The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts.

Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, and generally associate with more favorable prognosis. However, not all SF3B1 mutations are the same, and little is known about how distinct hotspots influence disease. Here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and a lack of the favorable survival seen with other SF3B1 mutations. Moreover, compared to other hotspot SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data have implications for our understanding of the functional diversity of spliceosome mutations, as well as the pathobiology, classification, prognosis, and management of SF3B1-mutant MDS.

Early transfusion patterns improve the Molecular International Prognostic Scoring System (IPSS-M) prediction in myelodysplastic syndromes.

BACKGROUND: The Molecular International Prognostic Scoring System (IPSS-M) is the new gold standard for diagnostic outcome prediction in patients with myelodysplastic syndromes (MDS). This study was designed to assess the additive prognostic impact of dynamic transfusion parameters during early follow-up. METHODS: We retrieved complete transfusion data from 677 adult Swedish MDS patients included in the IPSS-M cohort. Time-dependent erythrocyte transfusion dependency (E-TD) was added to IPSS-M features and analyzed regarding overall survival and leukemic transformation (acute myeloid leukemia). A multistate Markov model was applied to assess the prognostic value of early changes in transfusion patterns. RESULTS: Specific clinical and genetic features were predicted for diagnostic and time-dependent transfusion patterns. Importantly, transfusion state both at diagnosis and within the first year strongly predicts outcomes in both lower (LR) and higher-risk (HR) MDSs. In multivariable analysis, 8-month landmark E-TD predicted shorter survival independently of IPSS-M (p < 0.001). A predictive model based on IPSS-M and 8-month landmark E-TD performed significantly better than a model including only IPSS-M. Similar trends were observed in an independent validation cohort (n = 218). Early transfusion patterns impacted both future transfusion requirements and outcomes in a multistate Markov model. CONCLUSION: The transfusion requirement is a robust and available clinical parameter incorporating the effects of first-line management. In MDS, it provides dynamic risk information independently of diagnostic IPSS-M and, in particular, clinical guidance to LR MDS patients eligible for potentially curative therapeutic intervention.

Molecular and clinical presentation of UBA1-mutated myelodysplastic syndromes.

Mutations in UBA1, which are disease-defining for VEXAS syndrome, have been reported in patients diagnosed with myelodysplastic syndromes (MDS). Here, we define the prevalence and clinical associations of UBA1 mutations in a representative cohort of patients with MDS. Digital droplet PCR profiling of a selected cohort of 375 male patients lacking MDS disease-defining mutations or established WHO disease classification identified 28 patients (7%) with UBA1 p.M41T/V/L mutations. Using targeted sequencing of UBA1 in a representative MDS cohort (n=2,027), we identified an additional 27 variants in 26 patients (1%), which we classified as likely/pathogenic (n=12) and unknown significance (n=15). Among the total 40 patients with likely/pathogenic variants (2%), all were male and 63% were classified by WHO2016 as MDS-MLD/SLD. Patients had a median of one additional myeloid gene mutation, often in TET2 (n=12), DNMT3A (n=10), ASXL1 (n=3), or SF3B1 (n=3). Retrospective clinical review where possible showed that 83% (28/34) UBA1-mutant cases had VEXAS-associated diagnoses or inflammatory clinical presentation. The prevalence of UBA1-mutations in MDS patients argues for systematic screening for UBA1 in the management of MDS.

Embracing cancer complexity: Hallmarks of systemic disease.

The last 50 years have witnessed extraordinary developments in understanding mechanisms of carcinogenesis, synthesized as the hallmarks of cancer. Despite this logical framework, our understanding of the molecular basis of systemic manifestations and the underlying causes of cancer-related death remains incomplete. Looking forward, elucidating how tumors interact with distant organs and how multifaceted environmental and physiological parameters impinge on tumors and their hosts will be crucial for advances in preventing and more effectively treating human cancers. In this perspective, we discuss complexities of cancer as a systemic disease, including tumor initiation and promotion, tumor micro- and immune macro-environments, aging, metabolism and obesity, cancer cachexia, circadian rhythms, nervous system interactions, tumor-related thrombosis, and the microbiome. Model systems incorporating human genetic variation will be essential to decipher the mechanistic basis of these phenomena and unravel gene-environment interactions, providing a modern synthesis of molecular oncology that is primed to prevent cancers and improve patient quality of life and cancer outcomes.

Predicting cytopenias, progression, and survival in patients with clonal cytopenia of undetermined significance: a prospective cohort study.

