Genetic characterization of intramuscular myxomas.

Introduction: Intramuscular myxomas are benign tumors that are challenging to diagnose, especially on core needle biopsies. Acquired chromosomal aberrations and pathogenic variants in codon 201 or codon 227 in GNAS complex locus gene (GNAS) have been reported in these tumors. Here we present our genetic findings in a series of 22 intramuscular myxomas. Materials and methods: The tumors were investigated for the presence of acquired chromosomal aberrations using G-banding and karyotyping. Pathogenic variants in codon 201 or codon 227 of GNAS were assessed using direct cycle Sanger sequencing and Ion AmpliSeq Cancer Hotspot Panel v2 methodologies. Results: Eleven tumors carried chromosomal abnormalities. Six tumors had numerical, four had structural, and one had both numerical and structural chromosomal aberrations. Gains of chromosomes 7 and 8 were the most common abnormalities being found in five and four tumors respectively. Pathogenic variants in GNAS were detected in 19 myxomas (86%) with both methodologies. The detected pathogenic variants were p.R201H in nine cases (seven with abnormal and two with normal karyotypes), p.R201C in five cases, all with normal karyotypes, p.R201S in three cases (two with abnormal and one with normal karyotype), p.R201G in one case with a normal karyotype, and p.Q227E in one case with a normal karyotype. Conclusion: Firstly, our data indicate a possible association between chromosomal abnormalities and GNAS pathogenic variants in intramuscular myxomas. Secondly, the presence of the rare pathogenic variants R201S, p.R201G and p.Q227E in 26% (5 out of 19) of myxomas with GNAS pathogenic variants shows that methodologies designed to detect only the common "hotspot" of p.R201C and p.R201H will give false negative results. Finally, a comparison between Ion AmpliSeq Cancer Hotspot Panel v2 and direct cycle Sanger sequencing showed that direct cycle Sanger sequencing provides a quick, reliable, and relatively cheap method to detect GNAS pathogenic variants, matching even the most cutting-edge sequencing methods.

Identification of a novel PHIP::BRAF gene fusion in infantile fibrosarcoma.

INTRODUCTION: The ETV6::NTRK3 fusion is the most common gene alteration in infantile fibrosarcoma, a soft tissue tumor affecting patients under two years of age. Less frequently, these tumors harbor fusions of genes encoding other kinases, such as BRAF, which activates MEK in the mitogen-activated protein kinase pathway. The identification and characterization of these oncogenes are crucial to facilitate diagnosis, validate new treatments, and better understand the pathophysiology of these neoplasms. METHODS: Herein, we analyzed an ETV6::NTRK3-negative infantile fibrosarcoma from a 5-day-old patient by RNA-sequencing to identify new fusion transcripts. Functional exploration of the fusion of interest was performed by in vitro assays to study its activity, oncogenicity, and sensitivity to the MEK inhibitor trametinib. RESULTS: We identified a novel fusion involving the PHIP and BRAF genes. The corresponding fusion protein constitutively activated the mitogen-activated protein kinase pathway, resulting in fibroblast transformation. Treatment of transfected cells with trametinib effectively inhibited signaling by PHIP::BRAF. CONCLUSION: PHIP::BRAF is a novel fusion oncogene that can be targeted by trametinib in infantile fibrosarcoma.

CRTC1-SS18 Fusion Sarcoma With Aberrant Anaplastic Lymphoma Kinase Expression.

Undifferentiated small round cell sarcoma (USRCS) represents a highly heterogeneous group of tumors. A variety of specific gene fusions of USRCS have been reported, including CIC-FOXO4, CIC-NUTM1, BCOR-MAML3, and ZC3H7B-BCOR. Here we report a case of sarcoma harboring a rare recurrent CRTC1-SS18 gene fusion, which was considered as USRCS previously. This sarcoma was composed of nests of small round cells encapsulated in a fibrous stroma. Foci of necrosis and hemorrhage were observed in the tumor. Immunohistochemistry for anaplastic lymphoma kinase showed diffuse positivity. RNA-seq results revealed a chromosomal translocation of CRTC1 gene exon 1 on chromosome 19 with SS18 gene exon 2 on chromosome 18. Thereafter, fluorescence in-situ hybridization confirmed the presence of SS18 gene and CRTC1 gene break-apart, which manifested as the splitting of red and green signals into 2 parts. A previous study showed that CRTC1-SS18 fusion sarcoma and EWSR1-CREB1 fusion angiomatoid fibrous histiocytoma were clustered close in the expression profile. However, whether CRTC1-SS18 fusion sarcomas represent a high malignancy has been a matter of debate. Our study is a worthy addition to the series of rare rearrangements associated with sarcomas and may be of therapeutic relevance.

