Immunohistochemistry for the Diagnosis of Melanocytic Proliferations

29  Immunohistochemistry for the Diagnosis of Melanocytic Proliferations Achim A. Jungbluth and Klaus J. Busam

OUTLINE The Reagents, 348 Differentiation Markers, 348 Biomarkers, 354 Mutation Antigens, 358 Immune-Response Score, 359 Basic Applications of Immunohistochemistry, 359 Visualization of Melanocytes, 359

Immunohistochemistry (IHC) is an important ancillary method for the histopathologic diagnosis of melanocytic proliferations. Its purpose is to correlate the presence or absence of an antigen of interest with a cell or group of cells, such as to determine its line of differentiation. Antibodies to several antigens associated with melanocytic differentiation are available (see below). They help visualize melanocytes of normal skin (Fig. 29.1) and melanocytic tumors. Sometimes a single reagent may be sufficient; at other times a panel of multiple antibodies may be necessary both in support of melanocytic as well as alternate lines of differentiation to classify a tumor. IHC may also be used to look for the expression of miscellaneous antigens associated with biologic functions (so-called biomarkers), such as cell growth, and used to learn more about the nature of the tumor for either diagnosis or prognosis. Markers of interest for this purpose include p16, Ki-67, pHH3, PRAME, and 5-hydroxymethylcytosine. Finally, in the era of personalized medicine IHC is also used to identify antigens which can help predict treatment response, such as to determine mutation status of a melanoma for targeted therapy (BRAFV600E, RASQ61R). On occasion the mutation status can also be used for diagnostic purposes.

THE REAGENTS Differentiation Markers S100.  S100 protein is a family of calcium-binding proteins. The name “S100” derives from the original isolation procedure and refers to the protein’s solubility in a saturated (100%) ammonium sulfate solution of bovine brain.1 S100 proteins are involved in diverse intracellular and extracellular functions. S100 proteins are expressed in many different tissues and organs.2 Among the more than 20 known members of the S100 protein family, S100B is the most prevalent in melanoma. However, several other S100 proteins are also expressed in melanoma. Early immunohistochemical studies of S100 in melanoma were based on polyclonal reagents, which were not isoform specific. Currently, commercial reagents are available, which can distinguish between many 348

Immunohistochemistry for the Distinction of a Melanocytic from Nonmelanocytic Tumor, 360 Immunohistochemistry for the Distinction of a Melanocytic Nevus from Melanoma, 360 Immunohistochemistry for Treatment Selection and/or Staging Purposes, 362

S100 isoforms. However, since S100 is used for its sensitivity, specific isoform detection is rarely of interest. S100 negative melanomas are rare. S100P is positive in all subtypes of melanoma, including desmoplastic melanoma, for the diagnosis of which it remains an important reagent. Along with Sox10 and nerve growth factor receptor (NGFR), it is the most sensitive marker to visualize invasive melanoma (Fig. 29.2). In primary melanocytic tumors S100 protein usually uniformly stains nearly all neoplastic melanocytes. S100 protein is less suitable for visualizing intraepidermal melanocytes because it is both less sensitive (more melanocytes are highlighted by antibodies to melan-A, tyrosinase or Sox10 than by anti-S100) and less specific than other melanocyte markers. In contrast to other markers, S100 protein highlights also Langerhans cells in the skin and dendritic cells in lymph nodes. Metastatic tumors variably stain for S100 protein. The majority, all or nearly all, or only a minor population may be positive. On rare occasion, in the case of a dedifferentiated melanoma, a tumor may be negative for S100P.

GP100 (PMEL).  GP100 refers to the glycoprotein of 100 kDa, which is encoded by the PMEL (premelanosomal protein) gene, the human homologue of the mouse silver locus, and recognized by mAb HMB45. This antibody was generated by an immunization procedure employing preparations of a pigmented melanoma metastasis, as indicated by its acronym HMB referring to “human melanoma black.”3 The antigen is also referred to as Pmel17 (silv).4,5 GP100 is a structural component of the melanosomes. Its expression in melanosomes depends on the maturation stage. Some melanocytes of normal skin may express sufficient GP100 to be immunoreactive with HMB45, but others not. HMB45 is not a sensitive marker for the detection of normal intraepidermal melanocytes (see Fig. 29.1). Conventional intradermal and some compound melanocytic nevi are not infrequently negative for HMB45 (Fig. 29.3). In common junctional and compound melanocytic nevi, HMB45-positive cells are mostly present at the dermoepidermal junction, usually with diminished expression and/or loss from the top to the bottom of a melanocytic

CHAPTER 29  Immunohistochemistry for the Diagnosis of Melanocytic Proliferations

Keywords immunohistochemistry antbodies differentiation antigens biomarkers S100 Sox10 Melan-A MART1 tyrosinase HMB-45 ALK NTRK p16 BAP1 PNL2 PRAME BRAFV600E

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Fig. 29.1  Visualization of normal skin using antibodies to different markers: S100 protein, melanoma antigen (Melan-A), tyrosinase (TYR), GP100 (=antigen recognized by HMB45), microphthalmia transcription factor (MITF), and Sox10 (SOX).

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Fig. 29.2  (A) Mixed solid and desmoplastic melanoma (hematoxylin and eosin). (B) S100 highlights all tumor cells. (C) Melanoma antigen is expressed mainly in the solid tumor component and by in situ melanoma.

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Fig. 29.3  Melanocytic Nevus. The lesional melanocytes express melanoma antigen (Melan-A) and p16. They are negative for HMB45 and do not label for Ki-67.

Fig. 29.4  Only the junctional component and a few superficial dermal melanocytes of a compound melanocytic nevus stain with HMB45.

nevus (Fig. 29.4). Some melanocytic nevi, however, may stain uniformly for HMB45, such as blue nevi or deep penetrating nevi (Fig. 29.5). Staining for HMB45 is highly variable in congenital nevi or Spitz nevi. HMB45 is widely expressed in conventional melanomas (Fig. 29.6), especially those with epithelioid cells. In primary melanomas, HMB45 immunostaining is seen in at least 70% of tumors.3,6,7 Sensitivity of HMB45 in metastatic melanoma is somehow lower, with reports between around 60% of cases showing some form of immunoreactivity. In spindle cell melanomas, HMB45 is often less or not expressed. Most desmoplastic melanomas lack expression for HMB45 in the invasive spindle cell component. Expression may be homogeneous or spotty. HMB45 reactivity is usually confined to melanocytes and tumors of melanocytic differentiation, with rare exceptions. Angiomyolipomas

and perivascular epithelioid cell tumors (PEComas) often stain for HMB45. Focal staining for HMB45 may also occasionally be seen in atypical fibroxanthoma. Variations in reported staining results likely reflect heterogeneity in the IHC methods. At the time of its generation, HMB45 staining protocols involved enzyme digestion, which preceded heat-mediated antigen retrieval techniques. Currently, HMB45 is offered by several commercial providers. The specifications as to the proper antigen retrieval differ. Technical recommendations vary from no antigen retrieval (HMB45, #911501, BioLegend, San Diego, CA), enzymatic antigen retrieval (HMB45, ab787, Abcam, Cambridge, MA; NCL-LHMB45, Leica, Buffalo Grove, IL), and no enzyme digestion (HMB45, DAKO/Agilent, Santa Clara, CA) to heat-induced antigen retrieval using citrate or EDTA buffers (HMB45, 282M, Cell Marque/Merck, Darmstadt, Germany; HMB45, CM057, Biocare, Pacheco, CA). The variability of staining protocols is also reflected in the literature where no antigen retrieval, enzyme digestions, or heat-induced epitope retrieval were employed. This likely accounts for the significant variations in reported frequencies of immunoreactivity of melanocytic tumors in the literature. The authors of this textbook use heat-induced retrieval for immunostaining with HMB45. The difference between the various immunostaining pretreatments appears most significant in the assessment of melanocytes in normal skin and less so in melanomas.

Tyrosinase.  Tyrosinase is the key enzyme of melanin biosynthesis. It was first identified and named by the French chemist Gabriel Bertrand while studying the blackening of mushroom.8 It is a highly conserved molecule present in many species including microbial organisms, plants, and vertebrates to induce an enzymatic darkening process. Tyrosinase gained the interest of immunologists because it was known that melanoma patients could form an immune response to antigens related to

CHAPTER 29  Immunohistochemistry for the Diagnosis of Melanocytic Proliferations

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Fig. 29.5  Combined melanocytic nevus with a prominent pigmented deep penetrating nevus population (A–C). The pigmented melanocytes are positive with HMB45. All lesional melanocytes express BRAFV600E.

melanogenesis. A monoclonal antibody, clone T311, was developed for therapeutic purposes, but had also diagnostic value.9 In normal skin, T311 immunoreactivity is restricted to melanocytes in the epidermis and follicles. In acquired common melanocytic nevi, tyrosinase is predominantly expressed in junctional and/or superficial dermal melanocytes. Staining is usually minimal or absent in fusiform melanocytes of a neurotized nevus. In primary melanomas with an epithelioid cytomorphology, tyrosinase is expressed in most lesions. In metastatic melanoma, T311 immunoreactivity ranges between 60% and 90%. The variations in reported tyrosinase expression are in part related to tumor heterogeneity between various series and in part due to different methods. Good sensitivity for the detection of melanocytes can be obtained by using a high pH buffer and heating time of 30 minutes.10 As with HMB45, T311 shows little to no reactivity in many spindle cells, in particular desmoplastic melanomas.

TRP1.  TRP1 refers to tyrosinase-related protein. TRP1 is encoded by the TYRP1 gene. It is the human homologue of the brown (b)-locus in mice, the corresponding protein of which is associated with melanogenesis and pigmentation disorders of the skin. Other names for the protein include pigment-associated antigen and glycoprotein of 75 kDa (gp75).11 The exact function of TRP1 is still unknown. Besides its enzymatic activity, it may also play a structural role in the melanosome. Compared with other melanocyte differentiation antigens, little is known about the in situ protein expression of TRP1. Melanocytes of normal skin consistently express TRP1. Based on a limited number of cases studied, TRP1 expression in melanocytic nevi tends to parallel that of

tyrosinase. There is insufficient knowledge on the sensitivity and specificity of TRP1 for the diagnosis of melanoma, but most epithelioid melanomas seem to be immunoreactive for this marker.

TRP2.  Tyrosinase-related protein-2 (TRP2) is another melanocyte differentiation antigen.12 It is an enzyme, dopachrome tautomerase (DCT), which is involved in the modification of the pigment color. The absence of TRP2 does not lead to a loss of pigmentation, but rather to a color modification. Like other melanocyte differentiation antigens, TRP2 can elicit an autologous immune response in melanoma patients. Knowledge about TRP2 as a diagnostic reagent is currently still limited. A recent expression analysis in normal and tumor tissues using clone C-9, a novel commercial monoclonal antibody to TRP2, documented that except for some granular staining in hepatocytes and alveolar macrophages, reactivity of C-9 in normal tissues was only seen in melanocytes. In melanomas, TRP2 expression was found in 84% of primary 60% of metastatic melanomas. Desmoplastic melanomas are usually negative for TRP2. Melan-A/MART1.  Employing their “autologous T-cell epitope cloning” technique, Boon and colleagues identified a novel gene termed Melan-A (Melanoma Antigen).13 Shortly after, Kawakami and colleagues at the National Institutes of Health (NIH) identified the same gene analyzing the antigenic targets of tumor-infiltrating T-lymphocytes and named it MART1 (Melanoma Antigen Recognized by T-cells).14 Molecular analyses of Melan-A/MART1 showed an expression pattern characteristic of a melanocyte differentiation antigen. Melan-A/MART1 is a structural

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Fig. 29.6  Primary nodular melanoma homogeneously immunoreactive with HMB45, and negative for p16.

