Mass cytometry detects H3.3K27M-specific vaccine responses in diffuse midline glioma

Abstract

BACKGROUNDPatients with diffuse midline gliomas (DMGs), including diffuse intrinsic pontine glioma (DIPG), have dismal outcomes. We previously described the H3.3K27M mutation as a shared neoantigen in HLA-A*02.01+, H3.3K27M+ DMGs. Within the Pacific Pediatric Neuro-Oncology Consortium, we assessed the safety and efficacy of an H3.3K27M-targeted peptide vaccine.METHODSNewly diagnosed patients, aged 3-21 years, with HLA-A*02.01+ and H3.3K27M+ status were enrolled in stratum A (DIPG) or stratum B (nonpontine DMG). Vaccine was administered in combination with polyinosinic-polycytidylic acid-poly-I-lysine carboxymethylcellulose (poly-ICLC) every 3 weeks for 8 cycles, followed by once every 6 weeks. Immunomonitoring and imaging were performed every 3 months. Imaging was centrally reviewed. Immunological responses were assessed in PBMCs using mass cytometry.RESULTSA total of 19 patients were enrolled in stratum A (median age,11 years) and 10 in stratum B (median age, 13 years). There were no grade-4 treatment-related adverse events (TRAEs). Injection site reaction was the most commonly reported TRAE. Overall survival (OS) at 12 months was 40% (95% CI, 22%-73%) for patients in stratum A and 39% (95% CI, 16%-93%) for patients in stratum B. The median OS was 16.1 months for patients who had an expansion of H3.3K27M-reactive CD8+ T cells compared with 9.8 months for their counterparts (P = 0.05). Patients with DIPG with below-median baseline levels of myeloid-derived suppressor cells had prolonged OS compared with their counterparts (P < 0.01). Immediate pretreatment dexamethasone administration was inversely associated with H3.3K27M-reactive CD8+ T cell responses.CONCLUSIONAdministration of the H3.3K27M-specific vaccine was well tolerated. Patients with H3.3K27M-specific CD8+ immunological responses demonstrated prolonged OS compared with nonresponders.TRIAL REGISTRATIONClinicalTrials.gov NCT02960230.FUNDINGThe V Foundation, the Pacific Pediatric Neuro-Oncology Consortium Foundation, the Pediatric Brain Tumor Foundation, the Mithil Prasad Foundation, the MCJ Amelior Foundation, the Anne and Jason Farber Foundation, Will Power Research Fund Inc., the Isabella Kerr Molina Foundation, the Parker Institute for Cancer Immunotherapy, and the National Institute of Neurological Disorders and Stroke (NINDS), NIH (R35NS105068).

Keywords: Brain cancer; Cancer immunotherapy; Immunology; Oncology.

Conflict of interest statement

Conflict of interest: HO is an inventor of a utility patent application titled “H3.3 CTL peptides and uses thereof” (attorney docket no: 81906-938904-220400 US, client reference no. SF15-163), which has been exclusively licensed to Tmunity Therapeutics Inc.

Figures

Figure 1. Mass cytometry–based phenotyping of H3.3K27M-reactive CD8+ T cell subpopulations reveals associations between immunological responses and prolonged OS or PFS.

(A and B) Kaplan-Meier survival curves contrasting the (A) OS and (B) PFS of patients enrolled in stratum A (red) and stratum B (blue) using log-rank tests. (C) Left: PBMCs derived from a healthy donor showed an absence of H3.3K27M dextramer staining. Right: The H3.3K27M dextramer exhibited sensitive detection of H3.3K27M-specific T cell receptor–transduced (TCR-transduced) CD8+ T cells. (D) Heatmap visualizing the relative expression (Z score) of subpopulation markers in patient-derived H3.3K27M-reactive CD8+ T cells. (E) Heatmap visualizing the patient-specific presence (blue) or absence (red) of an expansion of H3.3K27M-reactive CD8+ T cells in a subpopulation-specific manner. (F and G) Kaplan-Meier survival curves contrasting the (F) OS and (G) PFS of patients who had an immunological response (blue) compared with patients who did not (red) using log-rank tests. (H and I) Kaplan-Meier survival curves contrasting the (H) OS and (I) PFS of patients who had an expansion of effector memory H3.3K27M-reactive CD8+ T cells (blue) compared with patients who did not (red) using log-rank tests.

Figure 2. Individual patient study course.

Depicted within this swimmer plot is the patient-specific timing of vaccine administration, dexamethasone treatment, detection of H3.3K27M-reactive T cell responses, tumor progression, and vital status (patients who withdrew consent were censored).

Figure 3. Mass cytometry–based analysis of circulatory MDSCs reveals an association between baseline levels of MDSCs and shortened OS and PFS.

(A) Representative gating strategy to identify myeloid cells from patient-derived PBMCs by mass cytometry. Cells that stained double-positive with iridium intercalator were identified as intact cells (far left). Cells with low cisplatin staining were identified as live cells (second from left). Cells that stained negative for CD45 or positive for CD3 were excluded (middle). Cells that stained positive for CD19 or positive for CD56 were excluded (second from right). Cells that stained positive for either CD11b or CD11c were identified as myeloid cells (far right). (B) Box plot shows the range of abundance of circulatory total MDSCs among patients with DIPG classified as MDSClo or MDSChi. MDSC classifications were established on the basis of a median threshold. (C and D) Kaplan-Meier survival curves contrasting (C) OS and (D) PFS of DIPG patients with baseline circulatory MDSC levels above (red) and below (blue) the median threshold, as determined by log-rank tests.

Figure 4. Administration of dexamethasone is positively associated with increased baseline MDSC levels and inversely associated with H3.3K27M-reactive CD8+ T cell responses and survival.

(A) Box plot demonstrates the abundance of circulatory MDSCs at baseline among patients with DIPG treated with dexamethasone (n = 6) for at least 3 days prior to treatment relative to patients who did not receive dexamethasone (n = 13). (B) Waterfall plot depicts the overall longitudinal percentage change of H3.3K27M-reactive CD8+ T cells at the final time point analyzed relative to baseline for each patient with DIPG who was treated with dexamethasone. Yellow bars indicate the pre-treatment period; red bars indicate patients who received dexamethasone after commencement of vaccination; and blue bars indicate patients who did not receive dexamethasone while in the study. (C and D) Kaplan-Meier survival curves contrasting the (C) OS and (D) PFS of patients with DIPG based on pretreatment dexamethasone administration, as determined by log-rank tests.

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.