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Pancreatic ductal adenocarcinoma cancer (PDAC) is a highly lethal malignancy requiring efficient detection when the primary tumor is still resectable. We previously developed the MxPancreasScore comprising 9 analytes and serum carbohydrate antigen 19-9 (CA19-9), achieving an accuracy of 90.6%. The necessity for 5 different analytical platforms and multiple analytical runs, however, hindered clinical applicability. We therefore aimed to develop a simpler single-analytical run, single-platform diagnostic signature.We evaluated 941 patients (PDAC, 356; chronic pancreatitis [CP], 304; nonpancreatic disease, 281) in 3 multicenter independent tests, and identification (ID) and validation cohort 1 (VD1) and 2 (VD2) were evaluated. Targeted quantitative plasma metabolite analysis was performed on a liquid chromatography-tandem mass spectrometry platform. A machine learning-aided algorithm identified an improved (i-Metabolic) and minimalistic metabolic (m-Metabolic) signatures, and compared them for performance.The i-Metabolic Signature, (12 analytes plus CA19-9) distinguished PDAC from CP with area under the curve (95% confidence interval) of 97.2% (97.1%-97.3%), 93.5% (93.4%-93.7%), and 92.2% (92.1%-92.3%) in the ID, VD1, and VD2 cohorts, respectively. In the VD2 cohort, the m-Metabolic signature (4 analytes plus CA19-9) discriminated PDAC from CP with a sensitivity of 77.3% and specificity of 89.6%, with an overall accuracy of 82.4%. For the subset of 45 patients with PDAC with resectable stages IA-IIB tumors, the sensitivity, specificity, and accuracy were 73.2%, 89.6%, and 82.7%, respectively; for those with detectable CA19-92 U/mL, 81.6%, 88.7%, and 84.5%, respectively; and for those with CA19-937 U/mL, 39.7%, 94.1%, and 76.3%, respectively.The single-platform, single-run, m-Metabolic signature of just 4 metabolites used in combination with serum CA19-9 levels is an innovative accurate diagnostic tool for PDAC at the time of clinical presentation, warranting further large-scale evaluation.

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Squamous penile cancer displays a rare human papillomavirus (HPV)–associated tumor entity. Investigations on the molecular pathogenesis of HPV-driven penile cancer are impaired by the rareness of clinical specimens and, in particular, are missing relevant cell culture models. Here, we identified in HPV-positive penile cancer cell lines that HPV16 oncoproteins control TP63 expression by modulating critical regulators, while integration into the TP63 open reading frame facilitates oncogene expression. The resulting feed-forward loop leads to elevated p63 levels that in turn enhance the release of the neutrophil-recruiting chemokine CXCL8. Remarkably, elevated CXCL8 amounts lead to the increased surface exposition of the Fc receptor of human IgA antibodies, FcαRI, on neutrophils and correlated with a higher susceptibility to antibody-dependent neutrophil-mediated cytotoxicity (ADCC) using an EGFR-specific IgA2 antibody. IHC staining of tissue microarrays proved that elevated expression of p63 together with neutrophil infiltration were significantly more frequent in HPV-positive penile cancer displaying a higher tumor grade. In summary, we identified a promising marker profile of patients with penile cancer at higher risk for worse prognosis. However, these patients may benefit from immunotherapeutic approaches efficiently engaging neutrophils for tumor cell killing.

Supplementary Figure 1 and Table 1 Legends from Anti–Tumor Necrosis Factor Therapy Inhibits Pancreatic Tumor Growth and Metastasis

Supplementary Figure 1 from Anti–Tumor Necrosis Factor Therapy Inhibits Pancreatic Tumor Growth and Metastasis

Chronic inflammation has been implicated in the pathogenesis of many severe autoimmune disorders, as well as in diabetes, pulmonary diseases, and cancer. Inflammation accompanies most solid cancers including pancreatic ductal adenocarcinoma (PDAC), one of the most fatal cancers with surgery being the only curative therapeutic approach currently available. In the present work, we investigated the role of the major proinflammatory cytokine tumor necrosis factor α (TNFα) in the malignancy of PDAC cells in vitro and in vivo. In vitro, TNFα strongly increased invasiveness of Colo357, BxPc3, and PancTuI cells and showed only moderate antiproliferative effect. TNFα treatment of mice bearing orthotopically growing PDAC tumors led to dramatically enhanced tumor growth and metastasis. Notably, we found that PDAC cells themselves secrete TNFα. Although inhibition of TNFα with infliximab or etanercept only marginally affected proliferation and invasiveness of PDAC cells in vitro, both reagents exerted strong antitumoral effects in vivo. In severe combined immunodeficient mice with orthotopically growing Colo357, BxPc3, or PancTuI tumors, human-specific anti-TNF antibody infliximab reduced tumor growth and metastasis by about 30% and 50%, respectively. Importantly, in a PDAC resection model performed with PancTuI cells, we found an even stronger therapeutic effect for both anti-TNF compounds. Infliximab and etanercept reduced the number of liver metastases by 69% and 42%, respectively, as well as volumes of recurrent tumors by 73% and 51%. Thus, tumor cell–derived TNFα plays a profound role in malignancy of PDAC, and inhibition of TNFα represents a promising therapeutic option particularly in adjuvant therapy after subtotal pancreatectomy. [Cancer Res 2008;68(5):1443–50]

Supplementary Table 1 from Anti–Tumor Necrosis Factor Therapy Inhibits Pancreatic Tumor Growth and Metastasis

Background: Pancreatic ductal adenocarcinoma (PDAC), which ranks forth on the cancer-related death statistics still is both a diagnostic and a therapeutic challenge. Adenocarcinoma of the exocrine human pancreas originates in most instances from malignant transformation of ductal epithelial cells, alternatively by Acinar-Ductal Metaplasia (ADM). RA96 antibody targets to a mucin M1, according to the more recent nomenclature MUC5AC, an extracellular matrix component excreted by PDAC cells. In this study, we tested the usability of multimodal nanoparticle carrying covalently coupled RA96 Fab fragments for pancreatic tumor imaging. Methods: In order to make and evaluate a novel, better targeting, theranostic nanoparticle, iron nanoparticles and the optical dye indocyanin green (ICG) were encapsulated into the cationic sphingomyelin (SM) consisting liposomes. RA-96 Fab fragment was conjugated to the liposomal surface of the nanoparticle to increase tumor homing ability. ICG and iron nanoparticle-encapsulated liposomes were studied in vitro with cells and (i) their visibility in magnetic resonance imaging (MRI), (ii) optical, (iii) Magnetic particle spectroscopy (MPS) and (iv) photoacoustic settings was tested in vitro and also in in vivo models. The targeting ability and MRI and photoacoustic visibility of the RA-96-nanoparticles were first tested in vitro cell models where cell binding and internalization was studied. In in vivo experiments liposomal nanoparticles were injected into a tail vain using an orthotopic pancreatic tumor xenograft model and subcutaneous pancreas cancer cell xenografts bearing mice to determine in vivo targeting abilities of RA-96-conjugated liposomes. Results: Multimodal liposomes could be detected by MRI, MPS and by photoacoustic imaging in addition to optical imaging showing a wide range of imaging utility. The fluorescent imaging of ICG in pancreatic tumor cells Panc89 and Capan-2 revealed increased association of ICG-encapsulated liposomes carrying RA-96 Fab fragments in vitro compared to the control liposomes without covalently linked RA-96. Fluorescent molecular tomography (FMT) studies showed increased accumulation of the RA96-targeted nanoparticles in the tumor area compared to non-targeted controls in vivo. Similar accumulation in the tumor sites could be seen with liposomal ferric particles in MRI. Fluorescent tumor signal was confirmed by using an intraoperative fluorescent imaging system which showed fluorescent labeling of pancreatic tumors. Conclusion: These results suggest that RA-96-targeted liposomes encapsulating ICG and iron nanoparticles can be used to image pancreatic tumors with a variety of optical and magnetic imaging techniques. Additionally, they might be a suitable drug delivery tool to improve treatment of PDAC patients.

Highlights • Elevated expression of Wnt pathway associated factors in HPV-positive penile cancer cells. • lacking nuclear beta-catenin translocation indicated an actively abrogated Wnt signaling. • elevated expression of the Wnt antagonist DKK1 in HPV-positive penile cancer cells. • DKK1-driven autocrine Wnt pathway inhibition in penile cancer cells. • DKK1+ penile cancers are with a higher frequency HPV+, less differentiated and grow more aggressively., Penile squamous cell cancer (PSCC) is the most frequent penile malignant disease. Infections with human papillomaviruses (HPV) are a major etiologic driver of PSCC. However, the molecular details of the underlying carcinogenesis are understudied because of rare clinical specimens and missing cell lines. Here, we investigated if the expression of high-risk HPV16 oncogenes causes an augmentation of the Wnt pathway using unique HPV-positive penile cancer (PeCa) cell lines in monolayer and organotypic 3D raft cultures as well as tissue micro arrays containing clinical tissue specimens. The HPV oncoproteins enhanced the expression of Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and the HPV-positive PeCa cells expressed a signature of Wnt target and stemness-associated genes. However, the notable lack of nuclear β-catenin in vitro and in situ raised the question if the enhanced expression of Wnt pathway factors is tantamount to an active Wnt signaling. Subsequent TOP-flash reporter assays revealed Wnt signaling as absent and not inducible by respective Wnt ligands in PeCa cell lines. The HPV-positive PeCa cells and especially HPV-positive PeCa specimens of the tumor core expressed the Wnt antagonist and negative feedback-regulator Dickkopf1 (DKK1). Subsequent neutralization experiments using PeCa cell line-conditioned media demonstrated that DKK1 is capable to impair ligand-induced Wnt signaling. While gene expression analyses suggested an augmented and active canonical Wnt pathway, the respective signaling was inhibited due to the endogenous expression of the antagonist DKK1. Subsequent TMA stainings indicated Dkk1 as linked with HPV-positivity and metastatic disease progression in PeCa suggesting potential as a prognostic marker.

Squamous penile cancer displays a rare human papillomavirus (HPV)–associated tumor entity. Investigations on the molecular pathogenesis of HPV-driven penile cancer are impaired by the rareness of clinical specimens and, in particular, are missing relevant cell culture models. Here, we identified in HPV-positive penile cancer cell lines that HPV16 oncoproteins control TP63 expression by modulating critical regulators, while integration into the TP63 open reading frame facilitates oncogene expression. The resulting feed-forward loop leads to elevated p63 levels that in turn enhance the release of the neutrophil-recruiting chemokine CXCL8. Remarkably, elevated CXCL8 amounts lead to the increased surface exposition of the Fc receptor of human IgA antibodies, FcαRI, on neutrophils and correlated with a higher susceptibility to antibody-dependent neutrophil-mediated cytotoxicity (ADCC) using an EGFR-specific IgA2 antibody. IHC staining of tissue microarrays proved that elevated expression of p63 together with neutrophil infiltration were significantly more frequent in HPV-positive penile cancer displaying a higher tumor grade. In summary, we identified a promising marker profile of patients with penile cancer at higher risk for worse prognosis. However, these patients may benefit from immunotherapeutic approaches efficiently engaging neutrophils for tumor cell killing.

Purpose HMGA2 has frequently been found in benign as well as malignant tumors and a significant association between HMGA2 overexpression and poor survival in different malignancies was described. In pancreatic ductal adenocarcinoma (PDAC), nuclear HMGA2 expression is associated with tumor dedifferentiation and presence of lymph node metastasis. Nevertheless, the impact of HMGA2 occurrence in other cell compartments is unknown. Methods Intracellular distribution of HMGA2 was analyzed in PDAC (n = 106) and peritumoral, non-malignant ducts (n = 28) by immunohistochemistry. Findings were correlated with clinico-pathological data. Additionally, intracellular HMGA2 presence was studied by Western blotting of cytoplasmic and nuclear fractions of cultured cells. Results HMGA2 was found in the cytoplasm and in the nucleus of cultured cells. In human tumor tissue, HMGA2 was also frequently found in the cytoplasm and the nucleus of tumor cells, however, nuclear staining was generally stronger. Direct comparison from tumor tissue with corresponding non-neoplastic peritumoral tissue revealed significantly stronger expression in tumors (p = 0.003). Of note, the nuclear staining was significantly stronger in lymph node metastatic cell nuclei compared to primary tumor cell nuclei (p = 0.049). Interestingly, cytoplasmic staining positively correlated with lymph vessel (p = 0.004) and venous invasion (p = 0.046). Conclusion HMGA2 is a prognostic marker in PDAC. Firstly, we found a positive correlation for cytoplasmic HMGA2 expression with lympho-vascular invasion and, secondly, we found a significantly stronger nuclear expression of HMGA2 in cancer-positive lymph node nuclei compared to primary tumor cell nuclei. So far, the role of cytoplasmic HMGA2 is nearly unknown, however, our data lend support to the hypothesis that cytoplasmic HMGA2 expression is involved in nodal spread.

Pancreatic cancer (PC) remains the fourth leading cause of cancer death; therefore, there is a clinically unmet need for novel therapeutics and diagnostic markers to treat this devastating disease. Physicians often rely on biopsy or CT for diagnosis, but more specific protein biomarkers are highly desired to assess the stage and severity of PC in a noninvasive manner. Serum biomarkers such as carbohydrate antigen 19-9 are of particular interest as they are commonly elevated in PC but have exhibited suboptimal performance in the clinic. MUC5AC has emerged as a useful serum biomarker that is specific for PC versus inflammation. We developed RA96, an anti-MUC5AC antibody, to gauge its utility in PC diagnosis through immunohistochemical analysis and whole-body PET in PC. Methods: In this study, extensive biochemical characterization determined MUC5AC as the antigen for RA96. We then determined the utility of RA96 for MUC5AC immunohistochemistry on clinical PC and preclinical PC. Finally, we radiolabeled RA96 with (89)Zr to assess its application as a whole-body PET radiotracer for MUC5AC quantification in PC. Results: Immunohistochemical staining with RA96 distinguished chronic pancreatitis, pancreatic intraepithelial neoplasia, and varying grades of pancreatic ductal adenocarcinoma in clinical samples. (89)Zr-desferrioxamine-RA96 was able to detect MUC5AC with high specificity in mice bearing capan-2 xenografts. Conclusion: Our study demonstrated that RA96 can differentiate between inflammation and PC, improving the fidelity of PC diagnosis. Our immuno-PET tracer (89)Zr-desferrioxamine-RA96 shows specific detection of MUC5AC-positive tumors in vivo, highlighting the utility of MUC5AC targeting for diagnosis of PC.

Simple Summary Death receptors may induce cellular death upon ligand binding but also activate other pro-inflammatory mechanisms in cancer cells, which may increase malignancy. The transcription factor NFkB works as a central hub in this system and connects the signaling of three major death ligands: TNF, CD95L and TRAIL. In this study, we analyzed the impact of a recombinant inhibitor of CD95L on the respective tri-lateral cross-talk using highly malignant pancreatic cancer cells as a model and in context of the standard chemotherapeutic agent, gemcitabine. We show that the specific “uni-lateral” inhibitor also clearly impacts the two other cytokines and importantly reduces the pro-inflammatory status of pancreatic cancer cells as well as their stem cell phenotype. Consequently, the local recurrent tumor growth after surgery of the primary tumor was reduced. Further, the unwanted, pro-inflammatory side-effects of gemcitabine are inhibited. Abstract Despite the potential apoptotic functions, the CD95/CD95L system can stimulate survival as well as pro-inflammatory signaling, particularly through the activation of NFκB. This holds true for the TNF/TNFR and the TRAIL/TRAILR systems. Thus, signaling pathways of these three death ligands converge, yet the specific impact of the CD95/CD95L system in this crosstalk has not been well studied. In this study, we show that gemcitabine stimulates the expression of pro-inflammatory cytokines, such as IL6 and IL8, under the influence of the CD95/CD95L system and the pharmacological inhibitor, sCD95Fc, substantially reduced the expression in two PDAC cell lines, PancTuI-luc and A818-4. The stem cell phenotype was reduced when induced upon gemcitabine as well by sCD95Fc. Moreover, TNF-α as well as TRAIL up-regulate the expression of CD95 and CD95L in both cell lines. Conversely, we detected a significant inhibitory effect of sCD95Fc on the expression of both IL8 and IL6 induced upon TNF-α and TRAIL stimulation. In vivo, CD95L inhibition reduced xeno-transplanted recurrent PDAC growth. Thus, our findings indicate that inhibition of CD95 signaling altered the chemotherapeutic effects of gemcitabine, not only by suppressing the pro-inflammatory responses that arose from the CD95L-positive tumor cells but also from the TNF-α and TRAIL signaling in a bi-lateral crosstalk manner.

Solid tumor biopsies are the current standard for precision medicine. However, the procedure is invasive and not always feasible. In contrast, liquid biopsies, such as serum enriched for extracellular vesicles (EVs) represent a non-invasive source of cancer biomarkers. In this study, we compared two EV isolation methods in the context of the protein biomarker detection in inflammatory bowel disease (IBD) and colorectal cancer (CRC). Using serum samples of a healthy cohort as well as CRC and IBD patients, EVs were isolated by ultracentrifugation and ExoQuick

To study novel treatment modalities for pancreatic ductal adenocarcinoma (PDAC), we need to transfer the knowledge from in vitro to in vivo. It is important to mirror the clinical characteristics of the typically local invasive growth of pancreatic cancer and the distant spread resulting in liver metastasis. Notably, for xenotransplant studies using human specimen, two models, i.e. subcutaneous (s.c.) and orthotopic (o.t.) transplantation are widely used.The subcutaneously and orthotopically inoculated Colo357 Bcl-xUpon TRAIL treatment, the primary tumors did not change their size, neither in the s.c. nor in the o.t. approaches. But when s.c. was compared to o.t., the size of the s.c. tumors was more than two-fold bigger than that of the o.t. tumors (P0.01). However, mice with orthotopically inoculated PDAC cells developed liver metastasis upon TRAIL treatment much more frequently (n = 13/17) than mice with subcutaneously inoculated PDAC cells (n = 1/11) (P0.01). As a likely driving force for this increased metastasis, a higher TNF-α staining intensity in the o.t. tumors was observed by immunohistochemistry.These data from a direct side-by-side comparison underline the importance of the proper inoculation site of the PDAC cells. Local invasion and liver metastases are a hallmark of PDAC in the clinic; the o.t. model is clearly superior in reflecting this setting. Moreover, a serious side-effect of a possible new therapeutic compound became obvious only in the o.t.

Ion channels and transporters (ICT) play important roles in almost all basic cellular processes. During last decades, abundant evidences have been provided that ICT (e.g., Ca

Hematogenic tumor cell spread is a key event in metastasis. However, the clinical significance of circulating tumor cells (CTC) in the blood and disseminated tumor cells (DTC) in bone marrow is still not fully understood. Here, the presence of DTC and CTC in esophageal cancer (EC) patients and its correlation with clinical parameters was investigated to evaluate the CTC/DTC prognostic value in EC. This study included 77 EC patients with complete surgical tumor resection. CTC and DTC were analyzed in blood and bone marrow using nested CK20 reverse transcription-nested polymerase chain reaction (RT-PCR) and findings were correlated with clinical data. Twenty-seven of 76 patients (36.5%) showed CK20 positivity in the blood, 19 of 61 patients (31.1%) in bone marrow, and 40 (51.9%) of 77 patients were positive in either blood or bone marrow or both. In multivariate analyses, only the DTC status emerged as independent predictor of overall and tumor specific survival. Our study revealed that, while the presence of CTC in blood is not associated with a worse prognosis, DTC detection in the bone marrow is a highly specific and independent prognostic marker in EC patients. Larger cohort studies could unravel how this finding can be translated into improved therapy management in EC.

Solid tumor biopsies are the current standard for precision medicine. However, the procedure is invasive and not always feasible. In contrast, liquid biopsies, such as serum enriched for extracellular vesicles (EVs) represent a non-invasive source of cancer biomarkers. In this study, we compared two EV isolation methods in the context of the protein biomarker detection in inflammatory bowel disease (IBD) and colorectal cancer (CRC). Using serum samples of a healthy cohort as well as CRC and IBD patients, EVs were isolated by ultracentrifugation and ExoQuickTM in parallel. EV associated protein profiles were compared by multiplex-fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and subsequent identification by mass spectrometry. Validation of gelsolin (GSN) was performed using fluorescence-quantitative western blot. 2D-DIGE resolved 936 protein spots in all serum-enriched EVs isolated by ultracentrifugation or ExoQuickTM. Hereof, 93 spots were differently expressed between isolation approaches. Higher levels of GSN in EVs obtained with ExoQuickTM compared to ultracentrifugation were confirmed by western blot (p = 0.0006). Although patient groups were distinguishable after both EV isolation approaches, sample preparation strongly influences EVs’ protein profile and thus impacts on inter-study reproducibility, biomarker identification and validation. The results stress the need for strict SOPs in EV research before clinical implementation can be reached.

Background The human pancreatic cancer cell line A818–6 can be grown in vitro either as a highly malignant, undifferentiated monolayer (ML) or as three-dimensional (3D) single layer hollow spheres (HS) simulating a benign, highly differentiated, duct-like pancreatic epithelial structure. This characteristic allowing A818–6 cells to switch from one phenotype to another makes these cells a unique system to characterize the cellular and molecular modifications during differentiation on one hand and malignant transformation on the other hand. Ion channels and transport proteins (transportome) have been implicated in malignant transformation. Therefore, the current study aimed to analyse the transportome gene expression profile in the A818–6 cells growing as a monolayer or as hollow spheres. Methods & Results The study identified the differentially expressed transportome genes in both cellular states of A818–6 using Agilent and Nanostring arrays and some targets were validated via immunoblotting. Additionally, these results were compared to a tissue Affymetrix microarray analysis of pancreatic adenocarcinoma patients’ tissues. The overall transcriptional profile of the ML and HS cells confirmed the formerly described mesenchymal features of ML and epithelial nature of HS which was further verified via high expression of E-cadherin and low expression of vimentin found in HS in comparison to ML. Among the predicted features between HS and ML was the involvement of miRNA-9 in this switch. Importantly, the bioinformatics analysis also revealed substantial number (n = 126) of altered transportome genes. Interestingly, three genes upregulated in PDAC tissue samples (GJB2, GJB5 and SLC38A6) were found to be also upregulated in ML and 3 down-regulated transportome genes (KCNQ1, TRPV6 and SLC4A) were also reduced in ML. Conclusion This reversible HS/ML in vitro system might help in understanding the pathophysiological impact of the transportome in the dedifferentiation process in pancreatic carcinogenesis. Furthermore, the HS/ML model represents a novel system for studying the role of the transportome during the switch from a more benign, differentiated (HS) to a highly malignant, undifferentiated (ML) phenotype.

Ion channels and transporters (ICT) play important roles in almost all basic cellular processes. During last decades, abundant evidences have been provided that ICT (e.g., Ca2+ and K+ channels) are notable for regulating physiological pancreatic duct cellular function and deregulation of ICT is closely associated with the widely accepted hallmarks of pancreatic ductal adenocarcinoma (PDAC) such as proliferation, apoptosis resistance, invasion, and metastasis. Hence this review focuses on the role of ICT malfunctions in context with the hallmarks of PDAC. After briefly introducing epidemiology and history of molecular oncology of PDAC and summarizing the recent studies on molecular classification systems, we focus then on the exocrine pancreas as a very active secretory gland which considerably impacts the changes in the ion transport system (the transportome) upon malignant transformation. We highlight multiplicity of ICT members (H+ transporters, Ca2+, K+, Na+ and Cl- channels) and their functional impact in PDAC. We also present some selective therapeutic options to interfere with transportome functions and thereby with key mechanisms of malignant progression. This will hopefully contribute to a better clinical outcome based on improved therapeutic strategies for this still extremely deadly disease.

Background ADAMs (a disintegrin and metalloproteinase) have long been associated with tumor progression. Recent findings indicate that members of the closely related ADAMTS (ADAMs with thrombospondin motifs) family are also critically involved in carcinogenesis. Gene silencing through DNA methylation at CpG loci around e.g. transcription start or enhancer sites is a major mechanism in cancer development. Here, we aimed at identifying genes of the ADAM and ADAMTS family showing altered DNA methylation in the development or colorectal cancer (CRC) and other epithelial tumors. Methods We investigated potential changes of DNA methylation affecting ADAM and ADAMTS genes in 117 CRC, 40 lung cancer (LC) and 15 oral squamous-cell carcinoma (SCC) samples. Tumor tissue was analyzed in comparison to adjacent non-malignant tissue of the same patients. The methylation status of 1145 CpGs in 51 ADAM and ADAMTS genes was measured with the HumanMethylation450 BeadChip Array. ADAMTS16 protein expression was analyzed in CRC samples by immunohistochemistry. Results In CRC, we identified 72 CpGs in 18 genes which were significantly affected by hyper- or hypomethylation in the tumor tissue compared to the adjacent non-malignant tissue. While notable/frequent alterations in methylation patterns within ADAM genes were not observed, conspicuous changes were found in ADAMTS16 and ADAMTS2. To figure out whether these differences would be CRC specific, additional LC and SCC tissue samples were analyzed. Overall, 78 differentially methylated CpGs were found in LC and 29 in SCC. Strikingly, 8 CpGs located in the ADAMTS16 gene were commonly differentially methylated in all three cancer entities. Six CpGs in the promoter region were hypermethylated, whereas 2 CpGs in the gene body were hypomethylated indicative of gene silencing. In line with these findings, ADAMTS16 protein was strongly expressed in globlet cells and colonocytes in control tissue but not in CRC samples. Functional in vitro studies using the colorectal carcinoma cell line HT29 revealed that ADAMTS16 expression restrained tumor cell proliferation. Conclusions We identified ADAMTS16 as novel gene with cancer-specific promoter hypermethylation in CRC, LC and SCC patients implicating ADAMTS16 as potential biomarker for these tumors. Moreover, our results provide evidence that ADAMTS16 may have tumor suppressor properties. Electronic supplementary material The online version of this article (10.1186/s12885-018-4701-2) contains supplementary material, which is available to authorized users.

Background The death receptors TRAIL-R1 and TRAIL-R2 are frequently overexpressed in cancer and there is an emerging evidence for their important role in malignant progression, also in the case of pancreatic ductal adenocarcinoma (PDAC). In their canonical localization at the plasma membrane, TRAIL-R1/−R2 may induce cell death and/or pro-inflammatory signaling leading to cell migration, invasion and metastasis. Although, they have repeatedly been found intracellular, in the cytoplasm and in the nucleus, their functions in intracellular locations are still not well understood. Likewise, studies dealing with the prognostic relevance of TRAIL-Rs located in particular cellular compartments are very rare. For PDAC, the correlation of nuclear TRAIL-R2 with worse patients’ prognosis has been shown recently. Corresponding data on TRAIL-R1 are not available so far. Methods In the present study we analyzed the expression of TRAIL-R1 in 106 PDACs and 28 adjacent, peritumoral non-malignant pancreatic ducts with special emphasis on its cytoplasmic and nuclear localization and correlated the immunohistochemical findings with clinico-pathological patient characteristics. Results TRAIL-R1 was found in 93.4% of all PDAC samples. Cytoplasmic staining was present with very similar intensity in tumor and normal tissue. In contrast, nuclear TRAIL-R1 staining was significantly stronger in tumor compared to normal tissue (p = 0.006). Interestingly, we found that the number of cells with cytoplasmic TRAIL-R1 staining negatively correlates with tumor grading (p = 0.043). No such correlation could be detected for nuclear TRAIL-R1. Neither, cytoplasmic nor nuclear TRAIL-R1 staining showed a correlation with other clinico-pathological parameter such as pTNM categories. However, Kaplan-Meier analyses revealed significantly prolonged median survival of patients with positive cytoplasmic TRAIL-R1 expression in more than 80% of tumor cells compared to patients with tumors containing a smaller quantity of cells positively stained for cytoplasmic TRAIL-R1 (20 vs. 8 months; p = 0.004). Conclusion Cytoplasmic TRAIL-R1 is a positive prognostic marker for patients with PDAC. Our findings indicate that loss of cytoplasmic TRAIL-R1 results in recurrent disease with more malignant phenotype thus suggesting anti-tumor activities of cytoplasmic TRAIL-R1 in PDAC.

Background In recent years, the concept of liquid biopsy diagnostics in detection and progress monitoring of malignant diseases gained significant awareness. We here report on a semi-quantitative real-time cytokeratin 20 RT-PCR-based assay, for detecting circulating tumor cells within a fraction of peripheral blood mononuclear cells in colorectal cancer patients. Methods In total, 381 patients were included. Prior to surgical tumor resection, a peripheral blood sample was drawn. Mononuclear cells were isolated by Ficoll centrifugation and a cytokeratin 20 qRT-PCR assay was performed. Quantitative PCR data was assessed regarding histopathological characteristics and patients´ clinical outcome. Results A cut-off value was determined at ≥ 2.77 [EU]. Stratifying patients by this cut-off, it represents a statistically highly significant prognostic marker for both the overall and disease-free survival in the entire cohort UICC I-IV (both p

Brown seaweeds have been used in phytotherapy since ancient times. A multitude of medical effects have been reported for their ingredients which also cover anticancer activities. Though being the most common brown seaweed in the Baltic Sea, nothing is known—apart from our recent publication (Geisen et al. Mar Drugs 13:4470-4491, 2015)—about the anticancer potential of the Bladderwrack, Fucus vesiculosus L., grown in this particular habitat with low salinity and nontidal shores. The aim of the present study was to investigate systematically the alga’s cytotoxic potential against human pancreatic cancer cells and to develop a purification procedure for the active compounds by using a bioassay-guided fractionation approach. Four out of six crude extracts showed considerable cytotoxic activity against Panc89 cells, three of which were considerably active against PancTU1 cells, as well. The most active crude extract revealed an effective half maximal concentration (EC50) of 72 μg mL−1 against Panc89 and of 77 μg mL−1 against PancTU1 cells after 72 h of treatment. The multistep purification procedure established for this extract resulted in a fourfold reduction of the EC50. Monitoring of each crude extract and fraction via 1H-NMR spectroscopy revealed a characteristic fingerprint which was significantly correlated with the activity. Structural analysis of the most active fractions revealed two similar molecules belonging to the group of polyphenols. Further separation is needed to present the chemical structure in all molecular details. The results show that F. vesiculosus from the Baltic Sea holds potent and novel anticancer substances.

