Fusobacterium nucleatum (Fn) is a gram-negative oral anaerobe and prevalent in colorectal cancer. Fn encodes a unique amyloid-like adhesin, FadA complex (FadAc), consisting of intact pre-FadA and cleaved mature FadA, to promote colorectal cancer tumorigenesis. We aimed to evaluate circulating anti-FadAc antibody levels as a biomarker for colorectal cancer. Circulating anti-FadAc IgA and IgG levels were measured by ELISA in two study populations. In study 1, plasma samples from patients with colorectal cancer (n = 25) and matched healthy controls (n = 25) were obtained from University Hospitals Cleveland Medical Center. Plasma levels of anti-FadAc IgA were significantly increased in patients with colorectal cancer (mean ± SD: 1.48 ± 1.07 μg/mL) compared with matched healthy controls (0.71 ± 0.36 μg/mL; P = 0.001). The increase was significant in both early (stages I and II) and advanced (stages III and IV) colorectal cancer. In study 2, sera from patients with colorectal cancer (n = 50) and patients with advanced colorectal adenomas (n = 50) were obtained from the Weill Cornell Medical Center biobank. Anti-FadAc antibody titers were stratified according to the tumor stage and location. Similar as study 1, serum levels of anti-FadAc IgA were significantly increased in patients with colorectal cancer (2.06 ± 1.47 μg/mL) compared with patients with colorectal adenomas (1.49 ± 0.99 μg/mL; P = 0.025). Significant increase was limited to proximal cancers, but not distal tumors. Anti-FadAc IgG was not increased in either study population, suggesting that Fn likely translocates through the gastrointestinal tract and interact with colonic mucosa. Anti-FadAc IgA, but not IgG, is a potential biomarker for early detection of colorectal neoplasia, especially for proximal tumors.

Significance:

Fn, an oral anaerobe highly prevalent in colorectal cancer, secretes the amyloid-like FadAc to promote colorectal cancer tumorigenesis. We report that circulating levels of anti-FadAc IgA, but not IgG, are increased in patients with both early and advanced colorectal cancer compared with the healthy controls, and especially in those with proximal colorectal cancer. Anti-FadAc IgA may be developed into a serological biomarker for early detection of colorectal cancer.

Colorectal cancer is the third-most common cancer worldwide and the fourth leading cause of cancer death (1). Many genetic and environmental risk factors have been identified for colorectal cancer such as genomic instability, gene mutations, Western diet, lifestyle, and obesity (2, 3). In addition, accumulating studies have now recognized dysbiosis of gut microbiome as a risk factor in the development and progression of colorectal cancer (4–6). Gut microbiome contributes to colorectal carcinogenesis through several mechanisms: (i) by directly inducing carcinogenesis using their virulence factors or metabolites (7–11), (ii) by altering host immune-surveillance systems (12–15), and (iii) by affecting the efficacy of anticancer therapies such as chemotherapy and immunotherapy (16–19).

Among the cancer-associated bacteria, Fusobacterium nucleatum (Fn), a gram-negative oral commensal anaerobe, is considered a key pathogen implicated in colorectal cancer (20, 21). The Fn level is not only significantly higher in tumor tissues compared with the normal controls (22–24), it is also highly correlated with lower survival rate (25, 26), advanced cancer stages (27–29), proximal location (6, 30–32), metastasis (33, 34), and recurrence (16, 35). In addition, increased level of Fn has been reported to be associated with microsatellite instability, CpG island methylator phenotype, and oncogenic mutations (26, 36).

FadA is a unique adhesin highly conserved among pathogenic Fusobacterium species and a major virulence factor responsible for bacterial adhesion, induction of inflammation, and colorectal cancer cell proliferation (7, 11). The fadA gene levels are 10- to 100-fold higher in colorectal tissues of patients with adenomas or colorectal cancer compared with the normal subjects (7). It is also significantly increased in the fecal microbiome in patients with colorectal cancer compared with the controls (21). FadA consists of two forms, the intact pre-FadA and mature FadA without signal peptide, both of which constitute the active and amyloid-like FadA complex (FadAc; ref. 37). The fadA mRNA is constitutively expressed (38); however, amyloid FadAc is only produced under stress and disease conditions, serving as a molecular switch to convert Fn from a benign commensal to a pathogen (39). Amyloid FadAc binds to extracellular domain of E-cadherin leading to the proliferation of cancer cells via activation of Annexin A1 and Wnt/β-catenin signaling (7, 11).

Early diagnosis and treatment of colorectal cancer is the most important determining factor for prognosis and patient survival. Although there are several commercially available serologic biomarkers for colorectal cancer such as carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), and cancer antigen (CA) 19-9 (40–42), it has become clear that these conventional biomarkers have low sensitivity and specificity and are not suited for early diagnosis and predicting prognosis of colorectal cancer (43–45). Because of the high correlation between Fn and colorectal cancer progression, there have been attempts to use Fn as a biomarker. In this study, we investigate the levels of circulating anti-FadA antibodies in the plasma and serum from two U.S. cohorts to evaluate its applicability as a novel colorectal cancer biomarker.