component of the melanosome without enzymatic function. It is outside the melanosome and localized to the Golgi apparatus. Various monoclonal antibodies to Melan-A/MART1 are available commercially. The oldest and most commonly used reagents are clones M2-7C10 (anti-MART1) and A103 (anti-Melan-A).15,16 They were generated at the NIH and the New York branch of the Ludwig Institute for Cancer Research (LICR), respectively, to evaluate the usefulness of Melan-A/MART1 as potential vaccine targets for cancer immunotherapy. Their potential as diagnostic marker was soon recognized.17,18 Both reagents show a similar staining pattern, except that A103 shows more consistent immunopositivity in steroid hormone producing cells and tumors.19 In normal skin, Melan-A/MART1 is usually expressed by intraepidermal and intrafollicular melanocytes (see Fig. 29.1). The number of Melan-A/MART-positive melanocytes is usually higher than for GP100 (as detected by HMB45) but lower than for TRP1 and tyrosinase. Melan-A/MART1 is expressed by most nevi.20 Different from HMB45, A103 immunoreactivity is homogeneously present in the dermal component of most compound and intradermal nevi of various subtypes as long as the melanocytes retain some epithelioid cell features (see Fig. 29.3). Neurotized or fusiform melanocytes may be negative (Fig. 29.7). Melan-A/MART1 is consistently expressed by most nodal melanocytic nevi. Most primary conventional melanoma, especially with an epithelioid cell phenotype, tend to express Melan-A/MART1 (Fig. 29.8). In most studies, 60% to 90% of melanomas are positive. In general, melanomas more likely express Melan-A/MART1 than GP100. The expression in primary or metastatic melanomas may be homogeneous (Fig. 29.9), but is not infrequently heterogeneous, with only a subpopulation of tumor cells immunoreactive for melan-A (Fig. 29.10). Spindle cell melanomas, in particular desmoplastic melanomas, are often negative for Melan-A/MART1. If they are immunoreactive for this antigen, staining is usually limited to associated in situ melanoma, superficial dermal tumor cells, or epithelioid cells of mixed conventional and desmoplastic melanoma. The invasive spindle cell component of pure desmoplastic melanomas is usually negative for Melan-A/MART1.

MITF.  Microphthalmia transcription factor (MITF) is a basic-loophelix-loop leucine zipper (bHLH-Zip) transcription factor encoded by the MITF gene.21 MITF is the human homologue of the mi gene in mice and can serve as an example how genetic research in mouse mutations resulted in immunohistochemical reagents useful for surgical pathology. MITF/mi was originally identified due to mutations that led to pigment disorders of the coat of mice. Genetic defects associated with MITF in humans are also known, such as Waardenburg 2A syndrome and Tietz-Albinism-Deafness syndrome. MITF homodimerizes or forms heterodimers with other transcription factors such as TFE3, TFEB, and TFEC with which it forms the MITF transcription factor family.22 Alternative splicing renders various MITF isoforms, some of which are widely distributed while others are tissue-restricted. Consequently, MITF plays a role in processes as diverse as melanogenesis, hematopoiesis, and bone formation. Isoform MITF-M refers to the isoform expressed in melanocytes. It activates the transcription of the key players of melanogenesis such as Melan-A, tyrosinase, TRP1, TRP2, and GP100. Several commercial reagents provided by various manufacturers for the immunohistochemical analyses of MITF are available. Most commonly used are clones D5 and C5,23,24 although other monoclonal and polyclonal antibodies are available such as clone 21D1418, clone EPR9731, clone MITF/915, clone 3F276, and several others. None of the currently available anti-MITF reagents was raised to the melanocyte-specific isoform MITF-M. Consequently, immunohistochemical studies employing current anti-MITF reagents will detect various MITF isoforms, and immunoreactivity will not be restricted to melanocytes or melanocytic lesions. As a result, immunoreactivity for MITF reagents has been demonstrated in various normal and tumorous nonmelanocytic tissues and lesions such as inflammatory cells, in particular mast cells and histiocytes, smooth muscle cells, and a number of lesions relevant for the differential diagnosis of melanocytic neoplasms, such as atypical fibroxanthoma or cellular neurothekeoma. Other D5-positive lesions include giant cell tumors. The monoclonal antibody D5 binds to melanocytes with a sensitivity comparable to other melanocyte differentiation antigens. MITF is homogeneously expressed by most melanocytic nevi and primary melanomas, except for desmoplastic melanoma and neurotized lesions.24 Due to the biology of MITF and the reactivity of the currently available reagents with various isoforms, the value of MITF in melanoma is limited and is mostly based on its sensitivity. SOX10.  SOX10 (sex-determining region Y-box 10) is a transcription factor.25 It is part of a gene family of approximately 20 members. Structurally they are characterized by a DNA-binding HMG (High Motility Group) box domain. The different SOX gene members exert various biological functions such as sex determination and neuronal development. Among the many SOX genes, SOX9 and SOX10 are those involved in melanogenesis. Older expression data were generated with a polyclonal goat antibody (N-20, Santa Cruz Biotechnology, Santa Cruz, CA), which since has been withdrawn from the market. SOX10 tends to be expressed by neural crest-derived cells (Schwann cells, melanocytes).26 Expression is found in melanocytes of normal skin (see Fig. 29.1), melanocytic nevi, and primary and metastatic melanoma. SOX10 tends to be homogeneously expressed in most melanomas. Like S100, positive labeling for SOX10 is found in most spindle cell melanomas, including desmoplastic melanomas, but a rare undifferentiated melanoma may lack SOX10 expression. SOX10 has high sensitivity for melanocytic neoplasms.26,27 However, SOX10 expression can also be found in nerve sheath neoplasms (Schwannoma, neurofibroma, malignant peripheral nerve sheath tumor), and in some epithelial tumors (myoepithelial neoplasms, pleomorphic adenomas, mammary carcinomas, and other tumors).26,27 Like other melanocyte markers, the “specificity” of SOX10

CHAPTER 29  Immunohistochemistry for the Diagnosis of Melanocytic Proliferations

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Fig. 29.7  (A) and (B) Compound melanocytic nevus with prominent neurotized dermal spindle cell component. (C) The epithelioid melanocytes express Melan-A. The fusiform melanocytes are negative.

is limited by its presence in cells and tissues other than melanocytes, such as mast cells. While SOX10 has been advocated for detecting residual melanocytic tumor, especially desmoplastic melanoma in a scar, some normal scars contain scattered SOX10-positive cells, which is why one cannot rely on immunohistochemical labeling alone, but needs to correlate the staining with the cytologic and histopathologic and clinical context.

