Ophthalmic Pterygium
2011; Elsevier BV; Volume: 178; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2010.10.037
ISSN1525-2191
AutoresJeanie Chui, Minas T. Coroneo, Lien Tat, Roger Crouch, Denis Wakefield, Nick Di Girolamo,
Tópico(s)Ocular Disorders and Treatments
ResumoPterygia are common ocular surface lesions thought to originate from limbal stem cells altered by chronic UV exposure. Traditionally regarded as a degenerative condition, pterygia also display tumor-like features, such as a propensity to invade normal tissue and high recurrence rates following resection, and may coexist with secondary premalignant lesions. This study was initiated to determine the rate of concurrent ocular surface diseases in patients with pterygia recruited from the practice of a single surgeon operating in a Sydney metropolitan hospital. One hundred pterygium specimens were histopathologically reviewed and selected cases were immunohistochemically assessed to confirm diagnosis. Along with previously documented typical features including epithelial proliferation, goblet cell hyperplasia, angiogenesis, inflammation, elastosis, stromal plaques, and Bowman's membrane dissolution, we identified five cases of ocular surface squamous neoplasia, six cases of primary acquired melanosis, two compound nevi (one suspect invasive melanoma), and one dermoid-like lesion. In 18 specimens, clusters of basal epithelial cells that coexpressed cytokeratin-15/-19 and p63-α were identified at the head of the pterygium, coinciding with clinical observation of Fuchs' flecks. Our data show that significant preneoplastic lesions may be associated with pterygium and that all excised pterygia should undergo histological examination. The presence of p63-α-positive epithelial cell clusters supports the hypothesis that pterygia develop from limbal epithelial progenitors. Pterygia are common ocular surface lesions thought to originate from limbal stem cells altered by chronic UV exposure. Traditionally regarded as a degenerative condition, pterygia also display tumor-like features, such as a propensity to invade normal tissue and high recurrence rates following resection, and may coexist with secondary premalignant lesions. This study was initiated to determine the rate of concurrent ocular surface diseases in patients with pterygia recruited from the practice of a single surgeon operating in a Sydney metropolitan hospital. One hundred pterygium specimens were histopathologically reviewed and selected cases were immunohistochemically assessed to confirm diagnosis. Along with previously documented typical features including epithelial proliferation, goblet cell hyperplasia, angiogenesis, inflammation, elastosis, stromal plaques, and Bowman's membrane dissolution, we identified five cases of ocular surface squamous neoplasia, six cases of primary acquired melanosis, two compound nevi (one suspect invasive melanoma), and one dermoid-like lesion. In 18 specimens, clusters of basal epithelial cells that coexpressed cytokeratin-15/-19 and p63-α were identified at the head of the pterygium, coinciding with clinical observation of Fuchs' flecks. Our data show that significant preneoplastic lesions may be associated with pterygium and that all excised pterygia should undergo histological examination. The presence of p63-α-positive epithelial cell clusters supports the hypothesis that pterygia develop from limbal epithelial progenitors. Pterygium is a wing-shaped ocular surface lesion traditionally described as an encroachment of bulbar conjunctiva onto the cornea.1Duke-Elder S. Diseases of the Outer Eye Part 1 System of Ophthalmology 8. Kimpton, London1965: 569-585Google Scholar Historically, pterygia were considered degenerative