RESUMO
Allelic variation of BAT-25 (a 25-repeat quasimonomorphic poly T) and BAT-26 (a 26-repeat quasimonomorphic polyA) loci as two mononucleotide microsatellite markers, were analyzed with high-performance liquid chromatography (HPLC) compared with Real-Time PCR using hybridization probes. BAT-26 and BAT-25 markers were used to determine an appropriate screening technique with high sensitivity and specificity to diagnose microsatellite instability (MSI) status in patients with colorectal cancer (CRC). One of the pathways in colorectal tumor genesis is microsatellite instability (MSI+). MSI is detected in about 15% of all CRCs; 3% are of these are associated with Lynch syndrome and the other 12% are caused by sporadic. Colorectal tumors with MSI have distinctive features compared with microsatellite stable tumors. Due to the high percentage of MSI+ CRC in Iran, screening of this type of CRC is imperative. Two markers were analyzed in tissues and sera of 44 normal volunteers and tumor and matched normal mucosal tissues as well as sera of 44 patients with sporadic CRC. The sensitivity and specificity of BAT-26 with real time PCR method (Hybridization probe) were 100% in comparison with sequencing method as the gold standard, while HPLC had a lower sensitivity and specificity. According to HPLC data, BAT-26 was more sensitive than BAT-25 in identifying MSI tumors. Therefore, MSI typing using the BAT-26 hybridization probe method compared to HPLC could be considered as an accurate method for diagnosing MSI in CRC tumors but not in serum circulating DNAs.
Assuntos
Neoplasias Colorretais/genética , Instabilidade de Microssatélites , Repetições de Microssatélites , Adulto , Idoso , Biomarcadores Tumorais , Estudos de Casos e Controles , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Neoplasias Colorretais/patologia , Feminino , Marcadores Genéticos , Mutação em Linhagem Germinativa , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Hibridização de Ácido Nucleico , Curva ROC , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Colorectal cancer (CRC) is the third most common cancer worldwide. Colorectal cancer incidence differs widely among different geographic regions. In addition to mutational changes, epigenetic mechanisms also play important roles in the pathogenesis of CRCs. O6-methylguanine-DNA methyltransferase (O(6)-MGMT) is a DNA repair protein and in the absence of MGMT activity, G-to-A transition may accumulate in the specific genes such as K-ras and p53. To identify which CpG sites are critical for its downregulation, we analyzed the methylation status of the MGMT gene promoter in two sites in CRC patients. Then we compared the frequency of their methylation changes with the results of our previously reported K-ras gene mutation, APC2 and p16 methylation. MGMT methylation was examined in 92 tumor samples. A methylation specific PCR (MSP) method was performed for two loci of MGMT gene which described as MGMT-A and MGMT-B. The prevalence of MGMT-A, and MGMT-B methylation was 49/91 (53.8%), and 83/92 (90.2%), respectively. We detected high frequency of MGMT-B but not MGMT-A methylation in tumor tissues with APC2 methylation. Our results showed that MGMT-B methylation is significantly associated with K-ras gene mutation rather than MGMT-A (p = 0.04). Simultaneously, an inverse correlation was found between p16 and MGMT-B methylation simultaneously (p = 0.02). Our study indicated that hypermethylation of the specific locus near the MGMT start codon is critical for cancer progression. MGMT-B assessment that is associated with K-ras mutation can have a prognostic value in patients with CRC.
Assuntos
Neoplasias Colorretais/genética , Metilação de DNA , O(6)-Metilguanina-DNA Metiltransferase/genética , Regiões Promotoras Genéticas , Adulto , Idoso , Estudos de Casos e Controles , Neoplasias Colorretais/patologia , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de NeoplasiasRESUMO
BACKGROUND: The pathogenesis of sporadic colorectal cancer (CRC) is influenced by the patient genetic background and environmental factors. Based on prior understanding, these are classified in two major pathways of genetic instability. Microsatellite instability (MSI) and CPG island methylator phenotype (CIMP) are categorized as features of the hypermethylated prototype, and chromosomal instability (CIN) is known to be indicative of the non-hypermethylated category. Secreted frizzled related protein 2 (SFRP2), APC1A in WNT signaling pathway and the DNA repair gene, O6-methylguanine-DNA methyltransferase (MGMT), are frequently hypermethylated in colorectal cancer. Detection of methylated DNA as a biomarker by easy and inexpensive methods might improve the quality of life of patients with CRC via early detection of cancer or a precancerous condition. AIM: To evaluate the rate of SFRP2 and MGMT hypermethylation in both polyp tissue and serum of patients in south Iran as compared with matched control normal population corresponding samples. MATERIALS AND METHODS: Methylation-specific PCR was used to detect hypermethylation in DNA extracted from 48 polypoid tissue samples and 25 healthy individuals. RESULTS: Of total polyp samples, 89.5% had at least one promoter gene hypermethylation. The most frequent methylated locus was SFRP2 followed by MGMT-B (81.2 and 66.6 percent respectively). Serologic detection of hypermethylation was 95% sensitive as compared with polyp tissue. No hypermethylation was detected in normal tissue and serum and its detection in patients with polyps, especially of serrated type, was specific. CONCLUSIONS: Serologic investigation for detection of MGMT-B, SFRP2 hypermethylation could facilitate prioritization of high risk patients for colonoscopic polyp detection and excision.