Identification of cancer/testis-antigen genes by massively parallel signature sequencing

Abstract

Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer͞testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 introncontaining genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family.germ line ͉ human ͉ transcript

Cancer/testis antigen CT45: Analysis of mRNA and protein expression in human cancer

“…25 In our previous study designed to identify new CT antigen gene by massively parallel signature sequencing (MPSS), for example, we chose to set the screening criteria for CT or ''CT-like'' genes as genes that had at least twice as many MPSS tags in testis than in all 32 normal somatic tissues combined. 18 Genes that fulfilled this initial criterion were further evaluated by RT-PCR, and only genes that showed mRNA expression in testis but in no more than 2 of the 15 normal somatic tissues tested were retained as CT candidate genes. Using this approach, more than 20 genes were identified, including CT45.…”

“…Primer and probe sequences for CT45 amplification were previously described. 18 Control amplification for housekeeping gene GAPDH was performed in all samples. Following the amplification, the same threshold was set for analyzing all experiments to compare Ct values derived from different experiments.…”

“…For a CT antigen to be a promising immunotherapeutic target, the following properties would be crucial: (i) the gene should have a testis-restricted, or at least highly testis-predominant, mRNA expression profile in normal tissues that is proven by experimental data such as RT-PCR; (ii) the protein expression should be documented by immunohistochemical analysis, and (iii) the gene should have a reasonable frequency of expression, ideally in at least 10-20% of at least 1 or 2 tumor types. Using these 3 criteria, we have evaluated the CT genes that we previously defined, [18][19][20] and CT45, a gene product encoded by a gene family on Xq26, was identified in this study as a potentially attractive CT vaccine target. By qRT-PCR and by immunohistochemical analysis with monoclonal antibodies, CT45 was found to be a nuclear antigen that is often expressed in lung cancer and ovarian cancer but rarely in breast cancer.…”

Cancer/Testis Genes in Multiple Myeloma: Expression Patterns and Prognosis Value Determined by Microarray Analysis

“…19) updated with three recently reported CT genes, i.e., CT45 (22), SLLP1/ SPACA3 (20), and KM-HN-1 (21). Of 93 CT genes belonging to 47 families, 64 could be interrogated by 104 probe sets with the Affymetrix HG-U133A or GNFH1 arrays.…”

Concise Review: Cancer/Testis Antigens, Stem Cells, and Cancer

“…Some CTAs can be expressed in nongametogenic tissues such as the pancreas, liver, and spleen at levels far below that observed in germ cells [39]; 2) their expression programs are strictly regulated by epigenetic mechanisms such as DNA methylation [40]; and 3) they are immunogenic. To date, nearly 40 distinct CTAs have been identified based on immunogenic properties [41], expression profiles [42], and by bioinformatic methods [43]. However, little is known about their specific functions, and their functional connection with stem cell biology and cancer is widely unexplored.…”