Results

Of the 159 colorectal cancers studied, 22 (14%) were found to be RER+ while 137 (86%) were RER− for two or more loci. The median age of patients with RER+ tumours was 67 years compared with 70 years for those with RER− cancers (p=0.89; table 1). RER+ tumours were found more commonly located in the right colon (p=0.006), tended to be larger than RER− tumours (p=0.01), and were more often poorly differentiated than RER− cancers (p=0.0008). A number of DNA based and immunohistochemical assays were used to assess the relation between tumour RER status and other molecular features (table 2). No significant associations were seen between RER status and the presence of a mutant c-Ki-ras gene, or between RER status and p53, c-erbB-2, or c-myc gene expression. Table 3 lists the clinical, molecular, and immunohistochemical findings for the 22 RER+ cases.

Univariate survival analysis showed that outcome was similar in RER+ and RER− cases (log rank test, p=0.68; fig 1). To allow for possible confounding factors, we entered RER status, patient age, tumour differentiation, tumour site, and tumour size into a multivariate survival analysis. This showed that the relative risk of death for patients with RER+ cancers was 0.95 that of patients with RER− cancers (95% confidence interval, 0.41–2.17; p=0.90).

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Figure 1

Survival of 159 patients with T3,N0,M0 colorectal cancer stratified by replication error (RER) status.

Discussion

Tumour development is a multistage process, involving many changes in the nuclear material of the cell. These changes result in significant alterations in the cellular phenotype including the control of cell growth and division. Vogelstein and colleagues1have outlined some of the genetic changes specific to colorectal tumour progression from adenoma to carcinoma including DNA hypomethylation, activation of oncogenes, and inactivation of tumour suppressor genes. The multistep nature of colorectal cancer progression involving some or all of the above genes is widely accepted.3 4 37However, there has been considerable speculation as to whether microsatellite instability reflected the presence of a fundamentally different pathway to malignancy, or if it merely represented a variation on a relatively consistent theme of oncogene activation and tumour suppressor gene loss. Based on an inverse association between microsatellite instability and the presence of c-Ki-ras and p53 mutations, Ionov and colleagues15 suggested that microsatellite instability might form the basis for an entirely new mechanism for carcinogenesis. Thibodeau and colleagues17 also showed a significant inverse relation between microsatellite instability and loss of heterozygosity at chromosomes 5q, 17p, and 18q. However, more recently, a considerable body of evidence has accumulated in the literature which suggests that microsatellite instability does indeed characterise a second parallel pathway to malignancy.38 Genomic instability has now been shown to give rise to genetic alterations in loci including TGFβ1RII39 and IGFIIR.40Indeed, a link has recently been reported between abnormal genomic imprinting and microsatellite instability in colon tumours.41

A study by Kim and colleagues42 showed that right sided RER+ tumours expressed lower levels of p53 product than RER− cases. However, these data appear to contradict the results of many other studies. Aaltonen and colleagues13 found c-Ki-ras and APC gene mutations in a similar proportion of RER+ and RER− colorectal cancers, while Watatani and colleagues43 and Ilyas and colleagues32 found that p53 gene mutation or overexpression was no more frequent in RER− than in RER+ colorectal cancers. Furthermore, although Konishiet al found that mutation of the APC, c-Ki-ras, and p53 genes occurred more frequently in HNPCC RER+ tumours than in sporadic RER+ tumours, they found no difference in the incidence of mutation in these genes between sporadic RER+ and RER− tumours.19 Loss of heterozygosity at chromosomes 5q, 8p, 17p, 18q, and 22q also occurred no more frequently in sporadic RER+ than in RER− tumours.19 Our data, indicating the fact that c-Ki-rasalterations and p53, c-erb, and c-myc protein overexpression occur with similar frequencies in sporadic RER+ and RER− cancers agree with these latter studies. However, table 2 indicates that there may be a trend towards a lower rate of K-rasalterations in RER+ tumours and a higher rate of c-erbB-2 immunostaining in these cases. Thus, we cannot exclude the possibility of a type 2 error in these data.

It is clear that the presence of an RER+ genotype has either some influence on, or at least an association with, a number of clinical and pathological characteristics of sporadic tumours. In accordance with almost all previous studies, we found that RER+ tumours tended to be right sided and poorly differentiated. In addition, RER+ tumours within this anatomically homogeneous series of colorectal cancers tended to be larger than in the RER− cases. The reason for this is unclear, although it is possible that the presence of an RER+ genotype may lead to enhanced growth characteristics without affecting the ability of a tumour to invade or metastasise to lymph nodes or distant organs.

The stage of the primary tumour at the time of initial operation is known to influence greatly the long term survival of colorectal cancer patients. Thus, we specifically omitted all but T3,N0,M0 (stage II) cases in an attempt to minimise any survival bias caused by variable anatomical staging of RER+ and RER− cases. In addition, we studied a series of patients with adequate follow up times to reduce the possibility of missing late, but potentially important, survival effects induced by RER status. Our 10 year survival analysis showed no difference between RER+ and RER− stage II cases. Even when entered into a multivariate analysis along with other possibly important confounding variables such as tumour grade, survival was almost identical in both RER+ and RER− cases. These results initially appear to be at variance with some earlier studies, but close examination shows that this is not the case. Lothe et al,16 in a series of 238 patients with colorectal cancer of variable stages, found that patients with RER+ tumours had a significantly better seven year survival rate than those with RER− cancers. However, this association between RER status and long term survival disappeared when the data were corrected for tumour stage in a multivariate analysis. Thibodeau and colleagues17 also found a positive association between the presence of microsatellite instability and outcome in the first four years after surgery, but no significant association in stage II or III cancers. It is probable that further studies examining a consecutive series of colorectal cancers of different tumour stages and assessing additional genetic alterations, will clarify the association between RER status, tumour progression, and clinical outcome. However, the above results suggest that if RER status does indeed have an important bearing on long term survival, then the effect is likely to be seen more prominently either in cases confined to the bowel wall or in those which have already metastasised to lymph nodes or to distant organs at the time of operation.