Circulating tumor cells (CTCs) can provide non-invasive insight into how a cancer patient responds to therapy. Their role in disease monitoring of advanced melanoma patients treated with immune checkpoint inhibitors (ICI) is unknown. CTC protein expression of human leukocyte antigen class-I (HLA I) and programmed death ligand-1 (PD-L1) may give insight into how a patient's disease evolves over the course of treatment. In our study, we utilize microfluidic Exclusion-based Sample Preparation (ESP) technology to isolate and characterize CTCs from patients with advanced-stage melanoma. CTC samples from melanoma patients are collected, captured, and stained. A range of 2 to 35 CTCs is observed in a cohort of 16 samples from 10 advanced-stage melanoma patients treated with ICI therapy. Single-cell protein expression data is generated from image cytometry analysis and used to calculate mean HLA I and PD-L1 expression. Using our ESP capture approach, we successfully detect phenotypic and numerical heterogeneity in CTCs from melanoma patients. Our assay shows sufficient capture sensitivity and promising prognostic and predictive information, as we illustrate in our case example. A greater clinical sample size will be necessary to confirm the diagnostic sensitivity and specificity of the assay in predicting clinical outcomes for patients with advanced-stage melanoma.

Extracellular vesicles (EVs) are cell-secreted lipid bilayer delimited particles that mediate cellular communication. These tiny sacs of cellular information play an important role in cell communication and alter the physiological process under both normal and pathological conditions. As such, tracking EVs can provide valuable information regarding the basic understanding of cell communication, the onset of early malignancy, and biomarker discovery. Most of the current EV-tracking strategies are invasive, altering the natural characteristics of EVs by modifying the lipid bilayer with lipophilic dyes or surface proteins with fluorescent reporters. The invasive labeling strategies could alter the natural processes of EVs and thereby have major limitations for functional studies. Here, we report an alternative minimally invasive EV labeling strategy using PicoGreen (PG), a small molecule that fluoresces at 520 nm when bound to dsDNA. We show that PG binds to dsDNA associated with small EVs (50-200 nm), forming a stable and highly fluorescent PG-DNA complex in EVs (PG-EVs). In both 2D cell culture and 3D organoid models, PG-EV showed efficient tracking properties, including a high signal-to-noise ratio, time- and concentration-dependent uptake, and the ability to traverse a 3D environment. We further validated PG-EV tracking using dual-labeled EVs following two orthogonal labeling strategies: (1) Bioconjugation via surface amine labeling and (2) donor cell engineering via endogenously expressing mCherry-tetraspanin (CD9/CD63/CD81) reporter proteins. Our study has shown the feasibility of using PG-EV as an effective EV tracking strategy that can be applied for studying the functional role of EVs across multiple model systems.

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.

Ewing sarcoma (EWS) is an aggressive pediatric malignancy of the bone and soft tissues in need of novel therapeutic options. To identify potential therapeutic targets, we focused on essential biological pathways that are upregulated by EWS-FLI1, the primary oncogenic driver of EWS, including mitotic proteins such as Aurora kinase A (AURKA) and kinesin family member 15 (KIF15) and its binding partner, targeting protein for Xklp2 (TPX2). KIF15/TPX2 cooperates with KIF11, a key mitotic kinesin essential for mitotic spindle orientation. Given the lack of clinical-grade KIF15/TPX2 inhibitors, we chose to target KIF11 (using SB-743921) in combination with AURKA (using VIC-1911) given that phosphorylation of KIF15S1169 by Aurora A is required for its targeting to the spindle. In vitro, the drug combination demonstrated strong synergy (Bliss score ≥ 10) at nanomolar doses. Colony formation assay revealed significant reduction in plating efficiency (1–3%) and increased percentage accumulation of cells in the G2/M phase with the combination treatment (45–52%) upon cell cycle analysis, indicating mitotic arrest. In vivo studies in EWS xenograft mouse models showed significant tumor reduction and overall effectiveness: drug combination vs. vehicle control (p ≤ 0.01), SB-743921 (p ≤ 0.01) and VIC-1911 (p ≤ 0.05). Kaplan–Meier curves demonstrated superior overall survival with the combination compared to vehicle or monotherapy arms (p ≤ 0.0001).

