Biologic markers of chronic graft vs. host disease

Abstract

Biologic markers of chronic graft vs. host disease (GVHD) may provide insight into the pathogenesis of the syndrome, identify molecular targets for novel interventions, and facilitate advances in clinical management. Despite extensive work performed to date largely focused on prediction and diagnosis of the syndrome, little synthesis of findings and validation of promising candidate markers in independent populations has been performed. Studies suggest that risk for subsequent chronic GVHD development may be associated with donor-recipient genetic polymorphism, deficiency in regulatory immune cell populations (NK, Treg, DC2), and variation in inflammatory and immunoregulatory mediators post-HCT (increased TNFα, IL-10 and BAFF, and decreased TGFβ and IL-15). Established chronic GVHD is associated with alteration in immune cell populations (increased CD3+ T cells, Th17, CD4+ and CD8+ effector memory cells, monocytes, CD86 expression, BAFF/B cell ratio, and deficiency of Treg, NK cells, and naïve CD8+ T cells). Inflammatory and immunomodulatory factors (TNFα, IL-6, IL-1β, IL-8, sIL-2R, and IL-1Ra, BAFF, anti-dsDNA, sIL-2Rα, and sCD13) are also perturbed. Little is known about biologic markers of chronic GVHD phenotype and severity, response to therapy, and prognosis.

Introduction

Chronic graft vs. host disease (GVHD) is a major source of morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Despite immune suppressive prophylaxis, most survivors develop chronic GVHD. Among those with established chronic GVHD, primary therapy provides durable complete remission in the minority of cases, and secondary therapy is associated with poor response, infectious complications, and mortality. Identification of biologic markers of the syndrome could facilitate significant advances in understanding to help guide prevention and treatment approaches. This review summarizes the literature in the field, with attention to both consistent findings across studies, as well as important divergent findings.

Biologic markers associated with risk for chronic GVHD development

Donor-Recipient non-HLA genetic polymorphism

One major area of investigation has focused on polymorphisms in donor and/or recipient inflammatory and immunoregulatory cytokines.^1–13 These largely demonstrate that polymorphism in IL-1, IL-6, IL-10, and TNFα are associated with increased risk for chronic GVHD. Other findings largely come from single gene candidate studies (Table 1).^{7, 14–22}

Table 1.

Donor-recipient gene polymorphism associated with risk for subsequent chronic GVHD

	Biologic marker	Risk for cGVHD
Increased risk
7Mullighan	IL-10 ATA promoter haplotype	Increased cGVHD
5Kim	IL-10 promoter haplotype ATA/ATA	Increased cGVHD and prolonged duration of IS
8Rocha	recipient IL-10 (GG) genotype	Increased cGVHD
10Takahashi	donor IL-10 allele number	Increased cGVHD
4Cullup	allele 2 IL-1a -889 and allele 2 IL-1α (VNTR) polymorphism in donor genotype	Increased cGVHD
8Rocha	recipient IL-1Ra genotype	Increased cGVHD
6Laguila	IL-6 position -174 (-174CC donor or recipient)	Increased cGVHD
3Cavet	IL-6 -174GG homozygous recipients	Increased cGVHD
4Cullup	recipient IL-6 -174 (GG)	Increased cGVHD
7Mullighan	TNF 488A (intronic polymorphism)	Increased cGVHD
1Bertinetto	absence of TNF-α 238 A allele	Increased limited and extensive cGVHD
11Viel	promoter gene TNFA-238GA: GA genotype at position -238 (in both donor and recipient)	Increased overall and extensive cGVHD
2Bogunia-Kubik	IFN-y 3/3 genotype	Increased cGVHD
18Inamoto	CCR9 genotype 926AG	Increased cutaneous cGVHD
21Ostrovsky*	Recipient heparanase SNP	Increased extensive cGVHD
15Arora	PARP1 SNP (rs1805410)	Increased cGVHD
14Ambruzova	MadCAM-1 rs2302217 AA homozygous recipients	Increased cGVHD and worsened OS
20McGuirk	haptoglobin polymorphism (HP 2-2 phenotype)	Increased cGVHD
19Kornblit*	donor HMGB1 2351insT Genotype	Increased cGVHD
16Boukouaci*	MICA-129 val allele	Increased cGVHD
Decreased risk
9Sivula	Donor-recipient matched predicted IL-10 production levels and recipient IL-10Rβ A/A homozygosity	Decreased extensive cGVHD
17Broen	donor homozygous for the CCR6 rs2301436 G allele or rs3093023 G allele	Decreased cGVHD
22Shimada	recipient FCRL3-169C/C genotype	Decreased cGVHD
7Mullighan	Recipient Fas -670A	Decreased cGVHD
12Clark*	BAFF	Late acute or no GVHD vs. chronic GVHD (classic or overlap)

^*Inamoto – CCR9 polymorphism was associated only with cutaneous cGVHD, not other organs and not overall cGVHD

^*Arora - greatest risk was observed in those with homozygous minor genotype for SNP rs1805

^*Ostrovsky - those with polymorphism associated with high heparanase levels had greatest risk

^*Kornblit – effect only observed in setting of myeloablative conditioning

^*Boukouaci - MICA-129 val allele increased risk for chronic GVHD in dose-dependent manner; increased soluble MICA (sMICA) post-HCT was associated with risk for chronic GVHD, while pre-HCT anti-MICA antibody had inverse association with chronic GVHD risk

^*Clark – recipient BAFF SNPs were associated with increased odds of late acute GVHD or no GVHD vs. chronic GVHD (including both classic chronic GVHD or overlap subtype of chronic GVHD). Among those with classic/overlap chronic GVHD, BAFF SNPs had no association with organ involvement, global severity of GVHD, or survival. These findings have been further addressed by Fore, et al.¹³

Negative findings reported by Lin (IL10 promoter genotype),²³ Cullup (IL-10 polymorphism),⁴ Bertinetto (IL-1RA, IL-10, and IL-1β),¹ Takahashi (donor-derived TNF2 allele (A)),¹⁰ Viel (IL-2),¹¹ and Laguila (TNF-α, IFN-γ, IL-10, or TGF-β1)⁶ should be noted. Together, these findings threaten confidence in association between cytokine polymorphisms and chronic GVHD risk.

Immune cell populations

Several studies have examined association of the cellular content of the donor allograft and risk for chronic GVHD (table 2).^24–33 While earlier studies have studied bone marrow, more recent studies focus on peripheral blood mobilized stem cell (PBSC) grafts. These studies support an inverse association between donor allograft nucleated cell and CD34+ cell dose, plasmacytoid dendritic cell (DC), and natural killer (NK) cell dose and subsequent risk for chronic GVHD. Examination of immune reconstitution from the recipient peripheral blood post-HCT supports deficiency in regulatory cell populations, including NK and regulatory T cells (Treg), as a determinant of subsequent chronic GVHD (table 2).

Table 2.

Immune cell populations associated with lower risk for subsequent chronic GVHD

	Source	Biologic marker
30Rocha	BM	Nucleated cell dose
28Morariu-Zamfir	BM	Nucleated cell dose
	BM	CD34+ cell dose
32Waller	BM	DC2 (CD3−CD4brightCD8−)
29Rajasekar*	Blood	DC2 (lin-HLA-DR1+CD123+)
25Larghero	BM	NK (CD16+CD56+)
24Kim	PBSC	NK (CD16+CD56+)
33Yamasaki	PBSC	NK (CD16/56+/CD34 ratio)
31Skert	blood	CD3+CD28+ T cells NK (CD16+CD56+) Treg (CD4+CD25+FoxP3+)
26Li	blood	Treg (CD4+CD25+CD127−)
27Miura*	Blood	Treg (FoxP3 mRNA expression in PBMC)

^*Rajasekar - found no association between PBSC graft DC2 content and subsequent risk for chronic GVHD; however, low plasmacytoid dendritic cell frequency on day 28 following HCT was significantly associated with increased risk for subsequent chronic GVHD.

^*Miura - FoxP3 mRNA expression in PBMC on serial measures post-HCT remained low in those that subsequently developed GVHD; conversely, this gradually increased, reaching levels of normal controls among those without GVHD.

Relevant negative findings include the following: Sierra, et al found no association between marrow cell dose and risk for chronic GVHD.³⁴ Baron, et al reported no effect of CD34+ dose in non-myeloablative conditioning and unrelated donor HCT.³⁵ Li, et al found no difference in CD4+ T cells or NK cells between those who subsequently developed chronic GVHD or not.²⁶ The findings of Ukena, et al challenge the inverse association of Treg and chronic GVHD risk: Those who developed chronic GVHD had comparable or increased CD4+CD25+CD127low Treg compared to those without chronic GVHD.³⁶

Inflammatory and immunoregulatory mediators

Prospective studies examining serial peripheral blood samples post-HCT support differences in inflammatory and immunoregulatory cytokines between those who do or do not develop chronic GVHD (table 3).^{26, 31, 37–39} Chronic GVHD development is associated with increased TNFα, IL-10 and BAFF, and decreased TGFβ and IL-15. Findings related to IFNγ are not consistent.

Table 3.

Inflammatory and immunoregulatory markers associated with risk for subsequent chronic GVHD

	Biologic marker	Risk for cGVHD
31Skert	TNFα IFNγ IL-10	Increased Decreased Increased
37Ritchie*	TNFα IFNγ	Increased Increased
26Li*	TGFβ TNFα	Decreased Increased
38Sarantopoulos*	BAFF BAFF/B cell ratio	Increased Increased
39Pratt*	IL-15	Decreased

^*Ritchie - increased (1.5 fold that present in baseline sample) expression of TNF-α and IFN-γ in peripheral blood T cells preceding the onset of extensive chronic GVHD

^*Li - peripheral blood samples within 2–4 weeks post-HCT

^*Sarantopoulos - BAFF levels, initially high within the first 3 months following HCT, declined among those who did not develop chronic GVHD. Conversely, those who developed chronic GVHD had persistently elevated BAFF levels, and 6 month BAFF levels ≥ 10ng/mL were significantly associated with development of chronic GVHD.

