ABSTRACT
Microarray analysis reaches increasing popularity during the investigation of prognostic gene clusters in oncology. The standardisation of technical procedures will be essential to compare various datasets produced by different research groups. In several projects the amount of available tissue is limited. In such cases the preamplification of RNA might be necessary prior to microarray hybridisation. To evaluate the comparability of microarray results generated either by amplified or non amplified RNA we isolated RNA from colorectal cancer samples (stage UICC IV) following tumour tissue enrichment by macroscopic manual dissection (CMD). One part of the RNA was directly labelled and hybridised to GeneChips (HG-U133A, Affymetrix), the other part of the RNA was amplified according to the "Eberwine" protocol and was then hybridised to the microarrays. During unsupervised hierarchical clustering the samples were divided in groups regarding the RNA pre-treatment and 5.726 differentially expressed genes were identified. Using independent microarray data of 31 amplified vs. 24 non amplified RNA samples from colon carcinomas (stage UICC III) in a set of 50 predictive genes we validated the amplification bias. In conclusion microarray data resulting from different pre-processing regarding RNA pre-amplification can not be compared within one analysis.
Subject(s)
Carcinoma/metabolism , Colorectal Neoplasms/metabolism , Gene Expression Profiling/methods , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis/methods , RNA, Neoplasm/metabolism , Carcinoma/genetics , Cluster Analysis , Colorectal Neoplasms/genetics , Humans , Microdissection , Neoplasm Staging , Nucleic Acid Amplification Techniques , Reproducibility of ResultsABSTRACT
BACKGROUND: Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) have been strongly implicated in the pathogenesis of many types of human cancer. We wanted to specifically define their role in established colorectal cancer liver metastases. PATIENTS AND METHODS: The MMP/TIMP expression profiles of N=9 colorectal primary tumour liver metastasis tissue pairs were determined using oligonucleotide-based arrays. Expression levels for the most relevant MMPs were confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). Additionally, unsupervised clustering using the MMP/TIMP profile of N=25 colorectal cancer liver metastases was performed and the response to palliative 5-fluorouracil (5-FU)-based chemotherapy was assessed using radiological response criteria. RESULTS: When comparing the primary tumors to their synchronous liver metastases, a statistically significant (p < 0.05) down-regulation of MMP1, -2, -3 and -12 was found in the metastases. Unsupervised clustering using the MMP/TIMP profiles of 25 liver metastases revealed two distinct subgroups with different responses to palliative, 5-FU-based chemotherapy (response rates: 22% vs. 56%, respectively). In particular, higher MMP7, TIMP1 and TIMP2 levels were found in the unfavourable group, while higher expression of MMP2, -9, -11 and -14 was associated with a more favourable response to chemotherapy. CONCLUSION: Colorectal cancer liver metastases show a distinctive MMP/TIMP profile with predictive implications.
