Measurements of Free and Total PSA, TissuePolypeptide-Specific Antigen (TPS), and CYFRA

21-1 in Prostate Cancer Patients UnderIntermittent Androgen Suppression Therapy

Gerhard Theyer,1* Alexander Durer,1 Ulrike Theyer,2 Ines Haberl,3Ernst Ulsperger,3 Gerhard Baumgartner,3 and Gerhard Hamilton4

1Department of Urology, Wilhelminenspital der Stadt Wien, Vienna, Austria2Department of Laboratory Medicine, Wilhelminenspital der Stadt Wien, Vienna, Austria

3Society for Research and Treatment of Tumor Diseases, Vienna, Austria4Ludwig Boltzmann Institute of Clinical Oncology, Vienna, Austria

BACKGROUND. The present study evaluated monthly measurements of free and total pros-tate-specific antigen (PSA), and the tumor proliferation markers tissue polypeptide-specificantigen (TPS) and cytokeratin fragment 21-1 (CYFRA 21-1) in patients with advanced prostatecancer receiving intermittent androgen suppression therapy (IAS).METHODS. Thirty-four men received alternating cycles of 8 month androgen suppressionand treatment cessation (mean duration, 10.3 months) until PSA increased to >20 mg/l.Measurements of testosterone, percentage of free PSA, TPS, and CYFRA 21-1 were performedusing ELISA and RIA assays.RESULTS. Periods of androgen suppression resulted in reversible reductions of testosterone(from 6 ± 0.8 to <0.58 ng/ml), PSA (from 31.2 ± 4.5 to <1.7 mg/l), and prostatic volume (meanreduction, 22.2 ± 4.6%), indicating apoptotic regression of the tumors. Upon treatment ces-sation, testosterone increased to 6.1 ± 0.56 ng/ml within 2 months, followed by an increase ofPSA to 5.8 ± 0.8 mg/l. The mean percentage of free PSA (15.1 ± 2.6%) exhibited no significantchange during the whole IAS cycle. TPS showed a decrease of 50% after 3 months, andCYFRA 21-1 a 25% decrease after 7 months of androgen suppression treatment. Duringtreatment cessation, TPS exceeded the normal cutoff value of 90 U/l late in tumor regrowth(9–11 months), whereas CYFRA 21-1 remained below the normal cutoff value of 3.3 ng/ml.CONCLUSIONS. PSA is the best and most sensitive marker of prostate cancer regression andregrowth during IAS cycles of the markers tested in this study. Free PSA constitutes approxi-mately 15% of total PSA (range, 5–32%), and its percentage showed no significant changeduring IAS cycles. The TPS and CYFRA 21-1 proliferation marker changes in IAS seem to berelated mainly to effects on normal androgen-dependent tissues. Prostate 41:71–77, 1999.© 1999 Wiley-Liss, Inc.

KEY WORDS: intermittent androgen suppression; PSA; free PSA; CYFRA 21-1; TPS;prostate cancer; tumor marker

INTRODUCTION

Prostate cancer has become the most commonnewly diagnosed cancer in men in recent years [1].Where there is tumor involvement outside the pros-tatic capsule, this disease is ultimately incurable in

Grant sponsor: Austrian National Bank; Grant number: 6960.*Correspondence to: Dr. Gerhard Theyer, Department of Urology,Wilhelminenspital der Stadt Wien, Montleartstrasse 37, A-1160 Vi-enna, Austria. E-mail: hamilton@netway.atReceived 8 June 1998; Accepted 16 October 1998

The Prostate 41:71–77 (1999)

© 1999 Wiley-Liss, Inc.

most cases. Palliative treatment consists of hormonalmanipulation by orchidectomy or estrogens, LHRHanalogues, and steroidal or nonsteroidal antiandro-gens to deprive the cancer cells of androgenic stimu-lation [2]. Androgen-dependent tumor cells exhibitapoptotic regression in a high percentage of cases inresponse to androgen deprivation. Unfortunately, thishigh initial response is temporary, due to the fact thatsurviving tumor cells progress to an androgen-independent growth [3]. All trials of chemotherapy forthese androgen-resistant tumors have been met withlimited success and have failed to result in significantprolongation of survival [4,5]. Continuous androgensuppression has been shown to accelerate the progres-sion to androgen independence in the Noble rat; there-fore, intermittent androgen suppression (IAS), whichaims to prolong androgen-dependent tumor growthby cycling between periods of androgen suppressionand treatment cessation until tumor regrowth to spe-cific levels, has been tested in clinical trials [6–8]. Theregrowing tumors have been shown to respond to an-drogen withdrawal for several cycles and IAS to resultin improved qualitaty of live and possible prolongedsurvival in pilot clinical trials [7–9].

