Frequent Telomerase Reverse Transcriptase Promoter and Fibroblast Growth Factor Receptor 3 Mutations Support the Precursor Nature of Papillary Urothelial Hyperplasia of the Urinary Bladder.

The precursor nature of papillary urothelial hyperplasia of the urinary bladder is uncertain. In this study, we investigated the telomerase reverse transcriptase (TERT) promoter and fibroblast growth factor receptor 3 (FGFR3) mutations in 82 patients with papillary urothelial hyperplasia lesions. Thirty-eight patients presented with papillary urothelial hyperplasia and concurrent noninvasive papillary urothelial carcinoma, and 44 patients presented with de novo papillary urothelial hyperplasia. The prevalence of the TERT promoter and FGFR3 mutations is compared between de novo papillary urothelial hyperplasia and those with concurrent papillary urothelial carcinoma. Mutational concordance between papillary urothelial hyperplasia and concurrent carcinoma was also compared. The TERT promoter mutations were detected in 44% (36/82) of papillary urothelial hyperplasia, including 23 (23/38, 61%) papillary urothelial hyperplasia with urothelial carcinoma and 13 (13/44, 29%) de novo papillary urothelial hyperplasia. The overall concordance of TERT promoter mutation status between papillary urothelial hyperplasia and concurrent urothelial carcinoma was 76%. The overall FGFR3 mutation rate of papillary urothelial hyperplasia was 23% (19/82). FGFR3 mutations were detected in 11 patients with papillary urothelial hyperplasia and concurrent urothelial carcinoma (11/38, 29%) and 8 patients with de novo papillary urothelial hyperplasia (8/44, 18%). Identical FGFR3 mutation status was detected in both papillary urothelial hyperplasia and urothelial carcinoma components in all 11 patients with FGFR3 mutations. Our findings provide strong evidence of a genetic association between papillary urothelial hyperplasia and urothelial carcinoma. High frequency of TERT promoter and FGFR3 mutations suggests the precursor role of papillary urothelial hyperplasia in urothelial carcinogenesis.

Comparison of Morphological and Histological Characteristics of Human and Sheep: Sheep as a Potential Model for Testing Midurethral Slings in vivo.

INTRODUCTION: The sheep was evaluated as a potential model for preclinical evaluation of urethral slings in vivo based on: (1) anatomical measurements of the sheep vagina and (2) histological tissue integration and host response to polypropylene (PP) slings. METHODS: Eight female, multiparous sheep were utilized. Three of 8 animals underwent surgery mimicking human tension-free vaginal tape protocols for midurethral slings and were euthanized at 6 months. The following measurements were obtained: vaginal length, maximum vaginal width with retraction, symphysis pubis length, and distance from the pubic bone to incision. Explanted sling samples from sheep and human were stained with hematoxylin and eosin for host reaction assessment. RESULTS: Geometric measurements were similar between humans and sheep. Sheep vaginal anatomy allowed sling placement similar to procedures in human surgeries, and all sheep recovered without problems. Comparative histology between the sheep and human indicated similar host reaction and collagen deposition around implants, confirming suitability of the sheep model for biomaterial response assessment. CONCLUSION: Sheep vaginal length is comparable to humans. Tissue integration and host response to PP slings showed chronic inflammation with rich collagen deposition around the material in both sheep and human specimens, highlighting the sheep as a potential animal model for preclinical testing of midurethral slings.

Development of a novel castration-resistant orthotopic prostate cancer model in New Zealand White rabbit.

