Preoperative detection of KRAS G12D mutation in ctDNA is a powerful predictor for early recurrence of resectable PDAC patients

www.nature.com/bjc ARTICLE Molecular Diagnostics Preoperative detection of KRAS G12D mutation in ctDNA is a powerful predictor for early recurrence of resectable PDAC patients Shiwei Guo1, Xiaohan Shi1, Jing Shen1, Suizhi Gao1, Huan Wang1, Shuo Shen1, Yaqi Pan1, Bo Li1, Xiongfei Xu1, Zhuo Shao1 and Gang Jin1 BACKGROUND: About 25–37% of resectable pancreatic ductal adenocarcinoma (PDAC) had a great chance of early recurrence after radical resection, which is mainly due to preoperative micrometastasis. We herein demonstrated the profiles of ctDNA in resectable PDAC and use of ctDNA to identify patients with potential micrometastasis. METHODS: A total of 113 and 44 resectable PDACs were enrolled in discovery and validation cohorts, separately. A panel containing 50 genes was used to screen ctDNA by an NGS-based assessment with high specificity. RESULTS: In the discovery cohort, the overall detection rate was 38.05% (43/113). Among positive ctDNA, KRAS mutation had the highest detection rate (23.01%, 26/113), while the others were <5%. Survival analysis showed that plasma KRAS mutations, especially KRAS G12D mutation, had significant association with OS and RFS of resectable PDAC. Plasma KRAS G12D mutation showed a strong correlation with early distant metastasis. In the validation cohort, survival analysis showed similar association between plasma KRAS G12D mutation and poor outcomes. CONCLUSIONS: This study demonstrated that plasma KRAS mutations, especially KRAS G12D mutation, served as a useful predictive biomarker for prognosis of resectable PDAC. More importantly, due to high correlation with micrometastasis, preoperative detection of plasma KRAS G12D mutation helps in optimising surgical selection of resectable PDAC. British Journal of Cancer (2020) 122:857–867; https://doi.org/10.1038/s41416-019-0704-2 BACKGROUND Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with a 5-year survival rate of <8%, and this is mainly due to late diagnosis and rapid progression.1 The majority, i.e., up to 80% of the patients have lost the chance of surgery at the time of diagnosis.2 Even for patients with resectable PDAC, a significant portion of them (25–37%) had great chances of early recurrence after undergoing radical resection.3,4 The presence of micrometastasis in patients that cannot be detected by current preoperative imaging techniques is the main reason for this quick relapse and as to why such patients cannot benefit from surgery.5,6 So, new-generation biomarkers are urgently needed to identify patients with micrometastasis, avoiding unnecessary surgical interventions. Liquid biopsy biomarkers are gaining interest in cancer research due to their advantages of convenient and noninvasive nature.7 Exosomes play an important role in PDAC progression and can be used for early detection, especially Glypican-1-positive exosomes can identify patients with latestage PDAC and distinguish from patients with precancerous pancreatic lesions.8 The value of circulating tumour cells (CTCs) in predicting prognosis has been broadly studied. The US Food and Drug Administration (FDA) has approved the use of CTC as a biomarker for prognostic evaluation in three types of cancers.9–11 Cell-free circulating tumour DNA (ctDNA) can be effectively distinguished from normal cell-free circulating DNA (cfDNA) by specific cancer-related mutations.12,13 Several studies have proved a high detection rate and potential prognostic value of ctDNA in PDAC.14–17 However, the data regarding the complexity of exosome isolation invalidated it in other centres, and heterogeneity as well as rarity of CTCs and ctDNA restrained the application mainly in advanced patients.18 Therefore, more stable methods and more validations are necessary before clinical application, especially for patients with early-stage PDAC who are commonly considered as resectable. In recent years, different procedures for plasma ctDNA processing, library construction and analytical methods have led to incomparable results and confusion in clinical application.19 For NGS techniques, random errors usually happened during the exponential amplification process, leading to high false-positive rate, and this has been one of the most important defects.20 However, some high-fidelity linear amplification sequencing methods and optimised analytical methods may help to improve.21 Hence, in this study, a highly specific ctDNA detection method was used to explore the clinical value of hotspot mutations in resectable PDAC patients and hoped that a 1 Department of Hepatobiliary Pancreatic Surgery, Changhai Hospital, Navy Military Medical University (Second Military Medical University), Shanghai, China Correspondence: Gang Jin () These authors contributed equally: Shiwei Guo, Xiaohan Shi, Jing Shen. Received: 25 June 2019 Revised: 30 October 2019 Accepted: 10 December 2019 Published online: 23 January 2020 © The Author(s) 2020 Published by Springer Nature on behalf of Cancer Research UK Preoperative detection of KRAS G12D mutation in ctDNA is a powerful. . . S Guo et al. 858 new ctDNA biomarker can be used to optimise the surgical selection of PDAC patients and eventually improve the survival time of PDAC. 1234567890();,: METHODS Patients Patients with resectable PDAC were collected from Changhai prospective database (Changhai Hospital, Shanghai, China). All patients underwent curative surgery for tumour resection and were histologically confirmed with PDAC. Patients who died of surgical complications within 1 month after surgery have been excluded. All patients provided written informed consent to use their clinical data. The study was conducted in accordance with the national guidelines, and acquired the approval of the Ethics Committee at Changhai Hospital. Clinical samples Approximately 0.5–1 cm3 tumour tissue was removed from the centre of the resected lesions, and then was rapidly maintained in liquid nitrogen until DNA extraction. In total, 10 mL of preoperative venous blood was collected from patients in ethylenediaminetetraacetic acid tubes (Vacutainer blood collection tubes, Becton, Dickinson and Company, NJ, USA). Within 2 h of collection, the blood was centrifuged at 1600g for 10 min at 4 °C to obtain plasma and a buffy coat layer (containing WBCs). The plasma was then further centrifuged at 16,000g for 10 min at 4 °C to pellet any remaining cells. Both plasma and the buffy coat layer were collected and stored at −80 °C. Patients were neither provided with the results of ctDNA sequencing nor any treatment decisions made based on ctDNA results. Tumour tissue, white blood cell genomic DNA and ctDNA extraction Before tumour tissue DNA extraction, at least one frozen resection of each sample was assessed by two specialist pathologists to confirm the tumour cellularity ≥20% (Supplementary Fig. 1). Tumour tissue genomic DNA was extracted from the freshly frozen mass using QIAamp DNA mini kit (Qiagen, Venlo, The Netherlan (...truncated)