Chronic Myelomonocytic Leukemia Patients Research Articles

Sequential Conditioning With FLAMSA Does Not Improve Outcomes of Allogeneic Stem Cell Transplantation in Chronic Myelomonocytic Leukemia Patients

As with myelodysplastic syndromes (MDS), effective treatment options for Chronic myelomonocytic leukemia (CMML) are limited, and the optimal treatment approach remains undefined. Allogeneic stem cell transplantation (allo-SCT) is potentially curative therapy for patients with CMML. Sequential conditioning with FLAMSA was initially developed for refractory acute myeloid leukemia (AML) and has since been applied in the treatment of MDS and CMML. Data on optimal allo-SCT conditioning in CMML Patients is scarce. This retrospective study from the Department of Stem Cell Transplantation at the University Medical Center Hamburg, Germany, compared allo-SCT outcomes in CMML patients across three conditioning regimes: Thiotepa-Busulfan (TB), Sequential FLAMSA-Busulfan Fludarabine (FLAMSA-FB), and Treosulfan-Fludarabine (Treo-Flu). Sixty-nine consecutive patients with CMML who underwent allo-SCT between the years 2006-2022 were included in the study. Twenty-two received TB, 27 received FLAMSA-FB, and 20 received Treo-Flu conditioning. Transplant sources included matched related donors (MRD, 8 patients), mismatched related donors (MMRD, 8 all in the TB group), matched unrelated donors (MUD, 31), and mismatched unrelated donors (MMUD, 22) with significant group variations (p<0.001). Most patients received anti-T lymphocyte Globulin (ATLG) for graft versus host disease (GVHD) prophylaxis (TB 68%, FLAMSA-FB 93%, Treo-Flu 85%, p=0.08). CPSS-Molecular score was comparable between the groups. One TB patient experienced primary graft failure, but engraftment times were comparable across the groups. Although not statistically significant, the TB group showed a trend towards improved 3-year OS rates (80%) compared to FLAMSA-FB (37%) and Treo-Flu (55%) (p=0.05). The TB group also displayed significantly higher 3-year PFS rates (80%) compared to FLAMSA-FB (33%) and Treo-Flu (both 39%), (p=0.02). No significant differences were observed in 3-year non-relapse mortality (NRM) across the TB (20%), FLAMSA-FB (30%), and Treo-Flu (26%) groups (p=0.8). Interestingly, no TB patients relapsed at 3 years, contrasting with the FLAMSA-FB (41%) and Treo-Flu groups (30%, p=0.02). Lastly, cumulative incidences of acute and chronic GVHD were similar across groups. Our analysis suggests FLAMSA-FB does not improve transplant outcomes, however TB represents the preferred conditioning regimen for CMML patients undergoing allo-SCT. It demonstrates notable advantages in relapse prevention and leads to improved OS and PFS compared to FLAMSA-FB and Treo-Flu protocols.

CBL mutations in chronic myelomonocytic leukemia often occur in the RING domain with multiple subclones per patient: Implications for targeting

Chronic myelomonocytic leukemia (CMML) is a rare blood cancer of older adults (3 in every 1,000,000 persons) characterized by poor survival and lacking effective mutation-specific therapy. Mutations in the ubiquitin ligase Cbl occur frequently in CMML and share biological and molecular features with a clonal disease occurring in children, juvenile myelomonocytic leukemia (JMML). Here we analyzed the clinical presentations, molecular features and immunophenotype of CMML patients with CBL mutations enrolled in a prospective Phase II clinical trial stratified according to molecular markers. Clinically, CBL mutations were associated with increased bone marrow blasts at diagnosis, leukocytosis and splenomegaly, similar to patients harboring NRAS or KRAS mutations. Interestingly, 64% of patients presented with more than one CBL variant implying a complex subclonal architecture, often with co-occurrence of TET2 mutations. We found CBL mutations in CMML frequently clustered in the RING domain in contrast to JMML, where mutations frequently involve the linker helix region (P&lt;0.0001). According to our comparative alignment of available X-ray structures, mutations in the linker helix region such as Y371E give rise to conformational differences that could be exploited by targeted therapy approaches. Furthermore, we noted an increased percentage of CMML CD34+ stem and progenitor cells expressing CD116 and CD131 in all CBL mutant cases and increased CD116 receptor density compared to healthy controls, similar to CMML overall. In summary, our data demonstrate that CBL mutations are associated with distinct molecular and clinical features in CMML and are potentially targetable with CD116-directed immunotherapy.

Impact of spliceosome mutation on outcomes of myelodysplastic syndrome and chronic myelomonocytic leukemia patients undergoing allogeneic hematopoietic cell transplantation

IntroductionAllogeneic Hematopoietic cell transplantation (allo-HCT) remains the only curative therapy for myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML). The impact of spliceosome mutations on allo-HCT outcome is unclear and further understanding is needed to assess the implications of this class of mutations on risk of relapse, overall survival (OS) and non-relapse mortality (NRM) in order to make decision regarding timing of allo-HCT. We examined the allo-HCT outcomes of MDS/CMML patients based on their spliceosome mutation profile to understand the impact of these mutations on transplant outcomes. ObjectiveTo compare outcomes of MDS/CMML patients with and without spliceosome mutations undergoing allo-HCT. MethodsThis is a single institution, retrospective study of MDS/CMML patients who underwent allo-HCT with myeloablative or reduced intensity conditioning (RIC) regimen at City of Hope from January 2016 to December 2021. Among them, patients who underwent molecular mutation profiling by NGS (Next Generation Sequencing) for a set of genes known to be mutated in myeloid neoplasms are included in this analysis. We compared OS, relapse free survival, NRM and acute/chronic graft versus host disease (GVHD) incidence between the spliceosome-mutated and unmutated groups. ResultsWe identified 258 consecutive MDS/CMML patients who underwent allo-HCT. Of these, 126 (48.8 %) patients had molecular profiling done among whom 57 (45.2 %) patients carried a spliceosome mutation. 84.9 % of patients had MDS and 55.6 % underwent a matched unrelated donor transplant. The median age for the whole cohort was 66 years (range 12–77).78.6 % and 73.7 % received RIC in the spliceosome and non-spliceosome groups, respectively. The 2-year OS for the whole cohort was 66.5 % (95 %CI 0.55–0.75) with a day 100 NRM of 7.1 % and 2-year cumulative incidence of relapse of 20 %. Grade II-IV acute GVHD at day 100 was 36.3 % (95 % CI 0.27–0.44) and any chronic GVHD at 2-years was 48.4 % (95 % CI 0.37–0.58). Patients who carried a spliceosome mutation had a significantly better 2-year survival of 83.8 % vs 55.9 % in the non-spliceosome group (P=0.002) and a better PFS of 73.7 % vs 50.0 % (P=0.007). There was no difference in the cumulative incidence of relapse at 2-years 15.9 % vs 18.5 % (P=0.59) between two groups but the spliceosome group had a significantly lower NRM at 2-years 10.4 % vs 31.5 % (P=0.009). There was no difference in incidence of acute or chronic GVHD between the two groups. ConclusionsAmong patients with MDS or CMML who underwent allo-HCT, our study shows better OS for patients who have spliceosome mutations due to lower NRM compared to those carrying non- spliceosome mutations. This favorable outcome of the spliceosome-mutated patients could have implications for timing of allo-HCT, particularly for patients in the intermediate MDS prognostic risk groups.

Chronic Myelomonocytic Leukemia and Atypical Chronic Myeloid Leukemia: A National Analysis

BackgroundMyelodysplastic/myeloproliferative overlap syndromes (MDS/MPN) are rare blood cancers characterized by concomitant myeloid hyperplasia and dysplasia. These heterogenous disorders include chronic myelomonocytic leukemia (CMML) and atypical chronic myeloid leukemia (aCML). MethodsUsing two large national cancer databases to examine a total of 15,704 CMML and 702 aCML patients, we report the largest study to date on the incidence, survival and demographic characteristics of CMML and aCML in the United States. ResultsOverall age-adjusted incidence of CMML and aCML was 0.63 per 100,000 Americans per year and 0.03 per 100,000 per year, respectively. CMML incidence in the U.S. was noted to rise steadily in the years between 2001 and 2019. Median patient age was 75 and 72 years for CMML and aCML, and the majority of CMML and aCML patients were male (62.9% and 62.0%) and White (90.1% and 86.3%). Median OS was 17.4 months for CMML, and 15.2 months for aCML. Multivariate Cox regression demonstrated features associated with reduced survival, including increasing age, comorbidities, Medicaid insurance status, and low-income residential zip code, highlighting survival disparities in underinsured and socioeconomically disadvantaged patients. In CMML, Black race was associated with inferior survival, while female sex, management at an academic center, and later calendar-year of diagnosis were associated with improved OS. ConclusionThese findings underscore the need to better understand the biological basis for such differences in survival and reflect the importance of access to specialized care for patients with these rare disorders.

Genomic evolution of patients with myeloid neoplasms and known antecedent clonal hematopoiesis.

6584 Background: Clonal hematopoiesis (CH) refers to the clonal expansion of hematopoietic stem cells (HSCs) with one or more somatic mutations in the absence of a hematologic malignancy. Although it is associated with normal aging, CH confers an increased risk of several comorbidities and hematologic malignancies, including myeloid neoplasms (MNs). However, the outcomes of patients with CH that transformed to MN are unknown. Here, we aim to evaluate the clinicopathologic characteristics and survival outcomes of patients with MN with known antecedent CH. Methods: We retrospectively evaluated patients seen at a tertiary cancer center from May 2016 to July 2023 with myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), or acute myeloid leukemia (AML) and identified those who had CH prior to MN diagnosis. At the time of MN transformation, genomic data was extracted from whole bone marrow aspirate samples and subject to 81-gene target PCR-based sequencing using a next-generation sequencing platform. Results: A total of 36 patients were identified; at the time of CH diagnosis, 79% had known cardiovascular comorbidities and 81% had a prior diagnosis of non-myeloid malignancies for which 72% received prior chemotherapy and 25% radiation. According to the Clonal Hematopoiesis Risk Score (CHRS), 22% were low, 56% deemed intermediate, and 22% at high risk of transformation to MNs. The most frequently observed CH-associated mutations were TET2 (41%), TP53 (38%), IDH2 (16%), EZH2 (16%), ASXL1 (13%), and DNMT3A (13%); 69% had more than one somatic mutation, and 61% had normal cytogenetics. The median time to transformation to MN was 15 months, 26 months, and 24 months for MDS (n=28), CMML (n=2), and AML (n=6) patients, respectively (p=0.72). The median overall survival from MN diagnosis was 54 months, not reached, and 2 months for MDS, CMML, and AML, respectively (p=0.0015). We further delved into the genomic evolution from CH to MN. In the 6 pts who transformed to AML, 4 had a TP53 mutation at CH with expansion of the clone at MN transformation, 2 acquired NPM1 mutations and 1 acquired FLT3 mutations. Both pts who progressed to CMML had expansion of their underlying CH clones (KRAS and ZRSR2). In those who developed MDS, 22 pts had expansion of their CH-mutated clone at the time of MN, most frequently in TET2 (36%), IDH2 (18%), and TP53 (14%); 5 pts acquired new driver mutations outside of their CH clone; and 1 pt lost their CH clone with no new somatic mutations detected. Conclusions: Individuals with CH are at risk of progressing to MN, especially with external selective pressures like antineoplastic therapy that are not accounted for in prognostic systems like CHRS. Patients often have underlying high-risk clones at the time of CH diagnosis that expand at the time of MN, though some patients acquired new driver mutations.

PTPN11variants in chronic myelomonocytic leukemia: Phenotypic and prognostic correlates.

6513 Background: PTPN11 (Ch12q24) is a proto-oncogene that codes for SHP2, a regulatory protein tyrosine phosphatase that impacts RAS/MAPK signaling. Germline variants have been described in juvenile myelomonocytic leukemia, while somatic variants have been associated with inferior survival in acute myeloid leukemia. PTPN11 variants are infrequent in chronic myelomonocytic leukemia (CMML), and their impact on disease phenotype and survival remains unclear. Methods: After IRB approval, 396 molecularly annotated CMML patients at Mayo Clinic were screened for somatic PTPN11 variants within 12 months of diagnosis while still in chronic phase disease. Overall (OS), acute myeloid leukemia (AML)-free survival (LFS), and predictors of survival were estimated using standard statistical measures. Results: 396 patients (median age 71 years; males 67%) with CMML were considered; pathogenic PTPN11 variants were seen in 12 (3%) with a median VAF of 20.5% (range: 4-50): A72T (n = 2), F285S (n = 2), and A72S (n = 2). Variants were most likely to be seen in the N-terminal SH2 domain (n = 7), followed by the PTP domain (n = 6), and the C-terminal SH2 domain (n = 1). PTPN11 variants clustered with CMML-2 (p = 0.02), higher absolute neutrophil count (p = 0.04), peripheral blood (p = 0.06) and bone marrow (p = 0.02) blast %, older age (p &lt;0.01), and were less likely to have SRSF2 comutations (p = 0.02). After a median follow-up of 18 months (range 0-193), 253 (64%) deaths and 68 (17%) AML transformations were recorded. PTPN11 variants negatively and independently impacted both OS (median 13 vs. 31 months; p &lt;0.01) and LFS (AML events: 42% (5/12) vs. 16% (63/384); p &lt;0.01). In age-adjusted multivariate analysis restricted to genetic risk factors, survival was negatively affected by abnormal karyotype and mutations involving PTPN11, DNMT3A, and SETBP1, and positively by TET2 mutations. All but SETBP1 retained significance in the presence of additional independent risk factors, namely male sex (p &lt;0.01) and leukocyte count (p &lt;0.01); HR in the latter analysis was 4.3 for PTPN11, 2.8 for DNMT3A, and 1.6 for abnormal karyotype. A similar analysis for LFS identified, as risk factors, PTPN11 (p = 0.01), DNMT3A (p &lt;0.01), and ASXL1 (p = 0.03) mutations, as well as bone marrow blast % (p &lt;0.01), absolute monocyte count ≥10 (p = 0.02), and immature circulating myeloid cells (p &lt;0.01). Conclusions: The current study identifies PTPN11 and DNMT3A mutations as independent risk factors for both OS and LFS in CMML. In regard to additional genetic risk factors, OS was positively affected by TET2 mutations and negatively by abnormal karyotype, while LFS was negatively affected by ASXL1 mutations.

