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1610 B Cell Immune Profiles in Essential Thrombocythemia with Calr Mutations: Clinical and Molecular Correlates

Myeloproliferative Syndromes: Clinical
Program: Oral and Poster Abstracts
Session: 634. Myeloproliferative Syndromes: Clinical: Poster I
Saturday, December 5, 2015, 5:30 PM-7:30 PM
Hall A, Level 2 (Orange County Convention Center)

Yu-Cheng Chang, MD1*, Ken-Hong Lim, MD1, Huan-Chau Lin, MD1*, Yi-Hao Chiang, MD1*, Ling Huang, MSc2*, Chen-Wei Kao2*, Chiao-Yi Chang, MSc2*, Ching-Sung Lin, MSc2*, Nai-Wen Su, MD1*, Johnson Lin, MD, MPH1*, Ruey-Kuen Hsieh, MD3*, Ming-Chih Chang, MD1*, Yi-Fang Chang, MD, PhD1*, Caleb Gon-Shen Chen, MD, PhD1*, Yuan-Yeh Kuo, PhD4* and Wen-Chien Chou, MD, PhD5

1Mackay Memorial Hospital, Taipei City, Taiwan
2Mackay Memorial Hospital, New Taipei City, Taiwan
3Department of Internal Medicine, Division of Hematology and Oncology, and Department of Medical Research Good Clinical Research Center Laboratory, Mackay Memorial Hospital, Taipei, Taiwan
4Graduate Institute of Oncology, College of Medicine, National Taiwan University, Taipei City, Taiwan
5Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan

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 JAK2V617F 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. JAK2V617F 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 JAK2V617F mutations. Compared to JAK2V617F 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.

*signifies non-member of ASH