Ocular surface squamous neoplasia – Review of etio-pathogenesis and an update on clinico-pathological diagnosis

Abstract

Ocular surface squamous neoplasia (OSSN) has a varied clinical presentation, the diagnosis of which rests on the histopathological examination of the excised lesion. The term OSSN includes mild dysplasia on one end of the spectrum and invasive squamous cell carcinoma on the other end. This lesion has a multi factorial aetiology with interplay of several factors like exposure to ultraviolet radiation, various chemical carcinogens and viral infections, however role of individual agents is not well understood. With the upsurge of infection with human immunodeficiency virus, a changing trend is seen in the clinical presentation and prognosis of patients of OSSN even in developed countries. Anterior segment optical coherence tomography (OCT) and confocal microscopy, hold promise in in-vivo differentiation of intraepithelial neoplasia from invasive squamous cell carcinoma. Variants of squamous cell carcinoma like Mucoepidermoid carcinoma, spindle cell carcinoma and OSSN associated with HIV infection should be suspected in a case of aggressive clinical presentation of OSSN or with massive and recurrent tumours. Surgery, chemotherapy and immunotherapy are the various treatment modalities which in combination show promising results in aggressive, recurrent and larger tumours.

Background

Ocular surface squamous neoplasia (OSSN) is a broad term encompassing conjunctival intraepithelial neoplastic lesions (CIN) and invasive squamous cell carcinoma (SCC) of conjunctiva and cornea.¹ CIN includes varying grades of dysplasia, ranging from mild, moderate, severe dysplasia to carcinoma in situ. When various intra epithelial lesions of squamous epithelium of conjunctiva were identified, numerous confusing terminologies such as epithelial plaque, intra epithelial epithelioma, dyskeratosis, dysplasia, precancerous epithelioma, Bowen’s disease of the conjunctiva and ‘Bowenoid epithelioma’ were used to describe this lesion. The term CIN, in vogue today was proposed by Pizarello and Jakobeic, derived from the terminology applied to the intraepithelial cervical malignancies.²

Advanced age, male gender, exposure to solar ultraviolet radiation, infection with human papilloma virus (HPV), immunosuppression and infection with human immunodeficiency virus (HIV) are the factors which play an essential role in the development of OSSN. OSSN, known as an indolent disease of adult males, has shown changing trends with regard to its clinical presentation and progression with upsurge of HIV infection.³ It presents more commonly in the interpalpebral area of perilimbal conjunctiva, and can extend across the limbus to involve the cornea. OSSN can show isolated corneal involvement also. Atypical manifestations of OSSN like massive surface tumours and scleral necrosis after prior surgical interventions have also been reported.^4,5 Atypical clinical manifestations of OSSN need impression cytology or incisional biopsy for definitive diagnoses for further management. While complete excision with histopathologically proven tumour-free margins is the preferred treatment for smaller localised lesions (<4 clock hours of limbus) alternative modalities like chemotherapy, immunotherapy have shown promise in recurrent and larger OSSN.⁶

Epidemiological aspects

The average incidence of OSSN of conjunctiva and cornea was estimated to be 0.13/100,000 in tribal groups in Uganda by Templeton in a study published in 1967,⁷ and 1.9/100,000 population per year in the Brisbane metropolitan area of Australia by Lee in 1992.⁸ Sun and co-workers reported an average incidence of 0.3 million per year in United States in 1997⁹ In a recent study published in 2012 an incidence of 37.3 per 10⁶ was reported for all eye cancers and 8.4 per 10⁶ for SCC.¹⁰ Highest risk of OSSN is seen in males, Caucasians and residents of lower latitudes (closer than 30° latitude to the equator).¹¹ OSSN occurs predominantly in adults, average age of occurrence of OSSN is reported as 56 years, with an age range of 4-96 years.¹ Younger age of presentation of OSSN is seen in addition to population residing at lower latitudes, amongst patients suffering from Xeroderma pigmentosum and HIV infection.^11–14

Predisposing factors

Genetic injury to a proliferating cell knocks out the control of basic cellular functions like division and differentiation and these cells become neoplastic. Several factors are implicated in the development of ocular surface squamous neoplasia. It has been speculated that OSSN may arise from dysfunctional limbal stem cells having been altered by various mutagenic agents, such as UV radiation.¹⁵ The mutagenic factors which have been best understood include exposure to ultraviolet B radiation, and infection with human papilloma virus. Other reported risk factors include exposure to petroleum products, heavy cigarette smoking, chemicals such as trifluridine, arsenicals, beryllium, ocular surface injury, vitamin A deficiency, light pigmentation of the hair and eye, defective DNA repair in Xeroderma Pigmentosum, family origin in the British Isles, Austria, or Switzerland, infection with HIV^16,17 and other immunocompromised states.

Role of ultraviolet B rays

Various studies have shown a linear relationship between exposure to ultraviolet radiation and development of OSSN. Residents of lower latitudes, male sex (outdoor activity), temporal lesions being more common and more aggressive than nasal lesions, higher incidence in fair skin individuals and history of actinic skin lesions make sunlight exposure as the prime aetiological trigger. The degree of risk depends on the type of UV rays, the intensity of exposure, total cumulative exposure and the quantity of the light-absorbing “protective mantle” of melanin. The UV portion of the solar spectrum can be divided into three wavelength ranges: UVA (320–400 nm), UVB (280–320 nm), and UVC (200–280 nm). Of these, UVB is believed to be responsible for the induction of various cutaneous and ocular surface cancers. Exposure to UVB light leads to the formation of pyrimidine dimers in DNA and also causes damage to the nucleotide excision repair pathway which plays a key role in repairing DNA damage caused by UV-B exposure. Unrepaired alterations in the DNA are the first essential step in the process of initiation These alterations become heritable when these altered and injured DNA undergoes one cycle of proliferation, this stimulus of proliferation could be provided by the UV rays themselves or could be provided by virus such as HPV or some chemical stimulus. In a study published in 2008 the role of matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinases in OSSN has been hypothesised with an altered pattern of expression of MMP-1 and MMP-3, following ultraviolet B radiation is implicated in the pathogenesis of OSSN.¹⁸

Xeroderma pigmentosum (XP) is an autosomal recessive disorder with defective DNA repair mechanism which also predisposes to OSSN and other mucosal and cutaneous cancers with aggressive clinical presentation at a younger age. OSSN has been reported as early as 3 years of age in a patient of XP.¹⁹ In the largest study with 87 participants of XP conducted at National Eye Institute, published in 2013, 10% of the patients of XP had ocular surface cancers,²⁰ age range of these patients was 5–28 years.

Human papilloma virus and OSSN

HPV is a nonencapsulated, icosahedral, non-lipid containing DNA virus that replicates in the nuclei of infected cell. There are several genetically distinct types of HPV, some types (e.g., 1, 2, 4, and 7) cause benign squamous lesions in humans and high-risk HPVs (e.g., types 16 and 18) have been implicated in the genesis of several cancers, particularly squamous cell carcinoma of the cervix, anogenital region, oropharyngeal region and ocular surface. Integration of the viral genetic material into the host genome is a critical event in the malignant transformation of the cell. Integration consistently interrupts the viral DNA within the E1/E2 open reading frame, leading to failure or loss of transcription of late E2 genes which functions as viral repressor proteins with absence of viral repressor proteins uncontrolled transcription of E6 and E7 gene occurs. This type of infection is called as non productive infection since life cycle of virus is interrupted and complete virions are not produced. E6 and E7 genes of high risk HPV types have a higher affinity of binding to various growth-regulating proteins encoded by proto-oncogenes and tumour suppressor genes.²¹ However, infection with HPV itself is not sufficient for carcinogenesis. In addition to genetic co-factors, HPV in all likelihood also acts in concert with other environmental factors. Evidence of the role of HPV 16 and 18 in the development of OSSN has been established by several studies, in contrast, our group reported that there is no association of high risk HPV in patients with OSSN at a tertiary eye care centres, as detected by in-situ hybridisation.²² There are other compelling studies which suggest that HPV does not play a role in the pathogenesis of OSSN.^23,24 In a study conducted by Scott and colleagues, published in 2001, HPV 16 or 18 DNA and mRNA corresponding to the E6 region were detected in all 10 CIN specimens examined using the reverse-transcriptase polymerase chain reaction.²⁵ Chauhan and associates in their study published in 2013, reported detection of HPV16 in 11% of their OSSN cases with a positivity of 9% in SCC patients and 15% in dysplastic cases using multiplex PCR with PGMY09/11 primer on fresh tumour tissues from OSSN cases. Authors also reported a better overall survival in patients with HPV infection.²⁶

HIV infection and OSSN

An increase in the incidence of OSSN, since the HIV pandemic, has suggested that HIV infection increases the risk for OSSN. In Africa, OSSN has been recognised to be strongly associated with HIV, the mean age at which the patients present with invasive squamous cell carcinoma ranges from 32 to 37 years, and the proportion of female patients ranges from 55% to 70%.²⁷ HIV infection is now established as a risk factor for the development of squamous cell neoplasia of the conjunctiva based on studies from Rwanda, Malawi, and Uganda.^28,29 A significant increase in the incidence of OSSN is also reported in patients with HIV/AIDS in the United States.³⁰ There are several studies which have reported OSSN as the first clinical presentation of HIV in young patients.^29,31 OSSN occurring in HIV patients are more aggressive and invasive requiring enucleation or even exenteration.³²