In patients who subsequently developed chronic GVHD, there was persistent elevation in BAFF and CD27+ B cell subsets together with decreased numbers of naïve B cells, resulting in a high BAFF:B cell ratio

^*Pratt – Low levels of IL-15 at day 7 was associated with increased likelihood of chronic GVHD requiring systemic immune suppressive therapy compared to those with higher IL-15 levels.

Predictors restricted to specific chronic GVHD phenotypic groups

Inamoto, et al found that CCR9 Genotype 926AG was associated with increased risk for cutaneous chronic GVHD.¹⁸ Shimura, et al demonstrated that CD34+CD133+VEGF-R2 endothelial progenitor cells remained low among those that developed sclerodermatous chronic GVHD.⁴⁰ Additionally, VEGF and b-FGF differed among HCT recipients vs. healthy controls, but no significant difference was observed between HCT recipients with or without sclerodermatous chronic GVHD. Mattson, et al demonstrated that low levels of clara cell secretory protein (CC16) predated diagnosis of bronchiolitis obliterans (BOS) by median of 10 months.⁴¹ This is a notable finding, as there are few established clinical predictors of the syndrome, and decline in pulmonary function can pre-date clinical recognition.

Biologic markers associated with chronic GVHD diagnosis

Immune cell populations

Examination of immune cell subsets in chronic GVHD has largely focused on T cell subsets: The evidence demonstrates that chronic GVHD is associated with increased CD3+ T cells,⁴² Th17,⁴³ and both CD4+ and CD8+ effector memory cells.^{44, 45} Monocytes are increased,⁴⁶ and CD86 expression is increased on monocytes and B cells (in response to CpG stimulation).^{46, 47} In contrast, chronic GVHD is associated with a deficiency of Treg,^{27, 43} NK cells,⁴² and naïve CD8+ T cells.⁴⁴ In one study Treg (defined by FoxP3 mRNA expression) were decreased, and T cell receptor gene rearrangement excisional circles (TREC) levels were reduced in chronic GVHD patients, suggesting that defective thymic function contributes to impaired Treg reconstitution.²⁷ Most investigation supports a reduction in total CD19+ B cells, but divergent findings have been reported on B cell subsets (naïve, transitional, memory).^48–51 B cell subpopulations may differ according to degree of humoral immunity impairment (table 4).⁵⁰ Recent data suggests that B cells in chronic GVHD patients have increased ERK and AKT signaling, and decreased levels of the pro-apoptotic Bim.⁵²

Table 4.

Immune cell populations associated with chronic GVHD diagnosis

	Biologic marker	Comparison	Finding in cGVHD
42Abrahamsen*	CD3+ T cells	extensive chronic GVHD vs. limited/no chronic GVHD	Increased
43Dander*	Th17 cells (CD4+IL-17+ cells by flow cytometry and IL-17 producing cells by ELISPOT assay)	Active cGVHD vs. inactive cGVHD	Increased
45Yamashita	CD4+ effector memory cells (CD4+CCR7-CD62Llow)	cGVHD vs. no cGVHD/healthy Donors	Increased
44D’asaro	CD8+ effector memory cells (CD8+CCR7-CD45RA+)	CGVHD vs. non- chronic GVHD HCT recipients, normal controls, and recipients of kidney transplant	Increased
	Naïve CD8 T cells (CD8+CCR7+CD45RA+)		Decreased
27Miura*	Treg (FoxP3 mRNA in PBMC)	cGVHD vs. no cGVHD	Decreased
43Dander	Treg (CD4+CD25+FoxP3+)	Active cGVHD vs. inactive cGVHD	Decreased
46Arpinati	BM monocytes	cGVHD vs. no CGVHD	Increased
	BM and blood monocyte CD86 expression		Increased
42Abrahamsen	NK cells (CD16/56+)	Extensive cGVHD vs. limited/no cGVHD	Decreased
	Total B cells (CD19+)		Decreased
48D’orsogna*	Total memory B cells (CD19+, CD27+)	cGVHD vs. no cGVHD	Decreased
	IgM memory B cells (CD19+, CD27+, IgM+)		Decreased
	Switched memory B cells (CD19+, CD27+, IgM−)		Decreased
49Greinix	CD19+/CD21− immature/transitional B cells	Active cGVHD vs. inactive/no cGVHD	Increased
	Non-class-switched IgD+ and class-switched IgD−CD27+ memory B cells		Decreased
47She*	B cell up-regulation of CD86 in response to PS-modified CpG	Early cGVHD cases vs. time-matched controls	Increased
51Sarantopoulos	BAFF/CD19+ B cell ratio	Active cGVHD vs. not/controls	Increased
	naïve B cells (CD19+IgD+CD38LoCD27−)		Decreased
	transitional B cells (CD19+IgD+CD38HiCD27−)		Decreased
	memory B cells (IgD+CD38lowCD27+ IgD+)		Increased
	Pre-GC B cells (IgD+CD38HiCD27+)		Increased
	post-GC memory B cells (IgDlow/-CD38lowCD27+)		Increased
	plasmablast/plasma cells (IgDlow/-CD38highCD27+)		Increased
50Kuzmina*	Immature (CD19+CD21low) B cells	Established cGVHD: hypo- vs. normo-/hyper-gammaglobulin groups	Increased
	transitional (CD19+CD21int− highCD38highIgMhigh) B cells		Increased
	naïve (CD19+CD10−CD27− CD21high) B cells		Decreased
	memory (both CD19+CD27+IgD+ non-class switched and CD19+CD27+IgD− class- switched) B cells		Decreased