A real-world experience of venetoclax combined with hypomethylating agents vs. monotherapy hypomethylating agents in patients with myelodysplastic syndromes and chronic myelomonocytic leukemia patients.

Current clinical research has reported the effectiveness and safety of venetoclax in combination with hypomethylating agents (VEN-HMA) in patients with myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). Thus, this study aimed to examine the effectiveness and safety of VEN-HMA therapy in patients with MDS and CMML and compared its short-term and long-term therapeutic effects with HMA monotherapy. We analyzed data from our center, comprising 19 patients with MDS and CMML who received VEN-HMA therapy, compared to 32 patients treated with HMA monotherapy. The overall response rate (ORR) in the VEN-HMA group was 73.7%, compared to 59.4% in the HMA group. The survival analysis revealed that the median overall survival (mOS) time in the VEN-HMA group was 16 months, with a median progression-free survival (mPFS) time of 9 months, both of which were longer than those observed in the HMA group (p < 0.05). Key adverse events (AEs) included grade 3-4 neutropenia (89.5% in VEN-HMA group vs. 87.5% in HMA group), grade 3-4 thrombocytopenia (73.7% vs. 71.9%), and anemia (73.7% vs. 90.6%). Infection of grade 3 or higher occurred in 63.2% of patients in the VEN-HMA group and 65.6% of patients in the HMA group. Our study has confirmed the effectiveness and safety of the combined treatment of HMAs and venetoclax, which offers significant advantages to patients due to the relatively high and rapid response rates.

Clinical Efficacy of Hypomethylating Agent Therapy in Patients with Chronic Myelomonocytic Leukemia

To observe the clinical efficacy and safety of hypomethylating agent therapy in chronic myelomonocytic leukemia (CMML). From February 2014 to June 2021, the clinical data, efficacy, survival time and safety of CMML patients diagnosed in the Second Hospital of Hebei Medical University and treated with hypomethylating agent therapy were retrospectively analyzed. A total of 25 CMML patients received hypomethylating agent therapy, including 18 cases treated with decitabine (DEC) and 7 cases treated with azacytidine (AZA) as the basic treatment. Among them, 20 patients responded, and 7 patients got complete remission (CR). All patients with CR were treated with DEC as the basic treatment. Five cases of CR occurred in the first 4 courses of treatment. After a median follow-up of 16.4 (9.4-20.5) months, 4 patients with CR progressed to acute myeloid leukemia (AML). The median overall survival (OS) time of 25 CMML patients was 17.4 months (95%CI: 12.437-22.363). According to MD Anderson prognostic scoring system (MDAPS), CMML-specific prognostic scoring system (CPSS), CPSS molecular (CPSS-mol), Mayo molecular model (MMM), risk stratification of patients was performed, and the difference only between different risk stratification of MDAPS and survival time was statistically significant. Common adverse reactions of hypomethylating agent therapy in CMML patients included infection, gastrointestinal reaction, hematological toxicity, skin allergy and liver function damage. All patients' symptoms were improved after corresponding treatment. Hypomethylating agent therapy is effective and safe for CMML patients. CR mostly occurs in the first 4 courses of treatment, and hypomethylating agent therapy combined with low-dose chemotherapy can be used for patients who do not respond. Hypomethylating agent therapy can delay the disease, but can't prevent progression.

Results from phase 1 of the MANIFEST clinical trial to evaluate the safety and tolerability of pelabresib in patients with myeloid malignancies

Pelabresib (CPI-0610), a BET protein inhibitor, is in clinical development for hematologic malignancies, given its ability to target NF-κB gene expression. The MANIFEST phase 1 study assessed pelabresib in patients with acute leukemia, high-risk myelodysplastic (MDS) syndrome, or MDS/myeloproliferative neoplasms (MDS/MPNs) (NCT02158858). Forty-four patients received pelabresib orally once daily (QD) at various doses (24–400 mg capsule or 225–275 mg tablet) on cycles of 14 d on and 7 d off. The most frequent drug-related adverse events were nausea, decreased appetite, and fatigue. The maximum tolerated dose (MTD) was 225 mg tablet QD. One patient with chronic myelomonocytic leukemia (CMML) showed partial remission. In total, 25.8% of acute myeloid leukemia (AML) patients and 38.5% of high-risk MDS patients had stable disease. One AML patient and one CMML patient showed peripheral hematologic response. The favorable safety profile supports the ongoing pivotal study of pelabresib in patients with myelofibrosis using the recommended phase 2 dose of 125 mg tablet QD. CLINICAL TRIAL REGISTRATION: NCT02158858

Clinical outcomes of allogeneic hematopoietic stem cell transplantation for chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) is a clonal disease derived from bone marrow hematopoietic stem cells, with a poor prognosis. Allogeneic hematopoietic stem cell transplantation (allo- HSCT) is one of the curable methods for CMML. The outcome of patient transplantation is influenced by various factors such as disease characteristics and comorbidities. Based on the existing prognostic stratification system, screening suitable CMML patients for transplantation and early transplantation is beneficial for their long-term survival. Doctors can evaluate the survival status of CMML patients after transplantation based on the newly developed transplant prognosis model and make targeted medical decisions.

Outcomes of intermediate or high-risk CMML patients treated with hypomethylating agents combined with venetoclax: A single center experience.

