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1.
Acta Cytol ; 64(1-2): 175-181, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31121596

RESUMO

Biliary brushing cytology has become the standard of practice for the investigation of strictures of the biliary and pancreatic duct systems. The methodology however has a limitation in that it has low diagnostic sensitivity when only cytologic evaluation is used. A number of testing methodologies have been applied to brushing specimens in an attempt to improve overall sensitivity without loss of specificity. These have included DNA ploidy analysis, immunocytochemistry, individual gene mutational analysis, fluorescence in-situ hybridization (FISH), and next generation sequencing (NGS). Currently, FISH coupled with routine cytology appears to be the method of choice for improving diagnostic sensitivity. NGS shows significant promise for improvement of diagnostic sensitivity.


Assuntos
Neoplasias dos Ductos Biliares/diagnóstico , Ductos Biliares Intra-Hepáticos/patologia , Sistema Biliar/patologia , Colangiocarcinoma/diagnóstico , Citodiagnóstico/métodos , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/metabolismo , Sistema Biliar/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Sensibilidade e Especificidade
2.
J Clin Pathol ; 73(1): 23-29, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31422372

RESUMO

AIMS: The histological distinction of intrahepatic cholangiocarcinoma (ICC) from metastatic adenocarcinoma remains a challenge. The primary goal was to evaluate the diagnostic value of morphology and albumin expression in the diagnosis of ICC. METHODS: We evaluated morphological patterns in 120 ICCs and 677 non-hepatic adenocarcinomas and performed in situ hybridisation (ISH) stain for albumin in the former cohort (retrospective cohort). We also identified 119 samples from primary and metastatic lesions, the validation cohort, in which albumin ISH was performed as part of the diagnostic workup. Targeted sequencing was performed on selected cases. We also mined existing expression profiling data including cases from The Cancer Genome Atlas (TCGA) (41 760 unique samples). RESULTS: In the retrospective cohort, 45% of ICCs and <1% of non-hepatic adenocarcinomas showed a cholangiolar pattern; albumin ISH was positive in 93% of ICCs with significant intratumorous heterogeneity. In the validation cohort, 29% of ICCs showed a cholangiolar pattern and 88% expressed albumin, while all metastatic non-hepatic neoplasms were negative (n=37) (sensitivity 88% and specificity 100%). Targetable genetic alterations (IDH mutations and FGFR2 fusions) were identified in 31% of ICCs (10 of 32). An analysis of the TCGA data validated the specificity of the albumin assay. CONCLUSIONS: The cholangiolar pattern and albumin RNA ISH distinguishes ICC from metastatic adenocarcinoma with high specificity. Given the high prevalence of targetable mutations in ICC, albumin RNA ISH is an essential component in the workup of tumours of uncertain origin. A specific diagnosis of ICC could trigger molecular testing and uncover targetable genetic alterations.


Assuntos
Albuminas/genética , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Biomarcadores Tumorais/genética , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Hibridização In Situ , Adenocarcinoma/genética , Adenocarcinoma/secundário , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias dos Ductos Biliares/secundário , Biópsia por Agulha , Diagnóstico Diferencial , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Retrospectivos , Transcriptoma , Adulto Jovem
3.
Gut ; 69(1): 52-61, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30971436

RESUMO

OBJECTIVE: Despite improvements in imaging, serum CA19-9 and pathological evaluation, differentiating between benign and malignant bile duct strictures remains a diagnostic conundrum. Recent developments in next-generation sequencing (NGS) have opened new opportunities for early detection and management of cancers but, to date, have not been rigorously applied to biliary specimens. DESIGN: We prospectively evaluated a 28-gene NGS panel (BiliSeq) using endoscopic retrograde cholangiopancreatography-obtained biliary specimens from patients with bile duct strictures. The diagnostic performance of serum CA19-9, pathological evaluation and BiliSeq was assessed on 252 patients (57 trainings and 195 validations) with 346 biliary specimens. RESULTS: The sensitivity and specificity of BiliSeq for malignant strictures was 73% and 100%, respectively. In comparison, an elevated serum CA19-9 and pathological evaluation had sensitivities of 76% and 48%, and specificities of 69% and 99%, respectively. The combination of BiliSeq and pathological evaluation increased the sensitivity to 83% and maintained a specificity of 99%. BiliSeq improved the sensitivity of pathological evaluation for malignancy from 35% to 77% for biliary brushings and from 52% to 83% for biliary biopsies. Among patients with primary sclerosing cholangitis (PSC), BiliSeq had an 83% sensitivity as compared with pathological evaluation with an 8% sensitivity. Therapeutically relevant genomic alterations were identified in 20 (8%) patients. Two patients with ERBB2-amplified cholangiocarcinoma received a trastuzumab-based regimen and had measurable clinicoradiographic response. CONCLUSIONS: The combination of BiliSeq and pathological evaluation of biliary specimens increased the detection of malignant strictures, particularly in patients with PSC. Additionally, BiliSeq identified alterations that may stratify patients for specific anticancer therapies.


Assuntos
Colangiopancreatografia Retrógrada Endoscópica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias dos Ductos Biliares/diagnóstico , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Doenças Biliares/diagnóstico , Doenças Biliares/genética , Doenças Biliares/patologia , Biomarcadores Tumorais/sangue , Antígeno CA-19-9/sangue , Constrição Patológica/diagnóstico , Constrição Patológica/genética , Diagnóstico Diferencial , Feminino , Humanos , Cirrose Hepática Biliar/diagnóstico , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/patologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Adulto Jovem
4.
APMIS ; 128(1): 3-9, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31628675

RESUMO

Biliary tract cancers (BTC) are a rare heterogeneous disease group with a dismal prognosis and limited treatment options. The mutational landscape consists of genetic aberrations both shared by and characteristic for anatomical location. Here, we present exome sequencing data on 22 genes from a phase 2 trial using a clinically validated panel used in patients with colorectal cancer. A total of 56 patients were included in a one-armed phase 2 trial investigating the treatment combination of capecitabine, gemcitabine, oxaliplatin, and cetuximab. Tissue DNA yield and quality allowed analysis of 30 patients on our panel including 22 genes. ARID1A (33%) and TP53 (33%) were found to be most frequently mutated followed by KRAS mutations found in 20% of the patients. Mutational aberrations in ARID1A were found more prevalent than expected, whereas TP53 and KRAS were in concordance with earlier reported data. Mutation in CTNNB1 was significantly associated with poor prognosis. Our panel is clinically validated and suitable for a high volume of samples to detect mutations in patients with BTC. However, it is reasonable to assume that the clinical utility could be optimized in this patient group by extending the panel to include BTC specific mutations with potential therapeutic consequences such as IDH1/2, FGFR fusions, ERBB3, and BRCA1/2.


Assuntos
Neoplasias dos Ductos Biliares/genética , Exoma , Mutação , Adulto , Idoso , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias dos Ductos Biliares/tratamento farmacológico , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Inclusão em Parafina , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de DNA , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , beta Catenina/genética
6.
Cancer Treat Rev ; 78: 1-7, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31255945

RESUMO

Cholangiocarcinoma is the most common aggressive biliary tract malignancy with dismal prognosis. Though surgical resection of the primary tumors yields better prognosis, majority of patients present at advanced, inoperable stages rendering systemic therapy as the only option. A significant progress has been made in understanding the cholangiocarcinoma tumorigenesis and molecular markers over the last decade, which opens doors to precision medicine in this dismal cancer. Intrahepatic cholangiocarcinomas are most likely to harbor mutations in isocitrate dehydrogenase genes (IDH1, IDH2), fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3), Eph receptor 2 (EPHA2), and BAP1 (gene involved in chromatin remodeling) genes, whereas ARID1B, ELF3, PBRM1, cAMP dependent protein kinase (PRKACA, and PRKACB) genetic mutations were implicated more commonly in distal and perihilar subtypes. Genomic studies have shown that FGFR2 aberrations are implicated in approximately 15% of intrahepatic cholangiocarcinomas, which make FGFR2 aberrations (Achilles heel) as potential novel targets in the management of cholangiocarcinoma. The current review comprehensively focuses on the role of FGFR2 inhibition either alone or in combination with other targeted therapy that act on down-stream and alternate kinase pathways in cholangiocarcinoma.


Assuntos
Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/terapia , Colangiocarcinoma/genética , Colangiocarcinoma/terapia , Aberrações Cromossômicas , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Animais , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , Gerenciamento Clínico , Humanos , Prognóstico
7.
Cancer Sci ; 110(10): 3197-3203, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31361379

