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Tips for Better EGFR Mutation Testing

Posted by Catherine Huang, PhD on Jan 10, 2019 12:00:00 AM

dnaTips for Better EGFR Mutation Testing

Molecular testing of genomic alterations in the EGFR gene is critical to personalized treatment decisions for patients with advanced non-small cell lung cancer (NSCLC). However, the testing landscape is complex. Some mutations confer sensitivity, and others confer resistance to anti-EGFR targeted therapies. Testing methodologies like NGS screen broadly for all potential mutations in a genomic region, while PCR-based methods specifically focus on known mutations. Both methods have their respective advantages and disadvantages. Testing is typically performed on multiple sample types including FFPE tissue samples, cytology samples, and cell-free tumor DNA (ctDNA) samples from peripheral blood, and each sample type presents unique challenges. While the complexities of EGFR mutation testing may be familiar, this guide will focus on how the right toolbox of NGS standards can help simplify and improve the quality and confidence of testing results.

Problem:  Many Critical EGFR Mutations Must Be Assessed

NSCLC patients with EGFR activating mutations (~40% of Asian and ~12% of Caucasian patients) are candidates for anti-EGFR therapies such as geltinib (Iressa®) and erlotinib (Tarceva®). Identification of these patients is key because their progression free survival (PFS) is significantly better on anti-EGFR therapy than on traditional chemotherapy.1,2

Tyrosine Kinase Inhibitors (TKIs) are small molecule drugs that bind to the ATP binding pocket of the EGFR gene blocking its activation and signaling functions. The two most common EGFR-activating mutations are small deletions in exon 19 (such as E746-A750del) and L858R substitution in exon 21, which represent >90% of the activating EGFR mutations. Other EGFR activating mutations are G719X in exon 18 and L861Q in exon 21.3 The EGFR activating mutations increase the affinity of the ATP-binding pocket for TKIs as compared with WT EGFR, which explains why the therapy isn’t effective for those patients without activating EGFR mutations.

Cancer cells respond to the first line EGFR TKIs by developing resistance, usually within 10-12 months.4 One primary driver of resistance is the development of the EGFR T790M mutation in exon 20, which accounts for 50-60% of disease progression while on TKI therapy.5 Resistance may develop due to pre-existing drug-resistant clones or de novo acquisition of T790M in cells initially drug sensitive. T790M mutation increases the affinity of EGFR for ATP and reduces its affinity for the TKI drug molecules.  However, osimertinib (TAGRISSO®) is a second line treatment for T790M positive patients that had disease progression while on first line EGFR TKI therapy.  Usually in T790M mutant EGFR protein, the bulky methionine residue blocks binding of small molecule drugs to the ATP binding pocket, but osimertinib binding requires this change, and therefore, identifying the T790M as the mechanism of resistance is critical for the choice of this second line therapy.

Second line therapies can lead to further mutation and resistance. Some patients develop tertiary mutations such as C797S.6 Studies from Niederst, et. al., have shown that the presence of T790M and C797S together in the same allele (cis) or in a different allele in the same cell (trans) confer different sensitivities to anti-EGFR TKIs.7 Determination of whether distinct variants are on the same or different molecules further complicates analysis of these tests.

Tip: Multiplexed NGS Standards Can Simplify QC for Multiplexed EGFR Mutation Tests 

SeraCare has combined the most critical EGFR mutations into a single reference material - Seraseq® ctDNA EGFR Panel – which contains the following mutations: p.T790M, p.L858R, p.E746_A750 del ELREA, and p.G719S. These mutations are detected by the Roche cobas® EGFR Mutation Test v2 and the reference material compatible with this test. Having a multiplexed NGS standard is important for sample quality control because these mutations represent different mutation types for which tests are likely to have different sensitivities and background levels. For even higher levels of multiplexing, Table 1 below provides a complete list of EGFR mutations in SeraCare’s VariantFlex Library for on-demand customization of EGFR-based reference standards.

Tips for better EGFR Mutation Testing-1

Table 1. EGFR mutations available in the VariantFlex Library.

Problem: Cell-Free DNA is a Challenging Sample Type

Tumor tissue biopsy for EGFR mutation testing is highly invasive and costly. Circulating cell-free tumor DNA (ctDNA) can be used for EGFR mutation testing from less-invasively collected peripheral blood, making it a preferable sample type.8 Studies have also indicated that use of ctDNA may better represent all the variants in a heterogenous tumor than tissue biopsy, which may sample only one or a few subclones.9

However, ctDNA is present in very limited quantities, especially from patients with early stage disease.  Concentrations as low as 1.12 ng/mL of plasma were measured in NSCLC patients.10 This low amount of material makes optimization of DNA extraction systems important and validation of minimum assay input amount becomes more critical.  Additionally, cell-free DNA is small-sized, with most molecules around 166 bp in length. Amplification-based systems, whether PCR or NGS, must be optimized for smaller amplicon size for efficient amplification from the small size templates.

These challenges are particularly relevant for EGFR T790M testing from blood samples. T790M results from a C>T conversion, but it has been documented that C>T can also be generated by heat-induced deamination,11 and both PCR and NGS assay workflows repeatedly expose DNA to high temperatures. For most sample types and mutation types, this “background” signal generated by the testing method is insignificant. However, the limited amount of input ctDNA often necessitates more amplification cycles and T790M at very low “background” levels can, if true, be clinically significant as a predictor of early resistance development.3

Tip: Use Patient-Like NGS Standards

Intact genomic DNA and sheared or sonicated DNA are not suitable control materials for ctDNA testing. Sonication of DNA results in a broad range of fragment sizes, and thus is not a direct mimic of the behavior of native cfDNA in downstream applications such as NGS library preparation. Additionally, sonication and shearing results in DNA damage that makes the DNA difficult to amplify resulting in poor NGS library efficiency (complexity). An additional problem is that use of these materials do not control for the entire testing workflow. 

Seraseq ctDNA reference materials have been demonstrated to have the same DNA size profile and “amplifiability” as native cfDNA (see A Novel Circulating Tumor DNA Reference Material Compared on Next-Generation Sequencing to Digital PCR Assays presented at AMP2017). Seraseq ctDNA EGFR Panel refence standards are available in two different formats (purified DNA and encapsulated DNA in plasma) to meet the different QC workflow control in testing of NSCLC patient samples. The Seraseq ctDNA EGFR Panel Mutation Mix is a 4-plex reference standard in purified DNA format for use in EGFR assay optimization, bioinformatics pipeline tuning, and assay validation, as well as assessment of sensitivity at different input amounts. The Seraseq ctDNA EGFR Panel Reference Material is a sample-to-result workflow process control reference standard in an encapsulated synthetic plasma matrix requiring sample extraction as part of the assay workflow QC. This format is most useful in end-to-end process QC, LoD determination, and as a routine positive run control. 

Use of patient-like reference materials can simplify assay optimization, troubleshooting, and routine QC measurements that ensure a robust and high quality test result. For example, when our R&D laboratory first evaluated the ArcherDx Reveal ctDNA 28 assay, we noted that the product insert document called for ~15-18 PCR-2 cycles which may be decreased depending on library yields. However, use of the maximum number of PCR cycles often resulted in an unacceptably high background detection of T790M.  Therefore, our lab used the Seraseq ctDNA v2 Mutation Mix (WT and AF0.125% to AF2%) to optimize the number of PCR cycles to maintain assay sensitivity and minimize background noise. Without highly patient-like reference materials, optimization studies would have led to wasted efforts as well as erroneous conclusions.

Problem: Multiple Assays and Multiple Sensitivities

In general, NGS assays are sensitive and can detect all mutations present in the regions of EGFR assayed. However, the tests have relatively high costs and longer turnaround times. PCR-based tests, especially CAST-PCR (Competitive allele-specific TaqMan PCR) can also be highly sensitive but can only detect the mutations for which primers/probes were designed, with somewhat lower costs and faster turnaround times. This means that many labs may want to have multiple EGFR mutation tests validated for use especially on challenging or very precious patient samples.  In some cases, NGS assays may be used at initial diagnosis/screening in tumor profiling, while disease progression due to EGFR T790M resistance mutation can be tested on an FDA-approved companion diagnostic test, such as the Roche Cobas EGFR test(s).

TIP: Reference Materials Allow Lab Personnel to Better Know Their Tests

The 4-plex Seraseq ctDNA EGFR Panel reference standards are available at 1% and 0.1% variant allele frequency, as determined by digital PCR and allele-specific TaqMan chemistry to establish a “ground truth” against which assays can be compared. Having reference materials near the assay’s lower limits of detection is helpful for establishing LoD and for comparing performance across assay systems or performance on the same assay systems in different laboratories. For example, a recent poster presented at the 2018 AACR Conference described the use of the Seraseq ctDNA v2 reference material as part of a multi-lab study and concluded that the contrived reference materials were useful in benchmarking assay sensitivity and selectivity across different NGS methods and bioinformatic pipelines. Contrived reference materials can help laboratory personnel to better understand the strengths and weaknesses of their assays, as well as standardize results across labs that will improve assay testing quality to enhance patient care decisions and benefit the patient (see poster presentation video by Stein et al.).

Learn more about SeraCare’s comprehensive liquid biopsy portfolio of products.

poster inset EricaSteinNISTScientific Poster & Video Download 

Multi-Laboratory Assessment of a New Reference Material for Quality Assurance of ctDNA Measurements

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Topics: SeraSeq, EGFR, ctDNA