Even though the adverse prognostic role of HER2 parameters (gene amplification and mRNA and protein overexpression) in patients with operable breast cancer is indisputable, that of TOP2A parameters remains controversial. Moreover, the optimal parameter(s) and respective methodologies for assessing HER2 [23, 34, 35] and TOP2A [7, 36] status are still not well defined. Concerning methods, in this study we employed CISH for the assessment of HER2 and TOP2A gene status. CISH is an accepted method for the evaluation of the HER2 gene  but it should be noticed that, while CISH and FISH results are highly concordant in true negative (diploid) and highly amplified tumors, the two methods yield discordant results in cases with low HER2 amplification . Further, CISH is not the method for reporting gene deletions [16, 38], hence we did not register any TOP2A deletions like those reported with FISH [33, 39–41]. In terms of TOP2A gene amplification, the incidence with CISH in our cohort (7%) was low but still comparable to previous reports assessing this parameter with FISH [33, 42],. while TOP2A amplified/HER2 non-amplified tumors have also been reported with FISH . It seems that, concerning the amplification part of TOP2A gene pathology, CISH results may be considered comparable with the much more abundant FISH results in the literature.
It is noteworthy that information in the literature regarding a thorough evaluation of both HER2 and TOP2A at all levels of molecular pathology (gene, RNA, protein) is very limited. To our knowledge the present study is one of the few attempting to define the relationship between TOP2A gene copy numbers with TOP2A RNA and TopoIIa protein expression and possibly the first in the context of an adjuvant phase III trial including patients with breast cancer treated with anthracyclines. In a report by Brase et al , the prognostic relevance of the HER2 and TOP2A genes was comprehensively studied at the DNA, RNA and protein level in three independent patient cohorts. It is of interest, that although this report differs from ours substantially in terms of design, patient population and treatment, both studies share a considerable number of common findings. In the Brase et al study , high TOP2A RNA levels were significantly associated with shorter metastasis-free survival in node-negative (low-risk) patients who did not receive adjuvant chemotherapy, but the same parameter seemed to be associated with better response to anthracyclines. In the present study, all patients had received epirubicin; in this "anthracycline-treatment homogeneous" population, where the parameter "anthracyclines" may be eliminated, high TOP2A mRNA expression was an adverse prognostic parameter for patient OS, a function that remained significant after adjustment for hormone receptor status, nodal involvement, Ki67 labelling and HER2 status. Thus, the present data support an unfavorable long-term prognostic role for TOP2A mRNA expression in high-risk patients who received adjuvant treatment.
In contrast to TOP2A mRNA expression, TOP2A gene amplification was independently associated with prolonged DFS in our patients, in line with repeated reports pointing out TOP2A gene amplification as a marker predicting for response to anthracyclines [42–47]. Of note, since, as shown, tumors with TOP2A gene amplification did not necessarily express high TOP2A mRNA levels and vice versa, the analyzed groups of patients with TOP2A gene amplification and high TOP2A mRNA expression overlapped to a small degree (8%) but certainly did not match. The effect of TOP2A amplification on patient outcome depends on the setting examined and seems relevant mainly in HER2-positive tumors, as revealed in the largest series tested so far for both HER2 and TOP2A gene amplification . The incidence of TOP2A-amplified tumors within the HER2-positive group was relatively small in the present study (21%), instead of the ~35% reported by Press et al . Even in this small group of patients, HER2/TOP2A co-amplification was revealed as an independent good prognostic factor for DFS in multivariate analysis; since DFS is treatment-related, this favorable prognostic value of HER2/TOP2A co-amplification might be considered in line with the earlier described positive predictive value of this marker for anthracyclines. Whether TOP2A itself or some other gene in the same chromosomal region, which is also detected by the large CISH/FISH probes, is responsible for the favourable prognostic value of HER2/TOP2A co-amplification remains to be elucidated.
As discussed above, two different TOP2A parameters, gene amplification and mRNA expression, seem to have a distinct impact on the outcome of breast cancer patients treated in the adjuvant setting. Although this appears as a paradox, it is fully compatible with the descriptive data presented in the present study concerning associations among TOP2A parameters: TOP2A mRNA and TopoIIa protein expression were strongly associated with each other but largely unrelated to TOP2A gene status. In comparison, all HER2 parameters (gene status, mRNA and protein expression) were strongly interrelated in our series, in accordance to most published data so far (comprehensively reviewed in ). The lack of associations between TOP2A gene status and TOP2A gene products (RNA and protein) has repeatedly been reported [27, 36, 43, 46, 49–51] and does not seem surprising considering key issues in TOP2A regulation and function. There is increased demand for TopoIIa protein during DNA replication , hence, the corresponding gene is transcribed independently of the existing copy numbers or of activated oncogenes . In addition, TOP2A transcription may be strongly downregulated by wild type p53 , or strongly upregulated by HMGB1 and HMGB2 , while the half-life of TOP2A mRNA may depend on redox-sensitive protein complexes . Biologically, the effective molecule is the protein and not its precursor nucleic acids but a prognostic role for TopoIIa protein could not be demonstrated so far in large patient series, probably because of differences in the performance and evaluation of immunohistochemical assessments (reviewed in ). Nevertheless, TOP2A mRNA levels were strongly associated with TopoIIa protein expression in our series, which at least indicates effective translation of TOP2A mRNA in the majority of the cases.
In line with the role of TOP2A in cell proliferation (reviewed in [12, 58]), we observed strong associations of the proliferation marker Ki67 and tumor grade (which is characterized by Ki67 labelling) with TopoIIa protein and TOP2A mRNA levels. High Ki67 scores coinciding with TopoIIa protein (over)expression have been reported for breast carcinomas as reflecting tumor proliferation status [50, 52, 57, 59], while the adverse prognostic impact of Ki67 in breast cancer is well established (reviewed in ). With respect to the strong association between Ki67 and TOP2A mRNA, which was also reported in a microarray profiling study , it might be argued that the herein observed adverse prognostic value of TOP2A mRNA reflects its strong association with high proliferation rates. This may in part be true, as previously suggested for TopoIIa protein as well . However, while Ki67 was an unfavorable prognosticator for both DFS and OS, the adverse prognostic effect of TOP2A mRNA was limited to OS. In fact, this latter finding was the only contrasting point between the present study involving high-risk early breast cancer in comparison to low-risk early breast cancer, where TOP2A mRNA was unfavourably associated with metastasis-free survival . To understand the long term influence of TOP2A expression on patient outcome, we need to consider newer functional aspects of TopoIIa protein and how these affect the fate of cancer cells when attacked by cytotoxic agents, such as anthracyclines (reviewed in [58, 63]). It seems that, according to its molecular environment (among other parameters, TopoIIb and DNA repair capacity), TopoIIa protein may contribute to the large array of genomic aberrations observed in advanced cancers, including alterations of the TOP2A gene itself, as they are found at increasing rates with progressing tumor size ( and present study). At present, several questions regarding TOP2A in cancer cells remain unanswered, such as, how are TOP2A amplified genes regulated, whether genes are amplified in one piece or in fragments, or whether they lack regulatory regions, what are the genomic profiles of relapsed/metastatic vs. primary tumors, especially if treatment has been administered, and so on. Genomic profiles of higher resolution than obtained with FISH/CISH probes (which detect additional genes than the ones they are meant for anyway) would be required for these studies, while interesting data on genomic patterns in relevance to breast cancer subtypes have already been offered  and await further evaluation.
In terms of predicting response to paclitaxel, none of the TOP2A parameters examined was related to patient outcome. This was somehow expected, since the addition of this drug to the epirubicin containing treatment schemes did not offer a significant advantage for disease-free or overall survival in the same patient cohort , while no association was observed between TOP2A status and response to taxanes in the neo-adjuvant setting as well . HER2 status (gene amplification and/or protein overexpression) and HER2 mRNA expression were not related to benefit from the taxane treatment in the present study. Relevant HER2 mRNA expression data could not be retrieved from the literature. The present HER2 status data seem to be in contrast to previous reports [8, 66], as well as to the findings from the CALGB 9344/INT0148 adjuvant trial on node-positive patients who were treated with doxorubicin/cyclophosphamide with or without paclitaxel, probably due to clinical context, methodological and treatment administration differences. In addition, Ki67 was also not associated with response to paclitaxel in our series, which does not support a previously suggested taxane-predictive role of this marker .
The adverse prognostic impact of high HER2 mRNA expression, for the same patient cohort, has already been published by our group . In comparison to HER2 mRNA, HER2 status was associated with patient outcome as a single variable but lost its prognostic significance upon multivariate analysis, in line with previous reports in the same treatment setting [39, 68, 69]. It seems that HER2 mRNA remains the most significant prognostic HER2 parameter, and various assays for the relative quantification of HER2 mRNA expression by qRT-PCR, in the single [26, 48, 70] or multiplex [71, 72] mode have already been developed and evaluated, while HER2 mRNA is included in the Recurrence Score obtained by Oncotype DX . In this study, high HER2 mRNA expression was an independent unfavorable prognostic factor, especially in terms of predicting relapse. Most interestingly, though, when examining high mRNA expression for both HER2 and TOP2A as a binary variable in the same tumor, this co-expression marker was strongly associated with shorter disease-free and overall survival, while it emerged as a new independent adverse prognostic factor in adjuvantly treated breast cancer patients. Of note, although the HER2 part of the high HER2/TOP2A mRNA co-expression marker is related to HER2 gene amplification, the TOP2A part is evidently not, as described throughout this manuscript. The biological background underlying this interaction is currently unknown and may be related to the conditions driving TOP2A (over)expression in the absence of gene amplification, as described above. It should also be noted that this combined marker is based on cohort-dependent cut-offs that were set for defining high mRNA expression for HER2 and TOP2A; hence, its validation in independent and larger patient cohorts is mandatory.