Bilateral I<eloid-lil<e Myofibroblastic Proliferations of the Cornea in Children LEONARD M. HOLBACH, MD,t RAMON L. FONT, MD,t IRA A. SHIVITZ, MD/ DAN B. JONES, MD1

Abstract: Two 6-month-old white boys developed localized multifocal white nodules of the anterior corneal stroma without evidence of antecedent trauma, inflammatory disease, or familial occurrence. The first patient had bilateral lesions initially; the second patient initially had unilateral involvement followed by a corneal nodule in the second eye at 11 years of age. Light and electron micro­scopic studies of the lesions in both patients were similar and disclosed stromal nodules composed of proliferating myofibroblasts, activated fibroblasts, and haphazardly arranged fascicles of collagen. Immunohistochemical studies re­vealed that the spindle cells within the nodules expressed immunoreactivity for virnentin and alpha-srnooth rnuscle actin. Management options for these slowly progressive corneal lesions include lamellar versus penetrating keratoplasty and topical corticosteroids. Ophthalmology 1 990; 97:1188-1193

Corneal keloids usually diffusely involve the entire stroma or appear as localized solitary nodules that tend to progress slowly. 1- 8 Multifocal corneal thickenings that often project above the corneal surface may occur in Salz­mann's nodular degeneration.9- 18 While corneal keloids have been documented as sequelae of trauma, inflam­matory disease, and in Lowe's syndrome, Salzmann's nodular degeneration has been associated with phlycten­ular keratoconjunctivitis, trachoma, vernal keratocon­junctivitis, measles, and scarlet fever.

We report two cases of bilateral keloid-like myofibro­blastic proliferations of the anterior corneal stroma oc­curring in children who became symptomatic at 6 months

Originally received: February 6, 1990. Revision accepted: April 25, 1990.

'Department of Ophthalmology, Cullen Eye Institute, Baylor College of Medicine, Houston.

2 Tennessee Arrowsmith Eye Institute, Nashville.

Supported by grants from the Retina Research Foundation, the Lions Eye Bank, Houston, Texas, Research to Prevent Blindness, Inc, New York, New York, and the Deutsche Forschungsgemeinschaft (DFG-Ho 11111 1-1 and 1-2), FRG.

Reprint requests to Ramon L. Font, MD, Ophthalmic Pathology Laboratory, Cullen Eye Institute, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

ofage. Neither patient had evidence of familial occurrence or preexisting corneal disease.

MATERIALS AND METHODS

The specimens were fixed in 10% buffered formalin, and sections were cut at 6 !lm from the paraffin-embedded material. The slides were stained with hematoxylin-eosin. Additionally, the following special staining techniques were used: periodic acid-Schiff (PAS), Masson trichrome, alcian blue, colloidal iron for mucopolysaccharides, and Congo red. The avidin-biotin complex (ABC) method was used on unstained sections to evaluate the presence of alpha-smooth muscle actin, desmin, and vimentin. Sec­tions were deparaffinized in xylene, rehydrated in an ethanol series, and washed in phosphate-buffered saline (PBS; pH 7.4). Endogenous peroxidase activity was neu­tralized using 100% methanol and 0.4% hydrogen per­oxide. The sections were incubated with normal horse serum (Vectastain ABC Kit for mouse lgG, Vector Lab­oratories, Burlingame, CA; diluted l :50) in a moist cham­ber for 30 minutes. All immunoreagents were diluted with PBS containing 2% bovine serum albumin.

The primary mouse monoclonal antibodies were ob­tained from Dako Laboratories (Santa Barbara, CA; des­min, l: 10; vimentin, l :20) and Accurate Chemical Sci­

