Abstract
BackgroundThe ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. In certain clinical and research situations, performing such analyses on archival formalin fixed paraffin-embedded (FFPE) surgical specimens would be advantageous as large libraries of such specimens with long-term follow-up data are widely available. However, FFPE tissue processing can cause fragmentation and chemical modifications of the RNA. A number of recent technical advances have been reported to overcome these issues. Our current study evaluates whether or not the technology is ready for clinical applications.MethodsA modified RNA extraction method and a recent DNA microarray technique, cDNA-mediated annealing, selection, extension and ligation (DASL, Illumina Inc) were evaluated. The gene profiles generated from FFPE specimens were compared to those obtained from paired fresh fine needle aspiration biopsies (FNAB) of 25 breast cancers of different clinical subtypes (based on ER and Her2/neu status). Selected RNA levels were validated using RT-qPCR, and two public databases were used to demonstrate the prognostic significance of the gene profiles generated from FFPE specimens.ResultsCompared to FNAB, RNA isolated from FFPE samples was relatively more degraded, nonetheless, over 80% of the RNA samples were deemed suitable for subsequent DASL assay. Despite a higher noise level, a set of genes from FFPE specimens correlated very well with the gene profiles obtained from FNAB, and could differentiate breast cancer subtypes. Expression levels of these genes were validated using RT-qPCR. Finally, for the first time we correlated gene expression profiles from FFPE samples to survival using two independent microarray databases. Specifically, over-expression of ANLN and KIF2C, and under-expression of MAPT strongly correlated with poor outcomes in breast cancer patients.ConclusionWe demonstrated that FFPE specimens retained important prognostic information that could be identified using a recent gene profiling technology. Our study supports the use of FFPE specimens for the development and refinement of prognostic gene signatures for breast cancer. Clinical applications of such prognostic gene profiles await future large-scale validation studies.
Highlights
The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens
Thirty nine blocks were selected for this study based on the following criteria: 1) sufficient invasive carcinoma remained in the formalin fixed paraffin-embedded (FFPE) block for RNA extraction and 2) > 70% malignant cells
We evaluated 50 FFPE blocks from the clinical surgical pathology, and total RNA was extracted from 39 FFPE blocks
Summary
The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. Gene expression profiling of breast cancers using DNA microarray technology is able to classify breast tumors into distinct biological subgroups and has been shown to predict treatment response and prognosis in several studies [3,4,5,6,7,8,9]. This high-throughput molecular technique requires fresh biospecimens to allow extraction of RNA of sufficient quantity and quality for analysis. FFPE is the most widely used standard of practice for tissue fixation for the purpose of diagnostic histology and long-term storage
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