Abstract
There is a lack of prospective data on the potential association of Fusobacterium nucleatum (F. nucleatum) and colorectal cancer risk. In this study, we assessed whether antibody responses to F. nucleatum are associated with colorectal cancer risk in prediagnostic serum samples in the European Prospective Investigation into Nutrition and Cancer (EPIC) cohort.
We applied a multiplex serology assay to simultaneously measure antibody responses to 11 F. nucleatum antigens in prediagnostic serum samples from 485 colorectal cancer cases and 485 matched controls. Conditional logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CI).
We observed neither a statistically significant colorectal cancer risk association for antibodies to individual F. nucleatum proteins nor for combined positivity to any of the 11 proteins (OR, 0.81; 95% CI, 0.62–1.06).
Antibody responses to F. nucleatum proteins in prediagnostic serum samples from a subset of colorectal cancer cases and matched controls within the EPIC study were not associated with colorectal cancer risk.
Our findings in prospectively ascertained serum samples contradict the existing literature on the association of F. nucleatum with colorectal cancer risk. Future prospective studies, specifically detecting F. nucleatum in stool or tissue biopsies, are needed to complement our findings.
Introduction
Several studies reported an overabundance of Fusobacterium nucleatum (F. nucleatum) in stool and tumor tissue of patients with colorectal cancer (1, 2). Although mechanistic studies suggest an etiologic role of the bacterium in colorectal carcinogenesis, it remains possible that it is simply an opportunistic passenger within developing tumors (1). To our knowledge, F. nucleatum has never been studied in relation to colorectal cancer risk using prediagnostic biological samples.
Multiplex serology provides an easy-to-apply tool to assess potential infection-associated cancers in large prospective epidemiologic studies that provide sufficient statistical power (3).
In this study, we assessed whether antibody responses to F. nucleatum proteins are associated with colorectal cancer risk in prediagnostic serum samples from 485 colorectal cancer cases and 485 controls in the European Prospective Investigation into Nutrition and Cancer (EPIC) cohort.
Materials and Methods
Study population
The colorectal cancer cases and controls in this nested case–control study are participants within the EPIC study (4). The study was approved by the International Agency for Research on Cancer Ethics Committee (Lyon, France) and the ethics committees of all local EPIC centers, and performed in accordance with the Helsinki Declaration.
This study included prediagnostic serum samples from 485 colorectal cancer cases with a median time between blood draw and diagnosis of 3.4 years (range 0.4–8.5 years; primary tumors coded C18-C20 according to the 10th revision of the International Statistical Classification of Diseases, Injury and Causes of Death). Controls were selected by incidence density sampling from all cohort members alive and cancer-free at the time of matching to cases (1:1) as described previously, including age and sex (5).
F. nucleatum multiplex serology
Serum samples were analyzed for antibodies against F. nucleatum proteins in a 1:1,000 dilution using the previously described multiplex serology method (3). We selected 11 F. nucleatum (strain ATCC25586) proteins as antigens (Table 1). Antigen-specific cut-off values were defined arbitrarily by visual inspection of percentile plots at the approximate inflection point of the curve. Antibody responses to F. nucleatum proteins were combined to overall F. nucleatum sero-positivity by assessing positivity to at least one, and to increase specificity, to at least two or three proteins.
F. nucleatum (strain ATCC25586) antigens and antigen-specific cut-off values (MFI)
| Name . | (Predicted) function . | Protein accession no.a . | Selected region (aa) . | Antigen-specific cutoff (MFI) . |
|---|---|---|---|---|
| Fn0131 | Type Vb secretion system (TpsB) | NP_603038 | 17–566 | 86 |
| Fn0253 | Outer membrane protein | NP_603160 | 37–132 | 30 |
| Fn0264 | Adhesin (FadA) | NP_603171 | 19–129 | 30 |
| Fn0387 | Type Va secretion system | NP_603291 | 1442–1714 | 30 |
| Fn1426 | Type Va secretion system | NP_604320 | 25–374 | 86 |
| Fn1449 | Type Va secretion system (Fap2) | NP_604343 | 2884–3155 | 33 |
| Fn1526 | Type Va secretion system (RadD) | NP_602353 | 1857–2135 | 30 |
| Fn1817_1 | Type Vb secretion system (TpsA) | NP_602617 | 205–276 | 42 |
| Fn1817_2 | Type Vb secretion system (TpsA) | NP_602617 | 839–909 | 112 |
| Fn1859 | Porin (FomA) | NP_602659 | 21–368 | 31 |
| Fn1893 | Type Va secretion system | NP_602692 | 1079–1351 | 30 |
| Name . | (Predicted) function . | Protein accession no.a . | Selected region (aa) . | Antigen-specific cutoff (MFI) . |
|---|---|---|---|---|
| Fn0131 | Type Vb secretion system (TpsB) | NP_603038 | 17–566 | 86 |
| Fn0253 | Outer membrane protein | NP_603160 | 37–132 | 30 |
| Fn0264 | Adhesin (FadA) | NP_603171 | 19–129 | 30 |
| Fn0387 | Type Va secretion system | NP_603291 | 1442–1714 | 30 |
| Fn1426 | Type Va secretion system | NP_604320 | 25–374 | 86 |
| Fn1449 | Type Va secretion system (Fap2) | NP_604343 | 2884–3155 | 33 |
| Fn1526 | Type Va secretion system (RadD) | NP_602353 | 1857–2135 | 30 |
| Fn1817_1 | Type Vb secretion system (TpsA) | NP_602617 | 205–276 | 42 |
| Fn1817_2 | Type Vb secretion system (TpsA) | NP_602617 | 839–909 | 112 |
| Fn1859 | Porin (FomA) | NP_602659 | 21–368 | 31 |
| Fn1893 | Type Va secretion system | NP_602692 | 1079–1351 | 30 |
Abbreviations: aa, amino acid; F. nucleatum, Fusobacterium nucleatum; MFI, median fluorescence intensity.
aNCBI reference sequence.
Statistical analysis
Conditional logistic regression models were applied to compute odds ratios (ORs) and 95% confidence intervals (CI) for the association of antibody responses to F. nucleatum proteins with colorectal cancer risk, overall and by time between blood draw and diagnosis. The following variables were considered as potential confounders and included in the model for adjustment: body mass index (BMI), smoking status, alcohol consumption, and highest education attained at baseline.
All statistical analyses were performed with SAS version 9.4 (SAS Institute). A P value below 0.05 was considered statistically significant.
Availability of data and materials
For information on how to submit an application for gaining access to EPIC data and/or biospecimens, please follow the instructions at http://epic.iarc.fr/access/index.php.
Results
Sero-prevalence to individual F. nucleatum proteins ranged between 3% and 10% among controls (Table 2). Fifty-three percent of controls were positive to any of the proteins; this number was reduced for positivity to at least two (21%) or three proteins (9%). We did not identify a higher sero-prevalence among colorectal cancer cases, neither to individual proteins (range 2%–11%), nor positivity to any (47%) or several proteins (≥2 proteins: 17%; ≥3 proteins: 9%). Thus, there was no significant positive association of antibodies to F. nucleatum proteins with colorectal cancer risk. Sero-positivity to FN0131 was inversely related with colorectal cancer risk (OR, 0.61; 95% CI, 0.38–0.98; P = 0.042; Table 2). However, this result is not significant after Bonferroni multiple-testing correction. Results did not vary for individuals diagnosed within or after more than 2 years of diagnosis (Table 2).
Antibody responses to F. nucleatum proteins and colorectal cancer risk, overall and by time between blood draw and diagnosis; the EPIC study, 1992–2003
| . | All . | ≤2 years follow-up time . | >2–8.5 years follow-up time . | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| . | Positive n (%) . | . | . | Positive n (%) . | . | . | Positive n (%) . | . | . | |||
| . | Controls . | Cases . | . | . | Controls . | Cases . | . | . | Controls . | Cases . | . | . |
| Antigen . | n = 485 . | n = 485 . | ORa (95% CI) . | P . | n = 130 . | n = 130 . | ORa (95% CI) . | P . | n = 355 . | n = 355 . | ORa (95% CI) . | P . |
| Fn0131 | 46 (9) | 31 (6) | 0.61 (0.38–0.98) | 0.042 | 13 (10) | 10 (8) | 0.66 (0.26–1.68) | 0.387 | 33 (9) | 21 (6) | 0.61 (0.34–1.09) | 0.096 |
| Fn0253 | 15 (3) | 10 (2) | 0.64 (0.28–1.45) | 0.283 | 3 (2) | 2 (2) | 0.73 (0.11–4.74) | 0.741 | 12 (3) | 8 (2) | 0.67 (0.27–1.68) | 0.393 |
| Fn0264 | 24 (5) | 32 (7) | 1.33 (0.75–2.34) | 0.331 | 5 (4) | 7 (5) | 1.08 (0.26–4.51) | 0.921 | 19 (5) | 25 (7) | 1.37 (0.73–2.56) | 0.334 |
| Fn0387 | 38 (8) | 42 (9) | 1.05 (0.66–1.67) | 0.847 | 10 (8) | 17 (13) | 1.39 (0.56–3.45) | 0.480 | 28 (8) | 25 (7) | 0.89 (0.50–1.58) | 0.688 |
| Fn1426 | 49 (10) | 53 (11) | 1.06 (0.69–1.63) | 0.777 | 15 (12) | 11 (8) | 0.79 (0.32–1.94) | 0.601 | 34 (10) | 42 (12) | 1.33 (0.80–2.20) | 0.273 |
| Fn1449 | 47 (10) | 41 (8) | 0.80 (0.51–1.27) | 0.347 | 12 (9) | 16 (12) | 1.26 (0.51–3.15) | 0.617 | 35 (10) | 25 (7) | 0.68 (0.39–1.18) | 0.165 |
| Fn1526 | 20 (4) | 15 (3) | 0.74 (0.37–1.49) | 0.401 | 4 (3) | 4 (3) | 0.87 (0.18–4.28) | 0.868 | 16 (5) | 11 (3) | 0.71 (0.32–1.61) | 0.414 |
| Fn1817_1 | 49 (10) | 40 (8) | 0.82 (0.52–1.29) | 0.384 | 17 (13) | 11 (8) | 0.63 (0.27–1.51) | 0.304 | 32 (9) | 29 (8) | 0.97 (0.55–1.70) | 0.907 |
| Fn1817_2 | 48 (10) | 46 (9) | 1.00 (0.66–1.52) | 0.994 | 14 (11) | 12 (9) | 0.87 (0.36–2.14) | 0.768 | 34 (10) | 34 (10) | 1.04 (0.64–1.69) | 0.888 |
| Fn1859 | 46 (9) | 34 (7) | 0.69 (0.43–1.11) | 0.128 | 8 (6) | 7 (5) | 0.80 (0.25–2.61) | 0.715 | 38 (11) | 27 (8) | 0.67 (0.39–1.14) | 0.137 |
| Fn1893 | 47 (10) | 45 (9) | 0.93 (0.60–1.45) | 0.757 | 11 (8) | 15 (12) | 1.14 (0.44–2.96) | 0.792 | 36 (10) | 30 (8) | 0.82 (0.49–1.39) | 0.468 |
| Any protein | 255 (53) | 230 (47) | 0.81 (0.62–1.06) | 0.130 | 71 (55) | 57 (44) | 0.61 (0.35–1.08) | 0.090 | 184 (52) | 173 (49) | 0.91 (0.66–1.24) | 0.546 |
| ≥2 Proteins | 103 (21) | 84 (17) | 0.73 (0.53–1.02) | 0.066 | 27 (21) | 26 (20) | 0.85 (0.44–1.64) | 0.618 | 76 (21) | 58 (16) | 0.72 (0.48–1.08) | 0.112 |
| ≥3 Proteins | 44 (9) | 46 (9) | 0.95 (0.61–1.47) | 0.818 | 10 (8) | 17 (13) | 1.23 (0.49–3.08) | 0.656 | 34 (10) | 29 (8) | 0.84 (0.50–1.42) | 0.523 |
