Genomic and Proteomic Analysis of Allogeneic Hematopoietic Cell Transplant Outcome. Seeking Greater Understanding the Pathogenesis of GVHD and Mortality

Success following allogeneic hematopoietic cell transplantation (HSCT) is ultimately determined by the ability to achieve sustained engraftment and immune reconstitution, eradication of the abnormal or malignant cells responsible for the patient’s disease, and control of graft-versus-host disease (GVHD). GVHD, an immune mediated reaction initiated by donor T cells in response to host alloantigen is the cause of significant morbidity and death in many patients. Genetic matching for HLA, the human major histocompatibility complex (MHC) located on chromosome 6p21 has for several years become well established as a requirement for optimal HSCT outcome [1]. Despite complete matching for all variation spanning 4Mb of DNA across the MHC, acute GVHD, chronic GVHD and transplant related mortality (TRM) occurs in a significant number of HLA identical sibling donor transplants.

The alloimmune reaction

Clinical GVHD results from an alloimmune reaction that occurs when immune competent donor T cells are transplanted to an immune compromised host and the genetic differences between donor and recipient are sufficient to induce T cell activation. [2] The genetic differences responsible for an allograft reaction encode polymorphic cellular proteins called histocompatibility antigens. [3] The strongest histocompatibility antigens are encoded by the class I and class II genes of the major histocompatibility complex (MHC), or HLA, but there are many other genes located throughout the genome that encode cellular peptides capable of generating significant alloimmune responses if variation in the gene product can be detected by T cells. These “allo” peptides are called minor histocompatibility antigens (mHA). [4] Although HLA identical siblings share 50% of their genome, the mHA disparity is sufficient to cause clinically significant acute GVHD in 30–40% of cases. Despite HLA matching among unrelated donor-recipient pairs, disparity for non-MHC mHA is greater because the recipient and donor do not share the same parental chromosomes. [5]

Pathogenesis of GVHD

The incidence, severity and duration of GVHD following HSCT vary substantially from patient to patient. [6–11] Differences in GVHD phenotype can result from both inherited and clinical or environmental factors including type of disease, disease status, treatment history, HLA match, age, sex of donor and patient (and sex match), and the type of conditioning therapy administered prior to transplant. [8,12,13]

High-dose cytotoxic conditioning can increase the risk of acute GVHD by causing mucosal injury which facilitates translocation of endotoxin into the bloodstream, suppressing T cell and NK cell mediated allograft resistance and activating antigen-presenting cells in the recipient. Within hours after transplantation, GVHD begins with the presentation of alloantigens by host dendritic cells, followed by activation of donor T cells which undergo clonal expansion and migration into various lymphoid organs and target tissues. [14] The anti-host alloimmune reaction is largely driven by the leakage of lipopolysaccharide (LPS) across the gut wall, [15] and further amplified by multiple components of the immune systems, including NK cells, monocytes, macrophages and proinflammatory cytokines. [16–20]

Proteomic analysis of acute GVHD and transplant-related mortality

Several studies have described changes in plasma proteins that correlate with the risk of acute GVHD, chronic GVHD and TRM (summarized in Table 1). Although there are common discoveries among several of these studies, there are also findings that have not independently replicated and for this reason true positives have not been clearly defined. There remains a great need for further and rigorously conducted studies that achieve both validation and also identification of the clinical covariables that in must be known to fully interpret and reliably implement biomarker data, both proteomic and genomic, into clinical risk assessment algorithms and pre-emptive therapies.

Table 1.

Summary of published changes in plasma proteins associated with acute GVHD, TRM and chronic GVHD. ¹

Protein	aGVHD	TRM	cGVHD
IL1-RN			Liem 1998 [54]
IL2R	Miyamoto 1996 [55]; Grimm 1998 [56]; Foley 1998 [57]; Nakamura 2000 [58]; Visentainer 2003 [59]; Shaiegan 2006 [60]; Paczesny 2008 [61]		Liem 1998 [54]; Fujii 2008 [62]
IL-6	Imamura 1994 [63]
IL-8	Uguccioni 1993 [64]; Paczesny 2008 [61]	Schots 2003 [65]
IL-10	Liem 1998 [54]		Liem 1998 [54]; Visentainer 2003 [59]
IL-12	Nakamura 2000 [58]; Mohty 2005 [66]
IL-15	Sakata 2001 [67]
IL-18	Nakamura 2000 [58]; Fujimori 2000 [68]; Shaiegan 2006 [60]; Luft 2007 [69]
BAFF			Fujii 2008 [62]
CD13			Fujii 2008 [62]
CCL8	Hori 2008 [70]
CXCL10	Piper 2007 [71]
HGF	Okamoto 2001 [72]; Paczesny 2008 [61]
IFNG	Imamura 1994 [63] ; Nakamura 2000 [58]
TNF	Holler 1990 [73]; Symington 1990 [74]; Imamura 1994 [63]
TNFR	Or 1996 [75]; Kitko 2008 [76]; Choi 2008 [77]; Paczesny 2008 [61]
Syndecan-1	Seidel 2003 [78]
anti-dsDNA			Fujii 2008 [62]

¹Refer to original publication for additional details.

