Chervin, Jordan

Abstract Background Myeloproliferative neoplasms (MPNs) can progress from chronic phase to accelerated or blast phase (MPN-AP/BP), a transition with poor prognosis and limited treatment options. While some abnormalities have been described, the genomic and clonal drivers of transformation remain poorly defined. We characterized mutational and structural alterations in MPN-AP/BP, leveraging paired samples to trace clonal evolution. Methods We profiled 186 samples from 160 individuals, including 159 MPN-AP/BP and 27 chronic-phase MPN samples (26 MF, 1 PV), using targeted sequencing of a 588 gene panel (mean depth: 709x). Paired pre- and post-transformation samples were available for 26 patients (n= 52). A consensus pipeline was used to detect somatic mutations, copy number variations (CNVs), and copy-neutral loss of heterozygosity (cnLOH). Clinical cytogenetic data were integrated when available. Single-cell validation is ongoing. Results Genomic complexity was widespread: 99% of patients harbored ≥1 oncogenic mutation (median 4; range 1–10). In total, 859 mutations were identified, most commonly in JAK2 (68%), ASXL1 (28%), TET2 (26%), SRSF2 (24%), TP53 (22%), and RUNX1 (22%). 684 structural alterations were identified in 86% of patients (median 3; range 1-20), including arm-level (22.5%), segmental (>5 Mb; 60%), and focal (<2 Mb; 9%) alterations. Recurrent events included 9p cnLOH (16%), del(5q) (13%), del(17p) (10%), and focal deletion of 21q22.12 involving RUNX1 (10%). Complex karyotypes (≥3 abnormalities) were observed in 55% of patients. Biallelic mutations accounted for 21% (183/859) of mutations, affecting 58% of patients across 21 genes, most frequently JAK2 V617F (28%), TP53 (17%) and TET2 (14%). Patients with biallelic mutations in these genes had significantly higher blast percentages compared to those with monoallelic mutations (p<0.05), highlighting an association with aggressive disease. In 26 patients with longitudinal samples (chronic phase MPN and MPN-AP/BP), 87% (125/144) of mutations were already detectable during the chronic phase, on average more than 7 years prior to transformation. Only 11% (16/144) of mutations were newly acquired, including additional RUNX1 and TP53 hits, or structural changes that converted pre-existing JAK2 V617F and TP53 R248Q mutations from monoallelic to biallelic via 9p cnLOH or 17p loss, respectively. These findings suggest transformation is often driven by outgrowth of pre-existing high-risk clones, indicating that these clones likely have a competitive advantage and that the mutations within these clones have a cooperative biological interaction which underlies this competitive advantage. To investigate this hypothesis, we examined whether patterns of recurrent co-mutations were identifiable in this cohort. We identified recurrent co-occurring mutations in SRSF2-IDH2, ASXL1-SRSF2, and ASXL1-EZH2, which were enriched at transformation (p < 0.05; Fisher exact test). Importantly, presence of these co-mutations was consistently associated with a higher transformation risk in two independent cohorts of MF patients from Mayo Clinic (n= 405) and University of Florence (n= 518). Clonal architecture analysis revealed that these co-mutations frequently arose within the same malignant clone: 100% for SRSF2-IDH2, 80% for ASXL1-SRSF2, and 85% for ASXL1-EZH2, and are often within the dominant clone. Prior studies have demonstrated mechanistic interplay between SRSF2 and IDH2 (Yoshimi et al.), as well as ASXL1 and SRSF2 (Sui et al.) mutations in myeloid malignancies. We assessed functional synergy of ASXL1-EZH2 using a murine model and identified that dual deletion caused a rapidly fatal myeloid neoplasm with shorter survival than single-gene loss. These results reinforce that functional synergy emerges within a shared cellular context, supporting clonal co-dependence as a driver of progression. Single-cell RNA sequencing of ASXL1-EZH2 mutant cases is ongoing and will be presented. Conclusions Transformation to MPN-AP/BP is driven by gradual clonal remodeling and expansion of pre-existing high-risk abnormalities. Biallelic and multi-hit alterations in TP53, JAK2, TET2, and RUNX1 promote clonal dominance and blast progression. Recurrent co-mutations such as IDH2-SRSF2 and ASXL1-EZH2 arise within the same clone, exhibit functional synergy, and are linked to poor outcomes. These findings support the clinical utility of early clonal and co-mutational profiling to guide risk-adapted intervention.

