Interobserver reproducibility of cribriform cancer in prostate needle biopsies and validation of International Society of Urological Pathology criteria.

AIMS: There is strong evidence that cribriform morphology indicates a worse prognosis of prostatic adenocarcinoma. Our aim was to investigate its interobserver reproducibility in prostate needle biopsies. METHODS AND RESULTS: A panel of nine prostate pathology experts from five continents independently reviewed 304 digitised biopsies for cribriform cancer according to recent International Society of Urological Pathology criteria. The biopsies were collected from a series of 702 biopsies that were reviewed by one of the panellists for enrichment of high-grade cancer and potentially cribriform structures. A 2/3 consensus diagnosis of cribriform and noncribriform cancer was reached in 90% (272/304) of the biopsies with a mean kappa value of 0.56 (95% confidence interval 0.52-0.61). The prevalence of consensus cribriform cancers was estimated to 4%, 12%, 21%, and 20% of Gleason scores 7 (3 + 4), 7 (4 + 3), 8, and 9-10, respectively. More than two cribriform structures per level or a largest cribriform mass with ≥9 lumina or a diameter of ≥0.5 mm predicted a consensus diagnosis of cribriform cancer in 88% (70/80), 84% (87/103), and 90% (56/62), respectively, and noncribriform cancer in 3% (2/80), 5% (5/103), and 2% (1/62), respectively (all P < 0.01). CONCLUSION: Cribriform prostate cancer was seen in a minority of needle biopsies with high-grade cancer. Stringent diagnostic criteria enabled the identification of cribriform patterns and the generation of a large set of consensus cases for standardisation.

Overexpression of miR-17-5p may negatively impact p300/CBP factor-associated inflammation in a hypercholesterolemic advanced prostate cancer model.

BACKGROUND: Previously, we demonstrated that cholesterol triggers the increase in p300/CBP-associated factor (PCAF), targeted by miR-17-5p. The p300, IL-6, PCAF, and miR-17-5p genes have important and contradictory roles in inflammation and prostate cancer (PCa). This study aimed to demonstrate the potential anti-inflammatory effect of miR-17-5 in an advanced PCa model with diet-induced hypercholesterolemia. METHODS AND RESULTS: In vitro, using the PC-3 cell line, we show that induction of miR-17-5p reduces p300 and PCAF expression, increases apoptosis, and decreases cell migration. Furthermore, we demonstrate that supplementing this same cell with cholesterol (2 µg/mL) triggers increased p300, IL-6, and PCAF. In vivo, after establishing the hypercholesterolemic (HCOL) model, xenografts were treated with miR-17-5p. Increased expression of this miR after intratumoral injections attenuated tumor growth in the control and HCOL animals and reduced cell proliferation. CONCLUSION: Our results demonstrate that inducing miR-17-5p expression suppresses tumor growth and inflammatory mediator expression. Further studies should be conducted to fully explore the role of miR-17-5p and the involvement of inflammatory mediators p300, PCAF, and IL-6.

The Complexity and Dynamics of the Tissue Glycoproteome Associated With Prostate Cancer Progression.

The complexity and dynamics of the immensely heterogeneous glycoproteome of the prostate cancer (PCa) tumor microenvironment remain incompletely mapped, a knowledge gap that impedes our molecular-level understanding of the disease. To this end, we have used sensitive glycomics and glycoproteomics to map the protein-, cell-, and tumor grade-specific N- and O-glycosylation in surgically removed PCa tissues spanning five histological grades (n = 10/grade) and tissues from patients with benign prostatic hyperplasia (n = 5). Quantitative glycomics revealed PCa grade-specific alterations of the oligomannosidic-, paucimannosidic-, and branched sialylated complex-type N-glycans, and dynamic remodeling of the sialylated core 1- and core 2-type O-glycome. Deep quantitative glycoproteomics identified ∼7400 unique N-glycopeptides from 500 N-glycoproteins and ∼500 unique O-glycopeptides from nearly 200 O-glycoproteins. With reference to a recent Tissue and Blood Atlas, our data indicate that paucimannosidic glycans of the PCa tissues arise mainly from immune cell-derived glycoproteins. Furthermore, the grade-specific PCa glycosylation arises primarily from dynamics in the cellular makeup of the PCa tumor microenvironment across grades involving increased oligomannosylation of prostate-derived glycoproteins and decreased bisecting GlcNAcylation of N-glycans carried by the extracellular matrix proteins. Furthermore, elevated expression of several oligosaccharyltransferase subunits and enhanced N-glycoprotein site occupancy were observed associated with PCa progression. Finally, correlations between the protein-specific glycosylation and PCa progression were observed including increased site-specific core 2-type O-glycosylation of collagen VI. In conclusion, integrated glycomics and glycoproteomics have enabled new insight into the complexity and dynamics of the tissue glycoproteome associated with PCa progression generating an important resource to explore the underpinning disease mechanisms.

Intratumoral Restoration of miR-137 Plus Cholesterol Favors Homeostasis of the miR-137/Coactivator p160/AR Axis and Negatively Modulates Tumor Progression in Advanced Prostate Cancer.

MicroRNAs (miRNAs) have gained a prominent role as biomarkers in prostate cancer (PCa). Our study aimed to evaluate the potential suppressive effect of miR-137 in a model of advanced PCa with and without diet-induced hypercholesterolemia. In vitro, PC-3 cells were treated with 50 pmol of mimic miR-137 for 24 h, and gene and protein expression levels of SRC-1, SRC-2, SRC-3, and AR were evaluated by qPCR and immunofluorescence. We also assessed migration rate, invasion, colony-forming ability, and flow cytometry assays (apoptosis and cell cycle) after 24 h of miRNA treatment. For in vivo experiments, 16 male NOD/SCID mice were used to evaluate the effect of restoring miR-137 expression together with cholesterol. The animals were fed a standard (SD) or hypercholesterolemic (HCOL) diet for 21 days. After this, we xenografted PC-3 LUC-MC6 cells into their subcutaneous tissue. Tumor volume and bioluminescence intensity were measured weekly. After the tumors reached 50 mm3, we started intratumor treatments with a miR-137 mimic, at a dose of 6 µg weekly for four weeks. Ultimately, the animals were killed, and the xenografts were resected and analyzed for gene and protein expression. The animals' serum was collected to evaluate the lipid profile. The in vitro results showed that miR-137 could inhibit the transcription and translation of the p160 family, SRC-1, SRC-2, and SRC-3, and indirectly reduce the expression of AR. After these analyses, it was determined that increased miR-137 inhibits cell migration and invasion and impacts reduced proliferation and increased apoptosis rates. The in vivo results demonstrated that tumor growth was arrested after the intratumoral restoration of miR-137, and proliferation levels were reduced in the SD and HCOL groups. Interestingly, the tumor growth retention response was more significant in the HCOL group. We conclude that miR-137 is a potential therapeutic miRNA that, in association with androgen precursors, can restore and reinstate the AR-mediated axis of transcription and transactivation of androgenic pathway homeostasis. Further studies involving the miR-137/coregulator/AR/cholesterol axis should be conducted to evaluate this miR in a clinical context.

The Effect of Gene Editing by CRISPR-Cas9 of miR-21 and the Indirect Target MMP9 in Metastatic Prostate Cancer.

