Mechanisms Of Gene Mutations And Gene Polymorphisms In Leukemia Relapse After Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation （allo-HSCT） is a potential effective curative therapy for a variety of malignant and nonmalignant hematologic diseases. The past30years have seen tremendous progress in allo-HSCT, such as availability of suitable donors, reduction in treatment-related mortality and improved supportive care measures. However there has been very little progress in reducing the incidence of relapse and improving the outcomes of patients who experience relapse following allo-HSCT. Hematologic malignancy relapse remains one of the leading causes of death following allo-HSCT. In our present study, we focused on the mechanisms of leukemia relapse after allo-HSCT including the biological characteristics of leukemia cells and disability of immune defence against leukemia relapse.Chapter1The Mechanisms of Donor Cell Leukemia Relapse after Allogeneic Hematopoietic Stem Cell Transplantation According to the origins of relapsed leukemic cells, the types of leukemia relapse after allo-HSCT can be divided into donor cell leukemia and patient’s leukemic cell relapse. Leukemia relapse arising in cells of donor origin in the transplant recipient, so called donor cell leukemia （DCL）, is a rare disease entity after allo-HSCT. The reported incidence of DCL has ranged from0.13%to5%, and only49cases of DCL after allo-HSCT were reported between1971and2006. The precise etiological mechanisms of DCL remain unknown, and no common mechanism can be identified in most of the cases reported in the literature. Careful analysis of the mechanisms with respect to the oncogenic transformation of donor-derived cells might provide a valuable insight into our understanding of leukemogenesis.According to the "two genetic hits （more or less） model", cooperation between two classes of genetic mutations contributes to leukemogenesis. One group （class Ⅰ） comprises mutations in the fins-related tyrosine kinase3gene （FLT3） or the neuroblastoma RAS viral oncogene homolog gene （NRAS）, which increase the proliferation and/or survival of hematopoietic stem/progenitor cells. The other complementation group （class Ⅱ） comprises mutations in CEBPA, the myeloid-lymphoid or mixed-lineage leukemia gene （MLL）, or the nucleophosmin gene （NPM1）, which cause impaired differentiation. We hypothesized that the "two genetic hits model" may contribute to the "leukemization" of donor cells and first evaluated these genetic mutations implicated in the development of acute myeloid leukemia （AML） in the DCL patient and donor. A series of stained archival slides of bone marrow from the patient were available, including specimens taken at the time of original diagnosis, at CR after one course of induction chemotherapy, during lasting CR before transplantation, and1,9and12months after HSCT, as well as samples of mononuclear-cell-enriched bone marrow taken at the time of relapse and at CR after relapse. A buccal mucosal swab specimen obtained from the patient during remission was also available. A buccal mucosal swab specimen and samples of mononuclear-cell-enriched peripheral blood and bone marrow were also available from the donor. Genomic DNA was extracted using a routine salting-out method and was analyzed for mutations in FLT3, NRAS, CEBPA, MLL and NPM1genes. We found that the patient and his donor-sister both harbored a germ-line mutation in CEBPA （584₅89dup）. Susceptible donor hematopoietic cells evolved to overt AML by developing two somatic CEBPA mutations （247dupC and914₉16dup） in the patient’s microenvironment. These were identical to the acquired mutations identified in leukemic cells originating from the patient during de novo AML. Our results provide the first report of multiple mutations of CEBPA contributing to the transformation of donor cells to the leukemic phenotype and provide clues to support the multiple genetic hit mechanism of DCL.Chapter2The Molecular Mechanisms of CEBPA Gene Multiple Mutations in the Development of LeukemiaIn our previous research, we have identified3different CEBPA gene mutant forms involved in donor cell leukemia, which disrupted3major functional domains of C/EBPa protein respectively. The patient and his donor-sister both harbored the N-terminal germ-line mutation （584₅89dup, disrupting the TAD2domain of protein）. Susceptible donor hematopoietic cells evolved to overt AML by developing two somatic CEBPA mutations, the N-terminal frameshift mutation （247dupC, resulting in overproduction of truncated30KDa isoform （p30） lacking the TAD1domain） and the C-terminal mutation （914₉16dup, disrupting the bZIP domain）, in the patient’s microenvironment. We used these3mutant forms, as well as CEBPA gene wide type to be subcloned into pLenti6.3/V5-DEST vector. Four leukemic cell lines （NB4, K562, Kasumi-1, HL60）, mouse myeloid progenitor cell line （32Dcl3） were transfected by different mutant forms. The distinct roles of different mutations in blockage of C/EBPa functions including inducing leukemic cell apoptosis, cell cycle arrest and promoting granulocyte differentiation were analyzed. We found that the N-terminal germ-line mutation （584₅89dup） retains the function of inducing apoptosis in leukemic cells and granulocyte differentiation, however, blockages the effect of cell cycle arrest of C/EBPa. The C/EBPa-p30protein mutant abrogates the effect of inducing apoptosis. The C-terminal mutation （914916dup） abrogates both the effects of inducing apoptosis in leukemic cells and of promoting G-CSF-induced differentiation of32Dcl3cells into mature neutrophile granulocyte. Our data provide clues to support that the N-terminal frameshift mutation of CEBPA works as a class â…  