Oncogene amplifications are manifestations of genetic instability and have been shown to play a part in the pathogenesis of many cancers. As a result of gene amplification, increased production of specific proteins leads cancer cells to acquire and maintain malignant potential. Her-2/neu gene is a protooncogene which is a member of the EGF receptor family and encodes a transmembrane receptor protein with tyrosine kinase activity and involves in normal cell growth. Her-2/neu protooncogene shows oncogenic conversion via gene amplification and protein overexpression which leads to abnormal proliferation and agressive behaviour of tumor cells[1,2].
Recently there has been a growing interest in Her-2/neu oncogene, expression / amplification which has been shown to correlate with a shorter survival in a variety of epithelial malignancies[3-7].
Since new therapeutic modalities are now available that specifically target Her-2/neu oncogene, diagnostic tests for identifying Her- 2/neu oncoprotein have been used as routine application in the management of breast cancer patients.
There are many studies reported in the literature investigating the role of Her-2/neu oncogene in prostate carcinomas which have provided contradictory results. This study was performed to test the hypothesis that Her-2/neu oncogene might have a role in the initiation and progression of prostate carcinomas.
The study included materials from 74 patients with prostate cancer who underwent radical prostatectomy with bilateral pelvic lymph node dissection at the urology department of Ankara University School of Medicine between 1993 and 2002. Hematoxylin and eosin stained histological sections from formalin fixed, paraffin embedded tissues were reviewed and histological grade was determined according to the Gleason system. Representative areas were selected for each tumor specimen and tissue array (TA) blocks were constructed by taking cores of 4 mm in diameter from these areas using punch biopsy needle. Two 6 cores (average 3.7 cores per case) were taken from each tumor specimen. Twenty three TA blocks, each containing 12 cores were obtained. Four-micrometer sections of these array blocks were cut and placed on charged slides. Three sections, one for Hematoxylineosin, one for immunohistochemistry, and one for flourescence in-situ hybridization were obtained from each TA block. Hematoxylin-eosin stained sections were used for histological verification of areas of benign prostate hyperplasia (BPH), low grade prostatic intraepithelial neoplasia (LGPIN), high grade prostatic intraepithelial neoplasia (HGPIN) and prostatic carcinoma. According to the Gleason grade system, one grade was determined in each core of the cases and the most frequent grade among the cores of the same case was defined as “dominant grade” and considered as the representative Gleason grade of the case. The highest Gleason grade among the cores was defined as the “maximum grade”.
Immunohistochemistry (IHC):
Sections (4 mµ) were deparaffinized and antigen retrieval was performed by immersing slides in 10 mmol/L citrate buffer (ph=6) and heating in a 95ºC water bath for 20 minutes. After application of hydrogen peroxide for 5 minutes, the slides were incubated with primary antibody which was a polyclonal rabbit anti-human HER2/neu antibody recognizing an intracellular domain of Her-2/neu oncoprotein (DAKO, Denmark, Code No: A 0485) (1/300 dilution) for 20 minutes. Then slides were incubated with biotin for 10 and with streptavidin for 10 minutes respectively. This was followed by immersion in substrate-chromogen solution for 8 minutes. Hematoxylin was used for counterstaining. Sections from a breast cancer specimen, which was known to be 3+ positive for Her-2/neu, were used as positive controls.
Both cytoplasmic and membranous staining were evaluated in the areas of cancer, LGPIN, HGPIN and BPH in each core and scored according to the intensity and the percentage of stained cells. Intensity was scored on a 0 to 3+ scale (0: no staining, 1: weak, 2: moderate, 3: strong staining). The percentage of stained cells was evaluated on a 0 to 3+ scale (for membranous staining: 0: no staining, 1+:1-5%, 2+:6- 20% and 3+:21-100%; for cytoplasmic staining: 0: no staining, 1+:1-30%, 2+:31-80%, 3+:81- 100%). Cytoplasmic staining score (CSS) and membranous staining score (MSS) were calculated separately by adding the intensity and percentage scores, and the total staining score (TSS) in each core was determined by adding CSS and MSS. The highest staining score (CSS: 0-6, MSS: 0-6, TSS: 0-12) among the cores of the same case was accepted as the representative staining score of the case.