^*Abrahamsen – blood samples obtained 3 months post-HCT; actual proximity of these samples to onset of chronic GVHD not described

^*Dander – demonstrated increased Th17 cells in cGVHD and infiltrating Th17 cells in GVHD target organs

^*Miura - Demonstrated that FoxP3 mRNA in PBMC at onset of chronic GVHD (prior to initiation of treatment) was significantly decreased compared to patients without chronic GVHD and healthy controls. TREC levels were also reduced in chronic GVHD patients, suggesting that defective thymic function contributes to impaired Treg reconstitution.

^*D’orsogna - this analysis considered history of acute and/or chronic GVHD together

^*She - Newly diagnosed extensive chronic GVHD (treated on phase III trial of cyclosporine/prednisone vs. cyclosporine/prednisone/hydroxychloroquine); Early onset chronic GVHD cases were 3–8mo post-HCT, while late onset cases were ≥ 9mo post-HCT. Early chronic GVHD cases were matched to 6 month post-HCT control samples, and late onset GVHD cases were matched to 12 month post-HCT control samples.

^*Kuzmina - Patients with established (median duration of chronic GVHD was 42 months) chronic GVHD were studied to discern differences in B cell subpopulations according to degree of humoral immunity impairment (serum immunoglobulin levels).

Relevant negative findings include the following: Clark, et al reported that chronic GVHD patients had increased numbers of CD4+CD25^high T cells; while FoxP3 was not studied, these cells lacked markers of activation, expressed CTLA-4, and demonstrated suppressive activity of CD4+CD25- T cells after polyclonal stimulus.⁵³ Arpinati, et al found that number and phenotype of BDCA1+CD19- myeloid and BDCA2+CD123+ plasmacytoid DC in the peripheral blood were similar in those with or without chronic GVHD; this finding contradicts previous reports suggesting allograft DC content was a predictor of chronic GVHD.⁴⁶ Finally, Greinix, et al found no significant differences in absolute B, T, and NK cell numbers between those with and without chronic GVHD; this finding challenges relative consensus among other reports.⁴⁹

Inflammatory and immunoregulatory mediators

The majority of studies have examined biologic markers of chronic GVHD diagnosis without attention to the time of chronic GVHD onset and sample acquisition after HCT or matching cases to controls at comparable time post-HCT (table 5).^{43, 54–58} The major exceptions to this are the reports from Fujii and Rozmus.^{59, 60} Another consideration is that most have examined a limited set of individual cytokines grounded in the Th1/Th2 paradigm, rather than more comprehensive discovery-based investigation. Finally, the control groups studied in comparison to chronic GVHD cases differ.

Table 5.

Inflammatory and immunoregulatory mediators associated with chronic GVHD diagnosis

	Biologic marker	Comparison	Finding in cGVHD
58Tanaka	IL-12	Extensive cGVHD vs. no cGVHD/controls	No difference
	IL-4		Decreased
54Barak	TNFα	cGVHD vs. no cGVHD/healthy controls	Increased
	IL-6		Increased
	IL-1β		Increased
43Dander	TNFα	cGVHD vs. healthy controls	Increased
	IL-6		Increased
	IL-8		Increased
55Kobayashi	sIL-2R	cGVHD onset vs. pre-cGVHD onset samples	Increased
56Liem	IL-1Ra (IL-1R antagonist)	cGVHD vs. no cGVHD	Increased
	sIL-2Rα		Increased
	IL-10		Increased
63Kohrt	IL-1R2	cGVHD vs. no cGVHD	Increased
62Kitko*	BAFF	cGVHD vs. no cGVHD	Increased
	sCD13		Increased
	elafin		Increased
	IL-2Rα		Increased
	MIG (CXCL9)		Increased
57Poloni*	CD8+ cells: IFNγ TNFSF10	cGVHD vs. no cGVHD	Increased Decreased
	CD14+ cells: TNFSF3 TNFSF10		Increased Increased
	CD4+ cells: TNFSF12 PDGFβ		Decreased Decreased
59Fujii*	Early onset cGVHD: sBAFF anti-dsDNA sIL-2Rα sCD13	cGVHD vs. time-matched controls	Increased Increased Increased Increased
	Late onset cGVHD: sBAFF anti-dsDNA		Increased Increased
60Rozmus*	Early onset cGVHD: IFNγ IL-2	cGVHD vs. time-matched controls	Decreased Decreased
	Late onset cGVHD: IL-4 IL-2 FoxP3		Decreased Decreased Decreased

