Integrative genomics highlights opportunities for innovative therapies targeting the tumor microenvironment in gallbladder cancer

Abstract

See Article, pages 1132–1144 See Article, pages 1132–1144 Gallbladder cancers (GBCs) together with intrahepatic and extrahepatic cholangiocarcinomas (CCAs) constitute rare malignancies of the biliary tract.[1]Wistuba II, Gazdar A.F. Gallbladder cancer: lessons from a rare tumour.Nat Rev Canc. 2004; 4: 695-706Crossref PubMed Scopus (317) Google Scholar Although all biliary tract cancers (BTCs) are associated with dismal outcome, GBC is the most common and aggressive, with an estimated incidence of 2.5 per 100,000 individuals worldwide and a 5-year overall survival (OS) rate of less than 5%.[2]Bray F. Ferlay J. Soerjomataram I. Siegel R.L. Torre L.A. Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Canc J Clin. 2018; 68: 394-424Crossref PubMed Scopus (37975) Google Scholar,[3]Rawla P. Sunkara T. Thandra K.C. Barsouk A. Epidemiology of gallbladder cancer.Clin Exp Hepatol. 2019; 5: 93-102Crossref PubMed Scopus (52) Google Scholar According to the GLOBOCAN 2018 data, GBC accounts for 1.2% of all diagnosed cancers, but 1.7% of all cancer-related deaths.[2]Bray F. Ferlay J. Soerjomataram I. Siegel R.L. Torre L.A. Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Canc J Clin. 2018; 68: 394-424Crossref PubMed Scopus (37975) Google Scholar,[3]Rawla P. Sunkara T. Thandra K.C. Barsouk A. Epidemiology of gallbladder cancer.Clin Exp Hepatol. 2019; 5: 93-102Crossref PubMed Scopus (52) Google Scholar GBC is associated with a dismal outcome as most patients are diagnosed too late for curative surgical treatment. Striking geographical and ethnicity-related variations in the incidence of GBC have been observed, suggesting that genetic variations contribute to GBC.[1]Wistuba II, Gazdar A.F. Gallbladder cancer: lessons from a rare tumour.Nat Rev Canc. 2004; 4: 695-706Crossref PubMed Scopus (317) Google Scholar GBC is also one of the few cancers more common in women than in men. Besides geographical and gender bias, chronic inflammation is the most important risk factor for GBC.[1]Wistuba II, Gazdar A.F. Gallbladder cancer: lessons from a rare tumour.Nat Rev Canc. 2004; 4: 695-706Crossref PubMed Scopus (317) Google Scholar Surgery is currently the only curative option, but most patients are diagnosed too late to be eligible for surgery, as GBC onset and progression are usually asymptomatic at early stages. Cytotoxic chemotherapy remains the mainstay of treatment in first-line metastatic BTC, including GBC, but has a modest effect on long-term survival.[4]Azizi A.A. Lamarca A. Valle J.W. Systemic therapy of gallbladder cancer: review of first line, maintenance, neoadjuvant and second line therapy specific to gallbladder cancer.Chin Clin Oncol. 2019; 8: 43Crossref PubMed Scopus (4) Google Scholar Combined chemotherapy did not improve OS compared to single-agent chemotherapy as second-line treatment in patients with advanced GBC.[4]Azizi A.A. Lamarca A. Valle J.W. Systemic therapy of gallbladder cancer: review of first line, maintenance, neoadjuvant and second line therapy specific to gallbladder cancer.Chin Clin Oncol. 2019; 8: 43Crossref PubMed Scopus (4) Google Scholar A better morpho-molecular characterization of GBC is urgently needed to better stratify patients into clinically relevant subgroups and to identify new actionable alterations. Thus, a deep and comprehensive characterization of the gene expression profile of GBC constitutes an important prerequisite to identify potential companion biomarkers to stratify patients, especially for first-line therapies.[4]Azizi A.A. Lamarca A. Valle J.W. Systemic therapy of gallbladder cancer: review of first line, maintenance, neoadjuvant and second line therapy specific to gallbladder cancer.Chin Clin Oncol. 2019; 8: 43Crossref PubMed Scopus (4) Google Scholar In this issue of Journal of Hepatology, Nepal and colleagues address this issue by establishing a comprehensive landscape of genetic, epigenetic and transcriptomic alterations in a large cohort of patients with GBC.[5]Nepal C. Zhu B. O'Rourke C.J. Bhatt D.K. Lee D. Song L. et al.Integrative molecular characterization of gallbladder cancer reveals microenvironment-associated subtypes.J Hepatol. 2021; 74: 1132-1144Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar Importantly, by integrating various data from DNA methylation changes, copy number analysis, RNA sequencing and whole exome sequencing, they highlight clinically relevant tumor subgroups associated with prognosis (Fig. 1). The study further suggests that targeting the tumor immune microenvironment (TIME), alone or in combination with cytotoxic drugs, may have therapeutic potential in GBC. To date, only few large-scale studies focusing on GBC have been performed. In some studies GBCs were included together with CCA, revealing that GBC and CCA exhibit both distinct and common molecular alterations.[6]Valle J.W. Lamarca A. Goyal L. Barriuso J. Zhu A.X. New horizons for precision medicine in biliary tract cancers.Canc Discov. 2017; 7: 943-962Crossref PubMed Scopus (224) Google Scholar,[7]Wardell C.P. Fujita M. Yamada T. Simbolo M. Fassan M. Karlic R. et al.Genomic characterization of biliary tract cancers identifies driver genes and predisposing mutations.J Hepatol. 2018; 68: 959-969Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar Common mutations included TP53, ARID1A and PI3KCA. In contrast, mutational frequencies of IDH1, BAP1 and FGFR fusions were very low or absent in GBC, while mutations of the ERBB family of proteins were higher in GBC than CCA.[8]Lamarca A. Barriuso J. McNamara M.G. Valle J.W. Molecular targeted therapies: ready for "prime time" in biliary tract cancer.J Hepatol. 2020; 73: 170-185Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,[9]Li M. Zhang Z. Li X. Ye J. Wu X. Tan Z. et al.Whole-exome and targeted gene sequencing of gallbladder carcinoma identifies recurrent mutations in the ErbB pathway.Nat Genet. 2014; 46: 872-876Crossref PubMed Scopus (240) Google Scholar Thus, morphologically and molecularly, GBC and CCA are clearly separate entities. A large integrative study of patients with GBC from Korea, India and Chile found that mutated genes not previously linked to GBC include the ETS domain-containing genes, ELF3 and EHF.[10]Pandey A. Stawiski E.W. Durinck S. Gowda H. Goldstein L.D. Barbhuiya M.A. et al.Integrated genomic analysis reveals mutated ELF3 as a potential gallbladder cancer vaccine candidate.Nat Commun. 2020; 11: 4225Crossref PubMed Scopus (13) Google Scholar Interestingly, ELF3 mutations were predominantly frame-shift alterations resulting in several neoantigens which may activate CD8+ T cells, indicating that they may serve as potential cancer vaccine candidates.[10]Pandey A. Stawiski E.W. Durinck S. Gowda H. Goldstein L.D. Barbhuiya M.A. et al.Integrated genomic analysis reveals mutated ELF3 as a potential gallbladder cancer vaccine candidate.Nat Commun. 2020; 11: 4225Crossref PubMed Scopus (13) Google Scholar Alterations in the Wnt pathway have been observed at the genomic or transcriptomic level in GBC and CCA.10Pandey A. Stawiski E.W. Durinck S. Gowda H. Goldstein L.D. Barbhuiya M.A. et al.Integrated genomic analysis reveals mutated ELF3 as a potential gallbladder cancer vaccine candidate.Nat Commun. 2020; 11: 4225Crossref PubMed Scopus (13) Google Scholar, 11Boulter L. Guest R.V. Kendall T.J. Wilson D.H. Wojtacha D. Robson A.J. et al.WNT signaling drives cholangiocarcinoma growth and can be pharmacologically inhibited.J Clin Invest. 2015; 125: 1269-1285Crossref PubMed Scopus (151) Google Scholar, 12Perugorria M.J. Olaizola P. Labiano I. Esparza-Baquer A. Marzioni M. Marin J.J.G. et al.Wnt-beta-catenin signalling in liver development, health and disease.Nat Rev Gastroenterol Hepatol. 2019; 16: 121-136Crossref PubMed Scopus (129) Google Scholar Mutations in the central Wnt pathway genes, CTNNB1 and APC, have been detected in GBC and may lead to constitutively increased Wnt signaling.[10]Pandey A. Stawiski E.W. Durinck S. Gowda H. Goldstein L.D. Barbhuiya M.A. et al.Integrated genomic analysis reveals mutated ELF3 as a potential gallbladder cancer vaccine candidate.Nat Commun. 2020; 11: 4225Crossref PubMed Scopus (13) Google Scholar Moreover, a study focusing on DNA methylation profiling revealed gradual hypermethylation and repressed gene expression of APC from non-neoplastic epithelia via dysplasia to invasive GBC.[13]Bragelmann J. Barahona Ponce C. Marcelain K. Roessler S. Goeppert B. Gallegos I. et al.Epigenome-wide analysis of methylation changes in the sequence of gallstone disease, dysplasia, and gallbladder cancer.Hepatology. 2020; Crossref PubMed Scopus (4) Google Scholar Highlighting the importance of Wnt signaling, the integrative analysis by Nepal and colleagues demonstrated that Wnt signaling is among the most significantly affected pathways in GBC. Furthermore, this study identified 3 GBC subtypes based on transcriptional profiles: subtype 2 was associated with relatively long survival, whereas subtypes 1 and 3 were associated with poor survival (Fig. 1). Besides patient survival and gene expression, multiple differences in histomorphology and the tumor microenvironment were observed between the 3 subtypes. However, no significant differences in mutation profiles were observed in the 3 subtypes possibly due to low mutational frequencies. Histomorphological analysis by Nepal and colleagues showed an enrichment of biliary, gastric foveolar and intestinal growth patterns in subtype 1, subtype 2 and subtype 3, respectively.