Expression and Gene Copy Number Analysis of ERBB2 Oncogene in Prostate Cancer

Cell Lines and Tumor Samples

Three prostate cancer cell lines (LNCaP, DU-145, PC-3) and 16 breast cancer cell lines (SK-BR-3, ZR-75-1, MDA-436, MDA-361, MDA-134, MDA-415, MDA-157, MCF-7, MCF-A10, BT-474, UACC-893, DU-4475, CAMA-1, HBL-100, HCC-1419) were obtained from the American Type Culture Collection (ATCC, Manassas, VA), and cultured according to the recommended conditions. For the analysis of ERBB2 expression by RT-PCR freshly frozen specimens of 5 benign prostate hyperplasias, 21 primary prostate carcinomas (prostatectomy specimens), and 8 locally-recurrent hormone-refractory prostate carcinomas (TURP specimens from patients treated with hormonal therapy) were obtained from Tampere University Hospital. For the immunohistochemical and CISH analyses formalin-fixed paraffin-embedded tumor samples of 54 primary untreated (prostatectomy specimens) and 50 locally recurrent hormone-refractory prostate carcinomas (TURP specimens) from Tampere University Hospital, as well as 22 untreated lymph-node metastases of prostate cancer from Lund University Hospital, were obtained. Freshly frozen and corresponding formalin-fixed paraffin-embedded tumor blocks from 30 untreated primary breast carcinomas obtained from Tampere University Hospital were also analyzed by RT-PCR, immunohistochemistry, and CISH. For the immunohistochemical and CISH analyses, tissue microarray blocks were constructed according to published guidelines. 28

Chromogenic in Situ Hybridization

CISH was done on 5-μm-thick archival formalin-fixed paraffin-embedded tissue sections with minor modifications to the previously described protocol. 29 In brief, the sections were deparaffinized and incubated in 0.1 mol/L of Tris-HCl (pH 7.3) in a temperature-controlled microwave oven (at 92°C for 10 minutes). The sections were then allowed to cool down for 20 minutes and washed with phosphate-buffered saline (PBS). Enzymatic digestion was done by applying 100 μl of digestion enzyme on to slides for 10 to 15 minutes at room temperature (Digest-All III solution; Zymed Inc., South San Francisco, CA). The slides were then washed with PBS and dehydrated with graded ethanol. The ready-to-use digoxigenin-labeled HER-2/neu probe (consisting of two contig BAC clones; Zymed, Inc.) was applied onto the slides. The slides were denatured on a hot plate for 3 minutes, and the hybridization was performed overnight at 37°C. After hybridization, the slides were washed with 0.5× standard saline citrate for 5 minutes at 75°C, followed by three washes in PBS/0.2% Tween20 (at room temperature). The HER-2/neu probe was detected with sequential incubations with mouse anti-digoxigenin (diluted 1:300; Roche Biochemicals, Mannheim, Germany), anti-mouse-peroxidase polymer (Powervision, Immunovision Inc., Daly City, CA), and diaminobenzidine chromogen. Tissue sections were lightly counterstained with hematoxylin and embedded.

The CISH hybridizations were evaluated using ×40 and ×60 dry objectives and using 10 × 22 wide-field oculars. Amplification was defined as six or more signals per nucleus in more than 50% of cancer cells, or when a gene copy cluster was seen. Cases with other types of copy number alterations were considered as aneuploid.

Immunohistochemistry

The sections were first deparaffinized, followed by antigen retrieval (autoclave cooking at 121°C for 2 minutes in 10 mmol/L of sodium citrate buffer, pH 6.0). p185^HER2 protein was detected with the monoclonal antibody CB11 (Novocastra Laboratories, Newcastle, UK). The antibody was diluted 1:200 in Powervision blocking solution (ImmunoVision Inc.) and incubated overnight at +4°C using Shandon Sequenza immunostaining cover plates (Shandon, Pittsburgh, PA). A conjugate of anti-mouse antibody and horseradish peroxidase was used for visualization (30 minutes at room temperature) with diaminobenzidine as a chromogen (PowerVision, ImmunoVision Inc.). Sections were counterstained with hematoxylin. Evaluation of the staining was done as described previously. 29 Briefly, intense membranous staining in >50% of cancer cells was considered as 3+. Staining present in a smaller proportion of the cells or lower intensity was considered as 2+ staining. No staining or faint staining in a few cells were scored as 0 and 1+, respectively.

RT-PCR

The freshly frozen specimens were first histologically examined for the presence of carcinoma cells using hematoxylin and eosin-stained slides. Only samples containing more than 60% of cancerous or hyperplastic tissue were selected for the gene expression analyses. One to three 20-μm frozen sections were cut using a cryotome. Total RNAs were isolated from the sections using Qiagen RNeasy MiniKit (Qiagen Inc., Valencia, CA), and used for the first-strand cDNA synthesis with Superscript II reverse transcriptase and oligo d(T)_12–18 primer according to the manufacturer’s protocol (Life Technologies, Inc., Gaithersburg, MD). Total RNA from prostate and breast cancer cell lines was isolated using Trizol reagent (Life Technologies, Inc.) according to the manufacturer’s instructions, and reverse-transcribed as described above. For preparing the standard curve, 5 μg of totRNA from prostate cancer cell line LNCaP (ATCC), was reverse-transcribed. After the first strand cDNA synthesis serial dilutions (one to five) were made to correspond cDNA transcribed from 500, 100, 20, 4, 0.8, and 0.16 ng of totRNA.

Primers for the ERBB2 and TATA box-binding protein (TBP) genes were designed with the assistance of the Primer3 program (available at http://www-genome.wi.mit.edu/cgi-bin/primer3.www.cgi). To avoid amplification of any genomic DNA, the forward and reverse primers for each gene were chosen from different exons. The sizes of PCR products were designed to be under 400 bp to optimize the RT-PCR measurements. Primer and probe sequences for the genes are given in Table 1 ▶ .

The PCR reactions were performed in the LightCycler apparatus 30 using the LC-FastStart DNA Hybridization Probes Kit (Roche Diagnostics, Mannheim, Germany). Thermocycling for each reaction was done in a final volume of 20 μl containing 2 μl of cDNA sample (or standard), 4 mmol/L of MgCl₂, 0.5 μmol/L of each primer, 0.2 μmol/L of fluorescein, and 0.4 μmol/L of LC Red640-labeled probes as well as 1× ready-to-use reaction mix including Taq DNA polymerase, reaction buffer, and dNTP mix. After 10 minutes of initial denaturation at 95°C, the cycling conditions of 50 cycles consisted of denaturation at 95°C for 3 seconds, annealing at 65°C (for ERBB2) or 58°C (for TBP), and elongation at 72°C for 7 seconds. The LightCycler apparatus measured the fluorescence of each sample in every cycle at the end of the annealing step. After proportional background adjustment, the fit point method was used to determine the cycle in which the log-linear signal was distinguished from the background, and that cycle number was used as the crossing-point value. The software produced the standard curve by measuring the crossing point of each standard and plotting them against the logarithmic values of concentrations. The expression level of ERBB2 was normalized by the housekeeping gene TBP. TBP was chosen for the reference gene, because there are no known retropseudogenes for it and the expression of TBP is lower than in many commonly used abundantly expressed reference genes. 31 After the PCR, every sample was also run in an agarose gel electrophoresis to ensure that a right size product was amplified in the reaction.

Statistical Analyses

The association of the gene copy number and tumor type with the expression level was calculated with nonparametric Kruskal-Wallis test with Dunn’s multiple comparisons test as well as with the Mann-Whitney U test.

ERBB2 Gene Amplification by CISH

Figure 1 ▶ shows an example of high-level amplification in a breast carcinoma as well as a prostate tumor without copy number alteration. None of the 54 untreated primary prostate cancers showed high-level amplification of ERBB2 oncogene (Table 2) ▶ . Fourteen (26%) cases showed an aneuploid gene copy number (three to five copies per cell). Eight (36%) of the untreated lymph node metastases were aneuploid, and rest of the cases (n = 14) showed two copies of the gene. Of the 50 hormone-refractory tumors 17 (34%) were aneuploid. One hormone-refractory tumor (2%) showed six to eight copies of ERBB2 gene (per cell), which is considered as a borderline amplification. The remaining 32 samples (64%) showed only two copies of the gene. Eight of the 30 (27%) breast cancer control specimens showed high-level amplification of ERBB2.

Expression of ERBB2 Protein by Immunohistochemistry

None of the androgen-dependent primary (n = 54) prostate tumors, lymph-node metastases (n = 20), or locally recurrent hormone-refractory tumors (n = 50) showed clearly positive immunostaining (with 2+ or 3+ intensity) (Table 2) ▶ . In contrast, all eight breast carcinomas containing ERBB2 amplification showed typical strong membranous staining indicating overexpression of the gene (Figure 1) ▶ .

Expression of ERBB2 Gene by RT-PCR

Figure 2 ▶ shows a standard curve of the real-time RT-PCR measurement of ERBB2 mRNA. The dynamic range was wide, corresponding to cDNA transcribed from 0.16 to 500 ng of total RNA and linear relationship between fractional cycle and fluorescent threshold was strong (r 2 ∼ 1). In addition to TBP, the expression of β-actin was measured and used alternatively for normalization in most of the samples (data not shown). The results were similar with both control genes. Because of the potential problems with the β-actin retropseudogenes, TBP was chosen for normalization in the whole material.

The breast cancer cell lines with ERBB2 amplification (BT-474, UACC-893, HCC-1419, SK-Br-3, MDA-361), 32-34 showed almost 20× higher expression of the gene (P = 0.0009) than cell lines without amplification (Figure 3) ▶ . ZR75-1 with only a gain of ERBB2 expressed approximately five times more mRNA of the gene than the other cell lines without high-level amplification, whereas MDA-361 with the gene amplification expressed only 2.5 times more of the gene than cell lines without the amplification. Of the cell lines with ERBB2 amplification, the level of amplification is lowest in the MDA-361. 32 Of the three prostate cancer cell lines (of which none contains ERBB2 amplification), only LNCaP expressed the gene in the range of ZR75-1 and MDA-361.

There were no significant differences in the expression levels of ERBB2 between the benign prostate hyperplasia and prostate cancers, either androgen-dependent or refractory, or the breast tumors without ERBB2 gene amplification (Figure 4) ▶ . The level of expression was below the lowest standard (0.16) in 33% of the benign prostate hyperplasia, 19% of the untreated and 25% of the hormone-refractory prostate carcinomas as well as in 32% of the untreated breast carcinomas. The median value of ERBB2 expression in the breast cancer tumors with the ERBB2 gene amplification was ∼20× higher than in the breast cancers without the amplification or prostate tumors (P < 0.0001).