A Plasma-Derived Protein-Metabolite Multiplexed Panel for Early-Stage Pancreatic Cancer.

Johannes F Fahrmann ,Ayumu Taguchi,Michele Yip-Schneider,Nikul Patel,Michela Capello,Jianjun Zhang,Eunice Murage,Ziding Feng,Anirban Maitra,Paul Brennan,Jody Vykoukal,Lenka Foretová ,Deepali L Kundnani ,Ivana Holcátová ,Vladimí­r Janout ,Eleonóra Fabiánovà ,Ghislaine Scélo ,Leonidas E Bantis ,Randall E Brand ,Jennifer B Dennison ,C Max Schmidt ,Samir Hanash

doi:10.1093/jnci/djy126

Abstract

BackgroundWe applied a training and testing approach to develop and validate a plasma metabolite panel for the detection of early-stage pancreatic ductal adenocarcinoma (PDAC) alone and in combination with a previously validated protein panel for early-stage PDAC.MethodsA comprehensive metabolomics platform was initially applied to plasmas collected from 20 PDAC cases and 80 controls. Candidate markers were filtered based on a second independent cohort that included nine invasive intraductal papillary mucinous neoplasm cases and 51 benign pancreatic cysts. Blinded validation of the resulting metabolite panel was performed in an independent test cohort consisting of 39 resectable PDAC cases and 82 matched healthy controls. The additive value of combining the metabolite panel with a previously validated protein panel was evaluated.ResultsFive metabolites (acetylspermidine, diacetylspermine, an indole-derivative, and two lysophosphatidylcholines) were selected as a panel based on filtering criteria. A combination rule was developed for distinguishing between PDAC and healthy controls using the Training Set. In the blinded validation study with early-stage PDAC samples and controls, the five metabolites yielded areas under the curve (AUCs) ranging from 0.726 to 0.842, and the combined metabolite model yielded an AUC of 0.892 (95% confidence interval [CI] = 0.828 to 0.956). Performance was further statistically significantly improved by combining the metabolite panel with a previously validated protein marker panel consisting of CA 19–9, LRG1, and TIMP1 (AUC = 0.924, 95% CI = 0.864 to 0.983, comparison DeLong test one-sided P= .02).ConclusionsA metabolite panel in combination with CA19-9, TIMP1, and LRG1 exhibited substantially improved performance in the detection of early-stage PDAC compared with a protein panel alone.

Highlights

Cells were grown in 1ml of DMEM + 10% FBS in 12-well dishes (Costar) to reach a 70% (5080%) confluency, 24 hours post initial seeding
Samples were separated using the following gradient profile: for the Hydrophilic Interaction Liquid Chromatography (HILIC) separation a starting gradient of 95% B and 5% D was increase linearly to 70% A, 25% B and 5% D over a 5min period at 0.4mL/min flow rate, followed by 1 min isocratic gradient at 100 % A at 0.4mL/min flow rate
Detected features exhibiting a relative standard deviation (RSD) less than 30 in quality control samples were considered for further statistical analysis

Summary

Introduction

Cells were grown in 1ml of DMEM + 10% FBS in 12-well dishes (Costar) to reach a 70% (5080%) confluency, 24 hours post initial seeding. The supernatant was further diluted with 90μL of 1:3:2 100mM ammonium formate, pH3 (Fischer Scientific): acetonitrile: 2-propanol and transferred to a 384-well microplate (Eppendorf) for lipids analysis using LCMS.

Results

Conclusion