[5]Nepal C. Zhu B. O'Rourke C.J. Bhatt D.K. Lee D. Song L. et al.Integrative molecular characterization of gallbladder cancer reveals microenvironment-associated subtypes.J Hepatol. 2021; 74: 1132-1144Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar Interestingly, this morpho-molecular subtyping resembles the pattern of intracholecystic papillary neoplasm, the known premalignant lesion of the gallbladder, raising interesting questions regarding the processes of tumor differentiation and malignant transformation. However, independent validation of this finding and identification of tissue biomarkers associated with these histological subtypes would provide additional support for an association of gastric foveolar GBC with good prognosis. Furthermore, subtype 2, which is associated with relatively good outcome, exhibited high levels of T cell infiltration, high tumor cell content and increased metabolism, whereas subtypes 1 and 3 (poor outcome group) showed a hypoxia signature, high levels of cancer-associated fibroblasts (CAFs), high levels of MDSCs (myeloid-derived suppressor cells), and high tumor immune dysfunction and exclusion (TIDE) scores indicating T cell exclusion and dysfunction (Fig. 1).[5]Nepal C. Zhu B. O'Rourke C.J. Bhatt D.K. Lee D. Song L. et al.Integrative molecular characterization of gallbladder cancer reveals microenvironment-associated subtypes.J Hepatol. 2021; 74: 1132-1144Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar MDSCs have been shown to exhibit strong immunosuppressive effects by upregulating regulatory T cells and immune suppressive cytokines, as well as supporting the conversion of fibroblasts to CAFs.[14]Veglia F. Perego M. Gabrilovich D. Myeloid-derived suppressor cells coming of age.Nat Immunol. 2018; 19: 108-119Crossref PubMed Scopus (696) Google Scholar Therefore, the study by Nepal and colleagues sheds new light on the tumor microenvironment of GBC in poor and good outcome subgroups, supporting the hypothesis that immunosuppression and microenvironment remodeling may account for poor patient survival and possibly poor response to immune checkpoint inhibitors (ICIs) (Fig. 1). Phase II trials studying anti-PD-(L)1 as monotherapy or combined with anti-CTLA-4 previously demonstrated disappointing response rates (5% to 16%), suggesting that only a minority of patients could benefit from immunotherapy.[15]Bang Y.-J. Ueno M. Malka D. Chung H.C. Nagrial A. Kelley R.K. et al.Pembrolizumab (pembro) for advanced biliary adenocarcinoma: results from the KEYNOTE-028 (KN028) and KEYNOTE-158 (KN158) basket studies.J Clin Oncol. 2019; 37 (4079-4079)Crossref PubMed Google Scholar,[16]Ioka T. Ueno M. Oh D.-Y. Fujiwara Y. Chen J.-S. Doki Y. et al.Evaluation of safety and tolerability of durvalumab (D) with or without tremelimumab (T) in patients (pts) with biliary tract cancer (BTC).J Clin Oncol. 2019; 37 (387-387)Crossref Google Scholar Moreover, PD-L1 expression was not suggested as a strong predictive factor of efficacy. Despite these disappointing results, large phase III trials were launched, based on a combination of standard of care chemotherapy with ICIs. These trials, run in an unselected population, are unlikely to identify the subgroup of patients that might benefit. The work of Nepal and colleagues, aiming at deciphering the different TIME present in GBC, could thus represent a major step forward for future clinical trial development.[5]Nepal C. Zhu B. O'Rourke C.J. Bhatt D.K. Lee D. Song L. et al.Integrative molecular characterization of gallbladder cancer reveals microenvironment-associated subtypes.J Hepatol. 2021; 74: 1132-1144Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar Similar analyses were previously done in CCA, highlighting clinically relevant subgroups of patients who may benefit from immunotherapies based on specific TIME.[17]Job S. Rapoud D. Dos Santos A. Gonzalez P. Desterke C. Pascal G. et al.Identification of four immune subtypes characterized by distinct composition and functions of tumor microenvironment in intrahepatic cholangiocarcinoma.Hepatology. 