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Human Molecular Genetics Advance Access originally published online on August 4, 2004
Human Molecular Genetics 2004 13(19):2303-2311; doi:10.1093/hmg/ddh238
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Human Molecular Genetics, Vol. 13, No. 19 © Oxford University Press 2004; all rights reserved

Distinct patterns of KRAS mutations in colorectal carcinomas according to germline mismatch repair defects and hMLH1 methylation status

Carla Oliveira1, Jantine L. Westra2, Diego Arango3, Miina Ollikainen4, Enric Domingo5, Ana Ferreira1, Sérgia Velho1, Renee Niessen2, Kristina Lagerstedt6, Pia Alhopuro3, Paivi Laiho3, Isabel Veiga7, Manuel R. Teixeira7, Marjolijn Ligtenberg8, Jan H. Kleibeuker9, Rolf H. Sijmons2, John T. Plukker10, Kohzoh Imai11, Pedro Lage12, Richard Hamelin14, Cristina Albuquerque13, Simo Schwartz, Jr5, Annika Lindblom6, Päivi Peltomaki4, Hiroyuki Yamamoto9, Lauri A. Aaltonen3, Raquel Seruca1,* and Robert M.W. Hofstra2

1Institute of Molecular Pathology and Immunology, The University of Porto, IPATIMUP, 4200-465 Porto, Portugal, 2Department of Medical Genetics, University of Groningen, 9713 AW Groningen, The Netherlands, 3Department of Medical Genetics, Haartman Institute, 4Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, 00014 Helsinki, Finland, 5Centre d'Investigacions en Bioquimica i Biologia Molecular (CIBBIM), Hospital Universitari Vall d'Hebron, Barcelona 08035, Spain, 6Department of Clinical Genetics, Karolinska University Hospital, S 171 76 Stockholm, Sweden, 7Department of Genetics, Portuguese Institute of Oncology (IPO), 4200-072 Porto, Portugal, 8Department of Human Genetics, UMC Nijmegen, 6500 HB Nijmegen, The Netherlands, 9Department of Gastroenterology, and 10Department of Surgery, University Hospital Groningen, 9713 AW Groningen, The Netherlands, 11First Department of Internal Medicine, Sapporo Medical University, Sapporo 060-8543, Japan and 12Serviço de Gastroenterologia, and 13Centro de Investigacao de Patobiologia Molecular-CIPM, Instituto Portugues de Oncologia Francisco Gentil, 1093 Lisbon, Portugal and 14INSERM U434 CEPH, 75010 Paris, France

Received May 17, 2004; Accepted July 22, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In sporadic colorectal tumours the BRAFV600E is associated with microsatellite instability (MSI-H) and inversely associated to KRAS mutations. Tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients carrying germline mutations in hMSH2 or hMLH1 do not show BRAFV600E, however no consistent data exist regarding KRAS mutation frequency and spectrum in HNPCC tumours. We investigated KRAS in 158 HNPCC tumours from patients with germline hMLH1, hMSH2 or hMSH6 mutations, 166 MSI-H and 688 microsatellite stable (MSS) sporadic carcinomas. All tumours were characterized for MSI and 81 of 166 sporadic MSI-H colorectal cancer (CRCs) were analysed for hMLH1 promoter hypermethylation. KRAS mutations were observed in 40% of HNPCC tumours, and the mutation frequency varied upon the mismatch repair gene affected: 48% (29/61) in hMSH2, 32% (29/91) in hMLH1 and 83% (5/6) in hMSH6 (P=0.01). KRAS mutation frequency was different between HNPCC, MSS and MSI-H CRCs (P=0.002), and MSI-H with hMLH1 hypermethylation (P=0.005). Furthermore, HNPCC CRCs had more G13D mutations than MSS (P<0.0001), MSI-H (P=0.02) or MSI-H tumours with hMLH1 hypermethylation (P=0.03). HNPCC colorectal and sporadic MSI-H tumours without hMLH1 hypermethylation shared similar KRAS mutation frequency, in particular G13D. In conclusion, we show that depending on the genetic/epigenetic mechanism leading to MSI-H, the outcome in terms of oncogenic activation may be different, reinforcing the idea that HNPCC, sporadic MSI-H (depending on the hMLH1 status) and MSS CRCs, may target distinct kinases within the RAS/RAF/MAPK pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hereditary non-polyposis colorectal cancer (HNPCC) accounts for ~1–8% of total colorectal cancer (CRC) burden on the basis of clinical criteria (1–4). When considering HNPCC as a syndrome linked to mismatch repair (MMR) mutations, the frequency range from 0.3% to 3% of the total CRC burden (5–7). Germline mutations in a number of MMR genes have been identified in patients with HNPCC. To date, germline mutations in the hMLH1 and hMSH2 genes account for about 90% of all identified MMR gene mutations. hMSH6 mutations are also found but the number of cases is considerably lower (10%) when compared with hMLH1 (~50%) and hMSH2 (~40%) (4,8). In particular tumours of patients with germline hMLH1 or hMSH2 mutations frequently show microsatellite instability (MSI-H) (5,9,10,11) (International Collaborative Group on HNPCC web site, available at http://www.nfdht.nl). The MSI-H phenotype is also found in ~15% of sporadic CRC. In contrast to the CRCs from HNPCC patients, the MMR deficiency seen in these tumours is not caused by (germline) mutations in the MMR genes but is due to hMLH1 promoter hypermethylation resulting in loss of the MLH1 protein (12). In MSI-H colorectal tumours, both sporadic and inherited forms, hundreds of thousands of mutations accumulate within repetitive sequences throughout the genome (13). These mutations occur not only in non-coding but also in coding sequences. As a consequence, genes involved in several signalling pathways, such as the WNT/APC/beta-catenin pathway and the TGFbeta pathway, are also subjected to mutation. The genes in these pathways encode proteins which play a key role in the development of MMR deficient and proficient CRCs [microsatellite stable (MSS) CRCs] (14).

Although genes with repetitive sequences are clear targets in tumours with a defective MMR system, mutations in non-repetitive sequences are also found in MSI-H tumours. An example of this is the occurrence of the V600E mutation in BRAF (15,16). BRAF is a member of the RAS/RAF/MAPK pathway, a crucial pathway in colorectal tumourigenesis (17,18). The BRAFV600E has been described in ~35% of sporadic MSI-H CRCs (16,19–24) and was found associated with hMLH1 promoter hypermethylation (21,22,25,26). BRAFV600E is, however, not restricted to MSI-H tumours as it also occurs in ~6% of CRCs without MMR deficiency (MSS CRCs) (16,19–24). KRAS another member of this signalling pathway is mutated in ~20% of sporadic MSI-H and 35% of MSS CRCs negative for the BRAFV600E mutation (16,20,21,24,27,28). These data demonstrate that point mutations in non-repetitive sequences occur frequently in both mismatch proficient and deficient tumours, but also that this pathway plays a pivotal role in the tumourigenesis of both types of sporadic CRCs. Furthermore, it was shown that mutations in both KRAS and BRAF in the same tumour sample are rarely found suggesting that the oncogenic capabilities of both genes are mutually exclusive in terms of clonal selection (16,20,21,24,27–29). Recently, it was found that tumours from HNPCC patients with germline mutations in hMSH2 or hMLH1 do not show BRAFV600E (22,26,30). Whether the absence of the BRAFV600E mutation in MSI-H CRC tumours from HNPCC patients is due to the presence of a KRAS mutations is not known as the occurrence of KRAS mutations in HNPCC CRCs is not well established.

The frequency of KRAS mutations in HNPCC families, fulfilling the Amsterdam criteria, was first determined by Aaltonen et al. (31), who found that KRAS mutations occur at similar frequency in familial and sporadic CRCs. The same findings were described latter in two other studies [11/37 (30%); 2/8 (25%)] (27,32). However other authors did not confirm this observation [4/23 (17%) in codon 12 (33), 2/20 (10%) in codon 12 (34) and 1/20 (5%) (28)]. In most of these studies, however, small numbers of HNPCC carcinomas were included and, in most of them, no demonstration of a hereditary defect in HNPCC patients was made. Moreover, it is not clear whether a specific spectrum of KRAS mutations might characterize tumours according to the presence of a germline underlying MMR defect and to the presence or absence of hMLH1 hypermethylation in sporadic MSI-H cases.

