ORIGINAL STUDY

HE4 as an Early Detection Biomarker of Epithelial

Ovarian Cancer
Investigations in Prediagnostic Specimens From the Janus Serumbank
Randi Elin Gislefoss, PhD,* Hilde Langseth, PhD,* Nils Bolstad, MD,
Kjell Nustad, MD, PhD, and Lars Mrkrid, MD, MSc, PhD

Objectives: Epithelial ovarian cancer is characterized by nonspecific signs and clinical

symptoms arising at late stages. Early detection is therefore important and may significantly
improve the survival rate. Cancer antigen 125 (CA125) has been the most extensively
studied serum biomarker in epithelial ovarian cancer, but low specificity limits its usefulness. A relatively novel biomarker, human epididymis protein 4 (HE4), has shown promise
in early detection of the disease. The aim of this study was to investigate how early the tumor
marker increases before diagnosis.
Methods/Materials: A nested case-control design was used to evaluate the performance of
HE4 and CA125 in prediagnostic serum samples from the Janus Serumbank. Serial specimens
from 120 women with invasive epithelial ovarian cancer were compared with healthy controls.
Serum level of CA125, HE4, and cotinine was measured. Spearman correlation and multiple
linear regression analyses were used to investigate impact of smoking, age, storage time, and lag
time (time from sampling until date of diagnosis).
Results: Spearman correlation showed a strong positive correlation between HE4 and
smoking in both cases and controls. Multiple linear regression analyses for pairwise differences between case and control showed that serum level of HE4 and CA125 was significantly increased (P = 0.002 and P G 0.001, respectively) 2 years before diagnosis and that
CA125 also was significantly increased up to 4 years before diagnosis (P = 0.002).
Conclusions: The present study showed that a difference between cases and controls in
serum concentration of HE4 seemed to be increased 2 years before diagnosis and that
CA125 was increased until 4 years before diagnosis.
Key Words: Ovarian cancer, Tumor marker, HE4, CA125, Early detection, Biobank
Received April 7, 2015, and in revised form June 10, 2015.
Accepted for publication June 10, 2015.
(Int J Gynecol Cancer 2015;25: 1608Y1615)

*Department of Research, Institute of Population-based Cancer

Research, Cancer Registry of Norway, Oslo, Norway; and Department
of Medical Biochemistry, Oslo University Hospital, RikshospitaletRadiumhospitalet, Oslo, Norway.
Address correspondence and reprint requests to Randi Elin Gislefoss,
PhD, Department of Research, Institute of Population-based
Copyright * 2015 by IGCS and ESGO
ISSN: 1048-891X
DOI: 10.1097/IGC.0000000000000532

1608

Cancer Research, Cancer Registry of Norway, Postbox 5313,

Majorstuen, N-0304 Oslo, Norway.
E-mail: randi.gislefoss@kreftregisteret.no.
Ethics: The study was approved by The Norwegian Regional Committees for Medical and Health Research REK 2009/1478.
Funding: This work was funded by The Norwegian Cancer Society.
Assay kits for the measurement of were provided free of charge
by Fujirebio Diagnostics.
The authors declare no conflicts of interest.

International Journal of Gynecological Cancer

& Volume 25, Number 9, November 2015

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International Journal of Gynecological Cancer

