Recent developments and future directions of first-line systemic therapy combined with immunotherapy for advanced or metastatic urothelial carcinoma: a historical perspective on treatment evolution.

Urothelial carcinoma presents significant treatment challenges, especially in advanced stages. Traditionally managed with platinum-based chemotherapy, the advent of immunotherapies, particularly immune checkpoint inhibitors, has revolutionized urothelial carcinoma treatment. This review explores the evolution of urothelial carcinoma management, focusing on the transition from immune checkpoint inhibitors monotherapy to innovative combination therapies. Pembrolizumab, following the KEYNOTE-045 trial, emerged as a pivotal ICI in pretreated metastatic urothelial carcinoma, outperforming traditional chemotherapy. However, limitations surfaced in untreated metastatic urothelial carcinoma patients, particularly in those with low PD-L1 expression, as evidenced by trials like IMvigor130 and KEYNOTE-361. These challenges led to the exploration of combination therapies, including immune checkpoint inhibitors with platinum-based chemotherapy, tyrosine kinase inhibitors, and antibody-drug conjugates. Notably, the CheckMate 901 trial demonstrated improved outcomes with a nivolumab-chemotherapy combination. A significant breakthrough was achieved with the combination of enfortumab vedotin, an antibody-drug conjugates, and pembrolizumab, setting a new standard in first-line treatment for locally advanced or metastatic urothelial carcinoma. Future directions involve further exploration of antibody-drug conjugates and immune checkpoint inhibitors, as seen in the TROPHY-U-01 and TROPiCS-4 trials. The review concludes that the locally advanced or metastatic urothelial carcinoma treatment landscape is rapidly evolving, with combination therapies offering promising avenues for improved patient outcomes, signaling a new era in urothelial carcinoma management.

Cumulative incidence and risk factors for medication-related osteonecrosis of the jaw during long-term prostate cancer management.

Bone-modifying agents (BMA) are extensively used in treating patients with prostate cancer with bone metastases. However, this increases the risk of medication-related osteonecrosis of the jaw (MRONJ). The safety of long-term BMA administration in clinical practice remains unclear. We aimed to determine the cumulative incidence and risk factors of MRONJ. One hundred and seventy-nine patients with prostate cancer with bone metastases treated with BMA at our institution since 2008 were included in this study. Twenty-seven patients (15%) had MRONJ during the follow-up period (median, 19 months; interquartile range, 9-43 months). The 2-year, 5-year, and 10-year cumulative MRONJ incidence rates were 18%, 27%, and 61%, respectively. Multivariate analysis identified denosumab use as a risk factor for MRONJ, compared with zoledronic acid use (HR 4.64, 95% CI 1.93-11.1). Additionally, BMA use at longer than one-month intervals was associated with a lower risk of MRONJ (HR 0.08, 95% CI 0.01-0.64). Furthermore, six or more bone metastases (HR 3.65, 95% CI 1.13-11.7) and diabetes mellitus (HR 5.07, 95% CI 1.68-15.2) were risk factors for stage 2 or more severe MRONJ. MRONJ should be considered during long-term BMA administration in prostate cancer patients with bone metastases.

The prognostic impact of peripheral blood eosinophil counts in metastatic renal cell carcinoma patients treated with nivolumab.

Although immune checkpoint inhibitors (ICIs) have gained approval for metastatic renal cell carcinoma (mRCC), the response rate is still limited. Therefore, it is urgent to explore novel markers of responses to ICIs that can help assess clinical benefits. Recently, it has been noted that peripheral blood eosinophil counts are an independent factor correlated with clinical outcome of ICIs in some types of cancer. We investigated peripheral blood absolute eosinophil counts (AECs) at baseline and 4 weeks after the initiation of nivolumab for mRCC patients between February 2016 and May 2022. In addition, we examined clinicopathological features including irAEs and analyzed the correlation between AECs and clinical efficacy of nivolumab. The median progression-free survival (PFS) and overall survival (OS) for all patients were 5.7 and 25.5 months, respectively. The median AECs in patients with irAEs were significantly higher at baseline and 4 weeks after the treatment compared to those without irAEs (p < 0.001 and p = 0.001). With the cutoff value of AECs of 329 cells/µL at 4 weeks after the treatment for prediction of irAEs, high-AECs groups had significantly higher number of responders compared with that in low-AECs group (p < 0.001). Accordingly, the PFS and OS were significantly better in patients with high-AECs group than those in low-AECs group (p = 0.03 and p = 0.009). High-AECs at 4 weeks after the treatment serve as the prominent surrogate marker associated with the incidence of irAEs and better clinical outcome in mRCC patients receiving nivolumab.

