CAP Companion Meeting at USCAP 2007 PATHOLOGIC EVALUATION OF MALIGNANT NEOPLASMS WITH INDETERMINATE MORPHOLOGICAL CHARACTERISTICS

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1 CAP Companion Meeting at USCAP 2007 PATHOLOGIC EVALUATION OF MALIGNANT NEOPLASMS WITH INDETERMINATE MORPHOLOGICAL CHARACTERISTICS Profess of Pathology University of Virginia Medical Center Room 3020 University Hospital 1215 Lee Street Charlottesville, VA Materials are used by the CAP with the permission of the faculty.

2 Not uncommonly, pathologists are confronted with neoplasms that have few distinguishing microscopic characteristics and, therefe, appear to be "undifferentiated." The differential diagnostic considerations in such cases are many, and special studies are almost always necessary to reach a definite conclusion. Determining the probable site of igin f a metastatic carcinoma and making a diagnostic separation between two differentiated but histologically similar neoplasms represent additional challenges in surgical pathology that require ancillary labaty analyses. This presentation will address approaches to these and other dilemmas that are particularly suited to resolution by immunodiagnosis. The following discussion is not intended to be exhaustive all-inclusive; rather, the auth s aim is to provide a framewk f the contempary use of adjuvant pathologic techniques f the diagnosis of solid tums. Immunohistochemistry is the principal procedure that is used in that context, but others will be considered as well. IMMUNOHISTOLOGIC REAGENTS FOR THE EVALUATION OF SOLID NEOPLASMS An extensive array of antibodies is available to the surgical pathologist to facilitate characterization of tums without histologically specific features. A highly select panel of immunostains, based on the histopathologic impression of the tum, can be extremely useful to narrow the diagnostic considerations, if not definitively identify the tum. The antibodies utilized f this purpose in our labaty are described briefly below. Immunohistochemical Reagents Intermediate Filaments Cytokeratins are constituents of the intermediate filaments (IFs) of epithelial cells expressed in various combinations depending on the epithelial type and the degree of differentiation. This class of IFs remains among the most commonly studied determinants in immunohistochemistry. Cytokeratin positivity helps crobate a diagnosis of sarcomatoid carcinoma and usually excludes the possibility of sarcoma, lymphoma melanoma. Notable exceptions include synovial sarcoma, chdoma, Ewing's sarcoma, and epithelioid sarcoma; some smooth muscle cells may also react f keratin (1). Vimentin is an IF that is present in most mesenchymal neoplasms and a variety of other classes of neoplasms (e.g., endometrial carcinoma). It is highly valuable as a marker of adequate tissue fixation and processing, in which positive and appropriate vimentin staining provides certainty of the ability of the tissue to react with antibodies in general. Desmin is another IF present in mesenchymal lesions, found principally in myogenous tums such as rhabdomyosarcoma and leiomyosarcoma (2). The neural IFs include neurofilament protein and GFAP. The first of these is restricted to neuronal and neuroendocrine cellular proliferations; however, it is suboptimally preserved in fmalin-fixed specimens. GFAP immuneactivity characterizes glial neoplasms such as astrocytomas and ependymomas. Expression of this reactant appears to be maintained even in poly differentiated tums of the nervous system (3). 2

3 CD45 CD45 is a surface antigen expressed by virtually all hematolymphoid proliferations, and monoclonal antibodies f this marker are reliably specific (4). The utility of CD45 is enhanced by concomitant staining with panels of antibodies to cytokeratin and Sl00 protein. These reagents are helpful in the resolution of such problems as whether not a polygonal small cell undifferentiated lesion is a carcinoma, lymphoma melanoma. Epithelial Membrane Antigen Since its description in 1979, epithelial membrane antigen (EMA) has become a widely used determinant in diagnostic immunocytochemistry. Providing that only membrane-based immuneactivity is regarded as valid, monoclonal antibodies to this discriminant are useful in determining the epithelial nature of undifferentiated tums. The tissue distribution is largely similar to that of keratin, with some caveats; in particular, not all epithelia are positive hepatocellular carcinomas, adrenoctical carcinomas and malignant germ cell neoplasms are EMA-negative (5). Also, EMA may be seen outside of non-epithelial lesions, including large cell anaplastic lymphoma, plasmacytoma, some T-cell lymphomas, epithelioid sarcoma, and synovial sarcoma; meningioma may also show EMA reactivity. The use of supplementary antibody panels that include other epithelial markers obviates potential misclassification. MOC-31 MOC-31 is a 41-kDa cell membrane glycoprotein that is widely distributed in epithelial cells and tums in many tissue sites. Monoclonal antibodies to this determinant have most often been used in the diagnostic distinction between serosal adenocarcinoma and mesothelioma (which typically lacks MOC-31) but they also fail to label a selected group of carcinomas that includes hepatocellular carcinoma, germ cell malignancies and renal cell carcinoma (6). Tum-Associated Glycoprotein (B72.3) The monoclonal antibody known as B72.3 labels a plasmalemmal glycoprotein designated tum-associated glycoprotein-72 (TAG-72). It was isolated from a human breast carcinoma cell line and appears to be virtually pan-carcinomatous in distribution. In likeness to MOC-31, however, TAG-72 is characteristically absent in mesotheliomas, adrenoctical carcinomas, hepatocellular carcinomas, renal cell carcinoma, nasopharyngeal carcinoma, thyroid carcinomas and malignant germ cell tums (7). Placental Alkaline Phosphatase The isoenzyme of alkaline phosphatase that is expressed by the nmal placenta (PLAP) is also evident as an oncofetal antigen in some genitourinary, gastrointestinal and pulmonary carcinomas. Meover, it is nearly universally seen in germ cell tums. Immunostains f PLAP therefe have their greatest application in separating germ cell neoplasms from somatic tums. We have found anti-plap to be an extremely useful screening reagent f possible germ cell differentiation in undifferentiated tums. When positive, stains f me specific oncofetal determinants, as well as keratin subclasses, EMA, and such markers as CD30, should be perfmed. CD30 is present in embryonal carcinoma and is only rarely if ever seen in seminoma. In contrast to germ cell tums, most PLAP-positive somatic tums unifmly express EMA and 3

