Differential expression and prognostic role of selected genes in colorectal cancer patients.

AIM: Colorectal cancer (CRC) is one of the most common malignant diseases. The aim of our study was to describe the expression status of 12 selected candidate genes, by comparing paired samples of healthy colon mucosa and tumour tissues and to correlate obtained data with clinical and pathological features, with the goal of revealing associations for individual gene expressions and tumour behaviour. MATERIALS AND METHODS: Samples from 53 patients with CRC were analyzed. Patients were divided into two groups based on the presence or absence of distant metastases at the time of primary tumour surgery. Expression levels were assessed by quantitative real-time polymerase chain reaction. RESULTS: We found changes in the expression of 10 out of 12 analyzed genes. Four genes were significantly up-regulated in tumour tissues: leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5; p<0.001), collagen triple helix repeat containing 1 (CTHRC1; p<0.001), visinin-like 1 (VSNL1; p<0.001) and versican (VCAN; p=0.001). Six genes were down-regulated: destrin (DSTN; p=0.004), mesoderm induction early response 1, family member 3 (MIER3; p<0.001), acyl-CoA synthetase long-chain family member 5 (ACSL5; p=0.002), mitogen-activated protein kinase 1/ERK (MAPK1; p<0.001), claudin 23 (CLDN23; p<0.001) and solute carrier family 26 (sulfate transporter), member 2 (SLC26A2; p<0.001). We recorded longer overall survival (OS) in the group of patients with higher expression of VSNL1 (p=0.032). Patients with more pronounced down-regulation of CLDN23 had shorter OS (p=0.045). In the group of patients without distant metastases, longer OS and disease-free interval (DFI) were found for patients with higher SLC26A2 expression in tumour tissues (p=0.036 and p=0.011, respectively). In the same group, lower expression of VSNL1 in healthy tissue corresponded to a longer DFI (p=0.020), smaller decrease of SLC26A2 and ACSL5 meant longer DFI (p=0.041 and p=0.040, respectively), as did greater increase of LGR5 expression (p=0.026). CONCLUSION: We identified differences in the expression of 10 genes in colorectal cancer tissue compared to healthy colon mucosa, and found prognostic significance for these changes which could be used for the development of a disease risk scoring system.

Detection of frequent ABCB1 polymorphisms by high-resolution melting curve analysis and their effect on breast carcinoma prognosis.

BACKGROUND: The ABCB1 gene encodes P-glycoprotein implicated in the development of cellular drug resistance. The aim of this study was to develop high-resolution melting (HRM) analysis for determination of ABCB1 polymorphisms and evaluate their associations with clinical data of breast carcinoma patients. METHODS: HRM analysis was designed to assess five single nucleotide polymorphisms (SNPs) in ABCB1 (rs2214102, rs1128503, rs2032582, rs2032583 and rs1045642) in genomic DNA from 103 breast carcinoma patients. Results were confirmed by direct DNA sequencing. RESULTS: HRM analysis revealed distinct patterns of melting curves for the respective genotypes of all followed SNPs. Sensitivity of HRM analysis compared with direct DNA sequencing was superior (97.1% vs. 93.9%). The overall accuracy of HRM was 97.6%. The coefficients of variation in replicate experiments encompassed the range 0.002%-0.038%. On the basis of the examined SNPs, one strong haplotype block containing rs2032582 and rs1128503 SNPs was identified. Significant associations of rs2032582 SNP with tumor size, negative HER-2/neu status, and family history of breast carcinoma were found. Patients carrying the ancestral homozygous genotype (GG) in rs2214102 had significantly worse progression-free survival in comparison with carriers of the non-ancestral allele (A) in the adjuvant set (p=0.005). CONCLUSIONS: A rapid, accurate, low-cost and time-effective method for screening ABCB1 SNPs was developed. Significant associations of ABCB1 rs2032582 and rs2214102 SNPs with prognostic factors and survival of patients were found.

Variation in the vitamin D receptor gene is not associated with risk of colorectal cancer in the Czech Republic.

PURPOSE: Increased levels of vitamin D may protect against colorectal cancer (CRC) development and recurrence. Accumulating epidemiologic evidence suggests these effects may be partly mediated by genetic variants of the vitamin D receptor (VDR) proposed to be associated with altered risk of CRC. We wished to determine if common VDR polymorphisms affected CRC risk in the Czech Republic, a homogenous European population with a high CRC incidence rate. METHODS: Frequencies of the common VDR gene polymorphisms rs2238136, rs1544410 (BsmI), rs7975232 (ApaI), and rs731236 (TaqI) were determined using allele-specific PCR in a case control analysis of a series of 754 CRC patients and 627 patients without malignant disease recruited from centers throughout the Czech Republic. Unconditional logistic regression was used to calculate odds ratios and 95% confidence intervals for the association between these variants and risk of CRC. RESULTS: None of the four polymorphisms tested had any significant effect on CRC risk. No significant differences were observed in susceptibility when the population was stratified by anatomical sub-site, sex, BMI, smoking, alcohol, or presence of polyps. CONCLUSIONS: We conclude that common variation in the VDR gene had little effect on its own on predisposition to sporadic CRC in the Czech population.

Association between exposure-relevant polymorphisms in CYP1B1, EPHX1, NQO1, GSTM1, GSTP1 and GSTT1 and risk of colorectal cancer in a Czech population.

Associations of functional single nucleotide polymorphisms in cytochrome P450 1B1, epoxide hydrolase 1, NAD(P)H:quinone oxidoreductase 1, glutathione S-transferase Pi-1 and deletions of glutathione S-transferases Mu-1 and θ-1 with colorectal cancer risk were investigated in a hospital-based case-control study on 495 matched pairs of Czech Caucasians. Polymorphisms were assessed by polymerase chain reaction restriction fragment length polymorphism-based methods, allele-specific multiplex and allelic discrimination by real-time polymerase chain reaction. Carriers of variant Ser allele in codon 453 of cytochrome P450 1B1 (rs1800440) were at a significantly lower risk of colorectal cancer compared to carriers of the wild-type allele (adjusted odds ratio, aOR=0.68, CI=0.51-0.89, p=0.006). The combination of polymorphisms in codons 453 and 432 (rs1056836) of cytochrome P450 1B1 further increased the protective effect (aOR=0.53, CI=0.34-0.83, p=0.005). The glutathione S-transferase Mu-1 deletion was associated with a moderately elevated colorectal cancer risk (aOR=1.30, CI=1.01-1.68, p=0.044). Combination of glutathione S-transferase Mu-1 and θ-1 deletion was associated with a significantly higher colorectal cancer risk compared to the presence of both full-length genes (aOR=1.58, CI=1.01-2.47, p=0.044). Genetic polymorphisms in glutathione S-transferase Pi-1, NAD(P)H:quinone oxidoreductase 1, epoxide hydrolase 1 and deduced epoxid hydrolase 1 activity did not modify the risk of colorectal cancer. These results provide further evidence that interaction between metabolic gene variants contributes to colorectal carcinogenesis.

CHEK2 gene alterations in the forkhead-associated domain, 1100delC and del5395 do not modify the risk of sporadic pancreatic cancer.

Checkpoint kinase 2 gene (CHEK2) alterations increase risk of several cancer types. We analyzed selected CHEK2 alterations in 270 Czech pancreatic cancer patients and in 683 healthy controls. The pancreatic cancer risk was higher in individuals who inherited rare alterations in CHEK2 region involving forkhead-associated domain other than I157T (OR=5.14; 95% CI=0.94-28.23) but the observed association was non-significant (p=0.057). The most frequent I157T mutation did not alter the pancreatic cancer risk and neither the followed deletion of 5395bp nor c.1100delC were found in any of pancreatic cases. We conclude that the I157T, other alterations in its proximity, del5395 and c.1100delC in CHEK2 do not predispose to pancreatic cancer risk in the Czech population.

