Publications by authors named "Frederike Dijk"

31 Publications

CCAAT/Enhancer-Binding Protein Delta (C/EBPδ): A Previously Unrecognized Tumor Suppressor that Limits the Oncogenic Potential of Pancreatic Ductal Adenocarcinoma Cells.

Cancers (Basel) 2020 Sep 7;12(9). Epub 2020 Sep 7.

Laboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Amsterdam University Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.

CCAAT/enhancer-binding protein δ (C/EBPδ) is a transcription factor involved in growth arrest and differentiation, which has consequently been suggested to harbor tumor suppressive activities. However, C/EBPδ over-expression correlates with poor prognosis in glioblastoma and promotes genomic instability in cervical cancer, hinting at an oncogenic role of C/EBPδ in these contexts. Here, we explore the role of C/EBPδ in pancreatic cancer. We determined C/EBPδ expression in biopsies from pancreatic cancer patients using public gene-expression datasets and in-house tissue microarrays. We found that C/EBPδ is highly expressed in healthy pancreatic ductal cells but lost in pancreatic ductal adenocarcinoma. Furthermore, loss of C/EBPδ correlated with increased lymph node involvement and shorter overall survival in pancreatic ductal adenocarcinoma patients. In accordance with this, in vitro experiments showed reduced clonogenic capacity and proliferation of pancreatic ductal adenocarcinoma cells following C/EBPδ re-expression, concurrent with decreased sphere formation capacity in soft agar assays. We thus report a previously unrecognized but important tumor suppressor role of C/EBPδ in pancreatic ductal adenocarcinoma. This is of particular interest since only few tumor suppressors have been identified in the context of pancreatic cancer. Moreover, our findings suggest that restoration of C/EBPδ activity could hold therapeutic value in pancreatic ductal adenocarcinoma, although the latter claim needs to be substantiated in future studies.
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http://dx.doi.org/10.3390/cancers12092546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564797PMC
September 2020

Macrophage-secreted MMP9 induces mesenchymal transition in pancreatic cancer cells via PAR1 activation.

Cell Oncol (Dordr) 2020 Dec 18;43(6):1161-1174. Epub 2020 Aug 18.

Center for Experimental and Molecular Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Purpose: Targeting tumor-infiltrating macrophages limits progression and improves chemotherapeutic responses in pancreatic ductal adenocarcinoma (PDAC). Protease-activated receptor (PAR)1 drives monocyte/macrophage recruitment, and stromal ablation of PAR1 limits cancer growth and enhances gemcitabine sensitivity in experimental PDAC. However, the functional interplay between PAR1, macrophages and tumor cells remains unexplored. Here we address the PAR1-macrophage-tumor cell crosstalk and assess its contributions to tumor progression.

Methods: PAR1 expression and macrophage infiltration were correlated in primary PDAC biopsies using gene expression datasets and tissue microarrays. Medium transfer experiments were used to evaluate the functional consequences of macrophage-tumor cell crosstalk and to assess the contribution of PAR1 to the observed responses. PAR1 cleavage assays were used to identify a macrophage-secreted PAR1 agonist, and the effects of candidate proteases were assessed in medium transfer experiments with specific inhibitors and/or recombinant agonist.

Results: PAR1 expression correlates with macrophage infiltration in primary PDACs, and macrophages induce mesenchymal transition of PDAC cells through PAR1 activation. Protease profiling identified macrophage-secreted matrix metalloprotease 9 (MMP9) as the relevant PAR1 agonist in PDAC. PAR1 and/or MMP9 inhibition limited macrophage-driven mesenchymal transition. Likewise, preventing mesenchymal transition by silencing ZEB1 or by pharmacological inhibition of the MMP9/PAR1 axis significantly reduced the ability of tumor cells to survive the anti-tumor activities of macrophages.

Conclusion: Macrophages secrete MMP9, which acts upon PDAC cell PAR1 to induce mesenchymal transition. This macrophage-induced mesenchymal transition supports the tumor-promoting role of macrophage influx, explaining the dichotomous contributions of these immune cells to tumor growth.
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http://dx.doi.org/10.1007/s13402-020-00549-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717035PMC
December 2020

Characterizing and classifying neuroendocrine neoplasms through microRNA sequencing and data mining.

NAR Cancer 2020 Sep 15;2(3):zcaa009. Epub 2020 Jul 15.

Laboratory of Translational RNA Biology, Department of Pathology and Molecular Medicine, Queen's University, 88 Stuart Street, Kingston, ON K7L 3N6, Canada.

Neuroendocrine neoplasms (NENs) are clinically diverse and incompletely characterized cancers that are challenging to classify. MicroRNAs (miRNAs) are small regulatory RNAs that can be used to classify cancers. Recently, a morphology-based classification framework for evaluating NENs from different anatomical sites was proposed by experts, with the requirement of improved molecular data integration. Here, we compiled 378 miRNA expression profiles to examine NEN classification through comprehensive miRNA profiling and data mining. Following data preprocessing, our final study cohort included 221 NEN and 114 non-NEN samples, representing 15 NEN pathological types and 5 site-matched non-NEN control groups. Unsupervised hierarchical clustering of miRNA expression profiles clearly separated NENs from non-NENs. Comparative analyses showed that miR-375 and miR-7 expression is substantially higher in NEN cases than non-NEN controls. Correlation analyses showed that NENs from diverse anatomical sites have convergent miRNA expression programs, likely reflecting morphological and functional similarities. Using machine learning approaches, we identified 17 miRNAs to discriminate 15 NEN pathological types and subsequently constructed a multilayer classifier, correctly identifying 217 (98%) of 221 samples and overturning one histological diagnosis. Through our research, we have identified common and type-specific miRNA tissue markers and constructed an accurate miRNA-based classifier, advancing our understanding of NEN diversity.
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http://dx.doi.org/10.1093/narcan/zcaa009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380486PMC
September 2020

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

Int J Cancer 2020 Oct 1;147(8):2065-2074. Epub 2020 May 1.

Genomic Epidemiology Group, German Cancer Research Center (DKFZ), Heidelberg, Germany.

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 ). 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 ). 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.
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http://dx.doi.org/10.1002/ijc.33004DOI Listing
October 2020

High-grade mesenchymal pancreatic ductal adenocarcinoma drives stromal deactivation through CSF-1.

EMBO Rep 2020 05 16;21(5):e48780. Epub 2020 Mar 16.

Laboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundance of stroma. Multiple molecular classification efforts have identified a mesenchymal tumor subtype that is consistently characterized by high-grade growth and poor clinical outcome. The relation between PDAC stroma and tumor subtypes is still unclear. Here, we aimed to identify how PDAC cells instruct the main cellular component of stroma, the pancreatic stellate cells (PSCs). We found in primary tissue that high-grade PDAC had reduced collagen deposition compared to low-grade PDAC. Xenografts and organotypic co-cultures established from mesenchymal-like PDAC cells featured reduced collagen and activated PSC content. Medium transfer experiments using a large set of PDAC cell lines revealed that mesenchymal-like PDAC cells consistently downregulated ACTA2 and COL1A1 expression in PSCs and reduced proliferation. We identified colony-stimulating factor 1 as the mesenchymal PDAC-derived ligand that deactivates PSCs, and inhibition of its receptor CSF1R was able to counteract this effect. In conclusion, high-grade PDAC features stroma that is low in collagen and activated PSC content, and targeting CSF1R offers direct options to maintain a tumor-restricting microenvironment.
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http://dx.doi.org/10.15252/embr.201948780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202203PMC
May 2020

Unsupervised class discovery in pancreatic ductal adenocarcinoma reveals cell-intrinsic mesenchymal features and high concordance between existing classification systems.

Sci Rep 2020 01 15;10(1):337. Epub 2020 Jan 15.

Laboratory for Experimental Oncology and Radiobiology, Amsterdam UMC, University of Amsterdam and Cancer Center Amsterdam, Amsterdam, Netherlands.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all common cancers. However, divergent outcomes exist between patients, suggesting distinct underlying tumor biology. Here, we delineated this heterogeneity, compared interconnectivity between classification systems, and experimentally addressed the tumor biology that drives poor outcome. RNA-sequencing of 90 resected specimens and unsupervised classification revealed four subgroups associated with distinct outcomes. The worst-prognosis subtype was characterized by mesenchymal gene signatures. Comparative (network) analysis showed high interconnectivity with previously identified classification schemes and high robustness of the mesenchymal subtype. From species-specific transcript analysis of matching patient-derived xenografts we constructed dedicated classifiers for experimental models. Detailed assessments of tumor growth in subtyped experimental models revealed that a highly invasive growth pattern of mesenchymal subtype tumor cells is responsible for its poor outcome. Concluding, by developing a classification system tailored to experimental models, we have uncovered subtype-specific biology that should be further explored to improve treatment of a group of PDAC patients that currently has little therapeutic benefit from surgical treatment.
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http://dx.doi.org/10.1038/s41598-019-56826-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962149PMC
January 2020

Time-Dependent Impact of Irreversible Electroporation on Pathology and Ablation Size in the Porcine Liver: A 24-Hour Experimental Study.

Technol Cancer Res Treat 2019 01;18:1533033819876899

Department of Pathology, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands.

Irreversible electroporation causes cell death through low frequency, high voltage electrical pulses and is increasingly used to treat non-resectable cancers. A recent systematic review revealed that tissue damage through irreversible electroporation is time-dependent, but the impact of time on the ablation zone size remains unknown. Irreversible electroporation ablations were performed hourly during 24 consecutive hours in the peripheral liver of 2 anaesthetized domestic pigs using clinical treatment settings. Immediately after the 24th ablation, the livers were harvested and examined for tissue response in time based on macroscopic and microscopic pathology. The impact of time on these outcomes was assessed with Spearman rank correlation test. Ablation zones were sharply demarcated as early as 1 hour after treatment. During 24 hours, the ablation zones showed a significant increase in diameter (rs = 0.493, = .014) and total surface (rs = 0.499, = .013), whereas the impact of time on the homogeneous ablated area was not significant (rs = 0.172, = .421). Therefore, the increase in size could mainly be attributed to an increase in the transition zone. Microscopically, the ablation zones showed progression in cell death and inflammation. This study assessed the dynamics of irreversible electroporation on the porcine liver during 24 consecutive hours and found that the pathological response (ie, cell death/inflammation), and ablation size continue to develop for at least 24 hours. Consequently, future studies on irreversible electroporation should prolong their observation period.
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http://dx.doi.org/10.1177/1533033819876899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753518PMC
January 2019

Unravelling the Diagnostic Dilemma: A MicroRNA Panel of Circulating MiR-16 and MiR-877 as A Diagnostic Classifier for Distal Bile Duct Tumors.

Cancers (Basel) 2019 Aug 15;11(8). Epub 2019 Aug 15.

Department of Surgery, Cancer Center Amsterdam, Amsterdam UMC, VU University Amsterdam, 1081 HV, The Netherlands.

Accurate diagnosis of pancreatic head lesions remains challenging as no minimally invasive biomarkers are available to discriminate distal cholangiocarcinoma (CCA) from pancreatic ductal adenocarcinoma (PDAC). The aim of this study is to identify specific circulating microRNAs (miRNAs) to diagnose distal CCA. In the discovery phase, PCR profiling of 752 miRNAs was performed on fourteen patients with distal CCA and age- and sex-matched healthy controls. Candidate miRNAs were selected for evaluation and validation by RT-qPCR in an independent cohort of distal CCA ( = 24), healthy controls ( = 32), benign diseases ( = 20), and PDAC ( = 24). The optimal diagnostic combination of miRNAs was determined by multivariate logistic regression analysis and evaluated by ROC curves with AUC values. The discovery phase revealed 19 significantly dysregulated miRNAs, of which six were validated in the evaluation phase. The validation phase confirmed downregulated miR-16 in patients with distal CCA compared to benign disease or PDAC ( = 0.048 and = 0.012), while miR-877 was significantly upregulated ( = 0.003 and = 0.006). This two-miRNA panel was validated as a CCA-specific profile, discriminating distal CCA from benign disease (AUC = 0.90) and from PDAC (AUC = 0.88). In conclusion, the present study identified a two-miRNA panel of downregulated miR-16 and upregulated miR-877 with promising capability to diagnose patients with distal CCA.
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http://dx.doi.org/10.3390/cancers11081181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721566PMC
August 2019

Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma.

Int J Cancer 2020 03 13;146(5):1445-1456. Epub 2019 Aug 13.

Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Circulating tumor DNA (ctDNA) is assumed to reflect tumor burden and has been suggested as a tool for prognostication and follow-up in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). However, the prognostic value of ctDNA and its relation with tumor burden has yet to be substantiated, especially in mPDAC. In this retrospective analysis of prospectively collected samples, cell-free DNA from plasma samples of 58 treatment-naive mPDAC patients was isolated and sequenced using a custom-made pancreatobiliary NGS panel. Pathogenic mutations were detected in 26/58 (44.8%) samples. Cross-check with droplet digital PCR showed good agreement in Bland-Altman analysis (p = 0.217, nonsignificance indicating good agreement). In patients with liver metastases, ctDNA was more frequently detected (24/37, p < 0.001). Tumor volume (3D reconstructions from imaging) and ctDNA variant allele frequency (VAF) were correlated (Spearman's ρ = 0.544, p < 0.001). Median overall survival (OS) was 3.2 (95% confidence interval [CI] 1.6-4.9) versus 8.4 (95% CI 1.6-15.1) months in patients with detectable versus undetectable ctDNA (p = 0.005). Both ctDNA VAF and tumor volume independently predicted OS after adjustment for carbohydrate antigen 19.9 and treatment regimen (hazard ratio [HR] 1.05, 95% CI 1.01-1.09, p = 0.005; HR 1.00, 95% CI 1.01-1.05, p = 0.003). In conclusion, our study showed that ctDNA detection rates are higher in patients with larger tumor volume and liver metastases. Nevertheless, measurements may diverge and, thus, can provide complementary information. Both ctDNA VAF and tumor volume were strong predictors of OS.
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http://dx.doi.org/10.1002/ijc.32586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7004068PMC
March 2020

Prognostic immunohistochemical biomarkers of chemotherapy efficacy in biliary tract cancer: A systematic review and meta-analysis.

