Ocular Surface Squamous Neoplasia

Review CORNEA 687 Volume 22, Number 7 October 2003 Ocular Surface Squamous Neoplasia A Review Surendra Basti, MD, and Marian S. Macsai, MD Abstract Even though ocular surface squamous neoplasia (OSSN) has been recognized for well over a century, the past decade has witnessed advances that have helped rewrite many of the paradigms for the diagnosis and management of these lesions. OSSN occurs predominantly in the elderly for whom they are the third most common oculoorbital tumors after melanoma and lymphoma. In addition to advanced age and male sex, other major risk factors linked to its pathogenesis are ultraviolet light, cigarette smoking, and the human papilloma virus. Although the latter has been linked to OSSN for nearly 4 decades, its identification and role in the pathogenesis of these tumors has been elucidated recently and is addressed in detail in this review. Newer techniques of impression cytology represent a noninvasive and reliable method of diagnosing OSSN and monitoring treated cases. The efficacy of chemotherapeutic agents such as mitomycin C and 5-fluorouracil have been proven in the recent past, making them a clear alternative to the time-tested treatment of surgical excision and cryotherapy. Early reports on the efficacy of topical Iterferon ␣ 2b indicate significant promise in providing another alternative for the treatment of some of these neoplasms. These advances thus represent a minimally invasive and highly successful approach to the diagnosis and treatment of OSSN. (Cornea 2003;22:687–704) S quamous lesions of the cornea and conjunctiva are uncommon but important because of their potential for causing ocular and even systemic morbidity and mortality. The clinical presentation of these lesions extends across a wide spectrum and differs based on the degree of pathologic involvement. The latter can range from mild to severe dysplasia to full-thickness epithelial dysplasia (carcinoma in situ) and invasive squamous cell carcinoma. Squamous neoplasms can involve the conjunctiva or the cornea individually but more commonly start in the conjunctiva and extend across the limbus to involve the adjacent cornea. Even though such lesions have been extensively reported since the first case described in 1860 by von Graefe,1 the understanding of the pathogenesis and the management of these lesions has changed significantly in the past decade. In this paper, we review the recent advances and present the current status of the diagnosis and management of squamous neoplasms of the cornea and conjunctiva. TERMINOLOGY Various terms have been proposed to describe the range of squamous neoplasms of the cornea and conjunctiva. The initial cases of squamous neoplasms described in the literature were cases of frank squamous cell carcinoma.1 Subsequently, it has been recognized that both invasive and noninvasive (intraepithelial) forms of squamous neoplasms occur.2–8 In the initial years following recognition of intraepithelial forms of squamous neoplasms, various terms were used to describe these, including epithelial plaque, Bowenoid epithelioma, and precancerous epithelioma. Pizzarello and Jakobiec9 proposed a terminology that parallels the gynecologic pathology terms for intraepithelial neoplasia. These authors classified conjunctival intraepithelial neoplasms as mild, moderate, and severe dysplasia based on the extent of involvement. Lesions that involve the basal one-third of the conjunctiva are classified as mild, those involving the inner two-thirds are classified as moderate, and lesions that are full thickness are termed severe dysplasia. Waring et al10 extended the term to include the Received for publication December 7, 2002; accepted July 1, 2003. From the Department of Ophthalmology (Drs Basti and Macsai), Feinberg School of Medicine, Northwestern University, Chicago, Illinois and Division of Ophthalmology (Dr Macsai), Evanston Northwestern Healthcare, Evanston, Illinois. Reprints: Marian S. Macsai, MD, Division of Ophthalmology, Glenbrook Hospital, 2050 Pfingsten Road, Suite 220, Glenview, Illinois 60025 (e-mail:[email protected]). Copyright © 2003 by Lippincott Williams & Wilkins 688 CORNEA Basti and Macsai Volume 22, Number 7 October 2003 cornea, and Erie et al11 further extended it to include invasive neoplasia. Lee and Hirst12 have proposed the term ocular surface squamous neoplasia (OSSN) to encompass the entire spectrum of dysplastic and carcinomatous lesions of the ocular surface. This term is gaining increasing acceptance at the present time and is used in this review. In the interest of clarity, we suggest qualifying OSSN lesions with a prefix such as benign, preinvasive, or invasive (Table 1). Such a system has the potential to provide a simple and accurate terminology for such neoplasms, especially if combined with a histopathologic hallmark of the lesion. For instance, benign OSSN— papillomatous type, preinvasive OSSN— grade III, invasive OSSN—squamous cell type, and so on. EPIDEMIOLOGY: INCIDENCE, RACIAL AND GEOGRAPHIC DISTRIBUTION, AGE AND SEX DISTRIBUTION Incidence OSSN is uncommon. Templeton13 studied tribal groups in Uganda between 1961 and 1966 and found an average incidence of 0.13/100,000. A more recent study conducted in Brisbane, Australia found the incidence to be 1.9/100,000 population.14 It is interesting to note that the incidence of squamous cell carcinoma of the skin was 600/100,000 in the latter geographic area. In a recent study from the United States, Sun and co-workers15 noted that the incidence was 0.3 per million per year. The number of cases of OSSN relative to the total number of oculoorbital tumors ranges from 4% to 29%. In the older population, OSSN is the third most common ocular tumor after melanoma and lymphoma.12 Benign lesions are at least a third as frequent as malignant lesions of the ocular surface. Racial and Geographic Distribution Several large studies of patients with OSSN have found a predominance in Caucasians ranging from 90% to 100%.8,9,11,16 Sun et al15 found the rate of OSSN to be 5-fold higher in Caucasian men. Darker-skinned populations in tropical climates close to the equator can also develop OSSN.13,17–19 Ni et al20 noted the age of onset was younger at latitudes less than 30 degrees from the equator as compared with the age of onset at 45 degrees from the equator. More recently, Newton and co-workers21 studied the geographic distribution of OSSN in relation to ambient solar ultraviolet radiation. They noted that the rate of squamous cell carcinoma declined by approximately 49% for each 10-degree increase in latitude. For instance, in Uganda, there are 12 cases per million per year in contrast to 0.2 cases per million per year in the United Kingdom. Age and Sex Distribution OSSN predominantly occurs in older men. Based on an evaluation of 18 studies reporting on such neoplasms, Lee and Hirst12 noted an average age of occurrence as 56 years with a range of 4 to 96 years. Several studies have shown that the average age of carcinoma in situ patients to be 5 to 9 years TABLE 1. Classification of Ocular Surface Squamous Neoplasms (OSSN) Benign OSSN Papilloma Pseudotheliomatous hyperplasia Benign hereditary intraepithelial dyskeratosis Ca, carcinoma. © 2003 Lippincott Williams & Wilkins Preinvasive OSSN Conjunctival/corneal intraepithelial neoplasms grades I–III Invasive OSSN Squamous Ca Mucoepidermoid Ca Ocular Surface Squamous Neoplasia CORNEA Volume 22, Number 7 October 2003 younger than those with invasive squamous cell carcinoma.11,20 This difference in age may represent the time taken for progression from intraepithelial neoplasia to invasive carcinoma. Patients with xeroderma pigmentosum develop OSSN at a younger age.21,23,24 Young patients with HIV are also more prone to develop aggressive OSSN.25 Based on the epidemiologic information