Anti-PD1 checkpoint inhibitor therapy in acral melanoma: a multicenter study of 193 Japanese patients.

BACKGROUND: Acral melanoma (AM) is an epidemiologically and molecularly distinct entity that is underrepresented in clinical trials on immunotherapy in melanoma. We aimed to analyze the efficacy of anti-programmed cell death 1 (anti-PD-1) antibodies in advanced AM. PATIENTS AND METHODS: We retrospectively evaluated unresectable stage III or stage IV AM patients treated with an anti-PD-1 antibody in any line at 21 Japanese institutions between 2014 and 2018. The clinicobiologic characteristics, objective response rate (ORR, RECIST), survival estimated using Kaplan-Meier analysis, and toxicity (Common Terminology Criteria for Adverse Events 4.0.) were analyzed to estimate the efficacy of the anti-PD-1 antibodies. RESULTS: In total, 193 patients (nail apparatus, 70; palm and sole, 123) were included in the study. Anti-PD-1 antibody was used as first-line therapy in 143 patients (74.1%). Baseline lactate dehydrogenase (LDH) was within the normal concentration in 102 patients (52.8%). The ORR of all patients was 16.6% (complete response, 3.1%; partial response, 13.5%), and the median overall survival (OS) was 18.1 months. Normal LDH concentrations showed a significantly stronger association with better OS than abnormal concentrations (median OS 24.9 versus 10.7 months; P < 0.001). Although baseline characteristics were similar between the nail apparatus and the palm and sole groups, ORR was significantly lower in the nail apparatus group [6/70 patients (8.6%) versus 26/123 patients (21.1%); P = 0.026]. Moreover, the median OS in this group was significantly poorer (12.8 versus 22.3 months; P = 0.03). CONCLUSIONS: Anti-PD-1 antibodies have limited efficacy in AM patients. Notably, patients with nail apparatus melanoma had poorer response and survival, making nail apparatus melanoma a strong candidate for further research on the efficacy of novel combination therapies with immune checkpoint inhibitors.

Origin of the metallic properties of heavily boron-doped superconducting diamond.

The physical properties of lightly doped semiconductors are well described by electronic band-structure calculations and impurity energy levels. Such properties form the basis of present-day semiconductor technology. If the doping concentration n exceeds a critical value n(c), the system passes through an insulator-to-metal transition and exhibits metallic behaviour; this is widely accepted to occur as a consequence of the impurity levels merging to form energy bands. However, the electronic structure of semiconductors doped beyond n(c) have not been explored in detail. Therefore, the recent observation of superconductivity emerging near the insulator-to-metal transition in heavily boron-doped diamond has stimulated a discussion on the fundamental origin of the metallic states responsible for the superconductivity. Two approaches have been adopted for describing this metallic state: the introduction of charge carriers into either the impurity bands or the intrinsic diamond bands. Here we show experimentally that the doping-dependent occupied electronic structures are consistent with the diamond bands, indicating that holes in the diamond bands play an essential part in determining the metallic nature of the heavily boron-doped diamond superconductor. This supports the diamond band approach and related predictions, including the possibility of achieving dopant-induced superconductivity in silicon and germanium. It should also provide a foundation for the possible development of diamond-based devices.

Anti-PD-1 antibody monotherapy versus anti-PD-1 plus anti-CTLA-4 combination therapy as first-line immunotherapy in unresectable or metastatic mucosal melanoma: a retrospective, multicenter study of 329 Japanese cases (JMAC study).

BACKGROUND: Anti-programmed cell death protein 1 (PD-1) antibody monotherapy (PD1) has led to favorable responses in advanced non-acral cutaneous melanoma among Caucasian populations; however, recent studies suggest that this therapy has limited efficacy in mucosal melanoma (MCM). Thus, advanced MCM patients are candidates for PD1 plus anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) combination therapy (PD1 + CTLA4). Data on the efficacy of immunotherapy in MCM, however, are limited. We aimed to compare the efficacies of PD1 and PD1 + CTLA4 in Japanese advanced MCM patients. PATIENTS AND METHODS: We retrospectively assessed advanced MCM patients treated with PD1 or PD1 + CTLA4 at 24 Japanese institutions. Patient baseline characteristics, clinical responses (RECIST), progression-free survival (PFS), and overall survival (OS) were estimated using Kaplan-Meier analysis, and toxicity was assessed to estimate the efficacy and safety of PD1 and PD1 + CTLA4. RESULTS: Altogether, 329 patients with advanced MCM were included in this study. PD1 and PD1 + CTLA4 were used in 263 and 66 patients, respectively. Baseline characteristics were similar between both treatment groups, except for age (median age 71 versus 65 years; P < 0.001). No significant differences were observed between the PD1 and PD1 + CTLA4 groups with respect to objective response rate (26% versus 29%; P = 0.26) or PFS and OS (median PFS 5.9 months versus 6.8 months; P = 0.55, median OS 20.4 months versus 20.1 months; P = 0.55). Cox multivariate survival analysis revealed that PD1 + CTLA4 did not prolong PFS and OS (PFS: hazard ratio 0.83, 95% confidence interval 0.58-1.19, P = 0.30; OS: HR 0.89, 95% confidence interval 0.57-1.38, P = 0.59). The rate of ≥grade 3 immune-related adverse events was higher in the PD1 + CTLA4 group than in the PD1 group (53% versus 17%; P < 0.001). CONCLUSIONS: First-line PD1 + CTLA4 demonstrated comparable clinical efficacy to PD1 in Japanese MCM patients, but with a higher rate of immune-related adverse events.

Long pulse production of high current D(-) ion beams in the JT-60 negative ion source.

The first long pulse production of high power D(-) ion beams has been demonstrated in the JT-60 U negative ion sources, each of which was designed to produce 22 A, 500 keV D(-) ion beams. Voltage holding capability and the grid power loading were examined for long pulse production of high power D(-) ion beams. From the correlation between voltage holding and the light intensity of cathodoluminescence from the Fiber Reinforced Plastic insulators, the acceleration voltage for stable voltage holding capability was found to be less than 320-340 kV where the light was sufficiently suppressed. By tuning the extraction voltage, the grid power loadings in the ion sources were decreased to the allowable levels for long pulse injection without a significant reduction of the beam power. After tuning the acceleration and extraction voltages, D(-) ion beams of 12.5 and 9.8 A were produced at 340 keV with cesium seeding at a rate of approximately 14 microg/s into the ion sources. The pulse duration of these D(-) ion beams was extended step by step, and then was successfully extended up to 18 s without degradation of the negative ion production. The D(-) ion beams were neutralized to yield 3.6 MW D(0) beams by a gas cell, and then injected into the JT-60 U plasma. Further, a slight reduction of D(-) ion beam power allowed the longer injection duration of 21 s at a D(0) beam power of 3.2 MW. The success in the long pulse production of a high power D(-) ion beam shows that negative ion beams can be produced during a few tens of seconds without degradations of negative ion production and the voltage holding in a large Cs-seeded negative ion source.

The effects of FasL on inflammation and tumor survival are dependent on its expression levels.

The apoptosis-inducing Fas ligand (FasL) is expressed in a variety of human cancers and has been implicated in tumor immune evasion. Paradoxically, ectopic expression of FasL in experimental tumors triggers a neutrophil-mediated inflammatory response and tumor rejection. To resolve these conflicting findings, we have established B16 melanoma and P29 Lewis lung carcinoma lines expressing different levels of FasL and examined their tumorigenicity in vivo. While tumors with a high level of FasL were rapidly rejected as previously reported, those expressing a low level of FasL were not rejected but grew faster than did FasL-negative parental cells. The growth enhancement of FasL(low) tumors was not observed in T-cell-deficient nude mice, suggesting that FasL expressed in tumors at low levels counteracted against T-cell-dependent antitumor responses. In support of this notion, FasL(low) tumors were found to grow faster than parental cells in mice that had acquired tumor-specific immunity. Furthermore, histological examinations revealed apoptosis of lymphocytes in tissue sections of FasL(low) tumors. These results collectively suggest that FasL on tumors is a double-edged sword: at high levels it triggers tumor rejection whereas at low levels it facilitates tumor growth possibly by suppressing antitumor immune responses.

