Colorectal cancer screening.

Colorectal cancer is one of the most common malignancies in Australia, and screening to detect it an earlier stage is cost-effective. Furthermore, detection and removal of precursor polyps can reduce incidence. Currently, there are limited data to determine the screening rate in Australia, but it is certainly lower than the 80% screening rate considered desirable. Whether colonoscopy is used as the screening test or to follow up positive results of an initial non-invasive test, it plays a fundamental role. Despite high sensitivity and specificity, it is expensive and invasive with measurable risk and is not acceptable as an initial test to many participants. It does not provide complete protection, and interval cancers between planned colonoscopies are associated with proximal location, origin in sessile serrated adenomas and operator-dependent factors. An essential component of colorectal screening is the measurement of colonoscopy quality indicators, such as caecal intubation and adenoma detection rates, which are known to be associated with the rate of interval cancer. The non-invasive screening test currently recommended in Australia is biennial testing for faecal occult blood between the ages of 50 and 75 using a faecal immunochemical test, with positives evaluated by colonoscopy. This is provided through the National Bowel Cancer Screening Programme, currently for those at the ages of 50, 55, 60 and 65 years, with full implementation of biennial screening by 2020. To improve screening in Australia, the most fruitful approach may be to acknowledge that there is a choice of screening tests and to focus on the goal of improving overall participation rate and being able to measure this.

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype.

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.

Copper removal strategies for Wilson's disease crisis in the ICU.

Wilson's disease is a rare, inherited, autosomal recessive disorder of copper metabolism which leads to an accumulation of copper in body tissues. If a patient develops a Wilson's crisis, mortality can approach 100%. The treatment of such patients is mostly organ support but a possible treatment goal is to try to rapidly remove copper from their system. We performed a literature search on methods for de-coppering strategies for patients in intensive care with known Wilson's disease. We found 11 case reports where therapeutic plasma exchange was used and six case reports where various forms of albumin dialysis were used as techniques for rapidly reducing serum copper levels. To date, the case reports are encouraging that therapeutic plasma exchange and albumin dialysis can either delay or prevent the need for liver transplantation in patients with fulminant hepatic failure due to Wilson's disease. However, these case reports are mainly in the paediatric or young adult population, thus further studies in adults are warranted.

DNA methylation within the normal colorectal mucosa is associated with pathway-specific predisposition to cancer.

There are two major molecular pathways to sporadic colorectal cancer, the chromosomal instability (CIN) and the CpG island methylator phenotype (CIMP) pathways. This study recruited 166 patients undergoing colonoscopy. Biopsy samples were collected from the cecum, transverse colon, sigmoid colon and rectum. DNA methylation was quantified at 'type A' (ESR1, GATA5, HIC1, HPP1, SFRP1) and 'type C' markers (MGMT, MLH1, CDKN2A, MINT2, MINT31, IGF2, CACNA1G, NEUROG1, SOCS1, RUNX3), and LINE-1. 'Type A' genes are frequently methylated in normal and neoplastic tissues, proportional to tissue age. 'Type C' methylation is more specific for neoplasia. The last five 'type C' markers comprise a CIMP panel. The mean 'type A' and CIMP-panel methylation Z-scores were calculated. In all, 88 patients had adenomatous lesions, 32 had proximal serrated polyps (PSPs) and 50 were normal. Most 'type A' genes showed direct correlations between methylation and age (ESR1, rho=0.66, P<0.0001), with higher methylation distally (ESR1, P<0.0001). On multivariate analysis, 'type A' methylation was inversely associated with colorectal adenomas (odds ratio=0.23, P<0.001), the precursor to CIN cancers. CIMP-panel methylation was significantly associated with advanced PSPs (odds ratio=5.1, P=0.009), the precursor to CIMP cancers. DNA methylation in normal mucosa varied with age and region and was associated with pathway-specific pathology. In the future, the colorectal field could yield important information and potentially inform clinical practice.

Methylation in p14(ARF) is frequently observed in colorectal cancer with low-level microsatellite instability.

Colorectal cancer (CRC) can be classified as high-level microsatellite instability (MSI-H), low-level MSI (MSI-L) and microsatellite stable (MSS) depending on levels of MSI. MSI-H CRC relies on a distinct molecular pathway due to the mismatch repair (MMR) deficiency and shows methylation in multiple gene promoters. The genetic pathway leading to MSI-L is unknown, although higher levels of promoter methylation are observed in this group compared with MSS CRCs. This study explored how promoter methylation affects MSI phenotype, by analysing the methylation status of eight CRC-related promoters, MSI phenotype and KRAS/BRAF mutations in a series of 234 CRCs. Promoter methylation of p14(ARF) was significantly related to MSI-L CRC with KRAS mutation. The MSI-H phenotype was related to methylation of MLH1 as expected, while the MSS phenotype was related to methylation of p16(INK4a) and O(6)-methylguanine-DNA methyltransferase, although this was not statistically significant. Thus, promoter methylation of p14(ARF) could be a significant alteration leading to CRC with MSI-L.

Epigenetic silencing of EphA1 expression in colorectal cancer is correlated with poor survival.

Aberrant expression of Eph and ephrin proteins has well-established functions in oncogenesis and tumour progression. We describe EphA1 expression in 6 colorectal cancer (CRC) cell lines, 18 controls and 125 CRC specimens. In addition, a well-characterised cohort of 53 paired normal colon and CRCs was also assessed. Expression of EphA1 mRNA was assessed by quantitative real-time PCR and correlated with protein expression by flow cytometry, immunoprecipitation, western blotting and immunohistochemistry. Significant upregulation (2- to 10-fold) of EphA1 was seen in over 50% of cases (P=0.005) whereas many of the remainder showed downregulation of EphA1. Intriguingly, EphA1 over-expression was more prevalent in stage II compared to stage III CRCs (P=0.02). Low EphA1 expression significantly correlated with poor survival (P=0.02). Epigenetic silencing appeared to explain the loss of EphA1 expression as methylation of the EphA1 CpG island strongly correlated with low EphA1 expression (P<0.01). Furthermore, EphA1 re-expression could be induced by treatment with demethylating agents. Our findings identify EphA1 as a potential prognostic marker in CRC. Although therapies targeting high EphA1 expression seem plausible in CRC, the loss of expression in advanced disease suggests a potential risk that targeted therapy, by selecting for loss of expression, might contribute to disease progression.

Promoter methylation of the mutated in colorectal cancer gene is a frequent early event in colorectal cancer.

The mutated in colorectal cancer (MCC) gene is in close linkage with the adenomatous polyposis coli (APC) gene on chromosome 5, in a region of frequent loss of heterozygosity in colorectal cancer. The role of MCC in carcinogenesis, however, has not been extensively analysed, and functional studies are emerging, which implicate it as a candidate tumor suppressor gene. The aim of this study was to examine loss of MCC expression due to promoter hypermethylation and its clinicopathologic significance in colorectal cancer. Correspondence of MCC methylation with gene silencing was demonstrated using bisulfite sequencing, reverse transcription-polymerase chain reaction and Western blotting. MCC methylation was detected in 45-52% of 187 primary colorectal cancers. There was a striking association with CDKN2A methylation (P<0.0001), the CpG island methylator phenotype (P<0.0001) and the BRAF V600E mutation (P<0.0001). MCC methylation was also more common (P=0.0084) in serrated polyps than in adenomas. In contrast, there was no association with APC methylation or KRAS mutations. This study demonstrates for the first time that MCC methylation is a frequent change during colorectal carcinogenesis. Furthermore, MCC methylation is significantly associated with a distinct spectrum of precursor lesions, which are suggested to give rise to cancers via the serrated neoplasia pathway.

Low level microsatellite instability may be associated with reduced cancer specific survival in sporadic stage C colorectal carcinoma.

