Fusobacterium nucleatumin Colorectal Carcinoma Tissue... : Clinical and Translational Gastroenterology (original) (raw)
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 segments26, 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 (_r_2>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).
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).
Characteristics of colorectal cancer cases according to the amount of tissue Fusobacterium nucleatum
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 (_P_trend<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.
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
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 (_P_trend=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 countries57, 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 epidemiology64, 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 (_r_2>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 studies8, 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
Acknowledgments.
We 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. We assume full responsibility for analyses and interpretation of these data. We have used HUGO (Human Genome Organisation)-approved official symbols for genes and gene products, including BRAF, CACNA1G, CDKN2A, CRABP1, IGF2, KRAS, MLH1, NEUROG1, PIK3CA, RUNX3, SLCO2A1, and SOCS1, all of which are described at www.genenames.org.
CONFLICT OF INTEREST
Guarantor of the article: Shuji Ogino, MD, PhD, MS.
Specific author contributions: All authors contributed to review and revision. Kosuke Mima, Caitlin A. Brennan, Danny A. Milner, Levi A. Garraway, Jeffrey A. Meyerhardt, Wendy S. Garrett, Curtis Huttenhower, Matthew Meyerson, Edward L. Giovannucci, Andrew T. Chan, Charles S. Fuchs, and Shuji Ogino developed the main concept and designed the study. Andrew T. Chan, Charles S. Fuchs, and Shuji Ogino wrote grant applications. Kosuke Mima, Yin Cao, Reiko Nishihara, Zhi Rong Qian, Jonathan A. Nowak, Yohei Masugi, Yan Shi, Annacarolina da Silva, Mancang Gu, Wanwan Li, Tsuyoshi Hamada, Keisuke Kosumi, Akiko Hanyuda, Li Liu, Mingyang Song, Jeffrey A. Meyerhardt, Edward L. Giovannucci, Andrew T. Chan, Charles S. Fuchs, and Shuji Ogino were responsible for collection of tumor tissue and acquisition of epidemiological, clinical and tumor tissue data, including histopathological and immunohistochemical characteristics. Kosuke Mima, Aleksandar D. Kostic, Susan Bullman, Caitlin A. Brennan, Wendy S. Garrett, Curtis Huttenhower, Matthew Meyerson, Charles S. Fuchs, and Shuji Ogino performed data analysis and interpretation. Kosuke Mima, Yin Cao, Reiko Nishihara, and Shuji Ogino drafted the manuscript. Yin Cao, Andrew T. Chan, Mingyang Song, Marios Giannakis, Caitlin A. Brennan, Hideo Baba, Wendy S. Garrett, Matthew Meyerson, Jeffrey A. Meyerhardt, Edward L. Giovannucci, Charles S. Fuchs, Reiko Nishihara, and Shuji Ogino contributed to editing and critical revision for important intellectual contents. All authors approved the final draft submitted.
Financial support: This work was supported by US National Institutes of Health (NIH) grants (P01 CA87969 to M.J. Stampfer; UM1 CA186107 to M.J. Stampfer; P01 CA55075 to W.C. Willett; UM1 CA167552 to W.C. Willett; P50 CA127003 to C.S.F.; R01 CA137178 to A.T.C.; R01 CA151993 to S.O.; R35 CA197735 to S.O.; and K07 CA190673 to R.N.); and by grants from the Project P Fund, The Friends of the Dana-Farber Cancer Institute, Bennett Family Fund, and the Entertainment Industry Foundation through National Colorectal Cancer Research Alliance. K.M. is supported by a grant from Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers from Japan Society for the Promotion of Science. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Potential competing interests: Chan previously served as a consultant for Bayer Healthcare, Millennium Pharmaceuticals, Pozen, and Pfizer. This study was not funded by Bayer Healthcare, Millennium Pharmaceuticals, Pozen, or Pfizer. This is redundant, and we will keep the last sentence. Meyerson has applied for a patent on Fusobacterium in colorectal cancer diagnosis and had ownership interest in and was a consultant and advisory board member for Foundation Medicine. He also receives research support from Bayer. The other authors declare no competing financial interest.
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Supplementary Information accompanies this paper on the Clinical and Translational Gastroenterology website (http://www.nature.com/ctg)
© 2016 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology