Fluorescent-based BAT-26 analysis for distinct screening of microsatellite instability in colorectal cancers

For easy screening of genetic instability in colorectal cancers, we tried BAT-26 and BAT-25 mononucleotide repeats using fluorescent analysis and evaluated their usefulness and problems compared with other markers: D5S346, D17S250, D2S123, and D2S391, D4S392 (located near BAT-26 and BAT-25 respectively). The high-frequency of MSI (MSI-H) tumours, defined as tumours having instability in more than two markers, were detected in 8/146 (5.5%). These MSI-H cases were younger ages at diagnosis, and showed significantly higher incidences of right side location, and poorly differentiated histology than other cases (p<0.05). Four cases (2.7%) showed a 1 bp size shift in BAT-26 and 2 of them showed loss of heterozygosity (LOH) at D2S391 near BAT-26 locus. Among 9 cases with a 1 bp size shift in BAT-25, 6 cases showed LOH at D4S392 near the BAT-25 locus (p=0.035). In all 4 cases, non-cancerous DNA had two analogous peaks of BAT-26, indicating the heterozygosity of BAT-26 in constitutional DNA. This phenomenon was also detected in the peaks of BAT-25 in some cases, in whose constitutional DNAs, 1 bp size shift was also detectable in three other markers. To elucidate the reasons for the alterations of the 1 bp size shift of peak of these markers, we examined by microsatellite analysis mixed samples of tumour DNA with complete loss of the one allele at the 1p loci and each constitutional DNA sample of neuroblastoma patients. One base shift of the peak signal of the microsatellite marker was clearly obtained in proportion to the ratio of cancerous DNA and constitutional DNA. Fluorescent-based analysis of BAT-26 or BAT-25 was easy and useful for detection of MSI-H in colorectal cancers without analyzing non-cancerous DNA. A 1 bp size shift in BAT-26 or BAT-25 was considered to be affected by LOH at these loci. Thus, it is important to distinguish MSI from LOH to evaluated MSI using these markers.