Biol Blood Marrow Transplant 21 (2015) 2079e2085

Biology of Blood and

Marrow Transplantation
journal homepage: www.bbmt.org

Biology: Biomarkers

Tear Cytokines as Biomarkers for Chronic

Graft-versus-Host Disease
Ji Won Jung 1, 2, Soo Jung Han 1, Mi Kyung Song 3, Tae-im Kim 1, Eung Kweon Kim 4,
Yoo Hong Min 5, June-Won Cheong 5, Kyoung Yul Seo 1, *
1
Department of Ophthalmology, Severance Hospital, Yonsei University College of Medicine, Seoul, South Korea
2
Department of Ophthalmology and Inha Vision Science Laboratory, Inha University School of Medicine, Incheon, South Korea
3
Biostatistics Collaboration Unit, Yonsei University College of Medicine, Seoul, South Korea
4
Department of Ophthalmology, Corneal Dystrophy Research Institute, Yonsei University College of Medicine, Seoul, South Korea/Institute of Vision Research, Severance
Biomedical Science Institute, Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea
5
Division of Hematology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea

Article history:
Received 16 June 2015
Accepted 14 August 2015
Key Words:
Graft-versus-host disease
Tear cytokines
Interleukin (IL)-10
Interleukin (IL)-6
Tumor necrosis factor (TNF)-a
Chronic ocular GVHD severity
a b s t r a c t
We investigated the tear cytokine profiles in patients who underwent stem cell transplantation (SCT) and
attempted to evaluate whether tear cytokines are associated with the presence of systemic chronic
graft-versus-host disease (GVHD), regardless of ocular GVHD status. We also tested tear cytokines as bio-
markers for chronic ocular GVHD severity. Forty-four patients who underwent SCT were enrolled and their
diagnosis of chronic GVHD was confirmed. Ocular surface parameters and tear cytokine profiles were eval-
uated and the correlations between concentrations of cytokines and ocular surface parameters or several
chronic ocular GVHD severity scales were evaluated. Tear interleukin (IL)-2, IL-10, IL-17a, interferon (IFN)-g,
IL-6, and tumor necrosis factor (TNF)-a were elevated in patients with chronic systemic GVHD compared with
patients without chronic systemic GVHD. Receiver-operating characteristic curve analysis revealed that area
under the curve (AUC) values for tear IL-10 (AUC ¼ .795), IL-17a (AUC ¼ .821), IL-6 (AUC ¼ .912), and TNF-a
(AUC ¼ .910) were significantly correlated with the presence of chronic GVHD (all P < .001). Tear IL-10, IL-6,
and TNF-a showed a stronger correlation with ocular surface parameters than other cytokines and these
cytokines also correlated with several chronic ocular GVHD severity scales (all P < .05). Our data suggest the
tear cytokines are useful biomarkers for the diagnosis of chronic GVHD after SCT and chronic ocular GVHD
severity.
Ó 2015 American Society for Blood and Marrow Transplantation.

INTRODUCTION and B cells, stimulate aberrant production of cytokines,

Chronic graft-versus-host disease (GVHD) is the main
cause of morbidity and mortality after allogeneic hemato-
poietic stem cell transplantation (SCT) [1]. More than 50% of
patients who receive allogeneic bone marrow transplants
develop chronic GVHD [2]. Furthermore, in peripheral blood
stem cell recipients, the risk of chronic GVHD is known to be
higher than that observed in bone marrow transplantation
patients [3]. Aberrant immune system recovery is thought to
play an important role in GVHD pathophysiology [4,5]. Im-
mune cells, including alloreactive and autoreactive T cells
Financial disclosure: See Acknowledgments on page 2084.
* Correspondence and reprint requests: Kyoung Yul Seo, Department of
Ophthalmology, Yonsei University College of Medicine, 50 Yonsei-ro,
Seodaemun-gu, Seoul 120-752, South Korea.
E-mail address: ellul@naver.com (K.Y. Seo).
which play important roles in chronic GVHD [6-8]. Several
biological markers, including these cytokines, have been re-
ported for the prediction and diagnosis of chronic GVHD, and
the markers were mainly identified in blood samples [9].
Recently, tear analysis has been demonstrated to be a
useful biomarker for various ocular surface diseases [10-12],
including dry eye disease, meibomian gland disease, and
allergies, as well as systemic diseases such as diabetes mel-
litus [13], cancer [14], and thyroid-associated disease [15].
These biomarkers also indicate disease progression and
response to treatment [16]. Although tear fluid analysis
needs clinical validation, its benefits lie in its fast, easy, and
noninvasive sampling [11]. A few studies have reported
increased levels of tear cytokines in patients with chronic
ocular GVHD [17,18].
The purpose of the present study was to investigate tear
cytokine profiles in patients who underwent SCT and to

