Driver mutations (JAK2V617F, MPLW515L/K or CALR), pentraxin-3 and C-reactive protein in essential thrombocythemia and polycythemia vera

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative neoplasms (MPNs) characterized by clonal expansion of an abnormal haematopoietic progenitor cell and a clinical course that is complicated by frequent cardiovascular complications as well as increased risk of transformation to myelofibrosis (MF) or acute leukaemia (AL) [ 1, 2]. The discovery of the Janus kinase 2 ( JAK2) V617F mutation significantly improved the understanding of the biology of these disorders [ 3]. The subsequent identification of other acquired somatic lesions, such as JAK2 exon 12 mutation [ 4], mutation in the gene encoding thrombopoietin receptor ( MPLW 515L/K) [ 5] and the recently discovered mutations in the exon 9 of calreticulin ( CALR) gene [ 6, 7] reinforced the central role of cytokine receptor/signal transduction lesions in promoting MPN phenotypes. In surveys of large MPN cohorts, JAK2V617F can be detected in 95% of patients with PV and JAK2 exon12 mutation in the remaining 5% of patients. Among ET patients, JAK2V617F can be detected in 60 to 65% of patients, MPLW515L/K in about 5% and CALR mutation in about 20 to 25%. The understanding that an array of somatic mutations contributes to the biology of these disorders [ 8, 9] prompted new studies to address the impact of patients’ mutation background on disease phenotype. These studies demonstrated that somatic mutations, by determining a more pronounced activation of platelets, leukocytes and endothelial cells [ 10, 11] and promoting an increased number of leukocytes [ 12] can sustain a condition of chronic inflammation, which may contribute not only to the premature atherosclerosis underlying the cardiovascular events but also to clonal evolution and second cancer [ 2, 13– 17]. Based on these data, there is an increasing interest in two inflammatory biomarkers, belonging to the superfamily of pentraxins, such as pentraxin 3 (PTX3) and high-sensitivity C-reactive protein (hs-CRP). We hypothesized that these inflammatory markers might be useful to improve prognostic classification of patients with ET and PV. In a previous paper, we showed that PTX3 and hs-CRP levels were correlated with JAK2 mutation allelic burden and associated with different risks of thrombosis, although in opposite directions [ 18]. Furthermore, elevated hs-CRP levels were associated with shortened leukaemia-free survival (LFS) in myelofibrosis [ 19]. However, whether or not somatic mutations, other than JAK2V617F, influence the blood levels of PTX3 and hs-CRP is unknown and their association with the main incident relevant outcomes in ET and PV patients has not been fully explored. Accordingly, in this cross-sectional study, we examined a large cohort of patients with PV and ET.

Blood samples of 477 patients with ET and PV, diagnosed according to the 2016 World Health Organization (WHO) criteria [ 20], were obtained from consecutive, well-characterized patients, regularly followed in two Italian haematological centers (Bergamo and Florence). Institutional review board approval was obtained from the two participating centres in the framework of the AIRC-Gruppo Italiano Malattie Mieloproliferative (AGIMM) project. Blood samples were obtained at diagnosis in 172 patients (36%) or during follow-up in 306 (64%) (median time from diagnosis was 4.2 years, range 0.03–29.8 years). The following previously published methods were used: real-time quantitative PCR for JAK2V617F [ 21] and high-resolution melting analysis followed by bidirectional Sanger sequencing or next-generation sequencing for MPLW515L/K and CALR mutations [ 22]. In particular, evaluation of JAK2V617F mutation was performed on the genomic DNA purified from granulocytes by qualitative or quantitative method. Qualitative assay was based on a allele-specific PCR starting from 100 ng DNA with a sensitivity defined as 0.5–2% [ 23]. Quantitative assay for the measurement of JAK2V617F allele burden was performed by a quantitative real-time PCR assay, using 40 ng DNA. All samples were analysed in triplicate, and the amount of JAK2V617F allele was calculated by comparison with serial dilutions of JAK2 plasmids. The sensitivity of the quantitative method is 0.08–0.008% [ 21]. JAK2V617F mutation was classified as having low allele burden (<50%, heterozygous) or high allele burden (≥50%, homozygous). High-sensitivity C-reactive protein (hs-CRP) was measured by a latex immunoassay (CardioPhaseHigh Sensitivity Siemens Healthcare Diagnostic Inc., Italy) and PTX3 plasma levels were measured by an in-house Sandwich ELISA as previously described [ 18].

Major outcomes recorded anytime during the follow-up period were major thrombosis, bleeding, evolution to MF or AL and death. Patients were treated according to current recommendations [ 24] and therapy included phlebotomy, hydroxyurea, pipobroman, busulfan and alpha interferon. Evolution to post-ET and post-PV MF and AL was diagnosed following the International Working Group for Myeloproliferative Neoplasms Research and Treatment and WHO criteria, respectively [ 20, 25].

The strong association between the levels of PTX3 and the JAK2V617F allele burden is consistent with our previous results [ 18] and considering the major role played by JAK2V617F mutation in the pathogenesis of inflammatory state in MPNs [ 10] is not surprising. Indeed, it is well established that JAK2V617F determines a more pronounced activation of platelets, leucocytes and endothelial cells [ 10– 12] and it might induce the accumulation of reactive oxygen species in the haematopoietic stem cell compartment [ 26], orchestrating the construction of the inflammatory microenvironment of MPNs [ 27]. The association of high PTX3 levels and JAK2V617F homozygous status with haematological evolution and all-cause mortality suggests that inflammation in the tumour microenvironment might contribute to the genetic instability and disease progression of MPNs [ 15, 28]. Although it could be argued that only hematological transformation in AML may be due to additional mutation acquisition, the relative small number of total events prevented a formal evaluation of differences by keeping a clear distinction between the MF and AML evolution. In multivariate analysis, after adjustment for JAK2V617 allele burden, the association between PTX3 levels and adverse outcomes was no longer statistically significant, suggesting that PTX3 levels might simply reflect inflammation derived from constitutively activated blood cells, belonging to the malignant clone harbouring JAK2V617F mutation. A second relevant finding, in keeping with our previous results [ 18], was that the major thrombosis rate tended to be lower at the highest PTX3 levels, suggesting that PTX3 might have a protective role against the detrimental effects of inflammation in the cardiovascular risk. Preclinical studies have shown that PTX3 is released from activated leukocytes and attenuates neutrophil recruitment at sites of inflammation [ 29], by limiting P-selectin-dependent inflammation [ 30]. Moreover, PTX3 deficiency was associated with increased fibrin deposition in different models of tissue damage and a direct fibrinolytic effect of PTX3 has been described in vitro [ 31] . In addition, PTX3 administration resulted to be protective in a model of arterial thrombosis [ 32], and in carcinogenesis models, it has been demonstrated that PTX3 acts as extrinsic oncosupressor by regulating complement-mediated and macrophage-sustained tumour inflammation [ 33]. While it is possible that JAK2V617F mutation is a potent driver of PTX3 release, the biologic and clinical correlates that can be attributed to high levels of circulating PTX3 remain to be defined and interpretations are mostly speculative. In this regard, that PTX3 might play a role in disease progression and development of fibrosis is a distinct possibility [ 34, 35].

In contrast to PTX3, hs-CRP levels were not associated with the JAK2V617F allele burden, supporting the hypothesis that hs-CRP and PTX3 reflect distinct aspects of the inflammatory process and that different mechanisms regulate the circulating levels of these proteins. Patients with a more pronounced chronic inflammation, as suggested by the high hs-CRP levels, exhibited an increased risk of haematological evolution and death of approximately three- and four fold, respectively, and to a lower extent also of thrombotic events compared to those in the lowest group. These findings are in keeping with previous studies in apparently healthy subjects, patients with cardiovascular disease and MPNs patients overall showing that higher levels hs-CRP are associated with an increased risk of all-cause mortality, malignant diseases and thrombotic events [ 18, 19, 35– 39]. A striking finding of our results is that the magnitude of predictive capacity of adverse outcomes of hs-CRP seems to be significantly greater in ET and PV patients than in subjects with and without cardiovascular disease in whom the risk was only about 1.5-fold increased in individuals with highest hs-CRP levels compared to those with lowest levels [ 37, 40, 41].

The strong correlation between JAK2V617F allele burden and PTX3 levels provides further evidence to support the role of the JAK2V617F mutation as a key driver of the MPN-associated chronic inflammation. These results may also represent the groundwork to perform prospective studies aimed at evaluating the impact of treatments targeting simultaneously the clonal haematopoiesis (for example, with interferon-alpha2) and the accompanying inflammatory status (with agents such as JAK1/2 inhibitors, statins and histone deacetylase inhibitors) on the risk of inflammation-driven disease progression and cardiovascular events, especially in patients with high PTX3 and hs-CRP levels.