Serum fucosylated haptoglobin in chronic liver diseases as a potential biomarker of hepatocellular carcinoma development.

BACKGROUND: Fucosylation is one of the most important glycosylation events involved in cancer and inflammation. We previously developed a lectin antibody ELISA kit to measure fucosylated haptoglobin (Fuc-Hpt), which we identified as a novel cancer biomarker. In this study, we investigated Fuc-Hpt as a biomarker in chronic liver diseases, especially in hepatocellular carcinoma (HCC). METHODS: We measured serum Fuc-Hpt levels using our ELISA kit in 318 patients with chronic liver diseases, including 145 chronic hepatitis (CH) patients, 81 liver cirrhosis (LC) patients, and 92 HCC patients. During a long-term follow-up period of 7 years (1996-2003), Fuc-Hpt levels were measured at three different time points in 19 HCC patients. Serum Fuc-Hpt levels were also examined with a short-term follow-up period of 3 years (2009-2012) in 13 HCC patients. RESULTS: Fuc-Hpt levels increased with liver disease progression. Patients with LC and HCC showed significantly increased Fuc-Hpt levels in comparison to CH patients or healthy volunteers. Fuc-Hpt levels tended to be higher in HCC patients than in LC patients. Fuc-Hpt was better than α-fetoprotein (AFP) and AFP-L3 for predicting HCC [diagnosed by computed tomography (CT) or ultrasound] in LC patients with long-term follow-up. More than 80% of LC patients with long-term follow-up showed increased Fuc-Hpt during hepatocarcinogenesis, and 38% of early-stage HCC patients with short-term follow-up showed a gradual increase in Fuc-Hpt before imaging diagnosis. CONCLUSIONS: These results suggest that Fuc-Hpt is a novel and potentially useful biomarker for predicting liver disease progression and HCC development.

Endoscopic haemostasis by polypectomy: a case of sigmoid colon tubular adenoma with arterial haemorrhage.

An 84-year-old woman was admitted to our hospital with a massive lower intestinal bleeding (LIB). The enhanced CT showed extravasation of blood in the sigmoid colon during the arterial phase. After discussion with the interventional radiologists, we proceeded to perform emergency colonoscopy that demonstrated massive gushing of blood from a pedunculated sigmoid colon polyp. The polyp was removed by snare polypectomy, which resulted in complete haemostasis. The pathological finding of the resected lesion was a tubular adenoma.

A case of spontaneous splenic rupture during chemotherapy for B-cell chronic lymphoid leukemia.

Spontaneous splenic rupture is a life-threatening disease and an important differential diagnosis of acute abdomen. Early clinical diagnosis and rapid intervention is required to ensure patient survival. Spontaneous splenic rupture may be induced by hematological, inflammatory or infiltrative diseases affecting the spleen. Splenomegaly may also significantly increase the risk of rupture. Other contributory factors include male, adulthood, rapid growth of the spleen and splenic abscess. Here, we present the case of a 69-year-old man who was undergoing chemotherapy for B-cell chronic lymphoid leukemia. He was admitted to our hospital after he suddenly developed persistent upper abdominal pain. Computed tomography and ultrasonography revealed accumulation of free fluid in and around the spleen. He was diagnosed as having spontaneous splenic rupture and an emergency operation was performed. During the operation, we found a massively enlarged spleen with several capsular tears, and performed a splenectomy. The patient made a good recovery. Pathological examination revealed that the spleen was infiltrated by CD20-, CD5- and CD23-positive lymphoid blasts. We encountered a case of spontaneous splenic rupture in a patient receiving chemotherapy for exacerbating B-cell chronic lymphoid leukemia. In a case of abdominal pain of acute onset in patients with hematological disease, spontaneous splenic rupture should be suspected.

[A long-term survival case of liver metastases from gastric cancer treated with radiofrequency ablation and radiotherapy].

A 64-year-old man was diagnosed as gastric cancer (cT4N1M0, Stage IIIB). Left upper abdominal evisceration was performed in July 2008. CT scan revealed liver metastases in the segments 6 and 8 about 4-month after the surgery. Liver metastases increased during postoperative adjuvant chemotherapy. We treated the metastases with local therapy. He received radiotherapy (total of 60 Gy) for a liver metastasis in the segment 8 in November 2009. He received radiotherapy (total of 50 Gy) for a liver metastasis in the segment 6 in November 2010 after a total of 3-radiofrequency ablation (RFA) was performed. Partial response was obtained. We have experienced a successful case of liver metastases from gastric cancer treated with RFA and radiotherapy.

[Synchronous double cancer of the gallbladder and rectum successfully treated with S-1 as second-line chemotherapy- a case report].

A 66-year-old man was referred to our hospital with obstructive jaundice. Computed tomography(CT)scan showed thickening of the gallbladder wall, invasion into the liver bed, and thickening of the rectal wall. Colonoscopy revealed a type 2 rectal cancer, in which adenocarcinoma was identified by endoscopic biopsy. He was diagnosed with double-cancer of the gallbladder and rectum. Because his gallbladder cancer was more life threatening than his rectal cancer, gemcitabine was administered at 1, 000 mg/m2 on days 1, 8, and 15 of a 28-day course. After 3 courses of gemcitabine, the CT scan showed that the lymph nodes in the hepatoduodenal ligament had been enlarged, and duodenal stenosis had occurred as a result of gallbladder cancer invasion. S-1 was administered orally at doses of 120 mg/day twice daily on days 1-28 of a 42-day course. Partial response was confirmed by CT scan. After 8 courses of S-1, the gallbladder cancer had progressed and liver metastases had appeared. He subsequently died of disease progression. He survived for 17 months after the first course of chemotherapy, and the progression-free survival with S-1 was 10 months. Therefore, S-1 could be an effective agent for synchronous double cancer of the gallbladder and rectum.

[Case of primary hepatic neuroendocrine carcinoma diagnosed by needle biopsy].

The patient was a 55-year-old man with a large hepatic tumor measuring 12 × 12 cm in the left lobe. To obtain the histological diagnosis, the target liver biopsy was performed. Histologically, the tumor revealed as a neuroendocrine carcinoma. After the diagnosis, he received the chemotherapy (CTX) with etoposide and cisplatin. Serum levels of NSE and the tumor size were decreased after the first course of CTX. We here report a case of primary hepatic neuroendocrine carcinoma treated with CTX following the diagnosis by the needle biopsy.

Poorly differentiated endocrine carcinoma of the pancreas responded to gemcitabine: Case report.

Poorly differentiated endocrine carcinoma (PDEC) of the pancreas is a rare and aggressive tumor. First-line treatment is commonly a combination of etoposide and cisplatin, but there is no consensus regarding further treatment recommendations. In this report, we describe a case of pancreatic PDEC treated with gemcitabine as third-line chemotherapy. A 62-year-old man with pancreatic PDEC was administered etoposide plus cisplatin as first-line treatment; he then received irinotecan for tumor relapse. However, because irinotecan induced ileus in this patient, we chose gemcitabine as third-line chemotherapy. After two cycles of gemcitabine (1000 mg/m(2) on days 1, 8 and 15 every 4 wk), a partial tumor response was noted by computed tomography (approximately 68% reduction in tumor size). Our patient survived for 15 mo after diagnosis. This is a rare case of unresectable pancreatic PDEC, which showed a partial response to gemcitabine after the failure of two other regimens. Gemcitabine could be an effective treatment option for pancreatic PDEC that is resistant to other treatments.

Overexpression of insulin receptor substrate-1 and hepatitis Bx genes causes premalignant alterations in the liver.

UNLABELLED: Activation of the insulin (IN)/insulin receptor substrate-1 (IRS-1)/mitogen-associated protein kinase (MAPK) and the Wnt/beta-catenin signaling cascades occurs frequently in hepatocellular carcinoma (HCC) associated with persistent viral infection. The aims of this study were to provide a chronic proliferative stimulus through IRS-1 in the context of hepatitis Bx (HBx) protein expression in transgenic mice and determine if constitutive expression of these genes is sufficient to cause hepatocyte dysplasia and cellular transformation. We generated transgenic mice in which the HBx (ATX), IRS-1, or both (ATX+/IRS-1) genes were expressed under a liver-specific promoter. We also assessed histology and oxidative damage as well as up-regulation of molecules related to these signal transduction cascades in the liver by quantitative reverse-transcriptase polymerase chain reaction. Whereas mice with a single transgene (ATX or IRS-1) did not develop tumors, ATX+/IRS-1+ double transgenic livers had increased frequency of hepatocellular dysplasia and developed HCC. All three transgenic lines had significantly increased insulin growth factor 1 (IGF-1), Wnt 1 and Wnt 3 mRNA levels, and evidence of DNA damage and oxidative stress. The ATX+/IRS+ double transgenic mice were distinguished by having the highest level of activation of Wnt 3 and Frizzled 7 and selectively increased expression of IGF-II, proliferating cell nuclear antigen, and aspartyl-(asparaginyl)-beta-hydroxylase, a gene associated with increased cell migration. CONCLUSION: These results suggest that continued expression of the ATX or IRS-1 transgenes can contribute to hepatocyte transformation but are not sufficient to trigger neoplastic changes in the liver. However, dual expression that activates both the IN/IRS-1/MAPK and Wnt/beta-catenin cascades is sufficient to cause dysplasia and HCC in a previously normal liver.

Effectiveness of real-time virtual sonography-guided radiofrequency ablation treatment for patients with hepatocellular carcinomas.

AIM: Real-time virtual sonography (RVS) can synchronize B-mode ultrasound (US) images with multiplanar reconstruction (MPR)-computed tomography (CT) images on the same screen in real time. The purpose of this study was to evaluate the effectiveness of RVS for radiofrequency ablation therapy (RFA) of hepatocellular carcinomas (HCC) in which it was difficult to identify contours or margins by B-mode US. METHODS: Sixty-three consecutive patients with a solitary HCC of less than 3.5 cm in diameter were enrolled in this study. Thirty-nine patients with HCC clearly detectable by B-mode US underwent conventional RFA, while the remaining 24 with obscure tumor lesions underwent RVS-guided RFA. A follow-up study of RFA treatment was performed every 3 months using enhanced CT imaging of the arterial and portal phase (at least 24 months). The accuracy of needle insertion was confirmed by measuring the gap between the needle insertion line and the center of the tumor from MPR-CT images. RESULTS: The local recurrence rate of the RVS-guided RFA group was similar to that of the conventional RFA group (8.3% vs 7.7%), despite the difficulty of detecting tumor lesions in the former group. The mean gap between the needle insertion line and the center of the tumor was 1.6 mm (0-3.2 mm) in eight patients treated with RVS-guided RFA. CONCLUSION: RVS-guided RFA can be useful for treating HCC that are difficult to detect by B-mode US.

Chronic ethanol consumption impairs cellular immune responses against HCV NS5 protein due to dendritic cell dysfunction.

BACKGROUND & AIMS: Alcoholic patients with and without chronic liver disease have a high incidence of infection with hepatitis C virus (HCV). Long-term ethanol consumption in mice has been associated with a strikingly reduced CD8(+) cytotoxic T-lymphocyte (CTL) response to HCV nonstructural proteins following DNA-based immunization. This study evaluated the effect of ethanol on dendritic cells (DCs) as a mechanism(s) for reduced CTL activity. METHODS: Mice were fed an ethanol-containing or isocaloric pair-fed control diet for 8 weeks, followed by DC isolation from the spleen. DCs were evaluated with respect to endocytosis properties, cell surface markers, allostimulatory activity, and cytokine production following stimulation. Immune responses to HCV NS5 protein were generated by genetic immunization. Syngeneic transfer was used to determine if DC dysfunction contributed to abnormal cellular immune responses. RESULTS: Long-term ethanol exposure resulted in a reduced number of splenic DCs but did not alter endocytosis capacity. There was an increase in the myeloid and a reduction in the lymphoid DC population. Ethanol reduced expression of CD40 and CD86 costimulatory molecules on resting DCs, which was corrected following stimulation with lipopolysaccharide or poly I:C. There was impaired allostimulatory activity. Cytokine profiles of DCs isolated from ethanol-fed mice were characterized by enhanced interleukin (IL)-1beta and IL-10 and decreased tumor necrosis factor alpha, IL-12, interferon gamma, and IL-6 secretion. Impaired CTL responses to NS5 were corrected by syngeneic transfer of control DCs. CONCLUSIONS: Altered DC function is one of the major changes induced by long-term ethanol consumption, which subsequently impairs the cellular immune response necessary for viral clearance.

Vaccination with protein-transduced dendritic cells elicits a sustained response to hepatitis C viral antigens.

