Case 1 -

Sarcomatoid Squamous Cell Carcinoma Steven D. Billings, M.D. Andrew L. Folpe, M.D.

Clinical History A 57-year-old man presented with a lesion on the nose. A biopsy was obtained. Morphology The biopsy demonstrated an obviously malignant-appearing, pleomorphic spindle cell neoplasm, without obvious differentiation. A focus of possible squamous dysplasia was also present. Solar elastosis was seen. By immunohistochemistry, the tumor was focally cytokeratin-positive, uniformly vimentin-positive, and S100 protein-negative. Diagnosis: Sarcomatoid squamous cell carcinoma Discussion This handout will focus on the use of immunohistochemistry (IHC) in the differential diagnosis of a relatively common scenario, the "histologically undifferentiated, pleomorphic spindle cell tumor involving the skin". Applications of IHC in the diagnosis of monomorphic spindle cell tumors (e.g., dermatofibrosarcoma protuberans) and histologically distinctive spindle cell tumors (e.g., neurothekeoma) are covered elsewhere in this course (see relevant handouts) Differential Diagnosis Most pleomorphic cutaneous spindle cell tumors occur in adults, and many involve sun-damaged skin. Thus, the typical differential diagnosis for these lesions is actually quite limited. Far and away the most common tumors encountered in this setting are sarcomatoid squamous cell carcinoma, sarcomatoid malignant melanoma and atypical fibroxanthoma/ superficial malignant fibrous histiocytoma. Less common tumors that also need to be included in the differential diagnosis include leiomyosarcoma and spindle cell variants of angiosarcoma, and very rarely unusual tumors such as rhabdomyosarcoma, pleomorphic liposarcoma, etc. An Approach to Evaluating Immunostains in the Skin As consultants, we have the opportunity to evaluate an enormous number of immunostains performed at outside laboratories. This affords us the opportunity to see what pathologists do correctly, and where we go wrong. Many, if not most, of the problems we see pathologists get into in evaluating IHC in the skin (and elsewhere) can be eliminated with careful attention to certain fundamental principles of IHC: Careful attention to positive and negative internal controls: This cannot be overemphasized. Every single IHC slide must be evaluated for appropriate staining of normal tissues for the antigen in question. Internal positive and negative controls completely "trump" external controls, whether those external controls are placed on the same glass slide or not. This is because specimen fixation is the single greatest variable in IHC. Fortunately for the pathologist, the skin is rich in normal internal positive controls for the vast majority of antigens (see Table 1). Evaluation of negative controls simply means making sure that tissues that shouldn't express an antigen aren't brown (or red)- to quote Dr. Allen Gown, "If everything is positive, nothing is positive". Inappropriate staining of negative controls usually is the result of inappropriate antibody concentration and/ or excessive epitope retrieval. Careful evaluation of all pieces of tissue on the entire slide: This should go without saying, but we have seen many cases in which referring pathologists simply missed the small HMB-45 positive focus in a sarcomatoid melanoma, for example. The use of a panel of immunostains, including both expected positive and negatives for all of the entities in the histologic differential diagnosis:A panel of immunostains, rather than single markers should always be performed. This approach greatly eliminates the potential for misdiagnosis owing to anomalous expression of antigens (e.g., cytokeratin in melanoma or angiosarcoma). The routine use of a small, carefully selected panel is also time and money effective. Table 1: Commonly evaluated antigens and their normal positive internal controls in the skin Antigen Normal Positive Internal Control in Skin Pan-cytokeratin and low molecular weight cytokeratins Suprabasal keratinocytes and adnexae High molecular weight cytokeratins Basal keratinocytes S100 protein Melanocytes, Langerhans cells, nerves, myoepithelial cells Melanocytic markers (e.g., HMB-45, Melan-A, MiTF, tyrosinase) Dermal melanocytes (may be HMB-45 negative) Smooth muscle actins Pilar smooth muscle Desmin Pilar smooth muscle Endothelial markers (e.g., CD31, CD34, FLI-1 protein, von Willebrand protein) Endothelial cells Cytokeratins Background: The cytokeratins are a family of 20 intermediate filament proteins, the expression of which is largely, but by no means entirely restricted to epithelial cells. There are many different ways to think about the cytokeratins, as individual cytokeratins (e.g., CK 7, CK20), as acidic and basic cytokeratins pairs (e.g., CK 8/18) or as "low" and "high" molecular weight cytokeratins. With regard to the last category, it is important to realize that the division of the cytokeratins into low and high molecular weights is entirely arbitrary. It is more useful to think of the low molecular weight cytokeratins as those of simple epithelia, such as simple ductules, and the high molecular weight cytokeratins as those of complex epithelia, such as urothelium or skin. For practical purposes, most of us tend to think about cytokeratins in terms of the antibodies we use to identify them. In our experience, the most widely used pan-keratin antibody is the AE1/AE3 cocktail. AE1 recognizes the acidic cytokeratins 10, 14, 15, 16, and 19, whereas AE3 recognizes the basic cytokeratins 1, 2, 3, 4, 5, 6, and 8. Both antibodies recognize a mixture of high and low molecular weight cytokeratins, and there is no point in running them individually. A recently developed wide spectrum cytokeratin antibody, OSCAR, appears to be a particularly sensitive marker, and has largely replaced AE1/AE3 as a screening cytokeratin at Mayo Clinic. The exact cytokeratins identified by OSCAR are not yet known, but appear to include an admixture of low and high molecular weight cytokeratins. The most widely used low molecular weight cytokeratin antibodies are Cam 5.2 (CK 8, 18, 19) and 35BH11 (CK 8 and 18). Almost all laboratories used the high molecular weight cytokeratin antibody 34BE12 (CK 1, 5, 10, 14/15), also known as "cytokeratin 903". Many other antibodies exist and one should always carefully read the product insert and any relevant literature before using them in one's own laboratory. Cytokeratin expression in normal skin: The skin serves as a superb control tissue for the evaluation of cytokeratin expression, as each epithelial cell type present within the skin expresses unique cytokeratins, as detailed below. Table 2: Cytokeratin expression in normal skin Cell Type Cytokeratins Expressed Suprabasal keratinocytes 1, 10, (11) Basal keratinocytes 5 and 14 Hair shaft 5, 6, (14), 15, 16, 17 Eccrine ductular cells 7, 8, 17, 18, 19 Eccrine myoepithelial cells 5, 6, 14 Pitfalls in the evaluation of cytokeratin expression in pleomorphic cutaneous spindle cell tumors: Falsely negative basal keratinocyte staining with broad spectrum cytokeratin antibodies3: Although these antibodies should react with basal keratinocytes (CK 5 and 14), these cells are frequently negative in many laboratories, for unknown reasons. This indicates that these high molecular weight keratins are not being identified. It is critical to realize that sarcomatoid squamous cell carcinomas may express almost exclusively high molecular weight cytokeratins such as CK 5 and CK 14. Therefore, the failure to appreciate negative basal cell staining with broad spectrum cytokeratin antibodies may result in the misdiagnosis of sarcomatoid squamous cell carcinoma. Extremely focal or absent cytokeratin expression in sarcomatoid squamous cell carcinoma: Sarcomatoid squamous cell carcinomas may show only extremely focal cytokeratin expression, may entirely lose cytokeratin expression in spindled areas, or may express only high molecular weight cytokeratins. The most sensitive markers of sarcomatoid SCC are 34BE12 and CK 5/6. It is also important to recognize that essentially all sarcomatoid SCC will express vimentin (see below). Vimentin Background: Vimentin is present in almost all embryonic cells and most cells of any