lesions, exemplified by degradation of Bowman's layer and elastosis. Currently, however, pterygia are described as a proliferative disorder resembling an aberrant wound healing response.2Di Girolamo N. Chui J. Coroneo M.T. Wakefield D. Pathogenesis of pterygia: role of cytokines, growth factors, and matrix metalloproteinases.Prog Retin Eye Res. 2004; 23: 195-228Crossref PubMed Scopus (267) Google Scholar Histopathologically, pterygia are characterized by a hyperplastic, centripetally directed growth of altered limbal epithelial cells accompanied by Bowman's layer dissolution, epithelial-mesenchymal transition, and an activated fibroblastic stroma with inflammation, neovascularization, and matrix remodeling, mediated through the concerted actions of cytokines, growth factors, and matrix metalloproteinases.2Di Girolamo N. Chui J. Coroneo M.T. Wakefield D. Pathogenesis of pterygia: role of cytokines, growth factors, and matrix metalloproteinases.Prog Retin Eye Res. 2004; 23: 195-228Crossref PubMed Scopus (267) Google Scholar, 3Di Girolamo N. McCluskey P. Lloyd A. Coroneo M.T. Wakefield D. Expression of MMPs and TIMPs in human pterygia and cultured pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2000; 41: 671-679PubMed Google Scholar, 4Di Girolamo N. Coroneo M.T. Wakefield D. Active matrilysin (MMP-7) in human pterygia: potential role in angiogenesis.Invest Ophthalmol Vis Sci. 2001; 42: 1963-1968PubMed Google Scholar, 5Di Girolamo N. Kumar R.K. Coroneo M.T. Wakefield D. UVB-mediated induction of interleukin-6 and -8 in pterygia and cultured human pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2002; 43: 3430-3437PubMed Google Scholar, 6Di Girolamo N. Coroneo M.T. Wakefield D. UVB-elicited induction of MMP-1 expression in human ocular surface epithelial cells is mediated through the ERK1/2 MAPK-dependent pathway.Invest Ophthalmol Vis Sci. 2003; 44: 4705-4714Crossref PubMed Scopus (91) Google Scholar, 7Di Girolamo N. Coroneo M. Wakefield D. Epidermal growth factor receptor signaling is partially responsible for the increased matrix metalloproteinase-1 expression in ocular epithelial cells after UVB radiation.Am J Pathol. 2005; 167: 489-503Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 8Di Girolamo N. Wakefield D. Coroneo M.T. UVB-mediated induction of cytokines and growth factors in pterygium epithelial cells involves cell surface receptors and intracellular signaling.Invest Ophthalmol Vis Sci. 2006; 47: 2430-2437Crossref PubMed Scopus (118) Google Scholar, 9Chui J. Di Girolamo N. Wakefield D. Coroneo M.T. The pathogenesis of pterygium: current concepts and their therapeutic implications.Ocul Surf. 2008; 6: 24-43Abstract Full Text PDF PubMed Scopus (187) Google Scholar Despite advances in understanding of its pathogenesis, pterygium remains an ophthalmic enigma. Intriguingly, pterygia have a predilection for the nasal limbus and affect only humans, possibly reflecting the unique ocular morphology of humans, compared with nonhuman primates and other animals.10Kobayashi H. Kohshima S. Unique morphology of the human eye.Nature. 1997; 387: 767-768Crossref PubMed Scopus (391) Google Scholar Although there is no consensus regarding the pathogenesis of pterygia, epidemiological evidence,11Moran D.J. Hollows F.C. Pterygium and ultraviolet radiation: a positive correlation.Br J Ophthalmol. 1984; 68: 343-346Crossref PubMed Scopus (331) Google Scholar, 12Threlfall T.J. English D.R. Sun exposure and pterygium of the eye: a dose-response curve.Am J Ophthalmol. 1999; 128: 280-287Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 13McCarty C.A. Fu C.L. Taylor H.R. Epidemiology of pterygium in Victoria, Australia.Br J Ophthalmol. 2000; 84: 289-292Crossref PubMed Scopus (169) Google Scholar, 14Tan C.S. Lim T.H. Koh W.P. Liew G.C. Hoh S.T. Tan C.C. Au Eong K.G. Epidemiology of pterygium on a tropical island in the Riau Archipelago.Eye. 2006; 20: 908-912Crossref PubMed Scopus (57) Google Scholar its association with sun-related disorders such as pinguecula and cataracts,15Lim R. Mitchell P. Cumming R.G. Cataract associations with pinguecula and pterygium: the Blue Mountains Eye Study.Am J Ophthalmol. 1998; 126: 717-719Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar climatic droplet keratopathy,16Taylor H.R. West S.K. Rosenthal F.S. Munoz B. Newland H.S. Emmett E.A. Corneal changes associated with chronic UV irradiation.Arch Ophthalmol. 1989; 107: 1481-1484Crossref PubMed Scopus (244) Google Scholar and squamous cell and basal cell carcinomas,17Clear A.S. Chirambo M.C. Hutt M.S. Solar keratosis, pterygium, and squamous cell carcinoma of the conjunctiva in Malawi.Br J Ophthalmol. 1979; 63: 102-109Crossref PubMed Scopus (93) Google Scholar, 18Kerkenezov N. A pterygium survey of the far north coast of New South Wales.Trans Ophthalmol Soc Aust. 1956; 16: 110-119PubMed Google Scholar together with our in vitro studies,5Di Girolamo N. Kumar R.K. Coroneo M.T. Wakefield D. UVB-mediated induction of interleukin-6 and -8 in pterygia and cultured human pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2002; 43: 3430-3437PubMed Google Scholar, 6Di Girolamo N. Coroneo M.T. Wakefield D. UVB-elicited induction of MMP-1 expression in human ocular surface epithelial cells is mediated through the ERK1/2 MAPK-dependent pathway.Invest Ophthalmol Vis Sci. 2003; 44: 4705-4714Crossref PubMed Scopus (91) Google Scholar, 7Di Girolamo N. Coroneo M. Wakefield D. Epidermal growth factor receptor signaling is partially responsible for the increased matrix metalloproteinase-1 expression in ocular epithelial cells after UVB radiation.Am J Pathol. 2005; 167: 489-503Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 8Di Girolamo N. Wakefield D. Coroneo M.T. UVB-mediated induction of cytokines and growth factors in pterygium epithelial cells involves cell surface receptors and intracellular signaling.Invest Ophthalmol Vis Sci. 2006; 47: 2430-2437Crossref PubMed Scopus (118) Google Scholar support the concept that UV radiation plays a major role in development of pterygium.19Di Girolamo N. Signalling pathways activated by ultraviolet radiation: role in ocular and cutaneous health.Curr Pharm Des. 2010; 16: 1358-1375Crossref PubMed Scopus (23) Google Scholar Furthermore, the limbal predilection may be explained by the phenomenon of peripheral light focusing, in which incidental light passes through the anterior chamber and is focused at the distal (nasal) limbus where limbal stem cells (LSCs) reside.20Coroneo M.T. Muller-Stolzenburg N.W. Ho A. Peripheral light focusing by the anterior eye and the ophthalmohelioses.Ophthalmic Surg. 1991; 22: 705-711PubMed Google Scholar A healthy corneal surface is maintained by self-renewing, lineage-specific stem cells (SCs) that reside in the limbus, a narrow annular transition zone that circumscribes the cornea. This regenerative capacity is regulated by exquisite programs that govern stem cell quiescence, proliferation, migration, and differentiation. Failure to maintain a normal microenvironment as a result of extrinsic (eg, UV radiation) or intrinsic (eg, cytokines) signals can result in the development of ocular disorders.2Di Girolamo N. Chui J. Coroneo M.T. Wakefield D. Pathogenesis of pterygia: role of cytokines, growth factors, and matrix metalloproteinases.Prog Retin Eye Res. 2004; 23: 195-228Crossref PubMed Scopus (267) Google Scholar, 3Di Girolamo N. McCluskey P. Lloyd A. Coroneo M.T. Wakefield D. Expression of MMPs and TIMPs in human pterygia and cultured pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2000; 41: 671-679PubMed Google Scholar, 4Di Girolamo N. Coroneo M.T. Wakefield D. Active matrilysin (MMP-7) in human pterygia: potential role in angiogenesis.Invest Ophthalmol Vis Sci. 2001; 42: 1963-1968PubMed Google Scholar, 5Di Girolamo N. Kumar R.K. Coroneo M.T. Wakefield D. UVB-mediated induction of interleukin-6 and -8 in pterygia and cultured human pterygium epithelial cells.Invest Ophthalmol Vis Sci. 2002; 43: 3430-3437PubMed Google Scholar, 6Di Girolamo N. Coroneo M.T. Wakefield D. UVB-elicited induction of MMP-1 expression in human ocular surface epithelial cells is mediated through the ERK1/2 MAPK-dependent pathway.Invest Ophthalmol Vis Sci. 2003; 44: 4705-4714Crossref PubMed Scopus (91) Google Scholar, 19Di Girolamo N. Signalling pathways activated by ultraviolet radiation: role in ocular and cutaneous health.Curr Pharm Des. 2010; 16: 1358-1375Crossref PubMed Scopus (23) Google Scholar, 21Di Girolamo N. Bosch M. Zamora K. Coroneo M.T. Wakefield D. Watson S.L. A contact lens-based technique for expansion and transplantation of autologous epithelial progenitors for ocular surface reconstruction.Transplantation. 2009; 87: 1571-1578Crossref PubMed Scopus (132) Google Scholar, 22Figueira E.C. Di Girolamo N. Coroneo M.T. Wakefield D. The phenotype of limbal epithelial stem cells.Invest Ophthalmol Vis Sci. 2007; 48: 144-156Crossref PubMed Scopus (100) Google Scholar The importance of an intact limbus and its stem cells was recognized four decades ago by Davanger and Evensen,23Davanger M. Evensen A. Role of the pericorneal papillary structure in renewal of corneal epithelium.Nature. 1971; 229: 560-561Crossref PubMed Scopus (519) Google Scholar who proposed that pterygia represent a specific zone of LSC deficiency. Our hypothesis for pterygium development takes into account peripheral light focusing2Di Girolamo N. Chui J. Coroneo M.T. Wakefield D. Pathogenesis of pterygia: role of cytokines, growth factors, and matrix metalloproteinases.Prog Retin Eye Res. 2004; 23: 195-228Crossref PubMed Scopus (267) Google Scholar, 9Chui J. Di Girolamo N. Wakefield D. Coroneo M.T. The pathogenesis of pterygium: current concepts and their therapeutic implications.Ocul Surf. 2008; 6: 24-43Abstract Full Text PDF PubMed Scopus (187) Google Scholar, 19Di Girolamo N. Signalling pathways activated by ultraviolet radiation: role in ocular and cutaneous health.Curr Pharm Des. 2010; 16: 1358-1375Crossref PubMed Scopus (23) Google Scholar, 20Coroneo M.T. Muller-Stolzenburg N.W. Ho A. Peripheral light focusing by the anterior eye and the ophthalmohelioses.Ophthalmic Surg. 1991; 22: 705-711PubMed Google Scholar at the nasal limbus, which activates and/or mutates LSCs, resulting in clonal expansion, local cell proliferation, and invasion into the cornea (Figure 1A). Alternatively, focal UV radiation may destroy the LSC repository, which acts as a barrier that segregates cornea from conjunctiva, thereby opening the flood gates for conjunctival ingress and pterygium formation. Furthermore, an intrinsic weakness in the LSC reserves is implied by less prominent limbal palisades in the nasal and temporal limbus,24Goldberg M.F. Bron A.J. Limbal palisades of Vogt.Trans Am Ophthalmol Soc. 1982; 80: 155-171PubMed Google Scholar, 25Shortt A.J. Secker G.A. Munro P.M. Khaw P.T. Tuft S.J. Daniels J.T. Characterization of the limbal epithelial stem cell niche: novel imaging techniques permit in vivo observation and targeted biopsy of limbal epithelial stem cells.Stem Cells. 