Loscocco, Giuseppe Gaetano

Background An increased risk of second cancers (SC), in patients with myeloproliferative neoplasms (MPN), has been suggested by a number of population-based and cancer registry studies (Blood 2011;118:6515). Such studies often included patients belonging to a broad category of MPN entities and did not always account for history of cancer prior to the diagnosis of MPN, associated comorbidities, mutations, or treatments received, in assessing risk association and prediction (Leukemia 2019;33:1996). The current study seeks to fill knowledge gaps, in this regard, in the context of a biologically more homogeneous MPN setting, vis-ẚ-vis polycythemia vera (PV) and essential thrombocythemia (ET). Methods The present 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. Diagnostic criteria were according to the International Consensus Classification (Blood 2022; 140:1200). Documented primary cancers were annotated in 17 categories, not including pre-cancerous conditions or MPN progressions. SC was defined as any cancer that developed after 3 months from the initial diagnosis of MPN. Cox proportional hazard regression model was used for univariable and multivariable analysis. Kaplan-Meier plots were used for time-to-event assessment (considering the first in case of multiple events). Results The study population included 1,968 consecutive patients (median age 60 years; 56% females) with PV (N=1,001) or ET (N=967), seen at the Mayo Clinic between 1970 and 2024. Median follow-up was 8.6 years. The incidences of arterial/venous thrombosis were14%/12% at diagnosis and 12%/10% after diagnosis. Co-morbid conditions included diabetes (12%), hypertension (52%), tobacco use (16%), and history of autoimmune/inflammatory disease (AID; 6%); hyperlipidemia was annotated in PV cases only (51%). Non-driver mutation information was available in 620 patients: TET2 (13%), ASXL1 (7%), DNMT3A (5%), SRSF2 (3%), SF3B1 (3%) and TP53 (2%). A history of another cancer was documented in 598 (30%) patients and included cancers documented before/at (N=291; 15%) or after (N=404; 20%) the diagnosis of MPN, with only the latter being referred to as “SC”. Most frequent cancer type was non-melanoma skin cancer (NMSC), accounting for 28% of cancers diagnosed before/at MPN, and 47% of SC; other SC types included lymphoid neoplasms (10%), melanoma (10%), prostate cancer (9%), and trachea/lung/bronchial cancer (8%), as the most frequent. On multivariable analysis, cancer history, before/at or after MPN diagnosis, clustered with age >60 years (OR 2.7), male sex (OR 1.5), and history of AID (OR 1.7). History of cancer prior to MPN diagnosis, NMSC or otherwise, was associated with inferior overall survival (p<0.01); by contrast, neither myelofibrosis-free (p=0.5) nor leukemia-free (p=0.8) survival was affected. Median time from MPN diagnosis to SC was 5.7 years. In multivariable analysis including all cancers, SC-free survival was adversely affected by age>60 years (HR 2.5; p<0.01), male sex (HR 1.7; p<0.01), history of previous cancer (HR 2.5, p<0.01), and AID history (HR 1.6; p<0.01); all these 4 risk factors remained significant after excluding NMSC. Interestingly, among informative cases, SRSF2 mutation also predicted SC (HR 2.6; p=0.02), even after NMSC cases were excluded (HR 3.4; p=0.02). Pre-MPN history of cancer did not affect either arterial (p=0.9) or venous thrombosis-free survival (p=0.7), in age- and sex-adjusted analysis. Conversely, after excluding post-SC thrombotic events, venous (p=0.02) but not arterial (p=0.1) events, occurring after MPN diagnosis, were predictive of SC. This association remained significant in multivariable analysis that included age, sex, and venous thrombosis history (p=0.03). Additional observations from this study, include an independent association between SC and hyperlipidemia in PV (p<0.01) and ruxolitinib monotherapy with NMSC (HR 9; p<0.01). Conclusions The risk of SC in patients with MPN is significantly higher in older patients, men, and in the presence of pre-MPN cancer history and AID. Incidental venous, but not arterial, thrombosis was associated with subsequent SC, not NMSC. The observed association of SC with SRSF2 mutations and hyperlipidemia is noted but requires further validation.

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 <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 <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<0.01). Survival was similar between groups 1, 2, and 3 (p>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 > 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 >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 Previous studies have suggested a higher prevalence of autoimmune or inflammatory diseases (AID) in patients with myeloproliferative neoplasms (MPN) (Haematologica 2010; 95:1216); more recent studies have suggested the absence of impact on tumor genetics or disease outcome (Leukemia 2023; 37:1741). In the current study, we conducted a before and after MPN diagnosis survey, in order to examine the prevalence of AID and its impact on overall and transformation-free survival as well as the risk of thrombosis or other co-morbidities, such as cancer. Methods The current study was conducted under an institutional review board approved minimum risk protocol. Diagnostic criteria were according to the International Consensus Classification (Blood 2022; 140:1200). AID was operationally classified as occuring before or after diagnosis, with the latter requiring first diagnosis occuring ≥3 months after MPN diagnosis. Non-driver mutations were screened by next-generation sequencing (NGS) and was available for 620 patients. Cox proportional hazard regression model was used for univariate and multivariable analysis. Kaplan-Meier plots were used for time-to-event assessment, using JMP Pro 17.0.0 software SAS Institute, Cary, NC, USA. Results The study population included 1,968 consecutive patients with polycythemia vera (PV; N=1,001) or essential thrombocythemia (ET; N=967), seen at the Mayo Clinic between 1970 and 2024. Mutation information was available in 620 patients. AID was diagnosed before or after MPN diagnosis in 157 (8%) patients, including 121 (6%) before and 43 (4%) after. 134 (85%) patients had history of only one AID while 23 (15%) had ≥2 AIDs. The most prevalent diagnoses included organ-specific AID (48 cases before and 21 after MPN diagnosis), inflammatory arthritis (37 before and 6 after), connective tissue diseases (17 before and 7 after) and inflammatory dermatosis (20 before and 4 after). For each one of these four categories, thyroid disorders (21 before and 6 after), rheumatoid arthritis (22 before and 2 after), antiphospholipid syndrome (8 before and 1 after), and psoriasis (11 before and 4 after) were the most frequent, respectively. Patients with pre-MPN AID history were older (p=0.02) and were more likely to have ET vs. PV and be females (p<0.01); they were also more likely to present with venous (18% vs. 12%, p=0.04) but not arterial (p=0.2) thrombosis. Pre-MPN AID history was significantly associated with pre-MPN cancer history, with the latter including (25% vs 14%, p<0.01) or excluding (18% vs 10%, p<0.01) non melanoma skin cancers (NMSC). Pre-MPN AID history did not correlate with driver or other mutations (including TET2, ASXL1, DNMT3A), leukocyte, neutrophil, monocyte, or lymphocyte counts (p≥0.1 in all instances). Pre-MPN history of AID did not affect overall (p=0.08), myelofibrosis-free (p=0.3), leukemia-free (p=0.3), arterial thrombosis-free (p=0.9), or venous thrombosis-free (p=0.6) survival. Second cancer-free survival (SC-FS), including (p<0.01) or excluding (p<0.01) NMSC, was negatively affected by pre-MPN history of AID. Multivariate analysis confirmed the independent detrimental effect of a pre-MPN history of AID (HR 1.7, p<0.01) on SC-FS; other independent risk factors for SC-FS were older age (p<0.01), male sex (p<0.01) and pre-MPN history of cancer (p<0.01). The association between pre-MPN history of AID and second cancer was also apparent when the latter was limited to lymphoproliferative neoplasms, although with borderline significance (p=0.08), which was further confirmed by adjusting for age and sex (p=0.09). Median time from MPN diagnosis to post-MPN AID was 9 years (range 1-23), which was similar between PV and ET (p=0.5). Post-MPN development of AID was more likely in females (HR 2.3; p=0.01) and in patients with pre-MPN history of AID (HR 4.8; p<0.01) or hypertension (HR 2.1; p<0.03). These risk factors remained significant during multivariable analysis of post-MPN AID-free survival. Conclusions The current study suggests an increased risk of post-MPN AID and second cancers, in patients with pre-MPN history of AID. The study also showed a significant association between pre-MPN history of AID and venous thrombosis at time of MPN diagnosis. These observations reinforce the widely suggested role of tumor-extrinsic inflammation in exacerbating MPN-intrinsic thrombophilia and the development of cancer.

Background Conventional thrombosis risk stratification in polycythemia vera (PV) considers age > 60 years and thrombosis history for assignment to low (no risk factor) or high (at least 1 risk factor) risk category (J Clin Oncol.2011;29(6):761-70). The current study exclusively focused on low-risk PV with the following objectives: i) estimate the incidence of arterial and venous thrombosis; ii) examine the prognostic relevance of clinical and genetic risk factors for thrombosis; and iii) assess the impact of treatment on thrombosis risk. Methods Patients were retrospectively recruited from the Mayo Clinic, USA, and University of Florence, Italy, PV databases. Diagnosis was according to the International Consensus Classification criteria (Blood 2022;140:1200). Only first major arterial (AT) and unprovoked venous thrombosis (VT) were considered. Conventional statistical methods were applied (JMP Pro 17.0.0, SAS Institute, Cary, NC, USA). Results Mayo Clinic cohort A total of 346 patients with low-risk PV were considered: median age 49 years; males 54%; median hemoglobin 17.7 g/dl; median leukocyte count 10.9 x 109/L; leukocyte count >11 x 109/L 49%; median platelet count 471 x 109/L; cardiovascular (CV) risk factors 47%; hypertension 29%; hyperlipidemia 20%; smoking history 12%; diabetes mellitus 5%. Most frequent mutations, other than JAK2, were ASXL1 (13%) and TET2 (13%). Median follow-up was 13.4 years (1-52) (4,636 patient-years) during which 25 (7%) AT and 20 (6%) VT were documented. The corresponding incidence rates were 5.4 and 4.3 per 1,000 patient-years. In multivariable logistic regression analysis, baseline hemoglobin >17.5 g/dl in women or >20 g/dl in men (15% vs 4%; OR 5.1; p<0.01) and absence of leukocytosis >11 x 109/L (10% vs 4%; OR 2.9; p=0.02) were associated with increased risk of AT; in addition, diabetes mellitus (19% vs 7%; OR 3.9; p=0.06), showed borderline significance; overall incidence of AT was 20% in the presence of ≥2 risk factors (n=61), 7% in the presence of one risk factor (n=177), and 1% in the absence of all three risk factors (n=108; p<0.01). Multivariable analysis for arterial thrombosis-free survival (TFS) confirmed the predictive performance of these risk factors: hemoglobin thresholds (HR 4.4; p<0.01); absence of leukocytosis >11 x 109/L (HR 2.7; p=0.03); and diabetes (HR 2.8; p=0.09); 20-year cumulative incidence of AT was 32% in the presence of ≥2 risk factors and 7% otherwise (p<0.01). In multivariable analysis, smoking history was found to be surprisingly protective against VT (0% vs. 7%; p=0.02) while female gender was associated with a borderline higher risk (8% vs 4%; OR 2.2; p=0.09); VT incidence rates were 9% in the presence of both risk factors (n=144), 4% with one risk factor (n=172), and 0% in the absence of both risk factors (n=40; p=0.02); 20-year cumulative incidence of VT was 10% in the presence of both risk factors and 3% otherwise (p=0.03). ASXL1 and TET2 mutations did not appear to modify the risk of either VT (p=0.59) or AT (p=0.66). Treatment at diagnosis included aspirin and cytoreductive therapy in 63% and 24% of patients, respectively. Aspirin but not cytoreductive therapy was associated with a lower incidence of AT (5% vs 11%; 2.3 vs 3.0 per 1,000 patient-years; p=0.04) and VT (4% vs 10%; 1.9 vs 2.8 per 1,000 patient-years; p=0.03). Thrombosis-free survival was not influenced by documented treatment at diagnosis or at the time of event (p>0.1). University of Florence cohort A total of 284 low-risk PV patients were followed for a median of 9.1 years (1-38) with 19 (7%) AT and 24 (8%) VT events recorded; the corresponding incidence rates were 7.4 and 9.3 per 1,000 patient-years. In this Florence cohort, risk factors for AT were diabetes mellitus (HR 3.9), hyperlipidemia (HR 3.8) and advanced age, with only age remaining significant during multivariable analysis (p=0.03). Predictors of VT included hyperlipidemia (HR 4.2) and hypertension (HR 2.1). Treatment with aspirin or cytoreductive therapy did not impact thrombosis-free survival (p>0.1). Conclusion The current study confirms the infrequent occurrence of AT and VT in low-risk PV but also exposes the challenges in identifying risk factors that are reproducible in different but otherwise well-characterized patient cohorts. Discrepancies were also apparent in retrospective comparison of treatment effects on thrombosis, which reinforces the need for prospective studies.

