Colorectal cancer-associated SNP rs17042479 is involved in the regulation of NAF1 promoter activity.

A novel risk locus at 4q32.2, located between the Nuclear Assembly Factor 1 (NAF1) and Follistatin Like 5 (FSTL5) genes, was associated with increased risk of developing colorectal cancer (CRC), with SNP rs17042479 being the most associated. However, the link between CRC development and the risk locus at 4q32.2 is unknown. We investigated the promoter activity of NAF1 and FSTL5 and analyzed the risk locus at 4q32.2 as gene regulatory region. Our results showed that the activity of the FSTL5 promoter was low compared to the NAF1 promoter. Analyses of the NAF1 promoter in conjunction with the region containing the risk locus at 4q32.2 showed that the region functions as gene regulatory region with repressor activity on NAF1 promoter activity. The SNP rs17042479(G) increased the repressor effect of the region. CRC patients' biopsies were genotyped for SNP rs17042479(A/G), and NAF1 expression profiles were examined. We found an association between SNP rs17042479(G), cancer stage and tumor location. Additionally, patients with SNP rs17042479(G) showed lower NAF1 expression in comparison to patients with SNP rs17042479(A) in tumor tissue and the NAF1 expression in tumor tissue was lower compared to healthy tissue. The results in the study imply that reduced NAF1 expression in the tumor contribute to a more aggressive phenotype. Furthermore, this study suggests that the SNP rs17042479(G) change the expression of NAF1 and thereby increases the risk of developing CRC.

Perioperative changes in cell-free DNA for patients undergoing surgery for colon cancer.

BACKGROUND: Various conditions with cellular decay are associated with elevated cell-free DNA (cfDNA). This study aimed to investigate if perioperatively measured cfDNA levels were associated with the surgical approach, complications, or recurrence. METHODS: Plasma was obtained from patients who underwent surgery for colon cancer at admission and at the time of discharge. Quantitative measurement of cfDNA was performed by amplifying two amplicons of 102 base pairs (bp) and 132 bp of Beta-2-Microglobulin (B2M) and Peptidyl-Prolyl cis-trans Isomerase A (PPIA), respectively. RESULTS: cfDNA was measured in 48 patients who underwent surgery for colonic cancer. Sixteen patients had recurrence during the follow-up period, fifteen developed a postoperative complication, and seventeen patients developed neither, acting as the control group. Postoperative cfDNA levels were significantly elevated from baseline samples, across all groups, with a median preoperatively B2M level of 48.3 alleles per mL and postoperatively of 220 alleles per mL and a median preoperatively level PPIA of 26.9 alleles per mL and postoperatively of 111.6 alleles per mL (p < 0.001 for B2M and p < 0.001 for PPIA). Postoperative levels of PPIA, but not B2M, were significantly higher in patients experiencing complications than in the control group (p = 0.036). However, a tendency towards an association between the surgical approach and the changes in cfDNA levels was found for PPIA (p = 0.058), and B2M (p = 0.087). CONCLUSIONS: Plasma cfDNA was increased after surgery in all patients with colon cancer. Postoperative PPIA levels were significantly higher in patients experiencing surgical complications but not in B2M levels.

Impact of in Vivo Ischemic Time on RNA Quality--Experiences from a Colon Cancer Biobank.

Considerable effort has been made to improve differentiated diagnostics as well as personalized treatment for colorectal cancer patients. High-quality fresh frozen tissue is often required to investigate relevant molecular signatures in these patients. In RNA expression studies, the "RNA integrity number" is widely accepted as a reliable marker of RNA quality. Here, we investigate the feasibility of obtaining high-quality tissue from a colon cancer biobank and the impact of in vivo ischemic time and various technical and clinicopathological factors on RNA quality. Biopsies were obtained immediately following the tumor removal. The time from clamping the main arterial supply to resection and removal of the tumor was used to estimate the in vivo ischemic time. We did not observe a significant difference in RNA quality between normal tissue and tumor tissue. We observed a significant correlation between in vivo ischemic time and RNA quality in normal tissue (r = -0.24, p<0.001) but not in tumor tissue. Male gender and laparoscopic procedure were also significantly associated with lower RNA quality in normal tissue only. In tumor tissue, poor differentiation was associated with low RNA quality. In conclusion, in vivo ischemic time, surgical procedure, and gender have minor but significant effects on the quality of RNA from normal colon tissue but not tumor tissue. Poorly differentiated tumors are associated with lower RNA quality. Although its impact is low, it can still be considered to note in vivo ischemic time in colon cancer specimen procurement.

