Tumor MLH1 Promoter Region Methylation in Lynch Syndrome
Tumor MLH1 Promoter Region Methylation in Lynch Syndrome
This study is the first large-scale assessment of specificity and sensitivity of pretesting MLH1 CRC loss for the presence of a constitutional MLH1 mutation. We have demonstrated the use of pyrosequencing-based testing of CRC tumour DNA for MLH1 promoter methylation in a large cohort of CRCs. A novel MLH1 promoter region methylation assay has been developed to good laboratory practice (GLP) standards and its clinical use demonstrated in the assessment of patients meeting Bethesda guidelines and in population-based prescreening for LS. A single assay is time and cost effective, and may encourage the introduction of prescreening into routine clinical practice. Unmethylated MLH1 has a sensitivity of 94.4% and a specificity of 87.7% for the identification of MLH1 mutation carriers from a group of cancers with MLH1 loss MMRd. As such methylation is more effective than BRAF testing.
MMR protein IHC should be used as the first-line LS prescreening test in patients meeting Bethesda guidelines and cancers with typical LS features histologically. If MSH2, MSH6 or PMS2 proteins are absent, this indicates high risk of LS and the individual should be tested for constitutional mutations in the relevant gene. If MLH1 protein is absent, tumour DNA should be subjected to MLH1 promoter methylation testing. If methylation is absent, this indicates high risk (>10%) of LS, and constitutional MLH1 mutation analysis should be conducted.
It was previously unknown whether BRAF mutation or MLH1 promoter region methylation or both, is best able to distinguish between sporadic MLH1 loss of CRCs and cancers from patients with constitutional MLH1 mutations. Previous studies have examined small numbers of patients, and thus, there is no guide for clinical practice.
Sporadic MMR-deficient cancers with loss of MLH1 (or MSI-high) are associated with MLH1 gene silencing through the epigenetic effect of promoter region methylation. In this study, MLH1 promoter region methylation was found in 64/73 (87.7%) sporadic MMR-deficient CRCs; 33/73 (45.0%) of the sporadic MMR-deficient CRCs are known to be negative for constitutional MLH1 mutations; 28/33 (84.8%) were found to have MLH1 promoter methylation. Methylation was consistent across all four cytosine residues in the functional area of the promoter region as described by Deng et al. Pyrosequencing allows accurate quantification of methylation; 9/73 (12.3%) sporadic MMR deficient CRCs were found to have normal MLH1 promoter region. Two were found to have BRAF mutation. Of the seven that were wt BRAF and normal MLH1 promoter region, four had been tested for constitutional MLH1 mutations by the ACCFR and were found to be negative. The remaining three had been classified as sporadic MMR-deficient due to the patient's age (over 50 years) and lack of family history. The aetiology of these cancers without promoter region hypermethylation is unclear, but possible factors include loss of protein expression, somatic mutation of MLH1 and loss of heterozygosity. It is feasible that the MLH1 promoter displayed mosaic or heterogeneous patterns of methylation for the CpGs dinucleotides captured in the pyrosequencing amplicon but enough of the surrounding CpGs dinucleotides were methylated to result in loss of MLH1 protein expression. Alternatively, the three untested patients may be carriers of constitutional MLH1 mutations.
It has been thought that MLH1 promoter methylation is found exclusively in sporadic MMR-deficient CRCs. The current study is the largest dataset of MLH1 mutation carriers tested for MLH1 promoter region methylation. MLH1 promoter methylation was found in 4/71 (5.6%) mutation carriers (see Table 4). Of these, 1/22 (4.5%) was a Manchester patient (from an Amsterdam family), and 3/49 (6.1%) were from the Australasian Colon Cancer Family Registry (two from Amsterdam families) A first-degree relative of the Manchester patient (with the same constitutional MLH1 mutation) has an unmethylated somatic MLH1.
This low MLH1 promoter methylation frequency in mutation carriers is supported by a recent literature review and meta-analysis which found eight positively methylated tumours in MLH1 mutation carriers taken from 12 studies (5.56%). It has been suggested that sporadic inactivation of the second normal MLH1 allele by hypermethylation may be the 'second hit' event in mutation carriers. While somatic MLH1 promoter region methylation is an infrequent event in constitutional MLH1 mutation carriers, this data demonstrates that it is not rare and supports the hypothesis that it may function as the second hit event. These findings also suggest that the discovery of MLH1 hypermethylation does not exclude the diagnosis of LS. While the sensitivity of MLH1 hypermethylation testing is adequate for low/moderate-risk individuals, it is not for high-risk patients (3/4 MLH1 mutation carriers who had MLH1 promoter methylation were from Amsterdam families).
