There is a growing interest in the use of high throughput microarray analysis for the molecular reclassification of diseases. This interest appears to be well founded, as many groups have reported consistent patterns of gene expression associated with pathologic phenotypes, clinical behaviors and outcomes [4
]. In the area of prostate cancer numerous groups [23
] have all reported significant differential gene expression between histologic tumor specimens and normal appearing prostate tissue from patients with tumor present elsewhere in the prostate. Recently, a group from our institution reported a 70 gene signature that may predict aggressiveness of prostate cancer [12
]. Comparison of the gene lists from published data sets with the results of our tumor versus adjacent normal analysis is complicated by the heterogeneity in samples, analysis platforms and analysis methods. Nevertheless, our study is qualitatively similar to other studies in the expression profile of tumors compared to adjacent normal tissue. A number of genes including hepsin
, fatty acid synthase SPARC1
coactivator show similar expression patterns across multiple prostate cancer studies [30
], and are also regulated in our study.
Our donors did not have prostate cancer or prostatic intraepithelial neoplasia (PIN) identified in their prostate and as such are good candidates for "true normals". Differential expression was much greater between tumor and donor tissue than between tumor and adjacent normal. The fact that tumor and adjacent specimens come from the same patients could possibly explain this difference but this was ruled out by our analysis. Another possibility is that tissue handing and processing differences could account for some or all of the differential expression seen when donor tissue is use as a baseline. In fact, data in the literature does suggest that tissue processing could effect the expression of genes such as fos
in prostate tissue [29
]. However, the same literature indicates that the effect warm ischemic time is limited to specific genes and in general, involves less than 1% of the regulated genes [29
]. Our studies emphasize the need for documentation and quality of all experimental processing steps, from sample acquisition to sample hybridization, in order to completely characterize gene expression differences between prostate donors, tumors and normal tissue adjacent to tumors.
In our experiment, tumor and adjacent normal specimens where taken from the same prostates and handled the same way. If differences in patient and donor tissue handling was the major issue driving differential expression in the tumor v donor and adjacent normal v donor comparisons, one would expect tumor v donor and adjacent normal to result in very similar expression profiles. However, we have shown that tumor v donor exhibits far greater differential expression than adjacent normal v donor (see Results). Furthermore, the differentially expressed genes seen in both tumor and adjacent normal include proto-oncogene and transcription factors that one might rationally expect to see in expectation of or in response to a local tumor. Therefore, while the possibility that some expression changes are due to differences in tissue handling cannot be formally ruled out, it is unlikely that the large and specific differences we observe in tumor v donor, tumor v adjacent normal and adjacent normal v donor are entirely due to processing differences. Clearly additional studies, including examination of patient process specimens that do not host prostate cancer (such as cystoprostatectomy for bladder cancer or prostates removed for benign hypertrophy) to examine this process further.
The most important finding from our analysis is the potential importance of the donor specimens and the possibility that a field effect exists around prostate tumors, resulting in significant molecular changes in histologically normal appearing tissue adjacent to prostate cancer. Significantly, evidence for such malignancy associated changes have been presented in other organs such as the cervix, bladder and breast [31
Furthermore, a variety of methods such as chromosomal analysis [13
], SAGE [14
], ploidy analysis [15
] have shown molecular abnormalities in normal appearing prostate adjacent to tumor. Image analysis has also been employed to identify consistent changes in "normal appearing" prostate tissue adjacent to tumor [17
]. In one study cases of prostatic adenocarcinoma was consistently detected by examining histologically normal tissue using high-resolution image cytometry [18
], and in another, combined highly sensitive and discriminating Fourier transform-infrared spectroscopy with statistical analysis was used to detect damaged DNA in normal appearing prostate tissue adjacent to cancer [34
In expression analysis, while most published prostate studies have used adjacent normals as the baseline tissue, Dhansekaran [23
], used both commercially available pooled donor normal tissue and adjacent normal tissue and noted differences in expression profile between the two specimen types. Genes that were differentially expressed in adjacent normals when compared to the pooled donor normals included signal transducers and transcription factors; and expression of these genes in adjacent normals was attributed to a field effect around tumors. Similarly, Yu [12
] have noted dysregulation of selected genes in both adjacent normals and tumors when compared to donors. Prakash [27
], found that gene expression in asymptomatic benign prostatic hyperplasia adjacent to tumors was different from asymptomatic BPH or symptomatic BPH not associated with tumors. The unique expression signature of BPH next to tumors included fos, jun, immediate early genes and this list was remarkably similar to the most up-regulated genes in the adjacent normals tissue in our study (see adjacent normal v donor, additional file 1
Finally, within archives of the University of Pittsburgh Pathology Tissue Bank, there was a donor prostate, which was found to harbor prostate cancer. When run on the Affymetrix arrays, the tumor classified with the tumors samples rather than the donor samples. Although this is clearly no more than an anecdotal event, it is an interesting finding.
Though microarray technology represents a major advance and provides a powerful tool for high-throughput expression analysis, the most effective use of this technology requires careful consideration of baseline normal tissue. Our results here emphasize the need for careful examination of what constitutes normal tissue and the importance of future studies to fully characterize normal appearing tissue adjacent to prostate cancers.