Erica Travaglino

Prognostic Factors and Life Expectancy in Myelodysplastic Syndromes Classified According to WHO Criteria: A Basis for Clinical Decision Making

PURPOSE The aim of this study was to evaluate the prognostic value of the WHO proposal, to assess the role of the main prognostic factors in myelodysplastic syndromes (MDSs) classified into WHO subgroups, and to estimate mortality (standardized mortality ratio [SMR]) and life expectancy in these groups as a basis for clinical decision making. PATIENTS AND METHODS Four hundred sixty-seven patients who were diagnosed as having de novo MDS at the Division of Hematology, University of Pavia (Pavia, Italy), between 1992 and 2002, were evaluated retrospectively for clinical and hematologic features at diagnosis, overall survival (OS), and progression to leukemia (leukemia-free survival). RESULTS Significant differences in survival were noted between patients with refractory anemia (RA), refractory cytopenia with multilineage dysplasia, RA with excess blasts, type 1 (RAEB-1), and RAEB-2. The effect of demographic factors on OS was observed in MDS patients without excess blasts (age, P = .001; sex, P = .006), as in the general population. The mortality of RA patients 70 years or older did not differ significantly from that of the general population (SMR, 1.62; P = .06). Cytogenetics was the only International Prognostic Scoring System variable showing a prognostic value in MDS classified into WHO subgroups. Transfusion-dependent patients had a significantly shorter survival than patients who did not require transfusions (P < .001). Developing a secondary iron overload significantly affected the survival of transfusion-dependent patients (P = .003). CONCLUSION These data show that the WHO classification of MDSs has a relevant prognostic value. This classification, along with cytogenetics, might be useful in decisions regarding transplantation. MDS with isolated erythroid lineage dysplasia identifies a subset of truly low-risk patients, for whom a conservative approach is advisable.

Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts.

BACKGROUND Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. METHODS We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. RESULTS We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. CONCLUSIONS Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).

Clinical significance of SF3B1 mutations in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms

In a previous study, we identified somatic mutations of SF3B1, a gene encoding a core component of RNA splicing machinery, in patients with myelodysplastic syndrome (MDS). Here, we define the clinical significance of these mutations in MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). The coding exons of SF3B1 were screened using massively parallel pyrosequencing in patients with MDS, MDS/MPN, or acute myeloid leukemia (AML) evolving from MDS. Somatic mutations of SF3B1 were found in 150 of 533 (28.1%) patients with MDS, 16 of 83 (19.3%) with MDS/MPN, and 2 of 38 (5.3%) with AML. There was a significant association of SF3B1 mutations with the presence of ring sideroblasts (P < .001) and of mutant allele burden with their proportion (P = .002). The mutant gene had a positive predictive value for ring sideroblasts of 97.7% (95% confidence interval, 93.5%-99.5%). In multivariate analysis including established risk factors, SF3B1 mutations were found to be independently associated with better overall survival (hazard ratio = 0.15, P = .025) and lower risk of evolution into AML (hazard ratio = 0.33, P = .049). The close association between SF3B1 mutations and disease phenotype with ring sideroblasts across MDS and MDS/MPN is consistent with a causal relationship. Furthermore, SF3B1 mutations are independent predictors of favorable clinical outcome, and their incorporation into stratification systems might improve risk assessment in MDS.

Impact of the degree of anemia on the outcome of patients with myelodysplastic syndrome and its integration into the WHO classification-based Prognostic Scoring System (WPSS)

Background Anemia is an established negative prognostic factor in myelodysplastic syndromes but the relationship between its degree and clinical outcome is poorly defined. We, therefore, studied the relationship between severity of anemia and outcome in myelodysplastic syndrome patients. Design and Methods We studied 840 consecutive patients diagnosed with myelodysplastic syndromes at the Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, and 504 patients seen at the Heinrich-Heine-University Hospital, Düsseldorf, Germany. Hemoglobin levels were monitored longitudinally and analyzed by means of time-dependent Cox’s proportional hazards regression models. Results Hemoglobin levels lower than 9 g/dL in males (HR 5.56, P=0.018) and 8 g/dL in females (HR=5.35, P=0.026) were independently related to reduced overall survival, higher risk of non-leukemic death and cardiac death (P<0.001). Severe anemia, defined as hemoglobin below these thresholds, was found to be as effective as transfusion-dependency in the prognostic assessment. After integrating this definition of severe anemia into the WHO classification-based Prognostic Scoring System, time-dependent regression and landmark analyses showed that the refined model was able to identify risk groups with different survivals at any time during follow up. Conclusions Accounting for severity of anemia through the WHO classification-based Prognostic Scoring System provides an objective criterion for prognostic assessment and implementation of risk-adapted treatment strategies in myelodysplastic syndrome patients.

