Imaging, Diagnosis, Prognosis

Calcium-Binding Proteins S100A8 and S100A9 as Novel Diagnostic Markers in Human Prostate Cancer Alexander Hermani,1Jochen Hess,2 BarbaraDeServi,1Senad Medunjanin,1Rainer Grobholz,3 Lutz Trojan,4 PeterAngel,2 and Doris Mayer1

Abstract Purpose: S100 proteins comprise a family of calcium-modulated proteins that have recently been associated with epithelial tumors. We examined the expression of two members of this family, S100A8 and S100A9, together with the S100 receptor RAGE (receptor for advanced glycation end products) in human prostate adenocarcinomas and in prostatic intraepithelial neoplasia. Experimental Design:Tissue specimens of 75 patients with organ-confined prostate cancer of different grades were analyzed by immunohistochemistry for expression of S100A8, S100A9, and RAGE. In addition, in situ hybridization of S100A8 and S100A9 was done for 20 cases. An ELISA was applied to determine serum concentrations of S100A9 in cancer patients compared with healthy controls or to patients with benign prostatic hyperplasia (BPH). Results: S100A8, S100A9, and RAGE were up-regulatedin prostatic intraepithelialneoplasia and preferentially in high-grade adenocarcinomas, whereas benign tissue was negative or showed weak expression of the proteins. There was a high degree of overlap of S100A8 and S100A9 expression patterns and of S100A8 or S100A9 and RAGE, respectively. Frequently, a gradient within the tumor tissue with an increased expression toward the invaded stroma of the prostate was observed. S100A9 serum levels were significantly elevated in cancer patients compared with BPH patients or healthy individuals. Conclusion: Our data suggest that enhanced expression of S100A8, S100A9, and RAGE is an early event in prostate tumorigenesis and may contribute to development and progression or extension of prostate carcinomas. Furthermore, S100A9 in serum may serve as useful marker to discriminate between prostate cancer and BPH.

Prostate cancer is the most frequently diagnosed malignancy in In an approach to identify genes that may play a role in men and the second leading cause of cancer deaths among men carcinogenesis (4, 5), we analyzed the expression of S100A8 in Western countries (1). There is an urgent need for and S100A9 in human prostate. The two proteins, S100A8 appropriate diagnostic and prognostic markers, in addition to (calgranulin A) and S100A9 (calgranulin B), also designated as the established serum protease prostate-specific antigen (PSA), MRP8 and MRP14, respectively, belong to the S100 multigenic allowing an early diagnosis and the prediction of the clinical family of calcium-modulated proteins of the EF-hand type behavior of individual tumors. Although the involvement of (6, 7). Most of the 20 thus far identified S100 genes, including certain genes and of chromosomal aberrations in prostate S100A8 and S100A9, are located in a gene cluster on carcinogenesis has been suggested (2, 3), the molecular chromosome 1q21, a region in which several rearrangements mechanisms underlying the initiation and progression of that occur during tumor development have been observed (8). prostate cancer are only poorly understood. Initially, S100A8 and S100A9 have been described mainly in neutrophils and macrophages and were shown to be involved in myeloid cell maturation (9, 10) and in inflammation (reviewed in ref. 11). The proteins were suggested to form 1 Authors’ Affiliations: Research Group Hormones and Signal Transduction and S100A8/S100A9 heterocomplexes (12, 13), which were shown 2Division of Signal Transduction and Growth Control, German Cancer Research Center, Heidelberg, Germany; and Departments of 3Pathology, and 4Urology, to be secreted by activated monocytes (14). Expression of University Hospital Mannheim, University of Heidelberg, Mannheim, Germany S100A8 and S100A9 in epithelial tissues was first described in Received 2/15/05; revised 4/11/05; accepted 4/29/05. context with squamous epithelia, e.g., various inflammatory Grant support:Tumorzentrum Heidelberg/Mannheim grant 781010. skin diseases (15), and with murine and human wound repair The costs of publication of this article were defrayed in part by the payment of page (16). More recently, an association of S100 protein expression charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. with adenocarcinomas in humans has emerged. Immunohis- Note: Supplementary data for this article are available at Clinical Cancer Research tochemical investigations have shown that S100A9 protein is Online (http://clincancerres.aacrjournals.org/). expressed in hepatocellular carcinomas, pulmonary adenocar- Requests for reprints: Doris Mayer, Research Group ‘‘Hormones and Signal cinomas, and invasive ductal carcinomas of the breast (17–19). Transduction,’’ Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Phone: 49-6221-423238; Fax: 49-6221-423237; In these tumors, elevated expression of S100A9 was correlated E-mail: [email protected]. with poor differentiation. In patients with ovarian carcinomas, F 2005 American Association for Cancer Research. S100A8 and S100A9 were found enriched in the cystic fluid and

Clin Cancer Res 2005;11(14) July 15, 2005 5146 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. S100A8 and S100A9 in Prostate Cancer in serum (20). Furthermore, overexpression of S100 mRNAs, antibodies (Santa Cruz, Heidelberg, Germany, and kindly provided by including S100A8 and S100A9, was reported for gastric cancers J. Roth, Institute of Experimental Dermatology, University of Mu¨nster, (21). In contrast, S100A8 and S100A9 are frequently down- Mu¨nster, Germany) and by the S100A9-specific mouse monoclonal regulated in poorly differentiated esophageal squamous cell antibodies S36.48 (a generous gift of Dr. P. Pfeifer, BMA Biomedicals, Augst, Switzerland) and 60B7 (kindly provided by R. Nozawa, carcinomas (22, 23). Recently, expression of S100A2 and Laboratory of Host Defenses, University of Shizuoka, Shizuoka, Japan). S100A4, two other proteins of the S100 family, was shown to A rabbit polyclonal antibody (Santa Cruz) was used for detection of be altered in human primary prostate cancer of different grades RAGE. A peroxidase 3,3V-diaminobenzidine system (DAKO, Hamburg, (24). For S100A2, a progressive loss with increasing tumor Germany) was used for the staining procedure. Finally, sections were grade was observed, whereas S100A4 showed increased counterstained with hematoxylin and mounted in glycerol/gelatin. expression in tumors with higher grades. For each antigen detected, staining intensity and area of positive For several members of the S100 protein family, a function as epithelial tissue were evaluated semiquantitatively. Staining intensities ligands for the receptor for advanced glycation end products were defined as moderate (1) or strong (2), whereas absent to faint (RAGE) has been discussed (25, 26). RAGE is a cell surface staining was considered as negative (0). For each section, areas with molecule that has been described as a multiligand receptor of similar staining intensity were evaluated together. The total areas with moderate and strong staining, respectively, were estimated as percentage the immunoglobulin superfamily. The interacting ligands of of the total area of a given epithelial growth pattern observed in a RAGE include advanced glycation end products, amphoterin, section. The cutoff for considerable positivity was set to 30%. When h -amyloids, and S100 proteins (25, 27–29). Direct interaction both moderate and strong staining was detected in the same section, the of S100 proteins with RAGE has been shown for S100A12 (EN- area representing the higher percentage of positivity, irrespective of the RAGE), S100B, S100A1, and S100P (25, 26, 30), and it is staining intensity, was used for statistical evaluation. Exact permutation suggested that also other S100 family members are able to test was used for comparison of benign prostatic tissue with tumor tissue modulate RAGE signaling. Engagement of RAGE by a ligand and Fisher’s exact test for comparison of low and high-grade tumors. triggers activation of central cellular pathways, including In situ hybridization. In situ hybridization of S100A8 and S100A9 mitogen-activated protein kinases, Cdc42/Rac, and nuclear mRNAs was done on 20 formaldehyde-fixed paraffin sections using factor nB signaling pathways, thereby influencing features like standard procedures. Sequences selected for the generation of ribo- probes specific for S100A8 and S100A9 transcripts, as verified by cell survival, cell motility, and inflammatory response (25, 31). BlastN2 search, were amplified by reverse transcription-PCR using gene Blockade of RAGE signaling function was reported to lead to specific primers for S100A8 (forward primer: 5V-ATTTCCATGCCGTC- decreased growth and metastasis of tumors in mice (31). TACAGG-3V; reverse primer: 5V-TGGCTTTCTTCATGGCTTTT-3V)and Expression of RAGE in prostate cancer was described with an S100A9 (forward primer: 5V-CAGCTGGAACGCAACATAGA-3V; reverse increased expression in metastatic compared with nonmeta- primer: 5V-CCACAGCCAAGACAGTTTGA-3V). PCR products represent- static cases (32). ing nucleotides 128 to 329 (S100A8, Genbank accession no. In the present study, we investigated the expression of NM_002964) and nucleotides 64 to 537 (S100A9, Genbank accession S100A8 and S100A9 and of their putative receptor RAGE in no. NM_002965) were cloned into pGEM-T vector (Promega, human prostatic tissue. In addition, we tested the value of Mannheim, Germany) and confirmed by sequencing. Digoxigenin- S100A9 as a serum marker for prostate cancer. labeled antisense and sense riboprobes were generated by in vitro transcription according to the digoxigenin application manual of Roche (Mannheim, Germany). Hybridization was done at 55jC Materials and Methods overnight. Hybridized probes were detected using an alkaline phosphatase–conjugated antidigoxigenin antibody (Roche) and an Tissue samples and patient sera. Prostate tissue was obtained with alkaline phosphatase reaction using nitroblue tetrazolium/5-bromo-4- consent from patients who underwent radical prostatectomy after chloro-3-indolyl phosphate as substrates. diagnosis of cancer. Tumors were diagnosed and classified according to ELISA. A sandwich immunosorbent assay for detection of S100A9 the Gleason system (33). H&E–stained paraffin sections serial to those (BMA Biomedicals) was used to determine S100A9 concentrations in submitted to in situ studies were used for verification of the diagnosis in serum. The immunoassay was carried out following the manufacturer’s the respective tissue specimens investigated. A total of 75 specimens instructions. For statistical comparison of groups of individual values, from 24 low-grade (Gleason score 5-6), 19 Gleason score 7, and 32 the Mann-Whitney test was applied and data were used for evaluation high-grade (Gleason score 8-10) organ-confined prostate cancers, scaled of a receiver operator characteristic analysis. according to Humphrey (34), were investigated. In these specimens, we observed 18 prostatic intraepithelial neoplasias (PIN). In 48 specimens, extended areas of benign prostatic tissue, including normal glands and Results single hyperplastic glands, were present. Serum samples were obtained before surgery with consent from Immunohistochemical staining of human prostate tissue patients with subsequently diagnosed prostate cancer (n = 56) and sections from 75 cases of prostate cancer revealed extensive from patients with benign prostatic hyperplasia (BPH, n = 56) as well expression of S100A8 and S100A9 proteins in adenocarcino- as from 18 healthy men. Serum PSA of patients with BPH or cancer was mas of 58 (77%) and 51 (68%) cases, respectively. Protein determined before surgery. At time of surgery, patients’ age ranged expression was evaluated semiquantitatively in tumor and between 55 and 82 years, and healthy men’s age ranged between 33 surrounding benign tissue. Both proteins showed a significant and 50 years. up-regulation (P < 0.0001, S100A8 and P < 0.0001, S100A9) Immunohistochemistry. For immunohistochemical staining of pro- in adenocarcinomas compared with benign prostatic tissue teins, dewaxed formalin-fixed paraffin sections (4 Am) were rehydrated and submitted to epitope retrieval by microwaving either in 0.1 mol/L (Table 1). Comparison of low-grade and high-grade cancers Tris (pH 9.5)/5% urea buffer for the detection of S100A8 and S100A9 revealed a preferential positivity of high-grade cancers (P = proteins or in 0.01 mol/L citrate buffer (pH 6.0), respectively, for the 0.053, S100A8 and P = 0.027, S100A9). Cancers diagnosed as detection of RAGE. After blocking with 5% bovine serum albumin in Gleason 7 showed an intermediate ratio of positive to total PBS, S100A8 and S100A9 were detected by specific rabbit polyclonal number of cases compared with low-grade and high-grade

