Section 4 -

Immunohistochemistry of Soft Tissue Sarcomas Antonio Llombart Bosch Samuel Navarro Fos Dept. Pathology University of Valencia Medical School Valencia Spain

Soft tissue sarcomas configure a heterogeneous and relatively uncommon group of neoplasms that frequently share similar morphologic features which complicate the establishment of a correct diagnosis which is necessary to determine the therapeutic approach as well as its prognostic impact. The importance of immunohistochemistry (IHC) in the histopathologic diagnosis of numerous neoplastic diseases is by now well established. Since its introduction in the early 1980's, the main role of IHC has been as an aid in the diagnosis of cases of STS. IHC has had a relevance in the classification of these tumors, determining their line of differentiation, elucidating their pathogenetic mechanisms and, to a lesser degree, predicting clinical behavior. Nevertheless, IHC should still be considered as an ancillary technique and does not replace routine light microscopic examination. Nowadays, a vast number of pathology laboratories have incorporated IHC as a routine procedure in the diagnosis of STS. [1, 2, 3, 4, 5] Some laboratories, especially in developing countries, still base the diagnosis of STS on H&E morphological pattern recognition, due to the limited availability of immunohistochemical techniques. In order to study the concordance between the morphologic diagnosis of STS and the immunophenotype, we have performed a collaborative study with the National Oncology Institute of Cuba (INO). [6] One hundred and nine cases of spindle and pleomorphic STS were retrieved from the files of the Cuban Registry of STS. Diagnosis was established separately using H&E examination followed by complimentary IHC. Regarding those cases with a spindle cell pattern, only 58% of global diagnostic agreement was obtained, being particularly low (33%) in the group of leiomyosarcomas. Within the pleomorphic sarcomas, 59% of cases were correctly typified using only conventional histopathology, whereas IHC allowed a reclassification of 100% of cases. This study confirms the need to use IHC for the correct typification of STS. For practical purposes, we summarize the immunophenotype of the most frequent types of STS based upon their phenotypic patterns. Spindle Cell STS . IHC is an important adjunct to the H&E examination in the diagnosis of spindle cell sarcomas. The diagnosis of leiomyosarcomas can be established using a panel of antibodies that include desmin, smooth muscle actin, pan muscle actin and h-caldesmon. We use all of the above-mentioned antibodies in order to establish a differential diagnosis with other spindle cell tumors, such as the myofibroblastic family of ST tumors that frequently express, to a lesser degree, some of these antibodies. This differential diagnosis is very important due to the fact that leiomyosarcoma is more aggressive than low grade myofibrosarcoma. In addition, desmin expression may be absent in poorly differentiated leiomyosarcoma. [7] The diagnosis of MPNST should be based upon S-100 positivity (50-70 % of cases), and other low-specificity markers such as hnk-1 or PGP 9.5, that can also be detected in other spindle cell STS. Nevertheless, in some poorly-differentiated MPNST the immunohistochemistry may be inconclusive. Malignant perineurial tumors are extremely rare, but present focal epithelial membrane antigen (EMA) and Claudin-1 positivity. [8] Monophasic synovial sarcoma can be discriminated given its expression of EMA (only in some cells), low molecular weight cytokeratin, (also located in a small group of isolated cells) and the cytoplasmic decoration of CD99. New markers for MFSS have been proposed recently, comprising E-cadherin, beta–Catenin, Bcl-2, EGFR and SALL-2. [9, 10, 11] It should be noted that some cases also focally express nuclear staining for S-100 a fact that may induce confusion with an MPNST. The diagnosis of fibrosarcoma is an exclusion diagnosis, based upon the microscopic appearance and the absence of expression of other markers except for vimentin. Dermatofibrosarcoma protuberans expresses a constant immunophenotype vimentin and CD 34 positivity, as is the case of solitary fibrous tumor. Finally, the diagnosis of a spindle cell variant of GIST can be based upon the expression of c-kit (CD 117), PDGFR a and CD 34. [12] Pleomorphic STS. Pleomorphic neoplasms configure a large number of soft tissue malignancies that were previously consistent with MFH. Since this tumor entity has been banned from the histological classification after the new WHO consensus [13] it is now mandatory to reclassify this heterogeneous group of tumors with an alternative methodology, due to their variable aggressive course. The use of IHC has also clarified some aspects in relation to the so-called Pleomorphic Malignant Fibrous Histiocytoma (MFH), allowing a reclassification of most cases according to their line of differentiation. IHC is also a useful tool for distinguishing pleomorphic sarcomas from anaplastic carcinoma (that express EMA and/or cytokeratin) lymphomas (CD45 +, ALK +, B/T cell markers) and melanomas (S-100 +, melan A +, HMB 45 +). [14] It is recommended to identify Pleomorphic Rhabdomyosarcoma because of its highly aggressive behavior. The diagnosis is based upon the expression of desmin, actin, MyoD1, myoglobin and myogenin. [15] Pleomorphic Liposarcomas tend to be negative for S-100, and the diagnosis of this neoplasm is based mainly on the identification of persistent isolated lipoblasts. Myxoid STS The value of IHC in the diagnosis of myxoid SFT is limited. Myxoid / round cell liposarcoma should be diagnosed upon the expression of S-100 protein. Myxoid MFH expresses only vimentin and occasionally pan actin HHF35, whereas the myxoid variant of leiomyosarcoma and MPNST express the same markers as their spindle cell counterparts. In addition, Myxoid Chondrosarcoma expresses S-100 as well as HNK 1 and SOX 9 [16]. Epithelioid STS [17] Despite the heterogeneity of this cytological pattern present in several tumor varieties Epithelioid sarcoma is the main representative of this group due to its specific morphology, including typical necrotic foci and cell palisading. IHC provides substantial support, expressing EMA, Cytokeratin, and in more than 50% of cases CD34 which is absent in carcinomas. The epithelioid variants of leiomyosarcoma, MPNST and angiosarcoma express also Cytokeratin in addition to their own specific markers. Epithelioid MPNST has also to be distinguished from melanoma due to the positivity of the last to HMB45 and melan-A. Small Round Cell Tumors. An accurate diagnosis of this group of neoplasms is very important in determining appropriate therapy. Morphologically, this group of tumors comprises small round undifferentiated cells and it is very difficult to establish a differential diagnosis when only routine morphologic light examination is performed. Rhabdomyosarcomas (embryonal, alveolar) can be distinguished upon the expression of muscle-related markers such as desmin, actin, myoglobin (low sensitivity), MyoD1 and Myf 4 (myogenin). The Ewing 's family of tumors / PNET express, in addition to a number of particular markers (NSE, synaptophysin, HNK 1), and specifically the cell surface glycoprotein p30 / 32, a product of the MIC2 gene. Antibodies to this glycoprotein, such as O13, MIC 2 and HBA 71, are very sensitive for Ewing 's / PNET. [18, 19, 20] However, this antigen is not specific, because its expression has also been shown in rhabdomyosarcomas, lymphoblastic lymphomas, synovial sarcomas, and mesenchymal chondrosarcomas. Recently, a new antibody that recognizes the Fli-1 gene product has been incorporated into the diagnostic panel for EFT. Nevertheless, Fli-1 is also detected in vascular neoplasms lymphomas, and to a lesser degree in rhabdomyosarcomas and synovial sarcomas. [21] Desmoplastic small round cell tumor can be diagnosed on the basis of WT-1 expression, desmin (dot-like staining) cytokeratin and EMA positivity. [22] Undifferentiated neuroblastic tumors enter into the differential diagnosis of SRCT. The expression of NB 84 and the negativity for CD99 helps in the correct typification of these tumors. Other Sarcomas Variants [1, 2, 3, 4, 5] This heterogeneous group should include clear cell sarcomas, alveolar soft part sarcomas and angiosarcomas. Clear cell sarcomas (malignant melanoma of soft tissue) express the same melanic markers S-100, HMB45 and melan- A. Moreover, almost 33% of cases express cytokeratin [23]. Angiosarcoma is characterized by the expression of vascular markers such as CD31 (the most sensitive and specific endothelial markers), CD34, Ulex lectin and recently Fli-1. [24] Finally, a great controversy exists regarding the hypothetical myogenic nature of Alveolar soft part sarcoma due to the reported expression of muscle markers such as desmin, actin, and MyoD1. Proliferation markers have been studied in order to complement or substitute the mitotic count. First, PCNA expression was discarded since it seems more likely to be an indicator of DNA repair. Ki67 (MIB 1) staining appeared to be a more reliable method of assessing proliferating cells, but we know that Ki67 stains all the cells that are in the different phases of the cell cycle. Other markers such as Topoisomerase IIa that stains only those cells in the proliferation phase of the cell cycle should be incorporated into the panel of antibodies in order to improve the grading of STS. The most commonly used immunomarkers for diagnostic and prognostic purposes in the group of STS have been summarized. Another controversial issue includes the establishment of the grading in SFS. Currently, the grading is performed according to major morphological features such as : tumor size, histology, cellular pleomorphism, mitotic index, necrosis extension and borders invasion, in which ICH is not specifically considered. Other Questions will be Additionally Discussed Such As: - WHICH ARE THE MOST USEFUL ANTIBODIES IN STS? - CAN WE RELY EXCLUSIVELY ON IHC FOR THE DIAGNOSIS OF ALL STS? - Are tissue array necessary for standardized the IHC diagnosis? References: Miettinen M. Immunohistochemistry of soft-tissue tumors. Possibilities and limitations in surgical pathology. Pathol. 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