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Purpose:Circulating tumor DNA (ctDNA) has the potential to guide therapy selection and monitor treatment response in patients with metastatic cancer. However, germline and clonal hematopoiesis–associated alterations can confound identification of tumor-specific mutations in cell-free DNA (cfDNA), often requiring additional sequencing of tumor tissue. The current study assessed whether ctDNA-based treatment response monitoring could be performed in a tumor tissue–independent manner by combining ultra-deep targeted sequencing analyses of cfDNA with patient-matched white blood cell (WBC)-derived DNA.Experimental Design:In total, 183 cfDNA and 49 WBC samples, along with 28 tissue samples, from 52 patients with metastatic colorectal cancer participating in the prospective phase III CAIRO5 clinical trial were analyzed using an ultra-deep targeted sequencing liquid biopsy assay.Results:The combined cfDNA and WBC analysis prevented false-positives due to germline or hematopoietic variants in 40% of patients. Patient-matched tumor tissue sequencing did not provide additional information. Longitudinal analyses of ctDNA were more predictive of overall survival than standard-of-care radiological response evaluation. ctDNA mutations related to primary or acquired resistance to panitumumab were identified in 42% of patients.Conclusions:Accurate calling of ctDNA mutations for treatment response monitoring is feasible in a tumor tissue–independent manner by combined cfDNA and patient-matched WBC genomic DNA analysis. This tissue biopsy-independent approach simplifies sample logistics and facilitates the application of liquid biopsy ctDNA testing for evaluation of emerging therapy resistance, opening new avenues for early adaptation of treatment regimens.

S1. Generation and characterization of AXL knockout cells. S2. Generation and characterization of AXL overexpression cells. S3. Aggregate characterization. S4. Characterization of bone signal in AXL-on mice at time of death. Supplementary Table 1: Differentially regulated genes in doxycycline-treated Tet-Axl 2 cells compared to Tet-Axl 1.

Supplementary Data 1 shows an overview of ctDNA dynamics, treatments, and radiological response measurements per patient.

Purpose:Circulating tumor DNA (ctDNA) has the potential to guide therapy selection and monitor treatment response in patients with metastatic cancer. However, germline and clonal hematopoiesis–associated alterations can confound identification of tumor-specific mutations in cell-free DNA (cfDNA), often requiring additional sequencing of tumor tissue. The current study assessed whether ctDNA-based treatment response monitoring could be performed in a tumor tissue–independent manner by combining ultra-deep targeted sequencing analyses of cfDNA with patient-matched white blood cell (WBC)-derived DNA.Experimental Design:In total, 183 cfDNA and 49 WBC samples, along with 28 tissue samples, from 52 patients with metastatic colorectal cancer participating in the prospective phase III CAIRO5 clinical trial were analyzed using an ultra-deep targeted sequencing liquid biopsy assay.Results:The combined cfDNA and WBC analysis prevented false-positives due to germline or hematopoietic variants in 40% of patients. Patient-matched tumor tissue sequencing did not provide additional information. Longitudinal analyses of ctDNA were more predictive of overall survival than standard-of-care radiological response evaluation. ctDNA mutations related to primary or acquired resistance to panitumumab were identified in 42% of patients.Conclusions:Accurate calling of ctDNA mutations for treatment response monitoring is feasible in a tumor tissue–independent manner by combined cfDNA and patient-matched WBC genomic DNA analysis. This tissue biopsy-independent approach simplifies sample logistics and facilitates the application of liquid biopsy ctDNA testing for evaluation of emerging therapy resistance, opening new avenues for early adaptation of treatment regimens.

The Supplementary Figures file consists of Supplementary Figures 1 to 15 belonging to the manuscript: "Metastatic colorectal cancer treatment response evaluation by ultra-deep sequencing of cell-free DNA and matched white blood cells"

The Supplementary Tables file consists of Supplementary Tables 1 to 15 belonging to the manuscript "Metastatic colorectal cancer treatment response evaluation by ultra-deep sequencing of cell-free DNA and matched white blood cells"

Contains fulltext : 287795.pdf (Publisher’s version ) (Open Access) OBJECTIVE: The objective of this study is to study (1) the relationship between patient-reported symptom burden and information needs in hospital-based palliative care and (2) differences in patient-reported needs during the disease trajectory. METHODS: Observational study: patient-reported symptom burden and information needs were collected via a conversation guide comprising assessment scales for 12 symptoms (0-10), the question which symptom has priority to be solved and a question prompt list on 75 palliative care-related items (35 topics, 40 questions). Non-parametric tests assessed associations. RESULTS: Conversation guides were used by 266 patients. Median age was 65 years (IQ-range, 57-72), 49% were male and 96% had cancer. Patients reported highest burden for Fatigue (median = 7) and Loss of appetite (median = 6) and prioritised Pain (26%), Fatigue (9%) and Shortness of breath (9%). Patients wanted information about 1-38 (median = 14) items, mostly Fatigue (68%), Possibilities to manage future symptoms (68%) and Possible future symptoms (67%). Patients also wanted information about symptoms for which they reported low burden. Patients in the symptom-directed phase needed more information about hospice care. CONCLUSION: Symptom burden and information needs are related. Patients often also want information about non-prioritised symptoms and other palliative care domains. Tailored information-provision includes inviting patients to also discuss topics they did not consider themselves.

Background: Treatment response monitoring of patients with metastatic colorectal cancer is currently performed by computed tomography (CT) imaging, which assesses tumor volume. Circulating tumor DNA (ctDNA) testing has the potential to replace or complement CT imaging by assessing the presence and abundance of tumor-specific mutations, allowing for personalized treatment and early adaptation of treatment regimens through risk stratification and the emergence of therapy resistance mutations. However, germline and clonal hematopoiesis-associated alterations can confound the identification of tumor-specific alterations in cell-free DNA (cfDNA), often requiring additional sequencing of tumor tissue. Aim: The present study assessed whether ctDNA-based treatment response monitoring could be performed in a tumor tissue-independent manner by combining ultra-deep targeted sequencing analyses of cfDNA with patient-matched white blood cell (WBC) derived DNA. Methods: In total, 183 cfDNA and 49 WBC samples, along with 28 tissue samples, from 52 metastatic colorectal cancer patients participating in the prospective phase III CAIRO5 clinical trial were analyzed using an ultra-deep targeted sequencing liquid biopsy assay. Results: The combined cfDNA and WBC analysis prevented the reporting of false positives due to germline or hematopoietic variants in 40% of patients. Patient-matched tumor tissue sequencing did not provide additional information. Longitudinal analyses of ctDNA were more predictive of overall survival than standard-of-care radiological response evaluation. ctDNA mutations related to primary or acquired resistance to the EGFR inhibitor panitumumab were identified in 42% of patients. Conclusions: Accurate ctDNA mutation detection by combined cfDNA and WBC genomic DNA analyses in a tumor tissue-independent manner mitigates sample logistics challenges in the clinical setting, and the application of this approach for evaluation of therapeutic response and resistance opens new avenues for early adaptation of treatment regimens. Citation Format: Iris van 't Erve, Jamie E. Medina, Alessandro Leal, Eniko Papp, Jillian Phallen, Vilmos Adleff, Elaine Jiayuee Chiao, Adith S. Arun, Karen Bolhuis, John K. Simmons, Aanavi Karandikar, Kenneth C. Valkenburg, Mark Sausen, Samuel V. Angiuoli, Robert B. Scharpf, Cornelis J. Punt, Gerrit J. Meijer, Victor E. Velculescu, Remond J. Fijneman. Ultra-deep targeted sequencing of cell-free DNA and patient-matched white blood cells for treatment response evaluation in patients with metastatic colorectal 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 5587.

Despite the growing body of clinical evidence to support the utility of comprehensive genomic profiling (CGP) for advanced cancer patients, only a small fraction of individuals receive precision oncology guided treatment strategies. To facilitate globally available CGP solutions, we have developed and validated the PGDx elio plasma complete cell-free DNA (cfDNA) kitted assay, which employs a hybrid capture-based 521-gene, 2.1 Mb targeted panel, combined with automated bioinformatics and reporting. PGDx elio plasma complete detects single nucleotide variants (SNVs), insertions and deletions (indels), amplifications, translocations, microsatellite instability (MSI), blood tumor mutation burden (bTMB), and loss of heterozygosity (LOH). Targets include clinically relevant alterations, such as those contained within ALK, BRAF, BRCA1/2, EGFR, ERBB2, FGFR1/2/3, KIT, KRAS, MET, NRAS, NTRK1/2/3, PIK3CA, RET, and ROS1. With as little as 10 ng of sample input of cfDNA, and sequenced on the NovaSeq 6000, PGDx elio plasma complete achieves deep coverage across the target region combined with automated bioinformatics and reporting algorithms. We assessed the analytical performance of this assay. Analytical specificity in a cohort of 20 non-cancerous donors was 100% for all clinically actionable variants and 99.9% panel-wide. The reportable range of the assay is ≥ 0.10% variant allele frequency (VAF) for SNVs and indels, ≥ 3 fusion reads for translocations, and ≥ 1.15-fold for amplifications. Furthermore, analytical sensitivity studies using dilution series with known positive alterations demonstrated a limit of detection ranging from 0.32-0.78% VAF for actionable mutations, 0.34-1.75% VAF for panel-wide mutations, 0.33% VAF for translocations, and 1.32-fold for amplifications. The assay produces highly reproducible results, with 100% average positive agreement (APA) for clinically actionable alterations and 92.5% APA for panel-wide alterations across operators, days, and runs. Compared to a CGP assay of a similar size, PGDx elio plasma complete achieved a 94.7% positive predictive value and >99.9% negative predictive value across all variant types assessed. The PGDx elio plasma complete assay can be processed manually or through automated liquid handling systems with high concordance, with an overall success rate of 97.8%. Taken together, these data demonstrate that the PGDx elio plasma complete assay is a sensitive, specific, accurate, reproducible, and robust approach to enable CGP to guide both translational biomarker discovery and CGP-informed precision oncology strategies. Citation Format: Kenneth C. Valkenburg, Vito Caropreso, Jesse Fox, Christopher Gault, Andrew Georgiadis, Kelly M. Gerding, Aanavi Karandikar, Cynthia Maddox, Paul McGregor, David Riley, Vuna Fa. Comprehensive liquid biopsy profiling enabled by PGDx elio plasmacomplete to facilitate precision oncology through decentralized access to testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 72.

The lack of validated, distributed comprehensive genomic profiling assays for patients with cancer inhibits access to precision oncology treatment. To address this, we describe elio tissue complete, which has been FDA-cleared for examination of 505 cancer-related genes. Independent analyses of clinically and biologically relevant sequence changes across 170 clinical tumor samples using MSK-IMPACT, FoundationOne, and PCR-based methods reveals a positive percent agreement of >97%. We observe high concordance with whole-exome sequencing for evaluation of tumor mutational burden for 307 solid tumors (Pearson r = 0.95) and comparison of the elio tissue complete microsatellite instability detection approach with an independent PCR assay for 223 samples displays a positive percent agreement of 99%. Finally, evaluation of amplifications and translocations against DNA- and RNA-based approaches exhibits >98% negative percent agreement and positive percent agreement of 86% and 82%, respectively. These methods provide an approach for pan-solid tumor comprehensive genomic profiling with high analytical performance.

Prostate cancer bone metastasis remains lethal and incurable, and often arises years after elimination of the primary tumor. It is unclear what underlies the decades-long clinical latency before recurrence, but evidence points to the existence of dormant residual tumor cells that disseminated before the primary tumor was eliminated. To design therapies to prevent progression of disseminated tumor cells (DTC) into lethal metastases, it is crucial to understand the mechanism(s) underlying this dormancy. The current study functionally validated our previous observation that implicated the GAS6/AXL axis in mediating DTC dormancy in the bone marrow. AXL-null and AXL-overexpressing prostate cancer cell lines were generated to determine if AXL was necessary and/or sufficient for dormancy. Characterization of these cells in vitro and using in vivo mouse models of DTC growth demonstrated that AXL was indeed sufficient to induce dormancy, but was unable to maintain it long-term and was not absolutely required for a dormancy period. Clinically, AXL expression correlated with longer survival in prostate cancer patients, and AXL was not expressed by cancer cells in primary or metastatic tissue. These data point to a tumor-suppressive role for AXL in prostate cancer, and future work is required to determine if AXL is expressed on human bone marrow DTCs. Implications: The ability of AXL to initiate but not maintain dormancy, coupled with its dispensability, suggests that targeting AXL alone will not prevent lethal metastatic outgrowth, and likely a cooperative network of factors exists to mediate long-term cellular dormancy.

Despite improvements in early detection and advances in treatment, patients with prostate cancer continue to die from their disease. Minimal residual disease after primary definitive treatment can lead to relapse and distant metastases, and increasing evidence suggests that circulating tumour cells (CTCs) and bone marrow-derived disseminated tumour cells (BM-DTCs) can offer clinically relevant biological insights into prostate cancer dissemination and metastasis. Using epithelial markers to accurately detect CTCs and BM-DTCs is associated with difficulties, and prostate-specific markers are needed for the detection of these cells using rare cell assays. Putative prostate-specific markers have been identified, and an optimized strategy for staining rare cancer cells from liquid biopsies using these markers is required. The ideal prostate-specific marker will be expressed on every CTC or BM-DTC throughout disease progression (giving high sensitivity) and will not be expressed on non-prostate-cancer cells in the sample (giving high specificity). Some markers might not be specific enough to the prostate to be used as individual markers of prostate cancer cells, whereas others could be truly prostate-specific and would make ideal markers for use in rare cell assays. The goal of future studies is to use sensitive and specific prostate markers to consistently and reliably identify rare cancer cells.

Approximately 29 000 men die of prostate cancer (PCa) each year in the United States, and 90% to 100% of them are due to incurable bone metastasis. It is difficult to determine (1) when PCa disseminates in the natural history of the disease; (2) where cancer cell disseminates before becoming overt metastatic lesions; and (3) which tumors are aggressive and which are indolent. Tumor tissue and liquid (blood and bone marrow) biopsies provide important information to answer these questions, but significant limitations exist for immunostaining strategies that assess protein expression in these tissues. Classic immunohistochemistry (IHC) assays can typically assess expression of one or two proteins per tissue section. We have developed a novel immunofluorescence staining protocol to detect a panel of seven proteins on PCa tissue from primary tumor biopsies and metastatic lesion autopsy tissue, as well as cancer cells from liquid biopsies. We used a tyramide‐based system to amplify the true signal and optimized the protocol to reduce background signal, thereby boosting the signal‐to‐noise ratio. Any protein‐specific antibody in this protocol can be exchanged for a different validated antibody. This protocol therefore, represents a highly informative and flexible assay that can be used to provide important information about cancer tissue for the purpose of improving detection, diagnosis, and treatment.

// Kenneth C. Valkenburg 1 , Angelo M. De Marzo 2, 3 and Bart O. Williams 1 1 Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, MI 49503, USA 2 Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD 21287, USA 3 Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD 21287, USA Correspondence to: Kenneth C. Valkenburg, email: kvalken1@jhmi.edu Keywords: mouse model, prostate cancer, basaloid, squamous, Apc Received: November 01, 2016 Accepted: May 03, 2017 Published: May 17, 2017 ABSTRACT Prostate cancer is the most diagnosed non-skin cancer in the US and kills approximately 27,000 men per year in the US. Additional genetic mouse models are needed that recapitulate the heterogeneous nature of human prostate cancer. The Wnt/beta-catenin signaling pathway is important for human prostate tumorigenesis and metastasis, and also drives tumorigenesis in mouse models. Loss of Smad4 has also been found in human prostate cancer and drives tumorigenesis and metastasis when coupled with other genetic aberrations in mouse models. In this work, we concurrently deleted Smad4 and the tumor suppressor and endogenous Wnt/beta-catenin inhibitor adenomatous polyposis coli (Apc) in luminal prostate cells in mice. This double conditional knockout model produced invasive castration-resistant prostate carcinoma with no evidence of metastasis. We observed mixed differentiation phenotypes, including basaloid and squamous differentiation. Interestingly, tumor cells in this model commonly lose androgen receptor expression. In addition, tumors disappear in these mice during androgen cycling (castration followed by testosterone reintroduction). These mice model non-metastatic castration resistant prostate cancer and should provide novel information for tumors that have genetic aberrations in the Wnt pathway or Smad4.

Cancers are not composed merely of cancer cells alone; instead, they are complex ‘ecosystems’ comprising many different cell types and noncellular factors. The tumour stroma is a critical component of the tumour microenvironment, where it has crucial roles in tumour initiation, progression, and metastasis. Most anticancer therapies target cancer cells specifically, but the tumour stroma can promote the resistance of cancer cells to such therapies, eventually resulting in fatal disease. Therefore, novel treatment strategies should combine anticancer and antistromal agents. Herein, we provide an overview of the advances in understanding the complex cancer cell–tumour stroma interactions and discuss how this knowledge can result in more effective therapeutic strategies, which might ultimately improve patient outcomes.

// Kenneth C. Valkenburg 1 , Sarah R. Amend 1 , James E. Verdone 1 , Emma E. van der Toom 1 , James R. Hernandez 1 , Michael A. Gorin 1 , Kenneth J. Pienta 1, 2, 3, 4 1 The James Buchanan Brady Urological Institute and Department of Urology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA 2 Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA 3 Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA 4 Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA Correspondence to: Michael A. Gorin, email: mgorin1@jhmi.edu Kenneth C. Valkenburg, email: kvalken1@jhmi.edu Keywords: disseminated tumor cells, mouse models, bone marrow, bone metastasis, cancer Received: June 17, 2016 Accepted: September 01, 2016 Published: September 13, 2016 ABSTRACT Bone metastasis is a lethal and incurable disease. It is the result of the dissemination of cancer cells to the bone marrow. Due to the difficulty in sampling and detection, few techniques exist to efficiently and consistently detect and quantify disseminated tumor cells (DTCs) in the bone marrow of cancer patients. Because mouse models represent a crucial tool with which to study cancer metastasis, we developed a novel method for the simple selection-free detection and quantification of bone marrow DTCs in mice. We have used this protocol to detect human and murine DTCs in xenograft, syngeneic, and genetically engineered mouse models. We are able to detect and quantify bone marrow DTCs in mice that do not have overt bone metastasis. This protocol is amenable not only for detection and quantification purposes but also to study the expression of markers of numerous biological processes or tissue-specificity.

BACKGROUND A clinical need to better categorize patients with prostate cancer exists. The Wnt/β-catenin signaling pathway plays important roles in human prostate cancer progression. Deletion of the endogenous Wnt antagonist adenomatous polyposis coli (Apc) in mice causes high grade prostate intraepithelial neoplasia, widely thought to be the precursor to prostate cancer. However, no metastasis occurrs in this model. New mouse models are needed to determine molecular causes of tumorigenesis, progression, and metastasis. METHODS To determine whether the overexpression of the prostate oncogene Hepsin could cause prostate cancer progression, we crossed a prostate-specific Hepsin overexpression model to a prostate-specific Apc-deletion model and classified the observed phenotype. RESULTS When Apc was deleted and Hepsin overexpressed concurrently, mice displayed invasive carcinoma, with loss of membrane characteristics and increase of fibrosis. These tumors had both luminal and basaloid characteristics. Though no metastasis was observed, there was evidence of adenomas and lung necrosis, inflammation, and chronic hemorrhage. CONCLUSIONS This work indicates that the Wnt/β-catenin pathway and the Hepsin pathway act in concert to promote prostate cancer progression. Both of these pathways are up-regulated in human prostate cancer and could represent chemotherapeutic targets. Prostate 75:1579–1585, 2015. © 2015 Wiley Periodicals, Inc.

The mouse is an important, though imperfect, organism with which to model human disease and to discover and test novel drugs in a preclinical setting. Many experimental strategies have been used to discover new biological and molecular targets in the mouse, with the hopes of translating these discoveries into novel drugs to treat prostate cancer in humans. Modeling prostate cancer in the mouse, however, has been challenging, and often drugs that work in mice have failed in human trials.The authors discuss the similarities and differences between mice and men; the types of mouse models that exist to model prostate cancer; practical questions one must ask when using a mouse as a model; and potential reasons that drugs do not often translate to humans. They also discuss the current value in using mouse models for drug discovery to treat prostate cancer and what needs are still unmet in field.With proper planning and following practical guidelines by the researcher, the mouse is a powerful experimental tool. The field lacks genetically engineered metastatic models, and xenograft models do not allow for the study of the immune system during the metastatic process. There remain several important limitations to discovering and testing novel drugs in mice for eventual human use, but these can often be overcome. Overall, mouse modeling is an essential part of prostate cancer research and drug discovery. Emerging technologies and better and ever-increasing forms of communication are moving the field in a hopeful direction.

Background Cancer metastasis is the primary cause of cancer-related deaths and remains incurable. Current clinical methods for predicting metastatic recurrence are not sensitive enough to detect individual cancer cells in the body; therefore, current efforts are directed toward liquid biopsy-based assays to capture circulating and disseminated tumor cells (CTCs and DTCs) in the blood and bone marrow, respectively. The most promising strategy is fluorescence-based immunostaining using cancer cell-specific markers. However, despite recent efforts to develop robust processing and staining platforms, results from these platforms have been discordant among groups, particularly for DTC detection. While the choice of cancer cell-specific markers is a large factor in this discordance, we have found that marker-independent factors causing false signal are just as critical to consider. Bone marrow is particularly challenging to analyze by immunostaining because endogenous immune cell properties and bone marrow matrix components typically generate false staining. For immunostaining of whole tumor tissue containing ample cancer cells, this background staining can be overcome. Application of fluorescent-based staining for rare cells, however, is easily jeopardized by immune cells and autofluorescence that lead to false signal. Results We have specifically found two types of background staining in bone marrow samples: autofluorescence of the tissue and non-specific binding of secondary antibodies. We systematically optimized a basic immunofluorescence protocol to eliminate this background using cancer cells spiked into human bone marrow. This enhanced the specificity of automated scanning detection software. Our optimized protocol also outperformed a commercial rare cell detection protocol in detecting candidate DTCs from metastatic patient bone marrow. Conclusions Robust optimization to increase the signal-to-noise ratio of immunofluorescent staining of bone marrow is required in order to achieve the necessary sensitivity and specificity for rare cell detection. Background immunofluorescent staining in bone marrow causes uncertainty and inconsistency among investigators, which can be overcome by systematically addressing each contributing source. Our optimized assay eliminates sources of background signal, and is adaptable to automated staining platforms for high throughput analysis. Electronic supplementary material The online version of this article (10.1186/s12575-018-0078-5) contains supplementary material, which is available to authorized users.

Background Wnt/β-catenin signaling is important for prostate development and cancer in humans. Activation of this pathway in differentiated luminal cells of mice induces high-grade prostate intraepithelial neoplasia (HGPIN). Though the cell of origin of prostate cancer has yet to be conclusively identified, a castration-resistant Nkx3.1-expressing cell (CARN) may act as a cell of origin for prostate cancer.

Introduction: Advances in precision medicine for solid tumors have led to an increase in the number of molecular biomarkers and signatures associated with clinical decisions and clinical trial eligibility. In advanced disease settings, timely clinical decision making informed by robust and accurate molecular biomarker testing is increasingly important. As such, there is a need for a comprehensive, decentralized and rigorously validated diagnostic test. To address this need, we developed and analytically validated PGDx elioTM tissue complete (ETC) assay, which reports somatic single nucleotide variants (SNVs), insertions and deletions (indels), amplifications, and rearrangements, as well as microsatellite stability (MSI) and tumor mutational burden (TMB). Methods: >500 FFPE tumor tissue specimens, across 35 tumor types and 9 organ systems, were analyzed using our 500+ gene targeted panel, ETC (assay in development). Accuracy of the results was compared to orthogonal methods, such as whole exome sequencing (WES), IHC, FISH, NGS, and other on-market IVD assays. Results for all variant types were analyzed for overall percent agreement (OPA). Additional analytical studies were performed to assess precision/repeatability for detection of somatic variants, evaluated across multiple operators, instruments and days. Results: 112 unique samples were evaluated for SNVs and indels by ETC and accuracy assessed via orthogonal validated NGS assays, demonstrating an OPA >99.9%. Detection of amplification events was assessed in 176 samples and concordance with orthogonal FISH and IHC assays demonstrated >93.0 % OPA. Rearrangement accuracy was evaluated across several clinically relevant translocations, with an >94.0 % OPA when compared to FISH and an RNA-based sequencing method (n=270). 115 pan cancer samples were analyzed for microsatellite status and accuracy with PCR was determined, with an overall agreement of 100.0%. Lastly, TMB was determined in 118 samples and results compared to WES-derived TMB, displaying a high level of concordance (Pearson correlation, p=0.903) across a range of TMB scores (0.2-89.7 muts/Mbp). Conclusions: The PGDx elio tissue complete assay system, including proprietary, automated bioinformatics, provides accurate and reproducible results for the detection of clinically relevant genetic alterations across tumor types. Further verification and validation studies of this gene panel are ongoing. The PGDx elio tissue complete assay will employ a decentralized, kitted model, increasing clinical accessibility to NGS and allow for delivery of highly accurate and timely results. Citation Format: Kelly M.R. Gerding, Kenneth C Valkenburg, Christina Oliveras, James R. White, Leila Ettehadieh, Gustavo Cerquiera, Christopher Gault, James Hernandez, Eric Kong, Samuel Angiuoli, John Simmons, Isabell Loftin, Abigail McElhinny. Analytical validation of a 500+ gene comprehensive NGS assay to detect genomic alterations across 35 tumor types [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B027. doi:10.1158/1535-7163.TARG-19-B027

Introduction: Genomic analysis using next-generation sequencing (NGS) enables simultaneous detection of targetable alterations and biomarkers with emerging clinical utility in non-small cell lung cancer (NSCLC) patients. Importantly, many of these alterations are mutually exclusive and tumor tissue for molecular testing is often limited. As such, we developed PGDx elioTM tissue complete (ETC) as a comprehensive NGS assay capable of detecting somatic single nucleotide variants (SNVs), insertions and deletions (indels), amplifications, and rearrangements, as well as microsatellite stability (MSI) and tumor mutational burden (TMB). Here, we present the performance of this assay in detecting key clinical variants in NSCLC. Methods: Studies comprising >300 NSCLC specimens (FFPE tissue and characterized cell lines) were analyzed using ETC, a 500+ gene assay (under development), across key clinically relevant variants in NSCLC, including tumor mutation burden (TMB). Accuracy of the results were compared to orthogonal methods (e.g. whole-exome sequencing (WES), IHC, FISH) and analyzed for the overall percent agreement (OPA). Additionally, archival FFPE samples from 46 NSCLC patients previously found to harbor ALK translocations, MET amplifications, MET exon 14 skipping mutations, EGFR mutations, and/or ROS1 translocations were also analyzed. The results were compared to orthogonal methods and the overall genomic landscape evaluated. Results: Clinical FFPE and characterized cell line specimens were evaluated for the following alterations: EGFR mutations (L858R, T790M, and Exon 19 deletions), BRAF V600E mutations, and ALK and ROS1 translocations. Compared to orthogonal methods, the NGS assay demonstrated >93% OPA across all variants. Comparison of TMB results to WES data demonstrated high accuracy and precision, across a range of DNA inputs (50-200 ng) and tumor purities (10-30%). In the 46 retrospective NSCLC cases, the NGS assay identified 15 ALK translocations, 6 MET amplifications, 1 MET exon 14 skipping mutation, and 5 EGFR mutations, with most being mutually exclusive. The majority of cases were confirmed by orthogonal assays, with the few apparent discordances likely due to tumor heterogeneity, assay distinctions, or analyte input. Higher TMB was found in cases without targetable alterations. Conclusions: ETC provides accurate and reproducible results for the detection of clinically relevant alterations in NSCLC. Further verification and validation studies of this gene panel are ongoing. Overall NGS showed excellent concordance with orthogonal variant detection methods. Importantly, ETC demonstrated added value in assessing all genomic alteration types in a single assay, as well as reporting composite genomic scores, suggesting that NGS may offer a comprehensive solution to molecular testing. Citation Format: Elizabeth Weingartner, Kelly M.R. Gerding, Gustavo Cerquiera, Christopher Gault, James Hernandez, Kenneth Valkenburg, Laurel Keefer, Eileen Sagini, Dorhyun Johng, Caitlin Gilley, Colby Ganey, Leila Ettehadieh, Diandra Denier, Christina Oliveras, Kartikeya Joshi, Eric Kong, Eniko Papp, Amy Greer, James R. White, Donna Nichol, John Simmons. Comparison of a comprehensive NGS profiling assay and conventional molecular testing approaches for detection of clinically relevant alterations in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B029. doi:10.1158/1535-7163.TARG-19-B029

Background Comprehensive genomic profiling in NSCLC has become increasingly important for clinical management of advanced stage patients. Alteration status of EGFR, ALK, BRAF, and ROS1 are validated biomarkers linked to approved drugs, with many clinical trials enrolling on the basis of additional biomarkers, including Tumor Mutation Burden (TMB). It is therefore important to develop accurate and sensitive tools for tumor profiling, particularly in NSCLC where biopsy materials are limited. Here, we present results from ongoing analytical performance studies with the PGDx elio tissue complete - IUO assay, verifying our capabilities to accurately identify a range of genomic alteration types, comprising SNVs, indels, translocations, and TMB. Methods >300 specimens in NSCLC, comprising FFPE tissue and characterized cell lines were analyzed on our >500-gene targeted panel. Accuracy of the results were compared to orthogonal methods, such as whole exome sequencing (WES), IHC, FISH, and on-market IVD assays. Results were analyzed for the positive percent agreement (PPA) and negative percent agreement (NPA) for all variants assessed. Analytical studies were performed to assess the limit of blank, limit of detection, and repeatability for detection of these variants. Results Clinical FFPE and characterized cell line specimens (representing EGFR and BRAF SNVs, EGFR exon 19 deletions, and ALK and ROS1 translocations) were evaluated and demonstrated high concordance between PGDx elio tissue complete - IUO and orthogonal methods, with high PPA and NPA for SNV, indel, and translocation alterations analyzed. Repeatability studies demonstrated 100% PPA and 100% NPA for all variants assessed across operators, instruments, and days. The accuracy for panel-wide sequence mutations were assessed by comparing the results of >50 clinical samples (representing >500 SNVs and ~40 indels) run on PGDx elio tissue complete IUO and a validated NGS method, resulting in high PPA and NPA across all alterations analyzed. Reported alterations between PGDx elio tissue complete IUO and the independent NGS method displayed high sensitivity for analyzed SNVs (≥4% MAF) and indels (≥6% MAF). Finally, comparison of TMB results to WES data demonstrated TMB can be accurately and consistently reported from this panel, across a range of DNA inputs (50-200 ng) and tumor purities (10-30%). Conclusions The PGDx elio tissue complete - IUO >500-gene assay system, including our proprietary bioinformatics, provides accurate and reproducible results for the detection of clinically relevant genetic alterations in this NSCLC study. Further verification and validation studies of this gene panel are ongoing. The PGDx elio tissue complete assay will employ a decentralized, kitted model, increasing clinical accessibility to NGS and allow for delivery of highly accurate and timely results. Citation Format: Kelly Gerding, Laurel Keefer, Christine McCord, Amy Greer, Shantanu Shewale, Nicole Barkley, Eileen Sagini, Dorhyun Johng, Kenneth Valkenburg, Caitlin Gilley, Colby Ganey, Alvis Hu, Diandra Denier, Lorenzo Jones, Christina Oliveras, Gregory Joseph, Kartikeya Joshi, James Hernandez, Christopher Gault, Eniko Papp, Peibing Qin, Sonya Parpart-Li, James White, Mark Sausen, Siân Jones. Analytical performance of a comprehensive genomic profiling system to detect actionable genetic alterations in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3534.

Skeletal metastases result in significant morbidity and mortality. This is particularly true of cancers with a strong predilection for the bone, such as breast, prostate, and lung cancers. There is currently no reliable cure for skeletal metastasis, and palliative therapy options are limited. The Wnt signaling pathway has been found to play an integral role in the process of skeletal metastasis and may be an important clinical target. Several experimental models of skeletal metastasis have been used to find new biomarkers and test new treatments. In this review, we discuss pathologic process of bone metastasis, the roles of the Wnt signaling, and the available experimental models and treatments.

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has been used as a surrogate for discoveries in human prostate development and disease. Prostate micro-dissection allows consistent study of lobe-specific prostate anatomy, histology, and cellular characteristics in the absence of contamination of other tissues. Testosterone affects prostate development and disease. Androgen deprivation therapy is a common treatment for prostate cancer patients, but many prostate tumors become castration-resistant. Surgical castration of mouse models allows for the study of castration resistance and other facets of hormonal biology on the prostate. This procedure can be coupled with testosterone reintroduction, or hormonal regeneration of the prostate, a powerful method to study stem cell lineages in the prostate. Together, prostate micro-dissection and surgical castration opens up a multitude of opportunities for robust and consistent research of prostate development and disease. This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse.

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

The ability of Wnt ligands to initiate a signaling cascade that results in cytoplasmic stabilization of, and nuclear localization of, β-catenin underlies their ability to regulate progenitor cell differentiation. In this review, we will summarize the current knowledge of the mechanisms underlying Wnt/β-catenin signaling and how the pathway regulates normal differentiation of stem cells in the intestine, mammary gland, and prostate. We will also discuss how dysregulation of the pathway is associated with putative cancer stem cells and the potential therapeutic implications of regulating Wnt signaling.

Introduction: In prostate cancer (PCa), DTCs are cells that have escaped the primary lesion and enter the metastatic site, most commonly the bone marrow (BM). The pelvis is a common metastatic site for prostate cancer metastases. However, it is unclear when PCa cells leave the prostate and travel to bone in clinically localized disease. Thus, we studied whether PCa DTCs can be detected at the time of radical prostatectomy (RP) by performing a bone marrow aspiration (BMA) from the anterior iliac crest (AIC) at the time of surgery. Methods: The study protocol was approved by the institutional review board at the Johns Hopkins Medical Institutions. Informed consent to perform BMA was obtained from 94 clinically localized PCa patients undergoing a robotic-assisted laparoscopic prostatectomy (RALP). In the supine position, patients underwent general anesthesia and were prepped and draped in a normal sterile fashion. Aspiration was performed by the attending surgeon prior to first incision and obtained percutaneously from the AIC using a BM biopsy needle (11G x 100 mm Hospital Trapsystem, HS Hospital Service S.p.A.). 8-20 mL of BM were aspirated into 1 or 2 10-mL syringes (BD) and transferred into collection tubes through an 18-gauge needle (PrecisionGlide, BD). Samples were distributed among 7 different principal investigators within 24 hours of collection for DTC analyses. One type of analysis included the AdnaTest, which first involved collecting 8 mL of BM from 43/77 (56%) patients. 5 mL of whole cell extract was then assayed with the AdnaTest ProstateCancerSelect kit (Qiagen) to enrich for epithelial-marker, EpCAM, positive cells using immunomagnetic beads. Reverse transcription (SensiScript RT kit, Qiagen) and real-time qPCR quantified the expression of RPL13A (control, ribosomal protein), EPCAM (epithelial), NKX3.1, prostate-specific antigen (PSA), androgen receptor full length (AR), and HOXB13 (prostate-specific). DTC detection was defined as positive prostate-specific marker expression in the BM. Quality control was performed with Sanger sequencing. Results: BM was successfully obtained from 77/94 (82%) patients. The median (range) age was 62 (43-76) years and the median (range) preoperative PSA was 6 (1.3-62.6) ng/mL. 8/77 (10%) patients had RP Gleason Score 6 disease, 37/77 (48%) were Gleason 3+4, 18/77 (23%) were 4+3, and 13/77 (17%) were Gleason 8-10. One patient had an undetermined RP Gleason due to effects from neoadjuvant chemotherapy. 65/77 (85%) had PSA 20. 50/77 (65%) had pT2, 21/77 (27%) had pT3a, and 6/77 (8%) had pT3b diseases. 3/77 (4%) had lymph node metastasis as confirmed in their pathology. The average time to collect BM and transfer into tubes was 2.5 minutes. The median volume collected was 10.5 mL. RALP was performed without BMA-associated complication and there were no reports of BMA-induced postoperative complications including discomfort at the BMA site. Patients were discharged on postoperative day one. For the AdnaTest, 1/43 (2%) BM expressed PSA and 43/43 (100%) expressed EpCAM. Conclusions: We demonstrated an efficient percutaneous approach to BMA from the AIC with 77/94 (82%) successful attempts to obtain BMA and an average collection time of 2.5 minutes. This technique is feasible and safe with no BMA-associated surgical complications or patient discomfort postoperatively. Samples are utilized to study PCa dissemination to bone. Putative PSA positive DTCs can be identified at the time of RP. Citation Format: Emily A. Caruso, Stephanie Glavaris, Heather J. Chalfin, Kenneth C. Valkenburg, Mohamad E. Allaf, Misop Han, Kenneth Pienta. Disease marker-specific disseminated tumor cells in bone are rare at the time of radical prostatectomy [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B083.

While the majority of prostate cancer (PCa) patients are cured by radical prostatectomy and/or radiation, 10-15% of these men eventually develop recurrent disease in the form of lethal metastases. The most common site for PCa metastasis is the bone marrow. The time from primary tumor removal to recurrence can range from 1-2 years to decades, and constitutes a period of minimal residual disease in which cancer cells remain in the body but are clinically undetectable. It is thought that these disseminated tumor cells (DTCs) are able to maintain a state of cellular dormancy in the bone marrow in which they are not proliferating or dying, and may be the seeds for lethal metastases. By understanding the mechanism(s) that regulate dormancy, we may be able to prevent metastasis altogether. Our group has previously found that the secreted bone marrow factor Gas6 and the expression of its receptor Axl are associated with dormant DTCs in xenograft mouse models. Thus, we hypothesize that Gas6 signaling through Axl mediates dormancy. In this study, we sought to functionally support this hypothesis both in vitro and in human PCa tissue. Overexpression of Axl in the C42B PCa cell line decreased proliferation in vitro, and this was independent of Gas6 when Axl was highly overexpressed. Axl overexpression also led to aggregation of C42B cells, suggestive of a possible role in circulating tumor cell (CTC) clustering to survive the circulation and inhabit the bone marrow. Furthermore, immunohistochemical staining of a PCa tissue microarray showed almost no Axl positivity in cancer cells, regardless of Gleason score and other clinical outcomes. Negative staining was also observed in sections from liver, lymph node, and tibial metastases. Consistent with this, Axl expression correlates with decreased incidence of biochemical recurrence and increased overall survival in the GRID and TCGA databases, respectively. These results indicate an antiproliferative role for Axl in PCa, and suggest its temporal expression during the natural history of PCa. Ongoing work includes assessing the expression of Axl on patient CTCs and DTCs, and using xenograft mouse models of metastasis to analyze the effects of Axl overexpression and knockout in PCa cell lines. Citation Format: Haley D. Axelrod, Kenneth C. Valkenburg, Jessica L. Hicks, Angelo M. DeMarzo, Kenneth J. Pienta. Axl may support dormancy of disseminated tumor cells by inducing cell clustering and decreasing proliferation of metastatic prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B026.

Approximately 30% of prostate cancer (PCa) patients develop biochemical recurrence after radical prostatectomy or radiation therapy; 40% of these patients develop metastasis. Importantly, 100% of men who die of PCa have cancer in the bone. Our goal is to determine whether we may identify disseminated tumors cells (DTCs) in the bone marrow (BM) of PCa patients. This will allow us to study and characterize the biology of BM-DTCs, allowing us to develop innovative strategies to target these cells before they develop overt lethal metastatic lesions. However, the identification of BM-DTCs has been hampered by strong background staining of non-tumor BM cells in immunofluorescence (IF) assays. This prevalent false positive staining calls into question the true identity of what had previously been classified as DTCs. The purpose of this study was to rigorously and systematically optimize our multiplex IF staining protocol so as to eliminate the background signal, thereby increasing confidence that positively stained cells are truly BM-DTCs. The IF protocol we employ involves applying BM (spiked with PCa cells as a positive control) onto positively charged agar-coated slides following a red blood cell lysis step. We then apply heat to promote proper seeding of the cells onto the slide, followed by fixation and permeabilization. We then use indirect IF with primary and fluorescently labeled secondary antibodies before staining the nucleus and mounting a coverslip. We primarily stained pan-cytokeratin (CK) to label cancer cells, CD45 to label white blood cells (WBCs), and DAPI to label the nuclei. This base protocol resulted in false positive staining of many WBCs in the fluorescent channel reserved for CK-positivity. In addition, we observed ubiquitous autofluorescent signal throughout the BM, making it difficult to detect true positives. We then went through the protocol step by step to determine the source of the background signal. We assessed fixation time, permeabilization, antigen retrieval, blocking reagents, primary antibody concentration, secondary antibody concentration, nuclear stain, wash reagents, and reagents to eliminate autofluorescence. Each step provided us with information about background signal. After these optimizations, our current novel protocol has eliminated autofluorescence and false positive signal. We now consistently observe high signal-to-noise ratio for CK-positivity in the tumor cells and no CK-positivity in the WBC population. We have also performed this protocol on a small number of PCa patient BM and blood samples and saw no background signal. These data lead us to conclude that we have, for the first time, a multiplex IF protocol that can be used to stain patient BM tissue to identify BM-DTCs. Future studies will determine the true number of BM-DTCs present at different time points during PCa progression, as well as study the biology of these rare cells in relation to development of metastasis and patient prognosis. Citation Format: Haley Axelrod, Kenneth J. Pienta, Kenneth C. Valkenburg. Systematic optimization of multiplex immunofluorescence of bone marrow eliminates false positives in the detection of disseminated tumor cells [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A030.

Prostate tumors are widely considered to be immunologically silent and resistant to immunotherapy. PCa is widely considered to be an immune desert, with little infiltration of cytotoxic T lymphocytes (CTLs). However, anti-tumor immune responses have been seen in PCa in the right context. We believe we can boost immune response and T cell infiltration into PCa tumors by inhibiting the stromal derived factor 1-CXC chemokine receptor 4 (SDF1-CXCR4) signaling axis and releasing tumor cells from the immune-suppressive bone marrow niche. We have shown that CXCR4 antagonism causes PCa disseminated tumor cells (DTCs) to leave the bone marrow niche and enter the circulation as released circulating tumor cells (CTCs). Our hypothesis is that the release of PCa cells from the tumor microenvironment will enhance chemotherapy-induced cell death and the subsequent presentation of tumor-associated antigens, allowing increased anti-tumor response to immunotherapies. I generated a novel selection-free immunofluorescence-based protocol to confirm this in dissemination models of cancer in mice. After CXCR4 inhibition, I observed an approximate five-fold increase in the number of released CTCs in the blood of mice 24 hours after treatment. Very little research has been performed on the proliferation of CTCs or DTCs, particularly when released from the bone marrow niche. I have been able to mark proliferating cells in blood and bone marrow with EdU and quantify proliferating CTCs. We determined that while released cancer cells are proliferating more than DTCs, the ratio of released proliferating cells is not sufficient to cause a robust response to chemotherapy alone. This led to the hypothesis that released cells could provide an excellent substrate for the immune system. Evidence to support this idea is that we consistently observe the engulfment of CTCs by CD45-positive immune cells from the blood of mice, indicating that immune cells may respond to CTCs but not DTCs. Our approach is unique in that we are using CXCR4 inhibition to release cancer cells into the bloodstream to expose them to immune cells and initiate an immune response via activated CTL infiltration into PCa tumors. We believe this represents an innovative polyvalent autologous vaccine approach as it does not rely on a single antigen to stimulate CTLs, and also relies on each individual patient's cancer cells and immune cells. We have also generated some immunofluorescent protocols to determine the immune composition of circulating cells in relation to CTCs. While we believe that CTLs are responsible for most of the anti-tumor immune response, we are using this opportunity to discover novel immune cell types that may be present before and after therapy. This approach, when combined with classic cancer therapy and immunotherapy, may represent a novel way to boost response to cancer therapies in metastatic PCa. Citation Format: Kenneth C. Valkenburg, Kenneth J. Pienta. Release of cancer cells from the prostate tumor microenvironment to promote neoantigen exposure and enhance anti-tumor immune response [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 1023.

Prostate cancer (PCa) remains the second most common cancer among men in the U.S., and kills nearly 30,000 men each year. While the majority of patients with localized disease are cured by surgery and/or radiation, 10-15% of these patients will develop metastasis most commonly in the bone marrow. The time from primary tumor removal to recurrence can range from years to decades, and constitutes a period of minimal residual disease in which cancer cells remain in the body but are clinically undetectable. These disseminated tumor cells (DTCs) are seeds for lethal bone metastases and are thought to maintain a long-term state of dormancy in the bone marrow. Very little is known about the mechanism(s) by which dormancy is regulated, but further investigation may lead to therapeutic strategies aimed at preventing recurrent metastasis altogether. In this effort, we have previously found the secreted bone marrow factor Gas6 and the expression of its receptor Axl to be associated with dormant bone marrow DTCs in xenograft mouse models. We hypothesize that bone marrow Gas6 signals through Axl to induce dormancy in prostate DTCs, and explored the functional consequences of altering Axl expression in these models. To determine if Axl is sufficient to induce dormancy, we overexpressed it in the Axl-negative human PCa C42B cell line and monitored tumor growth by BLI after intracardiac injection in NSG mice. By the time half of the control overexpression injected mice developed tumors, less than 10% of mice injected with Axl overexpression cells developed tumor signal. This delay in tumorigenesis indicates a prolonged period of dormancy that is mediated by Axl expression. To determine if Axl is necessary for dormancy, we knocked out Axl in the Axl-positive human PCa PC3 cells using CRISPR and monitored growth after intracardiac injection in the same manner as the overexpression cells. Knockout of Axl did not increase the time to tumor signal compared to control cells. Loss of Axl also did not decrease the fraction of EdU negative, non-proliferative cells present in the bone marrow at one week post-injection. These data indicate that Axl is likely not necessary for DTC dormancy. Altogether this study suggests that Axl overexpression is sufficient to induce dormancy, but endogenous Axl is not required for dormancy. This is further supported by negative immunohistochemical staining of Axl on cancer cells within growing primary and metastatic tumors from a PCa tissue microarray. Axl also correlated with decreased incidence of biochemical recurrence (Decipher GRID) and increased overall survival (TCGA). Ongoing work includes assessing the expression of Axl on patient CTCs and DTCs, and correlating it with proliferation status. Citation Format: Haley D. Axelrod, Kenneth C. Valkenburg, Jessica L. Hicks, Angelo M. DeMarzo, Kenneth J. Pienta. Axl is sufficient but not necessary for disseminated prostate tumor cell dormancy [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 53.

Approximately 28,000 men die of prostate cancer (PCa) each year in the US, and 90-100% of them are due to bone metastasis. It has been demonstrated that PCa cells detach from the primary tumor, “home” to bone, and develop into these incurable metastases. Existing methods of detecting cells early that can lead to bone metastasis have limited sensitivity and current efforts involve liquid biopsy assays to capture and detect disseminated and circulating tumor cells (DTCs and CTCs respectively). A significant challenge during this detection lies in the optimization of the signal-to-noise ratio during immunofluorescent staining. Noise during immunostaining can originate from endogenous immune cells and other bone marrow matrix components and lead to false staining which reduces sensitivity. We have developed an optimized protocol to reduce background noise from these factors while also enhancing signal using tyramide signal amplification (TSA). Following immuno-staining with PCa cell-specific markers, TSA amplifies beyond primary and secondary antibody binding using horseradish peroxidase (HRP) and fluorophore binding. To further apply this novel signal amplification protocol to the visualization of disseminated tumor cells in bone marrow, we will detect, quantify, and image parental and modified PCa cells injected into murine blood. We have previously shown that C-X-C chemokine receptor 4 causes PCa cells to home to bone. We have stably overexpressed CXCR4 in several PCa cell lines and will be able to determine the role of CXCR4 expression on the development of bone metastases. This robust protocol will be quickly modifiable for automated staining to yield higher throughput analysis. Citation Format: Sounak Roy, Kenneth C. Valkenburg, Kenneth J. Pienta. Detection of disseminated prostate cancer cells in bone marrow using tyramide signal amplification [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 5192.

Approximately 28,000 men die of prostate cancer (PCa) each year in the US, and 90-100% of them will be due to bone metastasis. It has been demonstrated that PCa cells detach from the primary tumor and “home” to bone. Osteoblasts (bone-forming cells) secrete the chemoattractant stromal-derived factor 1 (SDF-1, also known as CXCL12), and PCa cells express the receptors for SDF-1, C-X-C chemokine receptors 4 and 7 (CXCR4 and CXCR7). PCa cells use these receptors to enter the chemoprotective bone microenvironment, specifically, the hematopoietic stem cell (HSC) niche in the endosteum. While small molecule inhibition of CXCR4 can mobilize PCa cells from the bone marrow microenvironment, it is unknown whether CXCR7 plays a compensatory role when CXCR4 is antagonized. SDF-1 is known to bind with greater affinity to CXCR7 compared to CXCR4 indicating a possible role of CXCR7 in tumor migration to the endosteal niche. Therefore, it is crucial to determine the individual and combined roles of CXCR4 and CXCR7 in the formation and treatment of PCa bone metastases. To answer these questions, first we assessed CXCR4 and CXCR7 expression across a panel of prostate cancer cell lines. We then transiently overexpressed and knocked down CXCR4 and CXCR7 in PCa cells and found CXCR4 and CXCR7 expression in vitro to directly correlate to cell viability and migration in PC3 PCa cells. To further test dissemination and metastasis in bone, we are making stable overexpression and knockout cells and will perform in vivo metastasis experiments. We have developed a novel immunofluorescence protocol for detecting and quantifying tumor cells in murine blood and bone marrow and will use this technique to determine the ability of PCa cells to disseminate to bone in the presence or absence of CXCR4 and/or CXCR7. This will be quickly translatable because we are currently running a first-in-prostate cancer clinical trial to determine whether small molecule inhibition of CXCR4 to mobilize PCa tumor cells from the bone marrow, combined with docetaxel, will benefit metastatic PCa patients. If we determine that CXCR7 plays a compensatory role in dissemination and/or metastasis, we will need to add CXCR7 inhibition to our treatment strategy to obtain the most efficient benefit for patients. Citation Format: Sounak Roy, Kenneth C. Valkenburg, Kenneth J. Pienta. CXCR4 and CXCR7 play distinct and overlapping roles in prostate cancer dissemination to bone [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 903. doi:10.1158/1538-7445.AM2017-903

Bone metastasis results in the deaths of approximately 28,000 prostate cancer patients each year in the United States. Disseminated tumor cells (DTCs) go from the primary tumor site to the bone marrow, which provides a chemoprotective environment. C-X-C chemokine receptor 4 (CXCR4), highly overexpressed in prostate cancer, is at least partially responsible for bone homing. CXCR4 is also expressed in hematopoietic stem cells (HSCs), which occupy the same niche within the bone marrow as DTCs. HSCs can be mobilized from the bone marrow niche via CXCR4 antagonism. We have previously shown that human tumor cells can be similarly mobilized from the bone marrow niche via small molecule inhibition of CXCR4. We believe this represents an ideal strategy for treating bone metastasis and hypothesize that mobilized DTCs will proliferate in the vascular niche, causing them to become chemosensitive. Several outstanding questions still exist that we are in the process of answering. First is the percentage of DTCs that are mobilized with CXCR4 antagonism. Previously, we used a qPCR strategy to quantify tumor cells in the bone marrow and blood of mice. This was imperfect because we could not reliably count cells, nor could we query the cells further. Therefore, we developed a novel immunofluorescence protocol to detect, quantify, and query tumor cells residing in the bone marrow or blood of mice. We will use this technique to determine the percentage of bone marrow DTCs that are mobilized. The second outstanding question is whether or not mobilized DTCs proliferate once mobilized. This is crucial to know, considering most chemotherapy relies on tumor cell proliferation. Using our novel protocol, we will determine the percentage of bone marrow DTCs and mobilized DTCs that are proliferating. Finally, while there is evidence that combining CXCR4 antagonism with chemotherapy kills more tumor cells than chemotherapy alone, no one has shown definitively that it works specifically for prostate bone marrow DTCs or bone metastasis. Not only will we do this, but we will also be able to quantify how effective each therapy strategy is by quantifying the tumor cells present in each scenario. The data from these in vivo murine experiments, which would be impossible to get from humans, will go hand in hand with a first-in-prostate cancer clinical trial we are running that tests the same hypothesis. Combined, these data will be critically important in deciding whether mobilization therapy combined with chemotherapy is an effective strategy for treating metastatic prostate cancer. Citation Format: Kenneth C. Valkenburg, Emma E. van der Toom, Kenneth J. Pienta. Small molecule inhibition of CXCR4 mobilizes prostate disseminated tumor cells (DTCs) from the chemoprotective bone marrow niche, thereby sensitizing them to chemotherapy [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 4942. doi:10.1158/1538-7445.AM2017-4942

The majority of prostate cancer (PCa) deaths are attributed to bone metastases, which may not arise until years after the primary tumor has been removed. Disseminated tumor cells (DTCs) have been shown to already be present in the bone marrow of patients at the time of prostatectomy, and it is thought that they undergo a period of dormancy, allowing them to remain undetected for years before manifesting into clinically detectable metastases. It is not known what allows PCa DTCs to undergo and persist in this dormant state, but it is likely that factors in the bone marrow niche play an essential role. Previously our group has shown that one of these niche factors, Gas6, can regulate growth of PCa cells. The growth restraining effects of Gas6 on PCa cells is likely mediated through the tyrosine kinase receptor Axl, whose expression was found to be increased on dormant DTCs compared to proliferating metastases in a xenograft mouse model. Here, we utilized the TRAMP model, a genetically engineered mouse model of spontaneous PCa progression, to compare the frequency of Axl-hi and Axl-low bone marrow DTCs that are proliferating versus non-proliferating. Further, we also compared Axl expression and proliferation in matched primary tumors. To investigate the functional significance of Gas6/Axl signaling in mediating PCa dormancy, we have generated Axl knockout PCa cells to test the requirement of Axl in maintaining dormancy, and Axl overexpressing PCa cells to test the sufficiency of Axl in mediating dormancy. Determining the role of Axl and any other regulatory mechanisms of PCa DTC dormancy will be crucial in efforts to prevent lethal and incurable metastasis. Citation Format: Haley D. Axelrod, Kenneth C. Valkenburg, Brian W. Simons, Kenneth J. Pienta. Axl-hi DTCs in the bone marrow of a genetically engineered mouse model of prostate cancer exhibit decreased proliferation [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 4945. doi:10.1158/1538-7445.AM2017-4945

Wnt/β-catenin signaling is important for prostate development and cancer in humans. Activation of this pathway in differentiated luminal cells of mice induces high-grade prostate intraepithelial neoplasia (HGPIN). Though the cell of origin of prostate cancer has yet to be conclusively identified, a castration-resistant Nkx3.1-expressing cell (CARN) may act as a cell of origin for prostate cancer.To activate Wnt/β-catenin signaling in CARNs, we crossed mice carrying tamoxifen-inducible Nkx3.1-driven Cre to mice containing loxP sites in order to either conditionally knock out adenomatous polyposis coli (Apc) or constitutively activate β-catenin directly. We then castrated and hormonally regenerated these mice to target the CARN population.Loss of Apc in hormonally normal mice induced HGPIN; however, after one or more rounds of castration and hormonal regeneration, Apc-null CARNs disappeared. Alternatively, when β-catenin was constitutively activated under the same conditions, HGPIN was apparent.Activation of Wnt/β-catenin signaling via Apc deletion is sufficient to produce HGPIN in hormonally normal mice. Loss of Apc may destabilize the CARN population under regeneration conditions. When β-catenin is constitutively activated, HGPIN occurs in hormonally regenerated mice. A second genetic hit is likely required to cause progression to carcinoma and metastasis.

New concepts and enduring themes were the topic of conversation at the May 3, 2013, Origins of Cancer meeting. The 1-day symposium, titled “Hallmarks of Cancer,” showcased top scientific and translational discoveries on the origins and causes of cancer. The symposium got off to a strong start with an opening presentation by Peter Vogt (The Scripps Institute). Using the history of retroviruses as a guide, Vogt revisited the key findings that established the undeniable role of tumor virology in oncogenesis. While this concept is now deeply integrated into the field of cancer biology, it was not initially well received. Early findings in an avian system by Peyton Rous were largely dismissed as irrelevant to mammalian biology.1 However, the subsequent discovery of the cellular origins of viral oncogenes having the ability to transform mammalian cells gradually changed opinions and led to the 1989 Nobel Prize for Harold Varmus and J. Michael Bishop.2 This work has since been leveraged to develop effective clinical therapies for melanoma, breast cancer, and chronic myelogenous leukemia by targeting oncogenic proteins.3-5 Picking up the theme of genetic mutations or alterations as a driving force in cancer, Eric Holland (Memorial Sloan-Kettering Cancer Center) gave an elegant presentation showing how cluster analysis of transcriptional data could be leveraged to identify specific genomic aberrations that define molecular glioblastoma subclasses (proneural, mesenchymal, and classical). Interestingly, he showed that the proneural and classical variants of glioblastoma are commonly associated with aberrant growth factor signaling via amplification of receptors for platelet derived growth factor and epidermal growth factor, respectively.6 Armed with the knowledge of how genetic changes drive glioblastoma, Holland hopes to determine both the order of mutational events as well as the vulnerabilities of a given tumor type in order to develop novel therapies for malignant brain cancer. The question of which cells contribute to tumor growth was addressed in talks by Tannishtha Reya (University of California, San Diego) and Owen Witte (University of California, Los Angeles). Reya’s talk showed how she is pushing the field of stem cell and cancer stem cell research to new heights by elucidating the roles of Wnt and Hedgehog during asymmetric division of stem cells, and by revealing how cancer cells hijack these developmental pathways during tumorigenesis to create a heterogeneous population of tumor cells. One of the most fascinating aspects of Dr. Reya’s approach is the way she uses fluorescent molecular markers to visualize the way stem cells asymmetrically divide, orient their spindles, and become daughter cells with completely different transcriptomes and fates. Focusing on her work with the Musashi (Msi)–Numb signaling axis in chronic myelogenous leukemia (CML),7 Reya showed that the Msi–Numb pathway can push CML cells into an undifferentiated state, leading to advanced CML and blast crisis. Inhibiting Msi could lead to enhanced differentiation and impaired leukemic growth. This work has interesting implications for the cancer stem cell model in CML, but also in solid tumors, in which Msi is similarly expressed.8,9 Witte’s presentation centered on his work in prostate cancer and his efforts to decipher the combinatorial effects of multiple genetic alterations on tumor progression by altering distinct genetic pathways, such as p53, phosphatase and tensin homolog (PTEN), or AKT simultaneously with truncated erythroblast transformation-specific-related gene (ERG).10 He also talked about the controversy surrounding the cell-of-origin for prostate cancer.11 While several research groups, Witte’s included, have shown that the basal prostate compartment contains stem cells that act as the cell-of-origin for prostate cancer,12,13 others have shown that luminal cells are the cell-of-origin.14 Witte presented data that challenge the practice of using mouse models to study stemness and tumorigenic properties of murine prostate cells and outlined new approaches he is using to identify the cell-of-origin for prostate cancer. The topic of tumor metabolism was featured in several talks, notably those of Matt Vander Heiden (Massachusetts Institute of Technology) and Victor Velculescu (Johns Hopkins University). As first noted by Otto Warburg in the early 20th century, cancer cells leverage aerobic glycolysis over oxidative phosphorylation regardless of oxygen availability.15 However, the reason tumors chose to use a less efficient means of energy production remains a mystery. One popular theory suggests that aerobic glycolysis is employed to allow more rapid ATP generation and sustain high levels of cell growth, but several key observations suggest otherwise. A marginal difference in ATP is seen between nonproliferating and proliferating cells while an accrual of macromolecules is observed in the proliferative state. Vander Heiden used labeled glucose and gas chromatography–mass spectroscopy to show proliferating tumor cells use glucose alternatives, like glutamine, to synthesize essential macromolecules under specific environmental situations (e.g., hypoxia).16 Under hypoxic situations, cells are also glucose- and glutamine-limited. In this context, Vander Heiden hypothesized proteins may be consumed via macropinocytosis to execute macromolecule synthesis. In Ras-transformed cells, macropinocytosis was shown to be up-regulated in response to growth factor signaling and was required to rescue growth defects seen when glutamine was limited.17 From the perspective of genomic alterations in cancer, Velculescu presented the discovery of alterations in the key metabolic enzyme isocitrate dehydrogenase (IDH1) in gliomas. He noted only 5% of primary glioblastoma harbor mutations in this gene compared with 80% of secondary glioblastoma, indicating a differential metabolism state in advanced cancer. These talks illustrated the importance of continued research to identify novel ways to treat cancers by exploiting differences in altered metabolism of a tumor cell. Velculescu believes that every tumor, possibly in the world, is different at the genetic level. Therefore, he is improving methods of detection and treatment of cancer using the cancer genome to promote a customized approach over a “one size fits all” approach.18, 19 This requires a comprehensive analysis of cancer genomes, which includes identifying novel altered pathways such as IDH. As a cancer alters its metabolism to adapt to its environment, so too does the environment alter its composition to adapt to a tumor. Jeffrey Pollard (University of Edinburgh) provided an excellent discussion on the contribution of the stromal microenvironment during cancer progression and metastasis. Pollard used intravital multi-photon imaging20 to graphically demonstrate that macrophages make up large portions of tumors, secrete growth factors that regulate angiogenesis, stimulate invasion, and define a microenvironment that facilitates intravasation.21-24 His results support the emerging paradigm that a permissive, even contributive, microenvironment is necessary for malignant progression. From a clinical perspective, macrophages, or their unique signaling pathways, represent new therapeutic targets that may be efficacious in reducing cancer mortality. Yibin Kang (Princeton University) expanded the role of the tumor microenvironment in cancer progression by outlining how the intricate interactions between tumor cells and their stromal microenvironment lead to metastasis. Kang described the role of transcription factor Elf5, a protein that regulates luminal cell fate with known ties to cancer progression in breast cancer metastasis.25 Using an in vivo murine mammary model, he showed that loss of Elf5 drives an epithelial to mesenchymal transition through loss of adherens junctions and mislocalized vimentin, as well as an increase in overall migration. Animals heterozygous for Elf5 have increased lung metastases and Snail2 expression compared to wild-type Elf5 controls.26 In contrast, overexpression of Elf5 repressed migration and lung metastasis. Within estrogen receptor-negative breast cancer patient samples, high Elf5 expression reflects a better prognosis. Conversely, high Snail2 levels are associated with a worse prognosis.26 This work illustrates the functional insights that basic cancer research can provide for potential clinical applications. The Origins of Cancer symposium highlighted paradigm-shifting work that is being done to challenge the way researchers think about cancer etiology and treatment. Technological advances and multidisciplinary research efforts are driving the convergence of themes from different areas of study into a cohesive understanding of cancer biology emphasizing tumor metabolism and microenvironment in the progression of disease. Our challenge in the coming years is to match this understanding with equally impressive clinical successes.

Approximately 20-30% of prostate cancer patients develop disease recurrence and metastasis years after initial therapy. This is thought to be largely due to the presence of growth-arrested and chemoresistant disseminated tumor cells (DTCs) in secondary sites, such as bone. Bone metastasis is found in 90-100% of prostate cancer patients who succumb to the disease. There are still many gaps in knowledge about the biological mechanisms by which DTCs home to bone, resist chemotherapy, become dormant, and escape dormancy to grow into clinical metastases. As such, it is important to be able to detect, quantify, and study bone marrow DTCs. In particular, it must be possible to do this in metastatic cancer mouse models, which are critical to study the process of tumor dissemination. DTC detection techniques currently exist, usually as either a positive selection or negative selection methodology. Positive selection techniques use markers or cell size to isolate and purify tumor cells out of the bone marrow. Positive selection markers are generally epithelial-specific, such as EpCam, E-cadherin, or Cytokeratin, and therefore may miss cells that lose epithelial marker expression and may gain mesenchymal markers. DTCs can also be as small as or smaller than white blood cells, meaning that positive selection based on size may miss some DTCs. Negative selection enriches for DTCs by removing blood and bone marrow cells from the population, usually using cell-specific markers. A popular strategy is CD45-based depletion, which removes white blood cells, and theoretically leaves behind DTCs. In our hands, this strategy causes loss of DTCs in the depletion process. To capture these heterogeneous and rare DTCs, we have developed a strategy to detect DTCs in murine bone marrow in an un-biased manner. The procedure entails removal of the bone marrow via centrifugation from the long bones (femur and tibia) of mice that have been injected with cancer cells (the injection site may vary depending on the experimental setup). The bone marrow then undergoes red blood cell lysis, and further centrifugation. The white blood cells are then counted, and the bone marrow is spread onto glass slides. The cells on the slide are fixed, permeabilized, and stained (immunofluorescence and RNA fluorescent in situ hybridization can be used). The staining can include any type of marker, including epithelial, mesenchymal, disease-specific, species-specific, or other biologically interesting markers, such as cell cycle markers. The unbiased nature of this procedure is based on the lack of positive or negative selection based on cell size or protein expression. Some DTC loss is noted in this protocol, due to the centrifugation and staining steps, but the cell population on the slide should include all DTC types. Notably, this protocol can be used to detect human or mouse cells in the mouse bone marrow and can thus be used in immune-compromise and immune-competent mouse models of metastasis. Citation Format: Kenneth C. Valkenburg, James R. Hernandez, Sarah R. Amend, James E. Verdone, Michael A. Gorin, Kenneth J. Pienta. Unbiased detection of disseminated tumor cells in murine bone marrow. [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 4109.

The presence of detectable metastases in the bone marrow of prostate cancer patients represents lethal, incurable disease. These metastases that may emerge years or decades after prostatectomy are derived from small colonies of dormant tumor cells that disseminated from the primary tumor. Our group has previously shown that Gas6/Axl signaling is associated with disseminated tumor cell (DTC) dormancy in mouse models of prostate cancer metastasis. In this study we explored the functional significance of this association and tested the hypothesis that overexpression of Axl in prostate cancer cells would prolong their dormant phase in the bone marrow. Axl overexpression in the AR-positive Axl-negative human prostate cancer cell line C42B slowed proliferation in vitro. Select cells were completely dormant as detected by the lack of EdU incorporation over 40 hours, and this fraction was increased when co-cultured with bone-forming osteoblasts. To assess the functional effects of Axl on prostate cancer progression in vivo, we utilized a mouse model of prostate cancer metastasis in which subcutaneous tumors are established and surgically removed upon reaching 200mm3. At 10 weeks post-tumor removal, mice bearing C42B-Axl tumors did not have overt osteoblastic or osteolytic lesions in their femurs or tibiae, whereas mice bearing C42B-WT tumors had visible lesions by x-ray. Immunohistochemistry staining of mouse femurs and tibiae corroborated these findings. To confirm that the absence of metastases in mice with C42B-Axl tumors was not due to the inability of the cells to disseminate, we used immunofluorescence to detect human cells in the bone marrow of flushed femurs and tibiae. At 12 weeks post-tumor removal, fewer prostate cancer cells were detected in the bone marrow of mice with C42B-Axl tumors compared to those with C42B-WT tumors. Notably however, both groups had detectable prostate cancer cells in the bone marrow, indicating that the absence of C42B-Axl metastasis was not due to failure of primary tumor cells to disseminate. The presence of disseminated C42B-Axl cells in the bone marrow coupled with their failure to develop into clinical metastases at the time of C42B-WT metastatic outgrowth establishes a functional role for the association of Axl expression with DTC dormancy. Axl may be sufficient to induce and maintain dormancy, and ongoing experiments will address the requirement of Axl in this setting. Citation Format: Haley D. Axelrod, Kenneth C. Valkenburg, Kenneth J. Pienta. Overexpression of Axl delays metastatic outgrowth of prostate cancer bone marrow disseminated tumor cells. [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 1572.

The most common site of metastasis in prostate cancer (PCa) patients is the bone marrow, where disseminated tumor cells (DTCs) preferentially home to the hematopoietic stem cell niche and can undergo a state of dormancy. These PCa cells may lie dormant in the bone marrow for years after prostatectomy, but may eventually re-enter the cell cycle resulting in lethal metastatic disease. Using mouse models of PCa metastasis, our group has previously shown that Gas6/Axl signaling is associated with dormant bone marrow DTCs. In this study we investigated the requirement of Axl in initiating and maintaining the dormant phase. We hypothesized that overexpression of Axl in PCa cells would prolong dormancy. Overexpression of Axl in the bone-homing AR-positive Axl-negative human PCa cell line C42B slows proliferation in vitro. Subcutaneous injection of C42B-Axl cells in a xenograft mouse model resulted in delayed onset of tumor growth relative to WT controls. C42B-WT tumors reached an average volume of 200mm3 at 6.6 weeks, while C42B-Axl tumors reached the same volume at 11 weeks. Regions of the primary tumor with a high percentage of Axl+ cells correlated with a low number of Ki67+ cells, indicating that Axl may inhibit proliferation even in the primary tumor. Surprisingly, C42B-Axl tumors were more vascular as observed by eye, and regions of increased vasculature correlated with a high percentage of Axl+ cancer cells. Metastatic outgrowth in soft tissues reached a lethal burden ~12 weeks following C42B-WT tumor removal, while mice bearing C42B-Axl tumors did not have detectable metastases by eye at the same timepoint post-tumor removal and were apparently healthy. Metastatic outgrowth in the bone was detected by the presence of osteoblastic and osteolytic activity at 5-12 weeks post-tumor removal in the femurs and tibiae of mice bearing C42B-WT tumors. At 8-12 weeks after C42B-Axl tumor removal, however, there was no evidence of metastatic bone disease. While these experiments are still ongoing, we have shown that Axl expression in the primary tumor delayed metastatic burden in both soft tissues and in bone in a mouse model of PCa metastasis. In ongoing studies, we will compare DTC number and time to metastasis in the bone marrow of C42B-WT and C42B-Axl tumor-bearing mice. Citation Format: Haley D. Axelrod, Kenneth C. Valkenburg, Kenneth J. Pienta. Axl overexpression in a prostate cancer mouse model delays metastasis and may prolong dormancy of disseminated tumor cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A35.

Prostate cancer bone metastasis is a lethal incurable disease. Prostate cancer cells home to bone, where they are termed disseminated tumor cells (DTCs) and may reside dormant for years and/or grow into overt metastatic lesions. The bone homing process is largely thought to occur via local signaling of stromal-derived growth factor 1 (SDF1), secreted by osteoblasts within the bone marrow, to C-X-C chemokine receptor 4 (CXCR4), expressed on prostate cancer cells. Hematopoietic stem cells (HSCs) are also recruited to bone via SDF1/CXCR4 signaling. Specifically, HSCs reside in the osteoblastic endosteal niche, where they remain mostly quiescent. We have previously shown that prostate tumor cells occupy this HSC niche, and that the niche affords DTCs a chemoprotective status. Similar to HSCs, prostate DTCs can be mobilized from the niche by inhibiting the SDF1/CXCR4 signaling axis via small molecule CXCR4 inhibitors such as AMD3100 (trade name Plerixafor). Combining CXCR4 antagonism with the microtubule inhibitor docetaxel in mice injected subcutaneously with prostate cancer cells causes greater tumor regression than either compound alone. It is thought that mobilization of DTCs from the bone (we refer to mobilized DTCs as mobDTCs) causes them to proliferate in circulation, causing them to become susceptible to chemotherapy. We are using this strategy in a first-in-man clinical trial; we will treat bone metastatic prostate cancer patients with a potent next-generation CXCR4 antagonist coupled with standard docetaxel treatment. SDF1 also binds to the receptor CXCR7, which is overexpressed on prostate cancer cells as well. It is unknown whether CXCR7 will play a compensatory role when CXCR4 is antagonized. Therefore, it is crucial that we determine individual and overlapping roles of CXCR4 and CXCR7 in prostate cancer cells, particularly during bone metastasis. We hypothesized that CXCR7 can compensate for loss of CXCR4 function. We first assessed CXCR4 and CXCR7 expression across a panel of prostate cancer cell lines. We then stably overexpressed CXCR4 and CXCR7 in prostate cancer PC3 cells and determined classic cellular properties necessary for metastasis in vitro, such as proliferation, migration, and invasion. PC3 cells overexpressing either CXCR4 or CXCR7 had higher proliferation rates relative to PC3 parental cells. In addition, they were more migratory and invasive than parental cells. We are currently working to delete CXCR4 and/or CXCR7 from PC3 cells using the CRISPR-Cas9 system. We will perform in vivo xenograft metastasis experiments using prostate cancer cells with CXCR4 and/or CXCR7 overexpressed or knocked out. We hypothesize that loss of both receptors will be required to prevent bone metastasis. Future work will include small molecule inhibition of both CXCR4 and CXCR7 to mobilize prostate DTCs from bone. Citation Format: Kenneth C. Valkenburg, Sounak Roy, Kenneth J. Pienta. Targeting CXCR4 and CXCR7 to mobilize and kill prostate tumor cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A50.

The development and optimization of high-throughput screening methods has identified a multitude of genetic changes associated with human disease. The use of immunodeficient and genetically engineered mouse models that mimic the human disease has been crucial in validating the importance of these genetic pathways in prostate cancer. These models provide a platform for finding novel therapies to treat human patients afflicted with prostate cancer as well as those who have debilitating bone metastases. In this paper, we focus on the historical development and phenotypic descriptions of mouse models used to study prostate cancer. We also comment on how closely each model recapitulates human prostate cancer.

The Biomass Program develops design cases to understand the current state of conversion technologies and to determine where improvements need to take place in the future. This design case establishes cost targets for converting MSW to ethanol and other mixed alcohols via gasification.