Co-amplification Of ERBB2 Research Articles

Oral Etoposide and Trastuzumab Use for HER2-Positive Metastatic Breast Cancer: A Retrospective Study from the Institut Curie Hospitals.

Simple SummaryOral etoposide (VP16), an inhibitor of topoisomerase-II, has demonstrated clinical activity in metastatic breast cancer (MBC). To our knowledge, oral VP16 combined with trastuzumab (VP16-T) in HER2+ MBC has not been evaluated before. This combination is biologically relevant, as TOP2A, the gene encoding topoisomerase II, is often co-amplified with ERBB2. We report a retrospective analysis of the impact of oral VP16-trastuzumab on HER2+ MBC patients, together with TOP2A/ERBB2 co-amplification status, assessed through shallow whole genome sequencing. In addition to its low cost and convenience, the oral VP16-trastuzumab regimen has shown a satisfactory activity and excellent tolerability.Background: The TOP2A and ERBB2 genes are co-amplified in about 40% of HER2 positive (HER2+) breast cancers. Oral etoposide (VP16), an inhibitor of topoisomerase-II (encoded by TOP2A), has demonstrated clinical activity in metastatic breast cancer (MBC). The benefit of oral VP16 combined with trastuzumab (VP16-T) in HER2+ MBC has not yet been evaluated. Methods: Patients treated at the Institut Curie Hospitals with VP16-T for HER2+ MBC were retrieved by an in silico search. Progression-free survival (PFS), overall survival (OS), response rate, prolonged PFS (defined as at least 6 months), clinical benefit, and toxicity were assessed. The co-amplification of ERBB2 and TOP2A was assessed by shallow whole genome sequencing on tumor tissue whenever available. Results: Forty-three patients received VP16-T after a median number of six prior treatment lines for HER2+ MBC. Median PFS and OS were 2.9 months (95% CI [2.4–4.7]) and 11.3 months (95% CI [8.3–25.0]), respectively. Three patients had a complete response, while 12/40 (30%) experienced clinical benefit. Only three patients stopped treatment for toxicity. Seven (35%) patients displayed a TOP2A/ERBB2 co-amplification. No statistically significant correlation was found between outcome and TOP2A/ERBB2 co-amplification. Conclusion: Our analysis suggests a favorable efficacy and toxicity profile for VP16-T in patients with heavily pretreated HER2+ MBC.

Abstract P2-13-28: Inhibiting retinoic acid receptor signalling enhances the effect of neratinib in HER2 positive breast cancer cell lines

