Extracellular vesicles (EVs) are microvesicles secreted from various cell types. We aimed to discover a new biomarker for high Gleason score (GS) prostate cancer (PCa) in urinary EVs via quantitative ...proteomics. EVs were isolated from urine after massage from 18 men (negative biopsy n = 6, GS 6 PCa n = 6, or GS 8-9 PCa n = 6). EV proteins were labeled with iTRAQ and analyzed by LC-MS/MS. We identified 4710 proteins and quantified 3528 proteins in the urinary EVs. Eleven proteins increased in patients with PCa compared to those with negative biopsy (ratio >1.5, p-value < 0.05). Eleven proteins were chosen for further analysis and verified in 29 independent urine samples (negative n = 11, PCa n = 18) using selected reaction monitoring/multiple reaction monitoring. Among these candidate markers, fatty acid binding protein 5 (FABP5) was higher in the cancer group than in the negative group (p-value = 0.009) and was significantly associated with GS (p-value for trend = 0.011). Granulin, AMBP, CHMP4A, and CHMP4C were also higher in men with high GS prostate cancer (p-value < 0.05). FABP5 in urinary EVs could be a potential biomarker of high GS PCa.
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.
Proteomic analysis of urinary extracellular vesicles (EVs) is a powerful approach to discovering potential BCa biomarkers, however urine contains numerous EVs derived from kidney and normal urothelial epithelium that could dilute the information of cancer BCa cell‐derived EVs. In this study, we performed combined proteomic analysis of both urinary EVs and tissue‐extracted EVs (Te‐EVs) to identify reliable BCa biomarkers. This novel strategy presented here identified reliable urinary EV biomarker proteins exhibiting high levels of specificity and sensitivity for non‐invasive BCa detection.
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.
TERT promoter and FGFR3 hotspot mutations in urinary cell‐free DNA were analyzed. In combination with urine cytology, the sensitivity was 78.6%, and specificity was 96.0% for upper tract urothelial carcinoma diagnosis.
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.
We evaluated somatic mutations and fragmentation of circulating tumor DNA (ctDNA) using next‐generation sequencing and droplet digital PCR in renal cell carcinoma (RCC). ctDNA can be promising tools for monitoring and predicting prognosis of RCC.
High-fat diet (HFD) could induce prostate cancer progression. The aim of this study is to identify mechanisms of HFD-induced prostate cancer progression, focusing on inflammation.
We administered HFD ...and celecoxib to autochthonous immunocompetent Pb-Cre
;
(fl/fl) model mice for prostate cancer. Tumor growth was evaluated by tumor weight and Ki67 stain, and local immune cells were assessed by flow cytometry at 22 weeks of age. Cytokines which correlated with tumor growth were identified, and the changes of tumor growth and local immune cells after inhibition of the cytokine signals were evaluated in the mice. IHC analyses using prostatectomy specimens of obese patients were performed.
HFD accelerated tumor growth and increased the myeloid-derived suppressor cells (MDSCs) fraction and M2/M1 macrophage ratio in the model mice. Celecoxib-suppressed tumor growth, and decreased both local MDSCs and M2/M1 macrophage ratio in HFD-fed mice. HFD-induced tumor growth was associated with IL6 secreted by prostatic macrophages, as were phosphorylated STAT3 (pSTAT3)-positive tumor cells. Anti-IL6 receptor antibody administration suppressed tumor growth, and decreased local MDSCs and pSTAT3-positive cell fractions in HFD-fed mice. The tumor-infiltrating CD11b-positive cell count was significantly higher in prostatectomy specimens of obese than those of nonobese patients with prostate cancer.
HFD increased MDSCs and accelerated prostate cancer tumor growth via IL6/pSTAT3 signaling in the mice. This mechanism could exist in obese patients with prostate cancer. IL6-mediated inflammation could be a therapeutic target for prostate cancer.
