we will evaluate the safety and efficacy of pacritinib and azacitidine in newly diagnosed or previously treated CMML. Patients must be JAK inhibitor and HMA naïve (≤1 cycle of HMA allowed). Phase 1 will follow a ‘3+3‘ dose de-escalation design and will enroll 6-12 patients. The primary objective of this phase is to determine the recommended phase 2 dose (RP2D) of pacritinib in combination with a fixed dose of azacitidine. Pacritinib will be administered at 200mg twice daily (with a lower dose of 100mg twice daily being explored in the case of dose limiting toxicity) in combination with azacitidine 75mg/m 2 for 7 days. Phase 2 will utilize a Simon's two stage design to assess the preliminary efficacy of pacritinib in combination with azacitidine at the RP2D. In the first stage, after treating 6 patients at the RP2D, there will be an interim response assessment as part of the phase 2 portion. If at least 4 among these 6 patients show a response, the trial will proceed to a second stage and enroll an additional 12 patients. Patients will be treated for 6 cycles of 28 days each with an interim bone marrow biopsy after cycle 3. The primary endpoint of the phase 1 portion is the recommended RP2D through the assessment of dose limiting toxicities and of the phase 2 portion is the overall response rate (ORR) by the end of cycle 6 as defined by a clinical benefit or greater by myelodysplastic syndrome/myeloproliferative neoplasm International Working Group response criteria (Savona 2015). Secondary endpoints include duration of response, disease free survival, overall survival, changes in MPN-SAF total symptom scores, palpable splenomegaly, and adverse events. Several correlative studies are planned as part of this clinical trial. Modulation of biomarkers of disease activity with treatment including GM-CSF dependent STAT5 phosphorylation, MAPK, and PI3K signaling will be analyzed. We will also evaluate changes in mutational and cytogenetic burden with treatment as well as at baseline to identify predictive biomarkers of response.

Development of Novel Protein-Drug Conjugates for the Treatment of Chronic Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid stem cell neoplasm with few effective therapies and poor prognosis. We have developed CCL2-drug conjugates that specifically bind, internalize and eliminate cells of the disease clone via inhibition of the key metabolic enzyme NAMPT. CCL2 is a chemokine which binds CCR2, a receptor expressed predominantly on the classical monocytes canonically expanded in CMML, while CMML stem and progenitors display ~100-fold higher CCR2 expression compared with healthy equivalents ( Ferrall-Fairbanks et al, Blood Cancer Discov 2022 3(6):536-553). CCL2-NAMPTi conjugates thus represent an attractive and novel strategy, co-opting both lineage specificity and therapeutic window to deliver payload drugs for efficient and targeted cell killing. Methods: Four NAMPTi warheads were made in adaptable seven-step synthetic route and covalently conjugated to human CCL2 though TGase. We screened the range of CCL2-NAMPTi conjugates on CCR2-expressing cell lines (THP-1; MV411) for their ability to reduce NAD/NADH and induce cytotoxicity. The lead CCL2-NAMPTi conjugate was identified based on its ability to reduce NAD/NADH levels, induce cell death, and show impermeability when using the unconjugated warhead alone. Plasma stability studies confirmed conjugate stability in mouse and human plasma with no release of unconjugated active warhead. The lead compound was then tested on CD14+ cells isolated from CMML patient peripheral blood (PB); examining NAD/NADH levels, cell viability and induction of caspase 3/8. To better understand its specificity and target cell population, we conducted in vivo studies using fluorescently labelled CCL2 in wild type and CCR2 KO mice. Therapeutic effectiveness of the lead conjugate was evaluated by means of NAD/NADH reduction in THP1 subcutaneous models. Results: All CCL2-NAMPTi tested induced cell death in THP-1 cells (IC50 1.1-7.2 nM) following 72 hours incubation. The lead compound, MM015, effectively reduced NAD/NADH levels in CCR2+ cell lines (THP-1; MV411) 24-48 hours following treatment (IC 50 0.1-0.8 nM), with a corresponding decrease in cell viability at 72 and 96 hours (IC 50 1.1- 2.1 nM). No effect was observed on CCR2- cell lines (Jurkat; PC9). Specificity of the lead conjugate was confirmed by measuring NAD/NADH levels and cytotoxicity in presence of 100-fold excess of unconjugated CCL2. Efficacy studies on primary CMML PB samples confirmed that MM015 exposure specifically reduced NAD/NADH levels (n=6, IC 50 0.5-15 nM) and cell viability (n=6, IC 50 1.9-250 nM), and induced caspase 3/8 activation. Flow cytometry analysis of CMML PB confirmed CCR2 expression was largely restricted to monocytes and precursors, as compared with other immune cell subsets (n=6). Accordingly, efficacy measures in treated whole PB were exclusively seen in the CD14+ population, sparing lymphoid and other lineage cells, demonstrating ability of CCL2-NAMPTi to selectively target the CMML disease clone. In vivo experiments using fluorescently labelled CCL2 confirmed selective uptake of intravenously administered human CCL2 by CCR2+ cells within mouse blood, bone marrow and in THP-1 subcutaneous tumour cells. No uptake of fluorescently labelled CCL2 was observed in CCR2 KO mice, further confirming CCR2 specific uptake. Additionally, measurement of human CCL2 in CCR2 KO mouse peripheral blood by ELISA confirmed CCR2 dependent CCL2 uptake, 1 hour following intravenous administration. In vivo efficacy studies showed consistent reduction of NAD/NADH levels in xenografted tumor cells, and monocytic cells in the murine BM compartment, 24 hours following IV injection of MM015. Conclusion: We have developed a novel drug delivery strategy utilizing CCL2-NAMPTi conjugates to selectively target and eliminate key CMML cell populations. CCL2-NAMPTi can effectively reduce NAD levels and induce subsequent cell death in CCR2-expressing cell lines, primary CMML cells and in vivo models. This innovative approach holds therapeutic promise to address this major unmet clinical need.