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<0.01) during multivariable analysis (MVA) that also identified age >70 years (HR 16.6; p<0.01), age 50-70 years (HR 4.3; p<0.01), ANC ≥8 x 109/L (HR 1.9; p<0.01), ALC <1.7 x 109/L (HR 1.8; p<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 >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 <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<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 <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: 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<.001), higher hemoglobin (18.4 g/dL vs 17.5, p<.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<.001) at diagnosis. Pts with a VAF >50% were 69% in CC versus 24.1% in TT+CT pts (p<.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<.001), no difference was noted for arterial thrombosis nor for splancnic vein thrombosis. VT-FS by Kaplan Meyer was significantly shorter (p<.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<.001). In multivariable analysis, including JAK2VAF >50%, age >60y, history of VT, WBC >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<.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<.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<.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 In 2013, we published the first International Working Group (IWG) risk model for survival in polycythemia vera (PV), which included 3-tiered age categories, leukocytosis (≥15 x 109/L), and venous thrombosis, as risk variables (Leukemia. 2013;27:1874). In the current study, we examined the individual prognostic contribution from specific leukocyte components and mutations, in order to construct a more contemporary clinical risk model in PV and assess its interaction with genetic risk factors. Methods Study patients were retrospectively recruited from the Mayo Clinic, USA (N=1,007) and the University of Florence, Italy (N=514), in order to serve as discovery and validation cohorts, respectively. 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, absolute neutrophil (ANC), lymphocyte (ALC), and monocyte (AMC) counts were determined by Receiver Operating Characteristic (ROC) analysis. Akaike Information Criterion (AIC) and ROC plots were used to estimate predictive accuracy. Results Presenting features in the Mayo discovery cohort (N=1,007) included: median age 62.2 years; males 50.7%; median leukocyte count 10.8 x 109/L; leukocyte count ≥15 x 109/L 19%; palpable splenomegaly 32%; pruritus 34%; diabetes 16%; hypertension 61%; arterial thrombosis 14%; and venous thrombosis 14%. ANC, AMC, and ALC information was available in 736 patients with respective median (range) values of 7.6 (1.6-30), 0.6 (0-4.1), and 1.54 (0.19-6.9) x 109/L. At a median follow-up of 8.9 years for the Mayo discovery cohort, 372 (37%) deaths, 46 (4.6%) leukemic transformations, and 200 (20%) fibrotic progressions were recorded. Multivariable analysis (MVA) employing ROC-determined cutoff values revealed adverse survival impact for age >70 years (HR 18.3; p<0.01), age 50-70 years (HR 4.9; p<0.01), AMC ≥0.8 x 109/L (HR 1.5; p<0.01), ANC ≥8 x 109/L (HR 1.5; p<0.01), arterial thrombosis (HR 1.5; p<0.01), diabetes (HR 1.4; p=0.04), and male sex (HR 1.3; p=0.05). A similar MVA applied to the Florence validation cohort (N=514) confirmed significance for age (p<0.01), ANC (p<0.01), AMC (p=0.04), and arterial thrombosis (p<0.01), but not for male sex (p=0.8) or diabetes (p=0.15). Accordingly, age >70 years (4 points), age 50-70 years (2 points), ANC ≥8 x 109/L (1 point), AMC ≥0.8 x 109/L (1 point), and AT (1 point) were included in a revised IWG survival model using the Mayo cohort and validated by the Florence cohort: very low risk (0 points; N=67; median 37 years), low risk (1 point; N=55; median 29.2 years), intermediate risk (2-3 points; N=308; median 20.2 years), high risk (4-5 points; N=217; median 11.5 years), and very high risk (6-7 points; N=88; median 6.3 years). 20-year survival prediction performance of the revised IWG model (AIC 204; AUC 0.88) was superior to that of the 2013 IWG version (AIC 350; AUC 0.86). Mutation information at diagnosis/chronic phase was available in 270 Mayo patients: ASXL1 10%; SRSF2 4%, IDH2 2%, TP53 2%, RUNX1 1.4%. MVA of mutations identified SRSF2 (p<0.01), IDH2 (p=0.01), and ASXL1 (p=0.05), as risk factors for overall survival. The presence of any one adverse mutation was more likely in patients with AMC ≥0.8 x 109/L (24% vs. 7% incidence; p<0.01). In all-inclusive MVA, independent risk factors for OS were age >70 years (HR 14.7), age 50-70 years (HR 6.2), any one adverse mutation (HR 3.4), and ANC ≥8 x 109/L (HR 2.3), but not AMC ≥0.8 x 109/L (p=0.9), arterial thrombosis (p=0.24), or abnormal karyotype (p=0.4). Leukemia-free survival was adversely affected by SRSF2, RUNX1, TP53, and ASXL1 mutations and myelofibrosis-free survival by SRSF2 mutation. Conclusions: The currently proposed revised IWG risk model for survival in PV is globally applicable and based on four easily accessible clinical variables (Age, ANC, AMC, and arterial thrombosis). NGS-derived mutation information overshadows the prognostic relevance of monocytosis, provides additional prognostic information for overall survival, and assists in predicting leukemic and fibrotic transformations.

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<0.01). Females were represented more in JAK2-mutated ET (69%), compared to both PV (49%; p<0.01) and JAK2-WT ET (54%; p<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<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<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<0.01), male sex (p<0.01), higher leukocyte count (p<0.01), and arterial thrombosis history (p<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<0.01) and JAK2-WT (HR 1.4. 1.0-1.8; p=0.03) ET. In multivariable analysis, older age (p<0.01), higher leukocyte count (p<0.01), PV vs. JAK2-mutated ET (HR 1.8; p<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<0.01) and JAK2-mutated ET (HR 0.6; p=0.02) and the latter vs. PV (HR 0.6; p<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<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.