Abstract Introduction: Treatment strategies for HER2+ breast cancer (BC) involves first line treatment with the monoclonal antibody trastuzumab, however resistance to treatment can occur. The pan-HER tyrosine kinase inhibitor neratinib is an approved adjuvant treatment for early stage HER2+ BC after one year of treatment with trastuzumab. Recent studies have highlighted the potential benefits of using retinoic acid as an adjuvant treatment for BC. The retinoic acid receptor (RAR) family of nuclear transcription factors consists of RARα, RARβ and RARγ. Interestingly ~30% of HER2+ BC have a co-amplification of the RARA gene, which codes for RARα. We examined the effect of RARα agonists (Fenretinide & AM580) and antagonist (AGN194310) in combination with neratinib in two HER2+ cell lines: SKBR3, has co-amplification of ERBB2 and RARA, and HCC1569 has amplified ERBB2 but are not RARA-amplified. Methods: SKBR3 and HCC1569 cell lines were cultured in RPMI/10% FCS at 370C/5% CO2. 10mM stock concentrations of fenretinide (H7779-Sigma), AM580 (A8843-Sigma), AGN194310 (SML2665-Sigma) and neratinib (Puma Biotechnology, Inc) were prepared in DMSO. For proliferation assays, cells were seeded in 96 well plates at a density of 3 X 104 cells per well for 24h. Cells were treated with a drug alone (2X concentration) or combination of drugs (4X concentration) in 100μL of medium. Proliferation was measured using an acid phosphatase-based assay after 5 days as percentage growth versus DMSO control. The half maximal inhibitory concentration (IC50) was calculated for each drug, using CalcuSyn. The combination assays were performed using fixed ratios. The combination index (CI) values were calculated at the effective dose that inhibits 50% growth (ED50), using CalcuSyn. Values < 1 represent a synergistic effect, a value of 1 is additive and values > 1 represent an antagonistic effect. All data presented as the mean of biological triplicate experiments ± standard deviation. Results: This research has found that the synthetic retinoic acid fenretinide, a pan-RAR (α, β and γ) activator, was more potent in the HCC1569 cells compared to the SKBR3 cell line with an IC50 of 0.21 ± 0.03μM compared to 4.55 ± 0.87 μM, respectively. However, when combined with neratinib there was a strong antagonistic effect observed in the HCC1569 cell line (CI value: 15.63 ± 9.5). Conversely, fenretinide enhanced the effect of neratinib in the SKBR3 cell line (CI value: 0.83 ± 0.4). The RARα-specific activator AM580 had an IC50 >10μM for both cell lines. When combined with neratinib there was an additive effect observed in the RARα negative HCC1569 cells (CI value: 0.97 ± 0.4), and a synergistic effect observed in the RARα upregulated SKBR3 cell line (CI value: 0.78 ± 0.08). Next we wanted to determine if inhibiting RAR activity would have any effect on the proliferation of these cell lines. The IC50 for the pan-RAR (α, β and γ) antagonist AGN194310 was >10μM for each cell line. However, when combined with neratinib treatment there was a strong synergistic effect observed in the HCC1569 cell line (CI value: 0.52 ± 0.17) and the SKBR3 cell line (CI value: 0.66 ± 0.19). Conclusions: This study suggests a differential effect of RAR agonists when combined with neratinib in RARα amplified versus non-amplified HER2+ BC. Targeting RAR signalling, particularly with a RAR antagonist, in combination with the pan-HER inhibitor warrants further investigation in HER2+ BC. Citation Format: Debbie O'Reilly, Nicola Gaynor, Neil Conlon, Irmina Diala, Lisa D Eli, John Crown, Denis M Collins. Inhibiting retinoic acid receptor signalling enhances the effect of neratinib in HER2 positive breast cancer cell lines [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-28.

Gene amplification of CCNE1, CCND1, and CDK6 in gastric cancers detected by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization

New and effective treatments for advanced gastric cancer are urgently needed. Cyclins E and D1 form a complex with cyclin-dependent kinase 2, 4, or 6 to regulate G1-S transition. The G1-S regulatory genes encoding cyclin E (CCNE1), cyclin D1 (CCND1), and CDK6 (CDK6) are frequently amplified in gastric cancer and may therefore influence molecularly targeted therapies against ERBB2 or EGFR when coamplified. A total of 179 formalin-fixed and paraffin-embedded gastric cancer specimens were examined for these gene amplifications by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization. Amplification of at least 1 G1-S regulatory gene was found in 35 tumors (CCNE1 amplification, 15% of samples; CCND1, 6%; CDK6, 1%). In 13 of the 35 tumors, dual-color fluorescence in situ hybridization identified coamplification of the G1-S regulatory genes with ERBB2, EGFR, and/or KRAS in single cancer nuclei. The observation that cells with G1-S regulatory gene amplification contained clonal subpopulations with coamplification of ERBB2, EGFR, or KRAS in 5 early and 3 advanced cancers suggests that amplification of the G1-S regulatory genes represents an early event, which precedes ERBB2, EGFR, or KRAS amplification. Amplified CCNE1, CCND1, and CDK6 in advanced gastric cancer may be potentially useful as direct targets for molecular therapy or for combination therapy with ERBB2 or EGFR inhibitors. Multiplex ligation-dependent probe amplification could be a useful tool for identification of patients who would benefit from such therapies.