RESUMO

Intrahepatic cholangiocarcinoma is a rare malignant biliary neoplasm that causes a poor prognosis even after curative hepatectomy. Liver metastasis is the major recurrence pattern of intrahepatic cholangiocarcinoma; therefore, the prevention of liver metastasis is a desirable objective. The aim of this study is to identify gene(s) related to liver metastasis of intrahepatic cholangiocarcinoma and to examine the inhibitory effects on metastasis of intrahepatic cholangiocarcinoma by controlling such gene(s). We collected 3 pairs of intrahepatic cholangiocarcinoma frozen samples, and 36 pairs (primary and metastatic lesions) of intrahepatic cholangiocarcinoma formalin-fixed paraffin-embedded samples, from patients who underwent surgical resection at hospitals related to the Kyushu Study Group of Liver Surgery between 2002 and 2016. We carried out cDNA microarray analyses and immunohistochemistry to identify candidate genes, and evaluated one of them as a therapeutic target using human cholangiocarcinoma cell lines. We identified 4 genes related to liver metastasis using cDNA microarray, and found that CXCL12 was the only gene whose expression was significantly higher in liver metastasis than in primary intrahepatic cholangiocarcinoma by immunohistochemistry (P = .003). In prognosis, patients in the high CXCL12 group showed a significantly poor prognosis in disease-free (P < .0001) and overall survival (P = .0004). By knockdown of CXCL12, we could significantly suppress the invasive and migratory capabilities of 2 human cholangiocarcinoma cell lines. Therefore, CXCL12 might be associated with metastasis and poor prognosis in intrahepatic cholangiocarcinoma.


Assuntos
Neoplasias dos Ductos Biliares/patologia , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Colangiocarcinoma/patologia , Neoplasias Hepáticas/secundário , Idoso , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Prognóstico , Regulação para Cima
8.
Gastroenterology ; 157(4): 1067-1080.e9, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31229495

RESUMO

BACKGROUND & AIMS: Bile duct tumors are rare and have poor prognoses. Natural killer (NK) cells are frequent in human liver and infiltrate these tumors but do not control their progression. Responses of NK cells are regulated by NK immunoglobulin-like receptors (KIRs), which interact with HLA class I ligands. We aimed to characterize the features of the KIR gene loci and their ligands in patients with bile duct cancer (BDC). METHODS: We performed combined multidimensional characterization of genes that encode KIRs and their ligands in blood samples from patients with BDC from Sweden, followed for up to 8 years after diagnosis (n = 148), in 2 geographically matched cohorts of healthy individuals from Northern Europe (n = 204 and n = 900), and in healthy individuals from 6 geographically unrelated populations (n = 2917). We used real-time polymerase chain reaction, RNA sequencing, immunohistochemistry, and flow cytometry to evaluate NK-cell presence, as well as KIR and KIR-ligand expression in bile duct tumors and control tissues. RESULTS: Patients with bile duct tumors had multiple alterations at the KIR gene loci. KIR loci are grouped into genotypes that encode more inhibitory (group A) and more activating (group B) receptors, which can be subdivided into centromeric and telomeric fragments. Patients with BDC had a lower prevalence of KIR2DL3, which was linked to disequilibrium in centromeric A/B and B/B genotypes, compared with control individuals. The associations between KIRs and KIR ligands differed between patients with BDC and control individuals; patients had an altered balance between activating and inhibitory KIRs. KIR-positive NK cells infiltrated biliary tumors that expressed matched KIR ligands. CONCLUSIONS: In a multidimensional analysis of DNA from blood samples of patients with BDC in Europe, we found patients to have multiple alterations at the KIR and HLA gene loci compared with control individuals. These alterations might affect NK-cell tumor surveillance. NK cells from bile duct tumors expressed KIRs and were found in tumors that expressed cognate ligands. This should be considered in development of immune-based therapies for BDC.


Assuntos
Neoplasias dos Ductos Biliares/genética , Antígenos HLA/genética , Células Matadoras Naturais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Receptores KIR/genética , Idoso , Idoso de 80 Anos ou mais , Ásia , Neoplasias dos Ductos Biliares/sangue , Neoplasias dos Ductos Biliares/imunologia , Neoplasias dos Ductos Biliares/patologia , Estudos de Casos e Controles , Europa (Continente) , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Antígenos HLA/sangue , Antígenos HLA/imunologia , Humanos , Células Matadoras Naturais/patologia , Ligantes , Desequilíbrio de Ligação , Linfócitos do Interstício Tumoral/patologia , Masculino , Pessoa de Meia-Idade , América do Norte , Fenótipo , Prognóstico , Receptores KIR/sangue , Receptores KIR/imunologia , Receptores KIR2DL3/genética , Receptores KIR2DL3/imunologia , Fatores de Risco , América do Sul , Fatores de Tempo
9.
Hepatol Int ; 13(4): 490-500, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31214875

