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

Nature Cancer volume 2, pages 1372–1386 (2021)Cite this article

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A Publisher Correction to this article was published on 11 February 2022

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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.

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Fig. 1: ERK1/2 activation is a predictive biomarker of radiographic response to anti-PD-1 immunotherapy in patients with recurrent GBM.
Fig. 2: ERK1/2 phosphorylation evaluated by semiautomatic IHC quantification shows that is a predictive biomarker following PD-1 blockade in recurrent GBM.
Fig. 3: Validation of pretreatment p-ERK staining correlates with OS in an independent recurrent GBM cohort treated with adjuvant PD-1 blockade.
Fig. 4: Multiplex immunofluorescence of recurrent GBM samples shows p-ERK positivity in SOX2+ cells and associated myeloid cell infiltration.
Fig. 5: Spatial analysis of tumor cells expressing p-ERK and their associated myeloid cells.
Fig. 6: scRNA-seq in patients with GBM from high- and low-p-ERK groups.

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Data availability

Single-cell RNA-seq data supporting the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code nos. GSE103224 and GSE141383. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

Code availability

Code is available at https://github.com/RabadanLab/GBMsinglecell.

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Acknowledgements

This work was supported by NIH grant nos. 1R01NS110703-01A1 and 5DP5OD021356-05 (both A.M.S.); no. P50CA221747 SPORE for Translational Approaches to Brain Cancer (principal investigator M. S. Lesniak, with support to A.M.S.); developmental funds from the Robert H. Lurie Cancer Center Support Grant (no. P30CA060553, A.M.S.); Vagelos Precision Medicine Award (F.M.I., R.R. and J.Z.), U54CA193313 (R.R.), U54CA209997 (R.R.), R35CA253126 (R.R.); Keep Punching (F.M.I.); The William Rhodes and Louise Tilzer-Rhodes Center for Glioblastoma at New York-Presbyterian Hospital (F.M.I.); NIAID nos. 1R01AI099195 (U.B.) and R01AI134988 (U.B.). V.A.A. is financially supported by the Mexican government through the Mexican National Council for Science and Technology and the Plan of Combined Studies in Medicine of the National Autonomous University of Mexico. A.X.C. is funded by the Medical Scientist Training Program (no. T32GM007367). We thank T. Sudhakar for sample collection at Columbia University; K. McCortney, R. Javier and J. Walshon from the Nervous System Tumor Bank supported by the P50CA221747 SPORE for Translational Approaches to Brain Cancer; and B. Shmaltsuyeva for immunohistochemistry at the Northwestern University Pathology Core Facility funded by Cancer Center Support Grant (no. NCI CA060553). We thank L. Kai for technical support on multiplex staining performed at the Immunotherapy Assessment Core at Northwestern University and in the Flow Cytometry & Cellular Imaging Core Facility, which is supported in part by NIH through MD Anderson´s Cancer Center Support Grant no. CA016672, the NCI´s Research Specialist 1 (no. R50 CA23707, J.K.B.) and NIH grant nos. R01-CA120813 and 1R01-CA237418 (both A.B.H.).

Author information

Author notes

  1. These authors contributed equally: Víctor A. Arrieta, Andrew X. Chen.

  2. These authors jointly supervised this work: Fabio M. Iwamoto, Raul Rabadan, Adam M. Sonabend.

Authors and Affiliations

  1. Department of Neurosurgery, Lou and Jean Malnati Brain Tumor Institute, Robert H Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Víctor A. Arrieta, J. Robert Kane, Seong Jae Kang, Crismita Dmello, Catalina Lee-Chang, Joseph Shilati, Li Chen, Andrew Gould, Daniel Zhang, Christina Amidei, Jonathan T. Yamaguchi, Roger Stupp, Amy B. Heimberger, Craig Horbinski & Adam M. Sonabend

  2. PECEM, Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico

    Víctor A. Arrieta

  3. Program for Mathematical Genomics, Department of Systems Biology, Columbia University, New York, NY, USA

    Andrew X. Chen, Junfei Zhao & Raul Rabadan

  4. Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Cynthia Kassab & Xiaoyang Ling

  5. Department of Biomedical Informatics, Columbia University, New York, NY, USA

    Junfei Zhao, Jinzhou Yuan, Wenting Zhao, Peter Sims & Raul Rabadan

  6. Department of Preventive Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Kirsten B. Burdett & Hui Zhang

  7. Department of Neurosurgery, Columbia University, New York, NY, USA

    Pavan S. Upadhyayula & Jeffrey N. Bruce

  8. Robert Lurie Comprehensive Cancer Center and Department of Dermatology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Dinesh Jaishankar

  9. Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Jared K. Burks

  10. INSERM U1236, University of Rennes 1, Etablissement Français du Sang, Rennes, France

    Brice Laffleur

  11. Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Rimas V. Lukas & Roger Stupp

  12. Department of Neurology, Columbia University, Vagelos College of Physicians and Surgeons Columbia University Irving Medical Center, New York, NY, USA

