Hybrid Fusion FISH™ and Single-Cell Isolation: Genetic Interrogation of Circulating Tumor Cells (CTCs) via Capture By Hydrodynamic Trapping

Review Article: Part 3 of Series


Genetic interrogation at single-cell resolution is a highly sought model in cancer genomics and diagnostics. The ability to characterize the molecular genetic component of single cells in cancer biology is an emerging value proposition for clinical reference labs and pharmaceutical research organizations, particularly of importance for residual disease monitoring and biological modeling of patient-specific tumors. Single-cell genomic applications can differentiate between individual cancer cells from a homogenous sample population. Hybrid Fusion FISH™ with single-cell isolation allows for the genetic profiling of pre-cancerous and cancerous cells while retaining the overall morphology of the cell. The diagnostic utilities of such applications for Hybrid Fusion FISH on single cells include a) the identification of gene copy number variations (CNVs) and rearrangements including amplifications, deletions and translocations, b) the assessment of inter- and intratumor genetic heterogeneity, c) disease prediction and progression, d) characterization of clonal cell populations, and e) determining pharmacogenomics, therapeutic resistance and cellular plasticity.

While the above applications are appreciable, the main value is to preserve the single cell for complete molecular genetic characterization via Hybrid Fusion FISH™. Evaluating the heterogeneity of tumors at single cell resolution is increasingly valuable to pathologists. The ability to capture and culture rare single cells is also paramount. Traditional FISH assays and microfluidic systems are costly, time-consuming, and require considerable technician time for large-volume clinical reference laboratories. For large-scale economics and diagnostic adoptability, consumables (Hybrid Fusion FISH™ assays) paired with disposables (NeoPlate™ small volume liquid handling systems via high-throughput well plates) are clinically viable and cost-efficient. Such a system is presented below.

In the above simplified model, a cell suspension (i.e. blood, urine, cervical) is pipetted into a single inlet valve which automatically disperses single cells throughout multiple repetitive wells within the NeoPlate™. The wells are autonomously and automatically filled using hydrodynamic trapping — the design is such that the liquid-to-air surface tension prevents the cell fluid suspension from leaving the wells. No capital equipment or automated instruments are required for this process (i.e. does not require flow rate controls or elastomeric valves as seen in traditional liquid handling systems). Each NeoPlate™ well accommodates a volume of 0.1 ul. The wells can additionally serve as culture plates, which is particularly attractive for individual cancer cells to study differentiation and plasticity models (i.e. for CTCs and CTC clusters).

Hybrid Fusion FISH™ coupled to cost-efficient single-cell isolation systems such as the NeoPlate™ are clinically viable for residual disease monitoring. For example, a patient identified to benefit from a particular therapeutic agent will need to be monitored for residual disease over time. Such monitoring is conducted via liquid biopsies with cell-enrichment and isolation protocols. The ability to look at the morphology of individual cells while conducting Hybrid Fusion FISH™ for assessment of novel genetic aberrations enables the pathologist to adjust treatment protocols accordingly for therapies that will benefit from certain molecular characteristics. Molecular characterization of individual cancer cells (particularly for CTCs) and cancer stem cells is also important to predict the future course of disease. Individual cells that are captured can optionally be cultured within the same NeoPlate™ with the introduction of culture media through the inlet valve. Additionally, therapeutic agents can also be introduced via the inlet valve to study the effects of drugs on the isolated cells. This paradigm of cancer diagnostics is clinically attractive, giving rise to an avenue of precision diagnostics and medicine on a single-cell basis.

For such patient-tailored diagnostics, efficiency for laboratory implementation on a large scale is desired. The NeoPlate™ can be in the form of a well-plate (for cell culture and therapeutic reagent studies) or microscope slides (for analysis under a microscope). The plates are disposable and can be archived by the clinical reference lab for later review. Hybrid Fusion FISH™ probes are cost-efficient consumables that allow for increased multiplexed assays for single-cell analysis. Obtaining complex genetic information using single-cell disposable microfluidic devices and novel consumable diagnostic reagents is scalable for mass-prevention screening measures. With high-throughput and no capital expenditures, Hybrid Fusion FISH™ with the NeoPlate™ overcomes the cost hurdle for pathology labs to implement such tests.

Single cell analysis is an emerging field that provides significant insights on disease manifestation and progression for clinical and research use. Average cell measurements vs. single-cell analysis for highly heterogeneous tumors results in inconsistencies. In most cases, average cell measurements typically include the “normal” cell population which can considerably dilute the analytical validity of results. When using homogenous cell populations, the actual genomic status of an individual cell is not well represented or characterized. The ability to retain the morphological context of the surrounding tissue is important to the pathologist. In many cases, the morphological context of a single cell may be paired to the histopathological examination of the same cell to overcome clinical misinterpretation and allow for testing correlation. For liquid biopsies, the cellular diversity and heterogeneity can be resolved at the single-cell level to identify CTCs and peripheral mononuclear blood cells (PMBCs). With single cells that are isolated and cultured, cell-to-cell communication can be thoroughly investigated.

The field of single-cell genomics is a new era in biological research. With respect to isolation and genetic characterization, Hybrid Fusion FISH™ paired to various single-cell protocols is ushering the next generation of single-cell genomics for quickly studying the DNA of cells. Understanding biology at the single-cell level is removing the obscurities from typical “bulk” homogenous sample analyses. Studying individual genes at the single-cell level can reveal a wealth of information and provide a different perspective as opposed to broad analytical methods. With single-cell isolation and Hybrid Fusion FISH™, it is possible to understand the distribution of genetic mutations and chromosomal aberrations between cells. Clustering and clonal genetic effects are important in clinical pathology. There is an ordered lineage of mutations that can be tracked through single-cell genetic analysis of cancerous cells. Armed with such diagnostic information, a pathologist is able to determine genetic changes at an earlier time point and predict the course of disease for a future time point while implementing the most effective targeted therapies. Hybrid Fusion FISH™ offers cost-effective, single-cell genetic resolution for clinical and research utilities for identifying targeted therapies while advancing precision medicine.

While novel microfluidics applications are emerging (such as the NeoPlate™), Hybrid Fusion FISH™ with single-cell isolation is a prime example for such coupled utility in the diagnostic field. As liquid biopsies become more commonplace for residual disease monitoring and standard-of-practice for new patient diagnosis’, Hybrid Fusion FISH™ allows for efficient genetic interrogation for actionable outcomes. Specifically, warranting the use of therapeutic drugs based on the results of Hybrid Fusion FISH™ on single cells is economically valuable: the test can determine if a patient will benefit from a specific agent or rather, disqualify the patient from an expensive agent that will have no benefit (and may increase toxicity if implemented). The proposition is economically sound and yields improved clinical outcomes.

Today, single-cell capture systems are too expensive due to the capital equipment required and operational complexity. The Hybrid Fusion FISH™ model with single-cell isolation systems such as the NeoPlate™ utilizes a one-step dilution feature for multi-step assays using nanoliter reagent volumes. Such systems for clinical oncology applications are novel with high utility in the diagnostics setting. Molecular characterization of single cells is the foundation of disease, as Hybrid Fusion FISH™ aims to genetically characterize cells on a cost-efficient basis. Further assessment of single cells (post Hybrid Fusion FISH™) is also made possible (i.e. cytosolic and cell membrane characterization by IHC). The development of companion diagnostics that couple the screening tests to single cells is additionally attractive for pharmaceutical companies. This technology is an advancement in single-cell diagnostics coupled to disposable/consumable healthcare economics for large-volume reference laboratories.

LOOKING FORWARD

DISCLAIMER: The NeoPlate™ is a trademark and property of Neofluidics LLC. This article is part 3 of a series of articles dissecting, researching and evaluating Hybrid Fusion FISH™ applications within the clinical diagnostics and research environments. Prevnos Inc. is a cancer diagnostics company that is rapidly evolving the current cytogenetics and molecular cancer genetics markets. The company is the inventor of the world’s first Hybrid Fusion FISH™ tests (consumables) and the world’s most economical fluorescence microscope termed the Retina™ FISH scope (capital equipment). The company is also the inventor of GEN+ Connect (software), which enables digital pathology for molecular cytogeneticists around the world. Furthermore, the company explores clinical research with pharmaceutical companies for companion diagnostics. Prevnos engages in various forward-thinking R&D projects including, but not limited to, targeted cell-enrichment with subsequent FISH, CTC FISH™, non-disruptive FISH probe delivery vehicles, single-cell isolative technology for FISH, tyrosine kinase inhibitor master FISH assays for lung cancer, molecular characterization for stratification of prostate cancer patients, and COMET (chromatin organization mediated electrophoretic transfer) FISH™ assays for assessing DNA viability.

To learn more, visit Prevnos at www.prevnos.com