RUNX3 pathway signature predicts clinical benefits of immune checkpoint inhibition plus tyrosine kinase inhibition in advanced renal cell carcinoma.

BACKGROUND: Checkpoint inhibitor immunotherapy plus tyrosine kinase inhibitor (IO/TKI) have been recently recommended as standard first-line therapy for advanced renal cell carcinoma, while no clinical-available biomarker has been applied. This study aimed to investigate the associations between RUNX3 pathway signature and IO/TKI benefits in renal cell carcinoma (RCC). METHODS: Two IO/TKI cohorts (ZS-MRCC, JAVELIN-101) and one high-risk localized RCC cohort (ZS-HRRCC) were included. All samples were evaluated by RNA-sequencing, and RUNX Family Transcription Factor 3 (RUNX3) pathway were determined by single sample gene set enrichment analysis. Flow cytometry were applied for immune cell infiltration and function. RESULTS: RUNX3 signature was elevated in RCC samples, compared non-tumor tissues (P < 0.001). High-RUNX3 signature was associated with shorter progression-free survival (PFS) in both IO/TKI cohorts (ZS-MRCC cohort, P = 0.025; JAVELIN-101 cohort, P = 0.019). RUNX3 signature also predicted IO/TKI benefit in advanced RCC, compared with TKI monotherapy (interaction p = 0.027). RUNX3 signature was associated with decreased number of GZMB + CD8 + T cells (Spearman's ρ=-0.42, P = 0.006), and increased number of PD1 + CD8 + T cells (Spearman's ρ = 0.29, P = 0.072). Moreover, the integration of RUNX3 signature and GZMB expression showed predictive potential for TKI/IO (log-rank P < 0.001). In addition, the predictive value of RUNX3 signature for IO/TKI benefit was restricted in SETD2-wild type patients (log-rank P < 0.001). Finally, a risk score was established by random forest for IO/TKI benefit, showing remarkable predictive potency (Log-rank P < 0.001). CONCLUSIONS: RUNX3 pathway signature could be a potential predictive biomarker for IO/TKI treatment in advanced RCC, for both prognosis and treatment selection between IO/TKI and TKI monotherapy.

Unfavorable immunotherapy plus tyrosine kinase inhibition outcome of metastatic renal cell carcinoma after radical nephrectomy with increased ADAM9 expression.

Immunotherapy plus tyrosine kinase inhibitor (IO-TKI) has become the standard first-line therapy for advanced renal cell carcinoma (RCC). However, the modest response rate of IO-TKI therapy and the absence of biomarkers limited the selection of treatment strategies for RCC patients. There were three cohorts enrolled: two from our facility (ZS-MRCC and ZS-HRRCC) and one from a clinical study (JAVELIN-101). By RNA sequencing, the expression of ADAM9 in each sample was measured. By flow cytometry and immunohistochemistry, immune infiltration and T cell function were examined. Primary outcomes were established as treatment response and progression-free survival (PFS). Patients with low-ADAM9 expression had a higher objective response rate (56.5% vs 13.6%, P = 0.01) and longer PFS in both cohorts. In the ZS-HRRCC cohort, the expression of ADAM9 was associated with increased tumor-infiltrating T cells, which was proved by immunohistochemistry (P < 0.05) and flow cytometry (Spearman's ρ = 0.42, P < 0.001). In the high-ADAM9 group, CD8+ and CD4+ T cells revealed an exhausted phenotype with decreased GZMB (Spearman's ρ = - 0.31, P = 0.05, and Spearman's ρ = - 0.49, P < 0.001, respectively), and fewer Macrophages were identified. A predictive RFscore was further constructed by random forest approach, involving ADAM9 and immunologic genes. Only in the subgroup with the lower RFscore did IO-TKI outperform TKI monotherapy. High-ADAM9 expression was associated with immunosuppression and IO-TKI resistance. Expression of ADAM9 was also associated with the exhaustion and dysfunction of T cells. ADAM9-based RFscore has the potential to be used as a biomarker to distinguish the optimal patient treatment methods between IO-TKI and TKI monotherapy.

The prognostic significance of CDK6 expression in renal cell carcinoma treated by immune checkpoint plus tyrosine kinase inhibition.

