Realizing the Promise: NGS in Precision Cancer Medicine
Precision cancer medicine is driven by the latest advances in genetic technologies. In light of falling costs and increased accessibility sparking a greater uptake of next generation sequencing (NGS), Clinical Scientists Dr. Matthew Smith and Dr. George Burghel discussed the strengths and challenges of this approach.
Characterizing tumors at a molecular level can have huge implications for cancer therapy, overcoming the high level of inherent genetic complexity and heterogeneity to ensure patients only receive the most appropriate treatment. Known as precision medicine, this approach relies on actionable mutations possessing diagnostic, prognostic or predictive implications, and targeted mutations capable of identifying cancers sensitive to specific therapies.
A variety of genetic testing technologies are available for profiling these targets, as Smith commented: “Since the breadth of testing is currently limited to a handful of targets, the majority of routine diagnostic testing utilizes well-established techniques such as Sanger sequencing, pyrosequencing and qRT-PCR. This enables us to turn around tests in a clinically actionable timeframe, and provides a cost-effective strategy — but only as long as the number of tests are limited. This is becoming less feasible as the list of actionable genetic markers and targeted therapies expands.” Smith went on to explain: “With a reduction in cost and improvements in library preparation and sequencing, NGS has the capabilities for testing larger, multi-gene panels.”
How do current NGS approaches manage the notorious data load?
The data load generated by NGS is well-known as a bottleneck, requiring time and expert knowledge in extracting meaningful results. As a highly efficient alternative to whole genome sequencing, targeted sequencing is well suited to the clinical laboratory, where turnaround times and costs are a major priority. By capturing specific genomic regions from DNA samples prior to sequencing, only the regions of interest are sequenced. This approach also enables an increased depth of coverage, providing the sensitivity needed for heterogeneous samples and overcoming many of the challenges typically faced in NGS. In addition, numerous options exist for further streamlining the process of data analysis, from third party software to in-house solutions (Figure 2). “Cancer-specific gene panels and enrichment methods are becoming increasingly popular, and a number of laboratories and commercial companies have recently developed and validated these for clinical research use,” explained Burghel.
Read More: A Guide to Targeted NGS: Generating Accurate Data for Personalized Medicine
Since targeted NGS relies on the genomic regions analyzed, how is the content chosen for targeted sequencing panels?
When choosing content for a new panel, the current focus of molecular pathology labs is on delivering results that can be translated into clinical action. However, this can be complex, as Smith elaborated: “The content of any panel is a balancing act between trying to maximize the utility of the panel with expected sample numbers and desired throughput.” In general, for a diagnostic panel the focus is often very narrow, maximizing cost-efficiency and sample throughput, while limiting the amount of surplus sequencing data. The breadth of content can range from well-defined mutational hotspots (e.g. KRAS and BRAF) through to full exons (e.g. KIT and PDGFRA), depending on the target genes and clinical literature, and mutations can become clinically actionable following new discoveries. However, interpreting novel variants provides a significant challenge and there is a lag between discoveries in research and their clinical application. One way in which NGS technology has adapted in response to this is with the emergence of custom panels, enabling the user to select a chosen pool of relevant hybridization probes. The flexibility of such systems facilitates researchers in investigating variants relevant to their specific study, increasing the speed at which new content can be validated and decreasing the time lag from the laboratory to the clinic.
How can NGS be adapted for compatibility with tumor samples?
Due to tumor heterogeneity and the presence of DNA derived from non-tumor cells, a variant of interest may only occur at a relatively low allele frequency in the sample. Researchers are therefore particularly interested in NGS platforms that allow considerable depth (i.e. targeted panels). In addition, the use of DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue can present many technical challenges. Smith commented: “Due to the fixation process, the DNA can be degraded and we often have to work with very small amounts of DNA.” Because of this, a number of quality control matrices are analyzed, including the accurate measurement of low level DNA. Targeted enrichment methods are also carefully considered to ensure the uniform representation of all regions of interest.
How do target enrichment methods compare for targeted NGS?
The type of enrichment method is of utmost importance for targeted NGS, and the two main approaches fall into either the hybridization or amplicon-based categories. Utilizing PCR, amplicon strategies tend to be quick and easily integrated into existing laboratory workflows. However, data quality tends to be less robust when compared to a hybridization based approach, as it is very hard to determine and remove bias introduced by PCR. With the ability to easily capture larger target regions, hybridization methods are ideal for larger panels. Traditionally they have required more DNA input and hands on time. However, the method has been improving, and both of these factors have been addressed by OGT in the development of the SureSeq* NGS panels and Library Preparation Kit.
What do you see for the future of NGS in cancer medicine?
The fundamental premise of personalized cancer therapy is to ensure the right treatment for the right person at the right time, and with the area of genomic medicine growing at an unprecedented rate, it is becoming clear that targeted NGS is the key to this. As clinical genetics experts Dr. Matthew Smith and Dr. George Burghel have indicated, this technology is becoming increasingly embedded within the clinical research laboratory, with new panels emerging and evolving in response to the latest genetic discoveries. These panels provide the capability to detect low-level mutations from the ever-increasing catalog of clinically actionable aberrations and markers for directing cancer therapy, and in fact, Burghel commented that many of these genetic markers are already in use today. As Smith has affirmed, “Along with existing and emerging testing strategies, NGS has an extremely important role to play in the future of cancer characterization and treatment.”
*SureSeq: For Research Use Only; Not for Use in Diagnostic Procedures
Interviewees information:
Dr. George Burghel, HCPC Registered Clinical Scientist, Genomic Diagnostics Laboratory, The Manchester Centre for Genomic Medicine, Central Manchester University Hospitals NHS Foundation Trust
Dr. Matthew Smith, Principal Clinical Scientist, Molecular Pathology Diagnostic Service,Cellular Pathology - University Hospitals Birmingham NHS Foundation Trust, Queen Elizabeth Hospital Birmingham
