since serum prostate specific antigen ( psa ) was introduced as an early detection marker of prostate cancer ( pca ) in the 1980s , several trials involving novel and more precise biomarkers have failed to provide new markers with definitive clinical relevance and applicability . considering the low sensitivity of psa and the importance of distinguishing latent from clinically significant pca to optimize treatment , the need for specific diagnostic markers is apparent . recent investigations have pointed to caveolin-1 , exosomes , and pca-3   as diagnostic markers . however , their clinical applicability is still to be determined . this pilot study was designed to investigate the potential of chemokines as biomarkers for pca . chemokines are low molecular weight secretory proteins divided into four groups ( cxc , cc , c , and the cx3c family ) according to the arrangement of conserved cysteine ( c ) residues of the mature proteins , which interact as ligands ( cl ) with their corresponding receptors ( cr ) . due to their proinflammatory properties , chemokines regulate chemotaxis and metabolic activity of leukocytes circulating in the body and therefore significantly affect normal development , angiogenesis and atherosclerosis . they are involved in many aspects of oncogenesis , including regulation of cancer cell growth , dissemination and host - tumor response . hence , various tumor types differentially trigger a complex chemokine network determining the qualitative and quantitative degree of immunocellular infiltration . alterations of the chemokine expression level are significantly associated with more aggressive disease and poorer prognosis in diverse malignancies . however preliminary work pointed out the potential of cxc chemokines as prognostic biomarkers for pca . to assess the diagnostic relevance of chemokines in pca , the expression of a broad panel of chemokines in tumor tissue and their serum levels in patients with pca was investigated and associations tested with clinical and pathological parameters . patients undergoing radical prostatectomy for biopsyproven pca between january 2008 and october 2010 in the department of urology , goethe university , frankfurt , germany , were included in the study after signing informed consent for utilization of biomaterials and further scientific assessment . the tumor stage and grade was determined according to the sixth edition of the tnm classification . clinical parameters , including the patient s age and preoperative and postoperative psa values , were documented . ten milliliters of peripheral blood was drawn from patients the day before surgery and 5 to 7 days after surgery . as controls , 10 ml blood was drawn from 15 healthy , male , age - matched volunteers . blood samples were allowed to coagulate and then centrifuged at 3,000 rpm at 4c for 10 minutes . the human cytokine multiplex bead assay kit ( millipore , darmstadt , germany ) was used to determine cxcl12 , cxcl13 , cx3cl1 , ccl2 , ccl5 , and ccl20 in serum , according to the manufacturer s instructions . anti - chemokine antibody coated beads ( 25 l ) were pipetted into each well . samples ( serum ) or standards ( 25 l ) were then added to the wells and incubated with the beads for 2 hours . after washing , biotin conjugated detector antibody was added and incubated for 1 hour , followed by a 30-minute incubation with r - phycoerythrin conjugated streptavidin . analysis was carried out with a luminex 100 ( gurce , nivelles , belgium ) and expressed as pg / ml . immediately after resection , tissue was transferred to the department of pathology and reviewed by a pathologist . tumor tissue , as well as adjacent normal tissue , was fresh - frozen in liquid nitrogen . total rna from solid tumor tissue of patients with pca and adjacent normal tissue was extracted using rneasy kit ( qiagen , hilden , germany ) , according to the manufacturer s instructions . solid tissues were sliced into small pieces and transferred to a precellys tube pre - filled with 1.4-mm ceramic beads ( bertin technologies , saint - quentin - en - yvelines cedex , france ) containing lysis buffer . cells were lysed under rapid agitation at 6,500 rpm for 1 minute in three cycles using a precellys 24 homogenizer ( bertin technologies ) . cdna was synthesized from 1 g of total rna with oligo(dt ) primer using an affinityscript qpcr cdna synthesis kit ( stratagene , santa clara , ca ) , according to the manufacturer s instructions . the amount and quality of rna were analyzed using nanovue ( ge healthcare , piscataway , nj ) and bioanalyzer ( agilent technologies , palo alto , ca ) , respectively . real - time quantitative pcr ( qrt - pcr ) was performed using rt sybr - green / roxqpcr master mix and gene specific primers for cl and cr ( both from sabioscience , madison , wi ) ( table 1 ) , according to the manufacturer s protocol . gene expression ( fold change ) was calculated using the ct method , after normalizing the tumor sample with a corresponding normal sample . statistical significance of differences between parameters within the same group was tested by the wilcoxon matched pairs test and in different groups by the wilcoxon mann - whitney test . subgroup analyses employed