lineage re-express vimentin when grown in culture. This latter situation may be analogized to spindled change in neoplasms, inasmuch as essentially any spindled cell will express vimentin. Diagnostic uses: Vimentin is expressed by essentially all sarcomas, all melanomas, all spindled squamous cell carcinomas, all spindled angiosarcomas, etc. It is also expressed in almost all mesotheliomas, most gliomas, and a subset of non-spindled carcinomas. The diagnostic uses of vimentin are almost exclusively restricted to the subtyping of carcinomas, as certain carcinomas, such as endometrioid carcinomas, frequently co-express vimentin. This subject is beyond the scope of this course. S100 Protein Background : S100 protein is a 20 kd acidic calcium binding protein, which gets its name from its solubility in 100% ammonium sulfate. S100 protein is composed of 2 subunits, with 3 isotypes- αα (expressed in muscle), αβ (expressed in melanocytes, glia, chondrocytes, skin adnexae), ββ (expressed in Langerhans cells, Schwann cells). The commonly used polyclonal antibodies recognize all of these isotypes. Diagnostic uses: S100 protein is present in essentially 100% of normal melanocytes and nevi, and in approximately 97-98% of melanomas. S100 protein is thus the single best screening marker for melanocytic neoplasms. It is important to realize that very rare melanomas are S100 protein negative. S100 protein is not, however, completely specific for melanocytic tumors, and can be expressed in smooth muscle tumors (weakly), some carcinomas (but not squamous cell carcinoma), myoepithelial tumors, peripheral nerve sheath tumors and others. Melanocytic Markers HMB-45 Background: HMB-45 is a monoclonal antibody that identifies a premelanosomal protein, gp100. HMB-45 is often negative in resting melanocytes and nevi, but may be expressed in "reactive" melanocytes. HMB-45 is organelle specific but not lineage specific, meaning that non-melanocytic tumors that contain pre-melanosomes (e.g., Bednar tumor, angiomyolipoma) will be HMB-45 positive. HMB-45 should not be used as a screening marker for melanoma- use S100 protein instead. Diagnostic uses: Numerous studies have shown the sensitivity of HMB-45 in epithelioid melanomas to be approximately 85%. Sarcomatoid melanomas are less often positive (30-50%) and true desmoplastic melanomas are essentially never HMB-45 positive in the spindled/desmoplastic zones. Melan-A Background: Melan-A, the product of the MART-1 gene, is a 20-22 kd component of the premelanosomal membrane, of unknown function. Unlike HMB-45, Melan-A is expressed in resting melanocytes and nevi. As with HMB-45, Melan-A is organelle specific but not lineage specific. Melan-A should also not be used as a screening marker for melanoma. The most widely used antibody to Melan-A, A103, has a reproducible and diagnostically useful cross-reactivity with a cytoplasmic component of steroid producing cells. This is helpful in the diagnosis of adrenal cortical neoplasms, but is not typically of concern in the skin. Diagnostic uses: Essentially the same sensitivity and specificity as HMB-45 .Present in some HMB-45 negative melanomas, and vice versa. Microphthalmia Transcription Factor (MiTF) Background: MiTF is a nuclear regulatory protein critical for melanocyte development and for the expression of tyrosinase. It is expressed in essentially all resting melanocytes and nevi. It is the only nuclear marker of melanocytic differentiation. There are both melanocyte-specific and non-specific isoforms of this protein- unfortunately, the commercially available antibodies are not specific for the melanocytic isoforms. Diagnostic uses: MiTF was initially described as highly sensitive and specific marker of melanoma, and is expressed in well over 90% of epithelioid melanomas. Sarcomatoid melanomas are less often positive (40%) and true desmoplastic melanomas are very infrequently positive (<5%). Because the commercially available antibodies are not specific for the melanocytic MiTF isoforms, MiTF expression is not limited to melanomas, and may be seen in leiomyosarcomas, atypical fibroxanthomas, atypical lipomatous neoplasms, and very rare carcinomas. Thus, MiTF is best used for the confirmation of S100 protein-positive, HMB-45/Melan-A/ tyrosinase-negative tumors suspected of being melanomas. MiTF expression in the absence of S100 protein expression is not diagnostic of melanoma. MiTF is also expressed in "myomelanocytic tumors" such as angiomyolipoma and clear cell sugar tumor of the lung. Tyrosinase Background: Tyrosinase is an enzyme critical for melanin synthesis and is therefore melanosome-specific, but not melanoma specific. Tyrosinase is expressed in resting melanocytes and nevi. Diagnostic uses: The sensitivity of tyrosinase for melanoma is equal to or slightly better than that of HMB-45 or Melan-A. Its specificity is identical to that of HMB-45. Pitfalls in the immunohistochemical diagnosis of melanoma Cytokeratin and or desmin expression: Melanomas are notorious for anomalous expression of intermediate filament proteins, with some series documenting anomalous expression in over 30% of cases. Cytokeratin is the most common anomalously expressed intermediate filament in melanoma, obviously raising significant potential for misdiagnosis of cytokeratin-positive melanomas as carcinoma. Use of a panel of immunostains, including S100 protein, should greatly alleviate this problem. In general, anomalous intermediate filament expression tends to be focal in nature, although it may rarely be diffuse. We have seen desmin-positive melanomas misdiagnosed as rhabdomyosarcoma and leiomyosarcoma; additional IHC with markers such as HMB-45, myogenin and muscle actins is necessary for arriving at the correct diagnosis in these unusual cases. CD68 expression: CD68 (KP1) is a relatively non-specific marker of lysosomes, rather than a lineage specific histiocytic marker. CD68-positive melanomas may be mistaken for atypical fibroxanthomas, if one is using this marker in an attempt to confirm "fibrohistiocytic differentiation". CD68 has a very limited role in the diagnosis of cutaneous spindle cell tumors, as it may also be expressed in angiosarcomas, carcinomas, and leiomyosarcomas, among others. Entrapped smooth muscle actin-positive myofibroblasts: Myofibroblasts are frequently found intimately associated with the neoplastic cells of desmoplastic melanoma. We have seen such cases misdiagnosed as leiomyosarcoma or myofibroblastic lesions such as nodular fasciitis or fibromatosis, based on this staining. An S100 immunostain is very valuable here. Markers of Endothelial Differentiation CD31 (Platelet endothelial cell adhesion molecule-1) Background: CD31 is a130 kd transmembrane glycoprotein that is expressed on all endothelial cells, including lymphatic endothelial cells. CD31 is also routinely expressed by tissue macrophages and platelets. Diagnostic uses: CD31 is the single most sensitive and specific marker of endothelial differentiation, and is the single best endothelial marker to have in your own laboratory, if you can have just one. CD31 is expressed by essentially all benign vascular tumors and by over 90% of hemangioendotheliomas and angiosarcomas. CD31 expression in carcinomas is extraordinarily unusual, with only case reports in breast and thyroid carcinomas. Non-endothelial sarcomas, in particular epithelioid sarcoma, are CD31-negative. CD31-positive macrophages within non-endothelial tumors can be confused with positive tumor cells, leading to an erroneous diagnosis of an endothelial tumor. CD34 Background: CD34, a transmembrane glycoprotein of unknown function, is widely expressed on a number of normal tissues, including hematopoietic stem cells, interstitial cells of Cajal, endothelial cells, and dendritic interstitial cells in the skin and around nerves. CD34 is thus not a specific endothelial marker, but is a useful marker in the appropriate histologic context. Diagnostic uses: CD34 is a highly sensitive marker of benign, borderline and malignant endothelial tumors, being expressed by > 90% of cases. CD34 has also generally been regarded as the most sensitive marker for Kaposi sarcoma, although the KSHV-associated LANA