Utilização dos dados de seqüências expressas na identificação e caracterização de novos antígenos tumorais Cancer/Testi / Utilization of the expressed sequences data to the identification and characterization of new Cancer/Testis antigens

Os antígenos Cancer/Testis (CTs) são um subgrupo de antígenos tumorais com expressão restrita a testículo normal e diferentes tipos de tumores. Estes antígenos são capazes de induzir resposta imune humoral e celular em pacientes com câncer e, devido ao seu restrito padrão de expressão, são considerados candidatos ideais para o desenvolvimento de vacinas e imunização passiva. Neste trabalho, uma estratégia computacional baseada em dados de expressão provenientes de seqüências expressas (ESTs) foi utilizada para a identificação de novos antígenos CT. Seqüências expressas (mRNA e ESTs) foram alinhadas a seqüência genômica humana permitindo o agrupamento de seqüências expressas derivadas de um mesmo gene. ... Com base nesta análise, foram identificados cinco candidatos a antígenos CT freqüentemente em diferentes tipos de tumores. ... Anticorpos anti-CT101 [transcrito correspondente ao gene PASD1 (PAS domain containing protein 1)] foram encontrados em 41% das amostras de plasma de pacientes com câncer de útero e em 43,3% dos indivíduos sadios. A freqüência de anticorpos anti-CT802 [transcrito correspondente ao gene ASZ1 ou GASZ (Germ cell-specific ankyrin, SAM and basic leucine zipper domain containing protein 1)] observada nas amostras de plasma de pacientes com câncer de útero foi de 22,7% enquanto dentre os indivíduos sadios a freqüência foi de 6,7%. Já a freqüência de anticorpos anti-CT809, correspondente ao gene FAM46D (Family with sequence similarity 46, member D), foi de 7,7% nos pacientes com câncer de próstata e 6,7% nos indivíduos sadios. Além disso, utilizamos a técnica de RACE para caracterizar os candidatos CT704 e CT1001 que não apresentavam seqüência completa de mRNA disponível em banco de dados públicos. Em conjunto esses resultados demonstram que a estratégia in silico utilizada neste trabalho foi eficiente na identificação de 5 antígenos CTs sendo que para 3 destes foi possível detectar resposta imune humoral em pacientes com câncer.

Cancer/testis (CT) antigens are a subgroup of tumor antigens with a restricted expression in normal testis and in different types of tumors. These antigens are capable of eliciting humoral and cellular immune response in cancer patients and due to their restricted expression pattem they are considered promising candidates for the development of vaccines and passive immunotherapy. In this work, a computational approach based on expression data from expressed sequence tags (ESTs) was used to identify novel CT antigens. Expressed sequences (mRNA ans ESTs) were aligned against the human genome sequence, allowing the clustering of the sequences derived from a same gene. Considering the tissue origin of the expressed sequences corresponding to each gene it was possible to define an in silico expression pattem and to select novel CT antigen candidates. A total of 1255 candidate genes represented by spliced ESTs derived from testis and/or tumoral cDNA libraries were identified and 93 of them were selected for experimental validation of their expression pattem. The experimental validation of the expression pattem was carried out by RT-PCR in 21 normal tissues, 17 tumor cell lines and 160 samples deri ved from 9 different types of tumors. Based on this analysis we were able to identify five CT antigens candidates frequently expressed in different types of tumors. Candidate CT 1 O 1 was expressed in 41% o f tumor samples and the highest frequency of expression was observed in glioblastomas (70% ). Candidate CT704 was expressed in 65% of tumor samples with a high expression frequency among lung tumors (93%). Candidate CT802 was expressed in 20% of tumor samples with higher expression in uterus tumors (50%). Candidate CT809 was expressed in 24% of tumor samples and was predominantly expressed in lung tumors (50%). Candidate CT1001 was expressed in 18% of the tumor samples being more frequently expressed in gastric tumors (33.3%). Candidates CT101, CT802 and CT809 for which a full-length sequence were already available in public databases were selected to evaluate the presence o f hum oral immune response in cancer patients. The respective recombinant proteins were expressed in a bacterial system and were used in immunoblotting assays. Anti-CT101 antibodies [transcript corresponding to PASD1 gene (PAS domain containing protein 1)] were observed in 41% of plasma samples from patients with uterus tumor and in 43.3% of plasma from healthy individuais. The frequency of anti-CT802 antibodies [transcript corresponding to ASZ1 or GASZ gene (Germ cell-specific ankyrin, SAM and basic leucine zipper domain containing protein 1)] observed in plasma samples from patients with uterus tumor was 22.7% while among healthy individuais the frequency was 6.7%. Finally, the frequency of anti-CT809 antibodies, that corresponds to FAM46D gene (Family with sequence similarity 46, member D) was 7.7% among patients with lung tumors and 6.7% among healthy individuais. Moreover, we performed RACE experiments to characterize candidates CT704 and CT1001 that did not present a full-length mRNA sequence available in public databases. Taken together, these results showed that the in silico strategy used in this work was efficient in the identification of 5 CT antigens and for 3 of them it was possible to detect humoral immune response in cancer patients (AU)

Characterization of C21ORF100 as a Novel Prostate Differentiation Antigen

Differentiation antigens are immunogenic proteins expressed in specific cell lineages or at specific stages of differentiation ina particular tissue. Generally, their expression in normal cells is preserved after neoplasic transformation and this feature hasmade such molecules potential candidates for cancer immunotherapy. Using alignments between expressed sequence tags(ESTs) and the human chromosome 21 sequence, we have identified a novel gene, named C21orf100, which is exclusivelyexpressed in normal prostate and codes for a putative protein of 55 amino acids. Objective: To characterize C21orf100 as anovel prostate differentiation antigen. Material and Methods: C21orf100 mRNA expression was determined by RT-PCR in 22normal tissues and in 65 samples from melanomas and prostate, thyroid, stomach, uterus and breast tumors. The existenceof a humoral immune response against C21orf100 protein in prostate cancer patients was evaluated by immunoblotting usinga His-tagged recombinant protein. Results: As expected for a differentiation antigen, C21orf100 mRNA expression waspredominantly detected in both normal and tumor prostate samples. Antibodies against C21orf100 recombinant protein weredetected in 1 out of 50 (2%) plasma samples from prostate cancer patients and were not detected in the plasma from healthyblood donors. Conclusion: The restricted expression pattern and the detection of antibodies in prostate cancer patients suggestthat C21orf100 is a novel prostate differentiation antigen. However, due to the low frequency of antibody response againstC21orf100 detected among prostate cancer patients, further analysis is necessary to evaluate its potential for cancerimmunotherapy.

Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences

The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues.