Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms.

Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

Arterial thrombosis in Philadelphia-negative myeloproliferative neoplasms predicts second cancer: a case-control study.

Patients with Philadelphia-negative myeloproliferative neoplasm (MPN) are prone to the development of second cancers, but the factors associated with these events have been poorly explored. In an international nested case-control study, we recruited 647 patients with carcinoma, nonmelanoma skin cancer, hematological second cancer, and melanoma diagnosed concurrently or after MPN diagnosis. Up to 3 control patients without a history of cancer and matched with each case for center, sex, age at MPN diagnosis, date of diagnosis, and MPN disease duration were included (n = 1234). Cases were comparable to controls for MPN type, driver mutations and cardiovascular risk factors. The frequency of thrombosis preceding MPN was similar for cases and controls (P = .462). Thrombotic events after MPN and before second cancer were higher in cases than in controls (11.6% vs 8.1%; P = .013), because of a higher proportion of arterial thromboses (6.2% vs 3.7%; P = .015). After adjustment for confounders, the occurrence of arterial thrombosis remained independently associated with the risk of carcinoma (odds ratio, 1.97; 95% confidence interval, 1.14-3.41), suggesting that MPN patients experiencing arterial events after MPN diagnosis deserve careful clinical surveillance for early detection of carcinoma. This study was registered at www.clinicaltrials.gov as NCT03745378.

Impaired virus-specific T cell responses in patients with myeloproliferative neoplasms treated with ruxolitinib.

Ruxolitinib is effective in myeloproliferative neoplasms (MPN) but can cause reactivation of silent infections. We aimed at evaluating viral load and T-cell responses to human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in a cohort of 25 MPN patients treated with ruxolitinib. EBV-DNA and HCMV-DNA were quantified monthly using real-time polimerase chain reaction (PCR) on peripheral blood samples, and T-cell subsets were analyzed by flowcytometry. HCMV and EBV-directed T-cell responses were evaluated using the IFN-γ ELISPOT assay. Most patients had CD4+ and/or CD8+ T-cells below the normal range; these reductions were related to the duration of ruxolitinib treatment. In fact, reduced T-lymphocytes' subsets were found in 93% of patients treated for ≥5 years and in 45% of those treated for <5 years (P = .021). The former also had lower median numbers of CD4+ and CD8+ cells. Subclinical reactivation of EBV and HCMV occurred in 76% and 8% of patients. We observed a trend to an inverse relationship between EBV and CMV-specific CD4+ and CD8+ T-cell responses and viral load, and a trend to an inverse correlation with ruxolitinib dose. Therefore, our data suggest that the ruxolitinib treatment may interfere with immunosurveillance against EBV and HCMV.

Second cancers in MPN: Survival analysis from an international study.

One out of ten patients with Philadelphia-negative myeloproliferative neoplasms (MPN) develop a second cancer (SC): in such patients we aimed at assessing the survival impact of SC itself and of MPN-specific therapies. Data were therefore extracted from an international nested case-control study, recruiting 798 patients with SC diagnosed concurrently or after the MPN. Overall, 2995 person-years (PYs) were accumulated and mortality rate (MR) since SC diagnosis was 5.9 (5.1-6.9) deaths for every 100 PYs. A "poor prognosis" SC (stomach, esophagus, liver, pancreas, lung, ovary, head-and-neck or nervous system, osteosarcomas, multiple myeloma, aggressive lymphoma, acute leukemia) was reported in 26.3% of the patients and was the cause of death in 65% of them (MR 11.0/100 PYs). In contrast, patients with a "non-poor prognosis" SC (NPPSC) incurred a MR of 4.6/100 PYs: 31% of the deaths were attributed to SC and 15% to MPN evolution. At multivariable analysis, death after SC diagnosis was independently predicted (HR and 95% CI) by patient age greater than 70 years (2.68; 1.88-3.81), the SC prognostic group (2.57; 1.86-3.55), SC relapse (1.53; 10.6-2.21), MPN evolution (2.72; 1.84-4.02), anemia at SC diagnosis (2.32; 1.49-3.59), exposure to hydroxyurea (1.89; 1.26-2.85) and to ruxolitinib (3.63; 1.97-6.71). Aspirin was protective for patients with a NPPSC (0.60; 0.38-0.95). In conclusion, SC is a relevant cause of death competing with MPN evolution. Prospective data are awaited to confirm the role of cytoreductive and anti-platelet drugs in modulating patient survival after the occurrence of a SC.

The Genetic Basis of Primary Myelofibrosis and Its Clinical Relevance.

Among classical BCR-ABL-negative myeloproliferative neoplasms (MPN), primary myelofibrosis (PMF) is the most aggressive subtype from a clinical standpoint, posing a great challenge to clinicians. Whilst the biological consequences of the three MPN driver gene mutations (JAK2, CALR, and MPL) have been well described, recent data has shed light on the complex and dynamic structure of PMF, that involves competing disease subclones, sequentially acquired genomic events, mostly in genes that are recurrently mutated in several myeloid neoplasms and in clonal hematopoiesis, and biological interactions between clonal hematopoietic stem cells and abnormal bone marrow niches. These observations may contribute to explain the wide heterogeneity in patients' clinical presentation and prognosis, and support the recent effort to include molecular information in prognostic scoring systems used for therapeutic decision-making, leading to promising clinical translation. In this review, we aim to address the topic of PMF molecular genetics, focusing on four questions: (1) what is the role of mutations on disease pathogenesis? (2) what is their impact on patients' clinical phenotype? (3) how do we integrate gene mutations in the risk stratification process? (4) how do we take advantage of molecular genetics when it comes to treatment decisions?

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.).

CALR exon 9 mutations are somatically acquired events in familial cases of essential thrombocythemia or primary myelofibrosis.

Somatic mutations in the calreticulin (CALR) gene were recently discovered in patients with sporadic essential thrombocythemia (ET) and primary myelofibrosis (PMF) lacking JAK2 and MPL mutations. We studied CALR mutation status in familial cases of myeloproliferative neoplasm. In a cohort of 127 patients, CALR indels were identified in 6 of 55 (11%) subjects with ET and in 6 of 20 (30%) with PMF, whereas 52 cases of polycythemia vera had nonmutated CALR. All CALR mutations were somatic, found in granulocytes but not in T lymphocytes. Patients with CALR-mutated ET showed a higher platelet count (P = .017) and a lower cumulative incidence of thrombosis (P = .036) and of disease progression (P = .047) compared with those with JAK2 (V617F). In conclusion, a significant proportion of familial ET and PMF nonmutated for JAK2 carry a somatic mutation of CALR.

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.