The Prostate 49:140 ^144 (2001)

Molecular Stagingof SurgicalMarginsAfterRadicalProstatectomybyDetectionof TelomeraseActivity

Bernd Straub,* Markus MuÈ ller, Hans Krause, Carsten Goessl, Mark Schrader,RuÈ diger Heicappell, and Kurt Miller

DepartmentofUrology,Universita« tsklinikumBenjamin Franklin, FreieUniversita« t Berlin,Berlin,Germany

BACKGROUND. The further course of prostate cancer (PC) after radical prostatectomy (RPX)is decisively in¯uenced by the local tumor stage. Although it is thus far possible to assess therisk of local recurrence from the pathohistology, precise predictions cannot be made. A moreprecise evaluation would be desirable, mainly for early planning of adjuvant therapy. Otherauthors have shown that telomerase activity may be a marker for malignant potential. Weassessed the detection of telomerase activity using the telomeric repeat ampli®cation protocol(TRAP) in surgical margins compared to conventional histopathological examination.METHODS. Ninety-two patients with local PC who underwent RPX were examined. AfterRPX biopsies were obtained from four de®ned areas of the prostatic fossa and processed byTRAP assay for telomerase activity using a standard protocol.RESULTS. In 5 of 48 patients (10.4%) with organ-con®ned prostate carcinoma (pT2)telomerase activity could be detected. Seven of 47 patients (14.9%) with locally advancedPC (>pT2) had at least one positive specimen.CONCLUSIONS. The results obtained in our study indicate that detection of telomeraseactivity by TRAP assay may be a suitable parameter for molecular staging of surgical margins,because of the high tumor-speci®city. Further follow-up must clarify whether patients withpositive molecular detection have an increased risk of local recurrence. Prostate 49: 140±144,2001. # 2001 Wiley-Liss, Inc.

KEY WORDS: human prostate cancer; telomerase activity; polymerase chain reaction;staging

INTRODUCTION

The extent of local tumor extension in cases withnegative lymph node status is of considerable impor-tance for the further course of the disease after radicalprostatectomy (RPX) for prostate carcinoma (PC; [1]).At initial diagnosis, diagnostic measures such asprostate biopsies, digital rectal examination, and PSAserum levels are indicators of the extent of PC [2±6],but it is dif®cult to make a precise assessment onorgan-con®ned or locally advanced growth [7]. Basedon the current state of knowledge, RPX is usually anadequate therapeutic option in a number of thesecases. Local tumor extension, organ-con®ned orlocally advanced stage, pathological grading, Gleasonscore or the presence of positive surgical margins may

have a decisive in¯uence on the further course of thedisease [8±12]. Although it is possible to estimateprogression (over 40% in pT3 tumors) by the para-meters mentioned above, it has thus far not beenpossible to predict local recurrence in the individualpatient [13,14]. Surprisingly, about 20% of the patientswith primarily organ-con®ned tumor growth andnegative surgical margins are also affected by localprogression, but the risk cannot yet be accurately

*Correspondence to: Bernd Straub, MD, Department of Urology,UniversitaÈtsklinikum Benjamin Franklin, Freie UniversitaÈt Berlin,Hindenburgdamm 30, D-12200 Berlin, Germany.E-mail: straub@medizin.fu-berlin.de

Received 8 December 2000; Accepted 11 July 2001

ß 2001Wiley-Liss, Inc.

assessed in the immediate postoperative stage [15±17].If it were possible to identify patients with anincreased risk of local progression, they could besubmitted to adjuvant radiotherapy and/or hormoneablation therapy [18±22]. To improve local staging,surgical margins were examined by reverse transcrip-tase polymerase chain reaction (RT-PCR) for prostate-speci®c antigen (PSA) or prostate-speci®c membraneantigen by Theodorescu et al. [23]. This method seemsto be suitable for prognosis and assessment. However,there is one point of criticism, namely, that RT-PCR forPSA is unable to reliably differentiate between malig-nant and benign tissue [24±26].

Telomerase is a ribonucleoprotein enzyme thatadds telomeric repeats onto chromosomal ends usinga segment of its RNA component as a template.Telomerase activity is directly involved in telomeremaintenance and its activation plays a role in cell im-mortality. Telomerase activity has been detected in themajority of tumors [27], and nearly 90% of cancertissue from prostatectomy specimens evidenced telo-merase activity [28]. Other authors have shown thatthe level of telomerase activity correlates with thepathologic grade and have suggested that telomeraseactivity may be a marker for malignant potential[29]. In contrast, no telomerase activity was found inbenign prostatic hyperplasia (BPH) or normal pro-static tissue [30].

The aim of our study was to evaluate whethertelomerase activity can be detected in surgical marginsafter RPX.

MATERIALSANDMETHODS

Ninety-®ve consecutive patients with PC under-went RPX between June 1999 and August 2000 at ourinstitution. The investigations were conducted inagreement with the Helsinki Declaration. InstitutionalReview Board approval was obtained for this study.Each patient signed a consent form approved by theCommittee on Human Rights in Research of ourinstitution.

Following RPX, the gland was removed andbiopsies measuring 5 mm in diameter were obtained(removed with scissors) from four de®ned areas of theprostatic fossa (ventral urethra, median bladder neck,left and right lateral lobes in the middle of the lateralneurovascular bundle), frozen immediately in liquidnitrogen and stored at ÿ808C until further processing.Five slices, each 30 mm thick, were cut from thesebiopsies in the cryostat (Kryostat 2800, Leica Instru-ments GmbH, Nussloch, Germany). The surgicalspecimens were stored in formalin until regularhistopathological examination shortly afterwards andclassi®ed according to the TNM classi®cation of 1995after processing.

CHAPSExtraction [31]

Using the method described by Kim et al. [32],frozen slices were lysed in 200 ml of lysis buffer (10 mMTris-HCL (pH 7.5), 1 mM MgCl2, 1 mM EGTA,0.1 mM phenylmethylsulfonyl ¯uoride, 5 mMû-mercaptoethanol, 0.5% CHAPS, 10% glycerol(Roche, Mannheim, Germany)). The lysate was frozenin liquid nitrogen and stored at ÿ808C. The cells inCHAPS-lysat were incubated at 48C for 30 min andcentrifuged for 30 min at 55000 U/min at 48C.

Telomeric Repeat Amplif|cation Protocol(TRAP)Assay

The telomeric repeat ampli®cation protocol (TRAP)assay was performed according to the methoddescribed by Kim et al. [32] using a standard protocol(Roche, Mannheim, Germany) by applying 2 mgprotein each to the corresponding sample volume.Positive and negative controls were concomitantlyrun to con®rm that the samples were not cross-contaminated.

Variables were examined for Gaussian distribution(K-S and Lilliefors test) and by a one-sided Studentt-test; results were given as mean� SE using thestatistical package from STATISTICA

TM

(StatSoftTM

,Tulsa, Oklahoma, USA).

RESULTS

Histopathology

Forty-eight patients (50.5%) had an organ-con®nedtumor stage (pT2), 47 (49.5%) had locally advancedtumor growth with capsular penetration and/orpositive surgical margins (>pT2), 35 of them (66%)had positive pathohistological surgical margins, and6 patients (13%) also had lymph node metastases.Mean age in the pT2 group was 62.9 (�0.8) years, PSAwas 8.8 ng/ml (�1.1) at the time of diagnosis, and theGleason score 6.0 (�0.1) versus an age of 63.3 (�1.0),PSA of 21.4 ng/ml (�4.4; P> 0.001) and a Gleasonscore of 7.3 (�0.2; P< 0.001) in the >pT2 group(Table I). Four patients from the pT2 groups and 10patients from the >pT2 group were pretreated byneoadjuvant hormone ablation. Nerve-sparing RPXwas performed in 27 patients (28%), in two of thembilaterally. Ten of these patients (36%) had a histo-pathological >pT2 ®nding and three patients hadpositive contralateral surgical margins (11%).

