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Gene silencing through CpG island hypermethylation has been associated with genesis or progression of frequent microsatellite instability (MSI-H) cancers. To identify novel methylation sites unique to MSI-H colon cancers in an unbiased fashion, we conducted a global expression profiling-based methylation target search. We identified 81 genes selectively down-regulated in MSI-H cancers using cDNA microarray analysis of 41 primary colon cancers. Forty six of these 81 genes contained CpG islands overlapping their 5′untranslated regions. Initial screening of six genes in 57 primary colon cancers detected the following gene with MSI-H cancer-specific hypermethylation: RAB32, a ras family member and A-kinase-anchoring protein, was methylated in 14 of 25 (56%) MSI-H cancers but in none of 32 non-MSI-H cancers or 23 normal colonic specimens. RAB32 hypermethylation correlated with RAB32 mRNA down-regulation and with hMLH1 hypermethylation. In addition, the protein-tyrosine phosphatase receptor type Ogene, PTPRO, was frequently methylated in right-sided tumors. This methylation screening strategy should identify additional genes inactivated by epigenetic silencing in colorectal and other cancers.

Frequent microsatellite instability MSI (MSI-H) occurring in human tumors is characterized by defective DNA mismatch repair and unique clinical features. However, infrequent MSI (MSI-L) has not been attributable to any other defined molecular pathway, and its existence as a biologically distinct category has been challenged. Moreover, the global molecular phenotypes (GMPs) underlying MSI-H, MSI-L, or microsatellite-stable (MSS) tumors have never been evaluated. To evaluate the impact of MSI status on GMP and to determine the importance of MSI relative to other molecular and clinical features, cDNA microarray-derived data from 41 colon cancers were interpreted using principal components analysis. The clinically relevant principal component with the greatest impact on GMP was component 3, which distinguished MSI-H from non-MSI-H (i.e., MSI-L and microsatellite stable) tumors and was designated the MSI-H separator. Notably, MSI-L cancers were also clearly distinguished from non-MSI-L tumors by another principle component, component 10 (the "MSI-L separator"). This second finding validates the existence of MSI-L tumors as a distinct molecular phenotypic category. Thus, both components 3 and 10 reflected different aspects of MSI and helped to establish principal components analysis as a useful tool to identify and characterize distinct biological features of human malignancy.

Colorectal cancer (CRC) has traditionally been classified into two groups: microsatellite stable/low-level instability (MSS/MSI-L) and high-level MSI (MSI-H) groups on the basis of multiple molecular and clinicopathologic criteria. Using methylated in tumor (MINT) markers 1, 2, 12, and 31, we stratified 77 primary CRCs into three groups: MINT++ (2), MINT+ (1-2), and MINT- (0 markers methylated). The MSS/MSI-L/MINT++ group was indistinguishable from the MSI-H/MINT++ group with respect to methylation of p16(INK4a), p14(ARF), and RIZ1, and multiple morphological features. The only significant difference between MSI-H and non-MSI-H MINT++ cancers was the higher frequency of K-ras mutation (P0.004) and lower frequency of hMLH1 methylation (P0.001) in the latter. These data demonstrate that the separation of CRC into two nonoverlapping groups (MSI-H versus MSS/MSI-L) is a misleading oversimplification.