Background:Tumour budding, described as the presence of single cells or small clusters of up to five tumour cells at the invasive margin, is established as a prognostic marker in colorectal carcinoma. In the present study, we aimed to investigate the molecular signature of tumour budding cells and the corresponding tumour bulk.Methods:Tumour bulk and budding areas were microdissected and processed for RNA-sequencing. As little RNA was obtained from budding cells, a special low-input mRNA library preparation protocol was used. Gene expression profiles of budding as compared with tumour bulk were investigated for established EMT signatures, consensus molecular subtype (CMS), gene set enrichment and pathway analysis.Results:A total of 296 genes were differentially expressed with an FDR <0.05 and a twofold change between tumour bulk and budding regions. Genes that were upregulated in the budding signature were mainly involved in cell migration and survival while downregulated genes were important for cell proliferation. Supervised clustering according to an established EMT gene signature categorised budding regions as EMT-positive, whereas tumour bulk was considered EMT-negative. Furthermore, a shift from CMS2 (epithelial) to CMS4 (mesenchymal) was observed as tumour cells transit from the tumour bulk to the budding regions.Conclusions:Tumour budding regions are characterised by a phenotype switch compared with the tumour bulk, involving the acquisition of migratory characteristics and a decrease in cell proliferation. In particular, most tumour budding signatures were EMT-positive and switched from an epithelial subtype (CMS2) in the tumour bulk to a mesenchymal subtype (CMS4) in budding cells.

611 Background: Intratumour heterogeneity is a key challenge in colorectal cancer management. We have recently shown high levels of morphologic heterogeneity in microsatellite unstable (MSI) colorectal tumours (CRC). In this study we verified whether morphologic intratumour heterogeneity can be used as a guidance to test for molecular heterogeneity. Methods: 15 MSI CRCs tumour, characterized by a heterogenous morphology, were selected together with the associated metastasis. Macrodissection of the distinct morphologic tumour components was followed by MSI testing according to the Bethesda guidelines and Sanger sequencing for BRAF V600E mutations. In addition, 2 MSI CRC underwent macrodissection of the distinct morphologic components, followed by nanostring ncounter analysis. Results: MSI status varies between MSI-H and MSI-L in the primary tumours and 35% of the metastasis of MSI-H primary tumours presented as MSI-L or MSS. BRAF mutations status was constant throughout the primary tumours, but two BRAF mutated tumours presented with a BRAF wild type metastasis. Nanostring analysis revealed that ùorphologically identical clones clustered separately from tumour clones with a different morphologic display. Conclusions: Morphologically heterogeneous tumour compartments have less related gene expression profiles, linking morphologic homogeneity to molecular homogeneity. Our findings support the hypothesis that morphologic heterogeneity reflects molecular intratumour heterogeneity and can be used as a guidance for molecular testing of colorectal cancers.

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