Small intestinal neuroendocrine tumors (SI-NETs) are amine- and peptide-producing neoplasms. Most patients display metastases at the time of diagnosis; they have an unpredictable individual disease course and the tumors are often therapy resistant. Chromogranin A and 5-hydroxyindoleacetic acid are the biomarkers clinically used most often today, but there is a great need for novel diagnostic and prognostic biomarkers and new therapeutic targets. Sixty-nine biomarkers were screened in serum from 23 SI-NET patients and 23 healthy controls using the multiplex proximity ligation assay (PLA). A refined method, the proximity extension assay (PEA), was used to analyze 76 additional biomarkers. Statistical testing and multivariate classification were performed. Immunohistochemistry and ELISA were performed in an extended cohort. Using PLA, 19 biomarkers showed a significant difference in serum concentrations between patients and controls, and PEA revealed a difference in the concentrations of 17 proteins. Multivariate classification analysis revealed decoy receptor 3 (DcR3), trefoil factor 3 (TFF3), and midkine to be good biomarkers for the disease, which was confirmed by ELISA analysis. All 3 biomarkers were expressed in tumor tissue. DcR3 concentrations were elevated in patients with stage IV disease. High concentrations of DcR3 and TFF3 were correlated to poor survival. DcR3, TFF3, and midkine exhibited elevated serum concentrations in SI-NET patients compared to healthy controls, and DcR3 and TFF3 were associated with poor survival. DcR3 seems to be a marker for liver metastases, while TFF3 and midkine may be new diagnostic biomarkers for SI-NETs.

Keywords:
Small intestinal neuroendocrine tumor, Decoy receptor 3 , Trefoil factor 3, Midkine, Biomarkers, Proximity ligation assay, Proximity extension assay
1.
Bosman FT, Carneiro F, Hruban RH, Theise ND: WHO Classification of Tumours of the Digestive System, ed 4. Lyon, International Agency for Research on Cancer, 2010.
2.
Rindi G, Klöppel G, Couvelard A, Komminoth P, Körner M, Lopes JM, McNicol AM, Nilsson O, Perren A, Scarpa A, Scoazec JY, Wiedenmann B: TNM staging of midgut and hindgut (neuro) endocrine tumors: a consensus proposal including a grading system. Virchows Arch 2007;451:757-762.
3.
de Herder WW: Biochemistry of neuroendocrine tumours. Best Pract Res Clin Endocrinol Metab 2007;21:33-41.
4.
Arnold R, Wilke A, Rinke A, Mayer C, Kann PH, Klose KJ, Scherag A, Hahmann M, Müller HH, Barth P: Plasma chromogranin A as marker for survival in patients with metastatic endocrine gastroenteropancreatic tumors. Clin Gastroenterol Hepatol 2008;6:820-827.
5.
Janson ET, Holmberg L, Stridsberg M, Eriksson B, Theodorsson E, Wilander E, Öberg K: Carcinoid tumors: analysis of prognostic factors and survival in 301 patients from a referral center. Ann Oncol 1997;8:685-690.
6.
Modlin IM, Öberg K, Taylor A, Drozdov I, Bodei L, Kidd M: Neuroendocrine tumor biomarkers: current status and perspectives. Neuroendocrinology 2014;100:265-277.
7.
Öberg K: Circulating biomarkers in gastroenteropancreatic neuroendocrine tumours. Endocr Relat Cancer 2011;18(suppl 1):S17-S25.
8.
Turner GB, Johnston BT, McCance DR, McGinty A, Watson RG, Patterson CC, Ardill JE: Circulating markers of prognosis and response to treatment in patients with midgut carcinoid tumours. Gut 2006;55:1586-1591.
9.
Diebold AE, Boudreaux JP, Wang YZ, Anthony LB, Uhlhorn AP, Ryan P, Mamikunian P, Mamikunian G, Woltering EA: Neurokinin a levels predict survival in patients with stage IV well differentiated small bowel neuroendocrine neoplasms. Surgery 2012;152:1172-1176.
10.
Cui T, Hurtig M, Elgue G, Li SC, Veronesi G, Essaghir A, Demoulin JB, Pelosi G, Alimohammadi M, Öberg K, Giandomenico V: Paraneoplastic antigen Ma2 autoantibodies as specific blood biomarkers for detection of early recurrence of small intestine neuroendocrine tumors. PLoS One 2010;5:e16010.
11.
Modlin IM, Kidd M, Bodei L, Drozdov I, Aslanian H: The clinical utility of a novel blood-based multi-transcriptome assay for the diagnosis of neuroendocrine tumors of the gastrointestinal tract. Am J Gastroenterol 2015;110:1223-1232.
12.
Norlén O, Stålberg P, Öberg K, Eriksson J, Hedberg J, Hessman O, Janson ET, Hellman P, Åkerström G: Long-term results of surgery for small intestinal neuroendocrine tumors at a tertiary referral center. World J Surg 2012;36:1419-1431.
13.
Lundberg M, Thorsen SB, Assarsson E, Villablanca A, Tran B, Gee N, Knowles M, Nielsen BS, González Couto E, Martin R, Nilsson O, Fermer C, Schlingemann J, Christensen IJ, Nielsen HJ, Ekström B, Andersson C, Gustafsson M, Brunner N, Stenvang J, Fredriksson S: Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material. Mol Cell Proteomics 2011;10:M110.004978.
14.
Assarsson E, Lundberg M, Holmquist G, Björkesten J, Thorsen SB, Ekman D, Eriksson A, Rennel Dickens E, Ohlsson S, Edfeldt G, Andersson AC, Lindstedt P, Stenvang J, Gullberg M, Fredriksson S: Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability. PLoS One 2014;9:e95192.
15.
Blokzijl A, Nong R, Darmanis S, Hertz E, Landegren U, Kamali-Moghaddam M: Protein biomarker validation via proximity ligation assays. Biochim Biophys Acta 2014;1844:933-939.
16.
Tibshirani R, Hastie T, Narasimhan B, Chu G: Class prediction by nearest shrunken centroids, with applications to DNA microarrays. Stat Sci 2003;18:104-117.
17.
Breiman L: Random forests. Mach Learn 2001;45:5-32.
18.
Liaw A, Wiener M: Classification and regression by RandomForest. R News 2002;2:18-22.
19.
Lin WW, Hsieh SL: Decoy receptor 3: a pleiotropic immunomodulator and biomarker for inflammatory diseases, autoimmune diseases and cancer. Biochem Pharmacol 2011;81:838-847.
20.
Mild G, Bachmann F, Boulay JL, Glatz K, Laffer U, Lowy A, Metzger U, Reuter J, Terracciano L, Herrmann R, Rochlitz C: DCR3 locus is a predictive marker for 5-fluorouracil-based adjuvant chemotherapy in colorectal cancer. Int J Cancer 2002;102:254-257.
21.
Tsuji S, Hosotani R, Yonehara S, Masui T, Tulachan SS, Nakajima S, Kobayashi H, Koizumi M, Toyoda E, Ito D, Kami K, Mori T, Fujimoto K, Doi R, Imamura M: Endogenous decoy receptor 3 blocks the growth inhibition signals mediated by Fas ligand in human pancreatic adenocarcinoma. Int J Cancer 2003;106:17-25.
22.
Simon I, Liu Y, Krall KL, Urban N, Wolfert RL, Kim NW, McIntosh MW: Evaluation of the novel serum markers B7-H4, Spondin 2, and DcR3 for diagnosis and early detection of ovarian cancer. Gynecol Oncol 2007;106:112-118.
23.
Ao R, Du YQ, Wang Y, Chen YS, Wang BY: MMP-2 and DcR3 expression in esophageal cancer tissue and correlation with patient survival. Int J Clin Exp Med 2013;6:700-705.
24.
Zhou J, Song S, Li D, He S, Zhang B, Wang Z, Zhu X: Decoy receptor 3 (DcR3) overexpression predicts the prognosis and pN2 in pancreatic head carcinoma. World J Surg Oncol 2014;12:52.
25.
Zong L, Chen P, Wang DX: Death decoy receptor overexpression and increased malignancy risk in colorectal cancer. World J Gastroenterol 2014;20:4440-4445.
