Immunohistochemistry of the Skin

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1 CE update [immunology histology chemistry] Immunohistochemistry of the Skin David Tacha, HT(ASCP)HTL, PhD BIOCARE Medical, Walnut Creek, CA DOI: /PDPM26E5TLFQLUNG After reading this article the reader should understand the antigens, proteins, and molecular structures associated with the immunohistochemistry of the skin. Immunology questions and corresponding answer form are located after the Your Lab Focus section on p Immunohistochemistry (IHC) of the skin has progressed significantly over the last decade. This paper will discuss an overview of IHC of the skin with emphasis on commonly used antibodies, antibody panels, cytokeratins, and melanomas. The skin is the largest organ in the body and is highly complex. In dermatopathology, main areas of interest include: melanomas, squamous cell carcinoma, basal cell carcinoma, Paget s disease, spindle cell tumors, fibrous and fibrohistiocytic tumors, vascular neoplasms, and neuroendrocrine tumors. Several types of cutaneous lymphomas can also occur in the skin. In addition, the cutaneous manifestations of HIV [I1] Normal skin stained with AE1/AE3 and Romulin AEC. have presented a host of fungal, viral, and bacterial infections of the skin. In the last decade, immunohistochemistry has played an increasingly important role for routine diagnosis, prognosis, and research. The complexity of all skin disorders makes it imperative to understand IHC and the different disciplines that are required by today s dermatopathologists. An entire book could be dedicated to this subject. This paper will attempt to give a small overview of the use of commonly used antibodies and phenotyping in the modern dermatopathology laboratory. Antibodies Commonly Used Pan Cytokeratin Pan cytokeratin antibodies are useful in differentiating epithelial and mesothelial cells from mesenchymal cells in normal and tumor tissues. AE1/AE3 1 or Lu-5 antibodies serve as first-order pan cytokeratin antibodies for both acidic (type 1) and basic type (basic type 2). [I1] High Molecular Weight Cytokeratin (HMW CK) The HMW CK [34βE12] 2 recognizes cytokeratin 1, 5, 10, and 14. This antibody is reactive with squamous carcinomas and adenosquamous carcinomas, whereas adenocarcinomas are negative. In normal epithelia, 34bE12 stains stratified epithelia and myoepithelial cells. 34βE12 is useful as a differential marker for squamous carcinomas and adenocarcinomas and for benign and malignant tumors of the prostate gland. Low Molecular Weight Cytokeratin (LMW CK) Clone 5D3 recognizes CK 8/18 intermediate filament proteins. 3 In normal tissues, 5D3 recognizes all simple and glandular epithelium. In neoplastic tissues, 5D3 may prove useful for the identification of adenocarcinomas. It can be used in conjunction with a HMW CK to rule out squamous cell carcinoma. Keratinizing squamous carcinomas are generally negative. CD1a CD1a is a protein of 43 to 49 kd expressed on dendritic cells and cortical thymocytes. 4 CD1a staining has been shown to be useful in the differentiation of Langerhans cells from interdigitating cells. It has also proved useful for phenotyping Langerhans cell histiocytosis. CD4 CD4 is expressed in a T-cell subset (helper/inducer) and is found in approximately 80% of thymocytes and 45% of peripheral blood lymphocytes. CD4 is expressed in the majority of T-cell lymphomas, including mycosis fungoides. 5-7 CD7 CD7 molecule is a membrane-bound glycoprotein of 40kD, and is the earliest T-cell specific antigen to be expressed in lymphocytes. CD7 is expressed in the majority of thymocytes, peripheral blood T cells, and natural killer cells. Used in combination with CD4, CD7 and CD8 have been useful for differentiating mycosis fungoides and memory T cells