Introduction: Myelofibrosis (MF) is a clonal hematologic malignancy characterized by constitutive JAK-STAT activation and aberrant cytokine signaling. Allogeneic hematopoietic stem cell transplantation (allo-HCT) remains the only potentially curative therapy in MF. The contribution of disease-, patient- and donor-related factors to allo-HCT outcomes remains to be fully elucidated, as well as the presence of somatic mutations in genes such as ASXL1, EZH2, SRSF2, U2AF1 and IDH1/2 which are associated with worse prognosis in MF patients (pts). In this large multi- center retrospective analysis, we aimed to study the impact of clinical and molecular mutations on transplant outcomes. Methods: This analysis including 498 pts from 11 centers in North America and Canada. Univariate proportional-hazard Cox regression analysis stratified by center was used to assess the association of patient, transplant, and disease characteristics on overall survival (OS). Baseline patient demographic data and disease characteristics were selected as covariates in the multivariate analysis. Results: Pts transplanted between 2002-2021 were included in this analysis. Baseline and transplant characteristics are summarized in table 1. The 1- and 2-year OS was 77% (95% CI; 73-81) and 70% (95% CI; 66-74) and non-relapse mortality (NRM) 19% (95% CI: 17-21) and 23% (95% CI: 19-27), respectively. The cumulative incidence (CMI) of relapse at 1- and 2-years was 12% (95% CI; 10-14) and 16% (95% CI; 12-20). The CMI of acute graft versus host disease (aGVHD) grade II-IV was 31% (95% CI; 27-35) at 3 months and 42% (95% CI; 38-46) at 1-year post-transplant and grade was III-IV 13% (95% CI; 11-15) and 18% (95% CI; 14-22), respectively. The CMI of neutrophil engraftment at 1 and 3 months was 90% (95% CI: 87-93) and 97% (95% CI: 95-95) and for platelets 55% (95% CI: 50-59) and 80% (95% CI: 76-83). Univariate analysis included pts (age and gender), transplant (donor type and conditioning regimen intensity) and disease characteristics (DIPSS, mutations, cytogenetics (CG), blood counts, and spleen size). Hemoglobin > 10 g/dL (HR .5, 95% CI .34 -.73 p=3 x 10 -4) and platelets >50× 10 9/L (HR .59, 95% CI .42-.81, p=.0013) prior to transplant were associated with improved OS. High risk DIPSS was associated with decreased OS (p=0.0002). Mutations in ASXL1, EZH2, IDH1/2, U2AF1 and SRSF2 were not associated with decreased OS in this analysis (Figure 1). However, mutations in ZRSR2 were associated with inferior OS (HR 2.07, 95% CI 1.08-3.95, p=.0252). Lastly, JAK2 which had the most frequent mutations was not associated with OS (HR .0.93, 95% CI .65-1.31). There was no association between the CG risk groups and OS. Splenomegaly, measured radiographically, had no impact on OS but was strongly associated with engraftment (better engraftment defined as HR>1). For platelets, spleen size of 15.6-18.9 cm HR of 0.69 (95% CI .47-1, p=.0489) and size >18.9-22.5 cm HR of 0.48 ( 95% CI .33-.7, p=1x10^-4). For neutrophils, spleen size >19.9-22.5 cm had a HR of 0.68, (95% CI .48-.96, p=.0268) and size>22.5 cm had a HR of 0.65 (95% CI .46-.91, p=.0114). Mismatched unrelated (MMURD) and haplo identical donors had no impact on survival but were associated with delayed platelet engraftment; MMURD (HR .67, 95% CI .47-.96, p=.0298) and haploidentical donors (HR .56, 95% CI .39-.8, p=.0017). In a multivariate analysis adjusted for age, gender, highest DIPSS, donor type and conditioning regimen, platelets > 50× 10 9/L (HR .61, 95% CI .44-.87, p=.0053) hemoglobin >10 g/dL (HR .6, 95% CI .4-.9, p=.0244) and lower DIPSS at the time of transplant were associated with improved OS. Conclusions: In this large multicenter analysis only clinical variables; hemoglobin and platelets, were found to be associated with OS after allo-HCT in MF patients. Somatic mutations associated with poor prognosis were not associated with OS suggesting that transplant can overcome their negative impact. Splenomegaly was associated with delayed engraftment but not OS. Interestingly, using alternative donors (MMURD and haplo identical) didn't have a negative effect on OS, possibly reflecting the use of post-transplant cyclophosphamide for GVHD prophylaxis in recent years. These results highlight the importance of biologic factors in the prognostication of outcomes in MF pts undergoing allo-HCT independent of somatic mutations. Supported by the American Society of Hematology (ASH) and MPN Research Foundation

