Detection of Chitinase 3-Like 1 in Symptomatic Primary Care Patient Faecal Samples is Not a Reliable Biomarker of Colonic Lesions.

BACKGROUND: The current gold standard non-invasive test for detecting pre-cancerous changes is the faecal immunochemical test (FIT). However, this test can lack sensitivity and specificity and testing for another biomarker may address these limitations. Chitinase 3-like 1 (CHI3L1) is emerging as a potential biomarker of inflammation-associated carcinogenic changes in epithelial cells.  In this study CHI3L1 levels were analysed in patients and controls to determine their ability to improve detection of early CRC either alone or in combination with a FIT. METHODS: CHI3L1 levels were measured by ELISA in serum and stool samples from cohorts of CRC and healthy donors as well as stool samples from a cohort of symptomatic primary care patients. Faecal haemoglobin was also analysed in the same primary care samples using FIT. RESULTS: CHI3L1 levels were a good discriminatory marker of CRC, with no significant difference between levels detected in the stool and serum samples.  ROC curves that determined the optimal cut-point however identified that stool samples gave higher sensitivity (83% versus 69%) and specificity (89% versus 74%) than matched serum samples. Faecal CHI3L1 levels in the primary care patients were not significantly different (p=0.193) from those detected in the healthy controls. ROC curve analysis confirmed that faecal CHI3L1 levels had limited ability to discriminate between patients who did or didn't have evidence of lesions (AUC=0.52, p=0.74). Similarly, CHI3L1 levels did not reliably identify those symptomatic primary care patients who subsequently presented with early-stage disease (polyps and adenomas) or CRC. The discriminatory power of FIT was not increased by incorporating the CHI3L1 results in this setting. CONCLUSION: There was no evidence that measurement of faecal CHI3L1 has the potential to increase diagnostic accuracy, either alone or in combination with a FIT, in symptomatic primary care patients.

Detection of Fusobacterium nucleatum DNA in primary care patient stool samples does not predict progression of colorectal neoplasia.

BACKGROUND: Carriage of certain bacterial species may represent potential biomarkers of colorectal cancer (CRC). Prominent among these is Fusobacterium nucleatum. We explored the association of F. nucleatum DNA in stool samples with the presence of colonic neoplastic lesions in a cohort of primary care patients, and compared our findings with those from an unrelated cohort of colonoscopy patients followed clinically over time. METHODS: Carriage rates of F. nucleatum in stool samples were assessed in 185 patients referred for a faecal immunochemical test (FIT) by their general practitioners (GPs). Comparisons were made with stool samples from 57 patients diagnosed with CRC and 57 age-matched healthy controls, and with tissue samples taken at colonoscopy from 150 patients with a decade of subsequent clinical follow-up. FINDINGS: F. nucleatum DNA was found at a high rate (47.0%) in stool samples from primary care patients, and more often in stool samples from CRC patients (47.4%) than in healthy controls (7.0%), (P = 7.66E-7). No association was found between carriage of F. nucleatum and FIT positivity (P = 0.588). While evidence of stool-associated F. nucleatum DNA was significantly more likely to indicate a lesion in those primary care patients progressed to colonoscopy (P = 0.023), this finding did not extend to the progression of neoplastic lesions in the 150 patients with a decade of follow up. CONCLUSION: The finding of F. nucleatum DNA at similar rates in stool samples from patients diagnosed with CRC and in primary care patients with pre-cancerous lesions supports growing awareness that the presence of these bacteria may be a biomarker for increased risk of disease. However, molecular evidence of F. nucleatum did not predict progression of colonic lesions, which may lessen the utility of this bacterium as a biomarker for increased risk of disease.

APC Mutations Are Not Confined to Hotspot Regions in Early-Onset Colorectal Cancer.

While overall colorectal cancer (CRC) cases have been declining worldwide there has been an increase in the incidence of the disease among patients under 50 years of age. Mutation of the APC gene is a common early event in CRC but is reported at lower rates in early-onset colorectal cancer (EOCRC) than in older patients. Here we investigate the APC mutation status of a cohort of EOCRC patients in New Zealand using a novel sequencing approach targeting regions of the gene encompassing the vast majority of known APC mutations. Using this strategy we find a higher rate (72%) of APC mutation than previously reported in EOCRC with mutations being spread throughout the gene rather than clustered in hotspots as seen with sporadic mutations in older patients. The rate of mutations falling within hotspots was similar to those previously seen in EOCRC and as such our study has implications for sequencing strategies for EOCRC patients. Overall there were low rates of both loss of heterozygosity and microsatellite instability whereas a relatively high rate (40%) of APC promoter methylation was found, possibly reflecting increasing exposure of young people to pro-oncogenic lifestyle factors.

CDH1 gene mutation in early-onset, colorectal signet-ring cell carcinoma.

AIM: Colorectal signet-ring cell carcinomas (SRCC) are highly malignant tumours with poor prognosis that disproportionately affect younger patients. There is growing evidence of a unique set of molecular features that separate SRCC from conventional colorectal adenocarcinoma. Identification of these distinct features may have diagnostic and prognostic significance for patients and families. CDH1, which encodes E-cadherin, a cell adhesion protein, is commonly mutated in gastric SRCC and our study aimed to identify whether CDH1 mutation was also a common phenomenon in colorectal SRCC. METHODS: DNA was extracted from formalin-fixed paraffin embedded tumour tissue, the CDH1 gene was analysed by next generation sequencing and the pathogenicity of mutations assessed in silico. Sections cut from the same blocks were immunostained to identify the presence of the E-cadherin protein. RESULTS: We found 8 CDH1 mutations that meet our inclusion criteria in seven of 11 samples. Of these, five (from four patients), were likely to be germline mutations. E-cadherin staining was absent or markedly reduced in all of the seven samples with CDH1 mutation. CONCLUSION: Our finding of CDH1 mutations in a proportion of signet-ring cell carcinomas and associated reduction in E-cadherin in these tumours supports previous findings of a role for mutation of this gene in the development of this disease. In addition, the finding of likely germline mutations suggests that a subset of these tumours may be familial. Loss of E-cadherin staining in the absence of CDH1 mutations however also suggests a role for environmental factors in a subset of these tumours.

Faecal biomarkers do not always identify pre-cancerous lesions in patients who present in primary care with bowel symptoms.

AIM: To determine if tumour-derived M2-PK is potentially a more accurate biomarker of pre-cancerous bowel lesions in patients who present in primary care with bowel symptoms than the detection of faecal haemoglobin. METHODS: Patients requested by their general practitioners (GPs) to provide a stool sample to determine the presence of faecal haemoglobin consented for the same stool samples to be tested for the presence of M2-PK. For comparison M2-PK levels were also measured in stool samples from patients recently identified with colorectal cancer and healthy controls who self-reported no bowel problems at the time of sampling. RESULTS: M2-PK levels measured in 185 GP-derived samples were comparable to the control cohort (57 healthy controls) and notably lower than those in the 57 patients with CRC (2.6, 3.2 and 18.2U/ml-1, respectively). Sixty-seven of the GP patients were referred for colonoscopy. While 26 of these patients had a positive M2-PK, only 10 were found to have colonic lesions. Conversely, 18 of the 41 patients who had a negative M2-PK were found to have lesions that included one CRC, 13 adenomas and four other polyps. The FIT also failed to identify colonic disease in 19 of 48 patients referred for colonoscopy. There was, however, a significant association between lesions greater than 1cm and a positive FIT (p<0.02) that was not the case with M2-PK. A positive FIT identified one patient in the GP patient cohort subsequently diagnosed with colorectal cancer at follow-up colonoscopy whereas the same stool sample tested negative for M2-PK. CONCLUSIONS: Measurement of faecal M2-PK levels lacks the specificity and sensitivity (and therefore diagnostic accuracy) to identify the individuals who should be progressed for clinical follow-up. Accordingly, M2-PK is not is not a robust biomarker for identifying pre-cancerous bowel lesions in a primary care setting.

Helicobacter pylori infection perturbs iron homeostasis in gastric epithelial cells.

The iron deficiency anaemia that often accompanies infection with Helicobacter pylori may reflect increased uptake of iron into gastric epithelial cells. Here we show an infection-associated increase in total intracellular iron levels was associated with the redistribution of the transferrin receptor from the cell cytosol to the cell surface, and with increased levels of ferritin, an intracellular iron storage protein that corresponded with a significant increase in lysosomal stores of labile iron. In contrast, the pool of cytosolic labile iron was significantly decreased in infected cells. These changes in intracellular iron distribution were associated with the uptake and trafficking of H. pylori through the cells, and enhanced in strains capable of expressing the cagA virulence gene. We speculate that degradation of lysosomal ferritin may facilitate H. pylori pathogenesis, in addition to contributing to bacterial persistence in the human stomach.

Colonization with enterotoxigenic Bacteroides fragilis is associated with early-stage colorectal neoplasia.

BACKGROUND: Enterotoxigenic Bacteroides fragilis (ETBF) is a toxin-producing bacteria thought to possibly promote colorectal carcinogenesis by modulating the mucosal immune response and inducing epithelial cell changes. Here, we aim to examine the association of colonic mucosal colonization with ETBF and the presence of a range of lesions on the colonic neoplastic spectrum. METHODS: Mucosal tissue from up to four different colonic sites was obtained from a consecutive series of 150 patients referred for colonoscopy. The presence and relative abundance of the B. fragilis toxin gene (bft) in each tissue sample was determined using quantitative PCR, and associations with clinicopathological characteristics were analysed. FINDINGS: We found a high concordance of ETBF between different colonic sites (86%). Univariate analysis showed statistically significant associations between ETBF positivity and the presence of low-grade dysplasia (LGD), tubular adenomas (TA), and serrated polyps (P-values of 0.007, 0.027, and 0.007, respectively). A higher relative abundance of ETBF was significantly associated with LGD and TA (P-values of < 0.0001 and 0.025, respectively). Increased ETBF positivity and abundance was also associated with left-sided biopsies, compared to those from the right side of the colon. CONCLUSION: Our results showing association of ETBF positivity and increased abundance with early-stage carcinogenic lesions underlines its importance in the development of colorectal cancer, and we suggest that detection of ETBF may be a potential marker of early colorectal carcinogenesis.

The effect of polymeric formula on enterocyte differentiation.

Exclusive enteral nutrition is established as an initial therapy to induce remission in active Crohn's disease (CD), especially in children, but the mechanisms of action of this therapy are yet to be fully defined. Intestinal alkaline phosphatase (IAP), a recognised marker of enterocyte differentiation, is implicated in the innate gut immune response to enteric pathogens. Using the Caco-2 human adenocarcinoma cell line, this study showed that the incubation of human cells with a polymeric formula (PF) resulted in a dose-dependent increase in the expression of IAP on the cell surface. While further investigation is required to determine the pathway(s) involved, this finding suggests that cell surface-associated IAP may be an aspect of the gut's innate immune response to pathogenic bacteria that is strengthened by PF in the setting of CD.

Mean telomere length is not associated with current health status in a 50-year-old population sample.

OBJECTIVES: Telomeres are nucleoprotein complexes that cap the ends of linear chromosomes. Telomeric DNA decreases with age and shows considerable heterogeneity in the wider population. There is interest in the application of telomere length measures as a biomarker of general health or "biological age," and the possibility of using mean telomere length to gauge individual disease risk, and to promote lifestyle changes to improve health. This study examined the effectiveness of telomere length as a biomarker for an individual's current overall health status by assessing several measures of general health including SF-36v2 score, current smoking status and a comprehensive obesity phenotype. METHODS: Participants were from the Canterbury Health, Ageing and Lifecourse (CHALICE) cohort, a New Zealand population based multidisciplinary study of aging. Telomere length measurements were obtained on DNA from peripheral blood samples at age 49-51 (n = 351), using a quantitative polymerase chain reaction assay. RESULTS: No associations were found between telomere length measured at age 49-51 and any measures of current health status. The only significant association observed was between telomere length and gender, with females having longer telomere length than men. CONCLUSIONS: Our results suggest that telomere length measurements are unlikely to provide information of much predictive significance for an individual's health status.

Screening for enterotoxigenic Bacteroides fragilis in stool samples.