Chronic myelomonocytic leukemia (CMML) treatment remains a pressing clinical challenge. We conducted a retrospective analysis on 52 CMML cases, exploring the effectiveness of combining venetoclax (Vene) with hypomethylating agents (HMAs). The study's findings show promise: the HMAs plus Vene group (n = 13, 53.8%) demonstrated superior overall response rates compared to the HMA monotherapy (mono) group (n = 19, 31.6%) and HMA plus arsenic trioxide group (n = 9, 22.2%) by the second cycle, and notably higher response rates (53.8% vs. 15.7%, p = 0.04) compared to the HMA mono group after four cycles. Over a median follow-up of 14.7 months, the HMAs plus Vene group exhibited significantly lower cumulative mortality (23.1%) compared to the other two groups (p = 0.003 and p = 0.008, respectively). Furthermore, this group displayed extended overall survival compared to the others. The study also delved into the molecular mechanisms, revealing significant BCL2 mRNA overexpression in patients with CMML. These findings suggest the potential for HMAs combined with Vene therapy in CMML but emphasize the necessity for further prospective studies to determine its precise role in managing CMML.

Should oligomonocytic CMML be classified as an independent disease entity?

The 2022 WHO Classification revised the diagnostic criteria of CMML and included the variant, formerly known as oligomonocytic CMML (O-CMML), into the CMML disease. CMML is currently subtyped into MD-CMML and MP-CMML. In a recent study, Castaño-Díez etal. showed that the newly included O-CMML has unique clinical features and is an independent favourable risk factor, and proposed to view O-CMML as an independent subtype of CMML, parallel to MD-CMML and MP-CMML. Commentary on: Castaño-Díez et al. Characteristics and long-term outcome in a large series of chronic myelomonocytic leukaemia patients including 104 formerly referred to as oligomonocytic. Br J Haematol 2024;204:892-897.

NLRP3 inflammasome activation and symptom burden in KRAS-mutated CMML patients is reverted by IL-1 blocking therapy

Chronic myelomonocytic leukemia (CMML) is frequently associated with mutations in the rat sarcoma gene (RAS), leading to worse prognosis. RAS mutations result in active RAS-GTP proteins, favoring myeloid cell proliferation and survival and inducing the NLRP3 inflammasome together with the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which promote caspase-1 activation and interleukin (IL)-1β release. Here, we report, in a cohort of CMML patients with mutations in KRAS, a constitutive activation of the NLRP3 inflammasome in monocytes, evidenced by ASC oligomerization and IL-1β release, as well as a specific inflammatory cytokine signature. Treatment of a CMML patient with a KRASG12D mutation using the IL-1 receptor blocker anakinra inhibits NLRP3 inflammasome activation, reduces monocyte count, and improves the patient's clinical status, enabling a stem cell transplant. This reveals a basal inflammasome activation in RAS-mutated CMML patients and suggests potential therapeutic applications of NLRP3 and IL-1 blockers.

Venetoclax and Azacytidine Treatment for High Risk Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia As a Bridge Therapy to Transplant. a GESMD Study

Introduction Allogeneic hematopoietic cell transplantation (alloHCT) is the only potentially curative treatment for myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). Most common approach to bridge higher-risk MDS to alloHCT is to reduce tumor burden before alloHCT with azacytidine (AZA). Currently with this strategy less than 50% of patients achieve overall response rate (ORR) and less than 20% complete response (CR). Moreover, it is associated with a considerable rate of drop outs (approximately 50%) due to progression disease and mortality prior to alloHCT. Therefore, we need more effective treatments with faster and higher responses to facilitate more patients to undergo alloHCT. Venetoclax with AZA (VEN/AZA) is being investigated mostly in unfit MDS patients, it has shown higher ORR and early responses. Recently, a study suggested promising activity in 13 high-risk MDS patients who received VEN/AZA and proceeded to alloHCT (Komrokji et al, BCJ, 2022). The objective of our study is to analyze the feasibility, toxicity, and efficacy of VEN/AZA treatment in MDS and CMML patients who are intended to undergo alloHCT. Methods This is a retrospective multicenter study that includes high-risk MDS and CMML patients treated with VEN/AZA who are intended to undergo alloHCT. The response rate was assessed according to the IWG 2006, IWG 2023, ELN 2022. Savona criteria were used in CMML. Results A total of 23 patients were included, with a median age of 61 years (41-74), including 19 MDS and 4 CMML patients. Eight patients received VEN/AZA in a relapsed/refractory (R/R) setting, while 15 in first line (1L). Most patients received a 14-day scheme of VEN (n=16). The administration of VEN/AZA and other characteristics are described in Table 1. Grade 3/4 cytopenias were frequent (83% neutropenia 65% thrombocytopenia, 59% anemia), 17% experienced febrile neutropenia and 17% required hospitalization. G-CSF was used in 44% of patients, antifungal prophylaxis in 43%, and cycle delays due to cytopenias happened in half of them (47%). Thirty-day mortality was 0%, and one patient (4%) died within the first 60 days due to disease progression. The response rate following IWG 2023 criteria was: 7 CR (30%), 6 CR with limited count recovery (CR L, 26%), 4 CR with partial hematologic recovery (CRh, 17%), 3 partial remissions (PR, 13%), 1 hematologic improvement (HI, 4%), and 2 no response (9%). The ORR was 91.3% (21/23), and the rate of composite complete responses (CRc) including CR, CR L and CRh was 73.9% (17/23) . These responses occurred rapidly, all patients achieved the first response in 1 cycle and the median to best response was 1 cycle (range 1-3). In R/R patients the ORR was 87% and the CRc was 62%. Using the ELN 2022 versus IWG 2023 response criteria results in similar CRc, higher rate of CR (34% vs 22%) and lower ORR (86 vs 91%). The median follow-up since the start of treatment was 13.6 months (95% CI: 5.2-21). The 2-year overall survival (OS) was 63% (95% CI ± 24%). Currently, there are 2 patients awaiting alloHCT. Among the 21 evaluable patients, 19 underwent alloHCT, resulting in a alloHCT rate of 91%. The 2 patients who were not transplanted included one CMML patient with disease progression prior alloHCT and one refractory MDS. AlloHCT was performed with a median of 3 prior cycles (range: 1-9), and the median time from the start of VEN/AZA to transplantation was 4 months (range: 2.5-10). One patient who did not respond to VEN/AZA was treated with chemotherapy prior alloHCT achieving CR. Two patients progressed before alloHCT, one achieved CR after chemotherapy and the other one has received a sequential alloSCT. The two-year post-transplant OS was 67% (95% CI ± 28%) (Figure 1). Four patients relapsed after alloHCT, resulting in a two-year cumulative incidence of relapse (CIR) of 37% (95% CI ± 28%). One of these patients was treated with VEN/AZA post-transplant and he is currently in CR for 1.5 years. 2-year transplant-related mortality was 6% (95% CI ± 12%). Conclusion Treatment with VEN/AZA as a bridge to alloHCT in MDS and CMML patients is feasible with a high rate of patients undergoing alloHCT. It provides high and quick responses, even in R/R patients, with low treatment-related toxicity. Funding This study was supported by grants from the Institute of Health Carlos III (ISCIII), with grant numbers PI19/01476 and PI22/01660, co-financed by the European Union. Additional support was provided by Emili-Letang (HCPB).

