Important work published in ASCO JCO Precision Oncology suggest technical factors to be the major source of discordant ctDNA results
One major area of concern regarding wide use of liquid biopsy for therapy selection in precision medicine has been the discordance between the genetic analysis of tissue biopsy (the accepted “gold standard”) and mutation detection in plasma. While the need for liquid biopsy is clear in cases of insufficient tissue availability or the need for ongoing monitoring, if the mutation analysis from plasma is not reliable and trustworthy wider adoption will be certainly hampered.
Several publications give cause for concern – and uncertainty
Several publications (see references 1, 2 and 3 below) showed sharp differences between tissue genotypes and mutations detected in plasma; for example, in Kuderer et al. only 22% of the variants they detected in tissue (10 of 45) were detected in plasma.
Apart from technical factors, a few biological factors were attributed to the cause of false- negative (FN) and false-positive (FP) results in plasma. For the former, FNs were attributed to potentially be from ‘non-shedding’ tumors; these tumors are estimated in frequency to be found 20% to 30% of the time, (see reference 4). For FPs, white blood cell DNA can undergo clonal hematopoiesis, where its affect in plasma is called clonal hematopoiesis of indeterminate potential or CHIP.
Naturally in tissue, inherent tumor heterogeneity can be the source of FN in tissue genotyping. Particularly relevant with development of resistance while under therapy, tissue sampling issue may not capture the entire genetic variation of the entire tumor whereas liquid biopsy has that potential.
The aforementioned references, with discordance rates as high as 55% (reference 3), has caused caution around wide adoption of liquid biopsy. An influential report from ASCO/CAP (reference 5) “has put a damper on what some view as an overenthusiasm around liquid biopsy tests.” (GenomeWeb Premium, subscription required.)
A plasma ctDNA sequencing ‘bakeoff’ previewed in 2017
Over the course of 2017 and 2018, a group from AstraZeneca presented posters at various conferences (including the annual American Association for Cancer Research in 2017 and at an influential FDA/AACR workshop later that Fall) showing preliminary results of what they called a ‘Plasma-Seq Bakeoff’.
They took 24 samples from cancer patients to four different liquid biopsy test providers, and then comparing the results to each other as well as to matched tumor / normal tissue sequencing data. Of the 24 samples, 7 were breast cancer samples, 12 were lung cancer samples, 4 were ovarian cancer samples and 1 was prostate cancer sample; as the researchers wanted to challenge the test providers there were 8 Stage I samples and 13 Stage II samples; thus 21/24 samples were early stage. In addition, while the >8 mL of plasma from each patient was processed identically (collection in K2EDTA tubes, double- spun within 4 hours of collection), only 2 mL were provided to each test provider.
True positives were defined as a tumor tissue mutation matching a plasma mutation, or two plasma mutations matching each other regardless of the tissue data.
The Main findings of Stetson et al JCO Precision Oncology 2019 (see reference 6)
The researchers found ‘substantial variability among the ctDNA assays’, with sensitivities ranging from 38% to 89% and Positive Predictive Values (PPVs) ranging from 36% to 80%. The FP’s identified were novel mutations absent from somatic mutation databases. Of the 56 unique variants identified by all four ctDNA assays, a full 68% (41/56) were from technical discordance, rather than a biological source (e.g. the aforementioned CHIP).
Importantly, their analysis included the measure of minor allele frequency (MAF) from the plasma samples, and as BAM files were provided back to the researchers from the test providers a detailed and consistent bioinformatic analysis be undertaken.
They provide a striking figure (Figure 1) where the variant concordance with TP, FP and FN are all plotted across four different vendors, and the vast majority of FPs and FNs are less than 1% MAF.
Supplementary Fig. 1 | Variant tile plot. True positives (TP, green), false negatives (FN, orange), false positives (FP, red) not detected (blank) and not reported (gray) are plotted one variant per line. Not detected are variants not found in a vendors’ assay.
The authors comment: “50% (22 of 44) of all TP somatic variants had a VAF of less than 1%, underscoring the importance of analytically validated assays with sensitivity below 1%.” (Stetson et al. Reference 6)
How sensitive is your assay for liquid biopsy?
A relevant accompanying editorial, “Does Testing Error Underlie Liquid Biopsy Discordance?”, concludes with this:
“As the use of plasma NGS becomes increasingly widespread in cancer care, there remains a clear need for concordance studies such as this one. Future studies would ideally focus on actionable variants and would be limited to advanced cancer. In addition, we have found that orthogonal benchmarking against an established assay, such as digital droplet polymerase chain reaction, is a powerful way to establish a reference point for such analyses.”