The KrasG12D;Trp53fl/fl murine model of undifferentiated pleomorphic sarcoma is macrophage dense, lymphocyte poor, and resistant to immune checkpoint blockade.

Sarcomas are rare, difficult to treat, mesenchymal lineage tumours that affect children and adults. Immunologically-based therapies have improved outcomes for numerous adult cancers, however, these therapeutic strategies have been minimally effective in sarcoma so far. Clinically relevant, immunologically-competent, and transplantable pre-clinical sarcoma models are essential to advance sarcoma immunology research. Herein we show that Cre-mediated activation of KrasG12D, and deletion of Trp53, in the hindlimb muscles of C57Bl/6 mice results in the highly penetrant, rapid onset undifferentiated pleomorphic sarcomas (UPS), one of the most common human sarcoma subtypes. Cell lines derived from spontaneous UPS tumours can be reproducibly transplanted into the hindlimbs or lungs of naïve, immune competent syngeneic mice. Immunological characterization of both spontaneous and transplanted UPS tumours demonstrates an immunologically-'quiescent' microenvironment, characterized by a paucity of lymphocytes, limited spontaneous adaptive immune pathways, and dense macrophage infiltrates. Macrophages are the dominant immune population in both spontaneous and transplanted UPS tumours, although compared to spontaneous tumours, transplanted tumours demonstrate increased spontaneous lymphocytic infiltrates. The growth of transplanted UPS tumours is unaffected by host lymphocyte deficiency, and despite strong expression of PD-1 on tumour infiltrating lymphocytes, tumours are resistant to immunological checkpoint blockade. This spontaneous and transplantable immune competent UPS model will be an important experimental tool in the pre-clinical development and evaluation of novel immunotherapeutic approaches for immunologically cold soft tissue sarcomas.

A comprehensive molecular characterization of the 8q22.2 region reveals the prognostic relevance of OSR2 mRNA in muscle invasive bladder cancer.

Technological advances in molecular profiling have enabled the comprehensive identification of common regions of gene amplification on chromosomes (amplicons) in muscle invasive bladder cancer (MIBC). One such region is 8q22.2, which is largely unexplored in MIBC and could harbor genes with potential for outcome prediction or targeted therapy. To investigate the prognostic role of 8q22.2 and to compare different amplicon definitions, an in-silico analysis of 357 patients from The Cancer Genome Atlas, who underwent radical cystectomy for MIBC, was performed. Amplicons were generated using the GISTIC2.0 algorithm for copy number alterations (DNA_Amplicon) and z-score normalization for mRNA gene overexpression (RNA_Amplicon). Kaplan-Meier survival analysis, univariable, and multivariable Cox proportional hazard ratios were used to relate amplicons, genes, and clinical parameters to overall (OS) and disease-free survival (DFS). Analyses of the biological functions of 8q22.2 genes and genomic events in MIBC were performed to identify potential targets. Genes with prognostic significance from the in silico analysis were validated using RT-qPCR of MIBC tumor samples (n = 46). High 8q22.2 mRNA expression (RNA-AMP) was associated with lymph node metastases. Furthermore, 8q22.2 DNA and RNA amplified patients were more likely to show a luminal subtype (DNA_Amplicon_core: p = 0.029; RNA_Amplicon_core: p = 0.01). Overexpression of the 8q22.2 gene OSR2 predicted shortened DFS in univariable (HR [CI] 1.97 [1.2; 3.22]; p = 0.01) and multivariable in silico analysis (HR [CI] 1.91 [1.15; 3.16]; p = 0.01) and decreased OS (HR [CI] 6.25 [1.37; 28.38]; p = 0.0177) in RT-qPCR data analysis. Alterations in different levels of the 8q22.2 region are associated with manifestation of different clinical characteristics in MIBC. An in-depth comprehensive molecular characterization of genomic regions involved in cancer should include multiple genetic levels, such as DNA copy number alterations and mRNA gene expression, and could lead to a better molecular understanding. In this study, OSR2 is identified as a potential biomarker for survival prognosis.

Adipocyte-rich CTNNB1-mutated Intramuscular Gardner Fibroma Progressing to Desmoid Fibromatosis.

Gardner fibroma (GF) is a benign soft-tissue tumor that is associated with Gardner syndrome and can progress to, or co-occur with, desmoid fibromatosis (DF). Herein, we report a unique case of an 11-year-old boy who presented with a rapidly growing soft-tissue mass after biopsy of a stable fat-rich lesion present in the calf muscles since infancy, with Magnetic resonance imaging findings suggesting an intramuscular adipocytic tumor. The resection showed GF and DF. DF arising from a preexisting GF (the so-called "GF-DF sequence") is a well-documented phenomenon. Although immunohistochemistry was negative for nuclear ß-catenin expression, a CTTNB1 S45F mutation, which has been associated with aggressive behavior in DF, was identified in both components using a next-generation sequencing-based molecular assay. This is the first time a mutation in CTNNB1 has been identified in GF and the GF-DF sequence, thus expanding our knowledge of the molecular pathogenesis of the GF-DF sequence and highlighting the role of molecular testing in pediatric soft-tissue tumors. The histologic findings of an adipocyte-rich intramuscular GF also are unique, expanding the morphological spectrum of GF and adding GF to the differential diagnosis of intramuscular lesions with an adipocytic component.

Long noncoding RNA MIR31HG and its splice variants regulate proliferation and migration: prognostic implications for muscle invasive bladder cancer.

BACKGROUND: Growing evidence supports the pivotal role of long non-coding RNAs (lncRNAs) in the regulation of cancer development and progression. Their expression patterns and biological function in muscle invasive bladder cancer (MIBC) remain elusive. METHODS: Transcript levels of lncRNA miR-31 host gene (MIR31HG) and its splice variants were measured in our MIBC cohort (n = 102) by qRT-PCR, and validated in silico by the TCGA cohort (n = 370). Kaplan-Meier and multiple Cox regression analysis were conducted to evaluate the survival significance of MIR31HG and its splice variants. Functional experiments were performed to examine the proliferation and migration abilities of MIR31HG and its splice variants by knockdown approaches. RESULTS: In this study, a decreased expression of MIR31HG was found in bladder cancer cells and tissues, except in the basal subtype. Survival analysis showed that high expression of MIR31HG was associated with poor overall survival (OS) and disease-free survival (DFS) in patients with MIBC of basal subtype. Two splice variants of MIR31HG lacking exon 1 (MIR31HGΔE1) and exon 3 (MIR31HGΔE3) were identified to have specific expression patterns in different molecular subtypes of our MIBC cohort. MIR31HGΔE3 was highly expressed in basal subtype tumors. A high expression of MIR31HGΔE1 and MIR31HGΔE3 was associated with worse OS and DFS in our cohort. In vitro experiments revealed that knockdown of MIR31HG inhibits cell proliferation, colony formation, and migration in bladder cancer. Cell proliferation and migration assays after knockdown of splice variants of MIR31HG showed corresponding roles for the full-length transcript. CONCLUSIONS: Our study demonstrates that MIR31HG and its splice variants could serve as biomarkers for the classification and prognosis prediction of patients with MIBC.

Epigenetic profiling demarcates molecular subtypes of muscle-invasive bladder cancer.

