Genomic Aberrations Of Ph+ Chronic Myeloid Leukemia Patients With Additional Chromosomal Abnormalities

Objective: Chronic myeloid leukemia(CML)is genetically characterized by the Philadelphia(Ph)chromosome derived from a balanced translocation between chromosomes 9 and 22.The appearance of a variety of nonrandom chromosomal abnormalities besides the Ph chromosome was known as clonal evolution(CE).CE is often associated with the disease progression and a poor prognosis.We performed karyotyping,fluorescence in situ hybridization(FISH),array comparative genomic hybridization(aCGH)and whole exome sequencing(WES)on CML patients in order to discover the characteristics of the genomic changes when patients underwent clonal chromosomal aberrations and reveal the genes related to CML clonal evolution.These genes and their variations might play important roles in the causes and the progression of CML.We explored the significance of the genes,thereby increasing the understandings of cytogenetic and molecular genetic abnormalities in CML,and providing new ideas for the diagnosis and treatment of CML patients.Methods: We collected bone marrow or peripheral blood samples from patients diagnosed with CML in the genetics laboratory of the Oklahoma University Health Sciences Center from 2000 to 2019.Chromosome analysis and FISH were performed to detect patients' cytogenetic changes.CML patients with Ph chromosome as the sole anormaly were subsequently studied for their copy number variations(CNVs)using aCGH.We randomly selected ten patients diagnosed in the chronic phase(CP)with Ph chromosome only and the other ten CP-CML patients with additional chromosomal abnormalities(ACAs)other than Ph+ for further WES studies.The patients with ACAs were collected into the experimental group,and the patients with Ph+ only were in the control group.We performed bioinformatics method to filter and annotate gene variations related to CML and compared their frequencies between these two groups.Somatic variations in genes associated with ACAs in patients with CML were finally screened out and discussed.Results: In this study,we collected a total of 106 samples of CML patients.After routine cytogenetic analyses,84 of 106 CML patients were diagnosed with Ph+/BCR-ABL1 only,and additional chromosomal abnormalities were found in the remaining 22 patients.49 of84 patients were successfully extracted DNA for aCGH testings.A total of 17 pathogenic CNVs were observed in 11 patients.Partial deletion of the long arm of chromosome 9was the most common CNV,occurring in five patients(45.5%).The chromosome segment involved was 9q34.11-q34.12.The loss of ASS1 gene included in the del(9q)regions was confirmed in three patients(60%),and the loss of BCR-ABL1 fusion signal was observed in two of the three patients at the same time.A total of 122.05 Gb of raw data were obtained from twenty patients diagnosed with chronic phase CML after whole exome sequencing.The average sequencing depth of the samples was 42.41×(16.69×-72.37×),and the average sequence coverage of exons was 96.40% in all 20 patients.The number of overall genome variants,single nucleotide variants(SNVs)and insertion and deletion variants(INDELs)were higher in the experimental group than those of the control group,but the differences between the two groups were not found to be statistically significant(P>0.05).A total of 159 somatic variants that could cause amino acids' changes and affect their coding proteins' functions were identified in our study,the experimental group had more somatic variants than the control group(88 in the experimental group and 71 in the control group,P>0.05),of which 19 were further identified to be tumor related somatic variants commonly found in CML,including 10 missense SNVs,3 non frame-shift deletions,2 frame-shift deletions and 4 stop gains.Variant rs57875368(NM?007350:exon1:c.582?584del:p.194?195del)located in the gene PHLDA1 was significantly more frequent in the experimental group(P=0.043).Two new SNVs that had not been reported in the Catalogue of Somatic Mutations in Cancer(COSMIC)or the database of Single Nucleotide Polymorphisms(db SNP)were also found in the experimental group.They were located in the coding regions of the gene TLE1 and the gene CSMD2,respectively.Conclusion: Genome imbalanced changes in Ph+ CML patients are complex and diverse.Partial deletions of 9q were the most common copy number variations found in our study,which were often related to the poor prognosis in CML.Besides,the deficiency of ASS1 gene included in the deleted 9q regions could act as a potential suppressor of CML disease and affect patients' treatment and outcome.We also screened out a significantly high-frequency variant in chronic phase CML patients with ACAs,and found two new somatic variants that had not been reported by the COSMIC or db SNP databases.These three variants located in the gene PHLDA1,gene TLE1 and gene CSMD2 might contribute to the clonal evolution of CML.We hope the genetic information we have discovered could provide a new molecular basis for the future targeted drug research in CML patients with with additional chromosomal changes.