Reichard, Kaaren K.

ABSTRACT The clinical relevance of TP53 mutations ( TP53 MUT ) in myeloproliferative neoplasms (MPN) and their prognostic interaction with MPN subtype designation has not been systematically studied. In the current study, 114 patients with MPN harboring TP53 MUT (VAF ≥ 2%) were evaluated for overall survival (OS), calculated from the time of TP53 MUT detection: chronic phase myelofibrosis (MF‐CP; N = 61); blast‐phase (MPN‐BP; N = 31) or accelerated‐phase (MPN‐AP; N = 16) MPN, and polycythemia vera/essential thrombocythemia (PV/ET; N = 6). Sixty‐five (57%) patients harbored International Consensus Classification (ICC)‐defined multihit TP53 MUT and 56 (49%) monosomal/complex karyotype (MK/CK). Majority of MPN‐BP (90%) and MPN‐AP (81%) while 39% of MF‐CP and none of PV/ET patients harbored multihit TP53 MUT . OS in MPN‐BP and MPN‐AP was equally dismal (median 6 vs. 4.5 months, respectively; p = 1.0), regardless of multihit configuration ( p = 0.44), while OS in TP53 MUT MPN‐BP/AP ( N = 47; median 4 months) was inferior to that of a separate cohort ( N = 49) with TP53 wild‐type MPN‐BP/AP (median 11 months; p < 0.01). OS in MF‐CP was significantly shorter with multihit versus non‐multihit TP53 MUT (median 10 vs. 35 months; HR 2.9; p < 0.01), independent of other MF‐relevant genetic risk factors, including ASXL1/SRSF2/U2AF1 mutations. Multihit TP53 MUT was also associated with inferior survival following allogeneic stem cell transplant (ASCT): median 9 months versus “not reached” in patients with ( N = 9) versus without ( N = 8) multihit TP53 MUT ( p < 0.01). The presence of multihit or non‐multihit TP53 MUT in MPN‐BP/AP or multihit TP53 MUT in MF‐CP is associated with exceptionally poor prognosis and justifies inclusion into the ICC category of “myeloid neoplasms with mutated TP53 .” By contrast, detection of non‐multihit TP53 MUT , by itself, might not endanger short‐term survival in MF‐CP, PV, or ET.

Background: The majority of patients with chronic myelomonocytic leukemia (CMML) harbor somatic mutations with the most frequent being TET2 (56%), SRSF2 (42%), and ASXL1 (41%) (Leukemia 2020;34:1407). The prognostic relevance of mutations in CMML has been studied extensively with consistent observations suggesting favorable survival impact from TET2 and unfavorable from DNMT3A, ASXL1, NRAS, and SETBP1 mutations. Among the latter, DNMT3A mutation stands out as being the strongest predictor of inferior overall (OS) and leukemia-free (LFS) survival (AJH 2017;92:56). The current study was inspired by observations from exploratory analyses of a large Mayo Clinic CMML cohort. Methods: The current retrospective study was conducted under an institutional review board approved minimum risk protocol that allowed retrospective collection and analysis of data from patient records (Mayo Clinic, Rochester, MN; Scottsdale, AZ; and Jacksonville, FL). Mutations were screened by multi-gene next-generation sequencing (NGS). Diagnostic criteria were according to the International Consensus Classification (Blood 2022; 140:1200). Conventional statistical methods were employed for comparisons of groups and survival analysis (JMP Pro 17.0.0 software SAS Institute, Cary, NC, USA). Results: Exploratory analysis of a well-characterized cohort of 398 Mayo Clinic patients with CMML suggested an unusually long median survival of 96 months for 7 patients harboring PHF6 mutation vs. 27 months for those without the mutation. A subsequent institutional database search focusing on PHF6-mutated CMML identified 20 more cases, expanding the total number of study patients to 418, including 27 PHF6-mutated cases. PHF6 mutations clustered with (co-mutation frequency) TET2 (93%) and less likely to be co-mutated with TP53 (0%), PTPTN11 (0%) and SF3B1 (0%) mutations. PHF6-mutated patients often presented with thrombocytopenia (platelet count &lt;100 x 10(9)/L; 85% vs. 51% in PHF6 wild-type cases; p&lt;0.01). Median time from initial presentation with thrombocytopenia to CMML diagnosis was 4 years (0-14 years). In order to isolate the prognostic impact of PHF6 mutation without associated DNMT3A mutation and independent of TET2 mutation, we considered 4 mutational categories: i) DNMT3A-mutated (N=19; 2 co-expressed PHF6 and 7 TET2 mutations), ii) PHF6-mutated with wild-type DNMT3A (N=25; 23 co-expressed TET2 mutation), iii) TET2-mutated without DNMT3A or PHF6 co-expression (N=169), and iv) wild-type for DNMT3A, TET2, and PHF6 (N=205). Compared to the other groups, PHF6-mutated patients with wild-type DNMT3A were less likely to display leukocytosis (p&lt;0.01) or red cell transfusion need at presentation (p&lt;0.01). At a median follow-up of 18 months, 302 deaths, 78 leukemic transformations, and 44 allogenic stem cell transplants were documented. Univariate analysis for OS disclosed superiority in favor of PHF6-mutated patients with wild-type DNMT3A (median OS 90 months) vs. the other three groups with median OS ranging from 10 months for DNMT3A-mutated patients (HR 20.7, 95% CI 8.1-52.7; p&lt;0.01) to 40 months for TET2-mutated cases with wild-type DNMT3A (HR 3.5, 95% CI 1.7-7.6; p&lt;0.01). Results were similar for multivariable analysis of LFS, where, remarkably, none (0%) of the 25 PHF6-mutated patients with wild-type DNMT3A experienced leukemic transformation vs. 15-30% risk of leukemic transformation in the other three groups (p&lt;0.01). Multivariable analysis for both OS and LFS, which included other risk factors such as red cell transfusion need, leukocytosis (leukocyte count ≥13 x 109/L) and karyotype, confirmed the independent and favorable impact of PHF6 mutation/wild-type DNMT3A (p&lt;0.01 in both instances). Finally, all-inclusive multivariable analysis of karyotype and mutations, in DNMT3A wild-type patients, confirmed the highly favorable and independent prognostic impact of PHF6 mutation (HR 0.18, 95% CI 0.09-0.37). Conclusion: The current study proposes a 2-gene genomic signature (PHF6 mutated and DNMT3A wild-type) to identify a unique subset of CMML patients who often present with thrombocytopenia and whose clinical course is characterized by a very low risk of leukemic transformation, resulting in superior overall survival. Additional studies are warranted to validate these findings and include the information in contemporary risk models.

Introduction: VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is an adult-onset autoinflammatory disorder defined by somatic UBA1 mutations in hematopoietic cells. VEXAS is associated with a high frequency of clonal hematopoiesis (CH- 60%); with the clonal landscape being dominated by DNMT3A mutations. In our prior study (Gutierrez-Rodrigues Blood 2023), we showed that DNMT3A mutations in VEXAS associated with increased mortality, but without a higher risk of MDS/AML. As DNMT3A is a critical epigenetic regulator, we conducted this study to define the epigenetic deregulation and impact on mortality of DNMT3Amt (mutant) CH in VEXAS. Methods: After IRB approval, 88 VEXAS patients from the Mayo Clinic and the NIH were retrospectively assessed, including 81 screened for CH by error-corrected DNA sequencing (ECS). DNA methylation changes were identified using the Illumina Infinium MethylationEPIC BeadChip on peripheral blood samples (PB). Cellular indexing of transcriptomes and epitopes (CITE)-seq was performed using the 10x Chromium 3' GEX platform; 16,600 pooled cells were subjected to UMAP dimensionality reduction, followed by Azimuth based cell identification. Genotyping of targeted loci with chromatin accessibility (GoTChA) was conducted on two PB DNMT3Amt samples from CCUS individuals (Izzo et al Nature 2024). Results: Patients' median age was 70 years (range: 56 - 89); 95% were white. Twelve out of 81 patients screened by ECS were found with a DNMT3Amt (15%), with a median variant allele fraction (VAF) of 42% (range: 6-46%). In this cohort, while the presence of any CH mutation did not impact survival, DNMT3Amt CH had an age-independent negative impact on mortality (5.5 years vs 8.7 years, p =0.005), without an increase in risk for MDS/AML. Changes in DNA methylation were profiled in 19 and 9 VEXAS patients without CH (Median Age: 68) or with DNMT3Amt CH (Median Age: 74), respectively. VEXAS samples with DNMT3Amt CH had &gt;4,000 differentially methylated CpGs with 95% of the CpGs being hypomethylated compared to those without CH. Using the Roadmap Epigenomics project, we found that this hypomethylation was enriched at enhancer (Enh) sites and actively transcribed states (TxWk). Pathway analysis revealed that the hypomethylated sites were in or near genes involved in the development and differentiation of leukocytes, with IL-13 and IL-15 being identified as upstream regulators of the methylation changes. Bulk RNA-seq performed on PB of 2 and 3 VEXAS patients with and without DNMT3Amt, respectively, identified &gt;1,500 differentially expressed genes in DNMT3Amt samples, 78% of these being overexpressed. Pathway analysis was enriched in gene pathways involved in the development of leukocytes, with IL-13 and IL-15 once again being identified as upstream regulators. Integration of methylation and transcriptomic data identified upregulation of several genes within the IL-13 and IL-15 pathways, with these regions correlating with a decrease in methylation. To assess if the epigenetic dysregulation is linked to DNMT3A mutations, we performed GoTChA-seq on two DNMT3Amt clonal cytopenia samples (CCUS). The comparison of the number of cutsites in DNMT3A wild type vs mt cells from the same individual demonstrated increased open chromatin within the IL-13 and IL-15 pathways in mt cells, both globally and in a genomic locus specific manner. CITE-seq performed on 3 VEXAS samples (2 DNMT3Amt CH vs 1 no CH) further showed that expression of genes involved in IL-13 and IL-15 pathway is significantly increased in CD4+ and CD8+ T cells from DNMT3Amt samples, suggesting a paracrine regulatory effect of T lymphocytes (mostly UBA1 and DNMT3A wild-type) in VEXAS. Pathway analysis of DEGs in these cells also revealed an enrichment of genes involved in inflammation and lymphocyte proliferation. Conclusions: Our study validates the age-independent, negative prognostic impact of DNMT3Amt CH on survival in VEXAS syndrome. DNMT3Amt mediates epigenetic and transcriptional priming associated with upregulation of IL-13/15 pathway in CD4/CD8 T cells, which are mostly DNMT3A and UBA1 wild-type, suggesting that the contribution of T lymphocytes to inflammation may be secondary to CH in the myeloid compartment.

