Cancer-testis antigen BORIS is a novel prognostic marker for patients with esophageal cancer.

Esophageal squamous cell cancer (ESCC) is one of the most common lethal tumors in the world, and development of new diagnostic and therapeutic methods is needed. In this study, cancer-testis antigen, BORIS, was isolated by functional cDNA expression cloning using screening technique with serum IgG Abs from ESCC patients. BORIS was previously reported to show cancer-testis antigen like expression, but its immunogenicity has remained unclear in cancer patients. BORIS was considered to be an immunogenic antigen capable of inducing IgG Abs in patients with various cancers, including four of 11 ESCC patients. Immunohistochemical study showed that the BORIS protein was expressed in 28 of 50 (56%) ESCC tissues. The BORIS expression was significantly associated with lymph node metastasis in ESCC patients with pT1 disease (P = 0.036). Furthermore, the patients with BORIS-positive tumors had a poor overall survival (5-year survival rate: BORIS-negative 70.0% vs BORIS-positive 29.9%, log-rank P = 0.028) in Kaplan-Meier survival analysis and log-rank test. Multivariate Cox proportional hazard model demonstrated that BORIS expression was an independent poor prognostic factor (hazard ratio = 4.158 [95% confidence interval 1.494-11.57], P = 0.006). Downregulation of BORIS with specific siRNAs resulted in decreased cell proliferation and invasion ability of ESCC cell lines. BORIS may be a useful biomarker for prognostic diagnosis of ESCC patients and a potential target for treatment including by BORIS-specific immunotherapy and molecular target therapy.

Sentinel node mapping with thermoresponsive magnetic nanoparticles in rats.

BACKGROUND: In this study, we investigated the ability of magnetic resonance imaging (MRI) after interstitial administration of thermoresponsive magnetic nanoparticles to detect the sentinel lymph node (SLN). MATERIALS AND METHODS: Postcontrast MRI scans were acquired following subcutaneous injection of thermoresponsive magnetic nanoparticles into the thoracic wall of rats. The signal-to-noise ratio of axillary lymph nodes was calculated to assess whether the SLN could be detected by MRI. In a second experiment, after injecting thermoresponsive magnetic nanoparticles, i.e., Therma-Max 36, Therma-Max 42, Therma-Max 55, and Ferridex, into the subserosa of the cecum of rats, the injection sites, the SLNs, and the distant lymph nodes were resected and examined histologically in order to determine which nanoparticles, if any, were specifically retained in the SLN. RESULTS: MRI showed that the signal-to-noise ratio of axillary SLNs was significantly lower 24 h after injection of Therma-Max 42 than on the precontrast images (P < 0.05). Histologic evaluation revealed that Therma-Max 36 aggregated at body temperature and did not migrate to the SLN. Therma-Max 42, on the other hand, aggregated, and the particles became large enough to be retained in the SLNs. Therma-Max 55 and Ferridex did not aggregate, and they both migrated to the SLNs and the distant lymph nodes. CONCLUSIONS: The results of this study showed that thermoresponsive magnetic nanoparticles could be targeted to the SLN by adjusting the temperature at which they aggregate, and that they could be used as a contrast agent for SLN mapping by MRI.

Human liver-specific nanocarrier in a novel mouse xenograft model bearing noncancerous human liver tissue.

For the targeted delivery of genes and drugs to the human liver, hepatitis B virus (HBV) envelope L particles, which form hollow nanoparticles and display a peptide that is indispensable for liver-specific infection by HBV in humans, should be a useful tool. To test the efficacy of these particles in vivo, in the present study we generated a small animal model harboring a functional human liver tissue xenograft. An anti-asialo GM1 antibody was administered to SCID mice to induce the depletion of natural-killer-cell-dependent immune responsibility and then the mice underwent transplantation of a noncancerous liver tissue originating in humans into the kidney capsule. Interestingly, human liver tissues were engrafted in 58% of the animals at least for 14 days shown by a human hepatocyte-specific antibody. The engineered HBV nanoparticles which contained fluorescent chemicals could selectively bind to the xenograft in these immune-deficient mice when they were administered systemically. These results suggested that the model animal was usable to demonstrate the efficacy of the nanoparticles that could deliver chemicals specific to the normal human liver tissue by systemic administration, which will facilitate the study of human liver cell biology, drug metabolism and infections with hepatotropic viruses.

