Inflammatory Cell Infiltration Into Islets Without PD-L1 Expression Is Associated With the Development of Immune Checkpoint Inhibitor-Related Type 1 Diabetes in Genetically Susceptible Patients.

Immune checkpoint inhibitors (ICIs) could cause type 1 diabetes (T1D). However, the underlying mechanism remains unclear. We immunohistochemically analyzed pancreatic specimens from three individuals with ICI-related T1D, and their histopathological data were compared those from three patients who had received ICI therapy but did not develop T1D (non-T1D) and seven normal glucose-tolerant subjects as control subjects. All ICI-related T1D patients had susceptible HLA haplotypes. In ICI-related T1D, the ß-cell area decreased and the α-cell area increased compared with non-T1D and control subjects. The number of CD3-positive cells around islets increased in ICI-related T1D and non-T1D compared with control subjects, while the number of CD68-positive cells around islets increased in ICI-related T1D compared with non-T1D and control subjects. The expression ratios of programmed death-ligand 1 (PD-L1) on islets decreased in non-T1D and almost completely disappeared in ICI-related T1D, while PD-L1 expression was observed in most cells of pancreatic islets in control subjects. This study, therefore, indicates that ICI therapy itself could reduce PD-L1 expression on islets in all subjects, which may be related to ß-cell vulnerability. In addition, we showed that absence of PD-L1 expression on ß-cells, genetic susceptibility, and infiltration of macrophages as well as T lymphocytes around islets might be responsible for T1D onset.

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.

Anticancer maintenance chemotherapy prolonged prognosis of metastatic urothelial carcinoma patients: A single institute retrospective study using propensity score matching.

OBJECTIVES: To evaluate the therapeutic efficacy of anticancer maintenance chemotherapy for metastatic urothelial carcinoma. METHODS: We retrospectively compared the clinical outcomes of 74 patients with metastatic urothelial carcinoma who had been treated with or without anticancer maintenance chemotherapy between 2006 and 2020 at Osaka University Hospital. Progression-free survival and cancer-specific survival periods were calculated using the Kaplan-Meier method starting from the end date of induction chemotherapy. The backgrounds of patients who had treated with or without anticancer maintenance chemotherapy were adjusted using the propensity score matching method. RESULTS: Twenty-nine patients had undergone anticancer maintenance chemotherapy, whereas 45 patients had not. The median progression-free survival periods were 18.7 and 5.6 months (p = 0.0209), and the median cancer-specific survival periods were 25.1 and 15.2 months (p = 0.1299), in patients with or without anticancer maintenance chemotherapy respectively. In multivariate analysis, anticancer maintenance chemotherapy significantly prolonged both progression-free survival (hazard ratio 3.65, 95% confidence interval 1.96-6.78, p < 0.0001) and cancer-specific survival (hazard ratio 3.05, 95% confidence interval 1.62-5.76, p = 0.0006) in patients with partial response or stable disease after induction chemotherapy. Also, anticancer maintenance chemotherapy significantly prolonged both progression-free survival (13.1 months vs. 4.9 months, p = 0.0027) and cancer-specific survival (35.1 months vs. 11.8 months, p = 0.0044) in propensity score matched patients. CONCLUSIONS: Anticancer maintenance chemotherapy may be considered the treatment for metastatic urothelial carcinoma patients after induction chemotherapy.

EphA2 on urinary extracellular vesicles as a novel biomarker for bladder cancer diagnosis and its effect on the invasiveness of bladder cancer.