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Ta b l e 1. Evaluation of S100A8, S100A9, and RAGE positivity in human prostate cancer

Grading n S100A8 S100A9 RAGE Positive cases P* Positive cases P Positive cases P c Benign prostatic tissue 48 7/48 <0.0001 5/48 <0.0001 2/48 <0.0001 Low grade (Gleason score 5-6) 24 15/24 14/24 12/24 Gleason score 7 19 13/19 0.053 9/19 0.027 13/19 0.16 High grade (Gleason score 8-10) 32 28/32 28/32 23/32 PIN 18 8/18 0.053 8/18 0.016 6/18 0.013

NOTE: Positively stained tissue was evaluated. Low-grade cancers were compared withhigh-grade cancers and PIN with benign prostatic tissue using Fisher’sexact test. *P < 0.05 was considered statistically significant. cBenign prostatic tissue was compared with respective tumor tissue of the same cases using an exact permutation test. P values shown for benign prostatic tissue document significantly lower expression of S100A8, S100A9, and RAGE in benign prostatic tissue than in adenocarcinomas of all grades. cancers for S100A8. For S100A9, the relative amount of expression was detected in single benign prostate glands usually positive Gleason 7 cases was lower than in low-grade cancers. adjacent to tumor tissue and commonly restricted to the basal There was no obvious difference between cancer grades cell layer (Fig. 1C). Benign hyperplastic glands showed no, or concerning staining intensity. only weak, expression of the S100 proteins (Fig. 1D). In A marked overlap between S100A8 and S100A9 staining addition, positive staining was found in 8 of 18 PINs, patterns was apparent in corresponding tissue areas of serial indicating an up-regulation of S100A8 and S100A9 expression sections (Fig. 1A and B). S100A8 and S100A9 protein also in precancerous lesions (Fig. 1E).

Fig. 1. Immunohistochemical staining of S100A8 (A) and S100A9 (B-L)proteinin human prostate tissue. A and B, serial sections of an adenocarcinoma stained with S100A8 and S100A9 antibodies showing an overlapping expression pattern; C, benign gland showing S100A9-positive basal cells; D, weak to absent staining of S100A9 in hyperplastic glands; E, positive staining of PIN; F,expressiongradientwith increased positivity of tumor cells toward the stroma; G, S100A9 expression in a perineural tumor, nf, nerve fiber; H, high-grade adenocarcinoma with cribriform pattern; I,partiallyorcompletely stained glands beside negative glands; J, heterogeneous expression pattern of S100A9 in a low-grade tumor; K,tumor cells with nuclear staining; L, positive tissue histiocytes demonstrating specificity of antibody reaction. Magnifications: Â45 (A and B), Â12 0 (C, I, K ,andL), and Â60 (D-H and J).

