Abstract
Background
The association between prediagnostic antibody responses to Fusobacterium nucleatum (F. nucleatum) and subsequent risk of colorectal cancer (CRC) is not established.
Methods
We conducted a nested case-control study of 8,126 participants in a consortium of 10 prospective cohorts in the United States.
Results
Higher seroprevalence of any F. nucleatum antibody was observed among non-White participants (51.1%) compared to White participants (31.2%). We did not find any statistically significant association between seropositivity to any of the eight F. nucleatum proteins and CRC risk.
Conclusions
Prediagnostic antibody responses to F. nucleatum proteins were not associated with the risk of CRC.
Impact
Future studies may consider a more specific detection of the immunoglobulin isotypes or focus on examining F. nucleatum in stool or tissue samples.
Keywords: microbiome, Fusobacterium nucleatum, biomarker, epidemiology, colorectal cancer, serology
INTRODUCTION
The presence of Fusobacterium nucleatum (F. nucleatum) in the gut is associated with the development of colorectal cancer (CRC)(1). However, the influence of prediagnostic serologic testing of F. nucleatum on CRC risk remains unknown. We investigated the association of prediagnostic antibody responses to F. nucleatum proteins with CRC risk in a large diverse consortium in the United States. Given the higher burden of F. nucleatum-associated diseases in racial minorities, including periodontal disease and CRC(2,3), we performed detailed subgroup analysis by race/ethnicity.
MATERIALS AND METHODS
The consortium included nested case-control studies with available prediagnostic serum from 10 prospective cohorts(4). Serum samples were sent to the German Cancer Research Center (Heidelberg, Germany) and analyzed for antibodies against eight F. nucleatum (strain ATCC25586) proteins in a 1:1,000 dilution using the multiplex serology method (Supplementary Table 1)(5). Protein selection was based on previous findings describing immunogenicity or localization of the proteins on the outside of the bacterium(6). All samples were tested in a single batch of freshly antigen-loaded beads. Based on 20 daily replicates (one per 96-well plate), the median intra-day coefficient of variation (CV) (range) (i.e., intra-assay) for the F. nucleatum proteins was 18% (17%−25%); the between-day within-week CV was 20% (19%−20%); and the between-day between-week CV (i.e., inter-assay) was 26% (22%−30%). Protein-specific cut-off values for seropositivity were defined as described previously(5). For each case, we selected a matched control (1:1) based on age (± 1 year), sex, race/ethnicity, cohort, and date of blood collection (± 1 month) using incidence density sampling from all cohort members alive and cancer-free at the time of CRC diagnosis of cases.
We used chi-square tests to compare the seroprevalence of F. nucleatum antibodies in controls by race/ethnicity and conditional logistic regression to assess the association of F. nucleatum seropositivity with CRC risk. We examined whether the associations differed according to tumor location, time interval between blood draw and CRC diagnosis, and race/ethnicity.
RESULTS
This study included 4,063 pairs of cases and controls, of which 37.1% were male (Supplementary Table 2). The mean age at baseline was 63.6 years. White, African American, Asian American, and Latino participants represent 75.5%, 9.8%, 7.6%, and 5.2% of the population, respectively. The seroprevalence of F. nucleatum antibodies in controls ranged from 3.6% for FN0253 to 10.9% for FN1449 (Figure 1). In general, higher seroprevalence was observed among non-White participants compared to White participants (any F. nucleatum antibody: 51.1% vs 31.2%).
Figure 1. Seroprevalence of Fusobacterium nucleatum antibodies among controls according to race and ethnicity.
In this multiethnic consortium, we performed chi-square tests to compare the seroprevalence of F. nucleatum antibodies between White control participants (N = 3,067) and control participants of other racial groups (African Americans = 399, Asian Americans = 307, Latinos = 211, other/multiracial/unknown = 79). The seroprevalence was generally higher in non-White participants compared to White participants. Statistically significant comparisons are noted by * in the figure.
We did not find any statistically significant association between seropositivity to any of the F. nucleatum proteins and risk of CRC overall or by anatomic subsite (all P > 0.05) (Table 1). The associations did not differ between cases diagnosed within and beyond five years since blood draw (Supplementary Table 3). No differential association was observed by race/ethnicity (Supplementary Table 4).
Table 1.
