Proliferation, either as the main common denominator in genetic profiles, or in the form of single factors such as Ki67, is recommended for clinical use especially in estrogen receptor-positive (ER) patients. However, due to high costs of genetic profiles and lack of reproducibility for Ki67, studies on other proliferation factors are warranted. The aim of the present study was to evaluate the prognostic value of the proliferation factors mitotic activity index (MAI), phosphohistone H3 (PPH3), cyclin B1, cyclin A and Ki67, alone and in combinations. In 222 consecutive premenopausal node-negative breast cancer patients (87% without adjuvant medical treatment), MAI was assessed on whole tissue sections (predefined cut-off ≥10 mitoses), and PPH3, cyclin B1, cyclin A, and Ki67 on tissue microarray (predefined cut-offs 7th decile). In univariable analysis (high versus low) the strongest prognostic proliferation factor for 10-year distant disease-free survival was MAI (Hazard Ratio (HR)=3.3, 95% Confidence Interval (CI): 1.8-6.1), followed by PPH3, cyclin A, Ki67, and cyclin B1. A combination variable, with patients with MAI and/or cyclin A high defined as high-risk, had even stronger prognostic value (HR=4.2, 95%CI: 2.2-7). When stratifying for ER-status, MAI was a significant prognostic factor in ER-positive patients only (HR=7.0, 95%CI: 3.1-16). Stratified for histological grade, MAI added prognostic value in grade 2 (HR=7.2, 95%CI: 3.1-38) and grade 1 patients. In multivariable analysis including HER2, age, adjuvant medical treatment, ER, and one proliferation factor at a time, only MAI (HR=2.7, 95%CI: 1.1-6.7), and cyclin A (HR=2.7, 95%CI: 1.2-6.0) remained independently prognostic. In conclusion this study confirms the strong prognostic value of all proliferation factors, especially MAI and cyclin A, in all patients, and more specifically in ER-positive patients, and patients with histological grade 2 and 1. Additionally, by combining two proliferation factors, an even stronger prognostic value may be found.

SP6 was not superior to MIB1, but the two antibodies were comparable in the assessment of Ki67. Both MIB1 and SP6 could therefore be considered for prognostic use in primary breast cancer.

Background Proliferation is a key feature in breast cancer and also a clinical important factor for prognosis and treatment prediction. In the St Gallen Consensus of 2011, immunohistochemically analysed Ki67 was adopted as a surrogate marker to distinguish the “luminal A” from the “luminal B” subtype, in order to select patients with estrogen receptor positive disease expected to benefit from adjuvant chemotherapy. The mouse monoclonal antibody MIB1 is the generally accepted antibody for assessment of Ki67. However, other antibodies have been developed, e.g. the rabbit monoclonal antibody SP6. The assessment of Ki67 with MIB1 is unfortunately associated with a considerable lack of reproducibility. Rabbit monoclonal antibodies generally tend to have higher specificity without loss of sensitivity, compared to corresponding mouse monoclonal antibodies. SP6 has also been found to have reduced background staining compared to MIB1. According to these advantages, SP6 may be an alternative to MIB1 for routine staining of Ki67. Any methodological modification should, according to international recommendations, be compared against a clinically validated assay and demonstrate acceptable concordance before being introduced in clinical routine. The analysis of Ki67 with SP6 therefore needs to be evaluated and compared to MIB1 in a cohort of breast cancer patients with clinical follow-up. Aims To compare the antibodies SP6 and MIB1 for immunohistochemical assessment of Ki67 in primary breast cancer regarding prognostic strength and reproducibility of the evaluation. Methods Tissue microarray from a cohort of 237 premenopausal women with node-negative breast cancer was used for assessment of Ki67, with both SP6 and MIB1, by three different investigators. The 7th decile was applied for defining cut-off. Distant disease-free survival (DDFS) was used as endpoint and the follow-up was restricted to 5 years. Results Ninety per cent of the samples were classified into the same group, either high or low Ki67, irrespective of antibody used. Ki67 (high vs. low), analysed with both antibodies was associated to DDFS (34 events) in the univariable analyses (SP6: HR 2.6, 95% CI 1.3-5.2, p = 0.01 and MIB1: HR 2.8, 95% CI 1.4-5.7 p = 0.004) and showed borderline significance for DDFS in the multivariable analyses, also including HER2, age, and tumour size (SP6: HR 2.0, 95% CI 0.93-4.5, p = 0.074 and MIB1: HR 2.2, 95% CI 0.97-4.8, p = 0.058). The agreement between different assessors was somewhat higher for MIB1 than for SP6 (kappa-values 0.83-0.88 vs. 0.72-0.77). Conclusions SP6 was not superior to MIB1 and the two antibodies were comparable in the assessment of Ki67 for prognostic considerations in primary breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-15.