Treatment of Prostatic Carcinoma with Various Types of Estrogen Derivatives G. JONSSON, A. M. OLSSON, AND W. LUTTROP Department of Urology, University Hospital of Lund, Lund, Sweden AND
2. CEKAN, K. PURVIS,
AND
E. DICZFALUSY
Swedish Medical Research Council, Reproductive Endocrinology Research Unit, Karolinska Sjukhuset, Stockholm, Sweden
I. Clinical Results . . . 11. SteroidLevels . . . . A. Methods and Materials B. Results . . . . . . 111. Discussion . . . . . IV. Concluding Remarks . . References . . . . . Discussion . . . . .
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351 356 356 359 369 372 372 373
I. CLINICALRFSULTS Endocrine therapy of prostatic carcinoma has been the rule ever since Huggins and Hodges demonstrated the hormone dependence of the prostate. The methods used have varied from time to time and from one hospital to another. Opinions still differ on the mechanisms of the methods used, their suitability, side effects, etc. The therapeutic programs have included orchiectomy, estrogen administration, pituitary ablation, corticosteroid therapy, administration of antiandrogen, etc. Also nonendocrine methods such as radiotherapy as well as treatment with cytostatics have proved useful. The main purpose of endocrine therapy from the very beginning was to reduce the amount of androgen via inhibition of the secretion of the gonadotropins by the pituitary. The potency of the substances administered was generally estimated from determinations of the steroid substances in the urine, I n recent years, however, the mechanisms bringing about the effect of endocrine therapy have proved to be much more complex than was formerly supposed. This has been made clear by recent investigations with technically advanced methods. Among other things, the introduction of radioimmunoassay methods for determining steroidal and polypeptide hormones in the serum as well as demonstration of the 351
352
G. JONSSON
ET AL.
action of androgens on the prostate have shed light on certain points previously difficult to understand. Oral treatment with estrogenic compounds, such as stilbestrol, has several disadvantages in the form of dyspeptic symptoms, sodium retention, edema, cardiovascular complications, etc. To eliminate the above-mentioned disadvantages we began in 1955 to use a long-acting parenteral drug of low toxicity, Estradurinm (polyestradiol phosphate). The estrogenic effect of an intramuscular injection persists for a couple of months. In an initial series consisting of 105 patients, 40% had metastases before the beginning of treatment (Jonsson et d., 1963). Many of the patients had had symptoms for a fairly long time before they sought medical advice. In 97 patients it was possible to estimate the interval between the onset of symptoms and the beginning of treatment fairly accurately: the mean interval was 12 months. In all cases the diagnosis had been histologically confirmed by perineal puncture or transurethral resection. I n 47 of the 105 cases the urine was examined in respect to steroids, which included determinations of the total 17-ketosteroids, 17-hydroxycorticosteroids, individual 17-ketosteroid metabolites, such as androsterone (A) etiocholanolone (E) dehydroepiandrosterone, and estrone, l7/I-estradiol7 and estriol. The series was divided into two groups according to the dose of Estradurin: 80-120 mg monthly and 150-200 mg monthly in a single injection. The results of the steroid analysis are given in Table I. The data thus suggest that in the group treated with 8&120 mg of Estradurin per month
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TABLE I URINARY EXCRETION O F 17-KETOSXPROIDS (a), ANDROSTERONE ETIOCFIOLANOLONE (b), DERYDROEPIANDROSTERONE (c) BEFORE AND AFTER ESTRADURIN THERAPY
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Estradurin dosage (mg)
Before treatment (mg/24 hours)
After 4 months’ treatment (mg/24 hours)
80-120 160-200 b 80-120 160-200 c 80-120 160-200
4.93 4.72 2.79 2.69 0.29 0.20
4.86 3.92 2.86 2.16 0.28 0.20
a
ESTROGENIC TREATMENT OF PROSTATIC CARCINOMA
353
the mean excretion value of 17-ketosteroids did not differ significantly from the pretreatment value. On the other hand, in patients treated with 160-200 mg of Estradurin treatment resulted in a probably significant decrease in the excretion of 17-ketosteroids. Like the urinary total ketosteroids, the excretion of A E was not decreased after administration of 80-120 mg of Estradurin per month. But treatment with the larger dose of Estradurin produced a probably significant decrease in the elimination of A E. Subsequent orchiectomy, however, resulted in a highly significant decrease in the excretion of 17-ketosteroids1 androsterone, and etiocholanolone. Neither Estradurin therapy nor orchiectomy influenced the excretion values of dehydroepiandrosterone significantly. Although Estradurin is a fairly weak gonadotropin inhibitor, the clinical results were good: 40% of the patients were alive 5 years after the beginning of treatment. The good clinical results together with the determinations of the steroids suggested that Estradurin has a therapeutic effect directly on the malignant cells. The favorable clinical results obtained in a small group of patients with an initially low excretion of androgen metabolites lend additional support to this view. I n view of the aforementioned results of the determinations of steroids and the clinical response, Estradurin was combined with a stronger gonadotropin inhibitor, e.g. ethynylestradiol, in the next series (Jonsson, 1971). The material in this series consisted of 126 patients. The age distribution and the frequency of metastases were the same as in the first series. The patients in this series came from the same receiving area, the disease was equally advanced (75% thus belonged t o stage 111 IV, VACURG classification) , and had received the same supplementary therapy besides estrogen therapy. All the patients were reexamined after 3 months and afterward twice a year for a t least 5 years. The estrogen therapy in this series consisted of Estradurin given intramuscularly in a dose of 160 mg per month for 3 months, after which the dose was reduced to 80 mg per month. This treatment was combined with 1 mg of ethynylestradiol a day for 14 days. This was followed by a daily maintenance dose of 150 pg of ethynylestradiol. The effect of treatment was largely the same as that of estrogen therapy in general : substantial improvement of the patients’ general condition, and abatement or disappearance of urinary symptoms and of metastatic pain. I n several cases the number and spread of metastases became stationary, and in some cases roentgen examination showed regression.
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ET AL.
Of the 126 patients, 78 (62%) survived for more than 5 years. As expected, the 5-year survival rate of patients in stages I and I1 was high (74%) ; in stage IV it was 45%. The lower the differentiation, the higher the mortality from the basic disease. The present investigation produced no evidence in support of the assumption of an overmortality from cardiovascular diseases in patients treated with these estrogens, but patients with such diseases before treatment must be regarded as less good risks unless they receive adequate treatment of their cardiovascular complaints. Irrespective of the type of estrogen treatment given, most patients with prostatic carcinoma sooner or later deteriorate. These patients, like those primarily resistant to conventional estrogen treatment, constitute a serious therapeutic problem. In these two groups, increasing and sometimes very large doses of estrogen) radiation, corticosteroids, hypophysectomy, antiandrogens, cytostatics, etc., have been tried. In some cases palliation has been achieved, but, on the whole, the results are less encouraging, besides which several of the methods have serious side effects. It is not known why certain cases do not respond to antiandrogenic treatment or why they first respond but later become unsusceptible. In such cases, changes probably occur in the biological behavior of the carcinoma. During the last 7 years we have used estramustine phosphate (EstracyP) a nitrogen mustard derivative of estradiol, in the two abovementioned groups of prostatic carcinoma (Jonsson and Hogberg, 1971 ; Nilsson and Jonsson, 1975). Our own clinical material consisted of 128 patients with advanced prostatic carcinoma. One hundred and seventeen belonged to stage IV (metastases) and 11 to stage 111. All patients are followed for more than one year. Ninety patients had previously received some other antiandrogenic therapy (secondary treatment group) but had deteriorated during treatment. Judging from clinical experience, the expected duration of survival of these patients would be less than 6 months. Thirty-eight patients were given Estracyt from the very beginning (primary treatment group). All the patients were admitted to hospital for the treatment. Investigation on admission included histological and cytological examination of the prostate and sometimes of lymph nodes, roentgenological investigation of the skeleton, lungs, and urinary tract, analysis of the blood picture, determination of the acid and alkaline phosphatases, renal and liver function tests, etc. The examinations were repeated at certain intervals. Careful notes were made of the patient’s general condition, pain, use of analgetics, difficulty in urination, etc. )
ESTROGENIC TREATMENT OF PROSTATIC CARCINOMA
355
The histological and cytological investigation showed that 37 were poorly differentiated, 51 moderately differentiated, and 21 well diff erentiated; the degree of differentiation of 19 was uncertain. One hundred and seventeen patients had metastases, often in more than one site. The acid phosphatases were increased in 68%. Estracyt was given intravenously in a dose of 300450 mg/day for about 3 weeks. If the patient responded well, intravenous treatment with 300-450 mg twice a week was continued for a varying period depending on the patient’s response. If the patient was symptom free and the disease under control after 2-3 months’ treatment, the injections were stopped. In the event of exacerbations, intense treatment with daily injections was resumed. If no effect could be recorded after 3 weeks’ daily treatment, the preparation was withdrawn. We have also used Estracyt by mouth since 1971. The patients have received a dose of 600-900 mg a day either as primary treatment or as secondary maintenance therapy after intravenous treatment. It is sometimes not easy to assess the results of treatment, because it is difficult to evaluate the patients’ symptoms, especially pain. Estimation of the effect of treatment could, however, often be based on objective observations. For instance, previously bedridden patients could sometimes get up and even return to work. A most striking effect was the abatement of the pain and the improvement of the patient’s general condition. Regression or disappearance of metastases was noted in 33 of the 128 cases. During treatment the acid phosphatases fell in many cases, sometimes to normal level. There seems to be a close correlation between falling acid phosphatases and improvement of the patient’s general condition. Reelevation of the acid phosphatase levels in patients who had responded favorably was usually associated with a new deterioration. Changes were noted in the prostatic tumor in the form of cellular pycnosis, vacuolization, rupture of cell membranes, squamous epithelial metaplasia, and increased lipofuscin. On numerical evaluation of the results we found that about half of the patients who had got secondary therapy improved. Of the patients who received Estracyt as primary therapy, 80-90% improved. The results in these series suggest that in the group given secondary treatment the hitherto expected survival of less than 6 months might be prolonged to anything up to 5 years. The primary treatment group was too small to warrant any conclusions regarding the survival time. The side effects must be regarded as insignificant. There was occasionally thrombophlebitis in patients treated intravenously. Gastrointestinal disturbances, though usually only transient, occurred in some patients treated by mouth. A mild reaction of the liver, allergic reactions, leukopenia, and thrombocytopenia occurred in a few cases.
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11. STEROID LEVELS
A. METHODS AND MATERIALS A critical assessment of the endocrine effects of the three types of estrogen treatment discussed above, namely of Estradurin@ (polyestradiol phosphate, hereafter PEP), Estradurin ethynylestradiol (hereafter PEP EE) , and Estracyt@ (estramustine phosphate, hereafter EMP) necessitates serial measurements of a number of relevant hormonal parameters in the blood of previously untreated patients. Therefore, patients with histologically and/or cytologically diagnosed prostatic carcinoma and without any previous hormone therapy were allocated randomly to one of the above treatments, and the pretreatment values of a number of circulating steroids and pituitary hormones were then compared with those found monthly during treatment with the three estrogen regimens described.
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1. Clinical Material
This consisted of 24 patients with prostatic carcinoma stages I1 to IV (Veterans classification) diagnosed histologically and/or cytologically. The average age of the patients was 72.5 years (range 62-84 years). 2. Therapeutic Regimens and the Design of the Study
The patients were randomly allocated to one of the three regimens, consisting of (a) PEP (160 mg im monthly), ( b ) PEP EE (PEP 160 mg im monthly; and EE 150 pg per 0s daily), and ( c ) EMP (600 mg per 0s daily). The structural formulas of PEP and EMP are indicated in Fig. 1. Pretreatment (control) plasma samples were drawn from each patient
+
0 1
o r
0
0
O - p tP o H 'OH
E
=&
POLY ESTRADIOL PHOSPHATE (PEP) ESTRADURIN@
CICH,
- CH,
'
N'
CLCH, - c x,
-coo
ESTRAMUSTINE PHOSPHATE ( EMP) ESTRACYT @
FrG. 1. Structural formulas of polyestradiol phosphate (PEP) and estramustine phosphate (EMP).
ESTROGENIC TREATMENT OF PROSTATIC CARCINOMA
357
on each of three consecutive days. Then treatment was initiated immediately. Plasma samples were then obtained from each subject after 1 and 2 weeks, as well as after 1, 2, 3, 4, 5, and 6 months of treatment. Blood samples of 20 ml were withdrawn on each occasion from the antecubital vein into heparinized test tubes. The collection of plasma samples occurred invariably between 9:00 and 1O:OO AM. The blood was centrifuged immediately; the plasma was separated, frozen, and stored a t -2OOC until analyzed. 3. Hormonal Parameters Studied
The following unconjugated steroids were measured: pregnenolone (3p-hydroxy-5-pregnen-20-one) , 17-hydroxypregnenolone (3B,17-dihydroxy-5-pregnen-20-one),dehydroepiandrosterone (3p-hydroxyd-androsten-17-0ne)~ 20a-dihydroprogesterone (20a-hydroxy-4-pregnen-3-one), 17-hydroxyprogesterone ( 17-hydroxy-4-pregnene-3,20-dione), androstenedione (4-androstene-3,17-dione), testosterone (17p-hydroxy-4 androsten-3-one), dihydrotestosterone (17p-hydroxy-5a-androstan-3-one), cortisol (1lp,l7-dihydroxy-4-pregnene-3,20-dione),estradiol (1,3,5 (10) estratriene-3,17p-diol), and estrone (3-hydroxy-1,3,5 (10)-estratrien-17one). The following immunoreactive pituitary hormones were estimated : follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin. 4. Assay Procedures All steroids except cortisol were measured by radioimmunoassay procedures involving chromatographic purification and separation of steroids as described in detail in previous communications (Brenner et al., 1973; Purvis et al., 1975; Aedo et al., 1976). Cortisol was assayed by the protein-binding method described by Nugent and Mayes (1966). The assays of LH were performed according to Midgley (1966) as modified by Robyn et al. (1971), using the Second International Reference Preparation of human menopausal gonadotropin (2nd IRP-HMG) as standard. The measurements of FSH and prolactin were performed by the use of kits purchased from Biodata S.A., Rome, Italy. I n the FSH assays an H M G standard calibrated against the 2nd IRP-HMG was used, whereas the results of prolactin assays were expressed in terms of the NIH-1 human prolactin preparation.
5. Cateulation and Evaluution of Results I n all calculations, a log normal between-patients distribution of individual values was assumed (Gaddum, 1945). Hence in Figs. 2-14, geometric mean values and 95% confidence limits are indicated. In order to assess differences between control and treatment values for
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each treatment group, an analysis of variance was performed and the significance of the differences was computed, using appropriate contrasts. The percentage changes occurring during therapy and their statistical significance are indicated on p. 368 in Table 11. Furthermore, differences between control (pretreatment) values of all three groups were tested by an analysis of variance for each steroid or pituitary hormone assayed. If no significant difference was found, another analysis of variance was performed to compare the means of treatment results obtained by various treatment schedules (1-6 months of treatment) for each hormone. The significance of differences between group means was assessed by the q-test (Dixon and Massey, 1969). The results of these computations are shown on p. 369 in Table 111. In the case of the assays of 17-hydroxyprogesterone, estrone, estradiol and prolactin, a significant difference was found in the pretreatment (control) values of the 3 groups. In these instances, every individual measure-
1600 pgfmi 4
EMP
MONTHS
FIG.2. Circulating levels of pregnenolone in patients with prostatic carcinoma before and during treatment with polyestradiol phosphate (PEP given in monthly injections of 160 mg), pokyeskadiol phosphate ethynylegtradiol (PEP EE : MO mg im monthly and 150 pg per 0s daily, respectively) and estramustine phosphate (EMP: 600 mg per 0 s daily). Each column represents the geometric mean value of 8 subjects. Vertical bars indicate 95% confidence limits.
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ESTROGENIC TREATMENT OF PROSTATIC CARCINOMA
359
ment was transformed to a percentage, and differences between treatment groups were computed using an analysis of variance and the q-test, as indicated above.
B. RESULTS The results of the assays of three A5-steroids, pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone, are indicated in Figs. 2-4. Whereas the decrease in pregnenolone levels during PEP therapy was barely significant (cf. Table 11), a highly significant decrease was noted during the administration of EMP or of PEP EE. The levels of 17hydroxypregnenolone were reduced by each form of therapy ; however the effect of PEP was weaker than that of PEP EE,or EMP (cf. Table 111).Furthermore, whereas PEP did not induce a significant change in circulating dehydroepiandrosterone levels, a highly significant depression was noted in the patients treated with either EMP or PEP EE.Hence the two latter forms of estrogen therapy appeared to be more powerful inhibitors of these A5-steroids than was PEP.
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+
I*hi166p1m 6 :;Bs 6 * 6B
2000
1600
I
PEP
pg/ml
1200
66
400
'60 1 I
1200
-
CONTROLS
M
QS
1
I
3
L
6
FIG.3. 17-Hydroxypregnenolone levels in patients with prostatic carcinoma before and during treatment with various estrogen derivatives. For explanation, consult legend to Fig. 2.
360
1200
G. JONSSON
ET AL.
~
400 O-
CONTROLS 025
05
1
2
3
3600 1pg/ml
EMP
MONTHS
FIG.4. Dehydroepiandrosterone levels in patients with prostatic carcinoma before and during treatment with various estrogen derivatives. For explanation, consult legend to Fig. 2. Closer examination of Figs. 2 4 reveals a more or less conspicuous trend toward an increase in circulating steroid levels between the third and sixth treatment months in patients treated with PEP EE. Indeed, a regression analysis revealed that this increase was significant in both 17hydroxypregnenolone ( p < 0.001) and dehydroepiandrosterone levels ( p < 0.01). A similar trend has not been observed in patients treated with PEP alone or with EMP. The plasma levels of 20a-dihydroprogesterone (Fig. 5 ) were influenced only by PEP+EE and to a limited extent. However, the assay of 17hydroxyprogesterone revealed a highly significant suppression of this steroid during each therapeutic regimen studied (Fig. 6). Furthermore, the decrease in 17-hydroxyprogesterone levels was significantly greater during the administration of EMP or PEP EE than after the administration of PEP alone (cf. Table 111). As far as the levels of androstenedione were concerned, there was a limited (23-33%) but highly significant decrease in every group of patients (Fig. 7). In this respect, there was no difference between the various forms of estrogen treatment.
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361
ESTROGENIC TREATMENT OF PROSTATIC CARCINOMA
400
1
PEP
PEP
1
+EE
400 pg/ml
pg/ml
3w-
300-
200-
200100
-
0-
I T
4 0 0 pg/ml
300 200
~
1000-
il!
CONTROLS
EMP
7
3
b 6 MONTHS
FIG.5. 20 a-Dihydroprogesterone levels in patients with prostatic carcinoma before and during estrogen treatment. For explanation, consult legend to Fig. 2.
I n agreement with the findings shown in Figs. 3 and 4, a highly significant increase ( p < 0.001) was found between the third and sixth months of treatment in 17-hydroxyprogesterone levels and a significant increase ( p < 0.05) in androstenedione levels in patients treated with PEP EE (Figs. 6 and 7 ) . Again, no similar effect was observed during treatment with PEP or with EMP. The testosterone and dihydrotestosterone levels are shown in Figs. 8 and 9, respectively. All three estrogen regimens caused a highly significa.nt suppression of both testosterone and dihydrotestosterone levels. However, treatment with E M P or with PEP EE resulted in a much stronger suppression ( p < 0.001) than did treatment with PEP alone (Table 111).The testosterone levels in the latter group were approximately 2.0 ng/ml, whereas in the two other groups the values were in the vicinity of 150 pg/ml. Furthermore, dihydrotestosterone levels were diminished to values of the order of 300 pg/ml in the PEP-treated group, whereas they were approximately 100 pg/ml in the patients treated with PEP EE or with EMP.
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2200 1pg/mi
PEP
J
MONTMS
900 700
MONTHS
~
~
500 -
300. 100
CONTROLSW5
1600- pg/rnl
0.5
1
2
3
4
6
1600' pg/ml
1200
MONTHS
FIG.7. Androstenedione levels in patients with prostatic carcinoma before and during estrogen treatment. For explanation, consult legend to Fig. 2.
PEP
UONTUS
MONTHS
EMP
& & - m m - m 05 1 2 3 6
CONTROLS 0.2s
6
UONTHS
FIG.8. Testosterone levels in patients with prostatic carcinoma before and during estrogen treatment. For explanation consult legend to Fig. 2. PEP
PEP + E E
800
800
UONTHS
UONTHS
EMP
I -
1000 pg/ml
800-
600-
400200.
FIQ.9. Dihydrotestosterone levels in patients with prostatic carcinoma before and during estrogen treatment. For explanation, consult legend to Fig. 2.
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G. JOASSON ET AL.
PEP
PEP
700 a ng/ml
500
+
EE
700- ng/ml
-
500 -
300 -
CONTROLS 025
0.5
I
2
3
O-
I 6 MONTHS
CONTROLS 0.25
05
1
2
3
L 6 MONTHS
EMP
7001 ng/ml 500
300 100: O-
CONTROLS 025
0.5
1
2
3
09 0
6
MONTHS
FIG.10. Cortisol levels in patients with prostatic :arcinoma before and during estrogen treatment. n = 7. For explanation, consult legend to Fig. 2.
The assay of cortisol revealed major differences, as shown in Fig. 10. Whereas treatment with PEP did not alter circulating cortisol levels, the administration of PEP EE resulted in a marked elevation ( p < 0.001) of cortisol in plasma. The effect of E M P was even more marked; cortisol levels in EMY-treated patients greatly exceeded those found in subjects treated with PEP EE ( p < 0.001). The results of estrone and estradiol assays obtained during the administration of PEP and of PEP EE are indicated in Fig. 11. The higher values seen in the latter group should be viewed in the light of the slight but significant (0.1%) cross-reaction between EE and estradiol or estrone. The data of Fig. 11 indicate a progressive rise in estrone and estradiol values in YEP-treated patients from the first month onward. A regression analysis indicated that this gradual rise was significant ( p < 0.01). The same was true also in patients treated with PEP EE ( p < 0.001), suggesting an accumulation of these estrogens and/or of their precursors. I n the plasma samples of subjects treated with daily doses of 600 mg of EMP, the estrone-like immunoreactive material varied between 50 and 100 ng/ml and the estradiol-like material between 5 and 10 ng/ml. Also in these cases there was a highly significant increase in measured levels
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ESTROGENIC TREATMENT O F PROSTATIC CARCINOMA
(A)
1400 -
1400 -
1000 -
1000 -
MONTHS
700
~
365
MONTHS
700 a pg /ml
p g /ml
500
500 -
(B) 300 100 -
I353
FIG.11. Estronc ( A ) anti cstradiol (U) lcvrls in ptiticnts with Iirostatic c:ircinomn before and during treatment with PEP and PEP EE. For cxplanation, consult legend to Fig. 2.
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from the first to the sixth month of treatment ( p < 0.001). However, when another aliquot of some of these samples was partitioned between toluene and 1 N NaOH prior to chromatography, a considerable amount of this immunological activity rernaincti in the toluene phase. The chemical identity of the imrnunoreactivc cstronc- and cstratliol-like material is undcr investigation. Thc results of FSH and LH assays are depicted in Figs. 12 and 13. All three thcrapeutic regimens resulted in a marked reduction of circulating FSH and L H levels ( p < 0,001) in all instances. Howcver, the gonadotropin-sup?prcssing effect of both PEP EE and of EMP was much stronger ( p < 0.001) than that of PEP. Thc results of prolactin assays arc indicated in Fig. 14. Prolactin assays were performed only on two occasions during the pretreatment period and then l and 5 months following the initiation of estrogen treatment. There was no change in prolactin levels in subjects treated with PEP. However, combined treatment with PEP EE resulted in a significant, incrcasc ( p < 0.05). Moreover, treatment with EMP produced a much Inore marked ( p < 0.001) elevation. Indeed, the increase in prolactin levels in the E M P group was higher ( p < 0.01) than in patients treated with PEP EE.
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+
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PEP
PEP
30
1
30
mlU/ml
20 -
1
+ EE
rnlU/rnl
20 -
MONTHS
MONTHS
EMP
& 3
& A 4 6 MONTHS
FIG.12. Immunoreactire follicle-stimulating hormone (FSH) levels in patients with prostatic carcinoma before and during estrogen therapy. n = 7. For explanation, consult legend to Fig. 2. PEP
30
rntU/ml
30 20
20 10
0
&A&hl!lh66
CONTROLS 0.25
05
2
1
3
4
j m * B 0 2 5 0& 5 1& 2 * 3 &4 *6
6
CONTROLS
MONTHS
30
1
i
PEP * E E
rnlU/rnl
MONTHS
EMP
mlU/ml
FIG. 13. Immunoreactive luteinizing hormone (LH) levels in patients with prostatic carcinoma before and during estrogen treatment. n = 7. For explanation, consult legend to Fig. 2.
ESTROGENIC TREATMENT OF PROSTATIC CARCINOMA
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31
PEP + E E
PEP
1
15 ng/ml
O-
CONTROLS
MONTHS
EM P
1
15 ng/ml
T
YOUTHS
T
MONTHS
FIQ. 14. Immunoreactive prolactin levels in patients with prostatic carcinoma before and during estrogen therapy. For explanation, consult legend to Fig. 2.
Summarg of Hormonal Changes Table I1 presents the mean percentage change in each of the hormonal parameters studied, which was induced by the various therapeutic regimens administered. In the statistical analysis of these changes, the mean pretreatment values were compared with the mean of the values found 1, 2, 3,4,and 6 months after the initiation of the therapy, using appropriate contrasts in the analysis of variance. In the case of prolactin assays, the mean pretreatment value was contrasted with the mean of the first and fifth treatment months. The data of Table I1 indicate that the different degrees of gonadotropin suppression were closely correlated with similar decreases in testosterone, dihydrotestosterone, and 17-hydroxyprogesterone levels, but were poorly or not at all correlated with androstenedione and 20~dihydroprogesterone levels. Also the changes observed in prolactin and cortisol levels reflected major differences in estrogenic effect, whereas the three A5-steroids studied represented less-sensitive parameters of testicular endocrine function than testosterone, dihydrotestosterone, and 17-hydroxyprogesterone.
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G. JONSSON
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TABLE I1
EFFECTOF VARIOUSFORMSOF ESTROQEN THERAPY ON CIRCULATING HORMONE LEVELS IN PATIENTSWITH PROSTATIC CANCER
Estrogen treatmentn Hormones in plasma
PEP
Pregnenolone 17-Hydroxypregnenolone Dehydroepiandrosterone 20~Dihydroprogesterone 17-Hydroxypmgesterone Androstenedione Testosterone Dihydrotestosterone Cortisol Estrone Estradiol FSH
18.7b -23.8d + I .4a 0.0-50.3d -23.4d -55.0d -37.2d +1.6O +341d 443d -55.7d -30.7d +3.0a
+
LH
Prolactin ~
PEP
+ EE
-31. Id -41.4d -!23.7d -16.8' -73.5d -32.gd -96.7d -73.04 154d +966d*e +931dse -91.9d -87.64 +13.9*
+
EMP -37.2d -40. 4d -40. Id -4.9a -73.7d -23. -96.gd -73.7d +227d +2.1 x 106' +2.1 x 104j -98.6d -95.4d +46. gd
~~~~
Not significant. p < 0.05. ' p < 0.01. d p < 0.001. 0.1% Cross-reaction between EE and estrone or estradiol. Chemical identity not definitely established. 0 Figures indicate percentage changes during therapy. PEP, polyestradiol phosphate; EE, ethynylestradiol; EMP estramustine phosphate; FSH, follicle-stimulation hormone; LH, luteinizing hormone. a
f
I n Table I11 the significance of differences between treatment groups is assessed. The administration of PEP alone resulted in a significantly lower degree of suppression of circulating gonadotropin and steroid levels than treatment with either PEP EE or with EMP. This was especially true in the case of testosterone, dihydrotestosterone, and 17-hydroxyprogesterone, 17-hydroxypregnenolone, and dehydroepiandrosterone. An exception was androstenedione, which showed approximately the same degree of suppression in the different treatment groups. Also the cortisol and prolactin levels indicated a highly significant difference between the endocrine effect of PEP and of the two other regimens. Finally, the effect of EMP significantly exceeded that of PEP EE as far as their effect on cortisol and prolactin levels is concerned.
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ESTROGENIC TREATMENT OF PROSTATIC CARCINOMA
TABLE I11 SIGNIFICANCE OF DIFFERENCES IN HORMONAL CHANGES INDUCED BY DIFFERENT FORMS OF ESTROGEN THERAPY Contrastse Hormones in plasma Pregnenolone 17-Hydroxypregnenolone Dehydroepiandrosterone 20~Dihydroprogesterone 17-Hydroxyprogesterone Androstenedione Testosterone Dihydrotestosterone Cortisol Estrone Estradiol FSH LH Prolactin
PEP vs PEP E E
+
NS
p p
< 0.001 < 0.001 NS
PEP vs EMP
p
< 0.001 NS
p
2. CEKAN, K. PURVIS,
AND
E. DICZFALUSY
Swedish Medical Research Council, Reproductive Endocrinology Research Unit, Karolinska Sjukhuset, Stockholm, Sweden
I. Clinical Results . . . 11. SteroidLevels . . . . A. Methods and Materials B. Results . . . . . . 111. Discussion . . . . . IV. Concluding Remarks . . References . . . . . Discussion . . . . .
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351 356 356 359 369 372 372 373
I. CLINICALRFSULTS Endocrine therapy of prostatic carcinoma has been the rule ever since Huggins and Hodges demonstrated the hormone dependence of the prostate. The methods used have varied from time to time and from one hospital to another. Opinions still differ on the mechanisms of the methods used, their suitability, side effects, etc. The therapeutic programs have included orchiectomy, estrogen administration, pituitary ablation, corticosteroid therapy, administration of antiandrogen, etc. Also nonendocrine methods such as radiotherapy as well as treatment with cytostatics have proved useful. The main purpose of endocrine therapy from the very beginning was to reduce the amount of androgen via inhibition of the secretion of the gonadotropins by the pituitary. The potency of the substances administered was generally estimated from determinations of the steroid substances in the urine, I n recent years, however, the mechanisms bringing about the effect of endocrine therapy have proved to be much more complex than was formerly supposed. This has been made clear by recent investigations with technically advanced methods. Among other things, the introduction of radioimmunoassay methods for determining steroidal and polypeptide hormones in the serum as well as demonstration of the 351
352
G. JONSSON
ET AL.
action of androgens on the prostate have shed light on certain points previously difficult to understand. Oral treatment with estrogenic compounds, such as stilbestrol, has several disadvantages in the form of dyspeptic symptoms, sodium retention, edema, cardiovascular complications, etc. To eliminate the above-mentioned disadvantages we began in 1955 to use a long-acting parenteral drug of low toxicity, Estradurinm (polyestradiol phosphate). The estrogenic effect of an intramuscular injection persists for a couple of months. In an initial series consisting of 105 patients, 40% had metastases before the beginning of treatment (Jonsson et d., 1963). Many of the patients had had symptoms for a fairly long time before they sought medical advice. In 97 patients it was possible to estimate the interval between the onset of symptoms and the beginning of treatment fairly accurately: the mean interval was 12 months. In all cases the diagnosis had been histologically confirmed by perineal puncture or transurethral resection. I n 47 of the 105 cases the urine was examined in respect to steroids, which included determinations of the total 17-ketosteroids, 17-hydroxycorticosteroids, individual 17-ketosteroid metabolites, such as androsterone (A) etiocholanolone (E) dehydroepiandrosterone, and estrone, l7/I-estradiol7 and estriol. The series was divided into two groups according to the dose of Estradurin: 80-120 mg monthly and 150-200 mg monthly in a single injection. The results of the steroid analysis are given in Table I. The data thus suggest that in the group treated with 8&120 mg of Estradurin per month
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TABLE I URINARY EXCRETION O F 17-KETOSXPROIDS (a), ANDROSTERONE ETIOCFIOLANOLONE (b), DERYDROEPIANDROSTERONE (c) BEFORE AND AFTER ESTRADURIN THERAPY
+
Estradurin dosage (mg)
Before treatment (mg/24 hours)
After 4 months’ treatment (mg/24 hours)
80-120 160-200 b 80-120 160-200 c 80-120 160-200
4.93 4.72 2.79 2.69 0.29 0.20
4.86 3.92 2.86 2.16 0.28 0.20
a
ESTROGENIC TREATMENT OF PROSTATIC CARCINOMA
353
the mean excretion value of 17-ketosteroids did not differ significantly from the pretreatment value. On the other hand, in patients treated with 160-200 mg of Estradurin treatment resulted in a probably significant decrease in the excretion of 17-ketosteroids. Like the urinary total ketosteroids, the excretion of A E was not decreased after administration of 80-120 mg of Estradurin per month. But treatment with the larger dose of Estradurin produced a probably significant decrease in the elimination of A E. Subsequent orchiectomy, however, resulted in a highly significant decrease in the excretion of 17-ketosteroids1 androsterone, and etiocholanolone. Neither Estradurin therapy nor orchiectomy influenced the excretion values of dehydroepiandrosterone significantly. Although Estradurin is a fairly weak gonadotropin inhibitor, the clinical results were good: 40% of the patients were alive 5 years after the beginning of treatment. The good clinical results together with the determinations of the steroids suggested that Estradurin has a therapeutic effect directly on the malignant cells. The favorable clinical results obtained in a small group of patients with an initially low excretion of androgen metabolites lend additional support to this view. I n view of the aforementioned results of the determinations of steroids and the clinical response, Estradurin was combined with a stronger gonadotropin inhibitor, e.g. ethynylestradiol, in the next series (Jonsson, 1971). The material in this series consisted of 126 patients. The age distribution and the frequency of metastases were the same as in the first series. The patients in this series came from the same receiving area, the disease was equally advanced (75% thus belonged t o stage 111 IV, VACURG classification) , and had received the same supplementary therapy besides estrogen therapy. All the patients were reexamined after 3 months and afterward twice a year for a t least 5 years. The estrogen therapy in this series consisted of Estradurin given intramuscularly in a dose of 160 mg per month for 3 months, after which the dose was reduced to 80 mg per month. This treatment was combined with 1 mg of ethynylestradiol a day for 14 days. This was followed by a daily maintenance dose of 150 pg of ethynylestradiol. The effect of treatment was largely the same as that of estrogen therapy in general : substantial improvement of the patients’ general condition, and abatement or disappearance of urinary symptoms and of metastatic pain. I n several cases the number and spread of metastases became stationary, and in some cases roentgen examination showed regression.
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354
G. JONSSON
ET AL.