2007; 25: 1402-1409Crossref PubMed Scopus (248) Google Scholar suggesting that these regions might be more susceptible to damage and less likely to undergo effective repair. An analogous mechanism may occur in patients with total LSC deficiency,26Tseng S.C.G. Chen J.J.Y. Huang A.J.W. Kruse F.E. Maskin S.L. Tsai R.J.F. Classification of conjunctival surgeries for corneal diseases based on stem cell concept.Ophthalmol Clin North Am. 1990; 3: 595-610Google Scholar in which the absence of LSCs allows conjunctival invasion of the cornea to occur from 360 degrees (Figure 1B). In support of this posit, consecutive rounds of limbal excision affected wound healing, encouraged neovascularization, and promoted conjunctival ingress in rabbit corneas.27Huang A.J. Tseng S.C. Corneal epithelial wound healing in the absence of limbal epithelium.Invest Ophthalmol Vis Sci. 1991; 32: 96-105PubMed Google Scholar Ophthalmologists have traditionally regarded pterygia as benign lesions, because they grow slowly. Unless a pterygium is sufficiently large as to obscure the visual axis or causes astigmatism, decisions to treat are often based on a patient's cosmetic concerns. An argument against this view, however, is the local invasiveness and high rate of recurrence when pterygia are inappropriately managed.28Mourits M.P. Wyrdeman H.K. Jurgenliemk-Schulz I.M. Bidlot E. Favorable long-term results of primary pterygium removal by bare sclera extirpation followed by a single 90Strontium application.Eur J Ophthalmol. 2008; 18: 327-331PubMed Google Scholar Current management strategies for pterygia involve surgical excision, followed by wound closure with grafts or by application of adjunctive therapy to the bare scleral bed.29Hirst L.W. The treatment of pterygium.Surv Ophthalmol. 2003; 48: 145-180Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 30Ang L.P. Chua J.L. Tan D.T. Current concepts and techniques in pterygium treatment.Curr Opin Ophthalmol. 2007; 18: 308-313Crossref PubMed Scopus (165) Google Scholar Once excised, pterygia are commonly discarded without histological evaluation. This practice is not recommended, in the face of reported identification of unsuspected and potentially malignant secondary disorders in association with pterygia31Sevel D. Sealy R. Pterygia and carcinoma of the conjunctiva.Trans Ophthalmol Soc UK. 1969; 88: 567-578PubMed Google Scholar, 32Hirst L.W. Axelsen R.A. Schwab I. Pterygium and associated ocular surface squamous neoplasia.Arch Ophthalmol. 2009; 127: 31-32Crossref PubMed Scopus (74) Google Scholar, 33Perra M.T. Colombari R. Maxia C. Zucca I. Piras F. Corbu A. Bravo S. Scarpa A. Sirigu P. Finding of conjunctival melanocytic pigmented lesions within pterygium.Histopathology. 2006; 48: 387-393Crossref PubMed Scopus (17) Google Scholar (Table 1). These studies suggest that pterygia might have the propensity to evolve into precursors of squamous cell carcinoma and malignant melanoma of the ocular surface (Figure 1C).Table 1Premalignant Ocular Disease Reported in Association with Pterygium and PingueculaReferencesStudy populationTissuesSample sizeAssociated ocular disease%Sevel and Sealy31Sevel D. Sealy R. Pterygia and carcinoma of the conjunctiva.Trans Ophthalmol Soc UK. 1969; 88: 567-578PubMed Google ScholarCape Town, South Africapterygian = 100squamous cell carcinoma12carcinoma in situ17Clear et al17Clear A.S. Chirambo M.C. Hutt M.S. Solar keratosis, pterygium, and squamous cell carcinoma of the conjunctiva in Malawi.Br J Ophthalmol. 1979; 63: 102-109Crossref PubMed Scopus (93) Google ScholarMalawipinguecula and pterygian = 167hyperplasia or mild dysplasia75.4moderate dysplasia11.4carcinoma in situ12.6Erie et al34Erie J.C. Campbell R.J. Liesegang T.J. Conjunctival and corneal intraepithelial and invasive neoplasia.Ophthalmology. 