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Review
. 2015 Jul 29;7(1):80.
doi: 10.1186/s13073-015-0203-x. eCollection 2015.

Next-generation sequencing to guide cancer therapy

Jeffrey Gagan  1 , Eliezer M Van Allen  2
Affiliations
Review

Next-generation sequencing to guide cancer therapy

Jeffrey Gagan et al. Genome Med. .

Abstract

As a result of multiple technological and practical advances, high-throughput sequencing, known more commonly as "next-generation" sequencing (NGS), can now be incorporated into standard clinical practice. Whereas early protocols relied on samples that were harvested outside of typical clinical pathology workflows, standard formalin-fixed, paraffin-embedded specimens can more regularly be used as starting materials for NGS. Furthermore, protocols for the analysis and interpretation of NGS data, as well as knowledge bases, are being amassed, allowing clinicians to act more easily on genomic information at the point of care for patients. In parallel, new therapies that target somatically mutated genes identified through clinical NGS are gaining US Food and Drug Administration (FDA) approval, and novel clinical trial designs are emerging in which genetic identifiers are given equal weight to histology. For clinical oncology providers, understanding the potential and the limitations of DNA sequencing will be crucial for providing genomically driven care in this era of precision medicine.

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Figures

Fig. 1
Fig. 1
A summary of the workflow for NGS sequencing in oncology. The first row outlines selecting the appropriate sample and assay. Turning raw data into clinically actionable information is covered in the second row. The third row looks at how NGS may be used in the continued monitoring of disease. ctDNA circulating tumor DNA, FFPE formalin-fixed, paraffin-embedded specimen
Fig. 2
Fig. 2
The trade-off between coverage and amount of the genome covered. A hypothetical region of the genome contains an oncogene, a tumor suppressor and two genes of uncertain significance (GUS). For visual simplicity, we show ten reads, which will get sequencing depth at genes of interest. Whole-exome sequencing is able to cover each gene with fewer reads, whereas whole-genome sequencing rarely covers a specific base with more than one read. Bear in mind, this figure is vastly understating the relative size of intergenic regions. Realistic sequencing depth goals should be much higher
Fig. 3
Fig. 3
Amplicon-based and hybrid capture sequencing methods. The figure shows a hypothetical gene for which a clinical assay sequences exons 2 and 3. The DNA is sheared either in recovery from being formalin-fixed and paraffin-embedded, or deliberately to allow for sequencing adapter binding. Hybrid capture involves probes that are designed with homology to the gene of interest and bind cDNA. Notice that the fragmented DNA can contain information beyond the boundary of the exon. The probes are biotinylated and unbound DNA is washed away. In amplicon-based sequencing, only probes that contain the complementary sequence for both primers are amplified. Therefore, no information outside of the primers is sequenced
Fig. 4
Fig. 4
An illustration of new clinical trial designs. Basket and umbrella trials both incorporate genomic data into the basic construction of the trial. Basket trials are designed around specific mutations, regardless of the primary tumor site. Umbrella trials are first separated by primary tumor site and then split into conventional therapy and precision medicine arms
Fig. 5
Fig. 5
A representative clinical example of how NGS is utilized in recurrent lung adenocarcinoma. The illustrative case from the text has been fitted to the outline in Fig. 1. In a lung adenocarcinoma, there are a number of actionable mutations; this case shows a canonical EGFR mutation, treated with erlotinib. There are actually now two levels of resistance that can develop, illustrated in rows 3 and 4. FFPE formalin-fixed, paraffin-embedded specimen
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