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Abstract Background Thrombospondin-4 (THBS4) is a member of the extracellular calcium-binding protein family and is involved in cell adhesion and migration. The aim of this study was to evaluate the potential role of deregulation of THBS4 expression in colorectal carcinogenesis. Of particular interest was the possible silencing of expression by methylation of the CpG island in the gene promoter. Methods Fifty-five sporadic colorectal tumours stratified for the CpG Island Methylator Phenotype (CIMP) were studied. Immunohistochemical staining of THBS4 protein was assessed in normal and tumour specimens. Relative levels of THBS4 transcript expression in matched tumours and normal mucosa were also determined by quantitative RT-PCR. Colony forming ability was examined in 8 cell lines made to overexpress THBS4. Aberrant promoter hypermethylation was investigated as a possible mechanism of gene disruption using MethyLight. Methylation was also assessed in the normal colonic tissue of 99 patients, with samples biopsied from four regions along the length of the colon. Results THBS4 expression was significantly lower in tumour tissue than in matched normal tissue. Immunohistochemical examination demonstrated that THBS4 protein was generally absent from normal epithelial cells and tumours, but was occasionally expressed at low levels in the cytoplasm towards the luminal surface in vesicular structures. Forced THBS4 over-expression caused a 50-60% repression of tumour colony growth in all eight cell lines examined compared to control cell lines. Tumours exhibited significantly higher levels of methylation than matched normal mucosa, and THBS4 methylation correlated with the CpG island methylator phenotype. There was a trend towards decreased gene expression in tumours exhibiting high THBS4 methylation, but the correlation was not significant. THBS4 methylation was detectable in normal mucosal biopsies where it correlated with increasing patient age and negatively with the occurrence of adenomas elsewhere in the colon. Conclusions THBS4 shows increased methylation in colorectal cancer, but this is not strongly associated with altered gene expression, either because methylation has not always reached a critical level or because other factors influence THBS4 expression. THBS4 may act as a tumour suppressor gene, demonstrated by its suppression of tumour colony formation in vitro. THBS4 methylation is detectable in normal colonic mucosa and its level may be a biomarker for the occurrence of adenomas and carcinoma.

Abstract Background SnoN is an important regulator of the transforming growth factor beta (TGFβ) signalling pathway and has been shown to exhibit both tumour promotion and suppression activity. Methods To further explore the role of this complex molecule in colorectal tumorigenesis, we examined 52 paired normal and tumour colorectal specimens stratified by level of microsatellite instability; 18 with high-level microsatellite instability (MSI-H) and 34 microsatellite stable (MSS). SnoN transcript expression was quantitated by real-time PCR and analysed with respect to clinical indicators of prognosis. Results Within the MSI-H subgroup, SnoN was commonly either up-regulated (6/18, 33%) or down-regulated (7/18, 39%). A significantly different distribution of SnoN expression was observed in MSS cancers compared with MSI-H (P ≤ 0.001). Whilst 17/34 (50%) of MSS tumours demonstrated up-regulation, none showed down-regulated expression. Within the MSI-H subgroup, up-regulation was significantly correlated with lack of repeat tract mutation in the TGFβRII gene (P ≤ 0.025), suggesting that SnoN is more frequently up-regulated in the presence of functional TGFβ signalling. Conclusion Together these data support the notion that SnoN has both oncogenic and tumour suppressive properties depending on other genetic changes within the tumour, and that the MSI-H pathway of colorectal tumorigenesis presents an excellent model for the study of these opposing functions.

Adoption of organoid/tumoroid propagation of normal and malignant intestinal epithelia has provided unparalleled opportunities to compare cell growth factor and signaling dependencies. These 3D structures recapitulate tumours in terms of gene expression regarding the tumor cells but also allow deeper insights into the contribution of the tumour microenvironment (TME). Elements of the TME can be manipulated or added back in the form of infiltrating cytotoxic lymphocytes and/or cancer associated fibroblasts. The effectiveness of chemo-, radio- and immunotherapies can be explored within weeks of deriving these patient-derived tumour avatars informing treatment of these exact patients in a timely manner. Entrenched paths to colorectal cancer (CRC) from the earliest steps of conventional adenoma or serrated lesion formation, and the recognition of further sub-categorisations embodied by consensus-molecular-subtypes (CMS), provide genetic maps allowing a molecular form of pathologic taxonomy. Recent advances in organoid propagation and scRNAseq are reshaping our understanding of CMS and CRC.

Objective: Sessile serrated lesions (SSLs) are common across the age spectrum, but the BRAF mutant cancers arising occur predominantly in the elderly. Aberrant DNA methylation is uncommon in SSL from young patients. Here, we interrogate the role of ageing and DNA methylation in SSL initiation and progression., Design: We used an inducible model of Braf mutation to direct recombination of the oncogenic Braf V637E allele to the murine intestine. BRAF mutation was activated after periods of ageing, and tissue was assessed for histological, DNA methylation and gene expression changes thereafter. We also investigated DNA methylation alterations in human SSLs., Results: Inducing Braf mutation in aged mice was associated with a 10-fold relative risk of serrated lesions compared with young mice. There were extensive differences in age-associated DNA methylation between animals induced at 9 months versus wean, with relatively little differential Braf- specific methylation. DNA methylation at WNT pathway genes scales with age and Braf mutation accelerated age-associated DNA methylation. In human SSLs, increased epigenetic age was associated with high-risk serrated colorectal neoplasia., Conclusions: SSLs arising in the aged intestine are at a significantly higher risk of spontaneous neoplastic progression. These findings provide support for a new conceptual model for serrated colorectal carcinogenesis, whereby risk of Braf- induced neoplastic transformation is dependent on age and may be related to age-associated molecular alterations that accumulate in the ageing intestine, including DNA methylation. This may have implications for surveillance and chemopreventive strategies targeting the epigenome., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Aim: Colorectal cancer (CRC) is the third most common cancer in Australia, and survival after diagnosis of metastatic disease is improving. Our aim was to assess trends in epidemiology, treatment, molecular testing and survival in patients with metastatic CRC (mCRC)., Methods: Clinical data from February 2013 to December 2018 was recorded in a prospective, observational, multicenter cohort study conducted in Queensland, Australia, examining clinical and molecular biomarkers in cases of mCRC., Results: A total of 159 patients who had metastasis diagnosed after February 2013 were included in survival analysis. Median age at diagnosis was 63.9 years, but 29% had early-onset disease (diagnosis aged <50 years). Median overall survival was 2.5 years (95% confidence interval [CI], 2.2-3.0) for the 159 patients included in survival analysis. Independent factors correlated with poor prognosis included right-sided primary tumor, neutrophil-lymphocyte ratio >5, increased alkaline phosphatase level (ALP) and an increasing number of sites of metastatic disease. In contrast, metastasectomy was associated with improved overall survival (adjusted HR = 0.29' 95% CI, 0.16-0.54), with similar survival between patients who had liver and non-liver metastasectomy sites. Half (10/20) of the BRAF mutant CRC were also microsatellite unstable. The proportion of detected mutations amongst tested samples increased over time for Kirsten Rat Sarcoma (KRAS; OR [per year] = 1.19; 95% CI, 1.01-1.39). Concurrently, the methods of molecular genetics testing employed in routine clinical practice changed towards the adoption of next-generation sequencing., Conclusions: Metastasectomy in mCRC may be beneficial regardless of the anatomical site of metastasis. The adoption of next-generation sequencing techniques for molecular genetics testing coincided with a slightly increased rate of detection of KRAS and BRAF mutations, potentially reflecting greater test sensitivity. Further translational research is required in mCRC to define novel targets for treatment., (© 2020 John Wiley & Sons Australia, Ltd.)

Aims: Sessile serrated lesions (SSL) with dysplasia are uncommon polyps with a high risk of rapid malignant transformation. Most of these lesions have a BRAF mutation and 75% show loss of MLH1 expression in their dysplastic component. Different morphological patterns of dysplasia occurring in these polyps have recently been described. We hypothesised that a subset of SSLs with dysplasia mimicking the dysplasia seen in conventional adenoma (adenomatous dysplasia) may represent a collision lesion between an ordinary SSL and a conventional adenoma., Methods and Results: We selected 80 SSLs with dysplasia, including 19 with adenomatous dysplasia, 18 with serrated dysplasia and 43 with dysplasia not otherwise specified (NOS). BRAF mutation analysis was performed using molecular testing (allelic discrimination) and the mutation-specific BRAF-V600E immunohistochemistry (clone VE1). The overall BRAF-V600E mutation rate was 84% in all lesions, 68% in SSLs with adenomatous dysplasia, 89% in SSLs with serrated dysplasia and 88% in SSLs with dysplasia NOS. From the 63 SSLs with dysplasia that were positive for the BRAF-V600E mutation, a negative BRAF-V600E immunostaining was observed in the dysplastic component of 83% of SSLs with adenomatous dysplasia, 0% of SSLs with serrated dysplasia and 3% of SSLs with dysplasia NOS (P < 0.001)., Conclusions: These findings suggest that SSLs with adenomatous dysplasia may not represent advanced SSLs, but instead may be a collision between a non-dysplastic SSL and a conventional adenoma., (© 2019 John Wiley & Sons Ltd.)

