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We present the case of a 3-year-old girl with a recurrent melanocytic proliferation showing classical clinical and histological features of Spitz naevus/atypical Spitz tumour with evidence of a BRAF c.1799T>A, p.V600E (V600E) mutation.
A 2-year-old girl presented to her dermatologist with a papule on the right forearm. The lesion had been present for 6 months, with enlargement over the prior 2 months, but was otherwise asymptomatic. The patient was otherwise well. A shave biopsy was performed and reported elsewhere as Spitz naevus extending to the deep and lateral margins. The lesion continued to grow over the following 15 months and excision was performed.
The initial shave biopsy specimen showed hyperkeratosis overlying moderately acanthotic epidermis. There was expansion of the dermis by a melanocytic proliferation arranged in nests and cords. In areas the proliferation abutted the epidermis, but there was no clear evidence of an intraepidermal component. The constituent cells had an epithelioid to spindled morphology with a moderate volume of eosinophilic cytoplasm and a fine chromatin pattern with variably prominent nucleoli. There was sparse cytoplasmic pigmentation within the most superficial portions of the lesion, with occasional admixed melanophages. In most areas no pigmentation was apparent. Mitotic figures were identified in the superficial portions of the lesion, up to 2/mm2.
The subsequent excision specimen showed a compound melanocytic proliferation with features similar to those of the previous shave (Fig. 1). The intraepidermal component consisted of small nests of cells at the junction present centrally, overlying an area of established dermal scarring (Fig. 2A). These nests showed uniform size with mildly irregular distribution. There was no significant intervening lentiginous growth or pagetoid extension. The intradermal component consisted of cords and sheets of cells with a somewhat dumbbell-like configuration due to an expansile component with a pushing border in the superficial subcutaneous tissue. The lesional cells showed voluminous eosinophilic cytoplasm with a moderate degree of cytological variability, including some variation in size and chromasia with the majority of cells showing slightly irregular nuclei with vesicular chromatin and prominent nucleoli admixed with some cells showing a moderate degree of nuclear hyperchromasia (Fig. 2B). Once again, occasional mitotic figures were identified, up to 1/mm2, in this specimen present in the lower half of the lesion, although no deep marginal mitoses were identified. The lesion abutted one side margin.
Fig. 1The excision showed a compound melanocytic proliferation with an expansile component extending into the superficial subcutaneous tissue with a dumbbell configuration.
Fig. 2(A) There was a central area of scarring superficially with a limited overlying intraepidermal component consisting of small nests. (B) The constituent cells showed large nuclei and voluminous cytoplasm, with a moderate degree of cytological variability, notably including a scattered population of cells with hyperchromatic nuclei. (C) There was strong cytoplasmic staining of the lesional cells with BRAF V600E antibody.
Immunohistochemistry showed widespread positivity for Sox10 (BC34, 1:100; Biocare, USA), with variable staining of the lesional cells for HMB-45 (HMB45, 1:100; Agilent/Dako, USA), comprising strong staining of the intraepidermal component and areas of moderate staining scattered throughout the lesion, including staining of the expansile deeper component. The lesional cells showed strong positivity with BRAF V600E antibody (V600E; Abcam, UK) (Fig. 2C). There was a patchwork pattern of p16 positivity (E6H4; Ventana, USA). There was no staining with PRAME (EPR20330, 1:1000; Abcam). There was no staining with ALK1 (ALK-1; Ventana), ROS1 [D4D6(R), 1:50; Cell Signaling, USA] or pan-TRK (EPR17341, 1:100; Abcam) antibodies.
Array CGH testing was performed using the Agilent Oligonucleotide SurePrint high definition 60 k array as previously described.
An isolated segmental gain of chromosome 7q was detected, with no other copy number variation (Fig. 3). Next generation sequencing (NGS) was performed using the TruSight Oncology 500 (TSO500; Illumina, USA) targeted hybrid-capture based NGS assay. RNA and DNA were extracted from representative formalin-fixed, paraffin-embedded tissue microdissected from sections on glass slides. Extracted nucleic acid was quantified by Qubit Fluorometric Quantitation (ThermoFisher Scientific, USA). Library preparation and targeted capture were performed using the TSO500 kit and samples sequenced on NextSeq 550 (Illumina) using NextSeq V.2.5 hi output reagents, according to the manufacturer's instructions. Data analysis was performed using the Illumina BaseSpace TSO500 app V2.1.0 and a custom tertiary pipeline, the Clinical Genomics Workspace software platform (PierianDx, USA).
