ALBERT

Description: Key Points Genetically heterogeneous subclones with varying leukemia-initiating potential exist in neonatal transient abnormal myelopoiesis. This novel xenograft model of transient abnormal myelopoiesis may provide unique insight into the evolutionary process of leukemia.

Description: Abstract 3721 Background: Currently, umbilical cord blood cells (UCBCs) are used as the primary source of hematopoietic stem cells (HSCs) for transplantation instead of bone marrow cells (BMCs). UCBC transplantation has several advantages over BMC transplantation, including the much larger size of the available donor pool, the rich proportion of hematopoietic progenitor cells, and the low content of mature T cells that might cause a graft-versus-host reaction. However, the limited quantity of cord blood samples that can be obtained from a pregnant woman is considered to be a disadvantage of UCBC transplantation. To overcome quantity limitations, the use of mixed cord blood might be necessary. Due to the lack of appropriate animal models, the analyses of the differentiation capacity of mixed UCBCs in recipients have been limited to in vivo xenogeneic experiments and clinical observations. In this study, we evaluated the reconstitution and functioning of immune systems induced by mixed UCBC transplantation in mice. Method: To deplete NK cells, female C57BL/6 (B6) [H-2b] recipient mice were intraperitoneally administered rabbit anti-asialo GM1 antibody 1 day before transplantation. On the following day, the B6 recipients irradiated with a radiation dose of 9 Gray were transplanted with 3 different combinations of mixed UCBC {[1] GFP-Tg B6 (H-2b) and C3H (H-2k); [2] GFP-Tg B6 (H-2b) and BALB/c (H-2d); and [3] C3H (H-2k) and BALB/c (H-2d)}, with each combination containing equal number of cells. Engraftment of cells in the recipients' peripheral blood was detected by flow cytometric analysis using a specific antibody against lineage markers such as CD3e (T cells), CD45R/B220 (B cells), CD11b (macrophages), or Ly-6G (granulocytes) at intervals of 4 weeks for 16 weeks. In addition, the donor origin of each lineage population was determined by H-2Kk and/or H-2Kd antibody staining. GFP+ lineage cells were found to have B6 donor origin. The dermis samples harvested from the B6, C3H, and BALB/c mice were simultaneously skingrafted on the shaved backs of the recipients at over 16 weeks after transplantation. After the recipients rejected the skin graft, alloreactive T cell and antibody responses were examined to evaluate the functional maturity of the reconstituted immune system in the recipients. Result: The survival rates of the recipient mice at 16 weeks after transplantation were as follows: (1), 73%, 8/11; (2), 92%, 12/13; and (3), 100%, 1/1. Flow cytometric analysis showed that cells of all lineages were reconstituted by only GFP+ cells in almost all the B6 recipients transplanted with mixed UCBCs: (1), 50%, 4/8 and (2), 100%, 12/12. This findings indicated predominant bone marrow engraftment of UCBC-HSCs from MHC-matched B6 donors. Furthermore, the allogeneic UCBCs in the recipients transplanted with combination [3] were eliminated from the recipients, and their lineage cells (GFP− H-2Kk− H-2Kd−) in peripheral blood were derived from the B6 recipient's own X-ray-resistant HSC in the bone marrow. The recipient mice in which the immune system was reconstituted by UCBC-HSCs from the B6 donors accepted the skin graft from the B6 donors, but completely rejected the skin grafts from the C3H and BALB/c donors. This finding indicates that both CD8+ killer and CD4+ helper T cells were functionally mature in the recipients. Furthermore, the functional competence of both cellular and humoral immunity in the recipients that rejected the skin grafts from the C3H and BALB/c donors was determined by evaluating alloreactive T cell and antibody responses against H-2k and H-2d in an in vitro experiment. Conclusion: This study showed the promising potential of transplantation of mixed UCBCs for achieving high survival rates and functional immune reconstitution. We found that only MHC-matched HSCs were involved in hematopoiesis in the recipients' bone marrow. This finding suggests the presence of a surveillance system in bone marrow with the ability to distinguish self from non-self with different MHC antigens. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2995 Introduction: An increasing number of clinical trials have demonstrated the usefulness of cord blood as a source of hematopoietic stem cells for reconstitution of the hematopoietic system. Nevertheless, due to a lack of convenient animal models, information about the immunological competence of umbilical cord blood cell (UCBC)-derived lymphocytes has been relatively limited. Recently, we have established a murine model of UCBC transplantation, which reconstitutes the hematopoietic system of immunodeficient mice, and studied the correct immunological functions of UCBC-derived lymphocytes generated in an allogeneic environment. Materials and Methods: UCBC prepared from C57BL/6 (H-2b) mice were transferred into lethally irradiated BALB/c (H-2d) mice lacking T- and B-lymphocytes (RAG2 knockout). In order to evaluate the adaptive immune response in the recipient mice, rejection of skin grafts from third-party C3H/He (H-2k) mice and antibody responses to immunization with a T-dependent antigen, 2,4,6-trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH), were assessed. Additionally, the responsiveness of generated T cells was verified by mixed lymphocyte reaction (MLR) assay, cytotoxic