1986; 93: 176-183Abstract Full Text PDF PubMed Scopus (270) Google ScholarMayo Clinic, Minnesotapterygian = 92carcinoma in situ9.8Perra et al33Perra M.T. Colombari R. Maxia C. Zucca I. Piras F. Corbu A. Bravo S. Scarpa A. Sirigu P. Finding of conjunctival melanocytic pigmented lesions within pterygium.Histopathology. 2006; 48: 387-393Crossref PubMed Scopus (17) Google ScholarEcuadorpterygian = 80PAM without atypia6.3PAM with atypia2.5nevi2.5Hirst et al32Hirst L.W. Axelsen R.A. Schwab I. Pterygium and associated ocular surface squamous neoplasia.Arch Ophthalmol. 2009; 127: 31-32Crossref PubMed Scopus (74) Google ScholarQueensland, Australiapterygian = 533OSSN9.8PAM, primary acquired melanosis; OSSN, ocular surface squamous neoplasia. Open table in a new tab PAM, primary acquired melanosis; OSSN, ocular surface squamous neoplasia. In this study, we examined the histopathology of pterygia from patients treated by a single surgeon operating in a metropolitan hospital in Sydney, Australia. Histological features of pterygia and concurrent ocular diseases were recorded. All unusual cases were reviewed by an experienced anatomical pathologist and were further investigated by immunohistochemical methods. Additionally, we describe novel cell clusters in some pterygia that expressed putative LSC markers. These cell clusters may provide the first histological evidence supporting the view that pterygium is a disease of stem cell origin. Patients undergoing routine pterygium excision surgery by a single surgeon (M.T.C.) were recruited from Prince of Wales Hospital, Randwick, Sydney, Australia, and from the surgeon's private practice. Clinicodemographic features recorded included patient age and sex and location (nasal or temporal) and type of lesion (primary or recurrent) (Table 2). The study population was of mixed ethnic background, but the majority of patients were of European continental origin. Patients underwent routine ophthalmic examination and documentation of the pterygium, including anterior segment photography (iPIX camera; Designs For Vision, Ronkonkoma, NY) and in vivo confocal microscopy (HRT 3 Rostock cornea module; Heidelberg Engineering, Heidelberg, Germany). There were no clinical signs of dysplasia in any of the patients, although one patient demonstrated obvious pigmentation in the head of his pterygium. All patients underwent pterygium excision with reconstruction of the resulting wound using an autologous free limbal-conjunctival graft.35Coroneo M.T. Beheading the pterygium.Ophthalmic Surg. 1992; 23: 691-692PubMed Google Scholar Informed consent was obtained from each patient before tissue and data collection. This study was approved by the institutional Human Research Ethics Committee and adheres to the tenets of the Declaration of Helsinki.Table 2Clinicodemographic Data for 100 Patients with PterygiaVariableValueAge (years) Mean ± standard deviation50 ± 15 Range21–83Sex (no. of cases) Male62⁎Two females and one male had bilateral disease and pterygium surgery on separate occasions. Female35⁎Two females and one male had bilateral disease and pterygium surgery on separate occasions.Eye (no. of cases) Right45 Left55Location of lesion (no. of cases) Nasal96 Temporal4†All temporal pterygia were recurrent.Type of lesion (no. of cases) Primary59 Recurrent41 Two females and one male had bilateral disease and pterygium surgery on separate occasions.