Molecular classification and therapeutic targets in extrahepatic cholangiocarcinoma.

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 ∼25% 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 ∼25% of tumors with actionable genomic alterations, which has potential prognostic and therapeutic implications.

Molecular portrait of high alpha-fetoprotein in hepatocellular carcinoma: implications for biomarker-driven clinical trials.

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.

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.

Unique genomic profile of fibrolamellar hepatocellular carcinoma.

BACKGROUND & AIMS: Fibrolamellar hepatocellular carcinoma (FLC) is a rare primary hepatic cancer that develops in children and young adults without cirrhosis. Little is known about its pathogenesis, and it can be treated only with surgery. We performed an integrative genomic analysis of a large series of patients with FLC to identify associated genetic factors. METHODS: By using 78 clinically annotated FLC samples, we performed whole-transcriptome (n = 58), single-nucleotide polymorphism array (n = 41), and next-generation sequencing (n = 48) analyses; we also assessed the prevalence of the DNAJB1-PRKACA fusion transcript associated with this cancer (n = 73). We performed class discovery using non-negative matrix factorization, and functional annotation using gene-set enrichment analyses, nearest template prediction, ingenuity pathway analyses, and immunohistochemistry. The genomic identification of significant targets in a cancer algorithm was used to identify chromosomal aberrations, MuTect and VarScan2 were used to identify somatic mutations, and the random survival forest was used to determine patient prognoses. Findings were validated in an independent cohort. RESULTS: Unsupervised gene expression clustering showed 3 robust molecular classes of tumors: the proliferation class (51% of samples) had altered expression of genes that regulate proliferation and mammalian target of rapamycin signaling activation; the inflammation class (26% of samples) had altered expression of genes that regulate inflammation and cytokine enriched production; and the unannotated class (23% of samples) had a gene expression signature that was not associated previously with liver tumors. Expression of genes that regulate neuroendocrine function, as well as histologic markers of cholangiocytes and hepatocytes, were detected in all 3 classes. FLCs had few copy number variations; the most frequent were focal amplification at 8q24.3 (in 12.5% of samples), and deletions at 19p13 (in 28% of samples) and 22q13.32 (in 25% of samples). The DNAJB1-PRKACA fusion transcript was detected in 79% of samples. FLC samples also contained mutations in cancer-related genes such as BRCA2 (in 4.2% of samples), which are uncommon in liver neoplasms. However, FLCs did not contain mutations most commonly detected in liver cancers. We identified an 8-gene signature that predicted survival of patients with FLC. CONCLUSIONS: In a genomic analysis of 78 FLC samples, we identified 3 classes based on gene expression profiles. FLCs contain mutations and chromosomal aberrations not previously associated with liver cancer, and almost 80% contain the DNAJB1-PRKACA fusion transcript. By using this information, we identified a gene signature that is associated with patient survival time.

Precision Oncology and Systemic Targeted Therapy in Pseudomyxoma Peritonei.

PURPOSE: Pseudomyxoma peritonei (PMP) is a rare and poorly understood malignant condition characterized by the accumulation of intra-abdominal mucin produced from peritoneal metastases. Currently, cytoreductive surgery remains the mainstay of treatment but disease recurrence and death after relapse frequently occur in patients with PMP. New therapeutic strategies are therefore urgently needed for these patients. EXPERIMENTAL DESIGN: A total of 120 PMP samples from 50 patients were processed to generate a collection of 50 patient-derived organoid (PDO) and xenograft (PDX) models. Whole exome sequencing, immunohistochemistry analyses, and in vitro and in vivo drug efficacy studies were performed. RESULTS: In this study, we have generated a collection of PMP preclinical models and identified druggable targets, including BRAFV600E, KRASG12C, and KRASG12D, that could also be detected in intra-abdominal mucin biopsies of patients with PMP using droplet digital PCR. Preclinical models preserved the histopathological markers from the original patient sample. The BRAFV600E inhibitor encorafenib reduced cell viability of BRAFV600E PMP-PDO models. Proof-of-concept in vivo experiments showed that a systemic treatment with encorafenib significantly reduced tumor growth and prolonged survival in subcutaneous and orthotopic BRAFV600E-PMP-PDX mouse models. CONCLUSIONS: Our study demonstrates for the first time that systemic targeted therapies can effectively control PMP tumors. BRAF signaling pathway inhibition represents a new therapeutic opportunity for patients with BRAFV600E PMP who have a poor prognosis. Importantly, our present data and collection of preclinical models pave the way for evaluating the efficacy of other systemic targeted therapies toward extending the promise of precision oncology to patients with PMP.

DPPA3-HIF1α axis controls colorectal cancer chemoresistance by imposing a slow cell-cycle phenotype.