Clinical features

Clinically OSSN has myriad presentations. It usually appears as a sessile, fleshy, elevated lesion adjacent to the limbus in the inter-palpebral region. Contrary to general perception the thickness of the lesion is not always an indication of invasive SCC. Even reasonably thick tumours tend to be confined within the epithelium. The presentation of CIN and invasive SCC is very similar thus making clinical differentiation difficult. Usually the tumour presents as a circumscribed, gelatin-like, sessile, papillomatous lesion with variable degrees of leukoplakia (Fig. 1A–D). One often finds dilated conjunctival blood vessels feeding and draining the lesion. SCC is locally invasive and metastasis is seen in <2% of cases. It can invade intraocular tissues and orbit. Some lesions can be diffuse, flat, and poorly-demarcated without an obvious tumour making early diagnosis difficult. Massive tumours infiltrating the deeper corneal stroma and covering the entire ocular surface are also seen (Fig. 2A–F). Infiltrative variants of OSSN masquerading as necrotizing scleritis may pose a challenge in early diagnosis (Fig. 3 A–D).³³ Rarely pigmented variants of OSSN may be seen making differentiation from conjunctival melanoma difficult (Fig. 1C).³⁴

Figure 1.

Varied clinical presentation of OSSN (A). Slit lamp photograph under diffuse illumination shows papillary ocular surface tumour with prominent feeder (B). Figure shows a globular pink-coloured lesion arising with large feeder vessels. The lesion seems to be overlying a pterygium and was clinically mistaken to be a pyogenic granuloma (C). Figure shows a pigmented OSSN with feeder vessels (D). Slit lamp photograph under diffuse illumination showing large leukoplakic lesions with abnormal vessels.

Figure 2.

(A) Slit lamp photograph of the left eye under diffuse illumination shows a large overhanging conjunctivo-corneal mass with surface keratin, large intrinsic and feeder vessels, involving more than half of the corneal surface with invasion into deeper stroma (B). Schematic diagram showing cross-sectional view of the left eye of the patient and large tumour with deep stromal invasion of the cornea. Surgical plane of dissection is shown as a dotted line (C). Scanner view of the main limbal mass shows an invasive tumour involving conjunctival lamina propria and corneal stroma. The tumour is composed of nests and cords of tumour cells that invaded the conjunctival lamina propria and corneal stroma (Haematoxylin-eosin, X 20) (D). Microphotograph shows the tumour cells with marked dysplasia and prominent mitotic figures (Haematoxylin-eosin, X400) (E). Microphotograph shows the tumour invading the deeper corneal stroma but not reaching up to the Descemet’s (Haematoxylin-eosin, X 100) (F). Slit lamp photograph of the left eye under diffuse illumination shows the scleral corneal graft twelve months after treatment. The corneal graft shows mild stromal haze.

Figure 3.

(A) Slit lamp picture of the right eye under diffuse illumination shows scleral thinning and perforation from 7’0 clock to 11’0 clock on the temporal quadrant away from the limbus (B). Section of the eye after modified enucleation. Note the epibulbar nodular mass involving the peripheral cornea, limbus and sclera (C). Sections show squamous cell carcinoma with sheets of anaplastic tumour cells with focal keratinisation. (Haematoxylin-seosin X400) (D). Histopathology of the mass showing full thickness infiltration of sclera and involvement of choroidal tissue. There was focal retinal detachment and sub retinal exudates (Haematoxylin-eosin X100).

Aggressive variants of OSSN are less commonly seen but deserve mention. Mucoepidermoid carcinoma is seen in elderly individuals and has a propensity for intraocular and orbital invasion. Mucin producing cells give it a yellow cystic appearance. Spindle cell carcinoma is an aggressive variant with a tendency to metastasize. These are generally managed surgically with wider margins.