Prostate cancer (PCa) has a high prevalence and represents an important health problem, with an increased risk of metastasis. With the advance of CRISPR-Cas9 genome editing, new possibilities have been created for investigating PCa. The technique is effective in knockout oncogenes, reducing tumor resistance. MMP9 and miR-21 target genes are associated with PCa progression; therefore, we evaluated the MMP-9 and miR-21 targets in PCa using the CRISPR-Cas9 system. Single guide RNAs (sgRNAs) of MMP9 and miR-21 sequences were inserted into a PX-330 plasmid, and transfected in DU145 and PC-3 PCa cell lines. MMP9 and RECK expression was assessed by qPCR, WB, and IF. The miR-21 targets, integrins, BAX and mTOR, were evaluated by qPCR. Flow cytometry was performed with Annexin5, 7-AAD and Ki67 markers. Invasion assays were performed with Matrigel. The miR-21 CRISPR-Cas9-edited cells upregulated RECK, MARCKS, BTG2, and PDCD4. CDH1, ITGB3 and ITGB1 were increased in MMP9 and miR-21 CRISPR-Cas9-edited cells. Increased BAX and decreased mTOR were observed in MMP9 and miR-21 CRISPR-Cas9-edited cells. Reduced cell proliferation, increased apoptosis and low invasion in MMP9 and miR-21 edited cells was observed, compared to Scramble. CRISPR-Cas9-edited cells of miR-21 and MMP9 attenuate cell proliferation, invasion and stimulate apoptosis, impeding PCa evolution.

Cholesterol Triggers Nuclear Co-Association of Androgen Receptor, p160 Steroid Coactivators, and p300/CBP-Associated Factor Leading to Androgenic Axis Transactivation in Castration-Resistant Prostate Cancer.

BACKGROUND/AIMS: Cholesterol modulates intratumoral androgenic signaling in prostate cancer; however, the molecular mechanisms underlying these changes in castration-resistant prostate cancer (CRPC) are not fully elucidated. Herein, we investigated the effect of cholesterol on androgen receptor (AR) coactivators expression and tumorigenesis in vitro and in vivo. METHODS: Herein, we monitored the expression of AR coactivators (SRC-1, 2, 3 and PCAF) genes in PC-3 cells exposed to 2µg/mL of cholesterol for 8 hours by qPCR. We also performed cell migration at 0, 8, 24, 48 and 72h and flow cytometry assays (viability, apoptosis, and cell cycle) after a 24h exposure. Immunofluorescence assay was performed to evaluate the protein expression of the AR coactivators. Additionally, in vivo experiments were conducted using 22 male NOD/SCID mice. Mice were fed a standard (Control) or hypercholesterolemic (HCOL) diet for 21 days and then subcutaneously implanted with PC-3 cells. The tumor volume was calculated every two days, and after four weeks, the tumors were resected, weighed, and the serum lipid profile was measured. We also measured the intratumoral lipid profile and AR coactivators gene and protein expression by qPCR and Western Blot, respectively. Intratumor testosterone and dihydrotestosterone (DHT) concentrations were determined using ELISA. RESULTS: Cholesterol up-regulated the gene expression of coactivators SRC-1, SRC-2, SRC-3and PCAF, increasing AR expression in PC-3 cells. Next, cholesterol-supplemented PC-3 cells exhibited increased cell migration and altered cell cycle phases, leading to changes in proliferation and reduced apoptosis. We found that SRC-1, SRC-2, SRC-3 and PCAF proteins co-localized in the nucleus of cholesterol-supplemented cells and co-associate with AR. In the in vivo model, the hypercholesterolemic (HCOL) group displayed higher serum total and intratumoral cholesterol levels, increased testosterone and dihydrotestosterone concentrations, and up-regulated AR coactivator expression. The tumor volume of the HCOL group was significantly higher than the control group. CONCLUSION: Our findings revealed that increased nuclear translocation of the coactivators leads to up-regulated AR gene and protein expression, potentially influencing tumor progression. Studies targeting cholesterol-modulated changes in AR coactivator expression may provide insights into the molecular mechanisms associated with the CRPC phenotype.

miRNA and mRNA Expression Profiles Associated with Lymph Node Metastasis and Prognosis in Penile Carcinoma.

Penile cancer (PeC) is a rare disease, and no prognostic biomarkers have been adopted in clinical practice yet. The objective of the present study was to identify differentially expressed miRNAs (DEmiRs) and genes (DEGs) as potential biomarkers for lymph node metastasis and other prognostic factors in PeC. Tumor samples were prospectively obtained from 24 patients with squamous cell carcinoma of the penis. miRNA microarray analysis was performed comparing tumors from patients with inguinal lymph node metastatic and localized disease, and the results were validated by qRT-PCR. Eighty-three gene expression levels were also compared between groups through qRT-PCR. Moreover, DEmiRs and DEGs expression levels were correlated with clinicopathological variables, cancer-specific (CSS), and overall survival (OS). TAC software, TM4 MeV 4.9 software, SPSS v.25.0, and R software v.4.0.2 were used for statistical analyses. We identified 21 DEmiRs in microarray analysis, and seven were selected for validation. miR-744-5p and miR-421 were overexpressed in tissue samples of metastatic patients, and high expression of miR-421 was also associated with lower OS. We found seven DEGs (CCND1, EGFR, ENTPD5, HOXA10, IGF1R, MYC, and SNAI2) related to metastatic disease. A significant association was found between increased MMP1 expression and tumor size, grade, pathological T stage, and perineural invasion. Other genes were also associated with clinicopathological variables, CSS and OS. Finally, we found changes in mRNA-miRNA regulation that contribute to understanding the mechanisms involved in tumor progression. Therefore, we identified miRNA and mRNA expression profiles as potential biomarkers associated with lymph node metastasis and prognosis in PeC, in addition to disruption in mRNA-miRNA regulation during disease progression.

Prospective evaluation of a urinary biomarker panel to detect and predict recurrence of non-muscle-invasive bladder cancer.

PURPOSE: To perform a feasibility phase study of a panel of putative protein biomarkers and determine whether it can identify and predict tumor recurrence in patients with non-muscle-invasive bladder cancer (NMIBC) on follow-up. METHODS: We prospectively analyzed the urine of 152 patients previously treated for NMIBC. Quantitative expression of plasminogen activator inhibitor-1 (PAI-1), DJ-1, apolipoprotein A-I (apoA-1), matrix metallopeptidase-9 (MMP-9), and interleukin-8 (IL-8) was assessed by enzyme-linked immunosorbent assay and compared amongst patients with and without bladder cancer recurrence at urine collection and during 3 years of follow-up. Tumor recurrence was confirmed by pathologic analysis. We performed a prediction analysis, excluding patients with recurrence at the start of the study, and assessed the influence of previous use of intravesical BCG on the level of biomarkers. RESULTS: Median follow-up time was 47 months (interquartile range 39-50 months). Sixteen patients (10.5%) were diagnosed with recurrence at the start of the study, and 21 (15.4%) were diagnosed during the study. Three biomarker proteins (apoA-1, MMP-9, and IL-8) appear to hold diagnostic potential [odds ratio (OR) = 12.9; 95% CI 3.5-47.4]; while, PAI-1 and IL-8 predict recurrence (OR = 4.1; 95% CI 1.4-11.4). Previous use of intravesical BCG did not affect biomarker levels. CONCLUSION: In the feasibility phase, the panel of urine biomarkers analyzed detected and predicted recurrence of NMIBC and provided reliable results in patients who had previously used intravesical BCG. Validation studies are required to confirm the panel clinical utility.

Can increased expression of miR-Let-7c reduce the transition potential of high-grade urothelial carcinoma?

BACKGROUND: Bladder cancer is the leading transitional cell carcinoma affecting men and women with high morbidity and mortality rates, justifying the need to develop new molecular target therapies using microRNAs. This study aimed to evaluate the behavior of the T24 cell line after transfection with miR-Let-7c precursor mimic through invasion, migration, apoptosis, and cell cycle assays. METHODS AND RESULTS: T24 cell was transfected with the Let-7c mimic and its respective control and evaluated after 24 h. The expression levels of miR-Let-7c were analyzed by qPCR. We performed wound healing, Matrigel and flow cytometry, apoptosis, and cell cycle assays to determine its effect on cellular processes. Cells transfected with miR-Let-7c showed increased apoptosis rates (p = 0.019), decreased migration 24 h (p = 0.031) and 48 h (p = 0.0006), invasion potential (p = 0.0007), and cell proliferation (p = 0.002). CONCLUSIONS: Our results demonstrate that miR-Let-7c can act in different pathways of the carcinogenic cellular processes of muscle-invasive urothelial carcinoma cells, inhibiting cell proliferation and increasing apoptosis levels, consequently limiting their invasion potential. However, further studies should be carried out better to elucidate this microRNA's role in high-grade urothelial carcinomas and unveil which targets this microRNA may present, which are intrinsically related to the cancer survival pathways.

Artificial intelligence for diagnosis and grading of prostate cancer in biopsies: a population-based, diagnostic study.

BACKGROUND: An increasing volume of prostate biopsies and a worldwide shortage of urological pathologists puts a strain on pathology departments. Additionally, the high intra-observer and inter-observer variability in grading can result in overtreatment and undertreatment of prostate cancer. To alleviate these problems, we aimed to develop an artificial intelligence (AI) system with clinically acceptable accuracy for prostate cancer detection, localisation, and Gleason grading. METHODS: We digitised 6682 slides from needle core biopsies from 976 randomly selected participants aged 50-69 in the Swedish prospective and population-based STHLM3 diagnostic study done between May 28, 2012, and Dec 30, 2014 (ISRCTN84445406), and another 271 from 93 men from outside the study. The resulting images were used to train deep neural networks for assessment of prostate biopsies. The networks were evaluated by predicting the presence, extent, and Gleason grade of malignant tissue for an independent test dataset comprising 1631 biopsies from 246 men from STHLM3 and an external validation dataset of 330 biopsies from 73 men. We also evaluated grading performance on 87 biopsies individually graded by 23 experienced urological pathologists from the International Society of Urological Pathology. We assessed discriminatory performance by receiver operating characteristics and tumour extent predictions by correlating predicted cancer length against measurements by the reporting pathologist. We quantified the concordance between grades assigned by the AI system and the expert urological pathologists using Cohen's kappa. FINDINGS: The AI achieved an area under the receiver operating characteristics curve of 0·997 (95% CI 0·994-0·999) for distinguishing between benign (n=910) and malignant (n=721) biopsy cores on the independent test dataset and 0·986 (0·972-0·996) on the external validation dataset (benign n=108, malignant n=222). The correlation between cancer length predicted by the AI and assigned by the reporting pathologist was 0·96 (95% CI 0·95-0·97) for the independent test dataset and 0·87 (0·84-0·90) for the external validation dataset. For assigning Gleason grades, the AI achieved a mean pairwise kappa of 0·62, which was within the range of the corresponding values for the expert pathologists (0·60-0·73). INTERPRETATION: An AI system can be trained to detect and grade cancer in prostate needle biopsy samples at a ranking comparable to that of international experts in prostate pathology. Clinical application could reduce pathology workload by reducing the assessment of benign biopsies and by automating the task of measuring cancer length in positive biopsy cores. An AI system with expert-level grading performance might contribute a second opinion, aid in standardising grading, and provide pathology expertise in parts of the world where it does not exist. FUNDING: Swedish Research Council, Swedish Cancer Society, Swedish eScience Research Center, EIT Health.

Tissue Proteome Signatures Associated with Five Grades of Prostate Cancer and Benign Prostatic Hyperplasia.

The histology-based Gleason score (GS) of prostate cancer (PCa) tissue biopsy is the most accurate predictor of disease aggressiveness and an important measure to guide treatment strategies and patient management. The variability associated with PCa tumor sampling and the subjective determination of the GS are challenges that limit accurate diagnostication and prognostication. Thus, novel molecular signatures are needed to distinguish between indolent and aggressive forms of PCa for better patient management and outcomes. Herein, label-free LC-MS/MS proteomics is used to profile the proteome of 50 PCa tissues spanning five grade groups (n = 10 per group) relative to tissues from individuals with benign prostatic hyperplasia (BPH). Over 2000 proteins are identified albeit at different levels between and within the patient groups, revealing biological processes associated with specific grades. A panel of 11 prostate-derived proteins including IGKV3D-20, RNASET2, TACC2, ANXA7, LMOD1, PRCP, GYG1, NDUFV1, H1FX, APOBEC3C, and CTSZ display the potential to stratify patients from low and high PCa grade groups. Parallel reaction monitoring of the same sample cohort validate the differential expression of LMOD1, GYG1, IGKV3D-20, and RNASET2. The four proteins associated with low and high PCa grades reported here warrant further exploration as candidate biomarkers for PCa aggressiveness.

