Kosuke Yamaguchi

Antibody-Dependent Cellular Cytotoxicity Mediated by Cetuximab against Lung Cancer Cell Lines

Purpose: Epidermal growth factor receptor (EGFR) is commonly overexpressed in lung cancer. Cetuximab is a chimeric mouse-human antibody targeted against EGFR. Compared with its inhibitory properties, its immunologic mechanisms have not been well studied. In this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC) activity of cetuximab against lung cancer cell lines. Experimental Design: We studied the correlation between EGFR expression in lung cancer cell lines and the ADCC activity of cetuximab as well as the influence of interleukin-2 and chemotherapy on the ADCC activity. EGFR expression was measured by a quantitative flow cytometric analysis and immunohistochemistry. The ADCC activity was assessed by a 4-h 51Cr release assay. Peripheral blood mononuclear cells, purified T cells, natural killer (NK) cells, and monocytes from healthy donors or lung cancer patients were used as effector cells. Results: Fresh peripheral blood mononuclear cells exhibited cetuximab-mediated ADCC activity against lung cancer cell lines at a low concentration of cetuximab (0.25 μg/mL). A logarithmic correlation was observed between the number of EGFRs and ADCC activity. Even low EGFR expression, which was weakly detectable by immunohistochemistry, was sufficient for maximum ADCC activity, and further increases in EGFR expression on the target cells had no further effect on the ADCC activity. In addition, ADCC activity was enhanced by interleukin-2 mainly through activation of NK cells and was less susceptible to immunosuppression by chemotherapy than NK activity in lung cancer patients. Conclusions: These observations suggest the importance of ADCC activity as an immunologic mechanism of cetuximab in biological therapy for lung cancer patients.

Diagnostic and prognostic impact of serum‐soluble UL16‐binding protein 2 in lung cancer patients

UL16‐binding protein 2 (ULBP2) is one of the ligands for NKG2D (NKG2DL). ULBP2 expression is induced in transformed cells and is recognized by immune effector cells via the activating NKG2D immunoreceptor. Soluble forms of NKG2DL have been reported in the serum of patients with several types of cancer. The present study investigated the diagnostic and prognostic significance of serum‐soluble ULBP2 (sULBP2) in lung cancer patients. We used flow cytometry to evaluate the surface expression of NKG2DL by various lung cancer cells, while sULBP2 was measured using our original ELISA. In addition, the immunological effect of sULBP2 on peripheral blood mononuclear cells (PBMC) was examined by the 51Cr release assay. We found that ULBP2 was highly expressed and that the sULBP2 level was elevated in supernatants of cultured non‐small‐cell lung cancer (NSCLC) cells as well as in the serum of NSCLC patients. ULBP2 levels were especially high in squamous cell carcinoma (SQ) patients. Clinical stage IIIB and IV NSCLC patients with a sULBP2 level ≥8.7 pg/mL showed significantly shorter survival than patients with sULBP2 <8.7 pg/mL. In multivariate analysis, a sULBP2 level ≥8.7 pg/mL (hazard ratio [HR], 2.13; P = 0.038) and clinical stage IV (HR, 2.65; P = 0.019) were independent determinants of a poor outcome. As a possible mechanism, we demonstrated that sULBP2 directly suppresses the cytolytic activity of PBMC. In conclusion, ULBP2 is the most significant NKG2DL for lung cancer, and sULBP2 is useful in the diagnosis of SQ and as a prognostic indicator for patients with advanced NSCLC. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02330.x, 2012)

The 6-minute pegboard and ring test is correlated with upper extremity activity of daily living in chronic obstructive pulmonary disease

