Abstract
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.
Abbreviations
- 3D-PCA:
-
Three dimensional-principal component analysis
- ADJNT :
-
Adjacent non-tumour ductal epithelium
- β:
-
Beta
- µg:
-
microgram
- µl:
-
microliter
- µm:
-
micron
- Cq:
-
Quantitation cycle
- DCIS:
-
Ductal carcinoma in situ
- DMP:
-
Differentially methylated probe
- FCD:
-
Fibrocystic disease
- FFPE:
-
Formalin-fixed paraffin-embedded
- H&E:
-
Haematoxylin and eosin
- HIN-1 :
-
High in normal-1
- HM450K:
-
Infinium HumanMethylation450 Beadchip
- IDC:
-
Infiltrating ductal carcinoma
- MCCS:
-
Melbourne Collaborative Cohort Study
- ng:
-
Nanogram
- NKX6-2 :
-
NK6 Homeobox 2
- NOS:
-
Not otherwise specified
- QC:
-
Quality control
- qPCR:
-
Quantitative polymerase chain reaction
- RASSF1A :
-
RAS association domain family protein 1A
- SWAN:
-
Subset-quantile Within Array Normalisation
- TCGA:
-
The Genome Cancer Genomic Atlas Network
- TN:
-
Triple Negative
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Acknowledgements
This study was made possible by the contribution of many people including those taking part in the Melbourne Collaborative Cohort Study, the original investigators and the diligent team who manage the study and continue working on follow-up. Cohort recruitment was funded by VicHealth and Cancer Council Victoria.
Author’s Contributions
EMW performed all the laboratory experiments described in this manuscript. EMW and JEJ performed the analysis of the experimental data. CAM contributed histopathological expertise. LB, DRE, MCS, GS and JLH made prior contribution to the MCCS resource utilised in this report. LB, MCS and DRE provided grant support for this study. RM contributed to the MCCS resource and logistical aspects of the project. GGG is the principal investigator of the Melbourne Collaborative Cohort Study. MCS provided additional grant support, the initial idea of the research described and overall supervision of the laboratory and analytical approaches. EMW, JEJ and MCS wrote the first draft of the manuscript to which all authors contributed. MBT and HCW contributed to manuscript preparation. The final manuscript was read and approved by all the authors.
Funding Information
This work was further supported by the National Health and Medical Research Council (NHMRC; APP1011618 and APP1026892) and The Victorian Breast Cancer Research Consortium. MCS is a NHMRC Senior Research Fellow and JLH is a NHMRC Senior Principal Research Fellow. EMW is a recipient of the Dyason Fellowship.
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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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Informed consent was obtained from all participants in this research study.
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Ee Ming Wong and JiHoon E Joo have contributed equally to this work.
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Wong, E.M., Joo, J.E., McLean, C.A. et al. Analysis of the breast cancer methylome using formalin-fixed paraffin-embedded tumour. Breast Cancer Res Treat 160, 173–180 (2016). https://doi.org/10.1007/s10549-016-3971-0
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DOI: https://doi.org/10.1007/s10549-016-3971-0