The total amount of blood required for this purpose will be 10 cc. A comparison of pre -therapy lymphocyte functions to those at intervals after each immunization will be made. These tests may provide evidence for the development of immune responses following DC + xALT therapy and will play an important role in the design o f future immunotherapy clinical trials. 21.7 T cell Cytokine Production CD4+ and CD8+ T cells derived from patients prior to and after DC + xALT therapy will be cultured in 96- well plates with 0, 5, or 10 μg of PHA, TTRNA pulsed DCs, and cont rol (PBMC RNA) puls ed DCs. After 16 and 72 hours of culture, supernatants are harvested and processed in duplicate with a custom BioRad Bio- Plex 12-plex (IL -2, IL -4, IL -5, IL -6, IL -7, IL -10, IL -12 (p70), IL -15, IFN -γ, TNF -α, IP10) Cytokine Reagent Kit (BioRad) to differenti ate responses into TH 1 and TH 2 types. Briefly, supernatants are incubated with anti -cytokine conjugated beads, followed by incubation with biotinylated detection antibody. Reaction mixture is detected with streptavidin -PE and analyzed on a Luminex 100 machine (Luminex Corporation, Austin, Texas). Unknown cytokine concentrations are calculated by BioPlex Manager software using standard curves derived from a recombinant cytokine standard. A paired t -test will be used to compare cytokine differences between treatment groups as described above. A Bonferroni correction will be used to control for the multiple cytokines being evaluated such that P<0.007 will be required for statistical significance. 21.8 Cytokine Flow Cytometry The CFC analysis will be done at the U F Brain Tumor Immunotherapy Program . This process involves the rapid early detection and analysis of the production of CD107a, γ -IFN, TNFα, and IL -2 prior to cellular secretion following antigen -specific stimulation in vitro as determined by CFC. The fun ctional CD4+ and CD8+ immune response of the patients will be monitored using an 11 color CFC assay. Evidence for intra - and inter -molecular epitope spreading and evidence for the induction of autoimmunity will also be sought. For cellular immune respons es a polyfunctional analysis [9-11] of T cell function will be performed as previously described [11]. Subsets of naïve (CD45RO-CD27+), effector (CD45RO+CD57+), effector memory (CD45RO+CD27-), central memory (CD45RO+CD27+), and terminal effector (CD45RO-CD57+) cells will be analyzed for production of IFNγ, TNF α, and IL -2 as well as induction of the cytolytic granule marker CD107. Immediately preceding each vaccination , 90 mLs of peripheral blood will be drawn into vacutainer tubes containing ACD. PBMC s will be separated by density gradient centrifugation on ficoll -Hypaque. Cryopreserved PBMC samples are thawed and rested overnight at 370C/5% CO 2 in RPMI media containing 10% fetal calf serum. Cells are adjusted to 2x106/well and incubated with 1 μg/mL of e ach of the co -stimulatory MAbs αCD28 and αCD49d with or without stimulation with autologous tumor, mRNA -pulsed DCs, and autologous normal brain mRNA (when available)(2 μg/mL) in the presence of Brefeldin A (5 μg/mL Sigma -Aldrich, St. Louis, MO) monensin (1µg/mL; Golgistop, BD Biosciences, San Diego, CA), and αCD107a PE -Cy5 for 5- 6 hr at 370C and 5% CO 2. Following stimulation, cells will be treated with EDTA for 15 minutes at ambient temperature (AT, 18 - 22ºC). The cells will be washed and stained with MAbs specific for CD4 PerCP -Cy5.5 -PE, CD8 APC -Cy7, Re-MATC H Protocol Page 89 of 106 Version: 20180628 CD45RO PE -TR, CD27 APC, CD57 FITC, CD14/CD19 (PacBlue) and a vital -dye reagent (LIVE/DEAD Fixable Violet Dead Cell Stain Kit for Flow Cytometry; Invitrogen Corp., CA) for 20 minutes at RT. After two washes, 1xBD FACS Lysing solution (BD Biosciences, San Jose, CA) will be added and samples will be incubated for 10 minutes at RT. After one wash, 1xBD FACS Permeabilizing Solution 2 (BD Biosciences, San Jose, CA) will be added and samples incubated for 10 minutes at AT. After one wash, cells will be stained with αCD3 (AmCyan), αIFN -γ (PE -Cy7), αTNF -α (A700), and αIL2 (PE) for 30 minutes on ice, washed, and fixed in PBS containing 1% formaldehyde (Sigma -Aldrich, St. Louis, MO). In all experiments, a negative cont rol (αCD28/49d), and a positive control (SEB, 10 μg/mL, Sigma -Aldrich) will be included. The samples are acquired on a custom LSRII polychromatic flow cytometer (BD Immunocytometry System, San Jose, CA) equipped for detection of 17 fluorescent parameters. We are planning to collect a minimum of 500,000 total lymphocytes from each sample, because we expect the frequency of responding cells to be between 0.05 and 1.0%. This number of events is required based on calculations performed by Dr. Holden Maecker (BD