title: ImmunoRatio: a publicly available web application for quantitative image analysis of estrogen receptor (ER), progesterone receptor (PR), and Ki-67.
abstract: BACKGROUND
  Accurate assessment of estrogen receptor (ER), progesterone receptor (PR), and Ki-67 is essential in the histopathologic diagnostics of breast cancer. Commercially available image analysis systems are usually bundled with dedicated analysis hardware and, to our knowledge, no easily installable, free software for immunostained slide scoring has been described. In this study, we describe a free, Internet-based web application for quantitative image analysis of ER, PR, and Ki-67 immunohistochemistry in breast cancer tissue sections.
  
  
  METHODS
  The application, named ImmunoRatio, calculates the percentage of positively stained nuclear area (labeling index) by using a color deconvolution algorithm for separating the staining components (diaminobenzidine and hematoxylin) and adaptive thresholding for nuclear area segmentation. ImmunoRatio was calibrated using cell counts defined visually as the gold standard (training set, n = 50). Validation was done using a separate set of 50 ER, PR, and Ki-67 stained slides (test set, n = 50). In addition, Ki-67 labeling indexes determined by ImmunoRatio were studied for their prognostic value in a retrospective cohort of 123 breast cancer patients.
  
  
  RESULTS
  The labeling indexes by calibrated ImmunoRatio analyses correlated well with those defined visually in the test set (correlation coefficient r = 0.98). Using the median Ki-67 labeling index (20%) as a cutoff, a hazard ratio of 2.2 was obtained in the survival analysis (n = 123, P = 0.01). ImmunoRatio was shown to adapt to various staining protocols, microscope setups, digital camera models, and image acquisition settings. The application can be used directly with web browsers running on modern operating systems (e.g., Microsoft Windows, Linux distributions, and Mac OS). No software downloads or installations are required. ImmunoRatio is open source software, and the web application is publicly accessible on our website.
  
  
  CONCLUSIONS
  We anticipate that free web applications, such as ImmunoRatio, will make the quantitative image analysis of ER, PR, and Ki-67 easy and straightforward in the diagnostic assessment of breast cancer specimens.
authors.affiliation: Institute of Medical Technology, University of Tampere, Biokatu 6, 33014 Tampere, Finland.
authors.forename: Vilppu J
authors.initials: VJ
authors.lastname: Tuominen
authors.forename: Sanna
authors.initials: S
authors.lastname: Ruotoistenmäki
authors.forename: Arttu
authors.initials: A
authors.lastname: Viitanen
authors.forename: Mervi
authors.initials: M
authors.lastname: Jumppanen
authors.forename: Jorma
authors.initials: J
authors.lastname: Isola
mesh_terms: D000465:Algorithms; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D005260:Female; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007150:Immunohistochemistry / Q000379:methods*; D020407:Internet; D053208:Kaplan-Meier Estimate; D019394:Ki-67 Antigen / Q000032:analysis; D011379:Prognosis; D011960:Receptors, Estrogen / Q000032:analysis; D011980:Receptors, Progesterone / Q000032:analysis; D015203:Reproducibility of Results; D012984:Software; D046888:Tissue Array Analysis
chemical_list: D019394:Ki-67 Antigen; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone