[A Case of Spindle Cell Tumor in the Gastric Mucosa Treated with Laparoscopic Local Gastrectomy].

The patient was a 43-year-old man. An upper gastrointestinal endoscopic examination revealed a gastric submucosal tumor(SMT)-like, elevated 8-mm lesion in the greater curvature of the upper body of the stomach. It was diagnosed as spindle cell tumor on the basis of biopsy findings, and a gastrointestinal stromal tumor(GIST)was suspected. Various immunohistochemical staining techniques were used; however, a definitive diagnosis could not be achieved. There was no evidence of distant metastasis even on thoracoabdominal computed tomography imaging; thus, the patient was referred to our department for definitive diagnosis and surgical treatment. Laparoscopic local gastrectomy with concomitant intraoperative gastroscopy was performed. Pathological examination of the resected specimen showed a type Ⅱc-like lesion with a maximum diameter of 6 mm in the mucosal layer along with spindle cell proliferation. Immunostaining was negative for c- kit, DOG1, CD34, S-100, SMA, WT-1, desmin(N), EMA, and keratin(pan)and positive for ß-catenin, Bcl-2, and vimentin; furthermore, low Ki-67(MIB-1)expression was detected. Therefore, GIST, solitary fibrous tumor, leiomyoma, leiomyosarcoma, desmoid tumor, spindle cell carcinoma, and synovial sarcoma were excluded, and an unclassifiable spindle cell tumor arising from the gastric mucosa was diagnosed. The patient has remained recurrence-free for 1 year and 8 months post-operatively and is currently under careful outpatient follow-up.

[A Case of Large Cell Neuroendocrine Carcinoma of the Ampulla of Vater].

An 80-year-old women admitted to our hospital with jaundice. Abdominal contrast-enhanced CT scan revealed an enhanced tumor, measuring 10 mm, at the duodenal ampulla. Upper endoscopy showed a submucosal tumor-like lesion at the duodenal ampulla. Immunohistochemical findings showed positive for chromogranin A and synaptophysin, and neuroendocrine carcinoma was diagnosed. Subtotal stomach-preserving pancreaticoduodenectomy with regional lymph node dissection was performed. The final diagnosis was large cell neuroendocrine carcinoma(LCNEC). Multiple metastases of liver, lung and bone were occurred 14 months after the surgery, and she died 21 months after the surgery. LCNEC of the duodenal ampulla is very rare, and its prognosis is poor.

[A Case of Omental Desmoid Tumor after a Small Bowel Resection for Gastrointestinal Stromal Tumor].

An intra-abdominaldesmoid tumor, especially omentaldesmoid tumor, is rare. Here, we report a case of omentaldesmoid tumor after a smallbowelresection for gastrointestinalstromaltumor (GIST). A 73-year-old man underwent a partial resection of smallbowelfor GIST. He received adjuvant therapy with imatinib due to high risk of recurrence. After 2.5 years of treatment, a follow-up CT showed a 15mm nodule in the omentum near the splenic flexure. We considered the possibility of recurrence and imatinib failure, and laparoscopic tumor resection was performed for differential diagnosis. Immunohistochemicalstaining showed negative for c-kit, CD34, desmin, and S100. However, it was diagnosed as desmoid tumor because of positive b-catenin. Intra-abdominal desmoid tumor should be a differential diagnosis for a new single lesion in patients with GIST.

Tumor-induced CD11b+Gr-1+ myeloid cells suppress T cell sensitization in tumor-draining lymph nodes.