Bacteroides fragilis is a commensal bacterium found in the gut of most humans, however enterotoxigenic B. fragilis strains (ETBF) have been associated with diarrhoea and colorectal cancer (CRC). The purpose of this study was to establish a method of screening for the Bacteroides fragilis toxin (bft) gene in stool samples, as a means of determining if carriage of ETBF is detected more often in CRC patients than in age-matched healthy controls. Stool samples from 71 patients recently diagnosed with CRC, and 71 age-matched controls, were screened by standard and quantitative PCR using primers specific for the detection of the bft gene. Bacterial template DNA from stool samples was prepared by two methods: a sweep, where all colonies growing on Bacteroides Bile Esculin agar following stool culture for 48 h at 37 °C in an anaerobic environment were swept into sterile water and heat treated; and a direct DNA extraction from each stool sample. The bft gene was detected more frequently from DNA isolated from bacterial sweeps than from matched direct DNA extractions. qPCR was found to be more sensitive than standard PCR in detecting bft. The cumulative total of positive qPCR assays from both sample types revealed that 19 of the CRC patients had evidence of the toxin gene in their stool sample (27%), compared to seven of the age-matched controls (10%). This difference was significant (P = 0.016). Overall, ETBF carriage was detected more often in CRC patient stool samples compared to controls, but disparate findings from the different DNA preparations and testing methods suggests that poor sensitivity may limit molecular detection of ETBF in stool samples.

Comparison of Fecal Inflammatory Markers in Crohn's Disease.

BACKGROUND: Fecal biomarkers are used increasingly to monitor Crohn's disease (CD). However, the relative accuracy of different markers in identifying inflammation has been poorly evaluated. We evaluated fecal calprotectin (FC), lactoferrin (FL), and S100A12 (FS) using endoscopic validation in a prospective study of the progression of CD after intestinal resection. METHODS: Data were collected from 135 participants in a prospective, randomized, controlled trial aimed at preventing postoperative CD recurrence. Three hundred nineteen stool samples were tested for FC, FL, and FS preoperatively and 6, 12, and 18 months after resection. Colonoscopy was performed at 6 and/or 18 months. Endoscopic recurrence was assessed blindly using the Rutgeerts score. C-reactive protein (CRP) and Crohn's Disease Activity Index (CDAI) were assessed. RESULTS: FC, FL, and FS concentrations were elevated preoperatively (median: 1347, 40.9, and 8.4 µg/g, respectively). At 6 months postoperatively, marker concentrations decreased (166, 3.0, 0.9 µg/g) and were higher in recurrent disease than remission (275 versus 72 µg/g, P < 0.001; 5.7 versus 1.6 µg/g, P = 0.007; 2.0 versus 0.8 µg/g, P = 0.188). FC > 135 µg/g, FL > 3.4 µg/g, and FS > 10.5 µg/g indicated endoscopic recurrence (score ≥ i2) with a sensitivity, specificity, and negative predictive value (NPV) of 0.87, 0.66, and 91%; 0.70, 0.68, and 81%; 0.91, 0.12, and 71%, respectively. FC and FL correlated significantly with the presence and severity of endoscopic recurrence, whereas FS, CRP and CDAI did not. CONCLUSIONS: FC was the optimal fecal marker for monitoring disease activity in postoperative CD and was superior to CRP and CDAI. FL offered modest sensitivity for detecting recurrent disease, whereas S100A12 was sensitive but had low specificity and NPV.

Telomere length measurement on the Roche LightCycler 480 Platform.

BACKGROUND: The average length of telomeres as measured in genomic DNA from human peripheral blood leukocytes is proving to be a potential biomarker of great interest, particularly with respect to studies of aging, specific diseases, and the effects of various stresses on overall health. AIMS: The aim of this study was to establish an effective real-time quantitative polymerase chain reaction (qPCR) method for telomere length measurement on the Roche LightCycler® 480 (LC480) real-time PCR platform. METHODS: Measurement of relative average telomere length was achieved by comparing products amplified from telomere-specific primers and single copy reference gene primers in a ratio (T/S). RESULTS: Extensive testing led us to conclude that a modification of the original two-plate T/S assay was more compatible with this platform than the recently developed single-plate assay, and that choice of hot-start Taq polymerase and intercalating dye were critical factors. CONCLUSIONS: This modified assay generates reliable measurements as judged by correlation with data derived by the telomeric restriction fragment Southern blot-based method.

G-quadruplex structures and CpG methylation cause drop-out of the maternal allele in polymerase chain reaction amplification of the imprinted MEST gene promoter.

We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5' end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs) in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR) buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings.

Promoter methylation correlates with reduced Smad4 expression in advanced prostate cancer.

BACKGROUND: Transforming growth factor-beta (TGF-beta) is a potent growth inhibitor in a wide range of cell types. A transducer of TGF-beta signaling known as Mothers against decapentaplegic homologue 4 (Smad4) is a known tumor suppressor found on chromosome 18q21.1 and is typically inactivated by deletion or mutation in pancreatic and colorectal cancers. The purpose of the article is to investigate Smad4 expression, gene copy number and methylation status in advanced cases of prostate cancer. METHODS: We have employed Methylation Specific PCR (MSP) to identify methylation sites within the Smad4 promoter and combined this with quantitative real-time PCR to look for correlates between methylation status and Smad4 expression and to examine androgen receptor (AR) expression. Bacterial artificial chromosome-comparative genomic hybridization (BAC-CGH) has been used to look for genomic amplifications and deletions which may also contribute to expression changes. RESULTS: We fail to find evidence of genomic deletions or amplifications affecting the Smad4 locus on chromosome 18 but show a correlation between promoter methylation and the loss of Smad4 expression in the same material. We confirm that the AR locus on the X chromosome is amplified in 30% of the advanced clinical samples and that this correlates with increased transcript levels as previously reported by other groups. CONCLUSION: This indicates that epigenetic changes affect the expression of the Smad4 protein in prostate cancer and points to methylation of the promoter as a novel marker of and contributor to the disease warranting further study.

RASSF1A promoter methylation is frequently detected in both pre-malignant and non-malignant microdissected prostatic epithelial tissues.

BACKGROUND: The RASSF1A gene is a tumor suppressor gene inactivated by hypermethylation in a very wide variety of malignant tumors including prostate cancer. METHODS: In this study we have used laser capture microdissection to provide pure cell populations to investigate the methylation status of 16 CpG sites in the promoter region of this gene in prostatic intra-epithelial neoplasia, in histologically normal epithelial cells associated with these lesions and in epithelial cells from benign prostatic hyperplasia. RESULTS: Unexpectedly, frequent methylation, detected by sequence analysis following bisulphite treatment, was observed in benign epithelium as well as in the lesions associated with prostatic intra-epithelial neoplasia and at high risk of cancer formation. Fifty percent or more CpG sites were methylated in 7/14 prostatic intra-epithelial neoplasms, 8/11 histologically normal epithelial cells and 8/12 specimens of benign prostatic tissue. CONCLUSION: These observations suggest that methylation of the RASSF1A gene is present in both pre-malignant and benign epithelia and suggests quantitation is required for it to be an effective marker of early prostate cancer.

Targeted disruption of the 3p12 gene, Dutt1/Robo1, predisposes mice to lung adenocarcinomas and lymphomas with methylation of the gene promoter.

The DUTT1 gene is located on human chromosome 3, band p12, within a region of nested homozygous deletions in breast and lung tumors. It is therefore a candidate tumor suppressor gene in humans and is the homologue (ROBO1) of the Drosophila axonal guidance receptor gene, Roundabout. We have shown previously that mice with a targeted homozygous deletion within the Dutt1/Robo1 gene generally die at birth due to incomplete lung development: survivors die within the first year of life with epithelial bronchial hyperplasia as a common feature. Because Dutt1/Robo1 heterozygous mice develop normally, we have determined their tumor susceptibility. Mice with a targeted deletion within one Dutt1/Robo1 allele spontaneously develop lymphomas and carcinomas in their second year of life with a 3-fold increase in incidence compared with controls: invasive lung adenocarcinomas are by far the predominant carcinoma. In addition to the mutant allele, loss of heterozygosity analysis indicates that these tumors retain the structurally normal allele but with substantial methylation of the gene's promoter. Substantial reduction of Dutt1/Robo1 protein expression in tumors is observed by Western blotting and immunohistochemistry. This suggests that Dutt1/Robo1 is a classic tumor suppressor gene requiring inactivation of both alleles to elicit tumorigenesis in these mice.