Muscle-invasive bladder cancer (MIBC) is a heterogeneous disease that often recurs despite aggressive treatment with neoadjuvant chemotherapy and (radical) cystectomy. Basal and luminal molecular subtypes have been identified that are linked to clinical characteristics and have differential sensitivities to chemotherapy. While it has been suggested that epigenetic mechanisms play a role in defining these subtypes, a thorough understanding of the biological mechanisms is lacking. This report details the first genome-wide analysis of histone methylation patterns of human primary bladder tumours by chromatin immunoprecipitations and next-generation sequencing (ChIP-seq). We profiled multiple histone marks: H3K27me3, a marker for repressed genes, and H3K4me1 and H3K4me3, which are indicators of active enhancers and active promoters. Integrated analysis of ChIP-seq data and RNA sequencing revealed that H3K4 mono-methylation demarcates MIBC subtypes, while no association was found for the other two histone modifications in relation to basal and luminal subtypes. Additionally, we identified differentially methylated H3K4me1 peaks in basal and luminal tumour samples, suggesting that active enhancers play a role in defining subtypes. Our study is the first analysis of histone modifications in primary bladder cancer tissue and provides an important resource for the bladder cancer community.

Slow N-acetylation as a possible contributor to bladder carcinogenesis.

Bladder cancer (BCa) is an exophytic tumor that presents as either noninvasive confined to the mucosa (NMIBC) or invading the detrusor muscle (MIBC), and was recently further subgrouped into molecular subtypes. Arylamines, major BCa environmental and occupational risk factors, are mainly metabolized by the genetically polymorphic N-acetyltransferases 1, NAT1 and NAT2. In this study, we investigated the association between N-acetyltransferases genetic polymorphism and key MIBC and NMIBC tumor biomarkers and subtypes. A cohort of 250 males with histologically confirmed urothelial BCa was identified. Tumors were genotyped for NAT1 and NAT2 using real-time polymerase chain reaction (PCR), and characterized for mutations in TP53, RB1, and FGFR3 by PCR-restriction fragment length polymorphism. Pathology data and patients' smoking status were obtained from medical records. Pearson χ2 and Fisher exact tests were used to check for associations and interactions. Results show that NAT1 G560 A polymorphism is significantly associated with higher muscle-invasiveness (MIBC vs NMIBC; P = .001), higher tumor grade (high grade vs low grade; P = .011), and higher FGFR3 mutation frequency within the MIBC subgroup (P = .042; .027). NAT2 G857 A polymorphism is also found to be significantly associated with higher muscle-invasiveness (MIBC vs NMIBC; P = .041). Our results indicate that slow N-acetylation is a contributor to bladder carcinogenesis and muscle-invasiveness. These findings highlight NAT1 as a biomarker candidate in BCa and a potential target for drug development.

JUND-dependent up-regulation of HMOX1 is associated with cisplatin resistance in muscle-invasive bladder cancer.

The standard-of-care for metastatic muscle-invasive bladder cancer (MIBC) is platinum-based chemotherapy regimens. Acquired resistance that occurs frequently through unidentified mechanisms, however, remains the major obstacle for implementing therapeutic effectiveness. Here, using data mining and analysis on clinical samples, we show that expression of JUND, a core component of activator protein-1 family, was significantly induced in cisplatin (CDDP)-resistant MIBC. Accumulation of nuclear JUND was associated with low post-chemotherapy survival in MIBC patients. In both genetically engineered cell models and murine xenograft models, we provided evidence that bladder cancer (BC) cells with excessive JUND expression were less responsive to CDDP treatment. This CDDP resistance was further demonstrated to be mediated, at least in part, by transactivation of HMOX1 [the gene encoding heme oxygenase-1 (HO-1)], one of the most important antioxidant signalling pathways of cell adaptation to stress. One mutation within the HMOX1 promoter successfully abolished oxidative stress-enhanced and JUND-driven HMOX1 promoter activation, suggesting that this unique site synergized for maximal HO-1 induction in CDDP-challenged BC cells. Overall, our data highlight an indispensible role of JUND, both as a target as a modifier of the oxidative stress signalling, in conferring an adaptive response during the pathogenesis of CDDP resistance in MIBC.

An immune relevant signature for predicting prognoses and immunotherapeutic responses in patients with muscle-invasive bladder cancer (MIBC).

Immune checkpoint inhibitors (ICIs) are novel treatments that significantly improve the survival time of MIBC patients, but immunotherapeutic responses are different among MIBC patients. Therefore, it is urgent to find predictive biomarkers that can accurately identify MIBC patients who are sensitive to ICIs. In this study, we computed the relative abundances of 24 immune cells based on the expression profiles of MIBC patients using single-sample gene set enrichment analysis (ssGSEA). Unsupervised clustering analysis of the 24 immune cells was performed to classify MIBC patients into different immune-infiltrating groups. Genome (gene mutation and copy number variation), transcriptome (mRNA, lncRNA, and miRNA), and functional enrichment were found to be heterogeneous among different immune-infiltrating groups. We identified 282 differentially expressed genes (DEGs) associated with immune infiltration by comparing the expression profiles of patients with different immune infiltration profiles, and 20 core prognostic DEGs were identified by univariate Cox regression analysis. An immune-relevant gene signature (TIM signature) consisting of nine key prognostic DEGs (CCDC80, CD3D, CIITA, FN1, GBP4, GNLY, SPINK1, UBD, and VIM) was constructed using least absolute shrinkage and selection operator (LASSO) Cox regression analysis. Receiver operating characteristic (ROC) curves and subgroup analysis confirmed that the TIM signature was an ideal biomarker for predicting the prognosis of MIBC patients. Its value in predicting immunotherapeutic responses was also validated in The Cancer Genome Atlas (TCGA) cohort (AUC = 0.69, 95% CI = 0.63-0.74) and the IMvigor210 cohort (AUC = 0.64, 95% = 0.55-0.74). The TIM signature demonstrates a powerful ability to distinguish MIBC patients with different prognoses and immunotherapeutic responses, but more prospective studies are needed to assess its reliability in the future.

Tumor Infiltrating Neutrophils Are Enriched in Basal-Type Urothelial Bladder Cancer.

BACKGROUND: Urothelial bladder cancers (UBCs) are distinct in two main molecular subtypes, namely basal and luminal type. Subtypes are also diverse in term of immune contexture, providing a rationale for patient selection to immunotherapy. METHODS: By digital microscopy analysis of a muscle-invasive BC (MIBC) cohort, we explored the density and clinical significance of CD66b+ tumor-associated-neutrophils (TAN) and CD3+ T cells. Bioinformatics analysis of UBC datasets and gene expression analysis of UBC cell lines were additionally performed. RESULTS: Basal type BC contained a significantly higher density of CD66b+ TAN compared to the luminal type. This finding was validated on TCGA, GSE32894 and GSE124305 datasets by computing a neutrophil signature. Of note, basal-type MIBC display a significantly higher level of chemokines (CKs) attracting neutrophils. Moreover, pro-inflammatory stimuli significantly up-regulate CXCL1, CXCL2 and CXCL8 in 5637 and RT4 UBC cell lines and induce neutrophil chemotaxis. In term of survival, a high density of T celsl and TAN was significantly associated to a better outcome, with TAN density showing a more limited statistical power and following a non-linear predicting model. CONCLUSIONS: TAN are recruited in basal type MIBC by pro-inflammatory CKs. This finding establishes a groundwork for a better understanding of the UBC immunity and its relevance.

A TP53-Associated Immune Prognostic Signature for the Prediction of Overall Survival and Therapeutic Responses in Muscle-Invasive Bladder Cancer.

Background: TP53 gene mutation is one of the most common mutations in human bladder cancer (BC) and has been implicated in the progression and prognosis of BC. Methods: RNA sequencing data and TP53 mutation data in different populations and platforms were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database to determine and validate a TP53-associated immune prognostic signature (TIPS) based on differentially expressed immune-related genes (DEIGs) between muscle-invasive bladder cancer (MIBC) patients with and without TP53 mutations. Results: A total of 99 DEIGs were identified based on TP53 mutation status. TIPS including ORM1, PTHLH, and CTSE were developed and validated to identify high-risk prognostic group who had a poorer prognosis than low-risk prognostic group in TCGA and GEO database. The high-risk prognostic group were characterized by a higher abundance of regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages than the low-risk prognostic group. Moreover, they exhibited a lower abundance of CD56bright NK cells, higher expression of CTLA4, LAG3, PDCD1, TIGIT, and HAVCR2, as well as being more likely to respond to anti-PD-1, and neoadjuvant chemotherapy than the low-risk prognostic group. Based on TIPS and other clinical characteristics, a nomogram was constructed for clinical use. Conclusion: TIPS derived from TP53 mutation status is a potential prognostic signature or therapeutic target but additional prospective studies are necessary to confirm this potential.

Amplification of 7p12 Is Associated with Pathologic Nonresponse to Neoadjuvant Chemotherapy in Muscle-Invasive Bladder Cancer.

Pathologic downstaging (pDS) to neoadjuvant chemotherapy (NAC) is one of the most important predictors of survival in muscle-invasive bladder cancer (MIBC). The use of NAC is limited as pDS is only achieved in 30% to 40% of cases and predictive biomarkers are still lacking. We performed a comprehensive immunomolecular biomarker analysis to characterize the role of immune cells and inhibitory checkpoints, genome-wide frequencies of copy number alterations, mutational signatures in whole exome, and tumor mutational burden in predicting NAC response. Our retrospective study included 23 primary MIBC patients who underwent NAC, followed by radical cystectomy. pDS to NAC was a significant prognostic factor for better recurrence-free survival (P < 0.001), with a median time to recurrence of 41.2 versus 5.5 months in nonresponders. DNA damage repair alterations were noticed in 38.1% (n = 8), confirming a positive correlation with high tumor mutational burden (P = 0.007). Chromosomal 7p12 amplification, including the genes HUS1, EGFR, ABCA13, and IKZF1, predicted nonresponse in patients with a sensitivity, a negative predictive value, and a specificity of 71.4%, 87.5%, and 100%, respectively. Total count of CD3+ T cells/mm2 tumor was a significant predictor of NAC response. In conclusion, 7p12 amplification may predict nonresponse to NAC and worse survival in MIBC. Multicenter, prospective trials with sufficient statistical power may further fortify these findings.

Hypermethylation of FOXA1 and allelic loss of PTEN drive squamous differentiation and promote heterogeneity in bladder cancer.

Intratumoral heterogeneity in bladder cancer is a barrier to accurate molecular sub-classification and treatment efficacy. However, individual cellular and mechanistic contributions to tumor heterogeneity are controversial. We examined potential mechanisms of FOXA1 and PTEN inactivation in bladder cancer and their contribution to tumor heterogeneity. These analyses were complemented with inactivation of FOXA1 and PTEN in intermediate and luminal mouse urothelium. We show inactivation and reduced expression of FOXA1 and PTEN is prevalent in human disease, where PTEN and FOXA1 are downregulated by allelic loss and site-specific DNA hypermethylation, respectively. Conditional inactivation of both Foxa1 and Pten in intermediate/luminal cells in mice results in development of bladder cancer exhibiting squamous features as well as enhanced sensitivity to a bladder-specific carcinogen. In addition, FOXA1 is hypermethylated in basal bladder cancer cell lines, and this is reversed by treatment with DNA methyltransferase inhibitors. By integrating human correlative and in vivo studies, we define a critical role for PTEN loss and epigenetic silencing of FOXA1 in heterogeneous human disease and show genetic targeting of luminal/intermediate cells in mice drives squamous differentiation.

Long non-coding RNAs identify a subset of luminal muscle-invasive bladder cancer patients with favorable prognosis.

BACKGROUND: Muscle-invasive bladder cancer (MIBC) is a heterogeneous disease, and gene expression profiling has identified several molecular subtypes with distinct biological and clinicopathological characteristics. While MIBC subtyping has primarily been based on messenger RNA (mRNA), long non-coding RNAs (lncRNAs) may provide additional resolution. METHODS: LncRNA expression was quantified from microarray data of a MIBC cohort treated with neoadjuvant chemotherapy (NAC) and radical cystectomy (RC) (n = 223). Unsupervised consensus clustering of highly variant lncRNAs identified a four-cluster solution, which was characterized using a panel of MIBC biomarkers, regulon activity profiles, gene signatures, and survival analysis. The four-cluster solution was confirmed in The Cancer Genome Atlas (TCGA) cohort (n = 405). A single-sample genomic classifier (GC) was trained using ridge-penalized logistic regression and validated in two independent cohorts (n = 255 and n = 94). RESULTS: NAC and TCGA cohorts both contained an lncRNA cluster (LC3) with favorable prognosis that was enriched with tumors of the luminal-papillary (LP) subtype. In both cohorts, patients with LP tumors in LC3 (LPL-C3) were younger and had organ-confined, node-negative disease. The LPL-C3 tumors had enhanced FGFR3, SHH, and wild-type p53 pathway activity. In the TCGA cohort, LPL-C3 tumors were enriched for FGFR3 mutations and depleted for TP53 and RB1 mutations. A GC trained to identify these LPL-C3 patients showed robust performance in two validation cohorts. CONCLUSIONS: Using lncRNA expression profiles, we identified a biologically distinct subgroup of luminal-papillary MIBC with a favorable prognosis. These data suggest that lncRNAs provide additional information for higher-resolution subtyping, potentially improving precision patient management.

MicroRNAs in tumor samples and urinary extracellular vesicles as a putative diagnostic tool for muscle-invasive bladder cancer.

PURPOSE: The identification of biomarkers characterizing the invasive potential of bladder cancer could enhance the clinical management of individual patients and therefore improve prognosis. The aim of this study was to define a miRNA panel in tumor tissues as well as in urinary extracellular vesicles (EVs) for discriminating muscle-invasive bladder cancer (MIBC) from non-muscle-invasive bladder cancer (NMIBC). METHODS: miRNA expression was analyzed in 24 formalin-fixed, paraffin-embedded (FFPE) tumor samples by microarray analysis and was further validated by qRT-PCR in 56 FFPE tumor samples as well as in 37 urinary EV samples. RESULTS: Microarray analysis revealed 63 miRNAs that were significantly differentially expressed (P < 0.05) between tissues from MIBC and NMIBC tumors. Five selected miRNAs (miR-146b-5p, miR-155-5p, miR-138-5p, miR-144-5p, and miR-200a-3p) were validated by qRT-PCR. The expression of all except miR-144-5p was significantly associated with high tumor grade. In urinary EVs, a different expression was verified for miR-146b-5p (P = 0.004) and miR-155-5p (P = 0.036), which exhibited significantly higher expression in urinary EVs from patients with MIBC. CONCLUSIONS: miRNAs are promising biomarkers for the identification of invasive bladder carcinomas. Tissue samples as well as urinary EVs may serve as sources for miRNA analysis. This method, in addition to histopathology, could provide a new diagnostic tool and facilitate individual therapeutic decisions to select patients for early cystectomy.

Gene Editing of α6 Integrin Inhibits Muscle Invasive Networks and Increases Cell-Cell Biophysical Properties in Prostate Cancer.

Human prostate cancer confined to the gland is indolent (low-risk), but tumors outside the capsule are aggressive (high-risk). Extracapsular extension requires invasion within and through a smooth muscle-structured environment. Because integrins respond to biomechanical cues, we used a gene editing approach to determine if a specific region of laminin-binding α6ß1 integrin was required for smooth muscle invasion both in vitro and in vivo. Human tissue specimens showed prostate cancer invasion through smooth muscle and tumor coexpression of α6 integrin and E-cadherin in a cell-cell location and α6 integrin in a cell-extracellular matrix (ECM) distribution. Prostate cancer cells expressing α6 integrin (DU145 α6WT) produced a 3D invasive network on laminin-containing Matrigel and invaded into smooth muscle both in vitro and in vivo. In contrast, cells without α6 integrin (DU145 α6KO) and cells expressing an integrin mutant (DU145 α6AA) did not produce invasive networks, could not invade muscle both in vitro and in vivo, and surprisingly formed 3D cohesive clusters. Using electric cell-substrate impedance testing, cohesive clusters had up to a 30-fold increase in normalized resistance at 400 Hz (cell-cell impedance) as compared with the DU145 α6WT cells. In contrast, measurements at 40,000 Hz (cell-ECM coverage) showed that DU145 α6AA cells were two-fold decreased in normalized resistance and were defective in restoring resistance after a 1 µmol/L S1P challenge as compared with the DU145 α6WT cells. The results suggest that gene editing of a specific α6 integrin extracellular region, not required for normal tissue function, can generate a new biophysical cancer phenotype unable to invade the muscle, presenting a new therapeutic strategy for metastasis prevention in prostate cancer. SIGNIFICANCE: This study shows an innovative strategy to block prostate cancer metastasis and invasion in the muscle through gene editing of a specific α6 integrin extracellular region.

