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Epigenetics; Tumour-suppressor proteins Epigenética; Proteínas supresoras de tumores Epigenètica; Proteïnes supresores de tumors Colorectal cancers (CRCs) often display histological features indicative of aberrant differentiation but the molecular underpinnings of this trait and whether it directly drives disease progression is unclear. Here, we identify co-ordinate epigenetic inactivation of two epithelial-specific transcription factors, EHF and CDX1, as a mechanism driving differentiation loss in CRCs. Re-expression of EHF and CDX1 in poorly-differentiated CRC cells induced extensive chromatin remodelling, transcriptional re-programming, and differentiation along the enterocytic lineage, leading to reduced growth and metastasis. Strikingly, EHF and CDX1 were also able to reprogramme non-colonic epithelial cells to express colonic differentiation markers. By contrast, inactivation of EHF and CDX1 in well-differentiated CRC cells triggered tumour de-differentiation. Mechanistically, we demonstrate that EHF physically interacts with CDX1 via its PNT domain, and that these transcription factors co-operatively drive transcription of the colonic differentiation marker, VIL1. Compound genetic deletion of Ehf and Cdx1 in the mouse colon disrupted normal colonic differentiation and significantly enhanced colorectal tumour progression. These findings thus reveal a novel mechanism driving epithelial de-differentiation and tumour progression in CRC. This project was supported by NHMRC project grant (1107831), a Cancer Council Victoria Grant (1164674) and the Operational Infrastructure Support Programme, Victorian Government, Australia. JMM was supported by a NHMRC Senior Research Fellowship (1046092). IYL was supported by F J Fletcher Research Scholarship and Randal and Louisa Alcock Scholarship from the University of Melbourne. LJJ was supported by La Trobe University Australian Postgraduate Awards. IN was supported by La Trobe University Postgraduate Research Scholarship. JWTT was supported by the University of Melbourne Australian Postgraduate Awards. OMS is a National Health and Medical Research Council (NHMRC) Senior Research Fellow (APP1136119). Open Access funding enabled and organized by CAUL and its Member Institutions.

BACKGROUND: Aspirin reduces the incidence of conventional adenomas driven by APC mutation and thus colorectal cancer. The effect of aspirin on the ~20% of colorectal cancers arising via BRAF mutation is yet to be established. METHODS: Braf(V637E/+);Villin-Cre(ERT2/+) mice were allocated to a control (n = 86) or aspirin-supplemented (n = 83) diet. After 14 months the incidence of murine serrated lesions, carcinoma and distant metastases were measured by histological examination. RNA was extracted from carcinomas from each cohort and subjected to sequencing to identify differentially expressed genes and molecular pathways. RESULTS: Aspirin did not reduce the incidence of murine serrated lesions or carcinoma when compared to control, however, did significantly reduce lesion size (P = 0.0042). Among the mice with carcinoma there was a significant reduction in the incidence of distant metastasis with aspirin treatment (RR 0.69, 95% CI 0.48–0.90, P = 0.0134). Key pathways underlying metastasis of carcinoma cells include NOTCH, FGFR and PI3K signalling, were significantly downregulated in carcinomas sampled from mice on an aspirin-supplemented diet. CONCLUSIONS: Aspirin reduces the incidence of metastatic Braf mutant carcinoma, although this is not due to a reduction in primary disease. The reduction in metastasis could be attributed to a delay or prevention of molecular changes within the primary site driving metastatic growth.

Colorectal cancer is one of the world’s most prevalent and lethal cancers. Mutations of the KRAS gene occur in ~40% of metastatic colorectal cancers. While this cohort has historically been difficult to manage, the last few years have shown exponential growth in the development of selective inhibitors targeting KRAS mutations. Their foremost mechanism of action utilizes the Switch II binding pocket and Cys12 residue of GDP-bound KRAS proteins in G12C mutants, confining them to their inactive state. Sotorasib and Adagrasib, both FDA-approved for the treatment of non-small cell lung cancer (NSCLC), have been pivotal in paving the way for KRAS G12C inhibitors in the clinical setting. Other KRAS inhibitors in development include a multi-targeting KRAS-mutant drug and a G12D mutant drug. Treatment resistance remains an issue with combination treatment regimens including indirect pathway inhibition and immunotherapy providing possible ways to combat this. While KRAS-mutant selective therapy has come a long way, more work is required to make this an effective and viable option for patients with colorectal cancer.

Purpose: While evidence indicates that Fusobacterium nucleatum (F. nucleatum) may promote colorectal carcinogenesis through its suppressive effect on T-cell–mediated antitumor immunity, the specific T-cell subsets involved remain uncertain. Experimental Design: We measured F. nucleatum DNA within tumor tissue by quantitative PCR on 933 cases (including 128 F. nucleatum–positive cases) among 4,465 incident colorectal carcinoma cases in two prospective cohorts. Multiplex immunofluorescence combined with digital image analysis and machine learning algorithms for CD3, CD4, CD8, CD45RO (PTPRC isoform), and FOXP3 measured various T-cell subsets. We leveraged data on Bifidobacterium, microsatellite instability (MSI), tumor whole-exome sequencing, and M1/M2-type tumor-associated macrophages [TAM; by CD68, CD86, IRF5, MAF, and MRC1 (CD206) multimarker assay]. Using the 4,465 cancer cases and inverse probability weighting method to control for selection bias due to tissue availability, multivariable-adjusted logistic regression analysis assessed the association between F. nucleatum and T-cell subsets. Results: The amount of F. nucleatum was inversely associated with tumor stromal CD3+ lymphocytes [multivariable OR, 0.47; 95% confidence interval (CI), 0.28–0.79, for F. nucleatum–high vs. -negative category; Ptrend = 0.0004] and specifically stromal CD3+CD4+CD45RO+ cells (corresponding multivariable OR, 0.52; 95% CI, 0.32–0.85; Ptrend = 0.003). These relationships did not substantially differ by MSI status, neoantigen load, or exome-wide tumor mutational burden. F. nucleatum was not significantly associated with tumor intraepithelial T cells or with M1 or M2 TAMs. Conclusions: The amount of tissue F. nucleatum is associated with lower density of stromal memory helper T cells. Our findings provide evidence for the interactive pathogenic roles of microbiota and specific immune cells.

