Biology of Human Tumors Reduced Expression of SMAD4 Is Associated with Poor Survival in Colon Cancer Pu Yan 1 , Dirk Klingbiel 2,3 , Zenia Saridaki 4,5 , Paola Ceppa 6 , Monica Curto 6 , Thomas Alexander McKee 7 , Arnaud Roth 8 , Sabine Tejpar 5 , Mauro Delorenzi 9,10,11 , Fredrik T. Bosman 1 , and Roberto Fiocca 6 Abstract Purpose: SMAD4 loss is associated with the development of metastases and poor prognosis. We evaluated expression of SMAD4 protein and its association with tumor characteristics, including biomarkers and outcome in terms of relapse-free sur- vival and overall survival. Experimental design: We used 1,564 stage II/III colon cancer samples from PETACC-3 to evaluate SMAD4 expression by immu- nohistochemistry. SMAD4 protein expression was validated by assessing mRNA expression using available expression array data. SMAD4 expression was also studied on 34 adenomas and 10 colon cancer liver metastases with their primaries. Loss of SMAD4 immu- noreactivity was defined as focal or diffuse. Cases without SMAD4 loss were subdivided into those with strong and weak expression. Results: SMAD4 protein expression was informative in 1,381/1,564 cases. SMAD4 loss was found in 293/1,381 (21%) cases. Of 1,088 cases without SMAD4 loss (79%), 530 showed weak and 558 strong expression. SMAD4 loss occurred also in adenomas, but less extensively than in carcinomas. Liver metastases followed mostly the expres- sion pattern of the primary tumor. SMAD4 loss, including weak expression, identified patients with poor survival in stage II as well as III and in both treatment arms. SMAD4 loss was less frequent in tumors with microsatellite insta- bility and more frequent in those with loss of heterozygosity of 18q. Conclusions: We conclude that clonal loss of SMAD4 expres- sion in adenomas, carcinomas, and liver metastases increases with disease progression. SMAD4 loss, and to a lesser extent weak expression, is strongly associated with poor survival regardless of stage. Clin Cancer Res; 22(12); 3037–47. Ó2016 AACR. Introduction Colorectal cancer remains a major public health problem in the Western world with an estimated 136,830 new cases and 50,310 deaths occurring in 2014 in the United States alone (1). Despite the progress made in the management of metastatic colorectal cancer over the last few years, with the incorporation in combination chemotherapy of two monoclonal antibodies targeting the EGFR (2–8) and one targeting the VEGF (9–12), in the adjuvant setting, after introduction of oxaliplatin/fluo- ruracil (5-FU)/leucovorin(LV) era, innovative approaches are eagerly awaited (13, 14). One approach is the search for prognostic and predictive markers, which might serve in select- ing patients who are likely to benefit from adjuvant chemo- therapy. To date, the most important prognostic factor and treatment determinant remains disease stage (15). Identifica- tion of tissue biomarkers capable of improving outcome through better patient stratification and selection for specific treatment is gaining momentum, as reflected in studies on microsatellite instability (MSI), loss of heterozygosity (LOH) of 18q, copy-number aberrations (CNA) and the mutation status of KRAS, BRAF, and TP53 (16–19). In spite of increas- ingly detailed molecular mapping of colorectal cancer, only few markers have shown some promise in clinical practice in terms of capability to predict disease course. The detailed unraveling of molecular abnormalities that characterize oncogenesis of colon cancer is of profound impor- tance for understanding the biology and clinical behavior of this disease. As previously noted, one of the most important and frequently encountered events is allelic loss on chromo- some 18q (20, 21), where SMAD4 (also called deleted in pancreatic carcinoma 4, DPC4) gene is located (22–24). The SMAD4 tumor-suppressor gene (TSG) codes for the common intracellular mediator of the TGFb superfamily signaling path- way, one of the most commonly altered cellular signaling pathways in human cancers (25–27) and involved in the regulation of cell proliferation, differentiation, apoptosis, and cell migration (27–30). 1 Institute of Pathology, University of Lausanne, Lausanne, Switzerland. 2 Swiss Group for Clinical Cancer Research SAKK,Coordinating Center, Bern, Switzerland. 3 Bioinformatics Core Facility, University of Lau- sanne, Lausanne, Switzerland. 4 Laboratory of Tumor Cell Biology, School of Medicine, University of Crete, Voutes, Heraklion, Greece. 5 Digestive Oncology Unit, University Hospital Gasthuisberg, Leuven, Belgium. 6 Division of Anatomic Pathology, Department of Surgical and Diagnostic Sciences, University of Genoa and IRCCS S. Martino/ IST University Hospital, Genoa, Italy. 7 Department of Pathology, Geneva University Hospital, Geneva, Switzerland. 8 Oncosurgery Unit, Geneva University Hospital, Geneva, Switzerland. 9 Ludwig Center for Cancer Research, University of Lausanne, Lausanne, Switzerland. 10 Department of Oncology, Faculty of Biology and Medicine, Univer- sity of Lausanne, Lausanne, Switzerland. 11 Bioinformatics Core Facility, SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). P. Yan and D. Klingbiel contributed equally to this article. Corresponding Author: Roberto Fiocca, University of Genova and IRCCS Martino/IST University Hospital, Via De Toni 14, 16132 Genova, Italy. Phone: 39-34-7533-1928; Fax: 39-010-353-7803; E-mail: fiocca@unige.it doi: 10.1158/1078-0432.CCR-15-0939 Ó2016 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 3037 on May 23, 2020. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst February 9, 2016; DOI: 10.1158/1078-0432.CCR-15-0939