Identification of spatially-resolved markers of malignant transformation in Intraductal Papillary Mucinous Neoplasms.

The existing Intraductal Papillary Mucinous Neoplasm (IPMN) risk stratification relies on clinical and histological factors, resulting in inaccuracies and leading to suboptimal treatment. This is due to the lack of appropriate molecular markers that can guide patients toward the best therapeutic options. Here, we assess and confirm subtype-specific markers for IPMN across two independent cohorts of patients using two Spatial Transcriptomics (ST) technologies. Specifically, we identify HOXB3 and ZNF117 as markers for Low-Grade Dysplasia, SPDEF and gastric neck cell markers in borderline cases, and NKX6-2 and gastric isthmus cell markers in High-Grade-Dysplasia Gastric IPMN, highlighting the role of TNFα and MYC activation in IPMN progression and the role of NKX6-2 in the specific Gastric IPMN progression. In conclusion, our work provides a step forward in understanding the gene expression landscapes of IPMN and the critical transcriptional networks related to PDAC progression.

Unveiling the mechanistic link between extracellular amyloid fibrils, mechano-signaling and YAP activation in cancer.

The tumor microenvironment is a complex ecosystem that plays a critical role in cancer progression and treatment response. Recently, extracellular amyloid fibrils have emerged as novel components of the tumor microenvironment; however, their function remains elusive. In this study, we establish a direct connection between the presence of amyloid fibrils in the secretome and the activation of YAP, a transcriptional co-activator involved in cancer proliferation and drug resistance. Furthermore, we uncover a shared mechano-signaling mechanism triggered by amyloid fibrils in both melanoma and pancreatic ductal adenocarcinoma cells. Our findings highlight the crucial role of the glycocalyx protein Agrin which binds to extracellular amyloid fibrils and acts as a necessary factor in driving amyloid-dependent YAP activation. Additionally, we reveal the involvement of the HIPPO pathway core kinase LATS1 in this signaling cascade. Finally, we demonstrate that extracellular amyloid fibrils enhance cancer cell migration and invasion. In conclusion, our research expands our knowledge of the tumor microenvironment by uncovering the role of extracellular amyloid fibrils in driving mechano-signaling and YAP activation. This knowledge opens up new avenues for developing innovative strategies to modulate YAP activation and mitigate its detrimental effects during cancer progression.

IL-1ß+ macrophages fuel pathogenic inflammation in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with high resistance to therapies1. Inflammatory and immunomodulatory signals co-exist in the pancreatic tumour microenvironment, leading to dysregulated repair and cytotoxic responses. Tumour-associated macrophages (TAMs) have key roles in PDAC2, but their diversity has prevented therapeutic exploitation. Here we combined single-cell and spatial genomics with functional experiments to unravel macrophage functions in pancreatic cancer. We uncovered an inflammatory loop between tumour cells and interleukin-1ß (IL-1ß)-expressing TAMs, a subset of macrophages elicited by a local synergy between prostaglandin E2 (PGE2) and tumour necrosis factor (TNF). Physical proximity with IL-1ß+ TAMs was associated with inflammatory reprogramming and acquisition of pathogenic properties by a subset of PDAC cells. This occurrence was an early event in pancreatic tumorigenesis and led to persistent transcriptional changes associated with disease progression and poor outcomes for patients. Blocking PGE2 or IL-1ß activity elicited TAM reprogramming and antagonized tumour cell-intrinsic and -extrinsic inflammation, leading to PDAC control in vivo. Targeting the PGE2-IL-1ß axis may enable preventive or therapeutic strategies for reprogramming of immune dynamics in pancreatic cancer.

