Melbourne School of Population and Global Health - Research Publications
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ItemAnalysis of the breast cancer methylome using formalin-fixed paraffin-embedded tumour(SPRINGER, 2016-11)PURPOSE: Aberrant DNA methylation occurs frequently in breast carcinogenesis. Tools for translational epigenetic studies of breast cancer involving formalin-fixed paraffin-embedded (FFPE) human tissues have now been developed. Few studies have measured genome-wide methylation in DNA derived from paraffin-embedded tumour tissues and compared the DNA methylation in corresponding adjacent non-tumour ductal epithelium (ADJNT). These studies are technically challenging due to the spectrum of breast cancer pathologies, the variable suitability of DNA extracted from FFPE material and the difficulties in identifying ADJNT. We assessed the suitability of FFPE breast cancer material for genome-wide DNA methylation assessment of tumour and ADJNT. METHODS: Twenty-one archival breast tumour tissues with paired ADJNT obtained from separate blocks and at least 2 cm from the tumour were sourced from The Melbourne Collaborative Cohort Study (MCCS). DNA was prepared from macrodissected tissue samples and assessed for genome-wide methylation using the Infinium HumanMethylation450 Beadchip (HM450K) array. RESULTS: The 1000 most differentially methylated probes between tumour and ADJNT in this FFPE-derived dataset differentiated tumour and ADJNT in The Cancer Genome Atlas Network data (TCGA; derived from high molecular weight DNA using the same HM450K array). CONCLUSIONS: Large-scale studies of genome-wide DNA methylation using FFPE breast cancer specimens offer the opportunity to further refine the pathological classification of tumours, to include subtypes that are underrepresented in the TCGA data and provide the capacity to further explore intra-tumoural heterogeneity.
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ItemTools for translational epigenetic studies involving formalin-fixed paraffin-embedded human tissue: applying the Infinium HumanMethyation450 Beadchip assay to large population-based studies.(Springer Science and Business Media LLC, 2015-10-06)BACKGROUND: Large population-based translational epigenetic studies are emerging due to recent technological advances that have made molecular analyses possible. For example, the Infinium HumanMethylation450 Beadchip (HM450K) has enabled studies of genome-wide methylation on a scale not previously possible. However, application of the HM450K to DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumour material has been more challenging than application to high quality DNA extracted from blood. To facilitate the application of this assay consistently across a large number of FFPE tumour-enriched DNA samples we have devised a modification to the HM450K protocol for FFPE that includes an additional quality control (QC) checkpoint. RESULTS: QC checkpoint 3 was designed to assess the presence of DNA after bisulfite conversion and restoration, just prior to application of the HM450K assay. DNA was extracted from 474 archival FFPE breast tumour material. Five samples did not have a detectable amount of DNA with an additional 42 failing to progress past QC checkpoint 3. Genome-wide methylation was measured for the remaining 428 tumour-enriched DNA. Of these, only 4 samples failed our stringent HM450K data criteria thus representing a 99% success rate. Using prior knowledge about methylation marks associated with breast cancer we further explored the quality of the data. Twenty probes in the BRCA1 promoter region showed increased methylation in triple-negative breast cancers compared to Luminal A, Luminal B and HER2-positive breast cancer subtypes. Validation of this observation in published data from The Cancer Genome Atlas (TCGA) Network (obtained from DNA extracted from fresh frozen tumour samples) confirms the quality of the data obtained from the improved protocol. CONCLUSIONS: The modified protocol is suitable for the analysis of FFPE tumour-enriched DNA and can be systematically applied to hundreds of samples. This protocol will have utility in population-based translational epigenetic studies and is applicable to a wide variety of translated studies interested in analysis of methylation and its role in the predisposition to disease and disease progression.
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ItemThe use of DNA from archival dried blood spots with the Infinium HumanMethylation450 array(BIOMED CENTRAL LTD, 2013-03-15)BACKGROUND: Dried blood (Guthrie card) spots provide an efficient way to collect and store blood specimens. DNA from this source has been utilised for a number of molecular analyses including genome-wide association studies, but only few studies have tested the feasibility of using it for epigenetic applications, particularly at a genome-wide level. RESULTS: In this study, we demonstrate the successful use of DNA isolated from archived dried blood spots for the Infinium HumanMethylation450 Beadchip, along with DNA from matched frozen buffy coats. We obtained high quality and reproducible genome-wide DNA methylation profiles using both sample types. We also report high correlations (r > 0.9907) between DNA obtained from matched dried blood spots and frozen buffy coats, sufficient to distinguish between unrelated individuals. CONCLUSIONS: We, thus, demonstrate that DNA from archived dried blood spots is suitable for genome-wide DNA methylation profiling.