Long-read transcriptome sequencing of CLL and MDS patients uncovers molecular effects of SF3B1 mutations.

Mutations in splicing factor 3B subunit 1 (SF3B1) frequently occur in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS). These mutations have different effects on the disease prognosis with beneficial effect in MDS and worse prognosis in CLL patients. A full-length transcriptome approach can expand our knowledge on SF3B1 mutation effects on RNA splicing and its contribution to patient survival and treatment options. We applied long-read transcriptome sequencing (LRTS) to 44 MDS and CLL patients, as well as two pairs of isogenic cell lines with and without SF3B1 mutations, and found >60% of novel isoforms. Splicing alterations were largely shared between cancer types and specifically affected the usage of introns and 3' splice sites. Our data highlighted a constrained window at canonical 3' splice sites in which dynamic splice site switches occurred in SF3B1-mutated patients. Using transcriptome-wide RNA binding maps and molecular dynamics simulations, we showed multimodal SF3B1 binding at 3' splice sites and predicted reduced RNA binding at the second binding pocket of SF3B1K700E Our work presents the hitherto most complete LRTS study of the SF3B1 mutation in CLL and MDS and provides a resource to study aberrant splicing in cancer. Moreover, we showed that different disease prognosis most likely results from the different cell types expanded during carcinogenesis rather than different mechanisms of action of the mutated SF3B1 These results have important implications for understanding the role of SF3B1 mutations in hematological malignancies and other related diseases.

Molecular taxonomy of myelodysplastic syndromes and its clinical implications.

ABSTRACT: Myelodysplastic syndromes (MDS) are clonal hematologic disorders characterized by morphologic abnormalities of myeloid cells and peripheral cytopenias. Although genetic abnormalities underlie the pathogenesis of these disorders and their heterogeneity, current classifications of MDS rely predominantly on morphology. We performed genomic profiling of 3233 patients with MDS or related disorders to delineate molecular subtypes and define their clinical implications. Gene mutations, copy-number alterations, and copy-neutral loss of heterozygosity 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 loss of heterozygosity 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 (BM) blast percentage across groups ranged from 1.5% to 10%, and the median overall survival ranged 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 BM blasts depended on the genetic subtypes. Within genetic subgroups, therapy-related MDS and myelodysplastic/myeloproliferative neoplasms had comparable clinical and outcome profiles to primary MDS. In conclusion, genetically-derived subgroups of MDS are clinically relevant and might inform future classification schemas and translational therapeutic research.

DNA methylation profiling of myelodysplastic syndromes and clinical response to azacitidine: A multicentre retrospective study.

Real-world data have revealed that a substantial portion of patients with myelodysplastic syndromes (MDS) does not respond to epigenetic therapy with hypomethylating agents (HMAs). The cellular and molecular reasons for this resistance to the demethylating agent and biomarkers that would be able to predict the treatment refractoriness are largely unknown. In this study, we shed light on this enigma by characterizing the epigenomic profiles of patients with MDS treated with azacitidine. Our approach provides a comprehensive view of the evolving DNA methylation architecture of the disease and holds great potential for advancing our understanding of MDS treatment responses to HMAs.

MDS patient registries - achievements and challenges.

Since the late 1980s, patient registries have played a pivotal role in the elucidation of rare diseases. For myelodysplastic syndromes (MDS), they revealed the disease actually to be diverse rather than rare. Registry data enabled the definition of various MDS subtypes and prognostic scores tailoring therapy. These classifications have been revised and refined several times, and the differential diagnosis of MDS has become increasingly complex. At the same time, the diagnosis has been made more commonly and no longer by specialized centers of expertise only. Consequently, several registries have collected data with different focuses and from different patient subpopulations. The current review presents three MDS registries and their rationale, scope, design, and achievements. All three complement each other and will remain a mainstay to advance the knowledge on MDS as well as to validate the outcomes of clinical trials. However, delineation of subtypes after the most recent WHO and IPC revisions, as well as the determination of the newest risk score M of the International Prognostic Scoring System (IPSS-M), no longer just shift cut-offs but are based on multivariate compilations of highly specific genetic information. This paradigm shift involves challenging registries with respect to the assignment of all patients for whom this information has not yet been available.

