We have investigated the influence of genetic instability [replication error (RER) phenotype] on APC (adenomatous polyposis coli), a gene thought to initiate colorectal tumorigenesis. The prevalence ofAPC mutations was similar in RER and non-RER tumors, indicating that both tumor types share this step in neoplastic transformation. However, in a total of 101 sequenced mutations, we noted a substantial excess ofAPC frameshift mutations in the RER cases (70% in RER tumors versus 47% in non-RER tumors, P < 0.04). These frameshifts were characteristic of mutations arising in cells deficient in DNA mismatch repair, with a predilection for mononucleotide repeats in the RER tumors (P < 0.0002), particularly (A). tracts (P < -0.00007). These findings suggest that the genetic instability that is reflected by the RER phenotype precedes, and is responsible for, APC mutation in RER large bowel tumors and have important implications for understanding the very earliest stages of neoplasia in patients with tumors deficient in mismatch repair.Instability at simple repetitive DNA sequences has been observed in a subset of sporadic colorectal cancers (1, 2) and in essentially all tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC) (3-5). Deficiency in binding and repair of DNA mismatches has been demonstrated in tumor cell lines exhibiting such instability (6-10), and studies show an increased spontaneous mutation rate in selectable genes (8,(11)(12)(13). In most HNPCC tumors and in many of the sporadic cases, the underlying basis of this mutator or RER (replication error) phenotype is mutational inactivation of a human mismatch repair gene, five of which have so far been described (10,(14)(15)(16)(17)(18). With the exception of GTBP/pl60 (19), germ-line mutations have been identified in each of these genes in HNPCC families (5, 15-18, 20, 21

Two apparently independent mechanisms of instability are recognized in colorectal cancer, microsatellite instability and chromosomal instability. Evidence from colorectal cancer cell lines indicates the presence of either, or both, types of instability in the vast majority. Here, we sought to determine the prevalence of such instability in primary sporadic colorectal cancers. Microsatellite instability was established by demonstration of ovel clonal, nongerm-line alleles in at least two of four tested loci. Chromosomal abnormalities were identified by comparative genomic hybridization (CGH) and flow cytometric analysis of nuclear DNA content. Tumours harbouring chromosomal instability were distinguished from those with stable but aneuploid karyotypes by comparing chromosomal defects at multiple sites throughout each cancer. This analysis allowed assessment of both the number of chromosomal abnormalities and their heterogeneity throughout the tumour. The results confirm that microsatellite instability is consistently associated with multiple, repeated changes in microsatellites throughout the growth of the affected colorectal carcinomas. There were also several carcinomas in which major structural or numerical abnormalities in chromosomes had clearly continued to arise during tumour growth. However, a substantial subset of tumours showed neither microsatellite instability nor multiple, major chromosomal abnormalities. We suggest that the development of a proportion of colorectal cancers proceeds via a different pathway of carcinogenesis not associated with either of the currently recognized forms of genomic instability.

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