Supplementary Tables and Figures Supplementary Table 1. identified in the biomarker discovery screen

Acc. pH ANOVA N/P P/T N/T name kDa pI number range p-value (r,p-value) (r, p-value) (r, p-value) Epidermis development / epithelial Cornulin Q9UBG 3-7 0.004 -1.26, 0.68 -4.23, 0.017 -5.32, 0.016 54 5.8 , type I cytoskeletal 13# P13646 3-7 0.002 -1.15, 0.76 -5.08, 0.017 -8.99, 0.010 50 4.9 Keratin, type II cytoskeletal 4# P19013 3-7 0.008 1.07, 0.76 -4.68, 0.018 -9.54, 0.011 64 8.5 Small proline-rich protein 3# Q9UBC 6-11 0.006 -1.14, 0.81 -3.84, 0.078 -3.94, 0.033 18 8.9 Protein synthesis Elongation factor 1-alpha 1 P68104 6-111 0.018 1.11, 0.78 1.52, 0.173 1.63, 0.0356 50 9.1 Elongation factor 1-delta P29692 3-7 0.024 1.41, 0.58 1.20, 0.180 1.63, 0.013 31 4.9 Eukaryotic translation initiation factor 3 subunit I Q13347 3-7 0.008 1.43, 0.63 1.88, 0.036 2.50, 0.010 37 5.4 Eukaryotic initiation factor 4A-III P38919 3-72 0.009 1.11, 0.86 1.76, 0.015 1.88, 0.017 47 6.3 Protein folding and stress response DnaJ homolog subfamily B member 1 P25685 6-11 0.014 -1.04, 0.88 -1.50, 0.134 -1.59, 0.043 38 8.7 DnaJ homolog subfamily B member 11 Q9UBS4 3-7 0.000 1.19, 0.60 1.84, 0.015 2.28, 0.008 41 5.8 Endoplasmin P14625 3-7 0.017 1.48, 0.68 3.18, 0.031 4.58, 0.025 92 4.8 Endoplasmic reticulum protein ERp29 P30040 3-7 0.005 1.57, 0.44 -1.05, 0.250 1.51, 0.015 29 6.8 Peptidyl-prolyl cis-trans isomerase B P23284 6-11 0.002 1.1, 0.88 1.74, 0.052 1.90, 0.021 24 9.3 Protein disulfide-isomerase A4 P13667 3-7 0.005 1.21, 0.76 2.81, 0.026 3.08, 0.010 73 5.0 Serpin H1 precursor# P50454 6-11 0.000 1.38, 0.56 5.25, 0.057 6.54, 0.007 46 8.8 Stress-induced-phosphoprotein 1 P31948 3-7 0.006 1.89, 0.58 1.82, 0.047 2.84, 0.010 63 6.4 Thioredoxin domain-containing protein 5 Q8NBS9 3-7 0.008 1.35, 0.64 2.17, 0.044 2.97, 0.015 48 5.6 Cellular organization and structuring Annexin A1# P04083 3-7 0.001 -1.43, 0.59 -2.61, 0.024 -5.08, 0.009 39 6.6 Annexin A5# P08758 3-7 0.002 1.55, 0.58 2.37, 0.017 3.45, 0.010 36 4.9 -A/C P02545 3-7 0.002 1.41, 0.58 2.02, 0.014 2.88, 0.011 74 6.6 Lamin-B1 P20700 3-7 0.015 1.6, 0.58 1.62, 0.058 2.45, 0.022 66 5.1 light chain 1# P05976 3-7 0.002 -1.26, 0.59 -4.61, 0.016 -5.23, 0.014 21 5.0 Septin-7# Q16181 6-11 0.007 1.16, 0.78 1.68, 0.109 1.91, 0.030 51 8.7 P08670 3-7 0.002 1.65, 0.59 2.11, 0.017 3.07, 0.008 54 5.1 Other Adenylosuccinate synthetase isozyme 2 P30520 3-72 0.009 1.11, 0.86 1.76, 0.015 1.88, 0.017 50 6.1 Alcohol dehydrogenase class 4 mu/sigma chain P40394 6-11 0.035 1.04, 0.88 -1.86, 0.135 -2.18, 0.037 41 8.1 Bifunctional purine biosynthesis protein PURH P31939 3-7 0.005 1.37, 0.59 1.78, 0.024 2.29, 0.010 65 6.3 Glutathione S-transferase kappa 1 Q9Y2Q3 6-11 0.009 1.07, 0.97 -1.68, 0.057 -1.63, 0.033 25 8.5 Interleukin-1 receptor antagonist protein P18510 3-7 0.004 -1.29, 0.66 -2.33, 0.043 -4.56, 0.008 20 5.8 Isocitrate dehydrogenase [NADP]# P48735 6-11 0.010 1.05, 0.89 1.62, 0.063 1.73, 0.051 51 8.9 Lactoylglutathione lyase# Q04760, 3-7 0.008 -1.26, 0.65 -2.22, 0.021 -2.71, 0.020 21 5.1 L-lactate dehydrogenase A chain# P00338 6-11 0.009 1.05, 0.88 1.72, 0.096 1.80, 0.038 37 8.4 Phosphatidylinositol transfer protein beta P48739 3-7 0.034 1.56, 0.58 -1.19, 0.083 1.23, 0.130 32 6.4 Plastin-2 P13796 3-7 0.017 1.97, 0.58 1.77, 0.066 3.24, 0.024 70 5.2 Sulfide:quinone oxidoreductase Q9Y6N5 6-111 0.018 1.11, 0.78 1.52, 0.173 1.63, 0.036 50 9.1 Superoxide dismutase [Mn]# P04179 3-7 0.003 1.83, 0.37 2.17, 0.038 4.11, 0.013 25 8.4 Transforming growth factor-beta-induced protein Q15582 6-11 0.011 1.29, 0.84 2.84, 0.096 3.68, 0.043 75 7.6 alpha-4 chain (TPM3) P67936 3-7 0.004 1.55, 0.58 2.07, 0.043 3.11, 0.010 29 4.7 Tryptophanyl-tRNA synthetase P23381 3-7 0.017 1.94, 0.42 2.86, 0.060 4.65, 0.024 53 5.8 UMP-CMP kinase# P30085 3-7 0.008 -1.2, 0.68 -2.54, 0.025 -3.22, 0.017 26 8.1 Not identified 3-7 0.002 -1.53, 0.62 -3.94, 0.015 -6.41, 0.010 Not identified 3-7 0.002 1.24, 0.78 2.44, 0.013 2.70, 0.011 Not identified 3-7 0.009 1.01, 0.99 1.65, 0.018 1.64, 0.022 1 Not identified 6-11 0.080 27.84, 0.56 -1.26, 0.223 71.15, 0.170

Note:

1,2 Single spots with two proteins identified, the separate ratios could not be distinguished for these proteins

# Proteins identified in multiple spots, values derived from that spot with the median ANOVA p- value were chosen.

N = normal, P = precancerous, T = tumor, r = ratio (a positive value indicates an increase and a negative value indicates a decrease), p-values are FDR corrected

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Supplementary Table 2. Patient cohort for clinical validation TN- local follow- # of dysplasia score score pt gender age site treatment stage follow-up up time margins margins cornulin 1 m 68 oral cavity T1N0 surg df 6,3 4 moderate 23 47 2 m 58 oropharynx T1N0 surg loc 0,7 4 no 41 62 3 m 65 oral cavity T1N0 surg df 5,5 3 moderate 54 60 4 f 86 oral cavity T1N0 surg loc 2,4 4 no 31 46 5 m 71 oral cavity T1N0 surg df 4,8 4 no 69 64 6 m 48 oral cavity T1N0 surg spt 7,6 4 mild 50 44 7 m 39 oral cavity T1N0 surg df 12,0 5 no 67 65 8 m 63 oral cavity T1N0 surg loc 1,6 4 moderate 7 42 9 m 47 oral cavity T1N0 surg df 9,9 6 severe 48 63 10 m 67 oral cavity T1N0 surg loc 1,5 3 mild 6 46 11 f 49 oral cavity T1N1 surg df 13,9 3 mild 48 59 12 m 56 oral cavity T1N0 surg spt 3,9 5 moderate 38 40 13 m 58 oral cavity T2N1 surg df 4,4 7 mild 25 50 14 m 62 oral cavity T2N1 surg loc 1,0 6 no 18 38 15 f 54 oral cavity T2N1 surg df 8,5 4 mild 44 53 16 f 67 oral cavity T1N0 surg spt 6,1 3 moderate 34 44 17 f 56 oral cavity T2N1 surg df 12,2 4 no 58 60 18 m 38 oral cavity T2N0 surg loc 1,8 4 moderate 30 43 19 m 53 oral cavity T2N2 surg df 12,0 7 moderate 46 51 20 f 53 oral cavity T3N1 surg loc 1,4 5 mild 32 62 21 m 59 oral cavity T4N0 surg df 5,3 5 no 65 61 22 m 72 oral cavity T3N2 surg loc 0,5 6 no 34 50 23 f 54 oral cavity T2N0 surg+rt df 9,6 7 mild 27 49 24 m 40 oropharynx T1N2 surg+rt loc 1,5 3 mild 40 59 25 f 66 oral cavity T2N0 surg+rt df 7,4 4 no 62 58 26 f 59 oral cavity T1N0 surg+rt spt 0,5 2 severe 36 53 27 m 63 oral cavity T2N2 surg+rt df 9,2 5 mild 66 57 28 m 47 oral cavity T2N2 surg+rt loc 0,5 5 no 43 45 29 m 51 oropharynx T2N2 surg+rt df 11,2 6 moderate 54 58 30 f 66 oral cavity T2N0 surg+rt loc 1,6 4 mild 27 41 31 f 58 oral cavity T2N2 surg+rt df 6,2 5 no 35 48 32 m 60 oral cavity T2N2 surg+rt loc 1,1 5 mild 11 41 33 m 43 oral cavity T2N2 surg+rt df 11,2 4 severe 49 51 34 m 43 oral cavity T2N2 surg+rt loc 0,5 7 no 43 43 35 f 71 oropharynx T3N0 surg+rt df 7,8 5 mild 48 39 36 m 51 oropharynx T3N2 surg+rt loc 3,3 5 no 54 64 37 m 69 oral cavity T3N0 surg+rt df 10,0 6 no 53 49 38 m 51 oropharynx T3N2 surg+rt loc 2,0 4 severe 27 49 39 f 53 oropharynx T3N0 surg+rt df 7,6 6 severe 60 43 40 f 56 oropharynx T3N1 surg+rt loc 1,8 5 no 46 41 41 m 55 oral cavity T3N2 surg+rt df 9,5 7 no 68 62 42 f 46 oropharynx T4N0 surg+rt loc 3,0 4 severe 53 40 43 f 69 oral cavity T4N0 surg+rt df 6,9 5 mild 31 28 44 m 50 oral cavity T4N2 surg+rt loc 0,7 7 severe 60 59 45 m 46 oropharynx T4N1 surg+rt df 11,2 5 severe 33 56 46 f 65 oral cavity T4N2 surg+rt loc 0,9 6 no 12 47 Note: pt=patient number, m=male, f=female, surg=surgery, surg+rt=surgery and radiotherapy, loc=local recurrence, spt=second primary tumor, df=disease-free

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Supplementary Figure legends and Figures

Supplementary Figure 1. Immunohistochemical staining of independent FFPE tissue specimen

A: Typical examples of keratin 4, keratin 13, cornulin and small proline-rich protein 3 staining on normal epithelium (N), severe dysplasia (D), and tumor tissue.

B: Averaged semi-quantitative scoring results of immunostaining of keratin 4, keratin 13, cornulin and small proline-rich protein 3 on an independent set of 5 normal epithelia, 5 severe dysplasias and 5 tumors. Staining intensity was divided into no staining (=0), moderate staining intensity (=1) and strong staining intensity (=2). Staining results were calculated by multiplying the staining intensity with the estimated percentage of stained cells, resulting in a range from

0 to 200. The expression of all four protein differs significantly between both normal and tumor, and between normal and severe dysplasias (p<0.01). From these data keratin-4 and cornulin were selected for further prognostic validation.

Supplementary Figure 2. Correlations between quantitative assessments of the staining results of observer 1 with observer 2. The upper panel shows the correlation of the assessment of the individual margins, while the lower panel shows the results on the averaged percentages for every patient. The high correlation coefficients demonstrate the accuracy of the quantitative scoring.

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Supplementary Figure 3. ROC curves representing the relation between sensitivity and specificity for keratin 4 and cornulin immunostaining in the prognostic validation study. Curves were computed based on averaged staining percentages. Significant (p<0.05) areas under the curve (AUC) are indicated within the graph. Dashed lines specify the medians, which were used to categorize the patients into those with high and low keratin 4 and cornulin expression levels.

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Supplementary Figure 1

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Supplementary Figure 2

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Supplementary Figure 3

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