Tefferi, Ayalew

Abstract RAS pathway (RASp) mutations induce proliferative features, and promote transformation in chronic myelomonocytic leukemia (CMML). However, the unique clonal landscape and hierarchy of distinct RASp mutations remain unexplored. To characterize the landscape, architecture, and implications of unique RASp mutations in CMML, we evaluated a cohort of 814 patients with CMML. We identified 461 RASp mutations among 342 patients (42%). N/KRAS and CBL mutations were the most common, frequently involved the P-loop or RING domains, respectively, and frequently appeared as dominant events (63% and 65%, respectively). BRAF, NF1, and PTPN11 mutations spanned throughout the gene structure, and frequently appeared as subclonal events (75%, 64%, and 59%, respectively). CBL mutations frequently occurred in codominance with SRSF2 and multihit TET2, and were enriched for KIT mutations. PTPN11 mutations more frequently co-occurred with SETBP1 and DNMT3A mutations, and were infrequently codominant with TET2 or ASXL1. RASp mutations predicted for shorter overall survival (hazard ratio [HR], 1.55; 95% confidence interval [CI], 1.15-2.07; P = .0075) and leukemia-free survival (LFS; HR, 1.67; 95% CI, 1.26-2.20; P = .0011), influenced outcomes of myelodysplastic and TET2 mutant CMML, and cooperated with IDH2 and RUNX1 mutations to induce shorter LFS. These data set the bases for refined genomic classifications of CMML, and underscore the need to develop RAS-directed therapies for patients with CMML.

e18523 Background: Allogeneic hematopoietic stem cell transplant (AHCT) remains the only curative option for genetically high-risk acute myeloid leukemia (AML). Venetoclax plus hypomethylating agents (Ven+HMA) is approved as frontline treatment in elderly patients with AML ( NEJM 2020; 383); however, less is known on the post-transplant outcomes following such therapy. Methods: Newly diagnosed (ND) AML patients who received Ven+HMA and underwent AHCT at Mayo Clinic from 2019 to 2024 were retrospectively recruited. Patients who required salvage therapy following Ven+HMA were excluded. Post-transplant survival (PTS) and relapse incidence were computed. Results: 55 ND-AML patients (60% male, 49% de novo , median age 69 [38-77]) received Ven+HMA (median cycles 3 [2-11]). ELN 2022 cytogenetic risk at time of diagnosis was non-adverse in 67% of patients, while 33% had adverse karyotype. Common mutations at diagnosis included: K/NRAS (88%), TP53 (26%), RUNX1 (23%), ASXL1 (16%), and IDH1/2 (17%). At the time of AHCT, 98% ( n=54 ) were in CR/CRi or MLFS; ELN cytogenetic risk was non-adverse in 91%, and adverse in 5 (9%) patients. Common mutations included: TP53 (16%), TET2 (18%), SRSF2 (15%), ASXL1 (13%), RUNX1 (18%), and DNMT3A (9%). Donor types included HLA-matched unrelated (75%), haplo-identical (11%), matched related (7%), mismatched unrelated (7%). 58% received fludarabine/melphalan conditioning and 54% GVHD prophylaxis with cyclophosphamide. At a median follow up of 25 months (0.7-68 mo), 20 patients (36%) have died, due to relapse in 10 (18%) cases. 3-year cumulative incidence of non-relapse mortality (NRM) was 17%. Median PTS was not reached (1/2/3-year survival 74%/66%/62%). Univariate analysis identified Karnofsky Performance Score <70% (3 vs. 21 months, p<0.01 ), TP53 (14 vs. 21 months p=0.02 ) and TET2 mutation at time of transplant (11 vs. 21 months , p=0.02 ) as risk factors. Multivariable analysis confirmed the prognostic impact of TP53 (HR 2.8) and TET2 mutations (HR 2.9). Age, adverse karyotype at transplant, and donor type did not impact PTS (p>0.1). 13 patients (24%) experienced post-transplant relapse; 1/2/3-year cumulative incidence of relapse was 10%, 21% and 25%, respectively. ELN-defined adverse karyotype at transplant increased risk of relapse (3 year cumulative incidence 80% vs. 17% for non-adverse , p<0.01 ). Overall, 51% of patients developed GVHD; 54%, 39% and 7% were acute, chronic GVHD and both, respectively. GVHD was protective against relapse (3 year incidence 15% vs. 36% in absence of GVHD [p=0.04]). Conclusions: Ven+HMA frontline treatment is associated with favorable post-transplant outcome in elderly ND-AML (3 year PTS 62%/cumulative incidence of relapse 25%/NRM 17%). TP53 mutation and ELN 2022-defined adverse karyotype at the time of transplant independently predicted PTS and relapse incidence, respectively.

e18596 Background: The International Consensus (ICC), but not the 5 th edition of the WHO classification (WHO5), distinguishes mature from immature mast cell leukemia (MCL) and systemic mastocytosis (SM) associated with myeloid neoplasm (SM-AMN) from SM associated with myeloid or lymphoid neoplasm (SM-AHN). The objectives of the current study were i) to assess prognostic input from disease classification by ICC vs. WHO5, and ii) to compare the predictive performance of the Mayo Alliance Prognostic scoring (MAPS) with other prognostic models and its prognostic interaction with the ICC-based morphologic classification. Methods: Study patients met diagnostic criteria per ICC/WHO5. MAPS risk variables included i) age >60 years, ii) hemoglobin below sex-adjusted lower limit of normal, iii) platelets <150 × 10⁹/L, iv) alkaline phosphatase above upper limit of normal, and v) advanced, including MCL, SM-AMN, and aggressive (ASM) vs. indolent/smoldering (ISM/SSM) SM. ICC-defined MCL includes only MCL-immature whereas WHO-defined MCL includes both MCL-immature and MCL-mature. WHO-defined SM-AHN includes SM-AMN and SM associated with lymphoid neoplasm (SM-ALN). Results: 910 Mayo Clinic patients with SM were diagnosed between 1968 and 2024 (median age 56 years). ICC categories were ISM/SSM (N=552), ASM (N=115), SM-AMN (N=235), and MCL (N=8). At median follow-up of 4 years, 345 (38%) deaths and 35 (3.8%) leukemic transformations were documented. Overall survival (OS) predictive performance at 5 years was superior with ICC (AUC 0.85), compared to WHO5 (AUC 0.83); median survival was 24.3 years in ISM/SSM, 5.8 years for ASM, 2.0 years for SM-AMN, 2.3 years for SM-AHN, 1.8 years for MCL-mature and 0.08 years for MCL-immature; the difference in OS was significant between MCL-immature and SM-AMN (p<0.01) but not between MCL-mature and SM-AHN (p=0.3). Similarly, OS was similar between MCL-mature and ASM (p=0.8) and between SM-ALN and ASM (p=0.08) but different between MCL-immature and ASM (p<0.01), SM-AMN and ASM (p<0.01), and MCL-mature and MCL-immature (p<0.01). Predictive performance at 5 years was superior with MAPS (AUC 0.91) and MAPS-molecular (AUC 0.91), compared to the mutation-adjusted risk score (MARS; AUC 0.82). Median OS estimates were not reached, 24.2, 11.7, 5.7, 2.6, and 0.7 years, in the presence of 0, 1, 2, 3, 4, or 5 of the forementioned MAPS risk factors. Among informative cases, ASXL1 mutation was the only genetic risk factor with MAPS-independent prognostic relevance. Prognostic inter-independence between ICC and MAPS was confirmed by multivariable analysis. Conclusions: The current study confirms the superior performance of MAPS, in regard to survival prediction, with little additional contribution from mutations. SM sub-classification by the ICC is prognostically independent of MAPS and more valuable than classification by WHO5.