BACKGROUND: Prostate cancer (PCa) models in mice and rats are limited by their size and lack of a clearly delineated or easily accessible prostate gland. The canine PCa model is currently the only large animal model which can be used to test new preclinical interventions but is costly and availability is sparse. As an alternative, we developed an orthotopic human prostate tumor model in an immunosuppressed New Zealand White rabbit. Rabbits are phylogenetically closer to humans, their prostate gland is anatomically similar, and its size allows for clinically-relevant testing of interventions. METHODS: Rabbits were immunosuppressed via injection of cyclosporine. Human PC3pipGFP PCa cells were injected into the prostate via either (a) laparotomy or (b) transabdominal ultrasound (US) guided injection. Tumor growth was monitored using US and magnetic resonance imaging (MRI). Contrast-enhanced ultrasound (CEUS) imaging using nanobubbles and Lumason microbubbles was also performed to examine imaging features and determine the optimal contrast dose required for enhanced visualization of the tumor. Ex vivo fluorescence imaging, histopathology, and immunohistochemistry analyses of the collected tissues were performed to validate tumor morphology and prostate-specific membrane antigen (PSMA) expression. RESULTS: Immunosuppression and tumor growth were, in general, well-tolerated by the rabbits. Fourteen out of 20 rabbits, with an average age of 8 months, successfully grew detectable tumors from Day 14 onwards after cell injection. The tumor growth rate was 39 ± 25 mm2 per week. CEUS and MRI of tumors appear hypoechoic and T2 hypointense, respectively, relative to normal prostate tissue. Minimally invasive US-guided tumor cell injection proved to be a better method compared to laparotomy due to the shorter recovery time required for the rabbits following injection. Among the rabbits that grew tumors, seven had tumors both inside and outside the prostate, three had tumors only inside the prostate, and four had tumors exclusively outside of the prostate. All tumors expressed the PSMA receptor. CONCLUSIONS: We have established, for the first time, an orthotopic PCa rabbit model via percutaneous US-guided tumor cell inoculation. This animal model is an attractive, clinically relevant intermediate step to assess preclinical diagnostic and therapeutic compounds.

Role of class I histone deacetylases in the regulation of maspin expression in prostate cancer.

Maspin repression is frequently observed in prostate cancer; however, the molecular mechanism(s) causing the loss is not completely understood. Here, we demonstrate that inhibition of class I histone deacetylases (HDACs) mediates re-expression of maspin which plays an essential role in suppressing proliferation and migration capability in prostate cancer cells. Human prostate cancer LNCaP and DU145 cells treated with HDAC inhibitors, sodium butyrate, and trichostatin A, resulted in maspin re-expression. Interestingly, an exploration into the molecular mechanisms demonstrates that maspin repression in prostate tumor and human prostate cancer cell lines occurs via epigenetic silencing through an increase in HDAC activity/expression, independent of promoter DNA hypermethylation. Furthermore, transcriptional activation of maspin was accompanied with the suppression of HDAC1 and HDAC8 with significant p53 enrichment at the maspin promoter associated with an increase in histone H3/H4 acetylation. Our results provide evidence of maspin induction as a critical epigenetic event altered by class I HDACs in the restoration of balance to delay proliferation and migration ability of prostate cancer cells.

Distinct mutational landscape of inverted urothelial papilloma.

A recent study has identified gene mutations involving the MAPK/ERK pathway, particularly the HRAS gene, in all inverted urothelial papillomas (IUPs), in the absence of pathway mutations in TERT promoter, FGFR3, and TP53/RB1genes. Neither recurrence nor progression was observed in IUPs. These data support several longstanding hypotheses: (1) IUPs are benign and do not recur or progress; (2) they harbor mutations that are different from those of urothelial carcinoma; and (3) they arise through different molecular mechanisms than low- or high-grade urothelial carcinoma. As the most critical differential diagnosis in this context is inverted-type urothelial carcinoma, more comprehensive studies are needed to compare and contrast these entities. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Androgen Deprivation Induces Transcriptional Reprogramming in Prostate Cancer Cells to Develop Stem Cell-Like Characteristics.

Enzalutamide, an antiandrogen, is approved for therapy of castration resistant prostate cancer. Clinical applications have shown that approximately 30% of patients acquire resistance after a short period of treatment. However, the molecular mechanisms underlying this resistance is not completely understood. To identify transcriptomic signatures associated with acquisition of drug resistance we profiled gene expression of paired enzalutamide sensitive and resistant human prostate cancer LNCaP (lymph node carcinoma of the prostate) and C4-2B cells. Overlapping genes differentially regulated in the enzalutamide resistant cells were ranked by Ingenuity Pathway Analysis and their functional validation was performed using ingenuity knowledge database followed by confirmation to correlate transcript with protein expression. Analysis revealed that genes associated with cancer stem cells, such as POU5F1 (OCT4), SOX2, NANOG, BMI1, BMP2, CD44, SOX9, and ALDH1 were markedly upregulated in enzalutamide resistant cells. Amongst the pathways enriched in the enzalutamide-resistant cells were those associated with RUNX2, hedgehog, integrin signaling, and molecules associated with elastic fibers. Further examination of a patient cohort undergoing ADT and its comparison with no-ADT group demonstrated high expression of POU5F1 (OCT4), ALDH1, and SOX2 in ADT specimens, suggesting that they may be clinically relevant therapeutic targets. Altogether, our approach exhibits the potential of integrative transcriptomic analyses to identify critical genes and pathways of antiandrogen resistance as a promising approach for designing novel therapeutic strategies to circumvent drug resistance.

Ser-486/491 phosphorylation and inhibition of AMPKα activity is positively associated with Gleason score, metastasis, and castration-resistance in prostate cancer: A retrospective clinical study.

BACKGROUND: We previously demonstrated that adenosine monophosphate-activated protein kinase (AMPKα) activity is significantly inhibited by Ser-486/491 phosphorylation in cell culture and in vivo models of metastatic and castration-resistant prostate cancer, and hypothesized these findings may translate to clinical specimens. METHODS: In this retrospective, single-institution pilot study, 45 metastatic prostate cancer cases were identified within the University Hospitals Cleveland Medical Center Pathology Archive with both metastasis and matched primary prostate tumor specimens in formalin-fixed, paraffin-embedded blocks, and complete electronic medical records. Thirty non-metastatic, hormone-dependent prostate cancer controls, who were progression-free as defined by undetectable prostate specific antigen for at least 79.6 months (range 79.6-136.0 months), and matched metastatic cases based on age, race, and year of diagnosis. All specimens were collected from 1991 to 2014; primary tumor specimens were obtained via diagnostic biopsy or prostatectomy, and metastasis specimens obtained via surgery or perimortem. 5-µ sequential slides were processed for phospho-Ser-486/491 AMPKα1 /α2 , phospho-Thr-172 AMPKα, AMPKα1 /α2 , phospho-Ser-792 Raptor, phospho-Ser-79 acetyl-CoA carboxylase, and phospho-Ser-872, 3-hydroxy-3-methylglutaryl-CoA reductase immunohistochemistry to determine expression, phosphorylation pattern, and activity of AMPKα. RESULTS: Increased inhibitory Ser-486/491 AMPKα1 /α2 phosphorylation, increased AMPKα protein expression, decreased AMPKα activity, and loss of nuclear AMPKα and p-AMPKα are associated with prostate cancer progression to metastasis. Increased p-Ser-486/491 AMPKα1 /α2 was also positively correlated with higher Gleason grade and progression to castration-resistance. CONCLUSIONS: p-Ser-486/491 AMPKα1 /α2 is a novel marker of prostate cancer metastasis and castration-resistance. Ser-486/491 phosphokinases should be pursued as targets for metastatic and castration-resistant prostate cancer chemotherapy.

Molecular testing for BRAF mutations to inform melanoma treatment decisions: a move toward precision medicine.