BACKGROUND & AIMS: Professional antigen-presenting dendritic cells are capable of eliciting a vigorous antiviral response in naive T cells. The administration of antigen-loaded dendritic cells offers a potential approach to induce high-level immunity against hepatitis C virus. METHODS: The dendritic cell population in mice was expanded in vivo by hydrodynamic delivery of naked DNA that encoded the secreted form of human fms-like tyrosine kinase 3 ligand. The CD11c-enriched dendritic cell population obtained from the spleen was transduced in vitro with recombinant hepatitis C virus core and nonstructural 5 proteins by using macromolecular-based protein delivery. Vaccine efficacy was assessed with a cytotoxic T-lymphocyte assay, cytokine enzyme-linked immunosorbent assays, and intracellular cytokine staining in vitro and by a tumor challenge model in vivo. RESULTS: Relative to mice inoculated with nontransduced dendritic cells, splenocytes derived from mice immunized with either hepatitis C virus core-transduced or nonstructural 5-transduced dendritic cells showed 3- to 5-fold greater antigen-specific cytotoxic T lymphocyte activity. The CD4(+) T cells obtained from mice immunized with nonstructural 5-transduced dendritic cells produced interferon gamma, but not interleukin 4, when stimulated with nonstructural 5. In contrast, T cells derived from mice immunized with hepatitis C virus core-transduced dendritic cells produced neither interferon gamma nor interleukin 4 when stimulated with core protein. Mice vaccinated with nonstructural 5-transduced dendritic cells, but not a nonstructural 5-expressing plasmid, showed a sustained antiviral response to nonstructural 5 as evidenced by reduced growth of nonstructural 5-expressing tumor cells inoculated 10 weeks after vaccination. CONCLUSIONS: These findings suggest that vaccination with protein-transduced dendritic cells may constitute an important antiviral strategy for hepatitis C virus.

Quick generation of fully mature dendritic cells from monocytes with OK432, low-dose prostanoid, and interferon-alpha as potent immune enhancers.

Dendritic cells (DCs) are one of the promising tools for enhancing antigen-specific immune responses in clinical settings. Many studies have been performed thus far to verify the efficacy of the DC vaccine in cancer patients; however, the responses have not always been satisfactory, partly because of DC incompetence. To obtain DCs potentially applicable for vaccination of cancer patients, our group sought to establish the strategy of DC generation mainly by modulating culture periods and maturation stimuli. Novel mature DCs that can be generated from monocytes within 3 days by using a combination of OK432 (Streptococcus pyogenes preparation), low-dose prostaglandin E2 (PGE2), and interferon-alpha (OPA-DCs) were developed. They strongly express CD83, CD86, and CCR7 and have potent ability to migrate to CCL21. In addition, they were able to activate natural killer and T helper 1 (TH1) cells and to induce peptide-antigen-specific cytotoxic T lymphocytes more significantly than monocyte-derived DCs stimulated with a conventional cytokine cocktail of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and PGE2 (monocyte-conditioned medium [MCM]-mimic DCs). The profound ability of OPA-DCs to stimulate these effectors is attributable to their higher expression of IL-12p70, IL-23, and IL-27 than MCM-mimic DCs, which was supported by the findings that the neutralization of IL-12p70 and IL-23 reduced the TH1 priming ability of OPA-DCs. Even when from advanced gastric or colonic cancer patients, OPA-DCs displayed abilities of migration and TH1 induction comparable to those from healthy subjects. Therefore, OPA-DCs may serve as a feasible vaccine with the potential to enhance TH1-dominant and cytolytic immune responses against cancers.

p27kip1 deficiency confers susceptibility to gastric carcinogenesis in Helicobacter pylori-infected mice.

BACKGROUND & AIMS: Determining how Helicobacter pylori promotes gastric cancer and whether H pylori eradication decreases cancer risk would be helped by suitable murine models. Mice lacking the cyclin-dependent kinase inhibitor p27kip1 are susceptible to carcinogen-induced tumors. Furthermore, p27 stimulates gastric epithelial apoptosis and inhibits proliferation, expression is decreased by H pylori, and low levels are associated with a poor prognosis in gastric cancer. We therefore evaluated p27-deficient mice as a model for H pylori-associated gastric cancer. METHODS: Wild-type and p27-/- C57BL/6 mice were infected with H pylori mouse-adapted Sydney strain at 6-8 weeks of age and 6-10 mice of each type were euthanized 15, 30, 45, 60, and 75 weeks later. RESULTS: Uninfected p27-/- mice developed gastric hyperplasia. H pylori-infected p27-/- mice frequently developed intestinal metaplasia (40% at 30 weeks, 67% at 45 weeks), and after 60 weeks 7 of 12 mice developed significant dysplasia and gastric cancer, recapitulating human intestinal-type gastric carcinogenesis. Wild-type mice developed intestinal metaplasia only after 75 weeks of infection; significant gastric dysplasia was observed in 1 animal (P < .05 for each comparison with p27-/- mice). No disease developed in uninfected mice. H pylori infection in p27-/- mice was associated with significantly decreased apoptosis and increased epithelial proliferation, inflammation, and H pylori density compared with infection in wild-type mice. CONCLUSIONS: p27 loss and H pylori colonization cooperate to produce gastric cancer. The p27-deficient mouse affords opportunities to examine the pathogenesis of H pylori in gastric carcinogenesis and to test eradication and chemopreventive strategies.

Type 1 interferon augments DNA-based vaccination against hepatitis C virus core protein.

