Gastric adenocarcinoma of fundic gland type (chief cell predominant type) with unique endoscopic appearance curatively treated by endoscopic submucosal resection.

Gastric adenocarcinoma of fundic gland type [chief cell predominant type; (GA-FD-CCP)] is a rare gastric cancer variant arising from non-atrophic mucosa without Helicobacter pylori infection in the upper third portion of the stomach. GA-FD-CCP originates deep in the mucosal layer; hence, endoscopic lesion detection is often difficult at an early stage because of a minimal change in the mucosal surface. Here we present a 66-year-old man with an early stage of GA-FD-CCP showing characteristic endoscopic features. Esophagogastroduodenoscopy demonstrated a flat, slightly reddish area with black pigment dispersion and irregular micro-surface structure at the gastric fornix. The tumor was resected by endoscopic submucosal dissection and was pathologically diagnosed as GA-FD-CCP. Prussian blue staining revealed that the black pigment was a hemosiderin deposition. We reported a rare case of successfully treated GA-FD-CCP with black pigmentation that aided in early lesion detection.

A potential approach for electrochemotherapy against colorectal carcinoma using a clinically available alternating current system with bipolar snare in a mouse model.

BACKGROUND: Although electrochemotherapy appears promising for the treatment of superficial tumors, its usefulness against internal tumors, such as colorectal carcinoma (CRC), has not been well examined. Furthermore, since direct current electric pulses have been used for electropermeabilization of tumors in all in vivo electrochemotherapy studies, including clinical trials, the usefulness of alternating current systems has not been examined at all. In a mouse model it was examined whether the alternating current system with a bipolar snare, which has been employed already as a clinical endoscopic treatment modality, was useful for electrochemotherapy against CRC. METHODS: Murine CRC colon 26 cells were implanted subcutaneously into syngeneic BALB/c mice and electrochemotherapy with bleomycin (BLM) using the alternating current system was performed against established CRC tumors. RESULTS: Electrochemotherapy significantly suppressed the growth of established CRC tumors, resulting in significantly prolonged survival of animals with CRC. Histological examination revealed that electrochemotherapy caused massive necrosis of CRC tumors. Subsequent analysis revealed that the delivery of alternating current electric pulses to CRC tumors profoundly increased intratumoral amounts of BLM. CONCLUSIONS: Because the alternating current system using a bipolar snare has been used widely as an endoscopic treatment modality in clinical settings, these results indicate that electrochemotherapy using the alternating current system may be a promising approach for the treatment of CRC.

Retrovirus-mediated in vivo gene therapy using the herpes simplex virus thymidine kinase gene against carcinomatous peritonitis.

BACKGROUND: Carcinomatous peritonitis is characterized by massive malignant ascites, while peritoneally disseminated carcinomatosis is characterized by a large number of metastatic solid tumors in the peritoneal cavity. Although both are fatal end-stage manifestations of malignancies derived from the digestive system, the former is usually more serious than the latter due to massive malignant ascites. Although the effectiveness of gene therapy against peritoneally disseminated carcinomatosis has been shown in animal experiments, its effectiveness against carcinomatous peritonitis remains to be examined. METHODS: A carcinomatous peritonitis model was made by inoculating murine hepatocellular carcinoma cells, MH134, into the peritoneal cavity of syngeneic C3H/He mice, resulting in production of massive malignant ascites without development of intraperitoneal solid tumors. Model animals were injected intraperitoneally with retroviruses carrying the herpes simplex virus thymidine kinase (HSV-tk) gene followed by ganciclovir (GCV) treatment. RESULTS: Retrovirus-mediated in vivo gene therapy with the HSV-tk/GCV system was shown to have a significant impact on survival of animals with carcinomatous peritonitis not only at an early stage, but also at an advanced stage. Furthermore, repeated injections of HSV-tk-carrying retroviruses significantly prolonged the survival of animals with carcinomatous peritonitis compared with a single injection protocol. When intraperitoneal administration of recombinant interleukin-2 (IL-2) was added to the HSV-tk/GCV system, levels of IL-1beta and IL-2 in malignant ascites were significantly increased, resulting in significantly reduced ascite volume and prolonged survival. CONCLUSIONS: Our results indicate the feasibility of retrovirus-mediated in vivo gene therapy with the HSV-tk/GCV system plus IL-2 treatment against carcinomatous peritonitis.

Assessment of efficiency and safety of adenovirus mediated gene transfer into normal and damaged murine livers.

BACKGROUND: When recombinant adenoviruses are infused directly into the circulation, transgene expression is almost completely restricted to the liver. AIMS: Efficiency and safety of adenovirus mediated gene transfer into damaged livers were examined in mice with liver cirrhosis or fulminant hepatitis. METHODS: Liver cirrhosis and fulminant hepatitis were induced by intraperitoneal administration of thioacetamide and D-galactosamine followed by lipopolysaccharide, respectively. Mice were infused with adenoviruses carrying the Escherichia coli beta-galactosidase gene, lacZ gene, into the tail vein. Transduction efficiency of the lacZ gene was estimated histochemically by X-gal staining and quantitatively using a chemiluminescent assay. Activation of adenovirus specific T cells and development of neutralising antibodies against adenovirus were also examined. RESULTS: Histochemical evaluation revealed that approximately 40%, 80%, and 40% of cells in normal, cirrhotic, and fulminant hepatitis livers, respectively, were stained blue using X-gal staining. Quantitative analyses revealed that levels of lacZ expression in cirrhotic livers were approximately 2.5-fold and sixfold greater than those in normal and fulminant hepatitis livers, respectively. Although transgene expression in fulminant hepatitis livers was significantly lower than that in normal livers, marked levels of transgene expression were achieved even in fulminant hepatitis livers. Significant adverse effects of adenoviruses were not observed in damaged livers. There were no significant differences in cellular or humoral immune responses to adenoviruses among animals with normal, cirrhotic, and fulminant hepatitis livers. CONCLUSIONS: Our results suggest that gene therapy with adenoviruses may be used efficiently and safely, even in patients with severe liver disease.

Electrochemotherapy for colorectal cancer with commonly used chemotherapeutic agents in a mouse model.

We examined here the usefulness of electrochemotherapy against colorectal cancer (CRC) using a mouse model. Electropermeabilization profoundly increased the sensitivity of murine CRC, Colon 26, and MC38 cells to bleomycin (BLM) but not to 5-fluorouracil (5-FU) or to cisplatin (CDDP) in vitro. In vivo experiments revealed that electrochemotherapy with 5-FU, CDDP, or BLM was much more effective against CRC compared with the treatment of the drug alone. Electrochemotherapy with BLM or CDDP exhibited profound antitumor effects on subcutaneously established CRC in mice, and complete tumor regression was observed in five and four of eight animals, respectively. Electrochemotherapy with 5-FU also had an impact on CRC development, and complete cure was observed in one of eight animals. Subsequent analyses revealed that electropermeabilization significantly increased intratumoral amounts of BLM and CDDP but not 5-FU. These results indicate that electrochemotherapy may be a promising treatment modality against CRC.

Particle-mediated gene transfer into murine livers using a newly developed gene gun.

Although particle-mediated gene transfer using gene gun technology has been applied for gene transfer into epidermis, applications of this technology to visceral tissues have not been well investigated. Although all helium gas-driven gene gun instruments have used macrocarriers to discharge DNA-coated microprojectiles so far, we used a newly developed gene gun instrument, in which a hammering bullet is used to discharge microprojectiles. With the gene gun, gold particles coated with lacZ expression plasmid were discharged to murine livers. LacZ expression was induced much more profoundly in the liver by particle-mediated gene transfer than by simple plasmid injection and electroporation-mediated gene transfer. LacZ expression was broadly and randomly distributed throughout the bombarded livers, indicating that particle-mediated gene transfer can induce transgene expression even at relatively distant areas from the surface of the bombarded tissue. Furthermore, although transgene expression was at its peak on day 2 after the bombardment, it was still detectable even on day 28. These results indicate that particle-mediated gene transfer with a newly developed gene gun may provide a new approach to gene therapy for human diseases.

