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Figure.
The Amount of Fusobacterium nucleatum in Colorectal Cancer
The Amount of Fusobacterium nucleatum in Colorectal Cancer

A, Quantitative real-time polymerase chain reaction (RT-PCR) assays for F nucleatum and SLCO2A1 using 2-fold dilution series (20, 40, 80, and 160 ng) from the same DNA specimen. Symbols indicate mean, and error bars, standard deviation of cycle threshold values of duplicate runs. The coefficient of determination (r2) in the assays for F nucleatum and SLCO2A1 is shown. B, The amount of F nucleatum in 558 pairs of colorectal carcinoma and adjacent nontumor tissue samples. Dot plots represent the amount of F nucleatum in colorectal carcinoma tissue and paired adjacent nontumor tissue. Statistical analyses were performed using a 2-sided Wilcoxon signed rank test.

Table 1.  
Characteristics According to the Amount of Fusobacterium nucleatum in Colorectal Carcinoma Tissue
Characteristics According to the Amount of Fusobacterium nucleatum in Colorectal Carcinoma Tissue
Table 2.  
Distribution of Cases According to the Amount of Fusobacterium nucleatum and the Density of T Cells
Distribution of Cases According to the Amount of Fusobacterium nucleatum and the Density of T Cells
Table 3.  
The Amount of Fusobacterium nucleatum in Colorectal Carcinoma Tissue and the Density of T Cells
The Amount of Fusobacterium nucleatum in Colorectal Carcinoma Tissue and the Density of T Cells
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    Citation

    Kosuke Mima, Yasutaka Sukawa, Reiko Nishihara, Zhi Rong Qian, Mai Yamauchi, Kentaro Inamura, Sun A. Kim, Atsuhiro Masuda, Jonathan A. Nowak, Katsuhiko Nosho, Aleksandar D. Kostic, Marios Giannakis, Hideo Watanabe, Susan Bullman, Danny A. Milner, Curtis C. Harris, Edward Giovannucci, Levi A. Garraway, Gordon J. Freeman, Glenn Dranoff, Andrew T. Chan, Wendy S. Garrett, Curtis Huttenhower, Charles S. Fuchs, Shuji Ogino. Fusobacterium nucleatum and T Cells in Colorectal Carcinoma. JAMA Oncol. 2015;1(5):653–661. doi:10.1001/jamaoncol.2015.1377

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Original Investigation
August 2015

Fusobacterium nucleatum and T Cells in Colorectal Carcinoma

Author Affiliations
  • 1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
  • 2Department of Nutrition, Harvard T. H. Chan School of Public Health, Boston, Massachusetts
  • 3Department of Epidemiology, Harvard T. H. Chan School of Public Health, Boston, Massachusetts
  • 4Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
  • 5Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts
  • 6Department of Gastroenterology, Rheumatology, and Clinical Immunology, Sapporo Medical University School of Medicine, Sapporo, Japan
  • 7Department of Biostatistics, Harvard T. H. Chan School of Public Health, Boston, Massachusetts
  • 8Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts
  • 9Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts
  • 10Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts
  • 11Channing Division of Network Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts
  • 12Division of Gastroenterology, Massachusetts General Hospital, Boston, Massachusetts
JAMA Oncol. 2015;1(5):653-661. doi:10.1001/jamaoncol.2015.1377
Abstract

Importance  Evidence indicates a complex link between gut microbiome, immunity, and intestinal tumorigenesis. To target the microbiota and immunity for colorectal cancer prevention and therapy, a better understanding of the relationship between microorganisms and immune cells in the tumor microenvironment is needed. Experimental evidence suggests that Fusobacterium nucleatum may promote colonic neoplasia development by downregulating antitumor T cell–mediated adaptive immunity.

Objective  To test the hypothesis that a greater amount of F nucleatum in colorectal carcinoma tissue is associated with a lower density of T cells in tumor tissue.

