Ultra-Low DNA Input into Whole Genome Methylation Assays and Detection of Oncogenic Methylation and Copy Number Variants in Circulating Tumour DNA

Celina Whalley,Andrew Beggs,Jill Brooks,Rachel Spruce,S-Cort Consortium ,Karl Payne,Enric Domingo,Paul Nankivell,Hisham Mehanna,Susan Richman,Nikolaos Batis,Andrew Blake

doi:10.3390/epigenomes5010006

Celina Whalley, Andrew Beggs + Show 10 more

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https://doi.org/10.3390/epigenomes5010006

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Abstract

Background: Abnormal CpG methylation in cancer is ubiquitous and generally detected in tumour specimens using a variety of techniques at a resolution encompassing single CpG loci to genome wide coverage. Analysis of samples with very low DNA inputs, such as formalin fixed (FFPE) biopsy specimens from clinical trials or circulating tumour DNA is challenging at the genome-wide level because of lack of available input. We present the results of low input experiments into the Illumina Infinium HD methylation assay on FFPE specimens and ctDNA samples. Methods: For all experiments, the Infinium HD assay for methylation was used. In total, forty-eight FFPE specimens were used at varying concentrations (lowest input 50 ng); eighteen blood derived specimens (lowest input 10 ng) and six matched ctDNA input (lowest input 10 ng)/fresh tumour specimens (lowest input 250 ng) were processed. Downstream analysis was performed in R/Bioconductor for quality control metrics and differential methylation analysis as well as copy number calls. Results: Correlation coefficients for CpG methylation were high at the probe level averaged R2 = 0.99 for blood derived samples and R2 > 0.96 for the FFPE samples. When matched ctDNA/fresh tumour samples were compared, R2 > 0.91 between the two. Results of differential methylation analysis did not vary significantly by DNA input in either the blood or FFPE groups. There were differences seen in the ctDNA group as compared to their paired tumour sample, possibly because of enrichment for tumour material without contaminating normal. Copy number variants observed in the tumour were generally also seen in the paired ctDNA sample with good concordance via DQ plot. Conclusions: The Illumina Infinium HD methylation assay can robustly detect methylation across a range of sample types, including ctDNA, down to an input of 10 ng. It can also reliably detect oncogenic methylation changes and copy number variants in ctDNA. These findings demonstrate that these samples can now be accessed by methylation array technology, allowing analysis of these important sample types.

Highlights

Abnormal epigenetic change in the form of the loss or gain of normal 5-methylcytosine patterns is one of the most ubiquitous epigenetic changes in cancer epigenetics, controlling gene expression via changes in transcription factor binding [1].Methylation in cancer is of particular interest due to its association with specific phenotypes such as the CpG Island Methylator Phenotype (CIMP) that possess unique characteristics [2]
Our results demonstrate that lower input to the Illumina Infinium assay as utilised in the MethylationEPIC and unavailable Methylation 450 K assay are feasible and provide reliable and reproducible results
Because the core of the Infinium assay protocol is a whole genome amplification step, theoretical inputs into this kit could be as low as 1 ng [16], and we have shown by our experiments on fresh blood derived samples, FFPE tissue, and circulating tumour DNA

Summary

Introduction

Abnormal epigenetic change in the form of the loss or gain of normal 5-methylcytosine patterns is one of the most ubiquitous epigenetic changes in cancer epigenetics, controlling gene expression via changes in transcription factor binding [1].Methylation in cancer is of particular interest due to its association with specific phenotypes such as the CpG Island Methylator Phenotype (CIMP) that possess unique characteristics [2]. We present the results of low input experiments into the Illumina Infinium HD methylation assay on FFPE specimens and ctDNA samples. Conclusions: The Illumina Infinium HD methylation assay can robustly detect methylation across a range of sample types, including ctDNA, down to an input of 10 ng. It can reliably detect oncogenic methylation changes and copy number variants in ctDNA. These findings demonstrate that these samples can be accessed by methylation array technology, allowing analysis of these important sample types

Methods

Results

Conclusion