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Table of Contents
Year : 2019  |  Volume : 39  |  Issue : 1  |  Page : 43-52

Assessment of the concordance between fluorescence in-situ hybridization and immunohistochemistry in evaluating topoisomerase IIα in breast carcinoma

Pathology Department, National Cancer Institute, Cairo University, Giza, Egypt

Date of Submission05-Jan-2019
Date of Acceptance02-Feb-2019
Date of Web Publication29-Nov-2019

Correspondence Address:
Nabil El-Bolkainya
Pathology Department, National Cancer Institute, Cairo University, Giza, 11511
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/EGJP.EGJP_7_19

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Background Topoisomerase IIα (TOP2A), encoded by TOP2A gene, is a molecular target for anthracycline therapy; hence, it can serve as a predictive or prognostic factor. There are several techniques used for measuring TOP2A status. The relative value of TOP2A gene amplification evaluated by fluorescence in-situ hybridization (FISH) and protein expression analyzed by immunohistochemistry (IHC) is yet unclear in the literature.
Aim To evaluate the concordance between FISH and IHC for TOP2A status and to assess their relations with some prognostic clinicopathological parameters.
Materials and methods A total of 86 invasive breast cancer paraffin blocks were retrieved from the archives of Pathology Department, National Cancer Institute, Cairo University, and were subjected to IHC, with a cutoff value of 10%, and FISH analysis with TOP2A/centromere enumeration probe 17 ratio of at least two for TOP2A expressions. The concordance between the results of both techniques as well as relations with selected prognostic parameters was evaluated.
Results Of 86 invasive breast carcinomas, 43 (50%) revealed TOP2A protein overexpression, whereas 23 (26.7%) cases had TOP2A gene amplification. TOP2A gene amplification was recognized in 16 of 43 TOP2A protein-expressed cases, with 37.2% concordance rate and 0.209 κ statistics. TOP2A gene amplification was significantly correlated with human epidermal growth factor receptor-2 immuno-expressing tumors and with molecular subtypes (P<0.001), whereas TOP2A protein level was correlated with histopathological types (P=0.001).
Conclusion Discordance was evident between TOP2A gene amplification and protein expression. However, TOP2A gene amplification has a valuable prognostic influence, whereas protein expression is still a predictive factor useful to guide chemotherapy.

Keywords: fluorescence in-situ hybridization, immunohistochemistry, invasive breast carcinoma, topoisomerase IIα

How to cite this article:
Loay I, Badawy OM, EL-Bolkainy T, Hafez NH, El-Bolkainya N. Assessment of the concordance between fluorescence in-situ hybridization and immunohistochemistry in evaluating topoisomerase IIα in breast carcinoma. Egypt J Pathol 2019;39:43-52

How to cite this URL:
Loay I, Badawy OM, EL-Bolkainy T, Hafez NH, El-Bolkainya N. Assessment of the concordance between fluorescence in-situ hybridization and immunohistochemistry in evaluating topoisomerase IIα in breast carcinoma. Egypt J Pathol [serial online] 2019 [cited 2022 Jan 26];39:43-52. Available from: http://www.xep.eg.net/text.asp?2019/39/1/43/272009

  Introduction Top

Anthracyclines are effective chemotherapeutic agents used in adjuvant therapy for breast cancer (Chen et al., 2017). Nuclear enzyme DNA topoisomerase IIα (TOP2A) is involved in DNA replication, transcription, and cell division and is reported as a target for anthracycline response (Romero et al., 2011). Anthracyclines inhibit the enzyme by blocking its ability to repair DNA strands after being cleaved, so they interrupt cell reproduction (An et al., 2018). The encoding gene for this enzyme is situated on chromosome 17q12–q21, near human epidermal growth factor receptor-2 (HER-2) gene (Brase et al., 2010). Preceding works have mentioned that TOP2A gene amplification serves as another predictive marker of anthracyclines sensitivity (Romero et al., 2011). However, contradictory findings were reported (Du et al., 2011).