Colorectal cancer is a leading cause of cancer-related death worldwide and approximately 20% of cases can be attributed to a mutation in the BRAF oncogene. Curcumin is a promising chemopreventive agent with various anti-cancer benefits. Although curcumin has been reported to have poor bioavailability, this limitation has been overcome by the formulation of nano-carriers. In this preclinical study, we investigated the ability of an improved formulation of curcumin to reduce the incidence of Braf mutant carcinoma. To investigate curcumin as a chemopreventive for Braf mutant colorectal cancer in a preclinical study utilizing a murine model of serrated neoplasia. An intestine-specific Braf mutant murine model (BrafV637E/+/Villin-CreERT2/+) was administered curcumin micelles (240 mg/kg, n = 69) in normal drinking water. Mice in the control group consumed normal drinking water (n = 83). Mice were euthanized at 14 months and the incidence of murine serrated lesions and carcinoma in each cohort were determined by histologic examination. At completion of the study (14 months), it was found that curcumin did not reduce the incidence or multiplicity of murine serrated lesions but did significantly reduce the number of invasive carcinomas (RR 0.83, 95% CI 0.69–0.9985, P = 0.0360) compared to control. We have performed the first long-term study assessing curcumin’s effect on the development of serrated neoplasia. We found that curcumin significantly reduces the risk of developing Braf mutant colorectal cancer. Our data supports further investigation of curcumin as a chemopreventive to reduce the risk of colorectal cancer arising via the serrated pathway.

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).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.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.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.

At diagnosis, 22% of colorectal cancer (CRC) patients have metastases, and 50% later develop metastasis. Peptide receptor radionuclide therapy (PRRT), such as 177Lu-PSMA-617, is used to treat metastatic prostate cancer. 177Lu-PSMA-617 targets prostate-specific membrane antigen (PSMA), a cell-surface protein enriched in prostate cancer and the neovasculature of other solid tumors, including CRC. We performed 68Ga-PSMA-11 PET/CT imaging of 10 patients with metastatic CRC to assess metastasis avidity. Eight patients had lesions lacking avidity, and 2 had solitary metastases exhibiting very low avidity. Despite expression of PSMA in CRC neovasculature, none of the patients exhibited tumor avidity sufficient to be considered for 177Lu-PSMA-617 PRRT.

The serrated neoplasia pathway gives rise to a distinct subgroup of colorectal cancers distinguished by the presence of mutant BRAFV600E and the CpG Island Methylator Phenotype (CIMP). BRAF mutant CRC are commonly associated with microsatellite instability, which have an excellent clinical outcome. However, a proportion of BRAF mutant CRC retain microsatellite stability and have a dismal prognosis. The molecular drivers responsible for the development of this cancer subgroup are unknown. To address this, we established a murine model of BRAFV600E mutant microsatellite stable CRC and comprehensively investigated the exome and transcriptome to identify molecular alterations in signaling pathways that drive malignancy. Exome sequencing of murine serrated lesions (mSL) and carcinomas identified frequent hot spot mutations within the gene encoding β-catenin (Ctnnb1). Immunohistochemical staining of β-catenin indicated that these mutations led to an increase in the presence of aberrant nuclear β-catenin that resulted in gene expression changes in targets of β-catenin transcription. Gene expression profiling identified a significant enrichment for transforming growth factor-β (TGF-β) signaling that was present in mSL and carcinomas. Early activation of TGF-β suggests that this pathway may be an early cue directing mSL to microsatellite stable carcinoma. These findings in the mouse model support the importance of alterations in WNT and TGF-β signaling during the transition of human sessile serrated lesions to malignancy. Keywords: BRAF, Colorectal cancer, WNT, TGF-β, Microsatellite

Colorectal cancer is an important cause of morbidity and mortality worldwide. The current treatment landscape includes chemotherapy, targeted therapy, immunotherapy, radiotherapy, and surgery. A key challenge to improving patient outcomes is the significant inter-patient heterogeneity in treatment response. Tumour organoids derived from the patients’ tumours via surgically resected or endoscopically biopsied tissue, have emerged as promising models for personalised medicine. This review synthesises the findings, to date, of studies which have explored the efficacy of ex vivo organoid sensitivity testing for predicting treatment response. Most studies have focused on predicting the response to standard-of-care radiotherapy and chemotherapy options. There is strong evidence to support organoid sensitivity testing of ionising radiation, 5-fluorouracil, and irinotecan, and to a lesser extent, oxaliplatin and TAS-102. Fewer studies have used organoids to identify patients who are likely to benefit from novel treatment options that otherwise remain in clinical trials. This review also summarises recent advancements in organoid culture to include non-epithelial components of the tumour microenvironment, to allow testing of immunotherapy and certain targeted therapy options. Overall, further prospective trials will support the implementation of organoid-based personalised medicine for colorectal cancer patients in the future.

CD96 is a novel target for cancer immunotherapy shown to regulate NK cell effector function and metastasis. Here, we demonstrated that blocking CD96 suppressed primary tumor growth in a number of experimental mouse tumor models in a CD8+ T cell–dependent manner. DNAM-1/CD226, Batf3, IL12p35, and IFNγ were also critical, and CD96-deficient CD8+ T cells promoted greater tumor control than CD96-sufficient CD8+ T cells. The antitumor activity of anti-CD96 therapy was independent of Fc-mediated effector function and was more effective in dual combination with blockade of a number of immune checkpoints, including PD-1, PD-L1, TIGIT, and CTLA-4. We consistently observed coexpression of PD-1 with CD96 on CD8+ T lymphocytes in tumor-infiltrating leukocytes both in mouse and human cancers using mRNA analysis, flow cytometry, and multiplex IHF. The combination of anti-CD96 with anti–PD-1 increased the percentage of IFNγ-expressing CD8+ T lymphocytes. Addition of anti-CD96 to anti–PD-1 and anti-TIGIT resulted in superior antitumor responses, regardless of the ability of the anti-TIGIT isotype to engage FcR. The optimal triple combination was also dependent upon CD8+ T cells and IFNγ. Overall, these data demonstrate that CD96 is an immune checkpoint on CD8+ T cells and that blocking CD96 in combination with other immune-checkpoint inhibitors is a strategy to enhance T-cell activity and suppress tumor growth.