Epidermal growth factor receptor (EGFR) supports colorectal cancer progression via oncogenic signaling. Anti-EGFR therapy is being investigated as a clinical option for colorectal cancer, and an observed interaction between EGFR and Prion protein has been detected in neuronal cells. We hypothesized that PrP C expression levels may regulate EGFR signaling and that detailed understanding of this signaling pathway may enable identification of resistance mechanisms and new actionable targets in colorectal cancer. We performed molecular pathway analysis following knockdown of PrP C or inhibition of EGFR signaling via gefitinib to identify changes in expression of key signaling proteins that determine cellular sensitivity or resistance to cisplatin. Expression of these proteins was examined in matched primary and metastatic patient samples and was correlated for resistance to therapy and progression of disease. Utilizing three colorectal cancer cell lines, we observed a correlation between high expression of PrP C and resistance to cisplatin. Investigation of molecular signaling in a resistant cell line revealed that PrP C contributed to signaling via colocalization with EGFR, which could be overcome by targeting p38 mitogen-activated protein kinases (p38 MAPK). We revealed that the level of Krüppel-like factor 5 (KLF5), a target downstream of p38 MAPK, was predictive for cell line and patient response to platinum agents. Further, high KLF5 expression was observed in BRAF-mutant colorectal cancer. Our study indicates that the EGFR to KLF5 pathway is predictive of patient progression on platinum-based therapy., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Goals: To provide preliminary evidence that sessile serrated adenomas (SSA) are low-risk polyps in young patients., Background: SSAs are the dominant polyp of the serrated neoplasia pathway and as such are the precursor of up to 20% of colorectal carcinomas (CRC). Up to 90% of these cancers are expected to harbor a BRAF mutation. SSAs are being diagnosed with increasing frequency in young patients, placing a significant burden on colonoscopic services. Evidence to direct the surveillance intervals for these young patients is not available., Study: We utilized 2 patient cohorts comprising (1) a consecutive series of patients who underwent outpatient colonoscopy through a tertiary hospital and (2) a consecutive series of resection specimens for CRC processed through a gastrointestinal pathology service. The prevalence of SSAs by age was determined in the patients undergoing colonoscopy and compared with the ages of patients with BRAF mutated CRC in the pathology series., Results: The prevalence of SSAs was similar irrespective of age. By comparison, BRAF mutated CRCs were very rare (3.8% of cases) in patients younger than 50 years of age and uncommon (9.3% of cases) in patients younger than 60 years of age, but increased to 39.8% in patients older than 80 years of age., Conclusions: These results suggest that SSAs develop at a young age, but have a prolonged dwell time and are unlikely to develop into cancer in patients younger than 60 years of age. These findings highlight the need for further targeted research to determine the most appropriate surveillance intervals for young patients with sporadic SSAs.

