Context.-The Topoisomerase IIα (TOP2A) protein is the target of the anthracycline class of chemotherapeutic agents. TOP2A is frequently coamplified with c-er6-B2 and consequently might be a prognostic and/or predictive factor for breast cancer patients when anthracycline-based chemotherapy is a consideration. A total of 20% to 35% of breast carcinomas show amplification of the erb-B2 gene, some of which also have coamplification of the TOP2A gene. Investigation of the prognostic or predictive significance of these gene amplifications requires a reliable and sensitive method for the measurement of gene copy number in clinical tumor samples.
Objective.-To assess 2 different assay methods that might allow accurate, reproducible, quantitative, and highthroughput estimation of gene copy number in fresh, frozen, or paraffin-embedded breast cancer specimens.
Design.-We developed an assay and analyzed the gene copy numbers of the erb-B2 and TOP2A genes in 8 breast cancer cell lines, 6 fresh frozen samples, and 38 paraffinembedded breast tumor specimens by a novel real-time polymerase chain reaction (PCR) assay using hybridization probes. The results were compared with standard fluorescence in situ hybridization.
Results.-We discovered a 100% concordance between assessment of gene copy number of erb-B2 and TOP2A between quantitative PCR and fluorescence in situ hybridization (FISH). Quantitative PCR also had the additional feature of uncovering an erb-B2 gene polymorphism. Finally, we observed that TOP2A amplification only occurred in conjunction with erb-B2 amplification in our paraffinembedded cases of invasive breast carcinoma and that this event was present in 5 (42%) of 12 erb-B2 amplified cases.
Conclusions.-We conclude that the potentially automatic, real-time PCR analysis using hybridization probes is an efficient method to perform copy number analysis, with results that appear identical to the FISH technique and with the benefit of identifying HER-2 polymorphisms.
(Arch Pathol Lab Med. 2005;129:39-46)
Genetic alteration in gene copy number by amplification or deletion is a common mechanism that leads to deregulation of gene expression and finally to neoplastic transformation. Gene amplification plays an important role in the initiation and progression of many rumors.1^3 Erb-B2 (also referred to as HER-2 or neu) is an orphaned receptor tyrosine kinase and a member of the epidermal growth factor receptor family.4 Erb-B2 is highly expressed in many cancers and in human breast cancer appears dysregulated by increased expression.5 This increased expression usually is attributed to amplification of the erb-B2 (HER-2/neu) gene. The erb-B2 oncogene is the most frequently amplified oncogene in breast cancer. It is found to be amplified in 25% to 30% of primary breast cancers,3,6,7 and evidence suggests that this marker is an important adverse prognostic indicator and predictor of the clinical outcome of lymph node-positive patients with breast cancer.8-14 These patients tend to display highly aggressive disease with early distant relapse and progression to death, particularly when treated with classic cyclophosphamide, methotrexate, and fluorouracil-based chemotherapy.15,16 This has led to the suggestion that women with node-positive disease and Erb-B2 overexpressing cancers should be treated with the more active anthracycline-based combination therapies, such as cyclophosphamide, doxorubicin, and fluorouracil and cyclophosphamide, epirubicin, and fluorouracil.17 Data are still controversial as to whether these more toxic therapies are any more effective in Erb-B2-positive cancers than traditional cyclophosphamide, methotrexate, and fluorouracil-based treatments. For example, Erb-B2 expression has been noted as both a positive and negative predictor of the response to anthracycline-based chemotherapy,17-20 and its routine use as a predictor of response with these agents is still debated.21,22 Erb-B2 has also been reported as a positive predictor of response to paclitaxel therapy.23 Because many cancers that do not express Erb-B2 may still respond to these treatments, overexpression of this molecule is not an unequivocal predictor. Data also indicate that breast cancers that overexpress Erb-B2 in conjunction with estrogen receptor may have resistance to antihormonal agents such as tamoxifen.14,24
The approval of the humanized antibody trastuzumab (Herceptin), which appears to be only effective in cancer that overexpresses Erb-B2, has resulted in a sharply increased clinical demand for Erb-B2 measurement in clinical samples. Reanalysis of initial, pivotal, clinical trial data, which indicated that cases in which the gene is amplified may respond better than cases where there is protein overexpression but no gene amplification, has added speculation that gene copy number assessment is a better predictor of trastuzumab response than immunohistochemical assessment of protein expression level.25 Currently, fluorescence in situ hybridization (FISH) is the most common assay used for c-Erb-B2 copy measurement, but its cost, potential artifacts, and the necessity for visual examination may interfere with its routine use. One potential confounder that affects the predictive value of Erb-B2 overexpression in the consideration of anthracycline-based chemotherapy is the topoisomerase IIα (TOP2A) gene. This gene, located near the erb-B2 locus, produces a protein involved in DNA replication that is the actual target of the anthracyclines.6 Exposure of cells to DNA-intercalating drugs, such as anthracyclines, anthraquinones, ellipticines, and acridines, results in stabilization of the DNA-topoisomerase complexes and double-stranded DNA breaks, resulting in triggering of apoptosis. The TOP2A gene is frequently coamplified with the erb-B2 gene26 however, the events may be independent, resulting in tumors with erb-B2 amplification and no TOP2A amplification and vice versa.27,28 In vitro evidence suggests that tumors that overexpress TOP2A will have increased sensitivity to anthracycline-based chemotherapeutic regimens29,30; however, this is still controversial.11 This has led to an interesting question: why is erb-B2 (appearing to be an adverse marker) frequently coamplified with TOP2A (which should be a beneficial marker)? Could the TOP2A erb-B2 coamplified tumors actually have a better response to anthracycline therapy than tumors that show only erbB2 amplification? Furthermore, does the method of measuring TOP2A dysregulation affect the predictive value of the measurement, as it seems to do with erb-B2?32,33 Currently, erb-B2 expression and/or amplification is commonly detected by immunohistochemical staining methods or by molecular techniques such as Southern blotting, FISH, and/or conventional polymerase chain reaction (PCR). FISH is the most commonly used detection technique, because it is found to be more sensitive and specific compared with immunostaining.14 35 Although immunohistochemical staining is a quick detection method, there is frequent variability in the results, which can be attributed to the source and type of antibody used, the methods of staining, and the methods used to interpret the staining result.14 Southern blotting is time-consuming and requires a large quantity of tissue for analysis, whereas conventional PCR has several limitations, most common of which are contamination problems, false-positive or false-negative results, and inconsistency. Recently, reports of analysis of HER-2 or TOP2A using more quantitative PCR approaches have been published.36-39 Still, more traditional, nonquantitative approaches for assessment of these genes are being used when a PCR technique is chosen.40
We present herein our observations on a rapid, efficient, and consistent method for analyzing copy number of both HER-2 and TOP2A genes by quantitative real-time PCR using hybridization probes (HybProbe, Roche Diagnostics, Basel, Switzerland) compared with standard FISH methods.
MATERIALS AND METHODS
Cell Lines and Tumor Specimens
Breast cancer cell lines and archived breast cancer specimens (fresh frozen and paraffin embedded) were used for the present study. Eight breast cell lines (HTB19, HTB22, HTB26, HTB30, HTB126, HTB129, HTB132, and HTB133 obtained from American Type Culture Collection) were cultured and grown under recommended conditions. Six fresh frozen breast cancer samples were obtained from the Calgary Laboratory Services Research Tissue Resource, and 24 archival, formalin-fixed, paraffin-embedded breast tumor specimens were obtained from Calgary Laboratory Services and the Department of Pathology, University of Saskatchewan. Specimens were used without microdissection if they showed greater than 80% tumor cell purity by histologie analysis, or 10-µm hemotoxylin-eosin-stained sections were subjected to laser capture microdissection using our PixCell II by accepted protocols (Arcturus Bioscience, Inc, Mountain View, Calif). Genomic DNA was extracted from these cell lines and the tumor specimens following the recommended protocol of the Qiagen DNeasy kit (Qiagen, Hilden, Germany).
Copy Number Standard Preparation
Genomic PCR products for erb-B2 (450 base pairs [bp]) and TOP2A (260 bp) were prepared from control placenta! DNA using primers: 5'-TCCAGTCTAAGCAGAGAGAC-3' and 5'-TCGC GTAGGTGTCCCTTTGA-3' for erb-B2 and 5'-AAGCAGTCACA AGCAAGGTG-3' and 5'-AGACACTATATTCTTGGAGTT-3' for TOP2A The primers were designed using the PrimerS Output program from the Massachusetts Institute of Technology (www. broad.mit.edu/cgi-bin/primer/primer3-www.cgi). The Qiagenpurified PCR products were cloned using a TOPO-TA cloning kit (Invitrogen, Carlsbad, Calif). Positive clones were reconfirmed by digestion with EcoRI and by sequencing using an automated Licor sequencer and its software. Confirmed clones were then grown in large scale for obtaining DNA. Qiagen-purified plasmid DNA with the insert was measured by a UV absorbance, and molecule concentration was calculated for each DNA insert size. Serial dilutions ranging from 10^sup 8^ to 104 molecules were prepared. These were used as copy number standards for quantitative analysis of copy number of the unknown breast cancer samples, using a LightCycler instrument (Roche Diagnostics).
Quantitative Real-Time PCR
Internal primers were designed for crb-B2 to obtain a smaller PCR fragment (205 bp) using primers 5'-TGGGTCACCTTCTC TTGACC-3' and 5'-CAAGTACTCGGGGTTCTCCA-3'. The same primer set as described previously was used for TOP2A, which gave a 260-bp PCR fragment product. Fluorescent probes (HybProbe) were designed to hybridize to the antisense strand of the PCR product of each gene. For erb-B2, the probes used were 5'-TGCCTGCTGCCCGACCTGCT-3'-fluorescein and LC640-5'TGCCACTCTGGAAAGGGCCA-3' and for TOP2A the probes were 5'-AACAAGTTAAATATGCCAC-3'-fluorescein and LC6405'-TTTGGCCAATGGAAGAGTTGGCCT-3'. The probes were selected using the GENSET OLIGOS-Oligo parameter calculation program (www.gensetoligos.com). They were obtained from Idaho Technology (Salt Lake City, Utah).
Measuring the Levels of Amplification
To calculate the fold of gene amplification, a relative copy number value was calculated for each test sample as a ratio of the absolute copy number obtained from the quantitative PCR divided by the mean value of 3 normal samples within the sample set of the same run.
Preparation of Cells for FISH.-Fluorescence in situ hybridization using erb-B2 and TOP2A genomic probes was performed on nuclei prepared from tissue culture cells, fresh frozen samples, and paraffin-embedded breast cancer specimens. Confluent cell line cultures were harvested to obtain interphase nuclei from cells that were predominantly in the Gl phase of the cell cycle. The cells were fixed in 1:3 acetic acid-methanol and dropped on clean microscopic slides. Imprint touch preparations from fresh frozen tissue specimens were made by slightly pressing a semithawed frozen tumor piece onto clean microscopic slides. They were fixed in the acetic acid-methanol fix solution and air dried.41 For paraffin-embedded specimens, nuclei were isolated from laser capture microdissected tumor cells according to previously published protocols.42
Probes for FISH.-12P-labeled erb-B2 and TOP2A genomic PCR fragments (450-bp and 260-bp fragments, respectively, as mentioned herein) were used as probes to screen a Pl artificial chromosome (PAC) library obtained from the United Kingdom Human Genome Resource. Positive PAC clones were then obtained from the same source and were further confirmed by PCR sequencing of a short genomic fragment and by FISH for chromosome localization. Confirmed erb-B2 and TOP2A PAC clones were used as probes for FISH analysis. For ploidy analysis, an a-centromeric plasmid probe for chromosome 17 (pZ17-14) was obtained from Resources for Molecular Cytogenetics in Italy (http://www.biologia.uniba.it/rmc/).