Background The prognostic relevance of TP53 mutations (TP53MT) in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) has been extensively studied and the information accessed for defining the new International Consensus Classification (ICC) category of “myeloid neoplasms with mutated TP53” (Blood 2022;140:1200). The latter designation requires ≥10% TP53 variant allele frequency (VAF) for AML and MDS/AML and in case of MDS, multi-hit TP53 or ≥10% VAF associated with complex karyotype (CK). We examined the prognostic relevance of TP53MT in myeloproliferative neoplasms (MPN), specified by subtype and disease stage. Methods The current study was conducted under an institutional review board approved minimum risk protocol. Additional informative cases from the University of Florence, Italy and the South Australian Myeloid Neoplasm Registry were included. Diagnostic criteria and assignment of TP53 allelic state were according to the ICC. Next-generation sequencing (NGS) and cytogenetic studies were performed according to institutional standard protocols. Conventional statistical methods were employed. Survival analyses were censored for allogeneic stem cell transplantation (ASCT) and were calculated from the time of first detection of TP53MT. Results 115 patients with MPN and documented pathogenic TP53MT were considered (median age 68.5 years). ICC-defined subtype designations, at time of TP53MT detection, were polycythemia vera (PV) or essential thrombocythemia (ET) (N=9), chronic phase myelofibrosis (MF-CP; N=63), accelerated phase MPN (MPN-AP; N=16), and blast phase MPN (MPN-BP; N=27). Driver mutation distribution was JAK2 64%, CALR 18%, MPL 8%, and triple-negative 10%. Karyotype was monosomal (MK) in 42%, CK 9%, and normal (NK) 35%. Frequent co-mutations were ASXL1 (28%), TET2 (14%), and SRSF2 (14%). TP53MT were classified as ICC-defined multihit in 67 (58%) cases; karyotype in the latter was MK (58%), CK (16%), non-MK/CK (13%), or NK (13%). TP53 VAF was ≥50% in 27 (24%) patients and &lt;10% in 25 (22%). TP53MT included one (N=96) or ≥2 (N=19). MPN subtype- and stage-specific distributions for multihit TP53MT were 91% for MPN-BP, 88% MPN-AP, 44% MF-CP, and 0% ET/PV. In order to examine disease subtype- and stage-specific prognostic contribution of TP53MT, the study population was divided into 10 operational groups: 1) PV/ET non-multihit/non-MK/CK (N=8; VAF range 3-32%); 2) MF-CP non-multihit/non-MK/CK with TP53MT VAF &lt;10% (N=10); 3) MF-CP non-multihit VAF ≥10% and non-MK/CK (N=11); 4) PV/ET/MF-CP non-multihit with MK/CK (N=5); 5) MF-CP multihit/non-MK/CK (N=8); 6) MF-CP multihit and MK/CK (N=20); 7) MPN-BP/AP non-multihit/non-MK/CK (N=2); 8) MPN-BP/AP non-multihit with MK/CK (N=2); 9) MPN-BP/AP multihit/non-MK/CK (N=9); and 10) MPN-BP/AP multihit with MK/CK (N=30). The corresponding median survivals were “not reached”, 55, 42, 6, 18, 10, “not reached”, 4, 4, and 6 months (p&lt;0.01). Survival was similar between groups 1, 2, and 3 (p&gt;0.2 for all comparisons) now referred to as “PV/ET/MF-CP non-multihit/non-MK/CK (N=39)”. Survival was similarly dismal for groups 8, 9, and 10 (p values &gt; 0.3 for all comparisons) now referred to as “MPN-BP/AP with or without multihit TP53MT or MK/CK (N=43)”. Survival was also similar between groups 4, 5, and 6 (p values &gt;3 for all comparisons) now referred to as “PV/ET/MF-CP with multihit TP53MT or MK/CK (N=33)”. The three consolidated groups were subsequently compared for survival that showed a significantly worse outcome for “MPN-BP/AP with or without multihit TP53MT or MK/CK” with median survival of 4 months, compared to 12 months for “PV/ET/MF-CP with multihit TP53MT or MK/CK” (HR 2.3; 95% CI; 1.3-3.9) and 55 months for “PV/ET/MF-CP non-multihit/non-MK/CK” (10.9; 95% CI 5.3-22.4); survival in the latter two groups was also significantly different, in favor of PV/ET/MF-CP non-multihit/non-MK/CK (HR 4.8; 95% CI 2.3-10.1). Conclusions: The presence of multi-hit TP53MT or MK/CK, in MF-CP, and possibly in ET/PV, warrants their inclusion in the ICC category of “myeloid neoplasms with mutated TP53”. By contrast, non-multihit TP53MT in ET/PV/MF-CP, regardless of VAF and in the absence of MK/CK, does not appear to compromise short-term survival. The observations from the current study also suggest a prognostic hierarchy with blast percentage being the most unfavorable, followed by MK/CK and multihit TP53MT.

