Analysis Of Mutation Patterns And Clinical Features Of Driver Genes And Non-driver Genes In Patients With Myeloproliferative Neoplasms

SECTION I Mutation and clinical features of JAK2,MPL,and CALR genes in myeloproliferative neoplasms[Background and Objective]Myeloproliferative neoplasms(MPNs)are clonal disorders of the hematopoietic stem cell,characterized by persistent abnormal proliferation of one or more lines of myeloid cells(granulocyte,erythrocyte,megakaryocyte)in the bone marrow,which mainly include polycythemia vera(PV),essential thrombocythemia(ET),primary myelofibrosis(PMF)and chronic myeloid leukemia(CML).There are one or more abnormalities of blood cells in patients,so MPNs are characterized by bleeding tendency,thrombosis,bone marrow fibrosis and extramedullary hematopoiesis with progressive splenomegaly,cytopenias or cytosis.Most(>85%)patients harbour a disease-initiating or driver gene mutation,the most prevalent occurring in the Janus tyrosine kinase 2 gene(JAK2 V617F),followed by calreticulin(CALR)and myeloproliferative leukemia virus oncogene(MPL).Mutation gene frequency,distribution,clinical phenotype and laboratory indicators vary according to different MPN subtypes.The types of JAK2,CALR,MPL and mutational burden have significant value in judging the prognosis of patients with MPN.For example,high mutational burden of JAK2 V617F is significantly associated with higher hemoglobin level,higher white blood cells count,splenomegaly and thrombotic events.The risk of progression to myelofibrosis or leukemia in patients with high-load-driven gene mutations increases in PV and ET.The study was designed to reveal the prevalence of JAK2,CALR and MPL gene mutations and the mutation types in MPNs patients,and to compare the clinical characteristics of different mutation types with each other and negative group.[Methods]In this retrospective study,143 patients who were diagnosed with MPNs at Qilu Hospital of Shandong University from January 2017 to June 2019 were enrolled.These patients were referred for PV(n=46),ET(n=49)and MF(n=48).Clinical manifestations,laboratory tests,JAK2,MPL,and CALR gene mutations,as well as disease-related prognostic scores and treatment options of each patient were analyzed.Statistical analysis was performed using SPSS 25.0 software,and p<0.05 was considered statistically significant.[Results]1.The total mutation rate of JAK2 V617F gene was 75.52%,and the mutation rates in PV,ET,and MF patients were 80.43%、71.43%and 75.00%,respectively.The JAK2 exon 12 gene mutation was only detected in 3 patients with PV.Among the 143 patients,the total mutation rate of CALR gene was 10.49%,among which the mutation rates of ET,MF and PV were 20.41%,10.42%and 0%,respectively.None MPL gene mutation was detected in PV,ET,and MF patients.Any two types of driver gene mutations had not been detected to coexist by direct sequencing.2.The PV patients with JAK2 V617F gene mutation had higher age,white blood cell count,platelet count,LDH,prognostic score and larger spleen(p<0.05)compared with the patients without JAK2 V617F gene mutations.3.The ET patients with JAK2 V617F gene mutation had higher red blood cell count,hemoglobin level,and Prognostic Score of Thrombosis(p<0.05)compared with patients without JAK2 V617F gene mutations.Patients with JAK2 V617F gene mutation had higher Prognostic Score of Thrombosis(p<0.05)compared with patients with CALR gene mutations.Patients with JAK2 V617F gene mutation had higher red blood cell count,hemoglobin level,Prognostic Score of Thrombosis(p<0.05)and lower fibrinogen compared with triple negative MPN patients.Patients with CALR gene mutation had higher hemoglobin level and lower fibrinogen(p<0.05)compared with triple negative MPN patients.4.In MF patients with JAK2 V617F gene mutation had higher white blood cell count,red blood cell count,hemoglobin level(p<0.05)compared with patients without JAK2 V617F gene mutations.When compared with blood cell count,hemoglobin level and platelet count,there was no statistically significant difference between patients with JAK2 V617F and CALR gene mutations(p>0.05).Patients with JAK2 V617F gene mutation had higher blood cell count,red blood cell count,hemoglobin level(p<0.05)compared with triple negative MPN patients.And there was no statistically significant difference between patients with CALR gene mutations to the triple negative MPN patients in each feature(p>0.05).