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Low mitochondrial DNA (mtDNA) copy number in tumors has been associated with worse prognosis in colorectal cancer (CRC). This study further deciphers the role of mtDNA copy number in CRC by comparing mtDNA copy number between healthy, adenoma and carcinoma tissue, by investigating its association according to several clinicopathological characteristics in CRC, and by relating it to CRC-specific survival in CRC patients. A hospital-based series of samples including cancer, adenoma and adjacent histologically normal tissue from primary CRC patients (n = 56) and recurrent CRC (n = 16) was studied as well as colon mucosa samples from healthy subjects (n = 76). Furthermore, mtDNA copy number was assessed in carcinomas of 693 CRC cases identified from the population-based Netherlands Cohort Study (NLCS). MtDNA copy number was significantly lower in carcinoma tissue (P = 0.011) and adjacent tissue (P < 0.001) compared to earlier resected adenoma tissue and in primary CRC tissue compared to recurrent CRC tissue (P = 0.011). Within both study populations, mtDNA copy number was significantly lower in mutated BRAF (P = 0.027 and P = 0.006) and in microsatellite unstable (MSI) tumors (P = 0.033 and P < 0.001) and higher in KRAS mutated tumors (P = 0.004). Furthermore, the association between mtDNA and survival seemed to follow an inverse U-shape with the highest HR observed in the second quintile of mtDNA copy number (HR = 1.70, 95% CI = 1.18, 2.44) compared to the first quintile. These results might reflect an association of mtDNA copy number with various malignant processes in cancer cells and warrants further research on tumor energy metabolism in CRC prognosis., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Insulin-like growth factors (IGFs) have been associated with growth, body size, physical activity and colorectal cancer (CRC). We hypothesized that variants in IGF-related genes increase the CRC susceptibility associated with a larger body size and a lack of physical activity. We assessed this in The Netherlands Cohort Study. Participants (n = 120852) completed a baseline questionnaire on diet and cancer. ~75% returned toenail clippings. Using a case-cohort approach and 16.3 years of follow-up, toenail DNA from 3768 subcohort members and 2580 CRC cases was genotyped. We aggregated unfavorable alleles (potentially increasing CRC risk) for 18 single nucleotide polymorphisms in 8 genes into a sum score. The sum score (in tertiles) and an IGF1 19-CA repeat polymorphism (19/19, 19/non-19 and non-19/non-19 repeats) in combination with body size (mostly in tertiles) and (non-)occupational physical activity (>12, 8-12 and <8 kJ/min in the job and >90, >60-90, >30-60 and ≤30 min/day) were analyzed by Cox regression. Increasingly higher hazard ratios (HRs) for CRC were observed for a larger adult body mass index, larger trouser size and tallness in the presence of more unfavorable alleles in men. HRs (95% confidence intervals) for joint effects were 1.55 (1.06-2.25), 1.78 (1.29-2.46) and 1.48 (1.01-2.17), respectively. In women, variant repeat alleles halved CRC risk irrespective of body size and physical activity. Almost no interactions tested significant. To conclude, a larger body size was a CRC risk factor in men in the presence of an accumulation of unfavorable alleles in IGF-related genes, but interactions were generally nonsignificant., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Acrylamide, a probable human carcinogen, is present in heat-treated carbohydrate-rich foods. Epidemiological studies have not shown a clear association between acrylamide intake and colorectal cancer (CRC) risk. This may be due to the molecular heterogeneity in colorectal tumors, which was not taken into consideration before. Since the acrylamide metabolite glycidamide induces specific DNA mutations in rodents, we investigated whether acrylamide is associated with CRC risk characterized by mutations in Kirsten-ras (KRAS) and adenomatous polyposis coli (APC); key genes in colorectal carcinogenesis. This case-cohort analysis, within the Netherlands Cohort Study on diet and cancer, was based on 7.3 years of follow-up. Acrylamide intake was assessed with a food frequency questionnaire. Mutation analysis of codons 1286-1520 in exon 15 in APC and codons 12 and 13 in exon 1 in KRAS was performed on tumor tissue of 733 cases. Hazard ratios (HR) were calculated using Cox proportional hazards analysis. Among men, acrylamide intake was statistically significantly associated with an increased risk of particularly tumors with an activating KRAS mutation {HR fourth versus first quartile: 2.12 [95% confidence interval (CI): 1.16-3.87], P trend: 0.01}. Among women, acrylamide intake was statistically significantly associated with a decreased risk of particularly tumors with a truncating APC mutation (fourth versus first quartile: 0.47 (95% CI: 0.23-0.94), P trend: 0.02), but only in the highest quartile of intake. This is the first study to show that acrylamide might be associated with CRC with specific somatic mutations, differentially in men and women. More research is needed to corroborate or refute these findings.

