JAK2V617F Mutation And PRV-1 Expression In BCR/ABL Negative Chronic Myeloproliferative Disease

Objectives: The classical BCR/ABL negative chronic myeloproliferative diseases (cMPDs) consist of polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). Recently, a highly pathogenic acquired characteristic gene mutation, JAK2V617F, was discovered. This mutation has been detected in up to 90% of patients with PV and in a sizeable proportion of patients with other cMPDs such as ET and IMF. JAK2V617F is a constitutively active tyrosine kinase that is able to activate JAK-STAT signaling. On the other hand, polycythemia rubra vera-1 (PRV-1), a novel hematopoietic receptor, was reported to be overexpressed in patients with BCR/ABL negative myeloproliferative diseases. However, it is not clear whether there is a relationship between JAK2V617F mutation and the overexpression of PRV-1mRNA, and whether they both influence the clinical characteristic of the BCR/ABL negative cMPDs.The present study is thus designed to examine the JAK2V617F mutation and measure the expression level of PRV-1mRNA in cMPDs, to make a further study on the molecular biological pathogenesis of BCR/ABL negative cMPDs, and to try to provide theoretical basis for developing possible targeted medicine for the treatment of JAK2V617F mutation positive cMPDs patients.Methods: The peripheral blood or bone marrow samples were obtained after informed consent from 62 BCR/ABL negative cMPDs patients(26 PV cases, 26 ET cases, 9 IMF cases and one CNL case), 20 chronic myelogenous leukemia (CML) cases, 11 acute leukemia (AL) cases and 12 healthy volunteers. The 62 BCR/ABL negative cMPDs patients were designed as research group and the others were control group. All Patients'clinical characteristics were observed and compared. The JAK2V617F mutation was detected by allele-specific polymerase chain reaction (AS-PCR) method and confirmed by direct sequencing and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP). The expression level of PRV-1 mRNA was measured by semi-quantity reverse transcription polymerase chain reaction (RT-PCR).Results:1 The mutation state of JAK2V617F in each group.The JAK2V617F point mutation was detected in 44 BCR/ABL negative cMPDs patients, including 23/26(88.5%) PV patients, 15/26(57.7%) ET patients, 5/9(55.6%) IMF patients and one CNL patient. The JAK2V617F mutation was not detected in 20 CML and 11 AL patients as well as 12 health adults. The mutation rate in BCR/ABL negative cMPDs (71.0%) was significantly higher than that in CML patients (0.0%) and AL patients (0.0%) as well as normal control (0.0%)(P<0.001). Comparing the subgroups of BCR/ABL negative cMPDs, we found that the mutation rate in PV (88.5%) was significantly higher than that in ET (57.7%) (X2=6.256,P=0.012) , while no statistically significant difference was found between PV and IMF (55.6%) (P=0.055). There was no statistically significant difference in mutation rate between ET and IMF (P=1.000).2 All the 44 JAK2V617F positive samples and 10 JAK2V617F negative samples were re-examed by DNA sequencing and PCR-RFLP analysis. The mutation results were exactly the same, and all the 44 JAK2V617F-positive samples were subgrouped according to their JAK2V617F mutation type: 5 PV patients and 1 IMF patient were T/T homozygote mutation at 1849th cordon, others were T/G heterozygote mutation at 1849th cordon. In all JAK2V617F negative samples, the wild type sequence of JAK2-JH2 domain was G/G homozygote at 1849th cordon.3 The expression level of PRV-1mRNA in each group.In 12 normal controls, 10 persons expressed PRV-1mRNA. The expression level range was from 0.000 to 0.799. The average expression level was 0.387±0.231. The expression of PRV-1mRNA was detected in 42/44 (95.5%) JAK2V617F mutation positive cMPDs patients and 17/18(94.4%) JAK2 V617F mutation negative cMPDs patients. The expression level of PRV-1mRNA in JAK2V617F mutation positive cMPDs patients(0.695±0.274) was significantly higher than that in JAK2V617F mutation negative cMPDs patients(0.403±0.168) (P<0.001). Besides, the expression level of PRV-1 mRNA in JAK2V617F mutation positive cMPDs patients(0.695±0.274) was significantly higher than that in CML(0.285±0.246) patients, AL patients (0.262±0.143) and normal control (0.387±0.231)(P<0.001).While there was no significant difference between JAK2V617F mutation negative patients (0.403±0.168) and all other patients in control group (P>0.05). Comparing the expression level of PRV-1mRNA in CML, AL patients with that in health volunteers, no statistically significant difference was found (P>0.05). There was no statistically significant difference in the expression level of PRV-1mRNA among the three subgroups of BCR/ABL negative cMPDs patients (PV, ET and IMF) (P>0.05).4 The comparison of clinical characteristicsThe peripheral white blood cell counts (mean 18.2×10~9/L) and platelets counts (mean 479×10~9/L) in JAK2V617F mutation positive PV patients were statistically significant higher than that (7.6×10~9/L and 277×10~9/L, respectively) in JAK2V617F negative PV patients (P=0.035 and P=0.025, respectively) at diagnosis.Compared with the mutation negative ET patients, the mutation positive ET patients had statistically significant higher hemoglobin (122g/L vs 146g/L, P=0.001) and peripheral white blood cell counts (10.9×10~9/L vs 14.6×10~9/L, P=0.044) as well as a higher complication rate (P=0.034). But no statistically significant difference in the clinical data was found between JAK2V617F positive and negative IMF patients (P>0.05).Conclusion:1 JAK2V617F point mutation was detected in a sizeable proportion of BCR/ABL negative cMPDs patients,the mutation rate was 88.5% in PV, 57.7% in ET and 55.6% in IMF patients. This mutation was not detected in CML patients, AL patients and healthy volunteers. From the above mentioned, we can say this mutation might contribute to the genesis of BCR/ABL negative cMPDs, JAK2V617F may serve as an important molecular marker for the diagnosis and classification of BCR/ABL negative cMPDs.2 There was a statistically significant difference in the expression level of PRV-1mRNA in the patients between JAK2V617F mutation positive group and negative group. This is the first report in Chinese literature. The overexpression of PRV-1mRNA may be related to JAK2V617F mutation, which could activate JAK-STAT signaling pathway.3 AS-PCR method is very sensitive, precise and economic. This method can be widely used for detecting JAK2V617F point mutation, making molecular biology diagnosis and typing BCR /ABL negative cMPDs.4 Compared with the JAK2V617F negative ET patients, the mutation positive ET patients had statistically significant higher hemoglobin concentration and peripheral white blood cell counts at diagnosis as well as a higher complication rate, which indicate that JAK2V617F positive ET patients have a higher tendency to transform into PV and JAK2V617F maybe an unfavorable prognostic factor for ET patients. These hypothesis need to be confirmed by further studies.