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.

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.