The use of anthracyclines for cancer treatment can cause acute toxicity like arrhythmias and acute pericarditis, or cause chronic toxicity such as cardiomyopathy and heart failure as well as bone marrow dysfunction including acute leukemia and myelosuppression (Varga et al., 2012). Therefore, the accurate recognition of patients who will benefit most of this kind of therapy is mandatory (Romero et al., 2011).

There are several techniques in use for measuring TOP2A status, namely, (i) TOP2A gene detection by fluorescence in-situ hybridization (FISH), chromogenic in-situ hybridization, silver in-situ hybridization, or quantitative real-time PCR; (ii) TOP2A mRNA detection by quantitative reverse transcriptase-PCR; and (iii) TOP2A protein expression evaluated by immunohistochemistry (IHC) (Qiao et al., 2015). Protein expression evaluated by IHC and gene amplification specified by FISH are the two main accessible methods used to assess TOP2A status in clinical settings and select cases that might respond well to treatment with TOP2A inhibitors (Romero et al., 2011).

IHC is a favored technique for studying TOP2A enzyme as it is a simple and rapid procedure that is routinely carried out in all pathology laboratories. However, results are partially affected by interlaboratory differences, sensitivity of used antibodies, dilution rates, and requirement for definitive scoring systems to avoid interpretation subjectivity (Brase et al., 2010). FISH has good sensitivity and specificity in identifying gene amplification, but it is an expensive, time-consuming procedure, requires a fluorescent microscope, highly skilled personnel, with difficulty in detecting tumor area under fluorescent light. FISH technique is usually suggested as a modality in cases with equivocal IHC interpretation (Tubbs et al., 2009).

The agreement between TOP2A gene status and enzyme level is not completely understood in the literature. Some studies compared the concordance of different methods used in identification of TOP2A, but the outcomes were conflicting. Some reported no agreement (Schindlbeck et al., 2010; Romero et al., 2012; Zaczek et al., 2012), whereas others found a significant concordance (Cardoso et al., 2004; Arriola et al., 2007; Moelans et al., 2010). Using different methodological approaches for measuring TOP2A may account, to some extent, for these contradictory results (Chen et al., 2017).

In this study, our objectives were to detect TOP2A expression by FISH and IHC techniques in primary invasive breast carcinoma cases and to estimate the degree of agreement (concordance) between both techniques. Moreover, we also assessed the relations between TOP2A status detected by both techniques and some prognostic clinicopathological parameters.

  Materials and methods Top

Patient and tissue sample selection

This retrospective study included 86 patients diagnosed as having invasive breast carcinoma at National Cancer Institute (NCI), Cairo University, during the years 2012–2015. Cases underwent primary breast surgery including breast-conserving surgeries or modified radical mastectomies. Informed consents were obtained from all patients for the surgical procedures and the use of tissues for research following the regulations of ethical committee of NCI.

Clinicopathological data including age, histological type, tumor grade, tumor size, lymph mode status, estrogen receptors (ER), progesterone receptors (PRs), and HER-2/neu status as well as molecular subtypes were obtained from the pathological reports. Tumors were histopathologically classified based on the standards described by WHO (Lakhani et al., 2012). Tumor grade was categorized in keeping with the criteria of Nottingham modification of the Bloom–Richardson system (Elston and Ellis, 2002). Molecular subtypes of all tumors were classified according to previously published data (Eliyatkın et al., 2015). Hematoxylin and eosin-stained slides of the selected cases were restored from Pathology Department Archives and evaluated to confirm the diagnoses. The corresponding immunostained slides for hormonal receptors and HER-2/neu were also reviewed to verify the scoring. The assessment for hormonal receptor status was implemented in accordance with the American Society of Clinical Oncology/College of American Pathologists (Hammond et al., 2010). HER-2/neu staining was estimated according to Wolff et al. (2013), where positive cases included only score 3+ cases. Equivocal cases were further analyzed by FISH to detect HER-2 status.

All cases were selected from pathological electronic database. Inclusion criteria included women of any age with (a) final histopathological reports of primary invasive breast carcinoma, (b) available reports including almost all studied clinicopathological characteristics, (c) no administered therapy before tumor excision, and (d) available formalin-fixed paraffin-embedded blocks with adequate tumor tissue to perform FISH and IHC evaluation of TOP2A.