FISH Analysis.-Two-color FISH analysis was performed by our usual methods.41 The probes were labeled with either digoxigenin 11-deoxyuridine triphosphate or fluorescein-12 deoxyuridine 5-triphosphate by a standard nick translation labeling method using a Gibco nick translation labeling kit (Gibco/Invitrogen, Carlsbad, Calif). Briefly, slides were boiled at 80°C in 70% formamide and 2X standard saline citrate (SSC) to denature DNA and then dehydrated through a 70%, 90% and 100% ethanol series. The FISH probes in hybridization solution with Herring sperm and human cot DNA were also denatured at 80°C for 8 minutes. Hybridization was performed overnight at 37°C. After hybridization the slides were washed with 50% formamide in 2x SSC at 42°C for 10 minutes, followed by a wash in 2x SSC at 42°C. Hybridization was detected by Cy3 anti-sheep antibody or antifluorescein antibody. The slides were counterstained with 4,6diamidino-2-phenylindole (40 ng/mL) and mounted in an antifade solution (Molecular Probes, Eugene, Ore). Images for each fluorescence were captured separately using Electronic Photography software (BioDx, Rockville, Md) and a Photometries PXL1400 camera and then overlayed to obtain the final image.
Data were analyzed with SPSS statistical software, version 11.0 for Windows (SPSS Inc, Chicago, Ill). Data association for categorical variables was computed using cross-tabulation analysis and χ^sup 2^ analysis. P
Copy number analysis of erb-B2 and TOP2A was performed in 8 breast cancer cell lines, 6 fresh frozen samples, and 24 paraffin-embedded breast cancer specimens by real-time quantitative PCR method using hybridization probes, a fluorescence resonance energy transfer technique that allows detection of the specific PCR product only. This assay produced an expected exponential growth curve (log phase) of erb-B2 standards, which begins when sufficient product has accumulated and the fluorescence is detectable above background (Figure 1, A). Figure 1, B is a standard curve constructed from the data from Figure 1, A. The slope of the line is -1/log (efficiency). Figure 2 shows the relative copy number of erb-B2 and TOP2A in cell lines (A), fresh frozen tissues (B), and in paraffin embedded tumor tissues (C). A value of 3 or more was considered as amplification. For each PCR run, 5 copy number standards from 10s to 104 copies of the respective target were used. A buffer-only negative control was run to check for any contamination during PCR. The LightCycler melting curve (Figure 3, A) shows the inverse derivative of fluorescence to temperature. The peak represents the temperature at which the hybridization probe melts in the analysis of 8 cell lines for erb-B2. Mutations or polymorphisms in the area of the probe binding appear as a shifted melting peak, since the product melts at a lower temperature. Note the shift in the melting peak for one sample, HTB 30. This PCR product from HTB 30 was sequenced. Sequence analysis showed a C->G polymorphism (Figure 3, b). This is a known polymorphism in Erb-B2 (www. genome.utah.edu/genesnps/). The incidence and significance of this polymorphism, if any, in the general population is not yet known. Of the other samples analyzed for erb-B2, 3 (12%) of the 24 paraffin-embedded tissue'showed the presence of the same polymorphism (data not shown).
Fluorescence in situ hybridization was initially performed in 22 samples: 8 cell lines, 6 fresh frozen tissues, and 8 paraffin-embedded tissues using PAC probes for erb-B2 and TOP2A. To assess ploidy, an a-centromeric plasmid probe for chromosome 17 was used. In the presence of normal ploidy, more than 5 signals per nucleus were assessed as amplification. If ploidy was increased, a gene-centromere ratio of more than 2:1 was assessed as amplification. By these methods, 5 (23%) of 22 cases showed amplification of erb-W., whereas 2 (9%) of 22 showed amplification of TOP2A. The coamplification frequency was 40%. All of these cases validated the results of quantitative PCR. Figure 4 shows examples of ert>-B2 and TOP2A amplification by FISH. Fluorescence in situ hybridization analysis on paraffin-embedded specimens was performed on laser capture microdissected tumor cells from which nuclei were isolated as described earlier.42 Figure 4, A shows localization of erb-E2 and TOP2A on chromosome 17 of normal metaphase chromosomes. Figure 4, B shows normal copy number of FISH signals in a breast cancer sample. Figure 4, C is an example of coamplification of erb-V2 and TOP2A (low level) in the HTB 30 cell line, and Figure 4, D shows amplification of erb-B2 and diploid copy number of TOP2A in a breast cancer tissue sample. Figure 4, E shows erb-B2 amplification and normal centromeric copy number in a breast cancer specimen.