    David Cieremans & Fabio M. Iwamoto

  13. Department of Microbiology and Immunology, Columbia University, New York, NY, USA

    Gerson Rothschild & Uttiya Basu

  14. Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Matthew McCord, Daniel J. Brat, Lee A. D. Cooper & Craig Horbinski

  15. Division of Hematology/Oncology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Bin Zhang

  16. Department of Medical and Molecular Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA

    Tim F. Cloughesy & Robert Prins

  17. Department of Pathology and Cell Biology, Columbia University, New York, NY, USA

    Peter Canoll

  18. Division of Hematology/Oncology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Roger Stupp

Authors
  1. Víctor A. Arrieta
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  2. Andrew X. Chen
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  3. J. Robert Kane
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  41. Raul Rabadan
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  42. Adam M. Sonabend
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Contributions

V.A.A., A.X.C. and J.R.K. performed the majority of experiments and analyses. V.A.A., J.R.K., R.R., F.M.I. and A.M.S. conceptualized and designed the study. V.A.A. and L.A.D.C. quantified IHC images. V.A.A., A.X.C. and A.M.S. wrote the manuscript. R.S., P.U., J.Z., R.V.L., C.D., D.C., X.L., A.G., S.J.K., J.S., D.Z., J.N.B. and J.T.Y. compiled the clinical data for analysis. T.F.C. and R.P. provided tumor samples and clinical data for the validation cohort. C.A. and L.C. provided administrative support. V.A.A., A.X.C., K.B.B. and H.Z. performed survival and statistical analyses. P.C. and J.N.B. are responsible for the Tumor Bank at Columbia University. C.H. is responsible for the Tumor Bank at Northwestern University. P.U. acquired tumor specimens. C.H., M.M., D.J.B. and P.C. scored the tumor slides. J.Y., W.Z. and P.S. provided scRNA-seq data. A.X.C., J.Z. and R.R. performed scRNA-seq data analysis. D.J., B.Z., C.K., J.K.B., X.L. and A.B.H. performed Opal multiplex tissue staining. V.A.A., A.X.C. and C.L.-C. analyzed multiplex immunofluorescence images. V.A.A., C.L.-C., B.L., G.R. and U.B. acquired and analyzed flow cytometry data. V.A.A. and S.J.K. performed immunoblots and the peptide competition assay. A.M.S., R.R. and F.M.I. supervised the entire study.

Corresponding authors

Correspondence to Fabio M. Iwamoto, Raul Rabadan or Adam M. Sonabend.

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Competing interests

F.M.I. is a consultant for Merck, Novocure, Regeneron, Abbvie, Alexion, Guidepoint and Tocagen. He has received clinical or research support (including equipment or material) from Bristol Myers Squibb and Merck. A.M.S., F.M.I. and R.R. are co-authors for the following patent filed by Columbia University: Systems and methods for predicting clinical responses to immunotherapies. A.M.S., V.A.A, F.M.I. and R.R. are co-authors of the following patent filed by Northwestern University: 5. Methods for treating glioblastoma. A.M.S. has received a consulting honorarium from Abbvie. R.R. is a member of the SAB of AimedBio and a founder of Genotwin. R.V.L. has received support from Roche-Genentech for meeting travel to present study results; honoraria for advisory boards for AstraZeneca, Abbvie and Ziopharm; and honoraria for medical editing for EBSCO publishing, Medlink Neurology and American Physician Institute. He has also received honoraria for consultation with Eisai and Abbvie, and honoraria for creating and presenting CME Board review material for the American Physician Institute. R.V.L. received drug support (but no additional support) from BMS for an investigator-initiated trial. A.B.H. is a consultant for Caris Life Sciences and WCG Oncology Advisory Board, receives royalties and milestone payments from Celldex Therapeutics and DNAtrix and receives clinical or research support (including equipment and materials) from Celularity, Codiak Biosciences, Moleculin and Carthera. The remaining authors declare no competing interests.

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Peer review information Nature Cancer thanks Robert Manguso and Ignacio Melero for their contribution to the peer review of this work.

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Extended data

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.

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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.

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Extended Data Fig. 5 Progression-free survival of the validation cohort from the Cloughesy T et al.14 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 BRAFV600E 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.

Source data

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.

Supplementary information

Reporting Summary

Supplementary Tables

Table 1. Clinical characteristics of our GBM cohort treated with anti-PD-1 therapy. Table 2. Clinical characteristics of our GBM cohort that did not receive immunotherapy. Table 3. Clinical characteristics of the validation cohort treated with adjuvant PD-1 blockade. Table 4. GO term enrichment in myeloid cells from high-p-ERK GBMs versus myeloid cells from low-p-ERK GBMs.

Supplementary Data 1

Flow cytometry gating for GBM patients treated with PD-1 blockade.

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Unprocessed immunoblots.

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Unprocessed immunoblots.

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Arrieta, V.A., Chen, A.X., Kane, J.R. et al. ERK1/2 phosphorylation predicts survival following anti-PD-1 immunotherapy in recurrent glioblastoma. Nat Cancer 2, 1372–1386 (2021). https://doi.org/10.1038/s43018-021-00260-2

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