Fig. 29.8  (A) Inflamed melanoma. (B) Immunohistochemistry for Melan-A highlights the melanocytes and their growth pattern.

PNL2.  PNL2 is a monoclonal antibody that was raised to human somatostatin receptor and found to label melanocytes and melanomas instead of the intended target protein.28 Staining with PNL2 shows similar sensitivity as antibodies to Melan-A/MART1 for the detection of normal melanocytes, melanocytic nevi, and primary and metastatic melanomas.29 The vast majority of metastatic melanomas (90%) are immunoreactive with PNL2. Like antibodies to Melan-A/MART1, PNL2 usually does not label desmoplastic melanoma. It also labels tumors in the PEComa family.

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B Fig. 29.9  (A) and (B) Metastatic melanoma in lymph node displaying an epithelioid cytology (hematoxylin and eosin). The tumor cells homogeneously express Melan-A and BRAFV600E.

Biomarkers

Fig. 29.10  Primary nodular melanoma with expression of melanoma antigen (Melan-A) in only a subpopulation of tumor cells.

Ki-67.  Ki-67 is a marker of cell proliferation. It is recognized by the antibody MIB-1. One finds nuclear expression of Ki-67 in cells in cell cycle phases G1, M, G2, and S, but not in G0, the resting phase. Since Ki-67 is expressed by all cells, a dual labeling with a cytoplasmic marker, in particular Melan-A/MART1, is recommended for assessing Ki-67 expression in melanocytes. In general, Ki-67 tends to be more expressed by melanomas than nevi,30,31 but there are exceptions. While in most nevi less than 2% of lesional melanocytes express Ki-67, it may be higher in mitotically active nevi, such as during pregnancy or in early childhood. In particular, benign proliferative nodules may display a high Ki-67 labeling index. Furthermore, some melanomas, like pure desmoplastic melanomas, may label for Ki-67 in less than 2% of its tumor cells. Although there are major imitations to the diagnostic use of Ki-67 labeling, an elevated proliferation index can be helpful for the distinction of a nevus from nevoid primary or metastatic melanoma (see Figs. 29.3, 29.11). PHH3.  Histone H3 is phosphorylated in the late G2 and M phases of the cell cycle. IHC for PHH3 (phosphor-histone H3) has been advocated

CHAPTER 29  Immunohistochemistry for the Diagnosis of Melanocytic Proliferations

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Fig. 29.11  Blue Nevus-like Metastatic Melanoma. (A) Pigmented tumor cells in the dermis. (B) Several lesional melanocytes label for Ki-67 (arrows point to cells with dual nuclear expression of Ki-67 and cytoplasmic labeling for Melan-A). This would not be expected in a common blue nevus. The clinical findings (pigmented lesion near excision of previously excised melanoma), histopathologic and immunohistochemical findings fit best with a nevus-like in-transit melanoma metastasis.

to use for mitotic counting because it facilitates recognition of cells in mitosis, and their distinction from “pseudo-mitotic structures,” i.e., nuclei undergoing apoptosis or pyknosis resembling a mitotic figure.32 Since PHH3 stains cells already in the mitotic prophase, which cannot be recognized on a routine hematoxylin and eosin (H&E)-stained section, mitotic counts obtained using IHC for PHH3 tend to be higher than those obtained from counting well-developed mitotic figures on routine sections. As for Ki-67, a dual stain with a melanocyte differentiation marker is recommended to ensure that only melanocytes in mitoses are counted and not inflammatory or other cells. PHH3 has gained in popularity for counting mitotic figures in response to the seventh edition of the AJCC melanoma staging system, which elevated the importance of the mitotic count, as it became a crucial parameter for the distinction of pT1a from pT1b stage melanomas. The subsequent increase in reported mitotic counts in thin melanomas contributed to a revised staging system (AJCC, 8th edition), in which mitotic figures are no longer needed for staging. Accordingly, there is limited need for using PHH3.

P16.  p16 is a tumor suppressor protein encoded by the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, located on chromosome 9p21.3. It plays an important role in cell cycle regulation by decelerating cell progression from G1 phase to S phase. IHC for p16 shows a range of findings in melanocytic lesions, with conflicting reports in the literature about the value of IHC for p16 or lack thereof for the distinction of melanocytic nevi from melanoma.30 While most nevi label for p16, there is not uncommonly a checkered labeling pattern—some cells label, but not all. In general, complete loss of labeling for p16 is more likely seen in melanomas (see Fig. 29.6) than in nevi, but benign nevi may on occasion also lack expression of p16, and some melanomas retain expression of p16 (Fig. 29.12). Based on the observation that homozygous deletions in the majority of tumor cells of an atypical spitzoid melanocytic neoplasm is associated with a higher risk for recurrence and may assist in classifying a tumor as spitzoid melanoma of childhood, IHC for p16 is often used by dermatopathologists for the distinction of Spitz nevi from melanoma. Complete lack of labeling

Fig. 29.12  Primary melanoma with homogeneous expression of p16.

for p16 may prompt further assessment of a lesion by FISH for 9p, if the histopathologic findings are worrisome for melanoma.

BAP1.  The BRCA-associated protein-1 (BAP1) is a deubiquitinating enzyme encoded by the BAP1 gene located on chromosome 3p21.31p21.2. It is widely expressed in normal tissues and functions as a tumor suppressor. It is expressed in the nucleus of a cell. Genomic changes impacting BAP1 may be associated with complete loss of the protein and complete lack of labeling for BAP1 using IHC. At times, however, IHC for BAP1 may be associated with a granular cytoplasmic staining in a Golgi-like pattern. As long as the nucleus is negative, this also counts as a negative staining result. Loss of nuclear staining may be associated with a BAP1 mutation or allelic loss. Truncating mutations

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before the nuclear localization sequence may be responsible for cytoplasmic aggregation. In dermatopathology, IHC for BAP1 is most often used to confirm the impression of a BAP1-deficient epithelioid melanocytic nevus/tumor (Fig. 29.13).33 If a patient has multiple BAP1deficient lesions, consideration needs to be given to the possibility of an underlying tumor predisposition syndrome related to a germline mutation of BAP1. IHC for BAP1 can also be helpful for the distinction of a cellular blue nevus from melanoma arising in association with or simulating the appearance of a blue nevus. Most cellular blue nevi retain expression of BAP1. Selective loss in a tumor component with atypical histopathologic features is strongly associated with melanoma.34 BAP1 expression may also be diagnostically useful for the distinction of a melanocytic nevus (e.g., blue nevus), primary nodular conventional or metastatic cutaneous melanoma from metastatic uveal melanoma to the skin. Most metastasizing uveal melanomas display loss of labeling for BAP1. Thus, for uveal melanoma BAP1 is a prognostic biomarker. Loss of BAP1 implies a higher risk for metastasis.