Fig. 3Array CGH showed segmental gain of 7q, without other copy number variation.
A BRAF p.V600E mutation was detected. In addition, copy number gains of MET and BRAF (both located within the area of chromosome 7 showing segmental gain on array CGH testing) were identified. No other significant findings were demonstrated.
Given the combination of morphological and genetic features, this lesion was classified as falling within the spectrum of the recently described group of ‘BRAF mutated and morphologically Spitzoid (BAMS) naevi and tumours’,
Benign and intermediate-grade melanocytic tumors with BRAF mutations and Spitzoid morphology: a subset of melanocytic neoplasms distinct from melanoma.
conventionally classified as Spitz naevus/low risk atypical Spitz tumour in view of the atypical morphological features, reassuring cytogenetic findings and relatively limited amount of experience with lesions of this type showing BRAF V600E mutation. A further excision of this site was planned, but the patient was subsequently lost to follow up.
In recent years the definition of Spitz naevus/tumour has moved from one based purely on clinical and histological features to include molecular features. In particular, true Spitz tumours are partially defined by the presence of Spitz-associated genomic fusions or HRAS mutation.
Although lesions with BRAF mutation and abnormalities of BAP1 were formerly frequently classified as Spitz tumours, these lesions are currently grouped separately as BAP1-inactivated naevus/melanocytoma in the World Health Organization classification.
As a result, the presence of BRAF V600E mutation has recently been considered almost exclusive of true Spitz naevus/tumour.
However a group of lesions with typical morphological features of Spitz tumour but evidence of BRAF mutation, most commonly V600E, have recently been reported by Zhao et al.,
Benign and intermediate-grade melanocytic tumors with BRAF mutations and Spitzoid morphology: a subset of melanocytic neoplasms distinct from melanoma.
who described the morphological and molecular features of 36 lesions in this group. The morphological features were compared with a control cohort of Spitz tumours showing genomic fusions. Other than a higher rate of cytoplasmic pigmentation and an absence of Kamino bodies in the BAMS group, no other morphological differences were detected.
Detection of BRAF V600E mutation can be of value in the diagnosis of morphologically challenging melanocytic proliferations. In particular, in adolescent/young adult patients with a morphological differential diagnosis which includes superficial spreading melanoma and Spitz naevus, the presence of BRAF V600E mutation provides supportive evidence for the former classification; detection of a Spitz-associated fusion [either by immunohistochemistry, fluorescence in situ hybridisation (FISH) testing or NGS] provides evidence for a Spitz naevus/tumour. However, as with all adjunctive testing in melanocytic pathology, the results need to be interpreted in conjunction with a constellation of other features. In particular, BRAF V600E mutation is a common feature in deep penetrating naevus/melanocytoma
which frequently enter the differential diagnosis. Demonstration of either presumptive WNT pathway activation (e.g. by immunohistochemistry for β-catenin and cyclin D1) or loss of BAP1 expression by immunohistochemistry can be useful in elucidating these diagnoses.
We have illustrated a case with otherwise entirely classical traditional clinical, morphological and molecular features of Spitz naevus/atypical Spitz tumour but showing BRAF V600E mutation, highlighting an additional potential difficulty in the diagnostic use of BRAF V600E immunohistochemistry for challenging melanocytic proliferations. As always, close attention to the combination of clinical, morphological, immunohistochemical, cytogenetic and molecular features allows an accurate multiparametric classification of these lesions. Only with such classification will meaningful long-term follow-up data be able to be accrued to determine how the various constellations of features correlate with clinical outcome.
Conflicts of interest and sources of funding
The authors state that there are no conflicts of interest to disclose.
References
Mesbah Ardakani N.
Thomas C.
Robinson C.
et al.
Detection of copy number variations in melanocytic lesions utilising array based comparative genomic hybridisation.
Benign and intermediate-grade melanocytic tumors with BRAF mutations and Spitzoid morphology: a subset of melanocytic neoplasms distinct from melanoma.
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