Determination of enzalutamide long-term safety and efficacy for castration-resistant prostate cancer patients after combined anti-androgen blockade followed by alternative anti-androgen therapy: a multicenter prospective DELC study.

BACKGROUND: Alternative anti-androgen therapy has been widely used as a first-line treatment for castration-resistant prostate cancer, and it may affect treatment outcome of subsequent agents targeting the androgen receptor axis. We conducted the prospective observational DELC (Determination of Enzalutamide Long-term safety and efficacy for Castration-resistant prostate cancer patients after combined anti-androgen blockade followed by alternative anti-androgen therapy) study to evaluate the efficacy of enzalutamide in patients with castration-resistant prostate cancer who underwent prior combined androgen blockade with bicalutamide and then alternative anti-androgen therapy with flutamide. METHODS: The DELC study enrolled 163 Japanese patients with castration-resistant prostate cancer who underwent alternative anti-androgen therapy with flutamide following failure of initial combined androgen blockade with bicalutamide in multiple institutions between January 2016 and March 2019. Primary endpoint was overall survival. Administration of enzalutamide was started at 160 mg orally once daily in all patients. RESULTS: The rate of decline of prostate-specific antigen by 50% or more was 72.2%, and median overall survival was 42.05 months. Multivariate analysis revealed that higher pretreatment serum levels of prostate-specific antigen (≥11.3 ng/mL; P = 0.004), neuron-specific enolase (P = 0.014) and interleukin-6 (≥2.15 pg/mL; P = 0.004) were independent risk factors for overall survival. Fatigue (30.0%), constipation (19.6%) and appetite loss (17.8%) were the most common clinically relevant adverse events. The enzalutamide dose was not reduced in any patient under the age of 70, but adherence was decreased in those over 70. CONCLUSIONS: In the DELC study, the safety of enzalutamide was comparable to that in previous reports. Serum levels of neuron-specific enolase and interleukin-6 were suggested as prognostic factors for castration-resistant prostate cancer with potential clinical utility.

Diagnostic and prognostic significance of tartrate-resistant acid phosphatase type 5b in newly diagnosed prostate cancer with bone metastasis: A real-world multi-institutional study.

OBJECTIVES: Approximately, 90% of men with advanced prostate cancer will develop bone metastasis. However, there have been few reports about noninvasive biomarker to detect and predict clinical outcome of bone metastasis (BM) in prostate cancer patients. METHODS: We examined 1127 patients who underwent prostate biopsy from August 2012 to June 2017. We also investigated bone turnover markers such as bone-specific alkaline phosphatase, type I collagen cross-linked N-terminal telopeptide, C-terminal pyridinoline cross-linked telopeptide of type I collagen, and tartrate-resistant acid phosphatase type 5b (TRACP 5b). RESULTS: A total of 282 patients were diagnosed as prostate cancer with complete clinical data, and 34 patients with bone metastasis. Multivariate analysis revealed C-terminal pyridinoline cross-linked telopeptide of type I collagen, tartrate-resistant acid phosphatase type 5b, and prostate-specific antigen (PSA) were independent biomarkers in detection of BM (p < 0.05, respectively). Furthermore, we developed predictive model formula based on tartrate-resistant acid phosphatase type 5b and PSA, for which the area under the curve was 0.95. In patients with bone metastasis, multivariate cox proportional hazards analysis revealed that this model was significantly associated with poor clinical outcome of cancer-specific survival (p < 0.05). In validation cohort with 137 patients, we also confirmed the utility of this model for diagnosis of BM (the area under the curve = 0.95). CONCLUSIONS: Our developed formula of tartrate-resistant acid phosphatase type 5b in accordance with PSA may serve as the useful tool in diagnosis and prediction of clinical outcome for prostate cancer with bone metastasis.

Perioperative circulating tumor DNA enables the identification of patients with poor prognosis in upper tract urothelial carcinoma.