ALK.  Anaplastic lymphoma kinase (ALK), aka CD246, is a tyrosine kinase receptor encoded by the ALK gene located on chromosome 2p23.2-p23.1. Commonly used monoclonal antibodies to ALK include the clones 5A4 and D5F3. IHC for ALK may be used for the analysis of Spitz tumors, since they may be associated with ALK-rearrangement and overexpression.35 Spitz tumors expressing ALK tend to be amelanotic and display a growth pattern of intersecting fascicles (Fig. 29.14). ALK expression may also be seen in a small subset of primary and metastatic melanomas (Fig. 29.15), where it is usually associated with the presence of a truncated ALK isoform. IHC for ALK may be used as a screening method to identify rare ALK-positive melanomas to select patients for treatment by ALK inhibitors.36

NTRK.  NTRK comprises a family of tropomyosin receptor kinase receptors. Members include three transmembrane proteins referred to as Trk A, B and C receptors. The corresponding genes are NTRK1, NTRK2 and NTRK3, respectively. Gene fusions represent the main molecular aberration involving NTRK in tumorigenesis, and have been

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Fig. 29.13  BAP1-inactivated Combined Melanocytic Nevus. Melanocytic nevus with large epithelioid melanocyte (A) and (B). (C) The large epithelioid melanocytes lack nuclear labeling for BAP1. All other cells retain expression of BAP1.

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B Fig. 29.14  Spitz Nevus Expressing ALK. (A) Compound Spitz nevus with intersecting fascicles of amelanotic spindle and epithelioid melanocytes. (B) The tumor cells strongly express ALK.

CHAPTER 29  Immunohistochemistry for the Diagnosis of Melanocytic Proliferations

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Fig. 29.15  Metastatic Melanoma Expressing ALK. (A) Metastatic melanoma in dermis (hematoxylin and eosin-stained section). (B) The tumor cells express Melan-A. (C) The tumor cells express ALK. The staining intensity is heterogeneous in this tumor. (D) Most tumor cells display cytoplasmic labeling for ALK. Strong staining tends to be associated with nuclear labeling.

found in various malignancies across different histologic phenotypes. In melanocytic neoplasms NTRK1 and NTRK3 fusions have been reported in Spitz lesions. However, they can also rarely be found in conventional melanomas. Antibodies to NTRK can used for the classification of Spitz lesions and help identify rare patients with metastatic melanoma, in which NTRK may play a role in tumor growth and targeting NTRK could be therapeutically beneficial. NTRK expression cannot be used for the distinction of benign from malignant lesions, since it can be seen in Spitz nevi (Fig. 29.16), atypical Spitz tumors, and spitzoid melanomas.35

SAC.  Soluble adenylyl cyclase (sAC) is a regulatory cytosolic enzyme present in every cell. It is a source of cAMP separate from transmembrane adenylyl cyclase enzymes. R21 is a monoclonal antibody to sAC, which can be used to immunohistochemically assess protein levels in tissue.37 The melanocytes of most melanocytic nevi tend to display a dotlike Golgi staining pattern, while some melanomas demonstrate a pan-nuclear expression pattern. However, not all melanomas show this staining pattern, and on occasion benign nevi may also show nuclear labeling, which limits the use of this antibody for diagnosis. The type of melanoma that most consistently displays nuclear labeling with R21 is lentigo maligna, with a sensitivity of 88%. Only approximately 40% of other

melanomas show pan-nuclear labeling with R21. Accordingly, the use of R21 has been advocated mainly for the diagnosis and margin assessment of lentigo malignas. However, the value of using R21 for margin assessment of lentigo malignas is limited by the fact that benign melanocytes of sun-damaged skin (e.g., lesions of solar melanocyte hyperplasia) may also display nuclear labeling with R21.

5-hmC.  5-hydroxymethycytosine (5-hmC) is a DNA pyrimidine nitrogen base derived from cytosine. Decreased levels of 5-hmC have been reported in tumor progression from nevus to melanomas and suggested to be of potential diagnostic use.38 Relative changes of 5-hmC expression in a single lesion may be helpful for the assessment of melanoma associated with a nevus. Nuclear expression of 5-hmC has also been suggested as an ancillary method for the distinction of a nodal nevus (expression retained) from metastatic melanoma (expression lost). However, for lymph node analysis, a co-label with cytoplasmic melanocyte differentiation marker (e.g., Melan-A/MART1) is necessary to ensure that the right cell type is being assessed. H3K27me3.  H3K27me3 is the trimethylated lysine residue at position 27 in the histone protein H3. It is associated with repression of gene

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A A

B Fig. 29.16  (A) Compound Spitz nevus (hematoxylin and eosin). (B) The lesional melanocytes homogeneously express NTRK1.

expression. EZH2 protein of polycomb repressive complex 2 (PRC2) transfers the methyl group to H3K27. It is an epigenetic marker. Normal tissues are expected to show nuclear labeling with antibodies to H3K27me3. Loss of H3K27me3 expression has been suggested to be helpful for the distinction of proliferative nodules of congenital nevi (nuclear expression usually retained in most cells) from pediatric melanomas (expression often lost in many tumor cells). Loss of H3K27 has also been suggested for the distinction of malignant peripheral nerve sheath tumor from melanoma.36 While loss of labeling may be more frequent in MPNSTs, loss of labeling or retention thereof can be seen in both MPNSTs and melanomas,39 which limits the diagnostic use of this marker for this diagnostic problem.

