Human primary liver cancer–derived organoid cultures for disease modeling and drug screening

Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.M.H. is a Wellcome Trust Sir Henry Dale Fellow and is jointly funded by the Wellcome Trust and the Royal Society (104151/Z/14/Z). L.B. is supported by an EMBO Postdoctoral Fellowship (EMBO ALTF 794-2014) and Marie-Curie Postdoctoral Fellowship (grant no. 656193_H2020-MSCA-IF-2014). G.M. was supported by a Marie Curie Initial Training Network (Marie Curie ITN WntsApp 608180) and a H2020 LSMF4LIFE grant (ECH2020-668350). This work was funded by an NC3Rs International prize, a Beit Prize, a Cambridge Cancer Center-pump priming award (CRUK-RG83267) and, partially, by a NC3Rs project grant (NC/R001162/1), all of them awarded to M.H. Work at the L.J.W.v.d.L lab was funded by the research program InnoSysTox (project number 114027003), by the Netherlands Organisation for Health Research and Development (ZonMw), and part of the research program financed by the Dutch Digestive Foundation (MLDS-Diagnostics project number D16-26). Work in the M.J.G. lab is funded by the Wellcome Trust (102696), Stand Up To Cancer (SU2C-AACRDT1213) and Cancer Research UK (C44943/A22536)

Molecular landscape and functional characterization of centrosome amplification in ovarian cancer

High-grade serous ovarian carcinoma (HGSOC) is characterised by poor outcome and extreme chromosome instability (CIN). Therapies targeting centrosome amplification (CA), a key mediator of chromosome missegregation, may have significant clinical utility in HGSOC. However, the prevalence of CA in HGSOC, its relationship to genomic biomarkers of CIN and its potential impact on therapeutic response have not been defined. Using high-throughput multi-regional microscopy on 287 clinical HGSOC tissues and 73 cell lines models, here we show that CA through centriole overduplication is a highly recurrent and heterogeneous feature of HGSOC and strongly associated with CIN and genome subclonality. Cell-based studies showed that high-prevalence CA is phenocopied in ovarian cancer cell lines, and that high CA is associated with increased multi-treatment resistance; most notably to paclitaxel, the commonest treatment used in HGSOC. CA in HGSOC may therefore present a potential driver of tumour evolution and a powerful biomarker for response to standard-of-care treatment.We acknowledge funding and support from Cancer Research UK, and the Cancer Research UK Cambridge Centre: A22905 (C.M.S., J.B.D.); A25177 (M.A.V.R), A25117 (K.H.). L.M.G. was supported by the Wellcome Trust PhD programme in Mathematical Genomics and Medicine (grant number RG92770). This research was also supported by the Ovarian Cancer Action (grant number 006; I.A.M.), and the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014). Work in the Cancer Molecular Diagnostics Laboratory/Blood Processing Laboratory was supported by the NIHR Cambridge Biomedical Research Centre, Cancer Research UK Cambridge Centre and the Mark Foundation Institute for Integrated Cancer Medicine. The views expressed are those of the authors and not necessarily those of Cancer Research UK, the NIHR or the Department of Health and Social Care. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A pan-cancer compendium of chromosomal instability

Chromosomal instability (CIN) results in the accumulation of large-scale losses, gains and rearrangements of DNA1. The broad genomic complexity caused by CIN is a hallmark of cancer2; however, there is no systematic framework to measure different types of CIN and their effect on clinical phenotypes pan-cancer. Here we evaluate the extent, diversity and origin of CIN across 7,880 tumours representing 33 cancer types. We present a compendium of 17 copy number signatures that characterize specific types of CIN, with putative aetiologies supported by multiple independent data sources. The signatures predict drug response and identify new drug targets. Our framework refines the understanding of impaired homologous recombination, which is one of the most therapeutically targetable types of CIN. Our results illuminate a fundamental structure underlying genomic complexity in human cancers and provide a resource to guide future CIN research

