Objective
The purpose of this study was to establish normal ranges for human epididymis protein 4 (HE4) serum levels in healthy women.
Study Design
HE4 levels were measured in healthy women and analyzed by age, menopausal status, and pregnancy status. Upper 95th percentiles were determined for normal ranges.
Results
Serum samples from 1101 healthy women and 67 pregnant women were analyzed. Above the age of 40 years significant elevations in HE4 concentrations emerged with advancing age. The upper 95th percentile for HE4 levels was 89 pmol/L for premenopausal women, 128 pmol/L for postmenopausal women, and 115 pmol/L for all women. There was a significant difference in the median serum HE4 levels in premenopausal women (46.6 pmol/L) compared with postmenopausal women (57.6 pmol/L; P < .001). In pregnant women, median HE4 concentrations were significantly lower than their premenopausal counterparts ( P < .001).
Conclusion
HE4 serum concentrations vary significantly on the basis of age. These variations must be considered when the upper limit of normal for HE4 is determined.
Over the past 3 decades cancer antigen 125 (CA125) has provided a biomarker for the monitoring of women who receive a diagnosis of ovarian cancer during treatment and before disease recurrence. CA125 has also been studied extensively for a possible role in early detection and screening for ovarian cancer. Although promising, the role of CA125 in this area has yet to be defined.
Although CA125 is the current standard biomarker for the management of ovarian cancer, it is not without limitations. CA125 is elevated in only 50-60% of early stage cases and is not expressed by up to 20% of all ovarian cancers. CA125 specificity is also limited because levels can be elevated in several benign gynecologic disorders, such as endometriosis, pelvic inflammatory disease and benign neoplasms of the ovaries and uterus. CA125 can be elevated in many common nongynecologic conditions such as congestive heart failure, hepatic disease, and inflammatory diseases that affect pleural, peritoneal, and pericardial surfaces.
The novel serum biomarker human epididymis protein 4 (HE4) has been shown to be over-expressed in serous, endometrioid, and clear cell epithelial ovarian cancers. HE4 has also been demonstrated to be a sensitive and specific serum biomarker for ovarian cancer that is elevated less frequently by benign conditions that occur in premenopausal women. Recently it was shown that the addition of HE4 to CA125 increased the sensitivity and specificity of either marker alone for the detection of ovarian cancer.
The HE4 protein is a whey acid protein with a 4 disulfide core that originally was isolated in epithelial cells of the human epididymis and is expressed in numerous tissues throughout the body, including the female reproductive tract. Importantly, HE4 circulates in the bloodstream and can be detected through an immunosorbent assay (EIA) with a monoclonal mouse antibody directed at an HE4 epitope.
In 2009, the United States Food and Drug Agency (FDA) approved HE4 for the monitoring of women who have received a diagnosis of epithelial ovarian cancer with similar indications to the use of CA125. To date, however, there are no large trials that are examining serum HE4 levels in healthy premenopausal and postmenopausal women and healthy pregnant women. The upper 95th percentile of 150 pmol/L for both premenopausal and postmenopausal women is reported in the FDA package insert for the HE4 EIA Kit (Fujirebio Diagnostics Inc, Malvern, PA). This value does not take into consideration patient age or menopausal status; what actually constitutes normal levels in healthy women and whether these levels vary by subgroups have not been clearly evaluated and published. The purpose of this study was to examine serum levels of HE4 in healthy women on the basis of age, menopausal status, and pregnancy status to refine normative data for this novel biomarker.
