RESUMEN
BACKGROUND AND AIMS: Lenvatinib is an effective drug in advanced HCC. Its combination with the anti-PD1 (programmed cell death protein 1) immune checkpoint inhibitor, pembrolizumab, has generated encouraging results in phase Ib and is currently being tested in phase III trials. Here, we aimed to explore the molecular and immunomodulatory effects of lenvatinib alone or in combination with anti-PD1. APPROACH AND RESULTS: We generated three syngeneic models of HCC in C57BL/6J mice (subcutaneous and orthotopic) and randomized animals to receive placebo, lenvatinib, anti-PD1, or combination treatment. Flow cytometry, transcriptomic, and immunohistochemistry analyses were performed in tumor and blood samples. A gene signature, capturing molecular features associated with the combination therapy, was used to identify a subset of candidates in a cohort of 228 HCC patients who might respond beyond what is expected for monotherapies. In mice, the combination treatment resulted in tumor regression and shorter time to response compared to monotherapies (P < 0.001). Single-agent anti-PD1 induced dendritic and T-cell infiltrates, and lenvatinib reduced the regulatory T cell (Treg) proportion. However, only the combination treatment significantly inhibited immune suppressive signaling, which was associated with the TGFß pathway and induced an immune-active microenvironment (P < 0.05 vs. other therapies). Based on immune-related genomic profiles in human HCC, 22% of patients were identified as potential responders beyond single-agent therapies, with tumors characterized by Treg cell infiltrates, low inflammatory signaling, and VEGFR pathway activation. CONCLUSIONS: Lenvatinib plus anti-PD1 exerted unique immunomodulatory effects through activation of immune pathways, reduction of Treg cell infiltrate, and inhibition of TGFß signaling. A gene signature enabled the identification of ~20% of human HCCs that, although nonresponding to single agents, could benefit from the proposed combination.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Compuestos de Fenilurea/farmacología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Quinolinas/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Hepatocelular/inmunología , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Masculino , Ratones , Compuestos de Fenilurea/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinolinas/uso terapéutico , Escape del Tumor/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
BACKGROUND AND AIMS: Mutations in TERT (telomerase reverse transcriptase) promoter are established gatekeepers in early hepatocarcinogenesis, but little is known about other molecular alterations driving this process. Epigenetic deregulation is a critical event in early malignancies. Thus, we aimed to (1) analyze DNA methylation changes during the transition from preneoplastic lesions to early HCC (eHCC) and identify candidate epigenetic gatekeepers, and to (2) assess the prognostic potential of methylation changes in cirrhotic tissue. APPROACH AND RESULTS: Methylome profiling was performed using Illumina HumanMethylation450 (485,000 cytosine-phosphateguanine, 96% of known cytosine-phosphateguanine islands), with data available for a total of 390 samples: 16 healthy liver, 139 cirrhotic tissue, 8 dysplastic nodules, and 227 HCC samples, including 40 eHCC below 2cm. A phylo-epigenetic tree derived from the Euclidean distances between differentially DNA-methylated sites (n = 421,997) revealed a gradient of methylation changes spanning healthy liver, cirrhotic tissue, dysplastic nodules, and HCC with closest proximity of dysplasia to HCC. Focusing on promoter regions, we identified epigenetic gatekeeper candidates with an increasing proportion of hypermethylated samples (beta value > 0.5) from cirrhotic tissue (<1%), to dysplastic nodules (≥25%), to eHCC (≥50%), and confirmed inverse correlation between DNA methylation and gene expression for TSPYL5 (testis-specific Y-encoded-like protein 5), KCNA3 (potassium voltage-gated channel, shaker-related subfamily, member 3), LDHB (lactate dehydrogenase B), and SPINT2 (serine peptidase inhibitor, Kunitz type 2) (all P < 0.001). Unsupervised clustering of genome-wide methylation profiles of cirrhotic tissue identified two clusters, M1 and M2, with 42% and 58% of patients, respectively, which correlates with survival (P < 0.05), independent of etiology. CONCLUSIONS: Genome-wide DNA-methylation profiles accurately discriminate the different histological stages of human hepatocarcinogenesis. We report on epigenetic gatekeepers in the transition between dysplastic nodules and eHCC. DNA-methylation changes in cirrhotic tissue correlate with clinical outcomes.
