Aberrant Gene Promoter Methylation Associated with Sporadic Multiple Colorectal Cancer

Background

Colorectal cancer (CRC) multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect.

Methodology/Principal Findings

We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals) and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2), RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008) and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047) as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006). Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17), SFRP1 (r = 0.83, 0.06), HPP1 (r = 0.64, p = 0.17), 3OST2 (r = 0.83, p = 0.06) and GATA4 (r = 0.6, p = 0.24). Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant difference in any evaluated gene.

Conclusions

These results provide a proof-of-concept that gene promoter methylation is associated with tumor multiplicity. This underlying epigenetic defect may have noteworthy implications in the prevention of patients with sporadic CRC.     

Role of Toll-Like Receptor 4 in Colorectal Carcinogenesis: A Meta-Analysis

Objective

This meta-analysis was performed to evaluate the role of toll-like receptor 4 (TLR-4) in colorectal carcinogenesis.

Methods

The PubMed, CISCOM, CINAHL, Web of Science, Google Scholar, EBSCO, Cochrane Library, and CBM databases were searched from inception through November 1st, 2013 without language restrictions. Odds ratios (ORs) or standardized mean differences (SMD) with their 95% confidence intervals (CI) were calculated.

Results

Fourteen case-control studies met the inclusion criteria for this meta-analysis. A total of 1,209 colorectal cancer (CRC) cases and 1,218 healthy controls were involved in this meta-analysis. Two common polymorphisms (299 A>G and 399 C>T) in the TLR-4 gene, TLR-4 mRNA and protein expression were assessed. Our meta-analysis results revealed that the TLR-4 399 C>T polymorphism might increase the risk of CRC (allele model: OR = 1.77, 95%CI = 1.32∼2.36, P<0.001; dominant model: OR = 1.83, 95%CI = 1.32∼2.52, P<0.001; respectively). However, we found no correlation between the TLR-4 299 A>G polymorphism and CRC risk (all P>0.05). A subgroup analysis by ethnicity suggested that TLR-4 genetic polymorphisms were associated with an increased risk of CRC among Asians (allele model: OR = 1.50, 95%CI = 1.19∼1.88, P = 0.001; dominant model: OR = 1.49, 95%CI = 1.16∼1.92, P = 0.002; respectively), but not among Caucasians and Africans (all P>0.05). Furthermore, our results showed that TLR-4 mRNA and protein levels in CRC patients were higher than those in healthy controls (TLR-4 mRNA: SMD  = 2.51, 95%CI  = 0.98∼4.05, P = 0.001; TLR-4 protein: OR  = 4.75, 95%CI  = 1.16∼19.36, P = 0.030; respectively).

Conclusion

Our findings provide empirical evidence that TLR-4 may play an important role in colorectal carcinogenesis. Thus, TLR-4 is a promising potential biomarker for the early diagnosis of CRC.     

Genetic Variant rs7758229 in 6q26–q27 Is Not Associated with Colorectal Cancer Risk in a Chinese Population

Background

A recent genome-wide association study has identified a new genetic variant rs7758229 in SLC22A3 for colorectal cancer susceptibility in a Japanese population, but it is unknown whether this newly identified variant is associated with colorectal cancer in other populations, including the Chinese population.

Methods

We examined the associations between rs7758229 and colorectal cancer risk among 1,147 cases and 1,203 controls matched by age and sex. Logistic regression model was used to assess the associations.

Results

No significant association was found between rs7758229 and colorectal cancer risk (OR = 0.95, 95%CI  = 0.84–1.09, P = 0.463). Similar results were observed in the stratification of tumor location (OR  = 0.94, 95%CI = 0.80–1.11, P = 0.481 for colon cancer, and OR  = 0.96, 95%CI  = 0.82–1.13, P = 0.621 for rectum cancer).

Conclusions

Our findings did not support an association between rs7758229 in 6q26-q27 and the risk of colorectal cancer in a Chinese population.     