^*Tanaka - studied cytokine response to ConA stimulation of PBMC from chronic GVHD subjects, HCT recipients without chronic GVHD and healthy controls

^*Barak - compared cytokine levels of those post-onset chronic GVHD (5–27 months post diagnosis) vs. HCT recipients who did not develop cGVHD (5–12 months post-HCT at sample) and healthy controls

^*Kitko – proteomic discovery and subsequent validation

^*Poloni – analyzed gene expression in selected immune cell populations (CD4+, CD8+, CD14+)

^*Fujii and Rozmus - reported on biologic markers in the context of newly diagnosed extensive chronic GVHD (phase III trial of cyclosporine (CSA)/prednisone vs. CSA/prednisone/hydroxychloroquine) with particular attention to the time of chronic GVHD onset post-HCT: Early onset chronic GVHD cases were 3–8mo post-HCT, while late onset cases were ≥ 9mo post-HCT. Early chronic GVHD cases were matched to 6 month post-HCT control samples, and late onset GVHD cases were matched to 12 month post-HCT control samples. Fujii reported protein levels by ELISA. Rozmus studied mRNA expression of interferon (IFN)-y and interleukin (IL)-2, -4, and -10 by quantitative PCR after stimulation of PBMC with PMA-ionomycin or anti-CD3.

Acknowledging these considerations, most studies support increased pro-inflammatory cytokines in chronic GVHD cases, including TNFα, IL-6, IL-1β, IL-8, sIL-2R (soluble alpha chain of the IL-2 receptor shed by activated T cells), and IL-1Ra (IL-1R antagonist), among others. Validated proteomic work from suggests that BAFF, sCD13,⁶¹ elafin, IL-2Rα, MIG (CXCL9), and anti-dsDNA may distinguish chronic GVHD cases from non-chronic GVHD controls with high accuracy.⁶²

Work from Fujii, et al and Rozmus, et al suggests that diagnostic markers of chronic GVHD may differ according to the time of chronic GVHD onset.^{59, 60} Fujii, et al reported that early onset chronic GVHD had increased sBAFF, anti-dsDNA, sIL-2Rα, and sCD13. Late onset chronic GVHD cases had increased sBAFF and anti-dsDNA. Rozmus, et al reported that early onset chronic GVHD demonstrated decreased IFNγ and IL-2, while late onset chronic GVHD had decreased IL-4, IL-2, and FoxP3.

Few studies have tried to discern the impact of immune suppression treatment on observed findings between chronic GVHD and non-chronic GVHD groups, since patients with chronic GVHD are often treated with multiple immune suppressive agents whereas patients without chronic GVHD may be off medications. A notable exception is the work from Kohrt, et al, where specific analyses isolated and identified the effects of medications by comparing new onset chronic GVHD cases not on immune suppression and patients with chronic GVHD already on treatment.⁶³

Relevant negative findings include the following: Tanaka, et al did not detect differences in IL-12 production between chronic GVHD and control subjects. Since IL-12 is a major signal important for Th1 differentiation, Tanaka’s results combined with multiple other conflicting findings challenge efforts to classify chronic GVHD as either a pure Th1 or Th2 disease.⁵⁸ Fujii, et al found that IFN-γ and IL-6 did not significantly differ between early or late chronic GVHD and controls; this challenges other reports implicating IFN-γ and IL-6.⁵⁹ A number of important negative findings were reported by Rozmus, et al:⁶⁰ There was no significant difference in IL-10 between early- or late-onset chronic GVHD and control; this challenges multiple reports implicating IL-10 in prediction and diagnosis of chronic GVHD. There was increased FoxP3 mRNA expression (p=0.08) in early onset chronic GVHD, while FoxP3 expression was lower in late-onset chronic GVHD patients (p=0.04); this finding adds to the complexity of the relationship between FoxP3 expression (not exclusive to Treg) and chronic GVHD. There was no correlation between cytokine patterns and organ involvement by chronic GVHD, and there was no correlation between cytokine expression and immune cell subsets. However, it should be noted that the overall sample size in this analysis limits definitive conclusions.

