Alteration of topoisomerase II-alpha gene in human breast cancer: association with responsiveness to anthracycline-based chemotherapy

Abstract

Purpose: Approximately 35% of HER2-amplified breast cancers have coamplification of the topoisomerase II-alpha (TOP2A) gene encoding an enzyme that is a major target of anthracyclines. This study was designed to evaluate whether TOP2A gene alterations may predict incremental responsiveness to anthracyclines in some breast cancers.

Methods: A total of 4,943 breast cancers were analyzed for alterations in TOP2A and HER2. Primary tumor tissues from patients with metastatic breast cancer treated in a trial of chemotherapy plus/minus trastuzumab were studied for amplification/deletion of TOP2A and HER2 as a test set followed by evaluation of malignancies from two separate, large trials for changes in these same genes as a validation set. Association between these alterations and clinical outcomes was determined.

Results: Test set cases containing HER2 amplification treated with doxorubicin and cyclophosphamide (AC) plus trastuzumab, demonstrated longer progression-free survival compared to those treated with AC alone (P = .0002). However, patients treated with AC alone whose tumors contain HER2/TOP2A coamplification experienced a similar improvement in survival (P = .004). Conversely, for patients treated with paclitaxel, HER2/TOP2A coamplification was not associated with improved outcomes. These observations were confirmed in a larger validation set, where HER2/TOP2A coamplification was again associated with longer survival when only anthracycline-containing chemotherapy was used for treatment compared with outcome in HER2-positive cancers lacking TOP2A coamplification.

Conclusion: In a study involving nearly 5,000 breast malignancies, both test set and validation set demonstrate that TOP2A coamplification, not HER2 amplification, is the clinically useful predictive marker of an incremental response to anthracycline-based chemotherapy. Absence of HER2/TOP2A coamplification may indicate a more restricted efficacy advantage for breast cancers than previously thought.

Conflict of interest statement

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

Figures

Fig 1.

Specimen accountability in the H0648 test set clinical trial. This schematic diagram summarizes the number of women entered in each treatment arm of the H0648g pivotal clinical trial and the breast cancer specimens analyzed by fluorescent in situ hybridization (FISH) in each treatment arm. A, anthracycline (doxorubicin or epirubicin); C, cyclophosphamide; HER2-pos, HER2 gene amplification; HER2- neg, lacking HER2 gene amplification; TOP2A-pos, TOP2A gene amplification; TOP2A-neg, lacking TOP2A gene amplification including both TOP2A normals and TOP2A gene deletions.

Fig 2.

Overall survival of women with HER2-gene amplified metastatic breast cancer treated in the test set H0648 clinical trial with anthracycline-containing chemotherapy. (A) Women treated with doxorubicin and cyclophosphamide (AC) alone (n = 77) comparing those with TOP2A-amplified tumors (TOP2A fluorescent in situ hybridization [FISH] ratio ≥ 2.00; n = 27) with those whose tumors are not TOP2A amplified (TOP2A FISH ratio < 2.00; n = 50; log-rank test P = .004). (B) Overall survival of women with HER2-amplified metastatic breast cancer treated with AC alone chemotherapy compared with AC plus trastuzumab by TOP2A status. Three of the subsets have similar overall survival (women whose cancers were HER2/TOP2A coamplified and were treated with AC alone; women whose cancers were HER2/TOP2A coamplified and were treated with AC plus trastuzumab; women whose tumors were HER2 amplified but not TOP2A amplified and were treated with AC plus trastuzumab), which was significantly longer than the subset of women whose cancers were HER2 amplified but not TOP2A amplified and were treated with AC alone.

Fig 3.

Clinical outcome of women stratified by TOP2A status and by treatment arm in the Breast Cancer International Research Group (BCIRG) 006 clinical trial: comparison of disease-free survival (DFS) of women whose breast cancers lacked TOP2A gene coamplification and were treated on the doxorubicin, cyclophosphamide, and docetaxel (ACT) control treatment arm versus DFS of women whose breast cancers had TOP2A gene coamplification and were treated on the ACT control treatment arm and versus DFS of women whose breast cancers lacked TOP2A gene coamplification but were treated on either the doxorubicin, cyclophosphamide, docetaxel, and trastuzumab (ACTH) or the docetaxel, carboplatin, and trastuzumab (TCH) experimental treatment arms and with DFS of women whose breast cancers had TOP2A gene coamplification and were treated on either the ACTH or TCH experimental treatment arms. The number of patients and events in each treatment arm is listed for each arm. Comparisons of clinical outcome by TOP2A status (coamplified v not coamplified) and treatment arm are illustrated elsewhere. Table 3 provides the corresponding tests for treatment effects and interaction terms. The BCIRG-006 study compared two different experimental trastuzumab plus chemotherapy regimens with chemotherapy alone. The control arm (AC→T) consisted of four cycles of doxorubicin 60 mg/m2 plus cyclophosphamide 600 mg/m2 every 3 weeks followed by four cycles of docetaxel 100 mg/m2 every 3 weeks. Patients in the first experimental arm (ACTH) received the same chemotherapy with the addition of trastuzumab beginning with the first docetaxel dose followed by 2 mg/kg/week for 1 year. A second, nonanthracycline experimental arm (TCH) consisted of docetaxel 75 mg/m2 plus carboplatin at an area under the curve of 6 every 2 weeks for six cycles concurrently with trastuzumab. Trastuzumab was administered at 4 mg/kg for the first dose followed by 2 mg/kg/week until completion of chemotherapy, then at 6 mg/kg every 3 weeks to complete 1 year of treatment.

Fig A1.

Fluorescent in situ hybridization (FISH) of HER2 and TOP2A gene in breast cancer cells. (A) FISH of TOP2A gene in breast cancer without coamplification (TOP2A normal). (B) FISH showing HER2 gene amplification in the breast cancer from (A). (C) FISH showing chromosome 17 centromere in the breast cancer from (A). (D) Composite of (A), (B), and (C). (E) FISH of TOP2A gene in breast cancer with TOP2A coamplification. (F) FISH showing HER2 gene amplification in the breast cancer from (E). (G) FISH showing chromosome 17 centromere in the breast cancer from (E). (H) Composite of (E), (F), and (G).

Fig A2.

Specimen accountability for Breast Cancer International Research Group (BCIRG) 006 clinical trial. Although a total of 2,990 patients had TOP2A FISH data (1,057 coamplified + 1,788 TOP2A normal + 145 TOP2A deleted), only 2,948 patients (ie, safety population) were included in the outcome analyses. The safety population includes patients classified by treatment actually received; hence, this population differs slightly from the intent-to-treat (ITT) population. Forty-two patients (2,990 − 2,948 patients) were not included in the curves of Figure 3 or calculations shown in Table 3. The rationale for using the safety population instead of the ITT population was that for biomarker analyses, it makes sense to use the treatment patients actually received rather than that allocated by randomization. Nevertheless, because the difference between the ITT and safety populations was quite small (1% of patients), it did not make any substantial difference which of these two populations we used for analyses of TOP2A status compared with outcome; the conclusions are the same. ACT, doxorubicin, cyclophosphamide, and docetaxel; ACTH, doxorubicin, cyclophosphamide, docetaxel, and trastuzumab; TCH, docetaxel, carboplatin, and trastuzumab.