ALBERT

Beschreibung: The outcome of allogeneic hematopoietic cell transplantation is influenced by donor/recipient genetic disparity at loci both inside and outside the MHC on chromosome 6p. Although disparity at loci within the MHC is the most important risk factor for the development of severe GVHD, disparity at loci outside the MHC that encode minor histocompatibility (H) antigens can elicit GVHD and GVL activity in donor/recipient pairs who are otherwise genetically identical across the MHC. Minor H antigens are created by sequence and structural variations within the genome. The enormous variation that characterizes the human genome suggests that the total number of minor H loci is probably large and ensures that all donor/recipient pairs, despite selection for identity at the MHC, will be mismatched for many minor H antigens. In addition to mismatch at minor H loci, unrelated donor/recipient pairs exhibit genetic disparity at numerous loci within the MHC, particularly HLA-DP, despite selection for identity at HLA-A, -B, -C, and -DRB1. Disparity at HLA-DP exists in 80% of unrelated pairs and clearly influences the outcome of unrelated hematopoietic cell transplantation; the magnitude of this effect probably exceeds that associated with disparity at any locus outside the MHC.

Beschreibung: Abstract 2737 Synonymous SNPs may directly impact gene function through various translational or post-translational mechanisms. Further, such “silent” SNPs in the mutational hotspots of AML-associated genes have recently been reported to carry prognostic impact. We aimed to determine the prevalence, clinical associations, and prognostic significance of a known SNP in exon 4 of IDH1, near the location of the frequently-mutated R132 codon. Diagnostic marrow specimens from 253 pediatric AML patients (treated on the COG trial AAML03P1) and 274 adult AML patients (treated on SWOG trials S9031, S9333, or S9500) were analyzed for the presence of SNP rs11554137 via direct sequencing. In the pediatric cohort (median age 9.8 years), SNP rs11554137 was present in 27 of 253 (10.7%) patients. SNP+ pediatric patients did not differ significantly from wild-type patients in terms of sex, racial distribution (African American patients accounted for 23% of SNP+ patients vs. 14% of wild-type patients, P=0.24), bone marrow blast percentage, or age distribution, except in patients aged 0–2 years, who accounted for 44% of SNP+ patients vs. 23% of wild- type patients (P=0.013). Recurrent cytogenetic abnormalities occurred with similar frequencies in both the SNP+ and wild-type pediatric populations, as did FLT3/ITD, NPMc, and CEBPA mutations. Miscellaneous cytogenetic abnormalities accounted for 33% of SNP+ patients vs. 14% of wild-type patients, P=0.033. IDH1 SNP status had no prognostic impact on survival in the pediatric cohort, as SNP+ and wild-type patients had similar rates of five-year overall survival (OS, 76% vs. 63%, P=0.50), disease-free survival (DFS, 48% vs. 53%, P=0.97), and relapse rate (RR, 39% vs. 39%, P=0.94). In the adult cohort (median age 63 years), the IDH1 SNP was present in 30 of 274 (10.9%) patients. A slight female predominance for the SNP (63% vs. 37%, P=0.052) occurred among adult patients. The SNP was more prevalent in African American patients, who accounted for 30% of the SNP+ patients vs. 7% of wild-type patients, P=0.0046. SNP+ patients also had somewhat higher diagnostic bone marrow blast percentages (medians 80% vs. 70%, P=0.025). The normal karyotype subset accounted for similar proportions of SNP+ vs. wild-type patients (42% vs. 46%, P=0.83). Notably, SNP rs11554137 was not present in adult core-binding factor AML. Miscellaneous cytogenetic abnormalities were significantly more common in SNP+ patients (46% vs. 22%, P=0.022). SNP status was not significantly associated with FLT3/ITD status when all adult patients were considered (P=0.14). However, within the normal karyotype subset, FLT3/ITD was present in 90% of SNP+ patients vs. 59% of wild-type patients (P=0.0053). SNP+ patients had somewhat poorer 5 year OS (10% vs. 18%, hazard ratio [HR]=1.17) though this difference was not statistically significant (P=0.44). Among the 142 patients who achieved complete remission (CR), however, 5-year relapse-free survival (RFS) was significantly worse for SNP+ patients (0% vs. 25%, HR = 2.89, P=0.0014). Of the 14 SNP+ patients who achieved CR, 13 relapsed and the 14th patient died of sepsis in remission after 61 days. In multivariate analysis, after adjusting for the effects of age and cytogenetic group, SNP rs11554137 retained an independent prognostic effect (P=0.0062) regarding RFS. Notably, when FLT3/ITD status is included in multivariate analysis, SNP positivity loses independent prognostic significance (HR=1.72, P=0.18). Genome-wide expression profiling was performed on 134 pediatric AML specimens in whom IDH1 SNP status was known. By comparing SNP+ patients with wild-type patients, we derived a distinct gene-expression signature for patients with SNP rs11554137. Among the most upregulated probe sets in the SNP+ cohort were those representing PEX6 and NFYA, both of which interact with the TGF-beta/SMAD signaling network; the retinoid × receptor beta gene RXRB; and the FER gene, a tyrosine kinase critical to FLT3 signaling. The IDH1 SNP rs11554137 is present in approximately 11% of pediatric and adult AML patients, and gene expression profiling data suggests that leukemia in SNP+ patients may have unique biologic features. The SNP was an independent predictor of decreased RFS in adult AML in univariate analysis, but not in multivariate analysis when adjusting for FLT3/ITD status. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Background: Sclerotic graft-versus-host disease (GVHD) is a distinctive phenotype of chronic GVHD after allogeneic hematopoietic cell transplantation (HCT), characterized by fibrosis of the skin or fascia, and often associated with severe disability and morbidity. Sclerotic GVHD has clinical and histopathological similarities with systemic sclerosis, an autoimmune disease whose risk is influenced by genetic polymorphisms. Patients and methods: We identified 15 candidate single-nucleotide polymorphisms (SNPs) that have well-documented association with susceptibility to systemic sclerosis. We hypothesized that these SNPs would also have associations with risk of sclerotic GVHD if the two diseases have similar pathogenic mechanisms. We evaluated the candidate SNPs for association with the sclerotic phenotype in a cohort of 891 patients who were diagnosed with chronic GVHD and had recipient or donor samples available for genotyping. The candidate SNPs were located in BANK1, BLK, CD247, HLA-DRA, HLA-DQB1, HLA-DPA1, IL12RB2, IRF8, PTPN22, STAT4, TNFSF4, TNIP1 and TNPO3-IRF5. Genotyping assays used the Affymetrix 5.0 Human GeneChip and Illumina 1M Quad and Illumina 2.5M BeadArrays. Candidate SNPs not genotyped on the array were imputed using the 1000 Genomes Project Phase 1 SNPs as a reference panel. Cox regression models were used to examine associations of donor and recipient SNPs with risk of sclerotic GVHD. Each SNP was assessed for allelic (additive) and genotypic (dominant and recessive) models. A two-sided P-value of

