Human Kallikrein-2 , Prostate Specific Antigen and Free-Prostate Specific Antigen in Combination to Discriminate Prostate Cancer from Benign Diseases in Syrian Patients

Prostate cancer is the most prevalent cancer and the second leading cause of cancer mortality among men in the western world. In Middle East countries it may be less common; but is still one of the most common cancers in men with elevated incidence especially in Lebanon and Syria (Salim et al., 2009; Shamseddin et al., 2014). Take into consideration the possibility associations between Prostate Cancer with genetic, familial, environmental and other factors, also the effect of different measures or detection programs applied in the different countries and the awareness variation of people toward these programs (Boyle et al., 2003; Arafa et al., 2012; Ezzi et al., 2014; Baade et al., 2015; Choubey et al., 2015) The prostate cancer had few specific symptoms in its early stage, for that reason, detecting cancer holds several difficult. The standard practice of screening for prostate cancer includes PSA, with or without digital rectal exam test (DRE), starting at the age of 50 years in all males or


Introduction
Prostate cancer is the most prevalent cancer and the second leading cause of cancer mortality among men in the western world.In Middle East countries it may be less common; but is still one of the most common cancers in men with elevated incidence especially in Lebanon and Syria (Salim et al., 2009;Shamseddin et al., 2014).Take into consideration the possibility associations between Prostate Cancer with genetic, familial, environmental and other factors, also the effect of different measures or detection programs applied in the different countries and the awareness variation of people toward these programs (Boyle et al., 2003;Arafa et al., 2012;Ezzi et al., 2014;Baade et al., 2015;Choubey et al., 2015) The prostate cancer had few specific symptoms in its early stage, for that reason, detecting cancer holds several difficult.The standard practice of screening for prostate cancer includes PSA, with or without digital rectal exam test (DRE), starting at the age of 50 years in all males or RESEARCH ARTICLE Human Kallikrein-2, Prostate Specific Antigen and Free-Prostate Specific Antigen in Combination to Discriminate Prostate Cancer from Benign Diseases in Syrian Patients Dala-Maria Bachour 1 *, Emil Chahin 2 , Sahar Al-Fahoum 2 earlier in men with certain risk factors.However, only according to the prostate biopsy results we can confirm the prostate cancer diagnosis (Shariat et al., 2008;Madu et al., 2010;Heidenreich et al., 2014).
PSA levels influenced by many factors like age, race, the Metabolic Syndrome or even Body Mass Index (BMI) and alcohol consumption (Chiu et al., 2011;Bashir et al., 2014;Daniyal et al., 2014;Ozbek et al., 2014;Woo et al., 2014).But the most important disadvantage of the PSA test is its low specificity which means that a high percentage of the men are negative on initial biopsy (Jansen et al., 2009).This lack of specificity of PSA warrants the use of new biomarkers to diagnose prostate cancer more accurately and decreases the number of unnecessary biopsies (Madu et al., 2010;Murray et al., 2014;Esfahani et al., 2015).
Kallikrein-related peptidases represent a subgroup of the serine protease family consisting of 15 members located at q13.4 of chromosome 19.Of the members of the kallikrein family, hK2 has a valuable importance since its levels are significantly higher in men with prostate cancer compared with men with benign diseases.HK2 is a 28 kDa serine protease produced by prostatic epithelial cells.Structurally, hK2 and PSA (hK3) share the highest sequence homology (in KLKS family).The serum level of hK2 increases in many conditions including leaking from the disrupted prostatic cells and over expression in cancerous prostate tissue which increases with increasing grade (Herrala et al., 2001;Borgoño et al., 2004;Jansen et al., 2009).
Therefore, many studies indicate that measurement of serum hK2 with PSA can improve diagnosis of prostate cancer, importantly, the ratio of hK2/fPSA ratio alone or combined with fPSA/PSA ratio enhances the discrimination between prostate cancer and BPH and may also be useful in grading the cancer (Becker et al., 2000;Stephan et al., 2006;Raaijmakers et al., 2007).All these advantages suggest that a panel or multiple markers including hK2 is an important biomarker for the presence and recurrence of cancer (Martin et al., 2004;Vickers et al., 2008;Thorek et al., 2013).
In this study, we investigate the role of hK2 and its combinations with other markers to discriminate prostate cancer from benign diseases in Syrian patients.

Patient selection
Included in this prospective oriented cross-sectional cohort study, were 70 patients who were referred to either the Urology department in Al-Bairouni university Hospital or Al-Mouwasat university Hospital or Ibn Al-Nafees Hospital in Damascus, Syria.Between May 2011 and March 2012 and aged between 45 -83 years old.These patients underwent prostatic TRUS guided needle-core prostatic biopsies because of either PSA values of 4.0 ng/mL or abnormal digital rectal examination (DRE), later they were diagnosed with either PCa (35 patients) or BPH (35 patients) according to the histological tests results and Grade was evaluated by the Gleason scoring system according to it patients classified into three groups: low-grade group (GS=2-3-4), intermediate-grade group (GS=5-6-7) and high-grade group (GS=8-9-10).Patients present with previous cancer, previous prostate biopsy or metastatic diseases were excluded.
Blood samples were collected before DRE and biopsy procedure and any treatment.All participants agreed to provide blood samples and had signed a written informed consent; the study has been approved by the ethics committee.
Plasma was separated from blood samples and was stored at -70°C.PSA, fPSA and hK2 were analyzed at a later time in the frozen serum samples.Both fPSA and PSA levels were analyzed with commercially ELISA kits (R&D Systems) and hK2 levels were analyzed using a for-research only ELISA kit (Uscn Life Science Inc.), according to the manufacturer's recommendations.

Statistical analysis
Statistical analyses were done with commercially available software using SPSS program version 19 and Microsoft Excel 2007.The analyses of differences between these variables in the three groups were performed with the T-student test for normally distributed parameters.Relationships between different variables were assessed by the Spearman correlation coefficient.For all analyses, P< 0.05 was considered statistically significant.ROC curves were constructed for PSA, fPSA, the fPSA/PSA ratio, hK2 and hK2/fPSA ratio, plotting sensitivity vs.

Results
All 70 patients underwent prostatic TRUS guided needle-core prostatic biopsy, divided according the histological tests results into a cancer group (35 patients) and a BPH group (35 patients).PSA, fPSA and hK2 were measured and the fPSA/PSA and the hK2/fPSA ratios were calculated, the results are presented in Table I.
The range of age values from 47 to 83 years in PCa patients, with a mean±SE of 78±5.3 years and from 45 to 82 years in BPH patients, with the mean±SE value being 76±6.1 years.Values did not reach statistical significance between the PCa and BPH groups (P=0.12).The range of PSA values from 3.71 to 146.3 ng/ml in PCa patients, with a mean±SE of 21.1±13.8ng/ml and from 1.69 to 41.2 ng/ml in BPH patients, with the mean±SE value being 12.3±3.97ng/ml.Values did not reach statistical significance between the PCa and BPH groups (P=0.11).fPSA/PSA ratio values ranged from 0.69 to 47.5% in PCa patients, with a mean±SE of 13.4±5.13%and from 2.55 to 72.3% in BPH patients, with the mean±SE value being 25.1±7.53%.Values also did not reach statistical significance between the PCa and BPH groups (P=0.06).hK2/fPSA ratio values ranged from 6.11 to 539.8% in PCa patients, with a mean±SE of 156.6±60.1% and from 1.39 to 42.7% in BPH patients, with the mean±SE value being 51.9±18.6%.Values were significantly higher in PCa than BPH (P=0.01).
The Gleason score (GS) values ranged from 3 to 9 in 35 patients diagnosed of cancer, with a mean 6.8.Of them 8 (22.9%) patients classified in low-grade group, 16 (45.7%)patients classified in intermediate-grade group and 11 (31.4%)patients classified in high-grade group.
In general, the levels of PSA, fPSA/PSA ratio and hK2/fPSA ratio were higher in cancer patients; however, the differences in fPSA/PSA ratios values did not reach ROC analyses were performed to show the relative potential of the PSA, fPSA/PSA ratio and hK2/fPSA ratio for discriminating prostate cancer from benign prostatic hyperplasia are: PSA (AUC:0.74,95% CI, 0.64-0.84),fPSA/PSA ratio (AUC:0.41,95% CI, 0.29-0.53),hK2/ fPSA (AUC:0.96,95% CI, 0.92-1,0) presented in Figure 1; which assure that diagnostic characteristics of kh2/fPSA ratio is better and gives the best discriminatory power of the other tests.

