Calreticulin Mutation Does Not Modify the International Prognostic Score for Predicting the Risk of Thrombosis Among 1,150 Patients with Essential Thrombocythemia
Authors: Finazzi Guido , Carobbio Alessandra , Guglielmelli Paola , Rumi Elisa , Salmoiraghi Silvia , Vannucchi Alessandro M. , Cazzola Mario , Passamonti Francesco , Rambaldi Alessandro , Barbui Tiziano 
Published: October 2019

Abstract
Background
An International Prognostic Score for the risk of thrombosis (IPSET-thrombosis) in Essential Thrombocythemia (ET) was developed (Barbui et al. Blood, 2012;120:5128). Risk factors included: age >60 years (1 point), cardiovascular (CV) risk factors (1 point), previous thrombosis (2 points) and the presence of JAK2V617F mutation (2 points). Low, intermediate and high risk categories were identified by scores 0-1; 2; and ≥ 3, respectively. Mutations in the exon 9 of CALreticulin (CALR) gene were recently identified in about 50-60% of patients with JAK2V617F negative ET and associated with a reduced thrombotic risk as compared with JAK2V617F positive patients.
Aim
To evaluate whether the identification of CALRmutation in patients with ET has any impact on the IPSET-thrombosis score
Patients and Methods
Under the auspices of AGIMM (AIRC Gruppo Italiano Malattie Mieloproliferative), four Italian centers with recognized experience in myeloproliferative neoplasms participated in the current study. Overall, 1,150 patients who met the 2008 WHO criteria for ET and were molecularly characterized for JAK2V617F, MPLW515L/K and CALR exon 9 mutations were included. The JAK2 and MPL mutations were assessed by real-time quantitative polymerase chain reaction and by high-resolution melting analysis followed by bidirectional Sanger sequencing. Mutations in exon 9 of CALRwere assessed by bidirectional sequencing or next generation sequencing.
Results
Presenting features of the study population were: median age 58 years (range 5th-95th percentile 27-82 years; 65% females), median hemoglobin 14.1 g/dL (range 5th-95th percentile 11.8-16.3), median leukocyte count 8.7x109/L (range 5th-95th percentile 5.4-14.7), median platelet count 718x109/L (range 5th-95th percentile 486-1313). CV risk factors (at least one among smoke, diabetes and hypertension) were present in 568 (49%) patients. Arterial or venous thrombosis history before or at diagnosis was documented in 167 (15%) patients. JAK2V17F, MPLW515L/K and CALRmutations were detected in 744 (65%), 44 (4%) and 164 (14%) patients respectively. The remaining 198 patients (17%) were wild-type for all three mutations.
During a median follow-up of 4.1 years (range 0-29), 104 patients developed an arterial or venous thrombotic event, with a total incidence rate of 1.59% patients/year (pt-ys). The IPSET-thrombosis ability to discriminate the thrombotic risk was confirmed. In fact, in the low risk (reference category), the rate was 0.57% pt-ys; in the intermediate risk was 1.60% pt-ys (Hazard Ratio (HR) 3.10, 95% Confidence Interval (CI) 1.55-6.18, p=0.001) and in the high risk group was 2.34% pts-yr (HR 4.59, 95% CI 2.41-8.77 p<0.0001).
As to the impact of CALR mutation in the three categories of the IPSET-thrombosis score, we observed that CALR mutated patients were more frequently distributed in the low risk (48%) and intermediate risk (46%) than in the high risk IPSET groups (6%). In univariate analysis, patients carrying CALR mutation had a lower incidence of thrombosis than those with JAK2V617F (HR 0.61, 95% CI 0.34-1.09, p=0.093). However, CALR mutated patients were significantly younger (median age 53.5 versus 60.8 years, p=0.001) and presented with less previous thrombosis (8% versus 17%, p=0.005) than JAK2V617F mutated patients. This could explain why in multivariable models, CALR mutation did not retain the association with the risk of thrombosis. This was demonstrated in the whole population (HR 0.81, 95% CI 0.30-2.17, p=0.674), as well as in the low risk (HR 1.01, range 0.27-3.81, p=0.987) and intermediate risk categories (HR 1.80, range 0.57-5.72, p=0.317); the high risk category was not evaluable for the low proportion of CALRmutated patients in this group.
Conclusions
CALR mutation does not have a significant impact on the IPSET-thrombosis prognostic score. The score can be used as it is to predict the risk of thrombosis in molecularly-annotated, WHO-2008 diagnosed ET patients.

Disclosures
Vannucchi: Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding.