Assessment of chemical mixtures using biomarkers of combined biological activity: A screening study in human placentas.

Humans are simultaneously exposed to complex mixtures of chemicals with limited knowledge on potential health effects, therefore improved tools for assessing these mixtures are needed. As part of the Human Biomonitoring for Europe (HBM4EU) Project, we aimed to examine the combined biological activity of chemical mixtures extracted from human placentas using one in vivo and four in vitro bioassays, also known as biomarkers of combined effect. Relevant endocrine activities (proliferative and/or reporter gene assays) and four endpoints were tested: the estrogen receptor (ER), androgen receptor (AR), and aryl hydrocarbon receptor (AhR) activities, as well as thyroid hormone (TH) signaling. Correlations among bioassays and their functional shapes were evaluated. Results showed that all placental extracts agonized or antagonized at least three of the abovementioned endpoints. Most placentas induced ER-mediated transactivation and ER-dependent cell proliferation, together with a strong inhibition of TH signaling and the AR transactivity; while the induction of the AhR was found in only one placental extract. The effects in the two estrogenic bioassays were positively and significantly correlated and the AR-antagonism activity showed a positive borderline-significant correlation with both estrogenic bioassay activities. However, the in vivo anti-thyroid activities of placental extracts were not correlated with any of the tested in vitro assays. Findings highlight the importance of comprehensively mapping the biological effects of "real-world" chemical mixtures present in human samples, through a battery of in vitro and in vivo bioassays. This approach should be a complementary tool for epidemiological studies to further elucidate the combined biological fingerprint triggered by chemical mixtures.

Immunohistochemistry-based assessment of androgen receptor status and the AR-null phenotype in metastatic castrate resistant prostate cancer.

BACKGROUND: Molecular and immunohistochemistry-based profiling of prostatic adenocarcinoma has revealed frequent Androgen Receptor (AR) gene and protein alterations in metastatic disease. This includes an AR-null non-neuroendocrine phenotype of metastatic castrate resistant prostate cancer which may be less sensitive to androgen receptor signaling inhibitors. This AR-null non-neuroendocrine phenotype is thought to be associated with TP53 and RB1 alterations. Herein, we have correlated molecular profiling of metastatic castrate resistant prostate cancer with AR/P53/RB immunohistochemistry and relevant clinical correlates. DESIGN: Twenty-seven cases of metastatic castrate resistant prostate cancer were evaluated using histopathologic examination to rule out neuroendocrine differentiation. A combination of a hybridization exon-capture next-generation sequencing-based assay (n = 26), fluorescence in situ hybridization for AR copy number status (n = 16), and immunohistochemistry for AR (n = 27), P53 (n = 24) and RB (n = 25) was used to profile these cases. RESULTS: Of 27 metastatic castrate resistant prostate cancer cases, 17 had AR amplification and showed positive nuclear expression of AR by immunohistochemistry. Nine cases lacked AR copy number alterations using next-generation sequencing/fluorescence in situ hybridization. A subset of these metastatic castrate resistant prostate cancer cases demonstrated the AR-null phenotype by immunohistochemistry (five cases and one additional case where next-generation sequencing failed). Common co-alterations in these cases involved the TP53, RB1, and PTEN genes and all these patients received prior therapy with androgen receptor signaling inhibitors (abiraterone and/or enzalutamide). CONCLUSIONS: Our study suggests that AR immunohistochemistry may distinguish AR-null from AR-expressing cases in the metastatic setting. AR-null status informs clinical decision-making regarding continuation of therapy with androgen receptor signaling inhibitors and consideration of other treatment options. This might be a relevant and cost-effective diagnostic strategy when there is limited access and/or limited tumor material for molecular testing.

Striae distensae: Immunohistochemical assessment of hormone receptors in multigravida and nulligravida.

BACKGROUND: Striae distensae (SD), a type of dermal scarring, are psychologically disappointing. To date, information and scientific research behind the role of hormonal factors in the development of SD are still unclear. It is vital to understand striae to offer patients the best therapeutic options. OBJECTIVES: To investigate early alterations regarding the expression of estrogen, androgen, and glucocorticoid receptors (estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR) in skin samples of multigravida (MG) and nulligravida (NG) cases and to compare them with normal controls. METHODS: This study included 30 subjects (10 MG and 10 NG cases with early SD and 10 healthy controls). Biopsies from SD lesions, perilesional normal skin of cases and normal skin of controls were examined immunohistochemically for ER, AR, and GR expression using immune peroxidase technique. RESULTS: Comparing MG and NG with controls, ER expression appeared reduced in MG and NG (P-value<.001), AR was elevated in MG (P-value<.05) with no considerable difference in NG (P-value>.05), while GR was elevated in both MG and NG (P-value<.05). On comparing perilesional skin with SD lesions in each of MG and NG groups, SD lesions revealed reduced ER expression in both groups (P-value<.05), whereas in MG group: AR expression was elevated with no difference detected regarding GR (P-value˃.05); meanwhile in NG, elevated expression in both AR and GR expression was noted (P-Value<.001) CONCLUSIONS: Striae distansae lesions demonstrated a significant increase in the expression of AR and GR and a declined expression of ER indicating their involvement in the development of early SD.

Hormonal receptors in lung adenocarcinoma: expression and difference in outcome by sex.

BACKGROUND: Lung cancer seems to have different epidemiological, biomolecular and clinical characteristics in females than in males, with a better prognosis for women. The aim of the study is to determine gender differences in lung adenocarcinoma in terms of androgen (AR), estrogen (ER)α and progesterone (PgR) receptors expression and their impact on outcome. RESULTS: Overall survival was significantly better in ERα and in PgR positive lung adenocarcinoma patients (median survival 45 vs. 19 months).Eight out of 62 patients showed positive expression of nuclear (n) AR and 18 of cytoplasmic (c) AR with a significantly better survival (49 vs. 19 and 45 vs. 19 months, respectively). There was a significant difference in survival between patients with vs. without c-AR expression (30 vs. 17 months). Finally, in the subgroup of women, median survival was greater in positive expression of c-AR than for women with negative c-AR (45 vs. 21 months). MATERIALS AND METHODS: We conducted an analysis on a cohort of 62 patients with advanced NSCLC treated at our institution. We investigated the immunohistochemical expression of n/c AR, ERα and PgR in 62 NSCLC and we correlated it with patients' clinic-pathologic characteristics and with prognosis. CONCLUSIONS: Our results showed that the positive expression of one hormonal receptor could represent a prognostic factor.Furthermore our study suggests that AR should become object of close examination in a larger series of lung adenocarcinoma patients, also for selection of the patients with best prognosis that can perform more chemotherapy lines.

Co-assessment of cytoplasmic and nuclear androgen receptor location in prostate specimens: potential implications for prostate cancer development and prognosis.

OBJECTIVE: To address, by co-assessing cytoplasmic and nuclear androgen receptor (AR) expression in prostate tissues, the contribution of the AR throughout the stages of prostate cancer (PC) and its value as a marker for predicting biochemical recurrence (BCR) after radical prostatectomy (RP). PATIENTS AND METHODS: Archival prostate specimens from patients who were cancer-free (43), with hormone-sensitive prostate cancer (HSPC, 62), and with androgen-independent prostate cancer (AIPC, 30) were used to construct tissue microarrays (total 135). Prostatic intraepithelial neoplasia (PIN) and non-neoplastic tissues (NA) found adjacent to HSPC were also included. Nuclear and cytoplasmic AR expression was scored by two observers using a composite scale, after immunohistochemical detection of the AR. The nuclear/cytoplasmic AR expression ratio was also calculated. Univariate Kaplan-Meier plots, and multivariate Cox and survival-tree analyses, were then used to assess the ability of the AR to predict BCR in the patients with HSPC. RESULTS: There was markedly greater nuclear AR staining intensity in NA than in normal prostate tissues from cancer-free patients. Cytoplasmic AR expression was highest in AIPC and markedly more than in HSPC. The nuclear/cytoplasmic AR expression ratio was highest in NA and PIN. In univariate analyses, a low nuclear AR, low cytoplasmic AR, and a high nuclear/cytoplasmic AR expression ratio were associated with BCR. Although cytoplasmic AR was an independent predictor of BCR in a Cox multivariate model (hazard ratio 2.736, 95% confidence interval 1.228-6.091, P = 0.014), survival-tree analyses suggested a complex relationship between AR expression and clinicopathological features. CONCLUSION: We propose that increased nuclear AR expression might be a precursor to PC and that cytoplasmic AR could contribute to the AIPC phenotype. The predictive ability of the AR might be closely linked to clinicopathological features.

A chemiluminescence-based assessment of androgen-binding activity in a large pedigree affected with androgen insensitivity syndrome.

We report a novel chemiluminescence (CL)-based method for assaying the ligand-binding activity of the androgen receptor. The central parts of this method are the utilization of the steroid CL marker as the replacement of the radioactive label in the conventional ligand-binding assay and the determination of the binding activity by the light measurement of the bound CL-label under an H(2)O(2)-microperoxidase system. The properties and reliability of this assay were investigated and verified using genital skin fibroblasts (GSF) from seven normal males. The method is precise (CV < 7% for both B(max) and K(d)) with high correlation coefficients (r > 0.93) in each Scatchard linear regression analysis. This assay can determine the androgen binding properties using only a quarter of the cells (approximately 40 000 cells/data point) of that required by the radiolabelling approach. The utility of the method was illustrated by binding experiment on the GSFs of several patients from a large Chinese family affected with androgen insensitivity syndrome. The familial distinct feature is that all patients shared an identical Arg840Cys substitution in the androgen receptor but displayed high phenotypic variation in disorders of male sexual development. The patients selected for the present study represent a wide spectrum of this phenotypic variation. This study thus provides insights on the pleiotropic effects of the mutation. In conclusion, the CL-based method can serve as an effective, precise and reliable replacement for the radiolabelling approach and has the advantages of simplicity, cost-effectiveness and health and environmental safety over the counterpart.

Text-derived concept profiles support assessment of DNA microarray data for acute myeloid leukemia and for androgen receptor stimulation.

BACKGROUND: High-throughput experiments, such as with DNA microarrays, typically result in hundreds of genes potentially relevant to the process under study, rendering the interpretation of these experiments problematic. Here, we propose and evaluate an approach to find functional associations between large numbers of genes and other biomedical concepts from free-text literature. For each gene, a profile of related concepts is constructed that summarizes the context in which the gene is mentioned in literature. We assign a weight to each concept in the profile based on a likelihood ratio measure. Gene concept profiles can then be clustered to find related genes and other concepts. RESULTS: The experimental validation was done in two steps. We first applied our method on a controlled test set. After this proved to be successful the datasets from two DNA microarray experiments were analyzed in the same way and the results were evaluated by domain experts. The first dataset was a gene-expression profile that characterizes the cancer cells of a group of acute myeloid leukemia patients. For this group of patients the biological background of the cancer cells is largely unknown. Using our methodology we found an association of these cells to monocytes, which agreed with other experimental evidence. The second data set consisted of differentially expressed genes following androgen receptor stimulation in a prostate cancer cell line. Based on the analysis we put forward a hypothesis about the biological processes induced in these studied cells: secretory lysosomes are involved in the production of prostatic fluid and their development and/or secretion are androgen-regulated processes. CONCLUSION: Our method can be used to analyze DNA microarray datasets based on information explicitly and implicitly available in the literature. We provide a publicly available tool, dubbed Anni, for this purpose.

Comprehensive assessment of candidate genes and serological markers for the detection of prostate cancer.

We examined whether selected polymorphisms in 11 candidate genes and serum levels of insulin-like growth factor I (IGF-I) help predict the presence of prostate cancer among patients prescreened with prostate-specific antigen (PSA) and digital rectal exam (DRE). We studied 1031 consecutive men who underwent one or more prostate biopsies because of an elevated PSA level (>4 ng/ml) or an abnormal DRE. Eleven candidate genes were examined, including the androgen receptor, SRD5A2, CYP17, CYP3A4, vitamin D receptor, PSA, GST-T1, GST-M1, GST-P1, IGF-I, and IGF binding protein 3. We also measured serum IGF-I levels before biopsy. Of the 1031 men, 483 had cancer on any biopsy (cases) and 548 men had no cancer (controls). Age, ethnicity, total PSA, and DRE result were strongly predictive of the presence of prostate cancer. The mean IGF-I level for cases (119.4 ng/ml) was lower than for controls (124.4 ng/ml, P = 0.05) and were not predictive for the presence of prostate cancer. We found no associations between the androgen receptor, SRD5A2, CYP17, CYP3A4, vitamin D receptor, GST-M1, GST-P1, and IGF binding protein 3 genotypes and prostate cancer risk. The adjusted odds ratios for having prostate cancer for patients with the GST-T1 and IGF-I variant alleles were 1.64 (95% confidence interval, 1.1-2.4; P = 0.01) and 1.70 (95% confidence interval, 1.1-2.7; P = 0.02), respectively. Nine of 11 candidate genes were not significantly predictive for prostate cancer in a clinical setting. The GST-T1 and IGF-I polymorphisms demonstrated modest associations with prostate cancer risk. IGF-I levels were not helpful in identifying patients with prostate cancer at the time of biopsy.