the kruskal - wallis test with the iman - conover method ( bonferroni- holm correction ) . null hypotheses of no difference were rejected if p - values were less than 0.05 . patients undergoing radical prostatectomy for biopsyproven pca between january 2008 and october 2010 in the department of urology , goethe university , frankfurt , germany , were included in the study after signing informed consent for utilization of biomaterials and further scientific assessment . the tumor stage and grade was determined according to the sixth edition of the tnm classification . clinical parameters , including the patient s age and preoperative and postoperative psa values , were documented . ten milliliters of peripheral blood was drawn from patients the day before surgery and 5 to 7 days after surgery . as controls , 10 ml blood was drawn from 15 healthy , male , age - matched volunteers . blood samples were allowed to coagulate and then centrifuged at 3,000 rpm at 4c for 10 minutes . the human cytokine multiplex bead assay kit ( millipore , darmstadt , germany ) was used to determine cxcl12 , cxcl13 , cx3cl1 , ccl2 , ccl5 , and ccl20 in serum , according to the manufacturer s instructions . anti - chemokine antibody coated beads ( 25 l ) were pipetted into each well . samples ( serum ) or standards ( 25 l ) were then added to the wells and incubated with the beads for 2 hours . after washing , biotin conjugated detector antibody was added and incubated for 1 hour , followed by a 30-minute incubation with r - phycoerythrin conjugated streptavidin . analysis was carried out with a luminex 100 ( gurce , nivelles , belgium ) and expressed as pg / ml . immediately after resection , tissue was transferred to the department of pathology and reviewed by a pathologist . tumor tissue , as well as adjacent normal tissue , was fresh - frozen in liquid nitrogen . total rna from solid tumor tissue of patients with pca and adjacent normal tissue was extracted using rneasy kit ( qiagen , hilden , germany ) , according to the manufacturer s instructions . solid tissues were sliced into small pieces and transferred to a precellys tube pre - filled with 1.4-mm ceramic beads ( bertin technologies , saint - quentin - en - yvelines cedex , france ) containing lysis buffer . cells were lysed under rapid agitation at 6,500 rpm for 1 minute in three cycles using a precellys 24 homogenizer ( bertin technologies ) . cdna was synthesized from 1 g of total rna with oligo(dt ) primer using an affinityscript qpcr cdna synthesis kit ( stratagene , santa clara , ca ) , according to the manufacturer s instructions . the amount and quality of rna were analyzed using nanovue ( ge healthcare , piscataway , nj ) and bioanalyzer ( agilent technologies , palo alto , ca ) , respectively . real - time quantitative pcr ( qrt - pcr ) was performed using rt sybr - green / roxqpcr master mix and gene specific primers for cl and cr ( both from sabioscience , madison , wi ) ( table 1 ) , according to the manufacturer s protocol . glyceraldehyde 3-phosphate dehydrogenase and -actin were used as housekeeping gene and for normalization . gene expression ( fold change ) was calculated using the ct method , after normalizing the tumor sample with a corresponding normal sample . all experiments were performed three to six times . statistical significance of differences between parameters within the same group was tested by the wilcoxon matched pairs test and in different groups by the wilcoxon mann - whitney test . subgroup analyses employed the kruskal - wallis test with the iman - conover method ( bonferroni- holm correction ) . null hypotheses of no difference were rejected if p - values were less than 0.05 . the study population consisted of 39 patients ( table 2 ) with a mean age at tumor diagnosis of 68.95.6 years . none of the patients had evident clinical signs of infection or acute or chronic inflammation at the time of surgery . histologically , all tumors were conventional acinar adenocarcinomas . in four patients the final histopathological analysis revealed positive margins ( r1 ) . all patients with microscopic residual disease ( r1 ) and five other patients ( 2 pt3a , 2 pt3b and 1 pt2c with biochemical relapse after surgery ) received radiation therapy . the mean follow - up time was 18.115.1 months . at the study endpoint , all patients were alive without signs of biochemical or clinical recurrence . using qrt - pcr , significantly higher expression of ccl2 and ccr6 in tumor tissue was observed , compared to normal adjacent tissue ( fig . the expression of cxcr4 , cx3cr1 , ccr2 , ccr5 , ccr7 , ccl5 , and ccl20 was higher and that of cxcr3 , cxcr5 , cxcr7 , cxcl12 , cxcl13 , and cx3cl1 lower in the tumor tissue without reaching significance . the association of gene expression of the chemokines in the tumor tissue was tested with clinical and oncologic parameters ( fig . no association was found between the other investigated chemokines and t stage , gleason score , grade , r1- and pn1-status , preoperative and postoperative psa or biochemical relapse . ccl2 was significantly over - expressed both preoperatively and postoperatively in pca patients , compared to controls . significantly diminished concentrations of