Supplemental Figure 1. Interaction analysis network for the 22 selected candidate which were identified as being hypermethylated either in the non-HPV-driven (highlighted in blue) or in the HPV-driven tumors (highlighted in purple) (STRING confidence view, enhanced, predicted functional partners scores >0.997) (23). 0% 25% 100%

ALDH1A2_CpG_1 PROM1_CpG_23 ALDH1A2_CpG_2 PROM1_CpG_25.26.27 ALDH1A2_CpG_3.4 PROM1_CpG_28 ALDH1A2_CpG_5 PROM1_CpG_29 ALDH1A2_CpG_8 PROM1_CpG_30.31 ALDH1A2_CpG_9 PROM1_CpG_32.33 ALDH1A2_CpG_13.14 PROM1_CpG_34 ALDH1A2_CpG_15.16 PROX1_CpG_1.2.3 ALDH1A2_CpG_17.18 PROX1_CpG_4.5 ALDH1A2_CpG_19 PROX1_CpG_6 ALDH1A2_CpG_20.21 PROX1_CpG_9 FKBP4_CpG_2 PROX1_CpG_10.11.12 FKBP4_CpG_3.4 PROX1_CpG_13 FKBP4_CpG_5 PROX1_CpG_14 FKBP4_CpG_6 PROX1_CpG_15 FKBP4_CpG_8 PROX1_CpG_16 FKBP4_CpG_9.10 PROX1_CpG_18 FKBP4_CpG_12.13 PROX1_CpG_19 FKBP4_CpG_14 PROX1_CpG_20 FKBP4_CpG_15 PROX1_CpG_21 FKBP4_CpG_16.17 PROX1_CpG_23 GDNF_CpG_5.6 PROX1_CpG_24 GDNF_CpG_7.8 PROX1_CpG_25 GDNF_CpG_9 PROX1_CpG_26.27.28 GDNF_CpG_13.14 PROX1_CpG_29.30 GDNF_CpG_15.16 TLX1_CpG_1 GDNF_CpG_19 TLX1_CpG_2.3 GDNF_CpG_20.21 TLX1_CpG_4 GDNF_CpG_22.23 TLX1_CpG_5 HOXA6_CpG_6.7 TLX1_CpG_6.7 HOXA6_CpG_11 TLX1_CpG_8.9.10.11 HOXA6_CpG_18.19 TLX1_CpG_12 HOXA6_CpG_20 TLX1_CpG_24 HOXA6_CpG_21 TLX1_CpG_28.29 HOXA6_CpG_27 TLX1_CpG_31 HOXA6_CpG_28 TLX1_CpG_32.33 HOXA6_CpG_31.32.33 TLX1 PROX1 PROM1 TLX1_CpG_34 OSR2_CpG_4.5 TLX1_CpG_35 OSR2_CpG_6 TLX1_CpG_36 OSR2_CpG_7.8 TLX1_CpG_37 OSR2_CpG_9 TLX1_CpG_38 OSR2_CpG_10 TLX1_CpG_39.40 OSR2_CpG_20 TLX1_CpG_44 OSR2_CpG_26.27 TLX1_CpG_46 OSR2_CpG_35 TLX1_CpG_47.48 OSR2_CpG_36.37 UNCX_CpG_4 OSR2_CpG_38 UNCX_CpG_10 OSR2_CpG_47 UNCX_CpG_11 PAX9_CpG_1 UNCX_CpG_12 PAX9_CpG_2 UNCX_CpG_13.14 PAX9_CpG_5 UNCX_CpG_15.16 PAX9_CpG_6 UNCX_CpG_17.18 PAX9_CpG_7.8 UNCX_CpG_19.20.21.22 PAX9_CpG_10.11 UNCX_CpG_23.24.25 PAX9_CpG_12 UNCX_CpG_29 PAX9_CpG_19.20 UNCX_CpG_30.31.32.33.34 PAX9_CpG_23.24 UNCX_CpG_36 PAX9_CpG_25 UNCX_CpG_37.38 PAX9_CpG_26.27 UNCX_CpG_39 PAX9_CpG_28 UNCX_CpG_40 PAX9_CpG_29 UNCX_CpG_41 PAX9_CpG_30 UNCX_CpG_42.43 PAX9_CpG_31.32 UNCX_CpG_45.46 PAX9_CpG_36.37.38 UNCX_CpG_47.48 PAX9_CpG_39 WIF1_CpG_6 PROM1_CpG_1 WIF1_CpG_7.8 PROM1_CpG_2.3.4.5 WIF1_CpG_9.10 PROM1_CpG_6 WIF1_CpG_11.12 PROM1_CpG_7 WIF1_CpG_13.14.15 PROM1_CpG_9 WIF1_CpG_18 PROM1_CpG_10 WIF1_CpG_19 PROM1_CpG_11.12 WIF1_CpG_20.21 PROM1_CpG_13 WIF1_CpG_25.26