Suppression of KRAS and CBL Mutations and Hematological Improvement By Lenzilumab and Azacitidine Treatment in Proliferative Chronic Myelomonocytic Leukemia

Introduction: Mutations in RAS-pathway genes ( NRAS, KRAS, CBL) are amongst the most common somatic mutations in cancer and historically resistant to most therapies. Targeted therapies that impact the RAS-pathway constitute a major unmet need in oncology. The proliferative form of chronic myelomonocytic leukemia (CMML) is commonly associated with RAS-pathway mutations and has a high propensity to develop into acute myeloid leukemia. In pre-clinical models, Lenzilumab (LENZ; Humanigen, Inc., Short Hills, NJ), a proprietary Humaneered ® first-in-class monoclonal antibody with best-in-class off-rate and affinity that neutralizes GM-CSF, resulted in a reduction of colony numbers and viability of CMML cells, with the greatest sensitivity in cells possessing RAS-pathway ( NRAS/ KRAS/ CBL) mutations, suggesting benefit in the targeted treatment of CMML. The PREcision Approach to CHronic Myelomonocytic Leukemia (PREACH-M) trial assesses the efficacy of LENZ, in addition to azacytidine (AZA), in CMML subjects with RAS-pathway mutations and high dose sodium ascorbate (ASC) and AZA in CMML subjects without RAS-pathway mutations. Interim data from 11 subjects with RAS-pathway ( NRAS/ KRAS/ CBL) mutations in PREACH-M receiving LENZ/AZA demonstrate reductions in circulating GM-CSF and CRP with 8 subjects achieving complete response or optimal marrow response. This report describes improvements in variant allele frequencies (VAF) for RAS-pathway mutations and hematologic improvements associated with LENZ/AZA treatment in CMML. Methods: PREACH-M is a Phase 2/3 non-randomized, open-label trial in 72 subjects, aged at least 18 years, with newly diagnosed CMML based on the WHO 2016 criteria; and RAS-pathway mutations at a variant allele frequency ≥3%. Subjects received 24 cycles (every 28 days) of AZA (SC; 75 mg/m 2 for 7 days) and LENZ (IV; 552 mg; d1 &amp; d15 of cycle 1 and d1 only for all subsequent cycles). Subjects without RAS-pathway mutations received the same AZA regimen and sodium ascorbate (IV; 30g for 7 days (15g for 1st dose only; 30g thereafter if no evidence of tumor lysis syndrome); PO; 1.1g on all other days). VAF was determined from bone marrow mononuclear cells using a 41-myeloid panel using Illumina Hi-Seq with a depth of 1000x performed on bone marrow aspirates obtained at baseline and Day 1 of treatment cycles 4, 7, and 12. Results: As of July 2023, 11 subjects completed at least 3 months of LENZ/AZA treatments and follow-up sequencing data were available for 10 subjects. Five of 10 subjects showed &amp;gt;10% decrease in VAF for at least one mutation detected at baseline, including 5 decreased alleles in the RAS-pathway ( KRAS, CBL) out of 22 total VAF mutation responses. CBL mutations showed the largest change with a decrease in &amp;gt;50% VAF in three subjects. Decreases in RAS-pathway clones by LENZ/AZA were durable at 6 months. No decreases in VAF for any mutations were observed in 2 evaluable subjects following ASC/AZA treatment. Following 3 months LENZ/AZA treatment, blood monocyte count improved from a baseline of 11.0 ± 6.0 x 10 9/L to 2.0 ± 1.0 x 10 9/L (p=0.030). Blast differential improved from 10.5 ± 5.0% to 4.0 ± 3.5% (p=0.038). Platelet count increased from 90.0 ± 50.0 x 10 9/L to 150.0 ± 60.0 x 10 9/L (p=0.010). Statistical improvements remained durable for at least 6 (n=6) and 12 (n=3) months. Hemoglobin concentration increased 100.0 ± 22.0 g/L to 115.0 ± 10.0 g/L at 3 months (p=NS, n=11) and progressively increased to 122.0 ± 10.0 g/L at 12 months (p=0.024, n=3). Spleen length decreased from 15.0 ± 4.0 cm to 13.0 ± 4.0 cm (p=0.03) and decreased further to 11.0 ± 2.0 cm in subjects who received 12 months of treatment (n=3). No evidence of disease progression or relapse was observed in any subject. Conclusion: In 11 evaluable subjects with proliferative CMML and RAS-pathway mutations, GM-CSF neutralization with LENZ in addition to AZA standard of care, resulted in significant decreases in the proportion of KRAS and CBL mutant leukemic cells, accompanied by clinically significant hematologic improvements and a reduction in splenomegaly. Lenzilumab may have efficacy in preventing outgrowth of RAS-pathway mutations, specifically KRAS and CBL, in the context of CMML and other myeloid malignancies.