Talniflumate abrogates mucin immune suppressive barrier improving efficacy of gemcitabine and nab-paclitaxel treatment in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease. This is due to its aggressive course, late diagnosis and its intrinsic drugs resistance. The complexity of the tumor, in terms of cell components and heterogeneity, has led to the approval of few therapies with limited efficacy. The study of the early stages of carcinogenesis provides the opportunity for the identification of actionable pathways that underpin therapeutic resistance. METHODS: We analyzed 43 Intraductal papillary mucinous neoplasms (IPMN) (12 Low-grade and 31 High-grade) by Spatial Transcriptomics. Mouse and human pancreatic cancer organoids and T cells interaction platforms were established to test the role of mucins expression on T cells activity. Syngeneic mouse model of PDAC was used to explore the impact of mucins downregulation on standard therapy efficacy. RESULTS: Spatial transcriptomics showed that mucin O-glycosylation pathway is increased in the progression from low-grade to high-grade IPMN. We identified GCNT3, a master regulator of mucins expression, as an actionable target of this pathway by talniflumate. We showed that talniflumate impaired mucins expression increasing T cell activation and recognition using both mouse and human organoid interaction platforms. In vivo experiments showed that talniflumate was able to increase the efficacy of the chemotherapy by boosting immune infiltration. CONCLUSIONS: Finally, we demonstrated that combination of talniflumate, an anti-inflammatory drug, with chemotherapy effectively improves anti-tumor effect in PDAC.

Microfluidics and Organoids, the Power Couple of Developmental Biology and Oncology Studies.

Organoids are an advanced cell model that hold the key to unlocking a deeper understanding of in vivo cellular processes. This model can be used in understanding organ development, disease progression, and treatment efficacy. As the scientific world embraces the model, it must also establish the best practices for cultivating organoids and utilizing them to the greatest potential in assays. Microfluidic devices are emerging as a solution to overcome the challenges of organoids and adapt assays. Unfortunately, the various applications of organoids often depend on specific features in a device. In this review, we discuss the options and considerations for features and materials depending on the application and development of the organoid.

Clinical and Genomic Characterization of Pancreatic Ductal Adenocarcinoma with Signet-Ring/Poorly Cohesive Cells.

Signet-ring cell (SRC)/poorly cohesive cell carcinoma is an aggressive variant of pancreatic ductal adenocarcinoma (PDAC). This study aimed to clarify its clinicopathologic and molecular profiles based on a multi-institutional cohort of 20 cases. The molecular profiles were investigated using DNA and RNA sequencing. The clinicopathologic parameters and molecular alterations were analyzed based on survival indices and using a validation/comparative cohort of 480 conventional PDAC patients. The primary findings were as follows: (1) clinicopathologic features: SRC carcinomas are highly aggressive neoplasms with poor prognosis, and the lungs are elective metastatic sites; (2) survival analysis: a higher SRC component was indicative of poorer prognosis. In particular, the most clinically significant threshold of SRC was 80%, showing statistically significant differences in both disease-specific and disease-free survival; (3) genomic profiles: SRC carcinomas are similar to conventional PDAC with the most common alterations affecting the classic PDAC drivers KRAS (70% of cases), TP53 (55%), SMAD4 (25%), and CDKN2A (20%). EGFR alterations, RET::CCDC6 fusion gene, and microsatellite instability (3 different cases, 1 alteration per case) represent novel targets for precision oncology. The occurrence of SMAD4 mutations was associated with poorer prognosis; (4) pancreatic SRC carcinomas are genetically different from gastric SRC carcinomas: CDH1, the classic driver gene of gastric SRC carcinoma, is not altered in pancreatic SRC carcinoma; (5) transcriptome analysis: the cases clustered into 2 groups, one classical/exocrine-like, and the other squamous-like; and (6) SRC carcinoma-derived organoids can be successfully generated, and their cultures preserve the histologic and molecular features of parental SRC carcinoma. Although pancreatic SRC carcinoma shares similarities with conventional PDAC regarding the most important genetic drivers, it also exhibits important differences. A personalized approach for patients with this tumor type should consider the clinical relevance of histologic determination of the SRC component and the presence of potentially actionable molecular targets.

The tumor stroma influences immune cell distribution and recruitment in a PDAC-on-a-chip model.

The dense tumor stroma of pancreatic ductal adenocarcinoma (PDAC) and its secreted immune active molecules provide a barrier for chemotherapy treatment as well as for immune cell infiltration to the tumor core, providing a challenge for immunotherapeutic strategies. Consequently, the investigation of processes underlying the interaction between the tumor stroma, particularly activated pancreatic stellate cells (PSCs), and immune cells may offer new therapeutic approaches for PDAC treatment. In this study, we established a 3D PDAC model cultured under flow, consisting of an endothelial tube, PSCs and PDAC organoids. This was applied to study the role of the tumor microenvironment (TME) on immune cell recruitment and its effect on partly preventing their interaction with pancreatic cancer cells. We observed that stromal cells form a physical barrier, partly shielding the cancer cells from migrating immune cells, as well as a biochemical microenvironment, that seems to attract and influence immune cell distribution. In addition, stromal targeting by Halofuginone led to an increase in immune cell infiltration. We propose that the here developed model setups will support the understanding of the cellular interplay influencing the recruitment and distribution of immune cells, and contribute to the identification of key players in the PDAC immunosuppressive TME as well as support the discovery of new strategies to treat this immune unresponsive tumor.

