Upper Tract Urothelial Carcinoma Patients Research Articles

Effect of low FGFR3 but high CD8 T cell infiltrated immune micro-environment on treatment response with pembrolizumab in upper tract urothelial carcinoma.

69 Background: Although several evidence revealed that upper tract urothelial carcinoma (UTUC) with fibroblast growth factor receptor 3 (FGFR3) mutations and/or expressions follow favorable outcomes, the relationship between immune cell markers and FGFR3 expression remains unknown. We aimed to clarify FGFR3 based immune microenvironment and to investigate biomarkers to predict treatment response of Pembrolizumab (Pem) in UTUC patients. Methods: We conducted immunohistochemical (IHC) staining of 214 UTUC patients. FGFR3, CD4, CD8, CD68, CD163, CD204, and PDL-1 expressions were examined and we created immune clusters assessed with heatmap clustering. We assessed the relationships of these immune markers toward further prognosis. In addition, neutrophil-lymphocyte ratio (NLR) and absolute monocyte count (AMC) were evaluated as blodd markers for predicting treatment response from Pem. Results: Among the UTUC patients, 109 (50.9%) showed high FGFR3 expression, and showed favorable prognosis compared to the counterparts. Among the six immune markers, CD8 high expression was revealed as an independent favorable factor (HR=0.44) whereas CD204 high was shown as one of independent prognostic factors for cancer death (HR=1.80). From the FGFFR3 based immune clustering, three immune clusters were identified in UTUC tumor specimen. Cluster A showed FGFR3 low with TAM rich component (CD163 and CD204 high) followed with poor prognosis due to poor response to Pem. Cluster B showed FGFR3 low with immune hot component (CD8 high) followed with most favorable prognosis due to good response to Pembrolizumab. Cluster C showed high FGFR3 expression but immune cold component, followed with favorable prognosis due to FGFR3 high expression but poor response was confirmed with Pem. Regarding of blood biomarkers, NLR showed correlation with CD8 expressions while AMC showed correlation with CD204 expressions. By using validation cohort (n=138), NLR<3.3 showed significantly longer survival compared to the counterparts with Pem treatment (p=0.046). Conclusions: The present study demonstrated a unique immune profile found in UTUC patients. Although most of the patients show poor response to Pem, low FGFR3 expression with immune-hot status may receive clinical benefit from Pem treatment.

The efficacy and timing of adjuvant chemotherapy in upper tract urothelial carcinoma.

A recovery period between surgery and initiation of adjuvant chemotherapy (AC) is common in patients with upper tract urothelial carcinoma (UTUC), which can progress after a relatively long time. Therefore, the efficacy of AC initiated within 90 days after radical nephroureterectomy (RNU) was evaluated in UTUC patients at stage ≥pT2 (N0-3M0), in addition to the effect of delayed AC initiation on survival outcomes. Clinical data for 428 UTUC patients diagnosed with transitional cell carcinoma with postoperatively confirmed pathological stages, muscle-invasive or greater-stage (pT2-4) disease, any nodal status, and metastasis-free (M0) disease were retrospectively analyzed. All patients who received AC were treated within 90 days after RNU and underwent at least 4 cycles of the AC procedure. Then, patients receiving AC were divided into the "within 45 days" and "45 to 90 days" groups according to the time interval between RNU and AC initiation. Their clinicopathological characteristics were evaluated and the survival outcomes of the 2 groups were compared. Any adverse events that occurred during the AC process were also recorded. A total of 428 patients were analyzed in the study, including 132 individuals who underwent the AC procedure with platinum in combination with gemcitabine within 90 days after RNU and 296 patients who failed to initiate AC within 90 days. The median age of all patients was 68 years (mean 67, range 28-90 years), and the median follow-up was 25 months (mean 36, range 1-129 months). There were no significant differences in age, sex, lymph node metastasis, tumor location, hydronephrosis status, hematuria status, cancer grade, or multifocality between the 2 groups. Individuals undergoing AC initiated within 90 days of RNU showed a significantly decreased mortality relative to those patients who did not receive AC. Shorter intervals between RNU and AC initiation within 45 days vs. 45-90 days did not improve patient OS and cancer-specific survival (CSS) and may have increased the incidence of adverse events. The present study data supported the finding that a platinum-based combination with gemcitabine regimen initiated postoperatively significantly improved OS and CSS in patients with UTUC at stages ≥pT2 (N0-3M0). Furthermore, no survival benefit was evident in patients who started AC within 45 days after RNU compared to those who received AC within 45 to 90 days.

A dynamic nomogram integrated with blood inflammation markers for predicting overall survival in patients with upper tract urothelial carcinoma.

Upper tract urothelial carcinoma (UTUC) is a relatively rare disease with a poor prognosis. A growing body of evidence demonstrates that inflammation and the inflammatory microenvironment play a crucial role in tumorigenesis and tumor progression. Our aim was to evaluate the prognostic value of blood inflammation markers and develop a prediction model that incorporates inflammation markers in order to predict overall survival (OS) of UTUC. We included 304 localized UTUC patients from two medical institutions who had undergone radical nephroureterectomy (RNU) (167 in the training cohort, 137 in the validation cohort). Univariate and multivariate Cox regression analyses were performed to screen the prognostic factors, and a nomogram and a web-based calculator were generated based on these predictors. The Harrell's concordance index (C-index), the area under the receiver operating characteristic (ROC) curve, the calibration curve, and decision curve analysis (DCA) were used to evaluate the performance of the nomogram. Independent predictors incorporated in the nomogram were pathological stage, surgical margin, albumin-to-globulin ratio (AGR), and hemoglobin-to-red cell distribution width ratio (HRR). The c-index value was 0.726 in the training cohort and 0.761 in the validation cohort. The area under the ROC of the nomogram at 1-, 3- and 5-year in the training and validation sets were 0.765, 0.755, 0.763, and 0.791, 0.833, 0.802, respectively. Both the internal and external validation calibration plots showed a subtle distinction between the predicted and the actual probabilities. And it appears to provide incremental benefits for clinical decision-making in comparison to the American Joint Committee of Cancer (AJCC) staging system. In patients with UTUC after RNU, lower preoperative AGR and HRR were independent predictors of inferior survival. In addition, we created a novel blood inflammation marker-based dynamic nomogram that may be useful for surgeons or oncologists in risk stratification and patient selection for more intensive therapy and closer follow-up.