In the present study, we therefore evaluated KRAS mutations in a large series of HNPCC colon carcinomas of patients which were previously characterized for germline mutations in MMR genes, and in a series of sporadic CRCs characterized for the MSI status and hMLH1 promoter methylation. With this work, we aimed to verify whether a specific spectrum of KRAS mutations might characterize tumours according to the presence of a germline underlying MMR defect and to the presence or absence of hMLH1 promoter hypermethylation in sporadic MSI-H cases. In addition, we compared the frequency of KRAS mutations with the frequency of BRAFV600E in HNPCC, MSI-H and MSS sporadic carcinomas [data from the literature and from our collaborative group (16,19–24,26–30)] in order to clarify the importance of BRAF and KRAS activation in the development of all sets of colon carcinomas.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Frequency of KRAS mutations in HNPCC sporadic MSI-H and MSS CRCs
KRAS mutations in codons 12, 13 and 61 were determined in 1012 primary CRCs including 158 tumours from HNPCC patients harbouring pathogenic germline mutations in MMR genes, 166 sporadic MSI-H tumours and 688 sporadic MSS tumours. KRAS mutations were observed in 33% (335/1012) of all colorectal tumours. A higher frequency of KRAS mutations was detected in HNPCC tumours (40%) in comparison with sporadic CRCs (32%), although this difference did not reach statistical significance. However, when sporadic CRCs were divided according to the MSI status, KRAS mutations were significantly more frequent in HNPCC tumours than that in sporadic MSI-H CRCs (22%) (P=0.0006) but similar to MSS (34%) CRCs (P=0.17) (Table 1, Fig. 1).


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  		Table 1. KRAS mutations in HNPCC and sporadic MSI-H (MMR deficient) and sporadic MSS (MMR proficient) colorectal tumours
 


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  		Figure 1. Frequency and spectrum of KRAS mutations in CRCs of three different groups. (A) Sporadic MSS. (B) Sporadic microsatellite unstable tumours (MSI-H). (B1) Subgroup of sporadic microsatellite unstable tumours with hMLH1 promoter methylation. (B2) Subgroup of sporadic microsatellite unstable tumours without hMLH1 promoter methylation. (C) Tumours from HNPCC patients with MMR gene germline mutations in hMSH2, hMSH6 or hMLH1.

 
Out of the 166 sporadic MSI-H CRCs screened for KRAS mutations 81 were also analysed for hMLH1 promoter methylation. hMLH1 hypermethylation was seen in 50 (62%) cases hMLH1 hypermethylated tumours showed statistically significantly lower frequency of KRAS mutations when compared with tumours from HNPCC patients (P=0.005), but no differences were found when MSI-H tumours without promoter hypermethylation were compared with HNPCC CRCs (P=0.65) (Table 1, Fig. 1).

From the 158 HNPCC patients with germline pathogenic alterations (single nucleotide or deletions) in one of the MMR genes, 61 showed mutations in hMSH2 (39%), six in hMSH6 (4%) and 91 in hMLH1 (58%). Within HNPCC CRCs, the frequency of KRAS mutations varied depending on the MMR gene affected (P=0.01). CRCs from patients carrying germline mutations in hMSH2 or hMSH6 harboured a higher frequency of KRAS mutations in comparison with CRCs from patients carrying germline mutations in hMLH1 (P=0.02) (Table 1). No differences were found between the type of germline MMR gene defect and frequency of KRAS mutations in HNPCC tumours. Of 158 HNPCC families 63 were harboured distinct MMR germline mutations. In these 63 families, no association was found between the type of germline defect and the frequency or type of KRAS mutation (Table 2). All the other families included in this study harboured 21 different germline MMR mutations, every germline mutation was found in at least two families. Within every group of families sharing a specific germline mutation, no association was found between the frequency and type of KRAS mutation. For instance, in the 27 families with the hMLH1 founder mutation 1 (genomic deletion of exon 16 and flanking introns) four different types of KRAS mutations (G12A, G12D, G12V and G13D) were found.


