Optimisation of an immunohistochemistry method for the determination of androgen receptor expression levels in circulating tumour cells

Background: AZD3514 inhibits and down regulates the androgen receptor (AR) and has undergone clinical trials in prostate cancer. To provide proof-of-mechanism (POM) in patients, an immunohistochemistry (IHC) method for determination of AR in circulating tumour cells (CTC) was developed and validated. Methods: After an assessment of specificity validation focused on intra-and inter-operator reproducibility utilising a novel modification of incurred sample reanalysis (ISR). beta-Content gamma-confidence tolerance intervals (BCTI) and Cohen's Kappa (kappa) were employed in statistical analysis of results. Results: In a first set of IHC reproducibility experiments, almost perfect agreement was recorded (kappa=0.94) when two different operators scored CTC as overall positive or negative for AR. However, BCTI analysis identified a specific bias in scoring staining intensity, where one operator favoured moderate over strong assignments, whereas the reverse was the case with the second operator. After a period of additional training involving deployment of a panel of standardised images, a second set of validation experiments were conducted. These showed correction of the inter-operator bias by BCTI with. for scoring intensity increasing from 0.59 to 0.81, indicative of almost perfect agreement. Conclusions: By application of BCTI to the validation of IHC, operator bias and therefore poor reproducibility can be identified, characterised and corrected to achieve a level of error normally associated with a quantitative biomarker assay, such as an ELISA. The methodological approach described herein can be applied to any generic IHC techniques.