Adrie van Bokhoven

Cadmium induces p53-dependent apoptosis in human prostate epithelial cells.

Cadmium, a widespread toxic pollutant of occupational and environmental concern, is a known human carcinogen. The prostate is a potential target for cadmium carcinogenesis, although the underlying mechanisms are still unclear. Furthermore, cadmium may induce cell death by apoptosis in various cell types, and it has been hypothesized that a key factor in cadmium-induced malignant transformation is acquisition of apoptotic resistance. We investigated the in vitro effects produced by cadmium exposure in normal or tumor cells derived from human prostate epithelium, including RWPE-1 and its cadmium-transformed derivative CTPE, the primary adenocarcinoma 22Rv1 and CWR-R1 cells and LNCaP, PC-3 and DU145 metastatic cancer cell lines. Cells were treated for 24 hours with different concentrations of CdCl2 and apoptosis, cell cycle distribution and expression of tumor suppressor proteins were analyzed. Subsequently, cellular response to cadmium was evaluated after siRNA-mediated p53 silencing in wild type p53-expressing RWPE-1 and LNCaP cells, and after adenoviral p53 overexpression in p53-deficient DU145 and PC-3 cell lines. The cell lines exhibited different sensitivity to cadmium, and 24-hour exposure to different CdCl2 concentrations induced dose- and cell type-dependent apoptotic response and inhibition of cell proliferation that correlated with accumulation of functional p53 and overexpression of p21 in wild type p53-expressing cell lines. On the other hand, p53 silencing was able to suppress cadmium-induced apoptosis. Our results demonstrate that cadmium can induce p53-dependent apoptosis in human prostate epithelial cells and suggest p53 mutation as a possible contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis.

A study of high‐dose oral silybin‐phytosome followed by prostatectomy in patients with localized prostate cancer

Silibinin is a polyphenolic flavonolignan derived from milk thistle (Silybum marianium) with anti‐oxidant properties. The purpose of the current trial was to determine the tissue and blood effects of high‐dose silybin‐phytosome in prostate cancer patients.

Detection of circulating tumor cells in metastatic and clinically localized urothelial carcinoma.

OBJECTIVEnTo examine the incidence and prognostic value of circulating tumor cells (CTCs) in urothelial cancer (UC). The detection of CTCs is prognostic in several cancer types.nnnMETHODSnA total of 44 subjects with UC were assessed for CTCs using CellSearch Technology and 7.5 mL of peripheral blood, sorted by magnetic separation (epithelial cell adhesion molecule positive) and immunofluorescent staining (positive for cytokeratin 8, 18, or 19, negative for CD45, positive for 4,6-diamidino-2-phenylindole) to identify the CTCs.nnnRESULTSnFive (17%) of 30 subjects with clinically localized and 7 (50%) of 14 subjects with metastatic UC had ≥1 detectable CTC (range 1-177). Six subjects had ≥5 CTCs. Fluorescence in situ hybridization analysis was performed in 20 samples from 18 unique subjects using the UroVysion probe set. Copy number gains consistent with neoplasm were observed in those with measurable CTCs but not in any of the CTC-negative samples tested. With a median follow-up of 337 days, all 7 patients with metastasis and detectable CTCs had died compared with 3 (43%) of the 7 with metastasis but without detectable CTCs.nnnCONCLUSIONnCTCs are commonly observed in metastatic UC. CTCs were observed in 50% of the patients with metastatic UC tested. Fluorescence in situ hybridization analysis confirmed the aneusomic chromosomal content in the CTCs. These findings suggest that measurable CTCs might be prognostic for shortened survival in patients with metastatic UC, although the optimal threshold for a positive finding is unknown. CTCs were also detected in a subset of patients with clinically localized disease, identifying a potential high-risk, preoperative group for future study.

Associations of Serum Sex Steroid Hormone and 5α-Androstane-3α,17β-Diol Glucuronide Concentrations with Prostate Cancer Risk Among Men Treated with Finasteride