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In 27 of 75 cases (36%), a staining gradient for the two results obtained by immunohistochemistry. Positive reaction S100 proteins was observed with an increased staining was observed in basal cells of benign glands (Fig. 2A and D) intensity in tumor cells adjacent to the stromal compartment and in adenocarcinomas of different grades (Fig. 2B, E, G, and or the capsule of the prostate (Fig. 1F). This gradient pattern H). The staining showed varying intensity in different positive was observed in carcinomas independently from the tumor tumor areas, including PIN, the latter showing positive grade. In addition, S100A8 and S100A9 expression was staining both in basal cells and in intraepithelial neoplastic frequently observed in tumor tissue surrounding nerve fibers cells (Fig. 2I). (Fig. 1G). In high-grade tumors, as shown for a cribriform Expression of the S100 receptor RAGE in prostate cancer was tumor gland (Fig. 1H), and also in other growth patterns, a previously reported by Kuniyasu et al. (32). We were interested marked heterogeneity in immunoreactivity was generally whether expression of RAGE coincides with the expression of observed. Tumor glands showing no staining were found S100A8 and S100A9. Immunohistochemical analysis of RAGE adjacent to partly positive and strongly positive glands (Fig. on the same set of prostate cancer tissues used for detection of 1I). Typically, groups of tumor glands or individual glands as the S100 proteins revealed a marked overlap of RAGE with well as single cells within the glands showed positive reaction S100A8 or S100A9 staining patterns (Fig. 3A-D and Supple- (Fig. 1J). In 29 of 75 cases (39%), nuclear staining in addition mental Figs. S1 and S2). Also, for RAGE, a strong expression in to, or instead of, cytoplasmic staining was observed at least in tumor tissue invading the stroma or adjacent to connective subpopulations of cells (Fig. 1K), although without visible tissue of the prostate capsule was observed (Fig. 3E). In correlation with the tumor grade. The different antibodies used addition, similar to S100A8 and S100A9, strong positivity was for detection of S100 proteins yielded similar staining patterns. found in perineural tumor tissue (Fig. 3F). RAGE expression S100A8 and S100A9 were detected in tissue histiocytes with all was detected in adenocarcinomas of different grades with a antibodies tested (Fig. 1L) independent of the histopathologic weak trend to increased positivity of high-grade cancers (P = properties of different specimens, indicating a specific reaction 0.16; Table 1). under the experimental conditions applied. Additional stain- We next addressed the question of a putative diagnostic ing was found in blood vessels, whereas stromal cells usually value of the S100 proteins as serum markers. For this purpose, showed no specific staining reaction, although positively an ELISA that was specific for S100A9 was selected. stained areas where observed in the stromal compartment. Comparing S100A9 serum levels of 37 cases of prostate Expression of S100A8 and S100A9 by stromal cells, however, cancer with 18 healthy controls, we discovered increased was not detected by RNA in situ hybridization of S100A8 and concentrations of S100A9 in serum of cancer patients (P = S100A9 mRNAs on prostate tissue sections using specific 0.0037; Fig. 4A). This observation, together with the finding of probes. poor expression of S100A9 in benign hyperplastic tissue, In situ hybridization revealed a marked overlap in S100A8 prompted us to compare a larger number of prostate cancer and S100A9 mRNA expression patterns in agreement with cases (n = 56) with individuals diagnosed for BPH (n = 56).

Fig. 2. Analysis of S100A8 and S100A9 mRNA expression by in situ hybridization. A and D, normal glands showing expression of S100A8 (A)andS100A9(D) in basal cells; B and E, serial sections of an adenocarcinoma hybridized with S100A8 (B)andS100A9(E) antisense probes showing an overlapping expression pattern of S100A8 and S100A9; C and F, hybridization results with the respective labeled S100A8 (C) and S100A9 (F)sense probes as negative control; G to I, in situ hybridization of S100A9, tumor with an adjacent benign gland (G, arrow), high-grade adenocarcinoma (H), and PIN (I). S100A9 is expressed in basal cells and in intraepithelial neoplastic cells. Magnifications: Â12 0 (A, D,andG)and Â60 (B, C, E, F, H ,andI).

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Fig. 3. Detection of RAGE by immunohistochemistry. A to C, serial sections of prostate cancer tissue stained for RAGE (A), S100A8 (B),andS100A9(C) showing an overlapping expression pattern; D, expression pattern of RAGE in a section serial to Fig.1Aand B, which show similar patterns of S100A8 and S100A9 staining; E, increased RAGE staining intensity in tumor tissue adjacent to stroma; F, RAGE-positive perineural tumor; Magnifications: Â45 (A-E); Â60 (F).