Overcoming therapeutic resistance in pancreatic cancer: Emerging opportunities by targeting BRCAs and p53.

Pancreatic cancer (PC) is characterized by (epi)genetic and microenvironmental alterations that negatively impact the treatment outcomes. New targeted therapies have been pursued to counteract the therapeutic resistance in PC. Aiming to seek for new therapeutic options for PC, several attempts have been undertaken to exploit BRCA1/2 and TP53 deficiencies as promising actionable targets. The elucidation of the pathogenesis of PC highlighted the high prevalence of p53 mutations and their connection with the aggressiveness and therapeutic resistance of PC. Additionally, PC is associated with dysfunctions in several DNA repair-related genes, including BRCA1/2, which sensitize tumours to DNA-damaging agents. In this context, poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) were approved for mutant BRCA1/2 PC patients. However, acquired drug resistance has become a major drawback of PARPi. This review emphasizes the importance of targeting defective BRCAs and p53 pathways for advancing personalized PC therapy, with particular focus on how this approach may provide an opportunity to tackle PC resistance.

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.

LAMC2 Regulates Key Transcriptional and Targetable Effectors to Support Pancreatic Cancer Growth.

PURPOSE: The identification of pancreatic ductal adenocarcinoma (PDAC) dysregulated genes may unveil novel molecular targets entering inhibitory strategies. Laminins are emerging as potential targets in PDAC given their role as diagnostic and prognostic markers. Here, we investigated the cellular, functional, and clinical relevance of LAMC2 and its regulated network, with the ultimate goal of identifying potential therapies. EXPERIMENTAL DESIGN: LAMC2 expression was analyzed in PDAC tissues, a panel of human and mouse cell lines, and a genetically engineered mouse model. Genetic perturbation in 2D, 3D, and in vivo allograft and xenograft models was done. Expression profiling of a LAMC2 network was performed by RNA-sequencing, and publicly available gene expression datasets from experimental and clinical studies examined to query its human relevance. Dual inhibition of pharmacologically targetable LAMC2-regulated effectors was investigated. RESULTS: LAMC2 was consistently upregulated in human and mouse experimental models as well as in human PDAC specimens, and associated with tumor grade and survival. LAMC2 inhibition impaired cell cycle, induced apoptosis, and sensitized PDAC to MEK1/2 inhibitors (MEK1/2i). A LAMC2-regulated network was featured in PDAC, including both classical and quasi-mesenchymal subtypes, and contained downstream effectors transcriptionally shared by the KRAS signaling pathway. LAMC2 regulated a functional FOSL1-AXL axis via AKT phosphorylation. Furthermore, genetic LAMC2 or pharmacological AXL inhibition elicited a synergistic antiproliferative effect in combination with MEK1/2is that was consistent across 2D and 3D human and mouse PDAC models, including primary patient-derived organoids. CONCLUSIONS: LAMC2 is a molecular target in PDAC that regulates a transcriptional network that unveils a dual drug combination for cancer treatment.

