Exosome-like Vesicles of Uterine Aspirates Permit the Identification of Diagnostic and Stratification Biomarkers of Endometrial Cancer Eva Coll de la Rubia (Presenter) Vall Hebron Research Institute

Authors: Irene Campoy (1), Cristian P. Moiola (1), Marc Hirschfeld (2), Jasmin Asberger (2), Silvia Cabrera (3), Xavier Matias-Guiu (4), Eduard Sabidó (5), Antonio Gil-Moreno (1,3), Pierre Thibault (6), Eva Colás (1)
(1) Biomedical Research Group in Gynaecology, Vall Hebron Research Institute (VHIR), Universitat Autonoma de Barcelona, CIBERONC, Barcelona, Spain (2) Department of Obstetrics and Gynecology, University Medical Center, Albert-Ludwigs-University, Freiburg, Germany (3) Department of Gynecological Oncology, Vall Hebron University Hospital, Barcelona, Spain (4) Department of Pathology and Molecular Genetics/Oncologic Pathology Group, Hospital Universitari Arnau de Vilanova, Universitat de Lleida, IRBLleida, CIBERONC, Lleida, and Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain (5) Proteomics Unit, Universitat Pompeu Fabra (UPF) and Centre de Regulació Genòmica (CRG), Barcelona, Spain (6) Proteomics and Bioanalytical Mass Spectrometry research unit. IRIC (Institute of Research in Immunology and Cancer)

There is an urgent need to develop non-invasive tests that improve EC detection. In this study, we used exosome-like vesicles isolated from a uterine fluid to identify and verify protein signatures that can differentially diagnose EC subtypes. A discovery phase was performed using a super-SILAC approach on 60 patients (EC type 1, EC type 2, and controls), and a verification phase was done by targeted proteomics (SRM) in 107 patients. A 2-protein signature achieved an AUC=0.935 for EC diagnosis. In addition, we also report a new protein signature that can differentiate type1 versus type2 EC (AUC=0.932). This study has important implications in early detection of EC and in patient stratification.

Introduction

Endometrial cancer (EC) accounts for more than 10,000 deaths per year in the US alone. EC is divided into the more common and less aggressive type 1 and the type2. There is an urgent need to develop non-invasive tests that can provide early detection of EC and that can discriminate EC subtypes. This study focuses on the identification of protein biomarkers in exosome-like vesicles (ELVs) isolated from uterine aspirates. Uterine aspirates are collected by a minimally invasive procedure and it represents the ideal body fluid since it is the closest to the neoplasic endometrium cells.

Methods

Protein extracts from purified ELVs were obtained following ultracentrifugation of UAs from age-matched groups of control, type1 and type2 EC patients (10 patients/group). The quality of isolated ELVs was monitored by Nanoparticle Tracking Analysis, and immunoblots. To profile protein abundance across different groups, we develop a super-SILAC approach where ELV proteins from 3 different EC cell lines grown in heavy Lys and Arg amino acids were combined with ELV protein extracts of each patient. Proteins were separated by SDS-PAGE and 10 gel-isolated bands per patient were digested with trypsin and analyzed by Mass Spectrometry. From 325 proteins identified and quantified in more than 4 patients per group, we generated a list of 54 protein candidates that was further validated by selected reaction monitoring (SRM) in an independent cohort of 107 patients including type 1 EC (n=45) EC, type 2 EC (n=21), and healthy individuals (n=41). A total of 86 unique peptides matching the proteins of interest were monitored. Isotopically-labelled peptides were spiked in each sample as peptide standards, and protein quantitation was performed using a QTRAP 5500 Sciex instrument.

Results

Our targeted mass spectrometry approach confirmed that ELVs from uterine aspirates contain proteins that can discriminate between cancer patients and healthy individuals, and can classify EC in the different subtypes. A 2-protein signature achieved an AUC=0.935 for EC diagnosis. In addition, we also report a new protein signature that can differentiate type1 versus type2 EC (AUC=0.932). This study has important implications in early detection of EC and in patient stratification.

Conclusions & Discussion

A targeted mass spectrometry approach defines protein signatures for endometrial cancer diagnosis in ELVs isolated from uterine aspirates.