Background: Previously published risk variables for chronic myelomonocytic leukemia (CMML) included age, gender, CMML subtype, red blood cell (RBC) transfusion status, hemoglobin, leukocyte, monocyte, lymphocyte and platelet counts, immature myeloid cells, peripheral blood (PB) or bone marrow blast percentage, and mutations including ASXL1, RUNX1, NRAS, and SETBP1. Recent studies have reported on additional prognostically favorable (TET2) or unfavorable (DNMT3A, ASXL1, NRAS, SETBP1) mutations. In the current study, we took advantage of a clinically well-characterized and molecularly annotated cohort of CMML patients, in order to revisit the prognostic interaction between the aforementioned risk factors, with emphasis on mutations. Methods: The current study was conducted under an institutional review board approved protocol that allowed retrospective collection and analysis of data from Mayo Clinic patient records. Mutations were screened by multi-gene next-generation sequencing (NGS). Diagnostic criteria were according to the International Consensus Classification (Blood 2022; 140:1200). Conventional statistical methods were employed (JMP Pro 17.0.0 software SAS Institute, Cary, NC, USA). Survival analysis was censored for allogeneic stem cell transplantation (ASCT). Receiver Operating Characteristic (ROC) plots and Akaike Information Criterion (AIC) were used to compare predictive performance of the new model (MMMv2) vs. the Mayo Molecular Model (MMM) vs. the CMML-specific prognostic scoring system (CPSS). Results: The study cohort included 416 patients with CMML that were fully annotated for mutations. Cytogenetic information was available in 404 patients. At a median follow-up of 18 months, 302 (72%) deaths, 72 (18%) leukemic transformations, and 44 (11%) ASCTs were documented. Age-adjusted multivariable analysis (MVA) of mutations identified the following as risk factors: DNMT3A (HR 5.5; p&lt;0.01), PHF6 (HR 0.3; p&lt;0.01), TET2 (HR 0.6; p&lt;0.01), PTPN11 (HR 2.5; p=0.01), NRAS (HR 1.6; p&lt;0.01), ASXL1 (HR 1.3; p=0.02), and SETBP1 (HR 1.8; p=0.01). A separate MVA of clinical and cytogenetic variables identified RBC transfusion need, leukocyte count ≥13 x 109/L, peripheral blood blast ≥1%, abnormal karyotype, older age, and male sex to be independently associated with inferior survival (p&lt;0.04 in all instances). All-inclusive MVA identified the following as independent risk factors for survival (p &lt;0.01): DNMT3A mutation (HR 3.7; 95% CI 2.0-6.7), absence of PHF6 mutation (HR 2.8; 1.3-5.8), RBC transfusion need (HR 2.3; 1.7-3.1), leukocyte count ≥13 x 109/L (HR 2.0; 1.5-2.6), abnormal karyotype (HR 1.6; 1.2-2.2), and absence of TET2 mutation (HR 1.4,1.1-1.8). An HR based prognostic model was subsequently constructed by assigning 3 points to DNMT3A mutation, 2 points each for RBC transfusion need, leukocyte count ≥13 x 109/L, and absence of PHF6 mutation and one point each for abnormal karyotype and absence of TET2 mutation, resulting in a 4-tiered survival risk model (MMMv2): low risk (0-2 points; N=103) with median survival of 74 months; intermediate-1 (3 points; N=65) with median 37 months; intermediate-2 (4-5 points; N=126) with median 19 months; and high (6 points; N=88) with median 12 months (p&lt;0.01). Independent genetic risk factors associated with inferior leukemia-free survival included DNMT3A (HR 5.5), PHF6 (HR 0.2), abnormal karyotype (HR 2.0), PTPN11 (HR 5.2), ASXL1 (HR 2.0), and BCOR (HR 8.7). Leukocyte count ≥13 x 109/L (p=0.01) but not RBC transfusion need (p=0.2) was additionally significant. However, presence of immature myeloid cells (p&lt;0.01) and circulating blasts ≥1% (p&lt;0.01) were prognostically more relevant than leukocyte count ≥13 x 109/L and independently predicted leukemic transformation, along with DNMT3A, PTPN11 and ASXL1 mutations. Predictive performance was superior for MMMv2 (AUC 87, AIC 137) vs. MMM (AUC 74, AIC 194) vs. CPSS (AUC 76, AIC 177). Conclusion: The revised Mayo Molecular Model (MMMv2) provides a contemporary risk model for CMML that recognizes DNMT3A as the most unfavorable and PHF6 as the most favorable mutation and confirms the mutation-independent prognostic contribution from red cell transfusion need and sustained leukocytosis (≥13 x 109/L). Mutations were also important in predicting leukemic transformation, notably DNMT3A, PTPN11 and ASXL1.