4 lack CD30. Therefe, a panel that includes cytokeratin, EMA, CD30, and PLAP may be useful in distinguishing among these pleomphic neoplasms (8,9). Hep-PAR1 Hep-PAR1 is a marker of hepatocellular differentiation that is preserved in paraffin sections. It is superi to alpha-fetoprotein (AFP) in that respect, but suffers from some of the same problems attending AFP regarding specificity. Despite this drawback, recent studies indicate that Hep-PAR1 is a helpful marker f metastatic hepatocellular carcinoma in many clinical settings (10). Carcinoembryonic Antigen Carcinoembryonic antigen (CEA) has enjoyed its greatest recognition in clinical medicine as a serologic indicat of the growth of colectal cancer. Immunohistochemically, CEA is strongly expressed in colectal adenocarcinoma, but it may be found in other epithelial tums. F example, adenocarcinomas of the lung, breast and gastrointestinal tract show unifm reactivity with certain epitope specific anti-ceas and thereby may narrow the potential anatomic sites f metastatic glandular neoplasms (11,12). Prostate-Specific Antigen As its name suggests, prostate-specific antigen (PSA) is largely restricted in its tissue distribution to epithelial cells of the prostate and prostatic adenocarcinomas. This cytoplasmic determinant appears to be expressed at an early stage of embryonic development inasmuch as even poly differentiated prostatic malignancies display its presence. Hence, PSA represents one of exceedingly few tissue-specific markers available in diagnostic immunohistochemistry and has been widely utilized in the recognition of metastatic prostate cancer. Extraprostatic expression of PSA has also been noted in periurethral gland adenocarcinoma in women, rectal carcinoid, and extramammary Paget's disease. These entities can generally be easily addressed using a panel of immunostains that includes PSA, as well as neuroendocrine markers and CEA (13). Thyroglobulin Thyroglobulin is likewise restricted in its expression to the follicular thyroid epithelium and related neoplasms. The greatest utility of this marker is to confirm a thyroid igin f metastatic carcinomas in lymph nodes, lung bone. Thyroglobulin reactivity is also helpful in the diagnosis of struma ovarii. Unftunately, "anaplastic" thyroid cancer seldom demonstrates this determinant immunohistochemically, limiting its usefulness in the evaluation of undifferentiated tums to "solid" follicular carcinomas, including "insular" thyroid carcinoma (14). Thyroid Transcription Fact-l Thyroid transcription fact-1 (TTF-1) is a DNA-binding transcriptional regulat protein present in nmal thyroid follicular cells, subsets of respiraty and alveolar epithelium, and the diencephalon; it is commonly present in adenocarcinomas of the lung and thyroid; most anaplastic thyroid carcinomas lack this marker; some auths have found it in 25% of such cases, whereas thyroglobulin reactivity is virtually never detected. The unifm lack of TTF-1 in adenocarcinoma of the breast is useful to discriminate primary lung tums from metastases of a breast tum. In addition, small cell neuroendocrine carcinomas of the lung and selected extrapulmonary sites label f TTF-1. Because of mutually 4

5 exclusive staining patterns in this group of tums, it has been touted as a differential diagnostic discriminant in the separation of Merkel cell carcinoma of the skin and metastatic small cell carcinoma of the lung (15,16). Gross Cystic Disease Fluid Protein-15 Over the past 20 years, progress has been made in characterizing several proteins manufactured by mammary epithelial cells; in particular, one such soluble product that is found in the fluid contents of "fibrocystic" breasts has been studied extensively. This determinant is known as gross cystic disease fluid protein-15 (GCDFP15) and is strongly expressed by cells with apocrine characteristics. GCDFP-15 is present in the tum cells of approximately 55-70% of breast carcinomas, regardless of histologic subtype (17). Estrogen Recept Protein Intuitively, one would expect that estrogen recept protein (ERP) would be restricted to carcinomas of the breast and Müllerian tract. Although those sites certainly account f most ERP-positive tums, lesions such as thyroid carcinoma, transitional cell carcinoma of the bladder, prostatic carcinoma, and even rare examples of gastric, pulmonary and hepatocellular carcinoma may express ERP (12). CA-125 CA-125 has been detected immunohistochemically in neoplasms of the Müllerian but roughly one-half of tums of the biliary tree and pancreas also may also react f this marker (18). Carcinomas in other sites are uncommonly positive f CA-125. CA19-9 CA19-9 is a glycoprotein related to the Le blood group antigen, and it is labeled by a monoclonal antibody raised against a human colon carcinoma cell line. Most carcinomas of the gastrointestinal pancreas, biliary tree, urinary bladder, ovaries and endometrium manifest CA19-9 reactivity, whereas tums of other anatomic locations are only spadically positive. In particular, hepatocellular carcinoma and renal cell carcinoma are consistently negative f this marker (19). CD15 & CD30 CD15 is a hematopoietic differentiation antigen shared by granulocytes, monocytes, Reed- Sternberg cells and subsets of neoplastic B-cells and T-lymphocytes. Several epithelial cell lines (most of which are glandular neuroendocrine) also express CD15, and it may be detected immunohistochemically in their malignant counterparts. Reactivity f CD15 is applicable to the separation of metastatic adenocarcinomas from histologically similar lesions such as malignant mesotheliomas, since it is lacking in the latter tums. When used in combination with CD45, CD15 also provides infmation on the cell lineage of lympheticular neoplasms; CD15- reactive tums in this class predominantly include Hodgkin lymphoma and T-cell non-hodgkin lymphomas. CD30 is likewise a leukocyte antigen that preferentially labels a subset of large-cell non-hodgkin lymphomas ( Ki-1 lymphomas). In the context of non-hematopoietic neoplasms, 5

