An oncolytic HSV-1 armed with Visfatin enhances antitumor effects by remodeling tumor microenvironment against murine pancreatic cancer.

Oncolytic viruses (OVs) have shown potential in converting a "cold" tumor into a "hot" one and exhibit effectiveness in various cancer types. However, only a subset of patients respond to oncolytic virotherapy. It is important to understand the resistance mechanisms to OV treatment in pancreatic ductal adenocarcinoma (PDAC) to engineer oncolytic viruses. In this study, we used transcriptome RNA sequencing (RNA-seq) to identify Visfatin, which was highly expressed in the responsive tumors following OV treatment. To explore the antitumor efficacy, we modified OV-mVisfatin, which effectively inhibited tumor growth. For the first time, we revealed that Visfatin promoted the antitumor efficacy of OV by remodeling the tumor microenvironment, which involved enhancing CD8+ T cell and DC cell infiltration and activation, repolarizing macrophages towards the M1-like phenotype, and decreasing Treg cells using single-cell RNA sequencing (scRNA-seq) and flow cytometry. Furthermore, PD-1 blockade significantly enhanced OV-mVisfatin antitumor efficacy, offering a promising new therapeutic strategy for PDAC.

Reprogramming macrophage by targeting VEGF and CD40 potentiates OX40 immunotherapy.

The low clinical response rate of checkpoint blockades, such as PD-1 and CTLA-4, highlighted the requirements of agonistic antibodies to boost optimal T cell responses. OX40, a co-stimulatory receptor on the T cells, plays a crucial role in promoting T cell survival and differentiation. However, the clinical efficacy of anti-OX40 agonistic antibodies was unimpressive. To explore the mechanism underlying the action of anti-OX40 agonists to improve the anti-tumor efficacy, we analyzed the dynamic changes of tumor-infiltrating immune cells at different days post-treatments using single-cell RNA-sequencing (scRNA-seq). In this study, we found that tumor-infiltrating regulatory T (Treg) cells were reduced after two rounds of anti-OX40 treatment, but the increase of infiltration and activation of CD8+ effector T cells, as well as M1 polarization in the tumor were only observed after three rounds of treatments. Moreover, our group first analyzed the antitumor effect of anti-OX40 treatments on regulating the macrophages and discovered the dynamic changes of vascular endothelial growth factor (VEGF) and CD40 signaling pathways on macrophages, indicating their possibility to being potential combination targets to improve the anti-OX40 agonists efficacy. The combination of VEGFR inhibitors or anti-CD40 agonist antibody with anti-OX40 agonists exhibited more remarkable inhibition of tumor growth. Therefore, the mechanism-driven combination of anti-OX40 agonists with VEGFR inhibitors or anti-CD40 agonists represented promising strategies.

Reshaping the Immune Microenvironment by Oncolytic Herpes Simplex Virus in Murine Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the major type of pancreatic malignancy with very poor prognosis. Despite the promising results of immune checkpoint inhibitors (ICIs) in some solid tumors, immunotherapy is less effective for PDAC due to its immunosuppressive tumor microenvironment (TME). In this report, we established an immunocompetent syngeneic PDAC model and investigated the effect of oncolytic herpes simplex virus-1 (oHSV) on the composition of TME immune cells. The oHSV treatment significantly reduced tumor burden and prolonged the survival of tumor-bearing mice. Further, by single cell RNA sequencing (scRNA-seq) and multicolor fluorescence-activated cell sorting (FACS) analysis, we demonstrated that oHSV administration downregulated tumor-associated macrophages (TAMs), especially the anti-inflammatory macrophages, and increased the percentage of tumor-infiltrating lymphocytes, including activated cytotoxic CD8+ T cells and T helper (Th)1 cells. Besides, the combination of oHSV and immune checkpoint modulators extended the lifespan of the tumor-bearing mice. Overall, our data suggested that oHSV reshapes the TME of PDAC by boosting the immune activity and leads to improved responsiveness of PDAC to immunotherapy.