Phase 3b Study Design for a Comparison of Treatment Preference between Oral Decitabine/Cedazuridine and Azacitidine in Adult Patients with IPSS-R Intermediate Myelodysplastic Syndrome, IPSS Intermediate-2 or High-Risk Myelodysplastic Syndrome, Low-Blast Acute Myeloid Leukaemia, or Chronic Myelomonocytic Leukaemia

Myelodysplastic syndromes (MDS) are heterogeneous clonal bone marrow neoplasms characterised by marrow dysplasia, low blood counts and risk of progression to acute myeloid leukaemia (AML). Hypomethylating agents (HMAs) decitabine and azacitidine (AZA) are effective treatments for hematologic cancers including MDS. AZA is indicated to treat International Prognostic Scoring System (IPPS) defined Intermediate-2 (Int-2) and high-risk (HR) MDS, low blast AML (LB-AML; 20-30% blasts) with multi-lineage dysplasia, and chronic myelomonocytic leukaemia (CMML) with 10-29% blasts without myeloproliferative disorder. Oral decitabine/cedazuridine is currently the only oral hypomethylating agent indicated to treat IPSS Int-1, Int-2 or HR MDS and CMML, in the United States, Canada and Australia. AZA administration requires daily visits to a hospital or clinic for 7 days every 28 days, for either intravenous infusions, or large-volume subcutaneous (SC) injections for a minimum of 6 cycles.This places a burden on this patient population, resulting in low treatment adherence, early discontinuation, and subsequently low treatment uptake. Oral decitabine/cedazuridine is administered as one tablet daily for the first 5 days of a 28-day cycle for a minimum of 4 cycles. This allows patients to take their medication at a location of their choice. Alongside previously reported preference for oral therapy, patients may favour oral decitabine/cedazuridine over SC AZA due to improved treatment convenience and reduced discomfort. However, this is untested in a controlled setting. This study aims to evaluate preference between oral decitabine/cedazuridine and SC AZA treatment for adult patients with Revised International Prognostic Scoring System (IPSS-R) intermediate, IPSS Int-2 or HR MDS, LB-AML or CMML. This phase 3b, open-label, multi-centre study (NCT05883956) has sites planned in Australia (4 sites) and New Zealand (5 sites), to compare preference for oral decitabine/cedazuridine (Treatment A) and SC AZA (Treatment B). The study design includes 28 days of screening, four continuous 28-day cycles of study treatment, and a follow-up period with two 28-day cycles of continued therapy. Patients will be randomised to two balanced treatment sequences: ABBA or BAAB (Figure 1). Patients will express a preference twice in the study; after completing Cycle 1 and 2, and after completing Cycle 3 and 4. This cross-over design is twice the length of a typical 2-period AB vs BA design and allows patients to act as their own controls. The study design allows estimation of the treatment effect while mitigating factors that may influence preference. These include the order of treatments, and fluctuations in the patient's physical and disease state over the course of the trial. All available preferences expressed throughout the trial will be used for primary analysis. A total of 42 patients are planned for this study. The primary objective of this study is to compare patients' treatment preference for oral decitabine/cedazuridine and SC AZA for the treatment of MDS, LB-AML or CMML. Secondary objectives include evaluation of carers' and clinicians' treatment preference between oral decitabine/cedazuridine and SC AZA for the treatment and continued treatment of adult patients with MDS, LB-AML or CMML. The primary objective will be assessed by a treatment preference in myelodysplasia questionnaire (pTPMQ), which instructs patients to indicate a preference for injection, oral/tablets or no preference. The secondary objectives will be evaluated by a carer treatment preference in myelodysplasia questionnaire (cTPMQ) and a medical clinician treatment preference in myelodysplasia questionnaire (mTPMQ). Treatment discontinuation rates, quality of life and safety of SC AZA and oral decitabine/cedazuridine will also be compared. The primary analysis will be conducted with a multilevel logistic regression model analysing all available preferences. Descriptive statistics including frequencies, proportions, mean, standard deviation, median, and interquartile range will be used to summarize the study data. When complete, this study will address an evidence gap in the comparison of patient preference for oral decitabine/cedazuridine and SC AZA, and subsequently inform the value of oral decitabine/cedazuridine for the treatment of MDS, LB-AML and CMML.

