Martin Schalling

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.).

Ropeginterferon alfa-2b, a novel IFNα-2b, induces high response rates with low toxicity in patients with polycythemia vera.

In this prospective, open-label, multicenter phase 1/2 dose escalation study, we used a next-generation, mono-pegylated interferon (IFN) α-2b isoform, ropeginterferon alfa-2b. The unique feature of ropeginterferon alfa-2b is a longer elimination half-life, which allows administration every 2 weeks. We present data from 51 polycythemia vera patients. The main goal was to define the maximum tolerated dose and to assess safety and efficacy. A dose range of 50 to 540 µg was tested without the appearance of dose-limiting toxicities. All drug-related adverse events were known toxicities associated with IFN-α. The cumulative overall response rate was 90%, comprising complete response in 47% and partial response in 43% of patients; the best individual molecular response level was a complete response in 21% of patients and partial response in 47%. Notably, we did not observe any correlation between the dose level and the response rate or response duration, suggesting that already low levels of ropeginterferon alfa-2b are sufficient to induce significant hematologic and molecular responses. These data suggest promising efficacy and safety of ropeginterferon alfa-2b and support the development of the drug in a randomized phase 3 clinical trial. The study was disclosed at www.clinicaltrials.gov as #NCT01193699 before including the first patient.

Molecular responses and chromosomal aberrations in patients with polycythemia vera treated with peg‐proline‐interferon alpha‐2b

Fifty‐one polycythemia vera (PV) patients were enrolled in the phase I/II clinical study PEGINVERA to receive a new formulation of pegylated interferon alpha (peg‐proline‐IFNα‐2b, AOP2014/P1101). Peg‐proline‐IFNα‐2b treatment led to high response rates on both hematologic and molecular levels. Hematologic and molecular responses were achieved for 46 and 18 patients (90 and 35% of the whole cohort), respectively. Although interferon alpha (IFNα) is known to be an effective antineoplastic therapy for a long time, it is currently not well understood which genetic alterations influence therapeutic outcomes. Apart from somatic changes in specific genes, large chromosomal aberrations could impact responses to IFNα. Therefore, we evaluated the interplay of cytogenetic changes and IFNα responses in the PEGINVERA cohort. We performed high‐resolution SNP microarrays to analyze chromosomal aberrations prior and during peg‐proline‐IFNα‐2b therapy. Similar numbers and types of chromosomal aberrations in responding and non‐responding patients were observed, suggesting that peg‐proline‐IFNα‐2b responses are achieved independently of chromosomal aberrations. Furthermore, complete cytogenetic remissions were accomplished in three patients, of which two showed more than one chromosomal aberration. These results imply that peg‐proline‐IFNα‐2b therapy is an effective drug for PV patients, possibly including patients with complex cytogenetic changes. Am. J. Hematol. 90:288–294, 2015.

Pre‐fibrotic/early primary myelofibrosis vs. WHO‐defined essential thrombocythemia: The impact of minor clinical diagnostic criteria on the outcome of the disease

The 2016 revised WHO criteria for the diagnosis of pre‐fibrotic/early primary myelofibrosis (pre‐PMF) require at least one of the following four borderline expressed minor clinical criteria: anemia, leukocytosis, elevated lactate dehydrogenase and splenomegaly. In this study, we evaluated the relative frequency of these four criteria in a group of 170 pre‐PMF patients and compared them to 225 ET cases. More than 91% of pre‐PMF cases showed one or more of these features required for diagnosis, by contrast with only 48% of ET patients. According to clinical data the cumulative risk of progression to advanced/overt PMF in pre‐PMF was 36.9% after 15 years. After fitting cox regression models to analyze the impact of the minor criteria on overall survival, only leukocytosis remained as a significant predictor of survival in both pre‐PMF and ET. Molecular characterization showed differences in survival in pre‐PMF but not ET, with CALR being a more favorable mutation than JAK2. The different outcome of pre‐PMF versus ET and associated molecular genetic data supports the concept of two different entities, rather than a continuum of the same disease. Although slightly less than 50% of ET patients also show one or more minor clinical criteria, accurate distinction between ET and pre‐PMF is possible by following an integrated approach including histomorphological diagnosis and presence of minor clinical criteria.

