ERK1/2 phosphorylation predicts survival following anti-PD-1 immunotherapy in recurrent glioblastoma

Abstract

Only a subset of recurrent glioblastoma (rGBM) responds to anti-PD-1 immunotherapy. Previously, we reported enrichment of BRAF/PTPN11 mutations in 30% of rGBM that responded to PD-1 blockade. Given that BRAF and PTPN11 promote MAPK/ERK signaling, we investigated whether activation of this pathway is associated with response to PD-1 inhibitors in rGBM, including patients that do not harbor BRAF/PTPN11 mutations. Here we show that immunohistochemistry for ERK1/2 phosphorylation (p-ERK), a marker of MAPK/ERK pathway activation, is predictive of overall survival following adjuvant PD-1 blockade in two independent rGBM patient cohorts. Single-cell RNA-sequencing and multiplex immunofluorescence analyses revealed that p-ERK was mainly localized in tumor cells and that high-p-ERK GBMs contained tumor-infiltrating myeloid cells and microglia with elevated expression of MHC class II and associated genes. These findings indicate that ERK1/2 activation in rGBM is predictive of response to PD-1 blockade and is associated with a distinct myeloid cell phenotype.

Extended Data Fig. 1 |. Optimization of the staining technique and quantification of p-ERK.

a, Titration of the p-ERK antibody (clone: D13.14.4E) using different dilutions performed in GBM samples. We show the same region of a GBM sample stained with the indicated dilutions of the p-ERK antibody with a low and high magnification image for each dilution. b, (left) Microarray containing breast cancer tissues stained with p-ERK antibody (1:500 dilution) representing a positive control. (right) Magnification of one the breast cancer tissues showing specific staining in the endothelium (red rectangle). c, (left) Nontumoral brain tissue stained with p-ERK antibody (1:500 dilution) representing a negative control. (right) Magnification of the white matter showing p-ERK staining with minimal background. Dilution titration and staining of positive and negative controls were performed as a single experiment in one standardized run. d, Workflow used for the software-based quantification of p-ERK⁺ cells.

Extended Data Fig. 2 |. Quantification and cut point optimization of p-ERK+ cell density in tumoral regions.

a, Dot plot showing the distribution of p-ERK quantification of all GBM samples treated and nontreated with PD-1 blockade N = 62 tumors). b, From top to bottom, micrographs showing one high p-ERK tumor sample and two low p-ERK tumor samples with positive staining in the endothelial cells (red arrows). In the dot plot, the magenta dot represents CU100 patient, the green dot represents NU01688 patient, and the red dot represents CU110 patient. IHC images are representative of 62 independent GBM samples. c, Conditional inference trees analysis for cut-point optimization in the GBM cohort treated with PD-1 blockade reveals a cut-point value similar to the median of all tumor samples. d, Forest plot representing the univariable analysis using a Cox regression model evaluating the clinical and molecular prognostic factors that might confound the association between survival p-ERK and presented as Hazard ratio (95% CI). N = 29 GBM patients. P value by two-sided Wald test. e, Kaplan-Meier curve comparing OS of recurrent GBM patients scored as either high or low p-ERK by assessment of a neuropathologist counting from initiation of PD-1 blockade (anti-PD-1 therapy group, N = 29 GBM patients) and from surgery at recurrence (no-immunotherapy group, N = 33 GBM patients). p-ERK scores in tumor regions were designated as follows: 0–1 were considered as low, and 2–3 as high; P value by two-sided log-rank test.

Extended Data Fig. 3 |. Preservation of the p-ERK epitope and peptide competition assay neutralizing the p-ERK1/2 antibody tested in FFPE GBM samples.

a, Protein extraction from FFPE GBM tissues for assessment of selected phosphoproteins. b, Western blot targeting p-ERK, ERK1, ERK2, p-AKT, AKT, and β-actin in a subset of GBM samples used for survival analysis. Western blotting was done as a single experiment in 12 independent GBM samples and 2 GBM cell lines. c, Peptide competition assay in which p-ERK1/2 antibody was neutralized with a blocking peptide employing extracted proteins obtained from GBM samples. The peptide competition assay was assessed by western blot. One western blot was incubated with the neutralized p-ERK antibody and the other with the free p-ERK antibody. d, Peptide competition assay employing IHC using the same GBM samples used to perform western blot employing the neutralized and free p-ERK antibody to perform the staining. The experiments were done in 4 independent GBM samples and 2 GBM cell lines as a single experiment.

