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Basic Science
rognostic Significance of Thymidylateynthase Expression in Patients withrostate Cancer Undergoing Radicalrostatectomy
ongnan Li, Yoichi Mizutani, Takumi Shiraishi, Koji Okihara, Osamu Ukimura,kihiro Kawauchi, Norio Nonomura, Masakazu Fukushima, Toshiyuki Sakai, andsuneharu Miki
BJECTIVES Thymidylate synthase (TS), a key enzyme in DNA synthesis, is overexpressed in a variety ofcancer cells. 5-Fluorouracil (5-FU), an anticancer agent used clinically against various cancers,including prostate cancer, inhibits DNA synthesis by binding TS. In this study, we investigatedthe expression of TS in prostate cancer and its prognostic significance. Its association with theexpression of dihydropyrimidine dehydrogenase (DPD), a principal enzyme in the degradation of5-FU and pyrimidine nucleotides, was also examined.
ETHODS Fifty-two prostatic tissue specimens were obtained from patients who had undergone radicalprostatectomy for prostate cancer without neoadjuvant hormonal therapy. We analyzed thecancerous tissue and normal prostatic tissue specimens for TS expression using immunohisto-chemistry.
ESULTS TS was expressed at greater levels in the prostate cancer specimens than in the normal prostatictissue specimens. The patients with prostate cancer with negative TS expression had a longerpostoperative recurrence-free rate than did those with positive expression during the 5 years offollow-up. TS expression was significantly decreased in patients who received neoadjuvanthormonal therapy. No relationship was found between the expression of TS and DPD. Patientswith prostate cancer with either negative TS or DPD expression had a significantly longerpostoperative disease-free rate than those with positive expression of both during the 5 years offollow-up.
ONCLUSIONS The results of the present study have shown for the first time that TS expression could be aprognostic marker for patients with prostate cancer undergoing radical prostatectomy. In addi-tion, the combination of TS and DPD expression might also be helpful for the prediction of the
prognosis of patients with prostate cancer. UROLOGY 69: 988–995, 2007. © 2007 Elsevier Inc.c5twhtcicttsn
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he anticancer agent, 5-fluorouracil (5-FU), isused clinically against various cancers, includingprostate cancer.1,2 Single-agent infusion 5-FU has
emonstrated some efficacy against hormone-refractoryrostate cancer, and response rates up to 27% have beeneported.3 5-FU itself is inactive and requires intracellular
his work was supported in part by the NPO Tsukusi Fellowship and Researchoundation.From the Department of Urology, Kyoto Prefectural University of Medicine, Kyoto;
epartment of Urology, Osaka University Faculty of Medicine, Osaka; Cancer Re-earch Laboratory, Taiho Pharmaceutical Company, Limited, Saitama; and Depart-ent of Molecular-Targeting Cancer Prevention, Kyoto Prefectural University ofedicine, Kyoto, JapanReprint requests: Yoichi Mizutani, M.D., Department of Urology, Kyoto Pre-
ectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-Ku, Kyoto 602-
t566, Japan. E-mail: [email protected]: May 4, 2006, accepted (with revisions): February 8, 2007
88 © 2007 Elsevier Inc.All Rights Reserved
onversion to 5-fluoro-2=-deoxyuridine 5=-monophosphate.-Fluoro-2=-deoxyuridine 5=-monophosphate exerts its cyto-oxic activity through the formation of a ternary complexith thymidylate synthase (TS) and 5,10-methylene-tetra-ydrofolate, resulting in inhibition of TS and blockage ofhe DNA synthetic process.4,5 TS is overexpressed in tumorells, which show high proliferative activity.6 Several stud-es examining the importance of TS expression have indi-ated that TS expression predicts for overall outcome andhe response to 5-FU cytotoxic therapy in several majorumor types.7–9 Furthermore, the immunohistochemicaltaining results for TS and dihydropyrimidine dehydroge-ase (DPD) predict the response to 5-FU.10,11
Our previous studies on renal cell carcinoma and bladderancer showed that TS activity was greater in the cancerous
issue specimens than in the normal tissue samples and that0090-4295/07/$32.00doi:10.1016/j.urology.2007.02.015
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he TS activity level correlated with stage progression andncrease in bladder cancer grade.12 In addition, TS activitys a significant prognostic marker in patients with bladderancer or renal cell carcinoma.12,13 Reported data on TSctivity in prostate cancer are limited, and little is knownbout the significance of TS in the biology of prostateancer. The aim of this study was to define whether TSxpression is a prognostic marker for patients with prostateancer.
ATERIAL AND METHODS
atientse obtained 52 prostate cancer specimens with adjacent nor-al prostatic tissues from 1997 to 2005. The patients had not
ndergone preoperative androgen-deprivation therapy or radio-herapy. The mean patient age was 65 years (range 53 to 75).he 2002 TNM system was used for pathologic staging.14 Theathologic stage was T2 in 38 patients and T3 in 14. Theleason grading system was used to determine the Gleason
core.15 The Gleason score of the 52 specimens was grade 4/5 inpatient, 4/4 in 1, 4/3 in 9, 3/5 in 2, 3/4 in 18, 3/3 in 8, 3/2 in
, 2/3 in 3, 2/1 in 1, and 1/2 in 1 patient. In addition, prostateancer tissue from 48 patients who had undergone neoadjuvantormonal therapy was examined.The local human investigations committee approved this
igure 1. Immunohistochemical staining for TS in normal pissue samples with absent and strong TS expression weemonstrated by immunohistochemistry. Original magnificaB) Positive TS staining (arrows) observed in cytoplasm of
tudy, and all patients provided informed consent. w
ROLOGY 69 (5), 2007
mmunohistochemistry for TS and DPDS and DPD expression was examined by immunohistochem-
stry, as previously described.5,16 The sections were incubatedith monoclonal antibody TS106 (1:500, dilution, Taiho Phar-aceutical, Saitama, Japan) or incubated with polyclonal rabbit
ntibody against human DPD16 (1:2000 dilution, Taiho Phar-aceutical) overnight at 4°C. The secondary antibody was
isualized with diaminobenzedine.
valuation of TS and DPD Expressionhe intensity of the immunoreactivity for TS and DPD wasvaluated in normal prostatic tissue and prostate cancerissue from the same slide in each case. At least 10 high-ower fields at 400� magnification were chosen randomly,nd more than 1000 carcinoma cells were counted for eachection. A pathologist who was unaware of the clinicopath-logic data and clinical outcomes of the patients examinedytoplasmic TS and DPD staining results. The intensity of TS andPD was graded from 0 to 3, and the extent was graded as focal
less than 25% of tumor staining positive) or diffuse (more than5% of tumor staining positive).5 A score of 0 or 1 was regarded asegative expression, and a score of 2 or 3 as positive expression.igure 1 shows representative examples; Fig. 1A shows a TS-egative normal prostate specimen and Fig. 1B TS-positive pros-ate cancer.
tatistical Analysisor statistical analysis, the Student t test and chi-square test
tic tissues and prostate cancer. Representative images ofamined. TS expression confined to cytoplasm of cells as�200. (A) Negative TS staining in normal prostatic tissue.tate cancer tissue.
rostare extionpros
ere used. Biochemical recurrence was defined as a postopera-
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ive serum prostate-specific antigen (PSA) level of 0.1 ng/mL orore.17 The postoperative recurrence-free rate was determined
sing the Kaplan-Meier method. The influence of each variablen the recurrence-free rate was analyzed by multivariate anal-sis of a Cox proportional hazard model. P �0.05 was consid-red significant.
