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Purpose: On the basis of preclinical evidence of epigenetic contribution to sensitivity and resistance to immune checkpoint inhibitors (ICI), we hypothesized that guadecitabine (hypomethylating agent) and atezolizumab [anti–programmed cell death ligand 1 (PD-L1)] together would potentiate a clinical response in patients with metastatic urothelial carcinoma (UC) unresponsive to initial immune checkpoint blockade therapy. Patients and Methods: We designed a single arm phase II study (NCT03179943) with a safety run-in to identify the recommended phase II dose of the combination therapy of guadecitabine and atezolizumab. Patients with recurrent/advanced UC who had previously progressed on ICI therapy with programmed cell death protein 1 or PD-L1 targeting agents were eligible. Preplanned correlative analysis was performed to characterize peripheral immune dynamics and global DNA methylation, transcriptome, and immune infiltration dynamics of patient tumors. Results: Safety run-in enrolled 6 patients and phase II enrolled 15 patients before the trial was closed for futility. No dose-limiting toxicity was observed. Four patients, with best response of stable disease (SD), exhibited extended tumor control (8–11 months) and survival (>14 months). Correlative analysis revealed lack of DNA demethylation in tumors after 2 cycles of treatment. Increased peripheral immune activation and immune infiltration in tumors after treatment correlated with progression-free survival and SD. Furthermore, high IL6 and IL8 levels in the patients’ plasma was associated with short survival. Conclusions: No RECIST responses were observed after combination therapy in this trial. Although we could not detect the anticipated tumor-intrinsic effects of guadecitabine, the addition of hypomethylating agent to ICI therapy induced immune activation in a few patients, which associated with longer patient survival.

Supplementary Figure 12: HLA-DR and NKG2D abundance on peripheral lymphocytes associate with longer progression free survival.

Purpose:On the basis of preclinical evidence of epigenetic contribution to sensitivity and resistance to immune checkpoint inhibitors (ICI), we hypothesized that guadecitabine (hypomethylating agent) and atezolizumab [anti–programmed cell death ligand 1 (PD-L1)] together would potentiate a clinical response in patients with metastatic urothelial carcinoma (UC) unresponsive to initial immune checkpoint blockade therapy.Patients and Methods:We designed a single arm phase II study (NCT03179943) with a safety run-in to identify the recommended phase II dose of the combination therapy of guadecitabine and atezolizumab. Patients with recurrent/advanced UC who had previously progressed on ICI therapy with programmed cell death protein 1 or PD-L1 targeting agents were eligible. Preplanned correlative analysis was performed to characterize peripheral immune dynamics and global DNA methylation, transcriptome, and immune infiltration dynamics of patient tumors.Results:Safety run-in enrolled 6 patients and phase II enrolled 15 patients before the trial was closed for futility. No dose-limiting toxicity was observed. Four patients, with best response of stable disease (SD), exhibited extended tumor control (8–11 months) and survival (>14 months). Correlative analysis revealed lack of DNA demethylation in tumors after 2 cycles of treatment. Increased peripheral immune activation and immune infiltration in tumors after treatment correlated with progression-free survival and SD. Furthermore, high IL6 and IL8 levels in the patients’ plasma was associated with short survival.Conclusions:No RECIST responses were observed after combination therapy in this trial. Although we could not detect the anticipated tumor-intrinsic effects of guadecitabine, the addition of hypomethylating agent to ICI therapy induced immune activation in a few patients, which associated with longer patient survival.

Supplementary TablesSupplementary Table 1: Patient and tumor sample informationSupplementary Table 2: Tumor mutation burdenSupplementary Table 3: Unfiltered whole exome sequencing mutation analysisSupplementary Table 4: Filtered whole exome sequencing mutation analysisSupplementary Table 5: Immunohistochemistry scores of tumorsSupplementary Table 6: FACS panel for PBMC analysisSupplementary Table 7: Geometric mean fluorescence intensity (GMFI) from PBMC FACSSupplementary Table 8: Representativeness of Study Participants

Supplementary Figure 1. Quantitation and location of epigenetic changes in all 6 patient samples. Supplementary Figure 2. Significant accessibility changes between 6 patients. Supplementary Figure 3. Accessibility changes shared in at least two patient samples, plotted in heatmaps, either losing accessibility (Groups a and e) or gaining accessibility (Groups d and f) with or without DNA methylation. Supplementary Figure 4. ChIP-seq analyses of Patient 5. Supplementary Figure 5. Validation of AcceSssIble data from each Group a, d, e, and f using locus-specific bisulfite sequencing. Supplementary Figure 6. Correlation plots of chromatin accessibility and DNA methylation changes vs. gene expression changes. Supplementary Figure 7. Epigenetically-regulated genes in the HIF1A-signaling pathway and VHL status in each ccRCC patient. Supplementary Figure 8. Example diagram of M.SssI treatment efficacy. Supplementary Table 1. List of loci with methylation-dependent accessibility changes that positively correlate with expression changes from TCGA RNA-seq data. Supplementary Table 2. List of loci with methylation-independent accessibility changes that positively correlate with expression changes from TCGA RNA-seq data. Supplementary Table 3. List of genes overlapping between epigenetically-regulated genes from our study, and commonly mutated genes in ccRCC from TCGA.

Alterations in chromatin accessibility independent of DNA methylation can affect cancer-related gene expression, but are often overlooked in conventional epigenomic profiling approaches. In this study, we describe a cost-effective and computationally simple assay called AcceSssIble to simultaneously interrogate DNA methylation and chromatin accessibility alterations in primary human clear cell renal cell carcinomas (ccRCC). Our study revealed significant perturbations to the ccRCC epigenome and identified gene expression changes that were specifically attributed to the chromatin accessibility status whether or not DNA methylation was involved. Compared with commonly mutated genes in ccRCC, such as the von Hippel-Lindau (VHL) tumor suppressor, the genes identified by AcceSssIble comprised distinct pathways and more frequently underwent epigenetic changes, suggesting that genetic and epigenetic alterations could be independent events in ccRCC. Specifically, we found unique DNA methylation–independent promoter accessibility alterations in pathways mimicking VHL deficiency. Overall, this study provides a novel approach for identifying new epigenetic-based therapeutic targets, previously undetectable by DNA methylation studies alone, that may complement current genetic-based treatment strategies. Cancer Res; 76(7); 1954–64. ©2016 AACR.

Legend and description of Supplementary Figures 1-8

In many cancers, combination therapy regimens are successfully improving response and survival rates, but the challenges of toxicity remain. GRP78, the master regulator of the unfolded protein response, is emerging as a target that is upregulated in tumors, specifically following treatment, and one that impacts tumor cell survival and disease recurrence. Here, we show IT-139, an antitumor small molecule inhibitor, suppresses induction of GRP78 from different types of endoplasmic reticulum (ER) stress in a variety of cancer cell lines, including those that have acquired therapeutic resistance, but not in the non-cancer cells being tested. We further determined that IT-139 treatment exacerbates ER stress while at the same time suppresses GRP78 induction at the transcriptional level. Our studies revealed a differential effect of IT-139 on chaperone protein family expression at multiple levels in different cancer cell lines. In xenograft studies, IT-139 decreased BRAF inhibitor upregulation of GRP78 expression in the tumor, while having minimal effect on GRP78 expression in the adjacent normal cells. The preferential decrease in GRP78 levels in tumor cells over normal cells, supported by the manageable safety profile seen in the Phase 1 clinical trial, reinforce the value IT-139 brings to combination therapies as it continues its clinical development.

