G-Cimp Status Prediction Of Glioblastoma Samples Using mRNA Expression Data

Glioblastoma Multiforme (GBM) is a tumor with high mortality and no known cure. The dramatic molecular and clinical heterogeneity seen in this tumor has led to attempts to define genetically similar subgroups of GBM with the hope of developing tumor specific therapies targeted to the unique biology within each of these subgroups. Recently, a subset of relatively favorable prognosis GBMs has been identified. These glioma CpG island methylator phenotype, or G-CIMP tumors, have distinct genomic copy number aberrations, DNA methylation patterns, and (mRNA) expression profiles compared to other GBMs. While the standard method for identifying G-CIMP tumors is based on genome-wide DNA methylation data, such data is often not available compared to the more widely available gene expression data. In this study, we have developed and evaluated a method to predict the G-CIMP status of GBM samples based solely on gene expression data.     

microRNA in the control of stem-like phenotype of cancer cells

Current therapies against metastatic tumors are still ineffective. Cancer stem cells — a small subset of cells inside the tumor that possesses a self-renewal capacity — might be responsible for the recurrence of the tumor after anti-cancer therapies. Their immortality and unique drug resistance impede their eradication during therapy. The ‘stemness’ of these cells is controlled by microRNAs. These molecules possess the ability to downregulate gene expression by binding to the target mRNA. It turns out that microRNAs control the expression of approximately 60% of the genes in human cells. MicroRNA aberrant expression can lead to cancer development and progression. Therefore, recent research has focused on unraveling the role of microRNA in maintaining a stem-like phenotype in malignant tumors and cancer stem cells. This review summarizes our current knowledge about microRNAs that control the self-renewal capacity of cancer stem cells and indicates the importance of profound research aimed at developing efficient miRNA-targeted therapies.     

Modeling the Repertoire of True Tumor-Specific MHC I Epitopes in a Human Tumor

DNA replication has a finite measurable error rate, net of repair, in all cells. Clonal proliferation of cancer cells leads therefore to accumulation of random mutations. A proportion of these mutational events can create new immunogenic epitopes that, if processed and presented by an MHC allele, may be recognized by the adaptive immune system. Here, we use probability theory to analyze the mutational and epitope composition of a tumor mass in successive division cycles and create a double Pölya model for calculating the number of truly tumor-specific MHC I epitopes in a human tumor. We deduce that depending upon tumor size, the degree of genomic instability and the degree of death within a tumor, human tumors have several tens to low hundreds of new, truly tumor-specific epitopes. Parenthetically, cancer stem cells, due to the asymmetry in their proliferative properties, shall harbor significantly fewer mutations, and therefore significantly fewer immunogenic epitopes. As the overwhelming majority of the mutations in cancer cells are unrelated to malignancy, the mutation-generated epitopes shall be specific for each individual tumor, and constitute the antigenic fingerprint of each tumor. These calculations highlight the benefits for personalization of immunotherapy of human cancer, and in view of the substantial pre-existing antigenic repertoire of tumors, emphasize the enormous potential of therapies that modulate the anti-cancer immune response by liberating it from inhibitory influences.     

Challenges and prospects of immunotherapy as cancer treatment

The concept of cancer immunotherapy stems from the proposed function of the immune system, called immunosurveillance, to protect against growing tumors. Due to genetic aberrations, tumor cells display an altered repertoire of MHC-associated peptides that can lead to the activation of immune cells able to eliminate the transformed cells. In some instances, under the pressure of the immune system, both the tumor and its microenvironment are shaped and immune-resistant tumor variants are selected initiating the process of cancer immunoediting. This can impair not only host-generated immunosurveillance, but also attempts to harness the immune response for therapeutic purposes, namely immunotherapy. Rather than being an exhaustive review of the different approaches of cancer immunotherapy, the focus of this review is to provide the reader with future challenges of the field by proposing 'second generation' immunotherapy approaches that take into account immunosubversive mechanisms adopted by tumor cells. After an introduction on the process of immunosurveillance and immunoescape we will analyze why current immunotherapy approaches have not fulfilled their promise and will finish by summarizing what are the challenges for future approaches.     

The metabolic state of cancer stem cells—a valid target for cancer therapy?

In the 1920s Otto Warburg first described high glucose uptake, aerobic glycolysis, and high lactate production in tumors. Since then high glucose uptake has been utilized in the development of PET imaging for cancer. However, despite a deepened understanding of the molecular underpinnings of glucose metabolism in cancer, this fundamental difference between normal and malignant tissue has yet to be employed in targeted cancer therapy in the clinic. In this review, we highlight attempts in the recent literature to target cancer cell metabolism and elaborate on the challenges and controversies of these strategies in general and in the context of tumor cell heterogeneity in cancer.     

Tumor-associated antigen arrays for the serological diagnosis of cancer

The recognition that human tumors stimulate the production of autoantibodies against autologous cellular proteins called tumor-associated antigens (TAAs) has opened the door to the possibility that autoantibodies could be exploited as serological tools for the early diagnosis and management of cancer. Cancer-associated autoantibodies are often driven by intracellular proteins that are mutated, modified, or aberrantly expressed in tumor cells and hence are regarded as immunological reporters that could help uncover molecular events underlying tumorigenesis. Emerging evidence suggests that each type of cancer might trigger unique autoantibody signatures that reflect the nature of the malignant process in the affected organ. The advent of novel genomic, proteomic, and high throughput approaches has accelerated interest in the serum autoantibody repertoire in human cancers for the discovery of candidate TAAs. The use of individual anti-TAA autoantibodies as diagnostic or prognostic tools has been tempered by their low frequency and heterogeneity in most human cancers. However, TAA arrays comprising several antigens significantly increase this frequency and hold great promise for the early detection of cancer, monitoring cancer progression, guiding individualized therapeutic interventions, and identification of novel therapeutic targets. Our recent studies suggest that the implementation of TAA arrays in screening programs for the diagnosis of prostate cancer and other cancers should be preceded by the optimization of their sensitivity and specificity through the careful selection of the most favorable combinations of TAAs.     

The cancer stem cell: premises, promises and challenges

Over the last decade, the notion that tumors are maintained by their own stem cells, the so-called cancer stem cells, has created great excitement in the research community. This review attempts to summarize the underlying concepts of this notion, to distinguish hard facts from beliefs and to define the future challenges of the field.     

Stromal biology of pancreatic cancer

The genetic paradigm of cancer, focused largely on sequential molecular aberrations and associated biological impact in the neoplastic cell compartment of malignant tumors, has dominated our view of cancer pathogenesis. For the most part, this conceptualization has overlooked the dynamic and complex contributions of the surrounding microenvironment comprised of non-tumor cells (stroma) that may resist, react to, and/or foster tumor development. Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease in which a prominent tumor stroma compartment is a defining characteristic. Indeed, the bulk of PDAC tumor volume consists of non-neoplastic fibroblastic, vascular, and inflammatory cells surrounded by immense quantities of extracellular matrix, far exceeding that found in most other tumor types. Remarkably, little is known about the composition and physiology of the PDAC tumor microenvironment, in particular, the role of stroma in tumor initiation and progression. This review attempts to define key challenges, opportunities and state-of-knowledge relating to the PDAC microenvironment research with an emphasis on how inflammatory processes and key cancer pathways may shape the ontogeny of the tumor stroma. Such knowledge may be used to understand the evolution and biology of this lethal cancer and may convert these insights into new points of therapeutic intervention.     

Reconciling the Structural Attributes of Avian Antibodies

Antibodies are high value therapeutic, diagnostic, biotechnological, and research tools. Combinatorial approaches to antibody discovery have facilitated access to unique antibodies by surpassing the diversity limitations of the natural repertoire, exploitation of immune repertoires from multiple species, and tailoring selections to isolate antibodies with desirable biophysical attributes. The V-gene repertoire of the chicken does not utilize highly diverse sequence and structures, which is in stark contrast to the mechanism employed by humans, mice, and primates. Recent exploitation of the avian immune system has generated high quality, high affinity antibodies to a wide range of antigens for a number of therapeutic, diagnostic and biotechnological applications. Furthermore, extensive examination of the amino acid characteristics of the chicken repertoire has provided significant insight into mechanisms employed by the avian immune system. A paucity of avian antibody crystal structures has limited our understanding of the structural consequences of these uniquely chicken features. This paper presents the crystal structure of two chicken single chain fragment variable (scFv) antibodies generated from large libraries by phage display against important human antigen targets, which capture two unique CDRL1 canonical classes in the presence and absence of a non-canonical disulfide constrained CDRH3. These structures cast light on the unique structural features of chicken antibodies and contribute further to our collective understanding of the unique mechanisms of diversity and biochemical attributes that render the chicken repertoire of particular value for antibody generation.     

Expanding the concepts and tools of metabolic engineering to elucidate cancer metabolism

The metabolic engineer's toolbox, comprising stable isotope tracers, flux estimation and analysis, pathway identification, and pathway kinetics and regulation, among other techniques, has long been used to elucidate and quantify pathways primarily in the context of engineering microbes for producing small molecules of interest. Recently, these tools are increasingly finding use in cancer biology due to their unparalleled capacity for quantifying intracellular metabolism of mammalian cells. Here, we review basic concepts that are used to derive useful insights about the metabolism of tumor cells, along with a number of illustrative examples highlighting the fundamental contributions of these methods to elucidating cancer cell metabolism. This area presents unique opportunities for metabolic engineering to expand its portfolio of applications into the realm of cancer biology and help develop new cancer therapies based on a new class of metabolically derived targets. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Clonal structure of rapid-onset MDV-driven CD4+ lymphomas and responding CD8+ T cells

Lymphoid oncogenesis is a life threatening complication associated with a number of persistent viral infections (e.g. EBV and HTLV-1 in humans). With many of these infections it is difficult to study their natural history and the dynamics of tumor formation. Marek's Disease Virus (MDV) is a prevalent α-herpesvirus of poultry, inducing CD4+ TCRαβ+ T cell tumors in susceptible hosts. The high penetrance and temporal predictability of tumor induction raises issues related to the clonal structure of these lymphomas. Similarly, the clonality of responding CD8 T cells that infiltrate the tumor sites is unknown. Using TCRβ repertoire analysis tools, we demonstrated that MDV driven CD4+ T cell tumors were dominated by one to three large clones within an oligoclonal framework of smaller clones of CD4+ T cells. Individual birds had multiple tumor sites, some the result of metastasis (i.e. shared dominant clones) and others derived from distinct clones of transformed cells. The smaller oligoclonal CD4+ cells may represent an anti-tumor response, although on one occasion a low frequency clone was transformed and expanded after culture. Metastatic tumor clones were detected in the blood early during infection and dominated the circulating T cell repertoire, leading to MDV associated immune suppression. We also demonstrated that the tumor-infiltrating CD8+ T cell response was dominated by large oligoclonal expansions containing both "public" and "private" CDR3 sequences. The frequency of CD8+ T cell CDR3 sequences suggests initial stimulation during the early phases of infection. Collectively, our results indicate that MDV driven tumors are dominated by a highly restricted number of CD4+ clones. Moreover, the responding CD8+ T cell infiltrate is oligoclonal indicating recognition of a limited number of MDV antigens. These studies improve our understanding of the biology of MDV, an important poultry pathogen and a natural infection model of virus-induced tumor formation.     

Hybrid-cell vaccines for cancer immune therapy

Hybrid cells generated by fusing allogenic-presenting cells, such as dendritic cells, with tumor cells are a new tool in cancer immunotherapy which are designed to enhance the immunogenicity of antigenic tumors by presenting the whole spectrum of tumor-associated antigens, by providing the co-stimulatory molecules required for T-cell activation, and by the expression of allogenic MHC molecules for recruitment and activation of T-cell help. This approach has been successfully tested in animal models as well as in clinical phaseI/II trials with various tumors. Besides clinical repsonses, induction of tumor-specific cytolytic T-cells were observed. The electrofusion protocol described here has the advantage of high fusion efficiency, high hybrid-cell viability, as well as high reproducibility, and can be used for various tumor cell types after minor adjustments are made to the instrument settings in order to process large numbers of dendritic cells with consistent efficiencies.     

Microenvironment determinants of brain metastasis

Metastasis accounts for 90% of cancer-related mortality. Brain metastases generally present during the late stages in the natural history of cancer progression. Recent advances in cancer treatment and management have resulted in better control of systemic disease metastatic to organs other than the brain and improved patient survival. However, patients who experience recurrent disease manifest an increasing number of brain metastases, which are usually refractory to therapies. To meet the new challenges of controlling brain metastasis, the research community has been tackling the problem with novel experimental models and research tools, which have led to an improved understanding of brain metastasis. The time-tested "seed-and-soil" hypothesis of metastasis indicates that successful outgrowth of deadly metastatic tumors depends on permissible interactions between the metastatic cancer cells and the site-specific microenvironment in the host organs. Consistently, recent studies indicate that the brain, the major component of the central nervous system, has unique physiological features that can determine the outcome of metastatic tumor growth. The current review summarizes recent discoveries on these tumor-brain interactions, and the potential clinical implications these novel findings could have for the better treatment of patients with brain metastasis.     

Bioinformatics for cancer immunology and immunotherapy

Recent mechanistic insights obtained from preclinical studies and the approval of the first immunotherapies has motivated increasing number of academic investigators and pharmaceutical/biotech companies to further elucidate the role of immunity in tumor pathogenesis and to reconsider the role of immunotherapy. Additionally, technological advances (e.g., next-generation sequencing) are providing unprecedented opportunities to draw a comprehensive picture of the tumor genomics landscape and ultimately enable individualized treatment. However, the increasing complexity of the generated data and the plethora of bioinformatics methods and tools pose considerable challenges to both tumor immunologists and clinical oncologists. In this review, we describe current concepts and future challenges for the management and analysis of data for cancer immunology and immunotherapy. We first highlight publicly available databases with specific focus on cancer immunology including databases for somatic mutations and epitope databases. We then give an overview of the bioinformatics methods for the analysis of next-generation sequencing data (whole-genome and exome sequencing), epitope prediction tools as well as methods for integrative data analysis and network modeling. Mathematical models are powerful tools that can predict and explain important patterns in the genetic and clinical progression of cancer. Therefore, a survey of mathematical models for tumor evolution and tumor–immune cell interaction is included. Finally, we discuss future challenges for individualized immunotherapy and suggest how a combined computational/experimental approaches can lead to new insights into the molecular mechanisms of cancer, improved diagnosis, and prognosis of the disease and pinpoint novel therapeutic targets.     

Why the Outcome of Anti-Tumor Immune Responses is Heterogeneous: A Novel Idea in the Context of Immunological Heterogeneity in Cancers

The question as to why some hosts can eradicate their tumors while others succumb to tumor-progression remains unanswered. Here, a provocative concept is proposed that intrinsic differences in the T cell receptor (TCR) repertoire of individuals may influence the outcome of anti-tumor immunity by affecting the frequency and/or variety of tumor-reactive CD8 and/or CD4 tumor-infiltrating lymphocytes. This idea implicates that the TCR repertoire in a given patient might not provide sufficiently different TCR clones that can recognize tumor antigens, namely, “a hole in the TCR repertoire” might exist. This idea may provide a novel perspective to further dissect the mechanisms underlying heterogeneous anti-tumor immune responses in different hosts. Besides tumor-intrinsic heterogeneity and host microbiome, the various factors that may constantly shape the dynamic TCR repertoire are also discussed. Elucidating mechanistic differences in different individuals’ immune systems will allow to better harness immune system to design new personalized cancer immunotherapy.     

Exploring cancer stem cell niche directed tumor growth

The finding that only a sub-fraction of tumor cells, so called Cancer Stem Cells (CSC), is endowed with the capacity to initiate new tumors has important consequences for fundamental as well as clinical cancer research. Previously we established by computational modeling techniques that CSC driven tumor growth instigates infiltrative behavior, and perhaps most interesting, stimulates tumor cell heterogeneity. An important question that remains is to what extend CSC functions are intrinsically regulated or whether this capacity is orchestrated by the microenvironment, i.e. a putative CSC niche. Here we investigate how extrinsic regulation of CSC properties affects the characteristics of malignancies. We find that highly invasive growth in tumors dependent on a small subset of cells is not restricted to CSC-driven tumors, but is also observed in tumors where the CSC capacity of tumor cells is completely defined by the microenvironment. Importantly, also the high level of heterogeneity that was observed for CSC-driven tumors is preserved and partially even increased in malignancies with a microenvironmentally orchestrated CSC population. This indicates that invasive growth and high heterogeneity are fundamental properties of tumors fueled by a small population of tumor cells.     

Hallmarks of cancer: the next generation

The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors. The hallmarks constitute an organizing principle for rationalizing the complexities of neoplastic disease. They include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. Underlying these hallmarks are genome instability, which generates the genetic diversity that expedites their acquisition, and inflammation, which fosters multiple hallmark functions. Conceptual progress in the last decade has added two emerging hallmarks of potential generality to this list-reprogramming of energy metabolism and evading immune destruction. In addition to cancer cells, tumors exhibit another dimension of complexity: they contain a repertoire of recruited, ostensibly normal cells that contribute to the acquisition of hallmark traits by creating the "tumor microenvironment." Recognition of the widespread applicability of these concepts will increasingly affect the development of new means to treat human cancer.     

Tumor-infiltrating lymphocytes in colorectal tumors display a diversity of T cell receptor sequences that differ from the T cells in adjacent mucosal tissue

Tumors from colorectal cancer (CRC) are generally immunogenic and commonly infiltrated with T lymphocytes. However, the details of the adaptive immune reaction to these tumors are poorly understood. We have accrued both colon tumor samples and adjacent healthy mucosal samples from 15 CRC patients to study lymphocytes infiltrating these tissues. We apply a method for detailed sequencing of T-cell receptor (TCR) sequences from tumor-infiltrating lymphocytes (TILs) in CRC tumors at high throughput to probe T-cell clones in comparison with the TCRs from adjacent healthy mucosal tissue. In parallel, we captured TIL counts using standard immunohistochemistry. The variation in diversity of the TIL repertoire was far wider than the variation of T-cell clones in the healthy mucosa, and the oligoclonality was higher on average in the tumors. However, the diversity of the T-cell repertoire in both CRC tumors and healthy mucosa was on average 100-fold lower than in peripheral blood. Using the TCR sequences to identify and track clones between mucosal and tumor samples, we determined that the immune response in the tumor is different than in the adjacent mucosal tissue, and the number of shared clones is not dependent on distance between the samples. Together, these data imply that CRC tumors induce a specific adaptive immune response, but that this response differs widely in strength and breadth between patients.     

Estrogen receptor alpha negative breast cancer patients: estrogen receptor beta as a therapeutic target

Clinical management of breast cancer is increasingly guided by assessment of tumor phenotypic parameters. One of these is estrogen receptor (ER) status, currently defined by ERalpha expression. However with the discovery of a second ER, ERbeta and its variant isoforms, the definition of ER status is potentially more complex. In breast tumors there are two ERbeta expression cohorts. One where ERbeta is co-expressed with ERalpha and the other expressing ERbeta alone. In the latter subgroup of currently defined ER negative patients ERbeta has the potential to be a therapeutic target. Characterization of the nature and role of ERbeta in ERalpha negative tumors is essentially unexplored but available data suggest that the role of ERbeta may be different when co-expressed with ERalpha and when expressed alone. This review summarizes available data and explores the possibility that ERbeta signaling may be a therapeutic target in these tumors. Evidence so far supports the idea that the role of ERbeta in breast cancer is different in ERalpha negative compared to ERalpha positive tumors. However, cohort size and numbers of independent studies are small to date, and more studies are needed with better standardization of antibodies and protocols. Also, the ability to determine the role of ERbeta in ERalpha negative breast cancer and therefore assess ERbeta signaling pathways as therapeutic targets would be greatly facilitated by identification of specific downstream markers of ERbeta activity in breast cancer.     

New basic discoveries and frontiers in diagnosis, prognosis and prediction of response to therapy in human thyroid, urinary bladder, and prostate tumors

The fourth edition of this workshop mainly focused on three different human oncotypes, which included thyroid, urinary bladder, and prostate tumors as clinical models to gain new basic knowledge on tumor diagnosis, prognosis, and treatment. At the previous editions (Giordano et al., 2000, J Cell Physiol 183:284-287; Giordano et al., 2001, J Cell Physiol 188:274-280; Giordano et al., 2002, J Cell Physiol 191:362-365), leaders in the fields of pathology, clinical oncology, and basic research presented and discussed the most recent and prevalent findings in such neoplasms from a basic and clinical perspective. A concept that has been widely proposed is that the analysis of intrinsic biological factors displayed by primary tumors may be a valid method for diagnosing different neoplasias and for measuring both their aggressiveness and response to therapy. To date, however, no single prognostic factor, such as oncogenes, suppressor genes, or genes involved in the control of the cell cycle and/or apoptosis has yet proven to be potent enough to be used in clinical practice as a prognostic and predictive factor. The new possibility to simultaneously analyze the expression of the complete repertoire of human genes and a large number of proteins could offer a new scenario in tumor classification, allowing for the formulation of a list of genes able to define a "signature" of tumor outcome. Moreover, starting from data obtained from biomolecular tumor analyses, it has been demonstrated that with this approach, it is also possible to design future therapeutic strategies.