Key differences between HPV-positive and HPV-negative head and neck squamous cell carcinomas (HNSCC)

Head and neck squamous cell carcinomas (HNSCC) develop from the mucosal epithelium in the oral cavity, pharynx, and larynx and are the most common malignancies that arise in the head and neck.1 Increasingly, tumors in the oropharynx are linked to prior infection with oncogenic strains of human papillomavirus (HPV), primarily HPV-16 and, to a lesser extent HPV-18 and others.2,3 HNSCCs of the oral cavity and larynx are primarily associated with tobacco-derived carcinogens, excessive alcohol consumption, or both, and are collectively referred to as HPV-negative HNSCC.4 The median age of diagnosis for non-virally associated HNSCC is 66 years, whereas the median age of diagnosis for HPV-associated oropharyngeal cancer is about 53 years.5

HPV-positive and HPV-negative HNSCC are two distinct diseases with different sites of origin, etiological agents, risk factors, and contributions to the development of oropharyngeal squamous cell carcinoma (OPSCC). The demographics, cause, and prevalence of HPV-positive and NPV-negative cancers are summarized in Table 1.

Table 1: Demographics, Cause, and Prevalence of HNSCC.

Whereas the incidence of smoking-related HNSCC continues to decline worldwide, that of HPV-positive HNSCC is on the rise.6 During 2007-2016, HPV-associated cancers increased by 2.1% per year on average, whereas cancers not associated with HPV decreased by 0.4% per year on average.6

HPV-positive and HPV-negative HNSCCs present with different molecular characteristics, immune landscapes, and clinical prognosis (Table 2) and lead to two fundamentally different diseases with distinct pathogenesis in terms of gene expression, tumor microenvironment (TME), and mutational burden.

Table 2. Pathology and Disease Signature in HNSCC.

Genomic and epigenetic analyses reveal extremely high heterogeneity in HNSCC in terms of characteristic mutations, molecular signature, cellular phenotype, composition of TME, and immune landscape (Table 3).

Table 3. Characteristic mutations in HPV-positive and HPV-negative HNSCC.

Various promising vaccine targets have been identified and treatment options employed for the treatment of HPV-positive and HPV-negative cancers with varying degrees of success (Table 4).

Table 4. Treatment options and targets.
Treatment personalization and de-escalation

According to Dr. Nishant Agrawal, Chief of Otolaryngology-Head & Neck Surgery UChicago Medicine, “We have seen a significant increase in the incidence of HPV-associated oropharyngeal cancer in relatively younger patients, with the median age of diagnosis in the 50s, even patients in their 30s.”32 He added that “Even at 2 years after radiation therapy, 15% of patients had grade 2 swallowing dysfunction and 8% had progressive dysphagia, so their swallowing is going to continue to get worse. Patients may also have chronic xerostomia. The dry mouth improves but it never gets back to 100%.”33 In contrast to patients with HPV-negative HNSCC, who have a five-year survival rate of about 25%-40%, patients with HPV-positive HNSCC fare much better with a disease-free survival rate of 85%-90% over five years. According to Dr. Agrawal, the better prognosis for HPV-positive patients suggests a need to de-escalate treatment while preserving survival.


  1. Stein, A.P. et al. (2015) Prevalence of human papillomavirus in oropharyngeal cancer: a systematic review. Cancer J. (21);138-46.
  2. Isayeva, T. et al. (2012) Human papillomavirus in non-oropharyngeal head and neck cancers: a systematic literature review. Head Neck Pathol. (6);S104-20.
  3. Michaud, D.S. et al. (2014) High-risk HPV types and head and neck cancer. Int J Cancer. (135);1653-61.
  5. Windon, M.J. et al. (2018) Increasing prevalence of human papillomavirus-positive oropharyngeal cancers among older adults. Cancer (124);2993-99.
  6. Ellington, T.D. et al. (2020) Trends in incidence of cancers of the oral cavity and pharynx – United States 2007-2016. Morb Morta Wkly Rep. (69);433-38.
  9. Gillison, M.L. et al. (2015) Epidemiology of human papillomavirus positive head and neck squamous cell carcinoma. J Clin Oncol. (33);3235-42.
  10. Mahal, B.A. et al. (2019) Incidence and Demographic Burden of HPV-Associated Oropharyngeal Head and Neck Cancers in the United States. Cancer Epidemiol Biomark Prev. 28(10);1660-67.
  11. Kazuhiro, K. et al. (2018) A Review of HPV-Related Head and Neck Cancer. J Clin Med Sep. 7(9);241.
  14. Canning, M. et al. (2019) Heterogeneity of the Head and Neck Squamous Cell Carcinoma Immune Landscape and Its Impact on Immunotherapy. Front Cell Dev Bio.l vol7.
  15. Fakhry, C. et al. (2017) The prognostic role of sex, race, and human papillomavirus in oropharyngeal and nonoropharyngeal head and neck squamous cell cancer. Cancer (123);1566-75. Doi: 10.1002/cncr.30353.
  16. Pai, S.I. et al. (2009) Molecular pathology of head and neck cancer: implications for diagnosis, prognosis, and treatment. Annu Rev Pathol. (4);49-70.
  17. Keck, M.K. et al. (2015) Integrative analysis of head and neck cancer identifies two biologically distinct HPV and three non-HPV subtypes. Clin Cancer Res. (21);870-81. Doi: 10.1158/1078-0432.CCR-14-2481.
  18. Hanna, G.J. et al. (2018) Frameshift events predict anti-PD-1/L1 response in head and neck cancer. JCI Insight 3:98811. Doi: 10.1172/jci.insight.98811.
  19. Elpek, K.G. et al. (2014) The tumor microenvironment shapes lineage, transcriptional, and functional diversity of infiltrating myeloid cells. Cancer Immunol Res. (2);655-67. Doi: 10.1158/2326-6066.CIR-13-0209.
  20. Mandal, R. et al. (2016) The head and neck cancer immune landscape and its immunotherapeutic implications. JCI Insight 1: e89829. Doi: 10.1172/jci.insight.89829.
  21. Badoual, C. et al. (2013) PD-1-expressing tumor-infiltrating T cells are a favorable prognostic biomarker in HPV-associated head and neck cancer. Cancer Res. (73);128-38. Doi: 10.1158/0008-5472.CAN-12-2606.
  22. Hanna, G.J. et al. (2017) Defining an inflamed tumor immunophenotype in recurrent, metastatic squamous cell carcinoma of the head and neck. Oral Oncol. (67);61-69.
  23. Taberna, M. et al. (2017) Human papillomavirus-related oropharyngeal cancer. Ann Oncol. (28);2386-98.
  24. Coca-Pelaz, A. et al. (2020) The risk of second primary tumors in head and neck cancer: a systematic review. Head Neck. (42);456-66.
  25. Tomaic, V. (2016) Functional roles of E6 and E7 oncoproteins in HPV-induced malignancies at diverse anatomical sites. Cancers (8);95.
  26. Johnson, D.E. et al. (2020) Head and neck squamous cell carcinoma. Nat Rev Dis Primers. (6);92.
  27. The Cancer Genome Atlas Network [TCGA]. (2015) Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature (517) 576-82.
  28. Beck, T.N. et al. (2016) EGFR and RB1 as Dual Biomarkers in HPV-Negative Head and Neck Cancer. Mol Cancer Ther. 15(10);2486-97.
  29. Schreiber, R.D. et al. (2011) Cancer immunoediting: integrating immunity’s roles in cancer suppression and promotion. Science 331;1565-70.
  30. Skeate, J.G., et al. (2016) Current therapeutic vaccination and immunotherapy strategies for HPV-related diseases. Hum Vaccin Immunother. (12);1418-29.
  31. Seiwert, T.Y. et al. (2016) Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial. Lancet Oncol. (17);956-65.
  32. Dr. Nishant Agrawal, Chief of Otolaryngology-Head & Neck Surgery UChicago Medicine. Personal interview on 3/25/22.
  33. https:\\\\issues\\april-25-2017\\deintensifiying-treatment-of-hpv-positive-oropharyngeal-cancer-could-reduce-toxicity-while-maintaining-function-and-survival

New ASCO 2021 poster highlights HPV-SEQ test’s ultra-sensitive detection of HPV 16/18 in plasma

Download our new poster, presented at the recent ASCO 2021 Annual Meeting: “Ultra-sensitive detection and quantification of human papillomavirus (HPV) DNA in the plasma of patients with oropharyngeal squamous cell carcinoma (OPSCC) enrolled in the OPTIMA 2 treatment de-escalation trial”.


As patients with HPV-driven tumors often have a good prognosis, clinical investigators have recently explored new strategies for treatment de-escalation to avoid unnecessary side-effects caused by overtreatment.  Important clinical data for HPV-SEQ was generated while investigating induction chemoimmunotherapy followed by risk/response stratified de-escalated locoregional therapy for patients with HPV+ OPSCC. During the trial, HPV-SEQ was employed to evaluate levels of cfHPV-DNA alongside patients’ radiographic response to therapy to assess the future utility in guiding treatment de-escalation strategies. HPV-SEQ showed robust quantitative detection of HPV 16/18 across a broad dynamic range over five orders of magnitude with low quantitative variability. Importantly, a high correlation was observed between dynamic changes in patients’ cfHPV DNA levels and radiographic responses following induction therapy.

High tissue biopsy to liquid biopsy concordance with OncoBEAM enhanced digital PCR – Part III

A recent publication summarizes the results of RAS mutation status in colorectal cancer (CRC) from 19 available clinical trials with matched tissue and ctDNA; OncoBEAM technology shines

In Part I of our liquid biopsy concordance series, we summarized seven publications and an internal dataset correlating the RAS mutation status between CRC tissue biopsy and cell-free DNA liquid biopsy. Across 913 patient samples, an overall percent agreement (OPA) of 90.3% was reported, with a positive percent agreement (PPA) of 88.7% and a negative percent agreement (NPA) of 91.8%.

In a recent report by Galvano et al. 2019 (“Detection of RAS mutations in circulating tumor DNA: a new weapon in an old war against colorectal cancer. A systematic review and meta-analysis”) the authors examined 19 clinical trials comparing RAS mutational status via standard tissue biopsy and ctDNA analysis.

A summary of the concordance results between matched CRC tissue and plasma

For the 1,180 patients examined in this meta-analysis, RAS mutational status was determined in ctDNA using one of three methods: NGS in 786 patients, BEAMing in 807 patients, and standard PCR in 217 by PCR. The table below uses data from Table 2 of the paper (available here).

It is clear from this data the superior sensitivity and specificity of the BEAMing technology used in Sysmex Inostics OncoBEAM tests. The summary receiver-operator characteristic (sROC) plots diagrammed as Figure 5 in the paper (available here) graphically illustrates the differences between BEAMing, NGS and PCR.

OncoBEAM technology a clear lead in sensitivity and specificity

The first prospective study has recently been published in Elez et al. 2019 (“Impact of circulating tumor DNA mutant allele fraction on prognosis in RAS-mutant metastatic colorectal cancer”), evaluating the prognostic potential of measuring the prevalence of RAS mutations in plasma and their correlation with overall survival (OS). From a cohort of 37 CRC patients with non-resectable metastatic disease, lower RAS MAF at baseline correlated with significant correlation with longer OS.

A list of the available OncoBEAM tests for CRC and other cancer types by BEAMing are available here and if you have any inquiries please feel free to contact us.


  1. Galvano A, Taverna S, and Russo A et al. Detection of RAS mutations in circulating tumor DNA: a new weapon in an old war against colorectal cancer. A systematic review of literature and meta-analysis. Ther Adv Med Oncol. 2019 11:1758835919874653. doi:10.1177/1758835919874653. PubMed Central PMCID: PMC6737868.
  2. Elez E, Chianese C and Vivancos A et al. Impact of circulating tumor DNA mutant allele fraction on prognosis in RAS-mutant metastatic colorectal cancer. Mol Oncol. 2019 13(9):1827-1835. doi: 10.1002/1878-0261.12547. PubMed PMID: 31322322.

Sysmex Inostics industry-leading sensitivity for liquid biopsy

Enhanced OncoBEAM™ digital PCR and unique NGS-based Plasma-Safe-SeqS: Ultra-high sensitivity cell-free DNA detection delivers unparalleled dynamic range enabling high resolution of treatment response monitoring and earlier detection of disease recurrence.

There is undisputed potential for liquid biopsies to transform the treatment trajectory of patients with cancer. This is due to its flexibility and utility for treatment selection at diagnosis, monitoring of therapeutic response, and early detection of resistance to therapy. Analysis of circulating tumor DNA (ctDNA) in plasma reduces the risk of not detecting clinically-actionable mutations in cancer patients due to tumor heterogeneity. Moreover, as blood draws for liquid biopsy are minimally invasive there is little potential for either inadequate material for analysis or complications arising from obtaining the needed sample.