Lysosomal lipid switch sensitises to nutrient deprivation and mTOR targeting in pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with limited therapeutic options. However, metabolic adaptation to the harsh PDAC environment can expose liabilities useful for therapy. Targeting the key metabolic regulator mechanistic target of rapamycin complex 1 (mTORC1) and its downstream pathway shows efficacy only in subsets of patients but gene modifiers maximising response remain to be identified. DESIGN: Three independent cohorts of PDAC patients were studied to correlate PI3K-C2γ protein abundance with disease outcome. Mechanisms were then studied in mouse (KPC mice) and cellular models of PDAC, in presence or absence of PI3K-C2γ (WT or KO). PI3K-C2γ-dependent metabolic rewiring and its impact on mTORC1 regulation were assessed in conditions of limiting glutamine availability. Finally, effects of a combination therapy targeting mTORC1 and glutamine metabolism were studied in WT and KO PDAC cells and preclinical models. RESULTS: PI3K-C2γ expression was reduced in about 30% of PDAC cases and was associated with an aggressive phenotype. Similarly, loss of PI3K-C2γ in KPC mice enhanced tumour development and progression. The increased aggressiveness of tumours lacking PI3K-C2γ correlated with hyperactivation of mTORC1 pathway and glutamine metabolism rewiring to support lipid synthesis. PI3K-C2γ-KO tumours failed to adapt to metabolic stress induced by glutamine depletion, resulting in cell death. CONCLUSION: Loss of PI3K-C2γ prevents mTOR inactivation and triggers tumour vulnerability to RAD001 (mTOR inhibitor) and BPTES/CB-839 (glutaminase inhibitors). Therefore, these results might open the way to personalised treatments in PDAC with PI3K-C2γ loss.

CXCR1/2 dual-inhibitor ladarixin reduces tumour burden and promotes immunotherapy response in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with few therapeutic options available. Despite immunotherapy has revolutionised cancer treatment, the results obtained in PDAC are still disappointing. Emerging evidence suggests that chemokines/CXCRs-axis plays a pivotal role in immune tumour microenvironment modulation, which may influence immunotherapy responsiveness. Here, we evaluated the effectiveness of CXCR1/2 inhibitor ladarixin, alone or in combination with anti-PD-1, against immunosuppression in PDAC. METHODS: A set of preclinical models was obtained by engrafting mouse PDAC-derived cells into syngeneic immune-competent mice, as well as by orthotopically transplanting patient-derived PDAC tumour into human immune-system-reconstituted (HIR) mice (HuCD34-NSG-mice). Tumour-bearing mice were randomly assigned to receive vehicles, ladarixin, anti-PD-1 or drugs combination. RESULTS: CXCR1/2 inhibition by ladarixin reverted in vitro tumour-mediated M2 macrophages polarisation and migration. Ladarixin as single agent reduced tumour burden in cancer-derived graft (CDG) models with high-immunogenic potential and increased the efficacy of ICI in non-immunogenic CDG-resistant models. In a HIR mouse model bearing the immunogenic subtype of human PDAC, ladarixin showed high efficacy increasing the antitumor effect of anti-PD-1. CONCLUSION: Ladarixin in combination with anti-PD-1 might represent an extremely effective approach for the treatment of immunotherapy refractory PDAC, allowing pro-tumoral to immune-permissive microenvironment conversion.

Integrative characterization of intraductal tubulopapillary neoplasm (ITPN) of the pancreas and associated invasive adenocarcinoma.

Pancreatic intraductal tubulopapillary neoplasm (ITPN) is a recently recognized intraductal neoplasm. This study aimed to clarify the clinicopathologic and molecular features of this entity, based on a multi-institutional cohort of 16 pancreatic ITPNs and associated adenocarcinomas. The genomic profiles were analyzed using histology-driven multi-regional sequencing to provide insight on tumor heterogeneity and evolution. Furthermore, an exploratory transcriptomic characterization was performed on eight invasive adenocarcinomas. The clinicopathologic parameters and molecular alterations were further analyzed based on survival indices. The main findings were as follows: 1) the concomitant adenocarcinomas, present in 75% of cases, were always molecularly associated with the intraductal components. These data definitively establish ITPN as origin of invasive pancreatic adenocarcinoma; 2) alterations restricted to infiltrative components included mutations in chromatin remodeling genes ARID2, ASXL1, and PBRM1, and ERBB2-P3H4 fusion; 3) pancreatic ITPN can arise in the context of genetic syndromes, such as BRCA-germline and Peutz-Jeghers syndrome; 4) mutational profile: mutations in the classical PDAC drivers are present, but less frequently, in pancreatic ITPN; 5) novel genomic alterations were observed, including amplification of the Cyclin and NOTCH family genes and ERBB2, fusions involving RET and ERBB2, and RB1 disruptive variation; 6) chromosomal alterations: the most common was 1q gain (75% of cases); 7) by transcriptome analysis, ITPN-associated adenocarcinomas clustered into three subtypes that correlate with the activation of signaling mechanism pathways and tumor microenvironment, displaying squamous features in their majority; and 8) TP53 mutational status is a marker for adverse prognosis. ITPNs are precursor lesions of pancreatic cancer with a high malignant transformation risk. A personalized approach for patients with ITPN should recognize that such neoplasms could arise in the context of genetic syndromes. BRCA alterations, ERBB2 and RET fusions, and ERBB2 amplification are novel targets in precision oncology. The TP53 mutation status can be used as a prognostic biomarker.

