ANTICANCER RESEARCH 32: 3699-3706 (2012)

Site- and Grade-specific Diversity of LINE1 Methylation Pattern in Gastroenteropancreatic Neuroendocrine Tumours

INGO STRICKER, DIMITRI TZIVRAS, SANDEEP NAMBIAR, JUERGEN WULF, SVEN-THORSTEN LIFFERS, MARKUS VOGT, BERLINDA VERDOODT, ANDREA TANNAPFEL and ALIREZA MIRMOHAMMADSADEGH

Institute of Pathology, Ruhr University Bochum, Bochum, Germany

Abstract. Background: Recent data indicate that further supplemented by a grading system (5, 6). These gastroenteropancreatic neuroendocrine tumours (GEP-NETs) guidelines for GEP-NETs were adopted in the seventh edition have a hypomethylated long interspersed element (LINE1) of the TNM Classification in 2010 (7-10). Presently, apart promoter. To answer the question, of whether LINE1 may be of from the recent TNM staging, NETs are graded according to value in assessing the malignant potential of GEP-NETs, we their Ki-67 proliferation index or mitotic rate into G1 and G2, analysed LINE1 methylation in different organs. Materials and and neuroendocrine carcinoma (NEC) (4). Such a Methods: A total of 58 GEP-NETs of gastric (n=14), classification and grading has been reported to more pancreatic (n=15), small intestine (n=17), appendix (n=8), accurately predict outcomes (11, 12). However, in individual colorectal (n=4) and non-neoplastic tissues were analysed cases, the assessment of the actual malignant potential is using DNA isolation, bisulphite-treatment and pyrosequencing. difficult and uncertain (13). Results: LINE1 hypomethylation was detected in 50% of In addition to clinical or histopathological markers, distinct gastric, 100% pancreatic, 82% small intestine, 87.5% appendix abnormalities, such as point mutations, deletions, and 100% colorectal NETs. G1 (p<0.001) and G2 (p<0.05) chromosomal losses/gains and, most importantly, methylation, colorectal, and G1 (p<0.001) and G2 (p<0.001) pancreatic have also been reported as markers of differentiation and NETs exhibited significant LINE1 hypomethylation compared biological aggressiveness of GEP-NETs (14-19). Among with non-neoplastic tissues. Higher rates of LINE1 these, hypomethylation of long interspersed nucleotide hypomethylation in G2 pancreatic NETs than in G1 NETs element type 1 (LINE1) has been previously implicated in (p<0.05) were observed. NETs exhibited a significantly lower well-differentiated NETs (16). LINE1, the most common frequency of hypomethylation in cases with lymph node family of non-long terminal repeats (LTR) retrotransposons metastases (p<0.05). Conclusion: LINE1 hypomethylation may in the human genome, comprises about 17% of the genome serve as a marker of tumour grade and lymph node metastasis. and exhibits a distinctive pattern of hypomethylation in various neoplasms (20-22). LINE1 elements retain their Gastroenteropancreatic neuroendocrine tumours (GEP-NETs) competency to be active in the human genome, and their are heterogeneous and rare tumours of epithelial origin hypomethylation can lead to transcriptional activation of occurring in the gastrointestinal (GI) tract exhibiting intact LINE1 elements, induce retrotransposition, and neuroendocrine differentiation (1-3). Numerous classification facilitate genetic instability (23-25). In GEP-NETs, relative systems were used before the World Health Organization hypomethylation of LINE1 in tumours has been reported to (WHO) classifications in 2000, subsequently revised in 2004 be more common in patients whose tumours had loss of (4). The European Neuroendocrine Tumour Society (ENETS) chromosome 18 and methylation of RASSF1A, while this was proposed a TNM staging system based on tumour thickness associated with lymph node metastasis (16). and/or size, lymph node involvement and metastatic disease, In the current study, we analysed LINE1 methylation patterns in a large series of GEP-NETs of different locations, stages of disease and proliferative activity.

Correspondence to: Ingo Stricker, Institute of Pathology, Ruhr- Materials and Methods University Bochum, 44789 Bochum, Germany. Tel: +49 2343024800, Fax: +49 2343024809, e-mail: ingo.stricker@ruhr-uni- Patients and tissue samples. A collective of 58 archival formalin- bochum.de fixed paraffin-embedded (FFPE) biopsies and resected GEP-NETs samples from 58 patients diagnosed at the Institute for Pathology, Key Words: LINE1, gastroenteropancreatic neuroendocrine tumours, Ruhr University of Bochum between 2002 and 2008 were collected pyrosequencing, methylation pattern. for the retrospective study. The following tumour sites were

0250-7005/2012 $2.00+.40 3699 ANTICANCER RESEARCH 32: 3699-3706 (2012) analyzed: stomach (n=14), pancreas (n=15) and small intestine/ sample. The primers were High-performance liquid chromatography colorectum (n=29). The primary site of small intestine colorectum (HPLC)-grade purified and manufactured by Metabion international tumours was the duodenum (n=3), ileum (n=4), small intestine not AG (Martinsried, Bavaria, Germany). otherwise specified (n=4), appendix (n=8), rectum (n=2) and one patient for each sigmoidal colon (n=1), ampulla of vater (n=1) and PCR conditions for pyrosequencing. Fifty nanograms of bisulphite- colon not otherwise specified (n=1). treated DNA were used in the 25 μl PCR reaction with 400 pmol/l The clinical data, as well as localisation, TNM and ENET stage forward and reverse primers, respectively. PCR conditions for and grade, are shown in Table I. The corresponding non-neoplastic LINE1 were, 95˚C for 15 min (95˚C for 40, 55˚C for 40, and 72˚C mucosa from patients were defined as normal. for 40), 40 cycles and 1×72˚C for 10 min using HotStar Taq The study was approved by the local Ethics Committee of the Ruhr (Qiagen). University Bochum, Faculty of Medicine (register no. 3980-11). Statistical analysis. All statistical analyses were carried out with the Classification and histopathological grading. The GEP-NET Statistica version 9 software package. Significance testing with specimens were subjected to histological examination by two continuous variables was performed using the Student t-test (for independent pathologists (A.T, I.S) for confirmation of the grading, comparison of two groups). WHO classification of NETs and staging according to the ENETs and TNM system (4). The grading system based on mitotic rate and Results Ki-67 labelling index was as recently proposed by ENETS and adopted by TNM 2010: G1 [mitotic count <2 per 10 high power ENETs and TNM staging, grading and other clinicopatho- fields (HPF), Ki-67 index up to 2%], G2 (mitotic count 2-20 per 10 HPF, Ki-67 index 3-20%), G3 (mitotic count >20 per 10 HPF, Ki-67 logical features. The ENETs and TNM staging, grading, Ki-67 index up to >20%). By convention, NETs of the appendix are not labelling index and clinicopathological features of 58 patients described by the grading system. The discrimination of typical and with GEP-NETs are summarized in Table I. Representative atypical carcinoids is based on a mitotic count of up to 1 per 10 staining of G1 NET, G2 NET and NEC tumours for standard HPF and 2-10 per 10 HPF respectively. Nevertheless a grading of HE and immunohistochemical staining with Synaptophysin and the 8 tumours was performed. Ki-67 are shown in Figure 1. Data on positive lymph node Immunohistochemical analysis. Immunostaining was performed using involvement was available for 11 patients (pN1=11). a Leica BOND-MAX™ autostainer and Bond Polymer Refine Red Detection™ system (Leica Microsystems, Wetzlar, Hesse, Germany) Tumour site-specific LINE1 hypomethylation. LINE1 according to the manufacturer’s specifications. Briefly, 1-2 micron- methylation was assessed using the primers described by thin sections were subjected to heat treatment for 20 min and then Tellez et al., and a mean of four CpG sites was taken as an incubated with pre-diluted anti-Ki-67 (#M7240, MIB-1, mouse estimate of LINE1 methylation (26). When tumours exhibited monoclonal; Dakocytomation, Hamburg, Germany) or anti- synaptophysin 67 (#AM363-5M, mouse monoclonal; Biogenex, a significant decrease in % LINE1 methylation in comparison Fremont, CA, USA), for 20 min. Recommended positive and negative to normal tissues, they were defined to be hypomethylated. controls were used. Slides were evaluated using a Zeiss Axioplan 2 Levels of LINE1 methylation tumour samples and those of imaging system (objective, ×40). All tumour cell areas on the slide normal tissues are shown in Figure 2. The median LINE1 that stained positively were included as part of the evaluations methylation in pancreatic GEP-NET samples was 64% regardless of the degree of staining. Cases with Ki-67 compared with 73% in normal samples (p<0.001) and hence immunoreactivity of less than 1% were scored "0". was statistically significant (Figure 2C). The median LINE1 DNA isolation, bisulphite pyrosequencing and LINE1 analysis. The methylation in small intestine-NET samples was 63% histopathological lesions of interest were micro-dissected and used for compared with 69.5% in normal samples (p<0.05) and hence DNA extraction. For patient tumour samples, 10 μm sections were was statistically significant (Figure 2D). The median LINE1 made from archived FFPE tissues. Tumour tissues were microdissected methylation in all 21 small intestine and colorectal NET using a scalpel, with the corresponding HE-slide as a template. After samples excluding the 8 appendix NETs was 63.5% heating for 1 h at 62˚C, the samples were deparaffinized and compared with 69.5% in normal samples (p<0.001) and rehydrated by passing them through first xylene and then a graded isopropanol series. DNA isolation was performed using the Qiagen hence was statistically significant (Figure 2F). In contrast, FFPE tissue kit (Qiagen, Hilden, North Rhine-Westphalia Germany), the median LINE1 methylation in gastric NETs and appendix following the instructions of the manufacturer. DNA quantity was NET samples in comparison to their respective normal assessed spectrophotometrically. One microgram of genomic DNA samples were statistically insignificant (Figure 2B, E). was subjected to bisulphite conversion using the EpiTect Bisulphite kit All grades of GEP-NET tumour samples (G1 NET, G2 (Qiagen) according to the supplier’s protocol. The eluted DNA (20 μl NET and NEC) were significantly less methylated than volume) was used for the methylation-sensitive PCR analysis. normal tissues at LINE1 for all sites except the stomach Pyrosequencing was performed in a PSQ HS96 instrument (Qiagen) using previously described methylation-specific primers (Figure 2G). The median LINE1 methylation in small for quantitative sequencing (pyrosequencing) of LINE1 (26). The intestine/colonic G1 NET (p<0.001) and G2 NET (p<0.05) mean of % methylation at 4 individual CpG sites within the samples was 62% and 64%, respectively, compared to 69% sequenced region was taken as % LINE1 methylation for each in normal samples (Figure 2H). Median LINE1 methylation

