CT-X antigen expression in human breast cancer Anita Grigoriadisa,1,2, Otavia L. Caballerob,1,3, Keith S. Hoekc, Leonard da Silvad, Yao-Tseng Chene, Sandra J. Shine, Achim A. Jungbluthb, Lance D. Millerf, David Cloustong, Jonathan Cebong, Lloyd J. Oldb,3, Sunil R. Lakhanid, Andrew J. G. Simpsonb, and A. Munro Nevillea aLudwig Institute for Cancer Research, 605 Third Avenue, New York, NY 10158; bLudwig Institute for Cancer Research, New York Branch at Memorial Sloan–Kettering Cancer Center, 1275 York Avenue, New York, NY 10021; cDepartment of Dermatology, University Hospital of Zurich, Gloriastrasse 31/F2, CH-8091 Zurich, Switzerland; dMolecular and Cellular Pathology, UQ Centre for Clinical Research and The School of Medicine, Level 6 Building 71/918, University of Queensland, The Royal Brisbane and Women’s Hospital, Herston 4029, Brisbane, Queensland, Australia; eWeill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021; fDepartment of Cancer Biology, Wake Forest University School of Medicine, Medical Center Boulevard, Hanes Building, Winston-Salem, NC 27157; and gAustin Health and the Cancer Vaccine Laboratory at the Ludwig Institute for Cancer Research, Austin Hospital, Heidelberg 3084, Australia Contributed by Lloyd J. Old, June 18, 2009 (sent for review April 29, 2009) Cancer/testis (CT) genes are predominantly expressed in human the present study was to undertake a more comprehensive germ line cells, but not somatic tissues, and frequently become analysis of CT-X antigen expression in primary breast cancer in activated in different cancer types. Several CT antigens have the context of clinicopathological parameters. The results point already proved to be useful biomarkers and are promising targets to a restricted expression of members of the MAGEA and for therapeutic cancer vaccines. The aim of the present study was NY-ESO-1/CTAG1B gene families, primarily in ER negative to investigate the expression of CT antigens in breast cancer. Using tumors, some of which belong to the basal phenotype. Such previously generated massively parallel signature sequencing lesions have a poorer prognosis for which, currently, therapeutic (MPSS) data, together with 9 publicly available gene expression options are limited. CT-X-based immunotherapy strategies may datasets, the expression pattern of CT antigens located on the X thus represent an important therapeutic option for patients with chromosome (CT-X) was interrogated. Whereas a minority of these subtypes of breast tumors. unselected breast cancers was found to contain CT-X transcripts, a significantly higher expression frequency was detected in estrogen Results and progesterone receptor (ER) negative breast cancer cell lines Detection of CT-X Antigen Expression in Massively Parallel Signature MEDICAL SCIENCES and primary breast carcinomas. A coordinated pattern of CT-X Sequencing Data. As an initial step in exploring CT-X antigen antigen expression was observed, with MAGEA and NY-ESO-1/ expression in breast cancer, we interrogated our previously CTAG1B being the most prevalent antigens. Immunohistochemical published MPSS data (22). These data were derived from a pool staining confirmed the correlation of CT-X antigen expression and of normal human breast luminal epithelial cells, a pool of ER negativity in breast tumors and demonstrated a trend for their predominantly ER-positive epithelial enriched primary breast coexpression with basal cell markers. Because of the limited ther- tumors and 4 breast epithelial cell lines (22, 23). Sequence tags apeutic options for ER-negative breast cancers, vaccines based on corresponding to 6 of the 83 CT-X antigens, including those for CT-X antigens might prove to be useful. MAGEA (1,646 transcripts per million [tpm]), CSAG2 (680 tpm), CT45 (263 tpm), PASD1 (24 tpm), CSAG1 (15 tpm), and cancer/testis antigens ͉ estrogen receptor ͉ therapy FMR1NB/NY-SAR-35 (11 tpm), were detected in only one sample, an ER-negative breast cell line BT20. ancer/testis (CT) antigens are encoded by a unique group of Cgenes that are predominantly expressed in human germ line CT-X Antigen Expression in Breast Cancer Gene Expression Studies. To cells, have little or no expression in somatic adult tissues, but further examine the possible relationship of CT-X expression become aberrantly activated in various malignancies (1). A total with ER status, a list of 66 Affymetrix probe sets identifying 65 of 153 CT antigens has been described to date and are compiled different CT-X-encoding genes was prepared (supporting infor- in the CT database (www.cta.lncc.br/) (2, 3). Of these antigens, mation (SI) Table S1). Nine published microarray-based gene 83 are encoded by multigene families located on the X-chromo- expression