Frequent Epigenetic Silencing of the Folate-Metabolising Gene Cystathionine-Beta-Synthase in Gastrointestinal Cancer

Background

Both gastric and colorectal cancers (CRC) are the most frequently occurring malignancies worldwide with the overall survival of these patients remains unsatisfied. Identification of tumor suppressor genes (TSG) silenced by promoter CpG methylation uncovers mechanisms of tumorigenesis and identifies new epigenetic biomarkers for early cancer detection and prognosis assessment. Cystathionine-beta-synthase (CBS) functions in the folate metabolism pathway, which is intricately linked to methylation of genomic DNA. Dysregulation of DNA methylation contributes substantially to cancer development.

Methodology/Principal Findings

To identify potential TSGs silenced by aberrant promoter methylation in CRC, we analyzed tumor and adjacent tissues from CRC cases using the Illumina Human Methylation45 BeadChip. We identified hypermethylation of the CBS gene in CRC samples, compared to adjacent tissues. Methylation and decreased mRNA expression of CBS were detected in most CRC cell lines by methylation-specific PCR and semiquantitative RT-PCR, as well as in gastric cancer. Treatment with 5-aza-2''-deoxycytidine and/or trichostatin A reversed methylation and restored CBS mRNA expression indicating a direct effect. Aberrant methylation was further detected in 31% of primary CRCs (29 of 96) and 55% of gastric tumors (11 of 20). In contrast, methylation was seldom found in normal tissues adjacent to the tumor. CBS methylation was associated with KRAS mutations in primary CRCs (P = 0.04, by χ²-test). However, no association was found between CBS methylation or KRAS mutations with cancer relapse/metastasis in Stage II CRC patients.

Conclusion

A novel finding from this study is that the folate metabolism enzyme CBS mRNA levels are frequently downregulated through CpG methylation of the CBS gene in gastric cancer and CRC, suggesting that CBS functions as a tumor suppressor gene. These findings warrant further study of CBS as an epigenetic biomarker for molecular diagnosis of gastrointestinal cancers.     

Cryptochrome 1 Overexpression Correlates with Tumor Progression and Poor Prognosis in Patients with Colorectal Cancer

Background

Clock genes drive about 5–15% of genome-wide mRNA expression, and disruption of the circadian clock may deregulate the cell''s normal biological functions. Cryptochrome 1 is a key regulator of the circadian feedback loop and plays an important role in organisms. The present study was conducted to investigate the expression of Cry1 and its prognostic significance in colorectal cancer (CRC). In addition, the function of Cry1 in human CRC was investigated in cell culture models.

Methods

Real-time quantitative PCR, Western blot analysis and immunohistochemistry were used to explore Cry1 expression in CRC cell lines and primary CRC clinical specimens. MTT and colony formation assays were used to determine effects on cellular proliferation ability. The animal model was used to explore the Cry1 impact on the tumor cellular proliferation ability in vivo. Transwell assays were performed to detect the migration ability of the cell lines. Statistical analyzes were applied to evaluate the diagnostic value and the associations of Cry1 expression with clinical parameters.

Results

Cry1 expression was up regulated in the majority of the CRC cell lines and 168 primary CRC clinical specimens at the protein level. Clinical pathological analysis showed that Cry1 expression was significantly correlated with lymph node metastasis (p = 0.004) and the TNM stage (p = 0.003). High Cry1 expression was associated with poor overall survival in CRC patients (p = 0.010). Experimentally, we found that up-regulation of Cry1 promoted the proliferation and migration of HCT116 cells, while down-regulation of Cry1 inhibited the colony formation and migration of SW480 cells.

Conclusions

These results suggest that Cry1 likely plays important roles in CRC development and progression andCry1 may be a prognostic biomarker and a promising therapeutic target for CRC.     

Identification of Kininogen-1 as a Serum Biomarker for the Early Detection of Advanced Colorectal Adenoma and Colorectal Cancer

Background

Serum markers represent potential tools for the detection of colorectal cancer (CRC). The aim of this study was to obtain proteomic expression profiles and identify serum markers for the early detection of CRC.

Methods

Proteomic profiles of serum samples collected from 35 healthy volunteers, 35 patients with advanced colorectal adenoma (ACA), and 40 patients with CRC were compared using Clinprot technology. Using enzyme-linked immunosorbent assays (ELISAs), 366 sera samples were additionally analyzed, and immunohistochemistry studies of 400 tissues were used to verify the expression of kininogen-1 and its value in the early detection of CRC.

Results

Predicting models were established among the three groups, and kininogen-1 was identified as a potential marker for CRC using Clinprot technology. ELISAs also detected significantly higher serum kininogen-1 levels in ACA and CRC patients compared to controls (P<0.05). Furthermore, the area under the receiver operating characteristic curve (AUC) for serum kininogen-1 in the diagnosis of ACA was 0.635 (P = 0.003), and for serum carcinoembryonic antigen (CEA) was 0.453 (P = 0.358). The sensitivity, specificity, and accuracy of serum kininogen-1 for diagnosing Duke’s stage A and B CRC was 70.13%, 65.88%, and 67.90%, respectively, whereas serum CEA was 38.96%, 85.88%, and 63.58%, respectively. Moreover, immunohistochemistry showed that expression of kininogen-1 was significantly higher in CRC and ACA tissues than in normal mucosa (48.39% vs. 15.58% vs. 0%, P<0.05).

Conclusions

These results suggest that Clinprot technology provides a useful tool for the diagnosis of CRC, and kininogen-1 is a potential serum biomarker for the early detection of advanced colorectal adenoma and CRC.     

Prognostic Significance of Cyclin D1 Expression in Colorectal Cancer: A Meta-Analysis of Observational Studies

Objective

Cyclin D1 plays a vital role in cancer cell cycle progression and is overexpressed in many human cancers, including colorectal cancer (CRC). However, the prognostic value of cyclin D1 overexpression in colorectal cancer is conflicting and heterogeneous. We conducted a meta-analysis to more precisely evaluate its prognostic significance.

Methods

A comprehensive literature search for relevant studies published up to January 2014 was performed using PubMed, EMBASE, and ISI Web of Science. The pooled hazard ratio (HR) with 95% confidence intervals (CI) was used to estimate the effects.

Results

22 studies with 4150 CRC patients were selected to evaluate the association between cyclin D1 and overall survival (OS), disease-free survival (DFS) and clinicopathological parameters. In a random-effects model, the results showed that cyclin D1 overexpression in CRC was significantly associated with both poor OS (HR = 0.73, 95% CI: 0.63–0.85, P<0.001) and DFS (HR = 0.60, 95% CI: 0.44–0.82, P = 0.001). Additionally, cyclin D1 overexpression was significantly associated with more relative older patients (≥60 years) (OR 0.62, 95% CI 0.44–0.89, P = 0.009), T3,4 tumor invasion (OR 0.70, 95% CI 0.57–0.85, P<0.001), N positive (OR 0.75, 95% CI 0.60–0.95, P = 0.016) and distant metastasis (OR 0.60, 95% CI 0.36–0.99, P = 0.047) of CRC.

Conclusion

The meta-analysis results indicated that cyclin D1 is an unfavorable prognostic factor for CRC. Cyclin D1 overexpression might be associated with poor clinical outcome and some clinicopathological factors such as age, T category, N category and distant metastasis in CRC patients.     

Mechanisms of Topoisomerase I (TOP1) Gene Copy Number Increase in a Stage III Colorectal Cancer Patient Cohort

Background

Topoisomerase I (Top1) is the target of Top1 inhibitor chemotherapy. The TOP1 gene, located at 20q12-q13.1, is frequently detected at elevated copy numbers in colorectal cancer (CRC). The present study explores the mechanism, frequency and prognostic impact of TOP1 gene aberrations in stage III CRC and how these can be detected by fluorescent in situ hybridization (FISH).

Methods

Nine CRC cell line metaphase spreads were analyzed by FISH with a TOP1 probe in combination with a reference probe covering either the centromeric region of chromosome 20 (CEN-20) or chromosome 2 (CEN-2). Tissue sections from 154 chemonaive stage III CRC patients, previously studied with TOP1/CEN-20, were analyzed with TOP1/CEN-2. Relationships between biomarker status and overall survival (OS), time to recurrence (TTR) in CRC and time to local recurrence (LR; rectal cancer only) were determined.

Results

TOP1 aberrations were observed in four cell line metaphases. In all cell lines CEN-2 was found to reflect chromosomal ploidy levels and therefore the TOP1/CEN-2 probe combination was selected to identify TOP1 gene gains (TOP1/CEN-2≥1.5). One hundred and three patients (68.2%) had TOP1 gain, of which 15 patients (14.6%) harbored an amplification (TOP1/CEN-20≥2.0). TOP1 gene gain did not have any association with clinical endpoints, whereas TOP1 amplification showed a non-significant trend towards longer TTR (multivariate HR: 0.50, p = 0.08). Once amplified cases were segregated from other cases of gene gain, non-amplified gene increases (TOP1/CEN-2≥1.5 and TOP1/CEN-20<2.0) showed a trend towards shorter TTR (univariate HR: 1.57, p = 0.07).

Conclusions

TOP1 gene copy number increase occurs frequently in stage III CRC in a mechanism that often includes CEN-20. Using CEN-2 as a measurement for tumor ploidy levels, we were able to discriminate between different mechanisms of gene gain, which appeared to differ in prognostic impact. TOP1 FISH guidelines have been updated.     

Polymorphisms in Alcohol Metabolism Genes ADH1B and ALDH2, Alcohol Consumption and Colorectal Cancer

Background

Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Epidemiological risk factors for CRC included alcohol intake, which is mainly metabolized to acetaldehyde by alcohol dehydrogenase and further oxidized to acetate by aldehyde dehydrogenase; consequently, the role of genes in the alcohol metabolism pathways is of particular interest. The aim of this study is to analyze the association between SNPs in ADH1B and ALDH2 genes and CRC risk, and also the main effect of alcohol consumption on CRC risk in the study population.

Methodology/Principal Findings

SNPs from ADH1B and ALDH2 genes, included in alcohol metabolism pathway, were genotyped in 1694 CRC cases and 1851 matched controls from the Molecular Epidemiology of Colorectal Cancer study. Information on clinicopathological characteristics, lifestyle and dietary habits were also obtained. Logistic regression and association analysis were conducted. A positive association between alcohol consumption and CRC risk was observed in male participants from the Molecular Epidemiology of Colorectal Cancer study (MECC) study (OR = 1.47; 95%CI = 1.18-1.81). Moreover, the SNPs rs1229984 in ADH1B gene was found to be associated with CRC risk: under the recessive model, the OR was 1.75 for A/A genotype (95%CI = 1.21-2.52; p-value = 0.0025). A path analysis based on structural equation modeling showed a direct effect of ADH1B gene polymorphisms on colorectal carcinogenesis and also an indirect effect mediated through alcohol consumption.

Conclusions/Significance

Genetic polymorphisms in the alcohol metabolism pathways have a potential role in colorectal carcinogenesis, probably due to the differences in the ethanol metabolism and acetaldehyde oxidation of these enzyme variants.     

The Knowledge of Colorectal Cancer Symptoms and Risk Factors among 10,078 Screening Participants: Are High Risk Individuals More Knowledgeable?

Objectives

We tested the a priori hypothesis that self-perceived and real presences of risks for colorectal cancer (CRC) are associated with better knowledge of the symptoms and risk factors for CRC, respectively.

Methods

One territory-wide invitation for free CRC screening between 2008 to 2012 recruited asymptomatic screening participants aged 50–70 years in Hong Kong. They completed survey items on self-perceived and real presences of risks for CRC (advanced age, male gender, positive family history and smoking) as predictors, and knowledge of CRC symptoms and risk factors as outcome measures, respectively. Their associations were evaluated by binary logistic regression analyses.

