The Origin and Contribution of Cancer-Associated Fibroblasts in Colorectal Carcinogenesis.

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. METHODS: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting-purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. RESULTS: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor ß was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB-IL34/CCL8 signaling that promotes macrophage chemotaxis. CONCLUSIONS: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC.

Traditional serrated adenoma-like lesions in patients with inflammatory bowel disease.

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.

DNA methylation changes that precede onset of dysplasia in advanced sessile serrated adenomas.

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.

CD96 Is an Immune Checkpoint That Regulates CD8+ T-cell Antitumor Function.

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.

Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis.

OBJECTIVE: Serrated 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. DESIGN: We 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. RESULTS: Targeted 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. CONCLUSION: We 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.

CpG Island Methylation in Sessile Serrated Adenomas Increases With Age, Indicating Lower Risk of Malignancy in Young Patients.

Among sessile serrated adenomas (SSAs) with identical histologic features, some never progress, whereas others become dysplastic and develop into invasive cancers. Development of the CpG island methylator phenotype is a feature of SSA progression; we examined the CIMP status of 448 SSAs and examined the association with patient clinical data. Overall, 190 SSAs were CpG island methylator phenotype-positive. CpG island methylator phenotype positivity was associated with older patient age (P < .001) and proximal polyp site (P < .001), but not with patient sex (P = .94) or polyp size (P = .34). These results might be used to improve SSA surveillance guidelines.

Sessile serrated adenomas with dysplasia: morphological patterns and correlations with MLH1 immunohistochemistry.

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.

GNAS mutations are present in colorectal traditional serrated adenomas, serrated tubulovillous adenomas and serrated adenocarcinomas with adverse prognostic features.

AIMS: Activating mutations in GNAS are important in the development of a range of neoplasms, including a small proportion of conventional adenomas and colorectal carcinomas (CRCs). However, their contribution to serrated pathway neoplasia is unclear, as mutations have only been examined in small series of sessile serrated adenomas (SSAs) and traditional serrated adenomas (TSAs), and not in serrated tubulovillous adenomas (sTVAs). The aim of this study was to investigate the frequency and significance of GNAS mutations in colorectal adenomas and CRCs. METHODS AND RESULTS: Using a large, well-characterized series, we identified GNAS mutations in 9.2% (18 of 196) of TSAs, 7.1% (four of 56) of sTVAs and 2.0% (nine of 459) of CRCs. Mutations were absent in SSAs (none of 43), tubular adenomas (none of 50) and conventional tubulovillous adenomas (none of 50). A BRAF or KRAS mutation was seen in 77.4% of GNAS mutant lesions, suggesting a synergistic effect with the mitogen-activated protein kinase pathway. In CRCs, GNAS mutations were associated with mucinous differentiation and serrated morphological features. CONCLUSIONS: GNAS mutations contribute significantly to the development of a subset of serrated adenomas and CRCs.

RNF43 and ZNRF3 are commonly altered in serrated pathway colorectal tumorigenesis.

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<0.0001). ZNRF3 was mutated in 16/54(30%) BRAF mutant/MSI and 5/33(15%) BRAF mutant/microsatellite stable compared to 0/27 BRAF wild type cancers (p=0.004). An RNF43 frameshift mutation (X659fs) occurred in 80% BRAF mutant/MSI cancers. This high rate was verified in a second series of 25/35(71%) BRAF mutant/MSI cancers. RNF43 and ZNRF3 had lower transcript expression in BRAF mutant compared to BRAF wildtype cancers and less cytoplasmic protein expression in BRAF mutant/MSI compared to other subtypes. Treatment with a porcupine inhibitor reduced RNF43/ZNRF3 mutant colony growth by 50% and synergised with a MEK inhibitor to dramatically reduce growth. This study suggests inactivation of RNF43 and ZNRF3 is important in serrated tumorigenesis and has identified a potential therapeutic strategy for this cancer subtype.

Histopathological findings of extra-ileal manifestations at initial diagnosis of Crohn's disease-related ileitis.

Crohn's disease is a chronic inflammatory disorder that can affect any part of the gastrointestinal tract. Our objective was to review the histological findings in index biopsies from the terminal ileum and other gastro-intestinal tract sites of Crohn's disease patients prior any treatment and to compare them with the findings from patients with non-specific ileitis. A total of 111 consecutive Crohn's disease cases (55 females, median age 27 years) with extra-ileal biopsies were retrospectively selected. Upper gastrointestinal inflammatory changes were found in 68 % of gastric biopsies, 60 % of oesophageal biopsies and 43 % of duodenal biopsies with no significant difference in frequency between paediatric and adult cases. Crohn's colitis was more common in paediatric cases than adult cases (85 % versus 57 %). Granuloma in at least one extra-ileal site was observed in 40 %, more frequently in paediatric cases than in adults (78 vs 27 %). Compared with Crohn's disease cases, the group of 151 non-specific ileitis cases (75 females, median age 52 years) were more likely to have normal upper and lower gastrointestinal biopsies and to show less frequent crypt architectural changes in the terminal ileum. In summary, Crohn's disease ileitis is often associated with inflammation elsewhere in the gastrointestinal tract while non-specific ileitis was infrequently associated with inflammation elsewhere for both paediatric and adult patients. These findings support the role of systematic biopsies in multiple gastrointestinal sites to help distinguishing Crohn's ileitis from non-specific ileitis in paediatric and adult population.

