Sara Torrecilla

Tumour initiating cells and IGF/FGF signalling contribute to sorafenib resistance in hepatocellular carcinoma

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 (13 400 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.

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.

Molecular predictors of prevention of recurrence in HCC with sorafenib as adjuvant treatment and prognostic factors in the phase 3 STORM trial

Objective Sorafenib is the standard systemic therapy for advanced hepatocellular carcinoma (HCC). Survival benefits of resection/local ablation for early HCC are compromised by 70% 5-year recurrence rates. The phase 3 STORM trial comparing sorafenib with placebo as adjuvant treatment did not achieve its primary endpoint of improving recurrence-free survival (RFS). The biomarker companion study BIOSTORM aims to define (A) predictors of recurrence prevention with sorafenib and (B) prognostic factors with B level of evidence. Design Tumour tissue from 188 patients randomised to receive sorafenib (83) or placebo (105) in the STORM trial was collected. Analyses included gene expression profiling, targeted exome sequencing (19 known oncodrivers), immunohistochemistry (pERK, pVEGFR2, Ki67), fluorescence in situ hybridisation (VEGFA) and immunome. A gene signature capturing improved RFS in sorafenib-treated patients was generated. All 70 RFS events were recurrences, thus time to recurrence equalled RFS. Predictive and prognostic value was assessed using Cox regression models and interaction test. Results BIOSTORM recapitulates clinicopathological characteristics of STORM. None of the biomarkers tested (related to angiogenesis and proliferation) or previously proposed gene signatures, or mutations predicted sorafenib benefit or recurrence. A newly generated 146-gene signature identifying 30% of patients captured benefit to sorafenib in terms of RFS (p of interaction=0.04). These sorafenib RFS responders were significantly enriched in CD4+ T, B and cytolytic natural killer cells, and lacked activated adaptive immune components. Hepatocytic pERK (HR=2.41; p=0.012) and microvascular invasion (HR=2.09; p=0.017) were independent prognostic factors. Conclusion In BIOSTORM, only hepatocytic pERK and microvascular invasion predicted poor RFS. No mutation, gene amplification or previously proposed gene signatures predicted sorafenib benefit. A newly generated multigene signature associated with improved RFS on sorafenib warrants further validation. Trial registration number NCT00692770.

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.

Abstract 3820: Epigenetic re-expression of fetal IGF2 as therapeutic target in hepatocellular carcinoma

Background and aims Hepatocellular carcinoma (HCC) is a major health problem. Most patients are diagnosed at advanced stages when the only approved therapy is the multi-kinase inhibitor sorafenib. Consequently, there is a great need for the development of new effective treatments. IGF signaling pathway is aberrantly activated in HCC; however, its contribution to HCC pathogenesis is still unclear. Since IGF2 is overexpressed in HCC, we aimed to elucidate the oncogenic potential and mechanism of dis-regulation of this protein and determine the antitumoral efficacy of molecular abrogation of this ligand by targeted therapies. Methods Transcriptomic profiling, miRNAs expression, RNA- and whole exome- sequencing and methylation were analyzed in 228 HCCs with a focus on IGF-pathway. Gene Set Enrichment Analysis and Ingenuity Pathway Analysis were carried out in IGF2-overexpressing tumors. Stable HCC cell lines with knock-down and ectopic overexpression of IGF2 were generated. A chemically-induced mouse model of HCC, and two genetically-engineered mosaic mouse models (GEMM) overexpressing IGF2 specifically in the liver were generated to assess IGF2 oncogenicity in hepatocarcinogenesis. The therapeutic potential of a monoclonal-antibody against IGF-ligands (IGF1/2-mAb) alone or in combination with sorafenib was tested in a xenograft model of HCC. Results Here, IGF2-overexpression occurred in 15% of HCC patients as a result of the epigenetic reactivation of IGF2-fetal promoters, mainly through loss of promoters methylation (53% of cases) and deregulation of miR-216b, miR-483-5p and miR-let7-d (35% of cases). Re-expression of IGF2 was associated with a progenitor cell-like, poorly differentiated and aggressive subtype of HCC, and poor prognosis (p Conclusions IGF2 is the first validated epidriver in HCC and has a key role in the hepatocarcinogenic process. These results provide the rationale for testing IGF1/2-mAb in a selected subset of HCC patients. Citation Format: Iris Martinez-Quetglas, Roser Pinyol, Daniel Dauch, Sara Torrecilla, Victoria Tovar, Agrin Moeini, Clara Alsinet, Anna Portela, Augusto Villanueva, Manel Esteller, Lars Zender, Josep M Llovet. Epigenetic re-expression of fetal IGF2 as therapeutic target 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 3820.