High interleukin-6 mRNA expression is a predictor of relapse in colon cancer.

AIM: To investigate the expression of interleukin-6 (IL6) in colon cancer tissue, and to examine if the risk of relapse is influenced by IL6 expression. MATERIALS AND METHODS: Fresh-frozen biopsies from tumor and normal adjacent tissues were taken from patients with colon cancer during surgery and stored at -80 °C. mRNA expression for interleukin-6 was evaluated with reverse transcription real time quantitative polymerase chain reaction. Survival analyses were carried-out using a Cox competing risk regression model. RESULTS: IL6 mRNA was significantly more highly expressed in tumor tissue compared to normal adjacent tissue (p<0.001). We found no significant association with regard to IL6 expression and histological differentiation or cancer stage. We found a significant association between high IL6 expression and risk of relapse (Hazard Ratio=2.23, 95% CI=1.10-4.53; p<0.05), also when adjusted for clinicopathological characteristics (Hazard Ratio=2.16, 95% CI=1.07-4.40; p<0.05). CONCLUSION: Interleukin-6 is up-regulated in colon cancer tissue at the transcriptional level and is significantly associated with increased risk of relapse.

Gene expression profiles in stages II and III colon cancers: application of a 128-gene signature.

PURPOSE: A 128-gene signature has been proposed to predict outcome in patients with stages II and III colorectal cancers. In the present study, we aimed to reproduce and validate the 128-gene signature in external and independent material. METHODS: Gene expression data from the original material were retrieved from the Gene Expression Omnibus (GEO) (n = 111) in addition to a Danish data set (n = 37). All patients had stages II and III colon cancers. A Prediction Analysis of Microarray classifier, based on the 128-gene signature and the original training set of stage I (n = 65) and stage IV (n = 76) colon cancers, was reproduced. The stages II and III colon cancers were subsequently classified as either stage I-like (good prognosis) or stage IV-like (poor prognosis) and assessed by the 36 months cumulative incidence of relapse. RESULTS: In the GEO data set, results were reproducible in stage III, as patients predicted to be stage I-like had a significant lower risk of relapse than patients predicted as stage IV-like (P = 0.04, Gray test). Results were not reproducible in stage II patients (P > 0.05, Gray test). In the Danish data set, two of four stage III patients with relapse were correctly predicted as stage IV-like, and the remaining patients were predicted as stage I-like and unclassifiable, respectively. Stage II patients could not be stratified. CONCLUSIONS: The 128-gene signature showed reproducibility in stage III colon cancer, but could not predict recurrence in stage II. Individual patient predictions in an independent Danish material were unsatisfactory. Additional validation in larger cohorts is warranted.

Origin of acute appendicitis: fecal retention in colonic reservoirs: a case control study.

BACKGROUND: Appendicitis is encountered predominantly in Western, industrialized countries. Animal experiments and clinical studies have suggested an obstructive fecalith as a cause of acute appendicitis. It was hypothesized that patients with acute appendicitis would have a longer colonic transit time and more fecal retention reservoirs (coprostasis) than healthy control persons, thus favoring the occurrence of a fecalith in the appendix. METHODS: Sixty-eight patients scheduled for appendectomy were included in this approved study. Before surgery, a plain abdominal radiograph was taken; at surgery, the degree of inflammation of the appendix was recorded, along with the presence or absence of a fecalith. Six weeks postoperatively, the patients underwent a colonic transit study. A cohort of 44 control persons over 18 years of age was selected at random to undergo the same marker study as the patients. The parameters studied were the number of radiopaque markers (h), the fecal retention or load (score 0-3) in the right, left, and distal colonic segments, and the number of fecaliths. RESULTS: Twelve patients were excluded; i.e., 56 patients and 44 controls were eligible for most analyses. In the patient group, statistically significant correlations were found between fecal loading scores and the number of markers (transit time) both overall and within the left and distal colonic segments (all p < 0.05). In the control subjects, there was significance with regard to the distal segment. The median colon transit time was 25.0 h (range 1-107 h) in patients with appendicitis compared with 19.0 h (range 0-71 h) in controls (p = NS). The transit time was longer in the right, left, and distal colon in patients than in control subjects, although not to a statistically significant extent. The total and segmental fecal loads in the colon did not differ significantly between patients and controls. A fecalith occurred in 49.0% of the patients and was in most cases associated with a gangrenous or perforated appendix. If a fecalith was not found, this correlated to a significant extent with a high fecal loading score in the left colon (p = 0.04). CONCLUSIONS: An obstructive fecalith occurred in one-half of the patients with acute appendicitis. The appendicitis patients had a colonic transit time similar to that in healthy controls. Furthermore, there was no difference in colonic fecal loading between patients and controls. In consequence, the occurrence of a fecalith could not be attributed to delayed colonic transit or fecal loading. However, we discovered greater amounts of feces in the colon of both patients and controls than would have been expected physiologically, and the role of these fecal reservoirs has yet to be understood.

