NimbleGen Arrays and LightCycler 480 System: A Complete Workflow for DNA Methylation Biomarker Discovery and Validation.

Transcription

1 Cancer Research Application Note No. 9 NimbleGen Arrays and LightCycler 480 System: A Complete Workflow for DNA Methylation Biomarker Discovery and Validation Tomasz Kazimierz Wojdacz, PhD Institute of Human Genetics University of Aarhus, Denmark Department of Experimental Clinical Oncology Aarhus University Hospital, Denmark 1 Introduction Biomarkers related to epigenetic changes of DNA methylation are highly promising candidates for both early diagnosis and treatment of not only neoplastic, but also many other diseases. The biomarker development procedure consists of five steps: 1. Identification and discovery 2. Initial clinical assay validation 3. Retrospective validation on the archival material 4. Prospective validation in clinical settings 5. Use of the marker in clinical practice Each novel DNA methylation biomarker has to undergo the above procedure for which the first two steps are the most challenging and at the same time are the bottleneck of the process. We have combined NimbleGen DNA Methylation arrays and the Methylation Sensitive High Resolution Melting (MS-HRM) technology available on the LightCycler 480 System into a single workflow for fast and efficient discovery and initial validation of DNA methylation biomarkers. Tomasz Kazimierz Wojdacz Contributing Authors: Johanne Ageling Windeløv 1, Britta Boserup Thestrup 1, Jens Overgaard 2, Lise Lotte Hansen 1 1 Institute of Human Genetics, University of Aarhus, Wilhelm Meyers Alle 4, The Bartholin Building, DK-8000 Aarhus C, Denmark 2 Department of Experimental Clinical Oncology, Aarhus University Hospital, Nørrebrogade 44, DK-8000 Aarhus C, Denmark For life science research only. Not for use in diagnostic procedures.

2 In the first step of the workflow, a limited number of clinical research samples was analyzed for global DNA methylation changes using the NimbleGen Human DNA Methylation 2.1M Deluxe Promoter Array. This array provides comprehensive coverage of biologically important regulatory regions including 29,500 gene promoters, 27,891 CpG islands and mirna promoters that can exhibit changes in DNA methylation during disease progression. In the experiments described here, 20 DNA research samples extracted from fresh frozen breast cancer tissue, and five DNA research samples from normal breast tissues obtained from breast reduction surgery, were used in microarray experiments. On the basis of the microarray results, target loci displaying DNA methylation changes were identified and MS-HRM assays were designed for some selected targets. Since publishing of the MS-HRM technology three years ago, it has become the method of choice for DNA methylation studies with a limited number of target loci. The technique enables investigation of DNA methylation changes within loci of interest in both qualitative and semi-quantitative fashion and at the same time the protocol is labor, time and cost efficient. We have used MS-HRM assays to verify the microarray data and subsequently to make a decision with respect to the use of the selected target(s) in an extensive clinical research validation study. Only the markers showing highly significant correlation between microarray and MS-HRM results were subsequently included in the panel of potential novel biomarkers for retrospective and prospective clinical validation. What is epigenetics and why is it important? Epigenetics, a rapidly expanding segment of genetic research focused on diseases such as cancer, studies traits and attributes passed from generation to generation that are caused by mechanisms other than changes in the underlying DNA sequence. Epigenetic mechanisms include changes in chromatin structure, such as alterations in nucleosome positioning and composition or changes in DNA methylation within promoters, CpG islands, and genic/intergenic regions. Epigenetic changes are important because they affect numerous biological processes, such as gene expression, differentiation, development, chromosome stability, X chromosome inactivation, and disease development and progression. DNA Methylation and CpG Islands CpG methylation is the covalent attachment of methyl groups (CH 3 ) to the cytosine bases present on both strands of 5'-CG-3' dinucleotides. CpG islands are genomic regions that contain dense clusters of CG dinucleotides that are often associated with gene promoters. The methylation status of CpG islands and promoter regions can affect expression of the associated gene. 2 NimbleGen Arrays and LightCycler 480 System: A Complete Workflow for DNA Methylation Biomarker Discovery and Validation