ESULTS
S Expression in Prostate Cancer and Normalrostatic TissueS was expressed in the cytoplasm of both normal prostatic
issue and prostate cancer cells. TS expression was detectedn 35 (67%) of 52 prostate cancer samples (Fig. 2A). Inontrast, TS expression was detected in 5 (9%) of the 52ormal prostatic tissue specimens. In addition, the intensityf cells that reacted with TS antibodies was significantlyreater in the prostate cancer specimens than in the normalrostate samples (P �0.0001, data not shown).
S Expression in Relation to Pathologic Featuresnd Tumor Stagehe staining percentage of TS expression was greater inatients with Gleason score 7 or greater disease (26 of 31,3%) than that for patients with Gleason score less thandisease (9 of 21, 42%; Fig. 2B). It was also greater in
tage T3 tumor (14 of 14, 100%) than in Stage T2umors (21 of 38, 55%; Fig. 2C).
elationship Between TS Expression andostoperative Recurrence-Free Rate in Patientsith Prostate Canceratients with prostate cancer undergoing radical prosta-ectomy alone were evaluated to determine the postop-rative clinical course. From these results, patients withrostate cancer were divided into two groups—those withositive TS expression and those with negative TS ex-ression. At 5 years of follow-up, patients with negativeS expression had a greater recurrence-free rate com-ared with those with positive TS expression (P �.0183; Fig. 3A). Using Cox regression analysis for the 52
™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™igure 3. Relationship between TS expression and postopeelationship between TS and DPD expression and postopostoperative recurrence-free rate of 52 patients with petermined using Kaplan-Meier method. (A) Patients categreater for patients with negative TS expression than foategorized by TS expression of Gleason score less thanatients with negative TS expression than for those with poxpression of Gleason score 7 or worse. No significant diffeD) Patients categorized by TS expression of preoperative sbserved between two groups of patients (P � 0.2296). (ESA level greater than 10 ng/mL. Recurrence-free rate signihose with positive expression (P � 0.0344). (F) Patients (nPD (b) and all others (a). Significant difference in recurreleason score less than 7 disease. (H) Patients with Gleas
™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™3rative recurrence-free rate in patients with prostate cancer anderative recurrence-free rate in patients with prostate cancer.rostate cancer undergoing radical prostatectomy alone wasorized by TS expression. Recurrence-free rate was significantlyr those with positive expression (P � 0.0183). (B) Patients7 disease. Recurrence-free rate was significantly greater for
sitive expression (P � 0.0256). (C) Patients categorized by TSrence observed between two groups of patients (P � 0.2722).erum PSA level of 10 ng/mL or less. No significant difference) Patients categorized by TS expression of preoperative serumficantly greater for patients with negative TS expression than for� 52) categorized as having positive expression of both TS andnce rate between two groups (P � 0.0108). (G) Patients withon score of 7 or more. (I) Patients with tumor with preoperative
erum PSA level of 10 ng/mL or less. (J) Patients with tumor a
90
igure 2. Expression of TS in normal prostatic tissue androstate cancer. Percentage of TS expression detected bymmunohistochemical assay as described in Material andethods section. (A) *P �0.0001 versus normal prostatic
issue; (B) *P � 0.0017 versus Gleason score less than 7
nd preoperative serum PSA level greater than 10 ng/mL.
UROLOGY 69 (5), 2007
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ROLOGY 69 (5), 2007 991
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atients, TS expression seemed to be an independentrognostic indicator (P � 0.021 on multivariate analy-is). The patients were also divided according to theleason grade and preoperative serum PSA level at base-
ine, and the recurrence-free rate of the groups with differ-nt TS expression status was analyzed. In Gleason score lesshan 7 cancer, the TS-negative group had a significantlyreater 5-year recurrence-free rate than did the TS-positiveroup (Fig. 3B). In those with Gleason score 7 or greaterancer, no significant difference was found (Fig. 3C). Inhose with a PSA of 10 ng/mL or less at baseline, the 5-yearecurrence-free rate of TS-negative patients tended to bereater than that of TS-positive patients. However, statisti-al significance was not reached (Fig. 3D). In those with aSA level greater than 10 ng/mL, the TS-negative groupad a significantly greater 5-year recurrence-free rate com-ared with the TS-positive group (Fig. 3E). These findingsndicate that the TS expression level in prostate cancerould be a prognostic indicator, with negative TS expres-ion a good prognostic sign.
ffect of Neoadjuvant Hormonal Therapy on TSxpression in Prostate Cancerhe expression of TS in patients with prostate cancer wasreater after radical prostatectomy alone than that afteradical prostatectomy plus neoadjuvant hormonal ther-py (32 of 52, 61% versus 18 of 48, 37%; Fig. 4A). Foratients with Stage T2 prostate cancer, positive TS ex-ression in those who underwent neoadjuvant hormonalherapy (9 of 38, 23%) was less than that in patients whonderwent radical prostatectomy alone (21 of 38, 55%; Fig.B). However, no significant difference was found in thoseith Stage T3 prostate cancer (14 of 14, 100% versus 10 of0, 100%; Fig. 4C). With Gleason score less than 7 prostateancer, positive TS expression in patients with radical pros-atectomy alone (9 of 21, 42%) was significantly greaterhan that in patients who had undergone neoadjuvant hor-onal therapy (1 of 13, 7%; Fig. 4D). A significant differ-
nce was also observed in patients with Gleason score 7 orreater prostate cancer between those who underwent rad-cal prostatectomy alone (26 of 31, 83%) and those who alsoeceived neoadjuvant hormonal therapy (18 of 35, 51%;ig. 4E).
These findings suggest that neoadjuvant hormonalherapy might downregulate TS expression in prostateancer, especially Stage T2 prostate cancer and Gleasoncore less than 7 disease.
rognostic Significance of Combined TS andPD EvaluationPD is the initial and rate-limiting enzyme in the three-
tep pathway of pyrimidine nucleotide catabolism.18 Inontrast, TS is an important enzyme in pyrimidine nu-leotides synthesis. Kornmann et al.19 reported that theombination of TS and DPD expression correlated highlyith survival. We examined the association between TS
nd DPD expression in patients with prostate cancer. No e92
ignificant association was observed between the levels ofS and DPD expression using the chi-square test (dataot shown).We then examined the prognostic significance of a
ombination of TS and DPD expression using Kaplan-eier analysis. On the basis of TS and DPD expression,
he 52 patients were stratified categorized as those havingositive expression of both TS and DPD and all others.e found a significant difference between these two
roups (P � 0.0108; Fig. 3F). Patients with positivexpression of both TS and DPD had a greater rate ofostoperative recurrence. The patients also were dividedsing the Gleason grade and preoperative serum PSA levelt baseline, and the recurrence-free rate of the groups withifferent TS and DPD expression were analyzed. In thoseith Gleason score less than 7 cancer, the TS-negativeroup had a significantly greater 5-year recurrence-free ratehan did the TS-positive group (Fig. 3G). In patients withleason score 7 or greater cancer, no significant differenceas found (Fig. 3H). In patients with a PSA level of 10g/mL or less, the 5-year recurrence-free rate of the TS-egative group tended to be greater than that of the TS-ositive group. However, statistical significance was noteached (Fig. 3I). In those with a PSA level greater than 10g/mL, the TS-negative group had a significantly greater-year recurrence-free rate than that of the TS-positiveroup (Fig. 3J).
