Published OnlineFirst April 20, 2015; DOI: 10.1158/1078-0432.CCR-14-3141 Biology of Human Tumors Clinical Cancer Research Clinically Relevant Molecular Subtypes in Leiomyosarcoma Xiangqian Guo1, Vickie Y. Jo2, Anne M. Mills3, Shirley X. Zhu1, Cheng-Han Lee4, Inigo Espinosa5, Marisa R. Nucci2, Sushama Varma1, Erna Forgo1, Trevor Hastie6, Sharon Anderson1, Kristen Ganjoo7, Andrew H. Beck8, Robert B. West1, Christopher D. Fletcher2, and Matt van de Rijn1 Abstract Purpose: Leiomyosarcoma is a malignant neoplasm with histochemical markers; LMOD1 for subtype I leiomyosar- smooth muscle differentiation. Little is known about its molec- coma and ARL4C for subtype II leiomyosarcoma. A leiomyo- ular heterogeneity and no targeted therapy currently exists for sarcoma tissue microarray with known clinical outcome leiomyosarcoma. Recognition of different molecular subtypes was used to show that subtype I leiomyosarcoma is associ- is necessary to evaluate novel therapeutic options. In a previous ated with good outcome in extrauterine leiomyosarcoma study on 51 leiomyosarcomas, we identified three molecular while subtype II leiomyosarcoma is associated with poor subtypes in leiomyosarcoma. The current study was performed prognosis in both uterine and extrauterine leiomyosarcoma. to determine whether the existence of these subtypes could be The leiomyosarcoma subtypes showed significant differ- confirmed in independent cohorts. ences in expression levels for genes for which novel targeted Experimental Design: Ninety-nine cases of leiomyosarcoma therapies are being developed, suggesting that leiomyosar- were expression profiled with 30end RNA-Sequencing (3SEQ). coma subtypes may respond differentially to these targeted Consensus clustering was conducted to determine the optimal therapies. number of subtypes. Conclusion: We confirm the existence of 3 molecular sub- Results: We identified 3 leiomyosarcoma molecular sub- types in leiomyosarcoma using two independent datasets and types and confirmed this finding by analyzing publically show that the different molecular subtypes are associated available data on 82 leiomyosarcoma from The Cancer with distinct clinical outcomes. The findings offer an opportu- Genome Atlas (TCGA). We identified two new formalin-fixed, nity for treating leiomyosarcoma in a subtype-specific targeted paraffin-embedded tissue-compatible diagnostic immuno- approach. Clin Cancer Res; 1–11. Ó2015 AACR. Introduction of leiomyosarcoma originates in various soft tissue sites where it is thought to often arise from smooth muscle cells Leiomyosarcoma is reported to represent as many as 24% of in vessel walls. all sarcomas (1, 2). Currently, no targeted therapy exists for The successful stratification of several tumors (including leiomyosarcoma and the tumor responds poorly to conven- breast cancer, lung cancer, and colon cancer) into molecular tional chemotherapy or radiotherapy. A significant proportion subtypes in the past decades has significantly improved our of leiomyosarcoma originates in the uterus, and the remainder knowledge of these malignancies and has led to changes in the therapeutic approach to these cancers (3–12). In a previous 1Department of Pathology, Stanford University School of Medicine, study, our group proposed the existence of three molecular Stanford, California. 2Department of Pathology, Brigham and subtypes of leiomyosarcoma by using microarray-based gene Women's Hospital and Harvard Medical School, Boston, Massachu- expression profiling on 51 fresh frozen samples of leiomyo- setts. 3Department of Pathology, University of Virginia, Charlottesville, Virginia. 4Division of Anatomical Pathology, Royal Alexandra Hospital, sarcoma (13). To validate this finding, we analyzed 99 leio- Edmonton, Alberta, Canada. 5Department of Pathology, Hospital de la myosarcoma cases collected at different institutions from the Santa Creu i Sant Pau, Autonomous University of Barcelona, Barce- 6 years 1991 to 2012 as an independent cohort, and performed lona, Spain. Department of Statistics, Stanford University, Stanford, fi 0 California. 7Stanford Comprehensive Cancer Center, Stanford Univer- expression pro ling with 3 end RNA sequencing (3SEQ; sity, Stanford, California. 