A Magnetic Bead-Based Sensor for the Quantification of Multiple Prostate Cancer Biomarkers.

Novel biomarker assays and upgraded analytical tools are urgently needed to accurately discriminate benign prostatic hypertrophy (BPH) from prostate cancer (CaP). To address this unmet clinical need, we report a piezeoelectric/magnetic bead-based assay to quantitate prostate specific antigen (PSA; free and total), prostatic acid phosphatase, carbonic anhydrase 1 (CA1), osteonectin, IL-6 soluble receptor (IL-6sr), and spondin-2. We used the sensor to measure these seven proteins in serum samples from 120 benign prostate hypertrophy patients and 100 Gleason score 6 and 7 CaP using serum samples previously collected and banked. The results were analyzed with receiver operator characteristic curve analysis. There were significant differences between BPH and CaP patients in the PSA, CA1, and spondin-2 assays. The highest AUC discrimination was achieved with a spondin-2 OR free/total PSA operation--the area under the curve was 0.84 with a p value below 10(-6). Some of these data seem to contradict previous reports and highlight the importance of sample selection and proper assay building in the development of biomarker measurement schemes. This bead-based system offers important advantages in assay building including low cost, high throughput, and rapid identification of an optimal matched antibody pair

Elevated Tumor Lactate and Efflux in High-grade Prostate Cancer demonstrated by Hyperpolarized 13C Magnetic Resonance Spectroscopy of Prostate Tissue Slice Cultures.

Non-invasive assessment of the biological aggressiveness of prostate cancer (PCa) is needed for men with localized disease. Hyperpolarized (HP) 13C magnetic resonance (MR) spectroscopy is a powerful approach to image metabolism, specifically the conversion of HP [1-13C]pyruvate to [1-13C]lactate, catalyzed by lactate dehydrogenase (LDH). Significant increase in tumor lactate was measured in high-grade PCa relative to benign and low-grade cancer, suggesting that HP 13C MR could distinguish low-risk (Gleason score ≤3 + 4) from high-risk (Gleason score ≥4 + 3) PCa. To test this and the ability of HP 13C MR to detect these metabolic changes, we cultured prostate tissues in an MR-compatible bioreactor under continuous perfusion. 31P spectra demonstrated good viability and dynamic HP 13C-pyruvate MR demonstrated that high-grade PCa had significantly increased lactate efflux compared to low-grade PCa and benign prostate tissue. These metabolic differences are attributed to significantly increased LDHA expression and LDH activity, as well as significantly increased monocarboxylate transporter 4 (MCT4) expression in high- versus low- grade PCa. Moreover, lactate efflux, LDH activity, and MCT4 expression were not different between low-grade PCa and benign prostate tissues, indicating that these metabolic alterations are specific for high-grade disease. These distinctive metabolic alterations can be used to differentiate high-grade PCa from low-grade PCa and benign prostate tissues using clinically translatable HP [1-13C]pyruvate MR

Diagnosis of prostate cancer by desorption electrospray ionization mass spectrometric imaging of small metabolites and lipids

Accurate identification of prostate cancer in frozen sections at the time of surgery can be challenging, limiting the surgeon's ability to best determine resection margins during prostatectomy. We performed desorption electrospray ionization mass spectrometry imaging (DESI-MSI) on 54 banked human cancerous and normal prostate tissue specimens to investigate the spatial distribution of a wide variety of small metabolites, carbohydrates, and lipids. In contrast to several previous studies, our method included Krebs cycle intermediates (m/z <200), which we found to be highly informative in distinguishing cancer from benign tissue. Malignant prostate cells showed marked metabolic derangements compared with their benign counterparts. Using the "Least absolute shrinkage and selection operator" (Lasso), we analyzed all metabolites from the DESI-MS data and identified parsimonious sets of metabolic profiles for distinguishing between cancer and normal tissue. In an independent set of samples, we could use these models to classify prostate cancer from benign specimens with nearly 90% accuracy per patient. Based on previous work in prostate cancer showing that glucose levels are high while citrate is low, we found that measurement of the glucose/citrate ion signal ratio accurately predicted cancer when this ratio exceeds 1.0 and normal prostate when the ratio is less than 0.5. After brief tissue preparation, the glucose/citrate ratio can be recorded on a tissue sample in 1 min or less, which is in sharp contrast to the 20 min or more required by histopathological examination of frozen tissue specimens. prostate cancer | Krebs cycle | metabolism | desorption electrospray ionization | mass spectrometry P rostate cancer (PCa) is the most commonly diagnosed solidorgan cancer and the second leading cause of cancer death in men in the United States (1). Because of prostate-specific antigen (PSA) screening in the United States, most PCas are discovered when they are confined to the prostate (2). Many of these localized PCas are treated by surgical removal of the entire prostate (radical prostatectomy). The presence of cancer cells at the edge of the surgical resection, or positive surgical margins, is associated with higher rates of recurrence and death from PCa (3, 4). Therefore, an important clinical challenge in PCa management is to devise a rapid and highly accurate method to detect cancerous cells in real time to allow resection of additional periprostatic tissues and reduce cancer recurrence after surgery. Over the last decade, several innovative analytical techniques (5-12) have been developed to distinguish cancer from benign tissue in various organs. However, none has achieved wide clinical adoption for various reasons including inconvenience, narrow information content, unavailability, poor sensitivity, slowness of adoption, and operating room workflow incompatibility. In PCa, intraoperative frozen sections have been used to attempt to identify PCa at the margin based on analysis of histology. However, frozen sections have been shown to have poor sensitivity and specificity for the detection of PCa and currently are not recommended Recently, a label-free molecular imaging method called desorption electrospray ionization mass spectrometric imaging (DESI-MSI) has been developed (15-17). DESI-MSI can rapidly evaluate the tissue metabolome in situ by simultaneously characterizing hundreds of lipids and metabolites. In the last 5 y, reports from our group Given the known alterations in metabolic pathways in PCa, we tested whether DESI-MSI of metabolites and lipids could have utility in discriminating cancer from normal tissue obtained from radical prostatectomy. Using tandem and high-resolution mass spectrometry we have characterized the distinct metabolite and lipid profiles of normal and malignant prostate. Although many earlier DESI-MSI studies considered only lipid profiles in identification of cancer, here we report imaging small metabolite Significance Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is a label-free molecular imaging technique that provides a window into the biochemical processes present in benign and malignant prostate tissue. This is important both in improving the understanding of tissue biology and in achieving rapid cancer diagnosis. We applied DESI-MSI to record lipid, carbohydrate, and most importantly, small metabolite images from 54 normal and malignant prostate tissue specimens. Several Krebs cycle intermediates were present at different concentrations in prostate cancer compared with normal tissue. Statistical calculations identified panels of metabolites that could readily distinguish prostate cancer from normal tissue with nearly 90% accuracy in a validation set. The results also indicated that the ratio of glucose to citrate ion signals could be used to accurately identify prostate cancer

Discovery of PTN as a serum-based biomarker of pro-metastatic prostate cancer.

Distinguishing clinically significant from indolent prostate cancer (PC) is a major clinical challenge. We utilised targeted protein biomarker discovery approach to identify biomarkers specific for pro-metastatic PC. Serum samples from the cancer-free group; Cambridge Prognostic Group 1 (CPG1, low risk); CPG5 (high risk) and metastatic disease were analysed using Olink Proteomics panels. Tissue validation was performed by immunohistochemistry in a radical prostatectomy cohort (n = 234). We discovered that nine proteins (pleiotrophin (PTN), MK, PVRL4, EPHA2, TFPI-2, hK11, SYND1, ANGPT2, and hK14) were elevated in metastatic PC patients when compared to other groups. PTN levels were increased in serum from men with CPG5 compared to benign and CPG1. High tissue PTN level was an independent predictor of biochemical recurrence and metastatic progression in low- and intermediate-grade disease. These findings suggest that PTN may represent a novel biomarker for the presence of poor prognosis local disease with the potential to metastasise warranting further investigation

Elevated serum microRNA levels associate with absence of high-grade prostate cancer in a retrospective cohort.

To reduce treatment of indolent prostate cancer (PCa), biomarkers are needed to improve identification of patients with a low-risk of having aggressive disease. Over-treatment of these patients occurs because of uncertainty in the aggressiveness of the entire tumor based on the biopsies, which do not accurately sample multifocal tumors. Circulating microRNAs (miRNAs) are stable serum markers and differential miRNA levels occur in men with PCa. The goal of this study was to identify circulating miRNAs that were associated with aggressive or indolent PCa. We measured circulating miRNAs in 150 patients prior to surgery and compared the miRNA levels to the pathology of the entire radical prostatectomy specimen. For this study we used an exceptionally well-characterized cohort of patients who had benign prostatic hyperplasia (BPH), low-grade or high-grade PCa. Low-grade was defined as patients with 100% Gleason grade 3 tumor as determined by step-wise sectioning. High-grade PCa patients had 30-90% Gleason grade 4+5 in the tumor. BPH patients had at least two biopsies negative for PCa. Twenty one miRNAs were selected for analysis. The miRNAs were quantified by RT-qPCR and analyzed by logistic regression. High levels of 14 miRNAs were exclusively present in the serum from patients with low-grade PCa or BPH, compared to men with high-grade PCa who had consistently low levels. The expression levels of the 14 miRNAs were combined into a "miR Score" which had a negative predictive value (NPV) of 0.939 to predict absence of high-grade PCa among PCa and BPH patients. Biochemical recurrence (BCR) was known for the PCa patients and a combined "miR Risk Score" accurately classified a subset of patients with low risk of BCR (NPV 0.941). In summary, measurement of serum miRNAs may have pre-surgical utility in combination with clinical risk calculators to identify patients with low risk of harboring aggressive PCa

Elevated Serum MicroRNA Levels Associate with Absence of High-Grade Prostate Cancer in a Retrospective Cohort

To reduce treatment of indolent prostate cancer (PCa), biomarkers are needed to improve identification of patients with a low-risk of having aggressive disease. Over-treatment of these patients occurs because of uncertainty in the aggressiveness of the entire tumor based on the biopsies, which do not accurately sample multifocal tumors. Circulating microRNAs (miRNAs) are stable serum markers and differential miRNA levels occur in men with PCa. The goal of this study was to identify circulating miRNAs that were associated with aggressive or indolent PCa. We measured circulating miRNAs in 150 patients prior to surgery and compared the miRNA levels to the pathology of the entire radical prostatectomy specimen. For this study we used an exceptionally well-characterized cohort of patients who had benign prostatic hyperplasia (BPH), low-grade or high-grade PCa. Low-grade was defined as patients with 100% Gleason grade 3 tumor as determined by step-wise sectioning. High-grade PCa patients had 30-90% Gleason grade 4+5 in the tumor. BPH patients had at least two biopsies negative for PCa. Twenty one miRNAs were selected for analysis. The miRNAs were quantified by RT-qPCR and analyzed by logistic regression. High levels of 14 miRNAs were exclusively present in the serum from patients with low-grade PCa or BPH, compared to men with high-grade PCa who had consistently low levels. The expression levels of the 14 miRNAs were combined into a “miR Score” which had a negative predictive value (NPV) of 0.939 to predict absence of high-grade PCa among PCa and BPH patients. Biochemical recurrence (BCR) was known for the PCa patients and a combined “miR Risk Score” accurately classified a subset of patients with low risk of BCR (NPV 0.941). In summary, measurement of serum miRNAs may have pre-surgical utility in combination with clinical risk calculators to identify patients with low risk of harboring aggressive PCa.</p

The immunomodulatory anticancer agent, RRx-001, induces an interferon response through epigenetic induction of viral mimicry

Abstract Background RRx-001, a dinitroazetidine derivative, is a novel anticancer agent currently in phase II clinical trials. It mediates immunomodulatory effects either directly through polarization of tumor associated macrophages or indirectly through vascular normalization and increased T-lymphocyte infiltration. With multiple additional mechanisms of action including upregulation of oxidative stress, depletion of GSH and NADPH, anti-angiogenesis and epigenetic modulation, RRx-001 is being studied as a radio- and chemo-sensitizer to resensitize tumors to prior therapy and to prime tumors to respond to radiation, chemotherapy and immunotherapy in combination therapy studies. Here, we identified another mechanism, viral mimicry, which refers to the \u201cunsilencing\u201d of epigenetically repressed viral genes present in the tumor that provokes an immune response and may contribute to the anticancer activity of RRx-001. Results RRx-001 inhibited the growth of colon cancer cells (HCT 116) and decreased levels of the DNA methyltransferases DNMT1 and DNMT3a in a time and dose-dependent manner. Treatment of HCT 116 cells with 0.5\ua0\u3bcM RRx-001 for 24\ua0h significantly increased transcripts of interferon (IFN)-responsive genes and this induction was sustained for up to 4\ua0weeks after transient exposure to RRx-001. ELISA assays showed that RRx-001 increased secretion of type I and III IFNs by HCT 116 cells, and these IFNs were confirmed to be bioactive. Transcription of endogenous retrovirus ERV-Fc2 and LTRs from the ERV-L family (MLT2B4 and MLT1C49) was induced by RRx-001. The induction of ERV-Fc2-env was through demethylation of ERV-Fc2 LTR as determined by methylation-specific polymerase chain reaction and combined bisulfite restriction analysis. Immunofluorescence staining with J2 antibody confirmed induction of double-stranded RNA. Conclusions Transient exposure of HCT 116 cells to low-dose RRx-001 induced transcription of silenced retroviral genes present in the cancer cell DNA with subsequent synthesis of IFN in response to this \u201cpseudo-pathogenic\u201d stimulus, mimicking an antiviral defense. RRx-001-mediated IFN induction may have the potential to improve the efficacy of immunotherapies as well as radiotherapy, standard chemotherapies and molecularly targeted agents when used in combination. The striking safety profile of RRx-001 in comparison to other more toxic epigenetic and immunomodulatory agents such as azacitidine makes it a leading candidate for such clinical applications

Circulating miRNAs predict absence of high-grade disease.

<p><b>A,</b> negative predictive value (NPV, y-axis) for absence of Gleason 4 +5 in the PCa patients only (N = 100) across the range of miR Score1 (x-axis) reaching NPV = 1 at the miR Score1 threshold of 7.19 (arrow). <b>B</b>, NPV reaches 1 for absence of Gleason 4+5 in entire cohort (BPH, low-grade PCa, high-grade PCa) at the miR Score2 threshold of 7.85 (arrow). The equations for the miR Scores, in which the miRNA name represents the log2 RQ value: miR Score1 = (0.269 x let-7a) + (0.257 x miR-103) + (0.254 x miR-451) + (0.255 x miR-24) + (0.252 x miR-26b) + (0.255 x miR-30c) + (0.221 x miR-93) + (0.253 x miR-106a) + (0.274 x miR-223) + (0.188 x miR-874) + (0.200 x miR-146a) + (0.118 x miR-100) + (0.276 x miR-107) + (0.204 x miR-130b). miR Score2 = (0.289 x level Let7a) + (0.286 x level miR-103) + (0.308 x level miR-451) + (0.267 x level miR-24) + (0.256 x level miR-26b) + (0.282 x level miR-30c) + (0.231 x level miR-93) + (0.263 x level miR-106a) + (0.293 x level miR-223) + (0.161 x level miR-874) + (0.227 x level miR-146a) + (0.165 x level miR-125b) + (0.116 x level miR-100) + (0.295 x level miR-107) + (0.183 x level miR-130b).</p

Circulating miRNAs levels in patients with BPH, low-grade PCa and high-grade PCa.

<p>Waterfall plots (linear y-axis) and scatter plots (log10 y-axis) of RQ values for all patients. In the waterfall plots each patient is shown as a vertical line on the x-axis and sorted by RQ within each group, for the top 12 miRNAs in the miR Score; let-7a, miR-24, 26a, 30c, 93, 103, 106a, 107, 130b, 146a, 223, and 451 and by disease. For the scatter plots the y-axis is shown log10 to improve visualization of low range RQs. BPH = benign prostate hyperplasia (N = 50). Gleason 3 = 100% of the tumor was Gleason grade 3 (N = 50). Gleason 4+5 = 30–90% of the tumor was Gleason 4 and/or 5 (N = 50).*p<0.05, ** p<0.01, *** p<0.0001 by Kruskal–Wallis one-way analysis of variance with Dunn's multiple comparisons test.</p