original article

original article
Evaluation of Prostate Specific Antigen and Early Prostate Cancer Antigen 2 as Diagnostic Markers
for Prostate Cancer among Sudanese with Prostate Enlargement. A study in Khartoum State, Sudan
Evaluation of Prostate Specific Antigen and Early Prostate
Cancer Antigen 2 as Diagnostic Markers for Prostate Cancer
among Sudanese with Prostate Enlargement.
A study in Khartoum State, Sudan
Akram H. Awadalla1 MSc, Bader Eldien H. Elabid2 MD. Abdelgader A. Almugadam3 PhD
1) M.Sc Clinical Chemistry Faculty of medicine and health sciences, Kordofan University.
2) MBBS (U of K), MD Clinical Pathology, Associate Professor of Clinical Pathology, University of Science and Technology.
3) M.Sc, PhD in Clinical Chemistry, Assistant Professor of Clinical Chemistry, Sudan University of Science and Technology.
Correspondence
Akram Hamed Awadalla, Department of Clinical chemistry Faculty of Medicine and Health Sciences. Kordofan University- Sudan.
P. O. Box 160. Elobied. Sudan, Telephone No. 002490611825113. Fax No. 002490611825113 (mobile 0912884294-00966548597450),
E.mail: [email protected]
Abstract
Background: Prostate cancer is now recognized as one of the principal medical problems facing
the male population in Sudan. Objectives: To evaluate the serum levels of prostate specific antigen
and early prostate cancer antigen 2 as diagnostic markers for prostatic cancer among Sudanese with
prostate enlargement. Patients, Materials and Methods: This study was carried out in the Soba and
Omdurman teaching hospitals in Khartoum state during the period from January 2011- January 2012.
Two hundred Sudanese with prostate enlargement were selected as the “test group”, and one-hundred
healthy volunteers with matching age and sex and socioeconomic status were included as the “control
group”. Data was collected by using questionnaires and analyzed by using Statistical Package for Social
Sciences (SPSS). Results: The results of this study revealed a significant difference between the means
of the serum levels of both early prostate cancer antigen-2 and prostate specific antigen of the test group
when compared with the control group. Also the results revealed a significant difference between the
means of the serum levels of both early prostate cancer antigen-2 and prostate specific antigen of the
patients with malignant prostate tumour when compared with the control group. Conclusion: This study
finally concluded that the serum levels of early prostate cancer antigen-2 and prostate specific antigen are
important markers for evaluation of prostate cancer because they are significantly raised when compared
with healthy control subjects and serum early prostate cancer antign-2 can be used as a useful prognostic
and screening marker for prostate cancer and its determination may detect a considerable proportion of
tumours missed by PSA.
Key words: Prostate tumours, early prostate cancer antigen-2 and prostate specific antigen
Introduction:
Cancer of the prostate (CaP) is now recognized
as one of the principal medical problems facing
the male population. In Europe, an estimated 2.6
million new cases of cancer are diagnosed each year.
Prostate cancer constitutes about 11% of all male
cancers in Europe(1) Furthermore, CaP is currently
the second most common cause of cancer death in
men(2). Prostate cancer affects elderly men more
often than young men. It is therefore a bigger health
concern in developed countries with their greater
proportion of elderly men. Thus, about 15% of male
cancers are CaP in developed countries compared
SUDANESE JOURNAL OF PUBLIC HEALTH - April 2012, VOL. 7 No. 2
47
to 4% of male cancers in undeveloped countries(3)
.The most common cause of cancer in the year 2004,
according to the report from Radiation and Isotopes
Center of Khartoum is breast cancer with 17.4%.
In males, the most common cancers are prostate
cancer 3.3%, which is found around all Sudan(4).
PSA is a glycoprotein produced almost exclusively
in the epithelium of the prostate gland. In the
circulation PSA may be complexed to serum proteins
(complexed PSA, or cPSA) or may be free (fPSA). The
cPSA and fPSA together comprise total PSA (tPSA).
The tPSA is normally less than 4 ng/ml (normal
ranges vary depending upon which assay is used).
