Genís Campreciós

Identification of an Immune-specific Class of Hepatocellular Carcinoma, Based on Molecular Features

BACKGROUND & AIMS Agents that induce an immune response against tumors by altering T-cell regulation have increased survival times of patients with advanced-stage tumors, such as melanoma or lung cancer. We aimed to characterize molecular features of immune cells that infiltrate hepatocellular carcinomas (HCCs) to determine whether these types of agents might be effective against liver tumors. METHODS We analyzed HCC samples from 956 patients. We separated gene expression profiles from tumor, stromal, and immune cells using a non-negative matrix factorization algorithm. We then analyzed the gene expression pattern of inflammatory cells in HCC tumor samples. We correlated expression patterns with the presence of immune cell infiltrates and immune regulatory molecules, determined by pathology and immunohistochemical analyses, in a training set of 228 HCC samples. We validated the correlation in a validation set of 728 tumor samples. Using data from 190 tumors in the Cancer Genome Atlas, we correlated immune cell gene expression profiles with numbers of chromosomal aberrations (based on single-nucleotide polymorphism array) and mutations (exome sequence data). RESULTS We found approximately 25% of HCCs to have markers of an inflammatory response, with high expression levels of the CD274 molecule (programmed death-ligand 1) and programmed cell death 1, markers of cytolytic activity, and fewer chromosomal aberrations. We called this group of tumors the Immune class. It contained 2 subtypes, characterized by markers of an adaptive T-cell response or exhausted immune response. The exhausted immune response subclass expressed many genes regulated by transforming growth factor beta 1 that mediate immunosuppression. We did not observe any differences in numbers of mutations or expression of tumor antigens between the immune-specific class and other HCCs. CONCLUSIONS In an analysis of HCC samples from 956 patients, we found almost 25% to express markers of an inflammatory response. We identified 2 subclasses, characterized by adaptive or exhausted immune responses. These findings indicate that some HCCs might be susceptible to therapeutic agents designed to block the regulatory pathways in T cells, such as programmed death-ligand 1, programmed cell death 1, or transforming growth factor beta 1 inhibitors.

The exosome complex establishes a barricade to erythroid maturation

Complex genetic networks control hematopoietic stem cell differentiation into progenitors that give rise to billions of erythrocytes daily. Previously, we described a role for the master regulator of erythropoiesis, GATA-1, in inducing genes encoding components of the autophagy machinery. In this context, the Forkhead transcription factor, Foxo3, amplified GATA-1-mediated transcriptional activation. To determine the scope of the GATA-1/Foxo3 cooperativity, and to develop functional insights, we analyzed the GATA-1/Foxo3-dependent transcriptome in erythroid cells. GATA-1/Foxo3 repressed expression of Exosc8, a pivotal component of the exosome complex, which mediates RNA surveillance and epigenetic regulation. Strikingly, downregulating Exosc8, or additional exosome complex components, in primary erythroid precursor cells induced erythroid cell maturation. Our results demonstrate a new mode of controlling erythropoiesis in which multiple components of the exosome complex are endogenous suppressors of the erythroid developmental program.

FOXO3‐mTOR metabolic cooperation in the regulation of erythroid cell maturation and homeostasis

Ineffective erythropoiesis is observed in many erythroid disorders including β‐thalassemia and anemia of chronic disease in which increased production of erythroblasts that fail to mature exacerbate the underlying anemias. As loss of the transcription factor FOXO3 results in erythroblast abnormalities similar to the ones observed in ineffective erythropoiesis, we investigated the underlying mechanisms of the defective Foxo3−/− erythroblast cell cycle and maturation. Here we show that loss of Foxo3 results in overactivation of the JAK2/AKT/mTOR signaling pathway in primary bone marrow erythroblasts partly mediated by redox modulation. We further show that hyperactivation of mTOR signaling interferes with cell cycle progression in Foxo3 mutant erythroblasts. Importantly, inhibition of mTOR signaling, in vivo or in vitro enhances significantly Foxo3 mutant erythroid cell maturation. Similarly, in vivo inhibition of mTOR remarkably improves erythroid cell maturation and anemia in a model of β‐thalassemia. Finally we show that FOXO3 and mTOR are likely part of a larger metabolic network in erythroblasts as together they control the expression of an array of metabolic genes some of which are implicated in erythroid disorders. These combined findings indicate that a metabolism‐mediated regulatory network centered by FOXO3 and mTOR control the balanced production and maturation of erythroid cells. They also highlight physiological interactions between these proteins in regulating erythroblast energy. Our results indicate that alteration in the function of this network might be implicated in the pathogenesis of ineffective erythropoiesis. Am. J. Hematol. 89:954–963, 2014.

A Systems Approach Identifies Essential FOXO3 Functions at Key Steps of Terminal Erythropoiesis

Circulating red blood cells (RBCs) are essential for tissue oxygenation and homeostasis. Defective terminal erythropoiesis contributes to decreased generation of RBCs in many disorders. Specifically, ineffective nuclear expulsion (enucleation) during terminal maturation is an obstacle to therapeutic RBC production in vitro. To obtain mechanistic insights into terminal erythropoiesis we focused on FOXO3, a transcription factor implicated in erythroid disorders. Using an integrated computational and experimental systems biology approach, we show that FOXO3 is essential for the correct temporal gene expression during terminal erythropoiesis. We demonstrate that the FOXO3-dependent genetic network has critical physiological functions at key steps of terminal erythropoiesis including enucleation and mitochondrial clearance processes. FOXO3 loss deregulated transcription of genes implicated in cell polarity, nucleosome assembly and DNA packaging-related processes and compromised erythroid enucleation. Using high-resolution confocal microscopy and imaging flow cytometry we show that cell polarization is impaired leading to multilobulated Foxo3 -/- erythroblasts defective in nuclear expulsion. Ectopic FOXO3 expression rescued Foxo3 -/- erythroblast enucleation-related gene transcription, enucleation defects and terminal maturation. Remarkably, FOXO3 ectopic expression increased wild type erythroblast maturation and enucleation suggesting that enhancing FOXO3 activity may improve RBCs production. Altogether these studies uncover FOXO3 as a novel regulator of erythroblast enucleation and terminal maturation suggesting FOXO3 modulation might be therapeutic in disorders with defective erythroid maturation.

Mixed hepatocellular cholangiocarcinoma tumors: Cholangiolocellular carcinoma is a distinct molecular entity

BACKGROUND & AIMS Mixed hepatocellular cholangiocarcinoma (HCC-CCA) is a rare and poorly understood type of primary liver cancer. We aimed to perform a comprehensive molecular characterization of this malignancy. METHODS Gene expression profiling, DNA copy number detection, and exome sequencing using formalin-fixed samples from 18 patients with mixed HCC-CCA were performed, encompassing the whole histological spectrum of the disease. Comparative genomic analysis was carried out, using independent datasets of HCC (n=164) and intrahepatic cholangiocarcinoma (iCCA) (n=149). RESULTS Integrative genomic analysis of HCC-CCAs revealed that cholangiolocellular carcinoma (CLC) represents a distinct biliary-derived entity compared with the stem-cell and classical types. CLC tumors were neural cell adhesion molecule (NCAM) positive (6/6 vs. 1/12, p<0.001), chromosomally stable (mean chromosomal aberrations 5.7 vs. 14.1, p=0.008), showed significant upregulation of transforming growth factor (TGF)-β signaling and enrichment of inflammation-related and immune response signatures (p<0.001). Stem-cell tumors were characterized by spalt-like transcription factor 4 (SALL4) positivity (6/8 vs. 0/10, p<0.001), enrichment of progenitor-like signatures, activation of specific oncogenic pathways (i.e., MYC and insulin-like growth factor [IGF]), and signatures related to poor clinical outcome. In the classical type, there was a significant correlation in the copy number variation of the iCCA and HCC components, suggesting a clonal origin. Exome sequencing revealed an average of 63 non-synonymous mutations per tumor (2 mean driver mutations per tumor). Among those, TP53 was the most frequently mutated gene (6/21, 29%) in HCC-CCAs. CONCLUSIONS Mixed HCC-CCA represents a heterogeneous group of tumors, with the stem-cell type characterized by features of poor prognosis, and the classical type with common lineage for HCC and iCCA components. CLC stands alone as a distinct biliary-derived entity associated with chromosomal stability and active TGF-β signaling. LAY SUMMARY Molecular analysis of mixed hepatocellular cholangiocarcinoma (HCC-CCA) showed that cholangiolocellular carcinoma (CLC) is distinct and biliary in origin. It has none of the traits of hepatocellular carcinoma (HCC). However, within mixed HCC-CCA, stem-cell type tumors shared an aggressive nature and poor outcome, whereas the classic type showed a common cell lineage for both the HCC and the intrahepatic CCA component. The pathological classification of mixed HCC-CCA should be redefined because of the new molecular data provided.

Trunk mutational events present minimal intra- and inter-tumoral heterogeneity in hepatocellular carcinoma

BACKGROUND & AIMS According to the clonal model of tumor evolution, trunk alterations arise at early stages and are ubiquitous. Through the characterization of early stages of hepatocarcinogenesis, we aimed to identify trunk alterations in hepatocellular carcinoma (HCC) and study their intra- and inter-tumor distribution in advanced lesions. METHODS A total of 151 samples representing the multistep process of hepatocarcinogenesis were analyzed by targeted-sequencing and a single nucleotide polymorphism array. Genes altered in early lesions (31 dysplastic nodules [DNs] and 38 small HCCs [sHCC]) were defined as trunk. Their distribution was explored in: a) different regions of large tumors (43 regions, 21 tumors), and b) different nodules of the same patient (39 tumors, 17 patients). Multinodular lesions were classified as intrahepatic metastases (IMs) or synchronous tumors based on chromosomal aberrations. RESULTS TERT promoter mutations (10.5%) and broad copy-number aberrations in chromosomes 1 and 8 (3-7%) were identified as trunk gatekeepers in DNs and were maintained in sHCCs. Trunk drivers identified in sHCCs included TP53 (23%) and CTNNB1 (11%) mutations, and focal amplifications or deletions in known drivers (6%). Overall, TERT, TP53 and CTNNB1 mutations were the most frequent trunk events and at least one was present in 51% of sHCCs. Around 90% of mutations in these genes were ubiquitous among different regions of large tumors. In multinodular HCCs, 35% of patients harbored IMs; 85% of mutations in TERT, TP53 and/or CTNNB1 were retained in primary and metastatic tumors. CONCLUSIONS Trunk events in early stages (TERT, TP53, CTNNB1 mutations) were ubiquitous across different regions of the same tumor and between primary and metastatic nodules in >85% of cases. This concept supports the knowledge that single biopsies would suffice to capture trunk mutations in HCC. LAY SUMMARY Trunk alterations arise at early stages of cancer and are shared among all malignant cells of the tumor. In order to identify trunk alterations in HCC, we characterized early stages of hepatocarcinogenesis represented by dysplastic nodules and small lesions. Mutations in TERT, TP53 and CTNNB1 genes were the most frequent. Analyses in more advanced lesions showed that mutations in these same genes were shared between different regions of the same tumor and between primary and metastatic tumors, suggesting their trunk role in this disease.

Hepatocellular Carcinoma Arising in an HNF-1α-Mutated Adenoma in a 23-Year-Old Woman with Maturity-Onset Diabetes of the Young: A Case Report.

Hepatocyte nuclear factor-1α mutated hepatocellular adenomas (H-HCA) are thought to have no to minimal malignant potential. This report describes a 23-year-old woman with maturity-onset diabetes of the young who developed a 12.5-cm hepatic mass with a radiographically and pathologically distinct 3.0-cm region. Histologically and immunohistochemically, the bulk of the mass was an H-HCA with extensive pseudoglandular formation and only focal steatosis. The 3.0-cm nodule showed small cell change, thickened hepatocyte plates, pleomorphic and hyperchromatic nuclei, reticulin loss, and stromal and vascular invasion, diagnostic of hepatocellular carcinoma (HCC). Immunohistochemically, increased expression of glutamine synthetase in tumor cells and CD34 expression in sinusoidal endothelial cells were seen in the HCC component. Nuclear expression of β-catenin, and exon 3 of CTNNB1 and TERT promoter mutations were absent in this case. Thus, we report a HCC arising in an H-HCA; although cases appear exceedingly rare, they reinforce the potential of H-HCA for malignant transformation.

Impaired Endothelial Autophagy Promotes Liver Fibrosis By Aggravating The Oxidative Stress Response During Acute Liver Injury

BACKGROUND & AIMS Endothelial dysfunction plays an essential role in liver injury, yet the phenotypic regulation of liver sinusoidal endothelial cells (LSECs) remains unknown. Autophagy is an endogenous protective system whose loss could undermine LSEC integrity and phenotype. The aim of our study was to investigate the role of autophagy in the regulation of endothelial dysfunction and the impact of its manipulation during liver injury. METHODS We analyzed primary isolated LSECs from Atg7control and Atg7endo mice as well as rats after CCl4 induced liver injury. Liver tissue and primary isolated stellate cells were used to analyze liver fibrosis. Autophagy flux, microvascular function, nitric oxide bioavailability, cellular superoxide content and the antioxidant response were evaluated in endothelial cells. RESULTS Autophagy maintains LSEC homeostasis and is rapidly upregulated during capillarization in vitro and in vivo. Pharmacological and genetic downregulation of endothelial autophagy increases oxidative stress in vitro. During liver injury in vivo, the selective loss of endothelial autophagy leads to cellular dysfunction and reduced intrahepatic nitric oxide. The loss of autophagy also impairs LSECs ability to handle oxidative stress and aggravates fibrosis. CONCLUSIONS Autophagy contributes to maintaining endothelial phenotype and protecting LSECs from oxidative stress during early phases of liver disease. Selectively potentiating autophagy in LSECs during early stages of liver disease may be an attractive approach to modify the disease course and prevent fibrosis progression. LAY SUMMARY Liver endothelial cells are the first liver cell type affected after any kind of liver injury. The loss of their unique phenotype during injury amplifies liver damage by orchestrating the response of the liver microenvironment. Autophagy is a mechanism involved in the regulation of this initial response and its manipulation can modify the progression of liver damage.

Abstract 2936: Molecular characterization of the immune subclass of hepatocellualr carcinoma

Background: Immune checkpoint inhibitors have emerged as a promising therapeutic approach in different solid tumors, including hepatocellular carcinoma (HCC). Nonetheless, little is known about the immune-component of HCC or potential biomarkers of response to these therapies. Aims: To perform comprehensive characterization of the HCC immunological profile and to identify biomarkers to select immunotherapy candidates. Methods: We performed gene expression array deconvolution through non-negative matrix factorization in 228 resected HCCs. Characterization of the transcriptional landscape was conducted using >1,000 signatures representing distinct immune cells by gene set enrichment and nearest template prediction analyses. Presence of immune infiltration, tertiary lymphoid structure (TLS), PD-1 and PD-L1 immunostainings was investigated using immunohistochemistry. DNA methylation profile of 450K CpG sites was analyzed to identify those with significant differences for each group. Extensive validation of the immune classifier was performed in 728 independent HCC samples. Results: Overall, an immune-related subclass of HCC was identified in ~27% of patients. The immune subclass was characterized by gene signatures identifying immune cells (i.e. T cells, TLS, cytotox, p 0.2 Tukey test). Integration with HCC molecular classifications revealed significant enrichment of the Immune subclass with IFN and S1 (p Conclusions: Around 27% of HCC patients belong to the Immune class, characterized by activation of immune cells and signatures of response to immunotherapies. Within this subclass, two distinct types have been characterized by presenting active or exhausted immune responses, a feature that provides the rationale for precision medicine-based therapies. Note: This abstract was not presented at the meeting. Citation Format: Daniela Sia, Yang Jiao, Iris Martinez, Olga Kuchuk, Carlos Villacorta Martin, Manuel Castro de Moura, Juan Putra, Genis Camprecios, Swan Thung, Samuel Waxman, Vincenzo Mazzaferro, Manel Esteller, Augusto Villanueva, Josep Maria Llovet. Molecular characterization of the immune subclass of hepatocellualr carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2936. doi:10.1158/1538-7445.AM2017-2936

Abstract 2388: Molecular heterogeneity and trunk driver mutations in hepatocellular carcinoma

Background and aims: Molecular heterogeneity in hepatocellular carcinoma (HCC) is ill-defined since trunk drivers (early events; common to all cells), branch drivers (later events; present in a subset of cells) and passenger mutations (not relevant), have not been thoroughly described. Most FDA/EMA approved molecular drugs target trunk drivers. We explored heterogeneity by analyzing trunk vs branch mutations in different HCC regions within single and multinodular tumours. Methods: Intra-tumoral heterogeneity was assessed in 21 patients with single HCCs (size > 4cm; 2 regions/tumour: 42 samples) and inter-tumoral heterogeneity was studied in 17 patients with multinodular HCCs (2-3 nodules/patient; total: 39 samples). Gene expression profiling, SNP array and deep-sequencing (coverage ∼850x) assessing 6 oncodrivers (TERT promoter, TP53, CTNNB1, ARID1A, AXIN1-2 by TruSeqAmplicon, validated by sanger) were explored. Clonality differentiating metastatic (clonal) vs synchronic (non-clonal) tumours was defined by SNP profiles. Trunk mutations were defined as present in a) all regions of a given tumour, or b) in all nodules of metastatic-clonal tumours; all other were considered as branch. Results: Intra-tumoral heterogeneity assessed by sequencing identified at least 1 oncodriver in 19/21 patients with single tumours. Among those, trunk mutations accounted for 17/19 (90%), and branch for 2/19 cases. Overall 63 mutations were identified, 56 (90%) were identical in different tumoral regions (i.e. truncal; TERT promoter most prevalent). Inter-tumoral heterogeneity explored by SNP profiles defined metastases in 35% (6/17 multinodular cases) and synchronous tumors in 65% (11/17 cases). Genetic proximity confirmed clonality in all metastatic nodules. Regarding molecular subclasses, half of clonal tumours retained identical molecular fingerprint, but the other half switched to more aggressive subclass. All non-clonal tumours belonged to distinct molecular subclasses. Driver oncogenes were explored in 9 patients (5 metastasis and 4 synchronic). Metastatic tumours showed 13 mutations, among which 11 (85%) were truncal. Mutations in non-clonal synchronic tumours were distinct. Conclusions: Single large HCCs shared common trunk drivers at distinct regions (90%). Similarly, 40% of multinodular tumours were clonal (metastasis) and shared common trunk oncodrivers, while 60% were synchronic, with distinct genomic profile/oncodrivers. Further studies at single-cell sequencing level are recommended. Citation Format: Daniela Sia, Andrew Neelis Harrington, Sara Torrecilla, Zhongyang Zhang, Genis Camprecios, Agrin Moeini, Sara Toffanin, Maria Isabel Fiel, Ke Hao, Monica Higuera, Laia Cabellos, Helena Cornella, Milind Mahajan, Yujin Hoshida, Augusto Villanueva, Sander Florman, Myron Schwartz, Josep Maria Llovet. Molecular heterogeneity and trunk driver mutations in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2388.