Improving prognosis of surrogate assay for breast cancer patients by absolute quantitation of Ki67 protein levels using Quantitative Dot Blot (QDB) method

Purpose The separation of Luminal A-like from Luminal B-like breast cancer subtypes in surrogate assay relies on Ki67 scores assessed by immunohistochemistry (IHC), a method known to be associated with subjectivity and inconsistency. We attempted to measure Ki67 levels absolutely, quantitatively and objectively in Formalin Fixed Paraffin Embedded (FFPE) specimens, and evaluate its influence on the performance of surrogate assay for breast cancer patients. Methods The Ki67 protein levels were assessed using both IHC and Quantitative Dot Blot (QDB) methods respectively in 253 specimens. These patients were assigned into Luminal A-like and Luminal B-like subtypes using either Ki67 score of 14% as cutoff in surrogate assay, or 2.31 nmole/g from QDB method as cutoff in adjusted surrogate assay. These two subtyping methods were compared with the Kaplan-Meier, univariate and multivariate survival analyses of the overall survival (OS) of Luminal-like patients. Results Ki67 levels measured using QDB method was highly correlated with those by IHC analysis (r=0.7, p<0.0001). The survival prediction for Luminal A-like patients was improved significantly in adjusted surrogate assay than surrogate assay (p=0.03 vs p<0.00052). The prediction of Hazard Ratio (HR) was also improve from 2.14 (95%CI: 0.89-5.11, p=0.087) to 6.89 (95%CI: 2.66-17.84, p<0.00001) in multivariate survival analysis. Conclusion Our study demonstrated that the inherent subjectivity and inconsistency associated with IHC analysis has adverse effect on the performance of surrogate assay. This issue can be improved by objective and quantitative measurement of Ki67 levels with QDB method in daily clinical practice.