Materials and Methods
A metaanalysis was performed with data collected in 3 independent trials that measured HE4 levels in healthy women with the use of the HE4 EIA kit (Fujirebio Diagnostics Inc). Data from the following studies were included: (1) An institutional review board–approved study at Women and Infant’s Hospital (WIH) that obtained residual serum from healthy premenopausal women (n = 101) and postmenopausal women (n = 91) and residual serum samples from women during their first, second, and third trimesters of pregnancy (n = 67); (2) an institutional review board–approved trial through M.D. Anderson Cancer Center that enrolled postmenopausal women in a multicenter low-risk ovarian cancer screening trial through an ovarian Specialized Programs of Research Excellence grant (SPORE P50), from whom 143 samples were obtained; and (3) Fujirebio Diagnostics Inc obtained serum samples that had been collected from institutional review board–approved repositories that included samples from 374 premenopausal and 392 postmenopausal healthy women that had been banked (protocol FDI-53). Institutional review board review of the FDI-53 protocol found that the data were unlinked and deidentified and therefore did not require approval. All blood samples were centrifuged, and the serum was collected and frozen at −80°C until testing.
Menopausal status was determined with the following criteria for each of the individual studies: For the WIH samples, women who were ≥55 years old were considered postmenopausal, and women who were ≤45 years old were considered premenopausal. No samples were obtained from WIH for women between the ages of 46 and 54 years. All women who were entered from the M.D. Anderson Cancer Center trial were postmenopausal, which was determined by a medical interview and a history of amenorrhea for >1 year. The menopausal status for the patient samples that were obtained from the Fujirebio clinical trial sample banks was determined through medical history or chart review, which was reported by the serum banks that supplied these samples. Serum levels for HE4 were tested at each institution with an HE4 EIA assay kit (Fujirebio Diagnostics Inc).
Statistical analysis
The primary endpoint of this study was to describe serum concentrations of HE4 (picomoles per liter) to determine the normal ranges in healthy premenopausal and postmenopausal women and pregnant women. In each group, the median, range, mean, standard deviation, percent coefficient of variation, and the 90th, 95th, 97.5th and the 99th percentile for serum HE4 levels were determined. Normal serum levels for HE4 were defined with a cut point at the upper 95th percentile. Probability values for medians were derived with a continuity corrected Pearson’s chi-square median test and the Wilcoxon rank sum method. Log base 2–transformed scatter plots also were generated for HE4 levels by decadal age group and menopausal status; standard scatter plots were generated for HE4 levels in pregnant women. All HE4 values were derived by cubic spline interpolation.
Results
This study included serum samples from 1168 women with a total of 1101 healthy, nonpregnant women and 67 pregnant women. There were 475 premenopausal women (101 women from WIH Rhode Island; 374 women from Fujirebio Diagnostics Inc), with a mean age of 34.3 years (range, 15–57 years) and 626 postmenopausal women (91 women from WIH; 143 women from M.D. Anderson Cancer Center; 392 women from Fujirebio Diagnostics Inc), with a mean age of 62.8 years (range, 34–94 years). In addition, the study included 67 healthy pregnant women from WIH, with 25 women in the first trimester, 25 women in the second trimester, and 17 women in the third trimester of pregnancy.
HE4 levels by menopausal status
The mean, standard deviation, median and ranges for serum HE4 levels by age groups and menopausal status are shown in Table 1 .
| Variable | Age group, y | ||||||
|---|---|---|---|---|---|---|---|
| Premenopausal | Postmenopausal | ||||||
| <30 | 30-39 | ≥40 | <60 | 60-69 | 70-79 | ≥80 | |
| n | 170 | 159 | 146 | 256 | 236 | 102 | 32 |
| Mean, pmol/L | 55.5 | 49.9 | 61.4 | 56.0 | 66.9 | 76.6 | 137.8 |
| SD, pmol/L | 66.8 | 28.6 | 54.6 | 26.1 | 49.3 | 34.1 | 87.7 |
| Median, pmol/L | 46.2 | 43.5 | 50.5 | 50.7 | 59.9 | 66.9 | 113.4 |
| Range, pmol/L | 24.5-656.4 | 22.4-293.5 | 22.6-645.6 | 18.7-285.8 | 12.0-690.8 | 21.7-228.9 | 48.5-430.8 |
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