Abstract
For decades, the “death of cancer cytogenetics” has been repeatedly proclaimed and has grown louder in the era of omics. However, cytogenetics continues to evolve, retaining its central role in the study of chromosomes. From the microscopic visualization of numerical and structural aberrations using staining and banding techniques, the field has steadily incorporated new methodologies, including fluorescence in situ hybridization, array comparative genomic hybridization, high-throughput sequencing, and, most recently, optical genome mapping. These approaches have revealed unprecedented details of chromosome structure and behavior, uncovered catastrophic genomic events in cancer, and given rise to new concepts such as cytogenomics and chromosomics. Although cytogenetics is sometimes viewed as synonymous with chromosome banding, its scope, namely the study of chromosome structure and behavior, extends far beyond any single method. Chromosomes remain indispensable for understanding genome architecture, chromosomal instability, and fusion-gene mechanisms in neoplasia, irrespective of the technologies applied. Rooted in its past yet continually branching outwards with each scientific advance, today’s cytogenetics integrates classical and modern approaches, carrying forward the legacy of chromosome banding while adopting genome-wide technologies. These developments underscore the enduring importance of cytogenetics for interpreting the structural complexity of cancer genomes. This review aims to demonstrate that cytogenetics, far from being obsolete, remains a vital component of modern cancer research by highlighting essential insights into chromosome structure, genome architecture, and fusion-gene mechanisms in the era of -omics.
- Cancer cytogenetics
- chromosomal instability
- genome architecture
- fusion genes
- cytogenomics and chromosomics
- review
Introduction
For many years, at conferences, meetings, and in countless conversations with fellow scientists, the “death of cancer cytogenetics” has been repeatedly proclaimed. With the introduction of each new technology in cancer genetics, predictions emerged that cytogenetics would soon become obsolete. These forecasts have only grown louder over the past two decades, in the era of -omics, massively parallel sequencing (also known as next-generation or high-throughput sequencing), large-scale genomic data generation, and the rise of personalized medicine. Yassmine Akkari, Senior Director of the Clinical Laboratory at the Steve and Cindy Rasmussen Institute for Genomic Medicine, Nationwide Children’s Hospital, and Professor of Pathology at The Ohio State University College of Medicine, described a similar situation. She wrote: “For decades, cytogenetics has been declared dead. But for as long as I can remember, I have never understood this statement. In my mind, cytogenetics is the science of chromosomes – and how can a science die?” (1).
Zhang and colleagues (2) similarly noted: “Between 1950 and 1970, cytogenetics was stagnant because there were few advances in new techniques. There were suggestions that ‘cytogenetics is a dead science’ and that it was no longer needed because of advances in other disciplines of biology.”
Professor Thomas Liehr, founding editor and long-time Editor-in-Chief of Molecular Cytogenetics, has also encountered this notion. He recalls that when he entered the field in 1991, cytogenetics was already being dismissed as outdated and soon to be replaced by more modern approaches. He further noted that even his own supervisor, Professor Erich Gebhart (Erlangen, Germany), faced such skepticism when publishing his first articles in 1968 (3).
On the other hand, the College of American Pathologists, together with the American College of Medical Genetics and Genomics, provides and continues “to develop effective and timely proficiency testing in the practice of clinical cytogenetics, which includes both conventional and molecular cytogenetics and cytogenomics” (https://www.cap.org/member-resources/councils-committees/cap-acmg-cytogenetics-committee/). Likewise, the European Cytogeneticists Association continuous to organize courses in classical and molecular cytogenetics leading to a European diploma in clinical cytogenetics, which covers all aspects of the field in depth (https://www.e-c-a.eu/EN/default.asp). Such activities by these bodies clearly indicate the ongoing relevance of cytogenetics in both research and clinical practice.
This review aims to demonstrate that cytogenetics, far from being obsolete, remains a vital component of modern cancer research and diagnostics, and that chromosomal abnormalities still hold a central role in oncology, even in the era of omics and precision medicine.
Defining Cytogenetics: The Chromosomal Language of Life
In The Facts On File Dictionary of Biology, cytogenetics is defined as “the area of study that links the structure and behavior of chromosomes with inheritance” (4). In Henderson’s Dictionary of Biological Terms, cytogenetics is defined as “genetics in relation to cytology; the cytological aspects of genetics” (from the Greek kytos, meaning hollow, and genesis, meaning descent) (5). The Bantam Medical Dictionary offers a slightly broader definition: “a science that links the study of inheritance (genetics) with that of cells (cytology); it is concerned mainly with the study of chromosomes, especially their origin, structure, and functions” (6). Stedman’s Medical Dictionary for the Health Professions and Nursing defines cytogenetics as “the branch of genetics concerned with structure and function of the cell, especially the chromosomes” (7). The online Merriam-Webster Dictionary describes cytogenetics as “a branch of biology that deals with the study of heredity and variation by the methods of both cytology and genetics.” The National Human Genome Research Institute defines cytogenetics as “a branch of biology focused on the study of chromosomes and their inheritance, especially as applied to medical genetics.” It further explains in its glossary: “Cytogenetics is the study of chromosomes in any species” (https://www.genome.gov/genetics-glossary/Cytogenetics).
Many other dictionaries similarly describe cytogenetics as a branch of biology or genetics that merges cellular and hereditary perspectives. The term ‘cytogenetics’ emerged during the 1930s and 1940s, as the study of chromosome structure and behavior was increasingly integrated with the principles of Mendelian genetics. Although no single individual is credited with coining the term, it gained prominence through the work of early pioneers such as C.D. Darlington, who helped establish cytogenetics as a distinct scientific discipline (8). The field gained further significance in the late 1950s, following the discovery that the correct number of human chromosomes is 46 (9), and the subsequent identification of chromosomal abnormalities associated with human diseases, most notably, trisomy 21 in Down syndrome (10). Over time, cytogenetics has also become indispensable in other areas of biology, including evolutionary studies, developmental biology, and reproductive medicine. Its applications extend beyond human health to plant and animal breeding, species classification, and comparative genomics (11-13). Thus, while the term ‘chromosome’ belongs to the early, descriptive era of cell biology, ‘cytogenetics’ marks a conceptual shift toward understanding chromosomes as carriers of genetic function and as clinically relevant structures.
So, as Dr. Yassmine Akkari aptly asked: How can a science die?
Cytogenetic Methodologies and Periods: From Pre-banding to Cytogenomics and Chromosomics
Like every scientific discipline, cytogenetics relies on a range of methodologies that have evolved and expanded over time. These techniques have become increasingly sophisticated, largely due to advances in related technologies and adjacent fields. From the early use of basic staining techniques, the field progressed to chromosome banding methodologies, introduced in 1970-71, which became the gold standard techniques for several decades (14-17). These were followed by high-resolution banding, molecular cytogenetic tools such as fluorescence in situ hybridization and array comparative genomic hybridization, and, more recently, by high-throughput sequencing and optical genome mapping. Collectively, these advances have continually expanded the cytogenetic toolkit to meet evolving diagnostic and research challenges.
In 2002, Liehr and Claussen (18) proposed the division of cytogenetics into three historical periods, based on the central role of banding techniques: the pre-banding period, the pure banding period, and the molecular cytogenetics period. However, since then, major technological developments have transformed the field, enabling the investigation of chromosomes at submicroscopic resolution.
Recent advances, particularly high-throughput sequencing, single-cell analysis, and optical genome mapping, have led to the emergence of a fourth period, often referred to as the ‘cytogenomics’ or ‘chromosomics’ era (19-24). This phase is defined by the integration of cytogenetics with large-scale genomic and epigenomic data, enabling comprehensive analysis of structural chromosomal variations at unprecedented resolution. The term ‘chromosomics’ reflects this integrative approach, emphasizing not only the detailed characterization of chromosomal architecture, but also efforts to understand its functional implications in development, disease, and evolution (21, 22, 25). In this context, cytogenetics is no longer a stand-alone discipline, but a central component of multi-omics strategies, offering crucial insights into genome organization and its disruption in human disease.
The Pre-banding period. The pre-banding period of cytogenetics began, at least symbolically, in 1888, when the German anatomist Heinrich Wilhelm Gottfried von Waldeyer-Hartz coined the term chromosome, combining the Greek words chroma (χρώμα, meaning color) and soma (σώμα, meaning body) (26). This period continued until 1970, when chromosome banding techniques were introduced (27). During this time, chromosome preparations were typically stained using simple dyes such as Orcein or Feulgen, which produced a uniform coloration across chromosomes (28-31). As a result, individual chromosomes were only distinguishable by their relative size and centromere position, a method known as karyotyping, a term derived from the Greek karyon (κάρυον), meaning nut or nucleus, and typos (τύπος), meaning form or impression. This morphology-based classification system made it extremely difficult to detect subtle structural abnormalities, balanced translocations, or small deletions and duplications.
These technical developments occurred in parallel with important theoretical breakthroughs. The Boveri–Sutton chromosome theory of inheritance, proposed independently by Walter Sutton (1902-1903) and Theodor Boveri (1902), identified chromosomes as the physical carriers of genetic material, a unifying framework that linked cytology with Mendelian genetics (32-34). This theory laid the groundwork for modern cytogenetics by establishing that the behavior of chromosomes during meiosis corresponded with the laws of inheritance.
Boveri later extended his insights into the field of oncology. In 1914, he published “Zur Frage der Entstehung maligner Tumouren” (Concerning the Origin of Malignant Tumours), in which he hypothesized that cancer could arise from chromosomal abnormalities, specifically, unbalanced chromosomal complements that disrupted normal cellular regulation (33, 35). Although speculative at the time, Boveri’s ideas anticipated the chromosomal basis of cancer and are now recognized as visionary.
A key milestone of the pre-banding period was the correct determination of the human diploid chromosome number (9). Until the mid-1950s, it was widely believed that humans had 48 chromosomes, as unlit then, microscopes lacked the resolution to distinguish closely apposed chromosomes. In 1956, Joe Hin Tjio and Albert Levan, using improved techniques such as hypotonic treatment and better chromosome spreading, accurately established the human chromosome number as 46 (2n = 46), fundamentally revising our understanding of human genetics. This discovery laid the foundation for further standardization. In 1960, the Denver Conference proposed the first uniform system for naming and classifying human chromosomes (36). Chromosomes were grouped into seven categories (A-G) based on morphology, size and centromere position, and were assigned the now-standard numbering from 1 to 22, along with the X and Y chromosomes. This nomenclature system enabled more consistent karyotyping and interpretation, even in the absence of banding techniques.
Despite the technical limitations of the time, pioneering cytogeneticists were able to identify major numerical aberrations. Among the earliest and most significant discoveries were trisomy 21, responsible for Down syndrome (10), and abnormalities involving the sex chromosomes (37, 38). In 1959, Turner syndrome was described in individuals with a single X chromosome (45,X), and in the same year, Klinefelter syndrome was linked to the presence of an extra X chromosome in males (47,XXY). These findings not only demonstrated the relevance of chromosome number to human development and disease but also marked the beginning of clinical cytogenetics as a diagnostic field.
One of the most notable breakthroughs of this period was the discovery, in 1960, of a minute chromosome in the leukemia cells of patients with chronic granulocytic leukemia (now termed ‘chronic myeloid leukemia’) by Peter Nowell and David Hungerford (39, 40) (Figure 1A). They reported that this tiny chromosome replaced one of the four smallest autosomes in the chromosome complement of cultured peripheral blood cells and was absent from the normal chromosome constitution. This led them to propose a possible causal relationship between the observed abnormality and leukemia. This aberration, later designated the Philadelphia chromosome, represented the first cytogenetic link to cancer.