BRAF gene mutations are found in 5%–15% of all CRCs. They are more frequent in cancers from Jass's subtypes 1 (MSI-H, chromosome stable, CpG Island methylator phenotype (CIMP) high, methylated MLH1 promoter region; 13% of all CRCs) and 2 (MSI-low or stable, chromosome stable, CIMP-high, partial MLH1 methylation; 8% of all CRCs). Both are thought to originate in serrated lesions. BRAF mutation is thought to be an unequivocal marker of the serrated neoplasia pathway. The discovery of a BRAF mutation is thought to rule out LS. In the current study, BRAF mutation was found in 48/73 (66%) sporadic MLH1 loss CRCs. This is consistent with previous studies. A BRAF mutation was detected in 1/71 (1.4%) CRCs from MLH1 mutation carriers. BRAF mutations have previously been reported as a rare finding in patients with LS, and are thought to represent a mixed lineage of cancer predisposition. Walsh et al have reported two families with evidence of LS and probable additional constitutional factors causing serrated neoplasia. Senter et al investigated 99 probands with Lynch spectrum cancers that demonstrated loss of PMS2 on IHC. Constitutional PMS2 mutations were detected in 62%, and three (one exon 10 deletion, two c.736_741del6ins11) of these were found to have tumour BRAF mutation. It is likely that BRAF mutation is a rare finding in LS, and that its occurrence represents the influence of other molecular pathways, as suggested by Walsh.
It has been suggested that in MMR-deficient cancers, BRAF mutation is a surrogate marker for MLH1 promoter methylation. However, there is now evidence that BRAF mutation occurs in only 50%–75% of sporadic MLH1 loss cancers. In a series of 270 CRCs, Wang et al found BRAF mutations in 42/123 (34%) MMR-deficient cases. BRAF was closely associated with MLH1 methylation (30/36 (83.3%) MLH1 hypermethylated cases also had a BRAF mutation). In a large population-based study, Woods et al examined 68 MSI-H CRCs for BRAF and MLH1 methylation in order to prescreen for constitutional mutation testing. BRAF mutation was closely but not exclusively associated with MLH1 methylation; 31/40 (78%) of the hypermethylated tumours had BRAF mutations. In the current study, 46/73 (63.0%) sporadic MLH1 loss cancers had both BRAF mutation and MLH1 methylation, but 18/73 (24.7%) had only MLH1 methylation. This is consistent with previous studies.
A recent heath technology assessment (HTA) report has established that it would be cost effective for the NHS to introduce systematic testing for LS of all CRCs up to age 70 years. This report addressed the issue of excluding sporadic MLH1 cases from requiring unnecessary referral to clinical genetics services. The cost-effectiveness analysis allows for the increased costs of performing additional tests and the inherent reduction in sensitivity when more tests are performed serially in an attempt to increase specificity. Our data suggest that only 65% of cases without constitutional mutations will be identified by using BRAF alone. This is increased to 90% by adding a MLH1 methylation test. There are around 16 800 new cases of CRC up to age 70 years each year in the UK. Around 2200 (13%) of these will be sporadic MLH1 cases. The increased specificity of additional MLH1 methylation testing (90%) rather than BRAF alone (65%) would reduce the number of cases requiring genetic counselling and testing from around 780 to 220, a reduction of 550 cases each year. In our laboratory, MLH1 mutation testing costs around £483, MLH1 methylation testing costs around £138, and BRAF testing around £69. On average in the UK, a new person's appointment with a genetic counsellor or physician costs around £500 and a follow-up appointment around £350. Adding MLH1 methylation testing into systematic testing would cost around £300 000 per year. The cost saving each year would be around £700 000 (£450 000 for counselling and £250 000 for constitutional MLH1 analysis).
There are some limitations to the current study. A proportion of the sporadic samples did not undergo constitutional testing for MLH1. Even full sequencing and a dosage test of MLH1 may miss mutations such as deep intronic splicing mutations, and sensitivity may therefore be reduced. Clendenning et al have reported the discovery of an intronic MSH2 mutation, 478 bp upstream from exon 2 causing LS. As such, some of the 'sporadic' MLH1 loss CRCs may have had an undetected constitutional mutation. However, the rate of non-methylated MLH1 and wt BRAF in the 40 (4/40, 10%) sporadic tumours with mutation testing was not different to that in the 33 (3/33, 9.1%) untested sporadic cases. Additionally, the untested sporadic patients may harbour constitutional methylation.
Constitutional MLH1 promoter region methylation has been described as a rare (33 reported cases) finding in CRC. It is thought that this epimutation is usually erased in the gametes, but inheritance has been demonstrated in four cases. A recent study from the German hereditary non-polyposis colorectal cancer (HNPCC) consortium investigated 32 mutation-negative suspected Lynch cases with MSI-H and MLH1 loss CRC. They report one case of heritable partial MLH1 promoter methylation, which is induced by a large genomic duplication including the complete MLH1 gene and the promoter. This suggests that even in mutation-negative Lynch cases, the finding of constitutional methylation is low.
Constitutional MLH1 methylation is likely a rare cause for CRC tumour DNA MLH1 promoter region methylation, and a rare cause for LS, although the true incidence is unknown. In 10%–15% of suspected Lynch cases, no disease-causing mechanism can be detected. In these cases, it may be prudent to test for constitutional MLH1 promoter methylation.
Schofield et al reported a population-based screening programme using IHC, MSI and BRAF testing in CRCs in patients aged below 60 years. In the cohort of 270, 70 were MSI-H. 82 had loss of MMR protein expression. BRAF testing was conducted on 76 tumours. Twenty-five mutant BRAF tumours were excluded from further testing; 45 'Red Flag' cases were identified (MSI-H and loss of MSH2 or MSH6, OR MSH-H and loss of MLH1/PMS2 and wtBRAF); 31 were tested for constitutional mutations; 15 mutation carriers (7 MLH1, 2 MSH2, 3 PMS2 and 3 MSH6) were identified. The incidence of constitutional mutation in their 'Red Flag' cases is 48%. Our study has demonstrated that IHC followed by MLH1 methylation testing is likely to have a higher 'hit' rate due to the higher specificity of MLH1 methylation compared with BRAF (88 vs 66% in our study). Using IHC as the initial test avoids additional expense of MSI and allows the appropriate gene to be targeted for constitutional testing.
Identification of families with LS is vital to enable reduction in morbidity and mortality with screening. The use of population-based prescreening has been hampered by a lack of evidence for the specificity and sensitivity of MLH1 promoter region methylation analysis for the detection of mutation carriers. It is hoped that this current study provides that evidence.
Discussion
This study is the first large-scale assessment of specificity and sensitivity of pretesting MLH1 CRC loss for the presence of a constitutional MLH1 mutation. We have demonstrated the use of pyrosequencing-based testing of CRC tumour DNA for MLH1 promoter methylation in a large cohort of CRCs. A novel MLH1 promoter region methylation assay has been developed to good laboratory practice (GLP) standards and its clinical use demonstrated in the assessment of patients meeting Bethesda guidelines and in population-based prescreening for LS. A single assay is time and cost effective, and may encourage the introduction of prescreening into routine clinical practice. Unmethylated MLH1 has a sensitivity of 94.4% and a specificity of 87.7% for the identification of MLH1 mutation carriers from a group of cancers with MLH1 loss MMRd. As such methylation is more effective than BRAF testing.
MMR protein IHC should be used as the first-line LS prescreening test in patients meeting Bethesda guidelines and cancers with typical LS features histologically. If MSH2, MSH6 or PMS2 proteins are absent, this indicates high risk of LS and the individual should be tested for constitutional mutations in the relevant gene. If MLH1 protein is absent, tumour DNA should be subjected to MLH1 promoter methylation testing. If methylation is absent, this indicates high risk (>10%) of LS, and constitutional MLH1 mutation analysis should be conducted.
It was previously unknown whether BRAF mutation or MLH1 promoter region methylation or both, is best able to distinguish between sporadic MLH1 loss of CRCs and cancers from patients with constitutional MLH1 mutations. Previous studies have examined small numbers of patients, and thus, there is no guide for clinical practice.
