Kiyotsugu Kojima

Blockade of bulky lymphoma‐associated CD55 expression by RNA interference overcomes resistance to complement‐dependent cytotoxicity with rituximab

Recently, anti‐CD20 (rituximab) and anti‐Her2/neu (trastuzumab) antibodies have been developed and applied to the treatment of malignant lymphoma and breast cancer, respectively. However, bulky lymphoma is known to be resistant to rituximab therapy, and this needs to be overcome. Fresh lymphoma cells were collected from 30 patients with non‐Hodgkins lymphoma, the expression of CD20 and CD55 was examined by flow cytometry, and complement‐dependent cytotoxicity (CDC) assays were carried out. Susceptibility to CDC with rituximab was decreased in a tumor size‐dependent manner (r = –0.895, P < 0.0001), but not in a CD20‐dependent manner (r = –0.076, P = 0.6807) using clinical samples. One complement‐inhibitory protein, CD55, contributed to bulky lymphoma‐related resistance to CDC with rituximab. A decrease in susceptibility to CDC with rituximab was statistically dependent on CD55 expression (r = –0.927, P < 0.0001) and the relationship between tumor size and CD55 expression showed a significant positive correlation (r = 0.921, P < 0.0001) using clinical samples. To overcome the resistance to rituximab by high expression of CD55 in bulky lymphoma masses, small interfering RNA (siRNA) was designed from the DNA sequence corresponding to nucleic acids 1–380 of the CD55 cDNA. Introduction of this siRNA decreased CD55 expression in the breast cancer cell line SK‐BR3 and in CD20‐positive cells of patients with recurrent lymphoma; resistance to CDC was also inhibited. This observation gives us a novel strategy to suppress bulky disease‐related resistance to monoclonal antibody treatment. (Cancer Sci 2006; 97: 72–79)

Identification of CD20 C-Terminal Deletion Mutations Associated with Loss of CD20 Expression in Non-Hodgkin's Lymphoma

Purpose: Rituximab is commonly incorporated into CD20-positive B-cell lymphoma therapy to improve response and prognosis. With increasing use, resistance to rituximab is a continuing concern, but CD20 mutation as a cause of resistance has not previously been reported. Experimental Design: Freshly collected lymphoma cells from 50 patients with previously untreated or relapsed/resistant non-Hodgkins B-cell lymphomas (diffuse large B cell, n = 22; follicular, n = 7; mucosa associated lymphoid tissue, n = 16; chronic lymphocytic leukemia, n = 2; small lymphocytic lymphoma, n = 1; lymphoplasmacytic, n = 1; mantle cell lymphoma, n = 1) were assessed for CD20 expression by flow cytometry, and CD20 gene sequencing was done on extracted DNA. Results: CD20 mutations were found in 11 (22.0%) of 50 patients and could be grouped as C-terminal deletion (8.0%), early termination (10.0%), and extracellular domain (2.0%) or transmembrane domain (2.0%) mutations. The mean fluorescence intensity of CD20 on fresh lymphoma cells was significantly lower for the C-terminal deletion mutation [3.26; 95% confidence interval (95% CI), 0.09-6.89] compared with wild type (30.8; 95% CI, 22.4-39.2; P < 0.05). In contrast, early termination mutations did not show significant differences in CD20 expression compared with wild type (19.5; 95% CI, 10.7-28.4; P > 0.05). Conclusions: It is possible that C-terminal deletion mutations of CD20 may be related to relapse/resistance after rituximab therapy. These mutations should be examined in patients showing progression of disease after partial remission.

Functional in vivo optical imaging of tumor angiogenesis, growth, and metastasis prevented by administration of anti-human VEGF antibody in xenograft model of human fibrosarcoma HT1080 cells

Angiogenesis plays a crucial role in cancer progression and metastasis. Thus, blocking tumor angiogenesis is potentially a universal approach to prevent tumor establishment and metastasis. In this study, we used in vivo and ex vivo fluorescence imaging to show that an antihuman vascular endothelial growth factor (VEGF) antibody represses angiogenesis and the growth of primary tumors of human fibrosarcoma HT1080 cells in implanted nude mice. Interestingly, administering the antihuman VEGF antibody reduced the development of new blood vessels and normalized pre‐existing tumor vasculature in HT1080 cell tumors. In addition, antihuman VEGF antibody treatment decreased lung metastasis from the primary tumor, whereas it failed to block lung metastasis in a lung colonization experiment in which tumor cells were injected into the tail vein. These results suggest that VEGF produced by primary HT1080 cell tumors has a crucial effect on lung metastasis. The present study indicates that the in vivo fluorescent microscopy system will be useful to investigate the biology of angiogenesis and test the effectiveness of angiogenesis inhibitors. (Cancer Sci 2009)

High reproducible ADCC analysis revealed a competitive relation between ADCC and CDC and differences between FcγRllla polymorphism

The anti-CD20 chimeric monoclonal antibody rituximab mediates cytotoxicity in malignant B cells via multiple mechanisms, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and direct induction of apoptosis. To optimize treatment of non-Hodgkins lymphoma, a fuller understanding of these mechanisms and their relative contributions to clinical efficacy is required. Here, we report the characteristics of the mutual impact between ADCC and CDC, the two major effector functions through the Fc receptors. To compare ADCC induced under various conditions, we developed a highly reproducible method of estimating ADCC activity using immortalized effector cells. The set of the effector cells that we established was able to calculate net ADCC with high reproducibility by comparing the cytotoxicity of effector cells expressing exogeneous FcγRIIIa to those of mock effector cells. In addition, the different property of effector cells of two FcγRIIIa single-nucleotide polymorphisms (SNP) could be also evaluated in exactly identical background. ADCC assessment in the presence of human serum directly provided the evidence of the competitive interaction of ADCC and CDC. The inhibition of ADCC of effector cells having low affinity SNP of FcγRIIIa by active complement was more potent than those having high-affinity SNP at the rituximab-concentration comparable to the serum level obtained in patients. These findings could have a profound impact on optimization of the regimen of therapeutic antibodies and on the development of antibodies that will enhance effector function.

An Imaging-Based Rapid Evaluation Method for Complement-Dependent Cytotoxicity Discriminated Clinical Response to Rituximab-Containing Chemotherapy

Purpose: Rituximab has greatly improved the efficacy of chemotherapy regimens for CD20-positive non-Hodgkins lymphoma. However, although several mechanisms of action of rituximab have been identified, the exact therapeutic functions of these mechanisms remains to be clarified. In addition, there is no established prognostic marker to predict an individual response. This study verified the validity of ex vivo complement-dependent cytotoxicity (CDC) susceptibility as a predictor of pathologic tumor regression in patients undergoing rituximab-containing chemotherapy and examined whether CDC contributes to the mechanism of action of rituximab. Experimental Design: A rapid assay system was established to evaluate the tumoricidal activity of rituximab using a living cell–imaging technique. We analyzed lymph node biopsies obtained from 234 patients with suspected lymphomas and estimated the association between CDC susceptibility and the response to rituximab-containing chemotherapy in diffuse large B-cell lymphoma and follicular lymphoma. Results: This study revealed that CDC susceptibility of lymphoma cells freshly obtained from patients was strongly associated with response to rituximab-containing chemotherapy in both diffuse large B-cell lymphoma and follicular lymphoma. This correlation was not apparent in cases that received chemotherapy without rituximab. Conclusions: The system that we have established allows a successful assessment of rituximab-induced CDC and can distinguish cases refractory to rituximab-containing chemotherapy. The association between CDC susceptibility and therapy response suggests that CDC is pivotal in the ability of chemotherapy including rituximab to induce remission.

MEK-ERK is involved in SUMO-1 foci formation on apoptosis.

Small ubiquitin‐related modifier (SUMO) modification appears to regulate the activity, intracellular localization, and stability of the targeted proteins. To explore the relationship among sumoylation, antitumor reagent, and apoptosis, we treated green fluorescence protein (GFP)‐SUMO‐1‐overexpressed K562 cells (K562/GFP‐SUMO‐1) with mitoxantrone (MIT) as an antitumor reagent. By the treatment with MIT, GFP‐SUMO‐1 formed foci in nuclei. While by the treatment with a tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), GFP‐SUMO‐1 located homogeneously in nuclei. When K562/GFP‐SUMO‐1 cells were treated with TPA plus MIT, GFP‐SUMO‐1 foci became larger and apoptosis was induced more than with MIT alone. The apoptosis induced by TPA plus MIT was prevented by blockage of GFP‐SUMO‐1 foci by small interfering RNA (siRNA) against SUMO‐1. The formation of GFP‐SUMO‐1 foci was reduced by a MEK inhibitor U0126 or a nuclear export inhibitor leptomycin B, and endogenous SUMO‐1 foci were reduced in K562 cells expressing the dominant‐negative MEK1 mutant. These results suggest that the formation of SUMO‐1 foci is regulated by the MEK‐ERK pathway and may induce apoptosis. (Cancer Sci 2007; 98: 569–576)