In the present study, we investigated the timecourse of free/total PSA and the tumor proliferationmarkers tissue polypeptide-specific antigen (TPS) andcytokeratin fragment 21-1 (CYFRA 21-1) during pros-tate cancer progression in clinical IAS. Monitoring ofserum testosterone and prostate specific-antigen(PSA) has been used in experimental animal modelsand clinical studies for determination of the suppres-sion-free treatment period by defining the level of tu-mor regrowth [10]. However, the expression of PSA isandrogen-dependent and therefore this marker maybe not adequately reflect tumor mass during andro-gen-withdrawal therapy [11]. PSA circulates in plasmain several forms, most of which are complexes withalpha-1-antichymotrypsin (ACT-PSA), uncomplexedfree PSA enzymogen, and inactivated free PSA [12,13].The proportion of PSA-ACT complex is greater in pa-tients with prostate cancer than in those with benignprostatic hyperplasia (BPH), and the reverse is ob-served for free PSA [14]. Therefore, the percentage offree PSA in respect to total PSA is measured and usedfor the differential diagnosis for malignant vs. benignprostatic disease and planning of biopsies. However,the free/total PSA index still exhibits overlap betweenprostate cancer and BPH, making the discriminationstatistically and the factors determining the produc-tion of different isoforms are not understood. IAS mayoffer the possibility to study the cell biology of theformation of free PSA during tumor regression/regrowth.

Different epithelia express characteristic combina-

tions of cytokeratins, depending on their origin ortype of differentiation [15]. In contrast to the cytoker-atins themselves, their fragments are soluble inplasma and therefore can be detected with the help ofmonoclonal antibodies. In a previous study we inves-tigated TPS, recognizing fragments of cytokeratin-8and -18 as possible markers for tumor progressionduring IAS [16]. In the present study, we includedCYFRA 21-1 in addition to TPS in an attempt to detectrelease/production of these cytokeratins during re-gression and regrowth of the tumors during IAScycles. Measurement of the soluble cytokeratin-19fragment referred to as CYFRA 21-1 showed that se-rum CYFRA 21-1 is a useful tumor marker in somemalignancies including lung cancer, head and neckcancer, carcinoma of the uterine cervix, and ovariancancer [17,18]. CYFRA 21-1 is increased in advancedtumor stages and might reflect tumor burden, andsince cytokeratin-19 is expressed in prostatic epitheliaand PIN, expression of this marker is to be expected atleast in some prostate cancers [19]. Cytokeratin frag-ments may be released by tumor cell lysis or due todegradation by activated proteases during cell prolif-eration, constituting two different mechanisms whichcan be investigated in the tumor regression and pro-liferation phases of IAS cycles [17].

MATERIALS AND METHODS

Patients

All patients gave written informed consent accord-ing to local regulatory requirements. Between June1993–March 1995, 34 consecutive and nonpreselectedpatients with disseminated adenocarcinoma of theprostate, fulfilling the inclusion criteria of histologi-cally confirmed tumor, TNM classification [20] >T2(T2 for relapses following radical prostatectomy), per-formance status 0 or 1, no pretreatment by hormoneablation or chemotherapy, and PSA >6 mg/l, were re-cruited for a nonrandomized, open intermittent an-drogen suppression trial, consisting of an initial8-month course of androgen ablation (LHRH antago-nist Zoladex and cyproterone acetate), followed bytreatment cessation and resumption of therapy uponincreases of PSA >20 mg/l.7 Of the 34 men included inthe present study, 10 had tumor classification T2(32%), 19 patients had T3 (56%), and 5 patients had T4(12%). Serum testosterone and PSA were monitoredmonthly, and patients failing to show normalization ofPSA (<4 mg/l) after 24 and 32 weeks of androgen ab-lation were excluded. Loss of androgen dependence isdefined as three sequential increases of PSA abovenormal range under androgen suppression therapy.

72 Theyer et al.

Follow-up examinations included digital rectal exami-nation, transrectal sonography, and yearly chest X-rays and bone scans.