Evaluation of the diagnostic efficiency of voided urine fluorescence in situ hybridization for predicting the pathology of preoperative "low-risk" upper tract urothelial carcinoma.

To evaluate the clinical utility of voided urine fluorescence in situ hybridization (FISH) for predicting the pathology of preoperative "low-risk" upper tract urothelial carcinoma (UTUC). Information of patients preoperatively diagnosed with "low-risk" UTUC receiving radical nephroureterectomy (RNU) between May 2014 and October 2019 were retrospectively collected. All of the patients accepted the FISH test and then were divided into two groups according to the results of FISH. The diagnostic value of FISH was assessed through the receiver operating characteristics (ROC) curve and area under the curve. Logistic regression analysis was applied to examine FISH as a predictive factor of tumor final stage and grade of preoperative "low-risk" UTUC. In total, 129 patients were included. Of them, 70 (54.2%) were marked with positive FISH result. The difference at final pathology in tumor stage and tumor grade between these two groups of FISH (-) and FISH (+) had significantly statistical significance (p<0.001). Regarding to the tumor stage at final pathology, the sensitivity, specificity, positive predictive value and negative predictive value of FISH were 70.7 (58.9-80.3)68.5 (54.3-80.1)75.7 (63.7-84.8) and 62.7 (49.1-74.7), respectively. Regarding to the tumor grade at final pathology, the sensitivity, specificity, positive predictive value and negative predictive value of FISH were 64.7 (53.5-74.6), 65.9 (50.0-79.1), 78.6 (66.8-87.1) and 49.1 (36.5-62.3), respectively. The results of logistic regression analysis indicated that FISH could predict the pathologic characteristics of preoperative "low-risk" UTUC independently. FISH was qualified with relatively high diagnostic estimates for predicting tumor stage and grade of preoperative "low-risk" UTUC, and could be an independent predictive factor in clinical practice. For preoperative "low-risk" UTUC patients but with positive FISH result, choosing nephron-sparing surgery may require special caution.

Head-to-head comparisons of 68Ga-PSMA-11 and 18F-FDG PET/CT in evaluating patients with upper tract urothelial carcinoma: a prospective pilot study.

To prospectively compare the uptake of 68Ga-prostate specific membrane antigen (68Ga-PSMA)-11 and 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) in upper tract urothelial carcinoma (UTUC) and investigate the correlation between radiological parameters and pathological features of UTUC. Clinicopathologic and imaging data were collected from 10 UTUC patients who underwent preoperative 68Ga-PSMA-11 and 18F-FDG PET/CT scans. The diagnostic capabilities of both imaging techniques were analyzed and compared in UTUC. Angiogenesis in the malignancies was assessed using Chalkley counting and the expression of folate hydrolase 1 (FOLH1) and glucose transporter1 (GLUT1) in UTUC were evaluated in the surgical specimens. Double immunofluorescence staining of PSMA and CD34 was used to examine tumor neovascularization. Tracer uptake and expression were compared and explored. Additionally, 10 patients with clear cell renal cell carcinoma (ccRCC) were included for prospective, comparative research. Ten UTUC patients with 12 malignant lesions and another 10 ccRCC patients were included. 18F-FDG PET/CT demonstrated a more effective detection of UTUC foci compared to 68Ga-PSMA-11 PET/CT (the SUVmax of 18.48 ± 6.73 vs. 4.38 ± 1.45, P < 0.01). Immunohistochemical analysis revealed a statistically significant difference in the expression of PSMA and GLUT1 in UTUC (P = 0.048), with higher pathological grades showing more intense GLUT1 staining than PSMA (75% vs. 12.5%). The Chalkley counting of angiogenesis in ccRCC was significantly higher than that in UTUC (229.34 vs. 71.67), which was proportional to 68Ga-PSMA-11 PET/CT SUVmax (both P < 0.05). 18F-FDG PET/CT holds better clinical potential for evaluating UTUC and detecting lymph node metastasis compared to 68Ga-PSMA-11 PET/CT, likely due to the relatively scant expression of FOLH1 in tumor neovascular endothelium while the abundant expression of GLUT1 in malignancy. Furthermore, the lower neovascular density in UTUC should not be overlooked.

Differential expression of SLITRK6 gene as a potential therapeutic target for urothelial carcinoma in particular upper tract cancer

BackgroundUrinary bladder urothelial carcinoma (UBUC) and upper tract urothelial carcinoma (UTUC) harbor analogous morphology with comparable cytogenetic changes as well as prognostic factors but their similar biological activities still remain controversial. SLITRK6 gene has been demonstrated to have distinct role in urothelial cancers with a distinction between UTUC and UBUC. MethodThe study included a total of 80 patients of urothelial carcinoma including 60 UBUC and 20 UTUC cases. The tumor tissues from both the groups were evaluated for gene expression at mRNA level by qRT-PCR, and protein expression by immunohistochemistry (IHC) and western blot. ResultsSignificantly more than 4-fold high mRNA expression of SLITRK6 was observed in UTUC against 1.2-fold in UBUC (p < 0.0001). The overall SLITRK6 expression by IHC was observed in 80% of the UBUC cases in comparison to 100% strong expression in UTUC patients and among two groups expression exhibited a significant difference for moderate to strong expression (p = 0.0005). The protein expression by western blot analysis in UTUC samples was considerably higher as compared to UBUC samples (1.64 vs. 0.76 respectively: p = 0.01). A strong concordance exhibited for the higher mRNA and protein expression in both UTUC and UBUC cases (∼75%) wherein 80%, 75% and 70% higher expression of SLITRK6 was detected by qRT-PCR, Western blot and IHC respectively. ConclusionTo conclude, although SLITRK6 exhibits a strong expression in both UTUC and UBUC but was considerably observed higher in majority of UTUC cases. Therefore, SLITRK6 appears as a promising novel possible gene target for urothelial carcinoma in particular UTUC.

The poor antitumor effect of pembrolizumab in advanced upper urothelial carcinoma with renal parenchymal invasion.

We investigated poor prognosticators in advanced or unresectable urothelial carcinoma, focusing on renal parenchymal invasion (RPI). This study included 48 bladder cancer (BC) and 67 upper tract urothelial carcinoma (UTUC) patients treated with pembrolizumab from December 2017 to September 2022 at Kobe University Hospital. Medical records were retrospectively reviewed for clinical characteristics, objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). Multivariate analyses were performed using the Cox proportional hazard regression model to identify parameters associated with either PFS or OS. Of 67 UTUC patients, 23 had RPI and 41 patients did not, while 3 cases could not be evaluated. Patients with RPI were predominantly elderly and had liver metastases. ORR for patients with RPI was 8.7%, while it was 19.5% for those without RPI. PFS was significantly shorter for patients with RPI compared with those without RPI. Patients with RPI had significantly shorter OS than those without RPI. On multivariate analysis, performance status (PS) ≥ 2, neutrophil-lymphocyte ratio (NLR) ≥ 3, C-reactive protein ≥0.3 mg/dL and RPI were independent prognostic factors for PFS. PS ≥ 2, NLR ≥ 3, visceral metastasis and RPI were independent prognostic factors for OS. UTUC patient OS was significantly shorter than BC patient OS, while no significant difference in PFS or OS was observed between BC patients and UTUC patients without RPI. RPI was a poor prognostic factor in advanced urothelial carcinoma treated with pembrolizumab, possibly resulting in a poorer prognosis for UTUC compared with BC.

Impact of a novel immune and nutritional score on prognosis in patients with upper urinary tract urothelial carcinoma following radical nephroureterectomy.

This study aimed to evaluate the clinical significance of a novel immune and nutritional score combining prognostic values of the controlling nutritional status (CONUT) score and prognostic immune and nutritional index (PINI) on long-term outcomes in patients with upper tract urothelial carcinoma (UTUC) after radical nephroureterectomy (RNU). This study analyzed 437 consecutive patients with UTUC treated by RNU. Restricted cubic splines were used to visualize the relation of PINI with Survival in patients with UTUC. The PINI was stratified into low- (1) and high-PINI (0) categories. The CONUT score was divided into three groups: Normal (1), Light (2), and Moderate/severe (3). Subsequently, patients were grouped according to CONUT-PINI score (CPS) (CPS group 1; CPS group 2; CPS group 3; and CPS group 4).Survival curves were plotted using the Kaplan-Meier method and log-rank test.The Cox proportional hazards regression model was used to determine the risk factors associated with overall Survival (OS) and cancer-specific Survival (CSS). By comprising independent prognostic factors, a predictive nomogram was constructed. PINI and CONUT score were identified as independent prognostic factors for OS and CSS. Kaplan-Meier survival analysis showed that the high CPS group was associated with worse OS and CSS than the low CPS group. Multivariate Cox regression and competing risk analyses showed that CPS, LVI, T stage, margin, and pN were independent factors associated with OS and CSS. Based on these five significant factors, we constructed a prognostic model for predicting clinical outcomes. The receiver operating characteristic curve indicated that the model had excellent predictive abilities for survival. The C-index of this model for OS and CSS were 0.773, and 0.789, respectively. The nomogram for OS and CSS showed good discrimination and calibration. Decision curve analysis (DCA) showed that this nomogram has a higher net benefit. The CPS combined the prognostic capacity of PINI and CONUT score and wasabletopredictpatientoutcomes in our cohort of UTUC patients. We have developed a nomogram to facilitate the clinical use of the CPS and provide accurate estimates of survival for individuals.

Comparison of different prediction tools for the risk of intravesical recurrence after radical nephroureterectomy for upper tract urothelial carcinoma: a propensity-matched analysis.

To compare the predictive performance of the current clinical prediction models for predicting intravesical recurrence (IVR) after radical nephroureterectomy (RNU) in patients with upper tract urothelial carcinoma (UTUC). We retrospectively analysed upper tract urothelial carcinoma patients who underwent radical nephroureterectomy in our centre from January 2009 to December 2019. We used the propensity score matching (PSM) method to adjust the confounders between the IVR and non-IVR groups. Additionally, Xylinas' reduce model and full model, Zhang's model, and Ishioka's risk stratification model were used to retrospectively calculate predictions for each patient. Receiver operating characteristic (ROC) curves were generated, and the areas under the curves (AUCs) were compared to identify the method with the highest predictive value. We included 217 patients with a median follow-up of 41months, of which 57 had IVR. After PSM analysis, 52 pairs of well-matched patients were included in the comparative study. No significant difference was found in clinical indicators besides hydronephrosis. The model comparison showed that the AUCs of the reduced Xylinas' model for 12months, 24months, and 36months were 0.69, 0.73, and 0.74, respectively, and those of the full Xylinas' model were 0.72, 0.75, and 0.74, respectively. The AUC of Zhang's model for 12months, 24months, and 36months was 0.63, 0.71, and 0.71, respectively, the performance of Ishioka's model is that the AUC of 12months, 24months and 36months was 0.66, 0.71, and 0.74, respectively. The external verification results of the four models show that more comprehensive data and a larger sample size of patients are needed to strengthen the models' derivation and updating procedure, to better apply them to different populations.

Impact of diagnostic ureteral catheterization on intravesical tumour recurrence following radical nephroureterectomy for upper tract urothelial carcinoma.

