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BAP1 and BRAFV600E expression in benign and malignant melanocytic proliferations
Human Pathology (2015) 46, 239–245
www.elsevier.com/locate/humpath
Original contribution
BAP1 and BRAFV600E expression in benign and malignant melanocytic proliferations☆ Adriano Piris MD a,b,c , Martin C. Mihm Jr. MD a,b , Mai P. Hoang MD a,c,⁎ a
Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114 Department of Dermatology, Brigham and Women's Hospital, Massachusetts General Hospital, Boston, MA 02114 c Department of Pathology, Massachusetts General Hospital, Boston, MA 02114 b
Received 2 October 2014; revised 14 October 2014; accepted 24 October 2014
Keywords: BAP1; BRAFV600E; Immunohistochemistry; Nevi; Melanoma
Summary BAP1 (BRCA1-associated protein 1) is a tumor suppressor gene whose mutations have recently been reported to increase susceptibility for the development of uveal melanoma, cutaneous atypical and epithelioid melanocytic lesions, clear cell renal cell carcinoma, and other tumors. Screening for BAP1 mutation/loss/inactivation and BRAFV600E mutation can be done by immunohistochemistry. We investigated BAP1 and BRAFV600E expression in 193 sporadic melanocytic lesions (11 dermal nevi, 20 congenital nevi, 40 primary and nondesmoplastic melanomas, 40 desmoplastic melanomas, 23 metastatic melanomas, 17 Spitz nevi, 19 atypical Spitz nevi, 8 atypical Spitz tumors, 14 proliferative nodules arising in congenital nevi, 1 nevus during pregnancy) and 30 melanocytic lesions from 3 patients with family history of uveal melanoma and BAP1 germline mutation. Most sporadic melanocytic lesions exhibited positive BAP1 nuclear staining, except for 1 proliferative nodule arising in congenital nevus, 1 desmoplastic, 1 nevoid, and 2 metastatic melanomas. BRAFV600E positivity was demonstrated in 80% of dermal, 5% of congenital, 6% of Spitz, and 5.5% of atypical Spitz nevi; 29% of proliferative nodules arising in congenital nevi; and 24% of primary and nondesmoplastic and 35% of metastatic melanomas. Combined BAP1 loss and BRAFV600E staining was seen in 67% of BAP1 tumor syndrome–associated lesions and in none of the sporadic melanocytic proliferations including Spitz and atypical Spitz nevi and atypical Spitz tumors, with the exception of 1 primary melanoma. The combined BAP1-BRAFV600E+ immunoprofile appears to be a constant feature of BAP1 tumor syndrome–associated melanocytic lesions, and the designation of Spitz nevi or variants thereof appears to be inaccurate for this group of lesions. © 2015 Elsevier Inc. All rights reserved.
1. Introduction BAP1 (BRCA1-associated protein 1) is a tumor suppressor gene whose mutations have recently been reported to increase susceptibility for the development of uveal ☆
Disclosures: There was no external funding for this work. The authors have no conflict of interest to declare. ⁎ Corresponding author at: Department of Pathology, Massachusetts General Hospital, 55 Fruit St, Warren 820, Boston, MA 02114. E-mail address: [email protected] (M. P. Hoang). http://dx.doi.org/10.1016/j.humpath.2014.10.015 0046-8177/© 2015 Elsevier Inc. All rights reserved.
melanoma, cutaneous atypical and epithelioid melanocytic lesions, lung adenocarcinoma, meningioma, clear cell renal cell carcinoma, mesothelioma, and other tumors [1–6]. Loss of heterozygosity from a second mutation or deletion results in tumor formation. A BAP1 tumor predisposition syndrome (OMIM #614327) shows autosomal dominant inheritance, and individuals with heterozygous BAP1 mutation are at high risk for developing the above-mentioned tumors with varying degrees of penetrance [7]. The melanocytic neoplasms in individuals with germline BAP1 mutation have shown loss of BAP1 expression and
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often BRAF mutations [5]. They have been reported to follow a benign clinical course [2,4,5]. Clinically, they present as benign-appearing, skin-colored or pink papules or nodules with a distinct appearance. Histologically, these lesions are characterized by an expansile, nodular or sheetlike, dermal aggregate of epithelioid melanocytes with welldemarcated cytoplasmic borders and ample and amphophilic or eosinophilic cytoplasm. Their nuclei show vesicular chromatin with regular nuclear membrane and prominent nucleoli. These proliferations may or may not be associated with background ordinary dermal nevic components. In addition, they can have binucleated or multinucleated giant cells and associated lymphocytic infiltrate [2,4,5]. Currently, there is no standardized terminology regarding BAP1-mutated melanocytic lesions arising in individuals with or without germline mutation [2,4,5,8–11]. Wiesner et al [11] reported a subset of sporadic atypical Spitz tumors with epithelioid morphology to be BRAF mutated and BAP1 negative. Gammon et al [8] also reported loss of nuclear BAP1 expression in 15 of 19 sporadic epithelioid Spitz tumors (combined melanocytic tumor with nests of conventional nevus cells and nodule of epithelioid/spitzoid melanocytes). Busam et al [9] reported a series of 8 combined melanocytic lesions with BAP1 loss and BRAFV600E positivity. Yeh et al [10] reported an additional series of 29 cases of ambiguous melanocytic tumors with 3p21 loss and Spitzoid morphology, and 12 (71%) of 17 had BRAFV600E mutation. This suggests that BAP1-negative melanocytic lesions may occur in patients without the tumor predisposition syndrome. BAP1 protein may become nonfunctional by mechanisms other than gene deletion or mutation. Screening for BAP1 mutation/loss/inactivation and BRAFV600E mutation can be done by immunohistochemistry. There have been only a few studies evaluating BAP1 expression in cutaneous melanomas and other melanocytic
Table 1
lesions, and only one study using both BAP1 and BRAFV600E immunostains (Table 1) [9,12]. In order to assess the specificity and sensitivity of these 2 immunostains in detecting the BAP1 tumor syndrome–associated melanocytic lesions, we aim to investigate BAP1 and BRAFV600E expression in (1) benign as well as malignant sporadic melanocytic lesions, and (2) a series of melanocytic lesions from 3 patients with family history of uveal melanoma and BAP1 germline mutation [4].
2. Materials and methods The study has been approved by Massachusetts General Hospital institutional board review (2013-P-000312). A total of 223 cases were included in the study. Tissue microarrays were prepared from paraffin-embedded material of 125 sporadic melanocytic proliferations (11 dermal nevi, 20 congenital nevi, 31 primary melanomas, 40 desmoplastic melanomas, and 23 metastatic melanomas). Whole-slide sections were obtained from 17 Spitz nevi, 19 atypical Spitz nevi, 8 atypical Spitz tumors, 14 proliferative nodules arising in congenital nevi, 1 nevus during pregnancy, and 9 nevoid melanomas. In addition, we analyzed whole-slide sections of 30 melanocytic lesions from 3 patients (16, 11, and 3 from each patient) with BAP1 germline mutations. The ordinary and well-defined melanocytic entities in this group of lesions were categorized according to preestablished histologic criteria. Atypical Spitz nevi included lesions with overall features of a classic Spitz nevus without expansile growth but with one atypical feature such as hypercellularity, mitotic activity, or ulceration. The following criteria were used in selecting atypical Spitz tumors: an asymmetric and expansile nodule in the dermis and often the
Summary of the literature Current study BAP1 IHC
BAP1 tumor syndrome–associated lesions Dermal nevi Congenital nevi Spitz nevi Atypical Spitz nevi Atypical Spitz tumor Epithelioid Spitz tumor Proliferative nodules Nevi in pregnancy Combined nevi Primary and nondesmoplastic melanoma Desmoplastic melanoma Metastatic melanoma Uveal melanoma Abbreviation: IHC, immunohistochemistry.
30/30 (100%) 0/11 (0%) 0/20 (0%) 0/17 (0%) 0/19 (0%) 0/8 (0%)
Wiesner et al [11], BAP1 IHC
Murali et al [12], BAP1 IHC
Busam et al [9], BAP1 IHC
Gammon et al [8], BAP1 IHC
Wiesner et al [5], BAP1 mutation 51/51 (100%) 0/28 (0%) 0/17 (0%)
9/32 (28%)
2/18 (11%) 15/19 (79%)
1/13 (8%) 0/1 (0%) 8/8 (100%) 1/40 (2.5%) 1/40 (2.5%) 2/23 (9%)
4/135 (3%) 5/18 (22%)
3/60 (5%)
13/33 (40%)
BAP1 and BRAFV600E expression
241
subcutaneous fat, lack of maturation, monomorphic and atypical tumor cells, and presence of deep and often marginal mitoses (Fig. 1) [13,14]. The defining criteria of proliferative nodules (8 benign, 6 atypical) arising in a background of congenital nevi were described in a prior study [15]. The BAP1-mutated melanocytic proliferations were selected from 3 patients belonging to a family with known BAP1 germline mutation [4]. All of these atypical melanocytic proliferations were excised and none had recurred. None of these lesions were melanomas. The histologic features of the latter are described below.
For anti-BRAFV600E, positive staining was characterized by diffuse and moderate (2+) to strong (3+) cytoplasmic staining of the tumor cells. Isolated nuclear staining, weak staining of occasional cells, or faint diffuse staining were considered as negative staining as previously reported [16,17]. A malignant melanoma with confirmed BRAFV600E mutation by molecular analyses was used as positive control.
2.1. Immunohistochemistry
The immunohistochemical results are summarized in Table 2. All 30 BAP1 tumor syndrome–associated melanocytic lesions exhibited loss of BAP1 nuclear staining. Cytoplasmic staining was noted in most cases, with marked cytoplasmic staining seen in 7 cases (23%). In 8 lesions (27%), dotlike BAP1 cytoplasmic positivity was noted. Most sporadic melanocytic lesions exhibited positive BAP1 nuclear staining. The exceptions were 1 proliferative nodule arising in a congenital nevus, 1 desmoplastic melanoma, 1 nevoid melanoma, and 2 metastatic melanomas. None of these 5 patients had personal or family history of cutaneous or uveal melanomas. The 4 melanomas with loss of BAP1 expression did not have special morphologic features that would set them apart from their respective melanoma subtype. BRAFV600E positivity was demonstrated in 80% of dermal nevi, 5% of congenital nevi, 6% of Spitz nevi, 5.5% of atypical Spitz nevi, 29% of proliferative nodules arising in
Immunohistochemical studies were performed on 5-μm-thick sections of formalin-fixed, paraffin-embedded tissue in a Bond 3 automated immunostainer (Leica Microsystems, Bannockburn, IL) with primary antibodies against BAP1 (BP-1, 1:400; Santa Cruz Biotechnology, Santa Cruz, CA) and BRAFV600E (clone: VE1, 1:100; Spring Bioscience, Pleasanton, CA). Appropriate positive and negative controls were included. The immunohistochemical stains were evaluated by 2 of the authors (A. P. and M. P. H.), and disagreements were reviewed together to achieve a consensus score. For anti-BAP1, nuclei of keratinocytes of the epidermis and appendages, fibroblasts, and lymphocytes served as internal positive controls for BAP1 expression. Tumors were scored as positive or negative based on whether their nuclei stained with BAP1.
3. Results
Fig. 1 In this atypical Spitz tumor, an expansile proliferation of epithelioid and spindled neoplastic cells is seen infiltrating into the subcutaneous tissue (A, ×40; B, ×100). The tumor cells exhibit strong nuclear BAP1 staining (C, ×100) while negative for BRAFV600E (D, ×100).
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congenital nevi, 24% of primary and nondesmoplastic melanomas, and 35% of metastatic melanomas. Sixty-seven percent of BAP1 tumor syndrome–associated lesions had combined BAP1 loss and BRAFV600E staining. In most cases, the large epithelioid cells lacked nuclear BAP1 staining and exhibited cytoplasmic BRAF staining (Fig. 2). In 9 cases, the background smaller nevus cells showed nuclear BAP1 and cytoplasmic BRAF staining (Fig. 3).
