Sectioned slides from formalin-fixed, paraffin-embedded (FFPE) samples will be stained for the biomarker(s) of interest, and then evaluated by a pathologist for review of biomarker staining expression levels. This sample will be taken at time of surgery. At the end of the study, the BTTC CC Research Nurse will contact the participating site to request the slides. 5.1.3 Tissue will be interrogated by Next Generation Sequencing for tumor antigens Generation of the HSPPC-96 vaccine requires > 7 grams of tissue. All available tumor aside from what will be used for routine pathologic studies and diagnosis should be sent for HSPPC-96 vaccine preparation. At the end of the study, up to 18 FFPE unstained slides (formalin fixed 63 Abbreviated Title: GBM Pembro HSPPC Version Date: 03/26/2024 paraffin embedded) may be collected. If subsequent tumor biopsies are obtained, including at time of progression, additional tissue specimens may be collected following the same method described above. These tissue collections for next generation sequencing are at the investigator’s discretion. The FFPE Tumor Tissue Slide specimens are maintained at room temperature and shipped ambient by the site upon request by BTTC Coordinating Center to: Attn: BTTC Coordinating Center Research Nurse Coordinator NCI / Neuro-Oncology Branch / Brain Tumor Trials Collaborative National Institutes of Health Building 82 , Room 215 Bethesda, MD 20892 Phone: 240-858-7083 Email: ukeme.ikiddeh-barnes@nih.gov 5.1.4 Tissue Collection at NCI Tumor samples will be collected at time of surgery by the Laboratory of Pathology NCI. Tissue sections will be obtained and will be labeled with the Study Subject ID number. The relationship between the Study Subject ID number and the patient clinical information will be stored in a secure database that is maintained and regularly backed up by the Laboratory of Pathology NCI. The Laboratory of Pathology at NCI will process the tissue samples per Appendices 16.2, 16.3, 16.4, 16.5. 5.1.5 Sample Storage, Tracking and Disposition All specimens obtained in the protocol are used as defined in the protocol. Any specimens that are remaining at the completion of the protocol will be stored in the conditions described below. The study will remain open so long as sample or data analysis continues. Samples from consenting subjects will be stored until they are no longer of scientific value or if a subject withdraws consent for their continued use, at which time they will be destroyed. If the patient withdraws consent the participant’s data will be excluded from future distributions, but data that have already been distributed for approved research use will not be able to be retrieved. The PI will record any loss or unanticipated destruction of samples as a deviation. Reporting will be per the requirements of section 7.2. 5.2 SAMPLES FOR GENETIC/GENOMIC ANALYSIS 5.2.1 Scope of the analysis Patients’ tissue specimens will be analyzed for exploratory tumor-derived biomarkers. Assays performed may include, but are not limited to: a) immunohistochemistry (IHC) of FFPE samples to determine the frequency of tumor infiltrating lymphocytes (TILs) such as CD8 T cells and CD4 regulatory T cells, as well as the expression of predictive biomarkers such as PD-L1; b) whole exome sequencing (WES) and transcriptome analysis (RNAseq) of flash frozen/OCT 64 Abbreviated Title: GBM Pembro HSPPC Version Date: 03/26/2024 samples to evaluate mutational burden and expression of neo-epitopes; and c) functional and phenotypic characterization of vitally frozen immune cell subsets. 5.2.2 Certificate of Confidentiality As part of study efforts to provide confidentiality of subject information, this study has obtained a Certificate of Confidentiality, which helps to protect personally identifiable research information.