Immunohistochemical method

The most representative paraffin embedded block was selected for each studied case. Two positively charged unstained slides of 4-µm and 3-µm tumor thickness were prepared from each block and were used for IHC and FISH preparation, respectively.

IHC staining with TOP2A used ready to use monoclonal mouse antibody against TOP2A, clone Ki-S1 (Thermo Fisher Scientific; Labvision, Fremont, California, USA), dilution 1 : 40. Immunostaining was carried out based on the manufacturer’s guidelines using autostainer BenchMark IHC/ISH staining module (a product of Ventana Medical Systems, Ventana Oro Valley, Arizona, United States). Positive control section for TOP2A was normal tonsillar tissue. Negative control sections were also included by replacing the primary antibody with PBS.

IHC stained slides were interpreted independently by the pathologists, using Olympus binocular microscope, without knowledge of patients’ clinicopathological information and FISH results. Only nuclear staining, regardless of staining intensity, was considered as positive expression. Evaluation of TOP2A was performed by nuclear cell counting choosing the most cellular tumor area. The number of positively stained nuclei was recorded in consecutive fields at ×400 magnification. The percentage of tumor nuclei expressing TOP2A was determined by counting 1000 cells per slide (Zaczek et al., 2012).

Fluorescence in-situ hybridization technique

Deparaffinization of tissue sections was performed through immersion in xylene, followed by immersion in three descending gradient alcohol concentrations for hydration. Slides were pretreated using pretreatment solution supplemented in Zytolight FISH-tissue implementation kit 98°C for 15 min, then subjected to pepsin enzyme digestion for 10 min. Tissue sections were denatured at 75°C for 10 min. Ten microliter of triple color probe of Her-2/TOP2A/centromere enumeration probe 17 (CEP17) [Zytovision (CE marked), Fischkai, Bremerhaven, Germany] was used and left overnight for hybridization. Slides were washed to remove debris to minimize autofluorescence. Then DAPI was applied as nuclear counterstain. Slides were preserved in −20°C until visualization.

For FISH interpretation, the slides were visualized using Zeiss Axioscope (Carl Zeiss AG, Oberkochen, Germany) fluorescent microscope using suitable orange, green, DAPI, and aqua filters. Zeiss imaging software system was used. We evaluated TOP2A gene copy number through counting orange signals, and CEP17 was demonstrated by aqua signals. HER-2 gene copy number was evaluated using green filter and was discarded as it is not our target in this study. Representative images of gene were acquired with a CCD camera in monochromatic layers that were subsequently merged by Zeiss software. A minimum of 100 nonoverlapping interphase nuclei were scored. TOP2A gene was considered amplified if TOP2A/CEP17 ratio was at least two as recommended by many researchers (Hagen et al., 2007; Nielsen et al., 2010).

Statistical methods

Data were analyzed using SPSS statistical package version 17 (SPSS Inc., Chicago, Illinois, USA). Numerical data were expressed as mean and SD or median and range as appropriate. Qualitative data were expressed as frequency and percentage. χ2-Test or Fisher’s exact test was applied to check the relation between qualitative variables. For quantitative data, comparison between two groups was done using Mann–Whitney test (nonparametric t-test). Concordance between the two assays was identified by calculating the agreement and κ coefficient. The 95% confidence interval was estimated for κ. Spearman-ρ method was used to test correlation between numerical variables. P value up to 0.05 was considered significant.