Background & Aims: Colorectal cancer is an epigenetically heterogeneous disease, however, the extent and spectrum of the CpG island methylator phenotype (CIMP) is not clear., Methods: Genome-scale methylation and transcript expression were measured by DNA Methylation and RNA expression microarray in 216 unselected colorectal cancers, and findings were validated using The Cancer Genome Atlas 450K and RNA sequencing data. Mutations in epigenetic regulators were assessed using CIMP-subtyped Cancer Genome Atlas exomes., Results: CIMP-high cancers dichotomized into CIMP-H1 and CIMP-H2 based on methylation profile. KRAS mutation was associated significantly with CIMP-H2 cancers, but not CIMP-H1 cancers. Congruent with increasing methylation, there was a stepwise increase in patient age from 62 years in the CIMP-negative subgroup to 75 years in the CIMP-H1 subgroup (P < .0001). CIMP-H1 predominantly comprised consensus molecular subtype 1 cancers (70%) whereas consensus molecular subtype 3 was over-represented in the CIMP-H2 subgroup (55%). Polycomb Repressive Complex-2 (PRC2)-marked loci were subjected to significant gene body methylation in CIMP cancers (P < 1.6 × 10 -78 ). We identified oncogenes susceptible to gene body methylation and Wnt pathway antagonists resistant to gene body methylation. CIMP cluster-specific mutations were observed in chromatin remodeling genes, such as in the SWItch/Sucrose Non-Fermentable and Chromodomain Helicase DNA-Binding gene families., Conclusions: There are 5 clinically and molecularly distinct subgroups of colorectal cancer. We show a striking association between CIMP and age, sex, and tumor location, and identify a role for gene body methylation in the progression of serrated neoplasia. These data support our recent findings that CIMP is uncommon in young patients and that BRAF mutant polyps in young patients may have limited potential for malignant progression., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Traditional serrated adenoma (TSA) is the least common subtype of serrated colorectal polyp. Large protuberant lesions are easily recognised; however, the origins of TSAs are not known, and early forms have not been described. Some large TSAs present with a flat 'shoulder' component surrounding the central protuberant component. We hypothesised that small polyps with the same histology as these shoulder regions may represent early TSAs. Thus the primary aim of the study is to describe the histology of these presumptive early TSAs., Methods and Results: We collected 70 small (<10 mm) polyps that may represent early TSAs on the basis of typical TSA cytology covering the luminal surface. We also identified 12 large TSAs with a shoulder component resembling these small polyps. The study polyp patients had a mean age of 58 years, and 54% were female; the polyps had a mean diameter of 4.1 mm and were predominantly distal (71%). Morphologically, slit-like serrations were present in 81%, ectopic crypt formations were present in 67%, and a villous component was present in 47%. These histological features were similar to those of the 12 shoulder lesions. Immunohistochemical stains showed an absence of β-catenin nuclear expression in 96% of the small polyps, retained expression of MLH1 in 100%, and Ki67 positivity restricted to the crypt bases and ectopic crypt formations. BRAF and KRAS mutations were identified in 47% and 31% of the polyps, respectively. BRAF-mutated polyps were more likely than KRAS-mutated polyps to arise in a precursor polyp (82% versus 18%, P < 0.001), and were more likely to have slit-like serrations (100% versus 73%, P = 0.003)., Conclusions: These morphological, immunohistochemical and molecular findings are similar to what has been reported in large TSAs, and support the hypothesis that these polyps represent early forms of TSA., (© 2018 John Wiley & Sons Ltd.)

Conventional adenomas are initiated by APC gene mutation that activates the WNT signal. Serrated neoplasia is commonly initiated by BRAF or KRAS mutation. WNT pathway activation may also occur, however, to what extent this is owing to APC mutation is unknown. We examined aberrant nuclear β-catenin immunolocalization as a surrogate for WNT pathway activation and analyzed the entire APC gene coding sequence in serrated and conventional pathway polyps and cancers. WNT pathway activation was a common event in conventional pathway lesions with aberrant nuclear immunolocalization of β-catenin and truncating APC mutations in 90% and 89% of conventional adenomas and 82% and 70% of BRAF wild-type cancers, respectively. WNT pathway activation was seen to a lesser extent in serrated pathway lesions. It occurred at the transition to dysplasia in serrated polyps with a significant increase in nuclear β-catenin labeling from sessile serrated adenomas (10%) to sessile serrated adenomas with dysplasia (55%) and traditional serrated adenomas (9%) to traditional serrated adenomas with dysplasia (39%) (P=0.0001). However, unlike the conventional pathway, truncating APC mutations were rare in the serrated pathway lesions especially sessile serrated adenomas even when dysplastic (15%) and in the BRAF mutant cancers with microsatellite instability that arise from them (8%). In contrast, APC missense mutations that were rare in conventional pathway adenomas and cancers (3% in BRAF wild-type cancers) were more frequent in BRAF mutant cancers with microsatellite instability (32%). We conclude that increased WNT signaling is important in the transition to malignancy in the serrated pathway but that APC mutation is less common and the spectrum of mutations is different than in conventional colorectal carcinogenesis. Moderate impact APC mutations and non-APC-related causes of increased WNT signaling may have a more important role in serrated neoplasia than the truncating APC mutations common in conventional adenomas.