NEW YORK (GenomeWeb) – Stand Up To Cancer-sponsored research has shown that genotyping liquid biopsy and tumor biopsy does not yield identical results and concluded that no single diagnostic test can be considered a "gold standard."
The research was published earlier this month in Clinical Cancer Research and relied in part on the first stage of a two-year collaboration between EKF Diagnostics and Massachusetts Hospital Cancer Center.
The CCR study was performed by four major US cancer centers in the SU2C program, including members of the funding organization's circulating tumor cell Dream Team. For some of the experiments in the study, researchers used the PointMan DNA enrichment technology from Cardiff, Wales-based EKF Molecular Diagnostics.
Andrew Webb, CEO of that division of EKF, told GenomeWeb in an interview that the collaboration came about through his previous work with Dream Team member Daniel Haber of Massachusetts General Hospital on Iressa-sensitive EGFR mutations while Webb was with other firms.
The group at MGH focuses on an in-house platform called iChip, Webb said. As reported by GenomeWeb, circulating tumor cells obtained by the iChip microfluidic platform can be even grown in vitro to enable susceptibility testing.
But with any CTC isolation platform, "You're dealing with very low copy number, so the idea was to use PointMan enrichment to give the best possible analytical sensitivity for their system," Webb explained.
PointMan is a targeted amplification method that applies a simple reagent set to standard DNA extracts and runs on a real-time thermal cycler. The product of the PointMan enrichment assay can then be used for detection methods such as PCR, pyrosequencing, or Sanger sequencing.
In the CCR study, researchers did Sanger sequencing on the enriched product, "which is simple, straight-forward, and cost-effective," Webb said.
A heterogeneous mix of wild-type and tumor cells in a typical tissue biopsy means there can be uncertainty in how much of a tiny biopsy sample is actually tumor. And tumors may also be polyclonal.
"What's become clear in the early days of looking at liquid biopsy [is that] generally you won't get concordance with the results you get from the blood sample," Webb said.
Such was the case with the CCR study. The four institutions looked at a total sample of 40 patients with EGFR-mutant tumors who had acquired resistance to anti-tyrosine kinase inhibitor therapies.
The study used the PointMan enrichment on DNA from cells obtained with the iChip that had been extracted with a Qiagen kit. The enriched PCR product was then sequenced. In parallel, freely ciculating tumor DNA was extracted at Roche Molecular Diagnostics using the firm's EGFR mutation test.
The researchers detected the T790M genotype in 75 percent of tumor biopsies, 70 percent of circulating tumor cell samples, and 80 percent of ctDNA samples. The mutation was found in about half of patients using each of the assays, and the concordance among the assays ranged from 57 to 74 percent.
"While CTC- and ctDNA-based genotyping were each unsuccessful in 20 to 30 percent of cases, the two assays together enabled genotyping in all patients with an available blood sample, and they identified the T790M mutation in 35 percent of patients for whom a tissue biopsy was negative or indeterminate," the authors noted.
Because of discordant genotypes, complimentary approaches may provide the best assessment, and clinical utility should be the gold standard, the study concluded.
"All these technologies are showing great potential in terms of the level of feasibility, however it really is now down to the studies, with clinical outcomes ... analytical concordance probably isn't going to give you the best result," Webb said.
Webb further noted that blood-based testing might be a more effective way to detect metastasis, and will perhaps prove safer for very sick patients who may have failed previous treatments but can't risk additional tissue biopsies.
"Liquid biopsy is really seen as the safest preferred option for these patients ... We're looking at what is the simple PCR-based enrichment, which could also be used then to prepare the sample for a number of downstream tests," Webb said, adding, "Its something that could be easily done in the hospital but it doesn't meet the requirements of a tissue test; if we can show it has the equivalent clinical sensitivity, then I think it's going to really help improve standard of care."
Updates on EKF
Currently, the point of care division of EKF is being offered for sale. A Chinese firm, Jinjing, is currently in due diligence and EKF announced to the London Stock Market on Monday that the due diligence period was extended until October 23, Webb said.
"Essentially EKF is three businesses — it's the point of care, clinical chemistry, and molecular — so depending on what happens during the bid process [that] will decide what happens going forward," he said. "They could well take the whole company, but from the announcement this morning it's likely that molecular may have a level of independence, but I can't really comment further than that."
The PointMan technology is part of EKF Molecular Diagnostics, which is essentially a kit manufacturer. That division also has a sister company, Selah Genomics, a CLIA-certified reference laboratory that uses NGS technology and has partnered with other firms to develop a clinical support tool for colon cancer.
EKF Molecular Diagnostics' PointMan was also featured in a poster presented at the Circulating Nucleic Acids in Plasma and Serum conference in Berlin last month, Webb said. Work by a group at Kanazawa University Hospital in Japan on a small sample showed that about 91 percent of T790M mutations detected in tumor tissues had concordant mutations in plasma, concluding that "PointMan is a useful method for determining plasma EGFR T790M."
Ultimately, T790M detection may be important for future companion diagnostics for personalized third-generation chemotherapeutic drugs, such as Astra Zeneca's AZD9291 and Clovis' rociletinib. These are the first tyrosine kinase inhibitors that are believed to be effective when a patient has the T790M variant, and these drugs are in trials and are under expedited US Food and Drug Administration review, Webb said. Roche filed for pre-market approval for the Cobas EGFR Mutation Test v2 as a companion diagnostic for ADZ9291 in July, as previously reported. Meanwhile, Clovis was reported to be evaluating Sysmex Inostics' BEAMing assay for blood-based detection.
EKF Molecular Diagnostics will also seek FDA approval for PointMan. Webb said the firm is working on research and clinical trials, and plans to have the test available in Europe later this year, aiming to head down the path to 510(k) submission in the next 12 to 18 months.
"We're not the final endpoint detection, so we'd look to have this work probably done in partnership," Webb said, noting that the firm is in ongoing discussion with other companies.