Graft-Versus-Host Disease

Increased incidence of chronic GvHD and CMV disease in patients with vitamin D deficiency before allogeneic stem cell transplantation

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Abstract

Vitamin D has emerged as a central player in the immune system, with its deficiency being implicated in the pathogenesis of several autoimmune diseases, including chronic GvHD. This is a retrospective cohort analysis of 166 patients, who underwent allogeneic hematopoietic stem cell transplantation (HSCT) at the Karolinska University Hospital, evaluating GvHD, graft failure, infectious complications and survival after HSCT in relation to pre-transplantation vitamin D levels. Most of the patients were deficient in vitamin D before HSCT (median 42 nmol/L). In multivariate analysis, vitamin D level before HSCT was identified as a significant independent risk factor for development of cGvHD. The increased incidence of cGvHD was not coupled to better disease-free survival; instead there was a trend towards lower overall survival in the vitamin D-deficient patients. In addition, we found a significant correlation between vitamin D deficiency and incidence of CMV disease, with no case of CMV disease occurring in patients with sufficient levels of vitamin D before HSCT. Our results support a role of vitamin D in immune tolerance following HSCT. These findings could be highly relevant for the care of HSCT patients, and prospective, randomized studies on the effect of vitamin D supplementation are therefore needed.

Introduction

Allogeneic hematopoietic stem cell transplantation (HSCT) is an important and potentially curative treatment of haematological malignancies, but GvHD and infections affect outcomes.

Vitamin D has emerged as a central player in the immune system,1 affecting T and B cells, macrophages and dendritic cells (DCs).2, 3, 4 A vitamin D-enriched milieu maintains an immature DC phenotype associated with decreased ability to stimulate alloreactive T cells in MLCs.5 Vitamin D deficiency has been associated with an increased incidence of chronic GvHD,6 and supplementation with vitamin D to induce a tolerogenic DC population has been suggested for prevention of GvHD.7 One mechanism for this maintenance of an immature DC population has been shown to be the upregulation of indoleamine 2,3-dioxygenase (IDO),8 an enzyme converting tryptophan to kynurenine and a central feature in tolerizing DCs.9

Contrary to the suppressive effects on the immune system, vitamin D also has a protective effect against infections. This was first shown in tuberculosis, where the traditional treatments with sunlight and cod liver oil, rich in vitamin D, are viewed in a new light after the discovery of antimicrobial peptides induced by vitamin D. The two antimicrobial peptides under the influence of vitamin D are LL-37 (cathelicidin) and β-defensin 2,10, 11 which have activity against several bacteria, as well as viruses12 and fungi.13

We present here what we believe is currently the largest epidemiological study on adult HSCT patients examining clinical outcome with regards to vitamin D deficiency before HSCT, including both infectious and non-infectious complications.

Patients and methods

Patients

The study was conducted in accordance with the Helsinki Declaration upon approval by the regional ethics committee in Stockholm. Serum specimens were collected from 166 consecutive patients (>12 years of age) undergoing HSCT between 2005 and 2011 at the Karolinska University Hospital, Huddinge, (Table 1). Serum samples from 20 healthy donors were used as controls.

Table 1 Patient characteristics

All data were taken from the patient's medical records. A total of 139 patients were eligible for evaluation of chronic GvHD (cGvHD) excluding patients with graft failure (n=13) or with a survival after HSCT of <100 days (n=14). For analysis of infectious complications, only patients with a follow-up at the Karolinska University Hospital were selected (n=137). When calculating disease-free survival (DFS), only patients with haematological malignancies (n=156) were considered.

For IDO analysis, 24 patients with vitamin D levels above or below 50 nmol/L were selected; 14 with cGvHD and 10 without cGvHD (Table 2). They were selected based on the following criteria: clear presence or absence of classical chronic GvHD (grade moderate to severe), no donor lymphocyte infusions or stem cell booster and no photopheresis.

Table 2 Patients included in IDO analysis

Follow-up ranged between 36 and 107 months after HSCT, with a median follow-up of 71 months.

Definitions

For cGvHD diagnosis and scoring, the NIH consensus criteria were used14 and both classic chronic GvHD and overlap syndrome were included, but not late-onset acute GvHD. Only cGvHD of moderate and severe grades was considered in the analysis.

Graft failure was defined as the lack of engraftment, engraftment with recipient cells or later developing full (>95%) recipient chimerism in the absence of relapse of the underlying disease. DFS was defined as survival with no evidence of relapse or progression of malignant disease. Overall survival (OS) was defined as the time from HSCT to death, regardless of the cause.

