You Kawarada

CD8+ tumor-infiltrating lymphocytes together with CD4+ tumor-infiltrating lymphocytes and dendritic cells improve the prognosis of patients with pancreatic adenocarcinoma.

Objective Recent studies have demonstrated the importance of tumor immunity for a cancer patients prognosis. In some types of cancer, it has been shown through immunohistochemical analysis that the existence of CD8+ tumor-infiltrating lymphocytes (TILs) is a crucial factor in determining prognosis. In an experimental model, CD4+ lymphocytes together with CD8+ lymphocytes contributed significantly to tumor immunity. Methods Specimens were taken from 80 surgically resected pancreatic adenocarcinomas between 1992 and 1999. Immunohistochemical staining of CD4, CD8, and S100 protein was performed, and the levels of these proteins were determined by microscopic analysis. The percentages of patients in the CD4(+) and CD8(+) groups were 59% (47/80) and 25% (16/80), respectively. When separated into 4 groups, CD4/8(+/+), CD4/8(+/−), CD4/8(−/+) and CD4/8(−/−), the overall survival rate was significantly higher in CD4/8(+/+) patients (13 cases) compared with those in all other groups combined (67 cases; P = 0.0098). CD4/8(+/+) status was negatively correlated with tumor depth and TNM stage. Multivariate analyses showed that CD4/8(+/+) status was an independent favorable prognostic factor. The number of tumor-infiltrating S100 protein positive cells was also significantly higher in the CD4/8(+/+) group than in others (P = 0.0084). Conclusions In pancreatic adenocarcinoma, the presence of CD4+ TILs together with CD8+ TILs serves as a good indicator of the patients outcome after surgical treatment.

Liposome-encapsulated CpG oligodeoxynucleotides as a potent adjuvant for inducing type 1 innate immunity.

Unmethylated cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODNs) exhibit potent immunostimulating activity by binding with Toll-like receptor 9 (TLR9) expressed on antigen-presenting cells. Here, we show that CpG-ODN encapsulated in cationic liposomes (CpG-liposomes) improves its incorporation into CD11c+ dendritic cells (DCs) and induces enhanced serum interleukin (IL)-12 levels compared with unmodified CpG-ODN. CpG-liposome potently activated natural killer (NK) cells (84.3%) and NKT cells (48.3%) to produce interferon-γ (IFN-γ), whereas the same dose of unmodified CpG-ODN induced only low numbers of IFN-γ–producing NK cells (12.7%) and NKT cells (1.6%) to produce IFN-γ. In contrast with the NKT cell agonist α-galactosylceramide, which induces both IFN-γ and IL-4 production by NKT cells, CpG-liposome only induced IFN-γ production by NKT cells. Such potent adjuvant activities of CpG-liposome were absent in TLR9-deficient mice, indicating that CpG-liposome was as effective as CpG-ODN in stimulating type 1 innate immunity through TLR9. In addition to TLR9, at least two other factors, IL-12 production by DCs and direct contact between DCs and NK or NKT cells, were essential for inducing type 1 innate immunity by CpG-liposome. Furthermore, ligation of TLR9 by CpG-liposome coencapsulated with ovalbumin (OVA) caused the induction of OVA-specific CTLs, which exhibited potent cytotoxicity against OVA-expressing tumor cells. These results indicate that CpG-liposome alone or combined with tumor antigen protein provides a promising approach for the prevention or therapy of tumors.

Expression of Pigment Epithelium-Derived Factor Decreases Liver Metastasis and Correlates with Favorable Prognosis for Patients with Ductal Pancreatic Adenocarcinoma

Pigment epithelium-derived factor (PEDF) is expressed in several normal organs and identified as an inhibitor of neovascularization. In the present study, we screened the expression of PEDF immunohistochemically and investigated its correlation with clinicopathological features in patients who underwent surgery for ductal pancreatic adenocarcinoma. Of the 80 patients, 22 cases (27.5%) were positive for PEDF. A significant association was found between the PEDF expression and low microvessel density (P = 0.0003). No correlation was found between PEDF expression and age, gender, depth of invasion, tumor diameter, lymphatic invasion, venous, invasion or histopathological grading. The patients in pathological stage II had a significantly higher incidence of PEDF-positive expression than those in pathological stage III or IVA (P = 0.0418). PEDF immunoreactivity was inversely associated with liver metastasis (P = 0.0422). The survival of patients that were PEDF positive was significantly longer than that of those with negative expression (P = 0.0026). Multivariate analysis using the Cox regression model indicated that PEDF-positive expression was an independent favorable prognostic factor (risk ratio, 0.394; P = 0.0016). We conclude that PEDF expression suggests a more favorable prognosis than in patients whose carcinomas lack PEDF expression.

