title: Digital imaging in the immunohistochemical evaluation of the proliferation markers Ki67, MCM2 and Geminin, in early breast cancer, and their putative prognostic value.
abstract: BACKGROUND
  Immunohistochemical assessment of proliferation may provide additional prognostic information in early breast cancer. However, due to a lack of methodological standards proliferation markers are still not routinely used for determining therapy. Even for Ki67, one of the most widely-studied markers, disagreements over the optimal cutoff exist. Improvements in digital microscopy may provide new avenues to standardise and make data more reproducible.
  
  
  METHODS
  We studied the immunohistochemical expression of three markers of proliferation: Ki67, Mini-Chromosome Maintenance protein 2 and Geminin, by conventional light microscope and digital imaging on triplicate TMAs from 309 consecutive cases of primary breast cancers. Differences between the average and the maximum percentage reactivity in tumour cell nuclei from the three TMA cores were investigated to assess the validity of the approach. Time-dependent Receiver Operating Characteristic curves were utilized to obtain optimal expression level cut-offs, which were then correlated with clinico-pathological features and survival.
  
  
  RESULTS
  High concordance between conventional and digital scores was observed for all 3 markers (Ki67: rs = 0.87, P < 0.001; MCM2: rs = 0.94, P < 0.001; and Geminin: rs = 0.86, P < 0.001; Spearman's rank). There was no significant difference according to the number of TMA cores included for either Ki67 or MCM2; analysis of two or three cores produced comparable results. Higher levels of all three proliferation markers were significantly associated with higher grade (P < 0.001) and ER-negativity (P < 0.001). Optimal prognostic cut-offs for percentage expression in the tumour were 8 %, 12 and 2.33 % for Ki67, MCM2 and Geminin respectively. All 3 proliferation marker cutoffs were predictive of 15-year breast cancer-specific survival in univariable Cox regression analyses. In multivariable analysis only lymph node status (HR = 3.9, 95 % CI = 1.79-8.5, P = 0.0006) and histological grade (HR = 1.84, 95 % CI = 1-3.38, P = 0.05) remained significantly prognostic.
  
  
  CONCLUSIONS
  Here we show that. MCM2 is a more sensitive marker of proliferation than Ki67 and should be examined in future studies, especially in the lymph node-negative, hormone receptor-positive subgroup. Further, digital microscopy can be used effectively as a high-throughput method to evaluate immunohistochemical expression.
authors.affiliation: Department of Research Oncology, King's College London, Faculty of Life Science and Medicine, Division of Cancer Studies, Bermondsey Wing, Guy's Hospital, London, UK. drjoshishalaka@gmail.com.|Breast Cancer Now Unit, King's College London, Faculty of Life Science and Medicine, Division of Cancer Studies, Bermondsey Wing, Guy's Hospital, London, UK. drjoshishalaka@gmail.com.|Present address: Tata Memorial Centre, Dr E Borges Road, Parel, Mumbai, 400012, Maharashtra, India. drjoshishalaka@gmail.com.
authors.forename: Shalaka
authors.initials: S
authors.lastname: Joshi
authors.affiliation: Department of Research Oncology, King's College London, Faculty of Life Science and Medicine, Division of Cancer Studies, Bermondsey Wing, Guy's Hospital, London, UK. johnathan.watkins@kcl.ac.uk.|Breast Cancer Now Unit, King's College London, Faculty of Life Science and Medicine, Division of Cancer Studies, Bermondsey Wing, Guy's Hospital, London, UK. johnathan.watkins@kcl.ac.uk.
authors.forename: Johnathan
authors.initials: J
authors.lastname: Watkins
authors.affiliation: Department of Research Oncology, King's College London, Faculty of Life Science and Medicine, Division of Cancer Studies, Bermondsey Wing, Guy's Hospital, London, UK. patrycja.gazinska@kcl.ac.uk.|Breast Cancer Now Unit, King's College London, Faculty of Life Science and Medicine, Division of Cancer Studies, Bermondsey Wing, Guy's Hospital, London, UK. patrycja.gazinska@kcl.ac.uk.
authors.forename: Patrycja
authors.initials: P
authors.lastname: Gazinska
authors.affiliation: Department of Research Oncology, King's College London, Faculty of Life Science and Medicine, Division of Cancer Studies, Bermondsey Wing, Guy's Hospital, London, UK. john.brown@kcl.ac.uk.
authors.forename: John P
authors.initials: JP
authors.lastname: Brown
authors.affiliation: Department of Research Oncology, King's College London, Faculty of Life Science and Medicine, Division of Cancer Studies, Bermondsey Wing, Guy's Hospital, London, UK. cheryl.gillett@kcl.ac.uk.|King's Health Partners Cancer Biobank, King's College London, Faculty of Life Science and Medicine, Division of Cancer Studies, Bermondsey Wing, Guy's Hospital, London, UK. cheryl.gillett@kcl.ac.uk.
authors.forename: Cheryl E
authors.initials: CE
authors.lastname: Gillett
authors.affiliation: Department of Research Oncology, King's College London, Faculty of Life Science and Medicine, Division of Cancer Studies, Bermondsey Wing, Guy's Hospital, London, UK. anita.grigoriadis@kcl.ac.uk.|Breast Cancer Now Unit, King's College London, Faculty of Life Science and Medicine, Division of Cancer Studies, Bermondsey Wing, Guy's Hospital, London, UK. anita.grigoriadis@kcl.ac.uk.
authors.forename: Anita
authors.initials: A
authors.lastname: Grigoriadis
authors.affiliation: Department of Research Oncology, King's College London, Faculty of Life Science and Medicine, Division of Cancer Studies, Bermondsey Wing, Guy's Hospital, London, UK. sarah.pinder@kcl.ac.uk.|King's Health Partners Cancer Biobank, King's College London, Faculty of Life Science and Medicine, Division of Cancer Studies, Bermondsey Wing, Guy's Hospital, London, UK. sarah.pinder@kcl.ac.uk.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Pinder
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D003952:Diagnostic Imaging / Q000379:methods*; D055088:Early Detection of Cancer / Q000379:methods*; D005260:Female; D064248:Geminin / Q000378:metabolism*; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000379:methods; D019394:Ki-67 Antigen / Q000378:metabolism*; D008875:Middle Aged; D064111:Minichromosome Maintenance Complex Component 2 / Q000378:metabolism*; D011379:Prognosis; D012372:ROC Curve; D046888:Tissue Array Analysis
chemical_list: D014408:Biomarkers, Tumor; C577995:GMNN protein, human; D064248:Geminin; D019394:Ki-67 Antigen; C084859:MCM2 protein, human; D064111:Minichromosome Maintenance Complex Component 2