Tumor MLH1 Promoter Region Methylation in Lynch Syndrome
Background and aims Lynch syndrome (LS) patients have DNA mismatch repair deficiency and up to 80% lifetime risk of colorectal cancer (CRC). Screening of mutation carriers reduces CRC incidence and mortality. Selection for constitutional mutation testing relies on family history (Amsterdam and Bethesda Guidelines) and tumour-derived biomarkers. Initial biomarker analysis uses mismatch repair protein immunohistochemistry and microsatellite instability. Abnormalities in either identify mismatch repair deficiency but do not differentiate sporadic epigenetic defects, due to MLH1 promoter region methylation (13% of CRCs) from LS (4% of CRCs). A diagnostic biomarker capable of making this distinction would be valuable. This study compared two biomarkers in tumours with mismatch repair deficiency; quantification of methylation of the MLH1 promoter region using a novel assay and BRAF c.1799T>A, p.(Val600Glu) mutation status in the identification of constitutional mutations.
Methods Tumour DNA was extracted (formalin fixed, paraffin embedded, FFPE tissue) and pyrosequencing used to test for MLH1 promoter methylation and presence of the BRAF c.1799T>A, p.(Val600Glu) mutation 71 CRCs from individuals with pathogenic MLH1 mutations and 73 CRCs with sporadic MLH1 loss. Specificity and sensitivity was compared.
Findingss Unmethylated MLH1 promoter: sensitivity 94.4% (95% CI 86.2% to 98.4%), specificity 87.7% (95% CI 77.9% to 94.2%), Wild-type BRAF (codon 600): sensitivity 65.8% (95% CI 53.7% to 76.5%), specificity 98.6% (95% CI 92.4% to 100.0%) for the identification of those with pathogenic MLH1 mutations.
Conclusions Quantitative MLH1 promoter region methylation using pyrosequencing is superior to BRAF codon 600 mutation status in identifying constitutional mutations in mismatch repair deficient tumours.
Lynch syndrome (LS) is responsible for 3%–4% of all colorectal cancer (CRC) and is the most common cause of hereditary CRC. It is caused by mutations in one of the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. Mutations result in MSI-H (microsatellite instability high) cancers. Identification of families with LS is necessary to initiate screening and to reduce CRC mortality.
Diagnosis of LS is complicated by the expense and time-consuming nature of constitutional mutation analysis. Family history criteria and tumour-derived biomarkers are used to prescreen to select patients for germline testing. The Amsterdam II criteria were designed to select research families for linkage analysis. They are currently used, somewhat inappropriately, for clinical purposes to select individuals at high risk of having a MMR gene mutation. Patients who meet these criteria have at least 60% chance of a mutation. These criteria are inherently specific but consequently have low sensitivity. Much work has been done over the last decade to improve the identification of non-Amsterdam Lynch families. The revised Bethesda guidelines described in 2004 are sensitive but have low specificity. They have been criticised for being overly complicated and are little used in clinical practice. Tumour MSI and MMR protein immunohistochemistry (MMR IHC) are currently used in conjunction with the revised Bethesda guidelines (or other medium risk criteria). The sensitivity of MSI is 89% for MLH1 and MSH2, but less than 80% for MSH6 and PMS2, with a specificity of 90% for all genes. MSI testing is impractical for population-based screening due to the need for a molecular genetics laboratory. MMR IHC may be preferable in patients meeting Bethesda guidelines because of the low sensitivity of MSI for detecting MSH6 and PMS2 gene mutation carriers. MMR IHC has a sensitivity of 100% and a specificity 91.5% for the detection of MLH1 carriers, a sensitivity 87.5% and specificity of 88.5% for the detection of MSH2 carriers. Prescreening of all newly diagnosed CRCs (population-based) is used in some specialist centres in the USA and Europe (none in the UK) in order to identify families not meeting clinical criteria for LS. A multicentre study of over 10 000 newly diagnosed CRC probands, found that MMR tumour testing was the most effective strategy for the identification of mutation carriers (sensitivity 100%, specificity, 93.0%, diagnostic yield 2.2% compared to use of the Bethesda guidelines; sensitivity 87.8% specificity, 97.5%, diagnostic yield, 2.0% p<0.001).
There are two independent molecular pathways which lead to MSI-H (MMR deficient) CRC. MSI-H cancers occur in LS and also as a result of epigenetic silencing of the MLH1 gene through hypermethylation of its promoter. This occurs in around 13% of sporadic CRCs. These cancers are also associated with the BRAF c.1799T>A, p.Val600Glu mutation and are not familial. LS cancers are characterised by MSI-H, a normal (unmethylated) MLH1 gene promoter region, and wild-type (wt) BRAF (ie, c.1799T, p.Val600). MMR IHC is able to effectively identify patients for MSH2, MSH6 testing. Sporadic defects in these genes are rare, so protein loss is highly indicative of a constitutional abnormality. However, MSI and MMR IHC are not specific enough to identify MLH1 constitutional mutation carriers because of this large group of sporadic cancers with MLH1 deficiency. A method of differentiating between these groups of cancers is required.
