Mechanism of immune response during immunotherapy.

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. 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). 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.

Immune Monitoring of Cancer Vaccines

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.

Active-specific immunization against melanoma: is the problem at the receiving end?

The recent progress in tumor immunology is a striking example of the successful application of modern biotechnology to understand the complex phenomenon of immune-mediated cancer rejection. Tumor antigens were identified and successfully utilized in active immunization trials to induce tumor antigen-specific T cells. This achievement has left, however, clinicians and researchers perplexed by the paradoxical observation that immunization-induced T cells can recognize tumor cells in standard assays but cannot induce tumor regression. A closer look at T cell physiology and tumor biology suggests that this observation is not so surprising. Here, we argue that successful immunization is one of several steps required for tumor clearance while more needs to be understood about how T cells localize and are effective within a tumor microenvironment impervious to the execution of their effector function.