Background Abnormal karyotype is not infrequent in polycythemia vera (PV) and has been associated with shortened survival (BJH 2018;182:437). In the current study, we utilized a large institutional database, in order to describe the spectrum and prevalence of specific cytogenetic abnormalities in PV, during different stages of the disease, and examine their clinical, molecular, and prognostic correlates, in the context of previously established risk factors. Methods After institutional review board approval, we identified 669 cytogenetically-annotated patients with PV, evaluated at the Mayo Clinic between 1973 and 2023, and met the International Consensus Classification diagnostic criteria (Arber et al. Blood 2022;140;1200). Cytogenetic reporting was in accordance with the International System for Human Cytogenomic Nomenclature. Results i) Prevalence of cytogenetic abnormalities at diagnosis and at different stages of the disease 436 of 669 (65%) patients with PV underwent cytogenetic evaluation within one year of diagnosis, 116 (17%) within 1-10 years, 47 (7%) after 10 years, and 70 (10%) at time of fibrotic or leukemic transformation. In patients with cytogenetic information obtained within 1 year of diagnosis, karyotype was normal in 369 (85%), showed loss of Y chromosome (-Y) in 15 (3%), and other abnormalities in 52 (12%). Most common abnormalities included trisomy 9 (n=11; 3%), del(20q) (n=10; 2%), trisomy 8 (n=4; 1%), other sole abnormalities (n=16; 4%), and ≥ 2 abnormalities (n=11; 3%) including complex karyotype (n=4; 0.9%). Frequency of abnormal karyotype was higher with longer disease duration; 15% within a year of diagnosis, 28% in 1-10 years, 49% over 10 years after diagnosis, and 50% at time of fibrotic or leukemic transformation (p&lt;0.01). Sole del (20q), single abnormalities excluding 20q-, +8, +9, or -Y, and ≥ 2 abnormalities including complex karyotype were progressively acquired; respective frequencies within a year of diagnosis, over 10 years after diagnosis, and at time of fibrotic or leukemic transformation were 2%/21%/10% (del (20q), 4%/17%/14% (single abnormality excluding 20q-, +8, +9, or -Y), 3%/11%/19% (≥ 2 abnormalities), 1%/ 2%,/10% (complex karyotype) (all p-values &lt;0.01). In addition, sole +8 displayed highest frequency at fibrotic transformation compared to within a year of diagnosis (5% vs 0.9%; p=0.04). ii) Phenotypic and Molecular Correlates 436 patients with PV (51% males; median age; 64 years) who underwent cytogenetic testing within one year of diagnosis were examined for clinical and molecular correlations. Abnormal karyotype compared to normal karyotype, correlated with older age (median: 70 vs 63 years, p&lt;0.01), male gender (64% vs 48%, p=0.02) and lower platelet count (370 vs 465 × 109/L, p&lt;0.01). Sole del (20q) compared to normal karyotype was associated with older age (median: 76 vs 63 years, p&lt;0.01); sole +8 exclusively occurred in patients ≥ 60 years (p=0.03); sole +9 was associated with venous thrombosis history (45% vs 17%, p=0.03). TET2 and SRSF2 mutations were found to cluster with normal karyotype (18% vs 4%; p=0.04 and 5% vs 0%; p=0.14 in normal vs abnormal karyotype, respectively). iii) Prognostic Impact of Cytogenetic Abnormalities At a median follow-up of 7.4 years (range; 0.04-42.4 years), a total of 163 (37%) patients have died, 50 (11%) and 14 (3%) underwent fibrotic and leukemic transformation, respectively. In univariate analysis for overall survival (OS), abnormal karyotype compared to normal karyotype, was associated with inferior survival (median OS; 10.5 vs 16.3 years, p&lt;0.01). In multivariate analysis, the adverse impact of abnormal karyotype remained significant (p=0.02; HR 2.0, 95% CI 1.2-3.3), along with age ≥60 years, leukocyte count ≥15 × 109/L, and SRSF2 mutations. Abnormal karyotype was also an independent predictor of myelofibrosis-free survival (p&lt;0.01; HR 3.7, 95% CI 1.8-7.8), along with SRSF2 mutations. Analysis of leukemia-free survival revealed abnormal karyotype to be a significant risk factor in univariate (p&lt;0.01), but not multivariate analysis that included SRSF2 mutation (p&gt;0.1). Conclusions The current study provides the distribution of cytogenetic abnormalities at different stages of PV and affirms the prognostic significance of abnormal karyotype at the time of diagnosis, highlighting the importance of incorporating cytogenetic studies in the diagnostic evaluation of PV.

