Free-to-Total Prostate-Specific AntigenRatios 18–24 Months Following ExternalBeam Radiation for Adenocarcinoma of

the Prostate

JOHN F. WARD, MD,1* PETER A.S. JOHNSTONE, MD,2 AND CHRISTOPHER J. KANE, MD1

1Department of Urology, Naval Medical Center, San Diego, California2Department of Radiology, Radiation Oncology Division, Naval Medical Center,

San Diego, California

Background and Objectives:The purpose of this study was to evaluatefree-to-total prostate-specific antigen (PSA) ratios after definitive externalbeam radiation therapy for men with adenocarcinoma of the prostate(CaP).Methods: A prospective evaluation of percent free PSA in men followingdefinitive external beam radiation therapy for CaP was compared to menwith untreated CaP and men at very low risk for CaP. Statistical compari-son of clinical and pathologic parameters was performed.Results: There was no statistically significant difference in free-to-totalPSA ratios for men with newly diagnosed CaP and men with detectablePSA who were treated with external beam radiation therapy.Conclusions: Free-to-total PSA ratios after definitive external beam ra-diation therapy for CaP are consistent with percent free PSA in patientswith newly diagnosed CaP. This supports the theory that PSA from in situprostate tissue following external beam radiation therapy is produced bymalignant cells.J. Surg. Oncol. 1999;70:91–94. Published 1999 Wiley-Liss, Inc.†

KEY WORDS: prostate-specific antigen; percent free PSA; radiotherapy;prostatic neoplasms

INTRODUCTION

Prostate-specific antigen (PSA), a chymotrypsin-likeenzyme directed at liquefaction of the seminal coagulum,is believed to leak from the prostatic ductal system intothe prostatic stroma and then into the bloodstream viacapillaries and lymphatics. Unlike traditional tumormarkers, PSA is not found in larger amounts in tumorcells compared with healthy tissue. In fact, the oppositeis true: malignant prostate tissue actually produces lessPSA and PSA mRNA than normal prostate epithelialcells and benign prostatic adenomatous tissue [1–3]. Theserum concentration of PSA is usually below 2.5 ng/ml,far less than the 5.0 mg/ml found in the seminal fluid.

Original work from Lilja (Malmo, Sweden) [4] andStenman et al. (Helsinki, Finland) [5] in 1991 demon-strated that the PSA molecule could be measured to existin different molecular forms in serum. Monoclonal anti-bodies (Mabs) can be generated against the PSA mol-

ecule which recognize one or more of five non-overlapping conformational immunoreactive epitopegroups on the PSA molecule. Current laboratory mea-surements of free PSA rely on a single epitope remainingaccessible to binding by a specific Mab.

Serum PSA activity, at least in vitro, is regulatedmainly by two protease inhibitors—alpha-1-antichymo-trypsin (ACT) and alpha-2-macroglobulin (AMG)—both

The Chief, Bureau of Medicine and Surgery, Navy Department, Wash-ington, D.C., Clinical Investigation Programs, sponsored this report,S-96-017, as required by HSETCINST 6000.41A. The opinions andassertions contained herein are those of the authors and are not to beconstrued as official or representing the views of the United StatesNavy or Department of Defense.*Correspondence to: John F. Ward, MD, Department of Urology, Na-val Medical Center, 34800 Bob Wilson Drive, San Diego, CA 92134-5000. Fax: (619) 532-7234. E-mail: jfward@snd10.med.navy.milAccepted 24 November 1998

Journal of Surgical Oncology 1999;70:91–94

Published 1999 Wiley-Liss, Inc. †This article is a US Govern-ment work and, as such, is in the public domain in the United Statesof America.

present in the serum in amounts more than 100 times thatof PSA. We believe this same regulation occurs in vivo,although no studies have been performed observing thechanging levels of ACT and AMG in vivo and the effectsuch changes have on PSA activity in vivo. These mac-romolecules possess the ability to configure covalentcomplexes with the active form of PSA. After complex-ing with AMG, no PSA epitopes remain exposed. How-ever, several antigenic epitopes remain exposed after theformation of the PSA-ACT complex, making possible aninteraction with a series of anti-PSA Mabs.

