Congenital melanocytic naevi initiated by BRAF fusion oncogene with firmness, pruritus and desmoplastic stroma.

In all five giant CMN tested, the BRAF fusion gene was the only pathogenic alteration identified (Table S5 ; see Supporting Information ). Four patients had too many satellites to count, many of which presented as firm nodules with significant pruritus. Stromal desmoplasia was a distinguishing histopathological feature, which we previously observed in 93% ( n = 54/58) of acquired melanocytic naevi with BRAF fusion genes. 10 , 14 A 19-year-old nonbinary person (assigned male at birth) presented with a giant melanocytic naevus on their back and upper neck; it had been present since birth (Figure 1a ). The naevus was uniformly coloured with surface rugosity, multiple nodular areas and hypertrichosis (Figure 1b, c ). At birth, numerous satellite lesions were present on the trunk and extremities, but the face was spared. Up to the age of 19 years, additional satellite naevi continued to develop including on the face, ultimately numbering > 1000 in total. The satellite naevi progressively became more firm, raised and pruritic. Magnetic resonance imaging (MRI) at 3 years of age showed no evidence of neurocutaneous melanosis; neurological development was normal. Giant congenital melanocytic naevus (CMN) with a BRAF fusion gene and numerous satellite naevi (patient 1). (a) In infancy a giant CMN spanned the entire back and upper neck of patient 1 in a ‘bolero’ pattern distribution. The colour was uniform, dermal nodules were scattered and hypertrichosis was moderate. (b) Numerous satellite naevi were present in infancy, predominantly over the lower extremities and trunk. (c) By adolescence, numerous additional satellite naevi had developed, with many involving the upper extremities, neck and face. (d) Satellite naevi also increased in number on the lower extremities. (e) Haematoxylin and eosin-stained sections (scanned with × 40 objective) showed a plaque-like compound melanocytic proliferation of nests and cords of melanocytes within desmoplasia. (f) Cords of melanocytes with scant cytoplasm are present between sclerotic collagen bundles in the dermis (scanned with × 40 objective). Between the age of 6 months and 3 years, approximately 50% of the giant CMN was surgically debulked and several satellite melanocytic naevi were excised, which resulted in a decrease in pruritus. Evaluation of the histopathology of a satellite melanocytic naevus from the back showed a plaque-like compound proliferation of melanocytes arranged as nests and cords within a desmoplastic stroma (Figure 1e ). Melanocytes were either large and epithelioid with abundant pale cytoplasm like those typically observed in Spitz naevi or small with scant cytoplasm, similar to those typically observed in common acquired naevi or CMN (Figure 1f ). Pigmentation was moderate and mostly present in melanophages in the papillary dermis. Melanocytes were scattered within the upper levels of the epidermis (pagetoid scatter) and extended along adnexal epithelium. Maturation with descent into the dermis was seen and significant cytological atypia or increased mitoses were not apparent. DNA sequencing revealed an AKAP9 :: BRAF gene fusion, predicted to result in an in-frame fusion transcript joining exon 15 of AKAP9 to exon 8 of BRAF , as the consequence of a complex rearrangement (duplication and inversion). There were no copy number changes in the tumour genome. A 28-year-old woman initially presented with a giant CMN involving the occipital scalp and back. The giant CMN was partially excised during childhood. Satellite naevi were present on her extremities (Figure 2c ), tongue, palate and nasal mucosa, and had persisted and slowly increased in number over time, ultimately estimated to number > 1000. They were heterogeneous in pigmentation: some showed light-to-dark brown colouring, while others were flesh-coloured (Figure 2a, b ). Larger brown lesions had irregular contours, surface rugosity and some elevated, nodular areas. There was no hypertrichosis. The patient complained of intermittent and at times severe pruritus, particularly in the area of the giant naevus and also in several satellite naevi. MRI of the brain and spine at the age of 13 years did not show signs of neurocutaneous melanosis. Numerous satellite naevi from BRAF rearranged giant congenital melanocytic naevus with fibrosis (patient 2). (a) Numerous satellite naevi on the abdomen. (b) Fibrotic nodules within satellite naevi are firm and raised. (c) Histopathology of a satellite naevus showing a compound proliferation of small ovoid melanocytes extending through a fibrotic dermis. (d, e) Haematoxylin and eosin-stained sections show epidermal hyperplasia with clefting around nests and between superficial melanocytes, as seen in Spitz naevi (original magnification × 40). (f) Within the dermis, cords of small melanocytes are present between thickened collagen bundles (haematoxylin and eosin, original magnification × 40). (d–f) Scanned with × 40 objective with different zoom levels applied. Initial excisions at the age of 7 months from the thighs, legs, knees, neck and buttock demonstrated plaque-like compound proliferations of small and ovoid-to-spindled melanocytes arrayed in nests and cords with pigmentation, pagetoid scatter, maturation with depth and adnexal extension (Figure 2d, e ). The melanocytes were interspersed between desmoplastic collagen bundles in the dermis (Figure 2f ). There was an increase of small, thin-walled vessels in the dermis. Some melanocytes were multinucleated. DNA sequencing identified