Generation and Targeting of Human Tumor-Specific Tc1 and Th1 Cells Transduced with a Lentivirus Containing a Chimeric Immunoglobulin T-Cell Receptor

CD4+ Th cells, in particular IFN-γ-producing Th1 cells, play a critical role in the activation and maintenance of Tc1 cells that are essential for tumor eradication. Here, we report the generation of artificial tumor-specific Th1 and Tc1 cells from nonspecifically activated T cells using a lentiviral transduction system. Anti-CD3-activated T cells from healthy human donors were transduced with a lentivirus containing a chimeric immunoglobulin T-cell receptor gene composed of single-chain variable fragments derived from an anticarcinoembryonic antigen (CEA)-specific monoclonal antibody fused to an intracellular signaling domain derived from the cytoplasmic portions of membrane-bound CD28 and CD3ζ. These artificial tumor-specific Tc1 and Th1 cells, termed Tc1- and Th1-T bodies, respectively, could be targeted to CEA+ tumor cells independently of MHC restriction. Specifically, Tc1-T bodies demonstrated high cytotoxicity and produced IFN-γ in response to CEA+ tumor cell lines but not CEA− tumors. Although Th1-T bodies exhibited low cytotoxicity, they secreted high levels of IFN-γ and interleukin-2 in response to CEA+ tumor cells. Such CEA+ tumor-specific activation was not observed in mock gene-transduced nonspecific Tc1 and Th1 cells. Moreover, Tc1- and Th1-T bodies exhibited strong antitumor activities against CEA+ human lung cancer cells implanted into RAG2−/− mice. Furthermore, combined therapy with Tc1- and Th1-T bodies resulted in enhanced antitumor activities in vivo. Taken together, our findings demonstrate that Tc1- and Th1-T bodies represent a promising alternative to current methods for the development of effective adoptive immunotherapies.

Neoadjuvant imatinib in a gastrointestinal stromal tumor of the rectum: Report of a case

Gastrointestinal stromal tumors (GISTs) are rare tumors of the gastrointestinal tract, and of these, GISTs involving the rectum are uncommon. This report describes a case of effective neoadjuvant therapy for a rectal GIST expressing the c-kit gene, where a laparoscopic ultralow anterior resection was successfully performed, thus preserving the anus. A 57-year-old woman visited our hospital due to constipation and was found by a digital examination to have a soft mass on the right wall of the rectum. Computed tomography revealed an 8.0 × 5.0-cm mass with an unclear margin adjacent to the rectum. A biopsy specimen was positive for CD34 and the c-kit gene product, but it was not positive for smooth muscle actin or S-100 protein, and thus the tumor was diagnosed as GIST. An abdominoperineal resection is generally essential for large rectal GISTs; however, she refused this operation. Neoadjuvant treatment with Imatinib decreased the tumor size (4.0 × 3.5 cm) and the anus was preserved by a laparoscopic ultralow anterior resection with direct coloanal anastomosis. She had no evidence of disease for 24 months postoperatively. To preserve the anus, a rectal GIST expressing the c-kit gene is best treated with Imatinib as neoadjuvant therapy.

Interleukin-6 produced by pancreatic carcinoma cells enhances humoral immune responses against tumor cells : A possible event in tumor regression

Production of interleukin‐6 (IL‐6) by human pancreatic carcinoma cells inversely correlates with potentials for blood‐borne metastasis to the liver in nude mice. IL‐6 cDNA was transfected to PCI‐43, one of our cultured pancreatic carcinoma cell lines that does not produce IL‐6 and generates numerous metastases to the liver. An IL‐6 high‐producer clone (PCI‐43h) generated few metastases; IL‐6 production thus has a direct effect on metastasis, whereas other transfectants (PCI‐43I and PCI‐43n), which are IL‐6 low‐, and IL‐6 non‐producers, respectively, did generate metastases. Tumor‐reactive IgG, which mediated antibody‐dependent cellular cytotoxicity (ADCC) in vitro, was detected in sera from recipient nude mice inoculated with PCI‐43h but not in sera from mice given PCI‐43I, PCI‐43n or parent PCI‐43. Tumor‐reactive IgM was detected in sera from all mice, irrespective of inoculated PCI‐43 species, with a slight augmentation being noted in PCI‐43h‐inoculated nude mice. Severe combined immunodeficiency (SCID) beige mice were then used as recipients for PCI‐43 species, and tumorigeneity was examined by s.c. inoculation of a suboptimal number of PCI‐43 transfectants (1 × 106/0.1 ml). Only PCI‐43h formed palpable masses in SCID beige mice, whereas it first grew to be palpable but subsequently became not palpable in nude mice, thereby revealing a dual action of tumor‐derived IL‐6. Thus, tumor‐derived IL‐6 confers growth promotion in SCID beige mice, while the same cytokine exhibits anti‐tumorigenic functions, presumably through humoral immune responses, in nude mice. Int. J. Cancer 75:284–289, 1998.

