No evidence for dual role of loss of heterozygosity in ... - Nature

Oncogene (2007) 26, 2513–2517

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No evidence for dual role of loss of heterozygosity in hereditary non-polyposis colorectal cancer S Tuupanen1, A Karhu1, H Ja¨rvinen2, JP Mecklin3, V Launonen1 and LA Aaltonen1 1 Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland; 2The Second Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland and 3The Department of Surgery, Jyva¨skyla¨ Central Hospital, Jyva¨skyla¨, Finland

Hereditary non-polyposis colorectal cancer (HNPCC) is caused by germline mutations in mismatch repair (MMR) genes, mostly MLH1 and MSH2. Somatic inactivation of the wild-type allele of the respective MMR gene is required for tumor development. Unexpectedly, a recent study utilizing DNA from paraffin-embedded tissue material detected frequent loss of the mutant MMR gene allele in HNPCC tumors. Dual role for loss of heterozygosity (LOH) was proposed. If somatic loss of the wild-type MMR gene allele had occurred through point mutation or promoter hypermethylation, frequent somatic deletions at the region of the MMR gene locus, perhaps targeting other relevant cancer genes, could quite commonly lead to loss of the mutant allele. To test this hypothesis, we studied a population-based series of 25 fresh-frozen HNPCC tumors with a germline mutation in MLH1 or MSH2 for LOH. Fourteen of the 25 tumors (56%) showed LOH at the respective locus, and all 14 losses targeted the wild-type allele (P ¼ 0.00006). These results strongly support the traditional two-hit model of HNPCC gene inactivation. Oncogene (2007) 26, 2513–2517. doi:10.1038/sj.onc.1210038; published online 9 October 2006 Keywords: allele; LOH; HNPCC; MMR; tumor

Hereditary non-polyposis colorectal cancer (HNPCC) is a dominantly inherited cancer predisposition syndrome that is caused by germline mutations in mismatch repair (MMR) genes. The majority of the HNPCC patients have mutations in MLH1 or MSH2 genes (Peltoma¨ki and Vasen, 2004). MMR genes are considered as caretaker genes, and inactivation of both alleles is believed to be required for tumor development (Parsons et al., 1993; Hemminki et al., 1994). Deficiency in MMR machinery creates genomic instability that can be observed as frequently mutated microsatellite sequences Correspondence: Professor LA Aaltonen, Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, Room B520a, PO Box 63, Haartmaninkatu 8, Helsinki 00014, Finland. E-mail: lauri.aaltonen@helsinki.fi Received 15 June 2006; revised 4 August 2006; accepted 25 August 2006; published online 9 October 2006

(microsatellite instability (MSI)) in tumor cells of the HNPCC patients (Aaltonen et al., 1993). High frequency of MSI can also be seen in 12–20% of unselected colorectal cancers (Ionov et al., 1993; Thibodeau et al., 1993; Aaltonen et al., 1998; Salovaara et al., 2000). In HNPCC tumors, somatic inactivation of the remaining wild-type allele can occur by different mechanisms: somatic mutation, promoter hypermethylation or loss of heterozygosity (LOH). Evidence of these mechanisms in HNPCC tumor initiation can be found in the literature. Somatic mutations as second hits have been found in both MLH1- and MSH2-deficient tumors, albeit at low frequencies (Leach et al., 1993; Konishi et al., 1996; Lu et al., 1996; Cunningham et al., 2001). MLH1 promoter hypermethylation has been detected in 17–46% of the HNPCC tumors (Cunningham et al., 1998; Herman et al., 1998; Kuismanen et al., 2000; Potocnik et al., 2001). Loss of the wild-type allele in HNPCC tumors was proposed by Hemminki et al. (1994) and it has been found to be the major mechanism for somatic second hits in most of the studies. Frequency of LOH in published studies varies between 33 and 86% (Hemminki et al., 1994; Lu et al., 1996; Tannerga˚rd et al., 1997; Kuismanen et al., 2000; Potocnik et al., 2001; Yuen et al., 2002), although the lost allele has not always been specified. In contrast, sporadic MMR-deficient tumors are most commonly due to bi-allelic inactivation of MLH1 by promoter hypermethylation, and these tumors rarely show LOH or somatic mutations in MLH1 (Kane et al., 1997; Cunningham et al., 1998; Herman et al., 1998; Veigl et al., 1998; Wheeler et al., 1999; Kuismanen et al., 2000). Promoter of the MSH2 has not been found to show hypermethylation (Cunningham et al., 1998; Herman et al., 1998). It has been widely accepted that HNPCC tumors well fit the traditional two-hit hypothesis; germline mutation accompanied with somatic inactivation of the wild-type allele, typically through allelic loss, initiates tumorigenesis. However, the number of tumors that have actually been studied has not been extensive. In this respect, a recent study reported LOH analyses in 35 Spanish HNPCC tumors carrying a germline mutation in MLH1 or MSH2 (Sanchez de Abajo et al., 2006). LOH was detected in 57% (20/35) of the tumors with a germline mutation but interestingly in 40% (8/20) of these,

