B Cell Immune Profiles in Essential Thrombocythemia with Calr Mutations: Clinical and Molecular Correlates
Authors: Chang Yu-Cheng , Lim Ken-Hong , Lin Huan-Chau , Chiang Yi-Hao , Huang Ling , Kao Chen-Wei , Chang Chiao-Yi , Lin Ching-Sung , Su Nai-Wen , Lin Johnson , Hsieh Ruey-Kuen , Chang Ming-Chih , Chang Yi-Fang , Chen Caleb Gon-Shen , Kuo Yuan-Yeh , Chou Wen-Chien 
Published: October 2019

Abstract
Introduction: Essential thrombocythemia (ET) is a BCL-ABL1-negative myeloproliferative neoplasm (MPN), and is characterized by increased number of mature megakaryocytes (MKs) in the bone marrow and sustained thrombocytosis in the peripheral blood. We have reported that activated B cells are increased in patients with essential thrombocythemia, and can facilitate platelet production mediated by cytokines, such as interleukin-1beta (IL-1β) and interleukin-6 (IL-6) regardless JAK2 V617F mutational status (Thromb Haemost. 2014, 112: 537). Recently, Calreticulin (CALR) mutations were discovered in JAK2/MPL-unmutated essential thrombocythemia (ET) and primary myelofibrosis. Although CALR mutations may be associated with activated JAK-STAT signaling pathway, its exact molecular pathogenesis remains elusive in MPN. Interestingly, in vitro study has shown that CALR is capable of driving B cells activation through the toll-like receptor 4 (TLR4) pathway (J Immunol.2010; 185: 4561). Here we sought to evaluate the association between CALR mutations and B cell immune profiles in ET patients.
Methods: Fifty-four patients diagnosed with ET based on the 2008 WHO classification were enrolled into this study. CALR mutations were screened by high-resolution melting analysis and nucleotide sequencing. JAK2 V617F and MPL mutations were screened by allele-specific PCR and nucleotide sequencing, respectively. B cell populations, granulocytes/monocytes membrane-bound B cell-activating factor (mBAFF) and CALR levels, B cells TLR4 expression and intracellular levels of IL-1β/IL-6 and the expression of CD69, CD80, and CD86 were quantified by flow cytometry. Serum BAFF and plasma CALR concentrations were measured by ELISA. Forty-eight healthy adults and 17 patients with reactive thrombocytosis were used for comparison. The association between clinical, laboratory and molecular characteristics were studied. Statistical significance was defined as a two-sided p value <0.05 and SPSS version 22.0 (IBM, New York, USA) was used for all analyses.
Results: In this series, 19 (35.2%) patients harbored 8 types of CALR exon 9 mutations including 4 (7.4%) patients with concomitant JAK2 V617F mutations. Compared to JAK2 V617F mutation, CALR mutations correlated with younger age at diagnosis (p=0.04), higher platelet count (p=0.004), lower hemoglobin level (p=0.013) and lower leukocyte count (p=0.013). Among all ET patients, CALR mutations correlated with significantly lower serum BAFF level (median 1.6 ng/mL, p =0.049) and higher fraction of B cells with TLR4 expression (median 11.3%, p=0.021). Compared to healthy adults, patients with ET had statistically significant higher serum BAFF concentrations and higher mBAFF levels on both granulocytes and monocytes, and higher fraction of B cells with TLR4 expression and higher fractions of B cells with intracellular IL-1β and IL-6 expression irrespective of their genotypes. ET patients with both JAK2 and CALR mutations had statistically higher number of CD69-positive and CD86-positive activated B cells when compared with healthy adults. Among the three mutational groups of ET patients, there were no significant differences in granulocytes/monocytes mBAFF, in the fraction of B cells with intracellular IL-1β or IL-6 expression, and the numbers of CD80-positive and CD86-positive activated B cells. Granulocyte membrane-bound CALR levels were highest in patients with reactive thrombocytosis. Plasma CALR concentrations were highest in patients with reactive thrombocytosis (mean +/- SE: 9.04 +/- 0.59) and lowest in CALR -mutated ET patients (5.35 +/- 0.90, p <0.001).
Conclusions: Activation of B cells is universally present in ET. Both granulocyte membrane-bound CALR levels and plasma CALR concentrations were lower in CALR-mutated ET patients suggesting that CALR may not play a major role in the activation of B cells in these patients.

Disclosures
No relevant conflicts of interest to declare.