Modern Pathology (2013) 26, 502–510 502 & 2013 USCAP, Inc All rights reserved 0893-3952/13 $32.00 Transgelin is a novel marker of smooth muscle differentiation that improves diagnostic accuracy of leiomyosarcomas: a comparative immunohistochemical reappraisal of myogenic markers in 900 soft tissue tumors Yves-Marie Robin1, Nicolas Penel2,3, Gae¨lle Pe´rot4,5, Agnes Neuville4,5,6, Vale´rie Ve´lasco4,5, Dominique Ranche`re-Vince7, Philippe Terrier8 and Jean-Michel Coindre4,5,6 1Department of Biology, Unit of Morphological and Molecular Pathology, Centre Oscar Lambret, Lille Cedex, France; 2Department of General Oncology, Centre Oscar Lambret, Lille Cedex, France; 3Research Unit (EA 2694), Medical School University, Lille-Nord-de-France University, Lille Cedex, France; 4Department of Pathology, Institut Bergonie´, Bordeaux Cedex, France; 5INSERM U916, Institut Bergonie´, Bordeaux, France; 6Laboratory of Pathology, Universite´ Victor Segalen Bordeaux 2, Bordeaux, France; 7Department of Patholoy, Centre Le´on Be´rard, Lyon, France and 8Department of Pathology, Institut Gustave Roussy, Villejuif, France Immunohistochemical use of myogenic markers serves to define smooth or skeletal muscle differentiation in soft tissue tumors. Establishing smooth muscle differentiation in malignant lesions can be challenging in some cases. We immunohistochemically examined 900 soft tissue tumors selected from the French Sarcoma Group’s archived tissue collection, which contains a large number of leiomyosarcomas. The four most widely used smooth muscle diagnostic markers were evaluated (smooth muscle actin, desmin, h-caldesmon and calponin), and compared with a novel marker, transgelin. The diagnostic performance of each marker was statistically assessed in terms of sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV) and accuracy (A), in leiomyosarcomas versus all other sarcomas including gastrointestinal stromal tumors (GIST), and second in leiomyosarcomas versus specific tumor types. In leiomyosarcomas versus all other sarcomas including GIST, transgelin emerged as the best diagnostic marker (Se: 83%, Sp: 82%, PPV: 67%, NPV: 92%, A: 83%), compared with smooth muscle actin (Se: 75%, Sp: 83, PPV: 66%, NPV: 89%, A: 81%), desmin (Se: 45%, Sp: 88%, PPV: 62%, NPV: 79%, A: 75%), h-caldesmon (Se: 50%, Sp: 90%, PPV: 67%, NPV: 81%, A: 78%) and calponin (Se: 76%, Sp: 70, PPV: 52%, NPV: 87%, A: 71%). In leiomyosarcomas compared with other specific tumor types such as undifferentiated pleomorphic sarcoma and myxofibrosarcoma, the accuracy for transgelin varied from 80 to 87% whereas it was lower for all other markers (between 51 and 80%). These results indicate that transgelin could be used in practice as an additional marker useful for decision making, especially in those tumors with incomplete immunophenotypes. Modern Pathology (2013) 26, 502–510; doi:10.1038/modpathol.2012.192; published online 23 November 2012 Keywords: immunohistochemistry; smooth muscle differentiation; transgelin; tissue microarray Leiomyosarcoma is one of the most frequent sarco- that soft tissue sarcomas showing a myoid differe- ma histotypes and represents about 15% of all soft ntiation, most often a smooth muscle differentiation, tissue sarcomas.1 The diagnosis of leiomyosarcoma are more aggressive than those with no myoid is clinically important because it has been reported differentiation.2,3 However, the distinction between a poorly differentiated leiomyosarcoma and an undifferentiated spindle cell or pleomorphic Correspondence: Professor J-M Coindre, MD, Department of sarcoma based on morphological features only is Pathology and INSERM U916, Institut Bergonie´, Bordeaux, France. E-mail: [email protected] often difficult and difficult to reproduce. In this Received 11 June 2012; revised 26 September 2012; accepted 26 situation, immunohistochemistry is very helpful. September 2012; published online 23 November 2012 Although myogenin and MyoD1, myogenic www.modernpathology.org Transgelin in mesenchymal tumors Y-M Robin et al 503 transcriptional regulatory proteins, are considered comas, 10 embryonal rhabdomyosarcomas, 8 Ewing sensitive and specific markers for rhabdomyo- sarcomas, 6 soft tissue osteosarcomas, 6 clear cell sarcomas,4 there is no sensitive and specific sarcomas, 5 schwannomas, 5 leiomyomas, 5 angio- single marker for identifying a smooth muscle mas, 3 myxomas, 3 angiosarcomas, 3 soft part differentiation. Markers regularly used for the alveolar sarcomas, 3 extra-skeletal myxoid chondro- diagnosis of smooth muscle tumors are desmin, sarcomas, 2 desmoplastic small round cell tumors, an intermediate filament and smooth muscle actin, 2 neurofibromas, 2 perineurinomas, 1 sclerosing a thin filament,5 along with calponin and