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Oral presentation

High throughput gene expression profiling provides a powerful tool for discovery of prognostic and predictive markers for breast cancer [1-4]. The main limitation to this approach is the requirement for high-quality RNA, which is difficult in the multicenter clinical trial setting. One solution is to use RNAlater, which allows procurement and shipping of tissue specimens at room temperature [5,6]. The National Surgical Adjuvant Breast and Bowel Project (NSABP) has conducted a pilot study to procure pretreatement core biopsy specimens in a neoadjuvant study. Most of the samples in this study provided high-quality RNA, as determined by Bioanalyzer and Affymetrix GeneChip analyses. When combined with a RNA amplification method, quality data could be obtained from 10 ng of total RNA as starting material. NSABP currently has two neoadjuvant trials in which pretreatment specimens are procured in RNAlater. However, the typical practice pattern in the USA makes it difficult to procure tissue in the adjuvant setting even with the use of RNAlater. Therefore, methods that permit high throughput gene expression profiling of formalin-fixed, paraffin-embedded materials are in great need. Such methods will also allow interrogation of archived tissue banks with annotation established from previously finished trials and will therefore shorten the time for marker development and validation. Chemical modification by formalin and degradation during storage make RNA extracted from paraffin a poor substrate for gene expression profiling [7]. We have examined both microarray and RT-PCR platforms for this purpose. In general microarray analysis using the Arcturus Paradise system has been a disappointment in our hands, with high rate for assay failure for materials older than 3 years. However, there are RNA amplification and labeling methods in development that are not dependent on oligo-dT priming for cDNA synthesis and may provide better results. In collaboration with Genomic Health, Inc., we have explored the use of high-throughput real time RT-PCR for discovery and validation of prognostic markers for node negative and estrogen receptor positive breast cancer [8]. This has resulted in development of the OncotypeDx assay, which is offered as a commercial reference laboratory test. The disadvantage of real-time RT-PCR assays is relatively low throughput (less than 1000 genes, even at industrial scale). DASL assay from Illumina is a kind of hybrid between PCR and microarray platforms, and may provide relatively cost-efficient means by which to assay many candidate genes using degraded RNA obtainable from paraffin blocks [9].

References

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