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Abstract Introduction Ph-like B-ALL is a poor-risk B-ALL subtype defined by gene expression signatures that resemble the BCR::ABL1-associated transcriptional program in the absence of the BCR-ABL1 oncoprotein. Recent studies have demonstrated that up to 80% of Ph-like B-ALLs have recurrent gene fusions involving cytokine receptors or tyrosine kinases that can be identified via cytogenetics, fluorescence in situ hybridization (FISH) or RNA sequencing. However, the inability to rapidly and accurately identify these alterations in the clinical setting is a major impediment to developing therapeutic strategies that may have efficacy in this B-ALL subtype. Here we used 88 patient (pt) samples to validate a rapid, streamlined clinical WGS assay (ChromoSeq) for B-ALL and assessed the diagnostic yield for Ph-like-associated SVs in 136 pts enrolled in the Alliance A041501 clinical trial. Methods ChromoSeq is a clinical WGS assay for hematologic malignancies that uses rapid laboratory workflows and focused analysis methods to identify clinically relevant SVs, copy number alterations and gene-level mutations. We updated the ChromoSeq pipeline for use in B-ALL pts and validated it using 88 retrospective B-ALL samples (80 adult/young adult and 8 pediatric from Washington University School of Medicine and St. Louis Children's Hospital), 54 of which were analyzed via RNAseq for gene fusions and a 6-gene Ph-like B-ALL signature (CA6, SPATS2L, MUC4, JCHAIN, BMPR1B, ADGRF1, NRXN3 and CRLF2). The “ChromoSeqV2” assay was then used to analyze 136 pretreatment blood or bone marrow samples from the Alliance A041501 (trial NCT03150693). These results were compared to data from RNAseq obtained for research purposes (N=35), clinical cytogenetics (N=69) and Ph-like classification via low-density microarray (LDA) card analysis (all pts). Results ChromoSeqV2 was validated in a CLIA-licensed setting using cohort of 88 retrospective B-ALL samples, 45 of which had ICC and WHO class-defining SVs based on G-banded karyotyping, FISH or RNAseq. ChromoSeqV2 detected 100% of the non-Ph-like SVs (37/37), including BCR::ABL1 (N=20), KMT2Ar (N=9), ETV6::RUNX1 (N=3), TCF3::PBX1 (N=1), and rearrangements involving MYC (N=2) or MEF2D (N=2). Fifteen patients met criteria for Ph-like B-ALL via cytogenetics or RNAseq either due to rearrangements of CRLF2 (N=5), JAK2 (N=2) or ABL2 (N=1) or high expression of a 6-gene Ph-like signature (N=7). ChromoSeqV2 detected a Ph-like SV in 93% of these cases (14/15) with 100% specificity in samples negative by RNAseq (N=23). ChromoSeqV2 was then used to profile 136 samples from the Alliance A041501 trial, where it identified ICC and WHO class-defining SVs in 87 pts (64%; 95% CI: 55-72%), including Ph-like SVs (56/136, 41%), KMT2A rearrangements (N=5), TCF3::PBX1 (N=3) and SVs involving MYC (N=3), MEF2D (N=2), DUX4 (N=2), and ZNF384 (N=1). Ph-like SVs were dominated by CRLF2 rearrangements (N=45) and included SVs involving EPOR (N=4), JAK2 (N=3), ABL1/ABL2 (N=2) and PDGFRB (N=1). IGH was the most common CRLF2 partner (N=32), followed by P2RY8::CRLF2 (N=7) and other genes (N=5). Multiple CRLF2 SVs were detected in 4 pts, and 33% (15/45) of pts with a CRLF2 rearrangement had a co-occurring CRLF2 gene mutation. The yield of ChromoSeqV2 for SVs in pts with the Ph-like expression signature via LDA card analysis was 66% (54/82; 95% CI 55%-76%) compared to 24% (11/45; 95% CI 13%-40%) by cytogenetics when these studies were successful and 70% (12/17; 95% CI 44%-90%) vs RNAseq in pts with available data. The sensitivity of ChromoSeqV2 for Ph-like SVs identified by either cytogenetics, FISH or RNAseq was 100% (21/21; 95% CI 84%-100%) and the positive predictive value of Ph-like SVs for the Ph-like expression signature by the LDA card assay was 96% (54/56; 95% CI 88%-99%). Conclusions Genomic assessment of 136 B-ALL pts from the Alliance A041501 trial using the ChromoSeqV2 rapid clinical WGS assay was 100% sensitive for Ph-like SVs identified by other molecular methods and had a 96% PPV for the Ph-like expression signature via LDA card analysis. The diagnostic yield of SV pts with the Ph-like signature was 66% using ChromoSeqV2, which was similar to RNAseq and superior to cytogenetics. These results demonstrate that ChromoSeqV2 is an effective approach for identifying potentially targetable rearrangements in B-ALL pts suspected of having the Ph-like phenotype. Support: U10CA180821, U10CA180882; . Servier; Pfizer.

