Quantification of tumour budding, lymphatic vessel density and invasion through image analysis in colorectal cancer

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. 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. 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