A.F.P.M. de Goeij

Kirsten ras mutations in patients with colorectal cancer: The 'RASCAL II' study

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes’ C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes’ B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Surviving in a Marine Desert: The Sponge Loop Retains Resources Within Coral Reefs

Sponge Pump “Darwins Paradox” asks how productive and diverse ecosystems like coral reefs thrive in the marine equivalent of a desert. De Goeij et al. (p. 108) now show that coral reef sponges are part of a highly efficient recycling pathway for dissolved organic matter (DOM), converting it, via rapid sponge-cell turnover, into cellular detritus that becomes food for reef consumers. DOM transfer through the sponge loop approaches the gross primary production rates required for the entire coral reef ecosystem. Sponges take up dissolved organic matter and convert it into consumable cellular material. Ever since Darwin’s early descriptions of coral reefs, scientists have debated how one of the world’s most productive and diverse ecosystems can thrive in the marine equivalent of a desert. It is an enigma how the flux of dissolved organic matter (DOM), the largest resource produced on reefs, is transferred to higher trophic levels. Here we show that sponges make DOM available to fauna by rapidly expelling filter cells as detritus that is subsequently consumed by reef fauna. This “sponge loop” was confirmed in aquarium and in situ food web experiments, using 13C- and 15N-enriched DOM. The DOM-sponge-fauna pathway explains why biological hot spots such as coral reefs persist in oligotrophic seas—the reef’s paradox—and has implications for reef ecosystem functioning and conservation strategies.

Molecular genetic alterations in hamartomatous polyps and carcinomas of patients with Peutz-Jeghers syndrome

Aim—To investigate whether mutations in the STK11/LKB1 gene and genes implicated in the colorectal adenoma–carcinoma sequence are involved in Peutz-Jeghers syndrome (PJS) related tumorigenesis. Methods—Thirty nine polyps and five carcinomas from 17 patients (from 13 families) with PJS were analysed for loss of heterozygosity (LOH) at 19p13.3 (STK11/LKB1 gene locus), 5q21 (APC gene locus), 18q21–22 (Smad4 and Smad2 gene locus), and 17p13 (p53 gene locus), and evaluated for immunohistochemical staining of p53. In addition, mutational analysis of K-ras codon 12, APC, and p53 and immunohistochemistry for Smad4 expression were performed on all carcinomas. Results—LOH at 19p was seen in 15 of the 39 polyps and in all carcinomas (n = 5). Interestingly, six of the seven polyps from patients with cancer had LOH, compared with nine of the 31 polyps from the remaining patients (p = 0.01). In one polyp from a patient without a germline STK11/LKB1 mutation, no LOH at 19p or at three alternative PJS candidate loci (19q, 6p, and 6q) was found. No LOH at 5q was observed. However, mutational analysis revealed an APC mutation in four of the five carcinomas. LOH at 17p was not seen in polyps or carcinomas; immunohistochemistry showed expression of p53 in one carcinoma and focal expression in three polyps. At subsequent sequence analysis, no p53 mutation was found. One carcinoma had an activating K-ras codon 12 mutation and another carcinoma showed 18q LOH; however, no loss of Smad4 expression was seen. Conclusions—These results provide further evidence that STK11/LKB1 acts as a tumour suppressor gene, and may be involved in the early stages of PJS tumorigenesis. Further research is needed to see whether LOH in PJS polyps could be used as a biomarker to predict cancer. Differences in molecular genetic alterations noted between the adenoma–carcinoma sequence and PJS related tumours suggest the presence of a distinct pathway of carcinogenesis.

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)

Oestrogen-modulated gene expression in the human endometrium

Abstract.To identify key regulatory mechanisms in the growth and development of the human endometrium, microarray analysis was performed on uncultured human endometrium collected during menstruation (M) and the late-proliferative (LATE-P)-phase of the menstrual cycle, as well as after 24 h incubation in the presence of oestradiol (17β-E2). We demonstrate the expression of novel gene transcripts in the human endometrium. i.e. mucin-9, novel oestrogen-responsive gene transcripts, i.e. gelsolin and flotillin-1, and genes known to be expressed in human endometrium but not yet shown to be oestrogen responsive, i.e. connexin-37 and TFF1/pS2. Genes reported to be expressed during the implantation window and implicated in progesterone action, i.e. secretoglobin family 2A, member 2 (mammaglobin) and homeobox-containing proteins, were up-regulated in uncultured LATE-P-phase endometrium compared to M-phase endometrium. Some gene transcripts are regulated directly by 17β-E2 alone, others are influenced by the in vivo environment as well. These observations emphasise that the regulation of endometrium maturation by oestrogen entails more then just stimulation of cell proliferation.

Cell kinetics of the marine sponge Halisarca caerulea reveal rapid cell turnover and shedding.

SUMMARY This study reveals the peculiar in vivo cell kinetics and cell turnover of the marine sponge Halisarca caerulea under steady-state conditions. The tropical coral reef sponge shows an extremely high proliferation activity, a short cell cycle duration and massive cell shedding. Cell turnover is predominantly confined to a single cell population, i.e. the choanocytes, and in this process apoptosis only plays a minor role. To our knowledge, such fast cell kinetics under steady-state conditions, with high turnover by shedding in the absence of apoptosis, has not been observed previously in any other multicellular organism. The duration of the cell cycle in vivo resembles that of unicellular organisms in culture. Morphological and histochemical studies demonstrate compartmentalization of choanocytes in the sponge tissue, which corresponds well with its remarkable cellular kinetics. Coral reef cavity sponges, like H. caerulea, inhabit low nutrient tropical waters, forcing these organisms to filter large volumes of water and to capture the few nutrients efficiently. Under these oligotrophic conditions, a high cell turnover may be considered as a very useful strategy, preventing permanent damage to the sponge by environmental stress. Halisarca caerulea maintains its body mass and keeps its food uptake system up to date by constantly renewing its filter system. We conclude that studies on cell kinetics and functional morphology provide new and essential information on the growth characteristics and the regulation of sponge growth in vivo as well as in vitro and the role of choanocytes in tissue homeostasis.