Results

From 10,078 eligible participants (average age 59 years), the mean knowledge scores for symptoms and risk factors were 3.23 and 4.06, respectively (both score range 0–9). Male gender (adjusted odds ratio [AOR] = 1.34, 95% C.I. 1.20–1.50, p<0.01), self-perception as not having any risks for CRC (AOR = 1.12, 95% C.I. 1.01–1.24, p = 0.033) or uncertainty about having risks (AOR = 1.94, 95% C.I. 1.55–2.43, p<0.001), smoking (AOR 1.38, 95% C.I. 1.11–1.72, p = 0.004), and the absence of family history (AOR 0.61 to 0.78 for those with positive family history, p<0.001) were associated with poorer knowledge scores (≤4) of CRC symptoms. These factors remained significant for knowledge of risk factors.

Conclusions

Male and smokers were more likely to have poorer knowledge but family history of CRC was associated with better knowledge. Since screening of these higher risk individuals could lead to greater yield of colorectal neoplasm, educational interventions targeted to male smokers were recommended.     

IGFBP3 Methylation Is a Novel Diagnostic and Predictive Biomarker in Colorectal Cancer

Background and Aim

Aberrant hypermethylation of cancer-related genes has emerged as a promising strategy for the development of diagnostic, prognostic and predictive biomarkers in human cancer, including colorectal cancer (CRC). The aim of this study was to perform a systematic and comprehensive analysis of a panel of CRC-specific genes as potential diagnostic, prognostic and predictive biomarkers in a large, population-based CRC cohort.

Patients and Methods

Methylation status of the SEPT9, TWIST1, IGFBP3, GAS7, ALX4 and miR137 genes was studied by quantitative bisulfite pyrosequencing in a population-based cohort of 425 CRC patients.

Results

Methylation levels of all genes analyzed were significantly higher in tumor tissues compared to normal mucosa (p<0.0001); however, cancer-associated hypermethylation was most frequently observed for miR137 (86.7%) and IGFBP3 (83%) in CRC patients. Methylation analysis using the combination of these two genes demonstrated greatest accuracy for the identification of colonic tumors (sensitivity 95.5%; specificity 90.5%). Low levels of IGFBP3 promoter methylation emerged as an independent risk factor for predicting poor disease free survival in stage II and III CRC patients (HR = 0.49, 95% CI: 0.28–0.85, p = 0.01). Our results also suggest that stage II & III CRC patients with high levels of IGFBP3 methylation do not benefit from adjuvant 5FU-based chemotherapy.

Conclusion

By analyzing a large, population-based CRC cohort, we demonstrate the potential clinical significance of miR137 and IGFBP3 hypermethylation as promising diagnostic biomarkers in CRC. Our data also revealed that IGFBP3 hypermethylation may serve as an independent prognostic and predictive biomarker in stage II and III CRC patients.     

Germline Variation in Colorectal Risk Loci Does Not Influence Treatment Effect or Survival in Metastatic Colorectal Cancer

Background

Colorectal cancer (CRC) risk is partly conferred by common, low-penetrance single nucleotide polymorphisms (SNPs). We hypothesized that these SNPs are associated with outcomes in metastatic CRC.

Methods

Six candidate SNPs from 8q24, 10p14, 15q13, 18q21 were investigated for their association with response rate (RR), time to progression (TTP) and overall survival (OS) among 524 patients treated on a phase III clinical trial of first-line chemotherapy for metastatic CRC.

Results

rs10795668 was weakly associated with TTP (p = 0.02), but not RR or OS. No other SNPs carried statistically significant HRs for any of the primary outcomes (RR, TTP or OS).

Conclusion

Common low-penetrance CRC risk SNPs were not associated with outcomes among patients with metastatic CRC.     

