This study evaluated risk factors for ICVD among Ph-negative MPNs patients.The main finding of our study is that hypertension, smoking, increased percentage of neutrophils or lymphocytes are associated with the higher frequency of ICVD events in Ph-negative MPNs patients, although the relative role of each factor may vary in the individual subgroup.
As described by the 2016 WHO classiﬁcation, PV, ET and PMF, which are all subcategorized as the Ph-negative MPNs . ET (49.2-52.5%) is regarded as the most common subtype of Ph-negative MPNs, followed by PV (27.9-34.7%) and PMF (14.4-19.6%) [9,16-18]. But in the Mayo Clinic cohort of 3023 patients with Ph-negative MPNs, PMF was the most common diagnosis (42%). Our data showed the similar distribution of ET (82, 44.3%) and PV(78, 42.2%) which difference from others. A possible explanation for this might be that regional studies have a different distribution.
In our data, the pooled prevalence of ICVD with Ph-negative MPNs patients was 30.8% (57/185) which was higher than recently published data. The meta-analysis of 29 cohort studies with 13,436 patients with Ph-negative MPNs showed the prevalence of cerebrovascular disease was 7.4% (95% CI, 5.0–10.8%) and transient ischemic attack was 3.5% (95% CI, 1.9–6.4%). As showed in a German study, 19.3% (28/145) suffered from stroke . A possible explanation for this might be that those published studies reported the overall thrombosis and ischemic stroke as a subtype of thrombosic events, while we focused on the ICVD with Ph-negative MPNs. On the other hand, the difference may be related to the data we accessed was from the region where has higher risk of cerebrovascular disease in the northeast China. For the same reasons, regarding the subtype analysis in our data, especially in PV and ET, the occurrence of ICVD complications was higher than previous findings [9,18].
Established cerebrovascular risk factors such as hypertension, smoking, diabetes have been found to impart a consistently increased risk of vascular complications for patients with Ph-negative MPNs by some studies. The prevalence of hypertension (59.6% vs 32.0%), smoking (22.8% vs 6.3%), drinking (22.8% vs 6.3%), were significantly higher in the ICVD group compared with the non-ICVD group in our data, similar to the report by Zhang Y et al that showed 55.5% of patients with thrombosis had at least one cerebrovascular risk factor, and at least one cerebrovascular risk factor was also a risk factor for thrombosis in Chinese PV patients. Futhermore, the current results showed that hypertension and smoking were two independent risk factors for Ph-negative MPNs patients with ICVD as demonstrated by multivariate analysis. However, the other publications suggested that cerebrovascular risk factors did not encourage the development of thrombotic events in Ph-negative MPNs [15,21,22]. The difference may be due to the different subject cohorts (MPNs vs PV or ET) or number of cases. In our data, analysis of subgroups suggested the differences between the ET, PV and PMF. Smoking was independent risk factor in ET patients, on the other hand, smoking and hypertension were concomitant risk factors associated with increased risk for ICVD in PV patients. Whereas, it did not show any independent risk factor in PMF patients possibly due to the smaller number of analyzed patients.
In the last decade, growing evidence has highlighted the key role of leukocytes in the prothrombotic state and several studies have investigated the association between leukocytosis and tendency for thrombosis in patients with Ph-negative MPNs, but the conclusions were inconsistent. The uncertainty was thought due to inconsistencies in the definition of leukocytosis, lack of a clear cutoff value for WBC counts, and heterogeneity in methods for its assessment. Zhou et al found that neutrophil-to-lymphocyte ratio (NLR) was significantly associated with a higher risk of thrombosis in ET patients, which can predict the future thrombosis during follow-up.
In our data, leukocyte counts were increased in Ph-negative MPNs with ICVD, but not statistically different for patients who had ICVD or not. However, the percentage of neutrophils was significantly increased in Ph-negative MPNs patients with ICVD, as well as in ET and PMF patients. Importantly, increased percentage of neutrophils was independent risk factors for ICVD in patients with ET on multivariate analysis. The neutrophil is the most abundant leukocyte, which play central roles in the inflammatory response to the activation of blood coagulation. The activated neutrophils can produce molecules (such as reactive oxygen species, proteolytic enzymes, β2 integrin Mac 1 or CD11b) on their cell surface to affect the hemostatic system and induce a hypercoagulable state in vivo. Neutrophils from patients with Ph-negative MPNs display a number of features of enhanced activation. On stimulation, normal neutrophils can expel extracellular strands of decondensed DNA in complex with histones and other neutrophil granular proteins to produce neutrophil extracellular traps (NETs). Meng et al demonstrated that NETs were found within the thrombi. In the vasculature, NETs can form a scaffolding structure onto which platelets aggregate and form thrombi[12,28]. Our study seemed to support that neutrophils were related to an increased risk of ICVD events in Ph-negative MPNs.