Abstract Venetoclax (Ven) in combination with hypomethylating agents (HMA) is widely used as frontline treatment in older or unfit patients with acute myeloid leukemia (AML). Liposomal cytarabine and daunorubicin (CPX-351) is FDA-approved for the treatment of secondary AML, including therapy-related (t-AML) and AML with myelodysplasia-related changes (AML-MR). The current study aimed to provide a comprehensive comparison between the two treatment strategies to better inform clinical decisions. Methods The current retrospective analysis was performed under an institutional review board approved minimal risk protocol at Mayo Clinic (MN, AZ, FL). AML-MR gene mutations or cytogenetic abnormalities were categorized according to the International Consensus Classification (Arber et al. Blood 2022;140:1200). Treatment response was assessed according to the 2022 European LeukemiaNet (ELN) criteria (Dohner et al. Blood 2022;140:1345). Overall survival (OS) analysis was censored for allogeneic stem cell transplant (ASCT). Statistical analyses were performed using JMP Pro (Version 18.0.0). Results A total of 600 patients with newly diagnosed AML (ND-AML) treated with CPX-351 (N-112, 19%), or Ven-HMA (N=488, 81%) were included. AML subtypes included de novo (N=277, 46%), post-myelodysplastic syndrome (post-MDS, N=114,19%), post-myeloproliferative neoplasm (post-MPN, N=70, 12%), post-MDS/MPN (N=36, 6%), t-AML (N=103, 17%) and AML-MR (N=274, 46%). Compared with those treated with Ven-HMA, patients receiving CPX-351 were younger (median age 65 vs 73 years; p<0.01), more likely to be female (50% vs 38%; p=0.02), more frequently diagnosed withsecondaryAML (68% vs 51%; p<0.01), and less likely to have NPM1MUT (5% vs 12%; p=0.02) whereas ELN 2022 cytogenetic risk distribution was similar between the two (adverse risk; 43% vs 40%; p=0.5). Comparison of treatment response and survival Complete response rates with (CR) or without (CRi) count recovery were similar between CPX-351 and Ven-HMA overall (55% vs 60%) as well as specifically in patients with AML-MR (60% vs 63%), adverse karyotype (44% vs 44%), NPM1MUT (100% vs 89%), TP53MUT(32% vs 45%), IDH1MUT (60% vs 76%), IDH2MUT (63% vs 77%), SF3B1MUT(71% vs 45%), and CBLMUT (33% vs 69%) [p>0.05 in all instances]. Compared to those with CPX-351, CR/CRi rates with Ven-HMA were superior in males (60% vs. 45%; p=0.04) and in patients with de novo AML (68% vs. 50%; p=0.03), STAG2MUT (86% vs. 44%; p=0.02), or CEBPAMUT (88% vs. 50%; p=0.03); infectious complications were significantly more frequent with CPX-351 (83% vs 62%, p<0.01). At a median follow-up of 12 months (range; 1-88), 416 (69%) deaths, 179 relapses (44% in CPX-351 and 52% in Ven-HMA responders) and 149 ASCTs (53% in CPX-351 and 18% in Ven-HMA) were documented. OS rates were similar between CPX-351 and Ven-HMA, in general (median 10 vs 13 months), as well as in patients with adverse karyotype, TP53MUT, IDH1MUT, and IDH2MUT (p>0.05 in all instances). By contrast, OS was superior in Ven-HMA-treated patients with post-MDS AML (median 12 vs. 7 months; age-adjusted p=0.02) and in CPX-351-treated patients harboring SF3B1MUT(median not reached vs 14 months; age-adjusted p<0.01). In addition, a borderline-significant OS advantage for CPX-351 was apparent in patients with PTPN11MUT (13.5 vs 8 months; p=0.08). Comparison in ICC defined AML-myelodysplasia-related CR/CRi and OS rates were comparable between patients treated with CPX-351 (N=57) or Ven-HMA (N=217), with response rates of 60% vs 63% (p=0.68) and median OS of 10 vs 14 months (p=0.61), respectively. However, OS was superior with Ven-HMA in patients with post-MDS AML (15 vs 8 months; age-adjusted p=0.02) and with CPX-351 in those with SF3B1MUT (NR vs 14 months; age-adjusted p=0.02). Conclusions: The current study suggests that Ven-HMA was as effective and possibly less toxic than CPX-351 for the treatment of ND-AML, including AML-MR, despite the bias for younger and fitter patients treated with the latter regimen. Observed differences involving patients with SF3B1MUT or post-MDS AML require validation in prospective studies and provide parameters for developing treatment-specific predictive models that will be presented at the meeting.

Abstract Background: The primary goal of induction therapy in acute myeloid leukemia (AML) is to induce complete remission with (CR) or without (CRi) hematologic recovery. In order to accomplish this, re-induction might be necessary based on D14-residual AML. The current study examines the impact of re-induction on relapse, transplant-censored survival, and post-transplant survival in a consecutive series of AML patients who have achieved CR/CRi following intensive chemotherapy. The study also examined the impact of variations in consolidation chemotherapy including the use of anthracycline-based chemotherapy (AC) in the first consolidation cycle and the use of high (HiDAC) vs. intermediate (IDAC) dose cytarabine. Methods: The current study was conducted under IRB-approved minimum risk protocols that allowed retrospective collection and analysis of patient data. Formal criteria were used for AML diagnosis, genetic risk stratification, and adjudication of response (Blood 2022;140:1345). Patients who received less intensive induction regimens or failed to achieve CR/CRi were excluded. Relapse-free (RFS) and overall survival (OS) analysis was calculated from the date of documented CR/CRi. Patients who underwent allogeneic stem cell transplant (ASCT) were censored at the time of transplant. Conventional statistical methods were utilized (JMP Pro 17.1.0, SAS Institute, Cary, NC, USA). Results: A total of 279 AML patients diagnosed between 2004 and 2020 were included: median age 58 years, males 60%, de novo 75%, post-chronic myeloid neoplasm (post-CMN) 16%, and therapy-related 9%. Induction regimens included idarubicin (Ida) + cytarabine (Cyt) in 183 (66%) patients, daunorubicin (Da) 60 mg/m² + Cyt in 76 (27%), and Da 90 mg/m2 + Cyt in 19 (7%). Re-induction was utilized to achieve CR/CRi in 72 (27%) at a median of 18 days from start of first induction regimen. The indication for re-induction was based on residual bone marrow blast (median 30%; range 4-90) on day14 (N=53) or day 21-28 (N=15), persistent extramedullary disease (N=2) or the presence of circulating blast (N=2). At a median transplant censored follow-up of 26 months (mos), 93 (33%) deaths, 117 (42%) relapses, and 134 (48%) ASCTs were documented; ASCTs were performed in CR1 (50%) or CR2 (50%). Median RFS was 18 mos, with respective 1-, 2-, 3- year RFS of 63%, 42% and 35%. In multivariable analysis (MVA), age ≥65 years (HR 1.5, p=0.04), European LeukemiaNet (ELN) adverse karyotype (vs. intermediate HR 1.7, p=0.02; vs. favorable HR 2.8, p<0.01), and the need for re-induction to achieve CR/CRi (HR 1.7, p=0.01) were independently predictive of relapse. RFS was not impacted by NPM1 mutation ( HR 0.5, p=0.09) or FLT3-ITD (HR 1.3, p=0.3). The median OS was 80 mos, with 5-year OS of 53%. In MVA, age ≥65 years (HR 2, p=0.01), ELN adverse karyotype (vs. intermediate HR 3.3, p<0.01; vs. favorable HR 4.7, p<0.01), FLT3-ITD (HR 1.9, p=0.04), and the need for re-induction (HR 1.9, p=0.03) independently predicted inferior OS. RFS was similar among patients who received AC (N=84; Idarubicin 9 mg/m2 or Da 60 mg/m2 days 1 to 2/3 + 100 mg/m2 Cyt days 1 to 5/7; median 18 mos) as their first consolidation cycle vs. those who received HiDAC (N=99; 3 g/m2 q12 hours on days 1,3 and 5; median 16 mos) or IDAC (N=58; IDAC; 1-2 g/m2 q12 hours on days 1, 3 and 5; median 24 mos; p=0.9); the corresponding median OS were 125, 85, and 40 mos (p=0.4). In patients aged ≥65 years, but not younger patients, both RFS (p<0.01; median 23 (N=36) vs. 8 (N=24) mos) and OS (median 40 vs. 12 mos, p<0.01) were significantly longer with IDAC vs. AC as their first consolidation cycle. In the AC consolidation cohort, patients who received 2-3 additional HiDAC/IDAC cycles displayed longer OS (p<0.01) and RFS (p=0.047), compared to those who received less. In both the HiDAC and IDAC cohorts, RFS and OS were not significantly different between those who received 3-4 versus 1-2 consolidation cycles. The need for re-induction did not affect post-transplant survival (p=0.5), which was superior in patients receiving Ida + Cyt induction (vs. Da 60mg/m2 + Cyt, HR 0.6, p=0.04) and in those transplanted in CR1 (HR 0.5, p<0.01). Conclusions: The current study signals the need for re-induction to achieve CR1 as a negative predictor of outcome in AML patients achieving CR/CRi following intensive induction chemotherapy. The particular information is potentially actionable in terms of the choice of consolidation strategy.

Abstract Introduction: Isocitrate dehydrogenase (IDH) mutations are observed in ~20% of patients with acute myeloid leukemia (AML). In the U.S., ivosidenib (ivo) is approved with or without azacitidine (aza) in the frontline (FL) and as monotherapy in relapsed/refractory (R/R) IDH1-mutated (mut) AML. Olutasidenib (oluta) is approved as monotherapy for R/R IDH1-mut AML. Enasidenib (ena) is approved as monotherapy for R/R IDH2-mut AML. This study describes real-world IDH inhibitor (IDHi) administration patterns and evaluates their outcomes in on-label settings. Methods: A retrospective review of patients with IDH1/2-mut AML who received ivosidenib, olutasidenib, or enasidenib was conducted at 12 academic institutions in the U.S. Best response at any time during treatment, including complete response (CR) and complete response with partial hematologic recovery (CRh), were graded using European Leukemia Net (ELN) 2022 criteria. Results: A total of 387 patients with IDH1- and IDH2-mut AML were treated with an IDHi in the FL or R/R setting. Median follow-up was 9.6 months (range, 0.03-90.7). Demographics include 79% White, 14% Black, 3% Asian, and 4% other/unknown race; 5% were Hispanic ethnicity; 46% were female; the median age was 70 years (range, 28-95). 70% had an Eastern Cooperative Oncology Group (ECOG) performance status of 0-1, 26% had secondary AML, and 10% had therapy-related AML. Per the ELN 2024 less-Intensive risk classification, 58% were favorable risk, 29% intermediate, 7% adverse, and 6% unknown. Overall, 209 of 387 patients (54%) received IDHi regimens on-label. In FL IDH1-mut AML (n=45), 11 received ivo monotherapy, 25 aza/ivo, and 9 used off-label combinations: 3 hypomethylating agent (HMA) + venetoclax (ven) + ivo, 5 decitabine/ivo, 1 intensive chemotherapy (IC)/ivo. Among R/R IDH1-mut AML (n=109), 47 received ivo monotherapy, 7 oluta monotherapy, and 55 off-label (31 HMA/ivo, 16 HMA/ven/ivo, 3 IC/ivo, and 5 other). 44 received off-label ena for FL treatment of IDH2-mut AML (25 ena monotherapy, 9 HMA/ena, 8 HMA/ven/ena, and 2 IC/ena). In R/R IDH2-mut AML (n=189), 119 patients received ena monotherapy and 70 received off-label combinations (25 HMA/ena, 30 HMA/ven/ena, 7 IC/ena, 6 ena/gilteritinib, and 2 other). In on-label treatment of FL IDH1-mut AML, one of four patients with response assessment of FL ivo monotherapy had a CR/CRh. Of 19 patients with response assessment on FL aza/ivo, 13 patients (68.4%) had a CR as best response (95% CI, 46.0-84.6%). Median OS was 14.9 mo (95% CI, 4.17-NR) for ivo monotherapy (n=11) and 31.9 mo (95% CI, 9.1-NR) for aza/ivo (n=25), with 0 and 2 patients receiving allogeneic stem cell transplant (allo-SCT), respectively. In R/R IDH1-mut AML, the rate of CR/CRh was 35.5% (95% CI, 21.1-53.1) for on-label ivo monotherapy. All 4 patients with response assessment for on-label oluta monotherapy had a CR. Median OS was 14.0 mo (95% CI, 10.1-22.4) for ivo monotherapy and NR for oluta monotherapy (95% CI, 10.1-NR), with 10 and 0 