BRAF mutation testing has been suggested. The methodology is well established and is currently in use in some centres. However, BRAF testing has low specificity. MLH1 promoter region methylation testing is attractive as a better prescreen test. Methylation is thought to be the first step in pathogenesis of cancers with sporadic loss of MLH1, and is thought to be rare in Lynch cancers. Lack of methylation should, therefore, be more specific for the identification of constitutional mutation carriers. Constitutional MLH1 methylation has been reported as a rare cause of mutation-negative LS (four cases reported). This may confound the use of methylation as a prescreen, but the incidence of this is likely to be extremely low. Tumour MLH1 promoter region methylation has not previously been tested in a large group of patients. While a number of methods for MLH1 methylation analysis have been developed, most are technically difficult (particularly in formalin fixed, paraffin embedded (FFPE) tissue) and expensive.
Guidelines for constitutional mutation testing for cancer susceptibility genes suggest a threshold of 10% risk. Using Bayes theorem, specificity and sensitivity of any prescreen test can be applied to individuals with differing risk determined by their family history of cancer. Individuals who fulfil Amsterdam II criteria have a pretest probability of harbouring a mutation of 60%. Individuals who fulfil the revised Bethesda guidelines and have loss of MLH1 in their tumour have a pretest probability of at least 10.5%. Patients from the general population who have MLH1 loss (tumour) have a pretest probability of 4.0%. We have previously shown that MSI testing alone is not an appropriate prescreening tool in Amsterdam criteria (I and II) positive families. Even if their tumour is microsatellite stable, the risk of having a mutation remains greater than 10%. Given that a recent Health Technology Assessment study has recommended that all CRCs in patients aged 60 years of age or younger should be prescreened for tumour mismatch repair deficiency (MMRd), strategies need to be developed to deal with the large number of MMRd tumours most of which will be the result of MLH1 promoter methylation in the tumour and not be caused by constitutional mutations.
The aim of this study was to: (1) develop a simple, cheap, reproducible method for quantitative MLH1 promoter region methylation analysis in FFPE tissue, (2) compare this with BRAF c.1799T>A p.Val600Glu mutation testing in patients whose CRCs demonstrate loss of MLH1 protein expression and (3) assess additional benefit of adding a methylation assay to BRAF testing in order to select patients for constitutional MLH1 mutation testing.
Abstract and Introduction
Abstract
Background and aims Lynch syndrome (LS) patients have DNA mismatch repair deficiency and up to 80% lifetime risk of colorectal cancer (CRC). Screening of mutation carriers reduces CRC incidence and mortality. Selection for constitutional mutation testing relies on family history (Amsterdam and Bethesda Guidelines) and tumour-derived biomarkers. Initial biomarker analysis uses mismatch repair protein immunohistochemistry and microsatellite instability. Abnormalities in either identify mismatch repair deficiency but do not differentiate sporadic epigenetic defects, due to MLH1 promoter region methylation (13% of CRCs) from LS (4% of CRCs). A diagnostic biomarker capable of making this distinction would be valuable. This study compared two biomarkers in tumours with mismatch repair deficiency; quantification of methylation of the MLH1 promoter region using a novel assay and BRAF c.1799T>A, p.(Val600Glu) mutation status in the identification of constitutional mutations.
Methods Tumour DNA was extracted (formalin fixed, paraffin embedded, FFPE tissue) and pyrosequencing used to test for MLH1 promoter methylation and presence of the BRAF c.1799T>A, p.(Val600Glu) mutation 71 CRCs from individuals with pathogenic MLH1 mutations and 73 CRCs with sporadic MLH1 loss. Specificity and sensitivity was compared.
Findingss Unmethylated MLH1 promoter: sensitivity 94.4% (95% CI 86.2% to 98.4%), specificity 87.7% (95% CI 77.9% to 94.2%), Wild-type BRAF (codon 600): sensitivity 65.8% (95% CI 53.7% to 76.5%), specificity 98.6% (95% CI 92.4% to 100.0%) for the identification of those with pathogenic MLH1 mutations.
Conclusions Quantitative MLH1 promoter region methylation using pyrosequencing is superior to BRAF codon 600 mutation status in identifying constitutional mutations in mismatch repair deficient tumours.
Introduction
Lynch syndrome (LS) is responsible for 3%–4% of all colorectal cancer (CRC) and is the most common cause of hereditary CRC. It is caused by mutations in one of the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. Mutations result in MSI-H (microsatellite instability high) cancers. Identification of families with LS is necessary to initiate screening and to reduce CRC mortality.