Table 1 shows the results of FISH and quantitative PCR on cell lines, frozen sections, and paraffin-embedded sections, using assays for erb-B2 and TOP2A, respectively. Specimens that did not show amplification of either erbB2 or TOP2A had quantitative PCR ratios of up to 2.47. Specimens that showed amplification by FISH had much higher quantitative PCR ratios (lowest was 4.81 for paraffin specimen 6). We, somewhat arbitrarily, chose a cutoff quantitative PCR ratio of higher than 3.0 for amplification to resolve the nonamplified specimens. The results thus show complete concordance between FISH and quantitative PCR for erb-B2 and TOP2A copy number in the specimens. The initial results show that erb-B2 amplification was detected by quantitative PCR in 5 (23%) of 22 total samples, and TOP2A amplification was detected by quantitative PCR in 2 (9%) of 22. erb-B2 and TOP2A were coamplified in 2 of 5 cases (40% of erb-B2 amplified cases). Table 2 shows the evaluation of 38 paraffin-embedded cases of breast carcinoma by quantitative PCR. In these cases, 7 cases showed erb-B2 amplification (18.4%) versus 5 of TOP2A. All cases of TOP2A amplification were associated with erb-B2 amplification and 5 of 7 (71% of erb-B2 amplified cases) showed coamplification of TOP2A.
DNA amplification is likely the most frequent genetic alteration that causes deregulated protein expression of cerb-E2 and TOP2A. Amplification of the erb-B2 oncogene has recently become an important biomarker for identifying patients who respond to anti-erb-B2 therapy (Herceptin). TOP2A, located in close proximity to the erb-B2 gene at chromosome region 17q12-q21, is often found to be coamplified.26,27 However, variations in frequency have been reported, depending on the patient cohort under investigation, the histologie subset, and the method used.8354344 Techniques such as Southern blotting, PCR, and immunostaining are common methods used to detect gene amplification and overexpression in cancer tissues. However, these techniques are time consuming and have several limitations that lead to inconsistent results. Realtime quantitative PCR, using the LightCycler, is promising, because it is fast, reliable, quantitative, and potentially automatic, circumventing some of the limitations of the other methods.45-47 Because this method is still relatively new to anatomic pathology, its usefulness and potential applications are still emerging. Its ability to provide quantitative analysis of gene copy number and simultaneous detection of mutation and polymorphism with higher efficiency and reliability compared with the conventional PCR methods may lend this method to new diagnostic applications.
In the present study, we developed an assay to perform copy number analysis for both erb-B2 and TOP2A genes using hybridization probes. To assess the accuracy, robustness, and reproducibility of this technique, we evaluated copy number analysis of these potential predictive markers in a series of breast cancer samples, including cell lines, fresh frozen tissues, and paraffin-embedded specimens. We verified quantitative PCR results using conventional FISH analysis. Our results showed 100% concordance between FISH measurement and quantitative PCR in all 22 cases where both techniques were performed. Although gene copy number assessment is now acknowledged as a much more reliable measurement, at least for HER-2, than immunohistochemical evaluation, for interest sake we also evaluated the concordance of our results with immunohistochemical evaluation using the TAB250 (antiHER-2, Zymed Laboratories, Inc, South San Francisco, Calif) and NCL-TOPOIIA (anti-TOP2A, Novocastra Laboratories, Ltd, Newcastle upon Tyne, United Kingdom) antibodies and identified 22% false-positive and 29% falsenegative results and 8% false-positive and 33% false-negative results, respectively (data not shown).
