Supplementary Online Content

Hayes HB, Ahn J, Fan X, et al. Association of Oral Microbiome With Risk for Incident Head and Neck Squamous Cell Cancer [published online January 11, 2018]. JAMA Oncol. doi:10.1001/jamaoncol.2017.4777

eTable 1. Number of filtered sequence reads per sample

eTable 2. Sequence reads per bacterial phylum, by cohort

eTable 3a. Median counts and fold changes (FC) for the association1 between selected bacteria related to periodontal diseases and dental caries and risk of head and neck squamous cell cancer (HNSCC) in the 2 cohorts

eTable 3b. Carriage rate and odds ratios (OR) for the association between selected bacteria related to periodontal disease and dental caries and risk of head and neck squamous cell cancer (HNSCC) in the 2 cohorts

eTable 4. Median counts and fold changes (FC) for the association between selected taxa and risk of head and neck squamous cell cancer (HNSCC) according to smoking status in the 2 cohorts

eTable 5. Median counts and fold changes (FC) for the association between selected taxa and risk of head and neck squamous cell cancer (HNSCC) according to time from oral wash sample collection to diagnosis in the 2 cohorts

Table 6. Median counts and fold changes (FC) for the association between selected taxa and risk of head and neck squamous cell cancer (HNSCC) according to site of cancer (oral cavity, pharynx, and larynx) in the 2 cohorts

eFigure. Overall oral microbiota structure according to subsequent occurrence of squamous-cell head and neck cancer (SCHNC) and SCHNC of the oral cavity, pharynx, and larynx in a case–control study nested within 2 cohorts

This supplementary material has been provided by the authors to give readers additional information about their work.

1 1 Supplementary Table 1. Number of filtered sequence reads per sample

Total Cases Controls (N=383) (N=129) (N=254) Cohort Mean ± SD Mean ± SD Mean ± SD

CPS-II 9,145 ± 2,992 9,004 ± 2,767 9,217 ± 3,110 PLCO 7,744 ± 2,830 7,987 ± 3,460 7,620 ± 2,455

Total 8,373 ± 2,983 8,445 ± 3,196 8,337 ± 2,875 2 1. Poor-quality sequences were excluded using the default parameters of the QIIME script split_libraries.py 3 (minimum average quality score=25, minimum/maximum sequence length=200/1000 base pairs, no ambiguous 4 base calls, and no mismatches allowed in the primer sequence). 5

© 2017 American Medical Association. All rights reserved.

Downloaded From: https://jamanetwork.com/ on 09/28/2021 6 Supplementary Table 2. Sequence reads per bacterial phylum, by cohort CPS-II Cohort PLCO Cohort All Subjects (N=172) (N=211) (N=383) Phylum Reads Percent (%) Reads Percent (%) Reads Percent (%) Actinobacteria 131,083 8.3 128,293 7.8 259,376 8.1 Proteobacteria 172,884 10.9 121,240 7.4 294,124 9.1 Bacteroidetes 182,466 11.6 167,913 10.3 350,379 10.9 Firmicutes 1,015,937 64.4 1,164,938 71.1 2,180,875 67.8 Fusobacteria 70,595 4.5 51,536 3.1 122,131 3.8 Others 5,388 0.3 5,001 0.3 10,389 0.3 Total 1,578,353 1,638,921 3,217,274 7

© 2017 American Medical Association. All rights reserved.

Downloaded From: https://jamanetwork.com/ on 09/28/2021 8 Supplementary Table 3a. Median counts and fold changes (FC) for the association1 between selected bacteria related to 9 periodontal diseases and dental caries and risk of head and neck squamous cell cancer (HNSCC) in the 2 cohorts. 2 Transformed counts Median Maximum 3 4 Case Control FC 95% CI p q Cook’s D Porphyromonas;gingivalis 0.00 0.00 0.76 (0.42, 1.35) 0.3437 0.7767 8.83 Tannerella;forsythia 0.00 0.00 0.98 (0.59, 1.62) 0.9310 0.9796 5.60 Aggregatibacter;actinomycetemcomitans 0.00 0.00 0.97 (0.58, 1.63) 0.9099 0.9796 47.74 Streptococcus;mutans 5.49 7.82 0.66 (0.43, 1.02) 0.0635 0.4097 61.36 10 1. The association between taxonomic abundance and HNSCC was detected by DESeq function, adjusting for cohorts, matching factors [age at oral sample 11 collection (+/- 1 year), gender, and race (white, other)], smoking status (current, former, never), number of cigarettes per day for ever-smokers, alcohol 12 drinking status (never, ever), grams ethanol per day for ever-drinkers, and HPV-16 status. 13 2. Sequence read counts were normalized by dividing raw counts by DESeq size factors. 14 3. FDR-adjusted p value. FDR adjustment was conducted at each taxonomic level (i.e. class, genus) separately. 15 4. The maximum Cook’s Distance for DESeq model. 16

17

18 Supplementary Table 3b. Carriage1 rate and odds ratios (OR) for the association2 between selected bacteria related to 19 periodontal disease and dental caries and risk of head and neck squamous cell cancer (HNSCC) in the 2 cohorts

Carriage, N (%) Cases Controls 2 2 2 OR 95% CI p-value (n=129) (n=254) Porphyromonas gingivalis 36 (27.9) 74 (29.1) 0.91 (0.51,1.61) 0.7392 Tannerella forsythia 49 (40.9) 104 (38.0) 0.91 (0.54, 1.54) 0.7199 Aggregatibacter actinomycetemcomitans 6 (4.7) 18 (7.1) 0.74 (0.24, 2.26) 0.5937 Streptococcus mutans 96 (74.4) 211 (83.1) 0.63 (0.33, 1.20) 0.1594 20 1. Selected taxa were coded as present/absent [1/0] for sequence reads from mouthwash samples. 21 2. Odds ratios, 95% confidence intervals, and p-values were calculated from logistic regression after controlling for matching factors [age at oral sample 22 collection (+/- 1 year), gender, and race (white, other)], smoking status (current, former, never), number of cigarettes per day for ever-smokers, alcohol 23 drinking status (never, ever), grams ethanol per day for ever-drinkers, and HPV-16 status. 24 25

© 2017 American Medical Association. All rights reserved.

Downloaded From: https://jamanetwork.com/ on 09/28/2021 26 Supplementary Table 4. Median counts and fold changes (FC) for the association1 between selected taxa and risk of head 27 and neck squamous cell cancer (HNSCC) according to smoking status in the 2 cohorts Transformed 2 counts Taxon Median 3 Class;Order;Family;Genus;Species Case Control FC 95% CI p q Current smoker N=41 N=10 Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium 0.00 1.03 0.46 (0.11, 1.87) 0.2773 0.7571 Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;oral_taxon_170 0.00 0.00 2.84 (0.50, 15.98) 0.2368 0.7837 Betaproteobacteria 4.25 34.29 0.37 (0.15, 0.93) 0.0353 0.2434 Betaproteobacteria;Neisseriales 3.28 29.70 0.37 (0.16, 0.87) 0.0231 0.3935 Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;sicca 0.00 4.12 0.14 (0.03, 0.71) 0.0174 0.2147 Betaproteobacteria;Neisseriales;Neisseriaceae;Kingella 1.10 3.62 0.58 (0.18, 1.89) 0.3663 0.7571 Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;nanceiensis 2.37 5.45 0.17 (0.04, 0.69) 0.0128 0.1787 Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;leadbetteri 0.00 4.98 0.14 (0.04, 0.54) 0.0042 0.1505 Negativicutes Selenomonadales;Veillonellaceae;Selenomonas;sputigena 0.00 1.22 0.09 (0.02, 0.34) 0.0005 0.0296 Former smoker N=70 N=115 Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium 2.54 2.71 0.68 (0.44, 1.05) 0.0806 0.4238 Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;oral_taxon_170 0.00 0.65 1.67 (1.01, 2.76) 0.0444 0.4631 Betaproteobacteria 39.34 72.84 0.72 (0.55, 0.95) 0.0186 0.2046 Betaproteobacteria;Neisseriales 30.31 53.14 0.67 (0.48, 0.94) 0.0188 0.2765 Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;sicca 5.22 10.80 0.56 (0.34, 0.93) 0.0242 0.3953 Betaproteobacteria;Neisseriales;Neisseriaceae;Kingella 4.71 5.48 0.54 (0.37, 0.81) 0.0026 0.1287 Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;nanceiensis 2.93 4.93 0.79 (0.49, 1.28) 0.3404 0.7986 Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;leadbetteri 2.85 4.56 0.68 (0.44, 1.06) 0.0927 0.6192 Negativicutes Selenomonadales;Veillonellaceae;Selenomonas;sputigena 0.90 0.72 0.97 (0.61, 1.55) 0.8971 0.9709 Never smoker N=18 N=129 Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium 4.15 4.12 0.63 (0.35, 1.13) 0.1229 0.4902 Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;oral_taxon_170 1.97 1.95 0.96 (0.45, 2.04) 0.9075 0.9829 Betaproteobacteria 71.33 87.51 1.15 (0.83, 1.59) 0.3939 1.0000 Betaproteobacteria;Neisseriales 69.23 73.00 1.06 (0.68, 1.67) 0.7869 0.9598 Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;sicca 18.51 15.52 0.97 (0.43, 2.18) 0.9452 0.9829 Betaproteobacteria;Neisseriales;Neisseriaceae;Kingella 5.50 5.81 1.07 (0.59, 1.93) 0.8164 0.9973 Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;nanceiensis 18.74 9.42 0.78 (0.37, 1.66) 0.5216 0.9702 Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;leadbetteri 7.72 3.37 1.22 (0.61, 2.43) 0.5740 0.9702 Negativicutes Selenomonadales;Veillonellaceae;Selenomonas;sputigena 2.91 1.69 0.41 (0.19, 0.90) 0.0260 0.6494 28 Heterogeneity tests found no significant difference of fold changes between selected taxa-HNSCC associations according to smoking status by using Chi- 29 square test of Cochran’s Q statistic (p > 0.05). 30 1. The association between taxonomic abundance and HNSCC was detected by DESeq function, adjusting for cohorts, matching factors [age at oral sample 31 collection (+/- 1 year), gender, and race (white, other)], number of cigarettes per day for current- and former-smoker, alcohol drinking status (never, ever), 32 grams ethanol per day for ever-drinkers, and HPV-16 status. 33 2. Sequence read counts were normalized by dividing raw counts by DESeq size factors. 34 3. FDR-adjusted p value. FDR adjustment was conducted at each taxonomic level (i.e. class, genus) separately. 35

© 2017 American Medical Association. All rights reserved.