Approximately one-half of advanced (unresectable or metastatic) melanomas harbor a mutation in the BRAF gene, with V600E being the most common mutation. Targeted therapy with BRAF and MEK inhibitors is associated with significant long-term treatment benefit in patients with BRAF V600-mutated melanoma. Therefore, molecular testing for BRAF mutations is a priority in determining the course of therapy. A literature search was performed using MEDLINE/PubMed and scientific congress databases using the terms 'BRAF,' 'mutation,' and 'cancer/tumor.' These results were filtered to include manuscripts that focused on diagnostic tests for determining BRAF mutation status. Numerous BRAF testing methods were identified, including DNA-based companion diagnostic tests and DNA- and protein-based laboratory-developed tests. Herein we review the characteristics of each method and highlight the strengths and weaknesses that should be considered before use and when interpreting results for each patient. Molecular profiling has shown that mutation load increases with melanoma tumor progression and that unique patterns of genetic changes and evolutionary trajectories for different melanoma subtypes can occur. Discordance in the BRAF mutational status between primary and metastatic lesions, as well as intratumoral heterogeneity, is known to occur. Additionally, the development of acquired resistance to combination BRAF and MEK inhibitor therapy is still a formidable obstacle. Therefore, tumor heterogeneity and the development of acquired resistance have important implications for molecular testing and ultimately the treatment of patients with advanced-stage melanoma. Overall, this information may help community oncologists more accurately and effectively interpret results of diagnostic tests within the context of recent data characterizing melanoma tumor progression.

TMPRSS2-ERG gene fusion is rare compared to PTEN deletions in stage T1a prostate cancer.

T1a prostate cancers (cancer found incidentally in transurethral resection, <5% of the tissue) are indolent tumors of the transition zone. The overexpression of ERG and the inactivation of PTEN have been shown to be important drivers of carcinogenesis in large series of prostate cancer, but the genetics of transition zone tumors have not been well characterized. We evaluated the status of ERG and PTEN in formalin-fixed paraffin-embedded tissue using immunohistochemical and FISH analysis in 54 T1a transition zone tumors. The protein expression of ERG was determined using a rabbit monoclonal antibody and nuclear staining was scored as positive or negative. The genomic status of ERG was determined using three colored FISH using an ERG-TMPRSS2 tri-color probe set. The protein expression of PTEN was determined using a rabbit monoclonal antibody and cytoplasmic, and nuclear staining was scored as positive or negative. The genomic status of PTEN was determined using dual color FISH with a PTEN probe and a CEP10 probe. We found ERG rearrangement in 2 of 54 tumors (4%), one with protein overexpression by immunohistochemistry. PTEN inactivation was seen in 13 of 54 tumors (24%). Nine of the 13 PTEN alleles were inactivated by hemizygous deletion. No homozygous PTEN deletion was observed. PTEN deletion and ERG rearrangement were mutually exclusive. ERG rearrangement was rare compared to peripheral zone tumors and to PTEN inactivation in T1a transition zone tumors. © 2016 Wiley Periodicals, Inc.

TERT promoter mutation status in sarcomatoid urothelial carcinomas of the upper urinary tract.

AIM: To determine TERT promoter mutation status as well as the expression of PAX8, GATA3, p63, p40, p53 and uroplakin III in 17 patients with the upper urinary tract sarcomatoid urothelial carcinoma. METHODS & RESULTS:  TERT C228T mutations were found in six of 17 cases (35%). p53 was expressed in 77% of these tumors. PAX8, GATA3, p40 and uroplakin III are less frequently expressed. Lymph node metastases were present in ten cases (59%). Eight patients (47%), including all three patients with TERT mutation, died of cancer within 2 years after surgery. CONCLUSION: Sarcomatoid carcinoma of the upper urinary tract is an aggressive tumor and the presence of TERT mutation may portend poor prognosis.

Obesity-initiated metabolic syndrome promotes urinary voiding dysfunction in a mouse model.

OBJECTIVE: Accumulating evidences suggests that obesity and metabolic syndrome (MetS) contribute towards lower urinary tract symptoms (LUTS) through alterations in the phenotype of bladder and prostate gland. Clinical studies indicate a link between MetS and LUTS. Nevertheless, there is lack of suitable animal model(s) which could illustrate an association linking obesity to LUTS. We examined the lower urinary tract function in an obesity-initiated MetS mouse model. METHODS: Male C57BL/6N wild-type and obese B6.V-Lepob/J maintained on regular diet for 28 weeks were subjected to the assessment of body weight (BW), body length (BL), waist circumference (WC), body mass index (BMI), blood glucose (BG), plasma insulin (INS), plasma leptin (LEP), total cholesterol (CHO), free fatty acid (FFA), and measurement of urinary functions. Whole animal peritoneal and subcutaneous adipose tissue measurements as well as prostate and bladder volumes were analyzed by MRI followed by histological evaluation. These parameters were used to draw correlations between MetS and LUTS. RESULTS: Obesity parameters such as BW, WC, and BMI were significantly higher in B6.V-Lepob/J mice compared to C57BL/6N mice (P < 0.01). Higher levels of total CHO and FFA were noted in B6.V-Lepob/J mice than C57BL/6N mice (P < 0.05). These results were concurrent with frequency, lower average urine volume and other urinary voiding dysfunctions in B6.V-Lepob/J mice. MRI assessments demonstrate marked increase in body fat and prostate volume in these mice. Compared to C57BL/6N mice, histological analysis of the prostate from B6.V-Lepob/J mice showed increased proliferation, gland crowding, and infiltration of immune cells in the stroma; whereas the bladder urothelium was slightly thicker and appears more proliferative in these mice. The regression and correlation analysis indicate that peritoneal fat (R = 0.853; P < 0.02), CHO (R = 0.729; P < 0.001), BG (R = 0.712; P < 0.001) and prostate volume (R = 0.706; P < 0.023) strongly correlate with LUTS whereas BMI, WC, INS, and FFA moderately correlate with the prevalence of bladder dysfunction. CONCLUSION: Our results suggest that LUTS may be attributable in part to obesity and MetS. Validation of an in vivo model may lead to understand the underlying pathophysiological mechanisms of obesity-related LUTS in humans. Prostate 76:964-976, 2016. © 2016 Wiley Periodicals, Inc.

Telomerase reverse transcriptase (TERT) promoter mutation analysis of benign, malignant and reactive urothelial lesions reveals a subpopulation of inverted papilloma with immortalizing genetic change.

AIMS: To understand more clearly the genetic ontogeny of inverted papilloma of urinary bladder, we analysed telomerase reverse transcriptase (TERT) promoter mutation status in a group of 26 inverted papillomas in comparison with the mutation status of urothelial carcinoma with inverted growth (26 cases), conventional urothelial carcinoma (36 Ta non-invasive urothelial carcinoma, 35 T2 invasive urothelial carcinoma) and cystitis glandularis (25 cases). METHODS AND RESULTS: TERT promoter mutations in inverted papilloma, urothelial carcinoma with inverted growth, urothelial carcinoma and cystitis glandularis were found in 15% (four of 26), 58% (15 of 26), 63% (45 of 71) and 0% (none of 25), respectively. C228T mutations were the predominant mutations (97%) found in bladder tumours, while C250T aberrations occurred in approximately 3% of bladder tumours. In the inverted papilloma group, TERT mutation occurred predominantly in female patients (P = 0.006). Among urothelial carcinomas, TERT promoter mutation status did not correlate with gender, histological grade or pathological stage. CONCLUSIONS: TERT promoter mutations were found in 15% of inverted papillomas. Our data suggest that there is a subpopulation of inverted papilloma that shares a carcinogenetic pathway with urothelial carcinoma with inverted growth and conventional urothelial carcinomas. Caution is warranted in exploring TERT promoter mutation status as a screening or adjunct diagnostic test for bladder cancer.

Increased androgen receptor gene copy number is associated with TMPRSS2-ERG rearrangement in prostatic small cell carcinoma.

Small cell carcinoma of the prostate (PSCC) is a highly aggressive malignancy that often develops in patients previously treated with hormonal therapy for metastatic prostatic acinar adenocarcinoma. The TMPRSS2-ERG gene rearrangement is highly specific for prostate cancer and shared by PSCC; however, the role of androgen receptor (AR) gene alterations and interaction with TMPRSS2-ERG rearrangement are incompletely understood in PSCC. Sixty-one cases of PSCC were examined for AR gene copy number and TMPRSS2-ERG rearrangement by fluorescence in situ hybridization (FISH) and AR protein expression by immunohistochemistry. Of 61 cases of PSCC, 51% (31/61) demonstrated increased AR gene copy number (FISH+), 54% (33/61) were positive for TMPRSS2-ERG gene fusion, and 38% (23/61) showed AR protein expression. Of the 31 AR FISH+ cases, 23 also showed TMPRSS2-ERG gene fusion, and 16 expressed AR protein. Of the 33 cases with TMPRSS2-ERG fusion, 28 were AR FISH+ or expressed AR protein. Statistically significant correlations were observed between AR gene copy number or AR protein expression and TMPRSS2-ERG gene fusion (P = 0.001 and P = 0.03, respectively). In summary, high AR gene copy number emerges during the development of PSCC, often in association with TMPRSS2-ERG rearrangement. This potential mechanism warrants further study. Improvement will come from understanding the biology of the disease and integrating new therapies into the treatment of this rare and aggressive tumor.

