Multi-protein panel in pancreatic cyst fluid for improved risk stratification for pancreatic cancer

Abstract Pancreatic cystic lesions (PCLs) are the sole radiologically recognisable and highly heterogeneous precursors of pancreatic cancer (PC). The malignant potential of PCLs is inferred from their types, as determined by empirical clinical practice guidelines; however, accurate risk stratification of patients preoperatively presents an unmet clinical need. We performed deep proteomic profiling of pancreatic cyst fluid (PCyF) and identified a first-of-its-kind multi-protein (n=89) panel termed “ASSIGN1” - Early diagnosis and detection of pAncreatic cySt malignancy SIGNature. ASSIGN1 was used for the development and validation of a support vector machine-based model for predicting malignant potential (based on malignancy risk score, zero to one) of individual PCLs using discovery/training and validation/test cohorts. The diagnostic accuracy of the model was evaluated based on histopathology of resected cysts using sensitivity, specificity and area under the receiver-operating-characteristic (AUROC) curve measures and compared to Fukuoka guidelines-based preoperative assessment. ASSIGN1-based malignancy risk score was a cyst type-independent and accurate (sensitivity=1.00, specificity=1.00 and AUROC=1.00) predictor of (i) pancreatic carcinoma and (ii) malignant potential of PCLs, which outperformed international consensus Fukuoka guidelines-based preoperative assessment (sensitivity=1.00; specificity=0.38; AUROC=0.71). Our findings demonstrated that ASSIGN1 holds promise to replace current preoperative laboratory tests, complement existing standard-of-care practices and improve preoperative diagnosis of PCLs and early detection of PC. Competing Interest Statement D. Manolis, D. P. O'Brien, B. M. Kessler, D. K. Chang, A. Maraveyas and L.L. Nikitenko have a patent pending to the University of Hull. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding Statement The project study was supported in part by the Cancer Research UK Early Detection and Diagnosis Primer Award (grant EDDPMA-May22/100018 EARLY DIAPAC: EARLY DIAgnosis of Pancreatic Cancer combined proteomics and genomics testing of pancreatic cyst fluid to LN, HK, DC, DO'B, BMK and AM) and the Working Independently to Support Hull Hospitals (WISHH) Charity (grant: Quantitative proteomic analysis of pancreatic cyst fluid for early detection of cancer using University of Hull HPC Viper to LN, DM and AM). We are indebted to Dr Aseem Allam and his family for the support and funding of the umbrella biobank study (TEM-PAC; ClinicalTrials.gov registration: NCT03536793, adopted by the NIHR; Materials and methods), which has generated the clinical material accessed by the investigators of the EARLY DIAPAC consortium. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: PCyF samples (n=31; n=15 female; n=16 male; average age at diagnosis - 63 years; Supplementary Table S1) were collected in 2018-2020 for the ethically-approved Tumour Regulatory Molecules as Markers of Malignancy in Pancreatic Cystic Lesions study (TEM-PAC; ClinicalTrials.gov registration: NCT03536793; Integrated Research Application System (IRAS) Project ID: 236870; Research Ethics Committee Reference 18/LO/0736; 24.10.2018-28.02.2029; adopted by the National Institute for Health and Care Research (NIHR), portfolio ID 55297) in the Queens Centre for Oncology and Haematology, Castle Hill Hospital (Hull, UK) and made available for our single center retrospective study through IRAS Substantial Amendment 002 (15.01.2021). There was no perceived selection bias in patient recruitment. All individuals who agreed to participate provided informed consent. PCyF samples were obtained either preoperatively (n=22) using EUS or during surgery (n=9) by FNA along with corresponding follow-up details (clinical, imaging and diagnostic histopathological analysis; Supplementary Table S1). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All MS raw files were deposited in the PRoteomics IDEntifications (PRIDE; RRID: SCR_003411) Archive31 under the unique identifier PXD045289.