Erica D. Pratt

Capture of circulating tumor cells from whole blood of prostate cancer patients using geometrically enhanced differential immunocapture (GEDI) and a prostate-specific antibody.

Geometrically enhanced differential immunocapture (GEDI) and an antibody for prostate-specific membrane antigen (PSMA) are used for high-efficiency and high-purity capture of prostate circulating tumor cells from peripheral whole blood samples of castrate-resistant prostate cancer patients.

Functional characterization of circulating tumor cells with a prostate-cancer-specific microfluidic device.

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45− cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2–400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents.

Randomized, noncomparative, phase II trial of early switch from docetaxel to cabazitaxel or vice versa, with integrated biomarker analysis, in men with chemotherapy-naïve, metastatic, castration-resistant prostate cancer

Purpose The TAXYNERGY trial ( ClinicalTrials.gov identifier: NCT01718353) evaluated clinical benefit from early taxane switch and circulating tumor cell (CTC) biomarkers to interrogate mechanisms of sensitivity or resistance to taxanes in men with chemotherapy-naïve, metastatic, castration-resistant prostate cancer. Patients and Methods Patients were randomly assigned 2:1 to docetaxel or cabazitaxel. Men who did not achieve ≥ 30% prostate-specific antigen (PSA) decline by cycle 4 (C4) switched taxane. The primary clinical endpoint was confirmed ≥ 50% PSA decline versus historical control (TAX327). The primary biomarker endpoint was analysis of post-treatment CTCs to confirm the hypothesis that clinical response was associated with taxane drug-target engagement, evidenced by decreased percent androgen receptor nuclear localization (%ARNL) and increased microtubule bundling. Results Sixty-three patients were randomly assigned to docetaxel (n = 41) or cabazitaxel (n = 22); 44.4% received prior potent androgen receptor-targeted therapy. Overall, 35 patients (55.6%) had confirmed ≥ 50% PSA responses, exceeding the historical control rate of 45.4% (TAX327). Of 61 treated patients, 33 (54.1%) had ≥ 30% PSA declines by C4 and did not switch taxane, 15 patients (24.6%) who did not achieve ≥ 30% PSA declines by C4 switched taxane, and 13 patients (21.3%) discontinued therapy before or at C4. Of patients switching taxane, 46.7% subsequently achieved ≥ 50% PSA decrease. In 26 CTC-evaluable patients, taxane-induced decrease in %ARNL (cycle 1 day 1 v cycle 1 day 8) was associated with a higher rate of ≥ 50% PSA decrease at C4 ( P = .009). Median composite progression-free survival was 9.1 months (95% CI, 4.9 to 11.7 months); median overall survival was not reached at 14 months. Common grade 3 or 4 adverse events included fatigue (13.1%) and febrile neutropenia (11.5%). Conclusion The early taxane switch strategy was associated with improved PSA response rates versus TAX327. Taxane-induced shifts in %ARNL may serve as an early biomarker of clinical benefit in patients treated with taxanes.

Single-Cell Copy Number Analysis of Prostate Cancer Cells Captured with Geometrically Enhanced Differential Immunocapture Microdevices

Limited access to tumor tissue makes repeated sampling and real-time tracking of cancer progression infeasible. Circulating tumor cells (CTCs) provide the capacity for real-time genetic characterization of a disseminating tumor cell population via a simple blood draw. However, there is no straightforward method to analyze broadscale genetic rearrangements in this heterogeneous cell population at the single cell level. We present a one-step controllable chemical extraction of whole nuclei from prostate cancer cells captured using geometrically enhanced differential immunocapture (GEDI) microdevices. We have successfully used copy number profile analysis to differentiate between two unique cancer cell line populations of metastatic origin (LNCaP and VCaP) and to analyze key mutations important in disease progression.

Capture and analysis of prostate cancer circulating tumor cells (CTCS) using geometrically enhanced differential immunocapture (GEDI).

53 Background: EpCAM-based immunocapture of prostate cancer (PC) CTCs yields relatively low purity and specificity. We developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that incorporates flow dynamics and utilizes a mAb to prostate-specific membrane antigen (PSMA) to optimize isolation and analysis of CTCs from PC patients. METHODS GEDI microfluidic silicon chips, fabricated using standard photolithography techniques, were functionalized by chemical cross-linking ending with a neutravidin terminated surface to which anti-PSMA biotinylated-mAb J591 was bound. C4-2 (PSMA+) and PC-3 (PSMA-) cells were used for chip optimization. 1 mL of peripheral blood from PC patients was flowed (1mL/hour) over functionalized chips. Captured cells were washed with PBS × 30 min, fixed with 3.7% formaldehyde, immunofluorescently stained for DAPI, androgen receptor (AR), tubulin and EpCAM and analyzed by high resolution point-scanning confocal microscopy. PSMA+, DAPI+, and CD45- cells were manually scored. RNA was extracted from unfixed captured CTCs using lysis buffer flowed thru the chip. RESULTS ∼80% capture efficiency was achieved from 26 PSMA positive C4-2 cells spiked into 1 mL blood flowed through the GEDI chip. RNA extracted from 50 C4-2 cells in 1 mL blood flowed thru the chip detected a known AR point mutation by RT-PCR and Sanger sequencing. Immunofluorescence staining of PSMA+ cells captured on the chip detected changes in AR subcellular localization and microtubule structure following treatment with DHT or paclitaxel, respectively. 10 patients with metastatic PC were analyzed by CellSearch (range 0-201 cells/7.5 mL) and GEDI chip (range 35->1200 cells/mL) yielding a 7->350 fold enrichment using GEDI. Captured PC cells isolated from PC patient incubated in 50 nm paclitaxel (ex vivo) overnight demonstrated microtubule bundling, indicative of drug-target engagement. CONCLUSIONS PSMA based GEDI microfluidic CTC capture is highly specific and sensitive in capturing PSMA positive PC CTCs; and allows detailed CTC analysis including protein expression and subcellular localization, mutational analysis and drug sensitivity assessment. [Table: see text].

Abstract 4891: Molecular and functional analysis of circulating tumor cells in castrate resistant prostate cancer using a geometrically enhanced microfluidic device based on PSMA immunocapture

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Prostate cancer (PC) is the most common type of cancer in males and the second leading cause of male cancer deaths in the United States. Circulating tumor cells (CTCs) are commonly found in the blood of metastatic patients and the capture of these cells is important in the study of disease progression. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device coated with the prostate-specific membrane antigen (PSMA) which captures prostate CTCs with high purity and efficiency. This device is designed with a staggered obstacle array in order to maximize the capture efficiency (>80%) and purity (70%) of CTCs. We have used this device to capture, enumerate and molecularly characterize CTCs isolated from 1 ml of blood from patients with castrate resistant prostate cancer (CRPC). First, we compared the capture efficiency from 16 CRPC patients’ blood and subjected it to CTC enumeration by either the GEDI device or the FDA approved CellSearch technology (EpCAM-based immunocapture). Our results showed a 2-350 fold enrichment in CTC counts by the GEDI (range 15-1200 CTCs/ml, median 54). In addition, there was minimal false positive CTC detection in healthy donor blood analyzed by the GEDI. Next we sought to characterize the captured CTCs using multiplex immunofluorescence for proteins specific for PC like androgen receptor (AR), PSMA, TMPRSS2-ERG, EpCAM and other markers involved in epithelial to mesenchymal transition (EMT). We observed low or lack of EpCAM expression in the captured PSMA+/CD45- CTCs, concomitant with high vimentin expression, suggesting that our device may be preferentially capturing CTCs that have undergone EMT. Using the GEDI device, we are able to detect a known single point mutation in the AR using cDNA extracted from approximately 50 C4-2 cells spiked into 1 ml of blood from a healthy donor. Importantly, we have captured CTCs from different CRPC patients and treated them ex vivo with docetaxel to assess effective drug-target engagement by determining the extent of microtubule (MT) stabilization and apoptotic cell death. Our results showed non-uniform CTC response to drug treatment, such that a subset of PSMA+/CD45- CTCs showed MT bundling, indicating tumor heterogeneity and further suggesting that we can potentially use this device to predict patient response to taxane-based chemotherapy. In summary, the GEDI microfluidic device is a novel and specific technology to isolate and characterize PC CTCs at the molecular level. We plan to use this device in prospective clinical studies in order to understand the molecular basis of disease progression and response to taxane-based chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4891. doi:10.1158/1538-7445.AM2011-4891

Abstract 1145: Analysis of circulating tumor cells from castrate resistant prostate cancer patients captured via a microfluidic device using the prostate specific membrane antigen antibody

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Prostate cancer (PC) is the most common type of cancer in males and the second leading cause of male cancer deaths in the United States. Circulating tumor cells (CTCs) are commonly found in the blood of patients suffering from mestatic disease and the capture of these cells is important in the study of disease progression. Existing devices use the EpCAM antibody which can capture circulating epithelial cancer cells, but also leads to a large number of leukocyte contamination. We have developed a microfluidic chip functionalized with an antibody to the prostate-specific membrane antigen (PSMA), which captures prostate CTCs with high purity and efficiency. This chip is designed with a staggered obstacle array that maximizes the CTC-wall interactions while minimizing the interactions with the other cells in the blood. PSMA-positive or -negative PC cells were spiked into blood from healthy donors, and we calculated capture rates to be ∼80%. Next, blood samples from patients (n = 20) with castrate resistant prostate cancer (CRPC) were run through the device and CTCs were captured and enumerated. CTCs were found in 90% of samples tested with the number captured averaging 27 ± 4 cells per mL of blood with a capture purity of 62 ± 2%. We plan to use this device to elucidate the molecular basis of CRPC clinical response to taxane treatment, which is the only class of chemotherapy drugs shown to improve survival in this type of PC. Previous data from our laboratory has shown that taxane treatment of PC cells inhibits the ligand-induced androgen receptor (AR) nuclear accumulation and transcriptional activation of androgen response element (ARE) containing genes including the prostate-specific antigen (PSA). These results implicate microtubules (MTs) in AR trafficking and suggest that the clinical activity of taxanes in CRPC may be mediated in part by the drugs’ ability to stabilize MTs and inhibit AR transcriptional activity. We plan to capture CTCs using the PSMA-specific microfluidic device from CRPC patients receiving taxane treatment and analyze them for AR subcellular distribution and correlate clinical response to taxane treatment. Our microfluidic device is a novel, specific, and improved technology to capture prostate CTCs that will allow us to better define the molecular basis of taxane treatment in CRPC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1145.