Checkpoint inhibitor immunotherapy plus tyrosine kinase inhibitor (IO/TKI) has become the first-line treatment for metastatic renal cell carcinoma (RCC), despite the lack of biomarkers. Cyclin-dependent kinase 6 (CDK6) has shown a regulatory role in antitumour response. The study enrolled two cohorts of metastatic RCC treated by IO/TKI (Zhongshan Hospital [ZS]-MRCC, n = 45; JAVELIN-101, n = 726) and two cohorts of localized RCC (ZS-HRRCC, n = 40; TCGA-KIRC, n = 530). CDK6 was evaluated by RNA-sequencing. Progression-free survival (PFS) was the primary endpoint. The prognostic role of CDK6 was evaluated by survival analysis. The correlation between CDK6 and tumour microenvironment was assessed by immunohistochemistry and flow cytometry. The high-CDK6 group displayed a lower response rate (13.6%) than the low-CDK6 group (56.5%) (P = .002). High-CDK6 was associated with poor PFS in both the ZS-MRCC cohort (high-CDK6, median PFS 6.4 months; low-CDK6, median PFS not reached; P = .010) and JAVELIN-101 cohort (high-CDK6, median PFS 10.0 months; low-CDK6, median PFS 13.3 month; P = .033). High-CDK6 was associated with increased PD1+ CD8+ T cells (Spearman's ρ = .47, P < .001) and decreased Granzyme B+ CD8+ T cells (Spearman's ρ = -.35, P = .030). Finally, a random forest score (RFscore) was built by integrating CDK6 and immunologic genes, which was associated with survival benefits of IO/TKI (RFscore-low, TKI vs IO/TKI, HR = 2.47, 95% CI 1.82-3.35, P < .001; RFscore-high, TKI vs IO/TKI, HR = 0.99, 95% CI 0.75-1.32, P = .963). Elevated CDK6 expression indicated resistance and poor PFS under IO/TKI therapy, which was related to exhausted CD8+ T cells. Integrated RFscore could evaluate the benefits of IO/TKI.

Downregulation of microRNA-182-5p contributes to renal cell carcinoma proliferation via activating the AKT/FOXO3a signaling pathway.

BACKGROUND: Emerging evidence has suggested that dysregulation of miR-182-5p may contribute to tumor development and progression in several types of human cancers. However, its role in renal cell carcinoma (RCC) is still unknown. METHODS: Quantitative RT-PCR was used to quantify miR-182-5p expression in RCC clinical tissues. Bisulfite sequencing PCR was used for DNA methylation analysis. The CCK-8, colony formation, flow cytometry, and a xenograft model were performed. Immunohistochemistry was conducted using the peroxidase and DAB methods. A miR-182-5p target was determined by luciferase reporter assays, quantitative RT-PCR, and Western blotting. RESULTS: miR-182-5p is frequently down-regulated in human RCC tissues. Epigenetic modulation may be involved in the regulation of miR-182-5p expression. Enforced expression of miR-182-5p in RCC cells significantly inhibited the proliferation and tumorigenicity in vitro and in vivo. Additionally, overexpression of miR-182-5p induced G1-phase arrest via inhibition of AKT/FOXO3a signaling. Moreover, FLOT1 was confirmed as a target of miR-182-5p. Silencing FLOT1 by small interfering RNAs phenocopied the effects of miR-182-5p overexpression, whereas restoration of FLOT1 in miR-182-5p -overexpressed RCC cells partly reversed the suppressive effects of miR-182-5p. CONCLUSIONS: These findings highlight an important role for miR-182-5p in the pathogenesis of RCC, and restoration of miR-182-5p could be considered as a potential therapeutic strategy for RCC therapy.

Dietary carrot consumption and the risk of prostate cancer.

PURPOSE: Previous studies regarding the association between carrot intake and prostate cancer risk have reported inconsistent results. We conducted a meta-analysis to summarize evidence on this association and to quantify the potential dose-response relationship. METHOD: A systematic literature search of papers published in August 2013 was conducted using PubMed, EMBASE, Scopus, Web of Science, the Cochrane register, and the Chinese National Knowledge Infrastructure databases, and the references of the retrieved articles were screened. The summary risk estimates with 95% confidence intervals (CIs) for the highest versus the lowest intake of carrots were calculated. A dose-response meta-analysis was also conducted for the studies reporting categorical risk estimates for a series of exposure levels. RESULTS: We found a significantly decreased risk of prostate cancer associated with the intake of carrots (odds ratio 0.82, 95% CI 0.70-0.97). In addition, the dose-response meta-analysis indicated that for each serving per week, or 10 g per day increment of carrot intake, the risk estimate of prostate cancer was 0.95 (0.90-0.99) or 0.96 (0.94-0.99). There was no evidence of significant publication bias based on Begg's funnel plot (P = 1.000) or Egger's test (P = 0.804). CONCLUSION: Carrot intake might be inversely associated with prostate cancer risk. Because of the limited number of cohort studies and substantial heterogeneity observed between studies in this meta-analysis, further well-designed prospective studies are warranted to confirm the findings from our study.

Meta-analysis of nonsteroidal anti-inflammatory drug intake and prostate cancer risk.

BACKGROUND: Epidemiological studies of the association between nonsteroidal anti-inflammatory drug (NSAID) intake and the risk of prostate cancer still remain controversial. Therefore, we conducted a meta-analysis to evaluate the potential association between NSAID intake and prostate cancer risk. METHODS: Eligible studies were retrieved by both computerized searches and reviews of references. Subgroup analyses on country and design of study were also performed. Random or fixed-effect models were used to pool estimates of odds ratios (ORs) with 95% confidence intervals (CIs). RESULTS: We observed that the intake of aspirin was associated with a marginally decreased risk of prostate cancer (OR = 0.95, 95% CI = 0.93 to 0.98). A similar result was found between nonaspirin NSAIDs and prostate cancer risk (OR = 0.94, 95% CI =0.90 to 0.98). However, a positive relation between all-NSAID intake and prostate cancer risk was observed (OR = 1.18, 95% CI = 1.15 to 1.22). CONCLUSIONS: We observed a marginally inverse correlation between the intake of aspirin and prostate cancer risk. On the contrary, a positive relationship between all-NSAID intake and prostate cancer was detected. Further research needs to be conducted to better clarify potential biological mechanisms.

PSMD2 overexpression as a biomarker for resistance and prognosis in renal cell carcinoma treated with immune checkpoint and tyrosine kinase inhibitors.

BACKGROUND: Integrated immune checkpoint inhibitors (ICIs) plus tyrosine kinase inhibitors (TKIs) are now the recommended first-line therapy to manage renal cell carcinoma (mRCC). Proteasome 26S subunit non-ATPase 2 (PSMD2) overexpression in tumors has been correlated with tumor progression. Currently, mRCC lacks an established biomarker for the combination of ICI+TKI. METHODS: This study involved RNA sequencing of RCC patients from two cohorts treated with ICI+TKI (ZS-MRCC and JAVELIN-Renal-101). We utilized immunohistochemistry alongside flow cytometry, aiming at assessing immune cell infiltration and functionality in high-risk localized RCC samples. Response and progression-free survival (PFS) were evaluated relying upon RECIST criteria. RESULTS: PSMD2 was significantly overexpressed in advanced RCC and among non-responders to ICI+TKI therapy. Overexpressed PSMD2 was correlated with poor PFS in the ZS-MRCC and JAVELIN-101 cohorts. Multivariate Cox analysis validated PSMD2 as an independent PFS predictor. PSMD2 overexpression was related to a reduction in CD8+ T cells, especially GZMB+ CD8+ T cells, besides an increase in PD1+ CD4+ T cells. Additionally, tumors with high PSMD2 levels showed enhanced T cell exhaustion levels and a higher regulatory T cell presence. A Machine Learning (ML) model based on PSMD2 expression and other screened factors was subsequently developed to predict the effectiveness of ICI+TKI. CONCLUSIONS: Elevated PSMD2 expression is linked to resistance and decreased PFS in mRCC patients undergoing ICI+TKI therapy. High PSMD2 levels are also associated with impaired function and increased exhaustion of tumor-infiltrating lymphocytes. An ML model incorporating PSMD2 expression could potentially identify patients who may have a higher likelihood of benefiting from ICI+TKI.

Effect of galectin-1 on prognosis and responsiveness of immune checkpoint plus tyrosine kinase inhibition in renal cell carcinoma.

BACKGROUND: In renal cell carcinoma (RCC), no clinically available biomarker has been utilized for checkpoint inhibitor immunotherapy (IO) + tyrosine kinase inhibitor (TKI) combinations. Galectin-1 overexpression is found in tumors, with potential immune-regulating roles. METHODS: RNA-sequencing was performed in two cohorts of RCC treated with IO/TKI combination therapy (ZS-MRCC, JAVELIN-101). Immunohistochemistry and flow cytometry were performed to investigate immune cell infiltration and function in the tumor microenvironment of RCC. The RECIST criteria were used to define response and progression-free survival (PFS). RESULTS: Galectin-1 expression was elevated in RCC with higher stage (p < 0.001) and grade (p < 0.001). Galectin-1 expression was also elevated in non-responders of IO/TKI therapy (p = 0.047). High galectin-1 was related with shorter PFS in both ZS-MRCC cohort (p = 0.036) and JAVELIN-101 cohort (p = 0.005). Multivariate Cox analysis defined galectin-1 as an independent factor for PFS (HR 2.505; 95% CI 1.116-5.622; p = 0.026). In the tumor microenvironment, high galectin-1 was related with decreased GZMB+CD8+ T cells (Speraman's ρ = -0.31, p = 0.05), and increased PD1 + CD8+ T cells (Speraman's ρ = 0.40, p = 0.01). Besides, elevated number of regulatory T cells (p = 0.039) and fibroblasts (p = 0.011) was also found in high galectin-1 tumors. Finally, a random-forest score (RFscore) was built for predicting IO/TKI benefit. IO/TKI therapy showed benefit only in low-RFscore patients (HR 0.489, 95% CI 0.358-0.669, p < 0.001), rather than high-RFscore patients (HR 0.875, 95% CI 0.658-1.163, p = 0.357). CONCLUSIONS: High galectin-1 indicated therapeutic resistance and shorter PFS of IO/TKI therapy. High galectin-1 also indicated CD8+ T cell dysfunction. High galectin-1 could be applied for patient selection of IO/TKI therapy in RCC.

Effect of Annexin A2 on prognosis and sensitivity to immune checkpoint plus tyrosine kinase inhibition in metastatic renal cell carcinoma.

BACKGROUND: Immunotherapy (IO) plus tyrosine kinase inhibitor (TKI) therapy is the first-line recommendation for advanced renal cell carcinoma (RCC), but no biomarker has been approved for it. Annexin A2 (ANXA2) can induce immune escape in tumors. METHODS: Two independent cohorts of advanced RCC treated by IO + TKI were utilized for survival analysis (ZS-MRCC, n = 45; Javelin-101, n = 726). ANXA2 expression was determined by RNA-sequencing. The impact of ANXA2 on the tumor microenvironment was assessed by RNA-sequencing, flow cytometry and immunohistochemistry in two localized RCC datasets (ZS-HRRCC, n = 40; TCGA-KIRC, n = 530). RESULTS: ANXA2 was upregulated in non-responders of IO + TKI therapy (p = 0.027). High-ANXA2 group showed poor progression-free survival (PFS) in both the ZS-MRCC cohort (HR, 2.348; 95% CI 1.084-5.085; P = 0.025) and the Javelin-101 cohort (HR, 1.472; 95% CI 1.043-2.077; P = 0.027). Multivariate Cox regression determined ANXA2 as an independent prognostic factor (HR, 2.619; 95% CI 1.194-5.746; P = 0.016). High-ANXA2 was correlated with decreased proportion of granzyme B+ CD8+ T cells (Spearman's ρ = - 0.40, P = 0.01), and increased TIM-3+ (Spearman's ρ = 0.43, P < 0.001) and CTLA4+ (Spearman's ρ = 0.49, P < 0.001) tumor-infiltrating lymphocytes. A random forest (RF) score was further build by integrating ANXA2 and immune genes, which stratified patients who would benefit from IO + TKI therapy (low-RF score, IO + TKI vs TKI, HR = 0.453, 95% CI 0.328-0.626; high-RF score, IO + TKI vs TKI, HR = 0.877, 95% CI 0.661-1.165; interaction P = 0.003). CONCLUSIONS: Upregulated ANXA2 was associated with poor PFS and therapeutic resistance in RCC treated by IO + TKI therapy, and related with T cell exhaustion. The integrated RF score could stratify patients who would benefit from IO + TKI therapy.

SPP1 expression indicates outcome of immunotherapy plus tyrosine kinase inhibition in advanced renal cell carcinoma.

