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Tools for translational epigenetic studies involving formalin-fixed paraffin-embedded human tissue: applying the Infinium HumanMethyation450 Beadchip assay to large population-based studies.

Wong EM, Joo JE, McLean CA, Baglietto L, English DR, Severi G, Hopper JL, Milne RL, FitzGerald LM, Giles GG, Southey MC - BMC Res Notes (2015)

Bottom Line: Five samples did not have a detectable amount of DNA with an additional 42 failing to progress past QC checkpoint 3.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Melbourne, Melbourne, VIC, 3010, Australia. emwong@unimelb.edu.au.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Probe mean beta values at the BRCA1 gene. a Mean beta values measured from 419 tumour-enriched DNA in our dataset. Twenty probes overlapping exon 1 of BRCA1, exon 1 of NBR2 and their shared bi-directional promoter showed increased methylation in triple-negative breast cancers (TNBC) compared with Luminal A (Lum A), Luminal B (Lum B) and HER2-positive (HER2-pos) subtypes (* denotes probes not present in the TCGA dataset). b Mean beta values measured from 156 tumour-enriched DNA in TCGA. Twenty-two probes overlapping exon 1 of BRCA1, exon 1 of NBR2 and their shared bi-directional promoter showed increased methylation in basal-like breast cancers (TNBC) compared with Luminal A (Lum A), Luminal B (Lum B) and HER2-enriched (HER2-pos) subtypes (+ denotes probes not present in our dataset)
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Fig3: Probe mean beta values at the BRCA1 gene. a Mean beta values measured from 419 tumour-enriched DNA in our dataset. Twenty probes overlapping exon 1 of BRCA1, exon 1 of NBR2 and their shared bi-directional promoter showed increased methylation in triple-negative breast cancers (TNBC) compared with Luminal A (Lum A), Luminal B (Lum B) and HER2-positive (HER2-pos) subtypes (* denotes probes not present in the TCGA dataset). b Mean beta values measured from 156 tumour-enriched DNA in TCGA. Twenty-two probes overlapping exon 1 of BRCA1, exon 1 of NBR2 and their shared bi-directional promoter showed increased methylation in basal-like breast cancers (TNBC) compared with Luminal A (Lum A), Luminal B (Lum B) and HER2-enriched (HER2-pos) subtypes (+ denotes probes not present in our dataset)

Mentions: The HM450K assay measures methylation at 53 CpG sites (53 methylation probes) across the BRCA1 gene. Three probes (cg19088651, cg11126247 and cg16919093) did not meet the QC criteria and were removed from the analysis. The 423 remaining FFPE tumour-enriched breast DNA in our study consisted of the following subtypes: 238 Luminal A, 87 Luminal B, 63 triple-negative and 30 HER2-positive. The mean beta value, standard deviation and 95 % CI for each BRCA1-specific probe across each subtype were calculated and plotted as a line graph. Approximately 20 probes along the BRCA1 promoter region displayed higher mean beta levels in the triple-negative breast tumours compared to other subtypes (student’s t-test, p < 10−5) (Fig. 3a). This probe set overlapped BRCA1 exon 1, exon 1 of its’ neighbouring gene, NBR2 [14] and their shared bi-directional promoter [15].Fig. 3


Tools for translational epigenetic studies involving formalin-fixed paraffin-embedded human tissue: applying the Infinium HumanMethyation450 Beadchip assay to large population-based studies.

Wong EM, Joo JE, McLean CA, Baglietto L, English DR, Severi G, Hopper JL, Milne RL, FitzGerald LM, Giles GG, Southey MC - BMC Res Notes (2015)

Probe mean beta values at the BRCA1 gene. a Mean beta values measured from 419 tumour-enriched DNA in our dataset. Twenty probes overlapping exon 1 of BRCA1, exon 1 of NBR2 and their shared bi-directional promoter showed increased methylation in triple-negative breast cancers (TNBC) compared with Luminal A (Lum A), Luminal B (Lum B) and HER2-positive (HER2-pos) subtypes (* denotes probes not present in the TCGA dataset). b Mean beta values measured from 156 tumour-enriched DNA in TCGA. Twenty-two probes overlapping exon 1 of BRCA1, exon 1 of NBR2 and their shared bi-directional promoter showed increased methylation in basal-like breast cancers (TNBC) compared with Luminal A (Lum A), Luminal B (Lum B) and HER2-enriched (HER2-pos) subtypes (+ denotes probes not present in our dataset)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4595238&req=5

Fig3: Probe mean beta values at the BRCA1 gene. a Mean beta values measured from 419 tumour-enriched DNA in our dataset. Twenty probes overlapping exon 1 of BRCA1, exon 1 of NBR2 and their shared bi-directional promoter showed increased methylation in triple-negative breast cancers (TNBC) compared with Luminal A (Lum A), Luminal B (Lum B) and HER2-positive (HER2-pos) subtypes (* denotes probes not present in the TCGA dataset). b Mean beta values measured from 156 tumour-enriched DNA in TCGA. Twenty-two probes overlapping exon 1 of BRCA1, exon 1 of NBR2 and their shared bi-directional promoter showed increased methylation in basal-like breast cancers (TNBC) compared with Luminal A (Lum A), Luminal B (Lum B) and HER2-enriched (HER2-pos) subtypes (+ denotes probes not present in our dataset)
Mentions: The HM450K assay measures methylation at 53 CpG sites (53 methylation probes) across the BRCA1 gene. Three probes (cg19088651, cg11126247 and cg16919093) did not meet the QC criteria and were removed from the analysis. The 423 remaining FFPE tumour-enriched breast DNA in our study consisted of the following subtypes: 238 Luminal A, 87 Luminal B, 63 triple-negative and 30 HER2-positive. The mean beta value, standard deviation and 95 % CI for each BRCA1-specific probe across each subtype were calculated and plotted as a line graph. Approximately 20 probes along the BRCA1 promoter region displayed higher mean beta levels in the triple-negative breast tumours compared to other subtypes (student’s t-test, p < 10−5) (Fig. 3a). This probe set overlapped BRCA1 exon 1, exon 1 of its’ neighbouring gene, NBR2 [14] and their shared bi-directional promoter [15].Fig. 3

Bottom Line: Five samples did not have a detectable amount of DNA with an additional 42 failing to progress past QC checkpoint 3.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Melbourne, Melbourne, VIC, 3010, Australia. emwong@unimelb.edu.au.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus