title: Optimisation of an immunohistochemistry method for the determination of androgen receptor expression levels in circulating tumour cells.
abstract: 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). β-Content γ-confidence tolerance intervals (BCTI) and Cohen's Kappa (κ) were employed in statistical analysis of results.
  
  
  RESULTS
  In a first set of IHC reproducibility experiments, almost perfect agreement was recorded (κ=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 κ 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 technique.
authors.affiliation: Clinical and Experimental Pharmacology Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester Cancer Research Centre, Manchester M20 4BX, UK. jcummings@picr.man.ac.uk.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Cummings
authors.forename: Robert
authors.initials: R
authors.lastname: Sloane
authors.forename: Karen
authors.initials: K
authors.lastname: Morris
authors.forename: Cong
authors.initials: C
authors.lastname: Zhou
authors.forename: Matt
authors.initials: M
authors.lastname: Lancashire
authors.forename: David
authors.initials: D
authors.lastname: Moore
authors.forename: Tony
authors.initials: T
authors.lastname: Elliot
authors.forename: Noel
authors.initials: N
authors.lastname: Clarke
authors.forename: Caroline
authors.initials: C
authors.lastname: Dive
mesh_terms: D000368:Aged; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002452:Cell Count / Q000379:methods; D045744:Cell Line, Tumor; D006801:Humans; D007120:Immunochemistry / Q000379:methods*; D008297:Male; D009360:Neoplastic Cells, Circulating / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D011724:Pyridazines / Q000494:pharmacology*; D011944:Receptors, Androgen / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: C410798:AR protein, human; C583656:AZD3514; D000970:Antineoplastic Agents; D011724:Pyridazines; D011944:Receptors, Androgen