Abstract P3-06-08: Ki-67 mRNA as a predictor for response to neoadjuvant chemotherapy in primary breast cancer
Authors: Marme F , Schneeweiss A , Aigner J , Eidt S , Altevogt P , Sinn P , Wirtz RM 
Published: August 2014

Background: Pathologic complete response (pCR, ypT0/is) is one of the strongest prognostic factors for primary breast cancer (PBC) in the setting of neoadjuvant chemotherapy (NAC). Factors predictive for response to NAC can help to stratify for an individualized therapy. As chemotherapeutic agents are most active in highly proliferative tumors, it was the aim of the present study to evaluate the potential of Ki-67 as a single proliferation marker, quantitatively measured on the mRNA or protein level, to predict response of PBC to NAC.

Material and methods: Pretreatment core cut biopsies from n=72 patients with PBC treated within a randomized phase II trial (1) of anthracylin/taxane based NAC were examined. Immunohistochemistry was performed for the Ki67 antigen on an automated IHC platform (Dako Techmate 500). Ki-67 were assessed either by visual scoring (vIHC) or by quantitative image analysis (qIHC). For quantitative IHC analysis tumor proliferation was analyzed after tumor stroma segmentation using the Aperio Image Analysis toolbox. RNA was extracted from formalin-fixed, paraffin embedded (FFPE) routine biopsies using a bead-based extraction method (STRATIFYER XTRAKT kits). Ki-67, TOP2A and RACGAP1 as well as CALM2 as a house keeping gene were measured via a multiplex quantitative RT-PCR (qPCR). Kaplan-Meier survival estimates, patitioning test and correlation analyses were performed using the SAS JMP® 9.0.0 software.

Results: There was only a moderate correlation between Ki-67 mRNA or Ki-67 measured by immunohistochemistry (IHC) and histologic grade (Spearman r = 0.52 p &lt; 0.0001; r = 0.23 p = 0.033, respectively). Conventional visual scoring and qIHC correlated well (r = 0,78 p &lt; 0,0001), while qPCR and qIHC correlated moderately (r = 0,50 p &lt; 0,0001). For Ki-67 determined by visual scoring of standard IHC there was an optimal discrimination at a cut-off of 20% stained nuclei with regard to chemotherapy response. Yet, the difference between pCR-rates for high and low Ki-67 on IHC was non-significant, with 16.3% and 12.5% pCRs in the IHC Ki-67 high and low group, respectively. For Ki-67 IHC analysed by quantitative image analysis there was optimal cut-off at 35% stained nuclei resulting in 37,5% versus 8,7% pCR rate and 54% of pCRs classified correctly. For qPCR determination, high Ki-67 mRNA expression was associated with a high pCR-rate of 36.4% as opposed to 5.8% in tumors with low Ki-67 mRNA levels and 82% classified correctly.

Conclusion: High Ki-67 mRNA expression measured by RT-qPCR was predictive for the achievement of pCR to NAC and in this respect was superior to Ki-67 determined by vIHC or qIHC. This is consistent with data on Ki-67 IHC as a predictor of pCR on larger neoadjuvant cohorts (1). RT-qPCR based measurements of Ki-67 mRNA ensures an objective and highly reproducible quantification of proliferative activity from FFPE tissue from routine core cut biopsies. As such it seems to be more robust and meaningful compared to protein-based determination of Ki-67 by IHC, both by visual scoring or quantitative image analysis.

1) Schneeweiss et al, Ann Oncol 2011 2) Fasching et al., BMC Cancer 2011

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-08.