Flourescence in-situ hybridization (FISH):
Her-2/neu gene amplification was investigated in 56 of 74 prostate carcinomas by FISH and for each case 1-3 cores were evaluated. Deparaffinisation, sequential acid incubation, denaturation, proteinase K digestion steps were followed respectively. After formamide denaturation and formalin fixation, the slides were dehydrated in graded ethyl alcohol and air dried. FISH was performed on pretreated slides using Q-BIOgene Chromosome 17p12 (HER2/NEU)/ Alphasatellite 17 Cocktail (dual color) TM FISH probe (Cat. #: PONC1712). After hybridization, nuclei were stained with 6-diamidin 2- fenilindiol dihydrochloride antifade solution (DAPI) and the signals were visualized on a ZEISS Axiscope fluorescence microscope. Only the cells with at least one red and one green signal in their nuclei were evaluated. Red (Her 2) and green (Chromosome 17) signals were counted in 40 non-overlapping tumor nuclei in each core and the ratio of red and green signals was defined as the amplification score. Cores with an amplification score of >2 or with tight clustering signal pattern in ³50% of counted cells were considered as positive for Her-2/neu gene amplification. The highest amplification score among the cores of the same case was accepted as the representative amplification score of the case.
Statistical Analysis:
“Kruskal Wallis Test”, Mann Whitney test”, “Student's T test”, “Chi Square test”, “Spearmen's Correlation test” were used for statistical analysis. All analyses were performed using Statistica software and a p value of <0.05 was considered as statistically significant.
Histopathology, Immunohistochemistry and FISH:
The distribution of cases according to Gleason grade is shown in Table 1.
Table 1: Distribution of cases according to the dominant and maximum Gleason grade.
Of the 74 prostate cancers, 54 (%72.97) showed immunostaining, only in cytoplasmic (n=34; 45.94%), membranous (n=6; 8.1%) in mixed (cytoplasmic and membranous) patterns (n=14; 18.91%) respectively (Fig. 1). No correlation between immunostaining scores and stage of the tumor was found (p=2.91).
There was no statistically significant association between immunostaining scores and dominant or maximum Gleason grade and patients' ages or preoperative PSA levels as well.
Immunostaining was compared in areas of BPH, LGPIN, HGPIN and prostatic carcinoma. The percentage of cores with only cytoplasmic or mixed (cytoplasmic and membranous) immunostaining patterns significantly increased from areas of BPH to PIN and reached the highest degree in areas of prostatic carcinoma (Chi- Square test, p=0, p=0); whereas no such correlation was found for cores with only membranous staining (Table 2).
Table 2: The percentage of immunostained cores in areas of BPH, PIN and carcinoma.
The percentage of immunostaining (in cytoplasmic or membranous pattern) was compared between cores with different Gleason grades. The relationship between Gleason grade and the percentage of immunostained cores was investigated among the total number of 254 cores representing 74 patients. There was no relationship between Gleason grade and Her-2/neu protein expression.
The percentages of immunostained cores (in cytoplasmic or membranous pattern) were compared between groups with different Gleason grades. There was no relationship between Gleason grade and Her-2/neu protein expression.
In 56 of the 74 cases, a minimum of 40 tumor cells with at least one CEP17 and one Her2 Neu signal could be detected in one or more cores stained with FISH. Gene amplification was detected in only 3 tumors, one of which belonged to a patient with androgen independent disease (Fig. 2). Of the 3 FISH positive cases, in only one with a score of +1 was present There was no association between amplification score and dominant or maximum Gleason grades (p=0.291, p=0.513, respectively) or between amplification score and immunostaining scores (p=0.864, p=0.521, respectively).
Figure 2: Her-2/neu gene amplification by FISH (green / red signals >2).
Some studies detected Her-2/neu overexpression in prostate carcinomas and found correlation with Gleason grade, tumor stage and aneuploidy[9,12-14], whereas no such correlation was found in some of them[8,11].
