Clonal expansion of hepatocytes with a selective advantage occurs during all stages of chronic hepatitis B virus infection.

Hepatocyte clone size was measured in liver samples of 21 patients in various stages of chronic hepatitis B virus (HBV) infection and from 21 to 76 years of age. Hepatocyte clones containing unique virus-cell DNA junctions formed by the integration of HBV DNA were detected using inverse nested PCR. The maximum hepatocyte clone size tended to increase with age, although there was considerable patient-to-patient variation in each age group. There was an upward trend in maximum clone size with increasing fibrosis, inflammatory activity and with seroconversion from HBV e-antigen (HBeAg)-positive to HBeAg-negative, but these differences did not reach statistical significance. Maximum hepatocyte clone size did not differ between patients with and without a coexisting hepatocellular carcinoma. Thus, large hepatocyte clones containing integrated HBV DNA were detected during all stages of chronic HBV infection. Using laser microdissection, no significant difference in clone size was observed between foci of HBV surface antigen (HBsAg)-positive and HBsAg-negative hepatocytes, suggesting that expression of HBsAg is not a significant factor in clonal expansion. Laser microdissection also revealed that hepatocytes with normal-appearing histology make up a major fraction of the cells undergoing clonal expansion. Thus, preneoplasia does not appear to be a factor in the clonal expansion detected in our assays. Computer simulations suggest that the large hepatocyte clones are not produced by random hepatocyte turnover but have an as-yet-unknown selective advantage that drives increased clonal expansion in the HBV-infected liver.

IGFBP7 is associated with poor prognosis in oesophageal adenocarcinoma and is regulated by promoter DNA methylation.

BACKGROUND: We examined whether silencing of IGFBP7 was associated with survival in patients with oesophageal adenocarcinoma. METHODS: Protein expression of IGFBP7 was determined using immunohistochemistry in a tissue microarray representing tumours from 65 patients with oesophageal adenocarcinoma who had not had neoadjuvant therapy. DNA methylation of the IGFBP7 promoter was determined with the melt curve analysis in cell lines and patient tissues. RESULTS: Expression of IGFBP7 was observed in the oesophageal adenocarcinoma of 34 out of 65 (52%) patients and was associated with significantly reduced median (11 vs 92 months) and 5-year survival (25% vs 52%). Multivariate analysis identified expression as an independent prognostic indicator for survival (hazard ratio=3.24, 95% confidence interval=1.58-6.67, P-value=0.0014). Hypermethylation of IGFBP7 was associated with silencing of gene expression in cell lines and patient tissues (P-value=0.0225). Methylation was observed in the squamous mucosa of 2 out of 15 (13%) patients with Barrett's oesophagus and 3 out of 17 (18%) with oesophageal adenocarcinoma. Methylation was observed in 14 out of 18 (78%) of biopsies of Barrett's mucosa and 23 out of 34 (68%) patients with oesophageal adenocarcinoma. CONCLUSION: Reduced IGFBP7 protein expression was associated with longer survival in patients with oesophageal adenocarcinoma. Methylation of the IGFBP7 promoter was associated with silencing of gene expression and was frequent in Barrett's oesophagus and oesophageal adenocarcinoma.

Low expression of gamma-glutamyl hydrolase mRNA in primary colorectal cancer with the CpG island methylator phenotype.

