The real-world safety of atezolizumab as second-line or later treatment in Japanese patients with non-small-cell lung cancer: a post-marketing surveillance study.

BACKGROUND: We conducted a post-marketing surveillance study to evaluate the clinical tolerability and safety of atezolizumab in Japanese patients with non-small-cell lung cancer (NSCLC). METHODS: This prospective, observational post-marketing cohort study was conducted in NSCLC patients who received atezolizumab 1200 mg every 3 weeks at 770 facilities in Japan between April 18, 2018, and March 31, 2020 (study number UMIN000031978). Case report forms were completed, recording patient characteristics, treatment details, adverse events, adverse drug reactions (ADRs), their severity, onset and outcomes. Follow-up was for 12 months or until atezolizumab discontinuation. RESULTS: Overall, 2570 patients were included, median age was 69.0 years, and 69.9% were males. ADRs were reported in 29.1% of patients, most commonly pyrexia (4.2%). Grade ≥ 3 ADRs occurred in 9.7% of patients aged <75 and 9.7% of those aged ≥75 years. The incidence of Grade ≥ 3 ADRs was not affected by the number of lines of previous treatment or the presence or history of an autoimmune disorder. Immune-related ADRs of interest that occurred in >1% of patients were interstitial lung disease (ILD; 4.4%), endocrine disorder (4.3%), and hepatic dysfunction (2.8%). ILD was significantly more common in patients with a history of, or concurrent, ILD versus those without (P ≤ 0.001). Risk factors of Grade ≥ 3 ADRs were a history of, or concurrent, ILD. Grade 5 ADRs occurred in 35 patients, 11 of whom had concurrent ILD. CONCLUSIONS: This large cohort study confirmed the clinical tolerability of atezolizumab in a real-world group of Japanese patients with NSCLC.

Anxious about rejection, avoidant of neglect: Infant marmosets tune their attachment based on individual caregiver's parenting style.

Children's secure attachment with their primary caregivers is crucial for physical, cognitive, and emotional maturation. Yet, the causal links between specific parenting behaviors and infant attachment patterns are not fully understood. Here we report infant attachment in New World monkeys common marmosets, characterized by shared infant care among parents and older siblings and complex vocal communications. By integrating natural variations in parenting styles and subsecond-scale microanalyses of dyadic vocal and physical interactions, we demonstrate that marmoset infants signal their needs through context-dependent call use and selective approaches toward familiar caregivers. The infant attachment behaviors are tuned to each caregiver's parenting style; infants use negative calls when carried by rejecting caregivers and selectively avoid neglectful and rejecting caregivers. Family-deprived infants fail to develop such adaptive uses of attachment behaviors. With these similarities with humans, marmosets offer a promising model for investigating the biological mechanisms of attachment security.

Abundant occurrence of basal radial glia in the subventricular zone of embryonic neocortex of a lissencephalic primate, the common marmoset Callithrix jacchus.

Subventricular zone (SVZ) progenitors are a hallmark of the developing neocortex. Recent studies described a novel type of SVZ progenitor that retains a basal process at mitosis, sustains expression of radial glial markers, and is capable of self-renewal. These progenitors, referred to here as basal radial glia (bRG), occur at high relative abundance in the SVZ of gyrencephalic primates (human) and nonprimates (ferret) but not lissencephalic rodents (mouse). Here, we analyzed the occurrence of bRG cells in the embryonic neocortex of the common marmoset Callithrix jacchus, a near-lissencephalic primate. bRG cells, expressing Pax6, Sox2 (but not Tbr2), glutamate aspartate transporter, and glial fibrillary acidic protein and retaining a basal process at mitosis, occur at similar relative abundance in the marmoset SVZ as in human and ferret. The proportion of progenitors in M-phase was lower in embryonic marmoset than developing ferret neocortex, raising the possibility of a longer cell cycle. Fitting the gyrification indices of 26 anthropoid species to an evolutionary model suggested that the marmoset evolved from a gyrencephalic ancestor. Our results suggest that a high relative abundance of bRG cells may be necessary, but is not sufficient, for gyrencephaly and that the marmoset's lissencephaly evolved secondarily by changing progenitor parameters other than progenitor type.

