Ki-67, as a well-known proliferative marker, is suggested as a valuable predictor of survival, recurrence, and aggressiveness of breast cancer (22, 23). Besides, many studies have investigated the associations between Ki-67 levels and tumor’s grade, stage, lymph node, and estrogen receptor (ER) status (22). The stratification of breast tumors to different molecular subtypes (according to the hormone receptor, HER2, and Ki-67 expression) and also the usefulness of Ki-67 as a tool for selecting systemic treatment for early-stage breast cancers is established (22). However, Ki-67 faces some limitations. For instance, it is not expressed in cells that have the potential to enter the G1 phase of the cell cycle, which will pose a risk of the misidentification of tumoral cells (24). Besides, its functions are not well understood, and more importantly, it has no generally accepted cut-off values (25).
MCM proteins have key roles in the regulation of DNA replication in eukaryotes. MCM family members have some superiorities over Ki-67. They can be detected in cells that are in the resting phase of the cell cycle but still have the replication competency. Also, their expression is stable during the cell cycle (7). As a result, compared with Ki-67, a greater number of proliferative cells in different types of neoplasms would be identified by MCM (26). Also, in normal breast terminal duct-lobular units (TDLU), levels of MCM expression are higher than Ki-67 and are consistent with the high proportion of mammary epithelial cells residing in a licensed MCM expressing but the non-proliferating state (5, 7, 27).
In this study, we aimed to reveal whether the MCM6 expression score can be interpreted as a proliferative marker and be used as an alternative to ki-67 in differentiating molecular subtypes and also histologic grades of breast cancer. Therefore, we compared the expression of Ki-67 and MCM6 in 124 breast cancer samples with different grades and molecular subtypes.
In both normal and tumoral breast tissues, MCM6 had significantly higher levels of expression than Ki-67. The median of MCM6 and Ki-67 nuclear staining scores were significantly higher in tumors than in normal breast tissues. Hence, the influence of these markers on the carcinogenesis of breast tumors can be inferred. MCM6 can differentiate between histologic grades of invasive ductal carcinoma (P < 0.001) with a meaningful correlation with mitotic figures, which is also stronger than that of ki-67. Thereby, it seems that MCM6 is capable of more precise classification of breast tumors regarding histologic and mitotic grades. Both ki-67 and MCM6 revealed an association with ER status. HR negative tumors had higher MCM6 and Ki-67 expression.
The most important finding is that both MCM6 and Ki-67 can discriminate between luminal A and B molecular subtypes and between HR-positive and TNBC, which has a critical role in selecting the therapeutic strategies. As we have mentioned previously, we chose the cut-off point of 32% for the discrimination between luminal A and B subtypes.
We found no significant association between the expression levels of these two biomarkers and prognostic factors (such as lymphovascular invasion, lymph node involvement, tumor size, OS, and DFS), which most probably due to small number of assessed cases.
Many studies have evaluated the expression of various members of the MCM family besides other prognostic markers (such as P53) in several types of malignancies. For example, the upregulation of MCM gene expression in uterine cervical cancers is noted and reaffirms MCM as a proliferative marker in the DNA replication pathway, whereby the proliferation of dysplastic and cancer cells becomes increasingly dysregulated and proposed these markers as a valuable screening tool in detecting pre-cancerous cervical lesions (28).
In recent years, several studies have focused on the expression of various members of the MCM family in breast cancers and have reported relatively comparable results. In a study by Cobanoglu et al, the expression of MCM2 had a significant association with the histologic grade of breast carcinoma and the cell proliferation capacity (indicated by Ki-67). In addition, a negative correlation between MCM2 or Ki-67 expression and estrogen receptor expression was reported. They observed no significant association between MCM2 or Ki-67 expression and patients’ age, tumor size, lymph node status, clinical stage, and menopausal status (29). Our results for the expression of MCM6 and ki-67 are in concordance with the MCM2 expression in this study.
Another study showed that after a median follow-up of 5.3 years, fluorescence in situ hybridization (FISH) assessed MCM2 LI is not predictive of disease recurrence. However, they have reported MCM2 as a useful marker for distinguishing the aggressive-type HER2-amplified breast carcinomas (with high malignancy grade) from hormone receptor-negative subtypes (30). They also proposed that despite the acceptable correlation between MCM2 and Ki-67, MCM2 protein can be a superior proliferative marker in discriminating different histologic grades of breast cancer (31). In our study, we observed comparable results for MCM6 and its meaningful correlations with tumor grade, hormone receptor status, and molecular subtypes.
In a study designed by Issac et al, MCM2, MCM4, and MCM6 were assessed at the level of mRNA transcription and protein expression in breast cancers. They concluded that these markers can especially help to differentiate between luminal A and luminal B subtypes. Also, they found a meaningful correlation between these markers and Ki-67 and the histologic grade. Low expression of these markers was associated with an increased probability of relapse-free survival (32). To the best of our knowledge, this study is the only one that has evaluated MCM6 in breast cancers. Our results are concordant with the findings of this study.
MCM family members are one of the proliferative markers, and their role in the determination of proliferative activity and their relationship with prognostic and therapeutic factors, even in a stronger power rather to PCNA and Ki-67, is confirmed (33). MCM proteins are highly expressed in malignant human cancer cells and pre-cancerous cells undergoing malignant transformation, but not in the differentiated somatic cells. Therefore, these proteins are ideal diagnostic markers for cancer and serve as promising targets for anti-cancer drug development (6, 34).
There are limitations to this study. The retrospective design hampers reaching definite and casual associations. Until now, there are no valid and standardized methods for the assessment of MCM6 expression level. The low prevalence of HER2-enriched tumors in our samples was a major obstacle for MCM6 to differentiates them from other molecular subtypes. Lastly, the optimal cut-off points for MCM6 LI to differentiate between luminal A and luminal B subtypes led to a sensitivity of 50%, which generally is not acceptable for diagnostic tests. Larger prospective studies are warranted to further evaluate the importance of MCM6 as a diagnostic and prognostic marker for breast cancer.
In conclusion, our data support that MCM6 is a superior discriminator of tumor grade by better capturing the scoring differences in all three parameters of the Nottingham Score better than Ki-67. In addition, it can be taken as an alternative marker for Ki-67 in the classification of breast tumors into different molecular subtypes, especially luminal ones. Differentiating luminal A and B cancers remains a significant clinical question since it impacts therapeutic decisions. In this study, we tried to determine an appropriate cut-off value point for differentiating between these subtypes. Maybe in the future, more detailed data regarding these issues can help establish a more precise and accurate cut-off point with higher specificity and sensitivity. Surely the suitability of this marker for routine clinical use instead of Ki-67 has more unknown aspects to be investigated in the future.