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  		Table 2. KRAS mutation frequency and amino acid change of HNPCC colorectal tumours from families carrying distinct MMR germline mutations
 
Frequency of KRAS mutations in codon 12, 13 and 61 in HNPCC, sporadic MSI-H and MSS CRCs
From the 335 colorectal tumours, both HNPCC and sporadic tumours, harbouring KRAS mutations, we found that codon 12 was mutated in 77% (258/335), codon 13 in 21% (69/335) and codon 61 in only 2% (8/335) of the cases.

Frequencies of KRAS mutations in codons 12, 13 and 61 differ significantly between HNPCC, sporadic MSI-H and MSS tumours (P=0.0001) (Table 3, Fig. 1).


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  		Table 3. Frequency of KRAS mutations in codons 12, 13 and 61 in CRCs
 
Codon 13 was significantly more mutated in HNPCC tumours when compared with sporadic MSI-H or MSS CRCs (HNPCC versus MSI-H: P=0.004 and HNPCC versus MSS: P=0.0001). Within HNPCC CRCs, the frequency of KRAS mutations in codons 12 or 13 did not vary depending on the MMR gene affected (P=0.23). Codon 61 was never mutated in our series of HNPCC tumours and rarely in the sporadic CRCs [MSI-H: 5% (n=2) and MSS: 3% (n=6), Table 1, Fig. 1].

Of the 37 sporadic MSI-H CRCs harbouring KRAS mutations 20 were analysed for hMLH1 promoter methylation. Of the four MSI-H tumours without hMLH1 promoter methylation three proved to have a codon 13 mutation, whereas codon 12 was affected similarly in MSI-H tumours independent of the hMLH1 promoter methylation status (hMLH1met–: 8/16 and hMLH1met+: 8/16).

KRAS nucleotide substitution in HNPCC, sporadic MSI-H and MSS CRCs
The frequency of nucleotide substitutions varied significantly between HNPCC, MSI-H and MSS CRCs. The frequency of guanine:thymidine, guanine:cytosine and guanine:adenine nucleotide substitutions was significantly different between HNPCC and MSS sporadic cases (P=0.0001) but not between HNPCC and MSI-H cases (P=0.27) (Fig. 1).

KRAS amino acid change in HNPCC, sporadic MSI-H and MSS CRCs
The spectrum of amino acid changes in KRAS codons 12, 13 and 61 was distinct in the three groups of CRCs. MSS CRCs showed a wider range of amino acid changes in comparison with MSI-H and HNPCC CRCs. The amino acid changes G12D, G12V, G12A and G13D occurred in all three CRC groups. The amino acid changes G12C (9%), G12S (5%), G12R (1%), G13R (0.4%), Q61K (0.4%) and K61L (0.4%) were found only in MSS CRCs. The Q61H amino acid change was present in 2% of MSS and 1% of MSI-H CRCs (Fig. 1).

The frequency of KRAS amino acid changes varied significantly between HNPCC, MSI-H and MSS CRCs. The frequency of amino acid changes glycine to valine (G12V) or glycine to alanine (G12A) in codon 12 was significantly different between HNPCC and sporadic MSS cases (G12V: P=0.004 and G12A: P=0.0007), but not between HNPCC and sporadic MSI-H cases. The same was true when we compared the frequency of the amino acid change glycine to aspartic acid in both codon 12 and 13 between HNPCC and MSS sporadic cases (G12D+G13D: P=0.0005), but not between HNPCC and MSI-H cases. However, the frequency of the amino acid change glycine to aspartic acid in codon 12 and codon 13, when analysed separately, was significantly different in HNPCC in comparison with sporadic MSI-H (G12D, HNPCC versus MSI-H: P=0.02 and G13D, HNPCC versus MSI-H: P=0.004) (Table 4, Fig. 1).


