Tissue Antigens ISSN 0001-2815 BRIEF COMMUNICATION SSX2-4 expression in early-stage non-small cell lung cancer K. B. V. Greve 1 , M. Pøhl 2,3 , K. E. Olsen 2,4 , O. Nielsen 4 , H. J. Ditzel 1,3 & M. F. Gjerstorff 1 1 Department of Cancer and Inflammation Research, Institute for Molecular Medicine, University of Southern Denmark, Odense, Denmark 2 Institute of Clinical Research, University of Southern Denmark, Odense C, Denmark 3 Department of Oncology, Odense University Hospital, Odense, Denmark 4 Department of Clinical Pathology, Odense University Hospital, Odense, Denmark Key words cancer/testis antigen; immunotherapy; lung cancer; SSX Correspondence Morten F. Gjerstorff Institute for Molecular Medicine (IMM) University of Southern Denmark Winsloewparken 25 3., DK-5000 Odense C Denmark Tel: +45-20261563 Fax: +45-65503950 e-mail: mgjerstorff@health.sdu.dk and Henrik J. Ditzel Institute for Molecular Medicine (IMM) University of Southern Denmark Winsloewparken 25 3., DK-5000 Odense C Denmark Tel: +45-65503781 Fax: +45-65503950 e-mail: hditzel@health.sdu.dk Received 10 December 2013; revised 19 February 2014; accepted 20 February 2014 doi: 10.1111/tan.12340 Abstract The expression of cancer/testis antigens SSX2, SSX3, and SSX4 in non-small cell lung cancers (NSCLC) was examined, since they are considered promising targets for cancer immunotherapy due to their immunogenicity and testis-restricted normal tissue expression. We characterized three SSX antibodies and performed immunohistochemical staining of 25 different normal tissues and 143 NSCLCs. The antibodies differed in binding to two distinctive splice variants of SSX2 that exhibited different subcellular staining patterns, suggesting that the two splice variants display different functions. SSX2-4 expression was only detected in 5 of 143 early-stage NSCLCs, which is rare compared to other cancer/testis antigens (e.g. MAGE-A and GAGE). However, further studies are needed to determine whether SSX can be used as a prognostic or predictive biomarker in NSCLC. Lung cancer causes most cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for more than 85% of new lung cancer cases (1). Current treatment strategies have proven unsatisfactory, highlighting the need for new treatment options such as immunotherapy. The group of tumor antigens known as cancer/testis antigens are considered attractive targets for immunotherapy due to their expression in a wide range of human cancers, their immunogenicity, and especially because their expression in normal tissue is restricted to the immune-privileged germ cells of the testis (2, 3). One such antigen is the MAGE-A3 cancer/testis antigen that has shown promise as a target in NSCLC. MAGE- A3 is detected in 35%–40% of all NSCLC (4, 5) and a MAGE-A3 antigen vaccine is currently being investigated in a phase III clinical trial in patients with MAGE-A3-positive NSCLC (6). The SSX gene family of cancer/testis antigens consists of nine genes (SSX1-9) and ten pseudogenes (ψSSX1-10). The identity of the products of SSX1-9 ranges between 73% and 92% at the protein level, and 87%–96% at the cDNA level. Alternative splice variants have been shown for SSX2, -4, -5, and -7, but it is unknown if they are expressed (7, 8). Expression of the SSX family of cancer/testis antigens have been correlated with more advanced stages of disease and worse patient prognosis in a range of human cancers (4, 9–11). In addition, both antibody and CD8+/CD4+ T cell responses to SSX proteins have been identified in patients with tumors of varying histological origins (12). SSX mRNA expression has been investigated in a range of different cancers (4, 13–16), while reports on protein expression are limited [e.g. malignant melanoma (17), prostate cancer (18), hepatocellular carcinoma (19), and esophageal cancer (20)]. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 1