http://dx.doi.org/10.1016/j.bbmt.2015.08.020
1083-8791/Ó 2015 American Society for Blood and Marrow Transplantation.
2080 J.W. Jung et al. / Biol Blood Marrow Transplant 21 (2015) 2079e2085

determine the ability of tear-derived biomarkers to confirm determine the utility of tear cytokines as biomarkers to diagnose chronic
the presence of chronic GVHD. Furthermore, we evaluated GVHD regardless of ocular GVHD diagnosis. The secondary outcomes were
correlations between tear cytokines and ocular surface parameters or
tear cytokines as biomarkers for chronic ocular GVHD several criteria assessing the severity of ocular GVHD to evaluate the value of
severity by assessing the correlation of ocular surface pa- tear cytokines as biomarkers for chronic ocular GVHD severity.
rameters and several established chronic ocular GVHD All patients underwent slit-lamp examination to measure the ocular
severity scales. surface staining score, fluorescein tear film break-up time (TBUT), and
conjunctival injection grade. The patients underwent Schirmer’s test I, and
the ocular surface disease index (OSDI) questionnaire was completed. Tear
MATERIALS AND METHODS samples were collected and tear cytokine concentrations were measured.
Patients TBUT was measured by applying a single fluorescein strip (Haag-Streit,
This study followed the tenets of the Declaration of Helsinki, and the Koeniz, Switzerland) to the inferior palpebral conjunctiva after instilling a
prospective study protocol was approved by the Severance Hospital Insti- drop of normal saline. The mean time for 3 attempts was recorded. After
tutional Review Board, Seoul, South Korea. Informed consent was obtained measuring TBUT, ocular surface staining was graded from 0 to 5 based on the
from all patients after explaining to them the purpose and possible conse- pattern of fluorescein staining noted on slit-lamp biomicroscopy [21].
quences of the study. Conjunctival injection grade was scored as 0, 1, or 2 [20]. Schirmer’s test I
Forty-four patients were enrolled in this study and they consulted our was performed without topical anesthesia by placing a standard paper strip
ophthalmology clinic after SCT for mainly ocular GVHD screening without in the mid-lateral portion of the lower fornix. The amount of wetting was
ocular symptoms or ocular complaints. Male or nonpregnant female pa- recorded after 5 minutes, and patients were asked to keep their eyes lightly
tients ages 15 to 60 years who visited the clinic 3 to 12 months after SCT closed during the test. Subjective symptoms were graded on a numerical
were included in the study. We excluded patients with histories of ocular scale from 0 to 4, according to the validated 12-item OSDI questionnaire.
surgery; ocular injury; and ocular diseases such as ocular infection, allergy, Total OSDI was calculated using the following formula: OSDI ¼ (sum of
and autoimmune disease, as well as patients with long-term use of topical scores for all questions answered  100)/(total number of answered
ocular medications except artificial tears. Diagnostic criteria for chronic questions  4), which ranges from 0 to 100 [22]. Classification of conjunc-
GVHD used the National Institute of Health (NIH) consensus criteria [19] tivitis in chronic GVHD [23], the NIH eye score [19], and a recently reported
defined as follows: “diagnosis of chronic GVHD requires the presence of at severity scale by the international chronic ocular GVHD consensus group
least 1 diagnostic clinical sign (eg, polikiloderma or esophageal web) or the [20] were used as the criteria for assessing the severity of ocular GVHD to
presence of at least 1 distinctive manifestation (eg, keratoconjunctivitis evaluate the correlation with tear cytokine concentrations (Table 2).