Background: Finasteride, an inhibitor of 5α-reductase (type II), lowers intraprostatic dihydrotestosterone (DHT), which is reflected in serum as reduced 5α-androstane-3α,17β-diol glucuronide (3α-dG). It also modestly increases serum testosterone (T), estrone (E1), and estradiol (E2). In this altered hormonal milieu, it is unknown whether serum concentrations of these hormones are associated with prostate cancer risk. Methods: In this nested case–control study of men in the finasteride arm of the Prostate Cancer Prevention Trial, sex steroid hormones and sex hormone binding globulin were measured at baseline and approximately 3-year posttreatment in 553 prostate cancer cases and 694 controls. Results: Median posttreatment changes in concentrations of 3α-dG, T, E1, and E2 were −73.8%, +10.1%, +11.2%, and +7.5% (all P < 0.001), respectively. Neither the pre- nor posttreatment concentrations of 3α-dG, nor its change, were associated with risk. Pretreatment, high concentrations of E1 and low concentrations of T were associated with increased cancer risk [OR; 95% confidence interval (CI) quartile 4 vs. 1: 1.38 (0.99–1.93) Ptrend = 0.03; 0.64 (0.43–0.93) Ptrend = 0.07, respectively]. Posttreatment, high concentrations of both E1 and E2 were associated with increased cancer risk [OR; 95% CI quartile 4 vs. 1: 1.54 (1.09–2.17) Ptrend = 0.03; 1.49 (1.07–2.07) Ptrend = 0.02, respectively]. Conclusions: Among finasteride-treated men, concentrations of 3α-dG were not associated with total or Gleason grades 2 to 6, 7 to 10, or 8 to 10 cancer. High serum estrogens may increase cancer risk when intraprostatic DHT is pharmacologically lowered. Impact: Low posttreatment serum estrogens may identify men more likely to benefit from use of finasteride to prevent prostate cancer. Cancer Epidemiol Biomarkers Prev; 21(10); 1823–32. ©2012 AACR.

Androgen Receptor CAG Repeat Length and TMPRSS2:ETS Prostate Cancer Risk: Results From the Prostate Cancer Prevention Trial

OBJECTIVEnTo investigate the association between the length of the polymorphic trinucleotide CAG microsatellite repeats in exon 1 of the AR gene and the risk of prostate cancer containing TMPRSS2:ETS fusion genes.nnnMETHODSnThis nested case-control study came from subjects enrolled in the Prostate Cancer Prevention Trial and included 195 biopsy-proven prostate cancer cases with a known TMPRSS2:ETS status and 1344 matched controls.nnnRESULTSnThere was no association between the CAG repeat length and the risk of TMPRSS2:ETS-positive (odds ratio, 0.97; 95% confidence interval, 0.91-1.04) or TMPRSS2:ETS-negative prostate cancer (odds ratio, 1.04; 95% confidence interval, 0.97-1.11) and in patients with low- or high-grade disease.nnnCONCLUSIONnOur findings suggested that AR CAG repeats are not associated with TMPRSS2:ETS formation in prostate cancer.

Temporal Changes in the Pathologic Assessment of Prostate Cancer

Thirty years have witnessed dramatic changes in the manner in which we diagnose and manage prostate cancer. With prostate-specific antigen screening, there was a shift towards smaller, clinically localized tumors. Tumors are often multifocal and display phenotypic and molecular heterogeneity. Pathologic evaluation of tissue obtained by needle biopsy remains the gold standard for the diagnosis and risk assessment of prostate cancer. Years of experience with grading, along with changes in the amount of biopsy tissue obtained and diagnostic tools available, have produced shifts in grading practices among genitourinary pathologists. Trends in Gleason grading and advances in pathological risk assessment are reviewed with particular emphasis on recent Gleason grading modifications of the International Society of Urologic Pathology. Efforts to maximize the amount of information from pathological specimens, whether it be morphometric, histochemical, or molecular, may improve predictive accuracy of prostate biopsies. New diagnostic techniques are needed to optimize management decisions.

P2.09-01 Tumor-Associated Immune Cell Infiltration Patterns in Early Stage Squamous Lung Carcinoma

differentiating sarcomatoid mesothelioma from pleomorphic carcinoma of the lung, and poorly differentiated chest wall sarcoma. Hence it frequently poses a diagnostic challenge for pulmonary pathologists. In this pilot study we evaluated the diagnostic performance of two recently proposed IHC biomarkers, GATA-3 and MUC4, in conjunction with BAP1. Method: Sarcomatoid mesothelioma or sarcomatoidpredominant biphasic mesothelioma (10 cases), pleomorphic carcinoma of the lung (10 cases) and poorly differentiated primary or metastatic chest wall or pleural sarcoma (10 cases) were retrieved from our diagnostic archive. Resections or large biopsies were selected over small biopsies whenever possible. All the cases were diagnosed between 2009 and 2017 by a specialist pulmonary pathologist and discussed at the local multi-disciplinary team meeting in relation to final diagnosis. Whole slide GATA-3 (L50-823, pre-diluted), MUC4 (8G7, 1:50) and BAP1 (C-4, 1:50) immunohistochemistry was performed using Ventana Benchmark ULTRA system. Lymphocytes (GATA-3/ BAP1) and bronchiolar epithelium (MUC4) were used as internal positive controls. Loss of GATA-3/BAP1 and MUC4 staining was defined as complete loss of nuclear or membrane and cytoplasmic signals, respectively. Any staining intensity above the external negative controls was accepted as positive. Extent of positive staining was grouped as <1%, 1-50% and >50%. Result: GATA-3 was positive in 8/10 sarcomatoid mesothelioma, 6/10 chest wall/pleural sarcoma and 2/10 pleomorphic carcinoma of the lung. MUC4 positivity was observed exclusively in pleomorphic carcinoma of the lung (6/10), but only focally. BAP1 loss was infrequently observed in all three types of tumours. Conclusion: The combination of GATA-3 and MUC4 immunohistochemistry show promise as markers that would help in distinguishing these three tumours. The role of BAP1 is uncertain. These pilot results warrant an extended study that consists of a larger cohort to evaluate the utility of these biomarkers.

Correlation of PD-L1 expression with tumor mutation burden and gene signatures for prognosis in early stage squamous cell lung carcinoma

Objectives: Anti–programmed cell death 1 (PD‐1)/programmed death ligand 1 (PD‐L1) immunotherapy has demonstrated success in the treatment of advanced NSCLC. Recently, PD‐1/PD‐L1 blockade also has demonstrated interesting results in small trials of neoadjuvant treatment in stage IB to IIIA NSCLC. In addition, several clinical trials using anti–PD‐1/PD‐L1 immunotherapy as an adjuvant or neoadjuvant treatment in patients with resectable stage NSCLC are ongoing. However, few analyses of anti–PD‐1/PD‐L1 immunotherapy–related biomarkers in early‐stage squamous cell lung carcinoma (SqCLC) have been reported. In this study, we evaluated PD‐L1 protein expression, tumor mutation burden, and expression of an immune gene signature in early‐stage SqCLC, providing data for identifying the potential role for patients with anti–PD‐1/PD‐L1 treatment in early‐stage SqCLC. Methods: A total of 255 specimens from patients with early‐stage SqCLC were identified within participating centers of the Strategic Partnering to Evaluate Cancer Signatures program. PD‐L1 protein expression by immunohistochemistry was evaluated by using the Dako PD‐L1 22C3 pharmDx kit on the Dako Link 48 auto‐stainer (Dako, Carpinteria, CA). Tumor mutation burden (TMB) was calculated on the basis of data from targeted genome sequencing. The T‐effector and interferon gamma (IFN‐&ggr;) gene signature was determined from Affymetrix gene chip data (Affymetrix, Santa Clara, CA) from frozen specimens. Results: The prevalence of PD‐L1 expression was 9.8% at a tumor proportion score cutoff of at least 50%. PD‐L1 mRNA and programmed cell death 1 ligand 2 mRNA positively correlated with PD‐L1 protein expression on tumor cells (TCs) and tumor‐infiltrating immune cells. PD‐L1 protein expression on tumor‐infiltrating immune cells was correlated with the T‐effector and IFN‐&ggr; gene signature (p < 0.001), but not with TMB. For TCs, all of these biomarkers were independent of each other and neither PD‐L1 protein expression, TMB, or T‐effector and IFN‐&ggr; gene signatures were independently prognostic for patient outcomes. Conclusions: Evaluation of PD‐L1 expression, TMB, and T‐effector and IFN‐&ggr; gene signatures in the cohort with early‐stage SqCLC found them to be independent of each other, and none was associated with overall survival. Our results also support the hypothesis that PD‐L1 expression is regulated by an intrinsic mechanism on TCs and an adaptive mechanism on immune cells.

Abstract B11: Proteomic analysis of serum-derived exosomes: Identification of novel protein signature associated with African-American prostate cancer

