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The biological mechanisms and potential clinical impact of heterotopic ossification (HO) in colorectal neoplasms are not fully understood. This study investigates the clinicopathological characteristics of colorectal neoplasms associated with HO and examines the potential role of the bone morphogenetic protein (BMP) pathway in development of HO. An artificial intelligence (AI) based classification of colorectal cancers (CRC) exhibiting HO and their association with consensus molecular subtypes (CMS) is performed. The study included 77 cases via the Dutch nationwide Pathology databank. Immunohistochemistry for BMP2, SMAD4, and Osterix was performed. An AI algorithm assessed the tumour-stroma ratio to approximate the CMS. A literature search yielded 96 case reports, which were analysed and compared with our cases for clinicopathological parameters. HO was more frequently observed in our cohort in traditional serrated adenomas (25%), tubulovillous adenomas (25%) and juvenile polyps (25%), while in the literature it was most often seen in juvenile polyps (38.2%) and inflammatory polyps (29.4%). In both cohorts, carcinomas were mostly conventional (>60%) followed by mucinous and serrated adenocarcinomas. Higher expression of BMP2, SMAD4, and Osterix was observed in tumour and/or stromal cells directly surrounding bone, indicating activation of the BMP pathway. The tumour-stroma analysis appointed >50% of the cases to the mesenchymal subtype (CMS4) (59%). HO has a predilection for serrated and juvenile/inflammatory polyps, mucinous and serrated adenocarcinomas. BMP signalling is activated and seems to play a role in formation of HO in colorectal neoplasms. In line with TGFβ/BMP pathway activation associated with CMS4 CRC, HO seems associated with CMS4., Competing Interests: Declaration of competing interest No potential conflicts of interest relevant to this article were reported., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The current stratification of tumor nodules in colorectal cancer (CRC) staging is subjective and leads to high interobserver variability. In this study, the objective assessment of the shape of lymph node metastases (LNMs), extranodal extension (ENE), and tumor deposits (TDs) was correlated with outcomes. A test cohort and a validation cohort were included from 2 different institutions. The test cohort consisted of 190 cases of stage III CRC. Slides with LNMs and TDs were annotated and processed using a segmentation algorithm to determine their shape. The complexity ratio was calculated for every shape and correlated with outcomes. A cohort of 160 stage III CRC cases was used to validate findings. TDs showed significantly more complex shapes than LNMs with ENE, which were more complex than LNMs without ENE (P < .001). In the test cohort, patients with the highest sum of complexity ratios had significantly lower disease-free survival (P < .01). When only the nodule with the highest complexity was considered, this effect was even stronger (P < .001). This maximum complexity ratio per patient was identified as an independent prognostic factor in the multivariate analysis (hazard ratio, 2.47; P < .05). The trends in the validation cohort confirmed the results. More complex nodules in stage III CRC were correlated with significantly worse disease-free survival, even if only based on the most complex nodule. These results suggest that more complex nodules reflect more invasive tumor biology. As most of the more complex nodules were diagnosed as TDs, we suggest providing a more prominent role for TDs in the nodal stage and include an objective complexity measure in their definition., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Both lymph node metastases (LNMs) and tumour deposits (TDs) are included in colorectal cancer (CRC) staging, although knowledge regarding their biological background is lacking. This study aimed to compare the biology of these prognostic features, which is essential for a better understanding of their role in CRC spread. Spatially resolved transcriptomic analysis using digital spatial profiling was performed on TDs and LNMs from 10 CRC patients using 1,388 RNA targets, for the tumour cells and tumour microenvironment. Shotgun proteomics identified 5,578 proteins in 12 different patients. Differences in RNA and protein expression were analysed, and spatial deconvolution was performed. Image-based consensus molecular subtype (imCMS) analysis was performed on all TDs and LNMs included in the study. Transcriptome and proteome profiles identified distinct clusters for TDs and LNMs in both the tumour and tumour microenvironment segment, with upregulation of matrix remodelling, cell adhesion/motility, and epithelial-mesenchymal transition (EMT) in TDs (all p < 0.05). Spatial deconvolution showed a significantly increased abundance of fibroblasts, macrophages, and regulatory T-cells (p < 0.05) in TDs. Consistent with a higher fibroblast and EMT component, imCMS classified 62% of TDs as poor prognosis subtype CMS4 compared to 36% of LNMs (p < 0.05). Compared to LNMs, TDs have a more invasive state involving a distinct tumour microenvironment and upregulation of EMT, which are reflected in a more frequent histological classification of TDs as CMS4. These results emphasise the heterogeneity of locoregional spread and the fact that TDs should merit more attention both in future research and during staging. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Tumor budding (TB), the presence of single cells or small clusters of up to 4 tumor cells at the invasive front of colorectal cancer (CRC), is a proven risk factor for adverse outcomes. International definitions are necessary to reduce interobserver variability. According to the current international guidelines, hotspots at the invasive front should be counted in hematoxylin and eosin (H&E)-stained slides. This is time-consuming and prone to interobserver variability; therefore, there is a need for computer-aided diagnosis solutions. In this study, we report an artificial intelligence-based method for detecting TB in H&E-stained whole slide images. We propose a fully automated pipeline to identify the tumor border, detect tumor buds, characterize them based on the number of tumor cells, and produce a TB density map to identify the TB hotspot. The method outputs the TB count in the hotspot as a computational biomarker. We show that the proposed automated TB detection workflow performs on par with a panel of 5 pathologists at detecting tumor buds and that the hotspot-based TB count is an independent prognosticator in both the univariate and the multivariate analysis, validated on a cohort of n = 981 patients with CRC. Computer-aided detection of tumor buds based on deep learning can perform on par with expert pathologists for the detection and quantification of tumor buds in H&E-stained CRC histopathology slides, strongly facilitating the introduction of budding as an independent prognosticator in clinical routine and clinical trials., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumor budding is a long-established independent adverse prognostic marker in colorectal cancer, yet methods for its assessment have varied widely. In an effort to standardize its reporting, a group of experts met in Bern, Switzerland, in 2016 to reach consensus on a single, international, evidence-based method for tumor budding assessment and reporting (International Tumor Budding Consensus Conference [ITBCC]). Tumor budding assessment using the ITBCC criteria has been validated in large cohorts of cancer patients and incorporated into several international colorectal cancer pathology and clinical guidelines. With the wider reporting of tumor budding, new issues have emerged that require further clarification. To better inform researchers and health-care professionals on these issues, an international group of experts in gastrointestinal pathology participated in a modified Delphi process to generate consensus and highlight areas requiring further research. This effort serves to re-affirm the importance of tumor budding in colorectal cancer and support its continued use in routine clinical practice., (© 2021. The Author(s).)

