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This study describes a novel surgical method to remove urinary bladder-related urachal abscesses in a cow. Traditional methods, such as the midline or paramedian approach in calves and the preinguinal approach, can be challenging in adult cattle, especially for lesions situated near the pelvic cavity. In this case, a 4-year-old cow had a urachal abscess attached to the bladder apex, making it difficult to approach via abdominal wall incision. Colpotomy and pulling the abscess through a vaginal wall incision allowed for complete exteriorization of the lesion and comfortable accessibility of the abscess and the resection site. This report suggests that colpotomy is an effective new route in adult cattle for accessing urachal abscesses not accessible via traditional surgical approaches.

Many researchers have focused on the role of the autonomic nervous system in the tumor microenvironment. Autonomic nerves include the sympathetic and parasympathetic nerves, which are known to induce cancer growth and metastasis. However, in salivary duct carcinoma (SDC), a rare and highly malignant tumor, the issue should be investigated from both biological and therapeutic perspectives. We explored the clinicopathological and prognostic implications of the autonomic nerves in 129 SDCs. Immunohistochemistry was performed to determine the nature of each nerve using antibodies against S100, tyrosine hydroxylase (TH) as a sympathetic marker, and vesicular acetylcholine transporter (VAChT) as a parasympathetic marker. The area of each marker-positive nerve was digitized and evaluated quantitatively. Double immunofluorescence for TH and VAChT was performed in selected cases. The expression of the secreted neurotrophins was also examined. S100-positive nerves were present in the cancer tissue in 94 of 129 cases (72.9%). Among them, TH-positive sympathetic nerves and/or VAChT-positive parasympathetic nerves were identified in 92 cases (97.9%), and 59 cases (62.8%) had TH/VAChT-co-expressing nerves. Double immunofluorescence revealed a mosaic pattern of sympathetic and parasympathetic fibers in co-expressing nerve bundles. The presence of autonomic nerves, regardless of their area, was significantly associated with aggressive histological features, advanced T/N classification, and a poor prognosis, with shorter disease-free and overall survival. There was an association between some tumor immune microenvironment-related markers and the autonomic nerve status, but not the latter and the secreted neurotrophin expression. This study suggests that autonomic nerves might play a role in the progression of SDC., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: To elucidate the clinical characteristics and progression rates of pachychoroid and conventional geographic atrophy (GA)., Design: Retrospective, multicenter, observational study., Participants: A total of 173 eyes from 173 patients (38 eyes with pachychoroid GA and 135 with conventional GA) from 6 university hospitals in Japan were included. All patients were Japanese, aged ≥50 years and with fundus autofluorescence images having analyzable image quality. A total of 101 eyes (22 with pachychoroid GA and 79 with conventional GA) were included in the follow-up group., Methods: The studied eyes were classified as having pachychoroid or conventional GA; the former was diagnosed if the eye had features of pachychoroid and no drusen. The GA area was semiautomatically measured on fundus autofluorescence images, and the GA progression rate was calculated for the follow-up group. Multivariable linear regression analysis was used to determine whether the rate of GA progression was associated with GA subtype., Main Outcome Measures: Clinical characteristics and progression rates of pachychoroid and conventional GA., Results: The pachychoroid GA group was significantly younger (70.3 vs. 78.7 years; P < 0.001), more male-dominant (89.5 vs. 55.6%; P < 0.001), and had better best-corrected visual acuity (0.15 vs. 0.40 in logarithm of the minimum angle of resolution; P  = 0.002), thicker choroid (312.4 vs. 161.6 μm; P < 0.001), higher rate of unifocal GA type (94.7 vs. 49.6%; P < 0.001), and smaller GA area (0.59 vs. 3.76 mm 2 ; P < 0.001) than the conventional GA group. In the follow-up group, the mean GA progression rate (square-root transformation) was significantly lower in the pachychoroid GA group than in the conventional GA group (0.11 vs. 0.27 mm/year; P  < 0.001)., Conclusions: Demographic and ocular characteristics differed between GA subtypes. The progression rate of pachychoroid GA, adjusted for age and baseline GA area, was significantly lower than that of conventional GA. Japanese patients with conventional GA showed characteristics and progression rates similar to those in White populations. Some characteristics of GA in Japanese population differ from those in Waucasian populations, which may be due to the inclusion of pachychoroid GA., Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article., (© 2024 by the American Academy of Ophthalmology.)

Introduction: NUT carcinoma is a rare cancer associated with a poor prognosis. Because of its rarity, its diagnosis is challenging and is usually made by excluding other diagnoses. Immunohistochemical analysis is a reliable technique that contributes to a correct diagnosis, but overestimating the expression of neuroendocrine (NE) markers may result in an incorrect diagnosis. In this study, we established the immunohistochemical phenotypes of NUT carcinoma compared with tumors that mimic its phenotype to identify potential diagnostic pitfalls., Methods: Eight cases of NUT carcinoma were examined along with eight basaloid squamous cell carcinomas and thirteen cases of small cell carcinoma using an immunohistochemical panel consisting of various antibodies., Results: Of the eight NUT carcinomas, three patients had a smoking history. All the cases examined for INSM1 were positive (6/6, 100%), although the staining was somewhat weak. Among the NE markers, synaptophysin was variably positive in two NUT carcinomas (2/6, 33%); however, all cases were negative for ASCL1, chromogranin A, and CD56. Moreover, the squamous cell markers, p40 and CK5/6, were weakly expressed in 4/6 (67%) and 3/6 (50%) of the NUT carcinomas, respectively., Conclusions: For tumors with an ambiguous morphology, applying the neuroendocrine phenotype of NUT carcinoma may be misleading; particularly, when distinguishing it from small-cell carcinoma. Similarly, null or weak expression of squamous cell markers may be observed in NUT carcinoma, but this differs from squamous cell carcinoma, which consistently demonstrates strong positivity for squamous cell markers., (© 2024. The Author(s).)

The toxicity of chemical substances to algal growth is generally measured by the 72-96 h algal growth inhibition test. We have developed a method to assess the toxicity of chemicals in aquatic environments more quickly and simply than conventional testing methods by delayed fluorescence (DF), which reflects the photosynthetic capacity of algae. The DF method is based on a technique for evaluating the amount of change in the decay curve due to the effects of chemicals ([Formula: see text], DF inhibition). Various studies on DF have been reported; however, few reports have evaluated the decay curve of DF by approach using inductive modeling based on measurement data such as principal component analysis (PCA) and partial least squares regression analysis (PLS). Therefore, the purpose of this study is to examine methods for estimating the magnitude and type of toxicity of chemicals by means of a principal component model (PC model) and multiple regression model (MR model) derived from changes in the decay curves of DF of algae exposed to a wide range of 37 toxic substances that have an effect of clear magnitude on algal growth. The changes in the DF decay curves due to exposure the 37 toxic substances to algae were summarized in the PC model composed of eigenvectors and scores of four principal components. For validation of usefulness, a hierarchical cluster analysis (HCA) of the amount of change in four PC scores revealed that the growth inhibition rate was more influential than the chemical type. We also found the possibility of quantitatively predicting the growth inhibition of chemicals by MR model by the amount of change in the PC scores., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To elucidate the clinical characteristics and progression rate of geographic atrophy (GA) associated with age-related macular degeneration (AMD) in a Japanese population., Design: Retrospective, multicenter, observational study., Participants: A total of 173 eyes from 173 patients from 6 university hospitals in Japan were included. Of 173 study eyes, 101 eyes from 101 patients were included in the follow-up group. All patients were Japanese, aged ≥ 50 years and had definite GA associated with AMD in at least 1 eye., Methods: The GA area was measured semiautomatically using fundus autofluorescence (FAF) images. In the follow-up group followed for > 6 months with FAF images, the GA progression rate was calculated by 2 methods: mm 2 per year and mm per year using the square-root transformation (SQRT) strategy. Simple and multiple linear regression analyses were used to identify the baseline factors associated with the GA progression rate., Main Outcome Measures: Clinical characteristics of GA and the GA progression rate., Results: The mean age was 76.8 ± 8.8 years, and 109 (63.0%) were males. Sixty-two (35.8%) patients had bilateral GA. The mean GA area was 3.06 ± 4.00 mm 2 (1.44 ± 1.00 mm [SQRT]). Thirty-eight eyes (22.0%) were classified as having pachychoroid GA. Drusen and reticular pseudodrusen were detected in 115 (66.5%) and 73 (42.2%) eyes, respectively. The mean subfoveal choroidal thickness was 194.7 ± 105.5 μm. In the follow-up group (follow-up period: 46.2 ± 28.9 months), the mean GA progression rate was 1.01 ± 1.09 mm 2 per year (0.23 ± 0.18 mm/year [SQRT]). In the multivariable analysis, the baseline GA area (SQRT; P = 0.002) and the presence of reticular pseudodrusen (P < 0.001) were significantly associated with a greater GA progression rate (SQRT)., Conclusions: Certain clinical characteristics of GA in Asian populations may differ from those in White populations. Asian patients with GA showed male dominance and relatively thicker choroid than White patients. There was a group with GA without drusen but with features of pachychoroid. The GA progression rate in this Asian population was relatively lower than that in White populations. Large GA and reticular pseudodrusen were associated with a greater GA progression rate., Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article., (Copyright © 2023 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

CFTR (cystic fibrosis transmembrane conductance regulator) is a tightly regulated anion channel that mediates chloride and bicarbonate conductance in many epithelia and in other tissues, but whether its regulation varies depending on the cell type has not been investigated. Epithelial CFTR expression is highest in rare cells called ionocytes. We studied CFTR regulation in control and ionocyte-enriched cultures by transducing bronchial basal cells with adenoviruses that encode only eGFP or FOXI1 (forkhead box I1) +  eGFP as separate polypeptides. FOXI1 dramatically increased the number of transcripts for ionocyte markers ASCL3 (Achaete-Scute Family BHLH Transcription Factor 3), BSND , ATP6V1G3 , ATP6V0D2 , KCNMA1 , and CFTR without altering those for secretory ( SCGB1A1 ), basal ( KRT5 , KRT6 , TP63 ), goblet ( MUC5AC ), or ciliated ( FOXJ1 ) cells. The number of cells displaying strong FOXI1 expression was increased 7-fold, and there was no evidence for a broad increase in background immunofluorescence. Total CFTR mRNA and protein levels increased 10-fold and 2.5-fold, respectively. Ionocyte-enriched cultures displayed elevated basal current, increased adenylyl cyclase 5 expression, and tonic suppression of CFTR activity by the phosphodiesterase PDE1C, which has not been shown previously to regulate CFTR activity. The results indicate that CFTR regulation depends on cell type and identifies PDE1C as a potential target for therapeutics that aim to increase CFTR function specifically in ionocytes.

Salivary duct carcinoma (SDC) is an aggressive type of salivary gland carcinoma. Recently, immunotherapies targeting immune checkpoints, including PD1, PD-L1, CTLA4, and LAG3, have had a considerable prognostic impact on various malignant tumors. The implementation of such immune checkpoint inhibitor (ICI) therapies has also been attempted in cases of salivary gland carcinoma. The tumor immune microenvironment (TIME) is implicated in tumorigenesis and tumor progression and is closely associated with the response to ICI therapies. However, the TIME in SDC has not been fully explored. We examined the immunohistochemical expression of CD8, FOXP3, PD1, PD-L1, CTLA4, LAG3, and mismatch repair (MMR) proteins, tumor-infiltrating lymphocytes (TILs), and microsatellite instability (MSI) status in 175 cases of SDC. The associations between these TIME-related markers and the clinicopathological factors and prognosis were evaluated. An elevated expression of CD8, FOXP3, PD1, CTLA4, and LAG3 was associated with more aggressive histological features and an advanced N and/or M classification, elevated Ki-67 index, and poor prognosis. Furthermore, cases with a high PD-L1 expression exhibited more aggressive histological features and adverse clinical outcomes than those with a low expression. Alternatively, there was no significant correlation between TILs and clinicopathological factors. No SDC cases with an MSI-high status or MMR deficiency were found. The coexistence of both an immunostimulatory and immunosuppressive TIME in aggressive SDC might play a role in the presence of T-cell exhaustion. The contribution of multiple immune escape pathways, including regulatory T cells and immune checkpoints, may provide a rationale for ICI therapy, including combined PD1/CTLA4 blockade therapy., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study aimed to illustrate the dose-response relationships of the direct scavenging activity of amide-based local anesthetics against multiple free radicals in vitro . We have demonstrated that amide-type local anesthetics selectively and directly scavenge some free radicals. Three kinds of free radicals were eliminated by all the four local anesthetics examined. Mepivacaine, lidocaine, bupivacaine, and dibucaine scavenged hydroxyl radicals in dose-dependent manners. Ascorbyl free radicals were also scavenged in dose-dependent manners, and lastly singlet oxygen was scavenged in dose-dependent manners. Three other free radicals were not scavenged by all of the four local anesthetics; tert -butoxyl radical was scavenged by all the anesthetics examined but dibucaine, nitric oxide by mepivacaine but not by the other three, and tyrosyl radical by mepivacaine and lidocaine but not by the other two. Some free radicals (superoxide anion, tert -butyl peroxyl radical, DPPH) were not scavenged by any of the four local anesthetics. The local anesthetics were also shown to inhibit lipid peroxidation by TBARS assay. These results suggest that local anesthetics have antioxidant properties through their free radical scavenging activities., Competing Interests: No potential conflicts of interest were disclosed., (Copyright © 2023 JCBN.)

Candida albicans can cause two major types of infections: superficial infection and systemic candidiasis. C. albicans infects diverse host niches, owing to a wide range of virulence factors and attributes, such as morphological transitions and phenotypic switching. C. albicans uses glycolysis, followed by alcoholic fermentation or mitochondrial respiration to rapidly generate ATP under aerobic conditions. In this study, we quantified the mRNA expression of several glycolysis-related enzymes associated with the initial phase of environmental changes using two strains: a type strain, NBRC 1385, and a strain from a patient with auto-brewery syndrome, LSEM 550. Additionally, we analyzed the regulation of a rate-limiting enzyme in glycolysis, phosphofructokinase 1 (PFK1). Our results showed that the mRNA expression of enzymes in the middle and last stages of glycolysis and alcoholic fermentation increased, and that of mitochondrial respiration enzymes decreased under short-term anaerobic conditions. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) administration showed similar results under anaerobic conditions. Moreover, PFK1 maintained its regulatory effect under different conditions; no significant change was observed in its mRNA expression. Our results suggest that C. albicans obtains energy via carbohydrate catabolism in the early phase of environmental change and survives in various parts of the host., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Okabayashi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Molecular targets and predictive biomarkers for prognosis in salivary duct carcinoma (SDC) have not been fully identified. We conducted comprehensive molecular profiling to discover novel biomarkers for SDC. A total of 67 SDC samples were examined with DNA sequencing of 464 genes and transcriptome analysis in combination with the clinicopathological characteristics of the individuals. Prognostic biomarkers associated with response to combined androgen blockade (CAB) treatment were explored using mRNA expression data from 27 cases. Oncogenic mutations in receptor tyrosine kinase (RTK) genes or genes in the MAPK pathway were identified in 55 cases (82.1%). Alterations in the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway were identified in 38 cases (56.7%). Interestingly, patient prognosis could be predicted using mRNA expression profiles, but not genetic mutation profiles. The risk score generated from the expression data of a four-gene set that includes the ADAMTS1, DSC1, RNF39, and IGLL5 genes was a significant prognostic marker for overall survival in the cohort (HR = 5.99, 95% confidence interval (CI) = 2.73-13.1, p = 7.8 × 10 -6 ). Another risk score constructed from the expression of CD3E and LDB3 was a strong prognostic marker for progression-free survival for CAB treatment (p = 0.03). Mutations in RTK genes, MAPK pathway genes, and PI3K/AKT pathway genes likely represent key mutations in SDC tumorigenesis. The gene expression profiles identified in this study may be useful for stratifying patients who are good candidates for CAB treatment and may benefit from additional systemic therapies., (© 2022. The Author(s).)

Background: The efficacy and safety of human epidermal growth factor receptor 2 (HER2)-targeted therapy and androgen deprivation therapy (ADT) for locally advanced or recurrent or metastatic (LA/RM) salivary duct carcinoma (SDC) have been reported in prospective studies. However, the survival benefit of these therapies to conventional therapy remains controversial, and whether HER2-targeted therapy or ADT should be chosen in HER2- and androgen receptor (AR)-positive SDC patients remains unknown., Methods: Overall, 323 LA/RM SDC patients treated at seven institutions between August 1992 and June 2020 were retrospectively enrolled. The primary aim was to analyze the effect of HER2-targeted therapy and ADT on overall survival from the diagnosis of LA/RM disease to death from any cause (OS1). The secondary indicators included the overall response rate (ORR), clinical benefit rate (CBR), overall survival from therapy initiation for LA/RM disease (OS2), progression-free survival (PFS), time to second progression (PFS2), duration of response (DoR), and duration of clinical benefit (DoCB) of HER2-targeted therapy or ADT as first-line therapy for HER2-positive/AR-positive SDC., Results: Patients treated with HER2-targeted therapy or ADT had longer OS1 than those treated without these therapies (Median OS1: historical control, 21.6 months; HER2-targeted therapy, 50.6 months; ADT, 32.8 months; HER2-targeted therapy followed by ADT, 42.4 months; and ADT followed by HER2-targeted therapy, 45.2 months, p  < 0.001). Among HER2-positive/AR-positive SDC patients, although HER2-targeted therapy had better ORR, CBR, and PFS than those of ADT as first-line therapy, we found no significant differences between HER2-targeted therapy and ADT regarding OS2, PFS2, DoR, and DoCB., Conclusion: Patients treated with HER2-targeted therapy and ADT showed longer survival in LA/RM SDC. HER2-targeted therapy can be recommended prior to ADT for HER2-positive/AR-positive SDC. It is warranted to establish a biomarker that could predict the efficacy of clinical benefit or better response in ADT., Competing Interests: Competing interests: The authors declare that there is no conflict of interest., (© The Author(s), 2022.)

Membrane proteins often cluster in nanoscale membrane domains (lipid rafts) that coalesce into ceramide-rich platforms during cell stress, however the clustering mechanisms remain uncertain. The cystic fibrosis transmembrane conductance regulator (CFTR), which is mutated in cystic fibrosis (CF), forms clusters that are cholesterol dependent and become incorporated into long-lived platforms during hormonal stimulation. We report here that clustering does not involve known tethering interactions of CFTR with PDZ domain proteins, filamin A or the actin cytoskeleton. It also does not require CFTR palmitoylation but is critically dependent on membrane lipid order and is induced by detergents that increase the phase separation of membrane lipids. Clustering and integration of CFTR into ceramide-rich platforms are abolished by the disease mutations F508del and S13F and rescued by the CFTR modulators elexacaftor plus tezacaftor. These results indicate CF therapeutics that correct mutant protein folding restore both trafficking and normal lipid interactions in the plasma membrane. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Background: Miyazaki Prefecture is one of the hotspots of severe fever with thrombocytopenia syndrome (SFTS) cases and related deaths in Japan since 2013 and other pathogens of tick-borne diseases (TBDs). Japanese spotted fever and scrub typhus are also endemic in this region., Objectives: A total of 105 wild boars, hunted in 2009, were serologically examined as sentinels for TBDs to indirectly demonstrate the potential hazard of ticks transmitting pathogens to humans in the studied area., Methods: The collected blood and spleens of the wild boars underwent serological and molecular tests for SFTSV, Rickettsia japonica (Rj) [antibody to spotted fever group rickettsiae (SFGR) were tested by using species-common antigen], and Orientia tsutsugamushi (Ot)., Results: Seroprevalences of SFTSV, SFGR, and Ot were 41.9%, 29.5%, and 33.3%, respectively. SFTS viral RNA was identified in 7.6% of the sera, whereas DNA of Rj or Ot was not detected in any sample. In total, 43.8% of the boars possessed an infection history with SFTSV (viral gene and/or antibody). Of these, 23.8% had multiple-infection history with SFGR and/or Ot., Conclusions: The high prevalence of SFTSV in wild boars might reflect the high risk of exposure to the virus in the studied areas. In addition, SFTSV infection was significantly correlated with Ot infection, and so were SFGR infection and Ot infection, indicating that these pathogens have common factors for infection or transmission. These data caution of the higher risk of SFTSV infection in areas with reported cases of other TBDs., (© 2021 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Objective: Salivary duct carcinoma (SDC) is a highly aggressive and uncommon tumor arising not only de novo but also in pleomorphic adenoma. Androgen receptor (AR)- and HER2-targeted therapy have recently been introduced for SDC as promising treatment options; however, no predictive biomarkers have yet been established. EZH2 and H3K27me3 are closely linked to the development and progression of various cancers, and EZH2 is also expected to be a desirable therapeutic target. We therefore explored the clinicopathological and prognostic implications of EZH2 and H3K27me3 in a large cohort of SDC patients, focusing on their impact on the therapeutic efficacy of AR- or HER2-targeted therapy., Materials and Methods: The EZH2 and H3K27me3 immunohistochemical expression and EZH2 Y646 gain-of-function mutation status were examined in 226 SDCs, and the relationship with the clinicopathological factors as well as clinical outcomes were evaluated within the three groups depending on the treatment: AR-targeted (combined androgen blockade with leuprorelin acetate and bicalutamide; 89 cases), HER2-targeted (trastuzumab and docetaxel; 42 cases), and conventional therapy (112 cases)., Results: EZH2 and H3K27me3 were variably immunoreactive in most SDCs. A positive correlation was found between the expression of EZH2 and H3K27me3. The EZH2 expression in the SDC component was significantly higher than that in the pre-existing pleomorphic adenoma component. EZH2 Y646 was not identified in any cases. EZH2-high cases more frequently had an advanced clinical stage and aggressive histological features than EZH2-low cases. An EZH2-high status in patients treated with AR-targeted therapy was associated with a significantly shorter progression-free and overall survival as well as a lower objective response rate and clinical benefit rate. In addition, a H3K27me3-high status in patients treated with AR-targeted therapy was related to a shorter overall survival. Conversely, there was no association between the EZH2 and H3K27me3 expression and the clinical outcomes in the conventional or HER2-targeted therapy groups., Conclusions: A high expression of EZH2 and H3K27me3 in SDC might be a predictor of a poor efficacy of AR-targeted therapy. Our data provide new insights into the role of EZH2 and H3K27me3 in therapeutic strategies for SDC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Saigusa, Hirai, Tada, Kawakita, Nakaguro, Tsukahara, Kano, Ozawa, Kondo, Okami, Togashi, Sato, Urano, Kajiwara, Shimura, Fushimi, Shimizu, Okamoto, Okada, Suzuki, Imanishi, Watanabe, Sakai, Ebisumoto, Sato, Honma, Yamazaki, Ueki, Hanazawa, Saito, Takahashi, Ando, Kohsaka, Matsuki and Nagao.)

Background/aims: Cystic fibrosis transmembrane conductance regulator (CFTR), the anion channel that is defective in cystic fibrosis (CF), is phosphorylated and activated by cAMP-dependent protein kinase (PKA). cAMP levels are downregulated by a large family of phosphodiesterases that have variable expression in different cell types. We have previously observed high levels of PDE8A expression in well-differentiated primary human bronchial epithelial (pHBE) cells and thus aimed to assess whether it played a role in cAMP-dependent regulation of CFTR activity., Methods: We assessed the effect of the selective PDE8 inhibitor PF-04957325 (PF) on intracellular cAMP levels ([cAMP] i ) in well differentiated pHBE cells from non-CF or CF donors and also in CFBE41o- cells that stably express wild-type CFTR (CFBE41o- WT) using ELISA and FRET-FLIM microscopy. CFTR channel function was also measured using electrophysiological recordings from pHBE and CFBE41o- WT cells mounted in Ussing Chambers., Results: PDE8 inhibition elevated [cAMP] i in well-differentiated pHBE cells and stimulated wild-type CFTR-dependent ion transport under basal conditions or after cells had been pre-stimulated with physiological cAMP-elevating agents. The response to PDE8 inhibition was larger than to PDE3 or PDE5 inhibition but smaller and synergistic with that elicited by PDE4 inhibition. CRISPR Cas9-mediated knockdown of PDE8A enhanced CFTR gene and protein expression yet reduced the effect of PDE8 inhibition. Acute pharmacological inhibition PDE8 increased CFTR activity in CF pHBE cells (F508del/F508del and F508del/R117H-5T) treated with clinically-approved CFTR modulators., Conclusion: These results provide the first evidence that PDE8A regulates CFTR and identifies PDE8A as a potential target for adjunct therapies to treat CF., Competing Interests: Partial support for this research was provided by Verona Pharma and KAB was an employee of Verona Pharma plc when the study was undertaken. Verona Pharma plc and Pfizer Inc. did not influence the design of the experiments or interpretation of the results., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

There is evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is highly expressed at the apical pole of ciliated cells in human bronchial epithelium (HBE), however recent studies have detected little CFTR mRNA in those cells. To understand this discrepancy we immunostained well differentiated primary HBE cells using CFTR antibodies. We confirmed apical immunofluorescence in ciliated cells and quantified the covariance of the fluorescence signals and that of an antibody against the ciliary marker centrin-2 using image cross-correlation spectroscopy (ICCS). Super-resolution stimulated emission depletion (STED) imaging localized the immunofluorescence in distinct clusters at the bases of the cilia. However, similar apical fluorescence was observed when the monoclonal CFTR antibodies 596, 528 and 769 were used to immunostain ciliated cells expressing F508del-CFTR, or cells lacking CFTR due to a Class I mutation. A BLAST search using the CFTR epitope identified a similar amino acid sequence in the ciliary protein rootletin X1. Its expression level correlated with the intensity of immunostaining by CFTR antibodies and it was detected by 596 antibody after transfection into CFBE cells. These results may explain the high apparent expression of CFTR in ciliated cells and reports of anomalous apical immunofluorescence in well differentiated cells that express F508del-CFTR., (© 2021. The Author(s).)

