Modern targeted therapies have greatly advanced the treatment of many solid tumors. The rational use of these agents requires optimal strategies for the rapid and accurate detection of targetable genomic alterations, both upon initial diagnosis and when acquired resistance to targeted therapies develops. While tissue genotyping has traditionally been considered the standard of care for identifying such genomic alterations, this methodology is limited by often inaccessible or insufficient tumor tissue, and by the risks associated with undergoing serial tumor biopsies. In non–small-cell lung cancer (NSCLC), for example, where molecular testing to guide initial treatment decision making is considered standard of care, clinical studies show that up to 10% to 20% of biopsies are inadequate for molecular testing due to insufficient tissue or amplifiable DNA.[1,2] Furthermore, tumor biopsies are limited by tumor heterogeneity, particularly in the setting of disease resistance, and thus may yield false-negative results.
Plasma genotyping is an emerging technology that can noninvasively and rapidly detect and monitor genomic alterations in cancer patients over time, and which has the potential to overcome some of the limitations associated with tissue genotyping. To date, multiple platforms for plasma genotyping have been tested and validated to various degrees; each has different advantages and limitations, complicating the interpretation and routine integration of these tests into clinical practice.
Biology of Cell-Free DNA
The presence of cell-free DNA (cfDNA) in the plasma was first described in 1948 by Mandel and Metais. It was not until years later that tumor-derived cfDNA—also called circulating tumor DNA (ctDNA)—was discovered, stemming from the finding that cancer patients had higher levels of plasma cfDNA than normal controls.[4,5] The exact mechanism by which DNA is released into the peripheral blood has not been well described; however, it is thought to occur through multiple mechanisms, including tumor cell apoptosis, necrosis, and extracellular vesicle secretion.[6-8] In contrast to genomic DNA, ctDNA is highly fragmented, with most DNA fragments measuring approximately 150 bp in length; this supports the hypothesis that cfDNA originates through cell necrosis or apoptosis.[9,10] Also, peripheral cfDNA exists in low concentrations, with the majority of studies reporting absolute cfDNA levels of less than 100 ng/mL—and only a fraction of this total cfDNA (< 1% of total cfDNA) is actually tumor-derived.[9,12] Thus, highly sensitive methodologies are required to detect ctDNA (Figure).
The concept of ctDNA “shed” is critical to the understanding and interpretation of plasma genotyping assay results, since even the most sensitive assays may not detect a mutation if a tumor is not shedding ctDNA. The degree to which a tumor releases DNA into the peripheral circulation determines the concentration of ctDNA that may be detected in the blood. Factors associated with increased ctDNA shed include mitotic rate, cell death, and tumor vascularization. The extent of metastatic disease burden and sites of metastasis have also been associated with detection of increased ctDNA.[6,7] In particular, the presence of metastasis to the liver or bone has been associated with higher levels of ctDNA in clinical studies.
Current Assays and Validation
A variety of potential testing platforms for plasma genotyping have been utilized in the past. Each platform has been validated to a different degree and exhibits unique capabilities and test characteristics. Traditional DNA detection methods, such as Sanger sequencing, have generally lacked the sensitivity needed to detect the low levels of ctDNA present in the peripheral blood. Thus, more recent assays have utilized an allele-specific quantitative polymerase chain reaction (PCR)-based platform to enrich for mutant DNA, or have used massive parallel sequencing with a next-generation sequencing (NGS) platform. Most currently validated ctDNA assays have been compared against tissue genotyping. The specificities reported for these tests are consistently high, ranging between 90% and 100%, but their sensitivities are more variable, ranging between 30% and 85%, depending on the testing methodology (Table 1). While few prospective validation studies have been carried out to date, retrospective analysis of samples collected from patients enrolled in prospective clinical trials is a reliable alternative.
