Multiscale and multimodal imaging for three-dimensional vascular and histomorphological organ structure analysis of the pancreas.

Exocrine and endocrine pancreas are interconnected anatomically and functionally, with vasculature facilitating bidirectional communication. Our understanding of this network remains limited, largely due to two-dimensional histology and missing combination with three-dimensional imaging. In this study, a multiscale 3D-imaging process was used to analyze a porcine pancreas. Clinical computed tomography, digital volume tomography, micro-computed tomography and Synchrotron-based propagation-based imaging were applied consecutively. Fields of view correlated inversely with attainable resolution from a whole organism level down to capillary structures with a voxel edge length of 2.0 µm. Segmented vascular networks from 3D-imaging data were correlated with tissue sections stained by immunohistochemistry and revealed highly vascularized regions to be intra-islet capillaries of islets of Langerhans. Generated 3D-datasets allowed for three-dimensional qualitative and quantitative organ and vessel structure analysis. Beyond this study, the method shows potential for application across a wide range of patho-morphology analyses and might possibly provide microstructural blueprints for biotissue engineering.

DNA-methylation signature accurately differentiates pancreatic cancer from chronic pancreatitis in tissue and plasma.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Differentiation from chronic pancreatitis (CP) is currently inaccurate in about one-third of cases. Misdiagnoses in both directions, however, have severe consequences for patients. We set out to identify molecular markers for a clear distinction between PDAC and CP. DESIGN: Genome-wide variations of DNA-methylation, messenger RNA and microRNA level as well as combinations thereof were analysed in 345 tissue samples for marker identification. To improve diagnostic performance, we established a random-forest machine-learning approach. Results were validated on another 48 samples and further corroborated in 16 liquid biopsy samples. RESULTS: Machine-learning succeeded in defining markers to differentiate between patients with PDAC and CP, while low-dimensional embedding and cluster analysis failed to do so. DNA-methylation yielded the best diagnostic accuracy by far, dwarfing the importance of transcript levels. Identified changes were confirmed with data taken from public repositories and validated in independent sample sets. A signature of six DNA-methylation sites in a CpG-island of the protein kinase C beta type gene achieved a validated diagnostic accuracy of 100% in tissue and in circulating free DNA isolated from patient plasma. CONCLUSION: The success of machine-learning to identify an effective marker signature documents the power of this approach. The high diagnostic accuracy of discriminating PDAC from CP could have tremendous consequences for treatment success, once the result from still a limited number of liquid biopsy samples would be confirmed in a larger cohort of patients with suspected pancreatic cancer.

Sex Differences in the Systemic and Local Immune Response of Pancreatic Cancer Patients.

BACKGROUND: Mounting evidence suggests that sex plays a critical role in various aspects of cancer such as immune responses. However, a male bias exists in human and non-human studies including immunotherapy trials. The role of sex on immune responses in pancreatic ductal adenocarcinoma (PDA) is unclear. METHODS: Here, tumor tissues (tumor and stroma separately) and corresponding blood samples from patients with PDA (n = 52) were systematically analyzed by immunohistochemistry and multiplex cytokine measurements and compared by sex. RESULTS: Females showed a stronger systemic immune response with higher levels of CXCL9, IL1B, IL6, IL10 and IL13. Additionally, more peripheral white blood cells were detected in females. In the microenvironment, males showed higher tumoral levels of CXCL12. No differences were detected in the stroma. Females showed a tendency towards an anti-tumoral immune cell profile. CXCL12 blockade indicated a differential microenvironmental effect by sex in an independent immunotherapy trial cohort of patients with PDA (one female, five males). The overall survival did not differ by sex in our cohort. CONCLUSION: Systemic and local immune responses differ between sexes in PDA. Accordingly, sex-dependent differences need to be considered in human studies and for specific immunological interventions before clinical translation.

Potential role of the bitter taste receptor T2R14 in the prolonged survival and enhanced chemoresponsiveness induced by apigenin.

