ALBERT

Description: Abstract 1474 Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy with approximately 85% of cases being of B-cell lineage and 15% of T-cell lineage. There is growing evidence that epigenetic deregulation plays an important role in the pathogenesis of leukemia. Somatic mutations in polycomb repressive complex 2 (PRC2) components such as EZH2, SUZ12, EED, and JARID2 have recently been described in myeloid and lymphoid malignancies and seem to be associated with a poor prognosis. PRC2 is a highly conserved histone H3 lysine 27 methyltransferase that regulates the expression of developmental genes. We sought to determine the frequency and prognostic impact of mutations within the three PRC2 core component genes EZH2, SUZ12, and EED in a clinical well-defined cohort of 154 randomly selected childhood ALL patients (male, n=82; female, n=72; median age 6.7 years, range 0.2–25.9 years): B-cell lineage, n= 125; T-cell lineage, n=29. A total of 168 samples were investigated at diagnosis (n=109), relapse (n=31), or both (n=14) by analysis of genomic DNA isolated from bone marrow cells. PCRs were performed using standard conditions with primers covering all coding exons including intron-exon boundaries to detect potential splice mutations. Forty-nine amplicons were analyzed for each sample (8,232 amplicons in total). Mutation analysis was performed by Sanger sequencing. So far, the entire cohort has been analyzed for EZH2 and 100/168 samples for mutations in all three genes. The complete mutation status will be presented at the meeting. We identified four EZH2 mutations (missense mutations, n=3, frameshift deletion, n=1) in four patients classified as c-ALL (n=2), pro-B-ALL (n=1) or early T-cell precursor ALL (n=1), respectively. No SUZ12 or EED mutations have been identified so far. Remission material was available for all patients with missense mutations to test whether the mutation was acquired or inherited. Hereby, a somatically acquired EZH2 mutation was revealed in a 3-year-old boy diagnosed with c-ALL in 1982 and treated within the standard-risk group of the German ALL-VII/81 trial. He achieved complete remission but relapsed four years after initial treatment (late relapse). A second complete remission was not achieved under relapse treatment within the German ALL-REZ BFM 85 study and the patient died four weeks after relapse. The underlying mutation was a heterozygous missense mutation in EZH2 exon 17 that caused an amino acid change of aspartic acid to histidine at position 664 within the SET domain. No remission or constitutional material was available for the patient harboring a heterozygous frameshift deletion in EZH2 exon 6, however, analysis of serial samples at diagnosis and relapse revealed the same EZH2 mutant clone. Furthermore, this mutation has not been described as a polymorphism in large single nucleotide polymorphism databases so far. The patient was a 10-year-old girl diagnosed with pro-B-ALL in 1984 and treated within the high-risk group of the German ALL-VII/81 trial. She achieved complete remission but relapsed and died 6 months after start of treatment (very early relapse). In summary, although mutations of PRC2 components seem to be rare in childhood ALL our findings indicate that genetic defects in PCR2 might contribute to the disease in individual cases associated with a poor prognosis. Disclosures: No relevant conflicts of interest to declare.