A Phase II Study of Vibecotamab, a CD3-CD123 Bispecific T-Cell Engaging Antibody, for MDS or CMML after Hypomethylating Failure and in MRD-Positive AML

Background Leukemic stem cells have high expression of CD123 compared to normal hematopoietic stem cells and is therefore a therapeutic target in multiple leukemias including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and chronic myelomonocytic leukemia (CMML). Vibecotamab (formally XmAb14045) is a CD3-CD123 bispecific engaging antibody that has shown clinical activity in relapsed/refractory AML, particularly in low-blast disease. We therefore sought to evaluate vibecotamab in other low-blast states, including MDS or CMML after hypomethylating agent failure and MRD-positive AML. Methods In this two-arm, open-label, phase II study, adults with either MDS (IPSS-R intermediate or higher risk) or CMML (CMML-1 or CMML-2) after failure of hypomethylating agents or AML in first or second morphologic remission with detectable MRD at a level of ≥0.1% by flow cytometry were eligible. CD123 expression ≥20% on aberrant myeloid blasts was required for enrollment. Vibecotamab was given IV in a ramp-up dose schedule on days 1 (0.43µg/kg), 3 (0.75µg/kg), 5 (1.1µg/kg), and 8 (1.7µg/kg) in cycle 1, followed by weekly doses of vibecotamab at a dose of 1.7µg/kg. Patients (pts) received up to 4 cycles of vibecotamab in 28-day cycles. The primary endpoint of the MDS/CMML cohort was response rate (CR + mCR + PR + HI + clinical benefit) within 4 cycles. The primary endpoint of the AML MRD cohort was the MRD negativity rate within 4 cycles. Results Between May 2022 and July 2023, 23 pts were treated (11 MDS/CMML, 12 AML MRD). Baseline characteristics are shown in Table 1. In the MDS/CMML cohort, 7 pts (63%) had received two or more prior lines of therapy, 8 pts (73%) had prior venetoclax exposure, and 2 pts (18%) had prior hematopoietic stem cell transplant (HSCT). Six MDS pts (66%) were IPSS-R high or very high risk. In the AML cohort, 6 pts (50%) had received two or more lines of prior therapy, 11 pts (92%) had prior venetoclax exposure, and 5 pts (42%) had prior HSCT. Ten pts (83%) were ELN 2022 adverse risk. The baseline CD123 expression was 72% (range, 43%-99%) in the MDS/CMML cohort and 89% (range, 55%-99%) in the AML MRD cohort. The baseline MRD by flow cytometry in the AML MRD cohort was 1.1% (range, 0.1%-3.9%). Responses are shown in Table 2. In the MDS/CMML cohort, 7 pts responded (64%), with 6 pts (56%) achieving marrow complete remission (mCR) and 1 pt (9%) achieving hematologic improvement (HI) per International Working Group (IWG) 2006 criteria. Among the 9 MDS pts, 4 (44%) achieved mCR + HI (2 HI-N, 1 HI-P, and 1 HI-P + HI-N), and 1 (11%) achieved HI (HI-N + HI-P). Per revised IWG 2023 MDS response criteria, 5 of the 9 MDS pts (56%) achieved complete remission with limited count recovery (CR L). Two of 4 MDS pts (50%) with TP53 mutations achieved CR L. Both CMML pts achieved mCR, with one pt also achieving HI-N. Among 9 pts with baseline bone marrow blasts =&amp;gt;5% at trial enrollment, 6 (67%) achieved a mCR, with or without HI. Best response occurred after the first cycle in all pts. CD123 expression was not associated with likelihood of response. Of the 7 responders, 5 are in ongoing response (range 0.3-6.9 months), one died in CR L from non-hematologic complications (heart failure), and one relapsed 5 months after achieving CR L. Of the 12 pts in the AML MRD cohort, 3 (25%) achieved MRD negativity, all of which occurred after 1 cycle of vibecotamab. Among the 3 responders, all were ELN adverse risk and had prior venetoclax exposure, 2 had prior HSCT, and 1 had inv(3). The median MRD and CD123 expression in responders was 0.2% (range 0.1%-0.2%) and 98% (range 95%-99%) vs 1.8% (range 0.5%-3.9%) and 87% (range 66%-96%) in non-responders, respectively. At last follow-up, all 3 responders are still in MRD-negative remission (range 3.2-12.8 months). Vibecotamab was well-tolerated with no pts requiring dose reductions or being taken off study due to adverse events. Ten pts (44%) experienced grade 2 infusion reactions and 1 pt (4%) experienced a grade 3 infusion reaction. Myelosuppression was minimal, consistent with previous studies of vibecotamab. Conclusion Vibecotamab was safe and active in low-blast, high-risk myeloid diseases, with a response rate of 64% in MDS/CMML after HMA failure and 25% in MRD-positive AML. The clinical activity of vibecotamab, including in pts with prior venetoclax exposure and/or HSCT, and its lack of clinically significant myelosuppression provide rationale to combine it with other agents in AML, MDS, and CMML.