an ATAD2::BRAF fusion gene, wherein intron 19 of ATAD2 was fused to intron 8 of BRAF , resulting in a predicted in-frame fusion transcript. A subsequent biopsy of a lesion from the foot at 24 years of age had similar histomorphology, with the melanocytes in the papillary dermis showing a more epithelioid morphology with mild pleomorphism akin to Spitz naevus. A 19-month-old boy presented with a giant CMN on the lower back; it had been present since birth (Figure 3a, b ). His naevus continued to grow and change colour after birth. A few satellite lesions were also noted. He underwent partial excisions of the melanocytic naevus. The patient’s parents reported that the child was frequently scratching the lesions, probably from pruritus and/or pain. Patient 3 at 19 months old with a giant congenital melanocytic naevus of the lower back. (a, b). Clinical presentation. (c, d) Histopathology demonstrated a broad compound melanocytic proliferation of sheets, nests and cords of epithelioid melanocytes. (c) Haematoxylin and eosin, original magnification × 10, with inconspicuous nucleoli set in a desmoplastic stroma. (d) Haematoxylin and eosin, original magnification × 100. Histopathology revealed a broad plaque-like melanocytic proliferation in the dermis and subcutis, composed of sheets, nests and cords of epithelioid melanocytes with abundant cytoplasm and inconspicuous nucleoli (Figure  3c, d ). Multinucleated melanocytes and pigmented melanocytes were present. Pagetoid scatter, significant cytological atypia and increased mitotic activity were absent. DNA sequencing identified a ST13::BRAF fusion gene. ST13 is located on chromosome 22, and the fusion transcript is predicted to be in-frame, joining exon 3 of ST13 with exon 10 of BRAF . The copy number profile showed loss of portions of chromosome 9p, but the region including CDKN2A was preserved. Portions of chromosome 22 were also lost. A 7-year-old girl presented with a giant CMN covering her trunk with numerous satellites numbering in the thousands, involving most of the trunk, abdomen, left leg, right flank and suprapubic area (Figure 4a, b ). Throughout her first 7 years of life, the giant naevus grew progressively, thickened and developed nodules and prominent folds. MRI at the age of 6 years showed neurocutaneous melanosis. Patient 4 at 6 weeks old with a giant congenital melanocytic naevus involving the trunk, abdomen, left leg, right flank and suprapubic area. (a, b). Satellite naevi were present on the scalp, face, extremities and buttocks, along with melanonychia of several nails. (c) Histopathology showed a markedly thick compound proliferation of small, ovoid melanocytes (haematoxylin and eosin, original magnification × 10). (d) Melanocytes were present in storiform arrangements between desmoplastic collagen bundles (haematoxylin and eosin, original magnification × 100). An AKAP9::BRAF fusion gene was identified by DNA sequencing. Owing to significant pruritus and pain associated with her naevi, she was started on trametinib, an oral MEK inhibitor, and experienced a significant improvement in symptoms, as previously reported. 16 Histopathology of the lesions demonstrated a thick, plaque-like compound proliferation of small, ovoid melanocytes arrayed in storiform cords with single cells splaying between desmoplastic collagen bundles (Figure 4c, d ). Pigmentation, pagetosis and maturation with depth were present, but epithelioid melanocytes were absent. A 37-year-old man presented with a history of giant CMN involving his entire back with extension to the anterior trunk treated by dermabrasion as an infant. He reported developing multiple flesh-coloured to brown papules and nodules on most surfaces of his body, including the face, palms of his hands, and soles of his feet in his childhood and teenage years, numbering > 1000 (Figure 5 ). Giant congenital melanocytic naevus (CMN) with TRIM4 :: BRAF fusion gene (patient 5). (a) Dermabraded giant CMN and multiple satellite naevi on the back and neck with biopsy site marked. The patient had a history of dermabrasion in infancy. (b) Histopathology shows clusters of melanocytes that mature with descent within a dermis with sclerotic collagen bundles (haematoxylin and eosin, original magnification × 100). (c) Higher-power view shows melanocytes with round, darkly staining nuclei and occasional nuclear pseudoinclusions. The melanocytes have variable amounts of amphophilic cytoplasm (haematoxylin and eosin, original magnification × 200). (d) Polymerase chain reaction for TRIM4 :: BRAF fusion gene with positive controls ( GAPDH1 , GAPDH2 ). The specific band is not present in the negative control (293 T cells) but is detected in the satellite naevus and the peripheral blood. An excisional biopsy was performed on a nodule located on the right neck (Figure 5a ). Histopathology revealed predominantly intradermal collections of melanocytes, organized in nests and exhibiting decreasing cell size with increased dermal depth (Figure 5b, c ). Rare nests with melanocytic cells of similar appearance were found in the overlying epidermis without pagetoid scatter of melanocytes. TNA sequencing identified a TRIM4::BRAF fusion transcript joining exon 4 of TRIM4 and exon 10 of BRAF resulting in a predicted in-frame fusion transcript encoding the kinase domain of BRAF . Both genes are located on chromosome 7q, indicating a likely complex rearrangement event on this chromosome. We designed a PCR assay to detect the TRIM4 :: BRAF fusion gene and identified the rearrangement in DNA isolated from the satellite naevus, as well as peripheral blood (Figure 5d ).

Two patients were evaluated at University of California in San Francisco (UCSF; patients 1 and 2), two at University of Texas Southwestern (patients 3 and 4) and one at University of Colorado Anschutz Medical Campus (patient 5). DNA extracted from the melanocytic naevi was sequenced at UCSF for patients 1–3. Tumour DNA was analysed using capture-based next-generation sequencing with the UCSF500 Cancer Gene Panel, which examines the coding regions of 479 genes (Table S1 ; see Supporting Information ) and the select introns of 47 genes. Analysis was performed as previously described. 15 Patient 4 has been described elsewhere. 16 Total nucleic acid (TNA) was extracted from a satellite nodule in patient 5 and analysed by the Colorado Molecular Correlates Laboratory using the ArcherDx FusionPlex Solid Tumor panel, 17 which evaluates select exons and introns for 53 genes (Table S2 ; see Supporting Information ), and the TruSight Tumor 26 (Illumina, San Diego, CA, USA) gene panel (Table S3 ; see Supporting Information ). The TNA, as well as RNA purified from patient whole blood and human embryonic kidney 293 T cells (negative control), was used to generate cDNA. Polymerase chain reaction was performed using primers specific for GAPDH or the TRIM4 :: BRAF fusion (Table S4 ; see Supporting Information ).

We describe the unique clinical and histopathological features of five large/giant CMN initiated by BRAF fusion genes. Four of the five patients in our series had > 100 satellite lesions, including at extracutaneous sites such as the tongue or oral mucosa. A previous study had found that the number of satellite naevi in individuals with large or giant CMN conferred an increased risk of neurocutaneous melanosis (5.1-fold increased risk in patients with > 20 satellites). 4 One of the three patients who had undergone MRI had neurocutaneous melanosis (patient 4). Martin et al . found that giant CMN with BRAF fusion genes ( n = 10) frequently demonstrated multinodularity. 7 All the giant CMN in our series had ≥ 1 nodular areas that were firm to the touch with marked pruritus. The pruritus was often unresponsive to multiple treatment modalities. Importantly, the distinctive pattern of numerous fibrotic satellite lesions as seen in four of our patients has not previously been reported in studies of giant CMN with BRAF fusion genes. 7–9 BRAF fusion genes have been identified in Spitz tumours and are among the defining genetic alteration of Spitz tumours, according to the 2018 World Health Organization Classification of Skin Tumours, along with other kinase fusions involving ALK , ROS1 , RET , MET , MAP3K8 , NTRK1 or NTRK3 , and mutations in HRAS . 18 We have previously described the cytomorphology of Spitz tumours with BRAF fusion genes in a large series of 58 patients. 14 These cases showed diverse fusion partners, but only a few of these occurred in more than one patient ( MAD1L1 , n = 3; AKAP9 , n = 3; CLIP2 , n = 3; AGK , n = 1). In our current cohort, AKAP9 was the fusion partner in 2 cases, with TRIM4 ( n = 1), STI1 ( n = 1) and ATAD2 ( n = 1). These acquired melanocytic tumours typically, but not always, demonstrated one of three main morphological patterns (buckshot fibrosis, cords in whorled fibrosis and spindle-cell fascicles) with marked stromal desmoplasia. 14 Our five patients with CMN also demonstrated marked stromal desmoplasia and the closest morphological pattern we identified was that of ‘cords in whorled fibrosis’ (patients 1–3). Similarly, the morphological patterns of cases from the cohort of Martin et al . were similar and could be classified as one of these three patterns. 7 We must also caution that it may be difficult to draw definitive conclusions about morphological correlations in BRAF fusion giant CMN given the small size of this cohort. All lesions showed pigmentation, particularly within the papillary dermis, and cells were arrayed in cords, single cells or sheets. Adnexal extension, commonly encountered in congenital naevi, was also present. Melanocytes matured with depth but, except for patient 4, did not exhibit the abundant cytoplasm typical of Spitz naevi. The observation of desmoplastic stroma in BRAF fusion-driven CMN is intriguing and may share similarities with other conditions, such as melanocytic naevi with 15q gain, Spitz naevi and desmoplastic melanoma. 14 , 19 Desmoplasia might result from an epithelial–mesenchymal transition (EMT)-like process, where EMT-inducing transcription factors (e.g. SNAI1 and ZEB1 ) orchestrate interactions between neoplastic melanocytes and the microenvironment, akin to what occurs in desmoplastic melanoma, 20 but the mechanism is currently unknown. The relationship between BRAF fusion genes in congenital naevi and satellite lesions that are too numerous to count, as seen in four of our patients, remains to be determined. This feature and unusual clinical phenotype was not highlighted in previously reported cases of giant CMN with BRAF fusion genes. One possible explanation is haematogenous dissemination of neoplastic melanocytes, reminiscent of the phenomenon observed in other benign conditions such as benign metastasizing leiomyoma and endometriosis. 21 , 22 Another related phenomenon is the occurrence of nodal melanocytic naevi, wherein benign melanocytes are found in lymph nodes, likely to be due to lymphatic spread. For example, benign nodal naevi frequently harbour BRAF V600E mutations. 23 These parallels suggest that BRAF fusion genes may confer unique properties on melanocytes, such as increased survival, proliferation or dissemination potential after entering the circulation. Alternatively, genetic mosaicism arising from postzygotic mutations could also explain this phenomenon. In this scenario, the widespread distribution of satellite lesions might result from an early embryonic mutation leading to clonal expansion of genetically altered melanoblasts during development. The limitations of our study, particularly the absence of prospective genotyping of patients with numerous satellite naevi, preclude definitive conclusions, but the unique biology of BRAF fusion-driven giant CMN warrants further investigation. Detection of BRAF fusions is critical for guiding targeted treatment in certain instances, and several methods are available, each with strengths and limitations. Fluorescence in situ hybridization using break-apart or fusion probes is a reliable and specific method to identify structural rearrangements involving BRAF . 14 While it cannot determine the exact fusion partner, this distinction is not currently required for RAF inhibitor therapy. RNA sequencing is a powerful technique that can broadly detect fusion transcripts and provide detailed information about the genes involved and their expression patterns. 13 DNA-based next-generation sequencing panels can also offers broad genomic analysis, identifying gene fusions, as well as mutations in oncogenes that may guide treatment (e.g. ruling out a BRAF V600E mutation). For fusion detection by DNA sequencing, large intronic regions must be targeted and sensitivity is reduced. 24 The fusion partner can predict response to treatment due to varying expression levels and its ability to promote dimerization. 25 Selecting the appropriate method depends on factors such as sample type, cost and the clinical need to identify actionable BRAF fusions. Of note, BRAF V600E immunohistochemistry is specific to BRAF V600E mutations and cannot detect BRAF fusions. BRAF fusion kinases can be inhibited by RAF inhibitors alone or in combination with MEK or extracellular signal-regulated kinase inhibitors. 12 , 25–27 A patient with a giant CMN with BRAF fusion kinase responded to treatment with the MEK inhibitor trametinib. 9 Patient 4 in our series also showed a good response to trametinib treatment, as previously reported. 16 She was the most severely affected patient of our series, with intracranial melanocytosis and deep muscular and fascial invasion of the trunk and pelvis leading to intractable pruritus, pain and insomnia. Recently, based on the results of the FIREFLY-1 phase II clinical trial ( NCT04775485 ), 28 the U.S. Food and Drug Administration granted accelerated approval of tovarafenib, a selective type II RAF kinase inhibitor for paediatric refractory low-grade glioma with either BRAF V600 mutation or a BRAF fusion or rearrangement. Given its mechanism of action, this drug may also provide benefit to patients with giant CMN with activating BRAF rearrangements. The presence of satellite naevi that are too numerous to count and desmoplastic stromal alterations may help identify BRAF fusion genes as therapeutic targets in a subset of giant CMN.