International Hepato-Pancreato-Billiary Association (IHPBA) registry study on COVID-19 infections in HPB surgery patients.

BACKGROUND: In response to the pandemic, the International Hepato-Pancreato-Biliary Association (IHPBA) developed the IHPBA-COVID Registry to capture data on HPB surgery outcomes in COVID-positive patients prior to mass vaccination programs. The aim was to provide a tool to help members gain a better understanding of the impact of COVID-19 on patient outcomes following HPB surgery worldwide. METHODS: An online registry updated in real time was disseminated to all IHPBA, E-AHPBA, A-HPBA and A-PHPBA members to assess the effects of the pandemic on the outcomes of HPB procedures, perioperative COVID-19 management and other aspects of surgical care. RESULTS: One hundred twenty-five patients from 35 centres in 18 countries were included. Seventy-three (58%) patients were diagnosed with COVID-19 preoperatively. Operative mortality after pancreaticoduodenectomy and major hepatectomy was 28% and 15%, respectively, and 2.5% after cholecystectomy. Postoperative complication rates of pancreatic procedures, hepatic interventions and biliary interventions were respectively 80%, 50% and 37%. Respiratory complication rates were 37%, 31% and 10%, respectively. CONCLUSION: This study reveals a high risk of mortality and complication after HPB surgeries in patient infected with COVID-19. The more extensive the procedure, the higher the risk. Nonetheless, an increased risk was observed across all types of interventions, suggesting that elective HPB surgery should be avoided in COVID positive patients, delaying it at distance from the viral infection.

Macrophages direct cancer cells through a LOXL2-mediated metastatic cascade in pancreatic ductal adenocarcinoma.

OBJECTIVE: The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated in immunocompetent in vivo PDAC models. DESIGN: Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies, K-Ras +/LSL-G12D;Trp53 LSL-R172H;Pdx1-Cre mice (KPC) and the K-Ras +/LSL-G12D;Pdx1-Cre mice (KC) were crossed with Loxl2 allele floxed mice (Loxl2Exon2 fl/fl) or conditional Loxl2 overexpressing mice (R26Loxl2 KI/KI) to generate KPCL2KO or KCL2KO and KPCL2KI or KCL2KI mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation. RESULTS: Using these PDAC mouse models, we show that while Loxl2 ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling. Loxl2 overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects Osm and Loxl2 expression and collagen fibre alignment. CONCLUSION: Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic disease.

Pancreatobiliary Maljunction-associated Gallbladder Cancer Is as Common in the West, Shows Distinct Clinicopathologic Characteristics and Offers an Invaluable Model for Anatomy-induced Reflux-associated Physio-chemical Carcinogenesis.

OBJECTIVE: To determine the associations of pancreatobiliary maljunction (PBM) in the West. BACKGROUND: PBM (anomalous union of common bile duct and pancreatic duct) is mostly regarded as an Asian-only disorder, with 200X risk of gallbladder cancer (GBc), attributed to reflux of pancreatic enzymes. Methods: Radiologic images of 840 patients in the US who underwent pancreatobiliary resections were reviewed for PBM and contrasted with 171 GBC cases from Japan. RESULTS: Eight % of the US GBCs (24/300) had PBM (similar to Japan; 15/ 171, 8.8%), in addition to 1/42 bile duct carcinomas and 5/33 choledochal cysts. None of the 30 PBM cases from the US had been diagnosed as PBM in the original work-up. PBM was not found in other pancreatobiliary disorders. Clinicopathologic features of the 39 PBM-associated GBCs (US:24, Japan:15) were similar; however, comparison with non-PBM GBCs revealed that they occurred predominantly in females (F/M = 3); at younger (<50-year-old) age (21% vs 6.5% in non-PBM GBCs; P = 0.01); were uncommonly associated with gallstones (14% vs 58%; P < 0.001); had higher rate of tumor-infiltrating lymphocytes (69% vs 44%; P = 0.04); arose more often through adenoma-carcinoma sequence (31% vs 12%; P = 0.02); and had a higher proportion of nonconventional carcinomas (21% vs 7%; P = 0.03). Conclusions: PBM accounts for 8% of GBCs also in the West but is typically undiagnosed. PBM-GBCs tend to manifest in younger age and often through adenoma-carcinoma sequence, leading to unusual carcinoma types. If PBM is encountered, cholecystectomy and surveillance of bile ducts is warranted. PBM-associated GBCs offer an invaluable model for variant anatomy-induced chemical (reflux-related) carcinogenesis.

Clinical Outcomes After Total Pancreatectomy: A Prospective Multicenter Pan-European Snapshot Study.

OBJECTIVE: To assess outcomes among patients undergoing total pancreatectomy (TP) including predictors for complications and in-hospital mortality. BACKGROUND: Current studies on TP mostly originate from high-volume centers and span long time periods and therefore may not reflect daily practice. METHODS: This prospective pan-European snapshot study included patients who underwent elective (primary or completion) TP in 43 centers in 16 European countries (June 2018-June 2019). Subgroup analysis included cutoff values for annual volume of pancreatoduodenectomies (<60 vs ≥60).Predictors for major complications and in-hospital mortality were assessed in multivariable logistic regression. RESULTS: In total, 277 patients underwent TP, mostly for malignant disease (73%). Major postoperative complications occurred in 70 patients (25%). Median hospital stay was 12 days (IQR 9-18) and 40 patients were readmitted (15%). In-hospital mortality was 5% and 90-day mortality 8%. In the subgroup analysis, in-hospital mortality was lower in patients operated in centers with ≥60 pancreatoduodenectomies compared <60 (4% vs 10%, P = 0.046). In multivariable analysis, annual volume <60 pancreatoduodenectomies (OR 3.78, 95% CI 1.18-12.16, P = 0.026), age (OR 1.07, 95% CI 1.01-1.14, P = 0.046), and estimated blood loss ≥2L (OR 11.89, 95% CI 2.64-53.61, P = 0.001) were associated with in-hospital mortality. ASA ≥3 (OR 2.87, 95% CI 1.56-5.26, P = 0.001) and estimated blood loss ≥2L (OR 3.52, 95% CI 1.25-9.90, P = 0.017) were associated with major complications. CONCLUSION: This pan-European prospective snapshot study found a 5% inhospital mortality after TP. The identified predictors for mortality, including low-volume centers, age, and increased blood loss, may be used to improve outcomes.

Infiltration pattern predicts metastasis and progression better than the T-stage and grade in pancreatic neuroendocrine tumors: a proposal for a novel infiltration-based morphologic grading.

The advancing edge profile is a powerful determinant of tumor behavior in many organs. In this study, a grading system assessing the tumor-host interface was developed and tested in 181 pancreatic neuroendocrine tumors (PanNETs), 63 of which were <=2 cm. Three tumor slides representative of the spectrum (least, medium, and most) of invasiveness at the advancing edge of the tumor were selected, and then each slide was scored as follows. Well-demarcated/encapsulated, 1 point; Mildly irregular borders and/or minimal infiltration into adjacent tissue, 2 points; Infiltrative edges with several clusters beyond the main tumor but still relatively close, and/or satellite demarcated nodules, 3 points; No demarcation, several cellular clusters away from the tumor, 4 points; Exuberantly infiltrative pattern, scirrhous growth, dissecting the normal parenchymal elements, 5 points. The sum of the rankings on the three slides was obtained. Cases with scores of 3-6 were defined as "non/minimally infiltrative" (NI; n = 77), 7-9 as "moderately infiltrative" (MI; n = 68), and 10-15 as "highly infiltrative" (HI; n = 36). In addition to showing a statistically significant correlation with all the established signs of aggressiveness (grade, size, T-stage), this grading system was found to be the most significant predictor of adverse outcomes (metastasis, progression, and death) on multivariate analysis, more strongly than T-stage, while Ki-67 index did not stand the multivariate test. As importantly, cases <=2 cm were also stratified by this grading system rendering it applicable also to this group that is currently placed in "watchful waiting" protocols. In conclusion, the proposed grading system has a strong, independent prognostic value and therefore should be considered for integration into routine pathology practice after being evaluated in validation studies with larger series.

