Microsatellite marker PCR was performed on DNA isolated from each of the seventeen adenomas and associated carcinomas, using DNA from normal colonic mucosa from each corresponding patient as a control. Twenty-eight microsatellite markers were examined for allelic losses, including several which had previously been shown by us to be associated with intrachromosomal genomic instability in a separate group of 59 sporadic colorectal cancers (15
). Additional markers in adjacent regions were examined in order to better define recurrent LOH sites potentially associated with genome-wide instability. Fractional allelic loss (FAL) and microsatellite instability (FINST) rates were calculated as described in the methods section ( and ).
Figure 2 Comparison of fractional allelic loss rate in adenomas and carcinomas for all informative markers each of the 17 patients. X-axis represents FAL rate calculated as described in the methods section. Y-axis lists patient number. Black bars represent carcinomas, (more ...)
Figure 3 Comparison of fractional microsatellite instability rate in adenomas and carcinomas for all informative markers in each of the 17 patients. X-axis represents FINST rate calculated as described in the methods section. Y-axis lists patient number. Black (more ...)
For the markers examined in this study, the combined fractional allelic loss rate FAL in the adenomas ranged from 0 to 0.192 (mean = 0.052), while in carcinomas it ranged from 0 to 0.636 (mean = 0.135) (). Twelve of seventeen adenomas (71%) and sixteen of seventeen carcinomas (94%) demonstrated loss of heterozygosity (LOH) for one or more markers, while multiple events were seen in six of seventeen adenomas (35%) and eleven of seventeen carcinomas (65%). Thirteen of the seventeen carcinomas (76%) had increased FAL rates when compared to their adjacent adenomas. In carcinomas, the mean FAL rate of 0.135 was over 2.5 times that seen in adenomas, a statistically significant difference (Z=−2.59, Wilcoxon Signed Rank test, p=0.010 ). This difference remained significant (Z=−2.39, p=0.017) after a possible outlier (patient 13063) was excluded (see ).
Mean Fractional Allelic Loss And Microsatellite Instability For Adenomas and Carcinomasa
Cumulative FAL rates were calculated for each individual marker tested (). FAL rates in adenomas ranged from 0 to 0.267, while in carcinomas it ranged from 0 to 0.29. Fifteen of twenty-eight markers (54%) showed LOH events in one or more adenomas, while twenty-six of twenty-eight markers (93%) demonstrated LOH events in carcinomas. Multiple LOH events were observed for five markers (18%) in adenomas and fifteen (54%) in carcinomas. The five markers with frequent LOH in adenomas were located on chromosomes 11p15, 11q23, 14q24, 14q32, and 15q26. Twenty-two of the twenty-eight markers (79%) had higher FAL rates in carcinomas than in adenomas. These differences ranged from 0.005 to 0.23, with a mean of 0.104. Of the remaining six markers, five were increased in adenomas, by 0.008 to 0.084 (mean 0.04).
Figure 4 Comparison of fractional allelic loss rate in adenomas and carcinomas for each of the 28 microsatellite markers in all 17 patients. X-axis represents FAL rate calculated as described in the methods section. Y-axis lists marker number. Black bars represent (more ...)
Fractional microsatellite instability for the markers examined (FINST) in adenomas ranged from 0 to 76.9%, while that in carcinomas varied from 0 to 70.4% (). Using instability in 30% of informative markers as a cutoff to define microsatellite unstable (MSI) lesions, four of the seventeen (23%) patients demonstrated microsatellite instability in both adenomas and carcinomas. These four patients had microsatellite instability in 37, 52, 62, and 77% of informative markers in their adenomas and 33, 48, 64, and 70% of informative markers in their carcinomas. One additional adenoma showed instability in 50% of markers, but only 19% in the carcinoma; this patient, had a small number of informative PCR reactions in the adenoma and microsatellite instability therefore may be overestimated for this particular sample.
For the microsatellite stable tumors, the mean FINST rate was 0.063 for the adenomas and 0.057 for the carcinomas, which was not a significant difference (Z=−1.162, Wicoxon Signed Rank test, p=0.25). For the four tumors with frank microsatellite instability, the mean FINST value for the adenomas ( 0.57 ) was nearly identical to the mean FINST rate for the corresponding carcinomas ( 0.54), suggesting for this small sample that additional microsatellite alterations were not widespread as adenomas progressed to carcinomas.