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.

Diehl Review figure of the BEAMing digital PCR method
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)

Emulsion PCR

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

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)
  • Patient stratification
  • Mechanisms of therapeutic targets and resistance

BEAMing allows for the quantification of a sample’s mutant fraction, where GE stands for genome equivalents:

20190717 Equation for Blog 6

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.

BEAMing grew out of digital PCR technology and in 2003 this method was described in a PNAS publication from Vogelstein’s team. In 2005, the same team published their first clinical data applying BEAMing technology to analyze plasma samples from cancer patients. In these samples, mutant circulating tumor DNA (ctDNA) levels were analyzed in the plasma of patients with advanced colorectal cancer, and indicated that some proportion of early stage and not just metastatic cancers shed mutant ctDNA into the blood. This raised questions about whether or not BEAMing could be clinically useful for presymptomatic diagnosis.

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)
  • 2015 – Sysmex Inostics launches the OncoBEAM platform at certified centers throughout Europe, Japan, and Asian Pacific to educate and train customers on BEAMing technology.
  • 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 – The OncoBEAMTM RAS CRC test from Sysmex Inostics using BEAMing technology receives CE Mark approval thus becomes the first IVD liquid biopsy assay available to CRC patients.
  • 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)
  • 2017 – Sysmex Inostics releases an OncoBEAM EGFR RUO kit to allow for BEAMing blood-based analysis in lung cancer in Europe and Asia. http://www.prnewswire.com/news-releases/oncobeamtm-egfr-kit-ruo-at-jessenius-medical-faculty-of-comenius-university-and-its-university-hospital-in-martin-slovakia-647893363.html
  • 2017 – Leading Spanish oncologists issue an expert taskforce review in support of incorporating BEAMing technology into clinical practice for the management of CRC patients. (19)
  • 2018 – Symex Inostics releases an OncoBEAM EGFR Kit v2 (RUO) to allow for BEAMing blood-based analysis in colorectal and lung cancers in Europe and Asia.
  • 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

  1. 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).
  2. Diehl, F. et al. Circulating mutant DNA to assess tumor dynamics. Nat. Med. 14, 985–990 (2008).
  3. 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).
  4. Misale, S. et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 486, 532–536 (2012).
  5. Diaz, L. A. et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature 486, 537–540 (2012).
  6. 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).
  7. 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).
  8. 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
  9. 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).
  10. 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).
  11. 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).
  12. 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).
  13. 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
  14. 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).
  15. 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).
  16. 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).
  17. 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
  18. 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
  19. 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
  20. 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).
  21. 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).
  22. 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).

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’.

Digital PCR and BEAMing technology

In this backdrop, Dr. Bert Volgelstein and Dr. Kenneth Kinzler published in 1999 a paper simply entitled ‘Digital PCR’ to enable digital detection of rare mutations, with a proof of their concept analyzing the mutant RAS oncogene in human stool samples.

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).

Beginning as Inostics: “INdividual diagnOSTICS”

In 2008, with the publication (from the same group at Johns Hopkins) of this influential Nature Medicine paper “Circulating mutant DNA to assess tumor dynamics”, Inostics GmbH was founded in Hamburg Germany. A contraction of the words “individual diagnostics”, Inostics set out to offer the translational research and targeted pharmaceutical markets the first liquid biopsy testing service, first as an RUO service in 2008 followed by a CLIA (Clinical Laboratory Improvement Amendments) and GCP (Good Clinical Practice) laboratory service offering.

In 2011 Inostics established a testing laboratory in Baltimore (Maryland US) on the Johns Hopkins medical campus. Right about that time our head of operations shared this vision for the future of cancer detection as a brief video interview titled “What is the Symex Inostics Benefit for Patients?”, still valid today.

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.

A few years after the acquisition, Sysmex Inostics launched and subsequently received a CE mark for an IVD kit version of the OncoBEAM assay (available only outside the United States) for RAS mutation detection in colorectal cancer, packaged along with specialized software and a flow cytometer from Sysmex Partec called the Cube 6i.  The OncoBEAM RAS kit represented the first CE Mark IVD liquid biopsy assay available for routine patient care (For additional details, here’s the Sysmex Europe webpage describing OncoBEAM offerings for colorectal cancer.)

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 ctDNA testing (liquid biopsy) for therapy surveillance in melanoma

Research at Johns Hopkins University demonstrates potential clinical utility of targetable, real-time monitoring of response to melanoma cancer therapy

Skin cancer is the most common of all cancers (in the US affecting some 3.3 million individuals), yet “fortunately, most skin cancers are slow growing, easy to recognize, and relatively easy to treat when detected early”. (Source: American Cancer Society Fact Sheet PDF) Yet for invasive melanoma (affecting 1% of the cases or about 96,000 individuals in the US), the five-year survival rate is only 23%.

Liquid monitoring – optimizing melanoma detection using ctDNA

In a recent webinar, Dr. Evan Lipson of the Johns Hopkins University School of Medicine Sidney Kimmel Comprehensive Cancer Center presented work he titled “Liquid monitoring: optimizing melanoma detection using circulating tumor DNA (ctDNA)”. In his presentation, he laid out the obstacles for surveillance after initial metastatic melanoma treatment (typically surgical resection and follow-up adjuvant chemotherapy), the potential utility for liquid monitoring, and then showed data from what he termed targetable, real-time monitoring of disease.

The obstacles include local recurrence of metastatic disease, usually through the lymphatic system or the bloodstream, and the tools currently used for monitoring are expert radiology to examine Computerized Tomography (CT) and Magnetic Resonance Imaging (MRI) scans, as well as a protein-based marker called LDH (lactate dehydrogenase). LDH measures general tissue damage, and Dr. Lipson commented this test has poor predictive value for advanced Stage IV melanoma and limited value for monitoring disease burden in response to therapy.  Dr. Lipson highlighted that while many melanoma patients receive great benefit from immunotherapy, there is a need for clinical diagnostics to determine accurate disease clearance and residual tumor activity in order to more precisely tailor therapy.  Radiographic imaging has a limited resolution and LDH lacks the dynamic range to accurately determine micrometastatic disease. 

Dr. Lipson reviewed the molecular drivers of melanoma per work published in 2015 by The Cancer Genome Atlas network titled “Genomic Classification of Cutaneous Melanoma”, where ~50% of melanoma were driven by mutations in the BRAF gene, while another ~30% more were driven by NRAS, accounting for about 75% – 80% of melanoma cases. He called this slide “Capitalizing on hot-spot somatic gene mutations in cutaneous melanoma”, laying out the rationale for this work.

Prior work using digital PCR BEAMing technology

Citing prior work with 12 patients with metastatic melanoma undergoing immune-checkpoint blocking therapy with primary tumor analysis followed-up with every 2 to 4 weeks of regular sampling of blood plasma samples, he showed data from this Journal of Immunotherapy of Cancer article published in 2014 where individual patients’ increase of ctDNA correlate with radiographic disease progression.

Of interest was its measurement – in terms of percent of Genome Equivalents (GE) of circulating tumor DNA as a percentage of wild-type DNA and its correlation with a common standard of disease progression, the Sum of Longest Diameter (SLD, in mm) of the identified metastatic lesions. The quantitation of genomic equivalents was through use of a clinically-validated digital PCR method called BEAMing, first described in 2008 and commercialized by Sysmex Inostics.

Key goals for the current study and its methods

Dr. Lipson posed several goals in his evaluation of BEAMing technology: to estimate concordance with tissue mutation analysis, to measure the clinical limits of detection and influence of targetable tumor mutation recognition, to assess evidence of disease activity compared to radiography, and to evaluate clinical utility as a blood-based tumor marker in patients treated with systemic therapy including immune checkpoint blockers.

He then described their patient selection criteria, involvement of expert radiologists, the specific mutations in the OncoBEAM test used (for BRAF it was V600E/K, for NRAS Q61H/K/L/R), the limit of detection at 0.03% genomic equivalents, and described three experimental cohorts.

In brief the A cohort comprised of a single plasma sample of a group of 57 metastatic melanoma patients to see how many ctDNA mutations can be detected, and then calculate sensitivity and specificity of the ctDNA OncoBEAM method. The B cohort comprised of 29 patents with high risk, surgically-resected melanoma with one of the six hotspot mutations detectable with the OncoBEAM test, and serial plasma samples analysed coincident with clinical and radiographic evaluations. The last C cohort were 30 patients with unresectable or metastatic melanoma with one of the six mutations, treated with medical therapy (e.g. anti-PD-1, BRAFi etc.) and serial plasma samples analysed coincident with clinical and radiographic evaluations.