BACKGROUND: Somatic mutations are frequently reported in individuals with cytopenia but without a confirmed haematological diagnosis (clonal cytopenia of undetermined significance; CCUS). These patients have an increased risk of progression to a myeloid malignancy and worse overall survival than those with no such mutations. To date, studies have been limited by retrospective analysis or small patient numbers. We aimed to establish the natural history of CCUS by prospectively investigating outcome in a large, well defined patient cohort. METHODS: This prospective cohort study was conducted at the Haematological Malignancy Diagnostic Service, a diagnostic laboratory in Leeds, UK. Patients aged at least 18 years who were referred for investigation of cytopenia were eligible for inclusion; those with a history of myeloid malignancy were not eligible. Targeted sequencing was conducted alongside routine clinical testing. Baseline mutation analysis was then correlated with the main study outcomes: longitudinal blood counts, disease progression to a myeloid malignancy, and overall survival with a median follow-up of 4·54 years (IQR 4·03-5·04). Data were collected manually from hospital records or extracted from laboratory or clinical outcome databases. FINDINGS: Bone marrow samples from 2348 patients were received at the Haematological Malignancy Diagnostic Service between July 1, 2014, and July 31, 2016. Of these, 2083 patients (median age 72 years [IQR 63-80, range 18-99]; 854 [41·0%] female and 1229 [59·0%] male) met the inclusion criteria and had samples of sufficient quality for further analysis. 598 (28·7%) patients received a diagnosis on the basis of their biopsy sample, whereas 1485 (71·3%) samples were classified as non-diagnostic; of these, CCUS was confirmed in 400 (26·9%) patients (256 [64·0%] male and 144 [36·0%] female). TET2, SRSF2, and DNMT3A were the most frequently mutated genes in patients with CCUS, with 320 (80%) of 400 patients harbouring a mutation in at least one of these genes. Age (p<0·0001), sex (p=0·0027), and mutations in ASXL1 (p=0·0009), BCOR (p=0·0056), and TP53 (p=0·0055) correlated with a worse overall survival; however, the number of mutations was the strongest predictor for progression to a myeloid malignancy (two mutations, p=0·0024; three or more mutations, p=0·0004). Extended sequencing of samples from a subgroup of patients with sequential samples and no mutations in the initial myeloid gene panel showed recurrent mutations in both DDX41 and UBA1, suggesting that these genes should be included in clinical test panels. INTERPRETATION: Mutation analysis is advised in patients who have undergone bone marrow examination and have an otherwise-unexplained cytopenia. High-risk genetic mutations and increased numbers of mutations are predictive of both survival and progression within 5 years of presentation, warranting clinical surveillance and, when necessary, intervention. FUNDING: MDS Foundation.

Novel precision medicine approaches and treatment strategies in hematological malignancies.

Genetic testing has been applied for decades in clinical routine diagnostics of hematological malignancies to improve disease (sub)classification, prognostication, patient management, and survival. In recent classifications of hematological malignancies, disease subtypes are defined by key recurrent genetic alterations detected by conventional methods (i.e., cytogenetics, fluorescence in situ hybridization, and targeted sequencing). Hematological malignancies were also one of the first disease areas in which targeted therapies were introduced, the prime example being BCR::ABL1 inhibitors, followed by an increasing number of targeted inhibitors hitting the Achilles' heel of each disease, resulting in a clear patient benefit. Owing to the technical advances in high-throughput sequencing, we can now apply broad genomic tests, including comprehensive gene panels or whole-genome and whole-transcriptome sequencing, to identify clinically important diagnostic, prognostic, and predictive markers. In this review, we give examples of how precision diagnostics has been implemented to guide treatment selection and improve survival in myeloid (myelodysplastic syndromes and acute myeloid leukemia) and lymphoid malignancies (acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and chronic lymphocytic leukemia). We discuss the relevance and potential of monitoring measurable residual disease using ultra-sensitive techniques to assess therapy response and detect early relapses. Finally, we bring up the promising avenue of functional precision medicine, combining ex vivo drug screening with various omics technologies, to provide novel treatment options for patients with advanced disease. Although we are only in the beginning of the field of precision hematology, we foresee rapid development with new types of diagnostics and treatment strategies becoming available to the benefit of our patients.

Integrated Genomic and Transcriptomic Analysis Improves Disease Classification and Risk Stratification of MDS with Ring Sideroblasts.

