Prognostic relevance of topoisomerase II α and minichromosome maintenance protein 6 expression in colorectal cancer

Colorectal malignancies are a major cause of death in industrialised countries. Most colorectal neoplasms are histologically adenocarcinomas and develop through an adenoma-carcinoma sequence which was first described by Vogelstein and Fearon [1]. The development of a colorectal carcinoma depends on various factors and may often span over years before a manifest malignancy occurs. The macroscopic shape, histological type and grading seem to play key roles in the transformation process as defined by the adenoma-carcinoma sequence. Also, genetic mutations significantly affect the likelihood of colorectal cancer formation [2].

Mitosis within the neoplasia plays a key role in the histopathological analysis of the tumour. Assessment of the proliferation rate by means of proliferation markers is routinely implemented in histological diagnostics. Monoclonal antibodies against antigens associated with cell proliferation, such as Ki-67 [3, 4] and proliferating cell nuclear antigen (PCNA) are part of routine diagnosis in malignancies. Besides these mentioned proteins there are additional proliferation associated proteins, such as topoisomerase II α (Topo II α) and the minichromosome maintenance protein 6 (MCM6), that can be detected by immunohistochemistry (IHC) [5, 6]. The group of topoisomerases comprises up to four enzymes that are essential in the DNA topology and crucial for DNA replication [7]. By applying the monoclonal antibody Ki-S4, Topo II α can be detected by IHC [5, 8]. The prognostic significance of expressed Topo II α by Ki-S4 was shown in different studies [9‐11]. High rates of expressed Topo II α correspond to an unfavourable clinical outcome. However, only a few studies comprising CRC patients have been published so far. Within these studies, fluorescent in situ hybridization (FISH) was applied to detect the expression rate of Topo II α. IHC has not been exerted to evaluate the clinical outcome of patients suffering from colorectal neoplasm yet.

Minichromosome maintenance proteins also play a key role in DNA replication of eukaryotic cells. These proteins are a part of the pre-replication complex, which binds to chromatin and therefore represent an essential role in cell division [12]. Ki-MCM6 is a specific antibody targeting MCM6 that can be used in formalin fixed tissue [6, 13, 14]. Multiple studies have verified the clinical relevance of MCM proteins as proliferation markers in malignant tumours so far [15‐17]. Though, to the best of our knowledge, no investigation of the clinical relevance in terms of clinical outcome of MCM6 in colorectal carcinoma patients in a representative cohort has been published.

This publication aims to investigate the clinical relevance of topoisomerase II α and minichromosome maintenance protein 6 as proliferation markers in a representative large cohort of human colorectal carcinoma tissue. Results in terms of immunohistochemical expression are correlated to clinical follow-up data. Furthermore, it has to be investigated, whether the degree of expressed proliferation markers varies between clinical-pathological profiles.

Colorectal carcinoma is a major tumour entity and is accountable for the second greatest cause of death in tumour patients [19]. In assessment of the prognosis, prognostic markers are required in addition to the UICC-staging. Dysfunctional cell proliferation plays a key role in neoplasms. Evaluation of proliferative markers in the routine diagnosis of carcinomas is essential. For example, IHC of the proliferative marker Ki-67 is well accepted and executed on a regular basis. High levels of Ki-67 expression indicate rapid tumour growth and are associated with a poor clinical outcome [20‐23]. Regarding colorectal carcinoma, contradictory conclusions concerning the proliferation markers have been made. Multiple studies described high expression levels of Ki-67 to be a negative prognostic marker [23‐25], whereas other studies came to the opposite conclusion [26]. A few studies did not monitor any impact of the Ki-67 expression levels on the clinical outcome [27].

In this study we focused on two key player proteins in cell division, the topoisomerase II α (Topo II α) and minichromosome maintenance protein 6 (MCM6). We here applied IHC of Topo II α and MCM6 to a large and representative cohort of patients diagnosed with colorectal carcinoma.

