M. van Engeland

Dietary Folate, Methionine, Riboflavin, and Vitamin B-6 and Risk of Sporadic Colorectal Cancer

Adequate intake of folate, methionine, riboflavin, and vitamin B-6 may prevent aberrant DNA methylation and thereby protect against colorectal cancer (CRC). However, previous epidemiological studies investigating associations between dietary intakes of these nutrients and CRC have been inconsistent. We investigated the associations between intakes of folate, methionine, riboflavin, and vitamin B-6 and CRC risk, accounting for the sublocalization of the tumor. Within the Netherlands Cohort Study on diet and cancer (n = 120,852), 2349 cases and 4168 subcohort members were available for data analyses from a follow-up period of 13.3 y after baseline. Gender-specific adjusted incidence rate ratios (RR) were calculated over quintiles of dietary intake in case-cohort analyses. Folate intake was not associated with CRC risk in either men or women. However, methionine was associated with decreased risk of proximal colon cancer among men (RR = 0.57 for highest vs. lowest quintile of intake; P-trend = 0.03) and rectal cancer among women (highest vs. lowest quintile; RR = 0.45; P-trend = 0.05). Riboflavin tended to be associated with decreased proximal colon cancer risk among women (RR = 0.61; P-trend = 0.07). Conversely, there was a strong positive association between vitamin B-6 and rectal cancer among women (RR = 3.57; P-trend = 0.01). Our findings suggest that relatively high methionine intake may protect against proximal colon cancer in men and rectal cancer in women but that folate may not have a protective effect. This is the 2nd prospective cohort study in which vitamin B-6 intake was associated with increased risk of rectal tumors in women, which might suggest that this vitamin enhances rectal cancer in women.

Prognostic value of CpG island methylator phenotype among colorectal cancer patients: a systematic review and meta-analysis

BACKGROUND Divergent findings regarding the prognostic value of CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) patients exist in current literature. We aim to review data from published studies in order to examine the association between CIMP and CRC prognosis. MATERIALS AND METHODS A comprehensive search for studies reporting disease-free survival (DFS), overall survival (OS), or cancer-specific mortality of CRC patients stratified by CIMP is carried out. Study findings are summarized descriptively and quantitatively, using adjusted hazard ratios (HRs) as summary statistics. RESULTS Thirty-three studies reporting survival in 10 635 patients are included for review. Nineteen studies provide data suitable for meta-analysis. The definition of CIMP regarding gene panel, marker threshold, and laboratory method varies across studies. Pooled analysis shows that CIMP is significantly associated with shorter DFS (pooled HR estimate 1.45; 95% confidence interval (CI) 1.07-1.97, Q = 3.95, I(2) = 0%) and OS (pooled HR estimate 1.43; 95% CI 1.18-1.73, Q = 4.03, I(2) = 0%) among CRC patients irrespective of microsatellite instability (MSI) status. Subgroup analysis of microsatellite stable (MSS) CRC patients also shows significant association between shorter OS (pooled HR estimate 1.37; 95% CI 1.12-1.68, Q = 4.45, I(2) = 33%) and CIMP. Seven studies have explored CIMPs value as a predictive factor on stage II and III CRC patients DFS after receiving adjuvant 5-fluorouracil (5-FU) therapy: of these, four studies showed that adjuvant chemotherapy conferred a DFS benefit among CIMP(+) patients, one concluded to the contrary, and two found no significant correlation. Insufficient data was present for statistical synthesis of CIMPs predictive value among CRC patients receiving adjuvant 5-FU therapy. CONCLUSION CIMP is independently associated with significantly worse prognosis in CRC patients. However, CIMPs value as a predictive factor in assessing whether adjuvant 5-FU therapy will confer additional survival benefit to CRC patients remained to be determined through future prospective randomized studies.

