of years, clinical researchers, test developers, and others have begun to pay increasing attention to a relative lack of tools to ensure assays are validated and performed according to common, or at least equivalent standards.
Last fall, for example, saw the launch of a consortium called the Blood Profiling Atlas in Cancer (BloodPAC) under the White House's Cancer Moonshot Initiative, aimed at aggregating and harmonizing data on current liquid biopsy tests, and on standardizing protocols for sample collection, preparation, and analysis for future test development.
"While many blood proﬁling platforms exist for research use only (RUO), questions remain regarding the performance characteristics and clinical validation of these platforms, and standard protocols for sample collection, processing, and analysis remain to be established. This information is a prerequisite to the design of clinical studies to demonstrate clinical utility … assay reproducibility, and provide support for [US Food and Drug Administration] approval and payer reimbursement," authors wrote in a paper describing BloodPAC this spring.
In parallel to BloodPAC (and to other efforts by academic groups), companies have also begun to highlight new products this year, recognizing growing attention and appetite for these tools.
Test validation (and even more so post-launch quality control and proficiency monitoring) may not be the most exciting aspects of diagnostic development and clinical genomics, but they are nonetheless a crucial part of both proving analytical accuracy, and eventually building a case for clinical utility.
Reference standards, or materials, serve as truth sets that can be used to demonstrate the performance of an assay, test its limits, check to make sure it is being performed correctly, or to precisely compare different tests to one another.
With a much longer history, cancer tissue tests also enjoy a wider variety of basic tools for this validation and monitoring work. In liquid biopsy though, the production of contrived materials that hew close enough to what actual circulating tumor DNA looks like has been a challenge.
Actual clinical samples, while ideal for proving the clinical validity of a test, aren't available in enough quantity, and don't offer the manipulability that makes contrived references so useful for testing different aspects of an assay's performance.
"Although plasma samples collected from cancer patients would be the ideal source of quality control materials in terms of biochemical properties, it is impractical to obtain a sufficient volume of plasma from one or a few cancer patients for quality assurance programs involving a large number of laboratories," Chinese University of Hong Kong researchers Jason Tsang and K.C. Allen Chan wrote in a discussion piece last week in Clinical Chemistry.
"Furthermore, the absolute or fractional concentrations of mutations in the plasma samples cannot be easily adjusted to test the performance of the assays at clinically critical levels, for example, at the limit of detection," the two added.
But creating synthetic references that closely mimic actual biological samples is really hard, researchers say, due to a number of factors including the fact that circulating tumor DNA is not fragmented randomly — something that can affect different test technologies differently.
"Locus-specific PCR assays would favor the amplification of longer DNA fragments over shorter ones … whereas adaptor ligation-based methods commonly used in next-generation sequencing platforms would have preference toward shorter fragments. Therefore, quality control materials with similar fragment size distributions as the circulating DNA … would be required to reflect the analytical characteristics of different detection approaches," Tsang and Chan argued in their article.
More broadly, "the technical challenges of replicating the clinical sample really reflect the field's challenges," explained Russell Garlick, chief science officer at diagnostics tools manufacturer SeraCare.
In other words, the fact that liquid biopsy testing is inherently difficult — due to the need to detect mutations that occur at a very low frequency compared to background DNA — means that creating materials to test the tests themselves is also a hurdle.
As discussion around goals for optimal reference standards continue, the last few months have seen a number of announcements by commercial firms of new products in this vein.
SeraCare itself launched a second-version ctDNA reference material product this month, which encompasses 40 somatic mutations in more than 20 genes, like BRAF V600E, KRAS G12D, and various EGFR alterations. The company's references combine a genomic DNA background with synthetic variant-containing DNA sequences at defined ratios — in this case across a range of allele frequencies down to 0.125 percent, the company said.
According to SeraCare, its references are produced using a unique technology that creates a DNA fragment size-distribution which more closely mimics native cfDNA than other shearing methods.
The company is hoping that it can develop references that are full commutable to clinical samples, but Garlick said it's not clear yet whether they've gotten there with this latest version. In an abstract shared at this year's American Association for Cancer Research annual meeting, the company reported that when it submitted its reference materials to several testing laboratories along with similar material that didn't use its special approach to shearing, "variant detection was superior in the novel reference material … using hybrid/capture-based assays."
This suggests "greatly improved commutability compared to existing materials composed only of sonicated DNA," authors from SeraCare wrote.