Reduced transforming growth factor-beta receptor II expression in hepatocellular carcinoma correlates with intrahepatic metastasis.

Hepatocellular carcinoma (HCC) occurs mainly in the liver associated with chronic hepatitis and hepatic cirrhosis as a result of prolonged viral infection. Transforming growth factor-beta (TGF-beta) induces the fibrosis in hepatic cirrhosis, although it is also an inhibitor of hepatocyte proliferation. To understand the role of TGF-beta signaling in HCC progression, we analyzed gene expression in HCC cells in relation to TGF-beta signaling using a two-way clustering algorithm. By the analysis, five HCC cell lines were classified into two groups according to their metastatic capacity. TGF-beta receptor II (TGFBR2) was downregulated in metastatic cells, which did not show a response to TGF-beta. Immunohistochemistry demonstrated clear membrane distribution of TGFBR2 in noncancerous hepatocytes, whereas reduced TGFBR2 expression was observed in 34 of 136 HCCs. In clinical cases, reduced TGFBR2 expression correlated with larger tumor size (P<0.001), poor differentiation (P<0.001), portal vein invasion (P=0.002), intrahepatic metastasis (IM) (P<0.001), and shorter recurrence-free survival (P=0.022). In conclusion, reduced TGFBR2 expression was associated with aggressive features of HCC such as IM, and may represent an immunohistochemical biomarker to detect aggressive HCC.

Beneficial effects of 5-Fluorouracil and heparin-based portal infusion chemotherapy combined with mitomycin C and cisplatin after curative resection of pancreatic cancer.

AIMS: We retrospectively assessed the benefits of 5-fluorouracil (5-FU)- and heparin-based portal infusion chemotherapy combined with systemic administration of mitomycin C (MMC) and cisplatin (CDDP) for 4 weeks following surgery (PI4W). The goal was to determine if this treatment prevented liver metastasis and improved survival for patients with potentially curative resection of pancreatic cancer. METHODS: 68 patients who underwent pancreatectomy from January 1995 to August 2007 were treated. Of these cases, 22 patients received portal infusion with 5-FU (250 mg/day) for 2 weeks (PI2W) following surgery, while 25 patients received PI4W therapy (250 mg/day of 5-FU with 2,000 IU/day of heparin everyday for 4 weeks, 4 mg MMC on days 6, 13, 20, 27, and 10 mg CDDP on days 7, 14, 21, 28). The remaining 21 patients were treated without adjuvant therapy during the perioperative period. RESULTS: All patients except one completed the portal infusion chemotherapy without toxicity. The cumulative liver metastasis-free survival rate in the PI4W group was significantly higher than those in the other two groups. Furthermore, in the PI4W group, 3-year survival was 82.9% and 5-year survival was 63.8%, rates which were significantly better than those observed in the other two groups. CONCLUSION: PI4W therapy after surgery is feasible and could become a promising adjuvant therapy in patients with potentially curative resection of pancreatic cancer. and IAP.

Molecular diagnosis and translymphatic chemotherapy targeting sentinel lymph nodes of patients with early gastrointestinal cancers.

The sentinel lymph node (SLN) is defined as the lymph node(s) that first receives lymphatic drainage from the site of the primary tumor. Recent progress in molecular techniques has demonstrated the presence of micrometastatic tumor cells in SLNs. Quantitative real-time RT-PCR assay, which enables rapid analysis, is currently being undertaken for intraoperative molecular diagnosis of SLNs. We developed an intraoperative real-time RT-PCR assay to detect micrometastasis in SLNs for early gastric cancer. All SLNs and randomly selected non-SLNs in 96 cT1 or cT2 gastric cancer patients were biopsied intraoperatively and examined by routine hematoxylin and eosin staining, immunohistochemistry with anticytokeratin antibody (AE1/AE3), and multimarker real-time RT-PCR assay including cytokeratin (CK) 19, CK20, and carcinoembryonic antigen. All patients with histopathologically verified metastasis in their SLNs demonstrated positive results by RT-PCR assay. Forty percent of patients with histopathologically negative SLNs showed positive SLNs by RT-PCR assay. RT-PCR assay revealed that 4 patients (4%) with negative SLNs had positive non-SLNs; however, these positive non-SLNs were identified within each SLN basin. We recently developed a new drug delivery system targeting SLNs with a phospholipid polymer, using 2-methacryloyloxyethyl phosphorylcholine conjugated with paclitaxel. Our preliminary data suggest that this novel drug delivery system may be feasible for translymphatic chemotherapy targeting SLNs of patients with cN0 early gastrointestinal cancer, who have the potential for occult metastasis in SLNs. Endoscopic resection of the primary tumor followed by translymphatic chemotherapy targeting SLNs may become a promising minimally invasive multidisciplinary therapy.

