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Clinical Chemistry 49: 219-229, 2003; 10.1373/49.2.219
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(Clinical Chemistry. 2003;49:219-229.)
© 2003 American Association for Clinical Chemistry, Inc.

HER-2/neu Gene Copy Number Quantified by Real-Time PCR: Comparison of Gene Amplification, Heterozygosity, and Immunohistochemical Status in Breast Cancer Tissue

Melanie Königshoff1,2, Jochen Wilhelm1, Rainer M. Bohle2, Alfred Pingoud1 and Meinhard Hahn1a

1 Institut für Biochemie, FB 08, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.

2 Institut für Pathologie, FB 11, Justus-Liebig-Universität Giessen, Langhansstrasse 10, D-35392 Giessen, Germany.

aAddress correspondence to this author at: Abteilung Molekulare Genetik, H0700, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Fax 49-6221-424639; e-mail m.hahn{at}dkfz.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Amplification of the oncogene HER-2/neu influences breast cancer pathogenesis, and therapy and prognosis may be affected by the degree of amplification. The extent of amplification or protein overexpression typically is analyzed by fluorescence in situ hybridization or immunohistochemistry (IHC), but quantitative PCR techniques have been described that may provide alternatives to these methods.

Methods: We developed a rapid-cycle, real-time PCR assay for quantification of HER-2/neu gene status. We compared results obtained with this assay with short tandem repeat findings by capillary electrophoresis (CE) and with protein overexpression assessments by IHC. Accuracy and linearity were tested on cell lines and with simulation experiments. We analyzed the amplification of HER-2/neu in 51 clinical tissue samples from patients with suspected breast cancer.

Results: The intra- and interrun CVs for HER-2/neu quantification by real-time PCR were 12% and 18%, and the CV for different simulated amplification and deletion experiments was <7%. The results for HER-2/neu gene status in cell lines matched the values reported in literature. We detected HER-2/neu amplification by real-time PCR in 11 samples, all from patients with invasive ductal carcinoma. Allelic imbalances were found by CE analyses in three samples and by protein overexpression in six samples; five of these were also detected by real-time PCR. Comparison of the quantification results with known prognostic indices yielded results similar to those reported in several other published studies.

Conclusions: The assay is suitable for accurate and precise quantification of HER-2/neu copy numbers in tumor tissue samples obtained in routine clinical practice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The HER-2/neu protooncogene encodes a transmembrane glycoprotein with tyrosine kinase activity and structural homology to the human epidermal growth factor receptor (EGFR; ERBB1) (1). HER-2/neu is expressed in many cell systems, triggers a rich network of signaling pathways, and plays an important role in normal growth and development (1)(2). Oncogenic amplification of the HER-2/neu gene has been observed in 20–30% of breast cancer cases and predicts more frequent relapse and shorter survival time (3). More interestingly, the HER-2/neu status affects the response and resistance to therapies (4). Therefore, HER-2/neu gene status is currently considered important for clinical decisions, especially about treatment with humanized monoclonal antibody against HER-2/neu receptor (HerceptinTM) (5)(6). Because the clinical results obtained with Herceptin are promising, there is a need for a sensitive, precise, and reproducible method to screen breast cancer patients for amplification of the HER-2/neu gene.

The most common detection methods for HER-2/neu include measurement of protein overexpression by the immunohistochemical assay HERCEPTest (7) and the detection of gene amplification by fluorescence in situ hybridization (FISH)1 techniques (8), both approved by the US Food and Drug Administration. Recently, PCR-based methods have become important in clinical analyses, e.g., conventional competitive PCR (9) and competitive reverse transcription-PCR (RT-PCR) methods (10) or more advanced, quantitative real-time PCR methods for RNA (11)(12) and DNA (13)(14)(15) analysis. Although quantitative RT-PCR usually reveals only relatively large differences in numbers of transcripts, quantitative assays for gene amplification must be able to discriminate very small concentration differences: the duplication of one allele (i.e., three gene copies instead of two per cell) represents a concentration difference of only 33%. Unfortunately, in real-time PCR methods (kinetic PCR), template/primer combinations may be very sensitive to subtle temperature inhomogeneities that may exist in real-time PCR instruments, reflecting the specific melting behavior of the amplicon and its flanking genomic sequences, which can lead to unpredictable variation of the results by a factor of two or three (16) and make such assays unsuitable for the precise and reliable quantitative analysis of gene copy number.

We developed a rapid-cycle, real-time PCR assay (17) for HER-2/neu, evaluated it with clinical samples, and compared the results obtained with clinical prognostic indices.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patients and samples
Excised breast tissue samples from 51 women (41–86 years of age) with suspected breast cancer were analyzed. Surgery was done between 1999 and 2001. The following procedures were in accordance with the current revision of the Helsinki Declaration. All participants gave informed consent. Diagnoses and routine histologic examinations of frozen sections were performed at the Department of Pathology, Justus-Liebig-Universität Giessen. Immediately after selection of diagnostic tissue samples and dissection of the margins for diagnostic purposes, we processed samples of tumor/tumor-like tissue and tumor-free tissue for immunohistochemical or real-time PCR analyses. For immunohistochemical analyses, a carefully selected part of the tumor tissue was subjected to fixation in neutral-buffered formalin (Rotihistofix; Roth). After routine embedding, we prepared sections for hematoxylin/eosin (H&E) staining and for standard estrogen and progesterone receptor immunohistochemistry (IHC). For real-time PCR analyses, tumor and tumor-free tissue were embedded in Tissue-TekTM (Sakura), subsequently snap-frozen in liquid nitrogen, and stored at -70 °C. This cryo-conserved breast tissue was then sectioned for H&E staining. For genomic DNA preparation and PCR amplification, we used up to fifteen 10-µm sections. Tumor samples were considered suitable for this analysis if they contained >80% invasive tumor cells.

dna isolation
Blood lymphocytes.
Human genomic RNA-free DNA was isolated from lymphocytes of fresh EDTA-treated blood with the QIAamp DNA Blood reagent set (QIAGEN), including a RNase A incubation step, according to the manufacturer’s instructions.

Breast cancer cell lines.
Cell pellets of the human breast cancer cell lines MDA-468 [American Type Culture Collection (ATCC) no. HTB-132], MCF-7 Cl.18 (ATCC no. HTB-22), and SK-BR-3 (ATCC no. HTB-30), shock frozen in liquid nitrogen and stored at -70 °C, were derived from ATCC stocks and kindly supplied by B. Brandt (Münster, Germany). Genomic RNA-free DNA was extracted using the same reagent set as above.

Breast tissue.
On the basis of results for H&E-stained sections, we dissected and pooled homogeneous tumor regions of 15 consecutive sections of frozen tissue under microscopic guidance. Genomic RNA-free DNA was isolated using the QIAamp DNA Tissue reagent set and RNase A according to the QIAGEN protocol.

All DNA samples were eluted in PCR-grade water (Merck). The quality and concentrations of all isolated DNA samples were estimated based on their ultraviolet absorbance spectra between 220 and 320 nm on a Hitachi U-3000 spectrophotometer and the A260 nm for double-stranded DNA: 50 mg/L double-stranded DNA has an A260 nm of 1. Samples were considered for further analysis only when the DNA concentration was at least 2 mg/L and the DNA was of high purity as indicated by the ultraviolet absorbance spectrum.

real-time pcr
Oligonucleotide primers.
The sequences of the primer and probes used in this study are shown in Table 1 . Primers were obtained from MWG (HPSF®-purified), and all hybridization probes were from TIB MOLBIOL. The length of the amplified sequence was 99 bp for HER-2/neu and 90 bp for the reference gene, IGF-1. All oligonucleotide sequences were checked with the program Oligo 5.0 (National Biosciences) for absence of false priming sites, formation of primer-dimers, primer/probe hybrids, and secondary structures.


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  		Table 1. Oligonucleotides used in this study.

DNA amplification.
For amplification and data collection, we used the LightCyclerTM system (Roche Diagnostics). All reactions were carried out in a total volume of 10 µL/capillary. Each reaction mixture contained 0.5 g/L bovine serum albumin; 6 mM MgCl2; 0.5 µM each primer, 0.2 µM each hybridization probe, 0.2 mM each deoxynucleotide triphosphate, and 0.5 U of Taq DNA polymerase in 1x PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl). The concentrations of all reaction components were optimized for a high signal-to-noise ratio (18). All biochemicals, unless otherwise stated, were from Roche Diagnostics.