Protein Paucimannosylation Is an Enriched N-Glycosylation Signature of Human Cancers.

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man1-3 GlcNAc2 Fuc0-1 ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man2-3 GlcNAc2 Fuc1 , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-ß-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.

Polymorphism in the PBX1 gene is related to cystinuria in Brazilian families.

The aim of our study was to determine regions of loss of heterozygosity, copy number variation analysis, and single nucleotide polymorphisms (SNPs) in Brazilian patients with cystinuria. A linkage study was performed using DNA samples from six patients with cystinuria and six healthy individuals. Genotyping was done with the Genome-Wide Human SNP 6.0 arrays (Affymetrix, Inc., Santa Clara, CA, USA). For validation, SNPs were genotyped using a TaqMan® SNP Genotyping Assay Kit. The homozygote polymorphic genotype of SNP rs17383719 in the gene PBX1 was more frequent (P = 0.015) in cystinuric patients. The presence of the polymorphic allele for this SNP increased the chance of cystinuria by 3.0-fold (P = 0.036). Pre-B-cell leukaemia transcription factor 1 (PBX1) was overexpressed 3.3-fold in patients with cystinuria. However, when we compared the gene expression findings with the genotyping, patients with a polymorphic homozygote genotype had underexpression of PBX1, while patients with a heterozygote or wild-type homozygote genotype had overexpression of PBX1. There is a 3-fold increase in the risk of the development of cystinuria among individuals with this particular SNP in the PBX1 gene. We postulate that the presence of this SNP alters the expression of PBX1, thus affecting the renal absorption of cystine and other amino acids, predisposing to nephrolithiasis.

Duplex scan and histologic assessment of acute renal injury in a kidney-kidney crosstalk swine experimental model.

OBJECTIVE: The objective of this study was to identify the effect of two left renal vasculature occlusion strategies on the duplex ultrasound-assessed rheology and histology of the contralateral kidney. METHODS: Pigs were randomly assigned to one of two groups: left renal artery-only clamping (A group, n = 8) or left renal artery and vein clamping (AV group, n = 9). Bilateral renal parenchymal biopsy specimens were taken every 10 minutes for 90 minutes. Duplex ultrasound resistive index (RI) and pulsatility index (PI) were measured. Mixed models with normal distribution and first-order autoregressive correlation structure and generalized estimating equation models were used. Results are presented as adjusted means with standard errors, estimated proportions with standard errors, and line plots with 95% confidence intervals. RESULTS: RI and PI increased in the nonischemic kidney. In A group animals, RI values increased significantly (P < .01) after 30 minutes of ischemia and PI increased significantly (P < .04) from 30 to 60 minutes of ischemia. The number of histologic abnormalities was higher in A group than in AV group biopsy specimens. The percentage of lesions increased significantly after 10 minutes in A group nonischemic kidneys (P < .02) and between 50 and 80 minutes in AV group nonischemic kidneys (P < .01). CONCLUSIONS: Nonischemic kidneys were acutely affected by contralateral ischemia. Their function was more adversely affected by unilateral renal artery occlusion with preserved renal vein patency (A group).

Utility of Pathology Imagebase for standardisation of prostate cancer grading.

