The Efficacy of Using Patient-Derived Organoids to Predict Treatment Response in Colorectal Cancer.

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.

Comparative analysis of Illumina Mouse Methylation BeadChip and reduced-representation bisulfite sequencing for routine DNA methylation analysis.

Researching the murine epigenome in disease models has been hampered by the lack of appropriate and cost-effective DNA methylation arrays. Here we perform a comprehensive, comparative analysis between the Mouse Methylation BeadChip (MMB) and reduced-representation bisulfite sequencing (RRBS) in two murine models of colorectal carcinogenesis. We evaluate the coverage, variability, and ability to identify differential DNA methylation of RRBS and MMB. We show that MMB is an effective tool for profiling the murine methylome that performs comparably with RRBS, identifying similar differentially methylated pathways. Although choice of technology is experiment dependent and will be predicated on the underlying biology being probed, these analyses provide insights into the relative strengths and weaknesses of each approach.

Braf mutation induces rapid neoplastic transformation in the aged and aberrantly methylated intestinal epithelium.

OBJECTIVE: Sessile serrated lesions (SSLs) are common across the age spectrum, but the BRAF mutant cancers arising occur predominantly in the elderly. Aberrant DNA methylation is uncommon in SSL from young patients. Here, we interrogate the role of ageing and DNA methylation in SSL initiation and progression. DESIGN: We used an inducible model of Braf mutation to direct recombination of the oncogenic Braf V637E allele to the murine intestine. BRAF mutation was activated after periods of ageing, and tissue was assessed for histological, DNA methylation and gene expression changes thereafter. We also investigated DNA methylation alterations in human SSLs. RESULTS: Inducing Braf mutation in aged mice was associated with a 10-fold relative risk of serrated lesions compared with young mice. There were extensive differences in age-associated DNA methylation between animals induced at 9 months versus wean, with relatively little differential Braf-specific methylation. DNA methylation at WNT pathway genes scales with age and Braf mutation accelerated age-associated DNA methylation. In human SSLs, increased epigenetic age was associated with high-risk serrated colorectal neoplasia. CONCLUSIONS: SSLs arising in the aged intestine are at a significantly higher risk of spontaneous neoplastic progression. These findings provide support for a new conceptual model for serrated colorectal carcinogenesis, whereby risk of Braf-induced neoplastic transformation is dependent on age and may be related to age-associated molecular alterations that accumulate in the ageing intestine, including DNA methylation. This may have implications for surveillance and chemopreventive strategies targeting the epigenome.

Aspirin reduces the incidence of metastasis in a pre-clinical study of Braf mutant serrated colorectal neoplasia.

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: BrafV637E/+;Villin-CreERT2/+ 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.

APC Mutation Marks an Aggressive Subtype of BRAF Mutant Colorectal Cancers.

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 < 2 × 10-5), advanced stage (p < 0.01), and poor survival (p = 0.026). Apcmin/+/BrafV637 animals had more numerous and larger SI and colonic lesions (p < 0.0001 and p < 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.

Alterations in signaling pathways that accompany spontaneous transition to malignancy in a mouse model of BRAF mutant microsatellite stable colorectal cancer.

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 ß-catenin (Ctnnb1). Immunohistochemical staining of ß-catenin indicated that these mutations led to an increase in the presence of aberrant nuclear ß-catenin that resulted in gene expression changes in targets of ß-catenin transcription. Gene expression profiling identified a significant enrichment for transforming growth factor-ß (TGF-ß) signaling that was present in mSL and carcinomas. Early activation of TGF-ß 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-ß signaling during the transition of human sessile serrated lesions to malignancy.

Complementing chronic frailty assessment at hospital admission with an electronic frailty index (FI-Laboratory) comprising routine blood test results.

