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- Materials and methods
Objective: Genetic aberration such as the amplification of c-myc has been commonly found in advanced prostate cancer. The aim of this study was to elucidate chromosome 8 alteration, including a gain and amplification of 8q24 (c-myc gene), related to the progression and survival in advanced (Stage C) prostate cancer.
Materials and methods: We used dual-probe fluorescence in situ hybridization with a centromere-specific probe for chromosome 8 (8cen), and with a region-specific probe for c-myc (8q24) to evaluate genetic changes in tumor samples from 50 patients who had undergone radical retropubic prostatectomy from 1986 to 2001.
Results: We classified the 8cen and c-myc copy numbers as normal, gain and amplification. The carcinoma foci with extra copies of c-myc, which was defined in 35 cases (70%), were divided into two groups: (a) a simple gain of the whole chromosome 8 (no increase in the c-myc copy number relative to the chromosome 8 centromere), which was identified in 15 cases (30%); and (b) a substantial amplification of c-myc (additional increases [AI] in the c-myc copy number relative to the chromosome 8 centromere), which was detected in 20 cases (40%). AI-c-myc was strongly associated with higher histopathological grades and Gleason’s scores (P = 0.0330, 0.0190, respectively). Patients with the AI-c-myc had earlier disease progression (P = 0.0029) and earlier cancer death (P = 0.0087) than did patients with normal patterns.
Conclusion: Identification of an AI-c-myc may serve as a potential marker of prostate cancer progression.
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- Materials and methods
In Japan, the number of patients with prostate cancer has been increasing recently,1 and hence, its control has become an important issue. Currently, the prognosis of the disease depends mainly on serum prostate-specific antigen (PSA) levels, pathological stage, and the grade of pathological differentiation. However these methods have their own limitations to be regarded as accurate indicator to the disease progress. The reason for this is that prostate cancer exhibits a variety of biological behaviors. Prostate cancer has been reported2–6 to be a typical tumor presenting diversified histological manifestations, plural lesions, and a wide variety of genetic analysis results. It is thus necessary to elucidate the factors controlling the biological characteristics of prostate cancer and to develop a proper method for its prognostication.
One promising diagnostic factor has been suggested to be an increase in the expression levels of chromosome 8 genes.5 As the cancer gene c-myc is known to be located within 8q24, an increased expression level of chromosome 8 genes in this region is thought to result in an enhanced expression of c-myc. In fact, an association between an increased c-myc expression and the genesis and progress of prostate cancer has been reported.4,7 Furthermore, a few researchers4,7–12 have demonstrated by means of fluorescence in situ hybridization (FISH) that an increased 8q24 level appears to be related to the process by which prostate cancer acquires its malignancy. Namely, a chromosome 8 alteration could serve as a potentially useful clinical index to the biological features of the prostate cancer. Nevertheless, all studies exploring the possibility of this factor as a clinical index in relation to differentiation and prognosis have been previously reported only with the American and European patients had prostate cancer,7,13–15 but only a little with Japanese patients.12
Prostate cancer at Stage T3N0M0, that is, of locally invasive, non-metastatic type, shows various types of malignancy and progress, which thus necessitates deliberate considerations in choosing the best suited therapy. In the present investigation, we examined the clinical usefulness of c-myc anomalies detected by the FISH technique, while focusing on Japanese patients with T3N0M0 prostate cancer.
Materials and methods
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- Materials and methods
Fifty patients with prostate cancer at stage T3N0M0 underwent radical prostatectomy during the period of 1986 through 2001. The mean age was 68 years (range, 52–74 years) and the mean observation period was 3 years (1–16 years). None of the patients had undergone either endocrine or radiation therapy prior to the operation.
The disease stage classification was based on the TNM classification system.16 All patients had pathologic stage pT3N0M0. The grades of pathhistological differentiation were identified as well differentiated, moderately differentiated, and poorly differentiated, in 10, 25, and 15 cases, respectively. The tumors were also categorized by three Gleason’s score groups of 4–6, 7 and 8–10, in 15, 14, and 21 cases, respectively (Table 1).
Table 1. Histopathological characteristics
|Total cases||n = 50|
|Mean age (years)||68 (range, 52–74)|
Biochemical failure was defined as three consecutive increases higher than 0.4 ng/mL. Clinical failure was declared when a new lesion such as local relapse or metastasis was detected by diagnostic imaging. Thirteen (26.0%) out of the 50 patients postoperatively received adjuvant hormonal therapy.
The data were statistically evaluated by means of the χ2-test, along with Fisher’s exact probability test if necessary.
The clinical relapse rate and disease-specific survival rate were obtained by means of the Kaplan–Meier method. The level of significance was examined by the log–rank test; P-value was determined by two-sided tests and a P-value of less than 0.05 regarded as statistically significant. Commercially available software (JMP 4.0.5J, SAS Institute, Cary, NC, USA) was used for statistical analysis.
Fluorescence in situ hybridization and assessment
Fluorescence in situ hybridization was conducted on specimens embedded in 10% formalin paraffin.4,6 The specimens were limited to those included the tissue of the typical primary grade based on Gleason’s score. From these specimens, sections measuring 5 µm in thickness4,6 were prepared and subjected to hematoxylin–eosin stain. After the deparaffinization, the sections were re-hydrated and immersed in 10 mmol/L citric acid buffer at 80°C for 30 min and then in 0.2mol/L HCl at room temperature for 20 min. This was followed by deproteinization with proteinase K (Dako Corporation, Carpinteria, CA, USA) at room temperature for 15 min. Subsequently, 75°C heat was applied to the specimens for 5 min in the presence of 70% formamide/2× SSC (100% formamide 280 mL, 20× SSC 40 mL, distilled water 80 mL). Probe mix (10 µL) was added to each section (α-satellite DNA probe 1 µL, c-myc locus-specific probe 1 µL, hybridization buffer [Vysis, Inc., Downers Grove. IL] 7µL, DW 1 µL) and then the specimens were hybridized at 37°C overnight.
Dual-probe FISH was performed with an α-satellite DNA probe which was specific to chromosome 8 centromere (CEP8: Spectrum Green, Vysis, Downers Grove, IL, USA), and with a locus-specific probe for 8q24 (locus specific identifiers: LSI c-myc, Spectrum Orange, Vysis). When the hybridization was completed, the specimens were washed three times with 2× SSC at 45°C for 10 min. After antifade compound p-phenylenediamine (DAPI; Vysis) was added, the sections were observed with a fluorescence microscope (Olympus, Tokyo, Japan) through a triple-band pass filter (Vysis) and signals were counted.
The interphase nuclei only from a focus of adenocarcinoma with primary grade according to Gleason’s score were evaluated. The interstitious elements4,6 and any adjoining or overlapping nuclei were excluded in this study.
Three hundred clearly bordered, physically undamaged nuclei were enumerated. On each nucleus, green signals for chromosome 8 and orange signals for the c-myc gene were counted to obtain the relative number of the cells and the average ratio of c-myc signals/CEP8 signals.4,8
The signal patterns for quantitative chromosome abnormalities and the interpretation of signal counts were defined as follows4,7(Fig. 1):
Figure 1. (a) Dual color fluorescence in situ hybridization with a centromere probe for chromosome 8 (CEP8, spectrum green), and region-specific probe for c-myc (spectrum orange), in representative of the carcinoma foci. Nuclei are counterstained with DAPI. (b) A cancer epithelial nucleus has four signals for chromosome 8 and nine signals for the c-myc gene. The connecting signals were counted as one. (c) A cancer cell nucleus has three signals for chromosome 8 and seven signals for the c-myc gene.
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The definition of signal patterns:
Simple gain/nucleus (increase in chromosome): a nucleus with at least three signals for both centromere 8 and c-myc.
Simple loss CEP8/nucleus (loss of chromosome 8): a nucleus with no or one signal for centromere 8.
Simple loss c-myc/nucleus: a nucleus with a fewer signals for c-myc than those for centromere 8.
Additional increase (AI)-c-myc/nucleus: a nucleus with excessively amplified signals for c-myc than those for centromere 8.
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- Materials and methods
The genesis, growth, and development of a tumor result from accumulated gene aberrations. It is considered that such accumulation also plays an important role in determining the malignancy and prognosis of the tumor.17
Chromosome abnormalities in tumor cells have been studied as a factor indicative of gene aberrations as well as the malignancy and prognosis of tumors. Chromosome analyses have hitherto relied on examinations of the nuclear types in metaphase chromosome, but it is difficult to prepare metaphase chromosome specimens from a solid tumor, which is composed mostly of interphase cells. The recently developed FISH modality18 is an effective method for analysing interphase cells. This technique has been employed to study chromosome abnormalities in prostate cancer as well.4,5,8–15,19 Among these investigations, some have reported on the prognostic importance of the loss of 8p22,7,13–15 13q14,9 and 17p1314 as well as of the amplification of 8q24.4,7–12
The 8q24 region is the locus of the c-myc cancer gene, which was cloned for the first time in the human cellular nucleus.20 In addition, it is also known to be related to the progression of prostate cancer. Using a cellular model for prostate cancer, Facchini et al.21 and Amati et al.22 demonstrated the excessive expression of c-myc gene accelerated cytokinesis, inhibited apoptosis, thereby enhancing cancer development. It has been hypothesized that the increase and amplification of c-myc gene contributes to the development and the progression of prostate cancer.4,7,10–12,23
In the present investigation, we studied the 8q24 (c-myc) region using the FISH method in Japanese patients with locally invasive non-metastatic (T3N0M0) prostate cancer. As a result, the c-myc region was shown to have increased in 30.0% while it was excessively amplified in 40.0% of the subjects. An excessive c-myc amplification was found to be significantly related to progressive pathohistological differentiation and an advanced Gleason’s score. In comparison to cases of no detectable c-myc aberration, the excessively amplified cases had a significantly earlier relapse after the operation as well as a significantly poorer disease-specific survival rate.
These results confirmed that in Japanese patients, in line with the findings of other nations, that an over-amplification of the c-myc gene in 8q24 was clearly related to the malignancy and progress of prostate cancer.12 Correspondingly, these results provided new information to substantiate the possibility of c-myc as a clinically useful indicator in the prognosis of prostate cancer,7,12–15 since the data published so far remain insufficient.
On the other hand, a resent report from the USA7 described that an examination of the patients with prostate cancer at a similar pathological stage showed a c-myc gain was observed in 34.7% and an amplification of c-myc was observed in 19.4%. Our finding of the amplified c-myc region (40.0%) was much higher than their results. When comparing the 5-year non-relapse rate and 5-year survival rate, our study demonstrated rates of 14.5% and 31%, while their results showed 56% and 80%, respectively. These differences in prognostic data appear causally to reflect a characteristic of malignancy of the disease in the subjects rather than the ethnic difference.
Other findings also suggest an increase in malignancy of prostate cancer may be through the graded induction of what process has been shown to be related to the amplification of the c-myc region.23 Visakorpi et al.24 carried out a comparative genomic hybridization (CGH) analysis of prostate cancer and reported an increased 8q level in the relapsed lesion was detected in 89% of all subjects. Alers et al.11 showed an 8q increase was found in 44% of the subjects with bone metastasizing prostate cancer and he concluded that c-myc amplification was a relatively late genetic change involved in bone metastasis from prostate cancer. Van Den Berg et al.25 analysed 44 primary prostate cancer by means of FISH of which result indicated four (9%) cases had c-myc amplification, and three (75%) out of these four cases were defined as having lymph node metastases.
It has been suggested that when 8q24 amplification is concurrent with the loss of the 8p22 region, the carcinomatous progress is significantly accelerated.7,14,15 Our observations of amplified 8q24 are also considered to belong to such a tumor subset that gradually developed toward a higher malignancy within stage T3N0M0 prostate cancer. Further elucidation of other factors regulating the malignant properties of prostate cancer as well as appropriate combinations of such findings might allow a subset of tumors to be defined to improve the prognosis of the disease.
In conclusion, our results showed that c-myc gene amplification was related to both pathohistological differentiation and Gleason’s score, that might provide important information for the prognosis of prostate cancer. Amplification of the c-myc gene associated with the gradual enhancement of malignancy, seems to be a useful indicator for the patients requiring close observation and care as well as for the selection of additional therapeutic treatments.