Laboratory Measurements

Blood samples were taken from each patient previ-ous to treatment and at monthly intervals thereafter,and stored at -70°C until analysis. Serum testosteronewas measured using a microtiter plate ELISA (BiomarDiagnostics, Marburg, Germany) according to themanufacturer’s instructions (normal range, 2.4–12 ng/ml; detection limit, 0.1 ng/ml). Free and total PSAwere determined by respective ELISA tests (Enzy-mun-Test PSA and Enzymun-Test FREE PSA, Boeh-ringer Mannheim, Mannheim, Germany) involvingmonoclonal antibodies and streptavidin detection. Themonoclonal antibodies for total PSA recognize freePSA and ACT-PSA, and the antibody for free PSAshows <1% crossreactivity with ACT-PSA. The anti-bodies used for determination of PSA isoforms inthese assays fulfilled the criteria of equimolarity inrecognizing free and complexed PSA. The lower de-tection limit for both free and total PSA was 0.05 mg/l,and the standards for free PSA were calibrated usingthe standards of Stanford University.

TPS (Beki Diagnostics AB, Bromma, Sweden) wasdetermined with an ELISA assay consisting of poly-clonal horse antibody plastic beads and the M3 mono-clonal antibody, the results of which correlated wellwith the TPS-IRMA test. The standard curve was es-tablished with samples containing 0–2,500 U/ml TPA,and the assay was performed as an automated beadenzyme immunoassay (COBAS CORE ROCHE ana-lyzer). According to the manufacturer’s instructions,a cutoff value of 90 U/l was found for TPS in ahealthy control group of 195 probands (95th percen-tile). CYFRA 21-1 was measured by a radioimmuno-metric assay (ELSA-CYFRA 21-1, CIS Bio Interna-tional, Gif-sur-Yvette, France). Presumed normal sub-jects (n = 250) had a CYFRA 21-1 level below 3.3 ng/ml, and the detection limit of the assay was given as0.05 ng/ml according to the manufacturer’s instruc-tions.

Statistics

Nonparametric statistical analysis was used. Dif-ferences between two independent groups were de-termined with the help of the Kruskal-Wallis test;P < 0.05 was regarded as statistically significant. Thethree tumor classification groups were tested for sta-tistically significant differences using the Tukey mul-tiple comparison test. All calculations were done usingthe Statistica software package (StatSoft, Tulsa, OK).

RESULTS

IAS Treatment

Data were obtained from 34 prostate cancer pa-tients with a mean age of 75 years (range, 53–91 years),with a tumor classification of T2 for 10 patients, T3 for19 patients, and T4 for 5 patients. The majority of pa-tients showed grade 3 tumors (22 patients G3, 5 pa-tients G1, and 7 patients G2), and 6/34 patients hadproven metastatic disease. Mean duration of treatmentcessation was 10.3 months (range, 3–18 months). Ac-cording to the IAS study protocol, all patients re-sponded to androgen suppression and prostatic vol-ume decreased by 22.2 ± 4.6%, as measured by ultra-sonography. Of the 34 patients, 8 were in their secondIAS treatment cycle and due to the variable length oftreatment interruption, 82% of the patients were ob-served after 7 months (p8), and 50% of patientsreached 11 months of treatment cessation (p12). Pre-treatment values for PSA, free PSA, TPS, and CYFRA21-1 were compared for the three tumor classificationgroups T2–T4 (data not shown). Although the meanPSA values increased from 18.1 ± 6.2 mg/l for T2 to33.7 ± 12.5 mg/l for T4 and the TPS values from 67 ±9.8 U/l for T3 to 111 ± 12.9 U/l for T4, no statisticallysignificant differences were found between the groupsfor any of the four parameters studied.

Testosterone and PSA

Blood samples from 34 patients were obtainedmonthly, and tumor response following IAS wasmonitored using free and total PSA, serum testoster-one, and TPS. IAS cycles resulted in reversible reduc-tion in serum testosterone (from initial mean value ±SEM of 6 ± 0.8 ng/ml to 0.58 ± 0.05 ng/ml for s5–p1;Fig. 1A) and a corresponding rapid decline of PSA tobaseline levels (from initial mean value ± SEM of 31.2± 4.5 mg/l to 1.7 ± 0.7 mg/l for s4–p2; Fig. 1B), fol-lowed in each individual patient by a slow increase inPSA upon cessation of the antiandrogenic therapy(p3–p6) and intermediate PSA expression during pro-longed treatment cessation (p7–p12, 5.8 ± 0.8 mg/l).PSA values during off-treatment were limited by thereinitiation of androgen suppression in patients ex-ceeding 20 mg/l PSA. These observations are consis-tent with tumor regression and subsequent reappear-ance of hormone-responsive PSA-positive tumor cells,since all these patients responded to further treatmentcycles (data not shown). Serum testosterone levels re-covered within 2 months to mean values ± SEM of 6.1± 0.56 ng/ml (p3–p5), with significantly lower levels(P < 0.01) for patients with longer cessation of andro-gen suppression (p9–p12, 4.4 ± 0.26 nmol/l).

PSA, TPS, and CYFRA 21-1 in Prostate Cancer 73

Free and Total PSA

Free PSA exhibited a rapid decrease in response toandrogen suppression (Fig. 2A) from a mean value ±SEM of 1.98 ± 0.34 mg/l to 0.24 ± 0.05 mg/l (s6–p2),and an increase upon treatment cessation to maximallevels of 0.82 ± 0.13 mg/l (mean value ± SEM) <5months following the end of androgen suppression(p6–p12). Except for the quantitative differences, thetime course of free PSA closely matched that of totalPSA. The percentage of free PSA remained relativelyconstant during IAS cycles (Fig. 2B), varying at around15% of total PSA, and for these patients, the meanvalues of free PSA at different time points during IAScycles exhibited no statistically significant differences.

TPS

In IAS patients, serum concentrations of TPSmatched the time course of IAS treatment phases,showing a decrease during androgen suppression

from an initial concentration of 85.4 ± 19.1 U/l (mean± SEM) to the 43.1 ± 8.4 U/l minimum after 3 monthsof treatment at s4 (Fig. 3A). TPS remained at valuescomparable to the phase of initial androgen suppres-sion for p2–p9 (77.4 ± 10 U/l), and reached valuesexceeding the normal range cutoff of 90 U/l after p9(114 ± 14.8 U/l from p10–p12). No increased produc-tion and/or release of TPS was observed during an-drogen suppression-induced apoptotic regression,which was most likely to occur within the days andweeks following initiation of treatment (s2–s4).

CYFRA 21-1

Determinations of CYFRA 21-1 showed a slow de-crease of this tumor marker until the end of androgensuppression (initial mean concentrations ± SEM of1.72 ± 0.1 ng/ml to the minimal mean value of 1.3 ±0.16 ng/ml at s8; P < 0.04; Fig. 3B) and a recovery toinitial values during treatment cessation (1.80 ± 0.1ng/ml; P < 0.02). In this IAS group, the CYFRA 21-1

Fig. 1. Monthly determinations (mean ± SEM) of testosterone(A) and prostate-specific antigen (PSA) (B) during intermittentandrogen suppression cycles (androgen suppression, s1–s8; treat-ment cessation, p1–p10).

Fig. 2. Monthly determinations (mean ± SEM) of free prostate-specific antigen (fPSA) (A) and percentage of fPSA of total PSA (B)during intermittent androgen suppression cycles (androgen sup-pression, s1–s8; treatment cessation, p1–p10).

74 Theyer et al.

values were below the reported cutoff of 3.3 ng/ml inthe normal population, and compared to TPS, CYFRA21-1 decreased for the whole androgen suppressionphase and was significantly lower only at the end ofthis period.

DISCUSSION

Adenocarcinomas of the prostate in advanced stageare treated by surgical and/or antiandrogenic hor-mone ablation, aiming at elimination of production,metabolization, and use of androgens as far as pos-sible [2]. Despite very high initial responses, the tu-mors recur as highly androgen-resistant and chemo-resistant tumors not amenable to further treatment,within several years in most cases [4,5]. A new con-cept, i.e., intermittent androgen suppression (IAS),tries to prolong the hormone-dependency of tumorcells and possibly survival time by allowing for a lim-

ited regrowth of hormone-sensitive cells between sup-pression treatment cycles of 8-month duration [3,7].Support for this treatment modality comes from ex-perimental animal models and clinical pilot trials,demonstrating significant increases in time to progres-sion to androgen insensitivity and lack of adverse ef-fects on the survival of patients [7,8]. An open clinicaltrial studying IAS was initiated at our institution in1993 and was used as a model of controlled tumorproliferation to investigate the proliferation-associatedmarker TPS and the standard marker PSA for assess-ment of tumor progression during IAS [16].

PSA is widely used as tumor marker for adenocar-cinoma of the prostate. Regarding IAS, the discussionabout monitoring disease progression and the lengthof androgen suppression and when to reinitializetreatment is ongoing. An androgen suppression phaseof 8–9 months seems to be optimal in achieving hightumor regression and preserving androgen-dependen-cy of tumors [21]. The interpretation of PSA in ad-vanced prostate cancer may be complicated by the oc-currence of tumor cells with lower androgen respon-siveness and lower production of PSA, resulting pos-sibly in a poorer correlation of PSA with tumorprogression/volume [11]. Therefore, other markerssuch as free PSA and detection of circulating tumorcells by PCR have been discussed as progressionmarkers [22].