Association of seropositivity to Fusobacterium nucleatum proteins with colorectal cancer risk
| Colorectal cancer (Cases: 4063/ Controls: 4063) |
Proximal colon cancer (Cases: 2118/ Controls: 2118) |
Distal colon cancer (Cases: 899/ Controls: 899) |
Rectal cancer (Cases: 621/ Controls: 621) |
||||||
|---|---|---|---|---|---|---|---|---|---|
| Protein | No. of positive cases/controls | OR (95% CI)a | No. of positive cases/controls | OR (95% CI)a | No. of positive cases/controls | OR (95% CI)a | No. of positive cases/controls | OR (95% CI)a | Pheterogeneity by anatomic subsiteb |
| FN0131 | 299/300 | 1.00 (0.84–1.19) | 142/148 | 0.95 (0.74–1.23) | 74/66 | 1.14 (0.80–1.64) | 51/62 | 0.80 (0.54–1.19) | 0.43 |
| FN0253 | 151/148 | 1.02 (0.81–1.29) | 77/70 | 1.11 (0.79–1.54) | 28/39 | 0.71 (0.43–1.16) | 25/26 | 0.96 (0.56–1.67) | 0.34 |
| FN0264 | 200/236 | 0.84 (0.70–1.02) | 101/120 | 0.84 (0.64–1.10) | 56/54 | 1.04 (0.71–1.52) | 27/42 | 0.63 (0.39–1.04) | 0.29 |
| FN1426 | 285/270 | 1.06 (0.89–1.26) | 150/141 | 1.07 (0.84–1.36) | 54/67 | 0.79 (0.54–1.15) | 47/43 | 1.11 (0.71–1.74) | 0.36 |
| FN1449 | 442/443 | 1.00 (0.87–1.15) | 227/227 | 1.00 (0.82–1.22) | 105/111 | 0.94 (0.71–1.24) | 62/63 | 0.98 (0.68–1.43) | 0.94 |
| FN1526 | 165/189 | 0.87 (0.70–1.07) | 90/98 | 0.92 (0.68–1.23) | 38/35 | 1.09 (0.68–1.75) | 26/40 | 0.62 (0.37–1.05) | 0.27 |
| FN1817_1 | 208/247 | 0.83 (0.68–1.00) | 107/121 | 0.87 (0.66–1.15) | 44/48 | 0.90 (0.58–1.41) | 32/48 | 0.65 (0.41–1.03) | 0.51 |
| FN1859 | 144/163 | 0.88 (0.69–1.10) | 66/85 | 0.75 (0.53–1.06) | 40/33 | 1.21 (0.76–1.92) | 17/29 | 0.56 (0.30–1.04) | 0.10 |
Abbreviations: CI, confidence interval; F. nucleatum, Fusobacterium nucleatum; OR, odds ratio.
Conditional logistic regression matched according to age, sex (male, female), race/ethnicity (White, African American, Asian American, Latino, other/multiracial/unknown), and cohort (Cancer Prevention Study-II; Multiethnic Cohort Study; Prostate, Lung, Colorectal, and Ovarian cancer Screening Trial; Physician’s Health Study; Southern Community Cohort Study; Women’s Health Initiative; Nurses’ Health Study; Health Professionals Follow-up Study; New York University Women’s Health Study; Campaign Against Cancer and Stroke).
P for heterogeneity was calculated using a likelihood ratio test, comparing a model that allowed separate associations between seropositivity to F. nucleatum proteins and risk of colorectal cancer of different anatomic locations with a model that assumed a common association.
DISCUSSION
Non-invasive diagnostic methods offer readily accessible and potentially widely available approaches for cancer detection in a large population. Although postdiagnostic serological testing of F. nucleatum has the potential for detecting CRC(7), our results showed that prediagnostic antibody responses to F. nucleatum proteins were not predictive of subsequent CRC risk, consistent with a prior study from the European Prospective Investigation into Nutrition and Cancer (EPIC) cohort(5). Despite a higher seroprevalence in racial minorities in our multi-cohort pooled analysis, the association did not differ by race/ethnicity. There are several possible explanations for the overall null association. First, serology indirectly detects past and current infections independent of site of infection. F. nucleatum is most predominant in the oral cavity(1), making it difficult to differentiate between individuals positive due to F. nucleatum in the colon and those with F. nucleatum-related periodontitis. Second, different isotypes of immunoglobulin may also be differentially related to the presence of CRC. Notably, the secondary antibody applied in our method—multiplex serology—was directed against IgG, IgA, and IgM simultaneously. In a study by Wang et al.(7), CRC patients infected with F. nucleatum produced higher levels of IgA than IgG, suggesting that measuring the IgA class may have higher specificity for CRC. Third, we cannot exclude the possibility that the observed antibody responses partially result from cross-reactivity with other closely related bacteria. Finally, the use of antibiotics in treating F. nucleatum-related infection may also be an explanation as studies have suggested that antibiotics use may be associated with an increased CRC risk(8). One limitation of the study is that we could not formally determine the sensitivity and specificity of the multiplex serology as there is no established gold standard assay available that our data could be compared with.
