[CANCER RESEARCH 38, 4349-4352, November 1978] 0008-5472/78/0038-0000$02.00 Dihydrotestosterone Concentration in Prostate Cancer Tissue as a Predictor of Tumor Differentiation and Hormonal Dependency1

Jack Geller,2 Jerry Albert, Daniel de la Vega, Debra Loza, and Woods Stoeltzing

Mercy Hospital and Medical Center, Medical Research Facility, San Diego, California 92103

Abstract Materials and Methods Tissue dihydrotestosterone and 5a-reductase (A4-3-ke- Clinical. Patients with previously untreated Stage D pros tosteroid-5a-oxidoreductase) levels have been measured tate cancer who underwent transurethral resection of pros in prostates of patients with cancer and benign prostatic tate and in whom 75% or more of the resected tissue was hypertrophy; significant decreases in average values for cancer were selected for study. Tissue was utilized for both of these biochemical parameters were noted in histological study and biochemical assays. Patients were prostate cancer compared to benign prostatic hypertro followed, whenever possible, by objective parameters for phy, although individual values overlapped in both tumor responsiveness, as outlined by Schmidt ef al. (13). groups. Prostate cancer tissue dihydrotestosterone levels Antiandrogen therapy consisted of either castration, dieth- appeared to correlate better than did either histological ylstilbestrol (3 mg or more per day), or megestrol acetate tumor grading or 5a-reductase with the ultimate clinical (Megace; 160 mg/day). Parameters used to follow patients response to antiandrogen therapy. These results suggest included bone scan, prostatic acid phosphatase, and pros that assay of tissue dihydrotestosterone levels in prostate tate size. In some patients, objective data were not available cancer should be further explored as a possible marker and subjective evidence such as change in weight, pain, for tumor differentiation. appetite, and activity was utilized. Prostate DHT levels and histological grading were retrospectively correlated with Introduction tumor response. Tissue DHT and 5a-reductase (A"-3 ketosteroid-5a-oxi- Although measurement of the estrogen receptor in breast doreductase) concentrations were measured according to cancer tissue is now a well-established technique for pre previously reported methods (3). Prostate cancer was dicting responsiveness to endocrine or ablative treatment, graded histologically, with the use of a scale of 1 to 3, with a similar approach has not yet been developed for prostate 3 representing an undifferentiated tumor and 1 a well- tumors. Although there is abundant evidence that a high- differentiated tumor. affinity, low-capacity receptor protein similar to that found in the rat exists in human prostate cytosol and purified nuclei (6-10, 12), uniform detection of such a receptor in Results human BPH3 tissue has not yet been accomplished by any Comparison of 5a-Reductase and DHT Levels in Pa group that has studied more than 10 prostates (2). This is tients with Prostate Cancer and BPH. In Chart 1, it can be attributed to the difficulty of separating cytosol receptor from testosterone- and estradiol-binding globulin (7), re seen that tissue DHT levels were significantly less (p < 0.05) duction of [3H]DHT at 0-4°,the lability of DHT receptor in patients with cancer of the prostate, as compared to those with BPH, although there was a considerable overlap. complex to shearing (9) and temperature changes (9), the near saturation of endogenous cytosol receptor with DHT In 4 patients with prostate cancer (Chart 1), in whom both DHT levels and 5a-reductase were measured in the same (12), and the slow dissociation of DHT from receptor (2), negating a possible exchange assay. DHT, which can be tissue tumor samples, there was no difference in DHT levels measured in a 50-mg biopsy sample of prostate (1), may compared to those of BPH tissue, although 5<*-reductase represent a suitable alternative to the cytosol receptor as a levels were significantly reduced in 3 of these patients. biochemical marker for tissue differentiation, since DHT These 3 patients (7, 8, and 9 in Table 1), who have been levels are significantly higher in androgen target tissues followed clinically for at least 6 months, have responded than in other tissues (4). In this study, the usefulness of favorably to antiandrogen treatment. measuring DHT levels in prostate cancer tissue for predict Correlation of Tissue DHT Levels and Histological Grad ing clinical responsiveness to antiandrogen therapy has ing with Clinical Response to Therapy (Table 1; Chart 1, A been compared with histological tumor grading, the stan and B). In 9 patients with previously untreated Stage D dard technique currently being used for this purpose in prostate cancer, response to antiandrogen therapy has previously untreated patients with prostate cancer. been retrospectively correlated with both tissue DHT levels and histological grading. We have designated prostate DHT levels >2.0 ng/g as "differentiated," since this value is 1 Presented at the John E. Fogarty International Center Conference on more than 2 S.D. above the mean of non-androgen target Hormones and Cancer, March 29 to 31, 1978, Bethesda, Md. Supported by Grant 5 R26 CAÎ8003-02,awarded by the National Cancer Institute, Depart tissues. Histological tumor grading of 1 and 2 (Fig. '\A) was ment of Health, Education and Welfare. used to indicate well- and moderately differentiated tumors, 1To whom requests for reprints should be addressed. 3The abbreviations used are: BPH, benign prostatic hypertrophy; DHT, respectively (Figure 1X\);tumors with greater than Grade 2 17/3-hydroxy-5a-androstan-3-one. histological changes (Fig. 1B) were considered to be poorly

