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Original articleImmunohistochemical demonstration of gonadotropin-releasing

hormone receptors in prostate carcinoma

Janos Szabó, M.D.a, Attila Végh, M.D.a, Gergo Rácz, M.D.b, Béla Szende, M.D.b,*a Department of Urology, Central Hospital of the Hungarian Army, Budapest, Hungary

b 1st Department of Pathology and Experimental Cancer Research, and Research Group of Molecular Pathology, Hungarian Academy of Sciences,Semmelweis University, Budapest, Hungary

Received 14 December 2004; accepted 3 March 2005

bstract

The antiandrogen and gonadotropin-releasing hormone (Gn-RH) analogue treatment of prostate carcinoma is based on decreasedroliferative and increased apoptotic activity of tumor cells, induced by androgen ablation. Gn-RH analogues decrease the serum level ofndrogens by breaking the pituitary-gonadal axis, but increasing evidence points to the direct effect of Gn-RH analogues on tumor cells.mmunohistochemical demonstration of Gn-RH receptors recently became possible. Cryostat and paraffin sections of prostate biopsyamples of 10 untreated patients with prostate carcinoma (T2�T3, Gleason score 5�8) were investigated for Gn-RH receptors (Gn-RHR)nd androgen receptors, respectively, using the immunoperoxidase method. Membrane bound, focal Gn-RHR positivity was found in 5amples. Nuclear androgen receptor positivity appeared in 7 samples. No correlation between Gn-RHR and androgen receptor positivity wasound, neither receptor status and tumor-nodes-metastasis stage nor Gleason score could be related to each other. Correlation betweenn-RHR positivity and response to luteinizing hormone-releasing hormone analogue treatment will be investigated further. © 2005lsevier Inc. All rights reserved.

Urologic Oncology: Seminars and Original Investigations 23 (2005) 399–401

eywords: Gonadotropin-releasing hormone receptor; Prostate; Carcinoma; Immunoperoxidase

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. Introduction

Analogues of luteinizing hormone-releasing hormoneLH-RH) became important in palliative therapy of prostatearcinoma [1–3]. Theoretically, the effect of LH-RH ana-ogues on prostate carcinoma is based on inhibition of theituitary-gonadal axis (i.e., and suppression of LH and tes-osterone secretion). However, increasing evidence points tohe fact that LH-RH analogues also exert a direct inhibitoryffect on certain types of cancer cells [4,5]. Receptors forH-RH have been found in various androgen-dependentnd androgen-independent prostate carcinoma cell lines6–11]. High incidence of receptors for LH-RH and LH-RHeceptor gene expression has also been shown in humanrostate carcinoma specimens by in vitro ligand competitionssays as well as by reverse transcriptase polymerase chaineaction [12].

Microscopic demonstration of gonadotropin-releasing

* Corresponding author. Tel.: �36-1-266-0451; fax: �36-1-266-0451.

aE-mail address: bszende@korb1.sote.hu (B. Szende).

078-1439/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved.oi:10.1016/j.urolonc.2005.04.001

ormone (Gn-RH) receptors (Gn-RHR) was performed soar by in situ hybridization of messenger ribonucleic acidor LH-RH receptor [13,14] in prostate cancer cells. Be-ause affinity purified polyclonal antibodies to react withn-RHR of human origin became available, an attempt wasade to show LH-RH receptors in human prostate carci-

oma samples obtained by needle biopsies using the immu-operoxidase method.

. Materials and methods

Diagnostic needle biopsies of 10 patients with T2�T3tage untreated prostate cancer, known prostate-specific an-igen (PSA) values, and tumor-nodes-metastasis (TNM) sta-us were examined. For immunohistochemical studies,-�m thin frozen sections were cut in a cryostat. The re-aining tissue blocks were fixed in buffered neutral forma-

in and embedded in paraplast, and 8-�m thin sections wereut and stained with hematoxylin and eosin. Immunoperox-dase reaction was performed on paraffin sections using

ntiandrogen receptor antibody (DAKO, Glostrup, Den-

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400 J. Szabó et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 399–401

ark), and on frozen sections using the polyclonal anti-Gn-HR (C-18) Sc 8681 as well as anti-Gn-RHR (N-20) Sc.682 antibodies (Santa Cruz Biotechnology, Inc., Santaruz, CA). The dilution of the antiandrogen receptor anti-ody was 1:100 and that of the anti-Gn-RHR-s was 1:20.he antibodies were applied overnight at 37oC. The sectionsere treated with proteinase K and, to inactivate endoge-ous peroxidase, incubated in methanol and H2O2. Theecondary antibodies (antimouse immunoglobulin G andnti-goat immunoglobulin G, respectively, products ofAKO) were used in a dilution of 1:100 the next day.iaminobenzidine served as chromogen and methyl green

s counterstain. Gleason score, androgen receptor, and Gn-HR positivity were assessed using an Axiophot micro-

cope (Zeiss, Jena, Germany).

. Results

The age, TNM stage, serum PSA level, and Gleasoncore of the patients are shown in Table 1. Gn-RHR andndrogen receptor status is also indicated in Table 1. Nu-lear androgen receptor positivity was observed in 7 casesFig. 1). Positive tumor cells were evenly distributed in theumor tissue. The ratio of positive cells was between 30%nd 50% in 3 cases, between 50% and 70% in 3 cases, andore than 70% in 1 case.Gn-RHR positivity (for both antibodies) appeared in 5

ases. The positive reaction was membrane bound both inhe cell membrane and the cytoplasm. Positivity was focaln scattered gland-like structures of the tumor tissue, but, ift appeared, all cells of the gland-like structure were positiveFig. 2). It is noteworthy that when Gn-RHR positivity wasound in the tumor tissue, normal, nontumorous glands inhe sample were also positive.

Neither androgen nor Gn-RH receptors were detectablen 2 cases, and both were detectable in 3 cases. No corre-

able 1ge, TNM stage, Gleason score, serum PSA level, Gn-RHR and androge

arcinoma

atient number Age (yrs) TNM stage PSA*

1 59 T2c-Nx-Mx 12.52 81 T2c-Nx-Mx 13.93 75 T3c-N2-M1b 4004 52 T2c-N1-M1b 15005 79 T3b-N0-M0 80.66 72 T2c-Nx-Mx 257 71 T2b-Nx-Mx 21.48 77 T3c-Nx-Mx 209 75 T2b-N0-M0 5.80 61 T3-N0-M0 0.5

* Normal values: 0.00–4.00 ng/ml.a Androgen receptor positivity was: �, when 30% to 50% of the cells; �

ere positive.

ations among TNM stage, PSA level, Gleason score, and n

ndrogen receptor or Gn-RHR expression could be discov-red. The patients received total androgen blockade. One yearfter starting the treatment, patient No. 5 died of a stroke. Therostate tumor showed progression in patient No. 3; the otheratients have been in stable clinical condition.

. Discussion

The expression of Gn-RHR has been shown in severalnimal and human tumor cells, including prostate carci-oma. It became evident that Gn-RH analogues, in additiono their effect on the pituitary-gonadal axis, exert a directffect on the tumor cells possessing Gn-RHR, resulting in aecrease in cell proliferation and an increase in apoptoticell death [15–17].

The direct effect of Gn-RH analogues on prostate carci-oma is strongly supported by data obtained as a result of in

tor status of the tumor tissue of 10 patients with advanced prostate

Gn-RHR Androgen receptora Gleason score

Negative Negative 6� Negative 5Negative �� 8Negative Negative 6� � 5Negative �� 8� ��� 7� � 6� � 6Negative �� 8

en 50% to 70% of the cells; and ���, when more than 70% of the cells

ig. 1. Androgen receptor positivity in the nuclei of cells of a prostatearcinoma (patient No. 7) (antiandrogen receptor immunoperoxidase, mag-

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itro treatment of cultured prostate carcinoma cells [12] andlso by in vivo observations. Damyanov [18] reported thatH-RH agonist treatment was successful in a patient whonderwent orchiectomy with hormone-refractory prostatearcinoma. Our previous studies [17] showed that an in-rease in apoptosis ensued already 24 hours after the firstH-RH analogue injection, before suppression of androgenroduction could be supposed.

Regarding the mode of action of Gn-RH analogue onumor cells, the comprehensive work of Emons et al. [19]evealed that these compounds interfere with the signalransduction of growth factor receptors and related onco-ene products associated with tyrosine-kinase activity.n-RH induced activation of a phosphotyrosine phospha-

ase counteracting the effects of receptor associated tyrosineinase is the most probable mode of action.

However, modulations and mutations in androgen andonadotropin receptors may interfere with the aforemen-ioned processes, and influence the effect of either antian-rogen or Gn-RH therapy. From the practical point of view,mmunohistochemical detection of Gn-RHR in frozen sec-ions of prostate carcinoma tissue seems to be reliable andeproducible. The focal positivity of Gn-RHR may explainhe temporary regression and later the relapse of prostatearcinomas after LH-RH analogue treatment.

The correlation between Gn-RHR positivity and re-ponse to LH-RH analogue therapy remains to be examined.

large number of patients and a long observation periodre planned in our further studies.

eferences

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