A Rare Case of Scalp Metastasis from Encapsulated Angioinvasive Follicular Thyroid Carcinoma.

Cutaneous metastases from thyroid carcinomas are extremely rare; however, the scalp is a common site for cutaneous metastases from follicular thyroid carcinomas (FTCs). We report the case of a 77-year-old male patient with a blood-rich scalp lesion. Histopathological tests of punch biopsy specimens revealed subcutaneous well-formed follicular structures that were similar to those found in the thyroid gland. Immunohistochemistry using thyroid transcription factor-1 (TTF-1) and paired-box gene family 8 (PAX8) revealed an FTC metastasis. We performed total thyroidectomy and resection of the scalp lesion at the same time and administered postoperative radioactive iodine treatment. The primary thyroid lesion was diagnosed as an FTC based on extracapsular extension and vessel invasion. The patient has not experienced disease recurrence since the treatment. When scalp metastasis of thyroid carcinoma is suspected, we recommend total extirpation, including the primary tumor and scalp metastasis, for an improved prognosis.

Serum Levels of N- and C-ERC/Mesothelin and Clinicopathological Factors in Mesothelioma Patients and Those without Mesothelioma.

Objectives: ERC/mesothelin is a glycosylphosphatidylinositol (GPI)-anchor protein expressed in mesothelioma. A precursor protein is cleaved by proteases and an N-terminal fragment (N-ERC) is extracellularly secreted. A remaining C-terminal fragment (C-ERC) is tethered on cellular membranes by the GPI-anchor, but C-ERC is also released after cleavage by proteases. We and other groups reported that serum N-/C-ERC levels are associated with stages of mesothelioma and suggested the possibility of their usefulness as diagnostic markers. However, the N-ERC level is also influenced by renal functions that are not directly associated with conditions of mesothelioma. It is not known whether other clinical factors influence serum N-/C-ERC values. Furthermore, their relationship to the amount of ERC/Mesothelin in mesothelioma is not yet validated. The objective of this study is to clarify the relationship of serum N-/C-ERC levels and the status of mesothelioma and several clinical factors. Materials and Methods: We analyzed relations of serum N-/C-ERC levels and ages, gender and other clinical factors in 522 patients without mesothelioma and examined their relation to the amount of ERC/Mesothelin in mesothelioma tissues in 13 mesothelioma cases. Results: Serum N-ERC levels were influenced by renal functions. On the contrary, those of C-ERC were not influenced by any clinical factors examined in this study and were significantly correlated with the amount of ERC/Mesothelin in mesothelioma. Conclusion: Although both markers are good indicators of treatment-responses in individual patients with mesothelioma, only C-ERC reflected the amount of ERC/Mesothelin in mesothelioma among multiple patients, possibly because N-ERC was influenced by renal functions.

The Inhibitory Effects of Anti-ERC/Mesothelin Antibody 22A31 on Colorectal Adenocarcinoma Cells, within a Mouse Xenograft Model.

The expression of Renal Carcinoma (ERC)/mesothelin is enhanced in a variety of cancers. ERC/mesothelin contributes to cancer progression by modulating cell signals that regulate proliferation and apoptosis. Based on such biological insights, ERC/mesothelin has become a molecular target for the treatment of mesothelioma, pancreatic cancer, and ovarian cancer. Recent studies revealed about 50-60% of colorectal adenocarcinomas also express ERC/mesothelin. Therefore, colorectal cancer can also be a potential target of the treatment using an anti-ERC/mesothelin antibody. We previously demonstrated an anti-tumor effect of anti-ERC antibody 22A31 against mesothelioma. In this study, we investigated the effect of 22A31 on a colorectal adenocarcinoma cell line, HCT116. The cells were xenografted into BALB/c nu/nu mice. All mice were randomly allocated to either an antibody treatment group with 22A31 or isotype-matched control IgG1κ. We compared the volume of subsequent tumors, and tumors were pathologically assessed by immunohistochemistry. Tumors treated with 22A31 were significantly smaller than those treated with IgG1κ and contained significantly fewer mitotic cells with Ki67 staining. We demonstrated that 22A31 exhibited a growth inhibitory property on HCT116. Our results implied that ERC/mesothelin-targeted therapy might be a promising treatment for colorectal cancer.

Abnormal Exacerbation of Moderately Differentiated Gastric Adenocarcinoma in a Patient with TAFRO Syndrome: An Impaired Tumor Immunity?