Sample Description

Samples from two existing colorectal cancer sources were used following appropriate ethical guidelines. Study 1 samples were randomly selected from the Kentucky Colon Cancer Genetic Epidemiology Study, a population-base case–control study based on the Kentucky Cancer Registry (KCR; refs. 46, 47). Recruitment of participants was conducted between April 2003 and December 2010. A total of 1,040 incident colon cancer cases and 1,750 population-based controls completed the study with the collection of comprehensive lifestyle and epidemiological data, pathology information, and fasting blood samples. Cases were defined as individuals diagnosed with histopathologically confirmed incident primary colon cancer (excluding patients with rectal or syndromic cancers) who were invited to participate in the study within 3 months of KCR registration. Cases were eligible to participate if they: (i) had a nonrecurrent diagnosis; (ii) had no known family history or diagnosis of familial adenomatous polyposis (FAP), hereditary nonpolyposis colorectal cancer (HNPCC), Peutz-Jeghers, or Cowden disease; (iii) had no known diagnosis of inflammatory bowel disease such as Crohn disease or ulcerative colitis; (iv) were at least 21 years of age at the time of diagnosis; (v) had contact information listed in the KCR database; (vi) were willing to complete two questionnaires. The majority of participants completed data collection within 12 months (median of 5 months) of their colon cancer diagnosis.

Random digit dialing and friend referrals were utilized to recruit controls representative of the general Kentucky population. Controls consisted of frequency-matched individuals who have never been diagnosed with any cancer except nonmelanoma skin cancer and are over the age of 30, preferably ≥ 50 years old. For cases and controls, self-reported inflammatory bowel disease (e.g., Crohn disease or ulcerative colitis), family history of FAP, and HNPCC were excluded in the recruitment. The response rate for cases and controls who answered the phone and allowed eligibility determination was 70.8% and 66.7%, respectively. The study was approved by the Institutional Review Boards of the University of Kentucky (Lexington, Kentucky) and University of Virginia (Charlottesville, VA). All participants provided written informed consent.

Eligible cases and controls donated one blood sample and answered self-administered questionnaires. A two-step approach was used to collect blood samples and lifestyle risk factor data. First, a prepacked phlebotomy kit with detailed written instructions for blood sample collection and written consent forms was sent to each case subject. Participants were instructed to go to their physician offices or adjacent medical facilities for blood draw after overnight fasting. The samples were collected in purple-top (K3EDTA) blood collection tubes and shipped overnight on frozen ice pack. Upon receipt, the blood tubes were spun for 15 minutes at 600 × g and aliquots of plasma and concentrated buffy coat were prepared and frozen at −80°C. Second, a self-administered lifestyle risk factor questionnaire developed by the NCI Colon Cancer Familial Cancer Registry was sent to each participant to collect detailed information on demographics and lifestyle risk factors. The parent Kentucky Colon Cancer Genetic Epidemiology Study is not a matched (or paired) case–control study. Deidentified plasma samples from 25 randomly selected patients with colorectal cancer and 25 controls matched by gender and age (±1 year) were tested. The patient characteristics are summarized in Table 1.

Study 2 included randomly selected patients with advanced adenoma or adenocarcinoma who underwent surgical treatment at NYP/Weill Cornell Medical Center from 2013 to 2015. All patients who were seen in the colorectal group were offered participation in the clinical trial. Tissues were collected among those patients who provided consent, and tissue acquisition was feasible. An advanced adenoma was defined as a polyp that was greater than 3 cm in size, and was unable to be removed endoscopically. Deidentified serum samples from 50 patients with adenoma and 50 patients with adenocarcinoma were tested. Tumor stages were classified according to the Union for International Cancer Control classification. The patient characteristics are summarized in Table 1. For tumor locations in study 2, “proximal” include samples labeled as cecum, ascending, ascending/transverse, right colon, or right/cecum, while “distal” include samples labeled as sigmoid, descending, sigmoid/rectum, left colon, rectosigmoid junction, rectum. Samples from patients with mixed proximal and distal tumors were excluded. No tumor location was available for study 1. The study was approved by the Institutional Review Boards of the Weill Cornell Medical Center. All participants provided written informed consent.

TABLE 1

Patient characteristics

Study 1 (Kentucky)Normal (N = 25)CRC (N = 25)P
Gender Male
Female 
12 (48%)
13 (52%) 
12 (48%)
13 (52%) 
1.0 
Age Mean (yrs)
No. <65 yrs
No. >65 yrs 
59.7 ± 9.9
16 (64%)
9 (36%) 
59.6 ± 9.2
16 (64%)
9 (36%) 
0.953
1.0 
Tumor stage I+II
III+IV 

— 
11 (44%)
14 (56%) 
 
Study 2 (NYP/Cornell) Adenomas (N = 50) CRC (N = 50) P 
Gender Male
Female 
31 (62%)
19 (38%) 
18 (36%)
32 (64%) 
0.009 

Age 
Mean (yrs)
No. <65
No. >65 
64.9 ± 12.6
26 (52%)
24 (48%) 
67.6 ± 12.4
23 (46%)
27 (54%) 
0.291
0.548 
Tumor stage I+II
III+IV 

— 
30 (60%)
19 (38%) 
 
