Relationship between Topoisomerase 2A RNA Expression and Recurrence after Adjuvant Chemotherapy for Breast Cancer Free

Purpose: To perform an exploratory analysis of the relationship between gene expression and recurrence in operable hormone receptor (HR)–positive, human epidermal growth factor receptor 2 (HER2)–normal breast cancer patients treated with adjuvant doxorubicin-containing chemotherapy.

Experimental Design: RNA was extracted from archived tumor samples derived from 378 patients with stage I to III HR-positive, HER2-normal breast cancer and analyzed by reverse transcription-PCR for a panel of 374 genes, including the 21-gene recurrence score (RS). Patients were randomized to receive adjuvant doxorubicin plus cyclophosphamide or docetaxel in trial E2197, with no difference in recurrence seen in the treatment arms. All available recurrent cases were selected plus a nonrecurrent cohort. Cox proportional hazard models were used to identify relationships between gene expression and recurrence.

Results: TOP2A expression exhibited the strongest association with increased recurrence risk (P = 0.01), and was significantly associated with recurrence (P = 0.008) in a multivariate analysis adjusted for clinicopathologic features. Elevated TOP2A expression above the median was associated with a 2.6-fold increase (95% confidence interval, 1.3-5.2; P = 0.008) in risk of recurrence if the RS was <18, and a 2.0-fold increase (95% confidence interval, 1.2-3.2, P = 0.003) if there was an intermediate RS of 18 to 30.

Conclusions: In patients with HR-positive, HER2-normal breast cancer, a population known to have a low incidence of TOP2A gene alterations thought to be predictive of anthracycline benefit, there is a range of TOP2A RNA expression that is strongly associated with recurrence after adjuvant anthracyclines, which provides information complementary to RS, indicating that it merits further evaluation as a prognostic and predictive marker. (Clin Cancer Res 2009;15(24):7693–700)

Adjuvant cytotoxic chemotherapy, including anthracyclines and taxanes, reduces the relative risk (RR) of recurrence in women with operable breast cancer (1, 2). Anthracycline therapy has been associated with more acute toxicity, and serious delayed toxicities such as congestive heart failure and leukemia (3). Other adjuvant systemic therapies that reduce recurrence include endocrine therapy for hormone receptor (HR)–positive disease (4) and trastuzumab for disease that overexpresses the human epidermal growth factor receptor 2 (HER2) protein (5). A meta-analysis including nine randomized trials indicates that only patients whose tumors overexpress HER2 protein benefit from anthracycline therapy (6), suggesting that the 75% to 80% of patients with HER2-normal disease may be subjected to the risks of anthracyclines without deriving incremental benefit. It is believed that the preferential sensitivity of HER2-overexpressing disease may be due to coamplification of the topoisomerase 2 gene (TOP2A), which resides on the same amplicon as HER2 and serves as the therapeutic target for anthracyclines (7). Several studies have indicated that TOP2A amplifications and/or deletions are associated with sensitivity to anthracycline therapy; although they occur in ∼40% of HER2-overexpressing tumors (8), they are present in <10% of tumors that are HER2 normal and HR positive (9). Moreover, a recent trial showed that the combination of docetaxel and cyclophosphamide reduced the risk of recurrence and death compared with the standard combination of doxorubicin and cyclophosphamide (AC; ref. 10), whereas another trial in a similar population showed no benefit for the doxorubicin-docetaxel combination compared with AC (11, 12), raising additional questions regarding the effectiveness of anthracyclines in HR-positive, HER2-normal disease.

