IntroductionMaintenance of the hematopoietic system requires continual replenishment of mature blood cells from a rare population of bone marrow residing hematopoietic stem cells (HSCs). The alteration of the homeostatic control of hematopoiesis is considered to be a major culprit of drastic increase in pathologic incidences, such as bone marrow failure, anemia, and myeloid leukemia during aging. 1 However, the underlying mechanisms of pathogenesis of hematologic malignancy in elderly population remain poorly understood.Mounting evidence supports the idea that the accumulation of somatic DNA damage is a main cause of aging in multicellular organisms. [2][3][4][5] Mice with mutations in various DNA repair genes exhibit accelerated aging in the hematopoietic system because of the premature exhaustion of HSCs, indicating that DNA repair is crucial for the maintenance of HSC self-renewal and hematopoietic function. 6,7 DNA damage can directly result from genotoxic treatment such as ionizing radiation (IR), or may simply occur as a consequence of genome duplication infidelity or of genotoxic effects of reactive oxygen species (ROS). ROS, such as superoxide anions and hydrogen peroxide, are byproducts of normal oxidative metabolism in eukaryotic cells and are involved in many signaling process. However, they can be harmful to cellular components, including DNA. 3,8,9 An uncontrolled elevation of intracellular ROS levels is believed to contribute to cellular aging and the senescence process. 3 In fact, an abnormal elevation of intracellular ROS levels has been implicated in the pathogenesis of various diseases, such as ataxia telangiectasia and Fanconi anemia. 3 In that sense, the maintenance of ROS levels, through highly regulated mechanisms, is essential for cellular homeostasis. 10 Being continuously exposed to oxidants produced during metabolic activity and to external oxidants or oxidant-inducers through normal cellular physiology, DNAs within cells inevitably suffer the oxidative damage. Therefore, an accelerated proliferation of hematopoietic cells, which is expected to occur after clinical HSC transplantation, might lead to DNA damage through overexposure to oxidative stress generated on each cell cycle. Indeed, a hyperproliferation caused an accumulation of oxidative stress and resulted in functional exhaustion of murine HSCs, as shown by the failure to reconstitute hematopoiesis after serial transplantations. 11 Taken together, we hypothesize that the continuous production of ROS during long-term repopulation induces an accumulation of genomic damage that leads to exhaustion of human HSCs. We have previously developed a strategy that enables to examine the multipotency of a single human HSC using a reliable surrogate system. 12,13 By determining the in vivo repopulating dynamics of individual human HSCs, we demonstrated that the repopulating potential of the majority of human HSCs progressively deteriorated as they underwent extensive repopulation process. Furthermore, the self-renewing long-term repopulating...

Key Points• TP53 and RAS-pathway mutations predict very poor survival, when seen with CK and MDS/MPNs, respectively. • For patients with mutated TP53 or CK alone, long-term survival could be obtained with stem cell transplantation.Genetic alterations, including mutations and copy-number alterations, are central to the pathogenesis of myelodysplastic syndromes and related diseases (myelodysplasia), but their roles in allogeneic stem cell transplantation have not fully been studied in a large cohort of patients. We enrolled 797 patients who had been diagnosed with myelodysplasia at initial presentation and received transplantation via the Japan Marrow Donor Program. Targeted-capture sequencing was performed to identify mutations in 69 genes, together with copy-number alterations, whose effects on transplantation outcomes were investigated. We identified 1776 mutations and 927 abnormal copy segments among 617 patients (77.4%). In multivariate modeling using Cox proportional-hazards regression, genetic factors explained 30% of the total hazards for overall survival; clinical characteristics accounted for 70% of risk. TP53 and RAS-pathway mutations, together with complex karyotype (CK) as detected by conventional cytogenetics and/or sequencing-based analysis, negatively affected posttransplant survival independently of clinical factors. Regardless of disease subtype, TP53-mutated patients with CK were characterized by unique genetic features and associated with an extremely poor survival with frequent early relapse, whereas outcomes were substantially better in TP53-mutated patients without CK. By contrast, the effects of RAS-pathway mutations depended on disease subtype and were confined to myelodysplastic/myeloproliferative neoplasms (MDS/MPNs). Our results suggest that TP53 and RAS-pathway mutations predicted a dismal prognosis, when associated with CK and MDS/MPNs, respectively. However, for patients with mutated TP53 or CK alone, long-term survival could be obtained with transplantation. Clinical sequencing provides vital information for accurate prognostication in transplantation.

