Clonal Evolution Studies Of Chronic Myeloid Leukemia And Astrocytoma

By far, cancer is still one of the main reasons accounting for the worldwide death.With the in-depth studies, our understanding on cancer biology and genomics has changed. The cellular complexity and dynamic evolution characteristic of the disease should be taken into account when translating the genomics of cancer to the clinic treatment. The distinct genetic clones compete for space and resources in cancer evolution, and as a consequence the fittest clone drives their expansion into new cancer subpopulations. Cancer evolves over variable time frames and tempos, and in any cancer patient, the clonal structure, genotype and phenotype shifts over time. The clonal evolutionary study of cancer could provide more comprehensive knowledge on the cancer pathogenesis and progression, and those studies on understanding cancer evolution can also have important clinical applications, for instant finding more robust predictive biomarkers to improve personalize treatment and prevent drug resistance. And the evolutionary characteristics such as clonal structure or the mutation diversity may estimate the cancer prognosis.Chronic myeloid leukemia(CML) is a kind of clonal myeloproliferative disorder;the typical genetic aberration of the hematologic disease is the translocation between chromosome 9 and 22- t(9;22)(q22;q11), also known as Philadelphia chromosome(Ph), which generates the BCR/ABL1 fusion gene in molecular level. The product of this gene, the BCR/ABL1 protein, has a constitutive tyrosine kinase activity, which can promote cell proliferation in the absence of growth factors. Ph chromosome is the major chromosome aberration in CML which confers the cancer phenotype of the disease, and almost all CML patients harbor BCR/ABL1 fusion genes. The targeted treatment directing against the Ph chromosome can induce remission in CML patient.However, the formation mechanism of the Ph chromosome and the genetic clonal evolution structure after targeted treatment are still unclear.Glioma is a general term used to describe any tumor that arises from the glia tissue of the brain. And astrocytoma is most common tumor of neuroepithelial tissue.Although the low-grade astrocytoma usually grow slowly,those tumors progress to malignant high-grade tumors causing the relapse which decreases the entirely survivalrate. The astrocytoma extremely easily recur after the resection with the increased malignancy, however, the mechanism is still enigmatic. Most astrocytoma patients die from tumor recurs and malignant transformation after relapse, therefore the study of clone evolution in genetic level is of significance to understand the biological behavior as well as the targeted therapies.Objective Clonal evolution is an important characteristic of cancer. In this study, two kinds of cancer CML and astrocytoma were included to explore the influence of clonal evolution on disease progression and treatment. The CML exists typical Ph chromosome; while the disease character of astrocytoma is recurrence and malignancy after surgical resection. Though the whole genome sequence, we attempt to seek the formation mechanism of the BCR/ABL1 translocation, the genetic clonal evolution after Ph targeted treatment; and aim to reveal the genetic factors underlie the astrocytoma recurrence and malignancy.MethodsIn this study, the patient was in chronic phase after hematopoietic stem cell transplantation(HSCT) and the donor is her sister; then the patient developed in lymphoid blast crisis with BCR/ABL1 fusion positive; the patient obtained remission after targeted therapy. the bone marrow biopsies in chronic phase(Ph-) after allogeneic bone marrow transplantation, blast crisis phase(Ph+) and remission phase(Ph-) after targeted treatment were collected for a CML patient, and the skin biopsy of the patient was also obtained as normal control.The histopathologic examination showed astrocytoma WHO II grade for the patient in preliminary diagnosis, the tumor was removed by surgery followed by chemotherapy and radiation; the tumor recur after one year with the histopathologic examination showing glioblastoma accompanied local anaplastic astrocytoma WHO IV grade. The surgical resected tumor specimen of preliminary diagnosis(WHO II grade) and relapsed phase(WHO IV grade) were collected for a astrocytoma patient,and the peripheral blood of the patient was obtained as normal control.The DNA were extracted for the bone marrow biopsies, tumor tissue specimen,skin biopsy and peripheral blood; and whole genome resequencing were conducted using Illumina Hiseq XTen(depth≥50×). Raw data were filtered to remove low-quality reads to get the clean data. Then the clean data were aligned to human reference genome(UCSC hg19) by BWA. Software samtools(1.0),control-FREEC(v6.7), mu Tect(1.1.4), Strelka(v1.0.13), crest(V0.01) were used to detect the genetic aberration including mutation, copy number gain or loss and chromosome aberration. Based on the somatic single nucleotide variants and copy number changes of the tumor, EXPANDS predicts the number of subpopulations(SPs)that coexist in a tumor, the size of the SPs in the tumor bulk and the mutations that mark each SP. The Sci Clone software was used to conduct the subclone analysis for the paired specimen.ResultsThe CML patient involved in this study was in chronic phase after allogenic bone marrow transplant, following by lymphoid blast crisis phase and then obtained remission after targeted Ph therapy. There is a series of SNPs, INDEL, CNVs and SVs in the bone marrow specimen though analyzing the sequenced data; there are more genetic aberration including mutation, insertion, deletion and chromosome aberration in the blast crisis phase(Ph+); the analysis confirmed that the Ph chromosome in blast crisis phase originate from the patient herself(the bone marrow acceptor); some specified genetic aberration in the blast crisis phase(Ph+)disappeared after the targeted therapy; and there is BRIP1 frameshift deletion mutation in the blast crisis phase(Ph+). There were eight SPs for blastic phases and remission phase, and the evolution tree for both were “tree form”.The astrocytoma patient who was diagnosed with grade II astrocytoma preliminary then recurred with glioblastoma(grade IV) complicated with local anaplastic astrocytoma(grade III) was included in the study. The genomic DNA sequencing and sequenced data analysis were conducted for the tumor tissue specimen; there is NOTH1 missense mutation in both of preliminary and recurrent tumor; there is TP53 heterozygous nonsense mutation in preliminary tumor; while a TP53 heterozygous nonsense mutation was found in all tumor cells, and IDH1 heterozygosis missense mutation in the recurrent tumor.; more chromosome aberration were detected in relapsed tumor; and there are 4 and 3 SPs in preliminary and recurrent tumor respectively, the evolution tree are “tree form” and “parallel form” respectively which indicating there were higher heterogeneity in the recurrent tumor.Conclusion BCR/ABL1 translocation is the typical clonal evolution in the process of CML disease progression; the secondary mutations and chromosome aberration disappeared with the targeted Ph therapy which suggests that the Ph chromosome may influence the disease procession though the genetic clonal evolution. And the DNA repair gene(such as BRIP1) and chromosome instability gene(such as BRCA2) mutation play a significant role in the origin of BCR/ABL fusion gene.As for the astrocytoma, molecular classification can be on the basis of special mutations, however further studies are need for the relationship between the IDH1 mutation and the prognosis; the subclone structure analysis could provide evidence for the clinical prognosis; chemotherapy and radiation therapy may be the selection pressure for the tumor clonal evolution, and the results indicated that radiation therapy should be given cautiously after the initial surgery.