Clinicopathologic Study of Biliary Intraepithelial Neoplasia in Cholangiocarcinoma.

BACKGROUNDS AND AIMS: Biliary intraepithelial neoplasia (BilIN) is a precursor of cholangiocarcinoma (CC) and it has been associated with several chronic inflammatory conditions. This study aimed to elucidate the prevalence of BilIN in CC and its clinicopathological significance. METHODS: Medical records of 193 patients with histologically confirmed CC were analyzed. We reviewed the pathology findings of 48 patients who underwent curative surgery for CC. RESULTS: Of the 48 patients analyzed, 34 and 14 patients had extrahepatic and intrahepatic CC respectively. BilIN was detected in 28 patients (58%) and showed a significantly higher prevalence in extrahepatic CC (75%) than in intrahepatic CC (21%; p < 0.001). In the subgroup of 34 patients with extrahepatic CC, 25 and 9 patients were BilIN positive and negative respectively. Poor differentiation and T3 stage were significantly more common in the BilIN-negative group than in the BilIN-positive group (p < 0.05). The expression of MUC5AC, p53, and loss of Smad4 showed no difference between BilIN-positive CC and in BilIN-negative CC, but the Ki-67 expression was significantly higher (p < 0.05). CONCLUSION: BilIN-positive CC showed less invasiveness than negative cases. The Ki-67 expression was significantly higher in BilIN-positive CC.

Downregulation of spleen tyrosine kinase in hepatocellular carcinoma by promoter CpG island hypermethylation and its potential role in carcinogenesis.

Spleen tyrosine kinase (SYK) has predominantly been studied in hematopoietic cells, where it is involved in immunoreceptor-mediated signaling. However, SYK expression has been shown in numerous non-hematopoietic cells, and its downregulation has been shown to be involved in tumor formation and progression. SYK methylation has been demonstrated to identify a subset of hepatocellular carcinoma (HCC) cases with poor prognosis, but little is known regarding the biological role of SYK in HCC. We found that SYK methylation is a common event in HCC, and is inversely associated with its expression. We established stable HCC cell lines with inducible SYK expression vectors, and compared the differential RNA expression profiles of HCC cell lines with or without the induction of SYK. Gene ontology analysis revealed that the SYK-regulated genes were enriched for genes involved in cell adhesion. Accordingly, we found that the induction of SYK expression increased the adhesion of cells to fibronectin and decreased cell migration and invasion, and that cessation of SYK overexpression increased cell migration and invasion. Our findings suggest that SYK is involved in regulating cell to matrix adhesions, and that SYK loss affects the migration, and invasion of HCC cells.

Prognostic implications of and relationship between CpG island hypermethylation and repetitive DNA hypomethylation in hepatocellular carcinoma.

PURPOSE: This study aims to determine the relationship between CpG island DNA hypermethylation and global genomic DNA hypomethylation and their prognostic implications in hepatocellular carcinoma. The association of DNA methylation changes with clinicopathologic factors and the chronological ordering of DNA methylation changes along multistep hepatocarcinogenesis were also assessed. EXPERIMENTAL DESIGN: Hepatocellular carcinoma (n = 20) and nonneoplastic liver samples (n = 72) were analyzed for their methylation status at 41 CpG island loci and 3 repetitive DNA elements (LINE-1, ALU, and SAT2) using MethyLight or combined bisulfite restriction analysis. After selection of 19 CpG island loci showing cancer-specific DNA methylation, another set of 99 hepatocellular carcinoma samples was analyzed for these loci. RESULTS: The number of methylated genes in hepatocellular carcinoma was significantly higher in hepatocellular carcinoma patients with a cirrhotic liver than in hepatocellular carcinoma patients with a noncirrhotic liver (9.9 versus 7.0, P = 0.001). Hepatocellular carcinoma from female patients showed a higher number of methylated genes than hepatocellular carcinoma from male patients (11.2 versus 8.4, P = 0.006). The genes CRABP1 and SYK showed significant association between CpG island hypermethylation and patients' poor survival. SAT2 hypomethylation occurred earlier than LINE-1 or ALU hypomethylation along the multistep hepatocarcinogenesis. Depending on the type of CpG island locus, a direct, inverse, or no relationship between CpG island hypermethylation and repetitive DNA hypomethylation was observed in hepatocellular carcinomas. CONCLUSION: The varying relationships between the hypermethylation of individual CpG island loci and the hypomethylation of repetitive elements suggests that they are not mechanically linked. SYK and CRABP1 hypermethylation may serve as useful tumor markers for prognostication of hepatocellular carcinoma patients.

Prognostic value of apoptosis-related markers in urothelial cancer of the upper urinary tract.

We investigated the expression of apoptosis-related markers and their association with the clinical outcomes of patients with urothelial carcinoma of the upper urinary tract. A total of 112 patients with urothelial carcinoma of the upper urinary tract that had surgery from March 1998 to July 2005 were included in the study. Tissue microarray slides were used for immunohistochemistry, and immunohistochemical staining was performed to investigate the association of apoptosis-related markers with clinical outcome. Apoptosis was confirmed by the TdT-mediated DUTP nick-end labeling method to obtain the apoptotic index. Survival analysis was performed according to the Kaplan-Meier method, and the Cox proportional hazard regression model was used to compare the relative influence of different prognostic factors. Among the 112 patients, 32 (28.6%) had altered expression of p53, 30 (26.8%) of bcl-2, 62 (55.4%) of bax, 27 (24.1%) of caspase-3, and 23 (20.5%) of survivin. The expression of p53 and caspase-3 was associated with the pathologic grade (P = .035 and P = .004, respectively). Altered expression of caspase-3 was associated with the pathologic stage (P = .016). The multivariate analysis showed that the expression of survivin (hazard ratio 2.91, 95% confidence interval 1.07-7.90, P = .036) and the apoptotic index (AI) (3.35, 1.06-10.56, P = .039), as well as the T and N stages (P = .043 and P = .010, respectively) were significantly associated with the disease-specific survival. Our results suggest that survivin expression and a high apoptotic index were poor prognostic factors for survival in patients with urothelial carcinoma of the upper urinary tract. These results may help to identify a subset of patients who require adjuvant therapy or closer follow-up.

