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ACKNOWLEDGMENTS
ACKNOWLEDGMENTS
RESULTS AND DISCUSSION
RESULTS AND DISCUSSION
MATERIALS AND METHODS
MATERIALS AND METHODS
INTRODUCTION
INTRODUCTION
Sanja Kiprijanovska1, Selim Komina2, Gordana Petrusevska2, Natasha Chokrevska Zografska3, Momir Polenakovic1 and
Katarina Davalieva1*
1Research Centre for Genetic Engineering and Biotechnology “Georgi D Efremov”, Macedonian Academy of Sciences and Arts, Skopje,
Republic of Macedonia
2Institute of Pathology, Medical Faculty, University “St. Cyril and Methodius”, Skopje, Republic of Macedonia
3Biochemical laboratory, Clinical Hospital “Acibadem Sistina”, Skopje, Republic of Macedonia
*e-mail: katarina@manu.edu.mk
The principal driving goal currently within prostate cancer (PCa) research is to identify non-invasive biomarker(s) for early detection of aggressive
tumors with greater sensitivity and specificity than PSA. In previous study [1], we focused on identification of non-invasive biomarkers in urine by
testing urine samples from PCa and benign prostatic hyperplasia (BPH) patients by 2-D DIGE coupled with MS and bioinformatics analysis. Two
acute phase response proteins, haptoglobin (HP) and α-1-microglobulin/bikunin (AMBP), which expression differed from the defined expression in
the canonical pathway were measured by immunoturbidimetry in an independent validation set and their combination showed greater accuracy than
PSA (AUCHP+AMBP=0.848 vs AUCPSA=0.754) in detecting PCa (Figure 1).
In this study, we have evaluated the PCa specificity of urinary HP and AMBP in larger and independent set of PCa, BPH, bladder cancer (BC) and
renal cancer (RC) patients. HP and AMBP concentration were determined by immunoturbidimetry in urine samples from 20 PCa, 18 BPH, 15 BC
and 8 RC patients. The mean age was 68.1±4.8, 69.1±8.5, 67.6±7.1 and 63.1±5.1 years for PCa, BPH, BC and RC group. The mean Gleason and
serum PSA in PCa group were 7.1±1.1 and 17.9±11.4 ng/ml, while the mean serum PSA in BPH group was 5.4±3.1 ng/ml. The average cancer
stage in BC and RC group was stage II.
The nonparametrics Kruskal-Wallis test comparing AMBP and HP urine concentrations among the 4 groups, showed significantly different
distribution between groups for both AMBP (p=0.0004) and HP (p<0.0001). The concentrations of AMPB in the PCa group showed significantly
higher level compared to BPH group (Mann-Whitney U-test, p=0.015) and opposite, significantly lower level compared to BC group (Mann-Whitney
U-test, p=0.009) (Figure 2). There was no significant differences in AMBP concentrations between PCa and RC group (Mann-Whitney U-test,
p=0.258). The concentration of HP in the PCa group showed a significantly lower level compared to BPH (Mann-Whitney U-test, p=0.036), BC
(Mann-Whitney U-test, p<0.0001) and RC group (Mann-Whitney U-test, p=0.0002) (Figure 2). The levels of AMBP and HP in PCa vs BPH group in
this study, have confirmed the previously observed expression [1]. The expression level of AMBP was also increased in BC and RC group as in
PCa group, although the median levels between PCa and BC groups were significantly different. Contrary, the expression level of HP which was
decreased in PCa compared to BPH, showed increased expression in BC and RC group.
In conclusion, the results from evaluation of HP and AMBP urinary levels in urogenital cancers highlight the urinary HP as specific biomarker for
PCa.
1. Davalieva K, Kiprijanovska S, Komina S, Petrusevska G, Zografska NC, Polenakovic M. Proteomics analysis of urine reveals acute phase response proteins as
candidate diagnostic biomarkers for prostate cancer. Proteome Sci 2015;13:2.
This work was supported by the funds for Science of the Macedonian Academy of Sciences and Arts (grant no. 09-114/1, Biomarker detection in
prostate cancer with the use of 2D DIGE/MALDI MS technology)
Figure 2. HP and AMBP levels in urine of BC, RC, PCa and BPH patients, expressed as relative ratio to urine creatinine and obtained by immunoturbidimetry. The
concentration of HP in the PCa group showed a significantly lower level compared to BPH (Mann-Whitney U-test, p=0.036), BC (Mann-Whitney U-test, p<0.0001)
and RC group (Mann-Whitney U-test, p=0.0002). The concentrations of AMPB in the PCa group showed significantly higher level compared to BPH group (Mann-
Whitney U-test, p=0.015) and opposite, significantly lower level compared to BC group (Mann-Whitney U-test, p=0.009). In the box plot analysis, median, 25th and
75th percentiles, standard deviation (+) and outliers (♦) are shown. Box plots were constructed based on a series of 51 urine samples.
Figure 1. Validation of candidate biomarkers for the diagnosis of PCa. (A) 2-DE profiles of TF, AMBP and HP in BPH and PCa patients samples obtained by 2-D DIGE.
(B) TF, AMBP and HP levels in urine of PCa and BPH patients, expressed as relative ratio to urine creatinine and obtained by immunoturbidimetry. (C) The combination
of the AMBP and HP yielded highest diagnostic accuracy (AUC=0.848). ROC curve was based on series of 32 urine samples.
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True positive rate (Sensitivity)
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ROC Curve (AMBP+HP)
AUC=0.848
TF HPAMBP
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TF/ Creatinine (mg/g)
p=0.015
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AMBP/ Creatinine (mg/g)
p=0.040
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HP/ Creatinine (mg/g)
p=0.031
C