| Essential thrombocythemia(ET)and primary myelofibrosis(PMF)are two important subtypes of myeloproliferative neoplasms(MPN),which are malignant clonal diseases originating from hematopoietic stem cells.Their main pathogenesis is the sustained abnormal activation of the JAK-STAT pathway,leading to continuous abnormal proliferation of bone marrow hematopoietic cells.The driver genes include Janus kinase 2(JAK2)mutation,myeloproliferative leukemia virus(MPL)mutation,and Calreticulin(CALR)mutation.In the diagnostic criteria for MPNs in the 2016 WHO classification of hematopoietic and lymphoid neoplasms,complete blood count,driver gene analysis,and bone marrow histopathology are the main basis for diagnosing various subtypes of MPNs.In recent years,with the rapid development of next-generation sequencing(NGS)technology,the effect of non-driver gene mutations on the progression and transformation of MPN has been increasingly studied.Among them,ASXL1,EZH2,SRSF3,IDH1/IDH2,U2AF1,CBL,K/NRAS are considered high-risk molecular mutations(HMR)for the progression and transformation of MPN diseases.There is currently a lack of in-depth exploration on the morphological changes in bone marrow histopathology that may occur with different driver genes,especially with the addition of different non-driver genes.This study aims to explore the potential clinical value of bone marrow histopathology in the risk stratification and prognosis evaluation of ET and PMF diseases by analyzing the relationship between different molecular backgrounds and bone marrow histopathology in ET and PMF patients.Part One The impact of different driver genes(JAK2V617F mutation,CALR mutation)and risk stratification on the bone marrow histopathology morphology in ET and PMF patients.Objective: To collect clinical and laboratory data from ET and PMF(prePMF and overt PMF)patients,analyze the differences in age,gender,blood routine,and bone marrow histopathology morphology among ET,prePMF,and overt PMF patients with JAK2V617 F gene mutation and CALR gene mutation.By further stratifying the risk of ET,prePMF,and overt PMF patients,the potential value of bone marrow histopathology morphology in disease assessment is explored.Methods:1.Collecting clinical and laboratory data from 233 diagnosed ET and PMF(prePMF and overt PMF)patients from January 2017 to December 2019 in the Department of Hematology at the Second Hospital of Hebei Medical University.This includes 123 cases of ET,110 cases of PMF(51 cases of prePMF and 59 cases of overt PMF).Clinical and laboratory examination data were collected,including age,gender,splenomegaly status,blood routine,bone marrow histopathology morphology characteristics(marrow proliferation degree,number of megakaryocytes,number of megakaryocyte clusters,number of naked nuclei,and the proportion of large megakaryocytes and dysplastic megakaryocytes),and driver gene mutation types.2.Comparing the differences in age,gender,splenomegaly status,blood routine,and bone marrow histopathology morphology characteristics between ET,prePMF,and overt PMF patients carrying different driver gene mutations(JAK2V617F mutation and CALR mutation).3.Stratifying ET patients according to the International Prognostic Score for Essential Thrombocythemia(IPSET-survival),stratifying ET and prePMF patients according to the International Prognostic Score for Essential Thrombocythemia-thrombosis(IPSET-thrombosis),and stratifying overt PMF patients according to the Dynamic International Prognostic Scoring System(DIPSS).The differences in clinical and laboratory characteristics of patients with different driver genes(JAK2V617F mutation and CALR mutation)were analyzed under different stratifications in ET,prePMF,and overt PMF patients.Results:1.In ET patients,the JAK2V617 F mutation group showed significantly lower percentages of megakaryocytes and poorly lobulated megakaryocytes in bone marrow histological features compared to the CALR mutation group.No other statistically significant differences were found between the two groups(P =0.020,P =0.001,P>0.050).2.In prePMF patients,the JAK2V617 F mutation group showed significantly higher white blood cell counts than the CALR mutation group(P=0.000).In bone marrow histological features,the JAK2V617 F mutation group showed significantly higher proportions of patients with active bone marrow proliferation and higher percentages of megakaryocytes compared to the CALR mutation group.No other statistically significant differences were found between the two groups(P>0.050).3.In overt PMF patients,the JAK2V617 F mutation group showed significantly lower proportions of patients with extensive bone marrow fibrosis and poorly lobulated megakaryocytes in bone marrow histological features compared to the CALR mutation group(P=0.022,P=0.001).However,the JAK2V617 F mutation group showed significantly higher numbers of megakaryocytes and megakaryocyte clusters compared to the CALR mutation group(P=0.014).No other statistically significant differences were found between the two groups(P>0.050).4.According to the IPSET-survival risk stratification in ET patients,no statistically significant differences were found between patients with different driver gene mutations in the low-risk group(P > 0.050).In the intermediate-risk group,the proportion of poorly lobulated megakaryocytes in bone marrow histological features was significantly lower in the JAK2V617 F mutation group than in the CALR mutation group(P=0.016).In the high-risk group,the number of naked nuclei was significantly lower in the JAK2V617 F mutation group than in the CALR mutation group(P=0.008).Further analysis of ET patients with JAK2V617 F mutation in the high,intermediate,and low-risk groups showed that the proportion of poorly lobulated megakaryocytes was significantly higher in the high-risk group than in the low-risk group in terms of bone marrow histological features(P=0.039).No statistically significant differences were found between the low-risk and intermediate-risk groups or between the intermediate-risk and high-risk groups(P>0.050).Further analysis of ET patients with CALR mutation in the high,intermediate,and low-risk groups showed that the high-risk group had a significantly higher number of naked nuclei in bone marrow histological features than the low-risk group(P=0.029).No statistically significant differences were found between the low-risk and intermediate-risk groups or between the intermediate-risk and high-risk groups(P>0.050).5.According to the IPSET-thrombosis risk stratification in ET patients,all patients in the low-risk group had the CALR mutation,and all patients in the high-risk group had the JAK2V617 F mutation.In the intermediate-risk group,patients with JAK2V617 F mutations were significantly younger than those with CALR mutations(P=0.000).In terms of bone marrow histological characteristics,the proportion of poorly lobulated megakaryocytes in the JAK2V617 F mutation group was significantly lower than in the CALR mutation group(P=0.000).No other statistically significant differences were observed(P>0.050).Further analysis of ET patients with JAK2V617 F mutations in the high-risk and intermediate-risk groups showed that patients in the high-risk group were significantly older than those in the intermediate-risk group(P=0.000).In terms of bone marrow histological characteristics,the proportion of proliferative active patients and poorly lobulated megakaryocytes in the high-risk group was significantly higher than in the intermediate-risk group(P=0.048,P=0.033).No other statistically significant differences were observed(P>0.050).Further analysis of CALR mutation patients in the intermediate-risk and low-risk group showed that patients in the low-risk group were significantly younger than those in the intermediate-risk group(P=0.000).6.According to the IPSET-thrombosis risk stratification in prePMF patients,all patients in the low-risk group had the CALR mutation,and all patients in the high-risk group had the JAK2V617 F mutation.In the intermediate-risk group,the patients with the JAK2V617 F mutation were significantly younger than those with the the CALR mutation(P=0.000).No other statistically significant differences were found between the groups(P>0.050).Further analysis of the JAK2V617 F mutation prePMF patients in the high-risk and intermediate-risk groups showed that the patients in the high-risk group were significantly older than those in the intermediate-risk group(P=0.000).No other statistically significant differences were found between the groups(P>0.050).Further analysis of the CALR mutation prePMF patients in the intermediate-risk and low-risk groups showed that the patients in the low-risk group were significantly younger than those in the intermediate-risk group(P=0.000).No other statistically significant differences were found between the groups(P>0.050).7.According to the DIPSS risk stratification in overt PMF patients in this group,no patients were classified as low-risk,and only one patient in the intermediate-1-risk group had the CALR mutation.In the intermediate-2-risk group,no significant differences were found between patients with different driver gene mutations.In the high-risk group,the proportion of abnormal megakaryocytes was significantly lower in patients with the JAK2V617 F mutation than in those with the CALR mutation(P=0.001),while the number of megakaryocytes was significantly higher in JAK2V617F-mutated patients than in CALR-mutated patients(P=0.019).No other statistically significant differences were found(P>0.050).Further analysis of JAK2V617F-mutated patients in the intermediate-1-risk,intermediate-2-risk,and high-risk groups showed that the patients in the intermediate-1-risk group were significantly younger than those in the high-risk group(P=0.015),and had higher red blood cell counts and hemoglobin levels(P=0.003,P=0.002).No other statistically significant differences were found between the groups(P>0.050).No significant differences were found between the intermediate-2-risk and high-risk groups(P>0.050).Further analysis of CALR-mutated patients in the high-risk and intermediate-2-risk groups found no statistically significant