Background/Aims: The occurrence and clinical relevance of DNA hypermethylation and global hypomethylation in pancreatic neuroendocrine tumours (PanNETs) are still unknown. We evaluated the frequency of both epigenetic alterations in PanNETs to assess the relationship between methylation profiles and chromosomal instability, tumour phenotypes and prognosis. Methods: In a well-characterized series of 56 sporadic G1 and G2 PanNETs, methylation-sensitive multiple ligation-dependent probe amplification was performed to assess hypermethylayion of 33 genes and copy number alterations (CNAs) of 53 chromosomal regions. Long interspersed nucleotide element-1 (LINE-1) hypomethylation was quantified by pyrosequencing. Results: Unsupervised hierarchical clustering allowed to identify a subset of 22 PanNETs (39%) exhibiting high frequency of gene-specific methylation and low CNA percentages. This tumour cluster was significantly associated with stage IV (p = 0.04) and with poor prognosis in univariable analysis (p = 0.004). LINE-1 methylation levels in PanNETs were significantly lower than in normal samples (p < 0.01) and were approximately normally distributed. 12 tumours (21%) were highly hypomethylated, showing variable levels of CNA. Interestingly, only 5 PanNETs (9%) were observed to show simultaneously LINE-1 hypomethylation and high frequency of gene-specific methylation. LINE-1 hypomethylation was strongly correlated with advanced stage (p = 0.002) and with poor prognosis (p < 0.0001). In the multivariable analysis, low LINE-1 methylation status and methylation clusters were the only independent significant predictors of outcome (p = 0.034 and p = 0.029, respectively). Conclusion: The combination of global DNA hypomethylation and gene hypermethylation analyses may be useful to define distinct subsets of PanNETs. Both alterations are common in PanNETs and could be directly correlated with tumour progression.

1.
Jiao Y, Shi C, Edil BH, de Wilde RF, Klimstra DS, Maitra A, Schulick RD, Tang LH, Wolfgang CL, Choti MA, Velculescu VE, Diaz LA Jr, Vogelstein B, Kinzler KW, Hruban RH, Papadopoulos N: DAXX/ATRX, MEN1, and mTOR pathway genes are frequently altered in pancreatic neuroendocrine tumors. Science 2011;331:1199-1203.
2.
Arnold CN, Nagasaka T, Goel A, Scharf I, Grabowski P, Sosnowski A, Schmitt-Graff A, Boland CR, Arnold R, Blum HE: Molecular characteristics and predictors of survival in patients with malignant neuroendocrine tumors. Int J Cancer 2008;123:1556-1564.
3.
Arnold CN, Sosnowski A, Schmitt-Graff A, Arnold R, Blum HE: Analysis of molecular pathways in sporadic neuroendocrine tumors of the gastro-entero-pancreatic system. Int J Cancer 2007;120:2157-2164.
4.
Malpeli G, Amato E, Dandrea M, Fumagalli C, Debattisti V, Boninsegna L, Pelosi G, Falconi M, Scarpa A: Methylation-associated down-regulation of RASSF1A and upregulation of RASSF1C in pancreatic endocrine tumors. BMC Cancer 2011;11:351.
5.
Pizzi S, Azzoni C, Bottarelli L, Campanini N, D'Adda T, Pasquali C, Rossi G, Rindi G, Bordi C: RASSF1A promoter methylation and 3p21.3 loss of heterozygosity are features of foregut, but not midgut and hindgut, malignant endocrine tumours. J Pathol 2005;206:409-416.
6.
House MG, Herman JG, Guo MZ, Hooker CM, Schulick RD, Lillemoe KD, Cameron JL, Hruban RH, Maitra A, Yeo CJ: Aberrant hypermethylation of tumor suppressor genes in pancreatic endocrine neoplasms. Ann Surg 2003;238:423-431.
7.
Baylin SB, Herman JG: DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet 2000;16:168-174.
8.
Chan AO, Kim SG, Bedeir A, Issa JP, Hamilton SR, Rashid A: CpG island methylation in carcinoid and pancreatic endocrine tumors. Oncogene 2003;22:924-934.
9.
Schmitt AM, Schmid S, Rudolph T, Anlauf M, Prinz C, Kloppel G, Moch H, Heitz PU, Komminoth P, Perren A: VHL inactivation is an important pathway for the development of malignant sporadic pancreatic endocrine tumors. Endocr Relat Cancer 2009;16:1219-1227.
10.
Choi IS, Estecio MR, Nagano Y, Kim do H, White JA, Yao JC, Issa JP, Rashid A: Hypomethylation of LINE-1 and Alu in well-differentiated neuroendocrine tumors (pancreatic endocrine tumors and carcinoid tumors). Mod Pathol 2007;20:802-810.
11.
