Originally published as JCO Early Release 10.1200/JCO.2007.12.1897 on August 27 2007

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Single Nucleotide Polymorphisms in the NOD2/CARD15 Gene Are Associated With an Increased Risk of Relapse and Death for Patients With Acute Leukemia After Hematopoietic Stem-Cell Transplantation With Unrelated Donors

Neema P. Mayor, Bronwen E. Shaw, Derralynn A. Hughes, Hazael Maldonado-Torres, J. Alejandro Madrigal, Satish Keshav, Steven G.E. Marsh

From the Anthony Nolan Research Institute, Royal Free Hospital; Department of Haematology, Royal Free and University College London School of Medicine, Royal Free Campus; Royal Marsden Hospital; Department of Medicine, Royal Free and University College London School of Medicine, Royal Free Campus, London, United Kingdom

Address reprint requests to Steven G.E. Marsh, PhD, Anthony Nolan Research Institute, Royal Free Hospital, Pond St, London NW3 2QG, United Kingdom; e-mail: marsh{at}ebi.ac.uk

	ABSTRACT

TOP ABSTRACT INTRODUCTION Patients and Methods RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Purpose Hematopoietic stem cell transplantation (HSCT) is an important option in the management of acute leukemia, but the risk of disease relapse and death remains appreciable. Recent studies have suggested that nucleotide-binding oligomerization domain 2 (NOD2)/caspase recruitment domain 15 (CARD15) gene single nucleotide polymorphisms (SNPs), implicated in innate immunity and Crohn's disease, may also affect immune function post-HSCT.

Patients and Methods NOD2/CARD15 genotypes were analyzed in 196 patients diagnosed with acute leukemia and their unrelated donors. The pairs are part of a previously well-characterized cohort with a median follow-up of 2.2 years (range, 0.42 to 6.61 years). T-cell depletion was used in 83% of pairs.

Results NOD2/CARD15 SNPs were associated with a reduction in overall survival (44% v 22%; log-rank P = .0087) due to an increase in disease relapse (32% v 54%; Gray's test P = .001) as compared with wild-type pairs. In multivariate analyses, the two most significant factors impacting outcome were transplantation in relapse and the presence of SNPs. The incidence of acute graft-versus-host disease was low and there was no significant difference due to the presence of SNPs.

Conclusion These data indicate an unrecognized role for the NOD2/CARD15 gene in unrelated donor HSCT for acute leukemia. The increased risk of disease relapse suggests that the wild-type gene product may contribute to a graft-versus-leukemia effect. These data suggest that NOD2/CARD15 genotyping before transplantation may contribute to prognosis and influence clinical management.

	INTRODUCTION

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Hematopoietic stem-cell transplantation (HSCT) is currently the only curative treatment for certain individuals diagnosed with acute leukemia who are considered to be high risk.¹ While the use of unrelated donors is well accepted, it is known that some complications may be increased.² Although HLA matching improves outcomes, recipients remain susceptible to life-threatening post-transplantation complications, including disease relapse and the development of graft-versus-host disease (GVHD).³ Therefore, defining variables that predispose to either of these events is of critical importance.

Several recent studies have described a significant correlation between transplantation outcome and three single nucleotide polymorphisms (SNPs) in the caspase recruitment domain 15 (CARD15) gene (also known as the nucleotide-binding oligomerisation domain 2 [NOD2] gene).^4-7 Holler et al^5,6 associated increases in the incidence and severity of acute GVHD (aGVHD) and a reduction in overall survival (OS) with the presence of SNPs in two separate patient cohorts. A subsequent study also reported an association with these SNPs and the incidence of aGVHD, but failed to find any significant correlations with OS.⁴ Conversely, Granell et al⁷ have demonstrated that in a T-cell depleted setting, there was no impact of SNPs on aGVHD but a significant effect was reported on disease-free survival (DFS). The majority of patients reported received transplants from sibling donors.

The NOD2/CARD15 gene encodes the NOD2 protein, a member of a newly defined family of intracellular proteins^8,9 that are critical mediators of inflammation and participate in the formation of a protein complex termed the inflammasome, which has widespread effects on innate immunity, cytokine secretion, cell survival, and apoptosis.^10,11 NOD2 is expressed in circulating monocytes, in Paneth cells located in the epithelial crypts of the small intestine, and has also been identified in dendritic cells.^8,12 The cellular function of NOD2 is still uncertain and its physiological role is as yet undefined. SNPs within the NOD2/CARD15 gene have, however, been associated with functional defects^13-15 and with the occurrence of inflammatory bowel disorders.¹⁶

We report herein the findings of an investigation into the impact of NOD2/CARD15 gene SNPs on the outcomes of acute leukemia HSCT pairs using an unrelated donor. Our data show that pairs with NOD2/CARD15 gene SNPs have a highly significant reduction in OS when compared with wild-type (WT) pairs. The reduced survival is attributable to a significant increase in disease relapse. The data also show that NOD2/CARD15 SNPs may offer a protective effect toward aGVHD. They suggest that NOD2 plays an important role in protection against recurrence of leukemia after HSCT.

	Patients and Methods

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Patients
NOD2/CARD15 genotyping was performed on 196 recipients and their Anthony Nolan Trust volunteer unrelated donors, who underwent an HSCT at one of 25 transplant centers in the United Kingdom between 1996 and 2003. These individuals formed part of a well-characterized cohort for whom high resolution HLA typing and long-term clinical data had been collated. Diagnoses were acute myeloid leukemia (AML; 98 of 196; 50%) and acute lymphoblastic leukemia (ALL; 98 of 196; 50%; Table 1). Recipients were defined as having early-stage disease if in first complete remission. All others were designated as late-stage disease. The majority of recipients had myeloablative conditioning (78%). Eighty-three percent of recipients had T-cell depletion (TCD) included in their pretransplantation conditioning, with in vivo alemtuzumab (Campath; Schering Health Care Ltd, West Sussex, United Kingdom) being the preferred method. Bone marrow was used as a source of stem cells in 81% of transplantations with the remaining 19% using peripheral blood stem cells as a graft. Two forms of post-transplantation immunosuppression predominated, cyclosporine A combined with methotrexate (47%) and cyclosporine A alone (31%).

HLA Typing
Recipients and donors were previously HLA typed to an allele-level resolution, with 64% (125 of 196) being matched at HLA-A, -B, -C, -DRB1 and -DQB1 (ie, 10 of 10 match). Of these, 16% (20 of 125) were also matched at HLA-DPB1 (ie, 12 of 12 match; Table 1).

NOD2/CARD15 Genotyping
Donor and recipient genomic DNA was extracted from whole blood using the salting out technique.¹⁷ Three SNPs were analyzed, namely, SNP 8 (p.R702W), SNP 12 (p.G908R) and SNP 13 (p.L1007fs; numbering according to European Molecular Biology Laboratory accession number AJ303140; Appendix Table A1, online only). All genotyping was carried out by polymerase chain reaction (PCR) using sequence specific primers (SSP). DNA samples with a known NOD2/CARD15 genotype determined by the Taqman (Applied Biosystems, Foster City, CA) protocol¹⁶ were used for optimizing PCR-SSP experiments. The primer sequences for exon 4 genotyping were a sense WT primer 5'-CATCTGAGAAGGCCCTGCTCC, a sense SNP 8 primer 5'-CATCTGAGAAGGCCCTGCTCT, each of which were used in an individual reaction with the antisense primer 5'-GTGCCCAACATTCAGGCCAC. For exon 8, the common sense primer 5'-GATGGAGGCAGGTCCACTTTGC was used with either the antisense WT 5'-TCGTCACCCACTCTGTTGCC or SNP 12 5'-TCGTCACCCACTCTGTTGCG. Exon 11 WT and SNP 13 analysis were performed simultaneously in a multiplex reaction. The primers were adapted from Ogura et al.¹⁸ Briefly, the long sense primer 5'-TGGTACTGAGCCTTTGTTGATGAGCTC forms a product with the long antisense primer 5'-CATTCTTCAACCACATCCCCATTCCT. These in turn form additional products with either the antisense WT primer 5'-CAGAAGCCCTCCTGCAGGCCCT or the sense SNP 13 primer 5'-CGCGTGTCATTCCTTTCATGGGGC, respectively.

A proportion of samples were sequenced to confirm their genotype and to ensure the validity of the PCR-SSP system. All PCR-SSP results correlated with those obtained by sequencing.

