Study assessments . Patients were monitored by laboratory assess- ments and physical examination. Tumor evaluations were performed with MRI at baseline and every 12 weeks. Blood was collected to assess ctDNA at baseline and every 12 weeks (10). PBMCs were collected at baseline and at weeks 12, 18, and 24, and then every 12 weeks there - after for CyTOF-based immunomonitoring assessments.investigational or anticancer agents with the exception of temozolomide (maximum 90 mg/m2/dose) during focal RT. Patients must have been either off systemic steroids or have been on a stable dose of dexameth- asone not exceeding 0.1 mg/kg/day (maximum 4 mg/day). All patients were enrolled upon completion of focal RT. Participating PNOC sites received the H3.3K27M synthetic pep - tide vaccine combined with a TT peptide through a central supply from the Immunologic Monitoring and Cellular Products Laboratory at the University (IMCPL) of Pittsburgh. Pharmacists at each site were trained for the emulsification process and prepared each vaccine emulsion (300 μg/dose H3.3K27M synthetic peptide and 200 μg/dose TT peptide in Montanide). The vaccine was administered subcutane - ously in either the arm or thigh, beginning 2–8 weeks after completion Figure 5. A patient who demonstrated a continued expansion of H3.3K27M-reactive CD8 + T cells. (A) MR images of a patient at baseline (left), week 12 (second from left), week 24 (second from right), and week 36 (far right). The patient (PNOC007-25) was found to have local progression in week 36. (B) Plot shows the longitudinal percentage of H3.3K27M-reactive CD8+ T cell subpopulations among live PBMCs. (C) Plot depicts the longitudinal percentage of circulatory MDSC subsets among live PBMCs. The Journal of Clinical Investigation CLINICAL MEDICINE 6335 jci.org Volume 130 Number 12 December 2020 outcomes of DIPG patients. Time-to-event analyses were calculated using the Kaplan-Meier method from the date of diagnosis to the date of censoring or death. Patients who withdrew consent were lost to follow-up or were still alive at the study cutoff date were censored for OS-based survival analyses. The log-rank and likelihood ratio tests were used for comparisons between groups in Kaplan-Meier and Cox proportional HR analyses, respectively. OS was used as the primary survival parameter for the Cox proportional HR multivariate analyses, which were conducted using nonconfounding variables (P > 0.10) as determined by Fisher’s exact test. A P value of less than 0.05 was con- sidered statistically significant. For stratum B, pilot OS data were obtained in this specific subpop - ulation to support the design of the subsequent phase 2 trial (n = 10). An overall immunological response to the vaccine was defined as at least a 25% post-vaccination increase in the proportion of H3.3K27M-reactive CD8+ T cells among total live PBMCs relative to baseline. An analogous approach was used for classifying the expansion of tSNE-stratified bulk and H3.3K27M-reactive CD8+ T cell subpopulations. Patients were further classified by relative abundance of PBMC-derived MDSC subsets at baseline. Myeloid cells from all patients were pooled and stratified on a tSNE plot, revealing 32 discrete clusters (Supplemental Figure 14), of which E-MDSC and M-MDSC subsets (51) were quantified. Patients were assigned MDSChi and MDSClo designations on the basis of the abundance of these cell populations relative to median levels at baseline. This approach was extrapolated to incorporate total (sum of E-MDSCs and M-MDSCs) MDSC levels. Data analyses and visualization were performed using the ggplot2 (52) and cytofkit (47) R packages. Study approval. Prior to trial activation, the appropriate approvals were obtained from the relevant regulatory agencies, including the FDA, for use of the investigational new drug (IND no. 17070), as well as IRB approval from UCSF, Lurie Children’s Hospital of Chicago, Seat - tle Children’s Hospital (Seattle, Washington, USA), Oregon Health & Science University, UCSD and Rady Children’s Hospital, Dana-Farber Cancer Institute, Children’s National Medical Center (Washington, DC, USA), Washington University in St. Louis, The Children’s Hospital Of Philadelphia (Philadelphia, Pennsylvania, USA), St. Jude Children’s Research Hospital (Memphis, Tennessee, USA), Nationwide Children’s (Columbus, Ohio, USA), Texas Children’s Hospital (Houston, Texas, USA), Primary Children’s Hospital (Salt Lake City, Utah, USA), and Children’s Minnesota (Minneapolis, Minnesota, USA). All study partic- ipants or their parents provided informed consent and age-appropriate assent according to institutional IRB guidelines. Author contributions SM, MDP, and HO conceptualized and designed the study. JMT, TN, PBW, NDA, KO, LHB, and RDG analyzed PBMC and tumor tissue samples. JEVM conducted imaging data analyses. ERB and JN evaluated ctDNA. SM, RRL, SG, AB, SNC, NSW, JRC, KG, and KJN enrolled and managed the patients. AMS provided poly-ICLC. AMM supervised the statistical and analytical methods used. All authors participated in writing and revising the manuscript. SM and HO, the study chairs, and SM and MP, on behalf of the PNOC leadership, reviewed AEs, SAEs, and RLTs weekly per PNOC stan- dardized operating procedures for safety monitoring.Central imaging review. A central radiology review of all cases was conducted retrospectively using response assessment in neu- ro-oncology (RANO) (45) criteria. MRI with T1 (with and without contrast enhancement), T2, and T2 fluid-attenuated inversion recov- ery (FLAIR) was used to measure tumor size. Anterior-posterior and transverse dimensions were measured using the Horos (version 3.0) software package. Progressive disease on MRI was defined as a greater than 25% increase in the sum of perpendicular diameters and/or development of new enhancing or nonenhancing lesions by a board-certified neuroradiologist. Study endpoints. The study’s primary endpoints were the eval- uation of safety for all patients and OS at 12 months for patients enrolled in stratum A. The exploratory endpoints included CyTOF- based evaluation of H3.3K27M-reactive CD8+ T cell responses in PBMC samples, immunohistochemical analysis of available tumor tissue, and ctDNA analyses. Safety . Adverse events (AEs), serious adverse events (SAEs), and RLTs were reported from vaccine initiation out to 30 days after the last study treatment. Grading was based on Common Terminology Crite - ria for Adverse Events (CTCAE), version 4.0 (46). RLTs were defined as any grade-2 or greater autoimmune reaction; any grade-3 or greater hematologic or nonhematologic toxicity with the exception of lymph- openia; any life-threatening event; and any other unexpected grade-2 or greater neurological deficit that was related to the vaccine therapy and did not respond to a trial of dexamethasone or bevacizumab. AEs, SAEs, and RLTs were reviewed weekly by the study chairs and PNOC leadership according to PNOC standardized operating procedures for safety monitoring. The data monitoring and safety committee (DSMC) at UCSF monitored the trial. An early stopping rule was implemented on the basis of RLT inci- dences as follows: the first 3 patients in each stratum were observed for at least 12 weeks before the next set of patients were enrolled. If a RLT occurred in 1 of the first 3 patients, an additional 3 patients would be enrolled in that stratum and observed for at least 12 weeks. If fewer than 2 of the first 6 patients in each stratum experienced a RLT within at least 12 weeks of follow-up, the remaining patients would be enrolled. If a RLT occurred in 2 or more of the first 6 patients, accrual would be suspended. Therefore, with 6 patients enrolled, there was an 88% probability of detecting 1 or more RLTs with an underlying rate of 30%. There was a 74% probability of detecting 1 or more RLTs with an under- lying rate of 20%. In stratum A, if all 19 patients were enrolled, there was an 86% probability of detecting 1 or more RLTs with an underlying rate of 10% and a 62% probability with an underlying rate of 5%. Mass cytometric data processing. Data acquisition for H3.3K27M epitope–specific CD8+ T cell responses was conducted using the Flu- idigm Helios CyTOF system at the UCSF Parnassus Flow Cytometry Core. The sensitivity and specificity of the HLA-A *02:01-H3.3K27 dextramer (Immudex) were validated by positive staining of CD8+ T cells transduced with an H3.3K27M-specific T cell receptor (4) and negligible staining in nontransduced CD8+ T cells, respectively. H3.3K27M-reactive CD8+ T cells were pooled from all patients and displayed via a t-SNE plot in the cytofkit package (47). Statistics for OS and immune responses. For stratum A, with a null hypothesis that OS12 is 40%, accrual of 19 patients provided 80% power with a target overall type I error of 0.05 to detect a 30% differ- ence in OS12 using a 1-sided exact binomial test. A null OS12 rate of 40% was chosen on the basis of prior studies (48–50) and review of The Journal of Clinical Investigation CLINICAL MEDICINE 6336 jci.org Volume 130 Number 12 December 2020 olas Bayless and Pier Federico Gherardini at the Parker Institute for Cancer Immunotherapy (PICI) for computational analyses of the mass cytometric data; and the Ian’s Friends Foundation. Prior publication: This work was partially presented at the 5th Biennial Conference on Pediatric Neuro-Oncology Basic and Translational Research (May 4, 2019, San Francisco, California, USA), as well as the 24th Annual Meeting and Education Day of the Society for Neuro-Oncology (November 24, 2019, Phoenix, Arizona, USA). Address correspondence to: Hideho Okada, Neurological Surgery, Helen Diller Family Cancer Research Building, UCSF, HD 472, 1450 3rd Street, San Francisco, California 94158, USA. Phone: 415.476.1637; Email: hideho.okada@ucsf.edu.Acknowledgments The authors thank the following individuals and organizations: the study participants and their families; all PNOC staff for their assistance; all PNOC principal investigators supporting the study; members of the IMCPL at the University of Pittsburgh for prepa - ration and distribution of the vaccine (supported in part by award P30 CA047904); the UCSF Parnassus Flow Cytometry Core for mass cytometry–related services (P30 DK063720) and use of the CyTOF2 “Charmander” (S10 1S10OD018040-01); the USCF data safety monitoring committee; Lawrence Fong and the Cancer Immunotherapy Laboratory (CIL) at UCSF for the processing and banking of patient-derived peripheral blood samples; Maryam Shahin for contributions to the optimization of the CyTOF ana - lytical platform; Marie-Eve Koziol of SEPPIC Inc. for training research nurses in the preparation of Montanide emulsions; Nich- 1.