Interferon-alpha (IFN-alpha) has shown promise as an adjuvant therapy following surgical resection of high-risk melanoma tumors, however, it's cellular targets(s) and mechanism of action remain undefined. The applicant has recently shown that IFN-alpha directly stimulates the so called JAK-STAT signaling pathway in tumor samples obtained directly from melanoma patients, and that pre-treatment of melanoma cells with IFN-gamma results in a 10,000-fold decrease in the concentration of IFN-alpha required for STAT activation. Utilizing mice deficient in these signaling intermediates, the applicant determined that the effects of IFN-alpha on the host are also critical to its anti-tumor actions. An NCI-sponsored phase I trial was recently initiated in which pre-treatments of interleukin-12 are used to stimulate the endogenous production of IFN-gamma prior to the administration of low-dose IFN-alpha, in the hope that sensitization of the patient to the effects of IFN-alpha may lead to increased efficacy and decreased toxicity. Patient peripheral blood mononuclear cells, serum, and tumor tissue have been procured before and immediately following the administration of IL-12. The hypothesis is that administration of IL-12 followed by low-dose IFN-alpha will result in enhanced JAK-STAT signaling in patient PBMCs and tumor tissue. Furthermore, it is proposed that the induction of IFN-alpha-responsive genes will result in significant alterations in the function and competency of the host immune system over the course of this six-month treatment. To test these hypotheses, a series of novel correlative studies have been outlined designed to assess the effects of cytokine therapy on patients with cancer.
In Aim 1 the applicant proposes to analyze interferon-alpha-induced STAT signaling in tumor cells and peripheral blood mononuclear cells obtained from patients receiving interferon-alpha plus interleukin-12. Levels of STAT proteins will be analyzed using the electrophoretic mobility shift assay. These techniques have never been systematically applied to patients enrolled in a clinical trial or to frozen patient tissues. Thus, the applicant hopes to demonstrate the applicability of this approach to the study of cytokine signaling in cancer patients and at the same time gain insight into the anti-tumor actions of IFN-alpha. The utility of immunohistochemical techniques and flow cytometry will also be investigated in the analysis of cytokine signaling, since these antibody-based techniques are quantitative, use low numbers of cells, and exhibit a high degree of specificity.
In Aim 2, the effects of IL-12 plus IFN-alpha on the host immune system will be evaluated utilizing the novel techniques of Real-Time PCR and intracellular staining for the detection of immunomodulatory cytokines. The applicant will first determine if the subcutaneous administration of IL-12 is an effective means of stimulating IFN-gamma production at the transcript and protein levels. In addition, he plans to characterize the effects of long-term IFN-alpha therapy on the function and competency of the host immune response. This will involve the analysis of lymphocyte subsets, markers of activation, and helper T cell profiles using flow cytometry and PCR-based techniques. These studies will provide information on the actions of IL-12 plus IFN-alpha, enhance our ability to monitor the effects of exogenously administered cytokines, and permit the identification of specific markers of cytokine responsiveness.
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