The DNA extracted from the tissue samples was adjusted to a concentration of 2 ng/µL. For each experiment, we used 2 µL (4 ng) of DNA unless otherwise stated. Before the PCR reaction mixtures were prepared, the genomic DNA samples were completely denatured in a boiling water bath for 10 min (16). For each LightCycler run, comprising 32 capillaries, the complete reaction master mixture was prepared on ice and immediately distributed into the prechilled capillaries. The standard amplification protocol consists of an initial denaturation step at 95 °C for 30 s, followed by 50 amplification cycles at 95 °C for 0 s, 55 °C for 5 s, and 72 °C for 10 s (constant temperature ramp of 20 °C/s). Fluorescence measurements were taken at the end of the annealing phase at 55 °C. During the evaluation phase of the assay, each amplification reaction was checked for the absence of nonspecific PCR products by native polyacrylamide gel electrophoresis.

Data evaluation.
Raw data were analyzed with the LightCycler software, Ver. 3. The fractional cycle number where the amplification signal curve crossed the threshold (CT) was calculated from the fluorescence signal ratio of acceptor/donor fluorophores (channel 2/channel 1), using the previously described threshold method (18)(19)(20). The absolute target copy numbers were determined with use of 1:2 dilution series of genomic DNA as external standard.

For each clinical sample, the amounts of the target gene (HER-2/neu) and the reference gene (IGF-1) were measured in tumor tissue as well as in healthy control tissue. Finally, the relative copy number (Q) of HER-2/neu vs IGF-1 gene was calculated using the following equation:

1
Where qT is the ratio of HER-2/neu vs IGF-1 copy numbers in tumor tissue; qN is the ratio of HER-2/neu vs IGF-1 copy numbers in healthy control tissue; NHER-2/neuT is the absolute copy number of HER-2/neu in tumor tissue; NHER-2/neuN is the absolute copy number of HER-2/neu in healthy control tissue; NIGF-1T is the absolute copy number of IGF-1 in tumor tissue; and NIGF-1N is the absolute copy number of IGF-1 in healthy control tissue.

Statistics.
Sensitivity, reproducibility, and the quantification range were verified by a pairwise randomization test (21). A t-test is not applicable here, because the CT values are not normally distributed. The probability, P, calculated with this test reveals whether the measured differences between the mean values of two data sets are attributable to stochastic effects (P close to 1) or to real concentration differences of the analyzed targets (P close to 0).

To test the linearity of the assay, we carried out simulation experiments of HER-2/neu gene amplification/deletion that were similar in principle to previously described experiments (18). Briefly, HER-2/neu gene amplification is calculated from the ratio of the determined gene copy numbers of HER-2/neu and IGF-1, measured in separate PCR reactions. To simulate HER-2/neu gene amplification in the "tumor" sample, the DNA sample is used in different concentrations for the determination of HER-2/neu (5000, 2500, 500, and 250 copies per PCR) and is compared with a constant concentration of the DNA sample used for the quantification of IGF-1 (always 500 copies per PCR), determined in separate reactions. The ratio of HER-2/neu to IGF-1 for "normal" samples is calculated from two independent reactions containing 500 copies each. All samples were measured in triplicate. The mean values were used for the calculation of the Q values. Each Q value was determined for three different runs.

loss of heterozygosity analysis
Oligonucleotide primers.
The primer sequences for the d(AC)n dinucleotide short tandem repeat (STR) polymorphisms D17S800 and D17S1818 (distance from HER-2/neu gene locus is proximal 0.9 cM for D17S800 and distal 0.5 cM for D17S1818) are shown in Table 1Up . The nonfluorescent primer of each pair was designed with a specific 5'-terminal heptanucleotide tail for reduction of peak splitting (22). All STR primers were synthesized by Applied Biosystems.

DNA amplification.
PCR was performed on a GeneAmp PCR System 2400 (Applied Biosystems) in a total reaction volume of 15 µL, containing 25 ng of genomic DNA, 0.4 µM each primer, 0.2 mM each deoxynucleotide triphosphate, and 0.5 U of Taq DNA polymerase in 1x PCR buffer (see above); all reagents were from Roche Diagnostics. The following cycling conditions were used for both STR markers: initial denaturation step of 5 min at 95 °C, followed by 10 cycles of 15 s at 95 °C, 30 s at 56 °C, and 30 s at 72 °C, and 25 cycles of 15 s at 90 °C, 30 s at 56 °C, and 30 s at 72 °C, with a final extension step for 10 min at 72 °C. Each PCR amplification was monitored by electrophoretic separation on native 15% polyacrylamide gels.

Capillary electrophoresis.
PCR products were diluted 10- to 50-fold in PCR-grade water (Merck), and 1.5 µL of each diluted sample was mixed with 0.5 µL of GeneScan-500-TAMRA fragment size standard (Applied Biosystems) and 12 µL of Template Suppression Reagent (Applied Biosystems). This mixture was denatured at 95 °C for 4 min and snap-cooled on ice. The DNA fragments were separated by capillary electrophoresis (CE) and detected by laser-induced fluorescence on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) as described in detail elsewhere (23).

Analysis of CE data.
The loss of heterozygosity (LOH) analysis was carried out as described previously (24) but with use of the GENESCAN 500 6-carboxytetramethylrhodamine (TAMRA) fragment length standard (50-, 100-, 150-, and 200-nucleotide fragments). The accuracy achievable was assessed by the SDs of the peak-area ratios of 10 independent runs. The peak-area ratios used were calculated from the standard fragment pairs 50/100, 100/150, 150/200, and 100/200.

The reproducibility was tested by the repeated determination of the qLOH value for a DNA sample. Equal amounts of DNA from healthy tissue of a heterozygous individual were amplified for both markers, D17S800 and D17S1818. The amplification products were then analyzed in eight independent CE runs. The qLOH value is the ratio of the q values for the tumor (qtumor) and healthy tissue (qnorm) samples and corresponds here to the factor of HER-2/neu gene amplification. A q value for a sample (tumor or healthy tissue) was calculated by dividing the peak area of one allele by that of the other allele.

ihc
HER-2/neu IHC was performed in neutral-buffered, formalin-fixed, paraffin-embedded tissue from all breast cancer patients. We cut 5-µm sections from the tissue blocks and transferred them to glass slides (ChemMate capillary gap microscope slides, 75 µm; Dako). The paraffin sections were placed in an oven overnight at 37 °C. After deparaffinization in xylene, acetone, and acetone/Tris-buffered saline (100 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.5), each step for 10 min, the sections were washed twice with Tris-buffered saline. Staining was performed with the HERCEPTest reagent set (K 5204; Dako) exactly as specified by the manufacturer. Epitope retrieval was performed at 95 °C in a water bath for 40 min. Graded positive and negative controls from the reagent set were used. Counterstaining was performed with hematoxylin. The slides were dehydrated in graded alcohol and cleaned in xylene before being coverslipped in gelatin.

Invasive breast cancer cells were scored as 0, 1+, 2+, or 3+ according to the HERCEPTest guidelines. Scores of 0 or 1+ were regarded as immunohistochemically negative, whereas scores of 2+ and 3+ were regarded as immunohistochemically positive. Cytoplasmic staining was ignored.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
real-time pcr
Development of primer/probes systems.
The first goal of this study was to develop a robust primer/probes system for the precise and reliable quantification of HER-2/neu in clinical samples by rapid-cycle, real-time PCR. We paid special attention to the melting behavior of the amplicon regarding temperature inhomogeneities (16). We designed a stable primer/probes system within the exon 2/intron 2 sequence of HER-2/neu (GenBank accession no. M12036) using common PCR primer software and selection criteria, based on the data bank entry (18). The standard PCR conditions for this system were optimized for a high signal-to-noise ratio by adjusting Mg2+ and probe concentrations (data not shown). The signal and calibration curves of HER-2/neu amplifications are shown in Fig. 1 , A and C, respectively. Similarly, different genes (HBB, IGF-1, GAPDH, and PBGD) were tested for their suitability as internal controls/reference genes. Among these, IGF-1 and its primer/probes (see Table 1Up ) were best suited (lowest variation in CT values, high signal-to-noise ratio, optimum signal curves, and low variation of calibration curves; see Fig. 1 , B and D).



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  		Figure 1. Amplification profiles and mean calibration curves for HER-2/neu and IGF-1.

(A), amplification profiles for HER-2/neu; (B), amplification profiles for IGF-1. The DNA was serially diluted 1:2 with water from 8000 to ~250 genome-equivalents (3 ng were assumed to be 1000 haploid genome-equivalents). The negative controls without target DNA show no signal increase and are not visible. (C), mean calibration curve of six independent quantification experiments for HER-2/neu (twofold dilutions of human genomic DNA). Calibration curve function, including SDs: y = -3.7 (± 0.33)x + 40.4 (± 1.05). (D), mean calibration curve for IGF-1. Calibration curve function: y = -3.5 (± 0.23)x + 36.7 (± 0.74). The calibration curves, obtained by plotting log copy number vs CT and given in the form y = ax + b, were calculated with the fit points method with arithmetic background correction. Error bars, SD.

Statistical analysis.
The precision of the assay was determined by statistical analysis of the quantification results for HER-2/neu and IGF-1 copy numbers of a DNA sample (4 ng {cong} 1300 copies per reaction) that was measured six times in sixfold replicate runs on the same day and on 3 different days, respectively. The intrarun variation (CV) for HER-2/neu varied between 7.6% and 18% (mean, 12%; mean calculated copy number, 1357). The interrun CV was 11–24% (mean, 18%; mean calculated copy number, 1350). The observed CT values for repetitive measurements of a sample are not necessarily normally distributed; therefore, statistical analyses were done by randomization tests, which do not make any assumptions concerning the distribution pattern (21). The pairwise randomization test performed with nine aliquots of the same DNA sample (each measured four times) revealed a probability of nearly 100% (for both HER-2/neu and IGF-1) that all samples do have the same concentrations.

The linearity was determined in simulation experiments. The mean (± SD) Q values were 9.8 ± 0.5 for 10-fold amplification, 4.9 ± 0.3 for 5-fold amplification, 0.97 ± 0.06 for a balanced sample, and 0.51 ± 0.06 for the deletion of one allele of HER-2/neu.

Determination of cutoff values.
DNA samples from healthy volunteers contained the same copy numbers of the test and reference genes (HER-2/neu and IGF-1). Determination of these copy numbers would ideally produce a q value of 1 (see Eq. 1Up ); therefore, the expected Q value for two different DNA samples from the same individual would also be 1 (these two samples correspond to the DNA from tumor and healthy breast tissue of a patient in clinical studies). We tested this assumption using blood lymphocyte DNA from healthy volunteers. HER-2/neu and IGF-1 copy numbers were quantified in a 4-ng DNA sample. The mean (SD) Q value calculated from six experiments was 1.02 (0.06). Analysis of DNA isolated from six benign tissue samples produced a mean (SD) Q value of 1.0 (0.1). On the basis of the SDs determined for replicate measurements and the results of the statistical analysis, the Q cutoff value was set to 1.3 for amplifications and to 0.7 for deletions, corresponding to 3 SD of the mean for benign samples.

Analysis of breast tumor cell lines.
The HER-2/neu copy number was quantified in tumor cell lines with known but different amplification values for HER-2/neu (25)(26)(27)(28). The following cell lines were used: SK-BR-3 (high amplification), MDA-468 (little or no amplification), and MCF-7 Cl.18 (no amplification or allele deletion). The signal curves of the quantification runs are shown in Fig. 2 . The Q values, calculated using the mean CT values of samples measured in quadruplicate, were 9.0 for SK-BR-3, 0.86 for MDA-468, and 0.61 for MCF-7 Cl.18.



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  		Figure 2. Quantification of HER-2/neu and IGF-1 in three different human tumor cell lines.

In each PCR experiment, 4 ng of genomic DNA of the specific cell line was analyzed in quadruplicate. (A), HER-2/neu-specific amplification curves; (B), IGF-1-specific curves. Blood lymphocyte DNA of a healthy volunteer was used as a control.

Analysis of clinical samples.
We quantified the HER-2/neu copy number in DNA samples from 51 patients with suspected breast cancer. The histopathologic diagnosis was unknown at the time of the HER-2/neu analysis. In 39 of these samples, no amplification was detected (Q value, 1.0 ± 0.12), 1 sample showed a deletion (Q value, 0.51), and 11 samples showed a significant amplification, with Q values varying between 1.4 and 11 (mean, 2.9; median, 2.0). Typical signal curves for a patient with significant HER-2/neu gene amplification are shown in Fig. 3 .



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  		Figure 3. Quantification of HER-2/neu and IGF-1 in tissue samples from a breast cancer patient.

Homogeneous tumor (T) or healthy tissue (N) areas were dissected under microscopic control from a series of consecutive 10-µm cuttings of the breast cancer sample. DNA was extracted from these pooled tissue dissections. In each PCR experiment, 4 ng of genomic DNA from each specific sample was analyzed in quadruplicate. (A), HER-2/neu-specific amplification curves; (B), IGF-1-specific curves. The calculated Q value for this sample, based on the quantification results, is 3.1, corresponding to a threefold HER-2/neu gene amplification in the tumor tissue (i.e., in mean six copies per cell).