An additional benefit of liquid biopsy is the turn-around-time to test result: typically 5 to 7 days whereas for tissue biopsy the time to test result is 15-30 days. This reduced time to return a result enables clinicians to make more timely decisions to put the patient on an effective therapy trajectory earlier, and this alone may greatly benefit progression-free and overall survival. While liquid biopsy testing has clear advantages for improving patient management and transforming clinical trial development for novel therapies, not all liquid biopsy approaches demonstrate the same level of performance. The utility of any liquid biopsy for a specific clinical intended use must be considered within the context of an assay’s analytical and clinical performance in order to truly improve patient outcomes and expedite clinical trial development.

Table 1: Copies of mutant ctDNA per mL of plasma, data adapted from Bettegowda et al.1 across multiple tumor types.

The clinical need for high sensitivity liquid biopsy detection

In an influential report published in Science Translational Medicine in 2014 (“Detection of circulating tumor DNA in early- and late-stage human malignancies”)1, Plasma-Safe-SeqS and BEAMing technologies were used to measure ctDNA in a wide range of tumor types from both localized and metastatic disease. As one can see from Table 1, the range of copies of ctDNA in plasma vary significantly, and the Plasma-Safe-SeqS method approaches the sensitivity-level of single-molecule detection.

As Sysmex Inostics only requires 2mL of plasma, the range of copies of mutant DNA per 2mL have been calculated in the last column of Table 1. With 2mL of plasma for Plasma-Safe-SeqS circulating tumor DNA analysis, a 0.05% mutant allele frequency sensitivity is achieved. This Limit of Detection (LoD95) means the analyte is detected 95% of the time.

A recent publication compared four liquid biopsy service providers using identical plasma samples matched to tissue. These four tests were sensitive and specific to only 1% mutant-allele frequency (MAF), regardless of their claims, with almost all of the positive calls less than 1% identified to be false positives.2

With a highly sensitive technology like BEAMing and Plasma-Safe-SeqS it becomes clear that nearly 50% of patients have ctDNA detected below 1% MAF. A survey of plasma samples from patients with advanced, treated and/or recurrent disease shows the importance of sensitivity. In metastatic colorectal cancer (mCRC) a full 48% of patients have mutated RAS <1% MAF. For EGFR-mutated T790M in non-small cell lung carcinoma (NSCLC) the percentage of samples with <1% MAF was 42%. And for estrogen-receptor positive, HER2 negative (ER+/HER2-) breast cancer 45% of samples had mutated PIK3CA <1% MAF. This is illustrated in Figure 1.3

Figure 1: Percentage of patients with <1% mutant allele frequencies of RAS, EGFR or PIK3CA

The expensive consequences of low-sensitivity liquid biopsy

Assuming a conventional limit of 1% MAF for liquid biopsy, what are the economic consequences of using less-sensitive technologies? For head and neck squamous cell carcinoma (HNSCC) where a low-frequency HRAS mutation status (5% of the target population) is used for a clinical trial, a full 89% of the mutant HRAS patients have MAF less than 1%.4 (See Figure 2.) Therefore to obtain 50 patients with mutant HRAS, the difference between having a sensitivity of 1% versus a 0.05% sensitivity for Plasma-Safe-SeqS is an approximately nine-fold difference in screening populations – from over 9,000 to only 1,000 patients needing testing.

Figure 2: The expensive consequences of a low-sensitivity liquid biopsy assay, using head and neck squamous cell carcinoma and mutant HRAS screening status

Exaggerated commercial claims from liquid biopsy test providers

Several circulating tumor DNA tests on the market claim limits of detection (LoD) that are well-below 1%, often down to 0.1%. Yet for several NGS-based methods, an examination of their technical specifications or their analytical validation publications reveal that their analytical sensitivity drops precipitously at less than 0.5% MAF (see Figure 3). For example, ‘Competitor F’ has a 98.9% analytical sensitivity at >0.5%, but drops to only 67.3% at a target MAF of 0.1 to 0.49% (see Table 2).6

Both OncoBEAM and Plasma-Safe-SeqS technologies have demonstrated sensitivities down to 0.03% and 0.05% MAF respectively, and do not have this kind of drop-off in analytical sensitivity below 0.5% MAF.

Table 2: The target allele frequency, analytical sensitivity and detection limit across four competing 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-A2.9

Proven clinical sensitivity of OncoBEAM down to 0.03% MAF

With a publication record stretching back to 2003, the Sysmex Inostics  OncoBEAM enhanced digital PCR technology has the longest and largest set of clinical publications for any liquid biopsy method available today. One publication from 2018 compared Sysmex Inostics  OncoBEAM technology to droplet digital PCR (from Bio-Rad) and to an NGS method (56G Oncology Panel from Swift Biosciences) for both mCRC and NSCLC with both matched tissue and plasma samples to do the comparison. The authors state in their abstract:10

“Excellent matches between cfDNA/FFPE mutation profiles were observed. Detection thresholds were between 0.5–1% for cfDNA samples examined using ddPCR and NGS, and 0.03% with BEAMing. This high level of sensitivity enabled the detection of KRAS mutations in 5/19 CRC patients with negative FFPE profiles.”

In a recent publication11, investigators compared the performance of OncoBEAM RAS testing to Foundation’s liquid biopsy NGS method in patients with hepatocellular carcinoma, a tumor that is challenging to biopsy and has been shown to exhibit a lower ctDNA rate than other cancers. Investigators found that RAS mutations were not detected by NGS in over 60% of the samples with a MAF between 0.02% and 0.1% as determined by BEAMing. Above 0.1% MAF the plasma NGS method confirmed RAS mutational status in only 44% of patients (44.4%).11

In a study of ESR1 mutations in estrogen receptor-positive (ER+) metastatic breast cancer (mBC) patients receiving selective estrogen receptor degrador (SERD) therapy, OncoBEAM technology was used to test for hotspot mutations in the ESR1 and PIK3CA genes across 153 patients enrolled in a Phase II clinical study.12

The measured allele frequency distribution for the ESR1 mutations in the target patient population is striking (Figure 3). The median MAF is 0.45% with a large number of patients showing MAF of <0.25%. Indeed, of the 153 total samples analyzed, a full 31 mutations in either ESR1 or PIK3CA were measured using OncoBEAM at less than 0.1% (range between 0.020% and 0.096%) and their distribution is shown in Figure 4.

Figure 3: The mutant allele frequency of ESR1 mutations across 153 patient samples

Figure 4: The distribution of measured MAF of ESR1 and PIK3CA mutations from 31 cell-free DNA samples less than 0.1%. Derived from the Supplement12

Proven sensitivity of Sysmex Inostics Plasma-Safe-SeqS down to 0.05% MAF

Recently Sysmex Inostics has launched a next-generation sequencing (NGS)-based liquid biopsy technology called Plasma-Safe-SeqS, with several disease-specific panels available.

One panel called the Plasma-Safe-SeqS Head and Neck Cancer Panel analyses typically low-frequency mutations in the potential therapeutic targets HRAS and PIK3CA, in addition to truncal mutations (with the potential to add additional information to support a true negative finding) in the TP53 and CDKN2A genes. Analysis with reference standard materials show a 95% detection rate down to 0.05% MAF (Figure 5).

Figure 5: Sensitivity (LoD95) is established using SeraCare Seraseq ctDNA Mutation Mix v2 (6 mutations) with the Plasma-Safe-SeqS Head and Neck Cancer Panel. The indicated number of target mutant molecules is in a background of 10K wildtype GE, thus 5 mutant molecules has a corresponding MAF of 0.05%.

In another study, the Plasma-Safe-SeqS ER+/HER2- Breast Cancer Panel was used to analyze both clinical and contrived samples for ESR1, PIK3CA and AKT1 on both the NGS-based Plasma-Safe-SeqS method and the orthogonal OncoBEAM enhanced digital PCR technology. Across 35 clinical ER+/HER2- breast cancer plasma specimens and several reference material samples at a variety of allele frequencies, the measured R-squared value of 0.97 is as shown in Figure 613.

Figure 6: Clinical data derived from 35 samples of ER+/HER2- breast cancer specimens. For the reference material, data points represent averages for replicate testing at different DNA input levels and mutant allele frequency tiers.

Potential utility in real-time monitoring of disease status

Thanks to the ultra-high sensitivity of both the OncoBEAM and Plasma-Safe-SeqS technologies, new applications for liquid biopsy are possible.

For example, in the setting of breast cancer serial monitoring, utilization of a highly sensitive assay like Plasma-Safe-SeqS to detect disease recurrence after administration of adjuvant therapy may allow change in therapeutic decision-making (Figure 7).

Figure 7: Clinical development example using liquid biopsy for detectioin of disease recurrence after administration of adjuvant therapy

Sysmex Inostics’ OncoBEAM and Plasma-Safe-SeqS offerings are ideally suited for:

Highly sensitive mutation detection

  • Emerging therapeutic indications
  • Prevalent “truncal” mutations, driver mutations present in every tumor cell which help indicate the presence or absence and quantity of ctDNA

Highly sensitive, cost-effective serial testing

  • Molecular monitoring
  • Evaluation of minimal residual disease
  • Disease surveillance: detection of relapse and recurrence in patients that have been determined to have no evidence of disease by radiographic imaging

To learn more about OncoBEAM and Plasma-Safe-SeqS or other purpose-designed clinical oncology tests from Sysmex Inostics, please contact us today.


  1. Bettegowda et al (2014). Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies. Science Transl. Med. 6(224):224ra24. PMID:24553385
  2. 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.
  3. Kinde I and Vogelstein B. et al. (2011). Detection and quantification of rare mutations with massively parallel sequencing. Proc Natl Acad Sci USA. 108 (23) : 9530 – 5. PMID:21586637
  4. Wang X and Agrawal N et al (2015). Detection of somatic mutations and HPV in the saliva and plasma of patients with head and neck squamous cell carcinomas Sci Transl Med 7(293):293ra104 PMID:26109104
  5. cobas EGFR Mutation Test v2, Summary of Safety and Effectiveness Data, Table 15. Available at
  6. FoundationACT technical specifications, available at: retrieved July 18, 2019.
  7. Odegaard JI, Vincent JJ and Talasaz A et al. (2018) Validation of a Plasma-Based Comprehensive Cancer Genotyping Assay Utilizing Orthogonal Tissue- and Plasma-Based Methodologies. Clin Cancer Res. 24(15):3539-3549. PMID: 29691297.
  8. Plagnol, V. and Forshew T. et al. (2018) Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling. PLoS ONE 13(3): e0193802. PMID:29543828
  10. Garcia J and Payen L et al. (2018) Cross-platform comparison for the detection of RAS mutations in cfDNA (ddPCR Biorad detection assay, BEAMing assay, and NGS strategy) Oncotarget 9(30):21122-21131 PMID:29765524
  11. Lim H.Y. and Llovet J.M. et al. (2018). Phase II Studies with Refametinib or Refametinib plus Sorafenib in Patients with RAS-Mutated Hepatocellular Carcinoma. Clin. Cancer Res. 24(19),:4650–4661 DOI:10.1158/1078-0432.CCR-17-3588
  12. Spoerke JM and Lackner MR et al. (2016) Heterogeneity and clinical significance of ESR1 mutations in ER-positive metastatic breast cancer patients receiving fulvestrant Nature Comm 13(7):11579 PMID:27174596
  13. Rugo H.S. and Shapiro G.I et al. (2019). Palbociclib in combination with fulvestrant or tamoxifen as treatment for hormone receptor positive (HR+) metastatic breast cancer (MBC) with prior chemotherapy for advanced disease (TBCRC 035) A phase II study with pharmacodynamics markers. San Antonio Breast Cancer Symposium (2018) Abstract PD2-12.

High tissue biopsy to liquid biopsy concordance with OncoBEAM enhanced digital PCR – Part II

A summary of a group of publications demonstrating the high concordance between high-sensitivity OncoBEAM digital PCR and standard tissue biopsy across several different cancer types

In Part I, we covered the high tissue biopsy to liquid biopsy concordance across six different colorectal cancer studies (available here) with an Overall Percent Agreement (OPA) calculated across 913 clinical samples to be 90.3%. Here we review tissue and liquid biopsy concordance studies in non-small cell lung cancer (NSCLC), breast cancer, and melanoma.

High concordance between tissue and plasma EGFR activating and T790M mutations in NSCLC

Both activating and T790M resistance mutations in non-small cell lung cancer (NSCLC) were examined in a dual observational and Phase I trial of an EGFR inhibitor rociletinib in Karlovich et al. (2016 Clinical Cancer Res.)1 Tissue was tested using Standard-of-Care (SoC) real-time PCR, and of 63 patients tested the OPA of EGFR activating mutations between tissue and the OncoBEAM EGFR test on plasma was 81%.

Looking at the same 63 patients for the EGFR T790M resistance mutation, the OPA between tissue and plasma was 67%; however, an additional 9 patients detected EGFR T790M in plasma that went undetected in tumor tissue, and another 9 patients detected EGFR T790M in plasma where insufficient tissue was available for analysis. Overall the OncoBEAM test identified more T790M-positive patients (51) than did the tumor test (45).

Another study also examined EGFR mutations in NSCLC for a Phase I trial (“AURA”) of an EGFR inhibitor AZD9291 (approved as TAGRISSO® or osimertinib) in Thress et al. (2015 Lung Cancer)2. They used a cross-platform comparison across two real-time PCR platforms for EGFR (cobas EGFR Mutation Test and therascreen EGFR amplification refractory mutation assay) and two digital PCR platforms (Bio-Rad Droplet Digital™ PCR and OncoBEAM BEAMing digital PCR). Their preliminary assessment across 38 samples achieved 95% concordance between tissue testing and OncoBEAM for Exon 19 deletions and L858R mutations, and 70% concordance between tissue testing and OncoBEAM for T790M.

The authors note that the concordance between plasma testing with the cobas EGFR Mutation Test and OncoBEAM (instead of tissue against OncoBEAM and plasma) was over 90%, with 14/20 of the discordant tissue versus plasma results were in perfect agreement when comparing the two orthogonal plasma testing technologies; the remaining 6/20 discordant cobas plasma versus OncoBEAM plasma results all had MAF of <0.2%, below the detection limit of the cobas EGFR Mutation Test, and likely due to tumor heterogeneity to explain the tissue versus plasma discordance.

In a study examining third-generation tyrosine kinase inhibitor (TKI) activity against EGFR T790M resistance, Oxnard et al. (2016 J. Clinical Oncol)3 in their analysis determined Objective Response Rate (ORR) and Progression-Free Survival (PFS) in T790M-positive and T790M-negative patients. Their results concluded ORR and median PFS were similar in T790M-positive plasma and T70M-positive tumor; thus with a validated assay some patients could avoid a tumor biopsy for T790M tissue testing and use a plasma-based test instead.

Concordance between tissue and plasma testing in breast cancer for AKT1 and PIK3CA

In Rudolph et al. (2016 BMC Cancer)4, more than 600 clinical breast cancer samples were tested for a specific AKT1 mutation [G49A:E17K] and overall its mutation prevalence was 6.3%. All of the tissue samples via OncoBEAM for mutations in both the ATK1 and PIK3CA genes; a subset of 90 samples were also tested via a broad-panel NGS test.

Within this sample set, there were 179 breast cancer tissue samples that were AKT1E17K mutation-positive. Overall plasma concordance with OncoBEAM yielded an OPA of 81.6%. Of 121 metastatic breast cancer samples that were PIK3CA mutation-positive in tissue, the plasma concordance had an OPA of 86.8%.

In Higgins et al. (2012 Clin Cancer Res)5 49 retrospective tumor and temporally-matched plasma samples were compared for PIK3CA mutations, and achieved an OPA of 100%. With an additional 41 prospective cohort tumor samples with matched tissue and plasma another OPA of 100% was observed.

In Di Leo et al. (2018 Lancet Oncol.)6 a group of postmenopausal women with hormone-receptor positive, HER2-negative advanced breast cancer participated in an mTOR inhibition Phase III trial (called BELLE-3). ctDNA PIK3CA status was determined by OncoBEAM, while tissue was evaluated by real-time PCR. Of the 256 samples evaluated, an overall concordance of 83% was reported, with 80% sensitivity and 87% specificity.

Concordance of ctDNA and melanoma tissue and its implication for patient management

Using the OncoBEAM BRAF and NRAS assay, Rowe et al. (2018 Molecular Oncol.)7 determined the BRAF and NRAS tissue and plasma mutation status across 55 patient samples collected prospectively. This work investigated the clinical utility (i.e. impact on clinical outcomes and interpretation of radiographic images) of measuring ctDNA in patients with metastatic or high-risk resected melanoma.

Both tissue and plasma were tested with OncoBEAM BRAF and NRAS assay technology, and across the 55 patient samples the researchers determined an OPA of 90.9%.

In another prospective study Haselmann et al. (2018 Clinical Chem.)8 examined BRAF mutation status in both tissue and plasma to correlate with the clinical course of disease and with response to treatment. Of 187 patient tissue samples tested (all were Stage I or Stage II as part of ‘Study 1’ in their report), concordance between tissue testing by OncoBEAM compared to plasma testing achieved an OPA of 90.9%.

The correlation to the clinical course of disease and response to treatment across many more patients (n=1204, “Study 2 follow-up of patients”) achieved an OPA of 95.7%.

A summary table of OncoBEAM performance across NSCLC, breast cancer, and melanoma

If you would like to access our concordance data for colorectal cancer across tissue and plasma samples analyzed by OncoBEAM, you can find it here.

For more information about how OncoBEAM enhanced digital PCR can help in your liquid biopsy studies, please contact us here.


1. Karlovich C, Goldman JW, Sun JM, Mann E, Sequist LV, Konopa K, Wen W, Angenendt P, Horn L, Spigel D, Soria JC, Solomon B, Camidge DR, Gadgeel S, Paweletz C, Wu L, Chien S, O’Donnell P, Matheny S, Despain D, Rolfe L, Raponi M, Allen AR, Park K, Wakelee H. Assessment of EGFR Mutation Status in Matched Plasma and Tumor Tissue of NSCLC Patients from a Phase I Study of Rociletinib (CO-1686). Clin Cancer Res. 2016 22(10):2386-95. doi:10.1158/1078-0432.CCR-15-1260.

2. Thress KS, Brant R, Carr TH, Dearden S, Jenkins S, Brown H, Hammett T, Cantarini M, Barrett JC. EGFR mutation detection in ctDNA from NSCLC patient plasma: A cross-platform comparison of leading technologies to support the clinical development of AZD9291. Lung Cancer. 2015 Dec;90(3):509-15. doi:10.1016/j.lungcan.2015.10.004.

