Prostate Cancer Circulating Tumor Cells Research Articles

Intracellular MicroRNA Imaging and Specific Discrimination of Prostate Cancer Circulating Tumor Cells Using Multifunctional Gold Nanoprobe-Based Thermophoretic Assay.

Circulating tumor cells (CTCs) have emerged as powerful biomarkers for diagnosis of prostate cancer. However, the effective identification and concurrently accurate imaging of CTCs for early screening of prostate cancer have been rarely explored. Herein, we reported a multifunctional gold nanoprobe-based thermophoretic assay for simultaneous specific distinguishing of prostate cancer CTCs and sensitive imaging of intracellular microRNA (miR-21), achieving the rapid and precise detection of prostate cancer. The multifunctional gold nanoprobe (GNP-DNA/Ab) was modified by two types of prostate-specific antibodies, anti-PSMA and anti-EpCAM, which could effectively recognize the targeting CTCs, and meanwhile linked double-stranded DNA for further visually imaging intracellular miR-21. Upon the specific internalization of GNP-DNA/Ab by PC-3 cells, target aberrant miR-21 could displace the signal strand to recover the fluorescence signal for sensitive detection at the single-cell level, achieving single PC-3 cell imaging benefiting from the thermophoresis-mediated signal amplification procedure. Taking advantage of the sensitive miR-21 imaging performance, GNP-DNA/Ab could be employed to discriminate the PC-3 and Jurkat cells because of the different expression levels of miR-21. Notably, PC-3 cells were efficiently recognized from white blood cells, exhibiting promising potential for the early diagnosis of prostate cancer. Furthermore, GNP-DNA/Ab possessed good biocompatibility and stability. Therefore, this work provides a great tool for aberrant miRNA-related detection and specific discrimination of CTCs, achieving the early and accurate diagnosis of prostate cancer.

Abstract PR013: Unlocking the proteome of metastatic prostate cancer circulating tumor cells

Abstract In recent years, liquid biopsies have emerged to provide improved insights on the current disease state of a patient, but they generally rely on a small number of protein markers from immunofluorescent staining or cell-free DNA, which provides limited information especially around drug targets. As more targeted therapies and immunotherapies enter the market, selecting the optimal drug target could improve patient outcomes drastically. One way to achieve this goal is to generate multi-omic datasets from the cancer cells in circulation, known as circulating tumor cells (CTCs). In this study, we collaborated with Astrin Biosciences and used their patented isolation technology to enrich CTCs with high purity from the blood of 15 metastatic castration resistant prostate cancer (mCRPC) patients. The enriched CTCs and a patient matched control sample consisting primarily of white blood cells were divided such that RNA and protein could be interrogated from the same patient. Label free unbiased mass spectrometry-based proteomics was used to identify the CTC-enriched proteins and marks the first time a global proteomic assessment has been performed on CTCs from cancer patients. Initial analysis identified over 1,500 peptides corresponding to greater than 500 proteins from each patients’ CTCs; including increased epithelial cell markers in the CTC-enriched sample and increased WBC markers in the control sample. Hallmarks of androgen response and detection of the androgen receptor were also identified in the CTC-enriched proteome. Overall, this study was designed to show the feasibility of obtaining large scale proteomic data from a liquid biopsy and future studies will provide more insight into the clinical utility of such data with a focus on cancer signaling mechanisms and biomarkers. Importantly, as we analyze more patients, we plan to compare the proteomic data with future RNA signatures across mCRPC patients to eventually be able to correlate them with drug response with the overall goal to improve treatment selection and patient outcomes. Citation Format: Justin M. Drake, Zoi Sychev, Alec Horrmann, Kaylee Judith Smith, Catalina Galeano-Garces, Ali Arafa, Nicholas Heller, Mahdi Ahmadi, Jiarong Hong, Megan Ludwig, Justin Hwang, Emmanuel S. Antonarakis, Jayant Parthasarathy. Unlocking the proteome of metastatic prostate cancer circulating tumor cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR013.

Development and initial clinical testing of a multiplex assay evaluating histone modifier p300 acetylation in prostate cancer circulating tumor cells.

e17020 Background: Histone modifying enzymes (HMEs) hold promise as targets for drug discovery in cancer. Histone acetyltransferases and histone deacetylases dictate the presence of acetyl marks, which regulate chromatin and transcriptional activation. We previously developed a novel ‘histone array on a chip’ to measure HME dysregulation associated with the development of castration resistant prostate cancer (CRPC) (ACS Chem Biol 2017). This approach identified decreased SIRT2 expression and increased p300 activity leads to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in CRPC xenografts. These alterations were subsequently validated in castration-resistant tissues (BMC Cancer 2017). The goal of the current study was to determine if circulating tumor cells (CTCs) could be used as a therapeutic biomarker to identify patients with altered p300 acetylation activity. Methods: Blood was collected from 11 CRPC and 3 hormone-sensitive patients with advanced PC under an IRB-approved protocol. PBMCs were isolated with Ficoll-paque PLUS, and lymphocytes were depleted using anti-CD45 magnetic beads. CTCs were then captured with EpCAM labeled coated magnetic beads. Bound cells were isolated with Exclusion-based Sample Preparation (ESP) technology. Bound cells were moved through staining and washing wells prior to imaging at 20x on a fluorescent microscope. Images were analyzed with NIS Elements software. Results: We initially optimized the staining of acetyl-p300, total p300, H3K18ac, and SIRT2 in single cell prostate cancer cell cultures. Exposure to A-485, a selective p300/CBP catalytic inhibitor, reduced levels of p300 and H3K18 acetylation confirming assay specificity. CTCs from patient samples were then captured by EpCAM antibody binding and target staining revealed clear expression of acetyl-p300, acetyl-H3K18, SIRT2, and CK (cytokeratin). No expression of CD45+ cells (lymphocyte exclusion) is noted. Acetyl-p300 displayed a strong positive correlation with its target acetylated H3k18 (Pearson’s r = 0.61), and SIRT2 expression showed robust negative correlation with H3k18 (r = -0.60). A distinct group of 6/11 (55%) of CRPC patients demonstrated altered expression. In one subject with an untreated CSPC sample and subsequent CRPC sample ( > 12mo) a loss of SIRT2 expression, increased p300 activity, and histone H3 hyperacetylation are associated with the transition from hormone-sensitive to CRPC. Conclusions: Acetyl-p300, H3K18ac, and SIRT2 increase in a subset of CTC from patients with CRPC. These biomarkers are associated with increased CBP/p300 activity. This biomarker suite could potentially be used for noninvasive monitoring of outcomes and predicting tumor responses to CBP/p300 acetylation inhibitors in clinical development.

