Hereditary non-polyposis colorectal cancer (HNPCC) is perhaps the most common hereditary colon cancer syndrome, accounting for 3–6% of the total colorectal cancer burden.¹ These tumours show a particular molecular phenotype in which thousands of mutations within repetitive sequences accumulate throughout the genome of the tumour cells causing high microsatellite instability (MSI-H).^2–5 Accumulative data has provided evidence that the underlying event leading to HNPCC is an inherited mutation of a DNA mismatch repair (MMR) gene, mainly MLH1 or MSH2, which account for approximately 90% of the known mutations to date.^6,7 Further, mutations in MSH6 account for almost 10% of the cases and a small number of mutations in PMS2 have been also reported in a few cases.^6–8 However, mutations in these four MMR genes account for only half of the HNPCC cases identified by pedigree criteria only.^9–11 The molecular events causing HNPCC-like pedigrees in which tumours do not have MMR defects is the subject of continued investigation.

The most effective strategy for the diagnosis of HNPCC is the compilation of a thorough family history of colorectal cancer and fulfilment of the Amsterdam criteria.⁹ However, this information is often not available, about 20% of HNPCC families with germline mutations do not meet these criteria, and further, some colorectal cancer prone families remain mutation negative in MMR gene analyses.¹¹ Because microsatellite instability (MSI) is a hallmark of tumours arising in the HNPCC syndrome,^3,4 international criteria have been developed to combine clinical and molecular features to help identify those patients at high risk of a diagnosis of HNPCC.¹¹ Current recommendations include first testing tumours for the presence of MSI and the absence of protein expression for several of the DNA mismatch repair genes by immunohistochemistry (IHC). Subsequent gene testing can then be targeted to the relevant gene. In this context, available data show that an abnormal immunostaining of MSH2 and MSH6 proteins is most likely the result of germline mutations. When mutations are detected, specific cancer screening and follow up programs are offered to mutation carriers, which have proved profitable in terms of cost-effectiveness when compared to standard care.^12–14

Up to this point, it has not been considered cost-effective to perform MSI and/or IHC testing of all unselected patients with colorectal cancer.¹¹ Most sporadic colorectal tumours show no mismatch repair defects and of the 12–15% of unselected colorectal tumours that do demonstrate defective MMR, the MSI-H^2,5 phenotype is primarily due to hypermethylation of the MLH1 gene promoter,^15,16 and not to germline mutations. Thus, it would be desirable to exclude this latter group from further MMR gene testing. However, the age at onset of colorectal cancer in HNPCC might be older than previously thought and further, some HNPCC patients have minimal family history.^1,17 These data suggest that testing of MSI in all colorectal tumours might be considered when population based screenings are performed or whenever possible.

Recently, an oncogenic V600E (previously known as V599E) hotspot mutation in BRAF, a kinase encoding gene from the RAS/RAF/MAPK pathway, has been found in colorectal tumours that show MMR deficiency.^18–20 Further, it has been shown that these mutations occur almost exclusively in tumours located in the proximal colon and with hypermethylation of MLH1, the gene involved in the initial steps of development of these tumours.^21,22 However, in more detailed analyses, BRAF mutations were not detected in those cases with or presumed to have a germline mutation in either MLH1 or MSH2.^21,23 Because of these findings, we have further investigated the role of the BRAF mutation V600E in HNPCC cases harbouring known pathogenic mutations in MLH1 or MSH2. Furthermore, we have investigated whether analysis of the V600E mutation might be used as a primary marker to obviate further genetic testing of MLH1 and MSH2 in HNPCC diagnostics.

METHODS

Tumour specimens

Colon cancers were obtained from the Hospital Universitari Vall d’Hebron (Barcelona, Spain), Groningen University Hospital (Groningen, The Netherlands), Mayo Clinic (Rochester, MN, US), Sapporo Medical University (Sapporo, Japan), Hospital of S. João (Porto, Portugal), Saint-Antoine Hospital (Paris, France), and also from several different hospitals in Finland and Sweden. Sample collection was carried out in accordance with the previously established ethical protocols from each of the participating institutions, and the respective ethics committees approved the study. Collected tumours were either paraffin embedded or immediately frozen in liquid nitrogen for further analyses.