We evaluated both agent-specific and non-agent-specific infectious complications during the first year after HSCT, censoring the follow-up at relapse of the malignant disease. Agent-specific outcomes were CMV disease, EBV-associated post-transplantation lymphoproliferative disorder, influenza, invasive fungal disease and bacteraemia. Non-agent-specific outcomes were pneumonia and days on IV antibiotics. Bacteraemia and days on IV antibiotics were analysed separately for neutropenic and non-neutropenic episodes.

CMV disease was defined according to Ljungman et al.15 Invasive fungal disease was defined according to De Pauw.16 Only probable and proven infections were considered in the analysis. Diagnosis of pneumonia required either a combination of new pulmonary infiltrates on chest X-ray or CT scan with symptoms of respiratory infection such as cough, dyspnoea or fever, excluding idiopathic pulmonary syndrome or autopsy-verified infectious pneumonia. Bacteraemia was defined as the first positive blood culture during a 10-day time period. Repeated positive blood cultures >10 days after the first were considered new episodes.

Prophylaxis and surveillance

GvHD prophylaxis consisted of cyclosporin A in combination with methotrexate or prednisone, or tacrolimus in combination with sirolimus. Patients with an unrelated donor were given anti-T-cell therapy, most commonly ATG (see Table 1 for details). Patients with a related donor did not receive anti-T-cell therapy. Patients were routinely monitored for CMV reactivation with PCR and pre-emptively treated as described previously.17 Antifungal, antibacterial and Pneumocystis jiroveci prophylaxis was given as previously described.18

Serum analysis

Vitamin D levels were measured in serum specimens collected before the start of conditioning therapy and in 85 of the patients also at 6 and/or 12 months after HSCT. Specimens were collected and handled according to current routine standards at the hospital laboratory. In brief, blood samples collected in serum tubes were left for 30–120 min at room temperature, or up to 12 h at +8 °C, before serum separation by centrifugation. Specimens were stored in liquid nitrogen until analysis.

Serum vitamin D was measured as 25-OH-cholecalciferol and expressed as nmol/L, hereafter referred to as 25-OH-D3. Analyses were performed by the laboratory for clinical chemistry (Karolinska University Hospital, Solna) using a chemiluminescence method (CLIA) approved by SWEDAC. Vitamin D deficiency was defined as <25 nmol/L, insufficiency as 26–49 nmol/L and sufficiency as 50 nmol/L, in accordance with the Nordic Guidelines.19

Serum levels of tryptophan metabolites were analysed before and at 1, 2, 3, 6, 12 and 24 months post HSCT by liquid chromatography-tandem mass spectrometry.20 IDO activity was estimated by calculating the serum kynurenine-to-tryptophan ratio. A median of six out of seven time points per patient (range 2–7) were available for analysis.

Statistical analysis

This is a retrospective cohort study. Data were analysed as of date of the last data collection, 14 April 2014. Sample size was based on a power analysis using data from a pilot study (unpublished). Primary end point was chronic GvHD; secondary end points were OS, DFS, graft failure and incidence of infectious complications. Cumulative incidence functions were used to estimate GvHD, considering death and relapse to be competing events. Probabilities of DFS and OS were calculated using the Kaplan–Meier estimates. Univariate analyses were performed using Gray’s test for cumulative incidence functions and the log-rank test for DFS and OS. Associations of patient and graft characteristics with outcomes were evaluated in multivariate analysis, using Cox proportional hazards model for dichotomous variables or negative binomial regression analysis for outcomes with repeated events or continuous variables. For two-sample comparisons, the Wilcoxon rank-sum test or Fisher’s exact test was used. GraphPad Prism 6 (GraphPad software, San Diego, CA, USA), IBM SPSS version 21 (SPSS Inc./IBM, Armonk, NY, USA) and R 3.0.1 software were used.

Results

Serum levels of 25-OH-D3 in HSCT patients

The median level of 25-OH-D3 for all 166 patients before transplantation was 42 nmol/L (range 10–118), below the deficiency level of 50 nmol/L (20 ng/mL)21 and significantly lower than in the healthy controls (median 66.5 nmol/L, range 21–104, P<0.001). Only 59 patients (36%) had sufficient levels of 25-OH-D3 (50 nmol/L) before HSCT, whereas the majority (N=88, 53%) were 25-OH-D3 insufficient (26–49 nmol/L) and 19 (11%) were even deficient (<25 nmol/L). The baseline characteristics of the patients divided into three groups based on 25-OH-D3 levels are summarised in Table 1.

During the first 6 months after transplantation, 25-OH-D3 levels decreased significantly (P<0.001) to a median level of 39 nmol/L (range 10–116; Figure 1). Vitamin D levels are not regularly monitored in the clinic, and only 18 patients (11%) received any kind of vitamin D supplement during the first year after transplantation, with a median dose of 800 IE daily (range 400–4000 IE). From 10 patients receiving vitamin D supplementation, serum specimens at 6 months post HSCT were available for testing. In these patients, the 25-OH-D3 levels showed no increase at 6 months (median 38, range 10–101 nmol/L) compared with the pre-transplantation levels (median 50, range 13–83 nmol/L).

Figure 1
figure1

Alterations of 25-OH-D3 levels in HSCT patients. Serum levels of 25-OH-D3 (median with interquartile range) in HSCT patients before conditioning (w0), and 26 and 52 weeks, respectively, after transplantation, as well as in healthy donors.

Acute and chronic GvHD

The cumulative incidence of acute GvHD grade II–IV was 42% in patients with vitamin D deficiency (<25 nmol/L), 48% in patients with insufficiency (26–49 nmol/L), and 37% in patients with sufficient vitamin D levels (50 nmol/L), showing no correlation between the 25-OH-D3 level at the time of transplantation and incidence of aGvHD. For chronic GvHD (NIH grade moderate or severe), however, the 2-year cumulative incidence was 56% in patients with vitamin D deficiency, compared with 31% in patients with insufficiency, and 21% in the vitamin D sufficient group (P=0.01; Figure 2). This association between 25-OH-D3 levels and cGvHD was confirmed in a multivariate landmark analysis at 100 days with death and disease relapse as competing risks, including vitamin D status (deficient, insufficient, sufficient), age, stem cell source, anti-T-treatment, haematological malignancy and sex mismatch (female donor to male recipient) as covariates. This analysis identified vitamin D status before HSCT as a significant independent risk factor for development of moderate to severe cGvHD (P=0.04). HLA mismatch (<8/8) was not associated with cGvHD in this material, and including it in the analysis did not change the result. The effect of 25-OH-D3 levels appeared to be continuous at the lower intervals, with a threshold at 60 nmol/L. The relative risk of cGvHD at 2 years after HSCT, measured as risk ratio, was 2.66 (95% CI: 1.03–6.87) for 25-OH-D3 below 60 nmol/L as compared with above 60 nmol/L before HSCT.

Figure 2
figure2

Cumulative incidence of cGvHD. Cumulative incidence of chronic GvHD, grade moderate-severe, in cohorts stratified by levels of 25-OH-D3 before transplantation. (N=139)

Survival and relapse

The 2-year OS was 63% in patients with vitamin D deficiency (<25 nmol/L), 69% in patients with insufficiency (26–49 nmol/L) and 76% in patients with sufficient levels (50 nmol/L; P=0.24; Figure 3a). Vitamin D level was significantly associated with OS when adjusted for age (P=0.02; Table 3), and in a multivariate model including age, stem cell source, anti-T-cell treatment, sex mismatch and haematological malignancy, a lower 25-OH-D3 level was a significant risk factor for death (P=0.03). The 2-year DFS showed no significant differences between the groups, with 59% DFS in both the vitamin D-deficient and insufficient groups, versus 67% in the vitamin D sufficient group (Figure 3b). There was no significant difference in relapse incidence between the vitamin D-deficient (19%) and sufficient patients (21%).

Figure 3
figure3

(a) Overall survival. OS, in cohorts stratified by levels of 25-OH-D3 before transplantation. (N=166). (b) Disease-free survival. DFS, in cohorts stratified by levels of 25-OH-D3 before transplantation. Only patients with haematological malignancies (N=156).

Table 3 Results of multivariate analysis of cGvHD and overall survival

Graft failure

Patients developing graft failure showed a trend towards lower pre-HSCT levels of 25-OH-D3 with a median level of 34 nmol/L (range 16–65), as compared with 43 nmol/L (range 10–118) in the other patients (P=0.06). As 11 out of 13 cases of graft failure were in patients having received reduced intensity conditioning (RIC), and RIC has previously been shown to be a risk factor for graft failure,22 we chose this subgroup for a post hoc analysis (Figure 4). The incidence of graft failure in the RIC subgroup was significantly higher in patients with 25-OH-D3 below the median level of 42 nmol/L (P=0.03). Within this subgroup, 25-OH-D3 levels were not significantly associated with other known risk factors for rejection, anti-T-cell treatment, HLA mismatch or non-malignant disease.22 Low 25-OH-D3 levels were more common among patients receiving cord blood transplants, but removing these patients from the analysis did not change the result.

Figure 4
figure4

Graft failure. Molar levels (nmol/L) of serum 25-OH-D3 before transplantation in patients with and without graft failure. Median with interquartile range. Only patients with reduced intensity conditioning (N=98).

Infectious complications

From separate analysis of each outcome, we found that the serum levels of 25-OH-D3 before transplantation were significantly correlated to CMV disease (P=0.005) and to days on IV antibiotics during the non-neutropenic period (P=0.011). After a Bonferroni correction to account for multiple comparisons, only CMV disease remained statistically significant.

There were a total of nine confirmed cases (incidence 6.6%) of CMV disease in the cohort, presenting as retinitis (N=1), colitis (N=4), gastritis (N=1) and multi-organ disease, including the lung (N=3). In all these patients, 25-OH-D3 levels before HSCT were below the insufficiency level (50 nmol/L), and four were below the deficiency level of 25 nmol/L. The correlation between the 25-OH-D3 levels and CMV disease remained statistically significant (P=0.004) when adjusted for patient age, CMV serologic mismatch, stem cell source, anti-T-cell therapy, acute GvHD, chronic GvHD and graft failure in a Cox proportional hazards model, with death and relapse as competing risks.

Further evaluations of the effect of 25-OH-D3 levels on CMV immunity revealed no association between the 25-OH-D3 level and peak viral load, repeated or prolonged CMV reactivations or viral replication kinetics.

Correlation analysis between serum 25-OH-D3 levels and IDO activity

As tolerogenic DCs produce high levels of IDO,8, 9 we used IDO activity in serum as a surrogate marker for DC maturation. A subgroup of 24 patients (Table 2) was selected for analysis of serum IDO activity. There was no significant correlation between pre-HSCT 25-OH-D3 levels and post HSCT IDO activity (Figure 5a). In patients with cGvHD (n=14), IDO activity increased at 52 weeks, irrespectively of the serum level of 25-OH-D3, as compared with non-cGvHD patients (P=0.005; Figure 5b). At this time point, all of the 14 patients with cGvHD had developed at least mild symptoms of cGvHD, and 10 had moderate to severe cGvHD.

Figure 5
figure5

(a) IDO activity in relation to vitamin D level. IDO activity, measured as fraction kynurenine/tryptophan in serum, in groups by levels of 25-OH-D3 before transplantation. High vitamin D equals above 50 nmol/L, low vitamin D equals below 50 nmol/L. Bars showing minimum to maximum value. (b) IDO activity in relation to cGvHD. IDO activity, measured as fraction kynurenine/tryptophan in serum, in groups by cGvHD status. Bars showing minimum to maximum value. **=Significant difference at 52 weeks (P<0.005).

Discussion

Activation of the immune system requires a finely-tuned balance between pro- and anti-inflammatory mechanisms, with negative feedback machinery being an integral part. In the development and perpetuation of GvHD, this elegant system is out of balance, causing an excess activation of the immune responses. Substantially extending previous reports, our analysis confirms that the majority of patients are insufficient in vitamin D at time of HSCT, and that low levels of vitamin D before transplantation significantly increase the risk of developing cGvHD. In addition, our results further indicate that low vitamin D levels before transplantation could represent a significant risk factor for CMV disease, graft failure and death. This is in line with the notion that sufficient vitamin D levels during the early post-transplantation phase are important for establishing a healthy and balanced immune system. A so-called cytokine storm23 and the initial immune reconstitution coincide during this period emphasising the imperative of important immune co-factors including vitamin D being present. Circulating 25-OH-D3 has a half-life of about 2 weeks, but the functional half-life considering generation from cholecalciferol stores in the body is considered to be 2–3 months.24 Thus, we presume the pre-transplantation levels to reflect the milieu during this critical period, whereas unbiased by other early post transplantation events such as aGvHD.

The actions of vitamin D on the immune system are complex, and not fully understood.1 We measured IDO activity in the patient sera as a possible marker for tolerogenic DCs,9 and found an upregulation in patients with cGvHD consistent with previous findings in acute GvHD25 but not previously shown in cGvHD. This probably reflects an adequate but insufficient upregulation of immunosuppressive mechanisms in response to an inflammatory milieu. We failed to find an association between IDO activity and vitamin D levels. This might be because IDO activity in serum by circulating monocytes26 overshadows DC activity in the tissues, but more studies focusing on vitamin D effects in vivo are needed.

Previous studies have shown conflicting results; although one study on 53 adult patients suggests an association between vitamin D levels and cGvHD incidence,6 others have reported better outcomes in established cGvHD after vitamin D supplementation.27 A recent study on 123 pediatric patients failed to show an association between pre-transplantation vitamin D levels and cGvHD incidence.28 The general incidence of cGvHD in the pediatric study was very low, only 14%, which could explain why no association was seen. This study comprises a larger, but heterogeneous patient cohort with some relevant variables varying significantly between the groups split by vitamin D levels.

The main end point of this study was incidence of chronic GvHD. The findings of associations between pre-transplantation vitamin D levels and CMV disease as well as graft failure should be interpreted with caution due to the low incidence of these complications, and further studies are needed, particularly prospective, randomized studies of the effects of vitamin D supplementation. As this study was performed in Scandinavia, with a majority of patients being Caucasian and sun exposure being low for most of the year, it would also be interesting to see if the findings can be confirmed in other parts of the world.

Patients undergoing allogeneic HSCT have repeatedly been shown to suffer from vitamin D deficiency.29, 30 In line with this, we observed a median vitamin D level before transplantation below sufficiency levels and the deficiency being further aggravated in the post-transplant period. It is likely that hospitalization and the prevailing recommendations to avoid sun exposure to prevent triggering of GvHD partly explain the decline in vitamin D levels following transplantation observed in this study. Given the increased risk of skin cancer associated with HSCT and sun exposure,31 we would hesitate to recommend increased sunbathing for transplanted patients. An alternative is oral supplementation before or during immune reconstitution. Acute GvHD of the gut and mucositis can be expected to reduce the intestinal uptake of oral vitamins, and the amount of vitamin D in parenteral nutrition is negligible. Indeed, in the few patients in this study receiving vitamin D substitution, no increase in vitamin D levels was seen. Most probably high doses of oral vitamin D, possibly 4000–6000 IE daily,32 starting before transplantation, would be needed to significantly increase serum levels.

It still remains to be answered which cutoff value of serum vitamin D is clinically relevant. We have chosen to use 25 and 50 nmol/L, respectively, as these are the cutoffs used in the Nordic Guidelines for deficiency and insufficiency.19 However, these levels mainly reflect the risk of rickets and osteoporosis and might not be relevant for optimal immunological reconstitution.

The importance of this finding lies not only with HSCT patients, but has general implications for future interventional trials. Several observational studies indicate associations between vitamin D deficiency and disorders of the immune system, such as multiple sclerosis,33 type 1 diabetes,34 rheumatoid arthritis35 and rejection of solid organ allografts.36 Some studies have also shown beneficial effects of vitamin D supplementation, most notably in the prevention of type 1 diabetes,37 and amelioration of symptoms of multiple sclerosis by treatment with high dose vitamin D.38 However, the relevance of these findings is questioned due to the lack of prospective, randomized trials. Such trials are difficult to carry out in diseases with low incidence and slow onset, such as diabetes type 1. Hence, cGvHD presents an appealing model for testing the hypothesis of vitamin D supplementation in the prevention of immune disorders.

To summarise, in a large cohort of patients, we confirm previous smaller studies indicating an association between low vitamin D levels and an increased incidence of cGvHD. We also show indications of a possible impact on OS and CMV disease, as well as graft failure in patients receiving non-myeloablative conditioning. Whether substitution with vitamin D improves the outcome is unknown. If that were the case, these findings would be highly relevant for the care of HSCT patients, warranting prospective, randomized studies on the effect of vitamin D supplementation.

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Correspondence to L von Bahr.

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The authors declare no conflict of interest.

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Author Contributions

LvB was the principal investigator. LvB, DM and KLB planned the study. AB and K-JM collected the patient samples. LvB, OB and ALB collected the clinical data. PJO performed the IDO analysis. ML performed the statistical analysis. LvB, JA, AB, K-JM, DM, PL and KLB analysed the results and wrote the paper. KLB takes responsibility for the integrity of the data and the accuracy of the data analysis.

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