Discussion
More studies suggest that there is no single marker could be used for the detection of PCa, and the combination of multiple markers improves the specificity of detecting PCa and makes it statistically stronger, in order to achieve best PCa detection by reducing the number of the unnecessary biopsies.
Previous studies have indicated that hK2 has a valuable importance since its levels are significantly higher in men with prostate cancer; based in on these findings and considering many factors like the aging of the population, reporting delay and ethnicity, we decided to investigate the hK2 role and its combinations with PSA and fPSA in the detection of PCa program applied in some Syrian hospitals; witch include PSA tests with or without DRE examination.Our results were compatible with the previous studies as we found that hK2, fPSA, and PSA combinations are valuable in predicting PCa in our population.
As shown in Table I, PSA levels or fPSA/PSA ratio did not differ significantly between the two groups (P=0.11 and P=0.06 respectively).However the ratio hK2/fPSA did distinguish significantly between PCa and BPH patients (P=0.01).
Our findings are similar to the Scorilas et al. study which reported that some kallikreins are elevated in many malignancies, especially hK2 which increase in prostate cancer so it may be used as a marker in this cancer (Scorilas et al., 2003).Subsequently, Nam et al. and Martin et al studies demonstrate the utility of the ratio hK2/fPSA to discriminate prostate cancer patients from non-cancer men (P>0.0001),suggesting that this ratio may have a useful diagnostic role (Nam et al., 2000;Martin et al., 2004).
We also found that the ROC curves show a good diagnostic performance by high AUCs for both PSA levels and fPSA/PSA ratio alone (AUC:0.74 and AUC:0.41 respectively), but a much stronger effect for the hK2/ fPSA ratio (AUC:0.96).
These findings agrees with Raaijmakers et al. which confirm that the ratio hK2/fPSA was a better diagnosis marker than the ratio fPSA/PSA alone according to the ROC curves analysis and results (Raaijmakers et al., 2007).Also Martin et al. results who approves that hK2/ fPSA ratio (AUC: 08) was useful in discriminating the two diseases (PCa and BPH) (Martin et al., 2004).Similarly, Becker et al. and Scorilas et al. studies showed a higher specificity of hK2/fPSA ratio vs. fPSA/PSA ratio in low PSA levels (Becker et al., 2000;Scorilas et al., 2003).
An explanation of this effect may be found in the position of fPSA as denominator and hK2 as numerator.Because the levels of fPSA are lower in men with PCa and the levels of hK2 is higher in men with PCa, the ratio could result in a test with more discriminating power.
Hence, our results showed an improved specificity of PCa detection when combined hK2 with PSA and fPSA.
We need to mention that unlike PSA test, the DRE exam was not performed to all patients.Hence, we notice that most of the patients (16/35) were classified in intermediate-grade group, with only 8/35 patients in low-grade group.
Several limitations of our study deserve mentioning.The small number of patients included in our study mainly caused by financial limitations was an obstacle especially in having a more reliable statistical analysis data.
Also, the levels of fPSA may be influenced by freezing our samples at -80 o C; although the freezing was as soon as possible after centrifuging and the analysis was made immediately after thawing in order to avoid fPSA degradation in vitro.
As a conclusion, our results demonstrate that the measurement of serum hK2 levels is very important in prostate cancer detection because we have found that the use of ratio of hK2/fPSA is better than the use of fPSA/ PSA alone since hK2/fPSA offered the most statistical power in detecting prostate cancer, as measured by the AUC on ROC analysis.
We believe our study should be looked upon as a demonstration of the potential of hK2 assisting to predict the biopsy results for PCa.The hK2 has been shown to add important information in the detection of prostate cancer; especially the ratio hK2/fPSA improves specificity and hence reduces the number of unnecessary biopsies.

Table 1 . Mean Values and Ranges of the Variables
*PSA: Prostate Specific Antigen, fPSA: free Prostate Specific Antigen, PSA: Prostate Specific Antigen, hK2: Human Kallikrein-2, BPH: benign prostate hyperplasia, PCa: prostate cancer; * Significant statistical significance between the PCa and BPH groups (P=0.06).Values were significantly higher in PCa than BPH (P=0.01) for hK2/fPSA ratio in this study sample.