Tumoral LINE-1 hypomethylation is associated with poor survival of patients with intrahepatic cholangiocarcinoma

A total of 172 formalin-fixed archival tissue samples were obtained from patients who underwent surgical resection for ICC at the Seoul National University Hospital, Seoul, South Korea, from April 2005 to December 2012. Fifteen non-neoplastic gallbladder tissue samples were obtained from patients with chronic cholecystitis. Hilar cholangiocarcinomas, which arise from the left and right hepatic ducts at or near their junctions, were excluded from the study. Through meticulous histological examinations, combined hepatocellular carcinoma and cholangiocarcinoma were excluded from the study. The type of operative procedures included sectionectomy or segmentectomy in 46 patients (26.7%), lobectomy in 124 (72.1%), and total hepatectomy in 2 (1.2%). Among 172 patients, 6 (3.5%) received neoadjuvant chemotherapy and 47 (27.3%) received adjuvant chemotherapy and/or radiotherapy. Thirty-four (19.8%) patients received adjuvant chemotherapy and 13 (7.6%) patients received concurrent chemoradiation therapy after surgery. All cases were reviewed by experienced gastrointestinal pathologists (KBL and JJJ) to confirm the diagnosis of ICC and to re-evaluate histological findings and tumor-node-metastasis (TNM) stages according to the 4th edition 2010 WHO classification and the 7th edition 2009 AJCC/UICC staging system, respectively [ 20, 21]. Gross types of ICC were classified into three types according to gross appearance, including mass-forming (MF) type, periductal infiltrative (PI) type, and intraductal growth (IG) type [ 22, 23]. When more than one type was found in a tumor, the tumor was classified as mixed type. This retrospective study was approved by the Institutional Review Board at the Seoul National University Hospital (IRB No. H-1011-046-339).

Through microscopic examination, tumor areas in which 1) the tumor cells comprised >45% of total neoplastic and non-neoplastic cells and 2) represented the predominant histological type of the individual case were marked with a marker pen. For cases with ICC of mixed gross type, tumor areas with highest tumor density were marked in the individual cases. The corresponding areas were scraped from unstained tissue glass slides with a knife blade. Because epithelial cells are usually denuded in normal intrahepatic bile ducts of the formalin-fixed surgical specimens, cystic ducts of cholecystectomy specimens were taken as surrogates for normal controls. Cystic duct epithelia were scraped from the unstained tissue glass slides and collected into microtubes containing 50 μL of tissue lysis buffer and proteinase K. After incubation of the tubes for 48 h at 55 °C, the lysates were subjected to heating at 95 °C for 30 min. This prolonged heating was found to be necessary for lessening the formalin fixation-induced discrepancy in the measured value of LINE-1 methylation level [ 24]. With fixation of tissue samples in formalin solution, formaldehyde induces protein-DNA crosslinks and interstrand DNA crosslinks which may cause some difficulty in thermal and alkaline denaturation. Incomplete denaturation of double-stranded DNA results in potential under-conversion of non-methylated cytosines to uracils during bisulfite treatment, which might cause misleading results in the measured values of LINE-1 methylation for formalin-fixed tissue samples. In a previous study, we found that formalin fixation causes artificial increases in the measured value of LINE-1 methylation level, and that prolonged heat-treatment of DNA samples obtained from formalin-fixed tissue samples decreased the discrepancy in the measured values of LINE-1 methylation level between paired fresh-frozen and formalin-fixed tissue samples [ 24]. Following centrifugation of the tissue lysates, the supernatants were transferred into new tubes. DNA samples were subjected to bisulfite modification of DNA samples using the EZ DNA methylation kit (Zymo Research, Orange, CA, USA). LINE-1 methylation levels were measured using PCR pyrosequencing assay. The primers and PCR conditions were described previously [ 12]. The methylation level at each CpG site was the percentage of C nucleotides relative to the sum of C and T nucleotides at each CpG site. The four percentage values in the four serial CpG sites (nucleotide positions 328, 321, 318, and 306 of X58075 (GenBank)) were averaged and this mean value was taken as the overall LINE-1 methylation level in a given sample.

Because LINE-1 methylation data followed a normal distribution, we used parametric tests to compare groups. However, when the following criteria were not met, we used both parametric tests and non-parametric tests: when two or more groups were compared, each group n should be greater than 15. Parametric tests (student t-test and ANOVA) were performed for comparison of two groups and three or more groups, respectively. Non-parametric tests (Mann-Whitney test and Kruskal-Wallis test) were further performed for the comparison of two groups and three or more groups, respectively, when one group was not greater than 15. The cancer-specific survival was calculated as the time from the date of surgery to the date of death by ICC. The data from patients who did not experience cancer-specific death were censored at the last follow-up visit to obtain the cancer-specific survival. The Kaplan-Meier log rank test and Cox proportional hazard method were used for survival analysis. For multivariate analysis, variables that were found to be significant in univariate analysis were included in the Cox proportional hazard model, and statistically significant variables were then selected by backward elimination. All p values were two-sided, and the statistical significance was set at p < 0.05. SPSS software (IBM SPSS Statistics version 23; Chicago, IL, USA) was used for all statistical analyses.

In total, 172 ICC patients underwent hepatic resection between 2005 and 2012. Of these patients, 147 patients (85.5%) presented with a single tumor. The male to female ratio was 121:51, and average age was 62.7 years (median, 63 years; range, 38–80 years). Gross type was MF type in 141 patients, PI type in 8 patients, IG type in 18 patients, and MF plus PI type in 5 patients. Stage grouping was stage I in 40, stage II in 37, stage III in 30, and stage IV in 65. Grading was well differentiated in 23, moderately differentiated in 94, and poorly differentiated in 55. Demographic and clinicopathological findings are summarized in Table  1.

The LINE-1 methylation level was significantly lower in ICC tissue samples than in normal gallbladder tissue samples (Fig.  1). Tumoral LINE-1 methylation levels were not different in ICCs between male and female patients and between younger and older patients (<64 years old and ≥64 years). No association was found between tumoral LINE-1 methylation levels and gross types. Tumoral LINE-1 methylation levels were not different between ICCs of single-tumor and multiple-tumor types. However, a significant difference was noted between well-differentiated ICCs and moderately or poorly-differentiated ICCs: well-differentiated ICCs showed higher methylation levels than those of moderately or poorly differentiated ICCs. Tumoral LINE-1 methylation levels tended to be higher in T1 stage than in T2b or higher T stages. No difference in LINE-1 methylation levels was found between ICCs of N0 and N1, or between ICCs of M0 and M1. While LINE-1 methylation levels were significantly lower in ICCs with lymphatic tumor emboli than in ICCs without lymphatic tumor emboli, no significant difference was noted between ICCs with and without venous tumor emboli and between ICCs with and without perineural invasion.

When ICCs were grouped into four quadrants (Q1, Q2, Q3, and Q4 in order of increasing level of LINE-1 methylation) based on their tumoral LINE-1 methylation levels, Q1 and Q2 exhibited significantly lower cancer-specific survival than that of Q3 and Q4 (Fig.  2). As displayed in the Kaplan-Meier survival curve, cancer-specific survival curves of Q1 and Q2 are similar, while cancer-specific survival curves of Q3 and Q4 are similar. Thus, ICCs were further grouped into low methylation status subgroup (Q1 and Q2) and high methylation subgroup (Q3 and Q4). Low LINE-1 methylation status was associated with worse cancer-specific survival in Kaplan-Meier survival analysis. In addition to low LINE-1 methylation status, T staging, N staging, lymphatic emboli, perineural invasion, and histological differentiation were included into a multivariate analysis which revealed that a low LINE-1 methylation status was independently associated with worse cancer-specific survival in patients with ICC (Tables  2 and  3).