Abstract
Cancer is characterised by complex somatically acquired genetic aberrations that manifest as intra-tumour and inter-tumour genetic heterogeneity and can lead to treatment resistance. In this case study, we characterise the genome-wide somatic mutation dynamics in a metastatic melanoma patient during therapy using low-input (50 ng) PCR-free whole genome sequencing of cell-free DNA from pre-treatment and post-relapse blood samples. We identify de novo tumour-specific somatic mutations from cell-free DNA, while the sequence context of single nucleotide variants showed the characteristic UV-damage mutation signature of melanoma. To investigate the behaviour of individual somatic mutations during proto-oncogene B-Raf -targeted and immune checkpoint inhibition, amplicon-based deep sequencing was used to verify and track frequencies of 212 single nucleotide variants at 10 distinct time points over 13 months of treatment. Under checkpoint inhibition therapy, we observed an increase in mutant allele frequencies indicating progression on therapy 88 days before clinical determination of non-response positron emission tomogrophy-computed tomography. We also revealed mutations from whole genome sequencing of cell-free DNA that were not present in the tissue biopsy, but that later contributed to relapse. Our findings have potential clinical applications where high quality tumour-tissue derived DNA is not available.
Highlights
whole genome sequencing (WGS) sequencing quality To characterise the impact of therapy on tumour genetic diversity, we performed WGS using cfDNA from samples taken before vemurafenib and after ipilimumab treatment, a formalin-fixed paraffin-embedded (FFPE) tumour biopsy and genomic DNA from peripheral blood mononuclear cells (Fig. 1)
To address the low abundance of circulating tumour DNA (ctDNA) in cfDNA we developed a low input (50 ng) DNA library preparation method
The archival tumour tissue obtained from FFPE material performed poorly compared to the normal and cfDNA samples with much greater variability observed in coverage across the genome
Summary
Whole exome sequencing (WES) and whole genome sequencing (WGS) of DNA derived from tumour biopsies has generated atlases of somatic mutations across cancer types, revealing deep genetic heterogeneity within and between tumours.[1,2,3] These catalogues have improved the molecular classification of cancer types and are a resource for drug development.[4, 5] for WES and WGS, high quality DNA from fresh tumour material is required, which has limited their widespread application in clinical practice.[6,7,8] Establishing the acquired genetic profile of a cancer by using targeted sequencing of specific cancer genes is increasingly used to both help diagnosis and guide treatment decisions and commonly uses formalin fixed paraffin-embedded tumour material.
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