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Original research
Performance evaluation of a CRISPR Cas9-based selective exponential amplification assay for the detection of KRAS mutations in plasma of patients with advanced pancreatic cancer
  1. Yue Shen1,2,
  2. Xiaoling Zhang2,
  3. Liyi Zhang2,
  4. Zuoying Zhang1,2,
  5. Bao Lyu2,
  6. Qian Lai2,
  7. Qinglin Li2,
  8. Yuhua Zhang2,
  9. Jieer Ying2,
  10. Jinzhao Song1,2
  1. 1School of life sciences, Tianjin University, Tianjin, China
  2. 2The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou, Zhejiang, China
  1. Correspondence to Dr Jieer Ying; jieerying{at}aliyun.com; Dr Jinzhao Song; songjinzhao{at}ucas.ac.cn

Abstract

Aims Pancreatic ductal adenocarcinoma (PDAC) is highly malignant, with shockingly mortality rates. KRAS oncoprotein is the main molecular target for PDAC. Liquid biopsies, such as the detection of circulating tumour DNA (ctDNA), offer a promising approach for less invasive diagnosis. In this study, we aim to evaluate the precision and utility of programmable enzyme-based selective exponential amplification (PASEA) assay for rare mutant alleles identification.

Methods PASEA uses CRISPR-Cas9 to continuously shear wild-type alleles during recombinase polymerase amplification, while mutant alleles are exponentially amplified, ultimately reaching a level detectable by Sanger sequencing. We applied PASEA to detect KRAS mutations in plasma ctDNA. A total of 153 patients with stage IV PDAC were enrolled. We investigated the relationship between ctDNA detection rates with various clinical factors.

Results Our results showed 91.43% vs 44.83% detection rate in patients of prechemotherapy and undergoing chemotherapy. KRAS ctDNA was more prevalent in patients with liver metastases and patients did not undergo surgical resection. Patients with liver metastases prior to chemotherapy showed a sensitivity of 95.24% (20/21) with PASEA. Through longitudinal monitoring, we found ctDNA may be a more accurate biomarker for monitoring chemotherapy efficacy in PDAC than CA19-9.

Conclusions Our study sheds light on the potential of ctDNA as a valuable complementary biomarker for precision targeted therapy, emphasising the importance of considering chemotherapy status, metastatic sites and surgical history when evaluating its diagnostic potential in PDAC. PASEA technology provides a reliable, cost-effective and minimally invasive method for detecting ctDNA of PDAC.

  • DIAGNOSIS
  • ENZYMES
  • PANCREAS

Data availability statement

Data are available on reasonable request.

https://doi.org/10.1136/jcp-2023-208974

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    Data availability statement

    Data are available on reasonable request.

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    Footnotes

    • Handling editor Vikram Deshpande.

    • YS and XZ contributed equally.

    • Contributors All authors contributed to the article. JY and JS conceived the study; YS, XZ, LZ and ZZ performed the experiments; YS and XZ performed data analysis; YS wrote the original draft; BL, QLai, YZ and QLi provide resources; Guarantor of the manuscript: JS; All authors reviewed and approved the final version of the manuscript.

    • Funding This study was supported by Zhejiang Leading Innovation and Entrepreneurship Team (2022R01006).

    • Competing interests None declared.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.