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Carcinogénesis/genética , Carcinoma Hepatocelular/genética , Metilación de ADN , Cirrosis Hepática/genética , Neoplasias Hepáticas/genética , Anciano , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Hígado/patología , Cirrosis Hepática/patología , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
BACKGROUND AND AIMS: Non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is increasing globally, but its molecular features are not well defined. We aimed to identify unique molecular traits characterising NASH-HCC compared to other HCC aetiologies. METHODS: We collected 80 NASH-HCC and 125 NASH samples from 5 institutions. Expression array (n = 53 NASH-HCC; n = 74 NASH) and whole exome sequencing (n = 52 NASH-HCC) data were compared to HCCs of other aetiologies (n = 184). Three NASH-HCC mouse models were analysed by RNA-seq/expression-array (n = 20). Activin A receptor type 2A (ACVR2A) was silenced in HCC cells and proliferation assessed by colorimetric and colony formation assays. RESULTS: Mutational profiling of NASH-HCC tumours revealed TERT promoter (56%), CTNNB1 (28%), TP53 (18%) and ACVR2A (10%) as the most frequently mutated genes. ACVR2A mutation rates were higher in NASH-HCC than in other HCC aetiologies (10% vs. 3%, p <0.05). In vitro, ACVR2A silencing prompted a significant increase in cell proliferation in HCC cells. We identified a novel mutational signature (MutSig-NASH-HCC) significantly associated with NASH-HCC (16% vs. 2% in viral/alcohol-HCC, p = 0.03). Tumour mutational burden was higher in non-cirrhotic than in cirrhotic NASH-HCCs (1.45 vs. 0.94 mutations/megabase; p <0.0017). Compared to other aetiologies of HCC, NASH-HCCs were enriched in bile and fatty acid signalling, oxidative stress and inflammation, and presented a higher fraction of Wnt/TGF-ß proliferation subclass tumours (42% vs. 26%, p = 0.01) and a lower prevalence of the CTNNB1 subclass. Compared to other aetiologies, NASH-HCC showed a significantly higher prevalence of an immunosuppressive cancer field. In 3 murine models of NASH-HCC, key features of human NASH-HCC were preserved. CONCLUSIONS: NASH-HCCs display unique molecular features including higher rates of ACVR2A mutations and the presence of a newly identified mutational signature. LAY SUMMARY: The prevalence of hepatocellular carcinoma (HCC) associated with non-alcoholic steatohepatitis (NASH) is increasing globally, but its molecular traits are not well characterised. In this study, we uncovered higher rates of ACVR2A mutations (10%) - a potential tumour suppressor - and the presence of a novel mutational signature that characterises NASH-related HCC.
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Carcinoma Hepatocelular/genética , Biología Molecular/estadística & datos numéricos , Enfermedad del Hígado Graso no Alcohólico/genética , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/etiología , Femenino , Humanos , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/genética , Masculino , Persona de Mediana Edad , Biología Molecular/métodos , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Factores de RiesgoRESUMEN
BACKGROUND & AIMS: Cholangiocarcinoma (CCA), a deadly malignancy of the bile ducts, can be classified based on its anatomical location into either intrahepatic (iCCA) or extrahepatic (eCCA), each with different pathogenesis and clinical management. There is limited understanding of the molecular landscape of eCCA and no targeted therapy with clinical efficacy has been approved. We aimed to provide a molecular classification of eCCA and identify potential targets for molecular therapies. METHODS: An integrative genomic analysis of an international multicenter cohort of 189 eCCA cases was conducted. Genomic analysis included whole-genome expression, targeted DNA-sequencing and immunohistochemistry. Molecular findings were validated in an external set of 181 biliary tract tumors from the ICGC. RESULTS: KRAS (36.7%), TP53 (34.7%), ARID1A (14%) and SMAD4 (10.7%) were the most prevalent mutations, with â¼25% of tumors having a putative actionable genomic alteration according to OncoKB. Transcriptome-based unsupervised clustering helped us define 4 molecular classes of eCCA. Tumors classified within the Metabolic class (19%) showed a hepatocyte-like phenotype with activation of the transcription factor HNF4A and enrichment in gene signatures related to bile acid metabolism. The Proliferation class (23%), more common in patients with distal CCA, was characterized by enrichment of MYC targets, ERBB2 mutations/amplifications and activation of mTOR signaling. The Mesenchymal class (47%) was defined by signatures of epithelial-mesenchymal transition, aberrant TGFß signaling and