Background Recent whole-genome sequencing identified four molecular subtypes of gastric cancer (GC), of which the subgroup of Epstein–Barr virus-associated GC (EBVaGC) showed a significant enrichment of PIK3CA mutations. We here aimed to validate independently the enrichment of PIK3CA mutations in EBVaGC of a Central European GC cohort, to correlate EBV status with clinico-pathological patient characteristics and to test for a major issue of GC, intratumoral heterogeneity. Patients and methods In a first step, 484 GCs were screened for EBV and PIK3CA hot spot mutations of exon 9/20 using EBER in situ hybridization and pyrosequencing, respectively. Secondly, an extended sequencing of PIK3CA also utilizing next generation sequencing was carried out in all EBVaGCs and 96 corresponding lymph node metastases. Results Twenty-two GCs were EBER-positive, all being of latency type I. Intratumoral heterogeneity of EBER-positivity was found in 18% of EBVaGCs. Twenty-three GCs held PIK3CA mutations in hot spot regions of exon 9 or 20, being significantly more common in EBVaGCs (P

// Qi Ling 1, 2, * , Xiao Xu 1, 2, * , Panpan Ye 3, * , Haiyang Xie 1, 2 , Feng Gao 1, 2 , Qichao Hu 2 , Zhikun Liu 1, 2 , Xuyong Wei 1, 2 , Christian Roder 4 , Anna Trauzold 4 , Holger Kalthoff 4 , Shusen Zheng 1, 2 1 Department of Surgery, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China 2 Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, China 3 The Ophthalmology Center, The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China 4 Institute for Experimental Cancer Research, Comprehensive Cancer Center North, CAU, Kiel, Germany * These authors contributed equally to this work Correspondence to: Shusen Zheng, email: zyzss@zju.edu.cn Holger Kalthoff, email: hkalthoff@email.uni-kiel.de Keywords: anatomy, microRNA, pancreatic cancer, recurrence Received: January 22, 2016 Accepted: January 10, 2017 Published: January 20, 2017 ABSTRACT Different clinical presentations and prognoses have been implied between pancreatic head and body/tail cancers. We aimed to identify the prognostic relevance of primary tumor location in patients undergoing resection for pancreatic ductal adenocarcinoma (PDAC). Thirty-two pairs of patients with strictly matched early stage (II) pancreatic head and body/tail cancers were enrolled. The molecular feature of the two subtypes of PDAC was assessed on the level of miRNA expression. Out of the 64 patients, 34 (53.1%) had tumor recurrence after radical resection during the follow-up period (2.3 ± 0.8 years). Both overall and tumor-free survival were significantly higher in the patients with pancreatic body/tail cancer compared with those with pancreatic head cancer. Patient age and tumor location were the independent prognostic factors for tumor recurrence. A remarkably lower expression of miR-501-3p and higher expression of miR-375 were found and were further verified in pancreatic body/tail cancer tissues compared with pancreatic head cancer tissues. The low expression of miR-501-3p was significantly associated with a low risk of tumor recurrence. Both, subcutaneous and orthotopic PDAC mouse models presented highly invasive tumor phenotypes upon up-regulated miR-501-3p expression. An in vitro study showed that miR-501-3p promoted the invasiveness of PDAC cells possibly via suppressing E-cadherin. In summary, at resectable early stage, pancreatic body/tail cancer presents a less malignant phenotype associated with deregulation of miR-501-3p compared with pancreatic head cancer.

