Michael Gormley

Functional significance of macrophage-derived exosomes in inflammation and pain

Summary Macrophage‐derived exosomes attenuated complete Freunds adjuvant‐induced thermal hyperalgesia in mice. Exosomal microRNA signature from patients with complex regional pain syndrome suggests a potential therapeutic and biomarker utility for exosomes. ABSTRACT Exosomes, secreted microvesicles transporting microRNAs (miRNAs), mRNAs, and proteins through bodily fluids, facilitate intercellular communication and elicit immune responses. Exosomal contents vary, depending on the source and the physiological conditions of cells, and can provide insights into how cells and systems cope with physiological perturbations. Previous analysis of circulating miRNAs in patients with complex regional pain syndrome (CRPS), a debilitating chronic pain disorder, revealed a subset of miRNAs in whole blood that are altered in the disease. To determine functional consequences of alterations in exosomal biomolecules in inflammation and pain, we investigated exosome‐mediated information transfer in vitro, in a rodent model of inflammatory pain, and in exosomes from patients with CRPS. Mouse macrophage cells stimulated with lipopolysaccharides secrete exosomes containing elevated levels of cytokines and miRNAs that mediate inflammation. Transcriptome sequencing of exosomal RNA revealed global alterations in both innate and adaptive immune pathways. Exosomes from lipopolysaccharide‐stimulated cells were sufficient to cause nuclear factor‐&kgr;B activation in naive cells, indicating functionality in recipient cells. A single injection of exosomes attenuated thermal hyperalgesia in a murine model of inflammatory pain, suggesting an immunoprotective role for macrophage‐derived exosomes. Macrophage‐derived exosomes carry a protective signature that is altered when secreting cells are exposed to an inflammatory stimulus. We also show that circulating miRNAs altered in patients with complex regional pain syndrome are trafficked by exosomes. With their systemic signaling capabilities, exosomes can induce pleiotropic effects potentially mediating the multifactorial pathology underlying chronic pain, and should be explored for their therapeutic utility.

Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells

BACKGROUND Whole genome amplification (WGA) is required for single cell genotyping. Effectiveness of currently available WGA technologies in combination with next generation sequencing (NGS) and material preservation is still elusive. RESULTS In respect to the accuracy of SNP/mutation, indel, and copy number aberrations (CNA) calling, the HiSeq2000 platform outperformed IonProton in all aspects. Furthermore, more accurate SNP/mutation and indel calling was demonstrated using single tumor cells obtained from EDTA-collected blood in respect to CellSave-preserved blood, whereas CNA analysis in our study was not detectably affected by fixation. Although MDA-based WGA yielded the highest DNA amount, DNA quality was not adequate for downstream analysis. PCR-based WGA demonstrates superiority over MDA-PCR combining technique for SNP and indel analysis in single cells. However, SNP calling performance of MDA-PCR WGA improves with increasing amount of input DNA, whereas CNA analysis does not. The performance of PCR-based WGA did not significantly improve with increase of input material. CNA profiles of single cells, amplified with MDA-PCR technique and sequenced on both HiSeq2000 and IonProton platforms, resembled unamplified DNA the most. MATERIALS AND METHODS We analyzed the performance of PCR-based, multiple-displacement amplification (MDA)-based, and MDA-PCR combining WGA techniques (WGA kits Ampli1, REPLI-g, and PicoPlex, respectively) on single and pooled tumor cells obtained from EDTA- and CellSave-preserved blood and archival material. Amplified DNA underwent exome-Seq with the Illumina HiSeq2000 and ThermoFisher IonProton platforms. CONCLUSION We demonstrate the feasibility of single cell genotyping of differently preserved material, nevertheless, WGA and NGS approaches have to be chosen carefully depending on the study aims.

