title: Development of automated quantification methodologies of immunohistochemical markers to determine patterns of immune response in breast cancer: a retrospective cohort study.
abstract: BACKGROUND
  Lymph nodes are one of the main sites where an effective immune response develops. Normally, axillary nodes are the first place where breast cancer produces metastases. Several studies have demonstrated the importance of immune cells, especially dendritic cells, in the evolution of breast cancer. The goal of the project is to identify differences in the patterns of immune infiltrates, with particular emphasis on dendritic cells, in tumour and axillary node biopsies between patients with and without metastases in the axillary nodes at the time of diagnosis. It is expected that these differences will be able to explain differences in survival, relapse and clinicopathological variables between the two groups.
  
  
  METHODS
  The study will involve 100 patients diagnosed with invasive breast cancer between 2000 and 2007, 50% of whom have metastases in the axillary lymph node at diagnosis. In selected patients, two cylinders from biopsies of representative areas of tumour and axillary nodes (with and without metastasis) will be selected and organised in tissue microarrays. Samples will be stained using immunohistochemical techniques for different markers of immune response and dendritic cells. Two images of each cylinder will be captured under standardised conditions for each marker. Each marker will be quantified automatically by digital image procedures using Image-Pro Plus and Image-J software. Associations of survival, relapse and other clinicopathological variables with the automatically quantified levels of immune infiltrates in patients with and without axillary node metastasis will be sought.
  
  
  BACKGROUND
  The present project has been approved by the Clinical Research Ethics Committee of the Hospital Universitari Joan XXIII (Ref: 22p/2011). Those patients whose biopsies and clinical data are to be used will give their signed informed consent. Results will be published in peer-reviewed journals.
authors.affiliation: Unitat de Suport a la Recerca Terres de l'Ebre, IISPV, Institut Universitari d'Investigació en Atenció Primària (IDIAP) Jordi Gol, URV, UAB, Tortosa, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: López
authors.affiliation: Molecular Biology and Research Section, Hospital de Tortosa Verge de la Cinta, IISPV, URV, Tortosa, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Callau
authors.affiliation: Department of Pathology, Hospital de Tortosa Verge de la Cinta, IISPV, Tortosa, Spain.
authors.forename: Ramon
authors.initials: R
authors.lastname: Bosch
authors.affiliation: Laboratory of Processing Systems of Microscopic Image Information, Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Korzynska
authors.affiliation: Department of Pathology, Hospital de Tortosa Verge de la Cinta, IISPV, Tortosa, Spain.
authors.forename: Joaquín
authors.initials: J
authors.lastname: Jaén
authors.affiliation: Department of Pathology, Hospital de Jerez, Jerez de la Frontera, Cadiz, Spain.
authors.forename: Marcial
authors.initials: M
authors.lastname: García-Rojo
authors.affiliation: VISILAB, Engineering School, Universidad de Castilla-La Mancha, Ciudad Real, Spain.
authors.forename: Gloria
authors.initials: G
authors.lastname: Bueno
authors.affiliation: Department of Pathology, Hospital de Tortosa Verge de la Cinta, IISPV, Tortosa, Spain.
authors.forename: Ma Teresa
authors.initials: MT
authors.lastname: Salvadó
authors.affiliation: Department of Pathology, Hospital de Tortosa Verge de la Cinta, IISPV, Tortosa, Spain.
authors.forename: Tomás
authors.initials: T
authors.lastname: Álvaro
authors.affiliation: Department of Gynaecology, Hospital de Tortosa Verge de la Cinta, IISPV, Tortosa, Spain.
authors.forename: Montse
authors.initials: M
authors.lastname: Oños
authors.affiliation: VISILAB, Engineering School, Universidad de Castilla-La Mancha, Ciudad Real, Spain.
authors.forename: María del Milagro
authors.initials: Mdel M
authors.lastname: Fernández-Carrobles
authors.affiliation: Department of Oncology, Hospital de Tortosa Verge de la Cinta, IISPV, Tortosa, Spain.
authors.forename: Montserrat
authors.initials: M
authors.lastname: Llobera
authors.affiliation: Department of Informatics, Hospital de Tortosa Verge de la Cinta, IISPV, Tortosa, Spain.
authors.forename: Jordi
authors.initials: J
authors.lastname: Baucells
authors.affiliation: Molecular Biology and Research Section, Hospital de Tortosa Verge de la Cinta, IISPV, URV, Tortosa, Spain.
authors.forename: Guifré
authors.initials: G
authors.lastname: Orero
authors.affiliation: Molecular Biology and Research Section, Hospital de Tortosa Verge de la Cinta, IISPV, URV, Tortosa, Spain.
authors.forename: Marylène
authors.initials: M
authors.lastname: Lejeune
mesh_terms: D001365:Axilla; D015415:Biomarkers / Q000378:metabolism*; D001943:Breast Neoplasms / Q000276:immunology* / Q000473:pathology; D002277:Carcinoma / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D015331:Cohort Studies; D003713:Dendritic Cells / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D005260:Female; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry / Q000379:methods*; D008198:Lymph Nodes / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D008207:Lymphatic Metastasis; D012189:Retrospective Studies
chemical_list: D015415:Biomarkers
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