The CpG island methylator phenotype (CIMP+) in colorectal cancer (CRC) is defined as concomitant and frequent hypermethylation of CpG islands within gene promoter regions. We previously demonstrated that CIMP+ was associated with elevated concentrations of folate intermediates in tumour tissues. In the present study, we investigated whether CIMP+ was associated with a specific mRNA expression pattern for folate- and nucleotide-metabolising enzymes. An exploratory study was conducted on 114 CRC samples from Australia. mRNA levels for 17 genes involved in folate and nucleotide metabolism were measured by real-time RT-PCR. CIMP+ was determined by real-time methylation-specific PCR and compared to mRNA expression. Candidate genes showing association with CIMP+ were further investigated in a replication cohort of 150 CRC samples from Japan. In the exploratory study, low expression of gamma-glutamyl hydrolase (GGH) was strongly associated with CIMP+ and CIMP+-related clinicopathological and molecular features. Trends for inverse association between GGH expression and the concentration of folate intermediates were also observed. Analysis of the replication cohort confirmed that GGH expression was significantly lower in CIMP+ CRC. Promoter hypermethylation of GGH was observed in only 5.6% (1 out of 18) CIMP+ tumours and could not account for the low expression level of this gene. CIMP+ CRC is associated with low expression of GGH, suggesting involvement of the folate pathway in the development and/or progression of this phenotype. Further studies of folate metabolism in CIMP+ CRC may help to elucidate the aetiology of these tumours and to predict their response to anti-folates and 5-fluorouracil/leucovorin.

Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis.

BACKGROUND: Hyperplastic polyposis of the colorectum is a precancerous condition that has been linked with DNA methylation. The polyps in this condition have been distinguished from typical small hyperplastic polyps and renamed sessile serrated adenomas. Sessile serrated adenomas also occur sporadically and appear to be indistinguishable from their counterparts in hyperplastic polyposis. AIMS AND METHODS: The existence of distinguishing molecular features was explored in a series of serrated polyps and matched normal mucosa from patients with and without hyperplastic polyposis by assessing mutation of BRAF, DNA methylation in 14 markers (MINTs 1, 2 and 31, p16, MGMT, MLH1, RASSF1, RASSF2, NORE1 (RASSF5), RKIP, MST1, DAPK, FAS, and CHFR), and immunoexpression of MLH1. RESULTS: There was more extensive methylation in sessile serrated adenomas from subjects with hyperplastic polyposis (p<0.0001). A more clearcut difference in patients with hyperplastic polyposis was the finding of extensive DNA methylation in normal mucosa from the proximal colon. CONCLUSIONS: A genetic predisposition may underlie at least some forms of hyperplastic polyposis in which the earliest manifestation may be hypermethylation of multiple gene promoters in normal colorectal mucosa. Additionally, some of the heterogeneity within hyperplastic polyposis may be explained by different propensities for MLH1 inactivation within polyps.

CIB1 contributes to oncogenic signalling by Ras via modulating the subcellular localisation of sphingosine kinase 1.

CIB1 (calcium and integrin binding protein 1) is a small intracellular protein with numerous interacting partners, and hence has been implicated in various cellular functions. Recent studies have revealed emerging roles of CIB1 in regulating cancer cell survival and angiogenesis, although the mechanisms involved have remained largely undefined. In investigating the oncogenic function of CIB1, we initially found that CIB1 is widely up-regulated across a diverse range of cancers, with this upregulation frequently correlating with oncogenic mutations of KRas. Consistent with this, we found that ectopic expression of oncogenic KRas and HRas in cells resulted in elevated CIB1 expression. We previously described the Ca2+-myristoyl switch function of CIB1, and its ability to facilitate agonist-induced plasma membrane localisation of sphingosine kinase 1 (SK1), a location where SK1 is known to elicit oncogenic signalling. Thus, we examined the role this may play in oncogenesis. Consistent with these findings, we demonstrated here that over-expression of CIB1 by itself is sufficient to drive localisation of SK1 to the plasma membrane and enhance the membrane-associated enzymatic activity of SK1, as well as its oncogenic signalling. We subsequently demonstrated that elevated levels of CIB1 resulted in full neoplastic transformation, in a manner dependent on SK1. In agreement with our previous findings that SK1 is a downstream mediator of oncogenic signalling by Ras, we found that targeting CIB1 also inhibited neoplastic growth of cells induced by oncogenic Ras, suggesting an important pro-tumorigenic role for CIB1. Thus, we have demonstrated for the first time a role for CIB1 in neoplastic transformation, and revealed a novel mechanism facilitating oncogenic signalling by Ras and SK1.

The reliability of immunohistochemistry as a prescreening method for the diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC)--results of an international collaborative study.

Hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is an autosomal dominant condition accounting for 2-5% of all colorectal carcinomas as well as a small subset of endometrial, upper urinary tract and other gastrointestinal cancers. An assay to detect the underlying defect in HNPCC, inactivation of a DNA mismatch repair enzyme, would be useful in identifying HNPCC probands. Monoclonal antibodies against hMLH1 and hMSH2, two DNA mismatch repair proteins which account for most HNPCC cancers, are commercially available. This study sought to investigate the potential utility of these antibodies in determining the expression status of these proteins in paraffin-embedded formalin-fixed tissue and to identify key technical protocol components associated with successful staining. A set of 20 colorectal carcinoma cases of known hMLH1 and hMSH2 mutation and expression status underwent immunoperoxidase staining at multiple institutions, each of which used their own technical protocol. Staining for hMSH2 was successful in most laboratories while staining for hMLH1 proved problematic in multiple labs. However, a significant minority of laboratories demonstrated excellent results including high discriminatory power with both monoclonal antibodies. These laboratories appropriately identified hMLH1 or hMSH2 inactivation with high sensitivity and specificity. The key protocol point associated with successful staining was an antigen retrieval step involving heat treatment and either EDTA or citrate buffer. This study demonstrates the potential utility of immunohistochemistry in detecting HNPCC probands and identifies key technical components for successful staining.

99mTc-alafosfalin: an antibiotic peptide infection imaging agent.

The radiolabeled antibiotic peptide (99m)Tc-alafosfalin was assessed as an infection imaging agent in a rat model by comparison with (99m)Tc-DTPA and (99m)Tc-leukocytes. (99m)Tc-alafosfalin was prepared via an instant cold kit and (99m)Tc-leukocytes were prepared using (99m)Tc-stannous fluoride colloid in an ex vivo labeling procedure of whole blood. In separate experiments, the three radiotracers were administered to rats infected with staphylococcus aureus. Quantitative biodistribution studies were performed as well as scintigraphic images and histopathology. (99m)Tc-alafosfalin is a stable product, obtained in high radiochemical purity (>95%). This agent was mainly renally excreted, with low liver, spleen and bone uptake, and resulted in a mean ratio of infected/non-infected thighs of 4.3/1.0 at 4 hr post radiotracer injection. (99m)Tc-DTPA gave a corresponding ratio of 1.9/1.0 and (99m)Tc-leukocytes gave 20.0/1.0 at the same time point. An in vitro assay found the level of (99m)Tc-alafosfalin binding to staphylococcus aureas higher than (99m)Tc-DTPA (10% versus 1% respectively). (99m)Tc-alafosfalin accumulates at sites of infection in a rat model better than the perfusion molecule (99m)Tc-DTPA, yet less than (99m)Tc-leukocytes. The distribution characteristics of this (99m)Tc-antibiotic peptide would be an advantage in imaging abdominal and soft tissue infection.

Sudden death in pregnancy from congenital malformation of the coronary arteries.

We report the case of a 40 yr old previously healthy woman who died suddenly during her second pregnancy from unusual coronary artery anomalies. The left anterior descending coronary artery arose from the above of the right sinus of Valsalva 1.1 cm above the ridge in the aorta and coursed behind the pulmonary valve before it dipped deeply into the myocardium of the interventricular septum. The left circumflex coronary artery was hypoplastic. The right coronary artery arose also from above the right sinus of Valsalva 1.1 cm above the ridge in the aorta but was otherwise normal. A fourth small coronary artery arose between the origins of the left anterior descending and right coronary artery and supplied the outflow tract of the right ventricle. Mild fibrosis was present in the interventricular septum. The rest of the autopsy examination was unremarkable. Such combination of coronary artery anomalies causing sudden unexpected death in a pregnant woman have not previously been reported.