A novel approach for inducing enhanced and selective transgene expression in hepatocellular-carcinoma cells.

Utility of the alpha-fetoprotein (afp) promoter for gene therapy against hepatocellular carcinoma (HCC) is limited because of the weak promoter activity. To circumvent this, the 5.1-kb 5;-flanking sequence of the human afp gene including the entire enhancer and silencer regions as well as the promoter region was employed for achieving strong, HCC-selective transgene expression. To thoroughly inhibit the promoter activity of the 5;-flanking sequence of the human afp gene, the afp 5;-flanking region was inserted downstream of the human interleukin-2 (il-2) gene controlled by the simian-virus-40 (SV40) early promoter. il-2-production ability of HCC cells transduced with the construct was significantly enhanced compared with that transduced with the same construct lacking the afp 5;-flanking region. Importantly, il-2 production of non-HCC cells was substantially inhibited by the addition of the afp 5;-flanking region to the construct. When the afp 5;-flanking region was inserted downstream of the human tumor-necrosis-factor-alpha (tnf-alpha) gene controlled by the retrovirus long-terminal-repeat (LTR) enhancer/promoter, tnf-alpha production ability of HCC cells was significantly enhanced and that of non-HCC cells was significantly suppressed compared with that transduced with the same construct lacking the afp 5;-flanking region. Our results indicated that the afp 5;-flanking region gave the enhanced HCC-selective activity to the non-tissue specific SV40 early promoter and LTR enhancer/promoter. It is essential for successful gene therapy to induce strong, target-cell-selective transgene expression. This novel strategy, therefore, may contribute to the establishment of HCC-selective cancer gene therapy.

Electrochemotherapy can eradicate established colorectal carcinoma and leaves a systemic protective memory in mice.

Mice bearing subcutaneously established colorectal carcinoma (CRC) were given intratumoral, intravenous or intraperitoneal injection of various doses of bleomycin (BLM), followed by the delivery of direct current, square wave electric pulses to the tumor. Approximately 50% of animals treated with electrochemotherapy with BLM had completely eradicated established CRC tumors. Importantly, it was shown that CRC-specific cytotoxic T lymphocytes were elicited in the spleens of cured animals, resulting in the protection of the rechallenge with CRC. These results indicate that electrochemotherapy with BLM is promising for the treatment of metastatic CRC as well as the original lesion.

Immunomodulation with FK506 around the time of intravenous re-administration of an adenoviral vector facilitates gene transfer into primed rat liver.

Although adenoviruses are an attractive vehicle for gene transfer into tissues including various tumors, in vivo adenoviral administration elicits a neutralizing antibody response which eliminates or substantially reduces the efficacy of subsequent treatments. Transiently immunosuppressive strategies at the time of initial adenoviral exposure have shown to prevent the formation of neutralizing antibodies and permit the successful adenoviral readministration in animals. Initial treatment in humans may, however, correspond to adenoviral readministration into animals, because the exposure to wild-type adenoviruses is common in humans. In the present study, we infused Adex1CAlacZ adenoviruses carrying the lacZ gene into the tail vein of rats, and examined whether a transient treatment with the immunosuppressant FK506 around the time of i.v. readministration of adenoviruses could induce the re-expression of the lacZ gene in animals primed with adenoviruses. Although i.v. infusion of adenoviruses carrying the lacZ gene resulted in transiently high levels of transgene expression in rat liver, i.v. reinfusion of adenoviruses failed to induce detectable levels of transgene expression. Conversely, when animals were treated transiently with FK506 around the time of adenoviral reinfusion, development of neutralizing antibodies and antigen-specific T cell proliferation in response to adenoviral reinfusion were significantly suppressed, and re-expression of the transgene was achievable.

Electrochemotherapy with bleomycin against colorectal carcinoma in a mouse model: evaluations of the dose and administration route of the drug and the electric field intensity.