Aims: Outcomes of colorectal cancer (CRC) treatment and survival have steadily improved during the past decades, accompanied by an increased risk of developing second primary tumours and metastatic tumours at unusual sites. Metastatic CRC can show mucosal colonisation, thereby mimicking a second primary tumour. This potential confusion could lead to incorrect diagnosis and consequently inadequate treatment of the patient. The aim of this study was to differentiate between metastatic CRC and a second primary (gallbladder cancer, GBC) using a combination of standard histopathology and molecular techniques., Methods and Results: Ten consecutive patients with both CRC and GBC were identified in our region using the Dutch National Pathology Archive (PALGA). Two patients served as negative controls. Histology of GBC was reviewed by nine pathologists. A combination of immunohistochemistry, microsatellite analysis, genomewide DNA copy number analysis and targeted somatic mutation analysis was used to aid in differential diagnosis. In two patients, CRC and GBC were clonally related, as confirmed by somatic mutation analysis. For one case, this was confirmed by genomewide DNA copy number analysis. However, in both cases, pathologists initially considered the GBC as a second primary tumour., Conclusions: Metastatic CRC displaying mucosal colonisation is often misinterpreted as a second primary tumour. A combination of traditional histopathology and molecular techniques improves this interpretation, and lowers the risk of inadequate treatment., (© 2019 The Authors. Histopathology Published by John Wiley & Sons Ltd.)