Description: Adult pluripotent stem cells can be characterized as cells with both the capacity to self-renew and the ability to differentiate into distinct hematopoietic cell lineages and also into other tissue types. Because of the important course to stem cell research and clinical application the development of ex vivo culture systems is crucial for an effective expansion of these hematopoetic progenitor cells. After positive selection, CD34+-cells from peripheral G-CSF stimulated blood of healthy donors were cultured for 2 weeks in a serum-free medium with 2% of cord blood serum and the addition of early-acting cytokines (SCF, IL-3, Flt3-ligand, LIF, and TPO). The cells were grown in 6-well inserts on a feeder layer of mouse embryonic fibroblastic cells (MEF). Both cell types were separated from each other by a microporous membrane allowing only diffusion of soluble factors like cytokines but not cell migration. Additionally, the membrane was coated with Matrigel, a basement membrane matrix equivalent (MG), or with fibronectin (FN). Cultures were sampled at days 4, 7, 10 and 14 for cell count, colony-forming units-assay and immunophenotyping by flow cytometry. After two weeks of culture, the highest proliferation of stem cells was found in the control (medium without MEF, Matrigel and FN; 364-fold) followed by MEF- and FN-cultures (341- and 306-fold, respectively). The lowest clonal production was estimated in the cultures with MG (MG alone: 124,4-fold and MEF+MG: 48,2-fold, respectively). Examining the cells for colony-forming units the highest number of colony-forming unit-granulocyte-erythrocyte-macrophage (CFU-GEM), burst-forming unit-erythrocyte (BFU-E), colony-forming unit-granulocyte-macrophage (CFU-GM) and colony-forming unit-erythrocyte (CFU-E) were found in cultures with MEF and MEF+FN. We found multipotential colonies (CFU-GEM and BFU-E) at the highest expansion rate with FN (13,3-fold) and MEF (8,2-fold) at the fourth day of culture and with both additives in a MEF+FN-culture at the seventh day (17,2-fold). In addition to these results, there were sporadic undifferentiated progenitor cell colonies in MEF+MG-culture up to the day 14, despite an initially lower growth rate than that on MEF alone. The percentages of CD34+-cells estimated by flow cytometry were downregulated continiously during the culture period. At day 7, the highest number of CD34+-cells was found in the cultures with MG (46,0%) and MEF+MG (42,0%). The maximum number of CD34+-cells expressing no CD38 but CD61 was found at day 4 in FN culture (20,4%) and MG culture (15,9 %), respectively. In summary, we recognized in cultures with MG as a factor similar to the natural microenvironment a correlation between the diminished proliferation rate of stem cells in favor of an increased generation of multipotential colonies combined with their delayed differentiation into granulocyte-macrophage and erythrocyte colonies. Appropiate feeder cells as well as matrix proteins like Matrigel or fibronectin may helpful in improving the ex vivo expansion of hematopoietic progenitor cells.

Description: The proof for the prenatal origin of childhood acute lymphoblastic leukemia (ALL) comes from the detection of concordant leukemia in monozygotic twins and the identification of translocation breakpoint genomic sequences at birth in a limited number of ALL patients with t(4;11) or t(12;21) chromosomal translocation. However, most patients with childhood ALL lack leukemia-specific fusion gene sequences. Therefore, we have used the rearranged immunoglobulin heavy chain (IgH) genes as a marker for the detection of preleukemic clones at birth. Guthrie card blood spots of 32 children with B-lineage ALL treated at our institution were available for this retrospective study. The ALL patients had a median age of 5 years (range, 15 months to 14 years) and had median presenting white blood cell (WBC) counts of 10150/μl (range, 800 to 103800/μl). In all patients a monoclonal IgH gene rearrangement was obtained from diagnostic bone marrow and sequenced. Clone-specific primers were designed using the specific D-N-J and N-D-N sequences. A two-stage polymerase chain reaction (PCR) using a semi-nested approach was developed to improve sensitivity and specificity of amplification. In all 32 patients, one leukemic cell could be detected in a background of 105 normal blood mononuclear cells. Nineteen of the 32 patients (59%) had detectable IgH gene rearrangements at birth using the sensitive semi-nested PCR. Sequencing of the PCR products obtained from Guthrie card blood spots revealed the identical sequences identified from diagnostic leukemic cells. The fetal characteristics of the leukemic cells were indicated by the small numbers of nucleotides inserted into the N region and the shortened D germ line segments. Interestingly, five of the six children (83%) with hyperdiploid ALL had detectable preleukemic clones at birth. Four of the five children (80%) with pro-B ALL, 13 of the 21 children (62%) with cALL and only two of the six children (33%) with pre-B ALL had preleukemic clones on their cards. We did not observe any differences in age at diagnosis or presenting WBC count between the 19 patients with preleukemic clones at birth and the 13 patients whose Guthrie cards were tested negative. Our results suggest that the majority of children with B-lineage ALL has preleukemic clones already at birth indicating a prenatal origin of leukemia. In addition, postnatal factors are important in leukemogenesis as well because of the long latency periods until clinical diagnosis of leukemia.

