Adoptive cell therapy using tumor-infiltrating lymphocytes for melanoma refractory to immune-checkpoint inhibitors.

To evaluate the feasibility of adoptive cell therapy (ACT) using ex vivo-expanded tumor-infiltrating lymphocytes (TILs) in Japanese patients with melanoma who failed immune-checkpoint inhibitor therapy, an open-label, single-arm, pilot study was conducted. We investigated the immunological and genetic factors of the pretreatment tumor and expanded TILs that may be associated with the clinical response. The treatment protocol comprised preparation of TIL culture, lympho-depleting non-myeloablative preconditioning with cyclophosphamide and fludarabine, TIL infusion, and intravenous administration of low-dose IL-2. Three patients of clinical subtypes mucosal, superficial spreading, and acral melanoma underwent TIL-ACT. Most severe adverse events, including fever and leukopenia, were manageable with the supportive regimen specified in the protocol, suggesting that the TIL-ACT regimen is suitable for Japanese patients with melanoma. One patient showed a short-term partial response, one relatively long-stable disease, and one experienced disease progression. Whole-exome and transcriptional sequencing of isolated tumor cells and immunohistochemical analyses before TIL-ACT revealed various immunostimulatory factors, including a high tumor mutation burden and immune cell-recruiting chemokines, as well as various immunosuppressive factors including TGF-ß, VEGF, Wnt/ß-catenin, and MAPK signaling and epithelial-to-mesenchymal transition, which might influence the efficacy of TIL-ACT. Our results imply mechanisms for the antitumor effect of and resistance to TIL-ACT. Further studies of immune-resistant mechanisms of TIL-ACT are warranted. This study is registered with the UMIN Clinical Trial Registry (UMIN 000011431).

Comparative studies on the activities of collagenases from Grimontia hollisae and Clostridium hystoliticum in the hydrolysis of synthetic substrates.

The collagenase produced by a gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently than that produced by a gram-positive bacterium Clostridium histolyticum (Chcol), which is currently the most widely used collagenase in industry [Teramura et al. (Cloning of a novel collagenase gene from the gram-negative bacterium Grimotia (Vibrio) hollisae 1706B and its efficient expression in Brevibacillus choshinensis. J Bacteriol 2011;193:3049-3056)]. Here, we compared the Ghcol and Chcol activities using two synthetic substrates. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2, 4-dinitrophenyl)-L-2, 3-diaminopropioyl]-L-Ala-L-Arg-NH2, Ghcol exhibited 350-fold higher activity than Chcol in the absence of CaCl2 and NaCl. The Ghcol activity markedly decreased with increasing concentrations of buffer, CaCl2 or NaCl, while the Chcol activity did not, suggesting that the Ghcol activity was sensitive to solvent components. In the hydrolysis of N-[3-(2-furyl)acryloyl]-L-Leu-Gly-L-Pro-Ala, Ghcol exhibited 16-fold higher activity than Chcol in the absence of CaCl2 and NaCl, and both enzyme activities did not decrease with increasing concentrations of buffer, CaCl2 or NaCl. pH dependences of activity revealed that the ionizable group responsible for acidic pKe may be Glu for Ghcol and Chcol, while that for alkaline pKe may be His for Ghcol and Tyr for Chcol. These striking differences suggest that the catalytic mechanism of Ghcol might be considerably different from that of clostridial collagenases.

Comparison of Nutrition-Related Adverse Events and Clinical Outcomes Between ICE (Ifosfamide, Carboplatin, and Etoposide) and MCEC (Ranimustine, Carboplatin, Etoposide, and Cyclophosphamide) Therapies as Pretreatment for Autologous Peripheral Blood Stem Cell Transplantation in Patients with Malignant Lymphoma.

BACKGROUND The aim of this study was to compare nutrition-related adverse events and clinical outcomes of ifosfamide, carboplatin, and etoposide regimen (ICE therapy) and ranimustine, carboplatin, etoposide, and cyclophosphamide regimen (MCEC therapy) instituted as pretreatment for autologous peripheral blood stem cell transplantation. MATERIAL AND METHODS We enrolled patients who underwent autologous peripheral blood stem cell transplantation between 2007 and 2012. Outcomes were compared between ICE therapy (n=14) and MCEC therapy (n=14) in relation to nutrient balance, engraftment day, and length of hospital stay. In both groups, we compared the timing of nutrition-related adverse events with oral caloric intake, analyzed the correlation between length of hospital stay and duration of parenteral nutrition, and investigated the association between oral caloric intake and the proportion of parenteral nutrition energy in total calorie supply. Five-year survival was compared between the groups. RESULTS Compared with the MCEC group, the ICE group showed significant improvement in oral caloric intake, length of hospital stay, and timing of nutrition-related adverse events and oral calorie intake, but a delay in engraftment. Both groups showed a correlation between duration of parenteral nutrition and length of hospital stay (P=0.0001) and between oral caloric intake (P=0.0017) and parenteral nutrition energy sufficiency rate (r=-0.73, P=0.003; r=-0.76, P=0.002). Five-year survival was not significantly different between the groups (P=0.1355). CONCLUSIONS Our findings suggest that compared with MCEC therapy, ICE therapy improves nutrition-related adverse events and reduces hospital stay, conserving medical resources, with no significant improvement in long-term survival. The nutritional pathway may serve as a tool for objective evaluation of pretreatment for autologous peripheral blood stem cell transplantation.

Peptide-pulsed dendritic cell vaccine in combination with carboplatin and paclitaxel chemotherapy for stage IV melanoma.

In this study, we aimed to evaluate the feasibility and efficacy of peptide-pulsed dendritic cell (DC) vaccine in combination with carboplatin and paclitaxel chemotherapy (DCCP) for patients with stage IV melanoma previously treated with dacarbazine-containing regimen. Six HLA-A24 and 3 HLA-A02 patients were treated with carboplatin (area under the curve 5) and paclitaxel (175 mg/m) on day 1 and DCs (2×10 cells) pulsed with Wilms tumor gene 1 (WT1), gp100, tyrosinase, and either MAGE-A3 (for HLA-A24) or MAGE-A2 (for HLA-A02) peptides on days 8 and 22 in 28-day cycle for up to three cycles. DCCP was well tolerated, and median progression-free survival and median overall survival were 2.3 and 12.0 months, respectively. In four of nine patients, a WT1-specific immune response (WT1-IR) was detected using the interferon-γ enzyme-linked ImmunoSpot assay and WT1/HLA tetramer assay. DCCP was more likely to elicit a WT1-IR in patients who received DCs pulsed with the HLA-A24-restricted peptide (75%) compared with patients who received DCs pulsed with the HLA-A02-restricted peptide (0%, P=0.058). Furthermore, three (75%) of four patients with a WT1-IR survived longer than 12 months, whereas only one (20%) of five patients without a WT1-IR who received the BRAF inhibitor after DCCP survived longer than 12 months. These results suggest that DCCP may be beneficial for HLA-A24 melanoma patients with a WT1-IR.

Lack of XAGE-1b and NY-ESO-1 in metastatic lymph nodes may predict the potential survival of stage III melanoma patients.

The cancer-testis antigens (CTA) are a large family of tumor-associated antigens expressed by a variety of cancer cells and primitive germ cells of the adult testis and placenta. These tumor-restricted expressing patterns suggest that CTA would be ideal targets for tumor-specific immunotherapy. XAGE-1 is a CTA that was originally identified by computer-based screening, and four transcription variants, XAGE-1a, -1b, -1c and -1d, have been characterized to date. Although the presence of XAGE-1 transcripts has been reported in various cancers, the expression of XAGE-1b in melanoma has not been fully characterized. In this study, we performed immunohistochemical staining of XAGE-1b together with NY-ESO-1, a well-known CTA, in 113 melanoma samples obtained from 84 patients and evaluated their expression in tumor cells. The effects of expression on tumor progression and patient prognosis were analyzed. Both XAGE-1b and NY-ESO-1 were expressed at high levels in lymph node metastasis and skin metastasis samples compared with the primary site (P < 0.01 in XAGE-1b and P < 0.05 in NY-ESO-1). In a subgroup analysis of 22 patients with stage III lymph node metastasis, overall survival was significantly higher in the XAGE-1b and NY-ESO-1 double-negative group than in the other groups (P < 0.05). These results suggest that lack of XAGE-1b and NY-ESO-1 expression could have a positive influence on clinical outcome in patients with melanoma.

Development of immersion vaccine for bacterial cold-water disease in ayu Plecoglossus altivelis.

Flavobacterium psychrophilum (F. psychrophilum) is the causative agent of bacterial cold-water disease (BCWD) that occurs in ayu Plecoglossus altivelis. Formalin-killed cell of F. psychrophilum has long been studied as an immersion vaccine for BCWD. In this study, we explored the possibility of F. psychrophilum collagenase (fpcol) for use as the immersion vaccine. BCWD convalescent ayu sera contained specific IgM antibodies against somatic F. psychrophilum and fpcol, meaning that fpcol is a promising antigen for the vaccine development. The recombinant fpcol was successfully expressed in Escherichia coli and Brevibacillus chosinensis (B. chosinensis). The culture supernatant of the B. chosinensis was used as an immersion vaccine solution. The vaccinated ayu were then challenged by soaking into F. psychrophilum culture. In two experimental groups, the relative percentages of survivals were 63 and 38%, respectively, suggesting that fpcol is promising as the immersion vaccine for ayu-BCWD.

Overexpression of adenylate cyclase-associated protein 2 is a novel prognostic marker in malignant melanoma.

Malignant melanoma is one of the lethal malignant tumors worldwide. Previously we reported that adenylate cyclase-associated protein 2 (CAP2), which is a well-conserved actin regulator, was overexpressed in hepatocellular carcinoma; however, CAP2 expression in other clinical cancers remains unclear. The aim of the current study was to clarify the clinicopathological significance of CAP2 overexpression in malignant melanoma. Immunohistochemical analyses revealed that many melanoma cells exhibited diffuse cytoplasmic expression of CAP2, whereas no normal melanocytes showed detectable immunostaining for CAP2. A high level of CAP2 expression was seen in 14 of 50 melanomas and was significantly correlated with greater tumor thickness and nodular melanoma subtypes. In addition, a high level of CAP2 expression was associated with poor overall survival in univariate and multivariate analyses. For 13 patients, samples of primary and metastatic melanoma tissue were available: four patients exhibited higher levels of CAP2 expression in metastatic tumor compared to the primary site, whereas no patient showed lower levels of CAP2 expression in metastatic melanomas. Our findings show that CAP2 overexpression is a novel prognostic marker in malignant melanoma and that CAP2 expression seems to increase stepwise during tumor progression, suggesting the involvement of CAP2 in the aggressive behavior of malignant melanoma.

Molecular analysis of Dirofilaria repens removed from a subcutaneous nodule in a Japanese woman after a tour to Europe.

A premature female Dirofilaria species, subsequently identified as Dirofilaria repens by its morphological features and mitochondrial 12S ribosomal RNA (12S rRNA) gene sequence, was removed from a subcutaneous nodule of the right temporal region of the head in a Japanese woman 2 years after she noticed swelling of her left calf following an insect sting during a tour to Europe; headache symptoms were noticed a few months later. The sequences of the mitochondrial 12S rRNA and cytochrome c oxidase subunit I genes from the organism were almost identical to those of sequences AM779772 (100% homology, 337/337) and AM749233 (99.8% homology, 536/537) of D. repens isolated from humans in Italy. However, the phylogenetic position of the 18S rRNA-internal transcribed spacer 1-5.8S rRNA region was in the same cluster as that of sequence JX290195 of Dirofilaria sp. "hongkongensis" (96.7% homology, 348/360), which was recently reported from Hong Kong as a novel Dirofilaria species. Information on regional genetic variation in D. repens isolated from animals and humans remains scarce. We report the detailed genetic features of this filaria as a reference isolate from a specific endemic area, to enrich the genetic database of D. repens.

Eosinophil superoxide anion generation induced by adhesion molecules and leukotriene D4.