Red meat intake has been linked to increased colorectal cancer (CRC) risk. Although the underlying mechanisms remain unclear, experimental studies suggest a role for dietary heme iron. Because heme iron was shown to promote specific mutations, it would be insightful to link heme iron data to CRC with mutations in key genes in an observational, population-based study. We investigated the association between dietary heme iron intake and risk of CRC with mutations in APC (adenomatous polyposis coli) and KRAS (Kirsten ras) and P53 overexpression in the Netherlands Cohort Study. After 7.3 years of follow-up, excluding the first 2.3 years due to incomplete coverage of the pathology registry and to avoid preclinical disease, adjusted hazard ratios (including adjustment for total meat) and 95% confidence intervals were calculated, using 4026 subcohort members (aged 55-69 years at baseline), 435 colon and 140 rectal cancer patients. When comparing the highest with the lowest tertile of intake, heme iron intake was associated with an increased risk of CRC harboring activating mutations in KRAS (hazard ratio = 1.71, 95% confidence interval: 1.15-2.57; P for trend = 0.03) and CRC without truncating mutations in APC (hazard ratio = 1.79, 95% confidence interval: 1.23-2.60; P for trend = 0.003). We observed a positive association between heme iron intake and the risk of CRC with activating G>A mutations in KRAS (P for trend = 0.01) and overall G>A mutations in APC (P for trend = 0.005). No associations were found with CRC harboring G>T mutations in KRAS/APC. Heme iron intake was positively associated with the risk of P53 overexpressed tumors but not with tumors without P53 overexpression (Pheterogeneity = 0.12). Heme iron intake was associated with an increased risk of colorectal tumors harboring G>A transitions in KRAS and APC and overexpression of P53. These novel findings suggest that alkylating rather than oxidative DNA-damaging mechanisms are involved in heme-induced colorectal carcinogenesis.

Pulmonary carcinoids comprise a well-differentiated subset of neuroendocrine tumors usually associated with a favorable prognosis, but mechanisms underlying disease progression are poorly understood. In an explorative approach to identify pathways associated with progression, we compared gene expression profiles of tumors from five patients with a favorable and five with a poor disease outcome. Differentially expressed genes were validated using quantitative real-time PCR on 65 carcinoid tumors, in combination with survival analysis. One of the identified pathways was further examined using immunohistochemistry. As compared with other chromosomal locations, a significantly higher number of genes downregulated in carcinoids with a poor prognosis were located at chromosome 11q (P = 0.00017), a region known to be frequently lost in carcinoids. In addition, a number of upregulated genes were found involved in the mitotic spindle checkpoint, the chromosomal passenger complex (CPC), mitotic kinase CDC2 activity and the BRCA-Fanconi anemia pathway. At the individual gene level, BIRC5 (survivin), BUB1, CD44, IL20RA, KLK12 and OTP were independent predictors of patient outcome. For survivin, the number of positive nuclei was also related to poor prognosis within the group of carcinoids. Aurora B kinase and survivin, major components of the CPC, were particularly upregulated in high-grade carcinomas and may therefore comprise therapeutic targets for these tumors. To our knowledge, this is the first expression profiling study focusing specifically on pulmonary carcinoids and progression. We have identified novel pathways underlying malignant progression and validated several genes as being strong prognostic indicators, some of which could serve as putative therapeutic targets.