High-grade serous ovarian carcinoma organoids as models of chromosomal instability

High-grade serous ovarian carcinoma (HGSOC) is the most genomically complex cancer, characterized by ubiquitous TP53 mutation, profound chromosomal instability, and heterogeneity. The mutational processes driving chromosomal instability in HGSOC can be distinguished by specific copy number signatures. To develop clinically relevant models of these mutational processes we derived 15 continuous HGSOC patient-derived organoids (PDOs) and characterized them using bulk transcriptomic, bulk genomic, single-cell genomic, and drug sensitivity assays. We show that HGSOC PDOs comprise communities of different clonal populations and represent models of different causes of chromosomal instability including homologous recombination deficiency, chromothripsis, tandem-duplicator phenotype, and whole genome duplication. We also show that these PDOs can be used as exploratory tools to study transcriptional effects of copy number alterations as well as compound-sensitivity tests. In summary, HGSOC PDO cultures provide validated genomic models for studies of specific mutational processes and precision therapeutics

High-grade serous ovarian carcinoma organoids as models of chromosomal instability.

Peer reviewed: TrueHigh-grade serous ovarian carcinoma (HGSOC) is the most genomically complex cancer, characterized by ubiquitous TP53 mutation, profound chromosomal instability, and heterogeneity. The mutational processes driving chromosomal instability in HGSOC can be distinguished by specific copy number signatures. To develop clinically relevant models of these mutational processes we derived 15 continuous HGSOC patient-derived organoids (PDOs) and characterized them using bulk transcriptomic, bulk genomic, single-cell genomic, and drug sensitivity assays. We show that HGSOC PDOs comprise communities of different clonal populations and represent models of different causes of chromosomal instability including homologous recombination deficiency, chromothripsis, tandem-duplicator phenotype, and whole genome duplication. We also show that these PDOs can be used as exploratory tools to study transcriptional effects of copy number alterations as well as compound-sensitivity tests. In summary, HGSOC PDO cultures provide validated genomic models for studies of specific mutational processes and precision therapeutics

The Genomic Landscape of Early-Stage Ovarian High-Grade Serous Carcinoma.

PURPOSE: Ovarian high-grade serous carcinoma (HGSC) is usually diagnosed at late stage. We investigated whether late-stage HGSC has unique genomic characteristics consistent with acquisition of evolutionary advantage compared with early-stage tumors. EXPERIMENTAL DESIGN: We performed targeted next-generation sequencing and shallow whole-genome sequencing (sWGS) on pretreatment samples from 43 patients with FIGO stage I-IIA HGSC to investigate somatic mutations and copy-number (CN) alterations (SCNA). We compared results to pretreatment samples from 52 patients with stage IIIC/IV HGSC from the BriTROC-1 study. RESULTS: Age of diagnosis did not differ between early-stage and late-stage patients (median 61.3 years vs. 62.3 years, respectively). TP53 mutations were near-universal in both cohorts (89% early-stage, 100% late-stage), and there were no significant differences in the rates of other somatic mutations, including BRCA1 and BRCA2. We also did not observe cohort-specific focal SCNA that could explain biological behavior. However, ploidy was higher in late-stage (median, 3.0) than early-stage (median, 1.9) samples. CN signature exposures were significantly different between cohorts, with greater relative signature 3 exposure in early-stage and greater signature 4 in late-stage. Unsupervised clustering based on CN signatures identified three clusters that were prognostic. CONCLUSIONS: Early-stage and late-stage HGSCs have highly similar patterns of mutation and focal SCNA. However, CN signature analysis showed that late-stage disease has distinct signature exposures consistent with whole-genome duplication. Further analyses will be required to ascertain whether these differences reflect genuine biological differences between early-stage and late-stage or simply time-related markers of evolutionary fitness. See related commentary by Yang et al., p. 2730

The copy number and mutational landscape of recurrent ovarian high-grade serous carcinoma