Inhibitory effects of the antiangiogenic agent TNP-470 on establishment and growth of hematogenous metastasis of human pancreatic carcinoma in SCID beige mice in vivo

Effects on the liver of the antiangiogenesis agent O-(chloroacetyl-carbamoyl) fumagillol (TNP-470) on hematogenous metastasis of a human pancreatic carcinoma cell line were examined. One million PCI-43 cells, a human pancreas carcinoma cell line, were injected into the spleen of SCID beige mice, then TNP-470 at 30 mg/kg was administered subcutaneously every other day. The mice were killed 6 or 10 weeks thereafter and metastatic nodules in the liver were counted and measured microscopically. Metastases were inhibited and an ∼10% loss of weight was evident in the TNP-470-administered mice. There was no suppression in maximal size of metastatic colonies in mice given the agent for 6 weeks, while inhibition was apparent in mice given the drug for 10 weeks. Suppression of proliferation and an increase in apoptosis were evident in metastatic nodules in the TNP-470-administered groups, following stainings for proliferative cell nuclear antigen and terminal deoxytransferase-mediated dUTP-biotin nick endlabeling, respectively. TNP-470 inhibited the proliferation of human umbilical vein endothelial cells but not PCI-43 in vitro. TNP-470 did not suppress production of vascular endothelial growth factor by PCI-43 cells. Neovascularization in vivo induced by PCI-43 cells was suppressed in the TNP-470-administered mice, using a diffusion chamber placed in subcutaneous tissues of SCID beige mice. These observations suggest that inhibition of angiogenesis is effective in suppressing establishment and subsequent growth of hematogenous micrometastases of pancreatic adenocarcinoma to the liver.

Hand-Assisted Endoscopic Esophagectomy for Esophageal Cancer

Abstract.Radical esophagectomy is a highly invasive operation for esophageal cancer, and improved techniques are being sought to reduce the invasiveness of this procedure. We devised a method in which an assistant inserts their left hand into the thoracic cavity, and the operator inserts their left hand into the abdominal cavity through a small incision in the upper quadrant during an endoscopic procedure. Between 1996 and 1999, we performed endoscopic esophagectomy on 18 patients. The median number of mediastinal lymph nodes removed by thoracoscopic surgery was 20.1 ± 9.4 and the median number of abdominal lymph nodes removed by laparoscopic surgery was 11.1 ± 5.6. The number of nodes dissected by endoscopic surgery did not differ significantly from the number of nodes dissected by conventional thoracotomy with laparotomy. Our experience shows that endoscopic esophagectomy with reconstruction of the esophagus assisted by inserting the hand into the thoracic and abdominal cavity, for safety and certainty, is an effective technique that is much less invasive than radical esophagectomy performed by conventional thoracotomy with laparotomy.

Anti-angiogenic treatment for peritoneal dissemination of pancreas adenocarcinoma: a study using TNP-470.

We established peritoneal dissemination‐prone subcultures (PCI‐43p3) using nude mice by a repetitive in vivo selection of intraperitoneally inoculated PCI‐43, a pancreas adenocarcinoma cell line. The subcultures showed upregulated expression of matrix metalloproteinase (MMP)‐9, but not MMP‐2 in culture supernatants. They also produced increased amounts of vascular endothelial growth factor (VEGF), which was not associated with alterations in isoforms of VEGF mRNA. PCI‐43p3 cells attached to cultured mesothelial cell monolayers more readily than did the parent PCI‐43 cells. The angiogenesis inhibiting agent, TNP‐470, at 30 mg/kg was administered to the model mice, resulting in a prominent suppression of the establishment of peritoneal nodules. The suppression was dependent on the duration of TNP‐470 treatment. TNP‐470 treatment significantly suppressed proliferation of tumor cells in disseminated nodules, assessed in terms of immunostaining for proliferating cell nuclear antigen (PCNA). TNP‐470 did not affect the in vitro attachment between PCI‐43p3 and mesothelial cells. The combined data show that anti‐angiogenic treatment profoundly suppresses the in vivo process of peritoneal dissemination.

The Role of Sialylated Lewis Antigens on Hematogeneous Metastases of Human Pancreas Carcinoma Cell Lines in vivo

Previous studies have shown that sialyl Lewis a (SLea) and sialyl Lewis x (SLex) correlated to hematogenous metastasis of human cancers. Although SLea/SLex and E-selectin act as a set of adhesion molecules in vitro, it is not clear whether the in vivo correlation is exclusively mediated by the adhesion function. To address this issue, we investigated whether or not the role of SLea/SLex antigens on hematogenous metastasis to the liver in SCID mice was exclusively mediated by adhesion by using antibodies for these antigens and SLea/SLEx-negative, human pancreas adenocarcinoma cell line PCI-6. The absence of SLea/SLex expression was supported by the absent flow cytometric detection of the antigens as well as by the absent attachment augmentation to activated endothelial cells. PCI-6 cells are xenotransplantable to nude and SCID mice and produce vascular endothelial cell growth factor (VEGF) in a significant amount. PCI-6 cells, 1 x 10(6), were injected into the spleens of SCID mice, and resultant liver metastases were evaluated six weeks later. We observed an inhibitory effect on the establishment and growth of metastatic colonies when anti-SLea or anti-SLex antibody was administered. This indicates that SLea/x antigens have an important in vivo role, even in the metastasis of SLea/SLex-negative tumor cells. This implies that there may be an in vivo function of SLea/x antigens other than that of the attachment between tumor and endothelial cells.