Annalisa Pacilli

Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia.

Mutations in the calreticulin (CALR) gene were recently discovered in patients with essential thrombocythemia (ET) lacking the JAK2V617F and MPLW515 mutations, but no information is available on the clinical correlates. In this series, CALR mutations were found in 15.5% of 576 World Health Organization-defined ET patients, accounting for 48.9% of JAK2 and MPL wild-type (wt) patients. CALR-mutated patients were preferentially male and showed higher platelet count and lower hemoglobin and leukocyte count compared with JAK2- and MPL-mutated patients. Patients carrying the CALR mutation had a lower risk of thrombosis than JAK2- and MPL-mutated patients; of interest, their risk was superimposable to patients who were wt for the above mutations. CALR mutation had no impact on survival or transformation to post-ET myelofibrosis. Genotyping for CALR mutations represents a novel useful tool for establishing a clonal myeloproliferative disorder in JAK2 and MPL wt patients with thrombocytosis and may have prognostic and therapeutic relevance.

Type 1 versus Type 2 calreticulin mutations in essential thrombocythemia: A collaborative study of 1027 patients

CALR (calreticulin) trails JAK2 as the second most mutated gene in essential thrombocythemia (ET). Mutant CALR in ET is a result of frameshift mutations, caused by exon 9 deletions or insertions; type‐1, 52‐bp deletion (p.L367fs*46), and type‐2, 5‐bp TTGTC insertion (p.K385fs*47) variants constitute more than 80% of these mutations. The current study includes a total of 1027 patients divided into test (n = 402) and validation (n = 625) cohorts. Among the 402 ET patients in the test cohort, 227 (57%) harbored JAK2, 11 (3%) Myeloproliferative leukemia virus oncogene (MPL), and 114 (28%) CALR mutations; 12% were wild‐type for all three mutations (i.e., triple‐negative). Among the 114 patients with CALR mutations, 51 (45%) displayed type‐1 and 44 (39%) type‐2 variants; compared to mutant JAK2, both variants were associated with higher platelet and lower hemoglobin and leukocyte counts. However, male sex was associated with only type‐1 (P = 0.005) and younger age with type‐2 (P = 0.001) variants. Notably, platelet count was significantly higher in type‐2 vs. type‐1 CALR‐mutated patients (P = 0.03) and the particular observation was validated in the validation cohort that included 111 CALR‐mutated ET patients (P = 0.002). These findings, coupled with the recent demonstration of preferential expression of mutant and wild‐type CALR in megakaryocytes, suggest differential effects of CALR variants on thrombopoiesis. Am. J. Hematol. 89:E121–E124, 2014.

Calreticulin mutation-specific immunostaining in myeloproliferative neoplasms: pathogenetic insight and diagnostic value

Mutations in the gene calreticulin (CALR) occur in the majority of JAK2- and MPL-unmutated patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF); identifying CALR mutations contributes to the diagnostic pathway of ET and PMF. CALR mutations are heterogeneous spanning over the exon 9, but all result in a novel common protein C terminus. We developed a polyclonal antibody against a 17-amino-acid peptide derived from mutated calreticulin that was used for immunostaining of bone marrow biopsies. We show that this antibody specifically recognized patients harboring different types of CALR mutation with no staining in healthy controls and JAK2- or MPL-mutated ET and PMF. The labeling was mostly localized in megakaryocytes, whereas myeloid and erythroid cells showed faint staining, suggesting a preferential expression of calreticulin in megakaryocytes. Megakaryocytic-restricted expression of calreticulin was also demonstrated using an antibody against wild-type calreticulin and by measuring the levels of calreticulin RNA by gene expression analysis. Immunostaining using an antibody specific for mutated calreticulin may become a rapid, simple and cost-effective method for identifying CALR-mutated patients complementing molecular analysis; furthermore, the labeling pattern supports the preferential expansion of megakaryocytic cell lineage as a result of CALR mutation in an immature hematopoietic stem cell.