Long-term outcome of high risk patients with myelodysplastic syndromes or secondary acute myeloid leukemia receiving intensive chemotherapy.

Intensive chemotherapy (IC) used to be a common treatment approach for patients with higher-risk myelodysplastic syndromes (MDS) or acute myeloid leukemia after MDS (sAML). We conducted a retrospective analysis of 299 patients, including a matched pair analysis comparing 96 patients receiving IC with 96 patients not undergoing IC, in order to evaluate the impact of IC on overall survival (OS) and to identify factors that influence remission rates and OS. Complete remission (CR) after first induction chemotherapy was reached in 50% of patients. Parameters influencing the probability of achieving CR were blast count in the bone marrow (< 30%), age < 65 years, presence of Auer rods, duration of antecedent MDS shorter than 6 months, and timing of IC in relation to first diagnosis. The difference in survival time was not significantly better for patients receiving IC (median OS 12.7 months vs. 7 months). Parameters favorably influencing survival were the presence of Auer rods, age below 60 years, blast count below 30%, IC given shortly after first diagnosis, and achievement of CR. On multivariate analysis, achieving CR, presence of Auer rods, and percentage of blasts below or above 30% significantly influenced median survival. Relapse occurred in 63% of patients after a median of 9.9 months with a median survival of 7.6 months. Considering the high relapse rate and short survival, we conclude that intensive chemotherapy is not promising for high-risk MDS or sAML.

Effect of deferasirox + erythropoietin vs erythropoietin on erythroid response in Low/Int-1-risk MDS patients: Results of the phase II KALLISTO trial.

OBJECTIVES: Erythropoiesis-stimulating agents (ESAs) remain first-choice to treat symptomatic anemia and delay transfusion dependence in most patients with lower-risk myelodysplastic syndromes (MDS) without del(5q). Deferasirox increased erythroid responses in some lower-risk MDS patients in clinical trials, and adding low-dose deferasirox to ESA treatment may further improve erythroid response. METHODS: KALLISTO (NCT01868477) was a randomized, open-label, multicenter, phase II study. Lower-risk MDS patients received deferasirox at 10 mg/kg/d (dispersible tablets) or 7 mg/kg/d (film-coated tablets) plus erythropoietin (n = 11), or erythropoietin alone (n = 12) for 24 weeks. The primary endpoint was the between-group difference in erythroid response within 12 weeks. RESULTS: Erythroid response occurred in 27.3% of patients receiving deferasirox plus erythropoietin vs 41.7% of patients receiving erythropoietin alone within 12 weeks (difference 14.4%; 95% CI -24.0, 48.16). Within 24 weeks, the hematologic response rate was 27.3% with deferasirox plus erythropoietin vs 50% with erythropoietin alone, and hematologic improvement rates were 45.5% vs 100%. Deferasirox plus erythropoietin was generally well tolerated. CONCLUSIONS: In this small pilot study, combining low-dose deferasirox with erythropoietin did not improve erythroid response. It remains of interest to investigate early chelation approaches with even lower deferasirox doses plus erythropoietin in lower-risk MDS patients before the onset of transfusion dependence.

Cytogenetic clonal evolution in myelodysplastic syndromes is associated with inferior prognosis.

