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Bladder cancer (BLCA) genomic profiling has identified molecular subtypes with distinct clinical characteristics and variable sensitivities to frontline therapy. BLCAs can be categorized into luminal or basal subtypes based on their gene expression. We comprehensively characterized nine human BLCA cell lines (UC3, UC6, UC9, UC13, UC14, T24, SCaBER, RT4V6 and RT112) into molecular subtypes using orthotopic xenograft models. Patient-derived, luciferase-tagged BLCA cell lines were cultured in vitro and engrafted into bladders of NSG mice. Tumor growth was monitored using bioluminescence imaging and mRNA-based molecular classification was used to characterize xenografts into molecular subtypes. RNAseq analysis and basal, luminal, and epithelial-mesenchymal transition (EMT) marker expression revealed distinct patterns; certain cell lines expressed predominantly basal or luminal markers while others demonstrated mixed expression. SCaBER expressed high basal and EMT markers and low luminal markers, consistent with a true basal cell. RT4V6 was a true luminal cell line, displaying only high luminal makers. UC13, T24 and UC3 only showed increased expression of EMT markers. RT112, UC6, UC9 and UC14 expressed basal, luminal, and EMT markers. Immunohistochemical analysis validated our findings. Ki67 was assessed as a continuous percentage of positively stained cells. Morphological assessment of xenografts included H&E and α-SMA staining. These findings will allow for the rational use of appropriate models to develop targeted therapies to overcome or manipulate mechanisms of treatment resistance in BLCA.

IntroductionNadofaragene firadenovec (Ad-IFNα/Syn3) is now approved for BCG-unresponsive bladder cancer (BLCA). IFNα is a pleiotropic cytokine that causes direct tumor cell killing via TRAIL-mediated apoptosis, angiogenesis inhibition, and activation of the innate and adaptive immune system. We established an immunocompetent murine BLCA model to study the effects of murine adenoviral IFNα (muAd-Ifnα) gene therapy on cancer cells and the tumor microenvironment using a novel murine equivalent of Nadofaragene firadenovec (muAd-Ifnα).MethodsTumors were induced by instilling MB49 cells into the bladders of mice; luciferase imaging confirmed tumor development. Mice were treated with adenovirus control (Ad-Ctrl; empty vector), or muAd-Ifnα (3x1011 VP/mL), and survival analysis was performed. For single-cell sequencing (scRNAseq) analysis (72h), bladders were harvested and treated with collagenase/hyaluronidase and TrypLE for cell dissociation. Single cells were suspended in PBS/1% FBS buffer; viability was assessed with Vicell cell counter. scRNAseq analysis was performed using 10X genomics 3’ sequencing. Raw RNAseq data were pre-processed using Cell Ranger single-cell software. Seurat (R package) was used to normalize and cluster the scRNA data. Pooled differential gene expression analysis in specific cell clusters was performed with DESeq2.ResultsWe identified 16 cell clusters based on marker expression which were grouped into epithelial (tumor), uroplakin-enriched, endothelial, T-cells, neutrophils, and macrophage clusters. Top differentially expressed genes between muAd-Ifnα and Ad-Ctrl were identified. Within the specific cell clusters, IPA analysis revealed significant differences between muAd-Ifnα and control. IFNα signaling and hypercytokinemia/chemokinemia were upregulated in all clusters. Cell death pathways were upregulated in tumor and endothelial clusters. T-cells demonstrated upregulation of the immunogenic cell death signaling pathway and a decrease in the Th2 pathway genes. Macrophages showed upregulation of PD1/PD-L1 pathways along with downregulation of macrophage activation pathways (alternate and classical). Multiplex immunofluorescence confirmed increased infiltration with macrophages in muAd-Ifnα treated tumors compared to controls. PD1/PD-L1 expression was reduced at 72h.DiscussionThis single-cell analysis builds upon our understanding of the impact of Ad-IFNα on tumor cells and other compartments of the microenvironment. These data will help identify mechanisms to improve patient selection and therapeutic efficacy of Nadofaragene firadenovec.

Objective/Background: Drug-resistant strains of tuberculosis (TB) represent a major threat to global TB control. In low- and middle-income countries, resource constraints make it difficult to identify and monitor cases of resistance using drug susceptibility testing and culture. Molecular assays such as the GeneXpert Mycobacterium tuberculosis/rifampicin may prove to be a cost-effective solution to this problem in these settings. The objective of this study is to evaluate the use of GeneXpert in the diagnosis of pulmonary TB since it was introduced into two tertiary hospitals in Ghana in 2013. Methods: A 2-year retrospective audit of clinical cases involving patients who presented with clinically suspected TB or documented TB not improving on standard therapy and had samples sent for GeneXpert testing. Results: GeneXpert identified 169 cases of TB, including 17 cases of rifampicin-resistant TB. Of the seven cases with final culture and drug susceptibility testing results, six demonstrated further drug resistance and five of these were multidrug-resistant TB. Conclusion: These findings call for a scale-up of TB control in Ghana and provide evidence that the expansion of GeneXpert may be an optimal means to improve case finding and guide treatment of drug-resistant TB in this setting.

Aspergillomas are often misdiagnosed as tuberculosis (TB) in developing countries where the prevalence of TB is high, hemoptysis is often equated with TB, and most patients are diagnosed clinically. This report describes the case of a patient being treated for smear-negative TB who presented with hemoptysis and was found to have an aspergilloma.