Design, Setting, and Participants  A cross-sectional analysis was conducted on 598 rectal and colon carcinoma cases in 2 US nationwide prospective cohort studies with follow-up through 2006, the Nurses’ Health Study (participants enrolled in 1976) and the Health Professionals Follow-up Study (participants enrolled in 1986). Tissue collection and processing were performed from 2002 through 2008, and immunity assessment, 2008 through 2009. From 2013 through 2014, the amount of F nucleatum in colorectal carcinoma tissue was measured by quantitative polymerase chain reaction assay; we equally dichotomized positive cases (high vs low). Multivariable ordinal logistic regression analysis was conducted in 2014 to assess associations of the amount of F nucleatum with densities (quartiles) of T cells in tumor tissue, controlling for clinical and tumor molecular features, including microsatellite instability, CpG island methylator phenotype, long interspersed nucleotide element-1 (LINE-1) methylation, and KRAS, BRAF, and PIK3CA mutation status. We adjusted the 2-sided α level to .013 for multiple hypothesis testing.

Main Outcomes and Measures  Densities of CD3+, CD8+, CD45RO (protein tyrosine phosphatase receptor type C [PTPRC])+, and FOXP3+ T cells in tumor tissue, determined by means of tissue microarray immunohistochemical analysis and computer-assisted image analysis.

Results  F nucleatum was detected in colorectal carcinoma tissue in 76 (13%) of 598 cases. Compared with F nucleatum–negative cases, F nucleatum–high cases were inversely associated with the density of CD3+ T cells (for a unit increase in quartile categories of CD3+ T cells as an outcome: multivariable odds ratio, 0.47 [95% CI, 0.26-0.87]; P for trend = .006). The amount of F nucleatum was not significantly associated with the density of CD8+, CD45RO+, or FOXP3+ T cells (P fortrend = .24, .88, and .014, respectively).

Conclusions and Relevance  The amount of tissue F nucleatum is inversely associated with CD3+ T-cell density in colorectal carcinoma tissue. On validation, our human population data may provide an impetus for further investigations on potential interactive roles of Fusobacterium and host immunity in colon carcinogenesis.

Introduction

Accumulating evidence attests to an important role of T cell–mediated adaptive immunity in regulating tumor evolution, and immunotherapy has emerged as a promising strategy to treat various cancers.1,2 In colorectal carcinoma, high-level infiltrates of CD3+, CD8+, CD45RO (protein tyrosine phosphatase receptor type C [PTPRC])+, and forkhead box P3 (FOXP3)+ T cells have been associated with better clinical outcome.36 Evidence also indicates that molecular features of colorectal carcinoma, especially microsatellite instability (MSI), can influence antitumor T cell–mediated adaptive immunity.711

The human intestinal microbiome encompasses at least 100 trillion (1014) microorganisms, which can influence the immune system and health conditions.12 A growing body of evidence indicates a complex link between gut microbiome, immunity, and intestinal tumorigenesis.1317 Colorectal carcinogenesis represents heterogeneous processes with differing sets of genetic and epigenetic alterations, influenced by diet, environmental and microbial exposures, and host immunity.1822 Metagenomic analyses have shown an enrichment of Fusobacterium nucleatum in colorectal carcinoma tissue, which has been confirmed by quantitative polymerase chain reaction (PCR) for the 16S ribosomal RNA gene DNA sequence of F nucleatum.23,24 Studies have shown that a greater amount of F nucleatum in colorectal carcinoma tissue is associated with high degrees of MSI and CpG island methylator phenotype (CIMP).25 Experimental evidence suggests that virulence factors derived from F nucleatum inhibit T-cell activity26,27 and that in the ApcMin/+ mouse model, F nucleatum promotes colonic neoplasia development by downregulating antitumor T cell–mediated adaptive immunity.28 Therefore, we hypothesized that a greater amount of F nucleatum in colorectal carcinoma tissue might be associated with a lower density of T cells in tumor tissue. A better understanding of the relationship between F nucleatum and immune cells in the tumor microenvironment may open new opportunities to target the microbiota and immunity for colorectal cancer prevention and therapy.

