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.

Extracellular matrix production and degradation by adenoid cystic carcinoma cells: participation of plasminogen activator and its inhibitor in matrix degradation.

Adenoid cystic carcinoma (AdCC) is characterized by low mitogenic activity, high invasiveness, and vigorous production and accumulation of extracellular matrix (ECM). As it does in vivo, a cell line (ACCS) derived from a human AdCC grows very slowly and displays potential for production of a large amount of ECM. ACCS cells also produce a significant amount of proteolytic enzymes, including urokinase-type plasminogen activator (uPA), and M(r) 72,000 and 92,000 gelatinases. These cells can degrade considerable amounts of ECM elaborated by normal mesenchymal cells, including rat muscle cells and human fibroblasts, mainly through a uPA-plasmin cascade. However, ECM elaborated by ACCS cells themselves is resistant to degradation by either the tumor cells or purified uPA in the presence of plasminogen, whereas the degradation rates of ACCS ECM and mesenchymal ECM by plasmin are comparable. Treatment of ECM with glycine (pH 2.7), which removes plasminogen activator inhibitor from the matrix, results in an increase in the rate of ACCS ECM degradation by uPA. Moreover, fibrin agarose reverse zymography, autoradiography, and immunoblotting showed a high level of plasminogen activator inhibitor type 1 in ACCS ECM. These findings suggest that the plasminogen activator inhibitor type 1 in ECM produced by AdCC cells may play a role in preventing matrix destruction by the tumor itself, and thus the ECM components of tumor origin are stably accumulated in the intercellular spaces and may support or promote the growth of AdCC cells, which have primitively low growth activity.

Growth inhibition and differentiation of human salivary adenocarcinoma cells by medium conditioned with normal human fibroblasts.

The present study demonstrates that normal human fibroblasts (WI-38) exert a profound influence on the growth and differentiation of HSGc-C5, a clonal neoplastic epithelial cell line of human salivary gland origin. Coculture of HSGc-C5 with WI-38 resulted in a slowing of growth and an increase in glycosaminoglycan synthesis by an indirect effect involving a diffusible factor(s). Conditioned medium (CM) from WI-38 grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum affected HSGc-C5 as follows. The CM suppressed growth of monolayer cells; inhibited DNA synthesis; suppressed growth (decrease in size of colonies) in semisolid agar; stimulated glycosaminoglycan synthesis, induced expression of functional markers of the salivary gland, such as the secretory component, lactoferrin, and lysozyme; inhibited expression of alkaline phosphatase; and induced morphological alteration into elongated cells. These findings strongly suggest that WI-38 CM contains a factor(s) which inhibits growth and induces differentiation of HSGc-C5. The CM was also active on other human cancer cells as a growth inhibitor, but not on normal human fibroblasts. Partial purification and characterization of the factor(s) suggests that it may be a novel protein carrying both tumor inhibiting and differentiation inducing activities.

Biological characterization of pseudocyst-forming cell lines from human adenoid cystic carcinomas of minor salivary gland origin.

Two cell lines (ACCS and ACCY) were isolated from two individuals with adenoid cystic carcinoma (AdCC) using tissue culture techniques. Both cell lines have similar morphology, i.e., elongated and flattened cells with slender cytoplasmic processes. The two cell lines tend to form pseudocysts, which are a specific architectural feature of AdCC. Coexpression of cytokeratin and vimentin was found in the two cell lines, which occasionally also contained S-100 protein and lactoferrin or lysozyme immunoreactivity. Moreover, ACCS and ACCY displayed potential for the production of a large amount of extracellular matrix including basal lamina components such as fibronectin, laminin, and type IV collagen and glycosaminoglycans which are also part of the basal lamina. These findings suggest that the tumor cells, probably basal or myoepithelial like cells, are responsible for the formation of the peculiar stroma of AdCC consisting of a large amount of collagen-like fibers, basal lamina components, and mucopolysaccharides.

IL-12 plays a pivotal role in LFA-1-mediated T cell adhesiveness by up-regulation of CCR5 expression.

