Gene Expression Profile of Peripheral Blood Lymphocytes from Renal Cell Carcinoma Patients Treated with IL-2, Interferon-α and Dendritic Cell Vaccine

Medical Oncology Immunotherapy Group, Section of Hematology/Oncology, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire, United States of America
PLoS ONE (Impact Factor: 3.23). 12/2012; 7(12):e50221. DOI: 10.1371/journal.pone.0050221
Source: PubMed


Lymphocytes are a key component of the immune system and their differentiation and function are directly influenced by cancer. We examined peripheral blood lymphocyte (PBL) gene expression as a biomarker of illness and treatment effect using the Affymetrix Human Gene ST1 platform in patients with metastatic renal cell carcinoma (mRCC) who received combined treatment with IL-2, interferon-?-2a and dendritic cell vaccine. We examined gene expression, cytokine levels in patient serum and lymphocyte subsets as determined by flow cytometry (FCM). Pre-treatment PBLs from patients with mRCC exhibit a gene expression profile and serum cytokine profile consistent with inflammation and proliferation not found in healthy donors (HD). PBL gene expression from patients with mRCC showed increased mRNA of genes involved with T-cell and T(REG)-cell activation pathways, which was also reflected in lymphocyte subset distribution. Overall, PBL gene expression post-treatment (POST) was not significantly different than pre-treatment (PRE). Nevertheless, treatment related changes in gene expression (post-treatment minus pre-treatment) revealed an increased expression of T-cell and B-cell receptor signaling pathways in responding (R) patients compared to non-responding (NR) patients. In addition, we observed down-regulation of T(REG)-cell pathways post-treatment in R vs. NR patients. While exploratory in nature, this study supports the hypothesis that enhanced inflammatory cytotoxic pathways coupled with blunting of the regulatory pathways is necessary for effective anti-cancer activity associated with immune therapy. This type of analysis can potentially identify additional immune therapeutic targets in patients with mRCC.

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Available from: Benita Wolf
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    • "Peripheral blood is an important source of RNA for gene expression profiling studies (GEPs), because it can be used: to determine the toxicological or disease-related events that have occurred in inaccessible target tissues, to explore the interindividual GEP associated with donor sex, age, treatments and health conditions., and to describe the influence of technical procedures in GEPs, such as: the conditions of blood sample storage and protocols for RNA extraction and amplification [1], [2], [3], [4], [5]. "
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    ABSTRACT: ABSTRACT:Human peripheral blood is a useful material for biomedical research, since it can be used to investigate responses to treatment and predict diseases. However, variousbiological and technological factors produce a large degree of variation in blood of gene expression profiles. Our study wasbased on gene expression profiling analysis on peripheral blood of 45 healthy volunteers, 21 females and 24 men. The blood cells were concentrated, and the total RNA was isolated for the analysis of gene expression using the Affymetrix Gene Chip technology. The results were obtained by a fluorescent scanner, and the numerical data was analyzed using Bioconductor. Samples were clearly divided by gender throughthe unsupervised clustering analysis. 40 identified genes, differentiating samples by gender were analyzed according totheir biological function and chromosomal location. Most of them are located on the X and Y chromosomes. These results provide new insights into the genetic makeup which distinguishes both sexes probably associated with diseases and sex determination.
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    ABSTRACT: To evaluate CD4(+)CD25(+)FOXP3(+) T regulatory cells (T(REG)) and associated immune-regulatory pathways in peripheral blood lymphocytes (PBL) of metastatic renal cell carcinoma (mRCC) patients and healthy volunteers. We subsequently investigated the effects of immunotherapy on circulating T(REG) combining an extensive phenotype examination, DNA methylation analysis and global transcriptome analysis. Eighteen patients with mRCC and twelve volunteers (controls) were available for analysis. T(REG) phenotype was examined using flow cytometry (FCM). T(REG) were also quantified by analyzing the epigenetic status of the FOXP3 locus using methylation specific PCR. As a third approach, RNA of the PBL was hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays and the gene signatures were explored using pathway analysis. We observed higher numbers of T(REG) in pre-treatment PBL of mRCC patients compared to controls. A significant increase in T(REG) was detected in all mRCC patients after the two cycles of immunotherapy. The expansion of T(REG) was significantly higher in non-responders than in responding patients. Methylation specific PCR confirmed the FCM data and circumvented the variability and subjectivity of the FCM method. Gene Set Enrichment Analysis (GSEA) of the microarray data showed significant enrichment of FOXP3 target genes, CTLA-4 and TGF-ß associated pathways in the patient cohort. Immune monitoring of the peripheral blood and tumor tissue is important for a wide range of diseases and treatment strategies. Adoption of methodology for quantifying T(REG) with the least variability and subjectivity will enhance the ability to compare and interpret findings across studies.
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    ABSTRACT: To move forward with immunotherapy, it is important to understand how the tumor microenvironment generates systemic immunosuppression in patients with renal cell carcinoma (RCC) as well as in patients with other types of solid tumors. Even though antigen discovery in RCC has lagged behind melanoma, recent clinical trials have finally authenticated that RCC is susceptible to vaccine-based therapy. Furthermore, judicious coadministration of cytokines and chemotherapy can potentiate therapeutic responses to vaccine in RCC and prolong survival, as has already proved possible for melanoma. Although high-dose interleukin 2 immunotherapy has been superseded as first-line therapy for RCC by promiscuous receptor tyrosine kinase inhibitors (rTKIs) such as sunitinib, sunitinib itself is a potent immunoadjunct in animal tumor models. A reasonable therapeutic goal is to unite antiangiogenic strategies with immunotherapy as first-line therapy for RCC. This strategy is equally appropriate for testing in all solid tumors in which the microenvironment generates immunosuppression. A common element of RCC and pancreatic, colon, breast, and other solid tumors is large numbers of circulating myeloid-derived suppressor cells (MDSCs), and because MDSCs elicit regulatory T cells rather than vice versa, gaining control over MDSCs is an important initial step in any immunotherapy. Although rTKIs like sunitinib have a remarkable capacity to deplete MDSCs and restore normal T-cell function in peripheral body compartments such as the bloodstream and the spleen, such rTKIs are effective only against MDSCs, which are engaged in phospho-STAT3-dependent programming (pSTAT3+). Unfortunately, rTKI-resistant pSTAT3- MDSCs are especially apt to arise within the tumor microenvironment itself, necessitating strategies that do not rely exclusively on STAT3 disruption. The most utilitarian strategy to gain control of both pSTAT3+ and pSTAT3- MDSCs may be to exploit the natural differentiation pathway, which permits MDSCs to mature into tumoricidal macrophages (TM1) via such stimuli as Toll-like receptor agonists, interferon γ, and CD40 ligation. Overall, this review highlights the mechanisms of immune suppression used by the different regulatory cell types operative in RCC as well as other tumors. It also describes the different therapeutic strategies to overcome the suppressive nature of the tumor microenvironment.
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