Association of PD-L1 overexpression with activating EGFR mutations in surgically resected nonsmall-cell lung cancer.

BACKGROUND: Recent clinical trials have shown that immune-checkpoint blockade yields a clinical response in a subset of individuals with advanced nonsmall-cell lung cancer (NSCLC). We examined whether the expression of programmed death-ligand 1 (PD-L1) is related to clinicopathologic or prognostic factors in patients with surgically resected NSCLC. PATIENTS AND METHODS: The expression of PD-L1 was evaluated by immunohistochemical analysis in 164 specimens of surgically resected NSCLC. Cell surface expression of PD-L1 in NSCLC cell lines was quantified by flow cytometry. RESULTS: Expression of PD-L1 in tumor specimens was significantly higher for women than for men, for never smokers than for smokers, and for patients with adenocarcinoma than for those with squamous cell carcinoma. Multivariate analysis revealed that the presence of epidermal growth factor receptor gene (EGFR) mutations and adenocarcinoma histology were significantly associated with increased PD-L1 expression in a manner independent of other factors. Cell surface expression of PD-L1 was also significantly higher in NSCLC cell lines positive for activating EGFR mutations than in those with wild-type EGFR. The EGFR inhibitor erlotinib downregulated PD-L1 expression in the former cell lines but not in the latter, suggesting that PD-L1 expression is increased by EGFR signaling conferred by activating EGFR mutations. A high level of PD-L1 expression in resected tumor tissue was associated with a significantly shorter overall survival for NSCLC patients. CONCLUSIONS: High expression of PD-L1 was associated with the presence of EGFR mutations in surgically resected NSCLC and was an independent negative prognostic factor for this disease.

Assessment of the albumin-bilirubin grade as a prognostic factor in patients with non-small-cell lung cancer receiving anti-PD-1-based therapy.

INTRODUCTION: The albumin-bilirubin (ALBI) grade is a novel indicator of the liver function. Some studies showed that the ALBI grade was a prognostic and predictive biomarker for the efficacy of chemotherapy in cancer patients. The association between the ALBI grade and outcomes in patients with non-small-cell lung cancer (NSCLC) treated with cancer immunotherapy, however, is poorly understood. METHODS: We retrospectively enrolled 452 patients with advanced or recurrent NSCLC who received anti-programmed cell death protein 1 (PD-1)-based therapy between 2016 and 2019 at three medical centers in Japan. The ALBI score was calculated from albumin and bilirubin measured at the time of treatment initiation and was stratified into three categories, ALBI grade 1-3, with reference to previous reports. We examined the clinical impact of the ALBI grade on the outcomes of NSCLC patients receiving anti-PD-1-based therapy using Kaplan-Meier survival curve analysis with log-rank test and Cox proportional hazards regression analysis. RESULTS: The classifications of the 452 patients were as follows: grade 1, n = 158 (35.0%); grade 2, n = 271 (60.0%); and grade 3, n = 23 (5.0%). Kaplan-Meier survival curve analysis showed that the ALBI grade was significantly associated with progression-free survival and overall survival. Moreover, Cox regression analysis revealed that the ALBI grade was an independent prognostic factor for progression-free survival and overall survival. CONCLUSION: The ALBI grade was an independent prognostic factor for survival in patients with advanced or recurrent NSCLC who receive anti-PD-1-based therapy. These findings should be validated in a prospective study with a larger sample size.

Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus.

Several plasma membrane chloride channels are well characterized, but much less is known about the molecular identity and function of intracellular Cl- channels. ClC-3 is thought to mediate swelling-activated plasma membrane currents, but we now show that this broadly expressed chloride channel is present in endosomal compartments and synaptic vesicles of neurons. While swelling-activated currents are unchanged in mice with disrupted ClC-3, acidification of synaptic vesicles is impaired and there is severe postnatal degeneration of the retina and the hippocampus. Electrophysiological analysis of juvenile hippocampal slices revealed no major functional abnormalities despite slightly increased amplitudes of miniature excitatory postsynaptic currents. Mice almost lacking the hippocampus survive and show several behavioral abnormalities but are still able to acquire motor skills.

Immunoisolation of GABA-specific synaptic vesicles defines a functionally distinct subset of synaptic vesicles.

Synaptic vesicles from mammalian brain are among the best characterized trafficking organelles. However, so far it has not been possible to characterize vesicle subpopulations that are specific for a given neurotransmitter. Taking advantage of the recent molecular characterization of vesicular neurotransmitter transporters, we have used an antibody specific for the vesicular GABA transporter (VGAT) to isolate GABA-specific synaptic vesicles. The isolated vesicles are of exceptional purity as judged by electron microscopy. Immunoblotting revealed that isolated vesicles contain most of the major synaptic vesicle proteins in addition to VGAT and are devoid of vesicular monoamine and acetylcholine transporters. The vesicles are 10-fold enriched in GABA uptake activity when compared with the starting vesicle fraction. Furthermore, glutamate uptake activity and glutamate-induced but not chloride-induced acidification are selectively lost during immunoisolation. We conclude that the population of GABA-containing synaptic vesicles is separable and distinct from vesicle populations transporting other neurotransmitters.

Identification of differentiation-associated brain-specific phosphate transporter as a second vesicular glutamate transporter (VGLUT2).

Glutamate is the major excitatory neurotransmitter in mammalian CNS. In the presynaptic nerve terminal, glutamate is stored in synaptic vesicles and released by exocytosis. Previously, it has been shown that a transport protein originally identified as a brain-specific Na(+)-dependent inorganic phosphate transporter I (BNPI) functions as vesicular glutamate transporter and thus has been renamed VGLUT1. Recently, a protein highly homologous to VGLUT1, "differentiation-associated BNPI" (DNPI), has been discovered. Northern blot and in situ hybridization analyses indicate that DNPI mRNA is expressed in some brain regions in which VGLUT1 mRNA is not expressed. We now show that DNPI functions as vesicular glutamate transporter with properties very similar to VGLUT1 and propose to rename the protein VGLUT2. VGLUT2 is highly enriched in synaptic vesicles. Furthermore, VGLUT2 resides on a vesicle population that is distinct from vesicles containing the vesicular GABA transporter or VGLUT1, showing that the expression of VGLUT1 and VGLUT2 do not overlap. When VGLUT2 was expressed in BON cells, membrane fractions displayed ATP-dependent, carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive glutamate uptake. Overexpression of VGLUT2 in cultured autaptic GABAergic neurons yielded postsynaptic currents that were insensitive to the GABA(A) receptor antagonist bicuculline but blocked by the AMPA-receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[F]quinoxaline. Thus, expression of VGLUT2 suffices to cause GABAergic neurons to release glutamate in addition to GABA in a manner very similar to that reported previously for VGLUT1.

Subcellular localization of tetanus neurotoxin-insensitive vesicle-associated membrane protein (VAMP)/VAMP7 in neuronal cells: evidence for a novel membrane compartment.

The clostridial neurotoxin-insensitive soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors, tetanus neurotoxin-insensitive (TI)-vesicle-associated membrane protein (VAMP)/VAMP7, SNAP23, and syntaxin 3 have recently been implicated in transport of exocytotic vesicles to the apical plasma membrane of epithelial cells. This pathway had been shown previously to be insensitive to tetanus neurotoxin and botulinum neurotoxin F. TI-VAMP/VAMP7 is also a good candidate to be implicated in an exocytotic pathway involved in neurite outgrowth because tetanus neurotoxin does not inhibit this process in conditions in which it abolishes neurotransmitter release. We have now found that TI-VAMP/VAMP7 has a widespread distribution in the adult rat brain in which its localization strikingly differs from that of nerve terminal markers. TI-VAMP/VAMP7 does not enrich in synaptic vesicles nor in large dense-core granules but is associated with light membranes. In hippocampal neurons developing in vitro, TI-VAMP/VAMP7 localizes to vesicles in the axonal and dendritic outgrowths and concentrates into the leading edge of the growth cone, a region devoid of synaptobrevin 2, before synaptogenesis. After the onset of synaptogenesis, TI-VAMP/VAMP7 is found predominantly in the somatodendritic domain. In PC12 cells, TI-VAMP/VAMP7 does not colocalize with synaptobrevin 2, chromogranin B, or several markers of endocytic compartments. At the electron microscopic level, TI-VAMP/VAMP7 is mainly associated with tubules and vesicles. Altogether, these results suggest that TI-VAMP/VAMP7 defines a novel membrane compartment in neurite outgrowths and in the somatodendritic domain.

Ganglioside composition of GH3 cells: enhancement of fucoganglioside expression by estradiol, epidermal growth factor and insulin.

The GH3 cell line, a bipotential cell line secreting both prolactin (PRL) and growth hormone (GH), is a useful model for investigating GH/PRL cell lineage differentiation and anterior pituitary adenoma formation. In this study, we investigated the ganglioside composition of GH3 cells and identified two fucogangliosides as the major gangliosides expressed by these cells. Analyses by DEAE-Sephadex A-25 and thin-layer chromatography (TLC) revealed that the GH3 cells contained two major gangliosides, designated FG1 and FG2, respectively. Their structures were identified by fast atom bombardment mass spectrometry and proton nuclear magnetic resonance spectrometry: FG1 is IV2FUc alpha,II3NeuAc-GgOse4Cer and FG2 is IV2FUc alpha,IV3Gal alpha,II3NeuAc-GgOse4Cer. Expression of these fucogangliosides was enhanced by chronic treatment with 17 beta-estradiol (1 nM), epidermal growth factor (10 nM) and insulin (300 nM), which induced differentiation of GH3 cells to normal PRL-secreting cells. Interestingly, immunocytochemistry and flow cytometry revealed that the increased expression of these gangliosides reflected a quantitative change inside the cells but not on the cell surface. These results suggest that the intracellular distribution of fucogangliosides is closely related to the differentiation of GH3 cells.

Role of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism in mice.

In humans, mutations in the alpha-synuclein gene or exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produce Parkinson's disease with loss of dopaminergic neurons and depletion of nigrostriatal dopamine. alpha-Synuclein is a vertebrate-specific component of presynaptic nerve terminals that may function in modulating synaptic transmission. To test whether MPTP toxicity involves alpha-synuclein, we generated alpha-synuclein-deficient mice by homologous recombination, and analyzed the effect of deleting alpha-synuclein on MPTP toxicity using these knockout mice. In addition, we examined commercially available mice that contain a spontaneous loss of the alpha-synuclein gene. As described previously, deletion of alpha-synuclein had no significant effects on brain structure or composition. In particular, the levels of synaptic proteins were not altered, and the concentrations of dopamine, dopamine metabolites, and dopaminergic proteins were unchanged. Upon acute MPTP challenge, alpha-synuclein knockout mice were partly protected from chronic depletion of nigrostriatal dopamine when compared with littermates of the same genetic background, whereas mice carrying the spontaneous deletion of the alpha-synuclein gene exhibited no protection. Furthermore, alpha-synuclein knockout mice but not the mice with the alpha-synuclein gene deletion were slightly more sensitive to methamphetamine than littermate control mice. These results demonstrate that alpha-synuclein is not obligatorily coupled to MPTP sensitivity, but can influence MPTP toxicity on some genetic backgrounds, and illustrate the need for extensive controls in studies aimed at describing the effects of mouse knockouts on MPTP sensitivity.

Alteration of basal release of anterior pituitary hormones by pretreatment of primary cultured cells with trypsin.

The anterior pituitary (AP) gland secretes 6 different hormones. Prolactin (PRL) is secreted at a relatively high level without stimulation by the hypothalamus, while secretion of the others requires the action of stimulatory factors from the hypothalamus. In order to gain an insight into the mechanism underlying the different spontaneous release patterns of these hormones, we investigated their spontaneous release rate after pretreating rat anterior pituitary cells with trypsin. Rat AP cells were cultured on Cytodex microcarrier beads for 4 days and were then superfused with either control medium or medium containing trypsin (0.25%) for 5 min. The subsequent release rates of the AP hormones were monitored. The basal release of PRL was severely reduced to almost undetectable level and began to recover 120 min after the trypsin-pretreatment. Full recovery was attained over the next 100 min and was delayed by treatment with a protein synthesis inhibitor, cycloheximide (7 microM). In the trypsin-pretreated cells, basal release of PRL and growth hormone (GH) was severely reduced, while that of thyroid stimulating hormone (TSH) and adrenocorticotropic hormone (ACTH) was enhanced and luteinizing hormone (LH) and follicle stimulating hormone (FSH) was not markedly affected by the treatment, suggesting that the suppression of PRL release was not caused by nonspecific damage to the cells. Since trypsin does not readily enter cells, the altered secretion of AP hormones seems to be the result of restricted digestion of the external components of the cells. On the bases of these observations, we predicted that the mechanism of spontaneous release of hormones involves trypsin sensitive proteins (TSMP) on the plasma membranes of the anterior pituitary cells.

Experimental study of the histocompatibility of covered expandable metallic stents in the trachea.

STUDY OBJECTIVES: To evaluate the histocompatibility of four different materials used to cover expandable metallic stents. DESIGN: Prospective, randomized, unblinded study. SETTING: Animal research laboratory of Kurume University. PATIENTS OR PARTICIPANTS: Twenty 12- to 18-kg mongrel dogs. INTERVENTIONS: Handmade Gianturco-type stents with six bends, 20 mm in length, and 15 to 20 mm in diameter were covered using four different materials: polypropylene mesh, silicone-coated mesh, polyester mesh, and ePTFE. Covered stents were inserted into the trachea after i.v. anesthesia. Five animals were used in each group. MEASUREMENTS AND RESULTS: Postinsertion status was observed using clinical and bronchoscopy measures 7, 14, 28, and 56 days after intervention. Eight weeks after stent insertion, the tracheal specimens were studied pathologically. A scoring system was used to evaluate the histocompatibility of covered stents in each study group. The mean of clinical scores in the polypropylene group was 1.84+/-0.36, which was significantly higher than those in the groups of silicone, polyester, and ePTFE. The means of histopathologic scores in the polypropylene group and the silicone group were 1.60+/-0.55, which were significantly higher than those in groups of polyester and ePTFE. CONCLUSIONS: Polypropylene mesh cover was more suitable than silicone-coated mesh, polyester mesh, and ePTFE because of its excellent histocompatibility.

A preoperative alternating chemotherapy and radiotherapy program for patients with stage IIIA (N2) non-small cell lung cancer.

The objective of the present study was to evaluate the feasibility and toxicity of a preoperative alternating chemotherapy and radiotherapy program followed by surgery in stage IIIA non-small cell lung cancer (NSCLC). The tumor response, resection rate, tumor/lymph node downstaging, and survival were also evaluated. The positive predictive value (PPV) in the diagnosis of mediastinal lymph node metastasis was 81% using conventional magnetic resonance imaging (MRI) with short inversion-time inversion recovery (STIR) technique (STIR-MRI) on our criteria. Eligible patients had clinical N2 lesions (stage IIIA) and a World Health Organization (WHO) performance status of 0-2. The treatment program consisted of two courses of preoperative cisplatin, vindesine, and ifosfamide; alternating with radiotherapy, including two courses of 20 Gy radiation. Surgery was performed within 4 weeks after the treatment. Twenty-two patients with stage IIIA (N2) NSCLC (20 men and two women, age 35-71 years) were enrolled into the study. Hematologic and other toxicities were within an acceptable range. Surgery was not indicated for two patients because of distant metastasis; one patient with renal dysfunction and one with pancytopenia during this treatment underwent surgery subsequently. The clinical response rate was 50% (partial response in 11/22). Definitive surgery was indicated for 18 patients resulting in 17 patients with complete resection and one exploratory thoracotomy. A pathologic complete response of the primary tumor occurred in 41% of the patients (seven of 17; without residual tumor), whereas 58% (ten of 17) were pathologic N0. The median survival was 33 months with an actuarial 4-year survival rate of 33% in 17 patients with complete resection and 30 months with 28% 4-year survival rate in all entered patients. A randomized phase-III study using this approach for stage IIIA (clinical N-2 disease) is warranted.

Comparison between resected and irradiated small cell lung cancer in patients in stages I through IIIa.

The survival and recurrence of 37 patients with small cell lung cancer who underwent surgical resection were compared with those of 32 patients who were excluded from surgical resection but received radiotherapy. All but 2 patients received chemotherapy. The number of patients in the resected and nonresected groups in each pretreatment clinical stage were, respectively, as follows: 13 and 2 in stage I, 12 and 7 in stage II, and 12 and 23 in stage IIIa. The main reasons for exclusion from surgical resection were locally advanced disease in 15 patients, avoidance of pneumonectomy in 7, and poor pulmonary function in 5. In stage II, the mean tumor size was larger and there were fewer patients with peripheral tumors in the nonresected group than in the resected group. In stage IIIa, there were significantly more patients with overt N2 disease and central tumors in the nonresected group than in the resected group. The 5-year survival rate of the resected group in stage I was 67.7%. Although the nonresected group in stages II and IIIa had many adverse prognostic factors, there was no statistically significant difference between the survival of the resected and the nonresected groups. With respect to the site of first recurrence, a similar pattern was observed in the two groups in each stage, whereas local disease in stage I was completely controlled by surgical resection. These observations suggest that surgical resection can be considered a modality of treatment in clinical stage I. However, the treatment role of surgical resection in clinical stages II and IIIa, even in selected patients, remains unclear.

DNA ploidy pattern of each carcinomatous component in adenosquamous lung carcinoma.

The relationship between adenocarcinomatous and squamous carcinomatous components in 12 surgically resected adenosquamous lung carcinomas was analyzed using DNA flow cytometry. Well-preserved parts of the tumor showing either adenocarcinoma or squamous cell carcinoma were identified on paraffin blocks. The cells obtained from each component were stained with propidium iodide for DNA flow cytometry. In the analysis of DNA flow cytometry, both components in the same tumor were defined as being related to each other when they showed diploidy or when at least one DNA index of abnormal clones between two aneuploid components was identical. According to these criteria, 8 (67%) of the 12 tumors showed a relationship between adenocarcinomatous and squamous carcinomatous components. This suggests that, despite the different phenotypes, both components of some adenosquamous lung carcinomas may share similar biological characteristics.

Expandable metallic stents for tracheobronchial stenoses in esophageal cancer.

BACKGROUND: Tracheobronchial stenosis in patients with esophageal cancer can be life threatening. Few reports have discussed use of expandable metallic stents for central airway stenoses in patients with esophageal cancer. METHODS: Twelve patients with esophageal cancer underwent placement of expandable metallic stents for respiratory distress caused by tracheobronchial stricture. Single or double metallic stents were placed in the stenotic airways under fluoroscopic guidance. Improvement in respiratory symptoms and clinical outcome were assessed. RESULTS: Most stenoses were located in the trachea or the left main bronchus. From one to four expandable metallic stents were placed in each stricture site, with immediate relief of respiratory symptoms in 8 patients. One patient with tracheomalacia in alive 3 years after stent placement and another is alive 6 months after stent insertion. The other 10 patients lived from 10 to 70 days (mean; survival, 35 days) after stent placement. Death was due to progression of disease. CONCLUSIONS: Although metallic stents are useful for relieving respiratory distress in patients with advanced esophageal cancer, additional therapies should be considered.

Clinical investigation of endothelin-1 and nitric oxide in patients with portal hypertension focusing on plasma levels and immunohistological staining of liver tissues.