To provide preliminary evidence that sessile serrated adenomas (SSA) are low-risk polyps in young patients. 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. 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. 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. 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.

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 PrPC 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 PrPC 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 PrPC and resistance to cisplatin. Investigation of molecular signaling in a resistant cell line revealed that PrPC 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 Kruppel-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.

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. ispartof: CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY vol:8 issue:2 pages:269-290 ispartof: location:United States status: published

Background. The typical methylation patterns associated with cancer are hypermethylation at gene promoters and global genome hypomethylation. Aberrant CpG island hypermethylation at promoter regions and global genome hypomethylation have not been associated with histological colorectal carcinomas (CRC) subsets. Using Illumina’s 450 k Infinium Human Methylation beadchip, the methylome of 82 CRCs were analyzed, comprising different histological subtypes: 40 serrated adenocarcinomas (SAC), 32 conventional carcinomas (CC) and 10 CRCs showing histological and molecular features of microsatellite instability (hmMSI‐H), and, additionally, 35 normal adjacent mucosae. Scores reflecting the overall methylation at 250 bp, 1 kb and 2 kb from the transcription starting site (TSS) were studied. Results. SAC has an intermediate methylation pattern between CC and hmMSI‐H for the three genome locations. In addition, the shift from promoter hypermethylation to genomic hypomethylation occurs at a small sequence between 250 bp and 1 Kb from the gene TSS, and an asymmetric distribution of methylation was observed between both sides of the CpG islands (N vs. S shores). Conclusion. These findings show that different histological subtypes of CRC have a particular global methylation pattern depending on sequence distance to TSS and highlight the so far underestimated importance of CpGs aberrantly hypomethylated in the clinical phenotype of CRCs., This work was supported by two grants from Instituto de Salud Carlos III, Ministerio de Sanidad, Spain and FEDER funds (refs: PI12-1232, PI18-0144) and another from the European Union’s Horizon 2020 research and innovation program (ref. 848098)

PURPOSE: While evidence indicates that Fusobacterium nucleatum may promote colorectal carcinogenesis through its suppressive effect on T-cell-mediated antitumor immunity, the specific T-cell subsets involved remain uncertain. EXPERIMENTAL DESIGN: We measured F. nucleatum DNA within tumor tissue by quantitative PCR on 933 cases (including 128 F. nucleatum-positive cases) among 4,465 incident colorectal carcinoma cases in two prospective cohorts. Multiplex immunofluorescence combined with digital image analysis and machine learning algorithms for CD3, CD4, CD8, CD45RO (PTPRC isoform), and FOXP3 measured various T-cell subsets. We leveraged data on Bifidobacterium, microsatellite instability (MSI), tumor whole exome sequencing, and M1/M2-type tumor-associated macrophages [by CD68, CD86, IRF5, MAF, and MRC1 (CD206) multimarker assay]. Using the 4,465 cancer cases and inverse probability weighting method to control for selection bias due to tissue availability, multivariable-adjusted logistic regression analysis assessed the association between F. nucleatum and T-cell subsets. RESULTS: The amount of F. nucleatum was inversely associated with tumor stromal CD3(+) lymphocytes (multivariable odds ratio, 0.47, 95% confidence interval, 0.28–0.79, for F. nucleatum-high vs. negative category; P(trend)=0.0004) and specifically stromal CD3(+)CD4(+)CD45RO(+) cells (corresponding multivariable odds ratio, 0.52, 95% confidence interval, 0.32–0.85; P(trend)=0.003). These relationships did not substantially differ by MSI status, neoantigen loads, or exome-wide tumor mutational burden. F. nucleatum was not significantly associated with tumor intraepithelial T cells or with M1 or M2 tumor-associated macrophages. CONCLUSIONS: The amount of tissue F. nucleatum is associated with lower densities of stromal memory helper T cells. Our findings provide evidence for the interactive pathogenic roles of microbiota and specific immune cells.

Background Sessile serrated lesions (SSL) are common in both young and old individuals, but the BRAF mutant cancers arising occur predominantly the elderly. DNA Methylation is uncommon in SSL from young patients. Here we interrogate the role of aging and DNA methylation in SSL initiation and progression. Methods 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 aging, and histological and DNA methylation analysis was performed thereafter. We 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. Methylation analysis revealed extensive differences in age-associated DNA methylation between animals induced at 9 months versus wean; with relatively little differential Braf-specific methylation, implicating age-associated DNA methylation rather than Braf-specific DNA methylation in the heightened risk. 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 support a new conceptual model for serrated neoplasia whereby the risk of progression is related to the milieu of epigenetic alterations in the intestinal epithelium at the time of BRAF mutation, rather than the length of time since polyp initiation. This has implications for surveillance and chemopreventive strategies targeting the epigenome.

Colorectal cancer is a leading cause of cancer-related death worldwide and approximately 20% of cases can be attributed to a mutation in the BRAF oncogene. Curcumin is a promising chemopreventive agent with various anti-cancer benefits. Although curcumin has been reported to have poor bioavailability, this limitation has been overcome by the formulation of nano-carriers. In this preclinical study, we investigated the ability of an improved formulation of curcumin to reduce the incidence of Braf mutant carcinoma.To investigate curcumin as a chemopreventive for Braf mutant colorectal cancer in a preclinical study utilizing a murine model of serrated neoplasia.An intestine-specific Braf mutant murine model (BrafAt completion of the study (14 months), it was found that curcumin did not reduce the incidence or multiplicity of murine serrated lesions but did significantly reduce the number of invasive carcinomas (RR 0.83, 95% CI 0.69-0.9985, P = 0.0360) compared to control.We have performed the first long-term study assessing curcumin's effect on the development of serrated neoplasia. We found that curcumin significantly reduces the risk of developing Braf mutant colorectal cancer. Our data supports further investigation of curcumin as a chemopreventive to reduce the risk of colorectal cancer arising via the serrated pathway.