The interaction between Vav1 and EBNA1 promotes survival of Burkitt's lymphoma cells by down-regulating the expression of Bim.

Vav1 is a guanine nucleotide exchange factor (GEF) predominantly expressed in hematopoietic cells, and functions in the development and antigen-stimulated response of lymphocytes. Burkitt's lymphoma (BL) is characterized as transformed B cell lymphoma, and is highly associated with Epstein-Barr virus (EBV). EBV nuclear antigen 1 (EBNA1) is the only viral protein expressed across all three types of latency and essential for the persistence of EBV genome. It is not clear yet how EBNA1 contributes to the growth advantage of latently infected cells such as in EBV+ lymphoma B cells. Here, we reported that Vav1 interacts with EBNA1 via its C-terminal SH3 domain. This interaction suppresses the expression of a pro-apoptotic Bcl-2 family member, Bim, resulting in the resistance of the BL cells to apoptotic inductions. Our data uncovered Vav1 as a novel target for EBNA1, and suggested a pro-survival role of Vav1 in the pathogenesis of EBV associated BLs.

Smoking increased the risk of prostate cancer with grade group ≥ 4 and intraductal carcinoma in a prospective biopsy cohort.

OBJECTIVE: To investigate the association between smoking and different prostate cancer (PCa) pathological subtypes incidence in Chinese men. PATIENTS AND METHODS: We prospectively included 1795 patients who underwent prostate biopsies in one tertiary center between March 2013 and April 2016. Clinical data and biopsy outcomes were collected. Logistic regression was used to evaluate the association between cigarette smoking and PCa incidence. RESULTS: A total of 737 men, 480 men and 58 men were diagnosed with PCa, high-grade PCa (HGPCa, grade group ≥ 4 as accepted by the 2014 ISUP) and intraductal carcinoma of the prostate (IDC-P), respectively. Current smokers had a significantly higher risk of HGPCa than never smokers (OR = 1.89, 95%CI: 1.44-2.48). No such association was observed for low-grade disease and cigarette smoking (OR = 0.84, 95%CI: 0.61-1.16). In a sub-analysis, men who had smoked longer than 30 years had a higher risk of HGPCa, compared with men who had smoked fewer than 30 years (OR = 1.50, 95%CI: 1.09-2.06). Current smokers were more likely to develop IDC-P than never smokers (OR = 2.29, 95%CI: 1.14-4.59). CONCLUSION: Among men in this Chinese biopsy cohort, current smoking was associated with highly malignant PCa incidence, such as HGPCa and IDC-P. The duration of smoking may be associated with HGPCa.

Herpes Simplex Virus 1 Induces Phosphorylation and Reorganization of Lamin A/C through the γ134.5 Protein That Facilitates Nuclear Egress.

Herpes simplex virus 1 (HSV-1) remodels nuclear membranes during virus egress. Although the UL31 and UL34 proteins control nucleocapsid transit in infected cells, the molecular interactions required for their function are unclear. Here we report that the γ134.5 gene product of HSV-1 facilitates nucleocapsid release to the cytoplasm through bridging the UL31/UL34 complex, cellular p32, and protein kinase C. Unlike wild-type virus, an HSV mutant devoid of γ134.5 or its amino terminus is crippled for viral growth and release. This is attributable to a defect in virus nuclear egress. In infected cells, wild-type virus recruits protein kinase C to the nuclear membrane and triggers its activation, whereas the γ134.5 mutants fail to exert such an effect. Accordingly, the γ134.5 mutants are unable to induce phosphorylation and reorganization of lamin A/C. When expressed in host cells γ134.5 targets p32 and protein kinase C. Meanwhile, it communicates with the UL31/UL34 complex through UL31. Deletion of the amino terminus from γ134.5 disrupts its activity. These results suggest that disintegration of the nuclear lamina mediated by γ134.5 promotes HSV replication. IMPORTANCE: HSV nuclear egress is a key step that determines the outcome of viral infection. While the nuclear egress complex mediates capsid transit across the nuclear membrane, the regulatory components are not clearly defined in virus-infected cells. We report that the γ134.5 gene product, a virulence factor of HSV-1, facilitates nuclear egress cooperatively with cellular p32, protein kinase C, and the nuclear egress complex. This work highlights a viral mechanism that may contribute to the pathogenesis of HSV infection.