available at this time, it appears that the highest risk of OSSN is in older Caucasian men, especially those who live closer than 30 degrees latitude to the equator. CLASSIFICATION As described above, OSSN can be classified as benign, preinvasive, and invasive (Table 1). Of the benign OSSN, papillomas are by far the most frequent neoplasms. Other benign OSSN include pseudoepitheliomatous hyperplasia and benign hereditary intraepithelial dyskeratosis. Intraepithelial dysplastic lesions of the conjunctiva and cornea have malignant potential and are hence classified as preinvasive OSSN. Based on the extent of epithelium involved by the dysplastic process, these lesions may be graded as grades I to III (described below in the Pathology section). Of the invasive OSSN, squamous cell carcinoma is the most common tumor. Mucoepidermoid carcinoma can also occur on the ocular surface but is rare. ETIOPATHOGENESIS/ONCOGENESIS OSSN may represent the abnormal maturation of corneal and conjunctival epithelium as a result of a combination of factors such as ultraviolet B irradiation and human papilloma virus. Other reported risk factors include petroleum products, heavy cigarette smoking, chemicals such as trifluridine, arsenicals, beryllium, ocular surface injury, vitamin A deficiency, light pigmentation of the hair and eye, family origin in the British Isles, Austria, or Switzerland, and infection with the human immunodeficiency virus. There is considerable evidence to support the role of UV-B light and human papilloma virus in the pathogenesis of OSSN, and these are discussed below. UV-B Light UV light causes DNA damage and the formation of pyrimidine dimers.26 Failure or a delay in DNA repair can lead to somatic mutations and the development of cancerous cells such as occurs in xeroderma pigmentosum. Lee et al27 conducted a case-control study in which they identified risk factors for OSSN, which were pale skin, pale iris, propensity for sunburn, and spending over 50% of time outdoors in the first 6 years of life while living closer than 30 degrees latitude to the equator. Numerous other studies have also identified UV-B light as a major etiologic factor in the pathogenesis of OSSN.8,9,11,16–18,27–29 UV radiation is mutagenic for the p53 gene.30,31 Using immunohistochemical methods, Dushku and Reid32 evaluated p53 gene mutations in 5 cases of limbal tumors and found increased nuclear p53 mutations in all cases. This finding provides additional evidence for the causative role of UV light in OSSN. Human Papilloma Virus There is a well-recognized causal relationship between human papilloma virus (HPV) and squamous neoplasia of the uterine cervix. DNA of HPV types 6 and 11 are associated with benign genital warts and grade I cervical intraepithelial neoplasia.33,34 DNA of HPV types 16 and 18 are found in up to 80% of grades 2 and 3 cervical intraepithelial neoplasia and up to 90% of invasive squamous cell carcinoma.35–38 A similar causal relationship between OSSN and HPV has been suspected for over 2 decades. HPV type 6/11 has been detected in a large percentage of conjunctival papillomas,39 and HPV DNA has been isolated from dysplastic and malignant lesions of the conjunctiva and cornea.40–44 However, HPV DNA has also been found bilaterally in patients with unilateral ocular disease45 and in normal conjunctiva of patients 689 690 CORNEA Basti and Macsai Volume 22, Number 7 October 2003 with genital HPV.46 These last mentioned findings questioned the role of HPV in the pathogenesis of ocular surface squamous neoplasia. A recent study47 has helped put some of these findings into proper perspective. Scott and co-authors47 evaluated a test recognized to be a specific marker for active viral expression. They used reverse transcriptase polymerase chain reaction (PCR) to detect the presence of HPV mRNA corresponding to the E6-E7 region in histopathologically proven cases of conjunctival intraepithelial neoplasms (CIN) and in controls. HPV 16 or HPV 18 DNA and mRNA was demonstrated in all CIN specimens and was absent in all of the controls, indicating active HPV infection was present in the former group. It has been demonstrated that protein encoded by the E6 region of HPV 16 and 18 forms a complex with the protein encoded by the p53 tumor suppression gene in the host. The findings of the study by Scott et al are consistent with the findings reported for human cervical lesions and are strong evidence for a role of HPV in the pathogenesis of OSSN. It is likely that HPV does not act alone in the development of OSSN but may need a cofactor such as UV-B light or one of the other risk factors mentioned above.12 This fact might explain the absence of OSSN in spite of the presence of HPV in some clinically unremarkable conjunctival specimens. FIGURE 1. Leukoplakic appearance of a limbal squamous neoplasm (Courtesy Y. Y. Choong, MD, Malaysia). CLINICAL FEATURES lymph nodes, and has well-defined borders. The diffuse type (Fig. 4) is least common, invades adjacent conjunctiva, and in its early stages can present as persistent redness of the conjunctiva without associated follicles or papillae. Involvement of the palpebral conjunctiva can cause inward rotation of the lid margins. Thus, the diffuse type of OSSN can masquerade as a chronic conjunctivitis. These lesions are slow growing, and tumefaction occurs later in the disease process. The exact demarcation of the edge of these tumors may be difficult to determine clinically. Benign OSSN—papillomatous type lesions are exophytic, pink-red, strawberry-like papillary growths. They have a stippled red appearance, and these focal red areas correspond to the fibrovascular cores of the lesion. Papillomatous lesions have a biphasic age dis- Most conjunctival OSSN lesions are slightly elevated and have characteristic tufts of blood vessels and a pearly gray appearance. Their borders may or may not be well defined. They usually straddle the limbus but may be restricted to the conjunctiva and, less frequently, the cornea. The macroscopic appearance has been described as being one of three types: leukoplakic, gelatinous, or papilliform 9,11 (Figs. 1–3). Although circumscribed gelatinous lesions are most common, two other types of lesions have been described.48 The nodular type is rapidly growing, has a propensity for spread to adjacent FIGURE 2. Gelatinous appearing limbal squamous lesion. (Courtesy Shmuel Levartovsky, MD, Ashkelon, Israel). © 2003 Lippincott Williams & Wilkins Ocular Surface Squamous Neoplasia CORNEA Volume 22, Number 7 October 2003 FIGURE 3. Papilliform conjunctival lesions in a patient with squamous cell carcinoma with orbital involvement. (Courtesy Gary S. Lissner, MD, Chicago, IL). tribution and growth pattern. In children, they are most commonly multiple and pedunculated, involving the fornix, caruncle, or eyelid margin. In adults, they are usually single, sessile, and occur at the conjunctiva or limbus (Fig. 5) There is considerable overlap in the clinical features of the different OSSN lesions, and it is difficult to differentiate benign from preinvasive and invasive OSSN lesions based on the clinical appearance alone. Larger lesions that are fixated to underlying tissues are usually suggestive of malignancy. Growth patterns of OSSN lesions are variable, but most are slow growing, and patients present with redness, foreign body sensation, or mild irritation. Corneal OSSN lesions typically are usually preinvasive and appear as an opalescent FIGURE 4. Injected, diffuse involvement of both palpebral and bulbar conjunctiva in a patient with squamous cell carcinoma.