LINE-1 methylation in the peripheral blood mononuclear cells of cancer patients ☆ Nakarin Kitkumthorn a , Time Tuangsintanakul b, c , Prakasit Rattanatanyong b , Danai Tiwawech c, ⁎, Apiwat Mutirangura b, ⁎⁎ a Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Mahidol University, Bangkok 10400, Thailand b Center of Excellence in Molecular Genetics of Cancer and Human Diseases, Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand c Research Division, National Cancer Institute, Bangkok 10400, Thailand abstract article info Article history: Received 22 December 2011 Received in revised form 9 January 2012 Accepted 23 January 2012 Available online 1 February 2012 Keywords: LINE-1 methylation COBRA LINE-1 Level and pattern Peripheral blood mononuclear cells Cancer Background: Recently, we classified LINE-1 loci according to their methylation statuses and found that the percentage of hypomethylated LINE-1 loci ( u C u C) can differentiate between the peripheral blood mononucle- ar cells (PBMCs) of oral cancer patients and normal controls with a higher specificity and sensitivity than overall methylation levels. Here, we evaluated the LINE-1 methylation levels and patterns in PBMCs from patients with cancers of the nasopharynx, lung, liver, bile duct, breast and colon. Methods: Combined Bisulfite Restriction Analysis (COBRA) of LINE-1 loci was performed to examine the LINE- 1 methylation statuses of PBMCs from 216 cancer patients with 6 different types of cancer compared with 144 normal controls. Results: Only colorectal and nasopharyngeal cancer samples were found to have lower levels of overall LINE-1 methylation compared with normal controls (p b 0.0001 and p = 0.0022). However, % u C u C in cancers of the colon, liver, lung and nasopharynx was significantly higher compared with normal controls (p b 0.0001, p b 0.0001, p = 0.01 and p = 0.001, respectively). Furthermore, ROC curve analyses of these four cancer types also demonstrated the potential of % u C u C as a biomarker for cancer diagnosis. Conclusion: Changes in the levels and patterns of genome-wide methylation of PBMCs are associated with cancer risk. For LINE-1, % u C u C is a more effective tumour marker for determining cancer risk than overall methylation levels. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Epigenetic alterations are an important characteristic of carcino- genesis [1,2]. Hypermethylation of the promoters of specific genes and genome-wide DNA hypomethylation have been studied in most cancer types [3]. Genome-wide DNA hypomethylation is produced by the reduction of 5-methyldeoxycytosine at CpG-dinucleotide sites throughout the whole genome, particularly in repetitive DNA se- quences [4,5]. LINE-1 is one type of repetitive sequence that is dispersed throughout approximately 17% of the entire genome [6,7]. Consequent- ly, LINE-1 methylation is widely considered to comprise the significant proportion of genome-wide methylation [2,8,9]. LINE-1 hypomethyla- tion is correlated with genetic changes during carcinogenesis, including the following: genomic instability [10–14], hypermethylation and mu- tation of tumour-suppressor genes [12,15], alternate transcription of oncogenes [16] and the deregulation of cancer genes [17,18]. Therefore, many studies have hypothesised that LINE-1 methylation is a potential- ly useful tool as a universal tumour marker for the detection of cancer DNA [8]. Currently, LINE-1 methylation levels are mostly studied by com- paring DNA from tumour tissues to DNA from histologically normal tissues of the same original cell type. However, the processes used to obtain these tissues are difficult and invasive. On the contrary, some studies have used blood samples, which are capable of reflecting the genetic and epigenetic alterations in primary tissues [19–21]; obtaining blood samples is also a simple, gentle and noninvasive proce- dure. Therefore, blood samples may be a suitable “surrogate” specimen for assessing cancer susceptibility. Moreover, lower LINE-1 methylation levels in blood samples have been correlated with increased risks for various types of cancer such as the following: head and neck cancer, oral cancer [22,23], gastric cancer [24] and bladder cancer [25,28]; on the other hand, LINE-1 methylation levels are observed to be higher in renal cell carcinomas [26]. LINE-1 methylation has been shown to have a different level in each locus of genome [27]. For this reason, we classified the methylation statuses of LINE-1 loci using Combined Bisulfite Restriction Analysis (COBRA) to determine the methylation pattern of the 2 CpG dinucleo- tides in each LINE-1 sequence [23]. This technique differentiated LINE- Clinica Chimica Acta 413 (2012) 869–874 ☆ Disclaimers: None declared. ⁎ Correspondence to: D. Tiwawech, Research Division, National Cancer Institute, Rama VI Rd., Ratchathewi, Bangkok 10400, Thailand. Tel.: + 66 2354 7025. ⁎⁎ Correspondence to: A. Mutirangura, Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Rama IV Rd., Patumwan, Bangkok 10330, Thailand. Tel.: + 66 2256 4281x1713. E-mail addresses: tdnai@hotmail.com (D. Tiwawech), mapiwat@chula.ac.th (A. Mutirangura). 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.01.024 Contents lists available at SciVerse ScienceDirect Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim