Progress in omics research and preclinical models of gallbladder cancer / 中华外科杂志

Gallbladder cancer(GBC)is one common type of bile tract cancers with poor prognosis. This review summarizes the recent development of studies about somatic mutation, molecular subtype, microenvironment heterogeneity, organoid, orthotopic model, patient-derived xenograft and clinical translation on GBC in aspects of genomic,transcriptome,single cell omics and clinical translation. We expect this review will provide new ideas on dissecting molecular mechanisms underlying the development and emerging chemoresistance of GBC following therapy and promote GBC precision medicine.

Functional and genomic characterization of three novel cell lines derived from a metastatic gallbladder cancer tumor

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.

¿Es posible utilizar las muestras de colecistectomías con cáncer en investigación?: Calidad del ADN de muestras obtenidas del sistema público y privado de salud / Quality of DNA from archival pathological samples of gallbladder cancer

Background: The quality of the archival samples stored at pathology services could be a limiting factor for molecular biology studies. Aim: To determine the quality of DNA extracted from gallbladder cancer samples at different institutions. Material and Methods: One hundred ninety four samples coming from fve medical centers in Chile, were analyzed. DNA extraction was quantifed determining genomic DNA concentration. The integrity of DNA was determined by polymerase chain reaction amplification of different length fragments of a constitutive gene (β-globin products of 110, 268 and 501 base pairs). Results: The mean DNA concentration obtained in 194 gallbladder cancer samples was 48 ± 43.1 ng/µl. In 22% of samples, no amplification was achieved despite obtaining a mean DNA concentration of 58.3 ng/ul. In 81, 67 and 22% of samples, a DNA amplification of at least 110, 268 or 501 base pairs was obtained, respectively. No differences in DNA concentration according to the source of the samples were demonstrated. However, there were marked differences in DNA integrity among participating centers. Samples from public hospitals were of lower quality than those from private clinics. Conclusions: Despite some limitations, in 80% of cases, the integrity of DNA in archival samples from pathology services in our country would allow the use of molecular biology techniques.

Polimorfismo genético relacionado al metabolismo de la aflatoxina y el riesgo de cáncer vesicular en mujeres chilenas / Aflatoxin metabolism-related genetic polymorphisms and the risk of gallbladder cancer in Chilean women

Background/aim: High consumption of red chili pepper has been shown to be a risk factor for gallbladder cancer (GBC) in Chilean women with gallstones (GS). GS are the main cause of GBC, but not all patients with gallstones develop GBC. Since red chili pepper is a widely consumed spice among the Chilean population, the development of GBC in Chilean women cannot be completely explained by the presence of GS and red chili pepper consumption alone. Genetic factors in addition to these and other environmental factors may also be associated with an increased risk of GBC. We aimed to study whether genetic polymorphisms involved in aflatoxin metabolism are associated with the risk of GBC in Chilean women, because we detected aflatoxins B1 and B2 in red chili pepper purchased in Santiago, Chile. Methods: We conducted a hospital-based case-control study whose subjects were 57 patients with GBC, 119 patients with GS, and 70 controls. DNA was extracted from subjects’ blood or paraffin block samples using standard commercial kits. The statuses of the genetic polymorphisms of cytochrome P450 (CYP) 1A2 rs762551 and CYP3A4 rs2740574 were assayed using the TaqMan® SNP Genotyping Assay or the Custom TaqMan® SNP Genotyping Assay, respectively. Results: In the assay for the CYP1A2 polymorphism, of the 57 GBC patients, 23 (40.3 percent) had at least one minor allele (A/C or C/C). However, there were no significant differences in the genotypic or allelic frequencies among the three subject groups. In the assay for the CYP3A4 polymorphism, the minor G/G genotype was not detected in the three groups, and there were no significant differences in the genotypic or allelic frequencies among the three groups. Conclusion: These genetic polymorphisms were not related to the risk of GBC in Chilean women. Further studies including a greater number of controls and cases are needed to confirm this preliminary exploratory result.

Introducción/objetivo: El alto consumo de ají rojo ha demostrado ser un factor de riesgo de cáncer vesicular (CV) en mujeres chilenas con cálculos vesiculares. Los cálculos vesiculares son la causa principal de CV, no obstante, no todos los pacientes con cálculos vesiculares desarrollan CV. Debido a que el ají rojo es una especia ampliamente consumida entre la población chilena, el desarrollo de CV en las mujeres chilenas no puede ser explicado en su totalidad sólo por la presencia de cálculos vesiculares y consumo de ají rojo. Factores genéticos además de éstos y otros factores ambientales, también podrían estar relacionados con un aumento del riesgo de CV. Nuestro objetivo es estudiar si los polimorfismos genéticos involucrados en el metabolismo de la aflatoxina están relacionados con el riesgo de CV en mujeres chilenas, porque detectamos aflatoxinas B1 y B2 en ajíes rojos comprados en Santiago de Chile. Métodos: El estudio caso control, incluyó 57 pacientes con CV, 119 pacientes con cálculos vesiculares, y 70 controles. Se extrajo ADN de la sangre de los sujetos o de bloques de parafina, usando kits comerciales estándar. El estado de los polimorfismos genéticos del citocromo P450 (CYP) 1A2 rs762551 y CYP3A4 rs2740574 fueron estudiados usando el ensayo de genotipo SNP TaqMan® o el ensayo de genotipo SNP Custom TaqMan®, respectivamente. Resultados: En el ensayo para el polimorfismo CYP1A2, de los 57 pacientes con CV, 23 (40,3 por ciento) tuvieron al menos un alelo menor (A/C o C/C). No obstante, no hubo diferencias significativas en las frecuencias genotípicas o alélicas entre los tres grupos. En el ensayo para el polimorfismo CYP3A4, el genotipo menor G/G no fue detectado en los tres grupos, y no hubo diferencias significativas en las frecuencias genotípicas o alélicas entre los tres grupos. Conclusión: Estos polimorfismos genéticos no estaban relacionados con el riesgo de CV en mujeres chilenas...

Alteraciones genéticas en lesiones preneoplásicas y neoplásicas de la vesícula biliar / Genetic alterations in preneoplastic and neoplastic injuries of the gallbladder

This article aims to review the most relevant morphological and molecular aspects involved in gallbladder (GB) cancer. In Chile, gallbladder cancer is the main cause of death due to cancer, among women older than 40 years. However, there is almost none information about the morphological changes and the genetic alterations in-volved in the beginning and development of this neoplasia. Two carcinogenic ways have been described. The sequence adenoma-carcinoma is accepted to be less frequent and important. The most important is the sequence where a metaplasia evolves to displasia that progresses to carcinoma in situ and fnally it becomes invasive. This progress requires 10 to 15 years approximately. During this time, a continue progression of injuries have been described. Molecular research studies show genetic anomalies in some genes which are temporary events in preneoplastic injuries of the gallbladder. Some of them even exist before the frst morphological changes, while the expression of tumor suppressor genes like p53, adhesion molecules and oncogenes, among others, can be related to late GB carcinogenesis. The K-ras gene seems to play a role in this neoplasia, mainly in those that present an abnormal biliopancreatic union. The microsatelital instability has been found in a small subset of preneoplastic and neoplastic lesions. The existence of methylation in the promotor gene areas has been related to the cellular proliferation, invasion and metastasis and also in cases of chronic cholecystitis, suggesting that this epigenetic phenomenon represents a crucial early event in GB carcinogenesis.

Carcinoma subseroso de la vesícula biliar (pT2): valor pronóstico de la expresión de los genes supresores de tumores p53 y p27 en matrices de tejidos / p53 and p27 gene expression in subserosal gallbladder carcinoma

Background: Subserosal carcinoma is the stage that presents the greatest difficulty in the diagnosis therapeutic handling and prognosis evaluation. Aim To study the expression of p53 and p27 genes in subserosal gallbladder cancer. Material and methods: One hundred twenty seven tissue samples of subserosal gallbladder cancer (coming from 112 females aged 62 ± 13years and 15 men aged 67 ± 17years) and 50 control samples were selected to construct tissue arrays. p53 andp27genes were determined by immunohistochemistry. Results: Thirty eight percent of tumors were not detected at the macroscopic examination, 52 percent and 17 percent had lymph node and blood vessel involvement, respectively. Fifty six and 46 percent were positive for p53 and p27, respectively. No association between the expression of both genes and gender, degree of differentiation, lymph node or blood vessel involvement, was observed. Overall five years actuarial survival was 32 percent. Patients with positive or negative p53 expression had a 22 percent and 53 percent survival, respectively (p =0.05). No association between survival and p27 expression was observed. Conclusions: p53 gene expression is a prognostic factor for subserosal gallbladder cancer.

Estudio de patrones de metilación génica en tumores del tubo digestivo / Gene methylation patterns in digestive tumors

Background: The loss of tumor suppresor gene function damages the defensive mechanisms that protect the indemnity of genetic material. Promoter gene methylation is one of the inactivation mechanisms of suppressor genes. Aim: To study the methylation pattern of a group of genes in biopsy samples of gastrointestinal tumors. Material and methods: Forty eight gastric, 25 gallbladder, 24 colon and 6 pancreas cancer biopsy samples were randomly selected. The methylation pattern of CDH1, FHIT, CDKN2A, APC and MLH1 genes, was studied using a specific polymerase chain reaction test for methylation. Demographic, morphological and follow up variables of patients bearing the tumors were also analyzed. Results: The general methylation frequency of CDH1, FHIT, CDKN2A, APC and MLH1 genes was 64.1, 56, 39.8, 18.1 and 34 percent respectively. In gastric cancer samples there was a correlation between APC gene methylation and well differentiated tumors; between CDH1 methylation and Lauren diffuse type and the presence of three or more metastasic lymph nodes; between FHIT, CDKN2A and CDH1 gene methylation and male gender. In ¡ess differentiated gallbladder tumors, the frequency of CDH1 methylation was higher. There was a tendency towards a lower survival in colon and gastric cancer when MLH1 (p =0.07) y CDKN2A (p= 0.06) were methylated, respectively. Conclusions: An abnormal methylation pattern was associated with morphological features in gastric and gallbladder cancer and with a tendency towards a lower survival in colon and gastric cancer.

Mutación del gen K-ras en tumores pancreático-biliares / Frequency of K-ras mutation in biliary and pancreatic tumors

Background: The ras gene family (H-ras, N-ras and K-ras) are oncogenes that mutate frequently in human cancer, specially in tumors of the biliary tract and pancreas. Aim: To determine the frequency of K-ras gene codon 12 mutation in pancreatic and biliary tumors. Material and Methods: Samples of 35 gallbladder, 15 ampulla of Vater, 10 biliary tract and 9 pancreatic tumors, were analyzed. The tumor tissue was microdissected from paraffin embedded biopsies. The mutation was detected by a combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Results: Overall, 46% of samples had K-ras gene mutations. Mutation frequency was 80, 56, 50 and 29% for ampulla of Vater, pancreatic, biliary tract and gallbladder tumors, respectively. When compared with the rest, gallbladder tumors had a significantly lower frequency of the mutation. Median survival for biliary tract tumors was 6 months, compared with 65 months for gallbladder tumors (p <0.05). Conclusions: Gallbladder carcinoma had the lower frequency of K-ras mutation, when compared with pancreatic, biliary tract and ampulla of Vater tumors.

Expressão imuno-histoquímica e valor prognóstico da proteína p53 no carcinoma de vesícula biliar: estudo de 60 casos / Immunohistochemical expression and prognostic value of the p53 protein in gallbladder carcinoma: a 60-case study

BASE TEORICA/OBJETIVO: Poucos estudos, com pequenas amostras e resultados divergentes, têm sido realizados para avaliar a expressão da proteína p53 no carcinoma de vesícula biliar e sua relação com diferentes parâmetros clinicopatológicos. Baseados nesses fatos realizamos um estudo com objetivo de avaliar a expressão do p53 nessa afeccção e sua associação a fatores prognósticos. MATERIAL E MÉTODO: Amostras de 60 casos de carcinoma de vesícula biliar foram avaliadas por técnica imuno-histoquímica quanto à expressão da proteína p53 e analisadas em relação ao prognóstico, avaliado através do sistema de estádio TNM e Nevin, sobrevida e outros parâmetros clinicopatológicos. RESULTADOS: A expressão p53 foi positiva em 58,3 por cento dos tumores malignos de vesícula biliar. Não foi observada associação estatisticamente significante entre a expressão dessa proteína e fatores prognósticos, tipo ou grau histológico, presença de cálculos ou sobrevida. CONCLUSAO: A mutação do gene p53 está, muito provavelmente, envolvida na patogênese do carcinoma de vesícula biliar. Entretanto não parece haver, de acordo com nossos dados, associação entre a expressão da proteína p53 e o prognóstico ou a sobrevida dos pacientes portadores dessa afecção.

