Plasma Sex Hormone Binding Globulin in Patients With Prostatic Carcinoma Marco Grasso, MD, Arturo Buonaguidi, MD, Roberto Mondina, MD," Giovanni Borsellino, MD, Caterina Lania, MD, Giuseppe Banfi, MD,? and Patrizio Rigatti, MD

The concentrations of sex hormone binding globulin (SHBG) were measured in the plasma of 56 men, who were 47 to 85 years of age, by time-resolved immunofluorometric assay with a monoclonal antibody. Twenty-five of the men had untreated carcinoma of the prostate and 17 had untreated prostatic hyperplasia. There were 14 healthy control subjects. SHBG levels were significantly higher in patients with prostatic carcinoma (37.6 k 8.4 nmol/l) than in those with prostatic hyperplasia (24.5 -+ 5.2 nmol/l; P < 0.05) or control subjects (14.9 t 2.8 nmol/l; P < 0.01). It is not known why SHBG levels are higher in patients with carcinoma or hyperplasia of the prostate. The contradictory results obtained in other studies may be due to heterogeneity of the binding globulin causing its values to vary in the different assays used. Cancer 66:354-357,1990.

HE GROWTH AND FUNCTION Of the prostate gland, T like the growth and function of other organs of the male and female reproductive tracts, are under endocrine control, especially by the sex steroids. Because the most important of these for the prostate is the androgens, with dihydrotestosterone (DHT) being the most many attempts have been made to see whether or not the oc- currence and the development of prostatic carcinoma re- sult from abnormal androgenic stimulation. Currently, the results of these studies are controversial because men with prostatic cancer had plasma testosterone levels rel- atively lower than those of men of the same age without the d i~ease .~

In addition to assays of total or free hormone levels, the binding capacity of plasma proteins for steroids has been measured for these patients and for those with pathologic features of other hormone-dependent organs. Currently, however, none of these has provided results that are discriminant for carcinoma. Patients with prostate carcinoma who had not been treated had sex hormone binding globulin (SHBG) levels within the limits of normal

From the Department of Urology and tBiochemistry Laboratory, Is- tituto S. Raffaele, Milano; and the *Consiglio Nazionale delle Ricerche, Milano, Italy.

The authors thank Mr. Fabio Bravi (Centro Ricerche Italfarmaco, Cinisello, Milano) for statistical analysis of the data in this study.

Address for reprints: Marco Grasso, Divisione di Urologia, Via 01- gettina 60, 20 I32 Milano, Italy.

Accepted for publication January 3, 1990.

when assayed by the Rosner method5 and did not have higher levels than patients with prostatic hyperplasia.6 There also are no differences in SHBG levels in relation to the stage of carcinoma or whether or not metastases exist.

Recently, it was suggested that since the plasma binding globulin for steroids is not homogeneous, some method of assay might be able to show differences that others had not been able to show. The use of a suitable assay method has shown that for cancer of another hormone-dependent organ (the breast) there is an increase in steroid binding capacity that is great enough to differentiate the patients from the control subjects, and this assay has been proposed as an indicator for turn or^.^

Starting from this, we measured plasma SHBG con- centrations in patients with carcinoma and hyperplasia of the prostate and in healthy control subjects by a new immunofluorometric assay that uses a monoclonal anti- body.

Materials and Methods

Blood samples were drawn from the cubital vein be- tween 8:OO and 1O:OO AM (patients not necessarily fasting). The tripotassium salt of ethylenediamine tetraacetic acid (K3 EDTA) (25 mg/ml) was added and the samples were centrifuged at 3500 rpm for 25 minutes at 4°C. One-mil- Miter aliquots of plasma were stored at -25°C in plastic tubes and assayed within 4 months by time-resolved im-

354

No. 2 PLASMA SHBG IN PROSTATIC CA * Grasso d at. 355

munofluorometric assay (Delfia kit, Wallace, Pharmacia, Uppsala, Sweden). Assays were performed on single sam- ples and SHBG concentrations were measured in dupli- cate.

The anti-SHBG antibody is added to wells to bind the protein. The monoclonal anti-SHBG antibody labeled with lanthanide (europium) fluoresces when an enhance- ment solution is added.8 Because this label has a very long fluorescence decay time, a time-resolved fluorometer can differentiate between the fluorescence of the tracer and that of the background. The kit we used can assay con- centrations of SHBG from 6 to 200 nmol/l. We used an ARCUS 1230 fluorometer constructed for time-resolved fluorometry (LKB Wallace, Pharmacia). We also used both automatic and manual aspiration systems and an automatic shaker (LKB Wallace, Pharmacia).

Data were analyzed for statistical significance by one- way analysis of variance (ANOVA) followed by the New- man Keuls

Subjects

Fifty-six men 47 to 85 years of age without endocri- nologic and apparent liver disease were studied. None of them had been treated with drugs or hormonal therapy for any reason for at least 6 months. Twenty-five of these men had untreated prostatic carcinoma confirmed by his- tologic examination and 17 had untreated prostatic hy- perplasia. There were 14 normal control subjects of com- parable age and body mass.

The mean age of the 25 patients with prostatic carci- noma was 67.7 years (age range, 47 to 85 years). Their cancers were staged by transrectal prostatic echography, urography, lower abdominal computed axial tomography (CAT) scans, and bone scintigraphy; all stages, from A1 to D2, were represented. After blood collection for SHBG assay, 17 of these patients had had radical prostatectomy (Walsh procedure) and 6 had histologically documented lymph node metastases and were therefore treated by total androgenic block (GnRH analogues and flutamide). The other two patients were already in clinical stage D2 (bone scintigraphy positive) when we first saw them so they also were treated with GnRH analogues and flutamide.

The mean age of the I7 patients with untreated prostatic hyperplasia was 70 years (age range, 57 to 85 years). The hyperplasia was confirmed by histologic examination.

The 14 control subjects were all healthy volunteers with a mean age of 68 years (age range, 47 to 83 years), no subjective symptoms, and negative clinical examinations and transrectal prostatic echography.

Results

The SHBG levels for the patients with prostatic carci- noma were 37.6 k 8.4 nmol/l (range, 10.2 to 88.4 nmol/

1). In the patients with prostatic hyperplasia they were 24.5 k 5.2 nmol/l (range, 5.7 to 41.3 nmol/l) and in the normal subjects they were 14.9 k 2.8 nmol/l (range, 7.6 to 2 1.5 nmol/l).

The variance for the three groups was significantly ho- mogeneous ( P < 0.0 1) by the Fisher-Snedecor f value for probability (relative to the Levene test). The original data were converted to logarithms to the base 10 and the AN- OVA was repeated. This time the Levene f value was 1.18, which is not significant with 2 and 53 degrees of freedom. The f value for the ANOVA was 13.75, with 2 and 53 degrees of freedom. Therefore, the three groups differed significantly statistically (P < 0.0 I ). Multiple comparisons of the values of the differences between the means of the three groups according to the two-tailed Newman Keuls test yielded the following results: (1) the SHBG levels in normal patients differed from those in patients with hy- perplastic prostates ( P < 0.01) and prostatic carcinoma ( P < 0.01) and (2) the levels were different ( P < 0.05) between patients with benign prostatic hyperplasia and prostatic carcinoma.

We found no apparent correlations between the values and the clinical stages of prostatic cancer. Two of the three cases with values of less than 20 nmol/l were in Stage A 1 (tumor grades G1 Mostofi, G2-1 Gleason and G2 Mostofi, G1-2 Gleason) and one was in Stage CI (tumor grade G2 Mostofi, G3-4 Gleason). In addition, there were patients in these two stages who had values of 45 and 46 nmol/l.

In our cases, three normal patients (21%) had values that overlapped with three carcinoma patients ( I 2%). Six patients with prostatic hyperplasia (35%) had values that overlapped with the control subjects and three (1 7%) had values that overlapped with the carcinoma patients.

When the levels were analyzed for two age groups (younger than 69 years of age and older than 70 years of age), there was a modestly, not significantly, higher level in the older patients. For those with tumors the mean levels were 34.47 and 43.17 nmol/l (range, 47 to 85 nmol/ 1) and for those with hyperplasia the values were 23.14 and 26.02 nmol/l (range, 57 to 85 nmol/l). Similar dif- ferences were seen in the healthy control subjects.

Discussion

Theoretically, the same androgenic stimuli that control the development and function of the prostate also could influence the occurrence and growth of cancer in the gland. Clinical data have been reported showing that en- docrine treatment can be helpful in these cases." How- ever, attempts to identify levels or profiles of hormones that are characteristic of patients with carcinoma of the prostate or those at risk for it have not been successful. Hyperandrogenism has never been shown. In fact, patients with prostatic hyperplasia have high blood levels of DHT

356 CANCER July 15 1990 Vol. 66

and testosterone, but those with prostatic carcinoma have levels of these androgens that are equal to or lower than those of normal patient^.'^-'' These contraindications have been explained in several ways. First, it is difficult to find a really pure patient population because almost all men older than 60 years of age have benign prostatic hyperplasia and it is difficult to be certain that they have no microcarcinomas. Second, plasma testosterone levels are low in patients with advanced carcinoma so it is pos- sible that the endocrine condition is the result rather than the cause of the disease.I3

For many years the endocrine stimulus and its activity have been studied as the concentrations or amounts of individual steroids in biologic fluids. What has not been considered is the binding of these molecules, which cer- tainly affects the hormonal effect. Since the binding sys- tems were known, plasma assay methods have taken into account bound and free hormone levels and total levels. It is the free hormone that is biologically active. These studies are complicated by the fact that the equilibrium between bound and free hormone levels is dynamic, not fixed. For this reason, interest has shifted toward methods that define the characteristics of the plasma hormone binding system, both in dynamic terms (by kinetic studies of the pattern of plasma binder to hormone16317) and as a study of the binding protein itself under resting condi- tions and at eq~i l ibr ium. '~, '~

In addition, some data in the literature indicate that even the specific plasma steroid-transporting system is not

This suggests that the problem of de- fining or measuring the plasma binding system is complex, both practically and theoretically. In this situation every measurement must be interpreted in the light of the method used, and the results obtained by different meth- ods cannot always be compared. The binding capacity can be measured either as the amount of hormone that a volume of plasma binds under specified laboratory condition^'^^'^ or by immunoassay of the binding pro- tein.8.22 The former provides the actual activity and the latter measures the amino acid sequences that the antibody recognizes. Although there is usually a good correlation (for SHBG, too) between the biologic and immunologic measurements, the fact that this system may be nonho- mogeneous requires caution when generalizing this con- cept to all of the clinical cases and also indicates that one should not express the values in terms of concentration of a specific molecule without indicating how it was mea- sured.

All of this helps us to understand why the results of different investigators have not been in agreement. For example, with the Anderson modification of the Rosner method, no differences were found between plasma SHBG levels of patients with benign prostatic hyperplasia and patients with prostatic cancer. However, with the original

Rosner method the cancer patients had slightly lower lev- els (not significantly) than healthy control subjeck6 Within the population with tumors, there did not appear to be any differences in SHBG level in relation to stage of tumor spread or existence of metastases.

In our study we found significantly higher levels of SHBG in the prostatic cancer patients than in the healthy control subjects or patients with benign prostatic hyper- plasia. The latter group also had significantly higher levels than the control subjects. These data do not agree with those of other investigators who found no differences be- tween control subjects and patient^.^^^ Of the several pos- sible explanations for the discrepancy, the least likely, in our opinion, is that there is a racial difference between their study groups and ours. We think it is more likely that the difference is due to the nonhomogeneity of the SHBG and to the methods used, each of which detects a particular fraction or active site on the molecule. We used a solid phase immunofluorescent assay with a monoclonal anti-SHBG antibody, whereas other groups have mea- sured the binding capacity of the plasma globulin fraction for labeled DHT.