Biomarker Associations with Efficacy of Abiraterone Acetate and Exemestane in Postmenopausal Patients with Estrogen Receptor–Positive Metastatic Breast Cancer

Purpose: Abiraterone may suppress androgens that stimulate breast cancer growth. We conducted a biomarker analysis of circulating tumor cells (CTCs), formalin-fixed paraffin-embedded tissues (FFPETs), and serum samples from postmenopausal estrogen receptor (ER)+ breast cancer patients to identify subgroups with differential abiraterone sensitivity. Methods: Patients (randomized 1:1:1) were treated with 1,000 mg/d abiraterone acetate + 5 mg/d prednisone (AA), AA + 25 mg/d exemestane (AAE), or exemestane. The biomarker population included treated patients (n = 293). The CTC population included patients with ≥3 baseline CTCs (n = 104). Biomarker [e.g., androgen receptor (AR), ER, Ki-67, CYP17] expression was evaluated. Cox regression stratified by prior therapies in the metastatic setting (0/1 vs. 2) and setting of letrozole/anastrozole (adjuvant vs. metastatic) was used to assess biomarker associations with progression-free survival (PFS). Results: Serum testosterone and estrogen levels were lowered and progesterone increased with AA. Baseline AR or ER expression was not associated with PFS in CTCs or FFPETs for AAE versus exemestane, but dual positivity of AR and ER expression was associated with improved PFS [HR, 0.41; 95% confidence interval (CI), 0.16–1.07; P = 0.070]. For AR expression in FFPETs obtained <1 year prior to first dose (n = 67), a trend for improved PFS was noted for AAE versus exemestane (HR, 0.56; 95% CI, 0.24–1.33; P = 0.19). Conclusions: An AA pharmacodynamic effect was shown by decreased serum androgen and estrogen levels and increased progesterone. AR and ER dual expression in CTCs and newly obtained FFPETs may predict AA sensitivity. Clin Cancer Res; 22(24); 6002–9. ©2016 AACR.

A prospective genome-wide study of prostate cancer metastases reveals association of wnt pathway activation and increased cell cycle proliferation with primary resistance to abiraterone acetate–prednisone

Background Genomic aberrations have been identified in metastatic castration-resistant prostate cancer (mCRPC), but molecular predictors of resistance to abiraterone acetate/prednisone (AA/P) treatment are not known. Patients and methods In a prospective clinical trial, mCRPC patients underwent whole-exome sequencing (n = 82) and RNA sequencing (n = 75) of metastatic biopsies before initiating AA/P with the objective of identifying genomic alterations associated with resistance to AA/P. Primary resistance was determined at 12 weeks of treatment using criteria for progression that included serum prostate-specific antigen measurement, bone and computerized tomography imaging and symptom assessments. Acquired resistance was determined using the end point of time to treatment change (TTTC), defined as time from enrollment until change in treatment from progressive disease. Associations of genomic and transcriptomic alterations with primary resistance were determined using logistic regression, Fishers exact test, single and multivariate analyses. Cox regression models were utilized for determining association of genomic and transcriptomic alterations with TTTC. Results At 12 weeks, 32 patients in the cohort had progressed (nonresponders). Median study follow-up was 32.1 months by which time 58 patients had switched treatments due to progression. Median TTTC was 10.1 months (interquartile range: 4.4-24.1). Genes in the Wnt/β-catenin pathway were more frequently mutated and negative regulators of Wnt/β-catenin signaling were more frequently deleted or displayed reduced mRNA expression in nonresponders. Additionally, mRNA expression of cell cycle regulatory genes was increased in nonresponders. In multivariate models, increased cell cycle proliferation scores (≥ 50) were associated with shorter TTTC (hazard ratio = 2.11, 95% confidence interval: 1.17-3.80; P = 0.01). Conclusions Wnt/β-catenin pathway activation and increased cell cycle progression scores can serve as molecular markers for predicting resistance to AA/P therapy.

Characterization of an abiraterone ultraresponsive phenotype in castration-resistant prostate cancer patient-derived xenografts.