TAFRO syndrome is a relatively new disease entity first reported in 2010. We report a case of TAFRO syndrome accommodated by abnormal exacerbation of moderately differentiated gastric adenocarcinoma. The pathophysiology of TAFRO syndrome is largely unknown, but because the disease often responds to immunosuppressive therapy and also because T follicular helper (Tfh) cells are reported to be drastically decreased in TAFRO syndrome, involvement of a dysregulated immune system can be speculated. Growing evidence points toward a pivotal role of Tfh cells in tumor immunity through supporting ectopic lymphoid structures, which are recruitment sites for cells directly engaging in antitumor activity such as CD8+ T cells, NK cells, and macrophages. In fact, Tfh cells are reported to positively correlate with longer survival in human colorectal and breast cancer. Combined with our observations of hyperprogressive gastric cancer in the presented patient, an impaired tumor immunity is strongly indicated in TAFRO syndrome.

Bisecting-GlcNAc on Asn388 is characteristic to ERC/mesothelin expressed on epithelioid mesothelioma cells.

Mesothelioma is a highly aggressive tumour associated with asbestos exposure and is histologically classified into three types: epithelioid-type, sarcomatoid-type and biphasic-type. The prognosis of mesothelioma patients is poor and there is no effective molecular-targeting therapy as yet. ERC/mesothelin is a glycoprotein that is highly expressed on several types of cancers including epithelioid mesothelioma, but also expressed on normal mesothelial cells. This is a predicted reason why there is no clinically approved therapeutic antibody targeting ERC/mesothelin. In the present study, we focussed on the differential glycosylation between ERC/mesothelin present on epithelioid mesothelioma and that on normal mesothelial cells and aimed to reveal a distinct feature of epithelioid mesothelioma cells. Lectin microarray analysis of ERC/mesothelin using cells and patient specimens showed significantly stronger binding of PHA-E4 lectin, which recognizes complex-type N-glycans having a so-called bisecting-GlcNAc structure, to ERC/mesothelin from epithelioid mesothelioma cells than that from normal mesothelial cells. Further, liquid chromatography/mass spectrometry analysis on ERC/mesothelin from epithelioid mesothelioma cells confirmed the presence of a bisecting-GlcNAc attached to Asn388 of ERC/mesothelin. These results suggest that this glycoproteome could serve as a potential target for the generation of a highly selective and safe therapeutic antibody for epithelioid mesothelioma.

A New CD10 Antibody Inhibits the Growth of Malignant Mesothelioma.

Malignant mesotheliomas (MMs) are aggressive therapy-resistant tumors that generally have a poor prognosis. We previously reported the establishment of four new monoclonal antibodies (mAbs) for the diagnosis and treatment of MM. In this report, we characterized one of these antibodies, JMAM-1. The molecules whose antibodies were calibrated were picked up, transfected assuming CD10, and elucidated by fluorescence activated cell sorter. Survival experiments were performed using tumor-bearing mice model. JMAM-1 mAb was found to bind with CD10 antigen. The Kaplan-Meier survival curve showed a small but prolonged survival effect. JMAM-1 mAb-treated MSTO-211H cells showed increased cell cycle arrest involved by cyclin-dependent-kinase. JMAM-1 antibody has cytostatic effect and may be a candidate for the treatment of MM. Among mesothelioma, CD10-positive cases have been reported to have a poorer prognosis than negative cases, which can be used as a tool for diagnosis.

Expression status of PD-L1 and B7-H3 in mesothelioma.

Mesothelioma is a rare, aggressive malignancy with poor outcome, and has limited treatment options. The aim of this study was to perform a comprehensive analysis of programmed death ligand 1 (PD-L1) and B7 homolog 3 (B7-H3) expression in mesothelioma. We investigated the protein expression of PD-L1 and B7-H3 and their potential correlation with histological subtype, which might help to develop new therapies targeting these immune checkpoint molecules. Expression analysis of PD-L1 and B7-H3 was performed by immunohistochemistry using serial tissue sections of specimens obtained from 31 patients with mesothelioma. Tumors were classified into 22 epithelioid, 6 sarcomatoid, and 3 biphasic types. Of the 31 patients, 13 (41.9%) were positive for PD-L1 and 28 (90.3%) were B7-H3 positive. Twelve of the 13 PD-L1 positive patients were positive for B7-H3. PD-L1 and B7-H3 were widely co-expressed in biphasic and sarcomatoid type tumor cells. These findings might provide a rationale for the use of combination therapy for mesothelioma by targeting PD-L1 and B7-H3, as well as the development of anti-B7-H3 or anti-PD-L1 single agents.

Combined use of irinotecan and p53 activator enhances growth inhibition of mesothelioma cells.

Malignant mesothelioma (MM) is an aggressive malignant neoplasm which rapidly invades pleural tissues and has a poor prognosis. Here, we explore enhancement of the effect of irinotecan [camptothecin-11 (CPT-11)] by the p53-dependent induction of carboxylesterase 2 (CES2), a CPT-11-activating enzyme, in MM. The level of CES2 mRNA was greatly increased on treatment with nutlin-3a. A combination of CPT-11 and nutlin-3a inhibited the growth of MM cells more effectively than either drug alone. Knocking down CES2 in MM cells reduced the effect of the drug combination, and its forced expression in MESO4 cells enhanced the growth inhibitory activity of CPT-11 in the absence of nutlin-3a. Enhancement of the growth inhibitory activity of CPT-11 by nutlin-3a suggests a possible new combinatorial MM chemotherapy regimen.

Possible reversibility between epithelioid and sarcomatoid types of mesothelioma is independent of ERC/mesothelin expression.

BACKGROUND: Mesothelioma is histologically divided into three subgroups: epithelioid, sarcomatoid, and biphasic types. The epithelioid or sarcomatoid type is morphologically defined by polygonal or spindle-like forms of cells, respectively. The biphasic type consists of both components. It is not yet understood how histological differentiation of mesothelioma is regulated. ERC/mesothelin is expressed in most cases of the epithelioid type, but not in the sarcomatoid type of mesothelioma. Consequently, its expression is well correlated to the histological subtype. We hypothesized that ERC/mesothelin expression influences the histological differentiation of mesothelioma, and tested this hypothesis. METHODS: We performed studies using the overexpression or knockdown of ERC/mesothelin in mesothelioma cells to examine its effect on cellular morphology, growth kinetics, or migration/invasion activity, in vitro. We then transplanted ERC/mesothelin-overexpressing and control cells into the intraperitoneal space of mice. We examined the effect of ERC/mesothelin overexpression on mouse survival and tumor phenotype. RESULTS: In vitro cell culture manipulations of ERC/mesothelin expression did not affect cellular morphology or proliferation, although its overexpression enhanced cellular adhesion and the migration/invasion activity of mesothelioma cells. The survival rate of mice following intraperitoneal transplantation of ERC/mesothelin-overexpressing mesothelioma cells was significantly lower than that of mice with control cells. The histological evaluation of the tumors, however, did not show any morphological difference between two groups, and our hypothesis was not validated. Unexpectedly, both groups (ERC/mesothelin-overexpressing and control) of mesothelioma cells that were morphologically monophasic and spindle-like in vitro differentiated into a biphasic type consisting of polygonal and spindle-like components in the transplanted tumor, irrespective of ERC/mesothelin expression. CONCLUSIONS: These results suggested that the histological transition of mesothelioma between epithelioid and sarcomatoid types may be reversible and regulated not by ERC/mesothelin, but by other unknown mechanisms.

Usefulness of immunohistochemistry for mismatch repair protein and microsatellite instability examination in adenocarcinoma and background endometrium of sporadic endometrial cancer cases.

AIM: Microsatellite instability (MSI), which reflects loss of DNA mismatch repair (MMR) activity, and immunohistochemistry (IHC) for MMR proteins are employed as screening examinations for Lynch syndrome (LS). Recent studies revealed that there is a population of MSI-high tumors in sporadic endometrial cancer (EC). However, MSI data for Japanese EC patients are scarce. Furthermore, sporadic estrogen-dependent EC (type I) is generally considered to arise from hyperplasia. Because LS is usually associated with type I EC, we hypothesized that MSI might be involved in the oncogenic process in some sporadic EC. We conducted MSI testing to reveal MSI status in sporadic Japanese EC. IHC for MMR proteins was also performed. METHODS: Ninety-eight tissue samples of sporadic ECs from Japanese patients were used for IHC and MSI examinations. We also evaluated MMR protein expressions in the background normal endometrium. RESULTS: Microsatellite instability-high was observed in 10.2% of 98 cases with sporadic EC, a lower percentage than that in Western studies. Loss of some MMR proteins was observed in 23 cases (23.5%) and there was a significant correlation with MSI-high status (P < 0.001). Concerning the background endometrium, two cases showed partial loss of MLH1 and PMS2, corresponding to adjacent EC lesions, suggesting that MMR deficiency may already be present in the background endometrium. CONCLUSION: The MSI-high rate was low in our Japanese cohort. Our data confirmed the usefulness of MMR protein assessment for MSI screening in Japanese EC patients. Furthermore, IHC of the background endometrium might reveal the mechanism of MSI-high tumorigenesis.