Another aspect of our results that does not completely agree with those of others was the increase in SHBG level with age. In our subjects the increase was very modest (only 10% from 60 to 85 years of age). Because the control subjects and the patients in our study had similar mean ages and age distributions, the age factor did not affect the comparisons of our groups or the statistical analyses of the data.

In our study SHBG values were not correlated with severity or extent of the carcinoma, metastatic diffusion, or tumor grading.

The values for our three groups (cancer patients, hy- perplasia patients, and control subjects) were significantly different, but there was an overlap. This overlap, which has been found for most of the tumor markers proposed so far, limits the practical use of the test. There is overlap on both sides for the group with hyperplasia for whom values varied from 5 to 41 nmol/l. These patients with benign hyperplasia were diagnosed by clinical examina- tion, transrectal echography, and histologic examination of serial sections of material obtained at surgery. The question is: How many carcinomas might escape being detected by this combination of investigations or, in other words, how many of these patients with hyperplasia might have had subclinical carcinoma? The diagnostic limita- tions of digital exploration of the prostate are obvious and it is known that transrectal echographic diagnosis is based more on indirect signs (e.g., ratio of transverse to antero- posterior diameter or nonhomogeneity of the tissue) than on direct visualization of small carcinomas, which when smaller than a certain diameter can go undetected due to contingent factors (e.g., type of instrument or ability of

No. 2 PLASMA SHBG IN PROSTATIC CA - Grasso el al. 357

the technician).23324 The histologic examination of serial sections of material obtained at surgery should show car- cinomas that were not detected in the other examinations. The histologic examination should show all carcinomas at Stage A2, but perhaps only 89% of those at Stage A1 .25

Therefore, there is a small possibility that some of our patients with prostatic hyperplasia did have small carci- nomas (Stage A 1 ), which is not of great clinical importance in itself because the gland is completely removed, but which would cause a certain number of incorrect diag- noses. However, the expected number of false-negatives is limited to Stage A1 cases, which are certainly fewer than the number of cases of prostatic hyperplasia that had values overlapping with those of the carcinoma cases.

In conclusion, this test is currently of only limited use for detecting tumors, especially in cases that also may have benign prostatic hyperplasia. It is difficult to propose a theory for the increase in SHBG in patients with prostatic carcinoma on the basis of our knowledge of the endocri- nology of these cases because increased SHBG should be correlated with an increase in plasma estrogens and a fall in androgens, especially free androgens. This does not fit in with the theory, which has never been proven, that hyperandrogen is the basis of prostatic carcinoma.

In any case, prostate carcinoma, like breast cancer, is associated with a change in the plasma protein picture that can be detected as a change in SHBG; however, this requires further analysis and the use of the most suitable assay techniques. This might provide diagnostic methods that are discriminant for tumors and will certainly provide more information about the biochemistry of the tumor.

REFERENCES

I . OMalley BW. Mechanisms of action of steroid hormones. N Engl J Med 197 I ; 284:370-377.

2. Wilson JD. Recent studies on the mechanism of action of testos- terone. N Engl JMed 1972; 287:1284-1291.

3. Pasqualini JR. Hormonal regulation of the prostatic gland: Phys- iology and pathology. Triangle 1982; 21:21-26.

4. Meikle AW, Stanish WM. Familial prostatic cancer risk and low testosterone. J Clin Endocrinol Metab 1982; 54:1104-1107.

5. Karr JP, Wajsman Z, Kirdani RY, Murphy GP, Sandberg AA. Effects of diethylstilbestrol and estramustine phosphate on serum sex hormone binding globulin and testosterone levels in prostate cancer pa- tient. J Urol 1980; 124:232-236.

6. Houghton AL, Turner R, Cooper EH. Sex hormone binding glob- ulin in carcinoma of the prostate. Br J Urol 1977; 49:227-232.

