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Department of Molecular Oncology, John Wayne Cancer Institute, Santa Monica, California
Requests for reprints: Dave S.B. Hoon, Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Boulevard, Santa Monica, CA 90404. Phone: 310-449-5264; Fax: 310-449-5282. E-mail: hoon{at}jwci.org
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Abstract |
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Introduction |
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5% of cytosines are modified to 5-methylcytosine (1), and this modification has important regulatory effects on gene expression especially when it involves cytosine-guanine dinucleotide (CpG) rich areas (CpG islands) in promoter regions (2, 3). Epigenetic gene silencing by promoter hypermethylation of tumor suppressor genes is as significant as deletions or mutations (4-6), and it can play a significant role in malignant transformation and immortalization of cells. Therefore, assessment of promoter hypermethylation has become important for understanding the mechanisms of malignant transformation (7, 8). Sodium bisulfite modification (SBM) of genomic DNA is commonly used to detect methylation of CpG islands in promoter regions. Sodium bisulfite converts cytosines but not 5-methylcytosines to uracils (9). Therefore, the methylation status reflects the changes in the sequence of the DNA after modification. The modified DNA can be analyzed by sequencing (10), methylation-specific PCR (MSP; ref. 11), combined bisulfite restriction analysis (12), high-throughput quantitative methylation assay (MethyLight; ref. 13), quantitative analysis of methylated alleles (14), and other methods. The most widely available starting material for assessment of solid tumors is formalin-fixed, paraffin-embedded archived tissue (FF-PEAT) used for pathologic diagnosis after surgical removal of the tumor or after tumor biopsy. Use of FF-PEAT for epigenetic analysis, such as MSP, would have significant use to evaluate patients' specimens. As described in a previous report, there are critical issues with SBM of FF-PEAT (15, 16), formalin fixation causes fragmentation and cross-linking of the DNA that is dependent on the acidity of the formalin and soaking time. In addition, 84% to 96% of the source DNA may be lost during conventional SBM (17). This significant loss of template DNA hampers assessment of methylation; a previous study reported that 50 cycles of thermal cycling in MSP were needed for SBM DNA extracted from a small area of FF-PEAT by microdissection (18). This high number of PCR cycles would detect only a single copy of DNA, which increases the risk of false-positive results due to incomplete SBM, and may not be reproducible. Extracting DNA from a larger tumor area or whole-tumor sections increase yields of DNA would result in more reproducible results, but it increases the risk of contamination with DNA from noncancer cells.
On-slide SBM, in which DNA is modified in the tissues, was initially developed for MSP in situ hybridization (19), but its application has been very limited to date. However, on-slide SBM should minimize loss of DNA because it eliminates DNA purification steps. We have developed and optimized a protocol for on-slide SBM that can be easily adapted in laboratories to do histology-oriented methylation analyses. We validated the use of on-slide SBM using primary colorectal cancers with intratumoral adenoma component. Specificity was assessed by estrogen receptor 1 (ESR1) and thymine-DNA glycosylase (TDG) promoter methylation status. Intratumoral heterogeneity of p16 and Ras association domain family protein 1a (RASSF1a) promoter methylation status was also assessed. The feasibility of direct sequencing of a relatively long PCR product was tested to show that DNA modified by the on-slide SBM technique is degraded to a lesser extent compared with conventional SBM.
To evaluate the yield and conversion rate (i.e., percentage of modified DNA) of on-slide SBM, we have newly developed a highly sensitive quantitative real-time PCR (qPCR) assay for bisulfite-modified and unmodified DNA (i.e., genomic DNA). Alu, which is the most abundant repeat sequence in the human genome with a copy number of 1.4 x 106 in a cell (20), was used as a target of qPCR to maximize sensitivity. It allowed accurate quantification and precise evaluation for samples having very low amount of modified DNA. Bisulfite-modified DNA and the remaining unmodified DNA were separately quantified by Alu-qPCR with specifically designed primer sets.
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The Yield and Conversion Rate of Modification
The yield and conversion rate of modification was evaluated using eight tissue samples for on-slide, standard, and agarose-bead SBM by assessing the copy numbers of modified and unmodified Alu repeat sequences. Each sample was assessed in triplicate, and the median intersample coefficient of variance for modified and unmodified DNA was 6.8% and 11.2%, respectively.