3.1. Statistical analyses When the statistical association of each stain in BAP1 tumor syndrome–associated melanocytic lesions versus those not associated with BAP1 germline mutations was evaluated by Fisher exact test, a significant P value (.0001) was observed for BAP1 loss, BRAFV600E positivity, and combined BAP1 loss and BRAFV600E positivity (Table 2).
3.2. Histology of BAP1-mutated melanocytic tumors There were 2 observed growth patterns: (1) a single welldemarcated dermal nodule comprising epithelioid melanocytes (Fig. 2) and (2) more than 1 nest of epithelioid melanocytes in a background of a diffuse dermal melanocytic nevus, often with features of congenital onset (Fig. 3). In the second pattern, the number and density of these “atypical” nests were variable. When the nests were prominent, the pattern was a diffuse growth in the dermis. When only a few nests were seen, the pattern was that of a dermal nevus–like proliferation with features of congenital onset. A lentiginous junctional component was noted in 13 (43%) cases. Benign dermal nevic cells were seen at the periphery of 9 (30%) cases. The lesional cells were often epithelioid in appearance with ample cytoplasm and well-defined cytoplasmic border. Moderate cytologic atypia, multinucleated tumor cells, and Table 2
4. Discussion BAP1 is a deubiquitinating enzyme whose gene is located on chromosome region 3p12 [18]. BAP1 has been suggested to be a tumor suppressor gene with a role in cell proliferation and growth inhibition [19]. Monosomy of chromosome 3 is the most common somatic alteration in uveal melanoma with BAP1 somatic mutations in 47.4% of uveal melanomas [20]. In most cases, BAP1 loss is somatically acquired, but multiple tumors tend to be associated with a BAP1 germline mutation. The incidence of BAP1 germline mutation is 4 of 100 patients with ocular melanoma and 3 of 200 patients with a family history of cutaneous melanoma [4]. Consistent with prior studies, we found that the BAP1 tumor syndrome–associated melanocytic lesions are typically dome shaped, 5 mm in size, and located on the head and neck, trunk and extremities, and the number ranged from 5 to more than 50 [2,4,5]. Histologically, there appear to be 2 distinct histologic patterns—a single dominant nodular pattern and dermal nevus–like pattern often with features of congenital onset [2,4,5,9]. These lesions can present as large epithelioid cell proliferations or in combination with conventional nevi [9]. The lesional cells are epithelioid melanocytes with abundant amphophilic cytoplasm, well-defined cytoplasmic borders, vesicular chromatin, multinucleation, cytologic atypia, and minimal mitotic figures. Tumor-infiltrating lymphocytes are often identified [2,4,5,9]. In contrast to Spitz nevi, these lesions lack the epidermal hyperplasia and large epidermal nests with associated retraction artifact. In some cases, hypercellularity, nuclear pleomorphism, and prominent mitotic figures were seen, raising the concern of nevoid melanoma [4]. Often there was an associated proliferation of
Summary of immunohistochemical findings
Total BAP1 tumor syndrome–associated melanocytic lesions Sporadic melanocytic lesions Dermal nevi Congenital nevi Spitz nevi Atypical Spitz nevi Atypical Spitz tumor Proliferative nodules Nevi in pregnancy Primary melanoma Desmoplastic melanoma Metastatic melanoma Pa a
some degree of inflammation were noted in most cases. The host response was prominent in 9 (30%) cases.
n
Loss of BAP1 expression
BRAFV600E positivity
BAP1 loss and BRAFV600E positivity
223 30 193 11 20 17 19 8 14 1 40 40 23
35/222 (16%) 30/30 (100%) 5/192 (3%) 0/11 (0%) 0/20 (0%) 0/17 (0%) 0/19 (0%) 0/8 (0%) 1/13 (8%) 0/1 (0%) 1/40 (2.5%) 1/40 (2.5%) 2/23 (9%) .0001
53/218 (24%) 20/30 (67%) 33/188 (18%) 8/10 (80%) 1/20 (5%) 1/16 (6%) 1/18 (5.5%) 0/8 (0%) 4/14 (29%) 1/1 (100%) 9/38 (24%) 0/40 (0%) 8/23 (35%) .0001
21/217 (10%) 20/30 (67%) 1/187 (0.5%) 0/10 (0%) 0/20 (0%) 0/16 (0%) 0/18 (0%) 0/8 (0%) 0/13 (0%) 0/1 (0%) 1/38 (3%) 0/40 (0%) 0/23 (0%) .0001
BAP-1–associated vs non-BAP1 melanocytic lesions; statistically significant.
BAP1 and BRAFV600E expression
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Fig. 2 A single well-demarcated nodule is seen in dermis (A, ×40), which comprises epithelioid cells with ample cytoplasm and well-demarcated border (B, ×200). The tumor cells are negative for nuclear BAP1 staining (C, ×100) and strongly positive for BRAFV600E (D, ×100).