Trial in Progress: Venetoclax in Combination with ASTX727, an All-Oral Therapy for Chronic Myelomonocytic Leukemia and Other MDS/MPN with Excess Blasts (VICTORY MDS/MPN): A Randomized, Phase 2 Trial

Background and Significance: The rare nature and diagnostic difficulty associated with myelodysplastic/myeloproliferative neoplasms (MDS/MPN) has limited the inclusion of patients (pts) with these diseases, other than chronic myelomonocytic leukemia (CMML), in relevant clinical trials. This has resulted in the complete lack of approved agents for the treatment of pts with non-CMML MDS/MPN. Moreover, ASTX727 (oral decitabine-cedazuridine) was approved for CMML based on a study of only 16 pts with CMML. A larger study of CMML and non-CMML MDS/MPN is needed. MDS/MPNs appear to be BCL2-dependent (Bogenberger et al., 2015; Jilg et al., 2016), but data for venetoclax combination therapy are derived from retrospective analyses of relapsed/refractory disease (Cortes et al. 2019; Hammond et al., 2020) or single-arm studies not dedicated to MDS/MPN (NCT04655755, Venugopal et al. 2021). Venetoclax exposures are not affected by coadministration of ASTX727 and predict responses similar to historical data (Mannis et al. 2021). Based on these promising early data and the critical need, we proposed a randomized study of ASTX727 alone or in combination with venetoclax for pts with MDS/MPN and &amp;gt;5% blasts. Study Design and Methods: This is a National Cancer Institute-sponsored, multi-center, international, randomized phase 2 study (NCT05600894, NCI 10538) designed to evaluate the efficacy of ASTX727 alone or in combination with venetoclax in pts with newly diagnosed MDS/MPN with &amp;gt;5% marrow blasts. Key eligibility criteria include allowing hydroxyurea cytoreduction to a WBC &amp;lt;10,000/mm 3 for proliferative disease and excluding pts with need for emergent disease-directed therapy as well as prior therapy for myeloid malignancy other than hydroxyurea or thrombopoietic/erythropoietin stimulating agents. Pts will be randomized 1:1 to receive either ASTX727 alone (Days 1-5) or in combination with venetoclax, initially administered with a 3-day dosing ramp-up and given for 14 days per 28-day cycle (Figure 1). Pts are stratified by disease subtype, specifically CMML and non-CMML MDS/MPN. The primary endpoint is the rate of complete remission (adapted from the MDS/MPN-specific response criteria developed by Savona et al. 2015). Secondary endpoints include the overall response rate, overall survival, progression-free survival, allogeneic hematopoietic stem cell transplantation rate, clonality at time of hematologic remission, number of red cell and platelet transfusions required and toxicity of ASTX727 versus ASTX727 + venetoclax. Pts randomized to the ASTX727 monotherapy arm and without response after 4 cycles of therapy, or with progressive disease after an initial response, cross over to the ASTX727 + venetoclax arm. A futility analysis is planned for each cohort (first 18 and 13 pts in each treatment arm for the CMML and non-CMML cohorts, respectively). We assume that 85% and 90% of pts in the CMML and non-CMML MDS/MPN cohorts, respectively, randomized to the ASTX727 monotherapy arm will not achieve the primary endpoint and will cross over to the ASTX727 + venetoclax combination arm. The estimated accrual time of a total of 70 pts with CMML and 50 pts with non-CMML MDS/MPN will require about 2.9 and 2.1 years, respectively. Planned integrated studies include the use of an apoptosis multiplex immunoassay to evaluate how the addition of a BCL-2 inhibitor will influence the ability of ASTX727 to induce apoptosis and if particular thresholds of apoptosis are predictive of clinical response. Planned exploratory studies include the use of: 1) RNA-seq to evaluate if differences in baseline transcriptional programs appear to be predictive of response to ASTX727 and ASTX727+venetoclax, 2) next generation (Oncomine Comprehensive Assay (OCAv3)/whole exome sequencing to uncover biomarkers predictive of response/outcome, validate risk modeling to stratify these pts and detect/monitor measurable residual disease (MRD) on serial marrow samples that can predict depth of response with translation in improvement in event-based endpoints, and 3) circulating tumor DNA as a non-invasive means (i.e., without need for a marrow evaluation) of prospective MRD detection for prognostic use. This ongoing, randomized study may inform frontline therapeutic options, provide prospective data for venetoclax combination after HMA failure, and generate striking correlative science for pts with “high risk” MDS/MPN.

A Prediction Model for Early Recurrence after Allogeneic Hematopoietic Stem Cell Transplantation in Patients with CMML: A Nationwide Representative Multicenter Study

Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell malignancy. Some patients experience prolonged remission after aggressive chemotherapy, but the only current therapy with proven curative potential is allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, not all CMML patients benefit from allo-HSCT, and many experience relapse. Relapse is the leading cause of treatment failure for CMML treated with allo-HSCT, and treatment options for patients relapsing after allo-HSCT are limited. This study conducted a nationwide multicenter real-world study to identify risk factors and develop a novel prediction model for early recurrence in CMML patient posttransplantation. Clinicians can improve the survival of CMML patients with a high risk of early recurrence by intervening as early as possible. Methods: Patients ≥18 years old with CMML treated with allo-HSCT for the first time between 2005 and 2022 were included in this nationwide, multicenter, real-world study. Twenty-seven medical centers participated in this analysis, including the derivation cohort from 26 centers and the validation cohort from another center. Information collected included demographic and clinical characteristics, previous treatments and responses, and allo-HSCT treatment details. We first calculated the univariable association of each variable with 1-year relapse in the derivation cohort. Second, the variables with p values less than 0.1 in the univariate analysis were further included as candidate predictors in the multivariate analysis using a backward stepwise logistic regression model. The variables that remained in the final model based on the outcomes of the multivariate analysis in the derivation cohort were identified as independent prognostic factors. A scoring system to predict early recurrence after allo-HSCT was also established, and scores were assigned to the prognostic factors based on the regression coefficient. Results: Using multivariable logistic regression methods with stepwise variable selection, two highly significant independent prognostic factors for the early recurrence of CMML posttransplantation were identified: bone marrow blast cell count at diagnosis (p=0.026; odds ratio [OR], 1.149; 95% confidence interval [CI], 1.02-1.30), and leukocyte count pretransplantation (p=0.005; odds ratio [OR], 1.212; 95% confidence interval [CI], 1.06-1.39). A risk grading model was constructed according to the regression coefficients. Bone marrow blast cell count 0-5% is 0 point, 5%-10% is 1 point; 10-20% is 2 points, and &amp;gt;20% is 3 points. Leukocyte count 0-10×10 9/L is 0 point, 10-20×10 9/L is 2 points, and &amp;gt;20 ×10 9/L is 4 points. The points scored for each of these two factors were added to yield the overall risk score, which ranged from zero to seven. Patients were stratified into a low-risk group (0-1 point), a intermediate-risk group (2-3 points) and a high-risk group (4-7 points). The validated internal c-statistic was 0.790 (95% CI, 0.657-0.923), and the external c-statistic was 0.710 (95% CI, 0.580-0.840). The early recurrence rates of the derivation cohort in the low-risk, intermediate-risk, and high-risk groups were 5.9%, 17.9% and 31.8% (p&amp;lt;0.05). The Kaplan‒Meier estimations of relapse-free survival revealed good separation between these risk groups, and 91.9%, 79.4% and 66.1% (p&amp;lt;0.05), respectively. According to the calibration plots, the model-predicted probabilities showed a good correlation with the actual observed frequencies. Decision curve analysis indicated that the clinical implementation of the prognostic model could benefit early recurrence CMML patients. Conclusion: CMML patients who relapse after allo-HSCT have a poor prognosis. An integrated prediction model based on clinical biomarkers was developed and externally validated, and this is the first straightforward scoring model that incorporates clinical and laboratory risk factors to evaluate the early recurrence of CMML patients after receiving allo-HSCT. This model can be effectively utilized to help improve the survival and prognosis of CMML patients by accelerating the early identification of patients at a high risk of relapse and contributing to the appropriate implementation of urgent medical support.

Multi-Omics Reveals Azacitidine Partially Restores Hematopoiesis-Supportive Functions in Mesenchymal Stromal Cells from Patients with Chronic Myelomonocytic Leukemia

Background: Being the most standard first-line treatment option for chronic myelomonocytic leukemia (CMML), the mechanism by which hypomethylating agents exert therapeutic effects has not yet been elucidated. While it was previously believed that HMAs act primarily through inhibiting DNA methyltransferases and restoring the expression of tumour suppressor genes, recent findings suggested its therapeutic effects might involve remodelling the bone marrow mesenchymal niche. Bone marrow mesenchymal stromal cells (MSCs) were hypothesized to play a role in this process. Nevertheless, the lack of evaluations on MSC amenability to HMAs has hindered a complete understanding of the post-HMA treatment mechanisms in patients with CMML. Aims: The present study aimed to investigate the effects of azacitidine (AZA), the most commonly used HMA, on CMML patient-derived MSCs and to determine its associated clinical significance. The specific aims were to understand the AZA-induced modifications on the DNA methylome/transcriptome and, thus, to evaluate the molecular/functional impacts of AZA treatment on hematopoiesis-supportive functions in MSCs. At last, we attempted to evaluate the association between in vivo/ex vivo sensitivity of MSCs to AZA and post-HMA efficiencies in CMML patients. Methods: High-throughput multi-omic methods including DNA Methylation Profiling (Infinium HumanMethylation850K BeadChip, Illumina Inc), RNA-sequencing (RNA-seq), and exome sequencing were utilized to assess the regulatory effects of the treatment of AZA (10 μM for 48 hours) on the DNA methylome and transcriptome of CMML patient-derived MSCs. Molecular and functional investigations including DNA damage assessment assay and long-term culture initiating cell (LTC-IC) assay, were performed to validate the sensitivity of CMML patient-derived MSCs (Figure 1A). At last, using univariate survival analysis, we assessed the association between the sensitivity of patient-derived MSCs to AZA and the efficacy of HMA-treated patients. Results: AZA treatment induced global DNA demethylation in both CMML-MSCs and healthy MSCs, with CMML-MSCs showing a more complex and bidirectional response pattern to AZA than healthy MSCs. The combined multi-omic analysis identified 1395 “AZA-sensitive genes” as potential regulatory targets in CMML-MSCs (66.4% up-regulated and 37.5% down-regulated). These genes were associated with hematopoiesis support, cell adhesion, and immune activation. Functional assays confirmed that AZA restored the expression of hematopoiesis-supportive cytokines and improved the hematopoiesis-supportive abilities of CMML-MSCs. At last, the higher sensitivity of MSCs correlated with a significant superior progression-free survival (PFS; 21.8 months vs. 16.2 months, P=0.03) and higher overall response rate (77.8% vs. 22.2%) in CMML patients treated with at least 2 cycles of HMAs (Figure 1B). Conclusion: The study provides evidence that patient-derived MSCs are sensitive to AZA treatment at the DNA methylation level, suggesting a modulable bone marrow mesenchymal niche during HMA treatment. AZA treatment rejuvenated functionally defected CMML-MSCs and restored their ability to support normal hematopoiesis in vitro. The sensitivity of CMML-MSCs to HMA correlated with clinical outcomes, with patients having AZA-sensitive MSCs showing superior PFS and higher clinical response rates after HMA treatment. These findings support the theory that AZA may switch the dysfunctional MSCs from a “leukemic-supportive” phenotype to a “hematopoiesis-supportive” phenotype. However, further investigations are required to understand whether this recovery of the hematopoietic niche directly leads to the inhibition of malignant blast cells. Patient-derived xenograft models are recommended to further verify the potential therapeutic effects of HMA-associated modulations on the hematopoietic niche.

Preliminary Results of a Phase 2 Study of IMM01 Combined with Azacitidine (AZA) As the First-Line Treatment in Adult Patients with Chronic Myelomonocytic Leukemia (CMML)

Background: Patients with Chronic Myelomonocytic Leukemia (CMML) have poor prognosis and an inherent risk for transformation to acute myeloid leukemia. IMM01 is a recombinant signal regulatory protein α (SIRPα) IgG1 fusion protein that exerts anti-tumor activity via blocking “Don't eat me” signal and activating the “Eat me” signal to induce strong antibody-dependent cellular phagocytosis (ADCP). Furthermore, IMM01 activated macrophages could process and present tumor antigen to T cells and elicit long-last tumor-specific T cell immune response. Methods : This is an open-label, multi-center, phase 2 study (NCT05140811) that evaluated safety and efficacy of IMM01 in combination with AZA as the first-line treatment in patients with newly diagnosed CMML. We enrolled patients aged 18 years or older with the diagnosis of CMML, including subtypes CMML-1 and 2, not eligible for stem cell transplant. Patients with treatment-naive CMML were administered intravenous IMM01 at a dosage of 2.0mg/kg/week and subcutaneous AZA at a dosage of 75 mg/m 2 on D1-7 per 28-day cycle. Safety was assessed by CTCAE V5.0 and efficacy by IWG 2006 (MDS) criteria. Results : Between August 4, 2022 and July 7, 2023, 24 patients were enrolled: median age was 62 years, 15 (62.5%) males, 18(75.0%) ECOG≥1. Additionally, 1(4.2%), 7(29.2%) and 16(66.7%) patients were intermediate risk(IR)-1, IR-2 and high risk(HR) by CPSS-mol classification, respectively. According to World Health Organization (WHO) classification of CMML, patients enrolled included CMML-1(50.0%) and CMML-2(50.0%). Majority of patients had poor baseline hematologic conditions with a median Hb of 69.5 (32, 132) g/L and a median PLT of 73.5 (5, 667)×10 9/L. By the data cut-off date of July 7, 2023, the median follow-up for the study was 4 months (95%CI:3.3-4.8). Among the 16 efficacy evaluable patients who received initial treatment ≥3 months, overall response rate (ORR) was 87.5% (14/16), including 25.0% (4/16) complete response (CR), 18.8% (3/16) marrow CR (mCR) with hematologic improvement (HI), 6.3% (1/16) HI and 37.5% (6/16) mCR alone. Among the 9 efficacy evaluable patients who received initial treatment ≥4 months, ORR was 88.9% (8/9), including 44.4% (4/9) CR, 11.1% (1/9) mCR with HI and 33.3% (3/9) mCR alone. The CR rate of IMM01 combined with AZA is increasing with longer treatment duration. The median duration of response (DoR) was not reached. NGS analysis of bone marrow collected from responders were included in this study. The most frequent treatment related adverse events (TRAEs) (≥20%) were lymphopenia (70.8%), leukopenia (70.8%), neutropenia (70.8%), thrombocytopenia (58.3%), infusion related reaction (33.3%), anemia (25.0%), pyrexia (25.0%), pneumonia (25.0%), constipation (20.8%), nausea (20.8%), vomiting (20.8%) and leukocytosis (20.8%). The most common ≥Grade 3 TRAEs (≥10%) included lymphopenia (66.7%), neutropenia (54.2%), leukopenia (54.2%), thrombocytopenia (54.2%), anemia (20.8%) and pneumonia (16.7%), all of which were commonly reported in AZA monotherapy studies for treatment naive MDS/CMML patients in China. The study is ongoing. Conclusions: Preliminary data showed that IMM01 (without a low-dose priming) combined with AZA were well tolerated. The combo, when compared to the historical data of AZA alone, showed exciting efficacy result for patients with treatment-naive CMML1-2.