AIMS: Despite efforts to standardise grading of prostate cancer, even among experts there is still a considerable variation in grading practices. In this study we describe the use of Pathology Imagebase, a novel reference image library, for setting an international standard in prostate cancer grading. METHODS AND RESULTS: The International Society of Urological Pathology (ISUP) recently launched a reference image database supervised by experts. A panel of 24 international experts in prostate pathology reviewed independently microphotographs of 90 cases of prostate needle biopsies with cancer. A linear weighted kappa of 0.67 (95% confidence interval = 0.62-0.72) and consensus was reached in 50 cases. The interobserver weighted kappa varied from 0.48 to 0.89. The highest level of agreement was seen for Gleason score (GS) 3 + 3 = 6 (ISUP grade 1), while higher grades and particularly GS 4 + 3 = 7 (ISUP grade 3) showed considerable disagreement. Once a two-thirds majority was reached, images were moved automatically into a public database available for all ISUP members at www.isupweb.org. Non-members are able to access a limited number of cases. CONCLUSIONS: It is anticipated that the database will assist pathologists to calibrate their grading and, hence, decrease interobserver variability. It will also help to identify instances where definitions of grades need to be clarified.

miR-29b enhances prostate cancer cell invasion independently of MMP-2 expression.

BACKGROUND: The ability to metastasize is one of the most important characteristics of neoplastic cells. An imbalance between the action of some matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs drives the invasion process. Some studies have suggested that MMP-2 is involved in metastasis, while other studies have reported that collagen production by cancer cells might also contribute to motility. However, decreased expression of microRNA-29b (miR-29b), which may control MMP-2 and collagen gene expression, has been shown in prostate cancer (PCa). The objectives of the present study were to clarify whether MMP-2 as well as collagens I and III (encoded by COL1A1 and COL3A1, respectively) are controlled by miR-29b and to determine whether metastasis is altered by this relationship. METHODS: PCa DU145 and PC-3 cells were transfected with 100 µL of OPTI-MEM I containing 100 nmol of miR-29b (or its inhibitor) along with 1.5 µL of lipofectamine. Positive and negative controls were prepared using the same protocol. MMP-2, COL1A1 and COL3A1 messenger RNA (mRNA) levels were evaluated via real-time polymerase chain reaction (qRT-PCR). For qRT-PCR, 6 × 104 cells were used. Invasion studies were conducted with Matrigel assays, which simulate invasion of the extracellular matrix by neoplastic cells. After transfection of 3 × 104 cells, invasion was allowed to proceed for 48 h. Invasive cells were counted under an optical microscope. Each experiment was performed in triplicate. RESULTS: MMP-2 mRNA was not expressed in DU145 cells after transfection with miR-29b. After transfection of cells with the miR-29b inhibitor, COL1A1 (p = 0.02) and COL3A1 (p = 0.06) mRNA expression was increased in DU145 cells, and a large number of transfected DU145 and PC3 cells invaded the Matrigel membrane. CONCLUSIONS: In vitro studies showed that reducing the amount of miR-29b may lead to higher PCa cell invasion via a process that is independent of MMP-2. Collagen expression, controlled by miR-29b, may facilitate this motility process. Thus, the present study suggests that collagen production plays an active role in metastasis control and restoration of miR-29b levels may decrease metastasis. Altogether, these findings support further exploration of drug therapy targeting this aspect of the metastasis circuit.

miR-618: possible control over TIMP-1 and its expression in localized prostate cancer.