Prognostic and Therapeutic Implications of TP53 expression in Chronic Myelomonocytic Leukaemia: Results of a Multicentre Study

Thetumor suppressor TP53 is among the most mutated genes in cancer, including many hematological malignancies in which it usually imparts more aggressive, chemo-refractory, and poor prognosis diseases. TP53 mutations are, though, remarkably rare in chronic myelomonocytic leukemia (CMML): affecting 2.4% of cases in a recent study ( Gurney et al, Leukemia 2023). We found pathogenic TP53 mutations ( TP53MT) in only 1.1% of 640 genotyped UK CMML patients. As expected, TP53MT conferred worse leukemia-free (LFS: median 23 v 28 mo, p=0.031) and overall (OS: 26 v 29 mo, p=0.015) survival compared with TP53WTcases. The clinical relevance in CMML of TP53 expression, however, remains unexplored. To address this we studied 92 TP53WT CMML patients (discovery cohort) from the National Taiwan University Hospital (Taipei, Taiwan) with available RNA-seq on presentation bone marrow (BM), each with extensive longitudinal clinical, laboratory and outcome data. Validation cohorts comprised 33 RNA-sequenced patients from The Christie (Manchester, UK) and 24 from Hospital Morales Meseguer (Murcia, Spain). For all cohorts, RNA-seq data from healthy controls (HCs) were available. Patients in the discovery cohort were divided into low and high TP53 expression groups, determined by the maximally selected rank statistics. TP53low patients (n=15) displayed significantly lower expression than HCs, while TP53high (n=77) had expression levels comparable to HCs. Thus, although most CMML patients are TP53WT, a subset has down-regulated TP53 expression and potential for altered p53 (and downstream) functions. TP53low and TP53high patients did not differ in clinical features or concurrent genetic alterations, although in all cohorts TP53low patients showed a trend towards poorer response to hypomethylating agents (HMA). TP53 low patients displayed significantly worse outcomes than TP53high patients (LFS 7 v 19 mo, p=0.002; OS 11 v 26 mo, p=0.001). Moreover, TP53 expression further stratified prognosis within different CMML-specific prognostic scoring system (CPSS)-molecular risk groups (Fig 1) . Time-dependent ROC analysis indicated TP53 expression as complementary to current risk stratification systems. In multivariable analysis, low TP53 expression remained prognostically detrimental. Survival analyses were consistent in both validation cohorts. GSEA and IPA analyses revealed that compared with HCs or TP53high, TP53low cells exhibited depleted expression of p53-dependent pathways, including MYC targets, E2F targets, G2/M checkpoints, and DNA repair. Interestingly, these are all among the most upregulated pathways in TP53MT (v TP53WT) samples across multiple cancers in TCGA data ( Donehower et al, Cell Rep 2019), indicating distinct roles for p53 in regulating these pathways in a TP53WT context, and that the driving biology of TP53low CMML is distinct from (and not functionally equivalent to) that of oncogenic TP53 mutations. TP53low patients demonstrated significantly enhanced TNF-alpha and inflammatory response signals, highlighting other distinctive pathobiology of the TP53low subgroup. In line with our clinical observation, single-sample GSEA showed significant enrichment of HMA resistance signatures in TP53low patients, potentially contributing to the dismal prognosis in this group. We next investigated whether this could be exploited therapeutically. We treated primary CMML BM cells ex vivo with azacitidine and NSC-207895, an MDMX inhibitor with p53 activating properties, observing clear and substantial synergy for combination therapy in samples from 10 of 11 patients. There was a trend towards inverse correlation between TP53 expression and empirical synergy scores (r=-0.35), suggesting potential for p53 activation to enhance HMA sensitivity in CMML, with broad efficacy, perhaps preferentially in adverse risk TP53low cases. In summary, we have identified and validated, across three cohorts from three countries, that a subgroup of CMML patients display aberrantly low TP53 expression, associated with HMA resistance and worse survival. Transcriptomic analyses revealed distinctive biology driving this novel TP53low subgroup (Fig 2), mandating further mechanistic study. Importantly we report consistent and substantial synergy for combining pharmacological p53 activation with HMA, presenting a novel targeted approach to improve HMA response for this unmet clinical need.