The yield of modified DNA increased with the duration of incubation of on-slide SBM. When modified Alu copy numbers of on-slide and standard SBM were compared, the relative yield of modified DNA by on-slide SBM was higher by a mean factor of 1.3 ± 0.8 and 2.6 ± 1.0 (mean ± SD) at incubation settings of 60°C for 4 hours and 60°C for 8 hours, respectively (Fig. 3 ). The relative yield of modified DNA by agarose-bead SBM was only 0.9 ± 1.0 (mean ± SD) after 14 hours of incubation at 50°C.
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Optimization on Glass Slides
Because tissue sections did not adhere consistently enough to noncoated glass slides during on-slide SBM treatment, we initially tested the adhesive-coated, positive-charged slides (Superfrost Plus microscope slides, Fisher Scientific, Pittsburgh, PA) that are commonly used for immunohistochemistry. However, long incubation periods at increased temperatures in acidic or alkaline solutions sometimes caused tumor tissue sections to detach, and experiments had to be repeated. Therefore, we tested silane coated slides (Tekdon, Inc., Myakka City, FL) to improve the robustness of the protocol on 20 colorectal cancer tissue samples. Consequently, none of the tissue sections detached during the on-slide SBM process. The yield of modified DNA and conversion rate was equivalent to the other kinds of glass slides (data not shown). Because of the improvement of robustness of the protocol, we advocate use of silane coated slides for on-slide SBM.
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Discussion |
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The success of methylation analysis depends on yield of modified DNA and completeness of conversion. When the yield of modified DNA is low, it becomes difficult to detect the methylation status of small amounts of tissue. If the conversion rate is low, false-positive results for methylation can be observed in unmethylated DNA by PCR-based nonquantitative methods because of misgenerated DNA coincidentally having the similar sequence of methylated DNA. Our results showed that TDG, which was unmethylated by standard SBM in a pilot study using 26 colorectal cancer tissues and six colorectal cancer cell lines, was unmethylated by on-slide SBM in all 16 pairs of cancer and adenoma tissues. In addition, ESR1, which is reportedly methylated in all colorectal cancers (22), was methylated by on-slide SBM in all 16 pairs. Therefore, on-slide SBM is accurate enough for highly-sensitive methods, such as MSP, and does not cause false-negative or false-positive results. After incubation at 60°C for 8 hours, the conversion rate of on-slide SBM was similar to that of standard SBM. Furthermore, the yield was greater than that of agarose-bead SBM, which is commonly used for specimens containing minimal DNA, such as microdissected FF-PEAT tissue or blood serum.
We did not pretreat the tissue samples with a digestive enzyme, such as pepsin or proteinase K, that elevates permeability of reaction solution routinely done in a previous MSP in situ hybridization report (19), because predigestion can damage the microscopic tissue structure. Moreover, in our preliminary assessment, predigestion did not significantly increase the conversion rate or the yield of modified DNA (data not shown). Eliminating the predigestion step maintains the microscopic morphology of the tissue during SBM and allows subsequent hematoxylin staining and microdissection.
We initially planned to use a SBM direct sequencing technique to evaluate the conversion rates between three SBM methods. Assessment of 282 bp of the p16 promoter region of samples treated with the on-slide SBM protocol showed a complete, 100% conversion. Therefore, doing sequencing on modified DNA of a limited number of samples to evaluate the conversion rate was considered not fit to assess subtle differences among the three SBM methods. In addition, our preliminary assessment of SBM DNA revealed that unmodified cytosines are not randomly scattered but exist as clusters in the genome (data not shown) probably because of regional protection from bisulfite, such as fixed double helix. Therefore, we aimed to develop an assay to quantify bisulfite-modified and unmodified DNA using Alu-qPCR for evaluation of yield and conversion rate of SBM DNA. This assay can be widely used as a tool for preevaluation of quality of SBM DNA. In addition, it can be widely used to standardize the amount of template SBM DNA in each reaction of methylation assessment. Using this technique, we showed that on-slide SBM yielded 2.6 times more modified DNA than standard SBM. This is likely caused by loss of DNA in purification steps before and during the standard SBM. The high yield of on-slide SBM is a great advantage when the target tissue is small. In addition, on-slide SBM allowed 282-bp bisulfite direct sequencing from very small amount of FF-PEAT, reflecting the high quality of modified DNA.