Suppression of tumor-specific T cell sensitization is a predominant mechanism of tumor escape. To identify tumor-induced suppressor cells, we transferred spleen cells from mice bearing progressive MCA205 sarcoma into sublethally irradiated mice. These mice were then inoculated subdermally with tumor cells to stimulate T cell response in the tumor-draining lymph-node (TDLN). Tumor progression induced splenomegaly with a dramatic increase (22.1%) in CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSC) compared with 2.6% of that in normal mice. Analyses of therapeutic effects by the adoptive immunotherapy revealed that the transfer of spleen cells from tumor-bearing mice severely inhibited the generation of tumor-immune T cells in the TDLN. We further identified MDSC to be the dominant suppressor cells. However, cells of identical phenotype from normal spleens lacked the suppressive effects. The suppression was independent of CD4(+)CD25(+) regulatory T cells. Intracellular IFN-gamma staining revealed that the transfer of MDSC resulted in a decrease in numbers of tumor-specific CD4(+) and CD8(+) T cells. Transfer of MDSC from MCA207 tumor-bearing mice also suppressed the MCA205 immune response indicating a lack of immunologic specificity. Further analyses demonstrated that MDSC inhibited T cell activation that was triggered either by anti-CD3 mAb or by tumor cells. However, MDSC did not suppress the function of immune T cells in vivo at the effector phase. Our data provide the first evidence that the systemic transfer of MDSC inhibited and interfered with the sensitization of tumor-specific T cell responses in the TDLN.

Induction of peptide-specific immune response in patients with primary malignant melanoma of the esophagus after immunotherapy using dendritic cells pulsed with MAGE peptides.

Primary malignant melanoma of the esophagus (PMME) is a very rare disease with an extremely poor prognosis. Surgery is currently considered its best treatment, while any other measures are ineffective. We studied the effect of active specific immunotherapy using monocyte-derived dendritic cells (DCs) pulsed with the epitope peptides of melanoma-associated antigens (MAGE-1, MAGE-3) in patients with PMME after surgery, for the first time. The patient received passive immunotherapy with lymphokine-activated killer cells concomitantly. Two HLA-A24-positive patients with PMME were treated. Both patients initially received radical esophagectomy with regional lymphadenectomy, followed by adjuvant chemotherapy with dacarbazine, nimustine, vincristine and interferon-alpha. In the case 1 patient, active specific immunotherapy was used to treat a large abdominal lymph node metastasis that became obvious 21 months after surgery. The disease remained stable for 5 months, and the patient survived for 12 months after the initiation of immunotherapy. In the case 2 patient, immunotherapy was tried as post-operative adjuvant treatment after adjuvant chemotherapy. There was no tumor recurrence for 16 months after the immunotherapy. As of 49 months after esophagectomy, the patient is still alive. In both patients, the ability of peripheral lymphocytes to produce IFN-gamma in vitro in response to peptide stimulation was significantly enhanced and delayed-type hypersensitivity skin test response to MAGE-3 peptide was turned positive after immunotherapy. In conclusion, active specific immunotherapy for PMME with the use of DCs and MAGE peptides was safe and capable of inducing peptide-specific immune responses. This case report warrants further clinical evaluation of this immunotherapy for PMME.

Induction of cytotoxic T lymphocytes against human cancer cell lines using dendritic cell-tumor cell hybrids generated by a newly developed electrofusion technique.

Recently, dendritic cells (DCs) and DC-tumor cell hybrids (DC-tumor hybrids) have been used for cancer vaccine therapy in a clinical trial. DC-tumor hybrids combine the potent antigen-presenting capacity of DCs with the ability to present all tumor antigens expressed on tumor cells to T cells. We used DC-tumor hybrids as stimulator cells to induce tumor-specific cytotoxic T lymphocytes (CTLs) in vitro. DC-tumor hybrids were generated from human monocyte-derived DCs and human cancer-cell lines (GT3TKB, lung cancer; GCIY, gastric cancer) by our newly developed electrofusion technique, established and refined with the use of mouse cells. To evaluate the capacity of DC-tumor hybrids generated by our method to induce tumor antigen-specific CTLs, we performed a cytotoxic assay and an interferon-gamma release assay using CD8-dominant effector lymphocytes induced by them. DC-tumor hybrids more effectively induced tumor-specific primary T-cell response than did stimulation with DCs co-cultured with irradiated tumor cells overnight, irradiated tumor cells alone, or a mixture of DCs and irradiated tumor cells. DC-tumor hybrids were generated at a high fusion rate by our electrofusion technique. When CTLs were induced by DC-tumor hybrids in vitro, the high fusion rate did not contribute to the induction of CTLs with increased tumor-specific cytotoxicity. The addition of interleukin-12 to the culture medium did not augment the cytotoxicity of CTLs. Overall, our results suggest that DC-tumor hybrids effectively induce human tumor-specific CTLs and may thus be applicable for clinical trials of adoptive immunotherapy.