7. Mondina R, Borsellino G, Pirondini A et al. Breast carcinoma and plasma 17-beta-estradiol binding. Cancer 1989; 63:305-308.

8. Niemi S, Maentausta 0, Bolton NJ, Hammond GL. Time-resolved immunofluorometric assay of sex-hormone binding globulin. Clin Chem

9. Dixon WJ, Brown MB, Engelman L, Jennerich RI. BMDP Statis- tical Software Manual, vols. 1 and 2. Berkeley, CA: University of Cali- fornia Press, 1988; 187-209, 1013-1024.

10. Kirk RE. Experimental Design: Procedures for the Behavioral Sciences, ed. 2. Belmont, CA: Brooks/Cole, 1982; 123-125.

I I . Huggins C, Hodges CV. Studies on prostatic cancer: Effect of castration, of estrogen and androgen injection on serum phosphatases in metastatic carcinoma of the prostate. Cancer Res 1941; 1:293-297.

12. Hammond GL, Kontturi M, Vihko P, Vihko R. Serum steroids in normal males and patients with prostatic diseases. Clin Endocrinol

13. Hulka BS, Hammond JE, DiFerdinando G et al. Serum hormone levels among patients with prostatic carcinoma or benign prostatic hy- perplasia and clinic controls. Prostate 1987; 1 1 : 17 1 - 182.

14. Haapiainen R, Rannikko S, Alfthan 0, Adlercreutz H. Pretreat- ment plasma levels of testosterone and sex hormone binding globulin binding capacity in relation to clinical staging and survival in prostatic cancer patients. Prostate 1988; 12:325-332.

15. Meikle AW, Smith JA, Stringham JD. Production, clearance and metabolism of testosterone in men with prostatic cancer. Proslute 1987;

16. Mondina R, Capetta P, Cremonesi P, Grossi E, Polvani F. Protein binding and extraction of plasma steroids for clinical assays. In: Klopper A, Lerner L, van der Molen HJ, Sciarra F, eds. Research on Steroids, vol. 8. London: Academic Press, 1979; 331-333.

17. Mondina R, Cremonesi P, Polvani F. Patterns of transport and binding of sex steroids. Arch Androl 1979; 2:203-209.

18. Rosner W. A simplified method for the quantitative determination of testosterone-estradiol-binding globulin activity in human plasma. J Clin Endocrinol Metab 1972; 34:983-988.

19. Rudd BT, Duignan NM, London DR. A rapid method for the measurement of sex hormone binding globulin capacity of sera. Clin Chim Acta 1974; 55:165-178.

20. Murayama Y , Utsunomiya J, Takahashi 1, Kitamura M, Tomi- naga T. Sex hormone binding globulin as a reliable indicator of hormone dependence in human breast cancer. Ann Swg 1979; 190:133-138.

2 1. Mondina R, Bertulessi C, Capetta P, Grossi E, Polvani F. Fisio- patologia delle interazioni plasma-ormoni nell’aborto ricorrente. Atti Soc Ital Ostet Ginecol 1985; 63:159-169.

22. Hammond GL, Langley MS, Robinson PA. A liquid phase im- munoradiometric assay (IRMA) for human sex hormone binding globulin (SHBG). J Steroid Biochem 1985; 23:45 1-460.

23. Dahnert WF, Hamper UM, Eggleston JC, Walsh PC, Sanders RC. Prostatic evaluation by transrectal sonography with histopathologic correlation: The echopenic appearance of early carcinoma. Radiology

24. Lee F, Littrup PJ, Torp-Pedersen ST et al. Prostate cancer: Com- parison of transrectal US and digital rectal examination for screening. Genitourinary Radio1 1988; 168:389-394.

25. Rohr LR. Incidental adenocarcinoma in transurethral resections of the prostate. A m JSurg Pathol 1987; 11:53-58.

1988; 34163-66.

1978; 9.1 13-121.

10:25-31.

1986; 158:97-102.