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: 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: Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative approach for a variety of hematologic diseases but is still associated with substantial morbidity and mortality. Transplant-related mortality (TRM) after HSCT depends mainly on the toxicity of the conditioning regimen, infections, and graft-versus-host disease (GVHD). Polymorphisms at major histocompatibility complex MHC) have been proven to be critical for successful allogeneic HSCT. Polymorphisms at non-MHC loci have been associated with HSCT outcome as well. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is an inhibitory molecule that down-regulates T-cell activation. The purpose of this study was to identify an association between CTLA-4 single nucleotide polymorphisms (SNPs) and TRM in children undergoing allogeneic HSCT. Methods: 153 donors and 153 children (median age, 11 years) with acute lymphoblastic leukemia (n=90), acute myeloid leukemia (n=58) or juvenile myelomonocytic leukemia (n=5) who underwent allogeneic HSCT in a single center were genotyped of CTLA-4 gene for rs3087243 (CT60 A/G), rs231775 (+49 A/G) and rs4553808 using TaqMan real-time polymerase chain reaction. 49 children were transplanted in first, 40 children in second and 15 in third complete remissions. 49 patients did not reach complete remission at the time of transplantation. The donor was HLA-matched unrelated in 59% of transplants and HLA-identical related in 41% of transplants. Conditioning regimen was myeloablative in all cases and based on total body irradiation in 52% of transplants or busulfan in 44% of transplants. Two forms of post-transplant immunosuppression predominated, cyclosporine A and methotrexate in 64% of transplants and cyclosporine A alone in 25% of transplants. Results: We observed a significant association between the donor´s CTLA-4 genotype of rs3087243 and TRM in children undergoing allogeneic HSCT. Genotype AG of CTLA-4 rs3087243 SNP was found in 78 donors (51%), GG in 44 donors (29%), and 31 donors were homozygous for AA (20%). 30 patients died of transplant-related causes. 16 patients died of multi-organ failure, 7 of infection, 3 of acute GVHD, 2 of chronic GVHD, and 2 of hepatic sinusoidal obstruction syndrome. Interestingly, we observed a significantly reduced TRM in children who were transplanted from a donor with the CTLA-4 genotype GG in comparison to genotype AG or AA of rs3087243 (9% versus 19% versus 36%, p=0.013). In addition, we found significant differences of event-free survival (EFS) depending on the donor´s genotype. The EFS was 64%, 46% or 32% if the patient was transplanted from a donor with CTLA-4 genotype GG, AG or AA of rs3087243, respectively (p=0.043). In multivariate analysis, CTLA-4 genotype of rs3087243 was an independent risk factor for TRM (0.021). The CTLA-4 genotypes, in either donors or recipients, had no significant impact on relapse rate, acute and chronic graft-versus host disease. Conclusions: This study provides the first evidence that the CTLA-4 polymorphisms are significant risk factors for TRM and survival in children undergoing allogeneic HSCT and should be evaluated in further trials. Disclosures No relevant conflicts of interest to declare.

Description: Background:Hepatic sinusoidal obstruction syndrome (SOS), commonly known as veno-occlusive disease of the liver is a life-threatening early complication after hematopoietic stem cell transplantation (HSCT). Until now, examinations about the influence of genetic risk factors are extremely rare. Heparanase (HPSE), a pivotal endoglycosidase responsible for heparan sulfate degradation, is expressed by activated endothelial cells. HPSE has been shown to be involved in inflammation and may therefore play an important role in the pathogenesis of hepatic SOS. The purpose of this study was to identify an association between HPSE single nucleotide polymorphisms (SNPs) and hepatic SOS in children undergoing allogeneic HSCT. Methods:160 children (median age, 14 years) who underwent allogeneic bone marrow (n=91) or peripheral blood stem cell transplantation (n= 69) in a single center and their respective donors were genotyped of HPSE for rs4693608 and rs4364254 using TaqMan real-time polymerase chain reaction. The donor was HLA-matched unrelated in 63% of transplants and HLA-identical related in 25% of transplants. Conditioning regimen was myeloablative in all cases and based on busulfan in 46% of transplants or total body irradiation in 33% of transplants. Two forms of post-transplant immunosuppression predominated, cyclosporine A and methotrexate in 50% of transplants and cyclosporine A alone in 30% of transplants. Results:160 donor/patient pairs were analyzed. Cell samples from the patient were available in 155 cases and from the donor in 153 cases. Genotype AG of HPSE rs4693608 SNP was found in 82 patients (53%), AA in 49 patients (32%), and 24 patients were homozygous for GG (15%). Analysis of HPSE rs4364254 SNP revealed a similar distribution for TC (n=69, 44%) and TT (n=68, 44%) and a frequency of 18 patients (12%) for CC. Hepatic SOS was observed in 12 patients (8%). According to the modified Seattle criteria we identified ten patients with early-onset disease in the first 20 days after HSCT and two patients who developed hepatic SOS later on day +44 and day +83 after transplantation (late-onset SOS), respectively. If hepatic SOS was diagnosed all of our patients were treated with defibrotide as early as possible. Two patients (17%) developed severe hepatic SOS and died of multi-organ failure. The remaining ten patients with hepatic SOS (83%) could be successfully treated and survived. Patients with HPSE genotypes GG or AG of rs4693608 (G〉A) had a significantly reduced incidence of hepatic SOS on day 100 after HSCT compared to patients with genotype AA (5% vs. 14%, p=0.038). In addition, incidence of hepatic SOS in patients with genotype CC or CT of rs4364254 (C〉T) was significantly decreased in comparison to patients with genotype TT (2% vs. 15%, p=0.004). Interestingly, no patient with genotype CC developed hepatic SOS. Because both SNPs co-occur in vivo, we generated subsets: AA-TT, GG-CC and a group with remaining SNP combinations. We found significant differences between all three patient groups (p=0.035). Patients with AA-TT showed the highest incidence of hepatic SOS (17%), while hepatic SOS was not observed in patients with GG-CC (0%) and residual combinations were numerically in-between (5%). An impact caused by main patient and donor characteristics, established risk factors for hepatic SOS, and conditioning regimen could be excluded in multivariate analyses. Conclusions: This study provides the first evidence that HPSE polymorphisms are significant independent risk factors (p=0.030) for the development of hepatic SOS and should be evaluated in further trials. Disclosures No relevant conflicts of interest to declare.

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.