PURPOSE: Ring sideroblasts (RS) define the low-risk myelodysplastic neoplasm (MDS) subgroup with RS but may also reflect erythroid dysplasia in higher risk myeloid neoplasm. The benign behavior of MDS with RS (MDSRS+) is limited to SF3B1-mutated cases without additional high-risk genetic events, but one third of MDSRS+ carry no SF3B1 mutation, suggesting that different molecular mechanisms may underlie RS formation. We integrated genomic and transcriptomic analyses to evaluate whether transcriptome profiles may improve current risk stratification. EXPERIMENTAL DESIGN: We studied a prospective cohort of MDSRS+ patients irrespective of World Health Organization (WHO) class with regard to somatic mutations, copy-number alterations, and bone marrow CD34+ cell transcriptomes to assess whether transcriptome profiles add to prognostication and provide input on disease classification. RESULTS: SF3B1, SRSF2, or TP53 multihit mutations were found in 89% of MDSRS+ cases, and each mutation category was associated with distinct clinical outcome, gene expression, and alternative splicing profiles. Unsupervised clustering analysis identified three clusters with distinct hemopoietic stem and progenitor (HSPC) composition, which only partially overlapped with mutation groups. IPSS-M and the transcriptome-defined proportion of megakaryocyte/erythroid progenitors (MEP) independently predicted survival in multivariable analysis. CONCLUSIONS: These results provide essential input on the molecular basis of SF3B1-unmutated MDSRS+ and propose HSPC quantification as a prognostic marker in myeloid neoplasms with RS.

Therapy-related myelodysplastic syndromes in the genomics era.

Therapy-related myelodysplastic syndromes (t-MDS) represent a heterogeneous group of malignancies that arise as a late complication of prior exposure to chemotherapy and/or radiotherapy administered for a primary condition. T-MDS account for approximately 20% of all MDS and are characterized by resistance to current treatment strategies and poor prognosis. Our understanding of t-MDS pathogenesis has considerably improved over the last 5 years with the availability of deep sequencing technologies. T-MDS development is now considered as a multifactorial process resulting from complex interactions between an underlying germline genetic susceptibility, the stepwise acquisition of somatic mutations in hematopoietic stem cells, the clonal selection pressure exerted by cytotoxic therapies, and alterations of the bone marrow microenvironment. The survival of patients with t-MDS is generally poor. This can be explained by both patient-related factors including poor performance status and less tolerance to treatment and disease-related factors, such as the presence of chemoresistant clones, high-risk cytogenetic alterations and molecular features (e.g. high frequency of TP53 mutations). Around 50% of t-MDS patients are classified as high/very high risk based on IPSS-R or IPSS-M scores, versus 30% in de novo MDS. Long-term survival is only achieved in a minority of t-MDS patients who receive allogeneic stem cell transplantation, but the development of novel drugs may open new therapeutic opportunities, especially in unfit patients. Further investigations are needed to improve the identification of patients at higher risk of developing t-MDS and determine whether primary disease treatment can be modified to prevent the occurrence of t-MDS.

The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts.

Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, generally associate with more favorable prognosis, and serve as a predictive biomarker of response to luspatercept. However, not all SF3B1 mutations are the same, and here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and decreased survival. Moreover, in contrast to canonical SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data expand our knowledge of the functional diversity of spliceosome mutations, and they suggest that patients with E592K should be approached differently from low-risk, luspatercept-responsive MDS patients with ring sideroblasts and canonical SF3B1 mutations.

Patient-Derived iPSCs Faithfully Represent the Genetic Diversity and Cellular Architecture of Human Acute Myeloid Leukemia.

The reprogramming of human acute myeloid leukemia (AML) cells into induced pluripotent stem cell (iPSC) lines could provide new faithful genetic models of AML, but is currently hindered by low success rates and uncertainty about whether iPSC-derived cells resemble their primary counterparts. Here we developed a reprogramming method tailored to cancer cells, with which we generated iPSCs from 15 patients representing all major genetic groups of AML. These AML-iPSCs retain genetic fidelity and produce transplantable hematopoietic cells with hallmark phenotypic leukemic features. Critically, single-cell transcriptomics reveal that, upon xenotransplantation, iPSC-derived leukemias faithfully mimic the primary patient-matched xenografts. Transplantation of iPSC-derived leukemias capturing a clone and subclone from the same patient allowed us to isolate the contribution of a FLT3-ITD mutation to the AML phenotype. The results and resources reported here can transform basic and preclinical cancer research of AML and other human cancers. SIGNIFICANCE: We report the generation of patient-derived iPSC models of all major genetic groups of human AML. These exhibit phenotypic hallmarks of AML in vitro and in vivo, inform the clonal hierarchy and clonal dynamics of human AML, and exhibit striking similarity to patient-matched primary leukemias upon xenotransplantation. See related commentary by Doulatov, p. 252. This article is highlighted in the In This Issue feature, p. 247.

