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Minichromosome Maintenance Protein 2 Is a Strong Independent Prognostic Marker in Breast Cancer

Michael A. Gonzalez, Sarah E. Pinder, Grace Callagy, Sarah L. Vowler, Lesley S. Morris, Kate Bird, Jane A. Bell, Ronald A. Laskey, Nicholas Coleman

From the Medical Research Council Cancer Cell Unit, Hutchison/Medical Research Council Research Centre; Department of Histopathology, Addenbrooke’s Hospital; and Centre for Applied Medical Statistics, University of Cambridge, Cambridge; and Department of Histopathology, Nottingham City Hospital, Nottingham, United Kingdom.

Address reprint requests to Nicholas Coleman, MD, Medical Research Council Cancer Cell Unit, Hutchison/Medical Research Council Research Centre, Hills Rd, Cambridge CB2 2XZ, United Kingdom; e-mail: nc109{at}cam.ac.uk.

	ABSTRACT

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Purpose: To test the hypothesis that prognostic information in breast cancer may be derived from an accurate assessment of epithelial cell cycle entry, as indicated by expression of minichromosome maintenance (MCM) proteins.

Materials and Methods: We used immunohistochemistry to examine the distribution of Mcm-2 in breast tissue. Power calculations based on a pilot study of 67 whole tissue sections led to selection of an independent 347-core breast carcinoma tissue microarray validation set. We tested for associations between Mcm-2 (and Ki-67) labeling index (LI) and various clinicopathologic parameters.

Results: Mcm-2 was expressed more frequently than the standard proliferation marker Ki-67 in whole tissue sections of normal breast (P = .0003) and breast carcinoma (P < .0001). In 221 assessable cores of invasive carcinoma, the Mcm-2 LI showed a positive association with tumor size (P = .002), mitotic index (P < .0001), histologic grade (P < .0001), and the Nottingham Prognostic Index (NPI) score (P < .0001). Using a cutoff value of 50%, Mcm-2 LI was associated with overall survival (P = .0007), disease-free interval (P = .0002), and with the development of regional recurrence (P = .011) and distant metastases (P = .0016). Cox regression analysis suggested that the Mcm-2 LI is a strong prognostic factor in breast cancer that is independent and superior to histologic grade, lymph node stage, and Ki-67 LI, but not the NPI score.

Conclusion: Mcm-2 may be of utility as a prognostic marker to refine the prediction of outcome in breast cancer, for example when combined with parameters currently used in the NPI.

	INTRODUCTION

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PROGNOSIS IN breast cancer is determined by the extent of spread of the disease and by the inherent aggressiveness of the tumor, as defined by biologic characteristics such as the rate of tumor cell proliferation. Patient age, hormone receptor status, histologic type, and evidence of vascular invasion serve as useful predictors of outcome in routine clinical practice.¹ However, the most important independent prognostic indicators are the traditional pathologic parameters of lymph node stage,^2–4 histologic grade,^5,6 and tumor size,^2–4 which have been combined to refine prognostication further in the Nottingham Prognostic Index (NPI).^2,7 Patients can thereby be stratified into good (NPI score < 3.4; 15 year-survival rate, 80%), moderate (NPI score 3.4 to 5.4; 15 year-survival rate, 42%), and poor (NPI score > 5.4; 15-year survival rate, 13%) prognostic groups, on which therapeutic decisions can be based.¹

At present, the proliferative rate of breast tumors is assessed during histologic grading^5,6 by performing a mitotic count in a specified microscopic field area. This exercise provides a limited, cell cycle phase–specific estimation of the tumor growth rate. The frequency of entry of tumor cells into the cell cycle, regardless of phase, may associate independently with clinical course if adequately assessed. We have therefore tested the hypothesis that minichromosome maintenance proteins (MCMs), recently identified markers of cell cycle entry, may be of value in the clinical management of breast cancer.

