Extrinsic and intrinsic preanalytical variables affecting liquid biopsy in cancer

Liquid biopsy, through isolation and analysis of disease-specific analytes, has evolved as a promising tool for safe and minimally invasive diagnosis and monitoring of tumors. It also has tremendous utility as a companion diagnostic allowing detection of biomarkers in a range of cancers (lung, breast, colon, ovarian, brain). However, clinical implementation and validation remains a challenge. Among other stages of development, preanalytical variables are critical in influencing the downstream cellular and molecular analysis of different analytes. Although considerable progress has been made to address these challenges, a comprehensive assessment of the impact on diagnostic parameters and consensus on standardized and optimized protocols is still lacking. Here, we summarize and critically evaluate key variables in the preanalytical stage, including study population selection, choice of biofluid, sample handling and collection, processing, and storage. There is an unmet need to develop and implement comprehensive preanalytical guidelines on the optimal practices and methodologies

The Liquid Biopsy Consortium: Challenges and opportunities for early cancer detection and monitoring

The emerging field of liquid biopsy stands at the forefront of novel diagnostic strategies for cancer and other diseases. Liquid biopsy allows minimally invasive molecular characterization of cancers for diagnosis, patient stratification to therapy, and longitudinal monitoring. Liquid biopsy strategies include detection and monitoring of circulating tumor cells, cell-free DNA, and extracellular vesicles. In this review, we address the current understanding and the role of existing liquid-biopsy-based modalities in cancer diagnostics and monitoring. We specifically focus on the technical and clinical challenges associated with liquid biopsy and biomarker development being addressed by the Liquid Biopsy Consortium, established through the National Cancer Institute. The Liquid Biopsy Consortium has developed new methods/assays and validated existing methods/technologies to capture and characterize tumor-derived circulating cargo, as well as addressed existing challenges and provided recommendations for advancing biomarker assays

Automatic detection of circulating tumor cells and cancer associated fibroblasts using deep learning

Circulating tumor cells (CTCs) and cancer-associated fibroblasts (CAFs) from whole blood are emerging as important biomarkers that potentially aid in cancer diagnosis and prognosis. The microfilter technology provides an efficient capture platform for them but is confounded by two challenges. First, uneven microfilter surfaces makes it hard for commercial scanners to obtain images with all cells in-focus. Second, current analysis is labor-intensive with long turnaround time and user-to-user variability. Here we addressed the first challenge through developing a customized imaging system and data pre-processing algorithms. Utilizing cultured cancer and CAF cells captured by microfilters, we showed that images from our custom system are 99.3% in-focus compared to 89.9% from a top-of-the-line commercial scanner. Then we developed a deep-learning-based method to automatically identify tumor cells serving to mimic CTC (mCTC) and CAFs. Our deep learning method achieved precision and recall of 94% (± 0.2%) and 96% (± 0.2%) for mCTC detection, and 93% (± 1.7%) and 84% (± 3.1%) for CAF detection, significantly better than a conventional computer vision method, whose numbers are 92% (± 0.2%) and 78% (± 0.3%) for mCTC and 58% (± 3.9%) and 56% (± 3.5%) for CAF. Our custom imaging system combined with deep learning cell identification method represents an important advance on CTC and CAF analysis

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 (R 2 ¼ 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. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI

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 × 10^3), 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

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 °C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 °C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells

Multiwell Culture Devices with Perfusion and Oxygen Control

A microfluidic structure is formed with one or more microfluidic channels for receiving fluid and passing the fluid through a channel in communication with a collection chamber integrated with that channel through a transition stage that allows the collection chamber to gather targets within the fluid, such as cells, including circulating tumor cells in blood. The transition stage may be formed for an asymmetrical configuration with an obstacle and chamfered face configuration. A gas permeable membrane provides perfusion control for directing gas to the collection chamber. Porous elements provide bubble venting from the fluid flow

Molecular Histopathology Using Gold Nanorods and Optical Coherence Tomography

PURPOSE. To examine the novel application of a commercially available optical coherence tomography (OCT) system toward molecular histopathology using gold nanorod (GNR) linked antibodies as a functionalized contrast agent to evaluate ocular surface squamous neoplasia (OSSN). METHODS. GNRs were synthesized and covalently attached to anti–glucose transporter-1 (GLUT-1) antibodies via carbodiimide chemistry. Three specimens from each of three distinct categories of human conjunctival tissue were selected for analysis, including conjunctiva without epithelial atypia (controls); conjunctival intraepithelial neoplasia, carcinoma in situ (CIS); and conjunctival squamous cell carcinoma (SCC). Tissue sections were incubated initially with GNR tagged anti–GLUT-1 antibodies and then with a fluorescent-tagged secondary antibody. Immunofluorescence and OCT imaging of the tissue was performed and the results were correlated to the light microscopic findings on traditional hemotoxyin and eosin stained sections. RESULTS. No binding of the functionalized GNRs was observed within the epithelium of three normal conjunctiva controls. While immunofluorescence disclosed variable binding of the functionalized GNRs to atypical epithelial cells in all six cases of OSSN, the enhancement of the OCT signal in three cases of CIS was insufficient to distinguish these specimens from normal controls. In two of three cases of SCC, binding of functionalized GNRs was sufficient to produce an increased scattering effect on OCT in areas correlating to atypical epithelial cells which stained intensely on immunofluorescence imaging. Binding of functionalized GNRs was sufficient to produce an increased scattering effect on OCT in areas correlating to regions of erythrocytes and hemorrhage which stained intensely on immunofluorescence imaging within all nine tested samples. CONCLUSIONS. We have demonstrated the use of OCT for molecular histopathology using functionalized gold nanorods in the setting of OSSN. Our results suggest a threshold concentration of functionalized GNRs within tissue is required to achieve a detectable enhancement in scattering of the OCT signal

Identification and Quantitation of Circulating Tumor Cells

Circulating tumor cells (CTCs) are shed from the primary tumor into the circulatory system and act as seeds that initiate cancer metastasis to distant sites. CTC enumeration has been shown to have a significant prognostic value as a surrogate marker in various cancers. The widespread clinical utility of CTC tests, however, is still limited due to the inherent rarity and heterogeneity of CTCs, which necessitate robust techniques for their efficient enrichment and detection. Significant recent advances have resulted in technologies with the ability to improve yield and purity of CTC enrichment as well as detection sensitivity. Current efforts are largely focused on the translation and standardization of assays to fully realize the clinical utility of CTCs. In this review, we aim to provide a comprehensive overview of CTC enrichment and detection techniques with an emphasis on novel approaches for rapid quantification of CTCs