Inactivación del gen CDKN2A (p16) en cáncer de la vesícula biliar / Inactivation of CDKN2A gene (p16) in gallbladder carcinoma

Background: The CDKN2A gene encodes a cyclin dependent kinase inhibitor, p16, which promotes cell cycle arrest. Methylation of the promoter region trans-criptionally inactivates the gene. Aim: To study the relationship between methylation status of the prometer region of p16 gene, the immunohistochemical expression of p16 and clinical and morphological features of gallbladder carcinoma. Material and methods: We analyzed the methylation status of the promoter region of the CDKN2A gene in gallbladder adenocarcinomas using methylation specific PCR (MSP). We also used microsatellite markers near the CDKN2A gene to detect allelic imbalance (AI) and examined the tumors by immunohistochemistry (IHC) for p16 expression. Results: Of 38 gallbladder adenocarcinomas analyzed by IHC, 11 cases (29%) were negative for p16 protein. Nine (24%) had methylation of the promoter region of the CDKN2A gene. Twenty nine cases were negative for methylation, but four (14%) of these 29 exhibited AI at one or more of the microsatellite markers. CDKN2A promoter methylation was not associated with microsatellite instability (MSI-H). Conclusions: The inactivation of CDKN2A by methylation and/or deletion might play an important role in gallbladder carcinogenesis.

Malignant Gastrointestinal Stromal Tumor of the Gallbladder

Gastrointestinal stromal tumors (GISTs) of the gallbladder are representative of an extremely rare group of tumors. We have encountered a patient with a malignant GIST of the gallbladder and presented it with a review of some articles. A 72-yr-old woman initially presented with right upper quadrant abdominal pain, fever and chills. Emergency cholecystectomy was performed under the impression of gallbladder empyema. Liver metastasis was found at 7 months postoperatively and the patient expired 9 months after the surgery. At the time of cholecystectomy, the gallbladder showed a necrotic serosal surface with an irregular thickened wall. A mass, 6 cm in length and 3 cm in width, encircled the whole wall of the neck and upper body of the gallbladder. Microscopic findings revealed frequent mitotic figures (more than 20/50 HPF) and tumor necrosis. Hyperchromatic, pleomorphic and spindle shaped neoplastic cells that were arranged in a pattern of short fascicles infiltrated the entire layer of the gallbladder. The tumor cells were immunoreactive for CD117 antigen (c-kit protein) and vimentin. They were negative for desmin, smooth muscle actin and S-100 protein. Mutations of the c-kit proto-oncogene were not found in this case. These findings were sufficient to provide enough clinical, histopathological and immunohistochemicalevidence in diagnosing our case as a malignant GIST.

Mutación del gen p53 en el cáncer de la vesícula biliar / P53 gene mutation in gallbladder cancer

Background: Gallbladder cancer frequency and mortality renders it one of the most important neoplastic diseases in Chile. P53 tumor suppressor gene has been studied in most types of cancer, but there is scarce information about it in gallbladder cancer. Aim: To study the frequency of P53 gene mutation in gallbladder cancer in the ninth region of Chile. Material and methods: In 25 pathological samples of gallbladder cancer, the direct amplification and sequencing of p53 gene exons 5,6,7,8-8 was possible. Results: Seventeen punctual mutations were observed in 13 cases (52 percent). There were 10 transitions, 5 transversions, one insertion (codon 194) and one deletion (codon 186). Eight cases had mutations in exon 5, six had mutations in exon 6, two had mutations in exon 7 and one had mutations in exons 8-9. In 14 of 25 cases, gene p53 protein was positive. When immunohistochemical expression of gene p53 protein was positive in more than 20 percent of cells, there was a high correlation between genetic alterations and immunohistochemical expression of the protein, with a specificity, sensitivity, positive and negative predictive values over 80 percent. Conclusions: P53 gene mutation is observed in a high proportion of gallbladder cancers at it can be accurately detected with conventional immunohistochemical techniques. The importance of this gene in the genesis of this carcinoma should be determined studying preneoplastic lesions and early carcinomas

