Hitting the Target: Rapid Evolution of Plasma Assays Continues to Move Genomic Profiling Toward More Efficient Testing Methods

Using liquid biopsy assays to guide precision therapy in different cancer types is more relevant than ever before, and their use has even been incorporated into multiple national guidelines. Where these assays are particularly useful is for treatment selection in patients with little or no tumor tissue for sequencing or for whom repeatbiopsy is not feasible.

“Liquid biopsy is useful at any time when a physician needs genomic information about their patient’s cancer in order to make a treatment decision,” said Becky Nagy, MS, LGS, senior director of medical affairs at Guardant Health, in an interview withTargeted Therapies in Oncology. “Right now, this is largely used in patients with advanced cancers, although the potential uses will likely expand in the future.”

The ability to detect DNA derived from a tumor in the total cell-free DNA from all cells present in a patient’s blood is the basis of liquid biopsy assays.1As methods of detecting oncogenic drivers in circulating tumor DNA (ctDNA) advance, testing methods have been developed with increasingly enhanced clinical sensitivity (smallest amount of a substance in a sample that can accurately be measured by an assay) and specificity (ability of an assay to measure a particular substance versus others in a sample).²

Moreover, shed ctDNA is derived from cancer cells occurring anywhere in the body; this contrasts with tumor tissue, which can be usedto identify cancer-associated aberrations at only 1 sample location.³

There are concerns around tumor heterogeneity, where the tumor biopsy does not actually detect the different mutations that are in the patient’s tumor, whereas blood can overcomethat barrier,” Nagy said. Other advantages include a quicker turnaround time and avoidance of uninformative test results that frequently occur when insufficient tissue is collected for sequencing.

Two methods of ctDNA analysis, next-generation sequencing (NGS) and polymerase chain reaction (PCR), have emerged as the most widelyapplied techniques for use in the clinic. Realtime PCR assays are used to identify known mutations and are particularly applicable in scenarios in which a limited number of loci are evaluated. NGS technology holds promise in its ability to detect many cancer-associated mutations in a single patient sample.⁴

Real-Time PCR Companion Diagnostics With Plasma

Certain scenarios involving patients with select tumor types allow clinicians to use real-time PCR assays with FDA approval to determine whether tumors harbor specific mutations for which approved targeted therapies are available. These companion diagnostics have been verified in clinical trials against matched tumor tissue to determine concordance with traditionaltissue testing.

We call them ‘hot-spot’ tests because they are looking for just those [sites] that are frequently mutated, or so-called 'hot spots' in the gene,” Nagy said. “They are attractive [because] they are easy to do and set up. You can get them up and running in any pathology lab quickly. They are also relatively cheap and have a short turnaround time, such as days rather than weeks.”

Allele-specific PCR, alsoknown asamplif ication-refractory mutation system (ARMS), can be used to detect certain cancer-specific genetic aberrations. This type of test is sensitive enough to differentiate ctDNA from the much more abundant DNA from normal cells. The target mutation must be well characterized so that precise allele-specific PCR primers can be designed to detect small, even single-nucleotide, differences between normal and mutated alleles of a gene.⁵

A recent retrospective meta-analysis of 25 studies involving 4881 lung cancer cases evaluated the sensitivity and specificity of EGFR mutation detection by ARMS analysis of cell-free DNA compared with testing of matched tumor tissue biopsies. ARMS yielded a sensitivity of 65.3% (95% CI, 62.9%-67.6%) and specificity of 98.2% (95% CI, 97.6%-98.7%), with significantly greatersensitivity (P= .008) in later-stage tumors (73.7%; stage IIIB-IV) compared with early-stage tumors (64.2%;stageIA-IV). These results led investigators to conclude that ARMS can be used as an alternative to tissuegenotyping due to its high specificity and reasonable sensitivity.⁶

The first test to gain approval for clinical use on liquid biopsies was the PCR-based cobas EGFR Mutation Test v2.7,8This test provides an alternative for clinicians treating patients with non–small cell lung cancer (NSCLC) to test for EGFRexon 19 deletions or the exon 21 L858R point mutation, thereby determining whether they could receive targeted therapy in the frontline setting. Allele-specific PCR primers are used to expressly amplify the target mutant allele, but not its wild-type counterpart or other human DNA sequences.⁹

A previous version of this assay (v1) had been approved for detectingEGFRmutations in tumor tissue samples.9 The randomized phaseIII ENSURE trial (NCT01342965), which compared gemcitabine plus cisplatin versus erlotinib (Tarceva) for first-line treatment of patients with stage IIIB/IV NSCLC, served as the basis for the expanded indication in plasma samples. All patients enrolled in the study were required to have an EGFR exon 19 deletion or the L858R mutation by tumor tissue testing using the cobas EGFR Mutation Test v1.⁹

Of the 217 patients enrolled, 214 (98.6%) had plasmasamples availablefor testing.Concordancebetween the 2 versions ofthe cobas assay was high, with 76.7%positivepercent agreement between tissue and plasma and 98.2% negative percent agreement. Among patients with plasma samples positive for the specified EGFR alterations, targeted therapy with erlotinib resulted in improved progression-free survival (PFS) compared with treatment with the chemotherapy combination.⁹

Currently, the cobas EGFR assay has indications as a companion diagnostic for the tyrosine kinase inhibitors erlotinib and osimertinib (Tagrisso). Patients with EGFR exon 19 deletions or the L858R mutation may receive targeted therapy with erlotinib, and those with the T790M point mutations, EGFR exon 19 deletions, and L858R may receive osimertinib treatment. These EGFR alterations may be detected in plasma or tissue.^8-10

ARMS PCR techniques have recently been applied to the detection of mutations in other tumor types. In breast cancer, the therascreen PIK3CA RGQ PCR Kit was approved in 2019 as a companion diagnostic for alpelisib (Piqray), which is indicated for the treatment of patients with PIK3CA-mutated breast cancer. The kit is approved for use with either plasma or tissue samples.¹¹

The validity of the therascreen PIK3CA kit was verified in patients treated on the SOLAR1 trial (NCT02437318) that led to the approval of alpelisib in this patient population. Plasma samples collected prior to study treatment were retrospectively tested to determine PIK3CA mutation status using the therascreen test in the randomized population.¹²

In the patients analyzed (n = 543), the positive percent agreement between tissue and plasma was 54.6%; the negative percent agreement was 97.2%. Clinical efficacy of alpelisib in combination with fulvestrant for plasma PIK3CA-positive tumors was demonstrated, with a 46% reduction in the risk of disease progression or death (HR, 0.54; 95% CI, 0.33-0.88), which compared favorably with the PFS results in the tissue PIK3CA mutation–positive population (HR, 0.64; 95% CI, 0.48-0.85).¹²

False-negative results are a challenge with both of these approved assays. Low shedding of tumor DNA causing the allelic fraction to be lower than the test’s limit of detection may be the cause of the unreliable results,13 and this is especially true when the patient has a small tumor volume.³ Therefore, negative or uninformative results require validation by reflex tissue testing.^8,12

Added to that, Nagy said a “trade off” of these tests versus complete genotyping is the possibility of missing a significant proportion of mutations that if present, could open up new treatment options for patients with metastatic cancer.

Despite these limitations, these tests still have clinical utility for patients who do not have the option of (repeated) tissue biopsy, and they may be the only chance of identifying an important molecular marker in certain settings. Patients with a positive result by these assays can receive appropriate targeted therapy with high confidence, given the reliable specificity of the tests.^3,6

Will NGS Assays Replace Hot Spot Assays?

In January of this year, Guardant Health, Inc announced a strategic collaboration to develop and commercialize the blood-based NGS assay Guardant360 as a companion diagnostic for Amgen’s investigational AMG 510, an inhibitor of the KRAS G12C protein.¹⁴

The first-in-class targeted agent AMG 510 received orphan drug and fast track designations from the FDA for treating patients with colorectal cancer and previously treated NSCLC, respectively, with the KRAS G12C mutation. This mutation is present in close to 13% of patients with NSCLC.

"There are many different KRAS tests that are currently available,” Nagy said. “Some tests on the market are hot spot tests, and they only look for the most common mutations and might miss somewhere around 10% of mutations that actually are important to know about."

Nagy went on to explain that a more comprehensive assay in a patient with an advanced cancer such as NSCLC, where there are numerous actionable mutations, will be better for therapy selection.

"It’s true that you only need to test for [KRAS G12C] in order to determine if that patient would be sensitive to that drug,” she said. "However, as with many different drugs and genes, there are other mutations that will predict whether a patient will respond to a given targeted therapy. Having a more comprehensive view of that patient’s genomic profile will help clinicians not only pick the right drug but understand if that drug will be effective or if there would be other mutations or other types of genomic alteration that would predict a [better] response to therapy."

Previously, the Guardant360 assay received expedited access pathway designation from the FDA for use as a comprehensive liquid biopsy for patients with advanced cancer.¹⁵ Guardant Health has also partnered with AstraZeneca to develop a companion diagnostic test for the EGFR inhibitor osimertinib.¹⁶

Future Directions

In April 2020, the FDA released a guidance for industry encouraging expanded labeling for companion diagnostics used in precision oncology to facilitate broader application of assays with multiple drugs that target the same mutation in the same tumor type. One example of such a need identified by the FDA was the wide range of assays, for use in both tissue and plasma, that are intended to detect EGFR exon 19 deletions or the exon 21 L858R mutation in patients with NSCLC but have approvals as companion tests for only a subset of the drugs that target these alterations (TABLE).¹⁷

The field may see broader application of these FDA-approved plasma companion tests, however the full promise of this technology requires that patients undergo complete genotyping.

"If you are testing only for the tip of the iceberg and you are not looking underneath the surface for all the other mutations that could allow that patient to receive [the best drug available], it’s problematic,” she said. "The personalized medicine community wants to see a move towards more comprehensive genomic profiling."

Nagy wants clinicians across settings to be comfortable with the idea of using plasma tests reliably. "I want to dispel the myth that ctDNA testing is new and experimental,” she said. "Patients being treated with targeted therapies based on a ctDNA test have comparable outcomes to patients treated [based] on tissue tests, and we have also shown that [in clinical data].”

_References:

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_{2. Saah AJ, Hoover DR. “Sensitivity” and “specificity” reconsidered: the meaning of these terms in analytical and diagnostic settings. Ann Intern Med. 1997;126(1):91-94. doi: 10.7326/0003-4819-126-1-199701010-00026}

_{3. Shohdy KS, West HJ. Circulating tumor DNA testing—liquid biopsy of a cancer [published online ahead of print, March 26, 2020]. JAMA Oncol. doi: 10.1001/jamaoncol.2020.0346}

_{4. Calabuig-Fariñas S, Jantus-Lewintre E, Herreros-Pomares A, Camps C. Circulating tumor cells versus circulating tumor DNA in lung cancer—which one will win? Transl Lung Cancer Res. 2016;5(5):466-482. doi: 10.21037/tlcr.2016.10.02}

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_{6. Li C, He Q, Liang H, et al. Diagnostic accuracy of droplet digital PCR and amplification refractory mutation system PCR for detecting EGFR mutation in cell-free DNA of lung cancer: a meta-analysis. Front Oncol. 2020;10:290. doi: 10.3389/fonc.2020.00290}

_{7. FDA grants first liquid biopsy approval to the Roche cobas EGFR Mutation Test v2 [news release]. Indianapolis, IN: Roche Diagnostics Corporation; June 1, 2016. bit.ly/3euTfBN. Accessed April 17, 2020.}

_{8. cobas EGFR Mutation Test v2. FDA website. bit.ly/2KfVDyw. Published June 1, 2016. Accessed April 17, 2020.}

_{9. Summary of safety and effectiveness data (SSED): cobas EGFR Mutation Test v2. FDA website. bit.ly/3bAqKAV. Published November 13, 2015. Accessed March 9, 2020.}

_{10. cobas EGFR Mutation Test v2. Roche Diagnostics website. bit.ly/2yrdOi8. Accessed April 17, 2020.}

_{11. Qiagen launches first FDA-approved companion diagnostic PIK3CA biomarkers to enhance precision medicine in breast cancer [news release]. Germantown, MD, and Hilden, Germany: Qiagen NV; May 24, 2019. bit.ly/2RwvMWW. Accessed March 9, 2020.}

_{12. Summary of safety and effectiveness data (SSED): therascreen PIK3CA RGQ PCR Kit. FDA website. bit.ly/3cCYgXY. Published May 24, 2019. Accessed March 9, 2020.}

_{13. Tsui DWY, Blumenthal GM, Philip R, et al. Development, validation, and regulatory considerations for a liquid biopsy test. Clin Chem. 2020;66(3):408-414. doi: 10.1093/clinchem/hvaa010}

_{14. Guardant Health announces collaboration with Amgen to develop a global liquid biopsy companion diagnostic for AMG 510 KRAS G12C inhibitor [news release]. Redwood City, CA: Guardant Health, Inc; January 13, 2020. bit.ly/3eoHwoe. Accessed April 17, 2020.}

_{15. The Guardant360 assay receives expedited access pathway designation for breakthrough devices from FDA [news release]. Redwood City, CA: Guardant Health, Inc; February 15, 2018. prn.to/2yn0yLm. Accessed April 17, 2020.}

_{16. Guardant Health partners with AstraZeneca to develop blood-based companion diagnostic tests for Tagrisso and Imfinzi [news release]. Redwood City, CA: Guardant Health, Inc; December 13, 2018. bit.ly/3blS0TE. Accessed April 17, 2020.}

_{17. Developing and labeling in vitro companion diagnostic devices for a specific group of oncology therapeutic products: guidance for industry. FDA website. bit.ly/2VfLSqk. Published April 2020. Accessed April 17, 2020.}