& Volume 25, Number 9, November 2015

varian cancer is the sixth most common cause of cancerrelated death in women worldwide.1 In Scandinavia, the
incidence has been relatively stable the last 20 years and is
among the highest in the world.2 The disease is characterized
by vague and not easily recognizable physical symptoms
(bloated abdomen, pelvic and abdominal pain, and urinary
urgency). Ovarian cancer is typically diagnosed at advanced
stage, and the survival is relatively poor. In Europe and the
United States, patients diagnosed in stage I have a 5-year
survival rate of 90%. However, almost 90% of the patients
are diagnosed in advanced stages (International Federation of
Gynecology and Obstetrics IIIYIV), with an overall 5-year
survival rate of less than 30%.3 Inheritance of mutations in
the BRCA1 and BRCA2 gene is associated with increased
risk of ovarian cancer.4 Other factors that increase the risk
of ovarian cancer are age, obesity, and hormone replacement
therapy. Reproductive history with full-term pregnancy,
breastfeeding, and use of contraceptives reduces the risk
of developing ovarian cancer.5 The treatment of epithelial
ovarian cancer (EOC) has improved over the last decades by
more effective surgery in combination with optimized chemotherapy. Despite improvements in the treatment, the overall
cure rate is poor.6 Prognostic and predictive markers are
needed to optimize and personalize therapy.
The high survival rate in patients diagnosed at an early
stage warrants the need for improved diagnostic tools for early
detection. Measurement of the widely used serum tumor
marker cancer antigen 125 (CA125), followed by ultrasound
imaging of the ovaries, has been suggested as a screening
tool.7 CA125 is a high-molecular-weight glycoprotein,8
which is highly expressed in normal epithelium in the female
genital tract, mammary ducts, and in peritoneal and pleural
cavities.9Y11 Serum levels of this tumor marker vary with the
menstruation cycle12 and are often increased in pregnancy13
and common benign conditions such as endometriosis.14
Serum levels of CA125 are raised in approximately 90% of
patients with advanced EOC10 but is also elevated in patients
with other malignancies such as lung, endometrioid, breast,
and colon cancer.9 In addition, less than 50% of patients
with ovarian cancer stage I have increased serum levels of
CA125.3,9,15 The low sensitivity and specificity limit the
value of CA125 as a single screening tool, and combining it
with additional biomarkers to create a multiple biomarker
panel might be more effective.
Human epididymis protein 4 (HE4) is proposed as a
promising serum tumor marker in screening for early stage
of EOC.16 Moore et al17 investigated 8 different biomarkers
isolated and in combination with CA125. Of those, HE4
showed the highest sensitivity of 72.9% and a specificity of
95% as a single marker. In combination with CA125, the
sensitivity increased to 76.4% at 95% specificity.
HE4 is a glycoprotein that belongs to a whey acidic
protein gene family of protease inhibitors18; however, the
specific biological function remains unknown. Although HE4
is highly expressed in EOC compared with normal ovarian
epithelium, increased expression has also been demonstrated
in other malignancies and in normal tissue from the respiratory tract.16,17,19Y21 In a study among Nordic women, the
serum levels of HE4 were significantly increased by age and

HE4 and Ovarian Cancer

smoking,22 indicating the importance of including smoking

as a confounder. In the present study, serum cotinine was
measured to estimate the individual level of smoking.
Although some studies have used prediagnostic serum
samples23,24 to investigate candidate biomarkers of EOC,
most studies have used diagnostic material where the levels of
biomarkers are remarkably influenced by the disease, lacking
the opportunity to measure changes in serum levels before
diagnosis. In the present study, we used prediagnostic serum
samples from a prospective cancer biobank. The main objectives were to evaluate HE4 as an early detection biomarker
and to investigate how long before diagnosis the serum levels
of HE4 increase in patients with EOC.

MATERIALS AND METHODS

The study was performed using a nested case-control
design. The cases and controls were selected from the
Janus Serumbank cohort.

The Janus Serumbank Cohort

The cohort was established in 1972 and contains serum
samples from approximately 318,000 individuals. The Janus
cohort consist of 3 subgroups of donors: (I) persons participating in health examination surveys in different counties of
Norway (90%), (II) Red Cross blood donors from Oslo (10%),
and (III) previous Janus donors referred to Radium hospital for
cancer treatment (diagnostic samples collected before treatment). The members of subgroup I filled out a questionnaire
on risk factors for cardiovascular disease, for example, smoking
habits, alcohol use, and body mass index. The nationwide
health examinations were performed in the years 1972 to 2004.
Today only samples from subgroup III are being collected. A
large number of the cohort members have donated samples
several times in both groups (health examination and blood
donors). The serum samples have been collected in accordance
with procedures that have differed somewhat during the decades.25 To assess sample validity of the archived samples,
stability experiments have confirmed that a number of serum
components are stable after long-term storage.26Y28

The Cancer Registry of Norway

The Cancer Registry of Norway (CRN) holds the license
and has been the data handler for the Janus Serumbank since
establishment. It was founded in 1951 and has registered virtually all new cancer cases in Norway since 1953. Reporting
cancer cases is mandatory by law, and the information comes
from several independent sources, such as pathology laboratories, hospitals, the national patients discharge registry, and
causes of death registry, thus securing a high grade of completeness. The CRN is considered to capture close to 100% of
new cancer cases yearly, and its completeness and quality have
recently been evaluated.29,30 In a reevaluation of large series of
EOC cases, the completeness of reporting ovarian cancer to
CRN was 99.6%, and the accuracy of the diagnosis was found
to be 92%.31