The chemokine receptor CCR5 has been implicated in the recruitment of T cells to inflammatory sites. However, the regulation of CCR5 induction on T cells and its contribution to T cell adhesiveness are poorly understood. Using a Th1 clone, 2D6, that can be maintained with interleukin (IL)-12 or IL-2 alone (designated 2D6(IL-12) or 2D6(IL-2), respectively), we investigated how CCR5 is induced on T cells and whether CCR5 is responsible for up-regulating the function of adhesion molecules. 2D6(IL-12) grew, forming cell aggregates, in culture containing IL-12. This was due to lymphocyte function-associated antigen (LFA)-1-intercellular adhesion molecule (ICAM)-1 interaction, because 2D6(IL-12) expressed both LFA-1 and ICAM-1 and cell aggregation was inhibited by anti-ICAM-1 monoclonal antibody. Despite comparable levels of LFA-1 and ICAM-1 expression, 2D6(IL-2) cells did not aggregate in culture with IL-2. It is important that there was a critical difference in CCR5 expression between 2D6(IL-12) and 2D6(IL-2); the former expressed high levels of CCR5, and the latter expressed only marginal levels. Both types of cells expressed detectable albeit low levels of RANTES (regulated on activation, normal T expressed and secreted) mRNA. Unlike IL-12 or IL-2, IL-18 induced high levels of RANTES mRNA expression without modulating CCR5 expression. Therefore, combined stimulation with IL-12 and IL-18 strikingly up-regulated 2D6 cell aggregation. Notably, LFA-1-mediated aggregation of 2D6(IL-12) cells was suppressed by anti-CCR5 antibody. These results indicate that IL-12 plays a critical role in CCR5 expression on Th1 cells and consequently contributes to CCR5-mediated activation of LFA-1 molecules.

Establishment of bone morphogenetic protein 2 responsive chondrogenic cell line.

A clonal cell line named RMD-1 was established from the skeletal muscle of a 20-day fetal rat. RMD-1 represents a morphologically homogeneous population of undifferentiated mesenchymal cells, expressing alpha-smooth muscle actin and type I collagen, but no cartilage-associated genes. When cultured in agarose gel containing 100 ng/ml of recombinant human bone morphogenetic protein 2 (rhBMP-2; BMP-2), RMD-1 cells formed colonies and showed chondrocyte-like features as assessed by their ultrastructure, metachromatic staining with toluidine blue, and the production of large hydrodynamic-size proteoglycans. RMD-1 cells also differentiated into chondrocytes when the cells were plated at high density (over 2.5 x 10(5) cells/cm2) on type I collagen and incubated in medium containing 0.5% fetal bovine serum and 100 ng/ml of BMP-2. This chondrogenic differentiation was evidenced by a distinct morphological change into spherical cells, an increase in the levels of sulfated glycosaminoglycans, a decrease in type I collagen mRNA and the expression of cartilage-associated genes, including type II collagen, type IX collagen, aggrecan and alkaline phosphatase. In the presence of ascorbic acid and 10% serum, RMD-1 cells increased in size and expressed type X collagen as well as high alkaline phosphatase activity, then induced matrix mineralization. Thus, RMD-1 is a unique cell line that can differentiate from undifferentiated mesenchymal cells into hypertrophic chondrocytes.

Hedgehog proteins stimulate chondrogenic cell differentiation and cartilage formation.

Sonic hedgehog (Shh) and Indian hedgehog (Ihh) are important regulators of skeletogenesis, but their roles in this complex multistep process are not fully understood. Recent studies have suggested that the proteins participate in the differentiation of chondrogenic precursor cells into chondrocytes. In the present study, we have tested this possibility more directly. We found that implantation of dermal fibroblasts expressing hedgehog proteins into nude mice induces ectopic cartilage and bone formation. Immunohistological and reverse-transcription polymerase chain reaction (RT-PCR) analyses revealed that the ectopic tissues derived largely if not exclusively from host cells. We found also that treatment of clonal prechondrogenic RMD-1 and ATDC5 cells in culture with Ihh or recombinant amino half of Shh (recombinant N-terminal portion of Shh [rShh-N]) induced their differentiation into chondrocytes, as revealed by cytoarchitectural changes, Alcian blue staining and proteoglycan synthesis. Induction of RMD-1 cell differentiation by Ihh or rShh-N was synergistically enhanced by cotreatment with bone morphogenetic protein 2 (BMP-2) but was blocked by cotreatment with fibroblast growth factor 2 (FGF-2). Our findings indicate that hedgehog proteins have the ability to promote differentiation of chondrogenic precursor cells and that their action in this process can be influenced and modified by synergistic or antagonist cofactors.

Cloning and sequencing of the gene coding for dextranase from Streptococcus salivarius.

We cloned and sequenced the dextranase (Dex) (1,6-alpha-glucanhydrolase; EC 3.2.1.11)-encoding gene from Streptococcus salivarius (Ss) strain M-33. Recombinant clones from an Ss genomic library specifying Dex activity were identified as colonies surrounded by transparent halos on blue dextran plates. One of the clones had a 4.3-kb KpnI fragment containing the gene coding for an 826-amino-acid polypeptide with a molecular mass of 87.9 kDa, which corresponds well to that of native Dex from the Ss culture supernatant. There was no sequence homology between the gene encoding Ss Dex and the gene encoding dextran glucosidase of S. mutans, or between their protein products.

Physiologic and morphologic properties of motoneurons and spindle afferents innervating the temporal muscle in the cat.

Little is known about physiology and morphology of motoneurons and spindle afferents innervating the temporalis and on synaptic connections made between the two. The present study was aimed at investigating the above issues at the light microscopic level by using the intracellular recording and horseradish peroxidase or biotinamide labeling techniques and by the use of succinylcholine (SCh) for the classification of spindle afferents in the cat. Temporalis motoneurons had dendritic trees that ranged from a spherical form to an egg-shaped form. The shape deformation was more prominent for the dendritic trees made by motoneurons located closer to the nuclear border. No axon collaterals of the motoneurons were detected. On the basis of the values for the dynamic index after SCh infusion, temporalis spindle afferents were classified into two populations: presumptive groups Ia and II. The spindle afferents terminated mainly in the supratrigeminal nucleus (Vsup), region h, and the dorsolateral subdivision (Vmo.dl) of the trigeminal motor nucleus (Vmo). The proportion of group Ia afferent terminals was lower in the Vsup than that of group II afferents. In the Vmo.dl, the proportion of group Ia afferent terminals was nearly even throughout the nucleus, but that of group II afferent terminals increased in the more outlying regions. The proportion of terminal distribution in the central region of Vmo.dl was higher for group Ia than group II. The frequency of contacts (presumptive synapses) made by a single spindle afferent on a motoneuron was higher for group Ia than group II. The present study provided evidence that the central organization of spindle afferent neurons is different between groups Ia and II.

Effect of dexamethasone on invasion of human squamous cell carcinoma cells into collagen gel.

The effect of dexamethasone (Dex) on the ability to invade type I collagen gel was investigated in two cell lines of oral squamous cell carcinoma (SCC). At concentrations higher than 10(-8) M, Dex significantly suppressed the invasive growth of SCC cells into the gel. The same concentrations of Dex led to a decrease in urokinase type plasminogen activator (u-PA) synthesis and an increase in plasminogen activator inhibitor type 1 (PAI-1) synthesis by SCC cells. These findings suggest that Dex inhibits the invasiveness of SCC cells by decreasing their proteolytic activity.

Temporal patterns of trigeminal respiratory activity in rat brainstem-spinal cord in vitro.

Respiratory activity in trigeminal (V) motoneurons was studied in rhythmically active en bloc brainstem-spinal cord preparations isolated from neonatal rats (P0-P3). In the majority of preparations (83%), the temporal pattern of V activity consisted of spontaneous inspiratory phasic discharge with onset delayed or coincident with onset of phrenic motoneuron discharge. Blockade of alpha-2 noradrenergic receptor activation shifted onset of V respiratory discharges earlier than phrenic discharges, while elevation of extracellular potassium concentration or blockade of GABAergic and glycinergic inhibitory synaptic transmission had little effect on temporary pattern of V respiratory discharges. We conclude V motoneurons in the in vitro preparation generate respiratory activity during inspiratory phase, and their temporal patterns are modulated by inhibitory noradrenergic synaptic transmission.