To test our hypothesis, we used resources of 2 US nationwide prospective cohort studies (the Nurses’ Health Study and the Health Professionals Follow-up Study) and examined the amount of F nucleatum in relation to densities of CD3+, CD8+, CD45RO (PTPRC)+, and FOXP3+ T cells in tumor tissue of nearly 600 human colorectal carcinoma cases. To our knowledge, this is the first population-based study to examine the amount of F nucleatum in relation to the density of T cells in human colorectal carcinoma tissue.

Box Section Ref ID

At a Glance

  • High-level infiltrates of T cells in colorectal carcinoma have been associated with better patient survival.

  • Fusobacterium nucleatum may promote colonic neoplasia development by downregulating antitumor T cell–mediated immune response.

  • Using 598 colorectal carcinoma cases in 2 US nationwide prospective cohort studies, F nucleatum in colorectal carcinoma tissue was measured by means of quantitative polymerase chain reaction.

  • An inverse association between the amount of F nucleatum and the density of CD3+ T cells in colorectal carcinoma was observed (P for trend= .006).

  • On validation, the data may provide insights for strategies to target microbiota for colon cancer immune therapy and/or prevention.

Methods
Study Population

We used the databases of 2 US nationwide prospective cohort studies, the Nurses’ Health Study (with 121 700 women who enrolled in 1976) and the Health Professionals Follow-up Study (with 51 529 men who enrolled in 1986).29,30 Every 2 years, participants were sent follow-up questionnaires to gather information on health and lifestyle factors, and to identify newly diagnosed cancers and other diseases. The National Death Index was used to ascertain deaths of study participants and identify unreported lethal colorectal carcinoma cases. Medical records were reviewed, and the cause of death was assigned by study physicians. Formalin-fixed paraffin-embedded (FFPE) tissue blocks were collected from hospitals where participants with colorectal carcinoma had undergone tumor resection. We included both colon and rectal carcinoma cases, considering the colorectal continuum model.31 A single pathologist (S.O.), who was unaware of other data, reviewed hematoxylin-eosin–stained tissue sections from all colorectal carcinoma cases and recorded pathological features. Tumor differentiation was categorized as well to moderate or poor (>50% vs ≤50% glandular area). Based on the availability of data on F nucleatum and T-cell densities, a total of 598 colorectal carcinoma cases were included. Written informed consent was obtained from all study participants. Tissue collection and analyses were approved by the human subjects committee at the Harvard T.H. Chan School of Public Health and the Brigham and Women’s Hospital (Boston, Massachusetts).

Quantitative PCR for F nucleatum

Genomic DNA was extracted from colorectal carcinoma tissue and adjacent nontumor tissue in whole-tissue sections of FFPE tissue blocks using QIAamp DNA FFPE Tissue Kit (Qiagen). Custom TaqMan primer-probe sets (Applied Biosystems) for the 16S ribosomal RNA gene DNA sequence of F nucleatum and for the reference gene, SLCO2A1, were used as previously described.24 The primer-probe set for F nucleatum was designed to target the nusG gene of F nucleatum, and it has been demonstrated that the amount of F nucleatum measured by the quantitative PCR assay highly correlates with that measured by using transcriptome sequencing data (Pearson r = 0.97).24 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 minutes at 95°C and 45 cycles of 15 seconds at 95°C and 1 minute at 60°C. The primer and probe sequences for each TaqMan Gene Expression Assay were as follows: F nucleatum forward primer, 5′-CAACCATTACTTTAACTCTACCATGTTCA-3′; F nucleatum reverse primer, 5′-GTTGACTTTACAGAAGGAGATTATGTAAAAATC-3′; F nucleatum FAM probe, 5′-GTTGACTTTACAGAAGGAGATTA-3′; SLCO2A1 forward primer, 5′-ATCCCCAAAGCACCTGGTTT-3′; SLCO2A1 reverse primer, 5′-AGAGGCCAAGATAGTCCTGGTAA-3′; SLCO2A1 VIC probe, 5′-CCATCCATGTCCTCATCTC-3′. In colorectal carcinoma cases with detectable F nucleatum, the cycle threshold (Ct) values in the quantitative PCR for F nucleatum and SLCO2A1 decreased linearly with the amount of input DNA (in a log scale) from the same specimen (r2 > 0.99) (Figure, A). The interassay coefficient of variation of Ct values from the same specimen in 5 different batches was 1% or less for all targets in our validation study using 7 colorectal carcinomas (eTable 1 in the Supplement).

