Conjunctival Squamous Cell Carcinoma: A Clinical Review of Diagnostic Features, Genetics, Current Management and an Update on Targeted and Immunotherapies

1. Introduction

Invasive conjunctival squamous cell carcinoma (CSCC) has a worldwide incidence rate of 0.02 to 3.5 per 100,000 and showing increasing incidences in the last decades [1,2,3,4,5]. It typically presents at 60-70 years of age occurring more commonly in fair-skinned males, of increasing age, and following significant sunlight exposure; but presents at 30-40 year-old females in the tropics [2,4,5,6,7]. It has a high recurrence rate and local spread is common. Distant metastasis occur less commonly [8]. Invasive CSSC usually develops from conjunctival squamous intraepithelial neoplasia (CSIN) and in-situ squamous carcinoma. CSIN (also clinically known as ocular surface squamous neoplasia [OSSN]) is a preinvasive lesion and encompasses a spectrum of intraepithelial squamous dysplasia, from mild to severe dysplasia, and can progress to in-situ squamous carcinoma [8,9].

Invasive CSCC usually involves the interpalpebral conjunctiva and limbus (especially the nasal limbus) and can extend onto the cornea. It can also less frequently occur in the caruncle, tarsal, or forniceal conjunctiva. Invasive CSCC presents as a well-demarcated pinkish-grey nodule with keratinisation or ulceration [10]. In the African and Asian population, it often presents with brown surface pigmentation and feeder vessels [11]. It originates from conjunctival epithelium, shows squamous differentiation, and breaches the basement membrane to invade the underlying substantia propria/stroma [6,12,13].

Ultraviolet (UV) radiation, impaired immune surveillance, and immunosuppression (e.g. post-transplant or HIV infection) are well known risk factors. Opportunistic oncogenic viruses such as human immunodeficiency virus, human papilloma virus and Epstein Barr Virus, are linked with immunosuppression [2,4,13,14,15,16,17]. Mutations in TP53, TERT, EGFR, titin, HER2 and DNA repair genes and overexpression of matrix metalloproteinases have been identified in invasive CSCC [13,18,19,20,21,22,23,24,25,26,27,28,29].

The treatment for invasive CSCC varies between considerably between centres but mainly includes surgical excision and/or adjuvant cryotherapy, topical chemotherapy, brachytherapy, proton beam radiotherapy or photon external beam radiation and, in advanced cases with orbital invasion, orbital exenteration [1,6,30,31,32]. Invasive CSCC has a high rate of local recurrence (approx. 10–40%); incidence of up to 24% in regional lymph node metastases and distant metastases of ~6-18% [8,33,34,35,36].

Despite recent successes with targeted and immunotherapies in SCCs elsewhere, data on invasive CSCC treated with similar therapies (EGFR inhibitors, anti-VEGF, anti-PDL1) are promising but limited, often stemming from a single patient or small case series with inoperable or advanced disease prior to surgery [37,38,39,40,41,42,43,44,45,46,47]

This review aims to provide a comprehensive clinical overview of the current understanding of CSCC, its epidemiology, pathogenesis, clinical presentation, diagnosis, treatments, and explore the recent advances in novel biological therapies and future management.

2. Epidemiology

In-situ conjunctival squamous carcinoma is the most common preinvasive ocular surface malignancy and if untreated can progress to invasive CSCC. Its incidence varies geographically: in the UK, it is estimated at 0.4 cases/million/year, whereas both Australia and North America show incidences of 19-35 cases/million/year [48,49]. The mean age-standardised incidence rate worldwide is 0.18 and 0.08 cases/year/100 000 among males and females, respectively. However, the highest age-standardised incidence rate reported from Zimbabwe is 3.4 and 3.0 cases/year/100 000 population for males and females, respectively [2].