Description: Epstein-Barr virus (EBV)-associated lymphoproliferative disease (LPD) is a serious complication following hematopoietic stem cell transplantation (HSCT). In 86 patients undergoing HSCT in a single center we prospectively performed a quantitative EBV-specific polymerase chain reaction in peripheral blood mononuclear cells (PBMC) weekly after HSCT. Cellular immune reconstitution was monitored by flow cytometric analysis on days 30 and 60 after transplantation. We observed an EBV reactivation (〉103 EBV-genome copies/105 PBMC) only in patients who received antithymocyte globulin (ATG) for immunosuppression. 15 of 46 patients (33%) who received ATG experienced EBV reactivation. Interestingly, we found a similar incidence of EBV reactivation in 7 of 21 patients (33%) who received ATG (Fresenius) at a median total dose of 40 mg/kg and in 8 of 26 patients (31%) who received ATG (Sangstat) at a median total dose of 20 mg/kg. However, in patients with ATG (Fresenius) EBV reactivation was observed only in the range between 103 and 104 EBV-genome copies/105 PBMC and no EBV-associated LPD occurred. In contrast, in patients with ATG (Sangstat) EBV reactivation was always greater than 104 EBV-genome copies/105 PBMC. Median values for CD3+ cells were 157.7/μl versus 1.5/μl on day 30 and 327.0/μl versus 15.5/μl on day 60 in the group receiving ATG (Fresenius) or ATG (Sangstat), respectively. For CD3+CD4+ cells the corresponding numbers were 57.8/μl versus 0.5/μl on day 30 and 90.8/μl versus 10.0/μl on day 60. Median values for CD3+CD8+ cells were 89.4/μl versus 0.3/μl on day 30 and 237.3/μl versus 9.4/μl on day 60 in the group receiving ATG (Fresenius) or ATG (Sangstat), respectively. Patients with ATG (Sangstat) had significantly lower numbers of CD3+, CD3+CD4+, and CD3+CD8+ cells than patients with ATG (Fresenius) on day 30 (P

Description: The IL23R gene on chromosome 1p31 encodes a subunit of the receptor for the proinflammatory cytokine interleukin-23. Recently, IL23R could be identified as a novel inflammatory bowel disease susceptibility gene. The exchange of arginine with glutamine at position 381 of IL23R causes a blockade of the IL-23 signaling pathway which is associated with a reduced risk of both Crohn’s disease and ulcerative colitis. IL-23 is a pivotal cytokine in the differentiation of T cells into inflammatory, IL-17-producing T cells. Because of the requirement for IL-23 in autoimmune disease we hypothesized that IL23R Arg381Gln reduces the risk of graft-versus host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). 141 donors and 141 children (median age, 12 years) with acute lymphoblastic leukemia (n=63), acute myeloid leukemia (n=40), myelodysplastic syndrome (n=26) or chronic myeloid leukemia (n=12) who underwent allogeneic bone marrow (n=93) or peripheral blood stem cell transplantation (n=48; T-cell depleted: n=22) in a single center were genotyped of IL23R for rs11209026 (c.1142G〉A, p.Arg381Gln) using TaqMan real-time polymerase chain reaction. The donor was HLA-matched unrelated in 60% of transplants and HLA-identical related in 30% of transplants. Conditioning regimen was myeloablative in all cases. Two forms of post-transplant immunosuppression predominated, cyclosporine A and methotrexate in 65% of transplants and cyclosporine A alone in 24% of transplants. The IL23R Arg381Gln variant was present in 16 of the 141 donors (11.3%) and in 15 of the 141 patients (10.6%). Interestingly, we found a significantly reduced incidence of acute GVHD grade II–IV in patients who were transplanted from a donor with this specific variant (6.2% versus 33.6%; p=0.025). Furthermore, there was no severe acute GVHD grade III–IV if the variant occurred in the donor (0% versus 14.4%). In three of the donor-patient pairs the variant was present in both individuals and no acute GVHD was observed. The occurrence of the IL23R variant, in either donors or recipients, had no significant impact on chronic GVHD, relapse rate, treatment related mortality, and overall survival. In conclusion, IL23R Arg381Gln variant in the donor confers strong protection against acute GVHD after HSCT in children with hematological malignancies. Therefore, the IL23 signaling pathway seems to play an important role in the pathogenesis of acute GVHD. Blockade of the IL23 receptor by a monoclonal antibody could be a rational therapeutic strategy for acute GVHD.