At Sysmex Inostics we have both enhanced digital PCR and NGS-based liquid biopsy for testing patient samples, and a worldwide laboratory presence with clinically-validated assay sensitivity (depending on the particular mutations and assay technology) down to 0.03%.
Thress K., Barrett J.C. et al. Lung Cancer 90(3):509-15 (2015). EGFR mutation detection in ctDNA from NSCLC patient plasma: A cross-platform comparison of leading technologies to support the clinical development of AZD9291. PMID:26494259
Kuderer N.M., Blau C.A. et JAMA Oncol. 3(7):996-998 (2017). Comparison of 2 Commercially Available Next-Generation Sequencing Platforms in Oncology. PMID:27978570
Jovelet C., Lacroix L. et a Clin Cancer Res 22(12):2960-2968 (2016). Circulating Cell-Free Tumor DNA Analysis of 50 Genes by Next-Generation Sequencing in the Prospective MOSCATO Trial. PMID:26758560
Abbosh C. and Swanton C et Nat Rev Clin Oncol 12:1344-1356 (2017). Early stage NSCLC – challenges to implementing ctDNA-based screening and MRD detection. PMID:29968853
Merker J. and Turner N.C. et al. J Clin Oncol 36(16):1631-1641 (2018). Circulating Tumor DNA Analysis in Patients With Cancer: American Society of Clinical Oncology and College of American Pathologists Joint Review. PMID:29504847
Stetson D. and Dougherty B.A. JCO Precision Oncol (2019). Orthogonal Comparison of Four Plasma NGS Tests With Tumor Suggests Technical Factors are a Major Source of Assay Discordance. https://ascopubs.org/doi/10.1200/PO.18.00191
Sysmex OncoBEAM circulating tumor DNA testing in clinical practice
This white paper explores a brief history of cell-free DNA analysis, the sensitivity of alternative technologies, and the case for liquid biopsy for non-small cell lung cancer anti-EGFR therapy resistance
Evidence for “liquid biopsy” in clinical oncology continues to increase. Clinical practice guidelines now recommend plasma analysis alongside, and in specific situations in place of tumor tissue (references 1, 2). With the introduction of diverse new liquid diagnostics, it is now more complex than ever for physicians to select the right test for the right patient. A key starting point is to recognize that different types of assays excel for different clinical intended uses — matching the performance characteristics of a test with the clinical context of each patient is necessary to appropriately inform medical decisions.
Cell-free DNA (cfDNA) was first discovered in 1948 (reference 3) and is now known to originate from many different sources including infectious organisms, fetal DNA during pregnancy, genomic DNA from white blood cells, and tumor cells. Tumor-derived cfDNA originating from necrotic and apoptotic tumor and deposited into peripheral circulation is known as circulating tumor DNA (ctDNA) and was first described about 40 years after the initial discovery of cfDNA (reference 4). The first studies relating disease burden and cfDNA levels were completed in the early 2000s, and in landmark studies conducted in 2008, investigators at Johns Hopkins University (Baltimore, MD) showed that ctDNA levels in patients with colorectal cancer change in response to changes in tumor burden (reference 5). Discrimination of ctDNA from normal DNA is achieved by the presence of mutations. However, due to the fact that ctDNA typically represents a very small fraction of cfDNA present in the blood, use as a biomarker for evaluating tumor dynamics requires a quantitative assay with high analytical and clinical sensitivity to characterize accurately the relatively low number of mutant ctDNA fragments in a sample.
Different technologies have different strengths
Quantitative PCR (qPCR), next-generation sequencing (NGS), and digital PCR (dPCR) are the three most common technologies used for ctDNA analysis. Well-designed assays based on any of these methods can serve as useful diagnostic tools depending on the specific clinical needs of the patient.
PCR is an established technology that focuses on detection of one or a few mutations at a time with moderate sensitivity, which is best suited for cases where sample input is not limited. It has been adapted for in vitro diagnostic kits, as well as complete “sample-to-answer” instruments which may be able to offer patients improved access.
NGS provides coverage across many genomic targets and unique mutation types. High sensitivity is also possible, however panels that will be validated for clinical use must be carefully designed to the balance between sensitivity, coverage, cost, and sample requirements.
dPCR is widely regarded for possessing the highest accuracy, precision, and consistency of genetic diagnostic techniques. Its ability to cover known clinical indications at high sensitivity makes it ideal for cases where detection and quantification of low- frequency mutations can deliver key clinical information.
Clinical evidence shows that very low frequency ctDNA mutations (<0.1% allele frequency in plasma) may have important clinical implications across a variety of different cancer types. This evidence continues to accumulate at a rapid pace (references 6-8). In these cases, ultra-high sensitivity is essential to ensure vital information is not missed so that patient samples are appropriately characterized.
OncoBEAM analytical sensitivity
Sysmex OncoBEAM uses BEAMing technology (Beads, Emulsion, Amplification, Magnetics), a modified digital PCR method that interrogates millions of unique molecules within a sample to ensure detection of rare mutant molecules in the presence of many wildtype copies. The lower limit of detection is therefore consistent with low plasma mutant allele frequencies that are present for a significant proportion of cancer patients. Table 1 provides a comparison of analytical sensitivities for several leading ctDNA tests based on different technologies.
Table 1. Analytical sensitivities for several leading ctDNA tests. Notes:
For Competitor R, LoD95 is reported on a per-mutation basis as copies/ mL. Assuming an average of 5 ng DNA/ mL plasma and 3.3 pg/ genomic equivalent (GE)= ~1500 genomic equivalents per mL; 25 mutant copies/ 1500 GE= 1.7% MAF; 100 mutant copies/ 1500 GE= 6.7% MAF.
OncoBEAM Lung: DNA input in validation studies was ≥40 mutant molecules in order to minimize random sampling error. Analytical sensitivity and CI were calculated for LoD samples according to CLSI EP12-A2a. LoD95 was calculated according to CLSI EP17-A2 (reference 13).
Non-small cell lung cancer (NSCLC) anti-EGFR therapy resistance – an ideal case for liquid biopsy
Acquired resistance to first-line tyrosine kinase inhibitors (TKI) for NSCLC patients who harbor epidermal growth factor receptor (EGFR) sensitizing mutations (exon 19 deletions, L858R point mutation) presents a common clinical problem. Resistance often develops after 10-12 months and is most commonly driven by an acquired mutation in EGFR, T790M, which presents in up to 60% of patients (references 14, 15). Third-generation TKIs such as osimertinib (approved by the FDA in Nov 2015) have been shown to be effective in patients with T790M-mediated resistance and disease progression.
Though tissue is the preferred sample type for EGFR analysis for NSCLC, obtaining a tissue biopsy in patients with advancing disease presents significant challenges. In addition to some patients being unwilling or unable to undergo secondary biopsy, the complication rate for intrathoracic biopsies is nearly 20% (references 16, 17). Furthermore, even if secondary biopsy is feasible, the combined turn-around time for tissue acquisition and subsequent molecular analysis can be too long, which can significantly delay the administration of appropriate therapy (references 18, 19). Tissue sampling in the setting of secondary resistance is further confounded by molecular heterogeneity, wherein T790M may be present in only a subset of tumor cells. Thus, sampling of a single region of single metastatic lesion by tissue biopsy may fail to capture the T790M cells that are driving resistance/ progression (reference 20).
Because ctDNA analysis is minimally-invasive, faster than tissue analysis, easily repeatable via additional blood draws, and may better represent disease heterogeneity, it is ideally suited for EGFR analysis for NSCLC patients who have progressed on first-line therapy. However, a ctDNA test used in this setting must have high analytical sensitivity since T790M may be present in a small number of tumor cells, as well as at low concentration in the blood. Most importantly, clinical data must demonstrate that the diagnostic test can be used to predict meaningful patient outcomes for second-line EGFR therapy.
BEAMing demonstrates clinical utility for NSCLC
Based on the study by Oxnard et al. using BEAMing (reference 21), NCCN guidelines now recommend plasma testing for EGFR T790M for NSCLC patients who have progressed on a first or second-generation TKI1. Equivalent clinical outcomes were observed between patients treated with osimertinib who were plasma-positive for T790M, and patients for whom T790M was detected via tissue analysis.
Importantly, for patients who were positive for T790M in tissue, the median mutant allele frequency for T790M detected in plasma by BEAMing was <1%, with a number of patients exhibiting the mutation at <0.1%. This is below the threshold for reliable detection for many other ctDNA assays, including broad NGS panels that excel at generating data across many genomic locations, rather than very high-resolution data focused on regions with established clinical significance.
Mutant allele fraction distribution for EGFR T790M, n=158 plasma samples
For 40% of samples, T790M was below 1% MAF and may not be reliably detected by conventional NGS testing. (reference 22)
A highly sensitive and specific ctDNA diagnostic is essential to decrease risk and cost for the greatest number of patients
Since guidelines recommend reflex to tissue testing if T790M is not detected in plasma, use of a highly sensitive ctDNA assay can aid clinicians to accurately and rapidly identify T790M mutations in patients and thereby avoid a tissue biopsy. It has been shown that BEAMing is able to reliably detect approximately 20-40% more T790M-positive patients compared to other, less sensitive methods, which translates directly into more patients who are spared from tissue biopsy and its associated complications (references 21, 23, 24).
While a highly sensitive assay may raise concerns of false positive results, OncoBEAM testing also demonstrates exquisite specificity. In a blinded profiling of 100 EGFR mutation-negative NSCLC patient plasma samples, BEAMing yielded no false-positive results (reference 25). This suggests that tumor heterogeneity, and not BEAMing assay performance, is the likely cause of discordance between T790M-positive plasma results and tissue-negative reference results.
Overall, use of a reliable and simple blood test decreases risk and cost for the greatest number of advanced NSCLC patients, and extends access to those who would otherwise not receive testing at all if tissue analysis were the only option available.
National Comprehensive Cancer Network. Non-Small Cell Lung Cancer (Version 2.2018). Accessed February 8, 2018. Link to Guidelines.
Lindeman, N.I. and Yatabe Y. et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors. Arch. Pathol. Lab Med. 142(3):321-346 (2018). PMID:29355391
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Diehl, F. and Diaz L.A. et al. Circulating mutant DNA to assess tumor dynamics. Nat. Med. 14, 985-990 (2008). PMID:18670422
Schmiegel, W. and Fox S.B. et al. Blood-based detection of RAS mutations to guide anti-EGFR therapy in colorectal cancer patients: concordance of results from circulating tumor DNA and tissue-based RAS testing. Mol. Oncol. 11(2):208–219 (2017). PMID:28106345
Saunders, M.P. and Adams R.A. et al. Performance assessment of blood based RAS mutation testing: Concordance of results obtained from prospectively collected samples. Ann. Oncol. 27(6):149–206 (2016). https://doi.org/10.1093/annonc/mdw370.74
Barrull J.V and Lopez R. et al. Clinical applications of extended ctDNA RAS mutation determination in metastatic colorectal cancer. Journal of Clinical Oncology 35, no. 4_suppl (February 1 2017) 607-607. DOI: 10.1200/JCO.2017.35.4_suppl.607
Odegaard, J. and Talasaz A. et al. Validation of a plasma-based comprehensive cancer genotyping assay utilizing orthogonal tissue- and plasma-based methodologies. Clin Cancer Res. 24(15):3539-3549 (2018). DOI: 10.1158/1078-0432.CCR-17-3831.
Plagnol, V. and Forshew T. et al. Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling. PLoS ONE 13(3): e0193802 (2018). PMID:29543828
“Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures, 2nd Edition” (2012) Clinical and Laboratory Standards Institute, ISBN Number: 1-56238-796-0. Link to resource.
Steuer, C.E. and Ramalingam, S.S. Targeting EGFR in lung cancer: Lessons learned and future perspectives. Mol. Aspects Med. 45, 67-73 (2015). PMID:26022942
Yu, H.A. and Reily G.J. et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 19, 2240–2247 (2013). PMID:23470965
Overman, M.J. et al. Use of research biopsies in clinical trials: are risks and benefits adequately discussed? J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 31, 17–22 (2013). PMID:23129736
Lokhandwala, T. et al. Costs of Diagnostic Assessment for Lung Cancer: A Medicare Claims Analysis. Clin. Lung Cancer 18, e27–e34 (2017). PMID:27530054
Schwaederle, M. et al. Molecular tumor board: The University of California-San Diego Moores Cancer Center experience. The Oncologist 19, 631–636 (2014). PMID:24797821
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Piotrowska, Z. et al. Heterogeneity underlies the emergence of EGFRT790 wild-type clones following treatment of T790M-positive cancer with a third-generation EGFR inhibitor. Cancer Discov. 5, 713-722 (2015). PMID:25934077
Oxnard, G.R. et al. Association between plasma genotyping and outcomes of treatment with osimertinib (AZD9291) in advanced non-small-cell lung cancer. J. Clin. Oncol. 34(28):3375-3382 (2016). PMID:27354477
Stetson D. et al. Orthogonal Comparison of Four Plasma NGS Tests With Tumor Suggests Technical Factors are a Major Source of Assay Discordance. JCO Prec. Oncol. (2019). DOI: 10.1200/PO.18.00191.
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BEAMing enhanced digital PCR for liquid biopsy: its process, applications and history
Sysmex Inostics´OncoBEAM test uses BEAMing technology for liquid biopsy
BEAMing, which stands for beads, emulsion, amplification, magnetics, is a highly sensitive digital PCR method that combines emulsion PCR and flow cytometry to identify and quantify specific somatic mutations present in DNA. Developed by Bert Vogelstein at Johns Hopkins, it has been primarily used to isolate and analyze circulating tumor DNA (ctDNA) in the peripheral blood of patients with cancer. Vogelstein pioneered the idea that somatic mutations represent uniquely specific cancer biomarkers and developed BEAMing to take advantage of the distinct specificity inherent to these mutations. BEAMing does this by creating hundreds of millions of reaction compartments, enabling higher levels of sensitivity for ctDNA detection when compared to other digital PCR methods. Vogelstein’s early work developing BEAMing gave birth to the field of liquid biopsy. Since then, BEAMing enhanced digital PCR has become one of our core technologies and is now commercially available through Sysmex Inostics, called OncoBEAM.
BEAMING Technology Overview
DNA Isolation and Pre-amplification
BEAMing begins with the isolation of DNA from a patient’s blood or plasma sample. Target regions of the purified DNA undergo a pre-amplification step with conventional PCR utilizing primers of known sequences to amplify the genetic regions of interest.
Figure 1: (a) In analog assays, an average signal is acquired from the mutant and wild-type DNA molecules present in the sample. The ratio between the mutant and wild-type signal is an estimate of the mutation frequency. (b) In digital assays, the genotype of the individual DNA molecules is determined separately. Counting is used to quantify the mutant and wild-type DNA molecules present in the sample. (From Diehl & Luiz Curr Opin Oncol 2007 https://www.ncbi.nlm.nih.gov/pubmed/17133110)
The amplified DNA templates are then introduced to primers that are covalently bound to magnetic beads via streptavidin-biotin interactions and are compartmentalized into aqueous microdroplets of a water-in-oil emulsion. The aqueous phase is emulsified with the oil, creating millions of individual water droplets having a diameter of 3-10 microns. Within each droplet a separate PCR reaction will be performed. Statistically, through Poisson Distribution calculations each water droplet contains a single DNA molecule and a magnetic particle. In addition to the pre-amplified DNA, each emulsion droplet contains the necessary reagents and sequence-directed primer-coated magnetic beads to carry out the emulsion PCR reaction. The microemulsion droplets are temperature cycled using conventional PCR methods and each DNA template and bead, present together in a single aqueous compartment, are extended and amplified resulting in a bead coated with thousands of identical copies of the template DNA fragment. Since the amount of ctDNA in circulation in peripheral blood is extremely low relative to the amount of wild-type DNA and PCR may introduce artifact errors, a high-fidelity DNA polymerase is used in order to significantly limit errors normally introduced during PCR. This precaution limits the risk of false-positive detection and enables the accurate discrimination of target molecules.
Hybridization and Flow Cytometry
Following the emulsion PCR step, the water and oil phase are separated so that the microparticles can be collected in the aqueous phase. The microemulsion droplets are then broken to release the magnetic beads, which have the amplified copies of wild-type or mutant DNA attached. The beads are magnetically purified and base pair-specific fluorescent probes are hybridized to the DNA fragments on the beads to distinguish between wild-type and mutant DNA fragments. Each fluorescent probe binds specifically to the wild-type DNA and the other to specific mutant DNA, and can be differentiated by the dye color associated with wild-type and mutant alleles respectively. Each fluorescently labeled bead is analyzed in a flow cytometer resulting in the precise separation of mutant from wild-type DNA as well as the quantitation and ratio of mutant to wild-type DNA present in a sample.
Unique Feature of BEAMing
A key feature unique to BEAMing is the optimized and specific process of creating hundreds of millions of microscopic emulsion droplets to allow for the compartmentalization of every molecule of DNA in a particular sample into the collection of droplets. Emulsion PCR is run on the compartmentalized DNA, enabling hundreds of millions of PCR reactions to run in parallel. This massively parallel PCR platform delivers high levels of sensitivity for the detection of rare tumor DNA molecules amongst a large background of wild-type DNA. This method provides a digital readout of copy number and makes it possible to detect very rare mutant templates at copy ratios greater than 1:1,000 (0.1% sensitivity).