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  		Table 4. KRAS nucleotide substitution and amino acid change in HNPCC, sporadic MSI-H and MSS CRCs
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Frequency of KRAS mutations in HNPCC, sporadic MSI-H and MSS CRCs
Activating KRAS mutations represents the most common type of abnormality of a dominant oncogene in human cancer, with specificity and type of mutation varying in relation to tumour type (35). It is widely accepted that mutations in KRAS are among the critical transforming alterations occurring during colorectal tumourigenesis and occur early in the progression from adenoma to carcinoma (18). In this report, KRAS mutations were identified in 33% of all CRCs. This is consistent with previous studies that, with rare exceptions, have identified KRAS mutations in ~30–40% of CRC (36–38).

In agreement to previous reports we also found that tumours from sporadic CRC patients with an MSS phenotype show higher frequency of KRAS mutations than MSI-H CRC tumours (13,27,28,33,38–40). In contrast, other studies have reported similar frequencies of KRAS mutations in MSI-H and MSS CRCs (31,32,41).

We now report that KRAS is frequently mutated in tumours from HNPCC patients with germline MMR gene mutations. Furthermore, KRAS mutation frequency varies among HNPCC tumours depending on the MMR gene, which carried the germline mutation. The frequency of KRAS mutations is lower in tumours from patients with an hMLH1 germline mutation when compared with tumours from patients carrying either hMSH2 or hMSH6 germline mutations. In fact, tumours from patients with germline mutations in hMSH6 harbour the highest frequency of KRAS mutations (it should, however, be noted that only six tumours from hMSH6 HNPCC patients were included). This finding seems consistent with the role fulfilled by the MSH2:MSH6 complex (hMutS{alpha}) in DNA repair (8,42). As stated earlier, our data show different frequencies of KRAS mutations in tumours of patients with different MMR genes. It remains to be clarified whether or not this finding is an explanation for the apparently different clinical phenotypes seen in HNPCC families carrying mutations in specific MMR genes (43).

While HNPCC tumours did not differ from MSS sporadic CRCs regarding the frequency of KRAS mutations, they did differ significantly from sporadic MSI-H carcinomas. Our data are in accordance to previously reported findings (27) in a series of 24 HNPCC tumours with germline mutations in hMLH1 or hMSH2. These authors found KRAS mutations in 29% (7/24) of HNPCC tumours in comparison with 18% sporadic MSI-H (n=18) (27).

Our data and others show that sporadic MSI-H CRCs are mainly characterized by a relative low frequency mutation in KRAS when compared with HNPCC and MSS CRCs (27,28,40,44,45). However, when we compare HNPCC with sporadic MSI-H tumours, classified according to the methylation status of the hMLH1 promoter, differences were observed in KRAS mutation frequency. In MSI-H tumours, negative for hMLH1 promoter methylation, the frequency of KRAS mutations was similar to the frequency observed in HNPCC (Table 1). This subset of sporadic MSI-H may be explained by somatic mutations in MMR genes or gene loss, mimicking the inactivating events occurring in the hereditary counterpart (46,47). On the other hand, sporadic MSI-H colon carcinomas with hypermethylation of the hMLH1 promoter region showed the lowest frequency of KRAS mutations of all subsets of colon carcinomas. Similarly, preneoplastic lesions of the colon which show hMLH1 promoter hypermethylation or concurrent methylation also harbour low frequency of KRAS mutations (29). The majority of MSI-H CRCs with hMLH1 promoter hypermethylation are frequently mutated on the V600E hotspot of BRAF (71%), one of the downstream targets of KRAS (21,22,25,26). This finding and our data suggest that colon tumours progress by targeting distinct genes of the RAS/RAF/MAPkinase pathway, depending on the genetic/epigenetic event underlying MMR deficiency (mutation and loss versus hMLH1 promoter methylation). MSI-H tumours with MMR gene mutations (hereditary and sporadic forms) may preferentially target KRAS, whereas MSI-H tumours with hMLH1 methylation may preferentially target the BRAF gene (as depicted in Fig. 2). This assumption is in accordance to the absence of BRAF V600E mutations in HNPCC colorectal tumours (22,26,30).



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  		Figure 2. Summary of BRAFV600E and KRAS mutation frequency and type significantly associated with the distinct groups of CRCs. Grey (BRAF data) represents a summary of published data (16,19–24,26–30) and black (KRAS data) summarizes the results reported in this manuscript.

 
Codon, nucleotide and amino acid changes in KRAS in HNPCC, sporadic MSI-H and MSS CRCs
KRAS point mutations cluster at the guanosine triphosphate (GTP)-binding domain (codon 12/13) or at the GTPase domain (codon 61) and determine specific amino acid substitutions that lead to permanent activation of the p21 ras protein (35,48). In our series of 335 CRCs harbouring KRAS mutations, 98% carry mutations in codons 12 and 13, confirming that these two codons are preferentially affected in comparison with codon 61, as previously observed (35,48,49).

Several observations can be made concerning the frequency and type of KRAS mutations in codon 12 and 13 in sporadic MSS, sporadic MSI-H and HNPCC CRCs. The frequency of KRAS mutations in codon 12 or 13 significantly differs between sporadic and hereditary settings: KRAS mutations in codon 12 were more common in sporadic cases (overall MSI-H and MSS), whereas mutations in codon 13 were predominant in HNPCC. Moreover, within HNPCC the underlying MMR gene defect did not influence the position or nucleotide substitution of KRAS. Previously, an association was reported between the location of KRAS mutations and the underlying MMR gene involved (27). In contrast to our report, these showed that codon 13 was never affected in HNPCC tumours from patients with hMSH2 germline mutations (0/17), whereas codon 13 KRAS mutations were present in three out of seven cases in HNPCC tumours from patients with hMLH1 germline mutations (27). The low number of cases analysed in the aforementioned study may explain these differences (27).

Animal models have shown an association between chemical exposure and location and type of RAS activating mutations in resultant tumours (48). A good example to illustrate the presence of codon 12 mutations in sporadic carcinomas and its association with environmental factors is the predominance of codon 12 KRAS mutations in environmental associated carcinomas, such as lung and colon cancer associated with tobacco smoking and bladder cancer (50–54). Accordingly, to explain the differences found between the location of the mutation in sporadic and HNPCC tumours we hypothesise that sporadic carcinomas may be associated with an increased susceptibility to damage in specific DNA sequences by environmental factors, whereas hereditary tumours occur due to inherited predisposition.

Further, G:A transitions were the most common alterations found in CRCs. In addition, the frequency of G:A transitions was higher in MMR deficient tumours (HNPCC and sporadic MSI-H), than that in MSS colon carcinomas. This finding is in agreement with previous studies which showed that G:A alterations are particularly difficult to repair, even within a normal MMR background (55). Moreover, our data showed that G:T substitutions are far more frequent in MSS colon carcinomas than that in HNPCC or MSI-H tumours. Accordingly, the type of KRAS amino acid substitution varies among the three different groups of colon carcinomas analysed. The frequency of G12V, resulting from a G:T nucleotide substitution, was significantly higher in MSS sporadic than in HNPCC or sporadic MSI-H cases. In a large study of sporadic colon carcinomas, G12V was identified in 24% of all KRAS positive cases (49). The frequency of G13D, resulting from a G:A nucleotide substitution, was significantly higher in the group of HNPCC in comparison with sporadic carcinomas (MSI-H and MSS) in accordance with a previous report which showed that G13D was the most common type of KRAS mutation (55%) in HNPCC (27). The type of KRAS amino acid substitutions was shown to have different impacts on the outcome of colon cancer patients. Clinically, G12V mutations were associated with a 30% increased risk of recurrence or death (49). Whether or not the better prognosis of HNPCC and sporadic MSI-H patients is related to the low frequency of G12V mutations remains to be clarified.