Background Upper-extremity exercise is for pulmonary rehabilitation. The 6-minute pegboard and ring test (6PBRT) was developed to evaluate arm exercise capacity in patients with chronic obstructive pulmonary disease (COPD). The purpose of this study was to characterize the 6PBRT and evaluate its relationship with upper-extremity activities of daily living (ADLs) in COPD patients. Methods Twenty outpatients with mild to very severe COPD underwent the 6PBRT and spirometry, and their maximal inspiratory and expiratory pressures and grip strength were measured. For the 6PBRT, subjects were asked to move as many rings as possible in 6 minutes, and the score was the number of moved rings during the 6-minute period. Upper-extremity ADLs were evaluated with the upper extremity activities subdomain of the modified Pulmonary Functional Status and Dyspnea Questionnaire. Upper-extremity ADLs were also measured objectively by using a wrist accelerometer every day for 1 week. Results There was a positive correlation between 6PBRT score and inspiratory capacity (r = 0.71, P , 0.001), inspiratory capacity/total lung capacity predicted (r = 0.68, P , 0.01), and forced vial capacity (r = 0.57, P , 0.01). There was also a positive correlation between 6PBRT score and accelerometer count (r = 0.54, P , 0.05) and a negative correlation between 6PBRT score and arm activity score (ρ = −0.49, P , 0.05). Conclusion The 6PBRT may be a predictive test to maintain and improve upper-extremity ADL during pulmonary rehabilitation in patients with COPD.

Therapeutic antitumor efficacy of anti-epidermal growth factor receptor antibody, cetuximab, against malignant pleural mesothelioma

Epidermal growth factor receptor (EGFR) is commonly overexpressed in malignant pleural mesothelioma (MPM). Cetuximab is a chimeric mouse-human antibody targeted against EGFR and induces potent antibody-dependent cellular cytotoxicity (ADCC). The action of cetuximab against MPM cells has not been well studied. Therefore, in this study, we investigated the antitumor activity of cetuximab against MPM cell lines, particularly with respect to ADCC activity in vitro and in vivo. EGFR expression of MPM cells was measured by a quantitative flow cytometric analysis and immunohistochemistry. The effect of cetuximab on growth inhibition was assessed using a modified MTT assay. The ADCC activity was measured by a 4-h 51Cr release assay using fresh or IL-2-activated peripheral blood mononuclear cells. In vivo antitumor activity of cetuximab was evaluated using an orthotopic implantation mouse model. Cetuximab-mediated ADCC activity against MPM cells was observed at low concentration (0.25 mg/ml) and was enhanced by IL-2, whereas no direct effect on growth inhibition was detected. A logarithmic correlation was observed between the number of EGFRs on MPM cells and ADCC activity. Low EGFR expression on the MPM cells, which was weakly detectable by immunohistochemistry, was sufficient for maximum ADCC activity. In the mouse model, cetuximab treatment with or without IL-2 significantly inhibited intrathoracic tumor growth and prolonged their survival. Our study shows that cetuximab has potent anti-MPM activity both in vitro and in vivo, mainly through the immunologic mechanism of ADCC. Cetuximab has the potential to be used as a novel therapy for MPM patients.

Bronchodilator Reversibility Occurring during the Acute Phase of Paragonimiasis westermani Infection: A Case Report

A 43-year-old woman was referred to our hospital with peripheral blood hypereosinophilia and abnormal chest X-ray findings. Her pleural effusion revealed hypereosinophilia and a low glucose level. She was diagnosed with pulmonary paragonimiasis based on an elevated antibody level of Paragonimiasis westermani. Although she had no medical history of allergic disorders, a pulmonary function test revealed bronchodilator reversibility. After praziquantel therapy, her symptoms, hypereosinophilia in peripheral blood, and pleural effusion were improved. A repeated pulmonary function test after praziquantel therapy showed a negative bronchodilator response. Pulmonary paragonimiasis may induce bronchodilator reversibility during the acute phase of infection.