Cohort A findings: Specificity of 88.2%, Sensitivity of 95.2% at an LoD of 0.03%

Measuring concordance between tissue and ctDNA, of 57 patient samples 32 were positive in both tissue mutation and ctDNA mutation; five were positive in tissue but not detected in plasma; in the remaining 20 samples 19 were wild-type for the BRAF and NRAS genes, and 1 was wild-type in tissue but positive in plasma, and another sample insufficient tissue was available for testing but the ctDNA was positive for mutations. Overall, the sensitivity of the OncoBEAM assay was determined to be 86.8% with a specificity of 100%.

In these last two cases, interestingly they were both BRAF mutations detected in plasma-only and not in tissue; both patients received BRAFi therapy (dabrafenib and tramatenib) and both patients experienced a partial response (RECIST 1.1).

Using the aforementioned SLD measurement, they were able to determine that optimum sensitivity and specificity for tumor detection were achieved at 35.5mm SLD, and in 57 patient samples achieved a specificity of 88.2% and a sensitivity of 95.2% with a limit of detection (LoD) of 0.03%.

Cohort B findings: ctDNA may not be useful early-detection marker for high risk of local recurrence

Of the 29 high risk, resected melanoma patients with one of the six BRAF or NRAS mutations, 2 patients (7%) had locoregional recurrence via ordinary surveillance, however both patients had no measurable ctDNA.

Another 3 patients (10%) had distant recurrence, of which 2 were detected by ctDNA (liver and kidney metastasis respectively) and 1 were undetected by ctDNA (lung metastasis). Dr. Lipson suggested that ‘visceral metastases’ (in internal visceral organs) were more easily detected by ctDNA.

Cohort C findings: ctDNA detection precedes radiographic disease progression in a number of cases

Of the 30 patients with unresectable or metastatic melanoma, 17 patients (57%) experienced partial or complete response to therapy. The ctDNA measurement did not detect any evidence of disease activity after on-treatment assessment.

Of the 8 patients who developed radiographic disease progression, 4 patients had evidence of disease in ctDNA at the same time as radiographic detection of progression.

Yet in 4 patients (13%), ctDNA measurement detected disease progression by 8, 14, 25 and 38 weeks (average 21.2 weeks or over five months) before conventional radiography detected disease progression. ctDNA-based evidence of disease activity was seen in three of the four patients where expert radiographic evaluations were performed with no evidence of neoplastic disease.

Dr. Lipson said ‘ctDNA could be a helpful biomarker for radiologists, it warrants a second look at a radiographic scan’.

A case example of ctDNA predicting metastatic recurrence

He finished his presentation with a case example, a 68-year old female receiving anti-PD-1 (pembrolizumab) for metastatic melanoma, where there was detectable ctDNA at baseline of 0.95% genome equivalents MAF (minor allele fraction), but the imaging was ruled to be no evidence of disease; in retrospect, a 1.5 cm rim-enhancing centrally necrotic uterine mass is seen. After 3 months, no ctDNA was collected, but the mass grew in size, and this intra-fibroid region mass growing to 3cm ‘in an odd place for melanoma to show up” the CT images were deemed no evidence of disease.

Dr. Lipson notes the high level of experience of these radiologists, and how ‘tremendously difficult’ a region of the body it is to pick out metastasis. After 5 months, a PET-CT scan shows a 5cm hypermetabolic lesion within a fibroid uterus, coincident to a ctDNA measurement of 5.7% MAF. By biopsy, the lesion was confirmed to be metastatic melanoma.

A few conclusions

Dr. Lipson concluded that “ctDNA is a useful, non-invasive, blood-based biomarker that can detect targetable oncogenic mutations not seen in tumor analysis and can provide evidence of disease activity, predicting eventual disease progression and informing radiographic image interpretation”. In addition, he also suggested that longitudinal intrapatient blood-based detection of melanoma progression “may have implications in the setting of clinical trials where surrogate endpoints such as clinical / radiographic progression-free survival are used as markers of drug efficacy”.

You can access this published work for further details: Rowe and Lipson et al. 2018 Molecular Oncology “From validity to clinical utility: the influence of circulating tumor DNA on melanoma patient management in a real-world setting”. And for additional information about our OncoBEAM technology you can access it here, and a list of our current OncoBEAM-based ctDNA liquid biopsy tests here.