Targeting Oncogenic Enhancers in ASXL1-Mutant Chronic Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm with limited therapeutic options and dismal outcomes. Truncating mutations in the epigenetic modifier ASXL1 are common and lead to a proliferative disease phenotype, resistance to epigenetic therapies, and higher rates of AML transformation. ASXL1-mutant CMML is characterized by widespread epigenetic remodeling and up-regulation of key leukemogenic driver genes (e.g. HOXA9 and its co-factor MEIS1). There is a genotype-specific activation of enhancers in ASXL1-mutant CMML that is associated with up-regulation of these leukemogenic driver genes. Here, we test the hypothesis that disrupting the oncogenic interactions at these genotype-specific enhancers can be exploited for therapeutic benefit. Methods: We identified activated enhancers specific to ASXL1-mutant CMML as previously described ( Binder et al. Nat Commun 2022;13(1):1434). To disrupt the E2542605 enhancer, we created unique mapped single guide RNAs (confirmed by BLAT). We generated a ribonucleoprotein complex using the Alt-R CRISPR-Cas9 system (IDT Technologies, Coralville IA). We combined the RNP along with Electroporation Enhancer to 3x10^5 cells of the monocytic cell line U937 in 20uL strip-wells provided by the SF Cell Line 4D-Nucleofector X Kit (Lonza, Basel, Switzerland). To deliver the RNP, U937 cells were electroporated via listed cell line specific protocol by the Amaxa 4D-Nucleofector (Lonza). Forty-eight hours post-electroporation, cells were split and sequenced for E2542605 genomic structure disruption. In parallel, RNA was harvested, converted to cDNA and qPCR for HOXA9 and MEIS1 expression was performed using PrimeTime qPCR FAM labeled probe assays (IDT). Gene expression was determined using delta-delta Ct method. For assessment of selective sensitivity of ASXL1 / enhancer interactions, we performed progenitor colony forming assays using primary bone marrow mononuclear cells from CMML patients with or without ASXL1 mutations and healthy normal controls (n=2 in each category), exposed to increasing concentrations of the dual p300/CBP and BET bromodomain inhibitor EP31670. Briefly, we plated cells in methocellulose fortified with recombinant human cytokines supporting hematopoietic growth in duplicate, in a range of EP31670 concentrations. After 10 days, visible hematopoietic colonies were quantified and characterized. IC 50 values were generated using non-linear regression log(EP31670) vs. normalized response. Results: We computationally identified candidate transcriptional regulators enriched at the activated enhancers specific to ASXL1-mutant CMML, including BRD4 (FDR&amp;lt;2.57x10 -370), CBP (FDR=2.56x10 -217), and p300 (FDR=6.36x10 -93). We selected the ASXL1-mutant-specific enhancer E2542605 (predicted to drive HOXA9 expression), given the up-regulation of HOXA9 in ASXL1-mutant CMML (log 2-fold change +2.05, FDR=0.006, n=14). We disrupted E2542605 using two different CRISPR guides, leading to a PCR trace detection of a 14bp deletion and a 26bp indel in the E2542605 enhancer region, respectively. Both enhancer disruptions led to a down-regulation of its putative target gene HOXA9: Mean relative fold-change 0.36 (p=0.001, n=3) and 0.65 (p=0.046, n=3), respectively ( Fig. 1a). We then used a pharmacologic approach (EP31670) to target the candidate transcriptional regulators involved in the oncogenic interaction at these ASXL1-mutant-specific enhancers. We observed selective sensitivity to growth of CMML colonies (particularly ASXL1-mutant colonies) compared to ASXL1-wild type cases and normal bone marrow controls ( Fig. 1b). Conclusions: In CMML patients, truncating ASXL1 mutations are associated with the activation of genotype-specific enhancers and the recruitment of an extended repertoire of transcriptional regulators including BRD4, CBP, and p300. In a model system, the disruption of an enhancer predicted to govern the expression of the leukemogenic driver HOXA9, led to a significant down-regulation of HOXA9 expression. Pharmacologically disrupting the oncogenic interaction at such enhancers with EP31670 in primary patient samples led to significant therapeutic effects at nanomolar drug concentrations in a genotype-specific manner. Oncogenic interactions at activated enhancers are viable therapeutic targets in ASXL1-mutant CMML and warrant further clinical investigation.

Development of Oral Azacitidine with Cedazuridine for Myelodysplastic Syndrome (MDS) and Myeloproliferative Neoplasms (MPN) Including CMML (Chronic Myelomonocytic Leukemia) By Targeting Pharmacokinetic AUC Equivalence Vs Subcutaneous Azacitidine

Introduction/ Background: Hypomethylating agents (HMAs) such as azacitidine (AZA) and decitabine (DEC) are FDA-approved for patients with MDS/CMML and acute myeloid leukemia (AML). Treatment is generally administered parenterally as a daily injection or infusion over 5-7 days per four-week cycle. The oral fixed-dose combination of decitabine with cedazuridine, ASTX727, was approved by regulators in the USA and Canada for patients with MDS and CMML based upon the demonstration of biologically equivalent pharmacokinetics (PK) area under the curve (AUC) vs IV decitabine. ASTX030 is a combination of AZA and cedazuridine (CED), a cytidine deaminase inhibitor (CDAi), which enables oral AZA to achieve systemic AUC similar to that of parenteral (subcutaneous) AZA. Development and approval of an oral AZA providing equivalent PK exposure to the parenteral therapy has the potential to markedly reduce the treatment burden associated with parenteral treatment. This Phase I trial is designed to determine the dose combination for oral AZA plus CED to replicate SC AZA AUC exposures in subjects with MDS and MDS/MPN, including CMML. Methods: Adult patients with confirmed MDS, CMML, and other MDS/MPN or AML who are candidates to receive benefit from single agent AZA were enrolled in this open label Phase 1 trial. In addition to assessing safety and efficacy of each combination, the primary PK endpoint was to achieve oral AZA PK AUC 0-24 equivalence versus subcutaneous AZA (at the approved dose of 75 mg/m 2 over the span of 7 days). In the first cycle, patients received oral AZA alone on Day -3, followed by SC AZA at 75 mg/m 2 on Day 1. Subsequently, oral ASTX030 (combination of AZA with CED) was given at varying dose combinations of AZA and CED for each cohort. After cycle 1, patients received oral ASTX030 on Days 1-7 for each 28-day cycle. Dose levels for cohorts in escalation were determined by the data safety and review committee based on safety and PK results. Pharmacodynamic (PD) impact was assessed by changes in LINE-1 DNA methylation in peripheral blood cells. Clinical response assessments were based upon International Working Group (IWG) 2006 criteria for MDS patients and IWG 2015 criteria for MDS/MPN and CMML patients. Results: As of 05 JUL 2023, 65 patients were treated across 8 different dose combinations, with ≥6 patients per cohort. The median age was 72 years old (range 26-87), 36.9% (n=24) were female, 6.2% (n=4), had previously received HMA treatment (unlimited). Of the enrolled patients, 69% (n=45) had MDS, 25%(n=16) had CMML, and 6% (n=4) had MDS/MPN (other). Evaluated dose levels for AZA ranged from 60-136mg and CED dose ranged from 20-100 mg. In cohorts 1-2, tablet formulation was tested first (AZA ranging 80-100mg with CED ranging 80-100mg). Starting with cohort 3, capsule formulation was introduced for AZA to optimize the interaction effect of CDA inhibition by CED. In both tablet and capsule formulation, grade ≥3 AE's (regardless of causality) were observed in 75.9% of patients. The most common Grade ≥3 AE's regardless of relation to drug were leukopenia (25%), thrombocytopenia (20%), and anemia (20%). Gastrointestinal toxicity were similar (for example, nausea 70% ASTX030 vs 71% SC AZA) to those reported for SC AZA (VIDAZA USPI)). No dose limiting toxicity was observed in any of the cohorts. The CED dose of 20 mg resulted in ~100% bioavailability for AZA, suggesting inhibition in a linear range. LINE-1 demethylation results were similar to those reported historically with SC AZA. Although a clinical response comparable to SC AZA was observed, the immature nature of the data requires continued analysis to yield results on treatment efficacy. Conclusion: ASTX030 successfully achieved the primary endpoint of PK equivalence versus SC AZA based on total cycle AUC. The dose combinations evaluated were well tolerated, and the safety profile similar to that of SC AZA, with no unique AE's. LINE-1 demethylation and clinical response confirm the clinical activity of ASTX030. Phase 1B dose expansion for the ASTX030 study at the recommended dose combination of 144mg AZA with 20mg CED will begin soon to confirm PK equivalence for this dose level. An orally bioequivalent AZA based HMA offers significant opportunity for novel combinations and improved convenience for patients with high grade myeloid malignancy.