BACKGROUND: Colorectal cancers (CRCs) may be categorised according to the degree of microsatellite instability (MSI) exhibited, as MSI-high (MSI-H), MSI-low (MSI-L), or microsatellite stable (MSS). MSI-H status confers a survival advantage to patients with sporadic CRC. AIMS: To determine if low levels of MSI are related to the clinicopathological features and prognosis of sporadic stage C CRC. PATIENTS: A total of 255 patients who underwent resection for sporadic stage C CRC were studied. No patient received chemotherapy. Minimum follow up was five years. METHODS: DNA extracted from archival malignant and non-malignant tissue was amplified by polymerase chain reaction using a panel of 11 microsatellites. MSI-H was defined as instability at > or =40% of markers, MSS as no instability, and MSI-L as instability at >0% but <40% of markers. Patients with MSI-H CRC were excluded from analysis as they have previously been shown to have better survival. RESULTS: Thirty three MSI-L and 176 MSS CRCs were identified. There was no difference in biological characteristics or overall survival of MSI-L compared with MSS CRC but MSI-L was associated with poorer cancer specific survival (hazard ratio 2.0 (95% confidence interval 1.1-3.6)). CONCLUSIONS: Sporadic MSI-L and MSS CRCs have comparable clinicopathological features. Further studies are required to assess the impact of MSI-L on prognosis.

Promoter hypermethylation frequency and BRAF mutations distinguish hereditary non-polyposis colon cancer from sporadic MSI-H colon cancer.

BACKGROUND: Colorectal cancers resulting from defective DNA mismatch repair can occur in both hereditary non-polyposis colon cancer (HNPCC) and in the sporadic setting. They are characterised by a high level of microsatellite instability (MSI-H) and superficially resemble each other in that they are frequently located in the proximal colon and share features such as circumscribed tumour margins and tumour-infiltrating lymphocytes. However, significant differences can be demonstrated at the molecular level including widespread promoter hypermethylation and BRAF -activating mutations which occur significantly less often in HNPCC. AIMS: In this study, we sought to determine whether the presence of widespread promoter hypermethylation and BRAF mutations would exclude HNPCC. MATERIALS AND METHODS: We investigated the methylation status of four methylated in tumour markers (MINTs 1,2,12 and 31), and the promoter regions of 5 genes hMLH1, HPP1, MGMT, p16INK4A and p14ARF, in 21 sporadic MSI-H colorectal cancers and compared these with 18 cancers from HNPCC patients. The methylation status of CpG islands were determined by either methylation specific PCR (MSP) or combined bisulfite restricton analysis (COBRA). In addition we considered the BRAF mutation status of 18 HNPCC tumours and 19 sporadic MSI-H cancers which had been previously determined by RFLP analysis and confirmatory sequencing. RESULTS: Methylation of the promoter regions in target genes occurred less frequently within the HNPCC tumours (27% of analyses), compared with the sporadic MSI-H tumours (59% of analyses) (P < 0.001). Methylation of MINTs 1, 2, 12 and 31 occurred in 4% of analyses for HNPCC tumours contrasted with 73% for sporadic MSI-H tumours (P < 0.001). BRAF mutations were detected in 74% of sporadic tumours but none of the HNPCC cancers tested. CONCLUSIONS: The total number of genes and MINTs methylated in HNPCC was lower than in MSI-H colorectal tumours. No HNPCC tumour showed evidence of widespread promoter hypermethylation or BRAF mutation suggesting this feature could be used as a discriminator between familial and sporadic cases.

BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum.