Crit Rev Oncol Hematol 2019 Sep 8;141:82-94. Epub 2019 Jun 8.

Amsterdam UMC, University of Amsterdam, Dept. Medical Oncology, Cancer Center Amsterdam, Amsterdam, the Netherlands. Electronic address:

Introduction: Chemotherapy is the mainstay of systemic treatment of biliary tract cancer (BTC). However, the treatment response to chemotherapy varies between patients. Currently, no prognostic biomarkers for chemotherapy efficacy have been considered for use in clinical practice. A systematic review was conducted to evaluate the prognostic value of immunohistochemical biomarkers for chemotherapy in patients with resected as well as with advanced BTC.

Method: Medline and EMBASE databases were searched up to March 2017 for studies that evaluated biomarker expression by immunohistochemistry in resected or advanced BTC patients treated with chemotherapy. The primary endpoints were overall survival (OS) and disease or progression free survival (DFS or PFS).

Result: Twenty-six studies, including a total of 1348 patients and 26 different biomarkers, met the inclusion criteria and were included in this review. The most frequently studied prognostic biomarkers in BTC were the human Equilibrative Nucleoside Transporter 1 (hENT1), Ribonucleotide Reductase M1 (RRM1), and excision repair cross-complementation 1 (ERCC1). In the meta-analysis of patients treated with gemcitabine-based chemotherapy, high hENT1 expression was associated with longer OS (HR 0.43, 95% CI: 0.28 to 0.64) and DFS/PFS (HR 0.45, 95% CI: 0.33 to 0.61).

Conclusion: hENT1 is a promising prognostic biomarker for gemcitabine-based chemotherapy in resected as well as in advanced BTC and should be further validated for the selection of patients for chemotherapy.
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http://dx.doi.org/10.1016/j.critrevonc.2019.06.001DOI Listing
September 2019

Dilemmas for the pathologist in the oncologic assessment of pancreatoduodenectomy specimens : An overview of different grossing approaches and the relevance of the histopathological characteristics in the oncologic assessment of pancreatoduodenectomy specimens.

Virchows Arch 2018 Apr 27;472(4):533-543. Epub 2018 Mar 27.

Department of pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands.

A pancreatoduodenectomy specimen is complex, and there is much debate on how it is best approached by the pathologist. In this review, we provide an overview of topics relevant for current clinical practice in terms of gross dissection, and macro- and microscopic assessment of the pancreatoduodenectomy specimen with a suspicion of suspected pancreatic cancer. Tumor origin, tumor size, degree of differentiation, lymph node status, and resection margin status are universally accepted as prognostic for survival. However, different guidelines diverge on important issues, such as the diagnostic criteria for evaluating the completeness of resection. The macroscopic assessment of the site of origin in periampullary tumors and cystic lesions is influenced by the grossing method. Bi-sectioning of the head of the pancreas may offer an advantage in this respect, as this method allows for optimal visualization of the periampullary area. However, a head-to-head comparison of the assessment of clinically relevant parameters, using axial slicing versus bi-sectioning, is not available yet and the gold standard to compare both techniques prospectively might be subject of debate. Further studies are required to validate the various dissection protocols used for pancreatoduodenectomy specimens and their specific value in the assessment of pathological parameters relevant for prognosis.
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http://dx.doi.org/10.1007/s00428-018-2321-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924671PMC
April 2018

The Dutch Pancreas Biobank Within the Parelsnoer Institute: A Nationwide Biobank of Pancreatic and Periampullary Diseases.

Pancreas 2018 04;47(4):495-501

Objectives: Large biobanks with uniform collection of biomaterials and associated clinical data are essential for translational research. The Netherlands has traditionally been well organized in multicenter clinical research on pancreatic diseases, including the nationwide multidisciplinary Dutch Pancreatic Cancer Group and Dutch Pancreatitis Study Group. To enable high-quality translational research on pancreatic and periampullary diseases, these groups established the Dutch Pancreas Biobank.

Methods: The Dutch Pancreas Biobank is part of the Parelsnoer Institute and involves all 8 Dutch university medical centers and 5 nonacademic hospitals. Adult patients undergoing pancreatic surgery (all indications) are eligible for inclusion. Preoperative blood samples, tumor tissue from resected specimens, pancreatic cyst fluid, and follow-up blood samples are collected. Clinical parameters are collected in conjunction with the mandatory Dutch Pancreatic Cancer Audit.

Results: Between January 2015 and May 2017, 488 patients were included in the first 5 participating centers: 4 university medical centers and 1 nonacademic hospital. Over 2500 samples were collected: 1308 preoperative blood samples, 864 tissue samples, and 366 follow-up blood samples.

Conclusions: Prospective collection of biomaterials and associated clinical data has started in the Dutch Pancreas Biobank. Subsequent translational research will aim to improve treatment decisions based on disease characteristics.
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http://dx.doi.org/10.1097/MPA.0000000000001018DOI Listing
April 2018

Genome-wide meta-analysis identifies five new susceptibility loci for pancreatic cancer.

Authors:
Alison P Klein Brian M Wolpin Harvey A Risch Rachael Z Stolzenberg-Solomon Evelina Mocci Mingfeng Zhang Federico Canzian Erica J Childs Jason W Hoskins Ashley Jermusyk Jun Zhong Fei Chen Demetrius Albanes Gabriella Andreotti Alan A Arslan Ana Babic William R Bamlet Laura Beane-Freeman Sonja I Berndt Amanda Blackford Michael Borges Ayelet Borgida Paige M Bracci Lauren Brais Paul Brennan Hermann Brenner Bas Bueno-de-Mesquita Julie Buring Daniele Campa Gabriele Capurso Giulia Martina Cavestro Kari G Chaffee Charles C Chung Sean Cleary Michelle Cotterchio Frederike Dijk Eric J Duell Lenka Foretova Charles Fuchs Niccola Funel Steven Gallinger J Michael M Gaziano Maria Gazouli Graham G Giles Edward Giovannucci Michael Goggins Gary E Goodman Phyllis J Goodman Thilo Hackert Christopher Haiman Patricia Hartge Manal Hasan Peter Hegyi Kathy J Helzlsouer Joseph Herman Ivana Holcatova Elizabeth A Holly Robert Hoover Rayjean J Hung Eric J Jacobs Krzysztof Jamroziak Vladimir Janout Rudolf Kaaks Kay-Tee Khaw Eric A Klein Manolis Kogevinas Charles Kooperberg Matthew H Kulke Juozas Kupcinskas Robert J Kurtz Daniel Laheru Stefano Landi Rita T Lawlor I-Min Lee Loic LeMarchand Lingeng Lu Núria Malats Andrea Mambrini Satu Mannisto Roger L Milne Beatrice Mohelníková-Duchoňová Rachel E Neale John P Neoptolemos Ann L Oberg Sara H Olson Irene Orlow Claudio Pasquali Alpa V Patel Ulrike Peters Raffaele Pezzilli Miquel Porta Francisco X Real Nathaniel Rothman Ghislaine Scelo Howard D Sesso Gianluca Severi Xiao-Ou Shu Debra Silverman Jill P Smith Pavel Soucek Malin Sund Renata Talar-Wojnarowska Francesca Tavano Mark D Thornquist Geoffrey S Tobias Stephen K Van Den Eeden Yogesh Vashist Kala Visvanathan Pavel Vodicka Jean Wactawski-Wende Zhaoming Wang Nicolas Wentzensen Emily White Herbert Yu Kai Yu Anne Zeleniuch-Jacquotte Wei Zheng Peter Kraft Donghui Li Stephen Chanock Ofure Obazee Gloria M Petersen Laufey T Amundadottir

Nat Commun 2018 02 8;9(1):556. Epub 2018 Feb 8.

Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.

In 2020, 146,063 deaths due to pancreatic cancer are estimated to occur in Europe and the United States combined. To identify common susceptibility alleles, we performed the largest pancreatic cancer GWAS to date, including 9040 patients and 12,496 controls of European ancestry from the Pancreatic Cancer Cohort Consortium (PanScan) and the Pancreatic Cancer Case-Control Consortium (PanC4). Here, we find significant evidence of a novel association at rs78417682 (7p12/TNS3, P = 4.35 × 10). Replication of 10 promising signals in up to 2737 patients and 4752 controls from the PANcreatic Disease ReseArch (PANDoRA) consortium yields new genome-wide significant loci: rs13303010 at 1p36.33 (NOC2L, P = 8.36 × 10), rs2941471 at 8q21.11 (HNF4G, P = 6.60 × 10), rs4795218 at 17q12 (HNF1B, P = 1.32 × 10), and rs1517037 at 18q21.32 (GRP, P = 3.28 × 10). rs78417682 is not statistically significantly associated with pancreatic cancer in PANDoRA. Expression quantitative trait locus analysis in three independent pancreatic data sets provides molecular support of NOC2L as a pancreatic cancer susceptibility gene.
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http://dx.doi.org/10.1038/s41467-018-02942-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805680PMC
February 2018

Tumor manipulation during pancreatic resection for pancreatic cancer induces dissemination of tumor cells into the peritoneal cavity: a systematic review.

HPB (Oxford) 2018 04;20(4):289-296

Department of Surgery, OLVG, Amsterdam, The Netherlands; GastroIntestinal Oncology Center Amsterdam (GIOCA), The Netherlands. Electronic address:

Background: Intraoperative tumor manipulation may induce the dissemination of occult peritoneal tumor cells (OPTC) into the peritoneal cavity.

Methods: A systematic review was performed in the PubMed, Embase and Cochrane databases from inception to March 15, 2017. Eligible were studies that analyzed the presence of OPTC in peritoneal fluid, by any method, both before and after resection in adults who underwent intentionally curative pancreatic resection for histopathologically confirmed pancreatic ductal adenocarcinoma in absence of macroscopic peritoneal metastases.

Results: Four studies with 138 patients met the inclusion criteria. The pooled rate of OPTC prior to tumor manipulation was 8% (95% CI 2%-24%). The pooled detection rate of OPTC in patients in whom OPTC became detectable only after tumor manipulation was 33% (95% CI 15-58%). Only one study (28 patients) reported on survival, which was worse in patients with OPTC (median 11.1 months versus 30.3 months; p = 0.030).

Conclusion: This systematic review suggests that tumor manipulation induces OPTC in one third of patients with pancreatic cancer. Since data on survival are lacking, future studies should determine the prognostic consequences of tumor manipulation, including the potential therapeutic effect of 'no-touch' and minimally invasive resection strategies.
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http://dx.doi.org/10.1016/j.hpb.2017.08.018DOI Listing
April 2018

Do pancreatic cancer and chronic pancreatitis share the same genetic risk factors? A PANcreatic Disease ReseArch (PANDoRA) consortium investigation.

Int J Cancer 2018 01 16;142(2):290-296. Epub 2017 Oct 16.

Genomic Epidemiology Group, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive tumor with a five-year survival of less than 6%. Chronic pancreatitis (CP), an inflammatory process in of the pancreas, is a strong risk factor for PDAC. Several genetic polymorphisms have been discovered as susceptibility loci for both CP and PDAC. Since CP and PDAC share a consistent number of epidemiologic risk factors, the aim of this study was to investigate whether specific CP risk loci also contribute to PDAC susceptibility. We selected five common SNPs (rs11988997, rs379742, rs10273639, rs2995271 and rs12688220) that were identified as susceptibility markers for CP and analyzed them in 2,914 PDAC cases, 356 CP cases and 5,596 controls retrospectively collected in the context of the international PANDoRA consortium. We found a weak association between the minor allele of the PRSS1-PRSS2-rs10273639 and an increased risk of developing PDAC (OR  = 1.19, 95% CI 1.02-1.38, p = 0.023). Additionally all the SNPs confirmed statistically significant associations with risk of developing CP, the strongest being PRSS1-PRSS2-rs10273639 (OR  = 0.51, 95% CI 0.39-0.67, p = 1.10 × 10 ) and MORC4-rs 12837024 (OR  = 2.07 (1.55-2.77, p  = 0.7 × 10 ). Taken together, the results from our study do not support variants rs11988997, rs379742, rs10273639, rs2995271 and rs12688220 as strong predictors of PDAC risk, but further support the role of these SNPs in CP susceptibility. Our study suggests that CP and PDAC probably do not share genetic susceptibility, at least in terms of high frequency variants.
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http://dx.doi.org/10.1002/ijc.31047DOI Listing
January 2018

Prognostic value of occult tumor cells obtained by peritoneal lavage in patients with resectable pancreatic cancer and no ascites: A systematic review.

J Surg Oncol 2016 Nov 19;114(6):743-751. Epub 2016 Sep 19.

Department of Surgery, Onze Lieve Vrouwe Gasthuis, Amsterdam, The Netherlands.

The poor survival of patients with resectable pancreatic cancer might be related to the presence of occult peritoneal tumor cells (OPTC). This systematic review studies the prognostic value of cytology and carcinoembryonic antigen (CEA) by real-time polymerase chain reaction in peritoneal fluid. The results suggest that presence of OPTC is related to a worse survival in patients with resectable pancreatic cancer. Future studies should investigate its possible role in selecting patients for specific treatment strategies. J. Surg. Oncol. 2016;114:743-751. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jso.24402DOI Listing
November 2016

Functional single nucleotide polymorphisms within the cyclin-dependent kinase inhibitor 2A/2B region affect pancreatic cancer risk.

Oncotarget 2016 08;7(35):57011-57020

Genomic Epidemiology Group, German Cancer Research Center (DKFZ), Heidelberg, Germany.

