Nam-Yun Cho

DNA methylation profiles of gastric carcinoma characterized by quantitative DNA methylation analysis

Transcriptional silencing by CpG island hypermethylation is a potential mechanism for the inactivation of tumor-related genes. Virtually, all types of human cancers show CpG island hypermethylation, and gastric carcinoma (GC) is one of the tumors with a high frequency of aberrant CpG island hypermethylation. In this study, we prescreened DNA methylation of 170 CpG island loci in a training set of 8 paired GC and GC-associated non-neoplastic mucosae (GCN) using MethyLight technology and selected 27 DNA methylation markers showing higher methylation frequency or level in GC than in GCN. These markers were then analyzed in a tester set of 25 paired GC and GCN and 27 chronic gastritis (CG) from non-cancer patients to generate their DNA methylation profiles. We identified 17 novel methylation markers in GC, including SFRP4, SEZ6L, TWIST1, BCL2, KL, TERT, SCGB3A1, IGF2, GRIN2B, SFRP5, DLEC1, HOXA1, CYP1B1, SMAD9, MT1G, NR3C1, and HOXA10. Of the 27 selected CpG island loci, 23 were methylated in GC, GCN, and CG and the remainder four loci (DLEC1, CHFR, CYP1B1, and NR3C1) were only methylated in GC. We found that the number of methylated loci was significantly higher in GC than in GCN or CG and that Helicobacter pylori infection was strongly associated with aberrant CpG island hypermethylation in CG. Hypermethylation was more prevalent in Epstein–Barr virus (EBV)-positive GC than in EBV-negative GC and in diffuse-type GC than in intestinal-type GC. Through our large-scale screening of 170 CpG island loci, we found 17 new DNA methylation markers of GC, which may serve as useful markers that may identify a distinct subset of GC.

Clinicopathological features of CpG island methylator phenotype-positive colorectal cancer and its adverse prognosis in relation to KRAS/BRAF mutation

CpG island methylator phenotype (CIMP) is a recently described subset of colorectal cancers (CRC) with widespread methylation of multiple promoter CpG islands. But the prognostic implication of CIMP in CRC has not been clarified. Thus, the aim of the present study was to differentiate the unique characteristics of CIMP from those of microsatellite instability (MSI)‐high CRC, especially with regard to prognosis. CIMP, MSI, and mutations of KRAS codons 12 and 13 and of BRAF codon 600 were evaluated in 134 sporadic CRC. Patient survival and other clinicopathological variables were correlated with CIMP or genetic changes. High CIMP, high MSI, and mutations in KRAS or BRAF were detected in 31.3%, 14.2%, 33.6%, and 4.5% of overall CRC, respectively. High CIMP was closely associated with MSI and BRAF mutation but not with KRAS mutation. CIMP‐high, microsatellite‐stable (MSS) CRC were significantly associated with proximal location and nodal metastasis and had close but non‐significant associations with liver metastasis. A worse clinical outcome was found for CIMP‐high, MSS CRC with KRAS/BRAF mutation but not for those lacking KRAS/BRAF mutation. The findings support the contention that CIMP‐high CRC have distinct clinicopathological and epidemiological features and suggest that the alleged poor clinical outcome of CIMP‐high CRC patients is closely associated with the presence of KRAS/BRAF mutation.

Comparison of CpG island hypermethylation and repetitive DNA hypomethylation in premalignant stages of gastric cancer, stratified for Helicobacter pylori infection

CpG island hypermethylation and genomic DNA hypomethylation are found not only in gastric cancers but also in associated premalignant lesions. Helicobacter pylori infection induces aberrant CpG island hypermethylation in gastric mucosae. However, little is known about the relationship between H. pylori infection and aberrant methylation in premalignant lesions. The present study characterized methylation changes in a subset of genes and repetitive DNA elements (ALU, LINE‐1, SAT2) and examined their relationship with H. pylori infection in premalignant lesions of gastric cancers. We performed MethyLight analysis of 25 genes and SAT2 and COBRA analysis of ALU and LINE‐1 in 212 gastric tissue samples. H. pylori infection was closely associated with enhanced hypermethylation of CpG island loci in chronic gastritis samples, but this association was not found among intestinal metaplasias, gastric adenomas and gastric cancers. The number of methylated genes was greater in intestinal metaplasia and gastric adenoma samples than in chronic gastritis samples, regardless of H. pylori infection. Methylation of repetitive DNA elements in gastric lesions generally decreased with progression of the gastric lesion along the multistep carcinogenesis. No difference was noted in the number of methylated genes in chronic gastritis or intestinal metaplasia between gastric cancer patients and non‐cancer subjects. In conclusion, we found that there was no enhanced CpG island hypermethylation in gastric cancer and premalignant lesions in association with H. pylori infection and our findings suggest that CpG island hypermethylation and repetitive DNA hypomethylation are enhanced with progression of the gastric lesion through the multistep carcinogenesis, regardless of the status of H. pylori infection. Copyright

ALU and LINE‐1 hypomethylations in multistep gastric carcinogenesis and their prognostic implications

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.

CpG island methylator phenotype in colorectal cancers: comparison of the new and classic CpG island methylator phenotype marker panels.

CONTEXT CpG island methylator phenotype (CIMP) designates a subset of colorectal cancers featuring concordant hypermethylation of multiple promoter CpG islands. Little is known about the clinical outcome or histologic characteristics of CIMP-positive colorectal cancers defined by recently identified CpG island methylator phenotype panels. OBJECTIVE To investigate and compare the molecular and clinicopathologic features of CIMP-positive colorectal cancers defined by classic (p16, hMLH1, MINT1, MINT2, MINT31) and new (CACNA1G, IGF2, NEUROG1, RUNX3, SOCS1) CIMP panels. DESIGN We analyzed 130 colorectal cancers for hypermethylation of both panels using methylation-specific polymerase chain reaction. RESULTS With at least 2 markers methylated, both classic (39/130; 23.1%) and new (23.1%) CIMP-positive colorectal cancers were significantly associated with proximal tumor location, microsatellite instability, and BRAF mutation (all P values were less than .05). The new panel outperformed the classic panel in detecting these features. With at least 3 markers methylated, new CIMP-positive colorectal cancers (16.9%) were closely associated with proximal tumor location, low frequency of KRAS mutation, and high frequency of BRAF mutation (all P values were less than .05), whereas classic CIMP-positive colorectal cancers (18.5%) were closely associated with proximal tumor location, frequent microsatellite instability, and frequent BRAF mutation (all P values were less than .05). Analyzing a combination of CIMP and microsatellite instability status, CIMP-positive/microsatellite instability-negative colorectal cancers had the worst clinical outcomes. CONCLUSIONS Whereas the classic panel outperformed in predicting clinical outcome, the new panel was superior in detecting known clinicopathologic features of CIMP but inferior in prognostication power.

Genomic hypomethylation and CpG island hypermethylation in prostatic intraepithelial neoplasm.

Altered DNA methylation in cancer cells is characterized by focal CpG island hypermethylation and diffuse genomic hypomethylation. Both types of aberrant methylation are frequently found in human prostate adenocarcinoma (PCa). Prostatic intraepithelial neoplasm (PIN), a precursor lesion of PCa, has been demonstrated to contain CpG island hypermethylation, but little is known about the role of DNA hypomethylation. We analyzed the methylation status at 12 CpG island loci and at two repetitive DNA elements (LINE-1 and SAT2) from normal prostate (n = 20), PIN (n = 25), and PCa (n = 35) tissues using MethyLight assay or combined bisulfite restriction analysis. The methylation levels in LINE-1 and SAT2 decreased with progression of lesion types from normal prostate to PIN to PCa (P < 0.05), whereas promoter CpG island loci displayed increased methylation. Ten genes were found to be hypermethylated in a cancer-specific manner and were further analyzed in another set of PCa tissues (n = 64). The number of methylated genes was closely associated with TNM stage, Gleason sum, and preoperative serum PSA levels (P = 0.020, 0.073, 0.033, respectively). These results suggest that genomic hypomethylation and CpG island hypermethylation, common among PCas, are early events in prostate carcinogenesis and may be implicated in the development of PIN.

