Methods

This retrospective clinicopathologic study was performed under the auspices of the Massachusetts Eye and Ear Infirmary Institutional Review Board (Protocol 12-132H) and conducted in compliance with the rules and regulations of the Health Insurance Portability and Accountability Act and in adherence to the Declaration of Helsinki and all federal and state laws.

After the diagnosis of a conjunctival large cell acanthoma was made, the diagnostic files of the David G. Cogan Laboratory of Ophthalmic Pathology at the Massachusetts Eye and Ear Infirmary were reviewed from July 1, 2008 to June 30, 2012 to identify lesions diagnosed as conjunctival squamous or epidermoid dysplasia, carcinoma in situ, conjunctival intraepithelial neoplasia, and papilloma. Among the cases retrieved was a pigmented dysplasia, which was nonetheless selected for inclusion. Blocks were evaluated for adequacy of remaining tissue to allow a battery of immunohistochemical tests. In addition to the large cell acanthoma, 7 routine dysplasias and 4 papillomas were deemed appropriate for this study. Normal conjunctiva present in 11 out of the 12 tissue specimens that were selected served as a baseline for comparison and analysis of the data assembled from the lesions.

Only the conjunctival aspects of the lesions were systematically evaluated immunohistochemically, inasmuch as these portions constituted the majority of the specimens and only a few had small associated strips of what appeared to be corneal epithelium. In most dysplastic lesions, the corneal component had been scraped off, supplemented with an alcohol scrub of the denuded Bowman membrane; the scrapings were not submitted for microscopic examination. Clinical and hospital records as well as clinical photographs were renewed to glean demographic data and other pertinent information on the clinical history and examination findings. In addition to hematoxylin-eosin staining and periodic acid–Schiff (PAS) staining with and without diastase, 4- to 5-μm paraffin-embedded histopathologic sections were prepared that employed the immunoperoxidase method using the chromogen diaminobenzidine with hematoxylin counterstaining. Table 1 lists the immunohistochemical probes that were available for this investigation in the Diagnostic Immunopathology Division of the Department of Pathology at the Massachusetts General Hospital.

Positivity in the immunoperoxidase preparations was determined for the cytokeratins as an all-or-none phenomenon when any intensity of immunohistochemical staining was observed in 50% or more of cells constituting the entire thickness of the epithelium. An assessment of graded intensities of positivity was not attempted except for p53 immunostaining. Specific cell counts for nuclear positivity were performed for p53 and Ki-67 only. Two counts in different areas of each lesion were averaged and reported as a percent of positive cells among those counted per high-power field (so-called proliferation index for Ki-67). In cases of moderate dysplasia, Ki-67 counts were confined to the bottom half of involved epithelium, whereas the p53 counts were made throughout the full thickness of the epithelium in both moderate and severe dysplasias. Cytokeratin (CK) staining patterns of normal and lesional conjunctival epithelium were also evaluated based on selective basilar, suprabasilar, and superficial expression of positivity. The results were collected for each diagnostic category of lesion and the categories were compared with each other and with the staining results manifested by the attached normal segments of conjunctiva in the surgical specimens.

Results

Clinical Findings

The clinical features of all 12 lesions in this series are summarized in Table 2 .

Table 2

Clinical Characteristics of a Spectrum of 12 Conjunctival Intraepithelial Squamous Proliferations

Diagnosis	Age/Sex	Laterality, Location, a and Relevant History	Duration of Symptoms	Length of Follow-up	Recurrence
Large cell acanthoma	51/F	Left eye, nasal limbus; sessile	14 years	14 years	4 times
Dysplasia 1	79/M	Left eye, nasal limbus; sessile	“Many years”	4 years	None
Dysplasia 2	45/M	Right eye, temporal limbus; sessile	4 months	3 years	None
Dysplasia 3	79/M	Right eye, inferotemporal limbus; gelatinous, opalescent b ; positive rose bengal staining	3-4 months	2 years	None
Dysplasia 4	69/M	Right eye, nasal limbus; gelatinous, sessile	5-6 months	6 months	None
Dysplasia 5	83/M	Right eye, temporal limbus, gelatinous, sessile	3 months	6 months	None
Dysplasia 6	39/F	Right eye, temporal limbus; pigmented, gelatinous, sessile; African-American; HIV positive	8 years	4 months	None
Dysplasia 7	91/M	Left eye, nasal limbus; vascular, gelatinous, sessile; pterygium excision; glaucoma	2-3 years	7 months	None
Papilloma 1	56/M	Right eye, inferotemporal epibulbar; yellow-colored, fleshy, sessile	3-4 months	4 months	None
Papilloma 2	70/F	Left eye, nasal inferior fornix and caruncle/plica; punctate vessels; sessile; focal pigmentation due to coincidental nevus	3 months	1 year and 3 months	None
Papilloma 3	62/M	Left eye, lower palpebral near eyelid margin; sessile	3 months	6 years	2 times
Papilloma 4	39/M	Left eye, nasal bulbar and caruncle/plica; pedunculated	4 months	1 year and 7 months	Once (earlier treated with interferon for 3 months)

