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Performance validation of an amplicon-based targeted next-generation sequencing assay and mutation profiling of 648 Chinese colorectal cancer patients

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Abstract

Next-generation sequencing (NGS) has become a promising approach for tumor somatic mutation detection. However, stringent validation is required for its application on clinical specimens, especially for low-quality formalin-fixed paraffin-embedded (FFPE) tissues. Here, we validated the performance of an amplicon-based targeted NGS assay, OncoAim™ DNA panel, on both commercial reference FFPE samples and clinical FFPE samples of Chinese colorectal cancer (CRC) patients. Then we profiled the mutation spectrum of 648 Chinese CRC patients in a multicenter study to explore its clinical utility. This NGS assay achieved 100% test specificity and 95–100% test sensitivity for variants with mutant allele frequency (MAF) ≥ 5% when median read depth ≥ 500×. The orthogonal methods including amplification refractory mutation system (ARMS)-PCR and Sanger sequencing validated that NGS generated three false negatives (FNs) but no false positives (FPs) among 516 clinical samples for KRAS aberration detection. Genomic profiling of Chinese CRC patients with this assay revealed that 63.3% of the tumors harbored clinically actionable alterations. Besides the commonly mutated genes including TP53 (52.82%), KRAS (46.68%), APC (24.09%), PIK3CA (18.94%), SMAD4 (9.47%), BRAF (6.15%), FBXW7 (5.32%), and NRAS (4.15%), other less frequently mutated genes were also identified. Statistically significant association of specific mutated genes with certain clinicopathological features was detected, e.g., both BRAF and PIK3CA were more prevalent in right-side CRC (p < 0.001 and p = 0.002, respectively). We concluded this targeted NGS assay is qualified for clinical practice, and our findings could help the diagnosis and prognosis of Chinese CRC patients.

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Acknowledgments

We would like to thank Singlera Genomics for the precious technical help. We thank all participant departments for providing their specimens and relevant information.

Funding

This work was supported by the funding from the National Key Research and Development Plan of Precision Medical research in China (2016YFC0906000), National Natural Science Foundation of China (81401990), and Key Research and Development Project of Department of Science and Technology in Sichuan Province (2017SZ0005).

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Authors and Affiliations

  1. Department of Pathology, West China Hospital, Sichuan University, Chengdu, 610041, China

    Yajian Wang & Hong Bu

  2. Laboratory of Pathology, West China Hospital, Sichuan University, Chengdu, 610041, China

    Yajian Wang, Feng Ye & Hong Bu

  3. Department of Pathology, Peking University Third Hospital, Beijing, 100000, China

    Haijing Liu

  4. Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, 200032, China

    Yingyong Hou

  5. Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, 200032, China

    Xiaoyan Zhou

  6. Huayin Laboratory, Southern Medical University, Guangzhou, 510515, China

    Li Liang

  7. Department of Pathology, First Affiliated Hospital, Nanjing Medical University, Nanjing, 210000, China

    Zhihong Zhang

  8. Department of Pathology, Chinese PLA General Hospital and Chinese PLA Medical School, Beijing, 100000, China

    Huaiyin Shi

  9. Institute of Pathology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China

    Sanpeng Xu

  10. Department of Pathology, Xijing Hospital, Fourth Military Medical University, Xi’an, 710032, China

    Peizhen Hu

  11. Singlera Genomics Inc., Shanghai, 201318, China

    Zuyu Zheng, Rui Liu & Tingdong Tang

  12. Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041, China

    Feng Ye & Hong Bu

  13. Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100730, China

    Zhiyong Liang

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  1. Yajian Wang
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Contributions

FY and YW conceived and designed the experiments; HL, YH, XZ, LL, ZZ, HS, SX, PH, HB, and ZL organized the work of identifying clinical cases, reviewing the slides, collecting the clinical information, and performing the experiments in participating laboratories; the Singlera Genomics (ZZ, RL, TT) developed customized pipeline for variant identifying to analyze sequencing data from reads alignment to variant calling and annotation; YW analyzed the NGS results; the Singlera Genomics contributed reagents/analysis tools; YW wrote the manuscript. FY revised the paper. All authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Feng Ye.

Ethics declarations

The present study was approved by the Ethics Committee of Sichuan University (No. K2016031).

Conflict of interest

ZZ, RL, and TT are members of the Singlera Genomics, Shanghai, China. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; and in the decision to publish the results.

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Wang, Y., Liu, H., Hou, Y. et al. Performance validation of an amplicon-based targeted next-generation sequencing assay and mutation profiling of 648 Chinese colorectal cancer patients. Virchows Arch 472, 959–968 (2018). https://doi.org/10.1007/s00428-018-2359-4

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