Methylation and microsatellite status and recurrence following adjuvant FOLFOX in colorectal cancer

The prognostic impact of CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) on the treatment outcome of colon cancer patients receiving adjuvant 5‐fluorouracil/leucovorin/oxaliplatin (FOLFOX) is unclear. We investigated CIMP and MSI status in colorectal cancer patients treated with adjuvant FOLFOX. Stages II and III sporadic colorectal cancer patients who underwent curative resection followed by adjuvant FOLFOX were included. Eight CpG island loci (CACNA1G, CRABP1, IGF2, MLH1, NEUROG1, CDKN2A (p16), RUNX3 and SOCS1) and five microsatellite markers were examined. Disease‐free survival (DFS) was analyzed according to CIMP and MSI status. A total of 322 patients were included: male/female 192/130, median age 61 years (range 30–78), proximal/distal location 118/204 and Stages II/III 43/279. CIMP status was high in 25 patients (7.8%) and 21 patients (6.5%) had MSI‐high tumor. CIMP/MSI status was not significantly associated with DFS: 3‐year DFS 100% in CIMP(−)/MSI(+), 84% in CIMP(−)/MSI(−), 82% in CIMP(+)/MSI(−) and 75% in CIMP(+)/MSI(+) (p = 0.33). Results of exploratory analysis showed that concurrent methylation at NEUROG1 and CDKN2A (p16) was associated with shorter DFS: 3‐year DFS 69% in NEUROG1(+)/CDKN2A (p16)(+) versus 87% in NEUROG1(−)/CDKN2A (p16)(−) (p = 0.006). In conclusion, concurrent methylation of NEUROG1 and CDKN2A (p16) is associated with recurrence following adjuvant FOLFOX in Stages II/III colorectal cancer.

Loss of CDX2/CK20 expression is associated with poorly differentiated carcinoma, the CpG island methylator phenotype, and adverse prognosis in microsatellite-unstable colorectal cancer.

Several previous studies have demonstrated that the CDX2-negative (CDX2−) and/or CK20-negative (CK20−) phenotypes of colorectal cancers (CRCs) might be associated with high levels of microsatellite instability (MSI-H). The aim of this study was to investigate the clinicopathologic and molecular features of MSI-H CRCs with different CDX2/CK20 expression statuses. The CDX2 and CK20 expression statuses were immunohistochemically evaluated in 109 MSI-H CRC tissue samples, and the correlations of these statuses with clinicopathologic, molecular, and survival data were statistically analyzed. Of the 109 MSI-H CRCs, 15 were CDX2− (13.8%), and 19 were CK20− (17.4%). The simultaneous loss of CDX2 and CK20 expression (CDX2−/CK20−) was observed in 9 cases (8.3%). CDX2 loss was correlated with lymph node metastasis, poor differentiation, MLH1 loss, the mutation of BRAF, and CpG island methylator phenotype-high (CIMP-H) status. Right-sided tumor location, nodal metastasis, poor differentiation, and CIMP-H status were significant characteristics of CK20− tumors. The CDX2−/CK20− phenotype was associated with older age (above 56 y), higher stage (stage III or IV), deep invasion (pT3 or pT4), lymph node metastasis (pN1 or pN2), poor differentiation (nonmedullary/non–signet ring cell type), the mutation of BRAF, and CIMP-H status among MSI-H CRCs. Patients with CDX2−/CK20− tumors exhibited worse overall and disease-free survival compared with the patients with CDX2+ and/or CK20+ tumors (P<0.001). In the multivariate analysis for disease-free survival, the CDX2−/CK20− phenotype was an independent prognostic factor for MSI-H CRC (P=0.030, hazard ratio=3.288). The CDX2−/CK20− phenotype defines a distinct subgroup of MSI-H CRCs with poor differentiation, CIMP-H status, and unfavorable prognosis.

Methylation profiles of multiple CpG island loci in extrahepatic cholangiocarcinoma versus those of intrahepatic cholangiocarcinomas

CONTEXT CpG island hypermethylation is attracting attention because of its importance as a tumor marker and its potential mechanism for the development of human cancers. Extrahepatic cholangiocarcinoma has been poorly investigated with respect to CpG island hypermethylation, and the number of genes known to be methylated in extrahepatic cholangiocarcinomas is fewer than 20. OBJECTIVE To generate methylation profiles of 24 CpG island loci in extrahepatic cholangiocarcinomas, to correlate methylation findings with clinicopathologic findings, and to compare these findings with those of intrahepatic cholangiocarcinomas. DESIGN Sixty-three extrahepatic cholangiocarcinomas and 48 intrahepatic cholangiocarcinomas were investigated for hypermethylation in 24 CpG island loci by using methylation-specific polymerase chain reaction. RESULTS A total of 61 (96.8%) of 63 extrahepatic cholangiocarcinomas showed hypermethylation in at least one of the examined loci, and a high methylation frequency was seen in HOXA1 (95.2%), HPP1 (69.8%), and NEUROG1 (61.9%). The number of methylated CpG island loci was greater in extrahepatic cholangiocarcinomas with nodal metastasis than in those without nodal metastasis (P = .047), and hypermethylation of TIG1 was closely associated with nodal metastasis of extrahepatic cholangiocarcinomas (P = .007). CDH1 and NEUROG1 were more frequently methylated in extrahepatic cholangiocarcinoma than in intrahepatic cholangiocarcinoma, whereas CHFR, GSTP1, IGF2, MGMT, MINT31, p14, and RBP1 were more frequently methylated in intrahepatic cholangiocarcinoma: the differences was statistically significant (P < .05). CONCLUSIONS A close relationship exists between CpG island hypermethylation and nodal metastasis of extrahepatic cholangiocarcinomas. Methylation profiles of extrahepatic cholangiocarcinomas are somewhat similar to but distinct from those of intrahepatic cholangiocarcinomas.

Loss of CDX2 expression is associated with poor prognosis in colorectal cancer patients

AIM To investigate the clinicopathologic characteristics and prognostic implications associated with loss of CDX2 expression in colorectal cancers (CRCs). METHODS We immunohistochemically evaluated CDX2 expression in 713 CRCs and paired our findings to clinicopathologic and molecular characteristics of each individual. Endpoints included cytokeratin 7 and CK20 expression, microsatellite instability, CpG island methylator phenotype, and KRAS and BRAF mutation statuses. Univariate and multivariate survival analysis was performed to reveal the prognostic value of CDX2 downregulation. RESULTS CDX2 expression was lost in 42 (5.9%) patients. Moreover, loss of CDX2 expression was associated with proximal location, infiltrative growth, advanced T, N, M and overall stage. On microscopic examination, loss of CDX2 expression was associated with poor differentiation, increased number of tumor-infiltrating lymphocytes, luminal serration and mucin production. Loss of CDX2 expression was also associated with increased CK7 expression, decreased CK20 expression, CpG island methylator phenotype, microsatellite instability and BRAF mutation. In a univariate survival analysis, patients with loss of CDX2 expression showed worse overall survival (P < 0.001) and progression-free survival (P < 0.001). In a multivariate survival analysis, loss of CDX2 expression was an independent poor prognostic factor of overall survival [hazard ratio (HR) = 1.72, 95%CI: 1.04-2.85, P = 0.034] and progression-free survival (HR = 1.94, 95%CI: 1.22-3.07, P = 0.005). CONCLUSION Loss of CDX2 expression is associated with aggressive clinical behavior and can be used as a prognostic marker in CRCs.