title: Prostate cancer biomarker annexin A3 detected in urines obtained following digital rectal examination presents antigenic variability.
abstract: OBJECTIVE
  Annexin A3 (ANXA3) is a potential marker for prostate cancer (PCa). We aimed to develop robust immunoassays suitable for quantifying ANXA3 in urine samples obtained following digital rectal examination (DRE) in order to facilitate the diagnostic performance evaluation of this marker.
  
  
  METHODS
  Anti-ANXA3 monoclonal antibodies were generated and their epitopes mapped. Two different ANXA3 assay prototypes were established on the VIDAS® automated immunoanalyser and analytical validation was carried out using post-DRE urine samples obtained from patients with PCa (n=23) or benign prostate hyperplasia (n=31).
  
  
  RESULTS
  The assays had the same capture antibody (TGC44) but different detection antibodies (13A12 or 5C5), recognizing novel distinct epitopes. Both had a lower limit of quantification <1ng/mL and were highly specific for ANXA3, not cross-reacting with other annexins. Interassay imprecision was ≤11% and ≤15% for 13A12 and 5C5 assays, respectively. Surprisingly, a total lack of correlation was observed between ANXA3 levels measured by these two assays in post-DRE urines, indicating detection of distinct antigenic variants. Two freeze-thaw cycles did not affect analyte stability in either assay, whereas a lack of stability of antigenic variants was observed when samples were stored at -80°C for 1month.
  
  
  CONCLUSIONS
  Two different antigenic variants of ANXA3 are present in post-DRE urines and their clinical significance for diagnosis of prostate cancer should be further investigated. These variants are not stable over time in samples preserved at -80°C. Until this issue is resolved, ANXA3 should only be measured in freshly collected samples.
authors: Biomarkers Department, bioMérieux, Marcy l'étoile, France.
authors: Céline
authors: C
authors: Hamelin-Peyron
authors: Unité Médicale d'Oncologie Moléculaire et Transfert, Service de Biochimie biologie moléculaire Sud, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, Pierre Bénite, France; Faculté de Médecine Lyon Est, Université Lyon I, Centre de Recherche en Cancérologie de Lyon, INSERM 1052, CNRS 5286, Centre Léon Bérard, Lyon, France; Laboratoire Commun de Recherche HCL-bioMérieux, Pierre Bénite, France.
authors: Virginie
authors: V
authors: Vlaeminck-Guillem
authors: Department of Clinical Trials, bioMérieux, 69280 Marcy l'étoile, France.
authors: Hader
authors: H
authors: Haïdous
authors: ProteoSys AG, Mainz, Germany.
authors: Gerhard P
authors: GP
authors: Schwall
authors: ProteoSys AG, Mainz, Germany.
authors: Slobodan
authors: S
authors: Poznanović
authors: Biomarkers Department, bioMérieux, Marcy l'étoile, France.
authors: Emmanuelle
authors: E
authors: Gorius-Gallet
authors: Biomarkers Department, bioMérieux, Marcy l'étoile, France; Immunoassay R&D, bioMérieux, Marcy l'étoile, France.
authors: Sandrine
authors: S
authors: Michel
authors: Data and Knowledge Laboratory, bioMérieux, 69280 Marcy l'étoile, France.
authors: Audrey
authors: A
authors: Larue
authors: Immunoassay R&D, bioMérieux, Marcy l'étoile, France.
authors: Michèle
authors: M
authors: Guillotte
authors: Laboratoire Commun de Recherche HCL-bioMérieux, Pierre Bénite, France; Département d'Urologie, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, Pierre Bénite, France; Institut de Génomique Fonctionnelle de Lyon, Université Lyon I-CNRS-INRA, Ecole Normale Supérieure de Lyon, France.
authors: Alain
authors: A
authors: Ruffion
authors: Biomarkers Department, bioMérieux, Marcy l'étoile, France.
authors: Geneviève
authors: G
authors: Choquet-Kastylevsky
authors: Biomarkers Department, bioMérieux, Marcy l'étoile, France; Immunoassay R&D, bioMérieux, Marcy l'étoile, France. Electronic address: yasemin.ataman@biomerieux.com.
authors: Yasemin
authors: Y
authors: Ataman-Önal
mesh_terms: D000818:Animals; D017318:Annexin A3 / Q000652:urine*; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry; D000912:Antibodies, Neoplasm / Q000737:chemistry; D014408:Biomarkers, Tumor / Q000652:urine*; D015925:Cryopreservation; D051517:Digital Rectal Examination; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000652:urine; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009363:Neoplasm Proteins / Q000652:urine*; D011471:Prostatic Neoplasms / Q000175:diagnosis* / Q000652:urine*; D055550:Protein Stability
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D000912:Antibodies, Neoplasm; D014408:Biomarkers, Tumor; D000939:Epitopes; D009363:Neoplasm Proteins; D017318:Annexin A3
keywords: ANXA3; Antigenic variant; Prostate cancer; Sandwich immunoassay; Urinary biomarker