The Us3 Protein of Herpes Simplex Virus 1 Inhibits T Cell Signaling by Confining Linker for Activation of T Cells (LAT) Activation via TRAF6 Protein.

Herpes simplex virus 1 (HSV-1) is the most prevalent human virus and causes global morbidity because the virus is able to infect multiple cell types. Remarkably, HSV infection switches between lytic and latent cycles, where T cells play a critical role. However, the precise way of virus-host interactions is incompletely understood. Here we report that HSV-1 productively infected Jurkat T-cells and inhibited antigen-induced T cell receptor activation. We discovered that HSV-1-encoded Us3 protein interrupted TCR signaling and interleukin-2 production by inactivation of the linker for activation of T cells. This study unveils a mechanism by which HSV-1 intrudes into early events of TCR-mediated cell signaling and may provide novel insights into HSV infection, during which the virus escapes from host immune surveillance.

p32 is a novel target for viral protein ICP34.5 of herpes simplex virus type 1 and facilitates viral nuclear egress.

As a large double-stranded DNA virus, herpes simplex virus type 1 (HSV-1) assembles capsids in the nucleus where the viral particles exit by budding through the inner nuclear membrane. Although a number of viral and host proteins are involved, the machinery of viral egress is not well understood. In a search for host interacting proteins of ICP34.5, which is a virulence factor of HSV-1, we identified a cellular protein, p32 (gC1qR/HABP1), by mass spectrophotometer analysis. When expressed, ICP34.5 associated with p32 in mammalian cells. Upon HSV-1 infection, p32 was recruited to the inner nuclear membrane by ICP34.5, which paralleled the phosphorylation and rearrangement of nuclear lamina. Knockdown of p32 in HSV-1-infected cells significantly reduced the production of cell-free viruses, suggesting that p32 is a mediator of HSV-1 nuclear egress. These observations suggest that the interaction between HSV-1 ICP34.5 and p32 leads to the disintegration of nuclear lamina and facilitates the nuclear egress of HSV-1 particles.

Parthenolide induces autophagy via the depletion of 4E-BP1.

Parthenolide (PTL) is a sesquiterpene lactone isolated from feverfew and exhibits potent antitumor activity against various cancers. Many studies indicate that PTL treatment leads to apoptosis, however, the mechanism has not been defined. Here, we observed that cells underwent autophagy shortly after PTL treatment. Inhibition of autophagy by knocking out autophagy associated gene atg5 blocked PTL-induced apoptosis. Surprisingly, PTL decreased the level of translation initiation factor eIF4E binding protein 1 (4E-BP1) in correlation with autophagy. Ectopic expression or shRNA knockdown of 4E-BP1 further verified the effect of 4E-BP1 on PTL-induced autophagy. Meanwhile, PTL elevated the cellular reactive oxygen species (ROS) which located upstream of the depletion of 4E-BP1, and contributed to the consequent autophagy. This study revealed 4E-BP1 as a trigger for PTL-induced autophagy and may lead to therapeutic strategy to enhance the efficacy of anticancer drugs.

The N-terminal 20-amino acid region of guanine nucleotide exchange factor Vav1 plays a distinguished role in T cell receptor-mediated calcium signaling.

