This study was conducted primarily to define parameters that can reliably predict the presence of MMR gene mutations in two specific groups of patients with CRC suspected of having HNPCC—that is, patients with CRC before the age of 50 years and those with a CRC and at least one other HNPCC‐associated cancer, irrespective of age. The main conclusions that can be drawn from this study are as follows. (1) Immunohistochemical staining for the MMR proteins MLH1, MSH2 and MSH6 is the best single method to select patients with CRC, suspected of having an MMR gene mutation, for mutation analysis. (2) The low prevalence of MMR gene mutations in patients with CRC below the age of 50 years justifies preselection by IHC and/or MSI analysis before mutation analysis is carried out. (3) The occurrence of
2 HNPCC‐associated cancers in patients aged
60 years is rarely due to germline MMR gene mutations.
Recently, five large studies were published that also focused on the relative values of MSI analysis and IHC to detect potential germline MMR gene mutation carriers.7,8,9,11,28
The conclusion from all five studies and from several previous studies was similar to that from ours—namely, IHC is at least equally valuable for this goal as MSI analysis. There are, however, several differences between our study and the other five.
In three of the studies, immunostaining for the MSH6 protein and mutation analysis for germline mutations in MSH6
were not carried out.8,9,11
Although less prevalent in typical AC‐positive families, our results suggest that MSH6
mutations occur at about the same rate as those in MLH1
in a less selected patient population. In the study by Hampel et al
mutation analysis for MSH6
was carried out only in patients whose tumours showed MSI (low or high) or who were considered to be at high risk for HNPCC on the basis of clinical criteria and whose tumours lacked MMR gene expression. Although, probably, most mutations were detected by this approach, some MSH6
mutations could have been missed. Only Southey et al7
carried out IHC for MSH6 in all tumours.
Stormorken et al28
included only patients who were referred for genetic counselling. This may have influenced the resulting predictive values. The same is true for the sensitivity and the positive predictive value of the Amsterdam criteria, which can be expected to be high in a selected population like the one studied by Stormorken et al
. We observed that only 9 of 25 mutation carrier families fulfilled the revised Amsterdam criteria. Also, Hampel et al8
found only a small minority (3/23) of the mutation carriers' families fulfilling the Amsterdam criteria.
A limitation of many studies on this topic is that selection criteria are compared with each other, without establishing the gold standard—that is, the presence of mutations—in all participants. This is also true for four of the five studies mentioned above. Although the assumption that all pathogenic mutations of MLH1 and MSH2 lead to MSI can be well defended, a proper calculation of the sensitivity, specificity and predictive values requires a true gold standard. We carried out mutation analysis for all three genes in the first two thirds of included patients and did not find any clearly pathogenic mutation in MLH1 or MSH2 in patients with MSI‐low tumours.
The value of IHC depends partially on the quality of the nuclear stainings and the experience of the pathologist.12
The results are sometimes not interpretable because of absence or low intensity of the nuclear staining in tumour and normal cells.29
Mangold et al30
described the phenomenon of weak nuclear MLH1 staining in tumours of patients with MLH1
mutations, which can be observed as false‐positive staining. Experienced pathologists should be able to recognise these tumours with weakly positive MLH1 staining as tumours from possible MLH1
mutation carriers. On the other hand, MSI analysis gives difficulties as well. The quality of tumour DNA isolated from paraffin‐wax‐embedded tumours may be poor and not usable for MSI analysis. Although seldom mentioned, interobserver variation also occurs when scoring MSI. About 15% of sporadic colorectal cancers show MSI and MLH1‐negative stainings as a result of hypermethylation of the promoter region of MLH1
Table 4D shows that MLH1 staining has a low positive predictive value. The consequence of this is that more patients will be screened for MLH1
mutations with a negative outcome than is the case for the other two genes.
Apart from IHC and MSI analysis, we also evaluated the first‐degree family history for HNPCC‐associated cancers as an indicator of an MMR gene mutation. As expected, most mutation carriers had a first‐degree family member with such a tumour. Nevertheless, 6 of the 25 (almost 25%) mutation carriers had a negative first‐degree family history. This indicates that family history alone is an insufficient indicator of being a mutation carrier and we should not rely on it alone when deciding whether or not to carry out mutation analysis. We further calculated the predictive values for combinations of family history, MSI analysis and IHC. Although the specificity and positive predictive value both increased from this, the sensitivity decreased markedly. Therefore, when a patient fulfils one of the criteria used in this study, IHC or MSI analysis should be carried out without further selection. Both MSI and IHC analysis are proper selection tools, but when the family history raises high suspicion for HNPCC, both methods should be used to avoid missing mutation carriers.