Background: JAK2 mutations are invariably associated with polycythemia vera (PV); more than 50% of affected patients also harbor non-JAK2 mutations, some considered prognostically relevant (BJH 2020;189:291). We queried a large single institutional database of patients with PV, in order to examine the incidence and distribution of non-JAK2 mutations, across different disease stages, in order to assess their impact on disease phenotype and prognosis, in the context of other risk factors. Methods: Mayo Clinic databases (1998-2023) were searched to identify next-generation sequencing (NGS)-annotated patients with PV, with diagnosis retrospectively fitted to meet the 2022 International Consensus Classification (ICC) criteria (Blood 2022; 140:1200). NGS was performed either through research-based targeted capture assays or a clinically ordered 42-gene panel. Conventional statistical methods were used for group comparisons and survival analyses. Results: i) Mutational frequencies in chronic vs. transformed disease phases 319 patients with PV were considered; NGS was performed at time of i) diagnosis or before fibrotic or leukemic transformation (N=270; 84.6%), ii) fibrotic progression (N=37; 11.6%), or iii) leukemic transformation (N=12; 3.8%). Mutational frequencies in newly-diagnosed/chronic phase PV were 19% for TET2, 10% for ASXL1, 4% for SRSF2, ⁓3% each for SF3B1, DNMT3A, and SH2B3, and 1-2% each for TP53 and IDH2. Significant differences in mutation distribution were apparent for TP53/SRSF2/IDH1/U2AF1 in blast (50%/25%/17%/8%) vs. fibrotic (8%/0%/0%/5%) vs. chronic (2%/4%/2%/0.4%) phase (p&lt;0.01, &lt;0.01, =0.03, and =0.03, respectively). ii) Phenotypic correlates of specific mutations among patients with NGS information obtained during chronic phase (N=270) Median age was 61.4 years (51% males) and incidence rates included 22% for leukocytosis ( ≥15 x 10(9)/L) and 25%/14% for arterial/venous thrombosis history. Numerically prevalent or prognostically relevant mutations were selected for comparison of clinical or laboratory characteristics: TET2, ASXL1, SRSF2, TP53, IDH2. Significant associations were apparent for ASXL1 (younger age distribution), SRSF2 (older age distribution, leukocytosis), and TP53 (leukocytosis) mutations. Median age was 66.4, 58.7, 76.8, 63.9, and 68.6 years for TET2, ASXL1, SRSF2, TP53, IDH2 mutations, respectively (p&lt;0.01). The corresponding median leukocyte counts were 12.1, 11.4, 14.4, 16.7, and 11.5 x 10(9)/L (p&lt;0.01). iii) Prognostic impact of mutations detected at diagnosis or chronic phase (N=270) At median follow-up of 9.5 years (range 0.1-42.4), 115 (43%) deaths, 12 (4%) leukemic transformations, and 53 (20%) fibrotic progressions were documented. In univariate analysis of mutations, overall survival (OS) was adversely affected by SRSF2 and IDH2 mutations, leukemia-free survival (LFS) by ASXL1, SRSF2, IDH2, RUNX1, and CEBPA mutations, and myelofibrosis-free survival (MFS) by SRSF2 mutation. Age-adjusted (p&lt;0.01) multivariable analysis, including mutations and other risk variables, identified SRSF2 (HR 4.2, 95% CI 1.9-9.5), ASXL1 (HR 2.0, 1.1-3.7), IDH2 (HR 5.3, 1.8-15.3) mutations and leukocytosis ≥15 x 10(9)/L (HR 2.0, 1.3-3.1) as independent predictors of inferior OS. SRSF2 and ASXL1 mutations were also independently associated with inferior LFS (p=0.02 and 0.06, respectively) and SRSF2 (p&lt;0.01) and karyotype (p&lt;0.01) with inferior MFS (p&lt;0.01). Median survival for 235 patients without any adverse mutation (ASXL1, SRSF2, or IDH2) was 17.8 years vs. 8.8 years for 35 patients with any one of the three adverse mutations (p=0.01; HR 1.8, 95% CI 1.1-2.9). The number of non-JAK2 mutations, in general, was also associated with significantly different survival outcomes: median OS was 20, 15.1, and 9.3 years for patients with non-JAK2 mutations numbering zero, one, or two or more, respectively (p&lt;0.01) Conclusions: SRSF2 mutations in PV cluster with older and ASXL1 with younger age distribution. SRSF2 and TP53 mutational frequencies were higher in blast-phase disease. SRSF2, ASXL1, and IDH2 mutations were significantly associated with inferior OS, independent of each other and other risk factors. SRSF2 mutations were also detrimental to LFS and MFS and ASXL1 to LFS. Such information might allow further refinement of current risk models for PV, which in future studies should also account for variant allele frequency.