ObjectiveCurrent non-invasive diagnostic tests can distinguish between pancreatic cancer (pancreatic ductal adenocarcinoma (PDAC)) and chronic pancreatitis (CP) in only about two thirds of patients. We have searched for blood-derived metabolite biomarkers for this diagnostic purpose.DesignFor a case–control study in three tertiary referral centres, 914 subjects were prospectively recruited with PDAC (n=271), CP (n=282), liver cirrhosis (n=100) or healthy as well as non-pancreatic disease controls (n=261) in three consecutive studies. Metabolomic profiles of plasma and serum samples were generated from 477 metabolites identified by gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry.ResultsA biomarker signature (nine metabolites and additionally CA19-9) was identified for the differential diagnosis between PDAC and CP. The biomarker signature distinguished PDAC from CP in the training set with an area under the curve (AUC) of 0.96 (95% CI 0.93–0.98). The biomarker signature cut-off of 0.384 at 85% fixed specificity showed a sensitivity of 94.9% (95% CI 87.0%–97.0%). In the test set, an AUC of 0.94 (95% CI 0.91–0.97) and, using the same cut-off, a sensitivity of 89.9% (95% CI 81.0%–95.5%) and a specificity of 91.3% (95% CI 82.8%–96.4%) were achieved, successfully validating the biomarker signature.ConclusionsIn patients with CP with an increased risk for pancreatic cancer (cumulative incidence 1.95%), the performance of this biomarker signature results in a negative predictive value of 99.9% (95% CI 99.7%–99.9%) (training set) and 99.8% (95% CI 99.6%–99.9%) (test set). In one third of our patients, the clinical use of this biomarker signature would have improved diagnosis and treatment stratification in comparison to CA19-9.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by both, overexpression of transforming growth factor (TGF)β and resistance of the tumor cells to many apoptosis-inducing stimuli. The latter negatively impacts the outcome of therapeutic efforts and represents one important mechanism which tumor cells utilize to escape the immune surveillance. Since TGFβ acts as a tumor promoter in advanced tumor stages and suppression of apoptosis is a known driver of tumor progression, it is possible that TGFβ functions as a crucial determinant of tumor cell sensitivity to apoptosis in PDAC. Here, we have studied the impact of TGFβ on TNF-related apoptosis inducing ligand (TRAIL)-induced signaling in PDAC cells. In TGFβ-responsive Panc1 and Colo357 cells, TGFβ1 reduced total and plasma membrane-associated levels of TRAIL-R1 but not those of TRAIL-R2. Consistent with the known predominant role of TRAIL-R1 in TRAIL-mediated signaling in PDAC, TGFβ1 inhibited TRAIL-induced DISC formation and apoptosis as well as phosphorylation of MAPKs and IκBα. Similarly, it also reduced signaling of TRAIL-R1 following its specific activation with an agonistic antibody. In contrast, specific TRAIL-R2 signaling remained unchanged. The TGFβ1 effect on TRAIL-R1 expression was mimicked by ectopic expression of a kinase-active version of the TGFβ type I receptor ALK5 (ALK5-T204D) but not by ALK5 double mutant lacking the ability to phosphorylate Smad proteins (RImL45-T204D). Moreover, TGFβ regulation of TRAIL-R1 was absent in two PDAC cell lines lacking the Smad4 gene DPC4 and siRNA-mediated silencing of Smad4 in Smad4-positive Panc1 cells abolished the TGFβ-mediated decrease in TRAIL-R1 expression, together showing that ALK5/Smad4 signaling is crucial for TGFβ regulation of TRAIL-R1 expression. Our results suggest a novel tumor-promoting function of TGFβ1. By downregulating TRAIL-R1, TGFβ1 may not only promote tumor escape from immune surveillance but also negatively impact on TRAIL- or TRAIL-R1-based therapy regimens for treatment of PDAC.

Olga Maria Will,1,* Nicolai Purcz,2,* Athena Chalaris,3 Carola Heneweer,4,5 Susann Boretius,1 Larissa Purcz,2 Lila Nikkola,6 Nureddin Ashammakhi,6 Holger Kalthoff,7 Claus-Christian Glüer,1 Jörg Wiltfang,2 Yahya Açil,2 Sanjay Tiwari1 1Section Biomedical Imaging, Clinic for Radiology and Neuroradiology, MOIN CC, 2Department of Oral and Maxillofacial Surgery, University Hospital Schleswig-Holstein, 3Institute of Biochemistry, Christian-Albrechts-Universität zu Kiel, 4Clinic for Radiology and Neuroradiology, University Hospital Schleswig-Holstein, Kiel, 5Institute for Diagnostic and Interventional Radiology, University Hospital Cologne, Cologne, Germany; 6Department of Biomedical Engineering, Tampere University of Technology, Tampere, Finland; 7Institute for Experimental Cancer Research, University Hospital Schleswig-Holstein, Kiel, Germany *These authors contributed equally to this work Abstract: Despite aggressive treatment with radiation and combination chemotherapy following tumor resection, the 5-year survival rate for patients with head and neck cancer is at best only 50%. In this study, we examined the therapeutic potential of localized release of diclofenac from electrospun nanofibers generated from poly(d,l-lactide-co-glycolide) polymer. Diclofenac was chosen since anti-inflammatory agents that inhibit cyclooxygenase have shown great potential in their ability to directly inhibit tumor growth as well as suppress inflammation-mediated tumor growth. A mouse resection model of oral carcinoma was developed by establishing tumor growth in the oral cavity by ultrasound-guided injection of 1 million SCC-9 cells in the floor of the mouth. Following resection, mice were allocated into four groups with the following treatment: 1) no treatment, 2) implanted scaffolds without diclofenac, 3) implanted scaffolds loaded with diclofenac, and 4) diclofenac given orally. Small animal ultrasound and magnetic resonance imaging were utilized for longitudinal determination of tumor recurrence. At the end of 7weeks following tumor resection, 33% of mice with diclofenac-loaded scaffolds had a recurrent tumor, in comparison to 90%–100% of the mice in the other three groups. At this time point, mice with diclofenac-releasing scaffolds showed 89% survival rate, while the other groups showed survival rates of 10%–25%. Immunohistochemical staining of recurrent tumors revealed a near 10-fold decrease in the proliferation marker Ki-67 in the tumors derived from mice with diclofenac-releasing scaffolds. In summary, the local application of diclofenac in an orthotopic mouse tumor resection model of oral cancer reduced tumor recurrence with significant improvement in survival over a 7-week study period following tumor resection. Local drug release of anti-inflammatory agents should be investigated as a therapeutic option in the prevention of tumor recurrence in oral squamous carcinoma. Keywords: tumor recurrence, oral squamous cell carcinoma, head and neck cancer, NSAIDs, drug releasing polymers, mouse model&nbsp