  Results Top

Baseline clinicopathological characteristics

The clinicopathological characteristics of the studied patients as well as results of TOP2A status are demonstrated in [Table 1]. All patients were females, with a mean age of 52.3±12.3 years (range: 24–81 years). The histologic type invasive carcinoma of no special type predominated (70.9%) and 94.2% of cases were moderately and poorly differentiated (grade 2 and 3). Most cases (69.8%) were of large size (˃2 cm), and lymph node metastases were reported in 57% of cases. ER and PR were positive in 50% and 52.3% of cases, respectively. HER-2 was positive in 29.1%. The luminal molecular subtype was the most common (39.5%). The positivity rate of TOP2A as determined by IHC was higher (50%) than gene amplification detected by FISH (26.7%).
Table 1 Baseline clinicopathological characteristics of patients

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Topoisomerase IIα immunoreactivity

The percentage of tumor nuclei expressing TOP2A, in 1000 invasive breast carcinoma cells counted per sample, ranged from 0 to 90% with a median of 9.95%. Hence, a cutoff value of 10% was chosen to discriminate between positive and negative staining. Cases with TOP2A percentage of at least 10% were considered positive for TOP2A, whereas those with TOP2A percentage less than 10% were considered negative.

Of the studied 86 invasive breast carcinoma cases, 43 revealed TOP2A protein overexpression with a positivity rate of 50% ([Figure 1]a). The rest of cases (50%) were negative for protein expression ([Figure 1]b). Thirty-nine cases were associated with duct carcinoma in situ. TOP2A expression was detected in 37 (94.9%) cases of the in-situ components ([Figure 1]c). In the adjacent normal breast lobules, the expression of TOP2A was either completely negative or focal and patchy ([Figure 1]a). TOP2A expression was significantly overexpressed in tumor tissue compared with normal breast tissue (P<0.001).
Figure 1 (a) A case of medullary carcinoma with high immunoexpression for topoisomerase IIα (TOP2A). Note the completely negative breast lobules (red arrow) [immunohistochemistry (IHC), ×100]. (b) A case of IDC grade II with low expression for TOP2A (IHC, ×100). (c) A high-grade comedo DCIS strongly expressing TOP2A (IHC, ×200). DCIS, ductal carcinoma in situ; IDC, invasive duct carcinoma.

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The associations between TOP2A protein IHC status and the clinicopathological features are shown in [Table 2]. Among different histopathological subtypes, TOP2A level was more elevated in medullary carcinoma than in other subtypes; the difference was statistically significant (P=0.001). All cases of medullary carcinoma were positive for TOP2A. No significant relations between TOP2A IHC expression and the other studied parameters were detected.
Table 2 Relation between immunohistochemistry expression of topoisomerase IIα and clinicopathologic parameters

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Topoisomerase IIα gene amplification

TOP2A gene amplification was observed in 23 studied invasive breast carcinoma cases (26.7%) using FISH technique ([Figure 2]a). The median TOP2A/CEP17 ratio was at least 2. The remaining 63 (73.3%) carcinoma cases had nonamplified gene (either deleted or normal gene) ([Figure 2]b). TOP2A gene amplification was detected in 30% of the included carcinoma in-situ cases ([Figure 2]c).
Figure 2 (a) Fluorescence in-situ hybridization (FISH) analysis of medullary carcinoma showing amplified topoisomerase IIα (TOP2A) gene as expressed by clusters of orange signals and 2 centromere enumeration probe 17 (CEP17) aqua signals. (b) FISH analysis of invasive duct carcinoma showing nonamplified TOP2A gene as expressed by 2–3 orangeTOP2A signals, 1–2 aqua CEP17 signals, and 2 human epidermal growth factor receptor-2 (HER-2) green signals. In some nuclei, the red and green signals overlap giving yellow signals (red arrow). (c) FISH analysis of a case of high-grade DCIS showing amplified clusters of TOP2A orange signals and 2 aqua CEP17 signals. The red arrow points to the basement membrane bounding the ductal in-situ cells. DCIS, ductal carcinoma in situ.

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The results of FISH analysis were compared with the clinicopathological data ([Table 3]). TOP2A gene amplification was noted in 64% of HER-2+ cases (16/25) and in 11.5% of HER-2-negative tumors (7/61). TOP2A amplification was significantly higher in HER-2-expressing tumors than in those without (P<0.001).Overall, 16 of 86 (18.6%) studied cases showed simultaneous TOP2A amplifications and HER-2 protein positivity.
Table 3 Relation between topoisomerase IIα gene amplification and clinicopathological parameters

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TOP2A gene amplification was significantly related to the molecular subtypes (P<0.001). Amplification of the gene was reported in 76.9% of luminal HER-2+ and in 50% of HER-2-enriched molecular subtypes. Conversely, 91.2 and 85.2% of luminal and triple-negative subtypes, respectively, had nonamplified tumors. No statistical significance was obtained between TOP2A amplification and the other studied parameters.