PROM1_CpG_15 WIF1 UNCX WIF1_CpG_33

PROM1 PAX9 OSR2 HOXA6 GDNF FKPB4 ALDH1A2 OSR2 HOXA6 GDNF FKPB4 PROM1 PAX9 PROM1_CpG_17 WIF1_CpG_35.36 PROM1_CpG_18 WIF1_CpG_37 PROM1_CpG_20 WIF1_CpG_40.41 PROM1_CpG_21 WIF1_CpG_42 WIF1_CpG_43

Std0 WIF1_CpG_45 Tu11 Tu01 Tu02 Tu03 Tu04 Tu05 Tu06 Tu07 Tu08 Tu09 Tu10 Tu12 Tu13 Tu14 Tu15 Std40 Std100 Control WIF1_CpG_47 DNA-RNA- DNA+RNA- DNA+RNA+ Std0 Tu11 Tu01 Tu02 Tu03 Tu04 Tu05 Tu06 Tu07 Tu08 Tu09 Tu10 Tu12 Tu13 Tu14 Tu15 Std40 Std100 Control DNA-RNA- DNA+RNA- DNA+RNA+ Supplemental Figure 2. Confirmation of DMRs by quantitative methylation analysis. Methylation patterns of gene promoter DMRs, which were identified as hypermethylated in the non-HPV-driven tumors (Tu01-Tu10) by array analysis. A healthy mucosa sample served as control; standards for 0%, 40% and 100% methylation were included. 0% 17% 100%

