Ana Celma

Clinical significance of proliferative inflammatory atrophy finding in prostatic biopsies

Proliferative inflammatory atrophy (PIA) has been involved in prostatic carcinogenesis. However, little is known about the clinical significance of a PIA finding in prostatic biopsies (PBs). The aim of this study is to determine the incidence of prostate inflammatory atrophy (PIA) in prostate biopsies (PBs), its association to high‐grade prostatic intraepithelial neoplasia (HGPIN), prostate cancer (PCa), and tumor aggressiveness.

Targeted proteomics in urinary extracellular vesicles identifies biomarkers for diagnosis and prognosis of prostate cancer

Rapid and reliable diagnosis of prostate cancer (PCa) is highly desirable as current used methods lack specificity. In addition, identification of PCa biomarkers that can classify patients into high- and low-risk groups for disease progression at early stage will improve treatment decision-making. Here, we describe a set of protein-combination panels in urinary extracellular vesicles (EVs), defined by targeted proteomics and immunoblotting techniques that improve early non-invasive detection and stratification of PCa patients.We report a two-protein combination in urinary EVs that classifies benign and PCa patients (ADSV-TGM4), and a combination of five proteins able to significantly distinguish between high- and low-grade PCa patients (CD63-GLPK5-SPHM-PSA-PAPP). Proteins composing the panels were validated by immunohistochemistry assays in tissue microarrays (TMAs) confirming a strong link between the urinary EVs proteome and alterations in PCa tissues. Moreover, ADSV and TGM4 abundance yielded a high diagnostic potential in tissue and promising TGM4 prognostic power. These results suggest that the proteins identified in urinary EVs distinguishing high- and low grade PCa are a reflection of histological changes that may be a consequence of their functional involvement in PCa development. In conclusion, our study resulted in the identification of protein-combination panels present in urinary EVs that exhibit high sensitivity and specificity for PCa detection and patient stratification. Moreover, our study highlights the potential of targeted proteomic approaches–such as selected reaction monitoring (SRM)–as diagnostic assay for liquid biopsies via urinary EVs to improve diagnosis and prognosis of suspected PCa patients.

Behavior of chemiluminescent assays to measure serum testosterone during androgen deprivation therapy.

DOI: 10.1111/iju.13180 PCa guidelines recommend ST measurement during ADT in order to evaluate its efficacy and to diagnose the CR status. Regulatory agencies and PCa guidelines consider 50 ng/dL as the castration level of ST, although some studies suggest that lower levels are associated with better outcomes of the disease. During the 1980s, the FDA established the castration level of ST at 50 ng/dL, based on studies carried out in patients who had undergone surgical castration. This level corresponded to the LLOQ from the RIAs that had been used so far. In 2000, using a recently introduced CLIA, Oefelein et al. redefined the castration level of ST at 20 ng/dL in a group of 35 patients who had undergone surgical castration. The median level of ST was 15 ng/dL and the range 2–30 ng/dL. Surprisingly the level selected by the author corresponded to the 75th percentile of the distribution. In 2007, Morote et al. also using a CLIA declared 32 ng/dL to be “the castrate level of ST with clinical impact” because it represented the lowest level of ST capable of significantly discriminating the progression-free survival of CR in a group of 73 non-metastatic PCa patients subjected to LHRH agonist. Nowadays, CLIAs, which are usually integrated in automated platforms, are widely used in clinical practice to measure ST given that they are sensitive, fast and inexpensive. However CLIAs have a disturbing lack of accuracy and reproducibility, especially at low levels. That is why since 2007 the American Endocrine Society recommends only LC/GC MSMS to measure ST. These methods extract the testosterone in an initial step, avoiding its overdetection caused by contamination mainly with other steroids, and MSMS ensures an accurate and reproducible measurement in a second step. However, direct CLIAs continue to be the most widely used method to determine ST in clinical practice. Because of a recent change of the platforms used in our reference laboratory and the lack of information about the behavior of CLIAs to measure ST in PCa patients undergoing ADT, we carried out a prospective study in order to evaluate two commercial CLIAs. Between July and December 2015, ST measurement was requested in 249 patients with histologically confirmed PCa undergoing continuous LHRH agonist treatment as part of their routine control. Blood collection was carried out between 08.00 and 10.00 hours, and two aliquots of serum were processed in the automated Cobas 8000 (Roche Diagnostics Inc., Indianapolis, IN, USA) (PC8000) and the Advia-Centaur XPi (Siemens, Munich, Germany) (PA-CXPi) platforms, both using CLIA to measure ST directly. The LLOQ were 2.5 and 10 ng/dL, respectively. According to the manufacturers’ information, the intra-assay coefficients of variation ranged between 1.7% and 4.6% in the PC8000, and between 2.3% and 6.2% in PA-CXPi. The mean age of the study group was 74 years (range 48–89 years). ADT was indicated as the primary treatment in 89 patients (35.7%) with metastatic PCa, 118 (47.4%) patients who were treated with radiation therapy and 42 (16.9%) patients having a high risk of dissemination failure after primary treatment of PCa. The average period of LHRH agonist treatment was 19.6 months (range 4–57 months). The study was carried out in accordance with the ethical standards of the Helsinki Declaration II, and written informed consent was obtained. The levels of ST measured in the PC8000 were significantly lower than those measured in the PA-CXPi, P < 0.001, and the correlation between both measurements was only moderate, r = 0.55. The median levels and ranges were 7.8 ng/dL (range <2.5–54.6 ng/dL) and 33.4 ng/dL (range <10–91.6 ng/dL), respectively. The 95th percentiles corresponded to 34.6 and 75.9 ng/dL, respectively. These levels were below 20 ng/dL, between 20 and 50 ng/dL, and above 50 ng/dL in 193 (77.5%), 54 (21.7%) and two (0.8%) cases in the PC8000, and 62 (24.9%), 134 (53.8%) and 53 (21.3%) cases in the PA-CXPi (Fig. 1). The present study highlights the different behavior of direct CLIAs to measure ST in PCa patients undergoing ADT. This fact could compromise the ADT efficacy evaluation as well as the diagnosis of CR. As the automated CLIAs continue to be widely used to determine ST, because LC/GC MSMS technology is not available in most clinical laboratories, a careful selection of the CLIA used to measure ST in PCa patients under ADT is recommended.

Clinical Significance of Proliferative Inflammatory Atrophy in Negative Prostatic Biopsies: Pia in Negative Prostatic Biopsies

To analyze the association between prostatic proliferative inflammatory atrophy finding in negative prostate biopsies and future detection of prostate cancer (PCa) and its aggressiveness in men subjected to repeat biopsies, due to persistent suspicion of PCa.

Hormonal response recovery after long-term androgen deprivation therapy in patients with prostate cancer.

Abstract Objective: The aim of this study was to evaluate hormonal recovery after cessation of androgen deprivation therapy (ADT) in a group of elderly prostate cancer patients. Materials and methods: Forty patients with locally advanced or metastatic prostate cancer, with a mean age of 71.5 years [95% confidence interval (CI) 69.1–73.9], were treated with ADT for a mean duration of 74.6 months (95% CI 59.7–89.5 months). Mean follow-up time after ADT cessation was 36.5 months (95% CI 30.6–42.3 months). Serum testosterone and luteinizing hormone (LH) were determined at 6 month intervals after ADT cessation. Results: After 18 months of follow-up, all patients had recovered normal LH levels, while 38% of patients still presented castration levels of testosterone (< 50 ng/dl). A multivariate analysis was performed to find factors related to testosterone recovery (testosterone >50 ng/dl). Neither age at start of ADT nor clinical stage reached statistical significance. Only time under ADT was correlated with testosterone recovery (p = .031). Kaplan–Meier curves were obtained. Mean time for testosterone recovery was 14.5 months (95% CI 6.5–22.6 months) in patients treated with ADT for less than 60 months compared to 29.3 months (95% CI 19.6–39.1 months) in patients treated with ADT for more than 60 months (log-rank p = .029). Conclusions: Age did not correlate with testosterone recovery in a group of elderly prostate cancer patients in whom ADT was stopped. Testosterone recovery after ADT cessation was significantly correlated with time under ADT treatment. Significant implications related to economic aspects of the dosage schedule may be considered.

