Viswabandya, Auro

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

AbstractHaploidentical donors offer a potentially readily available donor, especially for non-White patients, for blood or marrow transplantation (BMT). In this collaboration across North America, we retrospectively analyzed outcomes of first BMT using haploidentical donor and posttransplantation cyclophosphamide (PTCy) in MDS/MPN-overlap neoplasms (MDS/MPN), an otherwise incurable hematological neoplasm. We included 120 patients, 38% of non-White/Caucasian ethnicity, across 15 centers with median age at BMT 62.5 years. The median follow-up is 2.4 years. Graft failure was reported in 6% patients. At 3-years, nonrelapse mortality (NRM) was 25%, relapse 27%, grade 3-4 acute graft versus host disease (GVHD) 12%, chronic GVHD requiring systemic immunosuppression 14%, progression-free survival (PFS) 48% and overall survival (OS) 56%. On multivariable analysis, statistically significant associations included older age at BMT (per decade increment) with NRM (sdHR 3.28, 95%CI 1.30-8.25), PFS (HR 1.98, 95% 1.13-3.45) and OS (HR 2.01, 95% CI 1.11-3.63), presence of mutation in EZH2/RUNX1/SETBP1 with relapse (sdHR 2.61, 95%CI 1.06-6.44), and splenomegaly at BMT/prior splenectomy with OS (HR 2.20, 95%CI 1.04-4.65). Haploidentical donors are a viable option for BMT in MDS/MPN, especially for those disproportionately represented in the unrelated donor registry. Disease-related factors including splenomegaly and high-risk mutations dominate outcomes following BMT.