Eradication of chronic hepatitis C virus (HCV) infection depends upon a broad-based cellular immune response. Genetic immunization stimulates such a response, but the resultant activity is generally weak. Type 1 interferons (IFNs), which are known for their direct anti-viral and anti-proliferative properties, possess vigorous immunomodulatory properties. The aim of this study was to assess the capacity of IFN-alpha to augment the cellular immune response to DNA vaccination against HCV core protein. Three types of IFN-alpha were investigated: the non-species-specific hybrid IFN A/D, human pegylated IFN-alpha, and a plasmid that expressed murine IFN-alpha. Low doses of hIFN-A/D and hPegIFN-alpha augmented three to fourfold the cellular immune response to DNA-based vaccination, determined in conventional CTL assays, as well as in an in vivo tumor challenge model. Importantly, augmentation occurred within a narrow concentration range; a further increase in IFN dosage suppressed the CTL response significantly. Humoral immunity showed a very similar pattern of augmentation. These findings demonstrate that the immunomodulatory properties of IFN-alpha can be exploited to augment DNA based immunization, but it is important to consider the effects of dose on both cellular and humoral immune response for optimal augmentation.

Helicobacter pylori increases proteasome-mediated degradation of p27(kip1) in gastric epithelial cells.

Helicobacter pylori infection is associated with increased gastric epithelial cell turnover and is a risk factor for noncardia gastric cancer. H. pylori reduces the expression of p27 protein, a cyclin-dependent kinase inhibitor of the G(1) to S-phase cell cycle transition and gastric tumor suppressor gene. Although cell cycle dysregulation associated with decreased p27 may contribute to gastric carcinogenesis, how H. pylori reduces p27 in gastric epithelial cells remains unknown. In the present study, we investigated the mechanisms of the p27 decrease, using AGS and MKN28 gastric epithelial cells cocultured with H. pylori strains under conditions of defined cell cycle distribution. The expression of p27 protein was reduced by H. pylori in a dose- and time-dependent manner. Northern blot and pulse-chase analyses revealed that this reduction was not regulated at a transcriptional level but by accelerated p27 degradation via a proteasome-dependent pathway. Despite up-regulation of the proteasome-dependent degradation of p27 protein, neither threonine 187-phosphorylated p27 nor skp2 (the ubiquitin ligase for p27) were increased. Furthermore, H. pylori impaired p27 ubiquitination and did not increase global proteasomal function. These results indicate that H. pylori increases the degradation of p27 through a proteasomal pathway distinct from the physiological pathway that degrades p27 during cell cycle progression. Putative virulence genes of H. pylori (cagA, cagE, or vacA) played no role in reducing p27 expression. Increased degradation of p27 by H. pylori through a proteasome-dependent, ubiquitin-independent pathway may contribute to the increased risk of gastric cancer associated with chronic H. pylori infection.

Impaired expression of proteasome subunits and human leukocyte antigens class I in human colon cancer cells.

BACKGROUND AND AIM: The presentation of human leukocyte antigens (HLA) class I requires the coordinated expression of numerous components involved in antigen processing and antigen presentation. Tumor cells may alter the expression of these components to decrease HLA class I expression, allowing them to escape immune surveillance. The aim of this study is to investigate the expressions of these components, including proteasome subunits, and their involvement in the expression of HLA class I in human colon cancer cells. METHODS: Four human colon cancer cell lines, HCT116, SW403, LoVo and DLD-1, were used to examine the expression of HLA class I by flow cytometry. Reverse transcription-polymerase chain reaction was performed to assess the expression of beta2-microglobulin, heavy chains, transporter subunits, immunoproteasomes subunits and proteasome activator 28 (PA28) subunits. RESULTS: Human leukocyte antigen class I was expressed highly in HCT116 and SW403 cells and weakly in LoVo cells, but was not expressed in DLD-1 cells. The DLD-1 cells were deficient in the expression of proteasome subunits including low molecular weight polypeptide proteasome subunit 2 (LMP2), multicatalytic endopeptidase complex-like-1 (MECL-1), PA28alpha and PA28beta, whereas other HLA class I-expressing cell lines expressed all components tested. gamma-Interferon (IFN-gamma) treatment of DLD-1 cells restored the expression of LMP2, MECL-1 and PA28beta, but not the expression of HLA class I. Enforced expression of PA28alpha induced the expression of HLA class I in IFN-gamma-treated DLD-1 cells, but not in untreated DLD-1 cells. CONCLUSION: These results suggest that the impaired expression of proteasome subunits is involved in the loss of HLA class I expression in human colon cancer cells.