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Amplification of HER-2/neu and Topoisomerase II in Primary and Metastatic Breast Cancer¹

Laboratory of Cancer Genetics, Institute of Medical Technology, Tampere University and Tampere University Hospital, FIN-33014 Tampere, Finland

	ABSTRACT

Top ABSTRACT Introduction Patients and Methods Results Discussion REFERENCES

 
Amplification of the HER-2/neu oncogene and amplification of the topoisomerase II gene are important determinators of the response to chemotherapy in advanced breast cancer. Assays of these genes are usually carried out using primary tumor samples, because biopsies from metastatic lesions are not usually taken. We studied the concordance of Her-2/neu and topoisomerase II amplification in primary breast tumors and their metastases by immunostaining and DNA in situ hybridization. HER-2/neu amplification, present in 28% of the primary tumors (n = 46), was always associated with amplification in its metastasis. Conversely, no metastases with HER-2/neu amplification were seen without amplification in the primary tumor. Topoisomerase II gene copy status (amplification/deletion/unaltered) remained generally unchanged in HER-2/neu-positive tumors, but in three cases, the predominant cell population in metastatic tissue was present only as a subpopulation in the primary tumor. We conclude that amplification of HER-2/neu measured in primary tumor reflects the status of metastases. Minor discrepancies between primary and metastatic tumors in topoisomerase II gene copy status may reflect evolvement of the amplicon structure in successive cell divisions.

	Introduction

Top ABSTRACT Introduction Patients and Methods Results Discussion REFERENCES

 
A number of studies have shown that the HER-2/neu oncogene undergoes gene amplification in 15–30% of cases of breast cancer. This amplification is associated with adverse clinicopathological features and shortened overall survival (reviewed in Ref. 1 ). Amplification of the HER-2/neu oncogene has recently become an important biomarker for identifying patients who respond to anti-HER2 therapy (trastuzumab) and also patients who are more likely to benefit from topo³ II inhibitors (doxorubicin and epirubicin; reviewed in Ref. 2 ). The latter association may be explained by concomitant amplification of the topo II gene, which is located adjacently to HER-2/neu at chromosome region 17q12–q21 (3 , 4) . Studies carried out in vitro indicate that topo II gene amplification leads to overexpression of topo II protein, which in turn makes the cells more sensitive to topo II inhibitors by providing an increased concentration of the drug target (4 , 5) . Our preliminary studies indicate that amplification of topo II in primary tumors is a strong predictor of clinical response and survival in metastatic disease (6) .

Assays for HER-2/neu and topo II gene amplification are usually carried out using primary tumor samples, although therapy targets metastases, which usually manifest years after the primary operation. Predicting the response to therapy in metastatic disease on the basis of biomarkers analyzed in the primary tumor is based on the assumption that the status of HER-2/neu and topo II genes does not change during metastatic progression. However, a survey of the literature does not provide convincing evidence for or against this hypothesis. There are only two published reports where HER-2/neu overexpression has been studied in primary and metastatic breast cancer (7 , 8) . No data are available on the persistence of amplification of topo II. Here we report results from a systematic study of HER-2 and topo II amplification in primary breast cancers and their metastatic tumors that developed later during follow up.

	Patients and Methods

Top ABSTRACT Introduction Patients and Methods Results Discussion REFERENCES

 
Patients and Tumors.
Forty-six breast cancer patients with formalin-fixed, paraffin-embedded tumor samples available from untreated primary tumors and later clinically manifested metastatic tumor deposits were identified in our breast cancer specimen database. The time period between surgical removal of the primary tumor and operation or biopsy of the metastatic tumor lesion ranged from 1 month to 19 years. Two separate metastatic lesions were available for this study from two patients. Cases with bilateral tumors and those with the possibility that the metastatic tumor could have originated from another primary cancer were excluded. The site of metastasis was local or regional in 33 cases, and 12 were hematogeneously-spread distant metastases (no data are available for three metastases). Most patients were diagnosed in the late 1980s and early 1990s, when adjuvant cytotoxic chemotherapy was not in widespread use in Finland.

