Retrospective Analysis of Topoisomerase IIa Amplifications and Deletions As Predictive Markers in Primary Breast Cancer Patients Randomly As
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《临床肿瘤学》
the Department of Oncology, Odense University Hospital, Odense
the Department of Pathology, Herlev Hospital, Herlev
the Department of Pathology, Roskilde County Hospital, Roskilde
the Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus
the DakoCytomation A/S, Glostrup
DBCG Secretariat, Copenhagen
the Department of Pathology, Odense University Hospital, Odense
the Department of Oncology, Copenhagen, Denmark
ABSTRACT
PURPOSE: The aim of the study was to evaluate the predictive value of HER2 and topoisomerase II gene (TOP2A) for the efficacy of epirubicin in the adjuvant setting of breast cancer patients.
PATIENTS AND METHODS: In the Danish Breast Cancer Cooperative Group trial 89D, 980 pre- and postmenopausal primary patients were randomly allocated to either CMF (cyclophosphamide, methotrexate, and fluorouracil; n = 500) or CEF (cyclophosphamide, epirubicin, and fluorouracil; n = 480) times 9, between January 1990 and November 1999. Tumor tissue was retrospectively identified from 805 patients and was analyzed for HER2-positivity and for TOP2A-amplifications and deletions.
RESULTS: HER2-positivity was found in 33% of the 805 investigated tumors and was not a predictive marker for epirubicin sensitivity. TOP2A changes were identified in 23% of the 773 investigated tumors: 12% had TOP2A amplifications and 11% had TOP2A deletions. We found that patients with TOP2A amplification had an increased recurrence-free (RFS; hazard ratio [HR], 0.43; 95% CI, 0.24 to 0.78) and overall survival (OS; HR, 0.57; 95% CI, 0.29 to 1.13), respectively if treated with CEF compared with CMF, and that patients with TOP2A deletions had an almost identical hazard ratio (RFS: HR, 0.63; 95% CI, 0.36 to 1.11; OS: HR, 0.56; 95% CI, 0.30 to 1.04). This is in contrast to patients with a normal TOP2A genotype for whom similar outcome was observed in both treatment arms (RFS: HR, 0.90; 95% CI, 0.70 to 1.17; OS: HR, 0.88; 95% CI, 0.66 to 1.17).
CONCLUSION: TOP2A amplification—and possibly deletion—seems to be predictive markers for the effect of adjuvant epirubicin containing therapy in primary breast cancer, but a final conclusion has to await a confirmative study or a meta-analysis.
INTRODUCTION
The Early Breast Cancer Trialists’ Collaborative Group’s overview analysis demonstrated a significant reduction in the risk of recurrence and death with anthracycline-containing regimens versus regimens without anthracyclines.1 The Danish Breast Cancer Cooperative Group (DBCG) trial DBCG-89-D is one such trial, and suggests that the substitution of methotrexate in the cyclophosphamide, methotrexate, fluorouracil (CMF) regimen, with epirubicin will lead to a further 25% to 30% proportional reduction in mortality.2 Despite the significant difference established in the group-wise comparison, the beneficial effect could be restricted to a minor part of the patients. Predictive factors would allow us to select those patients who would benefit the most from epirubicin, and thus spare the remaining patients from the additional toxicity.
A number of retrospective studies have investigated the predictive value of HER2 overexpression and/or amplification and have raised the hypothesis that patients with HER2-positive tumors have an inborn increased sensitivity toward anthracycline-containing therapy.3-5 But the results are somewhat nonconsistent and it has been hypothesized, that HER2 is rather to be viewed as a marker for the real anthracycline target topoisomerase II (topoII). The topoisomerase II gene (TOP2A) is located next to the HER2 gene on chromosome 17q12-q21.
TopoII is a key enzyme during cell division, where it creates a reversible double-strand DNA break allowing the two template stands to rotate freely and thus enable the passage of a second DNA double-strand through the break. Through binding to topoII the anthracyclines stabilize the DNA double-strand breaks which may lead to apoptosis, and topoII is probably the primary molecular target of anthracyclines.6 In vitro studies have hypothesized that amplification of TOP2A leads to overexpression of the topoII enzyme and increased sensitivity to anthracyclines7-9 and deletion to resistance.7 In a retrospective study, TOP2A changes were identified in 84% of 70 breast cancer patients with HER2 amplified tumors.10 Surprisingly deletions were almost as frequent as amplifications, and were observed concurrently in subsets of the tumors. The results from this pivotal study lead to exploration of the possible predictive value of TOP2A alterations for anthracycline-based chemotherapy in breast patients with HER2 amplified tumors, but a definitive answer has not been obtained.10-12
In a previous pilot-study, we observed TOP2A copy number changes in approximately 50% of HER2 amplified breast tumors and in 5% of nonamplified breast tumors.13 In the present study we investigated the predictive value of HER2-positivity and TOP2A changes in DBCG trial 89-D, a randomized comparison of intravenous cyclophosphamide, epirubicin, and fluorouracil (CEF) with CMF.
PATIENTS AND METHODS
January 1990 to November 1999, 1,195 patients were randomly assigned in the DBCG trial 89-D, hereof 980 Danish patients were eligible for this retrospective study, while data and tissue were not obtained from 215 Swedish patients. Patients were included if they were A: premenopausal, node-negative and with grade 2 or III tumors 5 cm; B: premenopausal with receptor-negative or unknown tumors, greater than 5 cm or with positive axillary lymph nodes; or C: postmenopausal with receptor-negative tumors, greater than 5 cm or with positive axillary lymph nodes. Following surgery (mastectomy, 79, 2% or lumpectomy, 19, 5% and axillary dissection) patients were randomly assigned in a factorial 2 x 2 design to either nine cycles of CMF (cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, and fluorouracil 600 mg/m2) given intravenously (IV) on day one every 3 weeks, nine cycles of CMF plus pamidronate, nine cycles of CEF (cyclophosphamide 600 mg/m2, epirubicin 60 mg/m2, and fluorouracil 600 mg/m2) given IV day once every 3 weeks, or nine cycles of CEF plus pamidronate. Pamidronate was given orally, 150 mg twice daily for 4 years. Six patients randomized to the CMF-arm received CEF and 26 patients randomized to CEF received CMF. Eighteen patients (13 randomly assigned to CEF and five randomly assigned to CMF) never received chemotherapy and were excluded from the predictive study. None of the remaining 962 patients received adjuvant tamoxifen. Locoregional radiotherapy was given after the first cycle of CMF or CEF, and during radiotherapy two cycles of CEF or CMF were substituted by single-agent cyclophosphamide (900 mg/m2). Radiotherapy was given against the residual breast following lumpectomy (48 Gy + boost 10 Gy) or chest wall following mastectomy if the tumor was greater than 5 cm (48 Gy), and against regional nodes in node-positive disease (48 Gy). In all cases, 2 Gy in five fractions per week. In the CMF and CEF groups, 206 (40.0%) and 173 (38.7%), respectively, received radiotherapy.
A number of variables were recorded prospectively, such as: age at surgery, histologic type (ductal carcinoma NOS, lobular carcinomas, and other types), malignancy grade,14 size (largest diameter of the invasive component), and numbers of removed and positive axillary lymph nodes.
Preparation of Tissue
Consecutive serial sections were cut at 4 μm from 805 available paraffin-embedded tumors for immunohistochemistry and FISH and stored cold until staining was performed. A slide stained with hematoxylin and eosin, prepared from each block, was used for confirmation of invasive carcinoma. All analyses were performed at the department of pathology, Roskilde Hospital, Denmark.
Immunohistochemistry
The sections were stained within 5 days from cutting. Certified laboratory staff using a Techmate immunostainer (DAKO) followed the procedure for the HercepTest (DakoCytomation, Glostrup, Denmark) according to the manufacturers protocol. Positive controls as supplied with the kit were included as well as in house controls together with a negative control for each case. The results were scored 0, 1+, 2+, and 3+ as recommended in the HercepTest.
FISH
Two new kits, HER2 FISH pharmDx and TOP2A FISH pharmDx (DakoCytomation, Glostrup, Denmark), were used according to the manufacturers’ manual, and as briefly described in the pilot study.13
In cases of ductal carcinoma with an in situ component, the negative control slide for HercepTest was used to mark the invasive areas avoiding any possibility of including the in situ component. Up to 60 gene signals (or the number closest to 60+) were counted in nuclei with identifiable boundaries. Optimally, only signals distinctly separated from each other were included, but in case of clusters due to high levels of amplification the number was estimated. The ratio was calculated as the number of signals for the gene probe (HER2 and TOP2A respectively) divided by the number of signals for the centromere 17. Cases were scored as HER2 or TOP2A FISH amplified when the ratio was 2. A TOP2A deletion is considered present when the ratio was less than 0.8 and this cut point was in the absence of a biologically well-defined ratio based on the work of Di Leo et al.11
HER2 FISH Analysis Was Performed on All HercepTest 2+/3+ Positive Specimens
All HER2 (3+) positive tumors and HER2 (2+) positive tumors with HER2 amplification investigated by FISH (fluorescence in situ hybridization; ratio 2) were considered HER2 positive.
Statistical Analysis
The primary outcome of interest was recurrence-free survival (RFS) calculated as the time from randomization until first locoregional recurrence, distant recurrence, second malignancy, or death, and overall survival (OS) calculated as the time from randomization until death. Follow-up time was quantified in terms of a Kaplan-Meier estimate of potential follow-up.15 Association between TOP2A-status and the classical prognostic variables and HER2 was investigated using contingency tables and 2 test. Survival curves were constructed according to the Kaplan-Meier product-limit method and compared using the log-rank test. Multivariate survival analysis was conducted using Cox proportional hazards models with backward selection.
The proportional hazard assumption was assessed graphically as well as by including a time-dependent component individually for each covariate. Finally, putative interactions of each covariate and the treatment parameter were investigated.
Receptor-status and malignancy grade were found to violate the proportional hazards assumption for both end points. Furthermore, nonproportionality was seen for TOP2A status and HER2-positivity with respect to the end point OS. For each end point, nonproportionality was taken into account by stratifying for those covariates that violated the proportional hazards assumption for that end point.
The backward selection procedure was applied separately to each analysis. All nonstratification variables, including treatment, were exposed to selection. If removed, treatment was re-entered into the reduced model after the selection procedure was completed.
The estimated effect of treatment with CEF within the three subgroups consisting of TOP2A amplified, TOP2A deleted, and TOP2A normal patients, and within the two subgroups consisting of HER2 positive and negative patients was investigated in a Cox proportional hazards models including parameters representing the interaction of treatment with CEF and patient subgroup and the estimated effect of CEF over CMF in these groups was compared.
The predictive value of a given characteristic was quantified by the hazard ratio (HR). The overall significance of interaction terms with two or more df was assessed by a Wald test.
All patient records were updated regarding disease status in August 2002 and death in December 2003.
With an event rate of 50% and an estimated 20% TOP2A positive patients,14 the number of events seems to be sufficient to detect a true clinical relevant difference. All tests were conducted with a 5% level of significance.
Ethics
The data used and presented were all collected and analyzed by the DBCG registry. The clinical study as well as the current biologic substudy was conducted according to the Helsinki declaration and approved by The Danish Scientific Ethical Committees (DBCG 89-D, v200.1616/89-V200.1623/89-12-089/01).
RESULTS
At the time of analysis, 217 (50%) patients treated with CMF and 207 (56%) treated with CEF and with available tumor tissue blocks (n = 805), were still alive without recurrence.
Median estimated potential follow-up time with respect to RFS was 8.06 years (95% CI, 7.77 to 8.46) for patients treated with CEF, and 8.09 years (95% CI, 7.61 to 8.63) for patients treated with CMF. Median potential follow-up time with respect to OS was 10.40 years (95% CI, 9.86 to 10.80) for patients treated with CEF and 10.28 years (95% CI, 9.71 to 10.61) for patients treated with CMF.
The classical prognostic variables were equally distributed in the two treatment arms (Table 1). The median relative dose-intensities of the chemotherapy were identical, 0.97, in the two treatment groups.
Blocks were unavailable for 175 (16%) of the 980 randomly assigned Danish patients (Fig 1). Premenopausal patients were overrepresented among patients where tumor blocks were unavailable as were patients with small tumors, receptor positive tumors, low malignancy grade, and one to three positive lymph nodes (2 test; P < .05). Age and treatment were distributed equally between the two groups. A log-rank test showed no difference in RFS or OS between the groups with and without available blocks (data not shown).
Amplification of TOP2A was seen in 93 of the 773 eligible patients (12.0%; CI, 9.9 to14.6) and deletion in 87 (11.3%; CI, 9.2 to 13.8; Table 2). Correlations between clinical and pathologic variables and TOP2A status are shown in Table 3. HER2 (3+) expression was seen in 246 (30.6%) of the 805 tumors and amplification of HER2 was seen in an additional 21% of the 81 HER2 (2+) positive tumors leading to 32.7% (CI, 29.5 to 36.0) HER2-positive tumors. Patients with HER2-positive tumors, were more often postmenopausal, had more than three positive lymph nodes and had negative ER-status (2 test; P < .001, data not shown). HER2-positivity was not correlated with increasing age, tumor size, or malignancy grade (2 test; P > .5, data not shown). TOP2A changes (amplifications/deletions) were seen in 140 (56.9% [CI, 50.5 to 63.1]) of the 246 HER2-positive TOP2A determined patients and in 40 (7.6% [CI, 5.5 to 10.3]) of the 527 HER2-negative TOP2A-determined negative patients. Thus 40 or 22% of the total number of TOP2A amplified or deleted patients would have been overlooked, if only HER2-positive samples had been analyzed for TOP2A alterations.
Univariate Analysis
Patients with TOP2A amplification or deletion and HER2-positivity had a significant reduction in RFS (Fig 2). Overall, the patients benefited from treatment with CEF (RFS; log-rank-test; P = .04, data not shown). However, univariate analysis could not identify any subgroup of patients (ie, patients with TOP2A-amplified or TOP2A-deleted positive tumors) that benefited specifically from treatment with anthracycline, although a trend was seen concerning patients with TOP2A-deleted and in particular amplified tumors (Fig 3). Only HER2-negative patients gained as a subgroup from treatment with CEF (Fig 4).
Multivariate Analysis
The Cox proportional hazards regression analysis of RFS was carried out according to treatment with CEF, menopausal status, tumor size, positive nodes, HER2-positivity and TOP2A status as selection variables. The putative interaction between TOP2A status and treatment with CEF with respect to RFS was investigated in a nonreduced Cox proportional hazards model for all patients that also included parameters representing the interaction.
In this model, the hazard ratio for treatment with CEF with respect to treatment with CMF was estimated at 0.63 (95% CI, 0.36 to 1.11) for TOP2A-deleted patients, 0.90 (95% CI, 0.70 to 1.17) for TOP2A-normal patients and 0.43 (95% CI, 0.24 to 0.78) for TOP2A-amplified patients (Fig 5). Pair-wise Wald tests show no difference in treatment HR between TOP2A deleted and TOP2A normal patients (W1 = 1.28; P = .26), whereas a significant difference in treatment HR was seen between TOP2A normal and TOP2A amplified patients (W1 = 5.09; P = .02). However, because of the low number of TOP2A altered patients an overall Wald-test fails to detect an overall TOP2A-status versus treatment interaction (W2 = 5.65; P = .06).
Corresponding analyses show benefit of treatment with CEF over CMF for both HER2-positive and negative patients, the hazard ratio is estimated at 0.79 (95% CI, 0.60 to 1.05) for HER2-negative patients and 0.75 (95% CI, 0.53 to 1.06) for HER2-positive patients. No interaction between treatment with CEF rather than CMF and HER2-positivity is seen (W1 = 0.6; P = .81)
After backwards selection was completed, only the following variables retained significance in the analysis of RFS for all patients: tumor size (cm), 1.147 (P < .0001; 95% CI, 1.081 to 1.216); positive nodes (1 to 3) 2.18 (P < .0001; 95% CI, 1.49 to 3.19); positive nodes (> 3) 5.03 (P < .0001; 95% CI, 3.47 to 7.27); HER2-positivity 1.37 (P = .007; 95% CI, 1.09 to 1.72); and treatment with CEF 0.78 (P = .03; 95% CI, 0.63 to 0.97).
The selection variables in the Cox proportional hazards analysis of OS were treatment with CEF, menopausal status, tumor size, and positive nodes. The computation of the hazard ratio with respect to OS for treatment with CEF rather than CMF within each TOP2A subgroup showed a nonsignificant increased OS for TOP2A amplified and TOP2A deleted patients, with HRs of 0.57 (95% CI, 0.29 to 1.13) and 0.56 (95% CI, 0.30 to 1.04), respectively (Fig 6). In the TOP2A-normal patients, no advantage of CEF compared with CMF was found (HR, 0.88; 95% CI, 0.66 to 1.17). Overall, no TOP2A-status versus treatment interaction was found (W2 = 2.56; P = .28).
Correspondingly, the hazard ratio for treatment with CEF rather than CMF for HER2-positive and HER2-negative patients was estimated at 0.73 (95% CI, 0.50 to 1.05) and 0.82 (95% CI, 0.59 to 1.13), respectively. No interaction between treatment with CEF and HER2 status was found (W1 = 0.23; P = .63).
DISCUSSION
In this large-scale study, we found a relative reduction in the risk of recurrence and death from breast cancer of around 50% in patients with primary operable and TOP2A amplified breast cancer that had been randomized to intravenous CEF compared to CMF. The results of the present study confirms the results of previous smaller studies that have suggested an increased sensitivity to anthracyclines in patients with TOP2A amplified breast tumors in the adjuvant11 and locally advanced setting.12,16,17 This in contrast to one negative neoadjuvant study16 using immunohistochemistry.
In addition we found an almost equally predictive value of TOP2A deletions, although with broad confidence-intervals—probably due to the low number of patients in this group. This finding was not expected, as the hypothesis was that deletions predicted anthracycline resistance. The clinical importance of TOP2A deletions has not been analyzed earlier, most likely since only a relatively small number of patients with TOP2A-deleted tumors have been identified in previous studies. The molecular mechanisms underlying the adjacent HER2 amplification and TOP2A gene deletion are unknown, as is the breakpoint, size, and functionality of the deleted TOP2A segment. The question of the predictive value of TOP2A deletion has to await a confirmative study or a meta-analysis, pooling data together from large-scaled phase III trails.
The predictive value of TOP2A-amplifications was not seen for the end point OS, probably because recurrence is a more precise measurement than death as patients often have received other treatment—containing anthracyclines—after their first relapse, and the effect of treatment therefore would confound the true predictive impact of the TOP2A status.
In the present study, the predictive value of TOP2A may have been less significant due to the use of methotrexate in the comparative regimen and the addition cyclophosphamide and fluorouracil in both treatment arms.
We found no evidence that the superiority of CEF over CMF with respect to RFS and OS was greater for HER2-positive patients than HER2-negative (ie, no interaction between HER2 status and treatment was seen.) Previous studies have reported inconsistent results concerning HER2 as a predictive marker for anthracycline-sensitivity, and the literature has recently been reviewed on several occasions.3-5 In brief, eight larger adjuvant studies have been published.11,18-25 Three studies found a significant interaction between treatment with anthracycline and HER2-status,19-2021 four studies found no interaction, but either a trend in the interaction analysis, or a risk reduction from anthracycline treatment in the HER2-positive patients.11,18,22-24 Two of the studies were repeated, with the same patient material, but with other antibodies, and no interaction was found.18,23
In the present study HER2 status was insufficient as a screening-tool for TOP2A amplifications or deletions, regardless of whether it was determined with IHC alone or a combination of IHC and FISH. Almost 8% of the HER2-negative patients had TOP2A changes and this would have been overlooked, had all the tumor samples not been analyzed. This is in accordance with the findings from our preceding pilot study where we found 4.4% (95% CI, 1.7 to 10.9) TOP2A amplifications or deletions among the 90 HER2 negative tumors.13 Prescreening with HER2 was originally proposed by J?rvinen et al26 in 1996, based on coexpression by IHC of topoII and HER2. Later the same group27 used FISH to study copy number aberrations of both TOP2A and HER2 in nine breast cancer cell lines and in 97 breast tumors, which were selected for the study according to their HER2 status by Southern blotting. Of 57 HER2-amplified primary breast carcinomas, 23 (44%) showed TOP2A coamplification and 24 (42%) showed a physical deletion of the TOP2A gene. No TOP2A alterations were found in 40 primary tumors without HER2 amplification. Di Leo et al11 estimated the prevalence of TOP2A amplification to be 6%, in a node-positive group of patients. We found the prevalence of TOP2A amplification in our study to be 12% and furthermore 11% had TOP2A deletions.
In conclusion, HER2-status gave no additional predictive information regarding anthracycline-containing chemotherapy. But in this large scaled randomized predictive study, we identified a group of patients with TOP2A amplifications, who benefited particularly from anthracycline-containing chemotherapy and found indications, although not statistically significant that an additional group of patients, the TOP2A deleted patients, also benefited from treatment with anthracycline. Based on the currently accepted biologic hypothesis, an increased sensitivity to anthracyclines was unexpected in patients with TOP2A deletions and confirmation should be awaited before implementation. A large-scale meta-analysis based on centralized reevaluation on TOP2A has already been organized, and samples from the current study will be included.
Authors’ Disclosures of Potential Conflicts of Interest
Acknowledgment
The skillful technical assistance of Vinni Bredahl and Birgit Hansen and the help from oncological and pathological departments in Denmark collecting patient data and tumor tissue is greatly appreciated.
NOTES
Supported by grants from Danish Cancer Society; Danish Medical Research Council; Danish Cancer Research Foundation; The Clinical Oncological Research Unit, Odense University Hospital; The A.P. Moeller Foundation; Dagmar Marshalls Foundation; Grosserer Georg Bjoerkner & Ellen Bjoerkners Fond; and Ambt Balslev’s Foundation. DakoCytomation, Glostrup, Denmark, made all kits available without costs and kindly provided a microscope.
Presented in part as an oral presentation at ECCO 12: The European Cancer Conference, Copenhagen, Denmark, September 21-25, 2003.
Authors’ disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Early Breast Cancer Trialists’ Collaborative Group: Polychemotherapy for early breast cancer: An overview of the randomised trials. Lancet 352:930-942, 1998
Mouridsen HT, Andersen J, Andersson M, et al: Adjuvant Anthracycline in Breast Cancer. Improved Outcome in Premenopausal Patients Following Substitution of Methotrexate in the CMF Combination with Epirubicin. Proc Am Soc Clin Oncol Oncol68a, 1999
Yamauchi H, Stearns V, Hayes DF: When is a tumour marker ready for prime time? A case study of c-erbB-2 as a predictive factor in breast cancer. J Clin Oncol 19:2334-2356, 2001
Ravdin PM: Is Her2 of value in identifying patients who particularly benefit from antracyclin during adjuvant therapy? A qualified yes. J Natl Cancer Inst 30:80-84, 2001
Jarvinen TA, Liu ET: Effects of HER-2/neu on chemosensitivity of tumour cells. Drug Resistance Updates 3:319-324, 2000
Cummings J, Smyth JF: DNA topoisomerase I and II as targets for rational design of new anticancer drugs. Ann Oncol 4:533-545, 1993
Withoff S, Keith WN, Knol AJ, et al: Selection of a subpopulation with fewer DNA topoisomerase II alpha gene copies in a doxorubicin-resistant cell line panel. Br J Cancer 74:502-507, 1996
Jarvinen TAH, Tanner M, Rantanen V, et al: Amplification and Deletion of Topoisomerase II{alpha} Associate with ErbB-2 Amplification and Affect Sensitivity to Topoisomerase II Inhibitor Doxorubicin in Breast Cancer. Am J Pathol 156:839-847, 2000
Jarvinen TA, Tanner M, Barlund M, et al: Characterization of topoisomerase IIa gene amplification and deletion in breast cancer. Genes Chromosomes Cancer 26:142-150, 1999
Jarvinen TA, Holli K, Kuukasjarvi T, et al: Predictive value of topoisomerase IIalpha and other prognostic factors for epirubicin chemotherapy in advanced breast cancer. Br J Cancer 77:2267-2273, 1998
Di Leo A, Larsimont D, Gancberg D, et al: HER-2 amplification and topoisomerase IIalpha gene aberrations as predictive markers in node-positive breast cancer patients randomly treated either with an anthracycline-based therapy or with cyclophosphamide, methotrexate, and 5-fluorouracil. Clin Cancer Res 8:1107-1116, 2002
Coon JS, Marcus E, Gupta-Burt S, et al: Amplification and overexpression of topoisomerase II predict response to antracycline-based therapy in locally advanced breast cancer. Clin Cancer Res 8:1061-1067, 2002
Olsen K, Knudsen H, Rasmussen BB, et al: Amplification of HER2 and TOP2A and deletion of TOP2A genes in breast cancer investigated by new FISH probes. Acta Oncol 43:35-42, 2004
Elston CW, Ellis IO: Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: Experience from a large study with long-term follow-up Histopathology 19:403-410, 1991
Schemper M, Smith TL: A note on quantifying follow-up in studies of failure time. Control Clin Trials 17:343-346, 1996
Park K, Kim J, Lim S, et al: Topoisomerase II-[alpha] (topoII) and HER2 amplification in breast cancers and response to preoperative doxorubicin chemotherapy. Eur J Cancer 39:631-634, 2003
Cardoso F, Durbecq V, Larsimont D, et al: Correlation between complete response to antracycline based chemotherapy and topoisomerase II- gene amplification and protein overexpression in locally advanced/metastatic breast cancer. Int J Oncol 24:201-209, 2004
Di Leo A, Larsimont D, Gancberg D, et al: Her-2 and topoisomerase II as predictive markers in a population of node-positive breast cancer patients randomly treated with adjuvant CMF or epirubicin plus cyclophosphamide. Ann Oncol 12:1081-1089, 2001
Muss HB, Thor AD, Berry D, et al: C-erbB-2 expression and response to adjuvant therapy in women with node-positive early breast cancer. N Engl J Med 330:1260-1266, 1994
Thor AD, Berry DA, Budman DR, et al: ErbB-2, p53, and efficacy of adjuvant therapy in lymph node-positive breast cancer. J Natl Cancer Inst 90:1346-1360, 1998
Paik S, Bryant J, Park C, et al: ErbB-2 and response to doxorubicin in patients with axillary lymph node-positive, hormone receptor-negative breast cancer. J Natl Cancer Inst 90:1361-1370, 1998
Paik S, Bryant J, Tan-Chiu E, et al: HER2 and choice of adjuvant chemotherapy for invasive breast cancer: National surgical adjuvant breast and bowel project B-15. J Natl Cancer Inst 92:1991-1998, 2000
Ravdin PM, Green S, Albain K, et al: Initial report of the SWOG biological correlative study of c-erbB-2 expression as a predictor of outcome in a trial comparing adjuvant CAF T with tamoxifen alone. Proc Am Soc Clin Oncol Oncol 17:1998
Moliterni A, Menard S, Valagussa P, et al: HER2 Overexpression and Doxorubicin in Adjuvant Chemotherapy for Resectable Breast Cancer. J Clin Oncol 21:458-462, 2003
Petit T, Wilt M, Velten M, et al: Comparative value of tumour grade, hormonal receptors, Ki-67, HER-2 and topoisomerase II alpha status as predictive markers in breast cancer patients treated with neoadjuvant anthracycline-based chemotherapy. Eur J Cancer 40:205-211, 2004
J?rvinen TA, Kononen J, Pelto-Huikko M, et al: Expression of topoisomerase IIalpha is associated with rapid cell proliferation, aneuploidy, and c-erbB2 overexpression in breast cancer. Am J Pathol 148:2073-2082, 1996
Tanner M, Jarvinen TA, Isola J: Amplification of HER-2/neu and topoisomerase IIa in primary and metastatic breast cancer. Cancer Res 61:5345-5348, 2001(Ann S. Knoop, Helle Knuds)
the Department of Pathology, Herlev Hospital, Herlev
the Department of Pathology, Roskilde County Hospital, Roskilde
the Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus
the DakoCytomation A/S, Glostrup
DBCG Secretariat, Copenhagen
the Department of Pathology, Odense University Hospital, Odense
the Department of Oncology, Copenhagen, Denmark
ABSTRACT
PURPOSE: The aim of the study was to evaluate the predictive value of HER2 and topoisomerase II gene (TOP2A) for the efficacy of epirubicin in the adjuvant setting of breast cancer patients.
