TOP2A Amplification Research Articles

Dual-colour CISH is a reliable alternative to FISH for assessment of topoisomerase 2-alpha amplification in breast carcinomas

Anthracyclines are among the most powerful antineoplastic drugs available for breast cancer treatment. Although HER2 amplification has been postulated to predict anthracycline benefit, numerous reports have demonstrated that HER2/TOP2A co-amplification is the clinically useful predictive marker of response to anthracyclines. The standard technique to evaluate gene status for target therapy selection is fluorescence in situ hybridization (FISH), but this technique has some disadvantages. Dual-colour chromogenic in situ hybridization (CISH) is an extension of the FISH protocol that allows bright-field microscopy and thus represents a user-friendly alternative to FISH. In order to evaluate whether dual-colour CISH is a reliable alternative to FISH in determining TOP2A gene amplification and to determine the frequency with which TOP2A and HER2 were co-amplified, we analysed 100 invasive breast cancer specimens (70 consecutive and 30 HER2-amplified samples) using tissue microarrays. Thus, a 99% agreement was found between TOP2A status determined by dual-colour CISH and FISH, as well as a high degree of correlation in TOP2A ratios using both techniques. TOP2A gene amplification was present in 8.6% of the 70 consecutive samples studied, all of which were HER2-amplified. Co-amplification of TOP2A was observed in 46.5% of the additional 30 HER2-amplified samples (no TOP2A amplification was seen in non-amplified HER2 samples). We conclude that dual-colour CISH represents an excellent alternative to FISH for determination of TOP2A gene status in invasive breast cancer. Our results showing TOP2A amplification only in HER2-amplified cases also add to the evidence that TOP2A determination should be restricted to those cases.

Role of Topoisomerases in Pediatric High Grade Osteosarcomas: TOP2A Gene Is One of the Unique Molecular Biomarkers of Chemoresponse

Currently, the treatment of pediatric high-grade osteosarcomas systematically includes one topoisomerase IIα inhibitor. This chemotherapy is usually adapted to the response to the neo-adjuvant therapy after surgery. The current and unique marker of chemoresponsiveness is the percentage of viable residual cells in the surgical resection. This late patient management marker has to be evaluated earlier in the therapeutic history of the patients on initial biopsy. Therefore, new biomarkers, especially those involved in the topoisomerase IIα inhibitor response might be good candidates. Therefore, our study was designed to target TOP1, TOP2A and TOP2B genes in 105 fresh-frozen diagnostic biopsies by allelotyping and real-time quantitative PCR. Our analyses in those pediatric osteosarcomas, homogeneously treated, highlighted the frequent involvement of topo-isomerase genes. The main and most important observation was the statistical link between the presence of TOP2A amplification and the good response to neo-adjuvant chemotherapy. Compared to adult cancers, the 17q21 amplicon, including TOP2A and ERBB2 genes, seems to be differentially implicated in the osteosarcoma chemoresponsiveness. Surprisingly, there is no ERBB2 gene co-amplification and the patients harboring TOP2A amplification tend to show a worse survival, so TOP2A analyses remain a preliminary, but a good molecular approach for the evaluation at diagnosis of pediatric osteosarcoma chemoresponsiveness.

Study of Her-2/neu and TOP2A expression in familial versus sporadic breast carcinoma

Introduction Genetic predisposition is one of the important risk factors that increase the incidence of breast cancer. The Her-2/neu gene is considered the most frequently amplified oncogene and is accompanied with either amplification or deletion of the TOP2A gene with an equal frequency. Aim of the study The aim of the study was to compare patients with familial breast cancer (FBC) with those having sporadic breast cancer (SBC) and also to evaluate the combined study on the hormonal status, immunostaining for Her-2/neu, and Her-2/neu and TOP2A copy number alterations by fluorescence in-situ hybridization (FISH). Patients and methods A total of 22 patients with invasive breast carcinomas were involved: 12 positive for the criteria that define FBC and 10 positive for SBC. Representative sections of formalin-fixed paraffin-embedded blocks were subjected to immunostaining for Her-2/neu (Hercep test), estrogen receptors, and progesterone receptors and also to FISH in order to evaluate TOP2A and Her-2/neu copy number alterations. Results FBC tumors were more aggressive than SBC tumors, as assessed by pathological parameters and immunohistochemical markers (lower estrogen receptor and progesterone receptor expression). However, the frequency of Her-2/neu gene amplification and protein overexpression was equal between the two groups. TOP2A gene amplification was observed in 25% of FBC patients compared with 20% of SBC patients. Coamplification of both Her-2/neu and TOP2A genes was observed in 8.3% of FBC patients and in 20% of SBC patients. In contrast, Her-2/neu amplification alone was observed in 25% of FBC patients compared with 10% of SBC patients. Importantly, TOP2A amplification without Her-2/neu gene amplification was observed in 16.7% of FBC patients but was not observed among SBC patients. Conclusion FBC tumors were more aggressive than SBC tumors. This study suggests that TOP2A gene amplification occurs both in FBC and in SBC patients but more frequently among FBC patients. Moreover, TOP2A gene amplification may occur independent of Her-2/neu amplification. We can conclude that this parameter should be determined on a routine basis in breast cancer patients, especially when FBC is suspected. In addition, a combined approach using immunohistochemical analysis and FISH can optimize Her-2/neu testing for breast carcinoma patients, especially among those with immunohistochemistry-equivocal tumors.

Evaluation of the prognostic role of centromere 17 gain and HER2/topoisomerase II alpha gene status and protein expression in patients with breast cancer treated with anthracycline-containing adjuvant chemotherapy: pooled analysis of two Hellenic Cooperative Oncology Group (HeCOG) phase III trials

BackgroundThe HER2 gene has been established as a valid biological marker for the treatment of breast cancer patients with trastuzumab and probably other agents, such as paclitaxel and anthracyclines. The TOP2A gene has been associated with response to anthracyclines. Limited information exists on the relationship of HER2/TOP2A gene status in the presence of centromere 17 (CEP17) gain with outcome of patients treated with anthracycline-containing adjuvant chemotherapy.MethodsFormalin-fixed paraffin-embedded tumor tissue samples from 1031 patients with high-risk operable breast cancer, enrolled in two consecutive phase III trials, were assessed in a central laboratory by fluorescence in situ hybridization for HER2/TOP2A gene amplification and CEP17 gain (CEP17 probe). Amplification of HER2 and TOP2A were defined as a gene/CEP17 ratio of >2.2 and ≥2.0, respectively, or gene copy number higher than 6. Additionally, HER2, TopoIIa, ER/PgR and Ki67 protein expression was assessed by immunohistochemistry (IHC) and patients were classified according to their IHC phenotype. Treatment consisted of epirubicin-based adjuvant chemotherapy followed by hormonal therapy and radiation, as indicated.ResultsHER2 amplification was found in 23.7% of the patients and TOP2A amplification in 10.1%. In total, 41.8% of HER2-amplified tumors demonstrated TOP2A co-amplification. The median (range) of HER2, TOP2A and CEP17 gain was 2.55 (0.70-45.15), 2.20 (0.70-26.15) and 2.00 (0.70-26.55), respectively. Forty percent of the tumors had CEP17 gain (51% of those with HER2 amplification). Adjusting for treatment groups in the Cox model, HER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with time to relapse or time to death.ConclusionHER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with outcome in high-risk breast cancer patients treated with anthracycline-based adjuvant chemotherapy.Trial registrationAustralian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12611000506998 and ACTRN12609001036202

BRCA1 and TOP2A gene amplification and protein expression in four molecular subtypes of breast cancer

We studied TOP2A amplification (using FISH methods), and TOP2A and BRCA1 protein overexpression (immunohistochemistry) in four molecular subtypes of breast cancer. Of 53 patients, 32 showed TOP2A and 38 showed BRCA1 overexpression. The highest percentage of TOP2A amplification (47.8%) and deletion (13%) was detected in Luminal B subtypes. Of 11 Luminal B tumors with TOP2A amplification, 9 (81.8%) overexpressed TOP2A. BRCA1 protein overexpression showed significant positive correlation with TOP2A protein expression. BRCA1 and TOP2A proteins exhibited similar patterns of expression in Luminal B and triple-negative breast cancer, suggesting the same prognosis in those patients.

Abstract P3-06-12: Effect of TOP2A and cMYC gene copy number on outcome in a Phase II trial of adjuvant TC (Docetaxel/Cyclophosphamide) plus trastuzumab (HER TC) in HER2-positive early stage breast cancer

Abstract Introduction: Approximately one-third of HER2+ early stage breast cancer (ESBC) patients have TOP2A-amplified breast cancer, the subpopulation known to benefit from anthracycline use (Press et al, JCO 2011). Data are needed to evaluate whether outcomes in ESBC patients treated with nonanthracycline-based regimens like docetaxel and cyclophosphamide + trastuzumab (HER TC) are affected by TOP2A or cMYC gene copy number. Methods: This was an open-label, phase II study of HER TC in HER2+ breast cancer patients. Outcome data have been previously reported (Jones et al, SABCS 2011, PD07-03). Tissue was collected to review HER2, cMYC, and TOP2A gene copy number at a central reference laboratory. HER2, cMYC, and TOP2A amplification was defined as FISH ratio >2, and deletion was defined as FISH ratio <1. Every 21 days, patients received T 75mg/m2 IV and C 600mg/m2 IV, plus weekly H 4mg/kg IV (loading dose) and 2mg/kg IV thereafter for a total of 4 cycles. After 4 cycles of TC+H, patients continued on H for 1 year on a 3-week schedule. The primary endpoint was disease-free survival (DFS) at 2 years with continued follow-up for 3 years. Secondary endpoints were overall survival (OS) and safety. Results: 493 patients with HER2+ ESBC were enrolled. From the 493 patients, 438 (89%) tissue samples were available and analyzed at Caris Diagnostics (Phoenix, AZ) to test for TOP2A, cMYC, and HER2 gene copy number by FISH. HER2 status was confirmed as positive in 87% of samples. Results for TOP2A, cMYC, and HER2 were generated in 438, 436, and 438 samples, respectively. TOP2A was classified as amplified in 43%, normal in 30%, deleted in 27%. cMYC was classified as amplified in 99(23%), normal in 246(56%), and deleted in 91(21%). Three-year DFS and OS in TOP2A and cMYC status as well as ER and Nodal status are depicted in Table 1. Multivariate analyses of age, nodal status, ER status, and gene expression shown below in Table 2 indicate that neither cMYC nor TOP2A status had an effect on outcome. Only nodal and ER status affected outcome. Conclusion: Outcome (DFS + OS) in a phase II study of a nonanthracycline regimen (TC) coupled with H was unaffected by cMYC or TOP2A gene copy number status. Only ER and nodal status showed an effect in a multivariate analysis. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-12.

202P - Biomarker (BM) Analyses of a Phase II Study of Neoadjuvant Pertuzumab and Trastuzumab With and Without Anthracycline (ATC)-Containing Chemotherapy for Treatment of HER2-Positive Early Breast Cancer (BC) (Tryphaena)

ABSTRACT Background TRYPHAENA showed that concurrent administration of trastuzumab (H) plus pertuzumab (P) with ATC in the neoadjuvant setting resulted in similar cardiac tolerability to sequential administration of ATC or to an ATC-free regimen. In addition, neoadjuvant P with H administered in combination with ATC- or carboplatin-based standard chemotherapy (CT) resulted in high pathological complete response (pCR, [ypT0/is]) rates, regardless of the CT regimen, in patients (pts) with HER2-positive BC. The molecular profile of pts who achieved a pCR was compared with those who did not. Methods Baseline core biopsies and sera from over 90% of the 225 pts enrolled in TRYPHAENA were available for BM analyses. The BMs and methods of assessment included: HER1/2/3, PTEN, and IGF1R protein expression by IHC (and FISH for HER2); HER1/2/3 mRNA expression by qRT-PCR; c-myc and TOP2A by FISH and PCR-based PIK3CA analyses detecting 8 mutations. Potential relationships between BMs and pCR were measured primarily using categorical analysis methods (chi-square test of association and Cochran–Mantel–Haenszel statistic). Median values were used as cut-offs except for TOP2A and PTEN where cut-offs were applied following a biologic rationale. Results Besides HER2 protein levels, no other measured BM was associated with pCR when BM samples from pts in each treatment arm were considered separately. TOP2A amplification (≥ 2.0 ratio) was observed in 32% of pts and was not associated with response to ATC regimen. There is no strong biologic rationale for correlation between BMs and pCR outcomes after treatment with different CT regimens; therefore, data from all pts were pooled. In the combined analysis, HER2 protein levels remained significantly correlated with pCR and high levels of HER2 mRNA were also indicative of improved pCR rates. PIK3CA mutations were seen in 24% of pts and were not associated with pCR. Conclusion This analysis supports the use of HER2 overexpression as the strongest predictive and clinically relevant BM for pCR after treatment with H + P + CT neoadjuvant regimen for pts with HER2-positive BC. Disclosure A. Schneeweiss: Prof. Schneeweiss is a member of an advisory board for F. Hoffmann La Roche and also has conducted reearch sponsored by F. Hoffmann La Roche. S. Chia: Dr Chia is a member of an advisory board for F. Hoffmann La Roche and also has conducted reearch sponsored by F. Hoffmann La Roche. R. Deb: -I have received consultancy fees from Roche Pharmaceuticals -I have received a fee for advisory board work for Roche. -Roche have paid for registration, travel and accommodation costs to attend courses. J. Ratnayake: Dr Ratnayake is an employee of F Hoffmann La Roche. A. Kiermaier: Dr Kiermeier is an employee of F Hoffmann-La Roche. V. McNally: Dr McNally is an employee of F Hoffmann-La Roche and owns stock in the company. G. Ross: Dr Ross is an employee of F Hoffmann-La Roche and owns stock in the company. J. Cortes: Dr Cortes has participated in advisory boards and received honoraria from F Hoffmann La-Roche. All other authors have declared no conflicts of interest.

TOP2A amplification in breast cancer is a predictive marker of anthracycline-based neoadjuvant chemotherapy efficacy

Anthracycline is a DNA topoisomerase 2-α (TOP2A) inhibitor and its concomitant over expression with Human Epidermal Growth Factor Receptor 2 (HER2) was investigated of being predictive for the response to anthracycline-based chemotherapies in breast cancer. 309 early and local advanced breast cancer patients were treated with anthracycline-based neoadjuvant chemotherapies in intense dose dense (IDD) (CE, Cyclophosphamide + Epirubicin) or conventional (TE, Paclitaxel + Epirubicin) regimens. HER2 proteins were qualitatively analyzed by immunohistochemistry (IHC) of primary tumor core biopsies, and TOP2A gene amplification levels of HER2 over-expressing cases were quantified by quantitative real-time polymerase chain reaction (qRT-PCR). Overall pathological complete response rate (pCR) was achieved in 14.3 %. HER2 was over expressed in 80/309 (25.9 %) cases, of which 61/80 cases have been tested for their TOP2A status. Over expression of HER2 was significantly positively correlated with higher pCR rates compared to low HER2 expression (27.5 % vs. 9.6 %, P < 0.001). Concurrent high TOP2A amplification led to a significantly higher pCR rate compared to low or no TOP2A amplification (56.3 % vs. 13.8 %, P = 0.001). HER2 over expression was associated with a significantly higher pCR rate only when TOP2A was also amplified (56.3 % vs. 9.6 %, P < 0.001), but not when it was deleted or normal (13.8 % vs. 9.6 %, P = 0.183) compared to HER2 low-expressing tumors. The interaction between HER2 or TOP2A and anthracycline-based regimen was observed in IDD and conventional neoadjuvant chemotherapies. The TOP2A amplification is related to anthracycline-based neoadjuvant chemotherapy sensitivity, and TOP2A should be included in future studies in breast cancer as a predictive marker.

HER2 amplification, HER2 overexpression,and TOP2A abnormalities in gastroesophageal cancers: Do they predict responsiveness to epirubicin-based chemotherapy?

e14672 Background: Abnormalities of TOP2A, a major target of anthracyclines, have been reported as positive predictive markers of response to anthracycline-based therapy in breast cancer. We determined abnormalities of TOP2A and HER2, and HER2 protein expression, in a cohort of patients with gastric or gastro-oesophageal junction adenocarcinomas treated with epirubicin-based chemotherapy. Methods: We studied 50 patients that received either adjuvant (74%) or perioperative (26%) epirubicin, cisplatin and 5-fluorouracil. All patients were screened for TOP2A amplification/deletion by fluorescent in situ hybridization (FISH), and for HER2 protein expression by immunohistochemistry (IHC). For each patient at least two paired surgical samples were examined. Tumors exhibiting a HER2 score of 2+ were further screened for gene amplification by FISH. We studied the association of TOP2A abnormalities and HER2 amplification/expression with recurrence free survival (RFS) and overall survival (OS). Results: By IHC, HER2 overexpression (score 3+) was detected in 9 patients. Intra-tumor heterogeneity of HER2 staining, resulting in discrepant HER2 scores, was detected in 4 of 50 (8%) paired samples analyzed. HER2 amplification was detected in 1 of 8 tumors with a HER2 score 2+. TOP2A amplification was observed in 1 of 50 tumors; none exhibited TOP2A deletions. In patients whose tumors showed HER2 overexpression or gene amplification (HER2-positive) and in patients with HER2-negative tumors, median RFS was 11.0 and 13.9 months, respectively (p= 0.35); median OS was 28.8 and 29.9 months, respectively (p= 0.56). A pathological complete response was observed in 1 of 4 HER2-positive patients that underwent perioperative chemotherapy. Conclusions: In this cohort of patients with early-stage gastroesophageal cancers who were treated with epirubicin-based chemotherapy, neither HER2 amplification nor HER2 overexpression were associated with outcome. TOP2A abnormalities are rare and their putative role as a determinant of responsiveness to anthracycline-based chemotherapy in gastroesophageal cancers is not supported by our findings.

Correlations of TOP2A gene aberrations and expression of topoisomerase IIα protein and TOP2A mRNA expression in primary breast cancer: A retrospective study of 86 cases using fluorescence in situ hybridization and immunohistochemistry

Our aim in this study was to assess the status of TOP2A gene aberrations (no change/amplification or deletion) and its correlations with topoisomerase IIα (Topo IIα) protein and TOP2A mRNA expression, respectively. TOP2A amplification, Topo IIα protein expression and TOP2A mRNA expression were assessed using samples of 86 cases of breast cancer by fluorescence in fluorescence in situ hybridization, quantitative real-time polymerase chain reaction and immunohistochemistry, respectively. Twenty two (22.57%) had amplification/deletion of TOP2A gene. Twenty eight (32.56%) tumor samples were 17q polysomy or monosomy. Topo IIα protein was expressed in 57 cases (66.27%, 57/86): 22 cases (38.62%, 22/57) and 35 cases (61.40%, 35/57) had amplification/deletion and no change of TOP2A gene, respectively. These three groups showed significant differences by one-way analysis of variance (P < 0.001). The average Ct values of TOP2A mRNA expression in the tumors with deletion, amplification and no change of TOP2A gene were 27.00, 27.33 and 31.66, respectively. We demonstrated that the TOP2A gene was amplified or deleted in breast cancer, with a significant correlation with high expressions of Topo IIα protein and TOP2A mRNA expression. Ki-67 expression index (mean = 14.9) decreased significantly in cases wherein TOP2A gene had no change and Her2/neu protein expression was weakly positive (0-1+, P < 0.001).

Prognostic Value of TOP2A Gene Amplification and Chromosome 17 Polysomy in Early Breast Cancer

The aim of this study was to analyze the occurrence of TOP2A gene amplification and chromosome 17 polysomy in patients with early breast cancer and to correlate the status of these alterations with the prognostic significance expressed as patients' clinical features and survival. Such concurrent analyses of TOP2A gene status and chromosome 17 polysomy have not been performed before. Study group included 149 consecutive stage I-III patients administered standard multimodality treatment. TOP2A abnormalities were examined by standard fluorescence in situ hybridization (FISH) and developed by our group quantitative real-time PCR (qPCR). TOP2A amplification and deletion assessed by FISH were found in 23% and 7% of the tumours, respectively, and by qPCR in 31% and 11% of the tumours, respectively. Chromosome 17 polysomy was detected in 40% of the cases. TOP2A amplification (by qPCR) correlated with shorter disease-free survival (p = 0.03) and overall survival (p = 0.047), and the prognostic value of TOP2A was confirmed in the multivariate analysis (HR = 3.22, 95% CI 1.09-9.56, p = 0.03). TOP2A gene amplification, but not chromosome 17 polysomy, carries negative prognostic information in early breast cancer. Given the aforementioned results, qPCR might serve as a prognostic tool in determining the patient's prognosis.

Carbonic anhydrase IX is a predictive marker of doxorubicin resistance in early-stage breast cancer independent of HER2 and TOP2A amplification

Background:In early-stage breast cancer, adjuvant chemotherapy is associated with significant systemic toxicity with only a modest survival benefit. Therefore, there is considerable interest in identifying predictive markers of response to therapy. Doxorubicin, one of the most common drugs used to treat breast cancer, is an anthracycline chemotherapeutic agent, a class of drugs known to be affected by hypoxia. Accordingly, we examined whether expression of the endogenous hypoxia marker carbonic anhydrase IX (CA IX) is predictive of outcome in early-stage breast cancer patients treated with doxorubicin.Methods:We obtained 209 early-stage pre-treatment surgically-resected breast tumours from patients, who received doxorubicin in their chemotherapeutic regimen and had >10 years of follow-up. Immunohistochemistry was used to detect CA IX, and we used fluorescence in situ hybridisation to detect both human epidermal growth factor receptor (HER2) and DNA topoisomerase II-alpha (TOP2A) gene amplification.Results:Carbonic anhydrase IX intensity was significantly correlated with progression-free survival (PFS) and overall survival (OS) in patients receiving 300 mg m−2 of doxorubicin (HR=1.82 and 3.77; P=0.0014 and 0.010, respectively). There was a significant, inverse correlation between CA IX score and oestrogen receptor expression, but no significant correlations were seen with either HER2 or TOP2A ratio.Conclusion:We demonstrate that CA IX expression is correlated with worse PFS and OS for breast cancer patients treated with doxorubicin, independent of HER2 or TOP2A gene amplification. This study provides evidence that using CA IX to detect hypoxia in surgically-resected breast tumours may be of clinical use in choosing an appropriate chemotherapy regimen.

HER2 and TOP2A in high-risk early breast cancer patients treated with adjuvant epirubicin-based dose-dense sequential chemotherapy

BackgroundHER2 and TOP2A parameters (gene status, mRNA and protein expression) have individually been associated with the outcome of patients treated with anthracyclines. The aim of this study was to comprehensively evaluate the prognostic/predictive significance of the above parameters in early, high-risk breast cancer patients treated with epirubicin-based, dose-dense sequential adjuvant chemotherapy.MethodsIn a series of 352 breast carcinoma tissues from patients that had been post-operatively treated with epirubicin-CMF with or without paclitaxel, we assessed HER2 and TOP2A gene status (chromogenic in situ hybridization), mRNA expression (quantitative reverse transcription PCR), as well as HER2 and TopoIIa protein expression (immunohistochemistry).ResultsHER2 and TOP2A amplification did not share the same effects on their downstream molecules, with consistent patterns observed in HER2 mRNA and protein expression according to HER2 amplification (all parameters strongly inter-related, p values < 0.001), but inconsistent patterns in the case of TOP2A. TOP2A gene amplification (7% of all cases) was not related to TOP2A mRNA and TopoIIa protein expression, while TOP2A mRNA and TopoIIa protein were strongly related to each other (p < 0.001). Hence, TOP2A amplified tumors did not correspond to tumors with high TOP2A mRNA or TopoIIa protein expression, while the latter were characterized by high Ki67 scores (p = 0.003 and p < 0.001, respectively). Multivariate analysis adjusted for nodal involvement, hormone receptor status, Ki67 score and HER2/TOP2A parameters revealed HER2/TOP2A co-amplification (21.2% of HER2 amplified tumors) as an independent favorable prognostic factor for DFS (HR = 0.13, 95% CI: 0.02-0.96, p = 0.046); in contrast, increased HER2/TOP2A mRNA co-expression was identified as an independent adverse prognostic factor for both DFS (HR = 2.41, 95% CI: 1.31-4.42, p = 0.005) and OS (HR = 2.83, 95% CI: 1.42-5.63, p = 0.003), while high TOP2A mRNA expression was an independent adverse prognostic factor for OS (HR = 2.06, 95% CI: 1.23-3.46, p = 0.006). None of the parameters tested was associated with response to paclitaxel.ConclusionsThis study confirms the favorable prognostic value of HER2/TOP2A co-amplification and the adverse prognostic value of high TOP2A mRNA expression extending it to the adjuvant treatment setting in early high-risk breast cancer. The strong adverse prognostic impact of high HER2/TOP2A mRNA co-expression needs further validation in studies designed to evaluate markers predictive for anthracyclines.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12611000506998.

Multiplication of Chromosome 17 Centromere Is Associated with Prognosis in Patients with Invasive Breast Cancers Exhibiting NormalHER2andTOP2AStatus

PurposeThis study aimed to investigate the clinical significance of chromosome 17 centromere (CEP17) multiplication (increased copy number of CEP17) related to human epidermal growth factor receptor 2 (HER2) and topoisomerase II alpha (TOP2A) status in patients with invasive breast cancer.MethodsWe constructed tissue microarrays using 594 invasive breast cancer samples and performed single-color silver-enhanced in situ hybridization (SISH) assay for HER2, TOP2A, and CEP17 to assess for copy number aberrations. The association of CEP17 multiplication with patient survival was analyzed according to HER2 and TOP2A status.ResultsAmong 567 informative cases, HER2 amplification was noted in 22.8%, TOP2A amplification in 8.3% and TOP2A deletion in 11.1%. CEP17 multiplication was identified in 33.2% and was significantly associated with worse overall survival (OS) (p=0.02) and disease-free survival (DFS) (p=0.02). CEP17 multiplication correlated with patient survival in patients with normal TOP2A or non-amplified HER2 status, but the prognostic significance was lost in those with altered TOP2A or amplified HER2. On multivariate analyses, CEP17 multiplication was an independent prognostic factor for poorer OS (p=0.02) and DFS (p=0.01) in patients with normal TOP2A and non-amplified HER2.ConclusionCEP17 multiplication was identified as a promising prognostic marker in patients with invasive breast cancer exhibiting either non-amplified HER2 or normal TOP2A status.

PD05-02: Effect of HER2/Topoisomerase II alpha (TOP2A) Gene Status or Protein Expression and Chromosome 17 (CEP17) Polysomy on the Outcome of Breast Cancer Patients Treated with Anthracycline-Containing Dose-Dense Sequential Adjuvant Chemotherapy with or without Paclitaxel – A Pooled Analysis of Two Hellenic Cooperative Oncology Group (HeCOG) Phase III Trials.

Abstract Background: The HER2 gene has been established as a valid biomarker in the treatment of breast cancer patients with trastuzumab and probably with other agents, such as paclitaxel or anthracyclines. The TOP2A gene has been associated with response to anthracyclines. The relationship of HER2/TOP2A gene status in the presence of CEP17 polysomy with patients’ outcome following adjuvant treatment with anthracyclines with or without paclitaxel is not established. Patients and methods: Formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks from 1,033 patients (61.5% of 1,681 randomized patients) with high-risk operable breast cancer enrolled in two sequential phase III trials1,2 were assessed in a central laboratory for HER2/TOP2A gene amplification and CEP17 polysomy by fluorescence in situ hybridization (FISH) and tumors were categorized according to the 2007 American Society of Clinical Oncology/College of American Pathologists guidelines. HER2 and TOP2A amplification was defined as a gene/CEP17 ratio of &amp;gt;=2.2 and &amp;gt;=2.0, respectively or a gene copy number of &amp;gt;6. Additionally HER2, TOP2A, ER/PgR, Ki67, CK5 and EGFR protein expression were assessed by immunohistochemistry (IHC) and all patients were classified according to their IHC phenotype. Treatment consisted of epirubicin-based dose-dense sequential adjuvant chemotherapy followed by hormonal therapy and radiation, as indicated. Results: Disease-free survival (DFS) and overall survival (OS) did not differ significantly between treatment groups. Median follow-up was 92 months, while 5-year DFS (OS) rates were 74% (88%), 69% (81%) and 75% (86%) for the E-T-CMF, E-CMF and ET-CMF groups, respectively. HER2 amplification was found in 24.1% of the patients and TOP2A amplification in 10.3%. In total, 46.7% of HER2 amplified tumors demonstrated TOP2A co-amplification. The median (range) of HER2, TOP2A and CEP17 copy numbers was 2.55 (0.70−45.15), 2.2 (0.50−26.15) and 2.05 (0.45−26.55), respectively. 21% of the tumors were considered to be polysomic (32.5% of those with HER2 amplification). Adjusting for treatment groups in the Cox model, TOP2A amplification, CEP17 polysomy and HER2/TOP2A co-amplification were not associated with either relapse or death. Treatment with paclitaxel was associated with improved survival in the HER2−amplified subgroup (HR=0.493, interaction p=0.036; adjusting for clinicopathological prognostic factors: HR=0.553, interaction p=0.054), an association that was not apparent for DFS. Conclusions: HER2 amplification was predictive for OS benefit from adjuvant treatment with paclitaxel in patients treated with epirubicin-based dose-dense sequential adjuvant chemotherapy, but not for DFS. TOP2A amplification, CEP17 polysomy and HER2/TOP2A co-amplification were not associated with outcome. 1. Ann Oncol 16:1762–71, 2005; 2. Ann Oncol 19:853–60, 2008. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD05-02.

P5-13-20: TOP2A Amplification Have Associated with Response to Anthracycline-Based Preoperative Chemotherapy in Primary Breast Cancer.

Abstract Background: Recent studies have suggested that HER2 gene amplification or overexpression have association with sensitivity of anthracycline-based chemotherapy. Preclinical studies found that TOP2A amplification or deletion only could be detected in HER2 amplification or over-expressive cases. And TOP2A protein is the target of anthracycline. In fact, the value of HER2 for predicting response to anthracycline-based chemotherapy in breast cancer may be more likely related to the concomitant amplification of the TOP2A gene. In this study we studied the association between TOP2A gene amplification and response to anthracycline-based preoperative chemotherapy. Methods: 309 early and local advanced breast cancer cases were enrolled in our study. HER2 proteins were qualitatively analysed by IHC, and TOP2A gene alterations were quantified by real-time polymerase chain reaction (RT-PCR) in primary tumor core biopsies from all HER2 over expressive cases. The enrolled patients received an intense dose dense (IDD) (CE, Cyclophosphamide + Epirubicin) or conventionally (TE, Paclitaxel+Epirubicin) scheduled anthracycline- based preoperative chemotherapy. The tumor was evaluated every two cycles. Median time on study for 309 patients with follow-up was 38 months. Results: The pCR was 14.3%. HER2 overexpression was found in 80/309 (25.9%) breast cancer cases, of which 61/80 cases have been tested for TOP2A status. 19/80 cases have not been tested for TOP2A status because of lacking core biopsies tumor tissue after pathological diagnosis. HER2 overexpression was associated with a significantly higher pCR rate compared to HER2 lower expression (27.5% vs. 9.6%, P&amp;lt;0.001). Further analysis was carried on and found the significantly higher pCR rate in TOP2A co-amplified cases compared to TOP2A deleted or normal cases (56.3% vs. 13.8%, P=0.001). HER2 overexpression was associated with a significantly higher pathologic complete response (pCR) rate only when TOP2A was co-amplified (56.3% vs. 9.6%, P&amp;lt;0.001), but not when deleted or normal (13.8% vs. 9.6%, P=0.183), compared to HER2 lower expression tumors. The interaction between HER2 or TOP2A and anthracycline-based regiment was observed not only in IDD but conventionally scheduled preoperative chemotherapy. Discussion: Previous studies demonstrate that TOP2A gene amplification may define a subtype of HER2−positive breast cancer, This subtype of breast cancer may be highly sensitive to anthracycline-based chemotherapy, which may improve progression in HER2 and TOP2A co-amplification cases. Howerver, this viewpoint needs suppose from preoperative chemotherapy. Using RT-PCR to test TOP2A statue from 61 primary breast cancer tumor tissue, we demonstrate that TOP2A amplification breast cancer have highly sensitive to anthracycline-based preoperative chemotherapy. This finding suggests that TOP2A as a predictive marker in breast cancer should be included in future studies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-20.

HER2 and TOP2A as predictive markers for anthracycline-containing chemotherapy regimens as adjuvant treatment of breast cancer: a meta-analysis of individual patient data

Prediction of response to anthracycline-based therapy for breast cancer is challenging. We aimed to assess the value of HER2 and TOP2A as predictive markers of response to anthracycline-based adjuvant therapy in patients with early breast cancer. We did a meta-analysis of individual patient data from five randomised adjuvant trials that compared anthracycline-based regimens with cyclophosphamide, methotrexate, and fluorouracil (CMF) regimens. We assessed the status of HER2 and TOP2A genes with fluorescent in-situ hybridisation. Tumour samples were submitted to an external laboratory for validation. We calculated hazard ratios (HR) to compare event-free survival (EFS) and overall survival in patients receiving anthracycline-based treatment with those receiving CMF in two HER2 cohorts (HER2 amplified and non-amplified tumours) and in three TOP2A cohorts (normal, amplified, and deleted tumours). We analysed data for 3452 patients for HER2 and 3102 patients for TOP2A. For EFS, HRs were 0·89 (95% CI 0·79-1·01) for HER2 non-amplified patients and 0·71 (0·58-0·86) for HER2-amplified patients (p(interaction)=0·0485); for overall survival, HRs were 0·91 (95% CI 0·79-1·05) for HER2 non-amplified patients and 0·73 (0·59-0·89) for HER2-amplified patients (p(interaction)=0·0718). In analysis of TOP2A status, HRs for EFS were 0·88 (0·78-1·00) for normal, 0·63 (0·46-0·87) for deleted, and 0·62 (0·43-0·90) for amplified (p(interaction)=0·0513); HRs for overall survival were 0·89 (0·78-1·03) for normal, 0·68 (0·49-0·95) for deleted, and 0·67 (0·46-0·98) for amplified (p(interaction)=0·1608). When patients with TOP2A-deleted and TOP2A-amplified tumours were grouped together (altered cohort) and compared with data from patients with normal TOP2A tumours, HRs for EFS were 0·64 (0·50-0·81) for altered and 0·88 (0·78-1·00) for normal (p(interaction)=0·0183); HRs for overall survival were 0·67 (0·52-0·86) for altered and 0·89 (0·78-1·03) for normal (p(interaction)=0·0455). Although HER2 amplification and combined TOP2A amplification and deletion may have some value in the prediction of responsiveness to anthracycline-based chemotherapy, our findings do not support the use of anthracyclines only in patients with HER2-amplified or TOP2A-aberrated tumours. Associazione Italiana Ricerca Cancro, Academy of Finland, Belgian Federation Against Cancer, Cancer Research UK, Les Amis de l'Institut Bordet, Scottish Breast Cancer Trials Group, NCIC Clinical Trials Group, Canadian Cancer Society Research Institute, Danish Council for Strategic Research, Pharmacia-Upjohn (now Pfizer), and Abbott Laboratories.

Anthracyclines, HER2, and TOP2A: the verdict

Should we restrict the use of anthracyclines in the adjuvant treatment of early breast cancer to patients with HER2-positive or TOP2A-amplified tumours? This question—which has been debated for a long time—is crucial for daily clinical practice because anthracyclines are one of the most active drugs in the treatment of breast cancer despite their related rare, but severe, late toxicities such as cardiotoxicity and secondary leukaemia. As far as HER2 is concerned, in 2008 the results of two meta-analyses 1 Gennari A Sormani MP Pronzato P et al. HER2 status and efficacy of adjuvant anthracyclines in early breast cancer: a pooled analysis of randomized trials. J Natl Cancer Inst. 2008; 100: 14-20 Crossref PubMed Scopus (325) Google Scholar , 2 Dhesy-Thind B Pritchard KI Messersmith H et al. HER2/neu in systemic therapy for women with breast cancer: a systematic review. Breast Cancer Res Treat. 2008; 109: 209-229 Crossref PubMed Scopus (76) Google Scholar suggested that the question might be answered and that only patients with HER2-positive tumours benefited from anthracyclines, but contradictory results were published the same year, 3 Bartlett JM Munro A Cameron DA et al. Type 1 receptor tyrosine kinase profiles identify patients with enhanced benefit from anthracyclines in the BR9601 adjuvant breast cancer chemotherapy trial. J Clin Oncol. 2008; 26: 5027-5035 Crossref PubMed Scopus (87) Google Scholar refuelling the debate. Regarding the predictive value of TOP2A, reported data are even more confusing. 4 O'Malley FP Chia S Tu D et al. Topoisomerase II alpha and responsiveness of breast cancer to adjuvant chemotherapy. J Natl Cancer Inst. 2009; 101: 644-650 Crossref PubMed Scopus (170) Google Scholar The good news is that the debate is now closed thanks to the excellent study published by Angelo Di Leo and colleagues 5 Di Leo A Desmedt C Bartlett JMS et al. HER2 and TOP2A as predictive markers for anthracycline-containing chemotherapy regimens as adjuvant treatment of breast cancer: a meta-analysis of individual patient data. Lancet Oncol. 2011; (published online Sept 13.)https://doi.org/10.1016/S1470-2045(11)70231-5 Summary Full Text Full Text PDF PubMed Scopus (159) Google Scholar in The Lancet Oncology. The bad news is that the results of this meta-analysis are negative and that, after more than 15 years of research, we still have no predictive markers of anthracycline sensitivity available for daily practice. HER2 and TOP2A as predictive markers for anthracycline-containing chemotherapy regimens as adjuvant treatment of breast cancer: a meta-analysis of individual patient dataAlthough HER2 amplification and combined TOP2A amplification and deletion may have some value in the prediction of responsiveness to anthracycline-based chemotherapy, our findings do not support the use of anthracyclines only in patients with HER2-amplified or TOP2A-aberrated tumours. Full-Text PDF Open Access