Chromosomal loss of 18q21 is a frequent event in colorectal cancer (CRC) development, suggesting that this region harbors tumor suppressor genes (TSGs). Several candidate TSGs, among which methyl-CpG-binding domain protein 1 (MBD1), CpG-binding protein CXXC1, Sma- and Mad-related protein 4 (SMAD4), deleted in colon cancer (DCC) and methyl-CpG-binding domain protein 2 (MBD2) are closely linked on a 4-Mb DNA region on chromosome18q21. As TSGs can be epigenetically silenced, this study investigates whether MBD1, CXXC1, SMAD4, DCC and MBD2 are subject to epigenetic silencing in CRC. Methylation-specific polymerase chain reaction and sodium bisulfite sequencing of these genes show that DCC, but not MBD1, CXXC1, SMAD4 and MBD2, has promoter CpG island methylation in CRC cell lines and tissues {normal mucosa [29.5% (18/61)], adenomas [81.0% (47/58)] and carcinomas [82.7% (62/75)] (P = 8.6 x 10(-9))} that is associated with reduced DCC expression, independent of 18q21 loss analyzed by multiplex ligation-dependent probe amplification. Reduced gene expression of CXXC1, SMAD4 and MBD2 correlates with 18q21 loss in CRC cell lines (P = 0.04, 0.02 and 0.02, respectively). Treatment with the demethylating agent 5-aza-2'-deoxycytidine, but not with the histone deacetylase inhibitor trichostatin A exclusively restored DCC expression in CRC cell lines. Chromatin immunoprecipitation studies reveal that the DCC promoter is marked with repressive histone-tail marks H3K9me3 and H3K27me3, whereas activity related H3K4me3 was absent. Only active epigenetic marks were detected for MBD1, CXXC1, SMAD4 and MBD2. This study demonstrates specific epigenetic silencing of DCC in CRC as a focal process not affecting neighboring genes on chromosomal region 18q21.

Intake of dietary factors that serve as methyl group donors may influence promoter hypermethylation in colorectal carcinogenesis. We investigated whether dietary folate, vitamin B2 and vitamin B6, methionine and alcohol were associated with mutL homologue 1 (MLH1) hypermethylation and the related molecular phenotypes of MLH1 protein expression, microsatellite instability (MSI) and BRAF mutations in patients with colorectal carcinomas. Within the Netherlands Cohort Study on diet and cancer (n = 120 852), 648 cases (367 men and 281 women) and 4059 subcohort members were available for data analyses from a follow-up period between 2.3 and 7.3 years after baseline. Gender-specific adjusted incidence rate ratios (RRs) were calculated over categories of dietary intake in case-cohort analyses. The intakes of folate, vitamin B2, methionine and alcohol were not associated with risk of tumors showing MLH1 hypermethylation, those lacking MLH1 protein expression or with MSI. Among men, we observed strong positive associations between folate and BRAF-mutated tumors (RR = 3.04 for the highest versus lowest tertile of intake, P(trend) = 0.03) and between vitamin B6 and tumors showing MLH1 hypermethylation (highest versus lowest tertile: RR = 3.23, P(trend) = 0.03). Among women, the relative risks of tumors with BRAF mutations or MLH1 hypermethylation were also increased in the highest tertiles of folate and vitamin B6 intake, respectively, but these did not reach statistical significance. The positive associations between folate intake and tumors harboring BRAF mutations and between vitamin B6 intake and those showing MLH1 hypermethylation were most pronounced among men and may suggest that these vitamins enhance colorectal cancer risk through genetic as well as epigenetic aberrations.