BDNF_CpG_6 IRX4_CpG_1 BDNF_CpG_9 IRX4_CpG_3 BDNF_CpG_12.13 IRX4_CpG_4 EOMES_CpG_1 IRX4_CpG_5.6 EOMES_CpG_2.3 IRX4_CpG_7 EOMES_CpG_12 IRX4_CpG_8 EOMES_CpG_13.14 IRX4_CpG_10 EOMES_CpG_15 IRX4_CpG_12.13.14.15 EOMES_CpG_16 IRX4_CpG_20 EOMES_CpG_17.18 IRX4_CpG_21 GATA4_CpG_1.2.3 IRX4_CpG_22 GATA4_CpG_5.6.7 IRX4_CpG_23 GATA4_CpG_12 IRX4_CpG_24 GATA4_CpG_13.14 IRX4_CpG_25 GATA4_CpG_22 IRX4_CpG_26 TA4 EOMES BDNF TA4 GATA4_CpG_23.24 IRX4_CpG_27 GATA4_CpG_29.30 PAX6_CpG_1 GATA4_CpG_31.32 PAX6_CpG_2 GATA4_CpG_35.36 PAX6_CpG_4 GATA4_CpG_39.40 PAX6_CpG_7.8 GATA4_CpG_41 PAX6_CpG_9.10 GFRA1_CpG_1 PAX6_CpG_11 GFRA1_CpG_2 PAX6_CpG_12.13 GFRA1_CpG_3 PAX6_CpG_14.15.16 GFRA1_CpG_4 PAX6_CpG_17.18 GFRA1_CpG_5 PAX6_CpG_19 GFRA1_CpG_6.7.8 PAX6_CpG_21.22.23 GFRA1_CpG_9 PAX6_CpG_24.25.26 GFRA1_CpG_10.11.12 PAX6_CpG_27 GFRA1_CpG_13 PHOX2B_CpG_2 GFRA1_CpG_18.19.20 PHOX2B_CpG_3 GFRA1_CpG_21 PHOX2B_CpG_5 GFRA1_CpG_22.23 PHOX2B_CpG_6.7 GFRA1_CpG_24.25 PHOX2B_CpG_9.10 GFRA1_CpG_28 PHOX2B_CpG_11.12.13 GFRA1_CpG_34 PHOX2B_CpG_14 GFRA1_CpG_35 PHOX2B_CpG_15 GRIA4_CpG_1 PHOX2B_CpG_16 GRIA4_CpG_2.3 PHOX2B_CpG_17 GRIA4_CpG_4.5 PHOX2B_CpG_19 GRIA4_CpG_7.8 SOX1_CpG_1.2 GRIA4_CpG_9.10 SOX1_CpG_3.4.5 GRIA4_CpG_11.12.13 SOX1_CpG_6 GRIA4_CpG_16 SOX1_CpG_8.9.10.11.12 GRIA4_CpG_17 SOX1_CpG_13.14 GRIA4_CpG_25 SOX1_CpG_15 GRIA4_CpG_30 SOX1_CpG_16 GRIA4_CpG_31 SOX1_CpG_17 GRIA4_CpG_33 SOX1_CpG_19 GRIA4_CpG_34.35.36 SOX1_CpG_20.21 HOXA13_CpG_7 SOX1_CpG_23.24 HOXA13_CpG_8 SOX1_CpG_25 HOXA13_CpG_10.11 SOX1_CpG_27.28.29 HOXA13_CpG_17.18 SOX1_CpG_30 HOXA13_CpG_19 TBX5_CpG_1.2

HOXA13 GRIA4 GFRA1 GA HOXA13_CpG_20.21 TBX5_CpG_3 TBX5_CpG_6 Std0 Tu11 Tu01 Tu02 Tu03 Tu04 Tu05 Tu06 Tu07 Tu08 Tu09 Tu10 Tu12 Tu13 Tu14 Tu15 Std40 TBX5_CpG_7.8 Std100 Control DNA-RNA- DNA+RNA- DNA+RNA+ TBX5_CpG_10.11 TBX5_CpG_12 TBX5_CpG_13 TBX5_CpG_20 TBX5_CpG_22.23.24.25.26

TBX5 SOX1 PHOX2B PAX6 IRX4 TBX5 SOX1 PHOX2B PAX6 TBX5_CpG_28.29.30 TBX5_CpG_31 TBX5_CpG_32 TBX5_CpG_33.34 TBX5_CpG_36 TBX5_CpG_37 TBX5_CpG_38.39 Std0 Tu11 Tu01 Tu02 Tu03 Tu04 Tu05 Tu06 Tu07 Tu08 Tu09 Tu10 Tu12 Tu13 Tu14 Tu15 Std40 Std100 Control DNA-RNA- DNA+RNA- DNA+RNA+

Supplemental Figure 3. Confirmation of DMRs by quantitative methylation analysis. Methylation patterns of gene promoter DMRs, which were identified as hypemethylated in the HPV-driven tumors (Tu11-Tu15) by array analysis. A healthy mucosa sample served as control; standards for 0%, 40% and 100% methylation were included. ALDH1A2** OSR2*** r = - 0.6607 r = - 0.7357 2DDCT value 2DDCT

Methylation (%) Methylation (%)

GATA4*** GRIA4*** IRX4*** r = - 0.8615 r = - 0.6500 r = - 0.7954 2DDCT value 2DDCT

Methylation (%) Methylation (%) Methylation (%)

