Biology of Blood and Marrow Transplantation 12:19-27 (2006) 䊚 2006 American Society for Blood and Marrow Transplantation 1083-8791/06/1201-0105$32.00/0 doi:10.1016/j.bbmt.2005.09.014
Genomic Polymorphism and Allogeneic Hematopoietic Transplantation Outcome Charles G. Mullighan,1 Effie W. Petersdorf
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1
Department of Pathology, St. Jude Children’s Research Hospital, Memphis, Tennessee; 2Department of Medicine, University of Washington, and Fred Hutchinson Cancer Research Center, Seattle, Washington Correspondence and reprint requests: Charles G. Mullighan, MBBS(Hons), MSc, MD, Department of Pathology, St. Jude Children’s Research Hospital, 332 N. Lauderdale, MS #342, Memphis, TN 38105 (e-mail: charles.
[email protected]). Received September 16, 2005; accepted September 29, 2005
ABSTRACT Polymorphisms in genes that encode antigen-presenting molecules, antigen receptors, and immune mediators are crucial determinants of the risk of complications after allogeneic hematopoietic cell transplantation (allo-HCT). Matching for HLA alleles remains the cornerstone of donor selection, and recent studies are refining our understanding and use of HLA typing in allo-HCT. High-resolution allelic HLA matching generally improves transplant outcome but may limit the donor pool and delay transplantation. Allelic mismatches may be permissible in certain circumstances without compromising outcome. There is growing interest in the role of natural killer (NK) cell–mediated immunity in allo-HCT. NK cells express an array of activating and inhibitory killer cell immunoglobulin-like receptors (KIR), and NK cell activation is negatively regulated by KIR interaction with HLA class I molecules. In haploidentical transplants, NK cell alloreactivity in the graft-versus-host direction can be predicted by the HLA class I and KIR genotypes of donor and recipient and has been associated with potent graft-versus-leukemic effects and low rates of graft-versus-host disease. KIR genotype and expression may influence transplantation outcomes in both HLA-matched and -mismatched transplants. Graft-versus-host disease and major infection remain problematic despite HLA matching, and there is mounting evidence that polymorphisms in non-HLA immune mediators and host defense genes influence the risk of these complications. The importance of non-HLA genomics in nonmyeloablative transplants is poorly understood and is under investigation. These findings suggest that tissue typing for allo-HCT is entering an exciting era in which both HLA and non-HLA genomic data may be used in a more sophisticated fashion to select donors, stratify risk, identify novel therapeutic targets, and ultimately improve outcome for allo-HCT recipients. © 2006 American Society for Blood and Marrow Transplantation
KEY WORDS Allogeneic hematopoietic transplantation ● Graft-versus-host disease killer cell ● KIR ● HLA ● Polymorphism
IMMUNOGENETICS OF UNRELATED HEMATOPOIETIC CELL TRANSPLANTATION Understanding the genetic basis of graft-versushost (GVH) and host-versus-graft allorecognition in unrelated donor allogeneic hematopoietic cell transplantation (allo-HCT) requires a full understanding of HLA genes and their interactions. The major histocompatibility complex is the most comprehensively studied multimegabase region of the human genome. Much of our knowledge of the immunogenetic basis of alloreactivity comes from long-standing clinical ex-
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perience in the use of myeloablative conditioning regimens and donor bone marrow as the grafting source. Under these circumstances, donor T-cell recognition of host HLA can give rise to graft-versus-host disease (GVHD), and host recognition of donor HLA may increase the risk of graft failure. Methodologic advances in tissue typing have revolutionized the HLA field and applications to transplantation. These data demonstrate that (1) high-resolution (allelic) DNA typing methods can uncover functionally relevant transplant determinants, and, therefore, these methods are required for donor evaluation; (2) the risks of 19
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graft rejection, GVHD, and mortality are increased with increasing numbers of HLA mismatches, and, therefore, selection of the donor with the fewest mismatches may reduce complications; and (3) the risks of graft rejection, GVHD, and mortality may be greater with mismatches detectable by low-level (antigen) resolution techniques than with mismatches detectable only by high-resolution methods, and, therefore, allele-mismatched donors should be prioritized over antigen-mismatched donors [1]. A minority of patients can find fully matched donors, but all patients have a mismatched donor. Current research priorities include the need for a more complete understanding of the properties of HLA mismatches that do not increase posttransplantation complications.
early chronic phase (CP) of chronic myeloid leukemia (CML) from a mismatched donor was similar to the survival of patients who received a transplant in late CP from an HLA-identical donor; this suggested that the potential benefit of HLA matching was offset by the negative effect of advanced disease [3]. Survival with an HLA-matched donor in late CP was similar to that with an HLA-mismatched donor in late CP. This retrospective analysis of CML suggests that the increased mortality associated with a longer time interval from diagnosis to transplantation must be weighed carefully against the increased mortality with earlier transplantation with a mismatched donor and also against the chance of disease progression to advancedphase CML during a prolonged donor search.
High-Resolution Donor Matching in Support of Unrelated HCT
Permissible Mismatches
The largest comprehensive analysis of donor-recipient low-resolution (antigen) and high-resolution (allele) HLA matching, performed by the National Marrow Donor Program, uncovered a statistically significantly increased risk of mortality associated with donor mismatching for HLA-A, -B, -C, and -DRB1 [2]. Allele mismatches at each of HLA-A, -B, -C, and -DRB1 were less detrimental when compared with matches at the appropriate locus than were antigen mismatches for mortality. Finally, when compared with HLA-A and -B antigen–matched and -DRB1– matched patients, those with an allele mismatch at HLA-A or -B or any mismatch at HLA-C had poorer survival and a higher incidence of acute GVHD. The National Marrow Donor Program analysis demonstrates that an optimal transplantation outcome can be achieved through comprehensive and precise donor matching for HLA genes. Many patients in need of an unrelated allo-HCT, however, do not have wellmatched donors; identification of permissive locus- and allele-specific mismatches will enable more patients to benefit from unrelated HCT with the judicious use of mismatched donors. In addition, the disease stage at the time of transplantation is an important factor in the overall success of transplantation. For patients with advanced or active malignancy who may not have time for a lengthy unrelated donor search, the risks and benefits of earlier transplantation from mismatched donors or deferral of transplantation until a better-matched donor can be identified are important clinical questions that remain to be addressed. Four outcomes can arise in this situation: (1) a bettermatched donor is identified, and the disease remains stable; (2) a better-matched donor is identified, but the disease advances; (3) a better-matched donor is not identified, but the disease remains stable; and (4) a better-matched donor is not identified, and the disease progresses. A single-center study found that the overall survival of patients who received a transplant in
Nucleotide substitutions that define unique HLA class I and II alleles are concentrated at residues that result in a change in the protein sequence and that contact either bound peptide or the T-cell receptor. One approach to better understand the clinical ramifications of HLA diversity on function has been to examine a wide array of HLA allele and antigen mismatches in racially diverse transplant populations (http://www. ihwg.org). An analysis by the International Histocompatibility Working Group in HCT of 2399 unrelated transplantations from North America, Europe, Asia, and Australia has enabled investigators to test the hypothesis that the permissibility of HLA mismatches is in part governed by the locus and the combination of mismatched alleles or antigens. Data contributed to the International Histocompatibility Working Group by the Japan Marrow Donor Program have provided a large group of transplantations for comparison with the data on white patients and donors. In both the Japanese and the white data sets, an increased hazard of death was associated with the number of mismatched alleles compared with 10/10 allele matches. Locus-specific effects on mortality were examined for transplantation pairs with a single HLA mismatch. Among white recipients, the presence of a single HLA-C mismatch conferred an increased hazard of mortality compared with a match, whereas an HLA-A mismatch was not associated with a statistically significant increase in risk. In contrast, Japanese recipients had an increased risk of mortality associated with an HLA-A mismatch and not with HLA-C mismatching. Examination of the specific HLA-A and HLA-C mismatch combinations represented in the Japanese and white recipients and donors revealed major differences between the allele combinations mismatched for a given antigen. Similar findings at each HLA locus were uncovered, and this indicates that the study of permissible mismatches will require large numbers of ethnically and racially diverse trans-
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plantation populations who have been characterized at high resolution and for whom complete clinical data are available. In conclusion, donor HLA disparity remains a major cause of increased morbidity and mortality after unrelated transplantation. The selection of prospective unrelated donors has historically been based on HLA matching for class I and II genes. Unmet needs include information on qualitative and quantitative measures of risk associated with HLA mismatching; this information is required to best address the needs of patients for whom a matched donor is not available. The importance of structure as a way to understand function requires a systematic approach— one that involves the examination of diverse populations to appreciate the full extent and nature of genetic variation and the application of this information to the analysis of clinical populations, with careful attention to the transplantation procedures and the nongenetic factors that also influence outcome.
NATURAL KILLER CELLS AND ALLO-HCT OUTCOME Studies showing marked variation in survival and GVHD in haploidentical transplants according to predicted natural killer (NK) cell alloreactivity have stimulated intense interest in the role of NK cells in allo-HCT. NK cells are lymphocytes of the innate immune system that act early in immune responses to lyse infected or malignant cells [4,5]. NK cells reconstitute early after allo-HCT and have been implicated in suppression of GVHD, promotion of engraftment, and graft-versus-leukemia (GVL) effects. Unlike T lymphocytes, NK cells do not rearrange antigen-receptor genes to generate highly antigen-specific receptors, but they express several groups of activating and inhibitory killer cell immunoglobulin-like receptors (KIR; Table 1). The KIR genes lie on chromosome 19. Diversity in the KIR genotype is determined by the number and type of KIR genes present in each KIR haplotype, allelic polymorphism in each KIR gene, and the combination of maternal and paternal haplotypes [6]. Many KIR recognize HLA class I molecules, but unlike T-cell antigen receptors, these interactions are inhibitory rather than activating. This inhibitory interaction is essential to prevent NK killing of autologous cells. The epitopes recognized by these inhibitory KIR are determined by specific amino acid residues of HLA class I molecules, particularly residue 80 of the ␣1 helix of HLA-C (Table 2). NK cells also express a lectin-like receptor, CD94-NKG2A, that recognizes HLA-E complexed with peptides derived from HLA class I molecules. The interaction between KIR and HLA class I KIR epitopes is an important determinant of NK alloreactivity and can be predicted by the HLA class I
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Table 1. NK Cell Receptors and Ligands Variable Inhibitory NK receptors KIR2DL1
KIR2DL2/3 KIR3DL1
KIR3DL2 KIR3DL3 CD94/NKG2A Activating NK receptors KIR2DS1 KIR2DS2 KIR2DS3 KIR2DS4 KIR2DS5 KIR3DS1 KIR2DL4 CD94/NKG2C NKG2D Unknown NKG2E, NKG2F, NKRP1A
Ligand HLA-C group 2 (amino acids Asn77 and Lys80), eg, Cw2, Cw4, Cw5, Cw6 HLA-C group 1 (Ser77 and Asn 80), eg, Cw1, Cw3, Cw7, Cw8 HLA-B alleles containing the Bw4 motif, eg, B5, B13, B17, B27, B44, B52, some B15 HLA-A3 and A11 Unknown HLA-E complexed with HLA-A, -B, -C, or G peptide HLA-C Unknown Unknown HLA-C Unknown Unknown HLA-G HLA-E complexed with HLA-A, -B, -C, or G peptide MICA, MICB, ULBP Unknown
MICA indicates MHC class I polypeptide-related sequence A; MICB, MHC class I polypeptide-related sequence B; ULBP, UL16 binding protein.
types of donor and recipient. Two dominant patterns of alloreactivity are determined by an asparagine/lysine dimorphism at HLA-C position 80: KIR2DL1 recognizes HLA-Clys80, and KIR2DL2/3 recognize HLA-CAsn80. In HLA-C–mismatched transplantations, NK alloreactivity may occur in the GVH (and GVL) direction if the donor possesses HLA-C motifs that are missing in the recipient; these are then sensed by donor NK cells as “missing self.” Similarly, hostversus-graft NK alloreactivity may occur if the recipient possesses HLA-C motifs that are absent in the donor. NK Alloreactivity in Haploidentical Transplantations
The potential importance of KIR ligand matching was identified by data from haploidentical transplantations, in which only 1 of 2 HLA haplotypes is shared between donor and recipient. The use of haploidentical donors has the potential to expand the donor pool, but such transplantations require more intensive immunosuppression (eg, T-cell depletion) to minimize the risk of graft rejection and severe GVHD. In the Perugia study of T cell– depleted unrelated haploidentical transplantations, NK alloreactivity was predicted by donor and recipient HLA-C typing and was also assessed by in vitro assays. Very low rates of 21
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Table 2. Summary of Non-HLA Genetic Associations with Allo-HCT Outcome Gene and Polymorphism TNF d VNTR, ⴙ488 SNP TNF a and d VNTRs, ⴚ1031 SNP TNF ⴚ1031T/ⴚ863C/ⴚ857C haplotype TNF ⴚ308 SNP TNFRSF1B 196 SNP IFNG intron 1 (CA)n VNTR IL1A ⴚ889 SNP IL1B ⴚ511, ⴙ3954 SNPs IL1RN*2 VNTR IL2 ⴚ330 SNP IL6 ⴚ174 SNP IL10 ⴚ1064 VNTR IL10 promoter SNPs IL10RB 238*G SNP IL18 promoter SNPs TGFB1 coding SNPs, TGFB1RII 1167 SNP CARD15 SNPs ESR1 intron 1 PX haplotype VDR ApaI SNP ACE D/D genotype FCGR2A aa131 SNP FCGR3B genotype GSTM1 null genotype MBL2 coding mutations, HYA haplotype MPO ⴚ463 SNP MTHFR 677 SNP SERPINE 4G/4G TNFRSF6 (Fas)
Association Acute and chronic GVHD, mortality Survival* Acute GVHD* Neutrophil engraftment* Relapse, acute GVHD* Acute GVHD,† chronic GVHD Chronic GVHD,* survival, and TRM* Hepatic acute GVHD, survival, and TRM* Acute and chronic GVHD Acute GVHD* Acute and chronic GVHD Acute and chronic GVHD Acute and chronic GVHD, survival‡ Acute GVHD Survival, TRM* Acute GVHD Acute GVHD, gut GVHD, mortality‡ Acute GVHD, survival Acute GVHD, survival Noninfectious pulmonary dysfunction Time to first infection Time to neutrophil recovery, mortality Hepatic VOD Major infection Bacterial infection Mucositis Catheter thrombosis† Acute GVHD, major infection