Clinical guidelines have recently advised combination therapy involving immunotherapy (IO) and tyrosine kinase inhibitors (TKI) as the first-line therapy approach for advanced renal cell carcinoma (RCC). Nevertheless, there is currently no available biomarker that can effectively distinguish the progression-free survival (PFS). RNA-sequencing and immunohistochemistry were conducted on our cohort of metastatic RCC patients, namely ZS-MRCC, who received combination therapy consisting of IO and TKI. We further applied RNA-sequencing, immunohistochemistry, and flow cytometry to examine the immune cell infiltration and functionality inside the tumor microenvironment of high-risk localized RCC samples. SPP1 expression was significantly higher in non-responders to IO-TKI therapy. Elevated levels of SPP1 were associated with poor PFS in both the ZS-MRCC cohort (HR = 2.73, p = .018) and validated in the JAVELIN Renal 101 cohort (HR = 1.61, p = .004). By multivariate Cox analysis, SPP1 was identified as a significant independent prognosticator. Furthermore, there existed a negative correlation between elevated levels of SPP1 and the presence of GZMB+CD8+ T cells (Spearman's ρ= -0.48, p < .001). Conversely, SPP1 expression is associated with T cell exhaustion markers. A significant increase in the abundance of Tregs was observed in tumors with high levels of SPP1. Additionally, a machine-learning-based model was constructed to predict the benefit of IO-TKI treatment. High SPP1 is associated with therapeutic resistance and unfavorable PFS in IO-TKI therapy. SPP1 expression have also been observed to be indicative of malfunction and exhaustion in T cells. Increased SPP1 expression has the potential to serve as a potential biomarker for treatment selection of metastatic RCC.

MicroRNA-490-5p inhibits proliferation of bladder cancer by targeting c-Fos.

MicroRNAs (miRNAs) are non-protein-coding sequences that play a crucial role in tumorigenesis by negatively regulating gene expression. Here, we found that miR-490-5p is down-regulated in human bladder cancer tissue and cell lines compared to normal adjacent tissue and a non-malignant cell line. To better characterize the function of miR-490-5p in bladder cancer, we over-expressed miR-490-5p in bladder cancer cell lines with chemically synthesized mimics. Enforced expression of miR-490-5p in bladder cancer cells significantly inhibited the cell proliferation via G1-phase arrest. Further studies found the decreased c-Fos expression at both mRNA and protein levels and Luciferase reporter assays demonstrated that c-Fos is a direct target of miR-490-5p in bladder cancer. These findings indicate miR-490-5p to be a novel tumor suppressor of bladder cancer cell proliferation through targeting c-Fos.

MicroRNA-101 suppresses motility of bladder cancer cells by targeting c-Met.

MicroRNAs (miRNAs) are small non-coding RNAs that play regulatory roles by repressing translation or cleaving RNA transcripts. Here, we report that the expression of microRNA-101 (miR-101) is down-regulated in human bladder cancer tissue versus normal adjacent tissue. To better characterize the role of miR-101 in bladder cancer, we conducted a gain-of-function analysis by transfecting the bladder cancer cell line T24 with chemically synthesized miR-101 mimics. We found that miR-101 directly targets c-Met via its 3'-UTR. Specifically, forced expression of miR-101 decreased c-Met expression at both mRNA and protein levels, consequently inhibiting T24 cell migration and invasion in a c-Met-dependent manner. In conclusion, we have shown miR-101 to be a novel suppressor of T24 cell migration and invasion through its negative regulation of c-Met.

MicroRNA-124-3p inhibits cell migration and invasion in bladder cancer cells by targeting ROCK1.

BACKGROUND: Increasing evidence has suggested that dysregulation of certain microRNAs (miRNAs) may contribute to human disease including carcinogenesis and tumor metastasis in human. miR-124-3p is down-regulated in various cancers, and modulates proliferation and aggressiveness of cancer cells. However, the roles of miR-124-3p in human bladder cancer are elusive. Thus, this study was conducted to investigate the biological functions and its molecular mechanisms of miR-124-3p in human bladder cancer cell lines, discussing whether it has a potential to be a therapeutic biomarker of bladder cancer. METHODS: Three human bladder cancer cell lines and samples from ten patients with bladder cancer were analyzed for the expression of miR-124-3p by quantitative RT--PCR. Exogenetic overexpression of miR-124-3p was established by transfecting mimics into T24, UM-UC-3 and J82 cells, after that cell proliferation and cell cycle were assessed by MTT assay, flow cytometry and Colony-forming assay. Cell motility and invasion ability were evaluated by wound healing assay and transwell assay. Tissue microarray, and immunohistochemistry with antibodies against ROCK1, MMP2 and MMP9 was performed using the peroxidase and DAB methods. The target gene of miR-124-3p was determined by luciferase assays, quantitative RT--PCR and western blot. The regulation of epithelial-to-mesenchymal transition by miR-124-3p was analyzed by western blot. RESULTS: miR-124-3p is frequently down-regulated in bladder cancer both in three bladder cancer cell lines, T24, UM-UC-3, J82 and clinical samples. Overexpression of miR-124-3p induced G1-phase arrest in T24, UM-UC-3 and J82 cell lines and suppressed cell growth in colony-forming assay. miR-124-3p significantly repressed the capability of migration and invasion of bladder cancer cells. In addition, ROCK1 was identified as a new target of miR-124-3p. ROCK1, MMP2, MMP9 were up-regulated in bladder cancer tissues. Furthermore, we demonstrated miR-124-3p could inhibit bladder cancer cell epithelial mesenchymal transfer, and regulated the expression of c-Met, MMP2, MMP9. CONCLUSIONS: miR-124-3p can repress the migration and invasion of bladder cancer cells via regulating ROCK1. Our data indicate that miR-124-3p could be a tumor suppressor and may have a potential to be a diagnostics or predictive biomarker in bladder cancer.