Clinical Outcomes and Mutational Profile Associated with Dysmyelopoiesis in Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML) is defined by absolute (≥ 0.5 x10 9/L) and relative (≥ 10%) monocytosis in peripheral blood. Presence of significant dysplasia in any of the three myeloid series, in particular dysgranulopoiesis, is an almost constant finding in CMML. In the absence of a clonal marker, a scenario very unlikely with the advent of next-generation sequencing (NGS), significant myeloid dysplasia becomes a crucial criterion for establishing the diagnosis. Although bone marrow dysplasia has demonstrated independent prognostic value in MDS, its significance in relation to CMML has never been assessed. The main interest of our work was to evaluate the genomic determinants of dysplasia and to assess its prognostic impact. We analyzed a series of 240 patients with CMML with clinical, morphological, immunophenotypic, cytogenetic and molecular information. Patients were diagnosed following WHO and ICC 2022 recommendations. As a novelty with respect to previous WHO classifications, the new definition of CMML includes those patients previously categorized as oligomonocytic CMML (OM-CMML). That is, myelodysplastic neoplasms (MDS) or unclassifiable myelodysplastic/myeloproliferative neoplasms with relative monocytosis and a monocyte count of 0.5 × 10 9/L to &amp;lt;1 × 10 9/L. A total of 167 (71%) patients were diagnosed with CMML-1 and 67 (29%) with CMML-2. Likewise, 193 (80%) patients were diagnosed with dysplastic CMML, 55 of whom would meet criteria for OM-CMML, and 47 (20%) were diagnosed with proliferative CMML. We assessed associations between dysmyelopoiesis and gene mutations by Spearman's rank correlation coefficient. Dysgranulopoiesis showed a significant positive correlation with NRAS, TET2 and ZRSR2 mutations; while a significant negative correlation with SETBP1 was observed. Dysmegakaryopoiesis showed a significant positive correlation with ASXL1 and TP53. Dyserythropoiesis showed a significant positive correlation with DNMT3A and SF3B1. The heatmap and matrix showing these correlations, as well as the differences in the degree of dysmyelopoiesis among these specific mutations are depicted in Figure 1-A &amp; 1-B. Later, we wanted to assess the prognostic impact of dysgranulopoiesis, dyserythropoiesis and dysmegakaryopoiesis in terms of overall survival (OS) and leukemia-free survival (LFS) in our series. By applying the MaxStat package in R, it allowed us to detect the best cut-off points for dysgranulopoiesis (&amp;gt;75%) and dysmegakaryopoiesis (&amp;gt;20%) to separate our series into two groups with significantly different outcomes (Figure 1-C). We were unable to detect a significant cut-off point for dyserythropoiesis and the presence of multilineage dysplasia also showed no prognostic impact. CMML patients with dysgranulopoiesis above 75% showed a significant shorter median OS (34.4 vs 67.8 months; Hazard Ratio (HR): 1,99 [1.24-3.19]; P=0.0036) and LFS (33.4 vs 66.6 months; HR: 1,87 [1.17-3]; P=0.008). Likewise, patients with dysmegakaryopoiesis above 20% also showed a significant shorter median OS (30.2 vs 67.1 months; HR: 2.52 [1.63-3.88]; P&amp;lt;0.0001) and LFS (30.4 vs 66.6 months; HR: 2.76 [1.78-4.28]; P&amp;lt;0.0001). Remarkably, dysmegakaryopoiesis above 20% and dysgranulopoiesis above 75% were identified as independent prognostic factors for both OS and LFS after adjustment by multivariable Cox regression analyses by CMML-specific prognostic scoring system (CPSS) and Mayo Prognostic Model. Furthermore, the addition of dysmegakaryopoiesis above 20% and dysgranulopoiesis above 75% to these prognostic indices improved their prognostic accuracy in terms of OS as assessed by a C-index for right-censored data (Figure 1-C). In conclusion, our study has allowed us to detect mutations in genes related to dysmyelopoiesis. In addition, both dysgranulopoiesis and dysmegakaryopoiesis have demonstrated a negative prognostic impact on the survival of patients in our series that was not captured by the CPSS and Mayo prognostic model. Their worse prognosis could be partially justified by their association with poor prognostic mutations (e.g. NRAS, ASXL1, TP53), which allows us to visualize them as a good surrogate of poor prognostic molecular profiles.

Cellular Interactions within Clonal Dendritic Cell Aggregates Drive Immune Tolerance in Chronic Myelomonocytic Leukemia Bone Marrow Microenvironment