a All lesions involved the corneal epithelium, which generally constituted 25% or less of the overall lesional size.

b No lesion in this series was leukoplakic.

Large cell acanthoma

Because of its rarity and disputed nature, the clinical history of the patient with the large cell acanthoma is presented in more detail. A 51-year-old woman of Lithuanian and Irish descent developed over a period of 1 year gradual blurring of vision of the left eye associated with a foreign body sensation and occasional eye redness. Her past medical history was remarkable for pre–Waldenstrom macroglobulinemia and hypertension. She had undergone excision of atypical skin lesions in the past, but had never received a diagnosis of skin cancer. She grew up in New England and had extensive sun exposure. Best-corrected visual acuity was 20/20 OU. In 1998, slit-lamp examination showed an irregular, elevated, opalescent lesion located at the nasal limbus OS (unfortunately, a clinical photograph is not available). There was positive punctate corneal and interpalpebral conjunctival epithelial staining with fluorescein and rose bengal. The Schirmer test was abnormal (OD 3 mm, OS 1 mm). The patient was initially diagnosed with keratitis sicca, OS>OD. Debridement of the limbal lesion, which prevented pathologic evaluation of the lesional margins, was done and the patient was given punctal plugs and artificial tears. The lesion recurred after 6 months and was noted to involve more of the corneal surface, prompting another excision (1998). One year later, it recurred for the second time, and was re-excised and submitted for histopathologic evaluation (1999), which we were able to reevaluate and which showed abnormal cells at the surgical margins. During the interim, most of the ocular surface remained stable. The lesion recurred, however, for a third time after 6 years, leading to a third excisional biopsy (2005), which we were unable to review. Seven years after the last excision, the lesion recurred once again (2012). The latest lesion was located at the nasal limbus from 7-11 o’clock, appeared fimbriated, and extended 3 mm onto the cornea. Ocular surface surgery was recommended. Alcohol-assisted superficial keratectomy was performed on the affected corneal epithelium with surgical removal of adjacent involved conjunctiva. The surgical specimen displayed an uninvolved conjunctival margin but the corneal margin was positive. The ocular surface was then reconstructed with an amniotic membrane graft. The patient was treated postoperatively with topical steroids and antibiotics, with rapid resolution of the epithelial defect. There has been no recurrence after 8 months of follow-up.

Squamous dysplasias

The clinical characteristics and epibulbar locations of the remaining 11 cases in this series are briefly described in Table 2 , which includes the 7 squamous dysplasias. There were 6 men and 1 woman with an average age of 69 years. All lesions arose at the limbus. The right eye was affected in 5 cases and the left in 2. The patients were symptomatic for an average of 24 months. All lesions were gelatinous or opalescent rather than leukoplakic, signifying the absence of keratinization. The lesions typically manifested a placoid or sessile papillary growth pattern with regularly distributed punctate vessels, indicating an underlying fibrovascular/papillary supporting architecture. The lesions bestrode the limbus extending onto the peripheral cornea as frosted epithelium, which constituted less than a quarter of their total size ( Figure 1 , Top left, Top right, and Middle left), with the largest portion situated in the juxtalimbal conjunctiva. One lesion in an African-American woman was partially pigmented ( Figure 1 , Middle left). The pigmentation extended within the epithelium onto the peripheral nonvascularized cornea. The average follow-up was 19 months, with no recurrences arising after a variety of therapies, including wide local excision of the conjunctival portion of the lesion, excision or scraping of the corneal extension, alcohol scrubbing of the denuded Bowman membrane, and adjuvant cryotherapy and/or topical antimetabolites to eradicate any remaining tumor cells.

