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.

Detecting Chromosome Condensation Defects in Gulf War Illness Patients.

BACKGROUND: Gulf War Illness (GWI) impacts 25-30% of gulf war veterans. Due to its heterogeneity in both etiology and symptoms, it has been challenging to establish the commonly accepted case definition for GWI. Equally challenging are the understanding of the general mechanism of GWI and the development of biomarkers useful for its clinical diagnosis and treatment. OBJECTIVE: We have observed that chromosome condensation defects can be detected in GWI patients. To document this phenomenon in GWI, we aim to describe and compare different types of chromosomal condensation defects in GWI patients, if possible. Since chromosomal condensation represents an important step of ensuring genome integrity, condensation defects could be used as a potential biomarker of GWI. METHODS: Lymphocytes from GWI patients have been used for short term cell culture followed by chromosome slide preparation. Both Giemsa staining and multiple color spectral karyotyping (SKY) were applied to study chromosome aberrations, focusing on different types of condensation defects. RESULTS: At least three subtypes of Defective Mitotic Figures (DMFs) were observed. Some individuals displayed elevated frequencies of DMFs. Another type of condensation defect identified as sticky chromosomes were also observed. CONCLUSION: Various types of condensation defects have been observed in GWI patients. It is rather surprising that some GWI patients exhibited a high level of chromosomal condensation defects. Previously, the elevated frequency of DMFs was only observed in cancer patients. Since chromosome condensation can be linked to other types of chromosome aberrations, as well as cellular stress conditions, the detailed mechanism and clinical impact should be further studied, especially with increased sample size.

A Postgenomic Perspective on Molecular Cytogenetics.

BACKGROUND: The postgenomic era is featured by massive data collection and analyses from various large scale-omics studies. Despite the promising capability of systems biology and bioinformatics to handle large data sets, data interpretation, especially the translation of -omics data into clinical implications, has been challenging. DISCUSSION: In this perspective, some important conceptual and technological limitations of current systems biology are discussed in the context of the ultimate importance of the genome beyond the collection of all genes. Following a brief summary of the contributions of molecular cytogenetics/cytogenomics in the pre- and post-genomic eras, new challenges for postgenomic research are discussed. Such discussion leads to a call to search for a new conceptual framework and holistic methodologies. CONCLUSION: Throughout this synthesis, the genome theory of somatic cell evolution is highlighted in contrast to gene theory, which ignores the karyotype-mediated higher level of genetic information. Since "system inheritance" is defined by the genome context (gene content and genomic topology) while "parts inheritance" is defined by genes/epigenes, molecular cytogenetics and cytogenomics (which directly study genome structure, function, alteration and evolution) will play important roles in this postgenomic era.

Evolutionary mechanism unifies the hallmarks of cancer.

The basis for the gene mutation theory of cancer that dominates current molecular cancer research consists of: the belief that gene-level aberrations such as mutations are the main cause of cancers, the concept that stepwise gene mutation accumulation drives cancer progression, and the hallmarks of cancer. The research community swiftly embraced the hallmarks of cancer, as such synthesis has supported the notions that common cancer genes are responsible for the majority of cancers and the complexity of cancer can be dissected into simplified molecular principles. The gene/pathway classification based on individual hallmarks provides explanation for the large number of diverse gene mutations, which is in contrast to the original estimation that only a handful of gene mutations would be discovered. Further, these hallmarks have been highly influential as they also provide the rationale and research direction for continued gene-based cancer research. While the molecular knowledge of these hallmarks is drastically increasing, the clinical implication remains limited, as cancer dynamics cannot be summarized by a few isolated/fixed molecular principles. Furthermore, the highly heterogeneous genetic signature of cancers, including massive stochastic genome alterations, challenges the utility of continuously studying each individual gene mutation under the framework of these hallmarks. It is therefore necessary to re-evaluate the concept of cancer hallmarks through the lens of cancer evolution. In this analysis, the evolutionary basis for the hallmarks of cancer will be discussed and the evolutionary mechanism of cancer suggested by the genome theory will be employed to unify the diverse molecular mechanisms of cancer.

Unstable genomes elevate transcriptome dynamics.

The challenge of identifying common expression signatures in cancer is well known, however the reason behind this is largely unclear. Traditionally variation in expression signatures has been attributed to technological problems, however recent evidence suggests that chromosome instability (CIN) and resultant karyotypic heterogeneity may be a large contributing factor. Using a well-defined model of immortalization, we systematically compared the pattern of genome alteration and expression dynamics during somatic evolution. Co-measurement of global gene expression and karyotypic alteration throughout the immortalization process reveals that karyotype changes influence gene expression as major structural and numerical karyotypic alterations result in large gene expression deviation. Replicate samples from stages with stable genomes are more similar to each other than are replicate samples with karyotypic heterogeneity. Karyotypic and gene expression change during immortalization is dynamic as each stage of progression has a unique expression pattern. This was further verified by comparing global expression in two replicates grown in one flask with known karyotypes. Replicates with higher karyotypic instability were found to be less similar than replicates with stable karyotypes. This data illustrates the karyotype, transcriptome, and transcriptome determined pathways are in constant flux during somatic cellular evolution (particularly during the macroevolutionary phase) and this flux is an inextricable feature of CIN and essential for cancer formation. The findings presented here underscore the importance of understanding the evolutionary process of cancer in order to design improved treatment modalities.

Chromosomal instability and transcriptome dynamics in cancer.

Whole transcriptome profiling has long been proposed as a method of identifying cancer-specific gene expression profiles. Indeed, a multitude of these studies have generated vast amounts of expression data for many types of cancer, and most have identified specific gene signatures associated with a given cancer. These studies however, often contradict with each other, and gene lists only rarely overlap, challenging clinical application of cancer gene signatures. To understand this issue, the biological basis of transcriptome dynamics needs to be addressed. Chromosome instability (CIN) is the main contributor to genome heterogeneity and system dynamics, therefore the relationship between CIN, genome heterogeneity, and transcriptome dynamics has important implications for cancer research. In this review, we discuss CIN and its effects on the transcriptome during cancer progression, specifically how stochastic chromosome change results in transcriptome dynamics. This discussion is further applied to metastasis and drug resistance both of which have been linked to multiple diverse molecular mechanisms but are in fact driven by CIN. The diverse molecular mechanisms that drive each process are linked to karyotypic heterogeneity through the evolutionary mechanism of cancer. Karyotypic change and the resultant transcriptome change alter network function within cells increasing the evolutionary potential of the tumor. Future studies must embrace this instability-induced heterogeneity in order to devise new research and treatment modalities that focus on the evolutionary process of cancer rather than the individual genes that are uniquely changed in each tumor. Care is also needed in evaluating results from experimental systems which measure average values of a population.

Chromosomal instability (CIN): what it is and why it is crucial to cancer evolution.

Results of various cancer genome sequencing projects have "unexpectedly" challenged the framework of the current somatic gene mutation theory of cancer. The prevalence of diverse genetic heterogeneity observed in cancer questions the strategy of focusing on contributions of individual gene mutations. Much of the genetic heterogeneity in tumors is due to chromosomal instability (CIN), a predominant hallmark of cancer. Multiple molecular mechanisms have been attributed to CIN but unifying these often conflicting mechanisms into one general mechanism has been challenging. In this review, we discuss multiple aspects of CIN including its definitions, methods of measuring, and some common misconceptions. We then apply the genome-based evolutionary theory to propose a general mechanism for CIN to unify the diverse molecular causes. In this new evolutionary framework, CIN represents a system behavior of a stress response with adaptive advantages but also serves as a new potential cause of further destabilization of the genome. Following a brief review about the newly realized functions of chromosomes that defines system inheritance and creates new genomes, we discuss the ultimate importance of CIN in cancer evolution. Finally, a number of confusing issues regarding CIN are explained in light of the evolutionary function of CIN.

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.

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.

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.

Why imatinib remains an exception of cancer research.

The archetype driving the drug targeting approach to cancer therapy is the success of imatinib against chronic phase chronic myeloid leukemia (CML-CP). Molecular targeting success of this magnitude has yet to be repeated for most solid tumors. To answer why imatinib remains an exception of cancer research, we summarize key features and patterns of evolution that contrast CML-CP from prostate cancer, an example of a solid tumor that also shares a signature fusion gene. Distinctive properties of CML-CP include: a large cell population size that is not geographically constrained, a highly penetrant dominant oncogene that sweeps the entire cell population, subsequent progressive and ordered clonal genetic changes, and the effectiveness of molecular targeting within the chronic phase, which is comparable to the benign phase of solid tumors. CML-CP progression resembles a clonal, stepwise model of evolution, whereas the pattern of solid tumor evolution is highly dynamic and stochastic. The distinguishing features and evolutionary pattern of CML-CP support why the success of imatinib does not carry over to most solid tumors. Changing the focus of cancer research from a gene-based view to a genome-based theory will provide insight into solid tumor evolutionary dynamics.

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.

Decoding the genome beyond sequencing: the new phase of genomic research.

While our understanding of gene-based biology has greatly improved, it is clear that the function of the genome and most diseases cannot be fully explained by genes and other regulatory elements. Genes and the genome represent distinct levels of genetic organization with their own coding systems; Genes code parts like protein and RNA, but the genome codes the structure of genetic networks, which are defined by the whole set of genes, chromosomes and their topological interactions within a cell. Accordingly, the genetic code of DNA offers limited understanding of genome functions. In this perspective, we introduce the genome theory which calls for the departure of gene-centric genomic research. To make this transition for the next phase of genomic research, it is essential to acknowledge the importance of new genome-based biological concepts and to establish new technology platforms to decode the genome beyond sequencing.

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.

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.