Study On ETS2 MRNA Expression And JAK2 V617F Mutation In BCR/ABL Negative CMPDs Patients

Objectives: The classical BCR/ABL negative chronic myeloproliferative diseases (cMPDs) consist of polycythemia vera (PV),essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). Differing from BCR/ABL positive chronic myelogenous leukemia (CML), neither a repeatable chromosome translocation was discovered in PV, ET and IMF patients nor was an effective medicine developed. Recently several studies discovered a highly pathogenic characteristic gene mutation, a G to T change in the 1849th codon of JAK2 gene, and the amino-isovaleric acid is accordingly replaced byβ-phenyl-α-aminopropionic acid in the 617th of JAK2 tyrosine kinase. This mutation is defined as JAK2 V617F(or JAK2 val617phe)point mutation and has been detected in up to 90% of PV patients and in a sizeable proportion of patients with other cMPDs such as ET and IMF, but rarely be found in other hematological disease. This finding is a landmark for understanding the molecular pathogenesis of BCR/ABL negative cMPDs.On the orther hand, erythroblastosis virus oncogene homolog 2 (ETS2) is a pro-oncogene, located in human chromosomal region 21q22.3, expresses in various tissues,including blood,breast and prostate. This gene encodes a 56 kD protein that is phosphorylated by a Ca2+-dependent mitogenic signal process. ETS2 protein is a member of the ETS family of transcription factors, involved in the regulation of cellular proliferation and differentiation and may play a critical role in T-cell activation and cytokines production. It had been reported that ETS2 gene was associated with the growth and invasion of breast carcinoma cells and was required to maintain the transformed state for human prostate cancer cells. It is also reported that ETS2 mRNA was over-expressed in cMPDs. However, it is not clear whether there is a relationship between the mutation of JAK2 V617F and ETS2 mRNA overexpression and whether they both influence the progression of BCR/ABL negative cMPDs. The present study is thus designed to examine the somatic JAK2 V617F mutation rate and measure the expression level of ETS2 mRNA in cMPDs,acute leukemia and normal control, 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 medicines for the treatment of JAK2 V617F mutation positive cMPDs.Methods:1 Heparinized peripheral blood or bone marrow was obtained after informed consent from 62 BCR/ABL negative cMPDs patients (26 PV cases, 26 ET cases,9 IMF cases and 1 CNL case), 20 CML cases, 10 acute leukemia (AL) cases and 15 healthy volunteers. The 62 of BCR/ABL negative cMPDs patients were designed as research group and the others were control group. All these patients'diagnosis fit the WHO criteria made in 2000.2 Total RNA and DNA was extracted from samples of the peripheral blood mononuclear cells (PBMNCs) or bone marrow mononuclear cells (BMMNCs) taken from patients in each group or healthy volunteers.3 The expression level of ETS2 mRNA was measured by RT-PCR method, which was established and widely used in our lab.4 JAK2 V617F mutation was detected by AS-PCR method and confirmed by direct DNA sequencing. Then the BCR/ABL negative cMPDs patients were divided into two subgroups: JAK2 V617F mutation positive group and mutation negative group.5 Patients'clinical characteristics including age, gender, clinical symptoms and signs, hemograms, bone marrow pictures, bone marrow pathological features and the reaction to conventional therapy and prognosis of each patient in different subgroup were observed and compared.6 Statistical analyses: Most statistic analysis was performed using the SPSS 11.0 version for windows. The statistically significant meaning of the difference in JAK2 V617F mutation rate was analyzed by Chi-square analysis. The statistically significant meaning of the difference in ETS2 mRNA expression level between JAK2 V617F mutation positive group and mutation negative group was analyzed by t-test. The results were shown as mean±SD(X—±S).Results:1 The JAK2 V617F point mutation was detected in 44 BCR/ABL negative MPD patients, including 23/26 (88.46%)PV patients,15/26(57.69%)ET patients, 5/9(55.56%)IMF patients and 1/1 (100% )CNL patient. While the somatic JAK2 V617F mutation was not detected either in the 20 CML patients, 10 AL patients or the 15 health volunteers.2 The relative expression level of ETS2 mRNA in BCR/ABL negative cMPDs group (mean value 0.2876, positive rate 81.96%), or in CML group (mean value 0.4133, positive rate 85.0%), or in AL group (mean value 0.5320, positive rate 80.0%) is statistically significant higher than that in health volunteer group (mean value 0.106 , positive rate 46.7%). P<0.05.3 The relative expression level of ETS2 mRNA in JAK2 V617F mutation positive group (mean value 0.323 , positive rate 86.0%) is statistically significant higher than that in negative group (mean value 0.203 , positive rate 72.2%). P=0.022.4 The comparison of clinical characteristics: The peripheral white blood cell counts (mean 18.2×109/L) and platelets counts (mean 479×109/L) in JAK2 V617F mutation positive PV patients were statistically significant higher than that (7.6×109/L and 277×109/L, respectively) in JAK2 V617F 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 (10.9×109/L vs 14.6×109/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 JAK2 V617F positive and negative IMF patients (P>0.05).Conclusions:1 A somatic JAK2 V617F point mutation was detected in a sizeable proportion of BCR/ABL fusion gene negative cMPDs patients: the mutation rate was 88.46% in PV, 57.69% in ET and 55.56% in IMF patients. This mutation was not detected in AL patients, CML patients or healthy volunteers.2 The expression level of ETS2 mRNA in JAK2 V617F mutation positive group is statistically significant higher than that in negative group.