Tumor locationa Proximal
Distal
Other 
17 (34%)
11 (22%)
22 (44%) 
23 (46%)
22 (44%)
5 (10%) 
<0.001 
Study 1 (Kentucky)Normal (N = 25)CRC (N = 25)P
Gender Male
Female 
12 (48%)
13 (52%) 
12 (48%)
13 (52%) 
1.0 
Age Mean (yrs)
No. <65 yrs
No. >65 yrs 
59.7 ± 9.9
16 (64%)
9 (36%) 
59.6 ± 9.2
16 (64%)
9 (36%) 
0.953
1.0 
Tumor stage I+II
III+IV 

— 
11 (44%)
14 (56%) 
 
Study 2 (NYP/Cornell) Adenomas (N = 50) CRC (N = 50) P 
Gender Male
Female 
31 (62%)
19 (38%) 
18 (36%)
32 (64%) 
0.009 

Age 
Mean (yrs)
No. <65
No. >65 
64.9 ± 12.6
26 (52%)
24 (48%) 
67.6 ± 12.4
23 (46%)
27 (54%) 
0.291
0.548 
Tumor stage I+II
III+IV 

— 
30 (60%)
19 (38%) 
 
Tumor locationa Proximal
Distal
Other 
17 (34%)
11 (22%)
22 (44%) 
23 (46%)
22 (44%)
5 (10%) 
<0.001 

Abbreviations: CRC, colorectal cancer; yrs, years.

aProximal includes patients with tumors in cecum, ascending and transverse colon; Distal includes patients with tumors in descending and sigmoid colon, and rectum; Other includes patients with tumors of unclear location or in both proximal and distal colons.

Purification of FadAc

The recombinant FadAc protein was prepared from Escherichia coli (E. coli) BL21 (DE3) carrying pYWH471-6 as described previously (37). E. coli BL21 (DE3) carrying pYWH471-6 was grown in Luria-Bertani broth containing 100 μg/mL ampicillin to mid-log phase followed by incubation with 0.1 mmol/L Isopropyl β-D-1-thiogalactopyranoside (Sigma-Aldrich) for 2.5 hours at 37°C to induce expression of the recombinant FadAc protein. The bacteria were harvested by centrifugation at 8,000 g for 5 minutes, the pellet (∼6 g) was incubated with 50 mL of buffer A (50 mmol/L NaH2PO4, 300 mmol/L NaCl, 8 mol/L Urea, pH 8.0) at 4°C overnight. After removing cells, debris, and insoluble material by centrifugation, the clear lysate was incubated with 5 mL TALON Metal Affinity Resins (Clontech Laboratories, Inc.) for 4 hours at 4°C. The mixture was transferred to a glass chromatography column (3 × 15 cm) and unbound materials were washed out with 150 mL of buffer A. The column was then eluted with 30 mL of buffer B (50 mmol/L NaH2PO4, 300 mmol/L NaCl, 8 mol/L Urea, pH 5.0) and the elute was serially collected in 3 mL aliquots. The column fraction containing recombinant FadAc were pooled and dialyzed against PBS (pH 7.2) in a dialysis tubing with MWCO of 6–8 kDa (Spectra/Por1; Spectrum Laboratories, Inc.). The concentration of purified recombinant FadA was measured by BCA protein assay kit (Thermo Fisher Scientific).

Preparation of Monoclonal Anti-FadA IgG

The mouse monoclonal (mAb) anti-FadA IgG (5G113G8) was prepared at the Hybridoma Core (Lerner Research Institute) described previously (37) and was used as a standard for determining the concentrations of anti-FadAc IgA and IgG in the samples. This approach is feasible because FadA is highly conserved in Fn (48).

ELISA

The levels of anti-FadAc IgA and IgG were determined by indirect ELISA. The 96-well ELISA plates were coated with 100 μL of purified recombinant FadAc (2 μg/mL) in 0.2 mol/L carbonate/bicarbonate buffer (pH 9.4) and incubated at 4°C overnight. After blocking, the wells were incubated with 100 μL of blood samples serially diluted in blocking buffer for 1 hour at room temperature. To obtain a standard curve, the wells were incubated with 100 μL of purified mouse mAb 5G113G8 at increasing concentrations. Each well was then incubated with 100 μL of goat anti-human IgA-HRP (1:6,000 dilution; PA1-74395, Thermo Fisher Scientific), goat-anti-human IgG-HRP (1:10,000 dilution; A2290, Sigma-Aldrich), or goat anti-mouse IgG-HRP (1:12,000 dilution; 62-6520, Thermo Fisher Scientific) for 1 hour at room temperature, followed by incubation with 100 μL substrate solution (1-Step Ultra TMB-ELISA; Thermo Fisher Scientific) for 30 minutes. The reaction was terminated with 2 mol/L H2SO4 and the absorbance at 405 nm was measured using an automated microplate reader. The levels of anti-FadAc IgA and IgG were determined by testing serial dilutions of the plasma or serum. The values that fell into the linear range of the standard curves generated using mouse mAb 5G113G8 were used to calculate the antibody concentrations. Wells with secondary antibodies alone were used to determine background values. All measurements were performed in duplicate. All assays were performed by one single individual. Intraplate and interplate coefficient of variation (CV) in study 1 and study 2 were as follow: study 1 intraplate CV = 8.2 ± 6.12%, interplate CV = 9.5 ± 0.6%; study 2 intraplate CV = 9.7 ± 6.7%, interplate CV = 10.8 ± 1.7%.