In eukaryotes, DNA replication occurs once and once only in each cell cycle. This is partly due to cyclical binding and release of a complex of MCM proteins (MCMs 2 through 7).^8,9 In the early G₁ phase of the cell cycle, the proteins ORC, Cdc6, and Cdt1 functionally interact with MCMs 2 through 7 at replication origins, resulting in the formation of the prereplication complex (pre-RC).^10–12 The hexameric MCM component of the pre-RC demonstrates helicase activity that may unwind DNA during replication.¹³ This allows the DNA replication machinery to access binding sites on DNA, making chromatin competent or licensed for DNA replication.

MCMs are specific markers of the cell cycle state in tissues. Expression is seen during all phases of the cell cycle, but MCMs are lost after exit from cycle; with rapid loss after differentiation and slower loss in quiescent (G₀) cells.^14–16 In contrast, proliferation markers in current use, such as proliferating cell nuclear antigen and Ki-67, provide only a limited assessment of cell cycle state.^17–23 The recent availability of antibodies to MCMs has provoked much interest, particularly in cancer research, where their application has demonstrated clinical value in screening, diagnosis, and estimation of prognosis of malignant disease.^24–37 Most studies have used antibodies against Mcm-2 and Mcm-5, although all MCMs show essentially similar distributions.^24,36

The aims of the present study were to characterize the pattern and frequency of expression of Mcm-2, as a prototype MCM, in normal and malignant breast tissue and to compare expression with that of Ki-67. We also aimed to investigate the association of the Mcm-2 labeling index (LI) with clinicopathologic features, including the NPI, and to assess the potential value of Mcm-2 in predicting clinical outcome for patients undergoing surgery for breast cancer. We used two independent sets of cases. In a pilot study we examined whole tissue sections of 67 cases, representing normal breast (n = 11) and breast carcinoma (n = 56). This enabled us to generate hypotheses concerning the expression of Mcm-2 in breast carcinoma that were subsequently tested in an independent 347-core breast carcinoma tissue microarray (TMA) validation set, after determination of a sample size that would provide appropriate statistical power.

	MATERIALS AND METHODS

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Clinical Specimens
Archival formalin-fixed, paraffin-embedded breast tissue was randomly selected from the Nottingham Tenovus Primary Breast Cancer Study, with local research ethics committee approval. The Nottingham study is based on a series of patients with primary operable breast carcinomas who are treated in a uniform manner. All carcinomas assessed were well characterized in terms of clinical and pathologic information. Tumor size was recorded in the fresh state and confirmed in the fixed specimen. Histologic grade,³⁸ tumor type,^39,40 and presence of vascular invasion⁴¹ were recorded. The mitotic index was determined as previously described.^5,6 Lymph node stage was assessed by low axillary node sampling (with sampling of internal mammary nodes for medial tumors); the nodes were then examined histologically at several levels.³⁸ For each case, the NPI score was calculated and estrogen receptor (ER) status determined by the dextran charcoal coated method, as previously described.⁴¹ All patients studied underwent regular postoperative clinical assessment, with a median follow-up period of 102.5 months (range, 2 to 192 months).

In our pilot study, we examined whole tissue sections of 56 randomly selected cases of invasive breast carcinoma (Table 1) from 56 patients with a median follow-up of 37 months (range, 2 to 47 months). We also examined 11 cases of randomly selected normal breast tissue obtained from 11 premenopausal women who had undergone reduction mammoplasty. To validate the results obtained from the pilot study in an independent data set, breast carcinoma TMAs were subsequently used for high-throughput analysis of a larger series of tumor samples with a longer follow-up period (median, 111 months; range, 4 to 192 months; Table 1). We used core tissue specimens of 0.6-mm diameter taken from 347 individual paraffin-embedded breast carcinoma donor blocks from the Nottingham Tenovus Primary Breast Cancer Study.

The frequencies of expression of Mcm-2 and Ki-67 in breast epithelium were determined by calculating an LI for each marker, representing the percentage of epithelial nuclei that stained positively. In the whole tissue sections, LIs were determined in areas of maximal frequency of expression identified at low magnification. All samples were examined independently by two observers (M.A.G. and G.C.), one of whom (G.C.) is consultant breast histopathologist. Approximately 20% of the samples were also examined independently by a second consultant breast histopathologist (S.E.P.). The final LIs for each case represented the means of the values obtained by the two or three observers. In the vast majority of cases, the LIs determined by the observers differed by less than 5% of the final values. In the rare cases where there was greater disagreement between observers, the final LI value was determined by consensus. Analysis was performed on at least 500 epithelial nuclei per whole tissue section and on all the neoplastic nuclei in a TMA core.

Statistical Analysis
To study the association with continuous variables, tests of hypotheses on the location parameter were performed using the Kruskal-Wallis test, with clinicopathologic details stratified according to histologic grade (1, 2, and 3),³⁸ lymph node stage (1, 2, and 3),³⁸ tumor type,⁴⁰ presence of vascular invasion (none, probable, and definite),⁴¹ mitotic index and NPI score; the Mann-Whitney U test was used for presence of distant metastases and menopausal status. LI values were compared using the Wilcoxon signed rank test for paired data. Box plots of the LIs by histologic grade and mitotic index were constructed.

Spearman’s rank correlation (denoted r) and 95% CIs were used to test the associations between two continuous variables. Kaplan-Meier cumulative survival curves were constructed, and the log-rank test was used to examine differences in survival in a phase I prognostic study (defined as a prognostic study that seeks to evaluate the association between a new marker and disease characteristics).⁴² In this analysis we defined overall survival time as the period from the date of operation for excision of the primary carcinoma to the date of the last clinic visit (if alive) or the date of death. We defined disease-free interval as the period from the date of operation to the date of any disease recurrence (local, regional, or distant) or the date of the last clinic visit. Univariate and multivariate Cox regressions were used to look at the effects of covariates on time to event data. All of the statistical tests were performed using SPSS v11.5 (SPSS Inc, Chicago, IL).

	RESULTS

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Pilot Study: Whole Tissue Sections of Normal and Malignant Breast Tissue
In the 11 cases of normal breast tissue examined, expression of Mcm-2 and Ki-67 was observed in both lobules and ducts (Fig 1A and 1B). The distribution of labeled cells seemed random, and no proliferating compartments could be characterized. There was a positive correlation between the Mcm-2 and Ki-67 LIs in normal breast (r = 0.85; 95% CI, 0.65 to 1), although expression of Mcm-2 (median LI, 35.8%; range, 1.1% to 83.7%) was significantly higher than that of Ki-67 (median LI, 4.1%; range, 0% to 35.4%; P = .003) and showed greater variability. The median difference in LIs was 21.8%. Stromal elements such as fibroblasts and endothelial cells in normal breast were negative for both Mcm-2 and Ki-67.

View larger version (62K): [in this window] [in a new window]   Fig 1. Expression of Mcm-2 and Ki-67. Mcm-2 labeling index (LI) exceeded Ki-67 LI in normal breast ([A] Mcm-2; [B] Ki-67) and invasive carcinoma ([C] Mcm-2 grade 2 ductal no special type [NST]; [D] Ki-67 grade 2 ductal NST). Mcm-2 LI was lowest in grade 1 ductal NST tumors (E), greatest in grade 3 ductal NST tumors (F), and intermediate in grade 2 ductal NST tumors (C). Tissue microarray cores illustrate the heterogeneity in Mcm-2 expression in breast carcinoma tissue cores (G).

View larger version (41K): [in this window] [in a new window]   Fig 2. Comparisons between Mcm-2 and Ki-67 labeling indices (LI), mitotic counts, and histologic grade in invasive carcinomas. Mcm-2 LI (green) exceeds Ki-67 LI (yellow) and shows a positive association with mitotic index and histologic grade in whole tissue sections (A, B) and tissue microarray (TMA) cores (C, D). Line, median; box, interquartile range; whiskers, expected range; circles, outliers; stars, extremes.

Survival curves were constructed following cut point analysis of the receiver operating statistics curves. Identical results were obtained with Mcm-2 LI cutoff values ranging from 50% to 80%. It was not possible to refine the cut point analysis further using the available data. There was only one death over a median of 37 months (range, 6 to 47 months) in 22 patients with a tumor Mcm-2 LI of less than 50%, compared with six deaths over a median of 35.5 months (range, 2 to 42) in 34 patients with a tumor Mcm-2 LI of more than 50% (Fig 3). However, this difference was not significant (P = .40). There was no significant difference in the length of survival between both groups (P = .18).