3. Oxnard GR, Thress KS, Alden RS, Lawrance R, Paweletz CP, Cantarini M, Yang JC, Barrett JC, Jänne PA. Association Between Plasma Genotyping and Outcomes of Treatment With Osimertinib (AZD9291) in Advanced Non-Small-Cell Lung Cancer. J Clin Oncol. 2016 Oct 1;34(28):3375-82. doi:10.1200/JCO.2016.66.7162.

4. Rudolph M, Anzeneder T, Schulz A, Beckmann G, Byrne AT, Jeffers M, Pena C, Politz O, Köchert K, Vonk R, Reischl J. AKT1 (E17K) mutation profiling in breast cancer: prevalence, concurrent oncogenic alterations, and blood-based detection. BMC Cancer. 2016 Aug 16:622. doi: 10.1186/s12885-016-2626-1. PubMed PMID: 27515171; PubMed Central PMCID: PMC4982009.

5. Higgins MJ, Jelovac D, Barnathan E, Blair B, Slater S, Powers P, Zorzi J, Jeter SC, Oliver GR, Fetting J, Emens L, Riley C, Stearns V, Diehl F, Angenendt P, Huang P, Cope L, Argani P, Murphy KM, Bachman KE, Greshock J, Wolff AC, Park BH. Detection of tumor PIK3CA status in metastatic breast cancer using peripheral blood. Clin Cancer Res. 2012 Jun 15;18(12):3462-9. doi:10.1158/1078-0432.CCR-11-2696.

6. Di Leo A, Johnston S, Lee KS, Ciruelos E, Lønning PE, and Bachelot T. et al. Buparlisib plus fulvestrant in postmenopausal women with hormone-receptor-positive, HER2-negative, advanced breast cancer progressing on or after mTOR inhibition (BELLE-3): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol. 2018 Jan;19(1):87-100. doi:10.1016/S1470-2045(17)30688-5.

7. Rowe SP, Luber B, Makell M, Brothers P, Santmyer J, Schollenberger MD, Quinn H, Edelstein DL, Jones FS, Bleich KB, Sharfman WH, Lipson EJ. From validity to clinical utility: the influence of circulating tumor DNA on melanoma patient management in a real-world setting. Mol Oncol. 2018 12(10):1661-1672. doi:10.1002/1878-0261.12373.

8. Haselmann V, Gebhardt C, Brechtel I, Duda A, Czerwinski C, Sucker A, Holland-Letz T, Utikal J, Schadendorf D, Neumaier M. Liquid Profiling of Circulating Tumor DNA in Plasma of Melanoma Patients for Companion Diagnostics and Monitoring of BRAF Inhibitor Therapy. Clin Chem. 2018 64(5):830-842. doi:10.1373/clinchem.2017.281543.

High tissue biopsy to liquid biopsy concordance with OncoBEAM enhanced digital PCR – Part I

A summary of an extensive set of publications demonstrating the high concordance between high-sensitivity OncoBEAM digital PCR and standard tissue biopsy in colorectal cancer

A long history of OncoBEAM enhanced digital PCR publications

Recently we published an OncoBEAM clinical milestones timeline infographic with the first paper  stretching back over a decade analyzing various clinical cancer samples for liquid biopsy. Several of the important references can be found on our Publications page

Here we review and summarize several concordance studies comparing RAS mutation status in colorectal cancer (CRC) patients as determined in tissue by Standard of Care (SoC) testing in plasma using OncoBEAM technology.

High RAS concordance between tissue and liquid biopsy in Colorectal Cancer (CRC)

The OncoBEAM Colorectal Cancer Panel 1 tests for 34 mutations in the BRAF, KRAS and NRAS genes, and was used in these studies to test plasma and occasionally tissue. The stated limit of detection (LOD) of the OncoBEAM test used is 0.02% MAF.

In Schmiegel et al. (Molecular Oncology 2017), a retrospective concordance study was performed across 98 patients from Australia and Germany. A total of 98 metastatic colorectal cancer (mCRC) patients had tissue testing via Sanger sequencing, pyrosequencing or NGS depending on the established institutional protocol, and OncoBEAM was used on tissue where the SoC method was discordant from the plasma OncoBEAM result.

This study found RAS mutations in tissue from 53% of the patient samples, and in plasma from 51% of the samples. The Positive Percent Agreement (PPA) between tissue and plasma was 90.4%, the Negative Percent Agreement (NPA) was 93.5%, and Overall Percent Agreement (OPA) was 91.8%. The authors conclude “The high concordance of plasma and tissue results demonstrates that blood‐based RAS mutation testing is a viable alternative to tissue‐based RAS testing.”

In Vidal et al. (Annals Oncol. 2017), a retrospective concordance study was performed in two institutions in Spain. RAS mutation status was determined in tissue by each institution’s SoC method (either the QIAGEN Therascreen KRAS RGQ PCR kit or the Roche cobas KRAS Mutation Test, both real-time PCR assays).

RAS mutations were detected in tissue from 48% of the 115 patient samples tested, and in plasma from 51% of the samples. The PPA between tissue and plasma was 96.4%, the NPA was 90.0%, and the OPA was 93.0%. The authors conclude “The high overall agreement between baseline plasma and tissue RAS mutation status demonstrated in more than 100 patients evaluated in our study supports the use of blood-based testing with OncoBEAM™ RAS CRC as a viable alternative to tissue SoC for determining RAS mutation status in mCRC patients treated in routine clinical practice.”

Internally at Sysmex Inostics we have conducted our own verification studies, and with prospectively collected samples from throughout Europe (specifically Germany, France, Belgium and Spain) of 135 patient samples where the Overall Percent Agreement (OPA) compared to OncoBEAM was 93.3%, and the PPA was 92.6% and the NPA was 94.0%. This data was presented at CSCO in 2017 (Jones et al.).

One additional study of concordance between tissue and liquid biopsy in CRC

Outside of the United States Sysmex Inostics offers the CE-marked OncoBEAM RAS CRC kit used in conjunction with a Sysmex readout platform. Two concordance studies were performed by individual laboratories using this OncoBEAM RAS CRC kit. (More information about this system can be found online here.)

In Grasselli et al. (Annals Oncol. 2017) a retrospective-prospective concordance study between tissue and plasma RAS mutation status was carried out in 146 metastatic CRC patients from three hospitals in Spain, as part of a Phase II TTD ULTRA clinical trial (NCT01704703). The SoC method was real-time PCR for tissue, and 130 of the 146 tissue samples were analyzed by OncoBEAM RAS CRC as well.