Purpose: There have been few reports on the evaluation of cancer cachexia based on skeletal muscle mass index (SMI) in patients with head and neck cancer., Patients and Methods: One hundred and ninety-two head and neck cancer patients were enrolled. In definitive and adjuvant chemoradiotherapy settings, clinical outcomes were compared between cachexia and non-cachexia patients., Results: Forty patients were diagnosed with cachexia (20.8%). In the definitive setting, overall survival (OS) was significantly shorter in the cachexia group (3-year OS: 50.0% vs 88.5%; p < 0.01), and multivariate analysis identified UICC stage IV, baseline albumin of <4 and cachexia as poor prognostic factors. However, cachexia was not significant in the adjuvant setting., Conclusion: Cancer cachexia was negatively associated with prognosis in patients with HNC who received definitive chemoradiotherapy. Nutritional intervention during chemoradiotherapy may improve survival in these patients., Competing Interests: NH reports personal fees from TAIHO Pharmaceutical Co., Ltd. YS reports personal fees from ONO Pharmaceutical Co., Ltd., Bristol-Myers Squibb Company, MSD K.K., and TAIHO Pharmaceutical Co., Ltd. NF reports personal fees from Eisai. JT reports personal fees from Eisai. ST reports grants and personal fees from ONO Pharmaceutical Co., Ltd., Bristol-Myers Squibb, MSD, AstraZeneca, Chugai, and BAYER. The authors report no other conflicts of interest in this work., (© 2021 Hayashi et al.)

Neuroendocrine carcinoma (NEC) of the head and neck is a rare type of malignancy, accounting for only 0.3% of all head and neck cancers, and its clinicopathological and genomic features have not been fully characterized. We conducted a retrospective analysis of 27 patients with poorly differentiated NEC of the head and neck seen at our institution over a period of 15 years. Patient characteristics, adopted therapies, and clinical outcomes were reviewed based on the medical records. Pathological analysis and targeted sequencing of 523 cancer-related genes were performed using evaluable biopsied/resected specimens based on the clinical data. The most common tumor locations were the paranasal sinus (33%) and the oropharynx (19%). Eighty-one percent of the patients had locally advanced disease. The 3-year overall survival rates in all patients and in the 17 patients with locally advanced disease who received multimodal curative treatments were 39% and 53%, respectively. Histologically, large cell neuroendocrine carcinoma was the predominant subtype (58% of evaluable cases), and the Ki-67 labeling index ranged from 59 to 99% (median: 85%). Next-generation sequencing in 14 patients identified pathogenic/likely pathogenic variants in TP53, RB1, PIK3CA-related genes (PREX2, PIK3CA, and PTEN), NOTCH1, and SMARCA4 in six (43%), three (21%), two (14%), two (14%), and one (7%) patients, respectively. Sequencing also detected the FGFR3-TACC3 fusion gene in one patient. The median value of the total mutational burden (TMB) was 7.1/Mb, and three patients had TMB ≥ 10. Regardless of the aggressive pathological features, our data revealed favorable clinical characteristics in the patients with locally advanced disease who received curative treatment. The lower TP53 and RB1 mutation prevalence rates compared to those described for small cell lung cancer suggests the biological heterogeneity of NEC in different parts of the body. Furthermore, the FGFR3-TACC3 fusion gene and mutations in genes encoding the components of the NOTCH and PI3K/AKT/mTOR pathways found in our study may be promising targets for NEC of the head and neck., (© 2021. The Author(s).)

Severe fever with thrombocytopenia syndrome (SFTS), caused by Dabie bandavirus , generally called SFTS virus (SFTSV), is an emerging zoonosis in East Asia. In Japan, 50-100 cases of SFTS have been reported each year since the first case was reported in 2013. SFTS is a tick-borne infectious disease, and SFTSV has been isolated from ticks in China and South Korea. Haemaphysalis longicornis and Amblyomma testudinarium are considered the primary vectors in Japan. However, the other tick species seldom feeding on humans might also play an important role in maintaining the virus in nature. In this study, we collected ticks on vegetation around the location where two SFTS patients were estimated to have been infected in Miyazaki Prefecture, Japan, isolated live SFTSV, and performed a phylogenetic analysis. A total of 257 ticks were collected, and SFTSV RNA was detected in 19.5% (9/46) of tick pools. A total of 10 infectious SFTSVs were successfully isolated from A. testudinarium , Haemaphysalis flava , Haemaphysalis formosensis , Haemaphysalis hystricis , and Haemaphysalis megaspinosa . Furthermore, the whole viral sequences isolated from ticks were highly homologous to sequences isolated from SFTS patients in the same sampling area in the past. These results suggest that SFTSVs are maintained in these tick species in the sampling area and sporadically transmitted to humans. Surveillance of SFTSV in ticks provides important information about the risk of incidental transmission to humans.

In-beam Mössbauer spectra of 57 Mn implanted into LiAlH 4 were measured at different temperatures between 17 and 300 K. The Mössbauer spectrum measured at 17 K showed two sets of doublets, which were assigned to 57 Fe atoms at substitutional sites at Al 3+ and Li + sites. The Debye temperatures θ M for the 57 Fe atoms at Al 3+ -substituted and Li + -substituted sites were estimated to be 194 K and 117 K, respectively. The assignments were confirmed by density functional theory calculations., (Copyright © 2020. Published by Elsevier Ltd.)

Two veterinary personnel in Japan were infected with severe fever with thrombocytopenia syndrome virus (SFTSV) while handling a sick cat. Whole-genome sequences of SFTSV isolated from the personnel and the cat were 100% identical. These results identified a nosocomial outbreak of SFTSV infection in an animal hospital without a tick as a vector.

Aspergillus fumigatus is an opportunistic mold that infects patients who are immunocompromised or have chronic lung disease, causing significant morbidity and mortality in these populations. While the factors governing the host response to A. fumigatus remain poorly defined, neutrophil recruitment to the site of infection is critical to clear the fungus. Galectin-3 is a mammalian β-galactose-binding lectin with both antimicrobial and immunomodulatory activities, however the role of galectin-3 in the defense against molds has not been studied. Here we show that galectin-3 expression is markedly up-regulated in mice and humans with pulmonary aspergillosis. Galectin-3 deficient mice displayed increased fungal burden and higher mortality during pulmonary infection. In contrast to previous reports with pathogenic yeast, galectin-3 exhibited no antifungal activity against A. fumigatus in vitro. Galectin-3 deficient mice exhibited fewer neutrophils in their airways during infection, despite normal numbers of total lung neutrophils. Intravital imaging studies confirmed that galectin-3 was required for normal neutrophil migration to the airspaces during fungal infection. Adoptive transfer experiments demonstrated that stromal rather than neutrophil-intrinsic galectin-3 was necessary for normal neutrophil entry into the airspaces. Live cell imaging studies revealed that extracellular galectin-3 directly increases neutrophil motility. Taken together, these data demonstrate that extracellular galectin-3 facilitates recruitment of neutrophils to the site of A. fumigatus infection, and reveals a novel role for galectin-3 in host defense against fungal infections., Competing Interests: The authors have declared that no competing interests exist.