Beschreibung: Background: Cytomegalovirus (CMV) infection is one of the most common complications after hematopoietic cell transplantation (HCT). Regardless of preemptive therapy for CMV infection, CMV disease still occurs in approximately 10% of seropositive HCT recipients, resulting in high mortality. A number of studies have reported the association between CMV infection or disease and single-nucleotide polymorphisms (SNPs), however the results have not been validated in independent cohorts. We performed a candidate gene validation study of previously reported SNPs using a large genome-wide association study (GWAS) cohort. Patients and methods: This GWAS cohort included donor-recipient (D/R) pairs who received the first allogeneic transplantation between 1990 and 2011 at the FHCRC. Of an overall cohort of 4855 D/R pairs, 2193 Caucasian CMV seropositive recipients and their donors (seropositve or seronegative) were analyzed. Two CMV phenotypes were analyzed: any infection within 100 days (only among 1585 recipients transplanted after 1995, antigenemia-based preemptive therapy administered) and CMV disease within 1 year after HCT. Published SNPs associated with CMV infection or disease were collected by a PubMed search using the keywords cytomegalovirus' and SNP' or polymorphism.' A p-value of less than 0.05 was required for validation. Genomic DNA samples from donors and recipients were analyzed using the Affymetrix GeneChip Genome-Wide Human SNP Array 5.0 (for one third of subjects) and Ilummina OmniExpress Beadchip Array (for the remainder). When the candidate SNPs were not included in the arrays, we imputed genotypes using IMPUTE software. Among genotyped and imputed SNPs, only SNPs with a minor allele frequency ≥0.05, call rate ≥90%, and Hardy-Weinberg equilibrium ≥0.0001 were analyzed. Donor serostatus, age, year of transplantation, donor type, HLA disparity, and intensity of conditioning regimen were used as adjustment factors as appropriate. Results: Among the 2193 HCT recipients in our cohort, 1009/1585 (64%) had CMV infection and 354/2193 (16%) had CMV disease. We identified 52 SNPs in 31 publications that reported significant associations between SNPs and CMV infection or disease (median number of subjects in these studies, 215; range, 37-1770). A total of 32 SNPs were available for the analysis. For CMV infection, six SNPs in four genes (TLR9, MCP1, CCR5, and CD209) in recipient or donor met our validation criteria (Table). For all SNPs but one, however, the direction of the association was opposite to that reported in the previous studies. We validated one SNP association (TLR9/rs5743836) in the recipient exactly as originally reported (adjusted HR, 1.16; 95% CI, 1.00-1.34, p=0.043). The donor genotype at this SNP was also significantly associated with CMV infection (adjusted HR, 1.47; 95% CI, 1.02-2.14, p=0.041) (Table), but an association with donor genotype was not assessed in the original publication. As for CMV disease, a different TLR9 SNP (rs352140) in donors was identified as significant (adjusted HR, 0.74; 95% CI, 1.02-2.14, p=0.029), although the ethnicity is different from the reported cohort (Table). None of the candidate SNPs in the recipient genome were associated with CMV disease. Conclusions: Of 32 SNPs that were previously reported to be associated with either CMV infection or disease, we were only able to validate two, both in TLR9. Most of the previously reported SNP associations for CMV infection or disease were not replicated in this large GWAS cohort. The inability to replicate these SNP associations does not necessarily negate the original discoveries, since many factors, including the sample size, could account for the different results. Our findings suggest that results of SNP association studies should be confirmed in multiple independent large cohorts and the biological mechanisms underlying the associations should be examined before clinical application. Unbiased discovery approaches are needed to determine the genetic associations of CMV infection and disease. Table 1 Table 1. Disclosures Boeckh: Chimerix Inc.: Consultancy, Research Funding; Viropharma Inc.: Research Funding; Genentech/Roche: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Gilead Sciences: Consultancy, Research Funding; Clinigen: Consultancy.