6 it also is expressed selectively by embryonal carcinomas in the germ cell tum group, as mentioned above (12,20,21). Calretinin Calretinin is a calcium-binding protein that is virtually universally expressed by mesothelial cells and malignant mesotheliomas. Calretinin is a recognized useful marker f discriminating mesothelioma of the epithelial type from adenocarcinoma in both paraffin sections and serous effusions. A limited number of cases of sarcomatoid mesothelioma may also show calretinin expression, separating these lesions from a spectrum of other spindle cell neoplasms of the pleura (22). S100 Protein S100 protein is a calcium flux determinant expressed by nmal melanocytes, Langerhans histiocytes, cartilaginous cells, adipocytes, Schwann cells, astrocytes, oligodendroglia, ependyma, eccrine sweat glands, reticulum cells, salivary glands and myoepithelial cells. It has its greatest use in the diagnosis of solid tums in identifying melanoma, clear-cell sarcoma, glioma and malignant peripheral nerve sheath tum. Also, it has become evident that certain poly differentiated epithelial malignancies have the ability to express Sl00 protein. Carcinomas of the breast, genitourinary pancreas, salivary glands and sweat glands are most notable f this antigenic expression (23). Other Melanocyte Markers Several other monoclonal antibody reagents with selectivity f melanocytic determinants have entered general practice as well. These include HMB-45, antityrosinase, MART-l (Melan- A) and KBA62. Such markers exhibit excellent labeling of fmalin-fixed tissues and show absolute nearly absolute specificity f melanocytes and melanocytic neoplasms. In our experience and that of others, HMB-45 and anti-tyrosinase do not label carcinomas lymphomas; on the other hand, MART-l reactivity is seen in most adrenoctical carcinomas, and KBA62 may decate occasional poly differentiated squamous and renal cell carcinomas as well as rare rhabdomyosarcomas (24,25). Chromogranin-A Chromogranin A (CgA) is a protein that is indigenous to the matrices of neurosecrety granules. It has a relatively ubiquitous distribution in neuroendocrine tissues, e.g., those of the anteri pituitary gland, thyroid C-cell system, parathyroid glands, paraganglion system, adrenal medulla, and pancreatic islets. Accdingly, antibodies to CgA are exceedingly specific in the delineation of neuroendocrine differentiation in epithelial tums. However, because CgA reactivity is directly related to the relative number of cytoplasmic endocrine granules in any given neoplasm, this marker is somewhat insensitive. F example, roughly 30% of small cell neuroendocrine carcinomas and neuroblastomas will exhibit labeling with anti-cga, and it is distinctly unusual f primitive neuroectodermal tums to do so (26). Synaptophysin Synaptophysin is an integral membrane glycoprotein of presynaptic vesicles that is detectable in a wide range of nmal and neoplastic neuroendocrine cells. In light of its subcellular associations one might assume that synaptophysin would have a tissue distribution synonymous to that of the chromogranins; however, in practicality, that is not true. A sizable proption of 6

7 neuroendocrine neoplasms will label f CGA, but not synaptophysin, and the converse of that relationship also applies (26). CD56 & CD57 CD56 and CD57 were initially described as membrane antigens of hematopoietic cells; namely, natural killer lymphocytes. Subsequent analyses have demonstrated a relative rarity of CD56/CD57-positive lymphomas, relegating this marker to a min role in hematopathology; it is most often seen in "large granular cell" lymphoproliferative disders and in the Reed - Sternberg-like cells of lymphocyte-predominant Hodgkin lymphoma. Unexpectedly, these determinants are also associated with myelin-associated glycoproteins, neural cell adhesion molecules and neurosecrety granule matrices. Hence, they are most often used today in solid tum pathology to label schwannian, neuroectodermal and neuroendocrine proliferations. Because of the variety of cell lineages involved in this group, it should be apparent that results of other immunostains are required to put the significance of CD56/CD57 reactivity into proper perspective, in any given case (26,27). CD99 (MIC-2; p30/32 Protein) The membranocytoplasmic protein known as CD99 in the hematopoietic antigen cluster designation is the same molecule that has been called MIC-2 p30/32 protein. The function of this moiety remains uncertain but is known to be expressed in virtually all primitive neuroectodermal tums (PNETs) and Ewing sarcomas. The specificity of CD99 antibodies f a neuroectodermal lineage is not absolute, however, because they may also label a minity (15%) of alveolar rhabdomyosarcomas as well as the overwhelming majity (90%) of lymphoblastic lymphomas. CD99 is observed in up to 20% of neuroendocrine carcinomas in some body sites as well. This is an imptant fact, because it may obscure the difference between NEC and PNET in selected instances (26,28). Muscle-Specific Actin The molecular family of actin proteins includes some moieties that are confined to cells with muscular differentiation, whereas others are seen in epithelia as well. In the diagnosis of mesenchymal neoplasms, monospecific antibodies to the fmer are highly desirable. One such reagent, HHF-35, demonstrates an excellent level of specificity and sensitivity f smooth and striated muscular proliferations, including leiomyosarcomas, hemangiopericytomas, and rhabdomyosarcomas. As such, it is a valuable adjunct to anti-desmin antibodies in the immunohistologic detection of myogenic tums (29-31). CD34 CD34 is expressed by embryonic cells of the hematopoietic system. These include lymphoid and myelogenous elements and also endothelial cells. In the setting of soft tissue tums, CD34 is a potential indicat of vascular differentiation. It is highly sensitive f endothelial differentiation, regardless of tum grade, and recognizes >85% of angiosarcomas and Kaposi's sarcomas. Nevertheless, the specificity of CD34 is a problem, inasmuch as it has been repted in some leiomyosarcomas, peripheral nerve sheath tums and epithelioid sarcomas, which could potentially simulate variants of angiosarcoma hemangioendothelioma. In addition, CD34 is so 7

8 commonly present in dermatofibrosarcoma protuberans (and its variants) and solitary fibrous tum that it is regularly used as an adjunct f the diagnosis of those neoplasms (31). CD31 The platelet-endothelial cell adhesion molecule-l (PECAM-l) is a l30-kda transmembrane glycoprotein shared by vascular lining cells, megakaryocytes, platelets and selected other hematopoietic elements. This marker is highly restricted to endothelial neoplasms among all tums of the soft tissue, and its sensitivity is also excellent. Virtually l00% of angiosarcomas are CD3l positive regardless of grade histotype, and the same statement applies to hemangioma and hemangioendothelioma variants (31). Thrombomodulin Thrombomodulin (TMN) is distributed among endothelial cells, mesothelial cells, osteoblasts, mononuclear phagocytic cells and selected epithelia. TMN may be present in some metastatic carcinomas and most mesotheliomas, both of which may be confused with epithelioid angiosarcomas; therefe, it should be interpreted with this caveat in mind and in the context of the results of other vascular markers. Nevertheless, TMN has proved to be a highly sensitive indicat of endothelial differentiation, particularly in poly differentiated vascular malignancies. Kaposi's sarcoma is likewise consistently immuneactive f this determinant (31). APPROACHES TO GENERIC CLASSIFICATION PROBLEMS IN SOLID TUMOR PATHOLOGY Small Round Cell Neoplasms Small cell neoplasms that cause potential diagnostic confusion include embryonal rhabdomyosarcoma, Ewing's sarcoma, neuroblastoma, PNET (peripheral neuroepithelioma), malignant lymphoma and small cell carcinoma. The anatomic locations and microscopic details of these tums and other aspects of their clinical presentations have a strong bearing on the relative likelihood of respective diagnoses, and immunohistochemical analyses may be tailed accding to such considerations. Small Cell Tums of Soft Tissue Embryonal rhabdomyosarcomas are typified by their immuneactivity f desmin and muscle-specific actin. When used in combination, antibodies to these determinants yield virtually absolute sensitivity f myogenic sarcomas and allow f exclusion of other diagnostic possibilities. Similarly, small cell lymphomas exhibit CD45 in a unifm manner, whereas other neoplasms in this categy are devoid of reactivity, with extremely rare exceptions. Neuroblastomas and neuroepitheliomas express synaptophysin and CD56/CD57. Rhabdomyosarcomas also share potential reactivity f CD56/CD57, but, excluding rare examples of PNET with divergent differentiation, primitive striated muscle sarcomas are distinguished from small cell neurogenic neoplasms by their desmin actin positivity. A particularly diagnostically challenging member of this tum group is, in fact, Ewing's sarcoma/pnet. Our approach is to make this unqualified diagnosis when a small cell neoplasm of soft tissue is reactive f CD99, with without vimentin, synaptophysin, and 8

9 CD56/CD57, and in the absence of desmin, actin, S100 protein and CD45. Some PNETs may also synthesize keratin in an aberrant fashion (Figure 1) (32). Small Cell Carcinomas Small cell carcinomas may exhibit neuroendocrine, squamous glandular differentiation. However, a determination of cell lineage is often difficult on conventional microscopy of these lesions, prompting the use of discriminating immunostains. CD56/CD57, synaptophysin and CGA are restricted to neuroendocrine neoplasms in this mphologic class of lesions; nevertheless, it should be reemphasized that they are seen in only 30-50% of cases (26). Thus, most of the time, immunohistologic studies will not successfully crobate a neuroendocrine lineage f small cell carcinoma, even though it is really present, and one could seriously question whether not such analyses are cost effective (33). Spindle Cell Neoplasms Spindle cell ("sarcomatoid") carcinomas have been described in a diversity of gans including the skin, aerodigestive kidney, bladder, female genital lung and other sites, and must be distinguished from melanomas and true sarcomas in such locations. In addition, spindle cell mesenchymal malignancies of soft tissue can be difficult to separate from one another diagnostically. Spindle Cell Carcinomas In cutaneous, mucosal gan-based locations, any spindle cell tum that displays reactivity f cytokeratin and EMA can be defined as carcinomatous (34). Most sarcomatoid carcinomas also coexpress vimentin, but the latter protein should be regarded as nonspecific when seen in conjunction with other intermediate filaments. Spindle Cell Amelanotic Malignant Melanomas Melanomas that are composed exclusively of fusifm cells retain the general immunohistochemical attributes of other malignant melanocytic neoplasms, with one exception. F unknown reasons, spindle cell melanomas react poly not at all with HMB-45, KBA62, MART-I and anti tyrosinase, making the conjoint use of antibodies to Sl00 protein, vimentin, EMA and cytokeratin paramount in this diagnostic context (34) (Figure 2). Positivity f either of the last two of these determinants excludes the possibility of malignant melanoma. Spindle Cell Sarcomas of Soft Tissue The malignant spindle cell neoplasms of soft tissue include fibrosarcoma, leiomyosarcoma, malignant peripheral nerve sheath tum, monophasic synovial sarcoma, malignant fibrous histiocytoma (MFH) and Kaposi's sarcoma. Among these, fibrosarcoma is characterized by its reactivity f vimentin, to the exclusion of desmin, actin, S100 protein, CD56/CD57, cytokeratin and EMA. In addition, it lacks endothelial markers. Leiomyosarcomas are unique in their diffuse reactivity f desmin and muscle-specific actin but may also express S100 protein and CD56/CD57. Malignant peripheral nerve sheath tums generally demonstrate only the last 3 of these four determinants. Synovial sarcoma represents the sole lesion in this categy that is capable of cytokeratin and EMA synthesis; it may also express CD56/CD57 and CD99. The immunohistologic features of Kaposi's sarcomas are controversial. Some investigats have 9

10 obtained consistent labeling of such neoplasms with D2-40, CD34 CD31 (31). Recently, immunostaining f herpesvirus-8 latent nuclear antigen has been helpful diagnostically (35). Epithelioid (Large Polygonal Cell) Neoplasms Metastatic carcinoma and melanoma, large cell malignant lymphoma, "syncytial" Hodgkin lymphoma, epithelioid sarcoma, clear cell sarcoma, epithelioid angiosarcoma, epithelioid leiomyosarcoma and epithelioid malignant peripheral nerve sheath tum are the differential diagnostic possibilities when dealing with polygonal cell malignancies in lymph nodes and soft tissues. Lymph Nodal Lesions In lymph nodes, epithelioid neoplasms most commonly represent large cell lymphomas metastases of visceral carcinomas and malignant melanomas. The separation of these differential diagnostic possibilities principally involves the use of antibodies to CD45, cytokeratin, EMA and S100 protein (Figure 3). Carcinomas are reactive f cytokeratin, with without EMA, whereas only lymphomas exhibit CD45. Malignant melanomas and some poly differentiated carcinomas exhibit S100 protein, but only the latter tums generally express cytokeratin. Soft Tissue Lesions An epithelioid malignancy of soft tissue is relatively unlikely to represent metastatic carcinoma melanoma, unless widespread dissemination by these neoplasms is clinically obvious. Primary non-hodgkin lymphomas of soft tissues do exist, but they are quite rare. Indeed, in this tissue compartment, epithelioid sarcoma, epithelioid angiosarcoma, epithelioid malignant Schwannoma, epithelioid leiomyosarcoma and clear cell sarcoma are the principal differential diagnostic considerations. Epithelioid sarcoma is a polyphenotypic neoplasm that may express vimentin, S100 protein and even desmin, overlapping with the immunohistologic attributes of other mesenchymal tums. However, it is unique among polygonal cell sarcomas in its consistent expression of cytokeratin; EMA reactivity is also observed in 75% of epithelioid sarcomas. Epithelioid angiosarcoma exhibits vimentin positivity, reactivity f CD3l, thrombomodulin and CD34. This profile usually includes a lack of keratin positivity, but rare examples of epithelioid angiosarcoma have demonstrated aberrant keratin expression, a potential pitfall in interpretation. Clear cell sarcoma is the only one of these lesions that binds HMB-45, KBA62 and antityrosinase; it displays Sl00 protein and vimentin as well but lacks cytokeratin, EMA, desmin, CD3l and CD34. HMB-45, anti-tyrosinase and KBA62 are imptant in distinguishing between clear cell sarcoma and epithelioid malignant peripheral nerve sheath tums, which are typically S100 protein positive but nonreactive f me melanocyte-selective markers. Lastly, only epithelioid leiomyosarcoma expresses desmin and muscle-specific actin (30,36). Another possible diagnostic entity in this categy is gastrointestinal stromal tum, which may assume the fm of an undifferentiated polyhedral-cell neoplasm. It is distinctive in its immuneactivity f vimentin and CD117 (C-kit protein) with without CO34, desmin, muscle-specific actin S100 protein (31). 10

