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Clinical Cancer Research 13, 3221, June 1, 2007. doi: 10.1158/1078-0432.CCR-06-3064
© 2007 American Association for Cancer Research
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Imaging, Diagnosis, Prognosis

Distinction of Hereditary Nonpolyposis Colorectal Cancer and Sporadic Microsatellite-Unstable Colorectal Cancer through Quantification of MLH1 Methylation by Real-time PCR

Marcus Bettstetter1, Stephan Dechant2, Petra Ruemmele1, Monika Grabowski3, Gisela Keller4, Elke Holinski-Feder3, Arndt Hartmann1, Ferdinand Hofstaedter1 and Wolfgang Dietmaier1

Authors' Affiliations: 1 Institute of Pathology and Molecular Diagnostics and 2 Department of Surgery, University of Regensburg, Regensburg, Germany; 3 Institute of Human Genetics, Ludwig-Maximilian-University; and 4 Institute of Pathology, University of Polytechniques, Munich, Germany

Requests for reprints: Wolfgang Dietmaier, Department of Pathology, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany. Phone: 49-941-944-6624; Fax: 49-941-944-6602; E-mail: Wolfgang.dietmaier{at}klinik.uni-regensburg.de.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Promoter hypermethylation occurs frequently in tumors and leads to silencing of tumor-relevant genes like tumor suppressor genes. In a subset of sporadic colorectal cancers (CRC), inactivation of the mismatch repair gene MLH1 due to promoter methylation causes high level of microsatellite instability (MSI-H). MSI-H is also a hallmark of hereditary nonpolyposis colorectal cancer (HNPCC) in which mismatch repair inactivation results from germ-line mutations. For differentiation of sporadic and hereditary MSI-H tumor patients, MLH1 promoter methylation analysis is a promising tool but is not yet used in daily diagnostics because only qualitative techniques without standardization are available. The aim of this study is to establish a reliable and quantitative MLH1 methylation analysis technique and to define valid MLH1 methylation cutoff values for HNPCC diagnostics.

Experimental Design: We developed a new real-time PCR–based technique to detect and quantify methylation of both proximal and distal hMLH1 promoter regions. We established and validated this technique in a cohort of 108 CRCs [94 MSI-H and 16 microsatellite stable (MSS) cases] comprising a reference (n = 58) and a tester tumor group (n = 50).

Results: The reference tumor group contained 28 HNPCC with proven germ-line mutations or positive Amsterdam I criteria (median age, 37 years) and loss of MLH1 expression, 14 sporadic MSI-H CRC tumors with loss of MLH1 expression and BRAF V600E mutation (median age, 80.5 years), and 16 sporadic MSS CRC (median age, 76.5 years). No MLH1 promoter methylation could be found in any MSS tumors. HNPCC patients showed no or low level of MLH1 promoter methylation. A cutoff value of 18% methylation extent could be determined in this study to define MLH1 hypermethylation specific for sporadic MSI-H cases. Methylation could also be verified qualitatively by melting point analysis. BRAF V600E mutations were not detected in any HNPCC patients (n = 22 informative cases).

Conclusion: According to the present data, quantitative MLH1 methylation analysis in MSI-H CRC is a valuable molecular tool to distinguish between HNPCC and sporadic MSI-H CRC. The detection of a BRAF V600E mutation further supports the exclusion of HNPCC.

Aberrant CpG methylation patterns have been reported in various diseases (1), especially in cancer (2, 3), wherein hypermethylation of regulatory CGI (CpG islands) can lead to inactivation of tumor suppressor genes (4, 5). Promoter hypermethylation of the mismatch repair gene MLH1 leads to a high level of microsatellite instability (MSI-H; refs. 6, 7), which can be found in ~15% of sporadic colorectal cancer (CRC; refs. 8–13). On the other hand, MSI-H is a hallmark of hereditary nonpolyposis colorectal cancers (HNPCC; refs. 8–10, 12, 13) and can arise by mismatch repair inactivation caused by germ-line mutations rather than by promoter hypermethylation (14, 15). Thus, examination of MLH1 promoter hypermethylation may be a promising tool to distinguish between sporadic MSI-H CRC and HNPCC candidates (6, 7, 16, 17). The MLH1 promoter methylation analysis could be combined with BRAF V600E mutation detection because BRAF V600E mutations are very frequent in MSI-H sporadic CRC but virtually not found in tumors of HNPCC patients (18, 19).

A variety of nonquantitative methods for the analysis of CpGs have been reported (15, 20–22), which are based on digestion of DNA with methylation-sensitive restriction enzymes or bisulfite modification of DNA and methylation-specific PCR primers. Because bisulfite treatment of genomic DNA converts all nonmethylated cytosines to uracils, whereas methylated cytosines remain unchanged, the methylation status of the genomic DNA is represented by the sequence of the bisulfite-modified DNA. DNA methylation can be determined by direct sequencing or sequencing of subclones (23, 24), methylation-specific PCR (25), combined bisulfite restriction analysis (26), single-strand conformational polymorphism (27), melting curve analysis (28, 29), or denaturing gel electrophoresis (30).

However, for diagnostic purposes, objective quantitative methods are required, which include clear validated cutoff values for methylation determination proven in a well-defined patient collective. Few quantitative techniques have been described [MethyLight (31), ConLight-MSP (32), MethylQuant (33)] which are not established for MLH1 methylation detection nor are they tested in a patient cohort of HNPCC cases with germ-line mutations in mismatch repair genes, sporadic MSI-H CRC, and microsatellite stable (MSS) CRC. In addition, these methods have several drawbacks. They use unstable bisulfite-modified DNA directly as template impairing reproducibility and robustness, or expensive and optimized internal fluorescent probes are required. In MethylQuant, only single methylated nucleotides are investigated (33), which is not sufficient for a loss of MLH1 expression (34, 35). We designed a quantitative method detecting only complete methylation of several CpGs in the regulatory regions of MLH1 without the need of fluorescence probes and studied a patient cohort of MLH1-negative HNPCC patients, sporadic MSI-H patients with loss of MLH1 expression and BRAF mutation V600E, and sporadic MSS CRC patients. The aim of the present study was to establish a robust, reliable, and cost-effective real-time PCR–based method to quantitatively and qualitatively analyze DNA methylation and to develop cutoff values of methylation extent, which can be used in the daily practice of HNPCC diagnostics.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Surgical specimens and cell lines
In a multicentric study, 108 formalin-fixed paraffin-embedded tumor samples were obtained from the pathology departments of the University of Regensburg (n = 56), the Ludwig-Maximilians-University of Munich (n = 40), and the Technical University of Munich (n = 12) and then analyzed for MLH1 promoter methylation, BRAF V600E mutation analysis, and immunohistochemistry of MLH1, MSH2, and MSH6 mismatch repair proteins. A reference group of 58 CRC patients were studied according to microsatellite status, BRAF V600E mutation, MLH1 germ-line mutation, and Amsterdam I criteria: (a) HNPCC patients (n = 28) were defined by (i) positive Amsterdam I criteria and/or pathogenic hMLH1 germ-line mutations, (ii) microsatellite instability (MSI-H), and (iii) negative hMLH1 immunohistochemistry (median age, 37 years); none of the 21 of 28 successfully BRAF codon 600 tested HNPCC cases showed a V600E mutation. (b) Sporadic MSI-H CRC patients (n = 14) were defined by (i) no evidence of fulfilled Amsterdam criteria (36) or hMLH1 germ-line mutations, (ii) BRAF V600E mutation, (iii) age ≥75 years, and (iv) negative hMLH1 immunohistochemistry (median age, 80.5 years). (c) Sporadic MSS-CRC patients (n = 16; median age, 76.5 years) showing MSS and intact expression of the mismatch repair proteins were used as control group. The institutional ethics committee approved the study.

The human colon cancer cell lines SW48 (MLH1 methylation positive) and SW480 (no MLH1 methylation) were obtained from American Type Culture Collection. All cell lines were maintained in DMEM with 10% fetal bovine serum at 37°C and 5% CO2.

DNA isolation
DNA was isolated from tissues and cell extracts using the High Pure PCR Template Preparation Kit (Roche) according to the supplier's recommendation. DNA was quantified photometrically.

Microsatellite analysis
Microsatellite analysis was done as previously described (37). Briefly, 50 to 100 ng of genomic DNA were used for multiplex microsatellite PCR amplification of the recommended Bethesda standard panel using the HNPCC Microsatellite Instability Test kit (Roche) according to the manufacturer's instruction. MSI-H was defined by a MSI frequency of >30% (38, 39) according to the Bethesda guidelines. One microliter of amplified PCR product was applied to the ABI PRISM 310 Genetic Analyzer using POP6 polymer. Automatic fragment analysis was done with GeneScan 3.1.2 software (Applied Biosystems).

Bisulfite modification
Bisulfite treatment was done to convert unmethylated cytosines into uracils whereas methylated cytosines remain unchanged. One hundred nanograms of genomic DNA were treated with sodium bisulfite using the EZ DNA Methylation Kit (ZYMO Research) according to the supplier's instructions. In brief, 5 µL of M-Dilution Buffer were added to the DNA sample and a volume of 50 µL was adjusted with sterile, nuclease-free water. After incubation at 37°C for 15 min, 100 µL of CT Conversion Reagent were added and the sample was incubated in the dark at 50°C for 16 h, and then chilled on ice for 10 min. Four-hundred microliters of M-Binding Buffer were added and then the sample was loaded into a Zymo-Spin I Column and centrifugated at 16,000 x g for 30 s. The modified DNA was washed with 200-µL M-Wash Buffer, treated with 200-µL M-Desulfonation Buffer, and washed twice with 200-µL M-Wash Buffer. Finally, the cleaned modified DNA was eluted with 12-µL M-Elution Buffer. Because bisulfite-modified DNA is very unstable, it was used immediately as template for PCR or stored at –80°C.

PCR preamplification of bisulfite-modified DNA
Principle. Bisulfite-modified DNA is very unstable, and results from methylation-specific quantitative real-time PCR with a bisulfite-modified DNA template are poorly reproducible. Therefore, we did an initial 15-cycle preamplification PCR using bisulfite-modified DNA and primers flanking the CpG stretches of interest. These flank primers contain preferably no CpGs and anneal therefore independently from methylation pattern (Fig. 1 ). flank primers may contain a wobble base if the sequence makes it unavoidable to include an eventually methylated cytosine within the primers. The preamplified promoter fragment generated with the flank primers is a stable DNA product and is used at 10-fold dilution as template in the subsequent methylation quantification.


Figure 1
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  		Fig. 1. Scheme of the methylation quantification. Bisulfite modification converts unmethylated cytosines into uracils whereas methylated cytosines remain unchanged. The CpG island of interest is preamplified using the bisulfite-modified DNA as template by the methylation-independent specific flank primer pair adjacent to the CpG island. The preamplified promoter is used as template in the methylation-specific real-time PCR: one reaction uses the methylation-specific (MS)-flank primer pair and amplifies only methylated template species whereas the other methylation-independent primer pair (flank-U-flank-D) amplifies the CpG-containing calibrator fragment.

 
MLH1 methylation analysis. Methylation of both proximal and distal located CpG islands of MLH1 promoter was analyzed. Each promoter region was separately preamplified by 15 PCR cycles using methylation-independent flank primers [Proxflank-up + Proxflank-down (proximal region), Distflank-up + Distflank-down (distal region)]. PCR was done in a total reaction volume of 20 µL, containing 5% (v/v) DMSO, 2.0 mmol/L Mg2+, 0.2 mmol/L deoxynucleotide triphosphates, 0.3 µmol/L of each flank primer, 10% (v/v) 10x PCR reaction buffer, 0.5 unit of Taq-Polymerase (Fermentas), and 4 µL (20 ng) of bisulfite-modified DNA. The sequences of primers are Proxflank-up, 5'-TTTTAAAAAYGAATTAATAGGAAGAG-3'; Proxflank-down, 5'-AAATACCAATCAAATTTCTCAACTC-3'; Distflank-up, 5'-GAGTGAAGGAGGTTAYGGGTAAGT-3'; and Distflank-down, 5'-AACRATAAAACCCTATACCTAATCTATC-3'.

An initial denaturation step of 20 s at 94°C was followed by 15 cycles of 20 s at 94°C, 1 s at 60°C, a temperature ramp-down of 0.2°C/s to 55°C, 1 min at 55°C, 1 min at 72°C, and an final elongation step at 72°C for 3 min. PCR products were purified from salts, primers, nucleotides, and enzyme with the MinElute PCR Purification Kit (Qiagen) according to the supplier's instructions and eluted in 12 µL of Tris-Cl (10 mmol/L, pH 8.5). The preamplified DNA was diluted by 1:10 with sterile, nuclease-free water and stored at 4°C.

Methylation-specific relative quantitative real-time PCR
The methylation status was determined by relative quantitative real-time PCR (LightCycler System, Roche). For each promoter region, two real-time PCR reactions with diluted preamplified regions as template were done: (i) a methylation-independent PCR using CpG flanking primers as a 100% amplification reference, and (ii) a methylation-specific PCR using a flank primer and a methylation-specific primer located inside the preamplified promoter fragments [for the proximal region: Proxflank-up and ProxMSr (5'-CGATTTTTAACGCGTAAGC-3'); for the distal region: Distflank-up and DistMSf (5'-GAACGACGAACGCGCG-3')]. The methylation-specific primers cover at least four eventually cytosines and have a 3' specificity for a methylated cytosine.

The preamplified promoter regions from the MLH1 methylation–positive CRC cell line SW48 (35) were used as calibrators and controls for 100% methylation in the methylation-specific PCR. The nonmethylated preamplified promoter regions from the CRC cell line SW480 served as controls for 0% methylation.

LightCycler PCR was carried out using the LightCycler FastStart DNA MasterPlus SYBR Green I Kit (Roche) with 2 µL of 1:10 diluted preamplified promoter fragments in a total volume of 20 µL, containing 0.5 µmol/L of each primer and 4 µL of 5x LightCycler-FastStart DNA MasterPlus SYBR Green I Mix (Roche). An initial denaturation for 10 min at 95°C was followed by 40 cycles of 95°C for 2 s, 55°C for 6 s, and 72°C for 8 s. Melting point analysis was done by heating the PCR products from 50°C to 95°C with an increase of 0.2°C/s whereas fluorescence was monitored continuously.

For relative quantification, the methylation values of samples were normalized to the methylation value of the calibrator, which is defined as 100%. The mathematical analysis was done with the RelQuant 1.01 software (Roche) using PCR efficiency correction, giving the proportion of methylated template.

BRAF mutation analysis
Mutation analysis of BRAF codon 600 was done by sequencing exon 15 using an ABI PRISM 3100 Genetic Analyser (Fig. 2 ). The following primers were used for BRAF V600E mutation: BRAF-600 up, 5'-TGTAAAACGACGGCCAGTTCATAATGCTTGCTCTGATAGGA-3'; BRAF-600 down, 5'-CAGGAAACAGCTATGACCCTTTCTAGTAACTCAGCAGC-3'. Amplifications were carried out according to ref. 40 using 0.02 unit/µL Taq DNA polymerase (Fermentas) and a PCR profile consisting of a 3-min initial denaturation at 94°C followed by 35 cycles of 60 s at 94°C, 60°C, and 72°C, followed by a 8-min final extension at 72°C.


Figure 2
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  		Fig. 2. DNA sequence analysis showing a representative BRAF V600E mutation.

 

   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Quantitative validation of MLH1 methylation analysis. The accuracy of the methylation quantification was tested by spiking experiments with purified preamplified promoter fragments from the MLH1 hypermethylated colon cancer cell line SW48 and the MLH1 unmethylated cell line SW480 in ratios of 1:1, 19:20, 4:5, 1:2, 1:5, 1:20, and 0:1 (corresponding to 100%, 95%, 80%, 50%, 20%, 5%, and 0% methylated DNA, respectively). Both distal and proximal MLH1 promoter regions were tested (Fig. 3 ). The methylation quantification analysis mirrored the ratios of the methylated templates in both promoter regions, thus showing the high accuracy of the method described.


Figure 3
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  		Fig. 3. Curves of methylation values showing the measured extent of methylation in percent of a spiking experiment with methylated and unmethylated samples using hMLH1 promoter fragments from CRC cell lines mixed in different ratios. The results of the methylation quantification mirror the real sample composition.

 
Qualitative methylation detection by melting point analysis. Quantification analyses were also verified by melting point analysis. The median melting point of distal flank PCR products was 85.0°C for methylated and 79.0°C for unmethylated DNA species. Proximal flank PCR products showed melting points of 78.5°C (methylated MLH1) and 73.0°C (unmethylated MLH1 promoter). If the sample contained both methylated and unmethylated promoters, both corresponding melting peaks appeared in the melting point analyses (Fig. 4 ).


Figure 4
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  		Fig. 4. Melting point analysis of flank PCR products. Representative melting curves of flank PCR products (distal promoter) from CRC patients. Case no. 97 shows a single peak at ~79°C resulting from unmethylated hMLH1 promoter fragment. Case no. 78 shows a high melting (85°C) point in a flank PCR product of a 74% methylation-positive patient. Besides the slight slope resulting from the 26% unmethylated promoter fragments at 79°C, a main peak is visible at 85°C originated from methylated PCR products. Case no. 89 with 44% methylated promoter fragments shows a melting peak of unmethylated (79°C) and methylated DNA (85°C) species.

 
Evaluation of quantitative MLH1 methylation in known HNPCC tumors and sporadic CRC (reference tumor group). We assessed methylation status of the distal and proximal regions of the hMLH1 promoter in three tumor cohorts: sporadic MSS CRC patients (n = 16), sporadic MSI-H CRC patients (n = 14), and HNPCC patients (n = 28). The methylation values ranged from 0% to 100%. None of the 16 MSS CRCs showed significant MLH1 promoter methylation. The median methylation values were distal, 2.5% (range, 0-6%); proximal, 0% (range, 0-1%); and mean of distal/proximal, 1.6% (range, 0-3%). All 14 sporadic MSI CRCs showed methylation at both MLH1 promoter regions [median methylation values: distal, 56% (range, 31-100%); proximal, 38% (range, 20-80%); mean of distal/proximal, 50.4% (range, 33-81%)]. The MLH1 methylation-positive sporadic MSI-H CRC patients had a mean age of 80.5 years; all of them showed BRAF V600E mutations and none of them fulfilled the Amsterdam criteria or carried hMLH1 germ-line mutations. The median values of MLH1 methylation in HNPCC were distal, 0% (range, 0-16%; five cases with values greater than 2%: 3%, 5%, 14%, 15%, 16%); proximal, 1% (range, 0-15%; three cases with values greater than 2% methylation: 3%, 10%, 15%); and mean of distal/proximal, 0.7% (range, 0-15%; four cases with values greater than 2% methylation: 5%, 7%, 8%, and 15%). According to these data, we defined a cutoff for methylation positivity of median values +5 SD. That is, (i) 18.3%, mean of distal/proximal median values (= 1.9 + 16,4); (ii) 26.5%, distal methylation: (= 2.2 + 23.3); or (iii) 17.8%, proximal methylation (= 1.6 + 16.2). Thus, in most of the HNPCC patients (86%; 24 of 28), no methylation [i.e., 0-2% methylation (mean of distal/proximal values)] was detected. However, 17% of HNPCC tumors show low-level methylation.

A quantile box plot of the median methylation values from the distal/proximal means shows that methylation is significantly different in each tumor group (PKruskal Wallis < 0.001; Fig. 5 ). Sporadic MSI-H CRCs show strong MLH1 promoter methylation (≥31%), whereas HNPCC tumors do not show methylation or only values up to 16% at most. MSS sporadic tumors, which are used as controls, are consistently MLH1 methylation negative.


Figure 5
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  		Fig. 5. Comparison of MLH1 promoter methylation in tumors from HNPCC, sporadic MSI-H CRC, and sporadic MSS CRC. The box for each tumor group represents the interquartile range (25-75th percentile); the line within each box shows the median value. Bottom and top bars of the whisker indicate the 10th and 90th percentiles, respectively. Outlier values are indicated (asterisks). The nonparametric Kruskal-Wallis test was used to examine differences between the three independent tumor groups (HNPCC, sporadic MSI-H tumors, and sporadic MSS tumors). The Mann-Whitney U test was used in case of two tumor groups. Statistical analysis was done using the SPSS13 software.

 
Validation of quantitative methylation in a MSI-H CRC test group. We tested the MLH1 methylation analysis using the 18% cutoff in further 50 MSI-H CRCs (test group, Table 1 ) in which a complete set of clinical and molecular data were not available. All cases with positive Amsterdam I (n = 4) or Amsterdam II (n = 2) criteria were methylation negative. All tumors with BRAF V600E mutations (n = 10) were methylation positive, independently from the Bethesda criteria. In 8 of 19 (42%) methylation-positive cases, no BRAF mutation was found. One of these patients (case no. 65) had a hemiallelic germ-line MLH1 methylation in nuclear blood cells (data not shown) and showed a nearly 50% mean methylation value (48%) also in tumor DNA. Overall, in 40 cases with informative methylation and BRAF data, a significant correlation of methylation positivity and BRAF V600E mutation was found (P = 0.005). Bethesda-positive patients with both BRAF mutation and MLH1 promoter methylation but without evidence of positive Amsterdam criteria or hMLH1 germ-line mutations (n = 4) showed a median age of 70 years (range, 66-74 years) and thus can be assigned to the sporadic MSI-H CRC group. Six cases with negative Amsterdam and positive (n = 4) or negative (n = 2) Bethesda criteria were methylation positive but did not carry a BRAF V600E mutation. Twenty-two patients showed neither methylation nor BRAF mutation, had a median age of 44.5 years (range, 31-78 years), and represent putative HNPCC patients. In 56% (28 of 50) of cases, the methylation level was higher in the distal than in the proximal promoter region. In one presumably sporadic tumor (case no. 84: BRAF mutation negative; age, 86 years), methylation positivity was detectable only in the distal but not in the proximal promoter region.


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  		Table 1. Clinical and molecular data of the tumor test group

 

   Discussion
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 Materials and Methods
 Results
 Discussion
References
 
In the daily routine practice, it is frequently difficult to identify likely candidates for HNPCC among CRC patients. To minimize the risk of overlooking HNPCC cases, the strict clinical criteria to diagnose HNPCC (Amsterdam criteria; ref. 36) were complemented by the Bethesda criteria (39, 41) to select patients for MSI analysis and immunohistochemical studies of the mismatch repair proteins. The drawback is that the higher sensitivity of the new clinical criteria is associated with a considerably lower specificity. In addition, although HNPCC patients are diagnosed with CRC at an earlier age, there are also studies showing that HNPCC-related CRC can also be diagnosed at age 60 years and older (42). Therefore, additional molecular methods are urgently needed to subdivide MSI-H–positive Amsterdam-negative patients into sporadic CRC or HNPCC cases.

The aim of this study was to establish a quantitative methylation analysis as a diagnostic tool that separates HNPCC candidates from sporadic MSI-H CRC. In this study, we analyzed the MLH1 promoter methylation, hMLH1 protein expression, microsatellite instability, and BRAF V600E mutation status in 108 CRCs and in 2 CRC cell lines.

Generally, several difficulties can complicate methylation analysis. Stromal and inflammatory cells within tumor tissues with methylation patterns different from the tumor methylation pattern may easily lead to false results if nonquantitative methylation analysis techniques such as methylation-specific PCR are used. Moreover, nonquantitative methylation detection techniques cannot distinguish between partial or monoallelic and complete biallelic methylation. Methylation analyses usually require a bisulfite modification step, which provides unstable modified DNA (bisulfite-modified DNA), impairing the reproducibility of analyses.

These drawbacks could be overcome by the quantitative methylation analysis technique presented here, which is highly reproducible because it uses a stabilized bisulfite-modified DNA generated by a 15-cycle preamplification PCR, providing sufficient stable template DNA for numbers of methylation analyses, but minimizes the risk of cross contaminations. The accuracy of this technique was proved by spiking experiments of methylated and unmethylated template DNA for both proximal and distal MLH1 promoter regions.

Because the melting point of double-stranded DNA increases with its CG content, melting points of the methylation-independent flank PCR products increase with each methylated cytosine because it resists bisulfite modification. Thus, methylation can be determined by melting point analysis (described here for the first time for MLH1; Fig. 4), which provides an additional valuable qualitative tool to control and verify the quantitative methylation analysis. Patients determined as methylation negative show only one melting peak (distal, ~80°C; proximal, ~72°C) whereas methylation-positive patients show melting peaks at a higher temperature (proximal, >78°C; distal, >85°C) dependent on the degree of methylation. Usually, the melting curves from methylation-positive CRC show two peaks (Fig. 4); the peak at the lower temperature (80°C) may be caused by nonmethylated promoter DNA from residual normal cells, unmethylated subpopulations of tumor cells, or unmethylated DNA in monoallelic methylated tumor cells.

In contrast to other methods like methylation-specific PCR, the methylation detection method described here allows the determination of methylation by using specific thresholds. Thus far, this is the first report providing cutoff values for MLH1 methylation to discriminate between HNPCC and sporadic CRC. Because the lowest value within the group of sporadic MSI-H tumors was 33% (Fig. 5A) and the highest value in HNPCC tumors was 15%, a cutoff value of 18% (= mean of proximal and distal median methylation values plus 5-fold SD) was defined for positive scoring. However, this value depends basically on purity grade of microdissected tumor samples. False-positive results could be excluded because no positive methylation was detected at any hMLH1-positive MSS CRC. Interestingly, three HNPCC patients from the reference group (25, 33, and 63 years old) with pathogenic MLH1 germ-line mutations and loss of MLH1 expression showed weak DNA hMLH1 methylation (16%, 14%, and 15%, respectively) in the distal promoter region and shifted melting peaks (data not shown). For this reason, we regard the analysis of the proximal region as more reliable for HNPCC diagnostics. We assume that these tumors carry monoallelic MLH1 promoter methylation (i.e., one allele is silenced by methylation), whereas the other allele carries the MLH1 germ-line mutation. Unfortunately, because there was no sequence polymorphism within the promoter regions, we were not able to discriminate between maternal and paternal alleles to verify monoallelic methylation by cloning and sequence analysis.

Within the MSI-H and MLH1 immunohistochemically negative tumor test group, we found a significant correlation of a BRAF mutation and MLH1 methylation in tumors apparently representing sporadic MSI-H CRC and loss of MLH1 protein expression (mean age, 69 years). This finding confirms the results from others (43, 44). Some patients (20%; 8 of 40) show MLH1 methylation but no BRAF mutation and may represent sporadic MSI-H tumors because there was no evidence for fulfilled Amsterdam criteria or pathogenic mutations and the median age of these patients was 69 years. However, in those cases, a hemiallelic germ-line MLH1 methylation (45–48) must be excluded by analysis of blood cells or normal tissue (e.g., case no. 65).

One case (no. 84), a 86-year-old patient and therefore most likely a sporadic CRC patient, had methylation only in the distal promoter region, showing that distal MLH1 promoter methylation alone is apparently sufficient for loss of MLH1 expression.

In summary, our data have a strong impact for HNPCC diagnostics. Accordingly, the analysis of both BRAF mutation and quantitative MLH1 methylation analysis should be done in any MSI-H CRC with loss of MLH1 expression for the following reasons:

(a) First, nonquantitative MLH1 methylation analysis detecting any MLH1 methylation level is not suited to differentiate between sporadic MSI-H and HNPCC patients because MLH1 methylation can occur not only in sporadic MSI-H CRC but also in some HNPCC patients (49).

(b) Second, quantitative MLH1 methylation analysis described here shows that HNPCC tumors show no or weak MLH1 methylation levels significantly lower than the high methylation levels in sporadic MSI-H CRCs. This difference can only be identified by quantitative MLH1 methylation analysis. To detect also HNPCC patients from, for example, small families not fulfilling the Amsterdam criteria or HNPCC patients not meeting the Bethesda criteria due to an age of more than 60 years, any MSI-H tumor with loss of MLH1 expression should be quantitatively analyzed for MLH1 methylation. Furthermore, not only tumor DNA but also DNA from normal or nuclear blood cells should be analyzed to identify also a potential monoallelic germ-line methylation (45–48) with MLH1 methylation values of ~50% both in tumor and blood cells.

(c) Third, BRAF V600E mutations virtually do not occur in HNPCC patients carrying pathogenic germ-line MLH1 mutations as shown in our study as well as in other reports (18, 19, 43, 44, 50). Vice versa, BRAF V600E mutations occur specifically in a part of sporadic MSI-H CRC (40-74%; refs. 19, 44) as well as in 4% to 12% of MSS CRC (current study; refs. 19, 43). According to the present data, the detection of a BRAF V600E mutation is a highly specific molecular marker to exclude HNPCC and should be used in parallel with quantitative MLH1 methylation analysis.


   Acknowledgments
 
We thank Stephanie Schilling, Irene Schardt, and Kerstin Meier for excellent technical assistance.


   Footnotes
 
Grant support: Dr. Mildred Scheel Foundation for Cancer Research (Deutsche Krebshilfe) and the German HNPCC project.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12/27/06; revised 3/13/07; accepted 3/21/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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