(Courtesy Gary S. Lissner, MD, Chicago, IL). FIGURE 5. Slow-growing, translucent conjunctival and limbal lesion with radiating fronds of vessels (arrowhead) in a young woman. ground-glass sheet whose surface is mottled. A characteristic feature of many corneal lesions is that they have sharply defined and fimbriated borders (Fig. 6) and are avascular. Less frequently, the edges may be ragged or even smooth. The convex leading edge spreads away from the corneoscleral limbus in an advancing arc. Fine white dots are often present over the gray epithelium. These lesions can sometimes appear as large, elevated, pearl-white mounds. Corneal OSSN lesions are slightly elevated compared with the adjacent normal epithelium. Rose Bengal staining produces a diffuse punctate stain over the gray sheet. The virulence of these corneal lesions themselves is low. Early involvement of the cornea adjacent to a conjunctival lesion may manifest as a mild opacification of the cornea. Such areas have dys- FIGURE 6. Fimbriated edges of a gray corneal lesion in a patient who spent prolonged periods of time outdoors. 691 692 CORNEA Basti and Macsai Volume 22, Number 7 October 2003 plastic corneal epithelium, which has a polycystic appearance that is best visualized by retroillumination. The etiology of these lesions is controversial, with some authors proposing a de novo dysplastic process in the cornea49 while others suggest a centripetal sliding of subsequently neoplastic cells from the limbus.50These lesions are typically indolent and slow growing. They have a particularly high tendency to recur.49,51,52 conjunctiva. • Pseudoepitheliomatous hyperplasia is a benign reactive or precancerous lesion of the conjunctiva that tends to develop rapidly over several weeks to months. These occur anywhere on the conjunctiva and have a whitish rather than gelatinous appearance. Rarely, they may have crater-like centers filled with keratin similar to a keratoacanthoma. DIFFERENTIAL DIAGNOSIS PATHOLOGY Differential diagnosis of OSSN includes pannus, actinic disease, vitamin A deficiency, benign intraepithelial dyskeratosis, pinguecula, pyogenic granuloma, keratoacanthoma, pseudoepitheliomatous hyperplasia, malignant melanoma, and nevi, especially in patients with racial melanosis. Pizzarello and Jakobiec9 suggest some important clinical features (Table 2) that may help differentiate some of these lesions. These are as follows: • Benign nevi occur in younger patients in the interpalpebral area and have distinctive cysts on slit-lamp examination. Poorly pigmented lesions may simulate a dysplastic process. • Malignant melanoma has a regular smooth surface, lacks gelatinous or leukoplakic surface disturbances, and may become ulcerated. • Papillomas may look similar to preinvasive OSSN or even invasive OSSN—squamous cell type. Hence, histopathologic examination is necessary to confirm the diagnosis. However, papillomas usually occur in younger patients, may be sessile or pedunculated, and can occur anywhere in the Histopathologic examination of papillomas demonstrates papillary fibrovascular fronds covered by acanthotic epithelium. (Fig. 7) Pediatric papillomas often have an admixture of goblet cells and neutrophils within the epithelium. A chronic inflammatory infiltrate may occupy the stroma. Adult papillomas may exhibit various degrees of epithelial dysplasia characterized by nuclear enlargement, increased nuclear-to-cytoplasmic ratio, and mitotic figures above the basal epithelial surface. The epithelial cells in papillomas have normal polarity, and the basal layers are usually unremarkable. Preinvasive OSSN lesions are classified as mild, moderate, or severe depending on the degree of involvement of the dysplastic epithelium. Mild dysplasia, or CIN grade I, is defined as dysplasia confined to the lower third of the conjunctival epithelial thickness. Moderate dysplasia (CIN grade II) extends into the middle third, and severe dysplasia (CIN III) to the upper third. Full-thickness dysplasia of the epithelium is also referred to as carcinoma-in-situ. The pathologic basis for a TABLE 2. Clinical Comparison of the Common Ocular Surface Neoplasms Lesion Onset Surface Location Growth Malignant melanoma Papilloma Nevi Pseudotheliomatous hyperplasia Adulthood Childhood/adulthood Childhood/young adulthood Any age Smooth/ulcerated Smooth/lobulated Small cysts White, smooth Conjunctiva Conjunctiva Interpalpebral Anywhere Slow Slow Slow Rapid © 2003 Lippincott Williams & Wilkins Ocular Surface Squamous Neoplasia CORNEA Volume 22, Number 7 October 2003 FIGURE 7. Thickening of the epithelium with fibrovascular fronds (arrow) within the acanthotic epithelium suggestive of squamous papilloma (Courtesy Paul J. Bryar, MD, Chicago, IL). diagnosis of preinvasive OSSN is based on the identification of epithelial disarray and abnormalities in maturation. In general, these conditions show an abrupt demarcation at the lateral edges of the lesion between neoplastic cells and the uninvolved benign epithelium. (Fig. 8) Invasive OSSN—squamous cell type (Fig. 9) shows nests of infiltrating cells that have penetrated the epithelial basement membrane and spread into the conjunctival stroma.53 Tumor cells may be well differentiated and easily recognizable as squamous, or poorly differentiated and difficult to distinguish from other malignanacies such as seba- FIGURE 8. Histopathologic appearance (hematoxylin-eosin, ⳯20 magnification) of conjunctival intraepithelial neoplasia. To the left is normal conjunctival epithelium. An abrupt transition (arrow) is seen between this area and dysplastic epithelial cells, which occupy the entire thickness of the epithelium to the right (CIN grade III) (Courtesy Paul J. Bryar, MD, Chicago, IL). FIGURE 9. Histopathology (hematoxylin-eosin, ⳯10 magnification) demonstrating dysplastic cells that have replaced the normal conjunctival epithelium. Invasion of the subepithelial area (arrow) by these cells confirms a diagnosis of invasive squamous cell carcinoma (Courtesy Paul J. Bryar, MD, Chicago, IL). ceous carcinoma. Well-differentiated tumors have large cells with hyperchromatic nuclei, prominent nucleoli, variable degrees of hyperkeratosis, and parakeratosis and mitotic figures. Less-differentiated tumors are uncommon but more aggressive. Two types of cells may be seen interspersed with squamous cells in such tumors. Spindle cells. These are pleomorphic cells with hyperchromatic elliptical nuclei with scant cytoplasm and frequent mitotic figures. These cells may be indistinguishable from fibroblasts, and hence spindle cell carcinomas are termed pseudosarcomas. Electron microscopy and immunohistochemical markers may be useful in differentiating these tumors from true sarcomas. Mucoepidermoid cells. These are cubiodal cells with mucicarmine-positive intracytoplasmic droplets. The cell nests are interspersed with pools of mucin in the extracellular space. Mucoepidermoid carcinomas exhibit a tendency to intraorbital extension as well as early recurrence if incompletely excised. For this reason it is important that their mucinous component be recognized and these tumors appropriately classified histopathologically. 