Sporadic colon tumours and MSI analysis

A total of 206 sporadic colorectal tumours with MSI from the available tumour collections were selected for this study. Genomic DNA was extracted with phenol-chloroform according to standard procedures. Further, tumours were analysed for MSI according to the international criteria for the determination of MSI, using various panels of dinucleotide and mononucleotide repeat sequences.^24,25 All tumours were classified as MSI-H and showed instability in at least 40% of the micro satellites analysed.²⁴ None of them had a family history suggestive of HNPCC.

HNPCC tumour samples and germline mutation analysis of MLH1 and MSH2

Extensive series of tumours from previously diagnosed HNPCC patients were analysed for germline mutations in the MLH1 and MSH2 genes in each of the institutions involved in this study. DNA was isolated as described above. Analyses were performed using several laboratory routines and mutations verified by automatic sequencing. In some cases, large deletions of MLH1 and MSH2 were detected by Southern blotting or alternative strategies.^26–28 A total of 111 HNPCC tumour cases which showed germline mutations of MLH1 or MSH2 were selected for this study. In addition, 45 cases showing abnormal immunostaining for MSH2 were also analysed. Immunohistochemistry for MSH2 was performed as previously described.²⁹ Although the germline mutation status of these cases was not determined, current available data strongly suggest that these cases are very likely to contain germline mutations in MSH2. These latter cases were referred for tumour analysis (MSI and IHC) because of either a positive family history or early onset of colon cancer.

Mutational analysis of the BRAF-V600E hotspot

Analysis of BRAF was performed by automatic sequencing. The fragment encompassing exon 15 was amplified by PCR in all carcinoma samples. Primer sequences and PCR conditions were based on those reported previously.¹⁹ Genomic DNA (25–100 ng) was amplified by PCR using the following cycling conditions: 30 s at 94°C, 30 s at 60°C, and 45 s at 72°C for 35 cycles. PCR products were purified and sequenced on an ABI Prism 377 Automatic sequencer (Perkin-Elmer, Foster City, CA) using the ABI Prism Dye Terminator Cycle Sequencing Kit (Perkin-Elmer).

RESULTS AND DISCUSSION

Overall, 77 tumours were obtained from a total of 67 HNPCC families showing 37 different germline mutations in MLH1 and 34 tumours accounting for 30 different germline MSH2 mutations were obtained from 33 HNPCC families (table 1). We found the V600E mutation of BRAF in 40% (82/206) of sporadic colon cancer cases showing MSI, which is in agreement with previous reports.^19,20 However, the V600E mutation was not detected in any of the 111 HNPCC tumours tested nor in any of the 45 additional analysed tumours showing abnormal IHC for MSH2 (table 1), indicating that this hotspot mutation is very rarely if ever involved in the tumourigenesis of colorectal cancer linked to HNPCC (p<0.001). In contrast to the commonly accepted concept that molecular pathways and genes involved in MSI are identical in HNPCC and sporadic colorectal tumours, these results suggest that genotypic differences might distinguish sporadic and hereditary MMR defective colorectal cancers, although the molecular explanation for such a difference is still to be unravelled.

Accordingly, future analysis of the BRAF-V600E mutation as part of the diagnostic protocols for HNPCC is an encouraging perspective. After completion of our study, however, two BRAF mutations have been reported in two mismatch repair deficient colon tumours showing germline alterations in MLH1 and MSH2.²¹ Nonetheless, further analyses of these cases showed that the BRAF mutation seen in the MSH2 tumour was a frameshift mutation caused by an insertion of an adenine within a repetitive sequence of five adenines and not V600E. In fact, this mutation generates a truncated protein and is most likely a background mutation of the underlying MMR defect. In addition, as stated by the authors, the alteration detected in the MLH1 case was likely to be a non-pathogenic variant.²¹ Furthermore, previous reports have shown that the BRAF-V600E mutation is detected in sporadic colorectal tumours with MSI, but only in those cases characterised by promoter hypermethylation of the MLH1 gene,^21,22 suggesting that mechanisms other than inactivation of the mismatch repair system itself might be modulating the tumourigenic advantage capabilities of the V600E mutation. These data are also in agreement with a recent report by Deng et al suggesting that BRAF might be used in a strategy to detect HNPCC families.²³