receiving allo-SCT, respectively. The CR/CRh rate for ivo was 38.1% (8 of 21 assessed; 95% CI, 20.8-59.1) in age < 70, and 30.0% (3 of 10 assessed; 95% CI, 10.8-60.3) in age ≥ 70. Median OS was 15.2 mo (95% CI 9.3-NR) in age < 70 (n=27) and 10.4 mo (95% CI 8.0-18) in age ≥ 70 (n=20). In R/R IDH2-mut AML, the rate of CR/CRh for on-label ena monotherapy was 38.2% (95% CI, 27.6-50.1). Median OS was 12.1 mo (95% CI, 8.2-16.6), with 5 having received allo-SCT. In demographic subgroups for ena monotherapy, 52 non-Hispanic White (NHW) patients with response assessment had a CR/CRh rate of 42.3% (95% CI, 0.30-0.56). Only 1 of 13 non-Hispanic Black (NHB) patients with response assessment had a CRh (7.7%; 95% CI, 1.4-33.0) as best response, with none attaining CR. Median OS was 13.7 mo (95% CI 10.3-26.3) in NHW (n=82), 11.2 mo (95% CI, 6.1-NR) in NHB (n=18), and 69.1 mo (95% CI, 52.6-NR) in Hispanic patients (n=8). The CR/CRh rate was 48.6% (17 of 35 assessed; 95% CI, 33.0-64.4) for age < 70, and 27.3% (9 of 33 assessed; 95% CI, 15.1-44.2) for age ≥ 70. Median OS was 12.0 mo (95% CI, 9.9-22.1) in age < 70 (n=48) and 5.8 mo (95% CI, 4.1-8.7) in age ≥ 70 (n=69). Conclusion: Practice patterns for IDH inhibitors in AML vary considerably, with high amounts of off-label use. Response rates for ivosidenib and enasidenib in the real-world setting are comparable to those of clinical trials. Descriptive analyses of demographic subgroups highlight areas in need of further research.

Abstract Background Momelotinib, a small-molecule Janus kinase 1 and 2 (JAK1/2) inhibitor (JAKi), is FDA-approved (September 15, 2023) for use in anemic patients with myelofibrosis (MF). The drug appears to have an edge over other FDA-approved JAKis in regard to its unique erythropoietic effect that has been attributed to its inhibitory activity on activin A receptor type-1 (ACVR1). In clinical trials, anemia response to momelotinib in JAKi-naïve MF patients has been reported in more than a third of patients with or without transfusion-dependent anemia (TDA). The current study examines the drug's efficacy and toxicity in JAKi-naïve patients treated outside of clinical trials and after the drug was commercially approved. Methods The current study was conducted under an institutional review board approved minimum risk protocol. Diagnostic criteria were according to the International Consensus Classification (Arber et al. Blood 2022; 140:1200). Study inclusion criteria included a hemoglobin level of <10 g/dL and absence of previous treatment with JAKi. Responses were adjudicated by the Revised International Working Group-European LeukemiaNet criteria (IWG-ELN; Blood 2024; 144:1813). Conventional statistical methods were used (JMP Pro 18.0.0 software SAS Institute, Cary, NC, USA). Results A Mayo Clinic enterprise-wide search identified 60 patients (median age 73 years; 55% males) with primary or secondary MF who received momelotinib outside of clinical trials as their first JAKi for treatment of anemia and associated symptoms. Driver mutation distribution included JAK2 42%, CALR 30%, MPL 18%, and triple-negative 10%. Table 1 outlines clinical and laboratory findings at the time of momelotinib treatment initiation; Dynamic International Prognostic Scoring System (DIPSS)-plus risk distribution included high (72%) and intermediate 1/2 (28%) risk. Twenty-seven (45%) patients were transfusion-requiring, 24 (40%) displayed peripheral blood (PB) blast ≥2%, 10 (17%) platelet count <100 x 109/L, and 14 (25%) marked palpable splenomegaly (≥20cm). Unfavorable karyotype was seen in 9 (16%) of 58 patients. NGS-derived mutations were assessed in 57 cases and included ASXL1 (35%), TET2 (19%), SF3B1 (16%), SRSF2 (14%) and 7% (N=4) each for EZH2, IDH2, SH2B3, and ZRSR2. Baseline plasma cytokine levels were assessed in 17 patients with elevated TNF-a in 88%, IL-2 soluble 65%, IL-6 29% and IL-18 53%. Anemia response and predictors Fourteen (23%) patients met revised IWG-ELN criteria for anemia response (TDA 15% and non-TDA 30%; p=0.15). The anemia response was within 3 months in 10 (71%) patients. Median (range) durations of momelotinib exposure and anemia response were 8 (1-23) and 5 (1-13) months, respectively. The drug was discontinued in 29 (48%) patients (39% due to side effects; Table 2). In age-adjusted univariate analysis, predictors of anemia response included circulating blasts <2% (33% [12/36] vs. 8% [2/24]; p=0.02), presence of SH2B3 mutation (100% [4/4] vs. 19% [10/53]; p<0.01), and elevated IL-2 soluble (36% [4/11] vs. 0% [0/6]; p=0.02). The correlation with SH2B3 mutation remained significant (p<0.01) after accounting for circulating blasts. Treatment emergent neuropathy and nephropathy Sensory peripheral neuropathy (PN) was reported by 12 (20%) patients with a median onset of 2 months (range 1-7); CTCAE v5.0 grades 1 (N=8), 2 (N=3), and 3 (N=1); reversibility was documented only for grade 1 severity and did not appear to be consistently affected by drug discontinuation or dose reduction (Table 3). Seventeen (29%) patients experienced a >20% treatment-emergent decrease in estimated glomerular filtration rate from baseline, including 14 (23%) whose serum creatinine level crossed from the normal to abnormal range with pre- and post-treatment median (range) values of 1.05 (0.60-1.26) and 1.53 (1.1-3.31), respectively, with the majority reverting back to the normal range upon drug discontinuation. Conclusion In routine practice, momelotinib therapy might alleviate anemia in about a quarter of JAKi-naive patients with MF but is associated with non-trivial rates of treatment-emergent peripheral neuropathy and nephropathy. The latter appeared to be reversible on treatment discontinuation. Our novel observation regarding association of anemia response with SH2B3 mutation requires validation in a larger group of informative cases.

Abstract Background A previous multicenter study of patients with TP53-mutated myeloproliferative neoplasms showed a significantly shorter survival in chronic phase myelofibrosis (MF-CP) that included primary (PMF) and secondary MF and was mostly based on research-based mutation screening and was not systematically controlled for patients without TP53 mutations (AJH 2025;100:552). The objective of the current study was to examine the prognostic impact of TP53 mutations (VAF ≥2%) that were discovered during clinical NGS testing among consecutive patients with PMF-CP. Impact on overall (OS) and leukemia-free (LFS) survival was analyzed in the context of other genetic risk factors and calculated from the time of mutation detection. Methods The current retrospective study includes consecutive patients with PMF-CP who underwent clinical NGS testing at the Mayo Clinic between 2015 and 2025. Diagnoses of PMF-CP and leukemic transformation (LT) were retrospectively fitted to be in accordance with the International Consensus Classification criteria (Blood 2022;140:1200). Mutation and cytogenetic analyses were performed according to institutional protocols for clinical use and reported in conformity with international guidelines. OS and LFS analyses were censored for treatment with allogeneic stem cell transplantation (ASCT) and calculated from the date of NGS. Conventional statistical methods were utilized (JMP Pro 17.1.0, SAS Institute, Cary, NC, USA). Results A total of 633 patients with PMF-CP underwent clinical NGS testing prior to LT and prior to ASCT (median age 65 years; males 59%). Median interval from initial diagnosis/referral to NGS testing was 2 months; 357 (56%), 392 (62%), and 429 (68%) patients had NGS performed within 3, 6 and 12 months of diagnosis, respectively. Risk distribution according to the dynamic International Prognostic Scoring System (DIPSS; Blood 2010;115:1703) were low (19%), intermediate-1 (44%), intermediate-2 (31%), or high (6%). TP53 mutations (TP53MUT) with variant allele frequency (VAF) of ≥2% were detected in 28 (4.4%) patients with a median VAF of 25.3% (range 2.4-85; ICC-defined multihit 43%). Patients with TP53MUT were more likely to harbor IDH2MUT (14% vs. 3%; p<0.01), EZH2MUT (14% vs. 4%; p=0.03), and unfavorable karyotype (36% vs. 19%; p=0.047) and less likely to harbor CALR type 1/like (4% vs. 18%; p=0.02) and SF3B1MUT (0% vs. 6%; p=0.07). The association with unfavorable karyotype was mainly driven by multihit configuration. No significant associations with clinical features were apparent. At a median follow-up of 40 months for surviving patients, 157 (25%) deaths, 48 (8%) LTs and 135 (21%) ASCTs were documented. A near-two-fold lower, albeit not statistically significant OS was observed in the presence of TP53MUT (median 123 vs. 228 months; HR 1.9, 95% CI 0.94-3.7; p=0.074); the difference reached significance for multihit TP53MUT (median 81 months; HR 3.6, 1.7-7.7; p<0.01) but not for non-multihit TP53MUT (median not reached; HR 0.7, 0.2-2.8; p=0.6), both compared to TP53WT (median 228 months). The significant impact of multihit TP53MUT on OS was confirmed in multivariable analysis (MVA) that included other high molecular risk (HMR) mutations and unfavorable karyotype: multihit TP53MUT (HR 2.5, 1.1-5.5; p=0.02), SRSF2MUT (2.4, 1.5-3.8; p<0.01), ASXL1MUT (1.8, 1.2-2.6; p<0.01), U2AF1MUT-Q157 (4.8, 2.6-8.6; p<0.01), and absence of CALR type 1/like (4.0, 2.0-7.8; p<0.01); additional prognostic contribution was not evident for unfavorable karyotype (p=0.17), EZH2MUT (p=0.3), IDH1MUT (p=0.6), IDH2MUT (p=0.7), or any other HMR mutation. Multihit TP53MUT was also associated with inferior LFS (10-year risk 62% vs. 32% for non-multihit TP53MUT vs. 15% for TP53WT; p=0.03) but significance was lost during MVA that included SRSF2MUT, which was the only HMR mutation showing a significant effect on LFS (HR 5.0, 2.5-9.8; p<0.01). Post-ASCT survival among 135 transplanted patients was significantly shorter for patients with multihit TP53MUT (N=4; median 5 months) vs. those with non-multihit TP53MUT (N=7; median not reached) vs. patients with TP53WT (N=124; median not reached) [p=0.02]. Conclusion The current study implicates multihit TP53MUT as an HMR mutation-independent prognostic factor for OS in PMF-CP. By contrast, prognosis in PMF-CP patients with non-multihit TP53MUT was more likely to be influenced by other HMR mutations, as was the case for those with TP53WT.