Tumor relapse is linked to rapid chemoresistance and represents a bottleneck for cancer therapy success. Engagement of a reduced proliferation state is a non-mutational mechanism exploited by cancer cells to bypass therapy-induced cell death. Through combining functional pulse-chase experiments in engineered cells and transcriptomic analyses, we identify DPPA3 as a master regulator of slow-cycling and chemoresistant phenotype in colorectal cancer (CRC). We find a vicious DPPA3-HIF1α feedback loop that downregulates FOXM1 expression via DNA methylation, thereby delaying cell-cycle progression. Moreover, downregulation of HIF1α partially restores a chemosensitive proliferative phenotype in DPPA3-overexpressing cancer cells. In cohorts of CRC patient samples, DPPA3 overexpression acts as a predictive biomarker of chemotherapeutic resistance that subsequently requires reduction in its expression to allow metastatic outgrowth. Our work demonstrates that slow-cycling cancer cells exploit a DPPA3/HIF1α axis to support tumor persistence under therapeutic stress and provides insights on the molecular regulation of disease progression.

Combination therapy for hepatocellular carcinoma: additive preclinical efficacy of the HDAC inhibitor panobinostat with sorafenib.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a heterogeneous cancer in which sorafenib is the only approved systemic therapy. Histone deacetylases (HDAC) are commonly dysregulated in cancer and therefore represent promising targets for therapies, however their role in HCC pathogenesis is still unknown. We analyzed the expression of 11 HDACs in human HCCs and assessed the efficacy of the pan-HDAC inhibitor panobinostat alone and in combination with sorafenib in preclinical models of liver cancer. METHODS: Gene expression and copy number changes were analyzed in a cohort of 334 human HCCs, while the effects of panobinostat and sorafenib were evaluated in three liver cancer cell lines and a murine xenograft model. RESULTS: Aberrant HDAC expression was identified and validated in 91 and 243 HCCs, respectively. Upregulation of HDAC3 and HDAC5 mRNAs was significantly correlated with DNA copy number gains. Inhibiting HDACs with panobinostat led to strong anti-tumoral effects in vitro and vivo, enhanced by the addition of sorafenib. Cell viability and proliferation declined, while apoptosis and autophagy increased. Panobinostat increased histone H3 and HSP90 acetylation, downregulated BIRC5 (survivin) and upregulated CDH1. Combination therapy with panobinostat and sorafenib significantly decreased vessel density, and most significantly decreased tumor volume and increased survival in HCC xenografts. CONCLUSIONS: Aberrant expression of several HDACs and copy number gains of HDAC3 and HDAC5 occur in HCC. Treatment with panobinostat combined with sorafenib demonstrated the highest preclinical efficacy in HCC models, providing the rationale for clinical studies with this novel combination.

MicroRNA-based classification of hepatocellular carcinoma and oncogenic role of miR-517a.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a heterogeneous tumor that develops via activation of multiple pathways and molecular alterations. It has been a challenge to identify molecular classes of HCC and design treatment strategies for each specific subtype. MicroRNAs (miRNAs) are involved in HCC pathogenesis, and their expression profiles have been used to classify cancers. We analyzed miRNA expression in human HCC samples to identify molecular subclasses and oncogenic miRNAs. METHODS: We performed miRNA profiling of 89 HCC samples using a ligation-mediated amplification method. Subclasses were identified by unsupervised clustering analysis. We identified molecular features specific for each subclass using expression pattern (Affymetrix U133 2.0; Affymetrix, Santa Clara, CA), DNA change (Affymetrix STY Mapping Array), mutation (CTNNB1), and immunohistochemical (phosphor[p]-protein kinase B, p-insulin growth factor-IR, p-S6, p-epidermal growth factor receptor, ß-catenin) analyses. The roles of selected miRNAs were investigated in cell lines and in an orthotopic model of HCC. RESULTS: We identified 3 main clusters of HCCs: the wingless-type MMTV integration site (32 of 89; 36%), interferon-related (29 of 89; 33%), and proliferation (28 of 89; 31%) subclasses. A subset of patients with tumors in the proliferation subclass (8 of 89; 9%) overexpressed a family of poorly characterized miRNAs from chr19q13.42. Expression of miR-517a and miR-520c (from ch19q13.42) increased proliferation, migration, and invasion of HCC cells in vitro. MiR-517a promoted tumorigenesis and metastatic dissemination in vivo. CONCLUSIONS: We propose miRNA-based classification of 3 subclasses of HCC. Among the proliferation class, miR-517a is an oncogenic miRNA that promotes tumor progression. There is rationale for developing therapies that target miR-517a for patients with HCC.

Functional patient-derived organoid screenings identify MCLA-158 as a therapeutic EGFR × LGR5 bispecific antibody with efficacy in epithelial tumors.

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.