Modern Pathology (2007) 20, 749–759 & 2007 USCAP, Inc All rights reserved 0893-3952/07 $30.00 www.modernpathology.org Gene expression analysis of soft tissue sarcomas: characterization and reclassification of malignant fibrous histiocytoma Robert Nakayama1,2,3, Takeshi Nemoto2,4, Hiro Takahashi2, Tsutomu Ohta2, Akira Kawai5, Kunihiko Seki6, Teruhiko Yoshida2, Yoshiaki Toyama3, Hitoshi Ichikawa1 and Tadashi Hasegawa6,7 1Cancer Transcriptome Project, National Cancer Center Research Institute, Tokyo, Japan; 2Genetics Division, National Cancer Center Research Institute, Tokyo, Japan; 3Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan; 4Department of Dermatology, Tokyo Medical and Dental University, Tokyo, Japan; 5Orthopedics Division, National Cancer Center Hospital, Tokyo, Japan; 6Pathology Division, National Cancer Center Hospital, Tokyo, Japan and 7Department of Surgical Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan In soft tissue sarcomas, the diagnosis of malignant fibrous histiocytoma (MFH) has been a very controversial issue, and MFH is now considered to be reclassified into pleomorphic subtypes of other sarcomas. To characterize MFH genetically, we used an oligonucleotide microarray to analyze gene expression in 105 samples from 10 types of soft tissue tumors. Spindle cell and pleomorphic sarcomas, such as dedifferentiated liposarcoma, myxofibrosarcoma, leiomyosarcoma, malignant peripheral nerve sheath tumor (MPNST), fibrosarcoma and MFH, showed similar gene expression patterns compared to other tumors. Samples from those five sarcoma types could be classified into respective clusters based on gene expression by excluding MFH samples. We calculated distances between MFH samples and other five sarcoma types (dedifferentiated liposarcoma, myxofibrosarcoma, leiomyosarcoma, MPNST and fibrosarcoma) based on differentially expressed genes and evaluated similarities. Three of the 21 MFH samples showed marked similarities to one of the five sarcoma types, which were supported by histological findings. Although most of the remaining 18 MFH samples showed little or no histological resemblance to one of the five sarcoma types, 12 of them showed moderate similarities in terms of gene expression. These results explain the heterogeneity of MFH and show that the majority of MFHs could be reclassified into pleomorphic subtypes of other sarcomas. Taken together, gene expression profiling could be a useful tool to unveil the difference in the underlying molecular backgrounds, which leads to a rational taxonomy and diagnosis of a diverse group of soft tissue sarcomas. Modern Pathology (2007) 20, 749–759; doi:10.1038/modpathol.3800794; published online 27 April 2007 Keywords: gene expression; malignant fibrous histiocytoma; myxofibrosarcoma; soft tissue sarcoma; reclassi- fication; undifferentiated pleomorphic sarcoma Malignant soft tissue tumors are a diverse group of recurrent genetic aberrations, such as chromosomal tumors of mesenchymal origin, which have gener- translocations resulting in gene fusions (SYT-SSX in ally been classified according to their histological synovial sarcoma, TLS-CHOP in myxoid/round cell resemblance to normal tissue. Understanding of liposarcoma) or somatic mutations (KIT in gastro- molecular pathology gained in recent decades intestinal stromal tumors), and they are now shows that some soft tissue tumors exhibit single classified by these molecular markers specific to each tumor.1 In contrast, other malignant soft tissue tumors, such as malignant fibrous histiocytoma Correspondence: Dr H Ichikawa, PhD, Cancer Transcriptome (MFH), fibrosarcoma and leiomyosarcoma, are Project, National Cancer Center Research Institute, 5–1–1 Tsukiji, characterized by numerous, nonrecurrent complex Chuo-ku, Tokyo 104-0045, Japan. E-mail: [email protected] chromosomal aberrations, and frequently show Received 9 January 2007; revised 20 March 2007; accepted 27 overlapping histological appearance and immuno- March 2007; published online 27 April 2007 histochemical phenotypes that are often difficult to Characterization of MFH by gene expression R Nakayama et al 750 interpret by pathologists.2 Among them, diagnosis of malignant peripheral nerve sheath tumor (MPNST) MFH has been the most controversial issue.3–5 MFH and fibrosarcoma and quantified similarities as has been considered the most common soft tissue distances between MFH samples and the five sarcoma of adults; it is manifested by a broad range sarcoma types. of histological appearances and consists of four subtypes: storiform and pleomorphic type, myxoid type, giant cell type and inflammatory type. Recent Materials and methods clinicopathological, ultrastructural and immunohis- Patients and Tumor Samples tochemical studies