Localization of oral-motor rhythmogenic circuits in the isolated rat brainstem preparation.

Using an in vitro isolated brainstem preparation from neonatal rat (0-2 days), the minimal circuitry for production of rhythmical oral-motor activity was determined. In the presence of the excitatory amino acid agonist, N-methyl-D,L-aspartate (NMA), and the GABAA antagonist, bicuculline (BIC), rhythmical oral-motor activity was recorded from the motor branch of the trigeminal nerve. In preparations where the brainstem was isolated in continuity between the rostral inferior colliculus and the obex, oral-motor activity was not observed. However, when the brainstem was serially transected in the coronal plane starting at the obex and proceeding rostrally, rhythmogenic activity emerged and became more stable until the level of the rostral facial nucleus (facial colliculus, FC) was approached. Transections more rostral than the FC produced rhythms that progressively deteriorated until the trigeminal motor nucleus (MoV) was reached, at which point all activities ceased. Surgical isolation of an ipsilateral quadrant of the brainstem encompassing the tissue between the FC and inferior colliculus, rostro-caudally, and the midline to lateral brainstem, medio-laterally, exhibited oral-motor activity as well. The remaining contralateral side of brainstem was devoid of rhythmical trigeminal activity. However, further coronal transection of the remaining brainstem at the level of the FC induced rhythmical oral-motor activity in the trigeminal nerve. The data suggest the existence of bilaterally coordinated rhythmogenic circuits in each half of brainstem between the rostral trigeminal nucleus and the rostral facial nucleus, which are tonically inhibited by brainstem circuits caudal to the facial nucleus.

Antagonism by glibenclamide of the effect of morphine in hippocampal preparations.

We previously showed that morphine lowered the affinity of Ca2+ antagonist binding and subsequently enhanced field potentials in hippocampal preparations. In the present study, the effect of various K+ channel antagonists on these actions of morphine was studied. Higher Kd value of [3H]nitrendipine binding was obtained for membranes prepared from slices treated with morphine. Concomitant treatment of slices with morphine and tetramethylammonium (TMA) or glibenclamide attenuated the effect of morphine. Apamin and mast cell-degranulating (MCD) peptide were without effect on morphine-induced change in [3H]nitrendipine binding. In those experiments, no change in concentration of binding sites was observed. Glibenclamide reduced the morphine enhancement of field potentials. These results suggested the regulation of Ca2+ channels by morphine through K+ channel opening.

Effect of lung inflation on levator veli palatini muscle activity.

Palatal movements play a critical role in regulating oropharyngeal airflow during breathing. We hypothesized that these movements are coordinated with breathing movements via afferent signals from the lung. However, the control of palatal movements in relation to the lung remains unclear. This study was designed to define, by electromyographic techniques, the relationship between palatal movement and lung action during respiration. We performed tracheotomies on 12 mongrel dogs anesthetized with sodium pentobarbital and found that lung inflation augmented the activity of the levator veli palatini muscle (LVP). Two kinds of discharges were recognized during the expiratory pause following lung inflation. One was a continuous, low-amplitude discharge induced during apnea following lung inflation. The other was a transient, high-amplitude discharge which appeared immediately after lung inflation. Both of these response activities were eliminated by bilateral vagotomy. We thus concluded that palatal movements, which can regulate expiratory airflow resistance and cause switching from nasal to oral airflow, are under the control of vagal afferent signals from the lung.

Measurement of velopharyngeal movements induced by isolated stimulation of levator veli palatini and pharyngeal constrictor muscles.

