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Samples were collected from all the patients prior to surgery using blood collection tubes without anticoagulants. Each blood sample was centrifuged at 3000 rpm and stored at −80 °C until use. Tumor markers including CA125 (ARCHITECT CA125 II, Abbott Japan LLC, Tokyo, Japan), CA19-9 (CL AIA-PACK^® SLa, Tosoh Corporation, Tokyo, Japan), CEA (CL AIA-PACK^® CEA, Tosoh Corporation, Tokyo, Japan), and HE4 (ARCHITECT HE4, Abbott Japan LLC, Tokyo, Japan) were measured using a chemiluminescence immunoassay, according to the manufacturer’s instructions. Serum samples in dry ice were transported to the Tosoh diagnostics product divisions (Tosoh Corporation, Kanagawa, Japan), and CA19-9 and CEA concentrations were determined immediately. HE4 and CA125 (ARCHITECT CA125 II) were measured at BML INC., Tokyo, Japan. In case of CA125 and HE4 levels under the limit, we recorded the lower limit of calibration as 1 (U/mL) and 20 (pmol/L), respectively. Measurements were performed by clinical laboratory technologists who were blinded to the study.

The sampling of the peripheral blood was performed under the standard procedure from the cubital vein, using 7.5 mL tubes of S-Monovette^® Serum Gel (Sarstedt) preoperatively, once or twice during the follow-up period and always when recurrence was suspected. The samples were transported to the Department of Clinical Biochemistry UH Brno, where the serum was separated by centrifugation, and the samples were analyzed either immediately (CA125, HE4) or stored frozen at −80 °C until analysis (DJ1 and L1CAM).
The quantitative assessments of L1CAM and DJ1 levels were performed by enzyme-linked immunosorbent assay (ELISA) using ELISA reader iMARK (Bio-Rad). For L1CAM, kit CN MBS 2023094 (MYBioSource, USA) was used. DJ1 serum levels were measured using kit CN CY-9050V2 (CircuLex MBL, UK). The serum concentrations of HE4 and CA125 were determined using quantitative, chemiluminescent microparticle immunoassay (CMIA) on the analyzer Architect i2000 (Abbott, Abbott Laboratories, USA). For CA125 measurement, the diagnostic set ARCHITECT Ca125 II (CN 2K45-24, Abbott) was used. HE4 serum level assessments were performed using the diagnostic set ARCHITECT HE4 (CN 2P51-25, Abbott).