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.

No abstract

In colorectal tumorigenesis, loss of function of the mismatch repair genes is closely associated with genomic instability at the nucleotide level whereas p53 deficiency has been linked with gross chromosomal instability. We have addressed the contribution of these two forms of genetic instability to tumorigenesis using mice mutant for Msh2 and p53. As previously reported, deficiency of both genes leads to rapid lymphomagenesis Here we show that heterozygosity for p53 also markedly reduces survival on an Msh2 null background. We characterized the patterns of genomic instability in a small set of tumours and showed that, as predicted p53 deficiency predisposes to aneuploidy and Msh2 deficiency leads to microsatellite instability (MSI). However, heterozygosity for p53 in the absence of Msh2 resulted in increased MSI and not aneuploidy. This implied role for p53 in modulating MSI was confirmed using a large cohort of primary fibroblast clones. The differences observed were highly significant (P50.01) in both the fibroblast clones (which all retained p53 functionality) and the tumours, a proportion of which retained p53 functionality. Our results therefore demonstrate a dose sensitive role for p53 in the maintenance of genomic integrity at the nucleotide level.

Activating mutations in the Ki‐ras2 oncogene are frequently observed in sporadic colorectal adenomas and their incidence is reported to rise in large and tubulovillous adenomas to values close to those in carcinomas. This study shows that this property is a feature of adenomas growing in large bowel that has already demonstrated its propensity to engender malignant tumours: i.e., bowel in which there is a synchronous carcinoma. Adenomas from cancer‐free bowel do not share this high incidence of Ki‐ras mutations. This difference in mutation incidence between adenomas from cancer‐free and cancer‐bearing patients does not appear to derive from sampling bias relative to adenoma size, site, or patient age, nor is it found in another gene (APC) known to be of importance in adenoma formation. Large, dysplastic adenomas from cancer‐bearing bowel, however, are particularly liable to carry Ki‐ras mutations when they arise in patients over 70 years old. The observations suggest that the role of Ki‐ras mutations may be more subtle than merely enhancing adenoma growth. Adenoma cells of cancer‐prone individuals may suffer more mutational events than those in persons selected as cancer‐free.

BAX gene mutations occur in approximately 50% of RER+ colorectal cancers. To determine the role of these mutations in tumour progression we analysed multiple di erent tumour sites from RER+ colorectal cancers for BAX mutations. Sixty colorectal carcinomas were analysed for microsatellite instability at loci BAT-26, L-myc, TGFbRII, D13S160 and D2S123. Twelve out of 60 tumours (20%) were RER+. Forty-®ve di erent tumour sites from the 12 RER+ carcinomas were analysed for BAX mutations at the [(G)8] tract in exon 3. Six out of 12 (50%) RER+ tumours showed BAX mutations, four of which showed a homogenous pattern of such mutations detected in all tumour sites. In the other two cases, BAX mutations were present in some but not all tumour sites sampled from the same patient. In contrast, TGFbRII mutations were found in 9/12 cases (75%) and in each of these were present in all the sampled sites. Two cases showed neither BAX nor TGFbRII mutation. These data suggest that mutations in TGFbRII may occur at a very early stage in tumour progression, perhaps in the founder clone. BAX mutations, however, are clearly not necessary for formation of the founder clone and can occur for the ®rst time later in tumour progression.

No abstract

Current opinion of the genetic events driving colorectal tumourigenesis focuses on genomic instability. At least two apparently independent mechanisms are recognized, microsatellite instability and chromosomal instability. The genetic defects underlying each type of instability are only partially understood and controversy remains as to the role of p53 in the generation of chromosomal defects in colorectal cancer. This study sought to clarify the relationships between chromosomal abnormalities and defects of both p53 and mismatch repair. Extensive chromosomal analysis was undertaken, using¯ow cytometry and comparative genomic hybridization, of a series of sporadic colorectal cancers which had been grown to early passage as subcutaneous xenografts in SCID mice. Overall levels of chromosomal defects were observed to be low in RER+ cancers compared with RERx and distinctive patterns of chromosomal anomalies were found to be associated with both the RER+ and RERx phenotype. No particular level or pattern of chromosomal anomalies appeared to be associated with p53 status, supporting recent observations that abnormal p53 function is not suf®cient to cause chromosomal anomalies in colorectal tumours.