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.

Germline Aberrations in Pancreatic Cancer: Implications for Clinical Care.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis and represents a major public health issue, as both its incidence and mortality are expecting to increase steeply over the next years. Effective screening strategies are lacking, and most patients are diagnosed with unresectable disease precluding the only chance of cure. Therapeutic options for advanced disease are limited, and the treatment paradigm is still based on chemotherapy, with a few rare exceptions to targeted therapies. Germline variants in cancer susceptibility genes-particularly those involved in mechanisms of DNA repair-are emerging as promising targets for PDAC treatment and prevention. Hereditary PDAC is part of the spectrum of several syndromic disorders, and germline testing of PDAC patients has relevant implications for broad cancer prevention. Germline aberrations in BRCA1 and BRCA2 genes are predictive biomarkers of response to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor olaparib and platinum-based chemotherapy in PDAC, while mutations in mismatch repair genes identify patients suitable for immune checkpoint inhibitors. This review provides a timely and comprehensive overview of germline aberrations in PDAC and their implications for clinical care. It also discusses the need for optimal approaches to better select patients for PARP inhibitor therapy, novel therapeutic opportunities under clinical investigation, and preclinical models for cancer susceptibility and drug discovery.

Interrupting the nitrosative stress fuels tumor-specific cytotoxic T lymphocytes in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors owing to its robust desmoplasia, low immunogenicity, and recruitment of cancer-conditioned, immunoregulatory myeloid cells. These features strongly limit the success of immunotherapy as a single agent, thereby suggesting the need for the development of a multitargeted approach. The goal is to foster T lymphocyte infiltration within the tumor landscape and neutralize cancer-triggered immune suppression, to enhance the therapeutic effectiveness of immune-based treatments, such as anticancer adoptive cell therapy (ACT). METHODS: We examined the contribution of immunosuppressive myeloid cells expressing arginase 1 and nitric oxide synthase 2 in building up a reactive nitrogen species (RNS)-dependent chemical barrier and shaping the PDAC immune landscape. We examined the impact of pharmacological RNS interference on overcoming the recruitment and immunosuppressive activity of tumor-expanded myeloid cells, which render pancreatic cancers resistant to immunotherapy. RESULTS: PDAC progression is marked by a stepwise infiltration of myeloid cells, which enforces a highly immunosuppressive microenvironment through the uncontrolled metabolism of L-arginine by arginase 1 and inducible nitric oxide synthase activity, resulting in the production of large amounts of reactive oxygen and nitrogen species. The extensive accumulation of myeloid suppressing cells and nitrated tyrosines (nitrotyrosine, N-Ty) establishes an RNS-dependent chemical barrier that impairs tumor infiltration by T lymphocytes and restricts the efficacy of adoptive immunotherapy. A pharmacological treatment with AT38 ([3-(aminocarbonyl)furoxan-4-yl]methyl salicylate) reprograms the tumor microenvironment from protumoral to antitumoral, which supports T lymphocyte entrance within the tumor core and aids the efficacy of ACT with telomerase-specific cytotoxic T lymphocytes. CONCLUSIONS: Tumor microenvironment reprogramming by ablating aberrant RNS production bypasses the current limits of immunotherapy in PDAC by overcoming immune resistance.

p53 mutations define the chromatin landscape to confer drug tolerance in pancreatic cancer.