Concordance analysis

TOP2A gene amplification was identified in 16 of 43 cases that had elevated TOP2A enzyme (37.2%). Twenty-seven (62.8%) cases did not show gene amplification despite TOP2A-positive immunostaining. Concordance rate was 37.2% and discordance rate was 62.8% ([Table 4]).
Table 4 Concordance between immunohistochemistry and fluorescence in-situ hybridization results for the detection of topoisomerase IIα

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The strength of association between FISH method and IHC for detection of TOP2A status was assessed. Fair agreement was obtained; κ statistics was 0.209. 95% confidence interval was from 0.026 to 0.392.

  Discussion Top

Breast cancer is the commonest tumor among female worldwide. It is a prevalent source of cancer-related death (An et al., 2018). At NCI, Cairo University, breast cancer constituted 19.3% of total primary malignant tumors presented in the period between 2000 and 2011 (Mohamed, 2016). However, breast cancer is a known treatable disease. Recognition of trustworthy prognostic and predictive indicators is a main challenge in clinical oncology practice (Chen et al., 2017). For that reason, selection of the best procedure for evaluating each indicator will influence the tumor therapy and prognosis (Brase et al., 2010).

TOP2A is a cell cycle-dependent enzyme. It reaches the highest level at the beginning of mitosis and decreases to the lowest amount at the end of mitosis. TOP2A expression in tumor cells is reported to be a direct molecular target for anthracycline-containing therapy. Cells with a low level of the enzyme are less sensitive to the drug, and cells containing a high amount are more responsive (Qiao et al., 2015). In addition, TOP2A is considered as a prognostic marker for aggressive tumors and survival in breast cancers (Rody et al., 2009).

However, the prognostic and predictive effect of evaluating TOP2A level in breast cancer is a subject of debate, and its usefulness in clinical practice is to be established. Moreover, the concordance between different methods used for measuring TOP2A level in tumor cells is still to be established (Tubbs et al., 2009; Brase et al., 2010).

In this study, we initially assessed the expressions of TOP2A in breast carcinoma cases by IHC and FISH techniques and evaluated the concordance between both techniques.TOP2A protein expression was observed in 43 of 86 (50%) studied cases. This was in agreement with previously published data where TOP2A expression was identified by IHC in up to 50–80% of cases (detection according to 10% cutoff) (Arriola et al., 2007; Zaczek et al., 2012; Iyikesici et al., 2014). This difference among studies could be explained by different antibody clones used, whether monoclonal or polyclonal with different specificity and sensitivity; number of the studied cases; and different cutoff values used to define overexpression, whether 5% (Fountzilas et al., 2012), 10% (Zaczek et al., 2012; Iyikesici et al., 2014), 15% (Chen et al., 2017), 25% (Arriola et al., 2007), or 30% (An et al., 2018). Another possible cause might be owing to either using automated quantitative image analysis (Chen et al., 2017) or using subjective analysis (Fountzilas et al., 2012; Zaczek et al., 2012) for measurement of protein expression.

In this study, TOP2A protein expression was significantly overexpressed in tumor tissue compared with normal adjacent breast tissue (P<0.001). We noticed that TOP2A IHC in adjacent normal breast tissue was either completely negative or focal and patchy. To our knowledge, studies concerning TOP2A status in normal tissues are limited. It was published that TOP2A was detected in normal proliferating tissues like spermatocytes, germinal lymphoid center, and proliferative endometrium. No expression was identified in terminally differentiated tissues like skeletal muscles and nerves, as TOP2A is not detected in G0 cell cycle phase. Normal breast, salivary gland, and kidney tissue revealed rare positive cells (Hadar et al., 2008).