Introduction: Clonal dendritic cell (DC) aggregates in chronic myelomonocytic leukemia (CMML) are seen in approximately 20-30% patients at diagnosis. Previously, these were thought to be comprised of CD123+ plasmacytoid DCs but our data has suggested that these are heterogenous and comprise of myeloid DCs, monocytes, and myeloid-derived suppressor cells. Furthermore, these aggregates uniformly express immune checkpoints such as indoleamine 2,3-dioxygenase-1 (IDO), associate with an IDO-specific systemic impact (increased trytophan catabolism), and expanded regulatory T cell population in CMML, suggesting a role in inducing immune tolerance. DC aggregates are also associated with increased risk of acute myeloid leukemia (AML) transformation and resistance to hypomethylating agents, highlighting that they are important for disease progression in CMML (Mangaonkar A et al. Blood Adv 2020). We performed this study to decipher the architecture of a CMML DC aggregate to understand the complex inter-cellular interactions, and assess their impact on the adaptive immune system. Methods: In this study, we used archival fresh-frozen paraffin-embedded (FFPE) bone marrow biopsy specimens obtained from CMML patients, and isolated monocytes (CD14+ magnetic bead sorting) and lymphocytes (CD14-ve fraction) from untreated CMML and age-matched normal control (NC) peripheral blood mononuclear cells (PBMCs). DCs were cultured from monocytes using a commercial protocol. Spatial architecture of the CMML BM microenvironment was interrogated through multiplex imaging of FFPE samples by co-detection by indexing (CODEX) using 30 markers (CCR6, CD107a, CD11c, CD20, CD21, CD31, CD3e, CD4, CD44, CD45, CD45RO CD56, CD68, CD8, E-Cadherin, FoxP3, HLA-DR, CD14, CD16, CD11b, CD33, CD15, CD66b, CD123, CD303, CD34, CD163 and IDO). Single-cell transcriptomic signatures were analyzed using the 10X single cell Fixed RNA profiling platform, and validated using bulk RNA seq (Illumina stranded mRNA prep) from isolated and cultured cells. Impact of DCs on lymphocytes was assessed via autologous mixed DC-T culture experiments. Results: BM FFPE samples from 5 CMML cases (3 with DC aggregates) were included for multiplex imaging via CODEX. Results confirm the significant interactions of CD8+ and CD4+ T cells with CD123+/HLA-DR+ DCs within a CMML DC aggregate (Fig. 1A). Upon quantitative assessment of cell-cell interactions, IDO+/CD8+T ( P=0.006), IDO+/MDSC ( P=0.005), and IDO+/macrophage ( P=0.001) interactions were significantly increased in CMML samples with versus without DC aggregates . Specific findings were validated using single-stain immunohistochemistry and multiplex immunoflourescence staining. Next, in order to assess transcriptomic signatures within BM at the single-cell level, we chose two cases; CMML (BM aspirate blast% 1, WBC 49.6 x 10 9/L, ASXL1/GATA2/SETBP1/SRSF2 mutations) and AML transformed from CMML (BM aspirate blast% 81, WBC 152 x 10 9/L, NPM1/DNMT3A/TET2 mutations). Approximately, 10,000 cells were obtained and analyzed with cell type annotation through human PBMC reference (Azimuth). Results show an expanded regulatory T cell population in AML compared to CMML (UMAP plot, Fig. 1B), suggestive of immune tolerance. In order to validate findings and perform functional experiments, we used sorted monocytes and cutured DCs from CMML and NC. DC phenotype was confirmed both through visual inspection and flow cytometry, and clonality was confirmed through Sanger sequencing (for previously identified mutations). Differential gene expression analysis through bulk RNAseq showed significant differences between CMML and NC lymphocytes with immunoinhibitory receptor, LILRB2 (log2fold change=7.05, adjusted P=0.0002) being one of the top five significantly upregulated genes, further confirming immune tolerance. Finally, mixed autologous DC-lymphocyte reaction (1:4 ratio) experiments demonstrated that DCs induce T cell tolerance in CMML (expansion of exhausted PD1/TIM3/LAG3+ CD8 T cells) when compared to NC. Conclusion: Clonal dendritic cell aggregates within the CMML bone marrow microenviroment interact with T cells and induce immune tolerance. This represents a novel mechanism of disease progression in CMML with potential therapeutic targets (IDO, LILRB2, and/or CD123).

A Large Cohort Study of 412 Patients with SRSF2/TET2 Co-Mutated Myeloid Neoplasms: The Molecular Landscape and Clinical Outcomes

Introduction: Serine and arginine-rich splicing factor 2 ( SRSF2) and tet methylcytosine dioxygenase 2 ( TET2) are early mutations that drive the development of myeloid neoplasms and monocytosis. SRSF2/ TET2 co-mutations are most common in chronic myelomonocytic leukemia (CMML). However, they can also be seen in myelodysplastic syndrome (MDS), acute myeloid leukemia from CMML (AML-CMML), other AML (AML-Other, mostly de novo AML), and myeloproliferative neoplasms (MPN). Previous studies have investigated these co-mutations in myeloid diseases separately and as a subset (CMML) or small subset (MDS or AML) of the patients. Additionally, these studies mainly compared the clinical difference between patients with and without these double mutations. A large cohort study of patients with both mutations, irrespective of the morphologic diagnosis, has not been reported. Methods: Out of the Moffitt NextGen sequencing database of more than 8000 patients, we identified 412 patients with both SRSF2 and TET2 mutations and with diagnosed or suspected myeloid neoplasms. The demographics and clinical parameters were collected. Overall survival (OS) was defined as the time from sequencing to the last visit at Moffitt or death. Two experienced hematopathologists confirmed the final diagnosis according to the 5 th edition of the World Health Organization classification of hematological malignancies. Chi-squared tests of independence were used to compare the association between gene mutation and diagnosis, and a post hoc test based on adjusted standardized residuals was used. Cox proportional hazards models were used to compare the OS within and across diagnoses and included gender, age, gene mutation, and diagnosis (when comparing across diagnoses) as covariates. Results: Of the 412 patients, 284 (69%) were male, 128 (31%) were female, and the average age was 75 years. The five most common diagnoses, in descending order, were CMML (41%), AML-Others (23%), MDS (12%), AML-CMML (9%), and MPN (6%). The ten most common additional mutated genes, in descending order, were ASXL1 (44%), RUNX1 (28%), CUX1 (13%), JAK2 (13%), CBL (11%), TP53 (10%), SETBP1 (9%), KRAS (9%), STAG2 (9%), and NRAS (9%). A comparison of the top 5 diagnoses and the top 10 additional gene mutations showed an association between diagnosis and gene mutation (chi2 = 131, 36 df, p=.000). A post hoc test showed CBL mutations were less frequent in patients with AML-Others (p=.029), STAG2 mutations were less common in CMML patients (p=.007), and STAG2 mutations were more frequent in patients with MDS (p=.000). Among all patients, RUNX1 mutation (HR=2.4, 95% CI [1.3, 4.4], p=.005), ASXL1 mutation (HR=2.3, 95% CI [1.3, 4.2], p=.005), and CBL mutation (HR=2.0, 95% CI [1.0, 3.8], p=.048) were associated with lower OS. Among patients diagnosed with CMML, KRAS mutation (HR=4.1, 95% CI [1.5, 11.3], p=.006) and ASXL1 mutation (HR=3.3, 95% CI [1.4, 8.3], p=.009]) were associated with lower OS. Among patients with low-grade myeloid diseases (CMML and MDS), ASXL1 mutation (HR=2.6, 95% CI [1.2, 5.9], p=.021) was associated with lower OS. For the five most frequently co-mutated genes ( ASXL1, RUNX1, CUX1, JAK2, and CBL), no difference in OS was found between patients with CMML vs MDS, or AML-CMML vs AML-Others. Conclusions: SRSF2 and TET2 co-mutations occurred in various myeloid diseases, including CMML, AML transformed from CMML, other types of AML, MDS, and MPN. RUNX1, ASXL1, and CBL, were commonly mutated and associated with lower OS compared to patients with only SRSF2 and TET2 mutations. When comparing OS for patients diagnosed with low-grade diseases (CMML or MDS), ASXL1 mutation was associated with an increased risk of death. No difference in OS was found between CMML vs MDS, or AML-CMML vs AML-Others when co-mutation with ASXL1, RUNX1, CUX1, JAK2, or CBL was present.