Evaluation of antigen‐positive toxin‐negative enzyme immunoassay results for the diagnosis of toxigenic Clostridium difficile infection

Clostridium difficile (C. difficile)-associated diarrhea (CDAD) is a challenging nosocomial infectious disease. C. DIFF Quik Chek Complete assay is widely used to detect glutamate dehydrogenase (GDH) antigen and toxin A/B of C. difficile simultaneously. However, the interpretation of GDH positive/toxin negative results is problematic. We performed a retrospective study of patients with GDH positive/toxin negative results to determine the probability of detecting toxigenic C. difficile and its risk factors. Between April 2012 and March 2017, we investigated cultures of fecal specimens followed by toxin detection tests. The clinical histories of patients with and without toxigenic C. difficile were compared using univariate- and multivariate-analyses. In total, 2675 patients were examined using C. Diff Quik Chek Complete assay. Among 356 GDH positive/toxin negative patients, cultures were performed in 220 cases and toxigenic C. difficile was recovered from 139 (63.2%) specimens. Patients with toxigenic C. difficile had significantly lower body mass index than those without. Over half the GDH positive/toxin negative patients were infected with toxigenic C. difficile. Lower BMI was a CDAD risk factor in this patient population. These data can be utilized to initiate isolation and clinical interventions before confirmatory test results are available. J. Med. Invest. 65:131-135, February, 2018.

Acute-phase reaction induced by zoledronate and its effect on prognosis of patients with advanced non-small cell lung cancer

OBJECTIVES Zoledronate (ZOL) is usually used for prevention of skeletal-related events in cancer patients with bone metastases. The first administration of ZOL is occasionally associated with development of acute-phase reaction (APR), which is due to activation of γδ T cells. ZOL-related APR was associated with better overall survival (OS) of patients with non-small cell lung cancer (NSCLC) in our previous retrospective study. However, it remains to be clarified whether γδ T cells are more activated in patients who experienced ZOL-related APR, and whether γδ T cell activation is involved in prolongation of OS. MATERIALS AND METHODS Twenty-three patients with advanced NSCLC were recruited between 2012 and 2014 in this study. We administered ZOL to participants with standard care. The patient characteristics, change in γδ T cell counts and cytokines, OS, and skeletal-related event-free survival were compared between patients with APR (APR group) and those without APR (non-APR group). RESULTS Ten patients (43.5%) experienced a ZOL-related APR. The number of γδ T cells at baseline in the APR group was significantly higher than that in the non-APR group. Serum interleukin-6 and tumor necrosis factor-α in the APR group were significantly increased, but no change in the number of γδ T cells was observed after the first administration of ZOL in both groups. OS in the APR group was significantly longer than that in the non-APR group (median survival time: 23.1 vs. 14.5 months, p < 0.01). CONCLUSION We showed that APR is related to higher numbers of γδ T cells at baseline and increased cytokines after the first ZOL administration, but not to proliferative responses of γδ T cells. In addition, better OS was observed in the APR group. Therefore, the number of γδ T cells might be a prognostic marker in patients with NSCLC.