The Roche cobas EGFR Mutation Test v2 is the only ctDNA assay that is currently approved by the US Food and Drug Administration (FDA) for the detection of EGFR-sensitizing and resistance mutations in NSCLC. This platform was originally approved by the FDA for the detection of EGFR-sensitizing mutations in tissue. Recently, this approval has been extended to include plasma genotyping for both EGFR-sensitizing mutations and the T790M resistance mutation. The approval of the plasma genotyping assay was based on data from the retrospective analysis of paired plasma and tissue samples from 517 NSCLC patients screened for the ENSURE study, which compared treatment with cisplatin/gemcitabine vs erlotinib in EGFR-mutant NSCLC patients. The cobas assay demonstrated moderate sensitivity and high specificity—76.7% (range, 70.5%–81.9%) and 98.2% (range, 95.4%–99.3%), respectively—for the detection of EGFR-sensitizing mutations. The approval for inclusion of EGFR T790M was based on a retrospective analysis of plasma samples from EGFR-mutant NSCLC patients with acquired resistance to initial kinase inhibitor therapy who were enrolled in the early-phase trials (AURA and AURA2) of the third-generation epidermal growth factor receptor (EGFR) kinase inhibitor osimertinib. When compared with an NGS plasma genotyping assay, the sensitivity and specificity of the cobas assay for detection of EGFR T790M were 91.5% and 91.1%, respectively; however, when compared with tissue genotyping, sensitivity and specificity were lower, at 61.4% and 78.6%, respectively.[15,16] The discordance of these results was hypothesized to be related to the heterogeneity of the presence of resistance mutations in tumor tissue.
Many of the earliest attempts at ctDNA genotyping utilized the Scorpion amplification refractory mutation system (Scorpion-ARMS) platform, which uses allele-specific primers with fluorescent probes to amplify target DNA alleles. Kimura et al evaluated the test characteristics of ctDNA analysis using a Scorpion-ARMS platform, comparing it against tissue genotyping in NSCLC patients treated with gefitinib. They demonstrated that cfDNA analysis had the ability to detect EGFR mutations with a sensitivity of 85.7% and a specificity of 94.3%. Similar results have been reported in several other retrospective validation studies that used Scorpion-ARMS technology to assess plasma EGFR mutations in the NSCLC population.[2,18]
Newer PCR methodologies, such as beads, emulsion, amplification, and magnetics (BEAMing) and digital droplet PCR (ddPCR), are characterized by higher sensitivity and specificity, and have the additional capability of quantitating mutant ctDNA levels. A recent prospective clinical study compared a ddPCR-based plasma assay against tissue genotyping for the detection of EGFR and KRAS mutations in patients with advanced NSCLC. This assay demonstrated 100% specificity for the detection of EGFR-sensitizing and KRAS mutations. The median turnaround time for ctDNA testing results was only 3 days (range, 1–7 days), highlighting another advantage of ddPCR technology. BEAMing is a highly specific and sensitive PCR methodology with quantitative capabilities. Bettegowda et al reported ctDNA test characteristics using a BEAMing platform in a cohort of 640 patients across all tumor types and stages, demonstrating variability based on stage and tumor type. Within the cohort of 206 patients with metastatic colorectal cancer, the sensitivity for detection of KRAS mutations was 87.2% and the specificity was 99.2%. ctDNA analysis using BEAMing technology has also been retrospectively validated in studies in NSCLC to detect both EGFR-sensitizing and EGFR resistance mutations.[20,21] One limitation of both ddPCR and BEAMing is their limited ability to assess for more complex alterations, such as translocations or copy number alterations.
NGS technologies are capable of sequencing millions of small DNA fragments in parallel, resulting in highly sensitive and specific detection capabilities. NGS platforms also have the ability to quantify allele frequency and can detect complex alterations such as translocations and copy number changes. Using a multiplex NGS PCR platform to investigate for a panel of 50 cancer-related genes, Couraud et al reported an assay specificity of 86% to 100% and a sensitivity of 58% for the detection of all 50 genes. The ability to detect complex alterations present at low allele frequencies using NGS technology was demonstrated in a study by Paweletz et al. Complex alterations—including rearrangements in ALK, ROS1, and RET; HER2 insertions; and MET amplifications—were detected with 100% specificity and 77% sensitivity. Several commercially available ctDNA assays, including Guardant360 (Guardant Health) and FoundationACT (Foundation Medicine), utilize NGS platforms to investigate for a panel of potentially actionable mutations. Lanman et al validated the Guardant360 platform in a study of 165 patients with advanced solid tumors and reported a sensitivity of 85% and a specificity of > 99%. A subsequent large retrospective analysis of ctDNA testing results from more than 15,000 patients in whom the Guardant360 platform was used reported an accuracy of 87% when plasma genotyping was compared with historical tissue genotyping data from the Cancer Genome Atlas.[25,26]
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