Quantitative PCR using hybridization probes allows a rapid analysis of gene copy number, making it feasible for analysis of routine clinical samples. It is important to first standardize the PCR conditions for maximum efficiency. If different probes for HER-2 or TOP2A are chosen from those presented herein, hybridization efficiency of both the probes should be comparable to ensure simultaneous annealing of the 2 oligomers. It is also important to consider that secondary structure of the primers and the target probes may have a substantial effect on hybridization and thus PCR sensitivity and specificity and should be considered in primer design.48 Fluorescent PCR must be optimized such that annealing of probes and primers is equally efficient. Unlike SYBR Green, which binds and detects any double-stranded DNA in a quantitative PCR run, hybridization probes bind only to the PCR product of interest and produce a fluorescent signal based on fluorescence resonance energy transfer (FRET). Thus, a positive signal is highly specific to the final product.4" We also observed that quantitative PCR assays using hybridization probes show considerably greater efficiency and better reproducibility than intercalating dye analysis (data not shown). Also, as for any other diagnostic technique, method of data analysis and its interpretation are important. Although use of internal copy number standards in each run enables one to potentially obtain the absolute copy number of the final PCR product of the unknown sample, we believe it is appropriate to calculate the level of amplification by the ratio of the absolute value of the unknown sample to the mean of at least 3 normal values within each run. Irrelevant (lymphoid) samples showing 2 to 4 signals by FISH were considered normal and were used as controls in the quantitative PCR assays. All 22 breast samples analyzed by quantitative PCR and FISH showed complete concordance between the 2 methods. Our findings confirm and build on the previous use of quantitative PCR to evaluate breast specimens for erb-B2 and TOP2A copy number by evaluating a different region of the 2 genes and using FRET-based probes, which can also identify mutations or polymorphisms.7
In summary, our present study shows that copy number analysis of the c-erb-K and TOP2A genes using hybridization probes (HybProbe) for qualitative PCR is an accurate method for quick and reliable gene copy number analysis in cancer tissues when compared with the generally accepted "gold standard" FISH method. Although the recent introduction of chromogenic in situ hybridization is very promising,49 the quantitative PCR assay offers the potential for high-throughput automation and can also identify polymorphisms, depending on localization of the FRET probe set. Quantitative PCR also offers the potential for multiplexing of numerous primer sets, even further increasing throughput.
This work was funded by the Canadian Breast Cancer Research Foundation (Alberta/NWT Chapter), which also provided salary support for Dr Murthy. Dr Demetrick gratefully acknowledges the Alberta Research Excellence Envelope, Partners in Health Fund, the Terry Fox New Investigator Fund, the Canadian Foundation for Innovation, and the Alberta Science and Research Authority for equipment funding and the Canadian Institutes of Health Research Clinician-Scientist Program for salary support. Drs Demetrick and Magliocco also thank Calgary Laboratory Services for salary support and specimen access. This work was performed using the resources of Alberta Cancer Board Histology Facility, and Sandy Eyton-Jones is acknowledged for her technical assistance.
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Sabita K. Murthy, PhD; Anthony M. Magliocco, MD; Douglas J. Demetrick, MD, PhD
Accepted for publication September 9, 2004.
From the Departments of Pathology (Drs Murthy, Magliocco, and Demetrick), Oncology (Drs Magliocco and Demetrick), and Medical Biochemistry (Dr Demetrick), The University of Calgary, Calgary Laboratory Services (Drs Magliocco and Demetrick), and Tom Baker Cancer Center (Drs Magliocco and Demetrick), Calgary, Alberta. Dr Murthy is currently with the Division of Medical Genetics, Al Wasl Hospital, Dubai, United Arab Emirates.
The authors have no relevant financial interest in the products or companies described in this article.
Reprints: Douglas J. Demetrick, PhD, MD, FRCPC, FCAP, The University of Calgary, Room 302, HMRB, 3330 Hospital Dr NW, Calgary, Alberta, Canada T2N 1N4 (e-mail: firstname.lastname@example.org).
Copyright College of American Pathologists Jan 2005
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