Perioperative systemic chemotherapy improves the prognosis of upper tract urothelial carcinoma (UTUC). The first objective of this study was to verify whether perioperative circulating tumor DNA (ctDNA) analysis using a pan-cancer gene panel and next-generation sequencing could identify patients with poor prognosis who require perioperative chemotherapy. Second, we investigated whether ctDNA is useful for minimal residual disease (MRD) detection and treatment monitoring in UTUC. This study included 50 patients with untreated UTUC, including 43 cases of localized UTUC. We performed targeted ultradeep sequencing of plasma cell-free DNA (cfDNA) and buffy coat DNA and whole-exome sequencing of cancer tissues, allowing exclusion of possible false positives. We attempted to stratify the prognosis according to the perioperative ctDNA levels in patients with localized UTUC. In patients with metastatic UTUC, ctDNA was evaluated before, during, and after systemic treatment. In total, 23 (46%) of 50 patients with untreated UTUC were ctDNA positive, and 17 (40%) of 43 patients with localized UTUC were ctDNA positive. Of the detected TP53 mutations, 19% were false positives due to clonal hematopoiesis of indeterminate potential. Among preoperative risk factors, only the preoperative ctDNA fraction>2% was a significant and independent risk factor associated with worse recurrence-free survival (RFS). Furthermore, the existence of ctDNA early points after the operation was significantly associated with worse RFS, suggesting the presence of MRD. ctDNA also showed a potential as a real-time marker for systemic therapy in patients with metastatic UTUC. Detection of ctDNA may indicate potential metastasis and guide decisions on perioperative chemotherapy.

Novel diagnostic model for bone metastases in renal cell carcinoma patients based on bone scintigraphy analyzed by computer-aided diagnosis software and bone turnover markers.

BACKGROUND: Computer-assisted diagnosis (CAD) systems for bone scans have been introduced as clinical quality assurance tools, but few studies have reported on its utility for renal cell carcinoma (RCC) patients. The aim of this study was to assess the diagnostic validity of the CAD system for bone scans and to construct a novel diagnostic system for bone metastases in RCC patients. METHODS: We evaluated bone scan images of 300 RCC patients. Artificial neural network (ANN) values, which represent the probability of abnormality, were calculated by BONENAVI, the CAD software for bone scans. By analyzing ANN values, we assessed the diagnostic validity of BONENAVI. Next, we selected 108 patients who underwent measurements of bone turnover markers and assessed the combined diagnostic validity of BONENAVI and bone turnover markers. RESULTS: Forty-three out of 300 RCC patients had bone metastases. The AUC of ANN values was 0.764 and the optimum sensitivity and specificity were 83.7 and 62.7%. By logistic analysis of 108 cases, we found that ICTP, a bone resorption marker, could be a diagnostic marker. The AUC of ICTP was 0.776 and the optimum sensitivity and specificity were 57.1 and 86.8%. Subsequently, we developed a novel diagnostic model based on ANN values and ICTP. Using this model, the AUC was 0.849 and the optimum sensitivity and specificity were 76.2 and 80.7%. CONCLUSION: By combining the high sensitivity provided by BONENAVI and the high specificity provided by ICTP, we constructed a novel, high-accuracy diagnostic model for bone metastases in RCC patients.

Early dynamics of circulating tumor DNA predict clinical response to immune checkpoint inhibitors in metastatic renal cell carcinoma.