To clarify the cooperative roles of recurrently identified mutations and to establish a more precise risk classification system in acute myeloid leukemia (AML), we comprehensively analyzed mutations in 51 genes, as well as cytogenetics and 11 chimeric transcripts, in 197 adult patients with de novo AML who were registered in the Japan Adult Leukemia Study Group AML201 study. We identified a total of 505 mutations in 44 genes, while only five genes, FLT3, NPM1, CEBPA, DNMT3A and KIT, were mutated in more than 10% of the patients. Although several cooperative and exclusive mutation patterns were observed, the accumulated mutation number was higher in cytogenetically normal AML and lower in AML with RUNX1-RUNX1T1 and CBFB-MYH11, indicating a strong potential of these translocations for the initiation of AML. Furthermore, we evaluated the prognostic impacts of each sole mutation and the combinations of mutations and/or cytogenetics, and demonstrated that AML patients could be clearly stratified into five risk groups for overall survival by including the mutation status of DNMT3A, MLL-PTD and TP53 genes in the risk classification system of the European LeukemiaNet. These results indicate that the prognosis of AML could be stratified by the major mutation status in combination with cytogenetics.

Interleukin IL-17 is a proinflammatory cytokine that has been implicated in the pathogenesis of various autoimmune diseases. The single nucleotide polymorphism (SNP), rs2275913, in the promoter region of the IL-17 gene is associated with susceptibility to ulcerative colitis. When we examined the impact of rs2275913 in a cohort consisting of 438 pairs of patients and their unrelated donors transplanted through the Japan Marrow Donor Program, the donor IL-17 197A allele was found to be associated with a higher risk of acute graft-versus-host disease (GVHD; hazard ratio [HR], 1.46; 95% confidence interval [CI], 1.00 to 2.13; P = 0.05). Next, we investigated the functional relevance of the rs2275913 SNP. In vitro stimulated T cells from healthy individuals possessing the 197A allele produced significantly more IL-17 than those without the 197A allele. In a gene reporter assay, the 197A allele construct induced higher luciferase activity than the 197G allele, and the difference was higher in the presence of T cell receptor activation and was abrogated by cyclosporine treatment. Moreover, the 197A allele displayed a higher affinity for the nuclear factor activated T cells (NFAT), a critical transcription factor involved in IL-17 regulation. These findings substantiate the functional relevance of the rs2275913 polymorphism and indicate that the higher IL-17 secretion by individuals with the 197A allele likely accounts for their increased risk for acute GVHD and certain autoimmune diseases.

Key Points• Significant HLA locus mismatches responsible for transplant-related events were determined in 7898 unrelated marrow donor transplants.• This information provides a rationale for use of an algorithm for unrelated donor selection.We hypothesized that the compatibility of each HLA loci between donor and patient induced divergent transplant-related immunologic responses, which attributed to the individualized manifestation of clinical outcomes. Here, we analyzed 7898 Japanese pairs transplanted with T-cell-replete marrow from an unrelated donor with complete HLA allele typing data. Multivariable competing risk regression analyses were conducted to evaluate the relative risk (RR) of clinical outcomes after transplantation. A significant RR of HLA allele mismatch compared with match was seen with HLA-A, -B, -C, and -DPB1 for grade III-IV acute graft-versus-host disease (GVHD), and HLA-C for chronic GVHD. Of note, only HLA-C and HLA-DPB1 mismatch reduced leukemia relapse, and this graft-versusleukemia effect of HLA-DPB1 was independent of chronic GVHD. HLA-DRB1 and HLA-DQB1 double (DRB1_DQB1) mismatch was revealed to be a significant RR for acute GVHD and mortality, whereas single mismatch was not. Thus, the number of HLA-A, -B, -C, -DPB1, and DRB1_DQB1 mismatches showed a clear-cut risk difference for acute GVHD, whereas the number of mismatches for HLA-A, -B, -C, and DRB1_DQB1 showed the same for mortality. In conclusion, we determined the biological response to HLA locus mismatch in transplant-related immunologic events, and provide a rationale for use of a personalized algorithm for unrelated donor selection. (Blood. 2015;125(7):1189-1197