‫ العدد الثاني‬- ‫ اجمللد السابع‬- ‫اجمللة السودانية للصحة العامة‬
original article
Evaluation of Prostate Specific Antigen and Early Prostate Cancer Antigen 2 as Diagnostic Markers
for Prostate Cancer among Sudanese with Prostate Enlargement. A study in Khartoum State, Sudan
A mildly increased tPSA in a patient with a very
large prostate can be due to nodular hyperplasia,
or to prostatitis, rather than carcinoma. The fPSA
correlates more closely with benign prostatic
conditions than the tPSA. The cPSA has a greater
sensitivity for prostatic adenocarcinoma at the low
ranges of elevation. A rising tPSA is suspicious for
prostatic carcinoma, even if the tPSA is in the normal
range. Transrectal needle biopsy, often guided
by ultrasound, is useful to confirm the diagnosis,
although incidental carcinomas can be found in
transurethral resections for nodular hyperplasia(5).
Early prostate cancer antigen (EPCA) is a nuclear
matrix protein originally identified through protein
profiling of rat prostate tissue. Alterations of nuclear
matrix proteins are considered to be associated
with carcinogenesis. Immunohistochemical studies
on prostate core biopsies using auto antibodies
against EPCA showed sensitivity and a specificity
superior to 80% for the diagnosis of PCa(6). Early
prostate cancer antigen-2 (EPCA-2) is a protein of
which blood levels are elevated in prostate cancer.
It appears to provide more accuracy in identifying
early prostate cancer than the standard prostate
cancer marker, PSA(7) “EPCA-2” is not the name of a
gene. EPCA-2 gets its name because it is the second
prostate cancer marker identified by the research
team. This earlier marker was previously known as
“EPCA” but is now called “EPCA-1”(8).
Patients, Materials & Methods
This study was conducted in Omdurman and Soba
teaching hospitals in urinary tract surgery division
in Khartoum state, during the period from January
2011 to January 2012.
A total of 200 Sudanese patients with prostatic
tumour were selected as test group after taking their
consent. Each volunteer in this study was asked to
come to Omdurman and Soba teaching hospitals
for medical assessment and sample collection. Onehundred healthy subjects were selected as a control
group who were age, sex and socioeconomic status
matched to the test group.
Clinical data was obtained from the patient’s
history and recorded on a questionnaire sheet.
Clinical assessment of the study group was done by
a medical doctor and they were not suffering from
prostatitis or exposed to recent needle biopsy or
received chemical, hormonal or radiation therapy
which may affect the levels of early prostate cancer
antigen-2 and prostate specific antigen.
After informed consent, venous blood sample (5ml)
was collected from the study subjects. After blood
SUDANESE JOURNAL OF PUBLIC HEALTH - April 2012, VOL. 7 No. 2
48
clotting, the samples were centrifuged within 20
minutes after collection at 3000 rpm for 5 minutes
and the sera were stored -20 ○C until analysis.
The serum was allowed to reach room temperature
and early prostate cancer antigen-2 was measured
by ELISA and the micro titer plate provided in the
kit had been pre-coated with a monoclonal antibody
specific to EPCA2. Standards or samples were then
added to the appropriate micro titer plate wells
with a biotin-conjugated polyclonal antibody
preparation specific for EPCA2. Next, Avidin
conjugated to Horseradish Peroxidase (HRP) was
added to each micro plate well and incubated. Then
a substrate solution was added to each well. Only
those wells that contain EPCA2, biotin-conjugated
antibody and enzyme-conjugated Avidin exhibited
a change in colour. The enzyme-substrate reaction
was terminated by the addition of a sulphuric acid
solution and the change in colour was measured
spectrophotometrically at a wavelength of 450nm ±
10nm. The concentration of EPCA2 in the samples
was then determined by comparing the O.D. of the
samples to the standard curve.
The serum levels of prostate specific antigen were
determined by full automated chemistry analyzer in
the following steps:
• 1st incubation: 20 µl of sample, a biotinylate
monoclonal PSA- specific antibody, and
monoclonal PSA- specific antibody labeled with
a ruthenium complex react to form a sandwich
complex.
• 2nd incubation: After addition of streptavidincoated micro-particles, the complexes became
bound to the solid phase via interaction of biotin
and streptavidin.
• The reaction mixture was aspirated into the
measuring cell where the micro-particles were
magnetically captured on to the surface of
the electrode. Unbound substances were then
removed Procell.
• Application of a voltage to the electrode then
induced chemiluminescent emission which was
measured by a photomultiplier.
• Results were determined via a calibration curve
which is an instrument- specifically generated by
2- point calibration and a master curve provided
via the reagent barcode.
Quality control:
The precision and accuracy of all methods used in
this study were checked including commercially
prepared control sera.