2 CD34 CD34 antigen is selectively expressed in human lymphoid and in myeloid hematopoietic progenitor cells. The antibody to CD34 also reacts with vascular endothelial cells in normal tissues, and in benign and malignant proliferations. The utility of CD34 has value in the studies of benign and malignant vascular tumors as well as characterization of acute leukemia in bone marrow. It is also used to differentiate dermatofibrosarcoma protuberans from fibrous histiocytoma. 8 CD56 CD56 (neural cell adhesion molecule, a natural killer cell marker) is part of a family of cell surface glycoproteins. CD56 plays a role in embryogenesis and contact-mediated interactions between neural cells. CD56 is expressed in a variety of normal and abnormal tissues including: skin, small cell carcinoma, neuroblastoma, neurons, astrocytes, Schwann cells, NK cells, and a subset of activated T-cell lymphomas. 9 Factor XIIIa This is a polyclonal antibody to the A-subunit of human coagulation factor XIII. It recognizes human Factor XIII A-chain in both reduced and nonreduced forms. Factor XIIIa is a dermal dendrocyte marker and shows variable reaction with these types of tumors. It can be used for histiocytic phenotyping and has been reported to mark capillary hemangiomas and tumors of the central nervous system. Factor XIII has also been used with CD34 to differentiate between dermatofibroma and dermatofibrosarcoma protuberans. 8 A is composed of alpha and beta chains whereas B is composed of 2 beta chains. Antibody stains melanocytes, schwannomas, peripheral neural tissue astrocytes, and benign and malignant melanomas and their metastases. 10,11,14 protein is also expressed in the antigen presenting cells such as the Langerhans cells in skin and interdigitating reticulum cells in the paracortex of lymph nodes. protein is highly soluble and may be eluted from frozen tissue during staining. HMB45 HMB45, a neuraminidase-sensitive oligosaccharide side chain of a glycoconjugate, is present in immature melanosomes. The HMB45-reactive antigen is present in cutaneous melanocytes, prenatal and infantile retinal pigment epithelium (RPE), and melanoma cells. It is also thought to be oncofetal in nature. This antibody has been shown to label the majority of melanomas. 10,11,14 The MART-1 The MART-1/Melan A recognizes a protein of 18 kda, identified at MART-1 (Melanoma Antigen Recognized by T- cells 1) or Melan-A. The MART-1 recognizes a subcellular fraction found in melanosomes. The antibody labels melanomas and tumors showing melanocytic differentiation. It does not mark neoplasms of epithelial origin, lymphomas, or mesenchymal tumors. Melan- A is a useful addition to melanoma panels as it is apparently specific for melanocytic lesions. Both HMB-45 and MART-1 are coexpressed in the majority of melanomas. Studies have shown MART-1 is more sensitive than HMB45 when labeling metastatic melanomas Tyrosinase Tyrosine is a key enzyme involved in the initial stages of melanin biosynthesis. Studies have shown tyrosinase to be a more sensitive marker when compared to HMB45 and MART It is also shown to label a higher percentage of desmoplastic melanomas than HMB45. Unlike HMB45, neither tyrosinase nor MART-1 discriminates between activated or resting melanocytes. Other studies have shown tyrosinase to be a very specific marker for melanomas, and it does not cross react with any tumors or normal tissues. In conclusion, tyrosinase appears to be a superior melanoma marker when compared to HMB45. [I2] 312 A B Pan Melanoma Cocktail (MART-1 and Tyrosinase) We have not observed any study or any case where HMB45 was positive and both MART-1 and tyrosinase were negative Tyrosinase and MART-1 are coexpressed in the majority of melanomas. Thus, the tyrosinase and MART-1 cocktail is more sensitive than HMB45 alone. [I3] The combination of MART-1 and tyrosinase make this antibody cocktail a firstorder pan melanoma screener, and may prove to be a valuble marker for metastatic melanoma in sentinel lymph nodes. [I2] A: Melanoma stained with tyrosinase and DAB; B: Melanoma stained with HMB45 and DAB. (Note the superior staining with tyrosinase when compared to HMB45.) Nerve Growth Factor (p75) Monoclonal antibody NGFR, p75 recognizes the 75 kd NGF receptor