Telomerase activity

Five of 48 patients with organ-con®ned prostatecarcinoma (pT2) had at least one positive sample oftelomerase activity (10.4%, Table I), three of them

SurgicalMargins After Radical Prostatectomy 141

had more than one positive sample. Seven of 47patients with locally advanced prostate carcinoma(14.9%, Table I) had at least one positive sample, fourof them had more than one positive sample. Thedetection of telomerase activity revealed no statisti-cally signi®cant correlation with clinical and histo-pathological parameters like the Gleason score orinitial PSA level for both organ-con®ned and locallyadvanced histopathological ®ndings (Table II). Four-teen patients pretreated by neoadjuvant hormoneablation had no detection of telomerase activity insurgical margins.

DISCUSSION

Theodorescu et al. [23] have recently demonstratedthe suitability of RT-PCR-assisted PSA and PSMAdetection in surgical margins for molecular stagingafter RPX. This procedure is controversial inasmuch asRT-PCR for PSA or PSMA cannot reliably differentiatemalignant tissue from benign tissue [25,26]. Thus, itwould be ideal to have a tumor marker speci®c formalignant tissue that is hardly or not at all detected inbenign prostatic hyperplasia (BPH). According to theresults reported in other studies, telomerase canindeed be considered a highly sensitive and speci®cmarker for the detection of carcinoma tissue [27±33].In PC, the detection of telomerase activity was shownto be a suitable tumor marker. The additionaldetection of telomerase activity in surgical marginsseemed ideal.

The results obtained in our study indicate thatdetection of telomerase activity by TRAP assay maybe a suitable parameter for molecular staging ofsurgical margins, because of the high tumor-speci®city[30,31,33].

Further follow-up must clarify if these patients withpositive molecular margins actually have an increasedrisk of local recurrence. The molecular status ofsurgical margins may become an important criterionin the decision for early adjuvant radiotherapy orhormone withdrawal therapy in patients with PC.Another important prognostic aspect may be anincrease of the PSA value from the zero level in PCpatients. In patients with positive molecular marginsat the time of surgery, this could be helpful in planningearly radiotherapy.

CONCLUSION

We could demonstrate detection of telomeraseactivity in surgical margins in a substantial numberof the patient population. The results obtained in ourstudy indicate that detection of telomerase activity byTRAP assay may be a suitable parameter for molecularstaging of surgical margins, because of the high tumor-speci®city. The clinical value of the results obtained inthis study will have to be assessed by further follow-up to determine whether patients with positivemolecular detection have an increased risk of localrecurrence. Only then is it possible to make a ®naldecision on the validity of the results.

TABLE 1. Patient Characteristics and Telomerase Activity of Prostate Cancer (PC)Patients.MeanValues (�SEM)

PC pT2 (n� 48) PC>pT2 (n� 47) t-test P-value

Age [years] 62.9 (�0.8) 63.2 (�1.0) 0.73PSA [ng/ml] 8.2 (�1.0) 20.5 (�4.0) < 0.001Gleason score 6.0 (�0.1) 7.3 (�0.2) < 0.001Nerve sparing surgery [%] 36.2 21.3 0.112TA positive patients (%) 5 (10.4) 7 (10.4) 0.51

TA, Telomerase activity in surgical margins.

TABLE II. Correlations of Clinical Histopathological Data With Molecular Staging forProstate Cancer (PC) Patients

Telomeraseactivity1 r (P-value)

Telomerase activity1 >pT2; %r(P-value)

PSA 0.09 (0.39) ÿ0.11(0.41)Gleason score ÿ0.076 (0.47) 0.069 (0.63)

r, Regression-coef®cient.1Detection of telomerase activity.

142 Straub et al.

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