No evidence for dual role of LOH in HNPCC tumors S Tuupanen et al

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mutant allele was lost. To explain the data, a model called dual role of LOH was presented. According to this, inactivation of the wild-type allele can arise by two alternative pathways. In certain tumors, LOH is a second hit (traditional two-hit model). In other tumors, the wild-type allele is first inactivated by somatic modification such as promoter hypermethylation, and LOH at the MMR gene locus may occur later on in tumor progression. This latter event might not target the respective MMR gene (already inactivated), but other genes in the region that may contribute to progression of the tumor. The favored pathway could differ by age of the patient. The traditional loss of the wild-type allele through somatic deletion could predominantly be seen

in young patients, whereas in older patients inactivation of the MMR gene by promoter hypermethylation could be more common. In the latter tumors, loss of the mutant allele would be visible more frequently, as a progression event. It was proposed that the dual-hit model could also fit the previously published data, because the patients in the early HNPCC studies might have been selected by young age – and thus would often display loss of the wild-type allele even if the dual role hypothesis was correct. To test this hypothesis, an LOH study using a population-based fresh-frozen HNPCC tumor material is ideal, because data can be created utilizing highquality DNA, from tumors that have not been selected

Figure 1 LOH in the 14 patients harboring point mutations was studied by direct sequencing. The mutation sites from the normal/ tumor DNA sample pairs were amplified by PCR and sequenced directly using Applied Biosystems (Foster City, CA, USA) BigDye v3.1 sequencing chemistry and ABI3730 Automatic DNA sequencer supplied with the sequencing analysis software v5.0. The PCR primers for the MLH1 and MSH2 exons have been designed previously (Chadwick et al., 2001) and are available upon request. The peak heights of the mutant and wild-type alleles in each sample were measured using Sequence Scanner software v1.0 (Applied Biosystems). LOH was scored if tumor sample displayed 40% decrease in allele strength (Canzian et al., 1996). See Table 1 for ratios. (a–g) Sequence chromatograms of the seven cases showing evidence of wild-type allele loss in cancer tissue. The sample ID and type of the germline mutation is shown above the pictures. All panels except (f) represent reverse direction of the sequence. N indicates normal tissue and T tumor tissue. Oncogene


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by age at onset. We have previously collected a population-based series of 1044 fresh-frozen colorectal cancers and respective normal tissue specimens, and analysed these for MSI and MSH2 and MLH1 mutations (Aaltonen et al., 1998; Salovaara et al., 2000). Twenty-five of 29 cases with pathogenic mutation were available for this study, whereas in four cases, either normal or tumor DNA had been exhausted. Of the 25, 23 attributed to a germline mutation in MLH1 and two in MSH2 (Aaltonen et al., 1998; Salovaara et al., 2000). In Finland, three founder mutations in MLH1 account for most of the HNPCC cases. These are a 3.5 kb deletion comprising exon 16, a splice site mutation in exon six (454-1 G>A), and a missense mutation in exon four (I107R). In our sample set, 11 (44%) of the patients carried the 3.5-kb deletion and 10 (40%) either of the other two founder mutations. In addition, two other germline mutations in MLH1 (Y126X and R659X) and two in MSH2 (1387-1 G >T, D603N) were seen in our samples (one case each). The age at the diagnosis varied between 36 and 75, 51.8 being the average. All of the tumor samples displayed at least 55% carcinoma tissue; however, in most of the cases, the proportion of tumor cells was 70–95%. Normal and tumor DNA samples of cases carrying point mutations were subjected to LOH analysis by direct sequencing, whereas the deletion comprising exon 16 was studied by a polymerase chain reaction (PCR)-based method designed for diagnosis of this founder mutation (Nystro¨m-Lahti et al., 1995), followed by agarose gel electrophoresis and signal intensity measurements. As expected, complete loss of the wild-type allele in tumor tissue was never seen due to normal tissue contamination. LOH was scored based on formula (Nwt Tmut)/(Nmut Twt) (Canzian et al., 1996), where wt is the wild-type allele and mut is the mutant allele. From the sequencing graphs (Figure 1), peak heights provided the respective signals, whereas after the agarose gel runs (Figure 2) band intensities were analysed by densitometry. No scoring was performed