Elucidation of inhibitory effects on metastatic sentinel lymph nodes of breast cancer during One-Step Nucleic Acid Amplification.

One-step nucleic acid amplification (OSNA) is an established method for intraoperative diagnosis of breast cancer metastasis in sentinel lymph nodes, based on quantification of CK19 mRNA, specific to breast epithelial cells. Inhibitors interfere with the PCR amplification process of PCR. Thus, OSNA, based on gene amplification without RNA purification, might be impacted by numerous factors persisting in a sample, and thereby potentially acting as PCR inhibitors. However, neither the characteristics of breast cancers showing inhibitory effects during OSNA, nor any of the possible inhibitors, have as yet been identified. Inhibitory effects detected during OSNA in 72 metastatic lymph nodes and the patients' clinicopathological features were examined. Left-over OSNA samples were analyzed with mass spectrometry to identify proteins possibly acting as inhibitors. Most tumors showed inhibitory effects, though to varying degrees. Large tumor, young age and high tumor-infiltrating lymphocyte counts were related to stronger inhibitory effects. Proteome analysis revealed elevations in RPB9 protein and EIF2 signaling upregulation in samples showing strong inhibitory effects. Tumors showing strong inhibitory effects had clinically relevant characteristics, including large size and extensive tumor-infiltrating lymphocyte involvement. Identifying inhibitors in OSNA might provide new insights into breast cancer biology as well as advancing the current technology.

Establishment of anti-mesothelioma monoclonal antibodies.

BACKGROUND: Mesotheliomas are aggressive, therapy-resistant tumors that are predicted to increase in incidence at least until 2020. The prognosis of patients with mesothelioma is generally poor because they are typically diagnosed at a late stage and their tumors are resistant to current conventional therapies. For these reasons, improved diagnosis and therapy are urgently required. To address these issues, the aim of our research was to develop novel mesothelioma-specific monoclonal antibodies (mAbs) as diagnostic and therapeutic agents. METHODS: To develop anti-mesothelioma mAbs useful for diagnosis and therapy, we repeatedly immunized a BALB/c mouse with viable mesothelioma cells, alternating between those from three mesothelioma cell lines. We hybridized the spleen cells from this immunized mouse with P3U1 myeloma cells. We then screened supernatants harvested from the hybridoma clones by assessing whether they bound to a mesothelioma cell line not used for immunization and altered its morphology. We designed this developmental strategy to reduce the risk of obtaining clonotypic mAbs against a single mesothelioma cell line. RESULTS: Our newly generated mouse anti-human mAbs immunostained clinical samples of mesotheliomas. One of the newly generated mAbs did not react with any other tumor cell line tested. Two other mAbs significantly inhibited the proliferation of mesothelioma cells. CONCLUSION: These newly generated anti-mesothelioma mAbs are potentially useful as diagnostic and therapeutic agents for mesothelioma. Moreover, our novel strategy for establishing antitumor mAbs may facilitate the development of new diagnostic and therapeutic techniques for mesotheliomas and other malignancies.

Ischemic Cardiomyopathy with a Rapid Progression from Thrombotic Thrombocytopenic Purpura.

An 83-year-old woman who complained of dizziness and nausea visited our hospital. An electrocardiogram showed ST-segment elevation in multiple leads and an echocardiogram showed severe hypokinesis of the anteroseptal wall of the left ventricle. However, emergency coronary angiography showed no stenotic lesions in any coronary arteries. A laboratory examination showed thrombocytopenia, renal dysfunction, and hemolysis. We therefore diagnosed the patient with thrombotic thrombocytopenic purpura (TTP). While we were preparing to initiate plasma exchange therapy, she suddenly developed cardiopulmonary arrest. A postmortem examination revealed microthrombi in the small vessels of the myocardium. We herein report a case of ischemic cardiomyopathy with a rapid progression from TTP.

Fulminant pneumococcal infection.

Fulminant pneumococcal infection is a fatal pneumococcal infection that tends to occur in immunocompromised hosts, such as patients who are asplenic or on immunosuppressant therapy. We experienced a case of a 73-year-old Japanese man with a medical history of coronary stent implantation and catheter ablation for atrial flutter who presented with dyspnoea at rest. The patient was diagnosed with streptococcal pneumonia based on a urine antigen test and CT. Despite the use of effective antibiotics and systemic therapies, his clinical course was rapidly progressive and he died 18 h after admission. This case of fulminant pneumococcal infection is reported along with the autopsy findings.

C-ERC/mesothelin provokes lymphatic invasion of colorectal adenocarcinoma.

BACKGROUND: Lymph node metastasis is a key event of colorectal cancer (CRC) progression. Mesothelin is expressed in various types of malignant tumor and associated with an unfavorable prognosis. The full-length mesothelin (Full-ERC) is cleaved by protease into membrane-bound C-ERC/mesothelin and N-ERC/mesothelin which is secreted into the blood. The aim of this study was to examine the biological role of mesothelin in CRC by clinicopathological analysis and in vitro lymphatic invasion assay. METHODS: Ninety-one cases of CRC specimens were immunohistochemically examined and the localization of mesothelin in luminal membrane and/or cytoplasm was also evaluated. Lymphatic invasion assay was also performed using the human CRC cell line, WiDr, which was transfected with Full-, N- and C-ERC/mesothelin expression plasmids (Full-WiDr, N-WiDr and C-WiDr). RESULTS: Immunohistochemically, "luminal membrane positive" of mesothelin was identified in 37.4 %, and correlated with lymphatic permeation and lymph node metastasis, but not with patients' prognosis. Interestingly, among the patients with lymph node metastasis (N = 38), "luminal membrane positive" of mesothelin significantly correlated with unfavorable patients' outcome. In addition, lymphatic invasion assay revealed that Full-WiDr and C-WiDr more significantly invaded human lymphatic endothelial cells than the Mock-WiDr (P < 0.01). CONCLUSION: The luminal membrane expression of mesothelin was associated with unfavorable prognosis of CRC patients with lymph node metastasis. Moreover, this is the first report to prove the biological function of C-ERC/mesothelin associated with lymphatic invasion of cancer in vitro.

ERC/mesothelin is expressed in human gastric cancer tissues and cell lines.

ERC/mesothelin is expressed in mesothelioma and other malignancies. The ERC/mesothelin gene (MSLN) encodes a 71-kDa precursor protein, which is cleaved to yield 31-kDa N-terminal (N-ERC/mesothelin) and 40-kDa C-terminal (C-ERC/mesothelin) proteins. N-ERC/mesothelin is a soluble protein and has been reported to be a diagnostic serum marker of mesothelioma and ovarian cancer. Gastric cancer tissue also expresses C-ERC/mesothelin, but the significance of serum N-ERC levels for diagnosing gastric cancer has not yet been studied. We examined the latter issue in the present study as well as C-ERC/mesothelin expression in human gastric cancer tissues and cell lines. We immunohistochemically examined C-ERC/mesothelin expression in tissue samples from 50 cases of gastric cancer, and we also assessed the C-ERC/mesothelin expression in 6 gastric cancer cell lines (MKN-1, MKN-7, MKN-74, NUGC-3, NUGC-4 and TMK-1) using reverse transcription-polymerase chain reaction, flow cytometry, immunohistochemistry and immunoblotting. We also examined the N-ERC/mesothelin concentrations in the supernatants of cultured cells and in the sera of gastric cancer patients using an enzyme-linked immunosorbent assay (ELISA). N-ERC/mesothelin was detected in the supernatants of 3 gastric cancer cell lines (MKN-1, NUGC-4 and TMK-1) by ELISA, but its concentration in the sera of gastric cancer patients was almost same as that observed in the sera of the normal controls. In the gastric cancer tissues, C-ERC/mesothelin expression was associated with lymphatic invasion. N-ERC/mesothelin was secreted into the supernatants of gastric cancer cell lines, but does not appear to be a useful serum marker of gastric cancer.

NP-1250, an ABCG2 inhibitor, induces apoptotic cell death in mitoxantrone-resistant breast carcinoma MCF7 cells via a caspase-independent pathway.

Chemoresistance is one of the main obstacles to successful cancer therapy and is frequently associated with multidrug resistance (MDR). One of the most studied mechanisms of MDR is the high expression of ATP-binding cassette (ABC) transporters. Here, we demonstrated that NP-1250, an ABCG2 inhibitor, induced apoptotic cell death in ABCG2-overexpressing multidrug-resistant MCF7/mitoxantrone-resistant (MX) human breast carcinoma cells via a caspase-independent pathway. Incubation of MCF7/MX cells with NP-1250 significantly reduced cell viability, while NP-1250 had little effect on the viability of drug-sensitive MCF7/wild-type cells. Although the target molecules of NP-1250 in cell death remain unknown, investigation of NP-1250 will aid in the elucidation of the molecular mechanism of drug resistance and NP-1250 may become a new therapy for MDR cancers.

