Fusobacterium nucleatum in Colorectal Carcinoma Tissue According to Tumor Location.

OBJECTIVES: Evidence suggests a possible role of Fusobacterium nucleatum in colorectal carcinogenesis, especially in right-sided proximal colorectum. Considering a change in bowel contents and microbiome from proximal to distal colorectal segments, we hypothesized that the proportion of colorectal carcinoma enriched with F. nucleatum might gradually increase along the bowel subsites from rectum to cecum.
METHODS: A retrospective, cross-sectional analysis was conducted on 1,102 colon and rectal carcinomas in molecular pathological epidemiology databases of the Nurses' Health Study and the Health Professionals Follow-up Study. We measured the amount of F. nucleatum DNA in colorectal tumor tissue using a quantitative PCR assay and equally dichotomized F. nucleatum-positive cases (high vs. low). We used multivariable logistic regression analysis to examine the relationship of a bowel subsite variable (rectum, rectosigmoid junction, sigmoid colon, descending colon, splenic flexure, transverse colon, hepatic flexure, ascending colon, and cecum) with the amount of F. nucleatum.
RESULTS: The proportion of F. nucleatum-high colorectal cancers gradually increased from rectal cancers (2.5%; 4/157) to cecal cancers (11%; 19/178), with a statistically significant linear trend along all subsites (P<0.0001) and little evidence of non-linearity. The proportion of F. nucleatum-low cancers was higher in rectal, ascending colon, and cecal cancers than in cancers of middle segments.
CONCLUSIONS: The proportion of F. nucleatum-high colorectal cancers gradually increases from rectum to cecum. Our data support the colorectal continuum model that reflects pathogenic influences of the gut microbiota on neoplastic and immune cells and challenges the prevailing two-colon (proximal vs. distal) dichotomy paradigm.

Introduction

An increasing body of evidence suggests possible roles of microorganisms in colorectal carcinogenesis.[1, 2, 3, 4, 5, 6] Among various microbial species, Fusobacterium nucleatum appears to inhibit antitumor immune response and potentiate colonic neoplasia development in animal models.[7, 8, 9, 10] In addition, an enrichment of F. nucleatum can be observed in a subset of human colorectal neoplasms, and a high amount of F. nucleatum in carcinoma tissue has been associated with proximal tumor location, high-level microsatellite instability (MSI-high), and lower density of T cells in tumor tissue.[11, 12, 13, 14, 15, 16, 17, 18]

As a long organ, the colorectum is typically divided into proximal colon (cecum to transverse colon), distal colon (splenic flexure to sigmoid colon), and rectum in clinical, pathological, and epidemiological studies.[19, 20, 21] However, multiple studies have demonstrated that proportions of colorectal cancers with specific molecular features such as MSI-high, CpG island methylator phenotype (CIMP)-high, and BRAF and PIK3CA mutations gradually increase along the bowel subsites from rectum to ascending colon.[22, 23, 24, 25] These findings are consistent with the fact that microbiota, bacterial metabolites, and other contents of the large intestine continually (rather than abruptly) change from the proximal to distal segments[26, 27, 28, 29] and support the colorectal continuum model rather than the dichotomy or trichotomy model.[19, 20, 21] Hence, we hypothesized that the proportion of colorectal cancer enriched with F. nucleatum might gradually change along the bowel subsites from cecum to rectum.

To test this hypothesis, we utilized a database of colorectal carcinoma cases in two US nationwide prospective cohort studies, the Nurses’ Health Study and the Health Professionals Follow-up Study and examined the amount of F. nucleatum in colorectal cancer tissue according to the bowel subsites.

