Recent breakthroughs in high-throughput or next generation sequencing (NGS) are resulting in progress that extends into the clinic. More and more scientists are relying on standard formalin-fixed paraffin-embedded (FFPE) samples rather than the previous practice of using samples that existed outside of traditional clinical workflows. Use of FFPE samples has resulted in gains in translational medicine whose results extend into clinical practice. FFPE tissue samples are now routinely used as the starting materials for NGS. Knowledge bases and tools for the interpretation of FFPE data are helping clinicians to enhance patient care acting on information culled from FFPE sources. The FDA has approved new therapies that target mutated genes identified via clinical NGS and new genetic tests that depend on identifiers from NGS are becoming increasingly prevalent. For clinicians providing genomically driven precision medicine, understanding the benefits and limitations of DNA sequencing is crucial.
FFPE has traditionally been associated with problems that make fresh frozen samples more appealing. The formalin used as a fixing agent introduces crosslinks that can fragment DNA and cause sequencing alterations. Early studies suggested that using FFPE samples for PCR-based sequencing led to more errors than frozen specimens. Great progress in DNA extraction methods for FFPE samples has resulted in samples that are just as useful as fresh frozen for NGS methods. Currently clinicians may use FFPE data and feel comfortable that the results will be as high quality as frozen sources.
A third emerging data source is the so-called liquid biopsy. Historically, standard diagnostic techniques have depended on samples of tissue collected from the patient. Increasingly, researchers have begun to look at circulating DNA in the blood. The circulating cells generally suffer from low frequency and must go through an amplification stage. These methods hold great potential to improve diagnosis by looking for mutations that are associated with specific disease types.
Tumor heterogeneity poses challenges to liquid biopsies but is also a reason why they hold promise than traditional tissue biopsies. The low amount of some DNA in the sample in conjunction with the low incidence of mutation can result in mutations with low allele fractions being missed by liquid biopsies. This makes distinguishing between low allele fraction mutants and the errors in high-throughput sequencing difficult. Sequencing minimally invasive samples repeatedly can help in this respect and will lead over time to faster recognition of resistance mutations.
Formalin-fixed paraffin-embedded and fresh frozen samples are both useful for next generation sequencing. Additionally, liquid biopsies are emerging as an increasingly important tool in the clinic. Problem that plagued FFPE data sources are being overcome making FFPE samples as useful as frozen tissue. Liquid biopsies, currently a growing area of clinical study, are a novel addition to the clinician’s toolkit. These newer methods are posed to facilitate advances that traditional methods do not offer.