Introduction: We report the results of a phase II trial testing the hypothesis that adding the hypomethylating agent G to the PDL1 inhibitor A in patients with mUC who developed primary or acquired resistance to checkpoint blockade (CB) will overcome this resistance by (1) eliciting viral mimicry in the tumor tissue to potentiate and reinvigorate anti-tumor immunity, (2) epigenetic reprogramming of T lymphocytes to overcome exhaustion. Methods: Pts with mUC resistant to CB accrued at 3 centers were treated with G 45mg/m2 daily days 1-5 every 6 wks and A 1200mg every 3 wks. After initial safety lead in with 6 patients, trial was designed to add 37 additional patients. The primary endpoint was ORR. Correlative analyses included analysis of peripheral blood T cells and tumor tissue collected at baseline and once during treatment. Results: 21 pts were enrolled. 20 pts were evaluable for response. Best response was PD (16), SD (4). 10 patients progressed clinically prompting earlier than scheduled (12 wk) imaging. Four pts exhibited a “hyperprogression” phenotype exhibiting rapid acceleration of tumor growth rate starting with initiation of therapy. At presepcified interim analysis it was determined that the trial would not meet its primary endpoint and it closed early. Median PFS 2.6 mo, median OS 8 mo. The 4 patients with SD maintained that status for median 13 months (range 9-15 mo). Global DNA methylome and transcriptome profiles from pre- and post-treatment tumor samples revealed a lack of transposable element-induced viral mimicry activation, which correlated with minimal DNA demethylation being induced in the tumors. Of note, flow cytometry-based immune profiling of peripheral blood from patients suggests a correlation between increased progression-free survival (PFS) with 1) lower expression of DNAM-1 on mature NK cells and 2) lower expression of CD39 on CD8+ effector T cells at time of inclusion on the trial. Conclusions: While no responses were seen, both prolonged SD and hyperprogression were seen. Further tissue and peripheral blood based analyses are ongoing to elucidate the biological determinates of this dichotomy. Citation Format: Elizabeth R. Plimack, Kerry Campbell, Jean-Pierre J. Issa, Noah M. Hahn, David I. Quinn, Hyo Sik Jang, Galen Hostetter, Peter W. Nichols, Woonbok Chung, Jozef Madzo, Hitoshi Ohtani, Hui Shen, Toshinori Hinoue, Stephen B. Baylin, Peter A. Jones. A Phase II trial of guadecitabine (G) plus atezolizumab (A) in patients with metastatic urothelial carcinoma (UC) progressing after initial checkpoint inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT121.

Street trees are commonly associated with pavement cracking and lifting, resulting in costly repair of the pavement and/or removal of the affected tree/s. This study used Ground Penetrating Radar (GPR) to evaluate the effectiveness of three different prototype permeable pavement designs in reducing pavement damage caused by street tree roots (Melaleuca quinquenervia). Initial tests conducted in a simulated test environment were then replicated in the field to examine the performance of the GPR. The initial test results were positive and indicated that this technique could be used to reliably determine tree root size and depth under pavements with minimal error. Positive root identification results were recorded for all three prototype pavements. The accuracy of the GPR results was found to vary according to soil type, depth of aggregate sub-base, and water mass contained within both the soil, and the buried objects during the calibration process. The three-dimensional nature (overlapping) of genuine tree roots also affected the detection accuracy of the GPR in the field. The study has shown that GPR may play an important role in tree root identification in future, and improve pre-emptive trip hazard reduction management. Increasing permeable pavement sub-base depths may also lead to less pavement damage by tree roots thereby preventing future trip hazards and avoiding costly repairs.

// Sameer Chopra 1,* , Jie Liu 2,* , Mehrdad Alemozaffar 1 , Peter W. Nichols 3 , Manju Aron 3 , Daniel J. Weisenberger 4 , Clayton K. Collings 1 , Sumeet Syan 1 , Brian Hu 5 , Mihir Desai 1 , Monish Aron 1 , Vinay Duddalwar 6 , Inderbir Gill 1 , Gangning Liang 1 and Kimberly D. Siegmund 2 1 Department of Urology, Norris Comprehensive Cancer Center, USC Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 2 Department of Preventive Medicine, Norris Comprehensive Cancer Center, USC Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 3 Department of Pathology, Norris Comprehensive Cancer Center, USC Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 4 Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, USC Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 5 Department of Urology, Loma Linda University, Loma Linda, CA, USA 6 Department of Radiology, Norris Comprehensive Cancer Center, USC Keck School of Medicine, University of Southern California, Los Angeles, CA, USA * These authors have contributed equally to this work Correspondence to: Gangning Liang, email: // Kimberly D. Siegmund , email: // Keywords : small renal mass, tumor classification, DNA methylation, kidney cancer Received : July 29, 2016 Accepted : September 20, 2016 Published : September 27, 2016 Abstract Purpose: The clinical management of small renal masses (SRMs) is challenging since the current methods for distinguishing between benign masses and malignant renal cell carcinomas (RCCs) are frequently inaccurate or inconclusive. In addition, renal cancer subtypes also have different treatments and outcomes. High false negative rates increase the risk of cancer progression and indeterminate diagnoses result in unnecessary and potentially morbid surgical procedures. Experimental Design: We built a predictive classification model for kidney tumors using 697 DNA methylation profiles from six different subgroups: clear cell, papillary and chromophobe RCC, benign angiomylolipomas, oncocytomas, and normal kidney tissues. Furthermore, the DNA methylation-dependent classifier has been validated in 272 ex vivo needle biopsy samples from 100 renal masses (71% SRMs). Results: In general, the results were highly reproducible (89%, n =70) in predicting identical malignant subtypes from biopsies. Overall, 98% of adjacent-normals ( n =102) were correctly classified as normal, while 92% of tumors ( n =71) were correctly classified malignant and 86% of benign ( n =29) were correctly classified benign by this classification model. Conclusions: Overall, this study provides molecular-based support for using routine needle biopsies to determine tumor classification of SRMs and support the clinical decision-making.

Alterations in chromatin accessibility independent of DNA methylation can affect cancer-related gene expression, but are often overlooked in conventional epigenomic profiling approaches. In this study, we describe a cost-effective and computationally simple assay called AcceSssIble to simultaneously interrogate DNA methylation and chromatin accessibility alterations in primary human clear cell renal cell carcinomas (ccRCC). Our study revealed significant perturbations to the ccRCC epigenome and identified gene expression changes that were specifically attributed to the chromatin accessibility status whether or not DNA methylation was involved. Compared with commonly mutated genes in ccRCC, such as the von Hippel-Lindau (VHL) tumor suppressor, the genes identified by AcceSssIble comprised distinct pathways and more frequently underwent epigenetic changes, suggesting that genetic and epigenetic alterations could be independent events in ccRCC. Specifically, we found unique DNA methylation–independent promoter accessibility alterations in pathways mimicking VHL deficiency. Overall, this study provides a novel approach for identifying new epigenetic-based therapeutic targets, previously undetectable by DNA methylation studies alone, that may complement current genetic-based treatment strategies. Cancer Res; 76(7); 1954–64. ©2016 AACR.

Field testing of a proprietary stormwater treatment device was undertaken over 14 months at a site located in Nambour, South East Queensland. Testing was undertaken to evaluate the pollution removal performance of a Stormceptor® treatment train for removing total suspended solids (TSS), total nitrogen (TN) and total phosphorous (TP) from stormwater runoff. Water quality sampling was undertaken using natural rainfall events complying with an a priori sampling protocol. More than 59 rain events were monitored, of which 18 were found to comply with the accepted sampling protocol. The efficiency ratios (ER) observed for the treatment device were found to be 83% for TSS, 11% for TP and 23% for TN. Although adequately removing TSS, additional system components, such as engineered filters, would be required to satisfy minimum local pollution removal regulations. The results of dry weather sampling tests did not conclusively demonstrate that pollutants were exported between storm events or that pollution concentrations increased significantly over time.

This study evaluated the pollution removal and hydrologic performance of five, 10-year old street-side bioretention systems. The bioretention basins were subjected to a series of simulated rainfall events using synthetic stormwater. Four different pollution concentrations were tested on three of the bioretention basins. The four concentrations tested were: A) no pollution; B) typical Australian urban pollutant loads; C) double the typical pollution loads, and; D) five times the typical pollution loads. Tests were also undertaken to determine the levels of contaminant and heavy metals build-up that occurred in the filter media over the 10 year operational life of the bioretention systems. Although highly variable, the overall hydrological performance of the basins was found to be positive, with all basins attenuating flows, reducing both peak flow rates and total outflow volumes. Total suspended solids removal performance was variable for all tests and no correlation was found between performance and dosage. Total nitrogen (TN) removal was positive for Tests B, C and D. However, the TN removal results for Test A were found to be negative. Total phosphorus (TP) was the only pollutant to be effectively removed from all basins for all four synthetic stormwater tests. The study bioretention basins were found to export pollutants during tests where no pollutants were added to the simulated inflow water (Test A). Heavy metal and hydrocarbon testing undertaken on the bioretention systems found that the pollution levels of the filter media were still within acceptable limits after 10 years in operation. This field study has shown bioretention basin pollution removal performance to be highly variable and dependant on a range of factors including inflow pollution concentrations, filter media, construction methods and environmental factors. Further research is required in order to fully understand the potential stormwater management benefits of these systems.

Background: While many studies have uncovered genetic mutations that drive tumorigenesis, far fewer have described epigenetic changes, such as nucleosome positioning and DNA methylation, which lead to the development of cancer. Therefore, accurately mapping these changes between normal and tumor tissue will provide novel information to identify genes that undergo epigenetic changes that drive tumorigenesis (“epigenetic driver genes”). In this study, we used an assay developed in our laboratory to investigate the epigenetic changes between clear cell renal cell carcinoma (ccRCC, the most common subtype of renal carcinoma) tumors and normal tissue to uncover genes that contribute to ccRCC tumorigenesis. Methods: Current methods to investigate epigenomic changes in clinical samples are expensive and require abundant biological sample material for analysis. We have developed a novel assay (“AcceSssIble”) to simultaneously determine DNA methylation and chromatin accessibility in clinical samples. It is rapid and cost-effective, only requiring 20 mg of tissue, the Infinium HumanMethylation450 BeadChip platform, and the CpG methyltransferase M.SssI. We used this method to measure the changes in DNA methylation and chromatin accessibility in 9 matched pairs of ccRCC tumors and adjacent normal tissue from different patients, and intersected this data with RNA-seq data of 72 matched ccRCC samples and DNA methylation data of 160 matched ccRCC samples from The Cancer Genome Atlas (TCGA). Genes that were revealed to have the most changes in chromatin structure and expression were then targeted by siRNA knockdown for functional validation in ccRCC. Results: From the AcceSssIble assay on 9 pairs of ccRCC patient tumor/normal samples, we uncovered 438 genes whose promoters change in chromatin accessibility in at least 2 ccRCC samples, both dependent and independent of DNA methylation changes, and have an accompanying change in gene expression in TCGA RNA-seq data. The results produce a striking figure in which chromatin accessibility changes are inversely correlated with DNA methylation but directly correlated with gene expression changes. Interestingly, loss of (DNA methylation change-dependent) accessibility preferentially occurred within CpG islands, while gain of (DNA methylation change-dependent) accessibility was strongly biased towards non-CpG islands. Meanwhile, chromatin accessibility changes independent of DNA methylation changes do not show preference in CpG content. Furthermore, pathway analyses reveal involvement of HIF1α signaling, cAMP-mediated signaling, and G-protein Coupled Receptor Signaling in the development of ccRCC. Lastly, we performed siRNA knockdown experiments on several top genes most changing in expression and accessibility, which revealed two genes, encoding type IV collagen and an RNA-binding protein, whose knockdown resulted in a significant increase in proliferation in normal kidney epithelial cells. Conclusions: Our study revealed a vast number of chromatin accessibility and accompanying gene expression changes that occur in gene promoters in the development of ccRCC, both dependent and independent of DNA methylation changes. Each individual tumor has a unique profile of epigenetic alterations. Moreover, almost none of the genes that were found to undergo epigenetic and resulting gene expression changes overlap with TCGA's findings of commonly mutated genes in ccRCC. Overall, these studies represent novel approaches that can help identify new therapeutic target genes and treatment strategies for ccRCC, including personalized approaches. Citation Format: Elinne Coral Becket, Christopher Duymich, Yin-Wei Chang, Kurinji Pandiyan, Peter Nichols, Peter Jones, Inderbir Gill, Gangning Liang. Elucidation of epigenetic driver genes in clear cell renal cell carcinoma using a newly developed assay, AcceSssIble. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-05.

Adequate infiltration through Permeable Interlocking Concrete Pavements (PICPs) is critical to their hydraulic and stormwater treatment performance. Infiltration is affected by clogging caused by the trapping of fines in the PICP surface, which, over time, reduces treatment performance. Clogging can be reduced by periodic maintenance such as vacuum sweeping and/or pressure washing. Maintenance requirements can be indicated by measuring reduced infiltration rates. This paper compared infiltration results using the standard test (C1781M-14a) with the results of a new stormwater infiltration field test (SWIFT) developed in Australia to evaluate the maintenance requirements of PICPs. A strong correlation (Pearson’s r = −0.714) was found between results using the two methods. This study found that the SWIF T was a reliable method for es timating the degree of clogging of PICPs while successfully overcoming some of the problems with the more technical existing test methodology such as horizontal water leakage (use of sealant), unrealistic pressure heads, speed of test, and portability. The SWIFT test is a simple, fast and inexpensive way for asset managers and local government employees to quickly assess the maintenance requirements of PICP installations in the field.

Water Sensitive Urban Design (WSUD) treatment devices are often used to restore natural drainage properties in developed catchments. WSUD can make positive contributions to the restoration of natural ecosystem processes, by supporting trees and habitats in urban areas without taking up limited urban space. This paper reports on the development and testing of a new WSUD device, the Wicking Tank. It is designed to supply sufficient volumes of water to urban trees through periods of drought via synthetic wicks from an underground storage tank to support adequate tree health. Relying on gravity fed stormwater, and the natural capillarity, adhesion, and cohesion properties of water and the process of hydraulic redistribution, water is transferred from the tank and into the rhizosphere of the tree. Water demand is controlled passively by the water potential differential across the root zone. Proof of concept testing of the Wicking Tank has shown the device to successfully draw water into soil to support the ongoing survival of a potted plant for over 20 weeks. Substantial differences are anticipated between this proof of concept test and an in-situ field trial. A field-based demonstration style version of the Wicking Tank is planned for construction and testing in 2015.

Adequate infiltration through Permeable Interlocking Concrete Pavements (PICPs) is critical to their hydraulic performance. Detected by monitoring infiltration performance, reduced infiltration rates can indicate that maintenance is required. Measurement of infiltration rates has previously been problematic on PICPs because of a lack of accepted standard methodologies and the practical difficulties in modifying existing testing methodologies. On large sites, standard methodologies necessitate multiple measurements to achieve accuracy. Standard methods also contend with practical issues such as sealing the rings to the surface to prevent lateral water flow. This study examined the performance of two PICP surface infiltration rate measurement methods: a modified double-ring infiltrometer (DRIT), and a specially designed rainfall simulation infiltrometer (RSIT). A positive correlation (R2 = 0.85) of results was found between the two, demonstrating that the RSIT was comparable to the DRIT. The modified DRIT produced surface infiltration results approximately 60% higher than the RSIT results. The RSIT provided lower variation between tests, requiring fewer measurements in large sites whilst still maintaining accuracy, thereby improving testing efficiency. The new RSIT method also eliminates some of the practical difficulties with existing methodologies such as unrealistic pressure heads artificially increasing infiltration rates, and the use of sealant under test measurement infiltration rings.