BACKGROUND: Acutely ill and frail older adults have complex social and health care needs. It is important to understand how this complexity affects acute outcomes for admission to hospital. We validated a frailty index using routine admission laboratory tests with outcomes after patients were admitted to hospital. METHODS: In a prospective cohort of older adults admitted to a large tertiary hospital in the United Kingdom, we created a frailty index from routine admission laboratory investigations (FI-Laboratory) linked to data comprising hospital outcomes. We evaluated the association between the FI-Laboratory and total days spent in hospital, discharge to a higher level of care, readmission and mortality. RESULTS: Of 2552 admissions among 1750 older adults, we were able to generate FI-Laboratory values for 2254 admissions (88.3% of the cohort). More than half of admitted patients were women (55.3%) and the mean age was 84.6 (SD 14.0) years. We found that the FI-Laboratory correlated weakly with the Clinical Frailty Scale (CFS; r 2 = 0.09). An increase in the CFS and the equivalent of 3 additional abnormal laboratory test results in the FI-Laboratory, respectively, were associated with an increased proportion of inpatient days (rate ratios [RRs] 1.43, 95% confidence interval [CI] 1.35-1.52; and 1.47, 95% CI 1.41-1.54), discharge to a higher level of care (odd ratios [ORs] 1.39, 95% CI 1.27-1.52; and 1.30, 95% CI 1.16-1.47) and increased readmission rate (hazard ratios [HRs] 1.26, 95% CI 1.17-1.37; and 1.18, 95% CI 1.11-1.26). Increases in the CFS and FI-Laboratory were associated with increased mortality HRs of 1.39 (95% CI 1.28-1.51) and 1.45 (95% CI 1.37-1.54), respectively. INTERPRETATION: We determined that FI-Laboratory, distinct from baseline frailty, could be used to predict risk of many adverse outcomes. The score is therefore a useful way to quantify the degree of acute illness in frail older adults.

Integrative Genome-Scale DNA Methylation Analysis of a Large and Unselected Cohort Reveals 5 Distinct Subtypes of Colorectal Adenocarcinomas.

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.

Retrospective descriptive observational study of patients who presented to an Australian hospital emergency department with neck soft tissue injury.

OBJECTIVE: To describe clinical presentation and management of neck soft tissue injury in an Australian ED. METHODS: This is a retrospective cohort study conducted in a tertiary hospital ED in Queensland, Australia. This study included all patients aged 18-65 years presenting with neck sprain/strain in 2016. Main outcome measures are patient demographics, comorbidities, presentation, acute management and follow up. RESULTS: Of 339 patients, 176 (52%) had cervical computed tomography (CT) scans and 3% plain radiographs. Two had fractures (CT yield of 2/176; 1.1%) and three were admitted with neurological symptoms, leaving 334 patients. Of 264 patients receiving medications in the ED, simple analgesia + oral opioid (146, 55.3%) was most frequently used, followed by simple analgesia (89, 33.7%) and opioid + benzodiazepine +/- simple analgesia (16, 6%). Opioids were prescribed for 169 (64%) (including i.v. opioids for 34 [12.9%] and for 85/97 (88%) with pain scores ≤4), and benzodiazepines for 22 (8.3%). Ten (3%) were referred for physiotherapy management in ED and eight (2.4%) for outpatient physiotherapy follow up. Of 113/334 (33.8%) receiving discharge prescription, 60 (53.1%) were prescribed oral opioid + simple analgesia, 37 (32.7%) oral opioids and seven (6.2%) opioids + benzodiazepines; 205 (61%) were discharged without a recorded follow-up plan. CONCLUSIONS: There is large practice variation in management of neck soft tissue injury in ED. Over half of the patients received CT scans with modest yield. Opioids were commonly used both in ED and on discharge. There is need for a standard management plan to be developed for patients presenting with acute neck soft tissue injury.

Copy number profiles of paired primary and metastatic colorectal cancers.