Each specimen was analyzed in duplicate for each target in a single batch, and we used the mean of the 2 Ct values for each target. Spearman rank correlation coefficients between the 2 Ct values (in duplicated runs) in each case with detectable target amplification in the quantitative PCR assays for F nucleatum (n = 76) and SLCO2A1 (n = 598) were 0.95 and 0.92, respectively. The amount of F nucleatum in each specimen was calculated as a relative unitless value normalized with SLCO2A1 using the 2-∆Ct method (where ∆Ct = the average Ct value of F nucleatum − the average Ct value of SLCO2A1) as previously described.32(p1107)

Analyses of MSI, DNA Methylation, and KRAS, BRAF, and PIK3CA Mutations

Genomic DNA was extracted from colorectal carcinoma tissue in whole-tissue sections from FFPE tissue blocks. Microsatellite instability status was analyzed with 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 at least 30% of the markers and MSI-low/microsatellite stable as instability in less than 30% of the markers. Methylation analyses of long interspersed nucleotide element-1 (LINE-1) and 8 promoter CpG islands specific for CIMP (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1) were performed as previously described.3437 Polymerase chain reaction and pyrosequencing were performed for KRAS (codons 12, 13, 61, and 146), BRAF (codon 600), and PIK3CA (exons 9 and 20).3840

Immunohistochemical Analysis and Quantification of the Density of T Cells in Tumor Tissue

We constructed a tissue microarray and conducted immunohistochemical analysis for CD3, CD8, CD45RO (PTPRC), and FOXP3. We used an automated scanning microscope and the Ariol image analysis system (Genetix) to measure densities (measured in cells per square millimeter) of CD3+, CD8+, CD45RO (PTPRC)+, and FOXP3+ T cells in tumor tissue. We evaluated up to 4 tissue cores from each tumor in tissue microarray and calculated the mean density of each T-cell subset as previously described.5

Statistical Analysis

All statistical analyses were conducted using SAS, version 9.3 (SAS Institute), and all P values were 2 sided. Neither the amount of F nucleatum, T-cell density, nor the log-transformed value of the amount of F nucleatum or T-cell density fit a normal distribution with the use of the Kolmogorov-Smirnov test for normality (P ≤ .022). Thus, our primary hypothesis testing was the linear trend test in an ordinal logistic regression model to assess associations of the amount of F nucleatum in colorectal carcinoma tissue (an ordinal predictor variable) with the density of CD3+, CD8+, CD45RO+, or FOXP3+ T cells in tumor tissue (an ordinal quartile outcome variable). Cases with detectable F nucleatum were categorized as low or high on the basis of the median cut point amount of F nucleatum, whereas cases without detectable F nucleatum were categorized as negative. The linear trend test was performed using the ordinal predictor variable of F nucleatum (negative, low, and high) as a continuous variable in an ordinal logistic regression model. Because we tested 4 primary hypotheses (for CD3+, CD8+, CD45RO+, and FOXP3+ T cells as outcome variables), we adjusted the 2-sided α level to .013 (= .05/4) by simple Bonferroni correction. All other analyses on F nucleatum, including evaluation of individual odds ratio estimates, represented secondary analyses. In those secondary analyses, in view of multiple comparisons, we interpreted our data cautiously, in addition to the use of the adjusted α level of .013.