Is elective cancer surgery feasible during the lock-down period of the COVID-19 pandemic? Analysis of a single institutional experience of 404 consecutive patients.

BACKGROUND: We aimed to assess the feasibility and short-term clinical outcomes of surgical procedures for cancer at an institution using a coronavirus disease 2019 (COVID-19)-free surgical pathway during the peak phase of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. MATERIALS AND METHODS: This was a single-center study, including cancer patients from all surgical departments, who underwent elective surgical procedures during the first peak phase between March 10 and June 30, 2020. The primary outcomes were the rate of postoperative SARS-CoV-2 infection and 30-day pulmonary or non-pulmonary related morbidity and mortality associated with SARS-CoV-2 disease. RESULTS: Four hundred and four cancer patients fulfilling inclusion criteria were analyzed. The rate of patients who underwent open and minimally invasive procedures was 61.9% and 38.1%, respectively. Only one (0.2%) patient died during the study period due to postoperative SARS-CoV2 infection because of acute respiratory distress syndrome. The overall non-SARS-CoV2 related 30-day morbidity and mortality rates were 19.3% and 1.7%, respectively; whereas the overall SARS-CoV2 related 30-day morbidity and mortality rates were 0.2% and 0.2%, respectively. CONCLUSIONS: Under strict institutional policies and measures to establish a COVID-19-free surgical pathway, elective and emergency cancer operations can be performed with acceptable perioperative and postoperative morbidity and mortality.

GLI1/GLI2 functional interplay is required to control Hedgehog/GLI targets gene expression.

The Hedgehog-regulated transcription factors GLI1 and GLI2 play overlapping roles in development and disease; however, the mechanisms underlying their interplay remain elusive. We report for the first time that GLI1 and GLI2 physically and functionally interact in cancer cells. GLI1 and GLI2 were shown to co-immunoprecipitate in PANC1 pancreatic cancer cells and RMS13 rhabdomyosarcoma cells. Mapping analysis demonstrated that the zinc finger domains of both proteins are required for their heteromerization. RNAi knockdown of either GLI1 or GLI2 inhibited expression of many well-characterized GLI target genes (BCL2, MYCN, PTCH2, IL7 and CCND1) in PANC1 cells, whereas PTCH1 expression was only inhibited by GLI1 depletion. qPCR screening of a large set of putative canonical and non-canonical Hedgehog/GLI targets identified further genes (e.g. E2F1, BMP1, CDK2) strongly down-regulated by GLI1 and/or GLI2 depletion in PANC1 cells, and demonstrated that ANO1, AQP1 and SOCS1 are up-regulated by knockdown of either GLI1 or GLI2. Chromatin immunoprecipitation showed that GLI1 and GLI2 occupied the same regions at the BCL2, MYCN and CCND1 promoters. Furthermore, depletion of GLI1 inhibited GLI2 occupancy at these promoters, suggesting that GLI1/GLI2 interaction is required for the recruitment of GLI2 to these sites. Together, these findings indicate that GLI1 and GLI2 co-ordinately regulate the transcription of some genes, and provide mechanistic insight into the roles of GLI proteins in carcinogenesis.

Frequency and clinicopathologic associations of DNA mismatch repair protein deficiency in ampullary carcinoma: Routine testing is indicated.

BACKGROUND: The significance of DNA mismatch repair (MMR) deficiency in ampullary cancers (ACs) has not been established. METHODS: In total, 127 ACs with invasive carcinomas measuring ≥3 mmthat had adequate tissue were analyzed immunohistochemically. RESULTS: MMR loss was detected in 18% of ACs (higher than in colorectal cancers). Twelve tumors with MLH1-PMS2 loss were negative for BRAF V600E mutation, suggesting a Lynch syndrome association. MMR-deficient tumors (n = 23), comparedwith MMR-intact tumors (n = 104), showed a striking male predominance (male:female ratio, 4.7). Although the deficient tumors had slightly larger invasion size (2.7 vs 2.1 cm), they also had more expansile growth and less invasiveness, including less perineural invasion, and they ultimately had lower tumor (T) classification and less lymph node metastasis (30% vs 53%; P = .04). More important, patients who had MMR-deficient tumors had better clinical outcomes, with a 5-year overall survival rate of 68% versus 45% (P = .03), which was even more pronounced in those who had higher Tclassification (5-year overall survival, 69% vs 34%; P = .04). MMR deficiencyhad a statistically significant association with medullary phenotype, pushing-border invasion, and tumor-infiltrating immune cells, and it occurred more frequently in ampullary-duodenal type tumors. Programed cell death-ligand 1 (PD-L1) levels analyzed in the 22 MMR-deficient ACs revealed that all medullary carcinomas were positive. Nonmedullary MMR-deficient carcinomas expressed PD-L1 in 33% of tumors cells according to the criteria for a combined positive score ≥1, but all were negative according to the tumor proportion score≥1 method. CONCLUSIONS: In ACs, MMR deficiency is even more frequent (18%) than in colon cancer and often has a Lynch-suggestive profile, thus routine testing is warranted. Male gender, pushing-border infiltration, ampullary-duodenal origin, medullary histology, and tumor-related inflammation have a significantly higher association with MMR deficiency. MMR-deficient tumors have less aggressive behavior. PD-L1 expression is common in medullary-phenotype ACs, thus immunotherapy should be considered at least for this group.

Inter- and intra-tumoural heterogeneity in cancer-associated fibroblasts of human pancreatic ductal adenocarcinoma.

Cancer-associated fibroblasts (CAF) are orchestrators of the pancreatic ductal adenocarcinoma (PDAC) microenvironment. Stromal heterogeneity may explain differential pathophysiological roles of the stroma (pro- versus anti-tumoural) in PDAC. We hypothesised that multiple CAF functional subtypes exist in PDAC, that contribute to stromal heterogeneity through interactions with cancer cells. Using molecular and functional analysis of patient-derived CAF primary cultures, we demonstrated that human PDAC-derived CAFs display a high level of inter- and intra-tumour heterogeneity. We identified at least four subtypes of CAFs based on transcriptomic analysis, and propose a classification for human PDAC-derived CAFs (pCAFassigner). Multiple CAF subtypes co-existed in individual patient samples. The presence of these CAF subtypes in bulk tumours was confirmed using publicly available gene expression profiles, and immunostainings of CAF subtype markers. Each subtype displayed specific phenotypic features (matrix- and immune-related signatures, vimentin and α-smooth muscle actin expression, proliferation rate), and was associated with an assessable prognostic impact. A prolonged exposure of non-tumoural pancreatic stellate cells to conditioned media from cancer cell lines (cancer education experiment) induced a CAF-like phenotype, including loss of capacity to revert to quiescence and an increase in the expression of genes related to CAF subtypes B and C. This classification demonstrates molecular and functional inter- and intra-tumoural heterogeneity of CAFs in human PDAC. Our subtypes overlap with those identified from single-cell analyses in other cancers, and pave the way for the development of therapies targeting specific CAF subpopulations in PDAC. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Delivery of hepato-pancreato-biliary surgery during the COVID-19 pandemic: an European-African Hepato-Pancreato-Biliary Association (E-AHPBA) cross-sectional survey.

BACKGROUND: The extent of the COVID-19 pandemic and the resulting response has varied globally. The European and African Hepato-Pancreato-Biliary Association (E-AHPBA), the premier representative body for practicing HPB surgeons in Europe and Africa, conducted this survey to assess the impact of COVID-19 on HPB surgery. METHODS: An online survey was disseminated to all E-AHPBA members to assess the effects of the pandemic on unit capacity, management of HPB cancers, use of COVID-19 screening and other aspects of service delivery. RESULTS: Overall, 145 (25%) members responded. Most units, particularly in COVID-high countries (>100,000 cases) reported insufficient critical care capacity and reduced HPB operating sessions compared to COVID-low countries. Delayed access to cancer surgery necessitated alternatives including increased neoadjuvant chemotherapy for pancreatic cancer and colorectal liver metastases, and locoregional treatments for hepatocellular carcinoma. Other aspects of service delivery including COVID-19 screening and personal protective equipment varied between units and countries. CONCLUSION: This study demonstrates that the COVID-19 pandemic has had a profound adverse impact on the delivery of HPB cancer care across the continents of Europe and Africa. The findings illustrate the need for safe resumption of cancer surgery in a "new" normal world with screening of patients and staff for COVID-19.

Dynamic landscape of pancreatic carcinogenesis reveals early molecular networks of malignancy.

OBJECTIVE: The initial steps of pancreatic regeneration versus carcinogenesis are insufficiently understood. Although a combination of oncogenic Kras and inflammation has been shown to induce malignancy, molecular networks of early carcinogenesis remain poorly defined. DESIGN: We compared early events during inflammation, regeneration and carcinogenesis on histological and transcriptional levels with a high temporal resolution using a well-established mouse model of pancreatitis and of inflammation-accelerated KrasG12D-driven pancreatic ductal adenocarcinoma. Quantitative expression data were analysed and extensively modelled in silico. RESULTS: We defined three distinctive phases-termed inflammation, regeneration and refinement-following induction of moderate acute pancreatitis in wild-type mice. These corresponded to different waves of proliferation of mesenchymal, progenitor-like and acinar cells. Pancreas regeneration required a coordinated transition of proliferation between progenitor-like and acinar cells. In mice harbouring an oncogenic Kras mutation and challenged with pancreatitis, there was an extended inflammatory phase and a parallel, continuous proliferation of mesenchymal, progenitor-like and acinar cells. Analysis of high-resolution transcriptional data from wild-type animals revealed that organ regeneration relied on a complex interaction of a gene network that normally governs acinar cell homeostasis, exocrine specification and intercellular signalling. In mice with oncogenic Kras, a specific carcinogenic signature was found, which was preserved in full-blown mouse pancreas cancer. CONCLUSIONS: These data define a transcriptional signature of early pancreatic carcinogenesis and a molecular network driving formation of preneoplastic lesions, which allows for more targeted biomarker development in order to detect cancer earlier in patients with pancreatitis.

Stromal heterogeneity in pancreatic cancer and chronic pancreatitis.

BACKGROUND/OBJECTIVES: An abundant stromal reaction is a hallmark of pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP). The cells mainly responsible for the stromal reaction are activated pancreatic stellate cells (PSCs). Despite their crucial role, PSCs are not well characterized. PSCs share characteristics with the better-known hepatic stellate cells (HSCs). The aim of this study was a detailed analysis of PSCs in PDAC and CP. METHODS: Whole-slide specimens of CP (n = 12) and PDAC (n = 10) were studied by histochemistry and immunohistochemistry. The stroma was evaluated using Movat's pentachrome stain. PSCs were tested by immunohistochemistry for PSC markers (α-SMA, CD34, desmin, NGFR, SPARC and tenascin C) and HSC markers (α-crystallin B, CD56, NGF, NT-3, synaptophysin and TrkC). Alpha-SMA, tenascin C, SPARC and NT-3 staining were verified on tissue micro arrays (TMAs) from a well-characterized cohort of 223 PDAC patients. PSCs isolated from human PDAC and CP tissue samples as well as HSCs were evaluated by immunofluorescence. RESULTS: While the stroma of CP cases was characterized by a collagen-rich fibrosis, PDAC stroma displayed higher mucin content (p = 0.0002). PSCs showed variable expression of tested markers. In PDAC samples, staining of most markers was found around tumor complexes, while CP samples showed a greater variety of localizations. Alpha-SMA staining correlated with collagen-rich fibrosis (p = 0.012), while NT-3 staining correlated with mucin-rich stroma (p = 0.008). A peritumoral staining was confirmed for α-SMA, tenascin C, SPARC and NT-3 in the PDAC TMA cohort (n = 223). In a subgroup of patients with pancreatic head tumors and UICC 2009 IIB (n = 144), α-SMA staining intensity was a prognostic factor for overall survival at uni- and multivariate analysis (p = 0.036 and p = 0.002). CONCLUSIONS: The close similarities between PSCs and HSCs were confirmed. Heterogeneous expression patterns of the tested markers might reflect different levels of activation or differentiation, or even multiple subpopulations of PSCs. Survival analysis suggests an impact of stromal composition on survival.

Pancreatic Premalignant Lesions Secrete Tissue Inhibitor of Metalloproteinases-1, Which Activates Hepatic Stellate Cells Via CD63 Signaling to Create a Premetastatic Niche in the Liver.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) metastasizes to liver at early stages, making this disease highly lethal. Tissue inhibitor of metalloproteinases-1 (TIMP1) creates a metastasis-susceptible environment in the liver. We investigated the role of TIMP1 and its receptor CD63 in metastasis of early-stage pancreatic tumors using mice and human cell lines and tissue samples. METHODS: We obtained liver and plasma samples from patients in Germany with chronic pancreatitis, pancreatic intra-epithelial neoplasia, or PDAC, as well as hepatic stellate cells (HSCs). We performed studies with Ptf1a+/Cre;Kras+/LSL-G12D;Trp53loxP/loxP (CPK) mice, Pdx-1+/Cre;Kras+/LSL-G12D;Trp53+/LSL-R172H (KPC) mice, and their respective healthy littermates as control, and Cd63-/- mice with their wild-type littermates. KPC mice were bred with Timp1-/- mice to produce KPCxTimp1-/- mice. TIMP1 was overexpressed and CD63 was knocked down in mice using adenoviral vectors AdTIMP1 or AdshCD63, respectively. Hepatic susceptibility to metastases was determined after intravenous inoculation of syngeneic 9801L pancreas carcinoma cells. Pancreata and liver tissues were collected and analyzed by histology, immunohistochemical, immunoblot, enzyme-linked immunosorbent assay, and quantitative polymerase chain reaction analyses. We analyzed the effects of TIMP1 overexpression or knockdown and CD63 knockdown in transduced human primary HSCs and HSC cell lines. RESULTS: Chronic pancreatitis, pancreatic intra-epithelial neoplasia, and PDAC tissues from patients expressed higher levels of TIMP1 protein than normal pancreas. The premalignant pancreatic lesions that developed in KPC and CPK mice expressed TIMP1 and secreted it into the circulation. In vitro and in vivo, TIMP1 activated human or mouse HSCs, which required interaction between TIMP1 and CD63 and signaling via phosphatidylinositol 3-kinase, but not TIMP1 protease inhibitor activity. This signaling pathway induced expression of endogenous TIMP1. TIMP1 knockdown in HSCs reduced their activation. Cultured TIMP1-activated human and mouse HSCs began to express stromal-derived factor-1, which induced neutrophil migration, a marker of the premetastatic niche. Mice with pancreatic intra-epithelial neoplasia-derived systemic increases in TIMP1 developed more liver metastases after injections of pancreatic cancer cells than mice without increased levels of TIMP1. This increase in formation of liver metastases from injected pancreatic cancer cells was not observed in TIMP1 or CD63 knockout mice. CONCLUSIONS: Expression of TIMP1 is increased in chronic pancreatitis, pancreatic intra-epithelial neoplasia, and PDAC tissues from patients. TIMP1 signaling via CD63 leads to activation of HSCs, which create an environment in the liver that increases its susceptibility to pancreatic tumor cells. Strategies to block TIMP1 signaling via CD63 might be developed to prevent PDAC metastasis to the liver.

Intracellular autofluorescence: a biomarker for epithelial cancer stem cells.

Cancer stem cells (CSCs) are thought to drive tumor growth, metastasis and chemoresistance. Although surface markers such as CD133 and CD44 have been successfully used to isolate CSCs, their expression is not exclusively linked to the CSC phenotype and is prone to environmental alteration. We identified cells with an autofluorescent subcellular compartment that exclusively showed CSC features across different human tumor types. Primary tumor-derived autofluorescent cells did not overlap with side-population (SP) cells, were enriched in sphere culture and during chemotherapy, strongly expressed pluripotency-associated genes, were highly metastatic and showed long-term in vivo tumorigenicity, even at the single-cell level. Autofluorescence was due to riboflavin accumulation in membrane-bounded cytoplasmic structures bearing ATP-dependent ABCG2 transporters. In summary, we identified and characterized an intrinsic autofluorescent phenotype in CSCs of diverse epithelial cancers and used this marker to isolate and characterize these cells.

Loss of Periostin Results in Impaired Regeneration and Pancreatic Atrophy after Cerulein-Induced Pancreatitis.

The extracellular matrix molecule periostin (POSTN, encoded by POSTN), which is secreted by activated pancreatic stellate cells, has important functions in chronic pancreatitis and pancreatic cancer. However, the role of POSTN in acute pancreatitis and subsequent regeneration processes has not been addressed so far. We analyzed the function of POSTN in pancreatic exocrine regeneration after the induction of a severe acute pancreatitis. Postn-deficient mice and wild-type control animals received repetitive cerulein injections, and a detailed histologic analysis of pancreatic tissues was performed. Although there was no difference in pancreatitis severity in the acute inflammatory phase, the recovery of the exocrine pancreas was massively impaired in Postn-deficient mice. Loss of Postn expression was accompanied by strong pancreatic atrophy and acinar-to-adipocyte differentiation, which was also reflected in gene expression patterns. Our data suggest that POSTN is a crucial factor for proper exocrine lineage-specific regeneration after severe acute pancreatitis.

A subset of metastatic pancreatic ductal adenocarcinomas depends quantitatively on oncogenic Kras/Mek/Erk-induced hyperactive mTOR signalling.

OBJECTIVE: Oncogenic Kras-activated robust Mek/Erk signals phosphorylate to the tuberous sclerosis complex (Tsc) and deactivates mammalian target of rapamycin (mTOR) suppression in pancreatic ductal adenocarcinoma (PDAC); however, Mek and mTOR inhibitors alone have demonstrated minimal clinical antitumor activity. DESIGN: We generated transgenic mouse models in which mTOR was hyperactivated either through the Kras/Mek/Erk cascade, by loss of Pten or through Tsc1 haploinsufficiency. Primary cancer cells were isolated from mouse tumours. Oncogenic signalling was assessed in vitro and in vivo, with and without single or multiple targeted molecule inhibition. Transcriptional profiling was used to identify biomarkers predictive of the underlying pathway alterations and of therapeutic response. Results from the preclinical models were confirmed on human material. RESULTS: Reduction of Tsc1 function facilitated activation of Kras/Mek/Erk-mediated mTOR signalling, which promoted the development of metastatic PDACs. Single inhibition of mTOR or Mek elicited strong feedback activation of Erk or Akt, respectively. Only dual inhibition of Mek and PI3K reduced mTOR activity and effectively induced cancer cell apoptosis. Analysis of downstream targets demonstrated that oncogenic activity of the Mek/Erk/Tsc/mTOR axis relied on Aldh1a3 function. Moreover, in clinical PDAC samples, ALDH1A3 specifically labelled an aggressive subtype. CONCLUSIONS: These results advance our understanding of Mek/Erk-driven mTOR activation and its downstream targets in PDAC, and provide a mechanistic rationale for effective therapeutic matching for Aldh1a3-positive PDACs.

Impaired autophagy induces chronic atrophic pancreatitis in mice via sex- and nutrition-dependent processes.

