STEAP1 protein overexpression is an independent marker for biochemical recurrence in prostate...

STEAP1 protein overexpression is an independent marker forbiochemical recurrence in prostate carcinoma

Shadia M Ihlaseh-Catalano,1,* Sandra A Drigo,2,3,* Carlos M N de Jesus,4 Maria

Aparecida C Domingues,1 Jos�e Carlos S Trindade Filho,4 Jo~ao Lauro V de Camargo1,* &

Silvia R Rogatto2,3,*1Department of Pathology, School of Medicine, UNESP, S~ao Paulo State University, Botucatu, Brazil, 2NeoGene

Laboratory, Department of Urology, School of Medicine, UNESP, S~ao Paulo State University, Botucatu, Brazil, 3CIPE,

AC Camargo Cancer Centre, S~ao Paulo, Brazil, and 4Department of Urology, School of Medicine, UNESP, S~ao Paulo

State University, Botucatu, Brazil

Date of submission 18 April 2013Accepted for publication 11 July 2013Published online Article Accepted 13 July 2013

Ihlaseh-CatalanoSM,DrigoSA, de JesusCMN,DominguesMAC,TrindadeFilho JCS , deCamargo J LV&Rogatto SR

(2013)Histopathology63, 678–685

STEAP1proteinoverexpression is an independentmarker for biochemical recurrence inprostatecarcinoma

Aims: To investigate the prognostic value of expres-sion levels of the genes STEAP1 and STEAP2, and ofSTEAP1 protein, in prostate carcinomas (PCa).Methods and results: STEAP1 and STEAP2 transcriptlevels were evaluated by RT-qPCR in samples from 35PCa, 24 adjacent non-neoplastic prostate (AdjP) tis-sues, five cases of benign prostatic hyperplasia (BPH),and two histologically normal prostates (N). STEAP1expression was assessed by immunohistochemistry insamples from 198 PCa, 76 AdjP, 22 BPH, and two N.The findings were compared with clinical and patho-logical parameters and patient outcome. STEAP1 andSTEAP2 transcript analysis showed no differences

between the groups tested. Although not significant,higher STEAP1 mRNA levels were detected in tumourswith high Gleason scores and in patients who pre-sented with biochemical recurrence (BCR). STEAP1overexpression was detected in PCa, and was signifi-cantly associated with high-grade Gleason scores, sem-inal vesicle invasion, BCR, and worse outcome(metastasis or PCa-specific death). STEAP1 overexpres-sion was significantly associated with shorter BCR-freesurvival. Multivariate analysis revealed that STEAP1 isan independent marker for BCR.Conclusions: These findings provide evidence thatSTEAP1 is a biomarker of worse prognosis in PCa patients.

Keywords: biological markers, prognosis, prostatic neoplasms, STEAP2, STEAP1

Introduction

Prostate carcinoma (PCa) is a heterogeneous diseasewith regard to its clinical, morphological and molecu-lar presentation. The currently used prognostic indi-cators, such as preoperative levels of prostate-specificantigen (PSA), Gleason score, and TNM staging, have

limitations in the accurate identification of patientswith aggressive tumours.1 Biochemical recurrence(BCR) has been widely used to monitor diseaserelapse,2,3 being considered as a risk factor for metas-tasis and PCa-specific mortality.4,5 Patients with BCRwith or without clinical evidence of local recurrenceare eligible for salvage therapy that might reduce therisk of metastasis.6 These indicators, however, identifyonly a subset of patients at risk, so the search fornovel prognostic markers and molecular markers fortargeted therapy remains relevant.Molecular studies have identified potential diagnos-

tic and prognostic markers and therapeutic targets for

Address for correspondence: S R Rogatto, NeoGene Laborat�orio,

Departamento de Urologia, FMB-UNESP, Distrito de Rubi~ao Jr, S/N,

Botucatu 18618-970, S~ao Paulo, Brazil. e-mail: rogatto@fmb.

unesp.br

*S.M.I, S.A.D., J.L.V.C and S.R.R. contributed equally to this work.

© 2013 John Wiley & Sons Ltd.

