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At the intersection of cytogenetics and genomics lies the exciting field of cytogenomics. It focuses on studying chromosomes at a molecular scale, involving techniques that analyze either the whole genome or particular DNA sequences to examine variations in structure and behavior at the chromosomal or subchromosomal level. By integrating cytogenetic techniques with genomic analysis, researchers can effectively investigate chromosomal abnormalities related to diseases, particularly cancer.
Traditional Techniques
In order to comprehend the current landscape of cytogenomics, it’s important to reflect on classical methodologies that led to the basic understanding of chromosomal structures and their roles in diseases. Alka Chaubey, CMO at Bioanano, described several key techniques such as karyotyping (KT), fluorescence in situ hybridization (FISH), and chromosomal microarray (CMA). These techniques were highly influential in shaping the foundations of cytogenomic research and continue to be utilized today. Chaubey explained that KT and FISH remain primary recommendations for heme malignancies and are integral in current global guidelines. In addition, CMA became the standard of care due to its cost-effectiveness and was later recommended for postnatal and prenatal analyses.
“Only a few new technologies have been added to standard clinical practice in recent years,” noted Ivan Liachko, CEO and Co-Founder of Phase Genomics. Among the most notable Liachko described are array-based technologies, such as comparative genomic hybridization (CGH) in the 1990s, and SNP-based microarrays later on in the 2000s. “Even though they are expected to completely replace older technologies, such as KT or FISH, they are considered complementary technologies working hand-in-hand with established standard-of-care diagnostic tools.” He explained that this is also true for NGS, which provides important insights but requires older techniques to detect large-scale rearrangements.
While each of these foundational techniques has facilitated important research and diagnostics, Chaubey pointed out that they were limited in their resolution and scope. Eventually, newer tools were needed to provide more comprehensive coverage and higher-resolution results.
Technological Advancements
Proximity Ligation
Conventional NGS-based technologies have often faced challenges with the detection of important features like structural variants (SVs). However, newer approaches like the OncoTerra™ Oncology Platform offered by Phase Genomics have addressed these limitations. “The OncoTerra Oncology Platform offers a scalable, high-resolution approach to profiling of structural rearrangement in human genome samples,” stated Liachko. “It takes advantage of the unique strengths of ultra-long-range sequencing to unlock the wealth of diagnostic and prognostic information contained in a sample using a cost-effective and scalable NGS-based assay.”
This platform employs proximity ligation to capture various ranges of DNA contacts from tumor samples. They are converted into sequencing libraries, and the OncoTerra Oncology Platform then interprets the resulting sequencing data to detect chromosomal abnormalities. “Our sequencing technology allows us to identify the interactions between genomic loci that are co-located in three-dimensional space but may be separated by significant distances in the linear genome,” stated Liachko. “Structural variants change this organization and therefore are easily detected with OncoTerra.” In addition, Phase Genomics has developed an AI-based analysis platform capable of interpreting these genome maps and providing a list of structural variants.
Liachko further elaborated that OncoTerra provides a comprehensive genomic perspective on the structural changes, both balanced and unbalanced, that play a role in cancer development. This is achievable across a range of sample types without the need for specialized equipment. OncoTerra is compatible with widely used short-read sequencing instruments (e.g., Illumina and Element) and standard molecular biology tools commonly found in labs.
Emphasizing OncoTerra's proficiency, Liachko noted that it excels in overcoming challenges intrinsic to conventional NGS methods, especially when breakpoints are in regions of repetitive sequences. In this case, the ultra-long-range sequencing allows researchers to “jump” over repetitive elements involved in structural rearrangements and provide results where normal NGS technologies have routinely failed.
This method not only identifies genetic variants more effectively than karyotyping, FISH, and microarrays in a single test but also overcomes a major challenge associated with cytogenetic technologies: the need for live/un-fixed cells or high-molecular-weight DNA. Many oncology samples are fixed in formalin, rendering those requirements unattainable. In contrast, Liachko underscored OncoTerra’s ability to process FFPE-fixed samples, making it a seamless addition to the workflows of numerous diagnostic labs.
Optical Genome Mapping
Optical genome mapping (OGM) visualizes long DNA molecules to detect SVs and provide a comprehensive view of the genome. “OGM can detect all classes of large genomic aberrations (structural and numerical) in a single, genome-wide workflow that KT, FISH, and microarrays can assess, but with 10,000x higher resolution than KT can provide,” stated Chaubey. “Additionally, OGM can reveal SVs not detected by current methods, increasing pathogenic findings in both genetic disease and cancer.”
OGM provides a detailed view of genomic structures through the use of Bionano's imaging tools, Saphyr® and Stratys®. Bionano also offers the Ionic® Purification System, which is available now for use with NGS applications. This system uses isotachophoresis (ITP) to separate, cleanse, and concentrate genomic DNA and RNA from cells, tissues, and FFPE samples via Ionic G2 chemistry, and is currently being adapted for future use with ultra-high molecular weight DNA. Bionano also employs DNA isolation and labeling kits and leverages their Variant Intelligence Applications™ (VIA) software for essential data analysis and interpretation tasks.
Due to its simple workflow, high resolution, and level of SV detection (particularly for hematological malignancy subtypes), Chaubey explained that OGM has become a highly utilized replacement for older techniques. “The main advantage of OGM over traditional cytogenetic methods for SV detection, such as FISH, CMA, and KT, is that it makes the detection of SVs easy and efficient.” Large SVs of all major types can be detected at variant allele fractions of 5% or more using a singular, comprehensive procedure with significantly higher resolution.
Results from various studies have also exemplified that OGM is not only strongly consistent with traditional cytogenetic methods but has also refined and better defined the findings by legacy methods. “OGM can reveal novel SVs that are not identified by current methods due to the inherent limitations of each technology,” stated Chaubey. “These novel SVs may impact genetic disease and cancer research by identifying pathogenic findings and resolving cryptic cases and complex events like chromothripsis, with higher precision and resolution. OGM offers digital, automated analysis and calling of variants, and does not require cell culture.”
This technology is further enhanced with Bionano’s VIA, which consolidates various data types for automated variant calling, interpretation, and annotation, optimizing operations and reducing turnaround times. While current genomic data analysis solutions are tedious and often require multiple software products, Chaubey emphasized that VIA integrates secondary and tertiary analysis, processing data from OGM, NGS, and microarrays to provide comprehensive insights into genome variation.
Direct Applications
Ever since they were developed, these two methods have paved the way for meaningful scientific studies. OncoTerra, in particular, has been used to analyze a wide range of cancer samples, successfully deciphering complex karyotypes in conditions such as acute myeloid leukemia (AML), ovarian cancer, and various other tumor types. “In a recent study with the Fred Hutchinson Cancer Center in Seattle, we analyzed 48 AML samples that were previously characterized by traditional cytogenetic techniques,” stated Liachko. “Not only did our analysis lead to the reclassification of 25% of cases based on additional structural variants found with OncoTerra, but it also resulted in the discovery of a novel recurrent genomic variant with apparent therapeutic potential.” These research results have clinical significance as AML patient treatment is informed by risk scores based on structural variants.
The advancements in OGM have been notably highlighted in recent research, particularly in assessing its potential for myelodysplastic syndrome (MDS) prognostication1. “Researchers from The University of Texas MD Anderson Cancer Center reported that when OGM was used instead of karyotyping, 17 to 21% of study subjects had different prognostic risk scores and in 13% of study subjects additional pathogenic variants were revealed,” explained Chaubey. Another important study evaluated OGM’s effectiveness in detecting cytogenetic abnormalities in MDS and acute myeloid leukemia (AML) samples2. The findings underscored OGM's potential greater impact on characterizing AML over MDS, prompting recommendations for its integration into prognostic scoring for both malignancies. “In 33% of MDS samples and 54% of AML samples, the study reported more clinically relevant variants were detected using OGM than were found by traditional cytogenetic methods and these variants were reported to be highly relevant to the understanding of pathogenesis of these disorders,” Chaubey noted.
Recent breakthroughs in cytogenomics are characterized by the emergence of these advanced technologies. These tools are not only surpassing traditional methods by overcoming the hurdles researchers have long dealt with but also offer more comprehensive insights into complex diseases.
References
- Yang H, Garcia-Manero G, Sasaki K, et al. High-resolution structural variant profiling of myelodysplastic syndromes by optical genome mapping uncovers cryptic aberrations of prognostic and therapeutic significance. Leukemia. 2022;36(9):2306-2316. doi:https://doi.org/10.1038/s41375022016528
- Balducci E, Kaltenbach S, Villarese P, et al. Optical genome mapping refines cytogenetic diagnostics, prognostic stratification and provides new molecular insights in adult MDS/AML patients. Blood Cancer Journal. 2022;12(9):126. doi:https://doi.org/10.1038/s41408022007181