OBJECTIVES: Detection of genomic alterations in circulating tumor deoxyribonucleic acid of peripheral blood can guide the selection of systemic therapy in cancer patients. The predictive significance of circulating tumor deoxyribonucleic acid in metastatic renal cell carcinoma remains unclear, especially for patients treated with immune checkpoint inhibitors. METHODS: In this study, we collected plasma samples before and 1 month after commencing nivolumab monotherapy or nivolumab plus ipilimumab therapy from 14 metastatic renal cell carcinoma patients. We performed circulating tumor deoxyribonucleic acid genomic profiling in plasma cell-free deoxyribonucleic acid by next-generation sequencing using a commercially available pan-cancer panel (Guardant360 CDx). Additionally, we also performed whole exome sequencing of tumor tissues and compared the concordance of genomic profiles with circulating tumor deoxyribonucleic acid. RESULTS: Nine patients had circulating tumor deoxyribonucleic acid in pretreatment plasma samples with a total of 20 mutations (15 single nucleotide variants, three insertions/deletions, and two copy number amplification). VHL (30.0%) was the most frequently mutated gene, followed by TP53 (20.0%), and 45.0% of circulating tumor deoxyribonucleic acid mutations were concordant with somatic mutations in tumor tissues. Patients with decreasing circulating tumor deoxyribonucleic acid mutant allele frequency had better progression free survival when compared to those with increasing mutant allele frequency (P = 0.0441). CONCLUSIONS: Our findings revealed that early circulating tumor deoxyribonucleic acid dynamics can serve as a predictive biomarker for response to immune checkpoint inhibitors in metastatic renal cell carcinoma patients.

Fragmentation of cell-free DNA is induced by upper-tract urothelial carcinoma-associated systemic inflammation.

Reliable biomarkers for upper-tract urothelial carcinoma (UTUC) have yet to be found. Plasma cell-free DNA (cfDNA) has been clinically applied as a minimally invasive blood biomarker for various types of cancer. We investigated the utility of plasma cfDNA as a blood biomarker in UTUC patients. The fragment size of plasma cfDNA was shorter and the concentration of plasma cfDNA was higher in UTUC patients than in healthy controls. The fragment size of plasma cfDNA had a moderate accuracy of diagnosing UTUC (area under the curve [AUC] = 0.72), and multivariate analysis indicated that the fragment size of plasma cfDNA was significantly associated with the presence of UTUC (odds ratio = 0.807, 95% confidence interval [CI] 0.653-0.955, P = .024). Furthermore, we found that the size of plasma cfDNA shortens alongside disease progression (P < .001). The fragment size of plasma cfDNA in UTUC patients may be an auxiliary tool for the diagnosis of UTUC patients. We also found a high correlation between the fragmentation of plasma cfDNA and serum levels of three inflammatory cytokines (TNFα [r = -.837], interleukin-6 [IL-6] [r = -.964], interleukin-1 receptor antagonist [IL-1ra] [r = -.911]), which were reported to associate with poor prognosis. Also, we found that the proportion of short fragments of cfDNA was significantly increased in the supernatant of peripheral blood mononuclear cells (PBMCs) from healthy controls cultured in media containing TNFα. These results supposed that cancer-associated systemic inflammation, especially tumor necrosis factor-α (TNFα), may contribute to the fragmentation of plasma cfDNA in UTUC patients.

Proteomic analysis of urinary and tissue-exudative extracellular vesicles to discover novel bladder cancer biomarkers.

Proteomic analysis of urinary extracellular vesicles (EVs) is a powerful approach to discover potential bladder cancer (BCa) biomarkers, however urine contains numerous EVs derived from the kidney and normal urothelial epithelium, which can obfuscate information related to BCa cell-derived EVs. In this study, we combined proteomic analysis of urinary EVs and tissue-exudative EVs (Te-EVs), which were isolated from culture medium of freshly resected viable BCa tissues. Urinary EVs were isolated from urine samples of 11 individuals (7 BCa patients and 4 healthy individuals), and Te-EVs were isolated from 7 BCa tissues. We performed tandem mass tag (TMT)-labeling liquid chromatography (LC-MS/MS) analysis for both urinary EVs and Te-EVs and identified 1960 proteins in urinary EVs and 1538 proteins in Te-EVs. Most of the proteins identified in Te-EVs were also present in urinary EVs (82.4%), with 55 of these proteins showing upregulated levels in the urine of BCa patients (fold change > 2.0; P < .1). Among them, we selected 22 membrane proteins as BCa biomarker candidates for validation using selected reaction monitoring/multiple reaction monitoring (SRM/MRM) analysis on urine samples from 70 individuals (40 BCa patients and 30 healthy individuals). Six urinary EV proteins (heat-shock protein 90, syndecan-1, myristoylated alanine-rich C-kinase substrate (MARCKS), MARCKS-related protein, tight junction protein ZO-2, and complement decay-accelerating factor) were quantified using SRM/MRM analysis and validated as significantly upregulated in BCa patients (P < .05). In conclusion, the novel strategy that combined proteomic analysis of urinary EVs and Te-EVs enabled selective detection of urinary BCa biomarkers.

Clinical importance of the expression of CD4+CD8+ T cells in renal cell carcinoma.

OBJECTIVE: CD4+CD8+ T cells are expressed in some cancer patients including those with renal cell carcinoma (RCC). However, no reports have mentioned the clinical importance of this expression. We evaluated the expression of CD4+CD8+ T cells in patients with various cancer types to clarify clinical characteristics and prognostic importance significantly correlating with these T cells. METHODS: Expression of CD4+CD8+ T cells was evaluated using flowcytometry in tissue-infiltrating lymphocytes extracted from 260 cancer tissues including 104 RCC samples. RNA sequencing and characterization and regression (Citrus) was used to determine characteristics. The prognostic importance of CD4+CD8+ T cells was evaluated by Cox regression analysis. RESULTS: Among eight cancer types, expression of CD4+CD8+ T cells was significantly highest in RCC patients. According to the expression of CD4+CD8+ T cells in adjacent normal tissue-infiltrating lymphocytes, 24 patients (23.1%) were defined as being positive for CD4+CD8+ with an expression higher than 9.29% in RCC patients. Citrus showed CD8+PD-1+TIM-3+CD103- T cells to be a specific subpopulation of CD4+CD8+ T cells. RNA sequencing revealed that CD4+CD8+ T cells had significantly lower diversity than the other T cells and shared most T-cell receptor clones with CD8+ not CD4+ T cells. Expression of CD4+CD8+ T cells was identified as an independent predictor of overall survival (hazard ratio: 0.11, 95% confidence interval: 0.01-0.86, P = 0.035) in multivariate analysis. CONCLUSIONS: The expression of CD4+CD8+ T cells was significantly up-regulated in RCC patients and correlated significantly with prognostic importance in surgically treated RCC patients.

The prognostic impact of immune-related adverse events in metastatic renal cell carcinoma patients treated with nivolumab: a real-world multi-institutional retrospective study.

BACKGROUND: Recent studies have shown that immune-related adverse events (irAEs) caused by immune checkpoint inhibitors (ICIs) were correlated with favorable clinical outcome in patients with melanoma. However, in metastatic renal cell carcinoma (mRCC) patients, there have been few reports about the correlation between irAEs and clinical efficacy of anti-programmed cell death protein-1 (PD-1) therapy. METHODS: We retrospectively investigated 160 mRCC patients who started nivolumab monotherapy between September 2016 and July 2019. IrAEs were defined as patients' AEs having a potential immunological basis that required close follow-up, or immunosuppressive therapy. We compared the data of patients who received nivolumab into two groups based on the occurrence of irAEs and assessed clinical efficacy in both groups. RESULTS: Of all mRCC patients, 47 patients (29.4%) developed irAEs. In patients who developed irAEs, the objective response rate and disease control rate were 38.8% and 77.6%, which were significantly higher when compared to that in patients without irAEs (p = 0.012 and p < 0.001, respectively). Furthermore, the incidence of irAEs was significantly associated with an increase in progression-free survival (PFS) [Hazard ratio (HR) = 0.4867; p = 0.0006] and overall survival (OS) (HR = 0.526; p = 0.0252). Importantly, PFS and OS seemed to be similar in patients who discontinued treatment because of irAEs and in those who did not discontinue because of irAEs (p = 0.36 and p = 0.35, respectively). CONCLUSION: Development of irAEs strongly correlates with clinical benefit for mRCC patients receiving nivolumab monotherapy in real-world settings.

[A Case of Para-Aortic Lymph Node Metastasis 10 Years after Transurethral Resection for Non-Muscle Invasive Bladder Cancer].

A 67-year-old man with non-muscle invasive bladder cancer (NMIBC) underwent transurethral resection (TUR) in January 2008. The pathological diagnosis was urothelial carcinoma (UC), grade 2, pT1. A second TUR was performed 2 months later, and no evidence of malignancy was found. After surgery, he was followed up via cystoscopy and urine cytology for 9 years, with no recurrence of the bladder tumor. In November 2017, he visited our orthopedic department complaining of pain in his left leg. Magnetic resonance imaging revealed an enlarged para-aortic lymph node (a suspected metastasis). Computed tomography (CT) revealed several enlarged lymph nodes but no recurrence in the bladder. A CT-guided biopsy was performed, and histopathological examination revealed a metastasis of the urothelial carcinoma. After definitive diagnosis, he received four cycles of gemcitabine-cisplatin chemotherapy. NMIBC with no local progression rarely causes distant metastases, but the possibility is always there.

Intratumoral and s.c. injection of inactivated hemagglutinating virus of Japan envelope (GEN0101) in metastatic castration-resistant prostate cancer.

Inactivated hemagglutinating virus of Japan envelope (HVJ-E) has an antitumor effect and tumor immunity. We undertook an open-label, phase I, dose-escalation study in patients with castration-resistant prostate cancer (CRPC) to determine the safety and efficacy of intratumoral and s.c. injection of HVJ-E (GEN0101). Patients with CRPC, who were resistant to or unable to receive standard of care, were included. GEN0101 was injected directly into the prostate and s.c. in two 28-day treatment cycles. The primary end-points were to evaluate the safety and tolerability of GEN0101 and determine its recommended dose. The secondary end-points were to analyze the antitumor effect and tumor immunity. Three patients received 30 000 mNAU GEN0101 and 6 received 60 000 mNAU. There was no dose-limiting toxicity, and the recommended dose of GEN0101 was defined as 60 000 mNAU. Radiographically, 1 patient had stable disease and 2 had progressive disease in the low-dose group, whereas 5 patients had stable disease and 1 had progressive disease in the high-dose group. Three patients in the high-dose group showed reduction in lymph node metastasis. Prostate-specific antigen increase rates in the high-dose group were suppressed more than those in the low-dose group. Natural killer cell activity was enhanced in 2 patients of the low-dose group and in 5 patients in the high-dose group. In conclusion, intratumoral and s.c. injections of GEN0101 were well-tolerated and feasible to use. The study is registered with the UMIN Clinical Trials Registry (no. UMIN000017092).

MicroRNA-92b-3p is a prognostic oncomiR that targets TSC1 in clear cell renal cell carcinoma.

Although several studies have reported that microRNA (miR)-92b-3p is involved in various cellular processes related to carcinogenesis, its physiological role in clear cell renal cell carcinoma (ccRCC) remains unclear. To clarify the role of miR-92b-3p in ccRCC, we compared miR-92b-3p expression levels in ccRCC tissues and adjacent normal renal tissues. Significant upregulation of miR-92b-3p was observed in ccRCC tissues. Overexpression of miR-92b-3p using a miRNA mimic promoted proliferation, migration, and invasion activities of ACHN cells. Functional inhibition of miR-92b-3p by a hairpin miRNA inhibitor suppressed Caki-2 cell growth and invasion activities in vitro. Mechanistically, it was found that miR-92b-3p directly targeted the TSC1 gene, a known upstream regulator of mTOR. Overexpression of miR-92b-3p decreased the protein expression of TSC1 and enhanced the downstream phosphorylation of p70S6 kinase, suggesting that the mTOR signaling pathway was activated by miR-92b-3p in RCC cells. Importantly, a multivariate Cox proportion hazard model, based on TNM staging and high levels of miR-92b-3p, revealed that miR-92b-3p expression (high vs. low hazard ratio, 2.86; 95% confidence interval, 1.20-6.83; P = .018) was a significant prognostic factor for overall survival of ccRCC patients with surgical management. Taken together, miR-92b-3p was found to act as an oncomiR, promoting cell proliferation by downregulating TSC1 in ccRCC.

The role of actinin-4 (ACTN4) in exosomes as a potential novel therapeutic target in castration-resistant prostate cancer.

Prostate cancer is the second leading cause of cancer death in men in the United States. Several novel therapeutic agents have been developed for castration-resistant prostate cancer (CRPC), but the prognosis for patients with CRPC remains poor. The identification of novel therapeutic targets for CRPC is an urgent issue. Exosomes are small vesicles secreted by a variety of cells, and exosomes derived from cancer cells have been reported to circulate in the patient's bodily fluids, promoting metastasis and invasion. We aimed to identify novel therapeutic targets for CRPC by proteomic analysis of serum exosomes. Exosomes were isolated by ultracentrifugation of sera from 36 men with metastatic prostate cancer: untreated (n = 8), well-controlled with primary androgen deprivation therapy (ADT) (n = 8), and CRPC (n = 20). We identified 823 proteins in the serum exosomes. Six proteins were increased in CRPC patients compared with untreated patients. In contrast, only ACTN4 was increased in the CRPC patients compared to the ADT patients. We focused on ACTN4 as a candidate for targeted therapeutics. ACTN4 was highly expressed in the prostate cancer cell line DU145 as well as exosomes from this line. RNA interference-mediated downregulation of ACTN4 significantly attenuated cell proliferation and invasion in DU145 cells. ACTN4 could be a potential therapeutic target for CRPC.

Failure to achieve castrate level of serum testosterone during luteinizing hormone-releasing hormone agonist therapy in a patient with prostate cancer.

We report the failure to achieve castrate level of serum testosterone during luteinizing hormone-releasing hormone agonist therapy in a patient with prostate cancer. A 76-year-old man was admitted to our hospital for evaluation of an elevated serum prostate specific antigen (PSA) level (191.10 ng/ml) in August 2011. He was diagnosed with T3aN0M1b prostate adenocarcinoma. A combined androgen blockade using luteinizing hormone-releasing hormone agonist (the 1-month depot of leuprorelin acetate) and antiandrogen was administered. Due to liver dysfunction, antiandrogens, both bicalutamide and flutamide, were stopped. The 1-month depot was switched to the 3-month depot in May 2013, but the patient complained of induration and abscess at the infection site. Leuprorelin acetate was replaced by goserelin acetate. Because no adverse event appeared after injection of the 1-month depot of goserelin acetate, the 3-month depot was administered in October 2013. The PSA level increased gradually, and the testosterone level was greater than 50 ng/dl, that is, above castrate range. The 3-month depot of both leuprorelin acetate and goserelin acetate was not effective for this patient. For this reason, the 1-month depot of leuprorelin acetate was started resulting in a rapid decrease in PSA and testosterone levels. Thereafter, androgen depriving therapy could be continued. Androgen deprivation therapy is the standard treatment for patients with advanced prostate cancer and luteinizing hormone-releasing hormone aims to suppress serum testosterone to castrate range. We recommend assessing the serum testosterone levels during luteinizing hormone-releasing hormone agonist therapy for monitoring treatment efficacy and verifying progression when the PSA level increases.

Expression of Nectin-4 and PD-L1 in Upper Tract Urothelial Carcinoma.