We found significantly increased S100A9 serum levels in that elevated S100A9 serum concentrations are connected with prostate cancer patients in comparison with BPH patients (P < cancer patients but not with patients suffering from a benign 0.0001; Fig. 4B), the latter exhibiting values that were similar prostatic disease. S100A9 levels of low-grade and high-grade to values obtained for healthy individuals. These data indicate cancer patients were both elevated without significant differences

Fig. 4. S100A9 serum concentrations in prostate cancer patients (CaP) compared with healthy controls and BPH. A,boxplot showing elevated S100A9 serum levels in patients with prostate cancer compared with healthy men (*P = 0.0037); B, S100A9 levels are significantly increased in cancer patients compared with BPH patients (**P =1.6Â 10 À7); C and D, receiver operator characteristic analysis of S100A9 and PSA comparing BPH and prostate cancer for all patients analyzed (C) and for patients with PSA < 10 ng/mL( D).

Clin Cancer Res 2005;11(14) July 15, 2005 5150 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. S100A8 and S100A9 in Prostate Cancer between the two groups. A comparison of S100A9 concen- adenocarcinomas and in PIN, suggesting an early involvement trations and the corresponding PSA serum levels in a receiver of the proteins in prostate cancer. It is intriguing that the operator characteristic analysis revealed that S100A9 values tumor cells, as precursors of which the luminal cells are provided a diagnostic reliability that was similar to the one considered (39), greatly restore the ability of S100A8 and based on PSA values in the cases analyzed (Fig. 4C). However, S100A9 expression, whereas in benign glands expression was PSA and S100A9 were independent parameters. The evaluation found only in the basal cell layer, which is responsible for the of S100A9 serum levels of patients with PSA values <10 ng/mL epithelial/stromal contact. Recently, different compounds that still allowed a highly significant discrimination of BPH and also participate in the formation of extracellular matrix of cancer by S100A9 (P < 0.0001), exceeding that achieved with connective tissue or basement lamina were found to associate PSA (P = 0.02; Fig. 4D; Table 2). with S100 proteins. For neutrophils, involvement of S100A8 The present data show that S100A8 and S100A9 mRNA and and S100A9 in the modulation of transendothelial migration proteins are up-regulated in PIN and in prostatic adenocarci- by interaction with glycosaminoglycans and carboxylated nomas of different histopathologic grades. The expression glycans has been shown (40, 41). In addition, S100A8 and patterns of both proteins are similar to that observed for the S100A9 have been described to increase adhesion of mono- S100 receptor RAGE. S100A9 was additionally detected with cytes to fibronectin (42). Worth mentioning in this context is significantly increased levels in sera of prostate cancer patients, the observed close spatial correlation of S100A8- and S100A9- whereas individuals with BPH displayed S100A9 levels within a positive basal cells and the stroma on the one hand and reference range. positive cancer cells and stroma on the other hand (see also Supplemental Fig. S2). Our finding of increased S100A8, S100A9, and RAGE expression toward the invaded connective Discussion tissue further suggests a functional relation with the extension of epithelial tissue. With respect to migration events, an S100 proteins are involved in a variety of biological involvement of RAGE engaged by S100B, S100A1, and processes and are considered to be expressed in a cell type– amphoterin has been associated with neurite outgrowth or tissue-specific manner. However, deregulated expression of (26, 28). Furthermore, RAGE has been shown to play an im- several members of the S100 family, including S100A8 and portant role for tumor growth and metastases formation in S100A9, under various pathologic conditions has been mice (31). Engagement of RAGE is known to trigger different reported (7, 35). Besides the well-documented expression in signaling cascades, usually leading to activation of the tran- inflammatory skin diseases (15), S100A8 and S100A9 and scription factor nuclear factor nB (25, 26, 43). Interestingly, other