e18600 Background: Recent studies have suggested that basophilia in myeloproliferative neoplasms (MPN), in general, and in primary myelofibrosis (PMF), in particular, might be associated with poor prognosis. In the current study, we examined for clinical and genotype correlates as well as the prognostic impact of absolute eosinophil (AEC) and basophil (ABC) counts in essential thrombocythemia (ET) and polycythemia vera (PV). Methods: Diagnostic criteria were according to the International Consensus Classification. Study patients were selected based on the availability of information on AEC and ABC. Institutional reference range for AEC was 0.03 to 0.48 x 10 9 /L and for ABC 0.01 to 0.08 x 10 9 /L. "Eosinophilia" was defined as an AEC of ≥0.5 x 10 9 /L. "Basophilia" was evaluated both at an ABC of ≥0.1 x 10 9 /L and ABC of ≥0.3 x 10 9 /L, the latter representing 3 x the upper limit of the normal reference range. Results: Essential thrombocythemia 660 patients were considered. Median (range) values for AEC and ABC were 0.2 x 10 9 /L (0-2.8) and 0.07 x 10 9 /L (0-1.7), respectively. On univariate analysis, AEC and ABC, as continuous variables, were predictive of inferior overall (OS; p<0.01 for both) but not leukemia-free (LFS), myelofibrosis-free (MFFS), venous thrombosis-free (VTFS), or arterial thrombosis-free (ATFS) survival. In univariable analysis, AEC ≥0.5 x 10⁹/L and ABC ≥0.1 x 10⁹/L negatively impacted OS (p<0.01 for both); significance was sustained during multivariate analysis that included IPSET risk variables: AEC ≥0.5 x 10⁹/L (p<0.01; HR 1.9) and ABC ≥0.1 x 10 9 /L (p<0.01; HR 1.7). Polycythemia vera 478 patients were considered. Median (range) values for AEC and ABC were 0.27 x 10 9 /L (0-6.5) and 0.12 x 10 9 /L (0-3.2), respectively. In univariable analysis, neither AEC nor ABC, as continuous variables were predictive of OS, LFS, MFFS, VTFS, or ATFS. AEC ≥0.5 x 10⁹/L also did not have any impact on disease progression or thromboses. However, ABC ≥0.1 x 10 9 /L was associated with a lower risk of post-diagnosis arterial thrombosis (p=0.03; HR 0.6), and ABC ≥0.3 x 10 9 /L was predictive of inferior LFS (p<0.01; HR 6.5) and MFFS (p<0.01; HR 2.5). ABC ≥0.3 x 10 9 /L sustained prognostic relevance in LFS (p<0.01; HR 7.6) during multivariable analysis that included leukocytosis, platelet count, basophil percentage, age, and palpable splenomegaly. Similar findings were noted with regards to MFFS for ABC ≥0.3 x 10 9 /L (p=0.01; HR 2.3) when compared against palpable splenomegaly and age. Conclusions: The current study suggests that AEC ≥0.5 x 10 9 /L and ABC ≥0.1 x 10 9 /L in ET are independently associated with shortened overall survival, while basophilia (ABC ≥0.3 x 10 9 /L) in PV is an independent predictor of leukemic and myelofibrotic disease progression.

Background Venetoclax + hypomethylating agent (Ven-HMA) therapy results in favorable response rates (CR/CRi 66%) and overall survival (OS; median 14.7 months) in unfit patients with newly-diagnosed acute myeloid leukemia (ND-AML; N Engl J Med 2020;383). However, there is substantial heterogeneity in individual patient outcomes based on response to treatment. The objective of the current study was to develop a genetic risk model to predict survival outcome in treatment responders and non-responders, analyzed separately, and also account for allogeneic stem cell transplant (ASCT). Methods Treatment-naïve AML patients receiving Ven-HMA, outside of a clinical trial, at the Mayo Clinic, USA (MN, AZ, FL) were retrospectively recruited. Molecular studies (48-gene panel) were performed by next-generation sequencing. Response was assessed according to the 2022 European LeukemiaNet (ELN) criteria (Blood 2022;140). Standard statistical analyses were performed using JMP Pro (Version 17.0.0). Results 400 ND-AML patients (median age 73 years, 64% male, 60% de novo) received Ven-HMA. ELN cytogenetic risk included favorable 2%, intermediate 60%, or adverse 38%. Mutations involved TP53 in 26%, TET2 19%, RUNX1 19%, SRSF2 18%, ASXL1 18%, DNMT3A 15%, K/NRAS 13%, NPM1 12%, IDH2 12%, FLT3-ITD 10%, IDH1 7% and DDX41 in 4% of informative cases. 153 (38%) patients achieved CR, 94 (24%) CR with incomplete count recovery (CRi), resulting in CR/CRi in 247 (62%) patients. CR/CRi rates were higher with NPM1 (86% vs 59%), IDH2 (77% vs 60%), and DDX41 mutations (93% vs 61%) and lower with TP53 (45% vs 67%), FLT3-ITD (41% vs 64%), RUNX1 (49% vs 65%) mutations and adverse karyotype (48% vs 71%), with all retaining significance in multivariable analysis. CR/CRi rate was highest (87%) in 75 patients with one or more favorable mutations (NPM1, IDH2, DDX41) and no unfavorable mutation (TP53, FLT3-ITD, RUNX1). CR/CRi rate was lowest in 171 patients with at least one unfavorable mutation and no favorable mutation (44%; p&lt;0.01). After a median follow up of 10.5 months (0.5-66), 105 relapses (43% of patients in CR/CRi), 248 (62%) deaths and 65 (16%) ASCT were documented. Univariate analysis for transplant-censored OS disclosed superior survival in patients achieving CR/CRi (median 21 vs 3.6 months; HR 5.4, 95% CI 4.1-7.1). Among patients achieving CR/CRi, IDH2 mutation was associated with superior (median not reached vs 20.5 months) and male gender (median 17.6 vs 29.2 months), TP53 mutations (median 11.9 vs 24.3 months) and adverse karyotype (median 12.6 vs 29.4 months) with inferior survival; multivariable analysis resulted in HRs (95% CI) of 2.1 (1.3-3.4) for male gender, 2.1 (1.2-3.8) for adverse karyotype, and 2.5 (1.1-5.7) for absence of IDH2 mutations. Male gender did not show an association with karyotype, or TP53 mutations (p&gt;0.1). Accordingly, an HR-weighted genetic risk model was generated with assignment of one point each to male gender, adverse karyotype, and absence of IDH2 mutation: high risk = 3 risk factors (n=38; median 9.6 months; 3-year survival 0%); intermediate risk = 2 risk factors (n=129; median 22 months; 3-year survival 38%); and low-risk = ≤1 risk factor (n=78; median not reached; 3-year survival 69%; p&lt;0.01). In patients bridged to ASCT (n=60), post-transplant survival was inferior in the presence of ≥ 2 risk factors (n=42; median 46 months) vs. ≤1 risk factor (n=18; median not reached; p=0.1). In patients not achieving CR/CRi, risk factors for OS included male gender (median 3.4 vs 6.1 months), adverse karyotype (median 3.3 vs 4.5 months) and TP53 mutation (median 3.1 vs 4.5 months) while those with IDH2 mutations lived longer (median 8.4 vs 3.4 months). In multivariable analysis, HRs (95% CI) were 1.9 (1.3-2.9) for male gender, 2.0 (1.4-2.9) for adverse karyotype, and 2.1 (1.1-4.3) for absence of IDH2 mutations. OS was shortest for non-responders harboring 3 risk factors (n=45; median 3 months; 1-year survival 0%) and longer for those harboring two (n=80; median 6 months, 1-year OS 15%) or ≤ 1 risk factor (n=25; median 14 months; 1-year OS 56%; p&lt;0.01). Conclusion The current study describes a molecular profile for predicting response to Ven-HMA in ND-AML and also introduces a novel approach to survival prediction that considers treatment response as the starting point. In both responders and non-responders, male gender and adverse karyotype were identified as unfavorable and IDH2 mutation favorable risk factor for OS.

Background Venetoclax + hypomethylating agent (Ven-HMA) therapy has demonstrated improved outcomes in elderly/unfit patients with newly-diagnosed acute myeloid leukemia (ND-AML) (N Engl J Med 2020;383). However, ~50% of patients who respond to Ven-HMA, eventually relapse (Am J Hematol 2024;99). In the current study we examined the prognostic contribution of gene mutations, karyotype, measurable residual disease (MRD) status and clinical risk factors for relapse in patients with ND-AML treated with Ven-HMA therapy. Methods Our study population of ND-AML was retrospectively recruited from Mayo Clinic (MN, AZ, FL), after institutional review board approval and based on documentation of complete remission with/without count recovery (CR/CRi) following Ven-HMA therapy. Relapse-free survival was determined from the time of remission to relapse or last follow-up. Survival analysis was censored for transplant. The 2022 European LeukemiaNet (ELN) criteria was used to assign cytogenetic risk and classify treatment response (Blood 2022;140). Standard statistical analyses were performed using JMP Pro (Version 17.0.0). Results 247 ND-AML patients treated with Ven-HMA (median age 73 years, 62% male, 64% de novo) who achieved CR (n=153), or CRi (n=94) were included. MRD by multiparameter flow cytometry (sensitivity 0.01%) performed at the time of CR/CRi, was negative in 117 (70%) of 166 informative cases. ELN cytogenetic risk categories at diagnosis included favorable 2%, intermediate 69%, or adverse 29%. Mutations involved TET2 21%, SRSF2 21%, TP53 19%, ASXL1 18%, DNMT3A 18%, NPM1 17%, IDH2 15%, RUNX1 15%, K/NRAS 12%, IDH1 8%, FLT3-ITD 7%, and DDX41 in 6% of informative cases. Relapse was documented in a total of 99 (40%) patients who achieved CR (39%) or CRi (41%) after a median remission duration of 6 months (1-31). Patients that relapsed within the first-year vs those that did not, were more likely to be males (75% vs 55%), MRD positive (44% vs 24%), and harbor TP53 mutations (23% vs 16%). Univariate analysis for RFS identified male gender (12 vs 29 months; p=0.02), secondary AML (10 vs 21 months; p&lt;0.01), adverse karyotype (9 vs 22 months; p&lt;0.01), TP53 mutations (6 vs 18 months, p&lt;0.01) and MRD positive remission (13 months vs not reached; p&lt;0.01) as risk factors for relapse; on the other hand, IDH2 mutations (not reached vs 15 months; p&lt;0.01) were associated with a lower risk of relapse. Multivariable analysis confirmed male gender (HR 2.2), TP53 mutations (HR 3.0) and MRD positive remission (HR 1.7) as unfavorable predictors of relapse. Subsequently, in a three-tiered relapse prediction model, RFS was significantly inferior in the presence of three risk factors (male gender, TP53 mutations, MRD positive remission) (n=10, median 4 months), vs two (n=34, median 10 months) vs ≤ 1 risk factor (n=122, median 31 months) (p&lt;0.01); 1-year cumulative incidence of relapse was 100%, 56%, and 26% in the presence of three, two and ≤ 1 risk factors, respectively. 50 of 99 (65%) patients received salvage therapy which included cladribine-cytarabine-Ven (n=19), intensive induction chemotherapy (n=9), FLT3 inhibitors (n=5), Ven-HMA-FLT3 inhibitor (n=1), IDH1/2 inhibitors (n=5), glasdegib-cytarabine (n=4), gemtuzumab (n=3), lenalidomide (n=1), or investigational therapies (n=3); in addition, Ven-HMA was resumed in 14 patients in whom treatment had been discontinued for a median of 5 months (2-12). Overall, 20 (31%) of patients achieved second CR/CRi and response rates were similar with intensive vs less intensive therapy (CR/CRi; 44% vs 29%; p=0.37). Median follow up after relapse was 3.4 months (1-66), during which 73 (74%) deaths, and 4 (5%) ASCT were recorded. Median transplant-censored survival was 4.2 months (1-year; 22%), and inferior in patients with adverse karyotype (median 4.6 vs 3.4 months; p=0.02), and TP53 mutations (3.6 vs 4.4; p=0.11); on the other hand, male gender (3.5 vs 6.8 months; p=0.51), MRD positive remission (4.6 vs 4.2; p=0.56), and IDH2 mutations (6.7 vs 4.2; p=0.28), did not influence post-relapse survival. Conclusions The current study identifies male gender, TP53 mutations, and MRD status as powerful predictors of relapse and proposes a relapse prediction model for ND-AML patients receiving Ven-HMA therapy. Furthermore, relapse following Ven-HMA was associated with uniformly poor outcomes, regardless of salvage treatment strategy.

Background: The JAK2 46/1, also referred to as the GGCC, haplotype, is represented by 4 main SNPs spanning a region of 250kb in chr9 including the JAK2 gene, that are in complete linkage disequilibrium. The GGCC haplo was found to confer susceptibility to JAK2 mutated myeloproliferative neoplasms (MPN) (Jones AV, 2009; Olcaydu D, 2009; Kilpivaara O, 2009) while association with JAK2-wildtype MPN remains controversial (Pardanani A, 2010; Jones AV, 2010; Guglielmelli P, 2010; Trifa A, 2016). The GGCC haplo was also found enriched in MPN pts with splancnic vein thrombosis (SVT) (Smalberg JH, 2011), and to correlate with higher JAK2V617F variant allele frequency (VAF) (Alvarez-Larran, A, 2012). Nullizigosity for GGCC haplo was associated with reduced survival (OS) in pts with myelofibrosis (MF) (Tefferi A, 2019), but no information on the impact of GGCC haplo status with phenotype and outcome in PV is available. Aim: To assess the clinical correlates and the prognostic significance of GGCC haplo status in a large cohort of pts with PV. Pts and Methods: A total of 399 PV pts, all JAK2V617F mutated, diagnosis revised according to 2022 ICC criteria, were genotyped for 46/1 haplotype by PCR allele discrimination with rs12343867 (with the “C” allele standing for 46/1 haplotype). Results: A total of 301 pts (75.4%) had the C allele, of which 143 (47.5%) were CC homo and 158 (52.5%) CT hetero; 98 (24.6%) were nullizygous for GGCC (TT). Since preliminary analysis disclosed no significant difference between CT and TT haplotype for all variables considered, homozygous CC pts were compared to TT+CT. The CC haplotype was associated with higher leukocytes (median 11.3x10e9/L vs 9.6, p&lt;.001), higher hemoglobin (18.4 g/dL vs 17.5, p&lt;.001), lower platelets (441x10e9/L vs 514, p=.002) more aquagenic pruritus (51.8% vs 33.2%, p=.001), and higher JAK2VAF (60.8+-24.8% vs 36.8+-19.7%, p&lt;.001) at diagnosis. Pts with a VAF &gt;50% were 69% in CC versus 24.1% in TT+CT pts (p&lt;.001). ASXL1 mutations were enriched among CC pts (20.3% vs 9.4%, p=0.04), otherwise no difference regarding additional myeloid mutations was noted. All above differences remained significant when considering CC versus TT and CT separately. More pts with CC haplotype suffered from venous thrombosis (VT) during the FU (16.1% vs 4.3%, p&lt;.001), no difference was noted for arterial thrombosis nor for splancnic vein thrombosis. VT-FS by Kaplan Meyer was significantly shorter (p&lt;.001) in CC pts; the rate of VT at 5, 10, 15y after diagnosis, was 5.5%, 12%, 20.2% for CC versus 2.6%, 3.4%, 6.4% for TT+CT, respectively (all p&lt;.001). In multivariable analysis, including JAK2VAF &gt;50%, age &gt;60y, history of VT, WBC &gt;15x10e9/L, only CC haplo remained significant (HR 3.6, 95%CI 1.4-9.5, p=.009). Progression to post-PV MF occurred in 81 pts (20.3%), significantly more among CC (n=45, 31.5%) than TT+CT (n=36; 14.1%; p&lt;.001). Myelofibrosis-FS was significantly shorter in CC vs other genotypes (p=.02). However, in multivariable analysis, only BM fibrosis G1 remained significant (HR 3.2, 1.7-5.9; p&lt;.001). Evolution to blast-phase occurred in 13 pts (3.2%), 9 (6.3%) with CC haplo vs 4 TT+CT (1.6%; p=.01). During the follow-up, 102 pts (25.5%) died, 50 (35%) CC and 52 TT+CT (20.3%; p=.001). In multivariable analysis, including MIPSS-PV variables (Tefferi A 2020), the CC haplo remained significant (HR 1.8, 1.04-3.2, p=.03) together with age (HR 3.0, 1.6-5.7; p&lt;.001), WBC (HR 2.6, 1.3-4.8; p=.004), and thrombosis history (HR 1.9, 1.0-3.7; p=.03). Conclusions. Homozygosity for the 46/1 or CC haplotype contributes to the phenotype diversity of PV, in part possibly mediated by associated higher JAK2V67F VAF, and predicts for incident VT and shorter OS. Supported by AIRC, 21267; MIUR,2022F4WMR3; IHM, PNRR-MAD-2022-12376695