11 Pleomphic Neoplasms Tums that are capable of assuming extremely bizarre, pleomphic cellular shapes include MFH, pleomphic liposarcoma, pleomphic rhabdomyosarcoma, "dedifferentiated" leiomyosarcoma, malignant peripheral nerve sheath tum and metastatic carcinoma melanoma (Figure 2). Metastatic Carcinomas and Melanomas Metastases of carcinomas and melanomas with a pleomphic microscopic appearance are delineated by their reactivity with anticytokeratin with melanocyte-selective markers, respectively. These immunophenotypes are not shared by any other pleomphic malignancy. Pleomphic Soft Tissue Tums Malignant peripheral nerve sheath tums are unique among pleomphic soft tissue sarcomas because they are the only lesions in this class that are capable of diffuse expression of S100 protein, with without CD56/CD57. Pleomphic rhabdomyosarcomas are globally positive f desmin and muscle-specific actin; in addition, they express myoglobin & myogenin, both of which are proteins that are restricted to striated muscle. Although it is a relatively insensitive marker in other fms of rhabdomyosarcoma, myoglobin assumes some imptance among pleomphic neoplasms because "dedifferentiated" leiomyosarcomas also exhibit desmin and actin reactivity but are devoid of myoglobin. Pleomphic MFH lacks all antigens except vimentin; pleomphic liposarcoma differs from this profile in showing focal S100 protein positivity in signet ring multi vacuolated tum lipoblasts. DIFFERENTIAL IMMUNOHISTOCHEMICAL DIAGNOSIS OF METASTATIC CARCINOMAS In the present climate of medical cost consciousness and cresponding limitations on the scope of radiographic procedures, the pathologist is often called on to aid in predicting the sources of metastatic non-small cell carcinomas of unknown igin. It is in this realm of inquiry that permutations in the expression of several tissue-restricted determinants achieve their greatest imptance. As mentioned previously, PLAP, CEA, S100 protein, AFP, EMA, TTF-1 and other tissue specific determinants are useful in this context. Among the latter, f example, anti-psa can be employed to detect metastatic prostatic carcinomas, anti-gcdfp-15 is specific f metastatic breast cancers and anti-thyroglobulin is effective in the recognition of metastatic carcinomas of the thyroid (Figure 4) (12,37,38). Other discriminants listed in the introducty sections of this chapter are found in carcinomas iginating in me than one tissue location. Nevertheless, because their distributions are not identical, combinations of reactivity can be used to generate probability tables that reflect the likelihood of anatomic igins. These, in turn, can be used to construct interpretative algithms. With infmation accumulated over a long period, the auth has had the opptunity to undertake validation studies of such algithms f metastatic carcinoma of unknown igin (MCUO). In those cases in which primary sites were found after initial biopsy of the tum, either by clinical evaluations by autopsy, overall crespondence with the pathologic prediction f the site of igin has been 67%. Others have had similar rates of success with immunohistochemical evaluations. This figure may not seem overly impressive, but it should be compared with the accuracy and efficiency of radiographic and other nonmphologic methods, 11

12 as outlined in the introduction of this presentation. Meover, when mistaken predictions occurred in our experience, they most often involved tums in the same general immunophenotypic class; f example, a tum felt to be most consistent with a pancreatic carcinoma actually proved to be a lung cancer with a largely superimposable set of immuneactivity patterns. Another practical aspect of our algithmic approach is still early in its evolution but is mentioned here f interest. Oncologists are well aware that only a minity of MCUO cases has definable primary tums that can be found during life. Accdingly, most patients in this group have been treated with a standard consensus panel of chemotherapeutic agents designed to have activity against a broad spectrum of malignancies. Nevertheless, that approach is probably not an optimal one compared with specific available treatments aimed at specific gan sites, judging by the dismal survival results that are realized. Thus, some oncologist colleagues have elected to administer chemotherapy regimens that are dictated by the predictive result of the MCUO algithm. In other wds, if lung carcinoma is the faved interpretation by the pathologist, a set of drugs tailed to that diagnosis would be given to the patient even if no pulmonary mass were evident by clinical evaluation. Very preliminary results of such a process have been promising, showing outcomes that are comparable to those of patients with high-stage tums of known anatomic derivation and somewhat better than those treated with generic MCUO therapy. Obviously, considerable wk will be required to decide whether this is ultimately the optimal means of treating MCUO cases. ELECTRON MICROSCOPY & OTHER STUDY MODALITIES Although electron microscopy has been used progressively less in recent years in the diagnosis of tums in surgical pathology, it still has considerable value in that context. In particular, there are several settings in the evaluation of possibly metastatic tums where ultrastructural studies are useful. Adenocarcinomas are characterized generically by the presence of sht, nonbranched plasmalemmal microvilli with a length-to-diameter ratio (LDR) of <10:1. Specialized features of specific lesions in that categy include the presence of laminated cytoplasmic granules (primitive surfactant bodies) in some examples of primary adenocarcinoma of the lung; cytoplasmic mucin granules and terminal webs of intermediate filaments that insert into the microvilli in adenocarcinomas with enteric differentiation; glycogen pools and lipid droplets in metastatic renal cell carcinomas; and tubular cristae in the mitochondria of steroid-producing tums such as adrenoctical carcinoma. Malignant mesothelioma, a simulant of adenocarcinoma at a light microscopic level, is typified by elongated, bushy, branching microvilli with a LDR of >10:1, together with complex desmosomal complexes and cytoplasmic tonofilaments. Mucin granules and surfactant bodies are absent in that tum type (39). Neuroendocrine carcinomas may be identified with certainty because of their synthesis of dense-ce (neurosecrety) granules measuring nm in diameter. Those inclusions have peripheral zones of lucency and tend to be clustered together in the cytoplasm, often near Golgi apparatus. Macular intercellular junctional complexes are also evident in such lesions, and small whls of perinuclear intermediate filaments are common. These characteristics are the same regardless of the site of igin of neuroendocrine tums; hence, electron microscopy cannot be used to distinguish primary pulmonary lesions from metastases. Germ cell tums exhibit electron microscopic characteristics that potentially simulate those of somatic carcinomas. Seminomas are undifferentiated at a fine structural level, showing only primitive appositional intercellular junctional complexes, prominent nucleoli, and the usual 12