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-Reduci ase A,'.. Endogenous Tissue DHT some of nature's experiments, including male pseudoher-

11109S!6 maphroditism Type II and testicular feminization, in which 5a-reductase in the former and cytosol receptor in the latter rao90807060SO403020100•A.•5± are virtually absent and prostate and male secondary sex structures are absent or ambiguous. It is presumed that DHT, the biologically active androgen in many target tis sues, is also very low in tissues of these patients. Tissue ûv.•!*-e-& DHT levels depend upon multiple factors. These include -v-••1 levels of plasma testosterone, a major substrate for DHT, b4 and 3-hydroxysteroid dehydrogenase, which removes DHT by reduction, in addition to previously mentioned 5«-reduc-

32i *••BPHA-••••AVVVÇaP<0tase and receptors. We have attempted to study the relative importance of some of these various biochemical factors as regulators of tissue DHT. In previously reported studies of patients with 0B•:•X••s BPH in which we acutely perturbed plasma testosterone, 05 Un points) 5<*-reductase, and cytosol receptor with either Megace, an p< 0 B llor A s only) antiandrogen, or polyestradiol phosphate (Estradurin), tis Chart 1..A, prostate levels of 5«-reductase for patients with BPH and sue DHT levels appeared to correlate best with change in cancer of the prostate. •¿,individualvalues for BPH; A, for cancer. Means cytosol receptor binding of DHT (5). This conclusion was are indicated by horizontal lines. B, tissue DHT levels for patients with BPH and cancer. Tissue DHT levels in 4 cancer tissues which were also assayed based on the fact that Megace and Estradurin had similar for 5a-reductase (see A in A) are shown as A, while other tissues are quantitative inhibitory effects on 5«-reductase and plasma indicated by •¿.Statisticalcomparisons of BPH and carcinoma group for both 5a-reductase activity and endogenous tissue DHT levels are shown at testosterone, but only Megace, which also decreased re the bottom of the chart. ceptor binding of DHT, significantly decreased tissue DHT levels. Table 1 In this study, we have shown in Chart 1 that among 4 Untreated prostate cancer (Stage D) patients with prostate cancer with DHT levels similar to those of the BPH group, 3 had low values of 5«-reductase DHT/g response to Patient123456''789Histologi-cal grade2232-32-332-32-32ngtissue"0.972.71.12.52.85.93.65.58.7ClinicaltherapyPartial (see Chart 1). The fact that these 3 patients responded regression6Objectivelyobjective favorably to antiandrogen therapy supports the hypothesis stable6Objective that DHT is a better biochemical marker of tumor differen progression''Partial tiation than is 5a-reductase. In 9 patients with prostate regression''Partialobjective cancer studied to date, tissue DHT also appears to correlate regression''Remission''Remission''Remission1'Partialobjective better with the ultimate response to antiandrogen therapy than histological grading (Table 1). Extension and confir mation of these studies in a much larger series of patients objective regression'' is necessary, however, before any final conclusions can be " Values > 2.0 ng/g indicate differentiated. reached. * Refers to use of objective criteria for evaluating response, It would thus appear that levels of DHT in prostate cancer including changes in bone scan, tumor mass, and prostatic acid tissue may provide a biochemical index for tissue differen phosphatase. r Lymph node replaced by prostate cancer tissue. tiation in previously untreated patients. Tumor DHT assay '' Refers to use of subjective and clinical criteria for response; also represents a practical approach to clinical limitations adequate data for objective evaluation were not available. on tissue availability, since assay of a 50-mg sample of tumor can accurately and precisely detect as little as 100 differentiated. Among 8 patients who had either partial pg of DHT or 2 ng of DHT per g wet weight of tissue, the objective regressions or subjective remissions, or who were lower limit set for DHT content in differentiated tumors. objectively stable on therapy, 7 had DHT levels >2.5 ng per Previous therapy for prostate cancer with drugs or surgery, g prostate (biochemically differentiated), while only 3 of which decrease plasma testosterone, invalidates the useful these 8 patients were classified as Grade 1 or 2 microscop ness of DHT for study of differentiation. In treated patients ically (histologically differentiated); one patient had objec with low endogenous steroid levels, cytosol receptor assay, tive progression of his disease on treatment and had a DHT when a reproducible method has been developed, might level below 1.8 ng/g and histological Grade 3 tumor. provide a reliable index of tumor differentiation, as recently suggested by Mobbs et al. (11). Discussion References DHT is found in significant amounts only in androgen 1. Albert, J., Geller, J., Stoeltzing, W., and Loza, D. An Improved Method target tissues, as demonstrated in previous studies (4). This for Extraction and Determination of Prostate Concentrations of Endog selectivity is due to requirements for 5a-reductase, DHT- enous Androgens. J. Steroid Biochem., in press, 1978. binding cytosol receptor, and nuclear acceptor in these 2. Geller, J. Differentiation of Prostate Cancer. In: E. B. Thompson and M. specialized tissues. That 5a-reductase and the receptor are Lippman (eds.). Steroid Receptors and the Management of Cancer, Ed. 1. Cleveland: CRC Press, Inc., in press, 1978. critical for biological activity of androgens is indicated by 3. Geller, J., Albert, J., Geller, S., and Lopez, D. Effect of Megestrol