sicca) confirmed by pertinent biopsy or other relevant tests in the same or
another organ” [19,20].
Tear Collection and Multiplex Bead Analysis
Patients with and without chronic GVHD were compared for de-
To collect tear samples, 30 mL of phosphate-buffered saline was instilled
mographic and clinical characteristics (Table 1). No significant differences
into the inferior fornix and 20 mL of tear fluid and buffer were collected with
were detected for age and period after SCT (P ¼ .919 and .173, respectively).
a micropipette at the medial and lateral canthus. To minimize ocular surface
irritation, we collected the mixture of tear fluid and buffer solution as soon
Outcome Measures as possible, without the use of anesthetic eye drops. The sample was
The primary outcome measures of this study were tear cytokine profiles. transferred into .5-mL Eppendorf tubes (Eppendorf, Fremont, CA) and stored
These results were compared for patients with and without chronic GVHD to at 70 C until further analysis by immunoassay. Cytokine concentrations
were measured using a multiplex immunobead assay (BDTM Cytometric
Bead Array Human Soluble Protein Flex Set; BD Biosciences, San Jose, CA).
Table 1 The analyzed cytokines were interleukin (IL)-2, IL-10, IL-17a, IL-4, interferon
Demographics of Patients with and without Chronic GVHD after SCT (INF)-g, IL-6, and tumor necrosis factor (TNF)-a. The measurements were
performed as previously described [24,25]. Data were acquired and analyzed
Characteristic No Chronic Chronic GVHD using BD Cytometric Bead Array software, which calculated the cytokine
GVHD after after SCT concentration on the basis of standard curves and a 4-parameter logistic
SCT (n ¼ 14) (n ¼ 30)
Age, yr* Table 2
Median (range) 36.5 (16-59) 39.0 (15-51) Chronic Ocular GVHD Severity Scales
Mean  SD 36.5  9.9 36.0  12.4
Classification of Conjunctivitis in Chronic GVHD [23]
Female, n (%) 6 (42.9%) 11 (36.7%)
0 None
Hematologic diagnosis, n (%)
1 Hyperemia
AML 5 (35.7%) 14 (46.7%)
2 Palpebral conjunctival fibrovascular changes with or without
ALL 6 (42.9%) 15 (50.0%)
epithelial sloughing
Non-Hodgkin’s lymphoma 0 (0.0%) 1 (3.3%)
3 Palpebral conjunctival fibrovascular changes involving 25% to 75% of
Aplastic anemia 3 (21.4%) 0 (0.0%)
total surface area
Stem cell source, n (%)
4 Involvement of >75% of total surface area with or without cicatricial
PB 13 (92.9%) 24 (80.0%)
entropion
BM 1 (7.1%) 6 (20.0%)
NIH-Defined Ocular Scoring in Chronic GVHD [19]
Manifestations of chronic GVHD at time
0 No dry eye symptoms
of tear sampling, n (%)
1 Dry eye symptoms not affecting ADL (eye drops 3 per day) or
Gastrointestinal tract 5 (16.7%)
asymptomatic signs of keratoconjunctivitis sicca
Lung 12 (40.0%)
2 Dry eye symptoms partially affecting ADL (eye drops >3 per day or
Liver 13 (43.3%)
punctual plugs) without vision impairment
Oral cavity 17 (56.7%)
3 Dry eye symptoms significantly affecting ADL (special eye wear to
Skin 24 (80.0%)
relieve pain) or unable to work because of ocular symptoms or loss
Eye 27 (90.0%)
of vision caused by keratoconjunctivitis sicca
Systemic immunosuppressive treatment
at time of tear sampling, n (%) Severity Scale in Chronic Ocular GVHD by the International Chronic
None 2 (14.3%) 2 (6.7%) Ocular GVHD Consensus Group [20]
Calcineurin inhibitor 3 (21.4%) 3 (10.0%)
Steroid 6 (42.9%) 17 (56.7%) Severity Schirmer’s CFS OSDI Conj
Calcineurin inhibitor þ steroid 3 (21.4%) 8 (26.7%) Scores Test (mm) (Points) (Points) (Points)
Period after SCT, monthsy (Points)
Median (range) 9.5 (4-12) 9.4 (3-12) 0 >15 0 <13 None
Mean  SD 8.4  3.6 9.9  2.7 1 11-15 <2 13-22 Mild/Moderate
2 6-10 2-3 23-32 Severe
AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; PB,
3 5 4 33
peripheral blood; BM, bone marrow.
* Using Mann-Whitney U test (P value ¼ .919). ADL indicates activities of daily living; CFS, corneal fluorescein staining;
y
Using Mann-Whitney U test (P value ¼ .173). Conj, conjunctival injection.
 J.W. Jung et al. / Biol Blood Marrow Transplant 21 (2015) 2079e2085
curve-fitting model. Flow cytometry was performed using the BD LSRII
system (BD Biosciences). The lower limits of detection were as follows: IL-2,
2.6 pg/mL; IL-10, 4.5 pg/mL; IL-17a, 18.9 pg/mL; IL-4, 4.9 pg/mL; IFN-g, 3.7
pg/mL; IL-6, 2.4 pg/mL; and TNF-a, 3.8 pg/mL. The value 0 was used for
statistical comparison between tear samples with concentrations below the
value of the lowest cytokine concentration (detection limit) in the linear
portion of the standard curve.
Statistical Analysis
The Mann-Whitney U test was used to compare the outcomes between
patients with and without chronic GVHD. The effect of tear cytokine con-
centration on the diagnosis of chronic GVHD was assessed after adjusting for
age, sex, and the period after SCT by a logistic regression model. Receiver
operating characteristic curves were generated to provide evidence of a
biomarker’s discriminating ability for chronic GVHD diagnosis and to esti-
mate the best level to be used as a cut-off value. Spearman’s rank correlation
coefficient was used to assess correlations between several variables stud-
ied. Statistical analyses were performed using SPSS for Windows (version
20.0; SPSS Inc., Chicago, IL) and MedCalc for Windows, version 14.0 (Med-
Calc Software, Ostend, Belgium). P values less than .05 were considered
significant.
RESULTS
Forty-four patients were evaluated for the diagnosis of
chronic GVHD and 30 of them were diagnosed using the NIH
consensus criteria [19]. Twenty-seven patients (90.0%) had
chronic ocular GVHD and many other organs were involved
Figure 1. Tear cytokine concentrations in patients with chronic graft-versus-host disease (GVHD) and without chronic GVHD. Tear cytokines were tested for
interleukin (IL)-2, IL-10, IL-17a, IL-4, interferon (IFN)-g, IL-6, and tumor necrosis factor (TNF)-a by multiplex immunobead assay. Horizontal bars indicate mean values
for each group. *P < .05, **P < .01, ***P < .001 using Mann-Whitney U test.
2081
(gastrointestinal tract, 16.7%; lung, 40.0%; liver; 43.3%; oral
cavity, 56.7%; skin, 80.0%) (Table 1).
Figure 1 shows comparisons of tear cytokine concentra-
tions between patients with and without chronic GVHD. All
measured cytokines, except IL-4, were elevated in patients
with chronic GVHD compared with those in patients without
chronic GVHD (IL-2, P ¼ .026; IL-10, P ¼ .007; IL-17a, P ¼ .008;
IL-4, P ¼.158; IFN-g, P ¼.02; IL-6, P <.001; and TNF-a, P <.001).
The diagnostic abilities of tear IL-10, IL-17a, IL-6, and TNF-a
were significant and independent in the logistic regression
model after adjusting for age, sex, and time period after SCT
(odds ratio for IL-10, 2.499; for IL-17a,1.038; for IL-6,1.036; and
for TNF-a, 1.024) (Table 3). Using receiver operating charac-
teristic curve analyses, these 4 cytokines showed that all the
area under the curve (AUC) values were greater than .70: IL-10,
.795; IL-17a, .821; IL-6, .912; and TNF-a, .910 (P < .001 for all).
For chronic GVHD diagnosis, the cut-off values were .82 pg/mL
for IL-10 (sensitivity 56.7%, specificity 85.7%), 20.26 pg/mL for
IL-17a (sensitivity 66.7%, specificity 78.6%), 81.08 pg/mL for
IL-6 (sensitivity 76.7%, specificity 100%), and 81.89 pg/mL
for TNF-a (sensitivity 73.3%, specificity 100%) (Figure 2).
The ocular surface parameters, including staining scores,
TBUT, conjunctival injection, Schirmer’s test I scores, and
OSDI scores were significantly higher in the chronic GVHD
2082 J.W. Jung et al. / Biol Blood Marrow Transplant 21 (2015) 2079e2085

