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From the Departments of Pathology,
* Medicine,
and Radiation Oncology,
|| Brigham and Women’s Hospital, Boston, Massachusetts; the Departments of Medical Oncology
and Radiation Oncology,
** Dana-Farber Cancer Institute, Boston, Massachusetts; Harvard Medical School,
Boston, Massachusetts; the Departments of Epidemiology and Biostatistics,
¶ Harvard School of Public Health, Boston, Massachusetts; and the Departments of Surgery and of Biochemistry and Molecular Biology,
University of Southern California Keck School of Medicine, Norris Comprehensive Cancer Center, Los Angeles, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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A number of methods to determine DNA methylation status in tumor tissues have been developed. For formalin-fixed, paraffin-embedded tissue, methylation-specific polymerase chain reaction (MSP) after sodium bisulfite conversion13
is widely used. However, because of the qualitative nature of the assay, MSP cannot reliably distinguish low levels of methylation from high levels of methylation. Furthermore, MSP does not appear to be highly reproducible for some samples that may have low levels of methylation, and it is difficult to assess performance characteristics. Thus, clinical use of qualitative MSP is a major concern in terms of quality control and assurance. Quantitative measurement of methylation is important because low levels of methylation (below the threshold of transcriptional silencing) may not be biologically important. Hence, a variety of quantitative assays to measure DNA methylation have been developed, including combined bisulfite restriction analysis,14
restriction ligation-mediated polymerase chain reaction (PCR),15
methylation-sensitive single nucleotide primer extension (Ms-SNuPE),16
ion-pair reverse-phase high performance liquid chromatography,17
denaturing high performance liquid chromatography,18
pyrosequencing,19, 20, 21
MALDI-TOF,22
and real-time PCR.5, 23, 24, 25, 26, 27, 28, 29, 30, 31
Most of these methods use DNA treated with sodium bisulfite. Therefore, sodium bisulfite treatment is a critical step in the measurement of DNA methylation. However, reproducibility of the sodium bisulfite conversion step has not been well investigated. Sodium bisulfite treatment presents a harsh environment for DNA molecules, and previous data suggest that 
84 to 96% of DNA is degraded during 4 hours of bisulfite treatment at 55°C.32
Assurance of reproducibility in this critical bisulfite treatment step is essential in applying quantitative methylation assays to clinical practice.
In this study, we evaluated the precision and performance characteristics of sodium bisulfite treatment and MethyLight, a real-time PCR assay.5, 26, 28, 33 Our data indicate that both sodium bisulfite conversion and MethyLight assays have good reproducibility and precision and can be effectively used to quantify DNA methylation in paraffin-embedded tumor tissue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Sodium Bisulfite Treatment and DNA Extraction
Hematoxylin and eosin (H&E)-stained slides of the tumors were reviewed, and areas of tumor were marked, to exclude pure normal tissue and enrich tumor DNA. H&E-stained tumor sections (10 µm x 14 sections) were scraped off slides from each of case 1 through case 4 and suspended in 140 µl of tissue lysate solution (100 mmol/L Tris, pH 8, 10 mmol/L ethylenediaminetetraacetic acid, pH 8, 1 mg/ml proteinase K, and 0.05 mg/ml tRNA). The lysate solutions were incubated overnight at 50°C. The lysate was aliquoted into seven tubes, each containing 18 µl of tissue lysate, and stored at –20°C until sodium bisulfite modification was performed. For methylation and immunohistochemistry study (using cohort colorectal cancer cases), H&E-stained tissue sections (depending on tissue and tumor size, in average, large tumor tissue 10 µm x 1 section) from each case were scraped off slides, suspended in 20 µl of the tissue lysate, and incubated at 50°C overnight. The tissue lysate was then stored at –20°C until sodium bisulfite modification was performed.