BACKGROUND & AIMS: Little is known about the mechanisms of the progressive tissue destruction, inflammation, and fibrosis that occur during development of chronic pancreatitis. Autophagy is involved in multiple degenerative and inflammatory diseases, including pancreatitis, and requires the protein autophagy related 5 (ATG5). We created mice with defects in autophagy to determine its role in pancreatitis. METHODS: We created mice with pancreas-specific disruption of Atg5 (Ptf1aCreex1;Atg5F/F mice) and compared them to control mice. Pancreata were collected and histology, immunohistochemistry, transcriptome, and metabolome analyses were performed. ATG5-deficient mice were placed on diets containing 25% palm oil and compared with those on a standard diet. Another set of mice received the antioxidant N-acetylcysteine. Pancreatic tissues were collected from 8 patients with chronic pancreatitis (CP) and compared with pancreata from ATG5-deficient mice. RESULTS: Mice with pancreas-specific disruption of Atg5 developed atrophic CP, independent of ß-cell function; a greater proportion of male mice developed CP than female mice. Pancreata from ATG5-deficient mice had signs of inflammation, necrosis, acinar-to-ductal metaplasia, and acinar-cell hypertrophy; this led to tissue atrophy and degeneration. Based on transcriptome and metabolome analyses, ATG5-deficient mice produced higher levels of reactive oxygen species than control mice, and had insufficient activation of glutamate-dependent metabolism. Pancreata from these mice had reduced autophagy, increased levels of p62, and increases in endoplasmic reticulum stress and mitochondrial damage, compared with tissues from control mice; p62 signaling to Nqo1 and p53 was also activated. Dietary antioxidants, especially in combination with palm oil-derived fatty acids, blocked progression to CP and pancreatic acinar atrophy. Tissues from patients with CP had many histologic similarities to those from ATG5-deficient mice. CONCLUSIONS: Mice with pancreas-specific disruption of Atg5 develop a form of CP similar to that of humans. CP development appears to involve defects in autophagy, glutamate-dependent metabolism, and increased production of reactive oxygen species. These mice might be used to identify therapeutic targets for CP.

Microenvironmental hCAP-18/LL-37 promotes pancreatic ductal adenocarcinoma by activating its cancer stem cell compartment.

OBJECTIVES: The tumour stroma/microenvironment not only provides structural support for tumour development, but more importantly it provides cues to cancer stem cells (CSCs) that regulate their self-renewal and metastatic potential. This is certainly true for pancreatic ductal adenocarcinomas (PDAC), where tumour-associated fibroblasts, pancreatic stellate cells and immune cells create an abundant paracrine niche for CSCs via microenvironment-secreted factors. Thus understanding the role that tumour stroma cells play in PDAC development and CSC biology is of utmost importance. DESIGN: Microarray analyses, tumour microarray immunohistochemical assays, in vitro co-culture experiments, recombinant protein treatment approaches and in vivo intervention studies were performed to understand the role that the immunomodulatory cationic antimicrobial peptide 18/LL-37 (hCAP-18/LL-37) plays in PDAC biology. RESULTS: We found that hCAP-18/LL-37 was strongly expressed in the stroma of advanced primary and secondary PDAC tumours and is secreted by immune cells of the stroma (eg, tumour-associated macrophages) in response to tumour growth factor-ß1 and particularly CSC-secreted Nodal/ActivinA. Treatment of pancreatic CSCs with recombinant LL-37 increased pluripotency-associated gene expression, self-renewal, invasion and tumourigenicity via formyl peptide receptor 2 (FPR2)- and P2X purinoceptor 7 receptor (P2X7R)-dependent mechanisms, which could be reversed by inhibiting these receptors. Importantly, in a genetically engineered mouse model of K-Ras-driven pancreatic tumourigenesis, we also showed that tumour formation was inhibited by either reconstituting these mice with bone marrow from cathelicidin-related antimicrobial peptide (ie, murine homologue of hCAP-18/LL-37) knockout mice or by pharmacologically inhibiting FPR2 and P2X7R. CONCLUSIONS: Thus, hCAP-18/LL-37 represents a previously unrecognised PDAC microenvironment factor that plays a critical role in pancreatic CSC-mediated tumourigenesis.