In the current study that is the subject of this report, we evaluated the relationship between recurrence and a panel of 374 genes, including TOP2A and 21-genes included in the Oncotype DX Recurrence Score (Genomic Health, Inc.; ref. 13). The study population included 378 patients with HR-positive, HER2-normal breast cancer and 0 to 3 positive axillary nodes who were treated with doxorubicin plus either cyclophosphamide or docetaxel in addition to standard hormonal therapy (12). We restricted our analysis to only patients with HR-positive, HER2-normal breast cancer because this is precisely that population that is believed not to derive benefit from adjuvant anthracycline therapy. After finding that TOP2A RNA expression exhibited the strongest association with recurrence in our initial exploratory analysis, we sought to further evaluate the relationship between TOP2A expression and classic clinicopathologic features. We also evaluated whether it provided information that is complementary to the 21-gene assay, which does not include TOP2A. We chose to focus on TOP2A not only because of the strong association with recurrence shown in our exploratory analysis, but also because multiple previous studies have suggested a relationship between response to adjuvant doxorubicin-containing therapy and TOP2A protein expression or gene amplifications and/or deletions (9). Moreover, in contrast to previous reports that evaluated protein expression or gene alterations, this report describes use of quantitative measurement of TOP2A RNA by quantitative reverse transcriptase-PCR, which has been shown to be a reliable, reproducible, and quantitative method for evaluating gene expression (14). We report herein not only a strong relationship between TOP2A RNA expression and recurrence in HR-positive, HER2-normal breast cancer treated with anthracycline-containing therapy, but also evidence that TOP2A RNA expression provides information that is complementary to both classic clinicopathologic features and the 21-gene assay. Moreover, our study provides validation of a recent independent report in another robust data set demonstrating a significant relationship between TOP2A RNA expression evaluated by microarray and recurrence in HR-positive breast cancer (15).

The study used tumor specimens and clinical information from patients enrolled on trial E2197 (ClinicalTrials.gov⁷

identifier NCT00003519), coordinated by the Eastern Cooperative Oncology Group (ECOG), details of which have been reported elsewhere (12). Patients were randomly assigned to receive four 3-wk cycles of doxorubicin 60 mg/m² and cyclophosphamide 600 mg/m² (AC) or docetaxel 60 mg/m² (AT), plus endocrine therapy for 5 y or longer if HR positive. Tamoxifen (20 mg daily for 5 y) was recommended for HR-positive disease beginning after completion of chemotherapy when the trial was initiated, although ∼40% eventually took an aromatase inhibitor at some point before or after 5 y when it was shown that these agents were more effective than tamoxifen. After a median follow-up of 76 mo, there was no significant difference between the AC and AT arms in disease-free survival (the primary study end point; defined as recurrence or contralateral breast cancer), relapse-free interval (the end point used in this analysis; defined as recurrence), or overall survival in the entire study group, and in the population included in this analysis. All patients included in this analysis provided written informed consent for participation in the trial and use of their tissue specimens that was approved by the local institutional review board.

Because of the low overall recurrence rate in E2197, a stratified sampling design was used, with recurrences sampled more heavily than nonrecurrences. Patients were sampled separately within groups defined by recurrence status, HR status (as determined by local laboratories), axillary nodal status (positive versus negative) and treatment arm (AC versus AT), giving eight sampling strata with separate sampling from recurrences and nonrecurrences in each stratum. E2197 included 2,952 patients with operable breast cancer, of whom 1,579 patients were potentially eligible for inclusion in this analysis, with reasons for exclusion summarized in the Consolidated Standards of Reporting Trials (CONSORT) diagram shown in Supplementary Fig. S1. Of the 1,579 potential patients eligible for the sampling and analysis, 191 (12%) had a recurrence and 1,388 (88%) did not have a recurrence. All patients with recurrence were included in the sampling (defined as “case” sample), plus a randomly selected sample of 641 patients without recurrence (defined as the “control” sample, based on the planned ratio of 1:3.5 for the case-control sampling), yielding a total of 832 patients for the analysis. Although the sampling stratification was based on HR expression determined in local laboratories, the final classification of HR status in this analysis is based on central HR expression testing. Of the 832 patients identified, samples from each patient were sent from the ECOG Pathology Coordinating Office (PCO) to Genomic Health. The case and control sample was selected by the coordinating statistician (RG), and all specimens were processed by the ECOG PCO and Genomic Health without knowledge of the recurrence status and clinicopathologic variables. Within the set of 383 subjects with centrally confirmed HR-positive, HER2-normal disease analyzed for the full set of 374 genes, 324 genes have complete values and 47 are missing values for a small number of cases (median, 1; range, 1-5), including 5 cases that were missing values for TOP2A, yielding 378 patients included in this analysis. The missing values for an individual gene were excluded from the computation of the statistic for that gene. There is thus some small variation in the case set for the individual gene statistics.

The E2197 protocol was approved by the institutional review boards of all participating institutions and was carried out in accordance with the Declaration of Helsinki, current Food and Drug Administration Good Clinical Practices, and local ethical and legal requirements. ECOG designed and coordinated the study and was responsible for all aspects of the data collection and analysis. Other members of the North American Breast Cancer Intergroup participated and contributed patients to the study, including the Southwest Oncology Group, Cancer and Leukemia Group B, and the North Central Cancer Treatment Group. Only patients who gave consent for future research of their tumor specimen were included in the analysis. The use of specimens for this project was approved the North American Intergroup Correlative Science Committee and by the Northwestern University Institutional Review Board (which oversees the ECOG PCO, where the specimens were banked and evaluated).

All specimens underwent analysis for tumor grade and, for estrogen, progesterone, and HER2 expression, in a central laboratory as previously described (16). Briefly, all formalin-fixed, paraffin embedded tissue specimens were processed by the ECOG PCO and Reference Laboratory at the Robert H. Lurie Comprehensive Cancer Center of Northwestern University (Chicago, IL). One section at 5 μm was cut, followed by three 10-μm sections using sterile conditions with fresh blade and water per tissue block. The 5-μm slide section was stained with H&E and was reviewed by Genomic Health pathology using the Oncotype DX breast cancer assay criteria for acceptance and macrodissection (13). Tumor specimens were evaluated for histologic grade using the modified Bloom-Richardson score by a single pathologist (FLB), using the H&E-stained tissue sections, and by two pathologists (FLB, SB) for estrogen, progresterone, and HER2 expression. RNA was extracted from formalin-fixed, paraffin embedded tissue specimens by cutting three 10-μm-thick sections without dissection, or from macrodissected tumor from six 10-μm sections. Cut sections were placed into 1.5 mL RNase/Dnase free microfuge tubes (PGC Scientific), labeled with a barcode (blinding the sample), and shipped at ambient temperature to the reference laboratory at Genomic Health, Inc.

Cases with no cancer (depleted by prior tissue studies) or with cancer cells occupying <5% of the section area were excluded from the study. Macrodissection to obtain enriched tumor tissue was done using a safety blade cleaned with RNaseZAP (Ambion) on sections having nontumor elements [such as smooth muscle, fibrosis, hemorrhage, normal breast stromal tissue, but not ductal carcinoma in situ (DCIS) or lobular carcinoma in situ (LCIS) or necrosis] that were both sufficiently localized to be amenable to macrodissection and constituted >50% of the overall tissue area of the section.

A total of 20 tissue microarray (TMA) blocks were constructed by the ECOG PCO using two 1.0-mm cores per each patient tumor. Thin sections (4 μm) from tissue microarrays were immunostained by the ECOG PCO using DakoCytomation EnVision+ System (Dako) in a two-step technique. After deparaffinization, sections were rehydrated, and endogenous peroxidase was blocked with 1% H₂0₂ in methanol. Following heat-induced epitope retrieval [BioCare pressure cooker; citrate buffer (pH 6.0)], sections were incubated with anti–estrogen receptor (ER) antibody (clone 1D5; dilution, 1:100; Dako) or anti–progesterone receptor (PR) antibody (clone 636; dilution, 1:200; Dako) at room temperature. The reaction was visualized using Envision+ kit (Dako) and 3,3′-diaminobenzidine as chromogen followed by light counterstaining with hematoxylin. Positive and negative controls were used in each staining run. ER and PR expression was determined on tissue microarray sections by two pathologists (FLG, SB) simultaneously designating a proportion score (range, 0-5), intensity score (range, 0-3), and Allred score (Allred score = proportion score + intensity score; range = 0-8) for each case; an Allred score of >2 was defined as positive as previously described (17). HER2 expression was defined if there was intense membrane staining in at least 30% of cells using the DAKO Herceptest consistent with ASCO-CAP guidelines (18). Only patients with HER2-normal disease [negative or equivocal by American Society of Clinical Oncology-College of American Pathology (ASCO-CAP) guidelines] were include in this analysis. Seven cases in the total sample were not evaluable on central immunohistochemistry; ER and PR status for these cases was determined using local results and HER2 status using genomic results.

Quantitative RNA expression levels were measured by real-time reverse transcriptase-PCR using gene-specific primers (14). All specimens were also analyzed for the 21 genes in the Oncotype DX Recurrence Score (in triplicate) as previously described (13), plus a panel of 353 other genes (run in single wells), including TOP2A. Gene amplification/deletion analysis was not conducted being already shown from other studies that the incidence is very low in this phenotype (<5-10%). The genes selected were assembled by searching the published literature, genomic databases, pathway analysis, and microarray-based gene expression profiling experiments performed in fresh-frozen tissue to identify genes likely to be associated with prognosis or response to chemotherapy (see Supplementary Table S1; includes the Human Gene Organization name).

Because there was no difference in the recurrence rate or overall survival between the two treatment arms, the analysis used samples from both arms. The primary end point for this analysis was recurrence-free interval, defined as the time from trial entry to the first evidence of breast cancer recurrence (which included invasive breast cancer in local, regional, or distant sites, including the ipsilateral breast, but excluded new primary breast cancers in the opposite breast; ref. 19).

Unlike the standard case-cohort design, only a subset of the recurrences from the E2197 study is included in the sample here. To estimate the magnitude of effects in the full E2197 population, sampling weights for each of the 16 groups in the sample are defined by the number of patients in the E2197 study in that group divided by the number in the sample. In the weighted analyses, contributions to estimators and other quantities, such as partial likelihoods, are multiplied by these weights. If the patients included in the case-control sample are randomly selected from the recurrences and nonrecurrences within each stratum, then the weighted estimators give consistent estimates of the corresponding quantities from the full E2197 sample. Because availability and analyzability of tissue samples was a factor in the selection, the possibility of systematic bias in the selection cannot be completely ruled out, but the comparisons in Table 1 of Goldstein et al. (11) using this same data set suggests that for many purposes, weighted analysis of the case-control sample should be representative of the full E2197 study. The weighted partial likelihood computed in this fashion is used for estimating hazard ratios and testing effects. The variance of the partial likelihood estimators is estimated using the general approach of Lin (20), which leads to a generalization of the variance estimator from Borgan et. al (21) to allow subsampling of cases. Weighted averages, with proportions estimated using weighted averages of indicator variables, are also used for estimating the distribution of factors and for comparing the distributions between the overall E2197 study population and the genomic sample. Tests comparing factor distributions are based on asymptotic normality of the difference in weighted averages. The adjusted variance based on Lin was used in the score statistics (20), and additional details of the weighted analysis methods are described by Gray (22).

Weighted Cox proportional hazards model score tests were used to rank genes by their individual significance for predicting recurrence risk by one of the collaborating statisticians (RG). The significance level was determined from Cox model score statistics using the available data for each gene as a single linear covariate without adjustment for other factors. Adjusted P values controlling the false discovery proportion (FDP) at ≤10% were computed using algorithm B* in Korn et al. (23), using 500 permutations. The P values were applied in a “step-down” fashion, which was the only approach guaranteed to give the stated level of control in the simulations of Korn et al. (24). The adjusted P values give the level of confidence that the false discovery proportion is ≤10%, in the sense that the P value is the proportion of experiments where the true false discovery proportion is expected to exceed the stated rate. Genes were ranked by significance levels, or equivalently by values of the test statistics. The test statistics give a ranking of strength of association that is invariant to differences in scale for different genes. The test statistics also directly relate to the strength of evidence of whether the genes affect the hazard rate for recurrence, although this criterion is somewhat arbitrary and not the only possible scale invariant measure of association. Weighted analysis of proportional hazards models was used to estimate hazard ratios and test for effects in joint models, and weighted Kaplan-Meier estimators of recurrence-free interval distributions were used to estimate recurrence rates; the weighting algorithm corrects for any potential bias in sampling of the nonrelapse cohort population. All P values are two sided.

There were 378 patients who had centrally confirmed HR-positive, HER2-normal disease and had genomic information available for TOP2A expression, of whom 77 had recurrences; their characteristics are shown in Table 1. The weighted distribution of recurrence scores (RS) was 51.0% in the RS < 18 group, 32.4% in the RS 18 to 30 group, and 16.6% in the RS ≥ 31 group. The median follow-up for the study population was 6.3 years, and the 5-year relapse-free interval and overall survival rates were 90.4% and 93%, respectively.

Continuous RS, without considering other variables, was a highly significant predictor of recurrence (P = 0.004 overall). When genes were considered individually, without adjusting for effects of other factors, there were 10 genes significantly (adjusted P < 0.05, controlling the false positive rate at 10%) associated with increased risk of recurrence (Table 2), including TOP2A, DEPDC1, NUSAP1, AURKB, KIFC1, GAPDH, BUB1B, BIRC5, TYMS, and PLK1. TOP2A exhibited the strongest association with increased risk of recurrence (unadjusted P = 0.000006; Korn's adjusted P = 0.01). There were 40 genes in the 374-gene set with correlations with TOP2A that were >0.5 (see Supplementary Table S2). The highest correlations were for proliferation related genes, including MKI67, BIRC5, CCNB1, MYBL2, and AURKA, which comprise all five of the genes included in the proliferation group of the Oncotype DX RS. The correlation coefficient between TOP2A and the proliferation group was 0.84, suggesting that some of the relationship between TOP2A and increased recurrence risk may be due to the association with proliferation. Furthermore, of the nine genes other than TOP2A for which increased expression correlated with an increased recurrence risk, expression of all but one gene (GAPDH) correlated with TOP2A expression, with six of the remaining eight genes being highly correlated (r ≥ 0.70). This high degree of overlap between genes associated with recurrence and genes whose expression correlated with TOP2A expression provides further support for our approach of focusing on TOP2A expression in our analysis.

The relationship between TOP2A expression and recurrence was evaluated as a continuous variable using a flexible family of curves (a natural spline with 3 degrees of freedom) to model the effect of TOP2A in a proportional hazards model for recurrence. The estimated curve, with 95% confidence interval (95% CI), shows substantial increasing trend over most of the range between TOP2A expression and increasing recurrence risk (Fig. 1).

The relationship between clinical and genomic features, recurrence, and TOP2A expression was also evaluated as a categorical variable. The median split (value = 6.23) for TOP2A was used because it appeared that the slope of the estimated recurrence log hazard ratio function was near its maximum at the median value (Fig. 1). The range of TOP2A values in the low expression group was 2.90 to 6.23, and in the high expression group, the range was 6.23 to 10.41, with 189 cases in each group. Clinical variables examined included age, nodal status, centrally determined tumor grade, and tumor size (Table 3). TOP2A expression was significantly more likely to be high in tumors associated with poor grade (P < 0.0001) and high RS (P < 0.0001). The relationship between TOP2A expression and centrally determined ER and PR expression quantitated by the Allred score was also evaluated. Tumors with a very low ER and PR Allred Scores of 0 to 2 were significantly more likely to have high TOP2A expression (P = 0.004), whereas tumors with a high PR score of 8 (P = 0.003) were more likely to exhibit low TOP2A expression (full data set not shown).

Cox proportional hazards models were fit to examine the joint effects of factors on recurrence rates (Table 4). The models included the factors of age, nodal status, centrally determined tumor grade, and tumor size. In model I, which did not include TOP2A expression, features associated with an increased risk of recurrence included young age (P = 0.05), two to three positive axillary lymph nodes (P = 0.001), and poor grade (P = 0.002). Model II added TOP2A as a continuous linear variable to model I; TOP2A x + 5 versus x was used for the hazard ratio, where x is an arbitrary value of TOP2A [comparable with the analysis of RS as continuous variable in the report by Paik et al. (13)]. In this model, TOP2A expression was a highly significant predictor for increased recurrence (hazard ratio, 5.01; 95% CI, 1.53-16.43; P = 0.008). Model III added TOP2A (high versus low, using the median split) to model I. In this model, there was a very strong trend of high TOP2A expression being associated with an increased risk of recurrence (hazard ratio, 1.76; 95% CI, 0.98-3.15; P = 0.06). The corresponding models with RS were also fit as both a continuous and categorical variable, as RS is used for clinical decision making using both scenarios. The models including RS yielded similar results, with TOP2A expression being significantly associated with recurrence when analyzed as a continuous variable, and showing a strong trend if evaluated as categorical variable (see Supplementary Tables S3 and S4).

The relationship between RS risk groups and TOP2A expression is shown in Table 5, including the standard and TAILORx risk group definitions (25). The 5-year risk of recurrence for all patients was 4.8%, 14.6%, and 14.5% for patients with low, intermediate, and high RS, respectively, using the classic definitions. The RR of recurrence was significantly higher in the high versus low risk (RR, 3.0; P < 0.0001), and intermediate versus low (RR, 3.0; P < 0.0001), but not high versus intermediate. Similar trends were noted if the TAILORx definitions were used. Thus, in this chemotherapy-treated population, recurrence rates were significantly higher for high or intermediate compared with low RS (but not when comparing high versus intermediate RS).

