The Importance of Monitoring Non-clonal Chromosome Aberrations (NCCAs) in Cancer Research.

Cytogenetic analysis has traditionally focused on the clonal chromosome aberrations, or CCAs, and considered the large number of diverse non-clonal chromosome aberrations, or NCCAs, as insignificant noise. Our decade-long karyotype evolutionary studies have unexpectedly demonstrated otherwise. Not only the baseline of NCCAs is associated with fuzzy inheritance, but the frequencies of NCCAs can also be used to reliably measure genome or chromosome instability (CIN). According to the Genome Architecture Theory, CIN is the common driver of cancer evolution that can unify diverse molecular mechanisms, and genome chaos, including chromothripsis, chromoanagenesis, and polypoidal giant nuclear and micronuclear clusters, and various sizes of chromosome fragmentations, including extrachromosomal DNA, represent some extreme forms of NCCAs that play a key role in the macroevolutionary transition. In this chapter, the rationale, definition, brief history, and current status of NCCA research in cancer are discussed in the context of two-phased cancer evolution and karyotype-coded system information. Finally, after briefly describing various types of NCCAs, we call for more research on NCCAs in future cytogenetics.

The New Era of Cancer Cytogenetics and Cytogenomics.

The promises of the cancer genome sequencing project, combined with various -omics technologies, have raised questions about the importance of cancer cytogenetic analyses. It is suggested that DNA sequencing provides high resolution, speed, and automation, potentially replacing cytogenetic testing. We disagree with this reductionist prediction. On the contrary, various sequencing projects have unexpectedly challenged gene theory and highlighted the importance of the genome or karyotype in organizing gene network interactions. Consequently, profiling the karyotype can be more meaningful than solely profiling gene mutations, especially in cancer where karyotype alterations mediate cellular macroevolution dominance. In this chapter, recent studies that illustrate the ultimate importance of karyotype in cancer genomics and evolution are briefly reviewed. In particular, the long-ignored non-clonal chromosome aberrations or NCCAs are linked to genome or chromosome instability, genome chaos is linked to genome reorganization under cellular crisis, and the two-phased cancer evolution reconciles the relationship between genome alteration-mediated punctuated macroevolution and gene mutation-mediated stepwise microevolution. By further synthesizing, the concept of karyotype coding is discussed in the context of information management. Altogether, we call for a new era of cancer cytogenetics and cytogenomics, where an array of technical frontiers can be explored further, which is crucial for both basic research and clinical implications in the cancer field.

Tracking Karyotype Changes in Treatment-Induced Drug-Resistant Evolution.

Karyotype coding, which encompasses the complete chromosome sets and their topological genomic relationships within a given species, encodes system-level information that organizes and preserves genes' function, and determines the macroevolution of cancer. This new recognition emphasizes the crucial role of karyotype characterization in cancer research. To advance this cancer cytogenetic/cytogenomic concept and its platforms, this study outlines protocols for monitoring the karyotype landscape during treatment-induced rapid drug resistance in cancer. It emphasizes four key perspectives: combinational analyses of phenotype and karyotype, a focus on the entire evolutionary process through longitudinal analysis, a comparison of whole landscape dynamics by including various types of NCCAs (including genome chaos), and the use of the same process to prioritize different genomic scales. This protocol holds promise for studying numerous evolutionary aspects of cancers, and it further enhances the power of karyotype analysis in cancer research.

The Digital World of Cytogenetic and Cytogenomic Web Resources.

The dynamic growth of technological capabilities at the cellular and molecular level has led to a rapid increase in the amount of data on the genes and genomes of organisms. In order to store, access, compare, validate, classify, and understand the massive data generated by different researchers, and to promote effective communication among research communities, various genome and cytogenetic online databases have been established. These data platforms/resources are essential not only for computational analyses and theoretical syntheses but also for helping researchers select future research topics and prioritize molecular targets. Furthermore, they are valuable for identifying shared recurrent genomic patterns related to human diseases and for avoiding unnecessary duplications among different researchers. The website interface, menu, graphics, animations, text layout, and data from databases are displayed by a front end on the screen of a monitor or smartphone. A database front-end refers to the user interface or application that enables accessing tabular, structured, or raw data stored in the database. The Internet makes it possible to reach a greater number of users around the world and gives them quick access to information stored in databases. The number of ways of presenting this data by front-ends increases as well. This requires unifying the ways of operating and presenting information by front-ends and ensuring contextual switching between front-ends of different databases. This chapter aims to present selected cytogenetic and cytogenomic Internet resources in terms of obtaining the needed information and to indicate how to increase the efficiency of access to stored information. Through a brief introduction of these databases and by providing examples of their usage in cytogenetic analyses, we aim to bridge the gap between cytogenetics and molecular genomics by encouraging their utilization.