BACKGROUND: The karyotype of bone marrow cells at the time of diagnosis is a strong prognostic parameter for overall survival as well as acute myeloid leukemia (AML) progression in patients with myelodysplastic syndromes (MDS). However, to the authors' knowledge, few data exist regarding the prognostic impact of cytogenetic clonal evolution during the course of MDS. METHODS: The authors evaluated follow-up karyotype analyses in 549 patients from the Dusseldorf MDS Registry. RESULTS: Clonal evolution was detectable in 24% of the entire cohort and in 18% of 294 patients receiving best supportive care. The authors noted a clear adverse effect of clonal evolution on the risk of leukemic transformation (hazard ratio, 2.233; P = .036) and overall survival (hazard ratio, 3.677; P<.001). The authors also analyzed the prognostic influence of subclones detectable at the time of diagnosis. Again, such a finding was associated with a significantly shorter overall survival and a higher 5-year-probability of acute myeloid leukemia progression (30% vs 22%). CONCLUSIONS: The results of the current study support the belief that follow-up karyotype analyses should be performed, especially in patients with lower-risk and intermediate-risk MDS, to identify those patients who are at higher risk of disease progression and therefore might benefit from earlier or more intensive treatment. Cancer 2017;123:4608-4616. © 2017 American Cancer Society.

A retrospective study evaluating the impact of infectious complications during azacitidine treatment.

Azacitidine has become an available therapy for high-risk myelodysplastic syndromes. Infectious complications (IC) may impede the success of therapy. Since most patients are managed in an outpatient setting, often with low level of clinical and microbiological documentation, the impact of IC remains unclear. We retrospectively evaluated the clinical course of 77 patients with MDS treated with azacitidine between 2004 and 2015 (median age 69 years). Clinical workup included severity and type of IC, days in the hospital and with antimicrobial therapy, response to azacitidine, and overall survival (OS). In total, 614 azacitidine cycles were administered, 81 cycles with at least one IC. The median number of administered cycles was 6 (range 1-43). Median OS after the start of azacitidine was 17 months (range 1-103). Infection rates were higher in the first 3 cycles with bacterial infections leading. The better patients' hematological response to azacitidine with less IC occurred, and fewer days with antimicrobial treatment were needed. Compared to progressive disease, stable disease made no significant improvement in occurrence of IC and days in the hospital. Older age was associated with more IC and longer time in the hospital. Comorbidities or IPSS-R had no influence on IC. The incidence of IC correlated with hematological response and age. Stable disease led to longer OS, but incidence of IC was comparable to progressive disease and survival seemed to be bought by a considerable number of IC. IC rates were highest in the first 3 cycles. We recommend response evaluation after 4-6 cycles.

Causes of death in 2877 patients with myelodysplastic syndromes.

Patients with myelodysplastic syndromes face a poor prognosis. The exact causes of death have not been described properly in the past. We performed a retrospective analysis of causes of death using data of 3792 patients in the Düsseldorf registry who have been followed up for a median time of 21 months. Medical files as well as death certificates were screened and primary care physicians were contacted. Death after AML evolution, infection, and bleeding was considered to be clearly disease-related. Further categories of causes of death were heart failure, other possibly disease-related reasons, such as hemochromatosis, disease-independent reasons as well as cases with unclear causes of death. Median age at the time of diagnosis was 71 years. At the time of analysis, 2877 patients (75.9 %) had deceased. In 1212 cases (42.1 %), the exact cause of death could not be ascertained. From 1665 patients with a clearly documented cause of death, 1388 patients (83.4 %) succumbed directly disease-related (AML (46.6 %), infection (27.0 %), bleeding (9.8 %)), whereas 277 patients (16.6 %) died for reasons not directly related with myelodysplastic syndromes (MDS), including 132 patients with cardiac failure, 77 non-disease-related reasons, 23 patients with solid tumors, and 45 patients with possibly disease-related causes like hemochromatosis. Correlation with IPSS, IPSS-R, and WPSS categories showed a proportional increase of disease-related causes of death with increasing IPSS/IPSS-R/WPSS risk category. Likewise, therapy-related MDS were associated with a higher percentage of disease-related causes of death than primary MDS. This reflects the increasing influence of the underlying disease on the cause of death with increasing aggressiveness of the disease.

In patients with myelodysplastic syndromes with del(5q), factors other than age and sex contribute to the prognostic advantage, which diminishes over time.

This study aimed to determine the extent to which the prognostic advantage of myelodysplastic syndromes (MDS) with del(5q) is due to the more favourable age and sex distribution of patients in that group when compared to other MDS subtypes. A total of 1912 MDS patients from the Duesseldorf registry with less than 5% blasts in the bone marrow were evaluable and had complete covariates. As endpoints, overall survival and progression to acute myeloid leukaemia (AML) were considered. Cox models were computed for both outcomes. A multivariate Cox model for survival confirmed higher age and male sex as risk factors. In addition, we found a survival advantage of 9·1 years for MDS del(5q) patients compared to refractory cytopenia with unilineage dysplasia, while the survival advantage of MDS del(5q) over refractory cytopenia with multilineage dysplasia was 18·6 years. Considering progression to AML, we did not find any significant differences between the World Health Organization classification subtypes. Our analyses show that the higher survival probabilities of MDS del(5q) patients are not only due to age and sex, although higher age and male sex were also important risk factors. Interestingly, it seems that the survival advantage of MDS del(5q) decreases over time.

Diagnosis and treatment of primary myelodysplastic syndromes in adults: recommendations from the European LeukemiaNet.

Within the myelodysplastic syndrome (MDS) work package of the European LeukemiaNet, an Expert Panel was selected according to the framework elements of the National Institutes of Health Consensus Development Program. A systematic review of the literature was performed that included indexed original papers, indexed reviews and educational papers, and abstracts of conference proceedings. Guidelines were developed on the basis of a list of patient- and therapy-oriented questions, and recommendations were formulated and ranked according to the supporting level of evidence. MDSs should be classified according to the 2008 World Health Organization criteria. An accurate risk assessment requires the evaluation of not only disease-related factors but also of those related to extrahematologic comorbidity. The assessment of individual risk enables the identification of fit patients with a poor prognosis who are candidates for up-front intensive treatments, primarily allogeneic stem cell transplantation. A high proportion of MDS patients are not eligible for potentially curative treatment because of advanced age and/or clinically relevant comorbidities and poor performance status. In these patients, the therapeutic intervention is aimed at preventing cytopenia-related morbidity and preserving quality of life. A number of new agents are being developed for which the available evidence is not sufficient to recommend routine use. The inclusion of patients into prospective clinical trials is strongly recommended.

Impaired cytotoxicity associated with defective natural killer cell differentiation in myelodysplastic syndromes.

Natural killer cells are well known to mediate anti-leukemic responses in myeloid leukemia but their role in myelodysplastic syndromes is not well understood. Here, in a cohort of newly diagnosed patients (n=75), widespread structural and functional natural killer cell defects were identified. One subgroup of patients (13%) had a selective deficiency of peripheral natural killer cells (count <10/mm(3) blood) with normal frequencies of T and natural killer-like T cells. Natural killer cell-deficient patients were predominantly found in high-risk subgroups and deficiency of these cells was significantly associated with poor prognosis. In the second subgroup, comprising the majority of patients (76%), natural killer cells were present but exhibited poor cytotoxicity. The defect was strongly associated with reduced levels of perforin and granzyme B. Notably, natural killer cell function and arming of cytotoxic granules could be fully reconstituted by in vitro stimulation. Further phenotypic analysis of these patients revealed an immature natural killer cell compartment that was biased towards CD56(bright) cells. The residual CD56(dim) cells exhibited a significant increase of the unlicensed NKG2A(-)KIR(-) subset and a striking reduction in complexity of the repertoire of killer cell immunoglobulin-like receptors. Taken together, these results suggest that the widespread defects in natural killer cell function occurring in patients with myelodysplastic syndromes are mostly due to either unsuccessful or inefficient generation of mature, functionally competent natural killer cells, which might contribute to disease progression through impaired immune surveillance.

Cellularity, characteristics of hematopoietic parameters and prognosis in myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes (MDS) present with a normo- or hyperplastic bone marrow in most cases. We aimed at a characterization of patients with different types of cellularity. METHODS: We assessed marrow cellularity both by histology and cytology in 1270 patients and analyzed hematologic, cytogenetic, and prognostic parameters accordingly. RESULTS: The concordance of the assessment of cellularity differed dramatically between histology and cytology as only 36.5% were described as hypocellular by both methods (P < 0.0005) (hypocellular 16.4%, normocellular 23.3%, hypercellular 60.3%). There were no major differences with regard to hematopoietic insufficiency. The presence of fibrosis was associated to hypercellular bone marrow. Median survival differed from 38 months in hypocellular, 42 months in normocellular, and 25 months in hypercellular MDS (P < 0.0005). AML progression rates were 33% for hypercellular MDS after 2 yr, whereas hypo- and normocellular had a progression rate of 19% after 2 yr (P = 0.018). IPSS and IPSS-R were able to identify different risk groups within all three cellularity groups. CONCLUSION: Based on our data, hypocellular patients obviously do not present as a separate entity, as there were no striking differences with regard to cytogenetics and WHO types. Assessment of cellularity should be performed by histopathology.

Imetelstat: Finally a disease-modifying treatment for lower-risk myelodysplastic syndromes?

Favorable results were achieved in a phase 3 clinical trial (IMerge) with the telomerase inhibitor imetelstat in transfusion-dependent patients with lower-risk myelodysplastic syndromes (MDSs) who relapsed or were refractory to erythropoiesis-stimulating agents.1 Imetelstat is likely to become a useful addition to our limited therapeutic options for patients with MDS.

Comparison of cytomorphology and histomorphology in myelodysplastic syndromes.

Gold standard for the establishment of the diagnosis of myelodysplastic syndromes (MDS) are cytomorphological features of hematopoietic cells in peripheral blood and bone marrow aspirates. There is increasing evidence that bone marrow histomorphology not only aids in the diagnosis of MDS but can provide additional prognostic information, particularly through assessment of fibrosis and cellularity. However, there is only sparse data on direct comparison between histological and cytomorphological findings within the same MDS patient cohort. Therefore, we performed such an analysis under exceptionally well-standardized conditions. We reexamined biopsy material of 128 patients from the Düsseldorf MDS registry who underwent bone marrow trephine biopsy (in addition to bone marrow aspiration) at the time of diagnosis, addressing the following items: a. Analysis of concordance of diagnoses made by histology and cytomorphology b. Analysis of additional information by histology with regard to the diagnosis and prognosis. The respective biomaterials were available at our institution and had been processed according to unchanged protocols between 1992 and 2010. Fresh histopathological sections were obtained from the tissue blocks, stained under identical conditions and re-assessed by a designated expert pathologist (C.B.) without knowledge of the previous histopathological report or the respective cytomorphological diagnosis. The latter, likewise, was uniformly made by the same expert cytomorphologist (U.G.). Histopathology of bone marrow trephine biopsies reliably captured the diagnosis of MDS. Assignment to the diagnostic WHO subgroup was not entirely concordant with cytomorphology, mainly due to incongruences between the proportion of CD34-positive cells on histopathology and the cytomorphological blast count. Histopathology provided additional diagnostic and prognostic information with high diagnostic and prognostic significance, such as fibrosis. Likewise, histopathology allowed more reliable estimation of bone marrow cellularity.

Validation of a novel algorithm with a high specificity in ruling out MDS.

INTRODUCTION: A previously published web-based App using Gradient-boosted models (GBMs) of eight laboratory parameters was established by Oster et al. to facilitate diagnosis or exclusion of myelodysplastic syndromes (MDS) in patients. METHODS: To validate their algorithm, we compared 175 anemic patients with MDS diagnosis from our German MDS Registry with 1378 non-MDS anemic patients who consulted various specialties in the Düsseldorf university hospital. RESULTS: Based on hemoglobin level, leukocyte and platelet count, mean corpuscular volume, absolute neutrophil count, absolute monocyte count, glucose and creatinine, plus the patients' gender and age, we could not reproduce a high negative predictive value (NPV), but confirmed a useful specificity of 90.9% and a positive predictive value (PPV) of 77.1%. 1192 of 1378 controls were correctly categorized as "probably not MDS (pnMDS)" patients. A total of 65 patients were wrongly classified as "probable MDS (pMDS)," of whom 48 had alternative explanations for their altered laboratory results. In a second analysis, we included 29 patients with chronic myelomonocytic leukemia (CMML) resulting in only one label as possible MDS, suggesting that highly proliferative bone marrow disorders are correctly excluded. CONCLUSION: The possibility of reliably excluding MDS from differential diagnosis based on peripheral blood lab work appears to be attractive for patients and physicians alike while the confirmation of MDS diagnosis still requires a bone marrow biopsy.