3 2 Methods DNA was extracted from freshly frozen research tumor tissue samples and methylated DNA fragments were enriched from each sample using the MeDIP protocol 1. Two fractions from each sample (MeDIP-enriched and input) were labeled with Cy5 and Cy3, respectively, and co-hybridized to NimbleGen Human DNA Methylation 2.1M Deluxe Promoter Arrays. NimbleScan v2.6 Software (Roche NimbleGen) was used for feature extraction and subsequent annotation of the enrichment regions. Subtraction of enrichment regions was used as a statistical approach for the selection of differentially methylated regions. Subtraction calculates differentially methylated regions by subtracting the regions that showed significant enrichment on the array data. The test can be performed in both directions by subtracting tumors from controls or controls from tumors and regions of relative hypermethylation or hypomethylation are calculated, respectively (Figure 1). Three other methods, congruence, t-test and Chi2 test, were used to support the subtraction-based analysis in identifying the differentially methylated regions. However, primary calculations were based on the subtraction method. The regions with the highest scores for methylation differences were used for data validation. The MS-HRM technique was used as the single PCR-based method for microarray data validation. The MS-HRM assays were designed to target the candidate regions 2,3 and HRM experiments were performed as described in the LightCycler 480 System Application Manual 4. Methylation-Sensitive High-Resolution Melting (MS-HRM) High-resolution melting (HRM) enables the identification of a PCR product by exploring its sequence-dependent melting properties. In principle, three technical advances enabled development of the HRM technology: a new generation of saturating but non-inhibitory DNA dyes, a highly precise fluorescence-monitoring optical system, and new algorithms allowing detailed analysis of the melting profiles. All three elements are provided on the LightCycler 480 System, thus constituting a powerful platform for time-, labor- and costefficient investigation of DNA sequence variations. HRM technology was initially described for studies of single base pair changes. In principle, single-nucleotide changes result in aberration of the melting profile of the screened PCR product that can be visualized during an HRM scan. The adaptation of HRM technology to single-locus methylation studies allows for the detection of the methylated template in an unmethylated background at sensitivity equal to, or in some cases higher than, bisulfite sequencing. It provides insights into the methylation pattern of the screened PCR product, and methylation levels in the sample. In the experimental protocol, sodium bisulfite is first used to induce methylation-dependent single-nucleotide changes in the DNA template. Sodium bisufite changes cytosines into uracil, leaving 5-methyl cytosines untouched, thereby preserving the methylation marks of the template DNA. The induced changes are, in principle, sequence variations; therefore, adaptation of the HRM technology to studies of locus-specific methylation changes is straightforward. Cancer Research Application Note No. 9 3

4 Mean log 2 ratios Mean P-values Normal-tumor p-values Tumor-normal p-values > DNA methylation in normal samples > DNA methylation in tumor samples Normal samples Breast tumor samples Figure 1: Subtraction method overview. Log 2 signal ratios of each probe were averaged within each class, and NimbleScan analysis generated probe-level relative p-values for each class. A positive difference between the relative p-values of one class and the other, is interpreted as a relative increase in methylation in one class versus the other. How does MeDIP-chip work? Methylated DNA regions are first enriched by Methylated DNA Immunoprecipitation (MeDIP) using an antibody that recognizes and binds methylated CpG dinucleotides. The enriched methylated DNA fraction and an aliquot of the starting input material are then amplified, and the amplified materials are labeled with Cy5 and Cy3, respectively. The labeled samples are then pooled and hybridized to a microarray optimized for DNA methylation analysis such as the NimbleGen Human DNA Methylation 2.1M Deluxe Promoter Array. After hybridization, the arrays are washed, scanned and analyzed. 4 NimbleGen Arrays and LightCycler 480 System: A Complete Workflow for DNA Methylation Biomarker Discovery and Validation

5 3 Results The straightforward validation of the accuracy of our approach is the rediscovery of the hypermethylated regions that have been previously shown to undergo aberrant methylation in breast cancer. Indeed, in our microarray experiments we identified hypermethylated DNA regions in the promoters for genes, such as TWIST, CDKN2, GSTP1, CCND2 and MYOD1, which are known to be hypermethylated during breast cancer progression. Since MS-HRM assays for a number of these genes are already available in our laboratory, we have started validating the array experiments by screening the samples used in the microarray experiments with these MS-HRM assays. For these selected targets, the MS-HRM results correlated strongly with the array data (e.g., the frequency of hypermethylation of CCND2 gene was highly significant (p=0.02, Chi2 test)) confirming the microarray results. However, from the biomarker discovery point of view, the novel genes showing differential methylation in our sample panel were much more interesting. The initial statistical analysis of the data showed that over 1,000 loci represented on the microarray gained DNA methylation, and of those, 25 novel regions were selected for the initial validation experiments. For each of those regions MS- HRM assays were designed and the DNA methylation status of the new candidate biomarkers was investigated in the same research samples as used in microarray experiments. For all novel biomarkers highly significant DNA methylation frequencies were observed in MS-HRM experiments e.g. gene X p=0.0001, gene Y p= (Chi2 test). For some of our new target loci we observed low level (background) DNA methylation in the normal samples when using MS-HRM assays. Background methylation levels have previously been reported for different genes in various tissues. Screening the tumor specimens with the same assays showed significant increase of the methylation levels at these loci (Figure 2 shows representative MS-HRM results). In conclusion, despite the presence of low level DNA methylation of these loci in normal samples, the microarrays were still capable of identifying them as differentially methylated between the sample types. The above highlights the power of our approach which is capable of detecting not only de novo DNA methylation, but also differences in DNA methylation levels between two screened samples. 4 Discussion With the use of NimbleGen Human DNA Methylation 2.1M Deluxe Promoter Arrays, we were able to rediscover loci that have previously been shown to undergo aberrant DNA methylation during breast cancer carcinogenesis. More importantly, we discovered new aberrantly methylated genomic regions which now have to undergo validation for their prospective clinical use. For our validation experiments we have chosen the top 25 loci that showed the most significant differential DNA methylation in the array experiments. However, the vast number of differentially methylated regions discovered using MeDIP enrichment with the NimbleGen array provides a wealth of additional targets for future additional clinical investigation and validation. Our MS-HRM based validation experiments showed excellent correlation with the array data highlighting the accuracy of the array platform in the identification of the differentially methylated regions. Overall, the NimbleGen Human DNA Methylation 2.1M Deluxe Promoter Array is a robust and efficient tool for the identification of novel regions undergoing aberrant methylation during carcinogenesis. In validation experiments we used MS-HRM technology. The LightCycler 480 System is a platform supporting HRM analyses and the system allowed us to quickly optimize a panel of new MS-HRM assays and efficiently perform PCR-based verification of the microarray results which is recommended for any microarray based study. Cancer Research Application Note No. 9 5

6 A MS-HRM Results for a Novel Biomarker Normalized Melting Curves Relative Signal (%) Temperature ( C) B MS-HRM Results for a Novel Biomarker displaying low levels of methylation in normal (control) samples Normalized Melting Curves Relative Signal (%) Temperature ( C) Figure 2: Representative MS-HRM results for one of the novel biomarkers. Colors represent melting profiles for the controls, blue 100% methylation, red 10% methylation, yellow unmethylated controls. (A) The black line shows the melting curve for a sample with no methylation (normal control), and the green line illustrates the melting curve of a heavily methylated cancer research sample identified by the microarray analysis. (B) The pink line shows the melting curve of a normal sample with low level methylation, and the light blue line shows the melting curve of a cancer research sample that is heavily methylated. 6 NimbleGen Arrays and LightCycler 480 System: A Complete Workflow for DNA Methylation Biomarker Discovery and Validation

7 5 Conclusions The post-discovery clinical retrospective and prospective biomarker validation process (as outlined in the Introduction) requires the use of large panels of clinical material and has to be performed for each of the biomarkers identified in the microarray research studies. Furthermore, all the experiments have to be performed in standardized conditions and platforms. The technical specifications of the LightCycler 480 make this platform highly suitable for time, cost and labor efficiency for large-scale MS-HRM experiments. Importantly the MS-HRM assays developed for the array verification process are, in principle, tests that could be applied to future clinical assays. In summary, the combination of the NimbleGen microarray platform and LightCycler 480 System constitutes a complete workflow for DNA methylation biomarker discovery and validation. These combined platform specifications make this workflow highly suitable for clinically driven research studies. References 1. NimbleGen Sample Preparation Instructions. in NimbleGen User s Guide: DNA Methylation Analysis Wojdacz, TK, et al., Methylation-sensitive high-resolution melting. Nature Protocols. 3(12) Wojdacz, TK, et al., (2008) A new approach to primer design for the control of PCR bias in methylation studies. (Translated from english) BMC Res Notes 2008, 1:54 (in english). 4. Wojdacz, TK et al., Methylation-Sensitive High Resolution Melting (MS-HRM) on the LightCycler 480 System. LightCycler Real-Time PCR systems, Application Manual (2009); available from Roche Applied Science (please contact your local Roche representative). Cancer Research Application Note No. 9 7

8 Ordering Information Product Cat. No. Pack Size NimbleGen Microarrays NimbleGen Human DNA Methylation 2.1M Deluxe Promoter Array NimbleGen Microarray Processing Accessories Delivery Cat. No Service Cat. No Reagents Cat. No. NimbleGen Dual-Color DNA Labeling Kit NimbleGen Hybridization Kit NimbleGen Hybridization Kit, LS NimbleGen Wash Buffer Kit NimbleGen Array Processing Accessories NimbleGen Sample Tracking Control Kit Equipment Cat. No. NimbleGen Hybridization System 4 (110V) NimbleGen Hybridization System 12 (110V) NimbleGen Hybridization System 4 (220V) NimbleGen Hybridization System 12 (220V) NimbleGen Microarray Dryer (110V) NimbleGen Microarray Dryer (220V) NimbleGen MS 200 Microarray Scanner Software Cat. No. NimbleScan Software Individual License NimbleScan Software Site License SignalMap Software Individual License LightCycler 480 System LightCycler 480 Instrument II, 96-well instrument plus accessories LightCycler 480 Instrument II, 384-well instrument plus accessories LightCycler 480 High Resolution Melting (HRM) Master x 1 ml 500 reactions, 20 µl each LightCycler 480 Gene Scanning Software software package Roche Microarray Technical Support: For life science research only. Not for use in diagnostic procedures. LIGHTCYCLER, and NIMBLEGEN are trademarks of Roche. Other brands or product names are trademarks of their respective holders. Published by Roche Diagnostics GmbH Sandhofer Straße Mannheim Germany 2011 Roche Diagnostics. All rights reserved