In situ Hybridization.
A digoxigenin-labeled genomic probe for HER-2/neu was obtained from Zymed, Inc. (South San Francisco, CA) and used in CISH (9) , using reagent kits from the same manufacturer. In brief, the sections were deparaffinized and incubated in pretreatment buffer in a temperature-controlled microwave oven (92°C for 15 min). Enzymatic digestion was carried out with pepsin (10–30 min at room temperature). The slides were washed with PBS and dehydrated in graded dilutions of ethanol. The HER-2/neu probe (5–10 µl/slide) was applied to the slides under coverslips. The slides were co-denatured on a hot plate (94°C for 3 min), followed by overnight hybridization at 37°C. The slides were stringency washed (0.5x SSC for 5 min), and the hybridized probe was detected using the CISH detection reagents (anti-digoxigenin-FITC, anti-FITC-peroxidase, and diaminobenzidine as chromogen), according to the manufacturer’s instructions. The sections were counterstained with hematoxylin and mounted. The slides were analyzed under an ordinary transmitted light microscope and x40 objective. Copy numbers of six copies/cell or more in at least 50% of cancer cells or the presence of large copy number clusters were considered indicative of HER-2/neu amplification.

Two-color FISH of topo II was carried out with a previously characterized PAC probe (3) and a chromosome 17 pericentromeric probe (p17H8), which was used as a reference to determine the overall copy number of chromosome 17. The pericentromeric probe for chromosome 17 was labeled with fluorescein-5-dUTP and the topo II probe with digoxigenin-11-dUTP, by standard nick-translation. Pretreatment and denaturation of tissue sections were carried out similarly as with HER-2/neu CISH (9) . A mixture of topo II and 17 centromere probes (30 and 10 ng of probe DNA, respectively) was diluted in 10 µl of hybridization buffer (2x SSC, 50% formamide, 10% dextran sulfate, and 10 µg of placental DNA), which was then applied to the slides under coverslips. After hybridization, the slides were stringency washed with 0.5x SSC (5 min at 75°C), followed by three washes in PBS, 0.2% Tween 20. The topo II probe was detected with anti-digoxigenin rhodamine (diluted 1:300; Roche-Boehringer, Mannheim, Germany). Nuclei were counterstained with 0.1 µM 4,6-diamidino-2-phenylindole in an antifade solution (Vectashield; Vector Laboratories, Burlingame, CA).

FISH was evaluated using an Olympus BX50 epifluorescence microscope equipped as described previously (3) . Signals from at least 50–500 nonoverlapping nuclei with intact morphology were evaluated to determine the mean number of signals/cell for each probe. Both absolute copy numbers and the relative copy number ratio (ratio between mean number of topo II signals and the mean number of chromosome 17 centromere signals) were determined. Control hybridizations to nonmalignant breast tissue and to normal peripheral blood lymphocytes were carried out to ascertain equal hybridization efficiencies of topo II and the chromosome 17 centromere. Amplification of topo II was defined as a relative copy number ratio of 1.5 or more and deletion as a ratio of 0.8 or less. All FISH analyses were carried out in a blinded fashion, i.e., unaware of the copy number status of the primary or metastatic counterpart.

In three cases where discordance in topo II amplification was found between primary tumor and metastasis with FISH, the analysis was repeated using CISH (as with HER-2/neu) to ensure that evaluation of in situ hybridization signals was carried out from histologically representative regions.

Immunohistochemistry.
Deparaffinized tissue sections were heat pretreated in sodium citrate buffer (pH 7.3, 20 min at 92°C in a temperature-controlled microwave oven) and immunostained with a labeled monoclonal antibody to the intracellular domain of HER-2/neu protein (dilution clone CB-11; Novocastra Laboratories, Newcastle, United Kingdom). A standard avidin-biotin-peroxidase complex (ABC) technique was used for visualization, with diaminobenzidine as the chromogen (Histostain Plus-kit; Zymed Laboratories, San Francisco, CA). Only an intense cell membrane immunoreaction present in >50% of cancer cells (3+ positivity) was considered as overexpression of p185^HER2 protein.