Selective targeting by preS1 domain of hepatitis B surface antigen conjugated with phosphorylcholine-based amphiphilic block copolymer micelles as a biocompatible, drug delivery carrier for treatment of human hepatocellular carcinoma with paclitaxel.

Using dithioester-capped 2-methacryloyloxyethyl phosphorylcholine (MPC) as a macro chain transfer agent, a diblock copolymer was synthesized with n-butyl methacrylate (BMA) as hydrophobic core-forming blocks. The MPC-BMA unit was copolymerized with an immobilizable unit, p-nitrophenylcarbonyloxyethyl methacrylate (NPMA). The NPMA moiety then was modified by the addition of preS1 domain of hepatitis B surface antigen (HBsAg). This micelle-forming nanoparticle, the poly (MPC-co-BMA-co-NPMA) (PMBN) conjugated with preS1 enables solubilization of paclitaxel (PTX) with increased hepatotropism. The 50% inhibitory concentration (IC(50)) values of PTX and PTX/PMBN-preS1 against the human hepatocellular carcinoma cell line, HepG2, were 1,008 and 131 nM, respectively (p < 0.05). Conjugation of preS1 to PMBN enhanced strongly the synergistic inhibitory effect of paclitaxel on HepG2 cells in vitro, whereas such a change in IC(50) was not detected against the human squamous cell carcinoma cell line, A431. Tumor growth rates of a HepG2 xenograft in Balb/c nude mice after intraperitoneal injection of PTX, PTX/PMBN and PTX/PMBN-preS1 were +97.9%, -74.3% and -96.2%*, respectively (*p < 0.05 versus PTX). The local paclitaxel levels after administration of the PMBN-preS1 conjugate were determined in the xenografts by high-performance liquid chromatography and were 8 times higher than that after administration of paclitaxel alone. No side effects attributable to PMBN-preS1 were observed histologically in vital organs, and body weight loss was significantly less in the PTX/PMBN-preS1 group. These studies demonstrate that PMBN-preS1 may be used as a human hepatocyte-specific drug delivery carrier without serious adverse effects.

Development of targeted therapy with paclitaxel incorporated into EGF-conjugated nanoparticles.

BACKGROUND: 2-Methacryloyloxyethyl phosphorylcholine (MPC) polymer is a suitable vehicle for paclitaxel (PTX) delivery. A new targeted therapy has been developed by conjugating epidermal growth factor (EGF) to MPC polymer and its growth inhibitory and antitumor effects on cancer cells overexpressing EGF receptors (EGFR) has been investigated. MATERIALS AND METHODS: EGF was conjugated to poly [MPC-co-n-butyl methacrylate-co-p-nitrophenyloxycarbonyl poly (ethylene glycol) methacrylate] (PMBN) and mixed with PTX. The cytotoxicity of the resulting PTX incorporated into EGF-conjugated PMBN (EGF-PMBN-PTX) on EGFR-overexpressing and EGFR-deficient cell lines was compared with PTX incorporated into PMBN alone (PMBN-PTX) and PTX alone. Suspensions of the cells were injected into nude mice subcutaneously. EGF-PMBN-PTX, PMBN-PTX, PTX or NaCl solution was injected intraperitoneally. RESULTS: The cytotoxicity and antitumor effect of EGF-PMBN-PTX were significantly greater than those of PMBN-PTX for EGFR-overexpressing cells but not for an EGFR-deficient line. CONCLUSION: These results suggest that EGF-PMBN-PTX may represent a more potent targeted therapy for tumors overexpressing EGFR.