2020; 72: 965-981Crossref PubMed Scopus (42) Google Scholar The subgroup of GBC with a better prognosis identified by Nepal and colleagues would, at first glance, appear to be a good target for evaluating ICIs. In contrast, poor prognosis GBC were associated with dysregulation of immune-related pathways, including dysfunctional T cells and T cell exclusion.[5]Nepal C. Zhu B. O'Rourke C.J. Bhatt D.K. Lee D. Song L. et al.Integrative molecular characterization of gallbladder cancer reveals microenvironment-associated subtypes.J Hepatol. 2021; 74: 1132-1144Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar In agreement with the presence of an immunosuppressive microenvironment, poor prognosis GBC also exhibited expression of markers linked to the activation of transforming growth factor beta (TGFβ), a potent immunosuppressive cytokine (Fig. 1). Indeed, bifunctional antibodies targeting TGFβ with either CTLA-4 or PD-L1 were suggested to improve results of anti-CTLA-4 or anti-PD-L1 antibodies, due to adequate local sequestration of TGFβ within the TIME.[18]Ravi R. Noonan K.A. Pham V. Bedi R. Zhavoronkov A. Ozerov I.V. et al.Bifunctional immune checkpoint-targeted antibody-ligand traps that simultaneously disable TGFbeta enhance the efficacy of cancer immunotherapy.Nat Commun. 2018; 9: 741Crossref PubMed Scopus (126) Google Scholar Interestingly, preliminary results of a clinical trial testing bispecific anti-PD-L1/anti-TGFβ suggested activity, with a 23% response rate.[19]Kang Y.-K. Doi T. Kondo S. Chung H.-C. Muro K. Helwig C. et al.M7824 (MSB0011359C), a bifunctional fusion protein targeting PD-L1 and TGF-β, in Asian patients with pretreated recurrent or refractory gastric cancer: preliminary results from a phase I trial.J Clin Oncol. 2018; 36 (100-100)Crossref Google Scholar Once again, selecting the appropriate population might greatly improve predictive testing and lead to positive clinical trial results. Finally, the authors studied the TIDE signature of potential response to ICIs.[20]Jiang P. Gu S. Pan D. Fu J. Sahu A. Hu X. et al.Signatures of T cell dysfunction and exclusion predict cancer immunotherapy response.Nat Med. 2018; 24: 1550-1558Crossref PubMed Scopus (529) Google Scholar This analysis suggested that subtype 2 GBC might be the focus of interest for the implementation of ICIs. Taken collectively, the study by Nepal and colleagues suggests that the prognosis of patients with GBC depends on tumor cell properties and/or the TIME. The study further suggests that a comprehensive analysis of the TIME might represent the best approach to identify the population of interest for the development of immuno-oncology drugs in GBC (Fig. 1). Combined therapies targeting actionable alterations in tumor cells as well as in the TIME are emerging as relevant strategies to treat GBC, and also liver cancer. We believe that testing combination therapies should be considered together with relevant biomarkers for specific GBC subtypes. Biomarker-driven clinical trials will help to guide treatment and improve disease management. CC’s team is supported by Inserm , Université de Rennes 1 , Ligue Contre le Cancer ( CD22 , CD35 , CD85 ), INCa , and ITMO Cancer AVIESAN (Alliance Nationale pour les Sciences de la Vie et de la Santé) dans le cadre du Plan cancer (Non-coding RNA in cancerology: fundamental to translational). SR and PS are supported by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project-ID 314905040 – SFB/TRR 209 Liver Cancer and SR is supported by German Cancer Aid (Deutsche Krebshilfe , project no. 70113922). All authors contributed equally to the intellectual content of the manuscript. All authors wrote the initial draft and contributed significantly to the final version. CC and SR declare no conflict of interest related to this work. JE provides expertise for MSD, Roche, AstraZeneca and BMS. PS received honoraries from BMS, Roche, MSD, AstraZeneca, Novartis, Ipsen, Incyte and Janssen. Please refer to the accompanying ICMJE disclosure forms for further details. The following is/are the supplementary data to this article: Download .pdf (.17 MB) Help with pdf files Multimedia component 1 Integrative molecular characterisation of gallbladder cancer reveals micro-environment-associated subtypesJournal of HepatologyVol. 74Issue 5PreviewGallbladder cancer (GBC) is the most common type of biliary tract cancer, but the molecular mechanisms involved in gallbladder carcinogenesis remain poorly understood. In this study, we applied integrative genomics approaches to characterise GBC and explore molecular subtypes associated with patient survival. Full-Text PDF