Analysis of the k-ras mutant alleles and lincrna-p21 expression in tumors and liquid biopsy as prognostic factors in surgical colorectal cancer patients
- Autores: Yan Li
- Directores de la Tesis: Alberto Biete Solà (dir. tes.), Alfons Navarro Ponz (codir. tes.)
- Lectura: En la Universitat de Barcelona ( España ) en 2020
- Idioma: español
- Tribunal Calificador de la Tesis: Núria Viñolas Segarra (presid.), Jorge Moisés Lafuente (secret.), David Páez López-Bravo (voc.)
- Programa de doctorado: Programa de Doctorado en Medicina e Investigación Traslacional por la Universidad de Barcelona
- Materias:
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- Resumen
Blood vessels, together with lymphatics vessels, are the pathways of choice that tumors use to colonize at a distance. In colorectal tumors (CRC), liver metastases are the most frequent of all. It is currently admitted that circulating tumor cells (CTCs) and biomarkers released by tumors are one of the most efficient mechanism for the formation of metastases. The vascular anatomy of the colon shows that tumors located in the right colon drain their products into the superior mesenteric vein while tumors of the left colon and sigma drain their products into the inferior mesenteric vein. Both veins form the portal vein that terminates in the liver. Hypothesis：In blood and serum obtained through mesenteric venipuncture, more CTCs and higher concentrations of biomarkers may be encountered. Detection of CTCs and biomarkers in mesenteric vein may indicate residual disease in patients with CRC surgery. Objectives:1.Obtention of CTCs from both mesenteric veins (MVs) and CTCs from peripheral vein(PV).2.Determination of the different K-RAS allelic mutations of CTCs by Digital-PCR (DGPCR) from MVs and PV.3.Ascertain if the different allelic K-RAS mutations of CTCs in VM correspond to those in matched VP.4.Finding out if the different K-RAS allelic mutations of CTCs in VM and VP correspond to the mutations of the matched primary tumor and are related to the clinicopathological characteristics of the patients, the overall survival and disease-free time.5.Verify if the expression levels of lincRNA-p21 in both normal and tumor tissue are related to clinicopathological characteristics of the patients, overall survival and disease-free time. 6.Ascertain if the lincRNA-p21 levels in serum of MV and PV are related to clinicopathological characteristics of the patients, overall survival and disease-free time. Material and Methods：From 52 surgically resected CRC patients who later relapsed, samples of tumor tissue, normal tissue, and blood from the peripheral vein (PV) and MV were obtained from each patient at the time of surgery. KRAS mutations were assessed by Sanger sequencing and digital PCR (DGPCR) in tissue samples and by DGPCR in CTCs. Mutant KRAS copy number was assessed in CTCs. Results were correlated with overall survival (OS). Tumor and paired normal tissue samples from 177 resected CRC patients were included in the study. LincRNA-p21 expression was determined by RTqPCR and correlated with clinical and pathological characteristics and outcome. LincRNA-p21 expression was also explored in plasma samples derived from both tumor-draining mesenteric (MV) and peripheral veins (PV) (n=20). Blood vessels, together with lymphatics vessels, are the pathways of choice that tumors use to colonize at a distance. In colorectal tumors (CRC), liver metastases are the most frequent of all. It is currently admitted that circulating tumor cells (CTCs) and biomarkers released by tumors are one of the most efficient mechanism for the formation of metastases. The vascular anatomy of the colon shows that tumors located in the right colon drain their products into the superior mesenteric vein while tumors of the left colon and sigma drain their products into the inferior mesenteric vein. Both veins form the portal vein that terminates in the liver. Hypothesis：In blood and serum obtained through mesenteric venipuncture, more CTCs and higher concentrations of biomarkers may be encountered. Detection of CTCs and biomarkers in mesenteric vein may indicate residual disease in patients with CRC surgery. Objectives:1.Obtention of CTCs from both mesenteric veins (MVs) and CTCs from peripheral vein(PV).2.Determination of the different K-RAS allelic mutations of CTCs by Digital-PCR (DGPCR) from MVs and PV.3.Ascertain if the different allelic K-RAS mutations of CTCs in VM correspond to those in matched VP.4.Finding out if the different K-RAS allelic mutations of CTCs in VM and VP correspond to the mutations of the matched primary tumor and are related to the clinicopathological characteristics of the patients, the overall survival and disease-free time.5.Verify if the expression levels of lincRNA-p21 in both normal and tumor tissue are related to clinicopathological characteristics of the patients, overall survival and disease-free time. 6.Ascertain if the lincRNA-p21 levels in serum of MV and PV are related to clinicopathological characteristics of the patients, overall survival and disease-free time. Material and Methods：From 52 surgically resected CRC patients who later relapsed, samples of tumor tissue, normal tissue, and blood from the peripheral vein (PV) and MV were obtained from each patient at the time of surgery. KRAS mutations were assessed by Sanger sequencing and digital PCR (DGPCR) in tissue samples and by DGPCR in CTCs. Mutant KRAS copy number was assessed in CTCs. Results were correlated with overall survival (OS). Tumor and paired normal tissue samples from 177 resected CRC patients were included in the study. LincRNA-p21 expression was determined by RTqPCR and correlated with clinical and pathological characteristics and outcome. LincRNA-p21 expression was also explored in plasma samples derived from both tumor-draining mesenteric (MV) and peripheral veins (PV) (n=20). expression. Similar results were obtained when analyzed in MV samples. Conclusions:1.DGPCR is more efficient than Sanger sequencing for detecting KRAS mutations. 2.KRAS G13D mutations and high mutant KRAS copy number are associated with shorter OS. 3.The analysis of KRAS mutations in CTCs from blood obtained at the time of surgery can identify patients with a higher risk of relapse.
4.LincRNA-p21 is a marker of advanced disease and worse outcome in CRC with a major impact in RC patients, where it is clearly overexpressed. 5.LincRNA-p21 acts as a marker of radiotherapy response, since RC patients with high lincRNA-p21 levels benefit from post-operative CRT. 6.The analysis of lincRNA-p21 levels in plasma from tumor- -draining vein is a prognostic biomarker for resected RC patients.
