Somatic Mutations of Calreticulin in Myeloproliferative Neoplasms

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Genomic profiling and genotype-phenotype correlations in myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are disorders of the stem cell, due to acquired mutations causing a clonal proliferation of one or more hemopoietic progenitor in the bone marrow. According to the World Health Organization (WHO) 2016 classification of myeloid neoplasms, Ph-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and prefibrotic myelofibrosis (prePMF). A somatic mutation in JAK2, CALR or MPL genes is found in the great majority of patients with MPNs. These driver mutations constitutively activate the JAK/STAT pathway, resulting in increased phosphorylation of its substrates and leading to increased cytokine responsiveness of myeloid cells. MPNs may progress to more aggressive diseases. This evolution is typically associated with the acquisition of somatic variants in genes involved in different pathways, including DNA methylation and regulation of chromatin structure, transcriptional regulators, signaling pathway and splicing factors. The phenotype of MPNs seems to be related to the combination of driver and subclonal variants and their order of acquisition but the molecular and clinical correlations are not yet completely understood. This work is a genotype-phenotype study aimed to correlate biological and clinical features in a cohort of 509 MPN patients, diagnosed with ET, prePMF and PMF at the UOC Ematologia, Fondazione IRCCS Policlinico San Matteo, between 1985 and 2019. DNA sequence variants were studied through a NGS approach using the Illumina Nextera Rapid Capture Custom Enrichment Kit and HiSeq2500 platform. The panel targeted the coding sequence of 81 genes known to be involved in myeloid neoplasms. Overall, 589 additional somatic variants were detected. Compared to ET and prePMF, PMF showed a larger proportion of patients carrying at least one additional somatic variant, a higher average number of variants per patient and a greater involvement of high molecular risk genes. ET, prePMF and PMF showed different mutational landscapes: in ET the most commonly involved pathway was DNA methylation genes, while in prePMF RNA splicing genes were often affected, together with DNA methylation; in PMF chromatin structure, DNA methylation and RNA splicing were the most recurrent mutated pathways. This finding suggests that prePMF and PMF share molecular features with MDS, since RNA splicing is often involved in MDS development. No significative association between driver mutation and additional variants was found, except for mutations in the spliceosome genes, which did not occur in CALR- mutated patients. The correlations of the additional variants with the clinical picture highlighted a negative impact of high-risk genes on OS and on the progression of the disease. In conclusion, these data suggest that not only clinical and histopathological criteria, but also distinct mutation patterns might differentiate ET, prePMF and PMF. In the era of precision medicine, an accurate diagnosis and prognostic stratification will be useful in the choice of a proper treatment for each patient

COVID-19 in Patients with Hematologic Diseases

The COVID-19 outbreak had a strong impact on people’s lives all over the world. Patients with hematologic diseases have been heavily affected by the pandemic, because their immune system may be compromised due to anti-cancer or immunosuppressive therapies and because diagnosis and treatment of their baseline conditions were delayed during lockdowns. Hematologic malignancies emerged very soon as risk factors for severe COVID-19 infection, increasing the mortality rate. SARS-CoV2 can also induce or exacerbate immune-mediated cytopenias, such as autoimmune hemolytic anemias, complement-mediated anemias, and immune thrombocytopenia. Active immunization with vaccines has been shown to be the best prophylaxis of severe COVID-19 in hematologic patients. However, the immune response to vaccines may be significantly impaired, especially in those receiving anti-CD20 monoclonal antibodies or immunosuppressive agents. Recently, antiviral drugs and monoclonal antibodies have become available for pre-exposure and post-exposure prevention of severe COVID-19. As adverse events after vaccines are extremely rare, the cost–benefit ratio is largely in favor of vaccination, even in patients who might be non-responders; in the hematological setting, all patients should be considered at high risk of developing complications due to SARS-CoV2 infection and should be offered all the therapies aimed to prevent them

Haematological malignancies in relatives of patients affected with myeloproliferative neoplasms

Abstract In a cohort of 3131 patients with myeloproliferative neoplasms (MPNs), we identified 200 patients (6.4%) who reported a second case of haematological malignancies (HM) in first‐ or second‐degree relatives. The occurrence of a second HM in the family was not influenced by MPN subtype, sex or driver mutation, while it was associated with age at MPN diagnosis: 8.5% of patients diagnosed with MPN younger than 45 years had a second relative affected with HM compared to 5.5% of those diagnosed at the age of 45 years or older (p = 0.003), thus suggesting a genetic predisposition to HM with early onset

Autoantibodies against type I IFNs in patients with Ph-negative myeloproliferative neoplasms

Autoantibodies against type I IFNs in patients with Ph-negative myeloproliferative neoplasm

Mutation Type As a Major Determinant of Clinical Phenotype in Myeloproliferative Neoplasms Associated with Mutant Calreticulin