A High Degree of LINE-1 Hypomethylation Is a Unique Feature of Early-Onset Colorectal Cancer

Objective

Early-onset colorectal cancer (CRC) represents a clinically distinct form of CRC that is often associated with a poor prognosis. Methylation levels of genomic repeats such as LINE-1 elements have been recognized as independent factors for increased cancer-related mortality. The methylation status of LINE-1 elements in early-onset CRC has not been analyzed previously.

Design

We analyzed 343 CRC tissues and 32 normal colonic mucosa samples, including 2 independent cohorts of CRC diagnosed ≤50 years old (n = 188), a group of sporadic CRC >50 years (MSS n = 89; MSI n = 46), and a group of Lynch syndrome CRCs (n = 20). Tumor mismatch repair protein expression, microsatellite instability status, LINE-1 and MLH1 methylation, somatic BRAF V600E mutation, and germline MUTYH mutations were evaluated.

Results

Mean LINE-1 methylation levels (±SD) in the five study groups were early-onset CRC, 56.6% (8.6); sporadic MSI, 67.1% (5.5); sporadic MSS, 65.1% (6.3); Lynch syndrome, 66.3% (4.5) and normal mucosa, 76.5% (1.5). Early-onset CRC had significantly lower LINE-1 methylation than any other group (p<0.0001). Compared to patients with <65% LINE-1 methylation in tumors, those with ≥65% LINE-1 methylation had significantly better overall survival (p = 0.026, log rank test).

Conclusions

LINE-1 hypomethylation constitutes a potentially important feature of early-onset CRC, and suggests a distinct molecular subtype. Further studies are needed to assess the potential of LINE-1 methylation status as a prognostic biomarker for young people with CRC.     

Association of caucasian-identified variants with colorectal cancer risk in singapore chinese

Background

Genome-wide association studies (GWAS) in Caucasians have identified fourteen index single nucleotide polymorphisms (iSNPs) that influence colorectal cancer (CRC) risk.

Methods

We investigated the role of eleven iSNPs or surrogate SNPs (sSNPs), in high linkage disequilibrium (LD, r²≥0.8) and within 100 kb vicinity of iSNPs, in 2,000 age- and gender-matched Singapore Chinese (SCH) cases and controls.

Results

Only iSNP rs6983267 at 8q24.21 and sSNPs rs6695584, rs11986063, rs3087967, rs2059254, and rs7226855 at 1q41, 8q23.3, 11q23.1, 16q22.1 and 18q21.1 respectively showed evidence of association with CRC risk, with odds ratios (OR) ranging from 1.13 to 1.40. sSNP rs827401 at 10p14 was associated with rectal cancer risk (OR = 0.74, 95% CI 0.63–0.88) but not disease prognosis (OR = 0.91, 95% CI 0.69–1.20). Interestingly, sSNP rs3087967 at 11q23.1 was associated with CRC risk in men (OR = 1.34, 95% CI 1.14–1.58) but not women (OR = 1.07, 95% CI: 0.88–1.29), suggesting a gender-specific role. Half of the Caucasian-identified variants, including the recently fine-mapped BMP pathway loci, BMP4, GREM1, BMP2 and LAMA 5, did not show any evidence for association with CRC in SCH (OR ∼1; p-value >0.1). Comparing the results of this study with that of the Northern and Hong Kong Chinese, only variants at chromosomes 8q24.21, 10p14, 11q23.1 and 18q21.1 were replicated in at least two out of the three Chinese studies.

Conclusions

The contrasting results between Caucasians and Chinese could be due to different LD patterns and allelic frequencies or genetic heterogeneity. The results suggest that additional common variants contributing to CRC predisposition remained to be identified.     

Association between MLH1 -93G>A Polymorphism and Risk of Colorectal Cancer

Background

The -93G>A (rs1800734) polymorphism located in the promoter of mismatch repair gene, MLH1, has been identified as a low-penetrance variant for cancer risk. Many published studies have evaluated the association between the MLH1 -93G>A polymorphism and colorectal cancer (CRC) risk. However, the results remain conflicting rather than conclusive.

Objective

The aim of this study was to assess the association between the MLH1 -93G>A polymorphism and the risk of CRC.

Methods

To derive a more precise estimation of the association, a meta-analysis of six studies (17,791 cases and 13,782 controls) was performed. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to evaluate the strength of the association. Four of these published studies were performed on subjects of known microsatellite instability (MSI) status. An additional analysis including 742 cases and 10,895 controls was used to assess the association between the MLH1 -93G>A polymorphism and the risk of MSI-CRC.

Results

The overall results indicated that the variant genotypes were associated with a significantly increased risk of CRC (AG versus GG: OR = 1.06, 95% CI = 1.01–1.11; AA/AG versus GG: OR = 1.06, 95% CI = 1.01–1.11). This increased risk was also found during stratified analysis of MSI status (AA versus GG: OR = 2.52, 95% CI = 1.94–3.28; AG versus GG: OR = 1.29, 95% CI = 1.10–1.52; AA/AG versus GG: OR = 1.45, 95% CI = 1.24–1.68; AA versus AG/GG: OR = 2.29, 95% CI = 1.78–2.96). Egger’s test did not show any evidence of publication bias.

Conclusion

Our results suggest that the MLH1 -93G>A polymorphism may contribute to individual susceptibility to CRC and act as a risk factor for MSI-CRC.     

A Predictive Model Combining Fecal Calgranulin B and Fecal Occult Blood Tests Can Improve the Diagnosis of Colorectal Cancer

Aim

Current fecal screening tools for colorectal cancer (CRC), such as fecal occult blood tests (FOBT), are limited by their low sensitivity. Calgranulin B (CALB) was previously reported as a candidate fecal marker for CRC. This study investigated whether a combination of the FOBT and fecal CALB has increased sensitivity and specificity for a diagnosis of CRC.

Materials and Methods

Patients with CRC (n = 175), and healthy individuals (controls; n = 151) were enrolled into the development (81 cases and 51 controls) and validation (94 cases and 100 controls) sets. Stool samples were collected before bowel preparation. CALB levels were determined by western blotting. FOBT and fecal CALB results were used to develop a predictive model based on logistic regression analysis. The benefit of adding CALB to a model with only FOBT was evaluated as an increased area under the receiver operating curve (AUC), partial AUC, and reclassification improvement (RI) in cases and controls, and net reclassification improvement (NRI).

Results

Mean CALB level was significantly higher in CRC patients than in controls (P<0.001). CALB was not associated with tumor stage or cancer site, but positivity on the FOBT was significantly higher in advanced than in earlier tumor stages. At a specificity of 90%, the cross-validated AUC and sensitivity were 89.81% and 82.72%, respectively, in the development set, and 92.74% and 79.79%, respectively, in the validation set. The incremental benefit of adding CALB to the model, as shown by the increase in AUC, had a p-value of 0.0499. RI in cases and controls and NRI all revealed that adding CALB significantly improved the prediction model.

Conclusion

A predictive model using a combination of FOBT and CALB may have greater sensitivity and specificity and AUC for predicting CRC than models using a single marker.     

Down-regulation of ECRG4, a candidate tumor suppressor gene, in human breast cancer

Introduction

ECRG4/C2ORF40 is a potential tumor suppressor gene (TSG) recently identified in esophageal carcinoma. Its expression, gene copy number and prognostic value have never been explored in breast cancer.

Methods

Using DNA microarray and array-based comparative genomic hybridization (aCGH), we examined ECRG4 mRNA expression and copy number alterations in 353 invasive breast cancer samples and normal breast (NB) samples. A meta-analysis was done on a large public retrospective gene expression dataset (n = 1,387) in search of correlations between ECRG4 expression and histo-clinical features including survival.