smaller nevus cells, and thus, they can be designated as a combined nevus. In the current study, we found that all BAP1 tumor syndrome–associated melanocytic lesions showed BAP1 nuclear loss. However, cytoplasmic staining was seen in most cases and perinuclear dotlike positivity was noted in 27% of cases, similar to findings previously reported [2,8]. Gammon et al [8] reported that 8 (53%) of 15 sporadic epithelioid Spitz tumors in their series exhibited BAP1 loss and clumped perinuclear staining. The authors hypothesized that this perinuclear staining pattern could be the result of aberrant protein accumulation in the Golgi region rather than in the nucleus [8]. The incidence of BAP1 loss of expression in melanomas was 5.6%, similar to the 5% BAP1 mutation rate reported in 60 sequenced melanomas (45 cutaneous and 15 mucosal) [5,12]. In a series by Murali et al [12], 22% (5/18) of desmoplastic melanomas showed loss of BAP1 expression in comparison with 3% (4/135) of nondesmoplastic melanomas. On the contrary, we noted BAP1 loss only in 1 case of desmoplastic melanoma. The expression/mutation of BAP1 in congenital nevi, proliferative nodules arising in congenital nevi, nevus in pregnancy, and nevoid melanoma (lesions in the differential diagnosis of BAP1 tumor syndrome– associated melanocytic lesions) has not been studied. The frequency of BAP1 loss (2%-11%) in these lesions was low in comparison with that of BAP1 tumor syndrome– associated melanocytic lesions (100%; Table 2). It has been reported that the BAP1 tumor syndrome– associated melanocytic lesions have loss of BAP1 expression
and BRAFV600E mutation [5]. Accordingly, we noted that 67% of BAP1 tumor syndrome–associated lesions had combined BAP1 loss and BRAFV600E staining. Similar to findings reported by Busam et al [9] in a series of 8 cases of combined melanocytic lesions (an epithelioid component and conventional nevus component), the cells within the nodules in our cases were BAP1 negative and BRAFV600Epositive, whereas the background nevus cells when present were BAP1 positive and BRAFV600E positive. In all studied categories, combined BAP1 loss and BRAF staining appears to be a feature of BAP1 tumor syndrome–associated melanocytic lesions. The loss of BAP1 expression has been shown to correlate with biallelic BAP1 mutation, with positive and negative predictive values of 100% and 98.6%, respectively [11,21]; thus, screening by immunohistochemistry is cost-effective. False-negative interpretation can be avoided if internal positive controls (lymphocytes, fibroblasts) are present. Proposed names for BAP1 tumor syndrome–associated melanocytic lesions include “melanocytic BAP1-mutated atypical intradermal tumors” (MBAITs) and “nevoid melanoma-like melanocytic proliferation” (NEMMPs) [2,4]. Llamas-Velasco et al [22] described these lesions as “Wiesner's nevus,” and Yeh et al [10] designate them as “Spitz nevi associated with BAP1 loss.” Furthermore, in their description of these “BRAF-mutated/BAP1-negative” lesions, Wiesner et al [11] use the term atypical Spitz tumors. These authors assessed BRAF mutation and BAP1 expression in 32 sporadic atypical Spitz tumors. Nine (28%) of 32 exhibited BAP1 loss and 8 of 9 lesions with BAP1 loss had
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Fig. 3 A nodular growth of melanocytes comprises multiple nests of melanocytes seen in a background sheet-like proliferation of melanocytes (A, ×40; B, ×200). The lesional cells are negative for nuclear BAP1 staining, although cytoplasmic staining is noted (C, ×200). The entire lesion is positive for BRAFV600E (D, ×200). A dermal nevus–like proliferation with features of congenital onset is seen in the dermis (E, ×40; F, ×200). Nuclear BAP1 staining is seen in the background nevus cells, whereas the epithelioid cells are negative (G, ×200). Both components are positive for BRAFV600E (H, ×100).
BRAF mutation. They concluded that BRAF mutation together with BAP1 loss define a distinct subset of atypical Spitz tumors. None of our 17 Spitz nevi, 18 atypical Spitz nevi, and 8 atypical Spitz tumors exhibited the combined
BRAF + BAP1− phenotype. In addition, Spitz nevi have not been reported to harbor BRAF mutation [23], whereas common acquired nevi frequently have BRAF mutation [24]. Long-term follow-up was not available for these series
BAP1 and BRAFV600E expression [5,11]; thus, the question remains as to whether these tumors are atypical Spitz tumors or sporadic BAP1-associated melanocytic lesions. In summary, in our series of benign and malignant melanocytic lesions, the combined BAP1 loss and BRAFV600E positivity by IHC appears to be a constant feature of BAP1 tumor syndrome–associated melanocytic lesions, and the designation of Spitz nevi or variants thereof appears to be inaccurate for this particular group of lesions.
Acknowledgment We thank Drs Yayan Chen and Fang Liu, Department of Pathology, Saint Vincent Hospital, Worcester Medical Center, Worcester, MA, for their contribution to the cases.
References [1] Abdel-Rahman MH, Pilarski R, Cebulla CM, et al. Germline BAP1 mutation predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers. J Med Genet 2011;48:856-9. [2] Carbone M, Ferris LK, Baumann F, et al. BAP1 cancer syndrome: malignant mesotheliomas, uveal and cutaneous melanoma, and MBAITs. J Transl Med 2012;30:179. http://dx.doi.org/10.1186/ 1479-5876-10-179. [3] Wadt K, Choi J, Chung JY, et al. A cryptic BAP1 splice mutation in a family with uveal and cutaneous melanoma, and paraganglioma. Pigment Cell Melanoma Res 2012;25:815-8. [4] Njauw CN, Kim I, Piris A, et al. Germline BAP1 inactivation is preferentially associated with metastatic ocular melanoma and cutaneous-ocular melanoma families. PLoS One 2012;7:e35295. http://dx.doi.org/10.1371/journal.pone.0035295. [5] Wiesner T, Obenauf AC, Murali R, et al. Germline mutations in BAP1 predispose to melanocytic tumors. Nat Genet 2011;43:1018-21. [6] Testa JR, Cheung M, Pei J, et al. Germline BAP1 mutations predispose to malignant mesothelioma. Nat Genet 2011;28:1022-5. [7] Wiesner T, Fried I, Ulz P, et al. Toward an improved definition of the tumor spectrum associated with BAP1 germline mutations. J Clin Oncol 2012;30:e337-40.