revealed that MFH shows no evidence of true histiocytic differentiation and that Characteristics of 105 soft tissue tumors used in this it is not a single entity but rather a heterogeneous study are shown in Supplementary data 1. Among collection of pleomorphic subtypes of other sarco- them, 35 samples were previously analyzed in a mas. Since each type of sarcoma other than MFH different method.13 All patients received histologi- shows distinct biological behavior, particularly in cal diagnosis of primary soft tissue tumor at local recurrence or metastasis rate, MFH showing a National Cancer Center Hospital, Tokyo, from 1996 variety of clinicopathological characteristics should to 2002. In this paper, we use the term ‘MFH’ to be further reclassified to correctly evaluate the describe samples diagnosed as storiform and pleo- malignant potential of each case. In the latest edition morphic type MFH showing predominant pleo- of the WHO classification, myxoid type MFH was morphic features without immunohistochemical classified as myxofibrosarcoma in the fibroblastic phenotypes characteristic of specific differentiation, category, and other subtypes of MFH without any and the term ‘myxofibrosarcoma’ to describe MFH evidence of differentiation were classified as un- with predominant (450%) myxoid features con- differentiated high grade pleomorphic sarcoma.6 ventionally diagnosed as myxoid type MFH. Before WHO classification also suggested that the term the gene expression analysis pathologists confirmed ‘MFH’ might disappear when criteria for the the diagnosis of MFH was appropriate at the time of diagnosis of pleomorphic sarcomas showing a primary diagnosis. Tumor samples were collected distinct differentiation state can be reproducibly from the part with macroscopically high tumor defined.6 In this work, we used the term ‘MFH’ to content by pathologists immediately after surgical identify tumors diagnosed as storiform and pleo- excision and cryopreserved in liquid nitrogen until morphic type MFH, and the term ‘myxofibrosarco- use. This study was approved by the ethics ma’ for so-called MFH with predominant (450%) committee of National Cancer Center and conducted myxoid features conventionally diagnosed as according to tenets of the Declaration of Helsinki. myxoid type MFH. Recently, several studies report gene expression profiling of soft tissue tumors using microarray Gene Expression Profiling technologies to provide new insights into the tumor Total RNA was isolated using TRIzol (Invitrogen, characterization. They described distinct patterns of Carlsbad, CA, USA) according to the manufacturer’s gene expression in respective tumors with single, instruction. Samples were analyzed with a Gene- recurrent genetic aberrations, such as synovial Chip Human Genome U133A array (Affymetrix, sarcoma, myxoid/round cell liposarcoma, clear cell Santa Clara, CA, USA) containing 22 283 probe sets. sarcoma or gastrointestinal stromal tumors, and Target cRNA preparation from total RNA, hybridiza- heterogeneous patterns in spindle cell and pleo- tion to the microarray, washing and staining with an morphic sarcomas which are generally characterized antibody amplification procedure and scanning by complex chromosomal aberrations.7–12 No further were all carried out according to the manufacturer’s detailed analysis of gene expression in spindle instructions. The expression value (Signal) of each cell and pleomorphic sarcomas have been reported probe set was calculated using GeneChip Operating so far. Software (GCOS) ver. 1.3 (Affymetrix), so that the In this study, we analyzed gene expression profile mean of expression values in each experiment was of total 105 cases representing 10 types of soft tissue set at 100 to adjust for minor differences between tumors to identify their molecular characteristics. experiments. We observed similarity in gene expression among spindle cell and pleomorphic sarcomas, forming a relatively loose cluster, which is separated from Statistical Analysis the distinct clusters of synovial sarcoma, myxoid/ round cell liposarcoma and lipoma þ well-differ- Gene expression data were subsequently imported entiated liposarcoma. Next, we primarily analyzed into GeneSpring GX7.2 software (Agilent Technolo- 64 samples of spindle cell and pleomorphic gies, Santa Clara, CA, USA) and normalized to the sarcomas and showed heterogeneity of MFH in median of all samples enrolled in the analysis and terms of gene expression. We selected genes that log-transformed for each gene. Hierarchical cluster- could clearly distinguish between dedifferentiated ing analysis was performed using Pearson’s correla- liposarcoma, myxofibrosarcoma, leiomyosarcoma, tion. To select appropriate probe sets defining five Modern