The classification working group of the International Society of Urological Pathology consensus conference on renal neoplasia was in charge of making recommendations regarding additions and changes to the current World Health Organization Classification of Renal Tumors (2004). Members of the group performed an exhaustive literature review, assessed the results of the preconference survey and participated in the consensus conference discussion and polling activities. On the basis of the above inputs, there was consensus that 5 entities should be recognized as new distinct epithelial tumors within the classification system: tubulocystic renal cell carcinoma (RCC), acquired cystic disease-associated RCC, clear cell (tubulo) papillary RCC, the MiT family translocation RCCs (in particular t(6;11) RCC), and hereditary leiomyomatosis RCC syndrome-associated RCC. In addition, there are 3 rare carcinomas that were considered as emerging or provisional new entities: thyroid-like follicular RCC; succinate dehydrogenase B deficiency-associated RCC; and ALK translocation RCC. Further reports of these entities are required to better understand the nature and behavior of these highly unusual tumors. There were a number of new concepts and suggested modifications to the existing World Health Organization 2004 categories. Within the clear cell RCC group, it was agreed upon that multicystic clear cell RCC is best considered as a neoplasm of low malignant potential. There was agreement that subtyping of papillary RCC is of value and that the oncocytic variant of papillary RCC should not be considered as a distinct entity. The hybrid oncocytic chromophobe tumor, which is an indolent tumor that occurs in 3 settings, namely Birt-Hogg-Dubé Syndrome, renal oncocytosis, and as a sporadic neoplasm, was placed, for the time being, within the chromophobe RCC category. Recent advances related to collecting duct carcinoma, renal medullary carcinoma, and mucinous spindle cell and tubular RCC were elucidated. Outside of the epithelial category, advances in our understanding of angiomyolipoma, including the epithelioid and epithelial cystic variants, were considered. In addition, the apparent relationship between cystic nephroma and mixed epithelial and stromal tumor was discussed, with the consensus that these tumors form a spectrum of neoplasia. Finally, it was thought that the synovial sarcoma should be removed from the mixed epithelial and mesenchymal category and placed within the sarcoma group. The new classification is to be referred to as the International Society of Urological Pathology Vancouver Classification of Renal Neoplasia.

The International Society of Urologic Pathology 2012 Consensus Conference on renal cancer, through working group 3, focused on the issues of staging and specimen handling of renal tumors. The conference was preceded by an online survey of the International Society of Urologic Pathology members, and the results of this were used to inform the focus of conference discussion. On formal voting a Z65% majority was considered a consensus agreement. For specimen handling it was agreed that with radical nephrectomy specimens the initial cut should be made along the long axis and that both radical and partial nephrectomy specimens should be inked. It was recommended that sampling of renal tumors should follow a general guideline of sampling 1 block/cm with a minimum of 3 blocks (subject to modification as needed in individual cases). When measuring a renal tumor, the length of a renal vein/caval thrombus should not be part of the measurement of the main tumor mass. In cases with multiple tumors, sampling should include at a minimum the 5 largest tumors. There was a consensus that perinephric fat invasion should be determined by examining multiple perpendicular sections of the tumor/perinephric fat interface and by sampling areas suspicious for invasion. Perinephric fat invasion was defined as either the tumor touching the fat or extending as irregular tongues into the perinephric tissue, with or without desmoplasia. It was agreed upon that renal sinus invasion is present when the tumor is in direct contact with the sinus fat or the loose connective tissue of the sinus, clearly beyond the renal parenchyma, or if there is involvement of any endothelium-lined spaces within the renal sinus, regardless of the size. When invasion of the renal sinus is uncertain, it was recommended that at least 3 blocks of the tumor-renal sinus interface should be submitted. If invasion is grossly evident, or obviously not present (small peripheral tumor), it was agreed that only 1 block was needed to confirm the gross impression. Other recommendations were that the renal vein margin be considered positive only when there is adherent tumor visible microscopically at the actual margin. When a specimen is submitted separately as "caval thrombus, "the recommended sampling strategy is to take 2 or more sections to look for the adherent caval wall tissue. It was also recommended that uninvolved renal parenchyma be sampled by including normal parenchyma with tumor and normal parenchyma distant from the tumor. There was consensus that radical nephrectomy specimens should be examined for the purpose of identifying lymph nodes by dissection/palpation of the fat in the hilar area only; however, it was acknowledged that lymph nodes are found in

The International Society of Urological Pathology 2012 Consensus Conference made recommendations regarding classification, prognostic factors, staging, and immunohistochemical and molecular assessment of adult renal tumors. Issues relating to prognostic factors were coordinated by a workgroup who identified tumor morphotype, sarcomatoid/rhabdoid differentiation, tumor necrosis, grading, and microvascular invasion as potential prognostic parameters. There was consensus that the main morphotypes of renal cell carcinoma (RCC) were of prognostic significance, that subtyping of papillary RCC (types 1 and 2) provided additional prognostic information, and that clear cell tubulopapillary RCC was associated with a more favorable outcome. For tumors showing sarcomatoid or rhabdoid differentiation, there was consensus that a minimum proportion of tumor was not required for diagnostic purposes. It was also agreed upon that the underlying subtype of carcinoma should be reported. For sarcomatoid carcinoma, it was further agreed upon that if the underlying carcinoma subtype was absent the tumor should be classified as a grade 4 unclassified carcinoma with a sarcomatoid component. Tumor necrosis was considered to have prognostic significance, with assessment based on macroscopic and microscopic examination of the tumor. It was recommended that for clear cell RCC the amount of necrosis should be quantified. There was consensus that nucleolar prominence defined grades 1 to 3 of clear cell and papillary RCCs, whereas extreme nuclear pleomorphism or sarcomatoid and/or rhabdoid differentiation defined grade 4 tumors. It was agreed upon that chromophobe RCC should not be graded. There was consensus that microvascular invasion should not be included as a staging criterion for RCC.

Background Slow-growing prostate cancer (PC) can be aggressive in a subset of cases. Therefore, prognostic tools to guide clinical decision-making and avoid overtreatment of indolent PC and undertreatment of aggressive disease are urgently needed. PC has a propensity to be multifocal with several different cancerous foci per gland. Results Here, we have taken advantage of the multifocal propensity of PC and categorized aggressiveness of individual PC foci based on DNA methylation patterns in primary PC foci and matched lymph node metastases. In a set of 14 patients, we demonstrate that over half of the cases have multiple epigenetically distinct subclones and determine the primary subclone from which the metastatic lesion(s) originated. Furthermore, we develop an aggressiveness classifier consisting of 25 DNA methylation probes to determine aggressive and non-aggressive subclones. Upon validation of the classifier in an independent cohort, the predicted aggressive tumors are significantly associated with the presence of lymph node metastases and invasive tumor stages. Conclusions Overall, this study provides molecular-based support for determining PC aggressiveness with the potential to impact clinical decision-making, such as targeted biopsy approaches for early diagnosis and active surveillance, in addition to focal therapy. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1129-3) contains supplementary material, which is available to authorized users.

history. Without doctor’s explanation the CRR was decreased in patients with urologic medical history. CONCLUSIONS: The RR of FVC was increased by doctor’s explanation in patients aged

Over the last two decades bioretention (biofiltration) systems have been commonly constructed in urban areas to manage stormwater runoff by moderating peak flows and reducing downstream pollution loads. Bioretention systems are generally soil-plant based systems which typically include a filter medium above a drainage layer. They are often either lined with a geofabric to support infiltration, or with an impermeable membrane to prevent infiltration and/or to allow stormwater harvesting and reuse. Bioretention systems are known to treat a range of stormwater pollutants through physical, chemical and biological processes such as mechanical filtering, sedimentation, adsorption, and plant and microbial uptake. However, the long-term pollution removal performance, particularly of heavy metals, remains largely unknown. It is generally accepted that the filter media used in bioretention systems has a finite life span, after which time it should be replaced. However, there is only very limited information available on when this should occur, or how to assess this. It is also recognised that contaminated filter media may require regulated disposal. This study presents results from a series of controlled field experiments conducted over two years which evaluated the pollution removal performance of a series of 10 year old bioretention systems located in an industrial state in Australia.;

Background The value of lymph node dissection (LND) in the treatment of bladder urothelial carcinoma is well established. However, standards for the quality of LND remain controversial. Objective We compared the distribution of lymph node (LN) metastases in a two-institution cohort of patients undergoing radical cystectomy (RC) using a uniformly applied extended LND template. Design, setting, and participants Patients undergoing RC at the University of Southern California (USC) Institute of Urology and at Oregon Health Sciences University (OHSU) were included if they met the following criteria: (1) no prior pelvic radiotherapy or LND; (2) lymphatic tissue submitted from all nine predesignated regions, including the paracaval and para-aortic LNs; (3) bladder primary; and (4) category M0 disease. The number and location of LN metastases were prospectively entered into corresponding databases. Measurements LN maps were constructed and correlated with preoperative and pathologic characteristics. Kaplan-Meier curves were constructed to estimate overall survival (OS) and recurrence free survival (RFS) among LN-positive (LN+) patients. Results and limitations Inclusion criteria were met by 646 patients (439 USC, 207 OHSU), and 23% had LN metastases at time of cystectomy. Although there was a difference in the median per-patient LN count between institutions, there were no significant interinstitutional differences in the incidence or distribution of positive LNs, which were found in 11% of patients with ≤pT2b and in 44% of patients with ≥pT3a tumors. Among LN+ patients, 41% had positive LNs above the common iliac bifurcation. Estimated 5-yr RFS and OS rates for LN+ patients were 45% and 33%, respectively, and did not differ significantly between institutions. Conclusions LN metastases in regions outside the boundaries of standard LND are common. Adherence to meticulous dissection technique within an extended template is likely more important than total LN count for achieving optimal oncologic outcomes.