245 [8] Gammon B, Traczyk TN, Gerami P. Clumped perinuclear BAP1 expression is a frequent finding in sporadic epithelioid Spitz tumors. J Cutan Pathol 2013;40:538-42. [9] Busam KJ, Sung J, Wiesner T, von Deimling A, Jungbluth A. Combined BRAFV600E-positive melanocytic lesions with large epithelioid cells lacking BAP1 expression and conventional nevomelanocytes. Am J Surg Pathol 2013;37:193-9. [10] Yeh I, Mully TW, Wiesner T, et al. Ambiguous melanocytic tumors with loss of 3p21. Am J Surg Pathol 2014;38:1088-95. [11] Wiesner T, Murali R, Fried I, et al. A distinct subset of atypical Spitz tumors is characterized by BRAF mutation and loss of BAP1 expression. Am J Surg Pathol 2012;36:818-30. [12] Murali R, Wilmott JS, Jakrot V, et al. BAP1 expression cutaneous melanoma: a pilot study. Pathology 2013;45:606-9. [13] Barnhill RL, Argenyi ZB, From L, et al. Atypical Spitz nevi/tumors: lack of consensus for diagnosis, discrimination from melanoma, and prediction of outcome. HUM PATHOL 1999;30(5):513-20. [14] Spatz A, Calonje E, Handfield-Jones S, Barnhill RL. Spitz tumor in children. A grading system for risk stratification. Arch Dermatol 1999; 135:282-5. [15] Phadke PA, Rakheja D, Le LP, et al. Proliferative nodules arising within congenital melanocytic nevi: a histologic, immunohistochemical, and molecular analyses of 43 cases. Am J Surg Pathol 2011;35:656-69. [16] Long GV, Wilmott JS, Capper D, et al. Immunohistochemistry is highly sensitive and specific for the detection of V600E BRAF mutation in melanoma. Am J Surg Pathol 2013;37:61-5. [17] Routhier CA, Mochel MC, Lynch K, Dias-Santagata D, Louis DN, Hoang MP. Comparison of 2 monoclonal antibodies for immunohistochemical detection of BRAF V600E mutation in malignant melanoma, pulmonary carcinoma, gastrointestinal carcinoma, thyroid carcinoma, and gliomas. HUM PATHOL 2013;44:2563-70. [18] Jensen DE, Proctor M, Marquis ST, et al. BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1mediated cell growth suppression. Oncogene 1998;16:1097-112. [19] Jensen DE, Rauscher III FJ. BAP1, a candidate tumor suppressor protein that interacts with BRCA1. Ann N Y Acad Sci 1999;886:191-4. [20] Harbour JW, Onken MD, Roberson ED, et al. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science 2010;330:1410-3. [21] Pena-Llopis S, Vega-Rubin-de-Celis S, Liao A, et al. BAP1 loss defines a new class of renal cell carcinoma. Nat Genet 2011;43:1018-21. [22] Llamas-Velasco M, Perez-Gonzalez YC, Requena L, Kutzner H. Histopathologic clues for the diagnosis of Wiesner nevus. J Am Acad Dermatol 2014;70:549-54. [23] Palmedo G, Hantschke M, Rutten A, et al. The T1796A mutation of the BRAF gene is absent in Spitz nevi. J Cutan Pathol 2004;31:266-70. [24] Pollock PM, Harper UL, Hansen KS, et al. High frequency of BRAF mutations in nevi. Nat Genet 2003;33:19-20.
www.elsevier.com/locate/humpath
Original contribution
BAP1 and BRAFV600E expression in benign and malignant melanocytic proliferations☆ Adriano Piris MD a,b,c , Martin C. Mihm Jr. MD a,b , Mai P. Hoang MD a,c,⁎ a
Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114 Department of Dermatology, Brigham and Women's Hospital, Massachusetts General Hospital, Boston, MA 02114 c Department of Pathology, Massachusetts General Hospital, Boston, MA 02114 b
Received 2 October 2014; revised 14 October 2014; accepted 24 October 2014
Keywords: BAP1; BRAFV600E; Immunohistochemistry; Nevi; Melanoma
Summary BAP1 (BRCA1-associated protein 1) is a tumor suppressor gene whose mutations have recently been reported to increase susceptibility for the development of uveal melanoma, cutaneous atypical and epithelioid melanocytic lesions, clear cell renal cell carcinoma, and other tumors. Screening for BAP1 mutation/loss/inactivation and BRAFV600E mutation can be done by immunohistochemistry. We investigated BAP1 and BRAFV600E expression in 193 sporadic melanocytic lesions (11 dermal nevi, 20 congenital nevi, 40 primary and nondesmoplastic melanomas, 40 desmoplastic melanomas, 23 metastatic melanomas, 17 Spitz nevi, 19 atypical Spitz nevi, 8 atypical Spitz tumors, 14 proliferative nodules arising in congenital nevi, 1 nevus during pregnancy) and 30 melanocytic lesions from 3 patients with family history of uveal melanoma and BAP1 germline mutation. Most sporadic melanocytic lesions exhibited positive BAP1 nuclear staining, except for 1 proliferative nodule arising in congenital nevus, 1 desmoplastic, 1 nevoid, and 2 metastatic melanomas. BRAFV600E positivity was demonstrated in 80% of dermal, 5% of congenital, 6% of Spitz, and 5.5% of atypical Spitz nevi; 29% of proliferative nodules arising in congenital nevi; and 24% of primary and nondesmoplastic and 35% of metastatic melanomas. Combined BAP1 loss and BRAFV600E staining was seen in 67% of BAP1 tumor syndrome–associated lesions and in none of the sporadic melanocytic proliferations including Spitz and atypical Spitz nevi and atypical Spitz tumors, with the exception of 1 primary melanoma. The combined BAP1-BRAFV600E+ immunoprofile appears to be a constant feature of BAP1 tumor syndrome–associated melanocytic lesions, and the designation of Spitz nevi or variants thereof appears to be inaccurate for this group of lesions. © 2015 Elsevier Inc. All rights reserved.
1. Introduction BAP1 (BRCA1-associated protein 1) is a tumor suppressor gene whose mutations have recently been reported to increase susceptibility for the development of uveal ☆
Disclosures: There was no external funding for this work. The authors have no conflict of interest to declare. ⁎ Corresponding author at: Department of Pathology, Massachusetts General Hospital, 55 Fruit St, Warren 820, Boston, MA 02114. E-mail address: [email protected] (M. P. Hoang). http://dx.doi.org/10.1016/j.humpath.2014.10.015 0046-8177/© 2015 Elsevier Inc. All rights reserved.
