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Many long noncoding RNAs (lncRNAs) are deregulated in cancer and contribute to oncogenesis. In urothelial carcinoma (UC), several lncRNAs have been reported to be overexpressed and proposed as biomarkers. As most reports have not been confirmed independently in large tissue sets, we aimed to validate the diagnostic and prognostic value of lncRNA upregulation in independent cohorts of UC patients. Thus, expression of seven lncRNA candidates (GAS5, H19, linc-UBC1, MALAT1, ncRAN, TUG1, UCA1) was measured by RT-qPCR in cell lines and tissues and correlated to clinicopathological parameters including follow-up data (set 1: N n = 10; T n = 106). Additionally, publicly available TCGA data was investigated for differential expression in UC tissues (set 2: N n = 19; T n = 252,) and correlation to overall survival (OS). All proposed candidates tended to be upregulated in tumour tissues, with the exception of MALAT1, which was rather diminished in cancer tissues of both data sets. However, strong overexpression was generally limited to individual tumour tissues and statistically significant overexpression was only observed for UCA1, TUG1, ncRAN and linc-UBC1 in tissue set 2, but for no candidate in set 1. Altered expression of individual lncRNAs was associated with overall survival, but not consistently between both patient cohorts. Interestingly, lower expression of TUG1 in a subset of UC patients with muscle-invasive tumours was significantly correlated with worse OS in both cohorts. Further analysis revealed that tumours with low TUG1 expression are characterized by a basal-squamous-like subtype signature accounting for the association with poor outcome. In conclusion, our study demonstrates that overexpression of the candidate lncRNAs is found in many UC cases, but does not occur consistently and strongly enough to provide reliable diagnostic or prognostic value as an individual biomarker. Subtype-dependent expression patterns of lncRNAs like TUG1 could become useful to stratify patients by molecular subtype, thus aiding personalized treatments.

The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy—CD19, CD33, and CD123—to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further.

In this study, we investigate the role of radiomics for prediction of overall survival (OS), locoregional recurrence (LRR) and distant metastases (DM) in stage III and IV HNSCC patients treated by chemoradiotherapy. We hypothesize that radiomic analysis of (peri-)tumoral tissue may detect invasion of surrounding tissues indicating a higher chance of locoregional recurrence and distant metastasis. Two comprehensive data sources were used: the Dutch Cancer Society Database (Alp 7072, DESIGN) and \"Big Data To Decide\" (BD2Decide). The gross tumor volumes (GTV) were delineated on contrast-enhanced CT. Radiomic features were extracted using the RadiomiX Discovery Toolbox (OncoRadiomics, Liege, Belgium). Clinical patient features such as age, gender, performance status etc. were collected. Two machine learning methods were chosen for their ability to handle censored data: Cox proportional hazards regression and random survival forest (RSF). Multivariable clinical and radiomic Cox/ RSF models were generated based on significance in univariable cox regression/ RSF analyses on the held out data in the training dataset. Features were selected according to a decreasing hazard ratio for Cox and relative importance for RSF. A total of 444 patients with radiotherapy planning CT-scans were included in this study: 301 head and neck squamous cell carcinoma (HNSCC) patients in the training cohort (DESIGN) and 143 patients in the validation cohort (BD2DECIDE). We found that the highest performing model was a clinical model that was able to predict distant metastasis in oropharyngeal cancer cases with an external validation C-index of 0.74 and 0.65 with the RSF and Cox models respectively. Peritumoral radiomics based prediction models performed poorly in the external validation, with C-index values ranging from 0.32 to 0.61 utilizing both feature selection and model generation methods. Our results suggest that radiomic features from the peritumoral regions are not useful for the prediction of time to OS, LR and DM.