Bitter taste receptors (T2Rs) are G protein­coupled receptors originally detected in the gustatory system. More recently, T2Rs have been shown to be expressed in extra­oral cells eliciting non­gustatory functions. Emerging evidence has suggested a potential role for T2R signaling in diverse pathophysiological conditions, including cancer. -The aim of the present study was to evaluate the expression of T2R14 in pancreatic ductal adenocarcinoma (PDAC) and to assess its involvement in the anticancer effects induced by apigenin, a natural ligand of T2R14. For this purpose, T2R14 expression was explored in PDAC tumor tissue and tumor­derived cell lines. Using the cell lines expressing the highest levels of T2R14, its effects on chemoresponsiveness and migration upon activation with apigenin were investigated in vitro. To the best of our knowledge, the present study was the first to confirm the expression of the T2R family member T2R14 in PDAC. Patients with relatively high levels of T2R14 expression exhibited significantly prolonged overall survival compared with that of patients with low T2R14 expression. Furthermore, novel functions for apigenin were revealed; notably, apigenin was shown to elicit cytotoxic, anti­migratory and chemosensitizing effects to 5­fluoruracil (5­FU) and to 5­FU, leucovorin, irinotecan and oxaliplatin in pancreatic cancer cells. In conclusion, the present study extended the evidence for the anticancer effects of apigenin and strongly indicated the functional relevance of T2R14 in PDAC, even though their respective underlying pathways appear to be independent of each other.

Blood-Based Diagnosis and Risk Stratification of Patients with Pancreatic Intraductal Papillary Mucinous Neoplasm (IPMN).

PURPOSE: Intraductal papillary mucinous neoplasm (IPMN) is a precursor of pancreatic ductal adenocarcinoma. Low-grade dysplasia has a relatively good prognosis, whereas high-grade dysplasia and IPMN invasive carcinoma require surgical intervention. However, diagnostic distinction is difficult. We aimed to identify biomarkers in peripheral blood for accurate discrimination. EXPERIMENTAL DESIGN: Sera were obtained from 302 patients with IPMNs and 88 healthy donors. For protein biomarkers, serum samples were analyzed on microarrays made of 2,977 antibodies. A support vector machine (SVM) algorithm was applied to define classifiers, which were validated on a separate sample set. For microRNA biomarkers, a PCR-based screen was performed for discovery. Biomarker candidates confirmed by quantitative PCR were used to train SVM classifiers, followed by validation in a different sample set. Finally, a combined SVM classifier was established entirely independent of the earlier analyses, again using different samples for training and validation. RESULTS: Panels of 26 proteins or seven microRNAs could distinguish high- and low-risk IPMN with an AUC value of 95% and 94%, respectively. Upon combination, a panel of five proteins and three miRNAs yielded an AUC of 97%. These values were much better than those obtained in the same patient cohort by using the guideline criteria for discrimination. In addition, accurate discrimination was achieved between other patient subgroups. CONCLUSIONS: Protein and microRNA biomarkers in blood allow precise diagnosis and risk stratification of IPMN cases, which should improve patient management and thus the prognosis of IPMN patients. See related commentary by Löhr and Pantel, p. 1387.

The HIF-prolyl hydroxylases have distinct and nonredundant roles in colitis-associated cancer.

Colitis-associated colorectal cancer (CAC) is a severe complication of inflammatory bowel disease (IBD). HIF-prolyl hydroxylases (PHD1, PHD2, and PHD3) control cellular adaptation to hypoxia and are considered promising therapeutic targets in IBD. However, their relevance in the pathogenesis of CAC remains elusive. We induced CAC in Phd1-/-, Phd2+/-, Phd3-/-, and WT mice with azoxymethane (AOM) and dextran sodium sulfate (DSS). Phd1-/- mice were protected against chronic colitis and displayed diminished CAC growth compared with WT mice. In Phd3-/- mice, colitis activity and CAC growth remained unaltered. In Phd2+/- mice, colitis activity was unaffected, but CAC growth was aggravated. Mechanistically, Phd2 deficiency (i) increased the number of tumor-associated macrophages in AOM/DSS-induced tumors, (ii) promoted the expression of EGFR ligand epiregulin in macrophages, and (iii) augmented the signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2 signaling, which at least in part contributed to aggravated tumor cell proliferation in colitis-associated tumors. Consistently, Phd2 deficiency in hematopoietic (Vav:Cre-Phd2fl/fl) but not in intestinal epithelial cells (Villin:Cre-Phd2fl/fl) increased CAC growth. In conclusion, the 3 different PHD isoenzymes have distinct and nonredundant effects, promoting (PHD1), diminishing (PHD2), or neutral (PHD3), on CAC growth.