Isolation and characterization of Cox17p from porcine heart by determining its survival-promoting activity in NIH3T3 cells.

We have found that the gel filtration fraction of porcine heart extract clearly promoted the survival of NIH3T3 fibroblast cells in the serum-free medium condition. A structural analysis showed that the active fraction contained a novel peptide, porcine Cox17p (p-Cox17p), which was recently reported by Chen et al. as dopuin (Z. W. Chen et al., Eur. J. Biochem. 249 (1997) 518-522). Porcine Cox17p/dopuin possesses high sequence homology to the product of human COX17 gene (h-Cox17p). Although Cox17p has been implied to be involved in copper recruitment to mitochondria and in the functional assembly of cytochrome oxidase in yeast, its role in mammalian cells is unknown. In this study, we chemically synthesized p-Cox17p to investigate its biological effects. Refolding experiments of synthesized linear p-Cox17p revealed the existence of mostly one pattern of three intrachain disulfide bridges similar to that of native p-Cox17p, because the main oxidized p-Cox17p was completely co-eluted with the natural product. The addition of heavy metal ions such as copper, zinc and cadmium significantly inhibited the formation of the oxidized form, suggesting that reduced p-Cox17p may interact directly with these metal ions. The reduced and oxidized forms of p-Cox17p were also confirmed to promote the survival of NIH3T3 cells in serum-free medium as observed with the natural product, indicating that Cox17p may be a bioactive peptide.

1H NMR study on the conformation of bacitracin A in aqueous solution.

The conformation of bacitracin A, a widely used cyclic dodecapeptide antibiotic in aqueous solution, has been investigated using 500 MHz 1H NMR and molecular modeling. Findings revealed that a region (residues 1-6) is folded over the cyclic ring, resulting in metal coordination sites, a thiazoline ring, and Glu4 and His10 being proximate to each other.

Partial N-terminal sequence analysis of human class II molecules expressing the DQw3 determinant.

HLA-DQ molecules were isolated from DRw9-homozygous and DR4-homozygous cell lines by using a monoclonal antibody HU-18, which recognizes class II molecules carrying the conventional DQw3 determinant. The partial N-terminal sequence analysis of the DQw3 molecules revealed that they have sequences homologous to those of murine I-A molecules. Within the limits of our sequence analysis, the DQw3 molecules from the two cell lines are identical to each other in both the alpha and beta chains. The DQ alpha as well as DQ beta chains were found to have amino acid substitutions when compared to other I-A-like molecules whose sequences have been reported. These differences may contribute to the DQw supertypic specificity. The polymorphic nature of DQ molecules is in marked contrast to that of DR molecules where DR alpha chains are highly conserved while DR beta chains have easily detectable amino acid substitutions.

Reduced tumorigenicity of human gastric carcinoma cells engineered to produce IL-2 in SCID mice reconstituted with peripheral blood cells from cancer patients.

We have examined the validity of a humanized immune system with an animal model to assess cytokine gene therapy for cancer patients. For that purpose, we prepared hematologically-reconstituted severe combined immunodeficiency mice by transferring patient's peripheral blood cells containing CD34+ cells. These animals were inoculated subcutaneously with human gastric cancer lines transduced with cytokine genes. Tumorigenicity of interleukin-2-producing cells was significantly reduced in reconstituted but not in non-reconstituted mice, whereas that of wild-type and interleukin-6 producer cells was not affected irrespective of the reconstitution status. An inability to induce protective immunity in the reconstituted mice, which had rejected interleukin-2-producers, suggested that the effector cells mediating the antitumor response were non-T cells of donor origin. The experimental system presented in this study seems to be a feasible model to investigate applicable cytokines for patients.

beta-Amyloid peptide, substance P, and SEC receptor ligand activate cytoplasmic Ca2+ in neutrophil-like HL-60 cells: effect of chemotactic peptide antagonist BocMLF.