Effectiveness of electrochemotherapy against colorectal carcinoma (CRC) was evaluated in a mouse model. When mice with a subcutaneously established CRC tumor were administered intratumorally, intravenously or intraperitoneally with bleomycin (BLM) ranging from 1/50 to 1/2 of the 50% lethal dose, significant suppression of tumor development and even some cures were observed. When various electric field intensities ranging from 500 to 2,000 V/cm were applied for electrochemotherapy with BLM, all treatment protocols were similarly effective. Furthermore, when electrochemotherapy with the lowest dose of BLM and the lowest electric field intensity was repeated, complete cures of CRC were achieved in all animals.

Comparison of gene therapy with the herpes simplex virus thymidine kinase gene and the bacterial cytosine deaminase gene for the treatment of hepatocellular carcinoma.

BACKGROUND: Bystander effects induced by suicide gene/prodrug systems play an essential role in achieving successful antitumor effects. Although it has been shown in several in vitro studies that the bacterial cytosine deaminase (CD) gene/5-fluorocytosine (5-FC) system is superior to the herpes simplex virus thymidine kinase (HSV-TK) gene/ganciclovir (GCV) system, we examined here which suicide gene system was more promising in vivo for the treatment of hepatocellular carcinoma (HCC). METHODS: BNL1ME A.7R.1 murine HCC cells were retrovirally transduced with the HSV-TK or CD gene, and bystander effects caused by the appropriate prodrug treatment were examined not only in vitro but also in vivo. RESULTS: The CD/5-FC system was superior to the HSV-TK/GCV system in HCC cell elimination in vitro. The bystander effect of the HSV-TK/GCV was shown to be substantially dependent on cell-to-cell contact, whereas that of the CD/5-FC was not. However, antitumor effects on HCC and tumor immunity to parental HCC induced by the HSV-TK/GCV system were not inferior and even superior to those induced by the CD/5-FC system. Bystander effects induced by the suicide gene/prodrug systems in immunocompetent syngeneic mice were much more profound than those induced in vitro. However, significant bystander effects were not observed in athymic nude mice. CONCLUSIONS: These results suggest that both HSV-TK/GCV and CD/5-FC systems are useful for the treatment of HCC. The results also suggest that T-cell-mediated immune responses elicited by the suicide gene/prodrug systems play a substantial role in antitumor effects in vivo.

Transient cyclophosphamide treatment before intraportal readministration of an adenoviral vector can induce re-expression of the original gene construct in rat liver.

Although adenovirus is an attractive vehicle for transferring therapeutic genes in vivo, animal studies have indicated that the clinical usefulness of adenoviruses may be limited by their immunogenicity. Although immunosuppressive strategies around the time of initial exposure of adenoviruses have been shown to prevent the formation of neutralizing antibodies and permit the successful readministration of adenoviruses in animals, the practicality of the approaches remains questionable. Because the majority of prospective gene therapy patients have already been infected with wild-type adenoviruses, initial treatment with adenoviruses in humans may correspond to readministration of adenoviruses into animals. It is shown here that although intraportal infusion of adenoviruses carrying a reporter lacZ gene resulted in transient high levels of transgene expression in the rat liver, intraportal readministration of adenoviruses failed to induce detectable levels of transgene expression. Conversely, when animals were treated transiently with cyclophosphamide before the intraportal readministration of adenoviruses, development of neutralizing antibodies and antigen-specific T cell proliferation in response to adenoviral readministration was significantly suppressed and successful re-expression of the transgene was achievable. These results may have important implications for efficacy considerations when adenoviral vectors are employed in clinical settings.

Cancer gene therapy with HSV-tk/GCV system depends on T-cell-mediated immune responses and causes apoptotic death of tumor cells in vivo.