Death following colchicine poisoning.

A 45-year-old male was admitted to hospital after 2 to 3 days of vomiting, nausea, and diarrhea following an apparent overdose of colchicine tables. During hospitalization his white blood cell count fell dramatically. At death, 33 h following initial hospitalization, pleural effusion with bilateral bronchopneumonia was evident, together with numerous bacterial colonies and marked hypocellularity of bone marrow and reduced megakaryocytes, erythroid, and myeloid cells. The most striking histological findings were numerous metaphasic mitotic figures in gastric and small bowel epithelia. Colchicine was detected, confirmed by high pressure liquid chromatography with photodiode array detection, and quantitated in antemortem plasma collected 3.3 h following hospitalization and in postmortem blood and bile. Colchicine was not detected in liver, vitreous humor, or stomach contents.

Serum concentrations of brain-derived neurotrophic factor (BDNF) are decreased in colorectal cancer patients.

OBJECTIVE: To determine the usefulness of brain-derived neurotrophic factor (BDNF) as a diagnostic biomarker for colorectal cancer (CRC). MATERIALS AND METHODS: ELISA immunoassay was used to examine BDNF concentrations in the sera of two different retrospective cohorts consisting of CRC patients and age/gender matched controls. Cohort 1 consisted of 99 controls and 97 CRC patients, whereas cohort 2 consisted of 47 controls and 91 CRC patients. RESULTS: In cohort 1, the median concentration of BDNF was significantly (p< 0.0001) lower in CRC patient samples (18.8 ng/mL, range 4.0-56.5 ng/mL) than control samples (23.4 ng/mL, range 3.0-43.1 ng/mL). This finding was validated in an independent patient cohort (CRC patients: 23.0 ng/mL, range 6.0-45.9 ng/mL; control patients: 32.3 ng/mL, range 14.2-62.4 ng/mL). BDNF concentrations did not differ significantly between Dukes' staging in the patient cohort, however patients with Stages A, B, C and D (p< 0.01 for each stage) tumours had significantly reduced BDNF levels compared to healthy controls. Receiver operating characteristic analysis was performed to determine the ability of BDNF to discriminate between healthy controls and those with CRC. At 95% specificity, BDNF concentrations distinguished CRC patients with 25% and 18% sensitivity, respectively, in cohorts 1 and 2 (cohort 1: AUC=0.79, 95% CI 0.70-0.87; cohort 2: AUC =0.69, 95% CI 0.61-0.76). CONCLUSION: The serum levels of BDNF were significantly lower in colorectal cancer patients when compared to a control population, and this did not differ between different Dukes' stages.

Different APC genotypes in proximal and distal sporadic colorectal cancers suggest distinct WNT/ß-catenin signalling thresholds for tumourigenesis.

Biallelic protein-truncating mutations in the adenomatous polyposis coli (APC) gene are prevalent in sporadic colorectal cancer (CRC). Mutations may not be fully inactivating, instead producing WNT/ß-catenin signalling levels 'just-right' for tumourigenesis. However, the spectrum of optimal APC genotypes accounting for both hits, and the influence of clinicopathological features on genotype selection remain undefined. We analysed 630 sporadic CRCs for APC mutations and loss of heterozygosity (LOH) using sequencing and single-nucleotide polymorphism microarrays, respectively. Truncating APC mutations and/or LOH were detected in 75% of CRCs. Most truncating mutations occurred within a mutation cluster region (MCR; codons 1282-1581) leaving 1-3 intact 20 amino-acid repeats (20AARs) and abolishing all Ser-Ala-Met-Pro (SAMP) repeats. Cancers commonly had one MCR mutation plus either LOH or another mutation 5' to the MCR. LOH was associated with mutations leaving 1 intact 20AAR. MCR mutations leaving 1 vs 2-3 intact 20AARs were associated with 5' mutations disrupting or leaving intact the armadillo-repeat domain, respectively. Cancers with three hits had an over-representation of mutations upstream of codon 184, in the alternatively spliced region of exon 9, and 3' to the MCR. Microsatellite unstable cancers showed hyper-mutation at MCR mono- and di-nucleotide repeats, leaving 2-3 intact 20AARs. Proximal and distal cancers exhibited different preferred APC genotypes, leaving a total of 2 or 3 and 0 to 2 intact 20AARs, respectively. In conclusion, APC genotypes in sporadic CRCs demonstrate 'fine-tuned' interdependence of hits by type and location, consistent with selection for particular residual levels of WNT/ß-catenin signalling, with different 'optimal' thresholds for proximal and distal cancers.