Validation of the differential prognostic impact of type 1/type 1-like versus type 2/type 2-like CALR mutations in myelofibrosis

The discovery of mutations in calreticulin (CALR) in patients with primary myelofibrosis (PMF)1, 2 prompted a reappraisal of the clinical correlates and prognostic impact of the so-called driver mutations that include JAK2V617F, MPLW515L/K/A and CALR in ~60%, 5–10% and 20–25% of patients, respectively. As compared with their JAK2V617F counterpart, PMF patients harboring CALR mutations showed younger age, higher platelet and lower hemoglobin and leukocyte counts. The cumulative incidence of anemia, leukocytosis and thrombocytopenia was significantly lower in CALR-mutated patients who were also less likely to be red cell transfusion-dependent;3, 4 in addition, they had significantly longer large splenomegaly-free survival compared with the other genotypes as well as patients lacking the three driver mutations (triple-negative (TN) patients).3, 4 Interestingly, spliceosome mutations were significantly less represented in CALR-mutated patients; however, no additional molecular or cytogenetic correlate was highlighted.3 These data suggested a milder disease in patients harboring the CALR mutation, and conceivably the presence of CALR mutation was associated with better overall survival (OS) when compared with JAK2V617F- and MPLW515-mutated patients, and particularly TN patients.3, 4 In multivariable analysis, CALR mutations had a favorable impact on survival that was independent of International Prognostic Scoring System (IPSS), Dinamic IPSS (DIPSS)4 or DIPSS-plus risk stratification,3 and also of ASXL1 mutation, known for its dismal impact on survival in PMF.5 At this regard, we found that DIPSS-plus-independent OS was significantly longer in CALR-mutated/ASXL1-unmutated compared with CALR-unmutated/ASXL1-mutated patients.6

MIPSS70: Mutation-Enhanced International Prognostic Score System for Transplantation-Age Patients With Primary Myelofibrosis

Purpose To develop a prognostic system for transplantation-age patients with primary myelofibrosis (PMF) that integrates clinical, cytogenetic, and mutation data. Patients and Methods The study included 805 patients with PMF age ≤ 70 years recruited from multiple Italian centers and the Mayo Clinic (Rochester, MN), forming two independent learning and validation cohorts. A Cox multivariable model was used to select from among a list of 22 variables those that were predictive of overall survival (OS). Integrated clinical and genetic prognostic models with (MIPSS70-plus) or without (MIPSS70) cytogenetic information were developed. Results Multivariable analysis identified the following as significant risk factors for OS: hemoglobin < 100 g/L, leukocytes > 25 × 109/L, platelets < 100 × 109/L, circulating blasts ≥ 2%, bone marrow fibrosis grade ≥ 2, constitutional symptoms, absence of CALR type-1 mutation, presence of high-molecular risk mutation (ie, ASXL1, EZH2, SRSF2, IDH1/ 2), and presence of two or more high-molecular risk mutations. By assigning hazard ratio (HR)-weighted points to these variables, three risk categories were delineated for the MIPSS70 model; 5-year OS was 95% in low-risk, 70% in intermediate-risk, and 29% in high-risk categories, corresponding to median OS of 27.7 years (95% CI, 22 to 34 years), 7.1 years (95% CI, 6.2 to 8.1 years), and 2.3 years (95% CI, 1.9 to 2.7 years), respectively. In the MIPSS70-plus model, which included cytogenetic information, four risk categories were delineated, with 5-year OS of 91% in low-risk, 66% in intermediate-risk (HR, 3.2; 95% CI, 1.9 to 5.2), 42% in high-risk (HR, 6.4; 95% CI, 4.1 to 10.0), and 7% very high-risk categories (HR, 17.0; 95% CI, 9.8 to 29.2). Both models remained effective after inclusion of older patients in the analysis. Conclusion MIPSS70 and MIPSS70-plus provide complementary systems of risk stratification for transplantation-age patients with PMF and integrate prognostically relevant clinical, cytogenetic, and mutation data.

Epidemiology and clinical relevance of mutations in postpolycythemia vera and postessential thrombocythemia myelofibrosis: A study on 359 patients of the AGIMM group.

Transformation to secondary myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PPV‐MF) and essential thrombocythemia (PET‐MF). Although primary (PMF) and secondary MF are considered similar diseases and managed similarly, there are few studies specifically focused on the latter. The aim of this study was to characterize the mutation landscape, and describe the main clinical correlates and prognostic implications of mutations, in a series of 359 patients with PPV‐MF and PET‐MF. Compared with PV and ET, the JAK2V617F and CALR mutated allele burden was significantly higher in PPV‐MF and/or PET‐MF, indicating a role for accumulation of mutated alleles in the process of transformation to MF. However, neither the allele burden nor the type of driver mutation influenced overall survival (OS), while absence of any driver mutation (triple negativity) was associated with significant reduction of OS in PET‐MF, similar to PMF. Of the five interrogated subclonal mutations (ASXL1, EZH2, SRSF2, IDH1, and IDH2), that comprise a prognostically detrimental high molecular risk (HMR) category in PMF, only SRSF2 mutations were associated with reduced survival in PET‐MF, and no additional mutation profile with prognostic relevance was highlighted. Overall, these data indicate that the molecular landscape of secondary forms of MF is different from PMF, suggesting that unknown mutational events might contribute to the progression from chronic phase disease to myelofibrosis. These findings also support more extended genotyping approaches aimed at identifying novel molecular abnormalities with prognostic relevance for patients with PPV‐MF and PET‐MF. Am. J. Hematol. 91:681–686, 2016.

