Yuji Heike

Vasculogenic mimicry and pseudo‐comedo formation in breast cancer

Tumors require a blood supply for growth and hematogenous metastases. Until recently, most research in this area has focused on the role of angiogenesis, the recruitment of new vessels into a tumor from preexisting vessels. Previously, in a study of breast cancer (IBC), in which we used established inflammatory breast cancer (IBC) xenografts (WIBC‐9) originating from a patient with IBC (Shirakawa et al., Cancer Res 2001:61:445–451), we reported observing vasculogenic mimicry (VM), a condition in which bloodstreams within cancer tissue are not accompanied by a lining of endothelial cells (ECs) (Shirakawa et al., Cancer Res 2002:62:560–566). In the present study, we examined 331 surgically resected breast cancer specimens for evidence of VM, using immunohistochemistry and laser‐captured microdissection (LCM) followed by nested reverse transcriptase polymerase chain reaction (RT‐PCR). Surprisingly, 7.9% (26 specimens) of the 331 specimens exhibited evidence of VM. Of these 26 VM specimens, 84.6% (22 specimens) exhibited pseudo‐comedo formation. RT‐PCR analysis of 8 microdissected typical VM specimens revealed expression of Tie‐2, Flt‐1, thrombin receptor and CD31 in 63, 50, 0 and 0% of specimens, respectively. In contrast, results of RT‐PCR analysis of 8 specimens from non‐VM tumors were negative for expression of these genes. The 26 VM cases tended to have a higher percentage of hematogenous recurrence (p = 0.059) and a lower percentage of 5‐year survival (p = 0.071) than the 305 non‐VM cases. However, there were no significant differences in tumor size, lymph node metastasis, estrogen receptors or progesterone receptors between the 2 groups (p > 0.1). Our results suggest that the existence of VM increases the likelihood of hematogenous metastases and is in inverse proportion to prognosis.

Inhibition of Multidrug‐resistant Human Tumor Growth in Athymic Mice by Anti‐P‐glycoprotein Monoclonal Antibodies

In an effort to devise an effective treatment for human drug‐resistant cancers, we have developed monoclonal antibodies, MRK16 and 17, reactive to the multidrug transporter protein, P‐glycoprotein. The monoclonal antibodies given intravenously effectively prevented tumor development in athymic mice inoculated subcutaneously with drug‐resistant human ovarian cancer cells 2780AD. Treatment with MRK16 induced rapid regression of established subcutaneous tumors and apparent cures of some animals. Complement‐dependent cytotoxicity (MRK16) and antibody‐dependent cell‐mediated cytolysis (MRK16 and 17) were observed with these antibodies. These monoclonal antibodies may have potential as treatment tools against multidrug resistant human tumors possessing the P‐glycoprotein.

Tumor‐infiltrating endothelial cells and endothelial precursor cells in inflammatory breast cancer

Inflammatory breast cancer (IBC) is a specific type of breast tumor that generally has a poor prognosis, in spite of recent advances in treatment. In the present study, semiquantitative reverse transcriptase polymerase chain reaction examination of resected specimens showed that angiogenic factors, not lymphangiogenic factors, are overexpressed in IBC tumors, compared with non‐IBC tumors. Immunohistochemical analysis of the specimens revealed a significantly higher population of tumor‐infiltrating (TI) endothelial cells (ECs) or endothelial precursor cells (EPCs) in tumor‐associated stroma of IBC specimens than in non‐IBC specimens. In a previous study, we examined the phenotype of host cells in response to transplanted IBC cells, using an established human IBC xenograft model (WIBC‐9) (Shirakawa et al., Cancer Res 2001;61:445–51). The data obtained in that study are consistent with the findings of the present study. To explore the therapeutic potential of blocking vascular endothelial growth factor (VEGF) and angiopoietin (Ang) pathways in IBC, established vectors encoding soluble Flt‐1 (sFlt‐1) and soluble Tie2 (sTie2) were injected directly into WIBC‐9. Both vectors produced growth inhibition ratios of WIBC‐9 that were significantly higher than those of a non‐IBC xenograft (MC‐5). Also, both vectors suppressed WIBC‐9 lung metastases. The efficacy correlated with the number of TI ECs/EPCs, which was determined by fluorescence‐activated cell sorting. These ECs/EPCs incorporated acetylated lipoprotein and were integrated within a HUVEC monolayer in vitro culture on day 5.

Hyperglycemia during the neutropenic period is associated with a poor outcome in patients undergoing myeloablative allogeneic hematopoietic stem cell transplantation.

Background. Recipients of allogeneic hematopoietic stem cell transplantation (HSCT) frequently require support with parenteral nutrition and immunosuppressive drugs, which introduce the risk of hyperglycemia. Van den Berghe et al. showed that the strict glucose control improved the outcome of patients treated in the intensive care unit, and this point was evaluated in this study in a HSCT setting. Methods. A cohort of 112 consecutive adult patients treated by myeloablative allogeneic HSCT between January 2002 and June 2006 was reviewed retrospectively. Twenty-one patients were excluded due to graft failure, preexisting infectious diseases, preexisting neutropenia or previous allogeneic HSCT. The remaining 91 patients were categorized according to mean fasting blood glucose (BG) level in the neutropenic period after conditioning: normoglycemia (BG <110 mg/dL, n=28), mild hyperglycemia (110 to 150 mg/dL, n=49), and moderate/severe (>150 mg/dL, n=14). The primary endpoint was the occurrence of febrile neutropenia (FN) and documented infection during neutropenia, and the secondary endpoints included organ dysfunction according to the definition used by van den Berghe, acute graft-versus-host disease (GVHD), overall survival, and nonrelapse mortality (NRM). Results. Although the incidence of FN or documented infections was similar between the three groups, hyperglycemia was significantly associated with an increased risk of organ dysfunction, grade II–IV acute GVHD, and NRM. Conclusions. While the results suggested an association between the degree of hyperglycemia during neutropenia and an increased risk of posttransplant complications and NRM, the possibility that intensive glucose control improves the outcome after HSCT can only be confirmed in a prospective randomized trial.

Phase 1 trial of Wilms tumor 1 (WT1) peptide vaccine and gemcitabine combination therapy in patients with advanced pancreatic or biliary tract cancer.

An open-labeled, dose-escalation phase 1 trial of Wilms tumor 1 (WT1) vaccine and gemcitabine (GEM) combination therapy for patients with advanced pancreatic cancer or biliary tract cancer was performed. The primary end point was evaluation of toxicity, safety, and optimal immunologic dose of vaccine. Human leukocyte antigen (HLA)-A 0201, HLA-A 0206, and/or HLA-A 2402-positive patients with inoperable advanced pancreatic or biliary tract cancer who had not previously been treated with GEM were eligible for this study. Six doses of GEM and 4 doses of WT1 peptide (1 or 3 mg) emulsified in Montanide adjuvant were administered over 2 months. Twenty-five patients (13 male and 12 female) were enrolled. Nine patients had inoperable advanced pancreatic cancer, 8 had gallbladder cancer, 4 had intrahepatic, and 4 had extrahepatic bile duct cancer. The adverse events were comparable to those with GEM alone. Delayed-type hypersensitivity test was positive after vaccination in 2 patients, and WT1-specific T cells in peptide-stimulated culture were detected by tetramer assay in 59% (13 of 22) of patients. The disease control rate at 2 months was 89% for pancreatic cancer and 50% for biliary tract cancer. With a median follow-up time of 259 days, the median survival time for biliary tract cancer was 288 days, and that for pancreatic cancer was 259 days. Although objective clinical efficacy was not apparent, the safety of WT1 vaccine and GEM combination therapy was confirmed in this study.