Clinical and pathologic features of conjunctival squamous proliferations. (Top left) A 79-year-old man had a history of a longstanding dysplastic limbal lesion that was removed with 4 years of disease-free follow-up. The thickened nasal papillary lesion overrides the limbus onto the peripheral cornea. Note the regularly spaced small blood vessels signifying an underlying papillary architecture. There are several feeding vessels toward the left. (Top right) A 45-year-old man was symptomatic for 4 months and recurrence-free after 3 years of follow-up. A thickened and elevated conjunctival lesion coexists with flat, opalescent, dysplastic epithelium extending onto the peripheral cornea (arrows). (Middle left) A 39-year-old African-American woman who was HIV positive developed a minimally elevated, partially pigmented thickening of the temporal conjunctiva that was established to be a carcinoma in situ with extension of partially pigmented frosted epithelium onto the peripheral cornea (crossed arrows). The superior aspect of the lesion (arrows) displays irregularity of the epithelial surface signifying neoplastic involvement. (Middle right) A 39-year-old man with a 4-month history of a reddened caruncle caused by a squamous papilloma displays an elevated and pedunculated pinkish lesion (arrows) with dot-like vessels, indicating an underlying papillary supportive stroma. The lesion is located on the medial slope of the caruncle to involve the plica. (Bottom left) Large cell acanthoma. The first biopsy (epithelial scraping) of a left nasal limbal lesion from 1999 shows cells with an abundance of eosinophilic cytoplasm manifesting widely spaced nuclei with some variability in size with rare dyskeratosis (arrow) but without prominent nucleoli. (Bottom right) Large cell acanthoma. The periodic acid–Schiff stain reveals the presence of abundant granular positivity in the cytoplasm (arrows), which was abolished by pretreatment with diastase, demonstrating that the material was glycogen. (Hematoxylin-eosin: bottom left, ×400; periodic acid–Schiff: bottom right, ×400.)

Squamous papillomas

The 4 benign squamous papillomas were found in 3 men and 1 woman with an average age of 57 years. They were usually symptomatic for 3-4 months. Lesions arose in the epibulbar, plica/caruncular ( Figure 1 , Middle right), forniceal, and palpebral regions of the conjunctival sac, but not at the limbus. Three were sessile and 1 was pedunculated. The average follow-up was 28 months, during which 2 lesions recurred, requiring re-excision without adjuvant therapy.

Histopathologic and Immunohistochemical Findings

The immunohistochemical findings for all types of squamous lesions are summarized in Table 3 .

Table 3

Immunohistochemical Staining Results for a Spectrum of 12 Conjunctival Intraepithelial Squamous Proliferations

	EMA	BerEP4	P53	Ki-67	AE1	AE3	CK7	CK8	CK10	CK14	CK17	CK18	CK19	CK20
Normal conjunctiva a (+/−)	11/0	0/11	1.2%	2.4%	11/0	11/0	11/0	0/11	0/11	11 b /0	4 b /7	4 b /7	11/0	0/11
Large cell acanthoma	+	−	59.6%	3.3%	+ c	+	−	−	−	+	−	−	+	−
Dysplasia 1	+	−	87.8%	37.2%	+	+	−	−	− d	+	− d	−	+	−
Dysplasia 2	+	−	91.9%	42.8%	+	+	+	−	− d	+	+	−	+	−
Dysplasia 3	+	−	77.1%	44.7%	+	+	−	−	−	+	+	−	+	−
Dysplasia 4	+	−	− e	32.6%	+	+	−	−	−	+	+	−	−	−
Dysplasia 5	+	−	83.9%	39.6%	+	+	−	− d	− d	+	− d	−	+	−
Dysplasia 6	+	−	70.2%	45.8%	+	+	+	−	− d	+	+	+	+	−
Dysplasia 7	+	−	84.9%	24.6%	+	+	−	− d	− d	+	− d	−	+	−
Papilloma 1	+	−	11.2%	17.5%	+	+	+	−	−	− b	−	−	+	−
Papilloma 2	+	−	21.5%	12.9%	+	+	+	−	−	− b	− b	−	+	−
Papilloma 3	+	−	7.5%	28.4%	+	+	+	−	−	− b	−	−	+	−
Papilloma 4	+	−	8.4%	27.5%	+	+	+	−	−	− b	−	−	+	−

CK = cytokeratin; EMA = epithelial membrane antigen.

a Number of specimens with normal control conjunctiva = 11.

b Basilar cell positivity only.

c Positivity for the various cytokeratins without an explanatory notation indicates staining of 50%-100% of cells throughout the entire epithelium; negativity indicates staining of fewer than 50% of cells.

d Focal positivity in suprabasilar cells and often in large atypical epidermoid cells.

e Negative staining was either an authentic finding or an artifact attributable to poor antigen preservation.