Studying the Dynamics of Tunneling Tubes and Cellular Spheres.

Cancer cytogenetic analyses often involve cell culture. However, many cytogeneticists overlook interesting phenotypes associated with cultured cells. Given that cytogeneticists need to focus more on phenotypes to comprehend the genotypes, the biological significance of seemingly trivial cellular variations deserves attention. One example is the formation of cellular tunneling tubes (TTs) in cultured cancer cells, which likely play a role in cell-to-cell communication and material transport. In this chapter, we describe protocols for studying these TTs as well as cellular spheres. In addition to diverse chromosomal variants, these different types of variations should be considered for understanding cancer heterogeneity and dynamics, as they illustrate the importance of various forms of fuzzy inheritance.

Karyotype as code of codes: An inheritance platform to shape the pattern and scale of evolution.

Organismal evolution displays complex dynamics in phase and scale which seem to trend towards increasing biocomplexity and diversity. For over a century, such amazing dynamics have been cleverly explained by the apparently straightforward mechanism of natural selection: all diversification, including speciation, results from the gradual accumulation of small beneficial or near-neutral alterations over long timescales. However, although this has been widely accepted, natural selection makes a crucial assumption that has not yet been validated. Specifically, the informational relationship between small microevolutionary alterations and large macroevolutionary changes in natural selection is unclear. To address the macroevolution-microevolution relationship, it is crucial to incorporate the concept of organic codes and particularly the "karyotype code" which defines macroevolutionary changes. This concept piece examines the karyotype from the perspective of two-phased evolution and four key components of information management. It offers insight into how the karyotype creates and preserves information that defines the scale and phase of macroevolution and, by extension, microevolution. We briefly describe the relationship between the karyotype code, the genetic code, and other organic codes in the context of generating evolutionary novelties in macroevolution and imposing constraints on them as biological routines in microevolution. Our analyses suggest that karyotype coding preserves many organic codes by providing system-level inheritance, and similar analyses are needed to classify and prioritize a large number of different organic codes based on the phases and scales of evolution. Finally, the importance of natural information self-creation is briefly discussed, leading to a call to integrate information and time into the relationship between matter and energy.

Challenges and Opportunities for Clinical Cytogenetics in the 21st Century.

The powerful utilities of current DNA sequencing technology question the value of developing clinical cytogenetics any further. By briefly reviewing the historical and current challenges of cytogenetics, the new conceptual and technological platform of the 21st century clinical cytogenetics is presented. Particularly, the genome architecture theory (GAT) has been used as a new framework to emphasize the importance of clinical cytogenetics in the genomic era, as karyotype dynamics play a central role in information-based genomics and genome-based macroevolution. Furthermore, many diseases can be linked to elevated levels of genomic variations within a given environment. With karyotype coding in mind, new opportunities for clinical cytogenetics are discussed to integrate genomics back into cytogenetics, as karyotypic context represents a new type of genomic information that organizes gene interactions. The proposed research frontiers include: 1. focusing on karyotypic heterogeneity (e.g., classifying non-clonal chromosome aberrations (NCCAs), studying mosaicism, heteromorphism, and nuclear architecture alteration-mediated diseases), 2. monitoring the process of somatic evolution by characterizing genome instability and illustrating the relationship between stress, karyotype dynamics, and diseases, and 3. developing methods to integrate genomic data and cytogenomics. We hope that these perspectives can trigger further discussion beyond traditional chromosomal analyses. Future clinical cytogenetics should profile chromosome instability-mediated somatic evolution, as well as the degree of non-clonal chromosomal aberrations that monitor the genomic system's stress response. Using this platform, many common and complex disease conditions, including the aging process, can be effectively and tangibly monitored for health benefits.

Questions to guide cancer evolution as a framework for furthering progress in cancer research and sustainable patient outcomes.

We appear to be faced with 'two truths' in cancer-one of major advances and successes and another one of remaining short-comings and significant challenges. Despite decades of research and substantial progress in treating cancer, most patients with metastatic cancer still experience great suffering and poor outcomes. Metastatic cancer, for the vast majority of patients, remains incurable. In the context of advanced disease, many clinical trials report only incremental advances in progression-free and overall survival. At the same time, the breadth and depth of new scientific discoveries in cancer research are staggering. These discoveries are providing increasing mechanistic detail into the inner workings of normal and cancer cells, as well as into cancer-host interactions; however, progress remains frustratingly slow in translating these discoveries into improved diagnostic, prognostic, and therapeutic interventions. Despite enormous advances in cancer research and progress in progression-free survival, or even cures, for certain cancer types-with earlier detection followed by surgical, adjuvant, targeted, or immuno- therapies, we must challenge ourselves to do even better where patients do not respond or experience evolving therapy resistance. We propose that defining cancer evolution as a separate domain of study and integrating the concept of evolvability as a core hallmark of cancer can help position scientific discoveries into a framework that can be more effectively harnessed to improve cancer detection and therapy outcomes and to eventually decrease cancer lethality. In this perspective, we present key questions and suggested areas of study that must be considered-not only by the field of cancer evolution, but by all investigators researching, diagnosing, and treating cancer.

Genome chaos: Creating new genomic information essential for cancer macroevolution.

Cancer research has traditionally focused on the characterization of individual molecular mechanisms that can contribute to cancer. Due to the multiple levels of genomic and non-genomic heterogeneity, however, overwhelming molecular mechanisms have been identified, most with low clinical predictability. It is thus necessary to search for new concepts to unify these diverse mechanisms and develop better strategies to understand and treat cancer. In recent years, two-phased cancer evolution (comprised of the genome reorganization-mediated punctuated phase and gene mutation-mediated stepwise phase), initially described by tracing karyotype evolution, was confirmed by the Cancer Genome Project. In particular, genome chaos, the process of rapid and massive genome reorganization, has been commonly detected in various cancers-especially during key phase transitions, including cellular transformation, metastasis, and drug resistance-suggesting the importance of genome-level changes in cancer evolution. In this Perspective, genome chaos is used as a discussion point to illustrate new genome-mediated somatic evolutionary frameworks. By rephrasing cancer as a new system emergent from normal tissue, we present the multiple levels (or scales) of genomic and non-genomic information. Of these levels, evolutionary studies at the chromosomal level are determined to be of ultimate importance, since altered genomes change the karyotype coding and karyotype change is the key event for punctuated cellular macroevolution. Using this lens, we differentiate and analyze developmental processes and cancer evolution, as well as compare the informational relationship between genome chaos and its various subtypes in the context of macroevolution under crisis. Furthermore, the process of deterministic genome chaos is discussed to interpret apparently random events (including stressors, chromosomal variation subtypes, surviving cells with new karyotypes, and emergent stable cellular populations) as nonrandom patterns, which supports the new cancer evolutionary model that unifies genome and gene contributions during different phases of cancer evolution. Finally, the new perspective of using cancer as a model for organismal evolution is briefly addressed, emphasizing the Genome Theory as a new and necessary conceptual framework for future research and its practical implications, not only in cancer but evolutionary biology as a whole.

Further illusions: On key evolutionary mechanisms that could never fit with Modern Synthesis.

In light of illusions of the Modern Synthesis (MS) listed by Noble (2021a), MS's key concept, that gradual accumulation of gene mutations within microevolution leads to macroevolution, requires reexamination too. In this article, additional illusions of the MS are identified therein caused by the absence of system information and correct history. First, the MS lacks distinction among the two basic types of information: genome-defined system and gene-defined parts-information, as its treatment was based mostly on gene information. In contrast, it is argued here that system information is maintained by species-specific karyotype code, and macroevolution is based on the whole genome information package rather than on specific genes. Linking the origin of species with system information shows that the creation and accumulation of the latter in evolution is the fundamental question omitted from the MS. Second, modern evidence eliminates the MS's preferred theory that present evolutionary events can be linearly extrapolated to the past to reconstruct Life's history, wrongly assuming that most of the fossil record supports the gradual change while ignoring the true karyotype/genome patterns. Furthermore, stasis, as the most prominent pattern of the deep history of Life, remains a puzzle to the MS, but can be explained by the mechanism of karyotype-preservation-via-sex. Consequently, the concept of system-information is smoothly integrated into the two-phased evolutionary model that paleontology requires (Eldredge and Gould, 1972). Finally, research on genome-level causation of evolution, which does not fit the MS, is summarized. The availability of alternative concepts further illustrates that it is time to depart from the MS.

