CIN Positivity and Clinicopathological AJCC Staging of Cancer Cohorts.

<p>CIN Positivity and Clinicopathological AJCC Staging of Cancer Cohorts.</p

Cox Proportional Hazard Models showing Hazard Ratio (HR) of Overall Survival for the interaction between <i>BRAF</i> and CIN for overall and individual genomic regions, with 95% Confidence Interval (CI) adjusted for stage (I/II and III/IV).

<p>Cox Proportional Hazard Models showing Hazard Ratio (HR) of Overall Survival for the interaction between <i>BRAF</i> and CIN for overall and individual genomic regions, with 95% Confidence Interval (CI) adjusted for stage (I/II and III/IV).</p

Clinicopathological and Molecular Characteristics of the <i>BRAF</i>mut/MSS and <i>BRAF</i>wt/MSS cohorts.

<p>Clinicopathological and Molecular Characteristics of the <i>BRAF</i>mut/MSS and <i>BRAF</i>wt/MSS cohorts.</p

Molecular Characteristics of Cohorts Relative to Presence or Not of Chromosomal Instability (CIN).

<p>CIN+ was indicated by the presence of LOH. This was scored for overall CIN+ if a cancer had 1 of the 12 markers showing greater than 40% loss in intensity of one allele compared to its paired normal. CIN- was assigned if 40% (5 of 12) markers were informative and none of these showed LOH. For individual regions, CIN+ was scored if 1 marker showed LOH, and CIN- if 2of the 3 markers were informative and neither had LOH. CIMP high was scored if 3 of the 5 markers were positive for methylation.</p>*<p>CIN and corresponding <i>p53</i> mutational status was analysed using CIN data at the 5q, 8p and 18q loci only.</p

Heat map showing the distribution of whole chromosome arm and focal copy number aberrations across the cohorts.

<p>Sample heterogeneity occurred within cohorts however a focal pattern is evident in the <i>BRAF</i>mut/MSS and a whole arm pattern is present in the <i>BRAF</i>wt/MSS cohort.</p

Average number of copy number aberrations (CNAs) and percentage of genome affected per MSS cohort.

<p>A) Average number of CNAs delineated by length per cancer in each MSS cohort. MSS cohorts had a similar number of overall CNAs occurring per cancer, however the <i>BRAF</i>mut/MSS cancers showed a greater number of focal CNAs, with the <i>BRAF</i>wt/MSS cancers having a greater number of whole arm events. <i>BRAF</i>mut/MSI cancers had considerably fewer CNAs of all types. B) Average percentage of genome affected by CNAs delineated by length in each MSS cohort. <i>BRAF</i>wt/MSS cancers had the greatest proportion of genome affected by CNA events, which was due to the higher number of whole arm events in this cohort. <i>BRAF</i>mut/MSS cancers showed a lower proportion of the genome affected by CNAs, which is reflective of the comparably lower rate of whole arm and higher rate of focal events that occurred compared to <i>BRAF</i>wt/MSS cancers.</p

Percentage of cancers per cohort that had an amplification or deletion copy number aberration at each chromosome arm.

<p>Asterisks indicate those chromosome arms where significant differences (p<0.05) in the rate of CNAs per cancer occurred between MSS cohorts (red for the <i>BRAF</i>mut/MSS cohort and green for the <i>BRAF</i>wt/MSS cohort to indicate which has a significantly greater rate of CNAs per cancer).</p

Minimal Common Regions (MCRs) affecting ≥20% of cancers in at least one of the <i>BRAF</i>mut/MSS or <i>BRAF</i>wt/MSS cohorts where differences in CNA frequencies were detected between MSS cohorts.

<p>(Benjamini-Hochberg method applied for adjusted p values).</p

Extent of CNAs per Cohort.

<p><b>* Adjusted for multiple comparisons.</b></p

Oncogenic <i>BRAF</i> mutation induces DNA methylation changes in a murine model for human serrated colorectal neoplasia

<p>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 <i>BRAF</i> mutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether <i>in vivo</i> induction of mutant <i>Braf</i> is sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The <i>Braf^V637E</i> 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, <i>Braf</i> mutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic <i>Braf</i> 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 <i>Braf</i> signaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.</p