Genetic variants in selenoprotein genes increase risk of colorectal cancer.

Low selenium (Se) status correlates with increased risk of colorectal cancer (CRC). Since Se exerts its biological roles through the selenoproteins, genetic variations in selenoprotein genes may influence susceptibility to CRC. This study analysed 12 single-nucleotide polymorphisms (SNPs) in selenoprotein genes [glutathione peroxidase 1 (GPX1), GPX4, 15 kDa selenoprotein (SEP15), selenoprotein S (SELS), selenoprotein P (SEPP1) and thioredoxin reductase 2 (TXNRD2)] and in genes that code for a key protein in Se incorporation [SECIS-binding protein 2 (SBP2)] and in antioxidant defence [superoxide dismutase 2 (SOD2)] in relation to sporadic CRC incidence. CRC patients (832) and controls (705) from the Czech Republic were genotyped using allele specific PCR. Logistic regression analysis showed that three SNPs were significantly associated with an altered risk of CRC: rs7579 (SEPP1), rs713041 (GPX4) and rs34713741 (SELS). The association of these SNPs with disease risk remained after data stratification for diagnosis and adjustments for lifestyle factors and sex. Significant two-loci interactions were observed between rs4880 (SOD2), rs713041 (GPX4) and rs960531 (TXNRD2) and between SEPP1 and either SEP15 or GPX4. The results indicate that SNPs in SEPP1, GPX4 and SELS influence risk of CRC. We hypothesize that the two-loci interactions reflect functional interactions between the gene products. We propose that these variants play a role in cancer development and represent potential biomarkers of CRC risk.

NBN 657del5 heterozygous mutations and colorectal cancer risk in the Czech Republic.

The most frequent Nijmegen breakage syndrome (NBS)-causing mutation is a 5-base pair deletion in gene coding for nibrin (NBN 657del5), which results in a non-fully functional protein product and is particularly frequent in Central and Eastern Europe. Recent studies have investigated whether NBN 657del5 carriage may predispose to an increased risk of different types of cancer. The Czech Republic has one of the highest incidences of colorectal cancer in the world as well as high incidence of NBS. To assess whether NBN 657del5 associates with an increased risk of sporadic colorectal cancer, we have screened 771 colorectal cancer patients, 614 controls with negative colonoscopy and 818 healthy blood donors from the Czech Republic. There were no significant differences between the frequencies of heterozygous carriers among the three groups. The present results do not provide any evidence that the exceeding risk of CRC in this population is attributable to the high frequency of heterozygous carriage of the NBN 657del5.

The CHEK2 gene I157T mutation and other alterations in its proximity increase the risk of sporadic colorectal cancer in the Czech population.

Checkpoint kinase 2 (CHEK2) gene codes for an important mediator of DNA damage response pathway. Its mutations increase risk of several types of cancer. We analysed selected CHEK2 mutations in 631 Czech colorectal cancer (CRC) patients. The increased risk of CRC was associated with mutations in CHEK2 gene region involving fork head-associated domain [39/631 (6.2%) cases versus 19/683 (2.8%) controls; odds ratio (OR)=2.3; 95% confidence interval (CI)=1.3-4.0; p=0.003], and with the most frequent I157T mutation [30/631 (4.8%) cases versus 17/683 (2.5%) controls; OR=2.0; 95% CI=1.1-3.6; p=0.03]. Prevalence of 1100delC mutation in CRC patients (4/631) did not differ from that in the control population (2/730; p=0.4). The deletion of 5395 bp was not found in any of the successfully analysed CRC cases. We observed no association of analysed mutations with CRC family history. We conclude that the I157T and other alterations in its proximity predispose to sporadic but not to familial CRC in the Czech population.