Diagnosis

The gold standard for the diagnosis of OSSN is the histopathological evaluation of the lesion after an incisional or excisional biopsy. However there are several occasions when the clinician may opt to do diagnostic tests to corroborate the clinical suspicion of OSSN. In 1980 Gelender reported that cytological features can be seen using a cytobrush to obtain a specimen for fixation and Papanicolaou staining.³⁵ Impression cytology on cellulose acetate paper as described by Nolan et al had a reasonably high predictability rate of 77% (55/71) in diagnosing moderate dysplasia to microinvasive carcinoma.³⁶ Diagnosis is based on the presence of the universal cytological criteria which included nuclear enlargement, hyperchromasia, irregular nuclear outline, coarse nuclear chromatin, and prominent nucleoli.³⁷ That impression cytology is less sensitive in diagnosing invasive sqaumous cell carcinoma has been often highlighted in the literature. Interestingly, Nolan et al noted that the presence of keratin, inflammatory cells, and fewer diagnostic cells on impression cytology should alert the clinician about possibility of the disease at the severe end of the spectrum.³⁶ It is advised that in cases of hyperkeratotic lesions with negative impression cytology, surgical incision biopsy be done to rule out OSSN.³⁶ Bio pore membrane used for impression cytology was reported to accurately correlate with histological diagnosis in 80% (20/25) by Tole et al. More significantly Bio pore device is easy and rapid to use in routine clinical practice compared to longer preparation and transport time in impression cytology with cellulose acetate paper.³⁷ Finally, clinicians need to keep in mind that despite its advantages in early diagnosis of OSSN impression cytology has its limitations in differentiating carcinoma-in-situ from infiltrating carcinoma.

Anterior segment optical coherence tomography (OCT) allows morphologic and even histological characteristics of the tissue to be examined in vivo. The recent introduction of anterior segment OCT has enabled the assessment of the conjunctiva and cornea with high axial resolution of tissue planes. With the help of technological advances from time-domain to spectral-domain and ultra-high resolution (UHR) OCT, axial resolutions of 2–3 microns allow an optical biopsy of ocular surface tissue. A recent report by Kieval et al evaluated UHR OCT as a non-invasive diagnostic tool in differentiating OSSN from pterygia in 34 eyes.³⁸ Apart from a good correlation between histopathology and UHR OCT findings in the OSSN group (17 eyes), a severely thickened hyper-reflective epithelium with an abrupt transition from abnormal to normal tissue were significant findings suggestive of OSSN. Significant omissions by UHR OCT which were picked on histopathology included poor delineation of the cleavage plane owing to shadow artefacts in a few lesions and a false-negative result in one case of muco-epidermoid SCC.³⁸ The authors of this study also derived a cut-off thickness of 142 microns as differentiating OSSN from pterygia with 94% sensitivity and 100% specificity. Superficial cytological analysis enabled by confocal microscopy is a simple, safe and effective tool for early diagnosis of OSSN.³⁸ Xu et al evaluated the efficacy of in-vivo confocal microscopy in diagnosing OSSN type in addition to performing anterior segment OCT and confirmation with histopathology in five cases.³⁹ Cellular anisocytosis, anisonucleosis and altered nuclear-cytoplasmic ratio were seen on confocal microscopy in three cases diagnosed as CIN. In addition nests formed by isolated keratinised mitotic cells extending beyond the basement membrane helped diagnose invasive SCC.³⁹

Epigenetics

One of the challenges in OSSN is to predict the recurrences and the invasiveness of the lesion. Similar to cervical carcinoma, our group made an attempt to see if epigenetic markers could differentiate the intra-epithelial vs. invasive tumours. In a pilot study of five cases, we noted that hypomethylation of the promoter region of DML epigenetic changes could possible differentiate the intraepithelial lesion from invasive lesion. This however warrants confirmation by a larger study.⁴⁰

Histopathology

Complete pictorial documentation of the ocular surface with the lesion should be made on the Whatman’s filter paper No. 16, with a mention of the laterality and position of the lesion. Excised lesion, mucosal surface facing up, should be placed on the filter paper with diagram with or without separate margins (Fig. 4A). Excised lesion mounted on the filter paper in the operating room should be allowed to dry adequately (approx. 2–3 min) on the filter paper which causes adhesion of the tissue on the filter paper. This tissue on the filter paper is then submitted in 10% buffered formal saline to histopathology laboratory. Margins when not sent separately by the surgeon should be submitted by the grossing pathologist as superior/inferior/medial and/or lateral in separate filter papers submitted in separate cassettes (Fig. 4B). Main lesion which is seen as a greyish white plaque, nodule or thickening should be excised into two and submitted for edge wise processing.

Figure 4.

(A) Excised ocular surface squamous lesion placed on the filter paper. Exact laterality and position of the lesion can be made out with this pictorial documentation (B). Margins from the excised tissue are placed on separate filter papers and submitted in different cassettes.