Depending on the assay, we have demonstrated sensitivity in the range of 2:10,000 (0.02% sensitivity) to 4:10,000 (0.04% sensitivity).
Applications BEAMing is often used in the context of cancer care to conduct what is known as a liquid biopsy. It is used in both research and clinical contexts, some of which include:
Screening and early detection
Real-time monitoring of therapy
Evaluation of early treatment response
Monitoring of minimal residual disease
Risk for metastatic relapse (prognostic)
Mechanisms of therapeutic targets and resistance
BEAMing allows for the quantification of a sample’s mutant fraction, where GE stands for genome equivalents:
This value can be tracked in real-time using serial plasma measurements. The use of flow cytometric analysis allows for this type of quantification as well as higher sensitivity levels due to binary (i.e.: digital) signal readouts. The use of flow cytometry for a readout platform allows precise for quantification of mutant and wild-type DNA populations and high levels of sensitivity due to the digital signal nature of the data, with a lower sensitivity threshold as low as 0.01%.
Commercialization of BEAMing
In 2008, Inostics GmbH was formed to commercialize BEAMing with the goal of delivering highly sensitive liquid biopsy technology to the clinical marketplace. Inostics, based in Hamburg, Germany, was led by Dr. Hartmut Juhl as CEO and Frank Diehl and Philipp Angenendt, as CSO and CTO respectively. Diehl and Angenendt both were former post-doctoral students from Vogelstein’s laboratory with Diehl having worked extensively with BEAMing during his time there, bringing it from technical concept to clinical tool. (Two main publications are Nature Med 2008 and PNAS 2005.) The founding of Inostics’ filled an immediate need for pharmaceutical companies who were interested in understanding molecular drivers of response and resistance to investigational therapeutics. The technology allowed plasma samples from clinical trials to be retrospectively analyzed to determine response rates to different therapies.
As the first company to commercialize liquid biopsy testing technology in the field of oncology, Inostics gained immediate commercial growth amongst pharmaceutical and biopharmaceutical companies for their targeted therapy approaches.
In 2011, Inostics formed a US-subsidiary and laboratory to offer BEAMing-based testing to clinicians. Dan Edelstein (who studied at the Ludwig Center for Cancer Genetics and Therapeutics was led by Drs. Vogelstein and Kinzler) heads the Baltimore Maryland facility. In 2013, Inostics’ US-based laboratory achieved CLIA-certification and became the first laboratory in the world to offer plasma-based ctDNA assays for clinical practice. In 2014, Inostics was acquired by Sysmex Corporation to form Sysmex Inostics.
Currently, Sysmex Inostics conducts BEAMing under the OncoBEAM™ product name at its CLIA and GCP qualified laboratory located in the Science and Technology Park at Johns Hopkins, its GCP laboratory in Hamburg, Germany, at a partner laboratory in Shanghai, China, and Sysmex´ headquarters in Kobe, Japan.
History of BEAMing
In the late 1990s, Vogelstein and Kinzler coined the term “digital polymerase chain reaction (PCR)” when conducting research into somatic mutations associated with and potentially causative for colorectal cancer. A fundamental challenge that digital PCR was designed to address was the detection of minor quantities of a pre-determined somatic mutation in larger cell populations. While both digital and classical PCR can be used in quantitative or qualitative analyses, digital PCR has single-molecule sensitivity to produce an all-or-nothing signal thereby increasing the signal-to-noise ratio and overall sensitivity to rate targets. The results from this research indicate digital PCR is able to reliably quantify the proportion of variant sequences in a DNA sample.
Other avenues of clinical utility were explored and in a 2008 Nature Medicine publication, BEAMing ctDNA measurements were determined to reliably monitor tumor dynamics enabling the detection of low levels of ctDNA where “most such previous studies had not used techniques sufficiently sensitive” to further the case for the clinical utility of liquid biopsy. In addition to being highly sensitive, BEAMing inherently enables the quantification of mutant ctDNA levels.