melanoma, cutaneous atypical and epithelioid melanocytic lesions, lung adenocarcinoma, meningioma, clear cell renal cell carcinoma, mesothelioma, and other tumors [1–6]. Loss of heterozygosity from a second mutation or deletion results in tumor formation. A BAP1 tumor predisposition syndrome (OMIM #614327) shows autosomal dominant inheritance, and individuals with heterozygous BAP1 mutation are at high risk for developing the above-mentioned tumors with varying degrees of penetrance [7]. The melanocytic neoplasms in individuals with germline BAP1 mutation have shown loss of BAP1 expression and
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often BRAF mutations [5]. They have been reported to follow a benign clinical course [2,4,5]. Clinically, they present as benign-appearing, skin-colored or pink papules or nodules with a distinct appearance. Histologically, these lesions are characterized by an expansile, nodular or sheetlike, dermal aggregate of epithelioid melanocytes with welldemarcated cytoplasmic borders and ample and amphophilic or eosinophilic cytoplasm. Their nuclei show vesicular chromatin with regular nuclear membrane and prominent nucleoli. These proliferations may or may not be associated with background ordinary dermal nevic components. In addition, they can have binucleated or multinucleated giant cells and associated lymphocytic infiltrate [2,4,5]. Currently, there is no standardized terminology regarding BAP1-mutated melanocytic lesions arising in individuals with or without germline mutation [2,4,5,8–11]. Wiesner et al [11] reported a subset of sporadic atypical Spitz tumors with epithelioid morphology to be BRAF mutated and BAP1 negative. Gammon et al [8] also reported loss of nuclear BAP1 expression in 15 of 19 sporadic epithelioid Spitz tumors (combined melanocytic tumor with nests of conventional nevus cells and nodule of epithelioid/spitzoid melanocytes). Busam et al [9] reported a series of 8 combined melanocytic lesions with BAP1 loss and BRAFV600E positivity. Yeh et al [10] reported an additional series of 29 cases of ambiguous melanocytic tumors with 3p21 loss and Spitzoid morphology, and 12 (71%) of 17 had BRAFV600E mutation. This suggests that BAP1-negative melanocytic lesions may occur in patients without the tumor predisposition syndrome. BAP1 protein may become nonfunctional by mechanisms other than gene deletion or mutation. Screening for BAP1 mutation/loss/inactivation and BRAFV600E mutation can be done by immunohistochemistry. There have been only a few studies evaluating BAP1 expression in cutaneous melanomas and other melanocytic
Table 1
lesions, and only one study using both BAP1 and BRAFV600E immunostains (Table 1) [9,12]. In order to assess the specificity and sensitivity of these 2 immunostains in detecting the BAP1 tumor syndrome–associated melanocytic lesions, we aim to investigate BAP1 and BRAFV600E expression in (1) benign as well as malignant sporadic melanocytic lesions, and (2) a series of melanocytic lesions from 3 patients with family history of uveal melanoma and BAP1 germline mutation [4].
2. Materials and methods The study has been approved by Massachusetts General Hospital institutional board review (2013-P-000312). A total of 223 cases were included in the study. Tissue microarrays were prepared from paraffin-embedded material of 125 sporadic melanocytic proliferations (11 dermal nevi, 20 congenital nevi, 31 primary melanomas, 40 desmoplastic melanomas, and 23 metastatic melanomas). Whole-slide sections were obtained from 17 Spitz nevi, 19 atypical Spitz nevi, 8 atypical Spitz tumors, 14 proliferative nodules arising in congenital nevi, 1 nevus during pregnancy, and 9 nevoid melanomas. In addition, we analyzed whole-slide sections of 30 melanocytic lesions from 3 patients (16, 11, and 3 from each patient) with BAP1 germline mutations. The ordinary and well-defined melanocytic entities in this group of lesions were categorized according to preestablished histologic criteria. Atypical Spitz nevi included lesions with overall features of a classic Spitz nevus without expansile growth but with one atypical feature such as hypercellularity, mitotic activity, or ulceration. The following criteria were used in selecting atypical Spitz tumors: an asymmetric and expansile nodule in the dermis and often the
Summary of the literature Current study BAP1 IHC
BAP1 tumor syndrome–associated lesions Dermal nevi Congenital nevi Spitz nevi Atypical Spitz nevi Atypical Spitz tumor Epithelioid Spitz tumor Proliferative nodules Nevi in pregnancy Combined nevi Primary and nondesmoplastic melanoma Desmoplastic melanoma Metastatic melanoma Uveal melanoma Abbreviation: IHC, immunohistochemistry.
30/30 (100%) 0/11 (0%) 0/20 (0%) 0/17 (0%) 0/19 (0%) 0/8 (0%)
Wiesner et al [11], BAP1 IHC
Murali et al [12], BAP1 IHC
Busam et al [9], BAP1 IHC
Gammon et al [8], BAP1 IHC
Wiesner et al [5], BAP1 mutation 51/51 (100%) 0/28 (0%) 0/17 (0%)
9/32 (28%)
2/18 (11%) 15/19 (79%)
1/13 (8%) 0/1 (0%) 8/8 (100%) 1/40 (2.5%) 1/40 (2.5%) 2/23 (9%)
4/135 (3%) 5/18 (22%)
3/60 (5%)
13/33 (40%)
BAP1 and BRAFV600E expression
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subcutaneous fat, lack of maturation, monomorphic and atypical tumor cells, and presence of deep and often marginal mitoses (Fig. 1) [13,14]. The defining criteria of proliferative nodules (8 benign, 6 atypical) arising in a background of congenital nevi were described in a prior study [15]. The BAP1-mutated melanocytic proliferations were selected from 3 patients belonging to a family with known BAP1 germline mutation [4]. All of these atypical melanocytic proliferations were excised and none had recurred. None of these lesions were melanomas. The histologic features of the latter are described below.
For anti-BRAFV600E, positive staining was characterized by diffuse and moderate (2+) to strong (3+) cytoplasmic staining of the tumor cells. Isolated nuclear staining, weak staining of occasional cells, or faint diffuse staining were considered as negative staining as previously reported [16,17]. A malignant melanoma with confirmed BRAFV600E mutation by molecular analyses was used as positive control.