| . | All . | ≤2 years follow-up time . | >2–8.5 years follow-up time . | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| . | Positive n (%) . | . | . | Positive n (%) . | . | . | Positive n (%) . | . | . | |||
| . | Controls . | Cases . | . | . | Controls . | Cases . | . | . | Controls . | Cases . | . | . |
| Antigen . | n = 485 . | n = 485 . | ORa (95% CI) . | P . | n = 130 . | n = 130 . | ORa (95% CI) . | P . | n = 355 . | n = 355 . | ORa (95% CI) . | P . |
| Fn0131 | 46 (9) | 31 (6) | 0.61 (0.38–0.98) | 0.042 | 13 (10) | 10 (8) | 0.66 (0.26–1.68) | 0.387 | 33 (9) | 21 (6) | 0.61 (0.34–1.09) | 0.096 |
| Fn0253 | 15 (3) | 10 (2) | 0.64 (0.28–1.45) | 0.283 | 3 (2) | 2 (2) | 0.73 (0.11–4.74) | 0.741 | 12 (3) | 8 (2) | 0.67 (0.27–1.68) | 0.393 |
| Fn0264 | 24 (5) | 32 (7) | 1.33 (0.75–2.34) | 0.331 | 5 (4) | 7 (5) | 1.08 (0.26–4.51) | 0.921 | 19 (5) | 25 (7) | 1.37 (0.73–2.56) | 0.334 |
| Fn0387 | 38 (8) | 42 (9) | 1.05 (0.66–1.67) | 0.847 | 10 (8) | 17 (13) | 1.39 (0.56–3.45) | 0.480 | 28 (8) | 25 (7) | 0.89 (0.50–1.58) | 0.688 |
| Fn1426 | 49 (10) | 53 (11) | 1.06 (0.69–1.63) | 0.777 | 15 (12) | 11 (8) | 0.79 (0.32–1.94) | 0.601 | 34 (10) | 42 (12) | 1.33 (0.80–2.20) | 0.273 |
| Fn1449 | 47 (10) | 41 (8) | 0.80 (0.51–1.27) | 0.347 | 12 (9) | 16 (12) | 1.26 (0.51–3.15) | 0.617 | 35 (10) | 25 (7) | 0.68 (0.39–1.18) | 0.165 |
| Fn1526 | 20 (4) | 15 (3) | 0.74 (0.37–1.49) | 0.401 | 4 (3) | 4 (3) | 0.87 (0.18–4.28) | 0.868 | 16 (5) | 11 (3) | 0.71 (0.32–1.61) | 0.414 |
| Fn1817_1 | 49 (10) | 40 (8) | 0.82 (0.52–1.29) | 0.384 | 17 (13) | 11 (8) | 0.63 (0.27–1.51) | 0.304 | 32 (9) | 29 (8) | 0.97 (0.55–1.70) | 0.907 |
| Fn1817_2 | 48 (10) | 46 (9) | 1.00 (0.66–1.52) | 0.994 | 14 (11) | 12 (9) | 0.87 (0.36–2.14) | 0.768 | 34 (10) | 34 (10) | 1.04 (0.64–1.69) | 0.888 |
| Fn1859 | 46 (9) | 34 (7) | 0.69 (0.43–1.11) | 0.128 | 8 (6) | 7 (5) | 0.80 (0.25–2.61) | 0.715 | 38 (11) | 27 (8) | 0.67 (0.39–1.14) | 0.137 |
| Fn1893 | 47 (10) | 45 (9) | 0.93 (0.60–1.45) | 0.757 | 11 (8) | 15 (12) | 1.14 (0.44–2.96) | 0.792 | 36 (10) | 30 (8) | 0.82 (0.49–1.39) | 0.468 |
| Any protein | 255 (53) | 230 (47) | 0.81 (0.62–1.06) | 0.130 | 71 (55) | 57 (44) | 0.61 (0.35–1.08) | 0.090 | 184 (52) | 173 (49) | 0.91 (0.66–1.24) | 0.546 |
| ≥2 Proteins | 103 (21) | 84 (17) | 0.73 (0.53–1.02) | 0.066 | 27 (21) | 26 (20) | 0.85 (0.44–1.64) | 0.618 | 76 (21) | 58 (16) | 0.72 (0.48–1.08) | 0.112 |
| ≥3 Proteins | 44 (9) | 46 (9) | 0.95 (0.61–1.47) | 0.818 | 10 (8) | 17 (13) | 1.23 (0.49–3.08) | 0.656 | 34 (10) | 29 (8) | 0.84 (0.50–1.42) | 0.523 |