members of the S100 family recently have been overexpression of p65, the active subunit of nuclear factor nB, associated with rheumatoid arthritis (36), psoriasis (37), was found to occur as an early event in the development of cutaneous wound repair (16), and various experimental and prostate cancer (44). Furthermore, active nuclear factor nB was human neoplasms (4, 17–19). It is noteworthy that the frequently detected in prostatic adenocarcinomas with peri- patterns of differential gene expression in cancer, particularly neural location and has been linked functionally to mecha- in prostate and liver cancer, strongly correlate with the pattern nisms that promote perineural invasion (45), which is the of differential gene expression during wound-healing process- major mechanism of prostate cancer spread outside the es (38). These observations suggest additional functions of the prostate (46). Together with our findings of enhanced S100 protein family in injury and disease pathogenesis. Our expression of RAGE and S100A8/S100A9 in prostate cancer findings on the expression of S100A8 and S100A9 in prostatic and of frequent positivity of perineural tumor tissue (see also adenocarcinomas support this hypothesis. Using immunohis- Supplemental Figs. S1 and S2), it is intriguing to speculate tochemistry and RNA in situ hybridization on human prostate that S100/RAGE signaling events may be involved in the samples of patients with diagnosed prostate cancer, we could development of survival or growth advantages via nuclear show that S100A8 and S100A9 are up-regulated in prostatic factor nB. Therefore, the S100/RAGE signaling pathway may represent a suitable target for prevention or treatment strategies for prostate cancer. In addition, our data provide Ta b l e 2 . Statistical evaluation of S100A9 and PSA evidence for a diagnostic value of the S100 proteins. We found a significant increase of S100A9 serum concentrations serum levels for dissection of BPH patients from in prostate cancer patients compared with healthy individuals cancer patients or to BPH patients. In comparison to PSA, currently the best diagnostic serum marker for prostate cancer, S100A9 distin- Patient group nPguished with a higher sensitivity between BPH and cancer CaP BPH S100A9 PSA patient groups with low PSA levels. Thus, S100A9 may represent a helpful candidate for the dissection of prostate PSA > 0 ng/mL56 56 1.6 Â 10 À7 1. 5 Â 10À6 cancer and BPH cases, where PSA measurement fails to PSA < 10 ng/mL34 56 5.1 Â10 À6 0.02 CaP provide reliable diagnostic information. Most recently, PSA < 10 ng/mL34 49 1.3 Â 10À5 0.0006 CaP/BPH S100A9 was discovered by another group in voided urine after prostatic massage from patients with prostate cancer NOTE: P values were calculated, using Mann-Whitney test, for comparison of using two-dimensional gel electrophoresis (47). Together with S100A9 and PSA in cancer patients with BPH patients grouped regarding PSA levels. the present study, this finding further supports the putative Abbreviation: CaP, prostate cancer. diagnostic value of S100A9 measurement in body fluids as a marker for prostate cancer.

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In summary, our data show that S100A8 and S100A9 are Acknowledgments up-regulated in adenocarcinomas of the prostate and in PIN. The expression of both proteins markedly overlaps We thank A. Benner (Central Unit of Biostatistics, German Cancer Research Cen- with the expression of the S100 receptor RAGE, which has ter, Heidelberg, Germany) for advice with the statistical analysis and C. Gebhardt for been associated with invasive potential of different tumors. helpful discussion; J. Roth (Institute of Experimental Dermatology, University of Mu« nster, Germany)for generously providing the rabbit polyclonal antibodies against The use of S100A9 as a serum marker for the early S100A8 and S100A9; P. Pfeifer (BMA Biomedicals AG, Augst, Switzerland) for the detection of prostate cancer additionally may help to gift of monoclonal antibody clone S36.48; and R. Nozawa (Laboratory of Host facilitate dissection of prostate cancer patients from patients Defenses, University of Shizuoka, Japan) for generously providing the monoclonal with BPH. antibody 60B7.

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