PATIENTS AND METHODS: In the Danish Breast Cancer Cooperative Group trial 89D, 980 pre- and postmenopausal primary patients were randomly allocated to either CMF (cyclophosphamide, methotrexate, and fluorouracil; n = 500) or CEF (cyclophosphamide, epirubicin, and fluorouracil; n = 480) times 9, between January 1990 and November 1999. Tumor tissue was retrospectively identified from 805 patients and was analyzed for HER2-positivity and for TOP2A-amplifications and deletions.
RESULTS: HER2-positivity was found in 33% of the 805 investigated tumors and was not a predictive marker for epirubicin sensitivity. TOP2A changes were identified in 23% of the 773 investigated tumors: 12% had TOP2A amplifications and 11% had TOP2A deletions. We found that patients with TOP2A amplification had an increased recurrence-free (RFS; hazard ratio [HR], 0.43; 95% CI, 0.24 to 0.78) and overall survival (OS; HR, 0.57; 95% CI, 0.29 to 1.13), respectively if treated with CEF compared with CMF, and that patients with TOP2A deletions had an almost identical hazard ratio (RFS: HR, 0.63; 95% CI, 0.36 to 1.11; OS: HR, 0.56; 95% CI, 0.30 to 1.04). This is in contrast to patients with a normal TOP2A genotype for whom similar outcome was observed in both treatment arms (RFS: HR, 0.90; 95% CI, 0.70 to 1.17; OS: HR, 0.88; 95% CI, 0.66 to 1.17).
CONCLUSION: TOP2A amplification—and possibly deletion—seems to be predictive markers for the effect of adjuvant epirubicin containing therapy in primary breast cancer, but a final conclusion has to await a confirmative study or a meta-analysis.
INTRODUCTION
The Early Breast Cancer Trialists’ Collaborative Group’s overview analysis demonstrated a significant reduction in the risk of recurrence and death with anthracycline-containing regimens versus regimens without anthracyclines.1 The Danish Breast Cancer Cooperative Group (DBCG) trial DBCG-89-D is one such trial, and suggests that the substitution of methotrexate in the cyclophosphamide, methotrexate, fluorouracil (CMF) regimen, with epirubicin will lead to a further 25% to 30% proportional reduction in mortality.2 Despite the significant difference established in the group-wise comparison, the beneficial effect could be restricted to a minor part of the patients. Predictive factors would allow us to select those patients who would benefit the most from epirubicin, and thus spare the remaining patients from the additional toxicity.
A number of retrospective studies have investigated the predictive value of HER2 overexpression and/or amplification and have raised the hypothesis that patients with HER2-positive tumors have an inborn increased sensitivity toward anthracycline-containing therapy.3-5 But the results are somewhat nonconsistent and it has been hypothesized, that HER2 is rather to be viewed as a marker for the real anthracycline target topoisomerase II (topoII). The topoisomerase II gene (TOP2A) is located next to the HER2 gene on chromosome 17q12-q21.
TopoII is a key enzyme during cell division, where it creates a reversible double-strand DNA break allowing the two template stands to rotate freely and thus enable the passage of a second DNA double-strand through the break. Through binding to topoII the anthracyclines stabilize the DNA double-strand breaks which may lead to apoptosis, and topoII is probably the primary molecular target of anthracyclines.6 In vitro studies have hypothesized that amplification of TOP2A leads to overexpression of the topoII enzyme and increased sensitivity to anthracyclines7-9 and deletion to resistance.7 In a retrospective study, TOP2A changes were identified in 84% of 70 breast cancer patients with HER2 amplified tumors.10 Surprisingly deletions were almost as frequent as amplifications, and were observed concurrently in subsets of the tumors. The results from this pivotal study lead to exploration of the possible predictive value of TOP2A alterations for anthracycline-based chemotherapy in breast patients with HER2 amplified tumors, but a definitive answer has not been obtained.10-12
In a previous pilot-study, we observed TOP2A copy number changes in approximately 50% of HER2 amplified breast tumors and in 5% of nonamplified breast tumors.13 In the present study we investigated the predictive value of HER2-positivity and TOP2A changes in DBCG trial 89-D, a randomized comparison of intravenous cyclophosphamide, epirubicin, and fluorouracil (CEF) with CMF.
PATIENTS AND METHODS
January 1990 to November 1999, 1,195 patients were randomly assigned in the DBCG trial 89-D, hereof 980 Danish patients were eligible for this retrospective study, while data and tissue were not obtained from 215 Swedish patients. Patients were included if they were A: premenopausal, node-negative and with grade 2 or III tumors 5 cm; B: premenopausal with receptor-negative or unknown tumors, greater than 5 cm or with positive axillary lymph nodes; or C: postmenopausal with receptor-negative tumors, greater than 5 cm or with positive axillary lymph nodes. Following surgery (mastectomy, 79, 2% or lumpectomy, 19, 5% and axillary dissection) patients were randomly assigned in a factorial 2 x 2 design to either nine cycles of CMF (cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, and fluorouracil 600 mg/m2) given intravenously (IV) on day one every 3 weeks, nine cycles of CMF plus pamidronate, nine cycles of CEF (cyclophosphamide 600 mg/m2, epirubicin 60 mg/m2, and fluorouracil 600 mg/m2) given IV day once every 3 weeks, or nine cycles of CEF plus pamidronate. Pamidronate was given orally, 150 mg twice daily for 4 years. Six patients randomized to the CMF-arm received CEF and 26 patients randomized to CEF received CMF. Eighteen patients (13 randomly assigned to CEF and five randomly assigned to CMF) never received chemotherapy and were excluded from the predictive study. None of the remaining 962 patients received adjuvant tamoxifen. Locoregional radiotherapy was given after the first cycle of CMF or CEF, and during radiotherapy two cycles of CEF or CMF were substituted by single-agent cyclophosphamide (900 mg/m2). Radiotherapy was given against the residual breast following lumpectomy (48 Gy + boost 10 Gy) or chest wall following mastectomy if the tumor was greater than 5 cm (48 Gy), and against regional nodes in node-positive disease (48 Gy). In all cases, 2 Gy in five fractions per week. In the CMF and CEF groups, 206 (40.0%) and 173 (38.7%), respectively, received radiotherapy.
A number of variables were recorded prospectively, such as: age at surgery, histologic type (ductal carcinoma NOS, lobular carcinomas, and other types), malignancy grade,14 size (largest diameter of the invasive component), and numbers of removed and positive axillary lymph nodes.
