Yuzo Fujii

Pilot study for preoperative administration of 1-OHP to patients with advanced scirrhous type gastric cancer

A new DACH platinum complex, l-OHP, was developed by Kidani as an anticancer agent. A clinical trial took place in Europe which demonstrated its therapeutic efficacy for colorectal cancer. An effective treatment, especially chemotherapy for patients with a advanced scirrhous type gastric cancer, has not yet been established. An in vitro study showed that l-HOP inhibited cell growth in human gastric cancer cell lines. Our pilot study determined the efficacy of preoperative administration of l-OHP, 67 mg/m2 to 100 mg/m2, every 2-3 weeks, for two to three cycles, in five patients with this disease (Stage III and IV) roentogenoscopically and histologically. The platinum concentration in the tissues was also measured. By X-ray examination of the stomach at the time of pre- and post-administration of l-OHP, extension of the lesional gastric wall was observed. Histologically three Grade 2 responses and two Grade 1a responses were obtained according to the criteria presented by Japanese Research Society for Gastric Cancer. The mean platinum concentrations in the lesional tissues were 0.98 ppm and 0.5 ppm in the patients administered l-OHP for three and two cycles respectively. There was no toxicity that prevented surgery. These preliminary results showed the possibility that 1-OHP would be effective for patients with advanced scirrhous type gastric cancer as a neoadjuvant therapy.

Alpha-fetoprotein-producing gastric carcinoma: Biological properties of a cultured cell line

We describe a gastric carcinoma cell line that has been maintained in vitro for more than 10 years and retains the capacity to produce a large amount of alpha-fetoprotein. This cell line was isolated from a metastatic lymph node of a 63-year-old male patient with advanced gastric carcinoma (T2N3P0H0M0) who showed high serum levels of alpha-fetoprotein. The primary tumor was moderately differentiated tubular adenocarcinoma and the lymph node was poorly differentiated adenocarcinoma without any particular pattern. The cultured cells grew as densely packed islet-like colonies with small polygonal cells. Electron microscopy revealed cells abundant in cytoplasmic organelles, with some cellular attachments being tight with junctional complexes and some being loose across intercellular spaces. The free cell surface had microvilli. The population doubling-time was 152 h at passage 58. Chromosomal analysis revealed the modal number to be 77, with numerous karyotype abnormalities. The tumorigenicity of the cultured cells in athymic nude mice was positive only when they were subcutaneously transplanted beneath a plastic plate, but when the cells were transplanted subcutaneously or administered by intrasplenic injection in intact or weakly irradiated nude mice, no tumorigenicty was shown. The cell line produced tumor-associated antigens, such as alpha-fetoprotein, carcinoembryonic antigen, and tissue polypeptide antigen. This cell line may be useful for comparative studies of different types of gastric carcinoma and alpha-fetoproteins of different origins.

Surgery in patients with HIV infection : indications and outcome

We reviewed the records of 12 patients with HIV infection (one stage I, three stage II, two stage III, six stage IV) who received 15 surgical procedures under general or lumbar/epidural anesthesia. We discussed surgical indications, their poor wound healing and precautions for preventing the risk of transmission of HIV to health care workers. Six emergency and nine elective operations were performed. Postoperative complications developed after three emergency and three elective operations. Ten patients showed delay of wound healing which was not directly correlated with the CD4+ cell count. No operative deaths occurred. In any stage of HIV infection, not only palliative but also curative operations can be performed as long as HIV infection, opportunistic infections and HIV-related neoplasms can be controlled. Late stage wound healing is poor, but the wound will heal without keloid formation, although it takes two to three times longer than usual. For operating on patients with HIV infection precautions for preventing needle sticks, sharp injuries and blood exposure should be learned and used by health care workers. As a result, surgical staff members will be able to perform operations safely on HIV-infected patients to improve both quality of life and the prognosis of their disease.

A targeting model of boron neutron-capture therapy to hepatoma cells in vivo with a boronated anti-(alpha-fetoprotein) monoclonal antibody.