Perineural invasion (PNI) is a possible route for metastatic spread in various cancer types, including colorectal cancer (CRC). PNI is linked to poor prognosis, but systematic analyses are lacking. This study systematically reviews the frequency and impact of PNI in CRC. A literature search was performed using PubMed database from inception to January 1, 2014. Data were analyzed using Review Manager 5.3. A quality assessment was performed on the basis of modified REMARK criteria. Endpoints were local recurrence (LR), 5-year disease-free survival (5yDFS), 5-year cancer-specific survival (5yCSS), and 5-year overall survival (5yOS). Meta-analysis was performed in terms of risk ratios (RR) and hazard ratios (HR) with 95% confidence interval (95% CI). In this meta-analysis, 58 articles with 22,900 patients were included. PNI was present in 18.2% of tumors. PNI is correlated with increased LR (RR 3.22, 95% CI, 2.33-4.44) and decreased 5yDFS (RR 2.35, 95% CI, 1.66-3.31), 5yCSS (RR 3.61, 95% CI, 2.76-4.72), and 5yOS (RR 2.09, 95% CI, 1.68-2.61). In multivariate analysis PNI remains an independent prognostic factor for 5yDFS, 5yCSS, and 5yOS (HR 2.35, 95% CI, 1.97-3.08; HR 1.91, 95% CI, 1.50-2.42; and HR 1.85, 95% CI, 1.63-2.12, respectively). We confirmed the strong impact of PNI for LR and survival in CRC. The prognostic value of PNI is similar to that of well-established prognostic factors as depth of invasion, differentiation grade, lymph node metastases, and lymphatic and extramural vascular invasion. Therefore, PNI should be one of the factors in the standardized reporting of CRC and might be considered a high-risk feature.

Background: Mucinous carcinoma (MC) is found in 10%-15% of colorectal cancer (CRC) patients. It differs from the common adenocarcinoma (AC) in histopathological appearance and clinical behavior., Methods: Genome-wide DNA copy number and survival data from MC and AC primary CRC samples from patients from two phase III trials (CAIRO and CAIRO2) was compared. Chromosomal copy number data from The Cancer Genome Atlas (TCGA) was used for validation. Altogether, 470 ACs were compared to 57 MCs., Results: MC showed a reduced amount of copy number aberrations (CNAs) compared with AC for the CAIRO/CAIRO2 cohort, with a median amount of CNAs that was 1.5-fold lower (P = 0.002). Data from TCGA also showed a reduced amount of CNAs for MC. MC samples in both cohorts displayed less gain at chromosome 20q and less loss of chromosome 18p. A high rate of chromosomal instability was a strong negative prognostic marker for survival in MC patients from the CAIRO cohorts (hazard ratio 15.60, 95% CI 3.24-75.05)., Conclusions: Results from this study indicate that the distinct MC phenotype is accompanied by a different genetic basis when compared with AC and show a strong association between the rate of chromosomal instability and survival in MC patients.

Approximately half of the colorectal cancer (CRC) patients develop metastatic disease. Fluoropyrimidine-based chemotherapy forms the backbone of treatment in these patients. However, the response to this therapy varies between individuals. Therefore, an important challenge in CRC research is to identify biomarkers that are predictive of this response. In this study, we explored the potential of miRNAs, and the miRNA producing protein Dicer, as biomarkers that can predict chemo-sensitivity to fluoropyrimidine chemotherapy in patients with metastatic colorectal cancer (mCRC). We analyzed the levels of 22 miRNAs and the Dicer protein in primary tumors from patients with mCRC who were treated with first-line capecitabine monotherapy within the CAIRO trial of the Dutch Colorectal Cancer Group. Correlation between the expression status of miRNAs or Dicer in primary tumors and the progression free survival (PFS) were investigated. Patients with low expression of miR-143 in their primary tumor had increased median PFS compared to those with high expression of miR-143. Furthermore, FXYD3, an ion transport regulator and a putative target of miR-143, also showed an association with PFS. These findings warrant further studies to investigate the relationship between miR-143, FXYD3 and fluoropyrimidines, and the clinical utility of miR-143 as biomarker.

Aberrant DNA methylation often occurs in colorectal cancer (CRC). In our study we applied a genome-wide DNA methylation analysis approach, MethylCap-seq, to map the differentially methylated regions (DMRs) in 24 tumors and matched normal colon samples. In total, 2687 frequently hypermethylated and 468 frequently hypomethylated regions were identified, which include potential biomarkers for CRC diagnosis. Hypermethylation in the tumor samples was enriched at CpG islands and gene promoters, while hypomethylation was distributed throughout the genome. Using epigenetic data from human embryonic stem cells, we show that frequently hypermethylated regions coincide with bivalent loci in human embryonic stem cells. DNA methylation is commonly thought to lead to gene silencing; however, integration of publically available gene expression data indicates that 75% of the frequently hypermethylated genes were most likely already lowly or not expressed in normal tissue. Collectively, our study provides genome-wide DNA methylation maps of CRC, comprehensive lists of DMRs, and gives insights into the role of aberrant DNA methylation in CRC formation.