Analysis of the cell kinetics of cancer cells in situ (for example, by Ki-67 antigen expression or mitotic counts) has been increasingly used to evaluate prognosis and/or biological behavior of various human malignancies
. TOP2A is a nuclear enzyme that controls DNA topological structure and cell cycle progression
. It mainly supports DNA decoiling, chromosome segregation during anaphase of the cell cycle, and DNA replication, by creating a DNA-linked protein gate through which another intact DNA duplex passes
. This enzyme is a marker of cell proliferation in normal and neoplastic tissues
. In malignant cells, overexpression of TOP2A protein might reflect not only the proliferative advantage of these cells, but also qualitative alterations caused by malignant transformation and dedifferentiation
. The IHC method for in situ determination of TOP2A has been extensively validated and shown to reflect closely the exact enzyme activity in formalin-fixed paraffin-embedded human tissues, leading to the prognostic and predictive importance of this test in other neoplasms
This is the first study that brings together a gene and protein assessment of TOP2A in the largest series of prostate adenocarcinoma. Also, fractal image analysis was performed in order to confirm digital assessment in a subset of randomly selected cases. Furthermore, for the first time we bring an association between all these data and BRFS curve.
Concerning IHC pattern of expression for TOP2A, positive nuclear staining was found. Also, additional diffuse weak cytoplasmic staining was seen in some cases, as Faggad et al.
 and Gotlieb et al.
 also reported in their respective studies. Corroborating most works on literature, we found that the expression of TOP2A was an indicative of poor prognosis. Patients who expressed higher levels of its protein had higher Gleason scores, higher levels of preoperative PSA, and shorter BRFS. Indeed, high proliferation rates in cancers are typically associated with worse clinical outcome
. According to Faggad et al.
, one explanation for the shorter survival rate associated with elevated TOP2A levels could be an enhancement of tumor cell proliferation, which results in increased tumor aggressiveness. In the multivariate analysis, TOP2A positivity remained an independent prognostic factor for BRFS, along with the presence of SV invasion.
Increased TOP2A is a common, though not specific, occurrence in malignant cells
. According to O’Connor et al.
, there are several mechanisms to explain this upregulation in these cells. First, both pRB and p53 are negative regulators of topoisomerase IIα, and both are well known to be inactivated or deleted in malignant cells
. Inactivated or deleted pRB or p53 would be expected to increase the expression of this enzyme
. Another possible mechanism for the overexpression of TOP2A is the amplification of a coding gene such as that for HER2/neu. Both HER2/neu and TOP2A reside on the long arm of chromosome 17 and amplification of one gene locus could simultaneously overexpress both of these genes
. In the present study, we did not find any alterations on TOP2A status in FISH. Schindlbeck et al.
 found that TOP2A amplification was not significant for outcome in women with primary breast cancer, protein expression only (IHC) was related to outcome in those patients. According to this work, protein expression might be more relevant than TOP2A amplification or deletion in predicting the outcome of breast cancer patients that received anthracycline-based chemotherapy
. In another study of our group
, TOP2A amplification did not correlate with FISH results in soft tissue sarcomas. According to Werneck et al.
, increased TOP2A expression does not appear to result solely from gene amplification. The explanation for this finding is still unclear; it might be due to posttranscriptional regulation
. This corroborates studies on other solid tumors, like breast cancer
[21, 22] and gastric carcinoma
. Werneck et al.
 suggested that gene amplification and protein expression should be evaluated separately when the prognostic or predictive value of TOP2A is examined in any neoplasia.
High levels of TOP2A expression are generally associated with high levels of cellular proliferation and poor histologic differentiation of tumors
. The relationship between overexpression of this enzyme and poor prognosis has been reported in different neoplasias, like breast cancer
, urothelial bladder carcinoma
, larynx cancer
, bladder cancer
, and ovarian cancer
. On the other hand, although most studies on literature correlates high levels of TOP2A expression with poorer survival rates and more aggressive tumors, there are studies showing the opposite. Bredel et al.
 concluded that high expression of TOP2A and Ki-67 appeared to be associated with prolonged survival in glioblastoma patients. In a recent work, Schindlbeck et al.
 showed that TOP2A IHC positivity predicted lower risk of metastases and death in breast cancer patients. Yan et al.
 demonstrated that high TOP2A expression was correlated with better disease-free survival for postoperative non-small cell lung cancer (NSCLC) patients who received adjuvant chemotherapy. One reason for these discrepant results is that these patients received adjuvant chemotherapy and high grade tumors tend to present better response to this type of therapy. Yan et al.