26.
Yang CR, Hsieh SL, Ho FM, Lin WW: Decoy receptor 3 increases monocyte adhesion to endothelial cells via NF-κB-dependent up-regulation of intercellular adhesion molecule-1, VCAM-1, and IL-8 expression. J Immunol 2005;174:1647-1656.
27.
Zhou J, Song S, He S, Wang Z, Zhang B, Li D, Zhu D: Silencing of decoy receptor 3 (DcR3) expression by siRNA in pancreatic carcinoma cells induces Fas ligand-mediated apoptosis in vitro and in vivo. Int J Mol Med 2013;32:653-660.
28.
Chen PH, Yang CR: Decoy receptor 3 expression in AsPC-1 human pancreatic adenocarcinoma cells via the phosphatidylinositol 3-kinase-, Akt-, and NF-κB-dependent pathway. J Immunol 2008;181:8441-8449.
29.
Samson MH: Quantitative measurements of trefoil factor family peptides: possibilities and pitfalls. Scand J Clin Lab Invest 2013;73:193-202.
30.
Casado E, Garcia VM, Sánchez JJ, Gómez Del Pulgar MT, Feliu J, Maurel J, Castelo B, Moreno Rubio J, López RA, García-Cabezas MÁ, Burgos E, de Castro J, Belda-Iniesta C, López-Gómez M, Gómez-Raposo C, Zambrana F, Sereno M, Fernández-Martos C, Vázquez P, Lacal JC, González-Barón M, Cejas P: Upregulation of trefoil factor 3 (TFF3) after rectal cancer chemoradiotherapy is an adverse prognostic factor and a potential therapeutic target. Int J Radiat Oncol Biol Phys 2012;84:1151-1158.
31.
Vestergaard EM, Nexø E, Tørring N, Borre M, Ørntoft TF, Sørensen KD: Promoter hypomethylation and upregulation of trefoil factors in prostate cancer. Int J Cancer 2010;127:1857-1865.
32.
Huang YG, Li YF, Wang LP, Zhang Y: Aberrant expression of trefoil factor 3 is associated with colorectal carcinoma metastasis. J Cancer Res Ther 2013;9:376-380.
33.
Im S, Yoo C, Jung JH, Choi HJ, Yoo J, Kang CS: Reduced expression of TFF1 and increased expression of TFF3 in gastric cancer: correlation with clinicopathological parameters and prognosis. Int J Med Sci 2013;10:133-140.
34.
Lasa A, Garcia A, Alonso C, Millet P, Cornet M, Ramón y Cajal T, Baiget M, Barnadas A: Molecular detection of peripheral blood breast cancer mRNA transcripts as a surrogate biomarker for circulating tumor cells. PLoS One 2013;8:e74079.
35.
Jones DR: Measuring midkine: the utility of midkine as a biomarker in cancer and other diseases. Br J Pharmacol 2014;171:2925-2939.
36.
Sueyoshi T, Jono H, Shinriki S, Ota K, Ota T, Tasaki M, Atsuyama E, Yakushiji T, Ueda M, Obayashi K, Mizuta H, Ando Y: Therapeutic approaches targeting midkine suppress tumor growth and lung metastasis in osteosarcoma. Cancer Lett 2012;316:23-30.
37.
Zhong J, Huang HL, Li J, Qian FC, Li LQ, Niu PP, Dai LC: Development of hybrid-type modified chitosan derivative nanoparticles for the intracellular delivery of midkine-siRNA in hepatocellular carcinoma cells. Hepatobiliary Pancreat Dis Int 2015;14:82-89.
38.
Krzystek-Korpacka M, Diakowska D, Neubauer K, Gamian A: Circulating midkine in malignant and non-malignant colorectal diseases. Cytokine 2013;64:158-164.
39.
Jia HL, Ye QH, Qin LX, Budhu A, Forgues M, Chen Y, Liu YK, Sun HC, Wang L, Lu HZ, Shen F, Tang ZY, Wang XW: Gene expression profiling reveals potential biomarkers of human hepatocellular carcinoma. Clin Cancer Res 2007;13:1133-1139.