3 p75 NGFR. The p75 receptor protein is a member of the nerve growth factor (NGF)/tumor necrosis factor superfamily of receptors. NGFR reacts with peripheral nerve sheath tumors and pheochromocytomas. Studies have also shown NGFR to be reactive with round cell, desmoplastic, and neurotropic melanomas. 15 A B is a polymorphic family of cell membrane glycoproteins that mediate cellmatrix and cell-cell interactions involved in the mechanisms of tumor invasion and metastasis, and are subject to differential regulation during normal and malignant cell growth. Various tumors including melanoma, Merkel cell carcinoma, and squamous cell carcinoma have been reported. 17,21,22 Basal cell carcinomas do not express in contrast to squamous cell carcinomas. Cytokeratin and Intermediate Filament Proteins 23 The expression of cytokeratins can be very complex, but if fully understood, can be very valuable for diagnosis of skin disease. Moll reclassified cytokeratins into a numbering chart that simplified its understanding. Cytokeratins are classified as intermediate filaments proteins (IFP). The expression of IFP is controlled by individual genes and follows a carefully regulated pattern of cell differentiation. Intermediate filaments proteins molecular weight of cytokeratins varies from 40 to 68 kd. They are found in most normal epithelium and carcinomas. Distribution of Cytokeratin in as Classified by Moll 23 Cytokeratins are also expressed in pairs comprising of type 1 (9-20) and type II (1-8), and acidic and basic comprising of 1-6 (ph 7.3 to 7.8) and 7-20 (ph 4.9 to 6.0). [T1] and [T2]. Common Panels for Dermatopathology [I3] A: Metastatic melanoma in lymph node stained with HMB45 and DAB. (No staining with HMB45); B: Metastatic melanoma in lymph node stained with pan melanoma cocktail (MART-1 and tyrosinase) Malignant Melanoma Malignant melanoma is a malignancy of nevus cells. Sunlight appears to be the predisposing factor. Primary and especially metastatic melanomas can be a great challenge to diagnose correctly. Different types of malignant melanomas include: primary lesions, distant metastasis, rhabdoid, and sarcomatoid, including desmoplastic, neurotropic. and myxoid subtypes. In the past, and HMB45 were the 2 main antibodies used for confirming melanomas. Recently, several other antibodies have been developed to properly phenotype malignant melanomas. T3 is a list of antibodies known to stain and to distinguish the majority of all types of melanomas. Basal Cell Carcinomas Basal cell carcinomas are composed of cells resembling the basal of the epidermis. Distribution of Cytokeratin Number MW (kd) Distribution in Tissues Basal cell carcinoma tends to occur in sunexposed skin and is one of the most common kinds of skin cancer. It tends to grow very slowly, and in most cases, basal cell carinoma can usually be diagnosed based on morphology alone. However, there are other types tumors such as adnexal tumors, trichoepithe-liomas, sebaceous eptheliomas and squamous cell carcinomas that can resemble basal cell carcinoma. Basal cell carcinoma stain diffusely positive with Bcl- 2 whereas in trichoepitheliomas only the periphery of the lobules stains. In basal cell carcinomas, the tumor stroma is CD34 negative, and in