A distinct subset of atypical Spitz tumors is characterized by BRAF mutation and loss of BAP1 expression

Abstract

We recently reported that germline mutations in BAP1 cause a familial tumor syndrome characterized by high penetrance for melanocytic tumors with distinct clinical and histologic features. Melanocytic neoplasms in affected individuals harbored BRAF mutations, showed loss of BAP1 expression, and histologically resembled so-called "atypical Spitz tumors" (ASTs). ASTs are an ill-defined and probably heterogenous group of melanocytic tumors that display histologic features seen in both Spitz nevi and melanomas. Their biological behavior cannot be reliably predicted. In view of the histologic similarities of the familial tumors and ASTs, we hypothesized that a subset of ASTs might harbor genetic alterations seen in the familial tumors. To address this hypothesis, we analyzed 32 sporadic ASTs for BRAF mutations and for BAP1 expression. Nine (28%) sporadic ASTs showed loss of BAP1 expression, of which 8 (89%) had concomitant BRAF mutations. Only 1 of the BAP1-positive ASTs (4%) had a BRAF mutation (P<0.0001). BRAF-mutated, BAP1-negative tumors were primarily located in the dermis and were composed entirely or predominantly of epithelioid melanocytes with abundant amphophilic cytoplasm and well-defined cytoplasmic borders. Nuclei were commonly vesicular and exhibited substantial pleomorphism and conspicuous nucleoli. The combination of BRAF mutation and loss of nuclear BAP1 expression thus characterizes a subset of ASTs with distinct histologic features. The typical morphology of these tumors and BAP1 immunohistochemistry provide pathologic clues that will enable accurate identification of this subset. Future studies are necessary to determine whether this subset has a predictable clinical behavior.

FIGURE 1

Skin tumor from a 49-year-old man harboring a germline mutation in BAP1. A, Dome-shaped to pedunculated, skin-colored tumor. B, Polypoid, relatively symmetrical, dermal tumor consisting of (C) melanocytes arranged in nests and sheets. D, The neoplastic cells are characterized by moderate amounts of amphophilic cytoplasm, well-defined cytoplasmic borders, and pleomorphic oval nucleoli. E, BAP1 IHC is negative in tumor cells but positive in epidermal keratinocyte nuclei and in scattered lymphocytes within the tumor. F, Fluorescence in situ hybridization shows loss of the second BAP1 allele in tumor cell nuclei: 1 red signal (chr. 3p21, BAP1 locus), 1 orange signal (chr. 3p25), and 2 green control signals per nucleus. G, Array CGH shows a loss of the whole chromosome 3 with no other chromosomal aberrations. H, The sequencing electropherograms show a BRAF^V600E mutation and (I) the germline BAP1 mutation. The wild-type BAP1 sequence is markedly reduced indicating a loss of the wild-type allele in the tumor.

FIGURE 2

AST from the shoulder of a 23-year-old man (case 1). A, Polypoid, predominantly dermal melanocytic tumor composed of (B) large epithelioid cells with numerous admixed lymphocytes. C, The tumor cells contain abundant amphophilic cytoplasm with well-defined cytoplasmic borders and pleomorphic, round-to-oval, vesicular nuclei with conspicuous nucleoli. TILs are prominent. D, The epithelioid cells do not express BAP1, whereas the admixed lymphocytes are strongly positive for BAP1. E, Sequencing electropherograms show a BRAF^V600E mutation and (F) an inactivating, frameshift BAP1 mutation (c.459del, p.E154Rfs*33) in the tumor.

FIGURE 3

AST from the back of 19-year-old patient (case 2). A, Shave biopsy of a relatively symmetric dermal tumor composed of (B) sheets and nests of large epithelioid cells. Inset: No BAP1 staining in the nuclei of epithelioid cells, whereas the admixed lymphocytes are strikingly positive. C, The tumor cells contain abundant amphophilic cytoplasm with well-defined cytoplasmic borders and very pleomorphic round/oval vesicular nuclei with conspicuous nucleoli. Occasional multinucleate tumor cells are present. Note the prominent TILs.

FIGURE 4

AST from the upper back of 51-year-old woman (case 8). A, Symmetrical dermal tumor composed of (B) sheets of large epithelioid cells. The tumor cells contain abundant amphophilic cytoplasm with well-defined cytoplasmic borders and pleomorphic vesicular nuclei with conspicuous nucleoli. Occasional multinucleate tumor cells are present. Only few TILs are present. Inset: Absent nuclear staining for BAP1 in the epithelioid cells, with some positive admixed lymphocytes. C, Array CGH of the tumor shows a focal loss of the BAP1 region on chr. 3 and a loss of the long arm of chr. 16. D, The sequencing electropherograms show a BRAF^V600E mutation and (E) an inactivating frameshift mutation of BAP1 (c.920dup, p.N308Qfs*90).

FIGURE 5

AST from a 59-year-old man (case 9). A, Symmetrical, melanocytic tumor (B) with no epidermal involvement. C, Medium-to-large epithelioid cells with abundant amphophilic cytoplasm and pleomorphic, vesicular nuclei with conspicuous nucleoli. Inset: Absent nuclear BAP1 staining in large epithelioid melanocytes with strong nuclear staining in admixed lymphocytes. D, Array CGH shows a loss of the whole chromosome 3 with no other chromosomal aberrations. E, Sequencing revealed no BRAF^V600E mutation (wild type) but (F) a missense BAP1 mutation (c.516C > G, p.S172R) in the tumor.

FIGURE 6

AST from the upper back of a 44-year-old woman (case 6). A, Small polypoid melanocytic tumor (B) with medium-to-large epithelioid cells and admixed lymphocytes. Inset: Absent BAP1 staining in melanocytes but strong nuclear staining in admixed lymphocytes. C, Sequencing electropherograms show a BRAF^V600E mutation and an inactivating, nonsense mutation of BAP1 (c.178C > T, p.R60*). D, Array CGH shows a balanced profile with no gains and losses.

FIGURE 7

AST from the arm of a 37-year-old patient (case 10). A, Dome-shaped melanocytic tumor (B) composed of sheets of medium-sized, spindle-shaped, oval, and epithelioid melanocytes with amphophilic cytoplasm but lacking distinct cytoplasmic borders. C, Strong expression of BAP1 in keratinocytes and melanocytes. This tumor showed a BRAF^V600E mutation (data not shown).