Extended Data Fig. 4 |. Evaluation of the ischemic time on p-ERK degradation by IHC and western blot, and comparison to the samples used in this study.

a, Representatives images of the analysis conducted to evaluate p-ERK degradation in endothelial cells of GBM samples at different periods of ischemic time. For this, 3 human tumor specimens were obtained during surgery, and immediately divided into similar size portions, which then were subjected to different ischemic times before processing. Specific endothelial cells subjected to analysis are labeled with colors assigned by the software. b, Blue bars represent p-ERK⁺ cells mm² in tumor regions, and dots represent p-ERK intensity on individual endothelial cells within the same samples used to evaluate the effect of ischemic time on p-ERK degradation, and tumor samples used for survival analysis (PD-1 immunotherapy cohort and no immunotherapy cohort). Each dot represents one ROI analyzing one endothelial cell. Green dots (N = 24, 20, 13 endothelial cells from NU02608, NU02617, and NU02609, respectively) represent a statistically significant group compared to the group of 0 hrs. of ischemic time represented as gray dots (N = 18, 19, 14 endothelial cells from NU02608, NU02617, and NU02609, respectively). All samples were normalized to the average of values of the three 0 hrs. groups. P values by two-sided Kruskal Wallis test with post hoc Dunn’s multiple comparison test. c, Western blot showing p-ERK and other phosphoproteins in samples subjected to different ischemic times. Densitometry analysis for p-ERK western blot was performed using ERK1 and ERK2 staining. For this densitometry, every patient had density normalized by 0 minutes of ischemic time. N = 3 GBM samples. Error bars represent SEM. Western blot was done as a single experiment in 3 independent GBM samples.

Extended Data Fig. 5 |. Progression-free survival of the validation cohort from the Cloughesy T et al. clinical trial.

a, b, Kaplan-Meier showing progression-free survival following PD-1 blockade based on p-ERK high vs low for pre-study (a) and on-study (b) tumor samples. N = 13 GBM patients. P values by two-sided log rank test.

Extended Data Fig. 6 |. Multiplex immunofluorescence staining of recurrent GBM samples employing GFAP marker.

a, Bar plot showing the comparison of GFAP⁺ p-ERK⁺ cells and other cells expressing p ERK⁺. N = 6 tumor samples. P value by two-sided Mann Whitney U test. Data is presented as mean ± s.d. b, Representative images of three different tumor samples derived form results in a. From top to bottom: a BRAF^V600E GBM sample having high p-ERK staining, a wild-type BRAF/PTPN11 GBM having high p ERK staining, and a wild-type BRAF/PTPN11 GBM displaying low p-ERK staining. For the three tumor samples: (left) H&E and p-ERK IHC images of the same tumor region. (middle), Multiplex immunofluorescence images showing the markers for GFAP, p-ERK, and DAPI. (right) Multiplex immunofluorescence images showing the markers for GFAP, CD163, and DAPI. Experiment was done using a tumor sample in one standardized run per patient.

Extended Data Fig. 7 |. Single-cell RNA seq of GBM patients with high and low p-ERK IHC staining.

UMAP representation of 28,194 individual cells from 10 GBM patients measured with scRNA-seq (left). UMAP graph showing the representation of 3,153 myeloid cells derived from the 10 GBM patients (right). Each dot represents an individual cell.

Figure 1.. ERK1/2 activation is a predictive biomarker of radiographic response to anti-PD-1 immunotherapy in patients with recurrent GBM.

a, Dot plot showing the quantification of p-ERK⁺ cells before PD-1 blockade initiation in responder and nonresponder patients as previously defined13 (n = 29 GBM tumors). P = 0.0029, two-sided Mann–Whitney U-test. Data are presented as mean ± s.d. Each dot represents an independent patient sample. b, Examples of responder and nonresponder patients from results in a showing the corresponding MRI, associated H&E staining and immunostaining for p-ERK in the pretreatment sample. Top: in a responder patient, a biopsy performed in a gadolinium-enhancing lesion 10 months after treatment with anti-PD-1 therapy showed few tumor cells and a profuse CD3⁺ T-cell infiltrate. Flow cytometry analysis in the CSF and brain tumor showed that many of these T cells were CD4⁺ and CD8⁺. This patient experienced stable disease for at least 21 months after immunotherapy initiation. Bottom: a nonresponder patient with the corresponding MRI, H&E and p-ERK immunostaining in the pretreatment sample. Arrows indicate p-ERK+ endothelial cells. The experiment was performed using 29 tumor samples in one standardized run per patient.

Figure 2.. ERK1/2 phosphorylation evaluated by semiautomatic IHC quantification shows that is a predictive biomarker following PD-1 blockade in recurrent GBM.

a, Kaplan–Meier curve comparing OS of patients with recurrent GBM defined as having either high- or low-p-ERK tumors, counting from initiation of PD-1 blockade (anti-PD-1 therapy group, n = 29 patients) and from surgery at recurrence (no-immunotherapy group, n = 33 patients); P values, two-sided log-rank test. b, Forest plots representing univariable and multivariable survival analyses using a Cox proportional hazard model evaluating prognostic variables and p-ERK⁺ cell density on survival in the anti-PD-1 therapy cohort (top) and the no-immunotherapy cohort (bottom), presented as HR (95% CI). P values by two-sided Wald test. c, ROC curve of sensitivity and 1 – specificity displaying mean AUC (95% CI) for the anti-PD-1 therapy and no-immunotherapy cohorts (n values as in a) d, Left: dot plot comparing the quantification of p-ERK⁺ cell density between tumors of patients with GBM harboring either BRAF/PTPN11 mutations (n = 4 tumors), wild-type BRAF/PTPN11 (n = 5 tumors) or unknown BRAF/PTPN11 status (n = 2 tumors) that had OS >12 months with those that had either wild-type tumors (n = 11) or unknown BRAF/PTPN11 status (n = 7 tumors) and lived <12 months after initiation of immunotherapy. Right: H&E and p-ERK immunostaining of three GBM samples. From top to bottom: a BRAF mutated tumor and a wild-type BRAF tumor from patients that lived >12 months, and a wild-type BRAF tumor from a patient that lived <12 months. P values by two-sided Mann–Whitney U-test. Data are presented as mean ± s.d. Each dot represents an independent patient sample.

Figure 3.. Validation of pretreatment p-ERK staining correlates with OS in an independent recurrent GBM cohort treated with adjuvant PD-1 blockade.

a, Cartoon showing the time points of surgical tumor acquisition relative to treatment with PD-1 blockade identified as prestudy or on-study tumor samples. b, Left: change in p-ERK cell density in pre-study and on-study tumor samples. n = 12 paired tumor samples. Red and blue dots represent high- and low-p-ERK tumors, respectively; dashed green line represent the cut-point value used to partition high- and low-p-ERK tumors. P value calculated using two-sided Wilcoxon signed-rank test. Each dot represents an independent patient sample. Right: representative IHC micrographs showing the change in p-ERK immunostaining between paired tumor samples. c,d, Kaplan–Meier plots showing OS in high- versus low-p-ERK groups treated with adjuvant PD-1 blockade evaluating pre-study (c) and on-study tumor samples (d). P values by two-sided log-rank test. e, Forest plots for on-study tumor samples representing univariable and multivariable survival analysis using a Cox proportional hazard model presented as HR (95% CI). n = 13 patients with GBM, P values by two-sided Wald test. f, ROC curve of sensitivity and 1 – specificity displaying mean AUC (95% CI) for the validation GBM cohort. n = 13 patients with GBM.

Figure 4.. Multiplex immunofluorescence of recurrent GBM samples shows p-ERK positivity in SOX2+ cells and associated myeloid cell infiltration.

a, Bar plot showing the contribution to p-ERK expression from SOX2⁺, TMEM119⁺, CD163⁺ and other cells (SOX2⁻TMEM119⁻CD163⁻ cells). Differences among cell types were evaluated using one-way ANOVA with post hoc Tukeýs multiple comparisons test (n = 13 tumors). b, Dot plot showing comparison of SOX2⁺ p-ERK⁺ cells mm² between high- and low-p-ERK tumors (n = 13 tumors). c, Representative images of three different tumor samples derived from results in a and b. From top to bottom: a BRAF^V600E GBM sample with high-p-ERK staining, a wild-type BRAF/PTPN11 GBM with high-p-ERK staining and a wild-type BRAF/PTPN11 GBM displaying low-p ERK staining. For the three tumor samples: left, H&E and p-ERK IHC images of the same tumor region; middle, multiplex immunofluorescence images showing the markers for SOX2, p-ERK and DAPI; right, multiplex immunofluorescence images showing the markers for SOX2, TMEM119, CD163 and DAPI. Arrowheads indicate SOX2⁺ p-ERK⁺ cells. The experiment was done using 13 tumor samples in one standardized run per patient. d, Dot plot showing comparison of TMEM119⁺ between high- and low-p-ERK tumors (n = 13 tumors). e, Dot plot showing comparison of CD163⁺ cells mm² between high- and low-p-ERK tumors (n = 13 tumors). f, Left, scatter plot showing the correlation of p-ERK⁺ cells mm² with Iba1⁺ cells mm² obtained by software-based quantification of IHC-stained tumor samples. Right, representative images of p-ERK and Iba1 immunostaining of the same tumor region (n = 12 tumors). P values by two-sided Mann–Whitney U-test (b,d,e) or Pearson’s correlation (f). Data are presented as mean ± s.d. (a,b,d,e). Each dot represents an independent patient sample (a,b,d–f).