Distinct roles of amylin and oxytocin signaling in intrafamilial social behaviors at the medial preoptic area of common marmosets.

Calcitonin receptor (Calcr) and its brain ligand amylin in the medial preoptic area (MPOA) are found to be critically involved in infant care and social contact behaviors in mice. In primates, however, the evidence is limited to an excitotoxic lesion study of the Calcr-expressing MPOA subregion (cMPOA) in a family-living primate species, the common marmoset. The present study utilized pharmacological manipulations of the cMPOA and shows that reversible inactivation of the cMPOA abolishes infant-care behaviors in sibling marmosets without affecting other social or non-social behaviors. Amylin-expressing neurons in the marmoset MPOA are distributed in the vicinity of oxytocin neurons in the anterior paraventricular nucleus of the hypothalamus. While amylin infusion facilitates infant carrying selectively, an oxytocin's inverse agonist, atosiban, reduces physical contact with non-infant family members without grossly affecting infant care. These data suggest that the amylin and oxytocin signaling mediate intrafamilial social interactions in a complementary manner in marmosets.

The common marmoset as suitable nonhuman alternative for the analysis of primate cochlear development.

Cochlear development is a complex process with precise spatiotemporal patterns. A detailed understanding of this process is important for studies of congenital hearing loss and regenerative medicine. However, much of our understanding of cochlear development is based on rodent models. Animal models that bridge the gap between humans and rodents are needed. In this study, we investigated the development of hearing organs in a small New World monkey species, the common marmoset (Callithrix jacchus). We describe the general stages of cochlear development in comparison with those of humans and mice. Moreover, we examined more than 25 proteins involved in cochlear development and found that expression patterns were generally conserved between rodents and primates. However, several proteins involved in supporting cell processes and neuronal development exhibited interspecific expression differences. Human fetal samples for studies of primate-specific cochlear development are extremely rare, especially for late developmental stages. Our results support the use of the common marmoset as an effective alternative for analyses of primate cochlear development.

Neuronal development in the cochlea of a nonhuman primate model, the common marmoset.

Precise cochlear neuronal development is vital to hearing ability. Understanding the developmental process of the spiral ganglion is useful for studying hearing loss aimed at aging or regenerative therapy. Although interspecies differences have been reported between rodents and humans, to date, most of our knowledge about the development of cochlear neuronal development has been obtained from rodent models because of the difficulty in using human fetal samples in this field. In this study, we investigated cochlear neuronal development in a small New World monkey species, the common marmoset (Callithrix jacchus). We examined more than 25 genes involved in the neuronal development of the cochlea and described the critical developmental steps of these neurons. We also revealed similarities and differences between previously reported rodent models and this primate animal model. Our results clarified that this animal model of cochlear neuronal development is more similar to humans than rodents and is suitable as an alternative for the analysis of human cochlear development. The time course established in this report will be a useful tool for studying primate-specific neuronal biology of the inner ear, which could eventually lead to new treatment strategies for human hearing loss.

Dynamic Spatiotemporal Expression Changes in Connexins of the Developing Primate's Cochlea.

Connexins are gap junction components that are essential for acquiring normal hearing ability. Up to 50% of congenital, autosomal-recessive, non-syndromic deafness can be attributed to variants in GJB2, the gene that encodes connexin 26. Gene therapies modifying the expression of connexins are a feasible treatment option for some patients with genetic hearing losses. However, the expression patterns of these proteins in the human fetus are not fully understood due to ethical concerns. Recently, the common marmoset was used as a primate animal model for the human fetus. In this study, we examined the expression patterns of connexin 26 and connexin 30 in the developing cochlea of this primate. Primate-specific spatiotemporal expression changes were revealed, which suggest the existence of primate-specific control of connexin expression patterns and specific functions of these gap junction proteins. Moreover, our results indicate that treatments for connexin-related hearing loss established in rodent models may not be appropriate for human patients, underscoring the importance of testing these treatments in primate models before applying them in human clinical trials.