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HOLBACH et al • CORNEAL LESIONS IN CHILDREN

entific Corp. (Westbury, NY; alpha-smooth muscle actin, 1 :300). As negative controls, the primary antibodies were omitted or substituted by different IgG mouse monoclonal antibodies. As positive controls, surgical specimens from vascular smooth muscle (alpha-smooth muscle actin and desmin), striated muscle (desmin), and a schwannoma (vimentin) were stained simultaneously. Subsequently, the sections were incubated with biotinylated horse anti­mouse IgG antiserum (Vectastain ABC kit; 1 :200) and the avidin-biotinylated peroxidase complex (Vectastain ABC kit; 1: 160). The sites of peroxidase activity were vi­sualized by incubation of freshly prepared 3,3'-diamino­benzidine tetrahydrochloride (5 mg in 10 ml of 0.05 M trishydroxymethylaminomethane-buffered saline; pH 7.6) containing 0.0015% hydrogen peroxide. The slides were counterstained with Gill's hematoxylin, dehydrated, cleared in xylene, and mounted in Histoclad medium (C. Adams, Parsippany, NJ).

Tissue blocks from representative areas of the lesion were removed from the paraffin block. The tissue was deparaffinized with xylene, rehydrated, and fixed over­night in phosphate-buffered 2% glutaraldehyde; it was then postfixed in 1% osmium tetroxide. Thick sections were stained with paragon. Thin sections were stained with uranyl acetate and lead citrate and examined with the electron microscope.

CASE REPORTS

Case 1. A 34-month-old white boy was evaluated for bilateral corneal opacities. At 6 months of age, his parents noted "white spots" in the central and peripheral corneal portions of both eyes. Scrapings from the cornea and cultures were negative. The parents noted that the spots had progressively increased in size and denied any antecedent or concurrent ocular inflammation or injury. The boy had been functioning normally with appar­ently normal vision. He was the product of a full-term gestation that was delivered by cesarean section and had had a normal postnatal development. At the age of 44 months, the results of a general physical examination disclosed a systolic heart murmur consistent with pulmonic stenosis and slight hepatosplenomeg­aly. A 12-year-old maternal half-brother had blindness in one eye that was believed to have been caused by Toxocara canis infection. There was no other family history of ocular disease.

Ophthalmologic examination of the right eye was within nor­mal limits except for the cornea. Slit-lamp examination of the right cornea showed a sharply demarcated, crescent-shaped area of white opacification that was located in the anterior stroma peripherally and extended from 11:30 to 4 o'clock (Fig 1 ). The opacification extended to the junction of the anterior one-third and deeper two-thirds of the stroma. Peripheral corneal vascu­larization was present between 11:30 and 3 o'clock. A sharply demarcated, round nodule measuring 1.5 mm in diameter was located just inferior to the pupillary axis (Fig I). The epithelium was intact over both lesions. No inflammatory infiltrates or edema were present in the adjacent clear cornea.

The left cornea had a similar crescent-shaped zone ofanterior stromal opacification extending from 12:30 to 5 o'clock (Fig 2). An oval, sharply demarcated nodule was present in the mid­peripheral region at 3 o'clock (Fig 2). The remainder ofthe ocular examination results were normal. Ocular examination of both parents was unremarkable.

The results of acid mucopolysaccharides in urine, blood chemistries, karyotyping, tuberculin skin test, and skin fibroblast cultures were negative. A left corneal biopsy was performed on July 31, 1985. The immediate postoperative course was un­eventful. At that time, routine cultures for aerobic and anaerobic bacteria and fungi were negative. One year later, ocular exam­ination revealed a visual acuity of less than 20/400 in the right eye and 20/80 in the left eye. Slit-lamp examination results re­vealed fibrovascular scarring in the nasal periphery of the right cornea. The nodule located inferiorly to the visual axis of the right eye had enlarged. For this reason, the referring ophthal­mologist performed a lamellar keratectomy ofthe stromal nodule in the right eye. The microscopic findings were essentially similar to those observed in the first biopsy specimen described below. With spectacle correction and occlusion therapy, visual acuity was 20/60 in the right eye and 20/40 in the left eye.

In February 1987, the peripheral crescent scarring and the central nodule had enlarged and almost coalesced in both eyes (Figs 3 and 4). Visual acuity in the right eye decreased to 20/ 200. In an attempt to restore vision, two lamellar keratectomies ofthe right eye were performed elsewhere (August and December 1987). Unfortunately, this material was not available for his­topathologic examination. The scarring continued to progress in the left eye. Visual acuity decreased to 20/400 bilaterally. In January 1988, a penetrating keratoplasty of the right eye was performed elsewhere. Postoperatively, the patient achieved a vi­sual acuity of 20/80 in the right eye.

