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Last Updated: 10/10/18

SPORE in Skin Cancer

University of Pittsburgh Cancer Institute

Principal Investigator:
John M. Kirkwood, M.D.

Principal Investigator Contact Information

John M. Kirkwood, M.D.
Usher Professor of Medicine and Dermatology
Vice Chairman/Clinical Research, Dept. of Medicine
Director, Melanoma and Skin Cancer Program
University of Pittsburgh Cancer Institute
Department of Medicine
5117 Centre Avenue
Hillman Cancer Center, Room 1.32
Pittsburgh, PA 15213
Tel: (412) 623-7707
Fax: (412) 623-7704
Email: kirkwoodjm@upmc.edu

OVERALL ABSTRACT

The incidence of melanoma has risen dramatically in recent years, but no therapy has improved overall survival for the majority of patients with unresectable metastatic disease. The University of Pittsburgh Cancer Institute (UPCI) Melanoma and Skin Cancer Program (MSCP) will continue to conduct a Specialized Program of Research Excellence (SPORE) to improve our understanding of molecular and immunologic mechanisms of cancer progression and to validate prognostic and predictive biomarkers for personalized treatment of advanced melanoma and cutaneous T cell lymphoma (CTCL). Of our 4 Projects, 1 is continued from the prior funding period, and 3 new projects have been derived from developmental research conducted in the last period; 1 of the prior SPORE projects will be continued with independent R01 funding. Our highly integrated approach leverages complementary expertise in melanoma, oncology, dermatology, immunology, biostatistics, bioinformatics, machine learning, genomics, proteomics, and biomarker discovery to test hypotheses central to the improvement of therapeutic outcome in skin cancers. Regardless of clinical outcomes, these Projects will also generate urgently needed mechanistic data to inform development of new therapeutic strategies and pathways through which to monitor relapse and progression. Our 4 Projects will evaluate: (1) the prognostic and predictive value of the pro-inflammatory response and markers of immune suppression in relation to ipilimumab and interferon (IFN)a adjuvant therapy (leveraging an Eastern Cooperative Oncology Group-led adjuvant trial); (2) an engineered, 3-antigen dendritic cell vaccine and IFNa boost in patients with metastatic melanoma; (3) the safety and efficacy of vemurafenib modulation of immunotherapy with IFNa2b in patients with metastatic melanoma; and (4) an entirely new personalized microneedle vaccination technology in patients with melanoma and CTCL. The Administrative Core (A) coordinates the clinical research and provides scientific and fiscal oversight of the entire SPORE. The Biospecimen Core (B) is housed in the UPCI Immunologic Monitoring and Cellular Products Laboratory, where all tissue banking, biospecimen processing, and immune assays are conducted. The Biostatistics Core (C) and Informatics Core (D) support all projects with data analysis and management, respectively. We will continue to solicit, review, and fund applications for the career development and developmental research programs, which in the prior funding period led to the design of a new full project in this application (Project 3) and the promotion of career development recipients to co-investigators and a project leader.

Project 1: BIOMARKERS OF THE PROINFLAMMATORY RESPONSE AND ELEMENTS OF IMMUNE SUPPRESSION

Project Co-Leaders & Co-Investigators:
Ahmad A. Tarhini, M.D.
Walter Storkus, Ph.D.
Lisa H. Butterfield, Ph.D.
John M. Kirkwood, M.D.
William LaFramboise, Ph.D.

Project 1 builds on our preliminary data and recent reports in the literature that have identified a series of biomarkers of a pro-inflammatory immune response and of immunosuppression in both tumor tissue and in circulating blood that have promising therapeutic predictive and disease prognostic value in melanoma patients treated with immunotherapy. These will be simultaneously evaluated as part of E1609 trial testing adjuvant ipilimumab at high dose and standard dose versus IFNa in patients with operable stage IIIB/C and M1a/b melanoma.

