Corneal biomechanical alterations in patients with chronic ocular Graft Versus-Host Disease
Corneal biomechanical alterations in patients with chronic ocular Graft Versus-Host Disease
Giuseppe GiannaccareID 0 2
Marco Pellegrini 0 2
Leonardo Taroni 0 2
Federico Bernabei 0 2
Carlotta Senni 0 2
Arianna Grendele 0 2
Vincenzo Scorcia 1 2
Emilio C. Campos 0 2
0 Ophthalmology Unit, S.Orsola-Malpighi University Hospital, University of Bologna , Bologna , Italy
1 Department of Ophthalmology, University of ?Magna Graecia? , Catanzaro , Italy
2 Editor: Andrew W. Taylor, Boston University School of Medicine , UNITED STATES
To compare corneal biomechanics between patients with ocular graft versus-host disease (oGVHD) and healthy subjects (controls), and to further correlate these values with ocular and hematological characteristics.
Materials and methods
The following procedures were performed in oGVHD patients and controls: Schirmer test
(ST), break-up time (BUT), corneal and conjunctival staining, tear matrix
metalloproteinase9 (MMP-9) assay (InflammaDry test, Rapid Pathogen Screening, Inc, Sarasota, FL).
Corneal biomechanics were calculated by using ocular response analyzer (ORA, Reichert
Instruments, Depew, New York, USA). The Mann-Whitney U test was used to compare
continuous variables between oGVHD patients and controls. Correlations of corneal
biomechanics with ocular and hematological parameters were examined using Spearman?s
A total of 45 oGVHD patients (mean age ? SD, 51.5 ? 7.1 years) and 34 controls (47.8 ? 6.1
years) were included. Patients with oGVHD showed significantly lower values of corneal
hysteresis (CH) and corneal resistance factor (CRF) compared to controls (respectively, 9.4
? 1.8 mmHg vs 11.6 ? 1.6 and 9.7 ? 1.4 mmHg vs 12.3 ? 1.3; always p<0.001). Twenty-nine
of the oGVHD eyes (64.4%) were strong-positive for MMP-9, while 16 (35.6%) were
weakpositive. Conversely, only 4 of the control eyes (11.8%) were weak-positive for MMP-9. In
patients with oGVHD, CH was significantly correlated with corneal staining (Rs = -0.316, p =
0.035), conjunctival staining (Rs = -0.437, p = 0.003), ST (Rs = 0.390, p = 0.008), BUT (Rs =
0.423, p = 0.004), oGVHD severity grade (Rs = -0.383, p = 0.009), and MMP-9 positivity
grade (Rs = -0.429, p = 0.003), while CRF was correlated only with corneal staining (Rs =
-0.317, p = 0.034).
Corneal biomechanics are reduced in patients with oGVHD, and CH is negatively correlated
with disease severity grade and MMP-9 tear levels.
Allogeneic hematopoietic stem cell transplantation (HSCT) is an established and potentially
curative treatment for a variety of malignant and non-malignant hematological disorders.
Graft versus-host disease (GVHD) is a multi-organ systemic disease caused by complex
interactions between donor and recipient immune systems and represents the leading cause of
morbidity following HSCT.[
] Chronic ocular GVHD (oGVHD) develops in 40 to 60% of
patients undergoing HSCT, and dry eye disease (DED) represents the hallmark of this
] The disease is thought to be the result of the progressive immune-mediate
inflammatory damage of ocular surface structures, which may lead to lacrimal and meibomian
glands dysfunction, conjunctival keratinization, corneal epitheliopathy, eyelid laxity and
scarring, and in more severe cases even corneal melting and perforation.[
Recent proteomic studies evaluated the various constituent compositions of tears
inflammatory mediators in eyes with oGVHD and detected increased levels of cytokines and matrix
] Among these, MMP-9 is a proteolytic enzyme involved in
the degradation of corneal extracellular matrix components, whose dysregulation is implicated
in impaired wound healing and stromal keratolysis.[
] The excessive proteolytic activity
in the cornea determines the degradation of stromal components, that are known to be the
main determinants of biomechanical corneal behavior. Thus, we hypothesized that higher
tears levels of MMP-9 might affect the ultrastructure of the corneal stroma, producing changes
in corneal biomechanics.
In the present study, we compared the corneal biomechanical properties in patients with
oGVHD and healthy matched subjects; in addition, we investigated whether these properties
were correlated with ocular surface and hematological characteristics of oGVHD patients.
Materials and methods
Study design and patients
This prospective case-control study was conducted at a single tertiary-referral Ocular Surface
Center (S.Orsola-Malpighi University Hospital, Bologna, Italy) between January 2018 and
October 2018. The study was performed in accordance with the principles of the Declaration
of Helsinki and was approved by the local Institutional Review Board. Written informed
consent was obtained from all subjects included in the study before any procedure.
Consecutive hematological patients who underwent allogeneic HSCT and came at our
Center for routinely ocular surface check-up visits were screened for enrollment. Inclusion criteria
were: age older than 18 years and a diagnosis of chronic oGVHD according to the
International chronic ocular GVHD Consensus Group.[
] The severity of oGVHD was graded as
none (grade 0), mild/moderate (grade I) and severe (grade II).[
] Healthy sex- and
agematched subjects acted as controls. All the study procedures were carried out in one eye for
both patients and controls according to an automatic randomization procedure (www.
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Exclusion criteria for both groups included any prior ophthalmologic surgery in the last
year, keratoconus, corneal dystrophy, contact lens wearing, glaucoma, spherical
equivalent 5 diopters, astigmatism 3 diopters and any ocular infection within 3 months
prior to enrollment.
Clinical data including demographic characteristics, ongoing systemic and topical
therapies, hematological diagnosis, duration of disease, type of HSCT donor, time from GVHD
diagnosis to ophthalmological evaluation, presence and localization of systemic GVHD, and
history of previous therapies (i.e. autologous HSCT, radiotherapy [RT], and chemotherapy
[CT]) were collected at the time of the ophthalmic evaluation.
The InflammaDry test (Rapid Pathogen Screening, Inc, Sarasota, FL) was performed according
to the manufacturer?s instructions to determine whether both oGVHD patients and control
subjects exhibited pathological levels of MMP-9.[
] The use of any topical ophthalmic
medications was discontinued two hours prior to examination. In brief, a tear sample was collected
by lowering the palpebral conjunctiva and gently dabbing the fleece of the sample collector 8
to 10 times in multiple locations, allowing the patient to blink between dabs to ensure
saturation. The sampling fleece glistened or turned pink when an adequate sample was collected,
and was then snapped into the test cassette. The absorbent tip was immersed into the buffer
vial for 20 seconds and laid flat on a horizontal surface for 10 minutes before interpretation of
test results. The presence of one blue line and one red line in the test result window indicated a
positive test result (MMP-9 40 ng/mL), whereas only one blue line indicated a negative test
result (MMP-9 < 40 ng/mL). In addition, the positive red line was compared with the grading
index to classify the result as weak-positive and strong-positive.[
] Three well-trained
observers (FB, CS & AG) individually interpreted the InflammaDry grading result, masked to both
subject characteristics and clinical diagnosis. The result was confirmed when the
interpretations of at least two of the observers were in agreement.
Ocular surface work-up
Subjective symptoms of ocular discomfort were scored by the Ocular Surface Disease Index
(OSDI) questionnaire. Slit-lamp examination with ocular surface staining was performed after
administration of 2mL of 2% fluorescein dye using the blue cobalt filter and a 7503 Boston
yellow filter kit to enhance staining details. Corneal and conjunctival staining were graded using
respectively the Oxford score[
], and the van Bijsterveld score[
]; Schirmer test type I and
break up time (BUT) measurements were performed according to the Dry Eye Workshop
All the ocular surface examinations were performed by the same operator (GG) blinded to
Ocular response analyzer measurements
The ocular response analyzer (ORA, Reichert Instruments, Depew, New York, USA) measures
two applanation pressure points during a dynamic bi-directional applanation process
generated by a precisely metered air pulse. The first pressure point occurs as an air puff pushes the
cornea inward, while the second pressure point as the cornea returns from the applanated
state to normal. The difference between these two pressures is defined as the CH, while CRF is
calculated as a linear function of the two values. The average of both pressures provides
Goldmann-correlated IOP (IOPg), while the corneal-compensated IOP (IOPcc) is the recalculated
IOP value using corneal biomechanical information provided by the CH measurement.[
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All the ORA examinations were performed by the same operator (LT) blinded to subject?s
characteristics. The use of any topical ophthalmic medications was discontinued two hours
prior to examination. Before each examination, central corneal thickness (CCT) values
obtained with an ultrasonic pachymeter (Dicon P55, Paradigm Medical Industries Inc., Salt
Lake City, UT, USA) were inserted in the software. Because of the potential confounding effect
of diurnal IOP variation, all measurements were obtained between 10 AM and 12 PM. All
included ORA measurements had a waveform score > 7.0.[
] The average values of four
measurements with desirable curves were recorded for statistical analysis.
The SPSS statistical software (SPSS Inc, Chicago, Illinois, USA) was used for data analysis.
Values are expressed as mean ? standard deviation (SD). The Mann-Whitney U test was used to
compare continuous variables between ocular GVHD patients and control subjects. The ?2
test was used to compare dichotomous variables between ocular GVHD patients and control
subjects. Correlations of corneal biomechanical properties with demographical, hematological
and ocular parameters in the oGVHD group were examined using Spearman?s correlation
analysis. A Bonferroni correction for multiple testing was used by multiplying the observed
pvalue with the number of comparisons within each analysis. A p value < 0.05 was considered
We screened a total of 51 post-HSCT patients during the study period. Of these, 45 patients
fulfilled the inclusion criteria, and were finally enrolled in the study: 5 of them (11.1% of the
total) belonged to the oGVHD severity grade 0, 11 (24.4%) to grade I and 29 (64.4%) to grade
II. The remaining 6 patients were excluded from the analysis because the diagnosis of oGVHD
was not reached (n = 3), or due to the presence of significant corneal alterations (neovessels)
hampering accurate ORA measurements (n = 3). Thirty-four healthy subjects were enrolled as
control group. The demographic and clinical parameters of patients included in the analysis
are reported in Table 1. No significant differences in age and sex distribution between
hematological patients and control subjects were found (always p > 0.05).
Ocular surface parameters of oGVHD patients and control subjects are reported in Table 2.
A significant difference of all parameters was found between the two groups (always p < 0.05).
Patients with oGVHD showed significantly lower values of CH and CRF compared to
controls (respectively, 9.4 ? 1.8 mmHg vs 11.6 ? 1.6 and 9.7 ? 1.4 mmHg vs 12.3 ? 1.3; always p <
0.001) (Table 3). In addition, CCT was significantly lower in oGVHD patients compared to
controls (510.7 ? 32.5 ?m vs 537.9 ? 29.8, p < 0.001).
The proportion of MMP-9 tear film positivity significantly differs between the two groups
(P < 0.001). In particular, 29 of the oGVHD eyes (64.4% of the total) were strong-positive for
MMP-9, while 16 (35.6%) were weak-positive. Conversely, only 4 of the control eyes (11.8%)
were weak-positive for MMP-9 (Fig 1).
In patients with oGVHD, CH was significantly correlated with corneal staining (Rs =
-0.316, p = 0.035), conjunctival staining (Rs = -0.437, p = 0.003), Schirmer test (Rs = 0.390,
p = 0.008), BUT (Rs = 0.423, p = 0.004), oGVHD severity grade (Rs = -0.383, p = 0.009), and
MMP-9 positivity grade (Rs = -0.429, p = 0.003), while CRF was correlated only with corneal
staining (Rs = -0.317, p = 0.034).
No significant correlations were found between CH and CRF values and hematological
characteristics (always p>0.05).
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51.2 ? 7.1
65.9 ? 54.9
GVHD: Graft versus-host disease; AML: Acute myeloid leukemia; ALL: Acute lymphoblastic leukemia; HL: Hodgkin
lymphoma; CML: Chronic myeloid leukemia; CLL: Chronic lymphocytic leukemia; MM: Multiple myeloma; G-I:
gastro-intestinal; HSCT: hematopoietic stem cell transplantation; RT: radiotherapy; CT: chemotherapy.
PLOS ONE | https://doi.org/10.1371/journal.pone.0213117
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GVHD: Graft versus-host disease; OSDI: Ocular surface disease index; BUT: break-up time; VB: van Bijsterveld.
The cornea is a complex biomechanical composite, and the collagen present in the Bowman?s
layer and stroma provides the major contribution to its biomechanical behavior.[
Frequently, oGVHD determines the damage of corneal epithelium that can be observed at slit
lamp examination as punctate keratopathy, filamentary keratitis, and persistent epithelial
defect. In more severe cases, the persistent impairment of wound healing alters the stromal
ultrastructure, with the onset of keratolysis, corneal melting and even perforation. These
alterations of both corneal epithelium and stroma might produce changes to a various extent
of biomechanical properties of the cornea of oGVHD patients.
To date, few previous studies have investigated corneal biomechanics in patients with DED
owing to different etiologies, but not oGVHD, by means of both ORA and CorVis ST.[
The former is a non-contact tonometer that provides the measure of two parameters of corneal
biomechanics: CRF that reflects the overall resistance of the cornea, and CH that measures the
viscoelastic properties of the corneal tissue detecting the changes in the organization of
collagen lamellae. CorVis ST is a device that employs an ultrahigh speed Scheimpflug camera to
record dynamic deformation of the cornea. Impaired values of corneal biomechanics have
been reported in DED patients with and without Sjo?gren?s syndrome[
]; conversely, Firat
et al reported no alterations of corneal biomechanical parameters in patients with DED.
However, this study did not evaluate comprehensively ocular surface parameters, and did not
stratify patients according to the severity of DED.
To the best of our knowledge, this is the first study evaluating corneal biomechanics in the
setting of oGVHD. This condition is a type of iatrogenic DED, whose prevalence is increasing
due to the widespread adoption of HSCT for the treatment of hematological disorders. We
found significantly lower values of CH and CRF in oGVHD patients compared to healthy
matched subjects. In addition, the severity of ocular surface impairment in these patients was
associated with a greater alteration of corneal biomechanical properties, in particular for CH.
CCT: Central corneal thickness; GVHD: Graft versus-host disease; CH, corneal hysteresis; CRF, corneal resistance factor; IOPg; Goldmann-correlated intraocular
pressure; IOPcc, corneal-compensated intraocular pressure.
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Fig 1. Ocular response analyzer measurement signals and InflammaDry assays of 3 representative subject. A, D healthy control, negative for MMP-9; B, E ocular
graft versus-host disease (oGVHD) patient (severity grade I), weak-positive for MMP-9; C, F oGVHD patient (severity grade II), strong-positive for MMP-9.
This parameter reflects the viscoelastic properties of corneal tissue, detecting the changes in
the organization of collagen lamellae. The most reasonable explanation to these findings is that
the ocular surface inflammation caused by oGVHD might affect the corneal stroma
ultrastructure by enhancing the rates of collagen and elastin enzymatic degradation. In particular,
MMPs are proteolytic enzymes produced by stressed ocular surface epithelial cells, as well as
by the immune cells that infiltrate these tissues. They are involved in the degradation of
extracellular matrix components, and contribute to inflammation, wound healing, and tissue
] Overexpression of MMPs have been linked with corneal complications
like ulcer and melting, that occur in the most severe stages of oGVHD, probably because of
their role in the remodeling of extracellular matrix.[
Among MMP-family, MMP-9 represents the primary matrix-degrading enzyme of the
ocular surface and is the only one that can be tested with a commercially available assay in clinical
] In the present study, we tested MMP-9 positivity in the tear film of
patients with oGVHD and healthy subjects. All oGVHD patients resulted positive to MMP-9
assays against the negligible percentage of positivity among control subjects. This result is
consistent with recent studies, which demonstrated increased tear levels of elastolytic enzymes,
including MMP-9 and neutrophil elastase, in patients with oGVHD.[
] In addition, in order
to overcome the drawback of the dichotomous result of MMP-9, which corresponds to either
one of ??negative? or ??positive?, we employed a semi-quantitative method of grading the
intensity of positivity, as already suggested by others. Thus, we demonstrated that oGHVD
patients exhibited different grades of MMP-9 positivity, and stronger positivity grade was
associated with higher impairment of corneal biomechanics. This finding confirms the possible
role of MMP-9 in the alteration of the stromal composition and organization also in the setting
Conflicting results are available in the literature regarding central corneal thickness in
DED. Although few studies showed similar values between DED patients and normal subjects
], we found significantly lower values in patients with oGVHD compared to controls, in
agreement with others.[
] The corneal thinning in DED is attributed to the increased
osmolarity of the tear film that induces the activation of the inflammatory cascade and
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stimulate the production of high levels of the cytokine and MMP-9 by the distressed epithelial
cells. These inflammatory events may lead to apoptotic death of surface epithelial cells of
cornea and to destructive keratolysis and thinning.
The cause-effect relationship between increased MMP-9 tear levels and corneal
biomechanical impairment cannot be ascertained by our study, and this issue represents the main
limitation. Although InflammaDry testing is the only assay commercially available, a proteomic
study with tear dosage of each type of MMPs would have provided a more accurate correlation
between ocular surface inflammation and corneal biomechanics.
The present study shows that oGVHD impairs biomechanical properties of the cornea, likely
as a result of the alterations of ultrastructure and architecture of stromal collagen fibrils. Since
corneal biomechanical alterations are closely related to the grade of ocular surface
inflammation, they might represent a possible new surrogate marker of oGVHD severity.
Conceptualization: Giuseppe Giannaccare.
Data curation: Leonardo Taroni.
Formal analysis: Marco Pellegrini.
Investigation: Marco Pellegrini, Leonardo Taroni, Federico Bernabei, Carlotta Senni, Arianna
Project administration: Giuseppe Giannaccare.
Validation: Vincenzo Scorcia, Emilio C. Campos.
Visualization: Federico Bernabei, Carlotta Senni, Arianna Grendele.
Writing ? original draft: Giuseppe Giannaccare, Marco Pellegrini.
Writing ? review & editing: Vincenzo Scorcia, Emilio C. Campos.
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