Statistical analysis:
Statistical Package for Social Science (SPSS
‫ العدد الثاني‬- ‫ اجمللد السابع‬- ‫اجمللة السودانية للصحة العامة‬
original article
Evaluation of Prostate Specific Antigen and Early Prostate Cancer Antigen 2 as Diagnostic Markers
for Prostate Cancer among Sudanese with Prostate Enlargement. A study in Khartoum State, Sudan
Table 2: Comparison of the means serum of EPCA2, PSA of the control group and patients with
malignant prostate tumour.
version 17) computer software was used for data
analysis. The mean and standard deviation of the
serum levels of early prostate cancer antigen-2
and prostate specific antigen were calculated and
t-test was used for comparison. Linear regression
was used to assess the correlation between the
serum levels of prostatic specific antigen and early
prostate cancer antigen 2 with the duration of
prostatic tumour since detection. P.value<0.05 were
considered statistically significant (P.value < 0.01
were considered highly significant) and the results
presented in the form of tables and figures.
Variables
EPCA-2 (ng/ml)
PSA (ng/ml)
Results:
Serum early prostate cancer antigen-2 and prostate
specific antigen.
Table 1 shows a significant difference between the
means of the serum level of early prostate cancer
antigen-2 in the test group and control group.
(Mean ± STD) (48.53 ± 37.47ng/ml) versus (3.99
± 4.73ng/ml)(P.value = 0.000) the mean of the test
group is significantly raised.Also shows a significant
difference of the means of the serum level of prostate
specific antigen between the test group and control
group. (mean±STD) (23.0432.26±) ng/mlversus
(1.230.89±) ng/ml (P.value = 0.000) the mean of the
test group is significantly raised.
Table 2 shows a significant difference between the
means of the serum levels of EPCA-2 in the control
group and patients with malignant prostatic tumour,
mean ±SD: (3.9± 4.7) ng/ml versus (63.2± 37.1) ng/
ml respectively (P = 0.000). Also shows a significant
difference between the mean serum levels of PSA
of the control group and patients with malignant
prostatic tumour, mean ±SD :( 1.23± 0.89) ng/ml
versus (5.2± 0.46) ng/ml respectively (P= 0.001).
Early prostate
cancer antigen-2
(ng/ml)
Prostate specific
antigen(ng/ml)
Test group
n= 200
Control group
P. value
n= 100
48.53 ± 37.47
3.99 ± 4.73
0.000
23.04 ± 32.26
(3 – 129)
1.23 ± 0.89
(0.1 – 3.6)
0.000
(10 – 235)
(0.5 – 29)
• The table shows the mean ± SD, range in brackets () and
probability (P. value).
• t-test was used for comparison.
• P. value ≤ 0.05 is considered significant.
SUDANESE JOURNAL OF PUBLIC HEALTH - April 2012, VOL. 7 No. 2
1.23± 0.89
(0.1 – 3.6)
5.2± 0.46
(1 – 155.4)
(0.5 – 29)
(10 – 230)
P
0.000
0.001
• The table shows the mean±S.D, range in brackets () and
probability (P).
• t.test was used for comparison.
Table1: Comparison of means of serum level of
early prostate cancer antigen-2 and prostate specific
antigen of test group and control group.
Variable
3.9± 4.7
Malignant
prostate
tumour
n= 132
63.2± 37.1
Control group
n= 100
49
Discussion
In Sudan, there is no related published data
regarding the serum levels of early prostate cancer
antigen-2 in Sudanese with prostate enlargement,
so this study tends to compare the results with that
obtained in other previous studies in none Sudanese
people.
In this study patients with prostatic tumour have
a significance increase in the mean of the serum
level of early prostate cancer antigen-2 (EPCA-2)
compared with the control subjects (P = 0.000). From
the previous results we realize that early prostate
cancer antigen-2 tumour marker can be used to
distinguish between patients with organ confined
and non organ confined prostate cancer.. This
agrees with the results of a study done by Leman,
et al 2007(7) who reported that, the geometric mean
levels of EPCA-2 were significantly greater in the
men with non-organ confined prostate cancer than
in those with organ-confined disease (P <0.0001).
Also in this study the mean serum level of early
prostate cancer antigen-2 is significantly elevated
in patients with malignant prostate tumour when
compared with healthy control subject and this
may be due to the abnormal leakage of EPCA-2 into
the circulation influenced by the level of EPCA-2
expression in malignant epithelium. This agrees
with other findings in a study done by Zhao Z, et
al 2011(8) who found the mean±S.D in patients
with prostate cancer significantly increased in
comparison to healthy subjects, P<0.001.