We evaluated the impact of TOP2A expression on recurrence rates in the RS risk groups. For patients with low TOP2A expression, the risk of recurrence for tumors with low, intermediate, and high RS using the standard RS definitions was 3.2%, 9.9%, and 13.7%, respectively. For tumors with high TOP2A expression, the corresponding risk of recurrence was 8.1%, 19.7%, and 14.8%, respectively. Therefore, high TOP2A expression was associated with a 2.6-fold increased risk (95% CI, 1.3-5.2; P = 0.008) of recurrence for tumors with a low RS of <18, and a 2.0-fold increased risk (95% CI, 1.2-3.2; P = 0.003) of recurrence if there was an intermediate RS of 18 to 30. TOP2A expression was increased in 33% of those with a low RS, and 50% of those with an intermediate RS. Notably, most tumors that had a high RS also exhibited high TOP2A expression. The results were similar if the TAILORx risk group definitions were used for the midrange RS group of 11 to 25 (2.2-fold increased risk; P = 0.0005) and high-risk RS group of >25 (no difference), but provided greater discrimination of the low RS group of <11 (9.3-fold increased risk; P = 0.005).

There was no difference in recurrence rate between the two treatment arms of AC and AT in the overall study population (12), nor in the subgroup included in this analysis. When TOP2A was analyzed as a continuous (linear) variable, then in a model with treatment, TOP2A, and their interaction, the interaction was not significant (P = 0.27). When TOP2A was analyzed as a categorical variable using the median split, the interaction was again not significant (P = 0.08). To the extent that there was a suggestion of interaction, the treatment difference favored AC for low TOP2A and favored AT for high TOP2A expression. Alternatively, the TOP2A effect is somewhat weaker with AT than with AC.

We performed an exploratory analysis evaluating the relationship between recurrence and panel of selected genes in patients with HR-positive, HER2-normal breast cancer who received standard doxorubicin-containing chemotherapy plus hormonal therapy, and who were followed for at least 5 years. The approach used was not a genome-wide screen, but rather was limited to a panel of 374 genes that were selected because of their known or postulated association with prognosis or response to chemotherapy; the genes were identified by a search of the published literature, genomic databases, pathway analyses, and microarray-based gene expression profiling experiments performed in fresh-frozen tissue. We found that of the 374 genes evaluated, increased expression of only 10 genes was associated with an increased risk of recurrence, with TOP2A exhibiting the strongest association, and with most of the other significant genes highly correlated with TOP2A expression. We also found that although increased TOP2A RNA expression correlated highly with poor tumor grade and high RS, its expression nevertheless remained highly associated with recurrence when adjusted for classic clinicopathologic features (including grade) and with RS. Our findings confirm the report by Rody et al. (15), who recently reported an analysis of Affymetrix microarray data from 1,681 breast cancer patients, which revealed that higher TOP2A expression significantly correlated with tumor size, poor grade, HER2 expression, and positive lymph nodes, and was associated with a poorer survival in ER-positive, HER2-normal disease (P = 0.001), but not ER-negative disease. The association between TOP2A gene expression and recurrence was independent of whether patients were untreated or had received adjuvant therapy, and was the single most important prognostic factor in a multivariate model (hazard ratio, 2.40; 95% CI, 1.68-3.43; P < 0.001). Our findings not only confirm this report, but also show that TOP2A RNA expression provided information that was complementary to prognostic information provided by the Oncotype DX Recurrence Score in those with a low and midrange score.

