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Simple Summary: The analytic appropriateness and general applicability of lymph node-related risk factors used to predict long-term outcomes in patients with papillary thyroid carcinoma need to be validated. This study aimed to assess detailed lymph node-related risk factors and suggest new risk categories. In the present study, using the K-means clustering algorithm, we determined new cutoffs for lymph node variables besides extranodal extension: 0.2 cm and 1.1 cm for the maximal diameter of metastatic lymph node foci, 4 and 13 for the number of metastatic lymph nodes, and 0.28 and 0.58 for the metastatic lymph node ratio; new lymph node risk categories were suggested. The recurrence-free survival curves of each subgroup classified by these newly determined cutoffs showed significant differences. These newly developed risk categories might be considered when redefining risk stratification or staging systems. Background: Lymph node (LN)-related risk factors have been updated to predict long-term outcomes in patients with papillary thyroid carcinoma (PTC). However, those factors' analytic appropriateness and general applicability must be validated. This study aimed to assess LN-related risk factors, and suggest new LN-related risk categories. Methods: This multicenter observational cohort study included 1232 patients with PTC with N1 disease treated with a total thyroidectomy and neck dissection followed by radioactive iodine remnant ablation. Results: The median follow-up duration was 117 months. In the follow-up period, structural recurrence occurred in 225 patients (18.3%). Among LN-related variables, the presence of extranodal extension (p < 0.001), the maximal diameter of metastatic LN foci (p = 0.029), the number of retrieved LNs (p = 0.003), the number of metastatic LNs (p = 0.003), and the metastatic LN ratio (p < 0.001) were independent risk factors for structural recurrence. Since these factors showed a nonlinear association with the hazard ratio of recurrence-free survival (RFS) rates, we calculated their optimal cutoff values using the K-means clustering algorithm, selecting 0.2 cm and 1.1 cm for the maximal diameter of metastatic LN foci, 4 and 13 for the number of metastatic LN, and 0.28 and 0.58 for the metastatic LN ratio. The RFS curves of each subgroup classified by these newly determined cutoff values showed significant differences (p < 0.001). Each LN risk group also showed significantly different RFS rates from the others (p < 0.001). Conclusions: In PTC patients with an N1 classification, our novel LN-related risk estimates may help predict long-term outcomes and design postoperative management and follow-up strategies. After further validation studies based on independent datasets, these risk categories might be considered when redefining risk stratification or staging systems. [ABSTRACT FROM AUTHOR]

Background: Long-term management and follow-up strategies in patients with intermediate-risk papillary thyroid carcinoma (PTC) according to the American Thyroid Association (ATA) are still controversial due to the paucity of data on unique risk factors or a risk stratification system predictive of long-term outcomes. Patients and Methods: This study included 649 patients with PTC who underwent an initial surgical treatment. Retrospectively enrolled patients were categorized according to the ATA risk stratification system. Intermediate-risk patients were further categorized into subgroups by the number of ATA intermediate risk factors. The recurrence-free survival (RFS) rates of these subgroups were compared with those of low- and high-risk patient groups. Additionally, the patients were classified according to their response to the initial therapy using the dynamic risk stratification (DRS) system, and the percentages of patients in each category were compared among the subgroups. Results: The median follow-up period was 102 months. Structural recurrence occurred in 9.2% of all enrolled patients (60/649) and in 13.0% of intermediate-risk patients (40/308). Patients with two or more current intermediate risk factors had a poorer RFS than patients with only one risk factor (p < 0.001) and showed a comparable RFS to high-risk patients (p > 0.050). The percentages of patients with an excellent response category for DRS significantly decreased with an increase in the number of intermediate risk factors. Conclusion: Subclassification according to the number of intermediate risk factors may be useful to better predict the RFS and the response to initial therapy in patients with intermediate-risk PTC. [ABSTRACT FROM AUTHOR]

Objective: Primary tumour size (PTS) is known to be a significant prognostic factor in patients with papillary thyroid carcinoma (PTC) undergoing thyroidectomy. Although cutoff values of 2 and 4 cm are widely used in surgical extent decision and long‐term outcomes predictions, the effectiveness of arbitrary cutoff values in stratifying patients for target outcomes is questionable. This study aimed to determine new optimal cutoffs of PTS. Design, Patient And Measurements: Patients (n = 529) with PTC who underwent total thyroidectomy were retrospectively reviewed. After risk factor analysis for structural recurrence, the optimal cutoffs of PTS were automatically calculated using restricted cubic spline analysis and X‐tile software. Subgroups were classified based on the newly determined cutoff values. Both the recurrence‐free survival (RFS) and the proportion of patients in each response‐to‐therapy category, using the dynamic risk stratification system (DRS), were compared between the subgroups. Results: PTS was confirmed as an independent risk factor for structural recurrence. The optimal cutoff values were calculated as 1.4 and 3.0 cm. The subgroups stratified using these newly determined cutoffs showed significantly different RFS and DRS based on the response to initial therapy, but the subgroups classified using the cutoffs of 2 and 4 cm did not. Conclusions: The newly determined cutoff values of PTS may be useful in better stratifying patients with PTC undergoing thyroidectomy for target outcomes and can be considered as a new definition in staging and risk stratification systems. [ABSTRACT FROM AUTHOR]

Backgrounds: Human papilloma virus (HPV) infection is an important factor in development and prognosis of head and neck squamous cell carcinoma (HNSCC). It is also known that HPV infection could affect anti-cancer immunity. Methods: We examined the association between cancer immunity-related molecules and HPV infection, using immunohistochemical (IHC) staining for p16, PD-L1, and FOXP3 and real-time polymerase chain reaction for HPV. Results: The IHC staining for PD-L1 was positive in 30 out of 122 cases (24.6%) and p16 showed positivity in 68 out of 122 cases (55.7%). HPV was detected in 48 out of 122 cases (39.3%). P16 expression was significantly associated with PD-L1 expression (P=0.004), HPV infection (P=0.032), and the number of FOXP3-positive tumor infiltrating lymphocytes (P=0.009). PD-L1-positive or p16-positive HNSCC showed a worse prognosis than PD-L1-negative or p16-negative HNSCC. Conclusion: P16 expression may predict PD-L1 expression, and p16 expression may also be associated with the immune evasion of HNSCC. [ABSTRACT FROM AUTHOR]

Background: Hürthle cell‐rich nodules (HCNs) encompass non‐neoplastic to malignant lesions. There is paucity of literature on the frequency distribution of HCNs among Bethesda categories, histologic follow‐up, risk of malignancy (ROM), and risk of neoplasia (RON). The objective of this retrospective, multi‐institutional study was to determine the prevalence of the cytologic diagnostic category and surgical outcomes of patients with HCN. Methods: Nine tertiary health centers representing 6 Asian countries participated. Cases were retrieved from respective databases. The Bethesda System for Reporting Thyroid Cytopathology was used. Cytology results were correlated with surgical diagnoses. Results: Of 42,190 thyroid aspirates retrieved, 760 (1.8%) had a Hürthle cell predominance. Most (61%) were categorized as atypia of undetermined significance/follicular lesion of undetermined significance, Hürthle cell type" (AUS‐H); 35% were categorized as follicular neoplasm, Hürthle cell type (FN‐H); and 4% were categorized as suspicious for malignancy (SFM). Histologic follow‐up was available for 288 aspirates (38%). Most were benign on resection (66%), and the most common histologic diagnosis was Hürthle cell adenoma (28.5%). The ROM for AUS‐H, FN‐H, and SFM, as calculated on resected nodules, was 32%, 31%, and 71%, respectively; and the RON was 47%, 81%, and 77%, respectively. The 5 institutions that had an AUS‐H:HCN ratio below 0.5 diagnosed HCN less frequently as AUS‐H than as FN‐H. Conclusions: This is the largest, contemporary, multi‐institutional series of HCNs with surgical follow‐up. Although there was wide interinstitutional variation in prevalence and surgical outcomes, there was no significant difference in the ROM among institutions. The categories AUS‐H and FN‐H had a similar ROM for resected nodules. In a large, multi‐institutional study of Hürthle cell–rich lesions with surgical follow‐up, the incidence of Hürthle cell nodules, their Bethesda categorization on aspiration cytology, and the risks of malignancy and neoplasm are highlighted. [ABSTRACT FROM AUTHOR]