Abstract Enasidenib (ENA) is an inhibitor of isocitrate dehydrogenase 2 (IDH2) approved for the treatment of patients with IDH2-mutant relapsed/refractory acute myeloid leukemia (AML). In this phase 2/1b Beat AML substudy, we applied a risk-adapted approach to assess the efficacy of ENA monotherapy for patients aged ≥60 years with newly diagnosed IDH2-mutant AML in whom genomic profiling demonstrated that mutant IDH2 was in the dominant leukemic clone. Patients for whom ENA monotherapy did not induce a complete remission (CR) or CR with incomplete blood count recovery (CRi) enrolled in a phase 1b cohort with the addition of azacitidine. The phase 2 portion assessing the overall response to ENA alone demonstrated efficacy, with a composite complete response (cCR) rate (CR/CRi) of 46% in 60 evaluable patients. Seventeen patients subsequently transitioned to phase 1b combination therapy, with a cCR rate of 41% and 1 dose-limiting toxicity. Correlative studies highlight mechanisms of clonal elimination with differentiation therapy as well as therapeutic resistance. This study demonstrates both efficacy of ENA monotherapy in the upfront setting and feasibility and applicability of a risk-adapted approach to the upfront treatment of IDH2-mutant AML. This trial is registered at www.clinicaltrials.gov as #NCT03013998.

Background: ENA is an oral, selective inhibitor of IDH2 approved for the treatment (Tx) of patients (pts) with relapsed/refractory IDH2 mutated (IDH2m) AML. Here we report the results of a Phase 2 expansion and Phase 1b of the Beat AML Master Trial Phase 2/1b sub-study to assess the efficacy of Tx of newly diagnosed (ND) IDH2m AML pts ≥ 60 years of age with ENA monotherapy (ENAm) and subsequent response-driven addition of AZA Tx. (ClinicalTrials.gov NCT03013998). Methods: The study initiated with a 3-outcome, 2-stage Phase 2 design, which enrolled patients on ENAm for up to 5 cycles. Pts without CR/CRi after 5 cycles of ENAm, or progression/intolerance prior to this time, were transferred to Phase 1b to receive ENA + AZA (Figure 1). Key eligibility included ND IDH2m AML pts with age ≥ 60 years and ECOG performance status 0-2. Pts received ENAm 100 mg/day in continuous 28-day cycles and ENA + AZA (75 mg/m2 days 1-7 every 28 days) for Phase 1b. Response was assessed using 2017 ELN AML criteria. The primary endpoint was CR/CRi rate. The 2-stage design required 24 pts and tested the null hypothesis (H0) that CR/CRi rate equaled 20% vs 50% and then expanded to test a revised H0 of 30% vs 50% in 60 pts (conditional alpha=0.025, power=77%). Expansion also allowed further assessment of safety of this treatment regimen. Results: At data cut off (06/18/2020), 60 pts enrolled, received ENAm, and were evaluable for the primary endpoint. Median age was 75 years and 52% were female (Table 1). Median time on ENAm was 4.7 months (mos). At data cut off, 12 pts were still on ENAm Tx. Most common reasons for discontinuing ENAm were Tx failure (defined as no response to treatment) (23 or 38%), disease progression (loss of response to treatment) (7 or 12%) and adverse events (AEs; 6 or 10%). Five pts (8%) went to transplant. CR/CRi was achieved in 28 pts (47%; adjusted 95% CI 28-59, unadjusted exact 95% CI 34-60) (Table 2). Responses were higher (p=0.04) among the 44 pts with IDH2 R140 (55%) as compared to the 16 with IDH2 R172 mutation (25%) further supporting distinct biology between these subsets. After a median follow