Regular Dose of Gemcitabine Induces an Increase in CD14+ Monocytes and CD11c+ Dendritic Cells in Patients with Advanced Pancreatic Cancer

OBJECTIVE Chemotherapy and immunotherapy often seem to contradict each other. However, recent reports suggested that the anticancer effects in some chemotherapeutic agents were concerned with immune response. This study was designed to evaluate the immunological reaction by gemcitabine for future clinical trial of combination therapy with gemcitabine and cancer vaccines. METHODS We evaluated several immunological parameters in patients with advanced pancreatic cancer who received a conventional dose of gemcitabine for 2 months. Twenty-eight patients with metastasis or locally advanced tumor, including 18 gemcitabine-naïve and 10 with a history of preceding gemcitabine treatment, were enrolled in this study. The patients received gemcitabine 1000 mg/m(2) for 3 weeks, followed by 1 week of rest. We monitored the kinetics of lymphocytes, natural killer cells, monocytes, dendritic cells (DC), human leukocyte antigen (HLA)-multimer conjugated with CMV or WT1 peptide, and intracellular cytokine production of interferon-gamma and interleukin-4 by flow cytometry. The T cell receptor (TCR) repertoire was also analyzed. RESULTS The absolute number and percentage of CD14(+) monocytes and CD11c(+) (myeloid) DC increased with gemcitabine treatment (P = 0.033 and P = 0.021). The percentage of CD123(+) (plasmacytoid) DC also increased (P = 0.034), whereas no significant change was observed in other immune parameters, including multimer, intracellular cytokine production and TCR repertoire. CONCLUSIONS Our finding that gemcitabine treatment induced the proliferation of CD14(+) monocytes and CD11c(+) DC could support combination therapy with gemcitabine and specific immunotherapy such as peptide vaccination against pancreatic cancers.

The Role of PGE2 in the Differentiation of Dendritic Cells: How Do Dendritic Cells Influence T‐Cell Polarization and Chemokine Receptor Expression?

The role of prostaglandin E2 (PGE2) in the function of dendritic cells (DCs), T‐cell polarization, and expression of chemokine receptors was evaluated in human cells. Immature DCs were generated from peripheral blood CD14+ cells using a combination of GM‐CSF and interleukin‐4 (IL‐4) with or without PGE2. On day 6, maturation of DCs was induced by the addition of tumor necrosis factor alpha with or without PGE2. DCs harvested on day 6 (immature DCs) or day 9 (mature DCs) were examined using functional assays. In the presence of PGE2, immature and mature DCs showed, phenotypically, a lower expression of CD1a and, functionally, a higher allostimulatory capacity at a high DC/T‐cell ratio than control cells cultured in the absence of PGE2. DCs cultured in the presence of PGE2 induced the differentiation of naïve T cells toward a helper T‐cell type 1 (Th1) response, which was independent of IL‐12 secretion in the basal state despite a slightly lower interferon gamma secretion compared with control cells. However, the function of cytotoxicity‐stimulating autologous T cells was not augmented by the addition of PGE2. Immature DCs expressed the inflammatory chemokine receptors, CCR1 and CXCR4, but not CCR6, regardless of the presence or absence of PGE2. Mature DCs expressed CCR7 equally, measured using a migration test and the measurement of calcium flux with macrophage inflammatory protein‐3β and reverse transcription‐polymerase chain reaction assay in all of the groups. All of these findings suggest that PGE2 affects the DC‐promoted differentiation of naïve T cells to a Th1 response in the basal state, without affecting chemokine receptor expression on DCs.

Antithymocyte globulin affects the occurrence of acute and chronic graft-versus-host disease after a reduced-intensity conditioning regimen by modulating mixed chimerism induction and immune reconstitution