datasets derived from a total of 1,259 primary breast some and are referred to as the CT-X antigens (1). Although tumors and 51 breast cancer cell lines were available, and the ER their possible involvement in chromosomal recombination, tran- status was known in most. Four hundred three of 1,310 samples scription, translation and signaling has been proposed, the were ER-negative (Table 1). Using the HG-U133A platform, we physiological function of the great majority of CT-X antigens interrogated for each dataset gene expression patterns differ- remains poorly elucidated (1, 4). entiating between ER-negative and ER-positive samples. Ap- The expression of CT-X antigens varies greatly between tumor plying multiple testing controls, a P value cut-off of 0.05 and a types, being most frequent in melanomas, bladder, non-small cell 2-fold change filter, this analysis identified a set of 147 probe sets lung, ovarian, and hepatocellular carcinomas. The occurrence of CT-X antigens is uncommon in renal, colon, and gastric cancers (4). Where present, CT-X expression is associated with a poorer Author contributions: A.G., O.L.C., A.J.G.S., and A.M.N. designed research; A.G., O.L.C., K.S.H., L.d.S., Y.-T.C., S.J.S., A.A.J., D.C., J.C., and S.R.L. performed research; Y.-T.C., S.J.S., outcome and tends to be more frequent in higher grade lesions L.D.M., and S.R.L. contributed new reagents/analytic tools; A.G., O.L.C., K.S.H., L.d.S., and advanced disease (5–8). Y.-T.C., L.D.M., S.R.L., A.J.G.S., and A.M.N. analyzed data; and A.G., O.L.C., K.S.H., L.d.S., The combination of their restricted expression, and in some Y.-T.C., L.J.O., S.R.L., A.J.G.S., and A.M.N. wrote the paper. cases potent immunogenicity, has led to intense research into The authors declare no conflict of interest. their utilization in therapeutic vaccines (9). Clinical trials of Freely available online through the PNAS open access option. vaccines containing the CT-X antigens MAGEA and NY-ESO- 1A.G. and O.L.C. contributed equally to this work. 1/CTAG1B are underway in patients with several cancers, 2Present address: Breakthrough Breast Cancer Research Unit, Guy’s Hospital, King’s Health including those of the lung, ovary, and melanoma (10–16). Partners AHSC, London, United Kingdom. Relatively few studies have explored the expression pattern of 3To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. CT-X antigens in breast cancer and the few cases studied to date This article contains supporting information online at www.pnas.org/cgi/content/full/ have focused on NY-ESO-1/CTAG1B (17–21). The objective of 0906840106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906840106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 30, 2021 Table 1. Datasets used in this study Samples Available annotations Name Accession no. ERneg ERpos Unknown ER PR p53 HER2 Reference Boersma GSE5847 26 21 1 Yes No No No 28 Desmedt GSE7390 64 134 — Yes No No No 46 Doane Website* 42 57 — Yes Yes No Yes 30 Hess Website† 51 82 — Yes Yes No Yes 29 Ivshina GSE4922 34 211 4 Yes No Yes No 44 Minn GSE2603 42 57 22 Yes No No Yes 31 Neve E-TABM-157 33 18 — Yes Yes Yes Yes 27 Sotiriou GSE2990 34 85 6 Yes No No No 47 Wang GSE2034 77 209 — Yes No No No 45 vandeVijver Website‡ 69 226 — Yes No No No 34 *https://caarraydb.nci.nih.gov/caarray/. †http://bioinformatics.mdanderson.org/pubdata.html. ‡http://microarray-pubs.stanford.edu/wound࿝NKI/. (131 genes) that showed significant differential expression be- tion of CT-X expression and PR status, p53 mutation and HER2 tween ER-negative and ER-positive breast tumors in at least 5 status where available (Table 1). For each metric, the complete of the 9 datasets investigated. This list represents the ER specific datasets of the above mentioned 9 breast tumor cohorts were expression (ERSE) set (Table S2). Many of the ERSE genes interrogated for a significant relationship with metric status, and have previously been identified as differentially expressed in probe sets with less than a 2-fold change were discarded. This breast tumors in an ER status-dependent manner, or to be direct analysis was repeated for the CT-X antigen list (Table S1). There ER target genes (24–26). is a very strong overlap between the Doane (30) and Minn (31) The ERSE set was used to cluster the samples in each dataset 2-fold PR significant lists (137 probe sets, PRSE) (Table S12). (Fig. S1), confirming that ER status annotation was consistent The PRSE list also shared 105 probe sets in common with the in each dataset and that a CT-X-specific analysis would probably ERSE list (P value Ͻ0.001), confirming that PR and ER not be confounded by extraneous errors of annotation or data status-specific gene expression patterns are tightly linked.