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1 Supplementary Online Content Bell EH, Pugh SL, McElroy JP, et al. Molecular-based recursive partitioning analysis model for glioblastoma in the temozolomide era: a correlative analysis based on NRG Oncology RTOG JAMA Oncol. Published online January 12, doi: /jamaoncol emethods ereferences etable 1. Frequency Tables of 22 Protein Markers etable 2. Comparison Between Patients With and Without Available Tissue from RTOG 0525 etable 3. Single marker Cox Proportional Hazards Model for Overall Survival Adjusted by RX, Age, KPS, and Surgery etable 4. Association of MGMT Tumor Mask Protein Expression and Nuclear Mask Protein Expression with MGMT Promoter Methylation etable 5. Overall Survival by Current RPA etable 6. Overall Survival by NRG-GBM-RPA etable 7.1. Unadjusted Model Comparisons etable 7.2. Explanation of Variance for MGMT Methylation versus NRG-GBM-RPA etable 7.3. NRG-GBM-RPA by MGMT Methylation etable 8. Clinical Characteristics for GBM Institutional Cohort (n=176) efigure 1. Survivin, Ki-67, and pmtor protein levels correlate with OS in randomized NRG Oncology RTOG 0525 study participants efigure 2. Scatter plot demonstrating correlation of MGMT tumor protein expression scores versus MGMT methylation status in RTOG 0525 patients efigure 3. Validation of the NRG-GBM-RPA classification in an independent GBM cohort (excluding known IDH-mutant glioblastomas, N=9) efigure 4. Protein expression of MGMT and c-met by immunohistochemistry This supplementary material has been provided by the authors to give readers additional information about their work.

2 emethods Additional Quantitative Immunofluorescence Methods Four TMAs containing paraffin-embedded tumor cores from the 452 RTOG 0525 patients were cut at 5-µm and sections were placed on positively-charged slides. As a surrogate for tumor co-localization, proteins were colocalized with GFAP (glial fibrillary acidic protein) (DAKO;1:100) to stain the cytoplasmic compartments of glial cells. Deparaffinization and retrieval were performed as previously described. 1-3 Slides were scanned by HistoRx PM-2000 and analyzed by AQUAnalysis software. Antibodies that were used for the 5 most significant proteins were: c-met (SP44) (Spring Bioscience; 1:200), MGMT (MT3.1) (Santa Cruz; 1:100), Ki-67 (MIB-1) (Dako; 1:5000), survivin (71G4B7) (Cell Signaling Technology; 1:1000) and VEGFR1 (Y103) (Epitomics; 1:200). Each protein was scored in the tumor, cytoplasm, and nuclear components of each TMA core using the HistoRx TM AQUA platform and fluorescent IHC. 1 To address core to core variability in the data from quantitative IHC when multiple cores per patient were analyzed, maximum scores from each patient were used for overexpressed proteins, minimum scores for underexpressed proteins, and average scores were used for proteins with high heterogeneity (e.g. Ki-67). Markers were evaluated defined as discrete groups formed by dividing at the median, tertile, or quartiles. Additional Confirmation Study Methods Four TMAs containing multiple paraffin-embedded tumor cores from 176 patients were placed on positivelycharged slides. Slides were then placed in a 60 C oven for 1 hour, cooled, deparaffinized and rehydrated through xylene and graded ethanol solutions to water. All slides were quenched for 5 minutes in a 3% hydrogen peroxide aqueous solution to block for endogenous peroxidase. Antigen retrieval was performed by Heat-Induced Epitope Retrieval (HIER) in which the slides were placed in a 1X solution of Target Retrieval Solution (Dako, S1699) for 25 minutes at 96 o C using a vegetable steamer (Black & Decker) and cooled for 15 minutes in solution. Slides were stained with the Intellipath Autostainer Immunostaining System. All incubations on the Autostainer were performed at room temperature. The two components of Mach 3 Rabbit HRP Polymer Kit (M3R531L, Biocare Medicals, Concord, CA) were applied sequentially for 20 minutes each. Staining was visualized with the DAB+ (K346811; 5 minutes development, Dako, Carpinteria, CA). Slides were then counterstained in Richard Allen hematoxylin (Thermo Scientific, Middletown, VA), dehydrated through graded ethanol solutions, cleared in xylene and

3 coverslipped. The IHC analysis was done independently by two pathologists, who scored each core according to the Allred Score (AS) 4, which evaluates proportion of positive cells (0- negative; 1- >0-1/100 tumor; 2- >1/100-1/10 tumor; 3->1/10-1/3 tumor; 4- >1/3-2/3 tumor; 5- >2/3-1 tumor) and the intensity of the expression (0- negative; 1- weak; 2- intermediate; 3- strong), resulting in a total sum of 0-8. As multiple cores of each tumor were scored, the average value was used for statistics. To test the concordance between the two pathologists analysis, we used Cohen s weighted kappa coefficient 5-7.

4 ereferences 1. Camp RL, Chung GG, Rimm DL. Automated subcellular localization and quantification of protein expression in tissue microarrays. Nat Med. Nov 2002;8(11): Escobar J, Klimowicz AC, Dean M, et al. Quantification of ER/PR expression in ovarian low-grade serous carcinoma. Gynecol Oncol. Feb 2013;128(2): Otsuka S, Klimowicz AC, Kopciuk K, et al. CXCR4 overexpression is associated with poor outcome in females diagnosed with stage IV non-small cell lung cancer. J Thorac Oncol. Jul 2011;6(7): Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol. Feb 1998;11(2): Cohen J. A coefficient of agreement for nominal scales. Educational and Psychological Measurement. 1960;20: Cohen J. Weighted kappa: Nominal scale agreement with provision for scaled disagreement or partial credit. Psychological Bulletin. 1968;70: Fleiss JL, Cohen, J., and Everitt, B.S. Large sample standard errors of kappa and weighted kappa. Psychological Bulletin. 1969;72:

5 etable 1. Frequency Tables of 22 Protein Markers Frequency Tables of 22 Protein Markers Not Randomized (n=72) ARM 1 (Standard TMZ) (n=190) ARM 2 Dose-dense TMZ (n=190) Total (n=452) EGFR No 11 ( 15.3%) 42 ( 22.1%) 36 ( 18.9%) 89 ( 19.7%) Yes 61 ( 84.7%) 148 ( 77.9%) 154 ( 81.1%) 363 ( 80.3%) IGF1R No 11 ( 15.3%) 42 ( 22.1%) 34 ( 17.9%) 87 ( 19.2%) Yes 61 ( 84.7%) 148 ( 77.9%) 156 ( 82.1%) 365 ( 80.8%) Ki-67 No 5 ( 6.9%) 19 ( 10.0%) 13 ( 6.8%) 37 ( 8.2%) Yes 67 ( 93.1%) 171 ( 90.0%) 177 ( 93.2%) 415 ( 91.8%) MGMT No 16 ( 22.2%) 49 ( 25.8%) 48 ( 25.3%) 113 ( 25.0%) Yes 56 ( 77.8%) 141 ( 74.2%) 142 ( 74.7%) 339 ( 75.0%) NFkBp65 No 5 ( 6.9%) 17 ( 8.9%) 11 ( 5.8%) 33 ( 7.3%) Yes 67 ( 93.1%) 173 ( 91.1%) 179 ( 94.2%) 419 ( 92.7%) pakt No 1 ( 1.4%) 14 ( 7.4%) 9 ( 4.7%) 24 ( 5.3%) Yes 71 ( 98.6%) 176 ( 92.6%) 181 ( 95.3%) 428 ( 94.7%) perk No 7 ( 9.7%) 19 ( 10.0%) 14 ( 7.4%) 40 ( 8.8%) Yes 65 ( 90.3%) 171 ( 90.0%) 176 ( 92.6%) 412 ( 91.2%) pmtor No 7 ( 9.7%) 20 ( 10.5%) 18 ( 9.5%) 45 ( 10.0%) Yes 65 ( 90.3%) 170 ( 89.5%) 172 ( 90.5%) 407 ( 90.0%) pnfkbp65 No 20 ( 27.8%) 53 ( 27.9%) 52 ( 27.4%) 125 ( 27.7%) Yes 52 ( 72.2%) 137 ( 72.1%) 138 ( 72.6%) 327 ( 72.3%) PTEN No 16 ( 22.2%) 57 ( 30.0%) 43 ( 22.6%) 116 ( 25.7%) Yes 56 ( 77.8%) 133 ( 70.0%) 147 ( 77.4%) 336 ( 74.3%) Src No 17 ( 23.6%) 47 ( 24.7%) 48 ( 25.3%) 112 ( 24.8%)

6 Frequency Tables of 22 Protein Markers Not Randomized (n=72) ARM 1 (Standard TMZ) (n=190) ARM 2 Dose-dense TMZ (n=190) Total (n=452) Yes 55 ( 76.4%) 143 ( 75.3%) 142 ( 74.7%) 340 ( 75.2%) Survivin No 15 ( 20.8%) 44 ( 23.2%) 33 ( 17.4%) 92 ( 20.4%) Yes 57 ( 79.2%) 146 ( 76.8%) 157 ( 82.6%) 360 ( 79.6%) CD24 No 50 ( 69.4%) 109 ( 57.4%) 107 ( 56.3%) 266 ( 58.8%) Yes 22 ( 30.6%) 81 ( 42.6%) 83 ( 43.7%) 186 ( 41.2%) CD44 No 51 ( 70.8%) 103 ( 54.2%) 100 ( 52.6%) 254 ( 56.2%) Yes 21 ( 29.2%) 87 ( 45.8%) 90 ( 47.4%) 198 ( 43.8%) c-met No 44 ( 61.1%) 93 ( 48.9%) 91 ( 47.9%) 228 ( 50.4%) Yes 28 ( 38.9%) 97 ( 51.1%) 99 ( 52.1%) 224 ( 49.6%) P16 No 46 ( 63.9%) 90 ( 47.4%) 93 ( 48.9%) 229 ( 50.7%) Yes 26 ( 36.1%) 100 ( 52.6%) 97 ( 51.1%) 223 ( 49.3%) p53 No 49 ( 68.1%) 98 ( 51.6%) 94 ( 49.5%) 241 ( 53.3%) Yes 23 ( 31.9%) 92 ( 48.4%) 96 ( 50.5%) 211 ( 46.7%) PARP1 No 51 ( 70.8%) 104 ( 54.7%) 112 ( 58.9%) 267 ( 59.1%) Yes 21 ( 29.2%) 86 ( 45.3%) 78 ( 41.1%) 185 ( 40.9%) psrcy419 No 45 ( 62.5%) 102 ( 53.7%) 101 ( 53.2%) 248 ( 54.9%) Yes 27 ( 37.5%) 88 ( 46.3%) 89 ( 46.8%) 204 ( 45.1%) psrcy529 No 53 ( 73.6%) 109 ( 57.4%) 105 ( 55.3%) 267 ( 59.1%) Yes 19 ( 26.4%) 81 ( 42.6%) 85 ( 44.7%) 185 ( 40.9%) VEGFR1 No 52 ( 72.2%) 108 ( 56.8%) 110 ( 57.9%) 270 ( 59.7%) Yes 20 ( 27.8%) 82 ( 43.2%) 80 ( 42.1%) 182 ( 40.3%) VEGFR2 No 52 ( 72.2%) 108 ( 56.8%) 110 ( 57.9%) 270 ( 59.7%) Yes 20 ( 27.8%) 82 ( 43.2%) 80 ( 42.1%) 182 ( 40.3%)

7 Frequency Tables of 22 Protein Markers Not Randomized (n=72) ARM 1 (Standard TMZ) (n=190) ARM 2 Dose-dense TMZ (n=190) Total (n=452)

8 etable 2. Comparison Between Patients With and Without Available Tissue from RTOG 0525 etable 2.1 Overall Survival by Tissue Availability Without Tissue With Tissue Estimate Cumulativ e Estimate Cumulativ e Month (%) 95% CI (%) Failures At Risk (%) 95% CI (%) Failures At Risk , , , , , , , , , , Total MST(95%CI) 16.3 (15.2,17.2) 15.4 (13.9,16.9) Hazard ratio (With Tissue/Without Tissue) and 95% CI: 1.02 (0.89 to 1.17) p-value two-sided log-rank test: 0.75 etable 2.2 RPA Class by Tissue Availability Without tissue (n=673) With tissue (n=452) RPA class III 115 ( 17.1%) 91 ( 20.1%) IV 418 ( 62.1%) 255 ( 56.4%) V 140 ( 20.8%) 106 ( 23.5%)

9 etable 3. Single marker Cox Proportional Hazards Model for Overall Survival Adjusted by RX, Age, KPS, and Surgery TRP Markers p-value Hazard Ratio (95%CI) Average Ki67 in Nuclear Mask (Continuous) (1.0001, ) Average Ki67 in Tumor Mask (Continuous) (1.0001, ) MGMT in Nuclear (Continuous) < (1.0001, ) MGMT in Cytoplasm (Continuous) < (1.0001, ) MGMT Tumor Mask (Continuous) < (1.0001, ) Maximum pakt Nuclear/Cytoplasm Ratio (Continuous) (1.0021, ) Maximum pmtor Nuclear/Cytoplasm Ratio (Continuous) (0.1442, ) Maximum Survivin Cytoplasm/Nuclear Ratio (Continuous) (1.0468, ) Minimum cmet Cytoplasm Mask (Continuous) (1.0001, ) Only TRP markers with p value less than 0.05 are listed in the above table Note: All markers were round off to 100 except pakt, pmtor, and Survivin that were round off to 0.01.

10 etable 4 etable 4.1: Association of MGMT Tumor Mask Protein Expression with MGMT Promoter Methylation n Mean of MGMT Tumor Mask Standard Deviation of MGMT Tumor Mask Methylated Unmethylated p-value (t-test) < etable 4.2: Association of MGMT Nuclear Mask Protein Expression with MGMT Promoter Methylation n Mean of MGMT Nuclear Mask Standard Deviation of MGMT Nuclear Mask Methylated Unmethylated p-value (t-test) < 0.001

11 etable 5. Overall Survival by Current RPA III IV V Month Estimate (%) 95% CI (%) Cumulative Failures At Risk Estimate (%) 95% CI (%) Cumulative Failures At Risk Estimate (%) 95% CI (%) Cumulative Failures At Risk , , , , , , , , , , , , , , , Total MST(95%CI) 29.9 (16.4, not reached) 16.6 (13.3,18.5) 13.6 (9.1,17.8) p-value (two-sided log-rank test) 0.006

12 etable 6. Overall Survival by NRG-GBM-RPA Overall Survival by NRG-GBM-RPA (I: MGMT < median or (MGMT median & age < 50) vs. II: MGMT median & age 50 & c-met < top quartile vs. III: MGMT median & age 50 & c-met top quartile) NRG-GBM-RPA I NRG-GBM-RPA II NRG-GBM-RPA III Cumulative Cumulative Cumulative Month Estimate (%) 95% CI (%) Failures At RiskEstimate (%) 95% CI (%) Failures At RiskEstimate (%) 95% CI (%) Failures At Risk , , , , , , , , , , , , , , , Total MST(95% CI) 21.9 (16.4, 29.9) 16.6 (13.3, 20.0) 9.4 (5.6, 11.6) p-value (two-sided log-rank test) < 0.001

13 etable 7.1. Unadjusted Model Comparisons Predictor AIC Hazard Ratio P-value MGMT Methylation (1.01, 2.474) NRG-GBM-RPA I vs. II 1.59 (1.00, 2.54) 0.05 I vs. III 4.56 (2.55, 8.17) < All patients with both NRG-GBM-RPA and MGMT Methylation available were used in both models (n=157). etable 7.2. Explanation of Variance for MGMT Methylation versus NRG-GBM-RPA Schemper-Henderson Predictive Measure Predictive Inaccuracy Without Covariates With Covariates % Variance Explained Overall Survival MGMT Methylation NRG-GBM-RPA Progression Free Survival MGMT Methylation NRG-GBM-RPA Only includes patients with known MGMT methylation

14 etable 7.3. NRG-GBM-RPA by MGMT Methylation Methylated (n=49) Unmethylated (n=108) Unknown (n=9) Total (n=166) NRG-GBM-RPA I 32 ( 65.3%) 58 ( 53.7%) 6 ( 66.7%) 96 ( 57.8%) NRG-GBM-RPA II 14 ( 28.6%) 30 ( 27.8%) 3 ( 33.3%) 47 ( 28.3%) NRG-GBM-RPA III 3 ( 6.1%) 20 ( 18.5%) 0 ( 0.0%) 23 ( 13.9%) p-value (Chi-square test) : 0.19

15 etable 8. Clinical Characteristics for GBM Institutional Cohort (n=176) Age (years) <50 41 (23.3%) (76.7%) Gender Male 109 (61.9%) Female 67 (38.1%) KPS <70 60 (34.1%) (64.8%) N/A 2 (0.1%) Surgery Biopsy 35 (19.9%) Debulking 141 (80.1%) Treatment None 50 (28.4%) RT only 37 (21.0%) RT+TMZ (Stupp) 87 (49.4%) N/A 2 (1.1%)

16 efigure 1. Survivin, Ki-67, and pmtor protein levels correlate with OS in randomized NRG Oncology RTOG 0525 study participants High levels of survivin (cytoplasm/nuclear ratio) when split by the median trend toward decreased OS (A). High Ki-67 nuclear protein staining when split by the median significantly associate with decreased OS (B).Low levels of pmtor (nuclear/cytoplasmic ratio) when split by the median trend toward decreased OS (C) American Medical Association. All rights reserved.

17 efigure 2. Scatter plot demonstrating correlation of MGMT tumor protein expression scores versus MGMT methylation status in RTOG 0525 patients A total of 320 patients are shown as described in etable American Medical Association. All rights reserved.

18 efigure 3. Validation of the NRG-GBM-RPA classification in an independent GBM cohort (excluding known IDH-mutant glioblastomas, N=9) The NRG-GBM-RPA class correlated to OS in all GBM patients with heterogeneous treatments (A) and the NRG-GBM-RPA class correlated to OS in GBM patients treated with radiation and temozolomide (B) American Medical Association. All rights reserved.

19 efigure 4. Protein expression of MGMT and c-met by immunohistochemistry Top line: positive (A) and negative (B) reaction for MGMT expression strong nuclear staining in >75% of neoplastic cells in the positive case. Bottom line: positive (C) and negative (D) reaction for c-met strong cytoplasmic staining in the positive case; weak staining in less than 10% of neoplastic cells (A-D 400x) American Medical Association. All rights reserved.