by George Karlin-Neumann AL
Improved Liquid Biopsies With Combined Digital PCR and Next-Generation Sequencing
The discovery that circulating tumor DNA (ctDNA) can be found in the blood, urine and other bodily fluids of cancer patients has led to a new type of biopsy that bypasses surgery—liquid biopsy. However, the tiny fraction of ctDNA within a liquid biopsy is difficult to measure amidst the background of non-tumor circulating cell-free DNA (cfDNA) sloughed from normal cells. It was recently shown that the cell-free liquid biopsy, especially of plasma cfDNA, is a valid assay for cancer genotyping and has potential to direct cancer treatment plans.1
Through the combination of next-generation sequencing (NGS) and digital PCR (dPCR), liquid biopsy is poised to become the standard in cancer management (see Table 1). These technologies together provide quantitative detection and sequencing of small amounts of DNA required to link cancer genotype with available cancer treatments.
NGS and digital PCR together provide a complete
picture of the cancer genome Because cancers vary in type and complexity, effective mutational analysis tools are needed for research and care management. The wide- angle panoramic view of whole-genome sequencing afforded by NGS combined with dPCR’s zoomed-in precision detection of DNA provide a comprehensive picture of a cancer’s genetic makeup. By applying these
Table 1 – Cancer management using NGS and dPCR
complementary techniques at the appropriate time based on the disease type and stage, doctors can treat cancers more quickly, precisely and cost-effectively.
Through gene panels or whole genome sequencing, NGS can reveal un- predictable genomic mutations and rearrangements critical for making informed treatment decisions. The information achievable with NGS also allows researchers to identify non-driver (“passenger”) mutations arising early in the development of the tumor that may assist in tracking tumor recurrence. Unfortunately, the NGS process is slow, which can lead to delays in obtaining patient results and is economically unattractive as an assay for serial monitoring.
Genotyping a smaller set of known gene markers is more sensitive, cost- effective and faster with the Droplet Digital PCR (ddPCR) QX200 system (Bio-Rad Laboratories, Hercules, Calif.). Through multiplexing, a single ddPCR reaction can interrogate multiple genes and at least 5–10 mutations simultaneously. ddPCR identifies and precisely quantifies target DNA within a liquid biopsy, and thus is often used to validate NGS results.
ddPCR provides results that specify (absolute) copies per milliliter of plasma, both of the cancerous mutation and its wild-type counterpart. This nonrelative reporting of ctDNA values is less subject to physiological variations in background cfDNA levels, and may allow healthcare provid- ers to more accurately evaluate cancer growth longitudinally, as well as a patient’s response to treatment. NGS, on the other hand, typically provides only a ratio of mutant to wild-type DNA copies due to the need for DNA library construction and preamplification prior to sequencing.
Monitoring driver mutations, resistance
and recurrence While some cancers have more elusive driver mutations, cancers such as melanoma have a small set of defining mutations that can be predicted in the majority of tumors.2
In these cases, a tissue or plasma genotyping
assay using ddPCR multiplexing can quickly determine if any of the most probable mutations in the cancer are present. The physician can then design and execute a treatment plan specifically for the patient based on these results. This genotyping tool is also useful for devising treat- ment plans for non-small cell lung cancer (NSCLC) patients, the majority of whom have mutations in either the epidermal growth factor receptor (EGFR) or KRAS.3
AMERICAN LABORATORY 17 MARCH 2016
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