Distinct separation of multiple CTCs using inertial focusing phenomena utilizing single-looped spiral microfluidic lab-on-chip

Looking towards the point-of-care diagnosis purposes, nowadays the most emerging method for separation of Circulating Tumor Cells (CTCs) from normal blood-cells is the inertial focusing phenomena utilizing microfluidic lab-on-chips. Keeping eye on the distinctly separating multiple CTCs from normal blood-cells, here we designed a single-looped spiral microfluidic lab-on-chip based on inertial focusing phenomena, which has been more efficient than double-looped and three-looped spirals. The performance of the device has been investigated using large MCF-7 (breast-cancer CTCs), PC3-9 (prostate-cancer CTCs) and normal blood-cells (white blood-cell, red blood-cell) and also larger multiple CTCs like largest MCF-7, PC3-9, A549 (lung-cancer CTCs) cells and normal blood-cells in three-dimensional numerical simulation utilizing COMSOL Multiphysics 5.4 software. In both cases the chip has accomplished efficient separation performance of nearly 100 % at the Reynolds number ranges from 55.9 - 65 (throughput 70 - 82 ml/h) and 114.74 (throughput of 147 ml/h) respectively in terms of cell separation purity and recovery.

Abstract 2309: Unlocking the proteome of metastatic prostate cancer circulating tumor cells

Abstract In recent years, liquid biopsies have emerged to provide improved insights on the current disease state of a patient, but they generally rely on a small number of protein markers from immunofluorescent staining or cell-free DNA, which provides limited information especially around drug targets. As more targeted and immune therapies enter the market, selecting the optimal drug target could improve patient outcomes drastically. One way to achieve this goal is to generate multi-omic datasets from the cancer cells in circulation, known as circulating tumor cells (CTCs). In this study, Astrin Biosciences’ patented isolation technology was used to enrich CTCs with high purity from the blood of metastatic castration resistant prostate cancer (mCRPC) patients. The enriched CTCs and a patient matched control sample consisting primarily of white blood cells were divided such that RNA and protein could be interrogated from the same patient. Label free unbiased mass spectrometry was used to identify proteins and marks the first time a global proteomic assessment has been performed in CTCs from cancer patients. Initial analysis identified over 1,500 peptides corresponding to greater than 500 proteins with increased epithelial cell markers in the CTC-enriched sample and increased WBC markers in the control sample. Hallmarks of androgen response and detection of the androgen receptor were also identified in the CTC proteome. In addition to the proteome, RNA sequencing will also be performed to generate a multi-omic platform to detect cancer signaling mechanisms and biomarkers. Overall, this study was designed to show the feasibility of obtaining multi-omic data from a liquid biopsy and future studies will provide more insight into the clinical utility of such data. As we analyze more patients, we plan to compare the protein and RNA signatures across mCRPC patients and eventually be able to map patient outcomes and correlate them with drug response with the overall goal to improve treatment selection and patient outcomes. Citation Format: Justin M. Drake, Zoi Sychev, Alec Horrmann, Kaylee J. Smith, Catalina Galeano-Garces, Ali Arafa, Nicholas Heller, Mahdi Ahmadi, Jiarong Hong, Megan Ludwig, Justin Hwang, Emmanuel S. Antonarakis, Jayant Parthasarathy. Unlocking the proteome of metastatic prostate cancer circulating tumor cells [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 2309.

What Do We Have to Know about PD-L1 Expression in Prostate Cancer? A Systematic Literature Review. Part 7: PD-L1 Expression in Liquid Biopsy.

