Chiara Elena

Somatic Mutations of Calreticulin in Myeloproliferative Neoplasms

BACKGROUND Approximately 50 to 60% of patients with essential thrombocythemia or primary myelofibrosis carry a mutation in the Janus kinase 2 gene (JAK2), and an additional 5 to 10% have activating mutations in the thrombopoietin receptor gene (MPL). So far, no specific molecular marker has been identified in the remaining 30 to 45% of patients. METHODS We performed whole-exome sequencing to identify somatically acquired mutations in six patients who had primary myelofibrosis without mutations in JAK2 or MPL. Resequencing of CALR, encoding calreticulin, was then performed in cohorts of patients with myeloid neoplasms. RESULTS Somatic insertions or deletions in exon 9 of CALR were detected in all patients who underwent whole-exome sequencing. Resequencing in 1107 samples from patients with myeloproliferative neoplasms showed that CALR mutations were absent in polycythemia vera. In essential thrombocythemia and primary myelofibrosis, CALR mutations and JAK2 and MPL mutations were mutually exclusive. Among patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 or MPL, CALR mutations were detected in 67% of those with essential thrombocythemia and 88% of those with primary myelofibrosis. A total of 36 types of insertions or deletions were identified that all cause a frameshift to the same alternative reading frame and generate a novel C-terminal peptide in the mutant calreticulin. Overexpression of the most frequent CALR deletion caused cytokine-independent growth in vitro owing to the activation of signal transducer and activator of transcription 5 (STAT5) by means of an unknown mechanism. Patients with mutated CALR had a lower risk of thrombosis and longer overall survival than patients with mutated JAK2. CONCLUSIONS Most patients with essential thrombocythemia or primary myelofibrosis that was not associated with a JAK2 or MPL alteration carried a somatic mutation in CALR. The clinical course in these patients was more indolent than that in patients with the JAK2 V617F mutation. (Funded by the MPN Research Foundation and Associazione Italiana per la Ricerca sul Cancro.).

JAK2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcomes

Patients with essential thrombocythemia may carry JAK2 (V617F), an MPL substitution, or a calreticulin gene (CALR) mutation. We studied biologic and clinical features of essential thrombocythemia according to JAK2 or CALR mutation status and in relation to those of polycythemia vera. The mutant allele burden was lower in JAK2-mutated than in CALR-mutated essential thrombocythemia. Patients with JAK2 (V617F) were older, had a higher hemoglobin level and white blood cell count, and lower platelet count and serum erythropoietin than those with CALR mutation. Hematologic parameters of patients with JAK2-mutated essential thrombocythemia or polycythemia vera were related to the mutant allele burden. While no polycythemic transformation was observed in CALR-mutated patients, the cumulative risk was 29% at 15 years in those with JAK2-mutated essential thrombocythemia. There was no significant difference in myelofibrotic transformation between the 2 subtypes of essential thrombocythemia. Patients with JAK2-mutated essential thrombocythemia and those with polycythemia vera had a similar risk of thrombosis, which was twice that of patients with the CALR mutation. These observations are consistent with the notion that JAK2-mutated essential thrombocythemia and polycythemia vera represent different phenotypes of a single myeloproliferative neoplasm, whereas CALR-mutated essential thrombocythemia is a distinct disease entity.

A prospective study of 338 patients with polycythemia vera: the impact of JAK2 (V617F) allele burden and leukocytosis on fibrotic or leukemic disease transformation and vascular complications

We studied the relationship between JAK2 (V617F) mutant allele burden and clinical phenotype, disease progression and survival in patients with polycythemia vera (PV). The percentage of granulocyte mutant alleles was evaluated using a quantitative real-time polymerase chain reaction-based allelic discrimination assay. Of the 338 patients enrolled in this prospective study, 320 (94.7%) carried the JAK2 (V617F) mutation. Direct relationships were found between mutant allele burden and hemoglobin concentration (P=0.001), white blood cell count (P=0.001), spleen size (P=0.001) and age-adjusted bone marrow cellularity (P=0.002), while an inverse relationship was found with platelet count (P<0.001). During the study period, eight patients progressed to post-PV myelofibrosis (MF) (all carrying >50% mutant alleles), while 10 patients developed acute myeloid leukemia (AML). The mutant allele burden was significantly related to the risk of developing myelofibrosis (P=0.029) and retained its significant effect also in multivariable analysis (P=0.03). By contrast, the risk of developing AML as well as that of thrombosis was not significantly related to mutant allele burden. Leukocytosis did not affect thrombosis, MF, leukemia or survival. In conclusion, a JAK2 (V617F) allele burden >50% represents a risk factor for progression to MF in PV.

Prognostic factors for thrombosis, myelofibrosis, and leukemia in essential thrombocythemia: a study of 605 patients