Of the 126 patients, 78 (62%) survived for more than 5 years. As expected, the 5-year survival rate of patients in stages I and I1 was high (74%) ; in stage IV it was 45%. The lower the differentiation, the higher the mortality from the basic disease. The present investigation produced no evidence in support of the assumption of an overmortality from cardiovascular diseases in patients treated with these estrogens, but patients with such diseases before treatment must be regarded as less good risks unless they receive adequate treatment of their cardiovascular complaints. Irrespective of the type of estrogen treatment given, most patients with prostatic carcinoma sooner or later deteriorate. These patients, like those primarily resistant to conventional estrogen treatment, constitute a serious therapeutic problem. In these two groups, increasing and sometimes very large doses of estrogen) radiation, corticosteroids, hypophysectomy, antiandrogens, cytostatics, etc., have been tried. In some cases palliation has been achieved, but, on the whole, the results are less encouraging, besides which several of the methods have serious side effects. It is not known why certain cases do not respond to antiandrogenic treatment or why they first respond but later become unsusceptible. In such cases, changes probably occur in the biological behavior of the carcinoma. During the last 7 years we have used estramustine phosphate (EstracyP) a nitrogen mustard derivative of estradiol, in the two abovementioned groups of prostatic carcinoma (Jonsson and Hogberg, 1971 ; Nilsson and Jonsson, 1975). Our own clinical material consisted of 128 patients with advanced prostatic carcinoma. One hundred and seventeen belonged to stage IV (metastases) and 11 to stage 111. All patients are followed for more than one year. Ninety patients had previously received some other antiandrogenic therapy (secondary treatment group) but had deteriorated during treatment. Judging from clinical experience, the expected duration of survival of these patients would be less than 6 months. Thirty-eight patients were given Estracyt from the very beginning (primary treatment group). All the patients were admitted to hospital for the treatment. Investigation on admission included histological and cytological examination of the prostate and sometimes of lymph nodes, roentgenological investigation of the skeleton, lungs, and urinary tract, analysis of the blood picture, determination of the acid and alkaline phosphatases, renal and liver function tests, etc. The examinations were repeated at certain intervals. Careful notes were made of the patient’s general condition, pain, use of analgetics, difficulty in urination, etc. )
ESTROGENIC TREATMENT OF PROSTATIC CARCINOMA
355
The histological and cytological investigation showed that 37 were poorly differentiated, 51 moderately differentiated, and 21 well diff erentiated; the degree of differentiation of 19 was uncertain. One hundred and seventeen patients had metastases, often in more than one site. The acid phosphatases were increased in 68%. Estracyt was given intravenously in a dose of 300450 mg/day for about 3 weeks. If the patient responded well, intravenous treatment with 300-450 mg twice a week was continued for a varying period depending on the patient’s response. If the patient was symptom free and the disease under control after 2-3 months’ treatment, the injections were stopped. In the event of exacerbations, intense treatment with daily injections was resumed. If no effect could be recorded after 3 weeks’ daily treatment, the preparation was withdrawn. We have also used Estracyt by mouth since 1971. The patients have received a dose of 600-900 mg a day either as primary treatment or as secondary maintenance therapy after intravenous treatment. It is sometimes not easy to assess the results of treatment, because it is difficult to evaluate the patients’ symptoms, especially pain. Estimation of the effect of treatment could, however, often be based on objective observations. For instance, previously bedridden patients could sometimes get up and even return to work. A most striking effect was the abatement of the pain and the improvement of the patient’s general condition. Regression or disappearance of metastases was noted in 33 of the 128 cases. During treatment the acid phosphatases fell in many cases, sometimes to normal level. There seems to be a close correlation between falling acid phosphatases and improvement of the patient’s general condition. Reelevation of the acid phosphatase levels in patients who had responded favorably was usually associated with a new deterioration. Changes were noted in the prostatic tumor in the form of cellular pycnosis, vacuolization, rupture of cell membranes, squamous epithelial metaplasia, and increased lipofuscin. On numerical evaluation of the results we found that about half of the patients who had got secondary therapy improved. Of the patients who received Estracyt as primary therapy, 80-90% improved. The results in these series suggest that in the group given secondary treatment the hitherto expected survival of less than 6 months might be prolonged to anything up to 5 years. The primary treatment group was too small to warrant any conclusions regarding the survival time. The side effects must be regarded as insignificant. There was occasionally thrombophlebitis in patients treated intravenously. Gastrointestinal disturbances, though usually only transient, occurred in some patients treated by mouth. A mild reaction of the liver, allergic reactions, leukopenia, and thrombocytopenia occurred in a few cases.
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G . JONSSON
ET AL.
11. STEROID LEVELS
A. METHODS AND MATERIALS A critical assessment of the endocrine effects of the three types of estrogen treatment discussed above, namely of Estradurin@ (polyestradiol phosphate, hereafter PEP), Estradurin ethynylestradiol (hereafter PEP EE) , and Estracyt@ (estramustine phosphate, hereafter EMP) necessitates serial measurements of a number of relevant hormonal parameters in the blood of previously untreated patients. Therefore, patients with histologically and/or cytologically diagnosed prostatic carcinoma and without any previous hormone therapy were allocated randomly to one of the above treatments, and the pretreatment values of a number of circulating steroids and pituitary hormones were then compared with those found monthly during treatment with the three estrogen regimens described.
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1. Clinical Material
This consisted of 24 patients with prostatic carcinoma stages I1 to IV (Veterans classification) diagnosed histologically and/or cytologically. The average age of the patients was 72.5 years (range 62-84 years). 2. Therapeutic Regimens and the Design of the Study
The patients were randomly allocated to one of the three regimens, consisting of (a) PEP (160 mg im monthly), ( b ) PEP EE (PEP 160 mg im monthly; and EE 150 pg per 0s daily), and ( c ) EMP (600 mg per 0s daily). The structural formulas of PEP and EMP are indicated in Fig. 1. Pretreatment (control) plasma samples were drawn from each patient
+
0 1
o r
0
0
O - p tP o H 'OH
E
=&
POLY ESTRADIOL PHOSPHATE (PEP) ESTRADURIN@
CICH,
- CH,
'
N'
CLCH, - c x,
-coo
ESTRAMUSTINE PHOSPHATE ( EMP) ESTRACYT @
FrG. 1. Structural formulas of polyestradiol phosphate (PEP) and estramustine phosphate (EMP).
ESTROGENIC TREATMENT OF PROSTATIC CARCINOMA
357
on each of three consecutive days. Then treatment was initiated immediately. Plasma samples were then obtained from each subject after 1 and 2 weeks, as well as after 1, 2, 3, 4, 5, and 6 months of treatment. Blood samples of 20 ml were withdrawn on each occasion from the antecubital vein into heparinized test tubes. The collection of plasma samples occurred invariably between 9:00 and 1O:OO AM. The blood was centrifuged immediately; the plasma was separated, frozen, and stored a t -2OOC until analyzed. 3. Hormonal Parameters Studied
The following unconjugated steroids were measured: pregnenolone (3p-hydroxy-5-pregnen-20-one) , 17-hydroxypregnenolone (3B,17-dihydroxy-5-pregnen-20-one),dehydroepiandrosterone (3p-hydroxyd-androsten-17-0ne)~ 20a-dihydroprogesterone (20a-hydroxy-4-pregnen-3-one), 17-hydroxyprogesterone ( 17-hydroxy-4-pregnene-3,20-dione), androstenedione (4-androstene-3,17-dione), testosterone (17p-hydroxy-4 androsten-3-one), dihydrotestosterone (17p-hydroxy-5a-androstan-3-one), cortisol (1lp,l7-dihydroxy-4-pregnene-3,20-dione),estradiol (1,3,5 (10) estratriene-3,17p-diol), and estrone (3-hydroxy-1,3,5 (10)-estratrien-17one). The following immunoreactive pituitary hormones were estimated : follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin. 4. Assay Procedures All steroids except cortisol were measured by radioimmunoassay procedures involving chromatographic purification and separation of steroids as described in detail in previous communications (Brenner et al., 1973; Purvis et al., 1975; Aedo et al., 1976). Cortisol was assayed by the protein-binding method described by Nugent and Mayes (1966). The assays of LH were performed according to Midgley (1966) as modified by Robyn et al. (1971), using the Second International Reference Preparation of human menopausal gonadotropin (2nd IRP-HMG) as standard. The measurements of FSH and prolactin were performed by the use of kits purchased from Biodata S.A., Rome, Italy. I n the FSH assays an H M G standard calibrated against the 2nd IRP-HMG was used, whereas the results of prolactin assays were expressed in terms of the NIH-1 human prolactin preparation.