Abstract Background In our first exploration of mutations in essential thrombocythemia (ET; N=183), we described somatic variants other than JAK2/CALR/MPL in 53% (N=96) of patients with the most frequent being TET2 (16%), ASXL1 (11%), DNMT3A (6%), and SF3B1 (5%) [Blood Adv 2016;1:21]. In a subsequent report (N=270; BJH 2020;189:291), we highlighted the prognostic relevance of SF3B1, SRSF2, and EZH2 mutations for overall survival (OS), SRSF2, EZH2, and TP53 for leukemia-free survival (LFS), and SF3B1 and U2AF1 for myelofibrosis-free survival (MFFS). We have now expanded our NGS-informative patient cohort to 500 and examined the distribution and frequencies of specific mutations at different stages of the disease and corresponding prognostic correlates. Methods Molecularly-annotated ET patients diagnosed at the Mayo Clinic between January 1980 and May 2025 were considered. Diagnoses of chronic phase ET (ET-CP), post-ET MF, and post-ET acute myeloid leukemia (AML) were retrospectively fitted to be in accordance with the International Consensus Classification criteria (Blood 2022;140:1200). Mutation and cytogenetic analyses were performed according to institutional protocols for clinical use that included multi-gene next-generation sequencing (NGS); in a subset of patients, historically archived samples were accessed for analysis in a research setting. Patients were monitored until death or time of last follow-up, determined through electronic medical records or direct communication with patients or healthcare providers. Conventional statistical methods were utilized (JMP Pro 17.1.0, SAS Institute, Cary, NC, USA). Results A total of 500 ET patients (median 57 years; Females 61%) were informative for NGS-derived mutations that were obtained in the chronic (N=401; 80.2%; group A), post-ET MF (N=88; 17.6%; group B), or post-ET AML (N=11; 2.2%; group C) phase of the disease; group C included 9 (1.8%) patients who transformed directly to AML without transitioning through the MF phase. The most frequent mutations with at least 1% (N=5) mutational frequency were (approximately) TET2 (10%), ASXL1 (10%), DNMT3A (7%), SF3B1 (4%), TP53 (5%), SRSF2 (3%), EZH2 (2%), U2AF1 (2%), CBL (1%), IDH1 (1%), IDH2 (1%), RUNX1 (1%), ZRSR2 (1%), and PPM1D (1%). Comparison of distribution of mutations and their frequencies based on timing of NGS (AML phase vs. MF phase vs. ET-CP) revealed significant differences for TP53MUT (72.7% vs. 11.4% vs. 1%; p<0.01), ASXL1MUT (18.2% vs. 25% vs. 7.2%; p<0.01), and EZH2MUT (18.2% vs. 6.8% vs. 0.3%; p<0.01), respectively; borderline significance was also apparent for RUNX1MUT (9.1% vs. 0% vs. 1.3%; p=0.10) and ZRSR2MUT (9.1% vs. 2.3% vs. 0.5%; p=0.08). Notably, among 9 patients in whom NGS was performed at the time of direct transition from ET to AML, without an intermediary MF phase, 8 (88.9%) displayed TP53MUT and only one (11.1%) EZH2MUT and none (0%) ASXL1MUT. At a median follow-up of 66 months, 93 (18.6%) deaths, 26 (5.2%) leukemic transformations (LT), and 108 (21.6%) MF transformations were reported. In group A patients, age-adjusted multivariable analysis (MVA) of mutations identified ASXL1MUT (N=29; HR 3.8, 1.9-7.7; p<0.01) and SF3B1MUT (N=12; HR 3.9, 1.4-11.0; p=0.01) to be associated with inferior OS, ASXL1MUT (HR 4.9, 1.01-24.2; p=0.048) and SRSF2MUT (N=11; HR 11.9, 1.3-104.8; p=0.03) with inferior LFS, and ASXL1MUT (HR 9.0, 2.8-29.5; p<0.01) and SF3B1MUT (HR 15.5, 3.1-77.5; p<0.01) with inferior MFFS, all calculated from time of NGS. A similar analysis in group B patients identified ASXL1MUT (N=22; HR 3.7, 1.2-11.7; p=0.03) to be associated with inferior OS and TP53MUT (N=10; HR 13.4, 1.7-108.6; p=0.02) with inferior LFS. Age-adjusted MVA combining groups A and B (N=489) confirmed the association between inferior OS and ASXL1MUT (N=51; p<0.01) and SF3B1MUT (N=18; p<0.01) while inferior LFS was associated with TP53MUT (N=14; p<0.01) and ASXL1MUT (p=0.03). Conclusions The majority (⁓73%) of patients with post-ET AML harbor TP53MUT, at the time of LT. By contrast, TP53MUT are infrequent in ET-CP (⁓1%) and did not appear to predict LT, as was the case with post-ET MF. ASXL1MUT was shown to be an independent risk factor for OS, MFFS, and LFS in ET-CP and for OS in post-ET MF. SF3B1MUT was associated with inferior OS and MFFS in ET-CP and SRSF2 with inferior LFS in ET-CP. Identifying ET patients with adverse mutations allows for closer surveillance and potentially more tailored therapeutic interventions.