Patient-specific modeling of stroma-mediated chemoresistance of pancreatic cancer using a three-dimensional organoid-fibroblast co-culture system.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are considered to play a fundamental role in pancreatic ductal adenocarcinoma (PDAC) progression and chemoresistance. Patient-derived organoids have demonstrated great potential as tumor avatars for drug response prediction in PDAC, yet they disregard the influence of stromal components on chemosensitivity. METHODS: We established direct three-dimensional (3D) co-cultures of primary PDAC organoids and patient-matched CAFs to investigate the effect of the fibroblastic compartment on sensitivity to gemcitabine, 5-fluorouracil and paclitaxel treatments using an image-based drug assay. Single-cell RNA sequencing was performed for three organoid/CAF pairs in mono- and co-culture to uncover transcriptional changes induced by tumor-stroma interaction. RESULTS: Upon co-culture with CAFs, we observed increased proliferation and reduced chemotherapy-induced cell death of PDAC organoids. Single-cell RNA sequencing data evidenced induction of a pro-inflammatory phenotype in CAFs in co-cultures. Organoids showed increased expression of genes associated with epithelial-to-mesenchymal transition (EMT) in co-cultures and several potential receptor-ligand interactions related to EMT were identified, supporting a key role of CAF-driven induction of EMT in PDAC chemoresistance. CONCLUSIONS: Our results demonstrate the potential of personalized PDAC co-cultures models not only for drug response profiling but also for unraveling the molecular mechanisms involved in the chemoresistance-supporting role of the tumor stroma.

Immune features of the peritumoral stroma in pancreatic ductal adenocarcinoma.

Background: The peritumoral stroma is a hallmark of pancreatic ductal adenocarcinoma (PDA) with implications for disease development, progression and therapy resistance. We systematically investigated immune features of the stroma in PDA patients to identify markers of clinical importance and potential therapeutic targets. Methods: Tissue and blood samples of 51 PDA patients with clinical and follow-up information were included. Laser Capture Microdissection allowed us to analyze the stromal compartment in particular. Systematic immunohistochemistry, followed by software-based image analysis were conducted. Also, multiplex cytokine analyses (including 50 immune-related molecules) were performed. Functional analyses were performed using patient-derived 3D bioprints. Clinical information was used for survival analyses. Intercompartmental IL9 and IL18 gradients were assessed in matched samples of tumor epithelium, stroma, and serum of patients. Serum levels were compared to an age-matched healthy control group. Results: Stromal IL9 and IL18 are significantly associated with patient survival. While IL9 is a prognostic favorable marker (p=0.041), IL18 associates with poor patient outcomes (p=0.030). IL9 correlates with an anti-tumoral cytokine network which connects regulation of T helper (Th) 9, Th1 and Th17 cells (all: p<0.05 and r>0.5). IL18 correlates with a Th1-type cytokine phenotype and stromal CXCL12 expression (all: p<0.05 and r>0.5). Further, IL18 associates with a higher level of exhausted T cells. Inhibition of IL18 results in diminished Th1- and Th2-type cytokines. Patients with high stromal IL9 expression have a tumor-to-stroma IL9 gradient directed towards the stroma (p=0.019). Low IL18 expression associates with a tumor-to-stroma IL18 gradient away from the stroma (p=0.007). PDA patients showed higher serum levels of IL9 than healthy controls while serum IL18 levels were significantly lower than in healthy individuals. The stromal immune cell composition is distinct from the tumor epithelium. Stromal density of FoxP3+ regulatory T cells showed a tendency towards improved patient survival (p=0.071). Conclusion: An unexpected high expression of the cytokines IL9 and IL18 at different ends is of significance in the stroma of PDA and relates to opposing patient outcomes. Sub-compartmental cytokine analyses highlight the importance of a differentiated gradient assessment. The findings suggest stromal IL9 and/or IL18 as markers for patient stratification and as potential therapeutic targets. Future steps include investigating e. g. the role of local microbiota as both cytokines are also regulated by microbial compositions.