Total reagent cost and labor cost for on-slide SBM were relatively low because it does not require DNA purification. This is particularly advantageous when multiple areas of a tissue section are assessed simultaneously because DNA modification is completed before microdissection. Another advantage of on-slide SBM is consistency of modification conditions, such as incubation temperature, incubation duration, and concentration of bisulfite within a slide. Therefore, on-slide SBM should be especially suitable for assessments of intratumoral heterogeneity.
In conclusion, 8-hour incubation at 60°C in 3 mol/L sodium bisulfite solution without predigestion maintained tissue architecture during on-slide SBM. Yield of modified DNA was 2.6-fold greater than that of standard SBM. Bisulfite direct sequencing of a 282-bp amplicon was possible. Conversion rate was 97.1%, and no false-positive or false-negative results in the subsequent MSP for ESR1 and TDG were observed. Intratumoral heterogeneity of p16 and RASSF1a gene methylation representing pathologic malignant transformation was shown. On-slide SBM should be applicable in most methylation studies using FF-PEAT especially in assessments of intratumoral heterogeneity in solid tumors. The approach provides a unique opportunity to assess retrospective archived procured tissues from clinical trials in which there is limited tissue sections availability.
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Materials and Methods |
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On-Slide SBM and Subsequent Microdissection
To evaluate the yield and conversion rate of on-slide SBM, deparaffinized and rehydrated tissue sections were incubated in 0.2 mol/L NaOH at room temperature for 15 minutes and then incubated in 3 mol/L sodium bisulfite solution with 0.5 mmol/L hydroquinone (pH 5) in the dark. We tested four incubation settings (60°C for 4 hours, 60°C for 8 hours, 70°C for 4 hours, and 70°C for 8 hours) for three colorectal cancer specimens in the initial evaluation set. Eight colorectal cancer specimens were used with two incubation settings (60°C for 4 hours and 60°C for 8 hours) to compare conversion rate and yield of modified DNA between on-slide SBM and conventional SBM methods. After incubation, sections were rinsed with distilled water, soaked in 0.3 mol/L NaOH for 15 minutes to desulfonate the modified cytosines, and then desalted in distilled water at 60°C for 2 hours. After light staining with hematoxylin, 25-mm2 cancer tissue was microdissected under a microscope from each section. The microdissected areas of each section were identical for each incubation setting. Microdissected tissues were digested in 50 µL lysis buffer containing 4 µg proteinase K, 2.5% Tween 20, 50 mmol/L Tris, and 1 mmol/L EDTA at 50°C for 5 hours followed by heat deactivation of proteinase K at 95°C for 10 minutes. In each subsequent qPCR, 2 µL lysate was used as a template without DNA purification.
To validate the use of on-slide SBM to study intratumoral heterogeneity in methylation status, paired cancer and adenoma tissues were microdissected from each on-slide SBM section treated at the conditions that were found to be optimal. From each specimen, 1-mm2 tissue was microdissected and consisted of 2 to 10 ducts of cancer or adenoma tissue. Each specimen was digested in 10 µL lysis buffer; 1 µL lysate was used as a template in subsequent MSP or direct sequencing without DNA purification.
Standard SBM on Purified DNA from Microdissected Tissue
For comparison of standard SBM with on-slide SBM DNA, purified DNA from microdissected FF-PEAT sections was bisulfite modified with standard SBM method as described previously (23). DNA was extracted from the same area (25 mm2) of each tissue section as on-slide SBM. DNA was purified with phenol-chloroform-isoamylalcohol from proteinase K–digested tissues microdissected from deparaffinized sections and modified by sodium bisulfite 60°C for 3 hours with 1 µg salmon sperm DNA as a carrier using the same bisulfite solution as described above. Modified DNA was recovered using the Wizard DNA Clean-Up System (Promega, Madison, WI), desulfonated with 0.3 mol/L NaOH, and resuspended in 50 µL Tris-EDTA buffer; 2 µL of the solution were used as a template in subsequent qPCR.
