Ilaria Ambaglio

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.

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.

Development and validation of a prognostic scoring system for patients with chronic myelomonocytic leukemia

The natural course of chronic myelomonocytic leukemia (CMML) is highly variable but a widely accepted prognostic scoring system for patients with CMML is not available. The main aim of this study was to develop a new CMML-specific prognostic scoring system (CPSS) in a large series of 558 patients with CMML (training cohort, Spanish Group of Myelodysplastic Syndromes) and to validate it in an independent series of 274 patients (validation cohort, Heinrich Heine University Hospital, Düsseldorf, Germany, and San Matteo Hospital, Pavia, Italy). The most relevant variables for overall survival (OS) and evolution to acute myeloblastic leukemia (AML) were FAB and WHO CMML subtypes, CMML-specific cytogenetic risk classification, and red blood cell (RBC) transfusion dependency. CPSS was able to segregate patients into 4 clearly different risk groups for OS (P < .001) and risk of AML evolution (P < .001) and its predictive capability was confirmed in the validation cohort. An alternative CPSS with hemoglobin instead of RBC transfusion dependency offered almost identical prognostic capability. This study confirms the prognostic impact of FAB and WHO subtypes, recognizes the importance of RBC transfusion dependency and cytogenetics, and offers a simple and powerful CPSS for accurately assessing prognosis and planning therapy in patients with CMML.

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.

Minimal morphological criteria for defining bone marrow dysplasia: a basis for clinical implementation of WHO classification of myelodysplastic syndromes.

The World Health Organization classification of myelodysplastic syndromes (MDS) is based on morphological evaluation of marrow dysplasia. We performed a systematic review of cytological and histological data from 1150 patients with peripheral blood cytopenia. We analyzed the frequency and discriminant power of single morphological abnormalities. A score to define minimal morphological criteria associated to the presence of marrow dysplasia was developed. This score showed high sensitivity/specificity (>90%), acceptable reproducibility and was independently validated. The severity of granulocytic and megakaryocytic dysplasia significantly affected survival. A close association was found between ring sideroblasts and SF3B1 mutations, and between severe granulocytic dysplasia and mutation of ASXL1, RUNX1, TP53 and SRSF2 genes. In myeloid neoplasms with fibrosis, multilineage dysplasia, hypolobulated/multinucleated megakaryocytes and increased CD34+ progenitors in the absence of JAK2, MPL and CALR gene mutations were significantly associated with a myelodysplastic phenotype. In myeloid disorders with marrow hypoplasia, granulocytic and/or megakaryocytic dysplasia, increased CD34+ progenitors and chromosomal abnormalities are consistent with a diagnosis of MDS. The proposed morphological score may be useful to evaluate the presence of dysplasia in cases without a clearly objective myelodysplastic phenotype. The integration of cytological and histological parameters improves the identification of MDS cases among myeloid disorders with fibrosis and hypocellularity.

Clinical significance of somatic mutation in unexplained blood cytopenia

Unexplained blood cytopenias, in particular anemia, are often found in older persons. The relationship between these cytopenias and myeloid neoplasms like myelodysplastic syndromes is currently poorly defined. We studied a prospective cohort of patients with unexplained cytopenia with the aim to estimate the predictive value of somatic mutations for identifying subjects with, or at risk of, developing a myeloid neoplasm. The study included a learning cohort of 683 consecutive patients investigated for unexplained cytopenia, and a validation cohort of 190 patients referred for suspected myeloid neoplasm. Using granulocyte DNA, we looked for somatic mutations in 40 genes that are recurrently mutated in myeloid malignancies. Overall, 435/683 patients carried a somatic mutation in at least 1 of these genes. Carrying a somatic mutation with a variant allele frequency ≥0.10, or carrying 2 or more mutations, had a positive predictive value for diagnosis of myeloid neoplasm equal to 0.86 and 0.88, respectively. Spliceosome gene mutations and comutation patterns involving TET2, DNMT3A, or ASXL1 had positive predictive values for myeloid neoplasm ranging from 0.86 to 1.0. Within subjects with inconclusive diagnostic findings, carrying 1 or more somatic mutations was associated with a high probability of developing a myeloid neoplasm during follow-up (hazard ratio = 13.9, P < .001). The predictive values of mutation analysis were confirmed in the independent validation cohort. The findings of this study indicate that mutation analysis on peripheral blood granulocytes may significantly improve the current diagnostic approach to unexplained cytopenia and more generally the diagnostic accuracy of myeloid neoplasms.

Integrating clinical features and genetic lesions in the risk assessment of patients with chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm with variable clinical course. To predict the clinical outcome, we previously developed a CMML-specific prognostic scoring system (CPSS) based on clinical parameters and cytogenetics. In this work, we tested the hypothesis that accounting for gene mutations would further improve risk stratification of CMML patients. We therefore sequenced 38 genes to explore the role of somatic mutations in disease phenotype and clinical outcome. Overall, 199 of 214 (93%) CMML patients carried at least 1 somatic mutation. Stepwise linear regression models showed that these mutations accounted for 15% to 24% of variability of clinical phenotype. Based on multivariable Cox regression analyses, cytogenetic abnormalities and mutations in RUNX1, NRAS, SETBP1, and ASXL1 were independently associated with overall survival (OS). Using these parameters, we defined a genetic score that identified 4 categories with significantly different OS and cumulative incidence of leukemic evolution. In multivariable analyses, genetic score, red blood cell transfusion dependency, white blood cell count, and marrow blasts retained independent prognostic value. These parameters were included into a clinical/molecular CPSS (CPSS-Mol) model that identified 4 risk groups with markedly different median OS (from >144 to 18 months, hazard ratio [HR] = 2.69) and cumulative incidence of leukemic evolution (from 0% to 48% at 4 years, HR = 3.84) (P < .001). The CPSS-Mol fully retained its ability to risk stratify in an independent validation cohort of 260 CMML patients. In conclusion, integrating conventional parameters and gene mutations significantly improves risk stratification of CMML patients, providing a robust basis for clinical decision-making and a reliable tool for clinical trials.

Inappropriately low hepcidin levels in patients with myelodysplastic syndrome carrying a somatic mutation of SF3B1

Somatic mutations of the RNA splicing machinery have been recently identified in myelodysplastic syndromes. In particular, a strong association has been found between SF3B1 mutation and refractory anemia with ring sider-oblasts, a condition characterized by ineffective erythropoiesis and parenchymal iron overload. We studied the relationship between SF3B1 mutation, erythroid activity and hepcidin levels in myelodysplastic syndrome patients. Erythroid activity was evaluated through the proportion of marrow erythroblasts, soluble transferrin receptor and serum growth differentiation factor 15. Significant relationships were found between SF3B1 mutation and marrow erythroblasts (P=0.001), soluble transferrin receptor (P=0.003) and serum growth differentiation factor 15 (P=0.033). Serum hepcidin varied considerably, and multivariable analysis showed that the hepcidin to ferritin ratio, a measure of adequacy of hepcidin levels relative to body iron stores, was inversely related to the SF3B1 mutation (P=0.013). These observations suggest that patients with SF3B1 mutation have inappropriately low hepcidin levels, which may explain their propensity to parenchymal iron loading.