Next generation sequencing of the cellular and liquid fraction of pancreatic cyst fluid supports discrimination of IPMN from pseudocysts and reveals cases with multiple mutated driver clones: First findings from the prospective ZYSTEUS biomarker study

Abstract

Approximately half of all pancreatic cysts are neoplastic, mainly comprising intraductal papillary mucinous neoplasms (IPMN), which can progress to invasive carcinoma. Current Fukuoka guidelines have limited sensitivity and specificity in predicting progression of asymptomatic pancreatic cysts. We present first results of the prospective ZYSTEUS biomarker study investigating (i) whether detection of driver mutations in IPMN by liquid biopsy is technically feasible, (ii) which compartment of IPMN is most suitable for analysis, and (iii) implications for clinical diagnostics. Twenty-two patients with clinical inclusion criteria were enrolled in ZYSTEUS. Fifteen cases underwent endoscopic ultrasound (EUS)-guided fine-needle aspiration and cytological diagnostics. Cellular and liquid fraction of the cysts of each case were separated and subjected to deep targeted next generation sequencing (NGS). Clinical parameters, imaging findings (EUS and MRI), and follow-up data were collected continuously. All IPMN cases (n = 12) showed at least one mutation in either KRAS (n = 11) or GNAS (n = 4). Three cases showed both KRAS and GNAS mutations. Six cases harbored multiple KRAS/GNAS mutations. In the three cases with pseudocysts, no KRAS or GNAS mutations were detected. DNA yields were higher and showed higher mutation diversity in the cellular fraction. In conclusion, mutation detection in pancreatic cyst fluid is technically feasible with more robust results in the cellular than in the liquid fraction. Current results suggest that, together with imaging, targeted sequencing supports discrimination of IPMN from pseudocysts. The prospective design of ZYSTEUS will provide insight into diagnostic value of NGS in preoperative risk stratification. Our data provide evidence for an oligoclonal nature of IPMN.

Molecular analysis of cyst fluids improves the diagnostic accuracy of pre-operative assessment of pancreatic cystic lesions

“…Past studies [36][37][38][39] and data from our retrospective and prospective cohorts show that NGS analyses can be performed successfully on cytology smears or even cell-free cyst fluid. When comparing cfDNA samples with DNA extracted from smear samples, we found higher mean allelic frequencies of gene variants in the smear samples, probably due to higher cellularity.…”

KRAS/GNAS‐testing by highly sensitive deep targeted next generation sequencing improves the endoscopic ultrasound‐guided workup of suspected mucinous neoplasms of the pancreas

“…The high sensitivity and specificity of the KRAS/GNAS ‐testing by dtNGS, in which all DNA‐molecules are labeled with individual barcodes before amplification, 23 is probably due to the very low limit of detection down to 0.01% VAF. Therefore, it might be more sensitive than previously used NGS approaches with a limit of detection of 3% to 5% or Sanger sequencing with a limit of detection of 10% to 20% VAF 21 .…”

“…The wall of the PCL/MPD was only passed once for needle access to minimize PCL/MPD injury and to reduce potential access tract cell seeding (Figure 1). As we have discussed in our feasibility study, 23 a minimum cyst size of 10 to 15 mm was necessary to obtain an aspirated volume of at least 1000 μL for analysis of both CEA (≥500 μL) and cfDNA (≥500 μL). The remaining content of the hollow needle was used for cytological smear preparation.…”

“…The tube was transferred at ambient temperatures within 1 hour for further workup to the Center for Molecular Pathology, Institute of Pathology at the University Hospital of Heidelberg. Approaches for cfDNA extraction, library preparation, semiconductor sequencing and data analysis were performed as previously described 23 . DtNGS was performed using the Oncomine Colon cfDNA Assay (Thermo Fisher, Waltham, MA) of 14 known gastrointestinal cancer genes covering >240 hotspots: AKT1 , BRAF , CTNNB1 , EGFR , ERBB2 , FBXW7 , GNAS , KRAS , MAP2K1 , NRAS , PIK3CA , SMAD4 , TP53 , and APC .…”

Observation or resection of pancreatic intraductal papillary mucinous neoplasm: An ongoing tug of war

“…Thus, other biomarkers sampled from cyst fluid or other sources are emerging and of interest. Particular interest is expressed in next generation sequencing of pancreatic cystic fluid[38-41]. Investigators have reported KRAS / GNAS mutations to be present in 100% of IPMNs[39] and to be highly sensitive and specific (89% and 100%, respectively) for IPMNs and mucinous cystic lesions[39].…”