Anomalías cromosómicas y citometría de flujo en adenocarcinoma de vesícula biliar / Chromosome rearrangements and flow cytometry in gallbladder carcinoma

Background: Gallbladder carcinoma is the first cause of cancer deaths among chilean women. The few cytogenetic studies performed in these tumors have not found specific or primary chromosome abnormalities. Also, no relationships with specific oncogenes have been found. Aim: To perform cytogenetic studies in gallbladder carcinoma. Material and Methods: A chromosomal study and measurement of dna content, was performed in 38 samples of advanced gallbladder carcinoma and in 40 samples of gallbladders without malignant changes. Results: Hyperploidies were found in the karyotype of 15 carcinomas (near triploidies and near teraploidies). These findings were confirmed in the cytometric study. Multiple structural chromosome abnormalities were found in 11 and 15 samples, such as translocations, deletions, inversions, isochromosomes, rings and markers. Some chromosome alterations such as interstitial deletion of chromosome 4, deletion of distal region of chromosome 12, deletion of distal segment of the short arm of chromosome 17 with a fracture point in p12 and rearrangement of chromosome 6 were repeated in 2 or more cases. Conclusions: Hyperploidies in gallbladder carcinoma are an alteration that appears in advanced stages of the tumor. Chromosomal abnormalities may be a primary or specific alteration of this tumor, whose prognostic or diagnostic role should be explored. There are ongogenes related to some mentioned chromosomal fracture points, that should be explored with molecular techniques

Genética y cáncer vesicular en Chile: distribución de grupos sanguíneos / Genetics and gallbladder cancer in Chile: distribution of blood groups

En países anglosajones se ha comunicado mayor frecuencia de grupo sanguíneo A en pacientes con cáncer gástrico, comparado con el resto de la población, a diferencia de otros como Japón y Chile con igual distribución en cancerosos gástricos que en la población general. La relación del cáncer vesicular en Chile no sólo con factores ambientales sino también genéticos, eventualmente hace valioso este tipo de estudio en nuestro país. La población de cáncer vesicular del área Sur Oriente en Santiago, tiene una distribución de genes sanguíneos diferente a la de población general chilena, con mayor proporción relativa de grupo 0 y menor relativa de grupo A. Es posible que las diferencias se deban al azar, pero es más probable que la variación de la distribución sanguínea se deba a la calidad de estrato socioeconómico bajo predominante, por su semejanza a lo establecido para una población similar según un estudio chileno reciente

Evaluación inmunológica e inmunogenética de pacientes con cáncer de la vesícula biliar: comunicación preliminar / Immunologic and immunogenetic evaluation of the patients with gallbladder cancer: preliminary comunication

Se estudiaron cinco pacientes con cáncer de la vesícula biliar (CV) con prolongada sobrevida postoperatoria (4-12 años): Grupo A. Se compara este grupo con cinco pacientes intervenidos recientemente, cuya enfermedad está avanzada: Grupo B y con cinco enfermos sometidos a colecistectomía por litiasis y sin cáncer: Grupo C (control). En el Grupo A hubo 2 casos con antecedentes de cáncer en otras localizaciones (mama, útero y recto), lo que hace suponer una predisposición neoplásica y una especial inmunidad contra el cáncer. En este estudio preliminar, la respuesta inmunitaria de los pacientes del Grupo A y C fue similar. En los enfermos del Grupo B, se observó reducción de la inmunocompetencia con la progresión de la enfermedad, especialmente en los test cutáneos. En el estudio histoquímico, en los Grupos A y B, las poblaciones celulares relacionadas con la inmunidad se situaron en la periferia del tumor pero sólo en las del Grupo A se encontraron en el interior de la masa tumoral. En la investigación inmunogénetica se observó que el antígeno HLA-DR4 se encuentra en una proporción mayor en los pacientes con CV (38%), en comparación con la población chilena nornal (29%). Se incluye que es necesario: a. Proseguir la investigación, aumentando el número de casos; b. trabajar en el estudio inmuno histoquímico sólo con vesículas frescas, y c. Seguir investigando los antígenos del sistema HLA, tanto en pacientes portadores de cáncer vesícular, como en aquéllos con simple colelitiasis