To examine the immunological mechanisms involved in cancer gene therapy using the herpes simplex virus thymidine kinase (HSV-tk) gene and ganciclovir (GCV), murine hepatocellular carcinoma (HCC) cells, BNL1ME A.7R.1, were transduced retrovirally with the HSV-tk gene. HSV-tk-transduced cells exhibited a more than 2,000-fold higher sensitivity to GCV compared with untransduced parental cells. When HSV-tk-transduced HCC cells were mixed with parental cells at a 50% ratio and implanted subcutaneously into immunocompetent syngeneic mice, complete inhibition of tumor formation was achieved by GCV treatment. Conversely, no significant inhibitory effects on tumor formation were observed in athymic nude mice. When established solid tumors in immunocompetent mice containing HSV-tk-transduced cells at an only 5% ratio were treated with GCV, marked infiltration by lymphocytes including CD4(+) and CD8(+) ones, and apoptotic death of tumor cells were induced, and significant reduction or even complete regression of tumors was achieved. Furthermore, such cured mice rejected rechallenge with parental HCC cells into the contraflank regions. Our results indicate that cancer gene therapy with the HSV-tk/GCV system can indeed induce efficient antitumor effects and protective immunity in immunocompetent mice but not in nude mice, indicating that T-cell-mediated immune responses may be a critical factor for achieving successful gene therapy against cancer using the HSV-tk/GCV system.

Sonographic estimation of liver tumor development induced by oral administration of thioacetamide in rat.

Animal models for various types of cancer are of great help in the study of tumors and antitumor effects. Subcutaneous models have been widely utilized because they can be produced easily by subcutaneously implanting tumor cells into animals. Although subcutaneous models are very convenient for examining tumor development, they are definitely different from clinical manifestations of original tumors. In orthotopic animal models for internal tumors, however, it is difficult to examine tumor development without sacrificing animals. We demonstrate here that the occurrence and growth of liver tumors induced by oral administration of thioacetamide into rats were clearly observable by ultrasonography, and that the sonographic estimation was accurate. It was observed sonographically that the number and volume of liver tumors increased proportionately with TAA treatment periods. These results indicate that sonography is a useful and non-invasive method to investigate liver tumor development in rats.

Electrochemotherapy against colorectal carcinoma: comparison of in vitro cytotoxicity of 5-fluorouracil, cisplatin and bleomycin.

Effectiveness of electrochemotherapy against colorectal carcinoma (CRC) was investigated in vitro using murine CRC cell lines. Electropermeabilization did not increase the sensitivity of CRC cells to 5-fluorouracil or cisplatin. Conversely, electropermeabilization markedly increased the sensitivity of CRC cells to bleomycin (BLM), resulting in 1,000-fold higher susceptibility. Subsequent analyses revealed that electropermeabilization significantly increased intracellular BLM levels of CRC cells. These results suggest that electrochemotherapy with BLM is a promising modality for the treatment of CRC, and that electrochemotherapy can be translated from the treatment of superficial tumors to the treatment of internal tumors including CRC.

In vivo gene transfer of a suicide gene under the transcriptional control of the carcinoembryonic antigen promoter results in bone marrow transduction but can avoid bone marrow suppression.

We constructed the CEA419/CD retrovirus vector carrying the cytosine deaminase (CD) gene directed by the carcinoembryonic antigen (CEA) promoter. pCD2 retrovirus vector carrying the CD gene directed by the retrovirus long terminal repeat promoter was also used. When mice bearing intraperitoneally disseminated colorectal carcinomas (CRCs) were infused intraperitoneally with pCD2 or CEA419/CD retrovirus-producing cells, a CD fragment was detected in CRCs and bone marrow cells. It was shown that the CD gene was expressed both in CRCs and in the bone marrow of animals infused with pCD2 retrovirus-producing cells, while the CD gene was expressed solely in CRCs of animals infused with CEA419/CD retrovirus-producing cells. These results indicate that the use of a tumor-selective promoter may warrant the safety of in vivo gene therapy using suicide genes.

Effects of "body compression" on parameters related to ascites formation: therapeutic trial in cirrhotic patients.

Decreased effective circulating blood volume is an important factor in ascites formation in liver cirrhosis. We designed a "body compression" apparatus as a means to restore effective blood volume and investigated its effectiveness in reducing ascites formation in cirrhotics in terms of its effect on parameters of ascites formation noted below. The subjects, eight cirrhotics with ascites and eight cirrhotics without ascites were given spironolactone (50-75 mg/day) and furosemide (40-80 mg/day) while they received a diet containing 85 mEq of sodium per day. All four limbs and the lower abdomen were compressed with constant pressure [height (cm) divided by 13.6 mmHg] once, for 3h, using stroke rehabilitation splints, while patients lay supine. In cirrhotics both with and without ascites, urine volume, urinary sodium excretion, and creatinine clearance during the body compression were greater than values during control (non-compression) periods (urine volume, means 285 vs 169 ml/3h; P < 0.001, urinary sodium excretion 15.8 vs 9.5 mEq/3h; p < 0.001, creatinine clearance 74 vs 59 ml/min, P < 0.001, respectively). The increased basal plasma renin activity, angiotensin II, aldosterone, and norepinephrine levels in all cirrhotics were significantly decreased by the body compression. In another group of six cirrhotics who received no diuretics or albumin, repeat body compression alleviated ascites in three with well preserved renal function, but was ineffective in three with markedly impaired renal function. These results suggest that the improvement in renal function brought about by the body compression is attributable to an increase in effective circulating blood volume. This maneuver may be a useful complementary therapy in patients with cirrhotic ascites with well preserved renal function.

Hepatocellular carcinoma in an orthotopic mouse model metastasizes intrahepatically in cirrhotic but not in normal liver.

Prognosis of hepatocellular carcinoma (HCC) still remains poor mainly because of intrahepatic metastasis. In the majority of cases, HCC is found in conjunction with liver cirrhosis. It is, therefore, of great importance to investigate the invasive and metastatic behavior of HCC in cirrhotic liver. To examine this, a liver cirrhosis model was produced by injecting thioacetamide i.p. into mice. Murine HCC cells were labeled with the fluorescent carbocyanine dye, DiI, and implanted directly under the capsule of cirrhotic and normal livers of syngeneic mice. DiI-labeled HCC cells in the liver were observed under fluorescent and confocal microscopy. Histological analysis of cirrhotic and normal livers revealed that implanted HCC cells migrated to and invaded the adjacent periportal regions, but not the adjacent centrolobular areas. This characteristic behavior of HCC was more evident in cirrhotic liver than in normal liver. Furthermore, intrahepatic metastasis to unimplanted hepatic lobes was observed in cirrhotic liver as early as 7 days after implantation, while it was not detected in normal liver even 4 weeks later. Thus, an orthotopic animal model for HCC with cirrhosis described here may be suitable for investigating the invasive and metastatic behavior of HCC. Importantly, labeling tumor cells with a fluorescent dye before orthotopic implantation may be a convenient and useful method to investigate the invasive and metastatic behavior of various types of cancer.

Antitumor effect of electrochemotherapy on colorectal carcinoma in an orthotopic mouse model.

Electropermeabilization was shown to markedly increase the sensitivity of murine colorectal carcinoma (CRC) cells to bleomycin (BLM), resulting in more than 2,500-fold higher susceptibility to BLM. Subsequent in vivo electrochemotherapy with BLM revealed profound antitumor effects on subcutaneous CRC tumors. Furthermore, when electrochemotherapy with BLM was employed for the treatment of orthotopic CRC tumors in mice, significantly prolonged survival priods were observed. These results indicate the feasibility of electrochemotherapy with BLM for the treatment of CRC and demonstrate that electrochemotherapy can be translated from the treatment of cutaneous and subcutaneous tumors to the treatment of internal cancers including CRC.

Bystander effect caused by cytosine deaminase gene and 5-fluorocytosine in vitro is substantially mediated by generated 5-fluorouracil.

Because it appears impossible to transfer toxic genes to all the cells of a cancer, the bystander effect is critical to induce effective antitumor effects. In the present study, possible in vitro mechanisms of the bystander effect by the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) were investigated. CD-transduced cancer cells exhibited much higher sensitivity to 5-FC compared to parental cells. CD-transduced cells caused killing of neighboring parental cells in the presence of 5-FC, irrespective of direct cell-to-cell contact. Media conditioned by CD-transduced cells and 5-FC contained considerable amounts of 5-fluorouracil (5-FU) and exhibited profound cytotoxicity on parental cells. Furthermore, this killing ability of conditioned media correlated well with 5-FU levels converted from 5-FC by CD-transduced cells. CD was shown not to be secreted into media from cells. These results indicate that diffusible 5-FU plays the substantially causative role in the in vitro bystander effect caused by the CD/5-FC system.