Background: Aetiology of acute myeloid leukaemia (AML) is not well understood, perhaps because of its distinct subtypes. High-dose ionising radiation is a known risk factor, but less is known about risk from low-dose exposure such as from diagnostic radiography. Methods: Subjects were 412 matched case-control pairs. Ten-year subject histories of diagnostic radiography were based on interview and medical records. Results: There was no convincing association between AML risk and ionising radiation exposure from diagnostic imaging procedures, either for AML overall or for any AML subtype. Conclusion: The association between diagnostic radiography and AML risk remains uncertain.

Few predictive markers exist for response to adjuvant chemotherapy in breast cancer. The 78-kDa glucose-regulated protein (GRP78) is a potent antiapoptotic factor, conferring drug resistance. Recently, we reported that high GRP78 expression in breast cancer specimens predicts a shorter recurrence-free survival in patients who received doxorubicin-based adjuvant chemotherapy. Interestingly, the opposite effect was observed in 25 patients who additionally received a taxane. To confirm this potentially paradigm shifting finding, we investigated whether GRP78 is associated with recurrence-free survival in an independent cohort of taxane-treated breast cancer patients. Immunohistochemical staining of GRP78 was performed on archival paraffin-embedded formalin-fixed tumor specimens obtained from 48 female breast cancer patients before chemotherapy treatment. These patients received doxorubicin and cyclophosphamide, followed by paclitaxel or docetaxel on a clinical trial. GRP78 expression level was evaluated by a pathologist, masked to all clinical and outcome data. Association between GRP78 expression and recurrence-free survival was evaluated. GRP78 positivity predicts a better recurrence-free survival, independent of other prognostic factors [hazard ratio (HR) for moderate positivity: 0.40 (95% confidence interval (CI): 0.087-1.83); HR for strong positivity: 0.16 (95% CI: 0.018-1.50); p(trend) = 0.053]. In a pooled analysis with the previous 25 patients, almost identical HRs were obtained with p(trend) = 0.024. This provides further evidence that GRP78 is a potential independent predictor for response to taxane-based adjuvant chemotherapy in breast cancer.

Planning for the restoration of degraded ecosystems has a strong basis in facilitation successional theory, which, as applied in restoration practice, states that planting of structurally dominant tree species will assist the entry of other native species into a restored community. In Australia, tree planting has been widely applied in restoration of grassy woodland ecosystems. Trees have been postulated to reduce the cover and diversity of weed species, thus facilitating recolonization of native woodland species (indirect facilitation). The expected outcomes of this process include reduced species richness and abundance of exotic plant species and increased species richness and abundance/dominance of natives in areas beneath tree canopies, with these trends strengthening with time. To assess whether this was occurring, we carried out a comparative analysis of species assemblages found underneath and outside of planted tree canopies in sites replanted with juvenile canopy tree species 3–5 or 8–10 years previously. We sampled revegetated stands of Cumberland Plain Woodland, an endangered ecological community in Western Sydney, Australia. We found that neither the number nor abundance of native ground layer species beneath canopies increased as a result of trees being planted at sites of both ages. Where seed is limited, we predicted an increase in abundance of existing native species under planted tree canopies. On this point, the results were mixed and showed some natives with an increased abundance while others decreased. Exotic species richness showed the reverse of the expected pattern, being greater under tree canopies. These findings lend no support to the theory of indirect facilitation. We conclude that simple facilitation models may be inadequate to support planning of grassy woodland restoration and that those models incorporating successional time lags and restoration barriers are likely to be more informative about the development of communities initiated by tree planting.

To use a clinical model of androgen-deprivation therapy (ADT) followed by radical prostatectomy (RP) to test the hypothesis that prostatic intraepithelial neoplasia (PIN, a premalignant lesion of the prostate causally linked to prostate cancer) is heterogeneous for hormone responsiveness, which might explain aspects of the heterogeneity of the natural history of prostate cancer, for although ADT has been used to reduce prostate cancer, there are controversial data on the effect of ADT on PIN.We assessed retrospectively patients with biopsy-confirmed prostate cancer who had RP; some patients had receivedor=3 months of ADT at the discretion of their surgeons, and patients from the same cohort who did not have ADT were used as controls. Patients were sequentially selected from the database and their pathology slides were reviewed by a pathologist unaware of the initial presence of PIN (assessed by an independent observer). Fisher's exact test was used to compare the proportions of patients who had residual PIN in the study and control groups. Exact logistic regression was used to evaluate the duration of ADT on PIN regression.Eighteen patients initially diagnosed with PIN who had no ADT were identified, and 28 with PIN who had ADT were also assessed. All patients who had had no ADT had residual PIN, whereas seven of 28 receiving ADT had no residual PIN (P=0.043). The evaluation of ADT between responders and nonresponders showed a statistically significant association between PIN regression and the duration of ADT (P0.001). However, the response of PIN to ADT was not uniform, as 16% of patients on ADT for6 months had residual PIN, suggesting variable sensitivity of PIN to ADT.These results show that ADT causes PIN to regress, and that there is heterogeneity in this effect with the duration of ADT. We propose future prospective, multicentre, randomized trials in which the effect of ADT on PIN is characterized further.

Objectives To determine whether any differences exist in neuroendocrine (NE) cell differentiation in normal prostates among various ethnic groups because the incidence and mortality from prostate cancer vary across racial groups. Methods Archived paraffin-embedded prostate samples not containing any malignancy were obtained from cystoprostatectomy specimens. Prostatic tissue was obtained from 15 African Americans, 13 Hispanics, 15 Asians, and 16 whites. NE cells were identified based on immunoreactivity for chromogranin A. The mean number of NE cells per high power field (HPF) for each patient was determined using a visual quantitative method by two observers who were unaware of the race of the patients. Results The geometric mean number of NE cells was 6.1/HPF for Asians, 5.6/HPF for whites, 4.0/HPF for Hispanics, and 0.7/HPF for African Americans. A highly significant difference was observed in the distribution of NE cells between African Americans and each of the other races (P ≤0.003). A trend toward greater NE expression was noted in Asians and whites compared with Hispanics; however, this difference did not reach statistical significance. Conclusions We found a fivefold to eightfold difference in the distribution of NE cells in the normal prostates of African-American men compared with that of other races. The comparatively low NE cell expression in African-American men may play a role in the greater rate of prostate carcinogenesis seen in this ethnic group.

Purpose: There is increasing evidence for a fundamental role for epigenetic silencing of apoptotic pathways in cancer. Changes in DNA methylation can be detected with a high degree of sensitivity, so we used the MethyLight assay to determine how methylation patterns of apoptosis-associated genes change during bladder carcinogenesis and whether DNA methylation could be detected in urine sediments. Experimental Design: We analyzed the methylation status of the 5′ regions of 12 apoptosis-associated genes (ARF, FADD, TNFRSF21, BAX, LITAF, DAPK, TMS-1, BCL2, RASSF1A, TERT, TNFRSF25, and EDNRB) in 18 bladder cancer cell lines, 127 bladder cancer samples, and 37 samples of adjacent normal bladder mucosa using the quantitative MethyLight assay. We also analyzed the methylation status in urine sediments of 20 cancer-free volunteers and 37 bladder cancer patients. Results: The 5′ regions of DAPK, BCL2, TERT, RASSFIA, and TNFRSF25 showed significant increases in methylation levels when compared with nonmalignant adjacent tissue (P ≤ 0.01). Methylation levels of BCL2 were significantly associated with tumor staging and grading (P ≤ 0.01), whereas methylation levels of RASSF1A and ARF were only associated with tumor stage (P ≤ 0.04), and TERT methylation and EDNRB methylation were predictors of tumor grade (P ≤ 0.02). To investigate clinical usefulness for noninvasive bladder cancer detection, we further analyzed the methylation status of the markers in urine samples of patients with bladder cancer. Methylation of DAPK, BCL2, and TERT in urine sediment DNA from bladder cancer patients was detected in the majority of samples (78%), whereas they were unmethylated in the urine sediment DNA from age-matched cancer-free individuals. Conclusions: Our results indicate that methylation of the 5′ region of apoptosis-associated genes is a common finding in patients with bladder carcinoma. The ability to detect methylation not only in bladder tissue, but also in urine sediments, suggests that methylation markers are promising tools for noninvasive detection of bladder cancers. Our results also indicate that some methylation markers, such as those in regions of RASSF1A and TNFRSF25, might be of limited use for detection because they are also methylated in normal bladder tissues.

Transitional cell carcinoma (TCC) of the bladder that extends directly into contiguous organs (pT4) portends a poor prognosis. The 2002 American Joint Committee on Cancer staging system does not include seminal vesicle involvement by primary TCC of the bladder. In this analysis we evaluated the clinical outcomes and prognostic significance of seminal vesicle involvement with TCC of the bladder after radical cystectomy.From 1971 to 2001, 1,682 patients underwent radical cystectomy and pelvic lymphadenectomy for bladder cancer. Only those tumors that involved adjacent organs through the bladder wall (pT4) were included. Overall 132 male patients with a median age of 68 years (range 36 to 98) qualified for analysis. Patients were stratified into 4 subgroups of 1) direct extravesical prostatic stromal involvement only in 37 patients (28%), 2) prostatic stroma and seminal vesicle involvement in 37 patients (28%), 3) seminal vesicle involvement only in 10 patients (8%) and 4) other contiguous pelvic organ involvement (stage pT4b) in 48 patients (36%). Overall 88 patients (67%) received some form of adjuvant therapy. At a median followup of 12.5 years (range 0 to 15.2) clinical outcomes were analyzed including overall and recurrence-free survival using Kaplan-Meier plots.There was no significant difference in clinical outcomes or prognosis for groups 2 and 3, thus they were combined for analysis. Five-year overall survival for any seminal vesicle involvement (10%) was significantly worse than prostatic stromal involvement only (38%) but was similar to pT4b tumors (7%, p0.0001). The 5-year recurrence-free survival for seminal vesicle involvement (14%) was also significantly worse than prostatic stromal involvement alone (68%) but similar to that pT4b disease (25%, p = 0.01). Results were controlled for lymph node status.Patients with extravesical tumor extension into seminal vesicles and contiguous pelvic organs are at high risk for recurrence and progression. Involvement of the seminal vesicles by direct extension of bladder TCC portends a prognosis similar to that of pT4b disease and should, therefore, be classified as such.

The benefits of nationally coordinated schemes to manage the introduction of new stormwater quality improvement devices (SQIDs) have been acknowledged in various countries. This review compares the existing frameworks used in Australia, Austria, Germany, New Zealand, Switzerland, and the United States. The review describes the framework and structure of each system, and their individual components are presented. The Australian and New Zealand protocols have yet to be formally adopted and remain largely untested. The Australian and New Zealand protocols are compared with successfully established processes operating in Austria, Germany, Switzerland, and the United States. Adequate resourcing of administrative processes and having the technical capability to review test results were found to be a critical component of the successfully adopted certification schemes. Based on the level of success of the introduction of alternative certification systems, the most successful have been integrated into the formal legislative frameworks found in each country. The creation of a successful SQID certification pathway in Germany has been driven by the implementation of new fees on the release of stormwater from private property, with exemptions where stormwater is actively managed at source. The review presented in this paper may be of benefit to countries considering the development of their own SQID evaluation and verification protocols.

The trapping of sediments within permeable interlocking concrete pavements (PICPs) during infiltration is a key process that contributes to their pollution removal performance. This process also leads to clogging which decreases infiltration capacity and reduces pollution removal performance. Previous studies have shown clogging to be caused by a number of factors, including the trapping of fine particles, construction techniques, the lack of maintenance, and the mass of the trapped sediments. However, results of these studies have been varied, and the processes governing clogging are not well understood. This field study, undertaken over 12 months, measured the infiltration rates of PICPs that had been in service for a number of years. The study examined sediment extracted from the surface joints of pavements in order to correlate reduced infiltration with sediment particle size distributions (PSDs). Principal component analysis (PCA) was used to compare the differences in infiltration rates and the differences in PSD size class groups between the sites and sub-sites. This study found it was the 251–550 μm particle sizes that explained the most variation at sites with lower measured infiltration rates. A better understanding of sediment PSDs and PICP clogging should help designers to install these systems with more confidence in their long-term infiltration and pollution reduction performance.;

Previous studies have attempted to quantify the clogging processes of Permeable Interlocking Concrete Pavers (PICPs) using accelerated testing methods. However, the results have been variable. This study investigated the effects that three different sediment types (natural and silica), and different simulated rainfall intensities, and testing durations had on the observed clogging processes (and measured surface infiltration rates) of laboratory-based, accelerated PICP testing studies. Results showed that accelerated simulated laboratory testing results are highly dependent on the type, and size of sediment used in the experiments. For example, when using real stormwater sediment up to 1.18 mm in size, the results showed that neither testing duration, nor stormwater application rate had any significant effect on PICP clogging. However, the study clearly showed that shorter testing durations generally increased clogging and reduced the surface infiltration rates of the models when artificial silica sediment was used. Longer testing durations also generally increased clogging of the models when using fine sediment (< 300 μm). Results from this study will help researchers and designers better anticipate when and why PICPs are susceptible to clogging, reduce maintenance and extend the useful life of these increasingly common stormwater best management practices.

BACKGROUND Downregulation of genes which negatively control cell cycle progression represents a possible mechanism for prostate tumorigenesis. We examined the expression levels of the p16, p15, p14, and retinoblastoma-susceptibility (RB) genes in primary prostate cancers and human prostate cancer cell lines, and correlated this with the DNA methylation levels of two loci in p16. METHODS The mRNA levels of p16, p15, and p14 were examined by reverse transcriptase-PCR (RT-PCR). DNA methylation of the p16 5′ CpG island was determined by bisulfite genomic sequencing, while methylation of exon 2 shared by the p16 and p14 genes was measured by a quantitative bisulfite-based technique, methylation-sensitive single-nucleotide primer extension (Ms-SNuPE). RB protein levels were assessed by immunohistochemical staining of histologic sections of normal and tumor prostate tissues, using a monoclonal antibody (mAB). RESULTS Overexpression of p16 mRNA was found in 6/9 (67) of prostate tumors compared to the adjacent normal prostate, whereas elevated p14 and p15 levels were only observed in 2/9 (22) and 1/6 (17) of prostate cases, respectively. There was no statistically significant association of grade (P = 0.18) and stage (P = 1.00) of prostate cancer to the elevated p16 levels in the tumors. The p16 5′ CpG island was completely unmethylated in these tissues. In contrast, exon 2 of p16/p14 was methylated in both the tumor and normal adjacent prostates, and was increased in 8/11 (73) of tumors relative to normal tissues. There was no association between p16 overexpression and increased p16/p14 exon 2 methylation in these tumors (P = 1.00). Diminished RB levels in prostate tumors that had upregulated p16 mRNA were found, although absent RB was also detected in tumors without elevated p16 levels. The expression levels of the two genes, RB and p16, were not correlated statistically (P = 0.16). CONCLUSIONS Our studies show that although the levels of the cell cycle regulators p16, p15, p14, and Rb are altered in prostate cancers, there is no apparent correlation to grade, stage, or any pattern of regulation between the related genes. Exon 2 of p16/p14 is methylated in a majority of prostate tumors compared to the unmethylated upstream 5′ region, and may be a potential tumor marker for human prostate cancer. Prostate 43:233–242, 2000. © 2000 Wiley-Liss, Inc.