Establishment of combined immuno-chemotherapy with systemically administered gemcitabine and intra-portal administration of interleukin-2 in murine models of liver metastases of pancreatic cancer.

Despite attempts to use multiple drug combinations that include gemcitabine (GEM), there is very little evidence that these combination regimens are superior to the single use of this agent. We therefore investigated the suppressive effect of the combination of systemically administered GEM and locally administered interleukin-2 (IL-2) on liver metastasis in pancreatic cancer. Tumor-bearing mice were randomly divided into four groups: a control group, an IL-2 intrasplenic (is) administration group, a GEM intraperitoneal (ip) administration group, and a GEM ip+IL-2 is group. Liver weight, liver metastases, and tumor diameter (as assessed by the Winn assay) were compared among groups. Liver weight was significantly lower in the GEM+IL-2 group than in the control and IL-2 groups. The number of liver metastases was significantly reduced in the GEM+IL-2 group compared with all other groups. Splenocyte production of interferon-gamma increased significantly in the GEM+IL-2 group after stimulation with Concanavalin A. Furthermore, tumor diameter was significantly reduced in the GEM+IL-2 group in the Winn assay when compared to that of the control group. These findings suggest that a combined regimen of GEM and portally administrated IL-2 might prevent liver metastasis in pancreatic cancer patients more effectively than current approaches and could prove useful as a postsurgical adjuvant therapeutic.

Co-expression of aFGF and FGFR-1 is predictive of a poor prognosis in patients with esophageal squamous cell carcinoma.

Overexpression of aFGF, bFGF and FGFR-1 has been reported in various cancers, and it has been suggested that it may be a poor prognostic factor in cases with solid tumors. Therefore, we attempted to determine whether overexpression of aFGF, bFGF and FGFR-1 might also be a poor prognostic factor in patients with esophageal squamous cell carcinoma, and examined the expression of aFGF, bFGF and FGFR-1 in esophageal cancer tissue specimens to clarify their clinical significance. Seventy-nine patients with squamous cell carcinoma of the esophagus who underwent resection at the Department of Surgery, Keio University Hospital, were enrolled as the subjects of this study. None of the patients had received any previous treatment. Formalin-fixed and paraffin-embedded sections of esophageal cancer tissue were stained by immunohistochemical methods and examined for expression of the angiogenetic factors and their receptors, and also to determine the microvascular density (MVD). We examined the correlations between the expression of aFGF, bFGF and FGFR-1, and the MVD, clinicopathological background factors and survival of the patients by conducting statistical analyses of the data. The results revealed that positive aFGF expression was associated with a larger tumor area (p=0.009), and co-expression of both aFGF and FGFR-1 was associated with a larger tumor area (p=0.01) and poorer prognosis (p=0.04). There were positive correlations between the expression of aFGF and FGFR-1 (p<0.0001), and between those of bFGF and FGFR-1 (p=0.04). aFGF may promote proliferation of esophageal cancer cells in an angiogenesis-independent and autocrine manner, and may contribute to rapid growth of esophageal cancer on recurrence after esophageal resection.

Efficacy of an MPC-BMA co-polymer as a nanotransporter for paclitaxel.

BACKGROUND: Paclitaxel (PTX) is administered as a solution in polyoxyethylated castor oil (CO) due to its low water solubility, but solvent-induced side-effects may be severe. MATERIALS AND METHODS: PMB30W is a co-polymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and butyl methacrylate (BMA). Cytotoxicities of PTX in PMB30W (PTX-PMB30W) were examined in cell culture and in vivo. RESULTS: PTX-PMB30W and PTX in dimethyl sulfoxide showed similar toxicity in breast cancer cell lines MCF-7, SK-BR-3 and MX-1. Antitumor efficacies of PTX-PMB30W and PTX in CO (PTX-CO) were similar following weekly intraperitoneal administration of 50 mg/kg PTX in nude mice transplanted with MX-1 cells. At 200 mg/kg PTX, all animals died within 1 minute of PTX-CO administration. However, all animals receiving PTX-PBM30W survived. Ulceration occurred following subcutaneous injection of PTX-CO, but injection of PTX-PMB30W did not cause skin changes. CONCLUSION: Our data suggest that PMB30W can act as an effective PTX nanotransporter.