Liver metastasis is the major cause of death following a diagnosis of colorectal cancer (CRC). In this study, we compared the copy number profiles of paired primary and liver metastatic CRC to better understand how the genomic structure of primary CRC differs from the metastasis. Paired primary and metastatic tumors from 16 patients and their adjacent normal tissue samples were analyzed using single nucleotide polymorphism arrays. Genome-wide chromosomal copy number alterations were assessed, with particular attention to 188 genes known to be somatically altered in CRC and 24 genes that are clinically actionable in CRC. These data were analyzed with respect to the timing of primary and metastatic tissue resection and with exposure to chemotherapy. The genomic differences between the tumor and paired metastases revealed an average copy number discordance of 22.0%. The pairs of tumor samples collected prior to treatment revealed significantly higher copy number differences compared to post-therapy liver metastases (P = 0.014). Loss of heterozygosity acquired in liver metastases was significantly higher in previously treated liver metastasis samples compared to treatment naive liver metastasis samples (P = 0.003). Amplification of the clinically actionable genes ERBB2, FGFR1, PIK3CA or CDK8 was observed in the metastatic tissue of 4 patients but not in the paired primary CRC. These examples highlight the intra-patient genomic discrepancies that can occur between metastases and the primary tumors from which they arose. We propose that precision medicine strategies may therefore identify different actionable targets in metastatic tissue, compared to primary tumors, due to substantial genomic differences.

MLH1-93 G/a polymorphism is associated with MLH1 promoter methylation and protein loss in dysplastic sessile serrated adenomas with BRAFV600E mutation.

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.

RNF43 is mutated less frequently in Lynch Syndrome compared with sporadic microsatellite unstable colorectal cancers.

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.

How the BRAF V600E Mutation Defines a Distinct Subgroup of Colorectal Cancer: Molecular and Clinical Implications.

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.

Oncogenic BRAF mutation induces DNA methylation changes in a murine model for human serrated colorectal neoplasia.

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.

The role of APC in WNT pathway activation in serrated neoplasia.

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 ß-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 ß-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 ß-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.

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.

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.

Microsatellite stable colorectal cancers stratified by the BRAF V600E mutation show distinct patterns of chromosomal instability.

The BRAF (V600E) mutation in colorectal cancers that are microsatellite stable (MSS) confers a poor patient prognosis, whereas BRAF mutant microsatellite-unstable (MSI) colorectal cancers have an excellent prognosis. BRAF wild type cancers are typically MSS and display chromosomal instability (CIN). CIN has not been extensively studied on a genome-wide basis in relation to BRAF mutational status in colorectal cancer. BRAF mutant/MSS (BRAFmut/MSS) cancers (n = 33) and BRAF mutant/MSI (BRAFmut/MSI) cancers (n = 30) were compared for presence of copy number aberrations (CNAs) indicative of CIN, with BRAF wild type/MSS (BRAFwt/MSS) cancers (n = 18) using Illumina CytoSNP-12 arrays. BRAFmut/MSS and BRAFwt/MSS cancers showed comparable numbers of CNAs/cancer at 32.8 and 29.8 respectively. However, there were differences in patterns of CNA length between MSS cohorts, with BRAFmut/MSS cancers having significantly greater proportions of focal CNAs compared to BRAFwt/MSS cancers (p<0.0001); whereas whole chromosomal arm CNAs were more common in BRAFwt/MSS cancers (p<0.0001). This related to a reduced average CNA length in BRAFmut/MSS compared to BRAFwt/MSS cancers (20.7 Mb vs 33.4 Mb;p<0.0001); and a smaller average percent of CIN affected genomes in BRAFmut/MSS compared to BRAFwt/MSS cancers (23.9% vs 34.9% respectively). BRAFmut/MSI cancers were confirmed to have low CNA rates (5.4/cancer) and minimal CIN-affected genomes (average of 4.5%) compared to MSS cohorts (p<0.0001). BRAFmut/MSS cancers had more frequent deletion CNAs compared to BRAFwt/MSS cancers on 6p and 17q at loci not typically correlated with colorectal cancer, and greater amplification CNAs on 8q and 18q compared to BRAFwt/MSS cancers. These results indicate that comparable rates of CIN occur between MSS subgroups, however significant differences in their patterns of instability exist, with BRAFmut/MSS cancers showing a 'focal pattern' and BRAFwt/MSS cancers having a 'whole arm pattern' of CIN. This and the genomic loci more frequently affected in BRAFmut/MSS cancers provides further evidence of the biological distinctions of this important cancer subgroup.