Acknowledgements: This work was supported by Ovarian Cancer Action (grant number OCA_006 to IAMcN); the National Institute for Healthcare Research (NIHR) Imperial Biomedical Research Centre (grant number P77646 to IAMcN); the Wellcome Trust PhD programme in Mathematical Genomics and Medicine (grant number RG92770 to LMG); Cancer Research UK (grant numbers DRCQQR-Jun22\100005, A22905, A15601 and A26204 to JDB, FM and IAMcN); a European Society of Medical Oncology (ESMO) Translational Fellowship (to G.G.); the Beatson Cancer Charity and Hutchison Whampoa Limited. Infrastructure support was provided by CRUK/NIHR Experimental Cancer Medicine Centres at Imperial, Cambridge, Glasgow and other participating sites. We also thank the Biorepository, Bioinformatics, Histopathology, IT & Scientific Computing and Genomics Core Facilities of the Cancer Research UK Cambridge Institute and the Pathology Core at the Cancer Research UK Beatson Institute for technical support. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.The drivers of recurrence and resistance in ovarian high grade serous carcinoma remain unclear. We investigate the acquisition of resistance by collecting tumour biopsies from a cohort of 276 women with relapsed ovarian high grade serous carcinoma in the BriTROC-1 study. Panel sequencing shows close concordance between diagnosis and relapse, with only four discordant cases. There is also very strong concordance in copy number between diagnosis and relapse, with no significant difference in purity, ploidy or focal somatic copy number alterations, even when stratified by platinum sensitivity or prior chemotherapy lines. Copy number signatures are strongly correlated with immune cell infiltration, whilst diagnosis samples from patients with primary platinum resistance have increased rates of CCNE1 and KRAS amplification and copy number signature 1 exposure. Our data show that the ovarian high grade serous carcinoma genome is remarkably stable between diagnosis and relapse and acquired chemotherapy resistance does not select for common copy number drivers

The copy number and mutational landscape of recurrent ovarian high-grade serous carcinoma

Acknowledgements: This work was supported by Ovarian Cancer Action (grant number OCA_006 to IAMcN); the National Institute for Healthcare Research (NIHR) Imperial Biomedical Research Centre (grant number P77646 to IAMcN); the Wellcome Trust PhD programme in Mathematical Genomics and Medicine (grant number RG92770 to LMG); Cancer Research UK (grant numbers DRCQQR-Jun22\100005, A22905, A15601 and A26204 to JDB, FM and IAMcN); a European Society of Medical Oncology (ESMO) Translational Fellowship (to G.G.); the Beatson Cancer Charity and Hutchison Whampoa Limited. Infrastructure support was provided by CRUK/NIHR Experimental Cancer Medicine Centres at Imperial, Cambridge, Glasgow and other participating sites. We also thank the Biorepository, Bioinformatics, Histopathology, IT & Scientific Computing and Genomics Core Facilities of the Cancer Research UK Cambridge Institute and the Pathology Core at the Cancer Research UK Beatson Institute for technical support. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.The drivers of recurrence and resistance in ovarian high grade serous carcinoma remain unclear. We investigate the acquisition of resistance by collecting tumour biopsies from a cohort of 276 women with relapsed ovarian high grade serous carcinoma in the BriTROC-1 study. Panel sequencing shows close concordance between diagnosis and relapse, with only four discordant cases. There is also very strong concordance in copy number between diagnosis and relapse, with no significant difference in purity, ploidy or focal somatic copy number alterations, even when stratified by platinum sensitivity or prior chemotherapy lines. Copy number signatures are strongly correlated with immune cell infiltration, whilst diagnosis samples from patients with primary platinum resistance have increased rates of CCNE1 and KRAS amplification and copy number signature 1 exposure. Our data show that the ovarian high grade serous carcinoma genome is remarkably stable between diagnosis and relapse and acquired chemotherapy resistance does not select for common copy number drivers

Molecular landscape and functional characterization of centrosome amplification in ovarian cancer.