Results

ECRG4 was underexpressed in 94.3% of cancers when compared to NB. aCGH data revealed ECRG4 loss in 18% of tumors, suggesting that DNA loss is not the main mechanism of underexpression. Meta-analysis showed that ECRG4 expression was significantly higher in tumors displaying earlier stage, smaller size, negative axillary lymph node status, lower grade, and normal-like subtype. Higher expression was also associated with disease-free survival (DFS; HR = 0.84 [0.76–0.92], p = 0.0002) and overall survival (OS; HR = 0.72 [0.63–0.83], p = 5.0E-06). In multivariate analysis including the other histo-clinical prognostic features, ECRG4 expression remained the only prognostic factor for DFS and OS.

Conclusions

Our data suggest that ECRG4 is a candidate TSG in breast cancer, the expression of which may help improve the prognostication. If functional analyses confirm this TSG role, restoring ECRG4 expression in the tumor may represent a promising therapeutic approach.     

The Functional Significance of MicroRNA-29c in Patients with Colorectal Cancer: A Potential Circulating Biomarker for Predicting Early Relapse

Background

The recurrence of colorectal cancer (CRC) is frequent within the first year of curative resection surgery and may be unavoidable. microRNAs have been suggested to play roles in carcinogenesis and cancer recurrence. We recently identified microRNA-29c (miRNA-29c) as a predictor of early recurrence in CRC. In the present study, we further investigated the functions and serum level of miRNA-29c in relation to early recurrence of CRC.

Methods

First we further confirmed overexpression of miRNA-29c in non-early relapse subjects. Gain-of-function in vitro studies were used to evaluate the effect of miRNA-29c on cell proliferation, migration, invasion, and cell cycle progression. The colon cancer cell line Caco2 and a stable clone overexpressing miRNA-29c were xenografted to evaluate the in vivo effect of miRNA-29c in null mice. Finally, circulating miRNA-29c was investigated as a potential biomarker for identifying early relapse.

Results

miRNA-29c expression significantly decreased during early relapse compared to non-early relapse in UICC stage II and III CRC patients (P = 0.021). In vitro studies showed that overexpression of miRNA-29c inhibited cell proliferation and migration. The cell cycle studies also revealed that miRNA-29c caused an accumulation of the G1 and G2 population. In vivo, miRNA-29c suppressed tumor growth in null mice. The serum miRNA-29c increased significantly in early relapsed patients compared to non-early elapsed patients (P = 0.012).

Conclusions

miRNA-29c shows anti-tumorigenesis activity, and preoperative circulating miRNA-29c levels can be used to predict postoperative early relapse of CRC.     

Comparative profiling of primary colorectal carcinomas and liver metastases identifies LEF1 as a prognostic biomarker

Purpose

We sought to identify genes of clinical significance to predict survival and the risk for colorectal liver metastasis (CLM), the most common site of metastasis from colorectal cancer (CRC).

Patients and Methods

We profiled gene expression in 31 specimens from primary CRC and 32 unmatched specimens of CLM, and performed Significance Analysis of Microarrays (SAM) to identify genes differentially expressed between these two groups. To characterize the clinical relevance of two highly-ranked differentially-expressed genes, we analyzed the expression of secreted phosphoprotein 1 (SPP1 or osteopontin) and lymphoid enhancer factor-1 (LEF1) by immunohistochemistry using a tissue microarray (TMA) representing an independent set of 154 patients with primary CRC.

Results

Supervised analysis using SAM identified 963 genes with significantly higher expression in CLM compared to primary CRC, with a false discovery rate of <0.5%. TMA analysis showed SPP1 and LEF1 protein overexpression in 60% and 44% of CRC cases, respectively. Subsequent occurrence of CLM was significantly correlated with the overexpression of LEF1 (chi-square p = 0.042), but not SPP1 (p = 0.14). Kaplan Meier analysis revealed significantly worse survival in patients with overexpression of LEF1 (p<0.01), but not SPP1 (p = 0.11). Both univariate and multivariate analyses identified stage (p<0.0001) and LEF1 overexpression (p<0.05) as important prognostic markers, but not tumor grade or SPP1.

Conclusion

Among genes differentially expressed between CLM and primary CRC, we demonstrate overexpression of LEF1 in primary CRC to be a prognostic factor for poor survival and increased risk for liver metastasis.