Overexpression of cyclase-associated protein 2 in multistage hepatocarcinogenesis.

PURPOSE: Hepatocellular carcinoma (HCC) associated with chronic liver disease is known to show an obvious multistage process of tumor progression. We previously identified heat shock protein 70 as a molecular marker of early HCC during investigation of expression profiling in multistage hepatocarcinogenesis. In this report, we examined cyclase-associated protein 2 (CAP2), which is also listed as an up-regulated gene in early HCC. EXPERIMENTAL DESIGN: We measured the level of CAP2 mRNA by real-time quantitative PCR. We raised a polyclonal antibody against CAP2 and we confirmed the expression of CAP2 by immunoblotting and immunohistochemistry in HCC cell lines and HCC tissues. RESULTS: According to real-time quantitative PCR, the level of CAP2 mRNA was up-regulated in early HCC when compared with noncancerous liver tissue, and it was further up-regulated in progressed HCC. We raised a polyclonal antibody against CAP2, which showed a single 53-kDa band of strong intensity in the human HCC cell lines and HCC tissues but only a weak band in the noncancerous liver tissues in Western blot analysis. Immunohistochemical examination of CAP2 revealed its significant overexpression in early HCC when compared with noncancerous and precancerous lesions and in progressed HCC when compared with early HCC. CONCLUSION: Our findings show that CAP2 is up-regulated in HCC when compared with noncancerous and precancerous lesions. This is the first report that proves that CAP2 is up-regulated in human cancers and that this is possibly related to multistage hepatocarcinogenesis.

Nanoparticles for the delivery of genes and drugs to human hepatocytes.

Hepatitis B virus envelope L particles form hollow nanoparticles displaying a peptide that is indispensable for liver-specific infection by hepatitis B virus in humans. Here we demonstrate the use of L particles for the efficient and specific transfer of a gene or drug into human hepatocytes both in culture and in a mouse xenograft model. In this model, intravenous injection of L particles carrying the gene for green fluorescent protein (GFP) or a fluorescent dye resulted in observable fluorescence only in human hepatocellular carcinomas but not in other human carcinomas or in mouse tissues. When the gene encoding human clotting factor IX was transferred into the xenograft model using L particles, factor IX was produced at levels relevant to the treatment of hemophilia B. The yeast-derived L particle is free of viral genomes, highly specific to human liver cells and able to accommodate drugs as well as genes. These advantages should facilitate targeted delivery of genes and drugs to the human liver.

Clinicopathological features and surgical outcome of isolated metastasis of renal cell carcinoma.

BACKGROUND/AIMS: The benefit of pancreatic resection for metastatic renal cell carcinoma (RCC) is poorly defined. Here, we investigate the clinicopathological features and surgical outcome of patients with pancreatic metastasis from RCC. METHODOLOGY: Among a total of 131 patients who underwent pancreatic resection at our center between November 2000 and November 2005, four patients (three men, one woman) with a median age of 57 years (range: 52-80 years) at the time of pancreatic tumor presentation, had histologically confirmed metastatic RCC to the pancreas. The medical records, imaging data, surgical records, and pathology findings of these patients were reviewed retrospectively. RESULTS: All patients underwent radical nephrectomy for primary RCC. The pathologic stage was TNM stage T2N0M0 (n = 1) or T3aN0M0 (n = 2) (no data were available on one patient). RCCs developed in the right (n = 2) or left (n = 2) kidney. The median interval between nephrectomy and detection of pancreatic metastasis was 84 months (range: 0-285 months). All patients were asymptomatic, and the pancreatic masses with a median tumor diameter of 2.0 cm (range: 1.5-4.0 cm) were detected during routine follow-up or screening examinations. All pancreatic tumors were smooth, well-demarcated, and hypervascular on imaging studies. None of them showed evidence of associated extrapancreatic disease. Complete resection with an adequate margin of safety was achieved by distal pancreatectomy (n = 3) or pylorus-preserving pancreatoduodenectomy (n = 1). Within a median follow-up period of 39 months (range: 4-41 months) after the surgery, three patients were alive with no evidence of recurrence, and one patient was alive with evidence of recurrence. The median survival from nephrectomy was 103 months (range: 40-326 months). CONCLUSIONS: RCCs may demonstrate very late metastasis to the pancreas, thus the possibility of pancreatic metastasis should be considered when a patient with a pancreatic tumor has a history of RCC, despite the interval since nephrectomy. The experience gained in this study suggests that pancreatic metastasectomy should be attempted for RCC patients without extrapancreatic disease.