The Clinical History of BEAMing
2003 – BEAMing is first described as a highly sensitive method to identify and quantify uncommon variants in genes or transcripts. (1)
2005 – The clinical application of BEAMing is confirmed for the first time through the ctDNA analysis of APC mutations in colorectal cancer (CRC) patients to determine appropriate patient cohorts and the mechanism of ctDNA release into peripheral circulation. (2)
2008 – Inostics GmbH is founded to offer BEAMing services to biopharmaceutical companies.
2008 – Clinical applications of BEAMing is extended to monitoring tumor dynamics through testing in patients undergoing surgery or chemotherapy. (2)
2012 – BEAMing analysis is first offered as a CLIA-certified test.
2012 – A Johns Hopkins research team uses BEAMing to detect PIK3CA mutations in advanced breast cancer patients with a 100% concordance between plasma and tissue analysis. (3)
2012 – Studies first describing the emergence of RAS mutations as mediators of resistance to anti-EGFR therapy utilize BEAMing ctDNA analysis to detect KRAS-mutant clones in patients with advanced CRC who had originally KRAS-wild-type biopsy-classified tumors. (4, 5)
2013 – BEAMing is used to detect IDH1 mutations in glioma patient serum and cerebrospinal fluid. (6)
2014 – BEAMing is used to analyze samples in the CRYSTAL, OPUS, and CALGB80405 trials, leading to new clinical practice guidelines to expand RAS testing beyond KRAS Exon 2 for newly diagnosed metastatic colorectal cancer patients. (7, 8, 9)
2014 – BEAMing is used to detect cKIT resistance mutations in gastrointestinal stromal cancer patients. (10)
2014 – BEAMing is used to monitor melanoma patients receiving immune checkpoint blockade inhibitors revealing that ctDNA analysis can provide a more accurate picture of tumor response than traditional radiography. (11)
2015 – BEAMing is used to detect low frequency RAS mutations in patients with metastatic colorectal cancer that is missed by standard of care sequencing methods. These mutations indicate poor response to anti-EGFR therapy. (12)
2015 – BEAMing reveals the heterogeneity of resistance mechanisms in the plasma of patients receiving EGFR T790M directed therapy. (13)
2016 – BEAMing clinical trial results of BRAF V600E and V600K reveal that higher levels of ctDNA tend to result in poorer outcomes. (14)
2016 – BEAMing is used to detect ESR1 mutations in ER+ metastatic breast cancer; the mutational spectrum of ESR1 was largely heterogeneous. This revealed that plasma may be a better representative of mutational status than tissue because of the site-specific nature of tissue biopsy. (15)
2016 – BEAMing is used in the AURA trials to determine plasma EGFR mutational status of non-small cell lung cancer patients receiving Tagrisso osimertinib due to its high sensitivity levels for EGFR L858R, del19, and T790M mutations. Outcomes of the trial were concordant across tumor and blood assays. (16)
2017 – The OncoBEAM RAS CRC testing was shown to be highly concordant with tumor tissue testing leading to equivalent results when determining tumor mutational status. (17, 18)
2018 – OncoBEAM demonstrates the clinical value of blood-based ctDNA mutation testing to complement standard-of-care management of patients with advanced melanoma. These patients were undergoing treatment with targeted therapy or immune checkpoint inhibitors as an adjunct to radiographic imaging to monitor disease activity. (20)
2018 – OncoBEAM ctDNA liquid biopsy demonstrates superior response prediction for advanced pancreatic cancer over standard-of-care protein biomarkers (CA 19-9, CEA and CYFRA 21-1). This finding suggests utility of ctDNA for evaluation of therapeutic response for pancreatic cancer exceeding the resolution of current established protein-based biomarkers. (21)
2019 – The enhanced digital PCR OncoBEAM method shows clinical validity and superior performance versus a ‘pan-cancer’ next-generation sequencing test for blood-based mutation detection in hepatocellular carcinoma. Specifically, OncoBEAM was used to determine RAS mutational status across a total of 1,318 patients. (22)
References for the Clinical History of BEAMing
Dressman, D., Yan, H., Traverso, G., Kinzler, K. W. & Vogelstein, B. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc. Natl. Acad. Sci. U. S. A. 100, 8817–8822 (2003).
Diehl, F. et al. Circulating mutant DNA to assess tumor dynamics. Nat. Med. 14, 985–990 (2008).
Higgins, M. J. et al. Detection of tumor PIK3CA status in metastatic breast cancer using peripheral blood. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 18, 3462–3469 (2012).
Misale, S. et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 486, 532–536 (2012).
Diaz, L. A. et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature 486, 537–540 (2012).