Adverse histological features in malignant colorectal polyps: a contemporary series of 239 cases.

AIMS: Screening colonoscopy has led to more colorectal carcinomas presenting at an early stage potentially curable by endoscopic resection. In this study, we examined the clinical and histological features of a contemporary series of malignant colorectal polyps (MCPs) with subsequent surgical resection. METHODS: We conducted a retrospective study on a consecutive series of MCPs from 239 patients, predominantly males (57.7%) with a median age of 66 years, and assessed histological parameters associated with residual disease on the surgical specimens. RESULTS: Median MCP size was 18.6 mm, with 23.1% polyps measuring ≤10 mm. From the 140 surgical resection specimens, residual disease was identified in 20 cases, including 12 cases with metastatic lymph nodes and/or 9 cases with residual carcinoma in the large bowel wall. Histological parameters associated with nodal metastases were greater width and greater depth of the invasive component (p=0.001 and 0.006, respectively), poor differentiation (p=0.003) and a cribriform pattern (p=0.01). The risk of nodal metastases was 23.3% if two or three of these features were identified, while it was 0% and 4.5% if none or one was present, respectively. A positive margin was not associated with nodal metastasis and might be adequately treated by local endoscopic resection. CONCLUSIONS: Surgical resection should be recommended if ≥2 of these adverse histological features are present and may be warranted if one feature is present. A positive margin may require additional local resection but not necessarily surgery if no other adverse factors are present.

Traditional serrated adenoma: an update.

Although recognized 25 years ago, the traditional serrated adenoma (TSA) remains an ongoing source of diagnostic and biologic debate. Recent research has greatly improved our understanding of the morphological and molecular aspects of these polyps. In particular, the recognition of ectopic crypt foci (ECFs) in combination with typical cytology and slitlike serrations improves diagnostic reproducibility. Awareness that many TSAs, particularly BRAF-mutated TSAs, arise in precursor microvesicular hyperplastic polyps and sessile serrated adenomas can aid in making this diagnosis and should not be confused with a sessile serrated adenoma with dysplasia. At a molecular level, TSAs can be divided into 2 groups based on their BRAF or KRAS mutation status. The development of overt cytologic dysplasia is accompanied by TP53 mutation, Wnt pathway activation, and, in some cases, silencing of CDKN2A. Importantly, however, mismatch repair enzyme function is retained. Thus, the TSA is an important precursor of aggressive molecular subtypes of colorectal carcinoma.

Methylation and expression of the tumour suppressor, PRDM5, in colorectal cancer and polyp subgroups.

BACKGROUND: PRDM5 is an epigenetic regulator that has been recognized as an important tumour suppressor gene. Silencing of PRDM5 by promoter hypermethylation has been demonstrated in several cancer types and PRDM5 loss results in upregulation of the Wnt pathway and increased cellular proliferation. PRDM5 has not been extensively investigated in specific subtypes of colorectal cancers. We hypothesized it would be more commonly methylated and inactivated in serrated pathway colorectal cancers that are hallmarked by a BRAF V600E mutation and a methylator phenotype, compared to traditional pathway cancers that are BRAF wild type. METHODS: Cancer (214 BRAF mutant, 122 BRAF wild type) and polyp (59 serrated polyps, 40 conventional adenomas) cohorts were analysed for PRDM5 promoter methylation using MethyLight technology. PRDM5 protein expression was assessed by immunohistochemistry in cancers and polyps. Mutation of PRDM5 was analysed using cBioPortal's publicly available database. RESULTS: BRAF mutant cancers had significantly more frequent PRDM5 promoter methylation than BRAF wild type cancers (77/214,36% vs 4/122,3%; p<0.0001). Serrated type polyps had a lower methylation rate than cancers but were more commonly methylated than conventional adenomas (6/59,10% vs 0/40,0%). PRDM5 methylation was associated with advanced stages of presentation (p<0.05) and the methylator phenotype (p=0.03). PRDM5 protein expression was substantially down-regulated in both BRAF mutant and wild type cancer cohorts (92/97,95% and 39/44,89%). The polyp subgroups showed less silencing than the cancers, but similar rates were found between the serrated and conventional polyp cohorts (29/59, 49%; 23/40, 58% respectively). Of 295 colorectal cancers, PRDM5 was mutated in only 6 (2%) cancers which were all BRAF wild type. CONCLUSIONS: Serrated pathway colorectal cancers demonstrated early and progressive PRDM5 methylation with advancing disease. Interestingly, PRDM5 protein expression was substantially reduced in all polyp types and more so in cancers which also indicates early and increasing PRDM5 down-regulation with disease progression. Methylation may be contributing to gene silencing in a proportion of BRAF mutant cancers, but the large extent of absent protein expression indicates other mechanisms are also responsible for this. These data suggest that PRDM5 is a relevant tumour suppressor gene that is frequently targeted in colorectal tumourigenesis.

A clinicopathological and molecular analysis of 200 traditional serrated adenomas.

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, ß-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 ß-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.