	Results

Top ABSTRACT Introduction Patients and Methods Results Discussion REFERENCES

 
Determination of HER-2 Status in Primary and Metastatic Tumors.
Primary tumors and metastases (n = 94) from the 46 patients were first screened by means of HER-2/neu immunohistochemistry and CISH (Fig. 1) . The results of these two methods were in full concordance and allowed us to identify 27 samples with gene amplification (by CISH) and high level (3+) overexpression (Table 1) . Moderate overexpression (2+) was found in one primary tumor, which showed no gene amplification in CISH. Amplification of HER-2/neu was found in the primary tumors of 13 patients (13 of 46; 28.2%). Metastatic lesions from these patients (n = 14) were all found to have gene amplification and p185^HER2 overexpression. No HER-2/neu amplification was found in any of the metastases of the 33 patients whose primary tumors did not show amplification of HER-2/neu (Table 2) .

View larger version (125K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. CISH of HER-2/neu oncogene in a pair of primary breast tumors and their metastases. Both primary (A) and metastatic (B) tumors show typical HER-2/neu amplifications appearing as a peroxidase-positive clusters of gene copies. Counterstained with hematoxylin.

Identification of topo II Gene Aberrations in Primary and Metastatic Tumors.

topo II

topo II

topo II

topo II

topo II

topo II

topo II

topo II

topo II

View this table: [in this window] [in a new window]   Table 3 Topo II copy number aberrations in 13 pairs of primary and metastatic HER-2-positive breast cancers

topo II

topo II

topo II

	Discussion

Top ABSTRACT Introduction Patients and Methods Results Discussion REFERENCES

 
Protein products of HER-2/neu and topo II genes are therapeutic targets for trastuzumab (an anti-HER-2/neu antibody) and topo II inhibitors (mainly doxorubicin and epirubicin), which are both widely used in the treatment of metastatic breast cancer. A prerequisite for successful therapy with trastuzumab is amplification and overexpression of the HER-2/neu oncogene in the tumor cells (10) . According to our preliminary data (6) , patients with topo II amplification obtain the best benefit from epirubicin-based chemotherapy. However, therapeutic decision-making in metastatic breast cancer is based on the presence of biomarkers assessed in primary tumor samples, because metastatic lesions are rarely surgically removed or biopsied. Thus, it is assumed that biomarker status does not change during metastatic progression, which usually takes several years. To the best of our knowledge, there are only two small studies of HER-2/neu amplification or overexpression in primary tumors that later developed metastatic breast cancer (7 , 8) . In a study of 23 patients, Iglehart et al. (7) showed HER-2 status to be equivalent in primary tumors and in simultaneously removed axillary lymph node metastases. Nesland et al. (8) demonstrated some differences in HER-2 immunostaining between primary tumors and metastases from multiple sites. A very high incidence of HER-2 protein overexpression (60%) has been found in occult micrometastatic cells in the bone marrow (11) . However, in that work, an immunohistochemical technique was used whose analytical validity was not carefully validated in their sample material (11) . Furthermore, it remains unclear whether the occult micrometastatic cells represented the stem cells for overt clinical metastases.

In the present study, we were able to show that HER-2/neu amplification status always remained the same between primary tumor and its metastasis, despite the fact that in some cases the metastases appeared >10 years after removal of the primary tumor. Considering the stepwise accumulation of genetic changes as a model for breast cancer pathogenesis, our results indicate that HER-2/neu amplification must occur very early in the genetic cascade, at least before dissemination of the cancerous cells into the lymphatic or blood circulation. When HER-2/neu-negative cells disseminate and metastasize, they are perhaps fully malignant and unable to acquire amplification for mechanistic reasons, or HER-2/neu overexpression does not provide any significant selective growth advantage at the metastatic site. Our results indicating that HER-2/neu amplification is an early genetic event are very well in line with data on HER-2/neu amplification in preinvasive lesions (ductal carcinoma in situ; reviewed in Ref. 1 ). HER-2/neu amplification has been shown recently to occur before aneuploidization of the tumor genome, which further indicates the earlyness of HER-2/neu amplification in the pathogenesis of breast cancer (12) . However, the CISH method used in our study is not suitable for providing information as to whether the number of gene copies in amplification-containing tumors increases (or decreases) during metastatic progression. HER-2 amplification usually shows up as a tight amplification cluster consisting of numerous closely packed dots that are impossible to distinguish and count (see the examples in Fig. 1 ). The other possibility for genetic evolution is that the proportion of cancer cells with amplification increases (or decreases) during metastatic progression. In the case of amplified HER-2, usually all or at least the vast majority of cells already show amplification in the primary tumor. Hence, it was not possible to see further enrichment of HER-2-positive cells in the metastases.

The identity of HER-2/neu status has a clear clinical diagnostic implication. Our results indicate that HER-2/neu assays carried out on primary tumors reliably predict the HER-2/neu status of the metastases, which are the targets of trastuzumab therapy. Hence, if biopsies of the metastatic sites are otherwise not needed, there is no need to subject patients to biopsies just because of HER-2/neu diagnostics. It is well known that HER-2/neu positivity of the primary tumor predicts the response to trastuzumab incompletely, but on the basis of our findings, the explanation for a lack of response must be other than a change in HER-2/neu status between primary tumor and metastasis.

The results of topo II analyses were in line with those of HER-2/neu. Although topo II amplification and deletion were identical in most of the paired tumors studied (10 of 13), differences were seen in three cases. We have described previously the intratumoral heterogeneity of topo II aberrations (amplification and deletion may occur in distinct subpopulations of cells; Refs. 3 and 4 ). In one case like this, we found evolvement toward topo II deletion in metastasis. The patient concerned did not receive any adjuvant chemotherapy with topo II inhibitors, which could explain selection toward a more chemoresistant tumor phenotype. Two patients predominantly showed topo II amplification in their metastases, although amplification was seen only in a small subpopulation of primary tumor cells. This phenomenon may reflect natural genetic evolution, because these patients had not received any kind of adjuvant therapy after primary surgery.

In our study, the identity of HER-2 status was confirmed both by using immunostaining and CISH, which together overcome the analytical limitations caused by variation in the length of formalin fixation between primary tumors and metastases, which is likely to cause variation in results if only HER-2/neu immunohistochemistry is used. CISH also has the advantage of revealing tissue histopathology, which was found particularly useful when analyzing the metastatic tumor lesions. Metastases often contain only small foci of cancer cells, which would have been difficult to find with FISH using darkfield fluorescence microscopy.

	ACKNOWLEDGMENTS

 
The skillful technical assistance of Anne Luuri and Sari Toivola is greatly appreciated.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ The present study was supported by the Scientific Foundation of Tampere University Hospital, the Academy of Finland, and the Finnish Cancer Society.

² To whom requests for reprints should be addressed, at Laboratory of Cancer Genetics, Institute of Medical Technology, University of Tampere, FIN-33014 Tampere, Finland. Phone: 358-3-2156729; Fax: 358-3-2158923; E-mail: jorma.isola{at}uta.fi

³ The abbreviations used are: topo, topoisomerase; CISH, chromogenic in situ hybridization; FISH, fluorescence in situ hybridization.

Received 3/22/01. Accepted 5/30/01.

	REFERENCES

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5. Smith K., Houlbrook S., Greenall M., Carmichael J., Harris A. L. Topoisomerase II co-amplification with erbB2 in human primary breast cancer and breast cancer cell lines: relationship to m-AMSA and mitoxantrone sensitivity. Oncogene, 8: 933-938, 1993.[Medline]
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