Imaging, Diagnosis, Prognosis A Signature Predicting Poor Prognosis in Gastric and Ovarian Cancer Represents a Coordinated Macrophage and Stromal Response Rita A. Busuttil 1,4,5 , Joshy George 1 , Richard W. Tothill 2 , Kylie Ioculano 1 , Adam Kowalczyk 6 , Catherine Mitchell 3 , Stephen Lade 3 , Patrick Tan 8,9,10,11 , Izhak Haviv 1,7,12 , and Alex Boussioutas 1,4,5 Abstract Purpose: Gene-expression profiling has revolutionized the way we think about cancer and confers the ability to observe the synchronous expression of thousands of genes. The use of putative genome-level expression profiles has allowed biologists to observe the complex interactions of genes that constitute recognized biologic pathways. We used gastric and ovarian datasets to identify gene-expression signatures and determine any functional significance. Experimental Design: Microarray data of 94-tumor and 45-benign samples derived from patients with gastric cancer were interrogated using Hierarchical Ordered Partitioning and Collapsing Hybrid analysis identifying clusters of coexpressed genes. Clusters were further characterized with respect to biologic significance, gene ontology, and ability to discriminate between normal and tumor tissue. Tumor tissues were separated into epithelial and stromal compartments and immunohistochemical analysis performed to further elucidate specific cell lineages expressing genes contained in the signature. Results: We identified a "stromal-response" expression signature, highly enriched for inflammatory, extracellular matrix, cytokine, and growth factor proteins. The majority of genes in the signature are expressed in the tumor-associated stroma but were absent in associated premalignant conditions. In gastric cancer, this module almost perfectly differentiates tumor from nonmalignant gastric tissue and hence can be regarded as a highly tumor-specific gene-expression signature. Conclusions: We show that these genes are consistently coexpressed across a range of independent gastric datasets as well as other cancer types suggesting a conserved functional role in cancer. In addition, we show that this signature can be a surrogate marker for M2 macrophage activity and has significant prognostic implications in gastric and ovarian high-grade serous cancer. Clin Cancer Res; 20(10); 2761–72. Ó2014 AACR. Introduction Cancer is characterized by the invasion of malignant cells into surrounding supportive tissue and distant sites (1). It is well recognized that the consequent tissue deformation leads to an inflammatory host response with elevated levels of infiltrating inflammatory and immune cells (2). Although this host reaction could merely represent a generic response of the host to injury, the genes that control heterotypic cell interactions in wound healing and cancer stroma are subject to germ line sequence variations (3–8), suggesting that the tumor microenvironment may vary among patients exhibit- ing a variety of phenotypic responses. One such response was reported recently suggesting an immunosuppressive role of fibroblast activation protein a (FAP-a)–expressing cells in the tumor stroma that was tumor permissive (9). The relative contribution of stromal cells, such as tumor-associated mac- rophage (TAM) and carcinoma-associated fibroblasts (CAF) to the cancer phenotype in different patients is of great interest (10). More importantly, the identification of the specific stromal cell lineage responsible for cancer promo- tion would enable therapeutic trials aimed at patient cohorts that would best respond to an agent targeted against specific stromal components. FAP-a–expressing cells may be one example as well as TAMs that are thought to promote cancer and interact with both the cancer cells (11, 12) and other stromal cells (13). Authors' Affiliations: 1 Cancer Genetics and Genomics Laboratory; 2 Molecular Genomics Core Facility; 3 Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne; 4 Sir Peter MacCallum Depart- ment of Oncology; 5 Department of Medicine, Royal Melbourne Hospital; 6 National ICT Australia (NICTA); 7 Department of Pathology, The University of Melbourne, Parkville, Australia; 8 Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School; 9 Cellular and Molecular Research, National Cancer Centre; 10 Cancer Science Institute of Singapore, National University of Singapore; 11 Genome Institute of Singapore, Singapore; and 12 Faculty of Medicine in the Galilee, Bar Ilan University, Zfat, Israel Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). R.A. Busuttil and J. George contributed equally to this article. Corresponding Author: Alex Boussioutas, University of Melbourne, Melbourne Hospital, Parkville 3050, Australia. Phone: 61-383446252; Fax: 61-393471863; E-mail: alex.boussioutas@petermac.org doi: 10.1158/1078-0432.CCR-13-3049 Ó2014 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 2761