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.

Key Points• FOXP1 is downregulated in germinal centers, inversely to BCL6, whereby it regulates a network of genes, half of which are also BCL6 targets.• In transgenic mice, constitutive FOXP1 expression impairs GC formation and function, which might contribute to B-cell lymphomagenesis.B-cell maturation and germinal center (GC) formation are dependent on the interplay between BCL6 and other transcriptional regulators. FOXP1 is a transcription factor that regulates early B-cell development, but whether it plays a role in mature B cells is unknown. Analysis of human tonsillar B-cell subpopulations revealed that FOXP1 shows the opposite expression pattern to BCL6, suggesting that FOXP1 regulates the transition from resting follicular B cell to activated GC B cell. Chromatin immunoprecipitation-on-chip and gene expression assays on B cells indicated that FOXP1 acts as a transcriptional activator and repressor of genes involved in the GC reaction, half of which are also BCL6 targets. To study FOXP1 function in vivo, we developed transgenic mice expressing human FOXP1 in lymphoid cells. These mice exhibited irregular formation of splenic GCs, showing a modest increase in naïve and marginalzone B cells and a significant decrease in GC B cells. Furthermore, aberrant expression of FOXP1 impaired transcription of noncoding g1 germline transcripts and inhibited efficient class switching to the immunoglobulin G1 isotype. These studies show that FOXP1 is physiologically downregulated in GC B cells and that aberrant expression of FOXP1 impairs mechanisms triggered by Bcell activation, potentially contributing to B-cell lymphomagenesis. (Blood. 2013;121(21):4311-4320)

Budding is a process during which individual or small clusters of up to five tumour cells detach from the main tumour mass and invade into the surrounding stroma. In colorectal cancer, this feature is observed in 20-40% of cases and is associated with lymphovascular invasion, lymph node and distant metastases, and poor prognosis. A variety of scoring systems for budding have been proposed but so far a gold standard is lacking, hampering implementation of a budding score in guidelines for pathological examination of colorectal cancer. Furthermore, little is known about the mechanisms which cause tumour cells to detach from the main tumour mass and obtain increased invasive potential. In this review, we present an overview of tumour budding including its definition, scoring systems, prognostic relevance and biological mechanisms involved.

In our case series, loss of FOXP1 was associated with reduced survival rates in CRC tissue. Also, FOXP1 affects proliferation and inflammatory reaction in colorectal neoplasia.

Background:An everyday clinical practice dilemma in the 20–30% of metastatic colorectal cancer (CRC) patients that have not been operated on their primary tumour, is, under which specific histopathology and molecular circumstances, an endoscopic biopsy could be considered adequate to provide a representative RAS/BRAF molecular status to guide treatment.Methods:A consecutive series of 193 paired biopsy and primary CRC tumour samples between August 2008 and 2010 available in the Department of Pathology archives, University Hospitals, KU Leuven were retrieved. For a pair to be included, in the endoscopic biopsy, 20% of invasive adenocarcinoma cells should be present and enough slides to yield an extracted DNA concentration of ⩾5 ng μl−1, and no <2 ng μl−1 should be available for cutting. Exons 2–4 KRAS/NRAS, BRAF, PIK3CA molecular evaluation was performed with RT–PCR and Sequenom.Results:From 165 deemed adequate by the pathologist pairs, 85 (51.5%) were concordantly mutated in at least one of the tested genes, 70 (42.5%) were wt and 10 (6%) were discordant, harbouring a mutation in the primary and not in the endoscopic biopsy. In the re-evaluation, when more slides were cut per discordant pair, mutational status changed in two of the six discordantly KRAS-mutated pairs. A strong strength of agreement for both runs was observed (Cohen's kappa, k=0.877, P<0.001 and k=0.901, P<0.001, respectively) between the surgically acquired and the endoscopic biopsy specimens' evaluation.Conclusions:Based on our results, an endoscopic biopsy could provide an accurate mutational profile and become a justified alternative to a surgically removed primary tumour specimen, as long as specific histopathology criteria are met.