Detection of the Philadelphia chromosome/BCR::ABL1 fusion gene in the pre-banding, banding, and molecular cytogenetic periods. (A) Pre-banding period: “Mitotic cell and karyotype from marrow examined without culture from a patient with chronic granulocytic leukemia to show the Ph1 chromosome (small chromosome in the G group)” − original description by Conen. The left image corresponds to what is now referred to as a metaphase spread. Reproduced with permission from CMAJ Group. [Conen PE: Clinical conference on leukemia: Chromosome studies in leukemia. Can Med Assoc J 96: 1599-1605, 1967 (Figure 3)]. (B) Banding period: Philadelphia chromosome detected by G-banding. Left: metaphase spread. Right: corresponding karyogram. Courtesy of Kristin Andersen, Oslo University Hospital-Radiumhospitalet. (C) Molecular cytogenetic period: Detection of the Philadelphia chromosome/BCR::ABL1 fusion by fluorescence in situ hybridization (FISH) using the CytoCell BCR/ABL (ABL1) translocation, dual fusion FISH probe. Left: metaphase spread. Right: interphase nucleus. The ABL1 probe, red signal, hybridizes to chromosome 9, and the BCR probe, green signal, hybridizes to chromosome 22. Courtesy of Kristin Andersen, Oslo University Hospital-Radiumhospitalet.
In addition to the Philadelphia chromosome, several other important tumor-associated chromosomal abnormalities were identified during the pre-banding period. In 1963, Lele, Penrose and Stallard reported a visible deletion on the long arm of chromosome 13 in a patient with retinoblastoma, marking one of the first cytogenetic links between a constitutional chromosomal aberration and a solid tumor (41). That same year, Jacobs and colleagues published one of the earliest cytogenetic studies of Burkitt lymphoma, noting recurrent involvement of chromosome 8 in tumor cells (42). In 1965, Brewster and Garrett described chromosomal abnormalities in neuroblastoma, although the changes were nonspecific due to the limitations of the techniques available at the time (43).
These early findings, though limited in resolution, collectively demonstrated that both constitutional and acquired chromosomal abnormalities were involved in cancer biology. They provided initial insights into the cytogenetic basis of cancer development and diagnosis and laid the groundwork for the more detailed characterizations that would follow in the banding and molecular cytogenetics periods. In retrospect, these discoveries offered some of the first experimental support for Boveri’s 1914 hypothesis that cancer could result from chromosomal imbalances that disrupt normal cellular regulation. What had once been a visionary theory was now gaining empirical footholds.
The pure banding period. The introduction of chromosome banding techniques in 1970 marked the beginning of what is often referred to as the pure banding period (3, 18), representing the second major phase in human cytogenetics. Banding refers to the alternating light and dark patterns visible along chromosomes under light or fluorescence microscopy. These reproducible patterns, unique to each chromosome, allow for their precise identification and help significantly to improve the detection of both numerical and structural abnormalities.
The period began with Lore Zech’s introduction of Q-banding, a technique using quinacrine mustard that revealed reproducible fluorescence patterns along chromosomes under ultraviolet light (15, 27). In 1971, Seabright introduced a more practical and widely adopted alternative: trypsin-Giemsa staining for G-banding. This method produced comparable banding patterns, but could be visualized with standard light microscopy and did not require fluorescence (17). These innovations quickly became the gold standard in clinical cytogenetics, enabling consistent and accurate identification of chromosomal abnormalities with unprecedented resolution (44).
A major milestone of this period was the Paris Conference in 1971, which produced the influential document “Standardization in Human Cytogenetics” (45). This publication established a universal system for designating human chromosomes, chromosomal regions, and individual bands. It laid the foundation for the banding-based nomenclature that remains in use today and marked the beginning of what would become the International System for Human Cytogenetic/Cytogenomic Nomenclature (ISCN) (46) (see below, Evolution of the International System for Human Cytogenetic/Cytogenomic Nomenclature).
The pure banding period witnessed a surge in the discovery of recurrent chromosomal aberrations in cancer, many of which remain diagnostic and prognostic hallmarks today (47). One of the most groundbreaking findings came in 1973, when Janet Rowley demonstrated that the Philadelphia chromosome resulted from a reciprocal translocation between chromosomes 9 and 22, designated t(9;22)(q34;q11) (Figure 1B) (48). This built on Lore Zech’s earlier identification of the small marker as chromosome 22 using Q-banding (14). Banding techniques soon led to the recognition of other recurrent translocations, including t(8;21)(q22;q22) in acute myeloid leukemia, t(15;17) (q24;q21) in acute promyelocytic leukemia, t(11;14) (q13;q32) in mantle cell lymphoma, and t(14;18)(q32;q21) in follicular lymphoma (49).
Recurrent cytogenetic abnormalities were also identified in solid tumors, such as t(11;22)(q24;q12) in Ewing sarcoma, t(X;18)(p11;q11) in synovial sarcoma, t(2;13)(q35;q14) in alveolar rhabdomyosarcoma, del(11) (p13) in Wilms tumor, del(13)(q14) in retinoblastoma, and monosomy 22 in meningiomas (49). These discoveries provided the first consistent cytogenetic links between specific chromosomal rearrangements and distinct tumor types.
To organize and disseminate this rapidly expanding body of knowledge, Felix Mitelman compiled the “Catalogue of Chromosome Aberrations in Cancer”, first published in print during the banding period (50, 51). This pioneering reference systematically listed chromosomal rearrangements observed in human malignancies and became an essential resource for both researchers and clinicians. Over time, it evolved into the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer, which remains a key publicly available repository for cytogenetic and molecular data in oncology (52).
Concurrent advances in molecular genetics during the banding period laid the groundwork for molecular cytogenetics. Techniques such as restriction enzyme digestion (53), Southern blotting (54), and northern blotting (55) enabled the analysis of DNA structure and gene expression. Radioactive labeling of probes became routine in hybridization assays (56), and the introduction of Sanger sequencing in the late 1970s allowed for direct determination of nucleotide sequence (57). A pivotal resource was the 1982 publication of “Molecular Cloning: A Laboratory Manual” by Maniatis, Fritsch and Sambrook, which standardized and codified many of these protocols, quickly becoming the definitive reference for molecular biologists (58).
The application of these molecular tools to tumor samples with known cytogenetic abnormalities led to landmark discoveries, and by the end of 1987, eight unique fusion genes had been reported. In Burkitt lymphoma, rearrangements involving the MYC oncogene and immunoglobulin loci were identified, including IGH::MYC [t(8;14)(q24;q32)], IGK::MYC [t(2;8)(p12;q24)], and IGL::MYC [t(8;22)(q24;q11)] (59-62). In follicular lymphoma, the t(14;18)(q32;q21) translocation generated IGH::BCL2 (63), while the t(11;14)(q13;q32) led to IGH::CCND1 in B-cell chronic lymphocytic leukemia (64), a rearrangement that was later recognized as the genetic hallmark of mantle cell lymphoma. In chronic myeloid leukemia, the hallmark translocation t(9;22) (q34;q11) was shown to generate the BCR::ABL1 fusion gene (65, 66). In T-cell acute lymphoblastic leukemia, the t(8;14)(q24;q11) gave rise to TRA::MYC (67-69), while in T-cell lymphoma, the inv(14)(q11q32) generated IGH::TRA (70). These discoveries revealed that chromosomal rearrangements were able to activate oncogenes or produce fusion genes, driving malignant transformation, and underscored the emerging synergy between cytogenetics and molecular biology (49).
The pure banding period remained dominant until the mid-1980s, when molecular techniques began to redefine the field. In 1982, Langer-Safer et al. demonstrated DNA hybridization on polytene chromosomes using fluorescently labeled antibodies, laying the groundwork for fluorescence in situ hybridization (71). In 1986, Pinkel et al. successfully applied high-sensitivity fluorescence in situ hybridization to human metaphase chromosomes, marking the transition from the pure banding era to the molecular cytogenetics period (72).
Collectively, the achievements of the pure banding period transformed cytogenetics from a largely descriptive discipline into a powerful diagnostic and investigative tool. By enabling precise chromosomal identification and linking specific rearrangements to distinct malignancies, this era laid the essential cytogenetic and molecular foundations for modern cancer genetics.
The molecular cytogenetics period. The third major phase in human cytogenetics, known as the molecular cytogenetics period, began in the mid 1980s as a natural progression from banding-based analysis. It was marked by the development of fluorescence in situ hybridization and its first high-sensitivity application to human chromosomes using directly labeled DNA probes (72). This transition was driven by the need for greater resolution and gene-level specificity than chromosome banding techniques offered. Fluorescence in situ hybridization enabled the precise localization of DNA sequences on both metaphase chromosomes and interphase nuclei (Figure 1C) using fluorescently labeled probes, eliminating the hazards and limitations associated with radioactive methods.
A decisive advance of the early 1990s was the introduction of bacterial artificial chromosome (BAC) libraries (73). Compared with cosmids or yeast artificial chromosomes, BAC clones carry large human DNA inserts of up to 300 kb while remaining structurally stable, are easy to propagate in Escherichia coli, and avoid the frequent chimerism of yeast artificial chromosomes. These features made BAC clones highly reliable probes for fluorescence in situ hybridization, enabling precise chromosomal mapping and the detection of subtle aberrations undetectable by banding. BAC clones also provided the physical framework for the Human Genome Project and supported the development of locus-specific probes and whole-chromosome paints. Comprehensive BAC probe sets also provided the practical basis for the development of multiplex fluorescence in situ hybridization and spectral karyotyping (74, 75), which enables the simultaneous visualization of all chromosomes in distinct colors using combinatorial labeling and spectral imaging. Both methods significantly improved the detection of complex rearrangements, marker chromosomes, and cryptic structural aberrations, particularly in tumors with highly abnormal karyotypes (74, 76-78).
Another key development of the molecular cytogenetics period was the introduction of comparative genomic hybridization in 1992 (79). This technique enabled genome-wide detection of DNA copy-number changes without requiring specific probes for each locus or metaphase spreads from the test sample (79). In comparative genomic hybridization, differentially labeled tumor and reference DNA are co-hybridized to normal metaphase chromosomes, and gains or losses in tumor DNA are visualized as shifts in fluorescence intensity. Although limited by the resolution of metaphase spreads, this method represented a conceptual breakthrough, offering the first global approach to identifying chromosomal imbalances (80-82).
During the late 1980s and early 1990s, the adoption of new molecular genetic techniques played a pivotal role in accelerating the transition from the banding era to that of molecular cytogenetics (83). Among the most transformative innovations was the polymerase chain reaction (PCR), which enables rapid and specific amplification of DNA sequences from minimal amounts of genetic material (83-85). PCR quickly evolved into several powerful variations, including reverse transcription PCR for analyzing RNA, PCR-based Sanger sequencing, and rapid amplification of cDNA ends (RACE) for identifying unknown transcript termini (83-88). These methods provide a level of sensitivity and resolution far beyond that of chromosome banding or even early fluorescence in situ hybridization.
A landmark breakthrough during this period was the use of reverse transcription PCR to detect the BCR::ABL1 fusion transcript in chronic myeloid leukemia (87). This was the first demonstration that it was possible to confirm a fusion gene, previously inferred from a chromosomal translocation, directly at the RNA level, representing a major conceptual advance in leukemia diagnostics. It established fusion transcript detection as a clinically relevant molecular tool and was soon extended to other oncogenic fusions in leukemias and sarcomas. By linking cytogenetic observations to gene-level events, these developments effectively bridged classical cytogenetics and molecular biology, solidifying the foundations of the molecular cytogenetics era.