Abstract Background: Polycythemia vera (PV) is a myeloproliferative neoplasm characterized by red blood cell overproduction. In the phase 2 REVIVE study (NCT04057040), rusfertide was superior to placebo in achieving hematocrit (Hct) levels <45% and reducing or eliminating the need for therapeutic phlebotomy (PHL) in patients (pts) with PV who were PHL dependent prior to study entry (Kremyanskaya M, et al. N Engl J Med. 2024;390(8):723-35). During the open-label extension portion of REVIVE (Part 3), combining rusfertide and PHL with or without cytoreductive therapy (CRT) controlled erythrocytosis, provided long-term control of Hct, and was associated with a greater than 8-fold reduction in the estimated annual PHL rate (Gerds AT, et al. Blood. 2024;144(Suppl. 1):4559). Pts who participated in REVIVE Part 3 were eligible to transition to the open-label phase 2 THRIVE extension study (NCT06033586) and receive up to 2 additional years of rusfertide treatment. Our objective is to present initial results describing the long-term durability of response and safety profile of rusfertide in pts with PV who participated in REVIVE and transitioned to THRIVE. Methods Pts with PV who participated in REVIVE, completed ≥12 months of rusfertide therapy, and had an end-of-treatment visit were eligible to transition to THRIVE. Pts who enrolled in THRIVE continued to receive subcutaneous rusfertide once weekly with or without CRT. Median Hct and the number of PHLs each pt received were assessed in all pts who received ≥1 dose of rusfertide and had ≥1 efficacy assessment on study. Safety data were also collected, including treatment-emergent adverse events (AEs), serious adverse events (SAEs), non-PV malignancies, and thromboembolic events (TEs). New-onset AEs (ie, AEs that were reported in THRIVE but not REVIVE) were also recorded. Results: Forty-six/58 pts (79%) who enrolled in Part 3 of REVIVE transitioned to THRIVE. At the data cutoff date (27 May 2025), 44 of the 46 pts (96%) who enrolled in THRIVE remained on study. Median (range) age was 58 (27-77), and the majority were male (74%). More than half (57%) had high-risk PV; 54% of pts were on concurrent CRT. Median (range) treatment duration in THRIVE was 48 (22-66) weeks; median (range) treatment duration in REVIVE and THRIVE combined was 198 (154-267) weeks, or 3.8 (3.0-5.1) years. At baseline, mean (standard deviation [SD]) Hct was 42.3% (3.7) in THRIVE. At Week 60, mean (SD) Hct was 43.0% (4.2). In addition, mean hemoglobin and erythrocyte levels remained stable throughout THRIVE. Prior to study entry in REVIVE, the mean (SD) annualized PHL rate (ie, PHL/year) for the 46 pts who eventually rolled over to THRIVE was 9.2 (5.2) PHL/year. The mean (SD) annualized PHL rate during REVIVE Part 3 and THRIVE was 0.6 (0.8) and 0.7 (1.1) PHL/year, respectively. Overall, the most common AEs in the pts who participated in REVIVE and THRIVE were Grade 1-2 injection site reactions (87%), fatigue (59%), COVID-19 (50%), pruritus (44%), arthralgia (39%), dizziness (35%), anemia (30%), paresthesia (28%), headache (26%), abdominal pain and nausea (24% each), and diarrhea, dyspnea, and upper respiratory tract infection (22% each). In THRIVE, the most common new-onset AEs were anemia (20%) and fatigue (15%). Twelve pts (26%) had a prior history of non-PV cancer. Six pts (13%) had a new-onset cancer event, including basal cell carcinoma (n=3; 7%), squamous cell carcinoma (n=2, 4%), non-invasive bladder cancer (n=1, 2%), and multiple myeloma (n=1; 2%). Of these 6 pts, 5 (83%) had a prior history of non-PV cancer. Nine pts (20%) had a history of TEs prior to enrolling in REVIVE. One patient with cardiovascular risk factors and a TE prior to enrollment in REVIVE had a new-onset TE of cerebrovascular accident in THRIVE. Overall, there were no TEs in pts with low-risk PV in either REVIVE or THRIVE. Conclusions: After transitioning to THRIVE, continued treatment with rusfertide with or without CRT demonstrated consistent, long-term Hct control with reduced therapeutic PHLs compared to baseline. With a median of 3.8 years of treatment, rusfertide's safety profile was consistent with prior observations. Support Protagonist Therapeutics, Inc.

Abstract Introduction Persistent symptom burden in classic myeloproliferative neoplasms (MPNs) i.e., polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) is linked to shorter survival, more thromboembolic events and impaired quality of life. Identifying demographic, clinical, laboratory, and treatment-related predictors of symptom improvement can inform risk-adapted care and guide supportive interventions. Methods Patients with PV, ET, and MF were identified through a retrospective chart review. Total symptom scores (TSS) and scores of individual symptoms were recorded at the time of first TSS assessment. Demographic (age, sex, race, ethnicity), seasonal (summer: April–September; winter: October–March), clinical (time since diagnosis, depression, obesity, spleen size), treatment-related (phlebotomy, hydroxyurea, interferon-alpha, anagrelide, JAK inhibitors, antidepressants) and laboratory (complete blood counts, serum chemistry) factors were captured at the baseline. Symptom improvement was defined as a ≥50% TSS reduction from baseline. Cumulative incidence analyses, accounting for death as a competing risk, were performed to estimate the probability and timing of symptom improvement. Multicollinearity was assessed using variance inflation factors (VIFs). Highly correlated (VIF ≥5) features and features with ≥15% missing observations were excluded. Multivariable Fine-Gray hazards regression was performed to identify predictors of symptom improvement. Sub-distribution hazard ratios (sHR) with 95% confidence intervals (CI) were computed with p-values <0.05, indicating a statistically significant association with symptom improvement. Results A total of 532 patients (PV: 195, ET: 180, MF: 157) were identified between 2019 and 2024. The median age was 65 (IQR: 57-74) for PV, 65 (52-75) for ET, and 69 (63-75) for MF. Most patients were White (PV: 178, ET: 164, MF: 145) and female (PV: 99, ET: 134, MF: 84). Fatigue was the most frequently reported symptom (PV: 161, ET: 155, MF: 136) whereas weight loss was the least reported (PV: 9, ET: 12, MF: 26). Baseline TSS was highest in MF (median: 15; IQR: 7-27) followed by PV (13; 6-25) and ET (13; 5-23). Symptom improvement was observed in 89 (46%) PV, 71 (39%) ET, and 44 (28%) MF patients. Among those who improved, the cumulative incidence of symptom improvement reached 100% by 55 months in PV, 53 months in ET, and 58 months in MF. At 6, 12, and 24 months, cumulative symptom improvement was 22%, 34%, and 57% in PV; 15%, 25%, and 43% in ET; and 19%, 28%, and 39% in MF. Median time to improvement was not reached in any group. Death before symptom improvement was observed in 5 (3%) PV, 6 (3%) ET, and 37 (24%) MF patients. Multivariable regression analyses in PV identified higher baseline TSS (sHR: 1.02; 95%CI: 1.01-1.03) as associated with symptom improvement (p = 0.02). In ET, lower white blood cell count (sHR: 0.89; 95%CI: 0.81-0.98) and less time since diagnosis (0.99; 0.98-0.99) were associated with symptom improvement (p = 0.02). In MF, more months since diagnosis (sHR: 1.01 95%CI: 1.01-1.01) was associated with symptom improvement (p = 0.01). Conclusions Symptom trajectories differed across MPN subtypes. In PV, a higher baseline symptom burden; in ET, a lower white blood cell count and more recent diagnosis and in MF, a longer disease duration were associated with symptom improvement. These readily available parameters may help inform supportive strategies, tailor response expectations, and identify patients for symptom-directed interventions or clinical trials.