[Conclusion]1.In MPN patients,JAK2 V617F gene mutation was the most common driver gene mutation,followed by CALR gene mutation,and MPL gene mutation rate was the lowest.No MPL mutation was detected in these 143 patients.2.The co-occurrence of any two types of driver gene mutations was not found.3.The triple negative MPN patients accounted for 11.89%of the total 143 patients,which were basically consistent with the literature reports.4.In PV patients,there were statistically significant difference between JAK2 V617F gene mutation and prognosis scores,suggesting that gene mutations are associated with prognosis;In ET patients,there were statistically significant difference between JAK2 V617F gene mutation,CALR gene mutation to Prognostic Score of Thrombosis,suggesting that gene mutation was associated with the risk of thrombosis.5.Mutation gene frequency,distribution,clinical phenotype and laboratory indicators vary according to different MPN subtypes.SECTION Ⅱ Gene mutation patterns of myeloproliferative neoplasms by targeted next-generation sequencing[Background and Objective]Some molecular abnormalities besides driver genes are of great value in judging the prognosis of patients with MPNs.It is reported in literatures that ASXL1,SRSF2,EZH2,and IDH1/2 gene mutations predict poor prognosis in those patients with MPNs.These gene mutations are defined as high-risk molecular mutations,which indicate shorter overall survival and higher risk of progressing to AML.Patients with more than 1 high-risk molecular mutations have a worse prognosis,and studies have shown that the median overall survival of these patients is significantly lower than patients with one or no mutations(2.6 years vs.7.0 years vs.12.2 years,respectively).With the extensive development of next-generation sequencing,the detection of non-driver gene mutations in MPN patients has been gradually carried out clinically,but there is still a lack of systematic large-scale case analysis.Therefore,the impact of non-driver genes on the clinical features,treatment and prognosis of MPNs remains to be seen.In order to provide more genetic support for MPN diagnosis and stratification,we performed targeted next-generation sequencing of 38 MDS/AML/MPN-related genes in 57 patients with MPN,combined with NCCN guidelines and actual testing methods in our hospital,to further understand the clinical features,treatment,prognosis and survival of patients with different numbers of driver genes in MPN patients,so that we can provide more abundant datas for MPN database in China for individualized treatment and precision medicine.[Methods]A retrospective study was performed on 57 patients with diagnosis of MPNs treated at Qilu Hospital of Shandong University from January 2017 to June 2019.These patients were referred for PV(n=14),ET(n=17)and MF(n=26).We collected general data,clinical manifestations,laboratory tests,driver gene and non-driver gene mutations,as well as disease-related prognostic scores and treatment options of each patient.Statistical analysis was performed using SPSS 25.0 software,and p<0.05 was considered statistically significant.[Results]Next-generation sequencing was conducted in 57 patients with MPNs.These patients were referred for PV(n=14),ET(n=17)and MF(n=26).1.Non-driver gene mutation was not detected in PV patients enrolled.2.Among the 17 ET patients observed,non-driver gene mutations were found in 10 patients.The frequency of mutation was 0-5.ASXL1,TP53 mutation frequency were both 17.65%.TET2,U2AF1,DNMT3A,and SF3B1 mutation frequencies were all 11.76%and SRSF2 was 5.88%.Among the detected mutations,the mutation types were mainly point mutations and insertion/deletion mutations.However,the recommended other non-driver gene mutations such as CSF3R,EZH2,IDH1/2,SH2B3,ABL1,and CBL were not detected.3.Among the 26 MF patients observed,17 patients had non-driver gene mutations.The frequency of mutation was 0-4.ASXL1 mutation frequency was 46.15%.SF3B1 mutation frequency was 15.38%.TP53,TET2,and SRSF2 mutation frequency were all 7.69%.CSF3R,IDH1,U2AF1,NRAS,KRAS,CEBPA,ETV6,AML1 mutation frequency were all 3.85%and no EZH2,SH2B3,ABL1,CBL,DNMT3A gene mutations were detected.Among the detected mutations,the mutation types were mainly point mutations and insertion/deletion mutations.The clinical characteristics of these MF patients with non-driver genes(ASXL1,SF3B1,TET2,SRSF2,TP53,CSF3R,IDH1,U2AF1)were analyzed,and there was no significant difference between the indexes(p>0.05).4.4 of the 26 patients carrying multiple non-driver genes or high-risk molecular mutations with MF transformed to leukemia.The disease of these patients progressed rapidly and did not respond well to the current treatment.[Conclusion]1.Non-driver gene mutations were not detected in the 14 PV patients observed.There were at least one non-driver gene mutation in 58.82%(10/17)of ET and 65.38%(17/26)of MF patients,and more than half of the patients carried only one non-driver gene mutation.2.Among 17 ET patients and 26 MF patients,reproducible gene mutations like ASXL1,TET2,SRSF2,TP53,U2AF1,SF3B1 were found,with the highest ASXL1 mutation ratio.The mutation types were mainly point mutation,deletion and insertion mutations,with the highest proportion of point mutations.3.Non-driver gene mutations are associated with disease progression and transformation,and the MIPSS70/MIPSS70-Plus score should be conducted in the newly diagnosed patients for individualized treatment options.