Metanephric adenoma: the utility of immunohistochemical and cytogenetic analyses in differential diagnosis, including solid variant papillary renal cell carcinoma and epithelial-predominant nephroblastoma.

Metanephric adenoma is a benign renal neoplasm that overlaps in morphology with the solid variant of papillary renal cell carcinoma and epithelial-predominant nephroblastoma. To aid in resolving this differential diagnosis, we investigated the utility of immunohistochemical and molecular analyses in distinguishing between these entities; the first study, to our knowledge, to use a combined approach in analyzing all three tumors. We analyzed 37 tumors originally diagnosed as metanephric adenomas (2 of which we reclassified as papillary renal cell carcinomas), 13 solid variant papillary renal cell carcinomas, and 20 epithelial-predominant nephroblastomas using a combination of immunohistochemistry and fluorescence in situ hybridization (FISH) assessing for trisomy of chromosomes 7 and 17 and loss of Y. Immunohistochemical staining was performed for CK7, AMACR, WT1, and CD57. The combination of CK7-, AMACR-, WT1+, and CD57+ was considered characteristic of metanephric adenoma. Most of the tumors originally diagnosed as metanephric adenomas (31/37) showed the expected staining pattern of metanephric adenoma (CK7-, AMACR-, WT1+, and CD57+). Of the six tumors with discordant immunophenotype, two tumors were reclassified as papillary renal cell carcinoma after cytogenetic workup. It is recommended that all adult cases histologically resembling metanephric adenoma have WT1, CD57, CK7, and AMACR immunohistochemical staining performed. If the staining pattern is characteristic for metanephric adenoma (CK7-, AMACR-, WT1+, and CD57+, including membranous staining), then no other diagnostic tests are indicated. However, if there is a different immunostaining pattern, then we recommend FISH analysis.

Inflammatory myofibroblastic tumour of the urinary bladder: the role of immunoglobulin G4 and the comparison of two immunohistochemical antibodies and fluorescence in-situ hybridization for the detection of anaplastic lymphoma kinase alterations.

AIMS: We examined gene rearrangement and the expression of anaplastic lymphoma kinase (ALK) in urinary bladder inflammatory myofibroblastic tumour (IMT) using fluorescence in-situ hybridization (FISH) and two immunohistochemical antibodies to ALK. We also investigated whether IMT represents an immunoglobulin (Ig)G4-related disease. METHODS AND RESULTS: The performance of the Dako FLEX ALK monoclonal antibody (CD246) and the Cell Signaling Technology ALK (D5F3) XP monoclonal antibody were compared. Overall, 11 of 16 tumours showed ALK expression by immunohistochemistry (69%). Ten demonstrated ALK expression with both stains and one was positive with D5F3 but not CD246 (91% correlation). The D5F3 antibody yielded a stronger staining intensity and a higher sensitivity. Nine tumours demonstrated ALK rearrangements (56%) by FISH. Three were ALK(+) by immunohistochemistry but negative for rearrangement by FISH, whereas one showed rearrangement by FISH but was negative by immunohistochemistry. In total, 12 tumours were positive for ALK abnormalities (75%). Using current criteria, no cases were classified as an IgG4-related disease. CONCLUSIONS: The ALK D5F3 immunohistochemical stain showed superior staining characteristics compared with ALK CD246. Discrepancies in the results between FISH and immunohistochemistry for ALK abnormalities may have causes that are multifactorial. By current criteria, IMT does not represent an IgG4-related disease.