Plasma concentrations of endothelin-1 (ET-1) and nitrite/nitrate of patients with portal hypertension were measured. Sixteen patients with liver cirrhosis (the LC group) and 14 patients with idiopathic portal hypertension (the IPH group) and 12 healthy subjects (normal controls) were included in this study. The peripheral venous plasma concentration of ET-1 was significantly higher in the LC group (6.69+/-2.44 pg/ml) than in the IPH group (3.07+/-0.84 pg/ml) and normal controls (1.79+/-0.36 pg/ml), while the value in the IPH group was also significantly higher than that in normal controls. The peripheral venous plasma concentration of nitrite/nitrate was significantly higher in the LC group (67.7+/-38.9 &mgr;Mol/l) than in the IPH group (32.3+/-24.4 &mgr;Mol/l) and normal controls (26.1+/-9.8 &mgr;Mol/l). Hepatic venous plasma concentrations of ET-1 and nitrite/nitrate were measured in 8 patients from the LC group and 10 patients from the IPH group. The plasma concentration of ET-1 in the hepatic vein was significantly higher than that in the peripheral vein in both the LC and the IPH groups. The plasma concentration of nitrite/nitrate in the hepatic vein was significantly higher than that in the peripheral vein in the LC group. We also investigated the localization of ET-1, endothelin receptor (ET receptor) and nitric oxide synthase (NOS) in the liver tissue of LC patients (n=10), IPH patients (n=10) and normal controls (n=10). The expressions of ET-1, ET A receptors, ET B receptors, and inducive NOS (iNOS) were detected in patients with LC, and the labeling index (LI) was significantly higher than that in patients with IPH and normal controls. The expressions of ET-1, ET A receptors, and ET B receptors were found in patients with IPH, and the LI was significantly higher than that in normal controls. The expression of endothelial NOS (eNOS) was scarce in both LC and IPH patients. From these results, overproduction of ET-1 in the liver was regarded as one of the causes of the high plasma concentration of ET-1 in patients with LC and IPH. One of the causes of the high plasma concentration of nitrite/nitrate in LC was considered to be overproduction of nitric oxide (NO) in the liver. And we suggested that ET-1 is at a relatively higher density than NO in the hepatic sinusoid in LC and IPH.

Primary hemangiopericytoma of the superior mediastinum: a case report.

We present a case of hemangiopericytoma arising in the superior mediastinum. A 48-year-old man demonstrated a superior mediastinal mass lesion on chest x-ray and was admitted to our hospital. A CT scan of the chest showed a well-circumscribed, heterogenously-enhanced mass, 7.0 x 6.5 x 6.5 cm in size, located in the right superior posterior mediastinum, compressing the trachea. The tumor was extirpated by surgical resection. Based on the histological findings, electron microscopic findings, and immunohistochemical studies, the tumor was diagnosed as hemangiopericytoma.

Natriuretic peptides in the lung modulated by pneumonectomy.

BACKGROUND: Natriuretic peptides are vasodilator hormones involved in the regulation of blood pressure and volume homeostasis. However, the mechanism of these peptides after pneumonectomy remains obscure. METHODS: We investigated changes in the pulmonary arterial pressure and the localization and changes in the atrial (A-type) natriuretic peptide (ANP) and the C-type natriuretic peptide (CNP) in the lung, using immunohistochemistry and radioimmunoassay (RIA) in anesthetized dogs. Furthermore, we examined guanosine 3', 5'-monophosphate (cGMP) levels in plasma and in the contralateral lung. RESULTS: Pulmonary arterial pressure was significantly increased after pneumonectomy. The immunoreactivities of both ANP and CNP were detected in the endothelium of the pulmonary artery. In the contralateral lung, the concentrations of ANP and CNP were both significantly increased. In plasma, only ANP levels were significantly increased. In contrast, the plasma and lung cGMP levels were significantly reduced after pneumonectomy. CONCLUSIONS: We postulate that the processes from secretion in the vascular endothelial cells to the action via ANP and CNP receptors are effected in the contralateral lung tissue at the acute stage of pneumonectomy.

Thoracoscopic resection of a giant leiomyoma of the esophagus with a mediastinal outgrowth.

We reported a case of a 20-year-old man with a giant leiomyoma of the esophagus resected under video-assisted thoracic surgery (VATS). The patient demonstrated an abnormal shadow on a chest x-ray and a posterior mediastinal tumor 11 cm in diameter on a computed tomogram (CT) and on magnetic resonance imaging (MRI). A leiomyoma or a neurogenic tumor of the esophagus was suspected, and VATS was performed. The resected tumor was pathologically confirmed to be a leiomyoma of the esophagus. A giant esophageal leiomyoma showing extraluminal outgrowth should be treated by VATS.