There are no predictive markers that reliably identify which patients with operable breast cancer benefit from adjuvant anthracycline therapy. In HR-positive, HER2-normal breast cancer, the most common phenotype that comprises about two thirds of all breast cancers, it has been suggested that anthracyclines are probably not beneficial because of the low incidence of TOP2A gene amplifications and/or deletions that are predictive of anthracycline benefit (9). In this analysis, we included only patients with HER2-normal disease as defined by immunohistochemistry in a central laboratory, a population expected to have a low likelihood of harboring TOP2A gene alterations. The strong association between TOP2A RNA expression with recurrence suggests that higher RNA expression of TOP2A measured by quantitative reverse transcriptase-PCR may be associated with resistance to anthracycline treatment in this population; alternatively, it may identify individuals who are resistant to chemotherapy in general, or destined to have a poor prognosis irrespective of therapy. In other settings such as colorectal and pancreatic cancer, high tumor expression of the target enzyme (thymidylate synthetase) has been associated with resistance to therapy (5-fluorouracil; refs. 26–28). The results were similar in this analysis, in that high TOP2A RNA expression was associated with apparent resistance to doxorubicin-containing therapy. It is noteworthy that although TOP2A expression correlated with the Oncotype DX proliferation genes, and higher TOP2A expression was associated with resistance to doxorubicin-containing chemotherapy, high proliferation scores in other studies have been associated with resistance to tamoxifen (13) and greater sensitivity to doxorubicin-containing (29) and nondoxorubicin-containing (30) chemotherapy regimens. Based on our results and other work, we hypothesize that low TOP2A expression may identify a group of tumors that may be particularly sensitive to anthracycline therapy, and that high TOP2A expression may identify tumors that are resistant to anthracyclines, and which may be more sensitive to taxanes. This hypothesis requires testing and validation in other trials.

In this analysis that included only patients with HR-positive, HER2-normal disease, we found a statistically significant association between the RS groups (i.e., low, intermediate, and high) and risk of recurrence (Table 5). Although there was no association between RS analyzed as a continuous variable over its entire range and recurrence in the multivariate analysis (Supplementary Table S3), a previously reported analysis of this same data set indicated a significant association between RS and recurrence when evaluated as a continuous variable between a RS of 0 and 40, but not above 40 (11). This is consistent with previous studies demonstrating greater chemotherapy benefit in patients with an elevated RS treated with nonanthracycline-containing (30) and anthracycline-containing adjuvant chemotherapy (29). This also explains its lack of association with recurrence when evaluated as a continuous variable over the entire range of RS, especially in a population with HER2-normal disease (because increased HER2 expression contributes to high RS). In particular, TOP2A expression level provided information for patients with an intermediate (“classic” definition) or midrange (TAILORx definition) RS, a range in which there is considerable therapeutic uncertainty regarding whether chemotherapy is beneficial; this uncertainty has led to initiation of the “TAILORx” trial (25, 31).

This analysis had a number of important strengths, including the central immunohistochemistry testing for HRs and HER2, the specific phenotype evaluated (HR positive, HER2 normal), the method for evaluating TOP2A (RNA expression), and treatment administered (anthracycline therapy). There are also several important limitations. First, the median follow-up of 6.3 years may be of insufficient duration in patients with HR-positive disease, who frequently have late recurrences. Second, absence of a control group that did not receive chemotherapy makes it impossible to determine whether TOP2A is prognostic, predictive of therapeutic benefit, or both. Third, the absence of a nonanthracycline treatment arm makes it impossible to determine whether increased TOP2A expression is prognostic, predictive of benefit for any standard cytotoxic regimen, or predictive of benefit specifically for anthracycline-containing chemotherapy. In addition, although the exploratory nature of our analysis was preplanned, the chosen cut off point for TOP2A expression (i.e., the median value 6.23) was not prespecified, and the predictive accuracy may be overestimated. Although these questions cannot be answered from this data set, they may be addressed by evaluation of archival specimens from other completed studies.

In conclusion, we have shown in this exploratory analysis that evaluation of TOP2A RNA expression in HR-positive, HER2-normal operable breast cancer treated with adjuvant anthracycline-containing therapy provides information that is complementary to classic clinicopathologic information, and to validated genomic predictors that include proliferation genes such as the Oncotype DX RS. Evaluation of TOP2A RNA expression may be particularly useful in situations whether there is therapeutic equipoise, such as patients with an intermediate RS. We view these finding as hypothesis generating, and believe that additional studies are warranted in this specific patient population and perhaps other settings.

J. A. Sparano, consultant, Sanofi-Aventis. S. Shak, F. Baehner, and R. Bugarini are employed by Genomic Health; B. Childs, D. Brassard, and S. Rowley are employed by Sanofi-Aventis. The other authors disclosed no potential conflicts of interest.

We thank Adekunle Raji and other staff members at the Eastern Cooperative Oncology Group PCO at the Robert H. Lurie Comprehensive Cancer Center, Chicago, IL.