Frequency and clinical significance of JAK2V617F gene mutation in philadelphia negative classical chronic myeloproliferative neoplasms

Aim: Philadelphia-negative (Ph-negative) myeloproliferative neoplasms (MPN) are a sub-category of MPN including polycthemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) and are clonal disorders of hematopoiesis resulting from the transformation of a hematopoietic stem cell, with abnormal proliferation of one or more of the myeloid lineages. PV is a MPN characterized by increased red blood cell mass and usually overproduction of granulocytes and platelets and increased spleen size. ET is characterized by an overproduction of platelets and usually accompanied by thrombotic or hemorrhagic symptoms. PMF is primarily characterized by abnormal cytokine expression, bone marrow fibrosis, extramedullary hematopoiesis and shortened survival. After discovery of the JAK2V617F mutation in 2005, molecular basis was unleashed in >95% of PV patients and >50-60% of PMF patients. Several studies reported that presence of higher V617F allele burden in ET is associated with defined haematological and clinical markers indicative of a more aggressive behavior while, in PMF, low allele burden was reported to be associated with shortened survival. Most studies examining V617F allele burden have limitations due to retrospective nature. The purpose of this study is to analyze the association of JAK2V617F mutation with laboratory characteristics and clinical phenotype in 410 Turkish patients (170 ET, 135 PV, 105 PMF) under follow up in Istanbul University Istanbul Medical Faculty, Department of Internal Medicine, Division of Hematology and Health Sciences University Medical Faculty İstanbul Bakırköy Dr. Sadi Konuk Training and Research Hospital Division of Hematology. In addition, our study aimed at assessing the predictive value of burden of V617F allele on ET and PMF patients. Material and methods: Our study group consisted of 410 patients (170 ET, 135 PV, 105 PMF) diagnosed as Ph-negative MPN according to the WHO criteria in 2016. Patients' demographic data, clinical, laboratory findings and OS were registered. In 228 of 410 patients (118 ET, 84 PMF, 26 PV) we used a real-time semiquantitative polymerase chain reaction (PCR) with 'JAK2 MutaScreen kit' to screen JAK2V617 mutation and the mutant allele burden. In 182 of 410 patients (109 PV, 52 ET, 21 PMF), the JAK2V617F mutation was detected by fluorescent resonance energy transfer (FFET) probes and LightCycler techniques. All statistical analysis including clinical and laboratory parameters was performed using SPSS. All p-values<0.05 were considered statistically significant. OS curves of ET and PMF were prepared by the 'Kaplan-Meier' method and compared by the long-rank test. In addition, leukemia free survival (LFS) in PMF patients was estimated using 'Kaplan-Meier' method. Results: A total of 170 ET (102 female, 68 male), 135 PV (88 male, 47 female) and 105 PMF patients (56 female, 49 male) were included. The mean age for ET, PMF and PV was 57.93 (SD 15.65), 63.09 (SD 13.64) and 61.22 (SD 14.13), respectively. The mean follow-up time of ET, PMF and PV patients was 87.01 months (SD 67.82), 68.03 months (SD 57.41) and 70.42 months (SD 56.39), respectively. The frequency of JAK2V617F mutation was higher in PV compared to ET patients (81.5%, 63.5%; respectively; p=0.001). Moreover, the frequency of JAK2V617F mutation was higher in PMF compared to ET patients (76.2%; 63.5%; respectively; p=0.0029). JAK2V671F positive patients with mutant allele burden in upper quartile ranges (allele burden >50%) was higher in PMF with respect to ET (21.4%; 4.2%; respectively; p=0.001). Moreover, there was a trend towards higher mutant allele burden in PV compared to ET (15.4%; 4.2%; respectively; p=0.055). In our study, JAK2V617 mutation positive PV group consisted of a significantly lower number of male patients (p=0.008). JAK2V617F mutation positive PV patients had significantly higher leukocyte and platelet counts and lower Hgb levels (p=0.001; p=0.001 and p=0.018, respectively). Rates of total thrombosis and arterial thrombosis were significantly higher in JAK2V617F mutation positive PV compared to JAK2V617F mutation negative PV (42.7%, 20%; respectively; p=0.035 and 26.4%, 8%; respectively; p=0.042). In our study, JAK2V617F mutation positive ET patients had significantly higher Hgb and Htc levels and lower platelet counts at initial diagnosis (p=0.001; p=0.001 and p=0.001, respectively). JAK2V617 mutation negative PMF group consisted of a significantly higher number of female patients (p=0.001). In PMF patients, the presence of JAK2V617F mutation was significantly associated with increased spleen size (p=0.042), and higher leukocyte count (p=0.019). There was a trend towards higher incidence of venous thrombosis in JAK2V617F mutation positive PMF compared to JAK2V617F mutation negative PMF (10%; 0; respectively; p=0.051). With regard to the JAK2V617F status, ET patients were divided into three groups: JAK2V617F mutation negative (n=54), JAK2V617F positive with mutant allele burden in the lower quartile (n=59) and upper quartile ranges (n=5). ET patients with upper quartile JAK2V617F allele burden had significantly lower Hgb levels (p=0.001), lower Htc levels (p=0.001) and higher LDH levels (p=0.008) as compared to the other two groups. Also, ET patients with upper quartile JAK2V617F allele burden had significantly increased spleen size and higher bleeding complications (p=0.034; 0.002; respectively). We observed significant association between ET patients with high JAK2V617F allele burden and higher death rates (p=0.007). There was a trend towards higher incidence of venous thrombosis in ET patients with upper quartile JAK2V617F allele burden (p=0.057). With regard to the JAK2V617F status, PMF patients were divided into three groups: JAK2V617F mutation negative (n=25), JAK2V617F positive with mutant allele burden in the lower quartile (n=41) and upper quartile ranges (n=18). Comparison across all three groups revealed significant associations between upper quartile allele burden and higher leukocyte counts (p=0.001). Also, PMF patients with upper quartile JAK2V617F allele burden had significantly increased spleen size compared to the other two groups (p=0.018). We observed significant association between PMF patients with low JAK2V617F allele burden and higher incidence of venous thrombosis (p=0.035). Comparison across all three groups revealed significant associations between JAK2 wild-type patients and higher number of female patients (p=0.005). OS and LFS was significantly shorter in the high risk PMF group as defined by 'DIPSS-plus' criteria (p=0.001; p=0.005; respectively). Conclusion: The field of Ph-negative MPNs has recently witnessed tremendous advances in the basic knowledge of disease pathophysiology that followed the identification of mutations in JAK2. These discoveries led to a revision of the criteria employed for diagnosis by the World Health Organization. The prognostic role of the JAK2V617F mutation has been the objective of intensive research. There are a limited number of studies that analyze and compare MPN patients according to their JAK2 allele burden. While a definitive position cannot yet been taken on all of the issues, there is a consensus that the presence of higher V617F allele burden, that is on the basis of a stronger activation of intracellular signalling pathways, is associated with the clinical phenotype of PV. Our study group includes a large series of patients and outlines the clinical phenotype and laboratory characteristics of the JAK2V617F mutation and allele burden in the Turkish population. Moreover, our study has a long period of follow-up and thus provides real life data for MPN patients. Our findings reconfirms the significance of JAK2V617F mutation and allele burden in MPNs.