Stricker I, Tzivras D, Nambiar S, Wulf J, Liffers ST, Vogt M, Verdoodt B, Tannapfel A, Mirmohammadsadegh A: Site- and grade-specific diversity of LINE1 methylation pattern in gastroenteropancreatic neuroendocrine tumours. Anticancer Res 2012;32:3699-3706.
12.
Deng G, Nguyen A, Tanaka H, Matsuzaki K, Bell I, Mehta KR, Terdiman JP, Waldman FM, Kakar S, Gum J, Crawley S, Sleisenger MH, Kim YS: Regional hypermethylation and global hypomethylation are associated with altered chromatin conformation and histone acetylation in colorectal cancer. Int J Cancer 2006;118:2999-3005.
13.
Matsuzaki K, Deng G, Tanaka H, Kakar S, Miura S, Kim YS: The relationship between global methylation level, loss of heterozygosity, and microsatellite instability in sporadic colorectal cancer. Clin Cancer Res 2005;11:8564-8569.
14.
Estecio MR, Gharibyan V, Shen L, Ibrahim AE, Doshi K, He R, Jelinek J, Yang AS, Yan PS, Huang TH, Tajara EH, Issa JP: LINE-1 hypomethylation in cancer is highly variable and inversely correlated with microsatellite instability. PLoS One 2007;2:e399.
15.
Frigola J, Sole X, Paz MF, Moreno V, Esteller M, Capella G, Peinado MA: Differential DNA hypermethylation and hypomethylation signatures in colorectal cancer. Hum Mol Genet 2005;14:319-326.
16.
Ehrlich M: Cancer-linked DNA hypomethylation and its relationship to hypermethylation. Curr Top Microbiol Immunol 2006;310:251-274.
17.
La Rosa S, Klersy C, Uccella S, Dainese L, Albarello L, Sonzogni A, Doglioni C, Capella C, Solcia E: Improved histologic and clinicopathologic criteria for prognostic evaluation of pancreatic endocrine tumors. Hum Pathol 2009;40:30-40.
18.
Rindi G, Falconi M, Klersy C, Albarello L, Boninsegna L, Buchler MW, Capella C, Caplin M, Couvelard A, Doglioni C, Delle Fave G, Fischer L, Fusai G, de Herder WW, Jann H, Komminoth P, de Krijger RR, La Rosa S, Luong TV, Pape U, Perren A, Ruszniewski P, Scarpa A, Schmitt A, Solcia E, Wiedenmann B: TNM staging of neoplasms of the endocrine pancreas: results from a large international cohort study. J Natl Cancer Inst 2012;104:764-777.
19.
Rindi G, Kloppel G, Alhman H, Caplin M, Couvelard A, de Herder WW, Erikssson B, Falchetti A, Falconi M, Komminoth P, Korner M, Lopes JM, McNicol AM, Nilsson O, Perren A, Scarpa A, Scoazec JY, Wiedenmann B: TNM staging of foregut (neuro)endocrine tumors: a consensus proposal including a grading system. Virchows Arch 2006;449:395-401.
20.
Van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL, Delabesse E, Davi F, Schuuring E, Garcia-Sanz R, van Krieken JH, Droese J, Gonzalez D, Bastard C, White HE, Spaargaren M, Gonzalez M, Parreira A, Smith JL, Morgan GJ, Kneba M, Macintyre EA: Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 concerted action BMH4-CT98-3936. Leukemia 2003;17:2257-2317.
21.
Joensuu EI, Abdel-Rahman WM, Ollikainen M, Ruosaari S, Knuutila S, Peltomaki P: Epigenetic signatures of familial cancer are characteristic of tumor type and family category. Cancer Res 2008;68:4597-4605.
22.
Verma M, Srivastava S: Epigenetics in cancer: implications for early detection and prevention. Lancet Oncol 2002;3:755-763.
23.
Laird PW: The power and the promise of DNA methylation markers. Nat Rev Cancer 2003;3:253-266.
24.
La Rosa S, Marando A, Furlan D, Sahnane N, Capella C: Colorectal poorly differentiated neuroendocrine carcinomas and mixed adenoneuroendocrine carcinomas: insights into the diagnostic immunophenotype, assessment of methylation profile, and search for prognostic markers. Am J Surg Pathol 2012;36:601-611.
25.
Furlan D, Sahnane N, Mazzoni M, Pastorino R, Carnevali I, Stefanoli M, Ferretti A, Chiaravalli AM, La Rosa S, Capella C: Diagnostic utility of MS-MLPA in DNA methylation profiling of adenocarcinomas and neuroendocrine carcinomas of the colon-rectum. Virchows Arch 2013;462:47-56.
26.
Homig-Holzel C, Savola S: Multiplex ligation-dependent probe amplification (MLPA) in tumor diagnostics and prognostics. Diagn Mol Pathol 2012;21:189-206.
27.
Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP: A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res 2004;32:e38.
28.
Raftery CF: Model-based clustering, discriminant analysis, and density estimation. J Am Stat Assoc 2002;97:611-631.