Methods

Study population

We utilized the database of colorectal carcinoma cases in two US nationwide prospective cohort studies, the Nurses’ Health Study (121,701 women enrolled in 1976) and the Health Professionals Follow-up Study (51,529 men enrolled in 1986)[30, 31] and conducted a retrospective, cross-sectional analysis to assess the association of the amount of F. nucleatum in colorectal cancer tissue with tumor location. Every 2 years, we sent participants follow-up questionnaires to collect information on lifestyle factors and asked whether they had received diagnoses of major disease, including cancers. Study physicians reviewed medical records for incident colorectal cancer cases and recorded cancer stage (Tumor, Node, Metastasis) and tumor location (cecum, ascending colon, hepatic flexure, transverse colon, splenic flexure, descending colon, sigmoid colon, rectosigmoid junction, and rectum).[22]

We collected formalin-fixed paraffin-embedded (FFPE) tissue blocks from hospitals where participants with colorectal carcinoma had undergone tumor resection. A single pathologist (S.O.), who was unaware of other data, conducted a centralized review of hematoxylin and eosin–stained tissue sections of all colorectal carcinoma cases and recorded pathological features. Tumor differentiation was classified into well to moderate or poor (>50% vs. ≤50% glandular area). Written informed consent was obtained from all study participants. The institutional review boards at the Harvard T.H. Chan School of Public Health and the Brigham and Women’s Hospital (Boston, MA) approved the cohort studies.

Quantitative PCR for F. nucleatum

We dissected colorectal cancer tissues from whole-tissue sections of FFPE tissue blocks, and DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA). After the quantitative PCR assay for F. nucleatum was developed and validated as previously described,[16] we measured the amount of tissue F. nucleatum DNA in 1,102 colorectal carcinoma cases, while blinded to data on tumor location and other clinical, pathological, and tumor molecular features. Custom TaqMan primer/probe sets (Applied Biosystems, San Diego, CA) for the nusG gene of F. nucleatum and for the reference human gene SLCO2A1 were used as previously described.[16] Each reaction contained 80 ng of genomic DNA and was assayed in 20 μl reactions containing 1 × final concentration TaqMan Environmental Master Mix 2.0 (Applied Biosystems) and each TaqMan Gene Expression Assay (Applied Biosystems) in a 96-well optical PCR plate. Amplification and detection of DNA was performed with the StepOnePlus Real-Time PCR Systems (Applied Biosystems) using the following reaction conditions: 10 min at 95 °C and 45 cycles of 15 s at 95 °C and 1 min at 60 °C.

Our validation study has previously shown that, in colorectal carcinoma cases with detectable F. nucleatum DNA, the cycle threshold (Ct) values in the quantitative PCR for F. nucleatum and SLCO2A1 decreased linearly with the log-transformed amount of input DNA from the same specimen (r2>0.99), and that the interassay coefficient of variation of Ct values from the same specimen in five different batches was ≤1% for all targets.[16] Each specimen was analyzed in duplicate for each target in a single batch, and we used the mean of the two Ct values for each target. Spearman’s rank-correlation coefficients between the two Ct values (in duplicated runs) in each of cases with detectable target amplification in the quantitative PCR assays for F. nucleatum and SLCO2A1 were 0.95 and 0.92, respectively.[16] The amount of tissue F. nucleatum DNA in each specimen was calculated as a relative unitless value normalized with SLCO2A1 using the 2−ΔCt method (where ΔCt=“the mean Ct value of F. nucleatum”−“the mean Ct value of SLCO2A1”).[16]

Cases with detectable F. nucleatum DNA were categorized as low or high based on the median cutpoint while cases without detectable F. nucleatum DNA were categorized as negative, to keep consistent classification system with our previous study.[32]

Analyses of MSI, DNA methylation, and KRAS, BRAF, and PIK3CA mutations

Using DNA extracted from FFPE colorectal carcinoma tissue, MSI status was analyzed with the use of 10 microsatellite markers (D2S123, D5S346, D17S250, BAT25, BAT26, BAT40, D18S55, D18S56, D18S67, and D18S487) as previously described.[33] We defined MSI-high as the presence of instability in ≥30% of the markers, and MSI-low/microsatellite stable (MSS) as instability in <30% of the markers. Methylation analyses of long interspersed nucleotide element-1 (LINE-1)[34] and eight promoter CpG islands specific for CIMP (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1) were performed as previously described.[35, 36] PCR reaction and pyrosequencing were performed for KRAS (codons 12, 13, 61, and 146),[37, 38] BRAF (codon 600),[33] and PIK3CA (exons 9 and 20).[39, 40]

Statistical analysis

All statistical analyses were conducted using SAS (version 9.3, SAS Institute, Cary, NC) and all P values were two-sided. For our primary hypothesis testing, we examined the relationship of the tumor location variable (the nine subsites) with the amount of F. nucleatum DNA in colorectal cancer tissue (as an outcome variable). There was an initial flexibility of the outcome variable, which could be raw continuous, log-transformed (continuous), ordinal three-tiered (high vs. low vs. negative), binary (high vs. low/negative), or binary (high/low vs. negative). Considering these five possibilities, we used adjusted two-sided α level of 0.01 (=0.05/5). Neither the amount of F. nucleatum DNA nor its log-transformed value fitted a normal distribution with the use of the Kolmogorov–Smirnov test for normality (P<0.01). Thus we used multivariable logistic regression analysis to examine the relationship of the nine subsites (as a predictor variable; tested for a linear trend with one degree of freedom) with categorical outcome variables of F. nucleatum. For the ordinal outcome variable, the proportionality of odds assumption was not satisfied in ordinal logistic regression models (P=0.019). According to the distribution of colorectal cancer cases by the subsites and the amount of F. nucleatum (Figure 1), we used a binary outcome variable (F. nucleatum-high vs. -low/negative) in the logistic regression model. For the subsite variable, we assigned population average distance from anal verge to each bowel subsite (either the midpoint or junction/flexure), which was calculated based on published data using computed tomographic colonography,[22, 41] as follows: rectum (the midpoint), 9.8 cm; rectosigmoid junction, 20 cm; sigmoid colon (the midpoint), 44 cm; descending colon (the midpoint), 85 cm; splenic flexure, 102 cm; transverse colon (the midpoint), 131 cm; hepatic flexure, 160 cm; ascending colon (the midpoint), 171 cm; and cecum (the midpoint), 186 cm. A significant P value by the Wald’s test on the bowel subsite variable indicated a linear relationship of the bowel subsite with tissue F. nucleatum, but a curvilinear relationship might exist. Thus we assessed the non-linearity by a likelihood ratio test comparing the model with squared and/or cubic subsite variables with the model without squared or cubic subsite variable; a significant likelihood ratio test result would indicate the presence of non-linearity (curvilinearity).

Figure 1

Proportions of Fusobacterium nucleatum-negative, F. nucleatum-low, and F. nucleatum-high colorectal carcinoma cases along the bowel subsites. P-value was calculated by the linear trend test across the bowel subsite variable (population average distance from anal verge to each subsite (cm)) as a continuous variable in the univariable logistic regression model to predict the amount of tissue F. nucleatum (as a binary outcome variable (high vs. low/negative)).

The multivariable logistic regression models were adjusted for clinical features, including age (continuous), sex, year of diagnosis (continuous), and family history of colorectal carcinoma in any first-degree relative (present vs. absent). Studies have shown an enrichment of F. nucleatum in colorectal adenomas (before progression to carcinomas),[9, 13, 15] suggesting that F. nucleatum may be involved in early colorectal carcinogenesis. Hence, pathological and tumor molecular features of colorectal carcinoma may be present downstream in the causal sequence of events after the Fusobacterium variable, and adjusting for the tumor pathological and molecular features might cause biased results. Thus we did not include the pathological and tumor molecular variables in the multivariable logistic regression models. For cases with missing information on family history of colorectal carcinoma in a first-degree relative (1.3%), we included those cases in a majority category of a given covariate to minimize the number of variables in multivariable logistic regression models. We confirmed that excluding the cases with missing information on family history of colorectal carcinoma in a first-degree relative did not substantially alter the results (data not shown).

To assess the associations between the amount of tissue F. nucleatum and other categorical variables, chi-square test was performed. To compare mean age and mean LINE-1 methylation levels, an analysis of variance was performed. These comparisons represented secondary analyses, and we used adjusted α level of 0.003 (=0.05/14) by simple Bonferroni correction for multiple hypothesis testing.

Results

F. nucleatum in colorectal carcinoma tissue

We measured the amount of tissue F. nucleatum DNA in 1,102 colorectal carcinoma cases within the two prospective cohort studies using the quantitative PCR assay that was previously validated.[16] F. nucleatum DNA was detected (positive) in colorectal carcinoma tissue in 138 (13%) of the 1,102 cases and undetectable (negative) in the remaining 964 cases (87%). We equally dichotomized the 138 cases with detectable F. nucleatum DNA levels into two groups to keep consistency with our previous study.[32] Clinical, pathological, and tumor molecular features according to the amount (high vs. low. vs. negative) of tissue F. nucleatum are summarized in Table 1. High-level F. nucleatum in colorectal cancer tissue was associated with proximal tumor location, poor tumor differentiation, MSI-high, MLH1 hypermethylation, CIMP-high, and BRAF mutation (P≤0.0004 with the adjusted α level of 0.003 for multiple hypothesis testing).

Table 1

Characteristics of colorectal cancer cases according to the amount of tissue Fusobacterium nucleatum

Characteristicsa	All patients (n=1,102)	The amount of tissue Fusobacterium nucleatum			P valueb
		Negative (n=964)	Low (n=69)	High (n=69)
Mean age±s.d. (years)	69.5±8.9	69.4±8.9	70.9±9.0	69.2±8.5	0.39
Sex					0.35
Men	466 (42%)	415 (43%)	27 (39%)	24 (35%)
Women	636 (58%)	549 (57%)	42 (61%)	45 (65%)
Year of diagnosis					0.026
Prior to 1995	352 (32%)	323 (34%)	12 (17%)	17 (25%)
1996–2000	301 (27%)	262 (27%)	19 (28%)	20 (29%)
2001–2008	449 (41%)	379 (39%)	38 (55%)	32 (46%)
Family history of colorectal carcinoma in a first-degree relative					0.26
Absent	877 (81%)	762 (80%)	60 (88%)	55 (81%)
Present	211 (19%)	190 (20%)	8 (12%)	13 (19%)
Tumor location					0.0004
Proximal colon	536 (49%)	452 (47%)	36 (53%)	48 (72%)
Distal colon	316 (29%)	292 (31%)	12 (18%)	12 (18%)
Rectum	241 (22%)	214 (22%)	20 (29%)	7 (10%)
Nine bowel subsites c					<0.0001d
Cecum	178	147 (83%)	12 (6.7%)	19 (11%)
Ascending colon	253	212 (84%)	18 (7.1%)	23 (9.1%)
Hepatic flexure	32	28 (88%)	2 (6.3%)	2 (6.3%)
Transverse colon	73	65 (89%)	4 (5.5%)	4 (5.5%)
Splenic flexure	29	27 (93%)	1 (3.5%)	1 (3.5%)
Descending colon	53	48 (91%)	2 (3.8%)	3 (5.7%)
Sigmoid colon	234	217 (93%)	9 (3.8%)	8 (3.4%)
Rectosigmoid junction	84	77 (92%)	4 (4.8%)	3 (3.6%)
Rectum	157	137 (87%)	16 (10%)	4 (2.5%)
Disease stage					0.006
I	247 (25%)	230 (26%)	10 (16%)	7 (11%)
II	331 (33%)	279 (32%)	23 (37%)	29 (45%)
III	286 (29%)	246 (28%)	25 (40%)	15 (24%)
IV	135 (13%)	117 (14%)	5 (7.9%)	13 (20%)
Tumor differentiation					<0.0001
Well to moderate	994 (90%)	887 (92%)	57 (84%)	50 (72%)
Poor	106 (9.6%)	76 (7.9%)	11 (16%)	19 (28%)
MSI status					<0.0001
MSI-low/MSS	885 (84%)	805 (87%)	44 (67%)	36 (54%)
MSI-high	171 (16%)	118 (13%)	22 (33%)	31 (46%)
MLH1 hypermethylation					<0.0001
Absent	869 (86%)	782 (89%)	50 (79%)	37 (58%)
Present	140 (14%)	100 (11%)	13 (21%)	27 (42%)
CIMP status					<0.0001
Low/negative	823 (82%)	737 (84%)	50 (79%)	36 (56%)
High	186 (18%)	145 (16%)	13 (21%)	28 (44%)
BRAF mutation					<0.0001
Wild type	892 (84%)	795 (85%)	52 (79%)	45 (66%)
Mutant	172 (16%)	135 (15%)	14 (21%)	23 (34%)
KRAS mutation					0.51
Wild type	569 (57%)	501 (57%)	30 (51%)	38 (61%)
Mutant	435 (43%)	382 (43%)	29 (49%)	24 (39%)
PIK3CA mutation					0.88
Wild type	841 (84%)	738 (84%)	49 (82%)	54 (83%)
Mutant	162 (16%)	140 (16%)	11 (18%)	11 (17%)
Mean LINE-1 methylation level, %±s.d.	63.5±10.2	63.3±10.2	65.0±10.6	65.4±8.9	0.14

CIMP, CpG island methylator phenotype; LINE-1, long interspersed nucleotide element-1; MSI, microsatellite instability; MSS, microsatellite stable.

Percentage (%) indicates the proportion of cases with a specific clinical, pathological, or tumor molecular feature according to the amount of tissue Fusobacterium nucleatum. There were cases which had missing values for any of the characteristics except for age, sex, and year of diagnosis.

To assess associations between the ordinal categories (negative, low, and high) of the amount of tissue F. nucleatum and categorical variables, the chi-square test was performed. To compare mean age and mean LINE-1 methylation levels, an analysis of variance was performed. We adjusted two-sided α level to 0.003 (=0.05/14) by simple Bonferroni correction for multiple hypothesis testing.

Percentage indicates the proportion of F. nucleatum-negative, F. nucleatum-low, or F. nucleatum-high cases among all tumors in a given bowel subsite.

P value was calculated by the linear trend test across the bowel subsite variable (population average distance from anal verge to each subsite (cm)) as a continuous variable in the univariable logistic regression model to predict the amount of tissue F. nucleatum (as a binary outcome variable (high vs. low/negative)).

The relationship of the bowel subsites with the amount of F. nucleatum in colorectal cancer tissue

The amount of F. nucleatum in colorectal cancer tissue according to the bowel subsites from rectum to cecum is shown in Figure 1. The proportion of F. nucleatum-high cancers gradually increased from rectal cancers (2.5%=4/157) to cecal cancers (11%=19/178). In contrast, the proportion of F. nucleatum-low cancers was higher in rectal cancers (10%=16/157), ascending colon cancers (7.1%=18/253), and cecal cancers (6.7%=12/178) than in cancers of middle segments.

We assessed the relationship of the bowel subsite (as a predictor variable) with the amount of F. nucleatum in colorectal cancer tissue (as a binary outcome variable (high vs. low/negative)) by multivariable logistic regression analysis that adjusted for potential confounders (Table 2). The bowel subsite variable was significantly associated with high-level F. nucleatum in colorectal cancer tissue (Ptrend<0.0001 for trend across the bowel subsites from rectum to cecum, with the adjusted α level of 0.01). We demonstrated little evidence of non-linearity (P≥0.61) using likelihood ratio test, which compared the model with squared and/or cubic subsite variables to the model without squared or cubic subsite variable.