Background DISC-0974 is a monoclonal antibody that targets hemojuvelin, a co-receptor to BMP-SMAD signaling that stimulates hepcidin production. In healthy volunteers, DISC-0974 has been shown to be safe and induce dose-dependent decreases in serum hepcidin and corresponding increases in serum iron and absolute reticulocyte count (JCP 2024). Serum hepcidin level is abnormally elevated in myelofibrosis (MF) and is believed to contribute to iron-restricted erythropoiesis (AJH 2013:88:312). In an ongoing phase 1/2 study in MF (NCT05320198), DISC-0974 was well tolerated and associated with marked reduction in serum hepcidin levels and improvement in anemia (Blood 2023;142/DOI 10.1182). The current investigator-initiated laboratory correlative study examined the impact of mutations and baseline cytokine levels on anemia response to DISC-0974, in non-transfusion-dependent (nTD) patients with MF. Methods The current study was conducted under an institutional review board approved minimum risk protocol that allowed retrospective collection and analysis of data from Mayo Clinic patient records (Rochester, MN, USA). Study patients were selected from the aforementioned ongoing phase 1/2 study, based on availability of information on mutations and baseline cytokine levels. Definitions of nTD and anemia response criteria were according to the revised international working group (IWG) criteria (Blood 2024; in press): i) nTD was defined as the presence of ≤2 units transfused in the 12 weeks prior to enrollment; ii) a 12-week rolling average increase of hemoglobin by ≥1.5 or 1-1.4 g/dL is required for major and minor response, respectively. Mutation and cytokine analyses were performed according to institutional standard protocols; next-generation sequencing panel included 48 genes and cytokine panel 12 inflammatory cytokines. Results A total of 21 nTD patients with MF (median age 71 years; males 62%) were considered: 15 primary and 6 secondary. Driver mutation distribution was JAK2 52%, MPL 14%, and CALR-1 33%. DIPSS risk distribution was high (10%) and intermediate-2 (90%). Baseline median (range) values were 8.0 g/dL (range 7.1-9.5) for hemoglobin, 266 (41-1221) for serum ferritin, 79 (32-130) for serum iron, 32 (13-49) for transferrin saturation, and 59 (10-913) for serum erythropoietin. Karyotype was favorable (N=18) or unfavorable (N=2). Other mutations, included ASXL1 (N=5), SF3B1 (N=4), SRSF2 (N=1), U2AF1 (N=1), and “none of the above” (N=5). Elevated cytokines at baseline included TNF (90%), IL-18 (43%), IL-2R alpha (34%), IL-6 (24%), and MCP-1 (14%). DISC-0974 dose levels were 28 mg (N=2), 50 mg (N=7), 75 mg (N=7), and 100 mg (N=5). Twelve (60%) patients were on DISC-0974 monotherapy while 9 (40%) received concomitant ruxolitinib (N=5), pacritinib (N=1), or hydroxyurea (N=3). At median follow-up of 6 months (range 2-14), one (5%) patient has discontinued DISC-0974 for lack of response. Details on drug safety are summarized in another abstract (Gangat et al. ASH 2024). Per IWG criteria, 9 (43%) patients achieved major (MR) and 3 (14%) minor responses, resulting in an overall response (OR) rate of 57%. Two additional patients have surpassed the hemoglobin threshold for major response (≥1.5 g/dL) but have not yet met IWG criteria. OR and MR rates were higher in patients receiving DISC-0974 as monotherapy (OR 75% and MR 58%) vs. combined with JAK2 inhibitor or hydroxyurea (OR 33% and MR 22%) (p=0.05 for OR and 0.09 for MR), ASXL1 wild-type (N=15; OR 67% and MR 53%) vs. mutated (N=6; OR 33% and MR 17%) (p=0.16 for OR and 0.11 for MR), no ASXL1/SRSF2/U2AF1/SF3B1 (N=5; OR 100% and MR 80%) vs. at least one mutated (N=16; OR 44% and MR 31%) (p&lt;0.01 for OR and 0.05 for MR). OR was also higher in the presence of elevated MCP-1 (p=0.05) or elevated IL-2R alpha (p=0.05) and MR in the presence of elevated IL-2R alpha (71% vs. 29%; p=0.06). Conclusions: The current study suggests that ASXL1 mutation might attenuate but not abrogate anemia response to DISC-0974, in MF. Elevated inflammatory cytokines in MF are often associated with increased hepcidin levels (AJH 2013:88:312) and this might explain their association with a higher response rate to DISC-0974, observed in the current study. Regardless, it is important to note that these observations represent preliminary data from a subset of patients from an ongoing clinical trial.

Background Absolute basophil count (ABC) might be increased in patients with myeloproliferative neoplasms and has been associated with poor prognosis in chronic myeloid leukemia (CML) and, possibly, primary myelofibrosis (PMF, Front. Oncol 2019;9:872). In a Mayo Clinic survey of 382 patients with BCR::ABL1-negative leukocytosis and ABC ≥0.09 x 109/L, 191 (50%) were associated with myeloid neoplasms, including 104 (27%) with CML, 23 (6%) essential thrombocythemia, 21 (6%) polycythemia vera (PV), and 18 (5%) PMF (AJH 2020;95:E216). Herein, we tapped into a well-characterized cohort of patients with PV, in order to describe the prevalence of basophilia, at specific ABC thresholds, and corresponding clinical, molecular, and prognostic correlates. Methods Study patients were retrospectively recruited from Mayo Clinic (USA) databases, after approval from IRB. Diagnostic criteria were according to the International Consensus Classification (Blood 2022;140:1200). Study inclusion criteria included the availability of information on ABC and absolute eosinophil count (AEC). Mayo Clinic institutional reference range for ABC was 0.01 to 0.08 x 109/L and for AEC 0.03 to 0.48 x 109/L. “Eosinophilia” was defined as an AEC of ≥0.6 x 109/L. Basophilia was evaluated both at an ABC of ≥0.1 x 109/L and ABC of ≥0.3 x 109/L, the latter representing 3 x the upper limit of the normal reference range. Cytogenetic information obtained within one year of diagnosis was available in 270 patients. Next-generation sequencing information collected before disease transformation was available in 99 patients. Conventional statistical methods were employed for comparisons of groups and survival analysis (JMP Pro 17.0.0 software SAS Institute, Cary, NC, USA). Results Information on ABC and AEC, obtained within one year of diagnosis, was available in 478 patients (median age 66 years; males 51%). Median values at presentation were 10.6 x 109/L for leukocyte count, 453 x 109/L platelet count, and 17.3 g/dL hemoglobin level. Presenting symptoms included palpable splenomegaly 24%, pruritus 31%, arterial thrombosis 12%, and venous thrombosis 17%. Median and range values for ABC and AEC were 0.12 x 109/L (0-3.2) and 0.27 x 109/L (0-6.5), respectively. ABC was ≥0.3 x 109/L in 80 (17%) patients and ≥0.1 x 109/L in 315 (66%). AEC was ≥0.6 x 109/L in 68 (14.6%) patients. ABC≥0.3 x 109/L was associated with AEC ≥0.6 x 109/L (p&lt;0.01), leukocytosis (p&lt;0.01), and palpable splenomegaly (p=0.02), but not platelet count, pruritus, smoking history, diabetes, or arterial or venous thrombosis; in multivariable analysis, the association with leukocytosis, but not that with splenomegaly, was confirmed. At a median follow-up of 5.7 years (range 0.03-32.8), 132 (28%) deaths, 17 (3.6%) leukemic and 40 (8.4%) fibrotic progressions were documented. Post-diagnosis events also included arterial (7%) and venous (9%) thrombosis. In univariate analysis, neither ABC (p=0.8, 0.7, and 0.96) nor AEC (p=0.7, 0.6, and 0.8), evaluated as continuous variables, affected overall (OS), leukemia-free (LFS), or myelofibrosis-free (MFFS) survival, respectively. Similarly, eosinophilia at AEC ≥0.6 x 109/L did not appear to influence OS (p=0.99), LFS (p=0.6) or MFFS (p=0.5). On the other hand, although basophilia at ABC≥0.3 x 109/L did not affect OS (p=0.27), it was significantly associated with inferior LFS (HR 6.5, 95% CI 2.3-18.0; p&lt;0.01) and MFFS (HR 2.3, 95% CI 1.2-4.5; p=0.01); the corresponding p values for ABC≥0.1 x 109/L were 0.7, 0.1, and 0.17. The detrimental impact of basophilia (ABC≥0.3 x 109/L) on LFS was confirmed (P&lt;0.01) with multivariable analysis that included other clinical variables, including leukocytosis (NS), platelet count (NS), basophil percentage (NS), age (p&lt;0.01), and palpable splenomegaly (p&lt;0.01). Results were similar during multivariable analysis for MFFS with palpable splenomegaly (p&lt;0.01), ABC≥0.3 x 109/L (p=0.03), and age (p=0.05) sustaining independent predictive value. The number of informative cases was too small to evaluate prognostic interaction with mutations and karyotype. Conclusions: Basophilia in PV is associated with palpable splenomegaly and leukocytosis and independently predicts leukemic and myelofibrotic transformation. Additional studies are needed to confirm these observations and examine prognostic interaction with mutations and karyotype.