All studies listed examined HLA-matched myeloablative transplantations unless otherwise indicated. ACE indicates angiotensin-conveting enzyme; CARD15, caspase recruitment domain family, member 15; ESR1, estrogen receptor 1 (␣); FCGR2A, FCGR3B, low-affinity receptor for Fc fragment of immunoglobulin G, types IIa and IIIb; GSTM1, glutathione-Stransferase M1; IFNG, interferon ␥; IL, interleukin; IL1RN, IL-1 receptor antagonist; MBL2, mannose-binding lectin; MPO, myeloperoxidase; MTHFR, methylene tetrahydrofolate reductase; SERPINE, serpin peptidase inhibitor, clade E, also known as plasminogen-activator inhibitor; TGFB, transforming growth factor ; TNF, tumor necrosis factor; TNFRSF1B, TNF receptor 2; TNFRSF6, Fas (Apo 1); TRM, treatment-related mortality; VNTR, variable number of tandem repeats (microsatellite); SNP, single nucleotide polymorphism; VOD, veno-occlusive disease. *Unrelated donor transplantations. †Myeloablative and nonmyeloablative transplantations. ‡Related and unrelated transplantations.
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graft rejection, disease relapse, and mortality were observed in transplants with KIR ligand incompatibility and NK alloreactivity in the GVH direction [7]. It is important to note that this was accompanied by low rates of GVHD that were attributed to NK-mediated depletion of recipient antigen-presenting cells. This effect was seen only in patients with acute myeloid leukemia (AML) and not acute lymphoblastic leukemia (ALL), a finding attributed to low lymphoblast expression of the adhesion molecules necessary for NK/target cell interaction. These results were updated in a larger group of AML patients (n ⫽ 80), again showing strikingly low relapse (17% versus 79%) and superior event-free survival (52% versus 7%) in transplantations from NK-alloreactive donors compared with transplantations with no NK alloreactivity [8]. Subsequent studies of haploidentical and unrelated transplantations (in which HLA-C mismatching was relatively common) have yielded conflicting data regarding the clinical importance of NK alloreactivity [9-14]. This may in part be due to differences in transplantation protocols, particularly the intensity of T-cell depletion [15], stem cell dose, and posttransplantation immunosuppression, all of which may influence NK cell dose, engraftment, and alloreactivity. Furthermore, consideration of other determinants of NK alloreactivity, such as KIR genotype, may be important, as discussed below. NK Alloreactivity in HLA-Matched Transplantations
The “missing self” model of NK alloreactivity predicted by HLA disparity described previously has been informative in HLA-C–mismatched transplantations but is not applicable to HLA-matched transplantations. Furthermore, HLA-C genotype alone is insufficient to predict NK alloreactivity in some donor-recipient combinations. The KIR genes are located at chromosome 19q13.4 and segregate independently from HLA on chromosome 6. Prediction of NK alloreactivity may be improved by considering the KIR genotype in addition to HLA class I typing. A recent study that examined donor KIR and recipient KIR ligand (ie, HLA class I) genotype in 178 HLA-matched sibling T cell– depleted transplantations found that 62.9% of transplant pairs lacked a recipient KIR ligand for donor KIR. Missing KIR ligand was an independent risk factor for relapse in patients who underwent transplantation for AML and myelodysplasia [16]. This “missing ligand” model suggests that NK alloreactivity can occur in HLA-matched transplantations if the recipient lacks an HLA ligand for donor KIR and suggests that KIR genotyping should be performed in addition to HLA genotyping. The potential importance of the interaction between donor KIR and recipient KIR ligand in HLA-matched
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Figure 1. Use of KIR and HLA-C type to predict GVL effects. NK alloreactivity in the GVH direction is depicted for 9 possible donor-recipient combinations of HLA-C (KIR) epitopes. For each combination, donors of 2 KIR types are considered: 1 having HLA-Clys80– specific (KIR2DL1) and HLA-Casn80–specific (KIR2DL3) KIRs and the other having only HLA-Casn80–specific (KIR2DL3) KIRs. Shown for each donor are the subpopulations of NK cells that express KIR2DL1, KIR2DL3, or both and that use these receptors as their only inhibitory receptors for autologous major histocompatibility complex (MHC) class I molecules. For example, in the donor who is homozygous for HLA-Clys80 but has only HLA-Casn80–specific (KIR2DL3) KIRs, there are no NK cells using HLA-C–specific KIRs as their inhibitory receptors for autologous MHC class I molecules. NK cell subpopulations that are predicted to mediate GVL reactions are colored green (for go); those that are predicted to be inhibited by the recipient’s HLA-C type are colored red (for stop). The donor-recipient combinations for which knowledge of the HLA-C type is insufficient to predict the probability of a GVL effect, and for which knowledge of the KIR type is also required, are emphasized in bold. For simplicity, the additional HLA-Casn80–specific KIR (KIR2DL2) is not included in this example, but similar considerations apply to it. The icons for KIRs and their cognate HLA-C ligands are color coded: KIR2DL1 and HLA-Clys80 are shown in orange, and KIR2DL3 and HLA-Casn80 are shown in purple. Reproduced with permission from Nature Reviews Immunology (5) © 2003 Macmillan Magazines Ltd. (http://www.nature.com/reviews).