Preparation of Tissue
Consecutive serial sections were cut at 4 μm from 805 available paraffin-embedded tumors for immunohistochemistry and FISH and stored cold until staining was performed. A slide stained with hematoxylin and eosin, prepared from each block, was used for confirmation of invasive carcinoma. All analyses were performed at the department of pathology, Roskilde Hospital, Denmark.
Immunohistochemistry
The sections were stained within 5 days from cutting. Certified laboratory staff using a Techmate immunostainer (DAKO) followed the procedure for the HercepTest (DakoCytomation, Glostrup, Denmark) according to the manufacturers protocol. Positive controls as supplied with the kit were included as well as in house controls together with a negative control for each case. The results were scored 0, 1+, 2+, and 3+ as recommended in the HercepTest.
FISH
Two new kits, HER2 FISH pharmDx and TOP2A FISH pharmDx (DakoCytomation, Glostrup, Denmark), were used according to the manufacturers’ manual, and as briefly described in the pilot study.13
In cases of ductal carcinoma with an in situ component, the negative control slide for HercepTest was used to mark the invasive areas avoiding any possibility of including the in situ component. Up to 60 gene signals (or the number closest to 60+) were counted in nuclei with identifiable boundaries. Optimally, only signals distinctly separated from each other were included, but in case of clusters due to high levels of amplification the number was estimated. The ratio was calculated as the number of signals for the gene probe (HER2 and TOP2A respectively) divided by the number of signals for the centromere 17. Cases were scored as HER2 or TOP2A FISH amplified when the ratio was 2. A TOP2A deletion is considered present when the ratio was less than 0.8 and this cut point was in the absence of a biologically well-defined ratio based on the work of Di Leo et al.11
HER2 FISH Analysis Was Performed on All HercepTest 2+/3+ Positive Specimens
All HER2 (3+) positive tumors and HER2 (2+) positive tumors with HER2 amplification investigated by FISH (fluorescence in situ hybridization; ratio 2) were considered HER2 positive.
Statistical Analysis
The primary outcome of interest was recurrence-free survival (RFS) calculated as the time from randomization until first locoregional recurrence, distant recurrence, second malignancy, or death, and overall survival (OS) calculated as the time from randomization until death. Follow-up time was quantified in terms of a Kaplan-Meier estimate of potential follow-up.15 Association between TOP2A-status and the classical prognostic variables and HER2 was investigated using contingency tables and 2 test. Survival curves were constructed according to the Kaplan-Meier product-limit method and compared using the log-rank test. Multivariate survival analysis was conducted using Cox proportional hazards models with backward selection.
The proportional hazard assumption was assessed graphically as well as by including a time-dependent component individually for each covariate. Finally, putative interactions of each covariate and the treatment parameter were investigated.
Receptor-status and malignancy grade were found to violate the proportional hazards assumption for both end points. Furthermore, nonproportionality was seen for TOP2A status and HER2-positivity with respect to the end point OS. For each end point, nonproportionality was taken into account by stratifying for those covariates that violated the proportional hazards assumption for that end point.
The backward selection procedure was applied separately to each analysis. All nonstratification variables, including treatment, were exposed to selection. If removed, treatment was re-entered into the reduced model after the selection procedure was completed.
The estimated effect of treatment with CEF within the three subgroups consisting of TOP2A amplified, TOP2A deleted, and TOP2A normal patients, and within the two subgroups consisting of HER2 positive and negative patients was investigated in a Cox proportional hazards models including parameters representing the interaction of treatment with CEF and patient subgroup and the estimated effect of CEF over CMF in these groups was compared.
The predictive value of a given characteristic was quantified by the hazard ratio (HR). The overall significance of interaction terms with two or more df was assessed by a Wald test.
All patient records were updated regarding disease status in August 2002 and death in December 2003.
With an event rate of 50% and an estimated 20% TOP2A positive patients,14 the number of events seems to be sufficient to detect a true clinical relevant difference. All tests were conducted with a 5% level of significance.
Ethics
The data used and presented were all collected and analyzed by the DBCG registry. The clinical study as well as the current biologic substudy was conducted according to the Helsinki declaration and approved by The Danish Scientific Ethical Committees (DBCG 89-D, v200.1616/89-V200.1623/89-12-089/01).
RESULTS
At the time of analysis, 217 (50%) patients treated with CMF and 207 (56%) treated with CEF and with available tumor tissue blocks (n = 805), were still alive without recurrence.
Median estimated potential follow-up time with respect to RFS was 8.06 years (95% CI, 7.77 to 8.46) for patients treated with CEF, and 8.09 years (95% CI, 7.61 to 8.63) for patients treated with CMF. Median potential follow-up time with respect to OS was 10.40 years (95% CI, 9.86 to 10.80) for patients treated with CEF and 10.28 years (95% CI, 9.71 to 10.61) for patients treated with CMF.
The classical prognostic variables were equally distributed in the two treatment arms (Table 1). The median relative dose-intensities of the chemotherapy were identical, 0.97, in the two treatment groups.
Blocks were unavailable for 175 (16%) of the 980 randomly assigned Danish patients (Fig 1). Premenopausal patients were overrepresented among patients where tumor blocks were unavailable as were patients with small tumors, receptor positive tumors, low malignancy grade, and one to three positive lymph nodes (2 test; P < .05). Age and treatment were distributed equally between the two groups. A log-rank test showed no difference in RFS or OS between the groups with and without available blocks (data not shown).
Amplification of TOP2A was seen in 93 of the 773 eligible patients (12.0%; CI, 9.9 to14.6) and deletion in 87 (11.3%; CI, 9.2 to 13.8; Table 2). Correlations between clinical and pathologic variables and TOP2A status are shown in Table 3. HER2 (3+) expression was seen in 246 (30.6%) of the 805 tumors and amplification of HER2 was seen in an additional 21% of the 81 HER2 (2+) positive tumors leading to 32.7% (CI, 29.5 to 36.0) HER2-positive tumors. Patients with HER2-positive tumors, were more often postmenopausal, had more than three positive lymph nodes and had negative ER-status (2 test; P < .001, data not shown). HER2-positivity was not correlated with increasing age, tumor size, or malignancy grade (2 test; P > .5, data not shown). TOP2A changes (amplifications/deletions) were seen in 140 (56.9% [CI, 50.5 to 63.1]) of the 246 HER2-positive TOP2A determined patients and in 40 (7.6% [CI, 5.5 to 10.3]) of the 527 HER2-negative TOP2A-determined negative patients. Thus 40 or 22% of the total number of TOP2A amplified or deleted patients would have been overlooked, if only HER2-positive samples had been analyzed for TOP2A alterations.
Univariate Analysis
Patients with TOP2A amplification or deletion and HER2-positivity had a significant reduction in RFS (Fig 2). Overall, the patients benefited from treatment with CEF (RFS; log-rank-test; P = .04, data not shown). However, univariate analysis could not identify any subgroup of patients (ie, patients with TOP2A-amplified or TOP2A-deleted positive tumors) that benefited specifically from treatment with anthracycline, although a trend was seen concerning patients with TOP2A-deleted and in particular amplified tumors (Fig 3). Only HER2-negative patients gained as a subgroup from treatment with CEF (Fig 4).