The findings from this study on a large series of patients treated according to current clinical practice provide reassurance that essential thrombocythemia is an indolent disorder and affected patients have a long survival. Background Essential thrombocythemia is a chronic myeloproliferative disorder; patients with this disorder have a propensity to develop thrombosis, myelofibrosis, and leukemia. Design and Methods We studied 605 patients with essential thrombocythemia (follow-up 4596 person-years) with the aim of defining prognostic factors for thrombosis, myelofibrosis, and leukemia during follow-up. Results Sixty-six patients (11%) developed thrombosis with a 10-year risk of 14%. Age >60 years (p<0.001) and a history of thrombosis (p=0.03) were independent risk factors for thrombosis. Progression to myelofibrosis occurred in 17 patients (2.8%) with a 10-year risk of 3.9%. Anemia at diagnosis of essential thrombocythemia was significantly correlated (p<0.001) with progression to myelofibrosis. Leukemia occurred in 14 patients (2.3%) at a median time of 11 years after the diagnosis of essential thrombocythemia; the risk was 2.6% at 10 years. Age >60 years (p=0.02) was significantly correlated with the development of leukemia. Cytotoxic treatment did not imply a higher risk of leukemia. At the time of the analysis, 64 of the 605 patients (10.6%) had died. The 10-year probability of survival was 88%, with a median survival of 22.3 years. Age >60 years (p<0.001) and history of thrombosis (p=0.001) were independent risk factors for survival. Conclusions The findings from this study on a large series of patients treated according to current clinical practice provide reassurance that essential thrombocythemia is an indolent disorder and affected patients have a long survival. The main risk is thrombosis, while myelofibrosis and leukemia are rare and late complications.

Molecular and clinical features of the myeloproliferative neoplasm associated with JAK2 exon 12 mutations.

Although approximately 95% of patients with polycythemia vera (PV) harbor the V617F mutation in JAK2 exon 14, several mutations in exon 12 have been described in the remaining patients. We conducted a European collaborative study to define the molecular and clinical features of patients harboring these mutations. Overall, 106 PVs were recruited and 17 different mutations identified. Irrespective of the mutation, two-thirds of patients had isolated erythrocytosis, whereas the remaining subjects had erythrocytosis plus leukocytosis and/or thrombocytosis. Compared with JAK2 (V617F)-positive PV patients, those with exon 12 mutations had significantly higher hemoglobin level and lower platelet and leukocyte counts at diagnosis but similar incidences of thrombosis, myelofibrosis, leukemia, and death. In a multivariable analysis, age more than 60 years and prior thrombosis predicted thrombosis. These findings suggest that, despite the phenotypical difference, the outcome of JAK2 exon 12 mutations-positive PV is similar to that of JAK2 (V617F)-positive PV.

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.

A phase 2 study of ruxolitinib, an oral JAK1 and JAK2 inhibitor, in patients with advanced polycythemia vera who are refractory or intolerant to hydroxyurea

Polycythemia vera (PV) is a myeloproliferative neoplasm associated with somatic gain‐of‐function mutations of Janus kinase‐2 (JAK2). Therapeutic options are limited in patients with advanced disease. Ruxolitinib, an oral JAK1/JAK2 inhibitor, is active in preclinical models of PV. The long‐term efficacy and safety of ruxolitinib in patients with advanced PV who are refractory or intolerant to hydroxyurea were studied in a phase 2 trial.

Familial Chronic Myeloproliferative Disorders: Clinical Phenotype and Evidence of Disease Anticipation

PURPOSE Chronic myeloproliferative disorders (CMDs) have sporadic occurrence. However, familial clustering is reported. The purpose of this study was to assess the prevalence and the clinical phenotype of familial CMDs, and to study the anticipation of disease onset in successive generations. PATIENTS AND METHODS Among 458 patients with apparently sporadic CMDs, an interview-based investigation of family history was performed to identify familial cases. The clinical phenotype of familial CMDs was compared with that of sporadic CMDs. Anticipation was studied evaluating age at diagnosis and telomere length in successive generations. RESULTS Among 458 patients with apparently sporadic CMDs, the prevalence of familial cases was 7.6% (35 pedigrees; 75 patients). Kolmogorov-Smirnov and two-tailed Fishers exact tests did not demonstrate significant differences in clinical presentation between patients with familial and sporadic CMDs. Within 544 person-years of follow-up, patients with familial CMDs developed similar complications and disease evolutions as those with sporadic CMDs. The comparison of second-generation and first-generation patients showed a significantly younger age at diagnosis (Wilcoxon matched-pair test, P = .001) and a significantly higher age-dependent hazard of CMD onset (Nelson-Aalen method, P < .001) in patients of the second generation. A significant shortening of telomere length was highlighted in offspring compared with parent (P = .043). CONCLUSION This study indicates that a thorough investigation of family history should be part of the initial work-up of patients with CMDs. Patients with familial CMDs show the same clinical features and suffer the same complications as patients with sporadic disease. Age distribution between parent and offspring and telomere length shortening provide evidence of disease anticipation.

A dynamic prognostic model to predict survival in post–polycythemia vera myelofibrosis

Post-polycythemia vera myelofibrosis (post-PV MF) is a late evolution of PV. In 647 patients with PV, we found that leukocytosis leukocyte count>(15x10(9)/L) at diagnosis is a risk factor for the evolution of post-PV MF. In a series of 68 patients who developed post-PV MF, median survival was 5.7 years. Hemoglobin level less than 100 g/L (10 g/dL) at diagnosis of post-PV MF was an independent risk factor for survival. The course of post-PV MF, however, is a dynamic process that implies a progressive worsening of clinical parameters. Using a multivariate Cox proportional hazard regression with time-dependent covariates, we found that a dynamic score based on hemoglobin level less than 100 g/L (10 g/dL), platelet count less than 100x10(9)/L, and leukocyte count more than 30x10(9)/L is useful to predict survival at any time from diagnosis of post-PV MF. The resulting hazard ratio of the score was 4.2 (95% CI: 2.4-7.7; P<.001), meaning a 4.2-fold worsening of survival for each risk factor acquired during follow up. In conclusion, leukocytosis at diagnosis of PV is a risk factor for evolution in post-PV MF. A dynamic score based on hemoglobin level, and platelet and leukocyte count predicts survival at any time from diagnosis of post-PV MF.