View larger version (12K): [in this window] [in a new window]   Fig 3. Kaplan-Meier curves for whole tissue sections. Cumulative survival data are for all patients with invasive carcinoma (n = 56). Patients were censored when they ceased to be followed up for any reason but had not died. Survival is assessed according to tumor Mcm-2 labeling index (LI) of more than 50% or less than 50%.

We stained 347 invasive breast carcinoma TMA cores, of which 221 (63.6%) proved suitable for evaluation of Mcm-2 and Ki67 expression (Fig 1G). One hundred three cases could not be assessed because of absence or low number (< 100) of malignant cells (41 cases), high levels of background staining for either marker, presumed to be due to poor tissue fixation (47 cases), or detachment of the cores from the glass slide during microwave antigen retrieval or immunohistochemistry (15 cases). Twenty-three cases of ductal carcinoma-in-situ were not included in the study because expression of Mcm-2 in ductal carcinoma-in-situ had not been evaluated in whole tissue sections, and the sample size was considered too small for survival analysis. The clinical and pathologic characteristics of the 221 cases studied are listed in Table 1.

The Mcm-2 LI was higher than the Ki-67 LI (P < .0001) in the invasive carcinoma TMA cores and both LIs showed a strong positive association (r = 0.73; P < .0001; Fig 2C and 2D; Table 2). As with the whole tissue sections, positive associations were found between the Mcm-2 LI and tumor size (r = 0.21; P = .002), mitotic index (P < .0001; Fig 2C), histologic grade (P < .0001; Fig 2D), and the NPI score (P < .0001). In addition, Mcm-2 was associated with the presence of distant metastases (P = .006) and with histological type (P < .0001) when tumors were categorized²⁰ according to excellent (tubular, tubulo-lobular, invasive cribriform, and mucinous), good (tubular mixed, alveolar lobular, mixed NST, and special type), moderate (classical lobular, medullary, and atypical medullary, invasive papillary) and poor prognostic groups (NST, mixed NST and lobular, lobular mixed and solid lobular). There was no association with any individual histological type. The negative association observed between the Mcm-2 LI and ER expression in the pilot study was weaker and not significant in the TMA cores (r = -0.089; P = .32). No association was observed between the Mcm-2 LI and patient age (P = .90), menopausal status (P = .22), lymph node stage (P = .26), or vascular invasion (P = .19).

Associations with clinical outcome were investigated after cutoff analysis of the receiver operating statistics curves for this data set. Analysis was only performed within the range of cutoff values of 50% to 80%, suggested by the pilot study. Whereas refinement of the cutoff analysis could not be performed in the pilot study, the larger number of cases and increased length of follow-up in the validation study did enable this exercise to be undertaken. An optimal cutoff value of 50% was determined.

Mcm-2 LI was associated with overall survival (LR = 11.38; degrees of freedom [df] = 1; P = .0007; Fig 4A), as was the NPI (Fig 4B). There were four deaths in 68 patients with an Mcm-2 LI of less than 50% (mean survival, 143 months; range, 136 to 150 months), compared with 38 deaths in 153 patients with an Mcm-2 LI of greater than 50% (mean survival, 149 months; range, 138 to 161 months). In addition, Mcm-2 LI was associated with disease-free interval (LR = 13.89; df = 1; P = .0002), patients with an Mcm-2 LI of less than 50% having a mean disease-free interval of 124 months (range, 112 to 135 months), in contrast with 104 months (range, 89 to 119 months) for patients with an Mcm-2 LI of greater than 50% (Fig 4C). An association with regional recurrence was also observed (LR = 6.55; df = 1; P = .011; Fig 4D), but not with time to its development (Mcm-2 LI < 50% = 140 months; range, 132 to 147 months; Mcm-2 LI >50% = 149 months; range, 137 to 162 months). Development of metastases was also associated with Mcm-2 LI (LR = 9.92; df = 1; P = .0016), with six patients developing metastases in the Mcm-2 LI less than 50% group (n = 68; mean time, 140 months; range, 132 to 148 months), compared with 42 patients in the Mcm-2 LI greater than 50% group (n = 153; mean time, 144 months; range, 131 to 156 months). No associations were observed between Mcm-2 LI and development or time to local recurrence (LR = 0.65; df = 1; P = .42).