Christensson and colleagues [6,7] were the first inves-tigators to determine that the free-to-total PSA propor-tion (percent free PSA) was significantly lower in pa-tients with prostate cancer (CaP) than in men with benignprostates (0.18 and 0.28, respectively), even at PSA lev-els lower than 10 ng/ml [1]. Multiple investigators havenow confirmed that the application of this parameter re-sults in an increase in specificity of 55–73% for the de-tection of prostate cancer without compromising the sen-sitivity of the PSA test for distinguishing prostate cancerfrom benign prostate tissue [1,8,9].

The reason why patients with prostate cancer displayan increased proportion of serum PSA complexed toACT has not been clarified. It does not appear that thelow percentage of free PSA in the serum of these patientsis dependent upon the ACT concentration in the serumsince ACT is normally present in the serum at muchhigher molar excess than free PSA. Neither the PSA-ACT concentration nor the percentage of free PSA cor-relates with the total ACT concentration in the serum[7,10].

It is not known whether the interaction between theenzymatically active PSA and ACT takes place at a peri-cellular level or in the general circulation. In vitro testingindicates that there is a very low rate of free PSA bindingto free ACT. This indirectly indicates that this reactionmay occur predominantly at a pericellular level beforePSA enters the bloodstream [10]. Immunocytochemistryand in situ hybridization techniques have demonstratedthat local PSA-producing prostatic epithelium can syn-thesize ACT. ACT transcripts and expressed proteins canbe detected in most prostate cancer cells [11,12].

While the determination of the clinical utility of freeand total PSA in the detection of prostate cancer contin-ues, the effects of different treatment modalities on per-cent free PSA have not been studied.

Many post-external beam radiation nadir levels of totalPSA best predicting subsequent freedom from prostatecancer have been proposed. Although lower nadirs gen-erally are associated with superior outcomes, the identi-fication of a single absolute nadir level was not selectedat a recent ASTRO consensus conference. What propor-tion of post-radiation therapy (XRT) PSA representsbound vs. unbound PSA has not been previously studied.

The role free-to-total PSA may play in predicting treat-ment outcome following XRT for CaP has also not beendetermined.

MATERIALS AND METHODS

A total of 42 men were prospectively studied withpatient informed consent under a protocol approved bythe Institutional Review Board.

Peripheral venous sampling of 8–10 ml blood into se-rum separating tubes (Becton-Dickinson, San Jose, CA)was performed. All samples were allowed to coagulatefor 15 min at room temperature, centrifuged at low speedfor 10 min, and then immediately frozen at −70°C. Over-night courier was used to ship the frozen samples on dryice to UroCor, Inc. (Oklahoma City, OK), for analysis.

Assay system consisted of an Immulite solid-phasechemiluminescent detection system (Diagnostic ProductsCorp., Los Angeles, CA) to quantitate free PSA. TotalPSA measurements were quantitated by the commer-cially applied Tosoh AIA-600 system (Tokyo, Japan),which utilizes an enzyme-conjugated Mab and fluoro-genic substrate.

Group 1 consisted of all patients who at the time of thestudy were 18–24 months from definitive external beamXRT for adenocarcinoma of the prostate, regardless oftheir Gleason grade or clinical stage at the initiation oftreatment (16 men total). All men had received four-fieldtreatment of the prostate and periprostatic tissue. Thedose was prescribed at the 100% isodense line of a com-puter-generated treatment plan. A mean dose of 6,800cGy was delivered over 48 days in 37 fractions.

Group 2 consisted of 13 men with biopsy-proven ad-enocarcinoma of the prostate. All men were at least 6weeks from prostate biopsy and had not received therapyfor their CaP prior to venous sampling.

Group 3 consisted of 13 men less than 40 years old,considered to be at low risk for CaP, and without a familyhistory of CaP.

Statistical analysis was performed using a statisticalsoftware program (GB-Stat 6.5, Dynamic Microsystems,Inc., Silver Spring, MD). The two-tailed Studentt-testassuming equal variance (alpha4 0.05) was used todetermine statistical significance.

RESULTS

No statistically significant difference existed for clini-cal tumor stage (P 4 0.696) or Gleason grade (P 40.353) between men with newly diagnosed CaP andthose treated with XRT.