Mature dendritic cells generated from patient-derived peripheral blood monocytes in one-step culture using streptococcal preparation OK-432 exert an enhanced antigen-presenting capacity.

Dendritic cells (DCs) have been shown to be potent in inducing cytotoxic T cell (CTL) response leading to the efficient anti-tumor effect in active immunotherapy. Myeloid DCs are conventionally generated from human peripheral blood monocytes in the presence of interleukin (IL)-4 and granulocyte/macrophage colony-stimulating factor (GM-CSF). Streptococcal preparation OK-432, which is known to be a multiple cytokine inducer, has been extensively studied as to its maturation effects on immature DCs using an in vitro culture system. The purpose of this study was to examine whether it could be possible to generate mature DCs directly from peripheral monocytes using OK-432. We specifically focused on the possibility that recombinant cytokines, which are considered to be essential for in vitro DC generation, could be substituted by OK-432. Human peripheral monocytes, which were obtained from patients with advanced cancer, were cultured with IL-4 and OK-432 for 7 days. Cultured cells were compared with DCs generated in the presence of IL-4 and GM-CSF with or without OK-432 with regard to the surface phenotype as well as the antigen-presenting capacity. As a result, the culture of monocytes in the presence of IL-4 followed by the addition of OK-432 on day 4 (IL-4/OK-DC) induced cells with a fully mature DC phenotype. Functional assays also demonstrated that IL-4/OK-DCs had a strong antigen-presenting capacity determined by their enhanced antigen-specific CTL response and exerted a Th1-type T cell response which is critical for the induction of anti-tumor response. In conclusion, human peripheral blood monocytes cultured in the presence of IL-4 and OK-432 without exogenous GM-CSF demonstrated a fully mature DC phenotype and strong antigen-presenting capacity. This one-step culture protocol allows us to generate fully mature DCs directly from monocytes in 7 days and thus, this protocol can be applicable for DC-based anti-tumor immunotherapy.

[The effects of direct hemoperfusion using the filtration column filled with the adsorption fiber for immunosuppressive substances on cell-mediated immunity in tumor-bearing rats].

The patients with advanced cancer often lose their anti-tumor immune responses due to the increase of some immunosuppressive substances in the blood, such as cytokines and proteins derived from cancer cells or immune cells. We developed the adsorption fiber in transforming growth factor (TGF)-beta for the treatment to remove immunosuppressive substances and investigated the effects of direct hemoperfusion using the filtration column filled with this adsorption fiber in tumor-bearing rats. On day 0, KDH-8 tumor cells (1 x 10(6) cells/rat) were implanted subcutaneously into the back of WKAH/Hkm rats. On day 21 after tumor implantation, the rats underwent the direct hemoperfusion with this filtration column for 60 minutes. On day 28, the rats were sacrificed and the natural killer (NK) activities of their spleen cells were examined. As a result, the rats that underwent this treatment showed a significant increase in their NK activities compared with those of rats who underwent direct hemoperfusion with an empty column or had no treatment. Therefore, we indicated the possibility of a new immunotherapy technique against cancer using a direct hemoperfusion column filled with an adsorption fiber for immunosuppressive substances.

[Immunotherapeutic reactivity of dendritic cells loaded with a variety of antigen preparations].

The findings summarized here provide a direct comparison of the immunogenicity of various DC loading strategies included pulsing with protein, peptide, tumor cell lysate, irradiated tumor cells and electrofusion of DCs and tumor cells. For the treatment of 3-day established pulmonary metastases, electrofusion of DCs and tumor cells generated a therapeutic vaccine far superior to other methods of DC loading. Consistent with their therapeutic activity, fusion hybrids stimulated the release of the largest amount of interferon-gamma from immune T cells. However, IL-10 secretion did not correlate with in vivo therapeutic reactivity. In conclusion, DC-tumor fusion hybrids were the most effective vaccine to eradicate existing tumors. These data support the use of DC-tumor electrofusion cells for the treatment of human cancer.

[Two case reports of complete regression of liver metastases from colorectal cancer after locoregional immunochemotherapy].