Statistical Analysis

Categorical variables were presented as numbers and percentages and were compared using χ2 test or Fisher exact test between two groups. Continuous variables were expressed as mean ± SD and were compared using two-tailed Student t test between two groups. The optimal cutoffs of plasma and serum levels of anti-FadAc IgA and IgG were identified with ROC curve analysis using Youden index criterion with the requirement that sensitivity and specificity were at least 0.50. To examine the effect of colorectal cancer status in relation to the levels of anti-FadAc IgA and IgG, a multivariable linear regression model was used to adjust for confounding factor(s) (e.g., gender) with P value <0.1 in univariable analysis, with the status being coded as binary dummy variables. Study 1 was matched for sex and age, but not study 2. Therefore, the adjustment of gender and age was only considered for study 2. Tumor stage and location were classified into two groups. Analysis with nonparametric methods Wilcoxon rank-sum test and Kruskal–Wallis test yielded similar results. The results with P value <0.05 were considered significant. The analysis was carried out with SAS software version 9.4 (SAS Institute Inc.) and figures were generated using Excel. Additional analyses are available in Supplementary Appendix.

Data Availability Statement

Data are available upon request.

Plasma Levels of Anti-FadAc IgA, but not IgG, are Significantly Increased in Patients with Colorectal Cancer Compared with Healthy Controls

In study 1, the plasma levels of anti-FadAc IgA were significantly increased in patients with colorectal cancer compared with healthy subjects (1.48 ± 1.07 μg/mL vs. 0.71 ± 0.36 μg/mL, P = 0.001; Fig. 1A). The Youden index criterion in the ROC curve analysis yielded the cutoff of 0.81 μg/mL for anti-FadAc IgA levels, with 76% sensitivity and 76% specificity. No difference in anti-FadAc IgG was detected between the two groups (1.20 ± 1.26 μg/mL vs. 1.33 ± 1.07 μg/mL, P = 0.71; Fig. 1B). Furthermore, when the IgA titers were stratified by cancer stage, significant increase was detected in both early (stages I and II) and advanced (stages III and IV) colorectal cancer, compared with the normal controls (Fig. 1C). No tumor location was available for study 1.

FIGURE 1

Comparison of anti-FadAc IgA or anti-FadAc IgG levels in plasma from normal subjects and patients with colorectal cancer in study 1. Anti-FadAc IgA (A) and anti-FadAc IgG (B) levels in plasma samples from 25 normal subjects (N) and 25 patients with colorectal cancer (Ca). C, Plasma anti-FadAc IgA levels in normal subject (N), patient with early stages of colorectal cancer (I+II), and patient with advanced stages of colorectal cancer (III+IV). Each symbol represents one human subject. Horizontal lines indicate median values, boxes show the 25th–75th percentiles, and whiskers show the minimal and maximal individual values. **, P < 0.01; ***, P < 0.001.

FIGURE 1

Comparison of anti-FadAc IgA or anti-FadAc IgG levels in plasma from normal subjects and patients with colorectal cancer in study 1. Anti-FadAc IgA (A) and anti-FadAc IgG (B) levels in plasma samples from 25 normal subjects (N) and 25 patients with colorectal cancer (Ca). C, Plasma anti-FadAc IgA levels in normal subject (N), patient with early stages of colorectal cancer (I+II), and patient with advanced stages of colorectal cancer (III+IV). Each symbol represents one human subject. Horizontal lines indicate median values, boxes show the 25th–75th percentiles, and whiskers show the minimal and maximal individual values. **, P < 0.01; ***, P < 0.001.

Close modal

Serum Levels of Anti-FadAc IgA, But not IgG, are Significantly Increased in Patients with Colorectal Cancer Compared with Advanced Precancerous Polyps

Similarly, serum anti-FadAc IgA titers from study 2 were also found to be significantly increased in patients with colorectal cancer compared with those with advanced adenomas (2.06 ± 1.47 μg/mL vs. 1.49 ± 0.99 μg/mL, P = 0.025; Fig. 2A). There was a significant gender difference between the adenoma and colorectal cancer groups in study 2 (Table 1). After adjusting for gender, anti-FadAc IgA levels remain significantly higher in colorectal cancer compared with the advanced adenoma group (estimate = 0.592 μg/mL, SE = 0.262, P = 0.026). No difference was detected in anti-FadAc IgG levels (4.87 ± 4.46 μg/mL vs. 7.04 ± 10.7 μg/mL, P = 0.190; Fig. 2B).