Emerging Role of Chimeric RNAs in Cell Plasticity and Adaptive Evolution of Cancer Cells.

Gene fusions can give rise to somatic alterations in cancers. Fusion genes have the potential to create chimeric RNAs, which can generate the phenotypic diversity of cancer cells, and could be associated with novel molecular functions related to cancer cell survival and proliferation. The expression of chimeric RNAs in cancer cells might impact diverse cancer-related functions, including loss of apoptosis and cancer cell plasticity, and promote oncogenesis. Due to their recurrence in cancers and functional association with oncogenic processes, chimeric RNAs are considered biomarkers for cancer diagnosis. Several recent studies demonstrated that chimeric RNAs could lead to the generation of new functionality for the resistance of cancer cells against drug therapy. Therefore, targeting chimeric RNAs in drug resistance cancer could be useful for developing precision medicine. So, understanding the functional impact of chimeric RNAs in cancer cells from an evolutionary perspective will be helpful to elucidate cancer evolution, which could provide a new insight to design more effective therapies for cancer patients in a personalized manner.

Karyotype coding: The creation and maintenance of system information for complexity and biodiversity.

The mechanism of biological information flow is of vital importance. However, traditional research surrounding the genetic code that follows the central dogma to a phenotype faces challengers, including missing heritability and two-phased evolution. Here, we propose the karyotype code, which by organizing genes along chromosomes at once preserves species genome information and provides a platform for other genetic and nongenetic information to develop and accumulate. This specific genome-level code, which exists in all living systems, is compared to the genetic code and other organic codes in the context of information management, leading to the concept of hierarchical biological codes and an 'extended' definition of adaptor where the adaptors of a code can be not only molecular structures but also, more commonly, biological processes. Notably, different levels of a biosystem have their own mechanisms of information management, and gene-coded parts inheritance preserves "parts information" while karyotype-coded system inheritance preserves the "system information" which organizes parts information. The karyotype code prompts many questions regarding the flow of biological information, including the distinction between information creation, maintenance, modification, and usage, along with differences between living and non-living systems. How do biological systems exist, reproduce, and self-evolve for increased complexity and diversity? Inheritance is mediated by organic codes which function as informational tools to organize chemical reactions, create new information, and preserve frozen accidents, transforming historical miracles into biological routines.

Two-phased evolution: Genome chaos-mediated information creation and maintenance.

Cancer is traditionally labeled a "cellular growth problem." However, it is fundamentally an issue of macroevolution where new systems emerge from tissue by breaking various constraints. To study this process, we used experimental platforms to "watch evolution in action" by comparing the profiles of karyotypes, transcriptomes, and cellular phenotypes longitudinally before, during, and after key phase transitions. This effort, alongside critical rethinking of current gene-based genomic and evolutionary theory, led to the development of the Genome Architecture Theory. Following a brief historical review, we present four case studies and their takeaways to describe the pattern of genome-based cancer evolution. Our discoveries include 1. The importance of non-clonal chromosome aberrations or NCCAs; 2. Two-phased cancer evolution, comprising a punctuated phase and a gradual phase, dominated by karyotype changes and gene mutation/epigenetic alterations, respectively; 3. How the karyotype codes system inheritance, which organizes gene interactions and provides the genomic basis for physiological regulatory networks; and 4. Stress-induced genome chaos, which creates genomic information by reorganizing chromosomes for macroevolution. Together, these case studies redefine the relationship between cellular macro- and microevolution: macroevolution does not equal microevolution + time. Furthermore, we incorporate genome chaos and gene mutation in a general model: genome reorganization creates new karyotype coding, then diverse cancer gene mutations can promote the dominance of tumor cell populations. Finally, we call for validation of the Genome Architecture Theory of cancer and organismal evolution, as well as the systematic study of genomic information flow in evolutionary processes.

Genome Chaos, Information Creation, and Cancer Emergence: Searching for New Frameworks on the 50th Anniversary of the "War on Cancer".

The year 2021 marks the 50th anniversary of the National Cancer Act, signed by President Nixon, which declared a national "war on cancer." Powered by enormous financial support, this past half-century has witnessed remarkable progress in understanding the individual molecular mechanisms of cancer, primarily through the characterization of cancer genes and the phenotypes associated with their pathways. Despite millions of publications and the overwhelming volume data generated from the Cancer Genome Project, clinical benefits are still lacking. In fact, the massive, diverse data also unexpectedly challenge the current somatic gene mutation theory of cancer, as well as the initial rationales behind sequencing so many cancer samples. Therefore, what should we do next? Should we continue to sequence more samples and push for further molecular characterizations, or should we take a moment to pause and think about the biological meaning of the data we have, integrating new ideas in cancer biology? On this special anniversary, we implore that it is time for the latter. We review the Genome Architecture Theory, an alternative conceptual framework that departs from gene-based theories. Specifically, we discuss the relationship between genes, genomes, and information-based platforms for future cancer research. This discussion will reinforce some newly proposed concepts that are essential for advancing cancer research, including two-phased cancer evolution (which reconciles evolutionary contributions from karyotypes and genes), stress-induced genome chaos (which creates new system information essential for macroevolution), the evolutionary mechanism of cancer (which unifies diverse molecular mechanisms to create new karyotype coding during evolution), and cellular adaptation and cancer emergence (which explains why cancer exists in the first place). We hope that these ideas will usher in new genomic and evolutionary conceptual frameworks and strategies for the next 50 years of cancer research.

Origins and Consequences of Chromosomal Instability: From Cellular Adaptation to Genome Chaos-Mediated System Survival.

When discussing chromosomal instability, most of the literature focuses on the characterization of individual molecular mechanisms. These studies search for genomic and environmental causes and consequences of chromosomal instability in cancer, aiming to identify key triggering factors useful to control chromosomal instability and apply this knowledge in the clinic. Since cancer is a phenomenon of new system emergence from normal tissue driven by somatic evolution, such studies should be done in the context of new genome system emergence during evolution. In this perspective, both the origin and key outcome of chromosomal instability are examined using the genome theory of cancer evolution. Specifically, chromosomal instability was linked to a spectrum of genomic and non-genomic variants, from epigenetic alterations to drastic genome chaos. These highly diverse factors were then unified by the evolutionary mechanism of cancer. Following identification of the hidden link between cellular adaptation (positive and essential) and its trade-off (unavoidable and negative) of chromosomal instability, why chromosomal instability is the main player in the macro-cellular evolution of cancer is briefly discussed. Finally, new research directions are suggested, including searching for a common mechanism of evolutionary phase transition, establishing chromosomal instability as an evolutionary biomarker, validating the new two-phase evolutionary model of cancer, and applying such a model to improve clinical outcomes and to understand the genome-defined mechanism of organismal evolution.

What Is Karyotype Coding and Why Is Genomic Topology Important for Cancer and Evolution?

While the importance of chromosomal/nuclear variations vs. gene mutations in diseases is becoming more appreciated, less is known about its genomic basis. Traditionally, chromosomes are considered the carriers of genes, and genes define bio-inheritance. In recent years, the gene-centric concept has been challenged by the surprising data of various sequencing projects. The genome system theory has been introduced to offer an alternative framework. One of the key concepts of the genome system theory is karyotype or chromosomal coding: chromosome sets function as gene organizers, and the genomic topologies provide a context for regulating gene expression and function. In other words, the interaction of individual genes, defined by genomic topology, is part of the full informational system. The genes define the "parts inheritance," while the karyotype and genomic topology (the physical relationship of genes within a three-dimensional nucleus) plus the gene content defines "system inheritance." In this mini-review, the concept of karyotype or chromosomal coding will be briefly discussed, including: 1) the rationale for searching for new genomic inheritance, 2) chromosomal or karyotype coding (hypothesis, model, and its predictions), and 3) the significance and evidence of chromosomal coding (maintaining and changing the system inheritance-defined bio-systems). This mini-review aims to provide a new conceptual framework for appreciating the genome organization-based information package and its ultimate importance for future genomic and evolutionary studies.