Improved predictability of pancreatic ductal adenocarcinoma diagnosis using a blood immune cell biomarker panel developed from bulk mRNA sequencing and single-cell RNA-sequencing

Cancer Immunology, Immunotherapy https://doi.org/10.1007/s00262-023-03458-8 RESEARCH Improved predictability of pancreatic ductal adenocarcinoma diagnosis using a blood immune cell biomarker panel developed from bulk mRNA sequencing and single‑cell RNA‑sequencing Sung Ill Jang1 · Hyung Keun Lee2,6 · Eun‑Ju Chang3 · Somi Kim4 · So Young Kim2,6 · In Young Hong1,6 · Jong Kyoung Kim4,7 · Hye Sun Lee5 · Juyeon Yang5 · Jae Hee Cho1 · Dong Ki Lee1 Received: 7 March 2023 / Accepted: 26 April 2023 © The Author(s) 2023 Abstract Background Pancreatic ductal adenocarcinoma (PDAC) remains a devastating cancer due to its poor survival rate, early detection, and resectability. This study aimed to determine the peripheral blood mononuclear cell (PBMC) immune biomarkers in patients with PDAC and investigate the PDAC-specific peripheral blood biomarker panel and validate its clinical performance. Methods In this prospective, blinded, case–control study, a biomarker panel formula was generated using a development cohort—including healthy controls, patients at high risk of PDAC, and patients with benign pancreatic disease, PDAC, or other gastrointestinal malignancies—and its diagnostic performance was verified using a validation cohort, including patients with ≥ 1 lesion suspected as PDAC on computed tomography (CT). Results RNA-sequencing of PBMCs from patients with PDAC identified three novel immune cell markers, IL-7R, PLD4, and ID3, as specific markers for PDAC. Regarding the diagnostic performance of the regression formula for the three biomarker panels, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 84.0%, 78.8%, 47.2%, 95.6%, and 79.8%, respectively. Based on the formula scores for the biomarker panel, the false-negative rate (FNR) of the biomarkers was 8% (95% confidence interval [CI] 3.0–13.0), which was significantly lower than that based on CT in the validation cohort (29.2%, 95% CI 20.8–37.6). Conclusions The regression formula constructed using three PBMC biomarkers is an inexpensive, rapid, and convenient method that shows clinically useful performance for the diagnosis of PDAC. It aids diagnoses and differential diagnoses of PDAC from pancreatic disease by lowering the FNR compared to CT. Clinical trial registration Clinical Research Information Service, KCT0004614 (08 January 2020). Keywords Pancreatic ductal adenocarcinoma · Interleukin-7 receptor · Phospholipase D4 · Inhibitor of DNA binding 3 · Cancer biomarkers Abbreviations AUC Area under the curve CI Confidence interval Sung Ill Jang and Hyung Keun Lee contributed equally. * Dong Ki Lee 1 Department of Internal Medicine, Gangnam Severance Hospital, Yonsei University College of Medicine, 712 Eonjuro, Gangnam‑gu, Seoul 135‑720, Korea 2 Department of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea 3 Department of Biomedical Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea 4 Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Korea 5 Biostatistics Collaboration Unit, Yonsei University College of Medicine, Seoul, Korea 6 AccurasysBio Co., Ltd., Seoul, Korea 7 Institute for Convergence Research and Education in Advanced Technology, Yonsei University, Seoul, Korea 13 Vol.:(0123456789) Cancer Immunology, Immunotherapy CT Computed tomography DEG Differentially expressed gene EUS Endoscopic ultrasound FNR False-negative rate value GI Gastrointestinal ID3 Inhibitor of DNA binding 3 IL-7R Interleukin-7 receptor mRNA-seq MRNA sequencing NPV Negative predictive value PBMC Peripheral blood mononuclear cells PDAC Pancreatic ductal adenocarcinoma PLD4 Phospholipase D4 PPV Positive predictive value qPCR Quantitative PCR RMA Robust multi-average RNA-seq RNA-sequencing scRNA-seq Single-cell RNA-seq WT Whole transcript Introduction Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and deadly disease with a mortality rate that closely parallels its incidence. Most studies report a mean 5-year-survival rate of < 10% [1–3] due to the fact that PDAC is resistant to therapies, and patients are diagnosed when cancer cells have already metastasized; most commonly, to the liver, lung and/or peritoneum. However, the diagnosis of PDAC lacks sensitive or specific biomarkers for early diagnosis [4, 5]. Currently used circulating biomarkers, such as CA19-9, lack sufficient sensitivity and specificity for diagnostic purposes. Therefore, establishing a non-invasive early detection method that can be used for cancer screening is important to improve the survival of patients with PDAC. However, despite numerous studies, there are still no PDAC-specific biomarkers with widespread clinical use [2, 5, 6]. Liquid biopsy holds great promise as a method for noninvasive cancer detection, particularly through the analysis of cell-free DNA, cell-free RNA fragments, extracellular vesicles (particularly exosomes), or circulating tumor cells [7, 8]. In cancer patients, these molecules and vesicles are released into the circulation by apoptosis, necrosis, and active secretion. However, the sensitive detection of usually very limited amounts of tumor-specific molecules in the blood of patients with early-stage cancers remains a challenge. Here, we used complementary approaches, bulk RNA sequencing (RNA-seq), and single cell RNA -sequencing (scRNA-seq) to investigate the transcriptional landscape of peripheral blood mononuclear cells (PBMCs) and to determine the specific immune cell markers that may help in the differential diagnosis of PDAC from other benign pancreatic 13 and gastrointestinal diseases. Although the peripheral blood immune landscapes of individual patients were quite heterogeneous, we selected some common PDAC-specific markers (e.g., IL-7R, PLD4, and ID3) from the blood samples of patients with PDAC that were identified to greatly contrast those of healthy controls and patients with benign pancreatic diseases, including chronic pancreatitis and cystic disease. Clinical performance was found to have remarkable value for the diagnosis and differential diagnosis of PDAC from other pancreatic diseases through examining these two cohorts. In addition, this study showcases differences in the immune landscape between PDAC and benign pancreatic diseases, which may provide a wealth of hypothesis-generating data to benefit pancreatic disease researchers. Materials and methods Study design and registration of clinical research This prospective case–control study was performed using two cohorts (Fig. 1). A biomarker panel formula was created using a development cohort, and its diagnostic performance was verified using a validation cohort. The study procedures were approved by the Institutional Review Board of Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea (no. 3-2018-0293 and 3-2020-0238) and registere (...truncated)