FIGURE 2

Comparison of anti-FadAc IgA or anti-FadAc IgG levels in serum from patients with advanced adenomas and colorectal cancer in study 2. Anti-FadAc IgA (A) and anti-FadAc IgG (B) levels in serum samples were obtained from 50 patients each with advanced adenomas (Ade) or colorectal cancer (Ca). C, Serum anti-FadAc IgA levels in patient with advanced adenomas (Ade), early stages of colorectal cancer (I+II), and advanced stages of colorectal cancer (III+IV). D, Serum anti-FadAc IgA levels in patients with different tumor stages [advanced adenoma (Ade), early colorectal cancer (I+II), advanced colorectal cancer (III+IV)] and tumor locations. Tumor locations were categorized into proximal (cecum, ascending, right colon) and distal (sigmoid, rectum, descending, left colon, rectosigmoid junction). Patients with tumors of unclear location or in both proximal and distal colons were excluded. Each symbol represents one human subject. Horizontal lines indicate median values, boxes show the 25th–75th percentiles, and whiskers show the minimal and maximal individual values. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIGURE 2

Comparison of anti-FadAc IgA or anti-FadAc IgG levels in serum from patients with advanced adenomas and colorectal cancer in study 2. Anti-FadAc IgA (A) and anti-FadAc IgG (B) levels in serum samples were obtained from 50 patients each with advanced adenomas (Ade) or colorectal cancer (Ca). C, Serum anti-FadAc IgA levels in patient with advanced adenomas (Ade), early stages of colorectal cancer (I+II), and advanced stages of colorectal cancer (III+IV). D, Serum anti-FadAc IgA levels in patients with different tumor stages [advanced adenoma (Ade), early colorectal cancer (I+II), advanced colorectal cancer (III+IV)] and tumor locations. Tumor locations were categorized into proximal (cecum, ascending, right colon) and distal (sigmoid, rectum, descending, left colon, rectosigmoid junction). Patients with tumors of unclear location or in both proximal and distal colons were excluded. Each symbol represents one human subject. Horizontal lines indicate median values, boxes show the 25th–75th percentiles, and whiskers show the minimal and maximal individual values. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Close modal

When the results were stratified with tumor stage and location, the anti-FadAc IgA levels were only increased in advanced colorectal cancer compared to the advanced adenoma group (Fig. 2C). However, for patients with proximal tumors, the IgA levels were significantly increased in both early and advanced stages of colorectal cancer compared with the advanced adenoma group (Fig. 2D).

In this investigation, we found that levels of circulating anti-FadAc IgA were significantly increased in patients with colorectal cancer compared with healthy controls or to patient with advanced adenomas. The anti-FadAc IgA levels were elevated in both early and advanced colorectal cancer suggesting that it may be useful for not only advanced-stage but also early-stage diagnosis. This is consistent with our previous finding that the fadA genes levels are increased stepwise from normal to adenomas, and from adenomas to carcinomas (7). The adenoma group in study 2 exhibited higher titers than the normal subjects of cohort 1. However, because different specimens (plasma vs. serum) were used for these two studies, the results are not directly comparable. The elevated anti-FadAc IgA is closely associated with proximal tumors, consistent with previous reports of enriched Fn in proximal colorectal cancer (30, 31). Interestingly, we did not observe changes in IgG levels. These findings suggest that Fn likely translocates through the digestive tract inducing mucosal immune responses.

Serologic diagnosis is desirable due to the noninvasive nature, as well as easy attainment of the specimens. Previous studies reported colorectal cancer diagnosis using serum CEA, AFP, and CA19-9, achieving sensitivities of 80.43%, 73.91%, and 69.57%, and specificities of 75.00%, 69.44%, and 61.11%, respectively (41). Using anti-FadAc IgA, we achieved sensitivity and specificity both at 76%, demonstrating its superiority over AFP and CA 19-9 as potential markers. Using whole bacteria Fn as antigen, a previous study tested anti-Fn IgA in colorectal cancer, with a low sensitivity of 36.43%. Even when combined with CEA, the sensitivity was still only 53.10% (49). This may be due to the presence of common bacterial components conserved among most bacterial species such as outer membrane proteins and lipopolysaccharides making it difficult to predict colorectal cancer. Given that FadA is uniquely conserved in Fn, anti-FadAc antibody is a more specific biomarker. Furthermore, we used the colorectal cancer–promoting form of FadA, that is, the amyloid FadAc (39), thus achieving significantly improved sensitivity. We used the Youden index with the requirement of both sensitivity and specificity greater than 0.5 for the selection of the cut-off points. By doing so, it guarantees the cutoff yields a better approach than a random decision with a fair coin toss for potential clinical diagnosis. We anticipate that combining circulating anti-FadAc IgA with additional biomarkers may further improve the diagnostic sensitivity and specificity.

Our study had a few limitations. First, our sample size was limited, especially for those with clearly defined tumor locations. Future studies with larger sample sizes and external validations are warranted to confirm the findings, and to compare with the above-mentioned serologic parameters to stratify to additional patient and tumor characteristics, such as smoking, tumor histology, and molecular types. Second, as a proof-of-concept study, we performed retrospective analysis using samples available to us. As a result, different specimens were used in the two studies, one with plasma and the other with serum, rendering the results from the two studies incomparable. There was no adenoma group in study 1 and no healthy controls in study 2; therefore, we do not know whether anti-FadAc IgA can also be used for early detection of advanced precancerous adenomas. Third, given the retrospective nature of study, we cannot dismiss the possibility of reverse causality. As such, caution must be exercised for causal interpretation of our results. Prospective cohort study is warranted.