 postulated that adjuvant chemotherapy might overcome the adverse biology of cancers that expressed high levels of TOP2A protein. According to this latter work, NSCLC patients with high expression of the enzyme might be able to obtain more benefits from adjuvant chemotherapy than those with low expression, which emphasizes the predictive importance of TOP2A for such patients
Proliferation measurements in PCa have generally been done by studying the Ki-67 molecule, which is present in actively cycling cells
. IHC for Ki-67 in PCa has been shown to have prognostic importance (tumors with high Ki-67 expression tend to have a poorer prognosis and high tumor Ki-67 value also appear to predict tumor recurrence after radical prostatectomy)
. Since TOP2A has been found to correlate well with Ki-67 in a number of human diseases, Willman et al.
 suggested that similar prognostic information might be obtained by TOP2A IHC, with an advantage that the enzyme is the target of drugs being used for treating PCa patients. However, there are few works in the literature correlating TOP2A and clinicopathological parameters of PCa. Sullivan et al.
 showed that the expression of this enzyme increased with both stage and grade, and advancing stage was the stronger predictor of TOP2A expression. Willman et al.
 and Hasby et al.
, in their respective works, demonstrated that the prostatic carcinomas with the highest expression of the enzyme were more poorly differentiated and had the highest Gleason scores. Hughes et al.
 showed that TOP2A expression increased with increasing Gleason score and with hormone insensitivity. Murphy et al.
 showed that increased TOP2A copy number was associated with adverse clinical features, including high Gleason score, high stage, androgen resistant, HER2 amplification, and decreased survival under multivariate analysis. In a recent study, Karnes et al.
 demonstrated that the time for PCa patients to develop systemic progression (SP) was significantly associated with TOP2A protein expression: higher 5-year SP rates were observed in patients with higher protein levels of the enzyme. Ida et al.
 showed that TOP2A protein expression was predictive of SP and death in PCa patients with Gleason score ≥7 treated surgically, especially in PCa without ERG overexpression. Malhotra et al.
 demonstrated that a tri-marker proliferation index (which included Ki-67, TOP2A, and E2F1) provided improved prognostic performance in PCa; it predicted biochemical recurrence after radical prostatectomy.
Alenda et al.
 showed that PCa patients with Gleason score 7(4 + 3) have higher chances of presenting biochemical recurrence compared to patients with Gleason score 7(3 + 4). In addition, they also showed that the primary Gleason pattern 4 remained as an independent prognostic factor for the occurrence of biochemical recurrence in PCa patients
. Although the clinical difference between patients from both groups of Gleason 7 [(3 + 4) and (4 + 3)] is largely known, we found no difference in TOP2A expression between them. We suppose that TOP2A may not be related (at least not by itself) to early stages of carcinogenesis and morphological undifferentiating, which may result in further aggressive tumor biological behavior. However, when analyzing all Gleason scores there was a significant difference in TOP2A expression, showing that this protein may come up in more discrepant lesions. Furthermore, the absolute relation between the presence of TOP2A in tumors of patients with biochemical recurrence is an evidence that this protein is related to late stages of tumor development.
Besides its important role as a proliferation marker, as shown above, TOP2A is also the molecular target of several chemotherapy agents, including anthracyclines such as doxorubicin and etoposide
. These cytotoxic agents bind the DNA topoisomerase II complex and inhibit the relegation of DNA
. This converts TOP2A into a physiologic toxin and introduces high levels of permanent double-stranded breaks, which are detected by proteins ensuring genomic integrity
. As a result of the activation of this machinery, the cells with abundant DNA breaks are eliminated by apoptosis
. The sensitivity or resistance of a malignant cell to these anti tumor drugs, also called topo II poisons, is proportional to the level of TOP2A expression
[11, 14]. In PCa, there are several phase II trials using etoposide or docetaxel in combination with other chemotherapy agents to treat androgen-sensitive metastatic prostate carcinoma and hormone-refractory PCa, with promising results
The biological behavior of PCa still challenges researchers and urologists. While some patients present indolent disease with no need for treatment, others present aggressive disease with inevitable progression. Therefore a good prognostic and predictive marker of adjuvant and target therapies would be of great utility. In this sense, TOP2A is emerging as an important molecular target for many anticancer drugs, and several experimental works have clearly showed that cellular sensitivity to this enzyme is dependent on its high levels
. In conclusion, we found that higher expression of TOP2A protein in PCa patients is a strong indicative of poor prognosis. Also, since TOP2A is a target for many anti-neoplastic drugs, the IHC evaluation of this marker in routine practice can be a powerful tool for selecting appropriately aggressive therapies (use of adjuvant chemotherapy), specific target therapies, and the most suitable surgery approach in order to improve outcome of patients with prostate cancer. Also, we show for the first time that TOP2A gene copy number alterations are not observed in this type of tumor. So, higher protein expression of TOP2A is not related to gene amplification in PCa. Furthermore, TOP2A protein assessment has prognostic importance and, due to its relation with poor outcome, TOP2A IHC evaluation in the biopsy can represent an important tool for selecting the most suitable surgical and clinical approach for patients with PCa.