Histopathologically, the stromal nodule excised on July 31, 1985 displayed thick, hyalinized, collagenous bundles that were haphazardly arranged with focal areas of fibroblastic proliferation (Fig 5). The corneal epithelium was of irregular thickness with mild intracellular edema. Bowman's layer was absent (Fig 5). Results of special stains for aician blue, colloidal iron for mu­copolysaccharides, and Congo red were not contributory.

Case 2. A 6-month-old white infant whose mother noticed a "white scar" on his left cornea was brought for consultation to an ophthalmologist. The scar progressed to cover his cornea completely. No history of injury or antecedent eye disease was present. The patient was in good health. Slit-lamp examination results of the corneas of both parents and his brother were un­remarkable. The patient underwent five local biopsies, all of which were followed by subsequent recurrences. This material was not available for histopathologic examination.

At the age of 3 years, a lamellar keratoplasty was performed in the left eye. Microscopically, the specimen disclosed stromal nodules that were covered by a thin epithelium centrally. The epithelium was thickened peripherally. Mild intracellular edema of the basal cell layer was noted. The epithelial basement mem­brane was thickened and focally disrupted. Bowman's layer was either fragmented or absent. The stroma contained haphazardly arranged, plump, proliferating fibroblasts intermixed with bun­dles of collagen fibers (Figs 6 and 7). Postoperatively, the graft remained clear.

When the boy was 11 years old, his mother noticed a white lesion involving the inferior portion of the right cornea. At this time, results of ophthalmologic examination showed a visual acuity of 20/20 in the right eye and 20/800 in the left eye. A left exotropia was present. Slit-lamp examination results dis­closed a solitary, subepithelial, whitish nodule involving the in­ferior midperiphery of the right cornea (Fig 8). The overlying epithelium was intact. No inflammatory signs were present. The corneal graft of the left eye was clear centrally. Moderate opaci­fication and vascularization were present at the nasal and tem­poral edges of the donor-host interface. The remainder of the ocular examination was unremarkable. Cultures of the corneal lesion in the right eye showed no growth for bacteria or fungal

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Fill I. Case I. Tnp /eli . the right cornea shows a grayish­white stromal nodule located just below the pupillary axis and a peripheral anterior stromal crescentic opacity extending from the II- to 4­o'clock positions. Peripheral corneal vascularization is observed superonasally (Au­gust 1985). Fig 2. Case I. Top rif(ht. clinical appearance of the left cornea displays a midperipheral. slightly ele­vated, white nodule merging with a crescentic peripheral opacity extending from the 12:.10 to 5:30 positions (Au­gust 1985). Fig 3. Case I. Second row lefi. 18 months later. the corneal lesions in the right eye have markedly enlarged and show coales­cence of the paracentral nod­ule with the peripheral ar­cuate opacity. (For compar­ison . see Fig 1.) Fig 4. Case I. Second row rif(ht, 18 months later, there is marked progression of the stromal opacity in the left eye which extends toward the central cornea. (For comparison, see Fig 2.) Notice the iron line along the edges of the nod­ules. Fig 5. Case I. Third row lefi. histologic appearance of a biopsy specimen from the left cornea excised 2 years and 4 months from onset of disease. The stromal nodule discloses thick hyalinized collagenous bundles that are hapha7ardly arranged with focal areas of fibroblastic proliferation and scattered blood vessels. The corneal epithelium is of irregular thick ness with mild intracel­lular edema. Bowman's layer has been focally disrupted and is absent in most areas. Descemet's membrane and the endothelium are absent (hematoxylin-eosin: original magnification. X40). Fig 6. Case 2. Third row rif(hl. lamellar graft of the left cornea depicts the edge of an elevated stromal nodule. The lesion is hypercellular and composed of fascicles of plump spindle-shaped cells. which exhibit tapering cytoplasmic processes. that represent activated fibroblasts and myofibroblasts. The nodule is sharply demarcated from the underlying stroma (Masson trichrome: original magnification, X40). Fig 7. Case 2. Bottom le.fi , opposite edge of the stromal nodule shown in Figure 6. Notice the fascicles of plump spindle-shaped cells which represent activated fibroblasts and myofibroblasts. Bowman's layer is absent (Masson trichrome: original magnification, X64). Fig 8. Case 2. Bollom riKht. slit-lamp view of the right eye depicts a slightly elevated, grayish-white, subepithelial nodule located in the inferior midperipheral cornea.