Prostate cancer is a serious public health problem in many industrialized countries.Androgens appear to play a critical role in its etiology. Specifically, the active androgen in the prostate, dihydrotestosterone (DHT) which is synthesized by the enzyme steroid 5 alpha-reductase from testosterone (T), acts as a mitogen. Hence androgen-deprivation is commonly used during prostate cancer therapy. Two isozymes for steroid 5 alpha-reductase have been reported. The type II enzyme is prostate-specific and encoded by the SRD5A2 gene. We have investigated a polymorphic (TA)n dinucleotide repeat in the 3′ UTR (untranslated region) of the SRD5A2 gene in 30 matched samples of constitutional (“germline”) DNA from peripheral blood lymphocytes and microdissected, pure tumor DNA. We report here 8 LOH (loss of heterozygosity) events and 9 cases of microsatellite instability at this marker. Therefore, almost 57% of the samples examined showed evidence of somatic mutations at the 3' UTR of the SRD5A2 locus. Our dat...

Purpose: We determined the extent of p53 immunoreactivity in pathological stage C prostate cancer as well as its correlation to tumor grade, substage, recurrence and proliferation rate. To define better the temporal relationship of p53 nuclear reactivity in prostate cancer p53 immunoreactivity was evaluated in all associated prostatic intraepithelial neoplasia lesions.Materials and Methods: Using immunohistochemistry p53 status and proliferation rate were determined in 96 tumors from patients with pathological stage C prostate cancer. Single strand conformational polymorphism in exons 5 to 8 was used in a subset of specimens to assess the association of p53 nuclear accumulation with mutations in the p53 gene.Results: p53 Nuclear reactivity was demonstrated in 10 tumors (10.4%), including 6 with high and 4 with low level nuclear reactivity. Of the tumors 86 (89.6%) had no evidence of p53 immunoreactivity. Each of the 6 tumors with high level p53 reactivity had associated areas of prostatic intraepi...

International audience; The International Society of Urological Pathology convened a consensus conference on renal cancer, preceded by an online survey, to address issues relating to the diagnosis and reporting of renal neoplasia. In this report, the role of biomarkers in the diagnosis and assessment of prognosis of renal tumors is addressed. In particular we focused upon the use of immunohistochemical markers and the approach to specific differential diagnostic scenarios. We enquired whether cytogenetic and molecular tools were applied in practice and asked for views on the perceived prognostic role of biomarkers. Both the survey and conference voting results demonstrated a high degree of consensus in participants' responses regarding prognostic/predictive markers and molecular techniques, whereas it was apparent that biomarkers for these purposes remained outside the diagnostic realm pending clinical validation. Although no individual antibody or panel of antibodies reached consensus for classifying renal tumors, or for confirming renal metastatic disease, it was noted from the online survey that 87% of respondents used immunohistochemistry to subtype renal tumors sometimes or occasionally, and a majority (87%) used immunohistochemical markers (Pax 2 or Pax 8, renal cell carcinoma [RCC] marker, panel of pan-CK, CK7, vimentin, and CD10) in confirming the diagnosis of metastatic RCC. There was consensus that immunohistochemistry should be used for histologic subtyping and applied before reaching a diagnosis of unclassified RCC. At the conference, there was consensus that TFE3 and TFEB analysis ought to be requested when RCC was diagnosed in a young patient or when histologic appearances were suggestive of the translocation subtype; whereas Pax 2 and/or Pax 8 were considered to be the most useful markers in the diagnosis of a renal primary.

A high frequency (24%) of acute promyelocytic leukemia (APL) was noted among acute myelocytic leukemia (AML) cases at the Los Angeles County-University of Southern California (LAC-USC) Medical Center, in comparison with the expected frequency of 5% to 15%. Because of the high proportion of Latinos in this center, we questioned if APL is more common in this ethnic group. The proportion of APL among the 80 AML patients of Latino origin was significantly higher (30; 37.5%) when compared with the 62 non-Latinos (4; 6.5%) (P = .00001). In an attempt to verify this finding on a larger group of patients, we analyzed 276 pathologically verified cases of AML in patients aged 30 to 69 years from the entire County of Los Angeles, registered on an ongoing population-based epidemiologic study of AML. APL was more frequent among the 47 Latinos (24.3%) than in the 229 non-Latinos (8.3%) (P = .0075). APL is seen in younger patients with AML, but Latino AML patients also had a higher frequency of APL after accounting for their younger age (age-adjusted odds ratio for APL among Latinos in LAC-USC Medical Center, 9.4 [95% confidence interval (CI) 2.9, 30] P = .0002; among Latinos in the population-based study, 3.0 [95% CI 1.3 to 6.9] P = .01). The different ethnic distribution of AML was found to be due to a higher proportion of APL cases per se, and not to a lower proportion of any other French-American-British subtype (P = .0004). These results, from two different populations of AML patients, indicate that Latinos with AML have a higher likelihood of the APL subtype of disease, which may suggest a genetic predisposition to APL and/or exposure to distinct environmental factor(s).

Background : Tumor stage, histologic grade, and regional lymph node status are currently used to obtain prognostic information about bladder cancers. However, additional prognostic indicators are needed to aid clinicians in selecting patients who would benefit most from specific therapies. A majority of studies assessing the prognostic value of measuring tumor angiogenesis (i.e., measurement of tumor microvessel densities) have found a positive association between increasing microvessel densities and worsening prognosis. Purpose : We explored the relationship between established prognostic indicators and the extent of tumor-associated angiogenesis in patients with invasive transitional cell carcinoma (TCC) of the bladder, and we determined whether tumor microvessel density measurement could be used independently to predict bladder tumor behavior. Methods : Tumor tissue was obtained from 164 patients with invasive primary TCC of the bladder. The extent of tumor-associated angiogenesis in this tissue was evaluated by immunohistochemical methods using HPCA-1, a mouse monoclonal antibody directed against the endothelial cell antigen, CD34. The number of microvessels in a 200x microscopic high-power field (hpf) containing the area of greatest neovascularization within or immediately adjacent to each tumor was determined. The patient population was then divided into three equivalently sized groups, with tumors containing low (≤64), intermediate (65-99), or high (≥100) numbers of microvessels per hpf. Kaplan-Meier product limit estimates of overall survival and the complement of cumulative incidence curves for recurrence-free survival were plotted. When analyzing survival or recurrence, the logrank test was used to compare groups of patients with and without stratification according to tumor stage. Analysis of variance was used to test for an association between microvessel density and established prognostic variables. Reported P values are from two-sided tests. Results : Microvessel density was significantly associated with disease-free (P

Background : In the Western Hemisphere, 90% of bladder cancers are transitional cell carcinomas, while only 7% are classified as squamous cell carcinomas. In contrast, in Egypt and regions of the Middle East and Africa, where infection by the trematode Schistosoma haematobium is endemic, squamous cell carcinoma is the most common bladder cancer as well as the most common cancer in men. Purpose : We planned experiments to understand the genetic defects underlying the development of squamous cell carcinoma and to determine if the morphologically and clinically distinct squamous cell carcinoma and transitional cell carcinoma of the bladder evolve following different genetic alterations. Methods : Squamous cell carcinoma specimens from high-risk (Egypt, n = 19) and low-risk (Sweden, n = 12) populations were examined for genetic defects known to be involved in transitional cell carcinoma tumorigenesis. Homozygous deletions of the CDKN2 tumor suppressor gene were detected by comparative multiplex polymerase chain reaction. Mutations in the CDKN2 and p53 (also known as TP53) genes were analyzed by single-strand conformation polymorphism and DNA sequencing. Immunohistochemical staining of p53 protein was also performed. Allelic losses in chromosome arms 9p, 9q, and 17p were determined by microsatellite analysis. Results : Homozygous deletions and sequence mutations in the CDKN2 gene were found in 67% (eight of 12) of squamous cell carcinoma specimens, a frequency three times higher than that reported for uncultured transitional cell carcinomas (P =.009). Hemizygous and homozygous deletions in 9p, where CDKN2 resides, were found in 92% (11 of 12) of uncultured squamous cell carcinomas, while only about 39% (35 of 90) of transitional cell carcinomas showed these losses (P =.001). Deletions in 9p with no change in 9q were found in 92% (10 of 11) of squamous cell carcinomas compared with only 10% (11 of 110) of transitional cell carcinomas (P

Field testing of a proprietary stormwater treatment device (GPT) was undertaken over a one year period at a commercial site located in Sippy Downs, Queensland. The focus of the study was primarily on evaluating the effectiveness of the GPT device in removing pollution in the form of nutrients (Total Suspended Solids, Total Nitrogen, Total Phosphorus) from stormwater runoff. Water quality analysis was performed on water samples taken from the inflow and outflow of the GPT during 15 natural rainfall events. A new testing protocol was developed to ensure a comprehensive investigation of the stormwater treatment performance of the GPT. Pollution treatment Efficiency Ratios (ER) calculated for the GPT were found to be 49.2% for TSS, 26.6% for TN and 40.6% for TP. Although the nutrient removal rates of the GPT observed in the study were below those specified by Queensland regulations, the results are considered notable for a stormwater treatment device that was not specifically designed to remove nutrients from stormwater.