Specific Aims:

The current paradigm of adjuvant therapy in melanoma involves the indiscriminate treatment of all patients clinically considered at high risk for melanoma recurrence and mortality, despite data showing that only a small proportion of patients will benefit. The primary goal of Project 1 is to identify baseline and/or early on-treatment predictive biomarker(s) capable of classifying patients according to the degree of benefit they will receive from treatment with ipilimumab or IFNa. Secondary goals are to identify biomarkers of significant prognostic value capable of identifying patients at highest risk of recurrence and mortality. During the last funding period, we tested the prognostic and predictive value of autoimmunity and candidate biomarkers in relation to IFNa as part of two Eastern Cooperative Oncology Group (ECOG)-led adjuvant trials (E1697 and E1694). We also studied biomarkers predictive of CTLA4 blockade therapeutic benefit within metastatic and neoadjuvant trials that laid the groundwork for this modified continuation of Project 1. We identified a series of potentially therapeutically predictive biomarkers of a pro-inflammatory immune response and of immunosuppression in both tumor tissue and in circulating blood, which we will evaluate simultaneously due to the common systems biology, in our quest for a predictive biomarker model.

The renewal Project 1 will be nested within the ongoing ECOG E1609 trial that is testing adjuvant ipilimumab (at high dose and standard dose) versus IFNa in patients with operable stage IIIB/C and M1a/b melanoma who are at high risk for recurrence and death. The large sample size (n=1600) and diverse biorepository for E1609 make it an ideal platform through which to evaluate these interrelated markers for their immunotherapeutic predictive value with regard to ipilimumab in reference to IFNa, and disease prognostic value, as assessed individually and in combination. We will also develop a therapeutic predictive model that links circulating markers of the pro-inflammatory response and immunosuppression with the tumor microenvironment.

We hypothesize that a baseline pro-inflammatory biomarker signature will be predictive of therapeutic benefit. We expect to discover unique biomarker differences between ipilimumab and IFNa due to the different mechanisms of action but overlapping predictive models, given the common pro-inflammatory biological impact. Thus, our renewal Project 1 will assess and validate candidate biomarkers predictive of relapse-free survival (RFS, primary outcome) and overall survival (OS, secondary) of melanoma patients in the context of E1609 and will develop and validate a predictive model for clinical outcome using these markers.

Specific Aim 1: Circulating biomarkers.

Based on preliminary data from the last funding period, we will examine:

(1a) Cellular populations We will test the hypothesis that baseline and/or early on-treatment IFNg+CD4+ and IFNg+CD8+ antigen specific T cell immunity as well as specific host suppressor elements (defined populations of regulatory T cells and MDSC) predict therapeutic benefit (RFS and OS). We will also assess the overall lymphocyte count as a potential predictive marker.

(1b) Serum proteins We will test the hypothesis that baseline and/or early on-treatment pro-inflammatory cytokine and chemokine profiles predict therapeutic benefit (RFS and OS). Secondarily, we will examine candidate serum biomarkers known to be associated with the host cellular immune response (in Aim 1a) to confirm preliminary findings from the last funding period.

Specific Aim 2: Tumor and tumor microenvironment biomarkers.

Based on multiple recent reports, we will first test the hypothesis that a pretreatment, pro-inflammatory, tumor microenvironment (high baseline expression levels of immune-related genes) predicts therapeutic benefit (RFS and OS). As secondary sub-aims, we will test the association of the tumor mutational status (BRAF, NRAS, and WT status for both) and clinical outcome. We will also assess the methylation levels of immune-related genes.

Specific Aim 3: Prediction model development and validation.

Using known clinical covariates and significant markers identified in Aims 1 and 2, we will use the entire data set from E1609 to develop and validate models capable of identifying which patients belong to different therapeutic predictive groups.

Impact: Project 1 has the potential to significantly impact the field of adjuvant immunotherapy, transforming our current model of care from a nonspecific population approach to a personalized and targeted therapeutic strategy. Further, our results will provide urgently needed mechanistic insights into the complex interactions between host and tumor by linking elements of inflammation and immunosuppression in circulation with the tumor microenvironment, ultimately leading to improved therapeutic targeting and patient outcomes. Our findings should provide the foundation for assessing these biomarkers in other stages of melanoma, and for other immunotherapies (e.g., IL-2), other immune checkpoint inhibitors, and more specific (vaccine) modalities.