Agarose-Bead SBM on Microdissected Tissue
Bisulfite modification of DNA in digested tissues trapped in agarose beads (24) does not require the DNA purification steps and therefore allows a relatively high recovery rate of DNA from FF-PEAT samples. Same area (25 mm2) of each tissue section was microdissected and treated. For bisulfite modification, digested tissues trapped in 2% agarose beads containing 0.2 mol/L NaOH were incubated in bisulfite solution, same as for on-slide and standard SBM, at 50°C for 14 hours. After modification, beads were desalted as described previously (24) and heat diluted up to 50 µL with Tris-EDTA buffer; 2 µL of the diluted agarose gel were directly used as a template in subsequent qPCR without DNA purification.
Quantification of Bisulfite-Modified and Unmodified DNA
To determine the yield and conversion rate of SBM DNA, bisulfite-modified and unmodified DNA was separately quantified by Alu-qPCR. Primers specific for bisulfite-modified Alu sequence independent to the methylation status were 5'-TTGTAATTTTAGTATTTTGGGAGGT-3' (forward) and 5'-TCACCATATTAACCAAACTAATCTC-3' (reverse); their amplicon size was 83 bp. Primers specific for unmodified Alu sequence were 5'-CCTGAGGTCAGGAGTTCGAG-3' (forward) and 5'-CCCGAGTAGCTGGGATTACA-3' (reverse); their amplicon size was 115 bp. The reaction mixture for each Alu-qPCR consisted of SBM DNA template, 0.2 µmol/L forward and reverse primers, 1 unit iTaq DNA polymerase (Bio-Rad Laboratories, Hercules, CA), 0.02 µL fluorescein calibration dye (Bio-Rad Laboratories), and 1x concentration of SYBR Gold (Molecular Probes, Eugene, OR) in a total reaction volume of 20 µL with 5 mmol/L Mg2+. Real-time PCR amplification was done with a precycling heat activation of DNA polymerase at 95°C for 10 minutes followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 59°C (primer set for modified Alu) or 64°C (primer set for unmodified Alu) for 30 seconds, and extension at 72°C for 30 seconds using iCycler iQ Real-time PCR Detection System (Bio-Rad Laboratories). The absolute copy numbers of modified and unmodified Alu sequence in each sample were determined from the threshold cycle numbers with a standard curve done by serial dilutions of bisulfite-modified or unmodified Alu fragment, which was purified from PCR product and quantified by UV absorption. Triplicate reactions of qPCR were done, and each reaction plate included a negative control (no template). PCR products were electrophoresed on 2% agarose gels to confirm product size and specificity of the PCR. The relative yield was calculated as Qos / Qstd for on-slide SBM and Qag / Qstd for agarose-bead SBM, where Qstd, Qos, and Qag are the absolute copy numbers of modified Alu of standard, on-slide, and agarose-bead SBM, respectively. The conversion ratio was calculated as Qmod / (Qmod + Qunmod), where Qmod and Qunmod are the absolute copy numbers of modified and unmodified Alu, respectively. Cross-reaction of Alu primers on the salmon sperm DNA used as a carrier in the standard SBM was assessed using a blank sample prepared without tissue DNA.
Bisulfite Direct Sequencing for p16 Gene Promoter Region
To show the feasibility of on-slide SBM for bisulfite direct sequencing of relatively long target, we sequenced 282 bp of the modified p16 gene promoter, which is usually difficult for DNA from FF-PEAT modified by standard SBM. Bisulfite direct sequencing was done as described previously (25). The primers were 5'-GGGTAGGTGGGGAGGAGTTTAGTTT-3' (forward) and 5'-AATAACCAACCAACCCCTCCTCTTT-3' (reverse). PCR amplification was done in a 50-µL reaction volume using 1-µL template with a precycling heat activation of DNA polymerase at 95°C for 10 minutes followed by 36 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds followed by final extension at 72°C for 7 minutes with 2.5 mmol/L concentration of Mg2+ and 0.2 µmol/L of each primer concentration. After PCR products were run on agarose gels and purified, they were directly sequenced using CEQ Dye Terminator Cycle Sequencing kit (Beckman Coulter, Inc., Fullerton, CA) according to the manufacturer's instructions. Cycling program includes 30 cycles of denaturation at 95°C for 20 seconds, annealing at 55°C for 40 seconds, and extension at 60°C for 4 minutes. Sequences were read by CEQ 8000XL CAE System (Beckman Coulter).
MSP for ESR1, TDG, p16, and RASSF1a
Methylation status of CpG islands in ESR1, TDG, p16, and RASSF1a gene promoter regions of paired cancer and adenoma tissues was assessed by MSP. Forward primers of MSP were labeled with D2, D3, or D4 WellRED dye-labeled phosphoramidites (Beckman Coulter). The methylation-specific primers for ESR1 were 5'-D4-TAAATAGAGATATATCGGAGTTTGGTACG-3' (forward) and 5'-AACTTAAAATAAACGCGAAAAACGA-3' (reverse); the unmethylation-specific primers for ESR1 were 5'-D3-TAAATAGAGATATATTGGAGTTTGGTATGG-3' (forward) and 5'-AACTTAAAATAAACACAAAAAACAAA-3' (reverse). The methylation-specific primers for TDG were 5'-D4-TGAGCGTGATTGGGTTGTTTTATATTAC-3' (forward) and 5'-TTCTACCGTCCCCCACGA-3' (reverse); the unmethylation-specific primers for TDG were 5'-D3-AGTGTGATTGGGTTGTTTTATATTATGT-3' (forward) and 5'-CTTCTACCATCCCCCACAAA-3' (reverse). The methylation-specific primers for p16 were 5'-D4-TTATTAGAGGGTGGGGCGGATCGC-3' (forward) and 5'-CGATTTCGGGTCGCGGTCGTGG-3' (reverse); the unmethylation-specific primers for p16 were 5'-D2-TATTAGAGGGTGGGGTGGATTGT-3' (forward) and 5'-TGATTTTGGGTTGTGGTTGTGG-3' (reverse). PCR conditions were optimized to achieve highest sensitivity without false-positive results using universal methylated control DNA and universal unmethylated control DNA as described previously (25). The optimized annealing temperatures for methylated-specific primers for ESR1, unmethylated-specific primers for ESR1, methylated-specific primers for TDG, and unmethylated-specific primers for TDG, methylated-specific primers for p16, and unmethylated-specific primers for p16 were 61°C, 58°C, 62°C, 61°C, 62°C, and 61°C, respectively. Primers and PCR settings used for amplification of the RASSF1a promoter region were published previously (23). PCR amplification was done in a 10-µL reaction volume using 1-µL template with a precycling heat activation of DNA polymerase at 95°C for 10 minutes followed by 36 cycles of denaturation at 95°C for 30 seconds, annealing at optimized annealing temperature for 30 seconds, and extension at 72°C for 30 seconds followed by final extension at 72°C for 7 minutes with 2.5 mmol/L concentration of Mg2+ and 0.2 µmol/L of each primer concentration. PCR products were detected and analyzed by CEQ 8000XL CAE System as described previously (26).
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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.
Note: N. Umetani and M.F.G. de Maat contributed equally to this work.
Received 10/11/05; revised 2/17/06; accepted 3/ 2/06.
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gene (GSTP1) CpG island is present in a subset of proliferative inflammatory atrophy lesions but not in normal or hyperplastic epithelium of the prostate: a detailed study using laser-capture microdissection. Am J Pathol 2003;163:923–33.This article has been cited by other articles:
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  M. F.G. de Maat, C. J.H. van de Velde, M. P.J. van der Werff, H. Putter, N. Umetani, E. M. Klein-Kranenbarg, R. R. Turner, J. H. J.M. van Krieken, A. Bilchik, R. A.E.M. Tollenaar, et al. Quantitative Analysis of Methylation of Genomic Loci in Early-Stage Rectal Cancer Predicts Distant Recurrence J. Clin. Oncol., May 10, 2008; 26(14): 2327 - 2335. [Abstract] [Full Text] [PDF]
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 M. F.G. de Maat, N. Umetani, E. Sunami, R. R. Turner, and D. S.B. Hoon Assessment of Methylation Events during Colorectal Tumor Progression by Absolute Quantitative Analysis of Methylated Alleles Mol. Cancer Res., May 1, 2007; 5(5): 461 - 471. [Abstract] [Full Text] [PDF]
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