Advanced Biliary Cancer Patient Derived Organoids as a novel tool for functional tumour characterization and drug selection

Supervisors: Chiara Braconi (University of Glasgow), Owen Sansom (CRUK Beatson Institute)

Biliary cancers (BC) represent an area of unmet need. Eighty percent of BC patients present at an advanced stage where therapeutic options are limited. All the genomic/transcriptomic studies have been performed on resected cancers while the understanding of advanced BC (ABC) molecular make-up is hampered by the paucity of diagnostic tissue. Moreover, functional genomic studies aiming at understanding molecular mechanisms of ABC progression and drug resistance are limited by the lack of disease models that recapitulate tumour features and are applicable within patients' life expectancy. We have shown that patients' derived organoids (PDOs) represent a promising tool that closely resembles phenotypic and molecular features of their parental tumour enabling expansion of primary cultures for molecular analyses. We have provided the first evidence that PDOs can be established from a single biopsy core in ABC and can be used for drug testing and disease modelling within 6-12 weeks. Ultimate goal of these studies is to integrate a global characterization at genomic and transcriptomic level with drug testing in pre- and post-treatment PDOs in order to understand the molecular landscape of advanced BCs, elucidate the mechanisms of chemo-resistance and explore the value of PDOs as predictive biomarkers of drug response in ABC.

Keywords: Biliary cancers, cholangiocarcinoma, chemotherapy, genomics, therapeutics

Background
Advanced Biliary cancers (ABC), also known as cholangiocarcinomas (CCA), are malignancies occurring in the biliary system, whose incidence has been steadily increasing, while treatment options remain confined to chemotherapy (Braconi, Liver Intern 2019). A better understanding of ABC biology and mechanisms of resistance to treatment is warranted to develop novel therapeutic strategies that can implement a personalized approach. A comprehensive characterization of the genomic and transcriptomic features of resected BC has highlighted the potential of targetable molecular alterations in BC (Nakamura, Nat Genet 2015). However, a high degree of tumour and genomic evolution has been observed as the tumour becomes metastatic and is exposed to drug treatment (Khan, Canc Disc 2018). Data from our previous collaborative project showed that only 26% of diagnostic biopsies were suitable for molecular analyses in ABC patients (Moorcrafft, Ann Oncol 2018). In addition, multiple molecular alterations were found in 87% of ABC patients, in comparison to 29% in resected BC (Verlingue Ann Oncol 2017), suggesting that a flexible functional model to reliably test drug sensitivity in "real time" should be favoured to an approach based on coupling one alteration with one targeted drug. We have previously shown that patient derived organoids (PDO) can be established from single core biopsies in 6-12 weeks (Lampis, Gastroenterology 2018, Vlachogiannis, Science 2018) and mimic drug response observed in the clinic (Vlachogiannis, Science 2018).

Hypothesis
We hypothesize that the generation of organoids from ABC will enable 1) the study of molecular drivers that promote progression and chemo-resistance in ABC, and 2) provide a tool for the implementation of personalized medicine.

Aims
1) Establishment of longitudinal PDO from ABC patients during treatment course.
2) Drug screening in baseline PDO to inform second line treatment choice.
3) Genomic and functional studies of longitudinal PDO to elucidate the mechanism of drug resistance.