PROJECT 2: MULTIPLE ANTIGEN-ENGINEERED DC IMMUNIZATION AND IFNa-2b BOOST FOR METASTIC MELANOMA

Project Co-Leaders & Co-Investigator:
Lisa H. Butterfield, Ph.D.
John M. Kirkwood, M.D.
Lazar Vujanovic, Ph.D.

This project tests an improved DC vaccine which is designed to promote in vivo cross presentation and determinant spreading. Based on results from our previous trials, we have made several important improvements: engineering the DC with 3 full-length, defined, tumor antigens to activate multiple CD8+ and CD4+ T cell clones (reducing the concern for antigen loss variants); providing antigen presentation for the life of the DC; providing cognate CD4+ T cell help to the CD8+ T cells (“helped” CTL); using a matured DC (a cocktail specifically matched to adenovirus (AdV) transduction signals); activating innate immunity via NK cell migration and activation; and boosting with systemic IFNa (for endogenous DC type 1 skewing, improved cross-priming and direct effects on T cells). Together, this vaccine strategy should more potently activate a polyclonal anti-tumor response incorporating multiple adaptive and innate effectors which we predict will lead to a higher frequency of patients who not only activate vaccine-encoded antigen-specific T cell responses, but also develop determinant spreading and a significant clinical response.

Specific Aims:

Specific Aim 1: Completion of the AdVTMM2/DC +/- IFNa clinical trial.

We will fully assess the clinical and immunologic impact of the vaccine and IFNa boost, and test: A) Clinical outcomes in the advanced stage patients; B) Immunologic outcomes (from blood and TIL; baseline, post-vaccine and post-IFNa), including CD8+ and CD4+ T cells specific for vaccine antigens (tyrosinase, MART-1 and MAGE-A6), NK cell activation and tumor infiltration (TIL and tumor), T cell response to “spreading” antigens (including gp100, NY-ESO-1 and vascular antigens), T cell responses to autologous tumor, antibody responses to melanoma antigens, and regulatory responses (Treg, MDSC); and C) AdV-specific responses, including CD4+ and CD8+ T cells specific for total viral proteins, total anti-AdV antibodies, and anti-AdV neutralizing antibodies.

Specific Aim 2: Mechanism and biomarkers of clinical response (new Aim).

To understand patient differences in tumor biology, in DC vaccines and in candidate biomarkers, we will examine: A) DC vaccine transcriptional profiling (+/- IFN? and LPS maturation, +/- AdVTMM2) and compare to DC vaccine phenotype, cytokine production and antigen expression; B) Tumor transcriptional profiling (baseline, post-vaccine and post-IFNa) to examine tumor modulation by DC vaccines and IFNa therapy; C) Peripheral blood signaling induced by IFNa (phospho-STAT-1); D) Serum profiling (autoimmunity antibodies, LDH, CRP, cytokines) to examine previously identified candidate serum biomarkers; E) Molecular mimicry with mycoplasma (impact of baseline memory to mycoplasma); and F) Germline DNA SNP analysis with a focus on identified immunologic melanoma SNPs (IL-12p40, Calreticulin, and CRP). These studies will identify predictive and prognostic biomarkers of immunologic and clinical response.

Specific Aim 3: Mechanism of NK cell “activation” in anti-melanoma immunity (new Aim).

NK cell activity may also be critical for clinical response. We will perform in vitro studies to further our recent findings in AdV/DC-NK cell cross-talk utilizing banked melanoma patient blood and tumor to examine: A) Direct NK cell interactions with melanoma tumor cells (cytotoxicity, cytokines); B) Helper/type 1 skewing by NK cells to shape adaptive CD8+ and CD4+ T cell responses; and C) Melanoma tumor-derived inhibitory impact on NK cell function. These studies will identify the mechanisms of innate NK cell anti-tumor activity and any potential suppression by tumor.

Summary: Completion of the AdVTMM2/DC +/- IFNa trial and these three aims will allow us to test novel hypotheses. We will determine: 1) Vaccine-induced clinical and immune response, correlation with subsequent induction of determinant spreading, and determine the impact of adding IFNa to the antigen-engineered DC vaccine; 2) Layers of potential mechanisms of response and biomarkers of immunologic and clinical response; and 3) Innate-adaptive cross-talk between DC vaccines, NK cells, vaccine-activated T cells, and systemic IFNa.