trichoepithelilmas, it is focally positive for CD34. T1 1 & 10, 2 68, 56.5, 65.5 Cornifying stratified; skin, endocervix 3 & 12 63, 55 Human cornea 4 & 13 59, 58 Non-cornifying stratified squamous epithelia; tongue, esophagus 5 & 14, 15 58, 50, 50 Simple stratified epithelium; epidermis, squamous cell carcinoma 6 & 16 56, 48 Fast turnover cells; hair follicles, suprabasal cells, activated keratocytes 7 & 19 54, 40 Simple and glandular epithelium; breast, lung, bladder 8, , 45 Simple and glandular epithelium 9, 10, 11 64, 56.5, 56 Cornified stratified epithelium, anal canal, epidermis, squamous cell carcinoma Basal cells of complex epithelia; trachea,larynx, cervix, bronchi Ductal and glandular epithelium; liver cancer, adenocarcinoma, hair follicles Gastric and intestinal, urothelium, Merkel cell 313

4 Moll Classification of Common Cytokeratin Antibodies 23 T2 Melanoma Panel: Antibodies Positive for Melanomas T3 Clone: AE1/AE3 Moll: 1, 2, 3, 4, 5, 6, 7, 8, 10, 14, 15, 16, 17 and 19 Clone: Lu-5 Moll: 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19 Clone: MAK-6 Moll: 8, 14, 15, 18, 19 (LMW) Clone: AE1 Moll: 10, 14/15, 16/17, 19 (HMW) Clone: AE3 Moll: 1, 2, 3, 4, 5, 6, 7, 8 Type: LMW cytokeratin Clone: 5D3 Moll: 8/18 Type: HMW cytokeratin Clone: 34bE12 (903) Moll: 1, 2, 5, 10, 14/15 Type: HMW cytokeratin Clone: DE-SQ Moll: 13, 14, 15, 16 Squamous Cell Carcinoma Squamous cell carcinomas arise in the skin and occur most often in older individuals on sun exposed areas. Squamous cell carcinoma typically stain strong for keratins. The high molecular HMB45 Tyrosinase MART-1 Microphathalmia Pan Melanoma (Tyrosinase and MART-1) p75 CD56 MAGE-1 weight cytokeratins are typically present. By contrasting the BerEP4, LMW CK, androgen receptor, CK15 20 [I4], CEA, and antibodies, basal cell carcinoma can be distinguished from squamous cell carcinoma. [T4] Merkel Cell Carcinoma The Merkel cells of the epidermis are neuroendocrine cells derived from the neural crest. Normal Merkel cells can undergo neoplastic changes, producing Merkel cell carcinoma. Immunohistochemical studies have shown both neuroendrocrine and LMW cytokeratins to be positive for Merkel cell carcininoma [T5]. Studies have shown that in Merkel cell carcinoma of the skin may correlate to metastatic risk. 20 Protuberans protuberans (DFSP) is the malignant counterpart of dermatofibroma (DF). protuberans are composed of a proliferation [I4] Basal cells stained with CK15. of fibroblasts that may be arranged in patterns resembling pinwheels (storiform pattern). Dermatofibroma is a common variant of benign fibous histiocytoma and is composed of spindle-shaped fibroblasts. Dermatofibroma is normally located in the mid-dermis. Immunohistochemistry can be helpful in distinguishing between the 2, especially early DFSP from DF [T6]. 8 Summary The utility of immunohistochemistry for skin pathology has evolved significantly over the past 2 decades. Immunohistochemical staining has provided assistance and confirmation in identifying primary sites of origin and for cutaneous metastases with an unknown primary. Most markers, however, 314 Comparison of Basal Cell Versus Squamous Cell Carcinoma Basal Cell Carcinoma T4 Squamous Cell Carcinoma AE1/AE3+ AE1/AE3 + 34βE12+ 34βE12+ BerEP4+ BerEP4 (-) LMW CK [8/18] (+) LMW CK [8/18] (50% to 60%) (-) (80% to 90%) Androgen Receptor Androgen (+) (60%) Receptor (-) CK15 (+/-) (25%) CK15 (-) CEA - CEA+/+ (-) (+) Merkel Cell Carcinoma Panel Positive AE1/AE3 BerEP4 CEA (P) CK20 CAM5.2 Chromogranin A or B T5 Negative CK7 CK5/6 Desmin Vimentin TTF-1 Comparison between and Dermatofibroma T6 Dermatofibroma Smooth Muscle Smooth Muscle Actin (+) Actin (+) Vimentin (+) Vimentin (+) CD34 (+) CD34 (-) Factor XIIIa (-) Factor XIIIa (+)