Total PSA in patients with newly diagnosed CaP washigher than the control group (P 4 0.00007).

Total PSA in patients with newly diagnosed CaP wasalso higher when compared to total PSA in the post-XRTpatients (P 4 0.034).

Percent free PSA in patients with newly diagnosed

92 Ward et al.

CaP was statistically significantly different from the con-trol group, consistent with previous investigators’ reports[9,10] (P 4 0.0003) (Table I).

Percent free PSA in patients 18–24 months post-XRTwas (1) significantly lower than the percent free PSA inthe control group despite their total PSA not being sig-nificantly different (P 4 0.0004); and (2) not statisticallydifferent from the percent free PSA in patients withnewly diagnosed prostate cancer (P 4 0.584).

DISCUSSION

Because the prostate remains in situ after radiationtherapy, a logical question has been frequently askedduring the 1990s: What level of PSA after XRT could beexpected from the in situ prostate and presumably benignglands?

Grob et al. [13] in 1994 performed immune peroxidasehistochemistry for expression of PSA on biopsy speci-mens obtained 12 months after radiation therapy. Resultsof this study showed that the benign glands identified inthese biopsy specimens did not stain the PSA. Onlyglands that were histologically identified as malignantshowed PSA staining. These findings supported the con-clusion that serum PSA levels after external-beam radia-tion therapy were more likely secondary to persistentcancer rather than residual benign prostatic hypertrophy.

Our current study has found that following definitiveXRT, free-to-total PSA ratios are consistent with the lev-els expressed by men with newly diagnosed prostate can-cer, and not with levels expressed by men with benignprostatic hypertrophy. This supports the work of Grob etal. [3] that only malignant prostatic epithelial cells ex-press PSA after XRT.

The long-term significance of this finding is unknown.

One cannot take these results to indicate a failure ofexternal beam radiation therapy to treat prostate cancer.Should a low free-to-total PSA ratio following XRT rep-resent the persistence of malignant prostatic epithelialcells as concluded in this study, this may not representclinical failure in the management of the patient’s pros-tate cancer. What critical volume of prostate cancer cellsis needed for the disease to be clinically significant isunknown either pre-treatment or post-treatment.

Clinical application of the newly available free-to-totalPSA assay to patients treated with external beam radia-tion therapy is not yet defined. While Pearson et al. [14]have been able to retrospectively predict CaP in men 8years in advance of the diagnosis using percent free PSAmeasurements of stored serum samples, this predictivevalue of percent free PSA has not been demonstrated inpatients who have received definitive external beamtherapy for their disease.

CONCLUSIONS

Percent free PSA (free-to-total PSA ratio) in men18–24 months after definitive external beam radiationtherapy for CaP is consistent with percent free PSA inpatients with newly diagnosed adenocarcinoma of theprostate. This supports the theory that PSA from in situprostate tissue following external beam radiation therapyfor adenocarcinoma of the prostate is produced by ma-lignant cells. The clinical implication of this observationis unknown.

ACKNOWLEDGMENTS

Our thanks to Dr. Robert Marley of UroCor, Inc., forproviding support for this research project.

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TABLE I. Percent Free PSA in Prostate Cancer PatientsTreated With Radiation Therapy

A. Total Vs. Percent Free PSA ResultsMean total

PSA (ng/ml)Mean percent

free PSA

Control 0.5 (±0.23)a 49 (±0.29)a

Newly diagnosedprostate cancer 11.8 (±8.6) 14 (±0.06)

Prostate cancer treatedwith XRT 3.2 (±5.8) 16 (±0.11)

B. Statistical Differences in Percent Free PSA

Control (P)Newly

diagnosed (P)

Control P 4 0.0003Newly diagnosed

prostate cancer P 4 0.0003Prostate cancer treated

with XRT P 4 0.0004 P 4 0.584

aStandard deviation in parentheses.

Percent Free PSA After Radiation Therapy 93

to total PSA (F/T) ratio to total PSA for distinguishing benignprostatic hyperplasia (BPH) from prostate cancer (CaP) using theAbbott AxSym system. J Urol 1996;155:370A.

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94 Ward et al.