Sodium metabisulfite (1.9 g) was dissolved in mixture of 3.2 ml of 0.44 mol/L NaOH at 50°C. Then, 0.5 ml of 1 mol/L hydroquinone was added to the dissolved sodium bisulfite mixture. An 18-µl aliquot of the tissue lysate was denatured at 100°C for 10 minutes and chilled on ice. Then, 2 µl of 3 mol/L NaOH was added and incubated at 42°C for 20 minutes. The bisulfite solution (120 µl) was added (total volume of 140 µl) and incubated at 50°C for 15 hours in the dark. The bisulfite-converted DNA was recovered using a Qiagen QIAamp viral RNA mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions with some modifications. Buffer AVL/carrier RNA (560 µl) was added to the 140 µl of bisulfite-converted DNA sample and incubated at room temperature for 10 minutes. Ethanol (560 µl) was then added, and after extensive mixing, the mixture was loaded onto the provided spin columns in consecutive 630-µl aliquots. After each loading, the columns were centrifuged at full speed (21,000 x g) for 1 minute. Both the filtrate and spin column were saved, and both filtrates were passed through the column a second time in the same manner to increase the yield of recovery. The spin column was then washed with 500 µl of buffer AW1, followed by centrifugation at 21,000 x g for 1 minute. Buffer AW2 (500 µl) was then added to the column, and the column was centrifuged at 21,000 x g for 4 minutes to wash the column and eliminate possible buffer AW2 carry over. DNA in the spin column was eluted by the addition of 40 µl of buffer AVE, followed by a 1-minute incubation at room temperature and centrifugation at 7600 x g for 1 minute. This elution step was repeated with a second 40-µl volume of buffer AVE. Fifty µl of 0.2 mol/L NaOH was added to the 80-µl pooled eluate for 15 minutes at room temperature to desulphonate the sample, and then 10 µl of 1 mol/L HCl was added to for neutralization. Buffer AVL/carrier RNA (560 µl) was then added to the 140-µl sample mixture, and the recovery procedure was repeated with a new spin column. The eluted DNA (80-µl volume) was then used for MethyLight analysis.
Quantitative Real-Time PCR (MethyLight)
Real-time PCR assays to measure DNA methylation (MethyLight) have been described.5, 26, 28
Briefly, three sets of primers and probes designed specifically for bisulfite-converted DNA were used: a set for the gene of interest and two sets for ACTB and COL2A1 to normalize for the amount of input DNA. We used an ABI 7300 real-time PCR instrument (Applied Biosystems, Foster City, CA). Primers and probes for CDKN2A, MGMT, ACTB, and COL2A1 were previously described.5
The MLH1 forward primer is 5'-AGG AAG AGC GGA TAG CGA TTT-3', the MLH1 reverse primer is 5'-TCT TCG TCC CTC CCT AAA ACG-3', and the MLH1 probe is 6FAM-5'-CCC GCT ACC TAA AAA AAT ATA CGC TTA CGC G-3'-BHQ1. The amount of methylated DNA (PMR, percentage of methylated reference36
) at a specific locus was calculated by dividing the GENE:ACTB (or COL2A1) ratio of a sample by the GENE:ACTB (or COL2A1) ratio of SssI-treated human genomic DNA (presumably fully methylated) and multiplying by 100. Reactions using SssI-treated DNA were used to normalize for any difference in amplification efficiencies between GENE and ACTB (or COL2A1).
Measuring the Precision of Sodium Bisulfite Conversion and MethyLight Assay
Figure 1
shows our strategy to measure precision and reproducibility of bisulfite conversion of DNA and quantitative MethyLight assay. We performed bisulfite conversion on seven different aliquots from each of the four cell lysate samples (case 1 through case 4), starting on 7 different days. Thus, from each cell lysate, we have seven separate bisulfite-converted DNA samples (designated as B1 through B7), which ideally should give at least similar values for methylation at a specific locus. We measured PMR for each locus by the MethyLight assay on each of these seven separate bisulfite-DNA samples. For each case, we measured the coefficient of variation (CV) of these seven PMR values on B1 through B7, which would primarily depend on variations in bisulfite conversion. We repeated the MethyLight assays five times (designated as M1 through M5) and determined PMR values on the exact same set of bisulfite-converted DNA samples (B1 through B7) on 5 different days. We calculated the CV of these five PMR values (M1 through M5) on each one of the seven bisulfite-converted DNA samples (B1 through B7). Thus, these seven CVs would primarily depend on day-to-day variations of each MethyLight assay.
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). To obtain PMR variations solely due to variations of standard curves, we also performed a separate MethyLight run with five standard curves in a single plate and calculated five different PMR values for each sample-marker combination, using each one of the five different standard curves.
DNA Mixing Study to Assess Assay Linearity
We performed a mixing study to assess linearity of sodium bisulfite treatment and quantitative MethyLight assays (Figure 2)
. We first treated human peripheral blood leukocyte DNA (Promega, Madison, WI) with SssI to methylate all CpG sites. Assuming near complete methylation and no loss of DNA during SssI treatment, we prepared DNA mixtures in triplicates (each containing 1 µg of template DNA) for SssI-treated DNA:untreated DNA as follows: 50:50, 30:70, 20:80, 10:90, and 5:95 (Figure 2)
. We used 100% SssI-treated DNA as control reference DNA to calculate PMR as described above. We treated DNA mixtures in triplicates with sodium bisulfite and duplicated MethyLight assays, resulting in six data points of PMR values for each dilution. Linear regression analysis was performed to assess assay linearity.
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Statistical Analysis
For statistical analysis, paired t-test to compare means, F-test to assess the equality of variances38
and linear regression analysis to assess assay linearity were performed using the Microsoft Excel 2000 Analysis ToolPak. For F-test, raw PMR values were log-transformed to make distributions close to normal. Interclass correlation coefficients were calculated to assess assay reproducibility using the SAS program (version 9.1; SAS Institute, Cary, NC). To assess the association between promoter methylation and loss of protein expression, Fisher’s exact test was performed using the SAS program. All P values were two-sided.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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. Case 1 showed positive reaction (Ct <40) in MLH1, cases 2 and 3 showed positive reaction in CDKN2A and MGMT, and case 4 showed positive reaction in CDKN2A and MLH1. Table 1
shows representative Ct values (case 2, samples B1 through B7) for each of the five different MethyLight analyses (M1 through M5) for CDKN2A. We designated SD(B) as bisulfite-to-bisulfite SD (SD) (within-run variation in seven bisulfite-treated samples B1 to B7) in a particular MethyLight run, and SD(M) as run-to-run SD (within-sample variation by five runs M1 to M5) on a particular sample (Figure 1
and Table 1
). Table 2
shows SD(B) and SD(M) for all positive markers and control genes ACTB and COL2A1 in cases 1 through 4, which appeared comparable with SDs of Ct values that have been described in various real-time PCR assays.39, 40, 41
SD(B) and SD(M) of Ct values for COL2A1 on the four cases were significantly smaller than those for ACTB (P = 0.0004 by paired t-test).
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(case 3, samples B1 to B7) in the five different MethyLight runs (M1 to M5) for MGMT. We designated CV(B) as bisulfite-to-bisulfite CV, ie, within-run variation in seven bisulfite-treated samples B1 to B7, and CV(M) as MethyLight-to-MethyLight run-to-run CV, ie, within-sample variation by five runs M1 to M5.
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4 indicated methylation-negative.33
In our analysis, negative markers typically showed PMR 1 and were most frequently undetected (Ct >45 and PMR = 0). There were a total of five case-negative marker combinations (case 1, CDKN2A; case 1, MGMT; case 2, MLH1; case 3, MLH1; and case 4, MGMT). Within a total of 175 PMR results (five case-marker combinations times seven bisulfite-treated samples times five MethyLight runs) for these negative reactions, when using COL2A1 as a control, 160 runs (91%) showed PMR values of 0, 14 runs (8%) showed a PMR <1, and only one run (0.6%) showed PMR >1 (PMR 1.01). However, when using ACTB as a control, 4 of 175 negative reactions (2.3%) showed PMR >1 (PMR ranging from 1.10 to 3.59). Notably with either reference, none of these 175 presumed negative reactions showed PMR >4.
Mean and variance of PMR values in MethyLight using ACTB as a control were compared to those using COL2A1 as a control (Table 4)
. For each case-marker combination, we obtained PMR values 35 times (seven different bisulfite-treated samples times five MethyLight runs) using each one of ACTB and COL2A1 as a control. Distributions of PMR values became closer to normal distributions after log transformation of raw PMR values. Thereafter, we assessed the equality of variances in the 35 repeated runs. PMR variances using ACTB were significantly larger than those using COL2A1 in four of seven case-marker combinations (P < 0.003) (Table 4)
, indicating COL2A1 as a superior control. CVs of PMR values in MethyLight using COL2A1 as a control are shown in Table 5
. Both bisulfite-to-bisulfite variation [CV(B)] and run-to-run variation [CV(M)] were similar, ranging from 0.05 to 0.6. Therefore, MethyLight assay showed fair reproducibility in terms of both bisulfite-to-bisulfite variations [CV(B)] and run-to-run variations [CV(M)]. To assess within-sample variation of PMR values, interclass correlation coefficients were calculated as 0.84, 0.74, and 0.79 for CDKN2A, MLH1, and MGMT, respectively, indicating fair reproducibility of MethyLight assays.
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Assay Linearity
We performed repeated measurements of PMR values on DNA mixtures containing 50%, 30%, 20%, 10%, and 5% of methylated (M-SssI-treated) DNA (Figures 2
and 3)
. Correlation coefficients and P values for the test of a linear association were as follows: r = 0.962, P = 3 x 10–17 for CDKN2A; r = 0.913, P = 2 x 10–12 for MLH1; and r = 0.904, P = 10–9 for MGMT. Thus, our MethyLight assay showed fair to good linearity for all three genes.
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. There were only rare cases with PMR values between 3 and 5. As such, the MethyLight assay for each gene appears to distinguish two distinct subsets of colorectal cancer, reflecting methylation-negative and -positive tumors. Our previous MethyLight analyses have defined methylated promoters as those with PMR >4 and unmethylated promoters as PMR 4.33
The bimodal distributions of PMR values in Table 6
appear to support a cutoff of 4.
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. Notably, intact protein expression was observed in 96% (156 of 163), 99% (224 of 227), and 86% (120 of 139) of samples with PMR 4 for the promoters of CDKN2A, MLH1, and MGMT, respectively. Loss of protein expression was observed in 62% (45 of 73), 96% (43 of 45), and 66% (69 of 104) of samples with PMR >4 for CDKN2A, MLH1, and MGMT, respectively. Using a PMR cutoff of 4, promoter methylation was significantly associated with loss of respective protein expression (P = 1 x 10–21 for CDKN2A, P = 6 x 10–44 for MLH1, and P = 1 x 10–17 for MGMT). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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In this study, we evaluated precision of sodium bisulfite conversion as well as subsequent real-time PCR (MethyLight) assays for the CDKN2A (p16), MLH1, and MGMT promoters. Advantages of MethyLight technology include its quantitative and high-throughput nature and relatively simple assay procedures, which do not require opening of tubes containing PCR products. All of these features are extremely beneficial in clinical molecular diagnostics. Any of these properties is not a feature of qualitative MSP, which has been widely used for methylation detection. As for disadvantages of MethyLight, it cannot quantify methylation at the individual nucleotide level; rather, it can assess methylation levels at the primer sites and/or a probe site as a whole. On the other hand, bisulfite-sequencing, such as bisulfite-pyrosequencing,19, 20, 21 can achieve resolution at the individual nucleotide level. However, in contrast to MethyLight, pyrosequencing involves end-point analysis of PCR products and requires procedures that need opening of PCR tubes.
To measure the relative amount of methylated DNA, we divided the measured amount of a methylated gene of interest by the amount of COL2A1 (or ACTB) for input DNA. One should also be aware of PCR bias in quantitative molecular assays,42 so we further normalized for a potential difference in PCR efficiencies between the gene of interest and COL2A1 by dividing the gene:COL2A1 ratio in a sample of interest by that ratio in SssI-treated DNA (presumably fully methylated). For all three genes tested, we showed good precision in threshold cycle (Ct) values in terms of both bisulfite-to-bisulfite variation (among seven bisulfite-treated aliquots B1 to B7) in the same run and MethyLight-to-MethyLight run-to-run variation (among five repeated MethyLight runs M1 to M5) in the same bisulfite-treated DNA sample. COL2A1 reactions, as well as PMR values based on COL2A1 reactions, showed much smaller variances than ACTB. Thus, COL2A1 is a superior control gene to ACTB in terms of precision of MethyLight assays.
We also demonstrated that run-to-run CVs of PMR values were larger than bisulfite-to-bisulfite CVs of PMR values. This likely reflects the introduction of other sources of variations when we measured run-to-run CVs of PMR, including different standard curves. Thus, we also examined variations of standard curves in a single plate as well as between different MethyLight runs. We demonstrated that variations of standard curves were small and acceptable. Other sources of run-to-run variations are independent control reactions (COL2A1 or ACTB) in different MethyLight runs. A gene that showed small variation in repeated amplification reactions, such as COL2A1, would be suitable for a control. Normalizing methylation measurement for input DNA by a control reaction is necessary to compare quantitative methylation data across samples. Quality and quantity of DNA and the degree of bisulfite conversion can vary from sample to sample.
To assess acceptability of the precision of sodium bisulfite conversion and subsequent MethyLight assays, we measured PMR values (degrees of methylation) for the CDKN2A, MLH1, and MGMT promoters in 272 cases of colon cancer. We demonstrated that overall distributions of PMR values for all three loci were bimodal (either PMR 1 or >10) with only rare cases showing PMR values between 3 and 5. For most cases, CVs of PMR were sufficiently low to definitively differentiate methylation-positive tumor from methylation-negative tumor, with a PMR cutoff of 4. Thus, our data indicate that precision of sodium bisulfite conversion and quantitative Methy-Light assays is acceptable, and we can reliably determine the status of methylation for most cases by a single MethyLight run.
In addition, for each of the three genes tested, promoter methylation was strongly associated with loss of respective protein expression. Low PMR values between 0 and 4 were principally unrelated to loss of expression and, therefore, seemed to represent biologically insignificant low levels of methylation. There were infrequent cases with loss of expression without promoter methylation and cases with intact protein expression despite PMR values >10. The former may reflect different mechanisms of gene silencing, including gene deletion or mutation, and the latter may reflect partial methylation or mono-allelic methylation. Difficulty in interpreting immunohistochemistry, particularly for MGMT and CDKN2A (p16), may also contribute to some of the discrepant results.
In conclusion, sodium bisulfite conversion is reproducible, and subsequent quantitative real-time PCR methylation assays have acceptable precision. Quantitative promoter methylation data are highly correlated with loss of protein expression. Carefully validated quantitative MethyLight assays will be useful in both research and clinical molecular diagnostics.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Acknowledgments |
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Footnotes |
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Supported by the National Institutes of Health (grants P01 CA87969-03 and P01 CA55075-13).
P.W.L. is shareholder and Scientific Advisory Board Member of Epigenomics, AG, which has a commercial interest in the development of DNA methylation markers for disease detection and diagnosis. None of the work described in this manuscript was supported by Epigenomics, AG.
Accepted for publication December 6, 2005.
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