In summary, our study sheds novel light on using specific bacterial virulence factors as biomarkers for detection of colorectal neoplasia. Anti-FadAc IgA is a potential novel biomarker for colorectal cancer, especially for the tumors located in the proximal colon.

Y.W. Han reports a patent to detect serum anti-FadA antibodies and related diagnostic methods, U.S. Patent No. 11, 506, 661. No disclosures were reported by the other authors.

J.E. Baik: Data curation, writing-original draft. L. Li: Resources, writing-review and editing. M.A. Shah: Resources, writing-review and editing. D.E. Freedberg: Writing-review and editing. Z. Jin: Formal analysis, writing-original draft. T.C. Wang: Resources, writing-review and editing. Y.W. Han: Conceptualization, supervision, funding acquisition, writing-original draft, writing-review and editing.

This study was supported in part by NIH grants R01CA192111 and R01DE029532 to Y.W. Han.

Note: Supplementary data for this article are available at Cancer Research Communications Online (https://aacrjournals.org/cancerrescommun/).

1.
Shang
FM
,
Liu
HL
.
Fusobacterium nucleatum and colorectal cancer: a review
.
World J Gastrointest Oncol
2018
;
10
:
71
81
.
2.
Grady
WM
,
Carethers
JM
.
Genomic and epigenetic instability in colorectal cancer pathogenesis
.
Gastroenterology
2008
;
135
:
1079
99
.
3.
Alsheridah
N
,
Akhtar
S
.
Diet, obesity and colorectal carcinoma risk: results from a national cancer registry-based middle-eastern study
.
BMC Cancer
2018
;
18
:
1227
.
4.
Garrett
WS
.
Cancer and the microbiota
.
Science
2015
;
348
:
80
6
.
5.
Sears
CL
,
Garrett
WS
.
Microbes, microbiota, and colon cancer
.
Cell Host Microbe
2014
;
15
:
317
28
.
6.
Dejea
CM
,
Wick
EC
,
Hechenbleikner
EM
,
White
JR
,
Welch
JLM
,
Rossetti
BJ
, et al
.
Microbiota organization is a distinct feature of proximal colorectal cancers
.
Proc Natl Acad Sci U S A
2014
;
111
:
18321
6
.
7.
Rubinstein
MR
,
Wang
X
,
Liu
W
,
Hao
Y
,
Cai
G
,
Han
YW
.
Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/β-catenin signaling via its FadA adhesin
.
Cell Host Microbe
2013
;
14
:
195
206
.
8.
Prorok-Hamon
M
,
Friswell
MK
,
Alswied
A
,
Roberts
CL
,
Song
F
,
Flanagan
PK
, et al
.
Colonic mucosa-associated diffusely adherent afaC+ Escherichia coli expressing lpfA and pks are increased in inflammatory bowel disease and colon cancer
.
Gut
2014
;
63
:
761
70
.
9.
Nougayrede
JP
,
Homburg
S
,
Taieb
F
,
Boury
M
,
Brzuszkiewicz
E
,
Gottschalk
G
, et al
.
Escherichia coli induces DNA double-strand breaks in eukaryotic cells
.
Science
2006
;
313
:
848
51
.
10.
Abed
J
,
Emgard
JE
,
Zamir
G
,
Faroja
M
,
Almogy
G
,
Grenov
A
, et al
.
Fap2 mediates fusobacterium nucleatum colorectal adenocarcinoma enrichment by binding to tumor-expressed Gal-GalNAc
.
Cell Host Microbe
2016
;
20
:
215
25
.
11.
Rubinstein
MR
,
Baik
JE
,
Lagana
SM
,
Han
RP
,
Raab
WJ
,
Sahoo
D
, et al
.
Fusobacterium nucleatum promotes colorectal cancer by inducing Wnt/beta-catenin modulator Annexin A1
.
EMBO Rep
2019
;
20
:
e47638
.
12.
Wu
S
,
Rhee
KJ
,
Albesiano
E
,
Rabizadeh
S
,
Wu
X
,
Yen
HR
, et al
.
A human colonic commensal promotes colon tumorigenesis via activation of T helper type 17 T cell responses
.
Nat Med
2009
;
15
:
1016
22
.
13.
Gur
C
,
Ibrahim
Y
,
Isaacson
B
,
Yamin
R
,
Abed
J
,
Gamliel
M
, et al
.
Binding of the Fap2 protein of Fusobacterium nucleatum to human inhibitory receptor TIGIT protects tumors from immune cell attack
.
Immunity
2015
;
42
:
344
55
.
14.
Viaud
S
,
Saccheri
F
,
Mignot
G
,
Yamazaki
T
,
Daillere
R
,
Hannani
D
, et al
.
The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide
.
Science
2013
;
342
:
971
6
.
15.
Vetizou
M
,
Pitt
JM
,