Salivary duct carcinoma (SDC) is an aggressive, uncommon tumor histologically comparable to high-grade mammary ductal carcinoma. SDCs are usually androgen receptor (AR)-positive and often HER2-positive. Recently, therapies targeting these molecules for SDC have attracted attention. Lipid metabolism changes have been described in association with biological behavior in various cancers, although no such relationship has yet been reported for SDC. We therefore analyzed the clinicopathological relevance of the immunohistochemical expression of adipophilin (ADP) and fatty acid synthase (FASN), representative lipid metabolism-related proteins, in 147 SDCs. ADP and FASN were variably immunoreactive in most SDCs (both 99.3%), and the ADP and FASN expression was negatively correlated (P = 0.014). ADP-positive (≥ 5%) SDCs more frequently exhibited a prominent nuclear pleomorphism and high-Ki-67 labeling index than those ADP-negative (P = 0.013 and 0.011, respectively). In contrast, a high FASN score, calculated by the staining proportion and intensity, (≥ 120) was correlated with the high expression of AR and FOXA1 (P < 0.001 and = 0.003, respectively). The ADP and FASN expression differed significantly among the subtypes based on biomarker immunoprofiling, as assessed by the AR, HER2, and Ki-67 status (P = 0.017 and 0.003, respectively). A multivariate analysis showed that ADP-positive expression was associated with a shorter overall and progression-free survival (P = 0.018 and 0.003, respectively). ADP was associated with an aggressive histopathology and unfavorable prognosis, and FASN may biologically interact with the AR signaling pathway in SDC. ADP may, therefore, be a new prognostic indicator and therapeutic target in SDC.

There are concerns regarding overtreatment in papillary thyroid carcinoma (PTC). BRAF V600E and TERT promoter mutations play important roles in the development of PTC. However, initial surgical approaches for PTC based on genetic characteristics remain unclear. The present study aimed to identify genetic mutations as predictors of prognosis and to establish proper indications for lobectomy (LT) in patients with 1-4 cm intrathyroidal PTC. Prospectively accumulated data from 685 consecutive patients with PTC who underwent primary thyroid surgery at the Cancer Institute Hospital, Tokyo, Japan, between 2001 and 2012 were retrospectively reviewed. Of the 685 patients examined, 538 (78.5%) had BRAF V600E mutation and 133 (19.4%) had TERT promoter mutations. Patients with TERT promoter mutations displayed significantly worse outcomes than those without mutations (10-year cause-specific survival (CSS): 73.7% vs. 98.1%, p < 0.001; 10-year disease-free survival (DFS): 53.7% vs. 93.3%, p < 0.001). As for extent of thyroidectomy among TERT mutation-negative patients with 1-4 cm intrathyroidal PTC, patients who underwent LT showed no significant differences in 10-year CSS and 10-year DFS compared to patients who had total thyroidectomy (TT) under propensity score-matching. Avoiding TT for those patients indicates a possible pathway to prevent overtreatment and reduce postoperative complications.

Background: In the CheckMate-141 trial, nivolumab conferred a survival benefit in patients with recurrent or metastatic refractory squamous cell carcinoma (SCC) head and neck cancer (HNC). Here, we examined the efficacy of nivolumab in patients with histological subtypes or primary sites of HNC not included in the CheckMate-141 trial., Methods: This was a retrospective analysis of data collected prospectively from 97 patients who were treated with nivolumab for recurrent or metastatic HNC at our institution. The patients were assigned to three groups based on HNC primary site: 1) oral cavity, pharynx, and larynx, which were included in CheckMate-141 (n = 68), 2) nasopharynx (excluded in CheckMate-141, n = 7) and 3) other primary sites excluded in CheckMate-141 (n = 22) and assigned to two groups according to histological subtype: 1) SCC (included in CheckMate-141, n = 83) and 2) non-SCC (all sites excluded in CheckMate-141, n = 14). Survival outcomes and nivolumab treatment response were compared between the primary site and histological subgroups., Results: The median number of nivolumab treatments was 7 cycles (range, 1-53 cycles) and the median follow-up time was 9.1 months (range, 0.66-33.0 months). There were no significant differences in response rates between the three primary site subgroups (CheckMate-141 sites 22%, nasopharynx 43%, others 18%; p=0) or the two histological subtype subgroups (SCC 25%, non-SCC 7%, p=0). Similarly, overall survival and progression-free survival were comparable for patients stratified by primary site or histological subtype., Conclusion: No significant difference in response rates or survival outcomes was detected between nivolumab-treated HNC patients with primary sites and histological subtypes that were included versus excluded in the CheckMate-141 trial. These data provide a potential rationale for nivolumab therapy for all HNC patients in clinical practice., Competing Interests: Dr Yasuyoshi Sato reports personal fees from ONO Pharmaceutical Co., Ltd., Bristol-Myers Squibb Company, MSD K.K., TAIHO Pharmaceutical Co., Ltd., outside the submitted work. Dr Shunji Takahashi reports grants, personal fees from ONO Pharmaceutica and Bristol-Myers Squibb, during the conduct of the study; grants, personal fees from MSD, AstraZeneca, Chugai, and BAYER, outside the submitted work. The authors report no other conflicts of interest in this work., (© 2020 Sato et al.)

Salivary duct carcinoma (SDC) is a rare, aggressive malignancy that histologically resembles high-grade mammary duct carcinoma. Because of the rarity of this entity, data verifying the association between histologic features and patient survival are limited. We conducted a comprehensive histologic review of 151 SDC cases and performed an analysis of the association between various histomorphologic parameters and the clinical outcome with the aim of developing a histologic risk stratification model that predicts the prognosis of SDC patients. A multivariate analysis revealed that prominent nuclear pleomorphism (overall survival [OS]: P=0.013; progression-free survival [PFS]: P=0.019), ≥30 mitoses/10 HPF (PFS: P=0.013), high tumor budding (OS: P=0.011; PFS: P<0.001), and high poorly differentiated clusters (OS: P<0.001; PFS: P<0.001) were independent prognostic factors. Patients with vascular invasion demonstrated a marginally significant association with shorter PFS (P=0.064) in a multivariate analysis. We proposed a 3-tier histologic risk stratification model based on the total number of positive factors among 4 prognostically relevant parameters (prominent nuclear pleomorphism, ≥30 mitoses/10 HPF, vascular invasion, and high poorly differentiated clusters). The OS and PFS of patients with low-risk (0 to 1 point) (23% of cases), intermediate-risk (2 to 3 points) (54% of cases), and high-risk (4 points) (23% of cases) tumors progressively deteriorated in this order (hazard ratio, 2.13 and 2.28, and 4.99 and 4.50, respectively; Ptrend<0.001). Our histologic risk stratification model could effectively predict patient survival and may be a useful aid to guide clinical decision-making in relation to the management of patients with SDC.

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) disrupt epithelial secretion and cause cystic fibrosis (CF). Available CFTR modulators provide only modest clinical benefits, so alternative therapeutic targets are being explored. The anion-conducting transporter solute carrier family 26 member 9 (SLC26A9) is a promising candidate, but its functional expression is drastically reduced in cells that express the most common CF-associated CFTR variant, F508del-CFTR, through mechanisms that remain incompletely understood. Here, we examined the metabolic stability and location of SLC26A9 and its relationship to CFTR. Compared with SLC26A9 levels in BHK cells expressing SLC26A9 alone or with WT-CFTR, co-expression of SLC26A9 with F508del-CFTR reduced total and plasma membrane levels of SLC26A9. Proteasome inhibitors increased SLC26A9 immunofluorescence in primary human bronchial epithelial cells (pHBEs) homozygous for F508del-CFTR but not in non-CF pHBEs, suggesting that F508del-CFTR enhances proteasomal SLC26A9 degradation. Apical SLC26A9 expression increased when F508del-CFTR trafficking was partially corrected by low temperature or with the CFTR modulator VX-809. The immature glycoforms of SLC26A9 and CFTR co-immunoprecipitated, consistent with their interaction in the endoplasmic reticulum (ER). Transfection with increasing amounts of WT-CFTR cDNA progressively increased SLC26A9 levels in F508del-CFTR-expressing cells, suggesting that WT-CFTR competes with F508del-CFTR for SLC26A9 binding. Immunofluorescence staining of endogenous SLC26A9 and transfection of a 3HA-tagged construct into well-differentiated cells revealed that SLC26A9 is mostly present at tight junctions. We conclude that SLC26A9 interacts with CFTR in both the ER and Golgi and that its interaction with F508del-CFTR increases proteasomal SLC26A9 degradation., (© 2019 Sato et al.)

Introduction : Cystic fibrosis (CF) is the most frequent lethal orphan disease and is caused by mutations in the CFTR gene. The most frequent mutation F508del-CFTR affects multiple organs; infections and subsequent infections and complications in the lung lead to death. Areas covered : This review focuses on new targets and mechanisms that are attracting interest for the development of CF therapies. The F508del-CFTR protein is retained in the endoplasmic reticulum (ER) but has some function if it can traffic to the plasma membrane. Cell-based assays have been used to screen chemical libraries for small molecule correctors that restore its trafficking. Pharmacological chaperones are correctors that bind directly to the F508del-CFTR mutant and promote its folding and trafficking. Other correctors fall into a heterogeneous class of proteostasis modulators that act indirectly by altering cellular homeostasis. Expert opinion : Pharmacological chaperones have so far been the most successful correctors of F508del-CFTR trafficking, but their level of correction means that more than one corrector is required. Proteostasis modulators have low levels of correction but hold promise because some can correct several different CFTR mutations. Identification of their cellular targets and the potential for development may lead to new therapies for CF.

Aims: Salivary duct carcinoma (SDC) is an uncommon, aggressive tumour that, histologically, resembles high-grade mammary ductal carcinoma, and is characterised by the expression of androgen receptor (AR). The androgen signalling pathway, a potential therapeutic target, can be regulated by FOXA1. This study aimed to evaluate the clinicopathological implications of FOXA1 in SDC., Methods and Results: We examined the relationship between the immunoexpression of FOXA1 and FOXA1 mutations and clinicopathological factors, including the biomarker status and clinical outcome, in 142 SDCs. FOXA1 was expressed in 128 SDCs (90.1%); the immunoexpression was heterogeneous. SDCs with a higher FOXA1 labelling index (LI) (≥20%) more frequently showed less advanced tumors on T classification (P = 0.002). FOXA1 LI was correlated positively with the AR expression value (r = 0.430, P < 0.001). PI3K and p-mTOR positivity, and intact-PTEN, were associated with a higher FOXA1 LI. Twenty-two of 121 SDCs (18.2%) harboured FOXA1 gene mutations at the flanking regions in and around the forkhead DNA binding domain; however, the given gene mutation and the expression of FOXA1 were not significantly correlated. A multivariate analysis revealed that SDCs with a higher FOXA1 LI were associated with longer overall survival and progression-free survival (P = 0.029 and 0.016, respectively)., Conclusions: In SDC, FOXA1, which may biologically interact with the AR and PI3K signalling pathways, is a putative biomarker that may be associated with a favourable prognosis. Further studies are needed to apply the findings to the development of targeted personalised therapy for patients with SDC., (© 2018 John Wiley & Sons Ltd.)

Hyalinizing clear cell carcinoma of the bronchial glands is a very rare tumor. Since only five reports describing six tumors have been published to date, only a little is known about specific histologic findings and clinical features. Because of its rarity, hyalinizing clear cell carcinoma has not been described in the latest WHO classification of pulmonary tumors yet. Here we present three cases of bronchial hyalinizing clear cell carcinomas, confirmed by both fluorescence in situ hybridization (FISH) and RT-PCR, focusing on histologic and immunohistochemical characteristics in a comparison with three cases of salivary gland origin. In addition, we compared immunohistochemical features with bronchial mucoepidermoid carcinoma, a lesion that needs to be taken into account in differential diagnosis of hyalinizing clear cell carcinoma. All our bronchial hyalinizing clear cell carcinoma cases were surgically resected. Histologically, tumor cells showed clear to eosinophilic cytoplasm with hyalinizing stroma in various proportions, resembling those of salivary gland origin. Immunohistochemically, tumor cells were positive for CK7, CK5/6, p40, p63, and ATF1, while they were negative for TTF1, Napsin A, HMB45, and SOX10. The CK5/6 staining pattern varied in mucoepidermoid carcinomas, while that of hyalinizing clear cell carcinoma was uniformly positive. FISH revealed EWSR1-ATF1 fusion, and RT-PCR with sequencing confirmed specificity of the chimeric gene for hyalinizing clear cell carcinoma. Clinically, bronchial hyalinizing clear cell carcinoma was characterized by occurrence in the fourth to sixth decades, no link with smoking history, and a predilection for the right lung, in line with previous reports. In summary, our study confirmed that the bronchial hyalinizing clear cell carcinoma is a histologically and genetically identical tumor to that of salivary gland origin, and that gene rearrangement analysis can play a critical role in distinction from mucoepidermoid carcinoma.

Salivary duct carcinoma (SDC) constitutes one of the most aggressive cancers in the salivary gland and is associated with a poor prognosis; however, no established systemic therapy options are available. SDC exhibits biological similarity to prostate and breast cancers, therefore anti-hormone therapy and molecular target therapies are available, however with limited beneficial effects. Galanin and galanin receptors (GALRs) are well established as molecular biomarkers to predict the survival rate and risk of recurrence of head and neck squamous cell carcinoma. The present study investigated the clinicopathological features of patients with SDC and the methylation status of their galanin and GALR genes to demonstrate the prognostic value for this disease. The median overall survival (OS) was 37.2 months. T-stage, N-stage, disease stage, tumor size, and preoperative facial paralysis were significantly associated with OS, whereas human epidermal growth factor receptor 2 (HER2) overexpression was not. GALR1 and GALR2 methylation rates in tumor tissues were significantly increased compared with normal tissues with 9.85- and 4.49-fold increase, respectively. p27 kip1 and p57 kip2 expression significantly inversely correlated with the methylation rate of GALR1 and GALR2 . In addition, the observed GALR1 and/or GALR2 methylation rates were significantly correlated with a decrease in OS. These results suggest that GALR1 and GALR2 may serve as potential prognostic factors and therapeutic targets in SDC.

MYB-NFIB and MYBL1-NFIB have been reported in ~60% of adenoid cystic carcinoma cases, but driver alterations in the remaining ~40% of adenoid cystic carcinoma remain unclear. We examined 100 adenoid cystic carcinoma cases for MYB and MYBL1 locus rearrangements by fluorescence in situ hybridization (FISH) with originally designed probe sets using formalin-fixed paraffin-embedded materials. Approximately one-third of samples were also analyzed by fusion transcript-specific RT-PCR and capture RNA sequencing. In the 27 cases with frozen materials, MYB-NFIB and MYBL1-NFIB fusion transcripts were detected in 9 (33%) and 6 cases (22%) by RT-PCR, respectively. Meanwhile, high expression of MYB (18 cases, 67%) or MYBL1 (9 cases, 33%) was detected in all 27 cases in a mutually exclusive manner, regardless of its form (full-length, truncation, or fusion transcript). Interestingly, genomic rearrangements around the corresponding highly-expressed gene were observed in all 27 cases by FISH, suggesting a causative relationship between genomic rearrangements and gene expression. Among the 100 cases, including additional 73 cases, 97 harbored genomic rearrangements in the MYB (73 cases) or MYBL1 locus (24 cases) including 10 cases with atypical FISH patterns undetectable through ordinary split FISH approaches: breakpoints far distant from MYB (5 cases) and a small NFIB locus insertion into the MYB (3 cases) or MYBL1 locus (2 cases). In clinicopathological analyses, histological grade, primary tumor size, and lymph node metastasis were identified as prognostic factors, whereas MYB/MYBL1 rearrangements were not, but were associated with histological grade. In the present study, MYB or MYBL1 locus rearrangement was detected in nearly all adenoid cystic carcinoma cases, and therefore it would be a good diagnostic marker for adenoid cystic carcinoma. However, fusion transcript-specific RT-PCR for MYB-NFIB and MYBL1-NFIB and ordinary split FISH assays for MYB and MYBL1 were less sensitive, and thus detection methods should be judiciously designed because of the diversity of rearrangement modes in adenoid cystic carcinoma.

Rationale: Pleomorphic adenoma, the most common tumor of the salivary glands, is usually benign. It is well known, however, that pleomorphic adenomas occasionally undergo malignant transformation to carcinoma ex pleomorphic adenoma and can metastasize. More rarely pleomorphic adenomas can metastasize without histological malignant transformation. We herein report an unusual case of pleomorphic adenoma with multiple metastases comprehensively demonstrated on F-18 fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT)., Patient Concerns: A 63-year-old woman was referred to our hospital because of urine occult blood and lung nodules detected on a health checkup. She had a history of resection of a pleomorphic adenoma in the parotid gland at the age of 40 years and tumor recurrence at the age of 53 years. CT scan and magnetic resonance imaging revealed bilateral renal tumors, multiple pulmonary nodules, and an osteolytic lesion in the first lumbar vertebra., Diagnoses: Ultrasonography-guided percutaneous biopsy of the right renal tumor revealed a myxoid epithelial tumor that was consistent with metastasis of the pleomorphic adenoma from the parotid gland., Interventions: The patient was carefully observed with regular imaging examinations., Outcomes: The multiple lesions gradually progressed, and FDG-PET/CT subsequently revealed additional metastases in the liver and perineum., Lessons: Metastases of pleomorphic adenomas may occur years after the initial disease in association with local recurrences. Careful observation with whole-body imaging such as FDG-PET/CT is necessary.

Objective: While the biological behavior of follicular thyroid carcinoma (FTC) has been studied in great detail using clinical experience, few studies have investigated pre- or intraoperative factors related to the risk of distant metastasis (DM) among patients with FTC. The aim of this study was to analyze the characteristics of FTC with DM., Methods: This study retrospectively investigated 102 patients with FTC who underwent surgery between 1988 and 2013. We compared clinicopathological characteristics between FTC with and without DM., Results: Univariate analysis revealed nodal metastasis (p=0.045), serum thyroglobulin (Tg) at initial operation (≥1000ng/ml; p<0.0001), widely invasive appearance according to macroscopic findings (p<0.0001), thick tumor capsule (≥1mm; p<0.0001), vascular invasion (p=0.0003), extrathyroidal invasion (p=0.047), and venous tumor embolism (p=0.045) as significant risk factors for DM. Multivariate analysis conducted using pre- and intraoperative factors identified thick tumor capsule (≥1mm), serum Tg at initial operation (≥1000ng/ml), and macroscopically widely invasive appearance as risk factors independently associated with development of DM., Conclusion: Patients with these risk factors should undergo total thyroidectomy and radioactive iodine ablation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Central mucoepidermoid carcinoma (MEC) poses a diagnostic challenge because of its rarity and histological overlap with glandular odontogenic cyst (GOC). In MEC of both salivary glands and jaws, MAML2 arrangement has been well known as the specific gene alteration. We report a case of central MEC arising from GOC diagnosed by MAML2 fusion gene. A 57-year-old male presented a multilocular cystic lesion in left molar region of the mandible. Histopathologically, multiple cysts lined by thin cuboidal or non-keratinized squamous epithelium with small duct-like structures, mucous cells and ciliated cells were present. It was diagnosed as GOC. The recurrent lesion after nine years showed the proliferation of many cystic and solid nests composed of epidermoid, mucous and intermediated cells. Nested PCR revealed CRTC3-MAML2 fusion gene in the recurrent lesion, but not in the primary one. Similarly, MAML-2 rearrangement by FISH analysis was positive in the recurrent lesion, while negative for the primary one, thus confirming the diagnosis of central MEC arising from GOC. Analysis of MAML2 rearrangement can be used as a supportive evidence to distinguish central MEC from GOC., (© 2017 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Algal growth inhibition tests are generally used to determine the toxic effects of chemical substances on algae growth. In this report, we describe a rapid and simple test procedure using delayed fluorescence (DF) to determine chemical toxicities more rapidly than the conventional 72h or 96h growth inhibition tests. We assess the suitability of DF to serve as an alternative endpoint for biomass production and determine the variability by an interlaboratory ring study using a typical reference toxicant 3,5-dichlorophenol (DCP). The results suggest that DF has the potential to be used as a surrogate measure of photosynthetically-active biomass in the algal growth inhibition tests. The half maximal effective concentration (EC 50 ) values of DCP determined from the DF inhibition test in 6h and 24h (1.2±0.3mg/L and 2.7±0.5mg/L respectively) are in reasonable agreement with the EC 50 value of DCP determined by the 72h conventional method (1.8mg/L). In the interlaboratory ring study, the intralaboratory and interlaboratory variabilities of the EC 50 of the DF inhibition test for a 24h exposure period are 12% and 28% respectively. DF intensity can be considered as a surrogate of living biomass with active photosynthesis, and we conclude that a 24h exposure duration better estimates the toxic effects measured using conventional surrogate measures for dry weight such as cell counts, volume, optical density or fluorescence., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The molecular characteristics of therapeutically-relevant targets and their clinicopathological implications in salivary duct carcinomas (SDCs) are poorly understood. We investigated the gene alterations and the immunoexpression of crucial oncogenic molecules in 151 SDCs. The mutation rates that were identified, in order of frequency, were as follows: TP53 , 68%; PIK3CA , 18%; H-RAS , 16%; BRAF , 4%; and AKT1 , 1.5%. PIK3CA/H-RAS/BRAF mutations were more common in de novo SDC than in SDC ex-pleomorphic adenoma. Furthermore, these mutations were mutually exclusive for HER2 overexpression/amplification. TP53 mutations were frequently detected in cases with the aberrant p53 expression, and TP53 missense and truncating mutations were associated with p53-extreme positivity and negativity, respectively. DISH analysis revealed no cases of EGFR amplification. The rates of PI3K, p-Akt, and p-mTOR positivity were 34%, 22%, and 66%, respectively; PTEN loss was observed in 47% of the cases. These expressions were correlated according to the signaling axis. Cases with PI3K negativity and PTEN loss appeared to show a lower expression of androgen receptor. In the multivariate analysis, patients with SDC harboring TP53 truncating mutations showed shorter progression-free survival. Conversely, p-Akt positivity was associated with a favorable outcome. This study might provide information that leads to advances in personized therapy for SDC., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest in association with the present study.

Thyroid papillary carcinoma is the most common endocrine neoplasm and generally carries a favorable prognosis. However, a small subset of papillary carcinomas transforms into anaplastic carcinoma, an undifferentiated cancer with a dismal prognosis. Recent studies using next-generation sequencing revealed the genomic landscape of papillary carcinoma and anaplastic carcinoma. However, risk factors for anaplastic transformation in papillary carcinoma remain obscure. In the present study, we investigated molecular alterations of papillary carcinoma and anaplastic carcinoma components in 27 tumors in which anaplastic carcinoma coexisted with antecedent papillary carcinoma. We conducted direct sequencing for BRAF, TERT promoter and PIK3CA, and immunohistochemistry for p53, TTF-1 and subunits of the SWI/SNF complex (ARID1A, ARID1B, ATRX, SMARCA2, SMARCA4, SMARCB1, and PBRM1). BRAF V600E and TERT promoter mutated at the rate of 90% and 95%, respectively, and these mutational statuses were almost identical between the papillary carcinoma and anaplastic carcinoma components. PIK3CA mutation was positive in 33% of our samples with a heterogeneous mutation pattern of the papillary carcinoma and anaplastic carcinoma components. Aberrant expression of p53 and loss of TTF-1 were present in 63 and 59%, respectively, and these two alterations were confined to the anaplastic carcinoma components. There was a loss of the SWI/SNF complex in a subset of the tumors with a heterogeneous pattern of the papillary carcinoma and anaplastic carcinoma components: SMARCA4 in 4% and PBRM1 in 4%. In a multivariate comparison between the antecedent papillary carcinoma components and control papillary carcinomas without anaplastic transformation, TERT promoter mutation was independently associated with anaplastic transformation. Collectively, papillary carcinoma-derived anaplastic carcinomas are characterized by BRAF and TERT promoter mutations, and these mutations occur prior to anaplastic transformation. Alterations of PIK3CA and the SWI/SNF complex are relatively rare and temporally heterogeneous. Of note, a papillary carcinoma harboring TERT promoter mutation is at higher risk for anaplastic transformation.

