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Background  Tumour budding (TB) and Tumour Stroma Ratio (TSR) may be rewarding in the treatment stratification of patients with stage II colon cancer. However, lack of standardization may exclude these parameters from being used in a clinical setting. The purpose of this methodologic study was to compare stereology with semi-quantitative estimations of TSR, to investigate the intra-tumoural heterogeneity of TB and TSR, and to assess the intra- and inter-observer agreement.    Methods  Three paraffin embedded tumour blocks, one of them representing the deepest invasive front, were selected from each of 43 patients treated for stage II colon cancer. TSR was estimated in H&E sections semi-quantitatively using conventional microscopy, and stereologically on scanned slides, using the newCAST stereology platform. TB was scored across 10 high power fields at the invasive front in cytokeratin AE1/AE3 stained sections.    Results  Subjective, semi-quantitative estimates of TSR significantly correlated to the stereological estimates, with the best correlation found for sections with the deepest invasive tumour penetration (σ = 0.621,  p  < 0.001). Inter-observer agreement was moderate to substantial for both TB (Κappa = 0.46–0.73) and TSR (Κappa = 0.70–0.75).  The Intraclass correlation coefficient (ICC) for TSR varied from 0.322 based on stereological hotspot estimation to 0.648 for the semi-quantitative evaluation. For TB, ICC varied from 0.646 based on continuous data to 0.698 based on categorical data (cut-off: 10 buds). Thus, the intra-tumoural heterogeneity for both TB and the semi-quantitative estimation of TSR was low.    Conclusion  We recommend using only one tissue section representing the deepest invasive tumour area for estimation of TSR. For TB we recommend using one tissue section; however due to low representation of high-budding tumours, results must be considered with caution.    Electronic supplementary material  The online version of this article (10.1186/s13000-018-0697-9) contains supplementary material, which is available to authorized users.

Does heterogeneity matter in the estimation of tumour budding and tumour stroma ratio in colon cancer?