View larger version (19K): [in this window] [in a new window]   Fig 4. Kaplan-Meier curves for tissue microarray (TMA) cores. Patients (n = 221) were censored when they ceased to be followed up for any reason but had not died. Survival is assessed according to Mcm-2 labeling index (LI; A) and Nottingham Prognostic Index (NPI; B). Mcm-2 LI is also associated with disease-free interval (C) and regional recurrence (D).

	DISCUSSION

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As defined by expression of Mcm-2, a variable percentage of cells in normal premenopausal breast tissue have entered the cell cycle in both lobular and ductal compartments. Stoeber et al¹⁵ have examined expression of Mcm-2 in normal breast and reported a labeling index similar to our findings for both Mcm-2 (median, 47%; range, 22% to 78%) and Ki-67 (median, 6%; range, 4% to 10%). The median difference between the Mcm-2 LI and Ki-67 LI in normal breast tissue in our study was 21.8%, with a greater range of Mcm-2 LI values than of Ki-67 LI values. Differences between Mcm-2 LIs and Ki-67 LIs may be accounted for by the presence of cells in the early G₁ phase of the cell cycle, which may not express Ki-67.²⁰ Hence, breast (and other) tissue with a high proportion of cells that are progressing slowly through G₁ phase or held in G₁ would be predicted to show a higher LI for Mcm-2 than for Ki-67.

Blow and Hodgson⁴³ have proposed that failure to downregulate the DNA replication licensing system might make transition to uncontrolled cellular proliferation easier to achieve. The clinical significance of high Mcm-2 expression in normal breast tissue, therefore, warrants further investigation, because a high Mcm-2 LI may confer a risk of subsequent malignant transformation. It would also be instructive to study the expression of Mcm-2 in high-risk benign breast disease to elucidate its potential role as a marker that predicts the development of breast cancer in potential precursor lesions.

Our study also shows that Mcm-2 identifies significantly more cells in cycle than Ki-67 for all histologic subtypes of breast tumor examined. There is an important need in breast cancer for a prognostic index that can be used on an individual patient basis to provide accurate predictive information regarding survival or response to treatment, particularly perioperative systemic adjuvant therapy.^1,44 Our findings in this phase I study of two independent data sets suggest that the Mcm-2 LI has the potential to serve as an independent prognostic marker in breast cancer. The Mcm-2 LI was a stronger predictor of survival than the Ki-67 LI and was of greater value than the individual parameters of lymph node stage and histologic grade. Assessment of the Mcm-2 LI was a relatively straightforward, objective, and reproducible exercise.

The Mcm-2 LI in our study was determined in randomly selected tissue blocks or cores. This is appropriate to any future prognostic test based on detection of markers using histology or immunohistochemistry. As only a portion of tissue from resected carcinomas is sampled for histopathology, the area of maximal proliferative activity in an individual tumor may not be represented in the tissue sections examined. Nevertheless, our data suggest that determination of Mcm-2 LIs in randomly selected regions of invasive breast carcinoma provides potentially useful prognostic information that is superior to that derived from other parameters assessed on the same sample.

In conclusion, we have shown that the Mcm-2 LI may prove to be of clinical value in breast cancer as an independent prognostic factor for improving estimation of prognosis and guiding therapeutic decisions. There is potential for Mcm-2 LI to be combined with existing individual prognostic factors or with integrated indices such as the NPI. There is now a requirement for a study investigating a larger independent series of breast tumors in which the prognostic value of the Mcm-2 LI is assessed alone and in combination with the NPI. Independent prospective studies will be required in due course.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicated no potential conflicts of interest.

	NOTES

 
Supported by the Medical Research Council and Cancer Research United Kingdom.

M.A.G. is a Medical Research Council Clinical Research Training Fellow.

	REFERENCES

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Submitted April 17, 2003; accepted August 25, 2003.

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