Background Few diagnostic and prognostic biomarkers are available for head-and-neck squamous cell carcinoma (HNSCC). Long non-coding RNAs (lncRNAs) have shown promise as biomarkers in other cancer types and in some cases functionally contribute to tumor development and progression. Here, we searched for lncRNAs useful as biomarkers in HNSCC. Methods Public datasets were mined for lncRNA candidates. Two independent HNSCC tissue sets and a bladder cancer tissue set were analyzed by RT-qPCR. Effects of lncRNA overexpression or downregulation on cell proliferation, clonogenicity, migration and chemosensitivity were studied in HNSCC cell lines. Results Data mining revealed prominently CASC9, a lncRNA significantly overexpressed in HNSCC tumor tissues according to the TCGA RNAseq data. Overexpression was confirmed by RT-qPCR analyses of patient tissues from two independent cohorts. CASC9 expression discriminated tumors from normal tissues with even higher specificity than HOTAIR, a lncRNA previously suggested as an HNSCC biomarker. Specificity of HNSCC detection by CASC9 was further improved by combination with HOTAIR. Analysis of TCGA pan-cancer data revealed significant overexpression of CASC9 across different other entities including bladder, liver, lung and stomach cancers and especially in squamous cell carcinoma (SCC) of the lung. By RT-qPCR analysis we furthermore detected stronger CASC9 overexpression in pure SCC of the urinary bladder and mixed urothelial carcinoma with squamous differentiation than in pure urothelial carcinomas. Thus, CASC9 might represent a general diagnostic biomarker and particularly for SCCs. Unexpectedly, up- or downregulation of CASC9 expression in HNSCC cell lines with low or high CASC9 expression, respectively, did not result in significant changes of cell viability, clonogenicity, migration or chemosensitivity. Conclusions CASC9 is a promising biomarker for HNSCC detection. While regularly overexpressed, however, this lncRNA does not seem to act as a major driver of development or progression in this tumor.

Treatment of B-cell malignancies with CD19-directed chimeric antigen receptor (CAR) T-cells marked a new era in immunotherapy, which yet has to be successfully adopted to solid cancers. Epigenetic inhibitors of DNA methyltransferases (DNMTi) and histone deacetylases (HDACi) can induce broad changes in gene expression of malignant cells, thus making these inhibitors interesting combination partners for immunotherapeutic approaches. Urothelial carcinoma cell lines (UCC) and benign uroepithelial HBLAK cells pretreated with the DNMTi decitabine or the HDACi romidepsin were co-incubated with CAR T-cells directed against EGFR or CD44v6, and subsequent cytotoxicity assays were performed. Effects on T-cell cytotoxicity and surface antigen expression on UCC were determined by flow cytometry. We also performed next-generation mRNA sequencing of inhibitor-treated UCC and siRNA-mediated knockdown of potential regulators of CAR T-cell killing. Exposure to decitabine but not romidepsin enhanced CAR T-cell cytotoxicity towards all UCC lines, but not towards the benign HBLAK cells. Increased killing could neither be attributed to enhanced target antigen expression (EGFR and CD44v6) nor fully explained by changes in the T-cell ligands PD-L1, PD-L2, ICAM-1, or CD95. Instead, gene expression analysis suggested that regulators of cell survival and apoptosis were differentially induced by the treatment. Decitabine altered the balance between survival and apoptosis factors towards an apoptosis-sensitive state associated with increased CAR T-cell killing, while romidepsin, at least partially, tilted this balance in the opposite direction. Knockdown experiments with siRNA in UCC confirmed BID and BCL2L1/BCLX as two key factors for the altered susceptibility of the UCC. Our data suggest that the combination of decitabine with CAR T-cell therapy is an attractive novel therapeutic approach to enhance tumor-specific killing of bladder cancer. Since BID and BCL2L1 are essential determinants for the susceptibility of a wide variety of malignant cells, their targeting might be additionally suitable for combination with immunotherapies, e.g., CAR T-cells or checkpoint inhibitors in other malignancies.

[This corrects the article DOI: 10.1371/journal.pone.0232639.].[This corrects the article DOI: 10.1371/journal.pone.0232639.].

Since rhinosinusitis is an inflammatory disease, cytokines as key regulators of inflammation play a central role in its pathophysiology. In acute rhinosinusitis, several proinflammatory cytokines of different types have been identified. Initial information about the involvement of the inflammasome in rhinosinusitis has been gained, but this area remains open for more detailed research. Although it has been accepted now that chronic rhinosinusitis (CRS) needs to be differentiated into CRS with and without nasal polyps, it has become clear that this distinction is insufficient to clearly define subgroups with uniform pathophysiology and cytokine patterns. While Th1-cytokines are mostly found in CRSsNP and Th2 cytokines in CRSwNP, there is a substantial overlap, and several other cytokines have also been detected. Attempts to identify CRS endotypes based on cytokines are ongoing but not yet generally accepted. Despite the central role of cytokines in rhinosinusitis, no specific cytokine-targeted therapies are currently available, and only very few studies have specifically addressed the effects of such biologicals in rhinosinusitis.

BACKGROUND. A key objective in managing HPV· oropharyngeal squamous cell carcinoma (OPSCC) is reducing radiation therapy (RT) doses without compromising cure rates. A recent phase 1/1! HNOOS trial revealed that clinical factors alone are insufficient to guide safe RT dose de-escalation. Our prior research demonstrated that the genomic adjusted radiation dose (GARD) predicts RT benefit and may inform dose selection. We hypothesize that GARD can guide personalized RT deescalation in HPV· OPSCC patients. METHODS. Gene expression profiles were analyzed in 191 HPV· OPSCC patients enrolled in an international, multi-institutional observational study (AJCC Eighth Edition, stages I-III). Most patients received 70 Gy in 35 fractions or 69.96 Gy in 33 fractions (median dose: 70 Gy; range: 51.0-74.0 ??). Overall survival (OS) was 94.1% at 36 months and 87.3% at 60 months. A Cox proportional hazards model assessed association between GARD and 0S, and time-dependent receiver operating characteristic analyses compared GARD with traditional clinical predictors. RESULTS. Despite uniform RT dosing, GARD showed wide heterogeneity, ranging from 15.4 to 71.7. Higher GARD values were significantly associated with improved OS in univariate (HR = 0.941, P = 0.041) and multivariable analyses (HR = 0.943, P = 0.046), while T and ? stages were not. GARD demonstrated superior predictive performance at 36 months (AUC = 78.26) versus clinical variables (AUC = 71.20). Two GARD-based RT de-escalation strategies were identified, offering potential survival benefits while reducing radiation exposure. CONCLUSION. GARD predicts OS and outperforms clinical variables, supporting its integration into the diagnostic workflow for personalized RT in ??\\/· OPSCC. FUNDING. This work was supported by the National Cancer Institute through the Cleveland Clinic/Emory ROBIN center (U54CA274513, project 2), the European Union Horizon 2020 Framework Programme (grant/award 689715), the Italian Association for Cancer Research (AIRC project ID 23573), and the European Research Area Network ERA PerMed JTC2019/Fondazione Regionale per la Ricerca Biomedica project SuPerTreat (Supporting Personalized Treatment Decisions in Head and Neck Cancer through Big Data).

BACKGROUNDA key objective in managing HPV+ oropharyngeal squamous cell carcinoma (OPSCC) is reducing radiation therapy (RT) doses without compromising cure rates. A recent phase II/III HN005 trial revealed that clinical factors alone are insufficient to guide safe RT dose de-escalation. Our prior research demonstrated that the genomic adjusted radiation dose (GARD) predicts RT benefit and may inform dose selection. We hypothesize that GARD can guide personalized RT de-escalation in HPV+ OPSCC patients.METHODSGene expression profiles were analyzed in 191 HPV+ OPSCC patients enrolled in an international, multi-institutional observational study (AJCC Eighth Edition, stages I-III). Most patients received 70 Gy in 35 fractions or 69.96 Gy in 33 fractions (median dose: 70 Gy; range: 51.0-74.0 Gy). Overall survival (OS) was 94.1% at 36 months and 87.3% at 60 months. A Cox proportional hazards model assessed association between GARD and OS, and time-dependent receiver operating characteristic analyses compared GARD with traditional clinical predictors.RESULTSDespite uniform RT dosing, GARD showed wide heterogeneity, ranging from 15.4 to 71.7. Higher GARD values were significantly associated with improved OS in univariate (HR = 0.941, P = 0.041) and multivariable analyses (HR = 0.943, P = 0.046), while T and N stages were not. GARD demonstrated superior predictive performance at 36 months (AUC = 78.26) versus clinical variables (AUC = 71.20). Two GARD-based RT de-escalation strategies were identified, offering potential survival benefits while reducing radiation exposure.CONCLUSIONGARD predicts OS and outperforms clinical variables, supporting its integration into the diagnostic workflow for personalized RT in HPV+ OPSCC.FUNDINGThis work was supported by the National Cancer Institute through the Cleveland Clinic/Emory ROBIN center (U54-CA274513, project 2), the European Union Horizon 2020 Framework Programme (grant/award 689715), the Italian Association for Cancer Research (AIRC project ID 23573), and the European Research Area Network ERA PerMed JTC2019/Fondazione Regionale per la Ricerca Biomedica project SuPerTreat (Supporting Personalized Treatment Decisions in Head and Neck Cancer through Big Data).