Bioscience, personal communication) to detect a statistically significant number of positive events that can be used for the analysis of the data and the characterization of the different populations. Ideally, we would be able to select a validated s urrogate immunologic response marker for clinical efficacy, but no such marker has been identified to date and studies to validate such a marker would need to be large and prospective and would be clearly beyond the scope of this protocol. It would be our intent, however, to incorporate any additional knowledge that becomes available at the time of data analysis to evaluate the relative biologic significance of the immune response markers that we have chosen. Tetramer studies will not be routinely perfor med because the HLA restriction of all antigens present in the mRNA- pulsed DCs has not been determined. Tetramer analysis will be used, however, in patients with appropriate genetic haplotypes to quantitate changes in immunologic response to tumor -associa ted antigens differentially expressed in malignant brain tumors before and after vaccination as follows: IL -13R2α 345-354, Her - 2369-377, survivin 96-104, gp100 209-217, and TRP -2180-188. 21.9 Statistical Analysis for Polyfunctional Response The analysis used here will be one that has been successful at distinguishing HIV progressors and non- progressors based on T cell phenotype [9-11]. All data will be background subtracted. For each measure, a lower threshold corresponding to two standard deviations above background is set to 0 based on a Poisson model essentially allowing T cells to be designated as positive or negative for a certain phenotypic marker. The number of positive phenotypic markers post -vaccination will be calculated for each patient. Additional exploratory analyses will be conducted using an exact binomial test that will compare treatment groups with respect to the proportion of patients with 5 positive markers, and 4 or more positive markers. 21.10 Sero logy Antibody levels to specific antigens will be determined by FACS analysis of antigen immobilized on beads. Cellular lysate autologous tumor cells will be immobilized separately on Dynal M280 magnetic beads according to manufactures directions. Beads are reacted with patient serum for 30 minutes at AT. Washed beads are then developed with a fluorochrome conjugated goat anti -human polyclonal for an additional 30 minutes at AT. Beads are analyzed for fluorescent intensity on a BD FACSCalibur™. Differe ntial levels of antibody binding from pre and post DC + xALT therapy samples to immobilized tumor lysate will be reported as humoral response to the tumor antigens. As a further test, pooled normal brain lysate (Zyagen, Inc.) will be used to demonstrate t umor - specificity or preabsorb normal brain binding antibodies. As a means of demonstrating specificity, serums will be pre-incubated with the same immobilized lysates prior to reacting with beads to absorb reactive antibodies. 21.11 Statistical Analysis All data will be background subtracted and compared using paired t -tests as described above. Re-MATC H Protocol Page 90 of 106 Version: 20180628 21.12 Toll-Like Receptor Agonist Activity Release of endotoxin, a TLR4 agonist, from gut microbial release into the bloodstream during myeloablative therapy has been shown to enhance the activation and expansion of adoptively transferred tumor - specific lymphocytes in murine models [12]. Endotoxin levels (LPS) in patients receiving HDC + PBCT and NMA salvage chemotherapy will be measured in the serum before induction of chemotherapy and serially afterwards during immune monitoring blood draws. The quantitative chromogenic Limulus amebocyte lysaste assay (QCL -1000, Whittaker M.A. Bioproducts, Walkersville, MD) will be used to measure serum LPS levels. Blood will be drawn aseptically into LPS -free tubes. Samples are subsequently diluted with pyrogen -free water and heat inactivated for 100 C for 10 minutes. Escherichia coli 055:B5 reference endotoxin will be used for standard curve analysis (Whittakeer M.A. Bioproducts) and LPS levels at varying time points of therapy will be determined compared to baseline with a paired t -test. Upregulation of TLR expression and TLR signaling pathway genes in serially collected PBMCs will be analyzed by Oligo GEArray ® Human Toll -Like Receptor Signaling Pathway Microarray allowing the profiling of 113 genes related to TLR -mediated signal transduction including all identified members of the TLR family [13- 15]. 21.13 Statistical Analysis Data analysis will be conducted using Genespring (Silicon Genetics) and intensity dependent normalization will be performed on entire dataset. Triplicate samples of RNA extracted from various portions of tumor tissue (wher e sufficient material available) will be used to control for sampling variability and a threshold of a 2 -fold change in expression relative to baseline, and a p- value cutoff of 0.05 will be performed to determine changes in TLR -specific gene expression. Da ta will be confirmed by real- time RT -PCR for a subset of TLR signaling pathway genes that show statistically significant changes after therapy. The primary analysis will focus on pre and post HDC + PBSCT and salvage chemotherapy time points. 21.14 Tumor Associ ated Antigen (TAA) Profile in Recurrent Tumors The identification and validation of antigens in MB/PNETs that can serve as potent tumor rejection antigens may be important for the development of potent and specific immunotherapy against these tumors. RNA amplified from tumors used in vaccine preparation and from 2 nd recurrences (where biopsy or resection is clinically indicated for confirmatory diagnosis and sufficient tissue obtained) will be analyzed for a panel of common TAAs to determine the frequency of expression of these antigens in reMB/PNETs and whether immunotherapy directed against total tumor RNA encoded antigens modulates the expression of these genes after treatment. The top 40 uniformly overexpressed transcripts in common malignancies that w ere not expressed in complementary normal tissues w ere determined using database mining of publically available SAGE and microarray analyses [16]. An analysis of MB/PNETs was not included in this data set but we will investigate the frequency of expression of these 40 TAAs (TAA -40) using a c ustom microarray chip containing the published Tag sequences for identity of each of these proteins ( Agilent Made -to-Order Arrays, Affymetrix, Inc., Santa Clara, CA). RNA (directly isolated where sufficient material or amplified) from first and second rec urrent tumors will be analyzed by the TAA -40 expression chip. Tumors will be considered positive for a TAA -40 is expressed at > 33 copies per cell and at least twofold higher than reference normal brain RNA from three donors (Zyagen, Inc.). 21.15 Statistical Analysis Data analysis will be conducted using Genespring (Silicon Genetics) and intensity dependent normalization will be performed on entire dataset. Triplicate samples of RNA extracted from various portions of tumor tissue (where sufficient material a vailable) will be used to control for sampling variability and a threshold of a 2 -fold change in expression relative to control (normal brain) , and a 2- way ANOVA with a p- value cutoff of 0.05 will be performed to determine changes in TAA -40 specific gene expression. Data will be confirmed by real - time RT -PCR for a subset of TAA -40 genes . Re-MATC H Protocol Page 91 of 106 Version: 20180628 22. DATA MANAGEMENT 22.1 Data Management and Quality Control The Department of Neurosurgery will be responsible for the management of clinical data . 22.2 Laboratory Data Transmission and Accountability In addition to the observational clinical data, other material is collected, such as blood, tissue, and images, which must be transmitted, tracked, and accounted for by linking it to the appropriate patient and follow -up sequence. Also, expectations for t he generation of these elements and the resultant data must be set and monitored closely to ensure their timely collection and movement through the system. In the case of sample management, tissue and tissue -related data is managed by the Brain Tissue Ban k. 23. DATA AND SAFETY MONI TORING PLAN 23.1 Data Safety Monitoring Board The University of Florida Health Cancer Center (UFHCC) is committed to the safety of patients participating in clinical trials at its institution. The UFHCC is also committed to data accuracy and protocol compliance. The UFHCC has established an institutional plan to assure data safety and monitoring for all clinical trials at the University of Florida Health Cancer Center. This plan is designed to comply with policies and guidelines regarding data and safety monitoring from the National Institutes of Health and the National Cancer Institute. Data and safety monitoring takes place within the established process of protocol review and monitoring by the UFHCC Protocol Review and Monitoring Commi ttee (PRMC), the Institutional Review Board (IRB), and the UFHCC’s Data and Safety Monitoring Board (DSMB). The DSMB is the primary agent for assuring data and safety monitoring. The DSMB includes two co- chairs and multidisciplinary representation from cl inical researchers, physicians and biostatisticians. The DSMB includes both voting and advisory members from the UF Health Cancer Center, UF Health Cancer Center -Orlando Health, and the UF Proton Therapy Institute – the three facilities it serves. The UFH CC DSMB meets bi -annually. If an emergent situation arises the committee will convene a meeting. All studies identified by the PRMC as requiring review by the DSMB will be monitored. The DSMB will report final recommendations to the PRMC who reviews and then forwards these recommendations, along with any additional recommendations, to the Principal Investigator. The Principal Investigator is responsible for forwarding PRMC’s recommendation to the Institutional Review Board. Final responsibility and author ity for closing or amending of such trials will rest with the PRMC and Institutional Review Board. Requirements for periodic data and safety monitoring are commensurate to the clinical trial’s risk and complexity. The IRB, PRMC, and/or DSMB may require more frequent and/or additional monitoring to assure patient safety and trial integrity. Data safety and monitoring activities continue until all patients have completed their treatment and until all patients have been followed beyond the time point at whic h study -related adverse events would likely be encountered. Circumstances that would prompt communication with the DSMB include: • Any unexpected death • Any circumstance felt by the medical monitor to warrant DSMB notification and/or input • A frequency of DLTs that require suspension of enrollment 23.2 Independent Medical Monitor For research involving more than minimal risk (as defined in 32 CFR 219.102(i)) to subjects, an independent medical monitor shall be appointed by name. The m edical monitor will oversee the progress of this research protocol, especially issues of individual subject management and safety. The m edical monitor shall be independent of the investigative team and shall possess sufficient educational and professional experience to serve as the subje ct advocate. The m edical monitor shall promptly report discrepancies or problems to the PI . They Re-MATC H Protocol Page 92 of 106 Version: 20180628 shall have the authority to stop a research study in progress, remove individual subjects from a study, and take whatever steps are necessary to protect the safety and well -being of research subjects until the P I can assess the medical monitor's report. The Medical Monitor will review the general progress of the study in regard to the safety for the subjects in a written report discussed with the PI biannually . 23.3 Independent External Monitor An independent external monitor will conduct data and safety monitoring for this study . The monitor will compare source documents with completed case report forms to ensure accuracy and tabulation of adverse events. The monitor will report all findings to the sponsor -investigator, who will notify the IRB when significant safety concerns are i dentified. The independent external monitor will c onduct monitoring visit s to ensur e subject safety and that the protocol is conducted, recorded and reported in accordance with the protocol, stand ard operating procedures (SOPs), Good Clinical Practice (GC P), and applicable regulatory requirements. The monitor also provides ongoing education and resources to investigators and study teams to enhance data quality. The frequency of monitoring will be based on accrual and additional monitoring may be prompted b y findings from monitoring visits, unexpected frequency of serious and/or unexpected toxicities, or other concerns and may be initiated upon request of an investigator or the IRB. 23.4 Principal Investigator This clinical research study will also be monitored internally by the PI. In terms of internal review , the PI will continuously monitor and tabulate adverse events. Appropriate reporting to the UF IRB will be made. If an unexpected frequency of grade III or IV events occur, depending on their nature, acti on appropriate to the nature and frequency of these adverse events will be taken. This may require a protocol amendment, dose de -escalation, or potentially closure of the study. The PI of this study will also continuously monitor the conduct, data, and safety of this study to ensure that: • Stopping rules for toxicity and/or response are met; • Risk/benefit ratio is not altered to the detriment of the subjects; • Appropriate internal monitoring of adverse events and outcomes is done; • Over -accrual does not occur ; • Under -accrual is addressed with appropriate amendments or actions; • Data are being appropriately collected in a reasonably timely manner. 24. RISKS/BENEFITS ASSESSMENT 24.1 Potential Benefits Based on experience with immunotherapy and our previous clinical trials, DC -based immunotherapy may be of benefit to subjects with reMB/PNETs . Of course, because individuals respond differently to therapy, no one can know in advance if it will be benefici al in an individual case.