To investigate whether even a minimally invasive diagnostic procedure for the upper tract such as ureteral catheterization (UCath) may substantially increase the risk of intravesical recurrence (IVR) in patients with upper tract urothelial carcinoma (UTUC) treated with radical nephroureterectomy (RNU). The present, retrospective study enrolled 163 patients undergoing RNU for UTUC between 2010 and 2021 at two, tertiary care hospitals. The primary endpoint was the association between UCath and IVR-free survival (IVRFS). The secondary endpoints were the association of ureterorenoscopy (URS) and URS biopsy (URSBx) with IVRFS. Directed acyclic graph (DAG)-guided multivariable models were used to adjust for potential confounders. Of the 163 patients, 128 (79%), 88 (54%), and 67 (41%) received UCath, URS, and URSBx, respectively. URS was performed concurrently with UCath. During the follow-up period (median: 47months), IVR developed in 62 patients (5-year IVRFS rate: 52%). A DAG included concurrent bladder cancer, tumour size, hydronephrosis, positive cytology, and multiple UTUCs as potential confounders of the association between UCath and IVR. Both DAG-guided and stepwise multivariable models revealed a significant association between UCath and IVR (hazard ratio: 17.8; P < 0.001). UCath was also associated with shorter IVRFS in a subset of 75 patients who had not received URS (P < 0.001). In contrast, URS and URSBx were not associated with IVR in patients who had received UCath and URS, respectively. Any diagnostic manipulations of the upper urinary tract, even a minimally invasive procedure like UCath, could confer a risk of post-RNU IVR in UTUC patients.

Advanced chronic kidney disease; A comparison between nephroureterectomy and nephron-sparing surgery for upper tract urothelial carcinoma.

Renal function outcomes between radical nephroureterectomy (RNU) and nephron-sparing surgery (NSS) for upper tract urothelial carcinoma (UTUC) patients are not well established. We sought to compare the incidence and factors associated with development of advanced chronic kidney disease (CKD) between RNU and NSS and examine the role of acute kidney injury (AKI) on renal function outcomes. We retrospectively analyzed an institutional database for patients who underwent either RNU or NSS for UTUC. Cumulative incidence of postoperative advanced CKD, defined as eGFR < 30 ml/min/1.73 m2, was compared between groups. Fine-Gray competing risk regression was used to identify predictors of advanced CKD. Locally weight scatterplot smoothing was used to assess postoperative eGFR trends. AKI events were counted, staged, and assessed for influence of progression to advanced CKD. Four hundred and twenty-six patients were included in analysis, with a median follow up of 6.68 years (IQR 3.4-12.2). Median preoperative eGFR was similar between the groups (NSS: 68 ml/min/1.73 m2, RNU: 65 ml/min/1.73 m2,P = 0.220). Cumulative incidence of advanced CKD was significantly lower in the NSS cohort (P = 0.009). Factors associated with advanced CKD included age, diabetes, recurrent AKI and RNU. Percent of patients with an AKI event differed between the groups (51.5% NSS, 72.7% RNU, P = <0.001), there was no between group difference in percentage of patients with recurrent AKI (25.6% NSS, 25.9% RNU, P =1). NSS provides a renal function benefit in UTUC. AKI is common among UTUC patients and recurrent AKI is a risk factor for development of advanced CKD.

Molecular Characterization of Microrna Interference and Aristolochic Acid Intoxication Found in Upper Tract Urothelial Carcinoma in Patients with Balkan Endemic Nephropathy: A Systematic Review of the Current Literature.

The term "aristolochic acid nephropathy" (AAN) is used to include any form of toxic interstitial nephropathy that is caused either by ingestion of plants containing aristolochic acids (AA) or by the environmental contaminants in food such as in Balkan endemic nephropathy (BEN). Aristolochic acid (AA) intoxication is strongly associated with the development of upper tract urothelial carcinoma (UTUC); however, the underlying molecular mechanism remains to be defined. MicroRNAs (miRNA) regulate several biological processes, including cell proliferation, differentiation, and metabolism, acting as oncogenes or tumor suppressors. A unique miRNA expression profile suggested that miRNAs could function as regulators in UTUC developmental processes. This review aimed to summarize data available in the literature about underlying molecular mechanisms leading to the expression of miRNAs in AA-UTUC patients with BEN. Strong correlation in AA-UTUC has a distinctive gene alteration pattern, AL-DNA adducts, and a unique tumor protein (TP53) mutational spectrum AAG to TAG (A: T→T: A) transversion in codon 139 (Lys → Stop) of exon 5 activates the p53 tumor suppressor protein. Further, p53 protein is responsible not only for the expression of miRNAs but also acts as a target molecule for miRNAs and plays a crucial function in the AA-UTUC pathogenicity through activation of cyclin-dependent kinase (CyclinD1) and cyclin protein kinase 6(CDK6) to support cell cycle arrest. This study, proposed a molecular mechanism that represented a possible unique relationship between AA intoxication, miRNAs expression, and the progression of UTUC in patients with BEN.

ERBB2 Amplification as a Predictive and Prognostic Biomarker in Upper Tract Urothelial Carcinoma.

Upper tract urothelial carcinomas (UTUCs) occur in about 5-10% of all urothelial carcinomas and are frequently discovered in high-stage disease. We aimed to evaluate human epidermal growth factor receptor 2 (ERBB2) protein expression immunohistochemically and ERBB2 amplification in UTUCs by fluorescence in situ hybridization, applying a tissue microarray technique. ERBB2 overexpression and ERBB2 amplification were defined according to the recommendations of the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) for breast cancer and gastric carcinoma (GC), revealing scores of 2+ and 3+ in 10.2% and 41.8% of UTUCs, respectively. The performance parameters showed obviously higher sensitivity of ERBB2 immunoscoring according to the ASCO/CAP criteria for GC. ERBB2 amplification was detected in 10.5% of UTUCs. ERBB2 overexpression was more likely to be found in high-grade tumors and was associated with tumor progression. Univariable Cox regression analysis revealed a significantly lower progression-free survival (PFS) in cases with ERBB2 immunoscores of 2+ or 3+ according to the ASCO/CAP guidelines for GC. UTUCs with ERBB2 amplification showed a significantly shorter PFS in the multivariable Cox regression analysis. Irrespective of their ERBB2 status, patients with UTUC treated with platin showed a significantly lower PFS than UTUC patients who had not received any platin-based therapy. In addition, UTUC patients with a normal ERBB2 gene status who had not received platin-based therapy showed significantly longer overall survival. The results suggest that ERBB2 is a biomarker for progression in UTUCs and may define a distinct subgroup of UTUCs. As previously shown, ERBB2 amplification is infrequent. However, the small number of patients diagnosed with ERBB2-amplified UTUC might benefit from ERBB2-targeted cancer therapy. In clinical-pathological routine diagnostics, the determination of ERBB2 amplification is an established method in some defined entities and also successful in small samples. Still, the simultaneous use of ERBB2 immunohistochemistry and ERBB2 in situ hybridization would be important in order to record the low rate of amplified UTUC cases as completely as possible.

The impact of histological variants in patients with upper tract urothelial carcinoma treated with radical nephroureterectomy.

To investigate the impact of histological variants (HV) in patients with upper tract urothelial carcinoma (UTUC) and analyze the potential association between HV and postoperative bladder recurrence. The medical records of UTUC patients treated with RNU at our center from January 2012 to December 2019 were retrospectively analyzed. Patients were grouped according to the types of HV. Clinicopathological features and prognostic factors were compared among groups. A total of 629 patients were included in the study: 458 (73%) patients had pure urothelial carcinoma (PUC) and 171 (27%) patients had UTUC with HV. Squamous differentiation was the most common type (124 cases, 19%), followed by glandular differentiation (29 cases, 5.0%). Patients with HV had a higher proportion of T3 and T4 pathologic stages (P < 0.001) as well as high-grade disease (P = 0.002). In the univariate analysis, squamous differentiation and glandular differentiation were significantly associated with worse cancer-specific survival (CSS) (HR 2.22, 95% CI 1.62-3.04, P < 0.001; HR 1.90, 95% CI 1.13-3.20, P = 0.016). However, the multivariate analysis showed that this association became non-significant. We found that HV were associated with recurrent muscle-invasive bladder cancer (MIBC) after RNU and all patients had T2 and T3 initial tumor stages (P = 0.008, P < 0.001). We found that UTUC patients with HV were associated with biologically aggressive disease and recurrent MIBC after RNU. The detection of bladder recurrence following surgery needs to be given more attention in advanced UTUC patients with HV.

Immune Co-inhibitory Receptors CTLA-4, PD-1, TIGIT, LAG-3, and TIM-3 in Upper Tract Urothelial Carcinomas: A Large Cohort Study.

Programmed cell death 1 ligand 1), programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), T-cell immunoglobulin and mucin-domain containing-3, lymphocyte activation gene-3, and T-cell immunoglobulin and ITIM domain (TIGIT) are considered major immune co-inhibitory receptors (CIRs) and the most promising immunotherapeutic targets in cancer treatment, but they are largely unexplored in upper tract urothelial carcinoma (UTUC). The aim of this Cohort Study was to provide evidence concerning expression profiles and the clinical significance of CIRs among Chinese UTUC patients. A total of 175 UTUC patients who received radical surgery in our center were included. We used immunohistochemistry to evaluate CIR expressions in tissue microarrays (TMAs). Clinicopathological characteristics and prognostic correlations of CIR proteins were retrospectively analyzed. TIGIT, T-cell immunoglobulin and mucin-domain containing-3, PD-1, CTLA-4, Programmed cell death 1 ligand 1, and lymphocyte activation gene-3 high expression was examined in 136(77.7%), 86(49.1%), 57(32.6%), 18(10.3%), 28(16.0%), and 18(10.3%) patients, respectively. Log-rank tests and Multivariate Cox analysis both implied CTLA-4 and TIGIT expression was associated with worse relapse-free survival. In conclusion, this is the largest Chinese UTUC cohort study, and we analyzed the Co-inhibitory receptor expression profiles in UTUC. We identified CTLA-4 and TIGIT expression as promising biomarkers for tumor recurrence. Furthermore, a subset of advanced UTUCs are probably immunogenic, for which single or combined immunotherapy may be potential therapeutic approaches in the future.

Abstract 3296: A urine-based DNA methylation marker test to detect upper tract urothelial carcinoma: a prospective pilot study

Abstract Objectives: Upper tract urothelial carcinoma (UTUC) is an uncommon, yet lethal tumor of the urinary tract. Diagnosis and preoperative risk stratification of these patients present distinct challenges given the limitations of current available diagnostic tools, including endoscopic biopsy and imaging. Herein, we explore the feasibility and clinical performance of a urine-based epigenetic assay in the diagnosis of patients with UTUC. Methods: Under an institutional review board-approved protocol, voided urine samples were prospectively collected using the Bladder CARE Urine Collection Kit (Pangea Laboratory LLC, CA, USA) from primary UTUC patients before any genitourinary manipulation between November 2019 and March 2022. All patients were confirmed to have UTUC in the final specimen. A 1:1 age/sex-matched group of cancer-free healthy donors (control group) was also included in the study. “Healthy” status was based on self-reporting and defined as no history of any type of tumor. Urine samples were analyzed with Bladder CARE, a urine-based test that measures the methylation level of 3 urothelial cancer-specific biomarkers (TRNA-Cys, SIM2, and NKX1-1) and two internal control loci using methylation-sensitive restriction enzymes coupled with qPCR. Results were reported as Bladder CARE Index (BCI) score and categorized as “positive” (BCI &amp;gt; 5), “high risk” (2.5 &amp;lt; BCI ≤ 5) or “negative” (BCI ≤ 2.5). Association between BCI score and category, and clinical and pathological findings was assessed. Results: A total of 50 patients (40 males and 10 females) with a median age of 72 (64-79) years were included in the final analysis. Six patients received neoadjuvant chemotherapy. Surgical procedures included RNU (n=40), ureterectomy (n=7), and URS (n=3). All patients who underwent URS were confirmed to have UTUC through biopsy. Among UTUC patients, Bladder CARE test resulted positive in 47, high-risk in 1, and negative in 2 patients. Comparing Bladder CARE test results with tumor pathologic features demonstrated a statistically significant correlation between BCI values and tumor size (p = 0.009). UTUC patients had significantly higher BCI values compared to the controls (mean BCI 189.3 vs. 1.6, respectively; p &amp;lt; 0.0001). ROC curve showed an AUC of 0.9662 (95% CI = 0.9296-1). Sensitivity, specificity, PPV, and NPV of Bladder CARE to detect UTUC were 96%, 88%, 88.9%, and 95.7%, respectively. In comparison, the sensitivity of urine cytology in our cohort was 37%. Conclusions: In this prospective pilot study, the proposed urine-based epigenetic test (Bladder CARE) showed high sensitivity and negative predictive value in the diagnosis of patients with UTUC. In addition, the sensitivity of this test was exceedingly higher than the standard urine cytology. A larger sample size study to validate the accuracy of this test is the next step. Citation Format: Paolo Piatti, Alireza Ghoreifi, Sanam Seyedian, Yap Ching Chew, Benjamin Jara, Lucy Sanossian, Jeffrey Bhasin, Michael Basin, Taikun Yamada, Gerhard Fuchs, Sumeet Bhanvadia, Rene Sotelo, Andrew Hung, Monish Aron, Mihir Desai, Inderbir Gill, Siamak Daneshmand, Gangning Liang, Hooman Djaladat. A urine-based DNA methylation marker test to detect upper tract urothelial carcinoma: a prospective pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3296.

PD46-02 BLOOD-BASED LIQUID BIOPSY FOR DIAGNOSIS AND SURVEILLANCE OF PRIMARY UPPER TRACT UROTHELIAL CARCINOMA: ROAD TO EMERGING NOVEL BIOMARKER

You have accessJournal of UrologyCME1 Apr 2023PD46-02 BLOOD-BASED LIQUID BIOPSY FOR DIAGNOSIS AND SURVEILLANCE OF PRIMARY UPPER TRACT UROTHELIAL CARCINOMA: ROAD TO EMERGING NOVEL BIOMARKER Alireza Ghoreifi, Stephanie Shishido, George Courcoubetis, Salmaan Sayeed, Amy Huang, Andrew Hung, Anne Schuckman, Monish Aron, Mihir Desai, Siamak Daneshmand, Inderbir Gill, Peter Kuhn, Jeremy Mason, and Hooman Djaladat Alireza GhoreifiAlireza Ghoreifi More articles by this author , Stephanie ShishidoStephanie Shishido More articles by this author , George CourcoubetisGeorge Courcoubetis More articles by this author , Salmaan SayeedSalmaan Sayeed More articles by this author , Amy HuangAmy Huang More articles by this author , Andrew HungAndrew Hung More articles by this author , Anne SchuckmanAnne Schuckman More articles by this author , Monish AronMonish Aron More articles by this author , Mihir DesaiMihir Desai More articles by this author , Siamak DaneshmandSiamak Daneshmand More articles by this author , Inderbir GillInderbir Gill More articles by this author , Peter KuhnPeter Kuhn More articles by this author , Jeremy MasonJeremy Mason More articles by this author , and Hooman DjaladatHooman Djaladat More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003359.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Blood-based liquid biopsy has emerged as a novel diagnostic and prognostic biomarker in cancer patients, yet the efficacy of this test in patients with upper tract urothelial carcinoma (UTUC) is unknown. The aim of this study is to explore the feasibility of a liquid biopsy in the diagnosis and surveillance of primary UTUC. METHODS: In this prospective pilot study, under an IRB-approved protocol (#HS-19-00886), blood samples were collected between May 2021 and September 2022 from primary UTUC patients before surgical intervention with curative intent as well as follow-up visits. The samples were analyzed using the third generation comprehensive high-definition single cell assay (HDSCA3.0) to detect rare cells (e.g., circulating tumor cells [CTCs]) and other rare events (e.g., large extracellular vesicles [LEVs]) (Figure 1-A). The findings of pre-surgery liquid biopsies were compared to the blood samples of normal donors (NDs) who had no known pathology. Follow-up samples were compared to the baseline findings. RESULTS: A total of 22 UTUC patients and 50 NDs were included in the analysis. Pathologic staging of UTUC patients revealed 13 non-muscle invasive (<T2) and 9 muscle-invasive (≥T2) tumors. Most cases (17/22) were high-grade. Eight patients received neoadjuvant chemotherapy as well. HDSCA3.0 analysis indicated that rare cells were detectable in the peripheral blood collected from UTUC patients with a high heterogeneity. Total rare events and total rare cells detected in UTUC samples before surgery were significantly higher than that from the NDs. Similar trend was seen in other CTC and rare cell populations, including D|V, D|CK|V|CD, D|V|CD, D, D|CK, and total CK+cells as well as total LEVs and CK+only LEVs (Figure 1-B). Additionally, 11 follow-up samples, collected within a median 8 months following surgery, were available for the analysis that showed significant decrease in the incidence of total LEVs, CK LEVs, and CK|CD LEVs (Figure 1-C). Among these patients, there were 4 bladder recurrences but no distant metastasis. CONCLUSIONS: This study demonstrates promising evidence for the possible clinical utility of the liquid biopsy in UTUC. Rare cells and LEVs may be used in the diagnosis and surveillance of patients with UTUC. Source of Funding: USC Urology Research Council © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1169 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alireza Ghoreifi More articles by this author Stephanie Shishido More articles by this author George Courcoubetis More articles by this author Salmaan Sayeed More articles by this author Amy Huang More articles by this author Andrew Hung More articles by this author Anne Schuckman More articles by this author Monish Aron More articles by this author Mihir Desai More articles by this author Siamak Daneshmand More articles by this author Inderbir Gill More articles by this author Peter Kuhn More articles by this author Jeremy Mason More articles by this author Hooman Djaladat More articles by this author Expand All Advertisement PDF downloadLoading ...