Table 2

Assessment of the linearity and non-linearity on the relationship of the bowel subsites with the amount of Fusobacterium nucleatum in colorectal cancer tissue by multivariable logistic regression analyses

Bowel subsite variable (distance from anal verge to each subsite (cm))	Squared subsite variable		Cubic subsite variable		Likelihood ratio test
P value (Wald’s test)a	Included	P value (Wald’s test)	Included	P value (Wald’s test)	Degree of freedom	P valueb
Model for the amount of tissue Fusobacterium nucleatum (as an outcome variable (high vs. low/negative))
All cases
<0.0001	No	—	No	—	—	Referent
0.87	Yes	0.61	No	—	1	0.61
0.65	Yes	0.74	Yes	0.67	2	0.80
Cases from sigmoid colon to cecum (excluding rectal and rectosigmoid cancers)
0.002	No	—	No	—	—	Referent
0.82	Yes	0.51	No	—	1	0.50
0.84	Yes	0.85	Yes	0.80	2	0.77

Multivariable logistic regression model included age, sex, year of diagnosis, family history of colorectal cancer in parent or sibling, and the bowel subsite variable with or without the squared and cubic subsite variable, as indicated in the Table. We adjusted two-sided α level to 0.01 (=0.05/5) for multiple hypothesis testing.

P value was calculated by the Wald’s test on the bowel subsite variable (population average distance from anal verge to each subsite (cm)) as a continuous variable in the multivariable logistic regression model to predict the amount of tissue Fusobacterium nucleatum (as a binary outcome variable (high vs. low/negative)).

A significant P value by the likelihood ratio test indicates a non-linear (curvilinear) relationship, and a combination of insignificant P values by the likelihood ratio test and a significant P value by the Wald test on the bowel subsite variable in the model without the squared or cubic subsite variable indicates a linear relationship.

To exclude a potential influence of preoperative chemotherapy and/or radiation therapy for rectal cancers as a secondary analysis, we excluded cancers in the rectum and rectosigmoid and performed a linearity test. The bowel subsite variable (from sigmoid colon to cecum) was significantly associated with high-level tissue F. nucleatum (Ptrend=0.002 for trend across the bowel subsites, with the adjusted α level of 0.01), and there was no evidence for non-linearity (P≥0.50; Table 2).

Discussion

We found that the proportion of F. nucleatum-high colorectal cancers increased linearly along the large intestine from rectum to cecum. Although differences in clinical, pathological, and epidemiological features between proximal and distal colon cancers and between colon and rectal cancers have been known for decades,[19, 20, 21] emerging evidence indicates a gradual change in the proportions of key molecular features of colorectal cancer along the bowel subsites.[22, 23, 24, 25] Our data challenge the common dichotomy model and support the colorectal continuum model that recently emerged.[42, 43]

Analyses of molecular pathology are increasing importance in cancer research.[44, 45, 46, 47, 48, 49, 50, 51] Accumulating evidence suggests that colorectal tumors arise with sets of genomic and epigenomic alterations through interactions between neoplastic cells, immune cells, and microbiota that vary along the proximal to distal axis of colorectum.[42] In the current study, high-level tissue F. nucleatum DNA was associated with the molecular features of colorectal cancer, including MSI-high, CIMP-high, and BRAF mutation. Some studies reported that butyrate might suppress colonic inflammation and tumor development.[52, 53] In contrast, emerging evidence suggests that butyrate may promote the growth of colonic tumors that exhibit DNA mismatch repair deficiency.[5, 54] F. nucleatum is known to be one of the microbes that produce butyrate.[1, 5] Thus F. nucleatum might promote the development and progression of colorectal tumors through the production of butyrate, although additional studies are needed to elucidate the exact mechanisms underlying the association between F. nucleatum and colorectal carcinogenesis.

Cecal carcinomas represent an interesting subgroup of colorectal carcinomas characterized by high prevalence of KRAS mutations.[22, 55] In addition, along with rectum, cecum shows the highest incidence of carcinoma occurrence per surface area of mucosa.[56] Our current study has shown that F. nucleatum-enriched carcinomas are most prevalent in cecum compared with other subsites. Future studies should investigate the role of microbiota in cecal carcinogenesis.

Epidemiological evidence indicates a recent increase in the proportion of proximal colon cancers in Western countries[57, 58] and the association between postcolonoscopy cancer and proximal tumor location.[59, 60] Because any experimental system cannot perfectly recapitulate the complex nature of human tumor or microorganisms, analyses of human cancer tissue in a large population are useful in elucidating the relationship between microorganisms and cancer. However, no previous study has examined the amount of specific microbial species in human colorectal cancers according to detailed subsites (using an enough sample size), as we did in this study. Our population-based human data would guide future mechanistic investigations. Considering that diet, lifestyle, pharmacological factors (including antibiotics), and probiotics and prebiotics can influence the composition of intestinal microbiota,[61, 62, 63] future investigations may be warranted to examine potential influences of those modifiable factors on the intestinal microflora and tumorigenic processes.

Strengths of this study include the use of our molecular pathological epidemiology[64, 65] database (of 1,102 colorectal carcinoma cases in the two US nationwide, prospective cohort studies), which integrates epidemiological exposures, clinical characteristics, and tissue F. nucleatum in colorectal carcinoma. The sample size and the comprehensiveness of the colorectal cancer database enabled us to examine the amounts of F. nucleatum in colorectal cancer tissue in each of the bowel subsites and test the linearity of the relationship of the bowel subsites with the amount of F. nucleatum, while adjusting for clinical features. Importantly, our data set of colorectal cancer cases represented a population-based sample derived from a large number of hospitals in diverse settings across the United States that increases the generalizability of our findings.

We recognize limitations of our study. First, routine histopathology processing might have influenced the performance of the quantitative PCR assay to detect microorganisms in FFPE tissue specimens. Although measurement errors in FFPE tissue specimens would have likely driven our results toward the null hypothesis, we cannot exclude unmeasured confounding factors. However, our validation study has demonstrated a high linearity (r2>0.99) and a high reproducibility (interassay coefficient of variation ≤1%) of the quantitative PCR assay for F. nucleatum with the use of FFPE tissue specimens.[16] In addition, our data on the relationships of F. nucleatum with clinicopathological characteristics and tumor molecular features, including MSI and CIMP status, are consistent with the study using a quantitative PCR assay for frozen tissue specimens.[14] Second, rectal cancers are commonly treated by preoperative chemotherapy and/or radiation, which might have changed the gut and tumor microbiota. Therefore, we excluded preoperatively treated rectal cancers in which adequate pretreatment biopsy specimens were unavailable. In addition, as a secondary analysis, we excluded rectal and rectosigmoid cancers and observed similar findings on the association of bowel subsites (from the sigmoid colon to cecum) with the amount of tissue F. nucleatum. In the current study, rectal cancers showed a high proportion of F. nucleatum-low cases (10%) compared with rectosigmoid (4.8%) and sigmoid colon cancers (3.8%). These findings need to be validated by additional studies. Third, we did not examine other microbes (including Escherichia coli and Bacteroides fragilis[66, 67]) in colorectal cancer tissue or data on stool microbiota. Certainly, future comprehensive metagenomic analyses on tissue and stool microbiota may provide further insights on roles of gut microorganisms in the development and progression of colorectal tumors. Fourth, as an observational study, we could not conclude on a potential causal effect of F. nucleatum. Nonetheless, given complex roles of interactions between microbial and host factors in human carcinogenesis, we believe that our novel data on F. nucleatum (which appears to have a role in carcinogenesis in experimental studies[8, 9, 10]) in >1,000 colorectal cancer cases along the bowel subsites represent valuable information.

In conclusion, utilizing the database of the 1,102 colorectal carcinoma cases in the US nationwide prospective cohort studies, we have found that the proportion of colorectal cancer enriched with F. nucleatum increases linearly along the bowel subsites from rectum to cecum. Our human population-based data suggest a continuum model of pathogenic influences of F. nucleatum on colorectal carcinogenesis, which may be targeted for colorectal cancer prevention and treatment in the future.

Study Highlights