Tumor protein p53 (TP53) is the most frequently mutated gene in cancer 1,2. In patients with myelodysplastic syndromes (MDS), TP53 mutations are associated with high-risk disease 3,4 , rapid transformation to acute myeloid leukemia (AML) 5 , resistance to conventional therapies 6-8 and dismal outcomes 9. Consistent with the tumor-suppressive role of TP53, patients harbor both mono-and biallelic mutations 10. However, the biological and clinical implications of TP53 allelic state have not been fully investigated in MDS or any other cancer type. We analyzed 3,324 patients with MDS for TP53 mutations and allelic imbalances and delineated two subsets of patients with distinct phenotypes and outcomes. One-third of TP53-mutated patients had monoallelic mutations whereas two-thirds had multiple hits (multi-hit) consistent with biallelic targeting. Established associations with complex karyotype, few co-occurring mutations, high-risk presentation and poor outcomes were specific to multi-hit patients only. TP53 multi-hit state predicted risk of death and leukemic transformation independently of the Revised International Prognostic Scoring System (IPSS-R) 11. Surprisingly, monoallelic patients did not differ from TP53 wild-type patients in outcomes and response to therapy. This study shows that consideration of TP53 allelic state is critical for diagnostic and prognostic precision in MDS as well as in future correlative studies of treatment response. In collaboration with the International Working Group for Prognosis in MDS (Supplementary Table 1), we assembled a cohort of 3,324 peridiagnostic and treatment-naive patients with MDS or closely related myeloid neoplasms (Extended Data Fig. 1 and Supplementary Fig. 1). Genetic profiling included conventional G-banding analyses (CBA) and tumor-only, capture-based, next-generation sequencing (NGS) of a panel of genes recurrently mutated in MDS, as well as genome-wide copy number probes. Allele-specific copy number profiles were generated from NGS data using the CNACS algorithm 7 (see Methods and Code availability). An additional 1,120 samples derived from the Japanese MDS consortium (Extended Data Fig. 2) were used as a validation cohort. To study the effect of TP53 allelic state on genome stability, clinical presentation, outcome and response to therapy, we performed a detailed characterization of alterations at the TP53 locus. First, we assessed genome-wide allelic imbalances in the cohort of 3,324 patients, to include arm-level or focal (~3 Mb) ploidy alterations and regions of copy-neutral loss of heterozygosity (cnLOH) (Extended Data Fig. 3, Supplementary Figs. 2-4 and Methods).

Clinical recommendations for Acute Myeloid Leukemia (AML) classification and risk-stratification remain heavily reliant on cytogenetic findings at diagnosis, which are present in <50% of patients. Using comprehensive molecular profiling data from 3,653 patients we characterize and validate 16 molecular classes describing 100% of AML patients. Each class represents diverse biological AML subgroups, and is associated with distinct clinical presentation, likelihood of response to induction chemotherapy, risk of relapse and death over time. Secondary AML-2, emerges as the second largest class (24%), associates with high-risk disease, poor prognosis irrespective of flow Minimal Residual Disease (MRD) negativity, and derives significant benefit from transplantation. Guided by class membership we derive a 3-tier risk-stratification score that re-stratifies 26% of patients as compared to standard of care. This results in a unified framework for disease classification and risk-stratification in AML that relies on information from cytogenetics and 32 genes. Last, we develop an open-access patient-tailored clinical decision support tool.

Stably acquired mutations in hematopoietic cells represent substrates of selection that may lead to clonal hematopoiesis (CH), a common state in cancer patients that is associated with a heightened risk of leukemia development. Owing to technical and sample size limitations, most CH studies have characterized gene mutations or mosaic chromosomal alterations (mCAs) individually. Here we leverage peripheral blood sequencing data from 32,442 cancer patients to jointly characterize gene mutations (n = 14,789) and mCAs (n = 383) in CH. Recurrent composite genotypes resembling known genetic interactions in leukemia genomes underlie 23% of all detected autosomal alterations, indicating that these selection mechanisms are operative early in clonal evolution. CH with composite genotypes defines a patient group at high risk of leukemia progression (3-year cumulative incidence 14.6%, CI: 7–22%). Multivariable analysis identifies mCA as an independent risk factor for leukemia development (HR = 14, 95% CI: 6–33, P < 0.001). Our results suggest that mCA should be considered in conjunction with gene mutations in the surveillance of patients at risk of hematologic neoplasms.

Myeloproliferative neoplasms (MPN) that have evolved into accelerated or blast phase disease (MPN-AP/BP) have poor outcomes with limited treatment options and therefore represent an urgent unmet need. We have previously demonstrated in a multicenter, phase 1 trial conducted through the Myeloproliferative Neoplasms Research Consortium that the combination of ruxolitinib and decitabine is safe and tolerable and is associated with a favorable overall survival (OS). In this phase 2 trial, 25 patients with MPN-AP/BP were treated at the recommended phase 2 dose of ruxolitinib 25 mg twice daily for the induction cycle followed by 10 mg twice daily for subsequent cycles in combination with decitabine 20 mg/m2 for 5 consecutive days in a 28-day cycle. Nineteen patients died during the study follow-up. The median OS for all patients on study was 9.5 months (95% confidence interval, 4.3-12.0). Overall response rate (complete remission + incomplete platelet recovery + partial remission) was 11/25 (44%) and response was not associated with improved survival. We conclude that the combination of decitabine and ruxolitinib was well tolerated, demonstrated favorable OS, and represents a therapeutic option for this high-risk patient population. This trial was registered at www.clinicaltrials.gov as #NCT02076191.

TP53 mutations are associated with poor clinical outcomes and treatment resistance in myelodysplastic syndromes. However, the biological and clinical relevance of the underlying mono-or bi-allelic state of the mutations is unclear. We analyzed 3,324 MDS patients for TP53 mutations and allelic imbalances of the TP53 locus and found that 1 in 3 TP53 -mutated patients had mono-allelic targeting of the gene whereas 2 in 3 had multiple hits consistent with bi-allelic targeting. The established associations for TP53 with complex karyotype, high-risk presentation, poor survival and rapid leukemic transformation were specific to patients with multi-hit state only. TP53 multi-hit state predicted risk of death and leukemic transformation independently of the Revised International Prognostic Scoring System, while mono-allelic patients did not differ from TP53 wild-type patients. The separation by allelic state was retained in therapy-related MDS. Findings were validated in a cohort of 1,120 patients. Ascertainment of TP53 allelic state is critical for diagnosis, risk estimation and prognostication precision in MDS, and future correlative studies of treatment response should consider TP53 allelic state.