Table 3 IL-6, and TNF-a. Classification of conjunctivitis in chronic

Logistic Regression Model for the Diagnostic Abilities of Tear Cytokine GVHD correlated with IL-10 (r ¼ .467, P ¼ .003), IL-6 (r ¼ .684,
Concentrations for Chronic GVHD Diagnosis after Adjusting for Age, Sex, and
the Period after SCT
P < .001), and TNF-a (r ¼ .533, P < .001). There were signif-
icant correlations between the NIH eye score and IL-10
Cytokine Odds Ratio 95% CI P Value (r ¼ .508, P ¼ .001), IL-6 (r ¼ .421, P ¼ .008), and TNF-a
IL-2 2.02 .978-4.170 .057 (r ¼ .444, P ¼ .005). The severity scale by international
IL-10 2.499 1.007-6.207 .048
chronic ocular GVHD consensus group correlated with IL-10
IL-17a 1.038 1.007-1.070 .018
IL-4 1.47 .734-2.943 .277 (r ¼ .464, P ¼ .003), IL-6 (r ¼ .598, P < .001), and TNF-a
IFN-g 1.397 .892-2.190 .145 (r ¼ .570, P < .001).
IL-6 1.036 1.009-1.065 .010
TNF-a 1.024 1.004-1.044 .018
DISCUSSION
CI indicates confidence interval. The focus of the present study was to compare the tear
Significant correlations are in bold font.
cytokine profiles in patients with or without newly diag-
nosed chronic GVHD after SCT. Levels of tear IL-2, IL-10,
group than in the group without chronic GVHD (P < .001 for IL-17a, IFN-g, IL-6, and TNF-a were elevated in patients with
all) (Table 4). The correlations between tear cytokines and chronic GVHD compared with patients without chronic
ocular surface parameters were assessed in all subjects GVHD. A few studies reported increased levels of tear cyto-
(Table 5). In particular, tear IL-10, IL-6, and TNF-a showed kines in patients with chronic ocular GVHD [17,18]. Riemens
moderate or strong correlation (r ¼ .4 or greater), and they et al. [17] demonstrated that tear IL-6 and IFN-g levels were
were more strongly correlated with several ocular surface elevated and correlated with dry eye symptoms. However,
parameters than other cytokines. they performed tear sampling by Schirmer’s test strips, and
Figure 3 shows that the severity of chronic ocular GVHD IL-6 and IFN-g were detected in 44% and 50% of SCT patients,
correlated significantly with tear cytokines, especially IL-10, respectively; other cytokines were not detected. To the best

Figure 2. Receiver operating characteristic (ROC) curve of tear cytokine concentrations for predicting the diagnosis of chronic GVHD. ROC curve of IL-10 (area under
the curve (AUC)  standard error (SE) ¼ .795  .071; 95% CI (.647 to .902); P < .001), IL-17a (AUC  SE ¼ .821  .066; 95% CI (.677 to .920); P < .001), IL-6
(AUC  SE ¼ .912  .042; 95% CI (.787 to .976); P < .001), and TNF-a (AUC  SE ¼ .910  .044; 95% CI (.784 to .975); P < .001).
 J.W. Jung et al. / Biol Blood Marrow Transplant 21 (2015) 2079e2085 2083