† All temporal pterygia were recurrent. Open table in a new tab Pterygia (n = 100) were FFPE and oriented such that sections were cut longitudinally through the head and the body of the pterygium (Figure 1D). Sections (4 μm) were stained with H&E, then evaluated by two experienced senior ocular scientists (J.C. and N.D.). Unusual, suspect, and atypical cases were reviewed by a pathologist (R.C.) to provide a histopathological diagnosis. Pterygia with atypical features were investigated further by immunohistochemistry. Tissues were stained with putative markers for melanocytes, LSCs, or cytokeratins (Table 3). Briefly, 4-μm paraffin sections were dewaxed in xylene and rehydrated through a graded series of ethanol baths. Sections were subjected to antigen retrieval by heating in a microwave oven in 0.1 mol/L sodium citrate buffer (pH 6.0), followed by incubation in 3% H2O2 in methanol to block endogenous peroxidase activity. After blocking in 20% normal goat serum in Tris-buffered saline (pH 7.6) for 30 minutes, sections were incubated overnight in primary antibody (Table 3) at 4°C. Tissues were next incubated in biotinylated goat anti-rabbit or goat anti-mouse IgG (1:200 dilution; Dako, Glostrup, Denmark) for 30 minutes, followed by streptavidin-conjugated horseradish peroxidase (1:100 dilution; Dako) for 1 hour at room temperature. Sections were thoroughly washed with Tris-buffered saline between each step. Immunoreactivity was visualized using 3-amino-9-ethylcarbazole (AEC; Sigma-Aldrich, St. Louis, MO), and nuclei were counterstained with Mayer's hematoxylin (Dako). Sections were mounted in aqueous mounting medium (Crystal Mount; Biomeda Corporation, Foster City, CA), then coverslipped in DPX mounting medium (VWR International, Poole, UK). For double-labeling, tissues were incubated in a mixture of primary antibodies (Table 3), followed by incubations in goat anti-mouseAlexa488 and goat anti-rabbitAlexa594 (Invitrogen, Carlsbad, CA) and counterstained with DAPI (0.3 μmol/L final). Sections were coverslipped in Vectashield anti-fade mounting medium (Vector Laboratories, Burlingame, CA), then imaged. Negative control reactions included tissues that were incubated with an isotype antibody instead of an epitope-specific primary antibody. Photomicrographs were taken with a DP70 digital camera system mounted on an Olympus BX51 microscope (Olympus; Sydney, Australia) and processed with Photoshop version 9 (Adobe Systems, San Jose, CA).Table 3Primary Antibodies Used for ImmunohistochemistryTargeted epitopeAntibody typeHost⁎M, mouse; R, rabbit.CloneManufacturer†DAKO, DakoCytomation; CST, Cell Signaling Technology; TFS, Thermo Fisher Scientific; USB, United States Biological; Zymed, Zymed Laboratories.Catalog no.DilutionHuman melanosomeIgG1, κMHMB-45DAKOM06341:50Melan AIgG1, κMA103DAKOM71961:100S100BIgGR—DAKOZ03111:900P63 (pan)IgG2aM4A4DAKOM72471:50P63-alphaIgGR—CST48921:20Keratin-15IgG2aMMS-1068TFSLHK151:150Keratin-19IgG1M4A36USBC9097-24B1:150Ki-67IgGR—TFSRB-1510R71:200Connexin 43IgG1, κMCX-1B1Zymed13-83001:100IsotypeIgG1M—DAKOX09311:100IsotypeIgGR—DAKOX09031:900 M, mouse; R, rabbit.† DAKO, DakoCytomation; CST, Cell Signaling Technology; TFS, Thermo Fisher Scientific; USB, United States Biological; Zymed, Zymed Laboratories. Open table in a new tab Common histological features observed included a proliferative and locally invasive front of pterygium epithelium that abruptly transitioned into corneal epithelium at the advancing edge (Figure 2A). At the junction between the pterygium epithelium and normal cornea, the stroma was often characterized by feeder blood vessels (Figure 2A, asterisk) that preceded the fibroblastic stroma. The advancing pterygium edge was demarcated by a fragmented Bowman's layer (Figure 2A, arrows). Goblet cell hyperplasia was prominent in pterygium epithelium (Figure 2B), compared with autologous normal conjunctiva (Figure 2C). Feeder vessels extending the length of the lesion were regularly noted (Figure 2D), as well as subepithelial neovascularization (Figure 2D, inset). Stromal elastosis (Figure 2E, double asterisk) and both intra- and subepithelial (Figure 2F) and intravascular inflammation were present in 