Multivariate Analysis
The Cox proportional hazards regression analysis of RFS was carried out according to treatment with CEF, menopausal status, tumor size, positive nodes, HER2-positivity and TOP2A status as selection variables. The putative interaction between TOP2A status and treatment with CEF with respect to RFS was investigated in a nonreduced Cox proportional hazards model for all patients that also included parameters representing the interaction.
In this model, the hazard ratio for treatment with CEF with respect to treatment with CMF was estimated at 0.63 (95% CI, 0.36 to 1.11) for TOP2A-deleted patients, 0.90 (95% CI, 0.70 to 1.17) for TOP2A-normal patients and 0.43 (95% CI, 0.24 to 0.78) for TOP2A-amplified patients (Fig 5). Pair-wise Wald tests show no difference in treatment HR between TOP2A deleted and TOP2A normal patients (W1 = 1.28; P = .26), whereas a significant difference in treatment HR was seen between TOP2A normal and TOP2A amplified patients (W1 = 5.09; P = .02). However, because of the low number of TOP2A altered patients an overall Wald-test fails to detect an overall TOP2A-status versus treatment interaction (W2 = 5.65; P = .06).
Corresponding analyses show benefit of treatment with CEF over CMF for both HER2-positive and negative patients, the hazard ratio is estimated at 0.79 (95% CI, 0.60 to 1.05) for HER2-negative patients and 0.75 (95% CI, 0.53 to 1.06) for HER2-positive patients. No interaction between treatment with CEF rather than CMF and HER2-positivity is seen (W1 = 0.6; P = .81)
After backwards selection was completed, only the following variables retained significance in the analysis of RFS for all patients: tumor size (cm), 1.147 (P < .0001; 95% CI, 1.081 to 1.216); positive nodes (1 to 3) 2.18 (P < .0001; 95% CI, 1.49 to 3.19); positive nodes (> 3) 5.03 (P < .0001; 95% CI, 3.47 to 7.27); HER2-positivity 1.37 (P = .007; 95% CI, 1.09 to 1.72); and treatment with CEF 0.78 (P = .03; 95% CI, 0.63 to 0.97).
The selection variables in the Cox proportional hazards analysis of OS were treatment with CEF, menopausal status, tumor size, and positive nodes. The computation of the hazard ratio with respect to OS for treatment with CEF rather than CMF within each TOP2A subgroup showed a nonsignificant increased OS for TOP2A amplified and TOP2A deleted patients, with HRs of 0.57 (95% CI, 0.29 to 1.13) and 0.56 (95% CI, 0.30 to 1.04), respectively (Fig 6). In the TOP2A-normal patients, no advantage of CEF compared with CMF was found (HR, 0.88; 95% CI, 0.66 to 1.17). Overall, no TOP2A-status versus treatment interaction was found (W2 = 2.56; P = .28).
Correspondingly, the hazard ratio for treatment with CEF rather than CMF for HER2-positive and HER2-negative patients was estimated at 0.73 (95% CI, 0.50 to 1.05) and 0.82 (95% CI, 0.59 to 1.13), respectively. No interaction between treatment with CEF and HER2 status was found (W1 = 0.23; P = .63).
DISCUSSION
In this large-scale study, we found a relative reduction in the risk of recurrence and death from breast cancer of around 50% in patients with primary operable and TOP2A amplified breast cancer that had been randomized to intravenous CEF compared to CMF. The results of the present study confirms the results of previous smaller studies that have suggested an increased sensitivity to anthracyclines in patients with TOP2A amplified breast tumors in the adjuvant11 and locally advanced setting.12,16,17 This in contrast to one negative neoadjuvant study16 using immunohistochemistry.
In addition we found an almost equally predictive value of TOP2A deletions, although with broad confidence-intervals—probably due to the low number of patients in this group. This finding was not expected, as the hypothesis was that deletions predicted anthracycline resistance. The clinical importance of TOP2A deletions has not been analyzed earlier, most likely since only a relatively small number of patients with TOP2A-deleted tumors have been identified in previous studies. The molecular mechanisms underlying the adjacent HER2 amplification and TOP2A gene deletion are unknown, as is the breakpoint, size, and functionality of the deleted TOP2A segment. The question of the predictive value of TOP2A deletion has to await a confirmative study or a meta-analysis, pooling data together from large-scaled phase III trails.
The predictive value of TOP2A-amplifications was not seen for the end point OS, probably because recurrence is a more precise measurement than death as patients often have received other treatment—containing anthracyclines—after their first relapse, and the effect of treatment therefore would confound the true predictive impact of the TOP2A status.
In the present study, the predictive value of TOP2A may have been less significant due to the use of methotrexate in the comparative regimen and the addition cyclophosphamide and fluorouracil in both treatment arms.
We found no evidence that the superiority of CEF over CMF with respect to RFS and OS was greater for HER2-positive patients than HER2-negative (ie, no interaction between HER2 status and treatment was seen.) Previous studies have reported inconsistent results concerning HER2 as a predictive marker for anthracycline-sensitivity, and the literature has recently been reviewed on several occasions.3-5 In brief, eight larger adjuvant studies have been published.11,18-25 Three studies found a significant interaction between treatment with anthracycline and HER2-status,19-2021 four studies found no interaction, but either a trend in the interaction analysis, or a risk reduction from anthracycline treatment in the HER2-positive patients.11,18,22-24 Two of the studies were repeated, with the same patient material, but with other antibodies, and no interaction was found.18,23
In the present study HER2 status was insufficient as a screening-tool for TOP2A amplifications or deletions, regardless of whether it was determined with IHC alone or a combination of IHC and FISH. Almost 8% of the HER2-negative patients had TOP2A changes and this would have been overlooked, had all the tumor samples not been analyzed. This is in accordance with the findings from our preceding pilot study where we found 4.4% (95% CI, 1.7 to 10.9) TOP2A amplifications or deletions among the 90 HER2 negative tumors.13 Prescreening with HER2 was originally proposed by J?rvinen et al26 in 1996, based on coexpression by IHC of topoII and HER2. Later the same group27 used FISH to study copy number aberrations of both TOP2A and HER2 in nine breast cancer cell lines and in 97 breast tumors, which were selected for the study according to their HER2 status by Southern blotting. Of 57 HER2-amplified primary breast carcinomas, 23 (44%) showed TOP2A coamplification and 24 (42%) showed a physical deletion of the TOP2A gene. No TOP2A alterations were found in 40 primary tumors without HER2 amplification. Di Leo et al11 estimated the prevalence of TOP2A amplification to be 6%, in a node-positive group of patients. We found the prevalence of TOP2A amplification in our study to be 12% and furthermore 11% had TOP2A deletions.
In conclusion, HER2-status gave no additional predictive information regarding anthracycline-containing chemotherapy. But in this large scaled randomized predictive study, we identified a group of patients with TOP2A amplifications, who benefited particularly from anthracycline-containing chemotherapy and found indications, although not statistically significant that an additional group of patients, the TOP2A deleted patients, also benefited from treatment with anthracycline. Based on the currently accepted biologic hypothesis, an increased sensitivity to anthracyclines was unexpected in patients with TOP2A deletions and confirmation should be awaited before implementation. A large-scale meta-analysis based on centralized reevaluation on TOP2A has already been organized, and samples from the current study will be included.