693 694 CORNEA Basti and Macsai Volume 22, Number 7 October 2003 Electron microscopic features of 10,54–56 OSSN include excessive number of organelles such as mitochondria, endoplasmic reticulum, and tonofilaments; reduction in number of desmosomes; alteration or absence of the epithelial basement membrane; and deposition of a fibrillogranular material between the basement membrane and Bowman’s layer. DIAGNOSTIC TESTS FOR OSSN A tissue diagnosis is almost always required before treatment of OSSN is begun. This has traditionally been performed with an excision biopsy in smaller lesions or a map biopsy in larger lesions. These are discussed in the next section, which describes surgical techniques for OSSN. An alternative technique for tissue diagnosis is cytologic sampling. This technique is being used more often for the diagnosis and monitoring of OSSN and is discussed below. Cytology Cytologic sampling is a technique that can be of great value in the management of OSSN. Various techniques may be used to sample the cells. The advantages and disadvantages of these techniques are summarized in (Table 3). Malignant cells are particularly well suited for exfoliative cytology because they have poor intercellular adherence and tend to desquamate when the malignant neoplasm is located on the mucosal surface. Exfoliative cytology can be performed using specimens obtained by gently scraping the lesion with a platinum spatula,57–59 a cytobrush,60,61 or by aspiration of the surface of the lesion with a fluid-filled tuberculin syringe.62 Alternatively, impression cytology can be performed using cellulose acetate paper63,64 or with the recently described technique using a Biopore membrane (MillicellCM, 0.4 µm PICM 012550, Millipore Corp, Bedford, MA).65 Both the cytobrush and the platinum spatula provide good samples. However, cellular overlap may obscure some of the details in specimens obtained with this technique.60,61 Also, drying artifacts can occur with this technique of sampling. The latter can be avoided using the technique of aspiration cytology.62 Impression cytology with cellulose acetate paper is inexpensive and easy to perform, even for inexperienced operators, compared with cytologic scraping.66 However, specimens collected with cellulose acetate paper need to be transported and processed immediately. This makes it cumbersome and time consuming for routine use in the outpatient clinic. Theil et al65 reported on the utility of the Biopore membrane device for impression cytology. This technique has all the advantages of impression cytology. The device is mechanically stable, allowing direct orthogonal sampling from the ocular surface. There is good adhesion of cells to the Biopore membrane so that a large layer of cells is easily harvested. Specimens obtained can be placed in 95% alcohol for several weeks before processing. Consequently, this technique is advantageous for use in routine clinical practice TABLE 3. Comparison of Techniques of Sampling Conjunctival Cells for Cytology Technique Advantages Cytobrush Inexpensive CAP imp cyt Biopore imp cyt Simple, inexpensive Simple Specimens can be stored for several weeks Cell-to-cell relationship maintained CAP, cellulose acetate paper; imp cyt, impression cytology. © 2003 Lippincott Williams & Wilkins Disadvantages Morphologic detail may be obscured Drying artifacts can occur Specimens need to be read immediately None Ocular Surface Squamous Neoplasia CORNEA Volume 22, Number 7 October 2003 and has been used in management of OSSN.67,68 Cytologic study of OSSN can be used for the diagnosis of corneal and conjunctival tumors, evaluation of tumors during medical treatment, and follow-up of patients after treatment to evaluate for recurrences. The cytomorphology of OSSN has been well described. In a recent review of 267 impression cytology specimens of lesions that also had a biopsy, Nolan and co-authors69 provide a detailed description of the cytologic changes in OSSN. In cases with preinvasive OSSN, three distinct types of cellular features were identified as being the predominant cytologic feature: Keratinized dysplastic cells with hyperkeratosis (55% cases) Syncytial-like groupings (35% cases) Nonkeratinized dysplastic cells (10% cases) In cases with invasive squamous cell carcinoma, significant abnormal keratinization was seen in 70% of cases (Fig. 10), and a greater degree of inflammation was noted in addition to the above changes described for intraepithelial neoplasia. In general, there is a paucity of cells in cases at the severe end of the spectrum of invasive squamous cell carcinoma. These findings were confirmed in a recent study by Tole et al.67 It was not pos- sible even with considerable experience to predict invasion based on impression cytology alone. OSSN is frequently being treated using immuno- and chemotherapeutic agents at the present time. Cytology can be particularly useful to evaluate such cases following initiation of treatment.68,70 It is important to note that cytologic changes mimicking malignancy (nuclear enlargement and nuclear hyperchromasia/smudging of chromatin) have been reported in conjunctival biopsies up to 6 weeks following topical mitomycin C therapy.71 Nevertheless, there are features that can help differentiate these from true dysplasia, as was highlighted by Tole et al.67 Other techniques have been attempted to monitor therapy for OSSN. Nadjari et al70 perfomed DNA cytometry in addition to cytology in patients treated with chemotherapeutic agents. They conclude that DNA cytometry is useful to provide an objective identification of tumor cell regression. Immunostaining has also been used for the same purpose. Investigators from Japan have performed immunostaining for proliferating cell nuclear antigens (PCNA) and Ki-67.71 Aoki et al73 studied immunostaining for PCNA, p53 immunostaining, and argyrophilic nucleolar organizer regions (AgNORs). Based on these studies, it appears that these tests can be potentially useful as prognostic markers in OSSN. Further studies are required to determine their exact role in the follow-up of treated cases of OSSN. TREATMENT: NONSURGICAL AND SURGICAL Surgery FIGURE 10. Impression cytology using Biopore membrane in a patient with squamous cell carcinoma. Densely packed cells with pleomorphic nuclei, scattered mitoses, and dyskeratosis (arrow) is seen (Courtesy Paul J. Bryar, MD, Chicago, IL). Surgical excision of lesions has traditionally been the method of choice for treatment of OSSN. Because a tissue diagnosis is mandatory before any kind of adjunctive treatment, most surgeons resort to an excisional biopsy except for extensive lesions. A wide surgical margin of 45 mm is required to increase the chances of complete removal.74 Rose Bengal staining may help delineate the 695 696 CORNEA Basti and Macsai Volume 22, Number 7 October 2003 margins of the lesion75 (Fig. 11). The conjunctival defect remaining following excision can be closed primarily if it is small (less than three clock hours in diameter). Larger defects need tissue replacement either from a transpositional conjunctival flap, free conjunctival flap from the other eye, or using amniotic membrane transplantation. Lamellar techniques have been described for excision of lesions that extend into the superficial cornea or sclera.76 More extensive surgery may be required for lesions with intraocular invasion.77 In all cases, a “no touch” method is used, and direct manipulation of the tumor is avoided to prevent tumor cell seeding into a new area.74 To minimize corneal epithelial disruption in cases of suspected corneal preinvasive OSSN, Shields et al74 recommend using absolute alcohol application to facilitate epithelial removal as a complete sheet. Conjunctival specimens have to be carefully placed on absorbent paper and air dried to prevent curling of the thin conjunctiva and a loss of orientation of the tissue. Recurrence rates for preinvasive and invasive OSSN following surgical excision range from 15% to 52%.11,12 Because the tumor grows like the roots of a tree on apparently normal conjunctiva, small tumor islands may remain after seemingly complete surgical excision of tumors, thus making histologic monitoring of surgical margins difficult. In cases of corneal preinvasive OSSN, the thinness and incohesive characteristics of the epithelium make margin analysis difficult. Studies have consistently shown that recur- FIGURE 11. Rose Bengal staining delineates the lesion in a patient with a gelatinous conjunctival lesion (Courtesy Gary S. Lissner, MD, Chicago, IL). © 2003 Lippincott Williams & Wilkins rence rates are particularly high if dysplastic tissues are left at the margins of the excised lesion.9,11 Recurrences usually occur within 2 years after removal.78 Partially excised tumors, especially