Data Storage and Statistical Methods
Data were compiled and maintained on a Filemaker Pro database (Filemaker Inc, Santa Clara, CA). Analysis was performed using SPSS version 11.4 (SPSS Inc, Chicago, IL) and R version 2.2.1 (http://www.r-project.org). The probabilities of OS and DFS were compared using log-rank statistics and calculated by the Kaplan-Meier method as there were no competing events. The probabilities of transplant related mortality (TRM), disease relapse, and chronic GVHD (cGVHD) were compared using Gray's test and analyzed using the cumulative incidence method. Competing events included in the cumulative incidence analysis for TRM, disease relapse, and cGVHD were relapse, death without relapse, and death without cGVHD, respectively. Of the 196 pairs in the study, four pairs had to be omitted from the TRM and disease relapse analysis due to a lack of data. The ² test was used to analyze data on aGVHD and the achievement of neutrophil engraftment. aGVHD was defined as occurring up to 100 days post-transplantation. cGVHD was defined as occurring after 100 days post-transplantation.

Multivariate analysis was performed using Cox-regression analysis. Factors found to be significant in univariate analyses ( .2) were included in the model. These included recipient age, disease stage at transplantation, and HLA-DPB1 matching status in all three outcomes, donor age, cytomegalovirus serostatus of the recipient in OS and DFS, and stem cell source in disease relapse. For both univariate and multivariate analysis, P values were two sided and outcomes were considered to be significant with an level of .05. A trend was identified when alpha levels were = .05 to .1.

	RESULTS

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NOD2/CARD15 SNP Frequencies
Three NOD2/CARD15 variants (SNPs 8, 12, and 13) were analyzed for 196 unrelated donor–HSCT pairs. The occurrence of any SNP in a recipient of an unrelated donor HSCT was found to be 13% and 17.3% in donors, resulting in an overall frequency of 27.6% (Table 2). The frequency of the individual SNPs were comparable in donors and recipients (Appendix Table A2, online only) and was similar to that previously published.^4,6

View larger version (12K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. The presence of nucleotide-binding oligomerisation domain 2 (NOD2)/caspase recruitment domain 15 (CARD15) single nucleotide polymorphisms (SNPs) is significantly associated with inferior transplant outcomes in acute leukemia (AL). In AL, (A) overall survival (OS) and (B) disease-free survival (DFS), analyzed using Kaplan-Meier, are reduced in those pairs with SNPs. (C) Disease relapse, analyzed using cumulative incidence, is significantly increased. WT, wild type.

View larger version (21K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. The presence of nucleotide-binding oligomerisation domain 2 (NOD2)/caspase recruitment domain 15 (CARD15) polymorphisms has a significant impact on the outcome in patients who received transplantation for acute lymphoblastic leukemia (ALL) but not acute myeloid leukemia (AML). In ALL, NOD2/CARD15 polymorphisms are associated with worse (A) overall survival (OS), (C) disease-free survival (DFS), and (E) with an increase in disease relapse. Conversely, in AML, NOD2/CARD15 polymorphisms result in a trend for (D) reduced DFS, but has no significant impact on (B) OS or (F) disease relapse. SNP, single nucleotide polymorphism; WT, wild type.

Further analyses were performed to establish whether the effect of recipient or donor genotype was individually significant in transplantation outcome (Fig 3). The presence of a NOD2/CARD15 SNP in the recipient genotype resulted in a reduced OS (log-rank, P = .01; Fig 3A) and an increased incidence of disease relapse (Gray test, P = .005; Fig 3C) when compared with those with a WT genotype. There was no significant impact of donor genotype on either of these variables (Figs 3B and 3D). Matching for NOD2/CARD15 genotype in a graft-versus-host direction also had similar effects on OS and relapse (P = .05 and P < .001, respectively) while matching in a host-versus-graft direction resulted in no significant differences (data not shown).

View larger version (16K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 3. Single nucleotide polymorphisms (SNPs) of the nucleotide-binding oligomerisation domain 2 (NOD2)/caspase recruitment domain 15 (CARD15) gene are significantly associated with inferior transplant outcomes when in the recipient genotype. In acute leukemia, the presence of NOD2/CARD15 SNPs in the recipient genotype is significantly associated with (A) decreased overall survival (OS; analyzed using Kaplan-Meier) and (C) increased disease relapse (analyzed using cumulative incidence). There is no significant effect of donor genotype on (B) OS or (D) disease relapse. SNP, single nucleotide polymorphism.

	DISCUSSION

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It has been shown by this study, and indeed by numerous others, that SNPs of the NOD2/CARD15 gene are implicated in immunological phenomena that affect either the onset or progression of malignant diseases.^20,21 The impact of the SNPs on relapse post-allograft is marked, but the exact mechanism of this effect is currently unknown. A second finding, although less marked, is that the presence of a SNP leads to a reduction in the occurrence of aGVHD. It is therefore possible that recipients with NOD2/CARD15 SNPs are incapable of mounting an efficient graft-versus-leukemia or GVHD response, resulting in increased disease relapse and protection from aGVHD. The graft-versus-leukemia effect is a complex series of events that involve the generation of tumor-specific cells targeting leukemic cells post-HSCT. The cells involved in this process are thought to be of T-cell origin although recent evidence points to a role for natural killer cells²² and even possibly natural killer T cells.²³ It is thought to be caused by an immune reaction to genetic disparity, not unlike GVHD. In fact, certain theories propose that the two events stem from a common phenomenon, namely the cytokine storm,^24-26 an extreme increase in the production of cytokines that occurs during the early post-transplantation stages.

With this in mind, we postulate three possible mechanisms that may explain these data. Firstly, recent data have suggested that NOD2/CARD15 gene SNPs prevent its expression on the surface of epithelial cells.²⁷ This suggests that WT NOD2 is expressed on the surface of cells where cytosolic expression has been detected and also potentially on further, as yet unidentified, cell types. It is possible that the failure of leukemic cells to express NOD2 in recipients with NOD2/CARD15 variant genotype may lead to their evasion of the immune system, resulting in disease relapse. A second, indirect mechanism, is the inability of the NOD2 variant protein to initiate cytokine production. NOD2 is known to function as a regulator of cytokine production and a mediator of pro-inflammatory responses on recognition of the bacterial ligand muramyl dipeptide. While the effect of NOD2 SNPs are not yet known, numerous theories have been proposed that appear to argue for a loss or gain of function mechanism.^28-30 It is generally accepted that the NOD2/CARD15 SNPs downregulate expression of cytokines via the nuclear factor B pathway (ie, cause a loss of function).³¹ However, there is much controversy over what impact the SNPs have on the synergistic relationship with toll-like receptor 2,^32,33 and more specifically what subsequent impact this has on the production of interleukin-12 (IL-12). Recent studies have suggested SNPs of the NOD2/CARD15 genes actually result in either reduced or depleted levels of IL-12.¹³ Interestingly, it has been reported that low levels of IL-12 are associated with an increase in disease relapse post-HSCT,³⁴ but without an increased incidence of aGVHD.^34,35 A third potential mechanism is that NOD2 acts as a minor histocompatibility antigen. We have been able to demonstrate that the impact of NOD2/CARD15 SNPs on transplantation outcome causes a more potent effect in a graft-versus-host direction. This may suggest that the mutated NOD2 protein initiates an immune response in the event of nonrecognition of host antigens.

The reduced incidence of both graft-versus-leukemia and GVHD responses in pairs with NOD2/CARD15 SNPs appear to be due predominantly to the recipient's genotype. The noticeable impact of recipient genotype suggests that expression of NOD2 protein in long-lived tissue cells that may survive conditioning chemotherapy, such as tissue macrophages, dendritic cells, and Paneth cells, is of critical importance particularly when other elements of the adaptive and innate immune systems have been compromised or inactivated by conditioning regimens and/or post-transplantation immunosuppression. These findings are further substantiated by evidence that host dendritic cells, which are known to express NOD2, are capable of causing aGVHD post-HSCT.³⁶ NOD2/CARD15 SNPs may directly affect antigen presentation on leukemic cells, which in turn would reduce graft-versus-leukemia and graft-versus-host responses. The near universal use of TCD, specifically in vivo alemtuzumab, in this cohort explains the low incidence of GVHD seen, and hence may mask a profound effect due to the NOD2/CARD15 SNPs.

We analyzed the data in AML and ALL separately. Interestingly, all of the results were highly significant in ALL but showed only trends in AML. A possible explanation may be that the number of AML cases in the study are insufficient to uncover an effect. Alternately, this may suggest that the mechanism and/or threshold for the graft-versus-leukemia effect differs between these two diseases. There were a greater number of transplantations performed in a late stage of disease in the ALL subgroup than in AML, 67% and 50%, respectively. This suggests that the effect of NOD2/CARD15 SNPs may be more prominent in high-risk disease. Cytogenetics are known to impact on transplantation outcome.³⁷ Unfortunately, cytogenetic data were not available in this cohort and future studies should aim to collect this data.

