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.

Molecular Taxonomy of Myelodysplastic Syndromes and its Clinical Implications.

Myelodysplastic syndromes/neoplasms (MDS) are clonal hematologic disorders characterized by morphologic abnormalities of myeloid cells and peripheral cytopenias. While genetic abnormalities underlie the pathogenesis of these disorders and their heterogeneity, current classifications of MDS rely predominantly on morphology. We performed genomic profiling of 3,233 patients with MDS or related disorders to delineate molecular subtypes and define their clinical implications. Gene mutations, copy-number alterations (CNAs), and copy-neutral loss of heterozygosity (cnLOH) were derived from targeted sequencing of a 152-gene panel, with abnormalities identified in 91, 43, and 11% of patients, respectively. We characterized 16 molecular groups, encompassing 86% of patients, using information from 21 genes, 6 cytogenetic events, and LOH at the TP53 and TET2 loci. Two residual groups defined by negative findings (molecularly not-otherwise specified, absence of recurrent drivers) comprised 14% of patients. The groups varied in size from 0.5% to 14% of patients and were associated with distinct clinical phenotypes and outcomes. The median bone marrow blast percentage across groups ranged from 1.5 to 10%, and the median overall survival from 0.9 to 8.2 years. We validated 5 well-characterized entities, added further evidence to support 3 previously reported subsets, and described 8 novel groups. The prognostic influence of bone marrow blasts depended on the genetic subtypes. Within genetic subgroups, therapy-related MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) had comparable clinical and outcome profiles to primary MDS. In conclusion, genetically-derived subgroups of MDS are clinically relevant and may inform future classification schemas and translational therapeutic research.

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.

Advanced phase chronic myeloid leukaemia (CML) in the tyrosine kinase inhibitor era - a report from the Swedish CML register.

OBJECTIVES: The primary goal in management of chronic phase (CP) chronic myeloid leukaemia (CML) is to prevent disease progression to accelerated phase (AP) or blast crisis (BC). We have evaluated progression rates in a decentralised healthcare setting and characterised patients progressing to AP/BC on TKI treatment. METHODS: Using data from the Swedish CML register, we identified CP-CML patients diagnosed 2007-2011 who progressed to AP/BC within 2 yrs from diagnosis (n = 18) as well as patients diagnosed in advanced phase during 2007-2012 (n = 36) from a total of 544 newly diagnosed CML cases. We evaluated baseline characteristics, progression rates, outcome and adherence to guidelines for monitoring and treatment. RESULTS: The cumulative progression rate at 2 yrs was 4.3%. All 18 progression cases had been treated with imatinib, and six progressed within 6 months. High-risk EUTOS score was associated to a higher risk of progression. Insufficient cytogenetic and/or molecular monitoring was found in 33%. Median survival after transformation during TKI treatment was 1.4 yrs. In those presenting with BC and AP, median survival was 1.6 yrs and not reached, respectively. CONCLUSION: In this population-based setting, progression rates appear comparable to that reported from clinical trials, with similar dismal patient outcome. Improved adherence to CML guidelines may minimise the risk of disease progression.

How to manage patients with germline DDX41 variants: Recommendations from the Nordic working group on germline predisposition for myeloid neoplasms.

Increasing recognition of germline DDX41 variants in patients with hematological malignancies prompted us to provide DDX41-specific recommendations for diagnosis, surveillance, and treatment. Causative germline variants in the DDX41 predispose to the development of myeloid neoplasms (MNs), especially myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Almost 3%-5% of all patients with MDS or AML carry a pathogenic or likely pathogenic germline DDX41 variant, while half of them acquire a somatic second hit in the other allele. DDX41-associated MNs exhibit unique clinical characteristics compared to other hematological malignancies with germline predisposition: MNs occur mostly at advanced age and follow an indolent clinical course. Male carriers are more prone to develop MDS or AML than females. DDX41-associated MN is often hypoplastic, and the malignancy may be preceded by cytopenias.

Erythroid Differentiation Enhances RNA Mis-Splicing in SF3B1-Mutant Myelodysplastic Syndromes with Ring Sideroblasts.

Myelodysplastic syndromes with ring sideroblasts (MDS-RS) commonly develop from hematopoietic stem cells (HSC) bearing mutations in the splicing factor SF3B1 (SF3B1mt). Direct studies into MDS-RS pathobiology have been limited by a lack of model systems that fully recapitulate erythroid biology and RS development and the inability to isolate viable human RS. Here, we combined successful direct RS isolation from patient samples, high-throughput multiomics analysis of cells encompassing the SF3B1mt stem-erythroid continuum, and functional assays to investigate the impact of SF3B1mt on erythropoiesis and RS accumulation. The isolated RS differentiated, egressed into the blood, escaped traditional nonsense-mediated decay (NMD) mechanisms, and leveraged stress-survival pathways that hinder wild-type hematopoiesis through pathogenic GDF15 overexpression. Importantly, RS constituted a contaminant of magnetically enriched CD34+ cells, skewing bulk transcriptomic data. Mis-splicing in SF3B1mt cells was intensified by erythroid differentiation through accelerated RNA splicing and decreased NMD activity, and SF3B1mt led to truncations in several MDS-implicated genes. Finally, RNA mis-splicing induced an uncoupling of RNA and protein expression, leading to critical abnormalities in proapoptotic p53 pathway genes. Overall, this characterization of erythropoiesis in SF3B1mt RS provides a resource for studying MDS-RS and uncovers insights into the unexpectedly active biology of the "dead-end" RS. SIGNIFICANCE: Ring sideroblast isolation combined with state-of-the-art multiomics identifies survival mechanisms underlying SF3B1-mutant erythropoiesis and establishes an active role for erythroid differentiation and ring sideroblasts themselves in SF3B1-mutant myelodysplastic syndrome pathogenesis.

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.

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.

[Two cases of VEXAS syndrome]. / VEXAS ­ nytt autoinflammatoriskt syndrom med bred symtombild.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is a newly discovered syndrome caused by a somatic mutation in the UBA1 gene, located in the X chromosome. The syndrome mainly affects older men, and presents with persistent inflammation and rheumatological symptoms like polychondritis, lung infiltrates and dermatitis. Related hematological disturbances are thromboembolic events, macrocytic anemia, myelodysplastic syndrome, and vacuoles found in bone marrow hematopoietic cells. A genetic test of the UBA1 gene confirms the diagnosis when a clinical suspicion of VEXAS is raised. Patients usually respond to prednisolone at a dose of 15-20 mg/day but an effective and well tolerated long-term treatment strategy is still to be defined. The only potentially curative treatment is allogeneic stem cell transplantation. In this case report we present two cases of VEXAS, one of which has undergone an allogeneic stem cell transplantation.

Molecular International Prognostic Scoring System for Myelodysplastic Syndromes.

BACKGROUND: Risk stratification and therapeutic decision-making for myelodysplastic syndromes (MDS) are based on the International Prognostic Scoring System­Revised (IPSS-R), which considers hematologic parameters and cytogenetic abnormalities. Somatic gene mutations are not yet used in the risk stratification of patients with MDS. METHODS: To develop a clinical-molecular prognostic model (IPSS-Molecular [IPSS-M]), pretreatment diagnostic or peridiagnostic samples from 2957 patients with MDS were profiled for mutations in 152 genes. Clinical and molecular variables were evaluated for associations with leukemia-free survival, leukemic transformation, and overall survival. Feature selection was applied to determine the set of independent IPSS-M prognostic variables. The relative weights of the selected variables were estimated using a robust Cox multivariable model adjusted for confounders. The IPSS-M was validated in an external cohort of 754 Japanese patients with MDS. RESULTS: We mapped at least one oncogenic genomic alteration in 94% of patients with MDS. Multivariable analysis identified TP53multihit, FLT3 mutations, and MLLPTD as top genetic predictors of adverse outcomes. Conversely, SF3B1 mutations were associated with favorable outcomes, but this was modulated by patterns of comutation. Using hematologic parameters, cytogenetic abnormalities, and somatic mutations of 31 genes, the IPSS-M resulted in a unique risk score for individual patients. We further derived six IPSS-M risk categories with prognostic differences. Compared with the IPSS-R, the IPSS-M improved prognostic discrimination across all clinical end points and restratified 46% of patients. The IPSS-M was applicable in primary and secondary/therapy-related MDS. To simplify clinical use of the IPSS-M, we developed an open-access Web calculator that accounts for missing values. CONCLUSIONS: Combining genomic profiling with hematologic and cytogenetic parameters, the IPSS-M improves the risk stratification of patients with MDS and represents a valuable tool for clinical decision-making. (Funded by Celgene Corporation through the MDS Foundation, the Josie Robertson Investigators Program, the Edward P. Evans Foundation, the Projects of National Relevance of the Italian Ministry of University and Research, Associazione Italiana per la Ricerca sul Cancro, the Japan Agency for Medical Research and Development, Cancer Research UK, the Austrian Science Fund, the MEXT [Japanese Ministry of Education, Culture, Sports, Science and Technology] Program for Promoting Research on the Supercomputer Fugaku, the Japan Society for the Promotion of Science, the Taiwan Department of Health, and Celgene Corporation through the MDS Foundation.)

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.

Co-mutation pattern, clonal hierarchy, and clone size concur to determine disease phenotype of SRSF2P95-mutated neoplasms.

Somatic mutations in splicing factor genes frequently occur in myeloid neoplasms. While SF3B1 mutations are associated with myelodysplastic syndromes (MDS) with ring sideroblasts, SRSF2P95 mutations are found in different disease categories, including MDS, myeloproliferative neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and acute myeloid leukemia (AML). To identify molecular determinants of this phenotypic heterogeneity, we explored molecular and clinical features of a prospective cohort of 279 SRSF2P95-mutated cases selected from a population of 2663 patients with myeloid neoplasms. Median number of somatic mutations per subject was 3. Multivariate regression analysis showed associations between co-mutated genes and clinical phenotype, including JAK2 or MPL with myelofibrosis (OR = 26.9); TET2 with monocytosis (OR = 5.2); RAS-pathway genes with leukocytosis (OR = 5.1); and STAG2, RUNX1, or IDH1/2 with blast phenotype (MDS or AML) (OR = 3.4, 1.9, and 2.1, respectively). Within patients with SRSF2-JAK2 co-mutation, JAK2 dominance was invariably associated with clinical feature of MPN, whereas SRSF2 mutation was dominant in MDS/MPN. Within patients with SRSF2-TET2 co-mutation, clinical expressivity of monocytosis was positively associated with co-mutated clone size. This study provides evidence that co-mutation pattern, clone size, and hierarchy concur to determine clinical phenotype, tracing relevant genotype-phenotype associations across disease entities and giving insight on unaccountable clinical heterogeneity within current WHO classification categories.

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.

Implications of TP53 allelic state for genome stability, clinical presentation and outcomes in myelodysplastic syndromes.

Tumor protein p53 (TP53) is the most frequently mutated gene in cancer1,2. In patients with myelodysplastic syndromes (MDS), TP53 mutations are associated with high-risk disease3,4, rapid transformation to acute myeloid leukemia (AML)5, resistance to conventional therapies6-8 and dismal outcomes9. Consistent with the tumor-suppressive role of TP53, patients harbor both mono- and biallelic mutations10. 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.