title: Automated quantitative analysis (AQUA) of in situ protein expression, antibody concentration, and prognosis.
abstract: BACKGROUND
  Disparate results in the immunohistochemistry literature regarding the relationship between biomarker expression and patient outcome decrease the credibility of tissue biomarker studies. We investigated whether some of these disparities result from subjective optimization of antibody concentration.
  
  
  METHODS
  We used the automated quantitative analysis (AQUA) system and various concentrations of antibodies against HER2 (1 : 500 to 1 : 8000 dilutions), p53 (1 : 50 to 1 : 800 dilutions), and estrogen receptor (ER; 1 : 100 and 1 : 1000 dilutions) to assess expression of HER2 and p53 in a tissue microarray containing specimens from 250 breast cancer patients with long-term survival data available. HER2 expression in the tissue microarray was also assessed by conventional immunohistochemistry. Relative risk (RR) of disease-specific mortality was assessed for every cutpoint with the X-tile program. Cumulative disease-specific survival was assessed by the Kaplan-Meier method. All statistical tests were two-sided.
  
  
  RESULTS
  For HER2 and p53 and an optimal cutpoint, when a high antibody concentration (i.e., 1 : 500 dilution) was used with the AQUA system, low expression was associated with poorer survival than high expression; however, when a low antibody concentration (i.e., 1 : 8000 dilution) was used, high expression was associated with poorer survival. For example, for a 1 : 8000 dilution of HER2 antibody and high expression defined as the top 15% of HER2 expression, high HER2 expression was associated with increased disease-specific mortality (RR = 1.98, 95% confidence interval [CI] = 1.21 to 3.23; P = .007), compared with low expression. However, for a 1 : 500 dilution of HER2 antibody and high expression defined as the top 85% of HER2 expression, high HER2 expression was associated with decreased disease-specific mortality (RR = 0.47, 95% CI = 0.29 to 0.76; P = .002), compared with low HER2 expression.
  
  
  CONCLUSIONS
  Biomarker antibody concentration appears to dramatically affect the apparent relationship between biomarker expression and outcome.
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT 06520-8023, USA.
authors.forename: Anthony
authors.initials: A
authors.lastname: McCabe
authors.forename: Marisa
authors.initials: M
authors.lastname: Dolled-Filhart
authors.forename: Robert L
authors.initials: RL
authors.lastname: Camp
authors.forename: David L
authors.initials: DL
authors.lastname: Rimm
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000912:Antibodies, Neoplasm / Q000032:analysis*; D014408:Biomarkers, Tumor / Q000032:analysis*; D045744:Cell Line, Tumor; D016001:Confidence Intervals; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D008875:Middle Aged; D009369:Neoplasms / Q000737:chemistry* / Q000401:mortality; D016017:Odds Ratio; D011237:Predictive Value of Tests; D011379:Prognosis; D040081:Protein Array Analysis / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis / Q000276:immunology; D011960:Receptors, Estrogen / Q000032:analysis / Q000276:immunology; D016019:Survival Analysis; D016896:Treatment Outcome; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000737:chemistry
chemical_list: D000912:Antibodies, Neoplasm; D014408:Biomarkers, Tumor; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; D016159:Tumor Suppressor Protein p53