8Department of Pathology, Beth Israel refs. 14–20). In addition, we analyzed the publically available Deaconess Medical Center and Harvard Medical School, Boston, expression profiling dataset from TCGA on 82 cases. Our ana- Massachusetts. lysis confirms the existence of three molecular subtypes in Note: Supplementary data for this article are available at Clinical Cancer leiomyosarcoma. Using two novel markers, we could distin- Research Online (http://clincancerres.aacrjournals.org/). guish the three subtypes by immunohistochemistry and corre- Corresponding Author: Matt van de Rijn, Department of Pathology, Stanford late the subtypes with clinical outcome. The recognition of University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305. Phone: these molecular subtypes in leiomyosarcoma may have clinical 650-498-7154; Fax: 650-723-1950; E-mail: [email protected] significance as the subtypes vary in their expression of a number doi: 10.1158/1078-0432.CCR-14-3141 of genes for which novel targeted therapies either already exist Ó2015 American Association for Cancer Research. or are being developed. www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst April 20, 2015; DOI: 10.1158/1078-0432.CCR-14-3141 Guo et al. of contingency analysis between one subtype and the other Translational Relevance 2 subtypes was measured by two-tailed Fisher exact test and c test Leiomyosarcoma is a malignant neoplasm with varying with GraphPad Prism5 software. Univariate and multivariate degrees of smooth muscle differentiation and complex genet- analysis by the Cox proportional hazard method was performed ic abnormalities. It can occur in a wide range of sites but is using the survival package in R. Analysis of gene ontology (GO) usually managed as a single disease in conventional therapy was done using DAVID Bioinformatics Resources version 6.7 and in clinical trials. The recognition of molecular subtypes (25, 26). CSF1-signature (27) positive, and CINSARC signature can lead to the identification of novel therapies that may (28) positive cases were identified as those cases that coordi- affect one of these subtypes preferentially. We confirmed nately highly expressed these signature genes with >0.3 correla- the results of an earlier study and determined the existence tion with the centroid as performed previously (19, 27, 29, 30). of 3 molecular subtypes in leiomyosarcoma using two Principal component analysis (PCA) was performed on square independent cohorts. We identified two new formalin-fixed, root transformed TPM with R package pcaMethods, ellipse con- paraffin-embedded (FFPE) tissue-compatible diagnostic tour of principal components (PC) was computed and drawn immunohistochemical markers for two of these subtypes: with R package car. LMOD1 for subtype I and ARL4C for subtype II, and showed that these molecular subtypes are associated with distinct TMA construction, immunohistochemistry staining, and clinical outcomes. Our study offers an opportunity for a scoring leiomyosarcoma subtype–specific targeted treatment. The tissue microarrays used in this study (TA-201 and TA-381) were constructed with Tissue Arrayer (Beecher Instru- ments). TA-201 included the leiomyosarcoma cases with clinic outcome data, while TA-381 was comprised of leiomyosarcoma Materials and Methods cases analyzed by 3SEQ. For immunohistochemical staining, sections underwent antigen retrieval and were stained with 3SEQ library construction and bioinformatics analysis anti-ARL4C antibody (1:120, Sigma, CAT#HPA028927) and fi Paraf n blocks of 99 leiomyosarcomas, 4 myometrium, 3 anti-LMOD1 antibody (1:20, Sigma, CAT#HPA028325) with leiomyoma, and 6 undifferentiated pleomorphic sarcomas from the EnVisionþsystem (Dako). The staining results were scored 1991 to 2012 from 9 hospitals were obtained with Institutional as follows: 3 (þþþ), strong staining (>30% positivity); 2 (þþ), review board approval and a waiver of consent due to the archival weak staining (10%–30%); 1 (þ), equivocal or uninterpretable nature of the specimens. Multiple 2 mm-diameter cores were re- (<10%); 0 (À), absence of any staining. Hierarchical clustering fi embedded into paraf n blocks longitudinally and sectioned of immunohistochemical (IHC) results was performed with again to ensure the purity of tumor cells. After RNA isolation, software "Cluster3" with "uncentered Correlation" and "Cen- – 3SEQ libraries for next-generation sequencing based expression troid Linkage" and a clustered heatmap was visualized with fi pro ling were sequenced and analysis was performed as described Java TreeView (31, 32). previously (14–20). All gene expression profiling data used for this study have been deposited in the Gene Expression Omnibus (GEO) and are publicly accessible through GSE45510, GSE53844 Results and GSE54511. Consensus clustering identifies three molecular subtypes of After filtering data to genes with a SD greater than 100, trans- leiomyosarcoma based on gene expression data forming the data by log2 and gene-based centering, the Consensus We analyzed 99 cases of leiomyosarcoma by 3SEQ, a next- Clustering (R package ConsensusClusteringPlus; ref. 21) was used generation sequencing–based approach that quantifies the to determine the number of subtypes and