Expression And Clinical Significance Of 31 Mutated Genes In Pediatric Leukemia

PART Ⅰ: Expression of 31 mutated genes in pediatric acutelymphoblastic leukemiaObjective To investigate relationship of molecular testing and clinical significance in pediatric acute lymphoblastic leukemia(ALL).Methods Clinical data and lab findings of 255 children suffered from ALL were collected and analyses.Bone marrow samples of these patients were collected,morphology,immunophenotype and chromosome karyotype of samples were detected,31 fusion genes were screened by multiple nested reverse transcription-polymerase chain reaction(mutiplex nested RT-PCR)and confirmed by split-out RT-PCR.Results 255 ALL children with mean age of 63.07±2.67 months,included 153 male and 102 female,were involved in the study.Among these 255 patients,104 patients(40.78%)were detected with 9 kinds of fusion genes which included 42 cases of ETV6/RUNX1,17 cases of E2A/PBX1,16 cases of MLL arrangement,15 cases of BCR/ABL,5cases of SIL/TAL1,5cases of HOX11,3 cases of EVI1 and 1 case of SET/CAN.The positive detection rate of T-ALL was lower than that of B-ALL(p<0.05).For subtype of B-ALL,fusion gene of ETV6/RUNX1 and BCR/ABL were dominant in C-B-ALL,fusion gene of E2A/PBX1 or MLL were common in pre-B-ALL or pro-B-ALL(p<0.05).Among age groups,ETV6/RUNX1,MLL and BCR/ABL gene were commonly detected in age group of 1-10 year,<1year and >10year(p<0.05).Conclusion Combine technique of multiplex nested RT-PCR and split-out RT-PCR could detected 31 kinds of fusion genes which were common in leukemia.Molecular testing was available for accurate classification,risk stratification and treatment of pediatric ALL.Lower positive detection rate of T-ALL showed that further studies will be required for molecular classification in T-ALL.Differential distribution of fusion gene in age groups indicated one of the reasons for the different prognosis in pediatric B-ALL of age groups.PART Ⅱ : Expression of 31 mutated genes in pediatric acutemyeloid leukemiaObjective To investigate relationship of molecular testing and clinical significance in pediatric acute myeloid leukemia(AML).Methods Clinical data and lab findings of 133 children suffered from AML were collected and analyses.Bone marrow samples of these patients were collected,morphology,immunophenotype and chromosome karyotype of samples were detected,31 fusion genes were screened by multiple nested reverse transcription-polymerase chain reaction(mutiplex nested RT-PCR)and confirmed by split-out RT-PCR.Results 133 children with AML were classified into 28 cases of acute promyelocytic leukemia(APL)and 105 cases of non-APL(mean age of 73.03±4.54 months,62 male and 43 female)were involved in the study.Among these 133 AML patients,98 patients(73.68%)were detected with 7 kinds of fusion genes: 31 cases of AML/ETO,28 cases of PML/Ra Rα,12 cases of MLL arrangement,1 case of HOX,1 case of TLS/ERG,22 cases of of EVI1(7 of them also detected MLL arrangement).Rare mutation of PML/Ra Rα gene was founded in 2 cases of APL.The positive detection rates of non-APL were AML/ETO and EVI1;statistical differences of positive genes distribution were not found in different age groups(p > 0.05).99 cases were successfully cultured and 6 cases failed,73 cases of abnormal karyotype were analysised among the 99 cases(73.74%).Positive rate of detected mutated genes and abnormal karyotype in AML was higher than that of ALL(p < 0.05).Conclusion Combine technique of multiplex nested RT-PCR and split-out RT-PCR could detected 31 kinds of fusion genes which were common in AML,Molecular testing was available for accurate classification of AML.Combine technique of multiplex nested RT-PCR and gene scanning were useful to detect rare mutation of fusion genes.Combine technique of cytogenetics and molecular genetics were more useful to indicate risk stratification and treatment of pediatric in AML than that in ALL.PART Ⅲ: Minimal residual disease monitoring by molecularrespond in pediatric acute lymphoblastic leukemia withETV6/RUNX1,E2A/PBX1 or MLL arrangementObjective To monitoring minimal residual disease(MRD)by flowcytometry(FCM-MRD)and molecular respond(PCR-MRD)in pediatric B cell acute lymphoblastic leukemia(B-ALL)and to investigate sensitivity and prognosis of PCR-MRD in B-ALL.Methods 61 children suffered from B-ALL were divided into three groups and received chemotherapy of CCLG-ALL-2008 protocol,which included group A(ETV6/RUNX1+),group B(E2A/PBX1+),group C(MLL arrangement).These patients received bone marrow asperation in different time piont(TP)and MRD level were monitored by FCM and PCR in TP2 and TP3.Results Statistical differences of positive incidence of MRD which monitored by FCM and PCR were not found on TP2 and TP3(P>0.05).Following-up to February 2017,mean survival of the 61 patients was 43.93±2.23 months and event free survival(EFS)of 3 years was 80.0±5.4%;mean survival of group A,B and C were 50.33±1.13 months,24.87±3.61 months and 31.85±7.20 monthsrespectively,EFS of 3 years for group A,B and C were 93.1±4.7%、60.0±12.6% and 62.9±17.9%,prognosis for group A was superior to group B and C(P < 0.05).Conclusion Molecular genetics was useful to indicate risk stratification and treatment of pediatric in pediatric B-ALL.Technique of FCM and PCR were both useful to monitor MRD level in pediatric B-ALL and to indicate risk stratification.Results of MRD monitoring by FCM and PCR were similar,but Technique of PCR might be more suitable in group B and C for MRD monitoring.