fibrosarcoma, 1 malignant peripheral nerve sheath h-caldesmon, both involved in smooth muscle tumor, 1 giant cell tumor of the tendon, 1 myofibro- contraction.6,7 Smooth muscle actin and desmin blastoma and 1 granular cell tumor. Diagnosis of are generally expressed in many different types of leiomyosarcoma was based on criteria derived from skeletal muscle, smooth muscle, myofibroblastic Fletcher et al’s2 and Deyrup et al’s3 reports: a tumor and myoepithelial tumors, as well as in other that at least focally showed the presence lesions. Therefore, rigorous criteria should be used of eosinophilic spindle cells with vesicular blunt- to interpret positive myogenic markers in the ended nuclei arranged in a fascicular pattern. Such diagnosis of leiomyosarcomas.5,8,9 H-caldesmon is foci always accounted for at least 5 to 10% of the reputedly specific to smooth muscle differentiation surface examined. Tumors with these characteristics but lacks sensitivity (Se).6,7 Calponin is quite useful were also required to show extensive positivity for to assess smooth muscle differentiation although it smooth muscle actin or calponin, or focal and strong is probably more frequently used to detect positivity for desmin or h-caldesmon. The leiomyo- myoepithelial or myofibroblast lineage.6,7 A novel sarcoma differentiation level has been evaluated marker, transgelin, a 22 kDa actin-binding protein of according to the differentiation score used for the the calponin family, also correlates with smooth French sarcomas grading system, which is based on muscle differentiation.10 The promoter of the gene histological aspects only.12 In all, 65 cases out of 184 is the target of the transcriptional activator serum (35%) were defined as poorly differentiated (differen- response factor of which myocardin acts as a tiation score of 3) and therefore needed immunohisto- cofactor.11 In an unpublished study, Aurias et al chemistry to obtain a diagnosis of leiomyosarcoma. (Institut Curie, France), isolated target genes of Undifferentiated pleomorphic sarcomas were defined myocardin through gene expression profiling using as pleomorphic/spindle cell sarcoma with sarcoma cell lines and found that transgelin was one no evidence of a specific line of differentiation.2,13 of the most represented in leiomyosarcomatous All tumor slides were reviewed in full sections for differentiation. In this study, we aimed to establish diagnosis confirmation before tumor selection. whether or not transgelin is a useful addition to the other better-known smooth muscle differentiation Tissue Micro-Array markers, in leiomyosarcomas versus all other sarcomas including gastrointestinal stromal tumors All samples were paraffin embedded and formalin (GIST), and secondly in leiomyosarcomas versus fixed. A total of 816 tumors were examined in tissue specific tumor types. The diagnostic performance micro-array blocks with three spots of 1 mm per of transgelin was compared with that of smooth sample. The rest were examined in full sections. muscle actin, desmin, h-caldesmon and calponin in In the dermatofibrosarcoma protuberans subgroup, a large series of benign and malignant mesenchymal 15 tumors were examined in tissue micro-array with lesions irrespective of the line of differentiation or all markers, and 30 in whole sections with transge- anatomic site. lin alone. Immunohistochemistry Materials and methods Slides of 4-um-thick tissue sections were incubated Tumor Selection at room temperature in standard CC1 buffer (Venta- na), revealed with ‘Ultra View’ Universal DAB kit Nine hundred soft tissue tumors were selected and stained with Hematoxylin ReaDi solution from the French Sarcoma Group’s archived paraf- (Ventana). They were treated on automate VENTA- fin-embedded tissue collection: 184 leiomyosarco- NA-Benchmark-XT with the following antibodies: mas, 143 undifferentiated pleomorphic sarcomas, 80 smooth muscle actin (clone: 1A4; pre-treatment: desmoid tumors, 68 myxofibrosarcomas, 65 dedif- CC1; prediluted; source: Ventana), desmin (clone ferentiated liposarcomas, 60 synovial sarcomas, 50 D33, pre-treatment: CC1 ct; dilution: 1/50, source: GIST, 45 dermatofibrosarcoma protuberans, 36 spin- DAKO), caldesmon (clone Hcd, pre-treatment: CC1 dle cell/pleomophic lipomas, 26 nodular fasciitis, std; dilution: 1/50; source: DAKO), calponin (clone 19 pleomophic liposarcoma, 13 well-differentiated CALP; pre-treatment: CC1 std, dilution: 1/100, lipoma-like liposarcomas, 12 pleomorphic rhabdo- source: Biogenex) and transgelin (clone SM22 myosarcomas, 11 myxoid/round cell liposarcomas, ALPHA; pre-treatment: none; dilution: 1/500, 10 adult fibrosarcomas, 10 alveolar rhabdomyosar- source: Abcam). Modern Pathology (2013) 26, 502–510 Transgelin in mesenchymal