Background Survival risk models in essential thrombocythemia (ET) include the International Prognostic Scoring System (IPSET; Blood 2012;120:1197) and the more recently described “triple-A” prognostic score that considers Age and absolute neutrophil (ANC) and lymphocyte (ALC) counts, as risk variables (AJH 2023;98:1829). Monocytosis, defined by absolute monocyte count (AMC) of ≥0.8 x 109/L, was recently reported to be associated with inferior survival in polycythemia vera (ASH 2024 abstract). The current study examined the prognostic contribution of monocytosis in ET, in the context of other clinical and genetic risk factors. Methods Study patients were recruited from Mayo Clinic (USA) databases, based on availability of information on ANC, ALC, and AMC (N=649). Diagnostic criteria were according to the International Consensus Classification (Blood 2022;140:1200). Mutations were screened by next-generation sequencing (NGS). Conventional statistical methods were employed (JMP Pro 17.0.0, SAS Institute, Cary, NC, USA) and optimal cut-points for age, ANC, ALC, and AMC were determined by Receiver Operating Characteristic (ROC) analysis. Akaike Information Criterion (AIC) and ROC plots were used to estimate predictive accuracy of risk models. Results A total of 649 patients (median age 60 years; females 65%) were informative for AMC, ANC, and ALC, with median (range) values of 0.6 x 109/L (0.2-2.5), 5.7 x 109/L (1.5-26.5), and 1.9 (0.4-5.7), respectively. At presentation, IPSET risk distribution was low in 34%, intermediate 47%, and high 19%; triple-A risk distribution was low 21%, intermediate-1 50%, intermediate-2 13%, and high 16%; driver mutation distribution was 63.8% JAK2, 24.0% CALR, 3.7% MPL, and 8.5% triple-negative; median for platelets were 728 x 109/L, hemoglobin 13.9 g/dL, and leukocytes 8.5 x 109/L ; leukocytosis (leukocytes ≥11 x 109/L) was documented in 18.8% of patients, palpable splenomegaly 7%, arterial thrombosis (AT) history 14.5%, venous thrombosis (VT) history 9.2%, and major hemorrhage 7.8%. At median follow-up of 7 years (range 0.02-31.4), 146 (22.5%) deaths, 52 (8%) fibrotic progressions, 22 (3.4%) leukemic transformations, 71 (11%) AT events, 36 (5.6%) VT events, and 64 (10%) major bleeds were recorded. In age-adjusted univariate analysis, higher AMC was significantly associated with inferior survival, with a ROC determined cutoff value of ≥0.8 x 109/L (N=154; 24%); significance was sustained (HR 2.3; p&lt;0.01) during multivariable analysis (MVA) that also identified age &gt;70 years (HR 16.6; p&lt;0.01), age 50-70 years (HR 4.3; p&lt;0.01), ANC ≥8 x 109/L (HR 1.9; p&lt;0.01), ALC &lt;1.7 x 109/L (HR 1.8; p&lt;0.01), and AT history (HR 1.6; p=0.01). A similar MVA conducted on an external cohort from the University of Florence (N=514) confirmed the significant contribution from all of these risk variables, except AT (p=0.8). An HR-based 4-variable prognostic model based on Age &gt;70 years (4 points) or 50-70 years (2 points), AMC ≥0.8 x 109/L (1 point), ANC ≥8 x 109/L (1 point), and ALC &lt;1.7 x 109/L (1 point), henceforth referred to as the AAA+A model, was subsequently developed using the Mayo Clinic cohort and validated by the Florence cohort: Low (0-1 points; N= 180; median not reached), intermediate-1 (2-3 points; N=266; median 21.6 years), intermediate-2 (4-5 points; N=151; median 12.2 years), and high (6-7 points; N=52; median 8.7 years); HR values were 2.1 for high vs. intermediate-2, 5.4 for intermediate-2 vs. intermediate-1, and 3.8 for intermediate-1 vs. low (p&lt;0.001in all instances). The 20-year survival predictive performance of AAA+A (AIC 131; AUC 0.88) was superior to that of AAA (AIC 143; AUC 0.85) and IPSET (AIC 149; AUC 0.84). In MVA that included AAA+A risk variables, AMC ≥0.8 x 109/L (p=0.04) and ALC &lt;1.7 x 109/L (p=0.06) appeared to affect leukemia-free survival and ANC ≥8 x 109/L (p=0.04) myelofibrosis-free survival. In univariate analysis, AMC ≥0.8 x 109/L was associated with AT (p=0.03) but not VT (p=0.14) history and showed borderline significance for venous (p=0.09) and arterial (p=0.07) thrombosis-free survivals; however, significance was lost during MVA. Conclusions: Monocytosis is an adverse prognostic factor in ET and its inclusion as a risk variable enhances the predictive performance of the AAA risk model and is equally simple to apply worldwide. Analyses that include mutations will be presented at meeting.

