Immunoevolution of mouse pancreatic organoid isografts from preinvasive to metastatic disease

Pancreatic ductal adenocarcinoma (PDA) has a highly immunosuppressive microenvironment, which is contributed by the complex interaction between cancer cells and a heterogeneous population of stromal cells. Therefore, facile and trackable models are needed for integrative and dynamic interrogation of cancer-stroma interaction. Here, we tracked the immunoevolution of PDA in a genetically-defined transplantable model of mouse pancreatic tumour organoids that recapitulates the progression of the disease from early preinvasive lesions to metastatic carcinomas. We demonstrated that organoid-derived isografts (ODI) can be used as a biological source of biomarkers (NT5E, TGFB1, FN1, and ITGA5) of aggressive molecular subtypes of human PDA. In ODI, infiltration from leukocytes is an early event during progression of the disease as observed for autochthonous models. Neoplastic progression was associated to accumulation of Maf+ macrophages, which inversely correlated with CD8+ T cells infiltration. Consistently, levels of MAF were enriched in human PDA subtypes characterized by abundance of macrophage-related transcripts and indicated poor patients' survival. Density of MAF+ macrophages was higher in human PDA tissues compared to preinvasive lesions. Our results suggest that ODIs represent a suitable system for genotypic-immunophenotypic studies and support the hypothesis of MAF+ macrophages as a prominent immunosuppressive population in PDA

Genetic alterations analysis in prognostic stratified groups identified TP53 and ARID1A as poor clinical performance markers in intrahepatic cholangiocarcinoma

The incidence and mortality rates of intrahepatic cholangiocarcinoma have been rising worldwide. Few patients present an early-stage disease that is amenable to curative surgery and after resection, high recurrence rates persist. To identify new independent marker related to aggressive behaviour, two prognostic groups of patient were selected and divided according to prognostic performance. All patients alive at 36 months were included in good prognostic performers, while all patients died due to disease within 36 months in poor prognostic performers. Using high-coverage target sequencing we analysed principal genetic alterations in two groups and compared results to clinical data. In the 33 cases included in poor prognosis group, TP53 was most mutated gene (p\u2009=\u20090.011) and exclusively present in these cases. Similarly, ARID1A was exclusive of this group (p\u2009=\u20090.024). TP53 and ARID1A are mutually exclusive in this study. Statistical analysis showed mutations in TP53 and ARID1A genes and amplification of MET gene as independent predictors of poor prognosis (TP53, p\u2009=\u20090.0031, ARID1A, p\u2009=\u20090.0007, MET, p\u2009=\u20090.0003 in Cox analysis). LOH in PTEN was also identified as marker of disease recurrence (p\u2009=\u20090.04) in univariate analysis. This work improves our understanding of aggressiveness related to this tumour type and has identified novel prognostic markers of clinical outcome

Long-term organoid culture of a small intestinal neuroendocrine tumor

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare and highly heterogeneous neoplasms whose incidence has markedly increased over the last decades. A grading system based on the tumor cells’ proliferation index predicts high-risk for G3 NETs. However, low-to-intermediate grade (G1/G2) NETs have an unpredictable clinical course that varies from indolent to highly malignant. Cultures of human cancer cells enable to perform functional perturbation analyses that are instrumental to enhance our understanding of cancer biology. To date, no tractable and reliable long-term culture of G1/G2 NET has been reported to permit disease modeling and pharmacological screens. Here, we report of the first long-term culture of a G2 metastatic small intestinal NET that preserves the main genetic drivers of the tumor and retains expression patterns of the endocrine cell lineage. Replicating the tissue, this long-term culture showed a low proliferation index, and yet it could be propagated continuously without dramatic changes in the karyotype. The model was readily available for pharmacological screens using targeted agents and as expected, showed low tumorigenic capacity in vivo. Overall, this is the first long-term culture of NETs to faithfully recapitulate many aspects of the original neuroendocrine tumor

