NEW YORK (GenomeWeb) – Next-gen sequencing-based liquid biopsy analysis has now been available for several years via central send-out vendors like Guardant Health and Foundation Medicine.
In contrast, NGS tools companies have been slower to enter this area, launching broad sequencing kits for circulating cell-free DNA only in the last year or so.
Although there continues to be debate about when and how liquid biopsies should be used in clinical practice, labs are already incorporating these new assays into their operations. At the annual meeting of the Association for Molecular Pathology last week, two labs who have adopted recently launched kits from Thermo Fisher and Roche presented on their experiences.
During the meeting's corporate workshop day, Charles Ma, associate director at Cancer Genetics, discussed his team's adoption of ThermoFisher's Oncomine Lung cfDNA assay, which provides the backbone for a test CGI calls Liquid::Lung-cfDNA.
According to Ma, CGI, like many other labs, is excited about the possibilities liquid biopsy offers for more rapid, less expensive, and safer cancer genotyping, as well as for real-time monitoring and eventually early cancer detection.
Thermo Fisher currently offers Oncomine kits for lung cancer, colorectal cancer, and breast cancer — each with a different targeted panel of hotspots. But CGI decided to begin in lung cancer, which is emerging as a front line in the advance of liquid biopsy into the clinic.
CGI has been running its test using the Oncomine lung cfDNA assay since about May of this year, Ma said at the AMP meeting. The kit, which covers hotspots in 11 genes, offers a compromise between breadth and actionability. As other researchers have highlighted in recent years, techniques like PCR can offer increased sensitivity for individual mutations, but they don't allow the unbiased detection that NGS does. But overly large NGS panels can be cumbersome and challenging for clinicians who are less familiar with genomic medicine.
After Thermo Fisher launched the Oncomine cfDNA panel in late 2016, a company-organized research group called the OncoNetwork Consortium began evaluating the kit, sharing initial results this spring at the AMP Global Congress in Berlin.
Using Horizon Dx cfDNA reference standard samples, the 11 participating laboratories reported that they were able to detect eight target mutations at three different allele frequencies, with an average sensitivity of 94.8 percent and an average specificity of 99.8 percent.
Ma said that in CGI's hands, the panel can detect variants down to between .05 and 1 percent allele fraction, which translates for the company into sensitivity of about 85 percent, and specificity near 99 percent in research and clinical use.
The analogy of a needle in a haystack has been frequently applied to liquid biopsy, but Ma said that he views the task as even harder than that — more like finding a needle in a pile of needles.
The Oncomine technology, a method Thermo Fisher calls tag sequencing, provides an especially strong platform for maintaining high specificity, Ma added, or, in other words, making sure you don't pick the wrong needle.
Like Thermo's AmpliSeq, tag sequencing relies on creating amplicons. But it also involves molecular tags added prior to amplification. Molecules with the same tag are grouped into families, which allows for cross checking that can help ensure that resulting variant calls are accurate.
In addition, Ma said during the AMP meeting that CGI's liquid biopsy workflow includes a requirement for two aliquots of blood. If a therapeutically actionable mutation is detected in one aliquot, the company tests the second to make sure that they can reproduce that finding.
"So far, we've never had to go to a third aliquot," Ma said. "But if it is not repeatable, I'd probably ask for a third sample and try a third time," he added. "Or you could try digital PCR or another [orthogonal assay] just for that mutation."
As an illustration of what CGI sees as the value of blood-based cancer sequencing, Ma highlighted a case study from a few months after the company rolled out the test this spring.
A physician sought out a blood-based analysis for a patient exhibiting signs of resistance to Genentech's Tarceva (erlotinib). The Oncomine assay detected the patient's initial sensitizing EGFR mutation, as well as the classic T790M resistance marker, allowing the patient, who was in poor health, to be moved to a new drug without needing to have a biopsy.
In a second industry-sponsored session at AMP last week, representatives from ResearchDx — a diagnostic development and clinical lab services provider — discussed their ongoing work with Roche's recently-launched Avenio liquid biopsy panels.
Unlike Thermo Fisher's Oncomine kits, which are each targeted to a specific tumor type, the Avenio assays are agnostic and vary in breadth, including a 17-gene "targeted" assay, a 77-gene "expanded" kit, and a 197-gene "surveillance" panel optimized for longitudinal monitoring.
Last November, Roche previewed two of the now three research-use-only panels and described an inter and intra-laboratory reproducibility study using contrived samples in which ResearchDx served as one of three early-evaluation labs.
At AMP this year, ResearchDx's chief medical officer Shelly Gunn described how she and her colleagues have now moved on from this early study to an ongoing internal evaluation focused on the associated bioinformatic pipeline and how it fits in with their overall lab workflow.
According to Gunn, ResearchDx's introduction of the Avenio technology to its menu "has empowered [the company] to enter the liquid biopsy era."
When it became part of the Roche early evaluation program, Gunn said that ResearchDx was already fielding requests from customers for liquid biopsy analyses.
Faced with this need, a lab can develop its own assays and protocols using available reagents, can contract with an outside test provider, or can seek out a commercial kit that provides an end-to-end solution. ResearchDx wanted the latter.
Gunn said that she and her colleagues were impressed with the CAPP-Seq technology, which demonstrated near-perfect concordance for a variety of different variant types.
"As a lab looking for a kit like this, Avenio was also very attractive," Gunn said, because it fit into ResearchDx's existing five- to seven-day NGS workflow. In fact, she added, lab techs running samples during the firm's participation in Roche's EEP didn't even know they were processing liquid biopsies at the time.
However, the early evaluation did not include in-house variant analysis and calling, so it was not a true test of the end-to-end solution that ResearchDx was hoping for. Roche did the analysis of output FASTQ files and then shared the resulting data with the labs after the fact.
Gunn said that her lab has now started an internal evaluation in which it is making sure that running the software for variant calling and reporting in-house gives it what it is expecting.
So far, she said, the answer is yes, but the company is still in the process of testing the system, especially at the lower limits of acceptable sample input — 15 ng of DNA in the case of the expanded panel.
During the AMP workshop, she shared a few examples from samples the lab has processed so far.
In one, a sample from a case of a tumor of unknown origin, Research Dx was able to isolate a "robust" 220 ng of DNA from a 4.7-ml blood plasma sample. But in the second case, also a tumor or undetermined origin, 4 ml of plasma only yielded 20 ng of DNA.
"We looked at this as an opportunity to test lower limits of input of the assay … so we used the 15-ng input for the expanded 77-gene panel," Gunn said.
Encouragingly, quality control metrics for things like coverage uniformity and sequencing depth looked "just as good as" they did for the 50-ng sample.
"We will definitely be testing this with more samples – but at least with the first few it's working quite well," Gunn added. "And we clearly have the information in our hands we were looking for at the end of this assay – not just mutations, but the ability to annotate and make some clinical research conclusions on what we found."