with prominent fibroblastic proliferation interspersed with ir­cornea was performed. Microscopically, the specimen was similar regular bundles of collagen . to that described in case I by displaying marked hypercellularity Two months postoperatively, the lesion recurred in the right

elements. An excisional biopsy of the stromal nodule in the right

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HOLBACH et a/ • CORNEAL LESIONS IN CHILDREN

Fig 9. Electron micrograph of activated fibroblast shows di­lated cisternae of rough-sur­faced endoplasmic reticulum (ER) containing granular de­posits and scattered mito­chondria (M). Patchy base­ment membrane formation is noted (arrow) (original mag­nification, Xl6,950).

eye. The nodule increased in size, and caused irritation and de­creased visual acuity. The patient undetwent an 8.0-mm lamellar keratoplasty without complications. Histopathologic examina­tion of the specimen showed similar findings to those described in the excisional biopsy specimen. Three months postoperatively, the patient developed two nodular areas at the graft-host inter­face associated with areas ofperipheral vascularization. Following suture removal and the administration of topical corticosteroids, no further progression of the corneal changes was observed. Currently, the patient's visual acuity is 20/40 in the right eye and 20/800 in the left eye. The right cornea shows subepithelial nodules located at the graft-host junction where the sutures were found. Both right and left grafts remain clear.

IMMUNOHISTOCHEMICAL AND ELECTRON MICROSCOPIC FINDINGS

Immunoperoxidase studies showed that the majority of the spindle cells within the stromal nodules expressed alpha-smooth muscle actin and vimentin. The cells were not immunoreactive for desmin. Electron microscopic studies disclosed that the spindle-shaped cells within the stromal nodules were either activated fibroblasts or myo­fibroblasts showing numerous tapering cytoplasmic pro­cesses that contained well-developed, rough-surfaced en­doplasmic reticulum and long bundles of 5- to 6-nm, ac­tin-like microfilaments with scattered fusiform densities, some of which had inserted on the plasma membranes (Figs 9-11). Approximately 60 to 70% of the spindle cells had the ultrastructural features of myofibroblasts.

DISCUSSION

Clinically, the lesions in our two patients resembled those seen in Salzmann's nodular degeneration.9- 11 The

median age of the reported patients with Salzmann's nodular degeneration at the time of diagnosis was 38 years.9

-17 Well-documented cases have not been reported

at such an early age as in our two patients. At least 57 patients with corneal keloids have been re­

ported in the literature. 1-8 Corneal keloids are found most

frequently among younger patients. The median age of 35 patients with corneal keloids whose age was known was 13 years (range, 2 months-72 years). Sixty-six percent (23 of 35) of patients with corneal keloids became symp­tomatic within the first two decades of life. 1- 5•

7•8 This is

not surprising since it has been well established that the proliferative capacity of fibroblasts is increased in younger individuals. At the time of first presentation, both of our patients were 6 months old. Corneal keloids have been observed after injuries, particularly perforating trauma and in childhood keratitis. The spectrum of preceding ocular inflammations associated with keloids includes perforated corneal ulcers, complicating measles, and pu­rulent ophthalmia neonatorum. 1•

4 In the latter condition, the corneal keloids have been reported to be bilateral. Bilateral corneal keloids also may occur in Lowe's syndrome7 and are thought to be characteristic of this disease.6 The fact that our second patient developed sub­epithelial nodules at the graft-host interface where the sutures were located favors their keloidal nature since cu­taneous keloids arc known to form after surgical trauma.