poor overall survival. Finally, tumors in the Immune class (11%) had a higher lymphocyte infiltration, overexpression of PD-1/PD-L1 and molecular features associated with a better response to immune checkpoint inhibitors. CONCLUSION: An integrative molecular characterization identified distinct subclasses of eCCA. Genomic traits of each class provide the rationale for exploring patient stratification and novel therapeutic approaches. LAY SUMMARY: Targeted therapies have not been approved for the treatment of extrahepatic cholangiocarcinoma. We performed a multi-platform molecular characterization of this tumor in a cohort of 189 patients. These analyses revealed 4 novel transcriptome-based molecular classes of extrahepatic cholangiocarcinoma and identified â¼25% of tumors with actionable genomic alterations, which has potential prognostic and therapeutic implications.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Terapia Molecular Dirigida/métodos , Análisis de Secuencia de ADN/métodos , Transducción de Señal/genética , Anciano , Antígeno B7-H1/genética , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/genética , Colangiocarcinoma/patología , Estudios de Cohortes , Descubrimiento de Drogas , Europa (Continente)/epidemiología , Femenino , Estudio de Asociación del Genoma Completo/métodos , Factor Nuclear 4 del Hepatocito/genética , Humanos , Inmunohistoquímica , Masculino , Pronóstico , Receptor de Muerte Celular Programada 1/genética , Receptor ErbB-2/genética , Estados Unidos/epidemiologíaRESUMEN
BACKGROUND & AIMS: Cirrhosis and chronic inflammation precede development of hepatocellular carcinoma (HCC) in approximately 80% of cases. We investigated immune-related gene expression patterns in liver tissues surrounding early-stage HCCs and chemopreventive agents that might alter these patterns to prevent liver tumorigenesis. METHODS: We analyzed gene expression profiles of nontumor liver tissues from 392 patients with early-stage HCC (training set, N = 167 and validation set, N = 225) and liver tissue from patients with cirrhosis without HCC (N = 216, controls) to identify changes in expression of genes that regulate the immune response that could contribute to hepatocarcinogenesis. We defined 172 genes as markers for this deregulated immune response, which we called the immune-mediated cancer field (ICF). We analyzed the expression data of liver tissues from 216 patients with cirrhosis without HCC and investigated the association between this gene expression signature and development of HCC and outcomes of patients (median follow-up, 10 years). Human liver tissues were also analyzed by histology. C57BL/6J mice were given a single injection of diethylnitrosamine (DEN) followed by weekly doses of carbon tetrachloride to induce liver fibrosis and tumorigenesis. Mice were then orally given the multiple tyrosine inhibitor nintedanib or vehicle (controls); liver tissues were collected and histology, transcriptome, and protein analyses were performed. We also analyzed transcriptomes of liver tissues collected from mice on a choline-deficient high-fat diet, which developed chronic liver inflammation and tumors, orally given aspirin and clopidogrel or the anti-inflammatory agent sulindac vs mice on a chow (control) diet. RESULTS: We found the ICF gene expression pattern in 50% of liver tissues from patients with cirrhosis without HCC and in 60% of nontumor liver tissues from patients with early-stage HCC. The liver tissues with the ICF gene expression pattern had 3 different features: increased numbers of effector T cells; increased expression of genes that suppress the immune response and activation of transforming growth factor ß signaling; or expression of genes that promote inflammation and activation of interferon gamma signaling. Patients with cirrhosis and liver tissues with the immunosuppressive profile (10% of cases) had a higher risk of HCC (hazard ratio, 2.41; 95% confidence interval, 1.21-4.80). Mice with chemically induced fibrosis or diet-induced steatohepatitis given nintedanib or aspirin and clopidogrel down-regulated the ICF gene expression pattern in liver and developed fewer and smaller tumors than mice given vehicle. CONCLUSIONS: We identified an immune-related gene expression pattern in liver tissues of patients with early-stage HCC, called the ICF, that is associated with risk of HCC development in patients with cirrhosis. Administration of nintedanib or aspirin and clopidogrel to mice with chronic liver inflammation caused loss of this gene expression pattern and development of fewer and smaller liver tumors. Agents that alter immune regulatory gene expression patterns associated with carcinogenesis might be tested as chemopreventive agents in patients with cirrhosis.
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Anticarcinógenos/farmacología , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/prevención & control , Neoplasias Hepáticas/genética , Transcriptoma , Animales , Aspirina/farmacología , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Estudios de Casos y Controles , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/patología , Clopidogrel/farmacología , Dietilnitrosamina , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Indoles/farmacología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones Endogámicos C57BL , Escape del Tumor/genética , Microambiente TumoralRESUMEN
The clinical utility of serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC) is widely recognised. However, a clear understanding of the mechanisms of AFP overexpression and the molecular traits of patients with AFP-high tumours are not known. We assessed transcriptome data, whole-exome sequencing data and DNA methylome profiling of 520 HCC patients from two independent cohorts to identify distinct molecular traits of patients with AFP-high tumours (serum concentration > 400 ng/ml), which represents an accepted prognostic cut-off and a predictor of response to ramucirumab. Those AFP-high tumours (18% of resected cases) were characterised by significantly lower AFP promoter methylation (p < 0.001), significant enrichment of progenitor-cell features (CK19, EPCAM), higher incidence of BAP1 oncogene mutations (8.5% vs 1.6%) and lower mutational rates of CTNNB1 (14% vs 30%). Specifically, AFP-high tumours displayed significant activation of VEGF signalling (p < 0.001), which might provide the rationale for the reported benefit of ramucirumab in this subgroup of patients.
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Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , alfa-Fetoproteínas/genética , Anticuerpos Monoclonales Humanizados/uso terapéutico , Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/tratamiento farmacológico , Ensayos Clínicos como Asunto , Metilación de ADN , Humanos , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/tratamiento farmacológico , Regiones Promotoras Genéticas , alfa-Fetoproteínas/análisis , beta Catenina/genética , RamucirumabRESUMEN
OBJECTIVE: Sorafenib is effective in hepatocellular carcinoma (HCC), but patients ultimately present disease progression. Molecular mechanisms underlying acquired resistance are still unknown. Herein, we characterise the role of tumour-initiating cells (T-ICs) and signalling pathways involved in sorafenib resistance. DESIGN: HCC xenograft mice treated with sorafenib (n=22) were explored for responsiveness (n=5) and acquired resistance (n=17). Mechanism of acquired resistance were assessed by: (1) role of T-ICs by in vitro sphere formation and in vivo tumourigenesis assays using NOD/SCID mice, (2) activation of alternative signalling pathways and (3) efficacy of anti-FGF and anti-IGF drugs in experimental models. Gene expression (microarray, quantitative real-time PCR (qRT-PCR)) and protein analyses (immunohistochemistry, western blot) were conducted. A novel gene signature of sorafenib resistance was generated and tested in two independent cohorts. RESULTS: Sorafenib-acquired resistant tumours showed significant enrichment of T-ICs (164 cells needed to create a tumour) versus sorafenib-sensitive tumours (13â 400 cells) and non-treated tumours (1292 cells), p<0.001. Tumours with sorafenib-acquired resistance were enriched with insulin-like growth factor (IGF) and fibroblast growth factor (FGF) signalling cascades (false discovery rate (FDR)<0.05). In vitro, cells derived from sorafenib-acquired resistant tumours and two sorafenib-resistant HCC cell lines were responsive to IGF or FGF inhibition. In vivo, FGF blockade delayed tumour growth and improved survival in sorafenib-resistant tumours. A sorafenib-resistance 175 gene signature was characterised by enrichment of progenitor cell features, aggressive tumorous traits and predicted poor survival in two cohorts (n=442 patients with HCC). CONCLUSIONS: Acquired resistance to sorafenib is driven by T-ICs with enrichment of progenitor markers and activation of IGF and FGF signalling. Inhibition of these pathways would benefit a subset of patients after sorafenib progression.