Other markers

Other diverse markers have been reported: Svegliati, et al detected anti-PDGFR antibodies in extensive chronic GVHD cases, but not in non-chronic GVHD cases or control subjects.⁶⁴ Lai, et al found that chronic GVHD patients had significant up-regulation of CD28 and PI3K compared to those without chronic GVHD.⁶⁵ Kohrt, et al reported that differential gene expression of several genes (ADAMTS2, IL-1R2, BCAT1, SRGAP1, ADAMTS3, AREG, DAP2IP, IRS2, CXCR7, and CPM) accurately distinguished chronic GVHD cases from controls.⁶³ Boutin, et al found that urinary collagen degradation markers were not different between chronic GVHD patients at onset prior to starting immune suppressive therapy vs. autologous HCT patients or normal controls.⁶⁶ McGuirk, et al demonstrated that serum haptoglobin (Hp) levels in patients with chronic GVHD were higher than in patients without chronic GVHD and normal controls.²⁰ While these findings largely do not coalesce in a theoretical framework with the other reported findings to date, they speak to potential heterogeneous mechanisms involved in chronic GVHD, the importance of discovery-based approaches, and the need for further validation studies.

Diagnosis restricted to specific chronic GVHD phenotypes

A small number of studies have examined biologic markers of certain organ manifestations: Westekemper, et al found that expression of the Th1-associated chemokines - chemokine (C-X-C motif) ligand (CXCL) 9 (Mig), CXCL10 (IP-10), and their receptor chemokine (C-X-C motif) receptor 3 (CXCR3) - were significantly increased in chronic GVHD conjunctival biopsies compared to controls.⁶⁷ However, no correlation was found between chemokine expression levels and severity of clinical findings. Compared to HCT recipients without oral chronic GVHD, Imanguli, et al found that patients with oral involvement had increased T-bet+ effector T cells, keratinocyte apoptosis, expression of CXCL9 (MIG) and CXCR3, and type I interferon-mediated signaling events including STAT1 phosphorylation.⁶⁸ Mattson, et al found that Serum CC16 (clara cell secretory protein) levels were significantly lower in BOS,⁴¹ and Kuzmina has reported higher percentage of CD19⁺CD21^low B cells in newly diagnosed BOS.⁶⁹

Biologic markers associated with chronic GVHD severity

Relatively little information exists on the relationship between chronic GVHD severity and biologic markers of chronic GVHD. The majority have been described in the context of a historic classification schema (limited vs. extensive disease): Barak, et al described that TNFα, IL-6, and IL-1β levels correlated with severity of established chronic GVHD.⁵⁴ Miura, et al found that FoxP3 expression inversely correlated with the extent of chronic GVHD.²⁷ Li, et al described that limited chronic GVHD had greater Treg, greater TGF-β, and lower TNF-α compared to extensive chronic GVHD.²⁶ Sarantopoulos, et al did not demonstrate significant difference in BAFF levels between limited and extensive chronic GVHD.^{38, 51}

More recent reports, however, have utilized the proposed NIH Consensus classification for chronic GVHD severity: Kuzmina, et al reported that those with severe chronic GVHD had significantly lower naïve B cells and non-class-switched memory B cells compared to those with mild and moderate chronic GVHD.⁵⁰ Imanguli, et al demonstrated that caspase-3, CD8, CD3, CD68, KI-67, IL-15, MxA levels were positively associated with increasing clinical chronic GVHD severity.⁶⁸

Biologic markers associated with chronic GVHD phenotype

Fujii, et al reported several correlations between studied biomarkers and sites of chronic GVHD organ involvement:⁵⁹ Hepatic involvement was associated with sBAFF and sCD13; ocular involvement was associated with anti-dsDNA and anticardiolipin antibody; joint involvement was associated with anti-dsDNA levels, sBAFF, IL-6, and MCP-1; sclerodermatous involvement was associated with anti-dsDNA; and lichenoid skin rash was associated with sBAFF. Svegliati, et al found that higher levels of anti-PDGFR antibodies were detected in those with extensive skin or lung involvement.⁶⁴ Kuzmina, et al reported multiple associations among B cell subsets and BAFF levels and chronic GVHD organ involvement:⁵⁰ Those with ocular involvement had the lowest CD19+ B cell and highest transitional B cell numbers; those with pulmonary involvement had lower total CD19+, elevated immature B cells, decreased naïve B cells and non-class switched memory B cells, and elevated BAFF levels; scleroderma was associated with higher immature B cells, lower non-class-switched and class-switched memory B cells, and higher BAFF levels.

