Virchows Arch DOI 10.1007/s00428-015-1749-0

ORIGINAL ARTICLE

Prognostic significance of promoter CpG island hypermethylation and repetitive DNA hypomethylation in stage I lung adenocarcinoma Ye-Young Rhee & Tae-Hun Lee & Young Seok Song & Xianyu Wen & Hyojin Kim & Sanghoon Jheon & Choon-Taek Lee & Jei Kim & Nam-Yun Cho & Jin Haeng Chung & Gyeong Hoon Kang

Received: 18 September 2014 / Revised: 15 January 2015 / Accepted: 24 February 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract In carcinogenesis of peripheral pulmonary carcinomas, multiple genetic and epigenetic alterations are involved. In this study, we quantified methylation levels of repetitive DNA elements (L1 and Alu) and six CpG island methylator phenotype (CIMP)-panel markers in various lesions representing steps in the development of lung adenocarcinoma (ADC), including atypical adenomatous hyperplasia, adenocarcinoma in situ, and invasive ADC. We then assessed methylation levels in

Electronic supplementary material The online version of this article (doi:10.1007/s00428-015-1749-0) contains supplementary material, which is available to authorized users. Y.0.693 and absence of methylation of CIMP markers correlated independently with shorter cancer-specific survival. In conclusion, our findings suggest that Alu hypomethylation is an early and L1 hypomethylation a later event during multistep pulmonary carcinogenesis. The prognostic significance of the combination of methylation status of L1 and CIMP markers must be validated in large-scale studies of pulmonary ADC. Keywords Adenocarcinoma . Alu . DNA methylation . LINE-1 . Premalignant lesion

J. Kim Department of Neurology, Chungnam National University School of Medicine, Daejeon, South Korea

Introduction

J. H. Chung (*) Departments of Pathology, Seoul National University Bundang Hospital, Bundang, South Korea e-mail: [email protected]

Lung cancer is a major cause of cancer-related death. Adenocarcinoma (ADC) is the most common histologic type of primary lung cancer. ADC is heterogeneous with respect to molecular, clinical, and pathological characteristics

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[1]. Peripheral pulmonary carcinogenesis involves multiple genetic and epigenetic alterations as well as morphological alterations of bronchioloalveolar epithelium. Atypical adenomatous hyperplasia (AAH) and adenocarcinoma in situ (AIS) are important targets for studying multistep progression of peripheral pulmonary ADC [2, 3]. Both AAH and AIS can be found as incidental findings in lungs resected to remove a primary lung ADC. However, with the advent of more sensitive radiological imaging, these lesions are now being individually detected using thin-section high-resolution computed tomography [4, 5]. Several molecular studies support the existence of an AAH-AIS-ADC continuum [6, 7]. The DNA methylation alterations that occur in human cancer include global DNA hypomethylation and regional CpG island promoter hypermethylation. Global DNA hypomethylation primarily affects CpG sites in repetitive DNA elements, which are normally methylated and make up 45 % of the human genome. The long interspersed nucleotide element 1 (LINE-1 or L1) and Alu retrotransposons, major constituents of interspersed DNA repeats, comprise a substantial portion of the human genome (approximately 28 %). The levels of L1 and Alu methylation are regarded as surrogate markers for global DNA methylation [8–10]. Global DNA hypomethylation appears to play an important role in genomic or chromosomal instability [11, 12]. CpG island methylator phenotype (CIMP) refers to a subset of cancers with widespread concurrent hypermethylation of numerous gene promoter CpG island loci. Brain gliomas and colon cancers are often CIMP-high. Recently, Kondo et al. reported that some lung ADC are CIMP-high, notably those associated with male gender, heavy smoking, wild-type EGFR, and worse prognosis [13]. However, no other studies have investigated CIMP-high lung ADC. Limited information is available about DNA methylation changes in AAH and AIS during multistep carcinogenesis of lung ADC. It is not known whether CIMP occurs already in premalignant lesions or only after malignant transformation. To our knowledge, only one study has explored changes in global DNA methylation in putative preinvasive stages of lung ADC using Alu and SAT2 MethyLight assays [14]. In this study, we analyzed AAH, AIS, and ADC tissue samples for L1 and Alu methylation level using a pyrosequencing methylation assay, and CIMP status using a MethyLight assay. In addition, we investigated methylation levels of L1 and Alu and methylation status of six CIMP markers in surgically resected stage I ADCs and correlated these with clinicopathological findings and clinical outcome. We aimed to elucidate when during multistep carcinogenesis of lung ADCs methylation levels of L1 and Alu change and CIMP occurs and to identify whether methylation status of L1, Alu, or CIMP markers might have prognostic implications in stage I lung ADC.

Materials and methods Patients and specimens representing multistep adenocarcinoma development For the analysis of ADC precursor lesions, 157 formalin-fixed paraffin-embedded (FFPE) archival tissues from 108 patients with peripheral neoplastic lesions and 26 control cases were collected from the Department of Pathology, Seoul National University Bundang Hospital, Korea. Patients with peripheral neoplastic lesions had undergone curative surgical treatment ( l o be c t o m y, n = 85 ; we dg e r es ec ti o n, n = 2 1 ; an d segmentectomy, n=2) between June 2003 and April 2009. The 157 tissue samples included normal lung tissue (n=36) from both patients with ADC (n = 10) and control cases (n=26), AAH (n=20), AIS (n=30), and invasive ADC with a lepidic growth pattern (n=71). Of these patients, eight had multiple synchronous lesions. Control cases (n=26) included bullae and blebs (n=5), bronchiectasis (n=3), pulmonary tuberculosis (n=3), aspergilloma (n=2), and pulmonary metastases from colorectal cancer (n=13). In addition, FFPE archival tissues from 100 patients with stage I ADC were collected from the Department of Pathology, Seoul National University Bundang Hospital, Korea. Two FFPE tissue blocks containing tumor or normal lung tissues were obtained from each patient. These patients had undergone curative surgical treatment between June 2003 and April 2008 (lobectomy, n = 93; wedge resection, n = 4; and segmentectomy, n=3). None of these patients had been treated with preoperative neoadjuvant chemotherapy or radiation therapy. Clinicopathological characteristics, including patient age, sex, smoking status, and tumor size, were obtained from the electronic medical records. Smoking history was obtained from the health interview questionnaire. Current and former smokers were classified as “smokers” and never smokers as “nonsmokers.” Histologic subtypes of adenocarcinoma were classified according to the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society (IASLC/ATC/ERS) pulmonary adenocarcinoma classification [15]. This study was approved by the Institutional Review Board of Seoul National University Bundang Hospital (B-1001-091-301), and patient consent for inclusion in this study was waived. DNA hypermethylation analysis Under a microscope, areas of tumor were marked on the glass tissue slides to exclude normal tissue and enrich tumor cells. As normal control, areas of normal lung tissue free of inflammatory cells or tumor cells were marked on the glass tissue slides. After removal of the cover glass, the marked areas were microdissected with a knife blade. The dissected tissue samples were subjected to tissue lysis using proteinase-K lysis

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buffer. Sodium bisulfate conversion was performed using the EZ DNA methylation kit (Zymo Co, Orange, CA, USA). Six CIMP panel genes were analyzed by the MethyLight assay as described previously [16]. Of these six markers, oligonucleotide sequences of primers and probe targeting CDKN2A (p16) has been described in the literature, while primers and probes for five markers were developed in our laboratory for promoter sequences or the first exon (Supplementary Table 1). Briefly, two sets of primers and probes designed specifically to bind to bisulfite-converted DNA were used in the reaction; one set of primers and a probe were designed for every methylated target to be analyzed (methylated reaction), and one set of primers and a probe were directed to the reference locus, Alu (normalization control reaction). The normalization control reaction refers to the methylation-independent measurement to normalize for input DNA and control for DNA amplification. We used M.SssI-treated, placental genomic DNA as a constant reference sample to determine the percentage of methylated reference (PMR) at a particular locus. PMR was defined as 100×(methylated reaction/control reaction)sample/ (methylated reaction/control reaction) M.SssI-Reference . MethyLight assay was repeated in triplicate, and of the three measured values, the median was regarded as a representative value of methylation level of each marker. Each marker was considered methylated when median PMR value was >4. DNA hypomethylation analysis Polymerase chain reaction (PCR) and subsequent pyrosequencing for L1 and Alu were performed as described previously [17]. The PCR conditions were 45 cycles of 95 °C for 20 s, 52 °C for 20 s, and 72 °C for 20 s, followed by 72 °C for 5 min. The PCR products were purified and quantified in the PyroMark Q24 System (Qiagen, Valencia, CA, USA). The degree of methylation was expressed as the percentage of 5’methylated cytosines among all cytosines. The ratio of C to T nucleotides was evaluated for L1 methylation, and the ratio of G to A nucleotides was evaluated for Alu methylation. The mean of the methylation levels of the three successive CpG sites adjacent to the sequencing primer was taken as the overall L1 or Alu methylation level in a given sample. The experiments were repeated twice, and the average values were taken from these separate experiments. Statistical analysis All statistical analyses were performed using SPSS software (SPSS version 15.0 Chicago, IL). All P values were twosided, and statistical significance was set at PT1a) (n=41), to determine methylation status of CIMP panel markers (CCNA1, ACAN, GFRA1, EDARADD, MGC45800, and p16 (CDKN2A)) using MethyLight assay. Methylation frequency of individual markers in each lesions type is summarized in Supplementary Fig. 1. The number of methylated markers was 0, 0, 0.07, 0.13, and 0.45, respectively, in tissue samples of normal lung, AAH, AIS, ADC (T1a), and ADC (>T1a). However, CIMP (defined as a tumor showing methylation in ≥5 of the CIMP panel markers) was not detected in any of these lesions. Another independent set of stage I ADC tissue samples (n= 100) was analyzed for the presence of CIMP. Clinicopathological features of the associated cases are summarized in Supplementary Table 2. CIMP was found in two tumors (2 %). Methylation frequency for individual markers was 14 %, 13 %, 2 %, 2 %, 2 %, and 1 % (for CCNA1, ACAN, GFRA1, MFC45800, CDKN2A, and EDARADD, respectively). CCNA1 or ACAN methylation did not show any significant association with sex, age, T stage, visceral pleura invasion, lymphatic invasion, venous invasion, or smoking history. However, CCNA1 methylation was more frequent in ADC