Background. There have been no detailed analyses of the induction of donor cell–type chimerism, the onset and incidence of acute and chronic graft-versus-host disease (GVHD), and the immune recovery kinetics after reduced-intensity stem cell transplantation (RIST). Methods. To address these, with particular emphasis on the impact of the use of antithymocyte globulin (ATG) in RIST, we compared 39 consecutively registered patients who underwent RIST from an HLA-matched related donor and 33 patients who underwent conventional marrow-ablative transplantation. Results. The incidences of grades II to IV acute and chronic GVHD tended to be less in RIST with ATG than in either RIST without ATG or conventional marrow-ablative transplantation. In a multivariate analysis, the predictive factors for acute and chronic GVHD included, respectively, ATG and grades II to IV acute GVHD. In a chimerism analysis, the achievement of complete donor chimera in T-cell lineage was delayed in RIST without ATG compared with RIST with ATG (P =0.038), which might explain the observed delayed onset of acute GVHD in RIST with ATG compared with the other two regimens. The ratio of type 1 and 2 dendritic cells did not affect the development of GVHD, whereas the number of naive CD4+ T cells did. No difference was observed in the incidence of clinically definitive infection, including cytomegalovirus, among the three cohorts, regardless of the use of ATG. Conclusions. We suggest that the conditioning regimen and immunosuppressive strategy after RIST should be carefully balanced against the risk of GVHD and of relapse of the basic disorder caused by the lack of a graft-versus-leukemia benefit.

Evaluation of cytomegalovirus-specific T-cell reconstitution in patients after various allogeneic haematopoietic stem cell transplantation using interferon-γ-enzyme-linked immunospot and human leucocyte antigen tetramer assays with an immunodominant T-cell epitope

Cytomegalovirus (CMV) infection is a major complication for patients who received allogeneic haematopoietic stem cell transplantation (HSCT). Accurate monitoring of CMV‐specific T‐cell reconstitution is required for appropriate decision on treatment, such as anti‐viral drugs, which have adverse effects. Although human leucocyte antigen (HLA) tetramer and interferon‐γ‐enzyme‐linked immunospot (IFN‐γ‐ELISPOT) assays have been used to measure CMV‐specific T cells, detailed comparison of these assays and kinetics of anti‐CMV T‐cell reconstitution between reduced‐intensity transplantation (RIST) and conventional HSCT has not yet been performed. In this study, we performed prospective comparative monitoring of CMV‐specific T cells using HLA tetramer and IFN‐γ‐ELISPOT assays with a single immunodominant CMV495 peptide in 28 HLA‐A*0201 and 9 HLA‐A*0206 patients after various allogeneic HSCTs. The IFN‐γ‐ELISPOT assay was more sensitive for evaluation of functional T cells than the HLA tetramer assay, and CMV‐specific T cells were reconstituted earlier in patients who received RIST without anti‐thymocyte globulin (ATG) than those receiving RIST with ATG or conventional HSCT. The threshold level for protection from CMV reactivation was estimated as over 1 × 106 cells/l peripheral blood with the IFN‐γ‐ELISPOT assay. These results demonstrate that the IFN‐γ‐ELISPOT assay with CMV495 provides more accurate evaluation on CMV immunity in HLA‐A*0201 and ‐A*0206 patients, and may be useful for determining timing of various treatments.

Monoclonal anti-P-glycoprotein antibody-dependent killing of multidrug-resistant tumor cells by human mononuclear cells

Mouse monoclonal antibodies (MRK16 and MRK17) against human multidrug‐resistant cancer cell lines were tested for antibody‐dependent cytotoxicity mediated by human blood mononuclear cells, using a 4‐h 51Cr release assay. MRK16 (IgG28 isotype) was shown to be more effective than MRK17 (IgG1 isotype). Moreover, when four pairs of drug‐resistant and their parent sensitive human cancer cells were tested for antibody‐dependent cell‐mediated cytolysis (ADCC) using MRK16, only the drug‐resistant cell lines were susceptible to ADCC reaction. When highly purified lymphocytes (>99%) and monocytes (>97%) were isolated from blood mononuclear cells by centrifugal elutriation and adherence, MRK16 promoted both lymphocyte‐ and monocyte‐mediated tumor cell killing, whereas MRK17 induced only a lymphocyte‐mediated ADCC reaction. These results suggest that MRK16 of IgG28 subtype may be a useful therapeutic agent in eradication of drug‐resistant cancer cells expressing P‐glycoprotein through ADCC reaction.