neu in human prostate cancer and benign hyperplasia

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CancerMters 99 (19%) 185-189

CACCOCER LETTERS

Overexpression of her-2/neu in human prostate cancer and benign hyperplasia Kefeng Gwb, Anne-Marie Mes-Massona,JeanGauthierc,Fred Saada,b3* %Zentre de Recherche Louis-Charles Simurd/lnstitut du cancer de Montrial, 1560 rue Sherbrooke est, Mont&l, Quibec, H2L 4M1, Canada bDepurtetnent d’llrology, Hapita Notre-Dame, 1560 rue Sherbrooke est, Montrial, Qudbec, H2L,4MI, Cunudu ‘DPpartement de Puthologie, Hcpitul Notre-Dame, 1560 rue Sherbrooke est, Mont&d, Quibec, H2L 4M1, Cunudu

Received27 June1995;revisionreceived3 November199.5;accepted6 November1995

Abstract

Overexpression of the neu oncoprotein has been describedin several tumor models including breast and prostatecancer. Overexpressionof neu has been reported to have prognostic significance in certain tumors but controversy continues regarding the role and frequency of lzeuoverexpressionin prostatic cancer.The objectives of the study were twofold. First, to characterize neu expression in prostate cancer in comparison to benign prostatic hyperplasia. Second,to determine whether neu expression correlateswith Gleason grade in prostatecancer.Thirty-nine prostatecancersobtained from radical prostatectomy specimensand 10 benign prostatic hyperplasia specimenswere included in the study. Specimenswere formalin fixed and pa&In-embedded. neu expression was studied by immunohistochemical staining using a monoclonal neu specific AB-3 antibody. All 39 specimens (100%) of prostate cancer showed positive immunostaining of variable degree while 2 (20%) benign prostatic hyperplasia specimensshowed positive staining. Thus, neu oncogeneis overexpressedin localized prostate cancercomparedto benign prostatic hyperplasia.The degreeof neu immunostaining did not correlate with Gleason grade and there appearedto be a tendency towards an inverse relationship. The prognostic significance of the varying overexpressionis unknown. Keywords: Neu oncoprotein; Prostatecancer; Immunohistochemistry

1. Introduction Prostate cancer is the most frequent neoplasia in males and the second cause of death. However, molecular genetic changes associated with the carcinogenesis and tumor progression of prostate cancer have not been well characterized [I]. The HER-Uneu -.

* Corresponding author.Tel.: +151487654%. fax:+15148765476.

gene is located on the long arm of chromosome 17 (17q21) and encodes a transmembrane glycoprotein (~185 neu) with extensive homology to the epidermal growth factor receptor [2]. The pattern of neu expression has been studied in a variety of human malignancies [3-l 11. While the analysis of neu overexpression in prostatic cancer has recently been considered [12-161, the frequency and significance of neu overexpression in prostate cancer remains to be es-

0304-3835/96/$12.000 1996ElsevierScienceIrelandLtd. All rightsreserved SSDI 03O4-3835(95’)04061-8

186

K Gu ef (II. / Cuncer Lrlters 99 (19Y6) 18.5-iNY

tablished. In the following study, we detected the overexpression of mu in prostatic cancer and benign prostatic hyperplasia (BPH) by immunohistochemical staining. The level of neu expression was also correlated with Gleason grade and pathologic stage.

Table I

lmmunoreactivityof neu in prostatic cancer and BPH Total

no

2. Materials and methods Tissue specimens from 39 patients with adenocarcinoma of the prostate and 10 patients with BPH made up the study sample. Tumor specimens were obtained by radical prostatectomy between January and December 1993. The age of patients ranged from 47 to 71 years (mean 62). Tumors were staged according to the TNM classification [17] and histologic grade was evaluated according to Gleason’s grading system [ 181. Specimens were fixed with formalin, embedded in paraffin, sectioned, and stained by an immunoperoxidase method. Tissue sections were immunostained for neu oncoprotein by the avidin-biotin-peroxidase complex (ABC) method [19] using the AB-3 monoclonal neu specific antibody (Oncogene Science Inc., Manhsset, NY) which recognizes carboxy-terminal sequences of neu protein. Tissue sections were deparaffmized, treated with 0.3% HzOz to eliminate endogenous peroxidase activity and incubated in 0.5% Saponin for 30 min at room temperature to enhance immunostaining. The sections were blocked with horse serum for 20 min and incubated with the AB-3 primary antibody at 4’C overnight. The optimal concentration of primary antibody (1 ,ug/ml) was determined using breast cancer specimens known to express neu. Tissue sections were then treated with a secondary biotinylated antibody for 30 min. followed by treatment with the avidin-biotin-peroxidase complex (neu ABC Kit, Oncogene Science Inc.) for 30 min at room temperature. Reaction products were developed using diaminobenzidine containing 1% H202 as a substrate for peroxidase. Nuclei were counterstained with hematoxylin. Substitution of the primary antibody with phosphate buffered saline served as the negative staining control. Positive staining was characterized by brown staining located on cell membranes and within the cell cytoplasm [12]. Staining was scored by light microscopy according to the percentage of immunopositive cells and scored from 0 to 3. A score of 0

Prostatic 39 cancer Prostatic 10 hyperplasis

Degreeof immunoreactivity(k) .-~-_-._ 3 2 I slrong

moderate

weak

(1 negative -

24 (62)

10 (2%

5 (13’1

0 (0)

0 (0,)

j(JW

) (10)

8 (80) -

indicates no positive staining and was considered a negative result. A score of 1 was classified as weak with only lo-20% of celis staining positively, a score of 2 was classified as moderate with 20-50% of cells staining positively, and a score of 3 was classified as strong with 50% of cells being immunopositive. All slides were independently analyzed in a blinded study. 3. Results While freu is normally expressed on epithelial cells, this expression is undetectable by immunohistochemistry and detection of neu expression in tumor samples is due to neu overexpression. Of the 39 prostatic cancers studied, all specimens (100%) demonstrated some degree of immunostaining (Table 1). In comparison. only 2 out the of 10 (20%) of the BPH specimens showed weak or moderate immuoopositive staining (Table 1). This difference was statistically significant (P < 0.005). Fig. 1 shows examples of the different immunostaining patterns. Tumors were analyzed according to Gleason grade and were sub-divided into three groups. Six tumors were well differentiated (Gleason grades 24). 29 tumors showed moderate differentiation (Gleason grades S-7) and 4 tumors were classified as poorly differentiated (Gleason grades 8-l 0). The correlation of neu staining to Gleason grade is presented in Table 3. In well differentiated tumors the degree of mu immunoreactivity was strong (67%) or moderate (33%) whereas moderately differentiated samples showed greater variability. In particular, 68% of samples showed strong immunoreactivity (score ot 3), 17% were only moderately reactive (score of 2).

K. Gu et al. I Cancer Letters 99 (1996) 185-189

187

D

Fig. 1. Different immunostaining patterns recognized in the prostate: (A) staining scorn 3 in a prostate cancer of Gleason grade 5, (B) staining score 2 in a prostate cancer of Gleason grade 6, (C) staining score 1 in a prostate cancer of Gleason grade 9, and (D) staining score 0 in BPH.

and 15% were poorly immunoreactive (score of 1). In contrast, poorly differentiated tumors generally showed less staining, with three out of four tumors having a score of 2 while the remaining tumor scored as 1. All tumors were either pathological stageT2 or Ts. neu staining in relation to tumor stage is presentedin Table 2 Immunoreactivity of mu classified by Gleason grade Total Degree of immunoreactivity (8) no. 3 2 1 strong moderate weak

Gleason grade

2-4 5-7 8-10

6 29 4

4 (67) 20 (68) 0 (0)

2 (33) 5 (17) 3 (75)

0 (0) 4 (15) 1(25)

Table 3. Comparison of different stagesprovided no significant correlation. 4. Discussion

Abnormal activation of cellular proto-oncogenes has been proposed to play an important role in the Table 3 Immunoreactivity of neu classified by pathological stage

0 negative 0 (0) 0 (0) 0 (0)

Pathological Total Degree of immunoreactivity (8) no. stage 3 2 1 moderate weak strong

0 negative

T2 T3

0 (0) 0 (0)

28 11

18 (64 ) 6 (55)

7 (25) 3 (27)

3(11)

2 (18)

18X

K. Gu el ul. /Cwtcer

initiation and development of human malignancies [2 I]. Overexpression of certain oncopmteins is immunohisto&micdIy de&table and may be the result of point mutations, amplifications or rearfagemerits in proto-oncogene sequences. Overexpression of the neu oncogene has been detected frequently in human breast, ovarian, lung, Madder, and gastric cancers. Moreover, neu 6 overcxpression has been reported to be a significant predictor of both overall survival and time to relapse in patients with breast cancers (46,20]. In prostate cancer, the correlation and role of neu overexpsion in the initiation and progression remains uncertain since conflicting results from several laboratories exist. In particular, while one study concluded that neu oncoprotein is overexpression in both benign and malignant glands of human prostate 1121, a separate study failed to detect any immunoactivity of neu oncopmtein in prostate cancers [ 111. In contrast, it has been reported that positive staining was detected in 80% of prostate cancers with no normal and benign prostate hyperplasia staining [IO] in one study while only 16% ot prostate cancers overexpressed neu by imageanalysis-assisted quantitative immunocytochemistry [ 161. Our results are compatible with a previously published report which indicates that neu oncoprotein overexpression exists not only in all grades of prostate cancer, but also in some BPH [ 141. In BPH. the rleu oncoprotein is expressed in areas where cells appear to be in active proliferation or showing his. tologic evidence of atypical hyperplasia and dysplasia. Moreover, the significantly different overexpression of rleu oncoprotein between prostate cancer and prostatic hyperplasia may indicate that neu oncoprotein is involved in growth stimulation and probabl) contributes to the oncogenic transformation of prostatic cells. Our study appears to indicate that tumors which overexpressed neu oncoprotein at the highest levels were generally more differentiated, while highly undifferentiated tumors showed weaker neu expression These results contrast with previous reports that indicated an association between increased expressions of neu oncoprotein in breast tumor with poor nuclear grade 15,201. In the prostate, it is possible that malignant cells are no longer dependent on the function of the neti oncoprotein and thus while neu may be involved in the initial neoplastic transformation, it>

L.trrer.t Y9 (19961 185%IXV

contribution in tumor progression and mtastasis may be more limited. In our study we also observed that positive staining was hctemgeneaus in its intensity With some areas staining motx5intensely than others. This may sug&st that prostatic tumors contain several subpop&tiOnS of cc&$that Wfy with lb& of neu overemon. Variabi&y of immu&&tochemical staining results for neu oncwotcin among different la-es may &e to ‘ti v&biIity of diiferent antito&@t neu -tin [22]. Moreover, various w in.irnmu&&&e&at staining techniques and classification of the degrees of the staining as well as normal variations in tumor populations may be responriiae far co&iiting results. 5. CoIutTlwb In conclusion, it appears that the neu oncoprotein plays a role in proli4erdon and transformation of prostatic cells. However, limited in$xmation on the prognosis of prostatic cancer can be obfained since overexpression did not corn&&e with known prognostic parameters. We are presently working on tumor suppressor genes that may be involved in prostate cancer and which may be implicated in tumor progression and me&static potential.

Acknowledgements We would like to thank Marie-Jo& Milot for technical assistance. This research was funded in part by the Centre de Recherche Louis-Charles Simard and by the Department of Surgery of the Notre-Dame Hospital. A.-M.M.-M. is an F.R.S.Q. fellow. F.S. is a recipient of the Canadian Urologic Association Research Scholarship. References [I]

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