In conclusion, HNPCC colorectal and sporadic MSI-H tumours without hMLH1 hypermethylation shared similar KRAS mutation frequency, in particular G13D. Although MSI-H colon carcinomas with methylation of hMLH1 show the lowest frequency of KRAS mutations of all subsets of colon carcinomas, they harbour the highest frequency of BRAF mutations. Further, we show that depending on the genetic/epigenetic mechanism leading to MSI-H, the outcome in terms of oncogenic activation may be different, reinforcing the idea that HNPCC, sporadic MSI-H (depending on the hMLH1 status) and MSS CRCs may target distinct kinases within the RAS/RAF/MAPK pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Tumour specimens
Tumour DNA samples were obtained from 1012 CRCs, originating from 158 HNPCC patients with pathogenic MMR germline mutations and 854 sporadic cases (MSI-H 166, MSS 688). A family history was obtained in every case and none of the patients reported in this study, as sporadic, had a family history suggestive of HNPCC. Samples were obtained from University Hospital, Groningen and University Medical Centre Nijmegen (The Netherlands), Hospital of S. João- Porto, IPO-Porto and IPO-Lisbon (Portugal), Saint-Antoine Hospital, Paris, (France), University of Helsinki, Helsinki (Finland), Sapporo Medical University, Sapporo (Japan), Karolinska University Hospital, Stockholm (Sweden) and from Hospital Universitari Vall d'Hebron, Barcelona (Spain). The study protocol was reviewed and approved by the appropriate Ethics Committees, and tumour samples were obtained with informed consent. All HNPCC families selected for this study have been previously characterized for the presence of germline MMR gene mutations and showed a germline mutation in hMSH2, hMSH6 or hMLH1. Only HNPCC patients, for whom the mutations were proven pathogenic, independent of the mutation type, were included. HNPCC families without germline mutations of hMSH2, hMSH6 or hMLH1 were not included in this study. Genomic DNA was isolated from macro-dissected frozen or paraffin-embedded tumour tissue using standard methods.

MSI-H analysis
All colorectal tumours from HNPCC patients and sporadic cases were analysed for MSI-H according to the international criteria for the determination of MSI-H, using various panels of dinucleotide and mononucleotide repeat sequences (12). Tumours were classified as MSI-H when two of the five standard markers (56) or when more than 30% of the total number of markers analysed showed instability, otherwise were classified as MSS (12). MSI-H sporadic carcinomas were not investigated for the presence of somatic MMR gene mutations.

Mutation analysis of KRAS gene
Mutational analysis of KRAS codons 12, 13 and 61 was performed by SSCP/DGGE and/or sequencing. The KRAS mutation screening was done according to the methods of analysis in the different collaborative centres.

Analysis of hMLH1 methylation
The DNA methylation status of the hMLH1 promoter region was partly determined as described previously (57,58). Briefly, samples were analysed by methylation-specific PCR on bisulphite treated DNA. The primers used for this analysis were: 5'-GAA GAG TGG ATA GTG ATT TTT AAT GT-3' and 5'-ATC TCT TCA TCC CTC CCT AAA ACA-3' for unmethylated hMLH1 and 5'-AGC GGA TAG CGA TTT TTA ACG C-3' and 5'-TCT TCG TCC CTC CCT AAA ACG-3' for methylated hMLH1 (57,58).

The other samples, all from IPATIMUP, were analysed according to previously described methods (59). In short, samples were amplified after treatment with bisulphite, using primers flanking the CpG sites in hMLH1: 5'-GTT AGA TAT TTT AGT AGA GGT ATA TAA GT-3' and 5'-ACC TTC AAC CAA TCA CCT CAA TA-3'. PCR products were digested by BstUI (New England Biolabs). This enzyme cleaves only the CGCG sequence, which is not converted by bisulphite treatment when methylated.

Statistical analysis
The statistical analysis was performed using the {chi}2 test or Fisher statistical test when appropriated. Differences were taken to be significant at P<0.05.


   ACKNOWLEDGEMENTS
 
The authors would like to thank the following agencies for grant support. FCT-Portugal: project POCTI/35374/CBO/2000; Dutch Cancer Society: RUG99-1962/RUG2002-2678; Academy of Finland, Finnish Cancer Foundation, Sigrid Juselius Foundation; Spanish Fondo de Investigaciones Sanitarias: grant 01/1350; Center of Excellence in Disease Genetics of the Academy of Finland: project number 44870; Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and from the Ministry of Health, Labor and Welfare of Japan and Association pour la Recherche sur le Cancer: grant 4448.


   FOOTNOTES
 
* To whom correspondence should be address at: IPATIMUP, Rua Roberto Frias S/N, 4200-465 Porto, Portugal. Tel: +351 225570700; Fax: +351 225570799; Email: rseruca{at}ipatimup.pt


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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