13 constituency of basic metabolic ganelles. One salient feature of those tums is the presence of cytoplasmic glycogen pools, but those are shared by many non-germinal tums as well. Embryonal carcinomas largely resemble somatic adenocarcinomas including the fmation of plasmalemmal microvilli and intercellular gland-like spaces and yolk sac tums share the same potentialities. Chiocarcinomas are indeed relatively distinctive ultrastructurally, in that they demonstrate cytoplasmic tonofibrils that are reminiscent of those seen in squamous tums. The latter lesions are not part of the light microscopic differential diagnosis of chiocarcinomas, and that discrepancy may serve as a clue to the proper interpretation. Non-epithelial malignancies with specific electron microscopic attributes are represented by rhabdomyosarcoma, leiomyosarcoma, alveolar soft parts sarcoma, neuroblastoma, melanoma (and clear-cell sarcoma), and endothelial sarcomas. These neoplasms manifest primitive sarcomeric differentiation, with thick and thin cytoplasmic myofilaments; cytoplasmic skeins of thin filaments punctuated by dense bodies; paracrystalline cytoplasmic inclusions; complex interdigitating cytoplasmic extensions containing microtubules; premelanosomes; and Weibel- Palade bodies, respectively. All of those structures are absent in primary pulmonary tums, except f sarcomatoid carcinomas. Those lesions exhibit a mixture of cells with epithelial characteristics (e.g., intercellular junctions, tonofibrils, microvilli) and others with mesenchymal attributes (39). The electron microscopic attributes of other mesenchymal neoplasms are nondescript even misleading diagnostically. F example, primitive neuroectodermal tums (PNETs) and other blastomas such as hepatoblastoma Wilms tum are composed of primdial round cells joined by macular attachment plaques and often containing only basic ganelles. In a minity of cases, glycogen deposits nascent cytoplasmic extensions may be seen, the latter of which contain neurosecrety granules synaptic-type vesicles. Despite the fact that they are soft tissue neoplasms, epithelioid sarcomas and synovial sarcomas mirr their immunohistologic features ultrastructurally, because they manifest the presence polygonal cells joined by wellfmed intercellular attachment plaques and may even demonstrate microvillous differentiation. Hematopoietic proliferations are perhaps the most primitive at an electron microscopic level. They demonstrate only basic cytoplasmic constituents often containing abundant dispersed ribosomes, with without rough endoplasmic reticulum but lacking other distinguishing features. Cytogenetic infmation is rapidly accumulating on a variety of tum types, and it holds the promise of serving as helpful differential diagnostic data in the context being discussed here. F example, several neoplasms have unique chromosomal abnmalities that serve to exclude other possibilities. These include deletions of the sht arm of chromosome 3 in renal cell carcinoma; an unrelated deletion in the same chromosomal segment in primary SCNC of the lung; the t(11;22) translocation of PNET/Ewing sarcoma; t(1;13) t(2;13) translocations in alveolar rhabdomyosarcoma; the t(12;22) translocation of clear-cell sarcoma; the der(17)t(x;17) translocation of alveolar soft parts sarcoma; the t(x;18) translocation of synovial sarcoma; isochromosome 12p in germ-cell malignancies; and a group of semispecific specific karyotypic abnmalities in malignant lymphomas and leukemias. These markers are best assessed using fresh tissue, but assays f them that are based on the polymerase chain reaction fluescent in-situ hybridization studies are also becoming available f use in hospital practice (40-44). 13

14 REFERENCES 1. Battifa H. Diagnostic uses of antibodies to keratins. Prog Surg Pathol 1988;8: Truong LD, Rangdaeng S, Cagle PT, Ro JY, Hawkins H, Font RL. The diagnostic utility of desmin: a study of 584 cases and review of the literature. Am J Clin Pathol 1990;93: Mrison CD, Prayson RA. Immunohistochemistry in the diagnosis of neoplasms of the central nervous system Semin Diagn Pathol 2000;17: Kurtin PJ, Pinkus GS. Leukocyte common antigen-a diagnostic discriminant between hematopoietic and nonhematopoietic neoplasms in paraffin sections using monoclonal antibodies: crelation with immunologic studies and ultrastructural localization. Hum Pathol 1985;16: Pinkus GS, Kurtin PJ. Epithelial membrane antigen-a diagnostic discriminant in surgical pathology. Hum Pathol 1985;16: Mrison C, Marsh W Jr, Frankel W. A comparison of CD10 to pcea, MOC-31, and hepatocyte f the distinction of malignant tums in the liver. Mod Pathol 2002;15: Loy TS, Nashelsky MB. Reactivity of B72.3 with adenocarcinomas: an immunohistochemical study of 476 cases. Cancer 1993;72: Wick MR, Swanson PE, Manivel JC. Placental-like alkaline phosphatase reactivity in human tums: an immunohistochemical study of 520 cases. Hum Pathol 1987;18: Suster S, Man CA, Dominguez-Malagon H, Quevedo-Blanco P. Germ cell tums of the mediastinum and testis: a comparative immunohistochemical study of 120 cases. Hum Pathol 1998;29: Lugli A, Tnillo L, Mirlacher M, et al. Hepatocyte paraffin 1 expression in human nmal and neoplastic tissues: tissue microarray analysis on 3,940 tissue samples. Am J Clin Pathol 2004;122: Sheahan K, O'Brien MJ, Burke B, et a1. Differential reactivities of carcinoembryonic antigen (CEA) and CEA-related monoclonal and polyclonal antibodies in common epithelial malignancies. Am J Clin Pathol 1990;94: DeYoung BR, Wick MR. Immunohistologic evaluation of metastatic carcinomas of unknown igin: an algithmic approach. Semin Diagn Pathol 2000;17: Bostwick DG. Prostate-specific antigen: current role in diagnostic pathology of prostate cancer. Am J Clin Pathol 1994;102 (4Suppl 1): S31-S DeMicco C, Ruf J, Carayon P, et a1. Immunohistochemical study of thyroglobulin in thyroid carcinomas with monoclonal antibodies. Cancer 1987;59: Ordonez NG. Thyroid transcription fact-1 is a marker f lung and thyroid carcinomas. Adv Anat Pathol 2000;7: Kaufmann O, Dietel M. Expression of thyroid transcription fact-l in pulmonary and extrapulmonary small cell carcinomas and other neuroendocrine carcinomas of various primary sites. Histopathology 2000;36: Wick MR, Lillemoe T, Copland GT, et al. Gross cystic disease fluid protein-15 as a marker f breast carcinoma. Hum Pathol 1989;20: Loy TS, Quesenberry JT, Sharp SC. Distribution of CA125 in adenocarcinomas: an immunohistochemical study of 481 cases. Am J Clin Pathol 1992;98: Gatalica Z, Miettinen M. Distribution of carcinoma antigens CA19-9 and CA153; an immunohistochemical study of 400 tums. Appl Immunohistochem 1994;2: Arber DA, Weiss L. CDI5: a review. Appl Immunohistochem 1993;1: Elgin J, Phillips JG, Reddy VV, Gibbs PO, Listinsky CM. Hodgkin's and non-hodgkin's 14

15 lymphoma: spectrum of mphologic and immunophenotypic overlap. Ann Diagn Pathol 1999:3: Dei Tos AP, Doglioni C. Calretinin: a novel tool f diagnostic immunohistochemistry. Adv Anat Pathol 1998;5: Matsushima S, Mi M, Adachi Y, Matsukuma A, Sugimachi, K. Sl00 protein-positive breast carcinomas: an immunohistochemical study. J Surg Oncol 1994;55: Wick MR, Swanson PE, Rocama A. Recognition of malignant melanoma by monoclonal antibody HMB-45. J Cutan Pathol 1998;15: Kaufmann 0, Koch S, Burghardt J, Andring H, Dietel M. Tyrosinase, Melan-A, and KBA62 as markers f the immunohistochemical identification of metastatic amelanotic melanomas on paraffin sections. Mod Pathol 1998;11: Wick MR. Immunohistology of neuroendocrine and neuroectodermal tums. Semin Diagn Pathol 2000;17: Chu PG, Chang KL, Arber DA, Weiss LM. Immunophenotyping of hematopoietic neoplasms. Semin Diagn Pathol 2000;17: Dehner LP. Primitive neuroectodermal tum and Ewing's sarcoma. Am J Surg Pathol 1993;17: Tsukada T, McNutt MA, Ross R, Gown AM. HHF35, a muscle actin specific monoclonal antibody. II. Reactivity in nmal, reactive, and neoplastic human tissues. Am J Pathol 1987;127: Rangdaeng S, Truong LD. Comparative immunohistochemical staining f desmin and muscle-specific actin: a study of 576 cases. Am J Clin Pathol 1991;96: Suster S. Recent advances in the application of immunohistochemical markers f the diagnosis of soft tissue tums. Semin Diagn Pathol 2000;17: Devoe R, Weidner N. Immunohistochemistry of small round-cell tums. Semin Diagn Pathol 2000;17: Guinee DG, Fishback NF, Koss MN, et al. The spectrum of immunohistochemical staining of small cell lung carcinoma in specimens from transbronchial and open lung biopsies. Am J Clin Pathol 1994;102: Wick MR, Fitzgibbon JF, Swanson PE. Cutaneous sarcomas and sarcomatoid neoplasms of the skin. Semin Diagn Pathol 1993;10: Hong A, Davies S, Lee C. Immunohistochemical detection of the human herpes virus 8 (HHV8) latent nuclear antigen-1 in Kaposi's sarcoma. Pathology 2003;35: Papas-Cden P, Zarbo RJ, Gown AM, Crissman JD. Immunohistochemical characterization of synovial, epithelioid, and clear-cell sarcomas. Surg Pathol 1989;2: Brown RW, Campagna LB, Dunn JK, et al. Immunohistochemical identification of tum markers in metastatic adenocarcinoma: a diagnostic adjunct in the determination of primary site. Am J Clin Pathol 1997;107: Man CA, Wick M R, Suster S. The role of immunohistochemistry in the diagnosis of malignant mesothelioma. Semin Diagn Pathol 2000;17: Tucker JA. The continuing value of electron microscopy in surgical pathology. Ultrastruct Pathol 2000;24: Weinstein MH, Dal Cin P. Genetics of epithelial tums of the renal parenchyma in adults and renal cell carcinoma in children. Anal Quant Cytol Histol 2001;23: Dennis TR, Stock AD. A molecular cytogenetic study of chromosome 3 rearrangements in small cell lung cancer: consistent involvement of chromosome band 3q13.2. Cancer Genet Cytogenet 1999;113:

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17 Figure 1 PNET with divergent myogenic differentiation differentiation + PNET with divergent myogenic + DES/MSA Neuroendocrine Neuroendocrine carcinoma (primary & carcinoma (primary & secondary) secondary) CD56/SYN/CGA Small-cell Small-cell adenocarcinoma adenocarcinoma CD15/TAG72/MOC31 CD15/TAG72/MOC31 Malignant Malignant lymphoma lymphoma + Malignant leukemia Malignant leukemia melanoma melanoma Small-cell Small-cell squamous squamous CA CA CK CK LCA LCA EMA S100/HMB45/MART1 EMA S100/HMB45/MART1 VIM VIM Rhabdomyosarcoma Rhabdomyosarcoma (PNET) (PNET) Undiff. Sarcoma Undiff. Sarcoma DES/MSA FLI-1/SYN/CD56/CD99 PNET PNET FLI-1/SYN/CD99 Technically Technically inadequate specimen inadequate specimen IMMUNOHISTOCHEMICAL ALGORITHM FOR SMALL-CELL MALIGNANT TUMORS Figure 2 S100 S100 protein FLI-1/CD31/CD34/Thrombomodulin HMB45/MART1 Desmin/Muscle-specific actin actin Malignant melanoma Malignant melanoma Leiomyosarcoma Leiomyosarcoma Sarcomatoid Sarcomatoid squamous squamous cell cell carcinoma carcinoma Malignant schwannoma Malignant schwannoma Neuroid Neuroid malignant melanoma malignant melanoma + Vimentin + Atypical Atypical fibroxanthoma fibroxanthoma MFH MFH Angiosarcoma Angiosarcoma Technically Technically inadequate inadequate specimen specimen Keratin & Epithelial membrane antigen IMMUNOHISTOCHEMICAL ALGORITHM FOR MALIGNANT SPINDLE-CELL & PLEOMORPHIC TUMORS Figure 3 17

18 Malignant melanoma S100/HMB45/MART-1 CD45 CD45 Malignant lymphoma leukemia VIMENTIN KERATIN Squamous carcinoma LELCS LELCS Sarcoma BER-EP4/CD15/CEA/S100 Adenocarcinoma Technically inferi specimen IMMUNOHISTOCHEMICAL ALGORITHM FOR MALIGNANT LARGE-CELL MALIGNANT TUMORS 18

19 Figure 4 PSA+ PSA+ TGB+ TGB+ PSA/TGB/TTF1 PSA/TGB/TTF1 + TTF1+ TTF1+ KERATIN KERATIN Mixture Mixture Seminoma (PLAP+; VIM+) Seminoma (PLAP+; VIM+) Adrenoctical Adrenoctical Carcinoma (VIM+; PLAP-) Carcinoma (VIM+; PLAP-) Prostatic Prostatic carcinoma carcinoma Thyroid Thyroid carcinoma carcinoma Thyroid Lung carcinoma Thyroid Lung carcinoma All All (-) (-) RCC RCC NPC NPC ACC ACC + B72.3/MOC-31 B72.3/MOC-31 Both CALRET VIM VIM + + EMA EMA PLAP PLAP Either both PLAP PLAP + + GCDFP GCDFP CEA-M CEA-M S100 S100 PLAP PLAP CA-125 CA-125 CA19-9 CA19-9 CK20 CK20 ER ER + Mesothelioma Mesothelioma NPC NPC HCC HCC + EMA EMA Embryonal Embryonal CA CA Embryonal Embryonal CA CA GCDFP+ alone Breast, salivary gland CEA+ GCDFP+ alone alone GI Breast, lung, salivary gland b S100+ CEA+ alone alone GI lung, breast, salivary PLAP+ S100+ alone alone GI breast, salivar lung, PLAP+ alone kidney, breast GI lung CA125+ alone kidney, breast biliary CA125+ alone pancreas, biliary lung, brea GI pancreas, liver lung, bre CA19-9+ alone GI pancreas, GI liver CA19-9+ alone GI bladder, pancreas, lung Mulleria CK20+ alone GI bladder, lung pancre ER+ CK20+ alone alone Breast, GI bladder panc li ER+ alone Breast, stomach bladder GCDFP+CEA+ Breast, stomach salivary gland GCDFP+S100+ GCDFP+CEA+ Breast, Breast, salivary salivary gland gland GCDFP+PLAP+ GCDFP+S100+ Breast Breast, salivary gland GCDFP+CA125+ GCDFP+PLAP+ Breast, Breast salivary gland GCDFP+CA19-9+ GCDFP+CA125+ Salivary Breast, salivary gland, breast gland GCDFP+CK20+ GCDFP+CA19-9+ Not Salivary described gland, in literature breast GCDFP+ER+ GCDFP+CK20+ Breast Not described in literature CEA+S100+ GCDFP+ER+ Breast, Breast salivary gland, Mulleria CEA+S100+ Breast, stomach, salivary biliary gland, tract Mulleri CEA+PLAP+ GI stomach, biliary biliary tract lung, Mul CEA+CA125+ CEA+PLAP+ GI biliary biliary lung, Mu CEA+CA125+ pancreas, biliary lung, GI tr CEA+CA19-9+ GI pancreas, pancreas, bladder, lung, GI M CEA+CA19-9+ GI lung pancreas, bladder, CEA+CK20+ GI biliary lung tracts, pancreas, CEA+CK20+ GI endocervix, biliary tracts, bladder, pancreas lung CEA+ER+ Breast, endocervix, stomach, bladder, tract lung S100+PLAP+ CEA+ER+ Breast, stomach, breast, stomac trac S100+CA125+ S100+PLAP+ breast, breast, biliary stoma S100+CA19-9+ S100+CA125+ biliary breast, tree, biliar s S100+CA19-9+ kidney biliary tree, S100+CK20+ kidney biliary tree, sto S100+ER+ S100+CK20+ Breast, tract biliary tree, st PLAP+CA125+ S100+ER+ Breast,, biliary, tract GI tracts PLAP+CA19-9+ PLAP+CA125+ Biliary,,, biliary, GI GI tract trac PLAP+CK20+ PLAP+CA19-9+ Biliary,GI, Biliary,, tracts; GI tra PLAP+ER+ PLAP+CK20+ Breast, Biliary,GI, tract tracts ALL PLAP+ER+ NEGATIVE Breast, Renal cell carcinoma tract ALL NEGATIVE Renal Germ cell cell carcinoma tum (rare exa Nasopharyngeal Germ cell tum carcinoma (rare ex Nasopharyngeal carcinom IMMUNOHISTOLOGICAL EVALUATION OF MALIGNANT EPITHELIAL TUMORS OF UNKNOWN ORIGIN IMMUNOHISTOLOGICAL EVALUATION OF MALIGNANT EPITHELIAL TUMORS OF UNKNOWN ORIGIN Key to Abbreviations in Figures All interpretations begin at the CK/keratin keratin/ema box and me up down and radially from that reference point. Abbreviations CK = Cytokeratin; DES = Desmin; MSA = Muscle-specific actin; SYN = Synaptophysin; CGA = Chromogranin-A; TAG72 = Tum-associated glycoprotein-72; LCA = Leukocyte common antigen (CD45); EMA = Epithelial membrane antigen; VIM = Vimentin; PNET = Primitive neuroectodermal tum; CA = Carcinoma; Undiff. = Undifferentiated; LELCS = Lymphoepithelioma-like carcinoma; MFH = Malignant fibrous histiocytoma; PSA = Prostate specific antigen; TGB = Thyroglobulin; TTF1 = Thyroid transcription fact-1; CALRET = Calretinin; GCDFP = Gross cystic disease fluid protein-15; CEA-M = Carcinoembryonic antigen (detected by monoclonal antibody); PLAP = Placental alkaline phosphatase; CD20 = Cytokeratin 20; ER = Estrogen recept protein; NPC = Nasopharyngeal carcinoma; HCC = Hepatocellular carcinoma; RCC = Renal cell carcinoma; ACC = Adrenoctical carcinoma; GI = Gastrointestinal 19