therapeutic vaccine based on a CT antigen to delay the recurrence of resected non-small cell lung cancer (1). A second CT antigen vaccine is currently in an international phase II clinical trial for patients with resected malignant melanoma (2). The ﬁrst cancer antigen was cloned from the cells of a melanoma patient, by Thierry Boon and his colleagues (3). The antigen in question was denominated melanoma anti- gen-1, or MAGE-1, and subsequently renamed as MAGE- A1. An international eﬀort to discover additional cancer antigens soon revealed an entire family expressed only in tumors and in the immunoprivileged gametogenic tissues. This group was collectively termed the CT antigens by Old and Chen (4). There are now more than 70 CT gene families, many of them promising vaccine candidates. Nevertheless, the range of discovery programs that have diversely reported these important therapeutic candidates have resulted in the terminology of a CT antigen being loosely deﬁned and applied, adding to importance of a single carefully curated database to be able to accurately assess the relevance of individual proteins. Due to their importance, there is a rapidly expanding body of knowledge concerning these genes widely in the literature and diverse databases. To gather and uniformly present the available information on CT antigens, we have created a user-friendly interface termed the Cancer-Testis database (CTdatabase). The database integrates hetero- geneous data including basic gene, protein and expression information in normal and tumor tissues as well as immu- nogenicity in cancer patients. The CTdatabase contains links to external databases although a priority has been to speciﬁcally process relevant data so that it can be stored locally. The information available was expertly curated and annotated, and regular updates are planned. CT ANTIGENS PRESENT IN THE DATABASE A list of CT antigens was ﬁrst compiled manually from the literature (see references used to compile the list at http:// www.cta.lncc.br, under the link ‘Gene annotations’ in the main page). Computational prediction was also used. The resultant CTdatabase comprises 204 genes. The CTdatabase has a straightforward interface where information for individual genes and their products is dis- played. Each entry is listed according to the oﬃcial gene symbol (or the name available at NCBI’ Gene Entrez database) and information is further sorted into ‘domains’, displayed as ‘tabs’ by subject: Summary, Gene, Protein, mRNA expression, protein expression, immune response, PubMed. The domains have been popu- lated using automatic recovery from public databases, manual annotation and novel data generated by RT-PCR. THE ‘GENE’ AND ‘PROTEIN’ TABS The ‘Gene’ tab contains general information extracted from NCBI Entrez Gene database including aliases, mRNA RefSeq accession numbers (5), gene structure, chromosomal localization, exon-intron structure, RefSeq splicing variants as well as links to the genome browsers Nucleic Acids Research, 2009, Vol. 37, Database issue D817 MapViewer (6), UCSC Genome Browser (7) and Single Nucleotide Polymorphisms (6), where available. The annotations extracted from NCBI Entrez Gene were manually curated. For example, the transcripts for SPANXE and SPANXD were found to align to a single locus. Both entries are available in the CTdatabase, but a warning is displayed for SPANXE indicating that this gene is identical to SPANXD and the user is thus directed to the SPANXD entry. Likewise, some aliases were also corrected, for example, MAGE-A4 and MAGE-A5 are diﬀerent genes, but NCBI Entrez Gene reports MAGE- A4 as an alias for MAGE-A5 (as of July, 2008). The CTdatabase also annotated gene names as splicing var- iants when they aligned to a single genomic locus, as is the case of LAGE-1A and LAGE-1B which are variants of CTAG2, or SSX2B and SSX2A which are variants of SSX2. Careful inspection of CT gene mRNA alignments revealed that 66 are virtually identical copies of each other, i.e. the mRNAs align with the same identity level to more than one locus (14 distinct genes, see website’s link ‘Gene annotations’). This information is available in the CTdatabase within the section termed ‘Gene copies’ under the ‘Gene’ tab. In addition to nearly identical gene copies, many CT antigen genes have a common evolutionary origin. We grouped genes with >40% sequence identity as belonging to the same family [blastp (8), E-value <0.001, complexity ﬁlter oﬀ and percent identity normalized for alignment length as % identity alignment length/length of the shorter protein]. This analysis resulted in 12 groups: CSAG, CT45, CT47, CTAG, CTAGE, GAGE, MAGE, NXF, SPANX, SSX, TSPY, XAGE1. Using these groups as a guide, multiple alignments (non-edited) were made with Clustalx (9). Phylogenetic trees were inferred with the program MEGA 3 (10) using neighbor joining, pair wise deletion, JTT matrix and bootstrapping 100 times. The sub-families thus iden- tiﬁed are reported in the ‘Gene’ tab under the section ‘Phylogenetic relationships with CT genes’. Note that the family information provided by the CTdatabase is a ﬁrst approach and should be used with caution. As with the ‘Gene’ tab, the ‘Protein’ tab also contributes general information on the protein products of CTgenes, such as RefSeq accession numbers, names [from UniProt, (11)], and known protein domains. It also contains manu- ally annotated sections on protein–protein interactions, protein localization and protein function. THE ‘mRNA Expression’ Tab CT antigens (or candidate CT antigens) were classiﬁed according to their expression patterns. Based on a collec- tive analysis of data from CAGE, MPSS, RT-PCR and ESTs (see website for details), genes are considered to be: (a) testis-restricted, (b) testis/brain-restricted, or (c) testis- selective (Annotation ﬁeld: ‘Gene expression pattern’). Since the principal importance of CT antigens lies in their restricted expression in normal tissues and ample expression in cancers, a central feature of the