60% of cases. Uncommon histological features included basophilic stromal plaques within the pterygium body in 6% of cases (Table 4) that localized to elastotic zones (Figure 3, arrows). These plaques varied in size and shape, and were generally lilac in color after H&E staining (Figure 3, A and B, arrows) or dark blue when stained with phosphotungstic acid (Figure 3C, asterisk). However, we could not identify their composition further with other histological stains, including tetrachrome, Safranin O, von Kossa stain, or alizarin crimson (data not shown).Table 4Histological Findings in 100 Cases of PterygiaPterygia (no.)Histological findingsPrimary (n = 59)Recurrent (n = 41)TotalPAM without atypia415PAM with atypia with a subconjunctival nevus101Epidermolysis bullosa nevus011OSSN325Dermoid–like lesion011Plaques426Basal stem cell–like clusters81018PAM, primary acquired melanosis; OSSN, ocular surface squamous neoplasia. Open table in a new tab PAM, primary acquired melanosis; OSSN, ocular surface squamous neoplasia. Small clusters of basal cells were observed in 18% of our pterygium specimens. Cells within these aggregates were smaller, had increased nuclear-to-cytoplasm ratio, consisted of 8 to 15 cells anchored to the basement membrane, and were invariably associated with corneal-like epithelium near the head of the pterygium (Figure 4A, ovals). Morphologically, these cells appeared primitive and less differentiated than their suprabasal counterparts, suggesting that they may be stem cell like. Such observations prompted us to partially phenotype these cells by immunostaining, using well-accepted markers of LSCs. Indeed, cells within these microclusters demonstrated immunoreactivity to CK-15, CK-19, p63 (pan), and p63α (Figure 4, C, D, F, and G, respectively) and were double-immunoreactive to CK-15/p63α (Figure 4H) and CK-19/p63α (not shown), but lacked immunoreactivity to Ki-67 (proliferation marker) (Figure 4E) or Cx43 (gap junction protein) (data not shown). Our findings suggest that, although these cells are not proliferating (lacked Ki-67 expression), they retain proliferative potential (strong p63α expression) and may become activated when appropriate signaling mechanisms are initiated during pterygium development. These cell clusters were documented by Ernst Fuchs36Fuchs E. Ueber das Pterygium [Concerning the pterygium] German.Graefes Arch Ophthalmol. 1892; 38: 1-89Crossref Scopus (26) Google Scholar more than a century ago in his seminal article “Ueber das Pterygium” at both a microscopic (Figure 4B) and macroscopic level (Figure 5A) as small spots or flecks at the head of pterygia. Commonly known as Fuchs' flecks (or Fuchs' patches, Fuchs' islets), these pterygium cell clusters could be visualized in our patients by slit-lamp examination (Figure 5, B and C) and by confocal microscopy (Figure 5, D and E).Figure 5Clinical appearance of Fuchs' flecks in pterygia. A: Original illustrations by Ernst Fuchs 36Fuchs E. Ueber das Pterygium [Concerning the pterygium] German.Graefes Arch Ophthalmol. 1892; 38: 1-89Crossref Scopus (26) Google Scholar show small spots or fleckchen in the cap region at the head of pterygia. B–E: Pterygia with Fuchs' flecks (arrows) under slit lamp (B and C) and in vivo confocal microscopy (D and E). Slit lamp photographs were taken with an iPIX camera (Designs For Vision, Ronkonkoma, NY) and confocal micrographs with an HRT 3 Rostock cornea module (Heidelberg Engineering, Heidelberg, Germany). Confocal images were taken with a 63× objective. The field of view is 400µm × 400µm for (D) and 300µm × 300µm for (E). Image A is reproduced with permission from Springer (original publication: Fuchs E. Ueber de
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