The effects of high-dose esomeprazole on gastric and oesophageal acid exposure and molecular markers in Barrett's oesophagus.

BACKGROUND: Acid reflux is often difficult to control medically. AIM: To assess the effect of 40 mg twice daily esomeprazole (high-dose) on gastric and oesophageal pH and symptoms, and biomarkers relevant to adenocarcinoma, in patients with Barrett's oesophagus (BO). METHODS: Eighteen patients, treated with proton pump inhibitors as prescribed by their treating doctor, had their therapy increased to high-dose esomeprazole for 6 months. RESULTS: At entry into the study, 9/18 patients had excessive 24-h oesophageal acid exposure, and gastric pH remained <4 for >16 h in 8/18. With high-dose esomeprazole, excessive acid exposure occurred in 2/18 patients, and gastric pH <4 was decreased from 38% of overall recording time and 53% of the nocturnal period to 15% and 17%, respectively (P < 0.001). There was a reduction in self-assessed symptoms of heartburn (P = 0.0005) and regurgitation (P < 0.0001), and inflammation and proliferation in the Barrett's mucosa. There was no significant change in p53, MGMT or COX-2 expression, or in aberrant DNA methylation. CONCLUSIONS: High-dose esomeprazole achieved higher levels of gastric acid suppression and control of oesophageal acid reflux and symptoms, with significant decreases in inflammation and epithelial proliferation. There was no reversal of aberrant DNA methylation.

DNA hypermethylation in the normal colonic mucosa of patients with colorectal cancer.

The CpG-island methylator phenotype (CIMP+) in colorectal cancer (CRC) is characterised by frequent hypermethylation of promoter regions in tumour suppressor genes. Low level methylation of some CpG islands is also seen in the normal colonic mucosa and increases with age; however, it is still unclear what other factors regulate this phenomenon. The first aim of our study was to determine whether the level of promoter methylation is elevated in the normal colonic mucosa of patients with CIMP+ tumours. The second aim was to investigate whether common, functional polymorphisms in genes involved in methyl group metabolism are associated with the level of methylation in this tissue. CpG islands within the ERalpha, MYOD, P16(INK4A), MLH1, APC, P14(ARF), DAPK and TIMP3 genes were quantitatively evaluated for methylation in normal colonic mucosa from a large series of CRC patients using the MethyLight assay. Genotyping was carried out for polymorphisms in the MTHFR, TS, MS, MTHFD1 and DNMT3b genes. Methylation of ERalpha and MYOD in normal colonic mucosa increased with age and was higher in female subjects. Methylation of P16(INK4A), MLH1, TIMP3 and DAPK in normal mucosa occurred at a lower level than ERalpha and MYOD but also increased with age and was significantly higher in patients with CIMP+ tumours. The DNMT3b C46359T polymorphism was associated with significantly less methylation of MYOD and MLH1 and with trends for lower methylation in each of the other CpG islands examined. Our results demonstrate that age, gender and genetic factors can influence the methylation level of CpG islands in gene promoter regions of normal colonic mucosa. Further work is required to determine whether such methylation is associated with the development of CIMP+ CRC.