Purpose. We report a rare case of severe hypercalcemia that was ultimately diagnosed as primary bone marrow diffuse large B-cell lymphoma (BCL). Case Report. A 74-year-old male patient visited our hospital complaining of tenderness and swelling of the left knee caused by supracondylar fracture of the left distal femur. His initial blood tests showed a serum calcium level of 13.9 mg/dL, inorganic phosphorus of 4.34 mg/dL, and a serum creatinine level of 1.54 mg/dL. A serum assay of intact parathyroid hormone showed 5.24 pg/mL, and the patient’s serum 25(OH)D level was 22.33 ng/mL. To exclude malignancy, we performed imaging studies, including abdomen or chest computed tomography and positron emission tomography-computed tomography; however, no suspicious lesion was found, although the serum PTH-related peptide level was elevated at 4.0 pmol/L. A bone marrow biopsy was performed to identify any hidden hematologic malignancy. As a result, the pathology of bone marrow confirmed the presence of atypical lymphocytes that stained positive for the CD20 marker, which is consistent with BCL involving the bone marrow. Conclusion. This case highlights the importance of pursuing a thorough workup for rare underlying causes of hypercalcemia when parathyroid-related etiologies can be excluded. [ABSTRACT FROM AUTHOR]

Background For patients with papillary thyroid carcinoma (PTC), lymph node metastasis is associated with an increased recurrence rate. The purpose of this study was to investigate whether the vascular endothelial growth factor (VEGF), microvessel density (MVD), and vascular index (VI) can predict lymph node metastasis in patients with PTC. Methods From January 2011 to October 2011, 202 patients with PTCs underwent preoperative staging ultrasound evaluation. To evaluate vascularity, we measured the VI, VEGF expression, and MVD. Results The VI was significantly correlated with MVD ( p = .009). On multivariate analysis, young age showed a significant correlation with lymph node metastasis ( p < .001; p < .001; p < .001). However, the other clinicopathologic features, VEGF, MVD, and VI failed to show any significant correlations with lymph node metastasis. Conclusion Although the VI showed significant correlation with MVD, it was not significantly correlated to lymph node metastasis. © 2016 Wiley Periodicals, Inc. Head Neck 39: 334-340, 2017 [ABSTRACT FROM AUTHOR]

Objective To evaluate the clinical significance and compare the imaging features according to the Thyroid Image Reporting and Data System ( TIRADS) between atypia of undetermined significance/follicular lesion of undetermined significance ( AUS/ FLUS) subcategories. Design & patients A total of 192 thyroid nodules in 188 patients (mean age: 50·2 ± 11·8 years) that had been initially diagnosed as AUS/ FLUS on US-guided fine needle aspiration ( US- FNA) were included. One cytopathologist retrospectively reviewed the cytology slides, subcategorizing cytology results into AUS and FLUS. A TIRADS category was assigned to each thyroid nodule according to the number of suspicious US features. Clinical, US features and malignancy rates were compared between the two subcategories. Results Of the 192 AUS/ FLUS lesions, 149 (77·6%) were subcategorized as AUS and 43 (22·4%) as FLUS. Of the 192 AUS/ FLUS nodules, 82 (42·7%) were malignant. The malignancy rates between AUS and FLUS subcategories were not significantly different, 45·6% to 32·6%, respectively ( P = 0·127). When applying TIRADS, significant differences were seen in TIRADS category between benign and malignant nodules in the AUS subcategory ( P < 0·001), but not in the FLUS subcategory ( P = 0·414). The malignancy rates in TIRADS categories 3, 4a, 4b, 4c and 5 were 15·4%, 22·2%, 33·3%, 57·1% and 80·0% ( P < 0·001) in AUS nodules and 40·0%, 50·0%, 23·5%, 22·2% and 0·0% ( P = 0·414) in FLUS nodules, respectively. Conclusion Suspicious US features are useful in predicting malignancy among AUS subcategories but not in FLUS subcategories. Subcategorization into AUS and FLUS cytology may be helpful in deciding upon treatment or management of thyroid nodules. [ABSTRACT FROM AUTHOR]

We investigated the clinicopathological implications of leptin-signaling proteins and Epstein-Barr virus (EBV)-infection status in gastric carcinomas. Immunohistochemistry for leptin signalling-related proteins (leptin, leptin-receptor, pSTAT3, ERK, pAkt, mTOR and HIF-1 alpha), and in situ hybridization for EBV-encoded small RNAs was performed in 343 cases of gastric carcinomas. The siRNA against leptin-receptor was transfected into three stomach cancer cell lines, and western blot for caspase 3 was performed. The TNM stage was a prognostic factor in all 343 patients, and was negatively correlated with expression of leptin, pSTAT3, ERK, pAkt, mTOR and HIF-1 alpha (P < 0.05). Leptin-receptor expression was correlated with poor survival in 207 patients of the advanced gastric cancer (AGC) subgroup, 139 of the Lauren diffuse group, and in 160 patients with lymph node metastasis (P < 0.05, respectively). Additionally, in stomach cancer cells, cleaved caspase 3 level increased by leptin-receptor inhibition, that is, apoptosis increased. Interestingly, EBV-positive AGC (n = 29) tended to show better survival of patients than EBV-negative AGC (n = 178) (P = 0.06). pAkt expression was related with a good survival of 32 patients (9%) in the EBV-positive subgroup, but was not an independent prognostic factor. Among, leptin signaling-related proteins, expressions of leptin-receptor and mTOR were different between EBV-positive subgroup and EBV-negative subgroup (P < 0.05, respectively). In conclusion, leptin-signaling proteins and EBV status show different significance on patient survival, according to subsets of gastric carcinomas. The leptin-receptor may predict poor patient prognosis in the AGC, Lauren diffuse and lymph node metastasis subgroups, while EBV-positive status can show a good prognosis in the AGC. Each leptin signaling-related protein may be differently involved in carcinogenesis of EBV-negative and EBV-positive subsets. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was to evaluate the diagnostic utility of PAI at detecting thyroid microcalcifications at 700 nm laser wavelengths. Methods: This study included 36 resected samples in 18 patients. To evaluate the PA manifestation of microcalcifications in PAI, gray level histogram and co-occurrence matrix (COM) texture parameters were extracted from the 3 fixed ROI US and PA images, respectively, per sample. We compared the textural parameters obtained from specimen PAIs between samples with punctate microcalcifications on specimen radiography and those without microcalcifications. Results: On specimen US, the mean value (2748.4±862.5) of samples with microcalcifications on specimen radiography was higher than that (1961.9±780.2) of those without microcalcifications (P = 0.007). However, there were no significant differences in textural parameters obtained from specimen PAIs between samples with punctate microcalcifications on specimen radiography and those without when applying both the mean value of the three slices of thyroid specimens and the value of the thyroid specimen slice which had the highest value of the mean values in specimen US. Conclusion: PAI did not show significant PA contrast on thyroid microcalcifications indicating that the experimental setup and protocols should be enhanced, e.g., method of complete blood rejection from ex vivo specimens, the multi-wavelength spectroscopic PA imaging method which can solely extract the PA signal from microcalcifications even with high spectral interferences, or PA imaging with narrower slice thickness using 2-dimensional array transducer, etc. [ABSTRACT FROM AUTHOR]

Focal CpG island hypermethylation and diffuse genomic hypomethylation signify the changes in the DNA methylation status in cancer cells. ALU and LINE-1 repetitive DNA elements comprise ∼28% of the human genome. PCR-based measurements of these repetitive DNA elements can be used as a surrogate marker of the genomewide methylation content. Our study aimed to identify the timing of ALU and LINE-1 hypomethylations during multistep gastric carcinogenesis and their prognostic implications in gastric cancer (GC). In our study, we analyzed the methylation statuses of ALU and LINE-1 in 249 cases of gastric biopsy samples and another independent set of 198 cases of advanced GC by pyrosequencing. Regardless of the Helicobacter pylori infection status, a significant decrease in the ALU methylation levels was noted during the transitions from chronic gastritis to intestinal metaplasia and from gastric adenoma to GC. LINE-1 methylation decreased during the transition from intestinal metaplasia to gastric adenoma and no further decrease occurred during the transition from gastric adenoma to GC. A low LINE-1 methylation status was strongly associated with poor prognosis in GC. A multivariate analysis revealed that LINE-1 methylation status was an independent prognostic factor. Our findings suggest that ALU and LINE-1 hypomethylations are early events during multistep gastric carcinogenesis. Furthermore, the LINE-1 methylation status can be used as a molecular biomarker to define a subset of GC patients with poor prognosis. [ABSTRACT FROM AUTHOR]