Our study represents the first effort to prospectively examine serological antibodies to F. nucleatum in relation to CRC risk in a racially diverse consortium. Future studies may consider a more specific detection of the immunoglobulin isotypes or focus on examining F. nucleatum in stool or tissue samples.
Supplementary Material
ACKNOLWEDGEMENTS
The Campaign Against Cancer and Stroke thanks the participants and staff for their contributions, as well as the Maryland Cancer Registry, Center for Cancer Surveillance and Control, Department of Health and Mental Hygiene. The authors would like to thank the participants and staff of the Nurses’ Health Study and the Health Professionals Follow-up Study for their valuable contributions, as well as the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, and WY. The authors assume full responsibility for analyses and interpretation of these data. The American Cancer Society funds the creation, maintenance, and updating of the Cancer Prevention Study-II cohort. The authors express sincere appreciation to all Cancer Prevention Study-II participants, and to each member of the study and biospecimen management group. The authors would like to acknowledge the contribution from central cancer registries supported through the Centers for Disease Control and Prevention’s National Program of Cancer Registries and cancer registries supported by the National Cancer Institute’s Surveillance Epidemiology and End Results Program.
Financial support:
The National Institutes of Health funds: this consortium (R01 CA190428, principal investigator [PI]: M. Epplein); the Southern Community Cohort Study (U01 CA202979, PI: W.J. Blot); the New York University Women’s Health Study (U01 CA182934, Principal Investigator [PI]: A. Zeleniuch-Jaquotte; P30 CA016087, PI: B.G. Neel); the Nurses’ Health Study/Health Professionals Follow-Up Study (U01 CA167552; P01 CA087969; UM1 CA186107; UM1 CA167552); the Physicians’ Health Study (R01 CA097193; R01 CA040360; R01 HL034595); and the Multiethnic Cohort Study (U01 CA164973, PI: L. Le Marchand). R.M. Peek is supported by the National Institutes of Health (R01 DK058587, R01 CA077955). T.L. Cover is supported by the National Institutes of Health (R01 AI039657, R01 AI118932, P01 CA116087) and the Department of Veterans Affairs BX004447. M. Song is supported by the American Cancer Society (MRSG-17-220-01-NEC) and the National Institutes of Health (R00 CA215314). The Women’s Health Initiative program is funded by the National Heart, Lung, and Blood Institute through contracts, HHSN268201600018C, HHSN268201600001C, HHSN268201600002C, HHSN268201600003C, and HHSN268201600004C. The development of Helicobacter pylori multiplex serology was funded in part by the Joint Initiative for Innovation and Research of the German Helmholtz Association. The funding sources played no role in the analysis or interpretation of the data. Study sponsors had no role in the study design or collection, analysis, or interpretation of the data.
Footnotes
Conflict of interest: The authors have no competing interests to declare.
REFERENCES
- 1.Brennan CA, Garrett WS. Fusobacterium nucleatum — symbiont, opportunist and oncobacterium. Nat Rev Microbiol. 2019;17:156–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Borrell LN, Crawford ND. Socioeconomic position indicators and periodontitis: examining the evidence. Periodontology 2000. 2012;58:69–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Jackson CS, Oman M, Patel AM, Vega KJ. Health disparities in colorectal cancer among racial and ethnic minorities in the United States. Journal of Gastrointestinal Oncology. 2016;7:S32–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Butt J, Varga MG, Blot WJ, Teras L, Visvanathan K, Le Marchand L, et al. Serologic response to Helicobacter pylori proteins associated with risk of colorectal cancer among diverse populations in the United States. Gastroenterology. 2019;156:175–186. e2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Butt J, Jenab M, Pawlita M, Overvad K, Tjonneland A, Olsen A, et al. Antibody responses to Fusobacterium nucleatum proteins in prediagnostic blood samples are not associated with risk of developing colorectal cancer. Cancer Epidemiol Biomarkers Prev. 2019;28:1552–5. [DOI] [PubMed] [Google Scholar]
- 6.Kapatral V, Anderson I, Ivanova N, Reznik G, Los T, Lykidis A, et al. Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586. JB. 2002;184:2005–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Wang H-F, Li L-F, Guo S-H, Zeng Q-Y, Ning F, Liu W-L, et al. Evaluation of antibody level against Fusobacterium nucleatum in the serological diagnosis of colorectal cancer. Sci Rep. 2016;6:33440. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Qu G, Sun C, Sharma M, Uy JP, Song EJ, Bhan C, et al. Is antibiotics use really associated with increased risk of colorectal cancer? An updated systematic review and meta-analysis of observational studies. Int J Colorectal Dis. 2020;35:1397–412. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.