Purpose: To identify the molecular signature associated with abiraterone acetate (AA) response and mechanisms underlying AA resistance in castration-resistant prostate cancer patient-derived xenografts (PDXs). Experimental Design: SCID mice bearing LuCaP 136CR, 77CR, 96CR, and 35CR PDXs were treated with AA. Tumor volume and prostate-specific antigen were monitored, and tumors were harvested 7 days after treatment or at end of study for gene expression and immunohistochemical studies. Results: Three phenotypic groups were observed based on AA response. An ultraresponsive phenotype was identified in LuCaP 136CR with significant inhibition of tumor progression and increased survival, intermediate responders LuCaP 77CR and LuCaP 96CR with a modest tumor inhibition and survival benefit, and LuCaP 35CR with minimal tumor inhibition and no survival benefit upon AA treatment. We identified a molecular signature of secreted proteins associated with the AA ultraresponsive phenotype. Upon resistance, AA ultraresponder LuCaP 136CR displayed reduced androgen receptor (AR) signaling and sustainably low nuclear glucocorticoid receptor (nGR) localization, accompanied by steroid metabolism alteration and epithelial–mesenchymal transition phenotype enrichment with increased expression of NF-κB–regulated genes; intermediate and minimal responders maintained sustained AR signaling and increased tumoral nGR localization. Conclusions: We identified a molecular signature of secreted proteins associated with AA ultraresponsiveness and sustained AR/GR signaling upon AA resistance in intermediate or minimal responders. These data will inform development of noninvasive biomarkers predicting AA response and suggest that further inhibition along the AR/GR signaling axis may be effective only in AA-resistant patients who are intermediate or minimal responders. These findings require verification in prospective clinical trials. Clin Cancer Res; 23(9); 2301–12. ©2016 AACR.

Abstract 2755: Comparison of blood based liquid biopsy methodologies for improved risk assessment of prostate cancer (PCa)

Approximately 28,000 men die from prostate cancer in the US each year. Predictive biomarkers can provide patient risk assessment to enable therapeutic decision making. Compared to tumor, blood based liquid biopsies offer greater flexibility for noninvasive sample collection and allows for continuous monitoring of treatment response. While various liquid biopsy sample types and methodologies are currently available, it is critical to identify which method offers the highest sensitivity and specificity for clinical decision making. In this study we systematically compared specificity and sensitivity of prostate cancer detection using a pre-selected panel of PCa specific mRNA transcripts using four different methodologies. In this prospective laboratory study, we collected five blood tubes from a cohort of 40 metastatic castrate resistant prostate cancer (mCRPC) patients and 20 age matched healthy volunteers (HV). Circulating tumor cells (CTCs) were enumerated and blood samples enriched for CTCs were isolated for mRNA evaluation. Two tubes of plasma samples were utilized to isolate exosomes using ExoRNAeasy columns and CellSearch EpCAM capture respectively, while RNA was extracted directly from whole blood collected in PAXgene tubes. RNA isolated from blood samples enriched for CTCs, two exosome preps and whole blood samples were tested using qPCR (Fluidigm) for a panel of 48 prostate specific mRNA transcripts and three endogenous controls previously shown to be detected in whole blood. Receiver operator analysis (ROC) was performed to compare expression of mRNA transcripts across all four methods. Results showed PCa specific genes were detected in whole blood, Exo-52 exosome prep and samples enriched for CTCs, while exosomes isolated using CellSearch EpCAM capture failed to detect any PCa transcripts. ROC showed ExoRNAeasy exosome prep detected PCa specific mRNA transcripts with significantly higher sensitivity and specificity compared to three other methodologies. Area under curve (AUC) for ExoRNA prep 0.65 to 0.75 (average of z score at 3.27 p= 0.0023), compared to AUC for PAXgene is 0.55 to 0.83, (average of z score at 2.64 and p=0.05) and CTC samples is 0.52 to 0.67, (average of z score at 1.63 and p= 0.19). For CellSearch exosome method, the AUC is 0.47 to 0.52, average of z score at 0.66 and p= 0.32). These early results support the utility of exosome derived mRNA assay as a viable alternative to whole blood and CTC based assays. Further testing for expression of markers associated with prognosis and treatment response is necessary to warrant its prognostic or predictive clinical utility. Citation Format: Yashoda Rajpurohit, Shibu Thomas, Jaymala Patel, Dong Shen, Michael Gormley, Denis Smirnov, Deborah Ricci. Comparison of blood based liquid biopsy methodologies for improved risk assessment of prostate cancer (PCa) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2755. doi:10.1158/1538-7445.AM2017-2755

Abstract 4313: Abiraterone acetate (AA) treatment of prostate cancer patient-derived xenografts (PDX) demonstrates heterogeneity of responses and identifies potential biomarkers of adaptive resistance

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA AA, a prodrug of the CYP17A1 inhibitor abiraterone, is the first second-line hormonal agent shown to improve survival in metastatic castration-resistant prostate cancer (CRPC); however, as with all available treatments, responding tumors eventually develop resistance. This highlights the importance of identification of mechanisms involved in tumor response and resistance to AA. We evaluated the efficacy of AA in LuCaP CRPC PDXs and investigated mechanisms of response and/or resistance, focusing on androgen receptor (AR) and glucocorticoid receptor (GR) signaling. Mice bearing CRPC LuCaP 35CR, 96CR, 77CR, and 136CR PDXs were treated with AA (0.5 mmol/kg/d), and tumor responses and body weights were monitored. Tumors were harvested 7 days after the beginning of the treatment (d7) and at end of study (EOS) (sacrifice when tumor > 1000 mg). Gene expression analyses using Affymetrix HG-U219 gene array and qPCR were performed to evaluate the validity of established biomarkers and identify novel biomarkers related to AA response. Linear regression model analyses showed that AA inhibited tumor progression in 3 of 4 LuCaP PDXs (96CR, 77CR, 136CR), but the initial response was followed by development of resistance. Corresponding analyses of serum prostate-specific antigen (PSA) levels demonstrated a decrease post AA (96CR: p = 0.051; 77CR: p = 0.001; 136CR: undetectable at baseline). AA improved median survival (77CR: 7 vs 9.5 weeks, p = 0.022; and 136CR: 6.8 vs 21.6 weeks, p < 0.0001), and highly variable responses were observed for 96CR (5.75 vs 10 weeks, p = 0.25). 35CR did not exhibit significant growth inhibition, reduction in PSA, or decreased median survival on AA. Evaluation of gene signatures revealed a negative association of proliferation-associated genes with AA at d7 for 35CR and 136CR, while at EOS these signatures were negatively associated with AA for 96CR and 136CR. Signatures of AR-regulated genes were negatively associated with AA at d7 for 35CR and 136CR, while at EOS they were negatively associated with AA for 96CR and 136CR. Targeted sequencing of the AR ligand binding domain coding sequence did not detect any mutations in any PDX at EOS. Trends toward increased expression of full length AR (ARFL) and splice variant ARv7 were associated with AA at d7 for 136CR. At EOS, trends toward increased ARFL and ARv7 expression were associated with AA for 35CR and 96CR. Significantly increased GR expression was associated with AA at EOS for 96CR and 77CR, and a trend was observed for 136CR. Our results demonstrate model-specific heterogeneity of physiological and molecular responses to AA in CRPC. However, our data indicate that increased ARFL, ARv7, and/or GR could be biomarkers of adaptive resistance and potentially play functional roles in conferring resistance. Citation Format: Hung-Ming Lam, Ryan McMullin, Holly M. Nguyen, Michael Gormley, Roman Gulati, Weimin Li, Deborah Ricci, Karin Verstraeten, Shibu Thomas, Elahe A. Mostaghel, Peter S. Nelson, Robert L. Vessella, Eva Corey. Abiraterone acetate (AA) treatment of prostate cancer patient-derived xenografts (PDX) demonstrates heterogeneity of responses and identifies potential biomarkers of adaptive resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4313. doi:10.1158/1538-7445.AM2015-4313