Apigenin promotes apoptosis, inhibits invasion and induces cell cycle arrest of T24 human bladder cancer cells.

BACKGROUND: Apigenin (4',5,7-trihydroxyflavone) was recently shown effective in inhibiting several cancers. The aim of this study was to investigate the effect and mechanism of apigenin in the human bladder cancer cell line T24 for the first time. METHODS: T24 cells were treated with varying concentrations and time of apigenin. Cell viability was evaluated by MTT assay. Cell motility and invasiveness were assayed by Matrigel migration and invasion assay. Flow cytometry and western blot analysis were used to detect cell apoptosis, cell cycle and signaling pathway. RESULTS: The results demonstrated that apigenin suppressed proliferation and inhibited the migration and invasion potential of T24 bladder cancer cells in a dose- and time-dependent manner, which was associated with induced G2/M Phase cell cycle arrest and apoptosis. The mechanism of action is like to involve PI3K/Akt pathway and Bcl-2 family proteins. Apigenin increased caspase-3 activity and PARP cleavage, indicating that apigenin induced apoptosis in a caspase-dependent way. CONCLUSIONS: These findings suggest that apigenin may be an effective way for treating human bladder cancer.

Quantitative assessment of the associations between XRCC1 polymorphisms and bladder cancer risk.

BACKGROUND: The XRCC1 polymorphisms have been implicated in bladder cancer risk, but individually published studies show inconsistent results. The aim of our study was to clarify the effects of XRCC1 variants on bladder cancer risk. METHODS: A systematic literature search up to September 13, 2012 was carried out in PubMed, EMBASE and Wanfang databases, and the references of retrieved articles were screened. Crude odds ratios with 95% confidence intervals were used to assess the associations between XRCC1 Arg194Trp and Arg399Gln polymorphisms and bladder cancer risk. Heterogeneity and publication bias were also evaluated. RESULTS: A total of 14 and 18 studies were eligible for meta-analyses of Arg194Trp and Arg399Gln, respectively. Regrouping was adopted in accordance with the most probable appropriate genetic models. No obvious heterogeneity between studies was found. For overall bladder cancer, the pooled odds ratios for Arg194Trp and Arg399Gln were 1.69 (95% confidence interval: 1.25 to 2.28; P = 0.001) and 1.10 (95% confidence interval: 1.03 to 1.19; P = 0.008), respectively. After excluding the studies that were not in Hardy-Weinberg equilibrium, the estimated pooled odds ratio still did not change at all. CONCLUSIONS: The meta-analysis results suggest that XRCC1 Arg194Trp and Arg399Gln polymorphisms may be associated with elevated bladder cancer risk.

Alternative Complement Pathway Signature Determines Immunosuppression and Resistance to Immunotherapy Plus Tyrosine Kinase Inhibitor Combinations in Renal Cell Carcinoma.

BACKGROUND: Latest guidelines recommended immunotherapy (IO) plus tyrosine kinase inhibitor (TKI) combination as standard first-line therapy in renal cell carcinoma (RCC), with no predictive biomarker being applied. Complement system shapes tumor microenvironment, which may influence TKI+IO benefit. METHODS: Two cohorts from our institute and 2 external cohorts were enrolled. RNA-sequencing was performed for each sample, and alternative complement pathway signature (ACPS) was defined by single sample gene set enrichment analysis. Immune infiltration and function were assessed by immunohistochemistry and flow cytometry. RESULTS: Under TKI+IO therapy, ACPS was elevated in non-responders (P<0.01), and high-ACPS predicted lower response rate and shorter progression-free survival (P=0.040). Moreover, TKI+IO, rather than TKI monotherapy, may benefit patients of low-ACPS combined with SETD2-wild type (HR=0.55, P<0.001). In RCC, ACPS was associated with increased tumor-infiltrating T cells (Spearman's ρ=0.50, P=0.001). However, in high-ACPS samples, CD8+ T cells revealed an exhausted phenotype with decreased GZMB (P<0.001) and increased PD1 (P=0.008) expression. Elevated PD1 expression in high-ACPS samples was confirmed by immunohistochemistry (P=0.046). Besides, macrophage infiltration was increased in high-ACPS samples (P=0.045), along with suppressive cytokines. CONCLUSIONS: Under TKI+IO, high-ACPS was linked to immunosuppression and treatment resistance. ACPS might be used as a biomarker for better treatment strategy between TKI+IO or TKI monotherapy.

HMOX1 pathway signature predicts clinical benefit from immunotherapy plus tyrosine kinase inhibitor therapy in advanced renal cell carcinoma.

BACKGROUND: Immunotherapy (IO) plus tyrosine kinase inhibitor (TKI) emerged as standard first-line therapy for advanced renal cell carcinoma (RCC). The heme Oxygenase 1 (HMOX1) pathway is involved in tumor development and treatment resistance, which may affect the efficacy of TKI + IO. METHODS: Two cohorts from our center (ZS-MRCC, ZS-HRRCC), one cohort from clinical trial (JAVELIN Renal 101) and the Cancer Genome Atlas (TCGA-KIRC) were enrolled. HMOX1 pathway signatures were determined for each sample by RNA-sequencing and gene set enrichment analysis. Immune infiltration was evaluated by flow cytometry. Response and progression-free survival (PFS) were set as primary endpoints. RESULTS: Patients of low-HMOX1 signature showed higher objective response rate (43.5% vs. 27.3%) in ZS-MRCC cohort and longer PFS in both cohorts (ZS-MRCC cohort, p = 0.019; JAVELIN-101 cohort, p = 0.036). Patients in the high-HMOX1 signature arm also showed greater clinical benefit from TKI + IO, rather than TKI monotherapy (p < 0.001). In high-HMOX1 signature RCC tissues, CD8+ T cells showed a dysfunctional phenotype with decreased GZMB expression (Spearman's ρ = -0.32, p = 0.045). A risk score based on HMOX1 signature was further constructed by random forest approach, involving HMOX1 signature and immunologic features. In patients with a low risk level, TKI + IO combination therapy demonstrated longer PFS than TKI monotherapy (p < 0.001), however in individuals with a high risk score group, these two regimens did not give different advantages. CONCLUSIONS: Our study identified the HMOX1 pathway signature was a potential prognostic factor of progression-free survival for TKI + IO combination therapy in the advanced RCC in different cohort, especially in first-line management of mRCC in the Javelin 101 cohort. Moreover, HMOX1 signature was associated with T-cell function in tumor environment.

Favorable Immunotherapy Plus Tyrosine Kinase Inhibition Outcome of Renal Cell Carcinoma Patients with Low CDK5 Expression.

PURPOSE: Immunotherapy (IO) plus tyrosine kinase inhibitor (TKI) has become the first-line treatment for advanced renal cell carcinoma, despite the lack of prognostic biomarkers. Cyclin-dependent kinase 5 (CDK5) affects the tumor microenvironment, which may influence the efficacy of TKI+IO. MATERIALS AND METHODS: Two cohorts from our center (Zhongshan Metastatic Renal Cell Carcinoma [ZS-MRCC] cohort, Zhongshan High-risk Localized Renal Cell Carcinoma [ZS-HRRCC] cohort) and one cohort from a clinical trial (JAVELIN-101) were enrolled. The expression of CDK5 of each sample was determined by RNA sequencing. Immune infiltration and T cell function were evaluated by flow cytometry and immunohistochemistry. Response and progression-free survival (PFS) were set as primary endpoints. RESULTS: Patients of low CDK5 expression showed higher objective response rate (60.0% vs. 23.3%) and longer PFS in both cohorts (ZS-MRCC cohort, p=0.014; JAVELIN-101 cohort, p=0.040). CDK5 expression was enhanced in non-responders (p < 0.05). In the ZS-HRRCC cohort, CDK5 was associated with decreased tumor-infiltrating CD8+ T cells, which was proved by immunohistochemistry (p < 0.05) and flow cytometry (Spearman's ρ=-0.49, p < 0.001). In the high CDK5 subgroup, CD8+ T cells revealed a dysfunction phenotype with decreased granzyme B, and more regulatory T cells were identified. A predictive score was further constructed by random forest, involving CDK5 and T cell exhaustion features. The RFscore was also validated in both cohorts. By utilizing the model, more patients might be distinguished from the overall cohort. Additionally, only in the low RFscore did TKI+IO outperform TKI monotherapy. CONCLUSION: High-CDK5 expression was associated with immunosuppression and TKI+IO resistance. RFscore based on CDK5 may be utilized as a biomarker to determine the optimal treatment strategy.

Effects of PYCR1 on prognosis and immunotherapy plus tyrosine kinase inhibition responsiveness in metastatic renal cell carcinoma patients.

BACKGROUND: Immunotherapy plus tyrosine kinase inhibitor (IO-TKI) has become the first-line management for metastatic renal cell carcinoma (RCC), despite the absence of biomarkers. Recently, pyrroline-5-carboxylate reductase 1 (PYCR1) and proline metabolism have been reported regulatory roles in the anti-tumor response. METHODS: There were three cohorts enrolled: two from our institution (ZS-MRCC and ZS-HRRCC) and one from a clinical trial (JAVELIN-101). The PYCR1expression in each sample was evaluated by RNA sequencing. Flow cytometry and immunohistochemistry were performed to assess immune infiltration. Single-cell RNA-seq (scRNA-seq) data was used for cluster analysis of T cells and macrophages. Primary endpoints were set as response and progression-free survival (PFS). RESULTS: Patients in the low-PYCR1 group had greater objective response rate (52.2% vs 18.2%) and longer PFS in both cohorts (ZS-MRCC cohort, P=0.01, HR=2.80; JAVELIN-101 cohort, P<0.001, HR=1.85). In responders, PYCR1 expression was decreased (P<0.05). In the high PYCR1 group, CD8+ T cells exhibited an exhausted phenotype with decreased GZMB (Spearman's ρ=-0.36, P=0.02). scRNA-seq revealed tissue-resident memory T (Trm) (P<0.05) and tissue-resident macrophage (P<0.01) were decreased in samples with high PYCR1 expression. A machine learning score was further built by random forest, involving PYCR1 and Trm markers. Only in the subgroup with the lower RFscore did IO+TKI show a favorable outcome, compared to TKI monotherapy. CONCLUSIONS: Immunosuppression and IO+TKI resistance were correlated with high PYCR1 expression. T cell exhaustion and dysfunction were also related with the expression of PYCR1. PYCR1 has the potential to be employed as a biomarker to discriminate between IO+TKI and TKI monotherapy as the optimal patient treatment strategy.

Exploration of Prognostic Biomarkers of Muscle-Invasive Bladder Cancer (MIBC) by Bioinformatics.

We aimed to discover prognostic factors of muscle-invasive bladder cancer (MIBC) and investigate their relationship with immune therapies. Online data of MIBC were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus database (GEO) database. Weighted gene co-expression network analysis (WGCNA) and univariate Cox analysis were applied to classify genes into different groups. Venn diagram was used to find the intersection of genes, and prognostic efficacy was proved by Kaplan-Meier analysis. Heatmap was utilized for differential analysis. Riskscore (RS) was calculated according to multivariate Cox analysis and evaluated by receiver operating characteristic curve (ROC). MIBC samples from TCGA and GEO were analyzed by WGCNA and univariate Cox analysis and intersected at 4 genes, CLK4, DEDD2, ENO1, and SYTL1. Higher SYTL1 and DEDD2 expressions were significantly correlated with high tumor grades. Riskscore based on genes showed great prognostic efficiency in predicting overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) in TCGA dataset (P < .001). The area under the ROC curve (AUC) of RS reached 0.671 in predicting 1-year survival and 0.653 in 3-year survival. KEGG pathways enrichment filtered 5 enriched pathways. xCell analysis showed increased T cell CD4+ Th2 cell, macrophage, macrophage M1, and macrophage M2 infiltration in high RS samples (P < .001). In immune checkpoints analysis, PD-L1 expression was significantly higher in patients with high RS. We have, therefore, constructed RS as a convincing prognostic index for MIBC patients and found potential targeted pathways.