Koutaro Yamamoto

Immunological evaluation of personalized peptide vaccination with gemcitabine for pancreatic cancer

The aim of the present study was to investigate the safety and immune responses of personalized peptide vaccination when administered with gemcitabine (GEM) in advanced pancreatic cancer (APC) patients. Thirteen patients with APC were enrolled. Pre‐vaccination with peripheral blood mononuclear cells and plasma was carried out to examine cellular and humoral responses to 25 or 23 peptides in human leukocyte antigen A24++ or A2+++ patients, respectively. Only the reactive peptides (maximum of four) were then administered weekly at three different dose settings: 1, 2 and 3 mg of peptide. GEM was administered at 1000 mg/m2 per week for 3 weeks, followed by 1 week of rest. The combination therapy was well tolerated. Grade 3 toxicities were: anemia (three patients), neutropenia (two patients) and thrombocytopenia (two patients). Of these 13 patients, 11 (85%) showed clinical responses, such as reduction in tumor size and/or level of tumor markers. Augmentation of peptide‐specific cytotoxic T lymphocyte activity against pancreatic cancer cells was observed at each dose level, whereas the increment of peptide‐specific IgG antibodies was dependent on peptide dose. GEM did not inhibit the immune responses induced by personalized peptide vaccinations, and this new type of immunochemotherapy combination is recommended for further clinical study in APC patients. (Cancer Sci 2007; 98: 605–611)

Detection of peptide‐specific cytotoxic T‐lymphocyte precursors used for specific immunotherapy of pancreatic cancer

The prognosis of pancreatic cancer is extremely poor with a 5‐year survival of approximately 3%. Thus, the development of new treatment modalities, including a specific immunotherapy, is required. Our study investigated whether cytotoxic T‐lymphocyte (CTL) precursors reacting to peptides with vaccine candidates (13 peptides for HLA‐A2+ or ‐A24+ patients, respectively) were detectable in the prevaccination peripheral blood mononuclear cells (PBMCs) of 15 pancreatic cancer patients. Peptide‐specific CTL precursors were detectable in the majority (11 of 15, 73%) of patients, with a mean positive number of 1.5 peptides (ranging from 0–5 peptides) per patient. Positive peptide profiles varied among patients. These results may provide a scientific basis for a new kind of cancer immunotherapy, namely, a CTL precursor‐oriented peptide vaccine, for pancreatic cancer patients.

The nm23-H1 gene as a predictor of sensitivity to chemotherapeutic agents in oesophageal squamous cell carcinoma

SummaryRecently, nm23-H1, an anti-metastasis gene, has been reported to correlate with sensitivity to chemotherapeutic agents including cisplatin in human breast and ovarian carcinoma cells. The aim of this study was to evaluate a role for nm23-H1 in responsiveness to cisplatin-based chemotherapy in patients with oesophageal squamous cell carcinoma (OSCC). The expression of nm23-H1 protein was examined immunohistochemically in 32 eligible patients with OSCC who underwent adjuvant chemotherapy with cisplatin, etoposide, and 5-fluorouracil after tumour resection. Fifteen (46.9%) of 32 patients were positive for nm23-H1 staining and 17 (53.1%) were negative. Both disease-free survival and overall survival rates of nm23-H1-negative patients were significantly shorter than in nm23-H1-positive patients (P < 0.01 for both). There was no significant difference in clinicopathologic characteristics between nm23-H1-positive and nm23-H1-negative groups. Multivariate analysis also showed that nm23-H1 expression was the most significant factor for overall survival of OSCC patients included in this study (P = 0.0007). To further study the role of nm23-H1, a human OSCC cell line (YES-2) was transfected with a plasmid containing a fragment of the nm23-H1 cDNA in an antisense orientation. Reduced expression of nm23-H1 protein in the antisense-transfected (AS) clones was found by Western blot analysis as compared to wild-type YES-2 and YES-2/Neo (clone transfected with the neomycin resistance gene alone). MTT (3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide) assay showed that reduced expression of the nm23-H1 protein in AS clones was consistent with the degree of increased resistance to cisplatin but not etoposide or 5-fluorouracil. These data support the conclusion that reduced expression of nm23-H1 may be associated with resistance to cisplatin, suggesting the value of nm23-H1 expression as a prognostic marker for OSCC patients who are to undergo cisplatin-based chemotherapy.

Successful immunogene therapy using colon cancer cells (colon 26) transfected with plasmid vector containing mature interleukin-18 cDNA and the Igκ leader sequence

IL-18 is a novel cytokine that induces interferon (IFN)-γ secretion and plays an important role in antitumor immunity. In the present study, we constructed plasmid vectors encoding the murine mature IL-18 cDNA linked with the Igκ leader sequence and the pro-IL-18 cDNA to estimate the efficacy of the mature IL-18 vector and to evaluate IL-18–producing tumor cells as a tumor vaccine. Colon 26 cells were transfected with the abovementioned vectors or with vector alone (mock). Reverse transcription-polymerase chain reaction analysis showed increased expression of murine IL-18 cDNA in both mature IL-18 and pro-IL-18 transfectants in comparison to that in mock transfected cells. The ability of the culture supernatants of mature IL-18 transfectants to induce IFN-γ secretion was extremely high (40–140 pg/106 cells) in comparison to that of pro-IL-18 transfectants (4–18 pg/106 cells). When injected into syngeneic BALB/c mice, the growth of mature IL-18 transfectants, but not pro-IL-18 transfectants, was significantly less than that in mock transfected cells (P<.01, by ANOVA and analysis of covariance). In addition, injection of colon 26 or Meth-A cells into mice immunized with a mature IL-18 transfectant revealed acquired immunity. Depletion of natural killer cells did not affect the growth of transfectants. However, the growth inhibitory effects were partially abrogated following treatment with anti-CD4+ and anti-CD8+ antibodies. These data suggest that the rejection of mature IL-18/colon 26 cells was mediated through T-cell activation. Gene therapy using mature IL-18 transfectants containing a plasmid vector and the Igκ leader sequence may be a useful tumor vaccine. Cancer Gene Therapy (2001) 8, 9–16

The development of a cell array and its combination with laser-scanning cytometry allows a high-throughput analysis of nuclear DNA content.

We developed a cell array device for analyzing cellular characteristics such as DNA ploidy, numerical chromosomal aberrations, and antigen expression in multiple specimens in a single experiment. Fifty (10 x 5) spots, 2 mm in diameter, were arrayed in an area of 30 x 16 mm on a glass slide, and approximately 1,000 cells were placed on each spot. To demonstrate the usefulness of the cell array, we measured nuclear DNA content using laser-scanning cytometry for DNA ploidy analysis in nine human tumor cell lines and normal lymphocytes. Combining the cell array with laser-scanning cytometry allows not only measurement of nuclear DNA content for 50 samples but also easy comparison of DNA ploidy among the samples in a single experiment. In addition, we used the cell array for fluorescence in situ hybridization using a DNA probe specific for the pericentromeric region of chromosome 11.

Laparoscopic resection of large leiomyomas of the gastric fundus

Two patients with a large leiomyoma arising from the gastric fundus underwent laparoscopic resection. In case 1, the tumor was located in the anterior wall of the gastric fundus. To prevent stenosis and preserve the volume of the residual stomach, intragastric resection was adopted. The tumor was markedly and resected with laparosonic coagulating shears with a 1-cm safety margin. In case 2, a large tumor was detected in the duodenal bulb. Serious hemorrhage mandated emergency resection. The tumor originated from the posterior wall of the fundus. Attempts at reduction with the forceps failed. Reduction by digital manipulation via laparoscopic port sites was successful. An endostapler was used to resect the tumor and close the anterior wall. Both patients recovered uneventfully.