mutation, while the C-terminal mutation works as both of class â…  and class â…¡ mutations in inducing leukemia.Chapter3The Genetic Basis of Leukemia Relapse Arising in Cells of Patient Origin after Allogeneic Hematopoietic Stem Cell TransplantationAbout90%of leukemia relapse after allo-HSCT arises in cells of patient origin. Although it is extensively accepted that leukemia stem cells and drug-resistant leukemic clones are the root, important biological and clinical differences have been identified between diagnostic and relapsed leukemic cells, including the acquisition of new chromosomal abnormalities, gene mutations, and reduced responsiveness to chemotherapeutic agents. Recent advances in massively parallel sequencing technologies have facilitated not only to suggest that addition of new mutations and clonal evolution involved in the development of relapse after chemotherapy in childhood acute lymphoblastic leukemia （ALL）, but also to identify an increasing frequency of recurring submicroscopic genetic alterations contributing to high risk of relapse. However, a detailed analysis of genetic alteration from diagnosis to relapse in adult ALL has not been performed.Furthermore, allo-HSCT is a distinct treatment option and has2important forms to eliminate and select on malignant cells. The malignant cells that go on to causing relapse must initially survive ablation of chemotherapy and conditioning regimen. Then, after allo-HSCT, they must survive the graft-versus-leukemia （GVL） reaction. We hypothesized that the multiple selective pressures may be more prone to inducing of genetic alterations and somatic evolution in the pathogenesis of relapse after allo-HSCT. To explore the genetic basis of leukemia relapse after allo-HSCT, we captured and sequenced whole exomes for germline DNA from3relapsed Ph-B-progenitor ALL at3specific time points during relapse progression including diagnosis, complete remission after induction chemotherapy before HSCT, relapse after allo-HSCT. We found three major clonal evolution patterns during ALL relapse after allo-HSCT. In the first pattern, the relapsed clone originated from the founding clone with recurrent mutations in the primary tumor, and the majority of identified somatic mutations were shared by de novo leukemic cells and relapsed cells. In the second pattern, a subclone of the founding clone survived initial therapy, gained additional new mutations and expanded at relapse, in this pattern, de novo leukemic cells and relapsed cells shared a few of same mutations, but have some different mutations. In the third pattern, the relapsed clone genetically distinct from de novo leukemic cells. We also identified a novel, recurrently mutated gene （PTPN21） associated with the risk of relapse of cytogenetically normal adult ALL after allo-HSCT. The present study is the first report to perform detailed analysis of clonal evolution and genetic alterations in relapsed ALL after allo-HSCT.Chapter4Association of Genetic Variations in Graft-versus-Leukemia Reaction Pathways and Leukemia Relapse after Allogeneic Hematopoietic Stem Cell TransplantationRelapse remains the most challenging obstacle in allogeneic hematopoietic stem cell transplantation （allo-HSCT）. Therefore, defining variables that predispose to this event is of critical importance. There is an intense ongoing investigation of biologic factors of hematologic malignancy responsible for the risk of relapse post-HSCT. However few studies focus on inter-patient variability in graft-versus-leukemia （GVL） reaction. To explore key informative predicators from donor and patient genetic basis, we investigated the influence of genetic variations in GVL reaction pathways on the risk of relapse post-HSCT. We analyzed33single nucleotide polymorphisms （SNPs） in19 genes involved in GVL reaction including NK cell activating receptor gene （NKG2D）, cytokine genes （TNFa, TNFβ and TNF receptor â…¡, TGFβ and TGFβ receptor â…¡, IL6, IL10）, T-cell co-stimulatory molecule genes （CD28, ICOS, CTLA-4）, T-cell effector pathway genes （Granzyme B, Fas and Fas Ligand） and the innate immune genes （toll-like receptors, TLR1,2,3,8,9）. The study included2independent cohorts. The initial cohort consisted of138pairs of patients and their unrelated donors. The second cohort consisted of102pairs of patients and their HLA-identical sibling donors. We found that two SNPs in donor side:NKG2D （rs1049174G/C） and CTLA-4CT60A/G （rs3087243）, and one SNP in patient side:Fas-670C/T （rs1800682）, significantly influenced the risk of relapse post-HSCT. In94patients with acute myelogenous leukemia （AML）, more than half （58.3%） of patients receiving grafts from donors with the CTLA-4CT60variant genotype （AA） experienced relapse, but the incidence decreased to22.4%in those receiving grafts from donors with the CTLA-4CT60wild-type allele （P=0.011）. AML patients with the Fas-670homogeneous variant allele （TT genotype） had a higher incidence of relapse compared with those with the wild-type allele （CC or CT genotype）（TT:43.9%vs. CC/CT:16.7%, P=0.02）. Interestingly, the effects of CTLA-4CT60and Fas-670polymorphic futures were highly significant in AML but did not show any trend in ALL patients. However, donor with NKG2D G allele-positive haplotype, a haplotype expected to induce greater NK cell activity, was an independent protection factor against relapse both in patients with AML and ALL. Our findings, which is the first report of genetic variations in GVL reaction pathways from donors and patients and relapse risk. Transplant patients would thus benefit from accurate risk assessment and individualization of immunosuppressive therapy, taking into account of donor and patient genetic data, as well as other risk factors.