Background: WNT activation is a hallmark of colorectal cancer. BRAF mutation is present in 15% of colorectal cancers, and the role of mutations in WNT signaling regulators in this context is unclear. Here, we evaluate the mutational landscape of WNT signaling regulators in BRAF mutant cancers. Methods: we performed exome-sequencing on 24 BRAF mutant colorectal cancers and analyzed these data in combination with 175 publicly available BRAF mutant colorectal cancer exomes. We assessed the somatic mutational landscape of WNT signaling regulators, and performed hotspot and driver mutation analyses to identify potential drivers of WNT signaling. The effects of Apc and Braf mutation were modelled, in vivo, using the Apcmin/+ and BrafV637/Villin-CreERT2/+ mouse, respectively. Results: RNF43 was the most frequently mutated WNT signaling regulator (41%). Mutations in the beta-catenin destruction complex occurred in 48% of cancers. Hotspot analyses identified potential cancer driver genes in the WNT signaling cascade, including MEN1, GNG12 and WNT16. Truncating APC mutation was identified in 20.8% of cancers. Truncating APC mutation was associated with early age at diagnosis (p &lt, 2 × 10−5), advanced stage (p &lt, 0.01), and poor survival (p = 0.026). Apcmin/+/BrafV637 animals had more numerous and larger SI and colonic lesions (p &lt, 0.0001 and p &lt, 0.05, respectively), and a markedly reduced survival (median survival: 3.2 months, p = 8.8 × 10−21), compared to animals with Apc or Braf mutation alone. Conclusions: the WNT signaling axis is frequently mutated in BRAF mutant colorectal cancers. WNT16 and MEN1 may be novel drivers of aberrant WNT signaling in colorectal cancer. Co-mutation of BRAF and APC generates an extremely aggressive neoplastic phenotype that is associated with poor patient outcome.

TheBRAFoncogene is an integral component of the MAP kinase pathway, and an activating V600E mutation occurs in 15% of sporadic colorectal cancer. This is an early event in serrated pathway tumourigenesis, and theBRAFV600E has been commonly associated with the CpG island methylator phenotype, microsatellite instability (MSI), and a consistent clinical presentation including a proximal location and predilection for elderly females. A proportion of theBRAFmutant lesions remain as microsatellite stable (MSS), and in contrast to the MSI cancers, they have an aggressive phenotype and correlate with poor patient outcomes. Recent studies have found that they have clinical and molecular features of both theBRAFmutant/MSI and the conventionalBRAFwild-type cancers and comprise a distinct colorectal cancer subgroup. This review highlights the importance of theBRAFmutation occurring in colorectal cancer stratified for molecular background and discusses its prognostic and clinical significance.

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.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%, P0.001), and were more likely to have slit-like serrations (100% versus 73%, P = 0.003).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.

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.

Colorectal cancer is a major cause of cancer death and approximately 20% arises within serrated polyps, which are under-recognized and poorly understood. Human serrated colorectal polyps frequently exhibit both oncogenic BRAF mutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether in vivo induction of mutant Braf is sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The BrafV637E mutation was induced in murine intestine on an FVB;C57BL/6J background and assessed for morphological and DNA methylation changes at multiple time points from 10 days to 14 months. Extensive intestinal hyperplasia developed by 10 days post-induction of the mutation. By 8 months, most mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months. From 5 months onwards, Braf mutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic Braf signaling is sufficient to induce widespread DNA methylation changes. This occurs over an extended period of time, mimicking the long latency followed by rapid progression of human serrated neoplasia. This study establishes for the first time that DNA methylation arises slowly in direct response to prolonged oncogenic Braf signaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.

Sessile serrated adenomas are the precursor polyp of approximately 20% of colorectal carcinomas. Sessile serrated adenomas with dysplasia are rarely encountered and represent an intermediate step to malignant progression, frequently associated with loss of MLH1 expression. Accurate diagnosis of these lesions is important to facilitate appropriate surveillance, particularly because progression from dysplasia to carcinoma can be rapid. The current World Health Organization classification describes two main patterns of dysplasia occurring in sessile serrated adenomas, namely, serrated and conventional. However, this may not adequately reflect the spectrum of changes seen by pathologists in routine practice. Furthermore, subtle patterns of dysplasia that are nevertheless associated with loss of MLH1 expression are not encompassed in this classification. We performed a morphological analysis of 266 sessile serrated adenomas with dysplasia with concurrent MLH1 immunohistochemistry with the aims of better defining the spectrum of dysplasia occurring in these lesions and correlating dysplasia patterns with MLH1 expression. We found that dysplasia can be divided morphologically into four major patterns, comprising minimal deviation (19%), serrated (12%), adenomatous (8%) and not otherwise specified (79%) groups. Minimal deviation dysplasia is defined by minor architectural and cytological changes that typically requires loss of MLH1 immunohistochemical expression to support the diagnosis. Serrated dysplasia and adenomatous dysplasia have distinctive histological features and are less frequently associated with loss of MLH1 expression (13 and 5%, respectively). Finally, dysplasia not otherwise specified encompasses most cases and shows a diverse range of morphological changes that do not fall into the other subgroups and are frequently associated with loss of MLH1 expression (83%). This morphological classification of sessile serrated adenomas with dysplasia may represent an improvement on the current description as it correlates with the underlying mismatch repair protein status of the polyps and better highlights the range of morphologies seen by pathologists.