Clinical Relevance of Bone Marrow Fibrosis and CD34-Positive Cell Clusters in Primary Myelodysplastic Syndromes

PURPOSE We studied bone marrow (BM) histologic abnormalities in myelodysplastic syndromes (MDS) classified according to WHO criteria to determine their clinical correlates and prognostic value. PATIENTS AND METHODS Three hundred one consecutive patients were retrospectively evaluated for BM fibrosis and CD34 immunoreactivity. Marrow fibrosis was assessed following the European consensus guidelines. RESULTS Moderate to severe BM fibrosis was detected in 17% of cases and was associated with multilineage dysplasia (P = .001), high transfusion requirement (P < .001), and poor-risk cytogenetics (P = .007). CD34+ cell clusters were found in 23% of patients and were associated with WHO categories with excess of blasts (P < .001) and poor-risk cytogenetics (P = .001). In multivariable analysis, BM fibrosis and presence of CD34+ cell clusters had independent negative impact on overall survival (P < .001 and P = .019, respectively) and leukemia-free survival (P < .001 and P = .004, respectively). A hierarchical clustering analysis identified three subsets of patients with distinct clinical features. One cluster consisted mainly of patients with BM fibrosis, multilineage dysplasia, and high transfusion requirement; these individuals had lower overall survival and leukemia-free survival (P = .001 and P < .001, respectively). Within patients stratified according to International Prognostic Scoring System and WHO classification-based Prognostic Scoring System categories, BM fibrosis involved a shift to a one-step more advanced risk group. CONCLUSION BM fibrosis identifies a distinct subgroup of MDS with multilineage dysplasia, high transfusion requirement, and poor prognosis and represents an independent prognostic factor that may be useful in clinical decision making. Furthermore, the presence of CD34+ cell clusters is an independent risk factor for progression to acute leukemia.

Risk stratification based on both disease status and extra-hematologic comorbidities in patients with myelodysplastic syndrome

The incidence of myelodysplastic syndromes increases with age and a high prevalence of co-morbid conditions has been reported in these patients. So far, risk assessment in myelodysplastic syndromes has been mainly based on disease status. We studied the prognostic impact of comorbidity on the natural history of myelodysplastic syndrome with the aim of developing novel tools for risk assessment. The study population included a learning cohort of 840 patients diagnosed with myelodysplastic syndrome in Pavia, Italy, and a validation cohort of 504 patients followed in Duesseldorf, Germany. Information on comorbidity was extracted from detailed review of the patients’ medical charts and laboratory values at diagnosis and during the course of the disease. Univariable and multivariable survival analyses with both fixed and time-dependent covariates were performed using Cox’s proportional hazards regression models. Comorbidity was present in 54% of patients in the learning cohort. Cardiac disease was the most frequent comorbidity and the main cause of non-leukemic death. In multivariable analysis, comorbidity had a significant impact on both non-leukemic death (P=0.01) and overall survival (P=0.02). Cardiac, liver, renal, pulmonary disease and solid tumors were found to independently affect the risk of non-leukemic death. A time-dependent myelodysplastic syndrome-specific comorbidity index (MDS-CI) was developed for predicting the effect of comorbidity on outcome. This identified three groups of patients which showed significantly different probabilities of non-leukemic death (P<0.001) and survival (P=0.005) also in the validation cohort. Landmark survival analyses at fixed time points from diagnosis showed that the MDS-CI can better define the life expectancy of patients with myelodysplastic syndrome stratified according to the WHO-classification based Prognostic Scoring System (WPSS).Comorbidities have a significant impact on the outcome of patients with myelodysplastic syndrome. Accounting for both disease status by means of the WPSS and comorbidity through the MDS-CI considerably improves risk stratification in myelodysplastic syndromes.

SF3B1 mutation identifies a distinct subset of myelodysplastic syndrome with ring sideroblasts

Refractory anemia with ring sideroblasts (RARS) is a myelodysplastic syndrome (MDS) characterized by isolated erythroid dysplasia and 15% or more bone marrow ring sideroblasts. Ring sideroblasts are found also in other MDS subtypes, such as refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS). A high prevalence of somatic mutations of SF3B1 was reported in these conditions. To identify mutation patterns that affect disease phenotype and clinical outcome, we performed a comprehensive mutation analysis in 293 patients with myeloid neoplasm and 1% or more ring sideroblasts. SF3B1 mutations were detected in 129 of 159 cases (81%) of RARS or RCMD-RS. Among other patients with ring sideroblasts, lower prevalence of SF3B1 mutations and higher prevalence of mutations in other splicing factor genes were observed (P < .001). In multivariable analyses, patients with SF3B1 mutations showed significantly better overall survival (hazard ratio [HR], .37; P = .003) and lower cumulative incidence of disease progression (HR = 0.31; P = .018) compared with SF3B1-unmutated cases. The independent prognostic value of SF3B1 mutation was retained in MDS without excess blasts, as well as in sideroblastic categories (RARS and RCMD-RS). Among SF3B1-mutated patients, coexisting mutations in DNA methylation genes were associated with multilineage dysplasia (P = .015) but had no effect on clinical outcome. TP53 mutations were frequently detected in patients without SF3B1 mutation, and were associated with poor outcome. Thus, SF3B1 mutation identifies a distinct MDS subtype that is unlikely to develop detrimental subclonal mutations and is characterized by indolent clinical course and favorable outcome.

