Clinically actionable mutation profiles in patients with cancer identified by whole-genome sequencing

Next-generation sequencing (NGS) efforts have established catalogs of mutations relevant to cancer development. However, the clinical utility of this information remains largely unexplored. Here, we present the results of the first eight patients recruited into a clinical whole-genome sequencing (WGS) program in the United Kingdom. We performed PCR-free WGS of fresh frozen tumors and germline DNA at 75× and 30×, respectively, using the HiSeq2500 HTv4. Subtracted tumor VCFs and paired germlines were subjected to comprehensive analysis of coding and noncoding regions, integration of germline with somatically acquired variants, and global mutation signatures and pathway analyses. Results were classified into tiers and presented to a multidisciplinary tumor board. WGS results helped to clarify an uncertain histopathological diagnosis in one case, led to informed or supported prognosis in two cases, leading to de-escalation of therapy in one, and indicated potential treatments in all eight. Overall 26 different tier 1 potentially clinically actionable findings were identified using WGS compared with six SNVs/indels using routine targeted NGS. These initial results demonstrate the potential of WGS to inform future diagnosis, prognosis, and treatment choice in cancer and justify the systematic evaluation of the clinical utility of WGS in larger cohorts of patients with cancer

KIT Mutation and Loss of 14q May Be Sufficient for the Development of Clinically Symptomatic Very Low-Risk GIST

<div><p>The aim of this study was to determine the minimal set of genetic alterations required for the development of a very low risk clinically symptomatic gastro-intestinal stromal tumour within the stomach wall. We studied the genome of a very low-risk gastric gastro-intestinal stromal tumour by whole-genome sequencing, comparative genomic hybridisation and methylation profiling. The studied tumour harboured two typical genomic lesions: loss of the long arm of chromosome 14 and an activating mutation in exon 11 of KIT. Besides these genetic lesions, only two point mutations that may affect tumour progression were identified: A frame-shift deletion in RNF146 and a missense mutation in a zinc finger of ZNF407. Whilst the frameshift deletion in RNF146 seemed to be restricted to this particular tumour, a similar yet germline mutation in ZNF407 was found in a panel of 52 gastro-intestinal stromal tumours from different anatomical sites and different categories. Germline polymorphisms in the mitotic checkpoint proteins Aurora kinase A and BUB1 kinase B may have furthered tumour growth. The epigenetic profile of the tumour matches that of other KIT-mutant tumours. We have identified mutations in three genes and loss of the long arm of chromosome 14 as the so far minimal set of genetic abnormalities sufficient for the development of a very low risk clinically symptomatic gastric stromal tumour.</p></div

Single nucleotide variations in exon 1 of ZNF407 in a panel of 52 GIST samples.

<p>Abbreviations: NT, nucleotide change in NM_017757; AA, amino acid change in NP_060227; Pred, PolyPhen2 HDIV score; MAF, minor allele frequency 1000 Genomes project; a, Germline mutation; b, Somatic mutation.</p><p>The last column shows in how many of the 52 samples the mutation was found.</p

Mapping plot of the sequenced GIST sample.

<p>Plotted is the fraction of the genome that is covered to at least a certain depth.</p

Pathological characteristics of a very low-risk GIST.

<p>This H&E staining shows a low (upper picture) and high power view (middle picture) of the GIST that was submitted to whole genome sequencing and how it invades the gut wall. The tumour was also stained with an antibody specific to c-kit (lower picture).</p

Potential founding mutations in protein-coding regions.

<p>¹, Fraction of sequenced reads showing the mutation; p, pseudogene; s, confirmed by Sanger sequencing.</p><p>Potential founding mutations in protein-coding regions.</p

Coding mutations in ZNF407 zinc fingers.

<p>^a, The C2H2 zinc finger motif comprises two cysteines and two histidines, shown in capital letters. Positions of amino acid changes are highlighted as [x/x]. Amino acid (AA) positions are internal to protein sequence NP_060227.</p><p>Coding mutations in ZNF407 zinc fingers.</p