Combined methylation of p16 and hMLH1 (CMETH2) discriminates a subpopulation with better prognosis in colorectal cancer patients with microsatellite instability tumors.

The aim of this study was to determine the frequency of p16 and hMLH1 genes simultaneous methylation in colorectal cancer patients with Microsatellite Instability (MSI) tumors. We also wanted to analyze the relationship with other clinical features, with BRAF gene V600E mutation and with prognosis. Samples from fifty one patients with MSI positive sporadic colorectal cancer were included. DNA was extracted from tumor samples. Promoter methylation was analyzed using bisulfite modification and was detected by quantitative methylation-specific PCR. BRAF gene was amplified using specific primers and mutations were detected by real time PCR. Simultaneous methylation was transformed in a new variable called CMETH2. Frequency of CMETH2 was analyzed and compared with other clinicopathological variables. 33.3 % of patients were positive for CMETH2 and 25 % had BRAF V600E mutation. CMETH2 was related with proximal location, with poorly differentiated tumors and with BRAF V600E mutation. CMETH2 only showed influence in the overall survival (OS) in patients with distal tumors. However, with regard to the disease free survival (DFS) measure, CMETH2 was independent prognostic factor. We were able to discriminate tumors with high methylation features using a transformation analysis of variables into a new computed one (CMETH2). CMETH2 has demonstrated to be a useful prognostic factor in MSI tumors. The prognostic value of CMETH2 in DFS was independent of other clinicopathological variables. The use of CMETH2 could help in the election of the best therapeutic alternative for CCR patients with MSI tumors.

Role of circulating tumor cells as prognostic marker in resected stage III colorectal cancer.

BACKGROUND: The prognostic role of circulating tumor cells (CTC) in early colorectal cancer (CRC) has not been determined yet. We evaluated the potential prognostic value of CTC in stage III CRC patients. PATIENTS AND METHODS: Prospective multicenter study of 519 patients with stage III CRC recruited between January 2009 and June 2010. CTC were enumerated with the CellSearch System after primary tumor resection and before the start of adjuvant therapy. A total of 472 patients were included in the analysis. RESULTS: CTC ≥1, ≥2, ≥3 and ≥5 were detected in 166 (35%), 93 (20%), 57 (12%) and 34 (7%) patients, respectively. Median follow-up was 40 months. In the overall population, CTC ≥1 (disease-free survival (DFS): HR 0.97, P = 0.85; overall survival (OS): HR 1.03, P = 0.89), ≥2 (DFS: HR 1.07, P = 0.76; OS: HR 1.02, P = 0.95), ≥3 (DFS: HR 0.96, P = 0.87; OS: HR 0.74, P = 0.41) and ≥5 (DFS: HR 0.72, P = 0.39; OS: HR 0.48, P = 0.21) were not associated with worse DFS and OS. No clinicopathological characteristics were significantly associated with the presence of CTC. In patients with disease relapse, the proportion with CTC ≥1 was not significantly different between those with single versus multiple metastatic locations (37.9% versus 31.4%, P = 0.761). In the multivariate analysis, CTC ≥1 was not an independent prognostic factor for DFS (HR 0.97, P = 0.87) and OS (HR 0.96, P = 0.89). CONCLUSION: CTC detection was not associated with worse DFS and OS in patients with stage III CRC. Given the scarcity of CTC in these patients, it is likely that CTC determined by CellSearch system does not have a prognostic role in this setting. However, a longer follow-up is needed.

Influence of A9 region mutation in EphB2 gene in the prognosis of patients with colorectal adenocarcinoma.

BACKGROUND: EphB2 is a transmembrane glycoprotein implicated in the regulation of cell growth, differentiation, and motility. It has been proposed as a tumor suppressor gene, and the role of EphB2 protein in tumorogenesis has been demonstrated. The aim of this study was to test the influence of mutation of A9 region in EphB2 gene in the prognosis of patients with sporadic CCR. MATERIALS AND METHODS: A total of 473 patients with colorectal cancer were included. A9 region in exon 17 of EphB2 was amplified using specific primer and analyzed using Genescan. All mutations were confirmed by direct sequencing. RESULTS: EphB2 mutation was detected in 13 of the 473 patients (2.7%). Mutation of EphB2 showed association with tumor site, 12 of 13 mutations were proximal tumors (P < 0.001). EphB2 mutation confers better prognosis in the adenocarcinoma group; 100% of patients carrying the mutation survived and were disease free after 72 months (P = 0.02 and 0.03, respectively). CONCLUSIONS: Despite the low frequency of EphB2 gene, we got promising results. It would be very interesting to increase the population size to verify our results. If these findings are confirmed, EphB2 could help discriminate patients with adenocarcinoma with different prognosis and to improve the election of the most suitable treatment in each case.

Vascular endothelial growth factor gene polymorphisms in patients with colorectal cancer.

BACKGROUND: Angiogenesis plays an important role in tumor progression. The vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis. In the present study we evaluated single nucleotide polymorphisms (SNPs) -2578C > A, -1154G > A, and +936C > T in the VEGF gene, and their prognostic value for patients operated on for colorectal cancer (CRC). PATIENTS AND METHOD: VEGF polymorphisms have been analyzed in 177 patients who had undergone surgical resection at Hospital Clínico San Carlos. The analysis of these polymorphisms was performed with specific probes for each nucleotide in a multiplex reaction using real-time PCR. RESULTS: We only found a statistically significant relationship for one of these three polymorphisms, +936C > T, with gender and tumor location; 10.7% of patients heterozygotes for this SNP had tumors located in proximal colon, 35.2% in distal segment and 54.1% in rectum (p = 0.03). Patients with the +936T/T genotype had 100% overall survival (OS). CONCLUSION: Patients with a +936T/T genotype showed increased survival, therefore the +936C > T SNP could be a useful marker in the follow-up and clinical management of patients with colorectal cancer.