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Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Resistencia a Antineoplásicos , Factores de Crecimiento de Fibroblastos/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea/uso terapéutico , Somatomedinas/metabolismo , Anciano , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/genética , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Niacinamida/uso terapéutico , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor IGF Tipo 1 , Receptores de Somatomedina/antagonistas & inhibidores , Receptores de Somatomedina/metabolismo , Transducción de Señal , Somatomedinas/antagonistas & inhibidores , Somatomedinas/genética , Sorafenib , Esferoides Celulares , Tasa de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND & AIMS: Cholangiocarcinoma, the second most common liver cancer, can be classified as intrahepatic cholangiocarcinoma (ICC) or extrahepatic cholangiocarcinoma. We performed an integrative genomic analysis of ICC samples from a large series of patients. METHODS: We performed a gene expression profile, high-density single-nucleotide polymorphism array, and mutation analyses using formalin-fixed ICC samples from 149 patients. Associations with clinicopathologic traits and patient outcomes were examined for 119 cases. Class discovery was based on a non-negative matrix factorization algorithm and significant copy number variations were identified by Genomic Identification of Significant Targets in Cancer (GISTIC) analysis. Gene set enrichment analysis was used to identify signaling pathways activated in specific molecular classes of tumors, and to analyze their genomic overlap with hepatocellular carcinoma (HCC). RESULTS: We identified 2 main biological classes of ICC. The inflammation class (38% of ICCs) is characterized by activation of inflammatory signaling pathways, overexpression of cytokines, and STAT3 activation. The proliferation class (62%) is characterized by activation of oncogenic signaling pathways (including RAS, mitogen-activated protein kinase, and MET), DNA amplifications at 11q13.2, deletions at 14q22.1, mutations in KRAS and BRAF, and gene expression signatures previously associated with poor outcomes for patients with HCC. Copy number variation-based clustering was able to refine these molecular groups further. We identified high-level amplifications in 5 regions, including 1p13 (9%) and 11q13.2 (4%), and several focal deletions, such as 9p21.3 (18%) and 14q22.1 (12% in coding regions for the SAV1 tumor suppressor). In a complementary approach, we identified a gene expression signature that was associated with reduced survival times of patients with ICC; this signature was enriched in the proliferation class (P < .001). CONCLUSIONS: We used an integrative genomic analysis to identify 2 classes of ICC. The proliferation class has specific copy number alterations, activation of oncogenic pathways, and is associated with worse outcome. Different classes of ICC, based on molecular features, therefore might require different treatment approaches.
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Colangiocarcinoma/genética , Colangiocarcinoma/mortalidad , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad/epidemiología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidad , Anciano , Neoplasias de los Conductos Biliares , Conductos Biliares Intrahepáticos , Biopsia con Aguja , Colangiocarcinoma/clasificación , Colangiocarcinoma/patología , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Bases de Datos Factuales , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Hibridación in Situ , Estimación de Kaplan-Meier , Neoplasias Hepáticas/clasificación , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Retrospectivos , Análisis de SupervivenciaRESUMEN
Management of hepatoblastoma (HB), the most frequent pediatric liver cancer, is based on surgical resection and perioperative chemotherapy regimens. In this study, we aimed to identify actionable targets in HB and assess the efficacy of molecular therapies in preclinical models of HB. Paired tumor and adjacent tissues from 31 HBs and a validation set of 50 HBs were analyzed using RNA-seq, SNP, and methylation arrays. IGF2 overexpression was identified as the top targetable HB driver, present in 71% of HBs (22/31). IGF2high tumors displayed progenitor cell features and shorter recurrence-free survival. IGF2 overexpression was associated in 91% of cases with fetal promoter hypomethylation, ICR1 deregulation, 11p15.5 loss of heterozygosity or miR483-5p overexpression. The antitumor effect of xentuzumab (a monoclonal antibody targeting IGF1/2) alone or in combination with the conventional therapeutic agent cisplatin was assessed in HB cell lines, in PDX-derived HB organoids and in a xenograft HB murine model. The combination of xentuzumab with cisplatin showed strong synergistic antitumor effects in organoids and in IGF2high cell lines. In mice (n = 55), the combination induced a significant decrease in tumor volume and improved survival compared with cisplatin alone. These results suggest that IGF2 is an HB actionable driver and that, in preclinical models of HB, the combination of IGF1/2 inhibition with cisplatin induces superior antitumor effects than cisplatin monotherapy. Overall, our study provides a rationale for testing IGF2 inhibitors in combination with cisplatin in HB patients with IGF2 overexpression.
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Hepatoblastoma , Neoplasias Hepáticas , Humanos , Animales , Ratones , Hepatoblastoma/tratamiento farmacológico , Hepatoblastoma/genética , Hepatoblastoma/patología , Cisplatino/farmacología , Cisplatino/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Metilación de ADN , Genómica , Factor II del Crecimiento Similar a la Insulina/genéticaRESUMEN
PURPOSE: Immune checkpoint inhibitors combined with antiangiogenic agents produce benefits in the treatment of advanced hepatocellular carcinoma (HCC). We investigated the efficacy and immunomodulatory activity of cabozantinib alone and combined with anti-PD1 in experimental models of HCC, and explored the potential target population that might benefit from this combination. EXPERIMENTAL DESIGN: C57BL/6J mice bearing subcutaneous Hepa1-6 or Hep53.4 tumors received cabozantinib, anti-PD1, their combination, or placebo. Tumor and blood samples were analyzed by flow cytometry, IHC, transcriptome, and cytokine profiling. Cabozantinib-related effects were validated in a colorectal cancer patient-derived xenograft model. Transcriptomic data from three human HCC cohorts (cohort 1: n = 167, cohort 2: n = 57, The Cancer Genome Atlas: n = 319) were used to cluster patients according to neutrophil features, and assess their impact on survival. RESULTS: The combination of cabozantinib and anti-PD1 showed increased antitumor efficacy compared with monotherapy and placebo (P < 0.05). Cabozantinib alone significantly increased neutrophil infiltration and reduced intratumor CD8+PD1+ T-cell proportions, while the combination with anti-PD1 further stimulated both effects and significantly decreased regulatory T cell (Treg) infiltration (all P < 0.05). In blood, cabozantinib and especially combination increased the proportions of overall T cells (P < 0.01) and memory/effector T cells (P < 0.05), while lowering the neutrophil-to-lymphocyte ratio (P < 0.001 for combination). Unsupervised clustering of human HCCs revealed that high tumor enrichment in neutrophil features observed with the treatment combination was linked to less aggressive tumors with more differentiated and less proliferative phenotypes. CONCLUSIONS: Cabozantinib in combination with anti-PD1 enhanced antitumor immunity by bringing together innate neutrophil-driven and adaptive immune responses, a mechanism of action which favors this approach for HCC treatment.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Anilidas , Animales , Carcinoma Hepatocelular/patología , Humanos , Inmunidad , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/patología , Receptor de Muerte Celular Programada 1 , PiridinasRESUMEN
BACKGROUND: It is a challenge to identify patients who, after undergoing potentially curative treatment for hepatocellular carcinoma, are at greatest risk for recurrence. Such high-risk patients could receive novel interventional measures. An obstacle to the development of genome-based predictors of outcome in patients with hepatocellular carcinoma has been the lack of a means to carry out genomewide expression profiling of fixed, as opposed to frozen, tissue. METHODS: We aimed to demonstrate the feasibility of gene-expression profiling of more than 6000 human genes in formalin-fixed, paraffin-embedded tissues. We applied the method to tissues from 307 patients with hepatocellular carcinoma, from four series of patients, to discover and validate a gene-expression signature associated with survival. RESULTS: The expression-profiling method for formalin-fixed, paraffin-embedded tissue was highly effective: samples from 90% of the patients yielded data of high quality, including samples that had been archived for more than 24 years. Gene-expression profiles of tumor tissue failed to yield a significant association with survival. In contrast, profiles of the surrounding nontumoral liver tissue were highly correlated with survival in a training set of tissue samples from 82 Japanese patients, and the signature was validated in tissues from an independent group of 225 patients from the United States and Europe (P=0.04). CONCLUSIONS: We have demonstrated the feasibility of genomewide expression profiling of formalin-fixed, paraffin-embedded tissues and have shown that a reproducible gene-expression signature correlated with survival is present in liver tissue adjacent to the tumor in patients with hepatocellular carcinoma.
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Carcinoma Hepatocelular/genética , Perfilación de la Expresión Génica/métodos , Neoplasias Hepáticas/genética , Recurrencia Local de Neoplasia/genética , Anciano , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Estudios de Factibilidad , Femenino , Regulación Neoplásica de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Hígado/patología , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Adhesión en Parafina/métodos , Reacción en Cadena de la Polimerasa , Pronóstico , Análisis de Supervivencia , Fijación del Tejido/métodosRESUMEN
BACKGROUND & AIMS: IGF signaling has a relevant role in a variety of human malignancies. We analyzed the underlying molecular mechanisms of IGF signaling activation in early hepatocellular carcinoma (HCC; BCLC class 0 or A) and assessed novel targeted therapies blocking this pathway. METHODS: An integrative molecular dissection of the axis was conducted in a cohort of 104 HCCs analyzing gene and miRNA expression, structural aberrations, and protein activation. The therapeutic potential of a selective IGF-1R inhibitor, the monoclonal antibody A12, was assessed in vitro and in a xenograft model of HCC. RESULTS: Activation of the IGF axis was observed in 21% of early HCCs. Several molecular aberrations were identified, such as overexpression of IGF2 -resulting from reactivation of fetal promoters P3 and P4-, IGFBP3 downregulation and allelic losses of IGF2R (25% of cases). A gene signature defining IGF-1R activation was developed. Overall, activation of IGF signaling in HCC was significantly associated with mTOR signaling (p=0.035) and was clearly enriched in the Proliferation subclass of the molecular classification of HCC (p=0.001). We also found an inverse correlation between IGF activation and miR-100/miR-216 levels (FDR<0.05). In vitro studies showed that A12-induced abrogation of IGF-1R activation and downstream signaling significantly decreased cell viability and proliferation. In vivo, A12 delayed tumor growth and prolonged survival, reducing proliferation rates and inducing apoptosis. CONCLUSIONS: Integrative genomic analysis showed enrichment of activation of IGF signaling in the Proliferation subclass of HCC. Effective blockage of IGF signaling with A12 provides the rationale for testing this therapy in clinical trials.
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Carcinoma Hepatocelular , Factor II del Crecimiento Similar a la Insulina/genética , Neoplasias Hepáticas , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor IGF Tipo 1/genética , Animales , Anticuerpos Monoclonales/farmacología , Apoptosis/fisiología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , División Celular/fisiología , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Hepatocitos/patología , Hepatocitos/fisiología , Humanos , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/metabolismo , Receptor IGF Tipo 1/inmunología , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Transducción de Señal/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND/AIMS: The success of sorafenib in the treatment of advanced hepatocellular carcinoma (HCC) has focused interest on the role of Ras signaling in this malignancy. We investigated the molecular alterations of the Ras pathway in HCC and the antineoplastic effects of sorafenib in combination with rapamycin, an inhibitor of mTOR pathway, in experimental models. METHODS: Gene expression (qRT-PCR, oligonucleotide microarray), DNA copy number changes (SNP-array), methylation of tumor suppressor genes (methylation-specific PCR) and protein activation (immunohistochemistry) were analysed in 351 samples. Anti-tumoral effects of combined therapy targeting the Ras and mTOR pathways were evaluated in cell lines and HCC xenografts. RESULTS: Different mechanisms accounted for Ras pathway activation in HCC. H-ras was up-regulated during different steps of hepatocarcinogenesis. B-raf was overexpressed in advanced tumors and its expression was associated with genomic amplification. Partial methylation of RASSF1A and NORE1A was detected in 89% and 44% of tumors respectively, and complete methylation was found in 11 and 4% of HCCs. Activation of the pathway (pERK immunostaining) was identified in 10.3% of HCC. Blockade of Ras and mTOR pathways with sorafenib and rapamycin reduced cell proliferation and induced apoptosis in cell lines. In vivo, the combination of both compounds enhanced tumor necrosis and ulceration when compared with sorafenib alone. CONCLUSIONS: Ras activation results from several molecular alterations, such as methylation of tumor suppressors and amplification of oncogenes (B-raf). Sorafenib blocks signaling and synergizes with rapamycin in vivo, preventing tumor progression. These data provide the rationale for testing this combination in clinical studies.