BACKGROUND AND AIMS: Mutations in BRAF have been linked with colorectal cancers (CRC) showing high level microsatellite instability (MSI-H). However, the distribution of BRAF mutations in MSI-H cancers remains to be clarified with respect to precursor lesions and the CpG island methylator phenotype (CIMP). METHODS: Forty three hyperplastic polyps (HP), nine mixed polyps (MP), five serrated adenomas (SA), 28 conventional adenomas (AD), 18 hereditary non-polyposis colorectal cancers (HNPCC), and 127 sporadic CRC (46 MSI-H and 81 non-MSI-H) were collected from patients undergoing colectomy for either CRC or hyperplastic polyposis. Twenty five of 57 serrated lesions were derived from four patients with hyperplastic polyposis. HP were further subdivided according to recently documented morphological criteria into 27 classical HP and 16 variant lesions described as "sessile serrated adenoma" (SSA). All tumours were screened for BRAF activating mutations. RESULTS: The BRAF mutation was more frequent in SSA (75%) and MP (89%) than in classical HP (19%), SA (20%), and AD (0%) (p<0.0001), and also in sporadic MSI-H cancers (76%) compared with HNPCC (0%) and sporadic non-MSI-H cancers (9%) (p<0.0001). The BRAF mutation was identified more often in CIMP-high serrated polyps (72%) and CIMP-high CRC (77%) than in CIMP-low (30%) and CIMP-negative (13%) polyps (p = 0.002) as well as CIMP-low (18%) and CIMP-negative (0%) CRC (p<0.0001). CONCLUSIONS: The BRAF mutation was frequently seen in SSA and in sporadic MSI-H CRC, both of which were associated with DNA methylation. Sporadic MSI-H cancers may originate in SSA and not adenomas, and BRAF mutation and DNA methylation are early events in this "serrated" pathway.

Methylation patterns define two types of hyperplastic polyp associated with colorectal cancer.

AIM: Hyperplastic polyps (HP) of the colorectum have traditionally been regarded as non-neoplastic lesions. Recent data implicate HP in the pathogenesis of colorectal cancers (CRC) characterised by extensive DNA methylation and microsatellite instability. The aim of this study was to identify morphological and molecular features that may characterise subtypes of HP with potential for neoplastic progression. MATERIALS AND METHODS: HP (22) clustering around distal CRC (group I) were compared with HP (58) in subjects with hyperplastic polyposis (group II). DNA methylation was tested in methylated in tumour (MINT) loci (1, 2, 12, 31) and genes HPP1, MGMT, p14ARF, p16INK4a, and hMLH1. RESULTS: Group II HP showed significantly more methylation than group I HP at all loci except MINT1 and MGMT. Group I showed the lowest frequency of DNA methylation but the highest frequency of K-ras mutation. Group II HP (termed HP variant) had the morphological features of the recently described "sessile serrated adenomas". Methylation of hMLH1 was found most frequently in group II polyps that included foci of dysplasia (7/10) and in no group I lesions. CONCLUSION: The findings support the existence of morphological and molecular heterogeneity among HP and highlight a subset that is likely to have significant malignant potential.

APC mutation and tumour budding in colorectal cancer.

AIM: To determine the frequency of tumour budding and somatic APC mutation in a series of colorectal cancers stratified according to DNA microsatellite instability (MSI) status. MATERIAL/METHODS: Ninety five colorectal cancers were genotyped for APC mutation in the mutation cluster region (exon 15) and scored for the presence of tumour budding at the invasive margin in haematoxylin and eosin stained sections. A subset was immunostained for beta catenin and p16. RESULTS: The frequency of both somatic APC mutation and tumour budding increased pari passu in cancers stratified as sporadic MSI high (MSI-H), hereditary non-polyposis colorectal cancer (HNPCC), MSI low (MSI-L), and microsatellite stable (MSS). Both budding and APC mutation were significantly less frequent in sporadic MSI-H cancers than in MSI-L or MSS cancers. Tumour buds were characterised by increased immunostaining for both beta catenin and p16. CONCLUSION: Tumour budding is associated with an adverse prognosis. The lack of budding in MSI-H colorectal cancer may account for the improved prognosis of this subset and may be explained by an intact WNT signalling pathway and/or inactivated p16(INK4a).

Distinction between familial and sporadic forms of colorectal cancer showing DNA microsatellite instability.