40.
Zhu WW, Guo JJ, Guo L, Jia HL, Zhu M, Zhang JB, Loffredo CA, Forgues M, Huang H, Xing XJ, Ren N, Dong QZ, Zhou HJ, Ren ZG, Zhao NQ, Wang XW, Tang ZY, Qin LX, Ye QH: Evaluation of midkine as a diagnostic serum biomarker in hepatocellular carcinoma. Clin Cancer Res 2013;19:3944-3954.
41.
Shimada H, Nabeya Y, Okazumi S, Matsubara H, Kadomatsu K, Muramatsu T, Ikematsu S, Sakuma S, Ochiai T: Increased serum midkine concentration as a possible tumor marker in patients with superficial esophageal cancer. Oncol Rep 2003;10:411-414.
42.
Meng Z, Tan J, Zhang G, Tian W, Fu Q, Li W, He X, Wu S, Yang Z, Liang X, Dong L, Zhang Q, Liu L, Zhang Y, Xu K, Liu B, Li N, Li X, Jia Q, He Y, Wang S, Wang R, Zheng W, Song X, Zhang J, Hu T, Liu N, Upadhyaya A: Evaluation of serum midkine as a biomarker in differentiated thyroid cancer. Life Sci 2015;130:18-24.
43.
Wu X, Zhi X, Ji M, Wang Q, Li Y, Xie J, Zhao S: Midkine as a potential diagnostic marker in epithelial ovarian cancer for cisplatin/paclitaxel combination clinical therapy. Am J Cancer Res 2015;5:629-638.
44.
Hu R, Yan Y, Li Q, Lin Y, Jin W, Li H, Lu Y, Pang T: Increased drug efflux along with midkine gene high expression in childhood B-lineage acute lymphoblastic leukemia cells. Int J Hematol 2010;92:105-110.
45.
Hao H, Maeda Y, Fukazawa T, Yamatsuji T, Takaoka M, Bao XH, Matsuoka J, Okui T, Shimo T, Takigawa N, Tomono Y, Nakajima M, Fink-Baldauf IM, Nelson S, Seibel W, Papoian R, Whitsett JA, Naomoto Y: Inhibition of the growth factor MDK/midkine by a novel small molecule compound to treat non-small cell lung cancer. PLoS One 2013;8:e71093.
46.
Dai LC, Yao X, Wang X, Niu SQ, Zhou LF, Fu FF, Yang SX, Ping JL: In vitro and in vivo suppression of hepatocellular carcinoma growth by midkine-antisense oligonucleotide-loaded nanoparticles. World J Gastroenterol 2009;15:1966-1972.
47.
Oshima Y, Yajima S, Yamazaki K, Matsushita K, Tagawa M, Shimada H: Angiogenesis-related factors are molecular targets for diagnosis and treatment of patients with esophageal carcinoma. Ann Thorac Cardiovasc Surg 2010;16:389-393.
48.
Dianat N, Le Viet B, Gobbo E, Auger N, Bièche I, Bennaceur-Griscelli A, Griscelli F: Midkine lacking its last 40 amino acids acts on endothelial and neuroblastoma tumor cells and inhibits tumor development. Mol Cancer Ther 2015;14:213-224.
49.
Kubo S, Kawasaki Y, Yamaoka N, Tagawa M, Kasahara N, Terada N, Okamura H: Complete regression of human malignant mesothelioma xenografts following local injection of midkine promoter-driven oncolytic adenovirus. J Gene Med 2010;12:681-692.
50.
Takagi-Kimura M, Yamano T, Tagawa M, Kubo S: Oncolytic virotherapy for osteosarcoma using midkine promoter-regulated adenoviruses. Cancer Gene Ther 2014;21:126-132.
51.
Maldonado AR, Klanke C, Jegga AG, Aronow BJ, Mahller YY, Cripe TP, Crombleholme TM: Molecular engineering and validation of an oncolytic herpes simplex virus type 1 transcriptionally targeted to midkine-positive tumors. J Gene Med 2010;12:613-623.
© 2016 S. Karger AG, Basel
2016
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.