RESUMEN
BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.
Asunto(s)
Humanos , Animales , Masculino , Persona de Mediana Edad , Antígenos de Carbohidratos Asociados a Tumores/genética , Indígenas Sudamericanos/genética , Neoplasias de la Vesícula Biliar/genética , Líquido Ascítico/metabolismo , Células Tumorales Cultivadas , Pruebas de Carcinogenicidad , Chile , Dermatoglifia del ADN , Proteína p53 Supresora de Tumor/genética , Cisplatino/farmacología , Ratones Endogámicos NOD , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Análisis de Secuencia de ARN , Receptor ErbB-2/genética , Genes erbB-2/genética , Perfilación de la Expresión Génica , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Células Epiteliales/metabolismo , Queratina-19/genética , Queratina-7/genética , Carcinogénesis/genética , Neoplasias de la Vesícula Biliar/metabolismo , Antineoplásicos/farmacologíaRESUMEN
Background: In colorectal cancer, BRAF and KRAS mutation are mutually exclusive, but both are independent prognostic factors for the disease. Aim: To determine the frequency of BRAF V600E mutation in colorectal cancer. Material and Methods: A KRAS mutation study was carried out in 100 tissue samples of primary and metastatic adenocarcinomas of colon and rectum from patients aged 61.1 ± 62 years (56 women). Negative KRAS mutation cases underwent study of BRAF V600E mutation by restriction fragment length polymorphism (RFLP) and direct sequencing. Results: Primary tumors were located in the colon and rectum in 88 and six cases respectively. Five were liver metastases and in one case, the sample location was undetermined. Forty two samples were KRAS positive (mutated). In 12 of the 58 KRAS negative (wild type) samples, the V600E mutation in codon 15 of the BRAF gene was demonstrated. No differences in the frequency and distribution of mutations, stratified by gender, age, primary tumor versus metastasis, or tumor location were observed. Conclusions: Twelve percent of KRAS negative colorectal cancer samples showed BRAF gene mutation. Considering that 42% of samples have a KRAS mutation, 54% of patients should not respond to therapies with monoclonal antibodies directed against epidermic growth factor (EGFR) pathway.