Myofibroblastic CAF Density, Not Activated Stroma Index, Indicates Prognosis after Neoadjuvant Therapy of Pancreatic Carcinoma.

Neoadjuvant therapy (NT) for advanced PDAC is an emerging concept, affecting both stroma and tumor. The Activated Stroma Index (ASI; ratio of activated cancer-associated fibroblasts (CAF) to collagen deposition) is a prognostic marker in upfront resected pancreatic adenocarcinoma (PDAC). We assessed ASI and its prognostic relevance after NT. Tissue from resection specimens of n = 48 PDAC patients after neoadjuvant chemotherapy with FOLFIRINOX (FOL; n = 31), gemcitabine + nab-paclitaxel (GEM; 7) or combination treatment (COMB; 10) was compared with upfront resected matched controls (RES; 69). Activated CAFs were assessed by immunohistochemistry for α-SMA, and collagen was stained with aniline blue; the stained area was then determined by computational imaging analysis and ASI was calculated. In GEM, ASI was significantly higher and collagen deposition lower than in controls and FOL. The lowest quartile of ASI values had significantly longer overall survival (OS) in RES, whereas in FOL, the highest quartile had the best prognosis. After NT, OS was significantly improved in the α-SMA-high group; in RES, however, survival was independent of α-SMA. Reversed prognostic association of ASI thus points to the differing significance of stromal composition after FOL, while improved prognosis with high CAF abundance suggests a synergistic effect of myofibroblasts with chemotherapy. These divergences impede usability of ASI after NT.

Toward 3D-bioprinting of an endocrine pancreas: A building-block concept for bioartificial insulin-secreting tissue.

Three-dimensional bioprinting of an endocrine pancreas is a promising future curative treatment for patients with insulin secretion deficiency. In this study, we present an end-to-end concept from the molecular to the macroscopic level. Building-blocks for a hybrid scaffold device of hydrogel and functionalized polycaprolactone were manufactured by 3D-(bio)printing. Pseudoislet formation from INS-1 cells after bioprinting resulted in a viable and proliferative experimental model. Transcriptomics showed an upregulation of proliferative and ß-cell-specific signaling cascades, downregulation of apoptotic pathways, overexpression of extracellular matrix proteins, and VEGF induced by pseudoislet formation and 3D-culture. Co-culture with endothelial cells created a natural cellular niche with enhanced insulin secretion after glucose stimulation. Survival and function of pseudoislets after explantation and extensive scaffold vascularization of both hydrogel and heparinized polycaprolactone were demonstrated in vivo. Computer simulations of oxygen, glucose and insulin flows were used to evaluate scaffold architectures and Langerhans islets at a future perivascular transplantation site.

Radiotherapy orchestrates natural killer cell dependent antitumor immune responses through CXCL8.

Radiotherapy is a mainstay cancer therapy whose antitumor effects partially depend on T cell responses. However, the role of Natural Killer (NK) cells in radiotherapy remains unclear. Here, using a reverse translational approach, we show a central role of NK cells in the radiation-induced immune response involving a CXCL8/IL-8-dependent mechanism. In a randomized controlled pancreatic cancer trial, CXCL8 increased under radiotherapy, and NK cell positively correlated with prolonged overall survival. Accordingly, NK cells preferentially infiltrated irradiated pancreatic tumors and exhibited CD56dim-like cytotoxic transcriptomic states. In experimental models, NF-κB and mTOR orchestrated radiation-induced CXCL8 secretion from tumor cells with senescence features causing directional migration of CD56dim NK cells, thus linking senescence-associated CXCL8 release to innate immune surveillance of human tumors. Moreover, combined high-dose radiotherapy and adoptive NK cell transfer improved tumor control over monotherapies in xenografted mice, suggesting NK cells combined with radiotherapy as a rational cancer treatment strategy.

Tertiary lymphoid structures and their association to immune phenotypes and circulatory IL2 levels in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is usually unresponsive to immunotherapeutic approaches. However, tertiary lymphoid structures (TLS) are associated with favorable patient outcomes in PDA. A better understanding of the B cell infiltrate and biological features of TLS formation is needed to further explore their potential and improve patient management. We analyzed tumor tissues (n = 55) and corresponding blood samples (n = 51) from PDA patients by systematical immunohistochemistry and multiplex cytokine measurements. The tissue was compartmentalized in "tumor" and "stroma" and separately examined. Clinical patient information was used to perform survival analyses. We found that the mere number of B cells is not associated with patient survival, but formation of TLS in the peritumoral stroma is a prognostic favorable marker in PDA patients. TLS-positive tissues show a higher density of CD8+ T cells and CD20+ B cells and a higher IL2 level in the peritumoral stroma than tissues without TLS. Compartmental assessment shows that gradients of IL2 expression differ with regard to TLS formation: TLS presence is associated with higher IL2 levels in the stromal than in the tumoral compartment, while no difference is seen in patients without TLS. Focusing on the stroma-to-serum gradient, only patients without TLS show significantly higher IL2 levels in the serum than in stroma. Finally, low circulatory IL2 levels are associated with local TLS formation. Our findings highlight that TLS are prognostic favorable and associated with antitumoral features in the microenvironment of PDA. Also, we propose easily accessible serum IL2 levels as a potential marker for TLS prediction.

Promoter Hypermethylation Promotes the Binding of Transcription Factor NFATc1, Triggering Oncogenic Gene Activation in Pancreatic Cancer.

Studies have indicated that some genes involved in carcinogenesis are highly methylated in their promoter regions but nevertheless strongly transcribed. It has been proposed that transcription factors could bind specifically to methylated promoters and trigger transcription. We looked at this rather comprehensively for pancreatic ductal adenocarcinoma (PDAC) and studied some cases in more detail. Some 2% of regulated genes in PDAC exhibited higher transcription coupled to promoter hypermethylation in comparison to healthy tissue. Screening 661 transcription factors, several were found to bind specifically to methylated promoters, in particular molecules of the NFAT family. One of them-NFATc1-was substantially more strongly expressed in PDAC than control tissue and exhibited a strong oncogenic role. Functional studies combined with computational analyses allowed determining affected genes. A prominent one was gene ALDH1A3, which accelerates PDAC metastasis and correlates with a bad prognosis. Further studies confirmed the direct up-regulation of ALDH1A3 transcription by NFATc1 promoter binding in a methylation-dependent process, providing insights into the oncogenic role of transcription activation in PDAC that is promoted by DNA methylation.

Peripheral blood and tissue assessment highlights differential tumor-circulatory gradients of IL2 and MIF with prognostic significance in resectable pancreatic ductal adenocarcinoma.

Various reports have pointed out the potential of cytokines as diagnostic and prognostic biomarkers for pancreatic ductal adenocarcinoma (PDA). Nonetheless, the evidence is contradictory and the role of chronic inflammation and relationship between circulatory and corresponding tumoral cytokine levels remain elusive. Utilizing a broad array of cytokines, we identified two opposing parameters: serum levels of interleukin 2 (IL2) and macrophage migration inhibitory factor (MIF) are diagnostic and prognostic factors. While low IL2 levels are associated with PDA, they also relate to a favorable prognosis of patients. In contrast, high MIF levels are associated with PDA and simultaneously related to an unfavorable outcome. MIF levels are associated with the intratumoral density of M2 macrophages (CD163+). Focusing on the tumor-to-serum gradient, we unveiled a different pattern of compartmental cytokine expression between IL2 and MIF. Our findings indicate that an extra-tumoral source of IL2 exists in PDA patients leading to increased detectability in the circulatory system. In case of MIF, the tumor microenvironment is presumably the main site of production and thereby reflected by serum measurements. Taken together, our study describes IL2 and MIF levels as biomarker candidates for diagnosis and prognosis of PDA, highlighting the need for compartmental cytokine analyses. From the perspective of tumor immunobiology, we identify multiple inflammatory states (proposed as types I-III) and see that systemic chronic dysregulation, independent of tumor microenvironment, can be measured and is a possible tool for stratification. Thus, direct correlation of local cytokine levels to peripheral blood levels needs to be regarded with caution.