The levator veli palatini (LVP) and superior pharyngeal constrictor muscles (PC) close the velopharynx. However, for the velopharyngeal movements to be understood in detail, each muscle contraction must be analyzed precisely. This study was performed to clarify the velopharyngeal movement which was induced by a single muscle contraction, LVP or PC. Using a nasopharyngeal fiberscope, we analyzed the velopharyngeal movement associated with the contraction of the LVP and PC muscles in mongrel dogs. To elicit the maximal contraction of each muscle, we applied repetitive electrical stimulation to each peripheral nerve efferent to the LVP or PC muscle. Stimulation with a frequency of 77 Hz and 83 Hz induced maximal tension in the LVP and PC muscles, respectively, in isometric contraction. In a second series of experiments, fiberscopic views of the velopharyngeal movements associated with each muscle's maximal contraction were recorded. The degree of closure was calculated at several sections. The LVP muscle pulled the caudal fourth of the soft palate, while the PC projected the posterior wall at the level of the caudal end of the soft palate. The PC muscle also projected the lateral wall of the velopharynx. The effect of LVP contraction on the lateral wall was very small. These results show that the velopharyngeal movement associated with LVP contraction is very different from that with PC contraction.

Effect of serotonin (5-HT) on trigeminal rhythmic activities generated in in vitro brainstem block preparations.

We used rat isolated brainstem block preparations to analyze the functional roles of serotonin receptors in the generation of trigeminal rhythmic activities. We previously reported that trigeminal rhythmic activities could be induced by some pharmacological applications in an isolated brainstem preparation with a rostral boundary at the border between the inferior and superior colliculus, and a caudal border at the level of the rostral facial nucleus. However, the same stimulation did not induce trigeminal rhythmic activities in a whole brainstem block preparation with the same rostral boundary and a caudal border at the obex level. In the present study, both the 5-HT(1A) phthalimido-butyl-piperazine, and the 5-HT(2C) agonist, 1-2,5-dimethoxy-4-iodophenyl-2-aminopropane, combined with N-methyl-D,L-aspartate and bicuculline, elicited trigeminal rhythmic activities in a whole brainstem block preparation. Our results suggest that serotonin has both facilitation and inhibition effects on the generation of trigeminal rhythmic activities in an isolated brainstem block preparation in vitro.

The innervation of the levator veli palatini muscle by the glossopharyngeal nerve.

We developed a novel method which enables bloodless exposure of the levator veli palatini muscle in rat in order to investigate the physiological properties of this muscle. The levator veli palatini muscle which is innervated by a branch of the glossopharyngeal nerve showed rhythmic spontaneous movement in rats. Cutting the branch supplying LVP of the glossopharyngeal nerve caused cessation of the spontaneous movement of the levator veli palatini muscle. The spontaneous discharges of the glossopharyngeal nerve were synchronized with those of the phrenic nerve. A mixture of 95% oxygen and 5% room air influenced the efferent discharges from the branch of the glossopharyngeal nerve supplying the levator veli palatini muscle. These findings indicate that the motor nerve supply to the levator veli palatini muscle is the glossopharyngeal nerve, and the levator veli palatini muscle is related to the respiratory system, in particular with inspiration in rats.

Effect of norepinephrine receptors on trigeminal rhythm generation in newborn rats.

N-methyl-D,L-aspartate acid and bicuculline are required to enhance the trigeminal rhythmic activities in an in vitro isolated brainstem block preparation. In this study, we analyzed the effect of norepinephrine on the trigeminal neural circuit underlying rhythmic jaw movements. Rhythmic trigeminal activity is observed in brainstem preparations (inferior colliculus to obex) only following blockade of alpha(2)-adrenoceptors with idazoxan. This observation, combined with the inhibition of rhythm by alpha(2)-adrenoceptor agonists suggests endogenous alpha(2)-adrenoceptor mediated inhibition of trigeminal networks. A complex noradrenergic modulation of trigeminal systems is further supported by the prazosin-sensitive potentiation of rhythm by bath application of the alpha(1)-adrenoceptor agonist phenylephrine.

A fiberscopic study of velopharyngeal closure in patients with operated cleft palates.

We studied the differences in how velopharyngeal closure is learned and obtained by operated cleft palate patients during various activities. Sixty-eight operated cleft palate patients, who had complete closure during swallowing, were examined with the nasopharyngeal fiberscope to determine the extent of velopharyngeal closure while they were producing pressure consonants or vowels, and during blowing. We concluded that the complete closure when producing vowels was the most difficult to obtain, and closure when producing pressure consonants was a little more difficult than that during blowing.