title: Quantification of tumour budding, lymphatic vessel density and invasion through image analysis in colorectal cancer.
abstract: BACKGROUND
  Tumour budding (TB), lymphatic vessel density (LVD) and lymphatic vessel invasion (LVI) have shown promise as prognostic factors in colorectal cancer (CRC) but reproducibility using conventional histopathology is challenging. We demonstrate image analysis methodology to quantify the histopathological features which could permit standardisation across institutes and aid risk stratification of Dukes B patients.
  
  
  METHODS
  Multiplexed immunofluorescence of pan-cytokeratin, D2-40 and DAPI identified epithelium, lymphatic vessels and all nuclei respectively in tissue sections from 50 patients diagnosed with Dukes A (n = 13), Dukes B (n = 29) and Dukes C (n = 8) CRC. An image analysis algorithm was developed and performed, on digitised images of the CRC tissue sections, to quantify TB, LVD, and LVI at the invasive front.
  
  
  RESULTS
  TB (HR =5.7; 95% CI, 2.38-13.8), LVD (HR =5.1; 95% CI, 2.04-12.99) and LVI (HR =9.9; 95% CI, 3.57-27.98) were successfully quantified through image analysis and all were shown to be significantly associated with poor survival, in univariate analyses. LVI (HR =6.08; 95% CI, 1.17-31.41) is an independent prognostic factor within the study and was correlated to both TB (Pearson r =0.71, p <0.0003) and LVD (Pearson r =0.69, p <0.0003).
  
  
  CONCLUSIONS
  We demonstrate methodology through image analysis which can standardise the quantification of TB, LVD and LVI from a single tissue section while decreasing observer variability. We suggest this technology is capable of stratifying a high risk Dukes B CRC subpopulation and we show the three histopathological features to be of prognostic significance.
authors.affiliation: Digital Pathology Unit, Laboratory medicine, Royal Infirmary of Edinburgh, Edinburgh EH16 4SA, UK. p.d.caie@sms.ed.ac.uk.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Caie
authors.forename: Arran K
authors.initials: AK
authors.lastname: Turnbull
authors.forename: Susan M
authors.initials: SM
authors.lastname: Farrington
authors.forename: Anca
authors.initials: A
authors.lastname: Oniscu
authors.forename: David J
authors.initials: DJ
authors.lastname: Harrison
mesh_terms: D000328:Adult; D015331:Cohort Studies; D015179:Colorectal Neoplasms / Q000473:pathology*; D005260:Female; D005500:Follow-Up Studies; D006801:Humans; D007091:Image Processing, Computer-Assisted; D053208:Kaplan-Meier Estimate; D042601:Lymphatic Vessels / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D015999:Multivariate Analysis; D009361:Neoplasm Invasiveness; D016016:Proportional Hazards Models