JAK2V617F complete molecular remission in polycythemia vera/essential thrombocythemia patients treated with ruxolitinib

To the editor: Polycythemia vera (PV) and essential thrombocythemia (ET) are characterized by JAK2 V617F mutation in 95% and 60% of the patients, respectively.[1][1] Ruxolitinib is a JAK1/JAK2 inhibitor approved for myelofibrosis (MF) and more recently for hydroxyurea resistant/intolerant PV

Prognostic Impact of Bone Marrow Fibrosis in Primary Myelofibrosis. A Study of the AGIMM Group on 490 Patients

The prognostic significance of bone marrow (BM) fibrosis grade in patients with primary myelofibrosis (PMF) is still debated. A fibrosis grade greater than 1 was shown to associate with higher risk of death, and addition of fibrosis grade to IPSS score resulted in a more accurate prediction of survival. The aim of this study was to analyze the prognostic impact of BM fibrosis in 490 patients with PMF, evaluated at diagnosis, molecularly annotated and with extensive follow‐up information. We found that fibrosis grade 2 and greater on a 0–3 scale was associated with clinical characteristics indicative of a more advanced disease, such as anemia, leukopenia, thrombocytopenia, constitutional symptoms, larger splenomegaly and a higher IPSS risk category. Patients with higher grade of fibrosis were also more likely to have additional somatic mutations in ASXL1 and EZH2, that are prognostically adverse. Median survival was significantly reduced in patients with grade 2 and 3 fibrosis as compared with grade 1; this effect was maintained when analysis was restricted to younger patients. In multivariate analysis, fibrosis grade independently predicted for survival regardless of IPSS variables and mutational status; the adverse impact of fibrosis was noticeable especially in lower IPSS risk categories. Overall, results indicate that higher grades of fibrosis correlate with unique clinical and molecular aspects and represent an independent adverse variable in patients with PMF; these observations deserve confirmation in prospectively designed series of patients. Am. J. Hematol. 91:918–922, 2016.

A life‐threatening ruxolitinib discontinuation syndrome

CRIMM, Centro di Ricerca e Innovazione per le Malattie Mieloproliferative, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy; Department of Experimental and Clinical Medicine, University of Florence, DenoThe Excellence Center, Florence, Italy; Hematology Unit, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy Correspondence Alessandro M. Vannucchi, MD, CRIMM, Centro di Ricerca e Innovazione per le Malattie Mieloproliferative, AOU Careggi, Dipartimento di Medicina Sperimentale e Clinica, 50134 Firenze, Italy. Email: amvannucchi@unifi.it Funding information Supported by a grant from AIRC Investigator Grant 2014 (AIRC; Milan, Italy) project #15967, Special Program Molecular Clinical Oncology 5x1000 to AIRCGruppo Italiano Malattie Mieloproliferative (AGIMM) project #1005, Bando Ricerca Finalizzata e Giovani Ricercatori Anno 2011–2012 (Ministero della Salute, Italy) project GR-2011-02352109.

Germline transmission of LNKE208Q variant in a family with myeloproliferative neoplasms

unknown, but identification and treatment is plausible and testable. In summary, OSA was common in symptomatic adult patients with SCD, and associated with BMI in patients with CH SCD, but not with other hematologic or physiologic parameters routinely measured by us in clinic. Our data support a heightened clinical suspicion for NHD in HbSS patients with an ARC >400,000 cells/mL, since NHD was identified in 21% of symptomatic HbSS patients and associated with significant reticulocytosis. We expect that careful evaluation of symptoms, linked with appropriate diagnostic and therapeutic interventions for sleep disordered breathing (OSA or NHD), will identify modifiable clinical syndromes that could influence morbidity and mortality in adult patients with SCD.