Microscopic examination of the excised lesion with safe margins shows an abrupt transition of the epithelial lesion from the adjacent uninvolved conjunctiva (Fig. 5). Lesion should be studied for architectural and cytological atypia. Margins and base of the lesion should also be studied for atypia. Abnormalities in a dysplastic lesion reflect the deranged cellular proliferation that is the abnormal/injured mitotic cells do not fully differentiate as they rise in the epithelium (disordered proliferation with incomplete/altered differentiation). Depending on the level and thickness of epithelial involvement varying grades of dysplasia are classified. When lower one third and lower two thirds of the epithelium shows abnormal transformation the lesion is termed as mild and moderate dysplasia respectively. When the abnormality involves more than 2/3rds of the epithelial thickness, however surface maturation is preserved, the lesion is termed as severe dysplasia. Involvement of full thickness of epithelium, however with retained integrity of epithelial basement membrane is termed as carcinoma in situ (Fig. 6A–D). As the lesion progresses from mild to severe dysplasia, the cells differentiate less and less, gradually losing their squamous features until eventually the full thickness of epithelium is made of undifferentiated/immature atypical cells which can even have basaloid like appearance. Cytologically the dysplastic cells show an increase in the size of nuclei, irregular nuclear membrane, hyperchromasia and fine to coarse chromatin. Degree of nuclear atypia, irrespective of level of epithelial thickness involved can also cause up gradation or down gradation of severity of dysplasia. Hyperkeratosis and dyskeratosis (single cell keratinisation) with the formation of squamous pearls is not a discrete feature of intra epithelial lesions. An increased mitosis with the presence of abnormal mitoses is observed, scattered throughout the lesion, depending on the thickness displaying abnormal maturation. Mild to moderate chronic lymphomononuclear cell infiltration may be observed and could cause inflammatory destruction of epithelial basement membrane. This should not be confused with invasive squamous cell carcinoma, especially in cases of carcinoma in situ.

Figure 5.

The conjunctival epithelium is markedly thickened with an abrupt transition noted between the involved conjunctiva and the adjacent normal appearing conjunctival epithelium (arrow marked). Base of the excision biopsy is free of tumour involvement (Periodic Acid Schiff’s stain, X200).

Figure 6.

(A) Moderate dysplasia-squamous intra epithelial lesion, abnormal transformation of epithelium involving more than 2/3rds of epithelial thickness, however the degree of cytological atypia is not sufficient to term this lesion as severe dysplasia (B). Severe dysplasia-there is almost full thickness replacement by abnormal epithelium, surface maturation is noted with keratinisation. Basement membrane is intact. (C). Carcinoma- in-situ- there is full thickness replacement by abnormal epithelium with marked nuclear pleomorphism. Basement membrane is intact, (arrow marked) (Haematoxylin-eosin, X 400) (D). Invasive Squamous cell carcinoma-invasion of stroma is seen as tumour nests and broad expansive tumour masses. Tumour is well differentiated with horn pearls and abundant keratinisation (Haematoxylin-eosin X 100).

Lesions associated with HPV would show cytopathic effect in some cells, resulting in the formation of a halo cell, known as the Koilocyte.⁴¹ Koilocytes, though not sensitive for diagnoses of HPV infection, are pathognomonic of HPV infection if strict diagnostic criteria are used to identify them. Koilocyte is a mature squamous cell with a characteristic halo surrounding the nucleus. Halo is distinct, surrounded by dense eosinophilic cytoplasm. Nucleus of the Koilocyte looks enlarged with wrinkled nuclear membrane, dense chromatin and inconspicuous nucleoli. There can be frequent binucleation, although there could be virus present in the nucleus, but there is neither intranuclear nor intracytoplasmic viral inclusion seen with light microscopy. The formation of koilocytes decreases with increasing severity of dysplasia, and they are rarely seen in invasive lesions.

Reactive, regenerative or reparative squamous epithelium (for example in response to trauma, inflammation or infection) may manifest atypical cytology. Excised margins of ocular surface lesion could also display reparative/inflammatory atypia. Such changes should be studied with caution and distinguished from CIN.

In a reparative/regenerative/inflammatory lesion, a disparity is seen between the abnormal appearing, pleomorphic nuclei with prominent nucleoli versus the preserved orderliness of the cells with bland and pale chromatin. Normal mitotic figures can be seen, however there would be absence of abnormal mitotic figures. Clinical history is also important in such cases.