Vav1 is a guanine nucleotide exchange factor (GEF) specifically expressed in hematopoietic cells. It consists of multiple structural domains and plays important roles in T cell activation. The other highly conserved isoforms of Vav family, Vav2 and Vav3, are ubiquitously expressed in human tissues including lymphocytes. All three Vav proteins activate Rho family small GTPases, which are involved in a variety of biological processes during T cell activation. Intensive studies have demonstrated that Vav1 is indispensable for T cell receptor (TCR)-mediated signal transduction, whereas Vav2 and Vav3 function as GEFs that overlap with Vav1 on TCR-induced cytoskeleton reorganization. T cells lacking Vav1 exhibited severe defect in TCR-mediated calcium elevation, indicating that the co-existing Vav2 and Vav3 did not compensate Vav1 in calcium signaling. What is the functional particularity of Vav1 in lymphocytes? In this study, we identified the N-terminal 20 amino acids of Vav1 in the calponin homology (CH) domain to be essential for its interaction with calmodulin (CaM) that leads to TCR-induced calcium mobilization. Substitution of the 1-20 amino acids of Vav1 with those of Vav2 or Vav3 abolished the association with CaM, and the N-terminal mutations of Vav1 failed to potentiate normal TCR-induced calcium mobilization, that in turn, suspended nuclear factor of activated T cells (NFAT) activation and IL-2 production. This study highlights the importance of the N-terminal 20 aa of Vav1 for CaM binding, and provides new insights into the distinguished and irreplaceable role of Vav1 in T cell activation and signal transduction.

Construction of an IL12 and CXCL11 armed oncolytic herpes simplex virus using the CRISPR/Cas9 system for colon cancer treatment.

Oncolytic viruses are an emerging cancer treatment modality with promising results in clinical trials. The new generation of oncolytic viruses are genetically modified to enhance virus selectivity for tumor cells and allow local expression of therapeutic genes in tumors. The traditional technique for viral genome engineering based on homologous recombination using a bacterial artificial chromosome (BAC) system is laborious and time-consuming. With the advent of the CRISPR/Cas9 system, the efficiency of gene editing in human cells and other organisms has dramatically increased. In this report, we successfully applied the CRISPR/Cas9 technique to construct an HSV-based oncolytic virus, where the ICP34.5 coding region was replaced with the therapeutic genes murine interleukin 12 (IL12, p40-p35) and C-X-C motif chemokine ligand 11 (CXCL11), and ICP47 gene was deleted. The combination of IL12 and CXCL11 in oncolytic viruses showed considerable promise in colorectal cancer (CRC) treatment. Overall, our study describes genetic modification of the HSV-1 genome using the CRISPR/Cas9 system and provides evidence from principle studies for engineering of the HSV genome to express foreign genes.

ICP34.5 protein of herpes simplex virus facilitates the initiation of protein translation by bridging eukaryotic initiation factor 2alpha (eIF2alpha) and protein phosphatase 1.

The ICP34.5 protein of herpes simplex virus type 1 is a neurovirulence factor that plays critical roles in viral replication and anti-host responses. One of its functions is to recruit protein phosphatase 1 (PP1) that leads to the dephosphorylation of the α subunit of translation initiation factor eIF2 (eIF2α), which is inactivated by infection-induced phosphorylation. As PP1 is a protein phosphatase with a wide range of substrates, the question remains to be answered how ICP34.5 directs PP1 to specifically dephosphorylate eIF2α. Here we report that ICP34.5 not only binds PP1 but also associates with eIF2α by in vitro and in vivo assays. The binding site of eIF2α is identified at amino acids 233-248 of ICP34.5, which falls in the highly homologous region with human gene growth arrest and DNA damage 34. The interaction between ICP34.5 and eIF2α is independent of the phosphorylation status of eIF2α at serine 51. Deletion mutation of this region results in the failure of dephosphorylation of eIF2α by PP1 and, consequently, interrupts viral protein synthesis and replication. Our data illustrated that the binding between viral protein ICP34.5 and the host eIF2α is crucial for the specific dephosphorylation of eIF2α by PP1. We propose that herpes simplex virus protein ICP34.5 bridges PP1 and eIF2α via their binding motifs and thereby facilitates the protein synthesis and viral replication.