Serum Testosterone Levels in Prostate Cancer Patients Undergoing Luteinizing Hormone-Releasing Hormone Agonist Therapy

Background Serum testosterone measurement is recommended to assess the efficacy of androgen deprivation therapy (ADT) and to diagnose castration resistance in patients with prostate cancer (PCa). Currently, the accepted castrate level of serum testosterone is 50 ng/dL. Liquid chromatography and tandem mass spectrometry (LC MSMS) is the appropriate method to measure testosterone, especially at low levels. However, worldwide, chemiluminescent assays (CLIAs) are used in clinical laboratories, despite their lack of accuracy and reproducibility, because they are automatable, fast, sensitive, and inexpensive. Materials and Methods We compared serum testosterone levels measured using LC MSMS and CLIAs in 126 patients with PCa undergoing luteinizing hormone‐releasing hormone (LHRH) agonist therapy. Results The median serum testosterone level was 14.0 ng/dL (range, 2.0‐67.0 ng/dL) with LC MSMS and 31.9 ng/dL (range, 10.0‐91.6 ng/dL) with CLIA (P < .001). The serum testosterone levels, measured using LC MSMS, were < 20 ng/dL in 83 patients (65.9%), 20 to 50 ng/dL in 40 (31.7%), and > 50 ng/dL in 3 patients (2.4%). These ranges were found in 34 (27%), 72 (57.1%), and 20 (15.9%) patients when testosterone was measured using CLIA (P < .001). The castrate level of serum testosterone using LC MSMS and CLIA was 39.8 ng/dL (95% confidence interval [CI], 37.1‐43.4 ng/dL) and 66.5 ng/dL (95% CI, 62.3‐71.2 ng/dL), respectively. Conclusion We found that CLIA overestimated the testosterone levels in PCa patients undergoing LHRH agonist therapy. Thus, the castration level was incorrectly considered inadequate with CLIA in almost 15% of patients. The true castration level of serum testosterone using an appropriate method is < 50 ng/dL. Micro‐Abstract Prostate cancer guidelines have recommended serum testosterone measurement during androgen deprivation therapy to assess its efficacy and diagnose castration resistance. The present study compared the testosterone levels from a widely used chemiluminescent assay (CLIA) and liquid chromatography tandem mass spectrometry method, which is the recommended method, in prostate cancer patients undergoing luteinizing hormone‐releasing hormone agonist therapy. The CLIA overestimated the testosterone levels and suggested, incorrectly, the presence of inadequate castration in ≤ 15% of patients.

MP85-16 STUDY OF ALTERED RATIOS OF PROTEIN KINASE CK2 CATALYTIC SUBUNITS AND REGULATORY SUBUNIT (CK2BETA) IN RENAL CELL CARCINOMA. RELATION WITH EPITELIAL-TO-MESENCHYMAL TRANSITION MARKERS (IL-6/STAT3)

INTRODUCTION AND OBJECTIVES: Epitelial-to-mesenchymal transition (EMT) is a well characterised process linked to tumour progression and metastasis in a number of carcinomas. EMT enables carcinoma cells to lose cell to cell contacts and endows them with stem cell-like properties to invade and initiate metastasis. Recent reports have identified EMT as potentially playing a significant role in RCC disease recurrence, invasion and metastasis. Several signalling pathways like HIF/2 and IL-6/STAT, are well known inducers of the EMT phenotype. Protein kinase CK2 is a constitutively active serine/threonine kinase consisting of two catalytic subunits (CK2alpha/alpha’) and two regulatory subunits (CK2Beta) and is present in the nucleus and cytoplasm of all eukaryotic cells. The imbalance of CK2 catalytic and regularory subunits, due to underexpression of CK2Beta subunit, has been correlated with the expression of EMT markers. In clear cell renal cell carcinoma (ccRCC), the alterations in the ratios between CK2 subunits during the neoplasic process seems to participate at different stages of tumour progression METHODS: We analyzed the expression and distribution of CK2 subunits in samples of clear cell renal carcinomas (ccRCC) and renal healthy tissue from the same patients by immunohistochemistry on TMAs and Western-blot in a total of 98 patients. Tumour registry data and patient outcome were retrospectivelly collected and correlates with clinicopathological data (F€ urhman grade, pT stage and Risk group) and with IL-6/STAT inmunoexpression RESULTS: We observed an increase of CK2 in tumors. Regarding the subcellular distribution in normal tissue CK2alpha is predominantly cytoplasmic whereas tumors markedly increased in the core, with only a slight decrease in the cytoplasm, indicating nuclear overexpression CK2alpha tumors. CK2Beta changes are more discreet and its nuclear accumulation in tumors could be due to translocation from the cytoplasm, which is a marked decrease. Using 786-O cells, derived ccRCC pVHL deficient, and 786-O cells stably transfected with an expression vector pVHL, we have observed that the presence of VHL not decrease but it increases CK2alpha levels by altering the ratio between CK2alpha/CK2Beta. It was observed higher survival in the subset patients with underexpression of CK2sBeta although log-rank was not significant (0,301). The combination of overexpression of STAT3 and underexpression ok CK2Beta seems to provide a higher survival hazard ratio of 4,252 (95% IC, 1,182-18.413) CONCLUSIONS: The results indicate CK2alpha overexpression in clear cell renal cell carcinoma by a mechanism that does not appear due to inactivation of VHL. In patients with underexpression of IL-6/STAT3, CK2Beta was no able to discriminate any behaviour, but the patients defined as poor prognostic when STAT3 was overexpressed has similar survival than those that had underexpression of STAT3. The combination of overexpression of STAT3 and underexpression of CK2Beta provided a higher survival rate

MP70-04 BONE MASS BEHAVIOR AFTER ONE YEAR OF DIFFERENT TREATMENT STRATEGIES IN PROSTATE CANCER PATIENTS SUBJECTED TO ANDROGEN DEPRIVATION THERAPY

Kazuhiro Matsumoto*, Ryuichi Mizuno, Tokyo, Japan; Nobuyuki Tanaka, Saitama, Japan; Hiroki Ide, Tokyo, Japan; Masanori Hasegawa, Kawasaki, Japan; Masaru Ishida, Yokohama, Japan; Nozomi Hayakawa, Yota Yasumizu, Masayuki Hagiwara, Tokyo, Japan; Satoshi Hara, Kawasaki, Japan; Eiji Kikuchi, Akira Miyajima, Ken Nakagawa, Tokyo, Japan; Yosuke Nakajima, Yokohama, Japan; So Nakamura, Jun Nakashima, Mototsugu Oya, Tokyo, Japan

MP41-07 PROLIFERATIVE INFLAMMATORY ATROPHY IS ASSOCIATED TO LESS AGGRESSIVE AND INSIGNIFICANT TUMORS IN RADICAL PROSTATECTOMY SPECIMENS

INTRODUCTION AND OBJECTIVES: Proliferative inflammatory atrophy (PIA) has been proposed to be involved in prostate carcinogenesis by suggesting that it may rise to prostatic carcinoma (PCa) either directly or indirectly by first developing into high grade prostatic intraepithelial neoplasm (HGPIN). We have studied the PIA genetic signature 2 and more recently we have learned that PIA incidence in prostatic biopsies is around 30%, and it is associated to lower PCa detection and less aggressive tumors . The objectives of this study were to assess the incidence of PIA lesion in radical prostatectomy (RP) specimens and to confirm the hypothesis that PIA is associated to less aggressive tumors. METHODS: A retrospective study conducted in 200 consecutive patients subjected to RP was done. A single pathologist identified PIA in all specimens and this finding was related with: age, serum PSA, prostate volume, Gleason score, pathologic stage, percentage of tumor in the gland, maximal length of index tumor, perineural invasion, multifocality, bilaterality, insignificant cancer, positive margins, and HGPIN. Mann-Witney U test and Chi-square Pearson test were used to compare data. RESULTS: PIA was present in 64 specimens (32%). Age, serum PSA and prostate volume were similar in patients with and without PIA. Respectively, high Gleason grade was present in 34.4% and 49.3%, p1⁄40.033; non-organ confine disease in 10.9% and 16.9%, p1⁄40.187; perineural invasion in 59.4 and 74.3%, p1⁄40.025; multifocal tumors in 59.4% and 74.3%, p1⁄40.001; bilateral tumors in 46.9% and 70.5%, p1⁄40.005; insignificant tumors in 17.2% and 6.6%, p1⁄40.022; HGPIN in 92.2% and 90.4%, p1⁄40.456. CONCLUSIONS: This study confirms that the incidence of PIA in RP specimens was similar to the one observed previously in prostatic biopsies (32% versus 30%). PIA was not associated to HGPIN. PIA was associated to many pathologic characteristics of less aggressive tumors. Of great clinical interest could be the finding that PIA is associated to unilaterial, unifocal, low grade, and insignificant tumors. 1 De Marzo et al. Am J Pathol 1999; 155: 1985-92. 2 Morote et al. Annual EAU Congress 2012; P-848. 3 Morote el al. Annual AUA Meeting 2013; P-2226.