Histologically, the tumor/tumor-like tissue samples could be classified as follows: 15 cases were nonneoplastic samples (mastopathy, n = 10; fibrosis, n = 3; fibroadenoma, n = 1; fat necrosis, n = 1), 26 samples were from patients with invasive ductal carcinoma (IDC) of the breast, 7 were from patients with invasive lobular breast cancer, 1 was from a patient with mucinous carcinoma, 1 was from a patient with tubular carcinoma, and 1 was from a patient with IDC relapse. Amplification and deletion of HER-2/neu were detected exclusively within the IDC samples.

loh analysis
In addition, the STR markers D17S800 and D17S1818, located close to the HER-2/neu gene locus, were quantitatively analyzed in all 51 clinical samples for allelic imbalances (LOH) by CE on the ABI PRISM 310 Genetic Analyzer.

Repetitive determinations of peak-area ratios for the calibrators and qLOH values for samples were performed as described in Materials and Methods to assess a cutoff value for the LOH analyses. The SDs of the peak-area ratios of the fragment length calibrator were <3.5% (n = 1920) in all cases. The division of two ratios (e.g., ratios of fragment pairs 50/100 and 100/150), as done for the calculation of the qLOH values, produced a SD <7%. On the basis of this SD, allelic imbalances are significant when the qLOH is outside a range of 0.79–1.21 (1.0 ± 3 SD). The SDs of the qLOH values obtained from analyses of the sample PCR products for the two STR markers were <4% (n = 40) for both D17S800 and D17S1818.

Of the 51 clinical samples, 49 were informative for at least one STR marker, i.e., the marker showed a heterozygous allelotype. The two noninformative samples were from IDC patients. Forty samples were informative for both markers, 3 for only D17S800, and 6 for only D17S1818. Allelic imbalances were detected in three clinical samples, which were all from IDC patients (an example is given in Fig. 4 ). Two of these cases showed amplification for both analyzed STRs; the third case showed amplification only for D17S800, whereas D17S1818 was not informative in this case.



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  		Figure 4. LOH analysis of the STR marker D17S800.

The DNA samples were amplified with fluorophore dye-labeled primers specific for the STR D17S800. The PCR fragments were separated and detected by CE with detection by laser-induced fluorescence. The detail of the electropherogram shows the relevant STR-specific fragments for healthy (Normal) and tumor DNA from the same patient as in Fig. 3Up .

ihc
Six samples from IDC patients were positive for HER-2/neu overexpression by HERCEPTest. None of the other 20 samples from IDC patients were immunohistologically positive, nor were the samples from the patients with invasive lobular breast cancer or patients with other cancer subtypes.

comparison between methods and clinical prognostic indices
All three LOH-positive cases showed overexpression of HER-2/neu protein as detected by IHC and could also be identified by quantitative real-time PCR. In five of six immunohistochemically positive samples, HER-2/neu gene amplification was detected by real-time PCR. In only one case did the real-time PCR assay not identify an amplification although the sample was immunohistochemically positive. Six additional samples that have not been identified as such by other means were identified by quantitative real-time PCR as having significant HER-2/neu amplification.

The real-time PCR results were compared with the results of LOH analysis and IHC. In addition, a correlation with several important clinicopathologic variables was investigated. For this purpose, a homogeneous group containing positive as well as negative samples is advantageous. Because all samples with detected HER-2/neu gene amplification, STR marker imbalances, or HER-2/neu protein overexpression were within the IDC entity, this group of 26 samples was used for the following comparisons. The results are shown in Table 2 . Both methods, LOH analysis and IHC, correlated very well with the real-time PCR method. The steroid receptor status (estrogen and progesterone receptors) was the clinical variable that showed the strongest correlation with HER-2/neu gene amplification. No correlation was found between HER-2/neu amplification and tumor size or node status.


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  		Table 2. Correlation of HER-2/neu amplification in IDC detected by real-time PCR with clinicopathologic indices and other detection methods.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The pathogenesis of many tumors is influenced by an increased concentration of oncogene products, which in many cases can be detected by IHC. For example, HER-2/neu overexpression in breast cancer is measured with the HERCEPTest (7). One reason for protein overexpression in tumor cells can be oncogene amplification. Such gene amplifications can be detected by FISH, a standard clinical procedure in cancer diagnosis (8). In the future, PCR methods are likely to become more widely used for the same purpose because they are more sensitive, faster, and easier to perform. RT-PCR can be used to determine the transcript concentrations of oncogenes, which show greater variations than gene copy numbers and, therefore, can be more easily detected. Problematic steps are sample stabilization to minimize RNA degradation and the suboptimal reliability of the reverse transcription reaction (29).