differences.Characteristics(P>0.050).Conclusions:1.In ET,prePMF,and overt PMF,there are differences in bone marrow histopathology morphology characteristics between different driver genes mutations,JAK2V617 F mutation and CALR mutation,suggesting that different driver genes have different effects on the progression of the disease in ET,prePMF,and overt PMF.2.According to the commonly used international prognostic scoring system for ET,prePMF,and overt PMF patients,stratified comparisons were made.The results suggest that the effects of JAK2V617 F mutation and CALR mutation on ET and overt PMF may be different,mainly occurring in intermediate-high risk patients.However,the quantitative indicators of bone marrow histology adopted in this study were not sensitive enough for stratification of ET,pre-PMF,and overt PMF.Part Two Molecular background differences of CALR type I/type II mutations and their impact on bone marrow histopathology morphology in ET and PMF patients.Objective: This study aims to explore the impact of different molecular backgrounds on the bone marrow histopathology morphology characteristics of ET and PMF by analyzing the mutation frequency of CALR type I/II mutations and whether they are accompanied by non-driver gene mutations.Methods:1.Collect 77 patients diagnosed with ET and PMF carrying CALR type I/II mutations from January 2017 to December 2020 in the Department of Hematology,the Second Hospital of Hebei Medical University.The patients include 25 cases of ET and 52 cases of PMF(32 cases of prePMF and 20 cases of overt PMF).Clinical and laboratory data were collected,including age,gender,splenomegaly,blood routine,bone marrow histopathology morphology characteristics,and CALR mutation type.2.Use second-generation sequencing technology(NGS)to perform mutation detection of 38 hotspot genes(including CALR,JAK2,MPL,ASXL1,EZH2,SRSF2,IDH1,IDH2,U2AF1,TET2,STAG2,BCOR,BCORL1,DNMT3 A,CBL,CEBPA,MLL,SF3B1,ATM,GATA2,NRAS,KRAS,RUNX1,PPM1 D,ZRSR2,KIT,SETBP1,NF1,TP53,NPM1,PDGFRA,PHF6,PIGA,SH2B3,FLT3,STAT3,CSF3 R,WT1)in the 77 ET and PMF patients in this group.3.Compare the differences in age,gender,splenomegaly,blood routine,bone marrow pathological histological characteristics,driver gene mutation frequency(VAF),and accompanying non-driver gene mutations between CALR type I and type II mutations in ET,prePMF,and overt PMF patients.Further analyze the impact of VAF of CALR type I and type II mutations and non-driver gene mutations on disease subtypes.Results:1.In ET patients,a comparison between CALR type I and type II mutations revealed that the VAF value of CALR type I mutation was significantly lower than that of type II mutation(P<0.001).In terms of bone marrow histopathology features,the proportion of dysplastic megakaryocytes and the number of naked nuclei in the CALR type I mutation group were significantly higher than those in the CALR type II mutation group(P=0.000).No statistically significant differences were found in other variables(P>0.050).2.In prePMF patients,a comparison between CALR type I and type II mutations revealed that the VAF value of CALR type I mutation was significantly lower than that of type II mutation(P<0.001).In terms of bone marrow histopathology features,the proportion of dysplastic megakaryocytes with abnormal nuclear lobulation in the CALR type I mutation group was significantly higher than that in the CALR type II mutation group(P=0.008).No statistically significant differences were found in other variables(P>0.050).3.In overt PMF patients,the proportion of CALR type I mutation patients was significantly higher than that of type II mutation patients,and NGS results showed that the VAF value of CALR type I mutation was significantly lower than that of type II mutation(P < 0.001).In terms of bone marrow histopathology features,66.6%(10/15)of patients in the CALR type I mutation group had extensive bone marrow fibrosis and cell depletion,while100%(5/5)of patients in the CALR type II mutation group had extensive bone marrow fibrosis and cell depletion.No statistically significant differences were found in other variables(P>0.050).4.In ET,prePMF,and overt PMF patients,a total of 23 non-driver gene mutations were detected,and the proportion of non-driver gene mutations in overt PMF patients was significantly higher than that in ET and prePMF patients.The most common non-driver gene mutations were in the epigenetic regulator genes ASXL1,TET2,and EZH2.5.In patients with CALR type I/type II mutations,no significant statistical differences were found in the VAF values of CALR type I/type II mutations among ET,prePMF,and overt PMF patients carrying the same CALR mutation subtype(P>0.050).Conclusions:1.In ET,prePMF,and overt PMF patients,CALR type I mutation may have a greater impact on disease.The mutation frequency of CALR mutations may not be the main cause of different MPN subtypes.2.In this study,non-driver gene mutations were mainly occurred in overt PMF patients and may be deeply involved in the occurrence and development of overt PMF in CALR type I/II mutated patients. |
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