Study Design and Selection of Participants

A nested case-control design was used to investigate the
difference between cases and controls in CA125 and HE4

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Gislefoss et al

International Journal of Gynecological Cancer

levels. A case was defined as an individual that had been diagnosed as having an ovarian cancer in the cohort, and a control
was an individual in the cohort that was alive and free of cancer,
except for nonmelanoma skin cancer, at the time of diagnosis.
Epithelial ovarian cancer cases were identified by
linking the Janus cohort to CRN. Cases with serum samples
donated more than 10 years before date of diagnosis or less
than 3 months before diagnosis were excluded from the study.
We identified 120 cases, of whom 77 women had donated
1 sample and 43 had donated 2 or more blood samples. The
study participants were enrolled mainly during the 1970s
(15%) and 1980s (72%; Fig. 1).

& Volume 25, Number 9, November 2015

The controls were matched to each case sample on

age at serum sampling (T1 year), date of sample collection (T2 months), and county of residence. Controls were
selected randomly among eligible cohort members, one
control sample for each case sample. The total number of
samples was 348 from 120 cases (174 case samples) and
174 healthy control samples.
Ovarian tumors were classified according to subgroups, that is, serous carcinoma, mucinous carcinoma, adenocarcinoma not otherwise specified (NOS), endometrioid
carcinoma, clear cell carcinoma, carcinoma unspecified,
and others (International Classification of Diseases, 10th

FIGURE 1. Times of blood collections and ovarian cancer diagnosis. Open circles represent times of blood collections;
solid circles, time of ovarian cancer diagnosis.

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International Journal of Gynecological Cancer

& Volume 25, Number 9, November 2015

TABLE 1. Distribution of tumor histology and

morphology in the study material
Histology and Morphology (ICD-10 Codes*)

No

Adenocarcinoma NOS (8140, 8440, 8490,

8570, 9110)
Serous carcinoma (8441, 8460)
Mucinous carcinoma (8470, 8480, 8481)
Endometrioid carcinoma (8380)
Clear cell carcinoma (8310)
Carcinoma unspecified (8010, 8020, 8041)
Other (8260, 8950, 8980)

61

51.7

18
12
15
4
7
3

15.5
9.5
11.2
3.5
6.0
2.6

*Registered codes (ICD-7, ICD-O-2, and ICD-O-3) are translated

to ICD-10 using a combination of topography and morphology.

Revision [ICD-10] codes 8260, 8950, 8980). Adenocarcinoma

NOS represented the major part of the material (Table 1).
The characteristics of the study population are presented in Table 2. Median age at enrollment was 50.2 years for
cases and controls. To control for the effect of smoking on
the HE4 serum levels, we analyzed serum cotinine, a biomarker for tobacco use. Based on the measured cotinine levels,
the individuals were classified in 4 categories: nonsmokers
(G5 nmol/L), passive smokers (5Y85 nmol/L), moderate smokers
(85Y1700 nmol/L), and heavy smokers (91700 nmol/L).32

Sample Processing
Samples were thawed at room temperature, mixed well,
distributed in aliquots, and refrozen on dry ice. To reduce
uncertainty from interassay variation, the samples were randomly placed in batches locating the matched case-control
sets into the same batch. Samples were shipped on dry ice
to the analyzing laboratories. All analyses were performed
on coded samples, blinding the laboratory staff to the casecontrol status.

Serum Analyses
Serum HE4 and CA125 were analyzed at Central
Laboratory, Oslo University Hospital. HE4 was measured in
duplicates using a manual HE4 EIA (Fujirebio Diagnostics
AB, Gothenburg, Sweden). The assay is a solid-phase, noncompetitive immunoassay based on a sandwich technique
using 2 mouse monoclonal antibodies. Two kit controls with
coefficients of variation (CVs) of 15.1% and 7.0% at concentrations of 45.4 and 376.5 pmol/L, respectively, were
analyzed both in the first and last sample positions for each
batch, in line with the manufacturers instructions. In addition,
1 serum control with a CV of 9.6% at a concentration of
51 pmol/L was analyzed once for each batch.
CA125 concentration was measured in duplicate using
an in-house routine assay run on the AutoDelfia platform
(PerkinElmer). This is a 3-step immunofluorometric assay
using 2 characterized monoclonal antibodies to CA125: the
OC125-like mAb K93 (biotinylated F(ab)2-fragment) and the
M11-like mAb K101 (Eu-labeled IgG). Three serum controls
with CVs of 4.7%, 3.6%, and 4.1% at mean concentrations of