Cutibacterium acnes-derived extracellular vesicles promote tumor growth in renal cell carcinoma.

Bacterial flora are present in various parts of the human body, including the intestine, and are thought to be involved in the etiology of various diseases such as multiple sclerosis, intestinal diseases, cancer, and uterine diseases. In recent years, the presence of bacterial 16S rRNA genes has been revealed in blood, which was previously thought to be a sterile environment, and characteristic blood microbiomes have been detected in various diseases. However, the mechanism and the origin of the bacterial information are unknown. In this study, we performed 16S rRNA metagenomic analysis of bacterial DNA in serum extracellular vesicles from five healthy donors and seven patients with renal cell carcinoma and detected Cutibacterium acnes DNA as a characteristic bacterial DNA in the serum extracellular vesicles of patients with renal cell carcinoma. In addition, C. acnes DNA was significantly reduced in postoperative serum extracellular vesicles from patients with renal cell carcinoma compared with that in preoperative serum extracellular vesicles from these patients and was also detected in tumor tissue and extracellular vesicles from tumor tissue-associated microbiota, suggesting an association between C. acnes extracellular vesicles and renal cell carcinoma. C. acnes extracellular vesicles were taken up by renal carcinoma cells to enhance their proliferative potential. C. acnes extracellular vesicles also exhibited tumor-promoting activity in a mouse model of renal cancer allografts with enhanced angiogenesis. These results suggest that extracellular vesicles released by C. acnes localized in renal cell carcinoma tissues act in a tumor-promoting manner.

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.

Prognostic significance of serum fucosylated pro-haptoglobin in advanced renal cell carcinoma patients treated with immune checkpoint inhibitors.

With the widespread use of immune checkpoint inhibitors (ICIs), identifying predictive biomarkers is critical. Recently, serum fucosylated haptoglobin (Fuc-Hp) was thought to play an important role in tumour immunity in several types of cancer. Therefore, evaluating serum Fuc-Hp in the peripheral blood can potentially identify non-invasive predictive biomarkers for the clinical efficacy of ICIs. In this study, 31 patients with advanced renal cell carcinoma (RCC) treated with nivolumab were enrolled and defined as responders or non-responders according to RECIST criteria. Serum samples were collected before and 1 month after treatment initiation, and an ELISA assay was performed using Aleuria Aurantia Lectin (AAL) and 10-7G monoclonal antibodies that recognise Fuc-mature Hp (Fuc-mHp) and Fuc-pro Hp (Fuc-pHp), respectively. We first measured AAL-haptoglobin (Fuc-mHp) and total haptoglobin levels before nivolumab and found that neither value could predict the clinical response. Notably, serum 10-7G levels were significantly lower in the responder group (p = 0.035). We also confirmed the use of serum 10-7G levels for predicting progressive disease after nivolumab (area under the curve, 0.816). Accordingly, low 10-7G levels were significantly correlated with better progression-free survival (p = 0.041). In conclusion, serum Fuc-pHp analysis may identify patients with advanced RCC who benefit from ICIs.

Bacteria-derived DNA in serum extracellular vesicles are biomarkers for renal cell carcinoma.

This is the first study to determine the clinical importance of circulating bacterial DNA in patients with renal cell carcinoma (RCC). We performed 16S rRNA metagenomic analysis of serum extracellular vesicles (EVs) from 88 patients with RCC and 10 healthy donors and identified three abundant bacterial DNA: Bacteroidia, TM7-1, and Sphingomonadales. Combining characteristic bacterial DNA information (three bacteria-derived DNA), a BTS index was created to diagnose patients with RCC. The BTS index showed high sensitivity not only in the discovery cohort, but also in the validation cohort, suggesting that it was useful as a screening test. Furthermore, in nivolumab treatment of RCC, patients with higher levels of Bacteroidia DNA in serum EVs had significantly poorer progression-free and overall survival than did those with lower levels. This study showed that circulating Bacteria-derived DNA could be used as a biomarker for RCC.

The presence of lymph node metastases and time to castration resistance predict the therapeutic effect of enzalutamide for castration-resistant prostate cancer.

