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Analytical and clinical validation of a novel amplicon-based NGS assay for the evaluation of circulating tumor DNA in metastatic colorectal cancer patients

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Published/Copyright: July 24, 2019
Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 57 Issue 10

Abstract

Background

Evaluating the tumor RAS/BRAF status is important for treatment selection and prognosis assessment in metastatic colorectal cancer (mCRC) patients. Correction of artifacts from library preparation and sequencing is essential for accurately analyzing circulating tumor DNA (ctDNA) mutations. Here, we assessed the analytical and clinical performance of a novel amplicon-based next-generation sequencing (NGS) assay, Firefly™, which employs a concatemer-based error correction strategy.

Methods

Firefly assay targeting KRAS/NRAS/BRAF/PIK3CA was evaluated using cell-free DNA (cfDNA) reference standards and cfDNA samples from 184 mCRC patients. Plasma results were compared to the mutation status determined by ARMS-based PCR from matched tissue. Samples with a mutation abundance below the limit of detection (LOD) were retested again by droplet digital polymerase chain reaction (ddPCR) or NGS.

Results

The Firefly assay demonstrated superior sensitivity and specificity with a 98.89% detection rate at an allele frequency (AF) of 0.2% for 20 ng cfDNA. Generally, 40.76% and 48.37% of the patients were reported to be positive by NGS of plasma cfDNA and ARMS of FFPE tissue, respectively. The concordance rate between the two platforms was 80.11%. In the pre-treatment cohort, the concordance rate between plasma and tissue was 93.33%, based on the 17 common exons that Firefly™ and ARMS genotyped, and the positive percent agreement (PPA) and negative percent agreement (NPA) for KRAS/NRAS/BRAF/PIK3CA were 100% and 99.60%, respectively.

Conclusions

Total plasma cfDNA detected by Firefly offers a viable complement for mutation profiling in CRC patients, given the high agreement with matched tumor samples. Together, these data demonstrate that Firefly could be routinely applied for clinical applications in mCRC patients.

Keywords: amplicon-based NGS; ctDNA; ddPCR; mCRC; molecular diagnostics
Corresponding authors: Prof. Tianshu Liu, MD, PhD, Department of Medical Oncology, Center of Evidence Based Medicine, Zhongshan Hospital, Fudan University, 111 Yi Xue Yuan Road, Shanghai 200032, P.R. China, Fax: +86-21-64041990; and Prof. Wei Guo, PhD, Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, 111 Yi Xue Yuan Road, Shanghai 200032, P.R. China, Fax: +86-21-64041990-2376
aBeili Wang and Shengchao Wu contributed equally to this work.

Funding source: National Natural Science Foundation of China

Award Identifier / Grant number: 81572064

Award Identifier / Grant number: 81772263

Award Identifier / Grant number: 81772511

Award Identifier / Grant number: 81602038

Funding source: Key Developing Disciplines of Shanghai Municipal Commission of Health and Family Planning

Award Identifier / Grant number: 2015ZB0201

Award Identifier / Grant number: 201440389

Funding source: Shanghai Science and Technology Commission

Award Identifier / Grant number: 16411952100

Funding source: Zhongshan Hospital

Award Identifier / Grant number: 2018ZSLC05

Funding statement: This study was supported by grants from the National Natural Science Foundation of China (no. 81572064, 81772263, 81772511, and 81602038, funder Id: http://dx.doi.org/10.13039/501100001809), the Key Developing Disciplines of Shanghai Municipal Commission of Health and Family Planning (2015ZB0201, 201440389), the Projects from the Shanghai Science and Technology Commission (16411952100, funder Id: http://dx.doi.org/10.13039/501100003399) and the Project from Zhongshan Hospital, Fudan university (2018ZSLC05).

Acknowledgments

We thank the team of Laboratory Medicine of Zhongshan Hospital who provided facilities, insight and expertise that greatly assisted the research.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Employment or leadership: None declared.

  3. Honorarium: None declared.

  4. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2019-0142).

Received: 2019-02-06
Accepted: 2019-06-25
Published Online: 2019-07-24
Published in Print: 2019-09-25

© 2019 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2019-0142
Keywords for this article
amplicon-based NGS; ctDNA; ddPCR; mCRC; molecular diagnostics

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Blood biomarkers in neurology: “a call to arms” for laboratory professionals
  4. Reviews
  5. Diagnostic accuracy of glycated hemoglobin for gestational diabetes mellitus: a systematic review and meta-analysis
  6. Laboratory medicine: health evaluation in elite athletes
  7. Prostate cancer screening: guidelines review and laboratory issues
  8. Opinion Papers
  9. Extra-analytical sources of uncertainty: which ones really matter?
  10. Benefits and harms of wellness initiatives
  11. Genetics and Molecular Diagnostics
  12. Analytical and clinical validation of a novel amplicon-based NGS assay for the evaluation of circulating tumor DNA in metastatic colorectal cancer patients
  13. General Clinical Chemistry and Laboratory Medicine
  14. Pre-analytical practices for routine coagulation tests in European laboratories. A collaborative study from the European Organisation for External Quality Assurance Providers in Laboratory Medicine (EQALM)
  15. Preanalytical robustness of blood collection tubes with RNA stabilizers
  16. Continual improvement of the pre-analytical process in a public health laboratory with quality indicators-based risk management
  17. Comparison of six commercial serum exosome isolation methods suitable for clinical laboratories. Effect in cytokine analysis
  18. A multicenter study to evaluate harmonization of assays for N-terminal propeptide of type I procollagen (PINP): a report from the IFCC-IOF Joint Committee for Bone Metabolism
  19. Correlations between serum and CSF pNfH levels in ALS, FTD and controls: a comparison of three analytical approaches
  20. Dynamics of extracellular matrix proteins in cerebrospinal fluid and serum and their relation to clinical outcome in human traumatic brain injury
  21. Free light chains in the cerebrospinal fluid. Comparison of different methods to determine intrathecal synthesis
  22. Reference Values and Biological Variations
  23. Reference interval by the indirect approach of serum thyrotropin (TSH) in a Mediterranean adult population and the association with age and gender
  24. Next-generation reference intervals for pediatric hematology
  25. Hematology and Coagulation
  26. Preliminary evaluation of a new flow cytometry method for the routine hematology workflow
  27. Diabetes
  28. Trueness assessment of HbA1c routine assays: are processed EQA materials up to the job?
  29. Infectious Diseases
  30. Utility of procalcitonin for differentiating cryptogenic organising pneumonia from community-acquired pneumonia
  31. A high C-reactive protein/procalcitonin ratio predicts Mycoplasma pneumoniae infection
  32. Letters to the Editor
  33. Evaluation of reference change values for a hs-cTnI immunoassay using both plasma samples of healthy subjects and patients and quality control samples
  34. Outlier removal methods for skewed data: impact on age-specific high-sensitive cardiac troponin T 99th percentiles
  35. Comparison of precision and operational performances across six immunochemistry analyzers
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  40. Stability of tubular damage markers epidermal growth factor and heparin-binding EGF-like growth factor in urine
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