HE4 and Ovarian Cancer

13.4, 32.4, and 60.9 kU/L, respectively, were analyzed both in

the first and last sample positions on each 96-well plate.33
Measurement of cotinine was performed at the laboratory
of Bevital AS, Norway, by use of an Liquid Chromatographytandem Mass Spectrometry (LC-MS/MS) method.34,35 The
samples were applied with no other preparation than preanalytical storage on ice and protection from exposure to ultraviolet light and direct sunlight. The assay had sensitivity of
1 nmol/L (0.18 ng/mL), a within-day CV of 2% to 3%, and a
between-day CV of 6%.

Sample Quality
Biospecimens stored for up to 10 years were used in the
present study; thus, investigation of the sample quality was
necessary. The stability of HE4 and CA125 was examined by
Spearman correlation test for dependence between serum level
of the biomarkers and storage time in the control group. Neither
of the components showed a statistically significant correlation
to storage time (results not shown). This indicates that only
negligible degradation of the biomarkers may have occurred.

Statistical Analyses
The statistical analyses were performed by using the
program SPSS (Software Package for Social Sciences) version 19.36 Spearman correlation was performed to examine
the dependence between HE4, CA125, cotinine, storage time,
age at sampling, and lag time. All serum components had to
be log transformed to obtain normal distribution. Pairwise
difference in lg (log-transformed) HE4 and lg CA125 between
case and control samples was analyzed by a linear regression
TABLE 2. Baseline characteristics of the study
participants
Cases

Control

Characteristics
No. individuals
120
174
No. individuals with
43
V
serial samples
No. samples
174
174
Age at enrollment, 50.2 (45.0Y53.6) 50.2 (45.1Y53.2)
median (IQR), y
Age at diagnosis,
55.9 (51.3Y60.7)
V
median (IQR), y
Follow-up time,
6.53 (3.9Y8.4)
V
median (IQR), y
Smoking status, cotinine level
57 (47.6%)
48 (40.0%)
Nonsmokers
G5 nmol/L
Passive smokers
25 (20.8%)
36 (30.0%)
5Y85 nmol/L
Moderate smokers
26 (21.6%)
26 (21.6%)
85Y1700 nmol/L
Heavy smokers
12 (10.0%)
10 (8.4%)
91700 nmol/L

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TABLE 3. Median with IQR and retransformed mean

with CI values for HE4 and CA125 at enrollment and by
lag time G2 years

Component
HE4, pmol/L
Median (IQR)
Mean (CI)
CA125, kU/L
Median (IQR)
Mean (CI)

HE4, pmol/L
Median (IQR)
Mean (CI)
CA125, kU/L
Median (IQR)
Mean (CI)

Cases at
Enrollment
(n = 120)

Controls at
Enrollment
(n = 120)

48.3 (41.1Y59.0)
50.7 (25.9Y99.2)

44.9 (36.8Y57.8)
46.7 (25.9Y84.7)

19.4 (11.3Y28.2)
19.3 (3.8Y97.0)

13.6 (8.5Y23.4)
14.2 (3.6Y55.9)

Cases With Lag

Time G2 y
(n = 21)

Controls Matched
to Cases With
Lag Time G2 y
(n = 21)

54.2 (47.6Y85.2)
64.8 (25.1Y167.2)

42.7 (35.2Y52.9)
43.7 (26.9Y71.2)

30.5 (16.3Y67.1)
38.0 (4.5Y323.8)

12.3 (8.1Y21.3)
11.8 (3.3Y42.8)

model using lag time and difference in lg cotinine as independent variables. Lag time up to 4 years was particularly
investigated by including only women with samples collected
less than 4 years before diagnosis in the model. A significance
level of 5% was used.

RESULTS
Two unexplained high values of CA125, 355.1 and
719.5 pmol/L sampled 7.5 and 9.4 years before diagnosis,
respectively, were excluded before statistical analyses. Median with interquartile range (IQR) and retransformed mean
with confidence intervals (CIs) for serum levels of HE4 and
CA125 are presented in Table 3.
The Spearman correlation analysis showed a high
significant positive correlation between HE4 and cotinine
for cases (0.411; P G 0.001) and controls (0.484; P G 0.001).
Age at sampling was correlated with HE4 for cases (0.204;

& Volume 25, Number 9, November 2015

P = 0.008), whereas for CA125, both cases and controls

(j0.234 [P = 0.002], j0.321 [P G 0.001], respectively) were
correlated. No statistically significant correlation between HE4
and CA125 was demonstrated (data not shown).
Linear regression analyses were performed using difference between case and control in lg HE4 or lg CA125 as a
dependent variable and difference in lg cotinine as a independent variable. All serial samples were included. The results are presented in Table 4. The linear regression analyses
showed statistical significant results (B [unstandardized coefficient] = 0.030 [P = 0.023] and B = 0.163 [P G 0.001] for
the difference in HE4 and CA125, respectively). Multiple
linear regression analyses for samples collected less than
2 years before diagnosis (Table 5) showed statistical significant results (B = 0.147 [P = 0.002] and B = 0.530 [P G 0.001]
for the difference in HE4 and CA125, respectively). Including
samples collected less than 4 years before diagnosis showed
significant differences for CA125 (0.267; P = 0.001) only.
The linear regression results showed high significant association for the differences in lg cotinine indicating a strong
effect of smoking on the serum level of HE4.
We used median serum level of cotinine for passive
smokers and moderate smokers (ie, 20.6 and 380.1 nmol/L),
and the mean linear regression coefficient (0.069) for the
difference in log cotinine and log HE4 in the control group to
investigate how much smoking contributes to the HE4 level.
The calculation showed that smoking in average contributes
to a raise in HE4 of 21.5% between the 2 smoking groups
(HE4 = (380.1/20.6)0.062).

DISCUSSION
This nested case-control study aimed to evaluate HE4
as a biomarker for early detection of EOC. The results indicated that serum levels of HE4 may rise up to 2 years before
diagnosis, whereas CA125 seemed to increase earlier.
Age and smoking have been reported as the 2 most
important determinants that influence the HE4 levels in
healthy women. Bolstad et al22 showed that serum levels of
HE4 in healthy Norwegian woman increased by age, an increase
of 9% and 20% at 40 and 50 years compared with 20-year level.
Smoking was related to an increase in HE4 serum levels. This
corresponds well to results from another study.24 In the present
study, we assessed the impact of age by matching controls by
age and smoking by including results from cotinine measurements in the Spearman correlation and in the linear regression
analyses. Both analyses demonstrated a statistically high

TABLE 4. Multiple linear regression for pairwise difference between cases and matched control samples
Multiple Linear Regression
Difference between invasive tumor and control

Samples (n = 174)
Lg HE4

Lg CA125

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Coefficient
P
Standardized A
Coefficient
P
Standardized A

Constant

Difference in Lg Cotinine

0.030
0.023

0.069
G0.001
0.555
j0.054
0.013
j0.189

0.163
G0.001

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International Journal of Gynecological Cancer

& Volume 25, Number 9, November 2015

HE4 and Ovarian Cancer

TABLE 5. Multiple linear regression for pairwise difference between cases with lag time 3 monthsY2 years and
matched control samples
Multiple Linear Regression
Difference between invasive tumor and control

Samples (n = 22)
Lg HE4

Lg CA125

significant correlation between cotinine and HE4 serum

concentrations (P G 0.001).
Previous studies have reported results on HE4 as a biomarker for early detection using prediagnostic samples37,38;
however, relatively few cancer cases (G35) were included. In the
present study, we had access to prediagnostic serum samples
from 120 women with EOC. Additional access to multiple
samples from most of the women gave the possibility to follow
the changes in the serum biomarker levels over time. However,
that the samples had a variety of collecting time points is a
weakness of the study and made it difficult to draw conclusion
with respect to any time trends in the biomarkers concentration.
The linear regression for pairwise difference between case and
control was significant after correction for cotinine for HE4 and
CA125 (0.030 [P = 0.023] and 0.163 [P G 0.001], respectively).
We investigated the samples collected close to diagnoses by
including cases with lag time up to 4 years in the model. Entering samples collected less than 4 year before diagnosis did
not result in significant differences for HE4 in contrast to
CA125 (0.267; P = 0.001). However, including samples collected less than 2 years preceding diagnosis showed statistical
significance (0.147; P = 0.002) for HE4 and suggests that serum
levels of HE4 may rise up to 2 years before diagnosis, whereas
CA125 seems to increase earlier.
Expression of CA125 is lacking in 20% of the EOCs in
the initial stage, and this is a drawback for using this cancer
marker alone, although some studies have demonstrated that
CA125 may be a marker of early detection of EOC.39Y41
Bjrge et al42 observed a higher risk of EOC in women with
prediagnostic elevated CA125 (odds ratio, 3.1) in samples
from the Janus Serumbank. In the present study, linear regression analysis indicated a rise in serum level of CA125 at
least 4 years before diagnosis which is in accordance with
Zurawski et al.41
A positive correlation between HE4 and CA125 can be
expected close to diagnosis43; however, in the present study,
no significant correlation to lag time was displayed, possible
due to the fact that a minor number of samples were collected
close to diagnosis. CA125 was significantly negatively correlated with age in the control group, which also is reported in
another study.44
In the present study, we used an HE4 kit from Fujirebio
Diagnostics. The kit insert proposes a reference limit of 0 to
150 pmol/L based on measurements from 76 premenopausal
and 103 postmenopausal healthy women. Bolstad et al22 suggested

Coefficient
P
Standardized A
Coefficient
P
Standardized A

Constant

Difference in Lg Cotinine

0.147
0.002

0.080
0.010
0.537
j0.079
0.269
j0.246

0.530
G0.001

a considerably lower age-related reference limit based on measurements from 801 healthy nonsmoking women and pointed out
that including smokers would have reduced the difference.
Smoking is highly correlated with the serum level of HE4. An
average increase of more than 20% in HE4 serum concentration
in moderate smokers compared with passive smoker confirms
that smoking has a significant impact on serum level of HE4.
The serum level of HE4 does not seem to be affected by menstrual cycle45 in contrast to CA12546; however, Anastasi et al52
reported nonsignificant menstrual fluctuation in serum HE4 and
CA125 levels in woman older than 35 years.
A potential weakness in studies based on archival
samples is the stability and general validity of the samples.
Selecting an appropriate study design is therefore important.
A nested case-control design was used to investigate the
difference in biomarker level between cases and controls. We
have matched for age at sampling as the levels of HE4 and
CA125 in serum are increased by age and time for blood draw
to secure equal storage time. Measurement of biomarkers in
archival samples may be influenced by analytic batch, storage,
and freeze-thaw cycles.47,48 The interassay variation was
minimized by placing the case and the matched control in the
same batch. Matching of cases and controls was important to
decrease bias of storage time and possibly differences in
preanalytical sample handling, assuming that both cases and
controls would be equally affected. The stability of biomarker
level was investigated by Spearman correlation test and
showed that neither of the components was statistically and
significantly correlated with storage time. The impact of
freeze-thaw cycles on HE4 and CA125 has not been investigated; however, unpublished data from the Janus Serumbank
show that many proteins are robust to several freeze-thaw
cycles. Although matching may introduce selection bias in
a biomarker study, a case-control design is the preferred approach to deal with batch effects, storage effects, and freezethaw cycles.48
The search for new and better markers for early detection of EOC is important, and although there are both
affirmative and negative results with regard to the diagnostic
performance of HE4, a recent meta-study of HE4 vs CA125
concludes that HE4 is superior to CA125 for identification
of EOC in women with suspected gynecologic disease.49 A
number of studies have observed that HE4 seems to be released from tumor earlier than CA125, and that the HE4
levels are significantly higher than CA125 in the initial stages

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(International Federation of Gynecology and Obstetrics IYII)

of EOC compared with healthy controls.17,50,51 The diagnostic sensitivity of HE4 is also considerably higher than that
for CA125 in early stages.51
The present study showed that differences between
cases and controls in serum concentration of HE4 and CA125
seemed to increase 2 and 4 years, respectively, before diagnosis. CA125 has a low sensitivity and specificity which
limits the value of the biomarker, and combining CA125
with HE4 may therefore have better efficacy in screening.
However, smoking status should be taken into consideration
when using HE4 as an early marker of ovarian cancer.

ACKNOWLEDGMENT
We express our gratitude to all persons who have
donated blood to the Janus Serumbank of Norway and to all
enthusiasts that made the sample collection possible. The
Norwegian Cancer Society provided funding for the cotinine
analyses.

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