BACKGROUND: The imbalance between the action of the tissue inhibitors of matrix metalloproteinases (TIMPs) and the matrix metalloproteinases (MMPs) is one component of metastasis physiology. TIMP-1 overrides MMP-9 activity in cancer and might be regulated by miR-618. The aims of this study were to clarify whether TIMP-1 expression is modified by miR-618 and to clarify the effect of miR-618 expression on the invasion of prostate cancer cells. We also studied miR-618 expression in surgical specimens of patients with localized prostate cancer submitted to open radical prostatectomy. METHODS: After transfection of miR-618 or its antagonist in DU145 cells, qRT-PCR for TIMP-1/MMP-9 and both ELISA and zymography for MMP-9 were performed. Total miRNA was extracted from surgical specimens of PCa, and miR-618 expression was examined for correlations with Gleason score, pathological status and biochemical recurrence. RESULTS: DU145 cells transfected with miR-618 had a 76% reduction in TIMP-1 expression relative to control cells (p = 0.003). miR-618 inhibition reduced MMP-9 expression by 31% (p = 0.032) and MMP-9 absorbance evaluated with ELISA assay (p = 0.06).Zymography suggested higher MMP-9 activity in DU145 cells transfected with miR-618 than those transfected with miR-618 inhibitor, but the difference was not significant (p = 0.55). However, miR-618 expression was lower in surgical specimens of patients with Gleason score > 7 (p = 0.08) and more advanced disease (p = 0.07). CONCLUSIONS: In vitro, miR-618 overexpression decreases TIMP-1 and miR-618 inhibition decreases MMP-9, suggesting that miR-618 might be an oncomiR. However, the analysis of clinical samples of localized prostate cancer revealed an inconsistent pattern, as increased miR-618 expression was associated with lower Gleason score and pathological status. Further studies are needed to address whether miR-618 is a context-dependent miRNA.

Pathology Imagebase-a reference image database for standardization of pathology.

AIMS: Despite efforts to standardize histopathology practice through the development of guidelines, the interpretation of morphology is still hampered by subjectivity. We here describe Pathology Imagebase, a novel mechanism for establishing an international standard for the interpretation of pathology specimens. METHODS AND RESULTS: The International Society of Urological Pathology (ISUP) established a reference image database through the input of experts in the field. Three panels were formed, one each for prostate, urinary bladder and renal pathology, consisting of 24 international experts. Each of the panel members uploaded microphotographs of cases into a non-public database. The remaining 23 experts were asked to vote from a multiple-choice menu. Prior to and while voting, panel members were unable to access the results of voting by the other experts. When a consensus level of at least two-thirds or 16 votes was reached, cases were automatically transferred to the main database. Consensus was reached in a total of 287 cases across five projects on the grading of prostate, bladder and renal cancer and the classification of renal tumours and flat lesions of the bladder. The full database is available to all ISUP members at www.isupweb.org. Non-members may access a selected number of cases. CONCLUSIONS: It is anticipated that the database will assist pathologists in calibrating their grading, and will also promote consistency in the diagnosis of difficult cases.

The role of urinary KIM-1, NGAL, CA19-9 and ß2-microglobulin in the assessment of ureteropelvic junction obstruction in adults.

PURPOSE: The objective of this study is to evaluate the diagnostic properties of urinary biomarkers in adults with ureteropelvic junction obstruction: KIM-1, NGAL, CA19-9, and ß2-microglobulin. We also assessed urinary biomarker concentrations following pyeloplasty. MATERIAL AND METHODS: We prospectively studied adults from December 2013 to February 2015. We included 47 patients with a mean age of 38.6 ± 12.7 years. Each patient provided four samples of voided urine for biomarker measurement, one at pre-operative consultation and the others at 1, 3, and 6 months of post-operative follow-up. The control group consisted of 40 healthy individuals with no hydronephrosis on ultrasound evaluation. RESULTS: KIM-1 had an area under the curve of 0.79 (95% CI 0.70-0.89), NGAL 0.71 (95% CI 0.61-0.83), CA19-9 0.70 (95% CI 0.60-0.81), and ß2-microgloblin 0.61 (95% CI 0.50-0.73). KIM-1 was the most sensitive marker with a cut-off of 170.4 pg/mg creatinine (sensitivity 91.4%, specificity 59.1%), whereas CA19-9 was the most specific with a cut-off of 51.3 U/mg creatinine (sensitivity 48.9%, specificity 88.0%). Urinary concentrations of biomarkers decreased after pyeloplasty. CONCLUSIONS: The evaluation of urinary biomarkers is useful in adults undergoing pyeloplasty. KIM-1, NGAL, and CA19-9 were elevated and significantly decreased after surgery.