To our knowledge, the present study is one of the first to evaluate the role of TOP2A gene amplification and TopoIIa protein expression in the outcome of patients treated with trastuzumab-based regimens for MBC. The most important evidence provided herein is that TOP2A gene amplification is a favorable prognostic factor in HER2-positive patients treated with trastuzumab. Patients with HER2-positive/TOP2A non-amplified or deleted tumors did not seem to benefit from trastuzumab-based regimens and had an unfavorable outcome compared to TOP2A amplified tumors, in line with recent reports on TOP2A gene dosage
 and TOP2A gene amplification
The role of the TOP2A gene has mainly been examined in relation to anthracycline treatment. Co-amplification of HER2 and TOP2A was associated with favorable response to anthracycline-based therapy of locally advanced breast cancer
. The results of our study did not appear, at first, to be associated with the administration of anthracyclines, since only 12% of our patients had received such treatment in the 1st line metastatic setting. However, 39% of all patients and 43% of the HER2-positive ones had received anthracyclines in the adjuvant setting. Upon further analysis of our data, taking into account adjuvant and/or 1st line anthracycline treatment, a significant interaction of TOP2A with anthracycline treatment was observed both for TTP and survival. In terms of survival among the TOP2A amplified subgroup, anthracycline-treated patients were associated with decreased risk for death. It appears therefore that the improvement in survival of the TOP2A amplified subgroup treated with trastuzumab is probably due to the concurrent or previous exposure of the patients to anthracyclines, rather than the effect of the trastuzumab treatment itself. Furthermore, when TOP2A amplified patients treated with anthracyclines in the adjuvant setting were analyzed separately, they were found to have decreased risk for death, suggesting that even history of adjuvant anthracycline treatment results in survival advantage of TOP2A amplified patients treated with trastuzumab.
There is a very recent report from the Breast Cancer International Research Group (BCIRG) 006 trial
 and an additional retrospective analysis of almost 5,000 patients
 regarding the efficacy of trastuzumab in breast cancer patients with HER2 and TOP2A co-amplification. The first study is one of the largest randomized trials, which confirmed the role of trastuzumab in the adjuvant setting. The most interesting aspect of the BCIRG 006 trial is that it included a non-anthracycline regimen (docetaxel, carboplatin and trastuzumab), which was compared to AC-T (doxorubicin, cyclophosphamide, followed by docetaxel) with or without trastuzumab. This study demonstrated that the DFS benefit conferred by AC-T without trastuzumab in HER2-positive breast cancer patients is actually restricted to TOP2A co-amplified malignancies, which constituted a subset (35%) of the HER2-positive cancers, and is virtually indistinguishable from the benefit achieved by the addition of trastuzumab. Importantly, this same benefit (found in the TOP2A co-amplified subset) could also be attained by a non-anthracycline regimen in combination with trastuzumab, thus avoiding the toxicities seen with anthracyclines. In our study, trastuzumab was given to advanced-stage HER2-positive breast cancer patients in the metastatic setting, our findings should not therefore be compared to those of the BCIRG 006 trial
Recent studies support the role of TopoIIa protein expression, rather than TOP2A gene amplification, as a predictor of response to anthracycline-based chemotherapy in the adjuvant setting
. It is of note, that TopoIIa protein overexpression has been reported in HER2-positive, as well as HER2-negative tumors, independently of TOP2A gene amplification
. The latter finding has also been shown in our study; TopoIIa protein overexpression however, was not associated with either TTP or survival.
TopoIIa protein overexpression was however associated with ER-positive status and high Ki67 expression, partly in line with previous reports, since TopoIIa protein expression had been shown to be associated with ER-positive status
 and the Ki67 proliferation index
. To the best of our knowledge, the associations between TopoIIa and PTEN protein expression, as well as PIK3CA mutation presence are new findings in breast cancer tissue series, meriting further investigation for their biological importance. Of note, TopoIIa protein is upregulated in proliferating normal and cancer cells, in order to participate in the cell duplication process
. Hence, with the widely used cut-off of 5% positive neoplastic cells to assess TopoIIa protein positivity, tumors are found to be positive for TopoIIa in the absence of underlying amplification of the corresponding gene.
Alterations of the TOP2A gene mostly happen in HER2-positive tumors, however TOP2A does not always follow the amplification fate or rate of the HER2 amplicon, since it is not always included in the so called “smallest region of amplification” next to HER2[36, 42], while it may also be deleted in the presence of HER2 amplification, as observed here and elsewhere
. Thus, at least in a subset of TOP2A amplified tumors, the mechanism driving TOP2A amplification may be different than the one resulting in HER2 amplification
[9, 10, 43], as shown by the far lower ratio of TOP2A signals in comparison to HER2 signals
. In addition, TOP2A may also be amplified or deleted in the absence of HER2 amplification, further supporting the view of distinct and possibly multiple mechanisms, resulting in alterations of this gene. The absence of TOP2A amplification and the presence of deletions may practically have the same unfavorable impact on the outcome of HER2-positive patients, as shown in this study. With respect to gene deletions, it should be noted that the way markers are scored with FISH on FFPE sections it is unavoidable to obtain false positive results (deletions), due to nuclear truncations that interfere with the number of fluorescent signals to be counted per nucleus in a mostly unpredictable manner. Hence, although TOP2A gene deletions may indeed occur, the results concerning this FFPE-FISH marker, in the present and in the previously published studies, should be interpreted with caution.
In most of the published series, TOP2A gene amplification or deletion was a rare event in HER2-negative patients
. Only in four studies
[10, 16, 18, 45], the rate of TOP2A alterations was considerably greater than the 1% to 2% range reported in all other studies. In line with the majority of the published data we did not find HER2-negative patients with TOP2A gene amplification.