Large cell acanthoma

The last excision of the recurrent large cell acanthoma measured 0.7 × 0.5 × 0.1 cm. The lesion grew as a diffuse placoid thickening of the epithelium without an underlying papillary structure. It was composed of large, polygonal, eosinophilic cells 2-3 times the size of normal conjunctival keratinocytes with commensurately large nuclei containing small nuceloli. Many intercellular attachments (desmosomes) were seen during high-power microscopy. These features were detected in the first biopsy (1999) that could be reviewed ( Figure 1 , Bottom left and Bottom right) and persisted up to the final excision ( Figure 2 , Top left, Top right, and Middle left). Some variability in the size of the nuclei was focally detected ( Figure 2 , Top right) without any mitotic activity among the basilar and suprabasilar cells. The large tumor cells formed an abrupt interface with the adjacent normal conjunctival epithelium ( Figure 2 , Middle right) but were present at the corneal edge. The PAS stain highlighted prominent cytoplasmic positivity in the first available and last specimens ( Figure 1 , Bottom right, and Figure 2 , Bottom left), which could be abolished by means of pretreatment with diastase ( Figure 2 , Bottom right). This established that the stainable material was glycogen. Subepithelial chronic inflammation was not noted. p53 and Ki-67 nuclear staining ( Figure 3 , Top left and Top right) were the lowest cell counts of all the neoplasms in this series. Most notable were CK7 negativity ( Figure 3 , Middle left), in contrast to its consistent positivity in normal conjunctival epithelium; CK18 negativity, which is sometimes positive among the normal conjunctival basilar cells’ epithelium and rarely in suprabasilar cells ( Figure 3 , Middle right); and full-thickness epithelial positivity for CK14, which is present only in the basilar cells of the normal epithelium.

Large cell acanthoma. (Top left) There is placoid thickening of the conjunctival epithelium without a supportive papillary stroma. The superficial vessels of the substantia propria are mildly ectatic. The tumor cell nuclei are widely spaced and monotonously repetitive and regular. The inset reveals the cytologic detail of centrally placed nuclei possessing small nucleoli and numerous intercellular bridges, which are brought out by the shrinkage of the cytoplasm during tissue processing. (Top right) Minimal variability in the size of the nuclei, which failed to display hyperchromasia or mitotic activity. The most superficial epithelial cells retain their nuclei and show focal increased cytoplasmic eosinophilia, implying early parakeratosis (arrow). (Middle left) Large acanthoma cells artifactually overhang normal conjunctival epithelium, demonstrating the dramatic difference in the size of the 2 cell populations. (Middle right) There is an abrupt demarcation (arrows) between the large tumor cells on the left and the smaller normal conjunctival epithelial cells on the right. (Bottom left) The cytoplasm of many tumor cells stains intensely with the periodic acid–Schiff stain. (Bottom right) Diastase pretreatment abolishes the cytoplasmic positivity. The nuclei are shown to excellent advantage and lack mitotic activity or prominent nucleoli. (Hematoxylin-eosin: top left, ×200, inset, ×600; top right, ×400; middle left, ×200; middle right, ×400; periodic acid–Schiff: bottom left, ×400; bottom right, ×400.)

Large cell acanthoma, squamous dysplasia, and carcinoma in situ. (Top left) Large cell acanthoma. p53 immunostaining reveals many positive-staining nuclei in the basilar and suprabasilar cells; the most superficial nuclei fail to react for the presence of this antigen. (Top right) Large cell acanthoma. Ki-67 immunostaining discloses negligible nuclear positivity (arrows) restricted to the basilar cell region. (Middle left) Large cell acanthoma. Immunostaining for cytokeratin 7 discloses that the normal conjunctiva on the right (arrow) is positive but not the tumor cells (T). (Middle right) Large cell acanthoma. Cytokeratin18 is not expressed in any of the cells within the tumor (T) but does show an affinity for the basilar and suprabasilar cells of the normal conjunctiva (arrows). (Bottom left) Moderately severe routine squamous dysplasia. The small dysplastic squamoid cells are disorganized and occupy the bottom half of the involved conjunctival epithelium (arrows). The cytoplasm is obviously eosinophilic but not copious, and the nuclei and nucleoli are relatively small. The upper half of the lesion is covered by benign-appearing squamous epithelium with somewhat elongated nuclei oriented parallel to the cell surface. (Bottom right) Severe squamous dysplasia (carcinoma in situ). Atypical tumor cells have replaced all of the conjunctival epithelium and manifest nuclear variability up to the superficial layers. Note the light chronic inflammation in the substantia propria (SP). (Immunoperoxidase: top left, ×400; top right, ×400; middle left, ×200; middle right, ×200; hematoxylin-eosin: bottom left, ×400; bottom right, ×200.)

Squamous dysplasias

For the 7 conventional dysplasias, the average size of the excised specimens was 1.4 × 0.6 × 0.1 cm (range, 2.8 × 0.6 × 0.1 cm – 0.5 × 0.3 × 0.1 cm). All grew in a placoid or low papillary sessile pattern. Two were interpreted as moderately severe dysplasias with approximately 50% of the epithelial thickness replaced by atypical cells, whereas the other 5 lesions were severe dysplasias or carcinomas in situ with near to complete epithelial replacement. The moderate dysplasias with both basilar and suprabasilar mitoses were composed of small epidermoid/squamoid cells with eosinophilic cytoplasm and small round or oval nuclei with small nucleoli ( Figure 3 , Bottom left). These cells occupied the bottom half of the epithelium above the basement membrane, while the upper or outer 50% of the epithelium displayed more elongated eosinophilic cells with narrower nuclei oriented parallel to the surface. In the severe dysplasias and carcinomas in situ (from a practical point of view, this distinction is more notional than applicable), virtually the entire thickness of the epithelium was replaced by varying grades of atypical cells containing multiform hyperchromatic nuclei ( Figure 3 , Bottom right, and Figure 4 , Top left). In 3 lesions, there were large, bizarre, pleomorphic, and sometimes multinucleated epidermoid cells forming small clusters or eosinophilic tracts within a sea of smaller basaloid cells ( Figure 4 , Top left; Figure 4 , Top right and Bottom left inset). No lesion was exclusively composed of basaloid cells with inconspicuous cytoplasm. In the pigmented dysplasia, dendritic melanocytes were interspersed among the basaloid cells and transferred negligible pigment granules to them ( Figure 4 , Top right, Bottom right inset), as highlighted by MART-1 immunostaining. There was an abrupt demarcation of the dysplastic cells from the adjacent normal epithelial cells ( Figure 4 , Middle left). The PAS stain with and without diastase disclosed prominent cytoplasmic accumulations of glycogen in 5 cases, present either in the small squamoid cells or in the large epidermoid cells; there was a complete absence of goblet cells within the lesions ( Figure 4 , Middle right and inset). Chronic inflammation in the form of a variably prominent but typically modest infiltrative band in the superficial substantia propria was present in all lesions. The most salient immunohistochemical results ( Table 3 ) were p53 and Ki-67 nuclear staining in the lower half of the epithelium of the moderately severe lesions, whereas there was full-thickness staining with these biomarkers in the severe lesions ( Figure 4 , Bottom left and Bottom right; Figure 5 , Top left and Top right). The average percent of positive nuclei was 82.6% for p53 and 38.2% for Ki-67. Curiously, the larger epidermoid cells tended not to stain with Ki-67 ( Figure 5 , Middle left) but did for p53. Some horizontally flattened superficial cells were both p53 and Ki-67 positive. The surrounding normal conjunctival epithelium had close to zero p53 counts and low Ki-67 expression ( Table 3 ). With respect to immunostaining for the various cytokeratins, the dysplasias were overwhelmingly CK7 negative, in contrast to normal conjunctiva ( Figure 5 , Middle right). In a few instances, CK7 positivity was discovered among the basilar cells, at the surface of the lesions, or in their midst, generally within the large epidermoid cells ( Figure 5 , Bottom left). The dysplasias were more likely to express CK17 ( Figure 5 , Bottom right) than the other lesions and were full-thickness CK14 positive, whereas this marker is present only in the basilar cells of normal conjunctiva.

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