if they are carcinomatous to begin with, tend to demonstrate recurrence with aggressive behavior,11,20,79 possibly because of the tissue disruption associated with the primary excision, which enhances the ability of tumor cells to enter the eye.20 The increased recurrence rates and technical difficulty associated with ensuring complete surgical excision have prompted surgeons to use adjunctive treatment with the surgical excision. Cryotherapy, radiotherapy, chemotherapy, and immunotherapy have been used and are discussed below. Cryotherapy is a commonly employed modality of treatment in combination with surgical excision. Studies with long-term follow-up have demonstrated an average recurrence rate of 12% with this combination of treatments.78,80–83 Cryotherapy acts by destroying cells with a thermal effect and also obliterates the microcirculation, leading to ischemic infarction.84,85 It has the advantage of reaching both superficial tumor islands and deeply infiltrated tumor cells. Consequently, such a treatment can obviate the need for radical surgery with large residual tissue defects. A short-duration freeze with slow thaw, repeated two to three times (freeze-thaw-refreeze technique), is the recommended technique of cryotherapy. For lesions clinically confined to the conjunctiva, after the lesion has been surgically excised, a cryoprobe is inserted under the resected edge of conjunctiva. The probe is directed away from the globe, and the freeze-thawrefreeze technique is used to obliterate any remaining tumor cells. If the limbus or the episclera is suspected to be involved, cryotherapy should be applied to these sites also, using a similar technique. A nitrous oxide cryoprobe tip (2.5 or 5 mm) is used to form an iceball extending 2 mm for the conjunctiva, 1 mm for episcleral tissues, and 0.5 mm for the cornea. It is important to include the limbal region during cryotherapy and not to Ocular Surface Squamous Neoplasia CORNEA Volume 22, Number 7 October 2003 apply the cryoprobe for more than 3 seconds. Care must be taken to avoid excess freezing because this can cause iritis, hypotony, sector iris atrophy, corneal hemorrhage, and neovascularization. Extensive surgical excision or limbal cryotherapy can lead to limbal stem cell insufficiency, and this may require limbal autotransplantation.86 Radiotherapy has been used for OSSN since the 1930s. Various sources such as strontium-90, ␤-irradiation, and ␥-radiation have been used.87–89 Because radiotherapy usually takes several weeks, is rather cumbersome for routine office use, and can infrequently have untoward side effects,90 it is not widely used at the present time. Radiotherapy may have a role in the treatment of diffuse lesions or extensive OSSN lesions in conjunction with other modalities of treatment. Chemotherapy with antimetabolic agents such as mitomycin C and 5-fluorouracil has shown considerable promise in the management of preinvasive and invasive OSSN (Table 4). Topical chemotherapy has several advantages when compared with traditional surgical excision and cryotherapy.91 These include (1) treatment of the entire ocular surface, thereby eliminating the need to ensure clear tissue margins, as is necessary with surgical excision, (2) apparent targeting of the tumor cells reducing the risk of limbal stem cell deficiency associated with the more extensive surgical excisions involving the limbus, and (3) simplicity of treatment and reduced patient cost by avoiding repeat surgery in patients with recurrence. Disadvantages of topical chemotherapy include the limited penetration of these agents and, consequently, their potential for failure in eradicating disease when used as the sole agent in invasive squamous cell carcinoma. Also, there is a potential for adverse changes in the nasopharyngeal epithelium if exposure occurs. This last-mentioned complication can be prevented by the placement of punctual plugs for the duration of treatment. Reversible side effects of topical chemotherapeutic agents include conjunctival discomfort and hyperemia, punctate epithelial keratopathy, and blepharospasm. It is important to recognize that mitomycin C has the potential for causing serious ocular complications if not used as prescribed.92 Since the first report93 on the use of topical mitomycin C, various reports94–99 have confirmed the efficacy of this agent in the treatment of recurrent and primary OSSN (Table 4). Mitomycin C is an antitumor antibiotic that selectively inhibits DNA synthesis and is a cell cycle–nonspecific agent that is most effective in the G1 and S phases.100 Mitomycin C is referred to as a parent bioreductive alkylating agent, a drug activated by intracellular metabolic reduction of its quinone group. This leads to the generation of alkylating species or to redox cycling that produces active oxygen species that cause DNA damage. Because the hypoxia required for intracellular metabolic reduction of mitomycin is more pronounced in tumor cells than in normal tissues, it preferentially takes place in tumor tissues, creating a certain level of selectivity.100 Mitomycin C is used in concentrations of 0.02% or 0.04% 4 times a day for 2 weeks. These cycles are repeated at 4- to 6-week intervals. 5-FU is a cell-cycle–specific antimetabolite that acts during the S phase of the cell cycle. It is converted to 5-F DUMP, which inhibits thymidylate synthetase, preventing DNA and RNA synthesis because of a lack of thymidine. This results in unbalanced cell growth and ultimately cell death. Several reports have confirmed the efficacy of topical 5-FU in treatment of preinvasive OSSN.91,101–105 Both 5-FU and mitomycin C are currently being used 4 times daily for 1–2 weeks, and treatment is repeated if necessary. To decrease toxicity while maintaining efficacy, Yeatts et al104 have described an alternate pulsed dosing regimen using four applications of 1% 5-FU for 4 days, repeated every month for 4 to 6 cycles. They observed that with this pulsed dosing, the efficacy remained excellent, treatment was better tolerated, and the side effects were minimal compared with the 2-week treatment with 5-FU. 697 698 CORNEA Basti and Macsai Volume 22, Number 7 October 2003 TABLE 4. Reported Series of Patients with OSSN Treated with Mitomycin C or 5-FU Author Patients Agent 93 3 Frucht-Pery94 17 Frucht-Pery Heigle95 3 Wilson96 7 Shields97 10 Regimen MMC 0.02% × 10–22 d MMC 0.02–0.04% MMC 0.04% tid × 1 wk, 3 cycles one wk apart MMC 0.04% qid × 7 d, rpted in 1–2 w if reqd. Total 4–21 d tt MMC 0.04% qid × 7 d, 1 wk on, 1 wk off MMC 0.02% qid × 2 wks MMC 0.02–0.04% qid × 14 days 5-FU 1% qid for 14–21 d. Repeated 3 times Haas98 1 Rozenman99 8 Yeatts102 6 Midena103 1 5-FU Yeatts104 7 5-FU Yamamoto105 1 5-FU Success 3/3 Recurrence/ Partial Response Follow-Up — 4–12 mos 3–40 mos 14/16 CCIN REC: 4/16 0/1 SCC CCIN after 1 cycle; 1/1 SCC after 2 cycles 3/3 6/7 6–9 mos 1/7 partially 2–16 m, mean 9 responded to tt Comments Large CIN 10 ccin one cycle; 2 CCIN two cycles 2 CCIN min rec. 2 CCIN rec after two cycles All recurrent CCIN lesions Rec or extensive CIN lesions Mean 22 m, Extensive recurrent range SCC 6–50 mos 10/10 1/1 9 mos CIN 7/8 1/8 regrowth 24–44 mos CIN <8 mm 4/6 2/6 3–30 mos One patient had deep invasion noted after 5 FU tt Diffuse SCC 1% qid for 4 1/1 wks qid for 2–4 7/7 Two of these days. needed Repeated rett with 2–6 times 5-FU and one with MMC quid for 2 1/1 wks 7 mos All CIN Mean 18.5 3/7 after lesions mos; regression range with 1 7–36 mos course of 5-FU 30 mos Pt. had 2 courses of MMC before 5-FU CCIN, conjunctival-corneal intraepithelial neoplasia; CIN, conjunctival intraepithelial neoplasia; SCC, squamous cell carcinoma; tt, treatment; mos, months; REC, recurrence. © 2003 Lippincott Williams & Wilkins Ocular Surface Squamous Neoplasia CORNEA Volume 22, Number 7 October 2003 For topical mitomycin, Shields