T-lymphocyte (CTL) assay and phytohemagglutinin (PHA) blast formation assay. Results: In this UCBC transplantation model, the overall survival of the recipient mice was dose-dependent, and all surviving recipient mice were hematopoietic chimeras possessing all lineages. These allogeneic chimeras specifically rejected skin grafts from third-party C3H/He mice (rejection time; 9–16 days) while showing tolerance to skin grafts from both donor-type and recipient type, although with a significant delay in comparison to normal BALB/c mice (8–12 days) and C57BL/6 mice (9–11 days). Although accurate antibody responses of the allogeneic chimeras against TNP-KLH immunization were not anticipated, substantial amounts of TNP-specific IgG were detected in their sera. Interestingly, the level of TNP-specific IgM was significantly higher than that of normal BALB/c mice after TNP-KLH immunization, indicating retarded immunoglobulin class-switching in the allogeneic chimeras. The mechanism responsible for the antibody production by allogeneic chimeras still remains unclear. CTL and MLR assay revealed cell-mediated third-party-specific activity of killer and helper T cells, consistent with the tendency for the skin graft rejection and antibody production capability of the allogeneic chimeras to be inferior to those of normal mice. T cells of the allogeneic chimeras also proliferated non-specifically in response to PHA stimulation. The T-cell proliferation indices of the allogeneic chimeras when those of normal mice were taken as 100 were 21 in the MLR assay and 48 in the PHA blast formation assay, suggesting relative inferiority of the recognition and response system of T cells generated in an allogeneic environment. In any event, it may be important to note that T-dependent antigen-specific antibody production and alloantigen-specific rejection were substantial in the allogeneic chimeras. Conclusions: Overall, the present findings suggest that UCBC-derived lymphocytes generated in HLA-mismatched recipients are immunologically functional and competent. Regarding the relative inferiority of immune responses in the allogeneic chimeras, we predict the causes originate from narrowing of the T- and B-cell receptor repertoire by the allogeneic environment, and this possibility is now being examined. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome (IBMFS) characterized by bone marrow failure and pancreatic insufficiency. Patients with SDS have propensity to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Although it is the third most common IBMFS following Fanconi anemia and Diamond-Blackfan anemia in western countries, the incidence of SDS has been considered to be much lower in Japan. Patients and methods:In addition to the cases of SDS diagnosed by SBDSgene mutation analysis at Kyoto University, we captured those from the central review system of the Japanese Society of Pediatric Hematology and Oncology and the targeted sequencing system for IBMFS at Nagoya University as well as the previous Japanese nationwide survey for SDS that was conducted in 2010. Results:A total of 47 cases from 43 families with biallelic SBDSgene mutation were captured. After the previous nationwide survey, 21 cases were accumulated in the past 8 years: 2.6 cases per year in average. Median age at presentation is 0 year, and median age at diagnosis is two years. Male-female ratio of the patients was 2.1:1. Birth weight was less than 2500g in approximately half of the patients. Most common mutation was 183-184TA〉CT/258+2T〉C (73%) followed by 258+2T〉C/258+2T〉C (6.6%). Notably, a patient with heterozygous 258+2T〉C mutation was shown to have exon 3 deletion in the SBDS gene by whole exome sequencing. Clinical features at initial visit were various kinds of cytopenia, failure to thrive, steatorrhea, liver dysfunction, short stature, and skeletal abnormalities. Pancreatic insufficiency and/or pancreatic abnormality in imaging studies were found in almost all patients. Neutropenia was documented in one-third of the cases at initial visit; however, eventually 89% of the patients had neutropenia. Other hematological abnormalities were anemia (64%), thrombocytopenia (69%), and pancytopenia (40%). Bone marrow examination revealed hypoplasia (60%), dysplasia (36%), and chromosome abnormalities (23%) including del(20q), i(7q), and complex chromosome abnormalities. Four male patients (8.5%) of the cohort were documented to develop AML. Age at onset of AML was more than 18 years in three patients. Three patients complicated with AML and two patients with severe bone marrow failure underwent hematopoietic stem cell transplantation (HSCT). Interestingly, one patient with AML harboring normal karyotype is alive without relapse 20 years after HSCT. Three patients (6%) died; the cause of death was AML in two and complication of HSCT in one. Conclusion: Along with growing recognition of the disease among physicians and establishment of mutation analysis system, newly diagnosed cases of SDS were accumulated constantly. SDS may be more common than previously thought in our country. Clinical characteristics are similar to those that were reported in previous studies. As shown in a recent study on a large MDS cohort from an international registry of HSCT, patients with SDS may develop AML in adulthood and have very poor prognosis. The SDS cohort in Japan may provide a platform to investigate clinical and genetic factors associated with severity and phenotypes of the disease under a relatively uniform ethnic background. Disclosures No relevant conflicts of interest to declare.