Abstract Background/rationale Primary myelofibrosis (PMF) and post-polycythemia vera/essential thrombocythemia (Post-PV/ET MF) (collectively termed MF) are chronic hematopoietic malignancies with progressive bone marrow collagen fibrosis, peripheral cytopenias, extramedullary hematopoiesis, splenomegaly, debilitating constitutional symptoms and heightened risk of transformation to acute leukemia. The only currently approved therapies for MF are JAK inhibitors (JAKi) which can provide significant improvements in splenomegaly and systemic symptoms but do not fundamentally change the pathogenesis of the disease. As a result, additional non-JAKi therapies for patients with MF after JAKi failure represent an important translational focus. Reparixin is a novel, potent and specific inhibitor of the chemokine CXCL8 (IL)-8. IL-8 is a member of a class of cytokines involved in leukocyte recruitment and activation in tissues. There are two known types of CXCL8 receptors – CXCR1 and CXCR2, which are expressed in neutrophils (Baggiolini M, Adv Immunol 1994). IL-8/CXCR1/2 signaling has been shown to be a key mediator of inflammatory signaling in MF patients. Hematopoietic stem/progenitor cells from patients with MF are enriched for a CXCL8/CXCR2 gene signature and display enhanced proliferation and fitness in response to an exogenous CXCL8 ligand in vitro. Genetic deletion or a small molecule inhibitor of CXCR2 in MF mouse adoptive transfer models ameliorates fibrosis, extends overall survival and improves hematologic parameters (Dunbar A, Blood 2023). This pre-clinical data validates targeting this pathway in clinical investigations. Study design This is an open-label, phase II study assessing the efficacy, safety, and tolerability of reparixin in patients with Dynamic International Prognostic Scoring System (DIPSS) intermediate-2, or high-risk PMF, post ET/PV MF either after prior treatment with, or those who are ineligible for treatment with a JAKi. Patients will receive oral reparixin 1200 mg three times daily on a four-week cycle for a core study period of 24 weeks. Patients who achieve at least clinical improvement (CI) or stable disease (SD) with one grade improvement in bone marrow fibrosis by International Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) criteria at the Cycle 7 response assessment may continue receiving reparixin until loss of response, disease progression, unacceptable toxicity, patients/physician withdrawal, or termination of study by sponsor. The study monitors both efficacy and toxicity using a Bayesian Optimal Phase 2 (BOP2) design. If response is observed in at least one patient out of 10 and safety is confirmed at the interim analysis, the trial will proceed to full enrollment of 26 patients. If more than three responses are observed in the total cohort, then a signal of activity is confirmed. Primary Objective: To estimate the efficacy of reparixin treatment in DIPSS intermediate-2 or high-risk subjects with PMF, post PV-MF, or post ET-MF. Secondary Objectives: To assess the efficacy of reparixin as determined by response assessment using IWG-MRT/ELN criteria at the end of Cycle 6 and Cycle 12. To assess the change in bone marrow fibrosis grade at the end of Cycle 6 and Cycle 12 with reparixin. To assess the safety of reparixin as measured by the adverse event profile of Common Terminology Criteria for Adverse Events v5.0. To assess spleen volume reduction by imaging after Cycle 6 and Cycle 12 as compared to baseline spleen volume. Key Exploratory Objectives: To evaluate changes in quality of life and patient-reported outcomes To conduct correlative studies to measure biomarkers of reparixin activity, such as Vascular Endothelial Growth Factor (VEGF) levels, bone marrow microvessel density, megakaryocyte number and peripheral blood CD34+ cell number, and patients with and without IL-8 secretion. Key inclusion criteria: Have a pathologically confirmed diagnosis of PMF, post-ET-MF, or post-PV-MF as per the WHO diagnostic criteria with intermediate-2 or higher risk disease by DIPSS Be refractory/resistant to or intolerant of/inappropriate for JAKi therapy Have adequate liver and kidney function Platelet count ≥ 25 x 109/L Bone marrow and peripheral blood blast count < 10% ANC ≥ 1000 mm3. MPN Research Consortium (MPN-RC) 120 is currently enrolling patients through the MPN-RC clinical sites throughout the United States. Clinical Trial Identifier NCT05835466