The ecology of brain tumors: lessons learned from neurofibromatosis-1

Traditionally, cancer studies have primarily focused on mutations that activate growth or survival pathways in susceptible pre-neoplastic/neoplastic cells. However, recent research has revealed a critical role for non-neoplastic cells within the tumor microenvironment in the process of cancer formation and progression. In addition, the existence of regional and developmental variations in susceptible cell types and supportive microenvironments support a model of tumorigenesis in which the dynamic symbiotic relationship between neoplastic and non-neoplastic cell types dictate where and when cancers form and grow. In this review, we highlight advances in neurofibromatosis type 1 (NF1) genetically engineered mouse brain tumor (glioma) modeling to reveal how cellular and molecular heterogeneity in both the pre-neoplastic/neoplastic and non-neoplastic cellular compartments contribute to gliomagenesis and glioma growth.     

Pathways to Tumorigenesis—Modeling Mutation Acquisition in Stem Cells and Their Progeny

Most adult tissues consist of stem cells, progenitors, and mature cells, and this hierarchical architecture may play an important role in the multistep process of carcinogenesis. Here, we develop and discuss the important predictions of a simple mathematical model of cancer initiation and early progression within a hierarchically structured tissue. This work presents a model that incorporates both the sequential acquisition of phenotype altering mutations and tissue hierarchy. The model simulates the progressive effect of accumulating mutations that lead to an increase in fitness or the induction of genetic instability. A novel aspect of the model is that symmetric self-renewal, asymmetric division, and differentiation are all incorporated, and this enables the quantitative study of the effect of mutations that deregulate the normal, homeostatic stem cell division pattern. The model is also capable of predicting changes in both tissue composition and in the progression of cells along their lineage at any given time and for various sequences of mutations. Simulations predict that the specific order in which mutations are acquired is crucial for determining the pace of cancer development. Interestingly, we find that the importance of genetic stability differs significantly depending on the physiological expression of mutations related to symmetric self-renewal and differentiation of stem and progenitor cells. In particular, mutations that lead to the alteration of the stem cell division pattern or the acquisition of some degree of immortality in committed progenitors lead to an early onset of cancer and diminish the impact of genetic instability.     

Breast cancer intra-tumor heterogeneity

In recent years it has become clear that cancer cells within a single tumor can display striking morphological, genetic and behavioral variability. Burgeoning genetic, epigenetic and phenomenological data support the existence of intra-tumor genetic heterogeneity in breast cancers; however, its basis is yet to be fully defined. Two of the most widely evoked concepts to explain the origin of heterogeneity within tumors are the cancer stem cell hypothesis and the clonal evolution model. Although the cancer stem cell model appeared to provide an explanation for the variability among the neoplastic cells within a given cancer, advances in massively parallel sequencing have provided several lines of evidence to suggest that intra-tumor genetic heterogeneity likely plays a fundamental role in the phenotypic heterogeneity observed in cancers. Many challenges remain, however, in the interpretation of the next generation sequencing results obtained so far. Here we review the models that explain tumor heterogeneity, the causes of intra-tumor genetic diversity and their impact on our understanding and management of breast cancer, methods to study intra-tumor heterogeneity and the assessment of intra-tumor genetic heterogeneity in the clinic.     

The initiation of colon cancer in a chronic inflammatory setting

Chronic inflammation predisposes to cancer. We used an inflammation-induced human model of tumorigenesis to explore how populations of mutated cells expand and initiate the earliest stages of cancer. Ulcerative colitis (UC) is a chronic inflammatory disease of the colon associated with an increased risk of colorectal cancer mediated through a process of genomic instability. In order to characterize the process of clonal expansion, arbitrary primed (AR) and inter-simple sequence repeat (ISSR) PCR DNA fingerprint mutation profiles of single crypts were compared with the mutational profiles from clusters of crypts and whole biopsies within the same individual. To provide information at the earliest steps of neoplastic progression, we examined histologically negative crypts, as well as dysplastic crypts. Crypts from UC dysplasia/cancer show alterations in 10-20% of DNA fingerprint sites, regardless of (i) whether the crypts were dysplastic or non-dysplastic and (ii) whether the DNA came from one crypt or thousands of crypts. Of the mutational changes in single crypts, almost half are clonally expanded to adjacent crypts and/or to the thousands of crypts in a single biopsy. Using fluorescent in-situ hybridization to examine p53 alterations in individual crypt cells, we demonstrate that the mechanism of clonal expansion can occur through crypt fission. DNA alterations are initiated in colonic crypts and expand to adjacent crypts through crypt fission. Our data suggest that a continuous process of DNA mutations, clonal expansion through crypt fission and clonal succession initiates the development of inflammatory-associated colon cancer; this mutational process is moderated by crypt cell turn-over and cell death. This paradigm may apply to other inflammatory-induced cancers.     

Cancer stem cells: Back to Darwin?

Current models of cancer propagation or ‘stem’ cells pay scant attention to the evolutionary dynamics of cancer or to the underlying genetic, mutational drivers. Recent genetic studies on acute lymphoblastic leukaemia at the single cell level reveal a complex non-linear, branching clonal architecture—with sub-clones having distinctive genetic signatures. Most cancers appropriately interrogated are found to have intra-clonal genetic heterogeneity indicative of divergent clonal evolution. These data further suggest that clonal architecture might be driven by genetic heterogeneity of propagating or ‘stem’ cells. When assayed for leukaemic regeneration in NOD/SCID/γ mice, genetically diverse ‘stem’ cells read-out, broadly reflecting the clonal architecture. This has suggested a ‘back to Darwin’ model for cancer propagation. In this, cells with self-renewal potency or ‘stem-ness’ provide genetically diverse units of evolutionary selection in cancer progression. The model has significant implications for targeted cancer therapy.     

Natural selection in neoplastic progression of Barrett's esophagus

Neoplasms progress to cancer through a process of natural selection. The rate of evolution, and thus progression is determined by three parameters: mutation rate, population size of the evolving neoplastic cells, and intensity of selection or rate of clonal expansion. All three parameters are reviewed in the context of Barrett's esophagus, a pre-malignant neoplasm. Although Barrett's esophagus is an ideal model for the study of neoplastic clonal evolution, similar studies may be carried out in a wide variety of human neoplasms. Evolutionary analyses provide insights for clinical management, including rates of progression to cancer and emergence of resistance to interventions.     

Tumor cell progression and differentiation in metastasis

The development and evolution of tumors is regulated by both genetic and epigenetic events. It is thought that these processes tend to drive neoplastic development in opposing directions so that tumor progression, predominantly as a consequence of mutational events, leads to increasing tumor aggression. Conversely the induction of differentiation, largely through epigenetic mechanisms, tends to cause tumors to evolve to a more benign phenotype. However, these generalizations are a simplistic view of a complex dynamic event where both processes can be overlaid within a single neoplasm. Using malignant melanoma as a model system the alterations in gene expression and their effects upon metastatic dissemination, that accompany some of these changes, both natural and induced, are described.     

The genomic evolution of human prostate cancer

Prostate cancers are highly prevalent in the developed world, with inheritable risk contributing appreciably to tumour development. Genomic heterogeneity within individual prostate glands and between patients derives predominantly from structural variants and copy-number aberrations. Subtypes of prostate cancers are being delineated through the increasing use of next-generation sequencing, but these subtypes are yet to be used to guide the prognosis or therapeutic strategy. Herein, we review our current knowledge of the mutational landscape of human prostate cancer, describing what is known of the common mutations underpinning its development. We evaluate recurrent prostate-specific mutations prior to discussing the mutational events that are shared both in prostate cancer and across multiple cancer types. From these data, we construct a putative overview of the genomic evolution of human prostate cancer.     

Cancer driver mutations in protein kinase genes

Recent studies investigating the genetic determinants of cancer suggest that some of the genetic alterations contributing to tumorigenesis may be inherited, but the vast majority is somatically acquired during the transition of a normal cell to a cancer cell. A systematic understanding of the genetic and molecular determinants of cancers has already begun to have a transformative effect on the study and treatment of cancer, particularly through the identification of a range of genetic alterations in protein kinase genes, which are highly associated with the disease. Since kinases are prominent therapeutic targets for intervention within the cancer cell, studying the impact that genomic alterations within them have on cancer initiation, progression, and treatment is both logical and timely. In fact, recent sequencing and resequencing (i.e., polymorphism identification) efforts have catalyzed the quest for protein kinase ‘driver’ mutations (i.e., those genetic alterations which contribute to the transformation of a normal cell to a proliferating cancerous cell) in distinction to kinase ‘passenger’ mutations which reflect mutations that merely build up in course of normal and unchecked (i.e., cancerous) somatic cell replication and proliferation. In this review, we discuss the recent progress in the discovery and functional characterization of protein kinase cancer driver mutations and the implications of this progress for understanding tumorigenesis as well as the design of ‘personalized’ cancer therapeutics that target an individual’s unique mutational profile.     

Somatic alterations and metabolic polymorphisms.

Cancer is a multistep process in which multiple genetic alterations occur, causing a cumulative adverse effect on the control of cell differentiation, cell division and growth control. Somatic alterations acquired at the level of the cell become fixed in the developing cancer as chromosomal translocations, deletions, inversions, amplifications or point mutations. Since the ultimate unit of susceptibility to carcinogens is at the level of the cell, somatic gene alterations play an important role in carcinogenesis, as all neoplastic tumours exhibit somatic alterations. The incorporation of somatic alterations, such as oncogenes and tumour-suppressor genes, into epidemiological research provides an opportunity to clarify the role of exposure, other genetic changes and prognosis in cancer pathogenesis. The manner in which environmental factors act to initiate, accelerate or retard neoplastic progression Is currently being investigated using somatic gene mutational spectra to identify specific etiological carcinogens. Exploring the relationships between germline and somatic alterations may help to identify the timing of genetic events, important etiological exposures and gene-gene epistatic phenomena. Examining somatic alterations within the context of carcinogen-metabolizing enzymes may elucidate specific carcinogenic mechanisms. The use of somatic alterations to predict prognoses for patients with various malignancies may also help to enhance our ability to define subgroups of patients with different disease courses and treatment responses.     

The biology of acute myeloblastic leukemia

Many questions about the biology of AML remain to be answered. The initial genetic lesions that inhibit differentiation and increase the likelihood of self renewal have yet to be identified. Given the heterogeneity of this neoplasm, it is possible that many different mutational events may be capable of triggering leukemia. Alternatively, there may be only a small number of possible initial leukemic mutations, and the heterogeneous phenotype of the disease is determined by the evolution of different subclones that have acquired different secondary mutations. Studies with retroviral oncogenes have suggested that a common secondary event in an evolving myeloid tumor is the development of growth factor independence by either leukemic cell production of CSF or possibly constitutive activation of a CSF receptor. These mechanisms have not yet been established as important in human AML, although there is intriguing evidence to suggest that CSF genes are inappropriately activated in many cases.     

The clonal evolution of metastases from primary serous epithelial ovarian cancers

Several models of evolution from primary cancers to metastases have been proposed; but the most widely accepted is the clonal evolution model proposed for colorectal cancer in which tumors develop by a process of linear clonal evolution driven by the accumulation of somatic genetic alterations. Various other models of cancer progression and metastasis have been proposed, including parallel evolution and the same gene model. The aim of this study was to investigate the evolution of metastases from primary cancer in 22 patients diagnosed with high‐grade serous epithelial ovarian cancer. We established somatic genetic profiles based on the pattern of loss of heterozygosity, in several different regions of tumor tissue within the primary tumor and metastatic deposits from each case. Maximum parsimony tree analysis was used to examine the evolutionary relationship between the primary and metastatic samples for each patient. In addition, we investigated the extent of genetic heterogeneity within and between metastatic tumors compared with primary ovarian tumors. Our data suggest that most, if not all, metastases are clonally related to the primary tumors. However, the data oppose a single model of linear‐clonal evolution whereby a late stage clone within the primary tumor acquires additional genetic changes that enable metastatic progression. Instead, the data support a model in which primary ovarian cancers have a common clonal origin, but become polyclonal, with different clones at both early and late stages of genetic divergence acquiring the ability to progress to metastasis. © 2009 Wiley‐Liss, Inc.     

Hepatosinusoidal leukaemia/lymphoma consisting of epstein-barr virus-containing natural killer cell leukaemia/lymphoma and T-cell lymphoma; mimicking malignant histiocytosis

Previously diagnosed cases of hepatosinusoidal T-cell lymphoma and malignant histiocytosis (MH) may include lymphoid neoplasms of natural killer (NK) cell lineage associated with Epstein-Barr virus (EBV). Such hepatosinusoidal neoplasms were found to demonstrate hepatomegaly but not lymphadenopathy, and all were diagnosed by a liver biopsy. Sixteen adult patients diagnosed with hepatosinusoidal leukaemia/lymphoma (six NK-cell leukaemia/lymphomas [NKLLs], five instances of MH, three T-cell malignant lymphomas [T-MLs], and two adult T-cell leukaemia/lymphomas [ATLLs] were examined for EBV by in situ hybridization, then were studied immunohistochemically and subjected to a DNA analysis. Among our five patients with MH, neoplastic cells showed T-cells, but no histiocytic markers, and they were considered to have either a T-cell or NK-cell lineage. All NKLLs, MHs and T-MLs, except for ATLLs accompanied by reactive hemophagocytic histiocytes, varied in number in each case. In situ hybridization revealed the presence of EBV in the nuclei of atypical cells in all of the six lymphoid neoplasms of NK-cell lineage. Each case of MH and each T-ML which represented EBV demonstrated no definite T-cell or histiocytic markers. Patients with ATLL did not reveal EBV. In all patients with hemophagocytosis, EBV was present in the nuclei of the neoplastic lymphocytes, but not in the hemophagocytic cells. Finally, the 16 cases were reclassified into eight cases with EBV -containing NKLLs, six T-MLs, and two ATLLs. In addition, no true histiocytic neoplasms were observed. The mechanism of hemophagocytosis may be therefore the production of lymphokines (macrophage-activating factors) by neoplastic lymphocytes. EBV-associated hepatosinusoidal leukaemia/lymphoma may thus contain a lymphoid neoplasm of NK-cell lineage, which made it difficult to be distinguished from the previously designated malignant histiocytosis.     

Progression from ductal carcinoma in situ to invasive breast cancer: Revisited

Ductal carcinoma in situ (DCIS) is an intraductal neoplastic proliferation of epithelial cells that is separated from the breast stroma by an intact layer of basement membrane and myoepithelial cells. DCIS is a non-obligate precursor of invasive breast cancer, and up to 40% of these lesions progress to invasive disease if untreated. Currently, it is not possible to predict accurately which DCIS would be more likely to progress to invasive breast cancer as neither the significant drivers of the invasive transition have been identified, nor has the clinical utility of tests predicting the likelihood of progression been demonstrated. Although molecular studies have shown that qualitatively, synchronous DCIS and invasive breast cancers are remarkably similar, there is burgeoning evidence to demonstrate that intra-tumor genetic heterogeneity is observed in a subset of DCIS, and that the process of progression to invasive disease may constitute an ‘evolutionary bottleneck’, resulting in the selection of subsets of tumor cells with specific genetic and/or epigenetic aberrations. Here we review the clinical challenge posed by DCIS, the contribution of the microenvironment and genetic aberrations to the progression from in situ to invasive breast cancer, the emerging evidence of the impact of intra-tumor genetic heterogeneity on this process, and strategies to combat this heterogeneity.     

Cancer prevention strategies that address the evolutionary dynamics of neoplastic cells: simulating benign cell boosters and selection for chemosensitivity.

Cells in neoplasms evolve by natural selection. Traditional cytotoxic chemotherapies add further selection pressure to the evolution of neoplastic cells, thereby selecting for cells resistant to the therapies. An alternative proposal is a benign cell booster. Rather than trying to kill the highly dysplastic or malignant cells directly, a benign cell booster increases the fitness of the more benign cells, which may be either normal or benign clones, so that they may outcompete more advanced or malignant cells in a neoplasm. In silico simulations of benign cell boosters in neoplasms with evolving clones show benign cell boosters to be effective at destroying advanced or malignant cells and preventing relapse even when applied late in progression. These results are conditional on the benign cell boosters giving a competitive advantage to the benign cells in the neoplasm. Furthermore, the benign cell boosters must be applied over a long period of time in order for the benign cells to drive the dysplastic cells to extinction or near extinction. Most importantly, benign cell boosters based on this strategy must target a characteristic of the benign cells that is causally related to the benign state to avoid relapse. Another promising strategy is to boost cells that are sensitive to a cytotoxin, thereby selecting for chemosensitive cells, and then apply the toxin. Effective therapeutic and prevention strategies will have to alter the competitive dynamics of a neoplasm to counter progression toward invasion, metastasis, and death.     

Survivin expression in ganglioglioma

Summary Gangliogliomas are unusual central nervous system (CNS) neoplasms occurring mainly in children and young adults and inducing chronic pharmacoresistant epilepsy. These are usually well differentiated neuroepithelial tumors composed of neurons in association with neoplastic glial cells. Gangliogliomas present with favorable outcome. However, some may recur and/or progress to anaplasia and be associated with a dismal prognosis. Since histopathological features do not consistently correlate with clinical outcome, reliable prognostic factors have yet to be defined in gangliogliomas. Survivin is an anti-apoptotic protein whose expression has been found to be of prognostic significance in many human cancers, including gliomas. The objective of this study was to assess survivin expression using immunohistochemistry in 15 gangliogliomas. Ten lesions were low-grade neoplasms whereas 5 were high-grade tumors. Survivin expression appeared restricted to the neoplastic glial component and was detected in 6/15 gangliogliomas. Two additional tumors expressed survivin upon relapse. Half survivin expressing lesions displayed less than 1% immunoreactive cells. Survivin expression in more than 5% neoplastic glial cells was detected only in malignant and/or recurrent gangliogliomas. Extended lifespan in survivin expressing cells might enhance aggressive behavior in these tumors through accumulation of mutations, thereby allowing progression to malignant phenotypes. Survivin expression may carry a negative prognostic value in gangliogliomas.     

A cascade of modules of a network defines cancer progression

Similar histologic subtypes of cancers often exhibit different spectrum of genetic and epigenetic alterations. The heterogeneity observed due to lack of consistent and defined alterations affecting a unique set of gene(s) or gene products in cancers derived from a specific tissue, or an organ, pose a challenge in unraveling the molecular basis of the disease. This dilemma also complicates diagnosis, prognosis, effective management, and treatment modalities. To streamline the available and emerging data into a coherent scheme of events, a multimodular molecular network (MMMN) cancer progression model is presented as a roadmap to dissect the complexity inherent to this disease. The fact that disruption/dysregulation of more than one alternate target gene could affect the functionality of each specific module of a cascade provides a molecular basis for genetic and epigenetic heterogeneity in any given cancer. Polymorphisms/mutations as well as the extracellular matrix and or the epigenetically/genetically conditioned surrounding stromal cells could also influence the rate of tumorigenesis and the properties of the tumor cells. The formulation of MMMN cancer progression models for specific cancers is likely to provide the blueprints for the markers and targets to aid diagnosis, prevention, and therapy of this deadly disease.     

Ultrastructural diagnosis of early neoplasia in man

Recent progress in cancer detection technology has resulted in the increasing need for pathological diagnosis of small cancers at their early stage of development. Electron microscopy of early cancers may reveal ultrastructural markers characteristic of certain cell lineage and help classify these cancers in terms of aberration in differentiation of neoplastic cells. Furthermore, electron microscopy of early cancers may detect certain cellular gene products at cellular as well as subcellular levels, and help understand the process of malignant progression in situ of transformed cells.     

Multistep skin cancer in mice as a model to study the evolution of cancer cells

Although much of cancer research relies on Nowell's clonal evolution hypothesis as a conceptual framework, large gaps remain in understanding how tumors develop. The multistage skin cancer model in mice provides continuing insight on fundamental aspects of tumor evolution. In this model, mutation of the oncogene Hras is frequently the initiating event while mutation of the tumor suppressor p53 is a late event, associated with malignant progression. Recent evidence demonstrates that intracellular signaling from the initial Hras mutation leads directly to the activation of p53, creating selective pressure in favor of cells with mutant p53. Thus, selection for subsequent mutations is mechanistically linked to the initial mutation, explaining the preferred order of mutational events observed. Analysis of this model also reveals that a diverse array of signals can selectively impair or enhance clonal expansion of Ras mutant cells into a visible neoplasm. These modifiers can be genetic, physiological, or environmental and are often highly specific to tumor cells. This indicates that tumor cells have an inherent reduced capacity to buffer against perturbations. Reduced buffering may play an important role in both tumor evolution and therapy response and may be a hallmark of cancer cells.