Successful pancreatectomy with en-bloc resection of the celiac artery and portal vein for pancreatic endocrine carcinoma.

Invasion to the celiac axis and portal vein is one reason for the unresectability of pancreatic carcinoma of the body and tail. Some authors advocate a radical distal pancreatectomy with en-bloc resection of the celiac artery and portal vein. However, long-term survival is still rare. We report here on a very rare, long-term survivor of a locally-advanced endocrine carcinoma of the body of the pancreas that was treated by distal pancreatectomy with en-bloc resection of the celiac artery and portal vein. The patient recovered well postoperatively, and has survived for 55 months without evidence of recurrence. The experience gained in the present case suggests that radical pancreatectomy with en-bloc resection of the celiac artery and portal vein is a potential approach that might increase tumor resectability and improve the prognosis of patients with locally-advanced endocrine carcinomas of the pancreas.

Operative indications for relatively small (2-5 cm) gastrointestinal stromal tumor of the stomach based on analysis of 60 operated cases.

BACKGROUND: Removal of the primary lesion with a clear operative margin is the standard treatment for gastrointestinal stromal tumor (GIST) of the stomach. However, there are few reports on the operative indications for relatively small GIST. METHODS: Clinicopathologic features and survival data of all 60 patients with GIST of the stomach treated at Keio University Hospital from 1993 to 2004 were analyzed. Laparoscopic wedge resection was used as the primary procedure for tumors between 2 to 5 cm. Tumors larger than 5 cm were resected by laparotomy or laparoscopy-assisted operation. RESULTS: Thirty-five lesions (58.3%) were resected by laparoscopic wedge resection, 3 by laparoscopic operation with a small skin incision and 22 by conventional open procedures. The mean size of the tumors was 42.5 mm, with a range of 18 to 150 mm and a median value of 35.5 mm. All operative margins were clear, but 1 patient had liver metastases at the time of resection of the primary lesion. The median follow-up period was 53 months and the 5-year disease-free survival rate (DFS) was 96.1%. No local recurrence or distant metastasis was encountered in patients with tumors smaller than 4 cm. A statistically significant correlation was observed between tumor size and mitotic count in this cohort (P = .010). Tumors from the intermediate- (n = 14) and high-risk (n = 10) groups as classified by the Risk Assessment Classification showed significantly worse DFS than the low-risk and very low risk group (n = 35) (89.9% vs 100% in 5-year DFS, P = .045). Even among tumors smaller than 3 cm, 2 of 14 cases (14.3%) were classified into the intermediate-risk group. CONCLUSIONS: Although a prospective randomized trial remains to be performed, this study provides additional evidence suggesting that the early removal of GIST, at 5 cm or less in size, provides better DFS than later removal of the tumor at a larger size.

Gallbladder carcinoma with osteoclast-like giant cells.

Extraskeletal tumors containing multinucleated, osteoclast-like giant cells (OGCs) are uncommon. These neoplasms are most frequently reported in the breast and pancreas. Recently, some authors have suggested that carcinomas containing OGCs may represent a distinct clinicopathological entity with a more favorable prognosis. Occurrence in the gallbladder is extremely rare, with only one previous case. We report here on an additional case of gallbladder carcinoma with an infiltrate of OGCs. A 72-year-old woman presented with postprandial abdominal pain and was found to have a mass in the body of the gallbladder with direct liver invasion. Histological examination showed an adenosquamous carcinoma with an infiltrate of benign OGCs. Immunohistochemical analysis demonstrated that the giant cells were of histiocytic origin. The patient survived for 6 years without evidence of recurrence. This case adds to a small body of literature on gallbladder carcinoma with OGCs. Further studies are required to clearly define the prognostic significance of these giant cells in gallbladder cancer and the differences between adenosquamous carcinoma with OGCs and other gallbladder carcinomas (such as adenocarcinoma and squamous cell carcinoma) with those cells.