Genomic analysis GVHD and TRM

Several studies over the last 10 years have identified genetic polymorphisms associated with GVHD and TRM (summarized in Table 2). Initially these investigations focused on well known genes encoding proinflammatory or immune modulating cytokines including IL1A, IL1B, IL1RN, IL6, IL10, INFG, TGFB and TNF. [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] Subsequent studies have examined additional genes for variation associated with HSCT outcomes including CTLA4, ESR1, IL2, IL7R, IL8, IL10RB, IL18, NOD2, VDR. [28,35,36] [37] [38] [39] [40] [41] [42] [43]

Table 2.

Genetic variation in immune response genes associated with acute GVHD, TRM and chronic GVHD.

Gene	HSCT associated phenotype	Study population (N) 1	VNTR or SNP				Discovery and supporting reference(s) 3	Investigators reporting non-significant results 3
			alias	location	alleles	rs number
CTLA4 (CD152)	aGVHD & survival	MRD (536)	+49 (d), CT60 (d) 2		A/G, G/A	rs231775, rs3087243	Perez-Garcia 2007 [41]
	cGVHD		+49/GG		A/G	rs231775	Azarian 2007 [42]
ESR1	aGVHD	MRD (108)	VNTR, PvuII & XbaI	intron 1	- -	- -	Middleton 2003 [35]
FAS	aGVHD	MRD (160)	−670 (p)	promoter	A/G	- -	Mullighan 2004 [29]
IFNG	aGVHD	MRD (49)	VNTR	intron 1	na	- -	Middleton 1998 [21]
	aGVHD	MRD (100)	874		T/A	- -	- -	Socie 2001 [23]
IL1A	cGVHD	MRD (115)	−899, VNTR	promoter, intron 6	C/T, allele 2	- - - -	Cullup 2003 [27]	- -
	aGVHD & TRM	URD( 426)	−889	promoter	C/T	- -	- -	Mehta 2007 [34]
IL1B	aGVHD	MRD	−511 (p)	promoter	T/C	rs16944	MacMillan BJH 2003[28]	- -
	aGVHD	MRD (570)	−511 (p/d)	promoter	C/T	rs16944	- -	Lin 2003 [26]
IL1RN	aGVHD	MRD (99)	VNTR	intron 1	- -	na	Cullup 2001 [24]	- -
	aGVHD & cGVHD	MRD (107)	VNTR (d)	intron 2	absence of allele *2	- -	Rocha 2002 [25]	- -
		MRD (570)	9261 (p/d)	intron 1	G/A	448341	- -	Lin 2003 [26]
IL2	aGVHD	URD (95)	−333 (p)	promoter	T/G	rs2069762	MacMillan Transplant 2003 [43]	- -
IL6	aGVHD	MRD (160)	−174 (d)	promoter	G/C	rs1800795	Mullighan 2004 [29]	- -
	aGVHD	ND (93)	−174 (p/d)	promoter	G/C	rs1800795	Karabon 2005 [31]	- -
	cGVHD		−174	promoter	G/C	rs1800795	Cavet 1999 [22]	- -
	cGVHD	MRD (100)	−174 (p)	promoter	G/C		Socie 2001 [23]	- -
	aGVHD, cGVHD, TRM	MRD (570)	−174 (d)	promoter	G/C	rs1800795	- -	Lin 2003 [26]
IL7R	TRM	MURD (75)	+1237(p)	cSNP	A/G	- -	Shamim 2006 [40]	- -
	TRM	MRD (100)	+510,+1237, +2087,+3101	cSNPs	C/T,A/G,C/T,A/G	- -	- -	Shamim 2006 [40]
IL10	aGVHD	MRD (49)	IL10G; 4	promoter	- -	- -	Middleton 1998 [21]	- -
	aGVHD	MRD (144)	IL10/−1082, IL10−1064 (4)	promoter	A/G	- -	Cavet 1999 [22]	- -
	aGVHD	MRD (100)	hap 5	promoter	G-C-C	- -	Socie 2001 [23]	- -
	aGVHD	MRD (993)	−592 (p) & hap (p) 5	promoter	C/A, A-T-A		Lin 2003 [26]	- -
	aGVHD	MRD (160)	ATA hap(p)	promoter			- -	Mullighan 2004 [29]
	cGVHD	MRD (107)	−1082*G/G(p)	promoter	A/G	- -	Rocha 2002 [25]	- -
	cGVHD		ATA hap(p)	promoter			Mullighan 2004 [29]	- -
	TRM	URD (182)	IL10R24-SNP hap (d) 5	promoter	G-C-C		Keen 2004 [30]	- -
IL10RB	aGVHD	MRD (993)	c238 (d)	exon			Lin 2005 [37]	- -
IL18	Survival	URD (157)	GCG hap(p)				Cardodo 2004 [38]	- -
NOD2	aGVHD & TRM		SNP8, 12, 3	intragenic	G/A, G/C, insertion	rs2066844, rs2066845, rs2066847	Holler 2004, 2006 [36,39]	- -
TNF	aGVHD	MRD (49)	TNFd 6,7		VNTR	na	Middleton 1998 [21]
	TRM	MRD (144)	TNFd 7		VNTR	na	Cavet 1999 [22]
	aGVHD	MRD (100) MRD ( 570) MRD (160)	−308 7	promoter	G/A	rs1800629	- -	Socie 2001 [23]; Lin 2003 [26]; Mullighan 2004 [29]
	TRM	URD (182)	TNFd & −1031	promoter: VNTR & SNP	TNFd*4/−1031*C hap	- -	Keen 2004 [30]	- -
	aGVHD & TRM	MRD (160)	488 7				Mullighan 2004 [29]	- -
TNFRII	cGVHD	MRD (104)	codon 196	exon 6	T/G		Stark 2003 [79]	- -
VDR	aGVHD & TRM	MRD (88)	VNTR (d)	intron 8			Middleton 2002 [80]	- -