Impact: We will test a potent vaccine combination therapy which may promote a higher frequency and duration of clinical response. The examination of several predictive biomarkers (tumor infiltration and gene expression, pathogen memory, SNPs, NK cell activation) and prognostic biomarkers (adaptive cellular and humoral immunity, determinant spreading, NK cell activation, serum biomarkers, autoimmunity) may identify patients able to benefit from immune-based approaches, as well as the mechanisms of that benefit.

PROJECT 3: PHASE 1 STUDY OF ANTI-PD-1 ANTIBODY MK-3475 AND PEGIFNa-2b FOR ADVANCED MELANOMA

Project Co-Leaders:
Hassane Zarour, M.D.
John M. Kirkwood, M.D.

The goal of this proposal is to test the hypothesis that the combination of pembrolizumab (anti-PD-1 antibody) and pegylated interferon (PEG IFNa-2b) is safe, therapeutically more effective than treatment with single agent anti-PD-1 antibody alone and immunogenic for patients with advanced melanoma irrespective of BRAF or NRAS mutation status. To this end, we have proposed the dose-seeking and efficacy study of the combination pembrolizumab and PEG IFNa-2b for advanced melanoma. This proposal benefits from our well-established expertise in: 1) IFN-based therapy of melanoma; 2) immune monitoring of T cell responses to melanoma both in the peripheral blood and at tumor sites, and; 3) preclinical studies of inhibitory pathways that in dampen T cell responses to melanoma. This clinical trial is partially funded by an academic-industrial award (Merck and the Melanoma Research Alliance). This spore project proposed to perform the immunological and biomarker studies in the context of the first-in-human trial with pembrolizumab and PEG IFNa-2b for patients with recurrent inoperable AJCC stage III and metastatic stage IV melanoma.

Specific Aims:

Specific Aim 1: Pembrolizumab and PEG IFNa-2b combination therapy promote CD8+ T cell tumor infiltration and the upregulation of PD-L1 as well as multiple co-inhibitory receptors at tumor sites. Here, we propose to test whether treatment with pembrolizumab and PEG IFNa-2b will induce and/or increase CD8+ T cell infiltrates as compared to pre-treatment biopsy evaluation. We will investigate whether CD8+ T cells upregulate co-inhibitory receptors including Tim-3, BTLA, and TIGIT. We will also investigate whether tumor cells upregulate of PD-L1 in tumor biopsies pre- and post-treatment.

Specific Aim 2: Anti-PD-1 antibody and PEG IFNa-2b in combination will promote the Th-1 type gene signature in non-inflamed tumors as well as increased expression of inhibitory ligands. Here, we propose to perform transcriptome studies of tumor samples taken prospectively pre- and post-treatment (12 weeks). We will investigate whether the proposed combinatory approach is successfully able to promote a Th-1 gene signature among non-inflamed tumors. We will also evaluate pre- and post-therapy the presence of gene signatures of T cell exhaustion and T cell anergy to investigate the mechanisms driving T cell dysfunction at tumor sites. Such approach will permit us to identify biomarkers of positive clinical outcome. They will also allow us to determine what inhibitory pathways beyond PD-1 may need to be targeted to enhance further the clinical efficacy of the proposed combinatorial therapy.

Specific Aim 3: Anti-PD-1 antibody and PEG IFNa-2b in combination promote clonal expansion of CD8+ T effector cells at tumor sites. Here, we propose to investigate whether the proposed combinatorial therapy will contribute to the expansion of the repertoire of TA-specific T cells in the TME. Such studies are important to determine whether the gain of function within TA-specific CD8 T+ TILs is best attributed to one or the other of two non-exclusive mechanisms: 1) functional improvement of persisting clonotypes; and 2) recruitment of particular clonotypes exhibiting superior functional capabilities.

Impact: This SPORE project will determine whether PD-1 blockade and PEG IFNa-2b can prime T cell responses in the non-inflamed tumors, which are less likely to respond to anti-PD-1 therapy alone. It will also determine what other inhibitory pathways may need to be targeted in combination with PD-1 blockade and PEG IFNa-2b to reverse melanoma-induced T cell dysfunction. Finally, it is also expected that the correlative gene signature studies at tumor sites will allow us to understand better the molecular mechanisms driving tumor-induced T cell dysfunction in the TME.

Project 4: AN IN SITU PATIENT-SPECIFIC CHEMO-IMMUNOTHERAPY FOR SKIN CANCERS

Project Co-Leaders & Co-Investigators:
Louis D. Falo, Jr., M.D, Ph,D.
Larisa Geskin, M.D.
Hussein Tawbi, M.D., Ph.D.
William LaFramboise, Ph.D.

This project will evaluate an entirely novel approach to skin cancer treatment applicable to a broad range of topically accessible skin cancers. We propose to co-deliver a potent chemotherapeutic agent and an immune modifier to kill topically accessible cancer cells and simultaneously convert a cutaneous neoplasm into a highly potent patient specific immunogen capable of inducing innate, adaptive, and tumor specific memory immune responses. The strategy is enabled by a unique microneedle array (MNA) delivery device developed and produced in our laboratory. The therapeutic approach is polyfunctional and synergistic. Chemotherapy and adjuvant therapy are combined temporally and spatially. Chemotherapy is used to kill existing tumor cells through immunogenic “good death” mechanisms that convert dying cells into a rich source of tumor antigen. Simultaneously, a potent adjuvant is delivered to the tumor bed to recruit and activate innate immune responses and antigen presenting cells. This combined therapy is expected to result in both chemotherapeutic and immune-mediated tumor lysis, providing a rich source of full-length autologous tumor antigens in a pro-immunogenic environment that will promote the development of antigenically broad tumor-specific cellular and humoral immunity. This approach has the additional advantage of affording patient and tumor specific immunity by utilizing the patient's tumor directly as a source of immunogen, obviating obstacles related to antigen and tumor heterogeneity. The dissolvable MNAs we have developed can simultaneously deliver chemotherapeutic agent(s) and immune adjuvant(s) to the same cutaneous microenvironment, enabling manipulation of the tumor microenvironment through low dose delivery, with virtually no systemic exposure or toxicity. Further, the biologically active cargos loaded into MNA devices are extremely stable, and eliminate dependency on the cold chain. Together with the ease of fabrication and minimal dosing, these features enable an economically feasible patient specific chemo-immunization.

In this project, we will test our hypothesis that in situ topical MNA-directed chemo-immunotherapy will kill tumor cells locally and alter the tumor microenvironment to induce durable systemic tumor specific immunity. In this, the first clinical application of this strategy, our goal is to evaluate MNA-directed chemo-immunotherapy in patients with in-transit melanoma and in CTCL patients through phase I/II clinical trials. We will evaluate clinical responses and therapy induced immune effects locally and systemically.

Specific Aims:

Specific Aim 1: To evaluate MNA-directed topical chemo-immunotherapy (MNA-CI) in patients with loco regionally advanced resectable in-transit melanoma. We will conduct a pilot phase II clinical trial to evaluate safety, feasibility, and efficacy of MNA-CI in the induction of local tumor regression and local and systemic immune responses. We will perform hypothesis driven and hypothesis generating studies to characterize and compare the tumor microenvironment before, during, and after therapy, and the nature of the immune response generated in patients in treated lesions and systemically.

Specific Aim 2: To evaluate MNA-directed topical chemo-immunotherapy (MNA-CI) in patients with the most common variant of CTCL, mycosis fungoides (MF). We will conduct a pilot phase I/II clinical trial to evaluate safety and efficacy (local and distant tumor regression) in patients with early stage (stage IA-IIA) MF. We will conduct hypothesis driven and hypothesis generating studies to characterize and compare the tumor microenvironment before, during, and after therapy, and the nature of the local and systemic immune response generated in treated and control cohorts.

CORE A: ADMINISTRATIVE CORE

Co-Directors:
John M. Kirkwood, M.D.
Louis D. Falo, Jr., M.D., Ph.D.

The Administrative Core A will oversee all scientific, fiscal, and organizational activities of the MSCP Skin Cancer SPORE, including oversight of scientific and translational progress, oversight of expenditures, and regular meetings of the SPORE investigators. Core A will be responsible for the organization of our participation in the annual Skin SPORE retreats and interactions with the National Cancer Institute and other translational investigators of the University of Pittsburgh Cancer Institute, the National Cooperative Groups, and International Collaborative Groups with which investigators of the MSCP SPORE are actively engaged. Core A will be responsible for internal communications regarding the weekly Pathology and Translational Team Conferences of SPORE investigators, alternate-weekly fiscal reviews of all Projects and Cores, and monthly data safety reviews of the investigations of the MSCP SPORE. The Core will oversee clinical coordination, data management, and research tumor registry annotation of tissue bank specimens of protocol-driven and routine tissue banking (Core B), and biostatistical analyses (Core C) of data managed by the Informatics (Core D). The Administrative Core will be responsible for all communications with the external and internal Scientific Advisory Boards, Patient Advocates, and NCI personnel. The Core will coordinate travel of MSCP SPORE investigators selected to present work to annual NCI-sponsored SPORE meetings, and assist investigators in the preparation and submission of manuscripts for publication, maintaining records of SPORE publications. The Core will oversee and administer the Developmental Research and Career Development Programs in relation to the Projects and other Cores of the SPORE to ensure their smooth function and flexible adaptation to new advances, unforeseen challenges, and obstacles that may require redistribution of resources of the SPORE. Core A will serve as the exponent of MSCP investigators in the UPCI and Departments relevant to this Interdisciplinary Program, to optimize research productivity, complementarity, and synergy in relation to the Cancer Institute, other SPOREs, and Cooperative Groups.

Specific Aims:

Specific Aim 1: Provide scientific management and oversight of all SPORE activities.

Specific Aim 2: Provide fiscal management and oversight of all SPORE activities.

Specific Aim 3: Serve as liaison with the NCI SPORE Program Office, National Cooperative Groups, International Cooperative Groups, industry, other translational researchers, melanoma patient advocates, and the community.

Specific Aim 4: Organize and lead SPORE planning process in partnership with the internal and external advisory boards (IAB and EAB).

Specific Aim 5: Optimize resource allocation to achieve the translational research mission of the SPORE, changing direction and discontinuing projects that successfully mature to the point of gaining independent extramural support or that do not adequately meet their translational goals and substituting new projects as appropriate.

To fulfill these aims, the Administrative Core will continue to provide scientific and fiscal oversight and enhance communication on multiple levels to ensure the success of the MSCP SPORE. The Core will oversee day-to-day operations, coordinate periodic administrative and scientific reviews, and work closely with the NCI Program Office. Core personnel will continue to coordinate MSCP SPORE activities with those of the University of Pittsburgh Cancer Institute (UPCI), the Departments and Schools whose faculty participate in MSCP SPORE projects, with outside collaborating institutions, and with advocacy groups. The Core will foster interactions among SPORE projects and with related research outside the MSCP SPORE regionally, nationally, and internationally. The Core will continue to oversee the Developmental Research Program and the Career Development Program requests for new applications, and their review and prioritization.

The Core will provide the administrative framework through which the SPORE Executive Committee, IAB, and EAB can evaluate program activities and redirect resources as needed to maximize translational progress toward the goals of the MSCP SPORE. The Core will work to raise melanoma cancer awareness and to increase philanthropic support of melanoma and skin cancer research to enhance and increase the impact of the MSCP SPORE research effort. Centralization of these efforts in the Core maximizes efficiency and ensures that SPORE guidelines are followed uniformly throughout the program. The Administrative Core will monitor budgets of the SPORE Research Projects and Cores to ensure that no duplication of effort occurs between the MSCP SPORE and the shared facilities at UPCI, other funded SPOREs at UPCI, or the national cooperative groups. The Core will be used equally by the 4 research projects, the other 3 cores, the Developmental Research Program, and the Career Development Program.

