The value of a tumor bank in the development of cancer research in Brazil: 13 years of experience at the a C camargo hospital.

This article discusses the importance of biobanking to health research advancement in developing countries by analyzing the impact of the establishment of a tumor bank at the A C Camargo Hospital, a cancer care and research center located in Sao Paulo, Brazil. For the past 13 years, the human biological samples provided by the tumor bank have been used by investigators to study various types of cancer. We analyze the impact of biobanking in the overall quality of research projects performed at our institution. We also summarize the main findings of these investigations focusing on breast, prostate, head-neck, and gastroesophageal tumors, as well as the lessons learned over these years. We conclude that biobanking should be part of the strategy employed by scientists and research institutions dedicated to the study of human diseases.

Effects of Oligo dT-T7 RNA Primer in RNA Amplification from Paraffin-Embedded Tissue for Microarray Experiments

Introduction: Formalin-Fixed Paraffin-Embedded Tissue samples (FFPET) represent a valuable source for studies of geneexpression comparisons, since a great number of these samples is available in archive and presents a long time of clinicalfollow-up. However, the quality of total RNA of these samples is known to be inferior to frozen samples, being many timesinadequate for studies of gene expression using conventional methodologies. Objective: This study aims to establish a protocolfor amplification of messenger RNA (mRNA) derived from FFPET samples for using in microarray experiments. Material andMethods: 4 tumoral samples of invasive ductal breast carcinoma FFPET-buffered 10% were used. Total RNA was extracted andthe mRNA was linearly amplified in two rounds based on T7 RNA polymerase methodology using different concentrations ofoligo dT-T7 Primer for first strand cDNA (1st-cDNA) synthesis. Amplified antisense RNA (aRNA) was labeled with cianine-Cy3through reverse transcription in the presence of random primers and co-hybridized with reference RNA (HB4a) labeled withcianine-Cy5 in a customized platform containing 4,608 cDNAs corresponding to human genes. Results: The amplified RNAquality was influenced by the relative amount of oligo dT-T7, showing better results for ratio of 1:0.1 (total RNA : oligo dT-T7).Hybridizations showed value of intensity signals for the most of cDNAs immobilized in the platform. Conclusion: This studyshowed that the control of the relative amounts of RNA derived from FFPET material and oligo dT-T7 is extremely important toobtain high-quality amplified RNA, allowing its use in microarray experiments.

Gene expression profile associated with response to doxorubicin-based therapy in breast cancer.

PURPOSE: This study was designed to identify genes that could predict response to doxorubicin-based primary chemotherapy in breast cancer patients. EXPERIMENTAL DESIGN: Biopsy samples were obtained before primary treatment with doxorubicin and cyclophosphamide. RNA was extracted and amplified and gene expression was analyzed using cDNA microarrays. RESULTS: Response to chemotherapy was evaluated in 51 patients, and based on Response Evaluation Criteria in Solid Tumors guidelines, 42 patients, who presented at least a partial response (> or =30% reduction in tumor dimension), were classified as responsive. Gene profile of samples, divided into training set (n = 38) and independent validation set (n = 13), were at first analyzed against a cDNA microarray platform containing 692 genes. Unsupervised clustering could not separate responders from nonresponders. A classifier was identified comprising EMILIN1, FAM14B, and PBEF, which however could not correctly classify samples included in the validation set. Our next step was to analyze gene profile in a more comprehensive cDNA microarray platform, containing 4,608 open reading frame expressed sequence tags. Seven samples of the initial training set (all responder patients) could not be analyzed. Unsupervised clustering could correctly group all the resistant samples as well as at least 85% of the sensitive samples. Additionally, a classifier, including PRSS11, MTSS1, and CLPTM1, could correctly distinguish 95.4% of the 44 samples analyzed, with only two misclassifications, one sensitive sample and one resistant tumor. The robustness of this classifier is 2.5 greater than the first one. CONCLUSION: A trio of genes might potentially distinguish doxorubicin-responsive from nonresponsive tumors, but further validation by a larger number of samples is still needed.

A transcript finishing initiative for closing gaps in the human transcriptome.

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms.

The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags.

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define approximately 23,500 genes, of which only approximately 1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers.