Improving prognostication for patients with myelodysplastic syndromes

Background and aims: MDS constitute a heterogenous group of myeloid malignancies mainly characterized by dysfunctional hematopoiesis. Although cytopenia, dysplastic features and evidence of clonality are essential criteria for the diagnosis of all MDS, the several subtypes of the disease have a highly variable prognosis. The increasing quality and accessibility of DNA sequencing techniques have enabled huge advances for molecular characterization of the disease, and the prognostic impact of specific molecular markers in MDS is now well established. Several prognostic scoring systems have been developed during the last two decades but none of these tools accounted for the effect of molecular markers on outcome. MDS with RS is easily recognizable by the intra-cellular presence of iron-loaded mitochondria and this subtype reflects the heterogeneity of MDS. Hence, while RS are classically associated with SF3B1 mutations and an indolent disease course, RS are sometimes found in aggressive subtypes of MDS or AML. Patients and diseases change over time, and evolution patterns themselves can tell us something about disease biology and outcome. Clinicians account for these variations in practice, but current prognostic models do not. This may partly explain remaining discrepancies between observed and predicted prognosis. Hence, in this thesis we aimed to i) develop a novel prognostic score including molecular markers to refine prognosis prediction at diagnosis, ii) study the prognostic impact of combined gene mutation and gene expression in MDS with RS and iii) assess whether changes in erythrocyte (E) transfusion patterns during the early disease course can refine outcome prediction. Methods: Study I – an international cohort of 2957 patients with MDS, MDS/myeloproliferative neoplasms (MPN) were retrospectively collected. DNA sequencing with a panel of 152 genes known to be involved in myeloid malignancies was performed on all samples. Clinical data, cytogenetic and molecular features were retrieved and their association with outcomes was studied. A Cox multivariable model was used to estimate relative weights of selected explanatory variables. The score was validated on an independent cohort of 754 Japanese patients with MDS. Study II – A total of 129 patients with MDS and RS (MDSRS+) was assembled. All samples underwent DNA sequencing according to study I and thereafter RNA sequencing of CD34 sorted bone marrow mononuclear cells. Supervised/unsupervised clustering analysis and digital sorting were performed. A Cox multivariable model was used to assess association between clinical and genomic/transcriptomic patterns and outcome. Study III – a cohort of 677 Swedish patients was gathered from study I. We collected complete information on administered E-transfusions through the nationwide SCANDAT3-S database. Cox regression analyses were used to assess associations between clinical, molecular and transfusion data, and outcome. A Markov multistate model was used to assess association between changes in transfusion patterns and outcome. Results: Study I – TP53multi, MLL-PTD and FLT3 mutations were shown to be predictive of dismal outcome. In contrast, SF3B1 mutations were associated with favorable prognosis, however this effect was significantly influenced by the co-mutation patterns. A total of 22 variables (clinical, cytogenetic, and molecular markers) were used to build the IPSS-M score, each of them carrying a specific mathematic weight according to their individual impacts on endpoints. The calculation of the IPSS-M resulted in a unique score for individual patients and assigned each case to one of the 6 IPSS-M risk categories. When compared to the IPSS-R, the IPSS-M score clearly improved outcome prediction and led to the restratification of 46% of patients. The IPSS-M is validated both in MDS/MPN with WBC count below 13x109/L and in therapy related MDS (t-MDS). Study II – Most (~90%) MDSRS+ cases were found to have a mutation in SF3B1, SRSF2 or TP53multi. Overall, TP53multi and splice factors mutations were mutually exclusive, and SF3B1 and SRSF2 mutations cooccurred in only 3% of the patients. The three genetic subgroups were shown to have very different outcomes. Supervised transcriptome analysis confirmed the distinction between SF3B1-, SRSF2- and TP53multi-mutated MDS with RS. Unsupervised clustering analysis found three transcriptomic groups, each with distinct erythroid/megakaryocytic progenitor fraction, which predicted OS independently of IPSS-M. Study III – Whereas TP53multi, poor cytogenetic and higher bone marrow blasts predicted shorter time to first E-transfusion event, higher hemoglobin level and SF3B1alpha only were associated with longer time to first E-transfusion event. Next, E-transfusion state at 8 months after diagnosis was shown to be a strong predictor of OS independently of IPSS-M. Our model based on E-transfusion state at 8 months and IPSS-M (model 2) improved significantly prognostic prediction compared to IPSS-M only (model 1). Finally, a multistate model revealed that individual transfusion trajectories during the early disease course impacted both future transfusion requirement and OS. Conclusion: This thesis provides evidence that integration of genomic data to clinical characteristics improves greatly prognostication in MDS and we suggest that the novel IPSS-M prognostic score is implemented in clinical practice to provide further guidance in therapeutic decision-making. Our work also indicates that the heterogeneity of outcome in MDS cannot be explain by genetic profiling only and that studies of gene expression and integration of dynamic parameters among other techniques will contribute to a better understanding of the clinical course. In general, this thesis advocates for the need of a holistic approach of the disease to deepen our understanding of underlying mechanisms and ultimately to improve the care of patients with MDS. Enormous efforts are currently put in the field of precision medicine in cancer. Future integrative multiomics studies will hopefully improve individualized care to increase survival and quality of life of patients with MDS

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

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

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

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

Co-mutation pattern, clonal hierarchy, and clone size concur to determine disease phenotype of SRSF2 P95-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

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

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

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