The data presented is in contrast to that published previously.^4-7 The reasons for this may be the numerous differences in the demographics of the cohorts, particularly that this study includes only unrelated donor and a high proportion of patients receiving bone marrow as the source of stem cells, both of which have been associated with a worse outcome in high-risk recipients.³⁸ Analysis was performed in order to establish whether there was a biased effect of SNPs in the different risk groups of recipients (ie, their stage of disease at transplant, bone marrow v peripheral blood stem cell). The presence of a SNP resulted in similar significant associations in all of these subgroups (data not shown). Another difference in this cohort was the high proportion of patients receiving TCD in conditioning regimens. This may in part explain the lack of significance between the presence of SNPs and aGVHD as the use of TCD is well known to result in significant reductions in GVHD. This theory is substantiated by data published by Granell et al⁷ who also found no correlation between SNPs and the incidence of clinically significant aGVHD in T-cell depleted sibling transplantations. In concordance with the data presented, this group also noted a significant reduction in DFS in pairs with NOD2/CARD15 SNPs.

Recent studies have highlighted the importance of gastrointestinal decontamination protocols in the prediction of clinically significant GVHD and TRM attributable to NOD2/CARD15 SNPs.^4,5 The clinical data evaluated for this study did not include details on decontamination methods and therefore we are unable to make any correlations between them and the results observed. However, as mentioned previously, both this study and that by Granel et al⁷ both reported a lack of association between NOD2/CARD15 SNPs and GVHD, which was attributed to a high use of TCD. It is possible that any association with gastrointestinal decontamination may have been masked again by the use of TCD.

The data presented confirm that the NOD2 protein is critically involved in the biology of transplantation. Further studies are required to ascertain whether the findings of this study can be extended to other disease groups or non-TCD transplantation protocols. In addition, recently identified SNPs^39,40 will require further studies to identify their relevance. We suggest that the prospective genotyping of recipients and donors will have a beneficial effect on transplantation outcome, not only reducing disease relapse, but also improving the chance of survival. Thus, genotyping may play a critical role in selecting donors and in the planning and administering conditioning regimens and post-transplantation immunosuppression.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The author(s) indicated no potential conflicts of interest.

	AUTHOR CONTRIBUTIONS

TOP ABSTRACT INTRODUCTION Patients and Methods RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Conception and design: Neema P. Mayor, Bronwen E. Shaw, Derralynn A. Hughes, J. Alejandro Madrigal, Satish Keshav, Steven G.E. Marsh

Provision of study materials or patients: Neema P. Mayor, Bronwen E. Shaw, J. Alejandro Madrigal, Satish Keshav, Steven G.E. Marsh

Collection and assembly of data: Neema P. Mayor, Bronwen E. Shaw, Hazael Maldonado-Torres, J. Alejandro Madrigal, Steven G.E. Marsh

Data analysis and interpretation: Neema P. Mayor, Bronwen E. Shaw, Derralynn A. Hughes, Hazael Maldonado-Torres, J. Alejandro Madrigal, Satish Keshav, Steven G.E. Marsh

Manuscript writing: Neema P. Mayor, Bronwen E. Shaw, Derralynn A. Hughes, Hazael Maldonado-Torres, J. Alejandro Madrigal, Satish Keshav, Steven G.E. Marsh

Final approval of manuscript: Neema P. Mayor, Bronwen E. Shaw, Derralynn A. Hughes, Hazael Maldonado-Torres, J. Alejandro Madrigal, Satish Keshav, Steven G.E. Marsh

	Appendix

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	ACKNOWLEDGMENTS

 
We thank United Kingdom transplant centers, EBMT Immunology Working Party, John Goldman and Stephen Mackinnon, Richard Szydlo, Lamya Saeed, Chrissy Roberts, and Marjorie Lawton.

	NOTES

 
published online ahead of print at www.jco.org on August 27, 2007.

Supported by an EU-FP6 project Allostem 503319 and by the Medical Research Council United Kingdom (S.K.).

Presented in part at the Annual Meeting of the American Society of Hematology, December 9-12, 2006; BMT Tandem Meetings, February 8-12, 2007; European Group for Blood and Marrow Transplantation Meeting, March 25-28, 2007; and the European Federation of Immunogenetics, May 5-8, 2007.

S.K. and S.G.E.M. contributed equally to this article.

Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted April 21, 2007; accepted June 27, 2007.

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