5. Cateulation and Evaluution of Results I n all calculations, a log normal between-patients distribution of individual values was assumed (Gaddum, 1945). Hence in Figs. 2-14, geometric mean values and 95% confidence limits are indicated. In order to assess differences between control and treatment values for
358
G . JONSSON
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each treatment group, an analysis of variance was performed and the significance of the differences was computed, using appropriate contrasts. The percentage changes occurring during therapy and their statistical significance are indicated on p. 368 in Table 11. Furthermore, differences between control (pretreatment) values of all three groups were tested by an analysis of variance for each steroid or pituitary hormone assayed. If no significant difference was found, another analysis of variance was performed to compare the means of treatment results obtained by various treatment schedules (1-6 months of treatment) for each hormone. The significance of differences between group means was assessed by the q-test (Dixon and Massey, 1969). The results of these computations are shown on p. 369 in Table 111. In the case of the assays of 17-hydroxyprogesterone, estrone, estradiol and prolactin, a significant difference was found in the pretreatment (control) values of the 3 groups. In these instances, every individual measure-
1600 pgfmi 4
EMP
MONTHS
FIG.2. Circulating levels of pregnenolone in patients with prostatic carcinoma before and during treatment with polyestradiol phosphate (PEP given in monthly injections of 160 mg), pokyeskadiol phosphate ethynylegtradiol (PEP EE : MO mg im monthly and 150 pg per 0s daily, respectively) and estramustine phosphate (EMP: 600 mg per 0 s daily). Each column represents the geometric mean value of 8 subjects. Vertical bars indicate 95% confidence limits.
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ESTROGENIC TREATMENT OF PROSTATIC CARCINOMA
359
ment was transformed to a percentage, and differences between treatment groups were computed using an analysis of variance and the q-test, as indicated above.
B. RESULTS The results of the assays of three A5-steroids, pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone, are indicated in Figs. 2-4. Whereas the decrease in pregnenolone levels during PEP therapy was barely significant (cf. Table 11), a highly significant decrease was noted during the administration of EMP or of PEP EE. The levels of 17hydroxypregnenolone were reduced by each form of therapy ; however the effect of PEP was weaker than that of PEP EE,or EMP (cf. Table 111).Furthermore, whereas PEP did not induce a significant change in circulating dehydroepiandrosterone levels, a highly significant depression was noted in the patients treated with either EMP or PEP EE.Hence the two latter forms of estrogen therapy appeared to be more powerful inhibitors of these A5-steroids than was PEP.
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+
I*hi166p1m 6 :;Bs 6 * 6B
2000
1600
I
PEP
pg/ml
1200
66
400
'60 1 I
1200
-
CONTROLS
M
QS
1
I
3
L
6
FIG.3. 17-Hydroxypregnenolone levels in patients with prostatic carcinoma before and during treatment with various estrogen derivatives. For explanation, consult legend to Fig. 2.
360
1200
G. JONSSON
ET AL.
~
400 O-
CONTROLS 025
05
1
2
3
3600 1pg/ml
EMP
MONTHS
FIG.4. Dehydroepiandrosterone levels in patients with prostatic carcinoma before and during treatment with various estrogen derivatives. For explanation, consult legend to Fig. 2. Closer examination of Figs. 2 4 reveals a more or less conspicuous trend toward an increase in circulating steroid levels between the third and sixth treatment months in patients treated with PEP EE. Indeed, a regression analysis revealed that this increase was significant in both 17hydroxypregnenolone ( p < 0.001) and dehydroepiandrosterone levels ( p < 0.01). A similar trend has not been observed in patients treated with PEP alone or with EMP. The plasma levels of 20a-dihydroprogesterone (Fig. 5 ) were influenced only by PEP+EE and to a limited extent. However, the assay of 17hydroxyprogesterone revealed a highly significant suppression of this steroid during each therapeutic regimen studied (Fig. 6). Furthermore, the decrease in 17-hydroxyprogesterone levels was significantly greater during the administration of EMP or PEP EE than after the administration of PEP alone (cf. Table 111). As far as the levels of androstenedione were concerned, there was a limited (23-33%) but highly significant decrease in every group of patients (Fig. 7). In this respect, there was no difference between the various forms of estrogen treatment.
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361
ESTROGENIC TREATMENT OF PROSTATIC CARCINOMA
400
1
PEP
PEP
1
+EE
400 pg/ml
pg/ml
3w-
300-
200-
200100
-
0-
I T
4 0 0 pg/ml
300 200
~
1000-
il!
CONTROLS
EMP
7
3
b 6 MONTHS
FIG.5. 20 a-Dihydroprogesterone levels in patients with prostatic carcinoma before and during estrogen treatment. For explanation, consult legend to Fig. 2.
I n agreement with the findings shown in Figs. 3 and 4, a highly significant increase ( p < 0.001) was found between the third and sixth months of treatment in 17-hydroxyprogesterone levels and a significant increase ( p < 0.05) in androstenedione levels in patients treated with PEP EE (Figs. 6 and 7 ) . Again, no similar effect was observed during treatment with PEP or with EMP. The testosterone and dihydrotestosterone levels are shown in Figs. 8 and 9, respectively. All three estrogen regimens caused a highly significa.nt suppression of both testosterone and dihydrotestosterone levels. However, treatment with E M P or with PEP EE resulted in a much stronger suppression ( p < 0.001) than did treatment with PEP alone (Table 111).The testosterone levels in the latter group were approximately 2.0 ng/ml, whereas in the two other groups the values were in the vicinity of 150 pg/ml. Furthermore, dihydrotestosterone levels were diminished to values of the order of 300 pg/ml in the PEP-treated group, whereas they were approximately 100 pg/ml in the patients treated with PEP EE or with EMP.
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2200 1pg/mi
PEP
J
MONTMS
900 700
MONTHS
~
~
500 -
300. 100
CONTROLSW5
1600- pg/rnl
0.5
1
2
3
4
6
1600' pg/ml
1200
MONTHS
FIG.7. Androstenedione levels in patients with prostatic carcinoma before and during estrogen treatment. For explanation, consult legend to Fig. 2.
PEP
UONTUS
MONTHS
EMP
& & - m m - m 05 1 2 3 6
CONTROLS 0.2s
6
UONTHS
FIG.8. Testosterone levels in patients with prostatic carcinoma before and during estrogen treatment. For explanation consult legend to Fig. 2. PEP
PEP + E E
800
800
UONTHS
UONTHS
EMP
I -
1000 pg/ml
800-
600-
400200.
FIQ.9. Dihydrotestosterone levels in patients with prostatic carcinoma before and during estrogen treatment. For explanation, consult legend to Fig. 2.
364
G. JOASSON ET AL.
PEP
PEP
700 a ng/ml
500
+
EE
700- ng/ml
-
500 -
300 -
CONTROLS 025
0.5
I
2
3
O-
I 6 MONTHS
CONTROLS 0.25
05
1
2
3
L 6 MONTHS
EMP
7001 ng/ml 500
300 100: O-
CONTROLS 025
0.5
1
2
3
09 0
6
MONTHS
FIG.10. Cortisol levels in patients with prostatic :arcinoma before and during estrogen treatment. n = 7. For explanation, consult legend to Fig. 2.
