Conjunctival combined deep penetrating naevus: a clinicopathological report

To the Editor,

Deep penetrating naevus (DPN) is a well-recognised subtype of melanocytic naevus in the skin. Cutaneous DPN is a dermal-based and deep-extending, often wedge-shaped, melanocytic proliferation with characteristic histological features including variable degree of often random cytological atypia and limited mitotic activity which at times can be confused with melanoma. Recently, the molecular pathway of neoplasia and genomic alterations in DPN have been elucidated, suggesting a step wise progression starting from a conventional melanocytic naevus, so it is not surprising that up to two-thirds of cutaneous DPNs histologically present as combined lesions with a component of conventional naevus. Deep penetrating naevi arising from the conjunctiva are probably as common as their cutaneous counterparts, however they have been rarely reported in the literature. Herein we report a case of combined DPN arising from the conjunctiva to raise awareness regarding the existence of this entity in this anatomical location and to prevent diagnostic confusion with its clinical and histological mimics, particularly conjunctival melanoma.

A 16-year-old female presented to an ophthalmology clinic and reported some recent change (mild enlargement and increased darkening), in a right eye caruncle pigmented lesion, which had been present for 5 years. The lesion showed uniform dark pigmentation on examination with regular borders and measured 3×3 mm clinically. Her visual acuity was intact and ophthalmic examination was otherwise unremarkable. The lesion was surgically removed and processed for histological examination.

Microscopic examination demonstrated a predominantly subepithelial melanocytic proliferation with a biphasic appearance (Fig. 1A). Centrally and deeply there was a nodular expansion of clusters and nests of large epithelioid melanocytes with round-to-oval nuclei, small nucleoli and moderately abundant amphophilic cytoplasm with relatively diffuse but variable melanin pigmentation (Fig. 1B). The epithelioid melanocytes were admixed with irregular aggregates of melanophages containing intense intracytoplasmic melanin pigment. While the epithelioid melanocytic component occupied the majority of the lesion, there was also a component of melanocytic cells showing rounded to ovoid appearance with bland nuclei and scant to moderate amphophilic to eosinophilic cytoplasm resembling conventional naevus cells, largely present at the superficial aspect of the lesion (Fig. 1C). There was no significant cytological atypia and no mitotic activity. There was some extent of concurrent reactive epithelial proliferation with slight expansion of the pre-existing conjunctival crypts.

By immunohistochemistry the component of large epithelioid melanocytes demonstrated diffuse nuclear, cytoplasmic, and membranous staining with β-catenin (Fig. 1D) and diffuse nuclear staining with Cyclin-D1 (Fig. 2A). However, the superficial conventional naevus component showed only membranous staining with β-catenin and was largely negative for Cyclin-D1. BRAF-VE1 immunohistochemistry was diffusely positive in both epithelioid and naevoid components (Fig. 2B).

Based on the overall immunohistochemical and morphological features a diagnosis of combined deep penetrating naevus of the conjunctiva was established.

Melanocytic naevi are the most common type of the broad range of conjunctival tumours, making up approximately 23% of such lesions. DPN is a well described subtype of melanocytic naevus in the skin, however, it appears to be largely under-diagnosed and under-reported in the conjunctiva. Histologically, the lesion is frequently characterised by a subepithelial melanocytic proliferation, often with deep stromal extension, with or without a concurrent intraepithelial component. Conventional forms of deep penetrating naevus are characteristically described as clusters of melanocytes showing slightly larger round-to-oval nuclei with inconspicuous nucleoli and moderately abundant amphophilic cytoplasm with variable melanin pigmentation. Often there is an admixture of intensely pigmented melanophages which can manifest as irregular pigmentation clinically. Some degree of cytologic atypia with random variability in nuclear size and shape and low mitotic activity (usually less than 2/mm²) can be seen. In addition, DPN can commonly wrap around pre-existing stromal structures such as nerve bundles and vessels with lymphatic space invasion have been reported in otherwise benign deep penetrating naevi. A component of a pre-existing conventional naevus can be seen adjacent to DPN which has been previously recognised as ‘clonal’ and more recently as ‘combined’ naevus phenomenon, which can create both border and colour irregularities clinically.

The molecular profile of DPN has been recently described. It has been demonstrated that DPN usually starts as a conventional naevus as a result of activating somatic mutations in mitogen-activated protein kinase (MAPK) pathway such as BRAF, NRAS and MAP2K mutations. However, subsequent additional mutations in wingless/integrated (WNT) pathway, including somatic CTNNB1 or APC mutations, is required for the development of a morphologically distinct clone of larger epithelioid cells with characteristic cytomorphological features, often combined with pre-existing conventional naevus cells. Therefore, it can be speculated that DPN is a partially transformed lesion from a conventional naevus, however it is still an indolent tumour and prognostically far from melanoma. DPN can be considered as an intermediate lesion which has a propensity to acquire further genomic alterations affecting genes involved in cell cycle regulation and replicative senescence such as CDKN2A and TERT and evolve to melanoma. Such genomic alterations are often induced by ultraviolet radiation due to sun exposure; therefore, the presence of sun induced stromal damage manifesting as solar elastosis is a finding commonly seen in cutaneous and conjunctival melanomas. In addition, DPN-like melanomas frequently show a more significant degree of cytomorphological atypia and increased mitotic activity. At the cytogenomic level, they are often associated with multiple gains and losses of chromosomes which can be detected by array comparative genomic hybridisation or fluorescence in situ hybridisation.

The largest series of conjunctival deep penetrating naevi has described 34 lesions in 361 conjunctival naevi examined histopathologically (9.4%). This cohort of patients showed variable ages ranging from 7 to 51 years and clinically presented as darkly pigmented lesions often with some increase in size or pigmentation. There was a slight female predominance and the lesions were mainly located on the bulbar conjunctiva, followed by caruncle, semilunar fold and eyelid margin. Not surprisingly, a significant proportion of lesions (47%) were initially reported as common conjunctival naevus and a minority of cases (6%) misdiagnosed as melanoma histologically. The majority of the lesions were subepithelial (61%) followed by compound lesions (35%). A concurrent inclusion cyst was identified in 56% of cases. Mitotic activity was very low with most lesions not showing any mitosis (97%). By immunohistochemistry all lesions stained showed nuclear and cytoplasmic staining with β-catenin and nuclear staining with Cyclin-D1 in the DPN component. BRAF-VE1 was diffusely positive in all lesions. Ki-67 proliferation index was very low.

Clinically, it is important to have an awareness of the differential diagnoses for conjunctival pigmented lesions. Most patients present after identifying a spot on the eye, with only a small number experiencing pain or inflammation. Naevi typically appear as well-defined, unilateral, brown/yellow lesions at the interpalpebral limbus; they sometimes include cysts. Typically, patients undergo annual observation under slit-lamp to observe for change. Indications for removal of conjunctival naevi can be seen in Table 1. Malignant transformation in conjunctival naevi including DPN is extremely rare, estimated at less than 1% of cases. Clinical indicators of possible malignant transformation include sudden increase in size, changes in colour, increased thickness and more prominent feeder vessels. Conjunctival melanomas typically arise from primary acquired melanosis (PAM), but can also arise de novo or from pre-existing naevi. They typically appear in light-skinned, older adults and can present in a challengingly wide range of clinical appearances and locations. Conjunctival melanoma can recur locally and has the potential to metastasise (most commonly to the brain, lung, liver and facial lymph nodes).

At times, the inherent clinical and histological atypia can create a diagnostic challenge in separating DPN from more aggressive lesions including atypical DPN, primary acquired melanosis (PAM), conjunctival melanoma and pigmented ocular surface squamous neoplasia. However, a comprehensive histological, immunohistochemical and, if required, molecular assessment should readily distinguish DPN from its mimics. Another histological differential diagnosis is conventional or cellular blue naevus. While the constituent melanocytes in blue naevi are often spindle to dendritic in shape, sometimes they can show an epithelioid appearance, closely mimicking DPN. Albeit the molecular profile of blue naevus is different from DPN, but similar to uveal melanocytic neoplasms, including somatic mutations in G protein genes such as GNAQ and GNA11 with a small subset showing mutations in other genes including PLCB4 and CYCLTR2.

In summary, deep penetrating naevus is a subtype of melanocytic naevus which can occur but appears to be under-recognised in the conjunctiva. DPN can mimic more significant melanocytic and non-melanocytic lesions clinically; however, a thorough clinicopathological assessment should adequately distinguish DPN from its mimics. Ophthalmologists and pathologists should be aware of this entity in conjunctiva to avoid misdiagnosis.