Authors’ Disclosures of Potential Conflicts of Interest
Acknowledgment
The skillful technical assistance of Vinni Bredahl and Birgit Hansen and the help from oncological and pathological departments in Denmark collecting patient data and tumor tissue is greatly appreciated.
NOTES
Supported by grants from Danish Cancer Society; Danish Medical Research Council; Danish Cancer Research Foundation; The Clinical Oncological Research Unit, Odense University Hospital; The A.P. Moeller Foundation; Dagmar Marshalls Foundation; Grosserer Georg Bjoerkner & Ellen Bjoerkners Fond; and Ambt Balslev’s Foundation. DakoCytomation, Glostrup, Denmark, made all kits available without costs and kindly provided a microscope.
Presented in part as an oral presentation at ECCO 12: The European Cancer Conference, Copenhagen, Denmark, September 21-25, 2003.
Authors’ disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Early Breast Cancer Trialists’ Collaborative Group: Polychemotherapy for early breast cancer: An overview of the randomised trials. Lancet 352:930-942, 1998
Mouridsen HT, Andersen J, Andersson M, et al: Adjuvant Anthracycline in Breast Cancer. Improved Outcome in Premenopausal Patients Following Substitution of Methotrexate in the CMF Combination with Epirubicin. Proc Am Soc Clin Oncol Oncol68a, 1999
Yamauchi H, Stearns V, Hayes DF: When is a tumour marker ready for prime time? A case study of c-erbB-2 as a predictive factor in breast cancer. J Clin Oncol 19:2334-2356, 2001
Ravdin PM: Is Her2 of value in identifying patients who particularly benefit from antracyclin during adjuvant therapy? A qualified yes. J Natl Cancer Inst 30:80-84, 2001
Jarvinen TA, Liu ET: Effects of HER-2/neu on chemosensitivity of tumour cells. Drug Resistance Updates 3:319-324, 2000
Cummings J, Smyth JF: DNA topoisomerase I and II as targets for rational design of new anticancer drugs. Ann Oncol 4:533-545, 1993
Withoff S, Keith WN, Knol AJ, et al: Selection of a subpopulation with fewer DNA topoisomerase II alpha gene copies in a doxorubicin-resistant cell line panel. Br J Cancer 74:502-507, 1996
Jarvinen TAH, Tanner M, Rantanen V, et al: Amplification and Deletion of Topoisomerase II{alpha} Associate with ErbB-2 Amplification and Affect Sensitivity to Topoisomerase II Inhibitor Doxorubicin in Breast Cancer. Am J Pathol 156:839-847, 2000
Jarvinen TA, Tanner M, Barlund M, et al: Characterization of topoisomerase IIa gene amplification and deletion in breast cancer. Genes Chromosomes Cancer 26:142-150, 1999
Jarvinen TA, Holli K, Kuukasjarvi T, et al: Predictive value of topoisomerase IIalpha and other prognostic factors for epirubicin chemotherapy in advanced breast cancer. Br J Cancer 77:2267-2273, 1998
Di Leo A, Larsimont D, Gancberg D, et al: HER-2 amplification and topoisomerase IIalpha gene aberrations as predictive markers in node-positive breast cancer patients randomly treated either with an anthracycline-based therapy or with cyclophosphamide, methotrexate, and 5-fluorouracil. Clin Cancer Res 8:1107-1116, 2002
Coon JS, Marcus E, Gupta-Burt S, et al: Amplification and overexpression of topoisomerase II predict response to antracycline-based therapy in locally advanced breast cancer. Clin Cancer Res 8:1061-1067, 2002
Olsen K, Knudsen H, Rasmussen BB, et al: Amplification of HER2 and TOP2A and deletion of TOP2A genes in breast cancer investigated by new FISH probes. Acta Oncol 43:35-42, 2004
Elston CW, Ellis IO: Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: Experience from a large study with long-term follow-up Histopathology 19:403-410, 1991
Schemper M, Smith TL: A note on quantifying follow-up in studies of failure time. Control Clin Trials 17:343-346, 1996
Park K, Kim J, Lim S, et al: Topoisomerase II-[alpha] (topoII) and HER2 amplification in breast cancers and response to preoperative doxorubicin chemotherapy. Eur J Cancer 39:631-634, 2003
Cardoso F, Durbecq V, Larsimont D, et al: Correlation between complete response to antracycline based chemotherapy and topoisomerase II- gene amplification and protein overexpression in locally advanced/metastatic breast cancer. Int J Oncol 24:201-209, 2004
Di Leo A, Larsimont D, Gancberg D, et al: Her-2 and topoisomerase II as predictive markers in a population of node-positive breast cancer patients randomly treated with adjuvant CMF or epirubicin plus cyclophosphamide. Ann Oncol 12:1081-1089, 2001
Muss HB, Thor AD, Berry D, et al: C-erbB-2 expression and response to adjuvant therapy in women with node-positive early breast cancer. N Engl J Med 330:1260-1266, 1994
Thor AD, Berry DA, Budman DR, et al: ErbB-2, p53, and efficacy of adjuvant therapy in lymph node-positive breast cancer. J Natl Cancer Inst 90:1346-1360, 1998
Paik S, Bryant J, Park C, et al: ErbB-2 and response to doxorubicin in patients with axillary lymph node-positive, hormone receptor-negative breast cancer. J Natl Cancer Inst 90:1361-1370, 1998
Paik S, Bryant J, Tan-Chiu E, et al: HER2 and choice of adjuvant chemotherapy for invasive breast cancer: National surgical adjuvant breast and bowel project B-15. J Natl Cancer Inst 92:1991-1998, 2000
Ravdin PM, Green S, Albain K, et al: Initial report of the SWOG biological correlative study of c-erbB-2 expression as a predictor of outcome in a trial comparing adjuvant CAF T with tamoxifen alone. Proc Am Soc Clin Oncol Oncol 17:1998
Moliterni A, Menard S, Valagussa P, et al: HER2 Overexpression and Doxorubicin in Adjuvant Chemotherapy for Resectable Breast Cancer. J Clin Oncol 21:458-462, 2003
Petit T, Wilt M, Velten M, et al: Comparative value of tumour grade, hormonal receptors, Ki-67, HER-2 and topoisomerase II alpha status as predictive markers in breast cancer patients treated with neoadjuvant anthracycline-based chemotherapy. Eur J Cancer 40:205-211, 2004
J?rvinen TA, Kononen J, Pelto-Huikko M, et al: Expression of topoisomerase IIalpha is associated with rapid cell proliferation, aneuploidy, and c-erbB2 overexpression in breast cancer. Am J Pathol 148:2073-2082, 1996
Tanner M, Jarvinen TA, Isola J: Amplification of HER-2/neu and topoisomerase IIa in primary and metastatic breast cancer. Cancer Res 61:5345-5348, 2001(Ann S. Knoop, Helle Knuds)