In this study patients with prostatic tumour have a
significance increase in the mean of the serum level
of prostate specific antigen(PSA) compared with
the control subjects (P = 0.000) and this reflects
‫ العدد الثاني‬- ‫ اجمللد السابع‬- ‫اجمللة السودانية للصحة العامة‬
original article
Evaluation of Prostate Specific Antigen and Early Prostate Cancer Antigen 2 as Diagnostic Markers
for Prostate Cancer among Sudanese with Prostate Enlargement. A study in Khartoum State, Sudan
the ability of PSA in distinguishing persons with
prostate cancer from other persons without PCa.
This agrees with a study done by Zhigang Zhao
2010(9) who found a significant difference in the
mean of serum level of prostate specific antigen at
a cutoff value 4 ng/ml between the patients group
(2.07±1.12) and control group (0.88±0.46) ng/ml
(P = 0.014).
Also in this study the mean serum level of prostate
specific antigen is significantly elevated in patients
with malignant prostate tumour (5.2± 0.46) ng/
ml, P = 0.000 when compared with healthy control
subjects; this may be due to the abnormal leakage of
PSA into the circulation which is influenced by the
level of PSA expression in malignant epithelium and
by distortion of prostatic glandular architecture..
This agrees with a study done by Eddy S et al
2009(10) who reported an elevation of mean serum
PSA level in patient with PCa (5.2±2.2) ng/ml while
in the control subject (1.1±0.6) ng/ml, P<0.004.
2001.Oct; 37(Suppl 8):S4-66.
4. Bomford CK etal: Textbook of radiotherapy,
radiation physics, therapy and oncology,
Churchill, Livingstone, London 6 edition 2004
5. Jung K, Lien M, Butz H, Stephan C, Loening
SA, Keller T. New insights into the diagnostic
accuracy of completed and total prostate
specific antigen using discordance analysis
characteristics. J Urol. 2006; 175:1275-1280.
6. Dhir R, Vietmeier B, Arlotti J, et al. “Early
identification of individuals with prostate
cancer in negative biopsies”. April 2004; J. Urol.
171 (4): 1419–23.
7. Leman ES, Cannon GW, Trock BJ, Sokoll LJ, Chan
DW, Mangold L et al. EPCA-2: a highly specific
serum marker for prostate cancer. Urology
2007; 69: 714-720.
8. Zhao Z, Ma W, Zeng G, Qi D, Ou L, et al. Serum
Early Prostate Cancer Antigen (EPCA) Level
and Its Association with Disease Progression in
Prostate Cancer in a Chinese Population. 2011;
PLoS ONE 6(5): e19284. doi:10.1371/journal.
pone.0019284.
9. Zhigang Zhao, Guohua Zeng, and Wen Zhong.
Serum Early Prostate Cancer Antigen (EPCA)
as a Significant Predictor of Incidental Prostate
Cancer in Patients Undergoing Transurethral
Resection of the Prostate for Benign Prostatic
Hyperplasia; the Prostate 2010;70:1788 -1798
10. Eddy S, et al. Analysis of a Serum Test for
Prostate Cancer That Detects a Second Epitope
of EPCA-2.2009; Prostate 69: 1188–1194.
Conclusion:
This study concluded that the serum levels of early
prostate cancer antigen-2 and prostate specific
antigen are significantly increased in patients with
prostate cancer and both can be used as effective
tumour markers in prostate cancer diagnosis, early
detection of prostate cancer recurrence, screening,
follow up and monitoring.
ACKNOWLEDGEMENTS
I would like to express my deep and sincere gratitude
an appreciation to my supervisor, Dr. Badreldien
Hassan Alabid for his encouragement, supervision,
wisdom, patience, effort, critical comment and
invaluable sound advice and skilful guidance.
My so special thanks and sincere respect to my
co-supervisor, Dr. Abdelgader Elmugadam, for
his suggestions, close supervision and guidance
throughout the study period.
Furthermore, I highly acknowledge the assistance
of all authors listed in the reference list for the
literature that I have reviewed.
References:
1. Bray F, Sankila R, Ferlay J, Parkin DM. Estimates
of cancer incidence and mortality in Europe in
1995. Eur J Cancer 2002; 38(1):99-166.
2. Jemal A, Siegel R, Ward E, et al. Cancer statistics,
2008. CA Cancer J Clin 2008 Mar; 58(2):71-96.
3. Parkin DM, Bray FI, Devesa SS. Cancer burden in
the year 2000: the global picture. Eur J Cancer
SUDANESE JOURNAL OF PUBLIC HEALTH - April 2012, VOL. 7 No. 2
50
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