Chen, W. W. et al. BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles. Mol. Ther. Nucleic Acids 2, e109 (2013).
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A brief background on Sysmex Inostics, a liquid biopsy service offering for pharmaceutical companies
From pioneer to gold standard in liquid biopsy
The Johns Hopkins University has a long history of pioneering work in genetics and genomics. As the first medical school, founded in 1873 by its namesake benefactor Johns Hopkins (the unusual first name came from his grandmother’s surname), medical education at that time was a trade school, no undergraduate degree or other training was required. Johns Hopkins, with a $7M gift (equivalent to $150M in today’s dollars) was at that time the largest philanthropic endowment in US history. A hospital, a university with training colleges, and an orphanage were all established; the school of medicine was established in 1890.
Interestingly, Johns Hopkins was a pioneer across multiple medical practices in common use today, such as the use of sterile technique (1890), the establishment of gynecology as a medical specialty and the first use of pathology specimens to be examined under a microscopy (1893), even Victor McKusick ushered in the age of genetic medicine with his pioneering work on the inherited disease called Marfan Syndrome and the establishment of the first Department of Medical Genetics. He is acknowledged as the ‘father of genetic medicine’.
A few years later the same group published in 2003 a paper extending the digital PCR concept from 96-well plates to water-in-oil droplet emulsions, which they called BEAMing, named after the four components of the method (beads, emulsions, amplification and magnetics). This method, not requiring microfluidics nor expensive specialized equipment but rather common laboratory reagents and equipment (a flow cytometer is used for the readout).
Many panels based upon the OncoBEAM technology were made available through the service laboratory’s offerings, and GCP laboratories were established in Kobe Japan and in Shanghai China. (A list of available OncoBEAM tests is online here.)
Acquisition by Symex Corporation of Japan
In 2013 Sysmex Corporation acquired Inostics along with a German flow-cytometry company Partec. For those not familiar with Sysmex Corporation, they have annual revenues of about $2.7B USD (298 B Japanese Yen), over 7,000 employees worldwide, and a very high market share of the hematology laboratory diagnostics market.
With decades of specialized diagnostic equipment design, manufacturing and support, Sysmex Corporation has many complementary offerings for analysis of blood.
Our main focus at Sysmex Inostics is liquid biopsy
For those not familiar with liquid biopsy, in brief the advantages over standard tissue biopsy are as follows:
Liquid biopsy is:
much faster than tissue testing, being minimally invasive with a simple blood draw versus a medical procedure to obtain a tissue biopsy specimen
comprehensive profiling of all tumor sites with a single blood draw, avoiding the potential problems of local sampling with single site tissue biopsies which may not capture all the genetic variation across tumor sites
involves minimal pain and risk, compared to the pain and risk of tissue biopsy
enables serial monitoring (multiple sampling) over time, for monitoring cancer recurrence or surveillance of treatment response and resistance while patients undergo treatment
Our focus here is on the clinical application of liquid biopsy as part of clinical trials or with patient testing. We have laboratories worldwide (Baltimore US, Hamburg Germany, Kobe Japan and Shanghai China) GCP qualified to accept samples as part of clinical trials and also one laboratory in the United States for patient testing as well (Baltimore US). (Physicians in the US can order OncoBEAM tests through this website portal.)
If you are a pharmaceutical company looking for a diagnostic partner for liquid biopsy, we may have exactly what you need – contact us today.
OncoBEAM and Plasma-Safe-SeqS performance shown at the Association for Molecular Pathology Global Conference – June 2019 Hong Kong
A poster available for download courtesy of SeraCare is entitled “A non-small cell lung cancer reference material”
SeraCare Life Sciences has produced an EGFR reference material for medium and low allele frequency mutations that mimics circulating tumor DNA. Synthetic fragments containing the EGFR mutations T790M, L858R, G719S and ex19del were mixed at allele frequencies of 1% and 0.1% with a wild-type well-characterized genomic DNA sample.
These EGFR mutations were assayed by a number of technologies and providers as follows:
Bio-Rad droplet digital PCR
Sysmex Inostics OncoBEAM (enhanced digital PCR)
Roche cobas EGFR Mutation Test v2 (real-time PCR)
ArcherDx Archer Reveal ctDNA 28 kit (NGS)
Sysmex Inostics Plasma-Safe-SeqS (NGS)
SeraCare has kindly made this poster available for our readers – access a PDF of this poster by clicking download below.