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.

Panels of immunohistochemical markers help determine primary sites of metastatic adenocarcinoma.

CONTEXT: Although identification of the primary tumor in patients with metastatic adenocarcinoma has a profound clinical impact, diagnosing the organ of origin is frequently difficult. Because none of the individual immunohistochemical markers used for tissue identification are both site specific and site sensitive, multiple markers are needed to improve the prediction of primary sites. OBJECTIVE: To develop an effective approach to immunohistochemically evaluate metastatic adenocarcinoma for the assignment of a likely primary site of origin. DESIGN: Expression profiles of CDX2, cytokeratin (CK) 7, CK20, thyroid transcription factor 1 (TTF-1), carcinoembryonic antigen (CEA), MUC2, MUC5AC, SMAD4, estrogen receptor (ER), and gross cystic disease fluid protein 15 (GCDFP-15) were generated in adenocarcinomas from 7 primary sites, followed by construction of a decision tree and design of multiple-marker panels. Expression of these markers was evaluated immunohistochemically in 314 primary adenocarcinomas (50 cases each of colorectal, gastric, lung, pancreatic, bile duct, and breast, and 14 cases of ovarian origin) using the tissue array method. Results were validated using 60 cases of metastatic adenocarcinoma with known primaries. RESULTS: Organ-specific immunostaining profiles using multiple markers provided high sensitivity, specificity, and positive predictive value in detecting primary adenocarcinomas, as follows: colorectal, TTF-1-/CDX2+/CK7-/CK20+ or TTF-1-/CDX2+/CK7-/CK20-/(CEA+ or MUC2+); ovarian, CK7+/MUC5AC+/TTF-1-/CDX2-/CEA-/GCDFP-15-; breast, GCDFP-15+/TTF-1-/CDX2-/CK7+/CK20- or ER+/ TTF-1-/CDX2-/CK20-/CEA-/MUC5AC-; lung, TTF-1+ or TTF-1-/CDX2-/CK7+/CK20-/GCDFP-15-/ER-/CEA-/ MUC5AC-; pancreaticobiliary, TTF-1-/CDX2-/CK7+/ CEA+/MUC5AC+; and stomach, TTF-1-/CDX2+/CK7+/ CK20-. Overall, these combined phenotypes correctly predicted the tester samples (metastatic adenocarcinomas with known primaries) in 75% of cases. CONCLUSIONS: Determination of tissue-specific immunostaining profiles is valuable in the diagnostic differentiation of metastatic adenocarcinomas from seven common primary sites and should help to correctly predict the organ of primary tumor origin.

Methylation profiles of CpG island loci in major types of human cancers.

Several reports have described aberrant methylation in various types of human cancers. However, the interpretation of methylation frequency in various human cancers has some limitations because of the different materials and methods used for methylation analysis. To gain an insight into the role of DNA hypermethylation in human cancers and allow direct comparison of tissue specific methylation, we generated methylation profiles in 328 human cancers, including 24 breast, 48 colon, 61 stomach, 48 liver, 37 larynx, 24 lung, 40 prostate, and 46 uterine cervical cancer samples by analyzing CpG island hypermethylation of 13 genes using methylation-specific PCR. The mean numbers of methylated genes were 6.5, 4.4, 3.6, 3.4, 3.1, 3.1, 3.1, and 2.1 in gastric, liver, prostate, larynx, colon, lung, uterine cervix, and in breast cancer samples, respectively. The number of genes that were methylated at a frequency of more than 40% in each tumor type ranged from nine (stomach) to one (breast). Generally genes frequently methylated in a specific cancer type differed from those methylated in other cancer types. The findings indicate that aberrant CpG island hypermethylation is a frequent finding in human cancers of various tissue types, and each tissue type has its own distinct methylation pattern.

BRAF and KRAS mutations in prostatic adenocarcinoma.

Constitutive activation of the kinase cascade involving RAS, RAF, MEK and ERK is common to human cancers, and mutations of KRAS and BRAF are mutually exclusive and serve as alternatives to activate the RAS/RAF/ERK signaling pathway. RAS mutations are known to occur in prostate adenocarcinomas, but little is known about BRAF mutations in these tumors. In the present study, BRAF and KRAS mutations were characterized in 206 prostate adenocarcinomas by enhanced PCR-RFLP and direct sequencing. The identified KRAS and BRAF mutations were then analyzed with respect to preoperative serum PSA levels, Gleason scores and tumor stages. Mutations in codon 600 of BRAF were identified in 21 (10.2%) of 206 prostate adenocarcinomas. KRAS mutations in codons 12 or 13 were found in 15 (7.3%) of 206 prostate adenocarcinomas. However, no tumor specimen contained both BRAF and KRAS mutations. Prostate adenocarcinomas with a BRAF mutation tended to show higher preoperative serum PSA levels, Gleason scores and tumor stages than prostate adenocarcinomas with a KRAS mutation. The results obtained show that BRAF mutations are as uncommon as KRAS mutations in prostate adenocarcinoma. Although BRAF and KRAS are members of the same RAS/ERK signaling pathway, prostate adenocarcinomas with a BRAF mutation showed clinicopathologic features that differed from those of prostate adenocarcinoma with a KRAS mutation.