Regarding TOP2A gene amplification assessed by FISH technique, 23 (26.7%) of 86 cases had amplified gene. In an earlier work on Egyptian breast carcinoma cases at the same institute, TOP2A gene amplification was detected in 23.9% of the included cases, which approached our present results (Badawy and Loay, 2017). In literature, TOP2A gene amplification rate ranged from 7 to 50% (Varga et al., 2012; Zaczek et al., 2012; Qiao et al., 2015; Chen et al., 2017). This wide variation among different studies could be attributed to heterogeneous study populations with different clinical stage and pathological types that express diverse gene amplification, using different scoring systems with different cutoff value, or different specificity of the probes used for assessment. Chen et al. (2017), demonstrated that the gene copy rate could vary considerably depending on the length of the used hybridized FIHS probe for TOP2A locus. They mentioned that the longer probe of DAKO pharmDX (Dako Via Real, Carpinteria, CA, United States), but not shorter PathVysion (Abbott Molecular Inc., Abbott Laboratories. Abbott Park, Illinois, USA) probes, was responsible for their false-positive interpretation. On the contrary, others mentioned that FISH probe length does not affect the gene analysis (Varga et al., 2012). Another factor that might be implicated in this wide variation of amplification rate is the intratumoural heterogeneity with presence of both TOP2A amplified and nonamplified tumor cells adjacent to each other in the same tumor (Fountzilas et al., 2012).

To establish if TOP2A gene amplification is a suitable indicator for identifying breast cancers with elevated enzyme levels, we evaluated the concordance between gene amplification assessed by FISH and enzyme status detected by IHC in breast carcinoma cases. We reported a concordance rate of 37.2% and κ statistics of 0.209, indicating a fair association (discordance) between both techniques. So, we suggested that gene amplification did not predict protein expression, and therefore, the enzyme level in tumors might not be connected directly to gene amplification.

Our results were along with some already published research studies (Romero et al., 2012; Varga et al., 2012; Zaczek et al., 2012; Chen et al., 2017). In contrast to our results, a considerable agreement between both techniques was reported in other works (Arriola et al., 2007; Moelans et al., 2010). A previous study of 59 females with an advanced and metastatic carcinoma signified a good concordance between gene amplification and protein expression as well as a better anthracycline response by concomitant use of both evaluating methods (Cardoso et al., 2004). These observations were supported by an earlier in-vitro work on breast cancer cell lines that indicated TOP2A gene extracopies were related to elevated enzyme expression and better efficacy of TOP2A inhibitors (Järvinen et al., 2000). In another research, authors mentioned that TOP2A protein expression was found in high-grade breast tumors, even in the lack of gene amplification. In low-proliferative tumors, a significant correlation was reported (Bhargava et al., 2005).

Some factors may be responsible for the poor association between IHC outcomes in tumors and gene amplification. Loss of antigenicity during tissue preparation could be a contributing factor (Romero et al., 2011). Bhargava et al. (2005), argued against this explanatory factor by observing that most of their studied HER-2 and TOP2A genes co-amplified tumors showed strong HER-2 protein expression (score 3+); however, less TOP2A overexpression was reported in such cases.

Another probable explanatory factor for the discrepancy between gene amplification and protein status could be related to the fact that TOP2A has been proposed to be a proliferation indicator. So its level alters by proliferative signal without a gene role (Qiao et al., 2015). Brase et al. (2010) and Romero et al. (2011) stated that proliferating cells expressed elevated TOP2A protein and mRNA levels than did nonproliferating cells. They indicated that TOP2A mRNA analysis was correlated well with protein expression but not with gene amplification. In the same line, Rody et al. (2009), indicated that TOP2A enzyme was regulated at the stage of transcription and translation, so gene amplification may not severely affect the TOP2A expression in cancer cells. Experience of pathologists who performed the IHC and FISH, as well as the standard of the cytogenetic laboratory might also affect the concordance results (Romero et al., 2005).

The influence of TOP2A on breast cancer prognosis is questionable. Some authors have reported a restricted prognostic effect (Chen et al., 2013), whereas others have proved a strong effect (An et al., 2018; Qiao et al., 2015). To help in such debate, we evaluated the associations of TOP2A gene amplification and enzyme expression with some prognostic clinicopathological parameters.