Supplemental Figure 4. Correlation between gene promoter methylation and transcript levels (2DDCT value) in Tu01-Tu15 samples. Statistical non-parametric comparison for correlation between methylation and expression values was performed by Spearman’s Rho method (Spearman’s rank correlation coefficient r value is depicted in each graph, significance is documented by stars and was derived by Spearman’s rho p-value/two-tailed test, **=p<0.01, ***=p<0.001). plots for overall survival GATA4, GRIA4 5. Figure Supplemental samples) methylation. P values derived byLogRank/Mantel-Cox test. all of median the (below low and samples) all of median the (above high in categorized and computed was tumors 100 all in A. high methylatio high methylatio high methylatio high methylatio high methylatio low methylatio low methylatio low methylatio low methylatio low methylatio Number atris Number atris Number atris Number atris Number atris

Overall survival Overall survival Overall survival Overall survival Overall survival n n n n n n n n n n k k k k k 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 50 50 50 50 50 50 50 50 50 50 ***p<0.001 ***p<0.001 0 0 0 0 0 ***p<0.001 ***p<0.001 ***p<0.001 and IRX4) and clinical outcome of OPSCC patients (cohort 1 and cohort 2, n=100, Heidelberg). Kaplan-Meier (A) and progression-free survival 14 21 12 23 50 50 50 22 13 50 50 16 18 17 19 LHA, OSR2, (ALDH1A2, DMRs promoter gene five of status methylation the between Association Time (months) Time (months) Time (months) Time (months) Time (months) ALDH1A GATA4 GRIA4 OSR2 IRX4 high methylation high methylation 100 100 100 100 100 11 10 10 4 6 9 6 9 5 5 low methylation low methylation low methylation high methylation low methylation high methylation high methylation 2 low methylation 150 150 150 150 150 0 2 2 0 2 0 0 2 1 1 200 200 200 200 200 0 0 0 0 0 0 0 0 0 0 (B). The mean methylation value (mean of CpG units per amplicon) B. high methylatio high methylatio high methylatio high methylatio high methylatio low methylatio low methylatio low methylatio low methylatio low methylatio Number atris Number atris Number atris Number atris Number atris

Progression free survival Progression free survival Progression free survival Progression free survival Progression free survival n n n n n n n n n n k k k 0.00 0.25 0.50 0.75 1.00 k 0.00 0.25 0.50 0.75 1.00 k 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 50 50 50 50 50 50 50 50 50 50 ***p<0.001 0 0 0 0 0 ***p<0.001 ***p<0.001 ***p<0.001 ***p<0.001 50 50 50 50 50 17 19 11 14 13 11 17 14 15 9 high methylation Time (months) Time (months) Time (months) Time (months) Time (months) ALDH1A2 GATA4 GRIA4 OSR2 IRX4 100 100 100 100 100 low methylation low methylation 6 3 6 3 3 6 3 6 5 4 high methylation high methylation high methylation low methylation high methylation low methylation low methylation 150 150 150 150 150 0 2 0 2 1 1 2 0 2 0 200 200 200 200 200 0 0 0 0 0 0 0 0 0 0 A. HPV status and Methylation Score B. HPV DNA/p16 status and Methylation Score

1.00 HPV-driven/MS:3−5

1.00 non-DNA+p16+ non-HPV-driven/MS:3−5 DNA+p16+ MS:3-5 0.75 0.75 0.50 0.50 Overall survival Overall survival 0.25 0.25 Cohort 1/2 non-HPV-driven/MS:0−2 Cohort 1/2 non-DNA+p16+ MS:0-2 (Heidelberg) (Heidelberg) ***p<0.001 HPV-driven/MS:0−2HPV-driven/MS:0−2 ***p<0.001 DNA+p16+ 0.00 0.00 0 50 100 150 200 0 50 100 150 200 Time (months) Time (months) Number at risk Number at risk non-HPV-driven/MS:0−2 45 10 4 2 0 MS:0-2/non-DNA+p16+ 39 10 3 1 0 non-HPV-driven/MS:3−5 23 11 5 0 0 MS:0-2/DNA+p16+ 6 2 1 0 0 HPV-driven/MS:0−2 8 4 1 0 0 MS:3-5/non-DNA+p16+ 25 13 7 0 0 HPV-driven/MS:3−5 24 10 5 0 0 MS:3-5/DNA+p16+ 20 7 3 0 0

HPV status and Methylation Score DNA+RNA+ C. DNA+RNA− MS:3-5 1.00 DNA-RNA− 0.75 DNA+RNA− MS:0-2 DNA−RNA− 0.50 Overall survival 0.25

Cohort 3 **p=0.008

(Leipzig) 0.00 0 10 20 30 40 50 Time (months) Number at risk DNA-RNA-/MS:0-2 31 25 7 3 3 0 DNA+RNA-/MS:0-2 14 11 3 2 0 0 DNA-RNA-/MS:3-5 10 8 3 1 1 0 DNA+RNA-/MS:3-5 4 4 3 1 1 0 DNA+RNA+/MS:3-5 11 8 5 1 1 0

Supplemental Figure 6. Methylation signature score predicts clinical outcome of OPSCC patients independent of various HPV and p16 status (A) Kaplan-Meier plot for overall survival with regard to methylation signature score (MS) and stratified for HPV status as defined by both HPV16 DNA positivity and HPV16 RNA positivity (12) (Cohort 1/2, Heidelberg University Hospital). HPV-driven: DNA+RNA+, non-HPV-driven: DNA-RNA and DNA+RNA-. (B) Kaplan-Meier plots for overall survival with regard to methylation signature score (MS) and stratified for HPV/p16 status (91/100 patients from cohort 1/2, Heidelberg). The non- DNA+p16+ cases correspond to DNA-p16- (n=28), DNA+p16- (n=6) and DNA+p16- (n=30) (see also Supplemental Table 25). (C) Kaplan-Meier plot for overall survival with regard to the methylation signature score (MS) and stratified for HPV status as defined by both HPV16 DNA positivity and HPV16 RNA positivity for cohort 3 (Leipzig). P values derived by Log Rank/Mantel-Cox test. A. RNA pattern 1: high E6*I/E1^E4 B. RNA pattern 2: high E1C/L1 DNA- DNA-

1.00 DNA+RNAp1+ 1.00 MS:3-5 DNA+RNAp2+ MS:3-5 DNA+RNAp1- DNA+RNAp2- 0.75 0.75 0.50 0.50 Overall survival Overall survival 0.25 0.25 DNA+RNAp2- DNA- DNA- MS:0-2 MS:0-2 DNA+RNAp1- DNA+RNAp2+ ***p<0.001 DNA+RNAp1- ***p<0.001 0.00 0.00 0 50 100 150 200 0 50 100 150 200 Time (months) Time (months) Number at risk Number at risk MS:0−2/DNA- 23 3 3 2 0 MS:0−2/DNA- 23 3 3 2 0 MS:0−2/DNA+RNAp1- 26 8 2 0 0 MS:0−2/DNA+RNAp2- 25 9 1 0 0 MS:0−2/DNA+RNAp1+ 4 3 0 0 0 MS:0−2/DNA+RNAp2+ 5 2 1 0 0 MS:3−5/DNA- 15 8 3 0 0 MS:3−5/DNA- 15 8 3 0 0 MS:3−5/DNA+RNAp1- 25 9 5 0 0 MS:3−5/DNA+RNAp2- 13 5 3 0 0 MS:3−5/DNA+RNAp1+ 7 4 2 0 0 MS:3−5/DNA+RNAp2+ 19 8 4 0 0

C. HPV DNA Viral Load D. Treatment DNA-

1.00 RC/MS:3-5

DNAhigh MS:3-5 1.00 DNAlow Surgery/MS:3-5 0.75 0.75 0.50 0.50 Overall survival OVerall survival

0.25 DNAlow 0.25 Surgery/MS:0-2 DNA- MS:0-2 DNAhigh ***p<0.001 RC/MS:0-2 0.00 ***p<0.001 0 50 100 150 200 0.00 Time (months) 0 50 100 150 200 Time (months) Number at risk MS:0−2/neg 23 3 3 2 0 Number at risk Surgery/MS:0-2 MS:0-2/low 25 9 1 0 0 34 12 5 2 0 MS:0-2/high 5 2 1 0 0 RC/MS:0-2 17 2 0 0 0 39 16 7 0 0 MS:3-5/neg 15 8 3 0 0 Surgery/MS:3-5 5 4 2 0 0 MS:3-5/low 12 6 4 0 0 RC/MS:3-5 MS:3-5/high 20 7 3 0 0