Figure 2 The 3.5-kb genomic deletion was detected by previously described PCR-based method (Nystro¨m-Lahti et al., 1995). Three primers in one PCR reaction create products of different size for mutant (634 bp) and wild-type allele (475 bp). PCR reactions were performed in normal and tumor samples of 11 cases harboring the deletion. The PCR products were visualized by 2% agarose gel electrophoresis (Cambrex, Rockland, ME, USA) and the band intensities were measured by densitometry (FluorChem software, Alpha Innotech Corporation, San Leandro, CA, USA). To study LOH, the ratios between mutant and wild-type allele intensities in each sample were calculated and compared in normal/tumor pairs, according to the formula presented by Canzian et al. (1996). See Table 1 for ratios. Wild-type and mutant bands are indicated by arrows, and the presence of LOH with þ .

by eye. Allelic loss was scored if the ratio was >1.67 or o0.6. This corresponds to 40% or more decrease in strength of an allele in the tumor. PCR reactions and LOH analysis for every sample pair was repeated twice to confirm reproducibility. Figure 1 shows the direct sequencing-based LOH analysis of the 14 patients carrying point mutations in either MLH1 or MSH2. Of these 14 paired normal/ tumor samples, seven cases show loss of the wild-type allele in the tumor sample, with the mathematical formula. Figure 2 shows LOH analysis in normal/tumor sample pairs of the 11 patients carrying the 3.5-kb genomic deletion in MLH1. Utilizing the mathematical formula, LOH was scored in seven of the 11 cases and again these events affected the wild-type allele. Altogether, we observed LOH in 56% (14/25) of the cases (Table 1). In 14 of the 14 cases, LOH affected the wild-type allele (P ¼ 0.00006). We investigated if there is any correlation between LOH and type of the germline mutation, age or sex of the patient. Only one tumor out of five (20%) showed LOH in patients carrying the splice site mutation in exon six (454-1 G>A) in contrast to seven out of 11 (64%) tumors harboring the exon 16 deletion (P ¼ 0.28, Fisher’s exact test). The difference was not statistically significant. No correlation between LOH and age or sex was detected. LOH was present in six out of 12 (50%) tumors diagnosed less than 50 years of age and in eight of 13 (62%) tumors diagnosed over 50 years (P ¼ 0.7). Five out of 10 (50%) female patients and nine out of 15 (60%) male patients (P ¼ 0.7) showed LOH. It was of particular interest to compare our results to the recent work proposing the dual LOH hypothesis for HNPCC (Sanchez de Abajo et al., 2006). The authors reported LOH in 57% (20/35) of HNPCC tumors with MMR germline mutation and showed that in 40% (8/20) of these LOH targeted the mutant allele. They observed wild-type allele loss more frequently in tumors diagnosed in young patients (o50 years) (76.9%, 10/13) than in old (>50 years) (28.6%, 2/7). Correlation between LOH and any clinical–pathological feature, or type of germline mutation in the tumors, was not seen. In our population-based series of 25 fresh-frozen HNPCC tumors, LOH occurred at the same frequency (56%, 14/25), but in contrast and in concordance with previous studies by us and others, LOH was targeted to the wild-type allele. All but two of our samples were MLH1 deficient, which is due to predominant role of the three MLH1 founder mutations in the Finnish population. We could not see a correlation between mutation type and LOH. Several studies support our finding of the wild-type allele loss in HNPCC tumor samples. We have previously reported wild-type allele loss in six out of 17 MLH1-linked tumors (Hemminki et al., 1994). Similarly, Tannerga˚rd et al. (1997) showed LOH in six out of seven tumors, of which two could be shown to affect the wild-type allele. In MSH2-linked tumors, Lu et al. (1996) detected LOH in four out of eight (50%) tumors and two wild-type allele losses were seen. Oncogene


2516 Table 1 A total of 25 HNPCC patients included in the study are part of a well-described series of 1044 Finnish CRC patients, which were collected at nine Finnish central hospitals between 1994 and 1998 (Aaltonen et al., 1998; Salovaara et al., 2000) Sample ID c558 c614 c661 c811 c883 c870 c64 c587 c615 c700 c899 c219 c145 c340 c564 c676 c698 c952 c955 c1027 c1077 c1095 c430 c54 c138