Human-specific ARHGAP11B increases size and folding of primate neocortex in the fetal marmoset.

The neocortex has expanded during mammalian evolution. Overexpression studies in developing mouse and ferret neocortex have implicated the human-specific gene ARHGAP11B in neocortical expansion, but the relevance for primate evolution has been unclear. Here, we provide functional evidence that ARHGAP11B causes expansion of the primate neocortex. ARHGAP11B expressed in fetal neocortex of the common marmoset under control of the gene's own (human) promoter increased the numbers of basal radial glia progenitors in the marmoset outer subventricular zone, increased the numbers of upper-layer neurons, enlarged the neocortex, and induced its folding. Thus, the human-specific ARHGAP11B drives changes in development in the nonhuman primate marmoset that reflect the changes in evolution that characterize human neocortical development.

The polymicrogyria-associated GPR56 promoter preferentially drives gene expression in developing GABAergic neurons in common marmosets.

GPR56, a member of the adhesion G protein-coupled receptor family, is abundantly expressed in cells of the developing cerebral cortex, including neural progenitor cells and developing neurons. The human GPR56 gene has multiple presumptive promoters that drive the expression of the GPR56 protein in distinct patterns. Similar to coding mutations of the human GPR56 gene that may cause GPR56 dysfunction, a 15-bp homozygous deletion in the cis-regulatory element upstream of the noncoding exon 1 of GPR56 (e1m) leads to the cerebral cortex malformation and epilepsy. To clarify the expression profile of the e1m promoter-driven GPR56 in primate brain, we generated a transgenic marmoset line in which EGFP is expressed under the control of the human minimal e1m promoter. In contrast to the endogenous GPR56 protein, which is highly enriched in the ventricular zone of the cerebral cortex, EGFP is mostly expressed in developing neurons in the transgenic fetal brain. Furthermore, EGFP is predominantly expressed in GABAergic neurons, whereas the total GPR56 protein is evenly expressed in both GABAergic and glutamatergic neurons, suggesting the GABAergic neuron-preferential activity of the minimal e1m promoter. These results indicate a possible pathogenic role for GABAergic neuron in the cerebral cortex of patients with GPR56 mutations.

Modern slice culture for direct observation of production and migration of brain neurons.

For the understanding of histogenetic events in the three-dimensional brain primordia, direct observation of progenitor cells and young neurons is required. Although slice culture, which is one of the tissue or organ culture methods, effectively preserves the in vivo microenvironment where normal developmental processes occur, conventional phase-contrast microscopic observation of brain slices fails to provide good visibility of single cells. However, a combination of slice culture with the use of fluorescent dyes and/or the introduction of fluorescent protein genes provides live, three-dimensional information on cytogenetic and histogenetic events at the individual cell level. Dynamic cellular behaviors can then be vividly captured without destroying tissue structures.

Expression of Musashi1, a neural RNA-binding protein, in the cochlea of young adult mice.

Musashi1 (Msi1) is an RNA-binding protein expressed in neural stem/progenitor cells, astroglial progenitor cells and astrocytes in the vertebrate central nervous system. We hypothesized that Msi1 is expressed in only some of the supporting cells in the cochlea, which could become hair cell progenitors under special circumstances after an injury. To observe this, we investigated Msi1 expression in young adult mouse cochlea by immunohistochemistry using monoclonal antibody against Msi1. Msi1 immunostaining was found in a variety of supporting cells but not in outer hair cells in the organ of Corti. Although an immunoreactive ring was found around the inner hair cells, it also seemed to originate from the supporting cells. We suppose that this wide expression of Msi1 in supporting cells indicates that those cells might have the potential to become hair cell progenitors if injured, but that some other mechanisms strictly inhibit this ability.