Supplemental Figure 7. Methylation signature score predicts clinical outcome of OPSCC patients independent of HPV RNA patterns, DNA viral load and treatment. (A) Kaplan-Meier plot for overall survival with regard to the methylation signature score (MS) and stratified for HPV16 RNA pattern 1 (high E6*I/E1^E4) (12). (B) Kaplan-Meier plot for overall survival with regard to the methylation signature score (MS) and stratified for HPV16 RNA pattern 2 (high E1C/L1) (Hozlinger et al. 2012). (C) Kaplan- Meier plot for overall survival with regard to the methylation signature score (MS) and stratified for DNA viral load. (D) Kaplan- Meier plot for overall survival with regard to the methylation signature score (MS) and stratified for first-line treatment (surgery versus radiotherapy/chemotherapy (RC); see also Supplemental Tables 25-26). 0 22 100 0 8 100 0 17 100 0 16 100 0 12 100 ALDH1A2_CpG_1 ALDH1A2_CpG_2 ALDH1A2_CpG_5 ALDH1A2_CpG_8 ALDH1A2_CpG_9 ALDH1A2_CpG_13.14 ALDH1A2_CpG_15.16 ALDH1A2_CpG_17.18 ALDH1A2_CpG_19 ALDH1A2_CpG_20.21 IRX4_CpG_1 IRX4_CpG_3 IRX4_CpG_4 IRX4_CpG_5.6 IRX4_CpG_7 IRX4_CpG_8 IRX4_CpG_10 IRX4_CpG_12.13.14.15 IRX4_CpG_20 IRX4_CpG_21 IRX4_CpG_22 IRX4_CpG_23 IRX4_CpG_24 IRX4_CpG_25 IRX4_CpG_26 IRX4_CpG_27 OSR2_CpG_4.5 OSR2_CpG_6 OSR2_CpG_7.8 OSR2_CpG_9 OSR2_CpG_10 OSR2_CpG_20 OSR2_CpG_26.27 OSR2_CpG_35 OSR2_CpG_36.37 OSR2_CpG_38 OSR2_CpG_47 GATA4_CpG_1.2.3 GATA4_CpG_5.6.7 GATA4_CpG_12 GATA4_CpG_13.14 GATA4_CpG_22 GATA4_CpG_23.24 GATA4_CpG_29.30 GATA4_CpG_31.32 GATA4_CpG_35.36 GATA4_CpG_39.40 GATA4_CpG_41 GRIA4_CpG_1 GRIA4_CpG_2.3 GRIA4_CpG_4.5 GRIA4_CpG_7.8 GRIA4_CpG_9.10 GRIA4_CpG_11.12.13 GRIA4_CpG_16 GRIA4_CpG_17 GRIA4_CpG_25 GRIA4_CpG_30 GRIA4_CpG_31 GRIA4_CpG_33 GRIA4_CpG_34.35.36 LZ01 LZ02 LZ03 LZ04 LZ05 LZ06 LZ07 LZ08 LZ09 LZ10 LZ11 LZ12 LZ13 LZ14 LZ15 LZ16 LZ17 LZ18 LZ19 LZ20 LZ21 LZ22 LZ23 LZ24 LZ25 DNA-RNA- LZ26 LZ27 LZ28 LZ29 LZ30 LZ31 LZ32 LZ33 LZ34 LZ35 LZ36 LZ37 LZ38 LZ39 LZ40 LZ41 LZ42 LZ43 LZ44 LZ45 LZ46 LZ47 LZ48 LZ49 LZ50 LZ51 LZ52 LZ53 LZ54 DNA+RNA- LZ55 LZ56 LZ57 LZ58 LZ59 LZ60 LZ61 LZ62 LZ63 LZ64 LZ65 LZ66 LZ67 LZ68 DNA+RNA+ LZ69 LZ70 Std0 Std20 Std40 St60 Std80 Std100 ALDH1A2 OSR2 GATA4 GRIA4 IRX4

