Exploration The Characteristics And Clinical Significance Of Plasma Exosome Metabolites In Acute Leukemia Based On GC-MS

PART ? Identification of plasma exosomes and preliminary exploration of their metabolite profiles and metabolic pathways in acute leukemia[Objective]To identify the presence of exosomes and metabolic profiling of exosome in acute leukemia(AL);To find new approachs of pathogenesis,diagnosis and prognosis of acute leukemia.[Methods]The plasma samples of 71 patients with newly-diagnosed acute leukemia(AL)and 14 of healthy controls were collected in the**Hospital from March 2018 to March 2019.Eight patients with newly diagnosed acute leukemia and four healthy controls randomly selected were used for plasma exosomes extraction.Western-blot(immunoblotting test)and electron microscopy were employed to identify exosomes.The t test was used to observe the difference in exosomes concentrations between patients and healthy controls.GC-MS(gas chromatography-mass spectrometry)was used to perform high-throughput detection of targeted metabolites in 156 plasma exosomes including 71 pairs of samples before and after treatment and 14 healthy controls.The GC-MS methodological stability of sample preparation was investigated by calculating the relative standard deviation(RSD)of quality control samples(QC).The substance identification was performed by the automatic mass spectrometry deconvolution qualitative system(AMDIS)and the National Institute of Standards and Technology(NIST)standard library in combination with the retention index.71 patients with acute leukemia were compsed of 51 acute myeloid leukemia(AML)and 20 acute B lymphocytic leukemia(B-ALL).The two types of patients were divided into discovery and validation group.The AML patient discovery group included 9 AML patients and 6 healthy controls,and the validation group included 42 AML patients and 8 healthy controls;the B-ALL patient discovery group included 9 B-ALL patients and 6 healthy controls,and the validation group included 11 B-ALL patients and 8 healthy controls.The partial metabolites of different types of acute leukemia patients and healthy people were screened by partial least squares discriminant method(PLS-DA)combined with t-test FDR correction for the two groups of discovery groups.Group samples were validated to identify meaningful differential metabolites.[Results]1?Identification of exosomes between patients and healthy controls.The marker proteins TSG101 and CD63 of exosomes were detected in 8 patients with AL and 4 healthy controls by western blot,and vesicles with a diameter of 30-150 nm were observed under an electron microscope.The presence of plasma exosomes was demonstrated in all.At the same time,it was found that the diameter of exosomes in healthy people was larger than that in patients with AL,and the degree of aggregation was denser.2?Identification of plasma exosomal metabolites.Quality control(QC)sample testing showed that the proportion of compounds with relative area deviation(RSD)below 15%reached 60%,suggesting that the experimental data are reliable.Meet analytical requirements.According to the AMDIS and NIST standard libraries and retention indices,a total of 65 metabolites were identified:isobutyric acid,isovaleric acid,pyruvate,lactic acid,hexanoic acid,glycolic acid,2-methylhexanoic acid,alanine,valine,sarcosine,oxalic acid,3-hydroxypropionic acid,Leucine,3-hydroxybutyric acid,valine,urea,D-lythose,phosphate,glycerol,proline,isoleucine,glycine,succinic acid,methylsuccinic acid,Fumaric acid,serine,threonine,methionine,malic acid,homoproline,pyroglutamic acid,aspartic acid,hydroxyproline,cysteine,threonine,phenylalanine acid,xylitol,glutamine,ornithine,citric acid,d-tagatose,N-?-acetyl-L-lysine,D-fructose,tyrosine,allose,glucose,Galactose,palmitic acid,inositol,octadecenedioic acid,octadecenoic acid,linoleic acid,stearic acid,glucose 6-phosphate,arachidonic acid,myristic acid,inositol,2-palmitoyl glycerol,docosa tetraenoic acid,glyceryl monopalmitate,D-sucrose,maltose,glyceryl monostearate,?-ketoglutarate,and cholesterol.3?Analysis of metabolic profiles in patients with acute leukemia and healthy controls.There were significant differences in metabolic profiles between patients with AL and healthy controls(p<0.05),Using principal component analysis,PLS-DA,and t-tests,screening through the discovery group and validation group consisting of pre-treatment and healthy control samples from AL patients identified 16 metabolites:pyruvate,lactic acid,valine,urea,proline,isoleucine,serine,threonine,homoproline,pyroglutamic acid,threonine,phenylalanine,ornithine,citric acid,d-tagatose,N-?-acetyl-L-lysine,which significantly decreased compared to healthy people in AML patients;At the same time,It was found after treatment,comparing to pre-treatment,the overall levels of pyruvate,lactic acid,proline,homoproline,pyroglutamic acid,threonine,Citric acid,tagatose,and lysine were decreased,while the overall expression levels of valine,urea,isoleucine,serine,threonine,phenylalanine,and ornithine were increased.11 metabolites were verified in B-ALL patients that Urea,glycerol,Valine,isoleucine,serine,threonine,homoproline,aspartic acid,phenylalanine,ornithine and inositol showed an dramatic upward trend(p<0.05).while compared with pre-treatment,in post-treatment that the expression of isoleucine,serine,homoproline,aspartic acid,ornithine,and inositol were decreased,and the expression of valine,urea,glycerol,threonine,phenylalanine were increased.4?Analysis of acute leukemia metabolic pathways.Analysis of the metabolic pathways involved in the differential metabolites of AL through MetPA network analysis indicates that the metabolic disorders of exosomes in AML patients are mainly related to the aminoacyl-tRNA biosynthesis,citrate cycle,alanine,aspartic acid and glutamic acid metabolism,Pyruvate metabolism,glycine,serine and threonine metabolism most closely related(p<0.05,impact>0.1),followed by glycolysis/gluconeogenesis,glycerolipid metabolism,phenylalanine metabolism,phenylalanine,casein Biosynthesis of tryptophan and tryptophan.Metabolic disorders of exosomes in B-ALL patients mainly involved glycolysis/gluconeogenesis,aminoacyl-tRNA biosynthesis,citric acid cycle,glyoxylate,and carboxylic acid esters,alanine,aspartic acid and glutamic acid metabolism,pyruvate metabolism,glycine,serine and threonine metabolism(p<0.05,impact>0.1),and glutathione metabolism,amino group Sugar and nucleotide sugar metabolism,phosphoinositide metabolism,glycerolipid metabolism,phenylalanine metabolism,phenylalanine,tyrosine and tryptophan biosynthesis are also related to some extent(impact>0.1).[Conclusion]The plasma exosome metabolism profile of patients with acute leukemia is different from that of healthy people,and there is defference of the exosomal metabolism profile and metabolic pathways between AML and ALL.PART ? Investigation of the correlation between plasma exosomal metabolites and clinical characteristics in patients with acute leukemia and its clinical significance in efficacy and prognosis[Objective]To explore the significance of differential metabolites and clinical features related to the diagnosis and prediction of acute leukemia(AL),and evaluate the clinical significance of differential metabolites in effect and prognosis.[Methods]We followed up 71 patients who were newly diagnosed.All patients received 1-2 courses of induction chemotherapy and consolidated 2-4 courses.Information regarding the clinical characteristics of bone marrow cell morphology,immunotyping,chromosome and gene mutation(MICM)data were collected.The treatment efficacy and survival prognosis were evaluated at the end of follow-up.The GC-MS technology method,samples and substance identification are the same as the first part.Based on clinical data,Spearman's rank correlation analysis(Spearman)and Mann-Whitney U test were performed on 51 AML patients and 20 B-ALL patients with corresponding clinical factors,respectively,to find out the diagnostic or predictive value of different metabolites.Kaplan-Meier survival analysis was used to analyze characteristics and biomarker metabolites on survival in all patients.Survival-related metabolites and clinical indicators were included in the COX proportional hazards model for multivariate analysis to find biomarker metabolites that affected survival.[Results]1.Clinical efficacy and prognosis of patients with AL.The median follow-up time of 71 patients was 11.63(1.17,20.9)months.Among them,the median follow-up time of 51 patients with AML was 11.57(1.17,20.47)months,all of whom were induced with chemotherapy after 1-2 courses.38 patients achieved remission after 1 course of induction chemotherapy,and 13 patients did not respond.After 2 courses of induction chemotherapy,48 cases of remission were achieved,and 3 cases of did not reached.By the end of follow-up,33 patients had remission and 11 cases relapsed.13 died(including 6 deaths after relapse).The cumulative recurrence rate was 39.66%.The overall survival rate(OS)rate was 70.24%.The progression-free survival(PFS)rate was 60.34%.In 20 patients with B-ALL,the median follow-up time was 12.43(2.87,20.9)months.There were 16 patients who achieved remission after 1 courses of induction chemotherapy,and 4 did not respond.20 cases achieved remission after 2 courses of