Anthony Williams

Portable Filter-Based Microdevice for Detection and Characterization of Circulating Tumor Cells

Purpose: Sensitive detection and characterization of circulating tumor cells (CTC) could revolutionize the approach to patients with early-stage and metastatic cancer. The current methodologies have significant limitations, including limited capture efficiency and ability to characterize captured cells. Here, we report the development of a novel parylene membrane filter-based portable microdevice for size-based isolation with high recovery rate and direct on-chip characterization of captured CTC from human peripheral blood. Experimental Design: We evaluated the sensitivity and efficiency of CTC capture in a model system using blood samples from healthy donors spiked with tumor cell lines. Fifty-nine model system samples were tested to determine the recovery rate of the microdevice. Moreover, 10 model system samples and 57 blood samples from cancer patients were subjected to both membrane microfilter device and CellSearch platform enumeration for direct comparison. Results: Using the model system, the microdevice achieved >90% recovery with probability of 95% recovering at least one cell when five are seeded in 7.5 mL of blood. CTCs were identified in 51 of 57 patients using the microdevice, compared with only 26 patients with the CellSearch method. When CTCs were detected by both methods, greater numbers were recovered by the microfilter device in all but five patients. Conclusions: This filter-based microdevice is both a capture and analysis platform, capable of multiplexed imaging and genetic analysis. The microdevice presented here has the potential to enable routine CTC analysis in the clinical setting for the effective management of cancer patients. Clin Cancer Res; 16(20); 5011–8. ©2010 AACR.

3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood

Detection of circulating tumor cells has emerged as a promising minimally invasive diagnostic and prognostic tool for patients with metastatic cancers. We report a novel three dimensional microfilter device that can enrich viable circulating tumor cells from blood. This device consists of two layers of parylene membrane with pores and gap precisely defined with photolithography. The positions of the pores are shifted between the top and bottom membranes. The bottom membrane supports captured cells and minimize the stress concentration on cell membrane and sustain cell viability during filtration. Viable cell capture on device was investigated with scanning electron microscopy, confocal microscopy, and immunofluorescent staining using model systems of cultured tumor cells spiked in blood or saline. The paper presents and validates this new 3D microfiltration concept for circulation tumor cell enrichment application. The device provides a highly valuable tool for assessing and characterizing viable enriched circulating tumor cells in both research and clinical settings.

Circulating Tumor Cells: From Bench to Bedside

Circulating tumor cells (CTCs) represent a surrogate biomarker of hematogenous metastases. In recent years, their detection has gained increasing interest. There is ample evidence regarding the ability to detect CTCs and their prognostic relevance, but their demonstrated predictive value in therapeutic response monitoring is clinically even more meaningful. Many clinical trials in the early and metastatic cancer setting now include CTCs as a monitoring parameter, and numerous translational studies attempting their molecular characterization are under way. There has been great progress in defining the clinical importance of CTCs, and it now seems likely that we may expect wider implementation of CTCs as a diagnostic oncology tool to monitor therapeutic response in real time. Novel technologies may further facilitate molecular characterization of CTCs and development of novel therapeutic targets, possibly leading to more powerful treatment strategies for cancer patients. As the detection and evaluation of CTCs are becoming an increasingly important diagnostic and prognostic tool, the goal of this review is to communicate the knowledge obtained through analysis of primary tumors and CTCs to oncologists and medical specialists in managing patients with cancer.

Predicting Recurrence and Progression of Noninvasive Papillary Bladder Cancer at Initial Presentation Based on Quantitative Gene Expression Profiles

BACKGROUND Currently, tumor grade is the best predictor of outcome at first presentation of noninvasive papillary (Ta) bladder cancer. However, reliable predictors of Ta tumor recurrence and progression for individual patients, which could optimize treatment and follow-up schedules based on specific tumor biology, are yet to be identified. OBJECTIVE To identify genes predictive for recurrence and progression in Ta bladder cancer at first presentation using a quantitative, pathway-specific approach. DESIGN, SETTING, AND PARTICIPANTS Retrospective study of patients with Ta G2/3 bladder tumors at initial presentation with three distinct clinical outcomes: absence of recurrence (n=16), recurrence without progression (n=16), and progression to carcinoma in situ or invasive disease (n=16). MEASUREMENTS Expressions of 24 genes that feature in relevant pathways that are deregulated in bladder cancer were quantified by real-time polymerase chain reaction on tumor biopsies from the patients at initial presentation. RESULTS AND LIMITATIONS CCND3 (p=0.003) and HRAS (p=0.01) were predictive for recurrence by univariate analysis. In a multivariable model based on CCND3 expression, sensitivity and specificity for recurrence were 97% and 63%, respectively. HRAS (p<0.001), E2F1 (p=0.017), BIRC5/Survivin (p=0.038), and VEGFR2 (p=0.047) were predictive for progression by univariate analysis. Multivariable analysis based on HRAS, VEGFR2, and VEGF identified progression with 81% sensitivity and 94% specificity. Since this is a small retrospective study using medium-throughput profiling, larger confirmatory studies are needed. CONCLUSIONS Gene expression profiling across relevant cancer pathways appears to be a promising approach for Ta bladder tumor outcome prediction at initial diagnosis. These results could help differentiate between patients who need aggressive versus expectant management.

Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78–83%), high retention of cell viability (71–74%), high tumour cell enrichment against leukocytes (1.7–2 × 103), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4–0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

Targeting 4-1BB Costimulation to the Tumor Stroma with Bispecific Aptamer Conjugates Enhances the Therapeutic Index of Tumor Immunotherapy

Schrand and colleagues report the efficacy in five murine tumor models of an immunotherapeutic approach whereby systemic administration of tumor stroma-targeted 4-1BB aptamer conjugates, which target disseminated tumor lesions, elicits potent antitumor immunity with minimal dose-limiting toxicity. Despite the recent successes of using immune modulatory Abs in patients with cancer, autoimmune pathologies resulting from the activation of self-reactive T cells preclude the dose escalations necessary to fully exploit their therapeutic potential. To reduce the observed and expected toxicities associated with immune modulation, here we describe a clinically feasible and broadly applicable approach to limit immune costimulation to the disseminated tumor lesions of the patient, whereby an agonistic 4-1BB oligonucleotide aptamer is targeted to the tumor stroma by conjugation to an aptamer that binds to a broadly expressed stromal product, VEGF. This approach was predicated on the premise that by targeting the costimulatory ligands to products secreted into the tumor stroma, the T cells will be costimulated before their engagement of the MHC–peptide complex on the tumor cell, thereby obviating the need to target the costimulatory ligands to noninternalizing cell surface products expressed on the tumor cells. Underscoring the potency of stroma-targeted costimulation and the broad spectrum of tumors secreting VEGF, in preclinical murine tumor models, systemic administration of the VEGF-targeted 4-1BB aptamer conjugates engendered potent antitumor immunity against multiple unrelated tumors in subcutaneous, postsurgical lung metastasis, methylcholantrene-induced fibrosarcoma, and oncogene-induced autochthonous glioma models, and exhibited a superior therapeutic index compared with nontargeted administration of an agonistic 4-1BB Ab or 4-1BB aptamer. Cancer Immunol Res; 2(9); 867–77. ©2014 AACR.

Progress in circulating tumor cell capture and analysis: implications for cancer management

The hematogenous dissemination of cancer and development of distant metastases is the cause of nearly all cancer deaths. Detection of circulating tumor cells (CTCs) as a surrogate biomarker of metastases has gained increasing interest. There is accumulating evidence on development of novel technologies for CTC detection, their prognostic relevance and their use in therapeutic response monitoring. Many clinical trials in the early and metastatic cancer setting, particularly in breast cancer, are including CTCs in their translational research programs and as secondary end points. We summarize the progress of detection methods in the context of their clinical importance and speculate on the possibilities of wider implementation of CTCs as a diagnostic oncology tool, the likelihood that CTCs will be used as a useful biomarker, especially to monitor therapeutic response, and what may be expected from the future improvements in technologies.

Fourier ptychographic microscopy for filtration-based circulating tumor cell enumeration and analysis

Abstract. Circulating tumor cells (CTCs) are recognized as a candidate biomarker with strong prognostic and predictive potential in metastatic disease. Filtration-based enrichment technologies have been used for CTC characterization, and our group has previously developed a membrane microfilter device that demonstrates efficacy in model systems and clinical blood samples. However, uneven filtration surfaces make the use of standard microscopic techniques a difficult task, limiting the performance of automated imaging using commercially available technologies. Here, we report the use of Fourier ptychographic microscopy (FPM) to tackle this challenge. Employing this method, we were able to obtain high-resolution color images, including amplitude and phase, of the microfilter samples over large areas. FPM’s ability to perform digital refocusing on complex images is particularly useful in this setting as, in contrast to other imaging platforms, we can focus samples on multiple focal planes within the same frame despite surface unevenness. In model systems, FPM demonstrates high image quality, efficiency, and consistency in detection of tumor cells when comparing corresponding microfilter samples to standard microscopy with high correlation (R2=0.99932). Based on these results, we believe that FPM will have important implications for improved, high throughput, filtration-based CTC analysis, and, more generally, image analysis of uneven surfaces.

Size-Based Enrichment Technologies for CTC Detection and Characterization

The degree of metastatic outspread in malignant disease is one of the leading factors in determining the appropriate course treatment. Circulating tumor cells (CTCs) represent the population of cells that have acquired the means to gain access to the circulatory system, and the cell population ultimately responsible for the development of metastases at distant sites in the body. While promising as a biomarker for metastatic disease, the widespread study of CTCs has been limited by their rarity, as CTCs are reported to occur as infrequently as 1/mL of whole blood. In this text, we will discuss current and emerging technologies for the size-based enrichment of CTCs from whole blood, and compare some of the advantages and disadvantages of using a size-based approach to CTC enrichment versus affinity-based CTC enrichment platforms.

Identification of cancer associated fibroblasts in circulating blood from patients with metastatic breast cancer

Metastasis is facilitated by cancer-associated fibroblasts (CAF) in the tumor microenvironment through mechanisms yet to be elucidated. In this study, we used a size-based microfilter technology developed by our group to examine whether circulating CAF identified by FAP and α-SMA co-expression (cCAF) could be distinguished in the peripheral blood of patients with metastatic breast cancer. In a pilot study of patients with breast cancer, we detected the presence of cCAFs in 30/34 (88%) patients with metastatic disease (MET group) and in 3/13 (23%) patients with localized breast cancer (LOC group) with long-term disease-free survival. No cCAFs as defined were detected in healthy donors. Further, both cCAF and circulating tumor cells (CTC) were significantly greater in the MET group compared with the LOC group. Thus, the presence of cCAF was associated with clinical metastasis, suggesting that cCAF may complement CTC as a clinically relevant biomarker in metastatic breast cancer.