Gene expression profiling of human cancer has proved valuable in cancer research leading not only to the identification of targets but also contributing to our understanding of the mechanisms of the process [23, 24]. The application of microarrays is limited by the availability of fresh frozen tissue or the tissue preserved in RNAlater reagent. As FFPE samples are available in almost all the pathological laboratories and are often available in conjunction with clinical and follow-up data, they would be considered as the most valuable sources for microarray analysis , provided similar information can be obtained as would be expected from analyzing the FF samples. Because of fragmentation [3, 26, 27] and some other chemical modifications of RNA in FFPE samples, currently gene expression studies are largely limited to immuno-histochemical (IHC) staining and RT-PCR, which allow only a few genes to be amplified at a time [3, 28, 29]. In this paper we mainly focus on the use of FFPE samples in genome-wide gene expression experiments. We have tried to analyze the data for differential expression from different angles - at individual gene level, at "gene set" level and also used different statistical methods. In follow-up paper we would focus more on gene selection and relevance to breast cancer biology.
Like other investigators [5, 28, 30, 31], we also observed high reproducibility across technical replicates regardless of the sample type. However, the concordance between the paired FF and FFPE samples was weaker in our study, which is also consistent with other studies [23, 32]. The tissue archival age, or the "FFPE block age" is another factor for consideration. Cronin et al. compared frozen section breast tissue with FFPE samples of "various block ages" by RT PCR for 92 genes and found a 90% signal loss in FFPE samples . Our data on the genome-wide level also suggested the significance of FFPE "block age" on gene expression data. Srinivasan et al. , and Karsten et al  and Masuda et. al  have reviewed in detail the effect of fixative and tissue processing on the content and integrity of nucleic acids. There are four types of reactions of formaldehyde with nucleic acids: (1) Addition reaction or methylolation - the N-H groups of primarily adenine and thiamine are converted to N-CH2-OH groups (methylol groups). The poly(A) tail on RNA is thus heavily methylolated leading to poor reverse transcription. This methylolation is possibly reversible through hydrolysis. (2) Cross-linking - methylolated bases can react with N-H groups (on proteins and nucleic acids) to form -N-CH2_N- cross-links, which are not easily hydrolyzed. (3) Formation of apyrimidinic/apurinic sites - the N-glycosidic bond between A, C, G, T or U and the sugar backbone is broken, leaving a blank space in the sequence. This is not base-specific, but is not reversible. (4) Fragmentation - Formaldehyde catalyzes the hydrolysis of phosphodiester bonds, fragmenting strands of nucleic acids, which is also not reversible. Therefore, it was not a surprise to find differential gene expression in FFPE samples compared to FF samples in the present study.
Bibikova et al. used a smaller panel of the DASL assay with 16 pairs of FF and FFPE samples from healthy and tumor breast tissue and healthy and colon cancer tissue . They found that FFPE samples had 50% less gene expression compared to matched FF samples, which may be due to RNA degradation related to fixation and storage . The present study is one of the first few adequately powered, whole-genome DASL assays interrogating more than 18,000 genes that has compared the results of paired tumor and normal tissue from FF and FFPE samples.
Our findings suggest that the analysis of FFPE samples does not identify the exact same genes that would have been identified by analyzing FF samples, but at least, the list shows some similarity in terms of enrichment of GO-terms representing the lead functional groups of genes. In other words, FFPE samples may not be ideal for picking individual target gene(s), but may be used to identify the lead functional group(s) of genes that are differentially expressed in tumor. Findings of the differentially expressed genes in breast cancer were biologically meaningful. On the one hand the "cell cycle" & "cell division" related genes were up-regulated and on the other hand, genes related to "regulation of epithelial cell proliferation" were down-regulated. Genes involved in metalloexopeptidase activity, transforming growth factor beta signaling pathway, BMP signaling pathway, were found to be down-regulated in breast cancer.
As mentioned in the results section, some of the genes (including PPARG and FGF2) were found repeatedly in the lists of different GO-terms. Peroxisome proliferator-activated receptor-γ (PPARG), is expressed in a large number of human cancers, including breast, colon, stomach, prostate, pancreas, bladder, placenta, lung, chondrosarcoma and in leukemia [34, 35]. Recently Jiang et al. showed that PPARG expression in immunohistochemistry was positively correlated to estrogen receptor status, inversely associated with histological grade and tumor size, and in survival analysis patients with higher PPARG expression had significantly better prognosis . In the same direction, the present study showed evidence of down-regulation of PPARG in breast cancer tissue. GWAS using germ line DNA showed a significant association of SNP in the FGFR2 gene with breast cancer . In the same line, in the present study, we also found FGF2 to be down-regulated in breast cancer. Another biologically relevant gene that we found to be differentially expressed in breast cancer was corticotropin releasing hormone binding protein (CRHBP). Corticotropin-releasing hormone is a potent stimulator of synthesis and secretion of preopiomelanocortin-derived peptides. Although CRH concentrations in the human peripheral circulation are normally low, they increase throughout pregnancy and fall rapidly after parturition. Maternal plasma CRH probably originates from the placenta. Human plasma contains a CRH-binding protein that inactivates CRH and may prevent inappropriate pituitary-adrenal stimulation in pregnancy.
An apparent weakness of the present study is the lack of gene expression data from breast tissue preserved in RNA stabilization buffer, which would have served as the gold standard against which FFPE samples could have been compared. However, 72% of the genes that we found to be differentially expressed in FFPE breast tissue compared to corresponding FF in present study were also differentially expressed in FFPE skin tissue compared to the "gold standard"- RNAlater preserved corresponding skin tissue sample (unpublished data from our group). One of the strengths of the present study is that it was adequately powered to detect differential expression arising from tissue storage (FFPE vs. FF) or disease status (tumor vs. adjacent normal tissue).