Driver somatic mutations identify distinct disease entities within myeloid neoplasms with myelodysplasia

Our knowledge of the genetic basis of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) has considerably improved. To define genotype/phenotype relationships of clinical relevance, we studied 308 patients with MDS, MDS/MPN, or acute myeloid leukemia evolving from MDS. Unsupervised statistical analysis, including the World Health Organization classification criteria and somatic mutations, showed that MDS associated with SF3B1-mutation (51 of 245 patients, 20.8%) is a distinct nosologic entity irrespective of current morphologic classification criteria. Conversely, MDS with ring sideroblasts with nonmutated SF3B1 segregated in different clusters with other MDS subtypes. Mutations of genes involved in DNA methylation, splicing factors other than SF3B1, and genes of the RAS pathway and cohesin complex were independently associated with multilineage dysplasia and identified a distinct subset (51 of 245 patients, 20.8%). No recurrent mutation pattern correlated with unilineage dysplasia without ring sideroblasts. Irrespective of driver somatic mutations, a threshold of 5% bone marrow blasts retained a significant discriminant value for identifying cases with clonal evolution. Comutation of TET2 and SRSF2 was highly predictive of a myeloid neoplasm characterized by myelodysplasia and monocytosis, including but not limited to, chronic myelomonocytic leukemia. These results serve as a proof of concept that a molecular classification of myeloid neoplasms is feasible.

Molecular and clinical features of refractory anemia with ringed sideroblasts associated with marked thrombocytosis

We studied patients with myeloid neoplasm associated with ringed sideroblasts and/or thrombocytosis. The combination of ringed sideroblasts 15% or greater and platelet count of 450 x 10(9)/L or greater was found in 19 subjects fulfilling the diagnostic criteria for refractory anemia with ringed sideroblasts (RARS) associated with marked thrombocytosis (RARS-T), and in 3 patients with primary myelofibrosis. JAK2 and MPL mutations were detected in circulating granulocytes and bone marrow CD34+ cells, but not in T lymphocytes, from 11 of 19 patients with RARS-T. Three patients with RARS, who initially had low to normal platelet counts, progressed to RARS-T, and 2 of them acquired JAK2 (V617F) at this time. In female patients with RARS-T, granulocytes carrying JAK2 (V617F) represented only a fraction of clonal granulocytes as determined by X-chromosome inactivation patterns. RARS and RARS-T patient groups both consistently showed up-regulation of ALAS2 and down-regulation of ABCB7 in CD34+ cells, but several other genes were differentially expressed, including PSIP1 (LEDGF), CXCR4, and CDC2L5. These observations suggest that RARS-T is indeed a myeloid neoplasm with both myelodysplastic and myeloproliferative features at the molecular and clinical levels and that it may develop from RARS through the acquisition of somatic mutations of JAK2, MPL, or other as-yet-unknown genes.

Flow cytometry evaluation of erythroid dysplasia in patients with myelodysplastic syndrome

Erythroid dysplasia is the pathologic hallmark of myelodysplastic syndromes (MDS). To develop a quantitative flow-cytometry approach to its evaluation, we analyzed the expression of CD71, CD105, cytosolic H-ferritin (HF), cytosolic L-ferritin (LF) and mitochondrial ferritin (MtF) in erythroblasts from 104 MDS patients, 69 pathologic control patients and 19 healthy subjects. Six-parameter, 4-color flow cytometry was employed, and data were expressed as mean fluorescence intensity. Compared with pathologic and healthy controls, MDS patients had higher expression of HF (P<0.001) and CD105 (P<0.001), and lower expression of CD71 (P<0.001). MtF was specifically detected in MDS with ringed sideroblasts, and there was a close relationship between its expression and Prussian blue staining (r=0.89, P<0.001). In vitro cultures of myelodysplastic hematopoietic progenitors showed that both HF and MtF were expressed at a very early stage of erythroid differentiation, and that MtF expression is specifically related to mitochondrial iron loading. A classification function based on expression levels of HF, CD71 and CD105 allowed us to correctly classify >95% of MDS patients. This flow-cytometry approach provides an accurate quantitative evaluation of erythroid dysplasia and allows a reliable diagnosis of sideroblastic anemia, and may therefore be a useful tool in the work-up of patients with MDS.