Funder: NIHR Cambridge Biomedical Research Centre (BRC-1215-20014). Ovarian Cancer Action (grant number 006).High-grade serous ovarian carcinoma (HGSOC) is characterised by poor outcome and extreme chromosome instability (CIN). Therapies targeting centrosome amplification (CA), a key mediator of chromosome missegregation, may have significant clinical utility in HGSOC. However, the prevalence of CA in HGSOC, its relationship to genomic biomarkers of CIN and its potential impact on therapeutic response have not been defined. Using high-throughput multi-regional microscopy on 287 clinical HGSOC tissues and 73 cell lines models, here we show that CA through centriole overduplication is a highly recurrent and heterogeneous feature of HGSOC and strongly associated with CIN and genome subclonality. Cell-based studies showed that high-prevalence CA is phenocopied in ovarian cancer cell lines, and that high CA is associated with increased multi-treatment resistance; most notably to paclitaxel, the commonest treatment used in HGSOC. CA in HGSOC may therefore present a potential driver of tumour evolution and a powerful biomarker for response to standard-of-care treatment.We acknowledge funding and support from Cancer Research UK, and the Cancer Research UK Cambridge Centre: A22905 (C.M.S., J.B.D.); A25177 (M.A.V.R), A25117 (K.H.). L.M.G. was supported by the Wellcome Trust PhD programme in Mathematical Genomics and Medicine (grant number RG92770). This research was also supported by the Ovarian Cancer Action (grant number 006; I.A.M.), and the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014). Work in the Cancer Molecular Diagnostics Laboratory/Blood Processing Laboratory was supported by the NIHR Cambridge Biomedical Research Centre, Cancer Research UK Cambridge Centre and the Mark Foundation Institute for Integrated Cancer Medicine. The views expressed are those of the authors and not necessarily those of Cancer Research UK, the NIHR or the Department of Health and Social Care. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The copy number and mutational landscape of recurrent ovarian high-grade serous carcinoma

Acknowledgements: This work was supported by Ovarian Cancer Action (grant number OCA_006 to IAMcN); the National Institute for Healthcare Research (NIHR) Imperial Biomedical Research Centre (grant number P77646 to IAMcN); the Wellcome Trust PhD programme in Mathematical Genomics and Medicine (grant number RG92770 to LMG); Cancer Research UK (grant numbers DRCQQR-Jun22\100005, A22905, A15601 and A26204 to JDB, FM and IAMcN); a European Society of Medical Oncology (ESMO) Translational Fellowship (to G.G.); the Beatson Cancer Charity and Hutchison Whampoa Limited. Infrastructure support was provided by CRUK/NIHR Experimental Cancer Medicine Centres at Imperial, Cambridge, Glasgow and other participating sites. We also thank the Biorepository, Bioinformatics, Histopathology, IT & Scientific Computing and Genomics Core Facilities of the Cancer Research UK Cambridge Institute and the Pathology Core at the Cancer Research UK Beatson Institute for technical support. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.The drivers of recurrence and resistance in ovarian high grade serous carcinoma remain unclear. We investigate the acquisition of resistance by collecting tumour biopsies from a cohort of 276 women with relapsed ovarian high grade serous carcinoma in the BriTROC-1 study. Panel sequencing shows close concordance between diagnosis and relapse, with only four discordant cases. There is also very strong concordance in copy number between diagnosis and relapse, with no significant difference in purity, ploidy or focal somatic copy number alterations, even when stratified by platinum sensitivity or prior chemotherapy lines. Copy number signatures are strongly correlated with immune cell infiltration, whilst diagnosis samples from patients with primary platinum resistance have increased rates of CCNE1 and KRAS amplification and copy number signature 1 exposure. Our data show that the ovarian high grade serous carcinoma genome is remarkably stable between diagnosis and relapse and acquired chemotherapy resistance does not select for common copy number drivers