NOTE: Control subjects were selected by incidence density sampling from all cohort members alive and free of cancer at the time of matching and matched to cases 1:1 by age at blood collection (±6 months–± 2 years), sex, study center, time of the day at blood collection (±2–4 hours interval), fasting status at blood collection (<3/3–6 hours); among women by menopausal status, and among premenopausal women by phase of menstrual cycle, and hormone therapy use at time of blood collection.
aConditional logistic regression model conditioned on the matching factors with multivariable adjustment for BMI, education, smoking, and alcohol intake at baseline; significant associations (P < 0.05) are marked in bold font.
Discussion
In this multi-center prospective study, we found that antibody responses to F. nucleatum proteins were not associated with colorectal cancer risk.
Our findings are discordant to the published literature that suggests a role for F. nucleatum in colorectal cancer development. Our study design varies from previous literature in two important ways. First, previous human studies mostly measured F. nucleatum abundance at the site of interest, that is, in stool or tumor tissue (1), while we apply serology, which is limited by being a systemic measure of past and/or current infection. Thus, it remains possible that the antibody responses resulted from other infection sites and/or cross-reactive antibody responses from infection with other closely related bacteria. Second and a major strength of our study is that we employed a prospective design, in contrast to the case–control designs of previous studies to assess whether F. nucleatum infection increases colorectal cancer risk. However, the natural history of a potential etiologic role of F. nucleatum in colorectal cancer development with respect to timing and molecular pathways is unknown. Longitudinal studies and analyses of molecular colorectal cancer subtypes are needed to address this question in more depth.
In conclusion, antibody responses to F. nucleatum proteins in prediagnostic serum samples of the EPIC study were not associated with an increased risk of developing colorectal cancer. Future prospective studies, specifically detecting F. nucleatum in stool or tissue biopsies, are needed to help clarify whether F. nucleatum plays a role in colorectal tumor development.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Disclaimer
Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy, or views of the International Agency for Research on Cancer/World Health Organization. The funding sources had no influence on the design of the study; the collection, analysis, and interpretation of data; the writing of the article; or the decision to submit the paper for publication.
Authors' Contributions
Conception and design: J. Butt, M. Jenab, D.J. Hughes, M. Pawlita, K. Overvad, A. Tjonneland, H. Boeing, R. Tumino, B. Bueno-de-Mesquita, E. Weiderpass, M.-J. Sánchez, E. Ardanaz
Development of methodology: J. Butt, M. Jenab, D.J. Hughes, M. Pawlita, K. Overvad, B. Bueno-de-Mesquita, E. Ardanaz, T. Waterboer
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Butt, M. Jenab, D.J. Hughes, M. Pawlita, K. Overvad, A. Tjonneland, M.-C. Boutron-Ruault, F. Carbonnel, R. Kaaks, T. Kühn, H. Boeing, A. Trichopoulou, A. Karakatsani, D. Palli, V.M. Pala, R. Tumino, C. Sacerdote, S. Panico, B. Bueno-de-Mesquita, R.C.H. Vermeulen, J.R. Quirós, M.-J. Sánchez, E. Ardanaz, B. Van Guelpen, M.J. Gunter, T. Waterboer
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Butt, M. Jenab, D.J. Hughes, M. Pawlita, R.C.H. Vermeulen, E. Weiderpass, E. Ardanaz, B. Van Guelpen, M.J. Gunter, H. Freisling
Writing, review, and/or revision of the manuscript: J. Butt, M. Jenab, D.J. Hughes, M. Pawlita, K. Overvad, A. Tjonneland, A. Olsen, M.-C. Boutron-Ruault, F.R. Mancini, T. Kühn, H. Boeing, A. Trichopoulou, A. Karakatsani, D. Palli, V.M. Pala, R. Tumino, C. Sacerdote, S. Panico, B. Bueno-de-Mesquita, C.H. van Gils, R.C.H. Vermeulen, E. Weiderpass, J.R. Quirós, E.J. Duell, M.-J. Sánchez, J.M. Huerta, M. Dorronsoro, E. Ardanaz, B. Van Guelpen, S. Harlid, A. Perez-Cornago, M.J. Gunter, N. Murphy, H. Freisling, D. Aune, T. Waterboer
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Butt, M. Jenab, D.J. Hughes, R. Kaaks, H. Boeing, R. Tumino, C. Sacerdote, C.H. van Gils, J.R. Quirós, E. Ardanaz
Study supervision: J. Butt, M. Jenab, D.J. Hughes, M. Pawlita
Acknowledgments
We thank Ute Koch, Monika Oppenländer, and Claudia Brandel for excellent technical assistance with the serological measurements. D.J. Hughes was supported by the Health Research Board of Ireland project grants HRA_PHS/2013/397 and HRA_PHS/2015/1142. The coordination of EPIC is financially supported by the European Commission (DG-SANCO) and the International Agency for Research on Cancer. The national cohorts are supported by the Danish Cancer Society (Denmark); Ligue Contre le Cancer, Institut Gustave Roussy, Mutuelle Générale de l'Education Nationale, and Institut National de la Santé et de la Recherche Médicale (France); Deutsche Krebshilfe, Deutsches Krebsforschungszentrum, and Federal Ministry of Education and Research (Germany); the Hellenic Health Foundation (Greece); the Sicilian Government, AIRE ONLUS Ragusa, AVIS Ragusa, Associazione Italiana per la Ricerca sul Cancro-AIRC-Italy, and National Research Council (Italy); Dutch Ministry of Public Health, Welfare and Sports (VWS), Netherlands Cancer Registry (NKR), LK Research Funds, Dutch Prevention Funds, Dutch ZON (Zorg Onderzoek Nederland), World Cancer Research Fund (WCRF), and Statistics Netherlands (the Netherlands); Nordic Centre of Excellence programme on Food, Nutrition and Health. (Norway); Health Research Fund (FIS), PI13/00061 to Universidad de Granada (Granada, Spain), Regional Governments of Andalucía, Asturias, Basque Country, Murcia (no. 6236) and Navarra, Regional Government of Asturias (Asturias, Spain), and ISCIII RETIC (RD06/0020; Spain); Swedish Cancer Society, Swedish Scientific Council, and County Councils of Skåne and Västerbotten (Sweden); Cancer Research UK (14136 to EPIC-Norfolk; C570/A16491 to EPIC-Oxford), Medical Research Council [1000143 to EPIC-Norfolk, MR/M012190/1 to EPIC-Oxford (United Kingdom)].
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