TOP2A Amplification in the Absence of That of HER-2/neu: Toward Individualization of Chemotherapeutic Practice in Breast Cancer

To investigate the relationship between human epidermal growth factor receptor (HER)-2/neu and the gene encoding topoisomerase IIα (TOP2A) in breast cancer, while elucidating their association with clinicopathological variables. Real-time quantitative polymerase chain reaction (RQ-PCR) was performed on a 96-patient study group to assess gene amplification, and levels were determined using the comparative cycle threshold approach and Taqman assays. An immunohistochemistry (IHC) microarray (n = 76) was then employed to check for correlation between gene amplification and protein expression levels. Amplification levels of TOP2A did not differ significantly according to HER-2/neu status by either RQ-PCR or IHC microarray. Of the HER-2/neu(-) patients, 29.1% demonstrated levels of TOP2A above the third quartile, whereas 22.9% of the HER-2/neu(+) patients had values in the first quartile (log TOP2A <0.62), thereby indicating low-level amplification. Of the 60 patients characterized as HER-2/neu(-) using IHC and fluorescence in situ hybridization (FISH), 22.9% were classified as TOP2A(+) on the IHC microarray. Of the 14 patients deemed HER-2/neu(+) using IHC and FISH, meanwhile, the majority (n = 10) were classified as TOP2A(+). Our results indicate that amplification of TOP2A in breast cancer is not confined to those who are concomitantly HER-2/neu(+), and suggest that a significant proportion of HER-2/neu(-) patients exhibit high levels of TOP2A.

Does Topoisomerase II–Alpha Gene Amplification Provide Useful Information for Treatment Selection in Patients With Breast Carcinoma?

TO THE EDITOR: Press et al 1 concluded that “TOP2A coamplification, not HER2 amplification, is the clinically useful predictive marker of an incremental response to anthracycline-based chemotherapy.” I do not believe that this conclusion is justified on the basis of the data they examined. To evaluate whether a marker is predictive for benefit from a drug, one should compare (either prospectively or by using archived specimens from a previous trial) outcomes for patients who were randomly assigned to receive a regimen containing the drug or the same regimen not containing the drug, and one should examine how this treatment effect (ie, hazard ratio) depends on marker status. 2 The studies Press et al 1 examined did not permit such an evaluation. The closest one could come to a direct analysis of whether TOP2A amplification is a predictive marker of anthracycline effectiveness was an analysis that Press et al did not comment on. In trial BCIRG-006 of the Breast Cancer International Research Group, patients with HER2amplified tumors were randomized to receive ACT (doxorubicin, cyclophosphamide, docetaxel), ACTH (ACT plus trastuzumab), or TCH (docetaxel, carboplatin, trastuzumab). A comparison of the ACTH regimen with the TCH regimen isolates the value of the anthracycline relative to carboplatin with both in the presence of trastuzumab. In Figure 3 of the article by Press et al, 1 outcomes for these two regimens seem equivalent both forTOP2A-amplified and forTOP2Aunamplified tumors. In the patients with amplified tumors, there were 35 events on treatment with ACTH compared with 42 events on treatment with TCH; in the patients with unamplified tumors, there were 87 events on ACTH compared with 92 on TCH. Consequently, on the basis of the only relevant direct randomized comparison of anthracycline use available in the authors’ data, there is no evidence that the efficacy of the anthracycline depends on TOP2A status. The authors base their conclusions on indirect evidence. In both the BCIRG-006 adjuvant study and the H0648g, the original trastuzumab metastatic disease licensing study, the investigators compared an anthracycline regimen not containing trastuzumab with the same anthracycline regimen administered with trastuzumab. They noted that trastuzumab did not seem to improve outcome in the small subset of patients with amplifiedTOP2A but that it did improve outcome for patients without TOP2A amplifications. The significant interaction in Table 3 is for the incremental effect of trastuzumab, not as stated for the incremental effect of anthracycline. They interpret this variation in trastuzumab treatment effect as indicating that the anthracycline is more effective in patients withTOP2A amplifications. The fact that trastuzumab is ineffective in patients with TOP2A amplifications receiving an anthracycline-based regimen does not necessarily mean that the anthracyclines are effective in that subset. Patients with large amplifications not focused on the HER2 gene may have tumors driven by genes other than HER2 and thus be less responsive to HER2 inhibition. The conclusion is also based on a limited number of events involved. This type of indirect analysis does not provide a strong level of evidence according to the scale introduced by Simon et al 2 for evaluating the medical utility of predictive biomarkers. The pragmatic question of importance is which patients should be considered good candidates for anthracycline-based regimens. Previous studies have indicated that patients without HER2-amplified tumors are unlikely to benefit from such regimens. 3,4 The data for BCIRG-006 indicates that, for patients with HER2-amplified tumors who will receive trastuzumab, the docetaxel/carboplatin regimen seems as effective as the ACT regimen, regardless of TOP2A status. This leaves little room for the use of anthracyclines and no reason for the measurement of TOP2A amplification status. The data for BCIRG-006 suggests that patients with amplified HER2 and TOP2A might do as well on ACT as on ACTH—ie, they might not benefit from trastuzumab in the presence of ACT. This conclusion of therapeutic equivalence requires independent confirmation in a larger study, however, as it is based on only 42 disease-free survival events on ACT compared with 35 on ACTH.