The CDKN2A (p16) gene plays a key role in pancreatic cancer etiology. It is one of the most commonly somatically mutated genes in pancreatic cancer, rare germline mutations have been found to be associated with increased risk of developing familiar pancreatic cancer and CDKN2A promoter hyper-methylation has been suggested to play a critical role both in pancreatic cancer onset and prognosis. In addition several unrelated SNPs in the 9p21.3 region, that includes the CDNK2A, CDNK2B and the CDNK2B-AS1 genes, are associated with the development of cancer in various organs. However, association between the common genetic variability in this region and pancreatic cancer risk is not clearly understood. We sought to fill this gap in a case-control study genotyping 13 single nucleotide polymorphisms (SNPs) in 2,857 pancreatic ductal adenocarcinoma (PDAC) patients and 6,111 controls in the context of the Pancreatic Disease Research (PANDoRA) consortium. We found that the A allele of the rs3217992 SNP was associated with an increased pancreatic cancer risk (ORhet=1.14, 95% CI 1.01-1.27, p=0.026, ORhom=1.30, 95% CI 1.12-1.51, p=0.00049). This pleiotropic variant is reported to be a mir-SNP that, by changing the binding site of one or more miRNAs, could influence the normal cell cycle progression and in turn increase PDAC risk. In conclusion, we observed a novel association in a pleiotropic region that has been found to be of key relevance in the susceptibility to various types of cancer and diabetes suggesting that the CDKN2A/B locus could represent a genetic link between diabetes and pancreatic cancer risk.
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http://dx.doi.org/10.18632/oncotarget.10935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302969PMC
August 2016

Long-term follow-up of neoplastic pancreatic cysts without high-risk stigmata: how often do we change treatment strategy because of malignant transformation?

Scand J Gastroenterol 2016 Sep 13;51(9):1138-43. Epub 2016 May 13.

a Department of Gastroenterology and Hepatology , Academic Medical Center, University of Amsterdam , Amsterdam , The Netherlands ;

Unlabelled: *

Objective: Patients with potentially premalignant neoplastic pancreatic cysts without high-risk stigmata usually enter a surveillance program. Data on outcomes of such surveillance programs are scarce. We aimed to evaluate the resection rate and malignancy rate during follow-up.

Material And Methods: From our prospective database (2006-2015) of patients with pancreatic cysts, we analyzed patients with pancreatic cysts without high-risk stigmata with at least six months follow-up.

Results: In total, 146 patients were followed for a median of 29 months (IQR 13.5-50 months). In 124 patients (84.9%), no changes in clinical or imaging characteristics occurred during follow-up. Thirteen patients (8.9%) developed an indication for surgery after a median follow-up of 25 months (IQR 12-42 months). Two patients did not undergo surgery because of comorbidity, 11 patients (7.5%) underwent resection. Indications for surgery were symptoms (n = 2), development of a pancreatic mass (n = 1), a new nodule (n = 2), thickened cyst wall (n = 1), pancreatic duct dilation (n = 3), and/or suspicion of mucinous cystic neoplasm (MCN) (n = 3). Postoperative histology showed one pancreatic malignancy not originating from the cyst, three mixed type-intraductal papillary mucinous neoplasm (IPMN), one side branch-IPMN, two MCN, one neuroendocrine tumor, one serous cystadenoma, one inflammatory cyst, and one lymphangioma. The highest grade of cyst dysplasia was borderline dysplasia.

Conclusions: Most neoplastic pancreatic cysts without high-risk stigmata at initial presentation show no substantial change during 1-4-year follow-up. Only 7.5% of patients underwent surgery and less than 1% of patients developed pancreatic malignancy. This indicates that additional markers are needed to tailor treatment of pancreatic cysts.
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http://dx.doi.org/10.1080/00365521.2016.1179338DOI Listing
September 2016

Establishment of patient-derived xenograft models and cell lines for malignancies of the upper gastrointestinal tract.

J Transl Med 2015 Apr 11;13:115. Epub 2015 Apr 11.

Laboratory for Experimental Oncology and Radiobiology, Academic Medical Center, Meibergdreef 9, Amsterdam, AZ, 1105, The Netherlands.

Background: The upper gastrointestinal tract is home to some of most notorious cancers like esophagogastric and pancreatic cancer. Several factors contribute to the lethality of these tumors, but one that stands out for both tumor types is the strong inter- as well as intratumor heterogeneity. Unfortunately, genetic tumor models do not match this heterogeneity, and for esophageal cancer no adequate genetic models exist. To allow for an improved understanding of these diseases, tissue banks with sufficient amount of samples to cover the extent of diversity of human cancers are required. Additionally, xenograft models that faithfully mimic and span the breadth of human disease are essential to perform meaningful functional experiments.

Methods: We describe here the establishment of a tissue biobank, patient derived xenografts (PDXs) and cell line models of esophagogastric and pancreatic cancer patients. Biopsy material was grafted into immunocompromised mice and PDXs were used to establish primary cell cultures to perform functional studies. Expression of Hedgehog ligands in patient tumor and matching PDX was assessed by immunohistochemical staining, and quantitative real-time PCR as well as flow cytometry was used for cultured cells. Cocultures with Hedgehog reporter cells were performed to study paracrine signaling potency. Furthermore, SHH expression was modulated in primary cultures using lentiviral mediated knockdown.

Results: We have established a panel of 29 PDXs from esophagogastric and pancreatic cancers, and demonstrate that these PDXs mirror several of the (immuno)histological and biochemical characteristics of the original tumors. Derived cell lines can be genetically manipulated and used to further study tumor biology and signaling capacity. In addition, we demonstrate an active (paracrine) Hedgehog signaling mode by both tumor types, the magnitude of which has not been compared directly in previous studies.

Conclusions: Our established PDXs and their matching primary cell lines retain important characteristics seen in the original tumors, and this should enable future studies to address the responses of these tumors to different treatment modalities, but also help in gaining mechanistic insight in how some tumors respond to certain regimens and others do not.
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http://dx.doi.org/10.1186/s12967-015-0469-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4419410PMC
April 2015

Molecular diagnosis of minimal residual disease in head and neck cancer patients.

Cell Oncol (Dordr) 2012 Oct 4;35(5):367-75. Epub 2012 Sep 4.

Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, PO Box 7057, 1007 MB, Amsterdam, The Netherlands.

Aim: Locoregional recurrences and distant metastases in adequately treated head and neck squamous cell carcinoma (HNSCC) patients have a dismal effect on survival. Tumor cells that escape histopathological detection might be the prime cause of this effect. We evaluated whether minimal residual cancer (MRC) in deep surgical margins and disseminated tumor cells (DTCs) in bone marrow aspirates are associated with clinicohistopathological parameters and outcome.

Methods: Submucosal samples of deep resection margins of 105 HNSCC patients with histopathologically tumor-free surgical margins were analysed for the presence of MRC using hLy-6D qRT-PCR. Bone-marrow aspirates of 76 of these patients were analysed for DTCs by immunocytochemical staining. Presence of molecular-positive deep surgical margins, presence of DTC in bone marrow aspirates, and clinicohistopathological parameters were tested for associations with survival parameters by univariate and multivariate analyses.

Results: In addition to lymph node stage, it appeared that vasoinvasive growth and particularly infiltrative growth pattern are significant predictors for locoregional recurrence (p = 0.041 and p = 0.006, respectively) and disease-free survival (p = 0.014 and p = 0.008, respectively). Remarkably, neither the presence of molecular-positive deep surgical margins nor that of DTC in bone marrow aspirates were significantly related to outcome.

Conclusions: The presence of vasoinvasive and infiltrative growth in HNSCC tumor specimens are significant risk-factors for locoregional recurrence and disease-free survival. At present there seems no role for molecular analysis of deep surgical margins and bone marrow aspirates in predicting outcome with the methods used.
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http://dx.doi.org/10.1007/s13402-012-0097-1DOI Listing
October 2012

Expression of the antiapoptotic protein BAG3 is a feature of pancreatic adenocarcinoma and its overexpression is associated with poorer survival.

Am J Pathol 2012 Nov 31;181(5):1524-9. Epub 2012 Aug 31.

Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Fisciano, Italy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers, being the fourth leading cause of cancer-related deaths. Long-term survival reaching 15% is achieved in less than 5% of patients who undergo surgery, and median survival is only 6 months in those with inoperable lesions. A deeper understanding of PDAC biologic characteristics as well as novel prognostic markers are therefore required to improve outcomes. Herein we report that BAG3, a protein with recognized anti-apoptotic activity, was expressed in 346 PDACs analyzed, but was not expressed in the surrounding nonneoplastic tissue. In a cohort of 66 patients who underwent radical resection (R0), survival was significantly shorter in patients with high BAG3 expression (median, 12 months) than in those with low BAG3 expression (median, 23 months) (P = 0.001). Furthermore, we report that BAG3 expression in PDAC-derived cell lines protects from apoptosis and confers resistance to gemcitabine, offering a partial explanation for the survival data. Our results indicate that BAG3 has a relevant role in PDAC biology, and suggest that BAG3 expression level might be a potential marker for prediction of patient outcome.
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http://dx.doi.org/10.1016/j.ajpath.2012.07.016DOI Listing
November 2012

Ischemic preconditioning alters the pattern of gene expression changes in response to full retinal ischemia.

Mol Vis 2007 Oct 5;13:1892-901. Epub 2007 Oct 5.

Department of Molecular Ophthalmogenetics, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences (KNAW), Amsterdam, The Netherlands.

Purpose: Ischemic conditions in the retina have been implicated in several retinopathological conditions. Experimentally induced ischemia for 60 min followed by reperfusion leads to a loss of neurons in the inner retina. In contrast, a 5 min ischemic episode triggers a series of alterations that protect the retina against the damaging effects of a subsequent 60 min ischemic insult. This phenomenon is called ischemic preconditioning (IPC). To study the changes altered by IPC, we assessed the gene expression patterns in the rat retina after ischemia (60 min) followed by reperfusion (I/R) and compared these to the gene expression patterns after ischemia/reperfusion in preconditioned animals (IPC-I/R).

Methods: Changes in gene expression were studied, by means of microarrays, at 1, 2, 6, and 12 h after I/R in naíve and preconditioned animals. To identify functional pathways of interest, we used significantly regulated genes as input for gene ontology analysis. Microarray results were validated by real-time quantitative PCR.

Results: Most genes that were altered by I/R showed a comparable change in both naíve and preconditioned animals. Differential expression was found for a total of 1312 genes of the 20,280 features (6.4%) present on the array with a differential change of 1.7 fold or more. The list of genes with a differential change was characterized by a statistically significant overrepresentation of genes associated to the gene ontology terms tRNA aminoacylation (with a decreased expression due to preconditioning), immune response (with most genes upregulated), and apoptosis (mixed direction of changes). The results of quantitative PCR assays were in agreement with the microarray data.

Conclusions: The response of several functional groups of genes on ischemia was altered by a preconditioning stimulus. Most prominent differences were found for the group of genes encoding for aminoacyl-tRNA synthetases (ARSs), which is in line with the previously observed decreased expression of ARSs after induction of preconditioning. Our observations indicate that activation of translational activity may be a mediator of ischemia-associated damage in the retina, and IPC may prevent activation of this mechanism. An altered expression of genes implicated in immune response and in apoptosis may also be involved in effectuating IPC.
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October 2007

GAP-43 expression is upregulated in retinal ganglion cells after ischemia/reperfusion-induced damage.

Exp Eye Res 2007 May 27;84(5):858-67. Epub 2007 Jan 27.

Molecular Ophthalmogenetics, Netherlands Institute for Neuroscience (NIN), KNAW, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands.

In response to injury, the adult mammalian retina shows signs of structural remodeling, possibly in an attempt to preserve or regain some of its functional neural connections. In order to study the mechanisms involved in injury-induced plasticity, we have studied changes in growth associated protein 43 (GAP-43) after retinal ischemia/reperfusion in the rat. GAP-43 is a marker for neuronal remodeling and is involved in synapse formation. Ischemic injury of the rat retina was induced by 60 min of ischemia followed by reperfusion times varying from 2h up to 4 weeks. GAP-43 mRNA levels were significantly increased between 12h and 72 h reperfusion with a peak around 24h. GAP-43 specific antibodies showed that the total amount of GAP-43 labeling in the inner plexiform layer was diminished after 12h of reperfusion by approximately 35% and remained at this level up to 1 week postischemia despite the reduction in thickness of this layer during this period resulting from the ischemia-induced cell loss. At 2 and 4 weeks reperfusion, the amount of labeling was reduced by 70%, simultaneously with a decrease of GAP-43 transcript level. Between 72 h up to 2 weeks postischemia, the induction of intense GAP-43 labeling was observed in NeuN- and beta-tubulin-positive ganglion cell somata and in horizontally and vertically oriented processes in the inner plexiform layer. Ischemia also induced GAP-43 expression in some GFAP-positive Müller cells. Double-labeling showed that in controls and after ischemia GAP-43 was expressed by some amacrine cells of the glycinergic (glycine transporter 1), calretinin-positive, and dopaminergic (tyrosine hydroxylase) subpopulations. No increase of GAP-43 expression levels was found in these amacrine cells. The results demonstrate that ganglion cells show an elevated expression of GAP-43 after ischemia-inflicted damage. These findings suggest a temporal window during which ganglion cells may remodel their neuronal network in the damaged retina.
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http://dx.doi.org/10.1016/j.exer.2007.01.006DOI Listing
May 2007

Transfer of lens-specific transcripts to retinal RNA samples may underlie observed changes in crystallin-gene transcript levels after ischemia.

Mol Vis 2007 Feb 8;13:220-8. Epub 2007 Feb 8.

Department of Molecular Ophthalmogenetics, Netherlands Institute for Neuroscience-KNAW, Amsterdam, The Netherlands.

Purpose: Retinal ischemia appears to lead to alterations in retinal transcript levels of a group of genes known to be abundantly expressed in the lens. Our purpose is to study whether these alterations are truly the result of retinal ischemia or whether they could be caused by contamination of the retinal tissue with trace amounts of lens tissue.

Methods: Changes occurring in the retinal gene expression profile after induction of retinal ischemia were assessed by oligonucleotide microarrays and by real-time quantitative PCR.

Results: Microarray analysis of the retinal gene expression profile after 5 or 60 min ischemia showed altered transcript levels for a group of genes with functions related to "structural constituent of eye lens" (23 genes, predominantly crystallins). Subsequent qPCR assays for this set of genes showed extremely high variations in transcript levels between individual animals of both control and ischemia-treated groups. However, the relative transcript levels, or expression profile, of these genes was constant in all samples. The transcript levels of these genes were on average 2624-times higher in tissue samples isolated from the superficial layers of the total lens. Moreover, all 23 genes had high expression levels in lens compared to retina as was shown by microarray.