Downloaded From: https://jamanetwork.com/ on 09/28/2021 36 Supplementary Table 5. Median counts and fold changes (FC) for the association1 between selected taxa and risk of head 37 and neck squamous cell cancer (HNSCC) according to time from oral wash sample collection to diagnosis in the 2 cohorts Transformed 2 counts Taxon Median 3 4 Class;Order;Family;Genus;Species Case Control FC 95% CI p q ≤3 years (Case n=64) Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium 2.30 3.62 0.62 (0.42, 0.92) 0.0172 0.4298 Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;oral_taxon_170 0.52 0.94 3.44 (2.05, 5.77) <0.0001 0.0005 Betaproteobacteria 54.13 81.28 0.92 (0.81, 1.05) 0.2070 0.8917 Betaproteobacteria;Neisseriales 39.02 63.07 0.82 (0.63, 1.07) 0.1454 0.8241 Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;sicca 2.78 12.40 0.42 (0.25, 0.71) 0.0013 0.0471 Betaproteobacteria;Neisseriales;Neisseriaceae;Kingella 4.31 5.59 0.78 (0.54, 1.12) 0.1747 0.6593 Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;nanceiensis 3.06 6.39 0.43 (0.26, 0.71) 0.0010 0.0471 Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;leadbetteri 3.30 3.87 0.64 (0.41, 1.01) 0.0565 0.4495 Negativicutes Selenomonadales;Veillonellaceae;Selenomonas;sputigena 0.72 1.19 0.44 (0.27, 0.73) 0.0014 0.0471 >3 years (Case n=65) Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium 0.00 3.62 0.5 (0.32, 0.77) 0.0019 0.0469 Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;oral_taxon_170 0.00 0.94 1.11 (0.66, 1.85) 0.6976 0.9054 Betaproteobacteria 19.07 81.28 0.58 (0.43, 0.79) 0.0005 0.0051 Betaproteobacteria;Neisseriales 14.37 63.07 0.54 (0.37, 0.79) 0.0015 0.0124 Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;sicca 0.00 12.40 0.44 (0.25, 0.78) 0.0043 0.1386 Betaproteobacteria;Neisseriales;Neisseriaceae;Kingella 1.78 5.59 0.57 (0.38, 0.86) 0.0073 0.1213 Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;nanceiensis 3.54 6.39 0.52 (0.31, 0.87) 0.0126 0.2284 Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;leadbetteri 1.42 3.87 0.51 (0.32, 0.82) 0.0056 0.1386 Negativicutes Selenomonadales;Veillonellaceae;Selenomonas;sputigena 0.55 1.19 0.63 (0.37, 1.06) 0.0837 0.3961 38 1. The association between taxonomic abundance and HNSCC was detected by DESeq function, adjusting for cohorts, matching factors [age at oral sample 39 collection (+/- 1 year), gender, and race (white, other)], smoking status (current, former, never), number of cigarettes per day for ever-smokers, alcohol drinking 40 status (never, ever), grams ethanol per day for ever-drinkers, and HPV-16 status. 41 2. Sequence read counts were normalized by dividing raw counts by DESeq size factors. 42 3. All 254 controls were included. 43 4. FDR-adjusted p value. FDR adjustment was conducted at each taxonomic level (i.e. class, genus) separately. 44

© 2017 American Medical Association. All rights reserved.