29.
Schwarz G: Estimating the dimension of a model. Ann Stat 1978;6:461-464.
30.
Baba Y, Huttenhower C, Nosho K, Tanaka N, Shima K, Hazra A, Schernhammer ES, Hunter DJ, Giovannucci EL, Fuchs CS, Ogino S: Epigenomic diversity of colorectal cancer indicated by LINE-1 methylation in a database of 869 tumors. Mol Cancer 2010;9:125.
31.
Yachida S, Vakiani E, White CM, Zhong Y, Saunders T, Morgan R, de Wilde RF, Maitra A, Hicks J, Demarzo AM, Shi C, Sharma R, Laheru D, Edil BH, Wolfgang CL, Schulick RD, Hruban RH, Tang LH, Klimstra DS, Iacobuzio-Donahue CA: Small cell and large cell neuroendocrine carcinomas of the pancreas are genetically similar and distinct from well-differentiated pancreatic neuroendocrine tumors. Am J Surg Pathol 2012;36:173-184.
32.
Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB, Issa JP: CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci USA 1999;96:8681-8686.
33.
Dejeux E, Olaso R, Dousset B, Audebourg A, Gut IG, Terris B, Tost J: Hypermethylation of the IgF2 differentially methylated region 2 is a specific event in insulinomas leading to loss-of-imprinting and overexpression. Endocr Relat Cancer 2009;16:939-952.
34.
Ehrlich M: DNA methylation in cancer: too much, but also too little. Oncogene 2002;21:5400-5413.
35.
Nosho K, Irahara N, Shima K, Kure S, Kirkner GJ, Schernhammer ES, Hazra A, Hunter DJ, Quackenbush J, Spiegelman D, Giovannucci EL, Fuchs CS, Ogino S: Comprehensive biostatistical analysis of CpG island methylator phenotype in colorectal cancer using a large population-based sample. PLoS One 2008;3:e3698.
36.
Eden A, Gaudet F, Waghmare A, Jaenisch R: Chromosomal instability and tumors promoted by DNA hypomethylation. Science 2003;300:455.
37.
Roman-Gomez J, Jimenez-Velasco A, Agirre X, Castillejo JA, Navarro G, San Jose-Eneriz E, Garate L, Cordeu L, Cervantes F, Prosper F, Heiniger A, Torres A: Repetitive DNA hypomethylation in the advanced phase of chronic myeloid leukemia. Leuk Res 2008;32:487-490.
38.
Gaudet F, Hodgson JG, Eden A, Jackson-Grusby L, Dausman J, Gray JW, Leonhardt H, Jaenisch R: Induction of tumors in mice by genomic hypomethylation. Science 2003;300:489-492.
39.
Saito K, Kawakami K, Matsumoto I, Oda M, Watanabe G, Minamoto T: Long interspersed nuclear element-1 hypomethylation is a marker of poor prognosis in stage IA non-small cell lung cancer. Clin Cancer Res 2010;16:2418-2426.
40.
Pattamadilok J, Huapai N, Rattanatanyong P, Vasurattana A, Triratanachat S, Tresukosol D, Mutirangura A: LINE-1 hypomethylation level as a potential prognostic factor for epithelial ovarian cancer. Int J Gynecol Cancer 2008;18:711-717.
41.
Watanabe Y, Maekawa M: Methylation of DNA in cancer. Adv Clin Chem 2010;52:145-167.
42.
Silver A, Sengupta N, Propper D, Wilson P, Hagemann T, Patel A, Parker A, Ghosh A, Feakins R, Dorudi S, Suraweera N: A distinct DNA methylation profile associated with microsatellite and chromosomal stable sporadic colorectal cancers. Int J Cancer 2012;130:1082-1092.
43.
Toyota M, Suzuki H: Epigenetic drivers of genetic alterations. Adv Genet 2010;70:309-323.
44.
Martinez JG, Perez-Escuredo J, Castro-Santos P, Marcos CA, Pendas JL, Fraga MF, Hermsen MA: Hypomethylation of LINE-1, and not centromeric SAT-α, is associated with centromeric instability in head and neck squamous cell carcinoma. Cell Oncol (Dordr) 2012;35:259-267.
45.
Furlan D, Sahnane N, Bernasconi B, Frattini M, Tibiletti MG, Molinari F, Marando A, Zhang L, Vanoli A, Casnedi S, Adsay V, Notohara K, Albarello L, Asioli S, Sessa F, Capella C, La Rosa S: APC alterations are frequently involved in the pathogenesis of acinar cell carcinoma of the pancreas, mainly through gene loss and promoter hypermethylation. Virchows Arch 2014;464:553-564.
46.
Stuppia L, Antonucci I, Palka G, Gatta V: Use of the MLPA assay in the molecular diagnosis of gene copy number alterations in human genetic diseases. Int J Mol Sci 2012;13:3245-3276.
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.