[Significance of both nuclear and cytosol androgen receptor (AR) in assessment of AR status in prostate carcinoma and hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: Androgen receptor (AR) is closely associated with the genesis,development,treatment and assessment of prognosis of prostate carcinoma (PC) and hepatocellular carcinoma (HCC). How to determine the AR status accurately has important clinical significance. This study was designed to investigate the significance of androgen receptor(AR) in cell nucleus for assessment of the AR status of patients with PC and HCC. METHODS: A total of 94 PC and 192 HCC tissues were analyzed for the affinity (KD),cytosol AR (AcR) and nuclear AR(AnR) using radioligand binding assay(RBA). RESULTS: In 94 PC tissues, the Bmax values of AcR and AnR were 58.82+/-34.73 and 543.70+/-249.44 fmol/mg protein which were significantly higher than those of the surrounding tissues (21.63+/-14.89 and 89.20+/-47.32 fmol/mg protein, P< 0.001). The KD values of AcR and AnR were 0.84+/-0.52 and 2.15+/-0.79 nmol/L which were not significantly different as compared with the surrounding tissues(0.78+/-0.49 and 2.24+/-0.84 nmol/L, P >0.50). In 192 HCC tissues, the Bmax values of AcR and AnR were 18.09+/-16.87 and 59.93+/-34.12 fmol/mg protein, which were significantly higher than those of the surrounding tissues (10.87+/-7.60 and 25.54+/-20.10 fmol/mg protein, P< 0.001 ). The KD values of AcR and AnR were 0.76+/-0.57 and 1.89+/-0.74 nmol/L)which were not significantly different as compared with the surrounding tissues(0.69+/-0.48 and 1.94+/-0.88 nmol/L, P >0.50). The ratio of AnR/AcR was also higher (P< 0.001). Of 94 PC tissues, 48.94% were both AcR and AnR positive, being lower than that of the tissues with positive AcR alone (77.66%). Of 192 HCC tissues, 34.09% were both AcR and AnR positive, being also lower than that of AcR positive alone (56.26%). However, 63.01% of PC and 62.03% of HCC AcR-positive tissues were accompanied by AnR-positive. CONCLUSION: Despite the fact that the Bmax of both AcR and AnR increased in PC and HCC tissues as compared to the surrounding tissues, the AnR showed more significant changes. In assessment of the AR status of PC and HCC tissues, it would be more accurate to analyze with both AcR and AnR than with AcR alone.

Microassay for prostatic androgen receptors correlated with quantitative histological assessment.

A new microassay in which cryostat sections of prostate tissue were used to provide the source of soluble androgen receptor for biochemical assay, was devised using an isoelectric focusing method, with [3H]-mibolerone as the androgenic radioligand. Adjacent cryostat sections from the same tissue block were stained for diagnostic and quantitative histological assessment. The assay was used to illustrate variations in tissue androgen receptor concentration for correlation with epithelial cell content in benign prostate hyperplasia and prostatic cancer, and to show the effects of androgen receptor concentration of resection of prostatic tissue by electroresection. The results indicate that the heat in electroresection renders prostatic tissue unsuitable for androgen receptor assays, and suggest that knowledge of the cellular composition of carcinomatous prostates may be of importance in the full assessment of androgen receptor assay results. This method incorporates both a biochemical assay and histological assessment of the assayed tissue on near-facsimile sections, an advantage over conventional biochemical assays.