Liquid Biopsies for Circulating Tumor DNA Detection May Reveal Occult Hematologic Malignancies in Patients With Solid Tumors.

PURPOSE: High-risk clonal hematopoiesis (CH) is frequently incidentally found in patients with solid tumors undergoing plasma cell-free DNA sequencing. Here, we aimed to determine if the incidental detection of high-risk CH by liquid biopsy may reveal occult hematologic malignancies in patients with solid tumors. MATERIALS AND METHODS: Adult patients with advanced solid cancers enrolled in the Gustave Roussy Cancer Profiling study (ClinicalTrials.gov identifier: NCT04932525) underwent at least one liquid biopsy (FoundationOne Liquid CDx). Molecular reports were discussed within the Gustave Roussy Molecular Tumor Board (MTB). Potential CH alterations were observed, and patients referred to hematology consultation in the case of pathogenic mutations in JAK2, MPL, or MYD88, irrespective of the variant allele frequency (VAF), or in DNMT3A, TET2, ASXL1, IDH1, IDH2, SF3B1, or U2AF1 with VAF ≥ 10%, while also considering patient cancer-related prognosis. TP53 mutations were discussed case-by-case. RESULTS: Between March and October 2021, 1,416 patients were included. One hundred ten patients (7.7%) carried at least one high-risk CH mutation: DNMT3A (n = 32), JAK2 (n = 28), TET2 (n = 19), ASXL1 (n = 18), SF3B1 (n = 5), IDH1 (n = 4), IDH2 (n = 3), MPL (n = 3), and U2AF1 (n = 2). The MTB advised for hematologic consultation in 45 patients. Overall, 9 patients of 18 actually addressed had confirmed hematologic malignancies that were occult in six patients: two patients had myelodysplastic syndrome, two essential thrombocythemia, one a marginal lymphoma, and one a Waldenström macroglobulinemia. The other three patients were already followed up in hematology. CONCLUSION: The incidental findings of high-risk CH through liquid biopsy may trigger diagnostic hematologic tests and reveal an occult hematologic malignancy. Patients should have a multidisciplinary case-by-case evaluation.

Postazacitidine clone size predicts long-term outcome of patients with myelodysplastic syndromes and related myeloid neoplasms.

Azacitidine is a mainstay of therapy for myelodysplastic syndrome (MDS)-related diseases. The purpose of our study is to elucidate the effect of gene mutations on hematological response and overall survival (OS), particularly focusing on their posttreatment clone size. We enrolled a total of 449 patients with MDS or related myeloid neoplasms. They were analyzed for gene mutations in pretreatment (n = 449) and posttreatment (n = 289) bone marrow samples using targeted-capture sequencing to assess the impact of gene mutations and their posttreatment clone size on treatment outcomes. In Cox proportional hazard modeling, multihit TP53 mutation (hazard ratio [HR], 2.03; 95% confidence interval [CI], 1.42-2.91; P < .001), EZH2 mutation (HR, 1.71; 95% CI, 1.14-2.54; P = .009), and DDX41 mutation (HR, 0.33; 95% CI, 0.17-0.62; P < .001), together with age, high-risk karyotypes, low platelets, and high blast counts, independently predicted OS. Posttreatment clone size accounting for all drivers significantly correlated with International Working Group (IWG) response (P < .001, using trend test), except for that of DDX41-mutated clones, which did not predict IWG response. Combined, IWG response and posttreatment clone size further improved the prediction of the original model and even that of a recently proposed molecular prediction model, the molecular International Prognostic Scoring System (IPSS-M; c-index, 0.653 vs 0.688; P < .001, using likelihood ratio test). In conclusion, evaluation of posttreatment clone size, together with the pretreatment mutational profile as well as the IWG response play a role in better prognostication of azacitidine-treated patients with myelodysplasia.

Unified classification and risk-stratification in Acute Myeloid Leukemia.

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.

Patient-specific MDS-RS iPSCs define the mis-spliced transcript repertoire and chromatin landscape of SF3B1-mutant HSPCs.

