Abstract
BackgroundImmunohistochemical assessment of proliferation may provide additional prognostic information in early breast cancer. However, due to a lack of methodological standards proliferation markers are still not routinely used for determining therapy. Even for Ki67, one of the most widely-studied markers, disagreements over the optimal cutoff exist. Improvements in digital microscopy may provide new avenues to standardise and make data more reproducible.MethodsWe studied the immunohistochemical expression of three markers of proliferation: Ki67, Mini-Chromosome Maintenance protein 2 and Geminin, by conventional light microscope and digital imaging on triplicate TMAs from 309 consecutive cases of primary breast cancers. Differences between the average and the maximum percentage reactivity in tumour cell nuclei from the three TMA cores were investigated to assess the validity of the approach. Time-dependent Receiver Operating Characteristic curves were utilized to obtain optimal expression level cut-offs, which were then correlated with clinico-pathological features and survival.ResultsHigh concordance between conventional and digital scores was observed for all 3 markers (Ki67: rs = 0.87, P < 0.001; MCM2: rs = 0.94, P < 0.001; and Geminin: rs = 0.86, P < 0.001; Spearman’s rank). There was no significant difference according to the number of TMA cores included for either Ki67 or MCM2; analysis of two or three cores produced comparable results. Higher levels of all three proliferation markers were significantly associated with higher grade (P < 0.001) and ER-negativity (P < 0.001). Optimal prognostic cut-offs for percentage expression in the tumour were 8 %, 12 and 2.33 % for Ki67, MCM2 and Geminin respectively. All 3 proliferation marker cutoffs were predictive of 15-year breast cancer-specific survival in univariable Cox regression analyses. In multivariable analysis only lymph node status (HR = 3.9, 95 % CI = 1.79-8.5, P = 0.0006) and histological grade (HR = 1.84, 95 % CI = 1–3.38, P = 0.05) remained significantly prognostic.ConclusionsHere we show that. MCM2 is a more sensitive marker of proliferation than Ki67 and should be examined in future studies, especially in the lymph node-negative, hormone receptor-positive subgroup. Further, digital microscopy can be used effectively as a high-throughput method to evaluate immunohistochemical expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1531-3) contains supplementary material, which is available to authorized users.
Highlights
Immunohistochemical assessment of proliferation may provide additional prognostic information in early breast cancer
In this series of 309 cases, 70.1 % of patients were over 50 years of age, 53.8 % had lymph node-negative disease, 75.6 % were ER-positive and 16.8 % were HER2-positive ( HER2 status was known for only 50 % of patients in this historical cohort). 43.4 % were of histological grade 2 and 55.4 % were between 2 and 5 cm in size
Correlation between proliferation markers and methodology To explore the information provided by the scores for each marker, we first compared them across the cohort
Summary
Immunohistochemical assessment of proliferation may provide additional prognostic information in early breast cancer. With earlier detection and improved treatment options, breast cancer-related mortality is decreasing, while the detection of early stage disease is on the rise [2]. Traditional prognostic and predictive factors such as lymph node status, histological grade, invasive tumour size, hormone receptor (ER and PR) and HER2 status may be insufficient for prognosticating early stage disease [3, 4]. Histological grade is an important prognostic marker, which reflects proliferation status by incorporating an assessment of mitotic rate. Since the development of the MIB-1 antibody, immunohistochemical expression of Ki67 in paraffin-embedded tissue has been shown in a number of studies to be prognostic and predictive of treatment response in breast cancer [8,9,10]
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