Attempts to classify colorectal cancer into subtypes based upon molecular characterisation are overshadowed by the classical stepwise model in which the adenoma-carcinoma sequence serves as the morphological counterpart. Clarity is achieved when cancers showing DNA microsatellite instability (MSI) are distinguished as sporadic MSI-low (MSI-L), sporadic MSI-high (MSI-H) and hereditary non-polyposis colorectal cancer (HNPCC). Divergence of the 'methylator' pathway into MSI-L and MSI-H is at least partly determined by the respective silencing of MGMT and hMLH1. Multiple differences can be demonstrated between sporadic and familial (HNPCC) MSI-H colorectal cancer with respect to early mechanisms, evolution, molecular characterisation, demographics and morphology. By acknowledging the existence of multiple pathways, rapid advances in the fields of basic and translational research will occur and this will lead to improved strategies for the prevention, early detection and treatment of colorectal cancer.

A case of myoepithelial carcinoma displaying biallelic inactivation of the tumour suppressor gene APC in a patient with familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is an autosomal dominant disorder caused by mutation of the APC gene. It is characterised by the appearance of hundreds to thousands of colorectal adenomas in adolescence and the subsequent development of colorectal cancer. Various extracolonic malignancies are associated with FAP, including desmoids and neoplasms of the stomach, duodenum, pancreas, liver, and brain. We present a family affected by FAP with an exon 14 APC mutation displaying two rare extracolonic lesions, a hepatoblastoma and a myoepithelial carcinoma. The hepatoblastoma was found in a male patient aged 2 years. The second lesion, a myoepithelial carcinoma of the right cheek, was found in a female patient aged 14 years. Inactivation of the normal APC allele was demonstrated in this lesion by loss of heterozygosity analysis, thus implicating APC in the initiation or progression of this neoplasm. This is the first reported case of this lesion in a family affected by FAP.

Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis.

High-level microsatellite instability (MSI-H) is demonstrated in 10 to 15% of sporadic colorectal cancers and in most cancers presenting in the inherited condition hereditary nonpolyposis colorectal cancer (HNPCC). Distinction between these categories of MSI-H cancer is of clinical importance and the aim of this study was to assess clinical, pathological, and molecular features that might be discriminatory. One hundred and twelve MSI-H colorectal cancers from families fulfilling the Bethesda criteria were compared with 57 sporadic MSI-H colorectal cancers. HNPCC cancers presented at a lower age (P < 0.001) with no sporadic MSI-H cancer being diagnosed before the age of 57 years. MSI was less extensive in HNPCC cancers with 72% microsatellite markers showing band shifts compared with 87% in sporadic tumors (P < 0.001). Absent immunostaining for hMSH2 was only found in HNPCC tumors. Methylation of hMLH1 was observed in 87% of sporadic cancers but also in 55% of HNPCC tumors that showed loss of expression of hMLH1 (P = 0.02). HNPCC cancers were more frequently characterized by aberrant beta-catenin immunostaining as evidenced by nuclear positivity (P < 0.001). Aberrant p53 immunostaining was infrequent in both groups. There were no differences with respect to 5q loss of heterozygosity or codon 12 K-ras mutation, which were infrequent in both groups. Sporadic MSI-H cancers were more frequently heterogeneous (P < 0.001), poorly differentiated (P = 0.02), mucinous (P = 0.02), and proximally located (P = 0.04) than HNPCC tumors. In sporadic MSI-H cancers, contiguous adenomas were likely to be serrated whereas traditional adenomas were dominant in HNPCC. Lymphocytic infiltration was more pronounced in HNPCC but the results did not reach statistical significance. Overall, HNPCC cancers were more like common colorectal cancer in terms of morphology and expression of beta-catenin whereas sporadic MSI-H cancers displayed features consistent with a different morphogenesis. No individual feature was discriminatory for all HNPCC cancers. However, a model based on four features was able to classify 94.5% of tumors as sporadic or HNPCC. The finding of multiple differences between sporadic and familial MSI-H colorectal cancer with respect to both genotype and phenotype is consistent with tumorigenesis through parallel evolutionary pathways and emphasizes the importance of studying the two groups separately.