ccl5 in preoperative and postoperative samples were detected , compared to controls . the concentration of ccl20 increased significantly in the control group , compared to both preoperative and postoperative serum concentration in pca patients . the serum concentration of cx3cl1 also increased significantly in the controls compared to that in patient serum , both preoperatively and postoperatively . the level of cxcl12 and cxcl13 did not significantly differ among any of the groups . the association of chemokine serum concentrations with t stage , gleason score , grade , r1- and pn1-status , preoperative psa as well as biochemical relapse were then tested . ccl2 was significantly negatively correlated with the preoperative psa value ( spearman s correlation coefficient rho , 0.48 ; p=0.035 ) . chemokines are small secreted chemotactic cytokines that coordinate immunological machinery by promoting leukocyte migration and cross - communication among different cell types of the immune system . due to similarities between leukocyte trafficking and cancer cell dissemination , recent studies regarding different tumor entities point to significant involvement of chemokines in facilitating tumor promotion and dissemination . based on the evaluation of 15 members of the chemokine family , evidence is provided here that the gene activity of particular molecules is altered in pca , compared to normal tissue . ccl2 and ccr6 mrna were overexpressed in the tumor specimens , compared to adjacent normal tissue . ccl2 , but not ccr6 , was also significantly augmented in serum from tumor patients compared to healthy controls , indicating a critical role of ccl2 in neoplastic progression . a meta - analysis of gene - expression data has identified ccl2 as a primary driver in pca development . immunohistochemistry and immunofluorescence analysis of the chemokine expression profile reveal ccl2 to be predominantly expressed at the tumor site , more than for other chemokines . tissue microarray analysis shows that levels of ccl2 expression in pca tissue are greater than in non - neoplastic epithelia . therefore , ccl2 could serve as a specific diagnostic marker , closely associated with pca , even though ccl2 did not reach control values postoperatively . a possible explanation is that immunologic processes are still active five to seven days after surgical intervention . studies incorporating blood sampling at later post - surgical dates should , therefore , more clearly define the specific diagnostic value of ccl2 . one problem in employing ccl2 to diagnose pca is the lack of association with tumor stage and grade , meaning that ccl2 may not allow evaluation of tumor dissemination . shirotake et al .   found differences in ccl2 levels only between very early localized , and advanced castration resistant pca . in contrast , the report from lu et al .   pointed to a significant relationship between ccl2 level and gleason score . different analytic techniques ( microarray ) or a selected patient cohort ( asian ) may account for this discrepancy . obviously , further patient studies are necessary to clarify the diagnostic value of ccl2 for pca . ccl5 , ccl20 , and cx3cl1 were all reduced in patient sera , whereas no differences were seen in the gene analyses . therefore , pca progression seems accompanied by alterations in ccl5 , ccl20 , and cx3cl1 protein release but not in transcriptional modification . , this study was limited to pca with serum psa values of < 10 ng / ml , which can not be applied to our cohort with 41% of patients with a preoperative psa > 10 ng / ml . immunohistochemistry of 80 pca cases revealed absent or weak ccl20 expression in 61% , whereas strong staining was evident in only 10% of patients . this may corroborate our ccl20 data , although serum probes were not analyzed in this study . cx3cl1 is down - regulated in high - risk pca patients who develop biochemical recurrence following prostatectomy . the authors of this case - control study recommend cx3cl1 as a potential indicator of future recurrence . cxcl12 is suggested to facilitate tumor cell recruitment to the bone marrow and a high cxcl12 expression is associated with lymph node metastatic prostate carcinoma compared with non  lymph - node metastatic cancer in an immunohistochemical analysis . semiquantitative pcr evaluation points to an enhanced cxcl12 mrna expression level in prostate tumor , compared to adjacent ' normal ' tissue . serum cxcl12 levels are significantly higher for men who were biopsy positive compared to those who were biopsy negative for pca . in contrast , agarwal et al . , who performed multiplex enzyme - linked immunosorbent assay assays on 272 men , found no association between cxcl12 and pca status . a final assessment of the role of cxcl12 in pca is difficult since different techniques have been applied in relevant studies . the ccl2 changes dominate other chemokine changes , since pca related ccl2 alterations were found at both gene and protein level . however , ccr6 , ccl5 , ccl20 , and cx3cl1 might be further candidates pointing to neoplastic transformation . further large - volume biomarker studies are required to expand and validate the preliminary findings of this pilot study .