Evolutionarily dynamic alternative splicing of GPR56 regulates regional cerebral cortical patterning.

The human neocortex has numerous specialized functional areas whose formation is poorly understood. Here, we describe a 15-base pair deletion mutation in a regulatory element of GPR56 that selectively disrupts human cortex surrounding the Sylvian fissure bilaterally including "Broca's area," the primary language area, by disrupting regional GPR56 expression and blocking RFX transcription factor binding. GPR56 encodes a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor required for normal cortical development and is expressed in cortical progenitor cells. GPR56 expression levels regulate progenitor proliferation. GPR56 splice forms are highly variable between mice and humans, and the regulatory element of gyrencephalic mammals directs restricted lateral cortical expression. Our data reveal a mechanism by which control of GPR56 expression pattern by multiple alternative promoters can influence stem cell proliferation, gyral patterning, and, potentially, neocortex evolution.

Regulation of myeloid zinc finger protein 2A transactivation activity through phosphorylation by mitogen-activated protein kinases.

The myeloid zinc finger protein (MZF)-2 is a C(2)H(2) zinc finger transcription factor that is expressed in myeloid cells and involved in the growth, differentiation, and tumorigenesis of myeloid progenitors. Here we describe a novel isoform of MZF-2, designated MZF-2A, and show that it is phosphorylated by the mitogen-activated protein (MAP) kinases. An in vitro phosphorylation experiment revealed that the transactivation domain (TAD) of MZF-2A was phosphorylated strongly by extracellular signal-regulated kinase (ERK) and phosphorylated weakly by p38 MAP kinase but not by Jun N-terminal kinase. Experiments using "add-back" mutants showed that three serine residues (Ser(257), Ser(275), and Ser(295)) in the TAD were phosphorylated in vitro by ERK. In myeloid LGM-1 cells, various extracellular stimuli induced the phosphorylation of these serine residues, which was differentially inhibited by the protein kinase inhibitors U0126 and SB203580. Substitution of these phosphorylation sites with alanines resulted in a strong enhancement of the ability of MZF-2A to activate transcription in a luciferase reporter assay. Taken together, these results indicate that MZF-2A is a novel target for the ERK and p38 MAP kinase signaling pathways, and its transactivation activity is negatively regulated by MAP kinase-mediated phosphorylation of the TAD.

Flow cytometric analysis of neural stem cells in the developing and adult mouse brain.

Despite recent progress in the neural stem cell biology, their cellular characteristics have not been described well. We investigated various characteristics of neural stem cells (NSCs) in vivo during CNS development, using FACS to identify the NSCs. We first examined stage-dependent changes in the physical parameters, using forward scatter (FSC) and side scatter (SSC) profiles, of NSCs from the developing striatum, where they appear to be active throughout the life of mammals. NSCs were divided into several fractions according to their FSC/SSC profile. With development, their number decreased in the FSC(high) fractions but increased in the FSC(low)/SSC(high) fraction, whereas NSCs were significantly concentrated in the fraction containing the largest cells (about 20 microm in diameter) at any stage, which were mostly the cells with the highest nestin-enhancer activity. Furthermore, we demonstrated that, at all stages examined, the "side population" (SP), defined as the Hoechst 33342 low/negative fraction, which is known to be a stem cell-enriched population in bone marrow, was also enriched for Notch1-positive immature neural cells (about 60%) from the developing striatum. However, these immature SP cells were not detected in the large-cell fraction, however, but were concentrated instead in the FSC(low/mid) fractions. FACS analysis showed that SP cells from adults were included to some extent in the CD24(low)/PNA(low) fraction, where NSCs were greatly concentrated. Collectively, the characteristics of NSCs were not uniform and changed developmentally.