Previous studies of chimeric animals and human tissues have shown the clonal nature of organ development, giving clues as to the normal development of organs and also to abnormal developments, such as atheromatous plaques. The clonal nature of bladder cancer in female patients has been demonstrated, but little has been known of the clonal development of the normal urothelium. Using an X chromosome inactivation analysis of cells microdissected from histologic slides from the female human bladder, macroscopic urothelial patches of monoclonality were detected. These patches are about 120 mm.2 in size, contain about 2 x 10(6) cells each and reflect the presence of coherent cellular families composed of stem cells and their differentiated derivatives. The large size of these patches was surprising when compared with previously reported patch sizes in other organ systems. The patches most probably are composed of the descendants of the original founder cells, which would suggest that only 200 to 300 cells participated in the formation of the urothelium. The limited number of stem cells, each giving rise to millions of cells may provide an explanation for the "field defect" that is often referred to in the pathogenesis of bladder cancer, as different cell patches may possess different predispositions to tumorigenesis.

For over a century there have been many studies that describe the use of rain drop measurement techniques. Initial manual measurement methods evolved due to improved technology to include photographic and, more recently, automated disdrometer and laser measurement techniques. Despite these numerous studies, there have been few comparative reviews of the range of methodologies, and their relative performance. This review explores the raindrop measurement techniques available, and summarizes and classifies the techniques according to the method or principle involved. The requirements of a robust raindrop measurement technique are suggested, and these are reviewed against existing rain drop measurement techniques to provide a comparative guide to the use of the range of techniques available for any research study. This review revealed that while advances in technology have allowed many of the deficiencies of early techniques to be eliminated, challenges remain in relation to the precision of the measurement of the size, shape, and velocity of rain drops.

The contribution of promoter DNA methylation to the alteration of caner related gene expression has been well studied, however, these genes can also potentially be altered by chromatin accessibility without involvement of DNA methylation and can be epigenetically inherited. In this study, we used an assay developed in our laboratory (AcceSssIble) that can simultaneously interrogate DNA methylation and chromatin accessibility allowing us to investigate the epigenetic changes in uncultured clear cell renal cell carcinoma (ccRCC) tumors and normal tissue to uncover genes that contribute to ccRCC tumorigenesis. AcceSssIble is both cost-effective and easily analyzed using simple and straightforward computational analysis. Our study revealed significant changes to the epigenome of ccRCC, especially identifying epigenetically up- (160) or down-regulated genes (180), which are dependent on accessibility changes with (30%) or without (70%) DNA methylation involvement, and which were validated by a cross-correlation of the identified genes with RNA-seq and DNA methylation data from larger cohorts of ccRCC samples from The Cancer Genome Atlas (TCGA). In addition, our findings also revealed these sets of genes not found to be commonly mutated in ccRCC and undergo epigenetic changes at higher frequencies than common ccRCC mutations. In addition, pathway analysis suggests that genetic and epigenetic alterations are independent events, and one such example includes a set of changes in HIF1α signaling pathway genes that are independent of the Von Hippel-Lindau (VHL) status. This suggests a novel epigenetic basis for HIF1αs role during tumorigenesis that may be a common occurrence in ccRCC. Thus, the AcceSssIble analysis on ccRCC samples revealed novel candidates for epigenetic driver genes which were previously undetectable by widely used methylation studies. Overall, this study provides a novel approach that can help identify new epigenetic therapeutic targets and treatment strategies complementing current approaches based primarily on the genetic makeup of primary tumors. Citation Format: Elinne Becket, Sameer Chopra, Christopher Duymich, Lin J. Justin, Jueng Soo You, Kurinji Pandiyan, Peter W. Nichols, Kimberly D. Siegmund, Peter A. Jones, Gangning Liang. Identification of epigenetic regulated genes through simultaneous analysis of DNA methylation and chromatin structure in uncultured tumors. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B03.

We have previously demonstrated a strong association between nuclear accumulation of p53 protein, as determined by immunohistochemical analysis, and mutations in the p53 gene. The purpose of this study was to determine the relation between nuclear accumulation of p53 and tumor progression in transitional-cell carcinoma of the bladder.Histologic specimens of transitional-cell carcinoma of the bladder (stages Pa, noninvasive disease, to P4, disease with direct extension into adjacent organs or structures) from 243 patients who were treated by radical cystectomy were examined for the immunohistochemical detection of p53 protein. Nuclear p53 reactivity was then analyzed in relation to time to recurrence and overall survival.The detection of nuclear p53 was significantly associated with an increased risk of recurrence of bladder cancer (P0.001) and with decreased overall survival (P0.001). In patients with cancer confined to the bladder, the rates of recurrence for stage P1, P2, and P3a tumors that had no detectable nuclear p53 reactivity at five years were 7, 12, and 11 percent, respectively, as compared with 62, 56, and 80 percent, respectively, for tumors that had p53 immunoreactivity. Similar results were obtained when the presence or absence of p53 in the nuclei of the tumor cells was studied in relation to overall survival. In a multivariable analysis stratified according to grade, pathological stage, and lymph-node status, nuclear p53 status was an independent predictor (and in cancer confined to the bladder, the only independent predictor) of recurrence and overall survival (P0.001).In patients with transitional-cell carcinoma confined to the bladder, an accumulation of p53 in the tumor-cell nuclei detected by immunohistochemical methods predicts a significantly increased risk of recurrence and death, independently of tumor grade, stage, and lymph-node status. Patients with transitional-cell carcinoma confirmed to the bladder that demonstrates nuclear p53 reactivity should be considered for protocols of adjuvant treatment.

Despite the fact that 45% of all human gene promoters do not contain CpG islands, the role of DNA methylation in control of non-CpG island promoters is controversial and its relevance in normal and pathological processes is poorly understood. Among the few studies which investigate the correlation between DNA methylation and expression of genes with non-CpG island promoters, the majority do not support the view that DNA methylation directly leads to transcription silencing of these genes. Our reporter assays and gene reactivation by 5-aza-2'-deoxycytidine, a DNA demethylating agent, show that DNA methylation occurring at CpG poor LAMB3 promoter and RUNX3 promoter 1(RUNX3 P1) can directly lead to transcriptional silencing in cells competent to express these genes in vitro. Using Nucleosome Occupancy Methylome- Sequencing, NOMe-Seq, a single-molecule, high-resolution nucleosome positioning assay, we demonstrate that active, but not inactive, non-CpG island promoters display a nucleosome-depleted region (NDR) immediately upstream of the transcription start site (TSS). Furthermore, using NOMe-Seq and clonal analysis, we show that in RUNX3 expressing 623 melanoma cells, RUNX3 P1 has two distinct chromatin configurations: one is unmethylated with an NDR upstream of the TSS; another is methylated and nucleosome occupied, indicating that RUNX3 P1 is monoallelically methylated. Together, these results demonstrate that the epigenetic signatures comprising DNA methylation, histone marks and nucleosome occupancy of non-CpG island promoters are almost identical to CpG island promoters, suggesting that aberrant methylation patterns of non-CpG island promoters may also contribute to tumorigenesis and should therefore be included in analyses of cancer epigenetics.