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Neoadjuvant anti-PD-1 immunotherapy promotes a survival benefit with intratumoral and systemic immune responses in recurrent glioblastoma


Glioblastoma is the most common primary malignant brain tumor in adults and is associated with poor survival. The Ivy Foundation Early Phase Clinical Trials Consortium conducted a randomized, multi-institution clinical trial to evaluate immune responses and survival following neoadjuvant and/or adjuvant therapy with pembrolizumab in 35 patients with recurrent, surgically resectable glioblastoma. Patients who were randomized to receive neoadjuvant pembrolizumab, with continued adjuvant therapy following surgery, had significantly extended overall survival compared to patients that were randomized to receive adjuvant, post-surgical programmed cell death protein 1 (PD-1) blockade alone. Neoadjuvant PD-1 blockade was associated with upregulation of T cell– and interferon-γ-related gene expression, but downregulation of cell-cycle-related gene expression within the tumor, which was not seen in patients that received adjuvant therapy alone. Focal induction of programmed death-ligand 1 in the tumor microenvironment, enhanced clonal expansion of T cells, decreased PD-1 expression on peripheral blood T cells and a decreasing monocytic population was observed more frequently in the neoadjuvant group than in patients treated only in the adjuvant setting. These findings suggest that the neoadjuvant administration of PD-1 blockade enhances both the local and systemic antitumor immune response and may represent a more efficacious approach to the treatment of this uniformly lethal brain tumor.

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Fig. 1: Neoadjuvant pembrolizumab confers significant improvement in overall and progression-free survival in patients with recurrent glioblastoma.
Fig. 2: Tumor gene expression profile altered by neoadjuvant PD-1 blockade.
Fig. 3: Multiplex immunofluorescence imaging of tumor samples demonstrates varying degrees of PD-L1 expression and CD8+ T cell infiltration.
Fig. 4: Neoadjuvant PD-1 blockade alters correlative relationships between blood and tumor repertoire features and alters circulating immune cell phenotypes.
Fig. 5: Proposed mechanism of neoadjuvant PD-1 blockade in recurrent glioblastoma multiforme (GBM).

Code availability

R code is available in packages as described in the manuscript.

Data availability

RNA sequencing data are available in the Gene Expression Omnibus under accession number GSE121810, which includes source data for Fig. 2b and Extended Data Figs. 4 and 5. The remainder of data that support the findings of this study are available from the corresponding author on reasonable request.


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This study was funded in part by the National Institutes of Health SPORE in Brain Cancer (grant no. P50CA211015), the Parker Institute for Cancer Immunotherapy (grant no. 20163828), the Cancer Research Institute, the Musella Foundation, the Ben and Catherine Ivy Foundation, the Uncle Kory Foundation, the Defeat GBM Program of the National Brain Tumor Society, the Ziering Family Foundation and by Merck & Co., Inc. Research and/or financial support was also provided by Adaptive Biotechnologies. The authors also thank A. Garcia, N. Akkad, M. Attiah, S. Khattab, J. Reynoso, M. Wong and M. Guemes for their contributions to the experiments. ImmunoSEQ assays are for research use only and not for use in diagnostic procedures.

Author information




The conceptualization, methodology and supervision were carried out by R.M.P., P.Y.W. and T.F.C.; the investigation was carried out by R.M.P., T.F.C., P.Y.W., J.R.O., A.H.L., A.Y.M., A.C.W., T.B.D., W.H.Y., J.L.C., I.C.A.-R., H.C., T.J.K., J.F.d.G., D.A.R., I.K.M., A.L.C., E.Q.L., P.L.N., B.J.O. and N.A.B.; writing of the original draft was handled by A.Y.M.; and the draft was reviewed and edited by all the authors; funding was acquired by L.M.L., R.M.P., T.F.C. and P.Y.W.; the data were curated by J.R.O. and S.C.G.; formal analysis was carried out by J.R.O., B.M.E., A.Y.M., G.L., L.D., E.S.K., W.H., C.M.S. and J.A.R.; and project administration was carried out by J.R.O. and S.C.G.

Corresponding authors

Correspondence to Timothy F. Cloughesy or Robert M. Prins.

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Competing interests

J.A.R. and C.M.S. have a financial interest in Adaptive Biotechnologies. T.F.C. and D.A.R. have received compensation from Merck as consultants on advisory boards. P.Y.W. and H.C. have received honoraria from Merck. J.F.d.G. has done consulting and/or received honoraria with Merck and Bristol-Myers Squibb. I.K.M. reports research funding from General Electric, Amgen and Lilly; advisory roles with Agios, Puma Biotechnology and Debiopharm Group; and honoraria from Roche for a presentation.