Consequently, during the 1990s, the field of cancer cytogenetics advanced rapidly through the combined use of chromosome banding and molecular techniques, both established and newly emerging. The structural insights provided by chromosome banding laid the groundwork for interpreting chromosomal rearrangements, while methods such as reverse transcription PCR, fluorescence in situ hybridization, and PCR-based Sanger sequencing enabled the detection and molecular characterization of fusion genes at unprecedented resolution. This integrated approach led to the identification of numerous clinically significant fusion genes, many of which have become essential diagnostic and prognostic markers (47). According to the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer, 167 unique fusion partner genes were identified during the period 1988-1999, underscoring the remarkable impact of molecular cytogenetic and early molecular methods in cancer gene discovery. Of these, 65 were discovered between 1988 and 1994, while 102 were identified in the subsequent five years (1995-1999), reflecting a marked acceleration in gene discovery during the latter half of the decade. This surge coincided with the broader utilization of emerging molecular technologies, including fluorescence in situ hybridization using BAC clones as probes, reverse transcription PCR, and Sanger sequencing, which enabled the detection and precise characterization of fusion genes at unprecedented resolution.
In leukemia, several discoveries during this period transformed classification and therapy. In acute myeloid leukemia, translocations such as t(8;21)(q22;q22)/RUNX1::RUNX1T1 and inv(16)(p13q22)/CBFB::MYH11 defined new cytogenetic subtypes (89-91), while the identification of PML::RARA from t(15;17)(q24;q21) in acute promyelocytic leukemia revolutionized therapy through the introduction of all-trans retinoic acid (92, 93). In acute lymphoblastic leukemia, the cryptic t(12;21) (p13;q22) remained invisible to chromosome banding and was first uncovered by fluorescence in situ hybridization, leading to the identification of the ETV6::RUNX1 fusion, the most common genetic lesion in childhood acute lymphoblastic leukemia, associated with an excellent prognosis under current treatment protocols (47, 94-97). Another major advance was the identification of KMT2A at 11q23 as a central gene in leukemia pathogenesis (98, 99). Rearrangements of KMT2A involve more than 90 partner genes across acute myeloid leukemia, acute lymphoblastic leukemia, infant, and therapy-related leukemia (100-102). Together, these breakthroughs underscored how molecular cytogenetics not only expanded the catalog of fusion genes but also reshaped classification, prognosis, and treatment.
In sarcomas, cytogenetic–molecular correlations likewise transformed diagnosis and classification. The recurrent t(11;22)(q24;q12)/EWSR1::FLI1 defined Ewing sarcoma (103), while t(12;16)(q13;p11)/FUS::DDIT3 was recognized as the hallmark of myxoid liposarcoma (104). In alveolar rhabdomyosarcoma, t(2;13)(q35;q14)/PAX3::FOXO1 and the variant t(1;13) (p36;q14)/PAX7::FOXO1 emerged as critical diagnostic markers (105, 106). Synovial sarcoma was characterized by SS18::SSX fusions from t(X;18)(p11;q11), providing a unifying genetic signature for histologically variable cases (107-109). These discoveries not only clarified tumor classification but also established molecular criteria that remain central to the diagnosis of pediatric and soft tissue sarcomas (47).
A central insight of this period was the recognition that some genes appeared repeatedly as fusion partners, giving rise to the concept of ‘promiscuous’ partners in fusion genes in cancer (47). In hematological malignancies, prominent examples of 5′ fusion partners included KMT2A (formerly MLL), ETV6, and NUP98, as well as the immunoglobulin loci (IGH, IGK, and IGL). As 3′ fusion partners, recurrent examples included BCL6, RARA, and ALK together with T-cell receptor loci (TRA, TRB, and TRD). In solid tumors, EWSR1 emerged as the paradigmatic promiscuous 5′ partner in mesenchymal tumors, and RET as a recurrent 3′ partner in thyroid carcinomas. These patterns revealed that certain genes were repeatedly involved in chromosome aberrations such as translocations, reflecting intrinsic features of their regulatory context or protein domains that predisposed them to recurrent involvement in oncogenic pathways. Several genes first identified in this period, including ALK, KMT2A, RET, ETV6, RUNX1, EWSR1, NTRK1, PDGFRB, PLAG1, and NTRK3, are now recognized as promiscuous fusion partners across many tumor types (47, 52). A systematic comparison of the genes found as fusion partners between the 1988-1999 period and the full dataset of the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (updated on July 10, 2025) illustrates the dramatic expansion of these genes, underscoring both their central role in the biology of fusion-driven neoplasia and the immense progress made in their identification and characterization (Table I).
Promiscuous fusion genes identified in the full dataset of the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (updated to July 2025). For each gene, the table lists the number (count) and distinct fusion partner genes. The Increase column indicates the net gain in unique fusion partners compared with those of 1988-1999 (underlined).
A further important observation from this period was that it was possible to identify the same fusion gene and its associated cytogenetic aberration in different types of neoplasms. The recurrent ETV6::NTRK3 fusion gene, resulting from t(12;15)(p13;q25), was first described in infantile fibrosarcoma (historically termed congenital fibrosarcoma) (110) and in congenital mesoblastic nephroma (111, 112), and subsequently in acute myeloid leukemia carrying complex chromosomal aberrations involving chromosome bands 12p13, 12q15, and 15q25 (113). Nowadays, the ETV6::NTRK3 fusion gene is recognized across a wide spectrum of neoplasms, including acute lymphoblastic leukemia, adenocarcinomas arising in multiple organs, tumors of the central nervous system, melanoma, and many others (114). Over time, fusion-gene promiscuity has been documented for EWSR1::ATF1/CREB1, YAP1::TFE3, ACTB/MALAT1/PTCH1::GLI1, and others (114). This demonstrated that certain fusion genes are not confined to a single histogenetic tumor type but can act as versatile oncogenic drivers across mesenchymal, epithelial, and hematological tumors (114).
Another important realization of this period was the interchangeability of members of the same gene family as fusion partners, although not always within the same tumor type. This was exemplified by FUS, EWSR1, and TAF15, which together constitute the FET/TET gene family and show overlapping partnering patterns with certain oncogenes (47). For instance, EWSR1::DDIT3 and FUS::DDIT3 were both found in myxoid liposarcomas, whereas EWSR1::NR4A3 and TAF15::NR4A3 characterized extraskeletal myxoid chondrosarcomas. Similarly, EWSR1 fused with ERG in Ewing sarcoma, while FUS::ERG was identified in acute myeloid leukemia and later also in Ewing sarcoma (115-117). These examples highlighted that related family members are able to substitute for one another in oncogenic fusion events, underscoring a broader principle of functional redundancy among certain RNA-binding proteins in tumorigenesis.
Beyond diagnostics and tumor classification, the association between chromosomal aberrations and fusion genes ushered in a therapeutic revolution. The culmination of this period was the development of imatinib, the first tyrosine kinase inhibitor in oncology (118). This landmark therapy was the direct harvest of decades of work that began with the discovery of the Philadelphia chromosome in 1960, advanced through the identification of t(9;22)(q34;q11) and the BCR::ABL1 fusion, and ultimately led to the rational design of a drug that selectively inhibited the oncogenic kinase (118, 119). Imatinib transformed chronic myeloid leukemia from a rapidly fatal disease into a manageable condition with long-term survival, becoming the first true paradigm of targeted cancer therapy (120, 121). Its success not only validated the central role of fusion genes in cancer biology but also opened the door to the development of targeted therapies against other kinase fusions. The breakthrough of imatinib was so profound that, as Dan Longo later summarized in an editorial in the New England Journal of Medicine, “imatinib changed everything” (122).
The molecular cytogenetics period marked a turning point in the evolution of cancer cytogenetics. By integrating chromosome banding with emerging molecular techniques, researchers were finally able to investigate chromosomal rearrangements not only at the structural level but also at the gene and transcript level. This convergence enabled a deeper understanding of oncogenic mechanisms, more accurate tumor classification, and improved diagnostic precision, laying the groundwork for modern cancer diagnostics. The foundations established during this period set the stage for the genome-wide, high-resolution approaches that would characterize the next era: Cytogenomics. A new chapter was about to begin.
Cytogenetics in the New Millennium: A New Era for the Study of Chromosome Structure and Behavior
At the dawn of the 21st century, cytogenetics entered a transformative new phase, fundamentally redefining how scientists study chromosome structure, function, and behavior. This transition was catalyzed by the availability of the human genome sequence, the rise of advanced informatics tools, and the convergence of cytogenetics with genome-wide molecular technologies. No longer confined to the visible alterations captured by banding techniques, the field embraced a sequence-informed framework that enabled the high-resolution detection and interpretation of chromosomal abnormalities. This integrated approach became known as cytogenomics.
The term cytogenomics (or cytogénomique, in its original French usage) was first introduced by Alain Bernheim in the context of cancer research in 1999 (123). It began appearing in English-language literature around 2008 (124, 125), gaining traction as an umbrella term for the integration of chromosome banding with genomic technologies such as fluorescence in situ hybridization, comparative genomic hybridization, DNA fiber analysis, high-throughput sequencing, and computational genomics. This approach enabled researchers and clinicians to study chromosome structure and function at both cytological and molecular levels, advancing precision diagnostics and targeted therapy. As Speicher and Carter noted, it “blurs the boundaries with molecular biology,” earning it the designation ‘new cytogenetics’ (126).
Closely related, although conceptually broader, is the term ‘chromosomics’, introduced by Claussen in 2005 (25). Chromosomics expands the scope of cytogenetic investigation beyond sequence and structural analysis to include the dynamic plasticity of chromosomes within the interphase nucleus. It emphasizes the three-dimensional organization of the genome, chromatin remodeling, and epigenetic modifications that collectively shape gene regulation, cellular differentiation, and disease phenotypes. More recently, Liehr has emphasized chromosomics as the study of how nuclear organization and chromatin-mediated processes influence cellular function, disease susceptibility, and human evolution (21). In parallel, Deakin and colleagues proposed chromosomics to be a unifying framework that integrates genome sequencing, cytogenetics, cell biology, and bioinformatics, positioning it as a powerful approach to addressing unresolved questions in genome architecture and regulation (127).
Although cytogenomics has become the prevailing term for integrated genomic and cytogenetic analysis in both clinical and research settings, chromosomics offers a complementary perspective that unites the structural, spatial, and regulatory dimensions of chromosomes in both basic and translational research. Whether through the lens of cytogenomics or chromosomics, these frameworks have reshaped the field, laying the groundwork for modern precision diagnostics and ushering in a new era in the study of chromosomes.
These new conceptual frameworks, cytogenomics and chromosomics, were greatly accelerated by the release of the first draft of the human genome in 2001, a landmark event that transformed biomedical research (128, 129). For the first time, it became possible to explore the human genome at base-pair resolution, allowing chromosome bands, previously defined solely by cytological features, to be linked to precise DNA sequences and genomic coordinates (130). The emergence of freely available tools such as the University of California, Santa Cruz, Genome Browser (131), Ensembl (132), the database resources of the National Center for Biotechnology Information (133), and other public genomic databases has enabled researchers and clinicians worldwide to integrate cytogenetic findings with detailed molecular data. Genes were increasingly being identified and precisely mapped to chromosomal bands, assigned genomic positions, and annotated with their transcriptional orientation, indicating whether they are located on the plus or minus strand of the chromosome.
This period also saw widespread access to BAC clone libraries, which contained well-characterized, long DNA inserts (134-138). These BACs, often fully sequenced or end-sequenced, became essential tools in fluorescence in situ hybridization applications, enabling highly specific, sequence-anchored probe design (139). This development allowed researchers to map chromosomal breakpoints with greater precision, characterize copy-number changes at the molecular level, and build custom probe sets targeting specific genomic regions. It became possible for fluorescence in situ hybridization, which once relied on chromosomal morphology for probe positioning, to be anchored to genome sequence coordinates, further strengthening the fusion of cytogenetics and genomics.
Building on the availability of BAC clones anchored to the genome, the early 2000s saw the development of array-based comparative genomic hybridization platforms, initially constructed with BAC clones spotted onto glass slides (140, 141). These BAC arrays enabled the first genome-wide assessment of DNA copy-number changes at submicroscopic resolution, representing a significant improvement over conventional karyotyping and metaphase comparative genomic hybridization. In cancer research, this approach proved particularly impactful. BAC array comparative genomic hybridization was instrumental in revealing segmental gains and losses in tumors that appeared karyotypically normal or highly complex, including in gliomas, breast cancer, neuroblastoma, and soft tissue sarcomas (142-146).
The use of tiling-path arrays, in which overlapping BAC clones covered entire chromosomal regions or even whole genomes, allowed for the fine-mapping of chromosomal breakpoints, the identification of recurrent minimal regions of gain or loss, and the detection of novel amplified oncogenes or deleted tumor suppressors. These tools led to a deeper understanding of genomic instability in cancer and established new diagnostic and prognostic biomarkers. Clinically significant findings, using BAC array comparative genomic hybridization, include amplification of MYCN, located on chromosome sub-band 2p24.3, in neuroblastoma; amplification of ERBB2 (also known as HER2), located on 17q12, in breast cancer; and co-deletion of chromosome arms 1p and 19q (referred to as the 1p/19q co-deletion) in oligodendrogliomas (142, 144-149).
As the technology matured, BAC arrays were gradually replaced by oligonucleotide arrays with much higher probe density and resolution, enabling the detection of microdeletions, microduplications, and complex genomic rearrangements across the genome (150-153).
In parallel, single nucleotide polymorphism (SNP) arrays were developed to assess both copy-number variation and allelic imbalances that are not detectable by conventional array comparative genomic hybridization or G-banding (154-156). In acute myeloid leukemia and myelodysplastic syndrome, they revealed uniparental disomy with diagnostic and prognostic significance (153, 157). Similarly, in acute lymphoblastic leukemia, SNP arrays identified microdeletions as a common feature in adult and adolescent cases, highlighting unexpected similarities to the pediatric form of the disease (158).
These advancements culminated in the development of combined oligonucleotide/SNP array platforms, such as Affymetrix CytoScan HD, Oxford Gene Technology CytoSure Cancer+SNP arrays, and Illumina Infinium CytoSNP-850K, which enable the simultaneous detection of copy-number changes and copy-neutral genomic alterations on a single chip (159, 160). Their integration into routine diagnostics has provided high-resolution, genome-wide evaluation of structural variation at submicroscopic resolution, uncovering clinically relevant aberrations invisible to traditional karyotyping or array comparative genomic hybridization (153, 157, 158, 161).
Thus, at the beginning of the millennium, cytogenetics was no longer confined to the analysis of visible chromosomes under the microscope but had evolved into a sequence-informed discipline capable of interpreting genomic architecture at the kilobase scale and even at the nucleotide level. The combination of high-resolution array platforms with genome assembly data brought unprecedented precision to the detection and characterization of chromosomal abnormalities. This transformation laid the foundation for integrating array-based data with sequencing technologies, paving the way for the next phase in the field’s evolution, the era of cytogenomics powered by high-throughput sequencing (162-164).
The emergence of high-throughput sequencing, also referred to as next-generation sequencing, massively parallel sequencing, or deep sequencing, extended chromosome analysis from the submicroscopic to the nucleotide level (Figure 2). This technology enabled comprehensive and unbiased interrogation of both the genome and transcriptome, expanding the scope of cytogenomic investigations beyond the capabilities of array-based methods. Pioneering efforts in cancer genomics demonstrated its power to detect somatic mutations, copy-number changes, structural variants, and fusion genes in tumor samples (162-164). It can reveal complex rearrangements, sub-clonal alterations, and cryptic events, all within a single assay, surpassing both the resolution and scope of prior technologies. Depending on whether DNA or RNA is used as template, high-throughput sequencing can be applied as whole-genome sequencing for unbiased structural variant detection, whole-exome sequencing for mutation analysis within coding regions, and whole-transcriptome sequencing (RNA sequencing or RNA-seq) for transcript-level profiling. The latter includes the detection of fusion transcripts, alternative splicing, gene expression patterns, and allele-specific expression (162, 164-168). High-throughput sequencing has been particularly impactful in oncology, enabling more accurate tumor classification, risk stratification, and identification of actionable genomic targets.
Detection of the Philadelphia chromosome/BCR::ABL1 fusion gene in the new millennium using high-throughput sequencing. In this period, cytogenetics expanded into cytogenomics and chromosomics, extending analysis far beyond chromosome subbands such as 22q11.23 and 9q34.12 to provide nucleotide-level and functional insights. High-throughput sequencing analysis performed with the Arriba algorithm, using the test dataset included with the STAR-Fusion distribution, identified breakpoints at BCR (chr22: 23290413, ENST00000305877.13) and ABL1 (chr9: 130854064, ENST00000372348.9), generating an in-frame BCR::ABL1 transcript. The translocation is visualized in a circos plot as a connecting line between chromosomes 22 and 9. Retained protein domains include the BCR-ABL oligomerization, RhoGEF, PH, C2, F-actin binding, kinase, SH2, and SH3 domains. Supporting evidence comprised 5 split reads at breakpoint 1, 8 split reads at breakpoint 2, and 10 discordant read pairs.
As sequencing costs declined and bioinformatic tools matured, high-throughput sequencing rapidly became integral to both cancer research and clinical diagnostics. Its implementation enabled a shift from targeted or karyotype-based assessments to genome-wide profiling, accelerating the discovery of novel driver events and refining the molecular classification of tumors. Today, depending on the available material, a combination of chromosome banding, fluorescence in situ hybridization, array comparative genomic hybridization, and high-throughput sequencing methodologies is employed to comprehensively investigate the genetic landscape of cancer. In many tumor types, the combined use of chromosome banding and whole transcriptome sequencing is especially effective for identifying potential driver fusion genes (169). These two methodologies operate at different levels of genomic organization: Karyotyping visualizes large-scale chromosomal structure, while whole-transcriptome sequencing captures transcript-level changes. When a fusion transcript detected by whole transcriptome sequencing aligns with a cytogenetically visible breakpoint, it provides strong evidence of biological relevance. This approach was first used to identify the recurrent WWTR1::CAMTA1 fusion gene in epithelioid hemangioendothelioma with the translocation t(1;3)(p36;q25) (170). A similar strategy was later applied to identify the IRF2BP2::CDX1 fusion in mesenchymal chondrosarcoma carrying a t(1;5) (q42;q32) chromosome aberration (171), to demonstrate that ZC3H7B::BCOR is a recurrent chimera in endometrial stromal sarcomas with an X;22 translocation (172), and to identify the SERPINE1::FOSB fusion in pseudomyogenic hemangioendothelioma harboring a balanced t(7;19) (q22;q13) (173). This combinatorial strategy has since been widely adopted to uncover novel, clinically relevant fusion genes in tumors with simple karyotypic changes, including the identification of NFIA::CBFA2T3 and ZMYND8::RELA in pediatric erythroid leukemias (174, 175).
High-throughput sequencing has also enabled the discovery of clinically significant fusion genes that were undetectable by chromosome banding. In leukemia, the CBFA2T3::GLIS2 fusion, associated with a cryptic inversion of chromosome 16, inv(16)(p13.3q24.3), was identified solely through high-throughput sequencing (176). This fusion defines a distinct subtype of leukemia occurring exclusively in children and has never been reported in adults (177). The IGH::DUX4 fusion, resulting from a translocation involving 14q32 and 4q or 10q that cannot be visualized by karyotyping, was readily identified by whole-transcriptome sequencing and defines a high-risk subtype of B-cell acute lymphoblastic leukemia with distinct gene expression and immunophenotypic features (178-180).
High-throughput sequencing revealed recurrent rearrangements of the PRDM10 gene (11q24.3) in low-grade undifferentiated pleomorphic sarcomas. These tumors exhibit a distinct gene expression profile and indolent clinical behavior, supporting their recognition as a separate sarcoma subtype (181-184). In another group of sarcomas, sequencing revealed rare but recurrent fusion genes such as YAP1::KMT2A::YAP1 and VIM2::KMT2A (185-188). These fusions expand the spectrum of KMT2A-driven neoplasia beyond hematological malignancies. Although infrequent, their recurrence suggests a pathogenetic role and underscores the power of high-throughput sequencing in uncovering cryptic oncogenic drivers in mesenchymal tumors.
An emerging addition to the cytogenomic toolbox is optical genome mapping, a high-resolution technique that enables genome-wide detection of structural variants without sequencing. Optical genome mapping works by labeling ultra-high-molecular-weight DNA and imaging individual molecules as they are linearized through nanochannels, generating detailed genome maps that identify insertions, deletions, duplications, inversions, and translocations at kilobase resolution (24). It has also proven to be a valuable tool for detecting catastrophic genomic events collectively referred to as chromoanagenesis (see below). These complex rearrangements often evade detection by conventional cytogenetic methods due to their localized and chaotic nature. Accordingly, optical genome mapping has been described as a next-generation cytogenetics or next-generation cytogenomic tool (189, 190).
Optical genome mapping has shown particular utility in hematological malignancies, where it can detect cryptic rearrangements, fusion genes, and complex structural variants that are not visible by traditional karyotyping (189, 191-197). It has already been applied to the diagnosis and risk stratification of both myeloid and lymphoid neoplasms (192, 194, 198, 199). In a recent study of 12 BCR::ABL1-positive cases, optical genome mapping was compared with chromosomal analysis, fluorescence in situ hybridization, and reverse transcription PCR (Figure 3). Optical genome mapping revealed additional abnormalities including chromoanagenesis and IKZF1 deletion, leading the authors to conclude that “in scenarios where optical genome mapping is feasible, chromosomal analysis and reverse transcription polymerase chain reaction may not offer additional diagnostic value” (194).
Detection of the Philadelphia chromosome/BCR::ABL1 fusion gene using the latest cytogenomic tool, optical genome mapping (OGM), which enables direct visualization of structural variants across ultra-long DNA molecules. This technology complements sequencing-based approaches by providing genome-wide, high-resolution maps of chromosomal rearrangements. The molecule map from the sample (blue) aligns to chromosome 9 (Ref 9, top) and chromosome 22 (Ref 22, bottom), with vertical connections visualizing the translocation breakpoints. The fusion involves ABL1 on chr9q34.12 (~130.8 Mb, hg38) and BCR on chr22q11.23 (~23.2 Mb, hg38). Courtesy of Dr. Gokce A. Toruner, MD Anderson Cancer Center.
To support clinical integration, an international consortium for optical genome mapping in hematological malignancies has issued expert recommendations for its implementation in routine diagnostics (200, 201). Furthermore, the standing committee for the ISCN recently recognized the need for a specific nomenclature tailored to genome mapping technologies. A standardized framework was proposed to accurately report structural and copy-number abnormalities detected by this technology in both research and clinical settings (202).
Optical genome mapping has also been applied to bone and soft-tissue tumors, despite challenges in obtaining high-quality DNA from these specimens (203-205). In one study, it succeeded in 38 out of 53 tumors (72%) and identified clinically relevant alterations in 91% of analyzable cases, including POU2AF3::EWSR1 in a round cell sarcoma and t(1;5)(p22;p15) in a myxoinflammatory fibroblastic sarcoma, as well as complex genomic events (203). Another study reported successful analysis in 62% of cases (33 of 53 cases), detecting diagnostic alterations in 95.2% of evaluable samples, including novel oncogenic events and tumor-suppressor gene alterations (204). A third investigation successfully analyzed all 60 examined tumors, with optical genome mapping accurately identifying structural and copy-number variants previously detected by karyotyping and fluorescence in situ hybridization, while also revealing complex rearrangements and three- to six-way translocations that were not detectable by karyotyping (205). A combination of optical genome mapping and high-throughput sequencing identified diagnostic, disease-associated aberrations in 98% of cases.
The use of optical genome mapping has also been reported in carcinomas (206, 207). In metastatic lung squamous cell carcinoma, integration with whole-genome sequencing revealed intratumoral heterogeneity, with optical genome mapping detecting structural variants missed by sequencing and variants restricted to metastases (206). In clear-cell renal cell carcinoma, optical genome mapping combined with epigenome profiling enabled comprehensive characterization of tumor and adjacent tissue, uncovering both known and novel somatic aberrations (207).
Optical genome mapping has also been applied to brain tumors. In gliomas, it showed concordance with SNP array results while uncovering additional clinically significant aberrations that escape detection by SNP array (208). In pediatric PATZ1-rearranged gliomas, the combination of optical genome mapping and chromosome microarray analysis indicated that chromothripsis of chromosome 22 was responsible for generating the PATZ1 fusion (209). In another pediatric brain tumor, optical genome mapping identified a rare ZNF532::NUTM1 fusion (210).
Despite its strengths, optical genome mapping has some limitations. It may fail to detect certain abnormalities that are identifiable by other methods. For example, a near-tetraploid subclone was detected only by karyotyping (191). False-positive and false-negative results have also been reported, particularly when breakpoints fall within large repetitive regions or when inconsistencies arise between genome assemblies such as GRCh37 and GRCh38 (211).
Nevertheless, optical genome mapping is increasingly viewed as a valuable complement to traditional cytogenetic methods, such as karyotyping and fluorescence in situ hybridization. In some settings, it may even serve as an alternative, particularly when high-resolution structural analysis is required. Already in 2021, it was proposed as a promising substitute for standard cytogenetic approaches in acute lymphoblastic leukemia (191). More recently, concerns about a shrinking workforce and diminishing expertise in G-banding karyotyping have prompted the consideration of this method as a potential solution for the next generation of cytogenomic diagnostics (212).
Together, the technologies introduced in the New Millennium have not altered the core of cytogenetics, the science devoted to the study of chromosomes, their structure, origin, and function, but they have vastly expanded its methodological reach. By moving beyond the limitations of banding patterns and light microscopy, modern cytogenetic tools now permit the detection of rearrangements invisible to earlier techniques, the reconstruction of derivative chromosomes shaped by complex or catastrophic events (see below), the identification of complete or partial gene alterations such as gains and losses, and even the mapping of nucleotide-level changes, such as insertions, deletions, or substitutions, onto chromosomal coordinates. Cytogenetics has thus evolved into a truly integrative discipline that lies at the intersection of chromosomal architecture, molecular function, and genomic sequence. It remains firmly rooted in its foundational principles yet has been redefined by the transformative power of contemporary genomic technologies.
More Greek Words in the Field of Cytogenetics
The explosive growth of genome analysis in cancer research over the past decade has led to the introduction of several striking new terms, all rooted in Greek, to describe complex genome rearrangements and mutational processes. All of these terms, with the exception of kataegis (see below), begin with the prefix chromo-underscoring their close association with chromosomes and cytogenetics.
These newer terms reflect a conceptual shift in cytogenetics from gradual models of genome evolution to sudden, catastrophic, and mosaic changes that shape cancer genomes and drive oncogenic processes, including the generation of fusion genes (213-218).
Chromothripsis. First described in 2011, chromothripsis, a compound of chromo- (chromosome) and the Greek thripsis (θρύψις, shattering), refers to a phenomenon in which chromosomes undergo massive fragmentation and chaotic reassembly in a single catastrophic event (213). In a seminal study of bone cancer and leukemia, localized clusters of tens to hundreds of chromosomal breakpoints suggested that entire chromosomes or chromosomal arms can shatter and subsequently be rejoined through error-prone repair mechanisms such as non-homologous end joining (213). Thus, a new paradigm in cancer genome evolution was introduced, challenging the traditional view of tumor progression as a gradual process: punctuated bursts of genomic chaos are capable of driving oncogenesis in a single step. Formal detection criteria were later established, including clustered breakpoints, oscillating copy-number states, and random fragment joins (219).
Large-scale analyses have identified chromothripsis in osteosarcomas, brain tumors, and certain leukemia types, often associated with TP53 inactivation and poor prognosis, highlighting the vulnerability of cells with compromised genome surveillance (220-222). In prostate cancer, chromothripsis has generated complex multi-gene fusions (223). In leukemia, it contributes to recurrent fusions and complex karyotypes, facilitating rapid disease progression and resistance to treatment. The catastrophic restructuring of chromosomes can produce deletions, duplications, and gene fusions simultaneously, dramatically altering the cellular transcriptome in a single event (224-228). Chromothripsis has also been linked to the formation of extrachromosomal DNA, which amplifies oncogenes and promotes rapid tumor evolution and therapy resistance (229).
In pediatric cancer, chromothripsis is increasingly recognized as a major driver of genomic complexity and fusion-gene formation. In central nervous system tumors, it contributes to fusions such as C11orf95::RELA in ependymomas (230) and complex rearrangements involving PATZ1 fusions (209, 231). In a pediatric undifferentiated small round cell neoplasm, an HNRNPM::LEUTX fusion arose through chromothripsis of chromosome 19 (232). Similar mechanisms have been implicated in lipoblastomas involving the PLAG1 gene (233) and in the formation of PAFAH1B1::USP6 fusions in periosteal solid aneurysmal bone cysts (234).
Overall, chromothripsis exemplifies a ‘punctuated’ model of genome evolution, contrasting with the gradualistic view that dominated classical cytogenetics. It highlights the dynamic and unpredictable nature of chromosomal instability, and the ability of cancer cells to acquire multiple oncogenic alterations in a single step. As sequencing and cytogenomic technologies continue to advance, our ability to detect and interpret chromothripsis will further elucidate its roles in cancer initiation, progression, and therapeutic resistance.
Chromoanasynthesis. First described in 2011, chromoanasynthesis, from chromo- (chromosome) and the Greek anasynthesis (ανασύνθεσις, meaning reconstitution or rebuilding), refers to a replication-based mechanism leading to complex chromosomal rearrangements (235). Chromoanasynthesis involves errors during DNA replication and repair, resulting in local duplications, triplications, and complex segmental rearrangements (236).
Accumulating data indicate that chromoanasynthesis occurs in human germline cells and during early embryonic development (236). It is characterized by clustered copy-number gains (including segmental duplications and triplications), microhomology at breakpoint junctions, and frequent complex chromosomal structures such as inverted duplications. These features reflect replication stress and errors in template switching rather than repair of double-strand breaks alone.
Replication stress can arise from various factors, including oncogene activation, DNA secondary structures, or difficult-to-replicate regions. Under such stress, the replication machinery may stall or collapse, forcing the cell to use alternative, error-prone pathways to complete DNA synthesis. During this process, segments of DNA may be aberrantly duplicated or rearranged through template switching, leading to chromoanasynthesis.
Righolt and Mai highlighted chromoanasynthesis alongside chromothripsis as a manifestations of ‘chromosome crises’ that contribute to cancer genome complexity (215). Multiple chromoanasynthesis events have been identified in a case of renal leiomyosarcoma, including in the 12q13.11-q21.2 region, which harbors crucial cell-cycle genes such as MDM2 and CDK4. Rearrangements in this region was speculated to trigger further chromosomal instability, including events in 6q21-q27 and 7p22.3-p12.1 (237).
Chromoanasynthesis has also been reported as a common mechanism underlying ERBB2 gene amplifications in early-stage ERBB2-positive breast cancers (238). Extensive local duplications and complex rearrangements can increase oncogene dosage, promoting tumor progression and potentially contributing to resistance to targeted therapies.
Chromoanasynthesis can be considered as a part of a broader spectrum of catastrophic events driving genome instability in both cancer and constitutional disorders (239). This ‘chromosome chaos’ model suggests that single, abrupt events may account for many complex rearrangements previously thought to accumulate gradually. Overall, chromoanasynthesis highlights the critical role of replication-based catastrophic events in shaping genome complexity and driving tumor evolution.
Chromoplexy. The term chromoplexy, consisting of chromo- and the Greek word plexis (πλέξις, meaning weaving or interweaving), was introduced in 2013 to describe a distinct class of complex structural rearrangements in cancer genomes (216). It refers to coordinated, interdependent chains of DNA rearrangements involving multiple chromosomes, typically occurring in a single catastrophic event.
In a seminal study of prostate cancer, whole-genome sequencing revealed intricate networks of balanced translocations, deletions, and inversions involving several chromosomes (216). These rearrangements often generated oncogenic fusions and disrupted tumor suppressor genes, suggesting that chromoplexy represents a punctuated mechanism accelerating tumor evolution. As a category of ‘genomic catastrophes’ distinct from chromothripsis, chromoplexy is thought to arise from the simultaneous breakage of spatially proximal chromatin regions, potentially facilitated by transcriptional activity or replication stress. The resulting DNA ends are aberrantly ligated in a coordinated yet tangled fashion, producing a ‘weaving’ of chromosomes rather than complete shattering (216, 217, 240).
Since its initial description, chromoplexy has been reported in a wide range of tumor types beyond prostate cancer. It has been identified in EWSR1-rearranged round cell sarcomas (241, 242), often giving rise to fusion oncogenes critical for sarcomagenesis, as well as in pediatric tumors such as Wilms tumor, rhabdomyosarcoma, and neuroblastoma (243).
Chromoplexy has also been observed in NUT midline carcinoma (244), esophageal adenocarcinomas (245), lung adenocarcinomas (246), mesotheliomas (247, 248), uterine leiomyosarcomas (249), myoepithelial carcinomas of soft tissue (250), acute myeloid leukemias (251), and multiple myelomas (252-254). Its diagnostic and therapeutic relevance is further illustrated by a case of acute promyelocytic leukemia lacking the canonical PML::RARA, in which chromoplexy, involving a complex 9-partner translocation, generated an FNDC3B::RARB fusion (255).
Chromoplexy embodies a shift from viewing chromosomal rearrangements as isolated, stepwise events to understanding them as complex, interconnected networks arising in single bursts. It complements concepts such as chromothripsis and kataegis (see below), contributing to a modern view of the cancer genomic landscape, characterized by punctuated, catastrophic changes that drive tumor heterogeneity, genomic instability, and promote the emergence of therapy-resistant clones. Chromoplexy highlights the importance of coordinated, multi-chromosome rearrangements in shaping tumor progression and genome complexity, and has been placed within the broader framework of chromoanagenesis (240).
Chromoanagenesis. From chromo- and the Greek anagenesis (ἀναγέννησις, meaning rebirth or regeneration), the term chromoanagenesis was introduced in 2012 to describe catastrophic one-step genomic events that result in complex, localized chromosomal rearrangements (214). It serves as an umbrella term encompassing chromothripsis, chromoplexy, and chromoanasynthesis. By integrating these diverse catastrophic rearrangements into a unifying concept of genomic chaos, chromoanagenesis captures events arising from defective mitosis, micronucleus formation, or failures in DNA repair, ultimately leading to abrupt chromosome shattering and reassembly (214, 256-258). Mechanistically, chromoanagenesis is tightly linked to mitotic errors and centrosome dysfunction. Abnormalities in centrosome number or structure promote chromosome instability by enabling multipolar mitoses and chromosome missegregation, conditions that favor sudden, catastrophic rearrangements. In other words, they create fertile ground for chromoanagenesis (259).
Chromoanagenesis has been described as a cataclysmic mechanism capable of driving macroevolution-like leaps in cancer cells. In sharp contrast to Darwinian gradualism, it embodies the idea that dramatic, large-scale remodeling can fundamentally reshape cancer genome architecture in a single event. Sudden, massive reorganizations may provide cancer cells with rapid adaptability, new oncogenic properties, and enhanced survival under selective pressure (256-258).
