Gomez-Arteaga, Alexandra

Abstract Background: Acute myeloid leukemia (AML) with TP53 alteration (TP53alt), including deletion (TP53del) detected by cytogenetics (CG) (karyotype, FISH, or genomic microarray) and/or TP53 mutation (TP53mut) identified by next-generation sequencing, is associated with poor prognosis. However, a subset of patients may achieve long-term survival after allogeneic hematopoietic cell transplantation (allo-HCT). Patient-, disease-, and transplant-related variables may inform risk stratification and improve treatment decision-making in this high-risk population. We used the CIBMTR registry to investigate outcomes of patients with TP53alt AML undergoing allo-HCT. Methods: We included adult patients with 2017 ELN adverse-risk AML who underwent first allo-HCT between 2013–2019 and had available CG and molecular data. TP53del was centrally reviewed by expert cytogeneticist. Patients were stratified into three TP53alt groups: Group 1 (TP53mut+/ TP53del+), Group 2 (TP53mut −/ TP53del+), and Group 3 (TP53mut+/ TP53del −). The control group comprised patients with 2017 ELN adverse-risk AML negative for both TP53mut and TP53del. Patients from embargoed centers, lacking consent or comprehensive research data, or missing CG and molecular information were excluded. Results: Of 6,474 patients with adverse-risk AML, 703 patients with TP53alt met inclusion criteria: Group 1 (n=203), Group 2 (n=271), Group 3 (n=229). The control group included 2,763 patients. Median age was 58 years (44% ≥60); 44% were female; 16% had Karnofsky score <90, and 52% had HCT-CI ≥3. Secondary AML was more frequent in TP53alt groups (32%, 27%, 27%) versus control (21%) (p<.01). Complex and monosomal karyotypes were most common in Group 1 (61%), followed by Group 2 (54%), Group 3 (31%), and control (6%), p<.01. More patients in Groups 1 and 2 (28% each) underwent HCT in primary induction failure/relapse (PIF/Rel) versus Group 3 (19%) and control (19%), p<.01. Median time from diagnosis to transplant was 5 months (1-331). Donor type was matched related in 23%, matched or mismatched unrelated in 50%, haploidentical in 18%, and umbilical cord blood in 7%. Conditioning intensity was myeloablative (MA) in 51%; reduced-intensity (RIC) in 36% , and 13% non-MA (NMA). Post-transplant cyclophosphamide (PTCy) was used in 27%; and in vivo T-cell depletion (ATG or alemtuzumab) in 26%. After median follow-up of 66 months (3-130), 5-yr OS was 18% (Group 1), 18% (Group 2), 35% (Group 3) vs 46% in controls (p<0.001). 5-yr LFS was 14%, 15%, and 28%, respectively, vs 38% (p<0.001). 5-yr CIR was 69%, 66%, and 52% vs 43% (p<0.001). Transplant-related mortality (TRM) was similar across groups (1-year: 12–16%). On multivariate analysis, compared with controls, TP53del (with or without TP53mut) was independently associated with higher relapse risk: Group 1 HR 1.97, Group 2 HR 1.91, Group 3 HR 1.45 (all p<0.001). These findings translated into inferior OS (HR 1.94, 1.87, and 1.45, respectively, p<0.001). PIF/Rel and CR2+ were also associated with worse survival (HR 2.58 (p<0.001) and 1.18 (p<0.001, respectively). No significant difference was observed between MA and RIC for relapse, while non-myeloablative regimens (HR 1.65; p<0.001) were associated with higher CIR. In vivo T-cell depletion increased relapse risk (HR 1.22; p=0.004). TRM was not influenced by TP53alt status; it was impacted by age, KPS, time from diagnosis to HCT, and GVHD prophylaxis regiment (non-PTCy). Conclusions: This large, registry-based study with long term median follow-up showed that among patients with adverse-risk AML, TP53alt is a strong independent predictor of poor post-HCT outcomes in AML, particularly when TP53del is present. Patients with TP53del , with or without concurrent TP53mut, experienced the worst outcomes, followed by those with isolated TP53mut. Despite the limitations of registry data including lack of information on timing of TP53mut, variant allele frequency (VAF), number of mutations, or accurate information on measurable residual disease (MRD), these findings reinforce the adverse prognostic impact of TP53alt and support its inclusion in risk stratification frameworks. While some patients with TP53alt —particularly those with isolated mutations—may benefit from allo-HCT, relapse remains the dominant cause of failure. Future studies incorporating TP53 functional status, VAF, clonal architecture, specific CG, MRD, and novel transplant approaches are warranted.

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.