¹Study population: “MRD”, indicates HLA matched related donor; “ND”, not defined; “URD”, unrelated donor.

²“p”, indicates patient; “d”, donor.

³Refer to original publication(s) for additional details.

⁴IL10G and IL10R are a microsatellites located in and nearby the IL10 gene.

⁵IL10 promoter region haplotype, positions: −1082/−819/−592

⁶TNFd is a microsatellite in the TBF region.

⁷MRD cases, because they are HLA identical, share the same TNF genotypes

Unfortunately, the results of most of these single Center studies have not been independently validated by others in separate patient populations. Lack of validation or inconsistency in these results may be due largely to lack of statistical power since many of the original studies were based on relatively small numbers (<200–300 cases). Nevertheless, these results have been sufficiently compelling to warrant additional study. Comprehensive critical reviews of this research have been recently published [44],[45] and other current papers have addressed the potential impact of developments in genomic sciences and the opportunity for expanding HSCT outcomes research to genome-wide discovery. [1,46]

Genome-wide association studies (GWAS)

The remarkable development in recent years of methods and tools for the characterization of the entire human genome has dramatically broadened the opportunity for the genetic analysis of disease. The recent completion of the human genome map[47,48] and the development of dense SNP marker maps of the genome, [49,50] as well as development of massively parallel genotyping technologies, [51–53] have made it possible to screen genes in an unbiased manner for polymorphisms that correlate with any well defined phenotype, disease status or relevant quantitative trait. This is particularly important when considering complex traits that characterize HCT complications and outcomes. Consideration of the entire genome in an unbiased fashion permits the discovery of genetic factors that would have never been considered otherwise.

State of the art genomic and proteomic studies of GVHD and mortality

The three papers that follow this Introduction are summaries of the oral presentations that will be given during the Genomics and Proteomics Scientific Session of the 2009 BMT Tandem meetings. These papers are each timely progress reports of our emerging understanding of the pathogenesis of GVHD and TRM. The paper by Sophie Paczesny, Jamie Ferrara and team provides model example of the rigorous two phase, discovery and validation, proteomic study of changes in plasma proteins associated with the development of acute GVHD. The technology used for the discovery phase used an antibody array containing antibodies specific for 120 human proteins including acute phase reactants, cytokines, angiogenic factors, tumor markers, leukocyte adhesion molecules and metalloproteinases and their inhibitors. The papers by Seishi Ogawa et al and Jason Chien et al describe preliminary discovery data from two of the first large whole genome scans performed on DNA from both recipient and donor as a comprehensive approach to examining genetic disparity and GVHD (Ogawa et al), and the association of genetic variation with transplant outcomes including gram-negative bacteremia and bronchiolitis obliterans.