transplantations is supported by recent data showing that patients who lack expression of HLA-C group 1 alleles have a higher risk of relapse and reduced survival [17,18]. Furthermore, because not all individuals with a given KIR genotype express the corresponding receptor at the cell surface [19], KIR phenotyping by flow cytometry may be required to provide the most accurate prediction of NK alloreactivity. The roles of HLA-C and KIR genotype in predicting NK alloreactivity are shown in Figure 1. The expression of activating KIR also varies between individuals, and it is possible that activating KIR genotype influences allo-HCT outcome. Current data are inconclusive. Several studies have examined donor activating KIR genotype in HLA-identical sibling transplantations; some have detected associations between activating KIR, such as KIR2DS1 and KIR2DS2, and the risk of relapse [20,21], but others have not [16,18]. It is interesting to note that recent studies have found that recipient KIR genotype may also influence the risk of relapse and GVHD [18,22].
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NK Alloreactivity in Clinical Practice
How might NK alloreactivity be exploited to improve transplantation outcomes? Existing data suggest that NK alloreactivity influences outcome in both HLAmismatched and HLA-matched transplantations and is determined by both HLA class I and KIR genotype. Donor selection based on NK alloreactivity may improve the efficacy and safety of haploidentical transplantations and considerably expand the pool of available donors for recipients who lack an HLA-matched donor. In this setting, HLA typing may be used to identify recipients who are potentially susceptible to NK-mediated alloimmune responses. Recipients who express all 3 major HLA class I KIR ligands are likely to be resistant to NK-mediated lysis. Donor HLA type can then be used to identify potential donors that express HLA class I not present in the recipient. KIR typing, KIR allelic typing, and immunophenotyping may also be required to more accurately determine donor KIR expression and activating KIR genotype. In the HLA-matched transplantations, it will be of 23
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great interest to examine the importance of KIR genotype in conjunction with other clinical and genetic variables, such as cytokine genotype. At present there are few data that examine KIR genomics in nonmyeloablative transplantations. Because NK reconstitution (and, thus, GVL responses) may be significantly different in nonmyeloablative transplantations compared with myeloablative transplantations, this area requires further study. It may also be possible to expand alloreactive NK cells ex vivo for subsequent therapeutic use [23]. This is of particular importance for patients who possess a full complement of inhibitory KIR ligands, who are less susceptible to donor NK-mediated GVL effects.
NON-HLA GENETIC DETERMINANTS OF TRANSPLANTATION OUTCOME Interest in the relationship between non-HLA immunogenetic polymorphisms and the risk of complications after allo-HCT has been driven by several observations. First, severe GVHD remains a common complication in HLA-matched transplantations. Second, the pathogenesis of GVHD is a complex, multistep process that involves conditioning-induced organ damage and the induction of high levels of inflammatory cytokines that augment tissue damage and donor lymphocyte activation. Polymorphisms in the genes that encode these inflammatory mediators influence the level of expression or activity of the encoded mediators and may influence GVHD pathogenesis. Third, the genetic factors that influence the risk of other complications, such as chronic GVHD and infection, are less well understood, and it is possible that polymorphisms in inflammatory mediators and host defense genes are important. Non-HLA Immunogenetics and GVHD
A widely accepted paradigm of acute GVHD pathogenesis is a 3-step model in which (1) pretransplantation conditioning induces recipient tissue damage, (2) alloreactive donor T cells are activated and expand, and (3) cellular and inflammatory effectors result in tissue damage [24]. Chemotherapy and radiotherapy damage organs such as the gut and liver, resulting in upregulation of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)–1, as well as translocation of microbial products such as lipopolysaccharide and CpG that further augment immune responses. These inflammatory cytokines increase the expression of HLA and costimulatory molecules on antigen-presenting cells and activate donor T cells, which secrete additional cytokines, such as IL-2 and interferon ␥. Several other proinflammatory and antiinflammatory cytokines have also been implicated in GVHD pathogenesis, including IL-4, IL-5, IL-10, 24
IL-12, IL-15, and IL-18. Secretion of chemokines may also be important in recruiting activated T cells to sites of tissue injury. A variety of effectors and pathways damage recipient tissues, including the direct action of cytokines, cytotoxic lymphocytes via Fas and perforin, NK cells, and nitric oxide. Cytokine genes were among the first non-HLA candidate genes to be studied in allo-HCT outcome. A wealth of data support the importance of cytokines in GVHD pathogenesis, including murine models of GVHD showing that blockade of or administration of cytokines modulates the severity of GVHD and clinical data showing that changes in blood cytokine levels are associated with the risk and severity of GVHD. The profound upregulation in cytokine synthesis in the peritransplantation period has been termed a cytokine storm. Most cytokine genes contain common inherited polymorphisms, many of which are located in upstream or intronic regulatory regions or in downstream regions that may influence messenger RNA stability. The polymorphisms are commonly short nucleotide sequences that display variable numbers of repetitions (variable numbers of tandem repeats, or microsatellites) or single nucleotide polymorphisms. Many of the polymorphisms studied in allo-HCT are associated with variation in cytokine synthesis in vitro and have been associated with a range of inflammatory, autoimmune, and infectious diseases. Most studies of non-HLA polymorphisms and allo-HCT outcome have examined the risk and severity of acute GVHD in HLA-matched related donor myeloablative transplantations. There are fewer data regarding unrelated donor and nonmyeloablative transplantations and other outcomes such as chronic GVHD, infection, interstitial pneumonitis, and venoocclusive disease. Associations between TNF and IL10 gene polymorphisms and acute GVHD risk and severity are among the earliest and most consistently reported observations (see reviews [25,26]). TNF is an important inflammatory cytokine that is upregulated during acute GVHD, and IL-10 antagonizes the release and many of the actions of TNF. The large number of studies implicating TNF and IL10 polymorphisms in GVHD risk, and other reports of associations with TNF and IL-10 receptor genes, suggest that these associations are biologically important. Several other cytokine and immunoregulatory gene polymorphisms have been repeatedly associated with GVHD, such as a functional polymorphism in the IL6 gene promoter, an intronic microsatellite polymorphism in the interferon ␥ gene, and polymorphisms in the IL1 gene family. Polymorphisms in several genes that encode noncytokine immune mediators, such as the vitamin D receptor and estrogen receptor, have also been associated with GVHD and survival. Polymorphisms in the TNF, TNFRSF1B (TNF receptor), IL1, IL6, and IL10 genes have also been associated
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with chronic GVHD. These studies are summarized in Table 2. Associations with Other Allo-HCT Outcomes
Fewer studies have examined associations with non-GVHD outcomes, perhaps in view of the difficulty in rigorously detecting and evaluating these end points. However, several potentially important associations have been reported. We and others have described associations between MBL2 gene polymorphisms and major infection after transplantation [27]. MBL2 encodes mannose-binding lectin (MBL), an innate host-defense molecule that directly recognizes a diverse array of pathogens and triggers complement activation. Up to 40% of healthy individuals have low MBL levels, and there is intense interest in MBL-replacement therapy to ameliorate infection in a variety of clinical contexts. Polymorphisms in the myeloperoxidase gene MPO and the immunoglobulin receptor gene FCGRIIA have also been associated with infection [28]. It is likely that studies examining additional host defense genes and infection will be informative. Several pharmacogenomic studies have also been reported. Methotrexate is a widely used immunosuppressive agent in allo-HCT and contributes to significant toxicity, such as mucositis. Methotrexate metabolites influence the activity of methylene tetrahydrofolate reductase, and polymorphisms in the MTHFR gene have been associated with mucositis, delayed platelet engraftment, and relapse. Polymorphisms in the GSTM1 gene, which encodes a glutathione-S-transferase involved in the metabolism of busulfan, have been associated with the risk of veno-occlusive disease. It is feasible that more detailed pharmacogenomic studies will permit individualized dosing to reduce the risk of these complications. Limitations of Current Non-HLA Genomic Data
Non-HLA immunogenetic data may be used clinically to refine donor selection, to identify recipients at the highest risk of GVHD and inform counseling before transplantation, or to modify immunosuppressive strategies. These data might also be used to design or guide the use of novel therapies, such as cytokine blockade or MBL-replacement therapy. However, there are several important limitations of most existing studies that must be addressed before non-HLA genotyping enters clinical use. Lack of reproducibility. Many reported associations have not been confirmed in subsequent studies. Sample issues. Many studies have examined small transplantation cohorts that are highly heterogeneous in terms of patient characteristics and transplantation parameters. Genotyping issues. All reported studies have used a
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limited candidate gene approach. Many potentially important genes and polymorphisms have not been investigated. Consequently, it is very difficult to determine which polymorphic marker represents the strongest association with an outcome and is the most suitable for clinical use. Donor versus recipient. Many studies have examined only recipient genotype. It is likely that both donor and recipient genotypes are important, depending on the clinical context and gene studied. Where both genotypes have been examined, the relative importance of donor versus recipient genotype has often differed between studies. Statistical issues. Many studies have not applied rigorous statistical methods, such as confirmation of results in independent cohorts, correction for multiple comparisons, or multivariable analysis. Many studies are likely to contain false-positive and falsenegative associations. Few studies have examined interaction between multiple non-HLA genetic associations. Nonmyeloablative transplantations. Reduced-intensity conditioning regimens are associated with less early toxicity; however, (delayed) acute and chronic GVHD remain major problems. In view of the differences in conditioning-induced organ damage and cytokine induction between nonmyeloablative and myeloablative transplantations [29], it is likely that distinct non-HLA associations will be seen in this setting.