5 show a variation in expression. It is highly recommended that each investigator use a panel of antibodies for each individual case. Each antibody should be studied and carefully characterized. Data sheets, publications, meetings, books, and literature are important tools for characterizing antibodies and for keeping up with state-of-the-art technology. 1. Cooper D. The use of a IF, AE1, and AE3 monoclonal antibodies for the identification and classification of mammalian epithelial keratins. Differentiation. 1984;28: Kong X, Deng F, Wang J. The application of high molecular weight cytokeratin in differential diagnosis of prostate adenocarcinoma. Chung Hua Ping Li Hsueh Tsa Chih. 1996;25: Angus B. NCL-5D3: A new monoclonal antibody recognizing low molecular weight cytokeratins effective for immunohistochemistry using fixed paraffin-embedded tissue. J Pathol. 1987;153: Krenacs L, Tiszalvicz L, Krenacs T, et al. Immunohistochemical detection of CD1A antigen in formalin-fixed and paraffin-embedded tissue sections. J Pathol. 1993;171: Giam YC, Ong BH. Clinicopathological and immunohistological correlation of malignant lymphomas of the skin. Ann Acad Med Singapore. 1994;23: Kim YH, Hoppe RT. Mycosis fungoides and the Sezary syndrome. Semin Oncol. 1999;26: Izban KF, Hsi ED, Alkan S. Immunohistochemical analysis of mycosis fungoides on paraffin- Embedded tissue sections. Mod Pathol. 1998;11: Diaz-Cascajo C. protuberans with fibrosarcomatous areas: A clinico-pathologic and immunohistochemic study in 4 cases. Am J Dermatopathol. 1997;19: Savoia P. CD56-positive cutaneous lymphoma: A poorly recognized entity in the spectrum of primary cutaneous disease. Br J Dermatol. 1997;137: Fernando SS, Johnson S, Bate J. Immunohistochemical analysis of cutaneous malignant melanoma: Comparison of S-100 protein, HMB45 monoclonal antibody and NKI/C3 monoclonal antibody. Pathology. 1994;26: Blessing K, Sanders DS, Grant JJ. Comparison of immunohistochemical staining of the novel antibody Melan-A with protein and HMB- 45 in malignant melanoma and melanoma variants. Histopathology. 1998;32: Orchard GE. Melan A (MART-1): A new monoclonal antibody for malignant melanoma diagnosis. Br J Biomed Sci. 1998;55: Jungbluth AA. T311-An anti-tyrosinase monoclonal antibody for the detection of melanocytic lesions in paraffin embedded tissues. Pathol Res Pract. 2000;196: Orchard GE. Comparison of immunohistochemical labelling of melanocyte differentiation antibodies melan-a, tyrosinase and HMB45 with NKIC3 and protein in the evaluation of benign naevi and malignant melanoma. Histochem J. 2000;32: Kanik AB, Yaar M, Bhawan J. p75 nerve growth factor receptor staining helps identify desmoplastic and neurotropic melanoma. J Cutan Pathol. 1996;23: Taylor, Cole. Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist; Immunohistology of the Skin: Melanoma, Intrmediated Filamemt, 2 nd ed. Philadelphia; W.B. Saunders; David J. Dabbs, MD. Diagnositic Immunohistochemistry: Immunohistochemistry of Skin Tumors. Philadelphia: Churchill Livingstone Bruce R Soller, MD. Dermatologic Clinics. Update on Dermatopatholoy; Immuohistochemistry in Diagnostic Dermatopathology. Philadelphia: W.B. Saunders; Nancy Standler. A Short Course in Pathology; Chapter 5: Skin; Philadelphia: Churchill Livingstone; Kurzen H. Cytokeratins as markers of follicular differentiation: an immunohistochemical study of trichoblastoma and basal cell carcinoma. Am J Dermatopathol. 2001;23: Penneys NS, Shapiro S. expression in Merkel cell carcinoma may correlate with risk of metastasis. J Cutan Pathol. 1994;21: Fernandez-Figueras MT. and melanocytic tumors: a possible role for standard in the epidermotropic spread melanoma. J Cutan Pathol. 1996;23: Tacha, D. Antibody Panels and Immunohistochemistry; NSH at Madison WI,