IHC analysis was performed in order to detect the expression levels of Topo II α using the primary antibody Ki-S4, developed in the Institute of Haematopatholgy at the University Hospital Kiel. The antibody was proven to be a specific marker for Topo II α [8]. The expression of Topo II α was previously shown to be a significant prognostic indicator in breast cancer and mantel cell lymphoma, where a high intensity of expression was linked to a poor clinical outcome [5, 28]. Data of IHC for the detection of Topo II α expression in large and representative cohorts of CRC patients are limited. Boonsong et al. performed IHC to detect Topoisomerase I levels in 249 CRC patients but was unable to find a correlation neither to histo-pathological characteristics, nor to OS [29]. However, another recent study does reveal a significant correlation in terms of prolonged DFS and OS in patients with high expression rates of Topoisomerase I [30]. Our analysis also revealed a highly significant correlation between the Topo II α expression and the OS and DFS. Synoptically, our data is partially contradictory to previous studies. Lacking analysis of Topo II α in reasonably sized cohorts of patients suffering of CRC, validation is critical. Regarding the patient age, a significant coherence to Topo II α expression was monitored. In young patients (≤65 years), the expression was significantly higher which is likewise a contrary result to the study of Boonsong et al. [29]. Furthermore, within the cohort of younger patients, we were able to identify Topo II α expression as a prognostic marker. High expression rates cohered with a beneficial clinical outcome. As to why the prognostic value is only in the subset of young patients must be further explored – experimental validation is currently lacking.

CRC localised at the right hemi colon is generally associated with an inferior prognosis, we therefore expected expression levels of Topo II α to be significantly lower. To our surprise, the locus of neoplasia (left vs right hemi colon) did not prove any difference in expression rates of Topo II α. Patients diagnosed with an adeno carcinoma and Topo II α expression levels above the cut-off showed a highly significant favourable outcome.

Locally advanced tumour progression is accompanied with lower rates of Topo II α expression. Comparing UICC I + II with UICC III, a significant decrease in expression was monitored. Between UICC III and UICC IV, no difference was asserted. Within each UICC stage, significant impact of Topo II α expression levels on the clinical outcome was observed. These findings prove the prognostic impact of assessing Topo II α expression levels using IHC. In conclusion, our data provides an additional tool to the UICC classification in terms of prognosis and clinical outcome to identify Patients at risk, which may be of benefit to an (neo-) adjuvant treatment.

In further analysis, we assessed the expression levels of MCM6 and clinical characteristics, as well as patient outcome. CRC tissue specimens of a large cohort of patients were studied using IHC with the primary antibody Ki-MCM6, that is highly specific to the MCM6. The relevance of MCM in malignancies has been affirmed in various studies [15‐17, 31]. An analysis of MCM6 in patients diagnosed with CRC was absent.

As expected, the mean expression level of MCM6 (83%) was significantly higher than with Topo II α (52%). MCM6 is involved in the early phase of cell cycle replication. The protein is partly involved in the G₁ phase. Hence, a larger quantity of cells (including cells in early stages of the cell cycle) is stained by IHC [13]. The above-mentioned finding may explain the different quantity of expression when comparing Topo II α with MCM6. Similar results have been demonstrated in other tumour entities [6]. Correlation of Topo II α and MCM6 was clearly demonstrated. Neoplastic tissue with low expression levels of MCM6 exhibited low levels of Topo II α expression.

We did not expect that MCM6 expression levels would negatively correlate with the UICC staging. In progressive tumours, lower expression levels of MCM6 were observed, which is contrary to the Topo II α expression levels in our cohort. We expected high levels of MCM6 in advanced tumours with rapid tumour growth and subsequent greater cell proliferation as previously described by Giaginis et al. in terms of MCM2 expression [32].

Concerning the OS and DFS, expression levels above the cut-off were associated with a favourable outcome. Furthermore, in young patients (≤65 years) with histologically graded G2 adeno carcinoma, MCM6 expression levels above the cut-off also demonstrated a significant marker for a beneficial outcome.

For the first time our study presents data of Topo II α and MCM6 IHC detected expression levels in a large representative cohort of patients diagnosed with CRC. Contrary to the expected outcome, high expression levels of the proliferative markers MCM6 and Topo II α represent a significantly negative prognostic marker.

Increased cell proliferation was generally thought to be responsible for tumour progression and metastasizing. Whereby, as suggested by our data, rather poorly differentiated tumours with scarce cell proliferation seem to be liable for a poor progression of the disease.

In summary, we propose that from a prognostic point of view, high proliferative cell turnover should not be equated with a poor histological tumour differentiation. We finally conclude that assessing the proliferative turnover could be used for risk stratification of CRC patients in the future. Undoubtedly, our data is controversial in context of other malignancies, but carcinomas are diverse, and should not all be investigated in analogy. In this MS we present genuine data exhibiting novel findings in MCM6 and Topo II alpha exploration, that truthfully cannot be elucidated in any manner. A more in-depth investigation is required in order to demonstrate and consolidate our findings in validation cohorts.