Abstract A2-37: Predictive modeling to identify novel associations between gene amplification status and cancer cell line chemosensitivity

Abstract Background: Substantial research effort has been directed towards identifying gene amplification events during cancer development, with the view that these represent promising therapeutic targets or otherwise highlight pathways for therapeutic intervention. We considered whether datasets generated by recent large-scale pharmacogenomic studies (1, 2) could be mined to uncover novel associations between amplification status at particular genes of interest and cell line chemo-sensitivity or -resistance. As a case study, we examined the known amplification targets, ERBB2 and MYC, as well as a candidate amplification target, TPD52. Experimental procedures: We accessed and curated datasets from the Cancer Cell Line Encyclopedia (1) and Cancer Therapeutics Response Portal (2). Multivariate linear regression modelling was performed to determine whether cell lines from diverse cancer types displayed differential responses to the same compound according to their gene copy number at ERBB2, MYC and TPD52. Associations between drug response and gene expression of ERBB2, MYC and TPD52 were also modelled to ascertain whether these recapitulated the gene-drug associations predicted by copy number. Both models were adjusted for cell lineage as a potential confounder by performing agglomerative hierarchical clustering of cell lines into 5 groups based on the 500 most highly varying genes. For each drug, chemo-sensitivity measures were discretised into three categories (resistant, intermediate and sensitive) using the waterfall method (1). Results: Predictive modelling was possible for 24 drugs across 487 cell lines (1) and for an additional 323 drugs across 230 cell lines (2). Multivariate linear regression yielded 82 significant associations (p<0.05) between drug response and copy number at ERBB2 (n=57), MYC (n=20) or TPD52 (n=5). This included the known sensitization of ERBB2-amplified cell lines to lapatinib, which was the compound most significantly associated with both ERBB2 copy number and gene expression. We identified 9 novel associations between chemo-sensitivity and copy number at ERBB2 (n=1), MYC (n=3) or TPD52 (n=5), however, increased copy number was more frequently associated with resistance (n=72, p<0.05). Modelling also yielded 108 significant associations (p<0.05) between drug response and gene expression of ERBB2 (n=28), MYC (n=42) or TPD52 (n=38). However, there was limited concordance between drugs associated with copy number and those identified through gene expression; approximately 25% concordance for ERBB2 and MYC, and zero concordance for TPD52. Grouping cell lines by global expression profiles yielded a significant contribution to drug sensitivity in ~25% drug-gene associations, confirming cell lineage as a potential predictor of drug response. Taken together, these analyses identified several lead compounds for experimental validation in a cohort of TPD52-amplified cancer cell lines, with and without co-amplification of ERBB2 and/or MYC. Conclusions: We have applied predictive models to data from large-scale pharmacogenomic studies (1, 2) to detect both known and novel associations between gene amplification status and cell line chemo-sensitivity or -resistance. Such analyses have the potential to link existing anti-cancer drugs with specific gene amplifications, which may then be applied as new biomarkers to predict efficacy.

Semi-comprehensive analysis of gene amplification in gastric cancers using multiplex ligation-dependent probe amplification and fluorescence in situ hybridization

The prognosis of patients with gastric carcinomas at an advanced stage still remains dismal, and therefore novel therapeutic modalities are urgently needed. Since the successful targeting of amplified ERBB2 with a humanized monoclonal antibody, the amplified genes of other receptor tyrosine kinases such as EGFR, FGFR2, and MET, as well as those of other cell regulator genes, are being considered as candidate targets of molecular therapy. The aim of the present study was to determine the amplification status of 26 genes, which are frequently amplified in solid cancers, in advanced gastric cancers. A total of 93 formalin-fixed and paraffin-embedded advanced gastric cancer tissues were examined by multiple ligation-dependent probe amplification, and 32 cases with ‘gain’ or ‘amplified’ status of 16 genes were further examined for the respective gene amplification by fluorescence in situ hybridization (FISH) and for the respective protein overexpression by immunohistochemistry. The frequencies of gene amplifications in advanced gastric cancers were as follows: ERBB2 (13 cases, 14%), FGFR2 (7 cases, 8%), MYC (7 cases, 8%), TOP2A (7 cases, 8%), MET (4 cases, 4%), MDM2 (4 cases, 4%), CCND1 (3 cases, 3%), FGF10 (2 cases, 3%), and EGFR (1 case, 1%). Amplification of the receptor tyrosine kinases genes occurred in a mutually exclusive manner except for one tumor in which ERBB2 and FGFR2 were both amplified but in different cancer cells. Co-amplification of ERBB2 and MYC, and EGFR and CCND1, in single nuclei but on different amplicons, was confirmed in one case each. Attempts at correlating the FISH status with the immunohistochemical staining pattern showed variable results from complete concordance to no correlation. In conclusion, combination of multiple ligation-dependent probe amplification and FISH analysis is a feasible approach for obtaining the semi-comprehensive genetic information that is necessary for personalized molecular targeted therapy.

1057 Synergistic Antitumor Activity of Lapatinib and Retinoids on a Novel Subtype of Breast Cancer With Co-amplification of ERBB2 and RARA

1057 Synergistic Antitumor Activity of Lapatinib and Retinoids on a Novel Subtype of Breast Cancer With Co-amplification of ERBB2 and RARA

Synergistic antitumor activity of lapatinib and retinoids on a novel subtype of breast cancer with coamplification of ERBB2 and RARA

All-trans retinoic acid (ATRA), the only clinically available cyto-differentiating agent, has potential for the therapy/chemoprevention of breast carcinoma. Given the heterogeneous nature of this tumor, a rational use of ATRA and derivatives (retinoids) in the clinic requires the identification of patients that would benefit from retinoid-based protocols. Here, we demonstrate that 23-32% of the human ERBB2(+) breast cancers show coamplification of retinoic acid receptor alpha (RARA), encoding the retinoic acid receptor, RARα. This represents a novel subtype of breast cancer characterized by remarkable sensitivity to ATRA and RARα agonists, regardless of positivity to the estrogen receptor, a known modulator of retinoid sensitivity. In estrogen-receptor-negative cellular models showing coamplification of ERBB2 and RARA, simultaneous targeting of the corresponding gene products with combinations of lapatinib and ATRA causes synergistic growth inhibition, cyto-differentiation and apoptosis. This provides proof-of-principle that coamplification of ERBB2 and RARA can be exploited for the stratified and targeted therapy of a novel subtype of breast cancer patients, with an approach characterized by tumor cell selectivity and low predicted toxicity. The available cellular models were exploited to define the molecular mechanisms underlying the antitumor activity of combinations between lapatinib and ATRA. Global gene expression and functional approaches provide evidence for three components of the antiproliferative/apoptotic responses triggered by lapatinib+ATRA. Induction of the retinoid-dependent RARRES3 protein by ATRA stabilizes the effect of lapatinib inhibiting ERBB2 phosphorylation. Upregulation and activation of the transcription factor FOXO3A integrates ATRA-dependent transcriptional and lapatinib-dependent posttranscriptional signals, controlling the levels of effector proteins like the antiapoptotic factor, BIRC5. Stimulation of the TGFβ pathway by ATRA mediates other components of the apoptotic process set in motion by simultaneous targeting of ERBB2 and RARα.

Abstract 2287: A sub-population of HER2+ breast carcinomas is characterized by co-amplification of the ERBB2 and RARA genes that renders cancer cells sensitive to retinoids and combinations of these agents with lapatinib

Abstract In breast cancer, HER2-positivity is often the result of an amplification of the corresponding gene (ERBB2) mappping to chr 17 close to the locus of the retinoid receptor, RARα (RARA). We set up a PCR assay and a FISH method to evaluate the proportion of patients with co-amplification of ERBB2 and RARA using sections obtained from 76 HER2+ cases. We observed that 20-28% of the cases showed co-amplification of ERBB2 and RARA (ERBB2+/RARA+). To evaluate whether RARA co-amplification translated into retinoid sensitivity, we turned to a panel of 12 breast carcinoma cell lines. The growth of 3 ERBB2+/RARA+ cell lines, SKBR3, UACC-812 and AU565, was inhibited by all-trans-retinoic acid (ATRA). In response to the retinoid, growth inhibition was accompanied by cell differentiation. ERBB2+/RARA− cell lines were totally refractory to ATRA. Interestingly, SKBR3, UACC-812, and AU565 showed IC50 values to the anti-proliferative effects of ATRA falling in the low nanomolar range. These values were two orders of magnitude lower than the IC50s calculated for the control cell lines MCF-7 and T47D (ERBB2−/RARA−), representing popular models of the well known association between ERα-positivity and sensitivity to retinoids in breast carcinoma. We evaluated whether targeting the retinoid and HER2 pathways simultaneously has the potential to represent a viable therapeutic strategy for this group of patients. The anti-proliferative, differentiating and apoptotic effects of combinations between ATRA and lapatinib, a tyrosine kinase inhibitor used for the treatment of HER2+ breast carcinoma, was assessed in our panel of cell lines. In agreement with retinoid sensitivity, lapatinib and ATRA determined synergistic anti-proliferative effects only in the case of the cell lines, SKBR3, UACC-812 and AU565. Furthermore, apoptosis was observed only in ERBB2+/RARA+ cells treated with the combination of lapatinib and ATRA. In SKBR3 cells, this effect was preceded by a lapatinib-dependent enhancement of the lactogenic differentiation afforded by ATRA. The molecular mechanisms underlying the cross-talk between lapatinib and ATRA were evaluated focusing specifically on the RARα and HER2 signal transduction pathways, as well as performing whole-genome gene expression studies in SKBR3 cells. These last studies and functional approaches demonstrated that the combination produced perturbations of the TGFβ, FGF and IGF pathways and indicated a role for RIG-I and FOXO3A in the cross-talk. Our results constitute the rationale for the use of combinations between lapatinib and ATRA in the treatment of ERBB2+/RARA+ breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2287. doi:10.1158/1538-7445.AM2011-2287

Abstract 1717: Combinations of retinoids and lapatinib in the treatment of Her2/Neu-positive breast carcinomas with co-amplification of the ERBB2 and RARA genes

Abstract The current study represents proof of principle for the use of all-trans retinoic acid (ATRA) alone or in combination with the tyrosine kinase inhibitor, lapatinib, in the management of a subset of Her2/Neu-positive breast carcinomas. First-line treatment of Her2/Neu-positive breast carcinoma is based on the monoclonal antibody, trastuzumab, while specific tyrosine kinase inhibitors, such as lapatinib, are used as second-line agents. Novel and more effective therapeutic strategies are called for this type of cancer. ATRA and derivatives (retinoids) have been proposed in the chemoprevention and treatment of breast carcinoma. Retinoids activate specific nuclear receptors (RARs and RXRs), which mediate the therapeutic effects of these agents. The beneficial activity of retinoids is limited to ER-positive breast cancer cells, possibly as a consequence of RARα induction by estrogens. A significant fraction of Her2/Neu-positive breast carcinomas are ER-negative, hence this type of cancer is predicted to be refractory to retinoids. Her2/Neu-positivity is very often the result of an amplification of the corresponding gene (ERBB2), which maps to chromosome 17, approximately 650 kilobases upstream of the locus encoding RARα (RARA). Given this background, we evaluated whether a proportion of Her2/Neu-positive and ER-negative breast carcinomas are characterized by co-amplification of ERBB2 and RARA, using real-time PCR on genomic DNA extracted from paraffin sections from 60 patients. We observed that 28% of the cases showed co-amplification of ERBB2 and RARA, while the amplicon did not contain the latter in the remaining fraction of patients. RARA amplification was associated with increased levels of the corresponding RARα protein, as determined by immuno-histochemistry. To evaluate whether RARA co-amplification and consequent RARα over-expression was associated with retinoid sensitivity, we turned to a panel of representative breast carcinoma cell lines. We observed that the growth of two cell lines, SKBR3 and UACC812, characterized by ERBB2 and RARA co-amplification, was inhibited by ATRA. Growth inhibition was accompanied by differentiating and pro-senescence responses to the retinoid. All these phenomena were mediated by RARα, as demonstrated by the use of selective retinoid agonists and by silencing of the receptor by siRNAs. Similar effects were not observed in MDA-MB-453 and HCC1569 cells, which carry a simple ERBB2 amplicon and are totally refractory to the various effects of ATRA. In SKBR3 and UACC812, inhibition of Her2/Neu tyrosine kinase activity by lapatinib, led to a dramatic enhancement of all the retinoid responses, demonstrating strong synergism between the two compounds. Gene expression profiling and mechanistic studies in SKBR3 and UACC812 indicated a two-way cross-talk between Her2/Neu and RARα. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1717.

Non-incidental coamplification of Myc and ERBB2, and Myc and EGFR, in gastric adenocarcinomas

This study was conducted to assess the frequencies of protein overexpression and gene amplification of Myc and to identify the mechanisms of Myc gene amplification, especially with regards to its possible coamplification with ERBB2 or EGFR in gastric adenocarcinomas. By immunohistochemical analysis of a total of 300 formalin-fixed and paraffin-embedded gastric adenocarcinomas, the nuclear overexpression of MYC was found in 47 tumors (16%). A fluorescence in situ hybridization (FISH) analysis revealed that nine (19%) of the 47 tumors with protein overexpression had cancer cells with high levels of Myc amplification, whereas only seven (6%) of the 122 tumors without protein overexpression showed high-level Myc gene amplification. Such Myc amplification was significantly correlated with positive nuclear protein overexpression. The coamplification of ERBB2 or EGFR with Myc that was found in six and four cases, respectively, is believed to be non-incidental because those frequencies were significantly higher than the individual frequencies observed for the total examined cases (ERBB2: 7%; EGFR: 4%). The high levels of gene amplification of these three genes, as visualized by FISH, could be broadly classified into two typical types, namely, ‘multiple scattered signals' and ‘large clustered signals'. Using two-color FISH, the coexistence of coamplified Myc and ERBB2, or Myc and EGFR, within single nuclei in various combinations of amplification types and copy numbers, could be ascertained in all nine cases, including one in which the synchronous ‘multiple scattered type' coamplification of Myc and ERBB2 was observed. In three tumors, coamplification of ERBB2 and EGFR was found; however, ERBB2- and EGFR-amplified cell populations were separate and mutually exclusive. We propose that the non-incidental coamplification of Myc and either ERBB2 or EGFR occurred through translocation and subsequent rearrangement.

Amplification of a 280-Kilobase Core Region at the ERBB2 Locus Leads to Activation of Two Hypothetical Proteins in Breast Cancer

Amplification of the ERBB2 oncogene at 17q12 is clinically the most relevant genetic aberration in breast cancer and several studies have linked ERBB2 activation to poor clinical outcome. The development of targeted antibody-based therapy for ERBB2-overexpressing tumors and the possible role of ERBB2 as a predictor of chemotherapy treatment response have further emphasized the essential role of ERBB2 in breast cancer. Here, we performed a detailed characterization of the molecular events occurring at the ERBB2 amplicon in primary breast tumors. Analysis of the amplicon structure in 330 breast tumors by fluorescence in situ hybridization to a tissue microarray revealed a 280-kb common region of amplification that contains 10 transcribed sequences, including eight known genes. The expression levels of these 10 transcripts were determined in 36 frozen samples of grade-matched ERBB2-amplified and -nonamplified (as determined by fluorescence in situ hybridization) primary breast tumors by using quantitativereal-time reverse transcriptase-polymerase chain reaction. A highly significant association between amplification and expression levels was observed for six of these genes, including ERBB2 and two uncharacterized hypothetical proteins, MGC9753 and MGC14832. These results support the recent findings on the influence of copy number on gene expression levels and highlight novel genes that might contribute to the clinical behavior of ERBB2-amplified breast tumors.

Co-amplification of erbB2, topoisomerase II α and retinoic acid receptor α genes in breast cancer and allelic loss at topoisomerase I on chromosome 20

The DNA topoisomerase enzymes are targets for the cytotoxic effects of a number of anticancer agents termed topoisomerase inhibitors. We have analysed breast cancer biopsy specimens for genetic alterations at and around topoisomerase loci in order to obtain molecular insight into factors which may determine how tumours respond to chemotherapy. We show that of 50 tumours examined, the topoisomerase II α locus is co-amplified in 3 cases out of 6 with erbB2 amplification and that amplification can be accompanied by high expression of topoisomerase II α. In our attempts to distinguish amplification from aneuploidy and define the limits of amplification, we also observed co-amplification of the retinoic acid-α receptor with erbB2 and topoisomerase II α in the same three samples. At the topoisomerase I locus on chromosome 20, we observed allelic loss in two out of 17 samples. Genetics abberations at topoisomerase loci, therefore, appear to be relatively common in breast cancer.