Liquid biopsy is an accessible, non-invasive diagnostic tool for advanced prostate cancer (PC) patients, potentially representing a real-time monitoring test for tumor evolution and response to treatment through the analysis of circulating tumor cells (CTCs) and exosomes. We performed a systematic literature review (PRISMA guidelines) to describe the current knowledge about PD-L1 expression in liquid biopsies of PC patients: 101/159 (64%) cases revealed a variable number of PD-L1+ CTCs. Outcome correlations should be investigated in larger series. Nuclear PD-L1 expression by CTCs was occasionally associated with worse prognosis. Treatment (abiraterone, enzalutamide, radiotherapy, checkpoint-inhibitors) influenced PD-L1+ CTC levels. Discordance in PD-L1 status was detected between primary vs. metastatic PC tissue biopsies and CTCs vs. corresponding tumor tissues. PD-L1 is also released by PC cells through soluble exosomes, which could inhibit the T cell function, causing immune evasion. PD-L1+ PC-CTC monitoring and genomic profiling may better characterize the ongoing aggressive PC forms compared to PD-L1 evaluation on primary tumor biopsies/prostatectomy specimens (sometimes sampled a long time before recurrence/progression). Myeloid-derived suppressor cells and dendritic cells (DCs), which may have immune-suppressive effects in tumor microenvironment, have been found in PC patients circulation, sometimes expressing PD-L1. Occasionally, their levels correlated to clinical outcome. Enzalutamide-progressing castration-resistant PC patients revealed increased PD-1+ T cells and circulating PD-L1/2+ DCs.

A morphological subset of circulating tumor cells in advanced prostate cancer reveals a potential biomarker for clinical outcomes.

e17008 Background: A morphological subset of prostate cancer (PCa) circulating tumor cells (CTCs) with particularly small nuclei (< 8.5 μm), named very-small-nuclear CTCs (vsnCTCs), were found to be correlated with the presence of visceral metastases. It is reported that the depletion of nuclear envelope protein emerin promotes PCa metastasis and is associated with nuclear shape instability. In this study, we hypothesize vsnCTCs as prognostic biomarkers in metastatic castration resistant PCa (mCRPC), and aim to investigate the correlation between emerin level and vsnCTCs. Methods: PCa CTCs were enriched using the NanoVelcro CTC Assay from 76 patients with mCRPC. The Kaplan-Meier analysis and log-rank test were used to estimate and compare the overall survival (OS) and progression free survival (PFS) of androgen receptor signaling inhibitor (ARSI), taxanes and other therapy in patients stratified by the presence of vsnCTCs. The correlation between the presence of vsnCTCs and OS and PFS were evaluated using the Cox proportional hazard regression. The expression level of emerin in patients with and without vsnCTC were compared using Mann-Whitney U test, and the correlation between emerin level and CTC nuclear size was tested by Pearson correlation coefficient. Results: Patients with vsnCTC (i.e, vsnCTC+) had significantly shortened OS and PFS compared with those without vsnCTC (i.e, vsnCTC-). The median OS was 34 (vsnCTC+, n= 49) vs. 149 (vsnCTC-, n= 27) weeks (hazard ratio [HR] = 2.6, 95% confidence interval [CI]: 1.5-4.5, P< 0.001). The median PFS was 12 (vsnCTC+, n= 32) vs. 26 (vsnCTC-, n= 18) weeks (HR = 2.2, 95% CI: 1.3 -4.0, P= 0.004). The emerin expression level was significantly higher in vsnCTC+ compared to vsnCTC- ( P= 0.009). In addition, we observed a significantly positive correlation between emerin expression and CTC nuclear size (r = 0.52, P< 0.001). Conclusions: This study casts light on the importance of the vsnCTCs in patients with mCRPC, as vsnCTC+ patients represented a group at risk for faster clinical progression who are at the highest risk for mortality. vsnCTC represents a new hallmark of an aggressive subtype of mCRPC and is related to emerin dysregulation, which promotes lethal progression and metastasis of PCa.

Abstract 4331: Nuclear size of circulating tumor cells is associated with prognosis in metastatic, castration-resistant prostate cancer

Abstract Background: Current risk stratification models in prostate cancer (PC) have been based on clinical and pathological variables. Beyond serum prostate-specific antigen (PSA) concentration measurements, there remain few new biomarkers to help identify patients at risk for poor clinical outcomes. Morphological analyses using Gleason scoring along with cell nuclear size and shape remains to be a fundamental pathological practice of PC that have been utilized to identify aggressive diseases and to associate with aggressive metastasis. In particular, changes in nuclear shape and composition have been associated with outcome in early stage disease. Circulating tumor cells (CTCs) have arisen as contemporary noninvasive prognostic biomarkers for PC. Previously, a subgroup of PC CTCs, with prominently small nuclei (< 8.5 μm), were found to be correlated with the presence of visceral metastases. This subgroup was named very-small-nuclear CTCs (vsnCTCs). We proposed vsnCTCs as a putative biomarker of a lethal subtype in metastatic castration resistant PC (mCRPC). Methods: In this study, 76 patients with mCRPC were recruited for overall survival (OS) analysis. Of the 76 patients, 50 had available pre-treatment blood specimens prior to the initiation of androgen receptor signaling inhibitor (ARSI, e.g. abiraterone and enzalutamide) or taxane or tyrosine kinase inhibitor therapy. Using the NanoVelcro CTC Enumeration Assay, CTCs were captured and subjected to immunofluorescence staining. CTCs were identified as DAPI+/CK+/CD45- with a round or oval nucleus. Additionally, CTC nuclear size was measured and defined as the square root of the product of the long axis and the short axis. Kaplan-Meier analysis and Cox proportional hazards model were conducted. Results: Patients with vsnCTC (i.e., vsnCTC+) had a significantly shortened OS compared with patients without vsnCTC (i.e., vsnCTC-). The median OS was 34 (vsnCTC+, n=49) vs. 149 (vsnCTC-, n=27) weeks (log-rank HR=2.6 with 95% CI 1.5 to 4.5, p=0.0006). Progression free survival (PFS) analysis was performed for the 50 patients with pre-treatment blood samples. The median PFS was 12 (vsnCTC+, n=32) vs. 26 (vsnCTC-, n=18) weeks (log-rank HR=2.2 with 95% CI 1.3 to 4.0, p=0.0038). We also found that the hazard ratio of overall survival increased significantly as the CTC nuclear size decreased using the p-spline plot. Conclusions: Our study showed that nuclear size reduction has importance in CTCs in a fashion similar to its utility in tissue. This study points toward the importance of the vsnCTC in patients with mCRPC, as vsnCTC+ patients represented a group at risk for faster clinical progression who are at the highest risk for morality. We posit that the vsnCTC represents a new hallmark of an aggressive subtype of mCRPC. This has potential importance in optimizing therapeutic choices. Citation Format: JASMINE J. WANG, Pai-Chi Teng, Yu Jen Jan, Jie-Fu Chen, Galen Cook-Wiens, Nu Yao, Gina C. Chu, Pin-Jung Chen, Yingying Yang, Yee Hui Yeo, Yi-Te Lee, Leland W. Chung, Sungyong You, Yazhen Zhu, Michael R. Freeman, Andre Rogatko, Ju Dong Yang, Hsian-Rong Tseng, Edwin M. Posadas. Nuclear size of circulating tumor cells is associated with prognosis in metastatic, castration-resistant prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4331.

Abstract 3535: Whole genome sequencing of single circulating tumor cells from localized high-risk prostate cancer patients: Laser microdissection and 3Dtelomere profile

Abstract Advances in the development of precision medicine for prostate cancer patients rely on accurate identification of the genomic features underlying a patient's tumor. Genomic analysis of circulating tumor cells (CTCs) could provide a unique and accessible representation of tumor diversity, however for the use of CTCs as a less-invasive sampling of prostate cancer and as biomarkers for patient stratification and selection of targeted therapy, complex platforms are necessary to ensure an efficient enrichment (isolation), detection (identification-imaging) and characterization strategies (molecular profiles). In this study, we combined a filtration-based CTC isolation technology with prostate cancer cell-specific antibodies to identify the prostate cancer CTCs. After identification, we performed the 3D telomere profiling prior to laser microdissection for whole genome sequencing of singles CTCs from 11 localized high-risk prostate cancer patient samples (36 CTCs in total). We identified unique and common somatic single nucleotide variants (SSNVs) and copy number alterations (CNAs) that can be used to predict high-risk lethal prostate cancer and treatment response in patients with clinically localized high- risk prostate cancers. The distribution of telomeres intensities show a range between 0 to 190.000 (a.u) and telomeres parameters were similar as described previously for high risk patients by our group. We analyzed 36 CTCs from 11 different patient samples and compared with normal lymphocytes from the same patient using whole genome sequencing (WGS). We identified 184 common mutated genes in all CTCs and two mutated genes with the same base pair (bp) mutation (PMS2 and EIF2AK4). We also identified 503 consensus mutations found in all patient but not in all CTCs and 1728 unique mutations found in all or any CTCs of only one patient, showing the heterogeneity that arises in single cells. The list of genes affected are all involved in potential pathways for tumor development such as immune system, signal transduction, homeostasis, gene expression, chromatin organization, DNA repair, cell cycle, programmed cell death, cell-cell communication and others. MCPH1, also known as BRIT1, is a key regulatory gene of the DNA damage response pathway and was mutated in all CTCs from prostate patients. MCPH1 deficiency promotes genomic instability and increases cancer susceptibility. The PMS2 gene is a member of a set of genes known as the mismatch repair (MMR) genes which promotes also genomic Instability. Those mutations can be closely linked with the genomic instability we found assessing the telomere profiles. The CDH17 gene is a member of the cadherin superfamily and in contrast to classic cadherins, such as E- and N-cadherin, LI-cadherin possess biological functions distinct from classic cadherins and reduced expression of LI-cadherin is closely associated with tumor progression and lymph node metastasis of human colorectal carcinoma. These mutations were also found in all CTCs. All this information is needed for individualized therapy and predict drug resistance, phenotypic transition which is heterogeneous from cell to cell from patient to patient. Citation Format: Aline Rangel Pozzo. Whole genome sequencing of single circulating tumor cells from localized high-risk prostate cancer patients: Laser microdissection and 3Dtelomere profile [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3535.

Genomic Analysis of Localized High-Risk Prostate Cancer Circulating Tumor Cells at the Single-Cell Level.

Accurate risk classification of men with localized high-risk prostate cancer directly affects treatment management decisions and patient outcomes. A wide range of risk assessments and classifications are available. However, each one has significant limitations to distinguish between indolent and aggressive prostate cancers. Circulating tumor cells (CTCs) may provide an alternate additional source, beyond tissue biopsies, to enable individual patient-specific clinical assessment, simply because CTCs can reveal both tumor-derived and germline-specific genetic information more precisely than that gained from a single diagnostic biopsy. In this study, we combined a filtration-based CTC isolation technology with prostate cancer CTC immunophenotyping to identify prostate cancer CTCs. Next, we performed 3-D telomere profiling prior to laser microdissection and single-cell whole-exome sequencing (WES) of 21 CTCs and 4 lymphocytes derived from 10 localized high-risk prostate cancer patient samples. Localized high-risk prostate cancer patient CTCs present a high number of telomere signals with lower signal intensities (short telomeres). To capture the genetic diversity/heterogeneity of high-risk prostate cancer CTCs, we carried out whole-exome sequencing. We identified 202,241 single nucleotide variants (SNVs) and 137,407 insertion-deletions (indels), where less than 10% of these genetic variations were within coding regions. The genetic variation (SNVs + indels) and copy number alteration (CNAs) profiles were highly heterogeneous and intra-patient CTC variation was observed. The pathway enrichment analysis showed the presence of genetic variation in nine telomere maintenance pathways (patients 3, 5, 6, and 7), including an important gene for telomere maintenance called telomeric repeat-binding factor 2 (TRF2). Using the PharmGKB database, we identified nine genetic variations associated with response to docetaxel. A total of 48 SNVs can affect drug response for 24 known cancer drugs. Gene Set Enrichment Analysis (GSEA) (patients 1, 3, 6, and 8) identified the presence of CNAs in 11 different pathways, including the DNA damage repair (DDR) pathway. In conclusion, single-cell approaches (WES and 3-D telomere profiling) showed to be useful in unmasking CTC heterogeneity. DDR pathway mutations have been well-established as a target pathway for cancer therapy. However, the frequent CNA amplifications found in localized high-risk patients may play critical roles in the therapeutic resistance in prostate cancer.

Ribonucleotide reductase small subunit M2 is a master driver of aggressive prostate cancer.

Although there are molecularly distinct subtypes of prostate cancer, no molecular classification system is used clinically. The ribonucleotide reductase small subunit M2 (RRM2) gene plays an oncogenic role in many cancers. Our previous study elucidated comprehensive molecular mechanisms of RRM2 in prostate cancer (PC). Given the potent functions of RRM2, we set out to determine whether the RRM2 signature can be used to identify aggressive subtypes of PC. We applied gene ontology and pathway analysis in RNA‐seq datasets from PC cells overexpressing RRM2. We refined the RRM2 signature by integrating it with two molecular classification systems (PCS and PAM50 subtypes) that define aggressive PC subtypes (PCS1 and luminal B) and correlated signatures with clinical outcomes in six published cohorts comprising 4000 cases of PC. Increased expression of genes in the RRM2 signature was significantly correlated with recurrence, high Gleason score, and lethality of PC. Patients with high RRM2 levels showed higher PCS1 score, suggesting the aggressive PC feature. Consistently, RRM2‐regulated genes were highly enriched in the PCS1 signature from multiple PC cohorts. A simplified RRM2 signature (12 genes) was identified by intersecting the RRM2 signature, PCS1 signature, and the PAM50 classifier. Intriguingly, inhibition of RRM2 specifically targets PCS1 and luminal B genes. Furthermore, 11 genes in the RRM2 signature were correlated with enzalutamide resistance by using a single‐cell RNA‐seq dataset from PC circulating tumor cells. Finally, high expression of RRM2 was associated with an immunosuppressive tumor‐immune microenvironment in both primary prostate cancer and metastatic prostate cancer using CIBERSORT analysis and LM22, a validated leukocyte gene signature matrix. These data demonstrate that RRM2 is a driver of aggressive prostate cancer subtypes and contributes to immune escape, suggesting that RRM2 inhibition may be of clinical benefit for patients with PC.

MP09-15 PROGRAMMED DEATH-LIGAND 1 POSITIVE AND NON-EPITHELIAL SUBTYPE IN CIRCULATING TUMOR CELLS PREDICT LOCALLY ADVANCED PROSTATE CANCER PROGRESSION AND GUIDE THE IMMUNOTHERAPY AFTER SURGERY

INTRODUCTION AND OBJECTIVE: Circulating tumor cells (CTCs) is considered as “liquid biopsy” for tumor prognosis, and the detection of programmed death-ligand 1 (PD-L1) expressing in CTCs could promote the predictive efficiency. However, there are not standard of cut-offs to discriminate PD-L1 positive/negative expression in CTCs in prostate cancer (PCa). This study aimed to evaluate the rates of PD-L1 positive and non-epithelial (NE) subset in CTCs isolated from patients with high-risk (HRPC) and locally advanced PCa (LAPC) before surgery, and explored the clinical utility of these markers. METHODS: Four PCa cell lines were applied in spiking experiments to establish the cut-offs of NE (hybrid and mesenchymal) and PD-L1+ CTCs. From June 2016 to December 2018, 166 and 79 patients with HRPC and LAPC were collected. All patients underwent radical prostatectomy. Preoperative CTCs were enumerated by Canpatrol CTC enrichment system. PD-L1 expressing in CTCs were identified by the RNA in situ hybridization. And the correlation between CTCs markers and other parameters in terms of clinical outcome, tumor tissues immunochemistry staining, and the effect of immunotherapy was analyzed. RESULTS: The lowest rates of NE and PD-L1+ CTCs were observed in 22Rv1 cell line, the cut-offs of NE and PD-L1+ CTCs were established in vitro, being 45% and 25%, respectively. In HRPC cohort, the NE CTCs positive group (≥45%) had a higher PSA level and more advanced pathological tumor stage than negative group (P=0.041 and P=0.004). Multivariate analysis revealed that NE CTCs rate, not total CTCs amount, was an independent prognostic factor for progression-free survival (PFS) (HR=4.68, P=0.013). In LAPC cohort, the PD-L1+ CTCs positive group (≥25%) displayed a higher PSA level and more frequently lymph node metastasis than negative group (P=0.027 and P=0.028), with median PFS of 15.6 mouths vs. 22.6 mouths, HR=2.70, P=0.018). PD-L1 expression in CTCs was not associated with its expression in tumor tissue staining. While a significant correlation was observed between PD-L1+ CTCs rate and ERG or PTEN expression in tissues (P=0.010 and P=0.009). Furthermore, we found that patients with PD-L1+ CTCs positive expression exhibited a better PSA response to Durvalumab treatment than those with negative expression. CONCLUSIONS: A novel cut-off value of PD-L1+ and NE CTCs could be utilized as powerful prognosis parameter for HRPC and LAPC patients after surgery, and adjuvant immunotherapy can be adopted accordingly. Source of Funding: None.

Association of very small nuclear circulating tumor cell (vsnCTC) with clinical outcomes in metastatic castration-resistant prostate cancer.

168 Background: Circulating tumor cells (CTCs) have arisen as contemporary noninvasive prognostic biomarkers for prostate cancer (PC). Previously, a subgroup of PC CTCs, with particularly small nuclei (<8.5 μm), were found to be correlated with the presence of visceral metastases. This subgroup was named very-small-nuclear CTCs (vsnCTCs). We proposed vsnCTCs as a putative biomarker of a lethal subtype in metastatic castration resistant PC (mCRPC). Methods: In this study, 76 patients with mCRPC were recruited for overall survival (OS) analysis. Of the 76 patients, 47 had available pre-treatment blood specimens prior to the initiation of androgen receptor signaling inhibitor (ARSI, e.g. abiraterone and enzalutamide) or taxane therapy. Using the NanoVelcro CTC Assay, CTCs were captured and subjected to immunofluorescence staining. CTCs were identified as DAPI+/CK+/CD45- with a round or oval nucleus. Additionally, CTC nuclear size was measured and defined as the square root of the product of the long axis and the short axis. Kaplan-Meier analysis and Cox proportional hazards model were conducted. Results: Patients with vsnCTC (i.e., vsnCTC+) had a significantly shortened OS compared with patients without vsnCTC (i.e., vsnCTC-). The median OS was 34 (vsnCTC+, n=49) vs. 149 (vsnCTC-, n=27) weeks (log-rank HR=2.6 with 95% CI 1.5 to 4.5, p=0.0006). Progression free survival (PFS) analysis was performed for the 47 patients with pre-treatment blood samples. The median PFS was 14 (vsnCTC+, n=29) vs. 26 (vsnCTC-, n=18) weeks (log-rank HR=2.2 with 95% CI 1.2 to 3.9, p=0.0069). We also found that the hazard ratio of overall survival increased significantly as the CTC nuclear size decreased using the p spline plot. Conclusions: Our study showed that nuclear size reduction has importance in CTCs in a fashion similar to its utility in tissue. This study points toward the importance of the vsnCTC in patients with mCRPC, as vsnCTC+ patients represented a group at risk for faster clinical progression who are at the highest risk for morality. We posit that the vsnCTC represents a new hallmark of an aggressive subtype of mCRPC. This has potential importance in optimizing therapeutic choices.

Epithelial-mesenchymal transition of circulating tumor cells in prostate cancer is promoted by survivin.

ObjectiveRecent studies demonstrated that circulating tumor cells (CTCs) contribute to the metastasis of prostate cancer. Survivin knockout could inhibit epithelial-mesenchymal transition (EMT) and suppress several metastatic tumors. In this study, we examined the potential involvement of survivin in EMT in CTCs.MethodsCTCs were isolated from the peripheral blood of 100 patients with prostate cancer as EpCAM+/CD45− cells via FACS sorting and identified by immunofluorescence staining of prostate-specific antigen (PSA). CTCs and DU145 cells were transfected with survivin siRNA. Then, the levels of survivin, E-cadherin, and vimentin in CTCs and DU145 cells were detected via immunofluorescence staining, and the invasiveness of CTCs and DU145 cells was examined using a Transwell chamber.ResultsThe results revealed the abundant expression of PSA in the cytoplasm of CTCs. Transfection of survivin siRNA significantly decreased the levels of survivin and vimentin in CTCs and DU145, whereas that of E-cadherin was significantly increased, suggesting survivin plays an important role in EMT of CTCs. In addition, survivin siRNA significantly inhibited the invasiveness of CTCs and DU145 cells.ConclusionsSurvivin plays an important role in EMT of CTCs in prostate cancer, which might mediate the metastasis and invasion of prostate cancer.

Abstract 436: Prediction of aggressive disease in metastatic castration-resistant prostate cancer: The role of very small nuclear circulating tumor cells

Abstract Background: Circulating tumor cells (CTCs) have arisen as a contemporary noninvasive prognostic biomarker for prostate cancer (PC). Previously, a subgroup of PC CTCs, with particularly small nuclei (<8.54 μm), were found to be correlated with poor prognosis and the emergence of visceral metastases (VM). This subgroup was named very-small nuclear CTCs (vsnCTCs). We proposed vsnCTCs as a biomarker of aggressive disease in metastatic castration-resistant PC (mCRPC). Previous Studies also showed that the disruption of emerin, a linker of nucleoskeleton and cytoskeleton complex proteins, increases the cancer cells' capacity of migration and invasion. We hypothesize that emerin mislocalization is potentially associated with vsnCTC formation and may be a critical step for metastasis. Methods: 35 blood samples were obtained from PC patients who failed first-line androgen deprivation therapy (ADT) and starting 2nd line treatment with abiraterone, enzalutamide, or taxane-based chemotherapy. Using our NanoVelcro CTC assay, we captured, subclassified, and enumerated the CTCs from patient samples with high-resolution imaging. Survival analyses were performed to exam the correlation between vsnCTC presence and patients’ prognosis. Concurrently, emerin staining was performed and the distribution and expression levels of emerin were analyzed in selected vsnCTC samples. Results: The presence of one or more vsnCTCs strongly correlated with inferior overall survival (OS), progression free survival (PFS), and time to VM (TTVM). We also observed lower emerin content in vsnCTCs compared to WBC, and more prominent emerin mislocalization in vsnCTCs compared to CTCs with larger nuclei. Conclusion: Our study strongly demonstrated the importance of morphologic characterization of CTCs and suggested that vsnCTC is a blood-borne biomarker for prediction of aggressive disease. Additionally, emerin mislocalization in vsnCTCs could be a potential biological pathway behind this morphologic phenomenon. Citation Format: Pin-Jung Chen, Yu Jen Jan, Pai-Chi Teng, Jie-Fu Chen, Shirley Cheng, Nu Yao, Mariana Reis-Sobreiro, Amber Lozano, Amy Gomez, Hsian-Rong Tseng, Michael Freeman, Edwin Posadas. Prediction of aggressive disease in metastatic castration-resistant prostate cancer: The role of very small nuclear circulating tumor cells [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 436.

A noninvasive prognostic biomarker for metastatic castration-resistant prostate cancer: Very small nuclear circulating tumor cells.

179 Background: Circulating tumor cells (CTCs) have arisen as a contemporary biomarker for prostate cancer (PC). A subgroup of PC CTCs, with particularly small nuclei ( < 8.54 μm), were found to be correlated with poor prognosis and the emergence of visceral metastases (VM). This subgroup was named very-small nuclear CTCs (vsnCTCs). The findings led us to explore vsnCTCs as an aggressive biomarker in metastatic castration-resistant PC (mCRPC). We also explored a biological pathway that potentially drives this morphologic phenomenon. Studies showed that the disruption of the linker of nucleoskeleton and cytoskeleton (LINC) complex proteins, such as emerin, results in nuclear envelope instability and drives cancer cells to an amoeboid phenotype with increasing capacity of migration and invasion. We hypothesized that emerin mislocalization is associated with vsnCTC formation and may be a critical step of metastasis. Methods: Using our NanoVelcro CTC assay, we are able to capture and enumerate CTCs from patients' blood and correlate this data with clinical outcomes. We collected samples from 35 mCRPC patients who failed first-line androgen deprivation therapy and started treatment with abiraterone, enzalutamide, or taxane-based chemotherapy. Survival analyses were performed to exam the correlation between vsnCTC counts and patients’ prognosis. Concurrently, emerin staining was performed and the distribution and expression levels were analyzed in selected vsnCTC samples. Results: The presence of one or more vsnCTCs correlated with worse overall survival (P = 0.00013), progression free survival (PSA progression: P = 0.012; radiographic progression: P = 0.0015), and faster time to VM (P = 0.024). We also observed lower emerin content in vsnCTCs compared to WBC, and more prominent emerin mislocalization in vsnCTCs compared to CTCs with larger nuclei. Conclusions: Our study demonstrated the importance of morphologic characterization of CTCs and suggested that vsnCTCs is a putative biomarker for prediction of worse outcome. Additionally, our findings of emerin mislocalization in vsnCTCs suggested a potential biological pathway behind this nuclear morphologic phenomenon.

Abstract 5230: Development of a multi-parameter immunofluorescence assay for simultaneous detection of androgen receptor and androgen receptor variant 7 in prostate cancer circulating tumor cells

Abstract Background. Androgen receptor (AR) signaling is the primary driver of prostate cancer. Expression of the AR splice variant AR-V7, is predictive of resistance to anti-androgen therapies enzalutimide and abiratirone. There is great interest to non-invasively identify circulating tumor cells (CTCs) and investigate their expression of AR and AR-V7 and monitor them over time. RareCyte has developed a platform for automated visual identification of CTCs by immunofluorescence (IF) that allows up to 6-parameter assessment. We have developed a set of prototype assays using tyramide amplification to detect AR and AR-V7 individually and in combination with prostate membrane-specific antigen (PSMA). Using a novel method, we also developed an assay to identify AR and AR-V7 simultaneously. Methods. Normal human whole blood samples were spiked with prostate cancer lines serving as model CTCs (mCTCs). Prostate cancer blood from a patient with advanced and known high CTC count were collected under an IRB-approved protocol. Blood was processed onto microscope slides and stained on an automated stainer with CTC detection assays incorporating 4 core parameters (nuclear dye, CD45, pan-cytokeratin, EpCAM) plus the following markers: (1) AR; (2) AR-V7; (3) AR and PSMA; (4) AR-V7 and PSMA; (5) AR and AR-V7. Each assay was applied to the cancer cell lines PC3, LNCaP, and 22RV1 and to the patient sample. An antibody denaturing process was used to sequentially amplify two rabbit monoclonals for the assay containing both AR and AR-V7. No-primary (diluent only) staining controls were run to confirm success of the denaturing step. Results. Staining of spike-in CTC models confirmed the reported AR and AR-V7 phenotype of the cell lines, supporting the specificity of the assays. When applied to an equivalent volume of the patient sample, the assays identified a mean (SD) of 36 (11) CTCs. In the assays staining AR, the mean percent of AR+ CTCs was 70% (range 61 - 86) and for the assays staining AR-V7, the percent of AR-V7+ CTCs was 30% (range 26 - 39). In the assay staining both AR and AR-V7, 10 of 38 CTCs were AR-V7+ (26%); all of these were AR+; 15 CTCs were only AR+ (39%). Conclusions. We have developed multi-parameter IF assays for detection of AR and AR-V7 in prostate CTCs, including the first assay we are aware of to simultaneously visualize AR and AR-V7. It has been validated in prostate cancer CTC models. The percentages of AR and AR-V7-positive CTCs identified in a patient sample with the assay are consistent with the percentages observed when AR and AR-V7 are detected in independent assays, supporting the accuracy of the simultaneous detection assay. All AR-V7-positive CTCs were also AR-positive, supporting specificity of AR-V7 staining. Citation Format: Yao Sun, Daniel Campton, Arturo Ramirez, Tanisha Mojica, Alisa Clein, Celestia Higano, Daniel Sabath, Stephen Plymate, Eric Kaldjian. Development of a multi-parameter immunofluorescence assay for simultaneous detection of androgen receptor and androgen receptor variant 7 in prostate cancer circulating tumor cells [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 5230.

Entrapment of Prostate Cancer Circulating Tumor Cells with a Sequential Size-Based Microfluidic Chip.

Circulating tumor cells (CTCs) are broadly accepted as an indicator for early cancer diagnosis and disease severity. However, there is currently no reliable method available to capture and enumerate all CTCs as most systems require either an initial CTC isolation or antibody-based capture for CTC enumeration. Many size-based CTC detection and isolation microfluidic platforms have been presented in the past few years. Here we describe a new size-based, multiple-row cancer cell entrapment device that captured LNCaP-C4-2 prostate cancer cells with >95% efficiency when in spiked mouse whole blood at ∼50 cells/mL. The capture ratio and capture limit on each row was optimized and it was determined that trapping chambers with five or six rows of micro constriction channels were needed to attain a capture ratio >95%. The device was operated under a constant pressure mode at the inlet for blood samples which created a uniform pressure differential across all the microchannels in this array. When the cancer cells deformed in the constriction channel, the blood flow temporarily slowed down. Once inside the trapping chamber, the cancer cells recovered their original shape after the deformation created by their passage through the constriction channel. The CTCs reached the cavity region of the trapping chamber, such that the blood flow in the constriction channel resumed. On the basis of this principle, the CTCs will be captured by this high-throughput entrapment chip (CTC-HTECH), thus confirming the potential for our CTC-HTECH to be used for early stage CTC enrichment and entrapment for clinical diagnosis using liquid biopsies.

Biological significance of novel populations of prostate cancer circulating tumor cells (CTC).

315 Background: Decisions regarding selection and sequencing of treatment in advanced prostate cancer (PC) are often made empirically, without information on the PC molecular profile at the time of treatment. CTC provide real time information regarding contemporaneous biologically relevant cancer subtypes. Current assays often rely on EpCAM or other epithelial markers and may underestimate or exclude other CTC populations of clinical relevance. We devised a novel, simple, rapid and cheap method for isolation of CTC subsets including those without epithelial markers. Methods: Five mL of blood was processed as follows: CD45+ depletion (RosetteSep Human CD45 Depletion Cocktail, Stemcell Technologies); Ficoll centrifugation; antibody staining (mix of EpCAM-PE, PSMA-APC, N-cadherin-AF488, E-cadherin-PerCP/Cy5.5, CD45-APC-H7); Sytox Blue live/dead cell stain; followed by sorting (Becton Dickinson FACSAria Fusion cell sorter). Live cells were sorted into 4 categories denoted E, E+P, P, N (Table). N and P are not identifiable using existing conventional CTC techniques. CTC were assayed for prostate markers ( AR, FOLH1 (PSMA), KLK3 (PSA), TMPRSS2, AMACR) and malignant markers ( PTEN copy number) using digital droplet PCR. Results: Preliminary data are derived from 62 patient samples providing 47 sorted populations. Details are in the Table. Spiking experiments reliably identified as few as 10 CTC / 7.5 mL of blood. P cells were detected in all patients, often outnumbering sum of E and E+P; E and N were never co-expressed. P and N samples frequently expressed prostate markers. Only 1/27 showed no expression of any of these five prostate markers. PTEN deletion assays are in progress. Conclusions: “Unconventional” EpCAMneg CTC appear to be derived from PC and can be detected using broader cell selection criteria. These CTC may provide information on treatment resistance or escape mechanisms. [Table: see text]

RNA Biomarkers: Glycan Stimulation Enables Purification of Prostate Cancer Circulating Tumor Cells on PEDOT NanoVelcro Chips for RNA Biomarker Detection (Adv. Healthcare Mater. 3/2018)

In article number 1700701, Hsiao-hua Yu and co-workers purified circulating tumor cells (CTCs) from blood samples of prostate cancer (PCa) patients. This platform not only captures CTCs with high efficiency and specificity, but also, released these cells gently by a competitive-binding mechanism. The purified CTCs provide well-preserved RNA transcripts for the analysis of the expression level of several PCa-specific RNA biomarkers, which could provide clinical insights into the disease and valuable information for cancer diagnosis and therapeutics.