Background: The effectiveness of the combination chemotherapy of weekly paclitaxel and cetuximab has not yet been compared to that of the current standard regimen, EXTREME (combination of 5-fluorouracil, cisplatin and cetuximab)., Methods: We retrospectively reviewed the clinical records of R/M SCCHN patients who received cetuximab-containing chemotherapy as a first-line therapy; from these, patients receiving a weekly paclitaxel and cetuximab regimen (cohort A) and the EXTREME regimen (cohort B) were extracted. The responses, prognoses and adverse events of these two cohorts were evaluated., Results: A total of 86 patients were included (cohort A, 49; cohort B, 36). Patients with histories of platinum-based chemotherapy were more frequently given the cohort A treatment. Though the response rates were similar in the two cohorts (45% in cohort A and 51% in cohort B; p=0.83), the progression-free survival (PFS) was significantly more favorable in cohort A by the log-rank test (6.0monthsvs 5.0months; p=0.027). In the Cox-regression hazard analyses, male gender (hazard ratio [HR]=2.1, p=0.010), older age (≥ 70 yo) (HR=5.0, p=0.018), PS 0 (HR=2.2, p=0.027), no history of platinum chemotherapy (HR=3.2, p=0.003) and the presence of a tracheostomy (HR=2.3, p=0.039) were favorable factors within cohort A., Conclusion: In selected R/M SCCHN patients, the combination of weekly paclitaxel and cetuximab could be the better treatment option than the EXTREME regimen., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Salivary duct carcinoma (SDC) is an uncommon, aggressive malignant neoplasm histologically resembling high-grade mammary ductal carcinoma. SDC can arise de novo or ex pleomorphic adenoma. To clarify the correlation of biomarker immunoprofile with clinicopathological findings and clinical outcome of SDC, we conducted immunohistochemistry for EGFR, HER2, HER3, AR, CK5/6, p53, and Ki-67, along with HER2 fluorescence in situ hybridization in 151 SDCs. SDCs ex pleomorphic adenoma more commonly overexpressed EGFR, HER2, HER3, and Ki-67 than de novo SDCs ( P = 0.015, < 0.001, 0.045, and 0.02, respectively). In multivariate analysis, AR- and CK5/6+ were associated with shorter progression-free survival ( P = 0.027 and 0.004, respectively). Moreover, patients with p53-extreme negative/positive demonstrated poorer overall survival ( P = 0.007). On assessing the revised classification by the combination of biomarker expression, the percentages of each subtype were as follows: 'apocrine A' (AR+/HER2-/Ki-67-low) (24%), 'apocrine B' (AR+/HER2-/Ki-67-high) (18%), 'apocrine HER2' (AR+/HER2+) (35%), 'HER2-enriched' (AR-/HER2+) (12%), and 'double negative' (AR-/HER2-) (11%). 'Double negative' was further subclassified into 'basal-like' (EGFR and/or CK5/6+) (7%) and 'unclassified' (3%). Consequently, patients with 'apocrine A' showed a better progression-free survival than those with any other subtypes. Our revised immunoprofiling classification was valuable for predicting the survival and might be useful in personalized therapy for patients with SDC., Competing Interests: CONFLICTS OF INTEREST None.

Galactosaminogalactan and Pel are cationic heteropolysaccharides produced by the opportunistic pathogens Aspergillus fumigatus and Pseudomonas aeruginosa , respectively. These exopolysaccharides both contain 1,4-linked N -acetyl-d-galactosamine and play an important role in biofilm formation by these organisms. Proteins containing glycoside hydrolase domains have recently been identified within the biosynthetic pathway of each exopolysaccharide. Recombinant hydrolase domains from these proteins (Sph3 h from A. fumigatus and PelA h from P. aeruginosa ) were found to degrade their respective polysaccharides in vitro. We therefore hypothesized that these glycoside hydrolases could exhibit antibiofilm activity and, further, given the chemical similarity between galactosaminogalactan and Pel, that they might display cross-species activity. Treatment of A. fumigatus with Sph3 h disrupted A. fumigatus biofilms with an EC 50 of 0.4 nM. PelA h treatment also disrupted preformed A. fumigatus biofilms with EC 50 values similar to those obtained for Sph3 h In contrast, Sph3 h was unable to disrupt P. aeruginosa Pel-based biofilms, despite being able to bind to the exopolysaccharide. Treatment of A. fumigatus hyphae with either Sph3 h or PelA h significantly enhanced the activity of the antifungals posaconazole, amphotericin B, and caspofungin, likely through increasing antifungal penetration of hyphae. Both enzymes were noncytotoxic and protected A549 pulmonary epithelial cells from A. fumigatus -induced cell damage for up to 24 h. Intratracheal administration of Sph3 h was well tolerated and reduced pulmonary fungal burden in a neutropenic mouse model of invasive aspergillosis. These findings suggest that glycoside hydrolases can exhibit activity against diverse microorganisms and may be useful as therapeutic agents by degrading biofilms and attenuating virulence., Competing Interests: Conflict of interest statement: A patent has been filed describing the utility of the glycoside hydrolases as antibiofilm therapeutics (CA2951152 A1, WO2015184526 A1). B.D.S., P.B., N.C.B., P.L.H., and D.C.S. are listed as inventors.

Objective: Clear cell odontogenic carcinoma (CCOC) is a rare malignant odontogenic tumor (MOT) characterized by sheets and lobules of vacuolated and clear cells. To understand the biology of CCOC, we established a new cell line, CCOC-T, with EWSR1-ATF1 fusion gene from a mandible tumor with distant metastasis and characterized this cell line., Materials and Methods: To detect the EWSR1-ATF1 fusion gene, we used three CCOC cases, including the present case, by RT-PCR and FISH analysis. We characterized established CCOC-T cells by checking cell growth, invasion and the expression of odontogenic factors and bone-related factors. Moreover, the gene expression profile of CCOC-T cells was examined by microarray analysis., Results: Histologically, the primary tumor was comprised of cords and nests containing clear and squamoid cells separated by fibrous septa. In addition, ameloblastomatous islands with palisaded peripheral cells were observed, indicating probable odontogenic origin. This tumor expressed the fusion gene EWSR1-ATF1, which underlies the etiology of hyalinizing clear cell carcinoma (HCCC) and potentially that of CCOC. We found a breakpoint in the EWSR1-ATF1 fusion to be the same as that reported in HCCC. Established CCOC-T cells grew extremely slowly, but the cells showed highly invasive activity. Moreover, CCOC-T cells expressed bone-related molecules, odontogenic factors, and epithelial mesenchymal transition (EMT)-related molecules., Conclusion: To the best of our knowledge, this is the first report on the establishment of a CCOC cell line. CCOC-T cells serve as a useful in vitro model for understanding the pathogenesis and nature of MOT., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background and Aim: Owing to increased awareness and use of narrow-band imaging, there are more opportunities to treat superficial pharyngeal cancer (SPC). The present study aimed to describe the short- and long-term outcomes of endoscopic resection (ER) for SPC., Methods: This study included 166 consecutive SPC in 113 patients treated during 2006 to 2013 at one referral cancer center. In the first period, we treated patients using endoscopic mucosal resection (EMR), in the second period using conventional ESD (cESD) and in the recent period using double-scope ESD (dsESD), which involves a second thin endoscope for assistance to produce traction. Median follow-up period was 30 months., Results: All lesions were diagnosed as squamous cell carcinoma. Complete resection rate of cESD and dsESD procedures was 56.4% and 82.3% (P < 0.01), and local recurrence rate was 2.6% and 0.0%, respectively. Procedure duration was significantly shorter for dsESD than for cESD (P < 0.05). Four cases of recurrent lymph node (LN) metastasis were observed; however, all patients with LN metastases survived to a 48-month median interval after neck dissection. Risk factors for LN metastasis included subepithelium invasion, tumor thickness >1000 μm, droplet infiltration, and lymphovascular invasion. Overall survival rate after 5 years was 79.5%; no patients died of SPC. Cumulative rate of metachronous SPC after 5 years was 46.5%., Conclusion: ER for SPC is a feasible and effective treatment, although metachronous SPC occurred frequently. For the technique of ER, dsESD was effective., (© 2016 Japan Gastroenterological Endoscopy Society.)

The prognostic role of modified Glasgow Prognostic Score (mGPS), neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) in patients with salivary duct carcinoma (SDC) remains unclear. We conducted a multi-institutional retrospective cohort study of 140 SDC patients. The survival impact of these hematological markers was evaluated using multivariate proportional hazard models.High mGPS (≥1) was significantly associated with worse survival (3-year overall survival (OS): 16.7% vs 66.1%, p-value=0.003; 3-year progression-free survival (PFS): 0.0% vs 27.9%, p-value<0.001). Additionally, high C-reactive protein (CRP) (≥0.39 mg/dl) was significantly associated with worse survival (3-year OS: 32.1% vs 68.2%, p-value=0.001; 3-year PFS: 7.1% vs 31.1%, p-value<0.001). These associations were consistent with multivariate analysis adjusted for established prognostic factors. Although we also found significant association of high NLR (≥2.5) with OS (HR 1.80; 95% confidence interval, 1.05-3.08) in multivariate analysis, this association were inconsistent with the results of PFS. In addition, we found no significant associations of PLR with survival. In conclusion, we found that mGPS, CRP and NLR were identified as prognostic factors associated with survival in SDC patients.