About 25% of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) carry a somatic mutation of CALR, the calreticulin gene [N Engl J Med. 2013;369:2379-90]. So far, more than 50 different indels in CALR exon 9 have been found, but a 52-bp deletion (type 1 mutation) and a 5-bp insertion (type 2) are the most common lesions. All indels generate a novel C-terminus of the mutant protein, in which the endoplasmic reticulum retention signal KDEL is lost and the negatively charged amino acids are replaced by neutral and positively charged amino acids, disrupting the Ca-binding site. This suggests that both cellular dislocation and impaired Ca-binding activity may be involved in the abnormal proliferation of cells expressing a mutant calreticulin. It is still unclear, however, why the same mutant gene is associated with 2 different disease phenotypes (ET and PMF). In particular, little in known about the effect of the mutant protein on megakaryocyte biology and bone marrow collagen deposition. We studied the relationships between CALR mutation type, megakaryocyte biology, and clinical phenotype in patients with myeloproliferative neoplasms. According to the 2008 WHO criteria, 716 out of 892 patients had ET and 176 had PMF. Overall, 578 (65%) patients carried JAK2 (V617F), 230 (26%) had a CALR indel, and 84 (9%) had nonmutated JAK2 and CALR. Patients with MPL mutations were excluded. Twenty-six different types of CALR lesions were identified: 120 (52%) patients had type 1 mutation, 75 (33%) had type 2, and 35 (15%) carried other indels. The frequency of type 1 mutation was significantly higher in PMF than in ET (71% vs 46%, P=.004). All these variants involved 3 different stretches of negatively charged amino acids, with an increase in the isoelectric points (pI) of the mutant protein. As type 1 and type 2 mutations affected stretch I and III, respectively, the 26 indels were categorized into 3 groups on the basis of the stretch they affected: i) type 1-like (61%), affecting stretch I; ii) type 2-like (36%), stretch III; iii) and other types (3%), stretch II. The pI values were significantly different in the 3 groups (P<.001). The frequency of type-1 like mutations was significantly higher in PMF than in ET (82% vs 55%, P=.001). In vitro differentiated megakaryocytes from CALR-mutant patients displayed a significant increase in the extent of both intracellular Ca2+ release from the endoplasmic reticulum and extracellular Ca2+ entry inside the cytoplasm, as compared with healthy controls. Megakaryocytes carrying type 1-like CALR mutations exhibited the highest amplitude of Ca2+ flows regardless of the type of disease. In ET, impaired Ca2+ homeostasis was accompanied by atypical proplatelet architecture (ie, more branches and bifurcations). With respect to clinical phenotype at diagnosis, ET patients with type 2-like CALR mutation showed a trend towards higher PLT count (P=.063) and lower age (P=.053), and significantly lower LDH values (P=.021) than those with type 1-like mutation. In a hierarchical cluster analysis including demographic, clinical and molecular data, CALR mutation type (1 vs 2) identified the 2 clusters with the highest dissimilarity. Considering all patients, those with type 2-like CALR lesions had a better survival than those with JAK2 (V617F) (96.1% vs 84.4% at 10 years, P=.039), while no difference was found between the 2 CALR mutation types. ET patients with type 2-like CALR mutations showed a lower risk of thrombosis than those with JAK2 (V617F) (P=.010). By contrast, ET patients with type 1-like CALR mutations had a higher risk of myelofibrotic transformation that those with type 2-like CALR mutations (P=.029) and especially those with JAK2 (V617F) (P=.011). Finally, PMF patients with type 1-like CALR variants had a better survival than those with JAK2 (V617F) (80.1% vs 48% at 10 years, P=.008). In summary, abnormalities in megakaryocyte calcium metabolism and proplatelet architecture are found in patients with CALR-mutant myeloproliferative neoplasms, and their extent is related to mutation type. Type 2-like CALR mutations are more likely to be associated with isolated thrombocytosis without bone marrow fibrosis, ie, with an ET phenotype. By contrast, type 1-like CALR mutations are generally associated with bone marrow fibrosis, ie, with a PMF phenotype. Thus, in CALR-mutant myeloproliferative neoplasms, the mutation type is a major determinant of the clinical phenotype

In contemporary patients with polycythemia vera, rates of thrombosis and risk factors delineate a new clinical epidemiology

In multivariable analysis, previous arterial event (hazard ratio [HR], 1.7; 95% confidence interval [CI], 1.2 to 2.4) and hypertension (HR, 1.6; 95% CI, 1.2 to 2.2) were significantly associated with subsequent arterial events whereas previous venous event (HR, 2.6; 95% CI, 1.5 to 4.4) and age ≥65 years (HR, 1.7; 95% CI, 1.2 to 2.5) were significant predictors of future venous thrombosis

Second cancer in Philadelphia negative myeloproliferative neoplasms (MPN-K). A nested case-control study

We conducted a large international nested case-control study including 1881 patients with Philadelphia-negative myeloproliferative neoplasms (MPN). Cases (n\u2009=\u2009647) were patients with second cancer (SC: carcinoma, non-melanoma skin cancer, hematological second cancer, and melanoma) and controls (n\u2009=\u20091234) were patients without SC, matched with cases for sex, age at MPN diagnosis, date of MPN diagnosis, and MPN disease duration. The aim was to evaluate the risk of SC after exposure to cytoreductive drugs. Patients exposed to hydroxyurea (HU) (median: 3 years) had a risk of SC similar to unexposed patients (OR\u2009=\u20091.06, 95% CI 0.82-1.38). In contrast, in cancer-specific stratified multivariable analysis, HU had two-fold higher risk of non-melanoma (NM) skin cancer (OR\u2009=\u20092.28, 95% CI 1.15-4.51). A significantly higher risk of NM-skin cancer was also documented for pipobroman (OR\u2009=\u20093.74, 95% CI 1.00-14.01), ruxolitinib (OR\u2009=\u20093.87, 95% CI 1.18-12.75), and for drug combination (OR\u2009=\u20093.47, 95% CI 1.55-7.75). These three drugs did not show excess risk of carcinoma and hematological second cancer compared with unexposed patients. Exposure to interferon, busulfan, and anagrelide did not increase the risk. In summary, while it is reassuring that no excess of carcinoma was documented, a careful dermatologic active surveillance before and during the course of treatments is recommended

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