et al97 use a 1-week-on and 1-week-off technique. They report that this regimen is well tolerated and retains good efficacy. For each of the abovementioned topical chemotherapeutic regimens, preplacement of punctal plugs protects the nasopharyngeal tissue from exposure to these potentially toxic agents and is recommended. Based on the current clinical experience, it is clear that mitomycin and 5-FU are effective for complete eradication of preinvasive OSSN. In cases of invasive OSSN, these agents have traditionally been used to decrease tumor size before planned excision and or cryotherapy. However, Shields et al97 recently reported their experience using a higher concentration of MMC (0.04%) in large (> 8 mm) recurrent conjunctivalcorneal invasive OSSN—squamous cell type. No recurrence was noted during a mean follow-up of 22 months (range 6–50 months) in any of the 10 eyes they treated. (Figures 12 to 14) represent a strategy that we suggest for the management of OSSN, based on a review of the literature and our own experience. For all lesions whose largest diameter is less than three clock hours, an excision biopsy is the initial treatment of choice. For lesions 3 to 6 clock hours, a biopsy for confirmation of diagnosis and determination of the invasiveness of the lesion is recommended. In preinvasive le- sions of this size, topical chemotherapy represents the best option. For invasive lesions, a trial of chemotherapy is a good first option. This may achieve either complete resolution or chemoreduction. In the latter situation, surgical excision and cryotherapy to the bed of the excised lesion are required. For invasive lesions larger than 6 clock hours, a trial of high-dose chemotherapy may potentially be a useful option to obtain chemoreduction or, as suggested in the above-mentioned study, 97 complete eradication. Palliative treatment with radiotherapy or extensive surgery such as enucleation or exenteration may be the only options for large invasive lesions that do not respond to chemotherapy. Immunotherapy Interferons (IFNs) are a family of naturally occurring glycoproteins that bind to cell surface receptors and trigger a cascade of intracellular events, thus promoting antiviral and antitumor properties through direct and indirect mechanisms. Topical interferons were first reported to be efficacious in a case reported by Maskin.106 Vann and Karp107 reported on the successful use of a combination of intralesional and topical interferon␣2b in six patients with biopsy-proven primary or recurrent CIN. Complete resolution was noticed within 6 weeks of commencement of treatment. The median follow-up in these patients was 7.2 months (range 2–11 FIGURE 12. Suggested management strategy for OSSN less than 3 clock hours in diameter. 699 700 CORNEA Basti and Macsai Volume 22, Number 7 October 2003 FIGURE 13. Suggested management strategy for OSSN 3–6 clock hours in diameter. months). More recently, Schecter et al108 reported success with the use of topical interferons in a series of 7 eyes with presumed primary conjunctival and corneal intraepithelial neoplasia. IFN-␣2b (1 million units/mL) was used 4 to 6 times a day until resolution was observed and continued for a month thereafter. The median time to resolution was 54 days, and the range was 28–188 days. The follow-up in this series ranged between 2.9 and 18 months. Success has been reported in the dermatologic literature109,110 on the intralesional use of interferons in squamous cell car- cinoma. The above-mentioned studies106–108 clearly suggest that local treatment with interferons might represent a promising alternative to the existing modalities of treatment of preinvasive OSSN. Larger studies with longer follow-up are awaited to better assess the dosing regimen and recurrence rates with the use of interferons for preinvasive OSSN. LONG-TERM OUTCOME OF MANAGEMENT Studies11,111,112 alluding to the longterm outcomes in patients with excised FIGURE 14. Suggested management strategy for OSSN larger than 6 clock hours. © 2003 Lippincott Williams & Wilkins Ocular Surface Squamous Neoplasia CORNEA Volume 22, Number 7 October 2003 OSSN lesions have consistently demonstrated that histopathologic involvement of the margin of the lesion is the most important factor in predicting recurrence. These studies demonstrate that lesions where dysplastic cells are present at the edge of excised lesions have a 2- to 4-fold increase in the recurrence rate and that recurrent lesions appear earlier in the former group. For instance, in the series reported by Tabin et al,111 recurrent lesions appeared earlier in eyes with incompletely excised margins as compared with those with healthy margins (mean 2.5 versus 3.8 years). Recurrence rates vary between 53% when pathology showed involved margins and 5% when pathology confirmed clear margins.11 Lesions confined to the cornea alone are extremely slow growing but have an increased risk of recurrence. Eyes with invasive OSSN—mucoepidermoid type have also been reported to have an increased risk of recurrence.113,114 The course of ocular surface squamous neoplasms may be evanescent, but more frequently, it is slowly progressive and, if untreated, can lead to orbital and intraocular spread and require exenteration. Systemic spread is extremely rare with OSSN. HORIZONS AND VISTAS Future refinements of modern therapies will allow cell-specific anticancer treatment of these lesions. Cell-specific immunotherapies will require ex vivo expansion of the tumor cell and development of immunespecific treatments. These cell-specific immunotherapies will allow for topical application of tumor-specific antibodies that result in targeted destruction of the tumor cells, in both the main tumor body and surrounding tumor cell nests, with no damage to the surrounding tissue and preservation of the limbal stem cells. Invasive tumors may require surgical excision; however, intravenous delivery of tumor-specific antibodies may obviate the need for additional chemotherapy, radiation therapy, or cryotherapy. In the event that surgical excision of the tumor is required, ex vivo expansion and reimplantation of the patient’s limbal stem cells will result in stabilization of the ocular surface. OSSN presents a unique challenge in the treatment of squamous cell neoplasias. Cell-specific immunotherapies will allow treatment of these tumors with ill-defined margins, hidden tumor cell nests, and a relatively high rate of recurrence. Limiting UV exposure in the Caucasian patient, especially those who reside with 30 degrees of the equator, may decrease the overall incidence of these tumors in at-risk patient populations. However, the overall decrease in the ozone layer and progressive longevity of the human race may result in further increases in the incidence of these tumors. Future therapies will require targeted application of agents that result in tumor cell death and ocular surface stem cell preservation resulting in ocular surface stabilization and sight preservation. REFERENCES 1. Duke-Elder S, Leigh AG. Diseases of the outer eye. In: Duke-Elder S, ed. Systems of Ophthalmology, Vol 7, Part 2. St Louis: CV Mosby, 1985:1154–1159. 2. Nichols JV. Epithelial plaques of the conjunctiva and cornea. Arch Ophthalmol. 1939;22:370–376. 3. McGavic JS. Intraepithelial epithelioma of the cornea and conjunctiva (Bowen’s disease). Am J Ophthalmol. 1942;25:167–176. 4. Ash JE, Wilder HC. Epithelial tumors of the limbus. Am J Opthalmol. 1942;25:926–932. 5. Janert H. Zur peracancerose der cornea und conjunctiva. Von Graefes Arch Ophthalmol. 1956;157: 380–396. 6. Lugossy G. Precancerous conditions of the bulbar conjunctiva. Am J Ophthalmol. 1956;42:112–125. 7. Winter FC, Kleh TR. Precancerous epithelioma of the limbus. Arch Ophthalmol. 1960;64:208–215. 8. Irvine AR Jr. Dyskeratotic epibulbar tumors. Trans Am Ophthalmol Soc. 1963;61:243–273. 