With the advance of molecular biology, the mutations in JAK2, MPL (trombopoietin receptor) and CALR genes are identified in PV, ET and PMF. The types of driver mutations are closely related with the clinical features of Ph-negative MPNs patients. These mutations are responsible for both the onset and the pathogenesis of the disease and alter importantly cellular signaling pathways. The JAK2 gene codes for the Janus kinase2 protein, which participates in the JAK-STAT signaling pathway that is important in cellular proliferation and differentiation. The proportion of patients with the mutation is variable among the studies, which is ranging from 46.7 to 100% in patients with PV, from 31.3 to 72.1% in patients with ET, and from 25.0 to 85.7% in those with PMF. Our data showed the frequency of the patients with JAK2V617F mutation was 68.11%, which was similar as the published data[31,32]. These analysis by subgroups in our cohorts showed that JAK2V617F mutation is more frequent in PV(79.5%) than in ET(61.0%) and PMF(56.0%). These showed homogeneous results in the proportion of patients with the mutation have been reported by Zhang X et al in China cohorts. Approximately 11.1% of analyzed 126 patients with JAK2V617F-mutated MPNs were PMF which is in accordance with recently published data from China, whereas PV was considerably lower (49.2% vs 60.6%) and ET was higher (39.7% vs 30.4%) in our report. These difference may be caused by a region bias (the northeast China vs the central China) and the number of patients (126 vs 1537).
As similar to the previous report by Ong et al, the percentage of JAK2V617F mutation was significantly higher in the ICVD subgroup than the non-ICVD subgroup(78.9% vs 63.3%) in our research. Meanwhile, the ICVD events were observed in 35.7% of patients with JAK2V617F mutation, compared with 20.3% of patients without JAK2V617F mutation. The frequency of thrombotic events was significantly higher in patients with JAK2V617F mutation than in those without，was similar to previous observations. Therefore, the presence of the JAK2V617F mutation was considered a possible risk factor for ICVD. However, the current results showed that the JAK2V617F mutation was not the independent risk factors for Ph-negative MPNs patients with ICVD as demonstrated by multivariate analysis.It was thought that the higher hemoglobin and neutrophil counts, and fibrotic transformation induced by JAK2V617F take part in the pathogenesis of Ph-negative MPNs. Wolach1 et al showed that JAK2V617F expression was associated with increased NETs formation in response to neutrophils stimulation in Ph-negative MPNs patients and in JAK2V617F mouse models. Our results demonstrated that the older age, hypertension, blood counts (WBC, RBC, Hb, Hct, neutrophils, eosinophils), the percentage of neutrophils and eosinophils tended to be higher in patients with the JAK2V617F mutation than in those without. These suggested it was more likely due to older age, hypertension, higher percentage of neutrophils or other factors which increased the incidence of IVCD in Ph-negative MPNs patients with JAK2V617F mutation.
Our study showed the ICVD group had lower prevalence of CALR mutation than the non-ICVD group (3.2% vs 22.2%). The CALR gene codes for the calreticulin protein, which functions in calcium homeostasis and as a chaperone. Mutations of CALR could result in the replacement of the C-terminal negatively charged amino acids of calreticulin which offers an opportunity for immunologic targeting. Calreticulin has been implicated in the survival of malignant cells and diverse biologic processes, including proliferation, apoptosis, and immunogenic cell death[39,40]. Some researches confirmed that patients with mutant CALR are with younger age, higher platelet count, lower hemoglobin, lower leukocyte count, and lower incidence of thrombosis compared to JAK2 mutated ones[12,41]. Similar to those studies, the lower leukocyte count and percentage of neutrophils were found in CALR-mutated patients as compared to without in our data.
Several limitations of our study should be acknowledged. First, patients’ data were obtained from our hospital, we cannot exclude a selection bias. Second, the number of patients was also a limiting factor of this study and might affect our results.