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Comparison of KRAS Liquid Biopsy Kits Confirms Differences at Low Mutant Allele Fraction

NEW YORK – A study comparing two CE-marked liquid biopsy KRAS assays has confirmed that the platforms differ in their results for samples with very low mutant allele frequencies, though the clinical implications of this difference, if any, have yet to be conclusively investigated.

The two blood-based assays compared in the study, which are both available for clinical use in Europe, but not in the US, use two different PCR methods: Sysmex Inostics' BEAMing — a magnetic bead-based digital PCR platform — and Biocartis' Idylla quantitative real-time PCR technology.

In particular, the study, published this June in Scientific Reports, focused on the performance of the two technologies in cases of low allele frequency, paying particular attention to a "gray zone" below 1 percent mutant allele fraction in which investigators found that the Sysmex OncoBEAM Ras test reliably detected KRAS mutations across 116 patient plasma samples while the Idylla KRAS kit was discordantly negative in a significant proportion of those samples.

Although the study authors argued that the results suggest that the Idylla technology could be negatively impacting therapy decision making, Biocartis countered that the clinical importance of RAS mutation detection below one percent MAF is a more complicated question, with other research suggesting that low allele frequency mutations should not inform the same clinical decisions as higher frequency findings.

Ana Vivancos, the study's first author and a researcher at Vall d’Hebron Institute of Oncology in Barcelona, said in an interview this week that the comparison study stemmed from questions raised at a recent scientific meeting about choosing between the two assays. 

"One of the other authors on the paper asked me about this new [Idylla] kit emerging … [and] asked me what should we use, "Vivancos said. "I said, 'I can't tell you.' All we can do is go to the lab and take samples side by side. So that's what we did.".

Vall d'Hebron served as the first research-use test center for the OncoBEAM test, and Vivancos has received consulting fees from Sysmex Inostics, but the authors said that the company had no role in the design of the current study, or the collection, analysis, or interpretation of data.

International guidelines now widely recommend RAS gene testing in colorectal cancer in order to exclude mutation-positive patients from receiving anti-EGFR drugs that fail to work meaningfully in this biomarker population.

Because of this clinical import, Vivancos and her coauthors argued, accurate detection is of high importance, and the use of blood-based tests that lack necessary sensitivity may lead to mischaracterization of wild-type patients, potentially leading to worse outcomes.

Liquid biopsy assays are only beginning to be used in this space, and the two studied platforms are CE-marked for clinical use in Europe, but don't currently have regulatory approval in the US.

According to Vivancos, KRAS mutant allele frequencies can vary widely from patient to patient in colorectal cancer. In earlier research, she and her colleagues have seen almost 50 percent of CRC patients presenting at baseline with KRAS mutant levels below 1 percent MAF. For the current study, she and her colleagues planned to specifically look at cases with MAF below 5 percent to try to examine assay performance across these lower ranges.

Cases for the comparison came from clinically tested individuals seen at four Spanish hospitals, and a total of 559 metastatic CRC patients met selection criteria. When their plasma samples were tested with the OncoBEAM RAS CRC assay, 265 (47.4 percent) were KRAS mutants, of which about half, 147, had a MAF below 5 percent. The Idylla requirements of 1 ml of plasma could only be met for 116 out of those 147 samples, so the final comparison group was 116.

A subset of the cohort had the lowest level of mutant alleles — less than 1 percent frequency — and of those, 43 had matched tissue test data available to re-check the results of the two liquid biopsy assays.

Overall, Vivancos and her team reported that the Idylla assay recapitulated the OncoBEAM positives in 81 out of the 116 compared patients. In the subset of 79 OncoBEAM-positive samples with under 1 percent MAF, Idylla agreed in 48 cases.

Looking at the low MAF cases with matched tissue, investigators reported that OncoBEAM results were in agreement with tissue in 31 out of 42 cases. Idylla recapitulated tissue KRAS status in only 20 of those 43. 

For the 12 out of 43 patients that were KRAS mutation-negative in tissue, but positive by OncoBEAM, nine were also positive via Idylla. According to the authors, this adds evidence to the possibility that liquid biopsy-positive, tissue-negative patients may in fact be mutation positive, with their tissue results confounded by intratumoral heterogeneity or sample insufficiency.

Reflecting on the results, Vivancos and her coauthors suggested that in not calling the positives that OncoBEAM did at low MAF, the Idylla technology may cause incorrect treatment decisions.

"Most of the samples with low MAF analyzed by the Idylla ctKRAS kit will result in false-negative KRAS [wild-type] results," the group wrote. "In this scenario, the use of Idylla may incorrectly select patients for anti-EGFR therapy, exposing patients to undue side effects, increased medical costs, and worse outcomes due to lack of demonstrated clinical benefit of anti-EGFR therapy in patients with RAS mutations."

But according to Biocartis, the question is not so simple. "The difference between research and clinical use is important to take into account here," Biocartis Chief Scientific Officer Geert Maertens said in an email. The below-1 percent MAF grey zone is not just one in which assay results differ, but one in which "clinical patient treatment decisions become uncertain," he wrote.

For example, a Clinical Cancer Research study by French investigators in 2015, found that patients very low-frequency KRAS mutations in tissue, even when those calls were truly positive, tended to respond to anti-EGFR therapy at rates much more like KRAS wild-type patients. 

Maertens also highlighted a report in JCO Precision Oncology from March of this year, which he said reiterated that Idylla liquid biopsy and tissue tests show the same association with patients' progression-free and overall survival. 

"The Idylla technology and the sensitivity 'cutoffs' of Idylla tests are based on the clinical guidelines and prospective clinical studies," he said. 

Even in the study from Vivancos and her coauthors, patients with higher abundance KRAS mutations — MAF of 1 percent or higher — showed worse outcomes than those carrying low abundance mutations. Rapid progressors similarly had higher MAF than slower progressors.

Finally, since the study set included no OncoBEAM wild-type samples there could be no evaluation of platform specificity. In other words, the researchers did not examine whether OncoBeam might be calling cases as negative that Idylla marked as positive.