RESUMO

BACKGROUND: Although molecular characterization of iCCA has been studied recently, integrative analysis of molecular and clinical characterization has not been fully established. If molecular features of iCCA can be predicted based on clinical findings, we can approach to distinguish targeted treatment. We analyzed RNA sequencing data annotated with clinicopathologic data to clarify molecular-specific clinical features and to evaluate potential therapies for molecular subtypes. METHODS: We performed next-generation RNA sequencing of 30 surgically resected iCCA from Korean patients and the clinicopathologic features were analyzed. The RNA sequences from 32 iCCA resected from US patients were used for validation. RESULTS: Patients were grouped into two subclasses on the basis of unsupervised clustering, which showed a difference in 5-year survival rates (48.5% vs 14.2%, p = 0.007) and similar survival outcome in the US samples. In subclass B (poor prognosis), both data sets were similar in higher carcinoembryonic antigen and cancer antigen 19-9 levels, underlying cholangitis, and bile duct-type pathology; in subclass A (better prognosis), there was more frequent viral hepatitis and cholangiolar-type pathology. On pathway analysis, subclass A had enriched liver-related signatures. Subclass B had enriched inflammation-related and TP53 pathways, with more frequent KRAS mutations. CCA cell lines with similar gene expression patterns of subclass A were sensitive to gemcitabine. CONCLUSIONS: Two molecular subtypes of iCCA with distinct clinicopathological differences were identified. Knowledge of clinical and pathologic characteristics can predict molecular subtypes, and knowledge of different subtype signaling pathways may lead to more rational, targeted approaches to treatment.


Assuntos
Neoplasias dos Ductos Biliares/mortalidade , Colangiocarcinoma/mortalidade , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/uso terapêutico , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Feminino , Genes Neoplásicos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Prognóstico , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , República da Coreia/epidemiologia , Estudos Retrospectivos , Estados Unidos/epidemiologia , Regulação para Cima
10.
Int J Oncol ; 55(1): 298-308, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115511

RESUMO

Leptin is an adipokine minimally known for its activities or underlying mechanisms in cholangiocarcinoma. The present study explored the effects of leptin on the epithelial­mesenchymal transition (EMT) and pro­angiogenic capability of cholangiocarcinoma cells, and investigated the underlying mechanisms. Cholangiocarcinoma cells were treated with leptin, and their migration and invasion rates were investigated using Transwell assays. Furthermore, conditioned medium was collected from cholangiocarcinoma cells following leptin treatment and applied to human umbilical vein endothelial cells to assess tube formation. The expression of EMT and pro­angiogenic factors was examined by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blot analyses. Mechanistically, the function of pyruvate kinase muscle isozyme M2 (PKM2) was assessed in leptin­induced phenotypes using siRNA targeting PKM2 (si­PKM2). Bioinformatics screening and luciferase reporter assays were used to reveal microRNA (miR)­122 as the potential mediator between leptin and PKM2. Finally, the associations between leptin and miR­122 or PKM2 levels in patients with cholangiocarcinoma were assessed by ELISA and RT­qPCR. Leptin significantly increased the EMT and pro­angiogenic capability of cholangiocarcinoma cells, visibly inhibited endogenous miR­122 expression, and upregulated PKM2. Furthermore, si­PKM2 inhibited leptin­induced migration, invasion, EMT­associated marker expression levels and the pro­angiogenic capability in cholangiocarcinoma cells. In addition, miR­122 negatively regulated the expression of PKM2. When applied together with leptin, miR­122 was sufficient to reverse the multiple malignancy­promoting effects of leptin. Consistently, the serum leptin level positively correlated with that of PKM2, but negatively with that of miR­122 in patients with cholangiocarcinoma. Leptin, by downregulating miR­122 and elevating PKM2 expression, acts as a pleiotropic pro­malignancy cytokine for cholangiocarcinoma. Therefore, increasing miR­122 expression and inhibiting PKM2 may be future approaches for cholangiocarcinoma treatment.


Assuntos
Neoplasias dos Ductos Biliares/metabolismo , Proteínas de Transporte/genética , Colangiocarcinoma/metabolismo , Leptina/metabolismo , Proteínas de Membrana/genética , MicroRNAs/genética , Hormônios Tireóideos/genética , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Meios de Cultivo Condicionados/química , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Leptina/sangue , Proteínas de Membrana/metabolismo , Hormônios Tireóideos/metabolismo
11.
Int J Oncol ; 55(1): 45-58, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31059014

RESUMO

The present study aimed to explore the long non­coding RNA (lncRNA) expression profiles and correlation of lnc­PKD2­2­3 with tumor features and prognosis, and to investigate its effect on regulating cancer­cell stemness and its potential as a cancer stem cell (CSC) marker in cholangiocarcinoma (CCA). lncRNA expression profiles were determined in 3 pairs of CCA tumors and adjacent tissues by microarray analysis, and lnc­PKD2­2­3 expression was then validated in 60 paired samples by reverse transcription­quantitative polymerase chain reaction (RT­qPCR). Expression of common CSC markers [(CD44, CD133 and octamer­binding transcription factor 4 (OCT4)], CD44+CD133+ cell proportions, sphere formation efficiency and drug resistance to 5­fluorouracil (5­FU) were measured following ectopic overexpression of lnc­PKD2­2­3 or silencing via small hairpin RNA lentivirus transfection into the TFK­1 and Huh­28 CCA cell lines. Finally, lnc­PKD2­2­3 expression was measured in CCA stem­like cells and normal CCA cells. The results from the microarray analysis identified a total of 4,223 upregulated and 4,596 downregulated lncRNAs between CCA tumor tissue and paired adjacent tissue, which were enriched in regulating cancer­associated pathways. RT­qPCR validation revealed that lnc­PKD2­2­3 was upregulated in CCA and associated with a higher Eastern Cooperative Oncology Group performance score, poor differentiation, advanced TNM stage, increased carcinoembryonic antigen and poor overall survival in CCA patients. In vitro, lnc­PKD2­2­3 increased CD44, CD133 and OCT4 expression as well as the CD44+CD133+ cell proportion, raised the sphere formation efficiency and enhanced drug resistance to 5­FU in TFK­1 and Huh­28 cells. In addition, lnc­PKD2­2­3 was positively correlated with CSC markers in CCA tumor tissues and was markedly upregulated in CCA stem­like cells compared with that in normal CCA cells. In conclusion, lnc­PKD2­2­3, selected by lncRNA expression profiling, was associated with pejorative tumor features and poor prognosis, enhanced cancer stemness and may serve as a CSC marker in CCA.


Assuntos
Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica/métodos , Células-Tronco Neoplásicas/patologia , RNA Longo não Codificante/genética , Neoplasias dos Ductos Biliares/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Colangiocarcinoma/genética , Feminino , Fluoruracila , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Estadiamento de Neoplasias , Células-Tronco Neoplásicas/química , Prognóstico , Análise de Sobrevida , Regulação para Cima
12.
Cas Lek Cesk ; 158(2): 64-67, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31109165

RESUMO

Cholangiocellular carcinoma is a relatively rare malignant tumor, originating from cholangiocytes, with poor prognosis and late diagnosis. It is a malignancy with a variable biological etiology, numerous genetic and epigenetic changes. Its incidence in the Czech Republic is about 1.4 per 100,000 people per year. For good prognosis and long-term survival, early diagnosis with surgical treatment is important. In these cases, a 5-year survival rate is about 20-40 %. In the early diagnosis imaging methods and histopathological verification play an essential role, whereas laboratory oncomarkers are not yet sufficiently accurate. The same applies for genetic markers. This leads to the search of new molecular targets and the high effort in the introduction of cytological and molecular-biological methods with high specificity and sensitivity into routine practice. Current early diagnosis is based on the use of efficient imaging methods. The use of genetic testing, and especially knowledge of the molecular basis of this disease, will be of a great benefit. The observation of the association between the genetic pathways, IDH1, RAS-MAPK etc., and genetic mutations of genes, such as TP53, KRAS, SMAD4, BRAF, IDH1/2, may be significant. From the molecular point of view, it is also interesting to monitor oncogenic potential in HBV/HCV infection.


Assuntos
Neoplasias dos Ductos Biliares , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma , Neoplasias dos Ductos Biliares/diagnóstico , Neoplasias dos Ductos Biliares/genética , Colangiocarcinoma/diagnóstico , Colangiocarcinoma/genética , República Tcheca , Genes Neoplásicos , Testes Genéticos , Humanos , Mutação , Patologia Molecular
13.
Cancer Sci ; 110(7): 2166-2179, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31111617

RESUMO

Cholangiocarcinoma (CCA) is a malignant tumor originating from bile duct epithelium and its incidence is increasing year by year. In recent years, long noncoding RNAs (lncRNAs) have been found to play an important role in the occurrence and progression of malignant tumors. In the present study, for the first time, abnormal expression of lnc-RNA component of mitochondrial RNA processing endoribonuclease (RMRP) and its possible role in CCA were found. We explored the effects of RMRP on various behaviors of CCA cells in vitro and in vivo. In addition, by second-generation sequencing, we explored the microRNA expression profiles that RMRP may affect in the HCCC-9810 cell line. We also validated and explored the role of microRNA-217 (miR-217) with high differential expression by in vitro experiments. Our findings indicated that RMRP can play a part in promoting cancer by regulating the expression of miR-217. RMRP is involved in the progression of CCA and can be a novel indicator of poor prognosis in patients with CCA.