Genetic Variants of Methyl Metabolizing Enzymes and Epigenetic Regulators: Associations with Promoter CpG Island Hypermethylation in Colorectal Cancer

Aberrant DNA methylation affects carcinogenesis of colorectal cancer. Folate metabolizing enzymes may influence the bioavailability of methyl groups, whereas DNA and histone methyltransferases are involved in epigenetic regulation of gene expression. We studied associations of genetic variants of folate metabolizing enzymes (MTHFR, MTR, and MTRR), DNA methyltransferase DNMT3b, and histone methyltransferases (EHMT1, EHMT2, and PRDM2), with colorectal cancers, with or without the CpG island methylator phenotype (CIMP), MLH1 hypermethylation, or microsatellite instability. Incidence rate ratios were calculated in case-cohort analyses, with common homozygotes as reference, among 659 cases and 1,736 subcohort members of the Netherlands Cohort Study on diet and cancer (n = 120,852). Men with the MTHFR 677TT genotype were at decreased colorectal cancer risk (incidence rate ratio, 0.49; P = 0.01), but the T allele was associated with increased risk in women (incidence rate ratio, 1.39; P = 0.02). The MTR 2756GG genotype was associated with increased colorectal cancer risk (incidence rate ratio, 1.58; P = 0.04), and inverse associations were observed among women carrying DNMT3b C→T (rs406193; incidence rate ratio, 0.72; P = 0.04) or EHMT2 G→A (rs535586; incidence rate ratio, 0.76; P = 0.05) polymorphisms. Although significantly correlated (P < 0.001), only 41.5% and 33.3% of CIMP tumors harbored MLH1 hypermethylation or microsatellite instability, respectively. We observed inverse associations between MTR A2756G and CIMP among men (incidence rate ratio, 0.58; P = 0.04), and between MTRR A66G and MLH1 hypermethylation among women (incidence rate ratio, 0.55; P = 0.02). In conclusion, MTHFR, MTR, DNMT3b, and EHMT2 polymorphisms are associated with colorectal cancer, and rare variants of MTR and MTRR may reduce promoter hypermethylation. The incomplete overlap between CIMP, MLH1 hypermethylation, and microsatellite instability indicates that these related “methylation phenotypes” may not be similar and should be investigated separately. (Cancer Epidemiol Biomarkers Prev 2009;18(11):3086–96)

A novel classification of colorectal tumors based on microsatellite instability, the CpG island methylator phenotype and chromosomal instability: implications for prognosis

BACKGROUND We studied the overlap between the major (epi)genomic events microsatellite instability (MSI), the CpG island methylator phenotype (CIMP) and chromosomal instability (CIN) in colorectal cancer (CRC), and whether specific (epi)genotypes were associated with CRC-related deaths. PATIENTS AND METHODS Molecular analyses using tumor DNA were successful in 509 CRC cases identified within the Netherlands Cohort Study in the period 1989-1993. Follow-up for the vital status until May 2005 was 100%. RESULTS MSI (12.6%), CIMP-only (5.3%), CIMP + CIN (13.4%), CIN-only (58.2%) and triple-negative tumors (10.6%) differed significantly regarding tumor localization, differentiation grade, initial adjuvant therapy (AT) use and genetic characteristics (P ≤ 0.03). CIMP-only, CIMP + CIN and triple-negative tumors, compared with CIN-only tumors, were significantly associated with a 3.67, 2.44 and 3.78-fold risk of CRC-related deaths after 2-year follow-up (95% confidence intervals, CIs, 1.70-7.91, 1.35-4.41 and 1.97-7.25, respectively), but not after late follow-up. MSI tumors were borderline significantly associated with a 0.40-fold risk of CRC-related deaths after late follow-up (95% CI 0.15-1.03). CONCLUSION(S) This is the first study to show that specific (epi)genotypes may hold a differential prognostic value that may vary over time. Although no specific treatment data were available, an explanation for the differential findings over time might be that (epi)genotypes modify therapy response.