Sporadic MMR-deficient cancers with loss of MLH1 (or MSI-high) are associated with MLH1 gene silencing through the epigenetic effect of promoter region methylation. In this study, MLH1 promoter region methylation was found in 64/73 (87.7%) sporadic MMR-deficient CRCs; 33/73 (45.0%) of the sporadic MMR-deficient CRCs are known to be negative for constitutional MLH1 mutations; 28/33 (84.8%) were found to have MLH1 promoter methylation. Methylation was consistent across all four cytosine residues in the functional area of the promoter region as described by Deng et al. Pyrosequencing allows accurate quantification of methylation; 9/73 (12.3%) sporadic MMR deficient CRCs were found to have normal MLH1 promoter region. Two were found to have BRAF mutation. Of the seven that were wt BRAF and normal MLH1 promoter region, four had been tested for constitutional MLH1 mutations by the ACCFR and were found to be negative. The remaining three had been classified as sporadic MMR-deficient due to the patient's age (over 50 years) and lack of family history. The aetiology of these cancers without promoter region hypermethylation is unclear, but possible factors include loss of protein expression, somatic mutation of MLH1 and loss of heterozygosity. It is feasible that the MLH1 promoter displayed mosaic or heterogeneous patterns of methylation for the CpGs dinucleotides captured in the pyrosequencing amplicon but enough of the surrounding CpGs dinucleotides were methylated to result in loss of MLH1 protein expression. Alternatively, the three untested patients may be carriers of constitutional MLH1 mutations.
It has been thought that MLH1 promoter methylation is found exclusively in sporadic MMR-deficient CRCs. The current study is the largest dataset of MLH1 mutation carriers tested for MLH1 promoter region methylation. MLH1 promoter methylation was found in 4/71 (5.6%) mutation carriers (see Table 4). Of these, 1/22 (4.5%) was a Manchester patient (from an Amsterdam family), and 3/49 (6.1%) were from the Australasian Colon Cancer Family Registry (two from Amsterdam families) A first-degree relative of the Manchester patient (with the same constitutional MLH1 mutation) has an unmethylated somatic MLH1.
This low MLH1 promoter methylation frequency in mutation carriers is supported by a recent literature review and meta-analysis which found eight positively methylated tumours in MLH1 mutation carriers taken from 12 studies (5.56%). It has been suggested that sporadic inactivation of the second normal MLH1 allele by hypermethylation may be the 'second hit' event in mutation carriers. While somatic MLH1 promoter region methylation is an infrequent event in constitutional MLH1 mutation carriers, this data demonstrates that it is not rare and supports the hypothesis that it may function as the second hit event. These findings also suggest that the discovery of MLH1 hypermethylation does not exclude the diagnosis of LS. While the sensitivity of MLH1 hypermethylation testing is adequate for low/moderate-risk individuals, it is not for high-risk patients (3/4 MLH1 mutation carriers who had MLH1 promoter methylation were from Amsterdam families).
BRAF gene mutations are found in 5%–15% of all CRCs. They are more frequent in cancers from Jass's subtypes 1 (MSI-H, chromosome stable, CpG Island methylator phenotype (CIMP) high, methylated MLH1 promoter region; 13% of all CRCs) and 2 (MSI-low or stable, chromosome stable, CIMP-high, partial MLH1 methylation; 8% of all CRCs). Both are thought to originate in serrated lesions. BRAF mutation is thought to be an unequivocal marker of the serrated neoplasia pathway. The discovery of a BRAF mutation is thought to rule out LS. In the current study, BRAF mutation was found in 48/73 (66%) sporadic MLH1 loss CRCs. This is consistent with previous studies. A BRAF mutation was detected in 1/71 (1.4%) CRCs from MLH1 mutation carriers. BRAF mutations have previously been reported as a rare finding in patients with LS, and are thought to represent a mixed lineage of cancer predisposition. Walsh et al have reported two families with evidence of LS and probable additional constitutional factors causing serrated neoplasia. Senter et al investigated 99 probands with Lynch spectrum cancers that demonstrated loss of PMS2 on IHC. Constitutional PMS2 mutations were detected in 62%, and three (one exon 10 deletion, two c.736_741del6ins11) of these were found to have tumour BRAF mutation. It is likely that BRAF mutation is a rare finding in LS, and that its occurrence represents the influence of other molecular pathways, as suggested by Walsh.
It has been suggested that in MMR-deficient cancers, BRAF mutation is a surrogate marker for MLH1 promoter methylation. However, there is now evidence that BRAF mutation occurs in only 50%–75% of sporadic MLH1 loss cancers. In a series of 270 CRCs, Wang et al found BRAF mutations in 42/123 (34%) MMR-deficient cases. BRAF was closely associated with MLH1 methylation (30/36 (83.3%) MLH1 hypermethylated cases also had a BRAF mutation). In a large population-based study, Woods et al examined 68 MSI-H CRCs for BRAF and MLH1 methylation in order to prescreen for constitutional mutation testing. BRAF mutation was closely but not exclusively associated with MLH1 methylation; 31/40 (78%) of the hypermethylated tumours had BRAF mutations. In the current study, 46/73 (63.0%) sporadic MLH1 loss cancers had both BRAF mutation and MLH1 methylation, but 18/73 (24.7%) had only MLH1 methylation. This is consistent with previous studies.