Background: Sessile serrated adenomas (SSA) are the polyp precursor of 15–20% of colorectal carcinomas. There is debate about their prevalence and increasing discussion about the need for a serrated polyp detection rate as a quality indicator for colonoscopy. Aims: To assess the prevalence of SSA at an outpatient gastroenterology service. Methods: This is a retrospective study of an unselected consecutive series of patients who had an outpatient colonoscopy between April 2013 and May 2014. The colonoscopy reports were reviewed to identify age, gender, indication for procedure, completion, withdrawal time, adequacy of bowel preparation, number, size and location of polyps. The pathology of all polyps was centrally reviewed by a gastrointestinal pathologist. Results: A total of 707 patients underwent colonoscopy within the study period. The mean age of the cohort was 58 years, and 50.6% were female. Polyp(s) were identified in 66.5% of patients. The SSA detection rate was 20.1%, and the adenoma detection rate was 48.0%. SSA detection was associated with longer withdrawal times. Conventional adenoma detection was associated with older age, male gender, longer withdrawal time and a positive faecal occult blood test result. Conclusion: SSA are highly prevalent in an unselected series of patients attending a gastroenterology outpatient department. Identifying and removing these polyps may help prevent interval colorectal carcinoma. This result may serve as a benchmark for a high-quality colonoscopy service.

Patients with inflammatory bowel disease (IBD) have an increased risk of colorectal carcinoma. The significance of serrated lesions resembling traditional serrated adenoma (TSA) in IBD patients is unclear. In this retrospective study, we analyzed 52 TSA-like lesions arising in 30 IBD patients and diagnosed in colectomy or endoscopic specimens. The 27 colectomy lesions presented predominantly as ill-defined areas with granular appearance, with a median size of 15 mm, located throughout the large bowel and associated with synchronous advanced colorectal lesions in 58%. Low-grade serrated dysplasia was present in 56%, high-grade serrated dysplasia in 37%, and TSA-type cytology in 7%. Increased Ki-67 immunostaining and abnormal p53 expression were identified in 96% and 48%, respectively; 74% had a KRAS mutation, and 4% had a BRAF mutation. Endoscopically resectable TSA-like lesions were all discrete polypoid lesions, smaller in size (median 9 mm), predominantly in the distal large bowel, with an adjacent precursor polyp in 24%, and associated with synchronous and metachronous advanced colorectal lesions in 6%. Most (92%) show TSA-type cytology. p53 overexpression was present in 4%, KRAS mutation in 41%, and BRAF mutation in 32%. None of the 52 TSA-like lesions demonstrated loss of MLH1 or SATB2 expression by immunohistochemistry. On follow-up, 4 patients were diagnosed with colorectal carcinoma or high-grade adenomatous IBD-associated dysplasia. None of the patients with lesions showing TSA-type cytology only developed an advanced lesion. Our findings suggest that some TSA-like lesions, essentially from colectomy, may represent a form of IBD-associated dysplasia associated with an increased risk of advanced neoplasia.

Background Sessile serrated adenomas (SSAs) are common polyps which give rise to 20–30% of colorectal cancer (CRC). SSAs display clinicopathologic features which present challenges in surveillance, including overrepresentation in young patients, proclivity for the proximal colon and rarity of histologic dysplasia (referred to then as SSAs with dysplasia, SSADs). Once dysplasia develops, there is rapid progression to CRC, even at a small size. There is therefore a clinical need to separate the “advanced” SSAs at high risk of progression to SSAD and cancer from ordinary SSAs. Since SSAs are known to accumulate methylation over time prior to the development of dysplasia, SSAD backgrounds (the remnant SSA present within an SSAD) likely harbour additional methylation events compared with ordinary SSAs. We therefore performed MethyLight and comprehensive methylation array (Illumina MethylationEPIC) on 40 SSAD backgrounds and 40 matched ordinary SSAs, and compared the methylation results with CRC methylation, CRC expression and immunohistochemical data. Results SSAD backgrounds demonstrated significant hypermethylation of CpG islands compared with ordinary SSAs, and the proportion of hypermethylated probes decreased progressively in the shore, shelf and open sea regions. Hypomethylation occurred in concert with hypermethylation, which showed a reverse pattern, increasing progressively away from the island regions. These methylation changes were also identified in BRAF-mutant hypermethylated CRCs. When compared with CRC expression data, SV2B, MLH1/EPM2AIP1, C16orf62, RCOR3, BAIAP3, OGDHL, HDHD3 and ATP1B2 demonstrated both promoter hypermethylation and decreased expression. Although SSAD backgrounds were histologically indistinguishable from ordinary SSAs, MLH1 methylation was detectable via MethyLight in 62.9% of SSAD backgrounds, and focal immunohistochemical MLH1 loss was seen in 52.5% of SSAD backgrounds. Conclusions Significant hyper- and hypomethylation events occur during SSA progression well before the development of histologically identifiable changes. Methylation is a heterogeneous process within individual SSAs, as typified by MLH1, where both MLH1 methylation and focal immunohistochemical MLH1 loss can be seen in the absence of dysplasia. This heterogeneity is likely a generalised phenomenon and should be taken into account in future methylation-based studies and the development of clinical methylation panels. Electronic supplementary material The online version of this article (10.1186/s13148-019-0691-4) contains supplementary material, which is available to authorized users.

Isothermal methods, such as helicase-dependent amplification (HDA), have an advantage over polymerase chain reaction for DNA amplification owing to their ease of operation. Here, we developed a new HDA method that is nanoparticle-assisted, termed nanoHDA. This method uses gold nanoparticles (AuNPs) to improve the sensitivity and specificity of the isothermal method. In HDA, the denaturation of DNA templates is mediated by helicases, but this method is limited by the low denaturation efficiency of helicases. In this report, AuNPs with preferential affinity for single-stranded DNA (ssDNA) were utilized to improve the denaturation efficiency of helicases. The same affinity property of nanoparticles can also enhance specificity by suppressing primer-dimer formation. This nanoHDA method was employed to genotype the KRAS gene in genomic DNA samples from colorectal cancer patients, as achieved by the hybridization of nanoHDA amplicons using the NanoBioArray chip.