Accordingly, our data suggest that if the V600E mutation is detected in a colorectal MSI-H tumour, further testing for germline mutations in MLH1 or MSH2 should be considered only if other significant findings point towards MMR associated HNPCC, such as fulfilment of the strict Amsterdam criteria. Such caution is necessary because colorectal cancer is a common disease and some cases seen in HNPCC families must arise through pathways not involving germline mutations in the MMR pathway. Furthermore, it is not clear yet whether other DNA mismatch repair genes associated with HNPCC, such as hMSH6, will still need to be testing if BRAF-V600E is detected. In this context, the combination of MSI and IHC analyses provides valuable information. Current data suggest that nearly all HNPCC cases involving MSH2 and MSH6 arise as a consequence of a germline mutation. Thus, identification of these cases by a combination of MSI and IHC is relatively straightforward and highly predictive of a germline alteration. Therefore, germline testing of MSH2 and MSH6 should be considered if IHC demonstrates the involvement of these genes, regardless of other risk factors (fig 1). On the other hand, because epigenetic inactivation of MLH1 is a common event in approximately 12% of all colon cancers, the predictive value of the absence of protein expression for MLH1 determined by IHC is not perfect. Although IHC for MLH1 is important for identifying the involvement of this gene, the addition of BRAF testing might provide important predictive information. Therefore, if IHC suggests the involvement of MLH1, then the presence of BRAF-V600E suggests that the defective MMR in the tumour is the result of a somatic alteration.

On the other hand, testing of unselected cohorts of colorectal tumours for MSI is currently not recommended by international criteria. Reyes et al¹⁴ have recently shown that germline testing of individuals who meet the Amsterdam criteria only, results in the detection of the fewest gene carriers at the lowest cost, whereas MSI testing of all colorectal patients, although having the highest cost, allows the detection of most gene carriers. In this context, application of the Bethesda guidelines (selection of high risk individuals for MSI analysis) represents a mixed strategy in terms of costs and sensitivity for HNPCC diagnostics. Nonetheless, calculations from available data^13,30,31 reveal that the number of candidate patients to be analysed for MSI and MMR genes could be five times higher if unselected cohorts of colorectal tumours are considered (table 2). Also, the number of newly HNPCC diagnosed cases is doubled when unselected cohorts of tumours are used instead of only those meeting the Bethesda guidelines. Further, it has been recently shown that MSI is a predictive factor of tumour response to 5-fluoruracil and irinotecan drug based chemotherapy in colorectal cancer, suggesting a future use of MSI status for directing colorectal cancer patients towards specific and more effective treatments.^32,33 These data suggest that MSI testing should be considered for all colorectal cancers and question the effectiveness of limiting genetic MSI testing only to individuals who fulfil the Bethesda guidelines, even though the main reason for not applying wide MSI testing strategies is the prohibitive cost. However, when unselected cohorts of tumours are analysed for MSI, family history of cancer must be included in the screening protocols for familial cancer because some colorectal cancer families do not show MSI-H. Therefore, counselling and surveillance has to be offered to those patients with a positive family history even if MSI-H is not detected.

Further, utilisation of the BRAF-V600E mutation status might improve the cost-effectiveness of the actual genetic tests for HNPCC^13,14 (table 2) and we therefore suggest including BRAF screening as a step prior to germline testing of MLH1 in colorectal MSI-H tumours. Further, we also suggest using the same strategy for MSH2 if no immunohistochemistry analysis can be performed previously (fig 1). Accordingly, introduction of the BRAF-V600E test together with IHC analysis of the MMR genes in a decision tree for testing MLH1 and MSH2 in these tumours might provide a reliable, fast, and low cost strategy that will help to simplify the current protocols for HNPCC genetic testing.