African-American men face a stark prostate cancer (PCA)-related health disparity in the United States, with the highest incidence and mortality rate compared to all other races. African-American men are more frequently diagnosed with high Gleason grade PCA at a younger age and with a poorer prognosis. In the past three decades, there has been little improvement in reducing this health disparity, demanding additional and innovative measures. Here, we focused on the identification of novel serum exosome-based ‘protein signature9 for potential use in the early detection and better prognosis of PCA in African-American men. Analysis of clinical serum samples showed that compared to healthy individuals, exosome concentration was increased by ~3.2 fold (p=0.047) in the sera of African-American men with PCA (Gleason score 7). Mass spectrometry based proteomic analysis of serum exosomal proteins showed 55 common proteins in African-American men with PCA (PAA) and normal African-American men without PCA (NAA). Interestingly, 7 unique proteins [Isoform 2 of Coiled-coil and C2 domain-containing protein 1A, Keratin type I cytoskeletal 10, UPF0728 protein C10orf53, DnaJ homolog subfamily C member 13, Prothrombin, Apolipoprotein(a) and Coiled-coil domain-containing protein 172] were present only in PAA, and not in NAA. The top upregulated proteins in PAA, as compared to NAA, included Keratin type II cytoskeletal 2 epidermal (64 fold), Serum amyloid P-component (39 fold), Keratin type II cytoskeletal 1 (13 fold), Keratin type I cytoskeletal 9 (12 fold), Isoform 2 of Filamin-A (2.6 fold), Isoform 3 of Vitamin D-binding protein (2.4 fold), Ig kappa chain V-II region Cum (2.4 fold) and Isoform 2 of Eukaryotic translation initiation factor 2-alpha kinase 4 (1.8 fold). The top downregulated proteins in PAA included Apolipoprotein A-I (0.04 fold), Complement component C8 alpha chain (0.07 fold), Plasminogen (0.08 fold), Complement component C7 (0.1 fold), and Isoform 2 of Inter-alpha-trypsin inhibitor heavy chain H4 (0.2 fold). Importantly, Ingenuity pathway analysis (IPA) showed that the proteins detected by proteomic analysis in PAA exosomes belonged to the acute-phase response signaling pathway proteins, which have been reported for their potential use as diagnostic biomarkers for PCA. Next, we compared proteins loaded in PAA serum exosomes with Gleason score-matched Caucasian PCA (PCC) serum exosomes. PAA and PCC exosomes shared 57 common proteins, while PAA serum exosomes showed six unique proteins (Isoform 2 of Coiled-coil and C2 domain-containing protein 1A, UPF0728 protein C10orf53, Ig kappa chain V-I region EU, Ig kappa chain V-II region Cum, DnaJ homolog subfamily C member 13, Coiled-coil domain-containing protein 172). Furthermore, top upregulated proteins in PAA, as compared to PCC, included Isoform 2 of Eukaryotic translation initiation factor 2-alpha kinase 4 (34 fold), Keratin type I cytoskeletal 10 (10 fold), SCO-spondin (5.3 fold), Serum amyloid P-component (4 folds) and Apolipoprotein(a) (3.9 fold). The top down regulated proteins in PAA, as compared to PCC, included Ig lambda chain V-I region WAH (0.02 fold), alpha-1B-glycoprotein (0.03 fold), Apolipoprotein A-I (0.03 fold), Complement component C8 alpha chain (0.06 fold) and Isoform 2 of Haptoglobin (0.09 fold). Overall, we have identified several unique and differentially expressed proteins in the serum exosomes of African-American men with PCA. These results also support further development and application of serum exosome-based proteomic signature for early detection and better prognosis of PCA in African- American men. Citation Format: Gagan Deep, Gatikrushna Panigrahi, Rakesh Singh, Kathleen Torkko, Adrie Bokhoven. Proteomic analysis of serum-derived exosomes: Identification of novel protein signature associated with African-American prostate cancer. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B11.

Abstract 1675: Variations in the composition of inflammatory infiltrates are associated with persistence or regression of bronchial dysplasia

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnINTRODUCTION: The clinical course of dysplastic bronchial lesions is variable. While some regress, others progress to invasive carcinoma. We sought to identify how variations in inflammatory infiltrates might influence the clinical outcome of dysplastic bronchial lesions.nnMETHODS AND RESULTS: Gene expression microarray analyses identified 318 genes that distinguish persistent from regressive bronchial dysplastic lesions. Pathway analysis utilizing this genelist was performed using Ingenuity© software. Gene expression data showed several inflammation related pathways with statistically different levels of activity in persistent versus regressive lesions. Expression of genes associated with CD4 positive T-cells and HLA-DRA positive macrophages were both increased in regressive lesions. To further investigate these findings, a similar set of progressive and regressive lesions were selected for immunohistochemical (IHC) analysis. For each biopsy, IHC for T-cell markers CD3, CD4 and CD8 and macrophage marker CD68 was performed. Immunohistochemically positive inflammatory cell subsets were counted in a single high power field corresponding to the focus of maximum inflammation. For each marker, separate counts for epithelium and stroma-associated inflammation were performed. Preliminary IHC data (n=6) shows a strong trend towards increased numbers of macrophages in the dysplastic epithelium of regressive lesions (4.6 fold increase, p=0.082).nnCONCLUSION: Our findings suggest that differences in inflammatory cell subsets may distinguish persistent and regressive bronchial dysplasia. Preliminary data points to CD4 positive T-cells and intraepithelial macrophages as differentiating factors that correlate with regression.nnCitation Format: Mary C. OKeefe, Lori Dwyer-Nield, Michael Edwards, Robert L. Keith, Wilbur A. Franklin, Michio Sugita, York E. Miller, Micah Friedman, Meredith Tennis, Kevin S. Choo, Gregory Hickey, Jeannine Porter, Storey Wilson, Andrea Osypuk, Mary Weiser, Adrie van Bokhoven, Mark Geraci, Raphael Nemenoff, Daniel T. Merrick. Variations in the composition of inflammatory infiltrates are associated with persistence or regression of bronchial dysplasia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1675. doi:10.1158/1538-7445.AM2014-1675