Daillere
R
,
Lepage
P
,
Waldschmitt
N
,
Flament
C
, et al
.
Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota
.
Science
2015
;
350
:
1079
84
.
16.
Yu
T
,
Guo
F
,
Yu
Y
,
Sun
T
,
Ma
D
,
Han
J
, et al
.
Fusobacterium nucleatum promotes chemoresistance to colorectal cancer by modulating autophagy
.
Cell
2017
;
170
:
548
63
.
17.
Oh
HJ
,
Kim
JH
,
Bae
JM
,
Kim
HJ
,
Cho
NY
,
Kang
GH
.
Prognostic impact of fusobacterium nucleatum depends on combined tumor location and microsatellite instability status in stage II/III colorectal cancers treated with adjuvant chemotherapy
.
J Pathol Transl Med
2019
;
53
:
40
9
.
18.
Yuan
L
,
Zhang
S
,
Li
H
,
Yang
F
,
Mushtaq
N
,
Ullah
S
, et al
.
The influence of gut microbiota dysbiosis to the efficacy of 5-Fluorouracil treatment on colorectal cancer
.
Biomed Pharmacother
2018
;
108
:
184
93
.
19.
Zhang
S
,
Yang
Y
,
Weng
W
,
Guo
B
,
Cai
G
,
Ma
Y
, et al
.
Fusobacterium nucleatum promotes chemoresistance to 5-fluorouracil by upregulation of BIRC3 expression in colorectal cancer
.
J Exp Clin Cancer Res
2019
;
38
:
14
.
20.
Thomas
AM
,
Manghi
P
,
Asnicar
F
,
Pasolli
E
,
Armanini
F
,
Zolfo
M
, et al
.
Metagenomic analysis of colorectal cancer datasets identifies cross-cohort microbial diagnostic signatures and a link with choline degradation
.
Nat Med
2019
;
25
:
667
78
.
21.
Wirbel
J
,
Pyl
PT
,
Kartal
E
,
Zych
K
,
Kashani
A
,
Milanese
A
, et al
.
Meta-analysis of fecal metagenomes reveals global microbial signatures that are specific for colorectal cancer
.
Nat Med
2019
;
25
:
679
89
.
22.
Castellarin
M
,
Warren
RL
,
Freeman
JD
,
Dreolini
L
,
Krzywinski
M
,
Strauss
J
, et al
.
Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma
.
Genome Res
2012
;
22
:
299
306
.
23.
Kostic
AD
,
Chun
E
,
Robertson
L
,
Glickman
JN
,
Gallini
CA
,
Michaud
M
, et al
.
Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment
.
Cell Host Microbe
2013
;
14
:
207
15
.
24.
Ahn
J
,
Sinha
R
,
Pei
Z
,
Dominianni
C
,
Wu
J
,
Shi
J
, et al
.
Human gut microbiome and risk for colorectal cancer
.
J Natl Cancer Inst
2013
;
105
:
1907
11
.
25.
Kunzmann
AT
,
Proenca
MA
,
Jordao
HW
,
Jiraskova
K
,
Schneiderova
M
,
Levy
M
, et al
.
Fusobacterium nucleatum tumor DNA levels are associated with survival in colorectal cancer patients
.
Eur J Clin Microbiol Infect Dis
2019
;
38
:
1891
9
.
26.
Mima
K
,
Nishihara
R
,
Qian
ZR
,
Cao
Y
,
Sukawa
Y
,
Nowak
JA
, et al
.
Fusobacterium nucleatum in colorectal carcinoma tissue and patient prognosis
.
Gut
2016
;
65
:
1973
80
.
27.
Yan
X
,
Liu
L
,
Li
H
,
Qin
H
,
Sun
Z
.
Clinical significance of Fusobacterium nucleatum, epithelial-mesenchymal transition, and cancer stem cell markers in stage III/IV colorectal cancer patients
.
Onco Targets Ther
2017
;
10
:
5031
46
.
28.
Yamaoka
Y
,
Suehiro
Y
,
Hashimoto
S
,
Hoshida
T
,
Fujimoto
M
,
Watanabe
M
, et al
.
Fusobacterium nucleatum as a prognostic marker of colorectal cancer in a Japanese population
.
J Gastroenterol
2018
;
53
:
517
24
.
29.
Suehiro
Y
,
Sakai
K
,
Nishioka
M
,
Hashimoto
S
,
Takami
T
,
Higaki
S
, et al
.
Highly sensitive stool DNA testing of fusobacterium nucleatum as a marker for detection of colorectal tumours in a Japanese population
.
Ann Clin Biochem
2017
;
54
:
86
91
.
30.
Mima
K
,
Cao
Y
,
Chan
AT
,
Qian
ZR
,
Nowak
JA
,
Masugi
Y
, et al
.
Fusobacterium nucleatum in colorectal carcinoma tissue according to tumor location
.
Clin Transl Gastroenterol
2016
;
7
:
e200
.
31.
Yu
J
,
Chen
Y
,
Fu
X
,
Zhou
X
,
Peng
Y
,
Shi
L
, et al
.
Invasive fusobacterium nucleatum may play a role in the carcinogenesis of proximal colon cancer through the serrated neoplasia pathway
.
Int J Cancer
2016
;
139
:
1318
26
.
32.
Ito
M
,
Kanno
S
,
Nosho