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with lepidic pattern than in ADC with acinar pattern (29 % vs. 6 %, Chi-square test, P=0.009) (Supplementary Table 3). None of the patients with ADC with methylation of any of these six markers died of disease, in contrast to patients with ADC without methylation of these markers (Supplementary Fig. 2). A significant difference in cancer-specific survival was noted between stage I ADC cases with no methylation in any of six markers and with methylation of one or more markers (n=77 vs. 23; P=0.0358) (Fig. 1). Different patterns of L1 and Alu methylation during multistep pulmonary carcinogenesis The patterns of methylation of L1 and Alu in lesions reflecting different steps of lung carcinogenesis were dissimilar (Fig. 2a, b). AAH, AIS, and ADC-T1a samples showed significantly higher levels of L1 methylation than normal lung samples from control cases, whereas advanced ADC samples (>T1a) exhibited significantly lower levels of L1 methylation than those of AIS (Fig. 2a). While L1 methylation increased in early lesions and decreased in ADC, Alu methylation gradually decreased from normal lung to AAH to AIS tissue samples but was not different between AIS and ADC-T1a. In comparison to ADC-T1a, Alu methylation level further decreased in ADC (>T1a) (Fig. 2b). Associations between the L1 and Alu methylation level and clinicopathological features Methylation levels of L1 and Alu were lower in lung ADC than in precursor lesions but varied widely between individual ADC samples. To determine whether L1 and Alu methylation

Fig. 1 Kaplan–Meier log-rank test of cancer-specific survival in patients with stage I adenocarcinomas. Cancer-specific survival of stage I adenocarcinomas harboring no methylation at any of six markers was significantly shorter than stage I lung adenocarcinomas with methylation at one or more of six markers (P=0.0358)

status had any prognostic significance, we analyzed methylation levels of L1 and Alu in tumor and adjacent non-neoplastic lung tissue samples in an independent set of stage I ADCs (n= 100). L1 and Alu methylation levels were significantly correlated (P52.4 % was strongly associated with a survival rate lower than that of stage I ADC with a tumor L1 methylation level 19.7 % was strongly associated with a lower survival rate than stage I ADC with a tumor Alu methylation level T1a, adenocarcinomas of the T1b stage or higher

Although methylation status of CIMP markers was a significant factor in Kaplan–Meier analysis, we could not include methylation status of CIMP markers into multivariate analysis because none of the patients with methylation at one or more CIMP markers died during follow-up. However, by Kaplan– Meier log rank test and multivariate analysis the combination of high T/NL1 methylation ratio status and absence of CIMP methylation status was an independent variable heralding poor prognosis in stage I ADC patients (Fig. 4 and Table 2).

Cancer cells are characterized by global genomic hypomethylation and regional CpG island hypermethylation, and these methylation changes also occur in premalignant lesions preceding various human cancers. In a previous study, we analyzed lung ADC and its precursor lesions for methylation status of 18 genes involved in cancer-specific methylation

and found an increase in the number of methylated genes from AAH to AIS to ADC (Supplementary Fig. 3) [18]. We found methylation of CIMP markers already in AIS and the number of methylated CIMP markers increased during progression towards malignancy but did not reach 0.5, even in ADC> T1a. CIMP was not found in AAH and AIS lesions. These findings suggest that CIMP is a late event during pulmonary carcinogenesis, in contrast to CIMP-high colorectal cancer, the precursor lesions of which (sessile serrated polyp/adenomas) are also CIMP-high [19]. Furthermore, low-grade gliomas are more often CIMP-high (G-CIMP) than glioblastomas [20], which indicates that G-CIMP is an early event. In the present study, we used Alu and L1 methylation levels determined by pyrosequencing as surrogate marker for the methylation content of genomic DNA, and analyzed precursor lesions of lung ADC. Alu methylation level decreased progressively, whereas L1 methylation level increased initially and later decreased along with progression of the lesions to ADC. This pattern in the level of L1 methylation (initial

Fig. 3 Kaplan–Meier log-rank test of cancer-specific survival in patients with stage I adenocarcinoma according to L1 or Alu methylation status or ratio of tumor to normal L1 methylation level status (a, b, and c,

respectively). Significant associations were found between cancerspecific survival and L1 methylation or Alu methylation status or the ratio of tumor to normal tissue L1 methylation level status

Discussion

Virchows Arch Table 1

Univariate and multivariate survival analyses of 100 stage I ADC patients No. of cases

Univariate analysis

Multivariate analysis

Mean survival (months) (95 % CI) Age

Prognostic significance of promoter CpG island hypermethylation and repetitive DNA hypomethylation in stage I lung adenocarcinoma.

In carcinogenesis of peripheral pulmonary carcinomas, multiple genetic and epigenetic alterations are involved. In this study, we quantified methylati...
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