Table 1 Consensus statements
From: Criteria for preclinical models of cholangiocarcinoma: scientific and medical relevance
Number | Question | Statement | Response yes/total responders | Gradea |
|---|---|---|---|---|
Histological assessment | ||||
1 | Which of the following are malignant features of biliary tumours? | Invasion of the basement membrane | 31/32 | A |
Increased nucleus to cytoplasm ratio | 18/31 | C | ||
Distant metastasis | 27/32 | B | ||
Tumorigenic capacity of isolated cells after subcutaneous injection in immunodeficient mice | 29/32 | A | ||
2 | What type of histological investigation(s) should always be done to characterize an early-stage tumour in a preclinical CCA model? | Morphological examination of H&E | 32/32 | U |
Immunohistochemistry | 27/30 | A | ||
Immunohistochemistry for at least one biliary cytokeratin (for example, CK19, CK7, pan-CK, etc.) | 16/25 | C | ||
Markers for inflammatory cells and CAFs | 12/26 | D | ||
PAS reaction for highlighting mucin | 13/26 | C | ||
A broad panel of markers for hepatobiliary malignancies and metastasis | 12/24 | C | ||
3 | To allow correlation with the anatomical classification of human tumours, a preclinical model of CCA should specifically classify tumours induced as: | Intrahepatic CCA, perihilar CCA and distal CCA | 25/30 | B |
Intrahepatic CCA and extrahepatic CCA | 12/25 | D | ||
No need for such classification | 1/23 | D | ||
4 | Which of the following morphological and/or immunophenotypic features must be present to classify a lesion as CCA in a preclinical model? | Location within the liver or extrahepatic biliary tree | 24/28 | B |
Absence of an extrahepatic bile duct primary lesion | 14/28 | C | ||
Epithelial cytological features (cohesive groups or structures and/or pan-CK immunopositivity) | 25/28 | B | ||
At least focal gland formation | 9/25 | D | ||
Absence of hepatocellular differentiation (bile production and canalicular CD10 or BSEP) | 14/24 | D | ||
Immunopositivity for CK7 or CK19 | 31/31 | U | ||
Focal desmoplastic stroma | 22/30 | B | ||
Presence of precursor lesions | 4/24 | D | ||
Primary origin within the intrahepatic or extrahepatic biliary tree | 19/28 | D | ||
Absence of primary hepatobiliary lesions | 0/28 | U | ||
5 | What histopathological features of human CCA must be verified in a preclinical model of CCA? | Intratumoural heterogeneity (high stroma, inflammatory response, epithelial phenotype) | 27/30 | A |
Intertumoural heterogeneity (large versus small bile duct tumour in intrahepatic CCA) | 20/26 | B | ||
Growth pattern (mass-forming, periductal infiltration, intraductal growth) | 25/28 | A | ||
Proportion of tumour showing gland formation | 17/25 | C | ||
Immunopositivity for CK7 or CK19 | 32/32 | U | ||
Focal desmoplastic stroma | 26/30 | B | ||
Presence of precursor lesions | 16/24 | C | ||
6 | It has been proposed that intrahepatic CCA may originate from several cells of origin. Which of the following cell types may be the cells-of-origin for intrahepatic CCA? | Mature hepatocytes | 27/32 | B |
Mature cholangiocytes | 23/32 | B | ||
Hepatic progenitor/oval cells | 32/33 | A | ||
Peribiliary glands | 29/30 | A | ||
In vivo models: xenograft models, genetically engineered mouse models (GEMM) | ||||
7 | Concerning newly developed patient-derived xenograft models | Should the model(s) be validated by an expert pathologist, and the histology of the tumour shown in publications? | 37/37 | U |
Should immune profiling also be reported? | 20/31 | C | ||
Should the model(s) be validated in more than one mouse strain? | 8/34 | D | ||
Should the expert pathologist specify what type of CCA is found in the model? | 33/36 | A | ||
Do orthotopic xenograft models represent the most disease-relevant tumour environment in which to test a drug, compared to ectopic xenograft models? | 27/35 | B | ||
Should a drug be tested in more than one model? | 35/37 | A | ||
In vitro models: 2D culture models | ||||
8 | Which cell culture procedures should be standardized in experiments with cell lines or primary 2D cultures and be reported in publications? | Choice of plastic support (for example, TPP, Falcon, Corning, +/− ECM layer, etc.) | 30/34 | B |
Choice of cell culture medium | 29/34 | B | ||
Level of confluence when performing the experiments | 27/33 | B | ||
Isolation protocol for culture of primary cells | 31/35 | B | ||
Passaging and subculturing methods (for example, enzymatic versus mechanical dissociation, etc.) | 29/34 | B | ||
9 | The origin of any cell line (previously established or new) should be stated for publication according to the new CCA classification (that is, intrahepatic, perihilar, distal) | NA | 37/38 | A |
In vitro models: 3D cultures | ||||
10 | Contaminating non-tumour organoids often grow in CCA organoid cultures. How should selection for tumour organoids be performed? | Specific tumour ‘enrichment’ medium (that is, tumour initiating medium, as described by Broutier et al. (2017)7) | 29/31 | A |
Hand-picking of organoids with a different phenotype/removing the ‘normal-looking’ organoids | 21/30 | B | ||
Xenotransplantation in mice to select for tumour clones | 22/30 | B | ||
11 | Which analyses should be done to confirm the malignant origin of established organoid lines and be reported in publications? | Full genomic profiling | 8/28 | D |
Mutation analysis (targeted genomic profiling using a diagnostic panel) | 28/31 | A | ||
Phenotypic analysis | 28/30 | A | ||
Histological analysis (immunohistochemistry of EpCAM, CK7) | 28/32 | B | ||
Xenotransplantation in mice | 26/32 | B | ||
12 | Should every organoid culture be characterized (as proposed in question 11) before clinical applications such as drug screening? | NA | 33/36 | A |
13 | Personalized medicine applications, such as drug screenings to find the best treatment for the patient, will cost time. How much time is acceptable to initiate, grow and expand the organoids for these analyses? In other words, what is the maximum time acceptable to be relevant to the clinics? | <1 month | 9/35 | D |
<3 months | 20/35 | C | ||
<6 months | 4/35 | D | ||
Other; as short as possible/<1 month first-line treatment and <3 months second-line treatment | 2/35 | D | ||
