Hybrid Fusion FISH™ vs Next-Generation Sequencing: A Comparison of Diagnostic Values Between Emerging Cancer Profiling Technologies

Review Article: Part 2 of Series


DNA FISH and DNA sequencing are two distinct molecular genetic techniques that are implemented in clinical diagnostics for interrogation of genes. It is important to note that each technology offers certain advantages for clinical utility, and both should be seen as complementary and hold particular value to the pathologist depending on the clinical purpose and immediate need. New genomic approaches are only as viable in the clinical field as the actionable information that such tests provide. Therefore, a comparison between each approach is warranted to highlight the pros and cons of each technology, taking into consideration the clinical purpose, cost, and feasibility in the clinical environment. Sanger sequencing and pyrosequencing technologies are evolving today towards Next-Generation Sequencing (NGS). Likewise, traditional FISH is evolving towards Hybrid Fusion FISH. Each emerging technology has critical value propositions depending on the clinical context and desired outcomes. Significant technical differences between these two methods are highlighted below.

Using the “alphabet” analogy above (where the alphabet represents DNA), each letter represents a nucleotide that is strung together in a particular order. Segments of such letters form genes, which are the units implicated in cancer as a result of genetic aberrations. NGS is able to identify each specific “letter” from a bulk DNA sample collection, yielding gene information at single-nucleotide resolution as compared to a control reference genome. Hybrid Fusion FISH is able to identify large stretches of letters inside of single cells, yielding gene information at single-cell resolution as compared to a control reference cell. This is an important difference between both technologies, as Hybrid Fusion FISH is unable to identify single-nucleotide polymorphisms (SNPs) whereas NGS can identify such variants. However, Hybrid Fusion FISH can quickly identify deletions, rearrangements, translocations, fusions, amplifications, duplications, gains and other genetic aberrations in intact whole chromosomes, preserved single cells, and interphase and metaphase nuclei while retaining the morphology of the cell with gene localization. Depending on the clinical context, it may be important to know information such as a single variant change in nucleotides (NGS) or large copy number variations in gene sequences (Hybrid Fusion FISH). The “alphabet” analogy illustrates the major DNA resolution differences between both techniques.

Genetic Interrogation on Single Cells

The molecular characterization of single cells is highly desired to understand how cells interact with each other as opposed to a homogenous representation of a bulk sample. Cells interact and create molecular networks, particularly in progressive disease models. Understanding these interactions at the single-cell level are important in molecular oncology for building models for cancer cell plasticity, therapeutic resistance, and differentiation studies. Cancer is a heterogeneic disease whereby successive and consecutive mutations build over time to enable cellular immortality (generally achieved by escaping cellular senescence). The ability to look at the genotype (Hybrid Fusion FISH) and phenotype (morphometric analysis) of a single cell in real-time is valuable.

Tracking Clonal Cell Populations

The ability to track clonality of cells can be achieved on a single-cell basis by tracking the genetic changes via Hybrid Fusion FISH. Identifying the genotype of a single cell can help to identify groups of cells within a homogenous population harboring the same genetic aberrations. This is useful to track clonality of cells, particular in cancer. Such changes and cell populations can be tracked over time. More importantly, the quantitative enumeration of each clonal population is an important tracking metric for new disease diagnosis’ and residual disease monitoring. With NGS, quantifying individual cells with retained morphology is not possible, as the cell is ruptured for DNA extraction and amplification. With Hybrid Fusion FISH, the entire cell is preserved and enumeration of both cells and independent genes is possible.

Identifying Genetic Tumor Heterogeneity of FFPE Tissues

Biopsied tissues are typically placed into 10% neutral-buffered formalin (NBF) for subsequent fixture into formalin-fixed paraffin-embedded (FFPE) tissue blocks. These blocks are grossed and serially sectioned onto microscope slides for further analysis by a pathologist. Such a process retains the tissue architecture, which is important to the pathologist for identifying glandular aspects of particular tissues (i.e. ductal morphologies of prostate and breast tissues). Glands within such tissues typically harbor the same clonal genotype, but it is possible to differentiate glands based on intra- and intertumor glandular heterogeneity. These can indicate particular stratification metrics such as biochemical recurrence and drug refractory periods. Hybrid Fusion FISH enables the preservation of the tissue cross-sections while assessing genetic variations between individual cells and glands. With NGS, bulk DNA is processed from the cross-section, which is a homogenous mixture of all cells (including those matrix cells which are not the test target which will dilute the analytical validity), in addition to yielding low-quality DNA due to the paraffin and formalin fixations.

Utilities for Liquid Biopsies

Liquid biopsies enable a non-invasive means of sample collection (typically peripheral blood) for the characterization of solid tumors by identifying target molecules within the liquid (suspension) sample indicative of tumor origin. For cancer genetics stratification, peripheral blood may contain circulating tumor cells (CTCs) and cell-free DNA (cfDNA) which can be analyzed for genes. Hybrid Fusion FISH enables characterization of whole CTCs in addition to other whole cells, whereas NGS enables sequencing analysis of cfDNA. Cell-free DNA is shed into the bloodstream as cells turnover (normal and malignant cells). Identification of CTCs requires morphology preservation such that those cells of interest can be identified for subsequent Hybrid Fusion FISH. Rare CTCs can be captured by targeted-cell enrichment followed by genetic analysis via Hybrid Fusion FISH. With NGS, sequencing can be done on the homogenous mixture of cfDNA or bulk whole cells that are subsequently lysed for DNA extraction. Therefore, Hybrid Fusion FISH and NGS can be complementary to this regard, as each technology analyzes a specific component of non-invasive liquid biopsies.

Cytogenetics Analysis of Chromosomes and Nuclei

The entirety of the human genome is in the form of DNA molecules that are intricately packed into the nucleus of each cell. DNA molecules form chromosomes, whereby humans possess 22 pairs of homologous (paired) chromosomes and 1 pair of sex chromosomes (XX for female or XY for male) for a total of 23 pairs of chromosomes. Specific genes are found within particular chromosomes. In the event of a chromosomal aberration, particular cancer genes may become oncogenic. Hybrid Fusion FISH aids in the visualization of chromosomal aberrations for cytogenetics analysis. NGS does not allow for this type of visual resolution at the macromolecular scale. Hybrid Fusion FISH allows for the spectral karyotyping (visual analysis of all 23 pairs of chromosomes) whereas NGS can sequence a bulk DNA sample extract. Whereas the resolution of DNA analysis is at the single nucleotide level for NGS, a Hybrid Fusion FISH karyotype can be done at the single-DNA molecule level for each chromosome. Such differences are important for cancer cytogenetics, particularly when cell culture is involved.

Bioinformatics, Analyses and Other Considerations

NGS requires a solid bioinformatics pipeline with computer-intensive resources to generate results. NGS typically utilizes a suite of associated software products for the resulting datasets which need to be analyzed. Hybrid Fusion FISH results are analyzed via colored (qualitative) dots (quantitative). The major informational value in NGS is on single-base changes and SNPs (single nucleotide polymorphisms). The major informational value in Hybrid Fusion FISH is on copy number variants (CNVs including amplifications and deletions), translocations (fusions), and the real-time tracking of clonal cell populations. “VUS” (variants of unknown significance) are commonplace on NGS pathology reports because those variants are still unknown. Hybrid Fusion FISH does not have VUS because very specific probes are utilized for target detection where the abnormalities are definitively linked to known clinical outcomes. The turn-around-time (TAT) for Hybrid Fusion FISH can be achieved in as little as 3–24 hours, whereas NGS results can take 5 days to 3 weeks to obtain an actionable end-result. Certain clinically actionable genes require rapid TATs (under 12 hours, i.e. PML/RARA, BCR/ABL) which can be readily accomplished by Hybrid Fusion FISH.

With Hybrid Fusion FISH, the resources and COGs (cost of goods) is very minimal (microscope slide, commonplace lab reagents/detergents, and sample cells). For NGS, specific reagents (primers and dNTPs), flow cells/cartridges, large capital equipment and intensive computational software may pose as a barrier for entry for most clinical labs. Additionally, some labs running clinical NGS utilize “parallel confirmation” methodologies as a protocol measure. NGS may be done by an orthogonal confirmational approach. For example, labs use multiple sequencing instruments (i.e. Illumina and Ion Torrent) in parallel to cross-reference the results for confirmation (Mayo Clinic). This is a resource-heavy approach. In a clinical reference lab, Hybrid Fusion FISH may be ordered with IHC and/or PCR to confirm results (gene to protein correlation). Additionally, the starting material is significantly different: Hybrid Fusion FISH requires a cell which has 6 picograms of DNA. NGS requires 5–50 nanograms of DNA as the starting material. This difference is 833–8,333-fold.

With respect to data storage, Hybrid Fusion FISH results are not data-intensive (“2R2G2Y2A” coding system for colored dots). NGS requires storage of the sequencing data — this requires hard drive space and computing power. In today’s market (Illumina) and at a 30x read coverage (average NGS coverage), the FASTQ files is ~200 GB. In the “perfect market”, where no short reads are created (which is not what NGS systems do), the entire human genome is approximately 715 MB (each diploid cell is about 1.5 GB of data). For variant files (only list of mutations), it is about ~125 MB which is the VCF file — the end-user will still need to preserve the raw data files. The resource intensity can be immediately appreciated.

Conclusion

Overall, NGS and Hybrid Fusion FISH technologies should be viewed as harmonious and complementary rather than competitive to each other. There are certain advantages for each technology and both can offer the pathologist a wealth of clinical value and actionable information. Both technologies will continue to evolve over time and present as viable clinical diagnostics in precision medicine. NGS and Hybrid Fusion FISH are two completely different techniques. Each has its own merits depending on the intended use and should be disease-specific, resource-efficient, and economically feasible for scaling to the masses.

LOOKING FORWARD

DISCLAIMER: This article is part 2 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