On the Absence of Calreticulin (CALR) Mutations in Chronic Myeloprolierative Neoplasms (MPNs) with Splanchnic Venous Thrombosis (SVT): Experience from a Single Institution

Background: According to recent findings, the management of myeloproliferative neoplasms (MPNs) is highly dependent on presence or absence of thrombotic events. The JAK2 mutation has been identified as a marker of MPNs. It is also an occult marker in several patients with splanchnic venous thrombosis (SVT), but its contribution as an additional thrombotic risk factor in MPNs is still under discussion. Moreover, a pro-thrombotic risk factor, either inherited or acquired (Factor V Leiden mutation, deficiencies in protein C, protein S and Prothrombin mutation 20210) can be identified in these patients. Recently, another milestone in the molecular diagnosis of MPNs, somatic mutations in the CALR gene, has been reported. A total of 36 types of frame-shifting insertions and deletions were detected in the exon 9 of CALR gene, which encodes a Ca2+ binding protein in endoplasmic reticulum called calreticulin. 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 These mutations were reported to have a incidence of over 60% to 80% in JAK2 and MPLmutation-negative Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) patients. Compared to those with JAK2 mutation, CALR-mutated ET patients are younger and have a lower leukocyte count and higher platelet count. CARL mutations have been also reported as a favorable prognostic factor on thrombosis-free survival (TFS) for ET patients.

Aims: In this study, we evaluated the incidence of SVT, JAK2 and CALR mutations, and prothrombotic risk factors in patients with MPNs observed in our center from January 2000 to January 2014.

Methods: We performed a retrospective review of clinical charts of 466 Ph1 negative MPN patients followed in our center, classified according to the WHO 2008 classification. Patient and disease characteristics, including JAK2V617F, MPL and CALR mutations and thrombotic risk factors were recorded.

Results: The median age of patients with diagnosis of MPN was 43 years. Fourteen patients (13 females, 1 male; 3%) of median age 46 years presented a SVT. Three had a Budd Chiari syndrome and 11 a portal venous thrombosis. According to a histological review, these patients were classified as follows: ET, 2 cases, PMF, 3 cases, Polycythemia Vera (PV) 1 case, Myelofibrosis in a prefibrotic phase (MF0) 8 cases. Classification of 11 cases with Myelofibrosis according to the IPSS identified 7 as INT1, 1 as INT2 and 3 as low risk. Among all 14 patients diagnosed with SVT, 12 were JAK2^V617F positive with a median allelic burden of 30%, 1 patient was MPL positive, and 1 patient was triple-negative. CALR mutation was not observed in any of the patients. Two cases were diagnosed with MPN 30 months after SVT, 3 patients experienced SVT after a median follow-up of 108 months from MPN diagnosis while in 9 patients the diagnosis of MPN was concomitant to SVT. In the latter patients, median Hb levels were 12.4 g/dL , WBC 8260 /µL, HCT 36.3%, PLT 337.000/ µL and a modest hepatomegaly and splenomegaly were documented. Prothrombotic risk factors were found in 9 of 13 patients. Two patients experienced a thrombotic episode prior to the diagnosis of SVT and two subsequently during the follow-up. Interestingly, 9 (70%) of MPN patients with SVT exhibited at least one prothromobtic risk factor, such as factor V Leiden, Protein C deficiency, hyperhomocystinemia and 50% had two or more associated defects. Thirteen of the 14 patients are currently being treated as follows hydroxyurea (9), interferon (1), and ruxolitinib (3). All patients received oral anticoagulant treatment except for three who are on antiplatelet therapy. MPN patients without SVT had a lower prevalence of prothrombotic risk factors and developed venous thrombosis in different anatomical sites: in these cases WBC count, platelet values and the presence of JAK2^V617F mutation correlated with the development of the thrombotic event.

Conclusions: Although SVT has a low incidence in MPN patients, a potential benefit of testing for mutations in CALR gene and for additional prothrombotic risk factors is suggested in the whole MPN population for the prevention and treatment of this complication.