Study On The Pediatric And Adult Acute Lymphoblastic Leukemia By Whole Exome Sequencing

Acute lymphoblastic leukemia(ALL)is a group of malignant neoplasm of immature lymphoid progenitors that is most commonly of B cell lineage.Since 1960s’ remarkable progress have been made in the therapy of childhood ALL,the cure rates now has exceeded 80%,while adult ALL continues to have a dismal prognosis,with the cure rate of only about 40%.The age,leukocyte count,immunophenotype,cytogenetic and molecular genetics abnormalities,the reaction to the inductive treatment and the detection of minimal residual disease(MRD)are essential in risk stratification and hence constitute the foundation to carry out the tailored therapy.Most ALL cases harbor a recurring chromosomal alteration detectable by karyotyping or molecular techniques;such as hyperdiploidy,hypodiploidy,and chromosomal rearrangements including BCR-ABL1,TEL-AML1,E2A-PBX1 and rearrangement of MLL to a diverse range of fusion partners.The percentage of these lesions is different between children and adults,and is associated with the prognosis of the ALL patients.However,some ALL patients couldn’t figure out relevant cytogenic abnormalities.In the recent years,the whole genome sequencing and whole exome sequencing have provided a number of key insights into the genetic basis of a variety of disease,such as solid tumors and hematologic malignancies.It is of critical importance to perform the genome-wide profiling of the submicroscopic events between children and adult patients of ALL,and to identify the susceptible genes or pathways that are associated with the leukemia cell transformation.This approach will be also of great clinical significance to personalized therapy in ALL.We therefore conducted whole exome sequencing of the paired genomic DNA from mononuclear cells by bone marrow aspirates at diagnosis and their matched peripheral blood samples during the complete remission phrase.First of all,the bone marrow cell morphology,karyotyping,immunophenotype and common molecular markers were analyzed in a total of 18 cases.We then collected those patients who meet our requirements to carry out the whole exome sequencing.After scanning and detecting the sequence differences and structural variations by bioinformatics methods,we have currently completed exome sequencing in 18 paired patients,and this approach led to the identification of a total of 179 somatic(non-inherited)mutations including 160 SNVs and 19 insertions and/or deletions(indels),the 160 SNVs occurred in 153 genes and the 19 indels were found in 16 genes respectively,with average 9.9 mutations per patient.Among all of the 179 gene mutations identified,we identified a group of genes which were previously implicated in the pathogenesis of ALL or other malignancies,such as NF1,MLL2,PAX5 and IKZF and etc.In addition to these known mutations,we also found some new mutations that have not been reported,which may shed lights into the mechanisim of leukemogenesis.Then we performed GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis and found that mutations discovered in our initial patients for exome sequencing participated in various kinds of synthetic and metabolitic activity,apoptosis pathways,the ubiquitin-proteasome pathway and epigenetic modifications,and also some signaling molecules involved in the Rho pathways,tyrosine kinase pathways,and insulin signaling pathway etc.By integrating the recurrency and the function of these mutations and combined the literatures reported in ALL patients,we firstly selected eight genes,namely ARHGEF37,DLC1,HDAC1,H3F3 A,CTCF,ETV6,PDE1 A and LPHN2,for a full exome screening in ALL patients.It has been found that ARHGEF37 and DLC1 exhibited a certain mutation frequencies in ALL patients,DLC1 accounting for about 3.1% while ARHGEF37 for 2%.Besides,we also found mutations in ARHGEF26,BAIAP2,and NF1(recurrent 3 times in our series,which have been reported frequently in ALL patients)and some other genes involved in Rho pathway which regulates the actin rearrangement,cell motility and migration.At present,the study on ALL with normal karyotype and adult ALL patients at genome level has not been reported yet.Only several descriptive sequencing results and susceptible pathways based on the data of childhood ALL were reported.It seems to us that focusing on one or a few susceptible genes may be insufficient to figure out the key factors of this disease,wihle several players of the same “driver pathway” or the joint effects of several abnormal “driver pathways” may lead to the malignant transformation.For example,the Rho GTPases are molecular switches that control a wide variety of signal transduction pathways in the regulation of the cell adherence,JNK activation and actin cytoskeleton organization.Therefore it is not surprising that deregulated Rho signalling can contribute to disturbed cellular phenotypes in a wide range of diseases which have been reported in renal cell carcinoma,breast cancer,and ovarian carcinoma.The role of Rho pathway in the development of ALL deserves further study.To validate the potential role of these mutations in leukemogenesis,we performed functional analysis on some of the above mentioned mutations.In the colony forming assay,the number of colonies bearing plasmids of ARHGEF37 mutants was higher than that of the wild-type ones,among which the number of colonies expressing P560 L mutant had statistically significant difference from that of the wild-type one.In the Rho Activation Assay,the RhoA-GTP status of cells bearing ARHGEF37 R547 C and P560 L mutations were decreased compared with the wild-type in 293 T cells.And the RhoA-GTP status of cells bearing DLC1 V555 A,N833K and H1390 Y mutations were slightly increased.Hoever,the activity of V714 M mutation showed no obvious changes.We also constructed the mutants of DLC1 and ARHGEF37 plasmids and carried out transfection experiments in Ba/F3 and 32 D cells,which were interleukin 3(IL-3)-dependent mouse pro-B cell line and myeloid cell line respectively.In Ba/F3 cells,over expression of R547 C and P560 L mutants increased the cell proliferation,while in the 32 D cells,the R547 C and P560 L mutants reduced cell proliferation in IL-3 conditional culture medium with significant differences.Nevertheless,these stably transfected cells with the corresponding mutants could not survive without the supplement of IL-3,suggesting these mutations could be necessary but not sufficient for the phenotypic transformation.It seemed that Rho pathway plays a different role in the lymphoid progenitor cells as compared to myeloid ones.The whole exome sequencing has therefor enabled the possibility to comprehensively identify the differences of the genes in the coding region between tumor cells and the normal cells,and allowed the changes that associated with the disease in the genomic level to be found.With the recurrently mutated genes known in B-ALL like NF1,KRAS,PAX5,IKZF1 and PTPN11,we have discovered that the somatic mutations involved in Rho pathway such as ARHGEF37,DLC1 and ARHGEF26 genes,might play important roles in the pathogenesis of ALL.