Biologic markers associated with chronic GVHD response to therapy

Kobayashi, et al reported that patients with response to chronic GVHD therapy had a decrease in sIL-2R levels.⁵⁵ Fujii, et al reported that responders had significantly lower sIL-2Rα at chronic GVHD diagnosis than non-responders. Change over time after initial therapy appeared to be relevant: The ratio of 2 month level to that at start of therapy was studied as predictor of 9 month response. A lower ratio of sBAFF was correlated (p=0.05) with response, while the other studied markers were not.⁵⁹ Sarantopoulos, et al reported that responders to anti-CD20 therapy demonstrated naïve B cell reconstitution and decreased BAFF/B cell ratios.⁷⁰ Dander, et al reported limited data to support that IL-17 producing CD4+ cells on ELISPOT assay were associated with change in acute and chronic GVHD activity; clinical progression associated with increase in Th17 cells, while decline in organ scores correlated with decrease in Th17 cells.⁴³ Greinix, et al demonstrated a decreased ratio of CD21- immature/transitional B cells to CD27+ memory B cells over time among those who achieved partial response (vs. no response or progression) to chronic GVHD therapy.⁴⁹

Biologic markers associated with prognosis following chronic GVHD

Very few studies have demonstrated an association between biologic markers and survival. Pre-HCT polymorphism in MadCAM-1 was associated with decreased overall survival.¹⁴ In a small number of chronic GVHD cases, Kobayashi found that responders to therapy had a decrease in sIL-2R after therapy, while progressive increase in sIL-2R was associated with mortality.⁵⁵ This minimal data is far from definitive, and much more investigation is needed in this domain.

Conclusions and future directions

Major shortcomings in current practice and clinical investigation emphasize the potential importance of biologic markers of chronic GVHD. However, despite extensive investigation, there is little evidence that findings have facilitated a cohesive understanding, have translated into clinically useful tools, or have informed therapeutic strategies. We aim here to synthesize previously reported findings with attention to challenges in their interpretation, and to identify gap areas for future investigation.

Validated markers for risk of chronic GVHD development may permit avoidance of donors prone to cause chronic GVHD or alteration of transplant or GVHD approaches to mitigate these risks. While polymorphism in several cytokines appears to have importance, most are countered by other negative findings. Further work is needed to understand the relative importance of these findings in the context of known and emerging data in the field of donor-recipient HLA matching,^71–76 MHC haplotype SNP, ⁷⁷ and minor histocompatibility mismatch.^78–80 Published evidence supports deficiency of important regulatory cell populations (NK, Treg, DC2), and changes in post-HCT cytokines (increased TNFα, IL-10 and BAFF, and decreased TGFβ) as markers of subsequent chronic GVHD development.

Diagnostic markers for chronic GVHD may have several practical applications, such as incorporation into current diagnostic criteria, distinguishing chronic from late acute GVHD, and identification of therapeutic targets. Current evidence supports that chronic GVHD is associated with increased CD3+ T cells, Th17, CD4+ and CD8+ effector memory cells, monocytes, CD86 expression, and BAFF/B cell ratio. In contrast, chronic GVHD is associated with a deficiency of Treg, NK cells, and naïve CD8+ T cells. Evidence regarding specific B cell subsets is conflicting across reports and requires further study. Inflammatory mediators including TNFα, IL-6, IL-1β, IL-8, sIL-2R, and IL-1Ra have been reported, but require validation. BAFF, anti-dsDNA, sIL-2Rα, and sCD13 are supported by work from Fujii, et al, and have been tested and validated (these together with also elafin and MIG) by Kitko, et al.⁶² These represent important candidates for further validation.

Validated markers for chronic GVHD severity and phenotype have great potential for several applications. However, investigation performed to date is limited, focused on limited individual candidates, and lacks validation. Dedicated work in this area, particularly in the era of NIH Consensus severity and phenotype classification is needed. The associations summarized in this review are preliminary and require validation in larger studies.

Biologic markers associated with response and prognosis following treatment for chronic GVHD could greatly augment current clinical practice. Rational therapy could target mechanisms of resistance. Validated early markers of non-response could be used to identify non-responders before they develop worsening clinical chronic GVHD. There has been little investigation in this area to date, and this represents a priority area for future research.

There are several major challenges to consider: First, there is heterogeneity in the chronic GVHD cases included in different studies. Most pre-date the NIH Consensus guidelines, and therefore likely include late acute GVHD. There has been little attention to the impact of chronic GVHD organ involvement and severity on the studied outcomes. Time from HCT to sample acquisition (and matching cases/controls for this variable), interval between chronic GVHD onset and sample collection, and degree and type of immune suppression likely also impact results. As well, the bulk of studies deal with peripheral blood markers. While this represents a clinically feasible approach, biopsies at sites of chronic GVHD involvement may be important. The non-chronic GVHD controls in these studies also vary. Finally, most of the reported studies are small, have examined a limited number of candidates, conflict with other published results, and lack validation; these challenges may explain major contradictory results in the existing literature.

Thus, the major challenge for future investigation in chronic GVHD biologic markers will be to perform adequately powered studies utilizing well-characterized clinical populations with attention to selection of chronic GVHD cases and controls, the impact of other clinical variables including immune suppression, and subsequent validation of findings. Translation of such findings should provide clinically useful tools for assessing risk for chronic GVHD and chronic GVHD therapeutic response, and identify novel therapeutic strategies.