Methodology
PDOs will be established from liver biopsies or fine needle biopsies (FNB) from patients with ABC being treated with palliative first line chemotherapy at the Beatson West of Scotland Cancer Centre (BWoSCC). From our previous experience of PDO-based co-clinical trials the success rate of PDOs from biopsy cores is 70% in gastrointestinal cancers (Vlachogiannis, Science 2018). We are planning to enrol 30 patients in order to have at least 20 baseline PDO cultures for this pivotal study. Whole genome sequencing will be performed and data analysed as previously described (Lote, Ann Oncol 2017) to assess genomic alterations, including mutations, mutational signatures and CNV. To achieve a full characterization of the functional effect of the findings observed above we will integrate DNA sequencing analyses with gene expression analyses. We demonstrated that the deregulation of gene expression is conserved in PDOs in comparison to matched parental biopsy (Lampis, Gastroenterology 2018) and that does not change over time during sequential passages of PDOs. Through the expansion of cancer cells via PDOs we expect to molecular characterize the genomic landscape of ABC, and to compare it with the molecular make up of resected early stage ABCs (data from 300 samples are already available through established collaborations within the European Network for the Study of Cholangiocarcinoma of which Dr Braconi is a member). Patients will be subjected to repeated biopsies at the end of their first line treatment in order to establish a biobank of clinically annotated PDOs for the study of their molecular make-up before and after drug exposure (from data of phase III clinical trials we expect to have 5 partial responders and 5 rapid progressors that can be used for the analysis in Aim 3). International guidelines for ABC recommend one line of chemotherapy with cisplatin-gemcitabine, while no standard second line treatments are accepted (Valle, Ann Oncol 2016), making ABC an ideal indication for a PDO-based personalised approach. Baseline PDOs will be screened against a library of drugs compounds used in the clinic and those that are available within the phase I clinical trial unit at the BWoSCC. We will pivotal the choice of second line treatment on the bases of the results of the ex vivo drug screening. Patients suitable for second line treatment will be referred for specific trials and/or considered for second-line therapy with drugs selected on the bases of the ex vivo results. A first phase will consist in choosing drugs that are available within the NHS (i.e. fluorouracil, oxaliplatin, irinotecan, paclitaxel, drugs within phase I trials), while a second phase will comprise the launch of an umbrella study with engagement of pharmaceutical companies. We have previously shown that PDO derived from repeated biopsies (before and after treatment) can inform on the signalling pathways that drive drug resistance in colorectal cancer (Vlachogiannis, Science 2018). In these studies we propose to delineate the portrait of primary and secondary chemo-resistance in ABC. We hypothesize that the modulation of the key pathways identified in these analyses will revert chemo-resistance in ABC. Having PDOs from chemo-sensitive tumours will enable us to use them as positive controls and to study the potential of our induced modulation to re-establish benefit from chemotherapy at a level that is comparable to that observed in clinic. We showed that PDOs can be genetically modulated to induce and/or disrupt the expression of a gene of interest and assess its biological relevance (Lampis, Gastroenterology 2017). We will modulate gene expression in chemo-resistant PDOs through CRISR and/or lentiviral-technologies [as we previously described (Carotenuto, NCRI 2018;Lampis, 2018), and will assess treatment response ex vivo and in vivo. PDOs with modulation of target gene will be infected with luciferase construct and inoculated within the liver of NSG mice to generate ABC xenograft models that will be treated with and without drugs to investigate mechanisms of drug resistance.

The project will create a unique platform consisting of non-murine, patients based pre-clinical models that can be exploited for personalized medicine and functional studies in biliary tract cancers with direct impact on ABC patients and on the advancement of novel therapeutic strategies for personalized oncology.

Publications
Lampis A, Carotenuto P, Vlachogiannis G, Cascione L, Hedayat S, Burke R, Clarke P, Bosma E, Simbolo M, Scarpa A, Yu S, Cole R, Smyth E, Mateos JF, Begum R, Hezelova B, Eltahir Z, Wotherspoon A, Fotiadis N, Bali MA, Nepal C, Khan K, Stubbs M, Hahne JC, Gasparini P, Guzzardo V, Croce CM, Eccles S, Fassan M, Cunningham D, Andersen JB, Workman P, Valeri N, Braconi C. MIR21 drives resistance to Heat Shock Protein 90 inhibition in cholangiocarcinoma. Gastroenterology 2018.

Vlachogiannis G; Hedayat S VA, Jamin Y, Fernández-Mateos J, Khan K, Lampis A, Eason K, Huntingford I, Burke R, Rata M, Koh DM, Tunariu N, Collins D, Hulkki-Wilson S, Ragulan C, Spiter I, Moorcraft SY, Chau I, Rao S, Watkins D, Fotiadis N, Darvish-Damavandi M, Lote H, Eltahir Z, Smyth EC, Begum R, Clarke P, Hahne JC, Dowsett M, de Bono J, Workman P, Sadanandam A, Fassan M, Sansom O, Eccles S, Starling N, Braconi C, Sottoriva A, Robinson S, Cunningham D, Valeri N. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science 2018.