The assay of cortisol revealed major differences, as shown in Fig. 10. Whereas treatment with PEP did not alter circulating cortisol levels, the administration of PEP EE resulted in a marked elevation ( p < 0.001) of cortisol in plasma. The effect of E M P was even more marked; cortisol levels in EMY-treated patients greatly exceeded those found in subjects treated with PEP EE ( p < 0.001). The results of estrone and estradiol assays obtained during the administration of PEP and of PEP EE are indicated in Fig. 11. The higher values seen in the latter group should be viewed in the light of the slight but significant (0.1%) cross-reaction between EE and estradiol or estrone. The data of Fig. 11 indicate a progressive rise in estrone and estradiol values in YEP-treated patients from the first month onward. A regression analysis indicated that this gradual rise was significant ( p < 0.01). The same was true also in patients treated with PEP EE ( p < 0.001), suggesting an accumulation of these estrogens and/or of their precursors. I n the plasma samples of subjects treated with daily doses of 600 mg of EMP, the estrone-like immunoreactive material varied between 50 and 100 ng/ml and the estradiol-like material between 5 and 10 ng/ml. Also in these cases there was a highly significant increase in measured levels
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ESTROGENIC TREATMENT O F PROSTATIC CARCINOMA
(A)
1400 -
1400 -
1000 -
1000 -
MONTHS
700
~
365
MONTHS
700 a pg /ml
p g /ml
500
500 -
(B) 300 100 -
I353
FIG.11. Estronc ( A ) anti cstradiol (U) lcvrls in ptiticnts with Iirostatic c:ircinomn before and during treatment with PEP and PEP EE. For cxplanation, consult legend to Fig. 2.
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from the first to the sixth month of treatment ( p < 0.001). However, when another aliquot of some of these samples was partitioned between toluene and 1 N NaOH prior to chromatography, a considerable amount of this immunological activity rernaincti in the toluene phase. The chemical identity of the imrnunoreactivc cstronc- and cstratliol-like material is undcr investigation. Thc results of FSH and LH assays are depicted in Figs. 12 and 13. All three thcrapeutic regimens resulted in a marked reduction of circulating FSH and L H levels ( p < 0,001) in all instances. Howcver, the gonadotropin-sup?prcssing effect of both PEP EE and of EMP was much stronger ( p < 0.001) than that of PEP. Thc results of prolactin assays arc indicated in Fig. 14. Prolactin assays were performed only on two occasions during the pretreatment period and then l and 5 months following the initiation of estrogen treatment. There was no change in prolactin levels in subjects treated with PEP. However, combined treatment with PEP EE resulted in a significant, incrcasc ( p < 0.05). Moreover, treatment with EMP produced a much Inore marked ( p < 0.001) elevation. Indeed, the increase in prolactin levels in the E M P group was higher ( p < 0.01) than in patients treated with PEP EE.
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+
+
PEP
PEP
30
1
30
mlU/ml
20 -
1
+ EE
rnlU/rnl
20 -
MONTHS
MONTHS
EMP
& 3
& A 4 6 MONTHS
FIG.12. Immunoreactire follicle-stimulating hormone (FSH) levels in patients with prostatic carcinoma before and during estrogen therapy. n = 7. For explanation, consult legend to Fig. 2. PEP
30
rntU/ml
30 20
20 10
0
&A&hl!lh66
CONTROLS 0.25
05
2
1
3
4
j m * B 0 2 5 0& 5 1& 2 * 3 &4 *6
6
CONTROLS
MONTHS
30
1
i
PEP * E E
rnlU/rnl
MONTHS
EMP
mlU/ml
FIG. 13. Immunoreactive luteinizing hormone (LH) levels in patients with prostatic carcinoma before and during estrogen treatment. n = 7. For explanation, consult legend to Fig. 2.
ESTROGENIC TREATMENT OF PROSTATIC CARCINOMA
367
31
PEP + E E
PEP
1
15 ng/ml
O-
CONTROLS
MONTHS
EM P
1
15 ng/ml
T
YOUTHS
T
MONTHS
FIQ. 14. Immunoreactive prolactin levels in patients with prostatic carcinoma before and during estrogen therapy. For explanation, consult legend to Fig. 2.
Summarg of Hormonal Changes Table I1 presents the mean percentage change in each of the hormonal parameters studied, which was induced by the various therapeutic regimens administered. In the statistical analysis of these changes, the mean pretreatment values were compared with the mean of the values found 1, 2, 3,4,and 6 months after the initiation of the therapy, using appropriate contrasts in the analysis of variance. In the case of prolactin assays, the mean pretreatment value was contrasted with the mean of the first and fifth treatment months. The data of Table I1 indicate that the different degrees of gonadotropin suppression were closely correlated with similar decreases in testosterone, dihydrotestosterone, and 17-hydroxyprogesterone levels, but were poorly or not at all correlated with androstenedione and 20~dihydroprogesterone levels. Also the changes observed in prolactin and cortisol levels reflected major differences in estrogenic effect, whereas the three A5-steroids studied represented less-sensitive parameters of testicular endocrine function than testosterone, dihydrotestosterone, and 17-hydroxyprogesterone.
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G. JONSSON
ET AL.
TABLE I1
EFFECTOF VARIOUSFORMSOF ESTROQEN THERAPY ON CIRCULATING HORMONE LEVELS IN PATIENTSWITH PROSTATIC CANCER
Estrogen treatmentn Hormones in plasma
PEP
Pregnenolone 17-Hydroxypregnenolone Dehydroepiandrosterone 20~Dihydroprogesterone 17-Hydroxypmgesterone Androstenedione Testosterone Dihydrotestosterone Cortisol Estrone Estradiol FSH
18.7b -23.8d + I .4a 0.0-50.3d -23.4d -55.0d -37.2d +1.6O +341d 443d -55.7d -30.7d +3.0a
+
LH
Prolactin ~
PEP
+ EE
-31. Id -41.4d -!23.7d -16.8' -73.5d -32.gd -96.7d -73.04 154d +966d*e +931dse -91.9d -87.64 +13.9*
+
EMP -37.2d -40. 4d -40. Id -4.9a -73.7d -23. -96.gd -73.7d +227d +2.1 x 106' +2.1 x 104j -98.6d -95.4d +46. gd
~~~~
Not significant. p < 0.05. ' p < 0.01. d p < 0.001. 0.1% Cross-reaction between EE and estrone or estradiol. Chemical identity not definitely established. 0 Figures indicate percentage changes during therapy. PEP, polyestradiol phosphate; EE, ethynylestradiol; EMP estramustine phosphate; FSH, follicle-stimulation hormone; LH, luteinizing hormone. a
f
I n Table I11 the significance of differences between treatment groups is assessed. The administration of PEP alone resulted in a significantly lower degree of suppression of circulating gonadotropin and steroid levels than treatment with either PEP EE or with EMP. This was especially true in the case of testosterone, dihydrotestosterone, and 17-hydroxyprogesterone, 17-hydroxypregnenolone, and dehydroepiandrosterone. An exception was androstenedione, which showed approximately the same degree of suppression in the different treatment groups. Also the cortisol and prolactin levels indicated a highly significant difference between the endocrine effect of PEP and of the two other regimens. Finally, the effect of EMP significantly exceeded that of PEP EE as far as their effect on cortisol and prolactin levels is concerned.
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ESTROGENIC TREATMENT OF PROSTATIC CARCINOMA
TABLE I11 SIGNIFICANCE OF DIFFERENCES IN HORMONAL CHANGES INDUCED BY DIFFERENT FORMS OF ESTROGEN THERAPY Contrastse Hormones in plasma Pregnenolone 17-Hydroxypregnenolone Dehydroepiandrosterone 20~Dihydroprogesterone 17-Hydroxyprogesterone Androstenedione Testosterone Dihydrotestosterone Cortisol Estrone Estradiol FSH LH Prolactin
PEP vs PEP E E
+
NS
p p
< 0.001 < 0.001 NS
PEP vs EMP
p
< 0.001 NS
p