3700 Stricker et al: LINE1 Hypomethylation in GEP-NETs

Table I. Clinicopathological features of the study cohort including European Neuroendocrine Tumour Society (ENETS) and Tumour lymph node metastasis (TNM) staging, grading and Ki-67 labelling index.

Gender Age (years) Site Ki-67 index Grading ENETS TNM 2010 N0/N1 M0/M1

F 82 Gastric <1% G1 NET T1 T1 N0 M0 F 57 Gastric 1% G1 NET T1 T1 N0 M0 F 65 Gastric 1% G1 NET pT2 pT2 pN0 M0 M 76 Gastric 1% G1 NET pT2 pT2 N0 M0 M 77 Gastric 1% G1 NET pT1 pT1 pN0 M0 F 52 Gastric 1% G1 NET pT1 pT1 pN0 M1 M 72 Gastric 4% G2 NET pT2 pT2 pN0 M0 F 86 Gastric 1% G1 NET T1 T1 N0 M0 M 55 Gastric 55% NEC T1 T1 N0 M0 M 70 Gastric 1% G1 NET T1 T1 N0 M0 M 74 Gastric 1% G1 NET T1 T1 N0 M0 F 75 Gastric 1% G1 NET T1 T1 N0 M0 M 61 Gastric 1% G1 NET pT1 pT1 N0 M0 M 56 Gastric 100% NEC T1 T1 N0 M0 M 87 Small intestine 2% G1 NET pT2 pT2 pN1 M1 M 74 Small intestine 1% G1 NET pT3 pT3 pN1 M0 F 72 Small intestine 2% G1 NET pT1 pT1 N0 M0 M 74 Small intestine 1% G1 NET pT2 pT2 N0 M0 F 76 Small intestine 30% NEC pT3 pT3 pN1 M0 M 58 Small intestine 1% G1 NET pT1 pT1 N0 M0 F 71 Small intestine 1% G1 NET pT2 pT2 pN1 M1 F 83 Small intestine 1% G1 NET pT3 pT3 pN1 M1 M 72 Small intestine 2% G1 NET pT2 pT2 N0 M0 F 53 Small intestine 5% G2 NET pT2 pT2 pN1 M0 M 69 Small intestine 5% G2 NET pT3 pT3 N0 M1 F 80 Small intestine 10% G2 NET T1 T1 N0 M0 F 81 Small intestine 5% G2 NET pT3 pT3 pN1 M1 M 73 Small intestine 5% G2 NET pT3 pT3 pN1 M0 M 75 Small intestine 20% G2 NET T1 T1 N0 M0 F 81 Small intestine 50% NEC pT4 pT4 N0 M0 M 41 Small intestine <1% G1 NET pT2 pT2 pN0 M0 M 90 Pancreas 1% G1 NET pT1 pT1 N0 M0 F 79 Pancreas 1% G1 NET pT2 pT2 pN1 M0 M 69 Pancreas 1% G1 NET pT1 pT1 N0 M0 F 60 Pancreas 1% G1 NET pT1 pT1 pN0 M0 F 75 Pancreas 1% G1 NET pT2 pT2 N0 M0 M 74 Pancreas 2% G1 NET pT2 pT2 pN0 M0 M 54 Pancreas 1% G1 NET pT1 pT1 pN1 M0 F 59 Pancreas 0% G1 NET pT1 pT1 N0 M0 F 62 Pancreas 1% G1 NET pT1 pT1 pN0 M0 F 24 Pancreas 5% G2 NET pT1 pT1 N0 M0 M 33 Pancreas 3% G2 NET pT2 pT2 N0 M0 M 59 Pancreas 10% G2 NET pT3 pT3 pN1 M1 F 59 Pancreas 1% G1 NET pT1 pT1 pN0 M0 F 48 Pancreas 8% G2 NET pT1 pT1 pN0 M1 M 52 Pancreas 3% G2 NET pT3 pT2 pN0 M0 M 83 Colorectal 1% G1 NET pT1a pT1a pN0 M0 F 58 Colorectal 20% G2 NET T1 T1 N0 M0 F 75 Colorectal 3% G2 NET T1 T1 N0 M0 M 76 Colorectal 5% G2 NET T1 T1 N0 M0 M 53 Appendix 1% G1 NET pT2 pT1b N0 M0 M 56 Appendix 1% G1 NET pT1 pT1a N0 M0 M 69 Appendix 2% G1 NET pT3 pT1a N0 M0 F 42 Appendix 1% G1 NET pT3 pT2 N0 M0 F 18 Appendix 1% G1 NET pT2 pT1b N0 M0 M 19 Appendix 1% G1 NET pT1 pT1b N0 M0 M 42 Appendix 4% G2 NET pT1 pT1a N0 M0 F 19 Appendix 1% G1 NET pT1 pT1a N0 M0

M: Male; F: female; NET: Neuroendocrine Tumour; T: size or direct extent of the primary tumor; N: degree of spread to regional lymph nodes; M: metastasis.

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Figure 1. Staining of G1 NET, G2 NET and neuroendocrine carcinoma (NEC) tumours. HE and immunohistochemical staining of synaptophysin and Ki-67 in G1 NET, G2 NET and NEC tumour samples.

in pancreatic G1 NET and G2 NET samples was 66.5% and (defined by UICC and ENET). Classified by TNM 2010, 63%, respectively, compared to 73% in normal samples median LINE1 methylation from 22 pT1 (range=57%-72%), (p<0.001) and hence was statistically significant (Figure 2I). 15 pT2 (range=54-73%) and 7 pT3 tumours (range=49%- 67%) was 66%, 63% and 64% respectively. LINE1 in 1 pT4 LINE1 hypomethylation and grading. In pancreatic G2 NETs tumour was methylated by 76%. No significant methylation (n=5), LINE1 methylation (range=56%-64%) was differences between pT1 and pT2 (p=0.121417) and between significantly lower compared with G1 NETs (n=10) pT2 and pT3 tumours (p=0.43855) were observed. No (range=61%-72%). The median LINE1 methylation in G2 significant differences were observed using the ENET pancreatic NET samples was 63% compared with 66.5% in classification (19 pT1, 15 pT2 and 10 pT3 tumours; pT1 vs. G1 pancreatic NET samples (p<0.05) (Figure 3). No pT2: p=0.285, pT2 vs. pT3: p=0.265). significant differences in LINE1 methylation between G1 According to the pN category, LINE1 methylation in the NETs, G2 NETs and NECs of gastric, small intestine/ primary tumours was significantly lower in pN1 samples colorectal or appendiceal NETs could be observed. (median methylation 62%), compared with pN0 samples (median methylation 67%) (p<0.05) (Figure 4). Site-specific LINE1 hypomethylation of GEP-NETs in relation LINE1 hypomethylation did not significantly correlate with to pT and pN category of TNM and ENETS classification and tumour size. The Pearson product-moment correlation tumour size. There was no significant correlation between coefficient (r) of LINE1 methylation vs. tumour diameter for LINE1 methylation and tumour stage and pT category gastric, colorectal and pancreatic GEP-NETs were –0.02, –0.31

3702 Stricker et al: LINE1 Hypomethylation in GEP-NETs

Figure 2. Long interspersed element (LINE1) methylation in healthy tissues and gastroenteropancreatic neuroendocrine tumour (GEP-NET) samples: A: all sites (p<0.001); B: gastric NET; C: pancreatic NET (p<0.001), D: small intestine NET (p<0.05); E: appendiceal NET ; and F: small intestine and colorectal NET without appendix (p<0.001). Comparison of LINE1 methylation in healthy tissues and G1 NET, G2 NET and neuroendocrine carcinoma (NEC) tumours: G: all sites (each p<0.001); H: small intestine (each p<0.05); and I: pancreatic tumours (each p<0.001).

and –0.28, respectively. The percentage explained variance (r2 (27). Although various epigenetic alterations, including LINE1 ×100) calculated for these correlations were 0.07%, 10.09% hypomethylation have been reported for GEP-NETs, their and 8.24% respectively and was, hence, insignificant. tumour site-specific relevance remained unexplored. Our study, to our knowledge, for the first time, demonstrated that Discussion LINE1 hypomethylation is a predictor of tumour progression in small intestine/colorectal and pancreatic NETs but not in Genome-wide losses of DNA methylation have been regarded gastric and appendiceal NETs. Previous reports from us and as a common epigenetic event in several malignancies other groups have shown that in general, gastric NETs and including GEP-NETs and may play an essential role in tumour appendiceal NETs are less malignant among GEP NETs (28- development and progression. GEP-NETs exhibit a wide and 30). Such site-specific variation in LINE1 hypomethylation in complex spectrum of clinical behaviours with site-specific or GEP-NETs is consistent with a model of site-specific growth site-dependent functional modulations in their behavior functional interactions between mesenchymal and

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Figure 3. Relation between long interspersed element (LINE1) Figure 4. Relation between long interspersed element (LINE1) methylation in pancreatic G1 neuroendocrine tumour (NET) and G2 methylation in pN0 and pN1 tumours. Comparison of LINE1 methylation NET: Comparison of LINE1 methylation in G1 NET and G2 NET in the primary tumours with known pN0 and pN1 status showing a (p<0.05). significantly lower hypomethylation rate in pN1 tumours (p<0.05).

neuroendocrine cells contributing to the malignancy of GEP- lymph node metastasis. Based on these results, we hypothesize NETs, depending on their growth site. Although GEP-NETs that this site-specific diversity of global hypomethylation in may possess a malignant potential, their biological behaviour NETs may explain the site-specific differences in genomic differs depending on their type and hence their malignancy instability and loss of heterozygosity (LOH) of chromosomes can be predominantly defined on the basis of cellular 11q, 16q and 18 observed in neuroendocrine tumours (33). proliferation rate/grade (mitotic count or Ki-67 index). Such a Interestingly, an association between global hypomethylation grading based on the Ki-67 index, has been reported not only and 18q LOH was demonstrated in colorectal carcinomas (34, to more accurately predict outcomes but also to be an 35), an association also characteristic of NETs (16). Similarly, independent predictor for survival (12, 31, 32). Therefore, the previous studies have demonstrated that in prostatic current study investigating tumour site-specific LINE1 adenocarcinomas, genome-wide DNA hypomethylation of hypomethylation also encompassed its diversity across LINE1 was associated with alterations of chromosome 8 (36). progressive grades. Our study revealed that although both Furthermore, grade-dependent variation in LINE1 small intestine/colorectal and pancreatic NETs demonstrate hypomethylation, as well as significantly greater LINE1 hypomethylation in G1 and G2 grades, the pattern of hypomethylation observed in N1 tumours relative to N0 hypomethylation is more progressive during successive grades tumours strongly indicate that global hypomethylation may of pancreatic NET. Furthermore, the current study also also contribute towards clonal changes and clonal evolution in demonstrated that LINE1 hypomethylation did not correlate GEP-NETs. Along similar lines, Choi et al. also observed that with size or depth of GEP-NETs but was associated with LINE1 hypomethylation was associated with lymph node

3704 Stricker et al: LINE1 Hypomethylation in GEP-NETs metastasis, and was independent of RASSF1A methylation and 10 Wittekind C and Tannapfel A: The current TNM system for inversely correlated with O-6-methylguanine-DNA gastrointestinal tumours part I. Pathologe 31(5): 344-347, 2010. methyltransferase (MGMT) gene methylation (16). Such a (in German). model is consistent with the role of epigenetic plasticity in 11 Scarpa A, Mantovani W, Capelli P, Beghelli S, Boninsegna L, Bettini R, Panzuto F, Pederzoli P, delle Fave G and Falconi M: tumour progression (37-40). Pancreatic endocrine tumours: improved TNM staging and In summary, the current study, to our knowledge, for the histopathological grading permit a clinically efficient prognostic first time, demonstrates a site- and grade-specific diversity stratification of patients. 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