Quantitative competitive PCR and RT-PCR are known to be reliable and yield precise results, but rather large amounts of template (e.g., 200 ng of DNA) are needed for the analysis of a sample (30). Both methods need post-PCR analytical steps that are associated with a risk of cross-contamination of samples with PCR products. Real-time PCR-based methods circumvent some of these disadvantages and yield very precise results for both RNA and, particularly, DNA quantification (31). Recently developed real-time PCR techniques demonstrate the increased abundance of HER2/neu transcripts as well as gene amplification in breast cancer samples [e.g., see Refs. (13)(14)(15)], which are correlated (12). In the literature, comparisons with other clinical indices were based on measurements of mRNA concentrations by real-time RT-PCR, although DNA is simpler and more reliable to isolate and quantify than mRNA.

In this study, we compared the HER-2/neu gene amplification detected by quantitative real-time PCR with the results of the HERCEPTest and the findings for other important clinical prognostic indices to demonstrate the suitability of this method for prognosis of breast cancer progression. Additional information about the kind of gene amplifications in the same samples were obtained by LOH analysis of two dinucleotide repeats on either side of the HER-2/neu gene locus, which yielded such information as the size of the amplified genomic region, differential involvement of the alleles, and aneuploidy of chromosome 17.

For the methods used here, good-quality sample material is crucial for exact quantification. The samples should be homogeneous and contain sufficient tumor cells. For real-time PCR, DNA concentrations of as low as 4 ng per 10-µL reaction are sufficient. This amount corresponds to {approx}1330 copies of a single-copy gene such as HER-2/neu or IGF-1. It is a major advantage of this protocol that small amounts of material are sufficient because, in clinical practice, frequently only limited amounts of sample are available (e.g., microdissected samples), which must be used for several different analyses. When we used 4 ng of DNA per PCR reaction, the scattering of results as a result of limited template amounts was negligible (<3% for 1330 template copies vs >30% for 100 template copies; data not shown). The analysis of larger amounts of DNA will not increase the accuracy significantly.

IGF-1 was chosen as reference gene, to our knowledge the first time in the context of HER2/neu gene quantification, because the chromosomal region (12q22) is known to be rarely involved in genomic alterations in breast cancer. Chromosomes 2, 6, and 12 are least frequently numerically altered in breast tumors. Of 1638 published reports of structural alterations, only 4 have found alterations in the IGF-1 promoter region (12q22) (32).

The primer sequences were designed to generate very short amplicons. The minimum length was given by the lengths of the primer and probe sequences. Short amplicons, as are typically used in rapid-cycle PCR (17), are amplified with high and reproducible efficiency in short extension times (<10 s). The complete PCR performed on the LightCycler System, including data evaluation, requires only 20–30 min, whereas classic 96-well block cycler-based real-time PCR systems require 2–4 h. The LightCycler sample carousel takes a maximum of 32 samples. Because of the shorter time per experiment, similar sample throughput rates are possible with both the LightCycler and 96-well block cyclers. A major advantage of the LightCycler system is higher flexibility in clinical practice for several independent molecular markers requiring different amplification protocols.

We validated the HER-2/neu assay by quantification of the HER-2/neu gene in three cell lines: The quantification results for all three cell lines agreed well with known data for different detection methods (25)(26)(27)(28). For MDA-468, our calculated value of 0.86 corresponded to the values obtained by Roetger et al. (25), who reported a value of 0.7 (measured by competitive PCR), and by Kraus et al. (27), who reported a value of 1 (detected by FISH). In addition, several recent publications dealing with quantitative real-time PCR reported values for SK-BR-3 that are similar to our findings of 9.0: Lyon and coworkers obtained values between 8.7 and 12 (13) and 7.8 (33), whereas Woods obtained values of 7.5 (real-time PCR) and 8.0 (FISH) (34). The quantification results for the cell line MCF-7 CL.18, which is supposed to be haploid for HER-2/neu (25), showed an apparent deletion. These results demonstrate the achievable accuracy of the assay.