Conjunctival intraepithelial neoplasia can progress to invasive squamous cell carcinoma with destruction of the epithelial basement membrane and extension into the underlying stromal tissue. Invading tumour is seen as cords, strands and clusters of malignant cells in the stroma (infiltrative pattern), but can also be seen as broad pushing fronds (expansive pattern). Dense collagenous scleral tissue in cases of conjunctival OSSN and Bowman’s membrane in corneal OSSN (Fig. 7) attempt to limit the infiltration.⁴² As the cells become infiltrative they accumulate dense, acidophilic cytoplasm with cytoplasmic accumulation of actin and myosin which like keratin filaments appear acidophilic. These serve as machinery for invasion of the cell (Apparent keratinisation). They also redifferentiate from the undifferentiated stage of carcinoma in situ to larger differentiated cells with a low N: C ratio. Individual squamous cells in an invasive lesion (cytological features) could be large polygonal, small to spindle in shape. The more the degree of maturation, the dense, eosinophilic and copious is the cytoplasm, the pyknotic is the nucleus. There could be single cell keratinisation or formation of squamous pearl. Differentiation of squamous cell carcinoma is in terms of degree of keratinisation termed as well-differentiated, moderately differentiated and poorly differentiated carcinoma. Keratinisation is more in well and moderately differentiated tumours, and these tumours cytologically display less nuclear pleomorphism. Poorly differentiated tumours have predominance of immature cells with higher nuclear-cytoplasmic ratio, more pleomorphic nuclei, increased typical and atypical mitoses and minimal keratinisation.

Figure 7.

(A) Corneal OSSN-slit lamp photograph under diffuse illumination shows minimally elevated lesion involving ¾ of the cornea-scleral limbus. The corneal surface is covered by a greyish membrane with abnormal vessels (B). Atypical epithelium of the cornea, infiltration of corneal stroma is limited by intact bowmans membrane (arrow marked). An inflamed fibrovascular membrane is seen above the corneal stroma (Haematoxylin-eosin X 400).

Invasive squamous cell carcinoma, clinically and histologically needs differentiation form pseudoepitheliomatous hyperplasia. Histologically pseudoepitheliomatous hyperplasia resembles well differentiated or moderately differentiated carcinoma with irregular invasion, horn pearl formation and numerous mitotic figures. Disparity between architectural and cytological abnormality is noted with minimal or absent nuclear hyperchromasia, hyperplasia and pleomorphism, which helps in differentiating from a true malignant process^43,44 (Fig. 8).

Figure 8.

Pseudo epitheliomatous hyperplasia-section shows an abnormally thickened epithelium with surface keratinisation and stromal invasion as epithelial cords displaying irregular, jagged edges. There is minimal presence of nuclear hyperplasia, hyperchromasia with absence of nuclear pleomorphism (Haematoxylin-eosin X 200).

Other ocular surface lesions which can clinically mimic OSSN such as actinic keratosis, pterygium, pinguecula and actinic granuloma can be easily distinguished from OSSN by studying the morphology. Our study reported three cases in young women which clinically presented as OSSN and histopathological evaluation of the excised tissue confirmed those lesions as actinic granuloma.⁴⁵ OSSN can uncommonly coexist with pterygium, ⁴⁶ therefore vigilant study of pterygium specimens should be executed to exclude dysplastic changes.

Clear cell change/hydropic change of the cytoplasm may be seen in squamous cell carcinoma which should be observed with caution as it could mimic an aggressive disease such as pagetoid extension in a sebaceous gland carcinoma, Mucoepidermoid carcinoma, or even metastatic renal cell carcinoma. Special stains, immunohistochemical work up with clinical history and radiological correlation may be warranted in difficult cases. Only two cases of Clear cell carcinoma, a very rare variant of squamous cell carcinoma have been reported in the literature.^47,48 Frequent local recurrences are known, however local or distant metastasis is not reported. Special stains for mucin, lipid and glycogen exclude Mucoepidermoid carcinoma, sebaceous gland carcinoma and metastatic renal cell carcinoma.⁴⁹

Variants of Invasive Squamous cell carcinoma include Mucoepidermoid carcinoma, Spindle cell carcinoma, papillary squamous cell carcinoma and Acantholytic/Adenoid squamous cell carcinoma.

Mucoepidermoid carcinoma (MEC) is a mucin–producing squamous cell carcinoma of conjunctiva and is a rare neoplasm, unlike the commonly encountered Mucoepidermoid carcinoma of salivary glands. In a large series of 287 cases of squamous cell carcinoma of conjunctiva published by Cervantes and associates in 2002, there was only one case of Mucoepidermoid carcinoma of conjunctiva⁵⁰ Only 21 cases of MEC have been reported in the English literature till 2006.⁵¹ A series of 71 cases of OSSN published by Alves and associates in 2011 included only one case of MEC. These tumours have the appearance of squamous cell carcinoma with variable number of mucin producing cells. Intermediate cells are also noted. Mucin producing cells are large, pale, stain positively with periodic acid Schiff’s stain and with mucicarmine stain which is resistant to hyaluronidase but sensitive to sialidase (epithelial sialo-mucin). MEC is a locally aggressive neoplasm which can have similar clinical presentation like classic squamous cell carcinoma, histopathology plays a key role in diagnosis of this rare and locally aggressive disease. MEC has a high rate of recurrence, ranging from 84.6% to 100% in different published data,⁵² however local and distant metastases are uncommon. Regional lymph node metastasis in MEC has been reported in a single case by Hwang et al and associates.⁵²