Glossary of abbreviated terms

ADAMTS2

A disintegrin and metalloproteinase with thrombospondin motifs 2

ADAMTS3

A disintegrin and metalloproteinase with thrombospondin motifs 3

Anti-dsDNA

anti-double stranded DNA antibody

AREG

amphiregulin

BAFF

B cell activating factor (tumor necrosis factor (ligand) superfamily, member 13b)

BCAT1

branched chain aminotransferase 1

BDCA1

CD1c molecule (T-cell surface glycoprotein CD1c)

BDCA2

C-type lectin domain family 4, member C (dendritic cell lectin b)

b-FGF

basic fibroblast growth factor

BOS

bronchiolitis obliterans syndrome

CC16

clara cell secretory protein

CCR6

chemokine (C-C motif) receptor 6

CCR7

chemokine (C-C motif) receptor 7

CCR9

chemokine (C-C motif) receptor 9

CD123

cluster of differentiation molecule 123 (interleukin 3 receptor, alpha)

CD127

cluster of differentiation molecule 127 (interleukin 7 receptor)

CD133

cluster of differentiation molecule 133 (prominin 1)

CD16

Fc fragment of IgG, low affinity receptor

CD19

cluster of differentiation molecule 19 (B-lymphocyte antigen CD19)

CD21

complement component (3d/Epstein Barr virus) receptor 2

CD25

cluster of differentiation molecule 25 (interleukin 2 receptor, alpha subunit)

CD27

cluster of differentiation molecule 27 (CD 27 antigen)

CD28

cluster of differentiation 28 (T-cell-specific surface glycoprotein CD28)

CD3

cluster of differentiation molecule 3 (T cell co-receptor; CD3-Tcell receptor complex)

CD34

cluster of differentiation molecule 34 (hematopoietic progenitor cell antigen CD34)

CD38

cluster of differentiation molecule 38 (CD38 molecule; ADP-ribosyl cyclase 1)

CD4

cluster of differentiation molecule 4 (T cell surface glycoprotein CD4)

CD56

cluster of differentiation molecule 56 (neural cell adhesion molecule 1)

CD68

cluster of differentiation molecule 68 (macrophage antigen CD68)

CD8

cluster of differentiation molecule 8 (T cell co-receptor)

CD86

cluster of differentiation molecule 86 (T-lymphocyte activation antigen CD86; B7-2 antigen)

CPM

carboxypeptidase M

CXCL10

chemokine (C-X-C motif) ligand 10

CXCR3

chemokine (C-X-C motif) receptor 3

CXCR7

C-X-C chemokine receptor type 7

DAP2IP

DOC-2/DAB2 interactive protein

DC

dendritic cell

EPC

circulating endothelial progenitor cells

FAS

TNF receptor superfamily, member 6

FCRL3

Fc receptor-like 3 gene

FoxP3

forkhead box P3

GVHD

graft vs. host disease

HCT

hematopoietic cell transplantation

HLA

human leukocyte antigen

Hp

haptoglobin

IFN-γ

interferon gamma

IgD

immunoglobulin D

IL-10

interleukin 10

IL-12

interleukin 12

IL-15

interleukin 15

IL-17

interleukin 17

IL-1R2

interleukin 1 receptor, type II

IL-1RA

interleukin 1 receptor antagonist

IL-1β

interleukin 1 beta

IL-2

interleukin 2

IL-4

interleukin 4

IL-6

interleukin 6

IL-8

interleukin 8

IRS2

insulin receptor substrate 2

KI-67

MKI67 (proliferation-related Ki-67 antigen)

MadCAM-1

Mucosal addressin cell adhesion molecule–1

MCP-1

monocyte chemoattractant protein 1

MICA

MHC class I–related chain A

MIG

chemokine (C-X-C motif) ligand 9

MxA

interferon-induced MxA GTPase

NK

natural killer cell

PARP1

poly (ADP-ribose) polymerase 1

PBSC

peripheral blood mobilized stem cells

PI3K

phosphatidylinositol-4,5-bisphosphate 3 kinase

sCD13

soluble CD13 (aminopeptidase-N)

sIL-2R

soluble alpha chain of the IL-2 receptor

SRGAP1

slit-robo GTPase 1

STAT1

signal transducer and activator of transcription 1

TGF-β

transforming growth factor beta

Th1

T helper 1 cells

Th17

T helper 17 cells

Th2

T helper 2 cells

TNFSF10

tumor necrosis factor (ligand) superfamily, member 10

TNFSF12

tumor necrosis factor (ligand) superfamily, member 12

TNFSF3

lymphotoxin beta (TNF superfamily, member 3)

TNFα

tumor necrosis factor alpha

TREC

T cell receptor gene rearrangement excisional circles

Treg

regulatory T cell

VEGF

vascular endothelial growth factor