Histopathology 2013, 63, 678–685. DOI: 10.1111/his.12226

different tumours. Array-comparative genomic hybrid-ization (aCGH) is a high-throughput procedure thatpermits the identification of genomic copy numberimbalances. With this technique, it is possible to defineamplicons and to identify candidate genes associatedwith prognosis in a particular group of tumours. UsingaCGH in PCa, our group identified an ampliconmapped to 7q21.13, which includes STEAP1 andSTEAP2 (unpublished data). The STEAP (six-trans-membrane epithelial antigen of prostate) gene family ispredominantly expressed in epithelial tissues, and theprotein products act as ion channels, transport pro-teins, or gap-junction proteins.7 STEAP1 overexpres-sion has been reported in human cancer tissues andcell lines, including those of prostate, bladder, colonand ovarian tumours, and Ewing’s sarcoma.7,8 Hightranscript and STEAP1 protein levels have beenreported in PCa samples.8 In addition, STEAP1 overex-pression was described in primary prostate tumoursand in cell lines obtained from PCa metastases estab-lished in lymph nodes and bones.9

STEAP1 is predominantly expressed at the plasmamembrane of the prostate secretory epithelium.7,8

Taken together with its six-transmembrane topology,these findings suggest that STEAP1 acts as a chan-nel/transporter protein at the cell–cell junction.7,9,10

The strategic localization of STEAP1 at the cell sur-face, its low expression in normal tissues and its over-expression in neoplasia mark this protein as apotential target for diagnosis and therapy.Similarly, STEAP2 is overexpressed in prostate can-

cer cell lines,11 and has been found to be up-regulatedin PCa samples by the use of high-density genomearrays.12 In normal prostate, the STEAP2 protein islocated in the Golgi membrane and cytoplasmic vesi-cles, suggesting that it may be involved in secretory/endocytic pathways.7,13

Although overexpression of STEAP1 and STEAP2has been reported in PCa, the clinical relevance ofthese genes as putative prognostic markers remainsuncertain.7 The aim of the present study was toinvestigate STEAP1 and STEAP2 expression in PCatissues, and to associate the findings with clinicopath-ological features in a large series of prostate tumourswith long-term follow-up.

Materials and methods

T I S S U E S A M P L E S A N D P A T I E N T

C H A R A C T E R I S T I C S

PCa patients (n = 198) were followed prospectively(1993–2008) at the School of Medicine Hospital, S~ao

Paulo State University (UNESP), Botucatu, SP.Patients were advised of the procedures, and providedwritten informed consent, as approved by the HumanResearch Ethics Committees of both institutions. Themain clinicopathological features of the PCa patientsare shown in Table 1.The mRNA analysis was carried out using fresh

frozen macrodissected PCa tissue from 35 patients(median age 64 years, range 48–74 years), and 24samples of histologically normal adjacent prostate(AdjP) tissue obtained from PCa patients who under-went radical prostatectomy. Five fresh frozen samplesof benign prostatic hyperplasia (BPH) were obtainedfrom prostate biopsies. Two normal prostate (N) tissuesamples from autopsies (40 and 49 years of age), his-tologically confirmed to be free of PCa, intraepithelialneoplasia, benign prostate hyperplasia or prostatitis,were included as controls.STEAP1 analysis was performed on formalin-fixed

paraffin-embedded (FFPE) tissue blocks from 35 PCasamples (also evaluated for mRNA expression) and in34 paired AdjP tissues (in conventional slides). Anindependent cohort of PCa (n = 163), AdjP (n = 51)and BPH (n = 22) FFPE tissues arranged in three tis-sue microarrays (TMAs) and two N samples were alsoanalysed by IHC. In this cohort, the median ages ofthe PCa (total n = 198), BPH and AdjP groups were63 years (range 41–75 years), 68 years (range 55–86 years) and 64 years (range 44–75 years), respec-tively. The median PSA levels detected in the BPHand AdjP groups were 4.8 ng/ml and 8.5 ng/ml,respectively.Radical prostatectomy was the primary treatment in

166 PCa patients. In 32 cases subjected to hormonalneoadjuvant therapy, biopsy samples were takenbefore treatment and used for protein analysis. Allsamples evaluated were from untreated patients. Themedian follow-up for PCa patients was 81 months(range 3–161 months). Tumour grading was per-formed according to the Gleason score.14 Pathologicalstages were determined according to the pTNM classifi-cation.15 Tumours were further grouped into organ-confined versus non-organ-confined (≤pT2c or ≥pT3a,respectively).