Instabilotyping: comprehensive identification of frameshift mutations caused by coding region microsatellite instability.

Coding region frameshift mutation caused by microsatellite instability (MSI) is one mechanism contributing to tumorigenesis in cancers with MSI in high frequency. Mutation of TGFBR2 is one example of this process. To identify additional examples, a large-scale genomic screen of coding region microsatellites was conducted. 1115 coding homopolymeric loci with six or more nucleotides were identified in an online genetic database. Mutational screening was performed at 152 of these loci in 46 colorectal tumors with MSI in high frequency. Nine loci were mutated in > or =20% of tumors, 10 loci in 10-20%, 24 loci in 5-10%, 43 loci in <5%, and 66 loci were not mutated in any tumors. The most frequently mutated novel loci were the activin type II receptor gene (58.1%), SEC63 (48.8%), AIM 2 (47.6%), a gene encoding a subunit of the NADH-ubiquinone oxidoreductase complex (27.9%), a homologue of mouse cordon-bleu (23.8%), and EBP1/PA2G4 (20.9%). This genome-wide approach identifies coding region MSI in genes or pathways not implicated previously in colorectal tumorigenesis, which may merit functional study or other additional analysis.

Proliferation, apoptosis, and survival in high-level microsatellite instability sporadic colorectal cancer.

Sporadic colorectal cancer (CRC) characterized by high-level DNA microsatellite instability (MSI-H) has a favorable prognosis. The reason for this MSI-H survival advantage is not known. The aim of this study was to correlate proliferation, apoptosis, and prognosis in CRC stratified by MSI status. The proliferative index (PI) was measured by immunohistochemical staining with the Ki-67 antibody in a selected series of 100 sporadic colorectal cancers classified according to the level of MSI as 31 MSI-H, 29 MSI-Low (MSI-L), and 40 microsatellite stable (MSS). The Ki-67 index was significantly higher in MSI-H cancers (P < 0.0001) in which the PI was 90.1 +/- 1.2% (mean +/- SE) compared with 69.5 +/- 3.1% and 69.5 +/- 2.3% in MSI-L and MSS subgroups, respectively. There was a positive linear correlation between the apoptotic index (AI) and PI (r = 0.51; P < 0.001), with MSI-H cancers demonstrating an increased AI:PI ratio indicative of a lower index of cell production. A high PI showed a trend toward predicting improved survival within MSI-H cancers (P = 0.09) but did not predict survival in MSI-L or MSS cancers. The AI was not associated with survival in any MSI subgroup. In conclusion, this is the first study to show that sporadic MSI-H cancers are characterized by a higher AI:PI ratio and increased proliferative activity compared with MSI-L and MSS cancers, and that an elevated PI may confer a survival advantage within the MSI-H subset.

Hyperplastic polyposis: association with colorectal cancer.

Hyperplastic polyposis is a loosely defined syndrome initially thought not to confer a clinically important predisposition to colorectal cancer. The aim of the current study was to examine the clinical, histologic, and molecular features of a prospective series of cases meeting a strict definition of the condition. Twelve patients were identified, seven of whom had developed colorectal cancer. Most polyps were hyperplastic, but 11 patients also had polyps containing dysplasia as either serrated adenomas. mixed polyps, or traditional adenomas. The mean percentage of dysplastic polyps in patients with cancer was 35%, and in patients without cancer, 11% (p < 0.05). Microsatellite instability (MSI) was present in 3 of 47 hyperplastic polyps and two of eight serrated adenomas. Kras was mutated in 8 of 47 hyperplastic polyps and two of eight serrated adenomas. No polyps showed loss of heterozygosity of chromosomes 5q, 1p, or 18q. Two of seven cancers showed a high level of MSI. It is concluded that hyperplastic polyposis is associated with a high risk of colorectal cancer. Hyperplastic polyps are the dominant type of polyp, but most cases have some dysplastic epithelium. A higher proportion of dysplastic polyps is associated with increased cancer risk. Clonal genetic changes are observed in some hyperplastic polyps and serrated adenomas.