CORE B: BIOSPECIMEN CORE/IMMUNOLOGIC MONITORING AND CELLULAR PRODUCTS LABORATORY

Director:
Lisa H. Butterfield, Ph.D.

Co-Directors:
Uma N. Rao, M.D.
Jonhan Ho, M.D.

Core B serves the MSCP SPORE 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 (#96-099), as well as clinical protocol-specific banking. For all of the projects proposed in this renewal, 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 Aims:

Core B will support each Project as follows:

Project 1 (Tarhini/Storkus) 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) IFN?+CD4+ and IFN?+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-1a, IL-1Β, IL-2, IL-2R, IL-6, IL-8, IL-10, IL-17, TNF-a, IFNa-2b, MIP-1a, MIP-1Β, IP-10,VEGF, CXCL11, CXCL9 and C-reactive protein (CRP) by multiplex Luminex assay.

Project 2 (Butterfield/Kirkwood) The Core will support 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.

Project 3 (Zarour/Ferrone) The Core 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).

Project 4 (Falo/Geskin/Tawbi) 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.

Together, the Biospecimen Core B will support the projects of the SPORE with tissue banking (all Projects), standardized immunologic monitoring (all Projects) and cGMP cellular product production for therapy (Project 2). 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.

CORE C: BIOSTATISTICS CORE

Director:
Yan Lin, Ph.D.

CORE C (Biostatistics) will reside within the University of Pittsburgh Cancer Institute’s (UPCI) Biostatistics Facility, which provides clinical and basic-science investigators in UPCI with statistical expertise in design, analysis, and reporting of cancer-related research studies. These cover basic-science studies; phase I and phase II oncology clinical trials; epidemiologic studies, including those related to cancer prevention and awareness; and investigations of behavioral and health sequelae of cancer treatment. In its role as Core C for this SPORE, the Biostatistics Core will support all four research projects. We will collaborate with investigators on statistical aspects of the design of in vitro and in vivo laboratory-based studies as new data come to light, and perform both exploratory and confirmatory statistical analyses of the resulting data from key experiments in Projects 1 - 4. We will perform interim analyses of safety for the SPORE’s clinical trials (Projects 2, 3, & 4), and final analyses of their data on safety, immune response, and treatment efficacy. We will contribute to the review of the SPORE’s developmental research-program proposals, and provide statistical support to those that are funded. We will provide statistical support to the career-development awardees. We will work with the project investigators, with Core D (Informatics), and with UPCI Clinical Research Services, a component of Core A, to ensure that the requisite laboratory and clinical-trial data are available for statistical analyses. We will collaborate with the project investigators in writing and preparing progress reports, abstracts, manuscripts, and presentations. We will also conduct methodological research motivated by the SPORE projects to improve the statistical efficiency in the study design and analysis.

Specific Aims:

Core C, the Biostatistics Core, will assist MSCP SPORE investigators with study planning (in vitro, in vivo, in silico, and clinical) and biostatistical and bioinformatic analyses. A lead statistician will be assigned to each SPORE Project, but all Core personnel (including two Masters-level biostatisticians) and the entire UPCI Biostatistics Facility will be available to support the MSCP SPORE (see letter of support from Biostatistics Facility Director, Dr. Daniel Normolle). Specifically, Core C will support each Project as follows:

Project 1 Dr. Lin will take the lead in conducting the correlative analysis of Eastern Cooperative Onocology Group (ECOG) trial E1609 data and build prediction models for progression-free survival (PFS) and overall survival (OS) for surgically resected stage IIIB/C and M1a/b melanoma patients treated with either ipilimumab or high-dose INFa (HDI). Dr. Chandran will analyze all sequencing data and help develop the prediction models.

Project 2 Dr. Lin will take the lead in conducting the interim and final analysis of UPCI 09-21 trial data as well as in vitro and in vivo laboratory data and high-throughput biomarker data.

Project 3 Dr. Wang will take the lead in designing and conducting interim and final analyses of phase I trial data for the combination of vemurafnib and HDI as well as the analysis of in vitro and in vivo laboratory data.

Project 4 Dr. Day will take the lead in conducting the interim and final analyses of the microneedle array clinical studies in patients with melanoma and CTCL and their correlative laboratory studies.

CORE D: INFORMATICS CORE

Co-Directors:
Michael J. Becich, M.D, Ph.D.
Melissa I. Saul, M.S.

Translational research such as that proposed in the Melanoma and Skin Program (MSCP) SPORE has a spectrum of needs that benefit from software solutions, including clinical information annotation, data warehousing and disease modeling/analysis tools.

Specific Aims:

The Informatics Core (Core D), led by Drs. Becich and Ms. Saul, will provide clinical research informatics services to Melanoma and Skin Cancer Program (MSCP) SPORE investigators to support study design and conduct and data collection and extraction through the application of such informatics tools as natural language processing, machine learning, and data integration. The Informatics Core will draw heavily from the grant-funded work of Drs. Becich and Ms. Saul, who will specifically support each Project/Core as follows:

Project 1 The Informatics Core will be responsible for the data extraction and collection of clinical annotations by the Research Information Service (RIS) for the E1609 patient cohort and ensure the integration of these data into the Research Data Warehouse (RDW) deployed by the Department of Biomedical Informatics (DBMI) (i2b2, which is an open-source tool developed by Harvard) for use by the Biostatistics Core (Core C).

Project 2 The Informatics Core will assist in studying the management of the toxicities associated with the treatment for this cohort using our clinical phenotyping tool. Project 2 also relies on Core D’s Clinical Research Management System (CRMS), called the Clinical Trials Management Application (CTMA), and clinical annotation expertise provided by the RIS.

Project 3 The Informatics Core will coordinate with Drs. Zarour and Ferrone to ensure access to clinical annotation data for patients enrolled in the proposed clinical trials. With the recent addition to our RIS of collecting BRAF status, we will make these data available to the Project team as well as the Biostatistics Core. The study will also analyze the use of IFN-a and we will collect this electronically from our Electronic Medical Record (EMR) systems and analyze it with cohort discovery tool. Research data generated by Project 3 will also be incorporated into our RDW.

Project 4 Ms. Saul will lead the development of algorithms to identify patients with in transit melanoma who may be eligible for enrollment in Project 4. She will also coordinate the collection of clinical annotation data from our RIS for this subset of patients.

Core A (Administrative) The Informatics Core will ensure the requisite laboratory and clinical trial data are recorded, stored, backed up, and available for review and annual reporting to the Internal and External Advisory Boards and the National Cancer Institute. In addition, the Informatics Core will facilitate all data sharing within the MSCP SPORE and for intra-SPORE collaborations. The Administrative Core as well as all projects and cores will require informatics consulting and support for network storage and data sharing services. Finally, cohort discovery and data extraction support for Developmental Research and Career Development Program projects is anticipated, as phenotyping and clinical annotation services have been provided in the past and currently, including the application of advanced data collection, annotation, and standardization methods.

Core B (Biospecimen) Ms. Saul will lead the effort to expand our ability to identify new patients who are eligible for inclusion in our biospecimen repository as well as existing patients who may have missing or incomplete information that could be extracted from our EMR system. The Informatics Core initially helped create and will continue to contribute to the management of Core B’s IMBase and StemLab databases and will integrate that data into the RDW. Core B will be heavy users of our CTMA and RIS.

Core C (Biostatistics) The Biostatistics Core will rely heavily on our CRMS and CTMA to support SPORE clinical trial (Projects 2, 3 & 4) data management. The Informatics Core will supply Core C with direct access to the CTMA and the RDW for data access and adverse events reporting. The Biostatistics Core will rely heavily on the Info Core for network storage and data sharing services.

The Informatics Core will work with MSCP SPORE investigators and the PI to ensure that the existing clinical (from EMRs) and cancer registry data are available for analysis and to develop new algorithms if required by the project. In particular, we will work with Core C as we deploy our RDW to coordinate the biostatistics analysis requirements. Informatics Core staff will ensure compliance with all security and privacy regulations with respect to data and will work with the entire SPORE team in preparing progress reports, abstracts, manuscripts, and presentations.