S T E A P 1 A N D S T E A P 2 m R N A A N A L Y S I S B Y

R E V E R S E T R A N S C R I P T I O N Q U A N T I T A T I V E

P O L Y M E R A S E C H A I N R E A C T I O N ( R T - Q P C R )

Total RNA was extracted using the Rneasy mini Kit(Qiagen, Hilden, Germany), according to the manu-facturer’s instructions. RNA isolation and cDNA syn-thesis were performed as described elsewhere.16

© 2013 John Wiley & Sons Ltd, Histopathology, 63, 678–685.

STEAP1 expression in prostate carcinoma 679

Primers and probes for STEAP1, STEAP2 and GAPDHwere designed using the TaqMan Gene ExpressionAssay (Applied Biosystems, Foster City, CA, USA).PCR was carried out in duplicate on the ABI Prism7000 Sequence Detection System (Applied Biosys-

tems). Relative quantification (RQ) of the target geneswas calculated according to the 2�DDCt method.17

S T E A P 1 A N A L Y S I S B Y I M M U N O H I S T O C H E M I S T R Y

( I H C )

TMA platforms were constructed as previouslydescribed.18 TMA and conventional FFPE tissueblocks were cut at 3 lm, and the sections weremounted on conventional glass slides with (3-amino-propyl)triethoxysilane (Sigma-Aldrich, St Louis, MO,USA). Briefly, the slides were deparaffinized and grad-ually rehydrated. Antigen retrieval was performed ina microwave oven using 10 mM citrate buffer (pH6.0). The sections were incubated overnight with theprimary polyclonal rabbit anti-STEAP antibody (Invi-trogen, Camarillo, CA, USA) (1:300 dilution). Afterbeing washed with phosphate-buffered saline (PBS),the sections were incubated for 30 min with the sec-ondary biotinylated antibody, and then for 30 minwith streptavidin–peroxidase complex (LSAB; Dako,Carpinteria, CA, USA). Staining was developed using3,3′-diaminobenzidine (DAB), and counterstainingwas performed with haematoxylin.Positive (normal prostate tissues) and negative con-

trols were included in the reactions. Scores weredetermined according to the intensity of staining:weak expression (score 1); moderate expression (score2); and strong expression (score 3). The analyseswere performed simultaneously by two observers(M.A.C.D. and J.L.V.C.), and the final scores weredetermined by consensus, blinded to patient clinicaldata.

S T A T I S T I C A L A N A L Y S E S

Kruskal–Wallis and Mann–Whitney tests were appliedto compare STEAP1 or STEAP2 transcript levels withclinicopathological characteristics. Correlation analy-sis for gene expression was performed using Spear-man’s rank test. The chi-square test was applied todetermine the association between the categoricalvariables.The biochemical recurrence-free survival (BCR-FS)

probability was calculated using the Kaplan–Meiermethod. BCR was assumed when postsurgical serumPSA levels were ≥0.2 ng/ml.3,19 Patients subjected toneoadjuvant treatment were excluded from the sur-vival analysis. Multivariate analysis was performedusing the Cox proportional hazards model. A signifi-cance level of 5% was adopted. The Bonferroni correc-tion for multiple comparisons was applied to adjust theP-value. PRISM 3 (GraphPad Prism, San Diego, CA,

Table 1. Clinical and histopathological characteristics ofprostate cancer (PCa) patients

Characteristics

Gene expressionanalysis*n (%)

Proteinanalysis†n (%)

Age (median/range) 64 (48–74) 63 (41–75)

Pre-surgical PSA (ng/ml)

<10.0 27 (77) 112 (57)

10.0–20.0 04 (11.5) 52 (26)

≥20.0 04 (11.5) 30 (15)

Unknown 0 4 (2)

Gleason scores

<7 7 (20) 108 (54)

7 21 (60) 73 (37)

>7 7 (20) 17 (9)

Pathological stages (pTNM)

Low ≤pT2c 20 (57) 97 (49)

High ≥pT3a 15 (43) 101 (51)

Extraprostatic extension

Negative 20 (57) 90 (45)

Positive 15 (43) 108 (55)

Surgical margin

Negative 21 (60) 135 (68)

Positive 14 (40) 63 (32)

Angiolymphatic invasion

Negative 27 (77) 155 (78)

Positive 08 (23) 43 (22)

Seminal vesicle invasion

Negative 33 (94) 173 (87)

Positive 2 (6) 25 (13)

*Gene expression analysis included all 35 tumour samplesevaluated by IHC on conventional slides, n = 35.†Immunohistochemical (IHC) protein analysis comprised 35tumour samples on conventional slides and 163 PCa tissuecores in the tissue microarray platforms, n = 198.


680 S M Ihlaseh-Catalano et al.

USA) and SPSS version 17.0 (SPSS, Chicago, IL, USA)were used for statistical analyses.

Results

S T E A P 1 A N D S T E A P 2 m R N A E X P R E S S I O N

STEAP1 and STEAP2 transcript levels were not sig-nificantly different between PCa, BPH and AdjP tis-sues (Figure 1A,B). Increased STEAP1 expressionlevels were observed in tumours with high-gradeGleason scores (Gleason score of >7) as comparedwith BPH; however, this difference was not signifi-cant (Figure 1A). A marginally significant associa-tion was observed between STEAP1 overexpressionand higher presurgical PSA levels (PSA ≥10.0 ng/mlversus PSA <10.0 ng/ml, P = 0.057; Figure 1C). Inaddition, although not significant, higher levels ofSTEAP1 mRNA were detected in tumours frompatients who presented BCR (P = 0.07; Figure 1D).No association was detected between STEAP1 andSTEAP2 mRNA levels and other clinicopathologicalfeatures (data not shown). A significant positivecorrelation between STEAP1 and STEAP2 transcript

levels was observed in PCa samples (r = 0.54, P =0.002; Figure 1E).

S T E A P 1 E X P R E S S I O N

STEAP1 protein expression was assessed in a subsetof tumour samples that were also evaluated by tran-scription analysis (35 cases) and in an independentcohort of 163 cases with long-term follow-up. Weakmembranous STEAP1 immunostaining was detectedin histologically normal prostate tissues (Figure 2A)and in BPH (Figure 2B). Prostate tumour samplesshowed predominantly moderate or strong membra-nous and cytoplasmic immunostaining (Figure 2C).In contrast, AdjP tissues showed weak STEAP1expression (Figure 2D). STEAP1 overexpression wassignificantly associated with PCa (82%) in compari-son with AdjP tissues (41%) and BPH samples (0%)(P < 0.0001; Table 2).Although STEAP1 overexpression was associated

with seminal vesicle invasion, high-grade Gleasonscores, and ‘lethal phenotype’ (metastasis occurrenceor PCa-specific death), after Bonferroni correction formultiple comparisons (P < 0.005), a significant associ-

100 A

C D E

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0.1No YesBiochemical recurrencePre-surgical PSA

<10 ng/ml ≥10 ng/ml

P = 0.07P = 0.057

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STEAP1 mRNA levels

AdjP BPH Gleason<7 Gleason 7 Gleason > 7

Figure 1. STEAP1 A and STEAP2 B, mRNA relative expression in benign prostatic hyperplasia (BPH), adjacent non-neoplastic prostate tis-

sues (AdjP), and prostate carcinomas (PCa), grouped according to Gleason scores. STEAP1 transcript levels in PCa tumours according to C,

presurgical PSA levels and D, biochemical recurrence. E, Positive correlation between STEAP1 and STEAP2 mRNA levels (r = 0.54,

P = 0.002). Transcript values are indicated on a log scale. Bars indicate median values.



ation was observed only for STEAP1 overexpressionand BCR (P < 0.0001) (Table S1). No association wasdetected between STEAP1 expression and other clini-cal variables. No correlation was observed betweenSTEAP1 transcript and STEAP1 protein levels in the 35PCa evaluated with both procedures (data not shown).Univariate analysis indicated that STEAP1 protein

overexpression was associated with decreased BCR-FS(P = 0.001; Figure 2E; Table S2). The establishedprognostic factors, such as PSA level, Gleason score,pathological stage, surgical margin involvement, andseminal vesicle invasion, were also significantly asso-ciated with BCR-FS (Table S2).

Further multivariate analysis revealed thatSTEAP1 expression was an independent prognosticfactor for BCR-free survival (Table 3). Patients withtumours showing STEAP1 overexpression had ahigher risk of BCR (P = 0.002, HR 4.23, 95% CI1.68–10.61). Presurgical PSA level, Gleasonscore and extraprostatic extension were also foundto be independent prognostic factors for BCR(Table 3).Furthermore, survival analysis showed an associa-

tion between STEAP1 overexpression and poor out-come. Patients with tumours showing STEAP1overexpression had decreased time to metastasis

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STEAP1 normal expression

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Figure 2. A, STEAP1 protein expression in normal prostate, showing apical membranes of luminal cells with weak immunostaining. B,

Negative immunostaining in benign prostatic hyperplasia (BPH) tissue. C, Prostate carcinoma with strong immunostaining in neoplastic

acini. D, Adjacent non-neoplastic prostate (AdjP) tissue showing weak immunostaining. E, Biochemical recurrence-free survival (BCR-FS)

curve according to STEAP1 protein expression. The P-value was determined using the log-rank test.



occurrence or PCa-specific death (P = 0.039; datanot shown).

Discussion

The present study showed that STEAP1 protein over-expression was significantly associated with featuresof worse prognosis in prostate cancer, such as high-grade tumours, seminal vesicle invasion, and BCR.Moreover, STEAP1 overexpression was found to bean independent prognostic marker for BCR. To ourknowledge, this is the first report indicating STEAP1as a meaningful prognostic marker in PCa.Significant STEAP1 overexpression was found in

PCa tissues as compared with BPH and AdjP tissues.Furthermore, distinct STEAP1 immunostaining pat-terns were observed between PCa and non-tumour

tissues. Both diffuse and marked membranous andcytoplasmic immunostaining were detected in PCa tis-sues; conversely, only a membranous pattern wasobserved in BPH, AdjP and normal prostate tissues.Hubert et al.8 reported moderate to strong STEAP1immunostaining in both neoplastic and non-neoplas-tic prostate tissues. These discordant results can, inpart, be explained by the different antibodies used todetect STEAP1 and by the small number of samplesevaluated by these authors. Interestingly, we detecteddifferent STEAP1 expression patterns in BPH andAdjP tissues. STEAP1 expression in BPH samplesshowed a similarity with that found in normal sam-ples (weak immunostaining), whereas 41% of AdjPtissues showed STEAP1 overexpression. These find-ings corroborate previous data showing that histolog-ically normal prostate tissue adjacent to prostatetumours may have molecular changes that can possi-bly be explained by field cancerization.20

STEAP1 transcript levels were marginally associ-ated with adverse prognostic parameters, such ashigh-grade Gleason score, high presurgical PSA lev-els, and BCR, although not significantly. Up-regula-tion of STEAP1 has been detected in high-gradeprostate tumours21 and prostate cancer metastases22

as compared with normal prostate tissues by large-scale expression analysis (data accessible at NCBIGEO database; accession GSE45016 and GSE38241,respectively). Although these data have not been vali-dated here, the findings corroborate those of ourstudy and suggest that STEAP1 is involved in prostatetumour aggressiveness.Furthermore, protein analysis of the large series of

cases with long-term follow-up presented hererevealed that STEAP1 overexpression was signifi-cantly associated with BCR, which indicates tumourrecurrence.14 STEAP1 overexpression was also signifi-cantly associated with shorter BCR-free survival. Fur-ther, multivariate analysis confirmed that STEAP1 isan independent prognostic marker for BCR. Althoughputative prognostic biomarkers have been describedin PCa,23 at present it is not possible to precisely pre-dict clinical outcome. The most commonly used mar-ker for outcome in PCa patients is currently serumPSA and its derivatives, PSA velocity or PSA doublingtime.23,24 Approximately 10–40% of patients whoundergo radical prostatectomy experience BCR, whichindicates an increased risk of metastasis and mortal-ity.4,5 Indeed, in the present study, patients withSTEAP1 overexpression showed decreased time tometastasis occurrence or death from cancer, suggest-ing that STEAP1 could be considered as a new prog-nostic marker in clinical practice.

Table 2. STEAP1 protein expression detected by immuno-histochemistry (IHC) in prostate carcinomas (PCa), adjacentnon-neoplastic prostate tissues (AdjP) and Benign ProstaticHyperplasia (BPH)

Samples n

STEAP1 protein expression (%)

P†Overexpressed* Normal

Tumour 198 163 (82%) 35 (18%) <0.0001

AdjP 85 35 (41%) 50 (59%)

BPH 22 0 22 (100%)

*Moderate or strong membrane and cytoplasm immuno-staining was considered as overexpression.†Chi square test.

Table 3. Multivariate analysis of biochemical recurrence-free survival (BCR-FS) in prostate tumour patients (Coxregression model)

Variable P HR (CI95%)*

STEAP1 (overexpressedversus normal)

0.002 4.23 (1.68–10.61)

Preoperative PSA (ng/ml)† 0.007 1.62 (1.14–2.29)

Gleason score(>7 versus 7 versus <7)

<0.001 1.93 (1.34–2.79)

Extraprostatic extension(positive versus negative)

0.013 1.90 (1.14–3.17)

*HR: hazard ratio; CI95%: 95% confidence interval.†PSA (ng/ml): (<10.0) versus (≥10.0–<20.0) versus (≥20.0ng/ml).



Interestingly, all patients who developed metastasisduring the follow-up period or died from the diseaseshowed STEAP1 overexpression in their primarytumours. Accordingly, strong STEAP1 expression waspreviously reported in lymph nodes and bone metas-tases from PCa patients.9 Moreover, with suppressionsubtractive hybridization (SSH), higher levels ofSTEAP1 cDNA were observed in cell lines of lymphnode and bone PCa metastases than in prostatichyperplasia cells.8 A recent report showed thatSTEAP1 overexpression was associated with pooroverall survival in patients with colorectal cancer, dif-fuse large B-cell lymphoma, acute myeloid leukaemia,and multiple myeloma.25

On the basis of these data, it is possible to hypothe-size that cell clones showing STEAP1 overexpressionin primary prostate tumours have a metastasizingpotential that is maintained in the metastatic colo-nies. However, further studies are necessary to betterunderstand the mechanisms underlying STEAP dereg-ulation in prostate cancer.Previous reports have suggested that STEAP1 is a

potential target for immunotherapy of human PCa,with data showing that STEAP is specifically targetedby human CD8+ T cells in vitro.26,27 Prophylacticand therapeutic vaccination with mouse STEAPcDNA (mSTEAP) delayed tumour growth and pro-longed the overall survival of C57BL/6 mice chal-lenged with prostate cancer cells from transgenicadenocarcinoma mouse prostate (TRAMP).28,29

STEAP1 contains a haem-binding domain called‘apoptosis, cancer and redox associated transmem-brane’ that seems to facilitate electron transfer andalter cell growth and metabolism.30 STEAP1 wasshown to be involved in intercellular communicationby use of a fluorescent dye transfer assay with pros-tate cancer cell lines; in addition, monoclonal anti-bodies against STEAP1 significantly inhibited tumourgrowth in a mouse model using patient-derivedLAPC-9 prostate cancer xenografts.9 The authorsspeculated that STEAP1 can facilitate intercellulartransport, and may support tumour growth by medi-ating nutrient, metabolite and/or signal processes.Overall, these data suggest that STEAP1 could be atarget for adjuvant therapy.7,31 Further studies maycontribute to a clearer understanding of the STEAP1-related pathways in prostate cancer.In conclusion, we suggest that STEAP1 overexpres-

sion in prostate tumour cells is a prognostic marker.STEAP1 differential expression in more aggressivePCa combined with its reduced expression in normaltissues makes this gene an ideal candidate for molec-ular targeted therapy in PCa.

Author contributions

S. M. Ihlaseh-Catalano, J. L. V. de Camargo, andS. R. Rogatto: conceived and designed the experi-ments. S. M. Ihlaseh-Catalano: performed the experi-ments. S. M. Ihlaseh-Catalano and S. A. Drigo:analysed the data. S. M. Ihlaseh-Catalano, S. R. Rog-atto, S. A. Drigo, and J. L. V. de Camargo: wrote thepaper. C. M. N. de Jesus and J. C. S. T. Filho: clinicaldata collection. M. A. C. Domingues and J. L. V. deCamargo: pathological revision.

Acknowledgements

This work was supported by grants from the NationalInstitute of Science and Technology in Oncogenomics(INCITO, Fundac�~ao de Amparo �a Pesquisa do Estadode S~ao Paulo, FAPESP 2008/57887-9, and ConselhoNacional de Desenvolvimento Cient�ıfico e Tec-nol�ogico, CNPq 573589/08-9) and FAPESP 2004/10028-0. The authors would like to thank RodrigoMattos dos Santos, Greicy Helen Ribeiro GambariniPaiva, Renata Bueno, Dr Francisco Paulo da Fonsecaand Department of Pathology staff (UNESP and ACCamargo Cancer Centre, S~ao Paulo) for their assis-tance. The authors declare that there are no conflictsof interest.

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Supporting Information

Additional Supporting Information may be found inthe online version of this article:Table S1. STEAP1 expression detected by immuno-

histochemistry (IHC) in prostate carcinoma samplesrelated to clinic-histopathological variables.Table S2. Univariate analysis of biochemical recur-

rence-free survival (BCR-FS) in prostate carcinoma(PCa) patients.



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