OMMENThe role of TS expression in prostate cancer has not beenreviously studied. Multivariate analysis revealed thathe TS expression profile was an independent prognosticndicator of prostate cancer. These results suggest that TSxpression in patients with prostate cancer might providedditional prognostic information beyond the orthodoxlinical and pathologic prognostic markers. Ichikawat al.20 reported that patients with colorectal tumor withoth low DPD and low TS survived longer than didatients with tumor having the other patterns of TS andPD expression. Consistent with our findings, the com-ined evaluation of TS and DPD expression might pre-ict the prognosis more accurately than using one markern patients with prostate cancer. These data also reflecthe report by Jakob et al.11 that immunohistochemistryuch as reverse transcriptase polymerase chain reaction issuitable method to determine the correlation betweenS and DPD expression and histopathologic tumor re-ression. However, the precise mechanisms for this rela-ionship remain unclear at present. Additional studies areecessary to examine the mechanisms.The benefit of hormonal therapy against advanced
rostate cancer has been widely accepted. In addition,he antitumor effect is very high. Because TS is the keynzyme in the process of DNA synthesis, we believe it iseasonable to assume that this was the reason the TS
xpression was greater in the prostate cancer specimensUROLOGY 69 (5), 2007
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f the patients treated without neoadjuvant hormonalherapy than in those treated with surgery and neoadju-ant hormonal therapy. Furthermore, hormonal therapyight directly downregulate TS expression. A study bygasawara et al.21 showed that in nude mouse MCF-7
reast cancer xenografts, tumor TS levels were reducedy treatment with a pure antiestrogen. Hung et al.22
igure 4. TS expression in prostate cancer with/without nrade of prostate cancer. TS expression in prostate canescribed in Material and Methods section. (A) *P � 0.004
*P � 0.0078 versus not administered). (C) Patients with St*P � 0.0259 versus not administered). (E) Patients withdministered).
lso reported that the Ki-67 proliferation index was b
ROLOGY 69 (5), 2007
ignificantly greater in TS-positive tumors than inS-negative tumors, suggesting that TS-negative tu-ors might have a low rate of cell proliferation.TS is a key enzyme for pyrimidine synthesis. DPD is an
mportant pyrimidine salvage enzyme. No correlation wasound between DPD and TS expression in colorectalancer.20,23 We reported that no correlation was found
juvant hormonal therapy according to stage and Gleasonspecimens evaluated by immunohistochemical assay asrsus not administered. (B) Patients with Stage T2 disease3 tumor. (D) Patients with Gleason score less than 7 tumorason score 7 or greater tumor (*P � 0.0036 versus not
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arcinoma12,24 or renal cell carcinoma.13 Our data inrostate cancer are consistent with these results.Immunohistochemical staining for TS and DPD pre-
ict the response to 5-FU.10,11 Previous studies of severalancers have demonstrated that the TS expression levelredicted the response to 5-FU-based chemotherapy.4,5
reater TS expression was accompanied by a greateresponse rate to 5-FU-containing chemotherapy. Most ofhe administrated 5-FU is degraded through the catabolicathway with DPD.18 DPD activity is highly associatedith 5-FU pharmacokinetics.25 The efficacy of 5-FU is
elated to the plasma level of this agent, which is in-ersely associated with the DPD activity level.25 Ourrevious report demonstrated that primary cultured renalell carcinoma cells with both high TS activity and lowPD activity were more sensitive to 5-FU than thoseith either low TS activity or high DPD activity.26 Thesendings suggest that the TS and DPD expression levels inrostate cancer could be important predictive indicatorsor 5-FU efficacy. However, other factors might be moremportant, including the rate of degradation, carrier pro-ein level, and so forth, although the principle TS andPD expression levels might predict the response to-FU.
ONCLUSIONShe results of our study have demonstrated that TSxpression was significantly greater in the cancerous pros-ate and that positive TS expression was associated withworse prognosis. These findings suggest that the assess-ent of TS expression might be useful in the manage-ent of prostate cancer. Because TS expression could be
sed as a prognostic parameter in patients with prostateancer, an accurate prediction of prognosis might help toelect patients for more intensive surgical, hormonal, orhemotherapeutic approaches, including 5-FU. Addi-ional prospective studies are warranted to define the rolef TS in selecting patients for adjuvant therapy for pros-ate cancer.
cknowledgment. To Hal Gold for assistance in the prepa-ation of this report.
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