The presence of myofibroblasts has been reported in nodular fasciitis of the ocular adnexa, 19 granulation tissue and wound healing,20 congenital isolated fibromatosis of the eyelid,21 and fibrous histiocytomas. Myofibroblast-like cells also have been described in eyes with proliferative vitreoretinopathi2·23 and in subcapsular cataracts.24 The histopathologic features of the stromal nodules varied in each patient. In case 1, the stromal nodules were pauci­

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cellular and appeared more collagenized with focal areas offibroblastic proliferation (Fig 5), and in case 2, the nod­ules were markedly hypercellular with prominent spindle cell proliferation and less collagen deposition (Figs 6 and 7). The latter finding may be related to the different stage and duration of the lesions at the time of keratectomy

Fig 10. Electron micrograph depicts mature myofibroblast displaying infoldings of the nuclear membrane with bundles of thin actin-like fil­aments (F) under the plasma lemma and scattered fusi­form densities (arrowheads). Focal basement membrane is present (arrows) (original magnification, X14,600).

Fig 11. High-power view of cell process of myofibroblast (adjacent to activated fibro­blast) depicts numerous cy­toplasmic thin filaments (F) and fusiform densities (ar­rowheads). Collagen (C) (original magnification, X29,000).

and to the multiple excisional biopsies previously per­formed as in case 2. The immunohistochemical identifi­cation of alpha-smooth muscle actin in the myofibroblasts of the corneal lesions in our two cases is consistent with their electron microscopic features.

Having recognized the clinical, histopathologic, im­1192

HOLBACH et al • CORNEAL LESIONS IN CHILDREN

munohistochemical, and electron microscopic features of these exuberant, keloid-like myofibroblastic proliferations, it becomes evident that they are different from those de­scribed in keratectomy specimens from patients with

10 12 14 17Salzmann's nodular degeneration.9• • • • To our knowledge, the presence of myofibroblasts has not been reported previously in Salzmann's nodular degeneration.

The differential diagnosis of nodular, acquired, non­neoplastic lesions of the anterior corneal stroma should also include circumferential nodulocystic keratopathy,25

familial band-shaped nodular keratopathy,26 bilateral ring dermoids,27 and spheroidal degenerations.28 These clini­copathologic entities, however, are quite distinct from the lesions described in our cases. Recently, a 34-year-old man with nodular-type corneal scarring ofautosomal dominant inheritance and cutaneous manifestations ofthe epidermal nevus syndrome was documented.29 In our two patients, no evidence of familial occurrence, dermatologic changes, or other coexisting ocular diseases was present.

The etiology of the keloid-like myofibroblastic prolif­erations in our patients remains unknown. Possible local factors include trauma, retained foreign bodies, and in­flammation. None ofour patients had a history of surgery, trauma, or any other evidence ofa chronic ocular surface disorder. One may speculate that a previously unrecog­nized corneal infection had introduced microbial antigens that led to an immune-mediated hypertrophic scar.

It is important to defer any surgery in these patients until there is evidence of visual impairment or symptoms due to alterations of the tear film. If, indeed, the lesions interfere significantly with visual acuity, a lamellar ker­atoplasty may be required. In older patients with pro­gressive lesions, penetrating keratoplasty·may be indicated to minimize the chances of recurrence. The presence of increased amounts of collagen produced by activated fi­broblasts and myofibroblasts in the stromal nodules of these patients suggests that topical steroids may have a beneficial effect by inhibiting fibroblastic proliferation and collagen deposition.

ACKNOWLEDGMENTS

Rhoads E. Stevens, MD, kindly provided Figures 3 and 4.

REFERENCES

1. Smith HC. Keloid of the cornea. Trans Am Ophthalrnol Soc 1940; 38: 519-38.

2. Fenton RH, Tredici T J. Hypertrophic corneal scars (keloids). Surv Ophthalmol1964; 9:561-6.

3. Farkas TG, Znajda JP. Keloid of the cornea. Am J Ophthalrnol1968; 66319-23.

4. Frederique G, Howard RO, Boniuk V. Corneal ulcers in rubeola. Am J Ophthalmol 1969; 68:996-1003

5. O'Grady RB, Kirk HO. Corneal keloids. Am J Ophthalrnol 1972; 73: 206-13.

6. Collier E. Discussion, 594. Of: Brown N. Lowe syndrome: identification of the carrier state. In: Bergsma 0, Bron AJ, Collier E, eds. The Eye and Inborn Errors of Metabolism. New York: Alan R. Liss, 1976; 579­95. (Birth Defects: Original Article Series; voL 12, no. 3).

