Acta Ophthalmologica 2014

Peripheral hypertrophic subepithelial corneal

degeneration – clinical and histopathological
features
Petri J. J€arventausta, Timo M. T. Tervo, Tero Kivel€a and Juha M. Holopainen
Helsinki University Eye Hospital, Helsinki, Finland

ABSTRACT. Gore et al. 2013; J€ arventausta et al.

Purpose: To refine the diagnostic criteria for peripheral hypertrophic subepi- 2014). It presents with superficial
thelial corneal degeneration (PHSD) and characterize its clinical phenotype, fibrosis between the epithelium and
histopathology and immunohistochemical features. Bowman’s layer, with focal, mostly
Methods: Diagnostic criteria were refined on the basis of literature data. inconspicuous superficial neovascular-
Fourteen patients (13 women and one man; median age 52 years, range 33–66) ization reaching the limbal margin of
were identified based on these criteria. Keratectomy specimens were evaluated the fibrous tissue (Maust & Raber
via routine and immunohistochemical stainings. The main outcome measures 2003; Jeng & Millstein 2006; Gore
were symptoms, clinical phenotype, immunological status and histopathologic et al. 2013; J€arventausta et al. 2014).
The fibrosis causes the flattening of the
results.
central cornea and induces regular and
Results: We defined the diagnostic criteria of typical PHSD as elevated
irregular astigmatism that can be more
circumferential and perilimbal subepithelial fibrosis with focal superficial corneal or less efficiently treated with keratec-
neovascularization, which were supported by female sex (93%), bilaterality tomy (Gore et al. 2013; J€ arventausta
(86%), the centre being in the upper quadrants (81%) and irregular astigmatism et al. 2014).
of two dioptres or more. The typical symptoms were reduced vision (86%) and The pathogenesis of PHSD is
the symptoms of ocular surface disease (64%). Light microscopy showed fibrosis unknown, but it is presumed to be a
with abundant collagen deposition but no inflammation in all patients. An degeneration resembling Salzmann’s
immunohistochemical analysis of nine patients showed uniform staining for nodular degeneration (SND) to some
vimentin in three distinct types of fibroblasts in variable proportions: keratocyte- extent, rather than a dystrophy.
like cells that were positive for CD34, myofibroblasts that were positive for Salzmann’s nodular degeneration is a
smooth muscle actin (SMA) and fibroblasts that were negative for CD34 and non-inflammatory, progressive, often
SMA. Small numbers of CD68-positive macrophages were also found. bilateral degenerative corneal disorder
Conclusions: Peripheral hypertrophic subepithelial degeneration is characteristic (Farjo et al. 2006). Its characteristic
of middle-aged women, in whom it is typically a bilateral idiopathic degeneration of findings are bluish-grey elevated cor-
neal nodules that vary in size, location
the cornea associated with ocular surface disease and reduced vision. The fibrotic
and number. Usually, they are scat-
lesions probably undergo remodelling, inducing changes in corneal contour. A
tered, occur in any meridian and often
smouldering low-grade inflammation favouring low TGF-b1 concentrations is extend to the central cornea, unlike in
postulated as the primary pathological process leading to PHSD. PHSD, and as SND progresses, the
nodules may become confluent. Salz-
Acta Ophthalmol. 2014: 92: 774–782 mann’s nodular degeneration affects
ª 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd patients of various ages and races.
doi: 10.1111/aos.12394
Both SND and PHSD are more com-
mon in females (Maust & Raber 2003;
Das et al. 2005; Farjo et al. 2006; Gore
been described in the literature or et al. 2013; J€arventausta et al. 2014).
Introduction textbooks, appears to be an under- The cause of SND is likewise
Peripheral hypertrophic subepithelial recognized corneal disease, given that unknown. It can follow ocular surface
degeneration (PHSD) of the cornea, a three series of six to 22 patients have disease, contact lens wearing or ocular
typically bilateral and slowly progres- been reported from three centres in surgery, but this is not always the case.
sive perilimbal disorder that has rarely recent years (Maust & Raber 2003; The excision of the nodules usually

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 Acta Ophthalmologica 2014

improves vision. Spontaneous remis-
sion has not been reported.
The initial histopathologic analysis of
three PHSD lesions from two patients
showed non-specific and non-inflamma-
tory fibrosis and the absence of Bow-
man’s layer (Maust & Raber 2003; Jeng
& Millstein 2006). These findings were
recently verified in twelve eyes from
seven patients, although the analysis of
this series was complicated by the pres-
ence of elastotic stromal degeneration
and squamous metaplasia from an adja-
cent pterygium in six eyes from four
patients (Gore et al. 2013). The histo-
pathologic findings were also reminis-
cent of SND (Gore et al. 2013). Via
histopathological analysis, SND is asso-
ciated with the thinning of the epithe-
lium and breaks in Bowman’s layer.
Here, we aim to refine the diagnostic
criteria for PHSD and shed further
light on its phenotype and pathogenesis
by reporting the clinical features of
nine additional patients and histopath-
ologic data from keratectomy speci-
mens from 14 typical PHSD patients
without concurrent corneal lesions.
Furthermore, we present further sup-
port for our previous observation that
the HLA-B*44 allele may be enriched
in patients with PHSD.
Patients and Methods
Ethical aspects
This study was approved by the Ethical
Committee of the Helsinki University
Central Hospital. Each patient signed
an informed consent form. The study
protocol followed the tenets of the
Declaration of Helsinki.
Diagnostic criteria
Based on previous reports (Maust &
Raber 2003; Jeng & Millstein 2006;
Gore et al. 2013; J€ arventausta et al.
2014), we refined the diagnostic criteria
for typical PHSD (Table 1) based on
clinical symptoms and signs (irregular
and regular astigmatism, symptoms of
ocular surface disease, peripheral sub-
epithelial fibrosis, focal superficial
neovascularization and a predilection
toward superonasal location, females
and bilateral disease) and the absence
of evidence of any simulating lesions
(corneal intraepithelial neoplasia,
SND, climatic droplet keratopathy,
corneal amyloidosis or keloid and
hereditary hypertrophic scarring).
Patients who met these criteria were
included in the study.
Patients
Fourteen Caucasian patients, all Finn-
ish, with lesions resembling PHSD,
who were referred to the Helsinki
University Eye Hospital between Feb-
ruary 2006 and August 2012 met the
diagnostic criteria for typical PHSD
(Table 1) and underwent keratectomy
to remove the corneal lesions. Of these
consecutive patients, nine were pro-
spectively diagnosed with PHSD from
2011 to 2012, and five were identified
retrospectively from our clinical and
histopathological patient records. Five
patients in this series also appeared in
our recent report on another 14
patients who were retrospectively diag-
nosed with PHSD (J€ arventausta et al.
2014). They were also enrolled in the
present study because they fulfilled the
criteria for typical PHSD and pro-
vided sufficient material for the immu-
nohistochemical studies available (#2,
3, 4, 5 and 11 in Table 2).
All patients underwent a comprehen-
sive preoperative ophthalmic examina-
tion, including slit lamp biomicroscopy,
applanation tonometry, indirect oph-
thalmoscopy and corneal topography
with a TMS 2-N Topographic Model-
ling System (version 2.4.2; Tomey,
Nagoya, Japan) or an Orbscan II
(Bausch & Lomb, Rochester, NY,
USA), and seven patients underwent
anterior segment optical coherence
tomography (OCT) with an SS-1000
Casia (Tomey). The preoperative eval-
uation included their history of systemic
diseases, previous eye surgeries and
injuries and contact lens wearing. Based
on topography, the power of corneal
astigmatism (cylinder) was recorded
pre- and postoperatively. Colour cor-
neal photographs were taken pre- and
postoperatively. Patients were exam-
ined at 1, 2–7 days and 1 month post-
operatively and then as needed. The
HLA-A*, HLA-B*, HLA-DRB1* and
complement C4 genes, as well as immu-
noglobulin levels, were determined as
previously described (J€ arventausta
et al. 2014).
In all cases, surgical treatment was
performed in the form of manual
keratectomy, using a Beaver 2.5 mm
Crescent blade (Beaver-Visitec Interna-
tional, Waltham, MA) and Vannas
scissors as recently reported (J€ arven-
tausta et al. 2014). After the removal of
fibrous tissue, the underlying corneal
stroma appeared to be essentially clear.
We did not encounter any problems in
re-epithelization after keratectomy.

Table 1. Proposed diagnostic criteria for typical peripheral hypertrophic subepithelial degeneration (PHSD).

Characteristic Diagnostic criteria for typical PHSD Differential diagnosis

Fibrous tissue Subepithelial confluent perilimbal fibrosis with thickening of the 1) Salzmann’s nodular degeneration.
cornea, which may or may not extend to midperipheral 2) Previous injury or keratitis
but not to the central cornea 3) Concurrent inflammatory disease
Neovascularization Superficial corneal neovascularization extending not further that the 1) Pterygium
base of the fibrous tissue without deep stromal vessels 2) Pseudo-pterygium
3) Conjunctival intraepithelial neoplasia
Astigmatism 2 D or more of irregular and regular astigmatism in topography 1) Map dot fingerprint dystrophy
2) Irregular epithelium from other causes
Symptoms Mild itching/burning and ocular surface irritation 1) Dry eye
from irregular surface of the cornea 2) Ocular allergy
3) Autoinflammatory diseases of the cornea
Supportive criteria 1) Fibrosis Located in superior, especially superior nasal cornea
Bilateral fibrosis
2) Female sex

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Acta Ophthalmologica 2014

Table 2. Demographic and immunological characteristics of 14 patients with typical peripheral hypertrophic subepithelial degeneration (PHSD).

Astig. Astig. Follow Systemic

ID Sex Age C4A- gene C4B-gene HLA-A HLA-B HLA-DRB1 pre.op post.op up/m diseases

1 F 59 2 2 *02*24 *15*40 *01*08 9.3 0.5 21 Diabetes

2* F 46 3 1 *03*11 *35*44 *08*16 2.0 1.2 44 None
3* M 45 2 2 *01*32 *08*39 *03*08 3.1 2.7 30 None
4* F 51 2 2 *02*03 *07*07 *13*15 3.5 1.5 71 None
5* F 61 2 1 *01*02 *08*44 *03*12 6.3 3.3 29 Horner, Glaucoma
6 F 53 2 2 *02*03 *15*40 *13*13 3.6 1.4 1 None
7 F 45 3 1 *02*03 *07*44 *13*15 4.2 1.8 17 Thyroid carcinoma
8 F 58 2 1 *02*03 *07*44 *01*12 3.7 6.0 4 None
9 F 48 3 1 *02*02 *13*27 *01*07 7.9 4.2 17 Psoriasis
10 F 33 2 2 *31*68 *18*44 *01*04 2.1 0.8 5 None
11* F 64 3 2 *02*02 *40*44 *12*13 5.6 1.6 39 Hypertension
12 F 46 2 1 *03*03 *07*35 *01*13 4.5 1.9 8 None
13 F 50 3 1 *01*02 *15*57 *04*13 4.0 0.8 8 None
14 F 49 1 2 *24*24 *40*50 *03*04 5.0 4.2 1 None

* Included also in J€
arventausta et al. 2014.

Histopathologic analysis subepithelial fibrosis of the cornea with tory of autoimmune disease (psoriasis).
adjacent superficial fairly inconspicu- The leading symptom was reduced
All samples were fixed in 10% neutral
ous limbal vasculature to the base of visual acuity in twelve patients (86%;
buffered formalin and embedded in
the fibrosis (Table 2; Fig. 1). Twelve CI 57–98). Ocular surface disease
paraffin. Sections (5 lm thick) were
patients [86%; 95% confidence interval symptoms were reported by nine
cut and stained with haematoxylin and
(CI), 57–98] had bilateral disease. The patients (64%; 95% CI, 35–87). Two
eosin (HE), periodic acid- Schiff (PAS)
fibrosis was limited to the perilimbal patients (14%; 95% CI, 2–43) did not
and Masson’s trichrome for routine
cornea in eight eyes and extended to report any ocular symptoms. None
light microscopy.
the midperipheral cornea in 17 eyes. It showed Meibomian gland dysfunction
Immunostaining was performed
was centred in the upper nasal quad- (0%; 95% CI, 0–23).
using the avidin-biotinylated peroxi-
rant in 21 of the 26 involved eyes (81%; Biomicroscopically, in 13 patients,
dase complex method. The following
95% CI, 61–93) and was never centred the fibrosis appeared as an elevated,
primary monoclonal antibodies were
in the inferior nasal or inferior tempo- greyish-white, solitary, confluent cor-
used: vimentin (clone V9; Dako Den-
ral quadrant, although it partially neal lesion. In one patient, a skip area
mark A/S, Glostrup, Denmark) char-
involved the inferior nasal quadrant was present bilaterally, and in a second
acteristic of all mesenchymal cells,
in six eyes (25%) and the inferior patient, a skip area was present unilat-
including normal and reactive human
temporal quadrant in two eyes (8%; erally (Fig. 2). The central border of
keratocytes and leucocytes; CD34
Fig. 2). the fibrosis could extend up to the
(clone QBEND/10; Roche, Basel,
The median age of the patients at margin of the undilated pupil in the
Switzerland) characteristic of normal
diagnosis was 52 years (range, 33–66; midperipheral zone of the cornea, but
human corneal keratocytes and also
Table 2). Only one patient had a his- it did not involve the central cornea
found in a population of bone marrow-
(Figs 1 and 3). This border was mostly
derived stem cells; alpha smooth
demarcated in a feathery yet sharp
muscle actin (SMA; clone 1A3; Dako
fashion from the clear central cornea.
Denmark A/S) characteristic of myofi-
Typically, an iron deposition, similar to
broblasts; CD68 (clone PG-M1) char-
Stocker’s line in the pterygium, was
acteristic of macrophages; and CD45,
found at the central border of the
also known as leucocyte common
lesion. Biomicroscopic evaluation did
antigen (LCA; clone 2B11+PD7/26;
not reveal any signs of active ocular
Roche), characteristic of inflammatory
inflammation. Superficial neovascular-
cells. A non-ocular tissue known to
ization was characteristically limited to
express each marker was used as a
the limbal border of the fibrotic tissue
positive control. The slides were anal-
and did not extend over it. No deep
ysed via a consensus of PJ, TK, and
stromal vessels were found. Because of
JMH. Fig. 1. Colour photograph of a typical periph- the stringent diagnostic criteria, no
eral hypertrophic subepithelial degeneration pterygium- or pseudopterygium-like
Results (PHSD; patient #4 right eye in Table 2). The
lesions were present in our series. No
image illustrates the typical signs of PHSD,
which are arcuate peripheral fibrosis and
corneal thinning was observed via
Clinical features of PHSD
adjacent abnormal limbal vasculature. The clinical or tomographic analysis.
The 14 patients represented 13 females fibrosis is typically located in the upper nasal Corneal topography showed irregu-
and one male and presented with per- quadrant underneath the upper eye lid. No lar astigmatism in all patients (100%;
ilimbal arcuate or boomerang-shaped active inflammation is observed. 95% CI, 77–100). Representative

776
 Acta Ophthalmologica 2014

corneal topography (Fig. 3) high-

lighted a high degree of astigmatism,
corneal irregularity and the flattening
and thickening of the cornea at the
location of the fibrosis. Corneal thick-
ening and the loss of corneal transpar-
ency are also clearly visible in the OCT
images in the form of increased corneal
reflectance (Fig. 3).

Immunological markers
Fig. 2. The location of the corneal fibrosis in peripheral hypertrophic subepithelial degeneration
(PHSD). The lesion site is depicted as a polar plot showing that the lesion was centred most often The HLA genes showed a preponder-
in the upper nasal quadrant and never in the infero-temporal or infero-nasal quadrant. Note that ance of the HLA-B*44 allele (Table 2,
PHSD is usually a bilateral disorder. The patients are depicted in the same order as in Table 2 with 21%; 95% CI 8–41); excluding the five
patient #1 representing the innermost circle. patients shared with our previous

Fig. 3. Colour corneal images before (upper row left) and after (right) successful keratectomy for peripheral hypertrophic subepithelial degeneration
(PSHD; patient #7 in Table 2). Corneal topography (middle row) of the same patient before (left) and after (right) successful treatment. In the lower
row, optical coherence tomography (OCT) images are shown before (left) and after (right) keratectomy.

777
Acta Ophthalmologica 2014
series, the frequency was 17% (95% CI
7–41), as compared with the previously
reported 23% (J€ arventausta et al.
2014). In contrast to our previous
findings (J€arventausta et al. 2014), a
higher than usual frequency of ances-
tral haplotype 8.1 (AH8.1) was not
evident; none of the nine more recent
patients shared AH8.1. As in previous
research, no notable differences
emerged regarding C4 deficiencies
(Table 2), immunoglobulin levels (not
shown) or the frequency of other
HLA-*A, HLA-*B or HLA–*DRB1
alleles (J€arventausta et al. 2014).
Treatment
The indication for keratectomy was the
progression of the fibrosis or substan-
tial astigmatism accompanied either by
the worsening of visual acuity or ocular
surface disease. In eyes in which the
fibrosis was distinctly elevated, the
identification of the surgical plane
between the stroma and fibrosis was
relatively simple, and the postoperative
result was generally favourable
(Fig. 3). In patients who had only
slightly elevated fibrosis, the identifica-
tion of the surgical plane was more
difficult, and the excision easily became
either unnecessarily superficial or deep
(not shown). We did not need to
perform any reoperations.
Histopathological analysis
The excised tissue was always sufficient
for light microscopy. For two patients,
specimens were available from both
eyes (#5 and 9 in Table 2). The findings
were similar in each patient. The thick-
ness of the epithelium varied and com-
pensated for the variable thickness of
the subepithelial fibrosis. The epithe-
lium was attenuated to between two
and five cell layers (Figs 4 and 5) and
was thinnest when the underlying fibro-
sis was thickest. Its basement mem-
brane was thin and mostly continuous,
but in some sections, it was discontin-
uous, fragmented or had a porous
appearance. Bowman’s layer was pres-
ent in one specimen only. There, it
partially adhered to the deep surface of
the excised subepithelial fibrosis
(Fig. 4), suggesting that in other eyes,
it remained in the surgical bed. The
subepithelial fibrosis strikingly recapit-
ulated the collagenous lamellar archi-
tecture of the cornea, with intervening
778
thin, quiescent-appearing cells with
thin nuclei, which resembled kerato-
cytes, although the lamellae were coar-
ser, wavy and not as regularly arranged
as normal lamellae sharing the features
of the scleral architecture. Judging by
the number of nuclei, the fibrosis
appeared hypocellular. At its limbal
border, which was reliably identified in
two specimens, the fibrotic tissue was
continuous, with slightly looser and
more cellular perilimbal connective
tissue and without a similarly clear
lamellar architecture, which contained
occasional superficial neovascular
vessels (Fig. 4), plumper, activated
myofibroblasts and a small number of
lymphocytes. We did not find acute or
pathogenetically significant chronic
inflammation in any specimen.
In five specimens from five patients,
the excised fibrotic tissue was scarce
and fragmentary, and we could not
produce adequate immunohistochemi-
cal stainings using it. The immunohis-
tochemical analysis of the remaining
eleven specimens of nine patients
(Table 3) revealed abundant flattened
cells that were immunoreactive for
vimentin between the collagenous
lamellae (Figs 4 and 5). In all speci-
mens, further stainings identified a
heterogenous population of mesenchy-
mal cells consisting of three main types
that varied in proportion among the
specimens and areas of the fibrotic
tissue. The main types were myofibro-
blast-like cells characterized by SMA
(Fig. 4), fibroblasts resembling kerato-
cytes in their being immunoreactive for
CD34 (Fig. 5) and bland fibroblast-like
cells that were immunopositive only for
vimentin (Figs 4 and 5). In three of the
nine patients, myofibroblast-like cells
predominated over CD34-positive ones
(Fig. 4), whereas in four patients,
CD34-positive cells predominated over
myofibroblasts (Fig. 5), and in two
patients, approximately equal propor-
tions of both cell types were observed.
In all specimens, some to many vimen-
tin-positive fibroblast-like cells, scat-
tered CD68-positive macrophages
(Fig. 5) and single CD45-positive lym-
phocytes (not shown) were identified.
Discussion
We have refined the diagnostic criteria
for typical PHSD in an effort to shed
light on its typical clinical and histo-
pathological features. It is possible and
even likely that a wider spectrum of
corneal diseases may eventually be
identified as belonging to the PSHD
spectrum, but the study of its patho-
genesis will be facilitated by initially
concentrating on the most typical
cases. Several less-typical cases in
which the subepithelial fibrosis was
multifocal, vascularized or extended
to the central cornea were included in
a recent retrospective series of 23
patients (Gore et al. 2013) with PHSD,
and some were also included in the
original description (Maust & Raber
2003).
The leading reason for patients to
seek an ophthalmic evaluation in cases
of typical PHSD was decreased visual
acuity due to irregular and regular
astigmatism. Frequent high or irregular
astigmatism was also found in a recent
British series of 23 patients, although
this was not discussed in depth (Gore
et al. 2013). In addition, two-thirds of
our patients, all patients in the initial
description (Maust & Raber 2003) and
almost one-half in the British series
(Gore et al. 2013) suffered from ocular
surface irritation, which may have been
caused by the irregular corneal surface
and unstable tear film, leading to
ocular surface disease, or have been
an underlying feature leading to PSHD
in predisposed individuals. Our series
has a very strong female predomi-
nance, and most patients were middle-
aged. These findings are also in line
with previous studies of PSHD that
were based on less stringent criteria
(Maust & Raber 2003; Jeng & Millstein
2006; Gore et al. 2013; J€ arventausta
et al. 2014).
None of the patients had a history or
signs of concurrent inflammation of the
conjunctiva or the cornea, which some-
times occur in SND (Farjo et al. 2006).
Only one patient had a history of
systemic autoimmune or auto-inflam-
matory disease, contact lens wearing or
injury, which again aided in differenti-
ating PSHD from SND. Although
Meibomian gland disease is found in
about 50% of SND patients (Farjo
et al. 2006), none in our series had this
disease, which is in line with previous
reports (Maust & Raber 2003; Gore
et al. 2013). By definition, the leading
objective sign of typical PSHD was
elevated perilimbal subepithelial fibrosis
with adjacent superficial neovascular-
ization. This fibrosis causes the thicken-
ing of the peripheral to midperipheral
 Acta Ophthalmologica 2014

(A) (B)

(C) (D)

(E) (F)

(H)
(G)

(I) (J)

Fig. 4. Keratectomy specimen (patient #8) representing peripheral hypertrophic subepithelial degeneration (PHSD). Toward corneal periphery,
relatively loose perilimbal tissue (plt) with superficial neovascular vessels (black arrowheads) and relatively more abundant and plumper fibroblast-
like cells adjacent to less cellular, irregularly lamellar fibrous tissue (ft) without blood vessels are present (Panels A, C, E, G, & I). Corneal epithelium
over the fibrous tissue is attenuated (open arrowhead, panels A & B) and basement membrane over the perilimbal tissue (white arrowheads, panel C)
is thicker than over the fibrous tissue. The fibrous tissue appears hypocellular as judged by the number of visible nuclei (Panels A & C), but antibodies
to vimentin identify many more cells (Panel E). CD34 epitope-positive cells resembling keratocytes in this specimen are limited to the perilimbal tissue
(Panel G) whereas cells in the superficial layers of the fibrous tissue are smooth-muscle actin-immunoreactive myofibroblasts (Panel J). The deeper
fibrous layers remain unlabelled. The deep surface of this specimen has a fragment of putative Bowman’s layer (arrowhead) at the centre of the fibrous
tissue (Panels A, C, E, G & I). Otherwise, the findings resemble those of the deceptively hypocellular appearing fibrous tissue in the deeper layers at
the limbal margin (Panels B & D). Only some of the abundant fibroblast-like cells identified with antibodies to vimentin (Panel F) are labelled either
for CD34 epitope (open arrowheads, panel H), smooth muscle actin (open arrowheads, panel J), as highlighted in the insets or CD68 epitope of
macrophages (not shown). Panels A–D are light microscopic images, panels E–J are immunohistochemical stainings. HE, haematoxylin-eosin; PAS;
periodic acid-Schiff, Vim, vimentin; CD, cluster of definition (epitope); and SMA, smooth muscle actin.