Figure 5.. Spatial analysis of tumor cells expressing p-ERK and their associated myeloid cells.

a, Cartoon representing distances from TMEM119⁺ and CD163⁺ cells to SOX2⁺ p-ERK⁺/p-ERK⁻ cells. b,c, Bar plots comparing the mean distances from TMEM119⁺ (b) and CD163⁺ (c) cells to SOX2⁺ p-ERK⁺/p-ERK⁻ cells in high- versus low-p-ERK tumors. Dots represent tumor samples (n = 13 tumors). d, Representative multiplex immunofluorescence images illustrating spatial dimensions between TMEM119⁺ cells and SOX2⁺ p-ERK⁺ in a high- and a low-p-ERK GBM. The experiment was done in 13 tumor samples in one standardized run per patient. e, Cartoon representing distances from CD163⁺ cells to GFAP⁺ p-ERK⁺/p-ERK⁻ cells. f, Bar plots comparing mean distances from CD163⁺ cells to GFAP⁺ p-ERK⁺/p-ERK⁻ cells in high- versus low-p-ERK tumors. Dots represent tumor samples (n = 6 tumors). g, Representative multiplex immunofluorescence images illustrating spatial dimensions between CD163⁺ cells and GFAP⁺ p-ERK⁺ in a high- (left) and a low-p-ERK GBM (right). The experiment was performed on six tumor samples in one standardized run per patient. P values by two-tailed unpaired t-test (b,c,f). Data are presented as mean ± s.d. (b,c,f).

Figure 6.. scRNA-seq in patients with GBM from high- and low-p-ERK groups.

a, Quantification of, and representative, p-ERK IHC images and multiplex immunofluorescence images of GBM samples used in the analysis. Dashed green line represent the cut-point value used to partition high- and low-p-ERK tumors in the discovery and validation cohorts. n = 10 tumor samples. b, UMAP graph showing the expression of cell markers for tumor cells (SOX2), myeloid cells (CD14), endothelial cells (VWF) and pericytes (PDGFRB). The color key indicates expression levels. Each dot represents an individual cell. n = 28,194 cells contained in ten tumor samples. c, UMAP graph showing the overlapping annotations derived from high- and low-p-ERK IHC-stained tumors obtained from software-based quantification in all cells (top) and in the myeloid cell compartment (bottom). d, GO terms analysis. Differentially expressed gene signatures of the myeloid cell population infiltrating high- and low-p-ERK GBM samples. Twenty-seven differentially expressed GO terms are represented in a dot plot, with dot size corresponding to the percentage of genes that matched the GO term. Dot color corresponds to the q-value of enrichment. e, Top: UMAP plot showing expression of the MHC II protein binding complex gene signature in myeloid cells. Bottom: GSEA plot showing enrichment of the GO term MHC II protein binding complex within myeloid cells. n = 3,153 myeloid cells from ten tumor samples. f,g, Beeswarm plots showing the cell density of TMEM119⁺ MHC II⁺ cells (f) and CD163⁺ MHC II⁺ cells (g) between high- and low-p-ERK tumors (n = 23). P values by two-sided Mann–Whitney U-test. h, Representative multiplex immunofluorescence images illustrating the expression of MHC II by TMEM119⁺ and CD163⁺ cells in a high- and a low-p-ERK GBM sample. SOX2, TMEM119, CD163, MHC II and DAPI are included as cell markers. The experiment was performed in 23 tumor samples in one standardized run per patient. Data presented as mean ± s.d. (f,g).