*e-mail: katarina@manu.edu.mk Potential use of HP and AMBP in urine for the screening of prostate cancer 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 INTRODUCTION 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). 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. C MATERIALS AND METHODS 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. RESULTS AND DISCUSSION 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. 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. ACKNOWLEDGMENTS 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) 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.