Impact of white blood cell counts at diagnosis and during follow‐up in patients with essential thrombocythaemia and prefibrotic primary myelofibrosis

The authors would like to thank Prof. Salam Al-Kindy (professor of Haematology) and Dr. Anil Pathere (senior consultant of Clinical Haematology) from the Faculty of Medicine of Sultan Qaboos University for their kind co-operation. Ms. Simone J. Wielders is gratefully acknowledged for her assistance in the measurement of FV levels. This work was supported by a grant of the Cardiovascular Research Institute Maastricht (to E.C.).

Decanucleotide insertion polymorphism of F7 significantly influences the risk of thrombosis in patients with essential thrombocythemia

There is strong evidence that certain thrombophilic single nucleotide polymorphisms (SNPs) account for an increased risk of thrombosis. The additive impact of inherited thrombotic risk factors to a certain disease‐ immanent thrombotic risk is vastly unknown. Therefore, we aimed to investigate the influence of three novel, preselected SNPs on the risk of thrombosis in patients diagnosed with myeloproliferative neoplasm (MPN).

Essential thrombocythemia vs. pre-fibrotic/early primary myelofibrosis: discrimination by laboratory and clinical data

Among several groups of clinicians and hematopathologists a conflict of opinion has been repeatedly expressed concerning the validity of bone marrow (BM) features characterizing myeloproliferative neoplasms (MPNs) . In this regard, controversy is mainly focused on the distinction between essential thrombocythemia (ET) and pre-fibrotic/early primary myelofibrosis (pre-PMF). Although other groups confirmed the characteristic BM features and emphasized the clinical impact to discriminate both MPN subtypes , the existence of prePMF has been questioned, including clinical usefulness and particularly reproducibility of the corresponding diagnostic guidelines. In this context, it has been criticized that the MPN classification proposed by the World Health Organization (WHO), updated in 2008 and revised in 2016, was focused on BM morphology as the gold standard of diagnosis. As has been highlighted until now, none of the various mutations identified so far have proven to be specific and therefore cannot be applied for a molecular classification of MPNs and especially not for the distinction between ET and pre-PMF. Given the very different outcome and treatment options in clinical practice, there is an active interest to evaluate whether laboratory or clinical parameters could help to distinguish WHO-defined ET and pre-PMF in patients presenting with thrombocytosis. A first step to elucidate if blood tests could exert a predictive power in patients presenting clinically with an ET-like phenotype was initiated by Carobbio et al. To identify pre-PMF cases mimicking ET, the laboratory parameters for hemoglobin (Hb), white blood cell (WBC) count, and serum lactate dehydrogenase (LDH) level were used in a dichotomized manner, resulting in a step-by-step algorithm. The cutoff values at each step in this algorithm were optimized to produce the desired specificity and sensitivity. The result was that nearly 50% of all patients mimicking an ET-like phenotype could correctly be attributed to the pre-PMF group. The aim of the present study was to extend and improve this investigation by expanding the algorithm described by Carobbio et al. (the so-called Bergamo algorithm), so that the discriminatory ability could be raised. The first step was to include splenomegaly as clinical parameter into this algorithm, since it is an important factor setting pre-PMF apart from ET patients 6, 8, . In this regard, left shift in the peripheral blood (presence of single erythroblasts or myelocytes, metamyelocytes, promyelocytes, or myeloblasts) was also tested as a presumptive parameter. The second and more important approach to this problem was to develop a model which utilizes these parameters in a continuous manner by applying a logistic regression model rather than looking at each parameter in a stepwise order. This study is based on an Austrian registry diagnosed for MPN according to the 2008 WHO diagnostic criteria between 1985 and 2015, which was created by clinicians and hematopathologists in the Departments of Hematology and Clinical Pathology at the Medical University of Vienna, Austria, in collaboration with other clinical centers throughout Austria. In close cooperation with the local hematopathologists, BM biopsies were centrally reevaluated under a multi-headed microscope by three of

Impact of white blood cells on thrombotic risk in patients with optimized platelet count in essential thrombocythemia

Risk of thrombosis is significantly enhanced by both elevated platelet (PLT) and white blood cell (WBC) counts according to a retrospective analysis of a large anagrelide registry in thrombocythemic MPN patients. We were interested in the impact of elevated WBC counts on thrombosis risk in patients where PLT counts were reduced below the calculated cutoff of 574.5 G/L by treatment with anagrelide.