Assuntos
Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , MicroRNAs/genética , RNA Longo não Codificante/genética , Análise de Sequência de RNA/métodos , Animais , Neoplasias dos Ductos Biliares/genética , Linhagem Celular Tumoral , Colangiocarcinoma/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Regulação para Cima
15.
Genome Med ; 11(1): 21, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30987660

RESUMO

CRISPR/Cas9 has revolutionized cancer mouse models. Although loss-of-function genetics by CRISPR/Cas9 is well-established, generating gain-of-function alleles in somatic cancer models is still challenging because of the low efficiency of gene knock-in. Here we developed CRISPR-based Somatic Oncogene kNock-In for Cancer Modeling (CRISPR-SONIC), a method for rapid in vivo cancer modeling using homology-independent repair to integrate oncogenes at a targeted genomic locus. Using a dual guide RNA strategy, we integrated a plasmid donor in the 3'-UTR of mouse ß-actin, allowing co-expression of reporter genes or oncogenes from the ß-actin promoter. We showed that knock-in of oncogenic Ras and loss of p53 efficiently induced intrahepatic cholangiocarcinoma in mice. Further, our strategy can generate bioluminescent liver cancer to facilitate tumor imaging. This method simplifies in vivo gain-of-function genetics by facilitating targeted integration of oncogenes.


Assuntos
Neoplasias dos Ductos Biliares/genética , Sistemas CRISPR-Cas , Colangiocarcinoma/genética , Técnicas de Introdução de Genes/métodos , Genes ras , Actinas/genética , Animais , Neoplasias dos Ductos Biliares/diagnóstico por imagem , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Colangiocarcinoma/diagnóstico por imagem , Colangiocarcinoma/patologia , Genes Reporter , Genes p53 , Humanos , Camundongos
16.
Biomed Res Int ; 2019: 3526407, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31019967

RESUMO

Background: Cholangiocarcinoma (CCA) is the second most common malignant primary liver tumor and has shown an alarming increase in incidence over the last two decades. However, the mechanisms behind tumorigenesis and progression remain insufficient. The present study aimed to uncover the underlying regulatory mechanism on CCA and find novel biomarkers for the disease prognosis. Method: The RNA-sequencing (RNA-seq) datasets of lncRNAs, miRNAs, and mRNAs in CCA as well as relevant clinical information were obtained from the Cancer Genome Atlas (TCGA) database. After pretreatment, differentially expressed RNAs (DERNAs) were identified and further interrogated for their correlations with clinical information. Prognostic RNAs were selected using univariate Cox regression. Then, a ceRNA network was constructed based on these RNAs. Results: We identified a total of five prognostic DEmiRNAs, 63 DElncRNAs, and 90 DEmRNAs between CCA and matched normal tissues. Integrating the relationship between the different types of RNAs, an lncRNA-miRNA-mRNA network was established and included 28 molecules and 47 interactions. Screened prognostic RNAs involved in the ceRNA network included 3 miRNAs (hsa-mir-1295b, hsa-mir-33b, and hsa-mir-6715a), 7 lncRNAs (ENSG00000271133, ENSG00000233834, ENSG00000276791, ENSG00000241155, COL18A1-AS1, ENSG00000274737, and ENSG00000235052), and 18 mRNAs (ANO9, FUT4, MLLT3, ABCA3, FSCN2, GRID2IP, NCK2, MACC1, SLC35E4, ST14, SH2D3A, MOB3B, ACTL10, RAB36, ATP1B3, MST1R, SEMA6A, and SEL1L3). Conclusions: Our study identified novel prognostic makers and predicted a previously unknown ceRNA regulatory network in CCA and may provide novel insight into a further understanding of lncRNA-mediated ceRNA regulatory mechanisms in CCA.


Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Neoplasias Hepáticas , MicroRNAs , RNA Longo não Codificante , RNA Mensageiro , RNA Neoplásico , Idoso , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Masculino , MicroRNAs/biossíntese , MicroRNAs/genética , Pessoa de Meia-Idade , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Análise de Sequência de RNA , Taxa de Sobrevida
17.
Neoplasia ; 21(5): 429-441, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30933885

RESUMO

Cholangiocarcinoma (CCA) is a malignant cancer with an unknown etiology and an unfavorable prognosis. Most patients are diagnosed at an advanced stage, thus making it essential to find novel curative targets for CCA. Metabolic reprogramming of the tumor cells includes metabolic abnormalities in glucose (known as the Warburg effect) and other substances such as amino acids and fats. Metabolic reprogramming produces anti-oxidant substances, reduces tumor oxidative stress, and finally promotes the proliferation of tumors. There is increasing evidence to imply that SIRT2, a histone deacetylase, and its downstream target cMYC, play metabolic regulatory roles in tumor cells. However, the role of the SIRT2/cMYC pathway in CCA is unclear. To assess the metabolic reprogramming function of the SIRT2/cMYC pathway in CCA and to determine the downstream targets as well as evaluate the therapeutic effect, the CCA RNA-Seq data were downloaded from the TCGA database. Differentially expressed genes were confirmed and KEGG pathway enrichment analysis was performed. Overall, 48 paired CCA samples were collected and subjected to immunohistochemical detection, and the clinical characteristics of participants were summarized. The CCA cells were suppressed or overexpressed with different downstream targets of SIRT2 and then subjected to apoptosis, immunoblotting, seahorse, and metabolites tracing analysis. In vivo experiments were also performed. We found that the SIRT2/cMYC pathway contributed to the proliferation of CCA cells and confirmed that the downstream target is PHDA1 and the serine synthesis pathway. The up-regulated SIRT2 and cMYC levels resulted in low levels of mitochondrial oxidative phosphorylation and increased conversion of glucose to serine and led to poor patient survival. The highly active SIRT2/cMYC pathway up-regulated the serine synthesis pathway pyruvate and increased antioxidant production, thus consequently protecting the CCA cells from oxidative stress-induced apoptosis. Our data revealed that the SIRT2/cMYC pathway plays a critical role in transforming glucose oxidative metabolism to serine anabolic metabolism, thus providing antioxidants for stress resistance. SIRT2/cMYC-induced metabolic reprogramming may represent a new therapeutic target for treating CCA.


Assuntos
Neoplasias dos Ductos Biliares/patologia , Reprogramação Celular , Colangiocarcinoma/patologia , Regulação Neoplásica da Expressão Gênica , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sirtuína 2/metabolismo , Animais , Apoptose , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Movimento Celular , Proliferação de Células , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Feminino , Glucose/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Fosforilação Oxidativa , Prognóstico , Proteínas Proto-Oncogênicas c-myc/genética , Piruvato Desidrogenase (Lipoamida)/genética , Piruvato Desidrogenase (Lipoamida)/metabolismo , Serina/metabolismo , Sirtuína 2/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Clin Epigenetics ; 11(1): 39, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30832707

RESUMO

BACKGROUND: Cholangiocarcinoma (CCA) is a fatal cancer of the bile duct epithelial cell lining. The misdiagnosis of CCA and other biliary diseases may occur due to the similarity of clinical manifestations and blood tests resulting in inappropriate or delayed treatment. Thus, an accurate and less-invasive method for differentiating CCA from other biliary diseases is inevitable. METHODS: We quantified methylation of OPCML, HOXA9, and HOXD9 in serum cell-free DNA (cfDNA) of CCA patients and other biliary diseases using methylation-sensitive high-resolution melting (MS-HRM). Their potency as differential biomarkers between CCA and other biliary diseases was also evaluated by using receiver operating characteristic (ROC) curves. RESULTS: The significant difference of methylation levels of OPCML and HOXD9 was observed in serum cfDNA of CCA compared to other biliary diseases. Assessment of serum cfDNA methylation of OPCML and HOXD9 as differential biomarkers of CCA and other biliary diseases showed the area under curve (AUC) of 0.850 (0.759-0.941) for OPCML which sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were 80.00%, 90.00%, 88.88%, 81.81%, and 85.00%, respectively. The AUC of HOXD9 was 0.789 (0.686-0.892) with sensitivity, specificity, PPV, NPV, and accuracy of 67.50%, 90.00%, 87.09%, 73.46%, and 78.75%, respectively. The combined marker between OPCML and HOXD9 showed sensitivity, specificity, PPV, and NPV of 62.50%, 100%, 100%, and 72.72%, respectively, which may be helpful to prevent a misdiagnosis between CCA and other biliary diseases. CONCLUSIONS: Our findings suggest the application of serum cfDNA methylation of OPCML and HOXD9 for differential diagnosis of CCA and other biliary diseases due to its less invasiveness and clinically practical method which may benefit the patients by preventing the misdiagnosis of CCA and avoiding unnecessary surgical intervention.