CONCLUSIONS AND FUTURE PROSPECTS We are entering an era when detailed HLA and non-HLA genomic information may be used to improve the outcome of allo-HCT. Large studies have documented the importance of allelic HLA matching in unrelated transplantations and are defining permissible mismatches that will allow transplantations to be performed expeditiously without increasing risk. Existing data suggest that non-HLA polymorphisms and genetically determined NK alloreactivity also influence allo-HCT outcome, and there is intense interest in using this information clinically. Before this is done, many of the limitations of the existing studies described previously must be addressed. Many conflicting results are likely due to relatively small cohorts and differences in transplantation practices. Carefully designed prospective studies of large, homogeneous transplantation cohorts examining multiple genomic variants are needed to definitively confirm or refute associations; to examine interaction between multiple genetic and clinical variables; and to construct predictive models of risk that can inform transplantation practice. These studies must be performed in multiple clinical settings: HLA-matched sibling, HLA25
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matched unrelated, and haploidentical transplantations; transplantations using myeloablative and nonmyeloablative conditioning regimens; and cord blood transplantation. It will be difficult for all but the largest centers to conduct studies in isolation that examine multiple genetic and clinical variables with sufficient power, and collaborative studies will be essential. It will be important for non-HLA genomic studies to genotype each gene as comprehensively as possible, at least including all variants that have been previously examined. Newer microarray-based strategies offer a convenient way to genotype many thousands of loci and polymorphisms either on a genome-wide basis or by providing high-density coverage of genes and polymorphisms within a particular functional group. For NK/KIR studies, genotyping of KIR ligand and KIR receptors in both donor and recipient will be important. Ideally, genotyping strategies will be coupled to studies that examine the functional effects of polymorphisms, for example, by measuring blood levels or tissue expression of encoded mediators or by in vitro assays of NK alloreactivity. As the number of associated genetic variants increases, it will remain important to confirm associations in clinically similar independent cohorts.
ACKNOWLEDGMENTS E.W.P. is supported by grant nos. CA18029, CA100019, CA15704, AI33484, and AI49213 from the National Institutes of Health.
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REFERENCES 1. Petersdorf EW, Malkki M. Human leukocyte antigen matching in unrelated donor hematopoietic cell transplantation. Semin Hematol. 2005;42:76-84. 2. Flomenberg N, Baxter-Lowe LA, Confer D, et al. Impact of HLA class I and class II high-resolution matching on outcomes of unrelated donor bone marrow transplantation: HLA-C mismatching is associated with a strong adverse effect on transplantation outcome. Blood. 2004;104:1923-1930. 3. Petersdorf EW, Anasetti C, Martin PJ, et al. Limits of HLA mismatching in unrelated hematopoietic cell transplantation. Blood. 2004;104:2976-2980. 4. Farag SS, Fehniger TA, Ruggeri L, Velardi A, Caligiuri MA. Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect. Blood. 2002;100:1935-1947. 5. Parham P, McQueen KL. Alloreactive killer cells: hindrance and help for haematopoietic transplants. Nat Rev Immunol. 2003;3:108-122. 6. Parham P. Immunogenetics of killer cell immunoglobulin-like receptors. Mol Immunol. 2005;42:459-462. 7. Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science. 2002;295:2097-2100. 8. Velardi A, Ruggeri L, Capanni M, et al. Impact of NK cell 26
18.
19.
20.
21.
22.