up of 14.6 mos, the median overall survival (mOS) was 24.4 mos (95% CI 10.6-not reached). The median duration of response was not reached with 12 mos estimation of 57% (95% CI 34-75). Overall, 20 ENA-related serious adverse events (SAEs) occurred in 15 pts, the most common was differentiation syndrome (12 or 20%) and 1 had ENA-related SAE of tumor lysis syndrome (1.7%). One pt had ENA-related Grade 5 AE (renal failure/death). Most common AEs of any grade (in ≥20%) were nausea, anemia, and low potassium (Table 3). The 7-day/30-day/60-day deaths observed with ENAm were 2%/5%/11%, respectively. Phase 1b: Seventeen pts had inadequate response to ENAm and transferred to Phase 1b to receive ENA + AZA. Median time on Tx (including ENAm) was 6.2 mos and median time on Tx after pts started ENA + AZA was 2.1 mos (Table 2). Most common reasons for discontinuing ENA + AZA included Tx failure (5 or 29%), disease progression (2 or 12%), transplant, death and AEs (each 2 or 12%). CR/CRi was 41% (exact 95% CI 18-67). After a median follow up of 12.7 mos, the mOS from start of ENA + AZA combination Tx was 8.9 mos. Four ENA-related SAEs occurred in 3 pts on ENA + AZA Tx and the most common was differentiation syndrome (2 or 12.5%). One dose-limiting toxicity (Grade 3 nausea) related to both Txs was observed. Most common AEs (≥20%) of any grade were anemia, low albumin and vomiting (Table 3). One death occurred at day 13 of ENA + AZA. Conclusions: In newly diagnosed pts ≥60 years old with IDH2m AML, ENA had a low early death rate, high CR/CRi rate (47%, adjusted 95% CI 28-59), and yielded durable remissions. The most common unique toxicity with ENA was differentiation syndrome that occurred in 20% of patients. In pts who did not achieve CR/CRi with ENAm, a subset of patients achieved CR/CRi with addition of AZA. This combined approach of serial therapy with ENA monotherapy followed by AZA addition in pts with sub-optimal response resulted in a mOS exceeding 2 years for pts enrolled on study. Further focus on improving response among patients with IDH2 R172 mutations, identifying subsets of pts not responding to ENA monotherapy, and integrating new targeted agents into this treatment regimen are warranted. Figure 1 Disclosures Stein: Syndax: Consultancy, Research Funding; Celgene Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Biotheryx: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy. Borate:Genentech: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Research Funding; AbbVie: Other: Investigator in AbbVie-funded clinical trials; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding. Baer:Takeda: Other: Institutional research funding; AbbVie: Other: Institutional research funding; Astellas: Other: Institutional research funding; Forma: Other: Institutional research funding; Kite: Other: Institutional research funding; Oscotec: Other: Institutional research funding; Incyte: Other: Institutional research funding. Kovacsovics:Agios: Honoraria; Astella: Honoraria; Pfizer: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Jazz: Honoraria. Schiller:Astellas Pharma: Honoraria, Research Funding; Celator: Research Funding; Constellation: Research Funding; Abbvie: Research Funding; Actinium: Research Funding; Ariad: Research Funding; Stemline: Speakers Bureau; Cyclacel: Research Funding; Daiichi Sankyo: Research Funding; Deciphera: Research Funding; DeltaFly: Research Funding; Bristol-Myers Squibb: Current equity holder in publicly-traded company, Research Funding; Forma: Research Funding; FujiFilm: Research Funding; Gamida: Research Funding; Genentech-Roche: Research Funding; Geron: Research Funding; Jazz Pharmaceuticals: Research Funding; Karyopharm: Research Funding; Kite Pharma: Research Funding; Mateon: Research Funding; MedImmune: Research Funding; Onconova: Research Funding; Pfizer: Current equity holder in publicly-traded company, Research Funding; Regimmune: Research Funding; Samus: Research Funding; Sangamo: Research Funding; Tolero: Research Funding; Trovagene: Research Funding; Kaiser Permanente: Consultancy; Johnson & Johnson: Current equity holder in publicly-traded company; Agios: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Current equity holder in publicly-traded company, Research Funding, Speakers Bureau; AstraZeneca: Consultancy; Incyte: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding; Ono Pharma: Consultancy; Celgene: Research Funding, Speakers Bureau; Sanofi: Speakers Bureau; Gilead: Speakers Bureau. Olin:Astellas: Other: Site PI; Genentech: Other: Site PI; Pfizer: Other: Site PI; Daiichi Sankyo: Other: Site PI; Genentech: Consultancy; Amgen: Consultancy. Foran:Trillium: Research Funding; Xencor: Research Funding; H3Biosciences: Research Funding; Agios: Honoraria, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Boehringer Ingelheim: Research Funding; Actinium: Research Funding; Aprea: Research Funding; Aptose: Research Funding; Kura Oncology: Research Funding; Takeda: Research Funding; Revolution Medicine: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees. Lin:Trovagene: Research Funding; Tolero Pharmaceuticals: Research Funding; Seattle Genetics: Research Funding; Prescient Therapeutics: Research Funding; Abbvie: Research Funding; Bio-Path Holdings: Research Funding; Astellas Pharma: Research Funding; Aptevo: Research Funding; Incyte: Research Funding; Pfizer: Research Funding; Mateon Therapeutics: Research Funding; Ono Pharmaceutical: Research Funding; Jazz: Research Funding; Gilead Sciences: Research Funding; Genetech-Roche: Research Funding; Celyad: Research Funding; Celgene: Research Funding. Patel:DAVA Pharmaceuticals: Honoraria; Celgene: Consultancy, Speakers Bureau; Agios: Consultancy; France Foundation: Honoraria. Foster:Daiichi Sankyo: Consultancy; Bellicum Pharmaceuticals: Research Funding; Macrogenics: Consultancy, Research Funding. Druker:Leukemia & Lymphoma Society: Research Funding; Henry Stewart Talks: Patents & Royalties; Iterion Therapeutics (formerly Beta Cat Pharmaceuticals): Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Patient True Talks: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millipore (formerly Upstate Biotechnology): Patents & Royalties; MolecularMD (acquired by ICON): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; McGraw Hill: Patents & Royalties; Merck & Co: Patents & Royalties; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Dana-Farber Cancer Institute: Patents & Royalties; EnLiven: Consultancy, Research Funding; Aptose Therapeutics Inc. (formerly Lorus): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Blueprint Medicines: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Aileron Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Oregon Health & Science University: Patents & Royalties. Byrd:Acerta Pharma: Research Funding; Syndax: Research Funding; Vincera: Research Funding; Pharmacyclics LLC, an AbbVie Company, Janssen, Novartis, Gilead, TG Therapeutics: Other; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, Novartis, Janssen: Speakers Bureau; Novartis: Research Funding; Kartos Therapeutics: Research Funding; Trillium: Research Funding; Leukemia and Lymphoma Society: Other; Janssen: Consultancy; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, BeiGene: Research Funding. Levine:Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria; Astellas: Consultancy; Morphosys: Consultancy; Novartis: Consultancy; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Lilly: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Gilead: Honoraria. Mims:Abbvie: Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Leukemia and Lymphoma Society: Other: Senior Medical Director for Beat AML Study; Jazz Pharmaceuticals: Other: Data Safety Monitoring Board; Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy; Novartis: Speakers Bureau. OffLabel Disclosure: Enasidenib is not approved for the treatment of newly diagnosed AML.