Mouse Models of Muscle-invasive Bladder Cancer: Key Considerations for Clinical Translation Based on Molecular Subtypes.

CONTEXT: In the past few years, research has suggested that molecular subtypes in muscle-invasive bladder cancer (MIBC) may be exploited to accelerate developments in clinical disease management and novel therapeutics. OBJECTIVE: To review MIBC mouse models from a molecular subtype perspective, their advantages and limitations, and their applications in translational medicine, based on a PubMed search for publications from January 2000 to February 2018. EVIDENCE ACQUISITION: Publications relevant to MIBC mouse models and their molecular subtypes were identified in a literature review. EVIDENCE SYNTHESIS: We classified the models according to the technique used for their establishment. For xenotransplant and allograft models, the inoculated cells and inoculated locations are the major determinants of molecular subtypes. Although the cell lines used in xenotransplant models can cover most of the basal-squamous and luminal subtypes, allograft models offer a more realistic environment in which to reconstruct aspects of the associated stromal and immune features. Autochthonous models, using genetic and/or chemical stimuli to induce disease progression, can also generate models with basal-squamous and luminal subtypes, but further molecular characterisation is needed since other mutational variants may be introduced in these models. CONCLUSIONS: We identified preclinical MIBC models with different subtype specifications and assessed their promise and current limitations. These models are versatile tools that can reproduce the molecular complexity of MIBC and support novel therapeutic development. PATIENT SUMMARY: Understanding which models of muscle-invasive bladder cancer most accurately represent the clinical situation is important for the development of novel drugs and disease management strategies. We review the different models currently available and their relevance to different clinical subtypes.

Tumor­infiltrating M2 macrophages driven by specific genomic alterations are associated with prognosis in bladder cancer.

The present study aimed to explore the mechanism by which the immune landscape of the tumor microenvironment influences bladder cancer. CIBERSORT and ssGSEA analyses revealed that M2 macrophages accounted for the highest proportion from 22 subsets of tumor­infiltrating immune cells and were enriched in higher histologic grade and higher pathologic stage bladder cancer and 'basal' subtype of muscle invasive bladder cancer (MIBC). Kaplan­Meier survival curve analysis indicated that patients with high numbers of infiltrating M2 macrophages had worse overall and disease­specific survival rates. RNA sequencing and immunohistochemistry results indicated that M2 macrophages were enriched in MIBC and promoted angiogenesis. M2 macrophage infiltration was higher in bladder cancer tissues with mutant TP53, RB transcriptional corepressor 1, phosphatidylinositol­4,5­bisphosphate 3­kinase catalytic subunit α, lysine methyltransferase 2A, lysine demethylase 6A and apolipoprotein B mRNA editing enzyme catalytic­polypeptide­like, but lower in tissues with mutant fibroblast growth factor receptor 3 (FGFR3), E74­like ETS transcription factor 3, PC4 and SFRS1 interacting protein 1 and transmembrane and coiled­coil domains 4. In addition, M2 macrophage infiltration was lower in the tissues with amplified FGFR3, erb­b2 receptor tyrosine kinase 2, BCL2­like 1, telomerase reverse transcriptase and tyrosine­3­monooxygenase/tryptophan­5­monooxygenase activation protein ζ, as well as in the tissues with deleted cyclin­dependent kinase inhibitor 2A, CREB binding protein, AT­rich interaction domain 1A, fragile histidine triad diadenosine triphosphatase, phosphodiesterase 4D, RAD51 paralog B, nuclear receptor corepressor 1 and protein tyrosine phosphatase receptor type D. Finally, seven micro (mi) RNAs (miR­214­5p, miR­223­3p, miR­155­5p, miR­199a­3p, miR­199b­3P, miR­146b­5p, miR­142­5p) which were expressed differentially in at least three mutant genes and were positively correlated with M2 macrophage infiltration as well as expressed highly in high grade bladder cancer were identified. Overall, the present study concluded that M2 macrophages are the predominant tumor­infiltrating immune cell in bladder cancer and differentially expressed miRNAs due to cancer­specific genomic alterations may be important drivers of M2 macrophage infiltration. These findings suggested that M2 macrophage infiltration may serve as a potential immunotherapy target in bladder cancer.