Human Retrotransposons and the Global Shutdown of Homeostatic Innate Immunity by Oncolytic Parvovirus H-1PV in Pancreatic Cancer.

Although the oncolytic parvovirus H-1PV has entered clinical trials, predicting therapeutic success remains challenging. We investigated whether the antiviral state in tumor cells determines the parvoviral oncolytic efficacy. The interferon/interferon-stimulated genes (IFN/ISG)-circuit and its major configurator, human endogenous retroviruses (HERVs), were evaluated using qRT-PCR, ELISA, Western blot, and RNA-Seq techniques. In pancreatic cancer cell lines, H-1PV caused a late global shutdown of innate immunity, whereby the concomitant inhibition of HERVs and IFN/ISGs was co-regulatory rather than causative. The growth-inhibitory IC50 doses correlated with the power of suppression but not with absolute ISG levels. Moreover, H-1PV was not sensitive to exogenous IFN despite upregulated antiviral ISGs. Such resistance questioned the biological necessity of the oncotropic ISG-shutdown, which instead might represent a surrogate marker for personalized oncolytic efficacy. The disabled antiviral homeostasis may modify the activity of other viruses, as demonstrated by the reemergence of endogenous AluY-retrotransposons. This way of suppression may compromise the interferogenicity of drugs having gemcitabine-like mechanisms of action. This shortcoming in immunogenic cell death induction is however amendable by immune cells which release IFN in response to H-1PV.

Blood biomarkers for differential diagnosis and early detection of pancreatic cancer.

Pancreatic cancer is currently the most lethal tumor entity and case numbers are rising. It will soon be the second most frequent cause of cancer-related death in the Western world. Mortality is close to incidence and patient survival after diagnosis stands at about five months. Blood-based diagnostics could be one crucial factor for improving this dismal situation and is at a stage that could make this possible. Here, we are reviewing the current state of affairs with its problems and promises, looking at various molecule types. Reported results are evaluated in the overall context. Also, we are proposing steps toward clinical utility that should advance the development toward clinical application by improving biomarker quality but also by defining distinct clinical objectives and the respective diagnostic accuracies required to achieve them. Many of the discussed points and conclusions are highly relevant to other solid tumors, too.

Aggressive PDACs Show Hypomethylation of Repetitive Elements and the Execution of an Intrinsic IFN Program Linked to a Ductal Cell of Origin.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive desmoplasia, which challenges the molecular analyses of bulk tumor samples. Here we FACS-purified epithelial cells from human PDAC and normal pancreas and derived their genome-wide transcriptome and DNA methylome landscapes. Clustering based on DNA methylation revealed two distinct PDAC groups displaying different methylation patterns at regions encoding repeat elements. Methylationlow tumors are characterized by higher expression of endogenous retroviral transcripts and double-stranded RNA sensors, which lead to a cell-intrinsic activation of an interferon signature (IFNsign). This results in a protumorigenic microenvironment and poor patient outcome. Methylationlow/IFNsignhigh and Methylationhigh/IFNsignlow PDAC cells preserve lineage traits, respective of normal ductal or acinar pancreatic cells. Moreover, ductal-derived Kras G12D/Trp53 -/- mouse PDACs show higher expression of IFNsign compared with acinar-derived counterparts. Collectively, our data point to two different origins and etiologies of human PDACs, with the aggressive Methylationlow/IFNsignhigh subtype potentially targetable by agents blocking intrinsic IFN signaling. SIGNIFICANCE: The mutational landscapes of PDAC alone cannot explain the observed interpatient heterogeneity. We identified two PDAC subtypes characterized by differential DNA methylation, preserving traits from normal ductal/acinar cells associated with IFN signaling. Our work suggests that epigenetic traits and the cell of origin contribute to PDAC heterogeneity.This article is highlighted in the In This Issue feature, p. 521.

SST gene hypermethylation acts as a pan-cancer marker for pancreatic ductal adenocarcinoma and multiple other tumors: toward its use for blood-based diagnosis.