Background A JAK2 mutation is detected in almost all patients with polycythemia vera (PV) and in ⁓50% of those with essential thrombocythemia (ET). The two diseases also share characteristic clinical and bone marrow morphological traits, suggesting they exist on a disease continuum rather than representing discrete entities. The objective of the current study was to compare clinical and genetic characteristics as well as long-term outcome between formally-defined PV vs. JAK2-mutated ET vs. JAK2-wild-type (WT) ET. Methods Study patients were retrospectively recruited from Mayo Clinic, USA databases (1967-2024). Diagnostic criteria were according to the International Consensus Classification (ICC; Blood 2022;140:1200). All study patients were fully annotated for JAK2, CALR, and MPL mutations while cytogenetic and additional next-generation sequencing (NGS)-derived mutation information was available in a subset of patients. Conventional statistical methods were employed for phenotypic comparisons and survival analysis. Results Among 2,010 study patients, 1,010 had PV, 617 (63%) JAK2-mutated ET, and 383 JAK2-WT ET (CALR 26%; MPL 3%; triple-negative 9%). Phenotypic comparisons revealed older age distribution in PV compared to JAK2-mutated ET (p=0.03) and the latter compared to JAK2-WT ET (p&lt;0.01). Females were represented more in JAK2-mutated ET (69%), compared to both PV (49%; p&lt;0.01) and JAK2-WT ET (54%; p&lt;0.01). The frequencies of leukocytosis, palpable splenomegaly, constitutional symptoms, diabetes, hypertension, and abnormal karyotype were all significantly higher in PV, compared to both groups of ET, while JAK2-mutated compared to JAK2-WT ET displayed higher leukocyte count and incidence of hypertension. The incidences of arterial thrombosis at/prior to diagnosis were 14%, 16%, and 10% in PV, JAK2-mutated and WT ET, respectively (P=0.03), and venous thrombosis 14%, 13%, and 6% (p&lt;0.01). After a median follow-up of 8.6 years, 632 (32%) deaths, 329 (16%) fibrotic progressions, 79 (4%) leukemic transformations, and 236 (12%) arterial and 202 (10%) venous thrombotic events were recorded, among all 2,010 study patients. Median overall survival was 19.3 years for PV vs. 17.8 years for JAK2-mutated ET vs. 23 years for JAK2-WT ET (p&lt;0.01); however, the significant difference in favor of JAK2-WT ET was no longer apparent when analysis was adjusted for age and sex (p=0.15) and subsequently for other risk factors (p=0.86). All-inclusive multivariable analysis identified older age (p&lt;0.01), male sex (p&lt;0.01), higher leukocyte count (p&lt;0.01), and arterial thrombosis history (p&lt;0.01), but not the distinction between PV, JAK2-mutated, and JAK2-WT ET (p=0.9), to be independently associated with shortened survival. Leukemia-free survival was similar between PV and either JAK2-mutated (p=0.5) or JAK2-WT (p=0.1) ET. On the other hand, myelofibrosis-free survival was shorter in PV, compared to both JAK2-mutated (HR 1.8, 1.4-2.4; p&lt;0.01) and JAK2-WT (HR 1.4. 1.0-1.8; p=0.03) ET. In multivariable analysis, older age (p&lt;0.01), higher leukocyte count (p&lt;0.01), PV vs. JAK2-mutated ET (HR 1.8; p&lt;0.01), and JAK2-WT vs. JAK2-mutated ET (HR 1.6; p=0.01) were associated with a higher risk of fibrotic progression, with no difference noted between PV and JAK2-WT ET (p=0.4). Arterial thrombosis-free survival was similar between PV and JAK2-mutated ET (p=0.8). In univariate analysis, JAK2-WT ET vs. PV was associated with superior arterial thrombosis-free survival (p=0.02), but significance was lost when analysis was adjusted for arterial thrombosis history (p=0.1). Venous thrombosis-free survival favored JAK2-WT ET vs. PV (HR 0.3; p&lt;0.01) and JAK2-mutated ET (HR 0.6; p=0.02) and the latter vs. PV (HR 0.6; p&lt;0.01); the significant differences between PV and both groups of ET were sustained when analysis was adjusted for age, sex, and venous thrombosis history. Conclusions The current study highlights significant demographic and phenotypic differences between PV and JAK2-mutated ET. However, risk factor-adjusted overall, leukemia-free, and arterial thrombosis-free survival was not inferior in PV, compared to ET, regardless of the presence or absence of a JAK2 mutation in the latter. JAK2-WT ET was distinctly associated with a lower risk of venous thrombosis while JAK2-mutated ET appeared to be less vulnerable to fibrotic progression.

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.