PSA was found to occur in plasma in different iso-forms, and differences in the proportions of these pro-teolysed and complexed forms of PSA were studiedfor their possible correlation with specific prostaticdisease [12,13]. Up to 85% of PSA may exist in thebound form, which was reported to be predominant inprostatic carcinoma in contrast to BPH, thus facilitat-ing differential diagnosis of malign vs. benign disease[14,23–25]. The protease inhibitor alpha-1-antichymo-trypsin is the binding component in the majority ofPSA complexes, and decreased synthesis of this pro-tein by BPH cells may be partially responsible for thehigher percentage of free PSA in benign prostatic dis-ease [26]. The fraction of free PSA is influenced byprostate volume and possibly age and tumor stage,but not by acute inflammations or treatment [26,27]. Inour study, we found a reversible decrease of free PSAduring the androgen suppression phase in IAS, whichclosely parallels the time course of total PSA, resultingin an almost constant percentage of free PSA (15.1 ±2.6% for s2–p12) during IAS cycles. These results con-firm and extend previous findings on the stability ofthe percentage of free PSA in individual patients dur-ing continuous androgen ablation and treatment ofBPH with finasteride [27,28]. Our observations pointto a similiar impairment of the production of PSA andalpha-1-ACT in tumor and normal prostatic tissue

Fig. 3. Monthly determinations (mean ± SEM) of TPS (A) andCYFRA 21-1 (B) during IAS cycle (androgen suppression, s1–s8;treatment cessation, p1–p10). *Significantly different from mea-surements at beginning of androgen suppression and end of treat-ment cessation.

PSA, TPS, and CYFRA 21-1 in Prostate Cancer 75

during androgen ablation. Free PSA offers no addi-tional information for monitoring of IAS cycles, butIAS may constitute an interesting clinical model tostudy the biology of free PSA and ACT during apop-totic regression and tumor regrowth.

An alternative technique for detecting progressionof different tumors relies on the detection of tissuepolypeptide antigen (TPA) released by cycling cellsduring the S and G2 phases. The M3 monoclonal an-tibody, recognizing a proliferation-associated epitopeof TPA, is used for the ELISA or RIA measurement ofthe polypeptide-specific antigen (TPS) and was dem-onstrated recently to identify TPS as a cytokeratin-18derivative [16,29]. In prostate cancer, this tumormarker discriminates benign prostatic hypertrophyfrom tumor, and increasing serum concentrations ofTPS were detected with increasing tumor grade [29].TPS is decreased during androgen suppression in IAScycles; however, values exceeding the normal rangeare observable late in tumor progression, during pro-longed treatment cessation, and the reduction duringhormone suppression may be due to decreased releaseof TPS by cytokeratin-18-expressing normal tissues.Measurements of cytokeratin-19 (CYFRA 21-1) neverexceeded the normal range, and therefore it is con-cluded that the reduction during androgen suppres-sion may also be caused by decreased release fromnormal tissues. TPS and CYFRA 21-1 seem to be ac-tively produced by the respective normal and malig-nant tissues, and tumor regression in IAS seems to beexclusively apoptotic and not necrotic, since no in-creased release of cytokeratins were observed duringthis phase [30]. TPS constitutes a useful marker formonitoring larger, metastatic prostate cancers, andCYFRA 21-1 was not detectable in our IAS patientgroup in significant levels, although expression of cy-tokeratin-19 has been described in normal prostate,prostatic intraepithelial neoplasia, and prostate cancer[19,31,32].

The three prostate cancer tumor classificationgroups studied here showed no significant differencesin pretreatment values of total PSA, free PSA, TPS,and CYFRA 21-1, due to the overlap of T2 tumorsexhibiting biochemical indication of tumor progres-sion with T3 patients and due to the small group ofpatients with T4 tumors included in this IAS trial. De-spite androgen dependency of PSA production, thismarker seems suitable and reliable for guiding clinicaldecisions in IAS studies; the percentage of free PSAoffers no additional information. Determinations ofprostate-specific membrane antigen (PMSA) and hu-man glandular kallikrein (hK2) in blood samples fromour ongoing IAS trial are in progress, in cooperationwith other groups. Newly developed sandwich assaysfor both PSMA and hK2 will allow for the evaluation

of these independent and reportedly more tumor-specific markers in prostate cancer patients during dif-ferent treatment modalities [33,34].

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