Estimation of NOD2/CARD15 Haplotypes and Their Impact on the Outcome of Unrelated Donor Hematopoietic Stem Cell Transplantation for Acute Leukemia
Authors: Mayor Neema P , Shaw Bronwen E. , Maldonado-Torres Hazael , Cooke Louise , Madrigal J. Alejandro , Marsh Steven GE 
Published: October 2019

Abstract
Abstract 2372
Three polymorphisms of the innate immune response gene NOD2/CARD15 (SNPs 8, 12 and 13) have been correlated with detrimental outcomes post stem cell transplantation. We have shown that the presence of any of the three SNPs in the combined genotype of the pair (that is in the patient, the donor or in both) was associated with significant increases in disease relapse and mortality in a cohort of Acute Leukemia (AL) Unrelated Donor (UD) Hematopoietic Stem Cell Transplant (HSCT) pairs. Several other published studies have described either a detrimental effect or no effect of SNPs 8, 12 and 13 on HSCT outcome in different cohorts. We hypothesised that, given the differing results from various studies, SNPs 8, 12 and 13 were actually only genetic markers for clinical outcome post HSCT and that the functionally relevant polymorphism(s) may be located elsewhere within the gene and may display Linkage Disequilibrium (LD) with the known SNPs in some populations. An exonic sequencing protocol for the entire NOD2/CARD15 gene was established and used to screen a cohort of AL UD-HSCT pairs. A total of 22 NOD2/CARD15 SNPs were identified with frequencies ranging from 1 – 43%. No significant differences in SNP frequency were noted between recipients and donors. Three of the polymorphisms were novel, although in each case the SNP was present in a single individual. In order to demonstrate the existence of LD between the polymorphisms and thus prove that SNPs 8, 12 and 13 were inherited on a single chromosome in combination with one or more other polymorphisms, haplotype estimation was performed on the cohort. Haplotypes were estimated using an in house program Cactus, which employs a gene counting method and the Expectation Maximisation algorithm. Several models were tested which included different combinations of NOD2/CARD15 SNPs. Of particular interest were the results of a model that included data for five polymorphic loci, SNPs 5, 6, 8, 12 and 13. A total of 20 haplotypes were predicted with this model, 13 of which were observed in the cohort. Three haplotypes (WT (Haplotype (H) 1); SNP 5 and 6 positive (H2); and SNP 5, 6 and 8 positive (H3)) comprised greater than 96% of all haplotypes identified (frequencies 70.8%, 21.1% and 4.6% respectively) and were used to determine the effects on HSCT outcome in a total of 238 AL UD-HSCT pairs. Transplants occurred between 1996 and 2005 at UK HSCT centres. Diagnoses were ALL (45%) and AML (55%). 68% of the cohort were 10/10 HLA matched, 10% were also matched at HLA-DPB1. Myeloablative conditioning regimens were used in 74% of transplants; T-cell depletion was included in 81% of protocols usually with in vivo Alemtuzumab. Bone marrow was used in 73% of transplants, 27% used peripheral blood stem cells. Two forms of post-transplant immunosuppression predominated, Cyclosporine A alone (36%) or with Methotrexate (46%). The probability of OS was 28% at 8 years. The estimated probability of OS at one year was higher in recipients with H1/H1 (WT) and H2/H2 (SNP 5 and 6 positive) haplotypes (55% and 67% respectively) as compared to those with H1/H2 (41%) or H1/H3 (H3, SNPs 5, 6 and 8 positive; 29%) haplotypes (p=0.016). The 8-year probability of Event-Free Survival (EFS) was 28%. Recipient NOD2/CARD15 haplotypes also significantly affected EFS (4 years: H1/H1 46%, H1/H2 33%, H2/H2 60%, H1/H3 29%; p=0.020). The 8-year probability of disease relapse was 41%. Significant differences in the risk of disease relapse were seen with individual donor NOD2/CARD15 haplotype combinations (1 year: H1/H1 25%, H1/H2 32%, H2/H2 60%, H1/H3 50%; p=0.013). There were no significant affects of recipient or donor NOD2/CARD15 haplotype combinations on non-relapse mortality, acute Graft-versus-Host Disease (GvHD) or chronic GvHD. The observed effects on OS and relapse persisted after multivariate analysis (OS: Relative Risk (RR) 1.54, p=0.016; EFS: RR 1.41, p=0.052; Relapse: RR 3.36, p=0.023). We conclude that distinct NOD2/CARD15 haplotypes exist and that they significantly affect UD-HSCT outcome. Significant differences in outcome were seen compared to the analysis of SNPs 8, 12 and 13 alone showing the advantage of combining NOD2/CARD15 haplotypes over individual SNPs. Importantly, the inclusion of additional polymorphic positions meant that 60% of individuals previously thought to be WT actually had at least one SNP. We suggest that the effect of NOD2/CARD15 haplotypes may be considerable and must be confirmed in other studies.

Disclosures:
No relevant conflicts of interest to declare.