cornea and the flattening of the central
cornea in the involved quadrant, and it
frequently occurs bilaterally in the supe-
rior nasal quadrant.
Peripheral hypertrophic subepitheli-
al degeneration seems to be a very slow
progressing disease (Jeng & Millstein
2006), and thus, an initially conserva-
tive wait-and-see attitude is acceptable
in most cases. However, lubricants did
not seem to have any beneficial influ-
ence, as reported previously (Maust &
Raber 2003; Jeng & Millstein 2006;
Gore et al. 2013; J€ arventausta et al.
2014), and thus, symptomatic patients
seem to be good candidates for surgery.
The excision of the fibrotic tissue, if
performed in the correct surgical plane,
will diminish the astigmatism by
roughly half in our experience
(Table 2). We recommend that if the
astigmatism is three dioptres or more
or shows considerable irregularity,
keratectomy should be considered.
Another indication for surgery is
unstable tear film and consequent dry
eye syndrome or ocular surface disease.
After excision, we use an anti-inflam-
matory therapy similar to that used
after phototherapeutic keratectomy to

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Acta Ophthalmologica 2014

Table 3. Semiquantitative immunohistochemical characteristics of keratectomy specimens from

typical peripheral hypertrophic subepithelial degeneration (PHSD). Number of immunolabelled
cells was graded as most (50–100%) +++; many (25–49%) ++, some (5–24%) +; few (1–4%) (+);
single ; and none .

ID Vimentin CD34 SMA CD68e CD45

(A) 1 NS ++  (+) 
2* +++ + + (+) 
5* +++ ++ + NS 
7 +++ ++ (+) 
8 +++ + + (+) (+)**
9 +++ + +++ + (+)**
10 +++ + ++ NS 
(B) 12 +++ (+) ++ + 
14 ++ + (+) 

* Included also in J€arventausta et al. 2014.