9. Pizzarello LD, Jakobiec FA. Bowen’s disease of the conjunctiva: a misomer. In: Jakobiec FA, ed. Ocular Adnexal Tumors, Birmingham, AL: Aesculapius, 1978:553–571. 10. Waring GO III, Roth AM, Ekins MB. Clinical and pathological description of 17 cases of corneal intraepithelial neoplasia. Am J Opthalmol. 1984;97: 547–559. 11. Erie JC, Campbell RJ, Leisgang J. Conjunctival and corneal intraepithelial and invasive neoplasia. Ophthalmology. 1986;93:176–183. 12. Lee GA, Hirst LW. Ocular surface squamous neoplasia. Surv Ophthalmol. 1995;39:429–450. 13. Templeton AC. Tumors of the eye and adnexa in Africans in Uganda. Cancer. 1967;20:1689–1698. 701 702 CORNEA Basti and Macsai Volume 22, Number 7 October 2003 14. Lee GA, Hirst LW. Incidence of ocular surface epithelial dysplasia in metropolitan Brisbane. A 10year survey. Arch Ophthalmol. 1992;119:525–527. 15. Sun EC, Fears TR, Goedert JJ. Epidemiology of squamous cell conjunctival cancer. Cancer Epidemiol Biomarkers Prev. 1997;6:73–77. 16. Ash JE. Epibulbar tumors. Am J Ophthalmol. 1950; 33:1203–1219. 17. Clear AS, Chirambo MC, Hutt MSR. Solar keratosis, pterygium and squamous cell carcinoma of the conjunctiva in Malawi. Br J Ophthalmol. 1979;63: 102–109. 18. Malik MOA, El Sheikh EH. Tumors of the eye and adnexa in Sudan. Cancer. 1979;44:293–303. 19. Olurin O, Williams AO. Orbito-ocular tumors in Nigeria. Cancer. 1972;30:580–587. 20. Ni C, Searl SS, Kriegstein HJ, et al. Epibulbar carcinoma. Int Ophthalmol Clin. 1982;22:1–33. 21. Newton R. A review of the etiology of squamous cell carcinoma of the conjunctiva. Br J Cancer. 1996;74:1511–1513. 22. Gasterland DE, Rodrigues MM, Moshell AN. Ocular involvement in xeroderma pigmentosum. Ophthalmology. 1982;89:980–986. 23. Hertle RW, Durso F, Metzler JP, et al. Epibulbar squamous cell carcinomas in brothers with xeroderma pigmentosa. J Pediatr Ophthalmol Strabismus. 1991;28:350–353. 24. Iliff WJ, Marback R, Green WR. Invasive squamous cell carcinoma of the conjunctiva. Arch Ophthalmol. 1975;93:119–122. 25. Winward KE, Curtin VT. Conjunctival squamous cell carcinoma in a patient with human immunodeficiency virus infection. Am J Ophthalmol. 1989; 107:554–555. 26. Trosko JE, Krause D, Isoun M. Sunlight-induced pyrimidine dimers in human cells in vitro. Nature. 1970;228:358–359. 27. Lee GA, Williams G, Hirst LW, et al. Risk factors in the development of ocular surface epithelial dysplasia. Ophthalmology. 1994;101:360–364. 28. Birge HL. Cancer of the eyelids. 1. Basal cell and mixed basal cell and squamous cell epithelioma. Arch Ophthalmol. 1938;19:700–708. 29. Tabbara KF, Kersten R, Dauk N, et al. Metastatic squamous cell carcinoma of the conjunctiva. Ophthalmology. 1988;95:318–321. 30. Brash DE, Rudolph JA, Simon JA, et al. A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc Natl Adad Sci USA. 1991;88:10124–10128. 31. Vogelstein B, Kinzler KW. Carcinogens leave fingerprints. Nature. 1992;355:209–210. 32. Dushku N, Reid TW. P53 expression in altered limbal basal cells of pingueculae, pterygia and limbal tumors. Curr Eye Res. 1997;16:1179–1192. 33. Gissmann L, deVilliers EM, zur Hausen H. Analysis of human genital warts (condylomata acuminata) and other genital tumors for human papillomavirus type 6 DNA. Int J Cancer. 1982;29:143–146. 34. Wilbur DC, Reichman RC, Stoler MH. Detection of infection by human papillomavirus in genital condylomata. A comparison study using immunocytochemistry and in situ nucleic acid hybridization. Am J Clin Pathol. 1988;89:505–510. © 2003 Lippincott Williams & Wilkins 35. Crum CP, Ikenberg H, Richart RM, et al. Human papillomavirus 16 and early cervical neoplasia. N Engl J Med. 1984;310:880–883. 36. deVilliers EM, Wagner D, Schneider A, et al. Human papillomavirus infections in women with and without abnormal cervical cytology. Lancet. 1987;2: 703–706. 37. Pater MM, Dunne J, Hogan G, et al. Human papillomavirus types 16 and 18 sequences in early cervical neoplasia. Virology. 1986;155:13–18. 38. Durst M, Gissmann L, Ikenberg H, et al. A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions. Proc Natl Acad Sci USA. 1983; 80:3812–3815. 39. Sjo NC, Heegaard S, Prause JU, et al. Human papillomavirus in conjunctival papilloma. Br J Ophthalmol. 2001;85:785–787. 40. Lass JH, Grove AS, Papale JJ, et al. Detection of human papillomavirus DNA sequences in conjunctival papilloma. Am J Ophthalmol. 1983;96:670– 674. 41. Pfister H, Fuchs PG, Völcker HE. Human papillomavirus DNA in conjunctival papilloma. Graefes Arch Clin Exp Ophthalmol. 1985;223:164–167. 42. Naghashfar Z, McDonnell PJ, McDonnell JM, et al. Genital tract papillomavirus type 6 in recurrent conjunctival papilloma. Arch Ophthalmol. 1986; 104:1814–1815. 43. McDonnell PJ, McDonnell JM, Kessis T, et al. Detection of human papillomavirus type 6/11 DNA in conjunctival papillomas by in situ hybridization with radioactive probes. Hum Pathol. 1987;18: 1115–1119. 44. Mäntyjärvi M, Syrjänen S, Kaipiainen S, et al. Detection of human papillomavirus type 11 DNA in a conjunctival squamous cell papilloma by in situ hybridization with biotinylated probes. Acta Ophthalmol (Copenh). 1989;67:425–429. 45. McDonnell JM, McDonnell PJ, Sun YY. Human papillomavirus DNA in tissues and ocular surface swabs of patients with conjunctival epithelial neoplasia. Invest Ophthalmol Vis Sci. 1992;33:184– 189. 46. McDonnell JM, Wagner D, Ng ST, et al. Human papillomavirus type 16 DNA in ocular and cervical swabs of women with genital tract condylomata. Am J Ophthalmol. 1991;112:61–66. 47. Scott IU, Karp CL, Nuovo GJ. Human papillomavirus 16 and 18 expression in conjunctival intraepithelial neoplasia. Ophthalmology. 2002;109:542– 547. 48. Blodi FC. Squamous cell carcinoma of the conjunctiva. Doc Ophthalmol. 1973;34:93–108. 49. Geggel HS, Friend J, Boruchoff SA. Corneal epithelial dysplasia. Ann Ophthalmol. 1985;17:27–31. 50. Cameron JA, Hidayat AA. Squamous cell carcinoma of the cornea. Am J Ophthalmol. 1991;111:571– 574. 51. Cruess AF, Wasan SM, Wills WE. Corneal epithelial dysplasia and carcinoma in situ. Can J Ophthalmol. 1981;16:171–175. 52. Roberson MC. Corneal epithelial dysplasia. Ann Ophthalmol. 1984;16:1147–1150. 53. Spencer WH. Conjunctiva—Neoplasms and related conditions. In: Spencer WH, ed. Ophthalmic Pa- Ocular Surface Squamous Neoplasia CORNEA Volume 22, Number 7 October 2003 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. thology: An Atlas and Textbook. CD-ROM version, New York: WB Saunders, 1998: Vol. I, Chapter 248. Dark AJ, Streeten BW. Preinvasive carcinoma of the cornea and conjunctiva. Br J Ophthalmol. 1980; 64:506–514. Tanaka M, Kanei A, Nakajima A. Electron microscope studies of Bowen’s disease. Folia Ophthalmol Jpn. 1974;25:1098–1111. Tanabe Y, Tanabe S. Electron microscopic studies of corneal cancer. Folia Ophthalmol Jpn. 1965;16: 137–146. Naib ZM, Clepper AS, Elliott SR. Exfoliative cytology as an aid in the diagnosis of ophthalmic lesions. Acta Cytol. 1967;11:295–303. Dykstra PC, Dykstra BA. The cytologic diagnosis of carcinoma and related lesions of the ocular conjunctiva and cornea. Trans Am Acad Ophthalmol Otolaryngol. 1969;73:979–995. Gelender H, Forster RK. Papanicolaou cytology in the diagnosis and management of external ocular tumors. Arch Ophthalmol. 1980;98:909–912. Tsubota K, Kajiwara K, Ugajin S, et al. Conjunctival brush cytology. Acta Cytol. 1990;34:233–235. Anagnostopoulou-Fotinopoulou I, Rammou-Kinia R. Cytobrush sampling in conjunctival cytology. Diagn Cytopathol. 1993;9:113–115. Mader TH, Stulting RD. A new method of obtaining cells from the cornea and conjunctiva for cytologic study (letter). Arch Ophthalmol. 1990;108:639. Egbert PR, Lauber S, Maurice DM. A simple conjunctival biopsy. Am J Ophthalmol. 1977;84:798– 801. Nolan GR, Hirst LW, Wright RG, et al. Application of impression cytology to the diagnosis of conjunctival neoplasms. Diagn Cytopathol. 