Enfortumab vedotin is a novel antibody-drug conjugate targeting Nectin-4, which is highly expressed in urothelial carcinoma. However, the expression status of Nectin-4 in upper tract urothelial carcinoma (UTUC) remains unclear. The relationship between Nectin-4 and Programmed Death Ligand 1 (PD-L1) in UTUC is also ambiguous. We performed immunohistochemical analysis of 99 UTUC tissue microarray to assess the expression of Nectin-4 and PD-L1 in UTUC. Nectin-4-positivity was detected in 65 (65.7%) samples, and PD-L1 was detected in 24 (24.2%) samples. There was no correlation between the expression of Nectin-4 and PD-L1. Patients with strong Nectin-4-expressing tumors had a significantly higher risk of progression (p = 0.031) and cancer-specific mortality (p = 0.036). Strong Nectin-4 expression was also an independent predictor of disease progression in the high-risk group (pT3 ≤ or presence of lymphovascular invasion or lymph node metastasis) (Hazard ratio, 3.32 [95% confidence interval, 1.20-7.98; p = 0.027]). In conclusion, we demonstrated that Nectin-4 expression rate in UTUC was 65.7% and independent of PD-L1 expression. Strong Nectin-4 expression was associated with worse progression-free survival in high-risk UTUC. These findings suggested that enfortumab vedotin may be effective in a broad range of patients with UTUC, regardless of PD-L1 expression.

Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma.

Reliable biomarkers for renal cell carcinoma (RCC) have yet to be determined. Circulating tumor DNA (ctDNA) is an emerging resource to detect and monitor molecular characteristics of various tumors. The present study aims to clarify the clinical utility of ctDNA for RCC. Fifty-three patients histologically diagnosed with clear cell RCC were enrolled. Targeted sequencing was carried out using plasma cell-free DNA (cfDNA) and tumor DNA. We applied droplet digital PCR (ddPCR) to validate detected mutations. cfDNA fragment size was also evaluated using a microfluidics-based platform and sequencing. Proportion of cfDNA fragments was defined as the ratio of small (50-166 bp) to large (167-250 bp) cfDNA fragments. Association of mutant allele frequency of ctDNA with clinical course was analyzed. Prognostic potential was evaluated using log-rank test. A total of 38 mutations across 16 (30%) patients were identified from cfDNA, including mutations in TP53 (n = 6) and VHL (n = 5), and median mutant allele frequency of ctDNA was 10%. We designed specific ddPCR probes for 11 mutations and detected the same mutations in both cfDNA and tumor DNA. Positive ctDNA was significantly associated with a higher proportion of cfDNA fragments (P = .033), indicating RCC patients with ctDNA had shorter fragment sizes of cfDNA. Interestingly, the changes of mutant allele frequency in ctDNA concurrently correlated with clinical course. Positive ctDNA and fragmentation of cfDNA were significantly associated with poor cancer-specific survival (P < .001, P = .011). In conclusion, our study shows the clinical utility of ctDNA status and cfDNA fragment size as biomarkers for prognosis and disease monitoring in RCC.

Diagnostic potential of TERT promoter and FGFR3 mutations in urinary cell-free DNA in upper tract urothelial carcinoma.

Most upper tract urothelial carcinomas (UTUC) are muscle invasive at the time of diagnosis. Current standard methods for the diagnosis of UTUC are invasive. Urine cytology is the only non-invasive test for detecting UTUC, but its sensitivity is low. A novel non-invasive assay for UTUC detection would improve patient outcome. This study aimed to investigate the mutation of cell-free DNA (cfDNA) in urine supernatant to develop a reliable diagnostic biomarker for UTUC patients. We studied urinary cfDNA from 153 individuals, including 56 patients with localized UTUC, and carried out droplet digital PCR assay for TERT promoter and FGFR3 hotspot mutations. We could detect mutations of TERT C228T in 22/56 (39.3%), TERT C250T in 4/56 (7.1%), and FGFR3 S249C in 9/56 (16.1%) patients. FGFR3 mutation was detected only in ≤pT1 tumors (positive predictive value: 100.0%). In combination with cytology results, the sensitivity was 78.6%, and the specificity was 96.0%. Although these data need to be validated in a larger-scale cohort, mutation analysis of TERT promoter and FGFR3 in urinary cfDNA has the potential to be a non-invasive diagnostic marker and reliable factor for tumor staging.

Results of weekday-on and weekend-off administration schedule of sunitinib therapy for advanced renal cell carcinoma.

BACKGROUND: Sunitinib is widely prescribed as first-line therapy for metastatic renal cell carcinoma. To reduce the ratio of severe adverse events and improve the relative dose intensity, we prospectively tried our own alternative medication schedule, which we called the "weekday-on and weekend-off regimen". Here we report the results of this regimen compared to the conventional medication schedule. METHODS: In total, 58 patients were enrolled in this study. Twenty patients were treated under the alternative schedule (group I: weekday-on and weekend-off regimen) and 38 patients were treated using the conventional schedule (group II: 4 weeks on and 2 weeks off regimen). The relative dose intensity (6W-RDI) and prognoses were compared between the two groups. RESULTS: Median 6W-RDI of all the patients was 75.0%. Group I patients demonstrated significantly higher 6W-RDI compared to group II (77.2 vs. 70.4%) (p = 0.019). Multivariate analysis showed that the alternative sunitinib administration schedule was significantly associated with maintaining 6W-RDI above 75% for RCC patients treated with sunitinib (OR 3.592, 95% CI 1.042-12.383, p = 0.043). On the other hand, there were no significant differences between 2 groups regarding occurrence rate of severe adverse events and prognosis by multivariate analysis. CONCLUSIONS: We report the results of an alternative medication schedule, the "weekday-on and weekend-off regimen", as a means of increasing 6W-RDI for metastatic RCC patients.

[A Case of Urethral Carcinoma in Situ Resected by Urethrectomy of Anterior Urethra].

A 62-year-old man had been treated for urethral stricture developed after his right kidney was harvested for donation to his sister 34 years ago. Transurethral biopsy was performed because of positive urinary cytology and squamous cell carcinoma was detected from the site of urethral stricture. The patient with the desire to preserve the penis was referred to our department. Magneticresonanc e imaging showed no evidence of invasion to subepithelial tissue. Re-biopsy from the site of urethral stricture revealed squamous cell carcinoma in situ. Under the diagnosis of urethral carcinoma cTisN0M0, urethrectomy of anterior urethra with perineal urethrostomy was performed. Histopathological diagnosis was squamous cell carcinoma of the urethra pTis and surgical margins were negative. The patient reported complete urinary continence, normal erections and ejaculation from his urethrostomy. He showed no evidence of recurrence at 28 months after surgery.

Immunological classification of renal cell carcinoma patients based on phenotypic analysis of immune check-point molecules.

OBJECTIVES: To clarify comprehensive immunological signature patterns of tumour tissue-infiltrating lymphocytes in patients with renal cell carcinoma and show its clinical significance. MATERIALS AND METHODS: We investigated the surface marker expressions of tumour tissue-infiltrating lymphocytes quantitatively and classified them based on their functional populations. We extracted 109 sets of tumour tissue-infiltrating lymphocytes from 80 patients who underwent surgical resection of renal cell carcinoma, of which 44 tumour tissue-infiltrating lymphocytes were multiply extracted from 15 patients. Each tumour tissue-infiltrating lymphocyte was characterised on the basis of functional T-cell populations using ten surface marker expressions measured by flow cytometry. RESULTS: All sets of the tumour tissue-infiltrating lymphocytes were classified into three groups, which correlated significantly with Fuhrman grade (OR 0.253, 95% CI 0.094-0.678, P = 0.006). Importantly, both overall metastasis-free survival (HR 0.449, 95% CI 0.243-0.832, P = 0.011) and recurrence-free survival (HR 0.475, 95% CI 0.238-0.948, P = 0.035) of the patients with the higher marker expressions were significantly inferior to those of the patients with the lower marker expressions by multivariate analysis. Six specific genes for this classification identified by microarray analysis verified our results using the TCGA KIRC data set. In addition, we discovered the presence of intra-tumoural diversity in the classification of 3 (20%) of the 15 patients. CONCLUSIONS: This study showed that the presence of classable diversity in the immunological signature of tumour tissue-infiltrating lymphocytes correlated with prognosis and tumour aggressiveness that was observed even within individual tumours in some patients with renal cell carcinoma.