It has been reported that a discrete peptide fragment of beta-amyloid protein, beta A(25-35), and neuropeptide substance P (SP) possessed sequence homology and could bind to the serine protease inhibitor (serpin) enzyme complex (SEC) receptor. Thus, it has been thought that these peptides and SEC receptor ligand might have similar biological activities. In the present study, we found that C-terminal amidated beta A(25-35)-NH2, SP, and the SEC receptor ligand, Phe-Val-Phe-Leu-Met(FVFLM), could induce an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in neutrophil-like human leukemic (HL-60) cells. Pretreatment with pertussis toxin (PTX) potently inhibited the increase in [Ca2+]i stimulated by these peptides, suggesting that these responses might be mediated by PTX-sensitive G-proteins. Furthermore, we examined the effect on these responses of t-butyloxycarbonyl-methionyl-leucyl-phenylalanine (BocMLF), which is a competitive antagonist of chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF) at its receptor. BocMLF scarcely inhibited the [Ca2+]i increase stimulated by beta A(25-35)-NH2. However, the increase in FVFLM-induced [Ca2+]i was potently inhibited by BocMLF. The results suggest that the [Ca2+]i activation of beta A(25-35)-NH2 may have a different mechanism from that of FVFLM in neutrophil-like HL-60 cells, which is not mediated by the SEC-receptor.

Amyloid beta-peptide-induced inhibition of MTT reduction in PC12h and C1300 neuroblastoma cells: effect of nitroprusside.

We have investigated the effect of amyloid beta-peptide (Abeta) in rat pheochromocytoma PC 12h and murine C 1300 neuroblastoma cells by using MTT ¿3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay. Exposure of the cells to Abeta peptides, Abeta1-40 and its fragment Abeta25-35, induced a concentration-dependent inhibition of MTT reduction in both cell lines, and MTT-dependent LDH release due to cell lysis in PC12h cells. We also found that sodium nitroprusside (SNP), a spontaneous nitric oxide (NO) generator, significantly prevented the inhibition of MTT reduction and MTT-dependent LDH release caused by Abeta peptides at 10-100 microM, although a high concentration of SNP (> or = 333 microM) was remarkably toxic by itself. Since the inhibition of MTT reduction caused by Abeta is known as one of the first indicators of its toxicity, these findings suggest that Abeta peptides have a toxic effect in these cell lines, and SNP may attenuate the Abeta peptide-induced toxicity. In regard the mechanisms of the actions of SNP, hydroxylamine which also generates NO and 8-Br-cGMP, a membrane-permeable analogue of cyclic GMP (cGMP), failed to prevent the inhibition of MTT reduction caused by Abeta25-35 in PC12h cells, implying that the effect of SNP may be mediated by the NO-independent pathway. Since potassium ferrocyanide showed a significant prevention at 333 microM although it had toxic effect at this concentration, it is considered that the ferrocyanide portion of the SNP metabolite may be partially involved. The cell death induced by other oxidative insults, such as glutamate and hydrogen peroxide (H2O2), could not be attenuated by SNP in both cell lines. Thus, the observed effect of SNP might not be due to its direct antioxidative action.

Structure, tissue distribution, and pharmacological characterization of Xenopus orexins.

We isolated the Xenopus gene encoding prepro-orexin to predict the structures of orexins in submammalian chordates. Putative mature Xenopus orexin-A and -B are highly similar to each mammalian counterpart. Especially, the C-terminal 10 residues were highly conserved among these species and isopeptides. Immunohistochemical examination of Xenopus brain revealed that orexin-containing neurons were highly specifically localized in the ventral hypothalamic nucleus. A rich network of immunoreactive fibers was found in various regions of the Xenopus brain. The distribution was similar to that of mammalian orexins. Xenopus orexin-A and -B specifically bind and activate human orexin receptors expressed in Chinese hamster ovary cells. Of interest, Xenopus orexin-B had several-fold higher affinity to human OX2R compared with human orexins. These results suggest that Xenopus orexin-B might be a useful pharmacological tool as an OX2R selective high-affinity agonist.

Experimental Helicobacter pylori gastric infection in miniature pigs.