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Extracellular vesicles are lipid bilayer vesicles containing protein, messengerRNA and microRNA. Cancer cell-derived extracellular vesicles may be diagnostic and therapeutic targets. We extracted ...extracellular vesicles from urine of urothelial carcinoma patients and the control group to identify cancer-specific microRNAs in urinary extracellular vesicles as new biomarkers.
microRNA from urinary extracellular vesicles extracted from 6 urothelial carcinoma patients and 3 healthy volunteers was analyzed. We verified candidate microRNAs in an independent cohort of 60 urinary extracellular vesicles samples. To normalize the microRNA expression level in extracellular vesicles, we examined the following in extracellular vesicles: protein concentration, CD9 intensity, amounts of whole miRNAs, RNA U6B small nuclear expression and the creatinine concentration of original urine correlating with the counts of extracted extracellular vesicles measured by the NanoSight™ system.
From the microarray results 5 microRNAs overexpressed in urinary extracellular vesicles of urothelial carcinoma patients were identified. Creatinine concentration of original urine correlated most with particle counts of extracellular vesicles, indicating that creatinine could be a new tool for normalizing microRNA expression. MiR-21-5p was the most potent biomarker in urinary extracellular vesicles (sensitivity, 75.0%; specificity, 95.8%) and was also overexpressed in urinary extracellular vesicles from urothelial carcinoma patients with negative urine cytology. For the subgroup with negative urine cytology, the sensitivity was 75.0% and specificity was 95.8%.
MiR-21-5p in urinary extracellular vesicles could be a new biomarker of urothelial carcinoma, especially for urothelial carcinoma patients with negative urine cytology.
Background
In metastatic renal-cell carcinoma (mRCC), recent clinical trials have shown efficacy of first-line combination therapy, as evidenced by better clinical outcome over target therapy. ...However, there are insufficient real-world evidences in mRCC patients in Japan.
Methods
We performed a multicenter retrospective study of 72 mRCC patients who received nivolumab plus ipilimumab as first-line treatment between September 2018 and July 2021. Patient’s characteristics, clinical outcomes and safety were retrospectively reviewed. We analyzed overall response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) in patients treated with combination therapy.
Results
Of all patients, the median age was 70 years (range, 36–86) and the major type of histology was clear cell RCC (
n
= 55; 76.4%). Progressive disease (
n
= 25; 34.8%) and irAEs (
n
= 22; 30.6%) were the most common causes for discontinuing treatment. Median PFS and OS seemed similar between patients who discontinued treatment because of irAEs and for patients who did not (
p
= 0.360 and
p
= 0.069, respectively). Importantly, for patients with synchronous metastatic disease at diagnosis (
n
= 56), nephrectomy before initiating nivolumab plus ipilimumab had a significantly positive impact on better OS when compared to that in patients without nephrectomy (
p
= 0.028).
Conclusion
This study confirms efficacy and safety of nivolumab plus ipilimumab for mRCC patients in real-world settings. Furthermore, nivolumab plus ipilimumab was associated with a better outcome in patients who had undergone nephrectomy at diagnosis for synchronous mRCC.
There are no blood biomarkers for the diagnosis of renal cell carcinoma (RCC) in routine clinical use. We focused on the gene expression profile of peripheral blood cells obtained from RCC patients ...to discover novel biomarkers for RCC diagnosis. Using microarray analysis and quantitative verification, CXCL7 was shown to be significantly upregulated in the peripheral blood cells of RCC patients. Importantly, aberrant CXCL7 expression was confirmed even in peripheral blood cells obtained from early stage (pT1a) RCC patients, and the expression level of CXCL7 in peripheral blood cells was a potential independent biomarker for the diagnosis of RCC by receiver operating characteristic curve analysis (sensitivity, 70.0%; specificity, 64.0%; area under the curve = 0.722; multiple logistic regression analysis: odds ratio, 1.07; 95% confidence interval, 1.03–1.11; P = 0.0004). Moreover, CXCL7 expression in peripheral blood cells significantly decreased after resection of the primary tumor. CXCL7 is more highly expressed in PBMCs than in neutrophils from both healthy controls and RCC patients. Interestingly, CXCL7 expression in PBMCs from healthy volunteers was significantly elevated following coculture with RCC cells compared to those cocultured with normal cells as a control. These results suggest that aberrant CXCL7 expression in peripheral blood cells is induced by RCC cells and may serve as a novel biomarker in the diagnosis of RCC.
The expression level of CXCL7 in peripheral blood cells was up‐regulated in RCC patients compared to healthy controls.
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.
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