1994;11:246– 249. Thiel MA, Bossart W, Bernauer W. Improved impression cytology techniques for the immunopathological diagnosis of superficial viral infections. Br J Ophthalmol. 1997;81:984–988. Connor CG, McGinn TT, Tirey WW. Comparison of cytologic techniques used in differntial diagnosis of conjunctivitis. Invest Ophthalmol Vis Sci. 1991;32: 734. Tole DM, McKelvie PA, Daniell M. Reliability of impression cytology for the diagnosis of ocular surface squamous neoplasia employing the Biopore membrane. Br J Ophthalmol. 2001;85:154–158. McKelvie PA, Daniell M. Impression cytology following mitomycin C therapy for ocular surface squamous neoplasia. Br J Ophthalmol. 2001;85: 1115–1119. Nolan GR, Hirst LW, Bancroft BJ. The cytomorphology of ocular surface squamous neoplasia by using impression cytology. Cancer. 2001;93:60–67. Nadjari B, Kersten A, Rob B, et al. Cytologic and DNA cytometric diagnosis and therapy monitoring of squamous cell carcinoma in situ and malignant melanoma of the cornea and conjunctiva. Analyt Quant Cytol Histol. 1999;21:387–396. Salamao DR, Mathers WD, Sutphin JE, et al. Cytologic changes in the conjunctiva mimicking malignancy after topical mitomycin C chemotherapy. Ophthalmology. 1999;106:1756–1761. Kodama T, Kawamoto K, Kono T, et al. Comparison of immunostainings for proliferating cell 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. nuclear antigen and the Ki-67 in human extraocular lesions. Graefes Arch Clin Exp Ophthalmol. 1997; 235:767–772. Aoki S, Kubo E, Nakamura S, et al. Possible prognostic markers in conjunctival dysplasia and squamous cell carcinoma. Jpn J Ophthalmol. 1998;42: 256–261. Shields JA, Shields CL, DePotter P. Surgical management of conjunctival tumors. Arch Ophthalmol. 1997;115:808–815. Wilson FM. Rose Bengal staining of epibulbar squamous neoplasms. Ophthalmic Surg. 1976;7:21–22. Zimmermann LE. The cancerous, precancerous and pseudocancerous lesions of the cornea and conjunctiva: The Pocklington Memorial Lecture. In: Rycroft PV, ed: Corneoplastic Surgery, New York: Pergamon Press, 1969;420. Char DH, Crawford JB, Howes EL, et al. Resection of intraocular squamous cell carcinomas. Br J Ophthalmol. 1992;76:123–125. Peksayar G, Soyturk MK, Demiryont M. Long-term results of cryotherapy on malignant epithelial tumors of the conjunctiva. Am J Ophthalmol. 1989; 107:337–349. Sanders N, Bedotto C. Recurrent carcinoma in situ of the conjunctiva and cornea (Bowen’s disease). Am J Ophthalmol. 1972;74:688–693. Divine RD, Anderson RL. Nitrous oxide cryotherapy for intraepithelial epithelioma of the conjunctiva. Arch Ophthalmol. 1983;101:782–786. Dutton JJ, Anderson RL, Tse DT. Combined surgery and cryotherapy for scleral invasion of epithelial malignancies. Ophthalmic Surg. 1984;15:289–294. Fraunfelder FT, Wingfield D. Management of intraepithelial conjunctival tumors and squamous cell carcinomas. Am J Ophthalmol. 1983;95:359–363. Fraunfelder FT, Wingfield D. Therapy of intraepithelial epitheliomas and squamous cell carcinomas of the limbus. Trans Am Ophthalmol Soc. 1980; 79:290–300. Soll DB, Harrison SE. Basic concepts and an overview of cryosurgery in ophthalmic plastic surgery. Ophthalmic Surg. 1979;10:31. Wilkes TD, Fraunfelder FT. Principles of cryosurgery. Ophthalmic Surg. 1979;10:21. Copeland RA Jr, Char DH. Limbal autograft reconstruction after conjunctival squamous cell carcinoma. Am J Ophthalmol. 1990;110:412–415. Cerezo L, Otero J, Aragon G, et al. Conjunctival intraepithelial and invasive squamous cell carcinomas treated with strontium 90. Radiother Oncol. 1990;17:191–197. Goldberg JR, Becker SC, Rosenbaum HD. Gamma radiation in the treatment of squamous cell carcinoma of the limbus. Am J Ophthalmol. 1976;55: 811–815. Seale ER. Carcinoma of the limbus. Report of 19 cases treated with radium and effect of gamma radiation upon eye. AMA Arch Dermatol Syph. 1953; 68:286–294. Phillipp W, Daxecker F, Langmayr J, et al. Spontaneous corneal rupture after strontium irradiation of conjunctival squamous cell carcinoma. Ophthalmologica. 1987;195:113–118. de Keizer RJW, de Wolff-Rouendall D, van Delft JL. Topical application of 5-fluorouracil in premalig- 703 704 CORNEA Basti and Macsai Volume 22, Number 7 October 2003 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. nant lesions of cornea, conjunctiva and eyelid. Doc Ophthalmol. 1986;64:31–42. Rubinfield RS, Pfister RR, Stein RM, et al. Serious complications of topical mitomycin C after pterygium excision. Ophthalmology. 1992;99:1647– 1654. Frucht-Pery J, Rozenman Y. Mitomycin C therapy for corneal intraepithelial neoplasia. Am J Ophthalmol. 1994;117:164–168. Frucht-Pery J, Sugar J, Baum J, et al. Mitomycin C treatment for conjunctival-corneal intraepithelial neoplasia—a multicenter experience. Ophthalmology. 1997;104:2085–2093. Heigle TJ, Stulting RD, Palay DA. Treatment of recurrent conjunctival epithelial neoplasia with topical mitomycin C. Am J Ophthalmol. 1997;124: 397–399. Wilson MW, Hungerford JL, George SM, et al. Topical mitomycin C for the treatment of conjunctival and corneal epithelial dysplasia and neoplasia. Am J Ophthalmol. 1997;124:303–311. Shields CL, Naseripour M, Shields JA. Topical mitomycin C for extensive recurrent conjunctivalcorneal squamous cell carcinoma. Am J Ophthalmol. 2002;133:601–606. Haas K, Ben-Dor D, Levartovsky S. Treatment of conjunctival corneal intraepithelial neoplasia with topical mitomycin C. Arch Ophthalmol. 1999;117: 544. Rozenman Y, Frucht-Pery J. Treatment of conjunctival intraepithelial neoplasia with topical drops of mitomycin C. Cornea. 2000;19:1–6. Crooke ST, Bradner WT. Mitomycin C: a review. Cancer Treat Rev. 1976;3:121–139. Verweij J. Mitomycins. In: Pinedo HM, Longo DL, Chabner BA, eds. Cancer Chemotherapy and Biological Response Modifiers Annual 17. Amsterdam: Elsevier Science, 1997;76. Yeatts RP, Ford JG, Stanton CA, et al. Topical 5-fluorouracil in treating epithelial neoplasia of the conjunctival and cornea. Ophthalmology. 1995;102: 1338–1344. © 2003 Lippincott Williams & Wilkins 103. Midena E, Boccato P, Angeli CD. Conjunctival squamous cell carcinoma treated with topical 5-fluorouracil. Arch Ophthalmol. 1997;115:1600–1601. 104. Yeatts RP, Engelbrecht NE, Curry CD, et al. 5-Fluorouracil for the treatment of intraepithelial neoplasia of the conjunctiva and cornea. Ophthalmology. 2000;107:2190–2195. 105. Yamamoto N, Ohmura T, Suzuki H, et al. Successful treatment with 5-fluorouracil of conjunctival intraepithelial neoplasia refractive to mitomycin C. Ophthalmology. 2002;109:249–252. 106. Maskin SL. Regression of limbal epithelial dysplasia with topical interferon. Arch Ophthalmol. 1994; 112:1145–1146. 107. Vann RR, Karp CL. Perilesional and topical interferon alfa 2 b for conjunctival and corneal neoplasia. Ophthalmology. 1999;106:91–97. 108. Schechter BA, Schrier A, Nagler RS, et al. Regression of presumed primary conjunctival and corneal intraepithelial neoplasia with topical interferon alpha-2b. Cornea. 2002;21:6–11. 109. Edwards L, Berman B, Rapini RP, et al. Treatment of cutaneous squamous cell carcinoma by intralesional topical interferon alfa 2-b therapy. Arch Dermatol. 1992;128:1486–1489. 110. Berman B, Sequiera M. Dermatologic uses of interferons. Dermatol Clin. 1995;13:699–711. 111. Tabin G, Levin S, Snibson G, et al. Late recurrences and the necessity for long-term follow-up in corneal and conjunctival intraepithelial neoplasias. Ophthalmology. 1997;104:485–492. 112. Tunc M, Char DH, Crawford B, et al. Intraepithelial and invasive squamous cell carcinoma of the conjunctiva: analysis of 60 cases. Br J Ophthalomol. 1999;83:98–103. 113. Brownstein S. Mucoepidermoid carcinoma of the conjunctiva with intraocular invasion. Ophthalmology. 1981;88:1226–1230. 114. Carrau RL, Stillman E, Canaan RE. Mucoepidermoid carninoma of the conjunctiva. Ophthal Plast Reconst Surg. 1994;10:163–168.