protein is probably the current marker of choice for Kaposi sarcoma. In the differential diagnosis of epithelioid angiosarcomas, it is important to remember that CD34 is expressed by up to 60% of epithelioid sarcomas, but not by carcinomas. It is also expressed by dermatofibrosarcoma protuberans, gastrointestinal stromal tumors, leiomyosarcomas, and solitary fibrous tumors. FLI-1 protein Background: FLI-1 protein is a member of ETS family of nuclear transcription factors and is expressed in all mature endothelia and during the earliest stages of endothelial differentiation. FLI-1 is best known as the partner of EWS in the Ewing sarcoma-specific t (11;22) (q12; q24) (EWS-FLI-1) fusion gene. It is also routinely expressed by small lymphocytes (probably T-cells). FLI-1 is the only nuclear marker of endothelium. Diagnostic uses: We have recently shown FLI-1 to be a highly sensitive marker of endothelial neoplasms, including hemangiomas, hemangioendotheliomas, angiosarcomas and Kaposi sarcoma. FLI-1 is not expressed by epithelioid sarcomas, melanomas or carcinomas. It is important not to mistake intratumoral FLI-1 positive endothelial cells and lymphocytes for positive tumor cells. Von Willebrand Factor (Factor 8- related protein) Background : vWF is a clotting factor that is theoretically present only in Weibel-Palade body of endothelium and in platelets, and which should be in theory the most specific marker of endothelial differentiation. Unfortunately, vWF is secreted into the serum, and the inevitable high background staining seen with this marker greatly reduces its "real world" utility. VWF staining in serum may be seen only around the outer surface of cells and closely mimic true membranous expression. Diagnostic uses: vWF is the least sensitive endothelial marker, particularly in angiosarcomas. Given the technical issues discussed above, it is my opinion that there is no real role for vWF in the era of CD31, CD34 and Fli-1 Human Herpesvirus 8 Latency Associated Nuclear Antigen (LANA) Background: An infectious etiology for Kaposi sarcoma has long been suspected, and epidemiological, serologic, and molecular genetic studies over the past 10-15 years have identified a novel herpesvirus, HHV-8, as the likely causative agent of KS. HHV-8 is known to latently infect endothelial cells, as well as peripheral blood monocytes and B-lymphocytes in patients with KS. LANA is one of the most highly expressed proteins during latent HHV-8 infection. The LANA protein is encoded by the open reading frame 73 (ORF73) of the HHV-8 genome, where it tethers viral DNA to host heterochromatin and is thereby required for persistence of viral DNA in dividing cells. LANA also has an essential role in maintenance of the episomal DNA during latent infection and cell division, and also regulates gene expression in infected cells. LANA expression in endothelial tumors: A relatively small number of studies have examined LANA expression by immunohistochemistry. Using a polyclonal antisera, Katano et al found LANA expression in 100% of KS cases studied. Three separate studies, all using the LNA53 monoclonal antibody, noted LANA expression in well over 90% of KS cases, as compared with 0% of non-KS controls. Using the 13B10 clone, 3 very recent studies have noted LANA expression in very close to 100% of KS, as compared with 0% of non-KS potential mimics. Using this same antibody, we have recently found LANA expression to be specific for KS in the HIV-positive population, as well as in the general population. LANA expression in non-endothelial tumors: LANA expression has not been reported in any non-KS tumor, with the notable exceptions of primary effusion lymphoma and Castleman's disease. Pitfalls in the Immunohistochemical Diagnosis of Angiosarcoma Cytokeratin expression: Approximately 10-30% of angiosarcomas express cytokeratins (low molecular weight only). Failure to appreciate this may result in an erroneous diagnosis of carcinoma. Technically this is not considered anomalous expression, as normal endothelial cells also express cytokeratins, at low levels. CD68 expression: As in melanoma (see above), angiosarcomas may express CD68, particularly when they have granular cell features (so-called granular cell angiosarcoma). Muscle Markers Smooth muscle actin Background: The actins are a ubiquitously distributed family of intracellular proteins that may be broadly divided into muscle and non-muscle isoforms. The muscle-specific isoforms can be divided again into smooth, skeletal and cardiac isoforms. From a practical perspective, the single most useful actin antibody is mAb 1A4, which recognizes only smooth muscle isoforms. In the skin, smooth muscle actin is routinely expressed by pilar and vascular smooth muscle, reactive myofibroblasts, a subset of pericytes, and myoepithelial cells. Diagnostic uses: Smooth muscle actin is a highly sensitive marker of smooth muscle and myofibroblastic tumors in the skin. Desmin Background: Desmin is an intermediate filament protein that is expressed by muscle cells of all types, as well as submesothelial fibroblasts, a subset of lymph node dendritic cells, and endometrial stromal cells. Desmin expression is uncommon in myofibroblasts. Diagnostic uses: Desmin is best used as a screening marker for tumors of skeletal muscle differentiation, which are extraordinarily rare in the skin. Although desmin expression is almost always present in pilar smooth muscle tumors, it may be absent in smooth muscle tumors of vascular smooth muscle origin (vascular smooth muscle less commonly expresses desmin, for unknown reasons). Smooth muscle actin is a much better screening marker for smooth muscle tumors. When the differential diagnosis includes true smooth muscle and myofibroblastic tumors, strong desmin expression supports true smooth muscle differentiation. Anomalous desmin expression may be seen in melanoma, schwannoma, tenosynovial giant cell tumor, Ewing sarcoma, and angiomatoid (malignant) fibrous histiocytoma. Atypical Fibroxanthoma/ Superficial Malignant Fibrous Histiocytoma Diagnostic criteria: AFX typically present as a rapidly growing mass in a sun-exposed region of an older adult. Histologically, most AFX are undifferentiated, pleomorphic spindle cells tumors, although relatively monomorphic variants do exist. The diagnosis of AFX should be reserved for small (<1-1.5 cm) lesions that are confined to the dermis and which are completely visualize. Larger lesions, or ones where the base has been transected should be designated "superficial malignant fibrous histiocytoma" (or sarcoma, NOS, depending on your political persuasion). Use of these strict criteria will maintain the excellent prognosis of lesions designated as AFX. identical to MFH, but with largely benign behavior Immunohistochemistry : AFX is a histologic and immunohistochemical diagnosis of exclusion. There are no markers or combinations of markers that establish the diagnosis of AFX/MFH. The lesional cells of AFX must be negative for cytokeratins and S100 protein; a small amount of actin expression, indicative of myofibroblastic differentiation is acceptable. AFX commonly contains S100 protein-positive Langerhans cells, CD31-positive endothelial cells and macrophages and Factor XIIIa-positive dendritic cells, and these must be rigorously identified as non-lesional cells. CD68 expression does not support or exclude the diagnosis of AFX. CD99 (MIC2 glycoprotein, O13, HBA71) expression is not at all specific for the diagnosis of AFX, and in our opinion has no role in this diagnosis. Table 3: Expected immunophenotypes of common pleomorphic cutaneous spindle cell tumors Cytokeratin S100 protein Melanocytic markers Smooth muscle actin Desmin Endothelial markers Sarcomatoid SCC + - - - - - Melanoma -/+ + -/+ - -/+ - AFX/MFH - - - -/+ - - Leiomyosarcoma -/+ -/+ - + +/- -/+ Angiosarcoma -/+ - - - - + Selected References Folpe AL, Gown AM. Immunohistochemistry for Analysis of Soft Tissue Tumors. In: Goldblum JR, Weiss SW. Soft Tissue Tumors, 4th ed. St. Louis: CV Mosby. 2001. Miettinen, M. Immunohistochemistry of Soft Tissue Tumors. In: Miettinen, M. Diagnostic Soft Tissue Pathology, 1st ed. Philadelphia: Churchill Livingstone. 2003.