2.1. Immunohistochemistry
The immunohistochemical results are summarized in Table 2. All 30 BAP1 tumor syndrome–associated melanocytic lesions exhibited loss of BAP1 nuclear staining. Cytoplasmic staining was noted in most cases, with marked cytoplasmic staining seen in 7 cases (23%). In 8 lesions (27%), dotlike BAP1 cytoplasmic positivity was noted. Most sporadic melanocytic lesions exhibited positive BAP1 nuclear staining. The exceptions were 1 proliferative nodule arising in a congenital nevus, 1 desmoplastic melanoma, 1 nevoid melanoma, and 2 metastatic melanomas. None of these 5 patients had personal or family history of cutaneous or uveal melanomas. The 4 melanomas with loss of BAP1 expression did not have special morphologic features that would set them apart from their respective melanoma subtype. BRAFV600E positivity was demonstrated in 80% of dermal nevi, 5% of congenital nevi, 6% of Spitz nevi, 5.5% of atypical Spitz nevi, 29% of proliferative nodules arising in
Immunohistochemical studies were performed on 5-μm-thick sections of formalin-fixed, paraffin-embedded tissue in a Bond 3 automated immunostainer (Leica Microsystems, Bannockburn, IL) with primary antibodies against BAP1 (BP-1, 1:400; Santa Cruz Biotechnology, Santa Cruz, CA) and BRAFV600E (clone: VE1, 1:100; Spring Bioscience, Pleasanton, CA). Appropriate positive and negative controls were included. The immunohistochemical stains were evaluated by 2 of the authors (A. P. and M. P. H.), and disagreements were reviewed together to achieve a consensus score. For anti-BAP1, nuclei of keratinocytes of the epidermis and appendages, fibroblasts, and lymphocytes served as internal positive controls for BAP1 expression. Tumors were scored as positive or negative based on whether their nuclei stained with BAP1.
3. Results
Fig. 1 In this atypical Spitz tumor, an expansile proliferation of epithelioid and spindled neoplastic cells is seen infiltrating into the subcutaneous tissue (A, ×40; B, ×100). The tumor cells exhibit strong nuclear BAP1 staining (C, ×100) while negative for BRAFV600E (D, ×100).
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congenital nevi, 24% of primary and nondesmoplastic melanomas, and 35% of metastatic melanomas. Sixty-seven percent of BAP1 tumor syndrome–associated lesions had combined BAP1 loss and BRAFV600E staining. In most cases, the large epithelioid cells lacked nuclear BAP1 staining and exhibited cytoplasmic BRAF staining (Fig. 2). In 9 cases, the background smaller nevus cells showed nuclear BAP1 and cytoplasmic BRAF staining (Fig. 3).
3.1. Statistical analyses When the statistical association of each stain in BAP1 tumor syndrome–associated melanocytic lesions versus those not associated with BAP1 germline mutations was evaluated by Fisher exact test, a significant P value (.0001) was observed for BAP1 loss, BRAFV600E positivity, and combined BAP1 loss and BRAFV600E positivity (Table 2).
3.2. Histology of BAP1-mutated melanocytic tumors There were 2 observed growth patterns: (1) a single welldemarcated dermal nodule comprising epithelioid melanocytes (Fig. 2) and (2) more than 1 nest of epithelioid melanocytes in a background of a diffuse dermal melanocytic nevus, often with features of congenital onset (Fig. 3). In the second pattern, the number and density of these “atypical” nests were variable. When the nests were prominent, the pattern was a diffuse growth in the dermis. When only a few nests were seen, the pattern was that of a dermal nevus–like proliferation with features of congenital onset. A lentiginous junctional component was noted in 13 (43%) cases. Benign dermal nevic cells were seen at the periphery of 9 (30%) cases. The lesional cells were often epithelioid in appearance with ample cytoplasm and well-defined cytoplasmic border. Moderate cytologic atypia, multinucleated tumor cells, and Table 2
4. Discussion BAP1 is a deubiquitinating enzyme whose gene is located on chromosome region 3p12 [18]. BAP1 has been suggested to be a tumor suppressor gene with a role in cell proliferation and growth inhibition [19]. Monosomy of chromosome 3 is the most common somatic alteration in uveal melanoma with BAP1 somatic mutations in 47.4% of uveal melanomas [20]. In most cases, BAP1 loss is somatically acquired, but multiple tumors tend to be associated with a BAP1 germline mutation. The incidence of BAP1 germline mutation is 4 of 100 patients with ocular melanoma and 3 of 200 patients with a family history of cutaneous melanoma [4]. Consistent with prior studies, we found that the BAP1 tumor syndrome–associated melanocytic lesions are typically dome shaped, 5 mm in size, and located on the head and neck, trunk and extremities, and the number ranged from 5 to more than 50 [2,4,5]. Histologically, there appear to be 2 distinct histologic patterns—a single dominant nodular pattern and dermal nevus–like pattern often with features of congenital onset [2,4,5,9]. These lesions can present as large epithelioid cell proliferations or in combination with conventional nevi [9]. The lesional cells are epithelioid melanocytes with abundant amphophilic cytoplasm, well-defined cytoplasmic borders, vesicular chromatin, multinucleation, cytologic atypia, and minimal mitotic figures. Tumor-infiltrating lymphocytes are often identified [2,4,5,9]. In contrast to Spitz nevi, these lesions lack the epidermal hyperplasia and large epidermal nests with associated retraction artifact. In some cases, hypercellularity, nuclear pleomorphism, and prominent mitotic figures were seen, raising the concern of nevoid melanoma [4]. Often there was an associated proliferation of
Summary of immunohistochemical findings
Total BAP1 tumor syndrome–associated melanocytic lesions Sporadic melanocytic lesions Dermal nevi Congenital nevi Spitz nevi Atypical Spitz nevi Atypical Spitz tumor Proliferative nodules Nevi in pregnancy Primary melanoma Desmoplastic melanoma Metastatic melanoma Pa a
some degree of inflammation were noted in most cases. The host response was prominent in 9 (30%) cases.
n
Loss of BAP1 expression
BRAFV600E positivity
BAP1 loss and BRAFV600E positivity
223 30 193 11 20 17 19 8 14 1 40 40 23
35/222 (16%) 30/30 (100%) 5/192 (3%) 0/11 (0%) 0/20 (0%) 0/17 (0%) 0/19 (0%) 0/8 (0%) 1/13 (8%) 0/1 (0%) 1/40 (2.5%) 1/40 (2.5%) 2/23 (9%) .0001
53/218 (24%) 20/30 (67%) 33/188 (18%) 8/10 (80%) 1/20 (5%) 1/16 (6%) 1/18 (5.5%) 0/8 (0%) 4/14 (29%) 1/1 (100%) 9/38 (24%) 0/40 (0%) 8/23 (35%) .0001
21/217 (10%) 20/30 (67%) 1/187 (0.5%) 0/10 (0%) 0/20 (0%) 0/16 (0%) 0/18 (0%) 0/8 (0%) 0/13 (0%) 0/1 (0%) 1/38 (3%) 0/40 (0%) 0/23 (0%) .0001
BAP-1–associated vs non-BAP1 melanocytic lesions; statistically significant.