Conclusions: From these data, it appears plausible that during isolation of the retina, trace amounts of lens tissue may end up in the studied retinal samples. This would explain the high level of variability in transcript levels of genes, the strong correlation of relative levels between samples, and the link with lens-specific function of the "altered" genes. Changes in crystallin gene expression in other models of retinal degeneration have been reported and a careful examination of the transcript level of other lens-specific genes is essential to rule out a possible confounding effect of lens-material transfer.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2610369PMC
February 2007

Circadian expression of clock genes and clock-controlled genes in the rat retina.

Biochem Biophys Res Commun 2005 Apr;330(1):18-26

Netherlands Ophthalmic Research Institute (NORI)-KNAW, Glaucoma Research Group, Department of Ophthalmogenetics, Graduate School for the Neurosciences Amsterdam, The Netherlands.

The circadian expression patterns of genes encoding for proteins that make up the core of the circadian clock were measured in rat retina using real-time quantitative PCR (qPCR). Transcript levels of several genes previously used for normalization of qPCR assays were determined and the effect of ischemia-reperfusion on the expression of clock genes was studied. Statistically significant circadian changes in transcript levels were found for: Per2, Per3, Cry2, Bmal1, Rora, Rorb, and Rorc with changes ranging between 1.6- and 2.6-fold. No changes were found for Per1, Cry1, Clock, Rev-erb alpha, and Rev-erb beta. Significant differences in transcript levels were observed for several candidate reference genes: HPRT, GAPDH, rhodopsin, and Thy1 and, consequently, the use of these genes for normalization purposes in qPCR or Northern blots may lead to erroneous conclusions. Ischemia-reperfusion leads to a persistent decrease of Per1 and Cry2, which may be related to the selective degeneration of amacrine and ganglion cells. We conclude that while all clock genes are expressed in the retina, only a few show a clear circadian pattern.
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http://dx.doi.org/10.1016/j.bbrc.2005.02.118DOI Listing
April 2005

An immunocytochemical study on specific amacrine cell subpopulations in the rat retina after ischemia.

Brain Res 2004 Nov;1026(2):205-17

Netherlands Ophthalmic Research Institute, KNAW, Glaucoma Research Group, Research Unit Molecular Ophthalmogenetics, Graduate School for the Neurosciences Amsterdam, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands.

Transient retinal ischemia leads to the loss of neurons in the inner retina. In an accompanying paper [F. Dijk, S. Van Leeuwen, W. Kamphuis, Differential effects of ischemia/reperfusion on amacrine cell subtype-specific transcript levels in the rat retina, Brain Res., 1026 (2004) 194-204] we present the results of a study on the effects of experimentally induced retinal ischemia on transcript levels of genes expressed by distinct subpopulations of amacrine cells. In response to 60-min ischemia, three different patterns of changes in transcript levels were found, indicating a differential vulnerability of amacrine subtypes: (i) a gradual decrease of transcript level without recovery (parvalbumin; PV); (ii) a gradual decrease, with varying rates and degrees, followed by partial recovery after 72 h of reperfusion (choline acetyltransferase (ChAT), calretinin (CR) and glycine transporter (Glyt1)); (iii) no significant changes (substance P (SP)). In order to verify whether the degree of cell loss can be predicted from the quantified alterations in gene expression level, immunocytochemical stainings were carried out. A 60-min ischemic period was administered to the rat eye by raising the intraocular pressure, followed by a reperfusion period lasting between 2 h and 4 weeks. Cryosections were immunostained for Glyt1, PV, ChAT, CR, and SP. Double-labelling with apoptosis marker TUNEL was used to demonstrate cell type-specific apoptosis. Following ischemia, the numbers of detected PV-, Glyt1, ChAT-, and CR-immunopositive somata showed a substantial, but differential, reduction at 1-4 weeks after ischemia. The total amount of immunoreactivity present in the inner plexiform layer (IPL) also decreased. The extent of alterations derived from immunocytochemical staining was greater than was anticipated from the decrease of transcript levels. Only for SP, no significant decrease in number of cells or in the intensity of immunoreactivity in IPL was observed, which is in agreement with the absence of significant changes in transcript levels. In conclusion, retinal ischemia/reperfusion differentially affects amacrine cell populations. Although both protein and mRNA levels are reduced, transcript levels are less attenuated. Caution must be applied in the use of real-time quantitative PCR (qPCR) screening as a tool to assess the cellular pattern of neurodegeneration in the retina.
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http://dx.doi.org/10.1016/j.brainres.2004.08.014DOI Listing
November 2004

Differential effects of ischemia/reperfusion on amacrine cell subtype-specific transcript levels in the rat retina.

Brain Res 2004 Nov;1026(2):194-204

Netherlands Ophthalmic Research Institute KNAW, Glaucoma Research Group, Research Unit Molecular Ophthalmogenetics, Graduate School for the Neurosciences Amsterdam, Meibergdreef 47, Amsterdam 1105 BA, The Netherlands.

Transient retinal ischemia induces loss of retinal ganglion cells, supporting the hypothesis that ischemic conditions contribute to the induction and progression of glaucoma. However, after 60 min of ischemia, also amacrine cells are lost from the inner nuclear layer. The main goal was to determine the relative vulnerability of various amacrine subpopulations by measuring the levels of transcripts that are known to be specifically expressed by different amacrine subpopulations. A 60-min ischemic period was administered to the rat eye by raising the intraocular pressure, followed by a reperfusion period lasting between 2 h and 4 weeks. Total RNA was isolated from the whole retina and expression levels were assessed by real-time quantitative polymerase chain reaction (qPCR). Retinal ischemia/reperfusion has differential effects on the levels of the various transcripts. Three main patterns of changes were identified. (i) A gradual decrease of transcript level without recovery was observed for parvalbumin; this transcript is expressed by the glycinergic AII cells. (ii) A gradual reduction to different levels at 72 h of reperfusion followed by a partial or complete recovery (glycine transporter 1, glutamate decarboxylase, calretinin, and several other transcripts). The glycinergic amacrine cell markers recovered to 65-75% of the control level, while the main GABAergic markers had completely recovered at 4 weeks. (iii) No significant changes of transcript levels were found for markers of several smaller GABAergic subpopulations [including substance P (Tac1), somatostatin, and others]. Expression levels of photoreceptor-, horizontal cell-, and bipolar cell-specific transcripts were not altered. These patterns were confirmed by a cluster analysis of the data. Based on gene expression levels, it may be concluded that amacrine cells are vulnerable to ischemic insults and that the glycinergic amacrine cells are relatively more sensitive to ischemia than the GABAergic population. In particular, the extensive loss of the parvalbumin-containing AII amacrine cells, which serve in the rod pathway, may have functional implications for vision under scotopic conditions. In the accompanying paper [F. Dijk and W. Kamphuis, An immunocytochemical study on specific amacrine subpopulations in the rat retina after ischemia, Brain Res. (2004).], the results are evaluated at the protein level by immunostaining for a selection of the amacrine cell markers.
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http://dx.doi.org/10.1016/j.brainres.2004.08.034DOI Listing
November 2004

Ischemia-induced alterations of AMPA-type glutamate receptor subunit. Expression patterns in the rat retina--an immunocytochemical study.