Serrated pathway colorectal cancers (CRCs) are characterised by a BRAF mutation and half display microsatellite instability (MSI). The Wnt pathway is commonly upregulated in conventional CRC through APC mutation. By contrast, serrated cancers do not mutate APC. We investigated mutation of the ubiquitin ligases RNF43 and ZNRF3 as alternate mechanism of altering the Wnt signal in serrated colorectal neoplasia. RNF43 was mutated in 47/54(87%) BRAF mutant/MSI and 8/33(24%) BRAF mutant/microsatellite stable cancers compared to only 3/79(4%) BRAF wildtype cancers (p

Background. Aspirin is a well-studied chemopreventive agent for the prevention of conventional colorectal adenomas and cancer. Post hoc analyses of cardiovascular prevention trials demonstrate a long-term reduction in colorectal cancer incidence and mortality with aspirin treatment. Randomized controlled trials have shown a long-term benefit for reducing colorectal cancer incidence in patients with Lynch Syndrome, a form of hereditary colorectal cancer. These findings have led to the recommendation of aspirin as a chemopreventive for colorectal cancer by the U.S. Preventive Services Task Force and Cancer Council Australia. However, future research questions have noted a lack of knowledge of the differential benefits of aspirin in preventing sessile serrated lesions versus conventional adenomas and cancer. In our study, we hypothesized that aspirin will be an effective long-term treatment for the prevention of colorectal cancer resulting from sessile serrated lesions. We aimed to investigate this in a preclinical study utilizing a murine model of Braf mutant serrated neoplasia with a primary end-point investigating cancer incidence. Methods. BrafV637E/+/Villin-CreERT2/+ mice were injected with tamoxifen (i.p. 75mg/kg) at 2 weeks of age to activate the Braf mutation within the intestine, then commenced on an aspirin-supplemented (n=78) or control standard (n=83) diet at wean. Aspirin was supplemented into the diet and provided ad libitum, at a human equivalent dose of 80-120mg per day, until sacrifice at 14 months. The gastrointestinal tract was excised and evaluated for the presence of carcinoma. In addition, the peritoneal and thoracic cavities were inspected for metastatic disease. A gastrointestinal pathologist (CL) assessed all histology sections. Results. At sacrifice, 14 months post induction of mutant Braf, all mice developed murine serrated lesions reminiscent of human sessile serrated lesions. We observed a carcinoma incidence of 31% (26/83) of which 35% (9/26) had metastasized to the liver or peritoneal cavity. When mice were treated with an aspirin-supplemented diet there was no significant reduction in carcinoma incidence (22/78, 28%, P = 0.7315). However, there was a significant reduction in the incidence of metastasis (1/22, 5%, P = 0.0134). Mice in the control group had lesions of a significantly greater size when compared to aspirin treated mice (P=0.0032). Conclusions. Aspirin resulted in a 7-fold decrease in metastases. This study demonstrates that the benefit of aspirin in preventing Braf mutant serrated neoplasia occurs at the carcinoma-metastasis phase of colorectal carcinogenesis. This could have potential clinical benefit in reducing recurrence and mortality. Citation Format: Alexandra Kane, Cheng Liu, Lochlan Fennell, Diane McKeone, Jennifer Borowsky, Barbara Leggett, Vicki Whitehall. Aspirin reduces the incidence of metastasis in a preclinical study of Braf mutant colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1103.

The BRAF oncogene is an integral component of the MAP kinase pathway, and an activating V600E mutation occurs in 15% of sporadic colorectal cancer. This is an early event in serrated pathway tumourigenesis, and the BRAF V600E has been commonly associated with the CpG island methylator phenotype, microsatellite instability (MSI), and a consistent clinical presentation including a proximal location and predilection for elderly females. A proportion of the BRAF mutant lesions remain as microsatellite stable (MSS), and in contrast to the MSI cancers, they have an aggressive phenotype and correlate with poor patient outcomes. Recent studies have found that they have clinical and molecular features of both the BRAF mutant/MSI and the conventional BRAF wild-type cancers and comprise a distinct colorectal cancer subgroup. This review highlights the importance of the BRAF mutation occurring in colorectal cancer stratified for molecular background and discusses its prognostic and clinical significance.

BACKGROUND:Colorectal cancer is an epigenetically heterogeneous disease, however the extent and spectrum of the CpG Island Methylator Phenotype (CIMP) is not clear.RESULTSAn unselected cohort of 216 colorectal cancers clustered into five clinically and molecularly distinct subgroups using Illumina 450K DNA methylation arrays. CIMP-High cancers were most frequent in the proximal colons of female patients. These dichotomised into CIMP-Hl and CIMP-H2 based on methylation profile which was supported by over representation of BRAF (74%, PKRAS (55%, P78). We identified oncogenes susceptible to gene body methylation and Wnt pathway antagonists resistant to gene body methylation. CIMP cluster specific mutations were observed for genes involved in chromatin remodelling, such as in the SWI/SNF and NuRD complexes, suggesting synthetic lethality.CONCLUSIONThere are five clinically and molecularly distinct subgroups of colorectal cancer based on genome wide epigenetic profiling. These analyses highlighted an unidentified role for gene body methylation in progression of serrated neoplasia. Subgroup-specific mutation of distinct epigenetic regulator genes revealed potentially druggable vulnerabilities for these cancers, which may provide novel precision medicine approaches.

Background Sessile serrated adenomas with BRAF mutation progress rapidly to cancer following the development of dysplasia (SSAD). Approximately 75% of SSADs methylate the mismatch repair gene MLH1, develop mismatch repair deficiency and the resultant cancers have a good prognosis. The remaining SSADs and BRAF mutant traditional serrated adenomas (TSA) develop into microsatellite stable cancers with a poor prognosis. The reason for this dichotomy is unknown. In this study, we assessed the genotypic frequency of the MLH1–93 polymorphism rs1800734 in SSADs and TSAs to determine if the uncommon variant A allele predisposes to MLH1 promoter hypermethylation. Methods We performed genotyping for the MLH1–93 polymorphism, quantitative methylation specific PCR, and MLH1 immunohistochemistry on 124 SSAD, 128 TSA, 203 BRAF mutant CRCs and 147 control subjects with normal colonoscopy. Results The minor A allele was significantly associated with a dose dependent increase in methylation at the MLH1 promoter in SSADs (p = 0.022). The AA genotype was only observed in SSADs with MLH1 loss. The A allele was also overrepresented in BRAF mutant cancers with MLH1 loss. Only one of the TSAs showed loss of MLH1 and the overall genotype distribution in TSAs did not differ from controls. Conclusions The MLH1–93 AA genotype is significantly associated with promoter hypermethylation and MLH1 loss in the context of SSADs. BRAF mutant microsatellite stable colorectal cancers with the AA genotype most likely arise in TSAs since the A allele does not predispose to methylation in this context.

ObjectiveSerrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein.DesignWe use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair.ResultsTargeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not BrafV600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting.ConclusionWe generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.

Here, we report a method that uses gold nanoparticles (AuNPs) to enhance the specificity of DNA hybridization without reducing its detection sensitivity. The conventional stringent wash method utilizes high-temperature/low-salt conditions to enhance the specificity of DNA hybridization-based assays. This method creates a destabilizing environment for base pairing that affects specific and nonspecific duplexes. Therefore, specificity is achieved at the expense of signal intensity or sensitivity. However, in the proposed wash method, AuNPs predominantly destabilize nonspecific duplexes, offering specificity without compromising sensitivity. This AuNP wash technique has proven to be effective in detecting single nucleotide polymorphisms (SNPs) in genomic samples even at room temperature in a CD-like NanoBioArray (CD-NBA) chip. This method is also robust with sequence variation and is compatible with multiplex DNA analyses on microarrays. Thus, the AuNP wash method could potentially be useful for improving the accuracy of DNA hybridization results.

Most colorectal polyps are classified readily, but a subset of tubulovillous adenomas (TVA) with prominent serrated architecture causes diagnostic confusion. We aimed to (i) identify histological features that separate serrated TVAs from both conventional TVAs and traditional serrated adenomas (TSA) and (ii) perform a clinicopathological and molecular analysis to determine if the serrated TVA has unique features.We collected 48 serrated TVAs, 50 conventional TVAs and 66 BRAF wild-type TSAs for analysis. For each polyp we performed a clinicopathological assessment, BRAF and KRAS mutation profiling, cytosine-phosphate-guanosine (CpG) island methylator phenotype status, MGMT methylation and immunohistochemical assessment of seven markers [MutL homologue 1 (MLH1), p16, p53, β-catenin, Ki67, CK7 and CK20]. We found that serrated TVAs can be diagnosed reliably, and have features distinct from both conventional TVAs and TSAs. Compared to conventional TVAs, serrated TVAs are larger, more often proximal, more histologically advanced, show more CpG island methylation and more frequent KRAS mutation. Compared to TSAs they are more often proximal, show less CpG island methylation, more frequent MGMT methylation and more frequent nuclear staining for β-catenin.The serrated TVA can be diagnosed reliably and has unique features. It represents a precursor of KRAS mutated, microsatellite stable colorectal carcinoma.

Abstract Molecular aberrations leading to colorectal cancer are diverse and heterogeneity exists both at a molecular level and in clinical behavior. Defective mismatch repair (dMMR) is a feature of 15% of colorectal cancers. These are hypermutated tumors, mostly right sided and histopathologically elicit a marked immune response. A proportion of these arise due to germline mutation of a mismatch repair gene giving rise to Lynch syndrome, while the majority arise sporadically due to somatic alteration of the MLH1 mismatch repair gene. Although dMMR is associated with an excellent patient prognosis, as tumor stage advances the frequency of dMMR declines and the association with improved prognosis dissipates. It is apparent that dMMR tumors do not represent a unique molecular subset. As the knowledge of the underlying biology evolves, the hope is for individualized therapy that goes well beyond the crude and oversimplified categorization of dMMR versus proficient MMR.

The traditional serrated adenoma is the least common colorectal serrated polyp. The clinicopathological features and molecular drivers of these polyps require further investigation. We have prospectively collected a cohort of 200 ordinary and advanced traditional serrated adenomas and performed BRAF and KRAS mutational profiling, CpG island methylator phenotype analysis, and immunohistochemistry for a panel of 7 antibodies (MLH1, β-catenin, p53, p16, Ki67, CK7, and CK20) on all cases. The mean age of the patients was 64 years and 50% were female. Of the polyps, 71% were distal. Advanced histology (overt dysplasia or carcinoma) was present in 19% of cases. BRAF mutation was present in 67% and KRAS mutation in 22%. BRAF mutant traditional serrated adenomas were more frequently proximal (39% versus 2%; P≤0.0001), were exclusively associated with a precursor polyp (57% versus 0%; P≤0.0001), and were more frequently CpG island methylator phenotype high (60% versus 16%; P≤0.0001) than KRAS mutant traditional serrated adenomas. Advanced traditional serrated adenomas retained MLH1 expression in 97%, showed strong p53 staining in 55%, and nuclear β-catenin staining in 40%. P16 staining was lost in the advanced areas of 55% of BRAF mutant traditional serrated adenomas compared with 10% of the advanced areas of KRAS mutant or BRAF/KRAS wild-type traditional serrated adenomas. BRAF and KRAS mutant traditional serrated adenomas are morphologically related but biologically disparate polyps with distinctive clinicopathological and molecular features. The overwhelming majority of traditional serrated adenomas retain mismatch repair enzyme function indicating a microsatellite-stable phenotype. Malignant progression occurs via TP53 mutation and Wnt pathway activation regardless of mutation status. However, CDKN2A (encoding the p16 protein) is silenced nearly exclusively in the advanced areas of the BRAF mutant traditional serrated adenomas. Thus, the BRAF mutant traditional serrated adenoma represents an important precursor of the aggressive BRAF mutant, microsatellite-stable subtype of colorectal carcinoma.

The WNT signaling pathway is commonly altered during colorectal cancer development. The E3 ubiquitin ligase, RNF43, negatively regulates the WNT signal through increased ubiquitination and subsequent degradation of the Frizzled receptor. RNF43 has recently been reported to harbor frequent truncating frameshift mutations in sporadic microsatellite unstable (MSI) colorectal cancers. This study assesses the relative frequency of RNF43 mutations in hereditary colorectal cancers arising in the setting of Lynch syndrome. The entire coding region of RNF43 was Sanger sequenced in 24 colorectal cancers from 23 patients who either (i) carried a germline mutation in one of the DNA mismatch repair genes (MLH1, MSH6, MSH2, PMS2), or (ii) showed immunohistochemical loss of expression of one or more of the DNA mismatch repair proteins, was BRAF wild type at V600E, were under 60 years of age at diagnosis, and demonstrated no promoter region methylation for MLH1 in tumor DNA. A validation cohort of 44 colorectal cancers from mismatch repair germline mutation carriers from the Australasian Colorectal Cancer Family Registry (ACCFR) were sequenced for the most common truncating mutation hotspots (X117 and X659). RNF43 mutations were found in 9 of 24 (37.5%) Lynch syndrome colorectal cancers. The majority of mutations were frameshift deletions in the G659 G7 repeat tract (29%); 2 cancers (2/24, 8%) from the one patient harbored frameshift mutations at codon R117 (C6 repeat tract) within exon 3. In the ACCFR validation cohort, RNF43 hotspot mutations were identified in 19/44 (43.2%) of samples, which was not significantly different to the initial series. The proportion of mutant RNF43 in Lynch syndrome related colorectal cancers is significantly lower than the previously reported mutation rate found in sporadic MSI colorectal cancers. These findings identify further genetic differences between sporadic and hereditary colorectal cancers. This may be because Lynch Syndrome cancers commonly arise in colorectal adenomas already bearing the APC mutation, whereas sporadic microsatellite unstable colorectal cancers arise from serrated polyps typically lacking APC mutation, decreasing the selection pressure on other WNT signaling related loci in Lynch syndrome.

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.

Formalin fixation and embedding of clinical tissue samples in paraffin is a common method for archiving biological material. These samples are often well annotated and provide an invaluable resource for research. However, this process of fixation and storage of tissue leads to DNA damage and fragmentation. The use of DNA from formalin fixed, paraffin-embedded (FFPE) tissue to interrogate methylation levels on a genome-wide scale can pose challenges. We compared fresh and matched FFPE tissue DNA samples using the Illumina Infinium HD Human Methylation 450K BeadChip platform with a companion application for repair and "restoration" of DNA from FFPE tissue. Our results showed good correlation between fresh and FFPE sample data. FFPE DNA captured 99% of the CpG sites on the array on average. Significant cancer subgroups based on the CpG island methylator phenotype (CIMP) were clearly distinguished for both fresh and FFPE sample sets with cluster and scaling analysis. The DNA methylation status for the five standard CIMP panel genes which was evaluated for all samples by the MethyLight assay was correctly assigned in both fresh and FFPE samples by the array data. We conclude that the "restoration" method followed by assay on the Infinium HD Human Methylation 450K microarray can produce good quality data for DNA from FFPE samples.

The sessile serrated adenoma (SSA) is a relatively recently described polyp that can present diagnostic difficulties for the practicing pathologist. The frequency of SSA diagnoses varies dramatically in the reported literature. In addition, the histologic interface between the microvesicular hyperplastic polyp (MVHP) and the SSA continues to be a diagnostic problem. The trend in recent years has been toward a lower threshold for SSA diagnosis. Herein, we have performed a cross-sectional study of 6340 colorectal polyps received at a high-volume community-based pathology practice over a 3-month period. After central review, with strict application of the diagnostic criteria outlined in the 2010 edition of the World Health Organization Classification of Tumours of the Digestive Tract, we found that SSAs represented 12.1% of all polyps. In addition, we developed novel diagnostic subcategories in an attempt to determine the most appropriate cutoff for the interface between the MVHP and the SSA. We found that serrated polyps (MVHPs or SSAs) with any SSA-like crypts had clinical features more in common with the SSA than the MVHP and that this diagnostic cutoff showed good reproducibility between pathologists. This supports the position of a recent consensus publication proposing that polyps with as few as 1 SSA-type crypt should be diagnosed as an SSA. Applying these criteria to our cohort yields an overall SSA rate of 14.7%. In summary, we believe that SSAs continue to be underdiagnosed in pathologic practice and that this may result in inadequate surveillance and thus contribute to interval colorectal carcinomas.

BACKGROUND AND 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 using the Illumina HM450 DNA methylation and HT12 V3 expression microarrays in 216 unselected colorectal cancers. Mutations in epigenetic regulators were assessed using CIMP-classified Cancer Genome Atlas exomes.RESULTS: CIMP-High cancers dichotomised into CIMP-H1 and CIMP-H2 based on methylation profile, which was supported by over-representation of BRAF (74%, P Citation Format: Vicki L. Whitehall, Lochlan Fennell, Troy Dumenil, Gunter Hartel, Katia Nones, Catherine Bond, Diane McKeone, Ann-Marie Patch, Stephen Kazakoff, John Pearson, Nicola Waddell, Pratyaksha Wirapati, Paul Lochead, Shuji Ogino, Sabine Tejpar, Barbara Leggett. BRAF and KRAS mutation define distinct subtypes of the CpG island methylator phenotype in colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 479.

Approximately 30 % of colorectal carcinomas develop via the serrated neoplasia pathway characterized by widespread DNA methylation and frequent BRAF mutation. Serrated polyps represent a heterogeneous group of polyps which are the precursor lesions to serrated pathway colorectal carcinomas. The histological classification of serrated polyps has evolved over the last two decades to distinguish three separate entities: hyperplastic polyp, sessile serrated adenoma (SSA), and traditional serrated adenoma (TSA). The malignant potential of SSAs and TSAs has been clearly demonstrated. SSAs are more challenging to detect by colonoscopy and are likely to account for some interval carcinomas of the proximal colon. Serrated polyposis syndrome is now widely recognized as conferring a high risk of colorectal carcinoma although its cause remains elusive. The current understanding of the actual malignant potential of each serrated polyp subtype is still limited due to the lack of large-scale prospective studies. Patient management guidelines have been recently updated although high-level evidence to support them is still required.