BACKGROUND: Enzalutamide is effective against castration-resistant prostate cancer (CRPC). However, it is unclear which patients would benefit more from enzalutamide treatment. Here, we analyzed patients who received enzalutamide as first-line therapy for CRPC and evaluated the factors that predict treatment response and prognosis. METHODS: We retrospectively analyzed 101 patients treated with enzalutamide for CRPC at our institution. As primary endpoints we regarded the prostate-specific antigen (PSA) response rate and PSA-progression-free survival (PSA-PFS) from the start of enzalutamide treatment. Laboratory and imaging data were analyzed to predict treatment efficacy. RESULTS: PSA reductions of ≥ 50% and ≥ 90% were observed in 78 (77%) and 47 (47%) patients, respectively, compared with the baseline. During the follow-up period, 67 (66%) patients showed PSA progression, with a median PSA-PFS of 11 months. Moreover, 31 patients (31%) died, with a median overall survival of 64 months. On multivariate analysis, lymph node metastases at the start of enzalutamide treatment [odds ratio (OR) 0.0575, 95% confidence interval (CI) 0.0105-0.316, p = 0.0010] and time to CRPC (OR 0.177, 95% CI 0.0428-0.731, p = 0.0167] were associated with ≥ 90% PSA response. Lymph node metastases (hazard ratio [HR] 3.00, 95% CI 1.48-6.09, p = 0.0023) and time to CRPC (HR 1.84, 95% CI 1.02-3.30, p = 0.0419) were also predictors of PSA-PFS on a multivariate model. CONCLUSIONS: Time to CRPC and lymph node metastasis were predictors of the PSA response rate and PSA-PFS.

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.

Bladder Cancer in a Long-term Survivor of the Prune Belly Syndrome.

Prune belly syndrome (PBS) is a rare but morbid complexity of congenital anomalies that consists of abdominal wall defect, bilateral cryptorchidism, and urinary tract dilation. With the recent advances in perinatal care, the life expectancy of patients with PBS has improved. Although renal and sexual outcomes are frequently discussed in the literature, little is known about urinary tract malignancies in adulthood. Herein, we present a case of advanced bladder cancer in a 38-year-old man with PBS. To the best of our knowledge, a malignant tumor arising in a nondefunctionalized or nonaugmented bladder in patients with PBS has not been reported previously.

FAN score comprising fibrosis-4 index, albumin-bilirubin score and neutrophil-lymphocyte ratio is a prognostic marker of urothelial carcinoma patients treated with pembrolizumab.

It is important to identify prognostic and predictive markers of metastatic urothelial carcinoma (mUC) treated with immunocheckpoint inhibitors. We sought to establish a prognostic marker for patients with mUC treated with pembrolizumab based on only blood test results. We included 165 patients with mUC in the discovery cohort and 103 with mUC who were treated with pembrolizumab in the validation cohort. Multivariate and Cox regression analyses were used to analyse the data. In the discovery cohort, the fibrosis-4 index (hazard ratio [HR]: 2.13, 95% confidence interval [CI] 1.20-3.76, p = 0.010), albumin-bilirubin score (HR 1.91, 95% CI 1.27-2.88, p = 0.002), and neutrophil-lymphocyte ratio (HR: 1.84, 95% CI 1.22-2.79, p = 0.004) were independent significant prognostic factors. We established a 'FAN score' that included these three aforementioned items, which were assigned one point each. We divided patients into the 0-1 point (n = 116) and 2-3 points (n = 49) groups. The FAN score was a significant prognostic marker for cancer-specific survival (CSS) (HR 1.48, 95% CI 1.19-1.83, p < 0.001) along with the Eastern Cooperative Oncology Group Performance Status. The FAN score was also a prognostic factor of progression-free survival (PFS) (HR: 1.25, 95% CI 1.01-1.54, p = 0.036) along with the presence of liver metastasis. In the validation cohort, the FAN score was a significant prognostic factor for CSS (HR: 1.48, 95% CI 1.19-1.85, p = 0.001) and PFS (HR: 1.29, 95% CI 1.02-1.62, p = 0.034). We established the FAN score as a prognostic marker for patients with mUC treated with pembrolizumab.

Gut Microbiota-Derived Short-Chain Fatty Acids Promote Prostate Cancer Growth via IGF1 Signaling.

Excessive intake of animal fat and resultant obesity are major risk factors for prostate cancer. Because the composition of the gut microbiota is known to change with dietary composition and body type, we used prostate-specific Pten knockout mice as a prostate cancer model to investigate whether there is a gut microbiota-mediated connection between animal fat intake and prostate cancer. Oral administration of an antibiotic mixture (Abx) in prostate cancer-bearing mice fed a high-fat diet containing a large proportion of lard drastically altered the composition of the gut microbiota including Rikenellaceae and Clostridiales, inhibited prostate cancer cell proliferation, and reduced prostate Igf1 expression and circulating insulin-like growth factor-1 (IGF1) levels. In prostate cancer tissue, MAPK and PI3K activities, both downstream of the IGF1 receptor, were suppressed by Abx administration. IGF1 directly promoted the proliferation of prostate cancer cell lines DU145 and 22Rv1 in vitro. Abx administration also reduced fecal levels of short-chain fatty acids (SCFA) produced by intestinal bacteria. Supplementation with SCFAs promoted tumor growth by increasing IGF1 levels. In humans, IGF1 was found to be highly expressed in prostate cancer tissue from obese patients. In conclusion, IGF1 production stimulated by SCFAs from gut microbes influences the growth of prostate cancer via activating local prostate MAPK and PI3K signaling, indicating the existence of a gut microbiota-IGF1-prostate axis. Disrupting this axis by modulating the gut microbiota may aid in prostate cancer prevention and treatment. SIGNIFICANCE: These results suggest that intestinal bacteria, acting through short-chain fatty acids, regulate systemic and local prostate IGF1 in the host, which can promote proliferation of prostate cancer cells.

MiR-30b-3p and miR-126-3p of urinary extracellular vesicles could be new biomarkers for prostate cancer.

BACKGROUND: Extracellular vesicles (EVs) including exosomes are present in blood, urine, and saliva and contain proteins, microRNAs, and messenger RNAs. We investigated microRNAs in urinary EVs to discover new biomarkers of prostate cancer (PCa). METHODS: We isolated EVs from urine obtained following digital rectal examination (DRE) of 14 men with elevated levels of serum prostate-specific antigen (PSA) [negative biopsy (n=4) and PCa with Gleason scores of 6 (n=3), 7 (n=3), and 8-9 (n=4)]. MicroRNAs extracted from EVs were analyzed by microRNA microarray. RESULTS: MicroRNAs miR-30b-3p and miR-126-3p were identified as being overexpressed in urinary EVs of the PCa patients versus the biopsy-negative men, but no microRNAs were associated with the Gleason score. In the independent cohort as well, these two microRNAs were overexpressed in urinary EVs from the PCa patients versus the negative-biopsy men. Logistic regression analysis adjusted by age and PSA showed that these two microRNAs were significantly associated with the prediction of PCa in biopsy specimens. Sensitivity and specificity of miR-30b-3p and miR-126-3p for the prediction of PCa were 46.4% and 88.0% and 60.7% and 80.0%, respectively, which were better than those of serum PSA (53.5% and 64.0%, respectively). CONCLUSIONS: MiR-30b-3p and miR-126-3p in urinary EVs could be potential biomarkers of PCa.

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.

Peripheral T cell receptor repertoire features predict durable responses to anti-PD-1 inhibitor monotherapy in advanced renal cell carcinoma.

Immune checkpoint inhibitors (ICIs) offer significant clinical benefits to a subset of cancer patients via the induction of a systemic T cell-mediated anti-cancer immune response. Thus, the dynamic characterization of T cell repertoires in the peripheral blood has the potential to demonstrate noninvasive predictive biomarkers for the clinical efficacy of ICIs. In this study, we collected tumor tissues and peripheral blood samples from 25 patients with advanced kidney cancer before anti-programmed cell death protein 1 (PD-1) treatment and 1, 3, and 6 months after treatment initiation. Furthermore, we applied a next-generation sequencing approach to characterize T cell receptor (TCR) alpha and beta repertoires. TCR repertoire analysis revealed that the responders to anti-PD-1 showed an expansion of certain T cell clones even in the blood, as evidenced by the significant decrease in the TCR diversity index and increase in the number of expanded TCR clonotypes 1 month after treatment. Interestingly, these expanded TCR clonotypes in the peripheral blood were significantly shared with tumor-infiltrating T cells in responders, indicating that they have many circulating T cells that may recognize cancer antigens. Expression analysis also revealed that 1 month after treatment, T cells from the peripheral blood of responders showed significantly elevated transcriptional levels of Granzyme B, Perforin, CD39, and PD-1, markers of cancer-associated antigen-specific T cells. Altogether, we propose that global TCR repertoire analysis may allow identifying early surrogate biomarkers in the peripheral blood for predicting clinical responses to anti-PD-1 monotherapy.

[A Case of Pelvic Lymph Node Recurrence Detected by PSMA PET/CT after Primary Proton Beam Radiotherapy for Prostate Cancer].

The patient is a 72-year-old man who was referred to our hospital with an elevated prostate specific antigen (PSA) level. He was diagnosed with prostate cancer (cT2aN0M0) at the age of 62 years. He had undergone radical proton beam radiotherapy. The PSA level decreased to a nadir of 0.217 ng/ml after 5 years, gradually increasing thereafter to 1.595 ng/ml during the next 5 years. Although magnetic resonance imaging of the prostate showed an abnormal signal area in the prostate, repeated biopsies of the prostate revealed no malignant findings. Contrast-enhanced abdominal computed tomography (CT), bone scintigraphy and fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET)/CT did not detect any abnormalities in the prostate or metastatic lesions. ¹8F-prostate specific membrane antigen (PSMA)-PET/CT showed no accumulation in the prostate, but some accumulation in a left obturator lymph node. Open pelvic lymph node dissection was performed, and pathological examination confirmed lymph node metastasis from the prostate cancer. The PSA level decreased from 2.482 ng/ml preoperatively to 0.391 ng/ml at 3 months postoperatively. PSMA-PET/CT might be useful for early localization of recurrent lesions in biochemical recurrence after radical treatment for prostate cancer.

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.

Clinical Significance of Hotspot Mutation Analysis of Urinary Cell-Free DNA in Urothelial Bladder Cancer.

Recent studies showed the clinical utility of next-generation sequencing of urinary cell-free DNA (cfDNA) from patients with urothelial bladder cancer (UBC). In this study, we aimed to develop urinary cfDNA analysis by droplet digital PCR (ddPCR) as a high-throughput and rapid assay for UBC detection and prognosis. We analyzed urinary cfDNA of 202 samples from 2 cohorts. Test cohort was designed for investigating clinical utility of urinary cfDNA, and was composed of 74 samples from patients with UBC, and 52 samples of benign hematuria patients. Validation cohort was designed for validation and assessment of clinical utility comparing urinary cfDNA with UroVysion (Abbott, Illinois, USA), and was composed of 40 samples from patients with UBC, and 36 prospectively collected samples from patients under surveillance after surgery for urothelial carcinoma. We performed ddPCR analysis of hotspot gene mutations (TERT promoter and FGFR3). In the test cohort, the sensitivity of urinary cfDNA diagnosis was 68.9% (51/74) and the specificity was 100% in patients with UBC. The sensitivity increased to 85.9% when used in conjunction with urine cytology. In addition, patients with high TERT C228T allele frequency (≥14%) had significantly worse prognosis in bladder tumor recurrence than patients with low TERT C228T allele frequency or negative TERT C228T (p = 0.0322). In the validation cohort, the sensitivity of urinary cfDNA was 57.5% (23/40) and the specificity was 100% in UBC patients. The sensitivity of the combination of urine cytology with our hotspot analysis (77.5%) was higher than that of urine cytology with UroVysion (68.9%). In the post-surgical surveillance group, patients positive for the TERT C228T mutation had significantly worse prognosis for bladder tumor recurrence than mutation negative patients (p < 0.001). In conclusion, ddPCR analysis of urinary cfDNA is a simple and promising assay for the clinical setting, surpassing UroVysion for detection and prognosis determination in UBC.

TERT C228T mutation in non-malignant bladder urothelium is associated with intravesical recurrence for patients with non-muscle invasive bladder cancer.

Telomerase reverse transcriptase (TERT) promoter mutations are frequently found in tumors or urine from patients with urothelial carcinoma (UC). TERT promoter mutations are also detected in urine from patients with no evidence of cancer but are associated with subsequent UC development. Mutations in the TERT promoter are thought to be present in nonmalignant urothelium (NMU) during early stages of tumor formation prior to pathological change, but this has not been proven directly. In this proof-of-concept study, we investigated the clinical utility of TERT promoter mutation analysis in NMU of patients with non-muscle-invasive bladder cancer (NMIBC). This single-institute study included 53 primary tumors and 428 systematic bladder biopsy specimens from 54 patients with NMIBC. All patients underwent systematic random biopsy and transurethral resection of the bladder tumor. Genomic DNA was analyzed for TERT C228T and C250T mutations using droplet digital PCR (ddPCR). The association between TERT promoter mutation of NMU and bladder recurrence was examined by the Kaplan-Meier method and Cox proportional hazards model. Of the 54 patients, 16 (29.6%) had a TERT C228T mutation and three (5.6%) had a TERT C250T mutation in NMU. Of 428 biopsy specimens, the TERT C228T mutation was detected in 9% (31/364) of normal urothelium, 27% (4/15) of urothelial dysplasia (UD), 50% (9/18) of UD suspicious for carcinoma in situ (CIS), and 58% (18/31) of CIS. During follow-up (median: 3.7 years), 22 (40.7%) patients experienced bladder recurrence and five (9.3%) experienced disease progression. Cox proportional hazard analysis showed that TERT C228T mutation in NMU was significantly associated with bladder recurrence after adjustment for cofounding factors (P = 0.0128). Thus, TERT C228T mutation was detected in NMU, which was a reliable independent prognostic factor of bladder tumor recurrence.