Hepatic arterial infusion (HAI) with pharmacokinetic modulating chemotherapy (PMC) has been well known to be one of the most effective protocols for unresectable liver metastases from colorectal cancer. PMC is a combination of oral UFT and continuous hepatic arterial 5-FU infusion. We present herein the cases of two patients with multiple liver metastases from colorectal cancer in whom complete regression (CR) was achieved by HAI with PMC in combination with Lentinan (immunostimulator). These patients received HAI via an implantable port system with a 4-24-hour continuous perfusion of 5-FU at 1,000 mg/m2 plus Lentinan at 2 mg/body once a week, and oral administration of UFT at 200-300 mg/m2/day everyday. CR of all metastatic lesions in the liver was achieved 4 months after the initiation of the treatment in both patients. One patient maintained CR for 3 months, but he died due to a recurrence of liver metastases and peritoneal dissemination 19 months after the initiation of the treatment. The other patient has been well without recurrence for 21 months. Because the liver is the largest immunologic organ, Lentinan could have activated lymphocytes and macrophages in the liver. Judging from the clinical experience of these two cases, HAI with PMC in combination with Lentinan could be one of the most promising treatment strategies for unresectable liver metastases from colorectal cancer.

Effect of hospital volume on long-term outcomes of laparoscopic gastrectomy for clinical stage I gastric cancer.

BACKGROUND: This study aimed to examine the effect of hospital volume on long-term outcomes of patients who underwent laparoscopic gastrectomy for clinical stage I gastric cancer. PATIENTS AND METHODS: A total of 420 patients with clinical stage I gastric cancer who underwent laparoscopic gastrectomy at our university hospital (high-volume group) and affiliated hospitals (low-volume group) were included in this study. Overall survival (OS) and cause-specific survival (CSS) rates were analyzed. RESULTS: No significant differences were observed in the number of lymph nodes retrieved (29.9 vs. 27.7, p=0.21) and CSS between the high- and low-volume groups (p=0.92), although the OS rate in the low-volume group was significantly less than that in the high-volume group (p=0.045). CONCLUSION: These results indicate no clinical impact of hospital volume on prognosis of patients who underwent laparoscopic gastrectomy for clinical stage I gastric cancer when performed by surgeons with sufficient experience in open gastrectomy.

Effective treatment of spontaneous metastases derived from a poorly immunogenic murine mammary carcinoma by combined dendritic-tumor hybrid vaccination and adoptive transfer of sensitized T cells.

Curative immunotherapy against spontaneous metastases of poorly immunogenic tumors has been difficult to demonstrate, but it is highly relevant to clinical disease conditions. The 4T1 mammary carcinoma shares many characteristics of human mammary cancer. Here, mice with 4T1 spontaneous metastases were treated effectively with a combination of dendritic (DC)-tumor hybrid vaccination and adoptive transfer of tumor-draining lymph node-derived immune T cells. This strategy significantly prolonged survival and cured some mice. In this model, the combined immunotherapy induced a dramatic increase of T cells in the lung where metastases were located and in the spleen where tumor was not present. The mechanism of increasing numbers of T cells is likely attributed to the ability of DC-tumor hybrids to stimulate vigorous proliferation of adoptively transferred T cells rather than to promote their infiltration into tumor-harboring and lymphoid organs. Taken together, the combined approach may be useful for clinical development of cancer immunotherapy.

Immunotherapy of established murine squamous cell carcinoma using fused dendritic-tumor cell hybrids.