Table 4 chronic GVHD, increased levels of IL-10 imply its applicability

Ocular Parameters of Patients with and without Chronic GVHD after SCT in the diagnosis of chronic GVHD [9], as seen in our results.
Ocular Surface Parameters No Chronic Chronic GVHD P Value* The NIH consensus of chronic GVHD indicates that dry eye
GVHD after after SCT is a distinctive sign of chronic GVHD but insufficient in itself
SCT (n ¼ 14) (n ¼ 30) to establish a diagnosis of chronic GVHD until confirmation
Ocular surface staining, 0-5 by biopsy or other tests in the same organ or other organ
Median (range) 0 1 (0-3) <.001
[19]. However, this requirement can interfere with the early
Mean  SD 0 1.3  0.7
TBUT, sec diagnosis of systemic chronic GVHD. Evidence supporting
Median (range) 8 (0-10) 0 (0-8) <.001 the classification of dry eye disease as a diagnostic clinical
Mean  SD 6.7  3.9 1.8  2.7 entity for chronic GVHD has been recently published [20]. In
Conjunctival injection, 0-2 our study, the results of tear cytokine analysis showed a
Median (range) 0 (0-1) 1 (0-2) <.001
Mean  SD 0.1  0.3 1.2  0.6
significant correlation with their scores for chronic GVHD
Schirmer I score (mm) diagnosis, and thus, the results can be significant for possible
Median (range) 11 (6-23) 2 (0-14) <.001 diagnosis of chronic GVHD, not limited to the chronic ocular
Mean  SD 11.4  4.9 3.4  4.0 GVHD diagnosis.
OSDI score
We also investigated the role of tear cytokines as bio-
Median (range) 2.08 (0-10.42) 33.96 (0-68.75) <.001
Mean  SD 3.2  3.6 17.6  7.7 markers for chronic ocular GVHD severity by showing their
significant correlations with ocular surface parameters and
SD indicates standard deviation.
* Using Mann-Whitney U test.
the previously known classifications of chronic ocular GVHD
severity. Specifically, tear IL-10, IL-6, and TNF-a showed
stronger correlations with ocular surface parameters than
of our knowledge, the present study is the first to show tear other cytokines, and these cytokines also correlated with
cytokines profile in patients with chronic GVHD, regardless several chronic ocular GVHD severity scales. Although rela-
of presence of ocular GVHD. tively little information exists on biomarkers for chronic
Few studies [26,27] have also suggested that biomarkers for GVHD severity using other samples [9], TNF-a, IL-6, and IL-1b
chronic GVHD could differ with regard to the time of chronic levels have been suggested as candidate biomarkers [9,29].
GVHD onset from SCT; therefore, we limited the time after SCT Tear IL-6 and TNF-a have been known to correlate with
to 3 to 12 months and tried to adjust the period for analysis. As symptoms and signs of ocular surface disease such as
a result, tear cytokines, especially IL-10, IL-17a, IL-6, and TNF-a, dysfunctional tear syndrome, Sjögren syndrome, and mei-
significantly correlated with the presence of chronic GVHD bomian gland dysfunction [10,17,25,33,34]. Increased levels
and their diagnostic abilities for chronic GVHD diagnosis were of proinflammatory cytokines, such as IL-6, can cause the
significant. Several studies have evaluated the biological differentiation of naive CD4þ cells to Th17 T cells, which have
markers of chronic GVHD diagnosis using mainly blood been identified as key effector cells in autoimmune processes
samples and not tears, and the findings of these studies sup- [35]. Th17 cells, with the characteristic production of IL-17,
port increased proinflammatory cytokines, including TNF-a, can mediate the development of GVHD in mice [36]. In
IL-6, IL-1b, IL-8, the soluble alpha chain of the IL-2 receptor multiple mouse strains, blocking IL-6 signaling dramatically
(sIL-2R), and IL-1Ra [9,28,29]. Because previous studies could attenuates GVHD and Th17 responses [37,38]. However, Th17
not classify chronic GVHD as either pure Th1 or Th2 disease responses may not be the only prerequisite for the devel-
[9,30], we used the human Th1/Th2/Th17 cytokine kit in the opment of GVHD [39]. Moreover, in humans, the role of IL-6
present study. We observed increased levels of all tear cyto- in ocular GVHD might not only be to promote Th17 but also
kines except IL-4, which indicates Th2 cell differentiation. A to promote the differentiation of B cells and the induction of
lack of Th2 shift has been known to activate B cells by antibody production [40]. Our study showed that the in-
up-regulating the expression of B celleactivating factor in crease in proinflammatory cytokines IL-6 and TNF-a and the
GVHD [9,30]. Although the role of IL-10 in GVHD pathogenesis increase in IL-17 coincided very well and were correlated
remains poorly understood, there are reports showing both with each other.
beneficial and adverse effects of this cytokine in GVHD pro- There are several criteria for assessing the severity of
gression [31,32]. Notwithstanding the unclear role of IL-10 in chronic ocular GVHD. We used the NIH eye score, which is a