Ghidini M, Cascione L, Carotenuto P, Lampis A, Trevisani F, Previdi MC, Hahne JC, Said-Huntingford I, Raj M, Zerbi A, Mescoli C, Cillo U, Rugge M, Roncalli M, Torzilli G, Rimassa L, Santoro A, Valeri N, Fassan M, Braconi C. Characterisation of the immune-related transcriptome in resected biliary tract cancers. Eur J Cancer 2017.

Carotenuto P, Fassan M, Pandolfo R, Lampis A, Vicentini C, Cascione L, Paulus-Hock V, Boulter L, Guest R, Quagliata L, Hahne JC, Ridgway R, Jamieson T, Athineos D, Veronese A, Visone R, Murgia C, Ferrari G, Guzzardo V, Evans TRJ, MacLeod M, Feng GJ, Dale T, Negrini M, Forbes SJ, Terracciano L, Scarpa A, Patel T, Valeri N, Workman P, Sansom O, Braconi C. Wnt signalling modulates transcribed-ultraconserved regions in hepatobiliary cancers. Gut 2017

Khan K, Cunningham D, Werner B, Vlachogiannis G, Vatsiou A, Spiteri I, Lampis A, Darvish Damavandi M, Lote H, Said-Huntingford I, Chau I, Tunariu N, Mentrasti G, Trevisani F, Rao S, Watkins D, Starling N, Thomas J, Peckitt C, Khan N, Rugge M, Begum R, Hezelova B, Bryant A, Fassan M, Hahne JC, Braconi C, Sottoriva A & Valeri N. Forecasting the evolution of resistance to targeted treatment in metastatic colorectal cancer using longitudinal matched tissue and liquid biopsies in a prospective clinical trial. Cancer Discovery 2018.

Gay DM, Ridgway RA, Müller M, Hodder MC, Hedley A, Clark W, Leach JD, Jackstadt R, Nixon C, Huels DJ, Campbell AD, Bird TG, Sansom OJ. Loss of BCL9/9l suppresses Wnt driven tumourigenesis in models that recapitulate human cancer. Nat Commun. 2019 Feb 13;10(1):723. doi: 10.1038/s41467-019-08586-3.

Huels DJ, Bruens L, Hodder MC, Cammareri P, Campbell AD, Ridgway RA, Gay DM, Solar-Abboud M, Faller WJ, Nixon C, Zeiger LB, McLaughlin ME, Morrissey E, Winton DJ, Snippert HJ, van Rheenen J, Sansom OJ. Wnt ligands influence tumour initiation by controlling the number of intestinal stem cells. Nat Commun. 2018

Miller BW, Morton JP, Pinese M, Saturno G, Jamieson NB, McGhee E, Timpson P, Leach J, McGarry L, Shanks E, Bailey P, Chang D, Oien K, Karim S, Au A, Steele C, Carter CR, McKay C, Anderson K, Evans TR, Marais R, Springer C, Biankin A, Erler JT, Sansom OJ. Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy. EMBO Mol Med. 2015

Faller WJ, Jackson TJ, Knight JR, Ridgway RA, Jamieson T, Karim SA, Jones C, Radulescu S, Huels DJ, Myant KB, Dudek KM, Casey HA, Scopelliti A, Cordero JB, Vidal M, Pende M, Ryazanov AG, Sonenberg N, Meyuhas O, Hall MN, Bushell M, Willis AE, Sansom OJ. mTORC1-mediated translational elongation limits intestinal tumour initiation and growth. Nature. 2015

Valeri N, Braconi C, Gasparini P, Murgia C, Lampis A, Paulus-Hock V, Hart JR, Ueno L, Grivennikov SI, Lovat F, Paone A, Cascione L, Sumani KM, Veronese A, Fabbri M, Carasi S, Alder H, Lanza G, Gafa' R, Moyer MP, Ridgway RA, Cordero J, Nuovo GJ, Frankel WL, Rugge M, Fassan M, Groden J, Vogt PK, Karin M, Sansom OJ, Croce CM. microRNA-135b promotes cancer progression acting as a downstream effector of oncogenic pathways in colon cancer. Cancer Cell 2014

 

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