induction chemotherapy.By the end of the follow-up,19 patients had sustained remission,3 had relapses,and 0 died.The cumulative recurrence rate is 15%and the progression-free survival(PFS)rate is 85%.2.Correlation between plasma exosomal metabolites and clinical characteristics.51 patients with AML were composed of 5 cases of M1,21 cases of M2,6 cases of M3,10 cases of M4,and 9 cases of M5.In immunotype,34 cases expressed CD117,but 17 cases did not expressed;41 cases were CD 13 positive and 10 cases were negative;42 cases expressed CD33,and the remaining 9 cases were not expressed;16 cases were DR positive,and 35 cases were negative;2 cases were found to have JAK2 gene mutation,and 49 cases did not have this mutation;20 cases had FLT3 mutation,while 31 cases did not;10 had CEB PA mutations and 41 had no CEB PA mutations;8 had NRAS gene mutations and 43 had no NRAS mutation.There were 20 cases of acute lymphocytic leukemia(6 cases of B-ALL,13 cases of ph+B-ALL,1 case of phlike-B-ALL).16 cases expressed CD10,and 4 cases were not expressed;17 cases were positive for CD 19 and 3 cases were negative;6 cases were positive for CD20,and14 cases were negative;the fusion gene EVI1(>250)had high expression in 3 cases and 17 cases had low expression.There were 2 cases of FLT3 mutations,while 18 cases did not occur;1 case of TP53 mutations,and 19 cases did not have this mutation.The independent analysis of the Mann-Whitney rank sum U test was used to analyze the correlation between clinical factors and metabolic differences.It was found that CEBPA gene mutations was related to increased isoleucine levels,while low serine levels were associated with NRAS gene mutations.The high expression of EVI1 fusion gene in B-ALL is related to the increase of isoleucine and threonine.3.Correlation of exosomal metabolites(pre-and post-treatment)and prognosis in patients with AL.We analyzed the metabolic changes in the same patients before and after treatment.Based on paired t test,in the complete remission group of 51 patients with AML,tagatose(P=0.012)was significantly reduced after treatment,while citric acid decreased significantly in the non-responding group(P=0.026).And no significant difference was seen in other metabolites(p>0.05).In the remission group of B-ALL patients,a significant reduction in inositol was found after treatment(P=0.016),while no such change was observed in the non-remission group(P>0.05).Univariate survival analysis(Kaplan-Meier survival analysis)was performed in 51 patients with AML.It was found that clinical factors WT1(WT1>200),MYC,RUNX1,PTPN,and BCORL1 affected the overall survival OS(p<0.05).Pyruvate,serine,threonine,phenylalanine,and ornithine had some effect on overall survival(p<0.1).The multivariate analysis with Cox regression model indicated that patients with high levels of Pyruvic acid,serine and ornithine were likely to experience disease death(p<0.1),the metabolism changes of which can be considered as independent risk factors for AML survival,according to the leave-one-out cross-validation,the ROC model was used to test the above models,and it was found that pyruvate(AUC=0.744,p=0.009)and serine(AUC=0.710,p=0.025)metabolic change indicators may have a better prognosis predicted value.Meanwhile,clinical factors CD33,MYC,DNMT3A,RUNX1,PTPN,CEB PA,and BCORL1 affected progression-free survival(p<0.1).Pyruvic acid,Serine,Threonine,Phenylalanine,Ornithine and Lysine were associated with disease progression(p<0.1).The multivariate analysis with Cox regression model indicated thatpatients with high levels of serine and ornithine were more likely to experience disease progression(p<0.1),So the metabolism change of serine and ornithine were also independent risk factors in progression-free survival(PFS)in AML,according to leave-one-out cross-validation,the prediction effect of ROC detection model is not significant(AUC<0.7,p>0.05).In 20 cases of B-ALL,no patients died by the end of the follow-up.Univariate survival analysis found that clinical factors CD7,CD25,and BCORL1 were associated with B-ALL progression-free survival(PFS),while the aspartate had an effect on progression-free survival(p<0.1).The inclusion of a single factor in the Cox regression model showed no significant difference.[Conclusion]There are differential metabolites related to clinical factors of AL.Pyruvate,Serine and ornithine level changes(high post-/pre-)may be an independent risk factor for overall survival(OS)of AML,Serine and ornithine metabolism changes(high post-/pre-)have better predictive effect on OS;the metabolic changes of serine and ornithine(high post-/pre-)may also be independent risk factors for PFS in AML patients.