This study was designed to evaluate the changes in promoter CpG islands hypermethylation during breast cancer progression from pre-invasive lesions [flat epithelial atypia (FEA), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS)] to invasive ductal carcinoma (IDC). We performed MethyLight analysis for the methylation status of 57 promoter CpG island loci in 20 IDCs and their paired normal breast tissues. After selecting 15 CpG island loci showing breast cancer-specific DNA methylation, another set of normal breast tissue (n = 10), ADH/FEA (n = 30), DCIS (n = 35), and IDC (n = 30) of the breast were analyzed for these loci. We found six new methylation markers of breast cancer, namely DLEC1, GRIN2B, HOXA1, MT1G, SFRP4, and TMEFF2, in addition to APC, GSTP1, HOXA10, IGF2, RARB, RASSF1A, RUNX3, SCGB3A1 (HIN-1), and SFRP1. The number of methylated genes increased stepwise from normal breast to ADH/FEA and DCIS, while IDC did not differ from DCIS. Methylation levels and frequencies of APC, DLEC1, HOXA1, and RASSF1A promoter CpG islands were significantly higher in ADH/FEA than in normal breast tissue. GRIN2B, GSTP1, HOXA1, RARB, RUNX3, SFRP1, and TMEFF2 showed higher methylation levels and frequencies in DCIS than in ADH/FEA. DICS and IDC did not differ in the methylation levels or frequencies for most CpG island loci except SFRP1 and HOXA10. Our findings showed that promoter CpG island methylation changed significantly in pre-invasive lesions, and was similar in IDC and DCIS, suggesting that CpG island methylation of tumor-related genes is an early event in breast cancer progression. [ABSTRACT FROM AUTHOR]

ABSTRACT Background and aim: Low gamma-glutamyltranspeptidase (γ-GTP) familial intrahepatic cholestasis is a broad-spectrum condition that ranges from mild to severe. Severe forms of low γ-GTP familial intrahepatic cholestasis, including progressive familial intrahepatic cholestasis type-1 and -2 (PFIC-1 and PFIC-2), present with symptoms of cholestasis early in life and may progress to cirrhosis. Methods: We included five patients with low γ-GTP, cholestasis and hyperbilirubinemia. We analyzed clinicopathological features of these five cases. Results: The age of the patients at diagnosis ranged from 1 month to 1 year old. All patients presented with jaundice, and one experienced pruritus. In contrast to hyperbilirubinemia, serum γ-GTP levels were relatively low, or within normal range. Microscopically, intracanalicular cholestasis, bile duct loss or atrophy and varying degrees of fibrosis were found in all of the cases, whereas giant cell formation of hepatocytes was detected in three cases. Anti-ABCB11 immunostaining revealed loss of expression in three cases with diffuse giant cell transformation of hepatocytes but retained expression in one case with no giant cell transformation. Conclusions: Low γ-GTP familial intrahepatic cholestasis (PFIC-1 and PFIC-2) should be considered a differential diagnosis when pediatric patients with cholestatic liver diseases associated with normal to low serum γ-GTP levels relative to the extent of cholestasis. [ABSTRACT FROM AUTHOR]

Ex-adenoma carcinoma (EAC) is a carcinoma with contiguous adenoma element in its vicinity which provides a morphological evidence for adenoma-carcinoma sequence. During multistep colorectal carcinogenesis, promoter CpG island hypermethylation has been known to increase in a stepwise manner whereas diffuse genomic hypomethylation has been known to be an early event and not progress. However, some controversies exist. EAC is a good model to study the timing of hypermethylation and hypomethylation changes during multistep carcinogenesis, which this study aimed to elucidate. We analyzed 39 cases of EAC for their methylation status in eight DNA methylation markers of CpG island methylator phenotype (CIMP) panel, ten CIMP-nonrelated, cancer-specific markers, and three repetitive DNA elements (ALU, LINE-1, and SAT2) using MethyLight assay or combined bisulfite restriction analysis. Twenty-two cases of cancers had contiguous tubulovillous adenomas and 17 cases had contiguous tubular adenomas. Regardless of CIMP markers or nonrelated markers, a significant increase in the number of methylated genes was found from normal mucosa to adenoma, whereas no increase was found from adenoma to carcinoma. Both ALU and LINE-1 showed a significant decrease of methylation levels from normal mucosa to adenoma (p < 0.05), but there is no difference between adenoma and cancer. However, SAT2 methylation level exhibited a stepwise decrease from normal mucosa to adenoma to cancer. Our findings suggest that morphological progression from traditional adenoma to carcinoma does not appear to be accompanied by increases in promoter CpG island hypermethylation or repetitive DNA hypomethylation, except for SAT2 hypomethylation which showed continuous progression during multistep carcinogenesis. [ABSTRACT FROM AUTHOR]

Gastric carcinoma (GC) is one of the human cancers in which promoter CpG island hypermethylation is frequently found. CpG island methylator phenotype (CIMP) refers to a subset of GCs which harbor concordant methylation of multiple promoter CpG island loci. However, little is known regarding clinicopathological features of CIMP-positive (CIMP-high) GC. Our study aimed to characterize clinicopathological features of CIMP-high GC. We analyzed 196 cases of GCs for their methylation status in 16 cancer-specific CpG island loci using MethyLight assay and arbitrarily defined CIMP-high GC as those with methylation at 13 or more CpG island loci. With exclusion of microsatellite instability-positive GC and EBV-positive GC from the analysis, CIMP-high GC (n = 10, 6.7%) demonstrated tendency toward higher cancer stage, infiltrative growth type, poor differentiation, and diffuse or mixed type of Lauren classification. CIMP-high GC showed significantly shortened survival compared with that of CIMP-negative GC. When CIMP-negative GC (methylation at 12 or less) was divided into CIMP-intermediate and CIMP-low (methylation at one or none), CIMP-low exhibited better clinical outcome than CIMP-intermediate. Hypermethylation at 14 CpG island loci or more was closely associated with poor clinical outcome and found to be an independent prognostic factor. Our findings that CIMP-high GCs were featured with characteristic clinicopathological parameters, including poor prognosis are distinct from previous studies. More extensive, large-scaled study is necessary to validate the findings of the present study. [ABSTRACT FROM AUTHOR]

ABSTRACT Background and aim: The three-panel marker, glypican-3, HSP70 and glutamine synthetase, is helpful for making a diagnosis of early hepatocellular carcinoma. To further characterize the diagnostic values of these three markers, we performed immunostaining studies in non-neoplastic hepatocytes (chronic hepatitis B) and hepatocellular carcinoma. Methods: Immunostaining was performed for all three markers. The cases were 60 needle biopsies of chronic hepatitis B (30 with low-grade activity and 30 with high-grade activity) and 40 hepatocellular carcinomas (HCCs). Results: Glypican-3 staining was detected in only one of the 60 chronic hepatitis B cases, and this case expressed moderate immunoreactivity for glypican-3 and showed high-grade inflammatory activity. HSP70 expression was not observed in any cases. Glutamine synthetase was strongly immunoreactive at peri-venular hepatocytes with cytoplasmic staining in all cases. These findings reflected a normal expression pattern. On the other hand, all three markers were positive in 15 (37.5%) HCCs. At least two of three markers were positive in 26 (65.0%). Moreover, all three markers were negative in 2 (5.0%) HCCs. Conclusions: The combination of these three markers is highly specific for detecting hepatocellular carcinoma. However, caution should be exercised because non-neoplastic hepatocytes in chronic hepatitis B with high necro-inflammatory activity tend to show focal positivity for glypican-3. Also we emphasize that all three markers can show negative immunoreactivity in HCC. [ABSTRACT FROM AUTHOR]

Biliary intraepithelial neoplasia (BilIN) is the premalignant lesion of extrahepatic cholangiocarcinoma (EHC), and there are no published data regarding epigenetic changes throughout disease progression from normal biliary epithelia to BilIN to EHC. The objective of this study was to identify the occurrence of CpG island hypermethylation and repetitive DNA hypomethylation in BilIN. A total of 50 EHCs, 31 BilINs, and 31 normal cystic duct samples were analyzed for their methylation status in seven genes and two repetitive DNA elements. The number of methylated genes increased with disease progression (normal bile duct, 0.6; BilIN, 2.0; EHC, 3.6; P < 0.001). The methylation level of examined genes was significantly higher in BilIN than in normal samples (TMEFF2, HOXA1, NEUROG1, and RUNX3, P < 0.05) and in EHC than in BilIN samples (TMEFF2, HOXA1, NEUROG1, RUNX3, RASSF1A, and APC, P < 0.05). Long interspersed nucleotide element-1 (LINE-1) and juxtacentromeric satellite 2 (SAT2) methylation levels were markedly lower in EHC than in normal duct and BilIN samples, and BilIN samples showed a decrease of SAT2 methylation levels but no decrease of LINE-1 methylation levels compared to normal samples. These findings suggest that most of cancer-specific CpG island hypermethylation occur in the stage of BilIN and that CpG island hypermethylation seems to occur earlier than repetitive DNA element hypomethylation. [ABSTRACT FROM AUTHOR]