Diagnosis of LS is complicated by the expense and time-consuming nature of constitutional mutation analysis. Family history criteria and tumour-derived biomarkers are used to prescreen to select patients for germline testing. The Amsterdam II criteria were designed to select research families for linkage analysis. They are currently used, somewhat inappropriately, for clinical purposes to select individuals at high risk of having a MMR gene mutation. Patients who meet these criteria have at least 60% chance of a mutation. These criteria are inherently specific but consequently have low sensitivity. Much work has been done over the last decade to improve the identification of non-Amsterdam Lynch families. The revised Bethesda guidelines described in 2004 are sensitive but have low specificity. They have been criticised for being overly complicated and are little used in clinical practice. Tumour MSI and MMR protein immunohistochemistry (MMR IHC) are currently used in conjunction with the revised Bethesda guidelines (or other medium risk criteria). The sensitivity of MSI is 89% for MLH1 and MSH2, but less than 80% for MSH6 and PMS2, with a specificity of 90% for all genes. MSI testing is impractical for population-based screening due to the need for a molecular genetics laboratory. MMR IHC may be preferable in patients meeting Bethesda guidelines because of the low sensitivity of MSI for detecting MSH6 and PMS2 gene mutation carriers. MMR IHC has a sensitivity of 100% and a specificity 91.5% for the detection of MLH1 carriers, a sensitivity 87.5% and specificity of 88.5% for the detection of MSH2 carriers. Prescreening of all newly diagnosed CRCs (population-based) is used in some specialist centres in the USA and Europe (none in the UK) in order to identify families not meeting clinical criteria for LS. A multicentre study of over 10 000 newly diagnosed CRC probands, found that MMR tumour testing was the most effective strategy for the identification of mutation carriers (sensitivity 100%, specificity, 93.0%, diagnostic yield 2.2% compared to use of the Bethesda guidelines; sensitivity 87.8% specificity, 97.5%, diagnostic yield, 2.0% p<0.001).
There are two independent molecular pathways which lead to MSI-H (MMR deficient) CRC. MSI-H cancers occur in LS and also as a result of epigenetic silencing of the MLH1 gene through hypermethylation of its promoter. This occurs in around 13% of sporadic CRCs. These cancers are also associated with the BRAF c.1799T>A, p.Val600Glu mutation and are not familial. LS cancers are characterised by MSI-H, a normal (unmethylated) MLH1 gene promoter region, and wild-type (wt) BRAF (ie, c.1799T, p.Val600). MMR IHC is able to effectively identify patients for MSH2, MSH6 testing. Sporadic defects in these genes are rare, so protein loss is highly indicative of a constitutional abnormality. However, MSI and MMR IHC are not specific enough to identify MLH1 constitutional mutation carriers because of this large group of sporadic cancers with MLH1 deficiency. A method of differentiating between these groups of cancers is required.
BRAF mutation testing has been suggested. The methodology is well established and is currently in use in some centres. However, BRAF testing has low specificity. MLH1 promoter region methylation testing is attractive as a better prescreen test. Methylation is thought to be the first step in pathogenesis of cancers with sporadic loss of MLH1, and is thought to be rare in Lynch cancers. Lack of methylation should, therefore, be more specific for the identification of constitutional mutation carriers. Constitutional MLH1 methylation has been reported as a rare cause of mutation-negative LS (four cases reported). This may confound the use of methylation as a prescreen, but the incidence of this is likely to be extremely low. Tumour MLH1 promoter region methylation has not previously been tested in a large group of patients. While a number of methods for MLH1 methylation analysis have been developed, most are technically difficult (particularly in formalin fixed, paraffin embedded (FFPE) tissue) and expensive.
Guidelines for constitutional mutation testing for cancer susceptibility genes suggest a threshold of 10% risk. Using Bayes theorem, specificity and sensitivity of any prescreen test can be applied to individuals with differing risk determined by their family history of cancer. Individuals who fulfil Amsterdam II criteria have a pretest probability of harbouring a mutation of 60%. Individuals who fulfil the revised Bethesda guidelines and have loss of MLH1 in their tumour have a pretest probability of at least 10.5%. Patients from the general population who have MLH1 loss (tumour) have a pretest probability of 4.0%. We have previously shown that MSI testing alone is not an appropriate prescreening tool in Amsterdam criteria (I and II) positive families. Even if their tumour is microsatellite stable, the risk of having a mutation remains greater than 10%. Given that a recent Health Technology Assessment study has recommended that all CRCs in patients aged 60 years of age or younger should be prescreened for tumour mismatch repair deficiency (MMRd), strategies need to be developed to deal with the large number of MMRd tumours most of which will be the result of MLH1 promoter methylation in the tumour and not be caused by constitutional mutations.
The aim of this study was to: (1) develop a simple, cheap, reproducible method for quantitative MLH1 promoter region methylation analysis in FFPE tissue, (2) compare this with BRAF c.1799T>A p.Val600Glu mutation testing in patients whose CRCs demonstrate loss of MLH1 protein expression and (3) assess additional benefit of adding a methylation assay to BRAF testing in order to select patients for constitutional MLH1 mutation testing.
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