The investigators foundRAS mutations in tissue from 37.0% of the patient samples by real-time PCR, whereas RAS mutations were detected in plasma in 39.0% of the patient samples by OncoBEAM RAS CRC. Interestingly, when 130 of the tissue samples were analyzed by OncoBEAM, 46.1% of the tissue samples harbored RAS mutations. The researchers note that a full 48% of their samples harbored RAS mutations at less than 1% MAF.

Of the 146 samples tested, OncoBEAM had 89.7% Overall Percent Agreement, with a Positive Predicted Value (PPV) of 84% and a Negative Predictive Value (NPV) of 93%. Their abstract concludes: “Plasma RAS determination showed high overall agreement and captured a mCRC population responsive to anti-EGFR therapy with the same predictive level as SoC tissue testing. The feasibility and practicality of ctDNA analysis may translate into an alternative tool for anti-EGFR treatment selection.”

A summary table and meta-analysis

This table summarizes these four datasets along with three others, and when analyzed together the meta-analysis determines an Overall Percent Agreement of 90.3% (PPA 88.7% and NPA 91.8%) across a total of 913 clinical samples.

For more information about how OncoBEAM enhanced digital PCR can help in your liquid biopsy studies, please contact us here.


  1. Schmiegel, W.; Scott, R. J.; Dooley, S.; Lewis, W.; Meldrum, C. J.; Pockney, P.; Draganic, B.; Smith, S.; Hewitt, C.; Philimore, H.; 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. Molecular Oncology 2017, 11 (2), 208–219.
  2. Vidal, J.; Muinelo, L.; Dalmases, A.; Jones, F.; Edelstein, D.; Iglesias, M.; Orrillo, M.; Abalo, A.; Rodríguez, C.; Brozos, E.; et al. Plasma ctDNA RAS Mutation Analysis for the Diagnosis and Treatment Monitoring of Metastatic Colorectal Cancer Patients. Annals of Oncology 2017, 28 (6), 1325–1332.
  3. Grasselli J, Elez E, Caratù G, Matito J, Santos C, Macarulla T, Vidal J, Garcia M, Viéitez JM, Paéz D, Falcó E, Lopez Lopez C, Aranda E, Jones F, Sikri V, Nuciforo P, Fasani R, Tabernero J, Montagut C, Azuara D, Dienstmann R, Salazar R, Vivancos A. Concordance of blood- and tumor-based detection of RAS mutations to guide anti-EGFR therapy in metastatic colorectal cancer. Ann Oncol. (2017) 28(6):1294-1301.
  4. García-Foncillas J, Alba E, Aranda E, Díaz-Rubio E, López-López R, Tabernero J, Vivancos A. Incorporating BEAMing technology as a liquid biopsy into clinical practice for the management of colorectal cancer patients: an expert taskforce review. Ann Oncol. 2017 Dec 1;28(12):2943-2949.
  5. P. Saunders, C. Cooney, D. Edelstein, S. Mullamitha, M. Braun, S. Moghadam, P. Ronga, F.S. Jones, A. Telaranta-Keerie, R.A. Adams. Performance assessment of blood based RAS mutation testing: Concordance of results obtained from prospectively collected samples. Annals of Oncology, Volume 27, Issue suppl_6, 1 October 2016, 526P,
  6. Normanno N, Esposito Abate R, Lambiase M, Forgione L, Cardone C, Iannaccone A, Sacco A, Rachiglio AM, Martinelli E, Rizzi D, Pisconti S, Biglietto M, Bordonaro R, Troiani T, Latiano TP, Giuliani F, Leo S, Rinaldi A, Maiello E, Ciardiello F; CAPRI-GOIM Investigators. RAS testing of liquid biopsy correlates with the outcome of metastatic colorectal cancer patients treated with first-line FOLFIRI plus cetuximab in the CAPRI-GOIM trial. Ann Oncol. 2018 Jan 1;29(1):112-118.
  7. Jones F.S., Edelstein D.L., Stieler K., Lenfert E., Boehm V., Lukas A., Macioszek J., Wichner K., Ross C., Stamm C., van Rahden V., Holtrup F., Diehl F. Blood-based testing RAS and EGFR mutation testing in colorectal and lung cancer patients using the OncoBEAM platform, Chinese Society for Clinical Oncology 2017, Xiamen China. Abstract: E0457, Poster:P-22 (Please contact us for this poster).

With liquid biopsy, sensitivity matters: OncoBEAM digital PCR video

This video presentation shows the many applications for liquid biopsy across a range of cancer types where OncoBEAM enhanced digital PCR demonstrates superior sensitivity

OncoBEAM liquid biopsy clinical milestones timeline infographic

First published in 2003, BEAMing technology has an extensive publication record for liquid biopsy across a variety of cancer types.

Here’s an annotated timeline of OncoBEAM liquid biopsy from 2003 to the present. Please contact us if you have any requests or questions here.


Performance of Streck cfDNA blood collection tubes (BCT) for liquid biopsy testing

Through use of length-dependent qPCR, OncoBEAM enhanced digital PCR, and Plasma-Safe-SeqS NGS, several questions regarding the optimum handling and shipping conditions are answered

A 2016 publication from Sysmex Inostics, ‘Performance of Streck cfDNA blood collection tubes for liquid biopsy testing’ (Medina Diaz et al. PLoS One, November 2016, DOI:10.1371/journal.pone.0166354)  has proven to be very popular, with over 18,000 views and 48 citations. It is unique in its approach in evaluating a method for transfer of blood samples prior to liquid biopsy analysis, utilizing both highly-sensitivity OncoBEAM enhanced digital PCR in addition to Plasma-Safe-SeqS, our sensitive NGS-based method, for background mutations introduced through preservation and storage.

Experimental design

Venous blood samples from healthy donors as well as colorectal cancer (CRC) patients were collected in Streck Blood Collection Tubes (BCT) as well as standard K2EDTA tubes, then stored at different temperatures for various lengths of time before plasma separation and DNA purification. Length-specific qPCR was used to measure cfDNA compared to contaminating genomic DNA, and high-sensitivity enhanced digital PCR OncoBEAM and NGS-based Plasma-Safe-SeqS technology was used to look for introduction of mutations. Additionally, some spike-in low-level synthetic mutant DNA was also added and measured.

The number of healthy donors for the five study cohorts totaled 103; an overview of the experimental conditions and analysis methodologies is in figure 1 below and available online.

Figure 1 from Medina Diaz et al. available online here. Per its figure legend:

Experimental setup for cfDNA BCT vs K2EDTA performance experiments. Cohort I: Time point experiments at room temperature including DNA quantification and mutation analysis using BEAMing and Safe-SeqS. Cohort II: BEAMing analysis of blood samples spiked with synthetic double-stranded mutant DNA fragments at different allele frequencies. Cohort III: BEAMing analysis of samples collected from colorectal cancer (CRC) patients. Cohort IV: Experiment evaluating effects of extreme storage temperatures on DNA quantity. Cohort V: Experimental evaluation of recommended temperature range.

The effect of time on cfDNA in Streck Blood Collection Tubes

In Study Cohort I, 60 healthy samples were compared between K2EDTA and BCT storage at room temperature for 3 and 5 days, and no significant differences were detected in the quantity and quality of the cfDNA samples.

The effect of temperature on cfDNA in Streck Blood Collection Tubes

Of particular interest is ‘Study Cohort IV’, where BCT storage at temperatures slightly outside the official manufacturer-recommended temperature range (e.g. 6 °C to 37 °C); 4 °C, RT and 40 °C for up to 5 days was used in this experiment, and visual differences between the samples were apparent after the first centrifugation step. RT-stored samples (stored at both the 3 and 5 day intervals) had a clear plasma fraction with a defined buffy coat; the 4°C samples had an expanded cellular interface layer (affecting 20-50% of the plasma fraction), while the 40 °C samples had a darker, hemolytic plasma fraction.

Length-dependent qPCR indicated a much higher ratio in these temperature-stressed samples, indicating much higher genomic DNA contamination compared to reference.

The authors conclude “exposure to extreme temperatures outside the recommended range of 6-37 °C needs to be avoided for cfDNA BCTs in order to prevent dilution of potential mutant cfDNA molecules with wild-type genomic DNA released from WBCs (white blood cells).” See figure 2.

Figure 2: Whisker-plot data derived from Medina Diaz et al. PLOS One 2016 showing the effect of storage temperature outside the manufacture-recommended temperature range, and photograph of a clear buffy coat layer under RT conditions, an increased interface cell layer at 4 °C, and hemolytic plasma at 40 °C.

No spurious low-level mutations introduced

Going back to ‘Study Cohort I’, these 60 samples were analyzed by both OncoBEAM enhanced digital PCR for six KRAS mutations, as well as by Plasma-Safe-SeqS NGS-based mutation detection for five c-KIT amplicons.

Figure 3, a slightly different representation of Figure 3 in the Medina Diaz et al reference. A) Enhanced digital PCR (BEAMing) data analyzing 6 mutations across 60 samples B) Targeted NGS (Plasma-Safe-SeqS) data analyzing >500 bases across 15 samples.

As you can see from Figure 3A, with an analytical cutoff of the OncoBEAM technology at 0.02% none of the five KRAS mutations assayed for were observed above the cutoff. In Figure 3B, the base change pattern observed with Plasma-Safe-SeqS did not change in any of the conditions tested.

A few key take-away conclusions

Through work like this Sysmex Inostics has established recognized and standardized conditions for blood collection and transport for ultra-sensitive ctDNA detection for liquid biopsy testing. Certainly K2EDTA-collected plasma can be spun down within four hours and stored at -70°C, however the use of Streck BCT collection tubes allows for flexibility in transport, as long as the 6°C to 37°C temperature boundaries are observed for up to 5 days.

More information about our OncoBEAM and Plasma-Safe-SeqS technologies are available here for OncoBEAM, and here for Plasma-Safe-SeqS.


Medina Diaz I, Nocon A, Mehnert DH, Fredebohm J, Diehl F, Holtrup F (2016) Performance of Streck cfDNA Blood Collection Tubes for Liquid Biopsy Testing. PLoS ONE 11(11): e0166354.

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.

  • 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.


  1. National Comprehensive Cancer Network. Non-Small Cell Lung Cancer (Version 2.2018). Accessed February 8, 2018. Link to Guidelines.
  2. 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
  3. Mandel P, Metais P. [Les acides nucleiques du plasma sanguin chez l’homme] C R Seances Soc Biol Fil. 142, 241–243 (1948). PMID:18875018
  4. Stroun, M. and Beljanski M. et al. Neoplastic characteristics of the DNA found in the plasma of cancer patients. Oncology. 46(5):318-22 (1989). PMID:2779946
  5. Diehl, F. and Diaz L.A. et al. Circulating mutant DNA to assess tumor dynamics. Nat. Med. 14, 985-990 (2008). PMID:18670422
  6. 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
  7. 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).
  8. 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 
  9. cobas EGFR Mutation Test v2, Summary of Safety and Effectiveness Data, Table 15. Available at
  10. FoundationONE Liquid technical specifications, available on their website here.
  11. 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.
  12. 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
  13. “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.
  14. 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
  15. 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
  16. 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
  17. Lokhandwala, T. et al. Costs of Diagnostic Assessment for Lung Cancer: A Medicare Claims Analysis. Clin. Lung Cancer 18, e27–e34 (2017). PMID:27530054
  18. 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
  19. Lim, C. et al. Biomarker testing and time to treatment decision in patients with advanced nonsmall-cell lung cancer. Ann. Oncol. Off. J. Eur. Soc. Med. Oncol. ESMO 26, 1415–1421 (2015). PMID:25922063
  20. 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
  21. 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
  22. 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.
  23. Wu, Y.L. et al. MA08.03 Osimertinib vs Platinum-Pemetrexed for T790M-Mutation Positive Advanced NSCLC (AURA3): Plasma ctDNA Analysis. J. Thorac. Oncol. 12, S386 (2017). Link to reference
  24. Mok, T.S. et al. Osimertinib or Platinum-Pemetrexed in EGFR T790M-Positive Lung Cancer. N. Engl. J. Med. 376, 629-640 (2017). PMID:27959700
  25. Thress K. et al. Levels of EGFR T790M in plasma DNA as a predictive biomarker for response to AZD9291, a mutant-selective EGFR kinase inhibitor. Poster presented at: European Society for Medical Oncology 2014 Congress; 2014 Sep 26-30; Madrid, Spain; #1270P. Link to resource
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