Smoking is a risk factor for incisional hernia.

HYPOTHESIS: A number of risk factors for incisional hernia have been identified, but the pathogenesis remains unclear. Based on previous findings of smoking as a risk factor for wound complications and recurrence of groin hernia, we studied whether smoking is associated with incisional hernia. DESIGN: Cohort study. Clinical follow-up study for incisional hernia 33 to 57 months following laparotomy for gastrointestinal disease. Variables predictive for incisional hernia were assessed by multiple regression analysis. SETTING: Department of Surgery, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark. PATIENTS: All 916 patients undergoing laparotomy from 1997 through 1998. Surgeons performed clinical examination in 310 patients; patients who failed to meet for examination, died, or were lost to follow-up were excluded. MAIN OUTCOME MEASURES: Thirty-four variables related to patient history, preoperative clinical condition, operative severity and findings, and the surgeon's training. RESULTS: The incidence of incisional hernia was 26% (81/310). Smokers had a 4-fold higher risk of incisional hernia (odds ratio [OR], 3.93 [95% confidence interval (CI), 1.82-8.49]) independent of other risk factors and confounders. Relaparotomy was the strongest factor associated with hernia (OR, 5.89 [95% CI, 1.78-19.48]). Other risk factors were postoperative wound complications (OR, 3.91 [95% CI, 1.99-7.66]), age (OR, 1.04 [95% CI, 1.02-1.06]), and male sex (OR, 2.17 [95% CI, 1.21-3.91]). CONCLUSION: Smoking is a significant risk factor for incisional hernia in line with relaparotomy, postoperative wound complications, older age, and male sex.

Converging signals synergistically activate the LAMC2 promoter and lead to accumulation of the laminin gamma 2 chain in human colon carcinoma cells.

The trimeric extracellular matrix molecule laminin-5 and its constituent chains (alpha 3, beta 3, gamma 2) are normally not detectable intracellularly in intestinal epithelial cells but the laminin gamma 2 chain can be detected in cancer cells at the invasive front of a subset of colon carcinomas. These cells are subjected to cytokines such as transforming growth factor beta 1 (TGF-beta 1) and hepatocyte growth factor (HGF), produced by the tumour cells or by the surrounding stromal cells. The purpose of the present work was to investigate whether TGF-beta 1 and HGF, known to stimulate the LAMC2 gene encoding the laminin gamma 2 chain, might synergize to activate the LAMC2 promoter, and to identify the promoter elements involved. We find evidence for synergy between TGF-beta and HGF with respect to laminin gamma 2 chain expression and promoter activation and demonstrate that this requires the 5' activator protein-1 (AP-1) element of the promoter and an additional upstream element which is also responsive to co-expression of the Smad3 protein from the TGF-beta signalling pathway. The transcripts encoding the other laminin-5 chains are not synergistically activated by HGF and TGF-beta. Thus the synergistic activation of the LAMC2 gene is mediated via different cis-elements and results in an overproduction of the laminin gamma 2 chain relative to the other laminin-5 constituent chains. This difference may explain why laminin gamma 2 chains accumulate in the cells at the invasive front of colon carcinomas.