RATIONALE: Eosinophils preferentially accumulate at sites of inflammation in the asthmatic airway. Participation of circulating eosinophils in the airway inflammation in asthma involves their interaction with adhesion molecules expressed on the endothelial cell surface and exposure to inflammatory mediators, such as cysteinyl leukotrienes (cysLTs). OBJECTIVE: To investigate whether interaction of eosinophils with adhesion molecules modifies the functions of these cells induced by cysLTs. METHODS: Eosinophils were isolated from the blood of healthy donors, incubated in the EIA plates coated with adhesion proteins, and then exposed to LTD4. The generation of superoxide anion (O2-), adhesion to the plates, and release of eosinophil-derived neutrotoxin (EDN) were evaluated. RESULTS: Neither VCAM-1 nor LTD4 (100 nM) independently induced eosinophil O2- generation, however, combined exposure to the two molecules synergistically induced eosinophil O2- generation. ICAM-1 by itself induced eosinophil O2- generation, which was enhanced by LTD4. On the contrary, P-selectin did not induce O2- generation, either in the presence or absence of LTD4. LTD4 significantly enhanced eosinophil adhesion to rh-VCAM-1 and rh-ICAM-1, but not to rh-P-selectin. Finally, we observed that combined exposure of eosinophils to LTD4 and VCAM-1 induced the release of EDN. CONCLUSION: Combined exposure to VCAM-1 or ICAM-1 and cysLT effectively induces the effector functions of eosinophils. Eosinophil adhesion to and migration across endothelial cells via these specific adhesion proteins and subsequent exposure to cysLTs may be mechanisms underlying activation of the effector functions of eosinophils in the asthmatic airway.

Local delivery of doxorubicin for malignant glioma by a biodegradable PLGA polymer sheet.

Implantable, biocompatible and biodegradable devices bearing an anticancer drug can provide promising local therapy to patients with malignant disorders. With the aim of treating brain tumors, especially gliomas, a membranous sheet containing doxorubicin was produced by co-polymerization to poly(D,L-lactide-co-glycolide) (PLGA). When release of the drug from the sheet was measured, sustained release continued until day 34. The data contrasted with the burst release from material containing a higher proportion of the drug. In terms of biodegradability, a subcutaneous 3 x 3-mm tetragonal sheet was almost completely absorbed by day 80. When a glioma was implanted subcutaneously and the tumor nodule exposed to the sheet, the device inhibited tumor growth significantly. The sheet consisted of an amorphous structure with cavities estimated to have a diameter of 0.5 - 3 microm by electron microscopic observation. Since the sheet is implantable, biodegradable and has a sustained-drug release property, the device may play a role in the local therapy of brain tumors.

Inhibition of poly (ADP-ribose) polymerase as a protective effect of nicaraven in ionizing radiation- and ara-C-induced cell death.

BACKGROUND: Nicaraven is a drug used for patients with a subarachnoid hemorrhage. It crosses the blood-brain barrier and has potent antivasospastic and brain-protective effects. While nicaraven scavenges the hydroxyl radical, the mechanism of its protection remains obscure. In addition to the hydroxyl radical scavenging effect, nicaraven also exhibits inhibitory action on poly (ADP-ribose) polymerase (PARP). The mechanism of the pharmacological action of nicaraven has not yet been clarified. MATERIALS AND METHODS: Human myeloid HL-525 cells were exposed to ionizing radiation or hydrogen peroxide and the effect of nicaraven on the activation of the Egr-1 promoter was measured. Next, the action of the drug on DNA fragmentation and inhibition of thymidine uptake caused by the genotoxic stimulation of ionizing radiation or cytosine B-D-arabinofuranoside (ara-C) were assessed. Finally, direct inhibition of the PARP enzyme by nicaraven was measured. RESULTS: Nicaraven did not inhibit the activation of the Egr-1 promoter caused by H2O2 and the activation caused by ionizing radiation. However, the drug repressed DNA fragmentation and increased thymidine uptake dose-dependently. Nicaraven had a direct inhibitory effect on PARP. DISCUSSION: The effect of nicaraven on the Egr-1 promoter was different from that of another free-radical scavenger, N-acetyl cysteine. Nicaraven demonstrated similar protection of the PARP inhibitors including 3-aminobenzamide. Since nicaraven directly inhibits the PARP enzyme, the drug might be useful in oncology as well as in studying tissue-damaging conditions characterized by increased PARP activity.