Supplemental Material
Supplemental Figure Legends Table S1: Immune marker panels used in NanoString DSP. CAB# is custom antibody ID, which uniquely identifies this reagent. Table S2: Clinicopathological characteristics of EGFR TKI treated NSCLC patients in Yale cohort A. Table S3: Clinicopathological characteristics of NSCLC patients in Yale cohort B. Table S4: Clinicopathological characteristics of ICIs treated melanoma patients in Yale cohort C. Table S5: Multivariate analysis of the predictive markers for PFS in Yale melanoma ICIs treated cohort C. Table S6: Multivariate analysis of the predictive markers for OS in Yale melanoma ICIs treated cohort C. Figure S1: Representative images of NanoString DSP and AQUA compartment selection in NSCLC. The epithelial compartment was defined by cytokeratin positivity (green) which allowed the measurement of targets by both assays in the same compartment. Stroma was defined by cytokeratin negative DNA positive regions (purple). Figure S2: NanoString DSP reproducibility across different cores in ITx melanoma cohort C. (A) CD8 reproducibility across 2 cores (B) CD68 reproducibility across 2 cores Figure S3: NanoString DSP sequential compartment assignment. CD8 counts by NanoString DSP in tumor, CD68+ and CD45+ compartment. Compartment definition accuracy is affected by the order of barcode collection from each compartment. In overlapping compartments, measurement of a marker can be assigned to only one compartment.
Table S1 Table S2 Table S3 Yale NSCLC Cohort A Yale NSCLC Cohort B n=43 % Age <70 31 72.1 ≥70 12 27.9 Sex Female 26 60.5 Male 17 39.5 Smoking history Never smoker 13 30.2 Former/ Current smoker 30 69.8 Stage I/II III/IV Histology ADC 41 95.3 SCC 2 4.7 EGFR mutation Yes 36 83.7 Exon 18 3 7.0 Exon 19 16 37.2 Exon 20 Exon 21 14 32.6 Unknown 1 2.3 No 6 EGFR TKI treatment Afatinib Erlotinib Osimertinib Regorafenib Yale NSCLC Cohort B n= 113 % Age <70 64 56.6 ≥70 49 43.4 Sex Female 59 52.2 Male 54 47.8 Smoking history Never smoker 7 6.2 Former/ Current smoker 106 93.8 Stage I/II 85 75.2 III/IV 28 24.8 Histology ADC 66 58.4 SCC 22 19.5 Other 20 17.7 Unknown 5 4.4 Table S3 Yale Melanoma Cohort C n= 58 % Age <70 38 65.5 ≥70 20 34.5 Sex Female 25 43.1 Male 33 56.9 Stage I 10 17.2 II 11 19.0 III 22 37.9 IV 12 20.7 Unknown 3 5.2 Itx treatment Pembro Nivo Ipi+Nivo Specimen Primary Met LN 15 25.9 Mutation None 32 55.2 BRAF 17 29.3 NRAS 8 13.8 CKIT 1 1.7
Table S4 Cocktail A – Yale NSCLC cohort A Cocktail B – Yale NSCLC cohort B Cocktail C - Yale Melanoma Cohort C # Target CAB # Working Conc. (ug/mL) 1 Beta-Catenin 963 2 0.5 2.5 CD8 1022 PMS2 934 3 B7-H3 959 4 CD4 941 5 FOXP3 268 6 Mouse IgG1 891 Phospho-p70S6K 336 7 CD68 1023 8 Phospho-RPS6 767 Rabbit IgG 836 9 CD14 397 10 S6 837 11 GZMB 1068 MLH1 937 12 Ki-67 314 CD34 1041 13 Beta-2-microglobulin 964 MSH2 919 14 Histone H3 387 1.3 15 Akt 962 16 CD3 1019 17 VISTA 940 18 PD1 1024 0.2 19 Phospho-STAT3 458 20 CD44 619 21 STAT3 957 MSH6 920 22 CD56 1020 23 PD-L1 1039 24 CD45 1018 P-AKT 624 25 CD19 642 26 CD20 706 27 Pan-Cytokeratin 886 28 CD45RO 77 29 Tim3 1038 Bcl-2 960 30 HLA-DR 1052 31 32 33 34 35 B7-H4 1053 36 Ik-Ba 907 37 BIM 894 38 IDO1 1033 39 CD11c 1036 40 Mouse IgG2a 1258 41 BCL6 880 42 c-Myc 878 43 Phospho-STAT5 882 44 CD163 1031
Table S5 Table S6 Predictive markers for PFS - Multivariate Analysis Tumor ROI Cutpoint Hazard Ratio 95% CI P value CD68+ ROI CD45+ ROI IDO1 Low (1st and 2nd tertile) vs High (3rd tertile) 0.42 0.14 to 1.20 0.1 PDL1 0.51 0.19 to 1.27 0.15 B2M Median 0.3869 0.1998 to 0.7494 0.0033 CD11c 0.7 0.31 to 1.48 0.36 CD8 0.62 0.25 to 1.43 0.27 HLADR 1.14 0.37 to 3.17 0.8 TIM3 0.79 0.33 to 1.79 0.58 0.91 0.42 to 1.91 0.82 0.96 0.36 to 2.35 0.93 0.86 0.19 to 3.07 0.83 CD3 0.87 0.20 to 3.48 0.85 Table S6 Predictive markers for OS - Multivariate Analysis Tumor ROI Cutpoint Hazard Ratio 95% CI P value CD68+ ROI CD45+ ROI CD20 Median 0.39 0.10 to 1.22 0.11 PDL1 Low (1st and 2nd tertile) vs High (3rd tertile) 0.2 0.03 to 0.88 0.032 B2M 0.38 0.12 to 1.03 0.059 HLADR 0.29 0.05 to 1.64 0.17 CD4 0.46 0.14 to 1.36 0.16 MSH2 1.98 0.62 to 7.66 0.25 IDO1 0.5 0.09 to 2.09 0.35 MYC 2.15 0.70 to 7.08 PMS2 1.3 0.33 to 4.54 0.68 CD8 1.88 0.3 to 10.24 0.48 1.81 0.63 to 5.52 0.26 1.21 0.38 to 3.40 0.72 0.88 0.24 to 3.10 0.85 CD3 1.18 0.18 to 7.79 0.86 0.93 0.14 to 5.33 0.94
Figure S1 Figure S2 A B
Figure S3