We described previously that10B atoms delivered by monoclonal antibody (mAb) exerted a cytotoxic effect on AH66 cells in a dose-dependent manner upon thermal neutron irradiationin vitro. In the present study, the delivering capacity of boronated anti-(α-fetoprotein) (AFP) mAb to carry10B atoms to AFP-producing tumor xenografts in nude mice was determined. Boronated mAb was prepared by conjugating 50 mM10B compound to an anti-AFP mAb (2 mg/ml) usingN-succinimidyl-3-) (2-pyridyldithio) propionate. The number of10B atoms conjugated directly to the mAb was estimated to be 459/antibody by prompt γ-ray spectrometry. Boron concentrations in tumor tissue obtained 12, 24, 72, and 120 h after injection of 3.0 mg10B-conjugated anti-AFP mAb were 11.10±3.12 (SD,n=6), 29.30±5.11, 33.02±11.8, and 12.91±5.62 ppm respectively. For control10B-conjugated anti-dinitrophenol (DNP) mAb, the values were 9.59±0.99, 10.37±2.86, 10.00±2.95, and 8.83±4.71 ppm respectively. The concentrations in blood were less than 0.40±0.10 ppm with anti-AFP mAb and less than 0.51±0.15 ppm with anti-DNP mAb at each sampling time (12, 24, 72, and 120 h). The number of10B atoms delivered to the tumor cells was calculated to be 0.62×109, 1.63×109, 1.84×109 and 0.72×109 at each sampling time after injection of10B-anti-AFP mAb. The amount of10B atoms necessary for effective boron neutron-capture therapy was estimated to be 109/tumor cell. We were able to carry 1.84×10910B atoms to AH66 tumor cells by using10B-anti-AFP mAb. The accumulation reached its peak 72 h after injection. These data indicated that the10B-conjugated antitumor mAb could deliver a sufficient amount of10B atoms to the tumor cells to induce cytotoxic effects 72 h after injection upon thermal neutron irradiation.

Establishment and Characterization of BALB/c Retroperitoneal Sarcoma with Spontaneous Liver Metastases

The objective of this study was to examine the identity and characteristics of a spontaneously occurring murine retroperitoneal tumor of BALB/c mouse origin that selectively metastasized to the liver. From the primary tumor, a permanent cell line, termed LMFS (liver metastasis from sarcoma) was established in vivo and in vitro. After a subcutaneous injection of more than 1 × 105 cells in the side back of mice, the LMFS cells proliferated at the inoculation site (100% take) and induced metastatic nodules spontaneously in the liver, but not in the lung. By the limiting dilution technique, a cloned cell line, LMFS‐1, was established in vitro. The LMFS‐1 cell line had similar morphological characteristics to the LMFS cells both in vitro and in vivo. The doubling time of the LMFS‐1 cell line was 10 h in passage 60. The number of chromosomes ranged from 71 to 108 and 93% of metaphases showed near‐tetraploidy. In microscopic examination, no specific arrangement of the LMFS tumor cells was seen; the LMFS cell had medium‐ to large‐sized atypical nuclei and clear and large cytoplasm. Electronmicroscopy showed that the cytoplasm of the LMFS cell had a moderate amount of rough‐surfaced endoplasmic reticulum but no desmosomes or microvilli. Immunohistochemically, the LMFS cells were positive for vimentin, but showed no reaction for keratin or cytokeratin. Therefore, the LMFS tumor was considered to be an undifferentiated sarcoma. The LMFS cell line should be a useful tool not only for studies of metastasis, but also for experiments on the therapy of hepatic tumors.

Inhibition of liver metastases and tumor cell invasion in spontaneous liver metastasis model (LMFS) by sodium D-glucaro-δ-lactam (ND2001)

We examined the inhibitory effect of sodium D-glucaro-delta-lactam (sodium 5-amino-5-deoxy-D-glucosaccharic acid-delta-lactam: ND2001) upon liver metastases of the LMFS tumor. A permanent cell line, LMFS, was established from a spontaneously occurring murine retroperitoneal tumor of BALB/c mouse origin, and after a subcutaneous injection, the LMFS cells proliferated at the inoculation site (100% take) with liver metastases. ND2001 had little effect on the cell growth, cell cycle and phagokinesis of the LMFS cells in vitro. However, when the invasive activity was measured by the Boydem chamber method, the number of LMFS cells was reduced, with inhibition rates of 98.0%. After the LMFS cells treated with ND2001 in vitro, the numbers of hepatic metastases of subcutaneous inoculation of treated cells were reduced dose-dependently, and those of intravenous inoculation were not found by microscopical study. When the LMFS tumor-bearing mice were treated with ND2001 (0, 30, 100 mg/kg/d) from day 1, ND2001 (30 mg/kg) inhibited the liver metastases with a rate of 56.4%, and when given from day 15, ND2001 (100 mg/kg) inhibited with a rate of 47.5%. But ND2001 showed neither cytocidal nor anti-tumor activity. Combination therapy of primary tumor resection and ND2001 administration revealed that preoperative use of ND2001 was more effective in preventing liver metastases. These results suggested that ND2001 might have a potential use as an anti-metastatic agent for operative patients without metastasis.

Application of Boronated Anti-Cea Immunoliposome to Boron Neutron Capture Therapy

Boron neutron capture therapy (BNCT) is based on irradiation of a tumour with thermal neutrons after accumulation of a large amount of boron-10(10B) atoms on the target tumour cells. 10B captures neutrons and produces α- and 7Li particles through the reaction 10B(n,α)7Li. This reaction is very efficient in cell killing.1 BNCT has been applied to the treatment of malignant brain tumours or melanoma by using 10B- compounds selectively taken up by tumour cells.2 Alam et al reported that 1300 boron atoms had been conjugated to a molecule of monoclonal antibody.3 However, in these experiments, antibody was shown to lose activity by the direct conjugation with 10B-compound.

Characterization of a murine monoclonal antibody, 5D-4, reacting with pancreatic cancers and islets of Langerhans

A murine monoclonal antibody (5D-4) was prepared by immunizing mice ip with human pancreatic cancer cell line (AsPC-1). The 5D-4 MAb reacted immunohistochemically with pancreatic and gastrointestinal tract tumors as well as pancreatic islets, and to a less extent with normal tissues. The 5D-4 MAb reacted not only with ca 50 KDa and 30 KDa solubilized protein from AsPC-1 cells by Western blot analysis but also with human insulin in a competition RIA. Double immunoperoxidase staining showed that the 5D-4 MAb cross-reacted with insulin but did not react with glucagon, somatostatin or pancreatic polypeptide. Immunoelectron micrograph of Langerhans island double-stained with the 5D-4 MAb and anti-insulin Ab revealed that the 5D-4 Mab recognized human insulin and ca 50 KDa and 30 KDa antigens in B-cells of islets of Langerhans. Thus, the 5D-4 Mab may be useful for the diagnosis of islet cell tumors as well as pancreatic cancers.

Surgical indications for HIV-infected patients

We proposed surgical indications for major operations on HIV-infected patients. According to our criteria, major surgery could be performed for HIV-infected patients as long as the surgical approach is preferred and surgery is not likely to exacerbate the subjects quality of life during the course of the disease.

Delivery of 10B atoms for effective boron neutron capture therapy using boronated polyethylene glycol-binding BSA

The cytotoxic effect of boron neutron capture therapy (BNCT) is due to the nuclear reaction between 10B and thermal neutrons. Recently, polyethylene glycol (PEG) has attracted attention due to its ability to avoid uptake by reticulo- endothelial systems, and to accumulate in the tumor cells. We prepared boronated PEG-binding bovine serum Albumin (BSA). Prompt g-ray spectrometry showed that 250.0 +/- 4.9 ppm 10B atoms were found to conjugate to PEG-BSA. 10B concentrations in AsPC-1, human pancreatic cancer cells obtained 0, 9, 24 hrs after incubation with 10B- PEG-BSA were 0, 4.97 +/- 0.49, and 13.01 +/- 1.74 ppm respectively. It is said that the 10B concentrations between 15 and 30 ppm are necessary for effective boron neutron-capture therapy. These data indicated that the 10B-PEG-BSA were getting close to the lowest acceptable limit of 15 ppm, and when using Na210B20H18 and increasing the 10B binding site of the BSA, we could increase the 10B uptake levels in the tumors for BNCT.