Brain Res 2004 Feb;997(2):207-21

Glaucoma Research Group, Netherlands Ophthalmic Research Institute-KNAW, Graduate School for the Neurosciences Amsterdam, Meibergdreef 47, 1105 BA, Amsterdam, The Netherlands.

This study investigates whether retinal ischemia/reperfusion leads to alterations in the expression of AMPA-type glutamate receptor (AMPAR) subunits GluR1-4. In ischemia-vulnerable hippocampal neurons, a subunit-specific downregulation of GluR2 precedes the actual neurodegeneration. Our purpose was to study whether retinal ischemia induces a similar downregulation of GluR2 preceding the loss of ganglion and amacrine cells. A 60-min ischemic period was followed by reperfusion lasting between 2 h and 7 days. Changes in the expression patterns of GluR1-4 were assessed using immunocytochemistry. In the same sections, alterations in cell density, thickness of retinal layers, and density of apoptotic cells were investigated. Two-hour post-ischemia, GluR1 immunoreactivity was nearly absent from the inner plexiform layer (IPL). Thereafter, labeling intensity recovered slowly and was close to control levels at 7 days, albeit in a thinner IPL. The decrease in GluR2/3 labeling intensity was most profound at 4 h. The recovery of GluR2/3 staining intensity was slow, and staining was still decreased at 7 days. GluR2 immunoreactivity was not attenuated after ischemia. GluR4 labeling showed a similar time course as observed for GluR1, but the decrease in immunoreactivity was less profound and the recovery was nearly complete. The immunostaining of PKCalpha, a rod bipolar cell marker, was unaffected at all reperfusion times. The reduction of GluR staining preceded both the typical thinning of the IPL and the peak of cell loss, but coincided with a significant swelling of the IPL. In conclusion, retinal ischemia/reperfusion leads to differential changes in the expression of the different AMPA-type GluR subunits, which may affect excitatory synaptic transmission in the inner retina. However, no evidence was found for a preferential loss of GluR2 immunoreactivity that could account for selective neurodegeneration of amacrine and ganglion cells after retinal ischemia.
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http://dx.doi.org/10.1016/j.brainres.2003.08.069DOI Listing
February 2004

Ischemia-induced changes of AMPA-type glutamate receptor subunit expression pattern in the rat retina: a real-time quantitative PCR study.

Invest Ophthalmol Vis Sci 2004 Jan;45(1):330-41

Netherlands Ophthalmic Research Institute-KNAW, Glaucoma Research Group, Graduate School for the Neurosciences Amsterdam, Amsterdam, The Netherlands.

Purpose: To investigate whether the previously observed decrease in immunoreactivity of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunits GluR1, -2, -3, and -4 after ischemia-reperfusion in the rat retina is associated with changes at the mRNA expression level. Furthermore, to study possible changes in the ratios of alternative splice variants of GluR2 and -4 and possible changes in the subunit composition of the receptor complex after ischemia-reperfusion. The ischemia-induced changes were related to expression levels of immediate early genes, c-fos and c-jun, and to expression levels of different cell-type-specific transcripts.

Methods: A 60-minute ischemic event was induced unilaterally in the rat eye by cannulating the anterior chamber and raising the intraocular pressure. Reperfusion was allowed to occur for 2 hours up to 28 days. Total RNA was isolated from the retinas and transcript levels were assessed by real-time quantitative PCR (qPCR).

Results: A differential decrease was observed in the expression levels of all AMPA-type GluR subunits 2 hours after ischemia-reperfusion, with a significant downregulation of GluR2 and -3 transcript levels. At the long-term (72 hours-4 weeks), expression levels for all four subunits were decreased by approximately 64%. No changes were observed, either in the expression ratio of GluR2 and -4 splice variants, or in the relative expression of the different subunits. Immediate early genes c-fos and c-jun were transiently upregulated. Expression levels of the ganglion-cell-specific transcripts Thy-1 and neurofilament and of the AII-amacrine-specific transcript parvalbumin decreased after ischemia-reperfusion, whereas the ON bipolar cell transcripts mGluR6 and PKCalpha did not show ischemia-induced changes.

Conclusions: Shortly after ischemia-reperfusion immunolabeling of GluR1, -2/3, and -4 is strongly decreased, whereas the corresponding mRNA levels are not affected, indicating degradation at the protein level. In contrast, the GluR2 mRNA level is reduced, whereas immunostaining is not yet affected, suggesting that the GluR2 protein is relatively stable under postischemia conditions. The long-term decrease in mRNA levels of all AMPA-type GluR subunits suggests that ischemia affects a main component of the excitatory retinal neurotransmission. It remains to be investigated whether these changes contribute to the subsequent neurodegeneration.
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http://dx.doi.org/10.1167/iovs.03-0285DOI Listing
January 2004

Gene expression of AMPA-type glutamate receptor subunits in rod-type ON bipolar cells of rat retina.

Eur J Neurosci 2003 Sep;18(5):1085-92

Graduate School for the Neurosciences Amsterdam, the Netherlands.

The retinal rod bipolar cell type is involved in the sign-inverting depolarizing ON-type response to light. This response is mediated by the metabotropic glutamate receptor type 6 (mGluR6) expressed on the rod bipolar dendrites. In a previous immunocytochemical study, an unexpected colocalization was reported [W. Kamphuis et al. (2003) J. Comp. Neurol., 455, 172-186] of mGluR6 with the ionotropic AMPA-type glutamate receptor subunit GluR2 in rod bipolar cells of rat retina. The aim of the present study was to investigate whether expression of both genes could be found at the single-cell level. Two approaches were followed. (i). Retinal cells were isolated by enzymatic and mechanical treatment. Single cells with a bipolar morphology were harvested, subjected to multiplex PCR with protein kinase C (PKC)-, mGluR6- and GluR1-4-specific primers, followed by a real-time quantitative PCR assay. Of 23 studied cells, 74% expressed PKC and 87% expressed mGluR6. Using the presence of both transcripts as the criterion for a rod bipolar cell signature (n = 15), 73% of these cells expressed GluR2, with a minor contribution of GluR1 (20%), GluR3 (7%), and GluR4 (20%). Quantification of the transcript levels demonstrated that mGluR6 and GluR2 genes are expressed at similar levels in rod ON-type bipolar cells. (ii). Rod bipolar cells were identified in retinal sections by immunolabelling with a protein kinase C antibody and isolated using laser pressure catapulting (LPC). Quantitative PCR was employed to assess gene expression levels of reference genes, PKCalpha, mGluR6 and the GluR subunits. However, in samples from PKCalpha-immunopositive somata no significant enrichment of PKCalpha transcript levels was observed when compared with control samples from immunonegative somata. We conclude that this approach lacks sufficient spatial specificity. In conclusion, the results show coexpression of mGluR6 and GluR2 in rod bipolar cells; this is in good agreement with the results of previous immunocytochemical studies. The functional implications of AMPA-type glutamate receptors for ON-type rod bipolar-mediated signal transduction remains to be elucidated.
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http://dx.doi.org/10.1046/j.1460-9568.2003.02841.xDOI Listing
September 2003