Aberrant DNA methylation is often involved in carcinogenesis. Our initial goal was to identify DNA methylation biomarkers associated with pancreatic cancer. A genomewide methylation study was performed on DNA from pancreatic ductal adenocarcinoma (PDAC) and endocrine pancreas tumors. Validation of DNA methylation patterns and concomitant alterations in expression of gene candidates was performed on patient samples and pancreatic cancer cell lines. Furthermore, validation was done on independent data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Finally, droplet digital PCR was employed to detect DNA methylation marks in cell-free (cf) DNA isolated from plasma samples of PDAC patients and cancer-free blood donors. Hypermethylation of the SST gene (encoding somatostatin) and concomitant downregulation of its expression were discovered in PDAC and endocrine tumor tissues while not being present in chronic pancreatitis (inflamed) tissues and normal pancreas. Fittingly, treatment with a somatostatin agonist (octreotide) reduced cell proliferation and migration of pancreatic cancer cells. Diagnostic performance of SST methylation in a receiver operating characteristic curve analysis was 100% and 89% for tissue and plasma samples, respectively. A large body of TCGA and GEO data confirmed SST hypermethylation and downregulation in PDAC and showed a similar effect in a broad spectrum of other tumor entities. SST promoter methylation represents a sensitive and promising molecular, pan-cancer biomarker detectable in tumor tissue, and liquid biopsy samples.

Genome-wide association study identifies an early onset pancreatic cancer risk locus.

Early onset pancreatic cancer (EOPC) is a rare disease with a very high mortality rate. Almost nothing is known on the genetic susceptibility of EOPC, therefore, we performed a genome-wide association study (GWAS) to identify novel genetic variants specific for patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) at younger ages. In the first phase, conducted on 821 cases with age of onset ≤60 years, of whom 198 with age of onset ≤50, and 3227 controls from PanScan I-II, we observed four SNPs (rs7155613, rs2328991, rs4891017 and rs12610094) showing an association with EOPC risk (P < 1 × 10-4 ). We replicated these SNPs in the PANcreatic Disease ReseArch (PANDoRA) consortium and used additional in silico data from PanScan III and PanC4. Among these four variants rs2328991 was significant in an independent set of 855 cases with age of onset ≤60 years, of whom 265 with age of onset ≤50, and 4142 controls from the PANDoRA consortium while in the in silico data, we observed no statistically significant association. However, the resulting meta-analysis supported the association (P = 1.15 × 10-4 ). In conclusion, we propose a novel variant rs2328991 to be involved in EOPC risk. Even though it was not possible to find a mechanistic link between the variant and the function, the association is supported by a solid statistical significance obtained in the largest study on EOPC genetics present so far in the literature.

The transcription factor FLI1 promotes cancer progression by affecting cell cycle regulation.

Binding of transcription factors to mutated DNA sequences is a likely regulator of cancer progression. Noncoding regulatory mutations such as those on the core promoter of the gene encoding human telomerase reverse transcriptase have been shown to affect gene expression in cancer. Using a protein microarray of 667 transcription factor DNA-binding domains and subsequent functional assays, we looked for transcription factors that preferentially bind the mutant hTERT promoter and characterized their downstream effects. One of them, friend leukemia integration 1 (FLI1), which belongs to the E26 transforming-specific family of transcription factors, exhibited particularly strong effects with respect to regulating hTERT expression, while the even better binding ELK3 did not. Depletion of FLI1 decreased expression of the genes for cyclin D1 (CCND1) and E2F transcription factor 2 (E2F2) resulting in a G1/S cell cycle arrest and in consequence a reduction of cell proliferation. FLI1 also affected CMTM7, another gene involved in G1/S transition, although by another process that suggests a balanced regulation of the tumor suppressor gene's activity via opposing regulation processes. FLI1 expression was found upregulated and correlated with an increase in CCND1 expression in pancreatic cancer and brain tumors. In non-neoplastic lung cells, however, FLI1 depletion led to rapid progression through the cell cycle. This coincides with the fact that FLI1 is downregulated in lung tumors. Taken together, our data indicate a cell cycle regulatory hub involving FLI1, hTERT, CCND1 and E2F2 in a tissue- and context-dependent manner.