Expressions of PD-L1 and Nectin-4 in urothelial cancer patients treated with pembrolizumab

ObjectivesRecently, the standard of care for advanced urothelial cancer (UC) has been changed by developing immune-checkpoint inhibitors (ICIs). However, its response rate is limited to 20–30%. The identification of biomarkers to predict the therapeutic effects of ICIs is urgently needed. The present study explored the association between immunohistochemical biomarkers and clinical outcomes in UC patients treated with pembrolizumab.Patients and methodsA total of 85 patients with UC who received pembrolizumab after chemotherapy from January 2018 to May 2020 were retrospectively reviewed. Tumor tissues were obtained for immunohistochemical study from 47 out of 85 patients. The protein expressions of PD-L1, WT1, Nectin-4, CD4, CD8, Foxp3, and CD68 in tumor cells and/or tumor infiltrating lymphocytes were immunohistochemically examined. The associations between protein expressions and overall survival (OS), progression-free survival (PFS), and disease control rate (DCR) were statistically analyzed.ResultsPatients with positive PD-L1 in tumor cells showed significantly worse OS (Log-rank test: HR 5.146, p = 0.001, Cox regression analysis: HR 4.331, p = 0.014) and PFS (Log-rank test: HR 3.31. p = 0.022), along with significantly lower DCR (14.3%) compared to the PD-L1 negative patients (67.5%). In addition, patients with strong expression of Nectin-4 in tumor cells showed significantly higher DCR (100%) than the other patients (50%).ConclusionPD-L1 expression in tumor cells was associated with poor prognosis (OS and PFS) and low DCR. Interestingly, the strong expression of Nectin-4 was correlated with high DCR. PD-L1 and Nectin-4 expression in tumor cells could be prognostic biomarkers useful for pembrolizumab in patients with advanced UC.

Expressions of PD-L1 and Nectin-4 in urothelial cancer patients treated with pembrolizumab.

OBJECTIVES: Recently, the standard of care for advanced urothelial cancer (UC) has been changed by developing immune-checkpoint inhibitors (ICIs). However, its response rate is limited to 20-30%. The identification of biomarkers to predict the therapeutic effects of ICIs is urgently needed. The present study explored the association between immunohistochemical biomarkers and clinical outcomes in UC patients treated with pembrolizumab. PATIENTS AND METHODS: A total of 85 patients with UC who received pembrolizumab after chemotherapy from January 2018 to May 2020 were retrospectively reviewed. Tumor tissues were obtained for immunohistochemical study from 47 out of 85 patients. The protein expressions of PD-L1, WT1, Nectin-4, CD4, CD8, Foxp3, and CD68 in tumor cells and/or tumor infiltrating lymphocytes were immunohistochemically examined. The associations between protein expressions and overall survival (OS), progression-free survival (PFS), and disease control rate (DCR) were statistically analyzed. RESULTS: Patients with positive PD-L1 in tumor cells showed significantly worse OS (Log-rank test: HR 5.146, p = 0.001, Cox regression analysis: HR 4.331, p = 0.014) and PFS (Log-rank test: HR 3.31. p = 0.022), along with significantly lower DCR (14.3%) compared to the PD-L1 negative patients (67.5%). In addition, patients with strong expression of Nectin-4 in tumor cells showed significantly higher DCR (100%) than the other patients (50%). CONCLUSION: PD-L1 expression in tumor cells was associated with poor prognosis (OS and PFS) and low DCR. Interestingly, the strong expression of Nectin-4 was correlated with high DCR. PD-L1 and Nectin-4 expression in tumor cells could be prognostic biomarkers useful for pembrolizumab in patients with advanced UC.

Properties of the feline tumour suppressor reduced expression in immortalized cells (REIC/Dkk-3).

Reduced expression in immortalized cells (REIC/Dkk-3), a member of the human Dickkopf (Dkk) family, is a growth suppressor in human and canine mammary tumours. Mammary gland tumours are common neoplasms with high malignancy in female cats. The purpose of this study was to clone the feline REIC/Dkk-3 homolog, investigate its expression in cell lines established from feline mammary gland tumours, and test its tumour suppressor function. Western blot analysis revealed that expression of the REIC/Dkk-3 protein was reduced in feline mammary carcinoma cell lines. Forced expression of REIC/Dkk-3 induced apoptosis in feline mammary tumour cell lines. These results demonstrate that REIC/Dkk-3 expression, which is downregulated in feline mammary tumour cell lines, results in the induction of apoptosis in these cells. Our findings suggest that feline REIC/Dkk-3 represents a potential molecular target for the development of therapies against feline mammary cancers.

Management of paediatric tuberculosis in provincial and district hospitals in Afghanistan.

Case detection, diagnosis and treatment of tuberculosis 1 B) in children are challenging issues vorldwide. This study in Afghanistan aimed to evaluate paediatric TB case management, including contact investigation, at health facilities where all diagnostic processes were available. In 7 out of 8 regions of the country 1 province was selected. Documents used for management of paediatric TB cases were reviewed in 15 distinct hospitals and 8 provincial hospitals in the selected provinces. The key issues which emerged were: a low suspect rate among total outpatients (0.4%) and a very low suspect rate among children aged < 5 years; low performance of suspect management (68.5% suspects received further examinations); low utilization of other diagnostic methods; a high early defaulter rate (14.0%); and insufficient coverage of contact management (74.0%). This survey indicated that the Afghanistan national TB programme needs to develop plans to improve the quality of diagnosis, suspect management and contact management in paediatric TB cases.

Management of paediatric tuberculosis in provincial and district hospitals in Afghanistan

Case detection, diagnosis and treatment of tuberculosis [TB] in children are challenging issues worldwide. This study in Afghanistan aimed to evaluate paediatric TB case management, including contact investigation, at health facilities where all diagnostic processes were available. In 7 out of 8 regions of the country 1 province was selected. Documents used for management of paediatric TB cases were reviewed in 15 distinct hospitals and 8 provincial hospitals in the selected provinces. The key issues which emerged were: a low suspect rate among total outpatients [0.4%] and a very low suspect rate among children aged < 5 years; low performance of suspect management [68.5% suspects received further examinations]; low utilization of other diagnostic methods; a high early defaulter rate [14.0%]; and insufficient coverage of contact management [74.0%]. This survey indicated that the Afghanistan national TB programme needs to develop plans to improve the quality of diagnosis, suspect management and contact management in paediatric TB cases

Association between the scores on the general health questionnaire-28 and the saliva levels of 3-methoxy-4-hydroxyphenylglycol in normal volunteers.

To access the saliva level of 3-methoxy-4-hydroxy-phenylglycol (sMHPG) as an index of mental health in normal volunteers, we investigated the relationship between the sMHPG and the scores on the general health questionnaire-28 (GHQ-28). A total of 270 normal volunteers answered the GHQ-28 and the sMHPG levels were determined. The sMHPG levels in women and men were comparable. There was a significant negative correlation between the social dysfunction score on the GHQ-28 and sMHPG levels in women (P=0.0035), but not in men. Moreover, the sMHPG levels also correlated with the total GHQ-28 score (P=0.0205), the anxiety and the insomnia score (P=0.0306) in women. These data indicate a high social dysfunction score on the GHQ-28 to be associated with a reduced noradrenergic neuronal tone thus possibly reflecting psychomotor retardation in women.

Involvement of the noradrenergic system in performance on a continuous task requiring effortful attention.

To determine the effects of noradrenergic neuronal activity on performance in continuous tasks requiring effortful attention, the performance of 23 male students in the Uchida-Kraepelin test (UKT) was examined. The UKT requires continuous arithmetic addition of single-digit figures for 25 min. The relationship of performance with saliva levels of 3-methoxy-4-hydroxyphenylglycol (sMHPG) was analyzed. Saliva samples were taken before, during and after test performance, and sMHPG levels determined by gas chromatography-mass spectrometry. There was no significant change in mean sMHPG as a result of test performance. However, when initial effort was calculated, defined as number of items completed during the 1st min subtracted from the average completed per minute in the 1st and the 2nd halves (blocks) of the test, significant correlations with sMHPG (p = 0.0002 for the 1st block and p < 0.0001 for the 2nd block) were found. Thus the data indicate that noradrenergic neuronal activity affects the performance on continuous tasks requiring effortful attention.

Non-functional C-cell adenoma in aged horses.

Thyroid tumours occur in older horses, and most such tumours have been considered to be of follicular epithelial origin. However, their immunohistochemical characterization has not been investigated. The purpose of this study was to confirm a suspicion that most of these tumours are in fact parafollicular cell (C cell)-derived adenomas, and to evaluate their pathogenesis and functional state. Thyroid glands from 38 horses aged 10-29 years were evaluated, all tissue samples being examined histologically, immunohistochemically and ultrastructurally. Nodular tumour masses were found in the thyroids of 12 of 38 horses older than 10 years (31.6%), and in nine of 12 horses older than 20 years (75.0%), regardless of sex or breed. Nodular lesions were composed of solid proliferations of polygonal cells with eosinophilic granular cytoplasm. Immunohistochemically, tumour cells were positive for calcitonin and neuron specific enolase, but negative for thyroglobulin. Ultrastructurally, few if any secretion granules were found in tumour cells. On the basis of these results it was concluded that the nodular lesions were C-cell adenomas, not follicular adenomas. It was suspected that the C-cell adenomas were non-functional and unlikely to lead to calcitonin hypersecretion-related diseases.

