Alternative genetic pathways in colorectal carcinogenesis

Abstract

The comparative typing of matched tumor and blood DNAs at dinucleotide repeat (microsatellite) loci has revealed in tumor DNA the presence of alleles that are not observed in normal DNA. The occurrence of these additional alleles is possibly due to replication errors (RERs). Although this observation has led to the recognition of a subtype of colorectal cancer with a high incidence of RERs (caused by a deficiency in DNA mismatch repair), a thorough analysis of the RER frequency in a consecutive series of colorectal cancers had not been reported. It is shown here that the extensive typing of 88 colorectal tumors reveals a bimodal distribution for the frequency of RER at microsatellite loci. Within the major mode (75 tumors, RER- subtype), the probability that a locus exhibited instability did not differ significantly among loci and tumors, being 0.02. The subsequent development of a statistical test for an operational discrimination between the RER- and RER+ subtypes indicated that the probability of misclassification did not exceed 0.001 in this series. The frequency of K-ras mutation was found to be equivalent in the two subtypes. However, in the RER+ tumors, the p53 gene mutation was less frequently detected, the adenomatous polyposis coli (APC) mutation was rare, and the biallelic inactivation of either of these genes was not observed. Furthermore, the concomitant occurrence of APC and tumor growth factor beta receptor type II gene alterations was found only once. These data suggest that the repertoires of genes that are frequently altered in RER+ and RER- tumors may be more different than previously thought.

Figure 1

Detection of instability at microsatellite loci. (a) The PCR products generated at D9S178 from normal (N) and matched tumor (T) DNAs are compared for five cases. No difference was observed for sample MOUa. Arrows indicate the presence of additional alleles (RER) in the four other cases. (b) The PCR product generated by primers spanning the coding A₁₀ tract of the TGF-βRII gene from the tumor DNA is shown for the same five cases. The product generated from normal DNA is 73 bp long. This is the only product observed for case MOUa. In the four other cases, additional product(s) demonstrate(s) the presence of frameshift mutation(s).

Figure 2

FUM distribution in colorectal cancers. A total of 46 tumors that had been typed on 110 dinucleotide repeat loci are ordered according to their FUM values. The tumors that have a FUM value less than or equal to 0.06 were classified in the RER− subtype. Although about 4,000 elementary genotypic data on microsatellite loci were available for this subgroup, statistical analysis revealed heterogeneity neither for FUM values nor for the occurrence of RER at specific loci.

Figure 3

Detection of truncating mutation in exon 15 of the APC gene. Left and Right show polypeptides derived from the in vitro transcription–translation test performed on fragments I and II of APC exon 15, respectively. Fragment I is expected to give rise to a normal 72-kDa polypeptide that is observed in all lanes due to contamination of tumor fragments with nonneoplastic cells. Observation of shorter polypeptides (arrowheads) is indicative of the presence of an APC mutation that was in all cases confirmed by sequencing. Fragment II encodes a normal 85-kDa polypeptide. Note that in sample BOG shorter polypeptides are generated from both fragments I and II. This observation was suggestive of a localization of the truncating mutation on the DNA portion overlapping I and II, a hypothesis that was confirmed by sequencing.