7. Cibis GW, Tripathi RC, Tripathi BJ, Harris OJ. Corneal keloid in Lowe's syndrome. Arch Ophthalmol1982; 100:1795-9.

8. Lahav M, Cadet JC, Chirambo M, et aL Corneal keloids-a histo­pathological study. Graefes Arch Clin Exp Ophthalmol 1982; 218: 256-61.

9. Salzmann M. Uber eine Abart der kni:itchenfOfi:irmigen Hornhautdys­trophie. Ztschr f Augenh 1925; 57:92-9.

10. Katz D. Salzmann's nodular corneal dystrophy: report of a case. Arch Ophthalmol 1930; 4:16-21.

11. Brown EVL, Katz D. Salzmann's nodular corneal dystrophy: its patho­logic process and a suggested therapy. Arch Ophthalmol 1935; 13: 598-613.

12. Muir EB. Salzmann's nodular corneal dystrophy: report of a case. Am J Ophthalmol1940; 23:138-44.

13. Katz D. Salzmann's nodular corneal dystrophy. Acta Ophthalmol1953; 31:377-83.

14. Vannas A, Hogan MJ, Wood L Salzmann's nodular degeneration of the cornea. Am J Ophthalrnol 1975; 79:211-9.

15. Abbott RL, Forster RK. Superficial punctate keratitis of Thygeson as­sociated with scarring and Salzmann's nodular degeneration. Am J Ophthalmol1979; 87:296-8.

16. Reinach NW, Baum J. A corneal pigmented line associated with Salz­mann's nodular degeneration. Am J Ophthalmol1981; 91:677-8.

17. Pau H, Schmitt-Graff A. Salzmannsche Hornhautdystrophie (Kni:itch­enfOrmige Hornhautdegeneration). Graefes Arch Clin Exp Ophthalmol 1982; 218161-7.

18. Wood TO. Salzmann's nodular degeneration. Cornea 1990; 9:17-22. 19. Font RL, Zimmerman LE. Nodular fasciitis of the eye and adnexa: a

report of ten cases. Arch Ophthalmol1966; 75:475-81. 20. Skalli 0, Schurch W, Seemayer T, et aL Myofibroblasts from diverse

pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins. Lab Invest 1989; 60:275-85.

21. Witschel H. Angeborene isolierte Fibrornatose des Lides. Ophthal­rnologica 1982; 185:1-6.

22. Macherner R, van Horn 0, Aaberg TM. Pigment epithelial proliferation in human retinal detachment with massive periretinal proliferation. Am J Ophthalmol 1978; 85:181-91.

23. Kampik A, Kenyon KR, Michels RG, et aL Epiretinal and vitreous membranes: comparative study of 56 cases. Arch Ophthalmol 1981; 99:1445-54.

24. Novotny GEK, Pau H. Myofibroblast-like cells in human anterior cap­sular cataract Virchows Arch [A]1984; 404:393-401.

25. Carpel EF, Cameron JD, Wick MR. Circumferential nodulocystic ker­atopathy: a case report Graefes Arch Clin Exp Ophthalrnol 1988; 226505-9.

26. Meisler OM, Tabbara KF, Wood IS, et aL Familial band-shaped nodular keratopathy. Ophthalmology 1985; 92:217-22.

27. Mattos J, Contreras F, O'Donnell FE Jr. Ring dermoid syndrome. A new syndrome of autosomal dominantly inherited, bilateral, annular limbal dermoids with corneal and conjunctival extension. Arch Ophthalmol1980; 98:1059-61.

28. Garner A, Fraunfelder FT, Barras TC, Hinzpeter EN. Spheroidal de­generation of cornea and conjunctiva. Br J Ophthalmol 1976; 60: 473-8.

29. Hirst LW, Farmer ER, Green WR, et al. Familial corneal scarring: a new dystrophy? Ophthalmology 1984; 91:17 4-8.

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