University of Pittsburgh Cancer Institute (UPCI)

Skin Cancer SPORE

Core B: Biospecimen Core/Immunologic Monitoring and Cellular Products Laboratory

Director:
Lisa H. Butterfield, PhD
Co-Directors:
Uma N. Rao, MD
Jonhan Ho, MD

Core B serves as the Biospecimen Core, as well as the Immunologic Monitoring and Cellular Products Laboratory (IMCPL). The laboratory supports the SPORE community with blood and tissue banking for patients submitting blood and/or tumor to the Melanoma Program tissue bank, as well as clinical protocol-specific banking. For all four SPORE projects, the Core will perform a wide variety of standardized, SOP-driven immunological monitoring assays. For Project 2, Core B will also prepare autologous dendritic cell vaccines.

Specific support for each Project is as follows:

Core B will support Project 1 Aims 1a and 1b, testing circulating cellular and serum biomarkers from the trial ECOG1609. Core B will assess host effector and suppressor cellular immune responses. We will perform multicolor flow cytometry to compare PBMC before and after treatment, focusing on circulating (1) regulatory T cells (Treg) and (2) myeloid-derived suppressor cells (MDSC), (3) IFNγ+CD4+ and IFNγ+CD8+ antigen specific T cell immunity (gp100, MART-1, NY-ESO-1 peptide pools), and (4) CD4+ and CD8+ ICOShi, and CXCL3+/VLA4+ T cells. Treg will be defined as cells expressing (a) CD4+CD25hi+FoxP3+ or (b) CD4+CD25hi+CD39+ T cells (CD3+CD4+CD25+) versus total activated CD4+ T cells. MDSC will be defined as cells expressing (c) Lin1-/HLA-DR-/CD33+/CD11b+ lymphoid gate MDSC (d) Lin1-/HLA-DR-/CD33+/CD11b+ monocyte gate MDSC or (e) HLA-DR+low/CD14+ monocyte gate MDSC. Baseline, early on treatment and within-patient changes in T-reg and MDSC will be tested and compared with clinical outcome (RFS, OS). Core B will also perform testing of candidate serum biomarkers (cytokines, chemokines and other soluble factors) including IL-1α, IL-1β, IL-2, IL-2R, IL-6, IL-8, IL-10, IL-17, TNF-α, IFNα-2b, MIP-1α, MIP-1β, IP-10,VEGF, CXCL11, CXCL9 and C-reactive protein (CRP) by multiplex Luminex assay.

Core B will support Project 2 Aim 1 (clinical trial UPCI #09-021) with autologous AdVTMM2-transduced dendritic cell vaccine manufacture, characterization and release for therapy for a total of 36 patients. The Core will process all blood, leukapheresis and tumor samples and perform all banking (3 time points per patient). For Aim 2, The Core will perform standardized immune response assays. These include: CD8+ and CD4+ T cells specific for vaccine antigens (tyrosinase, MART-1 and MAGE-A6, ELISPOT); NK cell activation (PBMC flow cytometry), tumor infiltration (TIL flow cytometry); T cell response to “determinant spreading” antigens (including gp100, NY-ESO-1 and vascular antigens); T and NK cell responses to autologous tumor; serum antibody responses to melanoma antigens (NY-ESO-1, CSPG-4); and circulating regulatory responses (Treg, MDSC). Results obtained will be correlated with clinical outcome (Biostatistics Core C). Core B will also prepare DC, tumor and PBMC samples for shipment to collaborating sites.

Core B will support Project 3 through banking of PBMC and testing for activation and expansion of shared melanoma antigen-specific T cells by flow cytometry (as in Project 1).

Core B will perform the systemic immunologic monitoring proposed in Project 4 for both CTCL and melanoma patients. This will include serum banking and testing for cytokines, chemokines and growth factors (Luminex). The Core will also process PBMC and tumor samples, and perform absolute counts and percentages for activated CD4+ and CD8+ T cells, NK cell activation, Treg and MDSC, at multiple time points.

Data generated in Core B will be analyzed in Biostatistics Core C and correlated to clinical parameters. The blood and tissue bank data and inventory are linked to the Clinical Trials Management Application and will be part of the Research Data Warehouse developed by and managed in Informatics Core D.