MGMT and MLH1 promoter methylation versus APC, KRAS and BRAF gene mutations in colorectal cancer: indications for distinct pathways and sequence of events

BACKGROUND To study how caretaker gene silencing relates to gatekeeper mutations in colorectal cancer (CRC), we investigated whether O6-methylguanine DNA methyltransferase (MGMT) and Human Mut-L Homologue 1 (MLH1) promoter hypermethylation are associated with APC, KRAS and BRAF mutations among 734 CRC patients. METHODS We compared MGMT hypermethylation with G:C > A:T mutations in APC and KRAS and with the occurrence of such mutations in CpG or non-CpG dinucleotides in APC. We also compared MLH1 hypermethylation with truncating APC mutations and activating KRAS and BRAF mutations. RESULTS Only 10% of the tumors showed both MGMT and MLH1 hypermethylation. MGMT hypermethylation occurred more frequently in tumors with G:C > A:T KRAS mutations (55%) compared with those without these mutations (38%, P < 0.001). No such difference was observed for G:C > A:T mutations in APC, regardless of whether mutations occurred in CpG or non-CpG dinucleotides. MLH1 hypermethylation was less common in tumors with APC mutations (P = 0.006) or KRAS mutations (P = 0.001), but was positively associated with BRAF mutations (P < 0.001). CONCLUSIONS MGMT hypermethylation is associated with G:C > A:T mutations in KRAS, but not in APC, suggesting that MGMT hypermethylation may succeed APC mutations but precedes KRAS mutations in colorectal carcinogenesis. MLH1-hypermethylated tumors harbor fewer APC and KRAS mutations and more BRAF mutations, suggesting that they develop distinctly from an MGMT methylator pathway.

Dietary Folate Intake in Combination with MTHFR C677T Genotype and Promoter Methylation of Tumor Suppressor and DNA Repair Genes in Sporadic Colorectal Adenomas

Methylation of the promoter region of tumor suppressor genes is increasingly recognized to play a role in cancer development through silencing of gene transcription. We examined the associations between dietary folate intake, MTHFR C677T genotype, and promoter methylation of six tumor suppressor and DNA repair genes. Patients with colorectal adenoma (n = 149) and controls (n = 286) with folate intake in the upper or lower tertile with the CC or TT genotype were selected from a case-control study. Methylation-specific PCRs were conducted on colorectal adenoma specimens. The percentages of promoter methylation ranged from 15.7% to 64.2%. In case-case comparisons, folate was inversely associated with promoter methylation, especially among TT homozygotes. Case-control comparisons suggested that folate was not associated with the occurrence of adenomas with promoter methylation, and increased the risk of unmethylated adenomas, especially in TT homozygotes. The interactions between folate and MTHFR genotype were most pronounced for O6-MGMT: compared with CC homozygotes with low folate intake, the adjusted odds ratios (95% confidence interval) of having a methylated O6-MGMT promoter were 3.39 (0.82-13.93) for TT homozygotes with low folate intake and 0.37 (0.11-1.29) for TT homozygotes with high folate intake (P interaction = 0.02); the odds ratios for the occurrence of adenomas without methylation were 0.57 (0.16-2.11) for TT homozygotes with low folate intake and 3.37 (1.17-9.68) for TT homozygotes with high folate intake (P interaction = 0.03). In conclusion, folate intake seems to be inversely associated with promoter methylation in colorectal adenomas in case-case comparisons, and was positively associated with the occurrence of adenomas without promoter methylation in case-control comparisons, especially for TT homozygotes. (Cancer Epidemiol Biomarkers Prev 2007;16(2):327–33)

Clonal analysis favours a monoclonal origin for serous borderline tumours with peritoneal implants

Serous borderline tumours (SBTs) of the ovary were originally classified as such because the vast majority behave in a remarkably indolent manner, even in the presence of widespread tumour deposits, termed implants, and/or lymph node involvement. The pathogenesis of the implants is currently unknown. Two major hypotheses have been proposed: the first favours a monoclonal origin, arguing that the peritoneal lesions derive from neoplastic cells that are shed from the primary ovarian tumour. The second hypothesis favours a polyclonal origin as a result of a field defect of susceptible Müllerian cells from which multiple independent tumours arise. To test both hypotheses, genome‐wide allelotyping and B‐RAF/K‐RAS mutation analyses were employed to assess clonality in 25 metachronous or synchronous tumours from ten SBT patients. Loss of heterozygosity (LOH) profiling and K‐RAS/B‐RAF mutation analysis showed concordance of the genetic changes in all sites in 21 tumours from eight patients who were informative. These results favour a common origin, underscored by a likelihood ratio (probability of common origin/probability of independent origin) ranging from 2.43 to 7 662 850. In conclusion, this study strongly supports the hypothesis that both non‐invasive and invasive implants arise as a consequence of spread from a single ovarian site. Copyright

Aberrant methylation of the Adenomatous Polyposis Coli (APC) gene promoter is associated with the inflammatory breast cancer phenotype.

Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter occurs in about 40% of breast tumours and has been correlated with reduced APC protein levels. To what extent epigenetic alterations of the APC gene may differ according to specific breast cancer phenotypes, remains to be elucidated. Our aim was to explore the role of APC methylation in the inflammatory breast cancer (IBC) phenotype. The status of APC gene promoter hypermethylation was investigated in DNA from normal breast tissues, IBC and non-IBC by both conventional and real-time quantitative methylation-specific PCR (MSP). APC methylation levels were compared with APC mRNA and protein levels. Hypermethylation of the APC gene promoter was present in 71% of IBC samples (n=21) and 43% of non-IBC samples (n=30) by conventional MSP (P=0.047). The APC gene also showed an increased frequency of high methylation levels in IBC (in 74% of cases, n=19) vs non-IBC (in 46% of cases, n=35) using a qMSP assay (P=0.048). We observed no significant association between APC methylation levels by qMSP and APC mRNA or protein expression levels. In conclusion, for the first time, we report the association of aberrant methylation of the APC gene promoter with the IBC phenotype, which might be of biological and clinical importance.

Resistance to sunitinib in renal cell carcinoma: From molecular mechanisms to predictive markers and future perspectives

The introduction of agents that inhibit tumor angiogenesis by targeting vascular endothelial growth factor (VEGF) signaling has made a significant impact on the survival of patients with metastasized renal cell carcinoma (RCC). Sunitinib, a tyrosine kinase inhibitor of the VEGF receptor, has become the mainstay of treatment for these patients. Although treatment with sunitinib substantially improved patient outcome, the initial success is overshadowed by the occurrence of resistance. The mechanisms of resistance are poorly understood. Insight into the molecular mechanisms of resistance will help to better understand the biology of RCC and can ultimately aid the development of more effective therapies for patients with this infaust disease. In this review we comprehensively discuss molecular mechanisms of resistance to sunitinib and the involved biological processes, summarize potential biomarkers that predict response and resistance to treatment with sunitinib, and elaborate on future perspectives in the treatment of metastasized RCC.

E2Fs mediate a fundamental cell-cycle deregulation in high-grade serous ovarian carcinomas

Several studies described a role for the E2F/Rb pathway in ovarian serous carcinomas (SCAs). Since E2F/Rb pathway deregulation is a general hallmark of human cancer, it remains unclear whether this deregulation is of particular importance in SCAs or whether it reflects a common oncological feature. Here, we have clarified this issue by the examination of microarray expression profiles of SCAs and particularly by the comparison with another, less malignant, ovarian cancer type, serous borderline tumours (SBTs). Results were validated by quantitative RT‐PCR, both on the microarray samples and on an independent panel, and TP53 mutation analysis was performed. This integrated analysis revealed a significant increase in the expression of the transcription factors E2F1 and E2F3 in SCAs, when compared to SBTs. This was associated with vast overexpression of E2F target genes in SCAs compared to SBTs. High‐grade SCAs in particular exhibited a major deregulated E2F target expression pattern. Generally, overexpression of E2F targets in SCAs appeared to be well structured since those targets considered negative regulators of the cell cycle or promoters of apoptosis were usually not overexpressed in SCAs. Similar to E2F target deregulation, TP53 mutations were identified in SCA3s, to a lesser extent in SCA1s, and not in SBTs. These results suggest that a structured, generally up‐regulated E2F transcription factor activity is associated with a global cell‐cycle disturbance in high‐grade SCAs and exceeds typical E2F/Rb pathway disruption in tumours, at least compared with SBTs. Copyright