Immunohistochemistry for the Diagnosis of Melanocytic Proliferations

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. S100 proteins are involved in diverse intracellular and extracellular functions. S100 proteins are expressed in many different tissues and organs. 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 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.” The antigen is also referred to as Pmel17 ( silv ). 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 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. 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-L-HMB45, 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. 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 melanogenesis. A monoclonal antibody, clone T311, was developed for therapeutic purposes, but had also diagnostic value. 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. 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). 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. 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). 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). Molecular analyses of Melan-A/MART1 showed an expression pattern characteristic of a melanocyte differentiation antigen. Melan-A/MART1 is a structural 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). 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. Both reagents show a similar staining pattern, except that A103 shows more consistent immunopositivity in steroid hormone producing cells and tumors.

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. 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-loop-helix-loop leucine zipper (bHLH-Zip) transcription factor encoded by the MITF gene. 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. 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, 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. 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. 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). 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. 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). Like other melanocyte markers, the “specificity” of SOX10 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.

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. 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. 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.

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, 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 ).