Background: Primary and secondary [post-essential thrombocythemia (ET) or polycythemia vera (PV)] Myelofibrosis (MF) are clonal hematopoietic neoplasms marked by constitutive JAK-STAT activation, bone marrow fibrosis, cytopenias, and constitutional symptom burden. Allogeneic hematopoietic cell transplantation (HCT) is the only curative option in this disease. Median overall survival (OS) after JAK inhibitor (JAKi) discontinuation is poor, ranging from 6-24 months (Harrision et al. Ann Hematol. 2020). Haploidentical (Haplo)-HCT with post-transplant cyclophosphamide (PTCy) has improved donor availability, but data on post-HCT outcomes in MF remain scarce. Herein, we describe clinical outcomes of patients (pts) with MF who underwent haplo-HCT with PTCy. Methods: We conducted a multi-institutional study where we retrospectively reviewed charts to obtain pt, disease, and treatment characteristics of MF pts who underwent haplo-HCT from 2000 to 2019. Graft-versus-host-disease (GVHD), relapse, and non-relapse mortality (NRM) were described as cumulative incidences. OS and relapse free survival (RFS) were estimated using the Kaplan-Meier method. Univariate analyses were conducted with Cox or Fine and Gray regression. Results: Fifty-eight adult pts from 11 centers underwent haplo-HCT and were included in the analysis. Pt, disease, and HCT characteristics are listed in Table 1. Thirty-four (59%) pts were over 60 years of age at the time of HCT. Of the 53 pts in whom driver mutation data was available, JAK2 was reported in 34 (64%), CALR in 10 (19%), MPL in 4 (8%), and 5 (9%) were triple-negative. Twenty-two (38%) pts had HCT-CI ≥ 3. Median CD34+ cell dose was 6.81 (range: 2.3-28.6) x 106/kg. All pts received PTCy as a part of GVHD prophylaxis regimen. Median follow-up in this study was 28 (range: 3.3-75.7) months. Neutrophil and platelet engraftment was reported in 53 (91%) and 47 (81%) pts at a median time of 20 (range: 14-70) and 31 (range: 15-225) days, respectively. Five pts (9%) had graft failure. The cumulative incidences of all-grade acute and chronic GVHD were in 44% (95% CI: 31-56%) at 6 months and 31% (95% CI: 18-44%) at 2 years. Grade 3-4 acute GVHD was seen in 5 (9%) pts. Post-HCT relapse/disease persistence occurred in 12 (21%) pts with a median of 416 (range: 28-917) days. The 2-year estimates of OS, RFS, relapse and NRM were 69% (95% CI: 55-80%), 52% (95% CI: 37-65%), 21% (95% CI: 11-34%) and 27% (95% CI: 16-39%) respectively (Fig 1). On univariate analyses (Table 2), older age (HR 2.17, P=.012) and a higher HCT-CI (HR 1.4, P<.001) was associated with higher NRM. Higher HCT-CI was also associated with higher all-cause mortality (HR 1.38, P=.003). Bone marrow as a graft source was associated with a higher risk of relapse (HR 5.04, P<0.001, Fig 2), but did not significantly affect OS (HR 0.78, 95% CI: 0.18-3.46), RFS (HR 1.89, 95% CI: 0.71-5.02) or NRM (HR 0.58, 95% CI: 0.09-3.84). JAKi use prior to HCT, spleen size or type of driver mutation did not significantly affect outcomes. Conclusions: Based on this study, we conclude that haplo-HCT with PTCy is a valid option in pts with MF. The graft failure rates appear to be similar to those reported with sibling and unrelated donors. Older age, higher HCT-CI and bone marrow as graft source were associated with inferior outcomes. JAKi use prior to HCT or type of driver mutation did not significantly affect outcomes. Disclosures Grunwald: Trovagene: Consultancy; Daiichi Sankyo: Consultancy; Merck: Consultancy; Agios: Consultancy; Janssen: Research Funding; Cardinal Health: Consultancy; Incyte: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; Forma Therapeutics: Research Funding; Amgen: Consultancy; Celgene: Consultancy; Incyte: Consultancy, Research Funding; Celgene: Consultancy; Celgene: Consultancy; Pfizer: Consultancy; Incyte: Consultancy, Research Funding; Merck: Research Funding; Astellas: Consultancy; Genentech/Roche: Research Funding; Premier: Consultancy; Premier: Consultancy; Astellas: Consultancy; Premier: Consultancy; Daiichi Sankyo: Consultancy; Agios: Consultancy; Abbvie: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Trovagene: Consultancy; Trovagene: Consultancy; Abbvie: Consultancy; Abbvie: Consultancy; Agios: Consultancy; Forma Therapeutics: Research Funding; Forma Therapeutics: Research Funding; Astellas: Consultancy; Amgen: Consultancy; Amgen: Consultancy; Merck: Consultancy; Cardinal Health: Consultancy; Pfizer: Consultancy; Cardinal Health: Consultancy; Janssen: Research Funding; Genentech/Roche: Research Funding; Genentech/Roche: Research Funding. Dholaria:J&J: Research Funding; Takeda: Research Funding; Angiocrine: Research Funding; bms: Research Funding; Poseida: Research Funding. Abedin:Jazz Pharmaceuticals: Honoraria; Agios: Honoraria; Helsinn Healthcare: Honoraria; Pfizer: Research Funding; Helsinn Healthcare: Research Funding; Actinium Pharmaceuticals: Research Funding. Gupta:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy; Incyte: Honoraria, Research Funding; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees. DeZern:MEI: Consultancy; Celgene: Consultancy, Honoraria; Astex: Research Funding; Abbvie: Consultancy. Gerds:Sierra Oncology: Research Funding; Pfizer: Research Funding; Celgene: Consultancy, Research Funding; CTI Biopharma: Consultancy, Research Funding; AstraZeneca/MedImmune: Consultancy; Incyte Corporation: Consultancy, Research Funding; Apexx Oncology: Consultancy; Roche/Genentech: Research Funding; Imago Biosciences: Research Funding; Gilead Sciences: Research Funding. Jain:Bristol Myer Squibb: Other: for advisory board participation; Takeda: Consultancy, Honoraria; CareDx: Other: Advisory Board.