Beschreibung: Candidate genetic associations with acute GVHD (aGVHD) were evaluated with the use of genotyped and imputed single-nucleotide polymorphism data from genome-wide scans of 1298 allogeneic hematopoietic cell transplantation (HCT) donors and recipients. Of 40 previously reported candidate SNPs, 6 were successfully genotyped, and 10 were imputed and passed criteria for analysis. Patient and donor genotypes were assessed for association with grades IIb-IV and III-IV aGVHD, stratified by donor type, in univariate and multivariate allelic, recessive and dominant models. Use of imputed genotypes to replicate previous IL10 associations was validated. Similar to previous publications, the IL6 donor genotype for rs1800795 was associated with a 20%-50% increased risk for grade IIb-IV aGVHD after unrelated HCT in the allelic (adjusted P = .011) and recessive (adjusted P = .0013) models. The donor genotype was associated with a 60% increase in risk for grade III-IV aGVHD after related HCT (adjusted P = .028). Other associations were found for IL2, CTLA4, HPSE, and MTHFR but were inconsistent with original publications. These results illustrate the advantages of using imputed single-nucleotide polymorphism data in genetic analyses and demonstrate the importance of validation in genetic association studies.

Beschreibung: Abstract 234 Therapy-related myelodysplasia or acute myeloid leukemia (t-MDS/AML) is a lethal complication of cancer treatment. Study of t-MDS/AML offers a unique opportunity to understand leukemogenesis since known genotoxic exposures can be temporally and causally related to genetic changes associated with development of leukemia. Although development of t-MDS/AML is associated with known genotoxic exposures, its pathogenesis is not well understood, and methods to predict risk of development of t-MDS/AML in individual cancer survivors are not available. To better understand the pathogenetic mechanisms underlying development of t-MDS/AML we performed microarray analysis of gene expression in patients who developed t-MDS/AML after autologous hematopoietic cell transplantation (aHCT) for Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL) and controls that did not develop t-MDS/AML after aHCT. Peripheral blood stem cell (PBSC) samples obtained pre-aHCT from patients who subsequently developed t-MDS post-aHCT (cases) and controls matched for primary diagnosis, age, race/ethnicity, and time since aHCT were studied. In a training set of 18 t-MDS/AML cases and 37 controls, CD34+ cells were selected from PBSC samples using flow cytometry, and gene expression evaluated using Affymetrix HG U133 plus 2.0 Arrays. Differences in gene expression in CD34+ cells from cases and controls were analyzed using conditional logistic model. Significant differences in gene expression were seen in PBSC obtained pre-aHCT from patients who later developed t-MDS/AML compared to controls.(Blood, 2009; 114: 677) PBSC obtained pre-aHCT from patients who subsequently t-MDS/AML after aHCT showed significant downregulation of gene sets related to mitochondria and oxidative phosphorylation, ribosomes, aminoacyl-tRNA biosynthesis, amino acid metabolism, cell cycle regulation, and hematopoietic differentiation. G-protein coupled receptors, hematopoietic regulation, and cell adhesion related genes were upregulated in PBSC from cases. There was reduced expression of genes with binding motifs for the transcription factor NRF2, which regulates oxidative stress and drug detoxification. We then sought to identify a smaller PBSC gene signature that would identify NHL and HL patients at the pre-aHCT timepoint at high risk for developing t- MDS/AML after aHCT (Figure 1). A cross-validated 38-gene classifier was derived from the training set using prediction analysis of microarray (PAM). This gene classifier was applied to an independent test set of PBSC obtained pre-HCT from 16 patients who developed t-MDS/AML after aHCT for NHL or HL, and 20 matched controls that did not develop t-MDS/AML. Application of the 38-gene signature to the test set correctly classified 19 of the 20 subjects (95%) who did not subsequently develop t-MDS/AML, and 14 of the 16 subjects (87.5%) who did develop t-MDS/AML, with significant correlation between predicted and true disease status (P