Technical factors as the type of examined tissue, fixation, staining protocols, antigen retrieval procedures, the type of anti-Her2 antibodies, heterogeneity in case selection and scoring systems may be responsible for this disparity[15,16]. The most frequently observed immunohistochemical staining pattern was cytoplasmic rather than membranous. The cytoplasmic immunostaining may be explained by the binding of antibody to the intracellular part of Her-2/neu transmembrane protein. Ross et al.[12], found significant correlation between Her-2/neu cytoplasmic immunostaining intensity and tumor grade or aneuploidy status. Differential staining of Her-2/neu in different subcellular organelles may be related to cancer stages and cell types[17]. In the present study, the frequency of membranous immunostaining inversely correlated with Gleason grade and no membranous immunostaining was seen in the areas of grade 5 carcinoma. Compatibly, Kameda et al.[18] has previously reported that Her-2/neu immunoreactivity in cells of poorly differentiated gastric tumors was detected most often within the cytoplasm, whereas the immunoreactivity in well differentiated tumor cells was detected on the cell membranes. However, Her-2/neu expression detected by Western blotting was very low or none in the cytoplasmic positive tumor cells. This finding suggests that cytoplasmic staining may be a non-specific finding. Alternatively there may be an error preventing the membranous localization of the protein.
The significant increase of Her-2 overexpression from areas of BPH to PIN and carcinoma may indicate that Her-2/neu oncoprotein is involved in growth stimulation and oncogenic transformation of prostatic cells. However, the role of Her-2/neu expression in the progression of prostate carcinoma is argueable, since no correlation with known prognostic parameters such as Gleason grade, stage of tumor was detected. Our results are compatible with a previously published report which suggested that Her- 2/neu oncoprotein may be involved in the initial neoplastic transformation but its contribution in tumor progression may probably be more limited[19].
According to the studies performed using FISH, one group has found amplification of the Her-2/neu oncogene in up to 44% of the cases, and this was associated with advanced pathological stage and higher Gleason score. However, more recent work by that group showed a lower amplification rate of 10-25%[20-22]. Another study on different stages of prostate carcinomafrom localised to metastatic- found no amplification at any stage[23]. The differences in amplification rates in these studies might be due to the definition of amplification used by the different investigators or increase in gene copy numbers due to chromosome 17 polysomy which might be mistaken for true amplification. In our series, most of the cases were clinically localized prostate cancer, Her-2/neu gene amplification was present only in 4.05%, in spite of an overexpression rate of 72.97%. Polisomy or any other anomaly involving chromosome 17 was not detected in any of cases which might be responsible for high Her-2/neu protein expression. The discordance between the expression and amplification of Her-2/neu oncogene suggests that Her-2/neu oncoprotein expression may not be associated with Her-2/neu gene amplification which makes it difficult to comment on the significance of Her-2/neu protein expression in the development of prostatic carcinoma. We are in the same opinion with Zhau et al.[17] who suggested that the underlying mechanism for higher Her-2/neu protein expression might be due to increased rate of Her-2/neu transcription, increased Her-2/neu mRNA stability, posttranscriptional processing, or all of them.
It has been suggested that Her-2/neu amplification was rare in clinically localized prostate carcinoma[24]. Her-2/neu oncogene amplification/ overexpression has been shown to be associated with the progression of prostate cancer from an androgen dependent to an androgen independent phenotype. Higher expression of Her-2/neu has been detected in androgen refractory and metastatic prostate carcinomas, in contrast to early stages[25-29]. This finding led to the use of a monoclonal antibody targeting Her- 2/neu in the treatment of androgen refractory carcinoma[30], however trials showed that it had a poor efficacy as a single agent[31]. Because nearly all prostate carcinomas included in this study were clinically localized and only five of them were in hormone refractory state, we cannot comment on the significance of Her- 2/neu oncoprotein in the progression of androgen dependent to androgen independent phenotype.
In conclusion, the significant increase of Her-2 overexpression from areas of BPH to PIN and carcinoma may indicate that Her-2/neu oncoprotein is involved in stimulation of the growth and oncogenic transformation of prostatic cells. However, the role of Her-2/neu expression in the progression of prostate carcinoma is argueable, since no correlation with known prognostic parameters such as Gleason grade, stage of tumor was detected. Furthermore, the discordance between the expression and amplification rates of Her-2/neu oncogene suggests that Her-2/neu oncoprotein expression may not be associated with true gene amplification which makes it difficult to comment on the significance of Her-2/neu protein expression in prostate carcinoma. We think that some other factors such as increased rate of Her-2/neu transcription, posttranscriptional processing may be the underlying mechanism for high Her- 2/neu protein expression in prostate cancer. Our limitation was that most of the patients included in this study had clinically localized prostate cancer and no assesment could be made on the significance of Her-2/neu oncoprotein in the progression of carcinoma to the androgen independent phenotype. Therefore, studies on larger series of prostate carcinoma representing different stages are necessary to determine whether Her-2/neu gene has a role in prostate carcinoma.
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