Colorectal carcinoma prognosis can be predicted by alterations in gene p53 exons 5 and 8.

BACKGROUND AND AIMS: Gene p53 alteration is a genetic event described in the progression from adenoma to colorectal carcinoma. Most of the p53 mutations occur in exons 5 to 8 in highly preserved regions and in the three main structural domains of the p53 protein. It is possible that mutations affecting different structural regions may present different effects on the p53 protein function and, due to this, they may have different prognostic meaning. MATERIALS AND METHODS: The study population consisted of 353 patients diagnosed with sporadic colorectal cancer. Mutations in 5-8 exons of p53 gene were detected by means of single strand conformation polymorphism (SSCP). All samples that showed different migration bands in SSCP were confirmed by sequencing. RESULTS: A total of 69 patients (19.7%) showed alterations of the gene p53. It was observed that mutation in codon 175 in exon 5 was related to tumors located in the colon (p = 0.01) and the mutation in the codon 288 in exon 8 was related to rectal tumors (p = 0.02). In the study of overall survival, mutation in codon 175 of exon 5 conferred a better prognosis and alterations of exon 8 were related to a worse prognosis in different population subgroups: in men, in patients younger than 71 years old, in the tumors located in the proximal colon, the ones moderately differentiated, and those that are mucinous. CONCLUSION: According to this study, mutations in different exons of p53 are related to different phenotypes in colorectal cancer. These phenotypes could mean differences in the clinical evolution of the patients.

Circulating tumor cells in colorectal cancer: correlation with clinical and pathological variables.

BACKGROUND: The CellSearch System is a technique to detect circulating tumor cells (CTCs) in patients with cancer. Few data have been published concerning the role of CTCs detection by this method in colorectal cancer. The aim of this study was to correlate the presence of CTCs with the commonest clinical and morphological variables. PATIENTS AND METHODS: Blood samples were collected from 97 patients and 30 healthy volunteers. Quantification of CTCs in 7.5 ml of blood was carried out with the CellSearch System. The results were expressed as number of CTCs/7.5 ml and the cut-off of >or=2 CTCs/7.5 ml was chosen to define the test as positive. RESULTS: Positive CTCs were detected in 34 of 94 patients (36.2%). Correlation was not found among positive CTCs and location of primary tumor, increased carcinoembryonic antigen level, increased lactate dehydrogenase level or grade of differentiation. Only stage correlated with positive CTCs (20.7% in stage II, 24.1% in stage III and 60.7% in stage IV, P = 0.005). CONCLUSIONS: CTCs detection by CellSearch is a highly reproducible method that correlates with stage but not with other clinical and morphological variables in patients with colorectal cancer. Colon cancer tumor cells are detectable in all stages. Further studies are warranted.

Telomerase activity in colorectal cancer, prognostic factor and implications in the microsatellite instability pathway.

AIM: To determine whether the telomerase activity is related to the Microsatellite instability (MSI) genetic pathway and whether it means a difference in the survival. METHODS: The population consisted of 97 colorectal cancer patients. MSI determination was performed in accordance with the NCI criteria using PCR and Genescan. Telomerase activity was determined by the TRAP-assay, an ELISA procedure based on the amplification of telomeric repeat sequences. RESULTS: 6.2% showed high MSI (MSI-H), 10.3% showed low MSI (MSI-L) and 83.5% did not show this alteration (MSS). Positive telomerase activity was detected in 92.8% of the patients. 83.3% of MSI-H tumors showed positive telomerase against 93.8% of MSS tumors. In the overall survival analysis the absence of telomerase activity conferred a better prognosis. CONCLUSION: Previous works have shown that tumors which develop via the MSI pathway present a better prognosis. No link between telomerase activity and MSI status is observed, although sample sizes are small. Patients with telomerase negative tumors had better overall survival than patients with telomerase positive tumors.

Role of the BRAF mutations in the microsatellite instability genetic pathway in sporadic colorectal cancer.

BACKGROUND AND AIMS: Between 10 and 15% of all cases of colorectal cancer are the result of microsatellite instability (MSI) in the genetic pathway due to an alteration in the DNA repair genes. Tumors with high MSI are characterized by a better prognosis. The BRAF oncogene has been linked to the MSI pathway in tumorogenesis. The objective of this study was to determine whether alterations in BRAF are related to MSI and whether they can result in differences in survival rates. METHODS: The study cohort comprised 351 patients diagnosed with sporadic colorectal cancer. MSI was determined in accordance with the National Cancer Institute's (NCI) recommendations by means of PCR and sequence analyses. Mutational analysis of the BRAF gene was performed by real-time PCR and subsequent sequencing of the altered samples. The methylation pattern of the hMLH1 gene was determined using methylation-specific PCR analyses of bisulfite-treated DNA and the results confirmed by sequencing. RESULTS: Of the patients tested, 6.9% showed high MSI and 3.7% showed a BRAF gene mutation. hMLH1 methylation was observed in 67.2% of the patients with MSI and/or the BRAF alteration. The BRAF mutation was related to the MSI genetic pathway (P < 0.0001) and with hMLH1 methylation. In the analysis of overall survival only MSI had an independent prognostic value for the risk of death. Patients with the BRAF mutation showed a higher risk of death, although this association was found not to be statistically significant. CONCLUSIONS: There is a subgroup of carcinomas which develop via the MSI pathway that carry an alteration of the BRAF gene. This alteration confers a poorer outcome on these patients within the total group of patients with MSI who have a better prognosis. This hypothesis should be further investigated in a larger study population due to the low incidence of BRAF mutations in colorectal cancer.