CD40L-armed oncolytic herpes simplex virus suppresses pancreatic ductal adenocarcinoma by facilitating the tumor microenvironment favorable to cytotoxic T cell response in the syngeneic mouse model.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant cancers worldwide. Despite the promising outcome of immune checkpoint inhibitors and agonist antibody therapies in different malignancies, PDAC exhibits high resistance due to its immunosuppressive tumor microenvironment (TME). Ameliorating the TME is thus a rational strategy for PDAC therapy. The intratumoral application of oncolytic herpes simplex virus-1 (oHSV) upregulates pro-inflammatory macrophages and lymphocytes in TME, and enhances the responsiveness of PDAC to immunotherapy. However, the antitumor activity of oHSV remains to be maximized. The aim of this study is to investigate the effect of the CD40L armed oHSV on the tumor immune microenvironment, and ultimately prolong the survival of the PDAC mouse model. METHODS: The membrane-bound form of murine CD40L was engineered into oHSV by CRISPR/Cas9-based gene editing. oHSV-CD40L induced cytopathic effect and immunogenic cell death were determined by microscopy and flow cytometry. The expression and function of oHSV-CD40L was assessed by reporter cell assay. The oHSV-CD40L was administrated intratumorally to the immune competent syngeneic PDAC mouse model, and the leukocytes in TME and tumor-draining lymph node were analyzed by multicolor flow cytometry. Intratumoral cytokines were determined by ELISA. RESULTS: Intratumoral application of oHSV-CD40L efficiently restrained the tumor growth and prolonged the survival of the PDAC mouse model. In TME, oHSV-CD40L-treated tumor accommodated more maturated dendritic cells (DCs), which in turn activated T helper 1 and cytotoxic CD8+ T cells in an interferon-γ-dependent and interleukin-12-dependent manner. In contrast, the regulatory T cells were significantly reduced in TME by oHSV-CD40L treatment. Repeated dosing and combinational therapy extended the lifespan of PDAC mice. CONCLUSION: CD40L-armed oncolytic therapy endues TME with increased DCs maturation and DC-dependent activation of cytotoxic T cells, and significantly prolongs the survival of the model mice. This study may lead to the understanding and development of oHSV-CD40L as a therapy for PDAC in synergy with immune checkpoint blockade.

The distinct role of guanine nucleotide exchange factor Vav1 in Bcl-2 transcription and apoptosis inhibition in Jurkat leukemia T cells.

AIM: To investigate a novel function of proto-oncogene Vav1 in the apoptosis of human leukemia Jurkat cells. METHODS: Jurkat cells, Jurkat-derived vav1-null cells (J.Vav1) and Vav1-reconstituted J.WT cells were treated with a Fas agonist antibody, IgM clone CH11. Apoptosis was determined using propidium iodide (PI) staining, Annexin-V staining, DNA fragmentation, cleavage of caspase 3/caspase 8, and poly (ADP-ribose) polymerase (PARP). Mitochondria transmembrane potential (ΔΨ(m)) was measured using DiOC(6)(3) staining. Transcription and expression of the Bcl-2 family of proteins were evaluated using semi-quantitative RT-PCR and Western blot, respectively. Bcl-2 promoter activity was analyzed using luciferase reporter assays. RESULTS: Cells lacking Vav1 were more sensitive to Fas-mediated apoptosis than Jurkat and J.WT cells. J.Vav1 cells lost mitochondria transmembrane potential (ΔΨ(m)) more rapidly upon Fas induction. These phenotypes could be rescued by re-expression of Vav1 in J.Vav1 cells. The expression of Vav1 increased the transcription of pro-survival Bcl-2. The guanine nucleotide exchange activity of Vav1 was required for enhancing Bcl-2 promoter activity, and the Vav1 downstream substrate, small GTPase Rac2, was likely involved in the control of Bcl-2 expression. CONCLUSION: Vav1 protects Jurkat cells from Fas-mediated apoptosis by promoting Bcl-2 transcription through its GEF activity.