PRAME.  PRAME (preferentially expressed antigen of melanoma) is a member of the cancer testis (CT) antigen family—a group of proteins present in a variety of cancers. In adult normal tissues their expression is usually restricted to testicular germ cells and occasionally placenta. CT antigens have gained interest as targets for vaccine therapy. PRAME is commonly expressed in primary and metastatic melanoma, but also in a number of other cancer types, such as myxoid liposarcoma. The monoclonal antibody EPR20330 works well on formalin-fixed and paraffin-embedded tissues (Fig. 29.17). PRAME expression can be used as an ancillary tool to support a diagnosis of melanoma for the distinction of an atypical nevus from primary melanoma or nodal nevus from metastatic melanoma; positive labeling is expected in melanoma but not in melanocytic nevi. However, due to limitations in sensitivity (approximately 10% of conventional and up to two thirds of desmoplastic melanomas do not express PRAME), lack of staining does not exclude melanoma. If a melanoma is known to express PRAME, IHC can also be used for margin mapping. Although most nevi lack diffuse expression of PRAME, focal PRAME expression is not uncommon and diffuse expression may also be seen in rare cases.

B Fig. 29.17  Melanoma Expressing PRAME. (A) Melanoma in situ with nuclear labeling for PRAME. (B) Contrast the periphery of the PRAMEpositive melanoma in situ with lack of nuclear labeling of adjacent normal, i.e., non-neoplastic melanocytes.

Mutation Antigens

BRAFV600E.  The monoclonal antibody VE1 recognizes BRAFV600E mutant protein with a high degree of sensitivity and specificity.40–43 It has therefore become a useful reagent for the workup of patients with newly diagnosed metastatic melanoma for treatment selection. Positive staining strongly correlates with the presence of BRAFV600E mutations and can be used as strong enough evidence to initiate treatment. Lack of staining for VE1 does not exclude the possibility that a tumor may carry other BRAF mutations, such as BRAFV600K, which may respond to currently available BRAF inhibitors. IHC for BRAF may sometimes be preferable to molecular studies when the target tissue contains only a small amount of tumor tissue and many normal cells (e.g., small tumor deposit in a lymph node) (Fig. 29.18). The antibody may also sometimes be used as a diagnostic adjunct, that is, for the distinction of lentigo maligna melanoma with an invasive nevoid component versus incidental melanocytic nevus, or for staging purposes (see below). Caution is necessary when interpreting IHC results using VE1 for heavily pigmented or necrotic tumors to avoid false positive or negative test reports, respectively.

RASQ61R.  The rabbit anti-human monoclonal antibody SNP174 recognizes NRASQ61R with high sensitivity, but it is not entirely specific for it.44,45 It also binds to KRASQ61R and HRASQ61R. On occasion, it may also cross-react with NRASQ61K. The antibody is useful for quick assessment of the mutation status of newly diagnosed melanoma (Fig. 29.19). On occasion, it may also be helpful for the assessment of disease stage (distinguishing a second primary melanoma from a metastasis).

CHAPTER 29  Immunohistochemistry for the Diagnosis of Melanocytic Proliferations

HRAS.  The monoclonal antibody recognizes HRAS. IHC for HRAS can assist in the evaluation of Spitz nevi (Fig. 29.20), in particular desmoplastic Spitz nevi (see below).

Immune-Response Score Immune checkpoint blockade using inhibitors of programmed death-1 (PD1) and programmed death-ligand 1 (PD-L1) have shown promised for the treatment of various metastatic cancers, including

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melanoma. The immunohistochemical expression of PD1 and/or PD-L1 has been suggested to be useful to predict tumor response and is currently being investigated to determine its utility for treatment selection.

PD1.  Programmed cell death protein 1 (PD1) is a cell surface immunecheckpoint receptor that mediates suppression of T-cell inflammatory activity, thereby guarding against autoimmunity.

PD-L1.  The programmed cell death ligand 1 (PD-L1) is a 40 kDa type 1 transmembrane protein that plays a role in suppressing the immune system.

BASIC APPLICATIONS OF IMMUNOHISTOCHEMISTRY Visualization of Melanocytes

Fig. 29.18  Micrometastatic melanoma in lymph node immunoreactive with VE1 (anti-BRAFV600E).

Although there is usually no need for immunohistochemical studies for the diagnosis of most melanocytic proliferation, it may sometimes be advantageous or even necessary to use IHC for better visualization of melanocytes, such as when there is a dense inflammatory cell infiltrate blurring the dermoepidermal junction (see Fig. 29.7) or the quality of the tissue material is suboptimal (e.g., cautery artifact). It is also helpful for the analysis of pigmented nail lesions, when it is difficult to assess the melanocyte density on a routine H&E-stained sections. Melan-A/MART1 or tyrosinase are excellent markers for visualizing intraepidermal melanocytes because of high sensitivity and specificity (see Fig. 29.1). Sometimes, when there is heavy melanization and/or when IHC strongly labels the dendritic processes of melanocytes wrapped around keratinocytes, it may be difficult to read an immunostain for

B

A

C

Fig. 29.19  Melanoma with NRASQ61R mutation. The primary acral melanoma (A) is immunoreactive with the antibody SP174 (B). The metastatic tumor in the regional lymph node is also positive (C).

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SECTION V  Ancillary Studies

A

B Fig. 29.20  (A) Compound Spitz nevus (hematoxylin and eosin). (B) The tumor cells express HRAS.

