Correlation of Somatic Mutations and Clinical Characteristics at Presentation in Patients with Essential Thrombocytemia
Authors: Eftimov Aleksandar , Panovska-Stavridis Irina , Popova-Simjanovska Marija , Ivanovski Martin , Stojanovic Aleksandar , Cevreska Lidija , Matevska Nadica Geshkovska , Dimovski Aleksandar J 
Published: October 2019

Abstract
Background
Most patients with essential thromocythemia (ET) have somatic mutations in janus kinase 2 (JAK2), calreticulin (CALR) and thrombopoietin receptor (MPL) genes. The clinical correlates at the presentation of patients with different mutations has not been established as yet.
Materials and methods
We evaluated the clinical, laboratory, and molecular features of 150 consecutive patients (93 females/57 males; average age 59,0+/- 14,2 years) with ET who were diagnosed at the University Clinic for Hematology in Skopje based on the revised WHO criteria. DNA from all patients was isolated from peripheral blood obtained at diagnosis using Qiagene DNA exctraction kit. The presence of JAK2V617F mutation was evaluated with fluorescent allele specific PCR/capillary electrophoresis. The CALR exon 9 insertions or deletions were initially detected with a fluorescent PCR/capillary electrophoresis and characterized with Sanger sequencing. The presence of mutations in exon 10 of the MPL gene was analyzed with direct sequencing of PCR amplified fragments. Informant consent was obtained from all participants. Statistical analysis was performed by Wilcoxon rank- sum test.
Results
The basic hematological parameters at diagnosis for all patients were as follows; Plt = 962.1x109 ±351,2 x109/L; Hb = 126,8 ±39,2 g/L; Le = 10,2x103 ±4,2 x103/µL. No correlation was found between any of these parameters and age at diagnosis or sex of the patients. JAK2V617F, CALR and MPL mutations were found in 89 (59.3%), 42 (28%) and 2 (1.3%) patients, respectively, while 17 (11.4%) patients did not have a mutation in the 3 evaluated genes (triple negative). Twelve different mutations were found in CALR gene, the most common being 52-bp deletion (c.1092_1143del) and 5-bp insertions (c.1154_1155insTTGTC) which were present in 11 (26,2%) and 20 (47,6%) patients, respectively. Ten different mutations, including 6 insertions (c.1088insTTGTC; c.1088delinsTTTGTC; c.1113_1123delinsTTGT; c.1154delinsTATGTC; c.1126_1131delinsTGCGT; c.1154_1155insGTGTC) and 4 deletions (c.1092_1137del; c.1096_1129del; c.1089_1141del; c.1100_1145del), were detected in the other 11 patients with CALR mutation. The JAK2V617F mutation and triple negative status were more prevalent in female patients (67% and 68% respectively), while no sex preference was detected in patients with the CALR mutation. The average age of patients with JAK2V617F, CALR insertions, CALR deletions and triple negative status were 60,7±13,9, 54,5±12,9, 59,0±15,1 and 58,8+/-14,8 years, respectively. Patients with CALR mutation had a higher platelet count at the time of diagnosis (1134x109 ±372,9 x 109 /L) compared to both patients with mutated JAK2 (952 x109 ±335,6 x109/L) (P=0.047) and to triple negative patients (mean 886 x109/L±303,2 x109/L) (P=0.024). Patients who carried CALR insertions had higher platelet counts (1222,9 ±396,8 x109/L) than patients with CALR deletions (mean 956,8 ±287,8 x109/L ) (P=0.0405). White blood cells counts were the highest in patients with JAK2V617F mutation (mean 11.1±4,5x103/µL) compared to both CALR positive (mean 8,8±2,7x103/µL) and triple negative patients (mean 7,9±2,2x103/µL) (P=0.0049). No difference in Hb levels were present between patients with various mutations.
Conclussion
ET patients with CALR insertions present with higher platelet counts and at an earlier age than patients with JAK2V617F, CALR deletions or triple negative patients, which might translate to differences in prognosis in these groups of patients. Higher incidence of JAK2V617F mutation and triple negative status in female patients point to a common mechanism for predisposition to these conditions.

Disclosures
No relevant conflicts of interest to declare.