No abstract available.
Freedom

One of the hallmarks of the adaptive immune system is the specificity of B and T cell receptors. Thanks to somatic recombination, a large repertoire of receptors can be generated within an individual that guarantee the recognition of a vast number of antigens. Monoclonal antibodies have limited applicability, given the high degree of diversity among these receptors, in BCR and TCR monitoring. Furthermore, with regard to cancer, better characterization of complex genomes and the ability to monitor tumor-specific cryptic mutations or translocations are needed to develop better tailored therapies. Novel technologies, by enhancing the ability of BCR and TCR monitoring, can help in the search for minimal residual disease during hematological malignancy diagnosis and follow-up, and can aid in improving bone marrow transplantation techniques. Recently, a novel technology known as next generation sequencing has been developed; this allows the recognition of unique sequences and provides depth of coverage, heterogeneity, and accuracy of sequencing. This provides a powerful tool that, along with microarray analysis for gene expression, may become integral in resolving the remaining key problems in hematology. This review describes the state of the art of this novel technology, its application in the immunological and hematological fields, and the possible benefits it will provide for the hematology and immunology community.
Allergy and Immunology* ; Antibodies, Monoclonal ; Bone Marrow Transplantation ; Diagnosis ; Gene Expression ; Genome ; Hematologic Neoplasms ; Hematology* ; Immune System ; Microarray Analysis ; Monitoring, Immunologic ; Neoplasm, Residual ; Population Characteristics ; Receptors, Antigen, T-Cell ; Recombination, Genetic ; Sensitivity and Specificity

Multiple myeloma is a malignancy of B-cells that is characterized by the clonal expansion and accumulation of malignant plasma cells in the bone marrow. This disease remains incurable, and a median survival of 3-5 years has been reported with the use of current treatments. Viral-based therapies offer promising alternatives or possible integration with current therapeutic regimens. Among several gene therapy vectors and oncolytic agents, adenovirus has emerged as a promising agent, and it is already being used for the treatment of solid tumors in humans. The main concern with the clinical use of this vector has been its high immunogenicity; adenovirus is often able to induce a strong immune response in the host. Furthermore, new limitations in the efficacy of this therapy, intrinsic to the nature of tumor cells, have been recently observed. For example, our group showed a strong antiviral phenotype in vitro and in vivo in a subset of tumors, shedding new insights that may explain the partial failure of clinical trials based on this promising new therapy. In this review, we describe novel therapeutic approaches that implement viral-based treatments in hematological malignancies and address the novelty as well as the possible limitations of these new therapies, especially in the context of the use of adenoviral vectors for treating multiple myeloma.
Adenoviridae ; B-Lymphocytes ; Bone Marrow ; Genetic Therapy ; Hematologic Neoplasms ; Humans ; Multiple Myeloma ; Phenotype ; Plasma Cells

The recent progress in tumor immunology exemplifies the successful application of modern biotechnology for the understanding of the complex natural or therapy-induced phenomenon of immune-mediated rejection of cancer. Tumor antigens recognized by T cells were identified and successfully utilized in active immunization trials for the induction of tumor-antigen specific T cells. This achievement has left, however, the clinicians and researchers perplexed by the paradoxical observation of the immunization-induced T cells can recognize tumor cells in standard assays but most often cannot induce tumor regression. In this presentation, we will argue that successful immunization is one of several steps required for tumor clearance but more work needs to be done to understand how T cells can localize and be effective at the receiving end within a tumor microenvironment in most cases not conducive to the execution of their effector function. In fact, metastatic melanoma stands out among human cancers because of its immune responsiveness. Yet, the reason(s) remain(s) unclear. We have previously suggested that a promising strategy for the understanding of melanoma immune responsiveness could consist of the study of tumor/host interactions ex vivo through genetic profiling of serial fine needle aspirate biopsies that allow direct correlation between experimental results and clinical outcome.1 By prospectively studying the transcriptional profile of melanoma metastases during immunotherapy we observed that immune responsiveness is pre-determined by an immune reactive micro-environment.2 Interestingly, the addition of systemic interleukin-2 therapy to active specific immunization seems to increase the frequency of immune rejections of cancer. Functional profiling of the effect of interleukin-2 in tumors suggested that this cytokine induces or enhances the effector function of immunization-induced T cells by causing an acute inflammatory process at the tumor site that can in turn recruit and activate T cells.3 Thus, we hypothesize that effective immune responses occur when a pro- inflammatory inflammatory threshold is reached at tumor site capable of maintaining active immunization induced-T cells.4 To search the reason for the erratic behavior of metastatic melanoma, we analyzed 62 melanoma metastases to identify functional signatures possibly responsible for immune responsiveness. Melanoma metastases were biopsied with a 23 gauge needle and anti- sense RNA was amplified to produce single stranded cDNA for hybridization to custom-made cDNA arrays.5 Genes specific for the tumor microenvironment were sorted (Wilcoxon test p-value< 0.001). Eisens's hierarchical clustering was applied to the resulting gene pool and two subsets of melanomas were identified. A smaller cluster including 15 samples (24%) was characterized by significantly higher expression of the inflammatory cytokines GRO-alpha MIP-1alphaand beta MPC-1, -3 and -4, IL-1beta IL-8, RANTES, Lymphotactin and Lymphotoxin. This signature strongly correlated with up-regulation of IFN- responsive elements. The same cluster displayed a higher expression of MMP-9, 11 and 15 (cytokine-dependent metalloproteinases), genes encoding growth and angio-regulatory factors and cell cycle regulatory sequences. These findings suggested that some melanoma metastases display a very heterogeneous immune environment that could variably modulate T cell function at the receiving end of the immune response against cancer and could co- operate with the pro- inflammatory effects of the systemic administration of intereleukin-2. Although these studies need to be confirmed in larger patient populations this report suggests that strategies are presently available for the efficient screening of biological principles and related biomarkers using high-throughput technology. References: 1. Wang E, Marincola FM. A natural history of melanoma: serial gene expression analysis. Immunol Today 2000; 21:619-23. 2. Wang E, Miller LD, Ohnmacht GA, Mocellin S, Petersen D, Zhao Y, et al. Prospective molecular profiling of subcutaneous melanoma metastases suggests classifiers of immune responsiveness. Cancer Res 2002;62: 3581-6. 3. Panelli MC, Wang E, Phan G, Puhlman M, Miller L, Ohnmacht GA, et al. Genetic profiling of peripharal mononuclear cells and melanoma metastases in response to systemic interleukin-2 administration. Genome Biol 2002;3: RESEARCH0035. 4. Marincola FM, Wang E, Herlyn M, Seliger B, Ferrone S. Tumors as elusive targets of T cell- directed immunotherapy. Trends Immunol 2003;24334-41. 5. Wang E, Miller L, Ohnmacht GA, Liu E, Marincola FM. High fidelity mRNA amplification for gene profiling using cDNA microarrays. Nature Biotech 2000;17:457-9.

Tumor immunology embraces an extensive array of biological phenomena that include interactions between neoplastic cells and the innate and adaptive immune response. Among immune cells, T cells have taken the center stage because they can be easily demonstrated to specifically recognize autologous cancer cells. However, their role is limited and other components of the immune response are likely necessary for the completion of cancer rejection. Metastatic melanoma and renal cell carcinoma (RCC) are malignancies strongly predisposed to regress in response to the systemic administration of high-dose interleukin (IL)-2. Several clinical Studies in extensive cohorts of patients have shown that this treatment can induce complete or partial clinical regressions of metastatic disease in 15 to 20% of patients who receive this treatment.1-6 Although IL-2 has direct pluri-potent effects on cells with immune and inflammatory function, it remains unexplained which cell subset is implicated in mediating tumor regression. In a quest to characterize the mechanism of action of IL-2 during the course of immunotherapy, we have investigated the early changes in transcriptional profiles of circulating mononuclear cells and microenvironment of melanoma metastases following high dose IL-2 administration (720,000 IU/kg) by serial sampling of blood cells and tumors in the form of fine needle aspirate (FNA).7 Furthermore, studies are currently ongoing to characterize the proteomic profiling of RCC patients undergoing the same treatment using protein arrays (manuscript in preparation). The predominant activation of genes related to inflammation and activation of mononuclear phagocytes lead us to further characterize this cell subset in the context of stimulation with a panel of soluble factors potentially present in the circulation and tumor microenvironment.
Antibody Formation ; Carcinoma, Renal Cell/metabolism/*therapy ; Gene Expression Profiling ; Humans ; *Immunotherapy ; Interleukin-2/*immunology/*therapeutic use ; Lipopolysaccharides/pharmacology ; Melanoma/genetics/immunology/secondary/*therapy ; Phagocytes/drug effects/physiology ; Proteomics