Table 5
Correlations between Tear Cytokine Levels and Ocular Surface Parameters in Patients Who Underwent SCT

Cytokine Ocular Surface Staining TBUT Schirmer I Score Conjunctival Injection OSDI Scores
IL-2 .123 .303 .205 .133 .340
P ¼ .455 P ¼ .060 P ¼ .212 P ¼ .421 P [ .034
IL-10 .327 .268 .237 .492 .542
P [ .042 P ¼ .099 P ¼ .147 P [ .001 P < .001
IL-17a .251 L.344 .211 .252 .238
P ¼ .123 P [ .032 P ¼ .196 P ¼ .122 P ¼ .145
IL-4 .011 .303 .131 .088 .271
P ¼ .948 P ¼ .061 P ¼ .428 P ¼ .596 P ¼ .095
IFN-g .130 .312 .241 .171 .336
P ¼ .429 P ¼ .054 P ¼ .139 P ¼ .298 P [ .036
IL-6 .630 .289 L.446 .586 .488
P < .001 P ¼ .074 P [ .004 P < .001 P [ .002
TNF-a .462 L.416 .296 .518 .603
P [ .003 P [ .008 P ¼ .067 P [ .001 P < .001

Numbers are Spearman’s correlation coefficients. Significant correlations are in bold font.
2084 J.W. Jung et al. / Biol Blood Marrow Transplant 21 (2015) 2079e2085

Figure 3. Scattergram showing the correlations between classification of conjunctivitis in chronic GVHD (A), the NIH eye scores (B), and the severity score by the
International Chronic Ocular GVHD Consensus Group (C) and tear cytokines, IL-10, IL-6, and TNF-a. Spearman’s correlation coefficients were used.

clinical scoring system proposed as NIH consensus criteria in
2005 as part of a global assessment of chronic GVHD severity
[19] and classification of conjunctivitis in chronic GVHD [23].
Although it is still controversial, conjunctival involvement in
chronic GVHD indicates a sign of severe chronic GVHD and
decreased survival of patients [41]. In this present study, tear
IL-10, IL-6, and TNF-a level significantly correlated with both
severity criteria. Moreover, tear cytokine concentrations
showed a significant correlation with the severity score of
the most current system of chronic GVHD diagnosis [20],
which was reported by the International Chronic Ocular
GVHD Consensus Group. Therefore, the measurement of
these tear cytokines is expected to be useful in assessing
chronic ocular GVHD severity.
This study had some limitations. Although we analyzed a
relatively large sample size in comparison to previous studies
and considered time differences, this study was a cross-
sectional observation case series with inherent selection bias
because patients were referred to an eye clinic. Thus, larger
scale prospective design studies are necessary to confirm the
role of tear cytokine biomarkers for chronic GVHD. Moreover,
the analysis of tear cytokines warrants additional clinical
validation and improvement in clinical utility.
In conclusion, tear cytokine profiles differed significantly
according to the presence of newly diagnosed chronic GVHD.
Our study demonstrated suggested certain tear cytokines as
promising new possible diagnostic markers of chronic GVHD
and criteria for chronic ocular GVHD severity. Because tear
sampling is noninvasive and simple, it is expected to be
applicable for the screening and diagnosis of chronic GVHD.
ACKNOWLEDGMENTS
Financial disclosure statement: This study was supported
by a grant the from Bio and Medical Technology Develop-
ment Program of the National Research Foundation funded
by the Ministry of Science, ICT & Future Planning, South
Korea (grant no. NRF-2013M3A9D5072551). The supporters
had no role in the design and conduct of the study; collec-
tion, management, analysis, and interpretation of the data;
preparation, review, or approval of the manuscript; and de-
cision to submit the manuscript for publication.
Conflict of interest statement: The authors have no financial
or proprietary interest in any of the products or techniques
mentioned in the article.
 J.W. Jung et al. / Biol Blood Marrow Transplant 21 (2015) 2079e2085
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