Somatic inactivation of p53 (TP53) mainly occurs as missense mutations that lead to the acquisition of neomorphic mutant protein forms. p53 mutants have been postulated to exert gain-of-function (GOF) effects, including promotion of metastasis and drug tolerance, which generally contribute to the acquisition of the lethal phenotype. Here, by integrating a p53R270H -dependent transcriptomic analysis with chromatin accessibility (ATAC-seq) profiling, we shed light on the molecular basis of a p53 mutant-dependent drug-tolerant phenotype in pancreatic cancer. p53R270H finely tunes chromatin accessibility in specific genomic loci, orchestrating a transcriptional programme that participates in phenotypic evolution of the cancer. We specifically focused on the p53R270H -dependent regulation of the tyrosine kinase receptor macrophage-stimulating protein receptor (MST1r). MST1r deregulation substantially impinged on drug response in the experimental model, recapitulating the p53R270H -dependent phenotype, and strongly correlated with p53 mutant and aggressive phenotype in pancreatic cancer patients. As cellular plasticity in the final stages of the evolution of pancreatic cancer seems to predominantly originate from epigenetic mechanisms, we propose that mutant p53 participates in the acquisition of a lethal phenotype by fine-tuning the chromatin landscape.

Non-functional pancreatic neuroendocrine tumours: ATRX/DAXX and alternative lengthening of telomeres (ALT) are prognostically independent from ARX/PDX1 expression and tumour size.

OBJECTIVE: Recent studies have found aristaless-related homeobox gene (ARX)/pancreatic and duodenal homeobox 1 (PDX1), alpha-thalassemia/mental retardation X-linked (ATRX)/death domain-associated protein (DAXX) and alternative lengthening of telomeres (ALT) to be promising prognostic biomarkers for non-functional pancreatic neuroendocrine tumours (NF-PanNETs). However, they have not been comprehensively evaluated, especially among small NF-PanNETs (≤2.0 cm). Moreover, their status in neuroendocrine tumours (NETs) from other sites remains unknown. DESIGN: An international cohort of 1322 NETs was evaluated by immunolabelling for ARX/PDX1 and ATRX/DAXX, and telomere-specific fluorescence in situ hybridisation for ALT. This cohort included 561 primary NF-PanNETs, 107 NF-PanNET metastases and 654 primary, non-pancreatic non-functional NETs and NET metastases. The results were correlated with numerous clinicopathological features including relapse-free survival (RFS). RESULTS: ATRX/DAXX loss and ALT were associated with several adverse prognostic findings and distant metastasis/recurrence (p<0.001). The 5-year RFS rates for patients with ATRX/DAXX-negative and ALT-positive NF-PanNETs were 40% and 42% as compared with 85% and 86% for wild-type NF-PanNETs (p<0.001 and p<0.001). Shorter 5-year RFS rates for ≤2.0 cm NF-PanNETs patients were also seen with ATRX/DAXX loss (65% vs 92%, p=0.003) and ALT (60% vs 93%, p<0.001). By multivariate analysis, ATRX/DAXX and ALT status were independent prognostic factors for RFS. Conversely, classifying NF-PanNETs by ARX/PDX1 expression did not independently correlate with RFS. Except for 4% of pulmonary carcinoids, ATRX/DAXX loss and ALT were only identified in primary (25% and 29%) and NF-PanNET metastases (62% and 71%). CONCLUSIONS: ATRX/DAXX and ALT should be considered in the prognostic evaluation of NF-PanNETs including ≤2.0 cm tumours, and are highly specific for pancreatic origin among NET metastases of unknown primary.

Cell Lineage Infidelity in PDAC Progression and Therapy Resistance.

Infidelity to cell fate occurs when differentiated cells lose their original identity and either revert to a more multipotent state or transdifferentiate into a different cell type, either within the same embryonic lineage or in an entirely different one. Whilst in certain circumstances, such as in wound repair, this process is beneficial, it can be hijacked by cancer cells to drive disease initiation and progression. Cell phenotype switching has been shown to also serve as a mechanism of drug resistance in some epithelial cancers. In pancreatic ductal adenocarcinoma (PDAC), the role of lineage infidelity and phenotype switching is still unclear. Two consensus molecular subtypes of PDAC have been proposed that mainly reflect the existence of cell lineages with different degrees of fidelity to pancreatic endodermal precursors. Indeed, the classical subtype of PDAC is characterised by the expression of endodermal lineage specifying transcription factors, while the more aggressive basal-like/squamous subtype is defined by epigenetic downregulation of endodermal genes and alterations in chromatin modifiers. Here, we summarise the current knowledge of mechanisms (genetic and epigenetic) of cell fate switching in PDAC and discuss how pancreatic organoids might help increase our understanding of both cell-intrinsic and cell-extrinsic factors governing lineage infidelity during the distinct phases of PDAC evolution.