Search

Figure S1: Examples of a decision boundary that separate class A (red) from class B (blue) in 1 dimension (figure 1A) and 2 dimensions (figure 1B); Figure S2: Schematic of the supervised learning process (A) and leave one out cross validation (B); Figure S3: Single iteration of Leave-One-Out Cross-Validation performed at the blood sample level; Figure S4: An example of concordance of AURKA amplification in tumor and CTCs; Figure S5: (A) Kernel Density Estimate (KDE) curves of the classifier output are plotted for each patient sample colored by their diagnosis: NEPC (red) and CRPC (blue); Figure S6: Cell-level NEPC classifier (A) Receiver-Operating-Characteristic (ROC) curve generated on the classifier's single-cell output after LOOCV; Figure S7: To address whether the CTC classifier is simply stochastic, a reflection of an overall higher CTC count, linearity was assessed with a Pearson's coefficient showing a weak relationship between frequency of NEPC CTCs and total cell count; Table S1: A summary of cell-level features utilized to train Random Forest cell-level classifiers in both the LOOCV and for the classification of CTCs in the test cohort; Table S2: Patient characteristics (discovery cohort) including prior systemic therapies, serum markers including PSA (ng/ml), Chromogranin (ng/ml), NSE (ng/ml) and CTC counts; Table S3: Liver metastases in NEPC vs. CRPC; Table S4: The median concentration of CK-negative and AR-negative CTCs in CRPC, atypical CRPC, and NEPC patients; Table S5: Confusion matrix for the ability of CD56 staining to discriminate patients diagnosed with Small Cell Carcinoma NEPC vs CRPC; Table S6: Example of results from a single tube of blood.

The heterogeneity of an individual patient's tumor has been linked to treatment resistance, but quantitative biomarkers to rapidly and reproducibly evaluate heterogeneity in a clinical setting are currently lacking. Using established tools available in a College of American Pathologists–accredited and Clinical Laboratory Improvement Amendments–certified clinical laboratory, we quantified digital pathology features on 9,225 individual circulating tumor cells (CTC) from 179 unique metastatic castration-resistant prostate cancer (mCRPC) patients to define phenotypically distinct cell types. Heterogeneity was quantified on the basis of the diversity of cell types in individual patient samples using the Shannon index and associated with overall survival (OS) in the 145 specimens collected prior to initiation of the second or later lines of therapy. Low CTC phenotypic heterogeneity was associated with better OS in patients treated with androgen receptor signaling inhibitors (ARSI), whereas high heterogeneity was associated with better OS in patients treated with taxane chemotherapy. Overall, the results show that quantifying CTC phenotypic heterogeneity can help inform the choice between ARSI and taxanes in mCRPC patients. Cancer Res; 77(20); 5687–98. ©2017 AACR.

Purpose: The transition of prostate adenocarcinoma to a predominantly androgen receptor (AR) signaling independent phenotype can occur in the later stages of the disease and is associated with low AR expression +/− the development of small-cell or neuroendocrine tumor characteristics. As metastatic tumor biopsies are not always feasible and are difficult to repeat, we sought to evaluate noninvasive methods to identify patients transitioning toward a neuroendocrine phenotype (NEPC).Experimental Design: We prospectively studied a metastatic tumor biopsy, serum biomarkers, and circulating tumor cells (CTC, Epic Sciences) from patients with castration-resistant prostate cancer (CRPC) including those with pure or mixed NEPC histology present on biopsy. CTCs labeled with the patient's clinical status were used to learn features that discriminate NEPC patients, which was then applied to an independent cohort.Results: Twenty-seven patients with CRPC including 12 NEPC and 5 with atypical clinical features suggestive of NEPC transition were studied. CTCs from NEPC patients demonstrated frequent clusters, low or absent AR expression, lower cytokeratin expression, and smaller morphology relative to typical CRPC. A multivariate analysis of protein and morphologic variables enabled distinguishing CTCs of NEPC from CRPC. This CTC classifier was applied to an independent prospective cohort of 159 metastatic CRPC patients and identified in 17/159 (10.7%) of cases, enriched in patients with high CTC burden (P < 0.01) and visceral metastases (P = 0.04).Conclusions: CTCs from patients with NEPC have unique morphologic characteristics, which were also identified in a subset of CRPC patients with aggressive clinical features potentially undergoing NEPC transition. Clin Cancer Res; 22(6); 1510–9. ©2015 AACR.

Introduction: Here we describe the development of a protein immunofluorescent assay for the detection of nuclear-localized androgen receptor variant 7 (AR-V7) protein within circulating tumor cells (CTCs) identified in patient blood samples. Used in the clinic, the test result serves as a validated biomarker of futility for patients with progressing metastatic castration-resistant prostate cancer (mCRPC) who are treated with androgen receptor targeted therapies (AATT) in whom nuclear-localized AR-V7 CTCs are identified and have received level 2A evidence in the 2019 National Cancer Center Network (NCCN) guidelines (v1.0). Methods: Assay development was completed on the Epic Sciences rare cell detection platform using control cell lines of known AR-V7 status and clinical testing of mCRPC patient samples obtained at the decision point in management. Results and conclusions: Using these samples, all assay parameters, scoring criteria, and clinical cutoffs for positivity were prospectively selected and locked. After assay lock, blinded clinical validation testing was initiated on multiple, independent, clinical cohorts as reported by Scher et al (JAMA Oncol. 2016;2:1441-1449; JAMA Oncol. 2018;4:1179-1186) and Armstrong et al (J Clin Oncol. 2019;37:1120-1129).

Background: Clonal hematopoiesis (CH), the presence of acquired mutations in leukemia driver genes, promotes systemic inflammation and is common among aging men. Prostate cancer, a subset of which is lethal, also develops in aging men. We hypothesized that CH contributes to development of lethal prostate cancer. Methods: We conducted nested case-cohort studies for metastatic prostate cancer and prostate cancer-specific death (lethal prostate cancer) within the prospective Health Professionals Follow-up Study. First, we followed 1155 men free of prostate cancer and cardiovascular disease at blood draw (1993-1995) for development of lethal prostate cancer over up to 26 years. Second, we followed 532 men with incident non-metastatic prostate cancer for development of lethal prostate cancer. We sequenced blood DNA from 1488 participants for putative CH driver mutations in the 9 most common CH-defining genes with a custom targeted panel (VariantPlex, Invitae, Inc.) at ultra-high depth (mean, 18,000x), employing unique molecular identifiers for error correction. CH variant calling used a novel ensemble calling approach, ArCCH, validated with in-silico tumor dilutions and blinded technical replicates, which had high accuracy for variant allele frequencies (VAFs) as low as 0.1%. We estimated hazard ratios (HRs) with 95% confidence intervals (CIs) in proportional hazards regression with Prentice case-cohort weights. Results: In a random sample of 968 men initially free of prostate cancer and cardiovascular disease (median age at blood draw 60 years, interquartile range 52 to 67), 80% of men had CH at a variant allele frequency (VAF) of >0.1%, 15% had VAFs >2%, and 3% had VAFs >10%; 75% of men had variants in epigenetic modifier genes (DNMT3A, TET2, ASXL1), and 21% had variants in DNA repair genes (PPM1D, TP53, CHEK2). CH burden was strongly age-associated, with approximately one additional CH variant per decade of age at blood draw (mean difference 1.07 variants, 95% CI 0.93 to 1.21). Among men initially free from prostate cancer, after adjusting for age at blood draw, CH clones between 0.1% and 10% VAF were not related to lethal prostate cancer (206 events total; HR 0.93, 95% CI 0.49-1.76 for VAFs 2-10% vs. no variants detected at >0.1% VAF). Results for epigenetic modifiers and DNA repair genes were similar. While inconclusive, data were compatible with positive associations among younger men (< 65 years) or for VAFs >10%. Among men initially diagnosed with non-metastatic prostate cancer, results were similarly null for progression to lethal prostate cancer (164 events). Conclusions: This large prospective study with long-term follow-up suggests that low-level CH is unlikely a major contributor to and not well suited for early detection of lethal prostate cancer. Citation Format: Konrad H. Stopsack, Irenaeus C. Chan, Evelyn Schmidt, Alex Panchot, Samantha McNulty, Nicole A. Schreiber, Yiwen Zhang, Kathryn L. Penney, Michael F. Berger, Luis A. Diaz, Ross L. Levine, Kelly L. Bolton, Lorelei A. Mucci, Philip W. Kantoff. Clonal hematopoiesis and risk of lethal prostate cancer: a prospective cohort study with long-term follow-up. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P011.

Thrombospondin-1 (THBS1) is involved in the process of angiogenesis and is down-regulated by insulin-like growth factor 1 (IGF1) in porcine granulosa cells (GC), but what other hormones regulate GC THBS1 and its role in follicular growth is unclear. Thus, six experiments were conducted to determine the influence of other hormones on THBS1 gene expression in porcine GC, and to determine if THBS1 mRNA changes during follicular development. For Exp. 1-5, small (1-5 mm) follicles from ovaries of abattoir gilts were aspirated, GC collected and treated with FSH, IGF1, fibroblast growth factor 9 (FGF9), Sonic hedgehog (SHH), estradiol, cortisol, and/or prostaglandin E2 (PGE2). FSH, IGF1 and FGF9 each decreased (P 0.05) THBS1 mRNA abundance. Alone, PGE2 increased (P 0.05) THBS1 mRNA abundance. PGE2 significantly attenuated the FSH-induced inhibition of THBS1 mRNA expression. Estradiol, cortisol, and SHH had no effect on THBS1 mRNA abundance. In Exp. 6, small (1-3 mm), medium (4-6 mm) and large (7-14 mm) follicles were aspirated to measure abundance of THBS1 mRNA in GC which did not differ (P 0.10) between small and medium-sized follicles but was threefold greater (P 0.05) in large compared to small or medium follicles. We hypothesize that the inhibitory effects of FSH, IGF1 and FGF9 on the antiangiogenic gene THBS1 could contribute to promoting angiogenesis in the developing follicle, while stimulation of THBS1 mRNA by PGE2 may help reduce angiogenesis during the preovulatory period when PGE2 and THBS1 mRNA are at their greatest levels.

Abundance of G protein-coupled receptor 34 (GPR34) mRNA is greater in granulosa cells (GC) of cystic vs. normal follicles of cattle. The present experiments were designed to determine if GPR34 mRNA in GC changes during selection and growth of dominant follicles in cattle as well as to investigate the hormonal regulation of GPR34 mRNA in bovine GC in vitro. In Exp. 1, estrous cycles of non-lactating cows were synchronized and then ovariectomized on either day 3-4 or 5-6 after ovulation. GPR34 mRNA abundance in GC was 2.8- to 3.8-fold greater (P < 0.05) in small (1–5 mm) and large (≥ 8 mm) estrogen-inactive dominant follicles than in large estrogen-active follicles. Also, GPR34 mRNA tended to be greater (P < 0.10) in F2 than F1 follicles on day 3-4 post-ovulation. In Exp. 2–7, ovaries were collected at an abattoir and GC were isolated and treated in vitro. Expression of GPR34 was increased (P < 0.05) 2.2-fold by IGF1. Tumor necrosis factor (TNF)-α decreased (P < 0.05) the IGF1-induced GPR34 mRNA abundance in small-follicle GC, whereas IGF1 decreased (P < 0.05) GPR34 expression by 45% in large-follicle GC. Treatment of small-follicle GC with either IL-2, prostaglandin E2 or angiogenin decreased (P < 0.05) GPR34 expression whereas FSH, cortisol, wingless 3A or hedgehog proteins did not affect (P > 0.10) GPR34 expression. In Exp. 6 and 7, two presumed ligands of GPR34, L-a-lysophosphatidylserine (LPPS) and LPP-ethanolamine, increased (P < 0.05) GC numbers and estradiol production by twofold or more in small-follicle GC, and this response was only observed in IGF1-treated GC. In conclusion, GPR34 is a developmentally and hormonally regulated gene in GC, and its presumed ligands enhance IGF1-induced proliferation and steroidogenesis of bovine GC.

Angiogenin is a member of the ribonuclease A superfamily of proteins that has been implicated in stimulating angiogenesis but whether angiogenin can directly affect ovarian granulosa or theca cell function is unknown. Therefore, the objective of these studies was to determine the effect of angiogenin on proliferation and steroidogenesis of bovine granulosa and theca cells. In experiments 1 and 2, granulosa cells from small (1 to 5 mm diameter) follicles and theca cells from large (8 to 22 mm diameter) follicles were cultured to evaluate the dose-response effect of recombinant human angiogenin on steroidogenesis. At 30 and 100 ng/ml, angiogenin inhibited (P < 0.05) granulosa cell progesterone production and theca cell androstenedione production but did not affect (P > 0.10) granulosa cell estradiol production or theca cell progesterone production, and did not affect numbers of granulosa or theca cells. In experiments 3 and 4, granulosa and theca cells from both small and large follicles were cultured with 300 ng/ml of angiogenin to determine if size of follicle influenced responses to angiogenin. At 300 ng/ml, angiogenin increased large follicle granulosa cell proliferation but decreased small follicle granulosa cell progesterone and estradiol production and large follicle theca cell progesterone production. In experiments 5 and 6, angiogenin stimulated (P < 0.05) proliferation and DNA synthesis in large follicle granulosa cells. In experiment 7, 300 ng/ml of angiogenin increased (P < 0.05) CYP19A1 messenger RNA (mRNA) abundance in granulosa cells but did not affect CYP11A1 mRNA abundance in granulosa or theca cells and did not affect CYP17A1 mRNA abundance in theca cells. We conclude that angiogenin appears to target both granulosa and theca cells in cattle, but additional research is needed to further understand the mechanism of action of angiogenin in granulosa and theca cells, as well as its precise role in folliculogenesis.

Fibroblast growth factor 9 (FGF9) has been suggested to act as an antidifferentiation factor in cattle by reducing steroidogenesis and increasing cell proliferation in granulosa (GC) and theca (TC) cells. The objective of this study was to characterize FGF9 mRNA abundance in GC and TC during development of dominant follicles in dairy cattle. Estrous cycles of nonlactating dairy cattle were synchronized, and ovaries were collected on either d 3 to 4 (n = 8) or 5 to 6 (n = 8) postovulation for GC and TC RNA extraction from small (1–5 mm), medium (5.1–8 mm), and large (8.1–18 mm) follicles for PCR analysis. The FGF9 mRNA abundance was greater in GC than in TC. In GC, FGF9 mRNA abundance was greater in small, medium, and large estrogen-inactive [i.e., concentrations of estradiol (E2) < progesterone (P4)] follicles than in large E2-active (i.e., concentrations of E2 > P4) follicles at both early (d 3–4) and late (d 5–6) growing phases of first dominant follicle. Abundance of FGF9 mRNA increased in medium-sized follicles from early to late growing phase of the dominant follicle. In TC, FGF9 mRNA abundance was greater in large E2-inactive follicles than in large E2-active follicles on d 3 to 4 postovulation; no significant differences in TC FGF9 mRNA existed among follicle types on d 5 to 6 postovulation. Correlations among levels of follicular fluid hormones and FGF9 mRNA levels revealed significant negative correlations between GC FGF9 mRNA abundance and follicular fluid E2 (r = −0.68), free IGF-1 (r = −0.63), and E2-to-P4 ratio (r = −0.58). In summary, abundance of FGF9 mRNA in GC and TC increases in medium-sized follicles during development of dominant follicles and is less in dominant E2-active than subordinate E2-inactive follicles, suggesting that FGF9 signaling could contribute to normal follicle development and steroidogenesis in dairy cattle.

Circulating tumor cell (CTC) number measured with the CellSearch assay is prognostic for survival in metastatic castration-resistant prostate cancer before and after therapy. Using a standard operating protocol for sample collection, processing, and analysis, we compared detection rates of CellSearch performed using US Food and Drug Administration-cleared methodology with a second positive selection assay, AdnaTest, and a nonselection polymerase chain reaction (PCR)-based (direct detection PCR [DDPCR]) assay in 55 blood samples from 47 men with progressive metastatic castration-resistant prostate cancer. AdnaTest requires processing within 4 hours of the draw and detects KLK3, PSMA, and EGFR transcripts in cells captured on magnetic beads. The DDPCR assay can be processed up to 7 days after a draw and detects KLK2, KLK3, HOXB13, GRHL2, and FOXA1 genes. AdnaTest and DDPCR were considered positive if at least 1 transcript was detected. AdnaTest detected CTCs in 34 samples (62%; 95% confidence interval [CI], 48%-75%), of which 23 (68%) had unfavorable CTC counts by CellSearch. A positive DDPCR result was seen in 38 cases (69%; 95% CI, 55%-81%), including 24 (63%) with unfavorable CellSearch CTC counts. CellSearch found unfavorable CTC counts in 25 samples (45%; 95% CI, 33%-58%). Sensitivities were similar between the AdnaTest and DDPCR assays, and both were more sensitive than CellSearch. Concordance probability estimates (possible values, 0.5-1.0) associating the biomarker result with survival were similar: 0.77 (SE, 0.07) for AdnaTest, 0.72 (SE, 0.08) for DDPCR, and 0.76 (SE, 0.06) for CellSearch. Overall detection rates between the AdnaTest and DDPCR assays were similar, and both were superior to CellSearch. The DDPCR assay required the lowest blood volume, least on-site processing, and longest stability for batch processing.

IMPORTANCE A blood test to determine whether to treat patients with metastatic castration-resistant prostate cancer (mCRPC) with an androgen receptor signaling (ARS) inhibitor or taxane is an unmet medical need. OBJECTIVE To determine whether a validated assay for the nuclear-localized androgen receptor splice variant 7 (AR-V7) protein in circulating tumor cells can determine differential overall survival among patients with mCRPC treated with taxanes vs ARS inhibitors. DESIGN, SETTING, AND PARTICIPANTS This blinded correlative study conducted from December 31, 2012, to September 1, 2016, included 142 patients with histologically confirmed mCRPC and who were treated at Memorial Sloan Kettering Cancer Center, The Royal Marsden, or the London Health Sciences Centre. Blood samples were obtained prior to administration of ARS inhibitors or taxanes as a second-line or greater systemic therapy for progressing mCRPC. MAIN OUTCOMES AND MEASURES Overall survival after treatment with an ARS inhibitor or taxane in relation to pretherapy AR-V7 status. RESULTS Among the 142 patients in the study (mean [SD] age, 69.5 [9.6] years), 70 were designated as high risk by conventional prognostic factors. In this high-risk group, patients positive for AR-V7 who were treated with taxanes had superior overall survival relative to those treated with ARS inhibitors (median overall survival, 14.3 vs 7.3 months; hazard ratio, 0.62; 95% CI, 0.28-1.39; P = .25). Patients negative for AR-V7 who were treated with ARS inhibitors had superior overall survival relative to those treated with taxanes (median overall survival, 19.8 vs 12.8 months; hazard ratio, 1.67; 95% CI, 1.00-2.81; P = .05). CONCLUSIONS AND RELEVANCE This study suggests that nuclear-localized AR-V7 protein in circulating tumor cells can identify patients who may live longer with taxane chemotherapy vs ARS inhibitor treatment.

The effects of deoxynivalenol (DON) and zearalenone (ZEA) on reproduction in ruminants are unclear. This study was performed to evaluate the impact of DON and ZEA hydroxylated metabolites, α-zearalenol (α-Zol) and β-zearalenone (β-Zol), on cell proliferation, steroidogenesis and gene expression using bovine granulosa cells (GC). Cell proliferation was negatively affected after exposure to β-Zol at 31 μM and after exposure to α-Zol (3.1 μM) alone and combined with DON (3.3 μM). DON and α-Zol decreased steroidogenesis, while β-Zol at high concentration had stimulatory effects. DON and β-Zol increased CYP19A1 mRNA abundance. CYP11A1 mRNA abundance was stimulated by DON, alone and combined with α-Zol and β-Zol, whereas was inhibited by β-Zol alone. Generally mycotoxins effects on cell proliferation, steroidogenesis and gene expression were influenced by the presence or absence of IGF1. In conclusion DON and ZEA metabolites may impair in vitro cell proliferation, steroid production and gene expression in cattle.

Domoic acid (DA) is a potent neurotoxin produced by alga Pseudo-nitzschia spp. and has been associated with reproductive disorders in mammals. The aim of this study was to investigate if DA can affect the reproductive system via direct action on ovarian function. Bovine granulosa and theca cells were used as in vitro models for evaluating DA effects on ovarian cell proliferation and steroid production. In small-follicle granulosa cells (SMGC), cell proliferation and estradiol (E2) production was not affected (P0.05) while progesterone (P4) production was inhibited (P0.05) by DA at all doses tested. In large-follicle granulosa cells (LGGC), DA had no effect (P0.05) on cell proliferation or P4 production while E2 production was stimulated by 1 and 5 µg/ml DA (P0.05). DA (1 µg/ml) attenuated (P0.05) insulin-like growth factor 1 (IGF-1)-induced P4 production by large-follicle theca cells (LGTC), but did not affect androstenedione (A4) production or proliferation of LGTC. In glutamate-free medium, DA inhibited (P0.05) SMGC E2 production and this inhibition was similar to inhibition of E2 by trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid monohydrate (ACPD; a selective metabotropic glutamate receptor subtype agonist) while kainic acid (KA; an ionotropic glutamate receptor subtype agonist) had no effect (P0.10) on E2 production. Collectively, these results show for the first time that DA has direct effects on ovarian GC and TC steroidogenesis. Because DA inhibited E2 and P4 production, DA has the potential to be an endocrine disruptor.

We reported previously that fibroblast growth factor 9 (FGF9) acts as an anti-differentiation factor, stimulating proliferation of granulosa (GC) and theca (TC) cells while suppressing hormone-induced steroidogenesis of these cells. How FGF9 acts to simultaneously suppress steroidogenesis and stimulate proliferation remains to be fully elucidated. Thus, this study was undertaken to clarify the effects of FGF9 on the TC transcriptome. Ovaries were obtained from beef heifers at a local abattoir, TC were isolated from large antral follicles, and cultured with or without 30 ng/mL of FGF9 for 24 h in the presence of LH and IGF-1. Following treatment, total RNA was extracted from TC and processed for microarray using Affymetrix GeneChip Bovine Genome Arrays (n = 4/group). Transcriptome analysis comparing FGF9-treated TC with control TC using 1.3-fold cut-off and a P < 0.05 significance level identified 355 differentially expressed transcripts, with 164 elements up-regulated and 191 elements down-regulated by FGF9. The Ingenuity Pathway Analysis (IPA) was utilized to investigate how FGF9 treatment affects molecular pathways, biological functions, and the connection between molecules in bovine TC. The IPA software identified 346 pathways in response to FGF9 in TC involved in several biological functions, and unveiled interesting relationships among genes related to cell proliferation (e.g., CCND1, FZD5 and MYB), antioxidation/cytoprotection (e.g., HMOX1 and NQO1) and steroidogenesis (e.g., CYP11A1 and STAR). Overall, genes, pathways, and networks identified in this study painted a picture of how FGF9 may regulate folliculogenesis, providing novel candidate genes for further investigation of FGF9 functions in ovarian follicular development.

The heterogeneity of an individual patient's tumor has been linked to treatment resistance, but quantitative biomarkers to rapidly and reproducibly evaluate heterogeneity in a clinical setting are currently lacking. Using established tools available in a College of American Pathologists–accredited and Clinical Laboratory Improvement Amendments–certified clinical laboratory, we quantified digital pathology features on 9,225 individual circulating tumor cells (CTC) from 179 unique metastatic castration-resistant prostate cancer (mCRPC) patients to define phenotypically distinct cell types. Heterogeneity was quantified on the basis of the diversity of cell types in individual patient samples using the Shannon index and associated with overall survival (OS) in the 145 specimens collected prior to initiation of the second or later lines of therapy. Low CTC phenotypic heterogeneity was associated with better OS in patients treated with androgen receptor signaling inhibitors (ARSI), whereas high heterogeneity was associated with better OS in patients treated with taxane chemotherapy. Overall, the results show that quantifying CTC phenotypic heterogeneity can help inform the choice between ARSI and taxanes in mCRPC patients. Cancer Res; 77(20); 5687–98. ©2017 AACR.

Fumonisin B1 (FB1) is a Fusarium mycotoxin frequently occurring in corn in combination with deoxynivalenol (DON) and zearalenone. The aim of this study was to determine if FB1, alone and combined with DON or α-zearalenol (ZEA), zearalenone major active metabolite, can affect granulosa cell proliferation, steroid production, and gene expression in swine. Porcine granulosa cells were cultured for 2 days in serum-containing medium followed by 1 or 2 days in serum-free medium with or without added treatments. Fumonisin B1 had inhibitory effects on granulosa cell proliferation. Deoxynivalenol strongly inhibited cell growth, and no significant difference was detected in combination with FB1. α-Zearalenol showed a stimulatory effect on granulosa cell numbers even in combination with FB1. Regarding steroid production, FB1 increased progesterone production, and FB1 had no effect on estradiol production. Deoxynivalenol strongly inhibited progesterone and estradiol production, and FB1 had no significant effect on this response. α-Zearalenol increased progesterone production, and its combination with FB1 produced additive effects. α-Zearalenol had no effect on estradiol production, whereas it decreased estradiol production when co-treated with FB1. Fumonisin B1 was found to decrease CYP11A1 messenger RNA abundance, and the stimulatory effect of FB1 on progesterone production was found to be not dependent on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity suggesting that FB1 increases progesterone production through a different mechanism. The results show that these Fusarium mycotoxins can influence porcine granulosa cell proliferation and steroid production, thereby demonstrating their potential reproductive effects on swine.

3050 Background: MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets), is a high throughput, targeted-DNA-sequencing panel for somatic mutations created by the Department of Pathology at Memorial Sloan Kettering Cancer Center (MSK). The MSK-IMPACT is FDA approved for tumor tissue profiling to guide treatment selection. Recognizing access to tumor tissue in many cancers is difficult and may harbor inter & intra lesional heterogeneity, we sought to evaluate concordance of sequencing single CTCs vs. paired biopsy analyzed by MSK-IMPACT, to assess CTC vs. tumor clonality, and their relationship to outcomes. Methods: 148 biopsy samples from 138 mCRPC pts were submitted for IMPACT analysis and a blood draw within 30-day prior to initiation of a new line of treatment. Blood samples were sent to Epic Sciences for CTC detection. The detected CTCs underwent single cell low pass whole genome sequencing for copy number variation (CNV). For each set of matched samples, DNA copy number profiles from IMPACT and CTC sequencing were compared for similarity. Results: Of the 114 successfully sequenced samples 58 were from lymph nodes, 23 from bone, 25 from liver or lung, and 8 from other soft tissue. Of the 111 patients with CTCs analyzed, a total of 1073 CTCs were sequenced (range 1-27, median = 4 per pt). Of patients with both IMPACT & CTC, >80% pts had multiple subclones with distinguishable CNV. Concordance of genomics clones was found in 63%, and discordant in 37% cases. The clonal concordance between tissue biopsy and CTC was higher when the biopsy was obtained from bone marrow or a visceral site (71% concordance) than from a lymph node (53% concordance). Conclusions: Single CTC sequencing is often concordant to metastatic tissue, but unique CTC clones and the presence of multi-clonal disease highlight the potential to be underrepresented through tissue biopsy, especially when taken from the lymph node.[Table: see text]

The objectives of this study were to investigate the effects of fibroblast growth factor 9 (FGF9) on hormone-stimulated porcine granulosa cell proliferation and steroid production and to further elucidate the hormonal and developmental control of FGFR2IIIc gene expression in granulosa cells. Porcine ovaries were collected from a local slaughterhouse and granulosa cells were collected from small to medium (1 to 5 mm) follicles for 5 in vitro studies that were conducted. Cells were cultured for 48 h in 5% fetal calf serum plus 5% porcine serum and then treated with various combinations of FSH, IGF-I, FGF9, Sonic hedgehog (SHH), cortisol, PGE2, and/or wingless-type mouse mammary tumor virus integration site family member 5A (WNT5A) in serum-free medium for an additional 24 or 48 h. Medium was collected for analysis of steroid concentration via RIA, or RNA was collected for gene expression analysis of FGFR2IIIc via quantitative reverse transcription PCR. Fibroblast growth factor 9 stimulated (P 0.10) whereas IGF-I increased (P 0.10) on FGFR2IIIc gene expression whereas FGF9 in the presence of FSH and IGF-I inhibited (P < 0.05) FGFR2IIIc gene expression. In an in vivo study, granulosa cells from large (7 to 14 mm) follicles had greater (P < 0.05) abundance of FGFR2IIIc mRNA than small (1 to 3 mm) or medium (4 to 6 mm) follicles. In conclusion, IGF-I-induced FGFR2IIIc mRNA may be a mechanism for increased responses to FGF9 in FSH plus IGF-I-treated granulosa cells. Fibroblast growth factor 9 and IGF-I may work together as amplifiers of follicular growth and granulosa cell differentiation by stimulating estradiol production and concomitantly stimulating granulosa cell growth in pigs.

Importance A critical decision in the management of metastatic castration-resistant prostate cancer (mCRPC) is when to administer an androgen receptor signaling (ARS) inhibitor or a taxane. Objective To determine if pretherapy nuclear androgen-receptor splice variant 7 (AR-V7) protein expression and localization on circulating tumor cells (CTCs) is a treatment-specific marker for response and outcomes between ARS inhibitors and taxanes. Design, Setting, and Participants For this cross-sectional cohort study at Memorial Sloan Kettering Cancer Center, 265 men with progressive mCRPC undergoing a change in treatment were considered; 86 were excluded because they were not initiating ARS or taxane therapy; and 18 were excluded for processing time constraints, leaving 161 patients for analysis. Between December 2012 and March 2015, blood was collected and processed from patients with progressive mCRPC immediately prior to new line of systemic therapy. Patients were followed up to 3 years. Main Outcomes and Measures Prostate-specific antigen (PSA) response, time receiving therapy, radiographic progression-free survival (rPFS), and overall survival (OS). Results Overall, of 193 prospectively collected blood samples from 161 men with mCRPC, 191 were evaluable (128 pre-ARS inhibitor and 63 pretaxane). AR-V7–positive CTCs were found in 34 samples (18%), including 3% of first-line, 18% of second-line, and 31% of third- or greater line samples. Patients whose samples had AR-V7–positive CTCs before ARS inhibition had resistant posttherapy PSA changes (PTPC), shorter rPFS, shorter time on therapy, and shorter OS than those without AR-V7–positive CTCs. Overall, resistant PTPC were seen in 65 of 112 samples (58%) without detectable AR-V7–positive CTCs prior to ARS inhibition. There were statistically significant differences in OS but not in PTPC, time on therapy, or rPFS for patients with or without pretherapy AR-V7–positive CTCs treated with a taxane. A multivariable model adjusting for baseline factors associated with survival showed superior OS with taxanes relative to ARS inhibitors when AR-V7–positive CTCs were detected pretherapy (hazard ratio, 0.24; 95% CI, 0.10-0.57; P = .035). Conclusions and Relevance The results validate CTC nuclear expression of AR-V7 protein in men with mCRPC as a treatment-specific biomarker that is associated with superior survival on taxane therapy over ARS-directed therapy in a clinical practice setting. Continued examination of this biomarker in prospective studies will further aid clinical utility.

Background Circulating tumor cells (CTCs) expressing AR-V7 protein localized to the nucleus (nuclear-specific) identify metastatic castration-resistant prostate cancer (mCRPC) patients with improved overall survival (OS) on taxane therapy relative to the androgen receptor signaling inhibitors (ARSi) abiraterone acetate, enzalutamide, and apalutamide. Objective To evaluate if expanding the positivity criteria to include both nuclear and cytoplasmic AR-V7 localization ("nuclear-agnostic") identifies more patients who would benefit from a taxane over an ARSi. Design, setting, and participants The study used a cross-sectional cohort. Between December 2012 and March 2015, 193 pretherapy blood samples, 191 of which were evaluable, were collected and processed from 161 unique mCRPC patients before starting a new line of systemic therapy for disease progression at the Memorial Sloan Kettering Cancer Center. The association between two AR-V7 scoring criteria, post-therapy prostate-specific antigen (PSA) change (PTPC) and OS following ARSi or taxane treatment, was explored. One criterion required nuclear-specific AR-V7 localization, and the other required an AR-V7 signal but was agnostic to protein localization in CTCs. Outcome measurements and statistical analyses Correlation of AR-V7 status to PTPC and OS was investigated. Relationships with survival were analyzed using multivariable Cox regression and log-rank analyses. Results and limitations A total of 34 (18%) samples were AR-V7-positive using nuclear-specific criteria, and 56 (29%) were AR-V7-positive using nuclear-agnostic criteria. Following ARSi treatment, none of the 16 nuclear-specific AR-V7-positive samples and six of the 32 (19%) nuclear-agnostic AR-V7-positive samples had ≥50% PTPC at 12 weeks. The strongest baseline factor influencing OS was the interaction between the presence of nuclear-specific AR-V7-positive CTCs and treatment with a taxane (hazard ratio 0.24, 95% confidence interval 0.078-0.79; p=0.019). This interaction was not significant when nuclear-agnostic criteria were used. Conclusions To reliably inform treatment selection using an AR-V7 protein biomarker in CTCs, nuclear-specific localization is required. Patient summary We analyzed outcomes for patients with metastatic castration-resistant prostate cancer on androgen receptor signaling inhibitors and standard chemotherapy. Patients with circulating tumor cells that had AR-V7 protein in the cellular nuclei were very likely to survive longer on taxane-based chemotherapy, and tests unable to distinguish where the protein is located in the cell are not as predictive of benefit.

Background: The incidence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections is increasing in the United States, and it is possible that municipal wastewater could be a reservoir of this microorganism. To date, no U.S. studies have evaluated the occurrence of MRSA in wastewater. Objective: We examined the occurrence of MRSA and methicillin-susceptible S. aureus (MSSA) at U.S. wastewater treatment plants. Methods: We collected wastewater samples from two Mid-Atlantic and two Midwest wastewater treatment plants between October 2009 and October 2010. Samples were analyzed for MRSA and MSSA using membrane filtration. Isolates were confirmed using biochemical tests and PCR (polymerase chain reaction). Antimicrobial susceptibility testing was performed by Sensititre® microbroth dilution. Staphylococcal cassette chromosome mec (SCCmec) typing, Panton-Valentine leucocidin (PVL) screening, and pulsed field gel electrophoresis (PFGE) were performed to further characterize the strains. Data were analyzed by two-sample proportion tests and analysis of variance. Results: We detected MRSA (n = 240) and MSSA (n = 119) in 22 of 44 (50%) and 24 of 44 (55%) wastewater samples, respectively. The odds of samples being MRSA-positive decreased as treatment progressed: 10 of 12 (83%) influent samples were MRSA-positive, while only one of 12 (8%) effluent samples was MRSA-positive. Ninety-three percent and 29% of unique MRSA and MSSA isolates, respectively, were multidrug resistant. SCCmec types II and IV, the pvl gene, and USA types 100, 300, and 700 (PFGE strain types commonly found in the United States) were identified among the MRSA isolates. Conclusions: Our findings raise potential public health concerns for wastewater treatment plant workers and individuals exposed to reclaimed wastewater. Because of increasing use of reclaimed wastewater, further study is needed to evaluate the risk of exposure to antibiotic-resistant bacteria in treated wastewater.

Resistin is an adipokine that has not been extensively studied in cattle but is produced by adipocytes in greater amounts in lactating versus non-lactating cattle. Seven experiments were conducted to determine the effect of resistin on proliferation, steroidogenesis, and gene expression of theca and granulosa cells from small (1-5mm) and/or large (8-22 mm) cattle follicles. Resistin had no effect on IGF-I-induced proliferation of large-follicle theca cells or small-follicle granulosa cells, but decreased IGF-I-induced proliferation of large-follicle granulosa cells. Resistin weakly stimulated FSH plus IGF-I-induced estradiol production by large-follicle granulosa cells, but had no effect on IGF-I- or insulin-induced progesterone and androstenedione production by theca cells or progesterone production by granulosa cells of large follicles. In small-follicle granulosa cells, resistin attenuated the stimulatory effect of IGF-I on progesterone and estradiol production of small-follicle granulosa cells. RT-PCR measuring abundance of side-chain cleavage enzyme (CYP11A1), aromatase (CYP19A1), FSH receptor (FSHR) and LH receptor (LHCGR) mRNA in large- and small-follicle granulosa cells indicated that resistin reduced the stimulatory effect of IGF-I on CPY11A1 mRNA abundance in large-follicle granulosa cells but had no effect on CYP19A1, FSHR or LHCGR mRNA abundance in large- or small-follicle granulosa cells. Resistin had no effect on CYP11A1, CYP17A1 or LHCGR mRNA abundance in theca cells. These results indicate that resistin preferentially inhibits steroidogenesis of undifferentiated (small follicle) granulosa cells and inhibits proliferation of differentiated (large follicle) granulosa cells, indicating that the ovarian response to resistin is altered during follicular development.

Importance Preferential delivery of docetaxel to tumors by prostate-specific membrane antigen (PSMA)–targeted nanoparticles is clinically effective, and the selective reduction of PSMA-positive circulating tumor cells (CTCs) after treatment has implications for patient selection and disease monitoring. Objective To determine the safety and efficacy of BIND-014, a PSMA-directed docetaxel-containing nanoparticle, in patients with metastatic castration-resistant prostate cancer (mCRPC). Design, Setting, and Participants A multicenter open-label, phase 2 clinical trial of 42 chemotherapy-naive patients with progressing mCRPC after treatment with abiraterone acetate and/or enzalutamide was conducted from June 24, 2013, to June 10, 2016. Intervention Treatment with BIND-014 at a dosage of 60 mg/m 2 was given intravenously on day 1 of 21-day cycles in combination with prednisone until disease progression or unacceptable toxic effects occurred. Main Outcomes and Measures The primary end point was radiographic progression-free survival according to Prostate Cancer Working Group 2 recommendations and Response Evaluation Criteria in Solid Tumors, version 1.1. Secondary end points included prostate-specific antigen (PSA) response (≥50% reduction from baseline) and changes in CTC number (from ≥5 to Results Among the 42 patients (81% white), the median age was 66 (range, 50-85) years, and median number of doses received was 6 (range, 1-21). A PSA response was observed in 12 of 40 patients (30%; 95% CI, 18%-45%), measurable disease response in 6 of 19 (32% [95% CI, 15%-54%]), and CTC conversions in 13 of 26 (50%; 95% CI, 32%-68%). Median radiographic progression-free survival was 9.9 (95% CI, 7.1-12.6) months. With use of the Epic Sciences non-EPCAM-based CTC detection platform, CTCs were detected in 16 of 18 patients (89%); 11 of 18 (61%) had CTCs with PSMA expression above the analytical threshold level (PSMA positive) at baseline (range, 0.4-72.4 CTCs/mL). After treatment, PSMA-positive CTCs were preferentially reduced. Treatment-related adverse events included grade 1 or 2 fatigue (29 of 42 patients [69%]), nausea (23 [55%]), neuropathy (14 [33%]), and neutropenic fever (1 [2%]). Conclusions and Relevance These findings suggest that treatment with BIND-014 is active and well tolerated in patients with chemotherapy-naive mCRPC. Antitumor activity may be related to PSMA expression levels on CTCs, which suggests that patients who are likely to benefit from this treatment can be identified before treatment is initiated. Trial Registration ClinicalTrials.gov Identifier:NCT01812746

5053Background: Biomarkers to predict outcomes for individual patients (pts) on standard of care Rx is an unmet medical need in the treatment of mCRPC. Using baseline (BL) blood draws, we previousl...

Brain ribonuclease (BRB) is a member of the ribonuclease A superfamily that is constitutively expressed in a range of tissues and is the functional homolog of human ribonuclease 1. This study was designed to characterize BRB gene expression in granulosa cells (GCs) during development of bovine dominant ovarian follicles and to determine the hormonal regulation of BRB in GCs. Estrous cycles of Holstein cows (n = 18) were synchronized, and cows were ovariectomized on either day 3 to 4 or day 5 to 6 after ovulation during dominant follicle growth and selection. Ovaries were collected, follicular fluid (FFL) was aspirated, and GCs were collected for RNA isolation and quantitative polymerase chain reaction. Follicles were categorized as small (1-5 mm; pooled per ovary), medium (5-8 mm; individually collected), or large (8.1-17 mm; individually collected) based on surface diameter. Estradiol (E2) and progesterone (P4) levels were measured by radioimmunoassay (RIA) in FFL. Abundance of BRB messenger RNA (mRNA) in GCs was 8.6- to 11.8-fold greater (P0.05) in small (n = 31), medium (n = 66), and large (n = 33) subordinate E2-inactive (FFL E2P4) follicles than in large (n = 16) dominant E2-active (FFL E2P4) follicles. In the largest 4 follicles, GCs BRB mRNA abundance was negatively correlated (P0.01) with FFL E2 (r = -0.65) and E2:P4 ratio (r = -0.46). In experiment 2, GCs from large (8-22 mm diameter) and small (1-5 mm diameter) follicles were treated with insulin-like growth factor 1 (IGF1; 0 or 30 ng/mL) and/or tumor necrosis factor alpha (0 or 30 ng/mL); IGF1 increased (P0.05) BRB mRNA abundance, and tumor necrosis factor alpha decreased (P0.001) the IGF1-induced BRB mRNA abundance in large-follicle GCs. In experiment 3 to 6, E2, follicle-stimulating hormone, fibroblast growth factor 9, cortisol, wingless 3A, or sonic hedgehog did not affect (P0.10) abundance of BRB mRNA in GCs; thyroxine and luteinizing hormone increased (P0.05), whereas prostaglandin E2 (PGE2) decreased (P0.05) BRB mRNA abundance in small-follicle GCs. Treatment of small-follicle GCs with recombinant human RNase1 increased (P0.05) GCs numbers and E2 production. In conclusion, BRB is a hormonally and developmentally regulated gene in bovine GCs and may regulate E2 production during follicular growth in cattle.

273 Background: A previous analysis of 161 patients (pts) tested for nuclear-localized AR-V7(+) CTCs showed a therapy interaction between AR-V7 positivity and improved overall survival (OS) on taxane chemotherapy vs. androgen receptor signaling inhibitors (ARSI). To validate the use of the biomarker result for physician decision making, we prospectively analyzed an independent, multicenter, blinded, cross-sectional cohort (n = 225) to confirm a therapy interaction with AR-V7. We corrected for possible pt selection bias by the treating physician by analyzing the association of therapy to OS in low and high risk groups defined by the test cohort. Methods: Two analyses were performed: (1) the validation of a therapy interaction between AR-V7 positivity and superior OS benefit for pts treated with taxanes in the context of use for 2nd+ line pts; and (2) as the choice between ARSI or taxanes was at the discretion of the attending physician, pt risk was incorporated into the predictive biomarker assessment. A pt-specific risk score was developed from line of therapy and other covariates to stratify pts as low and high risk, and the association of AR-V7 status and OS was performed within each risk group to correct for physician decision making and address possible confounding with treatment assignment. Results: (1) A therapy interaction between AR-V7(+) pts and lower risk of death on taxanes vs. ARSI (HR: 0.23, p = 0.003, 95% CI: 0.09 – 0.61) was validated. (2) In the validation cohort, high risk AR-V7(+) pts had an OS benefit when treated with taxanes (p = 0.02) and the AR-V7(-) pts had an improved OS with ARSI therapy (p = 0.02). AR-V7 incidence was low in the low risk pts, precluding the analysis for this sub-cohort. Conclusions: The results validate that the nuclear-localized AR-V7(+) biomarker has an interaction with therapy and improved survival on taxanes. Further, when adjusting for pt risk, the biomarker is predictive of OS in the high risk group. Nuclear-localized AR-V7 protein in CTCs can aid in the decision between ARSI and taxane chemotherapy in the 2nd or greater line of therapy for mCRPC.

Background: Epithelial Plasticity (EP) is a proposed mechanism of immune escape, resistance to programmed cell death, resistance to specific drugs, and promotion of metastasis. EP has been extensively explored in mathematical, cell biological, and animal models, which predict the presence of these tumor cells in circulation. Most studies on CTCs in human subjects are based on enrichment of cells expressing EpCAM prior to enumeration or molecular interrogation, which precludes analysis of cells that might be EP. The non-enrichment Epic Sciences platform was utilized to identify CTCs in metastatic castration-resistant prostate cancer patient samples phenotypically consistent with EP: CTCs that do not express cytokeratins (CK), but do express malignant or EP biomarkers: Androgen Receptor (AR), AR splice variant 7 isoform (AR-V7) or N-cadherin. Phenotypic and genotypic analyses of individual CTCs were performed to assess the malignant origin of CK- CTCs and their prevalence associated with overall patient survival. Methods: 221 pre-therapy mCRPC blood samples were collected at Memorial Sloan-Kettering Cancer Center and shipped to Epic Sciences. Patients represent a diverse number of previous therapies and tumor burdens. All nucleated cells were plated to glass slides and subjected to DAPI and immunofluorescent (IF) staining of cytokeratin (CK), CD45 and another tumor marker (AR, AR-V7, or N-cadherin), followed by CTC identification by fluorescent scanners and malignant morphology. All samples were stained for AR, with a subset of samples stained for AR-V7 or N-Cadherin as well. Additionally, 83 AR(+)/CK(-) and 9 AR-V7(+)/CK(-) CTCs were analyzed for copy number variation (CNV) by low pass whole genome single CTC sequencing. Abnormal genomes were identified by amplifications and deletions of known oncogenes and tumor suppressors. Results: 3931 AR(+) CTCs were detected, of which 2996 (76%) expressed CK and 935 (24%) did not. The AR(+)/CK(-) CTCs had a lower median AR signal, less nucleoli, and more nuclear speckles by digital pathology vs. CK(+) CTCs. AR(+)/CK(-) and AR-V7(+)/CK(-) CTCs had gross genomic alterations consistent with malignant prostate origin (AR gain, PTEN loss, RB1 loss, 8q gain, 8p loss, etc), often sharing the same genomic alterations with CK(+) CTCs in the same patients, with some having unique CNV profiles Presence of AR(+)/CK(-) CTCs was associated with worse overall survival (median 13.7 mo vs. 33.9 mo, p < 0.0001) and remained additive and independent to treatment Line, pre-therapy PSA, and therapy type in multivariate models. Conclusions: Similar to CTCs expressing epithelial markers, CK(-) CTCs have gross genomic aberrations consistent with metastatic prostate cancer and are a negative prognostic factor associated with worse overall survival in both iterative threshold and multivariable analyses. Citation Format: Ryon P. Graf, Yipeng Wang, Nicole Schreiber, Brigit McLaughlin, Stephanie Greene, Angel Rodriguez, Adam Jendrisak, Jerry Lee, Mark Landers, Ryan Dittamore, Howard I. Scher. Phenotypic, genomic, and clinical associations of Circulating Tumor Cells (CTCs) lacking epithelial biomarkers in metastatic Castration Resistant Prostate Cancer (mCRPC) [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 1740. doi:10.1158/1538-7445.AM2017-1740

5076 Background: Ra-223 prolongs life in mCRPC pts with symptomatic osseous metastasis with inconsistent effects on PSA. Survival times are prolonged further when combined with Abi/Enza. Data from preclinical studies suggest that Ra-223 may sensitize tumors to DDR agents and/or biologic therapies. But predictive biomarkers of benefit to each or both combinationso are lacking. We studied CTC counts and the prevelance of specific CTC subtypes in patients before and following Ra-223 therapy, both as a single agent and in combination, to identify biomarkers of sensitivity and treatement efficacy, and effects of Ra-223 on tumor biology. Methods: Pre and ~4 week post RA-223 therapy blood samples were collected from 35 pts (2 samples each) given as a single agent (n = 20 pts) or in combination with other therapies (n = 15 pts, 9 w/ Enza, 5 w/ Abi, 1 w/ Taxane). Samples were processed and CTCs analyzed using the Epic Sciences platform. Total CTC count and the prevalence of specific CTC phenotypes present pre and post Rx were identified utilizing high content digital pathology and associated with therapy type and post-treatment change. Results: CTC declines were observed in 55% (11/20) and 60% (9/15) of pts treated with single agent and combination respectively. In Ra-223 alone pts, a novel CTC subtype (high N/C ratio, high nuclear area) was identified at baseline 11/20 samples (med = 33% of CTCs). Which was no longer detected in 10 (90%) of the pts treated. This contrasts with a second novel CTC subtype present at baseline in 4 pts (med CTC = 9%) that increased to 9 cases (med CTC = 18%) at follow-up. Conclusions: A subset of pts demonstrate post-therapy CTC declines following Ra-223 alone or in combination. A novel CTC subtype resolved by RA-223 in conjunction with total CTC kinetics may indicate pt benefit from Ra-223. A novel emergent CTC subtype has also been identified in pts already receiving Ra-223. Single CTC sequencing and protein analysys of these CTC subtypes are ongoing, and may help describe tumor evolution and sensitization to novel therapuetics.

5070 Background: Prior response to A or E does not predict sensitivity to E following A or A following E. The detection of AR-V7 predicts insensitivity to either drug, but identifies only a portion of non-responders. We previously identified 15 CTC subtypes based on unique phenotypic features in mCRPC pts, each with unique biology and different degree of likelihood of predicting resistance to either drug. Here we explored the relationship between individual subtypes and sensitivity to A vs. E, but not both. Methods: 107 pre-treatment blood samples from mCRPC pts starting A (n = 47) or E (n = 60) as a 1st or 2nd line of Tx were analyzed for CTCs utilizing the Epic Sciences platform. Samples were assayed for CTC subtypes based upon 15 pre-defined phenotypic CTC classifiers (Type A-O). Treatment outcomes were assessed by serial PSA changes and landmarked percent time of therapy progression on radiographs, and overall survival following either A or E. Cell type prevalence was also analyzed in relation to clinical outcomes, and subsets of the CTC subtypes subject to single cell NGS to ascertain genomic drivers common to each subtype. Results: CTCs were identified in 94% (101/107) of pt samples. One, cell Type K, found in 25% (27/107) of pts, was associated with a statistically significant inferior outcomes on E for all measures. Whereas similar outcomes were seen between K+ & K- pts treated with A. The distinct features of Cell Type K include a large nucleus, high nuclear entropy and high Nuclear/cytoplasmic AR terminal ratio; and a unique genomic profile enriched for cell cycle and DNA repair alterations relative to other CTC subtypes. Conclusions: The presence of specific CTC subtypes in pre-Rx phlebotomy samples associated with outcomes on A or E. A CTC subtype (Cell Type K) helped to identify pts with poor outcomes on E but not A vs. those without the cell type. Further biologic interrogation of K cells and ongoing clincial validation of the CTC subtype is planned. [Table: see text]

139 Background: Genomic profiling of metastatic tumors can identify mCRPC pts w/ “actionable” targets. However, responses from this approach are infrequent and not durable, broadly attributed to intra-tumor and intrer-cellular het. We sought to understand relationships between single CTC phenotypic and genomic patterns present when starting ARSi or taxane therapy (Tx) to develop more effective biomarker-directed treatment approaches. Methods: 9225 CTCs from 319 blood samples from 179 mCRPC pts were detected and digital phenotypically profiled using the Epic Sciences platform. Unsupervised clustering revealed 15 phenotypic CTC subtypes based on digital pathology features (Type ‘A’-‘O’). Phenotypic diversity was determined by a Shannon index of these subtypes in pt samples. Next, 856 CTCs representing each subtype were individually whole genome sequenced for copy number variation and genomic instability, to define phenogenomic single cell profile and taxonomy. Tx outcomes were associated with CTC profiles. Results: Pre-therapy identification of certain CTC phenotypic subtypes was associated with poor overall survival (OS) following ARSi and/or taxanes. 5/15 subtypes existed primarily in pts w/ high CTC het (84-94%, Bonferroni adjusted p < 0.05, permutation test), and linked to amp of AR and cMYC, and loss of TP53 and PTEN. Clusters of genomic events were linked to phenotypic features (i.e. small cell CTC w/AuroraK & nMYC amp) and provide single cell phenogenomic associations with pathways such as AR/PTEN feedback and DNA repair machinery. Conclusions: Presence ofspecific CTC subtypes was associated w/ poor survival on ARSi and/or taxanes and may inform Tx selection. Identifying CTC subtypes present in high het samples can provide insight into individual pt disease evolution and potential approaches to maximize benefit. Longitudinal monitoring of CTC phenogenomics is being studied in novel Tx trials.[Table: see text]

Fibroblast growth factors (FGF) regulate folliculogenesis of several species, including cattle. The cellular responses to a particular FGF are influenced by the diversity of high affinity fibroblast growth factor receptors (FGFR). There are 4 distinct genes encoding FGFR in vertebrates and the occurrence of mRNA splicing in the immunoglobulin-like domain III generates a diversity of sequences, and results in various isoforms of FGFR1, FGFR2, and FGFR3 (but not of FGFR4). Because FGFR have different ligand specificities, the presence of FGFR in bovine antral follicles is of fundamental importance for the FGF to exert their effects in the ovary. Hence, the objective of this study was to determine if FGFR1c, FGFR2c, FGFR3c, and FGFR4 mRNA abundance in granulosa cells (GC) change according to follicular size, steroidogenic status, and days post-ovulation during growth of first-wave dominant follicles in cattle. Oestrous cycles of non-lactating dairy cattle were synchronized and ovaries were collected on either Day 3–4 (n = 8) or Day 5–6 (n = 8) post-ovulation (as assessed by rectal ultrasonography). Follicular fluid (FFL) was aspirated from small (1–5 mm), medium (5.1–8 mm), or large (8.1–18 mm) follicles for measurement of oestradiol (E2) and progesterone (P4) levels by radioimmunoassay, and GC were collected for mRNA extraction. Relative quantity of target gene mRNA was expressed as 2−ΔΔCt using the comparative threshold cycle (Ct) method. Data were transformed to natural log (x + 1), to correct for heterogeneity of variance, and analysed via factorial ANOVA with the general linear model procedure of SAS and are reported as least squares means ± s.e.M. Follicle group (based on steroidogenic status and size of follicles), but not days post-ovulation or their interaction, significantly affected FGFR1c, FGFR2c, and FGFR3c mRNA abundance, whereas FGFR4 mRNA abundance was not affected by follicle group or days post-ovulation. FGFR1c mRNA abundance was greater (P 1) follicles (10.5 ± 15.5; n = 16) and greater (P

Background: A critical decision in the management of patients (pts) with mCRPC is when to administer an androgen receptor signaling (ARS) directed or a taxane therapy. The detection of AR-V7 mRNA in CTCs has been shown to predict for resistance to ARS,but not to taxane chemotherapy. We evaluated the relationship between AR-V7 protein expression and localization on CTCs to treatment outcomes in a separate, larger cohort as a predictive biomarker for clinical decision making. Methods: 193 prospectively collected blood samples from 161 unique pts with progressive mCRPC about to start an ARS or taxane therapy were evaluated with an Epic Sciences CTC immunoflorescent assay that assesses CTC AR-V7 protein expression and localization in individual cells. Associations between the presence AR-V7(+) CTCs pre-therapy and anti-tumor effects post-therapy included prostate-specific antigen (PSA) changes, radiographic progression free survival (rPFS), time on therapy, and overall survival (OS). Results: 130 pre-ARS inhibitor and 63 pre-taxane samples were assessed of which 191 (99%) were evaluable. AR-V7(+) CTCs were found in 34 (18%) samples including 3% of the 1st, 18% of the 2nd and 31% of the 3rd+ line baseline pre-therapy samples. Patients with AR-V7 positive CTCs in a pre-ARS sample showed no PSA response and had shorter rPFS, time on therapy, and OS than those without AR-V7(+) CTCs. PSA resistance to ARS was also seen in 65 of 112 (58%) of the AR-V7(-) samples. There was no association between PSA response, rPFS, and time on therapy between AR-V7(+) and AR-V7(-) pts treated with taxane therapy. In a multivariate model adjusting for age, type of therapy, line of therapy, and pre-therapy LDH, Hgb, and presence of visceral metastasis, AR-V7(+) pts had a superior OS on taxane therapy relative to ARS (HR: 0.242, CI: 0.103 to 0.569, p = 0.0350). Conclusions: The results validate the expression of the AR-V7 protein in the nucleus of CTCs in men with mCRPC as a treatment specific biomarker that predicts resistance to ARS inhibitor therapy and separately, clinical benefit with taxane therapy over ARS-directed therapy in a clinical practice setting. Continued examination of this biomarker in prospective studies will further determine its clinical utility. Citation Format: Howard I. Scher, David Lu, Nicole A. Schreiber, Jessica Louw, Ryon P. Graf, Ann Johnson, Adam Jendrisak, Glenn Heller, Richard Bambury, Herbert A. Vargas Alverez, Brigit McLaughlin, Justin Wahl, Stephanie Greene, Martin Fleisher, Ryan Dittamore. Nuclear localized AR-V7 protein as a predictive biomarker for treatment selection in metastatic castration resistant prostate cancer (mCRPC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4954.

5038Background: Enzalutamide (ENZ) and abiraterone + prednisone (AA) have transformed the care of pts with mCRPC. Qualified predictive biomarkers are needed to guide treatment selections. Studies o...

5046Background: Upregulation of glucocorticoid receptor protein expression in metastatic biopsies from pts with CRPC has previously been shown to correlate with intrinsic and acquired resistance to...

5013Background: An unmet need in the management of mCRPC is how to use approved AR signaling inhibitors (AR tx) and taxanes (T) to maximize survival for the individual patient. Pts with AR-V7+ CTCs...

5044Background: Prediction of pt response to AR Tx or T is an unmet medical need, as pts may respond to both classes of drug, to one but not the other, or have no response to either. Molecular prof...

Hedgehog signaling is involved in regulation of ovarian function in Drosophila, but its role in regulating mammalian ovarian folliculogenesis is less clear. Therefore, gene expression of Indian hedgehog (IHH) and its type 1 receptor, patched 1 (PTCH1), were quantified in bovine granulosa (GC) or theca (TC) cells of small (1–5 mm) antral follicles by in situ hybridization and of larger (5–17 mm) antral follicles by realtime RT-PCR from ovaries of cyclic cows genetically selected (Twinner) or not selected (control) for twin ovulations. Expression of IHH mRNA was localized to GC and cumulus cells, whereas PTCH1 mRNA was greater in TC than in GC. Estrogen-active (E-A; follicular fluid concentration of estradiol . progesterone) versus estrogen-inactive follicles had a greater abundance of mRNA for IHH in GC and PTCH1 in TC. Abundance of IHH mRNA in GC was not affected by cow genotype, whereas TC PTCH1 mRNA was less in large E-A follicles of Twinners than in controls. In vitro, estradiol and wingless-type (WNT) 3A increased IHH mRNA in IGF1-treated GC. IGF1 and BMP4 treatments decreased PTCH1 mRNA in small TC. Estradiol and LH increased PTCH1 mRNA in IGF1treated TC from large and small follicles, respectively. In summary, functional status of ovarian follicles was associated with differences in hedgehog signaling in GC and TC. We hypothesize that as follicles grow and develop, increased free IGF1 may suppress expression of IHH mRNA by GC and PTCH1 mRNA by TC, and these effects are regulated in a paracrine way by estradiol and other intra- and extragonadal factors. follicular development, follicular maturation, granulosa cells, ovary, theca cells

Fibroblast growth factor 9 (FGF9) protein affects granulosa cell (GC) function but is mostly localized to theca cell (TC) and stromal cell of rat ovaries. The objectives of this study were to determine the 1) effects of FGF9 on TC steroidogenesis, gene expression, and cell proliferation; 2) mechanism of action of FGF9 on TCs; and 3) hormonal control ofFGF9mRNA expression in TCs. Bovine ovaries were collected from a local slaughterhouse and TCs were collected from large (8–22 mm) follicles and treated with various hormones in serum-free medium for 24 or 48 h. FGF9 caused a dose-dependent inhibition (PPLHCGR,CYP11A1, andCYP17A1mRNA (via real-time RT-PCR) in TCs. FGF9 had no effect (P>0.10) onSTARmRNA abundance. Furthermore, FGF9 inhibited dibutyryl cAMP-induced progesterone and androstenedione production in LH+IGF1-treated TCs. By contrast, FGF9 increased (PFGF9mRNA in GCs and TCs was several-fold greater (PPFGF9mRNA in TCs. In summary, expression ofFGF9mRNA in TCs is developmentally and hormonally regulated. FGF9 may act as an autocrine regulator of ovarian function in cattle by slowing TC differentiation via inhibiting LH+IGF1 action via decreasing gonadotropin receptors and the cAMP signaling cascade while stimulating proliferation of TCs.

Hedgehog signaling is involved in regulation of ovarian function in Drosophila, but its role in regulating mammalian ovarian folliculogenesis is less clear. Therefore, gene expression of Indian hedgehog (IHH) and its type 1 receptor, patched 1 (PTCH1), were quantified in bovine granulosa (GC) or theca (TC) cells of small (1-5 mm) antral follicles by in situ hybridization and of larger (5-17 mm) antral follicles by real-time RT-PCR from ovaries of cyclic cows genetically selected (Twinner) or not selected (control) for twin ovulations. Expression of IHH mRNA was localized to GC and cumulus cells, whereas PTCH1 mRNA was greater in TC than in GC. Estrogen-active (E-A; follicular fluid concentration of estradiolprogesterone) versus estrogen-inactive follicles had a greater abundance of mRNA for IHH in GC and PTCH1 in TC. Abundance of IHH mRNA in GC was not affected by cow genotype, whereas TC PTCH1 mRNA was less in large E-A follicles of Twinners than in controls. In vitro, estradiol and wingless-type (WNT) 3A increased IHH mRNA in IGF1-treated GC. IGF1 and BMP4 treatments decreased PTCH1 mRNA in small TC. Estradiol and LH increased PTCH1 mRNA in IGF1-treated TC from large and small follicles, respectively. In summary, functional status of ovarian follicles was associated with differences in hedgehog signaling in GC and TC. We hypothesize that as follicles grow and develop, increased free IGF1 may suppress expression of IHH mRNA by GC and PTCH1 mRNA by TC, and these effects are regulated in a paracrine way by estradiol and other intra- and extragonadal factors.

Gene expression of fibroblast growth factor-9 (FGF9) is decreased in granulosa cells (GC) of cystic follicles compared with normal dominant follicles in cattle. The objectives of this study were to investigate the effects of FGF9 on GC steroidogenesis, gene expression, and cell proliferation and to determine the hormonal control of GC FGF9 production. GC were collected from small (1–5 mm) and large (8–22 mm) bovine follicles and treated in vitro with various hormones in serum-free medium for 24 or 48 h. In small- and large-follicle GC, FGF9 inhibited (P < 0.05) IGF-I-, dibutyryl cAMP-, and forskolin-induced progesterone and estradiol production. In contrast, FGF9 increased (P < 0.05) GC numbers induced by IGF-I and 10% fetal calf serum. FGF9 inhibited (P < 0.05) FSHR and CYP11A1 mRNA abundance in small- and large-follicle GC but had no effect (P > 0.10) on CYP19A1 or StAR mRNA. In the presence of a 3β-hydroxysteroid dehydrogenase inhibitor, trilostane, FGF9 also decreased (P < 0.05) pregnenolone production. IGF-I inhibited (P < 0.05) whereas estradiol and FSH had no effect (P > 0.10) on FGF9 mRNA abundance. TNFα and wingless-type mouse mammary tumor virus integration site family member-3A decreased (P < 0.05) whereas T4 and sonic hedgehog increased (P < 0.05) FGF9 mRNA abundance in control and IGF-I-treated GC. Thus, GC FGF9 gene expression is hormonally regulated, and FGF9 may act as an autocrine regulator of ovarian function by slowing follicular differentiation via inhibiting IGF-I action, gonadotropin receptors, the cAMP signaling cascade, and steroid synthesis while stimulating GC proliferation in cattle.

163 Background: AR directed Tx, including Abiraterone (A) and Enzalutamide (E), prolong survival in patients with mCRPC and are FDA approved. After primary Tx with either A or E, the response to E or A is decreased as is the duration of response. NGS tissue sequencing of mCRPC shows a range of disease phenotypes and genotypes which are associated with increased tumor heterogeneity. Single site biopsy, may not represent the diversity of disease. Our objective is to develop biomarkers of heterogeneity from the analysis of single cells from patients with mCRPC. Methods: 221 blood samples from 179 unique patients (pts) were collected from pts about to begin AR Tx (n = 150) or Taxanes (n = 71). Samples were analyzed with the Epic Sciences platform. Analysis included digital pathology of 20 discrete phenotypic cell features inclusive of AR, CK, size and shape measures. 9225 single CTCs were characterized, data standardized, features clustered and categorized into 15 phenotypically distinct CTC subtypes. Individual pt samples were then analyzed for the frequency and heterogeneity (Shannon Index) of CTC subtypes and monitored for clinical endpoints. A subset of CTCs (n = 350) were individually sequenced and analyzed for clonality and CNV to assess genomic heterogeneity. Results: A diversity of CTC phenotypes were observed at each line of therapy. In AR Tx pts, high heterogeneity predicts 6mo PFS (OR = 2.57, p = 0.01) and shorter OS (HR = 4.5, p < 0.001). Separately, specific CTC phenotypes also predict 6mo PFS (OR = 5, p < 0.001) and shorter OS (HR = 5.8, p < 0.001). Heterogeneity scores were not associated with taxane resistance. NGS analysis identified subclonal drivers of disease progression including multiple private and/or clustered genomic drivers. Conclusions: Single CTC characterization of phenotype and genotype supports the concept of tumor heterogeneity as a driver of disease resistance. The heterogeneity observed supports the potential value of single cell characterization to identify rare somatic sub-clonal alterations and can aid in the development of rational therapeutic trials.

302 Background: CellSearch is an FDA cleared assay that enumerates intact CK+, DAPI+, CD45- cells via EpCAM capture. CTC number is prognostic for survival pre- and post- systemic therapy. These cells are a subset of confirmed genomically altered CTC’s in blood. We report the prognostic significance for OS in mCRPC of CTC subtypes not enumerated by CellSearch including apoptotic CTCs, CK- CTCs, small CTCs, and CTC clusters. Methods: 221 blood samples from 179 unique patients were collected from pts about to begin AR directed (n = 150) or taxane (n = 71) therapy for progressive CRPC. Samples were analyzed utilizing the Epic Sciences platform to enumerate traditional (CK+, abnormal morph), apoptotic (CK+, fragmented nuclei), CK- (CK-/abnormal morph), small (CK+, normal morph), and CTC clusters. Patients were followed for up to 2.3 yrs. Paired CellSearch and Epic traditional CTC counts were collected on 173 patient samples. Results: In paired analysis, Epic traditional CTCs were identified in 86% of patients vs. 67% by CellSearch, and any Epic CTC subtype was found in 96% of the patient samples. Increased frequency of any CTC subtype was a univariate predictor of OS (see table). Conclusions: Epic identified CTC subtypes not identified by CellSearch or other technologies that select or sort based upon size or epithelial expression. Cells were detected in 96% of the patient samples and all 5 subtypes were found to be prognostic for survival. The biologic characterization of CTC subtypes as a biomarker of drug sensitivity is ongoing. The increased clinical sensitivity of subtypes and prevalence of CTCs identified on the Epic platform provides a greatly expanded array of clinically relevant biomarkers to develop predictive liquid biopsy signatures in the metastatic setting. [Table: see text]

ADCs hold promise for enhancing the therapeutic index of cytotoxics. STEAP1 is overexpressed in mCRPC and is the target of an ADC currently under clinical development. To identify pts most likely to benefit from the ADC, we explored STEAP1 expression as a predictive biomarker in tumor tissue using a CLIA-certified IHC assay, and on circulating tumor cells (CTCs) in blood using the CellSearch and EPIC platforms. Sixty pts with progressive mCRPC received doses ranging from 0.3 to 2.8 mg/kg once every three weeks. Response was defined as a ≥50% decline in PSA from baseline, and time on study for >6 months was consistent with continued clinical benefit. At doses of ≥2 mg/kg, the response rate (RR) was 10/45 (22%, 95% CI 9.9-34.1) and was highest in the STEAP1 IHC 3+ group, as was the fraction of patients who remained on study for >6 months. At ≥2 mg/kg, 20/45 pts (44%, 95% CI 29.5-58.5) had unfavorable CTC counts of ≥5/7.5mL at baseline and were considered evaluable. After treatment, conversions from unfavorable to favorable CTC counts of STEAP1 IHC Score and Treatment ResponseSTEAP1 IHC ScorePts Observed/TotalPSA decline by ≥50% from baseline>6 months on treatment(%, 95% CI)(%, 95% CI)(%, 95% CI)1+5/45 (11%, 1.9-20.1)1/5 (20%, -15.1-55.1)0/5 (0%)2+27/454/276/27(60%, 45.7-74.3)(15%, 1.5-28.5)(22%, 6.4-37.6)3+13/455/136/13(29%, 15.7-42.2)(39%, 12.5-65.5)(46%, 18.9-73.1)CTC Analysis and Treatment ResponseCTC endpointTime Point & CTC Conversion vs. baselinePts Observed/TotalPSA decline by ≥50% from baseline>6 months on treatment(%, 95% CI)(%, 95% CI)(%, 95% CI)CTC countsBaseline ≥5/7.5mL20/456/206/20(at ≥2 mg/kg)(44%, 29.5-58.5)(30%, 9.9-50.1)(30%, 9.9-50.1)Baseline An immuno-fluorescence-based assay developed to measure STEAP1 expression levels on CTCs showed readily detectable signal in 18/42 samples (43%, 95% CI 28-58), with a range from 1-56% of STEAP1-positive CTCs per case. After treatment, a decrease in the fraction of patients with STEAP1-positive and an increase in the STEAP1-negative CTCs was observed compared to baseline (see Table). We are prospectively selecting pts with STEAP1 IHC 2+/3+ tumors and assessing STEAP1 levels on CTCs in an ongoing Ph1 expansion study with the goal of developing a companion diagnostic to enrich for mCRPC pts most likely to benefit from treatment with the STEAP1 ADC. Citation Format: Daniel C. Danila, Howard I. Scher, Edith Szafer-Glusman, Amrita Herkal, Rebecca Suttmann, Martin Fleisher, Nicole A. Schreiber, Kristen Curtis, Houston Gilbert, Daniel Maslyar, Bernard Fine, Ron Firestein, Michael Mamounas, Mark R. Lackner, Omar Kabbarah. Predictive biomarkers of tumor sensitivity to STEAP1 antibody-drug conjugate (ADC) in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC). [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 4310. doi:10.1158/1538-7445.AM2015-4310

The disclosure provides methods correlating intra-patient genomic heterogeneity of single CTCs with phenotypic heterogeneity in each of a population of prostate cancer (PCa) patients.

5029 Background: STEAP1 is overexpressed in mCRPC and is an ADC target. STEAP1 expression in tissue assayed by IHC, protein and mRNA expression levels in circulating tumor cells (CTC) and by 89Zr-l...

147 Background: The optimal sequence to administer approved life prolonging ARS inhibitors: Abiraterone (A) and Enzalutamide (E),and taxanes: Docetaxel (D) and Cabazitaxel (C), to maximize individual pt benefit is an unmet medical need. Sensitivity to an individual agent is dependent on the intrinsic biology of the tumor and the treatment to which it has been exposed. For each agent, response rates are lower in pts who have received prior treatment with any of the other drugs, but for the individual, sensitivity to one does not necessarily predict sensitivity to the other. We sought to develop response and resistance signatures to A & E or D & C from a baseline blood sample. Methods: 91 pt blood samples collected from 79 pts for CTC analysis with the Epic Sciences platform prior to treatment (27 pre-A, 28 pre-E, 28 pre-D, 8 pre-C). Epic analysis identified traditional CTCs (CK+, CD45-, intact nuclei, morph distinct), CK- CTCs (CK-, CD45-, intact nuclei, morph distinct), small CTCs (CK+, CD45-, intact nuclei, small cell size), and CTC clusters. IF staining for AR N, AR C exp were performed, digital pathology algorithms analyzed CTC morphology. A classifier was developed to associate clinical phenotypes with outcome to a specific agent. Results: A and E signatures included: AR N/C exp. and presence of CK+, AR+, Nucleoli+ CTCs. D and C signatures by: presence of CK+, small, AR-, Nucleoli+ CTCs. Multivariate algorithms for A and E and D & C were statistically associated with de novo resistance. Line of therapy was not a univariate predictor of response. Conclusions: Characterization of CTCs identified predictive biomarkers of sensitivity to ARS Tx and taxane chemotherapy in mCRPC pts. The proposed A&E signature differs from C and D, providing the opportunity to better guide treatment selection and improve patient outcomes using a real-time, non-invasive blood biopsy. Prospective trials to validate results are planned. [Table: see text]