Downloaded From: https://jamanetwork.com/ on 09/28/2021 45 Supplementary Table 6. Median counts and fold changes (FC) for the association1 between selected taxa and risk of head and neck 46 squamous cell cancer (HNSCC) according to site of cancer (oral cavity, pharynx, and larynx) in the 2 cohorts Transformed 2 counts Taxon Median Maximum 3 4 5 Class;Order;Family;Genus;Species Case Control FC 95% CI p q Cook’s D Oral cavity (Case n=41) Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium 2.16 3.62 0.61 (0.37, 1.02) 0.0600 0.2769 0.01 Betaproteobacteria 55.66 81.28 0.85 (0.67, 1.07) 0.1721 0.6312 0.42 Betaproteobacteria;Neisseriales 32.60 63.07 0.77 (0.58, 1.03) 0.0806 0.4565 0.39 Betaproteobacteria;Neisseriales;Neisseriaceae;Kingella 4.58 5.59 1.04 (0.64, 1.66) 0.8846 1.0000 2.12 Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;oral_taxon_170 0.00 0.94 3.17 (1.66, 6.08) 0.0005 0.0280 18.9 Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;sicca 0.83 12.40 0.34 (0.17, 0.67) 0.0016 0.0683 23.20 Clostridia;Clostridiales;Peptoniphilaceae;Parvimonas;micra 1.40 1.93 0.29 (0.15, 0.55) 0.0002 0.0155 22.18 Pharynx (Case n=30) Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium 1.93 3.62 0.63 (0.38, 1.06) 0.0830 0.5184 0.01 Betaproteobacteria 36.67 81.28 0.84 (0.62, 1.14) 0.2534 0.6969 0.37 Betaproteobacteria;Neisseriales 20.57 63.07 0.87 (0.62, 1.22) 0.4146 0.8187 0.81 Betaproteobacteria;Neisseriales;Neisseriaceae;Kingella 3.09 5.59 0.54 (0.33, 0.88) 0.0143 0.4044 5.55 Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;oris 5.22 10.27 0.22 (0.12, 0.39) <0.0001 0.0001 6.78 Negativicutes Selenomonadales;Veillonellaceae;Veillonella;denticariosi 0.00 0.92 0.30 (0.15, 0.61) 0.0007 0.0397 0.25 Larynx (Case n=58) Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium 0.00 3.62 0.45 (0.28, 0.73) 0.0011 0.0263 0.02 Betaproteobacteria 19.52 81.28 0.49 (0.34, 0.69) <0.0001 0.0005 0.33 Betaproteobacteria;Neisseriales 15.57 63.07 0.46 (0.31, 0.68) 0.0001 0.0019 0.65 Betaproteobacteria;Neisseriales;Neisseriaceae;Kingella 2.66 5.59 0.38 (0.24, 0.59) <0.0001 0.0007 2.72 Betaproteobacteria;Neisseriales;Neisseriaceae;Kingella;dentificans 0.00 0.55 0.30 (0.17, 0.54) <0.0001 0.0061 2.32 Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria 10.00 46.37 0.47 (0.30, 0.75) 0.0016 0.0263 0.66 Bacilli;Lactobacillaes;Aerococcaceae;Abiotrophia 1.32 9.50 0.50 (0.31, 0.81) 0.0048 0.0604 1.35 Bacilli;Lactobacillales;Streptococcaceae;Streptococcus sanguinis 21.07 48.75 0.47 (0.32, 0.69) 0.0001 0.0106 0.46 Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga 9.36 20.80 0.59 (0.40, 0.86) 0.0065 0.0653 0.32 47 No significant heterogeneity of the associations between the oral taxa and HNSCC risk according to cancer site, using Cochran’s Q statistic (p > 0.05). 48 1. The association between taxonomic abundance and HNSCC was detected by DESeq function, adjusting for cohorts, matching factors [age at oral sample collection (+/- 1 49 year), gender, and race (white, other)], smoking status (current, former, never), number of cigarettes per day for ever-smokers, alcohol drinking status (never, ever), grams 50 ethanol per day for ever-drinkers, and HPV-16 status. 51 2. Sequence read counts were normalized by dividing raw counts by DESeq size factors. 52 3. All 254 controls were included. 53 4. FDR-adjusted p value. FDR adjustment was conducted at each taxonomic level (i.e. class, genus) separately. 54 5. The maximum Cook’s Distance for DESeq model. 55

© 2017 American Medical Association. All rights reserved.

Downloaded From: https://jamanetwork.com/ on 09/28/2021 56 Supplementary Figure 1. Overall oral microbiota structure according to subsequent 57 occurrence of squamous-cell head and neck cancer (SCHNC) and SCHNC of the oral 58 cavity, pharynx, and larynx in a case–control study nested within 2 cohorts. 59 60 Principal coordinate analysis (PCoA) plots grouped by cancer status and cancer site, where the 61 X and Y axes represent the first and second coordinates in the unweighted and weighted 62 UniFrac distance matrices, containing pairwise comparisons of phylogenetic distance between 63 microbial communities of the study participants. SCHNC cases (a-b) and SCHNC at oral cavity, 64 pharynx, and larynx (c-d), and controls were depicted using different colors and shapes. Larger 65 shapes indicate centroids for each group. PERMANOVA models showed no significant 66 differences comparing all SCHNC cases and cases at each site with controls when using 67 unweighted UniFrac distance (p = 0.68, 0.46, 0.81, and 0.75 for all cases, oral cavity, pharynx, 68 and larynx, respectively) and weighted UniFrac distance (p = 0.69, 0.32, 0.32, and 0.80 for all 69 cases, oral cavity, pharynx, and larynx, respectively). Models were adjusted for cohorts, 70 matching factors [age at oral sample collection (+/- 1 year), gender, and race (white, other)], 71 smoking status (current, former, never), number of cigarettes per day for ever-smokers, alcohol 72 drinking status (never, ever), grams ethanol per day for ever-drinkers, and HPV-16 status. 73 74

© 2017 American Medical Association. All rights reserved.

Downloaded From: https://jamanetwork.com/ on 09/28/2021