A recent heath technology assessment (HTA) report has established that it would be cost effective for the NHS to introduce systematic testing for LS of all CRCs up to age 70 years. This report addressed the issue of excluding sporadic MLH1 cases from requiring unnecessary referral to clinical genetics services. The cost-effectiveness analysis allows for the increased costs of performing additional tests and the inherent reduction in sensitivity when more tests are performed serially in an attempt to increase specificity. Our data suggest that only 65% of cases without constitutional mutations will be identified by using BRAF alone. This is increased to 90% by adding a MLH1 methylation test. There are around 16 800 new cases of CRC up to age 70 years each year in the UK. Around 2200 (13%) of these will be sporadic MLH1 cases. The increased specificity of additional MLH1 methylation testing (90%) rather than BRAF alone (65%) would reduce the number of cases requiring genetic counselling and testing from around 780 to 220, a reduction of 550 cases each year. In our laboratory, MLH1 mutation testing costs around £483, MLH1 methylation testing costs around £138, and BRAF testing around £69. On average in the UK, a new person's appointment with a genetic counsellor or physician costs around £500 and a follow-up appointment around £350. Adding MLH1 methylation testing into systematic testing would cost around £300 000 per year. The cost saving each year would be around £700 000 (£450 000 for counselling and £250 000 for constitutional MLH1 analysis).
There are some limitations to the current study. A proportion of the sporadic samples did not undergo constitutional testing for MLH1. Even full sequencing and a dosage test of MLH1 may miss mutations such as deep intronic splicing mutations, and sensitivity may therefore be reduced. Clendenning et al have reported the discovery of an intronic MSH2 mutation, 478 bp upstream from exon 2 causing LS. As such, some of the 'sporadic' MLH1 loss CRCs may have had an undetected constitutional mutation. However, the rate of non-methylated MLH1 and wt BRAF in the 40 (4/40, 10%) sporadic tumours with mutation testing was not different to that in the 33 (3/33, 9.1%) untested sporadic cases. Additionally, the untested sporadic patients may harbour constitutional methylation.
Constitutional MLH1 promoter region methylation has been described as a rare (33 reported cases) finding in CRC. It is thought that this epimutation is usually erased in the gametes, but inheritance has been demonstrated in four cases. A recent study from the German hereditary non-polyposis colorectal cancer (HNPCC) consortium investigated 32 mutation-negative suspected Lynch cases with MSI-H and MLH1 loss CRC. They report one case of heritable partial MLH1 promoter methylation, which is induced by a large genomic duplication including the complete MLH1 gene and the promoter. This suggests that even in mutation-negative Lynch cases, the finding of constitutional methylation is low.
Constitutional MLH1 methylation is likely a rare cause for CRC tumour DNA MLH1 promoter region methylation, and a rare cause for LS, although the true incidence is unknown. In 10%–15% of suspected Lynch cases, no disease-causing mechanism can be detected. In these cases, it may be prudent to test for constitutional MLH1 promoter methylation.
Schofield et al reported a population-based screening programme using IHC, MSI and BRAF testing in CRCs in patients aged below 60 years. In the cohort of 270, 70 were MSI-H. 82 had loss of MMR protein expression. BRAF testing was conducted on 76 tumours. Twenty-five mutant BRAF tumours were excluded from further testing; 45 'Red Flag' cases were identified (MSI-H and loss of MSH2 or MSH6, OR MSH-H and loss of MLH1/PMS2 and wtBRAF); 31 were tested for constitutional mutations; 15 mutation carriers (7 MLH1, 2 MSH2, 3 PMS2 and 3 MSH6) were identified. The incidence of constitutional mutation in their 'Red Flag' cases is 48%. Our study has demonstrated that IHC followed by MLH1 methylation testing is likely to have a higher 'hit' rate due to the higher specificity of MLH1 methylation compared with BRAF (88 vs 66% in our study). Using IHC as the initial test avoids additional expense of MSI and allows the appropriate gene to be targeted for constitutional testing.
Identification of families with LS is vital to enable reduction in morbidity and mortality with screening. The use of population-based prescreening has been hampered by a lack of evidence for the specificity and sensitivity of MLH1 promoter region methylation analysis for the detection of mutation carriers. It is hoped that this current study provides that evidence.
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