Up-regulation of dbpA mRNA in hepatocellular carcinoma associated with metabolic syndrome.

PURPOSE: Metabolic syndrome (MS) is a group of recognized risk factors for the development of hepatocellular carcinoma (HCC) in patients with chronic liver disease. The aim of this study was to analyze the clinicopathological characteristics of HCC patients with MS and the risk factors for recurrence. Also, the aim was to investigate the cold shock protein: DNA-binding protein A (dbpA) expression in HCC patients with MS. METHODS: A total of 243 patients who underwent curative resections for HCC were classified into two groups. dbpA expression was investigated in 66 HCC patients with MS and in 30 patients without MS by using real-time RT-PCR. Promoter methylation status was examined by using MS-PCR. RESULTS: The incidence of metabolic factors affect the HCC significantly higher in non-B non-C patients than in hepatitis B virus (HBV) or hepatitis C virus (HCV) patients (P < 0.001). Univariate analysis of HCC patients with MS recurrence revealed aspartate amino transferase (AST), multiple tumors, liver damage, hepatic vein invasion, advanced cancer stages (P < 0.01), alpha-fetoprotein (AFP) and diabetes mellitus type II (P < 0.05) as risk factors. Multivariate analysis, AST, multiple tumors, and hepatic vein invasion (P < 0.01) were identified as independent factors for the recurrence. dbpA mRNA was higher in patients with MS than in those without MS (P = 0.016), and it was mostly upregulated in non-B non-C HCC patients with MS than in non-B non-C HCC patients without HBV or HCV. Especially, in HCC patients with diabetes mellitus type II, the mRNA and protein levels were highly upregulated. The dbpA expression was regulated by promoter methylation status (P < 0.05). CONCLUSIONS: This study identifies that dbpA may accelerate the hepatocarcinogenesis in HCC patients with MS via inflammation-induced and oxidative stress pathways. The demethylation-related epigenetic activation may be one of the regulating factors for HCC patients with MS.

A severe form of epidermal nevus syndrome associated with brainstem and cerebellar malformations and neonatal medulloblastoma.

Here we report a boy with epidermal nevus syndrome associated with brainstem and cerebellar malformations and neonatal medulloblastoma. The patient had epidermal nevi and complicated brain malformations including macrocephaly with polymicrogyria, dysmorphic and enlarged midbrain tectum, enlarged cerebellar hemispheres with small and maloriented folia. The patient died after surgical resection of medulloblastoma which was newly recognized on MRI at 51 days of age. Postmortem pathological examinations showed very unique and bizarre malformation of the midbrain and hindbrain. The cerebellar cortex exhibited a coarse, irregular and bumpy surface, blurred border between the Purkinje cell layer and internal granule cell layer, and many foci of heterotopia in the cerebellar white matter. The brainstem showed multiple anomalies, including enlargement of superior colliculi, hypoplasia of pyramidal tracts and dysplasia of inferior olivary nuclei. The unusual constellation of brain malformations of our patient will widen the spectrum of epidermal nevus syndrome.

DNA binding protein A expression and methylation status in hepatocellular carcinoma and the adjacent tissue.

We investigated the expression and promoter methylation of dbpA in human hepatocellular carcinoma (HCC) and examined their correlation with clinicopathological features. In 96 paired samples of HCC and adjacent non-tumorous liver, and 10 normal liver specimens, dbpA mRNA was quantified by real-time RT-PCR, and promoter methylation was examined by methylation-specific polymerase chain reaction and bisulfite sequencing. The results showed that dbpA mRNA expression levels were higher in HCC compared to corresponding non-tumor tissues (P<0.01) and higher in non-virus-associated HCC compared to virus-associated cases (P<0.01). dbpA promoter was methylated in 37.7% of HCC samples and the promoter methylation was significantly correlated with the low expression of dbpA in non-virus-associated HCC (P<0.01), but not in virus-associated HCC. Surprisingly, poor prognosis was more significantly associated with high dbpA expression in non-tumorous liver (P=0.018) but not with that in HCC. Non-tumorous tissues consist of chronic hepatitis or liver cirrhosis, and these conditions are the background of hepatocarcinogenesis, defined as the hypercarcinogenic state. Our results suggest that the high expression of dbpA in the hypercarcinogenic state is an indicator of poor prognosis.