After validation, we quantified the HER-2/neu copy numbers in clinical samples. HER-2/neu amplifications and deletions were found only in tumors that were later characterized as IDCs. The highest proportion of samples with amplification was expected to be found in the group of IDCs (35)(36). The samples classified as benign could also be used as controls with normal HER-2/neu gene dose to validate the accuracy and the determined Q cutoff value. The Q value for these 15 benign samples ranged between 0.78 and 1.3 (mean ± SD, 1.0 ± 0.1), demonstrating that this assay is at least two times more precise than others reported (12)(15). It is remarkable that the SDs for the DNA from blood lymphocytes were significantly smaller than those for DNA from healthy tissue samples of patients. This phenomenon was also observed by others [e.g., Ref. (15)]. The reasons for these discrepancies were not investigated. It is likely that the conditions during surgery and DNA isolation from tissue samples might influence the DNA quality and the quantification results (e.g., spreading of data for noncancerous clinical samples).

Imbalances of HER-2/neu alleles in tumor samples were determined by LOH analysis with the two most proximate known STR markers to the HER-2/neu gene locus. Most of the samples with HER-2/neu amplification detected by real-time PCR did not show allele imbalances as indicated by the STR marker analysis. There are two possible reasons for this observation: either the cells are polyploid or amplification is limited to more or less the gene locus only. Because polyploidy is unlikely in breast cancer, as reviewed by Revillion et al. (37), the LOH analysis performed in this study supports the hypothesis that the amplified region, including the HER-2/neu locus, is smaller than 1.4 cM. Overall, only a few of the informative cases with HER-2/neu amplification were positive by LOH analysis. These samples might have longer amplified regions including the STR markers or, more likely with respect to the results discussed above, they can be aneuploid for chromosome 17. One sample showed a positive but significantly smaller qLOH value than expected from the Q value (qLOH = 1.5; Q= 11), i.e., the allelic imbalances are smaller than the total amount of gene amplification. In this case, the genetic situation is not clear and might be a complex combination of aneuploidy and gene amplification.

Investigation of protein overexpression by IHC showed a smaller number of positive cases than were detected by real-time PCR. This could mean that the amplified region does not include the promoter, or it could be that amplification of HER-2/neu is an early event and, therefore, might precede in some cases detectable overexpression of the protein (30). Only one IHC-positive sample was not identified by real-time PCR. Such cases have been reported and are probably caused by transcriptional or posttranslational activation (8). All cases with allelic imbalances showed protein overexpression, and all these cases were detected by real-time PCR.

Comparisons of our data with important clinical prognostic indices are shown in Table 2Up . The relationship of HER-2/neu amplification/protein overexpression and prognostic indices is often described in the literature, where IHC or FISH are mostly used as detection methods (37). Despite the discrepancies observed between different studies, we noted several associations, which were mostly similar to our results (see Table 2Up ). A correlation to a higher grade was not observed because only one sample showed a low grade, but in this sample all three methods used detected a HER-2/neu amplification/protein overexpression.

Overall, the number of patients in this study is too small to draw final conclusions about correlations.

In conclusion, this study dealing with HER-2/neu gene copy number detection by quantitative real-time PCR is the first to comprise both a comparison of different detection methods and a correlation to known clinical prognostic indices. We showed that the PCR method used, like IHC and FISH, is suitable for quantification of gene amplification regarding prognosis of breast cancer. IGF-1 was found to be well suited as a new reference gene for molecular breast cancer analysis. Our assay does not require post-PCR processing and RNA handling and allows for rapid (20–30 min/experiment), accurate, and precise quantification of HER-2/neu copy number in clinical tissue samples that do not need to be specially prepared for the analysis. It therefore is best suited for use in future clinical studies with more patients.


   Acknowledgments
 
This work was supported by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg Molekulare Biologie und Pharmakologie), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Fö-Kz 03 11 412), and the Fonds der Chemischen Industrie. We thank PD Dr. Burkhard Brandt (Westfälische Wilhelms-Universität Münster) for the cell lines, Drs. Uwe Kullmer (Asklepios Klinik Lich) and Marie-Luise Wagener (St.-Josefs-Krankenhaus Giessen) for clinical samples, Drs. Ludger Fink and Annerose Anders (Justus-Liebig-Universität Giessen) for fruitful discussions, and Prof. Dr. Peter Lichter (DKFZ Heidelberg) for generous support.


   Footnotes
 
1 Nonstandard abbreviations: FISH, fluorescence in situ hybridization; RT-PCR, reverse transcription-PCR; H&E, hematoxylin/eosin; IHC, immunohistochemistry; ATCC, American Type Culture Collection; CT, threshold cycle; STR, short tandem repeat; CE, capillary electrophoresis; LOH, loss of heterozygosity; and IDC, invasive ductal carcinoma.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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