Terdiman et al32
noticed that referral for genetic counselling because of the suspicion of hereditary cancer was an independent risk factor for the presence of an MMR gene mutation when compared with population‐based patients with CRC. We could not confirm that finding, although we also found a higher prevalence of mutations in patients referred to the clinical geneticist. A positive family history in most of those referred to a clinical geneticist is probably the determinant of this finding.
Our group of 224 patients with CRC at <50 years of age is the largest group so far reported in a single study. Most of these patients were referred specifically to participate in this study, whereas a minority had been referred to a clinical geneticist because of a perceived high risk for hereditary cancer. All patients came from the same geographical area and had their tumours diagnosed in approximately the same period. We therefore think that this group can be considered representative of all patients with CRC before the age of 50 years. Pathogenic mutations were detected in 14 of the 224 (6%) patients. MLH1
mutations were identified in 11 (5%) patients. This is similar to the findings of Pinol et al11
and in accordance with prevalences of 0–12% in other small, otherwise unselected groups of such patients.33,34,35,36,37,38
Owing to the low mutation frequency, it is, in our view, justified to carry out only mutation analysis in such patients if a screening by MSI analysis or IHC analysis suggests a mutation.
This may be different for people with
2 HNPCC‐associated cancers, including at least one CRC. In this group of 79 people, we did not find any mutation in the 29 patients who had their first tumour diagnosed after age 60 years (19 of these had only CRCs and 10 had CRC and at least one other HNPCC‐associated cancer). It is therefore questionable whether such patients should be screened for HNPCC and thus should be included in the Bethesda criteria. However, our group of patients was still rather small and further studies should be conducted before an age criterion for these patients can be confidently introduced. Nevertheless, when this subgroup is excluded from consideration, mutations were found in 17 of 50 patients with
2 cancers, of which at least one was diagnosed before age 60 years. Such a high occurrence may justify direct mutation analysis, without a prescreen for MSI or IHC.
Recently it was reported that the mean age at diagnosis of CRC of mutation‐positive relatives of probands, identified in a population‐based study of patients with CRC, was 61.2 years, much higher than that reported so far for mutation‐positive people, which is about 45 years.39
It is striking though, that the mean age at diagnosis of the probands in Hampel et al
study was 44 years. As patients in our study were collected irrespective of their family history, a similarly higher age at diagnosis of CRC in the relatives of our patients might have been expected. This was not the case as the mean age was 47.4 years. The fact that we did not find any mutation carriers in patients with
2 cancers, all diagnosed above age 60 years, also does not support the findings of Hampel et al39
. Hence, their findings need confirmation in other populations, before any change in counselling practices is considered.
Only 8 of 25 supposedly mutation‐carrying parents had CRC at the time of the study. Most of these 25 parents were older (
70 years) at that time and it was not expected that many of them would still develop CRC. This observation is in accordance with recent new calculations on the CRC risk in HNPCC—that is, 26.7% and 22.4% for men and women, respectively.40
A limitation of our study is the number of patients for whom a complete set of variables required for the calculations was available (n
174). Although smaller than the populations in the studies by Engel et al9
1119) and Pinol et al11
287), we think that the number of patients in our study was large enough for reliable calculations. Another limitation might be the fact that we have missed mutations, as we included only the major HNPCC‐related genes in our genetic screening. Mutations in other MMR genes, such as PMS2
, cannot be ruled out.41,42,43,44,45
Furthermore, we did not screen for mutations in the regulatory sequences (such as the promoter regions) of MLH1
, nor did we look for genomic rearrangements in MSH6
However, as such mutations probably represent only a small proportion of disease‐causing MMR gene mutations, this will not markedly alter the estimated prevalence of germline MMR gene mutations among patients with CRC. Finally, we cannot exclude survival bias in our study. Patients with MSI‐high tumours have a better prognosis than those with MSI‐low tumours, and, as we included some of the patients retrospectively and inclusion for the study required a blood sample for mutation analysis, the real mutation frequency might be somewhat lower.
The equal or even better potency of IHC to recognise patients with an MMR gene mutation does not apply to the detection of missense variants of currently unknown pathogenicity. In our study, 5 of 18 patients with such a variant had an MSI‐high tumour, whereas IHC showed normal staining of the protein, corresponding with the variant gene, in all 14 cases where IHC was available (table A provided online). For some of the known missense variants, the evidence for pathogenicity is strong, but for most of them this is questionable. Functional assays of these variants may elucidate their pathogenic potential, but these have not yet been developed to a stage of routine application.
In conclusion, this study confirms that IHC staining for the MMR proteins is the best single method to select patients with CRC, suspected of having an MMR gene mutation, for mutation analysis. Such selection is mandatory for most patients with CRC before the age of 50 years, in view of the low prevalence of mutations in those patients. The value of genetic analysis in patients with multiple HNPCC‐associated cancers, all occurring above age 60 years, is questionable.