Apigenin inhibits prostate cancer progression in TRAMP mice via targeting PI3K/Akt/FoxO pathway.

Forkhead box O (FoxO) transcription factors play an important role as tumor suppressor in several human malignancies. Disruption of FoxO activity due to loss of phosphatase and tensin homolog and activation of phosphatidylinositol-3 kinase (PI3K)/Akt are frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits antiproliferative and anticarcinogenic activities through mechanisms, which are not fully defined. In the present study, we show that apigenin suppressed prostate tumorigenesis in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice through the PI3K/Akt/FoxO-signaling pathway. Apigenin-treated TRAMP mice (20 and 50 µg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate as well as completely abolished distant organ metastasis. Apigenin treatment resulted in significant decrease in the weight of genitourinary apparatus (P < 0.0001), dorsolateral (P < 0.0001) and ventral prostate (P < 0.028), compared with the control group. Apigenin-treated mice showed reduced phosphorylation of Akt (Ser473) and FoxO3a (Ser253), which correlated with its increased nuclear retention and decreased binding of FoxO3a with 14-3-3. These events lead to reduced proliferation as assessed by Ki-67 and cyclin D1, along with upregulation of FoxO-responsive proteins BIM and p27/Kip1. Complementing in vivo results, similar observations were noted in human prostate cancer LNCaP and PC-3 cells after apigenin treatment. Furthermore, binding of FoxO3a with p27/Kip1 was markedly increased after 10 and 20 µM apigenin treatment resulting in G0/G1-phase cell cycle arrest, which was consistent with the effects elicited by PI3K/Akt inhibitor, LY294002. These results provide convincing evidence that apigenin effectively suppressed prostate cancer progression, at least in part, by targeting the PI3K/Akt/FoxO-signaling pathway.

Protection against oxidative DNA damage and stress in human prostate by glutathione S-transferase P1.

The pi-class glutathione S-transferase (GSTP1) actively protect cells from carcinogens and electrophilic compounds. Loss of GSTP1 expression via promoter hypermethylation is the most common epigenetic alteration observed in human prostate cancer. Silencing of GSTP1 can increase generation of reactive oxygen species (ROS) and DNA damage in cells. In this study we investigated whether loss of GSTP1 contributes to increased DNA damage that may predispose men to a higher risk of prostate cancer. We found significantly elevated (103%; P < 0.0001) levels of 8-oxo-2'-deoxogunosine (8-OHdG), an oxidative DNA damage marker, in adenocarcinomas, compared to benign counterparts, which positively correlated (r = 0.2) with loss of GSTP1 activity (34%; P < 0.0001). Silencing of GSTP1 using siRNA approach in normal human prostate epithelial RWPE1 cells caused increased intracellular production of ROS and higher susceptibility of cells to H2 O2 -mediated oxidative stress. Additionally, human prostate carcinoma LNCaP cells, which contain a silenced GSTP1 gene, were genetically modified to constitutively express high levels of GSTP1. Induction of GSTP1 activity lowered endogenous ROS levels in LNCaP-pLPCX-GSTP1 cells, and when exposed to H2 O2 , these cells exhibited significantly reduced production of ROS and 8-OHdG levels, compared to vector control LNCaP-pLPCX cells. Furthermore, exposure of LNCaP cells to green tea polyphenols caused reexpression of GSTP1, which protected the cells from H2 O2 -mediated DNA damage through decreased ROS production compared to nonexposed cells. These results suggest that loss of GSTP1 expression in human prostate cells, a process that increases their susceptibility to oxidative stress-induced DNA damage, may be an important target for primary prevention of prostate cancer.

EGFR alterations and EML4-ALK rearrangement in primary adenocarcinoma of the urinary bladder.