Loss of FGFR4 promotes the malignant phenotype of PDAC

Transcriptomic analyses of pancreatic ductal adenocarcinoma (PDAC) have identified two major epithelial subtypes with distinct biology and clinical behaviours. Here, we aimed to clarify the role of FGFR1 and FGFR4 in the definition of aggressive PDAC phenotypes. We found that the expression of FGFR4 is exclusively detected in epithelial cells, significantly elevated in the classical PDAC subtype, and associates with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligns with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Conversely, FGFR1 has more promiscuous expression in both normal and malignant pancreatic tissues and is strongly associated with the EMT phenotype but not with the basal-like cell lineage. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlates with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably leads to the enrichment of basal-like/squamous gene programs and is associated with either partial or full switch of phenotype. In sum, we show that endogenous levels of FGFR4 limit the malignant phenotype of PDAC cells. Finally, we propose FGFR4 as a valuable marker for the stratification of PDAC patients

DNA methylation patterns identify subgroups of pancreatic neuroendocrine tumors with clinical association

Here we report the DNA methylation profile of 84 sporadic pancreatic neuroendocrine tumors (PanNETs) with associated clinical and genomic information. We identified three subgroups of PanNETs, termed T1, T2 and T3, with distinct patterns of methylation. The T1 subgroup was enriched for functional tumors and ATRX, DAXX and MEN1 wild-type genotypes. The T2 subgroup contained tumors with mutations in ATRX, DAXX and MEN1 and recurrent patterns of chromosomal losses in half of the genome with no association between regions with recurrent loss and methylation levels. T2 tumors were larger and had lower methylation in the MGMT gene body, which showed positive correlation with gene expression. The T3 subgroup harboured mutations in MEN1 with recurrent loss of chromosome 11, was enriched for grade G1 tumors and showed histological parameters associated with better prognosis. Our results suggest a role for methylation in both driving tumorigenesis and potentially stratifying prognosis in PanNETs

DNA methylation patterns identify subgroups of pancreatic neuroendocrine tumors with clinical association

Here we report the DNA methylation profile of 84 sporadic pancreatic neuroendocrine tumors (PanNETs) with associated clinical and genomic information. We identified three subgroups of PanNETs, termed T1, T2 and T3, with distinct patterns of methylation. The T1 subgroup was enriched for functional tumors and ATRX, DAXX and MEN1 wild-type genotypes. The T2 subgroup contained tumors with mutations in ATRX, DAXX and MEN1 and recurrent patterns of chromosomal losses in half of the genome with no association between regions with recurrent loss and methylation levels. T2 tumors were larger and had lower methylation in the MGMT gene body, which showed positive correlation with gene expression. The T3 subgroup harboured mutations in MEN1 with recurrent loss of chromosome 11, was enriched for grade G1 tumors and showed histological parameters associated with better prognosis. Our results suggest a role for methylation in both driving tumorigenesis and potentially stratifying prognosis in PanNETs

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Pathologist's Role in the Management of Gastroenteropancreatic Neuroendocrine Tumors (GEP-NETs)

The introduction of effective treatments into clinical practice has opened new horizons for planning tailored surgical or medical management of gastroenteropancreatic neuroendocrine tumor patients. The choice of the optimal therapeutic option strictly relies on complete histopathological diagnosis that comprehends 3 essential informations: (1) classification of the neoplasm, (2) evaluation of its biological aggressiveness, and (3) assessment of the extent of the disease. Recently developed classification, grading, and staging systems by World Health Organization, European Neuroendocrine Tumor Society, and Union for International Cancer Control have furnished appropriate tools for standardized neuroendocrine tumors diagnostic and prognostic stratification, and are the mainstays in addressing patient-specific therapeutical choices

A rare case of three different tumors in the same pancreatic specimen: a case report and brief review of the literature

Solid pseudopapillary tumors (SPT) of the pancreas are rare neoplasms mainly affecting young women. Pancreatic serous cystadenomas (SCAs) and pancreatic neuroendocrine tumors (PanNETs) account for about 2% of all pancreatic neoplasms. The combination of these three lesions, to our knowledge, has never been described in literature. Here we report a case of combined SPT, SCA and PanNET affecting a 33-year-old woman