Description: Background Acute lymphoblastic leukemia (ALL) in Down syndrome (DS) have uncommon genetic alterations such as mutations of JAK2, RAS, and overexpressions of CRLF2. These findings suggest DS-ALL may have unique biological features compared with non-DS-ALL. While recent studies implicated HMGN1 or DYRK1A in chromosome 21 were associated with molecular pathogenesis of DS-ALL, it remains to be elucidated what predispose DS children to develop ALL. Materials and Methods We performed whole transcriptome sequencing, targeted deep sequencing, and SNP array analysis in 25 DS-ALL samples, which included four ETV6-RUNX1 fusions and one high hyperdiploid. To compare with DS-ALL, we also performed whole transcriptome sequencing and whole exome sequencing to 118 non-DS-ALL samples, which included several subtypes such as ETV6-RUNX1 or BCR-ABL1. To cluster gene expression profiling, we applied the hierarchical clustering method. The detection of Ph-like signatures was performed by the hierarchical clustering by the gene set reported by Harvey. Results In expression analysis, we identified 19 fusions in 25 DS-ALL samples. These fusions included 15 recurrent fusions in pediatric BCP-ALL and 4 novel fusions, which including SSBP3-DHCR24, PDGFA-TTYH3, and NIN-NDUFA6. In novel fusions, PDGFA-TTYH3 fusions were detected in two DS-ALL samples. The hierarchical clustering analysis (Figure 1) combining 25 DS-ALL with 123 non-DS ALL samples. In our cohort, we defined samples with PAX5 alteration only such as a mutation or fusion as PAX5-altered. This clustering revealed ALL samples were divided into six clusters (cluster E1 to E6). Among six clusters, DS-ALL samples were divided into four clusters. In these four clusters, chi-square test revealed the significant enrichment of DS-ALL in E6 cluster. Importantly, our expression analysis revealed DS-ALL samples were highly heterogeneous and had the same expression pattern corresponding to each subtype same as non-DS-ALL. Cluster E3 included most samples with PAX5 fusions. All samples with ETV6-RUNX1 fusions were classified into cluster E4. Most samples of high hyperdiploid were classified into cluster E5. Cluster E6 was characterized by BCR-ABL1 fusions and Ph-like signatures. We detected 21 samples had Ph-like signatures, which included seven DS-ALL samples and 14 non-DS-ALL samples. Though we also analyzed differentially expressed genes between DS-ALL and non-DS-ALL, no genes on chromosome 21 such as HNGN1 or DYRK1A was significantly expressed. To investigate a relation between expression and genomic status, we further searched mutational analysis and copy number analysis (Figure 2). In 25 DS-ALL samples, six samples revealed JAK2 mutations and CRLF2 fusions. Interestingly, all of these six samples had Ph-like signatures. In cluster E5, one non-DS-ALL sample revealed JAK2 mutation and CRLF2 fusion and this particular sample was expected to have the Ph-like signature. To detect other Ph-like samples, we performed hierarchical clustering of 143 ALL samples based on the genes with a significantly (adjusted P value