PD46-07 URINARY COMPREHENSIVE GENOMIC PROFILING AIDS IN DETECTION AND RISK PROGNOSIS OF UPPER TRACT UROTHELIAL CARCINOMA: A CASE-CONTROLLED COHORT STUDY

You have accessJournal of UrologyCME1 Apr 2023PD46-07 URINARY COMPREHENSIVE GENOMIC PROFILING AIDS IN DETECTION AND RISK PROGNOSIS OF UPPER TRACT UROTHELIAL CARCINOMA: A CASE-CONTROLLED COHORT STUDY Paul Yonover, Robert Kaplinsky, Keyan Salari, Adam Feldman, Debasish Sundi, Kevin G. Phillips, Daniel Fisher, Ava Cherry, Brian C. Mazzarella, Vincent T. Bicocca, Joe Gray, Theresa M. Koppie, Trevor G. Levin, and Justin Cohen Paul YonoverPaul Yonover More articles by this author , Robert KaplinskyRobert Kaplinsky More articles by this author , Keyan SalariKeyan Salari More articles by this author , Adam FeldmanAdam Feldman More articles by this author , Debasish SundiDebasish Sundi More articles by this author , Kevin G. PhillipsKevin G. Phillips More articles by this author , Daniel FisherDaniel Fisher More articles by this author , Ava CherryAva Cherry More articles by this author , Brian C. MazzarellaBrian C. Mazzarella More articles by this author , Vincent T. BicoccaVincent T. Bicocca More articles by this author , Joe GrayJoe Gray More articles by this author , Theresa M. KoppieTheresa M. Koppie More articles by this author , Trevor G. LevinTrevor G. Levin More articles by this author , and Justin CohenJustin Cohen More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003359.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: A urine test capable of detecting upper tract urothelial carcinoma (UTUC) that provides diagnostic and prognostic information might aid diagnosis and improve risk stratification, which are major clinical challenges in UTUC. Urinary comprehensive genomic profiling (uCGP) via next-generation sequencing with the CLIA-validated UroAmp assay (Convergent Genomics) was developed to identify mutations, diagnose disease, assess molecular grade and stage, and predict recurrence risk. Here we verify that a urine-based uCGP approach can identify lesions in UTUC patients and we compare these genomic profiles to subjects with bladder cancer. METHODS: uCGP was performed on 69 specimens from individuals with de novo pathology-confirmed UC of the bladder (UCB) and 12 urine specimens from individuals with de novo pathology-confirmed UTUC. Urine DNA was sequenced, comprehensively profiled across 60 genes, and disease classification was predicted by a machine learned algorithm. Differential mutational signals of UTUC and UCB were compared through odds ratio (OR) analysis with statistical significance ascribed by Fisher’s exact test. RESULTS: Among UTUC patients, grade distribution was 83% HG, 17% LG; stage distribution was 25% Ta, 25% T1, 8% T2, 8% T3, 8% T4, 25% unknown; among UCB patients grade distribution was 70% HG, 30% LG; stage distribution was 45% Ta, 22% T1, 20% T2, 7% T4, 4% Tis, 2% unknown. uCGP correctly classified 100% of UTUC subjects and 97% (67/69) of UCB subjects as disease positive. UTUC lesions had a high prevalence of TP53 mutations (70%) and frequently demonstrated genomic instability with large scale aneuploidy (40%), loss of heterozygosity (30%), ERCC2 amplification (30%), and CDKN2A loss (10%). Additional recurrent mutations included KMT2C (40%), KMT2D (40%), FGFR3 (30%), and TERT (30%). OR analysis revealed significant enrichment of promoter mutations in TERT (OR=10.6) among high grade (HG) UCB lesions compared to HG UTUC. Further, mutations in ARID1A, PLEKHS1, were solely observed in HG UCB lesions. CONCLUSIONS: uCGP is a quantitative diagnostic with the ability to profile genomic lesions in UTUC and UCB. While further study is needed, distinct mutational patterns in UTUC could potentially serve to identify urologic origins of urine-based disease signals. Source of Funding: Research support by Convergent Genomics © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1172 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Paul Yonover More articles by this author Robert Kaplinsky More articles by this author Keyan Salari More articles by this author Adam Feldman More articles by this author Debasish Sundi More articles by this author Kevin G. Phillips More articles by this author Daniel Fisher More articles by this author Ava Cherry More articles by this author Brian C. Mazzarella More articles by this author Vincent T. Bicocca More articles by this author Joe Gray More articles by this author Theresa M. Koppie More articles by this author Trevor G. Levin More articles by this author Justin Cohen More articles by this author Expand All Advertisement PDF downloadLoading ...

MP56-15 DEVELOPMENT AND OUTCOMES OF UPPER TRACT UROTHELIAL CARCINOMA FOLLOWING TRIMODALITY THERAPY FOR MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023MP56-15 DEVELOPMENT AND OUTCOMES OF UPPER TRACT UROTHELIAL CARCINOMA FOLLOWING TRIMODALITY THERAPY FOR MUSCLE INVASIVE BLADDER CANCER Jillian Egan, Affan Zafar, Linda Nguyen, Matthew Wszolek, Niall Heney, Dimitar Zlatev, Richard Lee, Jason Efstathiou, and Adam Feldman Jillian EganJillian Egan More articles by this author , Affan ZafarAffan Zafar More articles by this author , Linda NguyenLinda Nguyen More articles by this author , Matthew WszolekMatthew Wszolek More articles by this author , Niall HeneyNiall Heney More articles by this author , Dimitar ZlatevDimitar Zlatev More articles by this author , Richard LeeRichard Lee More articles by this author , Jason EfstathiouJason Efstathiou More articles by this author , and Adam FeldmanAdam Feldman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003309.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Trimodality therapy (TMT) for muscle invasive bladder cancer (MIBC) is widely accepted as an alternative to radical cystectomy, as reflected by its inclusion in national guidelines.1,2 However, the development and outcomes of upper tract urothelial carcinoma (UTUC) after TMT have not been described in a contemporary cohort. METHODS: Our IRB approved, institutional database was retrospectively reviewed for all patients who underwent TMT for MIBC from 2000-2017. Upper tract recurrence was defined as detection of UTUC after the completion of TMT. Pathologic and follow-up data were recorded from review of the original pathology and clinical reports. Descriptive statistics were calculated, and Kaplan Meier time-to-event analysis was performed using STATA 17.0. RESULTS: 271 patients were included. Median follow up was 51.8 [interquartile range (IQR) 18.0-98.1] months (mo). 7 patients (2.6%) experienced upper tract recurrence at a median time of 46 [5-49] mo. Median upper tract recurrence free survival was not reached. There was one high grade (grade 3/3), two grade 2/3, and two low grade (Grade 1-2/3) tumors detected. 2 patients did not have pathology available. 2 patients were treated with nephroureterectomy, 3 were managed endoscopically and 2 received no treatment (1 due to comorbidities and 1 refused treatment). For those treated with nephroureterectomy, pathologic grade and stage were grade 2 pTaNx and grade 3 pT2Nx. Both had associated carcinoma in situ. Neither suffered complications. Of those who developed UTUC after TMT, 3 had ureteral stents placed for tumor or resection near or surrounding the ureteral orifice and no patients had hydronephrosis at the time of TMT for their bladder cancer. CONCLUSIONS: Upper tract recurrence after TMT is relatively rare, occurring in 2.6% of patients, consistent with prior data on upper tract recurrence following cystectomy for MIBC.3 The management of UTUC patients after TMT can proceed according to the standard of care for UTUC with no increased risk of complications or poorer outcomes. Source of Funding: None. © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e780 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jillian Egan More articles by this author Affan Zafar More articles by this author Linda Nguyen More articles by this author Matthew Wszolek More articles by this author Niall Heney More articles by this author Dimitar Zlatev More articles by this author Richard Lee More articles by this author Jason Efstathiou More articles by this author Adam Feldman More articles by this author Expand All Advertisement PDF downloadLoading ...

MP69-01 ESTABLISHMENT OF A NOVEL ANIMAL MODEL IN CARCINOGENESIS OF UPPER TRACT UROTHELIAL CARCINOMA AND ITS GENE PROFILE

You have accessJournal of UrologyCME1 Apr 2023MP69-01 ESTABLISHMENT OF A NOVEL ANIMAL MODEL IN CARCINOGENESIS OF UPPER TRACT UROTHELIAL CARCINOMA AND ITS GENE PROFILE Akinaru Yamamoto, Atsunari Kawashima, Kentaro Jingushi, Yuichi Motoyama, Satoshi Nojima, Sassi Nesrine, Yohei Okuda, Toshiki Oka, Toshihiro Uemura, Gaku Yamamichi, Eisuke Tomiyama, Kosuke Nakano, Makoto Matsushita, Yu Ishizuya, Yoshiyuki Yamamoto, Taigo Kato, Koji Hatano, Tsujikawa Kazutake, and Norio Nonomura Akinaru YamamotoAkinaru Yamamoto More articles by this author , Atsunari KawashimaAtsunari Kawashima More articles by this author , Kentaro JingushiKentaro Jingushi More articles by this author , Yuichi MotoyamaYuichi Motoyama More articles by this author , Satoshi NojimaSatoshi Nojima More articles by this author , Sassi NesrineSassi Nesrine More articles by this author , Yohei OkudaYohei Okuda More articles by this author , Toshiki OkaToshiki Oka More articles by this author , Toshihiro UemuraToshihiro Uemura More articles by this author , Gaku YamamichiGaku Yamamichi More articles by this author , Eisuke TomiyamaEisuke Tomiyama More articles by this author , Kosuke NakanoKosuke Nakano More articles by this author , Makoto MatsushitaMakoto Matsushita More articles by this author , Yu IshizuyaYu Ishizuya More articles by this author , Yoshiyuki YamamotoYoshiyuki Yamamoto More articles by this author , Taigo KatoTaigo Kato More articles by this author , Koji HatanoKoji Hatano More articles by this author , Tsujikawa KazutakeTsujikawa Kazutake More articles by this author , and Norio NonomuraNorio Nonomura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003332.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Upper urinary tract (UT) and bladder are known to differ from the embryology stage. There have been numerous reports of mouse models of bladder carcinogenesis, but there are few reports of mouse models of upper tract urothelial carcinogenesis. The aim of this study is to establish a novel mouse model of upper tract urothelial carcinogenesis. METHODS: We used two strains including C57/BL6 of male and female mice. We fed 0.05% N-butyl-N-(4-hydro-oxybutyl) nitorosamine (BBN) from 6 weeks old to the harvest day. The harvested organs were diagnosed by two different pathologists with histopathological evaluation using Hematoxylin and Eosin staining. Both sides of UT from mouse drinking BBN were subjected to whole transcriptome sequencing (WTS). In human samples, 74 upper tract urothelial carcinoma (UTUC) tissues and 73 adjacent normal (AN) tissues also subjected to WTS from 74 UTUC patients. RESULTS: Female mice of certain strain showed early weight loss compared to the others. They showed high incidence of UTUC with less incidence of bladder cancer (Figure 1), while the others rarely had UTUC. The total incidence of UTUC in this mouse strain was 83.3%, with the earliest UTUC observed at 8 weeks after the start of BBN drinking. The incidence of bilateral UTUC was 22.2%, and these individuals showed weight loss relatively early after the start of BBN drinking. The cancerous side of UT expressed a basal type of mRNA expression profile, with increased expression of Cd44, Cdh3, and Krt14, similar to previous reports of bladder carcinogenesis mouse model (Figure 2). We detected 593 differential expressed genes between cancerous side and non-cancerous side of UT in this mouse model, which could also classify human UTUC and AN samples. In pathway enrichment analysis of these genes, several pathways associated with DNA replication were commonly upregulated in cancer tissues between mouse and human samples. CONCLUSIONS: We have established the novel animal model developing UTUC with high probability. This model could reflect the human UTUC. Source of Funding: This work was supported by a KAKENHI grant (21K20968, 22K09523) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e963 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akinaru Yamamoto More articles by this author Atsunari Kawashima More articles by this author Kentaro Jingushi More articles by this author Yuichi Motoyama More articles by this author Satoshi Nojima More articles by this author Sassi Nesrine More articles by this author Yohei Okuda More articles by this author Toshiki Oka More articles by this author Toshihiro Uemura More articles by this author Gaku Yamamichi More articles by this author Eisuke Tomiyama More articles by this author Kosuke Nakano More articles by this author Makoto Matsushita More articles by this author Yu Ishizuya More articles by this author Yoshiyuki Yamamoto More articles by this author Taigo Kato More articles by this author Koji Hatano More articles by this author Tsujikawa Kazutake More articles by this author Norio Nonomura More articles by this author Expand All Advertisement PDF downloadLoading ...