Most cases of epithelial ovarian cancer (EOC) exhibit extensive molecular heterogeneity, presenting challenges in developing targeted therapies. Despite extensive efforts and incremental successes in developing targeted drugs and immunotherapies for other cancers, chemotherapies continue to be the most used treatment of ovarian cancer. Although seemingly simplistic, identification of drugs targeting cellular machinery independent of genomic and genetic status continues to be a strong clinical need. Previously, we performed an RNAi-based screen of the druggable genome across a diverse histological panel of EOC cell line representing both platinum-sensitive and -resistant tumors (PMID: 23056589). This screen elucidated KIF11 as essential in maintaining EOC cell viability. KIF11, a mitotic spindle assembly motor protein, has been targeted clinically. Although drugs are well tolerated, potent, and specific, the objective response rates to KIF11 inhibitors in clinical trials were commonly less than 10%. The efficacy of KIF11 inhibitors is blunted via a compensatory motor kinesin, KIF15. The overexpression of KIF15 has been shown to compensate for absent KIF11 in the formation of the bipolar spindle apparatus during mitosis. Silencing KIF15 significantly sensitizes cells to KIF11 inhibitors and resensitizes resistant cells to KIF11 inhibitors. We developed a high throughput screening approach using Alpha technology to identify compounds that inhibit the protein-protein interaction (PPI) between KIF15 and TPX2, a unique approach to inhibiting KIF15 from previous efforts. Of the nearly 200,000 compounds screened, 177 compounds were selected to be further characterized based on assay performance and chemical properties. These compounds were screened for TPX2 or KIF15 binding by STD-NMR and waterLOGSY. Three compounds across two chemotypes were confirmed to bind KIF15. No compounds were found to bind TPX2. Additionally, 168 of the 177 compounds were screened for drug synergism in vitro. The synergism assay yielded 32 strongly and 8 weakly synergistic hits. The lead compound in each of the two chemotypes revealed by NMR were classified as strongly synergistic (max. bliss score of 8.0 and 29.9). To expand the potential lead compounds identified for further development, an antibody-free cellular thermal shift assay (CETSA) is being completed with 168 compounds. Preliminarily, CETSA has shown that the two lead compounds significantly stabilize KIF15 (ΔTm = 5.6 °C and 9.6 °C). These two lead compounds behaved in a dose-dependent manner in the AlphaScreen (IC50= 2 µM and 6 µM), a favorable characteristic for further development. To date, two chemotypes have been identified as KIF15 inhibitors uniquely targeting the KIF15-TPX2 PPI. The data indicates KIF15 inhibition in combination with KIF11 inhibition is synergistic, thus demonstrating a potential novel treatment approach for people with EOCs. Citation Format: Benjamin K. Gibbs, Justin T. Douglas, Rebecca J. Wates, Peter R. McDonald, Amy M. Whitaker, Cornelius N. Ndi, Harsh B. Pathak, Laurie A. Harned, Sarah A. Neuenswander, Melinda A. Broward, Bret D. Freudenthal, Anuradha Roy, Frank J. Schoenen, Andrew K. Godwin. Targeting the KIF15-TPX2 PPI to overcome KIF11 inhibitor resistance in epithelial ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5334.

BACKGROUND:MiT-Renal Cell Carcinoma (RCC) is characterized by genomic translocations involving microphthalmia-associated transcription factor (MiT) family members TFE3, TFEB, or MITF. MiT-RCC represents a specific subtype of sporadic RCC that is predominantly seen in young patients and can present with heterogeneous histological features making diagnosis challenging. Moreover, the disease biology of this aggressive cancer is poorly understood and there is no accepted standard of care therapy for patients with advanced disease. Tumor-derived cell lines have been established from human TFE3-RCC providing useful models for preclinical studies.METHODS:TFE3-RCC tumor derived cell lines and their tissues of origin were characterized by IHC and gene expression analyses. An unbiased high-throughput drug screen was performed to identify novel therapeutic agents for treatment of MiT-RCC. Potential therapeutic candidates were validated in in vitro and in vivo preclinical studies. Mechanistic assays were conducted to confirm the on-target effects of drugs.RESULTS:The results of a high-throughput small molecule drug screen utilizing three TFE3-RCC tumor-derived cell lines identified five classes of agents with potential pharmacological efficacy, including inhibitors of phosphoinositide-3-kinase (PI3K) and mechanistic target of rapamycin (mTOR), and several additional agents, including the transcription inhibitor Mithramycin A. Upregulation of the cell surface marker GPNMB, a specific MiT transcriptional target, was confirmed in TFE3-RCC and evaluated as a therapeutic target using the GPNMB-targeted antibody-drug conjugate CDX-011. In vitro and in vivo preclinical studies demonstrated efficacy of the PI3K/mTOR inhibitor NVP-BGT226, Mithramycin A, and CDX-011 as potential therapeutic options for treating advanced MiT-RCC as single agents or in combination.CONCLUSIONS:The results of the high-throughput drug screen and validation studies in TFE3-RCC tumor-derived cell lines have provided in vitro and in vivo preclinical data supporting the efficacy of the PI3K/mTOR inhibitor NVP-BGT226, the transcription inhibitor Mithramycin A, and GPNMB-targeted antibody-drug conjugate CDX-011 as potential therapeutic options for treating advanced MiT-RCC. The findings presented here should provide the basis for designing future clinical trials for patients with MiT-driven RCC.

This supplementary file contains additional Matierials and Methods detailing the VERSA process. Figure S1. Longitudinal analysis of capture efficiency of Epcam antibody. Figure S2. Multi-parametric analysis of gene expression, genomic profiling and AR protein analysis from captured CTCs. Table S1. Catalog numbers of primers used for quantitative qPCR. Table S2. Patients characteristics for patients 1-17. Table S3. Patient characteristics from patients 18-43

Prolonged cellular hypoxia leads to energetic failure and death. However, sublethal hypoxia can trigger an adaptive response called hypoxic preconditioning. While prolyl-hydroxylase (PHD) enzymes and hypoxia-inducible factors (HIFs) have been identified as key elements of oxygen-sensing machinery, the mechanisms by which hypoxic preconditioning protects against insults remain unclear. Here, we perform serum metabolomic profiling to assess alterations induced by two potent cytoprotective approaches, hypoxic preconditioning and pharmacologic PHD inhibition. We discover that both approaches increase serum kynurenine levels and enhance kynurenine biotransformation, leading to preservation of NAD+ in the post-ischemic kidney. Furthermore, we show that indoleamine 2,3-dioxygenase 1 (Ido1) deficiency abolishes the systemic increase of kynurenine and the subsequent renoprotection generated by hypoxic preconditioning and PHD inhibition. Importantly, exogenous administration of kynurenine restores the hypoxic preconditioning in the context of Ido1 deficiency. Collectively, our findings demonstrate a critical role of the IDO1-kynurenine axis in mediating hypoxic preconditioning.

Although therapeutic options for patients with advanced renal cell carcinoma (RCC) have increased in the past decade, no biomarkers are yet available for patient stratification or evaluation of therapy resistance. Given the dynamic and heterogeneous nature of clear cell RCC (ccRCC), tumor biopsies provide limited clinical utility, but liquid biopsies could overcome these limitations. Prior liquid biopsy approaches have lacked clinically relevant detection rates for patients with ccRCC. This study employed ccRCC‐specific markers, CAIX and CAXII, to identify circulating tumor cells (CTC) from patients with metastatic ccRCC. Distinct subtypes of ccRCC CTCs were evaluated for PD‐L1 and HLA‐I expression and correlated with patient response to therapy. CTC enumeration and expression of PD‐L1 and HLA‐I correlated with disease progression and treatment response, respectively. Longitudinal evaluation of a subset of patients demonstrated potential for CTC enumeration to serve as a pharmacodynamic biomarker. Further evaluation of phenotypic heterogeneity among CTCs is needed to better understand the clinical utility of this new biomarker.

Papillary renal cell carcinomas (PRCC) are a histologically and genetically heterogeneous group of tumors that represent 15-20% of all kidney neoplasms and may require diverse therapeutic approaches. Alteration of the NF2 tumor suppressor gene, encoding a key regulator of the Hippo signaling pathway, is observed in 22.5% of PRCC. The Hippo signaling pathway controls cell proliferation by regulating the transcriptional activity of Yes-Associated Protein, YAP1. Loss of NF2 results in aberrant YAP1 activation. The Src family kinase member Yes also regulates YAP1 transcriptional activity. This study investigated the importance of YAP and Yes activity in three NF2-deficient PRCC cell lines. NF2-deficency correlated with increased expression of YAP1 transcriptional targets and siRNA-based knockdown of YAP1 and Yes1 downregulated this pathway and dramatically reduced cell viability. Dasatinib and saracatinib have potent inhibitory effects on Yes and treatment with either resulted in downregulation of YAP1 transcription targets, reduced cell viability, and G0-G1 cell cycle arrest. Xenograft models for NF2-deficient PRCC also demonstrated reduced tumor growth in response to dasatinib. Thus, inhibiting Yes and the subsequent transcriptional activity of YAP1 had a substantial anti-tumor cell effect both in vitro and in vivo and may provide a viable therapeutic approach for patients with NF2-deficient PRCC.

Heat shock protein 90 (HSP90) is a molecular chaperone necessary for the folding and proper function of multiple "client" proteins. HSP90 is involved in numerous biological processes and is critical to maintain proteostasis and to protect the cells from potentially harmful environmental stresses such as heat. However, in cancer, the role of HSP90, and other molecular chaperones, is corrupted as many of HSP90 clients are kinases and transcription factors whose aberrant activation or mutation drives tumor growth. Thus, developing a polytherapy, or combination therapy, that includes an HSP90 inhibitor in addition to targeting an oncogene or oncogenic pathway is an appealing therapeutic approach. This protocol will provide detailed methods on how to assess the potential synergy of polytherapy by viability assays in vitro.

Abstract Purpose: Circulating biomarkers are an emerging tool to monitor treatment response and the emergence of resistant phenotypes. However, studies of circulating biomarkers, including circulating tumor cells (CTCs), in patients with clear cell renal cell carcinoma (ccRCC) have been limited due to difficulty in biomarker identification. Platforms relying on EpCAM and cytokeratin to identify CTCs have been limited due to significant phenotypic and intrapatient heterogeneity in renal cancer. Carbonic anhydrase IX (CAIX) and XII (CAXII) are more broadly expressed in ccRCC and recently been shown to capture CTCs from patients with ccRCC. However, downregulation of these targets can also occur; EpCAM is also expressed on a subset of cells that could go undetected if only CAIX was used to capture these cells. The aim of this study is to optimize multi-marker capture and analysis of ccRCC CTCs using EpCAM, CAIX and CAXII for further molecular analysis. Methods: We utilized the VERSA platform, an integrated CTC capture and analysis technology, to optimize capture of multiple ccRCC cell lines using antibodies to CAIX and/or EpCAM. To maximize the capture efficiency of ccRCC CTCs, we altered the magnetic particle type, antibody concentration, and tested both direct and indirect capture methods. Once an optimal method of capture was determined, we captured CTCs in an initial cohort of ten patients with ccRCC. CTCs were identified as cells that were captured by either CAIX or EpCAM, had an intact nucleus, were negative for CD45/CD34/CD66b, and positive for cytokeratin. Results: Capture of cell lines show that a combined CAIX + EpCAM capture was more efficient than single antibody capture using either EpCAM or CAIX alone. The type of magnetic particle used in the assay also affected capture efficiency. Sera-Mag beads (GE Healthcare) captured significantly more cells than FlowComp Dynabeads (Life Technologies) (94% vs 76%). Further increases in efficiency were made by incubating with antibody prior to bead conjugation (indirect binding, 94%) when compared to incubating the cells with antibody-conjugated beads (direct binding, 98%). This may result from increased accessibility of free antibody to partially obstructed antigens that is unique to renal cell carcinoma. This optimized assay has now been applied to ccRCC patients and has identified CTCs in up to 90% of patients with metastatic disease. Conclusions: We have increased the capture efficiency and identification of ccRCC cells by capturing with a combination of CAIX and EpCAM antibodies and optimizing bead and binding conditions. In doing so, we are able to identify and interrogate populations of CTCs that would be lost in capture methods that rely on EpCAM alone. These assays are now being utilized in multiple biomarker and therapeutic trials for patients with clear cell renal cell carcinoma. Citation Format: Rory M. Bade, Benjamin K. Gibbs, Jamie M. Sperger, Christos Kyriakopolous, Hamid Emamekhoo, Rana R. McKay, Toni K. Choueiri, Joshua M. Lang. Development of multi-marker capture and analysis of circulating tumor cells in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4585.

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology V1 Apr 2017MP98-10 UTILITY OF HIGH THROUGHPUT SCREENING IN IDENTIFYING AND REPURPOSING SMALL MOLECULE INHIBITORS FOR UROTHELIAL CARCINOMA Louis Krane, Reema Railkar, Tom Sanford, Benjamin Gibbs, Carole Sourbier, Christopher Ricketts, Darmood Wei, Kai Hammerich, Abhinav Sidana, Brad Scroggins, Rajarshi Guha, Kelli Wilson, Craig Thomas, and Piyush K Agarwal Louis KraneLouis Krane More articles by this author , Reema RailkarReema Railkar More articles by this author , Tom SanfordTom Sanford More articles by this author , Benjamin GibbsBenjamin Gibbs More articles by this author , Carole SourbierCarole Sourbier More articles by this author , Christopher RickettsChristopher Ricketts More articles by this author , Darmood WeiDarmood Wei More articles by this author , Kai HammerichKai Hammerich More articles by this author , Abhinav SidanaAbhinav Sidana More articles by this author , Brad ScrogginsBrad Scroggins More articles by this author , Rajarshi GuhaRajarshi Guha More articles by this author , Kelli WilsonKelli Wilson More articles by this author , Craig ThomasCraig Thomas More articles by this author , and Piyush K AgarwalPiyush K Agarwal More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3075AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In this study we performed the first identified quantitative high throughput screening to identify potential targets in urothelial cancer cell lines. We noted a potential new therapy (bardoxolone methyl) and validated this compound with further in vitro studies in cell lines not included in the screen. METHODS We screened 8 bladder cancer cell lines against 1,912 oncology-focused drugs using a 48 hr cell proliferation assay with an ATP-based readout (CellTiterGlo), for activity and potency of the compounds in a dose response manner. We identified candidate drugs based on two parameters: 1) more than 70% inhibition at 48 hours 2) a curve class of -1.1/-1.2 indicating curve class with good fit (r2>0.9). Follow up assays in additional cell lines, including viability, spheroid culture, nuclear localization assay, invasion, cell cycle and murine xenograft models were used as confirmation of efficacy and mechanism of the bardoxolone methyl. RESULTS Ward clustering analysis of the initial cell lines (figure 1a) along with curve class demonstration (1b) and medication grouping efficacy (1c) is presented here. Among the candidate drugs which were most active in all compounds, bardoxolone methyl was the most attractive based on IC 50 and previous human safety studies. Invasion assays (Figure 2a) 3-dimensional culture (2b) cell cycle arrest (2c) demonstrated excellent in vitro efficacy. Murine models were then created which highlighted strong inhibition of tumor growth in murine xenograft. (Figure 2d) CONCLUSIONS Quantitative high throughput screening was successful in identifying bardoxolone methyl as a novel treatment of urothelial carcinoma in vitro. Repurposing of this molecule may allow for future patient trials in urothelial malignancies. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1315 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Louis Krane More articles by this author Reema Railkar More articles by this author Tom Sanford More articles by this author Benjamin Gibbs More articles by this author Carole Sourbier More articles by this author Christopher Ricketts More articles by this author Darmood Wei More articles by this author Kai Hammerich More articles by this author Abhinav Sidana More articles by this author Brad Scroggins More articles by this author Rajarshi Guha More articles by this author Kelli Wilson More articles by this author Craig Thomas More articles by this author Piyush K Agarwal More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Non-enzymatic protein modification driven by thioester reactivity is thought to play a major role in the establishment of cellular lysine acylation. However, the specific protein targets of this process are largely unknown. Here we report an experimental strategy to investigate non-enzymatic acylation in cells. Specifically, we develop a chemoproteomic method that separates thioester reactivity from enzymatic utilization, allowing selective enrichment of non-enzymatic acylation targets. Applying this method to cancer cell lines identifies numerous candidate targets of non-enzymatic acylation, including several enzymes in lower glycolysis. Functional studies highlight malonyl-CoA as a reactive thioester metabolite that can modify and inhibit glycolytic enzyme activity. Finally, we show that synthetic thioesters can be used as novel reagents to probe non-enzymatic acylation in living cells. Our studies provide new insights into the targets and drivers of non-enzymatic acylation, and demonstrate the utility of reactivity-based methods to experimentally investigate this phenomenon in biology and disease.

BACKGROUND: Renal cell carcinoma (RCC) is one of the top ten causes of cancer death in the United States. The last ten years have shown a dramatic increase in the number of available treatment options, however metastatic RCC remains largely incurable. The classes of drugs that have been developed can be divided into agents that target the Vascular Endothelial Growth Factor (VEGF) pathway (Sunitinib, Sorafenib, Pazopanib, Axitinib, Bevacizumab), those that target the mammalian Target of Rapamycin (mTOR) pathway (Everolimus, Temsirolimus) and immune based therapies (IL-2 and PD-1 inhibition). There are currently limited biomarkers to guide clinical decisions, mainly due to the lack of tumor cells for longitudinal molecular analysis. Circulating tumor cells (CTCs) are potential a source of tumor cells that can be identified from a blood draw for serial analysis. CTCs have not been reliably detected in RCC due to the significant heterogeneity and high rate of false positive events in this disease when EpCAM has been used. We sought to identify RCC CTCs with alternative markers, such as carbonic anhydrase IX (CAIX) which is found in greater than 90% of clear cell RCC tumors. METHODS AND RESULTS: To evaluate for the presence and subtypes of CTCs from patients with RCC, we utilized a multi-parametric flow cytometry assay. We evaluated heterogeneity across subpopulations of putative CTCs with Epithelial Cell Adhesion Molecule (EpCAM), Carbonic Anhydrase IX (CAIX), Carbonic Anhydrase XII (CAXII), PAX8, and Cytokeratin (CK). Negative controls for immune and endothelial events were performed with markers for CD45, CD14, CD34, CD11b and CD61. We tested twenty blood samples from patients with RCC at the Dana Farber Cancer Institute and University of Wisconsin Carbone Cancer Center. CTC frequency in RCC ranges from 0-5410 CAIX+/CK+ events with a median of 24.5 putative CTCs/7.5mL of blood. A subset of patients with radiographic progression had a higher number of CTCs with a median of 295.2 CTCs/7.5 mL. A range of 1-231 EpCAM+/CK+ events were identified with a median of 5.5 CTCs/7.5mL. There was low frequency of events being CAIX+/EpCAM+/CK+, with a median of 1 CTC/7.5mL of blood. Assay specificity was dramatically improved through the combination of multiple positive markers with stains for immune and endothelial cells given frequent non-specific staining for cytokeratin in RCC blood samples. CONCLUSIONS: CTCs can be identified in patients with RCC using non-traditional markers. CAIX is a more sensitive marker than EpCAM to identify putative CTCs from patients with RCC. Specificity in the assay is critical given the high frequency of false positive events identified if only CD45 is used as a marker for immune cells. Our investigation is ongoing for further molecular characterization of orthogonal endpoints in identified CTCs. Future directions include longitudinal monitoring of CTCs during treatment with VEGF inhibitors. Citation Format: Joshua A. Desotelle, Chorom Pak, Erika Heninger, Jennifer L. Schehr, Rana R. McKay, Benjamin K. Gibbs, Craig Norton, Toni K. Choueiri, Joshua M. Lang. Identification of circulating tumor cells from renal cell carcinoma patients by a multi-parameter flow cytometry assay. [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 3159.

Abstract Purpose: There is a critical clinical need for new predictive and pharmacodynamic biomarkers that evaluate pathway activity in patients treated with targeted therapies. A microscale platform known as VERSA (versatile exclusion-based rare sample analysis) was developed to integrate readouts across protein, mRNA, and DNA in circulating tumor cells (CTC) for a comprehensive analysis of the androgen receptor (AR) signaling pathway. Experimental Design: Utilizing exclusion-based sample preparation principles, a handheld chip was developed to perform CTC capture, enumeration, quantification, and subcellular localization of proteins and extraction of mRNA and DNA. This technology was validated across integrated endpoints in cell lines and a cohort of patients with castrate-resistant prostate cancer (CRPC) treated with AR-targeted therapies and chemotherapies. Results: The VERSA was validated in cell lines to analyze AR protein expression, nuclear localization, and gene expression targets. When applied to a cohort of patients, radiographic progression was predicted by the presence of multiple AR splice variants and activity in the canonical AR signaling pathway. AR protein expression and nuclear localization identified phenotypic heterogeneity. Next-generation sequencing with the FoundationOne panel detected copy number changes and point mutations. Longitudinal analysis of CTCs identified acquisition of multiple AR variants during targeted treatments and chemotherapy. Conclusions: Complex mechanisms of resistance to AR-targeted therapies, across RNA, DNA, and protein endpoints, exist in patients with CRPC and can be quantified in CTCs. Interrogation of the AR signaling pathway revealed distinct patterns relevant to tumor progression and can serve as pharmacodynamic biomarkers for targeted therapies. Clin Cancer Res; 23(3); 746–56. ©2016 AACR.

Abstract Novel therapies that target the PD-1/PD-L1 axis have shown great potential to improve survival for patients with advanced melanoma. However, there are limited predictive biomarkers to identify patients who may respond to this class of agents. Previous studies suggest that a subset of responders to PD-1/PD-L1 targeting therapies have expression of PD-L1 in tumor biopsies. The development of these predictive biomarkers is complicated by intratumoral heterogeneity across primary and metastatic lesions for PD-L1 expression. Circulating tumor cells (CTCs) are one potential source of tumor samples for serial analysis. An integrated CTC capture and analysis technology known as the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform permits evaluation of CTCs at the protein, DNA and mRNA level for biomarkers of both therapeutic response and resistance. Using this platform, we have developed a method to isolate circulating tumor cells from patients with advanced melanoma. Using a cocktail of capture monoclonal antibodies including targets of CD146, GD2 and NG2, we have recovered cells across multiple cell lines with high efficiency. Using single antibody capture of SK-Mel-28 cells, capture was 52%, 54%, and 45% with CD146, GD2 and NG2 targets respectively. However, when using a cocktail of capture antibodies containing CD146, GD2 and NG2, capture increased to 92%. Similar results were obtained with other melanoma cell lines including, 624 and M21 cell lines. Cells were also stained for PD-L1 as a potential predictive biomarker of response to PD-L1 and PD-1 targeting immunotherapies. We are now collecting patient samples with this capture platform to identify CTCs expressing PD-L1 as a potential predictive biomarker to PD-1/PD-L1 targeting therapies. Citation Format: Benjamin K. Gibbs, Lindsay N. Strotman, Stephanie M. Thiede, Jamie M. Sperger, Benjamin P. Casavant, Scott M. Berry, David J. Beebe, Joshua M. Lang. Development of an integrated analysis platform of circulating melanoma cells for PD-L1 expression as a predictive biomarker. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4816. doi:10.1158/1538-7445.AM2014-4816