Cancer genomics and inherited risk

Abstract

Next-generation sequencing (NGS) has enabled whole-exome and whole-genome sequencing of tumors for causative mutations, allowing for more accurate targeting of therapies. In the process of sequencing the tumor, comparisons to the germline genome may identify variants associated with susceptibility to cancer as well as other hereditary diseases. Already, the combination of massively parallel sequencing and selective capture approaches has facilitated efficient simultaneous genetic analysis (multiplex testing) of large numbers of candidate genes. As the field of oncology incorporates NGS approaches into tumor and germline analyses, it has become clear that the ability to achieve high-throughput genotyping surpasses our current ability to interpret and appropriately apply the vast amounts of data generated from such technologies. A review of the current state of knowledge of rare and common genetic variants associated with cancer risk or treatment outcome reveals significant progress, as well as a number of challenges associated with the clinical translation of these discoveries. The combined efforts of oncologists, genetic counselors, and cancer geneticists will be required to drive the paradigm shift toward personalized or precision medicine and to ensure the incorporation of NGS technologies into the practice of preventive oncology.

Fig 1.

Principles of next-generation sequencing (NGS) technology. For NGS library preparation, DNA is randomly fragmented into desired size ranges. Adaptors containing the universal priming sites are ligated to the target ends of the fragments. After ligation, the template is immobilized to a solid support. Immobilization strategies for clonally amplified templates include either using emulsion polymerase chain reaction (emPCR) or a solid-phase amplification. In emPCR, an oil-aqueous emulsion reaction mixture is created to encapsulate bead-DNA complexes into single aqueous droplets. PCR is then performed within the droplets to create beads that contain several thousand copies of the same template sequence. The emPCR beads can then be attached to a glass slide or loaded into PicoTiterPlate (Roche Applied Science, Indianapolis, IN) wells. Solid-phase amplification relies on bridge PCR, where both forward and reverse PCR primers are tethered to a solid substrate by a flexible linker such that the clonally amplified clusters remain immobilized, thereby localizing to a single physical location on an array. At the conclusion of bridge PCR, each clonal cluster contains approximately 1,000 copies of a single member of the template library. Regardless of platform used, amplification is a necessary step because it allows the sequencing reactions to produce sufficient signal for detection by the imaging system of the instrument. Single-molecule as opposed to clonally amplified templates can also be accomplished using a number of possible approaches for immobilization. On the basis of whether clonally amplified or single-molecule templates are used, different sequencing and imaging strategies need to be applied. Although the DNA sequencing reactions vary among platforms, the most popular technologies, such as cyclic reversible termination, pyrosequencing, and the pH-based/semiconductor sequencing, perform sequencing by synthesis to sequence the template. The ability to move away from optically based detection systems to more scalable semiconductor technology has drastically reduced the costs associated with sequencing. Other sequencing technologies exist, such as the sequencing by ligation method, referred to as the SOLiD (support oligonucleotide) platform (Table 2).

Fig 2.

Pedigree case 1.

Fig 3.

Pedigree case 2.

Fig 4.

Challenging the traditional model of cancer genetic counseling. (A) Traditional model of clinical cancer genetics. (B) Incorporating next-generation sequencing (NGS) into genetic cancer risk assessment. (C) NGS of tumors with incorporation of incidental germline findings. WGS, whole-genome sequencing.

Fig A1.

Gene discovery: the potential of next-generation sequencing (NGS) technology. GWAS, genome-wide association study.