OBJECTIVE: To investigate the therapeutic efficacy of fused dendritic-tumor cell hybrids against murine squamous cell carcinoma (SCC). DESIGN: Squamous cell carcinoma VII is a poorly immunogenic murine SCC tumor in C3H/HEN (H-2(K)) mice. Subdermal tumors were established by inoculation in the mid abdomen of mice. Tumor diameters were measured with a Vernier caliper and used as an indication of treatment efficacy. Survival studies were performed on mice with 3-day pulmonary metastasis or subdermal tumors. Dendritic cells were generated from bone marrow and cultured for 8 days. Dendritic cells were harvested and mixed with cultured tumor cells in a 1:1 ratio. Cell fusion was achieved by exposing the cell mixture to an alternate electrical current to bring cells into alignment and close together, followed by a short direct electrical current pulse. SUBJECTS: Female C3H/HEN mice aged 8 to 12 weeks. INTERVENTIONS: Mice with 3-day established SCCVII tumors were vaccinated by inguinal intranodal injection of fusion cells (0.3 x 10(6) per side). To support the development of antitumor immunity, mice were given adjuvant injections intraperitoneally. Anti-OX40R monoclonal antibodies or interleukin 12 were used. Treatment groups included no treatment, anti-OX40R monoclonal antibodies or adjuvant IL-12 alone, fusion cells alone, and fusion cells with adjuvant treatment. MAIN OUTCOME MEASURES: Tumor size and overall survival. RESULTS: Mice treated with adjuvant treatment or fusion cells alone did not show a statistical difference in tumor growth when compared with controls. In contrast, fusion cells with adjuvant treatment demonstrated a significant decrease in tumor size when compared with nontreated mice (P < .001). Treatment with fusion cells also resulted in increased survival in the pulmonary metastasis and subdermal tumor models. CONCLUSION: Immunotherapy with fused dendritic-tumor cell hybrids can significantly affect 3-day established sSCC VII tumor growth.

RAD inhibition of sarcoma growth: implications for laryngeal transplantation.

PURPOSE: Laryngeal transplantation has not been widely accepted because of concerns regarding accelerated tumor recurrences in the setting of nonspecific immunosuppression. Allotransplantation could potentially be offered to patients if immunosuppressive therapy could be demonstrated to exert tumor suppressive properties. Preliminary reports have demonstrated an antiproliferative effect of everolimus (RAD), a derivative of the immunosuppressant rapamycin. MATERIALS AND METHODS: Forty-five 10-week-old inbred C57BL/6N (B6) mice were injected subcutaneously with 1 x 10(6) MCA205 sarcoma cells. On the third postinoculation day, the mice were divided into 4 treatment groups, undergoing daily gavage with RAD at 0, 0.2, 1.0, and 5.0 mg/kg per day for 10 consecutive days. Thereafter, treatment with RAD was discontinued and tumor size was measured every 2 days during treatment and biweekly until sacrifice on the 31st postinoculation day. Whole-blood trough levels (C(min)) were measured for each group. RESULTS: Mean tumor diameter among the control animals and the mice treated with RAD 0.2 mg/kg per day demonstrated no significant difference (P > .07). Groups treated with RAD 1 and 5 mg/kg per day demonstrated significant growth inhibition between the 7th and the 23rd postinoculation days (P < .0001), with no significant differences being noted between these two groups (P > .09). Mean tumor suppressive whole-blood C(min)'s for the 1 and 5 mg/kg per day groups were 75.6 and 368.9 pg/microL, respectively. CONCLUSIONS: RAD delivered at immunosuppressive doses of 1 and 5 mg/kg per day resulted in significant growth restriction of a fibrosarcoma in a murine model.

Mechanism of third signals provided by IL-12 and OX-40R ligation in eliciting therapeutic immunity following dendritic-tumor fusion vaccination.

Dendritic-tumor heterokaryons generated by electrofusion are highly immunogenic. In animal studies, a single vaccination was therapeutic for tumors established in the lung, skin, and brain. However, effective therapy required a third signal which could be provided by exogenous IL-12 or the agonistic anti-OX-40R monoclonal antibody (mAb). In this study, we investigated the mechanism and mode of actions of these two seemingly distinct adjuvants. In immunotherapy of the MCA205 sarcoma, administration of the neutralizing anti-IL-12 mAb nearly completely blocked the adjuvant effect of IL-12, but had minimal inhibitory effects on anti-OX-40R mAb. By contrast, in vivo administration of the antagonistic anti-OX-40L mAb inhibited the adjuvant effects of both IL-12 and anti-OX-40R mAb. Thus, a common pathway of endogenous OX-40 interaction is critical for the development of a therapeutic immune response. Analysis of the third signal mechanism revealed that in the absence of an adjuvant, vaccination with fusion hybrids led to IL-10 production without eliciting IFN-gamma secreting cells. The addition of IL-12 to vaccination suppressed IL-10 production and initiated sensitization of specific IFN-gamma secreting cells, resulting in a type 1-like antitumor immunity. These findings underscore the significance of the third signal in the design of dendritic cell-based cancer vaccines.