Biological Effects Of MPL Or CALR Mutation On Neutrophil Alkaline Phosphatase In Patients With Myeloproliferative Neoplasms

Objective:The myeloproliferative neoplasms(MPN)are clonal hematopoietic stem cell cancers characterized by the malignant production of mature myeloid cells.JAK2V617 F,MPL and CALR gene mutations are major genetic mutations in patients with MPN.These mutations occur in the stage of myeloid stem cells,and involvement each myeloid cells.Neutrophil alkaline phosphatase(NAP)is the product of mature neutrophils,which is an indispensable marker of functionally mature neutrophils.JAK2V617 F mutation can enhanced neutrophil NAP expression,the NAP score of patients with JAK2V617 F mutations was significantly increased.In this study,we will show an association between MPL or CALR mutations in patients with MPN and the NAP score from the clinical and cellular levels.Methods:1.Analysis the association between MPL or CALR mutations in patients with MPN and the NAP score from clinical levels.Recruited myeloproliferative neoplasms with JAK2 V617 F,MPL or CALR gene mutation in the Second Hospital of Shanxi Medical University in July 2014 to July 2016,including polycythemia vera(PV),essential thrombocythemia(ET)and primary myelofibrosis(PMF).Collect the NAP score of peripheral blood(all the patients from the same laboratory),the NAP score of each mutant group was analyzed statistically.2.Analysis the association between MPL or CALR mutations in patients with MPN and the NAP score from cellular levels.We constructed the lentivirus expression vector of wild-type and mutant of MPL and CALR.NB4,a myeloid lineage cell line,was transduced with MPL or CALR mutant lentiviral vector.NB4 cells were cultured with All-trans retinoic acid(ATRA)and Granulocyte colony stimulating factor(G-CSF).The cells at the time of induction were collected at 48 h,72h,96 h,120h and 144 h.Neutrophil alkaline phosphatase expression was evaluated by the Senso LyteTM p-Nitro phenyl phosphate(pNPP)alkaline phosphatase assay kit.Results:1.Comparative analysis of NAP scores in MPN with patients JAK2V617 F,MPL and CALR gene mutations.A total of 125 patients with newly diagnosed MPN were collected,including 75 patients with ET,19 patients with PMF and 31 patients with PV.In the 75 patients with ET,including 51 patients with JAK2V617 F,4 patients with MPL,20 patients with CALR mutation.The median NAP score of the patients with patients JAK2V617 F mutations was significantly higher than the normal range,while the NAP score MPL or CALR positive patients was lower.There was a significant difference in NAP scores between JAK2V617 F positive patients and CALR(P <0.01)or MPL(P = 0.001),but the difference between MPL positive and CALR was not significant(P = 0.245).In the 19 patients with PMF,including 13 patients with JAK2V617 F,2 patients with MPL and 4 patients with CALR mutation.The median NAP score of the patients with patients JAK2V617 F mutations was significantly higher than the normal range,while the NAP score MPL or CALR positive patients was lower.There was a significant difference in NAP scores between JAK2V617 F positive patients and CALR(P=0.003)or MPL(P= 0.027),but the difference between MPL positive and CALR was not significant(P =0.814).31 patients with PV were JAK2V617 F positive,the NAP score higher than the normal range.2.Comparative analysis the NAP score of induced NB4 cells with wild-type or mutant of MPL gene.The cells were collected at each induction time point.Research findings show,with the induction of differentiation time,the NAP expression of NB4 group,MSCV group,and MPLWT group was gradually increased,but the expression of MPLA497-L498LVIAins4 and MPLW515 L group was not obvious.After induced differentiation for 96 h,the differences between MPLW515 L group ? MPL A497-L498LVIAins4 group and NB4 group?MSCV group?MPLWT group was significant(P <0.05).3.Comparative analysis the NAP score of induced NB4 cells with wild-type or mutant of CALR gene.The cells were collected at each induction time point.Research findings show,with the induction of differentiation time,the NAP expression of NB4 group,MSCV group,and CALRWT group was gradually increased,but the expression of CALRtype1 and CALRtype2 group was not obvious.After induced differentiation for 96 h,the differences between CALRtype1?CALRtype2 group and NB4 group?MSCV group?CALRWT group was significant(P <0.05).Conclusions:The alkaline phosphatase expression of neutrophils with JAK2V617 F gene mutation was increased,while the alkaline phosphatase expression of neutrophils with MPL and CALR gene mutation was decreased.These data suggest that JAK2 V617 F,MPL,CALR mutants are able to lead to MPN phenotype,but they are heterogeneous in the pathogenesis and clinical features.