Spindle cell carcinoma is another rare and locally more aggressive variant of squamous cell carcinoma of conjunctiva with only few case reports in the literature. In a clinical series of 1643 conjunctival tumours seen in an ocular oncology service, Shields and associates had 219 patients with epithelial tumours, none of which were spindle cell carcinoma.⁵³ Cervantes and associates in their large series of 287 cases of squamous cell carcinoma have reported only two cases of spindle cell squamous cell carcinoma.⁵⁰ Spindle cell carcinoma/Sarcomatoid carcinoma is characterised by a biphasic pattern of squamous cell carcinoma with a generally much larger component of malignant spindle cells (divergent differentiation) reminiscent of a sarcoma. The squamous component may be scanty or even inapparent on light microscopy (Fig. 9A–D). In the latter circumstance, immunohistochemical and electron microscopic evidence of squamous differentiation is required for diagnosis. By immunohistochemistry, sarcomatous component is characteristically vimentin positive, may be positive for actin and desmin, and often shows only limited or absent staining for traditional epithelial markers such as cytokeratin or epithelial membrane antigen. In such challenging cases, immunostaining for p63 is another useful marker for the identification of epithelial differentiation.⁵⁴ Spindle cell carcinoma on ultrastructural studies displays features of epithelial differentiation in spindle cells, such as desmosomes and tonofilaments.⁵⁵ Spindle cell carcinoma is much more likely to extend through the sclera and cornea into the interior of the globe.⁵⁶ Differential diagnoses of spindle cell carcinoma call for exclusion of malignant spindle cell lesions such as fibrosarcoma, leiomyosarcoma, malignant fibrous histiocytoma and amelanotic malignant melanoma. However in conjunctiva sarcomas are even rarer and spindle cell carcinoma is still more likely.

Figure 9.

(A) Extended enucleation in a massive ocular surface tumour-extended enucleation specimen with a massive ocular surface tumour, enucleated eyeball is sectioned into one central and two peripheral calottes (B). Conjunctival margins are sent separately mounted on filter paper (C). Low magnification photomicrograph of the lesion altered scleral collagen, angle structures (arrow marked) and iris tissue. Densely cohesive spindle cell tumour is noted involving anterior one third of scleral fibres (Haematoxylin-eosin X100) (D). High magnification photomicrograph shows malignant spindle cells. Atypical mitotic figures are noted (arrow marked) (Haematoxylin-eosin, X 400).

Papillary squamous cell carcinoma is an exophytic squamous cell carcinoma with a papillary configuration composed of tumour surrounding fibrovascular core (Fig. 10A–C). There are controversial reports regarding prognosis of head and neck and they are generally believed to have a better prognosis.⁵⁷ In our experience we have come across a case of papillary SCC squamous cell carcinoma with a massive ocular surface tumour (Fig. 10A–C). CT scan showed evidence of orbital invasion along the lateral rectus muscle.

Figure 10.

(A) Papillary variant of squamous cell carcinoma. Slit lamp photograph under diffuse illumination shows a massive papillary variant of invasive squamous carcinoma invading into the orbit along the lateral rectus muscle (B). Scanner photomicrograph showing an exophytic squamous cell lesion with papillary pattern (Haematoxylin-eosin X 40) (C). Low magnification photomicrograph shows tumour cells with marked cytological atypia surrounding central fibrovascular core. (Haematoxylin-eosin X 100).

Adenoid squamous cell carcinoma or Acantholytic squamous cell carcinoma is another rare variant of SCC. Mauriello and associates had conducted a review of 12 years of cases of SCC on the file at the AFIP and these revealed only 14 cases of Adenoid Squamous cell carcinoma.⁵⁸ This neoplasm is composed of SCC, but characterised by acantholysis of tumour cells, leading to the formation of pseudolumina which imparts a false glandular like appearance or sometimes even confused with angiosarcoma. Pseudo lumina contain acantholytic cells, dyskeratotic cells and debris material. Histochemical stains, immune profiling and electron microscopy help to exclude adeno squamous carcinoma and adenoid cystic carcinoma. Special stains for mucin would be negative, tumour cells would be positive for cytokeratin and EMA. They would express hemidesmosomes and attached tonofilaments. This tumour is locally aggressive, can recur and even metastasise and should, therefore, be histopathologically differentiated from the less aggressive conventional squamous cell carcinoma. In the largest series of 14 cases published in 1997, 5/14 cases recurred and 1/14 cases showed brain metastasis, despite aggressive therapy.^58,59 Regular follow up of this variant of SCC is recommended.

