Quantitative expression profiling in formalin-fixed paraffin-embedded samples by affymetrix microarrays.

To date, few studies have systematically characterized microarray gene expression signal performance with degraded RNA from fixed (FFPE) in comparison with intact RNA from unfixed fresh-frozen (FF) specimens. RNA was extracted and isolated from paired tumor and normal samples from both FFPE and FF kidney, lung, and colon tissue specimens and microarray signal dynamics on both the raw probe and probeset level were evaluated. A contrast metric was developed to directly compare microarray signal derived from RNA extracted from matched FFPE and FF specimens. Gene-level summaries were then compared to determine the degree of overlap in expression profiles. RNA extracted from FFPE material was more degraded and fragmented than FF, resulting in a reduced dynamic range of expression signal. In addition, probe performance was not affected uniformly and declined sharply toward 5' end of genes. The most significant differences in FFPE versus FF signal were consistent across three tissue types and enriched with ribosomal genes. Our results show that archived FFPE samples can be used to profile for expression signatures and assess differential expression similar to unfixed tissue sources. This study provides guidelines for application of these methods in the discovery, validation, and clinical application of microarray expression profiling with FFPE material.

Current treatment protocols have eliminated the prognostic advantage of type 1 fusions in Ewing sarcoma: a report from the Children's Oncology Group.

PURPOSE Ewing sarcoma family tumors (ESFTs) exhibit chromosomal translocations that lead to the creation of chimeric fusion oncogenes. Combinatorial diversity among chromosomal breakpoints produces varying fusions. The type 1 EWS-FLI1 transcript is created as a result of fusion between exons 7 of EWS and 6 of FLI1, and retrospective studies have reported that type 1 tumors are associated with an improved outcome. We have re-examined this association in a prospective cohort of patients with ESFT treated according to current Children's Oncology Group (COG) treatment protocols. METHODS Frozen tumor tissue was prospectively obtained from patients diagnosed with ESFT, and reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine translocation status. Analysis was confined to patients with localized tumors who were diagnosed after 1994 and treated according to COG protocols. Translocation status was correlated with disease characteristics, event-free survival (EFS), and overall survival (OS). Results RT-PCR identified chimeric fusion oncogenes in 119 of 132 ESFTs. Eighty-nine percent of identified transcripts were EWS-FLI1, and of these, 58.8% were type 1. Five-year EFS and OS rates for patients with type 1 and non-type 1 fusions diagnosed between 2001 and 2005 were equivalent (type 1: EFS, 63% +/- 7%; OS, 83% +/- 6%; non-type 1: EFS, 71% +/- 9%; OS, 79% +/- 8%). CONCLUSION Current intensive treatment protocols for localized ESFT have erased the clinical disadvantage that was formerly observed in patients with non-type 1 EWS-FLI1 fusions.

Experimental comparison and evaluation of the Affymetrix exon and U133Plus2 GeneChip arrays.

BACKGROUND: Affymetrix exon arrays offer scientists the only solution for exon-level expression profiling at the whole-genome scale on a single array. These arrays feature a new chip design with no mismatch probes and a radically new random primed protocol to generate sense DNA targets along the entire length of the transcript. In addition to these changes, a limited number of validating experiments and virtually no experimental data to rigorously address the comparability of all-exon arrays with conventional 3'-arrays result in a natural reluctance to replace conventional expression arrays with the new all-exon platform. METHODOLOGY: Using commercially available Affymetrix arrays, we assess the performance of the Human Exon 1.0 ST (HuEx) and U133 Plus 2.0 (U133Plus2) platforms directly through a series of 'spike-in' hybridizations containing 25 transcripts in the presence of a fixed eukaryotic background. Specifically, we compare the measures of expression for HuEx and U133Plus2 arrays to evaluate the precision of these measures as well as the specificity and sensitivity of the measures' ability to detect differential expression. SIGNIFICANCE: This study presents an experimental comparison and systematic cross-validation of Affymetrix exon arrays and establishes high comparability of expression changes and probe performance characteristics between Affymetrix conventional and exon arrays. In addition, this study offers a reliable benchmark data set for the comparison of competing exon expression measures, the selection of methods suitable for mapping exon array measures to the wealth of previously generated microarray data, as well as the development of more advanced methods for exon- and transcript-level expression summarization.

Explaining differences in saturation levels for Affymetrix GeneChip arrays.

The experimental spike-in studies of microarray hybridization conducted by Affymetrix demonstrate a nonlinear response of fluorescence intensity signal to target concentration. Several theoretical models have been put forward to explain these data. It was shown that the Langmuir adsorption isotherm recapitulates a general trend of signal response to concentration. However, this model fails to explain some key properties of the observed signal. In particular, according to the simple Langmuir isotherm, all probes should saturate at the same intensity level. However, this effect was not observed in the publicly available Affymetrix spike-in data sets. On the contrary, it was found that the saturation intensities vary greatly and can be predicted based on the probe sequence composition. In our experimental study, we attempt to account for the unexplained variation in the observed probe intensities using customized fluidics scripts. We explore experimentally the effect of the stringent wash, target concentration and hybridization time on the final microarray signal. The washing effect is assessed by scanning chips both prior to and after the stringent wash. Selective labeling of both specific and non-specific targets allows the visualization and investigation of the washing effect for both specific and non-specific signal components. We propose a new qualitative model of the probe-target hybridization mechanism that is in agreement with observed hybridization and washing properties of short oligonucleotide microarrays. This study demonstrates that desorption of incompletely bound targets during the washing cycle contributes to the observed difference in saturation levels.

Growth inhibition of breast epithelial cells by celecoxib is associated with upregulation of insulin-like growth factor binding protein-3 expression.

Several experimental and epidemiological studies have suggested a role for the use of cyclooxygenase (COX)-2 inhibitors in the prevention of breast cancer. The relative lack of toxicity associated with these compounds favors their use as chemopreventive agents, but the underlying mechanism of their chemopreventive effect remains unclear. We have observed that the COX-2 inhibitor celecoxib inhibits growth and induces apoptosis in the immortalized breast epithelial cell line 184htert. Microarray gene expression analysis of 184htert cells treated with 50 microM celecoxib for 6h revealed the modulation of several genes of interest, including a significant induction of expression of the mRNA encoding insulin-like growth factor binding protein-3 (IGFBP-3). IGFBP-3 is a potent pro-apoptotic protein and growth inhibitor of breast cancer cells, which acts mainly by inhibiting the access of the mitogens IGF-I and IGF-II to their cell surface receptor, but also via IGF-independent effects. Quantitative real-time RT PCR demonstrated that 50 microM celecoxib induced a approximately 3-fold increase in expression of IGFBP-3 mRNA after 6h. Furthermore, ligand blot analysis revealed that celecoxib treatment was associated with the upregulation of IGFBP-3 at the protein level. IGFBP-3 (500 ng/ml) treatment of 184htert cells inhibited IGF-I and serum-induced proliferation, but had no effect on cell growth under serum-free conditions, indicating that IGF-independent effects of IGFBP-3 are not observed in this system. Our results suggest that celecoxib may decrease IGF-I-associated breast cancer risk by a mechanism involving induction of expression of IGFBP-3 and subsequent reduced proliferation of at-risk breast epithelial cells.