Abstract: Prostate cancer is the most frequent malignancy and the second leading cause of cancer deaths among males in the Western world. A study of 40 cases of prostate cancer is conducted in an attempt to identify prognostic biomarkers that can distinguish aggressive cases that must be treated immediately. HER-2/neu oncogene amplification was initially studied because amplification of this gene has been reported in many other types of cancer. In this study, HER-2 gene amplification was assessed by fluorescence in situ hybridization (FISH) using a HER-2/neu gene probe with a chromosome 17 centromere control probe. The study was performed on formalin-fixed, paraffin-embedded tissues. FISH successfully analyzed all cases. Only 5 out of 40 (12.5%) were found to be amplified. This frequency was lower than the frequency of amplification found in other in other cancers studied. The level of amplification observed was correlated with the pathological grade. Our data indicate that HER-2/neu gene amplification status can be determined by FISH on archival prostate cancer specimens, significantly correlates with high tumor grade and is more frequently encountered in tumors with advanced pathological stage. Also, FISH is a sensitive technique for detection of abnormalities in the HER-2/neu gene and further studies should be undertaken to determine whether a FISH-based HER-2/neu detection method may prove of importance in the prediction of prognosis and planning of therapy in prostate cancer patients.
INTRODUCTION
Prostate Cancer (PC) is the most commonly diagnosed cancer in men in the Western world and the second leading cause of male cancer death[1,2]. Risk factors for PC include age, race, country of origin and familial history.
An identifying number of recurring chromosomal abnormalities, identified in various tumors during the last years, have resulted in major advances in the understanding of the pathogenesis of malignant transformation. Although prostate cancer is the most common malignant disease in men in Western countries, knowledge about cytogenetic data are still limited as compared to other solid tumors. This is due to the failure to obtain a sufficient number of dividing cancer cells in vitro, the low mitotic index of the original tumor in most cases and the overgrowth by normal cells of the cancer cells in culture[3].
No molecular markers are currently available to accurately predict clinical outcome or to discriminate between aggressive cancers that will grow quickly and kill and those that will grow slowly for several years without serious ill effects.
Oncogene amplifications are manifestations of genetic instability, which has been implicated in the pathogenesis of many cancers. It is a characteristic of cancer cells that allows an increased production of specific proteins used for the acquisition and maintenance of the malignant phenotype. For example, it is known that approximately 25-30% of breast and ovarian carcinomas have amplification of the HER-2/neu gene, although the prognostic value of HER-2/neu amplification or over expression in other cancers has been controversial[4-9]. The HER-2neu oncogene, which is located on chromosome 17q21, encodes a transmembrane tyrosine kinase receptor family[10]. The receptor's role in oncogenesis can be derived from its action on cellular cascades involving proliferation and differentiation of epithelial cells.
The present study exploits the availability of the fluorescence in situ hybridization (FISH) technology for studying the amplification of HER-2/neu oncogene in prostate cancer cases to determine a subgroup of patients who are at risk for rapid disease progression can be identified. Our ultimate goal is to define prognostic biomarkers that can predict clinical outcome. Biomarkers that are able to discriminate the fast progressing cancers from the slow growing cancers are most urgently needed not only to save on the cost of the therapy, but also to preserve the quality of life and to avoid unnecessary morbidity associated with therapy[11,12]. A routinely available method to successfully identified abnormal DNA markers will be important in modern oncology practice.
MATERIALS AND METHODS
The primary population for study was prostate cancer patients who underwent radical prostatectomy at National Cancer Institute, Cairo, Egypt. Formalin-a consulting pathologist identified fixed paraffin-embedded tissue blocks from 40 Egyptian patients. One case of these patients was classified as stage T1, 15 cases as T2, 23 were classified as stage T3 and one case as T4. The lymph node status on all patients was none (No). Histopathological evaluation was performed using the Gleason System[13]. Gene amplification was determined by FISH using the CEP-17/HER-2/neu dual-probe FISH protocol (Vysis Corp, Downer's Grove, IL). Briefly, formalin fixed, paraffin embedded tissue sections (4 μm thick) were deparaffinized in xylene, dehydrated in 100% ethanol and air dried. After a brief wash in phosphate buffer saline, the slides were dehydrated serially and air-dried in the dark. Hybridization with the Spectrum Green HER-2/neu DNA probe and Spectrum Orange CEP-17 DNA probe was carried out according to the manufacture’s instructions, using codenaturation block, overnight hybridization and rapid wash procedures. Nuclei were counterstained with DAPI 1 g mL-1 in Vectashield mounting media (Vector Laboratories, Burlingame, CA). Details of the FISH protocol have been discussed extensively elsewhere[14-17]. The procedure for oncogene amplification has been previously described[16]. Briefly, the raw data on the number of HER-2/neu gene copy number and chromosome 17 centromeric numbers were entered on a two-way table. The average copy numbers for both were calculated separately using the marginal totals in the table. Division can then calculate the amplification ratio. Slides with amplification ratios of less than 1.5 will be classified as “nonamplified”. Slides with amplification ratios = 1.5 will be classified as “amplified”. Ratios between 1.5 and 2.0 will be considered as “low amplification”. Ratios between 2.1 and 4.0 will be considered as "moderate amplification”. Ratios exceeding 4.0 will consider as “high amplification”.
RESULTS
Forty Egyptian patients with prostate cancer were included in this study. The histopathologic grade and clinical stage of the tumors studied are summarized in Table 1. One case of these patients was classified as stage T1, 15 cases as T2, 23 were classified as stage T3 and one case as T4. The median age of the patients was 63 years (range 47-73 years). Gleason scores were available for all patients and were 2-4 in 4 patient, 5-7 in 29 patients and 8-10 in 7 patients. No lymph node involvement or metastasis at the time of transurethral prostatectomy was detected.
| Table 1: | Results of FISH analysis of HER-2/neu oncogene amplification |
| Table 2: | Relationship of HER-2/neu amplification to pathological grade |
| a: n=40, b: Amplification, c: Normal cell | |
HER-2/neu gene amplification, as determined by FISH, was performed on all cases studied. Among all the cases successfully analyzed by FISH, 5 (12.5%) showed HER-2/neu oncogene amplification and 35 (87.5%) showed no amplification (Table 1). The level of amplification observed was correlated with the pathological grade. The only one case with grade T4 showed high-level amplification, 2 cases with grade T3 showed moderate-level amplification and 2 cases with grade T2 showed low-level amplification, according to previously defined criteria (Table 2).
DISCUSSION
The present study was conducted to determine whether HER-2/neu oncogene can serve as biomarker to predict patient prognosis for prostate cancer.
The HER-2/neu gene codes for a transmembrane 185 kDa class I tyrosine kinase receptor protein related to the epidermal growth factor receptor, the amplification or overexpression of which is implicated in the pathogenesis of many human cancers[18-20]. Currently available data suggest that there is an increased HER-2/neu expression in prostate cancer by Western and Southern blotting[21-23]. Moreover, the study of HER-2/neu and other oncogene amplifications using immunohistochemistry has not yielded unequivocal results. FISH technique may prove to be a superior technique to overcome this problem[24].
Our study is not in agreement to prior reports of frequent HER-2/neu gene amplification in patients with prostate carcinoma. Ross et al.[25, 26] previously reported that HER-2 overexpression in patients with prostate carcinoma by IHC was 29% and that gene amplification was observed in 41% of patients. Those authors also found that both overexpression and amplification were correlated with tumor grade. An earlier study by Kuhn et al.[27] reported from their studies that HER-2 overexpression is not a prognostic marker for prostate cancer. In contrast to breast cancer, FISH detects HER-2/neu amplification in a substantial proportion of prostate cancers that do not overexpress HER-2/neu by IHC[28]. Thus, further investigation is needed to clarify these apparently discordant results[29].
In the present study we evaluated 40 patient specimens for HER-2/neu oncogene amplification. All cases were successful. As noted above, the frequency of HER-2/neu gene amplification found in the present study is less than other previous studies of some of the other types of cancer (Table 1). Mark et al.[16] found 11% HER-2/neu amplification in rhabdomyosarcoma. In another study of 40 cases of stage I to stage IV breast cancer, the overall frequency of HER-2/neu oncogene amplification was 22.5%. Furthermore, the highest frequency of oncogene amplification was found to be associated with the highest stages. The highest level of amplification was also found in the higher stages. Based on these results, it is evident that a comparable frequency and level of HER-2/neu amplification in prostate cancer was found[30]. Moreover, Mark et al.[17] demonstrated that the frequency of gene amplification is much lower and the level of amplification is less intense in their study of prostate cancer.
Oxley et al.[31] concluded that the increased Her-2/neu oncogene copy number appears to be rare in clinically localized prostatic adenocarcinoma and is related to chromosome 17 polysomy rather than true amplification. As a result, it would not be a useful biomarker for identifying those patients who will have recurrences after radical prostatectomy.
It has been shown that HER-2 expression in archival tumor specimens in patients with prostate carcinoma that has been progressed to hormone-refractory prostate carcinoma is infrequent[32].
It was also demonstrated by Skacel et al.[33] that low-level amplification of HER-2/neu in prostate cancer was found in 26% of cases (3 to 5 signals per nucleus, corrected for chromosome 17 aneusomy). The presence of HER-2/neu amplification was associated with high tumor volume (>2.0 cm (3), p = 0.004). Also Edwards et al.[34] showed low levels of HER2 gene amplification (8, 7/89) using FISH technique and HER-2 protein expression (12%, 11/89) using IHC.
Nevertheless, high levels of Her-2/neu expression in androgen-independent tissue have not been observed universally. Morris et al.[35] reported that approximately 40% of 10 androgen-independent samples over-expressed Her-2 (compared with 14% of androgen-dependent samples) in their screening program for Phase II trastuzumab and paciltaxel trial. Reese et al.[36] using the Tab 250 antibody, reported an even lower rate of Her-2 positivity in patients with androgen-independent disease: Only 3 of 22 patients had intermediate to high expression levels (2+ or 3+). Other reports also suggest that HER2 protein over-expression and gene amplification are relatively uncommon in androgen-independent prostate cancer[37].
In conclusion, HER-2/neu gene amplification status can be determined by FISH on archival prostate cancer specimens, significantly correlates with high tumor grade and is more frequently encountered in tumors with advanced pathological stage. Also, FISH is more sensitive than IHC for detection of abnormalities in the HER-2/neu gene and further studies should be undertaken to determine whether a FISH-based HER-2/neu detection method may prove of importance in the prediction of prognosis and planning of therapy in prostate cancer patients. Exploration of other biomarkers in prostate cancer using FISH is warranted.