Malignant ameloblastic fibro-odontoma in a dog.

An 11-year-old male Collie was presented with a swelling of the face caused by tumor masses arising from the gingiva. Postmortem examination revealed metastases to the lymph nodes, lung, liver, and orbital cavity. Histologically, the tumor represented a combination of fibrosarcomatous proliferation, pulpal mesenchyme, and undifferentiated odontogenic epithelium, with a follicular or plexiform growth pattern. In addition, the follicular areas of the tumor showed a biphasic character, and there were numerous apoptotic cells in plexiform areas. Furthermore, acidophilic material resembling dysplastic dentine or enamel matrix was observed in the metastatic lesion in the lung. Based on the histological characters, the present case was diagnosed as malignant ameloblastic fibro-odontoma. This study is the first known description of a possible malignant ameloblastic fibro-odontoma in a dog with metastasis to distant organs.

Intramolecular epitope spreading among anti-caspase-8 autoantibodies in patients with silicosis, systemic sclerosis and systemic lupus erythematosus, as well as in healthy individuals.

Dysregulation of apoptosis through the Fas-Fas ligand pathway is relevant in autoimmune disease onset. We recently reported elevated serum levels of sFas in patients with silicosis, systemic sclerosis (SSC) and systemic lupus erythematosus (SLE), and proposed a block of apoptosis in the pathogenesis. The disturbance of apoptosis in lymphocytes including autoreactive clones could induce autoantibody production. Since autoantibodies directed against unknown antigens are present in the sera of these patients, the sera samples were examined for the presence of autoantibodies directed to caspase-8. Using Western blotting, autoantibodies against caspase-8 were detected in healthy individuals and in over 60% of patients. Using epitope mapping employing 12 amino acid polypeptides with SPOTs system, a minimum of 4 epitopes and a maximum of 13 were found, which implied that epitope spreading was in progress. It is noteworthy that two important catalytic cystein residues were included within the epitopes; firstly the active site cystein Cys287, and secondly Cys360 located in the unique pentapeptide motif QACQG. Using recombinant human caspase-8 linked protein chip array, autoantibodies were identified and molecular weight determined. The antibodies were mainly IgG; 80% were subclass IgG1(lambda); 20% were IgG4(kappa). Despite the ratio of human light chain kappa:lambda = 2:1, the predominance of IgG1(lambda) is noticeable. Anti-caspase-8 autoantibodies are detectable in healthy individuals and in patients suffering silicosis, SSc or SLE. A few epitopes were detected in healthy individuals compared to those suffering autoimmune diseases, indicating the intramolecular epitope spreading. Relationship of autoantibodies and the clinical background of the patients requires clarification.

Anti-leptin receptor antibody mimics the stimulation of lipolysis induced by leptin in isolated mouse fat pads.

An anti-leptin receptor polyclonal antibody (receptor antibody), as well as leptin, stimulated the release of free fatty acids from isolated mouse fat pads in a time-dependent manner. Following a 90-min incubation, maximal lipolysis was observed at 6 microg/ml receptor antibody and 0.1 nM leptin. The receptor antibody did not show any additive effect to the stimulation of lipolysis induced by leptin, suggesting that they exert their actions through a similar mechanism involving the leptin receptor. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), quin 2-AM, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and neomycin sulfate (neomycin) all potently inhibited the stimulation of lipolysis by the receptor antibody and leptin. Short-term incubation of the fat pads with the receptor antibody or leptin showed a transient increase in the cellular content of cAMP and myo-inositol 1,4,5-trisphosphate (IP3) in similar concentrations to the free fatty acid release. Quin 2-AM and W-7 also inhibited the increase in cAMP content, suggesting that a Ca(2)+/calmodulin-dependent process may be involved in a part of the mechanism in which the receptor antibody and leptin exert their effects. The increase in cellular IP3 content via phosphoinositide-specific phospholipase C (PLC) sensitive to neomycin appears to be a primary step to initiate intracellular events. Both the receptor antibody and leptin may stimulate the lipolysis through mechanisms involving a transient increase in the cellular IP3 content followed by cAMP production, which leads to the activation of cAMP-dependent protein kinase.

Orthovanadate decreases leptin secretion from isolated mouse fat pads.

When isolated mouse fat pads were incubated with orthovanadate (vanadate) or insulin for up to 4 h, the leptin secretion into the medium was decreased by vanadate and increased by insulin. Propranolol, a nonspecific antagonist of beta-adrenergic receptors, bupranorol, a specific antagonist of beta3-adrenergic receptor, and H-89, an inhibitor of cAMP-dependent protein kinase (PKA) all inhibited the decrease by vanadate to various extents. In contrast, no inhibition was observed with specific antagonists of beta1- and beta2-adrenergic receptors or with inhibitors of protein kinase C and Ca/calmodulin kinase. Short-term incubation of the fat pads with vanadate showed a transient increase in the cellular cAMP content; this increase was inhibited by propranolol and bupranolol. Vanadate had no effect on the incorporation of [3H]-leucine into proteins of the fat pads with a 4-h incubation, although insulin stimulated the incorporation. The decreasing effect of vanadate on the leptin secretion seems to be independent of the regulation of protein synthesis. These results suggest that vanadate decreases the leptin secretion through mechanisms involving the increase in cellular cAMP content via beta3-adrenergic receptor, probably leading to the activation of PKA.

Ultrastructure of aortic elastic fibers in copper-deficient Sika deer (Cervus nippon Temminck).

Light microscopic and transmission and scanning electron microscopic observations were performed on the aortas of two 4- and 6-year-old deer affected with cervine ataxia and two 6-month- and 4-year-old healthy deer. Examination of the aortas from affected deer by transmission electron microscopy revealed the absence of distinct elastic laminae in the internal elastic lamina and tunica media, but discontinuous and irregular clumps of elastin were present. Scanning electron microscopy disclosed immature architecture of elastic fibers in the aortas from the copper-deficient deer, and the architecture was similar to that of a 6-month-old healthy deer.

Reliability and validity of Kasahara's scale of melancholic type of personality (Typus melancholicus) in a German sample population.

We explored the reliability and validity of Kasahara's scale of melancholic type of personality (KMT) in a German sample population. Subjects comprised 66 patients diagnosed with an affective disorder (F3, ICD-10) and 94 controls. Concerning reliability, KMT scores showed internal consistency with Cronbach's alpha coefficients of 0.65 for patients and 0.67 for controls. The KMT items, except for number 13 in controls, showed significant item-total correlations. In a test-retest procedure, the KMT total score and individual item scores were statistically similar and correlated. These results indicate reliability of the KMT. Concerning validity, KMT scores were significantly higher in patients than in controls. By controlling the effects of age and sex, partial correlation coefficients in a comparison of KMT and Zerssen's F-List (F-List) scores were 0.40 in patients and 0.53 in controls. These results show both the constructive and concurrent validity of the KMT. Sufficient reliability and validity of the KMT were shown in this German sample population to encourage cross-cultural investigation of Typus melancholicus.

Discoid lupus erythematosus developing in areas where fragments of windshield glass had become embedded in the skin.

We observed a 26-year-old female patient with discoid lupus erythematosus on her left cheek skin, where fine fragments of windshield glass had been embedded in an automobile accident 8 years previously. She gradually developed general fatigue, morning stiffness of fingers and anti-nuclear antibody. As an etio-pathogenesis of this patient, we speculate that a long exposure to quartz (silica) could give rise to discoid lupus erythematosus in only local damaged areas and at the same time induce systemic immunological changes in some genetically restricted persons; such as production of anti-nuclear antibody. Silica has multi-potential biological effects, especially on immunological functions.

Immobilization of bisphosphonates on surface modified titanium.

The efficiency of surface modifications on the immobilization of bisphosphonates on titanium was investigated with Ca-ion implantation and thin hydroxyapatite coatings. The ALP activity of osteoblastic cells and the inhibitory effects on the initial adherence of P. gingivalis were also evaluated using bisphosphonate-immobilized titanium. X-ray photoelectron spectroscopy analysis suggested that titanium surfaces modified with Ca-ion implantation and thin hydroxyapatite coatings caused the immobilization of bisphosphonate on titanium plates. The ALP activity of osteoblastic cells cultured on plates immobilized with bisphosphonate was almost the same as that of cells cultured on titanium plates, indicating that the bisphosphonate-immobilization showed no toxic effect on osteoblastic cells, and that it provides a favorable micro-environment with osteogenetic ability. Data of the adherence of oral bacteria showed that a bisphosphonate-immobilized titanium surface inhibited the initial adherence of P. gingivalis. These results indicate that the immobilization of bisphosphonates on titanium modified with Ca-ion implantation and thin hydroxyapatite coatings are useful for dental implants.

Antidesmoglein autoantibodies in silicosis patients with no bullous diseases.

BACKGROUND: Pemphigus is an autoimmune bullous disease characterized by the presence of antidesmoglein autoantibodies. However, the mechanism of its autoantibody production remains unknown. In previous reports, we have described rare cases of pemphigus and pemphigoid associated with silicosis. It is well known that during long-term silicosis, some autoimmune diseases, such as systemic sclerosis, systemic lupus erythematosus or rheumatoid arthritis, can occur. OBJECTIVE: The aim of this study was to explore the presence of pemphigus or pemphigoid autoantibodies in silicosis patients without clinical bullous diseases or collagen diseases. METHOD: The presence of pemphigus antibodies was examined in 54 silicosis patients with no associated bullous diseases, using immunofluorescence, the enzyme-linked immunosorbent assay (ELISA) for desmoglein 1 and 3, and immunoblotting methods. In the antibody-positive cases, HLA genotyping of peripheral lymphocytes was performed with PCR-RFLP. RESULTS: Seven out of the 54 patients were found to be positive for pemphigus antibodies and 1 for bullous pemphigoid by immunofluorescence. In addition, by ELISA, 6 patients were found to be positive against the desmoglein 1 antigen, 2 against the desmoglein 3 antigen and 2 against both desmoglein 1 and desmoglein 3. CONCLUSION: The results of the present study strongly suggest the occurrence of pemphigus and pemphigoid autoantibodies in patients with silicosis. It remains unclear whether such patients will develop an autoimmune bullous disease in the future. Accordingly, long-term follow-up of antibody-positive patients is required.

Different distribution of HLA class II alleles in anti-topoisomerase I autoantibody responders between silicosis and systemic sclerosis patients, with a common distinct amino acid sequence in the HLA-DQB1 domain.

Autoantibodies against DNA topoisomerase I (anti-topo I) have been reported to be specific to systemic sclerosis (SSc), however, anti-topo I was detected in patients with silicone breast implants, SLE without features of SSc, and rheumatic diseases. We detected anti-topo I positive silicosis patients without any symptoms of autoimmune diseases. The correlation between anti-topo I autoantibody responses and HLA class II has been established. HLA-DRB1*1502; DQB1*0601 has been reported to be the most frequent anti-topo I associated haplotype among Japanese SSc patients. In this study, haplotype HLA-DR15; DQ6 was detected in all 4 anti-topo I positive Asian Japanese SSc patients randomly selected. Furthermore, HLA-DQB1*0402 was identified in 3 of 4 anti-topo I positive silicosis patients. These findings coincide with the results of a previous study, in which all 4 Japanese patients with anti-topo I had the DQB1*04 alleles, whereas no studies among Caucasian-Americans, African-Americans and Choctaw Indians found the involvement of DQB1*04. We investigated common features among various DQB 1 alleles. HLA-DQB I with a distinct characteristic is clearly involved in the anti-topo I response irrespective of ethnic groups, the main disease, or silica exposure. A common positioning of distinct amino acids, (i.e. positions 14, 30, 57 and 77 of the DQbeta1 domain are methionine, tyrosine, aspartic acid and threonine, respectively,) seems to be associated with anti-topo I response. The above-mentioned amino acid sequence is detected in alleles *0301, *0303, *0306, *0401, *0402, *0601 and *0602.

Bullous pemphigoid associated with silicosis.

Bullous pemphigoid (BP) has never before been reported to associate with silicosis, although there are numerous reports of silicosis accompanied by different autoimmune diseases, such as systemic sclerosis, systemic lupus erythematosus, dermatomyositis or rheumatoid arthritis. We report on a 63-year-old Japanese patient with silicosis who developed tensed bullae, erosions and macular pigmentation on the trunk and extremities. Indirect immunofluorescence revealed anti-basement-membrane-zone antibodies; immunoblotting analysis demonstrated that the patient's serum reacted with the 230-kD BP antigen in the epidermal extracts, as well as a recombinant protein of the NC16a domain of 180-kD BP antigen. Clinical symptoms improved after treatment with systemic steroids. To the best of our knowledge, this is the first reported case of BP associated with silicosis.