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Acetate (Megace) on Steroid Metabolism and Steroid-Protein Binding in 9. Mainwaring, W. I. P., and Milroy, E. J. G. Characterization of the Specific the Human Prostate. J. Clin. Endocrinol. Metab., 43. 1000-1008, 1976. Androgen Receptors in the Human Prostate Gland. J. Endocrinol., 57: Geller, J., Albert, J., Loza, D., Geller, S., and Stoeltzing, W. DHT 371-381, 1973. Concentrations in Human Prostate Cancer Tissue. J. Clin. Endocrinol. 10 Menon, M., Tananis, C. E.. McLoughlin. M. G., Lippman, M. E.. and Metab., 46: 440-444, 1978. Walsh, P. C. The Measurement of Androgen Receptors in Human Geller, J., Albert, J., Stoeltzing, W., and Loza, D. Sex Steroid Effects on Prostatic Tissue Utilizing Sucrose Density Centrifugation and a Prota- Dihydrotestosterone Levels in Human Prostate (Abstract 313). The En mine Precipitation Assay. J. Urol.. 117: 309-312, 1977. docrine Society Program and Abstracts. 59th Annual Meeting, June 8 to 11 Mobbs, B. G., Johnson, I. E., Connolly, J. G.. and Clark, A. F. Androgen 10, 1977. Receptor Assay in Human Benign and Malignant Prostate Tumor Cytosol Geller. J , Cantor, T., and Albert, J. Evidence for a Specific Dihydrotes Using Protamine Sulphate Precipitation. J. Steroid Biochem., 9: 289- tosterone Binding Cytosol Receptor in the Human Prostate. J. Clin. 301,1978. Endocrinol. Metab.. 47. 854-862, 1975. 12 Rosen, V., Jung. I., Baulieu, E. E.. and Robel, P. Androgen Binding Hansson, V., Tveter, K. J., Attramadal, A., and Torgersen, O. Androgenic Proteins in Human Benign Prostatic Hypertrophy. J. Clin. Endocrinol. Receptors in Human Benign Nodular Prostatic Hyperplasia. Acta Endo Metab., 41: 761-770, 1975. crinol., 68: 79-88, 1971. 13 Schmidt. J. D., Johnson, D. E.. Scott, W. W., Gibbons, R. P., Prout, G. Krieg, M., Bartsch. W., and Voigt, K. D. Androgen Binding, Metabolism R., and Murphy, G. P. Chemotherapy of Advanced Prostatic Cancer. and Endogenous Tissue Levels in Human Benign Prostatic Hypertrophy Evaluation of Response Parameters. Urology, 7. 602-610. 1976. (BPH) and Normal Prostate. Acta Endocrinol., 85: 47, 1977.

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Fig. 1.A, moderately well-differentiated prostate cancer (Grade 2). The tumor appears to be forming glands, cells are uniform without evidence of anaplasia or pleomorphism, and there is fibrosis of the stroma. B, a fully differentiated prostate cancer (Grade 3). The tumor appears as solid sheets of pleomorphic anaplastic cells. There is no gland formation.

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Jack Geller, Jerry Albert, Daniel de la Vega, et al.

Cancer Res 1978;38:4349-4352.

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