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) 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; 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). Interestingly, spliceosome mutations were significantly less represented in CALR-mutated patients; however, no additional molecular or cytogenetic correlate was highlighted. 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 JAK2V617Fand MPLW515-mutated patients, and particularly TN patients. In multivariable analysis, CALR mutations had a favorable impact on survival that was independent of International Prognostic Scoring System (IPSS), Dinamic IPSS (DIPSS) or DIPSS-plus risk stratification, and also of ASXL1 mutation, known for its dismal impact on survival in PMF. At this regard, we found that DIPSSplus-independent OS was significantly longer in CALR-mutated/ ASXL1-unmutated compared with CALR-unmutated/ASXL1-mutated patients. Two main types of CALR mutations have been described until now, known as type 1 (a 52-bp deletion; p.L367fs*46) and type 2 (a 5-bp TTGTC insertion; p.K385fs*47); more than 50 indels were subsequently reported that can be grouped as type 1or type 2-like based on their molecular characteristics. The frequency of type 1 mutation is reported to be higher in PMF (60–80%) compared with essential thrombocythemia (39–61%), a part for a series including Chinese patients where no difference was noticed. Interestingly, known mutations generate a novel aminoacid C terminus of the protein with loss of the KDEL motif and replacement of negatively with positively charged or neutral amino acids, whose proportion however varies according to the type of mutation and might underlie emerging differences in the clinical correlates and prognostic impact associated with the two types of mutations. In this regard, it has been shown recently that the phenotype of mice expressing type 1 or type 2 mutation by retroviral transfer differs in that type 1 was associated with marked thrombocytosis and rapid progression to a myelofibrosis-like disease (with subsequent thrombocytopenia, splenomegaly and anemia), whereas type 2 was a milder phenotype with less thrombocytosis and reduced propensity to evolve. In a study of 617 PMF patients, of whom 140 were CALR-mutated, we found that patients harboring type 1 CALR mutation (n= 101) had a better OS compared with those harboring JAK2V617F mutation (n= 399; Po0.001), whereas no difference was observed between CALR type 1 and type 2 (n= 22), and as well as between patients with CALR type 2 mutation and those with JAK2V617F; such differences remained also after adjustment for the DIPSS score. A significant impact of CALR type 1 versus type 2 was observed by Tefferi et al. in a comparison of 76 and 10 CALR-mutated patients, respectively, that also included 196 patients harboring JAK2V617F mutation. Survival was longer in CALR type 1 compared with both CALR type 2 (hazard ratio (HR) 2.5, 95% confidence interval (CI) 1.1–5.4) and JAK2V617F (HR 2.8, 95% CI 1.9–4.2); in multivariable analysis that included DIPSS and ASXL1 mutational status, JAK2V617F versus CALR type 1 mutation, DIPSS and ASXL1 all remained independently predictive of shortened survival. At variance with the above results, a shorter survival associated with CALR type 1 mutation (HR 36.3, 95% CI 4.0–324.7) was reported by Cabagnols et al. in a study that included 45 type 1 and 8 type 2 PMF patients. On the other hand, by comparing 98 type-1/type 1like with 15 type 2/type 2-like CALR-mutated patients, Tefferi et al. showed that the median survival of type 1/type 1-like patients (13.7 years) was significantly better than type 2/type 2like (3.5 years) ones, that on turn had survival superimposable to patients harboring JAK2V617F mutation (4 years). The difference between the two types of CALR mutation remained significant after adjusting for age, ASXL1 or EZH2 mutations. Owing to the above conflicting results, the aim of this study was to evaluate the prognostic impact, if any, of the two different types of CALR mutations in a series of 396 PMF patients with a diagnosis of PMF made according to the 2008 World Health Organization criteria seen in our center. The mutational status of CALR was determined at diagnosis using previously described procedure of high-resolution capillary electrophoresis of fluorescent dye-labeled PCR amplicon of CALR exon 9; samples with abnormal peaks were subjected to conventional bidirectional Sanger sequencing. The mutated CALR allelic burden was estimated by peak area integration from capillary electrophoresis plot. The JAK2V617F mutational status was determined using real-time polymerase chain reaction, as described. Comparisons of quantitative variables between groups of patients were carried out using the nonparametric Wilcoxon rank-sum test. The cumulative probability of survival (OS) was estimated using the Kaplan–Meier method. Differences in OS between the groups were compared by using a log-rank test in univariate analysis. The SPSS package (V.22; Chicago, IL, USA) was used for statistical analysis. Out of the entire series, we found 251 patients (63.4%) harboring JAK2V617F mutation, 21 (5.3%) with a MPLW515 mutation, 50 (12.6%) lacking any known driver mutation and 74 (18.7%) who harbored a CALR mutation, of whom 53 (71.6%) were type 1/type 1-like and 21 (28.4%) were type 2/type 2-like. There was no statistically significant difference between the two groups harboring CALR mutation as regards the various hematologic and clinical variables reported in Table 1. On the other hand, patients harboring both CALR mutation types differed from the JAK2V617FCitation: Blood Cancer Journal (2015) 5, e360; doi:10.1038/bcj.2015.90