Assuntos
Neoplasias dos Ductos Biliares/diagnóstico , Biomarcadores Tumorais/genética , Moléculas de Adesão Celular/genética , Colangiocarcinoma/diagnóstico , Metilação de DNA , Proteínas de Homeodomínio/genética , Proteínas de Neoplasias/genética , Área Sob a Curva , Doenças dos Ductos Biliares/diagnóstico , Doenças dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/genética , Ácidos Nucleicos Livres , Colangiocarcinoma/genética , Diagnóstico Diferencial , Feminino , Proteínas Ligadas por GPI/genética , Humanos , Masculino , Sensibilidade e Especificidade
19.
Oncogene ; 38(25): 4948-4961, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842588

RESUMO

SMYD3, a member that belongs to the SET and MYND-domain (SMYD) family, has also been proven to largely participate in gene transcription regulation and progression of several human cancers as a histone lysine methyltransferase. However, the role and significance of SMYD3 in both the clinic and progression of hepatocellular carcinoma (HCC) remain unclear. Herein, we find that SMYD3 is increased in cirrhotic livers, and strikingly upregulated in hepatocellular carcinoma (HCC) tissues and cell lines. Subsequent analyses suggest that high expression level of SMYD3 significantly correlates with the malignant characteristics of HCC, and predicts poor prognosis in patients. Our results show that overexpression of SMYD3 increases, while silencing of SMYD3 inhibits, cell proliferation, invasiveness and tumorigenicity both in vitro and in vivo. SMYD3 also promotes intrahepatic metastasis of HCC cells. For the mechanisms, we identify that SMYD3 bound to CDK2 and MMP2 promoter and increased H3K4me3 modification at the corresponding promoters to promote gene transcription. Importantly, pharmacological targeting of SMYD3 with BCI-121 inhibitor effectively repressed the tumorigenicity of HCC cells. Finally, our results show that gene locus amplification is a cause for SMYD3 overexpression in HCC. These findings not only uncover that SMYD3 overexpression promotes the tumorigenicity and intrahepatic metastasis of HCC cell via upregulation of CDK2 and MMP2, but also suggest SMYD3 could be a practical prognosis marker or therapeutic target against the disease.


Assuntos
Carcinogênese/genética , Carcinoma Hepatocelular/patologia , Quinase 2 Dependente de Ciclina/genética , Amplificação de Genes , Histona-Lisina N-Metiltransferase/genética , Neoplasias Hepáticas/patologia , Metaloproteinase 2 da Matriz/genética , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/secundário , Ductos Biliares Intra-Hepáticos/patologia , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Progressão da Doença , Feminino , Amplificação de Genes/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Ativação Transcricional , Células Tumorais Cultivadas , Regulação para Cima/genética
20.
Liver Int ; 39 Suppl 1: 7-18, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30882996

RESUMO

Cholangiocarcinoma constitutes a heterogeneous group of malignancies that can emerge at any point of the biliary tree. Cholangiocarcinoma is classified into intrahepatic, perihilar and distal based on its anatomical location. Histologically, conventional perihilar/distal cholangiocarcinomas are mucin-producing adenocarcinomas or papillary tumours; intrahepatic cholangiocarcinomas are more heterogeneous and can be sub-classified according to the level or size of the displayed bile duct. Cholangiocarcinoma develops through multistep carcinogenesis and is preceded by dysplastic and in situ lesions. Definition and clinical significance of precursor lesions, including biliary intraepithelial neoplasia, intraductal papillary neoplasms of the bile duct, intraductal tubulopapillary neoplasms and mucinous cystic neoplasm, are discussed in this review. A main challenge in diagnosing cholangiocarcinoma is the fact that tumour tissue for histological examination is difficult to obtain. Thus, a major clinical obstacle is the establishment of the correct diagnosis at a tumour stage that is amenable to surgery which still represents the only curable therapeutic option. Current standards, methodology and criteria for diagnosis are discussed. Cholangiocarcinoma represents a heterogeneous tumour with regard to molecular alterations. In intrahepatic subtype, mainly two distinctive morpho-molecular groups can currently be discriminated. Large-duct type intrahepatic cholangiocarcinoma shows a high mutation frequency of oncogenes and tumour suppressor genes, such as KRAS and TP53 while Isocitrate Dehydrogenase 1/2 mutations and Fibroblast Growth Factor Receptor 2-fusions are typically seen in small-duct type tumours. It is most important to ensure the separation of the given anatomical subtypes and to search for distinct subgroups within the subtypes on a molecular and morphological basis.


Assuntos
Neoplasias dos Ductos Biliares/diagnóstico , Ductos Biliares Intra-Hepáticos/patologia , Biomarcadores Tumorais/genética , Colangiocarcinoma/diagnóstico , Neoplasias dos Ductos Biliares/classificação , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/classificação , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Predisposição Genética para Doença , Humanos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/genética
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