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).
Lenz, H. et al. 501ocalgb/Swog 80405: Phase Iii Trial of Irinotecan/5-Fu/Leucovorin (folfiri) or Oxaliplatin/5-Fu/Leucovorin (mfolfox6) with Bevacizumab (bv) or Cetuximab (cet) for Patients (pts) with Expanded Ras Analyses Untreated Metastatic Adenocarcinoma of the Colon or Rectum (mcrc). Ann. Oncol. 25, mdu438.13 (2014).
Bokemeyer, C. et al. FOLFOX4 plus cetuximab treatment and RAS mutations in colorectal cancer. Eur. J. Cancer Oxf. Engl. 1990 (2015). doi:10.1016/j.ejca.2015.04.007
Van Cutsem, E. et al. Fluorouracil, leucovorin, and irinotecan plus cetuximab treatment and RAS mutations in colorectal cancer. J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 33, 692–700 (2015).
Yoo, C. et al. Analysis of serum protein biomarkers, circulating tumor DNA, and dovitinib activity in patients with tyrosine kinase inhibitor-refractory gastrointestinal stromal tumors. Ann. Oncol. Off. J. Eur. Soc. Med. Oncol. ESMO 25, 2272–2277 (2014).
Lipson, E. J. et al. Circulating tumor DNA analysis as a real-time method for monitoring tumor burden in melanoma patients undergoing treatment with immune checkpoint blockade. J. Immunother. Cancer 2, 42 (2014).
Morelli, M. P. et al. Characterizing the patterns of clonal selection in circulating tumor DNA from patients with colorectal cancer refractory to anti-EGFR treatment. Ann. Oncol. Off. J. Eur. Soc. Med. Oncol. ESMO 26, 731–736 (2015).
Piotrowska, Z. et al. Heterogeneity Underlies the Emergence of EGFRT790 Wild-Type Clones Following Treatment of T790M-Positive Cancers with a Third-Generation EGFR Inhibitor. Cancer Discov. (2015). doi:10.1158/2159-8290.CD-15-0399
Santiago-Walker, A. et al. Correlation of BRAF Mutation Status in Circulating-Free DNA and Tumor and Association with Clinical Outcome across Four BRAFi and MEKi Clinical Trials. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 22, 567–574 (2016).
Spoerke, J. M. et al. Heterogeneity and clinical significance of ESR1 mutations in ER-positive metastatic breast cancer patients receiving fulvestrant. Nat. Commun. 7, 11579 (2016).
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. Off. J. Am. Soc. Clin. Oncol. 34, 3375–3382 (2016).
Grasselli, J. et al. Concordance of blood- and tumor-based detection of RAS mutations to guide anti-EGFR therapy in metastatic colorectal cancer. Ann. Oncol. Off. J. Eur. Soc. Med. Oncol. (2017). doi:10.1093/annonc/mdx112
Vidal, J. et al. Plasma ctDNA RAS mutation analysis for the diagnosis and treatment monitoring of metastatic colorectal cancer patients. Ann. Oncol. Off. J. Eur. Soc. Med. Oncol. (2017). doi:10.1093/annonc/mdx125
García-Foncillas, J. et al. Incorporating BEAMing technology as a liquid biopsy into clinical practice for the management of colorectal cancer patients: an expert taskforce review. Ann. Oncol. doi:10.1093/annonc/mdx501
Rowe, S.P. et al. From validity to clinical utility: the influence of circulating tumor DNA on melanoma patient management in a real‐world setting. Mol Oncol 12, 1661-1672 (2018).
Kruger, S. et al. Repeated mutKRAS ctDNA measurements represent a novel and promising tool for early response prediction and therapy monitoring in advanced pancreatic cancer. Ann Oncol. Off. J. Eur. Soc. Med. Oncol. (2018).
Lim H S et al. Phase II Studies with Refametinib or Refametinib plus Sorafenib in Patients with RAS-Mutated Hepatocellular Carcinoma. Clin Cancer Res 24:4650-4661 (2018).