K
,
Sukawa
Y
,
Mitsuhashi
K
,
Kurihara
H
, et al
.
Association of fusobacterium nucleatum with clinical and molecular features in colorectal serrated pathway
.
Int J Cancer
2015
;
137
:
1258
68
.
33.
Bullman
S
,
Pedamallu
CS
,
Sicinska
E
,
Clancy
TE
,
Zhang
X
,
Cai
D
, et al
.
Analysis of fusobacterium persistence and antibiotic response in colorectal cancer
.
Science
2017
;
358
:
1443
8
.
34.
Shigefuku
R
,
Watanabe
T
,
Kanno
Y
,
Ikeda
H
,
Nakano
H
,
Hattori
N
, et al
.
Fusobacterium nucleatum detected simultaneously in a pyogenic liver abscess and advanced sigmoid colon cancer
.
Anaerobe
2017
;
48
:
144
6
.
35.
Wei
Z
,
Cao
S
,
Liu
S
,
Yao
Z
,
Sun
T
,
Li
Y
, et al
.
Could gut microbiota serve as prognostic biomarker associated with colorectal cancer patients' survival? A pilot study on relevant mechanism
.
Oncotarget
2016
;
7
:
46158
72
.
36.
Tahara
T
,
Yamamoto
E
,
Suzuki
H
,
Maruyama
R
,
Chung
W
,
Garriga
J
, et al
.
Fusobacterium in colonic flora and molecular features of colorectal carcinoma
.
Cancer Res
2014
;
74
:
1311
8
.
37.
Xu
M
,
Yamada
M
,
Li
M
,
Liu
H
,
Chen
SG
,
Han
YW
.
FadA from fusobacterium nucleatum utilizes both secreted and nonsecreted forms for functional oligomerization for attachment and invasion of host cells
.
J Biol Chem
2007
;
282
:
25000
9
.
38.
Ponath
F
,
Tawk
C
,
Zhu
Y
,
Barquist
L
,
Faber
F
,
Vogel
J
.
RNA landscape of the emerging cancer-associated microbe Fusobacterium nucleatum
.
Nat Microbiol
2021
;
6
:
1007
20
.
39.
Meng
Q
,
Gao
Q
,
Mehrazarin
S
,
Tangwanichgapong
K
,
Wang
Y
,
Huang
Y
, et al
.
Fusobacterium nucleatum secretes amyloid-like FadA to enhance pathogenicity
.
EMBO Rep
2021
;
22
:
e52891
.
40.
Kim
NH
,
Lee
MY
,
Park
JH
,
Park
DI
,
Sohn
CI
,
Choi
K
, et al
.
Serum CEA and CA 19–9 levels are associated with the presence and severity of colorectal neoplasia
.
Yonsei Med J
2017
;
58
:
918
24
.
41.
Wang
YR
,
Yan
JX
,
Wang
LN
.
The diagnostic value of serum carcino-embryonic antigen, alpha fetoprotein and carbohydrate antigen 19–9 for colorectal cancer
.
J Cancer Res Ther
2014
;
10
:
307
9
.
42.
Scurr
MJ
,
Brown
CM
,
Bento
DFC
,
Betts
GJ
,
Rees
BI
,
Hills
RK
, et al
.
Assessing the prognostic value of preoperative carcinoembryonic antigen-specific T-cell responses in colorectal cancer
.
J Natl Cancer Inst
2015
;
107
:
djv001
.
43.
Fietcher
RH
.
Carcinoembryonic antigen
.
Ann Intern Med
1986
;
104
:
66
73
.
44.
Shinkins
B
,
Nicholson
BD
,
Primrose
J
,
Perera
R
,
James
T
,
Pugh
S
, et al
.
The diagnostic accuracy of a single CEA blood test in detecting colorectal cancer recurrence: results from the FACS trial
.
PLoS One
2017
;
12
:
e0171810
.
45.
Nikolaou
S
,
Qiu
S
,
Fiorentino
F
,
Rasheed
S
,
Tekkis
P
,
Kontovounisios
C
.
Systematic review of blood diagnostic markers in colorectal cancer
.
Tech Coloproctol
2018
;
22
:
481
98
.
46.
Li
L
,
Weiss
HL
,
Li
J
,
Chen
Z
,
Donato
L
,
Evers
BM
.
High plasma levels of pro-NT are associated with increased colon cancer risk
.
Endocr Relat Cancer
2020
;
27
:
641
6
.
47.
Li
L
,
Plummer
SJ
,
Thompson
CL
,
Tucker
TC
,
Casey
G
.
Association between phosphatidylinositol 3-kinase regulatory subunit p85alpha Met326Ile genetic polymorphism and colon cancer risk
.
Clin Cancer Res
2008
;
14
:
633
7
.
48.
Han
YW
,
Ikegami
A
,
Rajanna
C
,
Kawsar
HI
,
Zhou
Y
,
Li
M
, et al
.
Identification and characterization of a novel adhesin unique to oral fusobacteria
.
J Bacteriol
2005
;
187
:
5330
40
.
49.
Wang
HF
,
Li
LF
,
Guo
SH
,
Zeng
QY
,
Ning
F
,
Liu
WL
, et al
.
Evaluation of antibody level against fusobacterium nucleatum in the serological diagnosis of colorectal cancer
.
Sci Rep
2016
;
6
:
33440
.
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