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Bencenosulfonatos/administración & dosificación , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Piridinas/administración & dosificación , Sirolimus/administración & dosificación , Proteínas ras/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Dosificación de Gen/efectos de los fármacos , Genes ras/efectos de los fármacos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Quinasas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Transducción de Señal/efectos de los fármacos , Sorafenib , Serina-Treonina Quinasas TOR , Trasplante HeterólogoRESUMEN
BACKGROUND & AIMS: The advent of targeted therapies in hepatocellular carcinoma (HCC) has underscored the importance of pathway characterization to identify novel molecular targets for treatment. We evaluated mTOR signaling in human HCC, as well as the antitumoral effect of a dual-level blockade of the mTOR pathway. METHODS: The mTOR pathway was assessed using integrated data from mutation analysis (direct sequencing), DNA copy number changes (SNP-array), messenger RNA levels (quantitative reverse-transcription polymerase chain reaction and gene expression microarray), and protein activation (immunostaining) in 351 human samples [HCC (n = 314) and nontumoral tissue (n = 37)]. Effects of dual blockade of mTOR signaling using a rapamycin analogue (everolimus) and an epidermal/vascular endothelial growth factor receptor inhibitor (AEE788) were evaluated in liver cancer cell lines and in a xenograft model. RESULTS: Aberrant mTOR signaling (p-RPS6) was present in half of the cases, associated with insulin-like growth factor pathway activation, epidermal growth factor up-regulation, and PTEN dysregulation. PTEN and PI3KCA-B mutations were rare events. Chromosomal gains in RICTOR (25% of patients) and positive p-RPS6 staining correlated with recurrence. RICTOR-specific siRNA down-regulation reduced tumor cell viability in vitro. Blockage of mTOR signaling with everolimus in vitro and in a xenograft model decelerated tumor growth and increased survival. This effect was enhanced in vivo after epidermal growth factor blockade. CONCLUSIONS: MTOR signaling has a critical role in the pathogenesis of HCC, with evidence for the role of RICTOR in hepato-oncogenesis. MTOR blockade with everolimus is effective in vivo. These findings establish a rationale for targeting the mTOR pathway in clinical trials in HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Proteínas Quinasas/genética , ARN Neoplásico/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Fosfatidilinositol 3-Quinasas , Proteínas Quinasas/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Serina-Treonina Quinasas TORRESUMEN
Hepatocellular carcinomas represent the third leading cause of cancer-related deaths worldwide. The vast majority of cases arise in the context of chronic liver injury due to hepatitis B virus or hepatitis C virus infection. To identify genetic mechanisms of hepatocarcinogenesis, we characterized copy number alterations and gene expression profiles from the same set of tumors associated with hepatitis C virus. Most tumors harbored 1q gain, 8q gain, or 8p loss, with occasional alterations in 13 additional chromosome arms. In addition to amplifications at 11q13 in 6 of 103 tumors, 4 tumors harbored focal gains at 6p21 incorporating vascular endothelial growth factor A (VEGFA). Fluorescence in situ hybridization on an independent validation set of 210 tumors found 6p21 high-level gains in 14 tumors, as well as 2 tumors with 6p21 amplifications. Strikingly, this locus overlapped with copy gains in 4 of 371 lung adenocarcinomas. Overexpression of VEGFA via 6p21 gain in hepatocellular carcinomas suggested a novel, non-cell-autonomous mechanism of oncogene activation. Hierarchical clustering of gene expression among 91 of these tumors identified five classes, including "CTNNB1", "proliferation", "IFN-related", a novel class defined by polysomy of chromosome 7, and an unannotated class. These class labels were further supported by molecular data; mutations in CTNNB1 were enriched in the "CTNNB1" class, whereas insulin-like growth factor I receptor and RPS6 phosphorylation were enriched in the "proliferation" class. The enrichment of signaling pathway alterations in gene expression classes provides insights on hepatocellular carcinoma pathogenesis. Furthermore, the prevalence of VEGFA high-level gains in multiple tumor types suggests indications for clinical trials of antiangiogenic therapies.