Alexandra Kane

Objectives: Ceruloplasmin (Cp) and non–ceruloplasmin-bound copper (NCC) are measured when evaluating liver disease and disorders of copper (Cu) metabolism. Cp mass concentration is typically determined by immunoassays that detect both apo- and holoceruloplasmin. In contrast, enzymatic assays measure Cp ferroxidase activity and are therefore specific for holoceruloplasmin. Their clinical use has been limited by the absence of a method to convert enzyme activity to molar Cp concentration and to estimate NCC. Methods: Cp ferroxidase activity and serum Cu concentrations were measured in a training dataset (n=924) to define their linear relationship and derive a conversion factor based on the stoichiometry of six Cu atoms per Cp molecule. This factor was applied to an independent validation cohort (n=12,635). Cp activity, Cp mass concentration, and serum Cu were compared, and reference intervals for Cp activity and NCC were established. Results: The calibration of Cp activity assay against Cu concentration yielded a conversion factor of 0.0030 for expressing Cp activity in molar units. Reference intervals for Cp activity were 1.5–3.7 μmol/L in males and 1.6–4.3 μmol/L in females. Comparison with nephelometric Cp measurements demonstrated a positive bias and evidence of analytical interference in a subset of samples. Conclusions: We describe a method to convert Cp ferroxidase activity to molar concentration and demonstrate its application in a large clinical cohort. This approach enables estimation of NCC, although with considerable uncertainty, and establishment of reference intervals using activity-based measurements. Measurement of Cp by enzymatic activity provides a cost-effective and analytically robust alternative to immunoassays, with advantages in specificity.

Background: Serrated neoplasia arises from serrated precursor lesions. Hyperplastic polyps commonly activate MAPK signalling, initiated by BRAF or KRAS mutation, but premalignant KRAS -mutant sessile serrated lesions are rare. Here, we model Kras- and Braf- mutant neoplasia in vivo comparing histological, transcriptomic, and epigenetic changes. Methods: Temporospatial activation of oncogenic Braf ^V637 or Kras ^G12D was induced in murine intestine. Differential expression, methylation and pathways analyses identified oncogene-specific alterations. Results: Prolonged exposure to oncogenic Braf is associated with a time-dependent accumulation of murine serrated precursors (mSP, P = 3 × 10 ⁻¹⁰ ), and murine serrated lesions (mSL) and invasive cancer (8 × 10 ⁻⁸ ). Kras- mutants acquired fewer mSPs ( P = 0.06) and lower probability of developing mSLs ( P = 0.004). Kras- mutant mSLs rarely develop aberrant WNT signalling (1/23). Transcriptomic profiles diverged, with Braf- mutant intestines showing enriched immune and inflammatory signalling. Deconvolution analysis revealed Braf- mutants had comparably higher macrophage infiltrate ( P = 0.025) and upregulation of M1 macrophage gene sets ( P = 0.0008). Both mutations showed accumulating DNA methylation, however, an attenuated rate in a subset of CpG sites (1306) was observed in Kras- mutant intestine. Conclusion: Kras mutation can induce serrated neoplasia, but with significantly greater latency period and lower penetrance compared to Braf . Kras -mutant neoplasms display an attenuated CIMP-like phenotype, rarely developing aberrant WNT signalling. These data refine our understanding of MAPK-induced intestinal neoplasia.

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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.

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.

Background 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. Aim To investigate curcumin as a chemopreventive for Braf mutant colorectal cancer in a preclinical study utilizing a murine model of serrated neoplasia. Methods An intestine-specific Braf mutant murine model (Braf^V637E/+/Villin-Cre^ERT2/+) 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. Results 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. Conclusions 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 Apc^min/+ and Braf^V637/Villin-Cre^ERT2/+ 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). Apc^min/+/Braf^V637 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.

Background & Aims: 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 analysed 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 Apc ^{min/ +} and Braf ^V637 /Villin-Cre ^{ERT2/ +} 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 (PApc ^{min/ +} /Braf ^V637 animals had more numerous and larger SI and colonic lesions (P−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. Synopsis: We have comprehensively evaluated the somatic mutation landscape of WNT signaling regulators in serrated colorectal cancers. We identified a mosaic of mutations that may be responsible for elevating WNT signaling in this context. Approximately 20% of serrated colorectal cancers harbor truncating APC mutation, and these cancers confer extremely poor prognoses.

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 Braf^V637E 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.