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Licensed Unlicensed Requires Authentication Published by De Gruyter July 24, 2019

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

  • Beili Wang , Shengchao Wu , Fei Huang , Minna Shen , Huiqin Jiang , Yiyi Yu , Qian Yu , Yihui Yang , Ying Zhao , Yiwen Zhou , Baishen Pan , Tianshu Liu EMAIL logo and Wei Guo EMAIL logo



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.


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.


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.


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.

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.

Award Identifier / Grant number: 81572064

Award Identifier / Grant number: 81772263

Award Identifier / Grant number: 81772511

Award Identifier / Grant number: 81602038

Award Identifier / Grant number: 2015ZB0201

Award Identifier / Grant number: 201440389

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:, 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: and the Project from Zhongshan Hospital, Fudan university (2018ZSLC05).


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 (

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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