References:

  1. Cancer Genome Atlas Network. Cell 161(7):1681-96. 2015 Genomic Classification of Cutaneous Melanoma. PubMed PMID: 26091043
  2. Lipson EJ and Diaz LA Jr. et al. J Immunother Cancer. 2(1):42. 2014 Circulating tumor DNA analysis as a real-time method for monitoring tumor burden in melanoma patients undergoing treatment with immune checkpoint blockade. PubMed PMID: 25516806
  3. Diehl F and Diaz LA Jr. et al. Nat Med. 14(9):985-90 2008 Circulating mutant DNA to assess tumor dynamics. PubMed PMID: 18670422
  4. Rowe SP and Lipson EJ et al. Mol Oncol. 12(10):1661-1672 2018 From validity to clinical utility: the influence of circulating tumor DNA on melanoma patient management in a real-world setting. PubMed PMID: 30113761

Introducing Sysmex Inostics’ Fluid Dynamics Liquid Biopsy Blog

For a patient to go through the invasive procedure of a traditional biopsy in order to obtain a conventional biopsy, we in the molecular diagnostics testing industry often do not think about what patients often have to go through to get a tissue biopsy. Often patients will be asked to fast the night before; to request a care partner to take a day off to accompany the patient; to learn the risks of complications with the procedure and signing the paperwork; to deal with the recovery and any complications that may arise.

This recent essay in Stat News was written by a board-certified patient advocate specializing, ironically enough, in advocating for cancer patients. And she states clearly, “I hope I never have to undergo a biopsy like that again. It’s why I’m incredibly excited at the prospect of what are being called liquid biopsies.”

The promise of blood-based cancer diagnostics

Blood-based cancer diagnostics, whether based on the analysis of cell-free circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs), hold tremendous promise for targeted therapy selection, response prediction, and monitoring resistance and recurrence of cancer. Increasingly the field is expanding into cancer prognosis and early-detection and screening applications, although these applications are a few years away from wider adoption.

Investors in liquid biopsy have made major investments, from Guardant Health’s successful IPO (and rising over three-fold since going public) to GRAIL’s Series B funding of $900M bringing its total valuation to an eye-watering $1.5B. Other success stories in non-invasive cancer diagnostics include Exact Science’s 79% growth in sample volume compared to the year earlier, and this popular consultancy  estimates $4B invested into liquid biopsy companies over the past three years..

A fast-moving field at the front of standard of care

In lung cancer, specifically non-small cell lung carcinoma (NSCLC), it has been known for many years that ‘activating mutations’ in the EGFR gene occur in 10%-30% of unselected lung cancer patients. Of these with EGFR mutations, the Exon 19 deletion mutations and the point mutation L858R in exon 21 are the most common and called ‘classical’ mutations (about 85% of all EGFR mutations). Patients with these mutations initially had standard-of-care treatment with the first-generation tyrosine kinase inhibitor (TKI) erlotinib or the second-generation TKI gefitinib. In 2016, the FDA approved the first ctDNA liquid biopsy test, the Roche cobas EGFR Mutation Test v2 in cases where insufficient tissue is available, or where obtaining tissue is not practical.

Upon generation of resistance to the first- or second-generation TKI’s by the emergence of the T790M mutation (approximately two-thirds of patients on first- or second-generation TKI’s progress with the T790M mutation), the FDA approved shortly afterwards the same EGFR Mutation Test v2 for the T790M resistance mutation as a companion diagnostic for the third-generation TKI osimertinib. In late 2017, or a little over a year after the first- and second-generation TKI’s were approved, was osimertinib recommended as first-line standard of care for advanced NSCLC by NCCN. The reason for the change was an almost doubling of the progression-free survival (PFS), a remarkable advance in the treatment of advanced NSCLC.

Sysmex-Inostics also provides a full menu of EGFR testing options through our CLIA-qualified service laboratory in the US and ISO 9001 laboratory in Germany (FRUO). Both laboratories adhere to Good Clinical Practices guidelines. Given the speed at which new molecularly-guided therapies are developed with companion diagnostics, there is a need for a flexible testing provider to keep up with the frequent changes in the practice of precision medicine.

A sampling of upcoming topics

We will use this blog to dive deep into technical topics, to react to news that affects the world of liquid biopsy testing, to look deeper into the best-fit of tests for its particular intended use, and to share product- and company-related information. For example, on technical topics we anticipate discussing how many copies can be reliably detected at 0.1% MAF from 10 ng input, given the sampling limitations of stochastic variation; and for best-fit of tests we can look into the available tests for minimal residual disease (MRD) monitoring.

And for company information, we will have an online web presentation called “OncoBEAM ctDNA testing for early response prediction and therapy surveillance in melanoma and pancreatic cancer” provided through our partner GenomeWeb.

Please consider registering here for periodic blog updates, and contact us for project inquiries.