** At perilimbal margin.
NS, not stained.

(C)
(D)
(E)
Fig. 5. Keratectomy specimen (patient #7)
representing peripheral hypertrophic subepi-
thelial degeneration (PHSD) from the centre of
the fibrous tissue (panels A–E). Panels A and B
show routine stainings and panels C–E are
immunohistochemical stainings. Corneal epi-
thelium over the fibrous tissue is attenuated
(arrowhead, panel A) and basement membrane
is thin (white arrowhead, panel B). No blood
vessels are found, and the deep surface has no
fragments of Bowman’s layer. Although the
fibrous tissue appears hypocellular as judged
by the number of visible nuclei (panel A & B),
antibodies to vimentin identify abundant fibro-
blast-like cells (panel C), most of which are
CD34 epitope-positive and resemble kerato-
cytes in this specimen (panel D), and only
single ones reacted either for smooth muscle
actin (not shown) or the CD68 epitope of
macrophages (panel E). HE, haematoxylin-
eosin; PAS; periodic acid-Schiff and Vim,
vimentin; and CD, cluster of definition
(epitope).
induce the rapid healing of the corneal
epithelium, as well as lubrication ther-
apy, for several months. We have not
had any recurrences so far after the
excision of typical PSHD, although in
the British series, a small number of
recurrences was reported (Gore et al.
2013). Based on our findings, we do not
currently recommend the use of mito-
mycin C or other antimetabolites at the
time of surgery.
The light microscopic findings con-
sisted of non-specific, apparently hyp-
ocellular fibrosis with abundant
depositions of collagen and a lack of
inflammation; they were identical to
those previously reported for the four-
teen eyes of nine patients (Maust &
Raber 2003; Jeng & Millstein 2006;
Gore et al. 2013). Immunohistochem-
istry, however, identified three distinct
populations of vimentin-immunoposi-
tive flat mesenchymal cells in various
proportions between the collagenous
lamellae, allowing our first insights into
the possible pathogenetic mechanisms
underlying PSHD (Fig. 6). In all spec-
imens except one, a population of the
cells reacted with antibodies to CD34,
which is characteristic of mature kera-
tocytes in adult human cornea (Toti
et al. 2002; Joseph et al. 2003; Espana
et al. 2004; Barbaro et al. 2006; Maat-
ta et al. 2006; Ebihara et al. 2007;
Perrella et al. 2007; Samimi et al.
2007; Thill et al. 2007; Builles et al.
2008). Keratocytes belong to a subset
of fibroblast-like, mesenchymal, den-
dritic interstitial cells on which this
heavily glycosylated transmembrane
sialomucin protein is selectively
expressed. It is also found on hemato-
poietic stem cells, embryonic fibro-
blasts and vascular endothelial cells.
CD34 is suspected of mediating the cell
adhesion of keratocytes, which are
elongated cells with processes that
extensively contact other keratocytes
and the extracellular matrix, although
the ligand of CD34 in the cornea
remains unknown (Joseph et al.
2003). A keratocyte would initially
need to migrate between the epithelium
and Bowman’s layer to cause PSHD in
which the stroma itself seems to be
normal. This could happen either
through an existing opening along a
nerve ending or after a breach has
developed in Bowman’s layer for some
reason.
Cell culture experiments have shown
that under most culture conditions,
keratocytes lose CD34 and other kera-
tocyte-specific markers and differenti-
ate into spindle-shaped, proliferating,
SMA-positive myofibroblasts (Espana
et al. 2004; Barbaro et al. 2006; Ebi-
hara et al. 2007). This is mediated by
transforming growth factor-b1 (TGF-
b1). A similar response is evident in
inflammatory and traumatic corneal
scars, including complicated keratoc-
onus, in which the fibroblast-like cells
are typically CD34-negative and SMA-
positive myofibroblasts (Toti et al.
2002; Maatta et al. 2006). Such myo-
fibroblastic cells were also identified in
all PHSD specimens but two, and they
were likely derived from activated
keratocytes (Fig. 6). This suggests that
the fibrotic lesions undergo active
remodelling as the fibrosis matures,
which may contribute to corneal flat-
tening and irregular astigmatism.
The activating stimulus in PHSD
remains unknown. Cell culture studies
show that under a high concentration
of TGF-b1, keratocytes rapidly down-
regulate CD34 and differentiate into
myofibroblasts (Espana et al. 2004).

780
 Acta Ophthalmologica 2014

keratocytes are mitotically quiescent,

this phenotype might contribute to the
slow progression of PSHD in many
patients.
Finally, data from cell cultures has
also shown that once keratocytes have
differentiated into myofibroblasts, they
may be induced to down-regulate
SMA, for example, by changing the
environment or via fibroblast growth
factor, although the cells do not regain
CD34-positivity (Maltseva et al. 2001;
Espana et al. 2004; Perrella et al.
Fig. 6. Potential pathogenetic pathways in peripheral hypertrophic subepithelial degeneration 2007). While such a down-regulation
(PHSD). CD34-positive keratocytes may become activated, migrate into the subepithelial space of SMA would provide a plausible
and proliferate following a microenvironmental change. In an environment with low TGF-b1 alternative explanation for the presence
concentration, such as low-grade inflammation, and possibly also insufficient extracellular of SMA- and CD34-negative fibro-
calcium, activated keratocytes may differentiate into collagen-producing CD34-negative fibro- blasts in PHSD and would also help
blasts. In an environment with high TGF-b1 concentration and sufficient extracellular calcium, to explain the rarity of recurrences, it
activated CD34-positive keratocytes or CD34-negative fibroblasts may differentiate into CD34-
does not explain the CD34-immuno-
negative, SMA-positive myofibroblasts, which modulate the fibrosis and induce astigmatism.
Possibly these cells may eventually revert back to SMA-negative quiescent fibroblasts, whereas positive cells in our PSHD specimens
later upregulation of CD34 is unlikely to account for the presence of CD34-positive keratocyte- (Fig. 6).
like cells in PSHD. Alternatively, bone marrow-derived stem cells in the corneal stroma or scleral Keratocytes are not the only poten-
fibroblasts could serve as the initiating CD34-positive cells, or a mutation might initiate the tial precursor cells of PHSD in the
cascade or prohibit efficient upregulation of TGF-b1. cornea (Fig. 6). CD34 is also found in
scleral and limbal fibroblasts (Toti
et al. 2002; Espana et al. 2004), and
However, when the concentration of synthesizing fibroblasts in PSHD these cells might migrate between Bow-
TGF-b1 is low, keratocytes only down- (Fig. 6). It should be noted, however, man’s layer and the epithelium to
regulate CD34 and do not transform that CD34- and SMA-negative kerato- initiate PHSD. More recently, a sub-
into myofibroblasts (Espana et al. cytes that apparently do not produce population of corneal stromal cells that
2004). The fibroblast-like cells that collagen also exist, as has been found co-expresses CD34 and CD133, a
have only lost CD34 are thought to in uncomplicated keratoconus and marker of bone marrow-derived stem
synthesize collagen (Samimi et al. around deposits of macular dystrophy cells, has been identified (Perrella et al.
2007). Such a cell type would not only (Toti et al. 2002). Another possibility is 2007; Thill et al. 2007). Studies of
account for the vimentin-positive, that the environment might quench a short-term cell cultures suggest that
CD34-negative and SMA-negative signal that would otherwise up-regulate about 10 to 20 per cent of CD34-
population identified in all PHSD spec- TGF-b1. Cell culture experiments show immunopositive corneal stromal cells
imens, but it would also help to explain that, e.g., low levels of extracellular might originate in the bone marrow
the abundant collagen deposition that calcium concentrations and culturing (Perrella et al. 2007; Thill et al. 2007)
is characteristic of this degeneration on the amniotic membrane will allow and that these can differentiate into
(Fig. 6). The activated quiescent kera- the proliferation of keratocytes in the keratocyte-like fibroblasts (Thill et al.
tocytes around the intracorneal ring absence of their transformation into 2007), which might contribute to repar-
segments that apparently produce col- CD34-negative, SMA-positive myofi- ative processes in the cornea. Thus, a
lagen do not express SMA and have broblasts (Espana et al. 2004; Kawak- third alternative precursor cell might be
lost CD34 (Samimi et al. 2007), reca- ita et al. 2006). Finally, it is possible a CD34-positive, bone marrow-derived
pitulating this particular phenotype of that a mutation might render a kera- stem cell previously resident in the
PHSD specimens. tocyte and its progeny unable to up- cornea or recently migrated into the
Potentially, the absence of a signif- regulate TGF-b1 efficiently, an event subepithelial space from the bone
icant inflammatory reaction after the that normally precedes differentiation marrow.
minimally invasive procedure (Samimi into a myofibroblast. Our immunohistochemical studies
et al. 2007) might account for the low Cell culture experiments have thus confirmed a lack of inflammatory cells
TGF-b1 levels and the failure of acti- shown that at least under certain con- expressing LCA (CD45) within PSHD
vated keratocytes to differentiate into ditions and for some time, keratocytes specimens. In the perilimbal tissue, a
myofibroblasts after ring segment can proliferate without losing CD34 small population of immunopositive
implantation. Similarly, a putative (Perrella et al. 2007), as seems to be the cells was present, along with focal
smouldering, low-grade inflammation case in many PHSD specimens based neovascular vessels. Likewise, CD68-
barely sufficient to up-regulate TGF-b1 on our immunohistochemical findings immunopositive macrophages, while
secretion from keratocytes might (Fig. 6). This was the predominant cell present in almost all specimens, were
maintain an environment favouring type in four of our specimens and typically few in number but were more
differentiation predominantly into formed a minority population in most numerous in the perilimbal tissue. The
CD34- and SMA-negative collagen- of the remaining cases. Because normal reason for the superficial limbal

781
Acta Ophthalmologica 2014
neovascularization and whether it pre-
cedes PHSD remain unexplained. No
limbal stem cell deficiency or conjunc-
tivalization was observed in our series.
The typical bilaterality and superior
nasal location in our series also remain
unexplained. The elusive triggering fac-
tor could be related to immunological
deviations observed in some patients.
None of our patients reported PHSD-
like symptoms or findings in first-
degree relatives. Accordingly, an idio-
pathic degeneration of the cornea
seems a more likely diagnosis than a
corneal dystrophy. In line with this, we
did not find any evidence of disease
clustering in certain geographical areas
of Finland (data not shown). It does
not appear likely that PHSD would be
caused by a mutation in a single gene.
Given the above findings, it seems
reasonable to search for a recessive
inheritance pattern or a biological
aetiology for PHSD. Because of female
predilection and typical age, hormonal
issues could be triggering factors.
In summary, PHSD affects young to
middle-aged female patients, is typi-
cally bilateral and is observed in the
superior nasal quadrant of the cornea.
The fibrotic lesions cause regular and
irregular astigmatism and secondary
ocular surface disease symptoms. The
recognition of this disorder is impor-
tant because of its good prognosis with
surgical management (J€ arventausta
et al. 2014) (Table 2).
Acknowledgements
This study was supported by grants
from the Finnish Eye Foundation, the
Finnish Eye and Tissue Bank Founda-
tion, the Mary and Georg C Ehrnrooth
Foundation, the Sigrid Juselius Foun-
dation, the Helsinki University Central
Hospital Research Fund and the Evald
and Hilda Nissi Foundation.
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Received on October 30th, 2013.
Accepted on February 15th, 2014.
Correspondence:
Juha Holopainen
Helsinki University Eye Hospital
Haartmaninkatu 4C
Helsinki 00290
Finland
Tel: +35894711
Fax: +358975100
Email: juha.holopainen@hus.fi