Melan-A/MART1. This may lead to a perception of a higher density of melanocytes. In those cases, immunostains for nuclear antigens (MITF or Sox10) are preferable and permit a more accurate assessment of intraepidermal melanocyte density. Immunostains for Melan-A/MART1, MITF, and Sox 10 are not uncommonly used for margin assessments, especially for lentigo maligna melanoma in situ, as IHC facilitates low power assessment of changes in cell density from the center to the peripheral of a lesion and its transition to normal background density. Correlation with H&E findings and other immunostains is necessary when examining positive staining results. S100 protein does not consistently label all junctional melanocytes. Melanocytes of the nail matrix, for example, are often negative for S100. Melan-A/MART1 is a more sensitive marker for melanocytes. When using IHC for better visualization of melanocytes in the skin, one also needs to be aware of false-positive labeling. Within the epidermis, S100 stains not only melanocytes but also Langerhans cells. In the dermis antibodies to MITF, Melan-A/ MART1, and Sox10 often label mast cells. S100 labels dendritic cells and the dermis. Histiocytes are consistently positive for MITF. Both Sox10 and S100 label sweat glands. IHC may be helpful to highlight the extent of tumor for prognostic metrics (e.g., for more precise measurement of tumor thickness, especially in the setting of a scar or inflammation) or margin assessment. Melan-A/ MART1 or tyrosinase are good markers for this purpose. They highlight the majority of melanomas. However, some primary melanomas are negative for these markers or only partially express them (see Figs. 29.2, 29.10). S100 protein or Sox10 are the markers of choice to highlight invasive spindle cell melanoma. IHC may also be used for the detection of tumor cells in lymph nodes, especially sentinel lymph nodes (SLN) with minimal deposits of amelanotic tumor cells. Because of its high sensitivity, S100 protein is a common choice for the immunohistochemical analysis of SLN. However, not uncommonly many nonmelanocytic cells (dendritic cells) are also positive for S100 protein. Thus, careful correlation with the morphology of immunoreactive cells is needed for interpretation of the findings. Melan-A/MART, tyrosinase, and/or GP100 (HMB45) are excellent markers for the detection of metastatic melanoma in lymph nodes. However, not all melanomas are positive for these markers. Furthermore, the detection of immunoreactive cells does not equal the presence of metastatic melanoma. Inflammatory cells, such as mast cells, may be immunoreactive, or immunostains may highlight benign melanocytes. Melan-A/MART1, S100, and Sox10 consistently label nodal nevus cells. While most nodal nevi do not express GP100, on occasion

some, especially pigmented nevi and nodal blue nevi, may stain with HMB45. Melan-A/MART1 and HMB45 are widely used for the analysis of SLN. The former is more sensitive for the detection of melanoma than the latter. While some laboratories use several markers for the detection of melanocytes in SLN, we do not advocate the routine use of more than 2 markers for lymph node analysis. We have found dual nuclear and cytoplasmic labeling for Sox10 and Melan-A or PRAME and Melan-A advantageous for the analysis of melanocytes in lymph nodes.

Immunohistochemistry for the Distinction of a Melanocytic from Nonmelanocytic Tumor Historically, this has been the leading application of IHC for the diagnosis of tumors, when the findings on routine hematoxylin and eosin-stained sections do not permit a definite assessment of the line of differentiation, such as in the scenario of an amelanotic spindle or pleomorphic epithelioid cell tumor with no associated precursor. Detecting expression of Sox10 and HMB45, for example, would then support melanocytic differentiation and help establish a diagnosis of melanoma. Problems that may require the use of IHC include the distinction of a nevi or melanomas from poorly differentiated carcinoma, lymphoma, or soft tissue mimickers (e.g., nevus vs. cellular neurothekeoma or histiocytoma; melanoma vs. sarcoma). IHC for S100, Sox10, and/or NGFR is commonly used for the diagnosis of spindle cell melanoma with or without desmoplasia as it facilitates distinction from nonmelanocytic spindle cell tumors (desmoplastic leiomyosarcoma, spindle cell carcinoma, undifferentiated sarcoma). In difficult cases a combination of stains for melanocytic, epithelial, mesenchymal, and hematolymphoid differentiation may be necessary to classify a tumor (Fig. 29.21). How many markers are necessary depends on the histopathologic and clinical context, i.e., whether the differential diagnosis is narrow or broad. Caution is necessary when only one or two stains are used with limited specificity. An amelanotic Sox10-positive tumor could be a myoepithelial carcinoma. An amelanotic tumor expressing melan-A could be metastatic adrenocortical carcinoma, and an HMB45-positive tumor could be a PEComa.

Immunohistochemistry for the Distinction of a Melanocytic Nevus from Melanoma IHC may sometimes be used for the distinction of a melanocytic nevus from melanoma. There is currently no known single marker that would distinguish nevus with melanoma with high sensitivity and specificity.

CHAPTER 29  Immunohistochemistry for the Diagnosis of Melanocytic Proliferations

A

C

B

D

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Fig. 29.21  Intradermal proliferation of large amelanotic epithelioid melanocytes (hematoxylin and eosin, A, B). (C) While normal melanocytes of the overlying epidermis express SOX10, the tumor cells are negative. (D) The tumor cells are positive for CD30. A pathologist had initially interpreted the lesion as melanoma, but then revised the diagnosis to anaplastic large cell lymphoma once results from immunostains became available.

However, some immunohistochemical expression patterns may be regarded as soft evidence that can provide additional albeit limited support in favor of a diagnosis.

Expression Profile of p16, HMB45 and Ki-67.  Some pathologists use the immunohistochemical pattern of expression of p16, HMB45, and Ki-67 for the distinction of nevus from melanoma.31 Classically, a nevus stains uniformly for p16, stains for HMB45 only within the epidermis or superficial dermis with a gradient of loss of labeling toward the bottom of a lesion, and is negative for Ki-67 (see Fig. 29.3). On the other hand, lack of staining for p16, diffuse labeling of intradermal melanocytes for HMB45 (no gradient from top to bottom) (see Fig. 29.6), and labeling of intradermal melanocytes for Ki-67 especially near the base of the lesion, would be more typical of melanoma. However, there are many exceptions to this “rule.” While p16 is usually strongly expressed by most nevi, some show partial loss, often in a checkered pattern. On occasion a benign melanocytic nevus may be entirely immunonegative for p16. On the other hand, some lethal melanomas retain strong expression of p16 (see Fig. 29.12). Likewise, some benign melanocytic nevi express HMB45 uniformly (e.g., blue nevi, deep penetrating nevi, miscellaneous other nevi) (see Fig. 29.5), while some melanomas do not label at all for HMB45

or only minimally so (which may be at the dermoepidermal junction). Since most melanocytic nevi are negative for Ki-67 and most melanomas contain Ki-67 positive tumor cells, an immunostain (best using a co-label for both Melan-A/MART1 and Ki-67) documenting melanocytes with an increased proliferation index may at times be helpful to distinguish a nevus from a nevus-like primary or metastatic melanoma (see Fig. 29.10). Some slow-growing melanomas may not label for Ki-67 or only in a small proportion of tumor cell, while some nevi, for example, if biopsied during a growth phase, may stain for Ki-67. In most nevi with Ki-67-positive melanocytes, the immunoreactive cells are located in the superficial portion of the lesion. Benign proliferative nodules, however, may diffusely label for Ki-67. Thus, the use of one or a combination of the markers discussed in this paragraph is to be applied with caution.