BAP1 and BRAFV600E expression
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Fig. 2 A single well-demarcated nodule is seen in dermis (A, ×40), which comprises epithelioid cells with ample cytoplasm and well-demarcated border (B, ×200). The tumor cells are negative for nuclear BAP1 staining (C, ×100) and strongly positive for BRAFV600E (D, ×100).
smaller nevus cells, and thus, they can be designated as a combined nevus. In the current study, we found that all BAP1 tumor syndrome–associated melanocytic lesions showed BAP1 nuclear loss. However, cytoplasmic staining was seen in most cases and perinuclear dotlike positivity was noted in 27% of cases, similar to findings previously reported [2,8]. Gammon et al [8] reported that 8 (53%) of 15 sporadic epithelioid Spitz tumors in their series exhibited BAP1 loss and clumped perinuclear staining. The authors hypothesized that this perinuclear staining pattern could be the result of aberrant protein accumulation in the Golgi region rather than in the nucleus [8]. The incidence of BAP1 loss of expression in melanomas was 5.6%, similar to the 5% BAP1 mutation rate reported in 60 sequenced melanomas (45 cutaneous and 15 mucosal) [5,12]. In a series by Murali et al [12], 22% (5/18) of desmoplastic melanomas showed loss of BAP1 expression in comparison with 3% (4/135) of nondesmoplastic melanomas. On the contrary, we noted BAP1 loss only in 1 case of desmoplastic melanoma. The expression/mutation of BAP1 in congenital nevi, proliferative nodules arising in congenital nevi, nevus in pregnancy, and nevoid melanoma (lesions in the differential diagnosis of BAP1 tumor syndrome– associated melanocytic lesions) has not been studied. The frequency of BAP1 loss (2%-11%) in these lesions was low in comparison with that of BAP1 tumor syndrome– associated melanocytic lesions (100%; Table 2). It has been reported that the BAP1 tumor syndrome– associated melanocytic lesions have loss of BAP1 expression
and BRAFV600E mutation [5]. Accordingly, we noted that 67% of BAP1 tumor syndrome–associated lesions had combined BAP1 loss and BRAFV600E staining. Similar to findings reported by Busam et al [9] in a series of 8 cases of combined melanocytic lesions (an epithelioid component and conventional nevus component), the cells within the nodules in our cases were BAP1 negative and BRAFV600Epositive, whereas the background nevus cells when present were BAP1 positive and BRAFV600E positive. In all studied categories, combined BAP1 loss and BRAF staining appears to be a feature of BAP1 tumor syndrome–associated melanocytic lesions. The loss of BAP1 expression has been shown to correlate with biallelic BAP1 mutation, with positive and negative predictive values of 100% and 98.6%, respectively [11,21]; thus, screening by immunohistochemistry is cost-effective. False-negative interpretation can be avoided if internal positive controls (lymphocytes, fibroblasts) are present. Proposed names for BAP1 tumor syndrome–associated melanocytic lesions include “melanocytic BAP1-mutated atypical intradermal tumors” (MBAITs) and “nevoid melanoma-like melanocytic proliferation” (NEMMPs) [2,4]. Llamas-Velasco et al [22] described these lesions as “Wiesner's nevus,” and Yeh et al [10] designate them as “Spitz nevi associated with BAP1 loss.” Furthermore, in their description of these “BRAF-mutated/BAP1-negative” lesions, Wiesner et al [11] use the term atypical Spitz tumors. These authors assessed BRAF mutation and BAP1 expression in 32 sporadic atypical Spitz tumors. Nine (28%) of 32 exhibited BAP1 loss and 8 of 9 lesions with BAP1 loss had
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Fig. 3 A nodular growth of melanocytes comprises multiple nests of melanocytes seen in a background sheet-like proliferation of melanocytes (A, ×40; B, ×200). The lesional cells are negative for nuclear BAP1 staining, although cytoplasmic staining is noted (C, ×200). The entire lesion is positive for BRAFV600E (D, ×200). A dermal nevus–like proliferation with features of congenital onset is seen in the dermis (E, ×40; F, ×200). Nuclear BAP1 staining is seen in the background nevus cells, whereas the epithelioid cells are negative (G, ×200). Both components are positive for BRAFV600E (H, ×100).
BRAF mutation. They concluded that BRAF mutation together with BAP1 loss define a distinct subset of atypical Spitz tumors. None of our 17 Spitz nevi, 18 atypical Spitz nevi, and 8 atypical Spitz tumors exhibited the combined
BRAF + BAP1− phenotype. In addition, Spitz nevi have not been reported to harbor BRAF mutation [23], whereas common acquired nevi frequently have BRAF mutation [24]. Long-term follow-up was not available for these series
BAP1 and BRAFV600E expression [5,11]; thus, the question remains as to whether these tumors are atypical Spitz tumors or sporadic BAP1-associated melanocytic lesions. In summary, in our series of benign and malignant melanocytic lesions, the combined BAP1 loss and BRAFV600E positivity by IHC appears to be a constant feature of BAP1 tumor syndrome–associated melanocytic lesions, and the designation of Spitz nevi or variants thereof appears to be inaccurate for this particular group of lesions.
Acknowledgment We thank Drs Yayan Chen and Fang Liu, Department of Pathology, Saint Vincent Hospital, Worcester Medical Center, Worcester, MA, for their contribution to the cases.
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