An experimental Helicobacter pylori infection in miniature pigs was developed and investigated. Eighteen miniature pigs were inoculated with an H. pylori strain that has high virulence in mice at c. 5 x 10(10) cfu. H. pylori infection in miniature pigs was achieved by the administration of agar 1% in brucella broth with fetal bovine serum 10% just before inoculation. The bacterial colonisation and distribution were analysed by mapping of viable cell counts in the stomach in pigs of three different ages. The mapping assay was achieved on post-infection day 3 for the 5-day-old and 2-week-old pigs, and between days 41 and 43 for 3-month-old pigs. The highest cell counts were observed in 5-day-old pigs, which averaged 4.9 x 10(6) cfu/g of mucosa (n = 4). The bacteria were colonised mainly in the cardiac and fundus gland region in the 5-day-old and 2-week-old pigs, whereas the colonisation sites did not depend on the region in the 3-month-old pigs. Biopsy assay of the antral mucosa of a 3-month-old pig after H. pylori infection showed that this infection persisted for >22 months. Serum antibody against H. pylori was detected in the infected pigs but not in the uninfected animal. Immunostaining demonstrated the presence of bacteria on the epithelial surface of the infected pigs. A microscopic finding common to all the infected pigs, focal gastritis with infiltration of lymphocytes detected on the lesser curvature of the stomach, resembled the microscopic appearance in H. pylori-infected human patients. These results suggest that miniature pigs might be a suitable model for studying H. pylori infection.

Inhibitory effects of beta-amyloid peptides on nicotine-induced Ca2+ influx in PC12h cells in culture.

Synthetic beta-amyloid peptides and the neuropeptide substance P (SP) were examined for their ability to modulate nicotinic response in PC12h cells, a subclone of PC12 cells, SP, beta A1-40 and its peptide fragment beta A25-35-NH2 significantly inhibited an increase in cytoplasmic calcium concentrations ([Ca2+]i) induced by nicotine in a dose-dependent manner. Furthermore, beta A1-40 was found to inhibit the [Ca2+]i increase induced by depolarization with a high concentration of potassium. These findings show that both beta A1-40 and beta A25-35-NH2 may mimic the function of SP on inhibition of nicotinic response through different mechanisms.

Serotonin increases cytoplasmic Ca2+ concentration in PC12h cells: effect of tachykinin peptides.

We report here that serotonin (5-hydroxytriptamine, 5-HT) induces an increase in intracellular Ca2+ concentration ([Ca2+]i) in rat pheochromocytoma PC12h cells, a subclone of PC12 cells, which was detected by using Ca2+ sensitive indicator dye fura-2. The [Ca2+]i increase completely disappeared when extracellular Ca2+ was chelated with excess EGTA and potently suppressed in Na+-free buffer. Nifedipine, a voltage-dependent L-type calcium channel blocker, significantly blocked the 5-HT response. Addition of another 4 mM Ca2+ to the cell suspension attenuated the [Ca2+]i increase induced by 5-HT, whereas the nicotinic action was remarkably potentiated. Furthermore, metoclopramide, a 5-HT3 receptor antagonist, inhibited the 5-HT response in a dose dependent manner. These findings suggest that the 5-HT-induced [Ca2+]i increase involves the mediation of a voltage-dependent Ca2+ channel, evoked by membrane depolarization via the activation of cation channel-type receptors, 5-HT3 receptors. We also noted the inhibitory action of tachykinin peptides on the 5-HT response, suggesting that the cell line is useful to investigate these neuromodulatory actions in the nervous system.

Prevention of cerebral vasospasm by actinomycin D.

The role of endothelin, a newly found vasoconstrictor peptide, is examined in the pathogenesis of cerebral vasospasm after experimental subarachnoid hemorrhage (SAH) in the dog. Endothelin immunoreactivity was overexpressed in the endothelium of the vasospastic basilar artery. Because endothelin synthesis is regulated at the messenger ribonucleic acid transcription level, the effect of actinomycin D, a ribonucleic acid synthesis inhibitor, was studied as a means of preventing vasospasm. It was found that treatment with intravenous actinomycin D for 5 days beginning on the day of SAH completely inhibited the development of vasospasm. This novel experimental therapy may lead not only to the elucidation of the pathogenesis of cerebral vasospasm but also to the availability of a prophylactic adjuvant therapy for patients with SAH.

Trophic effects of substance P and beta-amyloid peptide on dibutyryl cyclic AMP-differentiated human leukemic (HL-60) cells.

The neuropeptide substance P (SP) is a mediator of neurogenic inflammation. Also, beta-amyloid protein (beta AP) can directly activate the cell types involved in inflammatory processes. The relationship between SP and beta AP on their biological actions has attracted much interest. SP is trophic in neuronal cells and protected them against the death induced by beta AP. In this study, we examined the effects of SP and beta-amyloid peptide on cell viability in neutrophil-like HL-60 cells, by means of the WST-1 tetrazolium and lactate dehydrogenase (LDH) release assays. The results showed that SP promoted the cell survival on neutrophil-like HL-60 cells in serum-free conditions. Also, beta-amyloid peptide showed trophic effects rather than toxic in these cells in WST-1 assay, though it is reportedly toxic in neuronal cells.

Impaired in vivo tumor growth of human pancreatic carcinoma cells retrovirally transduced with GM-CSF gene.

We have examined the antitumor effect of human pancreatic carcinoma cells (AsPC-1) retrovirally transduced with mouse granulocyte macrophage-colony stimulating factor (GM-CSF) gene in nude mice. Growth retardation of the subcutaneous tumors of GM-CSF-producing AsPC-1 cells was observed, although their in vitro proliferation was not different from that of wild-type cells. Histological examination revealed infiltration of monocytic cells into the tumor of GM-CSF-producing cells, and they were shown to be mainly CD11b positive cells by immunohistochemical staining. The survival of the mice inoculated intraperitoneally with GM-CSF- producing AsPC-1 cells was significantly prolonged compared with that of the mice inoculated with wild-type AsPC-1 cells. Thus, the expression of GM-CSF gene in human pancreatic cells induced an antitumor effect in vivo even in the mature T cell-deficient condition.

Establishment and characterization of a clear-cell sarcoma (malignant melanoma of soft parts) cell line.

A clear cell sarcoma (CCS) cell line, designated as NCS-1, was established in monolayer culture from a xenograft line originating from a metastatic CCS. Marked karyotypic aberrations and tumorigenicity in nude mice revealed the malignant derivation of the NCS-1 cell line. These cells contained abundant glycogen and were amelanotic by light microscopy. By electron microscopy, however, melanosomes in various developmental stages were seen, and some of them were partially melanized. The electron microscopic dopa reaction revealed the presence of tyrosinase activity. Enzyme-linked immunoadsorbent assay revealed that NCS-1 cells expressed a 75-kDa glycoprotein which was identified as a marker of highly differentiated melanoma cells. From these results, NCS-1 cells were found to retain both cytochemical and morphological properties of CCS. Application of NCS-1 cells to a panel of monoclonal antibodies recognizing melanocytic differentiation antigens showed that they corresponded approximately to highly differentiated melanoma cells. In conclusion, the present study strongly supports the close relationship between CCS and malignant melanoma.

Expression of cutaneous fatty acid-binding protein and its mRNA in rat skin.

Cutaneous fatty acid-binding protein (C-FABP) has been purified from rat skin. Since there was little information about the role of C-FABP in the skin, we investigated the expression of C-FABP and its mRNA in normal rat skin using an immunohistochemical technique and in situ hybridization. In the epidermis, C-FABP mRNA was found to be expressed in basal cells and highly in prickle cells, while C-FABP itself was strongly expressed in the upper prickle and the granular cell layers. In sebaceous glands, both C-FABP and its mRNA were expressed in both peripheral and differentiating cells, although the expression of C-FABP mRNA gradually reduced during differentiation of sebocytes. Since epidermis and sebaceous glands are active sites of fatty acid synthesis, these results suggest that C-FABP may have important roles in the transport and synthesis of fatty acids.