BACKGROUND: Urinary extracellular vesicles (uEVs) secreted from bladder cancer contain cancer-specific proteins that are potential diagnostic biomarkers. We identified and evaluated a uEV-based protein biomarker for bladder cancer diagnosis and analysed its functions. METHODS: Biomarker candidates, selected by shotgun proteomics, were validated using targeted proteomics of uEVs obtained from 49 patients with and 48 individuals without bladder cancer, including patients with non-malignant haematuria. We developed an enzyme-linked immunosorbent assay (ELISA) for quantifying the uEV protein biomarker without ultracentrifugation and evaluated urine samples from 36 patients with and 36 patients without bladder cancer. RESULTS: Thirteen membrane proteins were significantly upregulated in the uEVs from patients with bladder cancer in shotgun proteomics. Among them, eight proteins were validated by target proteomics, and Ephrin type-A receptor 2 (EphA2) was the only protein significantly upregulated in the uEVs of patients with bladder cancer, compared with that of patients with non-malignant haematuria. The EV-EphA2-CD9 ELISA demonstrated good diagnostic performance (sensitivity: 61.1%, specificity: 97.2%). We showed that EphA2 promotes proliferation, invasion and migration and EV-EphA2 promotes the invasion and migration of bladder cancer cells. CONCLUSIONS: We established EV-EphA2-CD9 ELISA for uEV-EphA2 detection for the non-invasive early clinical diagnosis of bladder cancer.

Urine Cell-Free MicroRNAs in Localized Prostate Cancer Patients.

Prostate cancer (PCa) is the most common cancer in men. Prostate-specific antigen screening is recommended for the detection of PCa. However, its specificity is limited. Thus, there is a need to find more reliable biomarkers that allow non-invasive screening for early-stage PCa. This study aims to explore urine microRNAs (miRs) as diagnostic biomarkers for PCa. We assessed cell-free miR (cfmiR) profiles of urine and plasma samples from pre- and post-operative PCa patients (n = 11) and normal healthy donors (16 urine and 24 plasma) using HTG EdgeSeq miRNA Whole Transcriptome Assay based on next-generation sequencing. Furthermore, tumor-related miRs were detected in formalin-fixed paraffin-embedded tumor tissues obtained from patients with localized PCa. Specific cfmiRs signatures were found in urine samples of localized PCa patients using differential expression analysis. Forty-two cfmiRs that were detected were common to urine, plasma, and tumor samples. These urine cfmiRs may have potential utility in diagnosing early-stage PCa and complementing or improving currently available PCa screening assays. Future studies may validate the findings.

Circulating extracellular vesicles carrying Firmicutes reflective of the local immune status may predict clinical response to pembrolizumab in urothelial carcinoma patients.

Bacterial flora has clinical significance for the host. The metabolic environment created by this flora influences immunotherapy in urothelial carcinoma. However, there are no reports on the clinical significance of bacterial flora in the host bloodstream. We aimed to clarify the correlation between extracellular vesicle (EV)-derived blood microflora information and tumor immunological status in urothelial carcinoma (UC) patients. Serum samples were collected from 20 healthy donors, 50 patients with localized UC, and 31 patients with metastatic UC (mUC) who had undergone pembrolizumab treatment. Bacterial DNA in EVs was extracted from each sample. Metagenomic sequencing was performed after amplification of the V1-V2 region of the bacterial 16S rRNA gene. Using the matched tumor tissue and serum samples, we revealed that the smaller amount of peripheral EVs carrying Firmicutes DNA was significantly correlated with the higher number of infiltrating T cells within tumor tissues (CD3; p = 0.015, CD4; p = 0.039, CD8; p = 0.0084) and the higher expression of activation markers on their surface (ICOS on both CD4; p = 0.0013 and CD8 T cells; p = 0.016 and 4-1BB on CD4 T cells; p = 0.016). In terms of circulating metabolic information, L-Ser and L-Pro levels, which play important roles in T cell expansion and proliferation, were significantly higher in the Firmicutes-low group (p = 0.010). All of the patients with higher Firmicutes abundance had disease progression without any clinical response (p = 0.026) and significantly inferior prognosis for pembrolizumab therapy (p = 0.035). This is the first study on the importance of peripheral bacterial EVs in cancer patients treated with cancer immunotherapy.

Biological distinction between grades 2 and 3 with respect to intravesical recurrence in T1 high-grade bladder tumors: a retrospective study.

BACKGROUND: The pathological grading system for non-muscle-invasive bladder cancer is based on the WHO 2004/2016 classification system (low-grade: LG/high-grade: HG) and the WHO 1973 classification system (Grade 1: G1/Grade 2: G2/Grade 3: G3). Recently, the usefulness of combining both systems and classifying the tumors as LG/G1, LG/G2, HG/G2, and HG/G3 has been demonstrated. In this study, we compared the prognosis of intravesical recurrence in relation to different treatment intensities between HG/G2 and HG/G3 bladder cancers. METHODS: We retrospectively evaluated the clinical and therapeutic outcomes of 145 patients diagnosed with T1 HG bladder cancer between 2000 and 2020. We classified 145 patients into three groups: (1) patients with T1 HG/G2 and HG/G3 who received intravesical instillation therapy (n = 76), (2) patients with T1 HG/G2 who did not receive intravesical instillation therapy (n = 32), and (3) patients with T1 HG/G3 who did not receive intravesical instillation therapy (n = 37). RESULTS: The median intravesical recurrence-free survival for all patients was 34.2 months. The number of tumors, the presence of intravesical instillation therapy, and tumor grade were significant prognostic factors for intravesical recurrence in all cases. Groups 2 and 3 showed significantly worse prognosis than group 1 in the multivariate analysis. CONCLUSIONS: Regarding intravesical recurrence, intravesical instillation therapy is necessary for both T1 HG/G3 and T1 HG/G2 bladder cancers.

High-fat diet promotes prostate cancer growth through histamine signaling.

Western high-fat diets (HFD) are regarded as a major risk factor for prostate cancer (PCa). Using prostate-specific Pten-knockout mice as a PCa model, we previously reported that HFD promoted inflammatory PCa growth. The composition of the gut microbiota changes under the influence of diet exert various effects on the host through immunological mechanisms. Herein, we investigated the etiology of HFD-induced inflammatory cancer growth and the involvement of the gut microbiome. The expression of Hdc, the gene responsible for histamine biosynthesis, and histamine levels were upregulated in large prostate tumors of HFD-fed mice, and the number of mast cells increased around the tumor foci. Administration of fexofenadine, a histamine H1 receptor antagonist, suppressed tumor growth in HFD-fed mice by reducing the number of myeloid-derived suppressor cells and suppressing IL6/STAT3 signaling. HFD intake induced gut dysbiosis, resulting in the elevation of serum lipopolysaccharide (LPS) levels. Intraperitoneal injection of LPS increased Hdc expression in PCa. Inhibition of LPS/Toll-like receptor 4 signaling suppressed HFD-induced tumor growth. The number of mast cells increased around the cancer foci in total prostatectomy specimens of severely obese patients. In conclusion, HFD promotes PCa growth through histamine signaling via mast cells. Dietary high-fat induced gut dysbiosis might be involved in the inflammatory cancer growth.

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.

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.

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.

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.

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.

A Potential Mechanism of Anticancer Immune Response Coincident With Immune-related Adverse Events in Patients With Renal Cell Carcinoma.

BACKGROUND/AIM: Some reports showed encouraging efficacy of immune checkpoint inhibitors among patients who experienced immune-related adverse events (irAEs). Thus, characterization of T-cell repertoire and immune signatures in peripheral blood mononuclear cells (PBMCs) and tumors before and after immune checkpoint inhibitors treatment should contribute to better understanding of irAE-provoked anticancer immune responses. MATERIALS AND METHODS: We applied expression analysis of immune-related genes and T-cell receptor sequencing in tumor and PBMCs from five patients with renal cell carcinoma before combined immunotherapy and at the onset of severe irAEs. RESULTS: We found that the cluster of differentiation 8 (CD8)/forkhead box P3(FOXP3), granzyme B(GZMB)/CD3, perforin 1(PRF1)/CD3 and programmed cell death 1(PD1)/CD8 expression ratios were significantly elevated in PBMCs at the onset of irAEs. In addition, we found expansion of certain T-cell clones in metastatic tissue after irAEs, which were already increased in peripheral blood at the onset of irAEs. CONCLUSION: irAE-provoked T-cells may also circulate and attack distant tumors, leading to durable response in patients with irAEs.

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.

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.

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.