Evaluation of tumor marker HE 4 assay on the Elecsys 2010 analyzer

© 2014 Jozo Æoriæ et al.; licensee University of Sarajevo Faculty of Health Studies. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. UNIVERSITY OF SARAJEVO FACULTY OF HEALTH STUDIES ABSTRACT


INTRODUCTION
Epithelial ovarian cancer (EOC) is the fourth most common cause of cancer mortality in women and the leading cause of death from gynecological malignancies.Th e majority of women have advancedstage disease at initial diagnosis and a 5-year survival of 10-30 %.Th e median survival after recurrence is only 2 years despite the advances in chemotherapy and secondary debulking surgery in selected patients (1).Th e human epididymis protein 4 (HE4, also known as WFDC2) belongs to the family of whey acidic four-disulfi de core (WFDC) proteins with suspected trypsin inhibitor properties.Th e corresponding gene codes for a 13 kD protein.In its mature glycosylated form the protein has a molecular weight of approximately 20-25 kD and consists of a single peptide chain containing two WFDC domains (2).HE4 was fi rst determined in the epithelium of the distal epididymis.It showed low expression in epithelia of respiratory and reproductive tissues including ovary, but high expression in ovarian cancer tis-sue.High secreted levels can also be found in the serum of ovarian cancer patients (3).Th e measured HE4 value of a patients sample can vary depending on the testing procedure used.Th e laboratory fi nding must therefore always contain a statement on the HE4 assay method used.HE4 values determined on patient samples by diff erent testing procedures cannot be directly compared with one another and could be the cause of erroneous medical interpretations (4,5).Th e assay is to be used as aid in monitoring recurrence or progressive disease in patients with epithelial ovarian cancer.Serial testing for patient HE4 assay values should be used in conjunction with other clinical methods used for monitoring ovarian cancer (6).Moore and colleagues (7) have reported that the serum biomarker algorithm ROMA (Risk of Ovarian Malignancy Algorithm), which utilizes the dual marker combination of HE4 and CA125, along with menopause status, shows good performance for risk prediction of ovarian malignances (7).

METHODS
Blood samples were collected from 36 female patients with epithelial ovarian cancer (mean age: 56 years, range 48-69 years).Control samples (N=20) were collected from healthy females.Th e blood was collected into the non-heparinized tubes and kept 30 min at room temperature, then centrifuged for 15 min at 3000 rpm.Th e sera were then stored at -20 o C until use.Th e serum levels of HE4 were determined using a chemiluminescent enzyme immunoassay on the Elecsys 2010 Analyzer (Roche Diagnostics, Germany) according to manufacturer, s instructions.Th e HE4 assay is a two-step immunoassay for quantitative determination of HE4 antigen in human serum using CMIA technology.In the fi rst step, sample and 2H5 anti-HE4 coated paramagnetic micropaticles are combined.HE4 antigen present in the sample binds to the anti-HE4 coated microparticles.After washing, 3D8 anti-HE4 acridinium-lebeled conjugate is added to create a reaction mixture in the second step.Following another wash cycle, pre-trigger and trigger solutions are added to the reaction mixture.Th e resulting chemiluminiescent reaction is measured as relative light units (RLUs).A direct relationship exists between the amount of HE4 antigen in the sample and RLUs.For quality control ElecsysPreciControl HE4 1 and 2 (Roche Diagnostics, Germany) were used.Statistical analysis Th e data were analyzed in SPSS version 18 (Chicago, IL, USA).Descriptive statistics and Pearson correlation were used.Th e diff erence between groups were considered signifi cant if p<0.05.

RESULTS
Th e results of the HE4 assay precision within-run and between-run analyses are showed in Table 1.Th e coeffi cients of variation (CV%) values for the within-run precision were 6.9 -8.2 %, with those for between-run ranging 7.9 -9.8%.Th e results variation was greater at lower concentrations.Th e HE4 assay accuracy results are presented in Table 2. Th e statistically signifi cant correlation between labeled and measured HE4 values was obtained (r=0.99),presented by the following equation: y= -1.20+0.99x,where y represents the measured HE4 levels, and x labeled HE4 levels.An intercept (-1.20) presents the systematic errors of the method, which was not statistically signifi cant (p>0.05).

DISCUSSION
Human Epididymis Protein 4 (HE4) is a 25 KD single peptide chain with a low expression in epithelia of repertory and reproductive tissues, but highly expressed in ovarian cancer tissue.HE4 is a novel serological marker used especially for ovarian cancer diagnosis because of its high sensitivity (8).Th e percentage of increase in HE4 values has been used as an aid in monitoring recurrence or progressive disease in patients with invasive epithelial ovarian cancer.Th ere is no clinically accepted cut-off for use in monitoring cancer progression in epithelial ovarian cancer (9).Currently, several biomarker panels are being evaluated in increase the sensitivity and specifi city of ovarian cancer diagnosis.Th e combination of CA125 and HE4 has been evaluated to improve ovarian cancer diagnosis.Th e data suggests that by combining these markers the predictive accuracy in ovarian malignancy is better than by applying any of the markers alone (10).As a single tumor marker, HE4 had the highest sensitivity for detecting ovarian cancer, especially in stage I disease.Th e analysis of HE4 by competitive chemiluminescent immunoassay on Elecsys 2010 analyser is rapid and easy to perform.Th e linearity range of assay is from 20 to 1000 pmol/L, which is acceptable for routine clinical use.Th e CV for precision in this assay is not greater than 10% at the lowest measurable concentration.Th e obtained CV% values for precision were for the within-run precision were 6.9 -8.2 %, with those for between-run ranging 7.9 -9.8%.

TABLE 1 .
Within-run and between-run precision of HE4 assay
*Mean of two measurements of calibrators

TABLE 3 .
HE4 measurements EOC, epithelial ovarian cancer 7. Moore RG, McMeekin DS, Brown AK.A novel multiple marker bioassay utilizing HE4 and CA125 for the prediction of ovarian cancer in patients with a pelvic mass.Gynecol Oncol 2009;112:40-6.8. Galgano MT, Hampton GM, Frierson HF.Comprehensive analysis of HE4 expression in normal and malignant human tissues.Mod Pathol 2006; 19:847-53.9. Allard J, Somers E, Theil R, Use of a novel biomarker HE4 for monitoring of patients with epithelial ovarian cancer ASCO Annual Meeting Proceedings 2008;26:5535.10.Montagnana M, Danese E, Ruzzenente O.The ROMA for estimating the risk of epithelial ovarian cancer in women presenting with pelvic mass: is it really useful.Clin Chem Lab Med 2011; 49(3):521-525.