Abstract 2385: Identification of a molecular marker for cetuximab sensitivity and development of a novel combination therapy for non-small cell lung cancers.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The epidermal growth factor receptor (EGFR) is an important molecular target for non-small-cell lung cancers (NSCLCs). A recent phase III study (the FLEX study) revealed that the combination of the anti-EGFR antibody cetuximab with conventional chemotherapy improved the survival of patients with NSCLCs. However, the survival benefit was only about 5 weeks. Therefore, the identification of a molecular marker for cetuximab sensitivity is urgently needed. In this study, we investigated a new molecular marker for cetuximab sensitivity and developed a combination therapy to improve the effectiveness of cetuximab. First, we established a NSCLC cell line that showed resistance to cetuximab by culturing a parental sensitive NSCLC cell line in the presence of increasing concentrations of cetuximab. Next, we performed microarray analysis to compare the gene expression patterns of cetuximab-sensitive and -resistant cell lines. Using pathway analysis, we found that the Akt and Wnt pathways were significantly activated in the resistant cell line as compared to their activation in the sensitive cell line. We confirmed this result by performing western blot analysis, which showed Akt phosphorylation and beta-catenin dephosphorylation in the resistant cell line. Therefore, the activation status of either the Akt or Wnt pathway is the candidate pathway that influences cetuximab sensitivity. Finally, we focused on the Akt pathway and treated both resistant and parental sensitive cell lines with the PI3-K inhibitor LY294002 in combination with cetuximab. This combination therapy induced a synergistic growth-inhibiting effect in both cell lines. The data suggest that the activation status of the Akt pathway is one of the markers for cetuximab sensitivity and the combination of an Akt pathway inhibitor and cetuximab would be a promising anti-EGFR therapy for NSCLCs. Citation Format: Miyako Takata, Hiroki Chikumi, Kosuke Yamaguchi, Jun Kurai, Masaki Nakamoto, Naoto Burioka, Tadashi Igishi, Eiji Shimizu. Identification of a molecular marker for cetuximab sensitivity and development of a novel combination therapy for non-small cell lung cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2385. doi:10.1158/1538-7445.AM2013-2385 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 454: ADAM proteases shed UL-16-binding protein 2 in human lung cancer cell lines.

NKG2D is an activating receptor that is expressed in immune effector cells, and the ligands for which are expressed in transformed cells. NKG2D-NKG2D ligand system might play an essential role in the tumor immunity of host cells. We have previously reported that a member of NKG2D ligand family, UL-16-binding protein 2 (ULBP2), is predominantly expressed on the surface of lung cancer cells, and is shed and solubilized into the sera of the lung cancer patients. In addition, we showed that the soluble form of ULBP2 (sULBP2) aggravates the cytotoxicity of host immune effector cells. Therefore, shedding of ULBP2 from the surface of cancer cells might be a critical component of the immune escape mechanisms employed in lung cancer. In this study, we investigated the mechanism of ULBP2 shedding from the cell surfaces in human lung cancer cell lines. First, we compared the amount of ULBP2 expression on the surface of normal cells and that in the culture medium of various lung cancer cell lines. We observed a significant correlation among non-small-cell lung cancer (NSCLC) cell lines (r=0.835, P Citation Format: Kosuke Yamaguchi, Hiroki Hikumi, Shinji Matsumaga, Miyako Takata, Naoki Kinoshita, Kiyoshi Hashimoto, Masaki Nakamoto, Jun Kurai, Masanari Watanabe, Akira Yamasaki, Tadashi Igishi, Naoto Burioka, Eiji Shimizu. ADAM proteases shed UL-16-binding protein 2 in human lung cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 454. doi:10.1158/1538-7445.AM2013-454

Abstract LB-88: A new rapid diagnostic test to identify epidermal growth factor receptor mutations in non-small cell lung cancer.

An epidermal growth factor receptor (EGFR) mutation is the best marker of sensitivity to the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Polymerase chain reaction (PCR)-based methods combined with the use of fluorescence probes, such as the Scorpion Amplification Refractory Mutation System (ARMS) or the PCR-Invader method, are frequently used to detect EGFR mutations before therapy. The sensitivities and specificities of these methods are satisfactory; however, there is potential to further improve the cost and detection time. We developed a new method for the rapid and reliable detection of EGFR mutations by using a combination of mutation-specific primers and the newly developed ultra-rapid real-time PCR (Hyper-PCR) amplification method that can be accomplished in Citation Format: Hiroki Chikumi, Miyako Takata, Keiji Matsunami, Shingo Matsumoto, Masahiro Kotani, Tomohiro Sakamoto, Hirokazu Touge, Kosuke Yamaguchi, Jun Kurai, Naomi Miyake, Masaki Nakamoto, Tadashi Igishi, Eiji Shimizu. A new rapid diagnostic test to identify epidermal growth factor receptor mutations in non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-88. doi:10.1158/1538-7445.AM2013-LB-88