Analysis of more than 10,000 tumors from The Cancer Genome Atlas revealed that chromoanagenesis is a frequent event, affecting a broad spectrum of cancer types (260). Independent studies have also identified chromoanagenesis as a major driver of genome disruption in osteosarcoma, and as being strongly associated with highly abnormal karyotypes, extensive clonal diversity, and treatment resistance in acute myeloid leukemia (251, 261).
Case reports further illustrate the role of chromoanagenesis in diverse malignancies and highlight its clinical relevance. For example, in B-cell lymphoma, complex rearrangements consistent with chromoanagenesis have been described (262). In acute myeloid leukemia, a KMT2A::AR-HGEF12 fusion was generated through chromoanagenesis, demonstrating how such catastrophic rearrangements can directly give rise to oncogenic fusions (263). Together, these findings support the view that chromoanagenesis is a central contributor to cancer genome complexity and intratumoral heterogeneity.
Beyond cancer, chromoanagenesis has also been recognized as an underestimated cause of rare constitutional chromosomal disorders, contributing to complex rearrangements in patients with developmental syndromes and congenital anomalies (264-266).
Altogether, chromoanagenesis emerges as a unifying force behind both somatic and constitutional genomic complexity.
Kataegis. Kataegis (καταιγίς), the Greek word for thunderstorm, has been adopted into cancer genomics to describe clusters of localized hypermutations observed in tumor genomes. This striking mutational pattern was first reported in 2012 in a whole-genome sequencing study of breast cancer, where mutation ‘showers’ appeared as clustered C>T and C>G substitutions at TpC dinucleotides (267). The term was chosen to reflect the visual and conceptual similarity to a genomic storm.
Kataegis is strongly associated with the activity of cytidine deaminases, particularly members of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like family, such as APOBEC3A and APOBEC3B (268-270). These enzymes preferentially deaminate cytosines in single-stranded DNA regions, leading to localized hypermutation. DNA repair intermediates, such as those generated during double-strand break repair or break-induced replication, provide vulnerable single-stranded DNA substrates for APOBEC activity (271, 272). Studies have shown that telomere crisis, resulting in dicentric chromosomes and extensive DNA damage, can trigger both chromothripsis and kataegis, highlighting their interconnected nature (239, 273, 274). Bioinformatics tools such as the R packages kataegis and Katdetectr have been developed to systematically identify and visualize kataegis events in sequencing data (275, 276).
Kataegis has been detected across a wide range of cancer types, including breast, lung, pancreas, thyroid, and prostate, as well as pediatric leukemia (267, 277-283). In breast cancer, it is observed in approximately 55-60% of cases and has been associated with later onset, ERBB2 amplification, and better prognosis (277, 282). The presence of kataegis is often linked to regions near genomic rearrangements, suggesting a role in structural genome instability and tumor heterogeneity, which may influence therapeutic responses (267). In a pan-cancer study involving 8,475 samples from 32 different cancer types, high kataegis activity was associated with increased expression of CD274 (PD-L1) and PDCD1LG2 (PD-L2), which encode ligands for the immune checkpoint receptor programmed cell death protein 1 PDCD1 (PD-1) (278, 284). The increased expression of CD274 and PDCD1LG2 may affect responses to immunotherapies and the use of Food and Drug Administration-approved immune checkpoint inhibitors (285).
In ovarian clear-cell carcinoma, APOBEC-mediated kataegis was found in the early phases of tumor pathogenesis. Tumors expressing APOBEC3B showed favorable prognosis and significant immunogenicity, as evidenced by marked cytotoxic T-cell infiltration (286, 287).
In lung adenocarcinomas, APOBEC mutational signatures are more frequent in receptor tyrosine kinase-driven cancer, such as those with EGFR, ALK, RET, and ROS1 alterations (about 25%). These signatures became even more pronounced under the selective pressure of targeted therapy, such as osimertinib in EGFR-mutant tumors, and are strongly associated with kataegis and structural rearrangements, including duplications, deletions, inversions, and translocations (280).
In prostate cancer, kataegis has been linked to aggressive presentation, higher structural variant burden, and extensive copy number loss in high-risk tumors in African men. These tumors showed increased APOBEC3 activity and unique evolutionary trajectories, suggesting that kataegis may contribute to greater tumor heterogeneity and influence clinical outcomes (281).
In pediatric B-cell acute lymphoblastic leukemia, kataegis occurs at high frequency, especially in subtypes carrying ETV6::RUNX1 and ETV6::RUNX1-like fusions, indicating a possible role for localized hypermutation in the pathogenesis of specific pediatric leukemia subgroups (283).
In summary, kataegis represents a distinct mutational phenomenon driven largely by APOBEC-mediated cytosine deamination during DNA repair processes. It has emerged as an important marker of genomic instability and tumor evolution, with potential implications for prognosis and treatment strategies across multiple cancer types.
Concluding Reflections on the Conceptual Shift in Cytogenetics through Modern Greek-derived Terms
Together, these modern Greek-derived terms represent more than just linguistic continuity. They encapsulate major conceptual advances in our understanding of chromosomal instability and structural variation, and they now form an integral part of what can be considered modern cytogenetics.
While traditional methods such as chromosome banding, fluorescence in situ hybridization, and array comparative genomic hybridization have long focused on detecting visible abnormalities or copy-number alterations, terms like chromothripsis, chromoplexy, and chromoanasynthesis, all beginning with chromo-, show that chromosomes remain the central unit of study. However, their analysis has shifted from chromosome banding alone to a resolution enabled by genome-wide sequencing, optical genome mapping, and advanced cytogenomic approaches.
Chromoanagenesis unifies these catastrophic events under the broader concept of single-step genome remodeling, challenging the classical view that complex rearrangements arise through slow, stepwise accumulation. The incorporation of the terms chromothripsis (cth) and chromoanasynthesis (cha) in the International System for Human Cytogenomic Nomenclature (ISCN) reflects the growing recognition of these genomic catastrophes as legitimate cytogenetic entities, reinforcing their place within the science of studying chromosomes: cytogenetics.
Kataegis, though lacking the chromo- prefix, describes a parallel phenomenon involving localized hypermutation. It often occurs together with structural rearrangements and contributes to tumor heterogeneity and evolution. APOBEC-mediated kataegis, as well as ultramutated phenotypes arising from defects in DNA polymerases such as POLE and POLD1, illustrate how mutational bursts can shape tumor biology, immunogenicity, and treatment response (288, 289). Although kataegis operates primarily at the sequence level, its co-occurrence with chromosomal instability places it firmly within the expanding framework of cytogenetics and cytogenomics.
Collectively, chromothripsis, chromoplexy, chromoanasynthesis, chromoanagenesis, and kataegis can be seen as core components of the emerging field of chromosomics. This evolving discipline integrates chromosome structure, nuclear organization, gene regulation, and replication timing into a systems-level interpretation of genome function and dysfunction.
Overall, these modern Greek-derived terms mark a major transition in cytogenetics: from static, structural descriptions to dynamic, mechanistic, and genome-integrated models of chromosomal behavior in health and disease.
Evolution of the International System for Human Cytogenetic/Cytogenomic Nomenclature
The inclusion of new methodologies in the field of cytogenetics, and its transformation over recent decades into cytogenomics, is clearly reflected in the successive editions from 1995 to the current edition of the ISCN (290). Each edition has documented the gradual integration of novel technologies, expanding the field from the analysis of visible chromosome morphology to the detection of molecular and submicroscopic genomic alterations (46, 291-300).
A major turning point came with ISCN 1995, which introduced standardized nomenclature for fluorescence in situ hybridization (291). This inclusion reflected the growing importance of fluorescence in situ hybridization in both clinical diagnostics and research, as a methodology that complemented chromosome banding by enabling the identification of cryptic rearrangements and gene-level abnormalities. The incorporation of fluorescence in situ hybridization into ISCN marked the beginning of molecular cytogenetics, bridging banding-based analysis with DNA-based techniques. In ISCN 2005, fluorescence in situ hybridization nomenclature was further modernized, simplified, and expanded, and a basic nomenclature for recording the results of array comparative genomic hybridization was introduced (292). The ISCN 2009 edition formally integrated array-based technologies into the nomenclature system, reflecting their widespread adoption in clinical and research laboratories. The array nomenclature was revised and expanded to accommodate all platform types, and the symbol ‘arr’ was introduced to denote copy-number variants detected by array comparative genomic hybridization and SNP arrays. Additional updates included expanded rules for describing mosaicism, marker chromosomes, and uniparental disomy, as well as updated idiograms based on refined banding resolution (293, 297).
During the preparation of ISCN 2013, the Committee emphasized that “the primary purpose of the ISCN is to foster communication among cytogeneticists using a standard nomenclature that can be used to describe any genomic rearrangement identified either by standard karyotyping or molecular methodologies.” Reflecting this principle, ISCN 2013 incorporated genome build references into array-based results to ensure compatibility with genomic databases (294, 298). It introduced a standardized representation for complex genomic events such as chromothripsis and added the generic term ‘region-specific assay’ (rsa) for describing targeted methods, regardless of platform. This edition laid the groundwork for bridging chromosome banding with high-resolution molecular technologies and emphasized unified reporting principles across different methodologies.
The ISCN 2016 edition marked an important conceptual transition. To reflect technological advances and the integration of genome-wide data, the term ‘Cytogenetic’ in its title was replaced with ‘Cytogenomic’, formally renaming it the International System for Human Cytogenomic Nomenclature (295). In collaboration with the Human Genome Variation Society Sequence Variant Description Working Group, a unified nomenclature was developed that combined ISCN-like descriptions of chromosomal rearrangements with Human Genome Variation Society-like nucleotide variant descriptions. The term ‘seq’ was introduced to indicate that an abnormality had been detected using a sequence-based technology. This sequence-based nomenclature established a shared descriptive framework for both the molecular genetics and cytogenetics communities.
These conceptual and structural refinements paved the way for ISCN 2020, which further expanded sequence-based nomenclature to describe chromosomal abnormalities at nucleotide-level resolution (46, 299). The edition fully supported genome assembly–based reporting and harmonized descriptions across karyotyping, fluorescence in situ hybridization, array technology, genome mapping, and sequencing technologies. Clear annotations of all changes were included to facilitate transition from earlier editions, and the system promoted interoperability with genomic databases and clinical informatics tools.
The ISCN 2024 edition represents one of the most comprehensive revisions in the history of the system (296, 300). Building on the foundational changes introduced in 2016 and 2020, ISCN 2024 further consolidates general rules for clarity, resolved ambiguities from previous editions, and harmonized terminology across all cytogenomic platforms. New nomenclature was introduced for genome mapping technologies (including optical and electronic mapping) (202), shallow sequencing (sseq), and methylation-specific assays (rsa-ms). The representation of complex structural rearrangements was refined, nomenclature for fusion genes was clarified and standardized (301), and all examples were revised to promote consistent application in both clinical diagnostics and research. Overall, ISCN 2024 promotes platform-independent, integrative cytogenomic reporting, reflecting the continued evolution of chromosome analysis into high-resolution, genome-wide diagnostics.
The evolution of ISCN mirrors the transformation of cytogenetics, the science of studying chromosome structure and function, from its roots in chromosome banding methodology to a fully integrated scientific field that continuously embraces and incorporates new technologies while still utilizing established approaches. By harmonizing traditional and modern methods, ISCN enables comprehensive, precise, and universally interpretable reporting, supporting both research and clinical diagnostics in an era where genome-wide technologies are redefining the boundaries of modern medicine.
Digital Evolution of Cytogenetics: Software Solutions for Chromosome Analysis
In addition to general genome browsers such as the University of California, Santa Cruz, Genome Browser (131), Ensembl (132), and the National Center for Biotechnology Information Genome Data Viewer (302), which provide detailed genomic information alongside chromosomal positions and cytogenetic banding patterns, a wide range of specialized software tools and web-based databases have been developed for cytogenetic and cytogenomic analysis. These resources, which are reviewed elsewhere (303), play an essential role in storing, accessing, comparing, and visualizing chromosomal and genomic data. Software tools developed for array-comparative genomic hybridization have also illustrated how the digital evolution of cytogenetics has been driven by the need to interpret increasingly complex genomic data (304).
Regarding chromosome visualization, Cytogenetic Data Analysis System (CyDAS) stands out as a tool for analyzing and interpreting karyotypes, simulating chromosomal aberrations, and generating human chromosome ideograms in accordance with ISCN standards (305). RIdeogram, an R package, enables the generation of high-quality chromosome ideograms and allows users to overlay diverse genomic or transcriptomic data (306). Additional tools such as Idiographica and ChromDraw support the clear presentation of cytogenetic data and gene localization through customizable idiograms (307, 308). Several other R-based tools have further enriched chromosome visualization capabilities, including karyoploteR (309), chromoMap (310), shinyChromosome (311), and GenoPlotR (312), which enable flexible, customizable, and interactive rendering of chromosome structures and annotations.
Tools such as PhenoGram (313), Circos (314), and Circa (from OMGenomics Labs) are widely used to display genetic variants and complex structural rearrangements. Circos and Circa, in particular, represent chromosomes in a circular layout and are highly versatile, capable of displaying translocations, fusion genes, and other large-scale genomic events in human samples. Furthermore, fusion-gene detection software such as Arriba includes extensions that map genomic breakpoints onto chromosome ideograms, providing an integrated view of structural alterations (315). The tools FusionInspector and Chimeraviz also provide visualization of fusion events (316, 317). Chimeraviz, an R package, is particularly notable for its ability to generate chromosome ideograms and fusion diagrams from outputs of multiple callers, aiding in the interpretation of structural rearrangements (317).
Programs focused on copy-number and structural variant analysis, such as the Integrative Genomics Viewer (318), CNVkit (319), and Chromothripsis Explorer (320), offer also ideogram-based visualizations of chromosomal aberrations and help bridge molecular data with cytogenetic interpretation. In parallel, optical genome mapping has emerged as a next-generation cytogenetic technique that enables high-resolution visualization of structural variants using long DNA molecules, complementing digital karyotyping and sequence-based methods.
In clinical cytogenetic laboratories, digital karyotyping is undergoing a transformation through the integration of artificial intelligence (321). Several companies have introduced artificial intelligence-guided systems that automate key steps in chromosome analysis, including metaphase detection, chromosome segmentation, classification, and preliminary karyogram generation. Applied Spectral Imaging offers HiBand version 8.4, which uses convolutional neural networks to learn chromosome identification, pairing, and arrangement. BioView has developed an Artificial Intelligence Karyotyping Application Suite with real-time karyotype correction, high-definition imaging, and abnormality detection. Diagens, based in China, provides the AutoVision platform, also using convolutional neural networks, with an advertised classification accuracy of over 99%. Finally, MetaSystems’ Ikaros version 6.3 employs deep neural networks and has demonstrated 97% accuracy across specimen types in clinical settings (321). These artificial intelligence-enabled systems significantly reduce analysis time and manual workload, allowing cytogeneticists to focus more on interpretation and less on repetitive technical tasks. While still in the early stages of widespread adoption, artificial intelligence-guided karyotyping represents a promising and potentially transformative advancement in the practice of digital cytogenetics.
As the number of specialized tools and database interfaces continues to grow, standardizing data formats and enabling interoperability across platforms will be key to maximizing their utility in both research and clinical workflows.
All in all, the continued development and refinement of these specialized software tools underscore the dynamic and evolving nature of cytogenetics. The need to analyze, interpret, and visualize chromosomal data in increasingly detailed and integrative ways has driven the creation of these programs. This highlights that cytogenetics, as a branch of genetics dedicated to the study of chromosomes, remains an active field that continues to expand and adapt alongside advances in molecular and computational biology. Together, these developments illustrate how digital tools are not only supporting but actively shaping the future of cytogenetic research and diagnostics.
The Legacy of Chromosome Banding in Today’s Oncology
Despite the emergence of powerful new technologies for studying chromosomes in cancer, chromosome banding analysis continues to play an indispensable and complementary role in both cancer diagnostics and research. New methods may challenge conventional approaches (322, 323), but chromosome banding remains especially valuable in hematological malignancies and in tumors of bone and soft tissue. In fact, the absence of chromosome banding results, whether due to technical failure or omission, has been associated with poor prognosis in acute myeloid leukemia (324-326). The fifth edition of the World Health Organization Classification of Hematolymphoid Tumors incorporates chromosomal aberrations, along with other genetic abnormalities, as key elements for defining disease entities (327, 328). Similarly, the fifth edition of the World Health Organization Classification of Bone and Soft Tissue Tumors includes chromosomal aberrations, fusion genes, and additional genetic features as integral components of tumor pathogenesis (329).
In cancer research, chromosome banding remains a valuable starting point for the discovery of novel fusion genes, which are subsequently characterized by high-throughput sequencing. Several studies published in 2024 and 2025 illustrate this approach and report fusion genes linked to specific chromosomal abnormalities (330-334).
Chromosomal abnormalities also continue to be reported at major scientific conferences such as the annual meetings of the American Society of Hematology (335, 336). Moreover, retrospective studies based on banding analysis, primarily in hematologic malignancies but also in bone and soft tissue tumors, remain a valuable source of data, providing biological and clinical insight. Recent large-scale retrospective analyses of chromosomal rearrangements in acute leukemia (337-341) and multiple myelomas (often in combination with fluorescence in situ hybridization) (342-345) have provided important insights into the patterns and the clinical implications of these aberrations.
A large retrospective study of gastrointestinal stromal tumors investigated chromosomal aberrations and the extent of intratumor cytogenetic heterogeneity (346). A subsequent study demonstrated that genomic complexity, defined by the number of chromosomal aberrations, may serve as a biomarker to guide de-escalation of adjuvant imatinib treatment in high-risk gastrointestinal stromal tumor (347).
In the context of this legacy, The Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer illustrates the remarkable evolution of cancer cytogenetics, transitioning from static documentation to dynamic, genome-informed analysis. First introduced in 1983 as a printed volume titled Catalog of Chromosome Aberrations in Cancer, and published in the journal Cytogenetics and Cell Genetics, it included 3,844 cases (50). As knowledge rapidly expanded, updated editions of the catalog followed: the 1985 edition documented 5,345 cases; the 1988 edition included 9,069, the 1991 edition listed 14,141, the 1994 edition reached 22,076, and by 1998, the catalog had grown to 30,541 cases (348-351). Over the 15-year period from 1983 to 1998, a total of 26,697 new cases were added, corresponding to an average of approximately 1,780 cases per year. The growth was exponential, with an estimated compound annual growth rate of 14.8% in reported chromosomal aberrations (Figure 4).
Annual number of cytogenetic entries reported in the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (as of July 10, 2025). The diagram shows the number of newly registered cytogenetic entries per year from 1971 to 2025. The steep rise throughout the 1980s and 1990s mirrors the exponential expansion documented in the printed catalog editions published between 1983 and 1998, reflecting the intense activity in chromosome banding-based cancer cytogenetics. After this period of rapid growth, annual contributions became more variable, influenced by evolving diagnostic strategies, shifts in publication and reporting practices, and the growing impact of genome-wide technologies on cancer cytogenetics.
In 2000, this growing body of work was transformed into a publicly accessible online database, hosted by the U.S. National Cancer Institute, enabling regular updates, advanced search capabilities, and global access (52). From that point forward, the database not only expanded in volume but also integrated emerging genomic data and tools, reflecting the shift from chromosome banding to modern sequence-based interpretation (52). The database now integrates CytoConverter, which translates conventional karyotype descriptions into genomic coordinates (352), and annotations from the Matched Annotation from the National Center for Biotechnology Information and EMBL-EBI (MANE) project, which provides a unified transcript set for clinical and research use (353). Additionally, through its incorporation into Google BigQuery, the dataset supports advanced, interactive analyses at scale (52).
Since the launch of the online version in 2000, the database has continued to grow, albeit at a slower pace. From 30,541 cases in 1998 to 80,008 by July 2025, the number of cytogenetically analyzed cancer cases has more than doubled, corresponding to an average of 1,832 new cases per year and a modest compound annual growth rate of 3.6% (Figure 4). Although modern technologies such as array-comparative genomic hybridization, genome-wide sequencing, and optical genome mapping are now widely used in cancer cytogenetics, new case studies based on chromosome banding continue to be documented and curated, underscoring the enduring clinical and research value of this foundational technique.
In contrast to cytogenetic case entries, which have accumulated steadily over decades from numerous studies worldwide, the fusion-gene data in the Mitelman Database reveal a striking imbalance (Figure 5A and B). As of the July 10, 2025, update, the database contains 52,669 fusion-gene entries in the standard Gene1::Gene2 format, corresponding to 34,398 unique gene fusions and 14,105 genes involved in them. Remarkably, 41,749 of these entries (approximately 79%) originate from just five large-scale studies: Gao et al. 2018 (354) with 21,065 entries, Hu et al. 2018 (355) with 10,150 entries, Yoshihara et al. 2015 (356) with 7,700 entries, Calabrese et al. 2020 (357) with 2,309 entries, and Cleynen et al. 2017 (358) with 525 entries. The remaining 10,920 entries (approximately 21%) are derived from 3,266 other publications (52).
Fusion-gene entries in the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (update of July 10, 2025). (A) Annual number of “Gene1::Gene2” fusions and unique Gene1::Gene2 fusions from 1982 to 2025. The sharp peaks in 2015, 2018, and 2020 correspond to a few large-scale transcriptome-wide studies, rather than a steady accumulation over time. (B) Cumulative totals of Gene1::Gene2 fusions and unique fusions over the same period. The near-vertical rises in 2015, 2018, and 2020 show that the apparent expansion of fusion-gene diversity is driven mainly by these large sequencing studies, with comparatively modest contributions from other years. ‘Unique’ Gene1::Gene2 fusions represent fusion pairs appearing for the first time in the database; reciprocal orientations (Gene1::Gene2 and Gene2::Gene1) are considered a single fusion event.
Likewise, this trend extends to the unique gene fusions, which also originate mainly from the same five large-scale studies. Analysis of the Mitelman Database (July 2025) shows that the number of newly reported fusion genes peaks sharply in years corresponding to the publication of these large-scale sequencing studies (Figure 5A and B).
For the purpose of this review, fusion genes appearing for the first time and never reported previously were identified for each year in the database (up to the July 10, 2025 update). Reciprocal Gene1::Gene2 and Gene2::Gene1 were considered as a single event, as both arise from the same underlying genomic aberration or its variants, and the format reported for the first time was retained.
Based on these criteria, 33,436 unique fusions were listed in the July 2025 version of the database. The years 2015, 2017, 2018, and 2020 account for 7,585, 884, 19,230, and 1,151 unique fusion genes, respectively, together representing 28,850 of the 33,436 unique fusions (≈86%). In 2015, the study by Yoshihara et al. (356) contributed 7,325 unique fusions; in 2017, Cleynen et al. (358) added 478; in 2018, the two studies by Gao et al. (354) and Hu et al. (355) contributed 9,263 and 9,794, respectively (jointly 19,057); and in 2020, Calabrese et al. (357) added 822. In total, 27,682 unique fusions (≈83%) originate from these five studies. These calculations confirm that the apparent expansion of fusion-gene diversity primarily reflects the influx of data from a few comprehensive transcriptome-wide studies rather than a continuous accumulation of independent cytogenetic discoveries (Figure 5A and B).
The Mitelman Database is extensive and widely regarded as the principal reference for chromosomal aberrations and gene fusions in cancer, but it does not include all published cases identified through chromosome banding analysis. Cases reported in conference abstracts (359), individual case reports (360, 361), smaller or larger case series (346, 362), and studies presenting cytogenetic data solely in supplementary materials (363-365) may be underrepresented. The database also excludes neoplasms carrying nonclonal chromosomal aberrations; for example, elastofibromas are characterized by chromosomal instability and predominantly nonclonal changes, whereas the few reported clonal aberrations in elastofibroma are believed to represent secondary rather than primary events (366-368). This biological nuance is not reflected in the database, which currently lists only two elastofibroma cases with clonal aberrations (52).
With regard to fusion genes, several of the most fusion-rich studies in the database draw upon overlapping The Cancer Genome Atlas tumor cohorts (354-357). As a result, the apparent diversity of fusion events in the database is partly inflated by multiple reports of the same underlying samples, underscoring the need for systematic curation, separating the wheat from the chaff, to accurately delineate the landscape of genuine, non-redundant fusion genes. Another limitation of the database is that it does not include fusions involving non-coding RNA or microRNAs. This omission may lead to misinterpretation of the frequency and distribution of certain fusion genes. Notable examples are the recurrent fusion gene MALAT1::GLI1 reported in plexiform fibromyxoma, gastroblastoma, and epithelioid neoplasms (369-372), and the fusion of microRNA 143 with NOTCH1 or NOTCH2 in glomus tumor (373-376).
Nevertheless, the Mitelman Database remains an indispensable resource for both cancer research and diagnostics, providing a curated and continually updated view of the cytogenomic landscape in human malignancies. It is frequently used in combination with other major databases in cancer genomics, such as the Catalogue of Somatic Mutations in Cancer, which focuses on somatic mutations, and ChimerDB, which integrates experimentally validated as well as computationally predicted gene fusions (377, 378). Together, these resources offer a more comprehensive understanding of cancer-associated structural variants, supporting efforts in fusion-gene discovery, the classification and interpretation of novel aberrations, and the development of targeted therapeutic strategies.
An important aspect missing from curated resources, including the Mitelman Database, is the documentation of non-clonal chromosome aberrations (NCCAs). Traditionally dismissed as technical artifacts or background noise (379), NCCAs are now recognized as visible manifestations of chromosomal instability and a major source of karyotypic heterogeneity in both malignant and pre-malignant conditions (23, 379-381). Each NCCA represents a unique genomic cnfiguration that can drive macroevolutionary changes in tumor cell populations, providing the diversity from which stable, disease-defining CCAs may arise (23, 379, 381). In inherited instability syndromes such as Fanconi anemia, NCCAs frequently appear years before the emergence of overt clonal evolution, and their burden may precede the development of complex karyotypes (382). Moreover, studies in cytopenic patients without hematologic malignancy have shown that the presence of NCCAs can be associated with adverse prognosis, in some cases exceeding the impact of CCAs (383). Systematically recording NCCAs in cytogenetic analyses, as proposed by Heng and colleagues (23, 379, 381), would improve our understanding of tumor heterogeneity and cancer evolution, and it could also strengthen the prognostic value of cytogenetic data. Furthermore, the development of a dedicated database capturing NCCA patterns across tumor types has been suggested as a potentially invaluable resource for both research and clinical practice (23, 379, 381).
In light of the above, despite the rise of high-throughput genomic technologies, chromosome banding retains its diagnostic and scientific value. It continues to serve not only as a complementary method but also as a foundational element in the legacy of cancer cytogenetics, supporting and informing modern genomic discovery. In the expanding territory of cytogenomics, chromosome banding remains a compass, guiding interpretation, anchoring discovery, and reminding us that even the most advanced journeys begin with a well-drawn map.
Is Cytogenetics Indeed a Dinosaur?
In a recent article published in the August 2025 issue of Prenatal Diagnosis, summarizing the International Society for Prenatal Diagnosis 2024 Debate 3, provocatively titled “Cytogenetics Is a Dinosaur and Should Be Replaced by Molecular Technologies”, Michael E. Talkowski and Yassmine M. N. Akkari presented opposing views under the moderation of Amy M. Breman (384). Dr. Talkowski argued that whole-genome sequencing and related platforms such as exome sequencing now provide a far more comprehensive and higher-resolution genetic screen than karyotyping, chromosomal microarray, and fluorescence in situ hybridization, and should replace them as the first-line approach in prenatal diagnosis. He envisioned a future driven by unbiased, genome-wide, non-invasive sequencing capable of detecting nearly all clinically relevant variants. His position is “that with more training in sequencing technologies, clinicians and patients alike will demand the most comprehensive approaches available, and this is clearly ES/GS testing”.
Dr. Akkari countered that cytogenetics is not merely a technique, but a science devoted to the study of chromosome behavior that continues to evolve with new tools such as optical genome mapping. She emphasized the enduring role of cytogenetics in education, in the interpretation of chromosomal structure, and in ensuring equitable access to diagnostics, noting that in many parts of the world, advanced sequencing remains a distant prospect: “In Lebanon, we can barely give Gleevec when a patient is diagnosed with chronic myeloid leukemia. Genome sequencing is not even on their radar.” She further stated that “as much as we want people to understand next-generation sequencing, we also need them to understand the structure and behavior of chromosomes,” and mentioned a discussion in which she was asked whether she would encourage her children to go into cytogenetics. Her answer was very genuinely “yes,” because “the science of cytogenetics will make them understand chromosomes. Understanding chromosomes will make them understand behavior. Understanding everything about this science will make them able to fully understand the data that labs like Dr. Talkowski’s are able to generate at large scale.”
In the concluding remarks, Dr. Breman observed that both sides ultimately agreed on one central point: Understanding chromosomes remains fundamental to clinical genetics. The debate thus reflected not extinction but transformation and the continuing evolution of cytogenetics through and within the framework of modern technologies.
In considering the arguments presented in this debate, my perspective aligns more closely with that of Dr. Akkari. Cytogenetics is the field of genetics studying the structure and behavior of chromosomes. So, paraphrasing Dr. Akkari’s question “How can a science die?” I would ask: How can a methodology, such as whole-genome sequencing, replace a science? Cytogenetics is hardly a dinosaur as a scientific discipline; it is less than 150 years old, whereas many scientific fields are far older. The comparison in the debate was not truly about the science of cytogenetics but rather about chromosome banding versus high-throughput technologies.
Like Dr. Akkari, I remain acutely aware that access to high-throughput sequencing and other advanced genomic technologies is far from universal. In fact, gel electrophoresis together with ethidium bromide staining (now largely replaced by less toxic intercalating fluorescent dyes such as SYBR Green or GelRed), which is the methodological counterpart to chromosome banding in molecular biology, is still routinely used to visualize DNA fragments, for example after PCR amplification. Developed in the early 1970s by two independent research groups (385-388), gel electrophoresis/ethidium bromide staining is almost the same age as chromosome banding, and yet it remains widely used in many laboratories despite the availability of more advanced technologies. The continued use of these methods despite five decades of technological progress illustrates a broader truth: Methods do not become obsolete simply because newer ones exist but only when they cease to be informative.
In the same way that gel electrophoresis/ethidium bromide staining remains a basic structural tool in molecular biology, chromosome banding remains a basic structural tool in cytogenetics; each continues to support and guide the interpretation of high-throughput genomic data. In many regions, both methods, chromosome banding and gel electrophoresis/ethidium bromide staining continue to serve as practical and informative research and diagnostic approaches.
And even when using the most advanced genomic technologies and the most sophisticated genomic analyses, all genetic findings, whether indels, pathogenic variants, or complex rearrangements, are ultimately positioned within chromosomal coordinates. In the end, every layer of genomic information returns to the same foundational structure: the chromosome, which remains the enduring framework of genome organization and function and the unifying theme of cytogenetics.
Concluding Remarks
Cytogenetics has undergone successive methodological revolutions, from chromosome banding to high-resolution molecular and genomic technologies. Today, platforms such as array comparative genomic hybridization, next-generation sequencing, and optical genome mapping enable the interrogation of chromosomes with unprecedented precision. New terms like cytogenomics and chromosomics have emerged to reflect this evolution. Others, such as chromothripsis, chromoplexy, and chromoanasynthesis, now unified under the umbrella of chromoanagenesis, capture the notion that catastrophic genomic events can reshape cancer genomes in a single step. Likewise, the term kataegis describes clusters of localized hypermutations, revealing yet another layer of chromosomal complexity in cancer.
Yet despite these advances, the core mission of the field, the study of chromosomes, remains unchanged. Whether called cytogenetics or cytogenomics, the field broadly encompasses all efforts to understand chromosomes across species. Just as molecular genetics has embraced new tools over time, replacing Southern blotting and restriction enzymes with PCR and sequencing without losing its identity, so too has cytogenetics persisted, adapting to new methods while maintaining its core focus. For some, cytogenetics is synonymous with chromosome banding, yet its scope and spirit extend far beyond any single method.
Like a tree, it stands rooted in its past, branching outward with each scientific advance. It has been nourished, bent, and reshaped by successive methods, yet never losing its core identity. Today, cytogenetics is both graceful and strong, revealing the hidden architecture of life in ever-greater detail, and carrying the legacy of chromosome banding into the omics era.
The future may see the replacement of chromosome banding with genome-wide approaches, but this will not be the end of cytogenetics. It will simply be its natural evolution. The field will continue to thrive by integrating new technologies to deepen our understanding of chromosome structure, function, and pathology. As long as we seek to understand chromosomes, by any method, cytogenetics will endure.
Acknowledgements
The Author thanks the CMAJ Group for kindly granting permission to reproduce Figure 1A. The Author also acknowledges the courtesy of Kristin Andersen (Oslo University Hospital – Norwegian Radium Hospital) for providing Figure 1B and C. Figure 2 was generated using the publicly available test dataset included with the STAR-Fusion distribution and analyzed with the Arriba algorithm. The Author gratefully acknowledges Professor Zhenya Tang for arranging access to the optical genome mapping image and Dr. Gokce A. Toruner (MD Anderson Cancer Center) for kindly providing it (Figure 3).
Footnotes
Conflicts of Interest
The Author declares no competing interests.
Author’s Contributions
I.P. conceived the topic, designed the structure of the review, performed the literature search, analyzed and synthesized the relevant scientific developments, wrote the manuscript, prepared all figures and tables, and approved the final version.
Funding
The Author’s research is financially supported by Barnekreftforeningen (The Norwegian Childhood Cancer Society).
Artificial Intelligence (AI) Disclosure
The Author used the AI language model ChatGPT (OpenAI, San Francisco, CA, USA) for editorial assistance, limited to grammar correction and improvement of English readability. The AI tool had no role in study design, data collection, analysis, or interpretation. The Author takes full responsibility for the content of this manuscript.
- Received December 22, 2025.
- Revision received February 10, 2026.
- Accepted February 16, 2026.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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In this issue
Jump to section
- Article
- Abstract
- Introduction
- Defining Cytogenetics: The Chromosomal Language of Life
- Cytogenetic Methodologies and Periods: From Pre-banding to Cytogenomics and Chromosomics
- Cytogenetics in the New Millennium: A New Era for the Study of Chromosome Structure and Behavior
- More Greek Words in the Field of Cytogenetics
- Concluding Reflections on the Conceptual Shift in Cytogenetics through Modern Greek-derived Terms
- Evolution of the International System for Human Cytogenetic/Cytogenomic Nomenclature
- Digital Evolution of Cytogenetics: Software Solutions for Chromosome Analysis
- The Legacy of Chromosome Banding in Today’s Oncology
- Is Cytogenetics Indeed a Dinosaur?
- Concluding Remarks
- Acknowledgements
- Footnotes
- References
- Figures & Data
- Info & Metrics
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