Supplemental Figure 8. Methylation heatmap of promoter DMRs associated with genes ALDH1A2, OSR2, GATA4, GRIA4, and IRX4 in cohort 3 (Leipzig) with n=70 OPSCC patients (DNA-RNA-: n=41; DNA+RNA-: n=18; DNA+RNA+: n=11). Standards (Std) for different percentages of methylation are also decicted. Chicago validation cohort (cohort 4). DNA methylation patterns of gene promoter DMRs for MS=0). Gray/Blackbarsindicatesurvivalstatus(OS:disease-specificdeathornot). and 9. Figure Supplemental cohort 3 (Leipzig). Color bar indicate methylation score (MS) of each patient starting from low to high (none of the patients showed IRX4 0

ALDH1A2 ALDH1A2_CpG_1 ALDH1A2_CpG_2 in cohort 4 (Chicago, n=50). Analysis was performed by MassARRAY as described for cohorts 1/2 (Heidelberg) and (Heidelberg) 1/2 cohorts for described MassARRAYas by performed was n=50). Analysis (Chicago, 4 cohort in ALDH1A2_CpG_5 ALDH1A2_CpG_8 ALDH1A2_CpG_9 22 ALDH1A2_CpG_13.14 ALDH1A2_CpG_15.16 ALDH1A2_CpG_17.18 100 ALDH1A2_CpG_19 ALDH1A2_CpG_20.21 0 OSR2_CpG_4.5 OSR2_CpG_6 OSR2 OSR2_CpG_7.8 h fv-ee ehlto sgaue s soitd ih lncl ucm o OSC n the in OPSCC of outcome clinical with associated is signature methylation five-gene The OSR2_CpG_9

OSR2_CpG_10 8 OSR2_CpG_20 OSR2_CpG_26.27 OSR2_CpG_35 100 OSR2_CpG_36.37 OSR2_CpG_38 0 GATA4_CpG_1.2.3OSR2_CpG_47 GATA4_CpG_5.6.7 GATA4 GATA4_CpG_12 GATA4_CpG_13.14 GATA4_CpG_22 17 GATA4_CpG_23.24 GATA4_CpG_29.30 GATA4_CpG_31.32 GATA4_CpG_35.36 100

GATA4_CpG_39.40 0 GRIA4_CpG_1GATA4_CpG_41 GRIA4_CpG_2.3 GRIA4_CpG_4.5 GRIA4 GRIA4_CpG_7.8 GRIA4_CpG_9.10

GRIA4_CpG_11.12.13 16 GRIA4_CpG_16 GRIA4_CpG_17 GRIA4_CpG_25 GRIA4_CpG_30

GRIA4_CpG_31 100 GRIA4_CpG_33

GRIA4_CpG_34.35.36 0 IRX4_CpG_1 IRX4_CpG_3 IRX4_CpG_4 IRX4_CpG_5.6 IRX4_CpG_7 IRX4 IRX4_CpG_8

IRX4_CpG_10 12 IRX4_CpG_12.13.14.15 IRX4_CpG_20 IRX4_CpG_21 IRX4_CpG_22 IRX4_CpG_23 IRX4_CpG_24 100

ALDH1A2, OSR2, GATA4, GRIA4 IRX4_CpG_25 IRX4_CpG_26 CH25 CH12 CH10 CH06 CH04 CH02 CH40 CH37 CH35 CH34 CH32 CH28 CH26 CH23 CH21 CH19 CH17 CH14 CH09 CH08 CH07 CH05 CH03 CH45 CH41 CH38 CH33 CH31 CH24 CH20 CH18 CH16 CH13 CH11 CH01 CH49 CH44 CH42 CH36 CH30 CH29 CH15 CH50 CH48 CH47 CH46 CH43 CH39 CH27 CH22 IRX4_CpG_27 Survivor Disease-associated death Methylation Score=5 Methylation Score=4 Methylation Score=3 Methylation Score=2 Methylation Score=1

PFS (PD/DSD)