Management

Management modalities in OSSN range from complete excision in well delineated tumours to chemotherapy in diffuse unresectable lesions.

Surgery

Complete excision with adequate margins is the treatment-of-choice for most localised lesions. Alcohol assisted kerato-epitheliectomy and lamellar sclerokeratoconjunctivectomy are indicated for the corneal and infiltrative components respectively. There seems to be a consensus on the beneficial role of double-freeze cryotherapy to the resected margins and base of the lesion amongst most clinicians. Complete extirpation is confirmed by histopathological examination of conjunctival margins which are sent separately. Negative surgical margin is the most important predictor for tumour recurrence. Recurrence rates are reported to range from 5% to 33% after negative margins to as high as 56% in those where margins were found to be positive.⁵⁹ The residual defect after excision may be covered by amniotic membrane transplant or buccal mucous membrane graft.⁶ Rare reports exist in the literature describing massive corneal OSSN infiltrating deeper stromal layers where globe and vision were salvaged by penetrating sclerokeratoplasty (Fig. 2A–F).⁶⁰ The availability of noninvasive techniques like UHR, OCT and ultrasound biomicroscopy has made it possible to evaluate the extent of corneoscleral invasion in OSSN.^38,39

In cases with suspected intraocular invasion by OSSN there may be cells in the anterior chamber simulating “uveitis” associated with raised intraocular pressure. These cases need local eyewall resection or modified enucleation to extirpate the tumour. Treatment in OSSN with orbital invasion may range from local resection and/or irradiation to eyelid sparing orbital exenteration.

Chemotherapy

Medical alternatives in the form of topical applications 5 Fluorouracil (5FU) and mitomycin C (MMC) have been extensively reported in the literature. Primary treatment with chemotherapeutic agents for OSSN is largely limited to localised OSSN. Both MMC and 5FU have also been used as adjuvant therapy for recurrent lesions. Extensive OSSN with a mean diameter of 40 mm have shown 57% reduction in tumour base after chemoreduction with MMC. However the cumulative and delayed toxicity of MMC on the ocular surface make these agents less preferred in the treatment of OSSN.^61,62

Immunotherapy

In 1994 Maskin was the first to report to report the use of topical interferon (IFNα2b) in a multi-focal limbal OSSN.⁶³ Karp reported complete response in five cases of OSSN measuring <8 mm with IFNα2b.⁶⁴ Over the past decade several authors have reported on the beneficial effects of IFNα2b in the treatment of OSSN. Owing to a better toxicity profile, IFNα2b currently seems to be the treatment of choice for wider and extensive OSSN involving >4 clock hours of the limbus.⁶⁰ Overall topical IFNα2b is preferred for OSSN which are relatively thinner for complete tumour control (immunotherapy) while combination therapy with topical and injection IFNα2b is reserved for partial reduction of thicker and extensive OSSN (immunoreduction). Occasional flu-like symptoms and ocular surface irritation are sometimes seen in patients after treatment with IFNα2b. Cost of IFNα2b remains to be a major disadvantage of IFNα2b therapy especially in the developing world.³⁴

Prognosis

The overall prognosis in OSSN is good. Modern treatment strategies are effective with local recurrence rates reported to be 5% and regional lymph node metastasis at <2%. Aggressive variants like muco-epidermoid and spindle cell carcinoma and OSSN in immunocompromised patients have a worse prognosis.

Conclusion

Histopathology is the gold standard in the diagnoses of various grades and types of OSSN, and gives a fair understanding of disease prognosis. Conjunctival impression cytology could be a diagnostic aid or to confirm flat corneal–limbal lesions.

Future studies are needed to understand the pathogenesis of OSSN and the role of various carcinogens specifically HPV and HIV in varied manifestations of the disease process and their role in prognosis of disease. Further work is required in the re diagnoses of OSSN with the aid of UHR, OCT and confocal microscopy. While complete excision with cryotherapy gives excellent results with localised lesions, newer modalities like immunotherapy hold a promise in the larger, unresectable and recurrent lesions.

Authors’ contributions

Dr. RM designed the manuscript, wrote the etio-pathogenesis, pathological diagnoses, and compiled the pictures. Dr. SR wrote the clinical diagnoses, management and prognosis, and contributed the clinical pictures. Dr. GKV designed the manuscript, contributed in writing the etio-pathogenesis, pathological diagnosis and contributed to the histopathology pictures and approved the final picture composites.

Conflict of interest

The authors declared that there is no conflict of interest.