In this study, we first evaluated TOP2A protein expression in relation to some clinicopathological parameters. The outcome verified that TOP2A protein expression was considerably more elevated in medullary carcinoma than in other histopathological subtypes. The difference is statistically significant (P<0.001). No statistically significant associations were noticed with the remaining clinicopathological parameters. Some previous studies reported that TOP2A protein was significantly connected to high-tumor grade (Rody et al., 2009; An et al., 2018), Ki67 index (Arriola et al., 2007; Fountzilas et al., 2012; Qiao et al., 2015; An et al., 2018), HER-2 status (Qiao et al., 2015), node metastasis and tumor size (Rody et al., 2009; Iyikesici et al., 2014), ER and PRs (Fountzilas et al., 2012; Iyikesici et al., 2014; Qiao et al., 2015), or molecular subtypes (Arriola et al., 2007).In this study, we also evaluated TOP2A gene amplification in relation to the clinicopathological parameters. We reported that TOP2A gene amplification was presented more in HER-2+ tumors (P<0.001) as well as in Luminal HER-2 and HER-2-enriched molecular subtypes than other studied subtypes (P<0.001). Therefore, our data showed an association between TOP2A amplification and HER-2-expressing cases. These results were in accordance with other previous studies that have revealed that 33–60% of HER-2-expressing tumors had synchronized TOP2A amplifications (Zaczek et al., 2012; Engstrøm et al., 2014). Romero et al., 2011, reported an elevated TOP2A gene expression in basal-like, luminal B, and HER-2-enriched tumors, when compared with luminal A tumors. They explained their results to the fact that these tumors are highly proliferative subtypes and TOP2A is considered as a proliferation marker.

Overall, 16 of 86 (18.6%) studied cases in the current study showed simultaneous TOP2A amplifications and HER-2+. This frequency is slightly higher than the reported range by others, 3.7–17% (O’Malley et al., 2009; Engstrøm et al., 2014).

In this study, we detected that 11.5% of HER-2-negative, 8.8% of luminal HER-2-negative, and 14.8% of triple-negative molecular subtypes revealed TOP2A gene amplification. Other authors reported much lower TOP2A gene amplification rate of ∼6.4% in the HER-2 negative cases; thus, they concluded that HER-2 positivity has been used to choose women for TOP2A status evaluation (Konecny et al., 2010; Di Leo et al., 2011). On the contrary, some authors reported 27% TOP2A gene amplification in HER-2-negative breast carcinoma (Zaczek et al., 2012); therefore, they thought that assessing TOP2A status only in HER-2+ cases could fail to spot TOP2A abnormalities in a considerable fraction of HER-2-negative cases that might benefit from anthracycline-containing therapy. No significant relations between TOP2A amplification and the remaining clinicopathological parameters were reported in this study. As opposed to the present study, others concluded a statistically significant association between gene amplification and high-tumor grade as well as ER expression (Arriola et al., 2007), tumor size and stage (Romero et al., 2011), or patients’ age at diagnosis (Chen et al., 2017).

Interestingly, we noticed that the associations between the studied clinicopathological characteristics and TOP2A gene amplification were considerably different from the associations with TOP2A enzyme level. These results might confirm our conclusion that FISH and IHC results for assessing TOP2A are not concordant, and TOP2A protein-overexpressing tumors and gene-amplified tumors are biologically unlike.

  Conclusion Top

This results demonstrated a fair agreement (discordance) between TOP2A gene amplification and TOP2A protein expression, confirming that gene amplification is not a proper indicator for recognizing breast cancers that express high TOP2A enzyme. Our results also pointed out to the presence of a considerable difference in the relations between TOP2A status evaluated by FISH or IHC techniques and some prognostic clinicopathological characteristics. These results confirmed our conclusion that FISH and IHC results are not concordant. An association between TOP2A amplification, not the protein level, and HER-2 expressing cases was noticed, indicating a significant prognostic influence of TOP2A gene amplification.[34]

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Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2], [Table 3], [Table 4]


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