The identification of mutations in epidermal growth factor receptor (EGFR) and translocations involving anaplastic lymphoma kinase (ALK) in lung adenocarcinoma has drastically changed understanding of the disease and led to the development of targeted therapies. Adenocarcinoma of the urinary bladder is rare and poorly understood at the molecular level. We undertook this study to determine whether EGFR mutations, increases in EGFR copy number, or ALK translocations are present in these tumors. Twenty-eight cases of primary bladder adenocarcinoma were analyzed. For EGFR mutational analysis, PCR-amplified products were analyzed on the Q24 Pyrosequencer with Qiagen EGFR Pyro Kits. All cases were analyzed via fluorescence in situ hybridization (FISH) using Vysis ALK Break Apart FISH Probes for detection of ALK chromosomal translocation and Vysis Dual Color Probes to assess for increased gene copy number of EGFR. None of the 28 cases examined showed mutational events in EGFR or ALK rearrangements. EGFR polysomy was seen in 10 out of 28 (36%) cases. No correlation with EGFR polysomy was seen in the tumors with respect to age, histologic subtypes, pathologic stage, or lymph node metastasis. In summary, EGFR mutations and ALK rearrangements do not appear to be involved in the development of primary adenocarcinoma of the urinary bladder. A subgroup of cases (36%), however, demonstrated increased gene copy number of EGFR by FISH.

Intraductal carcinoma of the prostate: interobserver reproducibility survey of 39 urologic pathologists.

The diagnosis of intraductal carcinoma (IDC) of the prostate remains subjective because 3 sets of diagnostic criteria are in use. An internet survey was compiled from 38 photomicrographs showing duct proliferations: 14 signed out as high-grade prostatic intraepithelial neoplasia (HGPIN), 17 IDC, and 7 invasive cribriform/ductal carcinoma. Each image was assessed for the presence of 9 histologic criteria ascribed to IDC. Thirty-nine respondents were asked to rate images as (1) benign/reactive, (2) HGPIN, (3) borderline between HGPIN and IDC, (4) IDC, or (5) invasive cribriform/ductal carcinoma. Intraclass correlation coefficient was 0.68. There was 70% overall agreement with HGPIN, 43% with IDC, and 73% with invasive carcinoma (P < .001, χ(2)). Respondents considered 19 (50%) of 38 cases as IDC candidates, of which 5 (26%) had a two-thirds consensus for IDC; two-thirds consensus for either borderline or IDC was reached in 9 (47%). Two-thirds consensus other than IDC was reached in the remaining 19 of 38 cases, with 15 supporting HGPIN and 4 supporting invasive carcinoma. Findings that differed across diagnostic categories were lumen-spanning neoplastic cells (P < .001), 2× benign duct diameters (P < .001), duct space contours (round, irregular, and branched) (P < .001), papillary growth (P = .048), dense cribriform or solid growth (both P = .023), and comedonecrosis (P = .015). When the 19 of 38 images that attained consensus for HGPIN or invasive carcinoma were removed from consideration, lack of IDC consensus was most often attributable to only loose cribriform growth (5/19), central nuclear maturation (5/19), or comedonecrosis (3/19). Of the 9 histologic criteria, only 1 retained significant correlation with a consensus diagnosis of IDC: the presence of solid areas (P = .038). One case that attained IDC consensus had less than 2× duct enlargement yet still had severe nuclear atypia and nucleomegaly. Six fold nuclear enlargement was not significant (P = .083), although no image had both 6× nuclei and papillary or loose cribriform growth: a combination postulated as sufficient criteria for IDC. Finally, 20.5% of respondents agreed that an isolated diagnosis of IDC on needle biopsy warrants definitive therapy, 20.5% disagreed, and 59.0% considered the decision to depend upon clinicopathologic variables. Although IDC diagnosis remains challenging, we propose these criteria: a lumen-spanning proliferation of neoplastic cells in preexisting ducts with a dense cribriform or partial solid growth pattern. Solid growth, in any part of the duct space, emerges as the most reproducible finding to rule in a diagnosis of IDC. Comedonecrosis is a rarer finding, but in most cases, it should rule in IDC. Duct space enlargement to greater than 2× the diameter of the largest, adjacent benign spaces is usually present in IDC, although there may be rare exceptions.