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Extended data

Extended Data Fig. 1 CONSORT diagram.

Flow diagram of disposition of patients enrolled in the study.

Extended Data Fig. 2 Kaplan–Meier plot of progression-free survival.

Median progression-free survival (PFS) for patients who received pembrolizumab only in the adjuvant setting was 72.5 d; patients who received neoadjuvant and adjuvant pembrolizumab had a median PFS of 99.5 d (hazard ratio 0.43, 95% confidence interval 0.20–0.90; two-sided P = 0.03 by log-rank test).

Extended Data Fig. 3 Eighteen gene interferon-γ-related signature scores in neoadjuvant versus adjuvant-only groups.

Line in middle of box represents the median; box extends from the 25th to 75th percentiles; whiskers represent minimum and maximum values; n = 28 independent biological samples; P = 0.025, U = 49 by two-sided Mann–Whitney U-test. *: P < 0.05.

Source Data

Extended Data Fig. 4 RNA sequencing comparison to other recurrent glioblastoma samples.

We combined our RNA sequencing dataset to that of GSE79671 (an RNA sequencing dataset of recurrent glioblastoma pre- and post-bevacizumab treatment; only pre-treatment (Pre-Tx) samples were used, and The Cancer Genome Atlas (TCGA) glioblastoma samples. We applied appropriate batch correction on log-transformed, normalized mRNA expression values using the removeBatchEffect function in the R package limma to estimate the fraction of glioblastoma patients with positive enrichment of cell cycle/cancer proliferation signatures (GSVA score ≥ 0.2). The proportion of positive enrichment of cell cycle/cancer proliferation signatures in our dataset as a whole is similar to GSE79671 (14 out of 29 (48%) versus 11 out of 20 (55%)). The number of samples with positive enrichment in the TCGA GBM is lower, at 41%. We observed that the neoadjuvant PD-1 monoclonal antibody therapy group is associated with a lower fraction of tumors with cell cycle signatures. Only 3 out of 14 tumors in the neoadjuvant group demonstrated positive enrichment, with 11 of 15 tumors in the adjuvant group and 11 of 20 tumors in the GSE79671 set (one-sided Fisher exact test, P = 0.01 and P = 0.05, respectively). GSVA, gene set variation analysis.

Source Data

Extended Data Fig. 5 RNA sequencing comparison to TCGA.

We combined our RNA sequencing dataset to the TCGA glioblastoma dataset, with appropriate batch correction, to estimate the fraction of glioblastoma patients with positive enrichment of cell cycle/cancer proliferation signatures (GSVA score ≥ 0.2). Three out of 14 tumors in the neoadjuvant group demonstrated positive enrichment, with 11 of 15 tumors in the adjuvant group and 73 of 166 tumors in The Cancer Genome Atlas set. TCGA: The Cancer Genome Atlas. GSVA: gene set variation analysis.

Source Data

Extended Data Fig. 6 Mass cytometry dimension reduction.

a, Diffusion map of peripheral blood mononuclear cells (PBMCs) sampled from n = 28 patients at baseline, the time of surgery and on-treatment. Phenotypically similar cells are depicted in an unsupervised manner along the same continuous axes in a pseudotemporal progression. b, t-distributed stochastic neighbor-embedding (tSNE) plot of PBMCs from n = 28 patients at all three time points. Phenotypically similar cells are clustered in an unsupervised manner. All represented cells in both panels are colored by algorithmically assigned cluster numbers using the FlowSOM package. CD3+CD4+, CD3+CD8+, CD3CD19+ and CD3CD14+CD16+CD11b+CD11c+ cells are labeled to demonstrate how clustered cells in close proximity to one another are plotted.

Supplementary information

Source data

Source Data Fig. 2

Statistical source data for panel b

Source Data Extended Data Fig. 3

Statistical source data

Source Data Extended Data Fig. 4

Statistical source data

Source Data Extended Data Fig. 5

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Cloughesy, T.F., Mochizuki, A.Y., Orpilla, J.R. et al. Neoadjuvant anti-PD-1 immunotherapy promotes a survival benefit with intratumoral and systemic immune responses in recurrent glioblastoma. Nat Med 25, 477–486 (2019).

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