Invasive CSCC, although rare compared to SCCs of other sites, is a common ocular surface malignancy. It is the most prevalent ocular surface carcinoma representing approximately 4% to 29% of tumours found in the ocular region [50] [51]. There has been a disconcerting increase in incidence in the last decades with markedly advanced cases presenting following the COVID pandemic. Worldwide the incidence is 2–35 per million [1,2,3,4] occurring more commonly in fair-skinned males (with a ratio of 1.83:1 compared with females), of increasing age, and following significant sunlight exposure [4,5,7] ]. Ultraviolet (UV) radiation is the strongest environmental etiological factor with incidence rate decreasing by 49% per each ten degrees increase in latitude [3].

In the African continent, the disease mainly affects women, at a younger age, and is strongly associated with human immunodeficiency virus (HIV) infection and variably with human papilloma virus (HPV) infection [2,17,52]. The combination of low latitude and high prevalence of HIV and HPV-infections most likely explains the higher incidence of invasive CSCC observed in Africa. A higher prevalence of CSCC is also seen in patients with impaired immune surveillance, certain autoimmune conditions (atopy, ocular cicatricial pemphigoid and xeroderma pigmentosum) and/or immunosuppression with susceptibility to oncogenic viruses HPV and Epstein Barr Virus (EBV) [2,13,14,15,16]. Iatrogenic immunosuppression is also a risk factor.

3. Aetiology and Pathogenesis

The aetiology of SCC is classically understood to be triggered by UV radiation exposure and immunosuppression (due to HIV-infection or post organ transplantation) [4]. Mutations in TP53, TERT, EGFR, titin, and DNA repair genes and overexpression of matrix metalloproteinases, Human Epidermal growth factor Receptor 2 (HER2) and programmed death ligand1 (PD-L1) have been identified in invasive CSCC [18,19,20,21,22,23,24,25,26,27,28,29]. However, none of these are pathognomonic, and have been reported in other conjunctival epithelial neoplasms e.g., pterygia and pingueculae. One study revealed similar UV-mediated driver mutations in both preinvasive and invasive cutaneous SCC and CSCC. TP53 mutations were the most frequent, followed by CDKN2A and PIK3CA [28].

The vulnerability of limbal stem cells to the oncogenic effects of UV radiation and certain viruses is postulated to give rise to most invasive CSCC [13]. UVB exposure may induce preinvasive disease by increase in pyrimidine dimers which damage DNA and disrupt its repair pathways. Proliferation of unrepaired DNA by continued excess UVB exposure or immunosuppression, can manifest CSIN/in-situ carcinoma (preinvasive disease) of the conjunctiva [21,53]. UV exposure also activates c-Jun N-terminal kinase 1 (JNK1), which plays a role in tumour development through phosphorylation and oncogenic signal transduction pathways [54].

UVB radiation has also been implicated in upregulated expression of matrix metalloproteinases (MMPs), particularly MMP-9 and -11) and tissue inhibitors of metalloproteinases (TIMPs) such as TIMP-2 and -3, which promote the tumourigensis via antiapoptotic, proliferative, invasive and metastatic properties [21].

UV-mediated driver mutations in telomerase reverse transcriptase (TERT) promoter and TP53 have been reported in up to 48% of in-situ and invasive CSCC cases. An overexpression of telomerase in the conjunctival epithelium affects telomere length with proliferative and anti-apoptotic functions [20,55]. TP53 mutations disrupt pyrimidine dimer repair pathways [53]. Furthermore, the role of stratifin acting as a p53 mediated inhibitor is also implicated in UV induced p53 pathogenesis [56].

Titin and EGFR mutations have also been reported in both in situ and invasive CSCC. Structural disturbances in nuclear proteins and EGFR translocation from the membrane into the cytoplasm, respectively, are thought to facilitate oncogenic activity [18]. The cytoplasmic staining of EGFR compared with membranous staining has been shown to be an inverse prognostic marker with increased number of orbital exenteration cases [19].

Overexpression of HER2 has been reported in higher grades of CSIN and invasive CSCC suggesting its main role in tumour progression rather than early oncogenic initiation. Anti-HER2 therapy may therefore be a considered in for select patients with advanced disease when conventional topical or surgical treatments are contraindicated [24]. However, there are no clinical trials investigating the role of anti-HER2 treatment on invasive CSCC.

Increased vascular endothelial growth factor (VEGF) and alpha-B crystallin protein expression has also been shown in invasive CSCC. VEGF is an established potent angiogenic factor and alpha-B crystallin is known to promote angiogenesis as a molecular chaperone of VEGF. Furthermore, treatment of invasive CSCC with topical mitomycin C reduced the expression of both [57].

PD-L1 is expressed in around 47% of CSSC, with higher expression levels correlating with invasive and advanced stage (≥T3) tumours and with increased densities of tumour-infiltrating lymphocytes [58,59].

These results are encouraging and may potentially mirror some success in a subset of invasive CSCC as established targeted therapies and PDL-1 targeted treatment blockades used in head and neck and cutaneous SCC.

Since the 1990s, increased incidence rate of invasive CSCC in Africa has been strongly associated with the high prevalence of HIV primarily as well as EBV and HPV (particularly HPV-16 & -18 subtypes, and less commonly HPV-5 & -8) in HIV-infected patients. HIV has an immunosuppressive effect on the limbal stem cells making them vulnerable to oncogenic viruses leading to aggressive invasive CSCC at a younger age with worse prognosis [60]. HPV driven invasive CSCC in immunocompetent patients has been controversial but a recent meta-analysis concluded an eight-fold increase in development of CSIN or invasive CSCC in HPV-infected patients in comparison to healthy control subjects [61]. HPV-16 with early gene expression (active HPV mRNA transcripts confirmed via RNAscope) was shown to cause in-situ carcinoma in a subset of patients who had atopy [62].Another study demonstrated that patients with HPV-positive tumours were significantly younger and had a higher recurrence rate compared to patients with HPV-negative tumours. The HPV-positive tumours also showed a non-keratinising morphology [63].

Other risk factors include: cigarette smoking, chronic inflammation and vitamin A-deficiency. Also there is an increased risk of CSIN/invasive CSCC with autoimmune ocular surface disorders such as ocular cicatricial pemphigoid/mucous membrane pemphigoid, and genetic conditions including and xeroderma pigmentosum [2,14,15].

4. Clinical Presentation and Assessment

CSIN and invasive CSCC most commonly occur in the interpalpebral conjunctiva and limbus (usually nasal limbus). Invasive CSCC frequently invades into the cornea. The caruncle, tarsal, or forniceal conjunctiva are less frequently involved. Advanced or recurrent invasive CSCC may extend to the tarsal and forniceal conjunctiva. The latter locations are also seen in immunosuppressed/immunocompromised patients and in those with atopy associated squamous carcinoma [62].

Most CSIN lesions appear as unilateral, gelatinous and minimally elevated but can also be papilliform or a leukoplakic plaque (due to keratinisation). However, intraepithelial lesions can be clinically indistinguishable from the invasive disease and hence histopathological diagnosis remains the ‘gold’ standard. Invasive CSCC usually presents as a unilateral, elevated, immobile, well-demarcated pearly pink-to-grey nodule, which can be ulcerated, leukoplakic, gelatinous, or papilliform. They often have feeder blood vessels and intrinsic vasculature (Figure 1). Pigmentation is variable, depending on the Fitzpatrick skin type. Patients may present on noticing a mass with/without pigmentation on their eye but can also have significant visual morbidities, such as irritation/burning with redness and reflex tearing, dry eye, pain, vision disturbance, double vision or even vision loss [35,36,64,65,66,67].

Differential diagnoses of invasive CSCC, include: sebaceous cell carcinoma, naevus, melanoma, lymphoma, or eyelid basal cell or squamous cell carcinoma invading the conjunctiva. CSIN can develop in pinguecula, pterygium, viral squamous papillomas and other inflammatory conjunctival conditions. Diagnosis is confirmed on histopathological assessment. Histological assessment in a specialist centre regularly reporting ophthalmic specimens is recommended for accurate diagnosis and grading. [8,50,68]

Clinical examination involves slit lamp biomicroscopy and regular colour photograph-documentation of the anterior segment (including with eversion of eyelids). Toluidine blue (0.05%) vital stain has been shown to be a good screening tool for OSSN/CSIN and can assist in identifying the tumour borders, but is not in widespread use due to high false positive rates [69]. Anterior segment optical coherence tomography (AS-OCT) has been used to distinguish OSSN from other conjunctival lesions. In OSSN/CSIN, the AS-OCT features are those of a hyperreflective lesion that has thickened epithelium, and an abrupt transition between normal and abnormal tissue [70]. However, squamous metaplasia is indistinguishable from OSSN on AS-OCT, highlighting the need for biopsy and histopathological analysis of lesions especially where there is overlap [71]. Other modalities such as ultrasound biomicroscopy and confocal microscopy can be helpful in clinical assessment and to look for intraocular invasion. Magnetic resonance imaging is required to assess orbital invasion. Diffusion and perfusion-weighted MR imaging can help in differentiating invasive CSCC from other eyelid masses [70,72,73,74].

5. Histomorphological Features

Macroscopically CSIN lesions look as flat, cream/white epithelial thickenings/plaques. They may also be mildly raised and papillomatous. Invasive CSCC are usually gelatinous grey/white nodular or papilliform masses without/with surface keratinisation, respectively, or ulcerated/erythematous elevated lesions. Lesions in the tropics can show pigmentation and a lot more surface keratinisation [11,35,36]. Depending on the specimen type, invasion of the cornea and other ocular structures and/or orbital soft tissue can be seen on macroscopic examination.

Microscopically CSIN lesions show a range of epithelial dysplasia (cytological atypia, architectural atypia and lack of maturation), which is graded mild (confined to lower third of epithelium), moderate (involves up to middle third), and severe (extends to upper third but surface differentiation preserved). In-situ squamous carcinoma shows full-thickness epithelial dysplasia. CSIN, including in-situ carcinoma, do not breach the epithelial basement membrane (Figure 2). At low magnification, an abrupt demarcation with the background normal conjunctival epithelium, and with goblet cell loss can be appreciated. At higher magnification, cytological atypia comprises nuclear enlargement and pleomorphism with increased nuclear-to-cytoplasmic ratio, hyperchromatic chromatin and prominent nucleoli. Abnormal mitotic figures are seen at varying levels of the epithelium. Dyskeratosis and apoptotic cells may also be present. Architecturally, the epithelium shows abnormal stratification with loss of normal polarity, and surface keratosis or parakeratosis. The underlying substantia propria/stroma often shows elastotic degeneration from chronic sun exposure. UV-associated cases can resemble skin actinic keratoses [35,36,75,76,77,78,79,80,81,82,83]. High-risk HPV-associated cases usually lack surface keratinisation or parakeratosis, show full thickness epithelial dysplasia and have a basaloid appearance [62,63].

Invasive CSCC usually arises from conjunctival in-situ squamous carcinoma but breaches the basement membrane invading the underlying stroma (Figure 2). The invasive carcinoma can be infiltrative or exhibit pushing borders and show stromal desmoplasia. Perineural and lymphovascular invasion may be present. Histological classification of invasive CSCC includes: conventional (showing various degrees of keratinisation); basaloid (poorly differentiated with pleomorphic cells with scant basophilic cytoplasm); spindle cell (poorly differentiated with pleomorphic spindled cells); pigmented (CSCC with melanosis and melanophages); acantholytic (acantholytic and dyskeratotic cells with apoptotic debris); and with mucinous differentiation (intracytoplasmic and extracellular mucin but no true gland formation, overlying in situ component with mucinous differentiation). Immunohistochemistry is usually only required for diagnosis in poorly differentiated tumours. For intraepithelial lesions, p53 is expressed in the dysplastic cells. In poorly differentiated invasive carcinomas, CSCC is positive for broad-spectrum/pan and high molecular weight cytokeratins. Diffuse nuclear and cytoplasmic ‘block-like’ p16 positivity can be predictive of high-risk HPV infection but requires molecular techniques to confirm HPV DNA or RNA presence [35,36,62,63,65,69,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91].

In-situ conjunctival squamous carcinoma and invasive CSSC are staged by the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC) T (tumour size), N (nodal spread), and M (metastasis) eighth edition classification system, which has been validated for the risk of tumour recurrence, development of metastasis and survival [92,93].