Molecular characterization of organoids derived from pancreatic intraductal papillary mucinous neoplasms

Abstract

Intraductal papillary mucinous neoplasms (IPMNs) are commonly identified non-invasive cyst-forming pancreatic neoplasms with the potential to progress into invasive pancreatic adenocarcinoma. There are few in vitro models with which to study the biology of IPMNs and their progression to invasive carcinoma. Therefore, we generated a living biobank of organoids from seven normal pancreatic ducts and ten IPMNs. We characterized eight IPMN organoid samples using whole genome sequencing and characterized five IPMN organoids and seven normal pancreatic duct organoids using transcriptome sequencing. We identified an average of 11,344 somatic mutations in the genomes of organoids derived from IPMNs, with one sample harboring 61,537 somatic mutations enriched for T!C transitions and T!A transversions. Recurrent coding somatic mutations were identified in 15 genes, including KRAS, GNAS, RNF43, PHF3, and RBM10. The most frequently mutated genes were KRAS, GNAS, and RNF43, with somatic mutations identified in six (75%), four (50%), and three (37.5%) IPMN organoid samples, respectively. On average, we identified 36 structural variants in IPMN derived organoids, and none had an unstable phenotype (> 200 structural variants). Transcriptome sequencing identified 28 genes differentially expressed between normal pancreatic duct organoid and IPMN organoid samples. The most significantly upregulated and downregulated genes were CLDN18 and FOXA1. Immunohistochemical analysis of FOXA1 expression in 112 IPMNs, 113 mucinous cystic neoplasms, and 145 pancreatic ductal adenocarcinomas demonstrated statistically significant loss of expression in lowgrade IPMNs (p < 0.0016), mucinous cystic neoplasms (p < 0.0001), and pancreatic ductal adenocarcinoma of any histologic grade (p < 0.0001) compared to normal pancreatic ducts. These data indicate that FOXA1 loss of expression occurs early in pancreatic tumorigenesis. Our study highlights the utility of organoid culture to study the genetics and biology of normal pancreatic duct and IPMNs.

Post‐translational Wnt receptor regulation: Is the fog slowly clearing?

“…Several reports revealed potential genetic cooperation of somatic mutations in RNF43 and KRAS in pancreatic neoplasms. [ 57,89 ] This genetic cooperation is also observed in pancreatic cancer cell lines (Table 1). While, in MSS‐CRC, concurrent somatic mutations are observed in APC , KRAS, SMAD4 , and TP53 , supporting Bert Vogelstein's multi‐step tumorigenesis model, MSI‐high CRC commonly shows gene mutations in BRAF and RNF43 .…”

Historical evolution of spheroids and organoids, and possibilities of use in life sciences and medicine

“…[95] 3D pancreatic cultures are usually generated from small amounts of biopsy samples, can be long-term expanded, and can be used for additional testing in case of patient-specific culture, very helpful for pathological cases, including cancer. [96] There is hope that they will soon allow replacement cell therapy for endocrine pancreatic disfunctions, like diabetes mellitus. [97]…”

Organoids in cancer research: a review for pathologist‐scientists

“…Organoids represent an efficient culture approach to expand and enrich neoplastic cellularity of limited human samples to obtain adequate material for molecular analysis. For example, organoids were recently used to generate material for whole genome sequencing and RNA sequencing from small pieces of human intraductal papillary mucinous neoplasms of the pancreas, a premalignant lesion for which limited material can be harvested for research investigations [39]. The use of expanded organoids to provide material for molecular analysis has utility not only in the research space but also potentially in molecular pathology.…”

“…In addition, because of the maintenance of cellular connections and typical growth factor enriched media, organoid culture is an efficient tool that allows growth of cell types that have been historically challenging to culture using traditional 2D approaches. These approaches have allowed culture of normal cells, including normal human airway [40], normal mouse and human liver [41], and normal human pancreatic duct [39]. In addition, premalignant neoplasms, which mostly lack traditional 2D culture models, have also been efficiently cultured as organoids, for example human pancreatic intraductal papillary mucinous neoplasm [39,42].…”