Prognostic Relevance of Truncating ASXL1 Mutations and ASXL1-Integrated Molecular Models in Proliferative CMML

Introduction Chronic myelomonocytic leukemia (CMML) can be classified into dysplastic (dCMML) and proliferative (pCMML) subtypes based on a white blood cell count (WBC) ≥ 13 x 10 9/L for pCMML. Though this cutoff may seem arbitrary, there is evidence that pCMML is a unique biological entity with a distinct molecular profile and worse outcomes (Carr RM, et al. Nat Commun. 2021). We hypothesized that contemporary CMML prognostic scoring systems - such as the Mayo Molecular Model (MMM; Patnaik MM, et al. Leukemia. 2014) and the CPSS-Molecular Model (CMM; Elena C, et al. Blood. 2016) - would not adequately risk-stratify pCMML patients. Moreover, while truncating ASXL1 mutations are considered a high-risk feature in CMML, their prognostic relevance in pCMML is unknown. Methods After IRB approval, we compiled a large (n = 888), molecularly-annotated database of CMML patients seen at two US medical centers (Mayo Clinic and MD Anderson) via retrospective chart review. All statistical analyses considered the clinical and laboratory parameters obtained at the time of presentation. The cohort was divided into dCMML and pCMML subgroups, which were analyzed independently alongside the entire cohort. Categorical variables were compared by Fisher exact or Pearson χ 2 tests and continuous variables by Mann-Whitney U tests. Univariate and multivariate analyses were performed using Cox proportional hazards regression models. Survival was assessed via the Kaplan-Meier method. P-values &amp;lt; 0.05 were considered significant. Calculations were performed using the BlueSky Statistics (v10.3.1) interface for R. Results We identified 474 (54%) dCMML cases and 410 (46%) pCMML cases with median age 71 years and 67% males. Patients with pCMML were more likely to have higher AMC (5.1 vs 1.5 x 10 9/L, p &amp;lt; 0.0001), circulating immature myeloid cells (IMC; 65% vs 36%, p &amp;lt; 0.0001) and abnormal karyotypes (37% vs 30%, p = 0.0187). They were also overrepresented in higher-risk categories of the MMM and CMM compared to dCMML (p &amp;lt; 0.0001 for each model). The number of somatic mutations was higher in pCMML than dCMML (mean 3.0 vs 2.6, p = 0.0050). Mutations in ASXL1 (53% vs 39%), NRAS (21% vs 11%), CBL (18% vs 12%), SETBP1 (14% vs 7%), JAK2 (10% vs 3%), CEBPA (5% vs 2%), GATA2 (3% vs 0%), and FLT3 (3% vs 1%) were more frequent while mutations in TET2 (38% vs 48%), SF3B1 (2% vs 8%), ZRSR2 (2% vs 8%), and NPM1 (1% vs 3%) were less frequent in pCMML compared to dCMML, respectively (p &amp;lt; 0.05 for all). Though the MMM and CMM effectively stratified the dCMML group, they did not provide meaningful risk stratification in the pCMML group (MMM p &amp;lt; 0.05 overall but could not distinguish between low and intermediate-1/2 or intermediate-1 and high risks; CMM p &amp;gt; 0.05). Likewise, there was no difference in the median overall survival (OS) or leukemia free survival (LFS) when pCMML patients were stratified by truncating ASXL1 mutations( Figure 1A). We then sought to better risk stratify pCMML. Univariate analysis identified increasing age, male gender, abnormal karyotype, and elevated WBC, ANC, and AMC as adverse predictors for OS. In addition, the presence of IMC and peripheral blood (PB) blast % were adverse factors for LFS, while age was not significant for LFS. None of the aforementioned mutations were significant in the univariate model for OS, whereas GATA2 and NPM1 mutations were significant for LFS. In multivariate analysis for OS, only male gender and AMC ≥ 10 x 10 9/L retained significance. In multivariate analysis for LFS, male gender, AMC ≥ 10 x 10 9/L, and peripheral blasts ≥ 5% remained significant; GATA2 and NPM1 mutations were not significant. These variables risk-stratified pCMML patients based on the presence of 0, 1, or ≥ 2 risk factors ( Figure 1B). In comparison, OS was 40.7 months (95% CI 35.2 - 50.1 mo.) for dCMML patients, 20.1 months (95% CI 18.2 - 22.7 mo.) for pCMML patients, and 31.8 months (95% CI 28.4 - 33.5 mo.) for the entire cohort. At last follow up, 586 deaths (66%) and 168 AML transformations (19%) were recorded in the cohort. Deaths in dCMML vs pCMML were 294 (62%) vs 291 (71%; p = 0.0067), with AML transformations in 83 (18%) vs 84 (21%; p = 0.2055) patients, respectively. Conclusions Truncating ASXL1 mutations, along with ASXL1-mutation weighted CMML prognostic models failed to effectively risk stratify pCMML patients. In pCMML, male gender and AMC ≥ 10 x 10 9/L, effectively risk stratified for OS, while these factors along with PB blasts ≥ 5% effectively risk stratified for LFS.