23.
alloreactivity on mismatched hematopoietic transplantation: an update on donor selection criteria and on transplantation outcomes [abstract]. Blood. 2003;102:527. Giebel S, Locatelli F, Lamparelli T, et al. Survival advantage with KIR ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors. Blood. 2003;102:814819. Bornhauser M, Schwerdtfeger R, Martin H, Frank KH, Theuser C, Ehninger G. Role of KIR ligand incompatibility in hematopoietic stem cell transplantation using unrelated donors. Blood. 2004;103:2860-2861. De Santis D, Bishara A, Witt CS, et al. Natural killer cell HLA-C epitopes and killer cell immunoglobulin-like receptors both influence outcome of mismatched unrelated donor bone marrow transplants. Tissue Antigens. 2005;65:519-528. Schaffer M, Aldener-Cannava A, Remberger M, Ringden O, Olerup O. Roles of HLA-B, HLA-C and HLA-DPA1 incompatibilities in the outcome of unrelated stem-cell transplantation. Tissue Antigens. 2003;62:243-250. Beelen DW, Ottinger HD, Ferencik S, et al. Genotypic inhibitory killer immunoglobulin-like receptor ligand incompatibility enhances the long-term antileukemic effect of unmodified allogeneic hematopoietic stem cell transplantation in patients with myeloid leukemias. Blood. 2005;105:2594-2600. Aversa F, Terenzi A, Tabilio A, et al. Full haplotype-mismatched hematopoietic stem-cell transplantation: a phase II study in patients with acute leukemia at high risk of relapse. J Clin Oncol. 2005;23:3447-3454. Cooley S, McCullar V, Wangen R, et al. KIR reconstitution is altered by T cells in the graft and correlates with clinical outcomes after unrelated donor transplantation. Blood. In press. Hsu KC, Keever-Taylor CA, Wilton A, et al. Improved outcome in HLA-identical sibling hematopoietic stem-cell transplantation for acute myelogenous leukemia predicted by KIR and HLA genotypes. Blood. 2005;105:4878-4884. Giebel S, Locatelli F, Wojnar J, et al. Increased risk of relapse for patients undergoing HLA-C matched URD-HSCT and lacking ligands for KIR2DL2/3. Bone Marrow Transplant. 2005; 35:S152-S153. Loiseau P, Chen C, Appert M-L, et al. The presence and number of activatory KIR (R⫹D⫺) impact the infection and survival status of patients transplanted with an HLA matched sibling donor [abstract 3339]. Blood (ASH Annual Meeting Abstracts). 2004;104:912a. Leung W, Iyengar R, Triplett B, et al. Comparison of killer Ig-like receptor genotyping and phenotyping for selection of allogeneic blood stem cell donors. J Immunol. 2005;174:65406545. Cook MA, Milligan DW, Fegan CD, et al. The impact of donor KIR and patient HLA-C genotypes on outcome following HLA-identical sibling hematopoietic stem cell transplantation for myeloid leukemia. Blood. 2004;103:1521-1526. Verheyden S, Schots R, Duquet W, Demanet C. A defined donor activating natural killer cell receptor genotype protects against leukemic relapse after related HLA-identical hematopoietic stem cell transplantation. Leukemia. 2005;19:1446-1451. Sun JY, Gaidulis L, Dagis A, et al. Killer Ig-like receptor (KIR) compatibility plays a role in the prevalence of acute GVHD in unrelated hematopoietic cell transplants for AML. Bone Marrow Transplant. 2005;56:525-530. Imai C, Iwamoto S, Campana D. Genetic modification of primary natural killer cells overcomes inhibitory signals and
American Society for Blood and Marrow Transplantation Plenary Session: February 17, 2006
induces specific killing of leukemic cells. Blood. 2005;106: 376-383. 24. Ferrara JLM, Cooke KR, Teshima T. The pathophysiology of graft-vs.-host disease. In: Ferrara JL, Cooke KR, Deeg HJ, eds. Graft-vs.-Host Disease. New York: Marcel Dekker; 2005:1-34. 25. Mullighan CG, Bardy PG. Advances in the genomics of allogeneic haemopoietic stem cell transplantation. Drug Dev Res. 2004;62:273-292. 26. Dickinson AM, Middleton PG, Rocha V, Gluckman E, Holler E. Genetic polymorphisms predicting the outcome of bone marrow transplants. Br J Haematol. 2004;127:479-490.
BB&MT
27. Mullighan CG, Heatley S, Doherty K, et al. Mannose-binding lectin gene polymorphisms are associated with major infection following allogeneic hemopoietic stem cell transplantation. Blood. 2002;99:3524-3529. 28. Rocha V, Franco RF, Porcher R, et al. Host defense and inflammatory gene polymorphisms are associated with outcomes after HLA-identical sibling bone marrow transplantation. Blood. 2002; 100:3908-3918. 29. Mohty M, Gaugler B, Faucher C, et al. The “cytokine storm” at the era of reduced intensity conditioning allogeneic stem cell transplantation. Bone Marrow Transplant. 2005;35:S79-S80.
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