SF3B1K700E is the most frequent mutation in myelodysplastic syndrome (MDS), but the mechanisms by which it drives MDS pathogenesis remain unclear. We derived a panel of 18 genetically matched SF3B1K700E- and SF3B1WT-induced pluripotent stem cell (iPSC) lines from patients with MDS with ring sideroblasts (MDS-RS) harboring isolated SF3B1K700E mutations and performed RNA and ATAC sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from them. We developed a novel computational framework integrating splicing with transcript usage and gene expression analyses and derived a SF3B1K700E splicing signature consisting of 59 splicing events linked to 34 genes, which associates with the SF3B1 mutational status of primary MDS patient cells. The chromatin landscape of SF3B1K700E HSPCs showed increased priming toward the megakaryocyte- erythroid lineage. Transcription factor motifs enriched in chromatin regions more accessible in SF3B1K700E cells included, unexpectedly, motifs of the TEA domain (TEAD) transcription factor family. TEAD expression and transcriptional activity were upregulated in SF3B1-mutant iPSC-HSPCs, in support of a Hippo pathway-independent role of TEAD as a potential novel transcriptional regulator of SF3B1K700E cells. This study provides a comprehensive characterization of the transcriptional and chromatin landscape of SF3B1K700E HSPCs and nominates novel mis-spliced genes and transcriptional programs with putative roles in MDS-RS disease biology.

Molecular International Prognostic Scoring System for Myelodysplastic Syndromes.

MDS Molecular International Prognostic Scoring SystemSamples from over 2500 patients with MDS were profiled for gene mutations and used to develop the International Prognostic Scoring System-Molecular (IPSS-M). TP53multihit, FLT3 mutations, and MLLPTD were identified as top genetic predictors of adverse outcomes. IPSS-M improves prognostic discrimination across all clinical end points versus prior versions.

ZBTB33 is mutated in clonal hematopoiesis and myelodysplastic syndromes and impacts RNA splicing.

Clonal hematopoiesis results from somatic mutations in cancer driver genes in hematopoietic stem cells. We sought to identify novel drivers of clonal expansion using an unbiased analysis of sequencing data from 84,683 persons and identified common mutations in the 5-methylcytosine reader, ZBTB33, as well as in YLPM1, SRCAP, and ZNF318. We also identified these mutations at low frequency in myelodysplastic syndrome patients. Zbtb33 edited mouse hematopoietic stem and progenitor cells exhibited a competitive advantage in vivo and increased genome-wide intron retention. ZBTB33 mutations potentially link DNA methylation and RNA splicing, the two most commonly mutated pathways in clonal hematopoiesis and MDS.

Allogeneic Hematopoietic Stem Cell Transplantation for Chronic Myelomonocytic Leukemia: Clinical and Molecular Genetic Prognostic Factors in a Nordic Population.

Chronic myelomonocytic leukemia (CMML) is an aggressive disease in which survival after allogeneic hematopoietic stem cell transplantation (HCT) remains relatively poor. An assessment of prognostic factors is an important part of treatment decision making and has the potential to be greatly improved by the inclusion of molecular genetics. However, there is a significant knowledge gap in the interpretation of mutational patterns. This study aimed to describe outcomes of allogeneic HCT in patients with CMML in relation to clinical and molecular genetic risk factors. This retrospective study included 64 patients with CMML who underwent allogeneic HCT between 2008 and 2018, with a median follow-up of 5.4 years. Next-generation sequencing using targeted myeloid panels was carried out on saved material from 51 patients from the time of transplantation. Kaplan-Meier and Cox regression were used for analysis of overall survival (OS), and cumulative incidence with competing risks and Fine and Gray models were used for analysis of relapse and nonrelapse mortality (NRM). Mutations were detected in 48 patients (94%), indicating high levels of minimal residual disease (MRD) positivity at transplantation, even among those in complete remission (CR) (n = 14), 86% of whom had detectable mutations. The most frequently mutated genes were ASXL1 (37%), TET2 (37%), RUNX1 (33%), SRSF2 (26%), and NRAS (20%). Risk stratification using the CMML-specific Prognostic Scoring System molecular score (CPSS-Mol) resulted in 45% of patients moving to a higher risk-group compared with risk stratification using the CPSS. High leucocyte count (≥13 × 109/L), transfusion requirement, and previous intensive chemotherapy were associated with higher incidence of relapse. Being in CR was not linked to better outcomes. Neither ASXL1 nor RUNX1 mutation was associated with a difference in OS, relapse, or NRM, despite being high risk in the nontransplantation setting. TET2 mutations were associated with a significantly higher 3-year OS (73% versus 40%; P = .039). Achieving MRD-negative CR was rare in this CMML cohort, which may explain why we did not observe better outcomes for those in CR. This merits further investigation. Our analyses suggest that the negative impact of ASXL1 and RUNX1 mutations can be overcome by allogeneic HCT; however, risk stratification is complex in CMML and requires larger cohorts and multivariate models, presenting an ongoing challenge in this rare disease.