Dephosphorylation of eIF2alpha mediated by the gamma134.5 protein of herpes simplex virus 1 facilitates viral neuroinvasion.

The gamma(1)34.5 protein, a virulence factor of herpes simplex viruses, redirects protein phosphatase 1 to dephosphorylate the alpha subunit of translation initiation factor 2 (eIF2alpha). Additionally, it inhibits the induction of antiviral genes by TANK-binding kinase 1. Nevertheless, its precise role in vivo remains to be established. Here we show that eIF2alpha dephosphorylation by gamma(1)34.5 is crucial for viral neuroinvasion. V(193)E and F(195)L substitutions in gamma(1)34.5 abrogate viral replication in the eye and spread to the trigeminal ganglia and brain. Intriguingly, inhibition of antiviral gene induction by gamma(1)34.5 is not sufficient to exhibit viral virulence.

The gamma 1 34.5 protein of herpes simplex virus 1 is required to interfere with dendritic cell maturation during productive infection.

The gamma(1)34.5 protein of herpes simplex virus 1 is an essential factor for viral virulence. In infected cells, this viral protein prevents the translation arrest mediated by double-stranded RNA-dependent protein kinase R. Additionally, it associates with and inhibits TANK-binding kinase 1, an essential component of Toll-like receptor-dependent and -independent pathways that activate interferon regulatory factor 3 and cytokine expression. Here, we show that gamma(1)34.5 is required to block the maturation of conventional dendritic cells (DCs) that initiate adaptive immune responses. Unlike wild-type virus, the gamma(1)34.5 null mutant stimulates the expression of CD86, major histocompatibility complex class II (MHC-II), and cytokines such as alpha/beta interferon in immature DCs. Viral replication in DCs inversely correlates with interferon production. These phenotypes are also mirrored in a mouse ocular infection model. Further, DCs infected with the gamma(1)34.5 null mutant effectively activate naive T cells whereas DCs infected with wild-type virus fail to do so. Type I interferon-neutralizing antibodies partially reverse virus-induced upregulation of CD86 and MHC-II, suggesting that gamma(1)34.5 acts through interferon-dependent and -independent mechanisms. These data indicate that gamma(1)34.5 is involved in the impairment of innate immunity by inhibiting both type I interferon production and DC maturation, leading to defective T-cell activation.

Metastatic cell detection using a phage-peptide-modified light-addressable potentiometric sensor.

Detection of metastatic cells is clinically demanded to diagnose metastasis in the early stage and access the therapeutic response to anticancer drugs. We applied phage display technology to cultured cells with different metastasis potentials and obtained four metastasis-associated peptides. The association between peptides and metastatic cells was validated by ELISA as well as biosensor studies. The selected phage-peptides not only bound SW620, the metastatic cell against which the peptides were screened, but were also able to capture breast cancer cells of high metastasis. The phage-peptide-modified LAPS (light-addressable potentiometric sensor) was able to distinguish metastatic cells from non-metastatic cells and detect as few as 100 metastatic cells per ml of blood. Thus LAPS modified with specific phage-peptides may be developed to provide a new diagnostic approach that can aid the treatment of cancer.

A conserved domain of herpes simplex virus ICP34.5 regulates protein phosphatase complex in mammalian cells.

ICP34.5, encoded by herpes simplex virus 1, is a protein phosphatase 1 (PP1) regulatory subunit that mediates dephosphorylation of the alpha subunit of translation initiation factor 2 (eIF2alpha). However, the mechanism of its action remains poorly understood. Here, we show that amino acid substitutions in the arginine-rich motif have differential effects on ICP34.5 activity. The phenotypes parallel with viral protein synthesis and cytopathic effects in virus infected cells. Besides the consensus PP1 binding motif, the Arg-motif appears to enhance the interaction between ICP34.5 and PP1. These results suggest that concerted action between the PP1 binding domain and the effector domain of ICP34.5 is crucial for eIF2alpha dephosphorylation and viral protein synthesis.

Preoperative prostate health index predicts poor pathologic outcomes of radical prostatectomy in patients with biopsy-detected low-risk patients prostate cancer: results from a Chinese prospective cohort.

OBJECTIVE: To determine the performance of the prostate health index (PHI) in predicting pathologic outcomes of radical prostatectomy (RP) in Chinese patients with low-risk prostate cancer (PCa). METHODS: Of all consecutive patients who underwent RP in one tertiary center from September 2013 to January 2017, we prospectively examined the data of 140 patients with low-risk PCa based on the Prostate Cancer Research International: Active Surveillance (PRIAS) criteria. All patients were eligible for active surveillance, but underwent RP. Clinical and pathological data were collected. Logistic regression was used to evaluate the associations between the PHI and outcome of RP. The area under the receiver operating curve (AUC) was used to evaluate the accuracy of different models. Decision curve analysis was used to evaluate the potential clinical usefulness of making model-based decisions. RESULTS: Only 44 (31.4%) patients were finally confirmed to have organ-confined Gleason ≤6 PCa. A low PHI was significantly predictive of organ-confined Gleason ≤6 PCa (p = 0.001), while tPSA and f/tPSA were not associated with final pathology. In the multivariate analyses, addition of the PHI significantly increased the predictive accuracy (AUC = 0.767, 95% Cl 0.685-0.849, p < 0.001). CONCLUSION: The PRIAS criteria for active surveillance may not suitable for Chinese patients with PCa. Addition of the PHI to the PRIAS models improved the prognostic performance. If confirmed in future larger and multicenter studies, PHI may help us to identify patients eligible for AS in China.

Validation of Urine-based Gene Classifiers for Detecting Bladder Cancer in a Chinese Study.

Background: Current standard methods used to detect and monitor bladder cancer (BC) are invasive or have low sensitivity. We have previously reported in an international European study four non-invasive tests for BC diagnosis based on the gene expression patterns of urine. Objective: to validate the tests in an independent Asian cohort. Design, setting and participants: Prospective blinded study in which consecutive voided urine samples from BC patients and controls (n=520) were collected in the Fudan University Shanghai Cancer Center from 2014-2016. Gene expression values were quantified using TaqMan Arrays. The same cut-off as previously reported for discrimination between tumours and controls was used in this validation study. Results and limitations: Finally, a total of 257 tumour and 132 control urine samples were analysed. We found a high accuracy for the four gene classifiers in this independent Asian set, the classifiers composed of 5 and 10 genes achieved the best sensitivity (80.54% and 81.32%, respectively) maintaining a high specificity (91.67% and 85.61%, respectively). Sensitivity of 5-gene (GS_D5) and 10-gene (GS_D10) expression classifiers in recurrent BC cases (78 and 79%, respectively) is comparable to that of primary BC cases (82%). Cytology and NMP22 identified 67% and 40%, respectively, of tumours that have been diagnosed with our tests. In addition, influence of each studied gene was analyzed and showed similar gene rank between Chinese and Caucasian population. Conclusions: Our study proves that our non-invasive diagnostic BC tests can be reproduced in independent cohorts and in an external laboratory. All the four gene classifiers have shown equal or superior performance to the current gold standard in the present and previously reported validation studies. Consequently, they may be taken for consideration as molecular tests applicable to clinical practice in the management of BC. Patient summary: Our gene classifiers achieve sensitivities up to 90% in HR NMIBC and MIBC patients, while this achievement is comparatively lower in LR NMIBC ones.