Infectious Complications in Patients with Myelodysplastic Syndromes: A Report from the Düsseldorf MDS Registry.

Despite notable advancements in infection prevention and treatment, individuals with hematologic malignancies still face the persistent threat of frequent and life-threatening complications. Those undergoing chemotherapy or other disease-modifying therapies are particularly vulnerable to developing infectious complications, increasing the risk of mortality. Myelodysplastic syndromes (MDS) predominantly affect the elderly, with the incidence rising with age and peaking at around 70 years. Patients with MDS commonly present with unexplained low blood-cell counts, primarily anemia, and often experience varying degrees of neutropenia as the disease progresses. In our subsequent retrospective study involving 1593 patients from the Düsseldorf MDS Registry, we aimed at outlining the incidence of infections in MDS patients and identifying factors contributing to heightened susceptibility to infectious complications in this population.

Introduction of a New Satellite Model for Participation in Clinical Trials in a Consortial Comprehensive Cancer Center with Four University Hospitals in Germany.

INTRODUCTION: The trend toward personalized medicine leads to very small study cohorts for clinical trials, which makes it difficult to recruit patients in a single study center. On the other hand, the administrative effort required to initiate a clinical trial is very high. As a result, Germany runs the risk of falling behind other countries as a trial location. For this reason, the Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD) has been working on the challenge of a new satellite model in which the main trial center is the only one to conclude a trial center contract with the sponsor and also handles all formalities with it. The remaining sites constitute the satellites. In contrast to former satellite models, the entire study-related interventions are carried out at each site in the present model. METHODS: In order to evaluate the approvability of the model, contact was made with both higher federal authorities and the responsible inspectorate, and none of them declared themselves responsible for a possible basic approval. The four ethics committees contacted agreed to the model subject to certain framework conditions. In addition, the model was validated by the preparation of several legal opinions on various issues (medical, labor, antitrust law). CONCLUSION: Study participation close to home is a decisive advantage for multimorbid patients. As up to four locations form a trial site in the model, a large catchment area can be covered with reduced administrative costs. The satellite model developed is intended to give patients broader access to medical innovations in cancer therapy.

Nilotinib is effective in patients with chronic myeloid leukemia in chronic phase after imatinib resistance or intolerance: 24-month follow-up results.

Nilotinib is a potent selective inhibitor of the BCR-ABL tyrosine kinase approved for use in patients with newly diagnosed chronic myeloid leukemia in chronic phase (CML-CP), and in CML-CP and CML-accelerated phase after imatinib failure. Nilotinib (400 mg twice daily) was approved on the basis of the initial results of this phase 2 open-label study. The primary study endpoint was the proportion of patients achieving major cytogenetic response (CyR). All patients were followed for ≥ 24 months or discontinued early. Of 321 patients, 124 (39%) continue on nilotinib treatment. Overall, 59% of patients achieved major CyR; this was complete CyR (CCyR) in 44%. Of patients achieving CCyR, 56% achieved major molecular response. CyRs were durable, with 84% of patients who achieved CCyR maintaining response at 24 months. The overall survival at 24 months was 87%. Adverse events were mostly mild to moderate, generally transient, and easily managed. This study indicates that nilotinib is effective, with a manageable safety profile, and can provide favorable long-term benefits for patients with CML-CP after imatinib failure.