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.

Meat consumption and K-ras mutations in sporadic colon and rectal cancer in The Netherlands Cohort Study

Case–cohort analyses were performed on meat and fish consumption in relation to K-ras mutations in 448 colon and 160 rectal cancers that occurred during 7.3 years of follow-up, excluding the first 2.3 years, and 2948 subcohort members of The Netherlands Cohort Study on diet and cancer. Adjusted incidence rate ratios and 95% confidence intervals were computed for colon and rectal cancer and for K-ras mutation status subgroups. Total fresh meat, most types of fresh meat and fish were not associated with colon or rectal cancer, neither overall nor with K-ras mutation status. However, several weak associations were observed for tumours with a wild-type K-ras, including beef and colon tumours, and an inverse association for pork with colon and rectal tumours; for meat products, an increased association was observed with wild-type K-ras tumours in the colon and possibly with G>A transitions in rectal tumours.

Quality control in immunocytochemistry: experiences with the oestrogen receptor assay.

AIMS: To evaluate the feasibility of an interlaboratory quality control programme in immunohistochemistry. METHODS: Several pathology laboratories were asked to carry out immunohistochemical oestrogen receptor staining on a set of freeze dried cryostat sections of breast cancer tissue. The sections and protocols for staining and semi-quantitative scoring were mailed to the participating laboratories in two trials. The oestrogen receptor content of the breast cancer samples was determined by radioligand binding assay on the tumour cytosol. RESULTS: In the first trial 11 laboratories (response rate 60%) participated. Eight (73%) of the participants scored within a 95% confidence interval and all but one correctly classified the tumour as receptor positive. In the second trial all 20 participating laboratories (response rate 55%) correctly scored one tumour sample as negative and 18 of them (90% of respondents) correctly classified the two other tumour samples as receptor positive. In a quantitative evaluation a histochemical score within 95% confidence interval limits was provided by eight (40%) and 12 (60%) of the participants. CONCLUSIONS: Semiquantitative scoring of immunocytochemical staining is valuable for performing correlative inter-laboratory studies, although this scoring protocol may not be required for diagnosis or prognosis. Significant inter-laboratory variability exists, leading to qualitatively correct receptor classification in 100% of receptor negative and 80% of receptor positive cases, and quantitative agreement in only about half of the cases. The perceived variability is not caused by systematic differences in the choice of the immunocytochemical technique, or the mailing of freeze dried sections. Quality control programmes should be included in the standard procedures of each diagnostic immunohistochemistry laboratory.

Cigarette smoking and KRAS oncogene mutations in sporadic colorectal cancer: results from the Netherlands Cohort Study

Since a KRAS oncogene mutation is an early event in colorectal cancer development and cigarette smoking is thought to have an effect on early stages of colorectal tumorigenesis, smoking, especially long-term smoking, may be associated with the risk for colorectal cancer with KRAS oncogene mutations. In the Netherlands Cohort Study on diet and cancer (n=120,852 men and women), using a case-cohort design, adjusted incidence rate ratios (RR) and 95% confidence intervals (CI) were computed for colorectal tumors with wild-type and with mutated KRAS gene, and with specific G:C-->T:A or G:C-->A:T point mutations in KRAS, according to cigarette smoking status, frequency, duration, pack years, age at first exposure, years since cessation, inhalation and filter usage. After 7.3 years and excluding the first 2.3 years, 648 cases and 4083 sub-cohort members were included in the analyses. Ex-smokers, but not current smokers, were at increased risk for colorectal cancer with wild-type KRAS gene tumors when compared with never smokers, albeit not statistically significant (RR 1.26, 95% CI 0.96-1.66). This was not observed for KRAS mutated tumors when comparing ex-smokers with never smokers (RR 1.15, 95% CI 0.79-1.66). The highest category of smoking frequency (>20 cigarettes/day) and inhalation of smoke were associated with an increased risk for colorectal cancer with wild-type KRAS gene tumors, though not statistically significant, when compared with never smoking (frequency: RR 1.24, 95% CI 0.90-1.71 and inhalation: RR 1.25, 95% CI 0.94-1.67). These associations were strongest in men (ex-smokers: RR 1.79, 95% CI 1.00-3.20; frequency: RR 1.91, 95% CI 1.03-3.52; inhalation: RR 1.69, 95% CI 0.94-3.04). No associations were observed between any of the smoking characteristics and the risk for colorectal cancer with mutated KRAS gene tumors, nor where there any clear associations with tumors with specific G:C-->A:T transitions or G:C-->T:A transversions. These results suggest that, in contrast to the hypothesis, smoking does not increase the risk for colorectal tumors with a mutated KRAS gene. Some smoking characteristics, i.e. being an ex-smoker, frequency and inhalation, may be associated with risk for colorectal cancer characterized by the wild-type KRAS gene, especially in men.