Our followup study of 48 patients with primary aldosteronism concerns the results of 2 different operative methods. After preoperative localization of the unilateral solitary tumor 22 patients underwent unilateral adrenalectomy and 26 underwent enucleation of aldosterone-producing adenoma. Both operative methods improved hypertension, hypokalemia, the low urinary sodium-to-potassium ratio, suppressed plasma renin activity, high plasma aldosterone concentration, high urinary aldosterone excretion and high urinary kallikrein excretion in similar orders of magnitude for 5 years. Levels of plasma cortisol and plasma adrenocorticotropic hormone following respective operations were also identical. Five years postoperatively, ambulation and furosemide administration under low sodium diet stimuli remarkably enhanced plasma renin activity and plasma aldosterone concentration in the aldosterone-producing adenoma enucleation group (p < 0.001), almost similar to that of normal subjects but increment magnitudes were slight (p < 0.05 to < 0.01) in the adrenalectomy group. Preoperatively, angiotensin II infusion failed to increase plasma aldosterone concentration in patients with primary aldosteronism. After respective operations, responses of plasma aldosterone concentration to angiotensin II infusion and of plasma cortisol to adrenocorticotropic hormone administration in the aldosterone-producing adenoma enucleation group were more sensitive than those in the adrenalectomy group. There was no remission of recurrent hyperaldosteronism in either group throughout the study. These results suggest that angiotensin II induces aldosterone release by an activation of tumor uninvolved cortical cells and that the enucleation of aldosterone-producing adenoma is more preferable than unilateral adrenalectomy.

The IL12RB2 gene acts as a tumor suppressor in human B cell malignancies. Indeed, Il12rb2 knockout (KO) mice develop spontaneously B cell tumors, but also lung epithelial tumors. This latter phenotype may be related to (i) impairment of host IL-12-mediated immunosurveillance and/or (ii) IL-12 inability to inhibit directly the growth of IL-12 unresponsive malignant cells. To address this issue, we transplanted IL-12R ؉ B16 melanoma cells into syngeneic Il12rb2 KO mice with the following rationale: (i) these mice have severe defects in IFN-␥ production, as well as in cytotoxic T lymphocyte and natural killer cell cytotoxicity, and (ii) they produce but do not use IL-12 that can potentially bind to and target tumor cells only. Il12rb2 KO mice displayed higher endogenous serum levels of IL-12 and developed smaller B16 tumors than WT animals. These tumors showed reduced proliferation, increased apoptosis, and defective microvessel formation related to down-regulated expression of a set of proangiogenic genes previously unrelated to IL-12. Such effects depended on direct activity of endogenous IL-12 on tumor cells in KO mice, and hydrodynamic delivered IL-12 caused further reduced tumorigenicity of B16 cells in these mice. A previously undescribed mechanism of the IL-12 antitumor activity has been here identified and characterized.angiogenesis ͉ tumor immunology ͉ cytokines I nterleukin 12 is a heterodimeric cytokine bridging innate and adaptive immunity (1). IL-12, which belongs to a family of structurally related cytokines including IL-23 and IL-27 (2, 3), is formed by the IL12p35 and IL12p40 subunits (4) and binds to the IL-12R, composed of the ␤1 and the ␤2 chains. Both chains are needed for high affinity binding of the cytokine and initiation of signal transduction (5, 6). IL12p40 associates with the p19 subunit to form IL-23, which binds to a receptor composed of IL12R␤1 and IL-23R (2, 3). Therefore, the IL12p35 and the IL12R␤2 subunits are unique components of IL-12 and IL-12R, respectively. IL-12, which is produced predominantly by antigen-presenting cells, drives T helper (Th1) responses, enhances T and natural killer (NK) cell cytotoxicity, and induces IFN-␥ production by T and NK cells (4,[7][8][9]. In addition, we have shown that IL-12 contributes to the regulation of normal human B cell function through stimulation of IgM synthesis and IFN-␥ production (10).IL-12 exerts antitumor activity through IFN-␥-dependent and independent mechanisms (11-15), which include modulation of the immune system and antiangiogenesis (16)(17)(18). We have recently demonstrated that the IL12RB2 gene acts as a tumor suppressor in human chronic B cell malignancies (14) and, accordingly, that aging IL12rb2 knockout (KO) mice develop spontaneously B cell tumors. The same mice showed high incidence of lung epithelial tumors. This latter finding may be related to reduced immune surveillance, because of their deficient T and NK cell-mediated cytotoxicity (19), Th2-biased T cell differentiation (20), and/or lack of IL-12-mediated dir...

To understand the mechanisms underlying bone marrow metastasis precisely, we established the highly metastatic 4T1E/M3 murine breast cancer cell line. 4T1 murine breast cancer cells were transfected with the neomycin resistance gene, selected in G418, intravenously injected into mice, and harvested from bone marrow. By repeating this protocol three times, we established the 4T1E/M3 cells. The clonality of 4T1E/M3 cells was markedly high confirmed by genomic southern analysis using neo-gene probe. When tissues harvested from mice after intravenous injection of 4T1E/M3 cells were examined histologically, markedly enhanced bone marrow metastasis was observed; 77% of spines from 4T1E/M3-injected mouse showed metastasis as compared to 14% metastasis seen with the parent cells. In vitro, 4T1E/M3 cells attached more strongly to the plastic plate and to bone marrow-derived endothelial cells. DNA micro arrays, real time RT-PCR and FACS analyses revealed that the expression of ICAM-1 and beta2 integrin was upregulated in 4T1E/M3 cells at both the mRNA and cell surface protein levels. 4T1E/M3 cells also showed greater anchorage-independent proliferation in soft agar, and migrated markedly faster than the parent cells in wound healing assays. Anti-ICAM-1 antibodies strongly inhibited both the colony formation and the migration activity of 4T1E/M3 suggesting the importance of the role of ICAM-1. Our newly established highly metastatic 4T1E/M3 cells may provide a potentially powerful tool to study the molecular mechanisms of bone marrow metastasis and to identify new molecular targets for therapeutic interventions.

IL-12 is an excellent candidate for the treatment of cancer due to its ability to drive strong antitumor responses. Recombinant IL-12 protein is currently used in cancer patients; however, systemic expression of rIL-12 presents disadvantages including cost and dose limitation due to its toxicity. In this study, we used hydrodynamic shear of cDNA as a tool to achieve systemic expression of IL-12. We found that sustained but toxic levels of serum IL-12 could be generated in 6- to 7-wk-old B6 mice after a single injection of the cDNA. Unexpectedly, we observed that when IL-12 cDNA is coinjected with IL-18 cDNA, IL-12 antitumor activity was maintained, but there was a significant attenuation of IL-12 toxicity, as evidenced by a greater survival index and a diminution of liver enzymes (ALT and AST). Interestingly, after IL-12 plus IL-18 cDNA administration, more rapid and higher IL-10 levels were observed than after IL-12 cDNA treatment alone. To understand the mechanism of protection, we coinjected IL-12 plus IL-10 cDNAs and observed an increase in survival that correlated with diminished serum levels of the inflammatory cytokines TNF-α and IFN-γ. Confirming the protective role of early IL-10 expression, we observed a significant decrease in survival in IL-10 knockout mice or IL-10R-blocked B6 mice after IL-12 plus IL-18 treatment. Thus, our data demonstrate that the high and early IL-10 expression induced after IL-12 plus IL-18 cDNA treatment is critical to rapidly attenuate IL-12 toxicity without affecting its antitumor capacity. These data could highly contribute to the design of more efficient/less toxic protocols for the treatment of cancer.

Bone is the most frequent site of breast cancer metastasis, and once such metastasis occurs, complete remission is extremely difficult to achieve. In an effort to define the mechanisms underlying metastatic spread of breast cancer to bone, we previously developed and characterized the highly bone metastatic 4T1E/M3 mouse breast cancer cells. We found that following injection into mice, 4T1E/M3 cells exhibited greater bone metastasis and greater in vitro anchorage-independent growth and cell migration than their parental cells (4T1E). We also found that expression of intracellular adhesion molecule-1 (ICAM-1) is crucially involved in these metastatic activities of 4T1E/M3 cells. In the present study, our analysis of gene and protein expression revealed that production of chemokine CCL2 (MCP-1) is dramatically reduced in 4T1E/M3 cells, and that restoration of CCL2 expression in 4T1E/M3 cells diminishes their metastasis to bone and lung. Overexpression of CCL2 in 4T1E/M3 cells significantly reduced not only in vitro anchorage-independent cell growth and cell migration, but also mRNA and cell surface expression of ICAM-1. Conversely, knocking down CCL2 in 4T1E parental cells augmented their metastatic spread to spine and lung. The expression of ICAM-1 was also upregulated in 4T1E-derived CCL2 knockdown cells. Taken together, these results suggest that CCL2 expression may negatively regulate breast cancer metastasis to bone marrow and lung in our model and that expression of ICAM-1 plays a crucial role in that process.

Background Avelumab is a human anti-PD-L1 IgG1 monoclonal antibody that has shown antitumor activity in several advanced cancers. We report results from JAVELIN Solid Tumor JPN, a phase 1 trial of avelumab in Japanese patients with advanced solid tumors with expansion in patients with advanced gastric cancer/gastroesophageal junction cancer. Methods In the dose-escalation part, eligible patients had various previously treated metastatic or advanced solid tumors. In the dose-expansion part, patients had stage IV gastric cancer/gastroesophageal junction adenocarcinoma and disease progression after prior therapy that included a platinum and fluoropyrimidine agent. Patients received avelumab every 2 weeks intravenously at 3, 10, or 20 mg/kg during dose escalation and 10 mg/kg during dose expansion. Results Among 17 patients who received avelumab in the dose-escalation part, no dose-limiting toxicities occurred, and the maximum tolerated dose was not reached. 40 patients were enrolled in the dose-expansion part, of whom 21 (52.5%) had received ≥ 3 prior lines of therapy for advanced disease. In these patients, the objective response rate was 10.0% (95% CI, 2.8–23.7%) and median overall survival was 9.1 months (95% CI, 7.2–11.2 months). Three of 40 patients (7.5%) had a grade 3 treatment-related adverse event (alanine aminotransferase increase, anemia, and hyponatremia), and no grade ≥ 4 treatment-related adverse events occurred. Five patients (12.5%) had an immune-related adverse event (all grade 1/2). Conclusions Avelumab showed acceptable safety in Japanese patients with advanced solid tumors and clinical activity in patients with advanced gastric cancer/gastroesophageal junction cancer and disease progression after chemotherapy. Electronic supplementary material The online version of this article (10.1007/s10120-018-0903-1) contains supplementary material, which is available to authorized users.

Dendritic cells (DC) that have been genetically modified to express cytokine genes may be novel tools for inducing antitumor immune responses. In the present study, the pMX retroviral vector was modified to express the mouse IL-2 (mIL-2pMX) and mouse IL-12 (mIL-12pMX) genes. Supernatants from 293 cells transfected with pMX retroviral vectors were harvested and used to transduce mouse lin − bone marrow (BM) progenitor cells. After 48 h co-culture with pseudotype retrovirus, BM cells were cultured for 12 days in the presence of mGM-CSF, mSCF and mTNF-␣ to obtain a DC-enriched fraction. Flow cytometric analysis showed that GFP protein expression in these cultures was 20-40% and that 40-50% of the cultured BM cells were positive for the DC marker, DEC205. About 60% of cells sorted for DEC205 also expressed GFP. The supernatants of DC-mIL-2 and DC-mIL-12 cultured for 48 h contained 5.2 ± 0.15 and 33.9

Objectives Tepotinib (MSC2156119J) is an oral, potent and highly selective small molecule mesenchymal-epithelial transition factor (MET) inhibitor for which the recommended Phase II dose of 500 mg once daily has been defined, based on the first-in-man trial conducted in the USA and Europe. We carried out a multicenter Phase I trial with a classic `3 + 3' design to determine the recommended Phase II dose in Japanese patients with solid tumors (NCT01832506). Methods Patients aged ≥20 years with advanced solid tumors (refractory to standard therapy or for whom no effective standard therapy was available) received tepotinib at 215, 300 or 500 mg once daily in a 21-day cycle. Occurrence of dose-limiting toxicities during cycle 1 was used to determine the maximum tolerated dose. Efficacy, safety and pharmacokinetics were also evaluated to support the dose assessment. Results Twelve patients were treated. Tepotinib was generally well tolerated with no observed dose-limiting toxicities; treatment-related adverse events were mainly grades 1–2. The tolerability profile of tepotinib was similar to that observed in non-Japanese populations. Pharmacokinetics in Japanese and Western patients was comparable. One patient with gastric cancer and one patient with urachal cancer had stable disease of ≥12 weeks in duration. The observed safety profile and pharmacokinetics are comparable with those in patients from the USA and Europe, and the recommended Phase II dose of tepotinib in Japanese patients was confirmed as 500 mg once daily. Conclusions These results, including initial signals of antitumor activity, support further development of tepotinib in Japanese patients with cancer.