Expression of Cancer Testis Antigens for the Distinction of Nevi from Melanoma.  Because of their unique expression patterns, conceptually, CT antigens should be an ideal marker for the distinction of melanoma from nevus; positive labeling should only be seen in a melanoma (see Fig. 29.16). Melanocytic nevi should be negative. While a positive test supports the diagnosis of melanoma, due to limitations in sensitivity, a negative test result is not informative. Among CT antigens,

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SECTION V  Ancillary Studies

PRAME is one of the most frequently expressed in melanoma, and therefore of potential diagnostic utility in selected cases. Approximately 90% of primary and metastatic melanomas express PRAME, with the exception of desmoplastic melanoma. Unfortunately, expression of PRAME can also be seen in nevi (usually focal), which limits its diagnostic value. However, it appears valuable for the assessment of margin clearance and for the distinction of nodal nevi from metastatic melanoma.

For the Distinction of a Desmoplastic Spitz Nevus from Desmoplastic Melanoma.  In contrast to desmoplastic melanomas, most desmoplastic melanocytic nevi express Melan-A/MART1 and often also p16. Thus, when confronted with a sclerosing melanocytic neoplasm, positive labeling for these markers favors a nevus, but correlation with the clinical and histopathologic context is important for diagnosis, since a rare desmoplastic melanoma may also display some positive staining for these markers. A different marker can be used for the distinction of desmoplastic Spitz nevi from desmoplastic melanoma. A subset of Spitz nevi, in particular desmoplastic Spitz nevus, is associated with copy gains for 11p, which includes the HRAS locus and is usually associated with overexpression of HRAS. Since HRAS is not a mutation associated with desmoplastic melanoma, on occasion immunohistochemical expression of HRAS can provide additional support for the distinction of desmoplastic Spitz nevus from desmoplastic melanoma. For the Distinction of Sclerosing Blue Nevus from Desmoplastic Melanoma. Most desmoplastic melanomas strongly express S100P and are usually negative or predominantly for Melan-A or GP100, in sclerosing blue nevi staining for S100 is often weak and focal, while the majority of cells homogeneously label with antibodies to Melan-A/MART1 or HMB45.

For the Distinction of Nodal Nevus from Metastatic Melanoma.  A combination of melanocyte markers can also be used as an adjunct for the distinction of a nodal melanocytic nevus from metastatic melanoma. Nodal nevomelanocytes tend to express S100P, Sox10, and Melan-A/ MART1. They tend to be positive for p16 and 5-hmC. They are usually negative for GP100 (HMB45) and do not stain for Ki-67. However, some nodal nevi, especially pigmented nodal nevi, may be positive for HMB45. Furthermore, just as with primary melanomas, some metastatic melanomas retain expression of p16 and may have a very low Ki-67 labeling index. Positive staining for PRAME would also support a diagnosis of metastatic melanoma.

Distinction of Proliferative Nodule from Pediatric Nodular Melanoma.  Immunostains cannot provide a definitive answer to the complicated diagnostic issue of when to distinguish a nodular melanoma of childhood from a benign proliferative nodule associated with a congenital nevus. However, IHC can provide supportive evidence. In a nodular melanocytic proliferation retained expression of 5-hmC and H3K27me37, a tumor in question is more likely a benign proliferative nodule than a melanoma. Pediatric melanomas arising in the setting of a large congenital nevus tend to lose expression for those markers. However, there are exceptions to this rule.

Immunohistochemistry for Treatment Selection and/or Staging Purposes While molecular methods, in particular next-generation sequence analysis, are the preferred way of tumor mutation analysis because a number of highly specific oncogenic targets can be identified in one assay, IHC plays an important role for treatment selection and should be viewed as complimentary to molecular testing. IHC provides a rapid method for selection of patients with metastatic melanoma for targeted

therapy at relatively low cost. It also has the advantage of high sensitivity in a mixed cell environment; IHC is more likely to detect a few metastatic tumor cells in a lymph node than molecular methods, since the nonneoplastic DNA may dilute the tumor cell DNA so much as to lead to a false negative molecular test result. Using IHC for determining the mutation status is relevant for patients with tumors carrying a BRAFV600E mutation, since the monoclonal antibody VE1 is highly specific and very sensitive in detection the mutant protein. While a positive test result opens a therapeutic opportunity, the lack of staining does not exclude the presence of other targetable BRAF mutations. IHC can also confirm the presence of an N- or KRASQ61R mutation using the antibody SP174. IHC for determining the BRAF or NRAS mutation status is not only helpful for treatment selection but can on occasion also be used diagnostically or for staging purposes. If a patient with a history of a prior melanoma, for example, develops a new superficial dermal melanoma nodule, the question may arise as to whether the new nodule is a recurrence of the former or a new primary nodular melanoma. Discordance of the mutation status (e.g., the prior melanoma was known to carry the BRAFV600E mutation; the new nodular melanoma is positive for NRASQ61R) could assist in the decision and favor two separate melanomas. A similar IHC application exists for BAP1 when, for example, a patient with a history of uveal melanoma develops a melanoma in the skin. If the uveal melanoma was associated with loss of BAP1 and the skin tumor stained positive for BAP1 and possibly also expressed BRAFV600E, it would unlikely be metastatic uveal melanoma to the skin and most likely a separate primary cutaneous melanoma. Using IHC for treatment selection is not restricted to documenting the mutation status. As in the case of ALK, it may also identify a truncated isoform that may be responsive to ALK inhibition.35 With the emerging dominance of immunologic therapy, in particular checkpoint blockade inhibition, IHC will likely play a role in predicting the probability of treatment response and accordingly in the selection of patients for therapy.

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