Colorectal cancer (CRC) is a complex and heterogeneous disease in which genomic instability and DNA promoter methylation play important roles. The aim of this study was to investigate the relationship between chromosomal instability (CIN), microsatellite instability (MSI) and promoter methylation of CRC-associated genes. Therefore, 71 CRCs were analysed for CIN and MSI by comparative genomic hybridization and the mononucleotide marker BAT-26, respectively. Promoter methylation of the tumour suppressor and DNA repair genes hMLH1, O(6)-MGMT, APC, p14(ARF), p16(INK4A), RASSF1A, GATA-4, GATA-5 and CHFR was analysed using methylation-specific polymerase chain reaction. These integrative analyses showed that in CIN+ CRCs, promoter methylation of GATA-4 and p16(INK4A) was inversely related to chromosomal loss at 15q11-q21 and gain at 20q13, respectively (P values: 3.8 x 10(-2) and 4.5 x 10(-2), respectively). Interestingly, promoter methylation of RASSF1A, GATA-4, GATA-5 and CHFR, as well as a high methylation index (MI), was positively related to chromosomal gain at 8q23-qter (P values: 1.5 x 10(-2), 3.8 x 10(-2), 3.9 x 10(-2), 4.9 x 10(-2) and 8.2 x 10(-3), respectively). MSI was associated with BRAF mutation, promoter methylation of hMLH1, APC and p16(INK4A) and a high MI (total number of methylated genes) (P values: 2.4 x 10(-2), 2.5 x 10(-3), 1.8 x 10(-2), 4.6 x 10(-2) and 1.0 x 10(-2), respectively). Therefore, we conclude that promoter methylation of pivotal tumour suppressor and DNA repair genes is associated with specific patterns of chromosomal changes in CRC, which are different from methylation patterns in MSI tumours.

A subset of sporadic colon cancers has been shown to have microsatellite instability caused by an epigenetic inactivation of the MLH1 gene by hypermethylation of the the CpG island in its promoter region. We report here that in colorectal cancer, inactivation of the MLH1 gene is frequently accompanied by hypermethylation of the CpG island in the promoter of the mitotic gene checkpoint with forkhead and ring finger domains (CHFR). This was first observed in the colon cancer cell lines HCT-116, DLD-1, RKO and HT29. Among the 61 primary colon cancer samples studied, hypermethylation of the MLH1 and the CHFR promoter was found in 31% of the tumors. In 68% of all primary cancers (13/19) with MLH1 promoter hypermethylation, hypermethylation of the CHFR promoter was observed as well (P-value < 0.0001, Fisher's two-sided exact). Hypermethylation of the HLTF, MGMT, RASSF1, APC, p14 and p16 promoter regions were also frequent events, being observed in 48% (28/58), 40% (26/64), 21% (14/64), 50% (31/62), 43% (26/60) and 56% (35/63), respectively. However, methylation of these genes was not associated with methylation of either MLH1 or CHFR. The observed methylation profile was unrelated to Duke's stage. The coordinated loss of both mismatch repair caused by methylation of MLH1 and loss of checkpoint control associated with methylation of CHFR suggests the potential to overcome cell cycle checkpoints, which may lead to an accumulation of mutations.

The adenomatous polyposis coli (APC) gene is considered to be a gatekeeper in colorectal tumourigenesis. Inactivating mutations in APC have been reported in 34-70% of sporadic colorectal cancer patients, the majority of which occur in the mutation cluster region (MCR). In this study, tumour tissue from 665 incident colorectal cancer patients, who originate from 120 852 men and women (55-69 years of age at baseline) participating in The Netherlands Cohort Study, was evaluated for the occurrence and type of APC mutations with regard to age at diagnosis, gender, family history of colorectal cancer, Dukes' stage, tumour differentiation and sub-localization. Mutation analysis of the MCR, which spans codons 1286-1513, was performed on archival adenocarcinoma samples using macrodissection, nested PCR and direct sequencing of purified PCR fragments. A large number of genetic aberrations (n = 978), including point mutations (n = 833), deletions (n = 126) and insertions (n = 19) was detected in the MCR in 72% of patients (479/665). In particular, we observed a large number of missense mutations, more than reported previously. This may indicate involvement in colorectal carcinogenesis, although their significance for APC functions is unclear. Truncating mutations were found in 37% of patients (248/665). Patients with rectosigmoid and rectum tumours relatively more frequently harboured C > T nonsense mutations and truncating frameshift mutations as compared with patients with proximal and distal colon tumours (P = 0.009 and P = 0.045, respectively). Differences in occurrence of truncating mutations with regard to tumour sub-localization suggest a different aetiology of tumourigenesis in colon and rectum.