The science of metrology is tightly coupled with biological science, in tandem with biology becoming less descriptive and more quantitative over the past half-century. The US National Institute of Standards and Technology (NIST), an agency of the US Department of Commerce, has been established to ‘promote U.S. innovation and industrial competitiveness by advancing measurement science, standards and technology in ways that enhance economic security and improve our quality of life’.
Photo courtesy of Flickr user Marni-
To this end, the Genome in a Bottle consortium was established in 2012 to develop “reference materials, methods data and reference methods needed to assess performance of human genome sequencing”. At a recent meeting, held at the National Institute of Science and Technology (NIST) headquarters in Gaithersburg, Maryland on August 27-28, 2015, a discussion arose concerning the need for standards for circulating free DNA (cfDNA). But first, a little background on what cfDNA is.
For those not familiar with cfDNA, it is degraded DNA found in the bloodstream of individuals that is regularly and routinely cleared from the circulation. In the late 1980’s, it was discovered (ref. 1) that fetal DNA could be detected as early as a gestational age of 9 weeks, and, with the advent of next-generation sequencing methods a non-invasive prenatal test, was developed for non-invasive, sensitive and specific detection of fetal aneuploidies from maternal plasma (ref. 2). Subsequent clinical studies have shown conclusively that non-invasive prenatal testing (NIPT) has significantly lower false-positive rates compared to standard screening for trisomies 21 and 18 and significantly higher positive predictive values (ref. 3). Currently there are several commercial companies offering these NGS-based laboratory-developed tests, including Natera, Sequenom, Ariosa Diagnostics (now part of Roche) in the United States; and BGI-Shenzhen, Berry Genomics, and CapitalBio in China. While currently in the US this test is only for pregnancies considered high-risk, it is expected in the coming years to become not only for average risk pregnancies but also covered by insurance.
The concept of a ‘liquid biopsy’ can trace its history to 1948, when DNA in circulation was first reported by Mandel and Metais in a French journal (ref. 4). Today, the term ‘liquid biopsy’ mixes two distinct concepts, including not only the detection of circulating tumor DNA (ctDNA) but also circulating tumor cells (CTCs), lending to confusion in the popular press as to whether it is DNA or tumor cells that are being detected and characterized. While CTCs will remain an active area of research, their presence at very low levels (in advanced cancers about 1 cell per 1 mL of blood, which also contains about 1 million white blood cells and 1 billion red blood cells) translates into limited clinical usefulness until better purification and characterization methods are developed.
To elaborate, Janssen Pharmaceuticals (a division of Johnson and Johnson) received FDA approval in 2004 for their CellSearch™ technology as a prognostic test for metastatic breast cancer, based upon a cell surface adhesion marker called EpCAM. However, with limited clinical sensitivity, this test was not widely adopted for this purpose. This has not limited a recent explosion of circulating tumor-cell analysis companies launching systems, among them Janssen’s upcoming iChip (ref. 5), Silicon Biosystems’ DEPArray™, Fluxion Bio’s Isoflux™, Clearbridge Biomedics ClearCell®, Cynvenio Biosystems’ LiquidBiopsy and many others. However due to their rarity and difficulty in determining causative mutations (it has been estimated that single-cell whole-genome sequencing or whole-exome sequencing would need equivalent read-depths on the order of 10,000-fold) (ref. 6), the CTC approach to a liquid biopsy is still several years away from routine clinical application.
Returning to the NIST meeting in August, Dr. Ken Cole of NIST’s Bioassay Methods Group shared a presentation on his group’s work looking at HER2 copy number measurements using both real-time qPCR and digital PCR on the same Standard Reference Material 2370, and how similar the measurements were across platforms. Controlling for pre-analytical variables (collection of the blood sample, isolation of the plasma, storage and extraction) is a difficult task, and one that deserves attention (and more research) in the future. As far as the utility of ctDNA for its prognostic value, he pointed to recent work published in Science Translational Medicine using ctDNA as an early detection marker for relapse, nicely summarized in this BBC report.
The following discussion certainly was instructive; the lack of standards means that developers of these assays can claim sensitivity that may strain credulity. cfDNA typically yields 10-30 ng per 10 mL of blood (that in turn yields 4-5 mL of plasma). In 30 ng of DNA, fragmented into 166-170 base-pair pieces, there are only about 5,000 copies of a diploid human genome in it. If a sensitivity of 0.1% is claimed, that is 1 part in 1,000. In that 30 ng of DNA, of 5,000 natural (‘wild-type’) sequences there are only 5 mutant copies available for detection. At 0.02% sensitivity, there is only 1 copy out of 5,000 available for detection.
At the annual Association for Molecular Pathology conference in Austin Texas, SeraCare announces the early-access of a new Seraseq Circulating Tumor DNA Reference Material, that contains five important mutations to either assist cancer treatment or monitor disease resistance (BRAF V600E, EGFR T790M, EGFR delL747-P753insS, ERBB2 dup, KRAS G12D). We show sequencing results (in cooperation with Swift Biosciences) of mutants at 10% allele frequency (to wild-type), 5% AF, 2.5% AF, and 1.25% AF (with plans to go much lower), and have the ability to not only expand a customized collection of mutations, but also adjust the relative allele frequency to whatever limit of detection is required.
If you are planning to attend the Association of Molecular Pathology meeting in Austin, Texas November 4-7 2015, we will be presenting early data about the performance of our circulating tumor DNA reference material (in addition to a new multiplexed fusion RNA product also in early access) at the Corporate Workshop Day November 4 2015 at 8am in Room 15, Level 4 of the Austin Convention Center.
For further information (or to request a trial), please feel free to contact us. These are certainly exciting times.
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