Chromosomal instability in BRAF mutant, microsatellite stable colorectal cancers.

The BRAF oncogene is mutated in 15% of sporadic colorectal cancers. Approximately half of these BRAF mutant cancers demonstrate frequent frameshift mutations termed microsatellite instability (MSI), but are diploid and chromosomally stable. BRAF wild type cancers are typically microsatellite stable (MSS) and instead acquire chromosomal instability (CIN). In these cancers, CIN is associated with a poor outcome. BRAF mutant cancers that are MSS, typically present at an advanced stage and have a particularly poor prognosis. We have previously demonstrated clinical and molecular similarities between MSS cancers with or without a BRAF mutation, and therefore hypothesised that CIN may also be frequent in BRAF mutant/MSS cancers. BRAF mutant/MSS (n = 60), and BRAF wild type/MSS CRCs (n = 90) were investigated for CIN using loss of heterozygosity analysis over twelve loci encompassing chromosomal regions 5q, 8p, 17p and 18q. CIN was frequent in BRAF mutant/MSS cancers (41/57, 72%), which was comparable to the rate found in BRAF wild type/MSS cancers (74/90, 82%). The greatest loss in BRAF mutant/MSS cancers occurred at 8p (26/44, 59%), and the least at 5q (19/49, 39%). CIN in BRAF mutant/MSS cancers correlated with advanced stage (AJCC III/IV: 15/17, 88%; p = 0.02); showed high rates of co-occurrence with the CpG Island Methylator Phenotype (17/23, 74%); and CIN at 18q and 8p associated with worse survival (p = 0.02, p<0.05). This study demonstrates that CIN commonly occurs in advanced BRAF mutant/MSS colorectal cancers where it may contribute to poorer survival, and further highlights molecular similarities occurring between these and BRAF wild type cancers.

p53 mutation is common in microsatellite stable, BRAF mutant colorectal cancers.

The majority of "serrated pathway" colorectal cancers have mutation of the BRAF oncogene and display the CpG island methylator phenotype (CIMP). Half these cancers have microsatellite instability (MSI) and an excellent prognosis. In the absence of MSI (microsatellite stable, MSS), BRAF mutation has been associated with a particularly poor prognosis. "Traditional pathway" cancers are BRAF wild type. Mutation of p53 is common and this correlates with advanced stage. We therefore hypothesized that p53 mutation would be common in MSS/BRAF mutant colorectal cancer. One thousand and eighty-one colorectal cancers were screened for BRAF mutation to identify two BRAF mutant study groups (MSI: n = 77; MSS: n = 69) and a BRAF wild type control group (n = 101). These were screened for p53 mutation by high resolution melt analysis and classified for CIMP and MGMT methylation by quantitative methylation specific PCR. Molecular data were compared to patient age, gender, tumor location and stage. p53 was mutated significantly more frequently in MSS/BRAF mutant (28/69, 40.6%) compared to MSI/BRAF mutant cancers (13/77, 16.9%), but this mutation rate did not differ from MSS/BRAF wild type cancers (47/101, 46.5%)(p < 0.0001). CIMP was less common in MSS/BRAF mutant (26/47, 55.3%) compared to MSI/BRAF mutant cancers (41/54, 75.9%), but was more common than in MSS/BRAF wild type cancers (3/85, 3.5%) (p < 0.0001). MSS/BRAF mutant cancers were more commonly proximal (38/54, 70.3%), but were similar to MSS/BRAF wild type cancers in terms of patient age, gender distribution and stage at presentation. MSS/BRAF mutant cancers share molecular and clinical features of both the serrated and traditional pathways of colorectal tumorigenesis.