Timothy B. Lannin

Detection of Circulating Pancreas Epithelial Cells in Patients With Pancreatic Cystic Lesions

Hematogenous dissemination is thought to be a late event in cancer progression. We recently showed in a genetic model of pancreatic ductal adenocarcinoma that pancreas cells can be detected in the bloodstream before tumor formation. To confirm these findings in humans, we used microfluidic geometrically enhanced differential immunocapture to detect circulating pancreas epithelial cells in patient blood samples. We captured more than 3 circulating pancreas epithelial cells/mL in 7 of 21 (33%) patients with cystic lesions and no clinical diagnosis of cancer (Sendai criteria negative), 8 of 11 (73%) with pancreatic ductal adenocarcinoma, and in 0 of 19 patients without cysts or cancer (controls). These findings indicate that cancer cells are present in the circulation of patients before tumors are detected, which might be used in risk assessment.

Microfluidic immunocapture of circulating pancreatic cells using parallel EpCAM and MUC1 capture: characterization, optimization and downstream analysis

We have developed and optimized a microfluidic device platform for the capture and analysis of circulating pancreatic cells (CPCs) and pancreatic circulating tumor cells (CTCs). Our platform uses parallel anti-EpCAM and cancer-specific mucin 1 (MUC1) immunocapture in a silicon microdevice. Using a combination of anti-EpCAM and anti-MUC1 capture in a single device, we are able to achieve efficient capture while extending immunocapture beyond single marker recognition. We also have detected a known oncogenic KRAS mutation in cells spiked in whole blood using immunocapture, RNA extraction, RT-PCR and Sanger sequencing. To allow for downstream single-cell genetic analysis, intact nuclei were released from captured cells by using targeted membrane lysis. We have developed a staining protocol for clinical samples, including standard CTC markers; DAPI, cytokeratin (CK) and CD45, and a novel marker of carcinogenesis in CPCs, mucin 4 (MUC4). We have also demonstrated a semi-automated approach to image analysis and CPC identification, suitable for clinical hypothesis generation. Initial results from immunocapture of a clinical pancreatic cancer patient sample show that parallel capture may capture more of the heterogeneity of the CPC population. With this platform, we aim to develop a diagnostic biomarker for early pancreatic carcinogenesis and patient risk stratification.

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.

Automated electrorotation shows electrokinetic separation of pancreatic cancer cells is robust to acquired chemotherapy resistance, serum starvation, and EMT

We used automated electrorotation to measure the cytoplasmic permittivity, cytoplasmic conductivity, and specific membrane capacitance of pancreatic cancer cells under environmental perturbation to evaluate the effects of serum starvation, epithelial-to-mesenchymal transition, and evolution of chemotherapy resistance which may be associated with the development and dissemination of cancer. First, we compared gemcitabine-resistant BxPC3 subclones with gemcitabine-naive parental cells. Second, we serum-starved BxPC3 and PANC-1 cells and compared them to untreated counterparts. Third, we induced the epithelial-to-mesenchymal transition in PANC-1 cells and compared them to untreated PANC-1 cells. We also measured the electrorotation spectra of white blood cells isolated from a healthy donor. The properties from fit electrorotation spectra were used to compute dielectrophoresis (DEP) spectra and crossover frequencies. For all three experiments, the median crossover frequency for both treated and untreated pancreatic cancer cells remained significantly lower than the median crossover frequency for white blood cells. The robustness of the crossover frequency to these treatments indicates that DEP is a promising technique for enhancing capture of circulating cancer cells.

Comparison and optimization of machine learning methods for automated classification of circulating tumor cells.

Advances in rare cell capture technology have made possible the interrogation of circulating tumor cells (CTCs) captured from whole patient blood. However, locating captured cells in the device by manual counting bottlenecks data processing by being tedious (hours per sample) and compromises the results by being inconsistent and prone to user bias. Some recent work has been done to automate the cell location and classification process to address these problems, employing image processing and machine learning (ML) algorithms to locate and classify cells in fluorescent microscope images. However, the type of machine learning method used is a part of the design space that has not been thoroughly explored. Thus, we have trained four ML algorithms on three different datasets. The trained ML algorithms locate and classify thousands of possible cells in a few minutes rather than a few hours, representing an order of magnitude increase in processing speed. Furthermore, some algorithms have a significantly (P < 0.05) higher area under the receiver operating characteristic curve than do other algorithms. Additionally, significant (P < 0.05) losses to performance occur when training on cell lines and testing on CTCs (and vice versa), indicating the need to train on a system that is representative of future unlabeled data. Optimal algorithm selection depends on the peculiarities of the individual dataset, indicating the need of a careful comparison and optimization of algorithms for individual image classification tasks.

Abstract 3492: Using CTCs to interrogate mechanisms of taxane resistance in the prospective TAXYNERGY clinical trial in prostate cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Prostate cancer progression into castration-resistant prostate cancer (CRPC) is driven by continued androgen receptor (AR) signaling despite surgical and chemical androgen ablation. The taxanes represent the only class of chemotherapy that improves overall survival in CRPC patients. Despite their success, CRPC patients do progress on taxane treatment rendering taxane-resistant tumors. The molecular mechanisms underlying clinical taxane resistance in CRPC have not been well elucidated due to the lack of available tumor tissue to study. Circulating tumor cells (CTCs) represent a liquid biopsy of the original tumor and isolation of them can lead to their molecular characterization potentially revealing predictive biomarkers for taxane sensitivity or resistance. Here, we use a geometrically enhanced differential immunocapture (GEDI) microfluidic device that couples an anti-prostate specific membrane antigen (PSMA) antibody with optimized 3D geometry to capture and isolate live CTCs from whole blood of CRPC patients. The GEDI-microfluidic device was shown to have a 2-400 fold higher sensitivity for CTC capture than the FDA-approved CellSearch® system. We have previously shown that CRPC patient CTCs can be used to derive functional information that correlates to clinical response to taxane chemotherapy, namely AR subcellular localization status. We have developed a suite of other functional assays that can be performed on live GEDI-captured CTCs that enable their molecular characterization and allow us to test specific mechanistic hypotheses based on our extensive preclinical data. Included, and herein described, are the determination of AR subcellular localization, extent of effective drug-target engagement assessed by microtubule bundling, identification of RNA species relevant to the mechanism of taxane resistance and computer vision algorithms that will allow for enriched and automated analysis of high-volume image sets of GEDI-captured CTCs. In addition, we will be testing the hypothesis that distinct AR splice variants may affect patient sensitivity to taxane-based chemotherapy. This suite of assays are being rigorously applied in a phase II clinical trial in which chemotherapy-naive CRPC patients will be initially treated with either docetaxel or cabazitaxel and clinically evaluated for an early switch to the other taxane following disease progression. This prospective, randomized, multi-site clinical trial will enroll 100 CRPC patients within one year. Patients will be followed until relapse and each patient will have 15 independent GEDI assays performed across five time points from baseline to chemotherapy crossover to relapse. The depth of coverage this suite of assays provides will offer unique insights for potential mechanisms of clinical taxane resistance and predictive biomarkers for taxane sensitivity in CRPC patient CTCs. Citation Format: Matthew S. Sung, Ada Gjyrezi, Guang Yu Lee, Alexandre Matov, Giuseppe Galletti, Matthew Loftus, Yusef Syed, Timothy Lannin, Atanas Hristov, Christopher Mason, Scott Tagawa, Brian Kirby, David Nanus, Paraskevi Giannakakou. Using CTCs to interrogate mechanisms of taxane resistance in the prospective TAXYNERGY clinical trial in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3492. doi:10.1158/1538-7445.AM2013-3492