Age at onset

Sex

Gene

Exon

Germline mutation

Ratio

LOH

65 75 54 58 40 61 43 45 39 61 59 49 42 66 41 41 43 66 65 57 56 56 36 35 39

F F F M M F F M M M M M F F M M M F M M M M F F M

MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MSH2 MSH2

4 4 4 4 4 4 6 6 6 6 6 17 16 16 16 16 16 16 16 16 16 16 16 9 12

I107R (T>G) I107R (T>G) I107R (T>G) I107R (T>G) I107R (T>G) Y126X (C>G) 454-1G>A 454-1G>A 454-1G>A 454-1G>A 454-1G>A R659X (C>T) 3.5-kb genomic deletion 3.5-kb genomic deletion 3.5-kb genomic deletion 3.5-kb genomic deletion 3.5-kb genomic deletion 3.5-kb genomic deletion 3.5-kb genomic deletion 3.5-kb genomic deletion 3.5-kb genomic deletion 3.5-kb genomic deletion 3.5-kb genomic deletion 1387-1 G->T D603N (G>A)

3.36 2.02 0.86 1.59 4.16 2.48 0.94 0.88 1.36 1.89 1.3 2.66 0.78 2.75 2.6 1.75 5.14 1.59 3.15 3.97 4.96 1.25 1.1 2.3 1.05

wt wt — — wt wt — — — wt — wt — wt wt wt wt — wt wt wt — — wt —

Abbreviations: HNPCC, hereditary non-polyposis colorectal cancer; LOH, loss of heterozygosity; wt, wild-type allele. LOH was scored based on formula (Nwt Tmut)/(Nmut Twt), presented by Canzian et al. (1996).

Kuismanen et al. (2000) studied the role of LOH and promoter hypermethylation in 27 HNPCC tumors with a known mutation in MLH1 or MSH2, and because these two phenomenons were never seen in the same patient, they suggested that the wild-type rather than the mutant allele was the target for somatic modification. However, that work as well as many others does not specify whether indeed the wild-type allele was the target of observed losses, leaving some space for ambiguity. We thus revisited the subject and found that our original results (Hemminki et al., 1994) could be reproduced in this new and more extensive sample set. The emerged discrepancy calls for an explanation. The opposite result obtained from the Spanish study could be explained by the phenomenon detected in the adenomatous polyposis coli tumor suppressor gene, in which the site of the germline mutation determines the type of the second hit (Lamlum et al., 1999). The genotypes involved could play a role, but because no significant correlation was seen in the studies (Sanchez de Abajo et al., 2006, this study) between LOH and the germline genotype, this is unlikely. Another factor explaining the differences in the results is coincidence; eight cases of loss of the mutant allele were seen in the Spanish study, and it is possible that coincidence has played some part in the results. Perhaps most likely, the differing results obtained from the Spanish and Finnish data sets could be explained by the sample material. In the Spanish study, tumor DNA was extracted from small amounts of paraffin-embedded tissue material, and in ours from abundant fresh-frozen Oncogene

tumor samples. It is known that DNA extracted from paraffin-embedded formalin-fixed tissue material is fragmented and the success of PCR amplification is reduced (Ben-Ezra et al., 1991). Williams et al. (1999) performed a PCR and sequencing-based study on frozen and formalin-fixed material and detected a high frequency of non-reproducible sequence alterations in formalin-fixed material but none in frozen tissues. We have observed multiple examples of allele-specific amplification owing to low concentration and/or quality of DNA, and in such cases, the amplified allele may also vary between consecutive PCR reactions. Paraffinembedded tissue samples are used very widely in cancer research and the importance of these is definite, as in many cases the paraffin blocks are the only available tumor material. However, the results obtained from such material, in particular if contrasting previous literature, should be interpreted with some caution. In conclusion, we showed in this study that in agreement with previous work by us and most others, LOH in MLH1- and MSH2-associated HNPCC tumors targeted the wild-type allele. We propose that the classical two-hit model is valid for HNPCC tumorigenesis. Acknowledgements We thank Païvi Peltoma¨ki and Saila Saarinen for the primers. This study was supported by grants from Academy of Finland (Grant numbers 213183, 214268, 203610 and Center of Excellence in Translational Genome-Scale Biology 2006–2011), Finnish Cancer Society and Sigrid Juselius Foundation.


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