We performed multivariable ordinal logistic regression analysis to adjust for potential confounders. The multivariable model initially included age (continuous), sex, year of diagnosis (continuous), family history of colorectal carcinoma in a first-degree relative (present vs absent), tumor location (proximal colon vs distal colon vs rectum), tumor differentiation (well to moderate vs poor), MSI (high vs MSI-low or microsatellite stable), CIMP (high vs low or negative), KRAS (mutant vs wild type), BRAF (mutant vs wild type), PIK3CA (mutant vs wild type), and LINE-1 methylation level (continuous). For cases with missing information in any of the covariates, we assigned a separate (“missing”) indicator variable. A backward stepwise elimination with a threshold of P < .05 was used to select variables in the final models. We assessed the proportional odds assumption in an ordinal logistic regression model, which was generally satisfied (P > .05).

All cross-sectional univariable analyses for clinical, pathological, and molecular associations (with variables listed in Table 1) were secondary exploratory analyses, and we adjusted the 2-sided α level to .003 (= .05/14) by simple Bonferroni correction for multiple hypothesis testing. To assess associations between the ordinal (negative, low, and high) categories of the amount of F nucleatum and categorical data, a Fisher exact test was performed. To compare mean age and mean LINE-1 methylation levels, an analysis of variance assuming equal variances was performed.

Results
F nucleatum in Colorectal Carcinoma Tissue

We analyzed tumor tissues of 598 colorectal carcinoma cases within the Nurses’ Health Study and the Health Professionals Follow-up Study, using the quantitative PCR assay to detect the 16S ribosomal RNA gene DNA sequence of F nucleatum, as previously described.24F nucleatum was detected in colorectal carcinoma in 76 (13%) of 598 cases and in adjacent nontumor tissue in 19 (3.4%) of 558 cases analyzed. In the 558 pairs of colorectal carcinoma and adjacent nontumor tissues, the amount of F nucleatum was higher in colorectal carcinoma tissue than in paired adjacent nontumor tissue (Wilcoxon signed rank test, P < .0001; Figure, B).

We categorized colorectal carcinoma cases with detectable F nucleatum as low or high on the basis of the median cut point amount of F nucleatum. Clinical, pathological, and molecular features are summarized according to the amount of F nucleatum in colorectal carcinoma tissue in Table 1. A greater amount of F nucleatum in colorectal carcinoma tissue was associated with stage II to IV disease, poor differentiation, MSI-high status, MLH1 hypermethylation, and CIMP-high status (P ≤ .003 for all comparisons by Fisher exact test; with adjusted α level of .003 for multiple hypothesis testing).

Associations of the Amount of F nucleatum With T-Cell Densities in Tumor Tissue

Using tissue microarray, we quantified densities of CD3+, CD8+, CD45RO+, and FOXP3+ T cells in tumor tissue by means of immunohistochemical analysis and the Ariol image analysis system. Correlations between densities of CD3+, CD8+, CD45RO+, and FOXP3+ T cells in tumor tissue (with Spearman rank correlation coefficients ranging from 0.14 to 0.42; P ≤ .002) are given in eTable 2 in the Supplement.

Table 2 shows the distribution of colorectal carcinoma cases according to the amount of F nucleatum and densities of CD3+, CD8+, CD45RO+, and FOXP3+ T cells. In our primary hypothesis testing, we conducted univariable and multivariable ordinal logistic regression analyses to assess associations of the amount of F nucleatum in colorectal carcinoma tissue (as an ordinal predictor variable) with the density of CD3+, CD8+, CD45RO+, or FOXP3+ T cells in tumor tissue (as an ordinal quartile outcome variable) (Table 3 and eTable 3 in the Supplement). The amount of F nucleatum in colorectal carcinoma tissue was associated with a lower density of CD3+ T cells in univariable (P for trend= .012) and multivariable ordinal logistic regression analysis (P for trend= .006). Compared with F nucleatum–negative cases, F nucleatum–high cases were inversely associated with the density of CD3+ T cells (for a unit increase in quartile categories of CD3+ T cells as an outcome: multivariable odds ratio, 0.47 [95% CI, 0.26-0.87]) (Table 3). The association of the amount of F nucleatum with the density of CD8+, CD45RO+, or FOXP3+ T cells in tumor tissue was not statistically significant (P for trend > .013; with adjusted α level of .013) (Table 3). We also used 4 ordinal categories of the amount of tissue F nucleatum (F nucleatum–negative, low, middle, and high) and observed similar findings in terms of the associations with the density of T cells (eTable 4 in the Supplement).

In an exploratory analysis, we did not observe a significant association of tissue F nucleatum with a Crohn-like reaction, peritumoral lymphocytic reaction, intratumoral periglandular reaction, or presence of tumor-infiltrating lymphocytes (P ≥ .11) (eTable 5 in the Supplement).

Tissue F nucleatum and Colorectal Cancer Mortality

In our exploratory analysis, we did not observe a significant association of tissue F nucleatum with colorectal cancer–specific mortality (P for trend= .45) or overall mortality (P for trend= .64) (eTable 6 in the Supplement).

Discussion

We conducted this study to test the hypothesis that the amount of F nucleatum in colorectal carcinoma tissue might be inversely associated with the density of T cells in tumor tissue. Using the database of the 598 colorectal carcinoma cases in the 2 US nationwide prospective cohort studies, we found that a greater amount of F nucleatum was associated with a lower density of CD3+ T cells in colorectal carcinoma tissue.

High densities of CD3+ pan-T cells and T-cell subpopulations (CD8+, CD45RO+, and FOXP3+ T cells) in colorectal carcinoma have been associated with better patient survival, indicating a major role of T cell–mediated adaptive immunity in inhibiting colorectal tumor progression.41 Virulence factors derived from F nucleatum have been shown to inhibit T-cell activity.26,27 Previous studies have shown that a virulence factor derived from Helicobacter pylori, which has been shown to cause gastric adenocarcinoma, can inhibit T-cell activity.42 This immunosuppressive effect may be similar to the potential immunosuppressive effect of F nucleatum. In the ApcMin/+ mouse model, F nucleatum promotes colonic neoplasia development through the recruitment of myeloid-derived suppressor cells into the tumor microenvironment.28 Myeloid-derived suppressor cells inhibit T-cell proliferation and induce T-cell apoptosis.43 These lines of experimental evidence are consistent with our finding of the inverse association between the amount of F nucleatum and the density of CD3+ T cells in tumor tissue. Additional studies are needed to investigate myeloid-derived suppressor cells in relation to F nucleatum in colorectal carcinoma tissue.

Studies have shown significant differences in the composition of intestinal microbiota along bowel subsites, likely leading to differences in colonic mucosal immunity.44,45 Consistent with a continuum of changes in intestinal microbiota and luminal contents, proportions of specific molecular features in colorectal cancer (namely, MSI-high, CIMP-high, and BRAF and PIK3CA mutations) continuously decrease from ascending colon to rectum.46,47 In the present study, the amount of F nucleatum in colorectal carcinoma tissue seemed to be associated with proximal tumor location, consistent with the previous study.25 Because epidemiologic evidence suggests that colonoscopy screening may be less effective for preventing proximal colon cancer than distal colon cancer,30,48 more effective prevention strategies may need to be developed for proximal colon cancer. Diet, antibiotics, and probiotics and prebiotics have been shown to influence the composition of intestinal microbiota.49 In light of our findings, it would be intriguing for future investigations to explore the potential influence of diet, lifestyle factors, and environmental exposures on F nucleatum and its immunosuppressive effect, which may have important implications for the development of colorectal cancer prevention strategies through targeting microbiota and immune cells.

We acknowledge limitations of our study. With the use of FFPE tissue specimens, routine histopathology procedures including tissue fixation, paraffin embedding, and storage may influence the quantitative PCR assay to detect microorganisms. However, technical artifacts, if any, would have affected our results likely toward the null hypothesis. In our limited validation study, we did detect F nucleatum in both paired FFPE and frozen tissue specimens in 2 colorectal carcinoma cases by means of the quantitative PCR assay. Our validation study also showed 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. In addition, our data on the relationship between F nucleatum and tumor MSI and CIMP status are in agreement with the recent study that used a quantitative PCR assay for Fusobacterium in frozen tissue specimens.25 These results suggest an acceptable performance of the quantitative PCR assay for F nucleatum in FFPE tissue specimens. Another limitation is the cross-sectional nature of our study. Hence, we cannot exclude a possibility of reverse causation. Although it is possible that immune cells may eradicate F nucleatum, experimental evidence indicating an immunosuppressive effect of F nucleatum on T-cell activity2628 formed a basis for our specific hypothesis. Because any experimental system cannot perfectly recapitulate the complex nature of the human tumor or immune system, analyses of human cancer tissue in a large population are useful in elucidating the relationship between microbiota and immunity in cancer.

Strengths of this study include the use of our molecular pathological epidemiology database (of nearly 600 colorectal carcinoma cases in the 2 US nationwide, prospective cohort studies), which integrates key tumor molecular features, the amount of F nucleatum, and immune reaction status in colorectal carcinoma tissue. The sample size and comprehensiveness of this population-based colorectal cancer database enabled us to assess the association between the amount of F nucleatum and the density of T cells, controlling for potential confounders.

Conclusions

In this cross-sectional analysis of 2 US nationwide prospective cohort studies, a greater amount of F nucleatum was associated with a lower density of CD3+ T cells in colorectal carcinoma tissue. These findings need to be validated by additional studies. On validation, our findings may provide insights for strategies to target microbiota and immune cells for colorectal cancer prevention and treatment.

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Article Information

Accepted for Publication: April 13, 2015.

Corresponding Author: Shuji Ogino, MD, PhD, MS, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 450 Brookline Ave, Room M422, Boston, MA 02215 (shuji_ogino@dfci.harvard.edu).

Published Online: June 4, 2015. doi:10.1001/jamaoncol.2015.1377.

Author Contributions: Drs Mima and Ogino had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Mima, Harris, Chan, Garrett, Huttenhower, Fuchs, Ogino.

Acquisition, analysis, or interpretation of data: Mima, Sukawa, Nishihara, Qian, Yamauchi, Inamura, Kim, Masuda, Nowak, Nosho, Kostic, Giannakis, Watanabe, Bullman, Milner, Giovannucci, Garraway, Freeman, Dranoff, Chan, Garrett, Fuchs, Ogino.

Drafting of the manuscript: Mima, Chan, Ogino.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: Mima, Nishihara, Giovannucci, Ogino.

Obtained funding: Chan, Huttenhower, Fuchs, Ogino

Administrative, technical, or material support: Nishihara, Kim, Masuda, Nosho, Kostic, Watanabe, Chan, Garrett, Fuchs, Ogino.

Study supervision: Qian, Milner, Harris, Chan, Huttenhower, Fuchs, Ogino.

Conflict of Interest Disclosures: Dr Chan previously served as a consultant for Bayer Healthcare, Millennium Pharmaceuticals, Pozen Inc, and Pfizer Inc. No other disclosures are reported.

Funding/Support: This work was supported by National Institutes of Health (NIH) grants (P01 CA87969 to S. E. Hankinson, UM1 CA186107 to M. J. Stampfer, P01 CA55075 and UM1 CA167552 to W. C. Willett, K07 CA190673 to Dr Nishihara, R01 CA137178 to Dr Chan, P50 CA127003 to Dr Fuchs, R01 CA151993 to Dr Ogino) and by grants from the Paula and Russell Agrusa Fund for Colorectal Cancer Research, the Friends of the Dana-Farber Cancer Institute, Bennett Family Fund, and the Entertainment Industry Foundation through the National Colorectal Cancer Research Alliance. Dr Mima is supported by a fellowship grant from Uehara Memorial Foundation. Dr Kim is supported by an Early Exchange Postdoctoral Fellowship Grant from Asan Medical Center.

Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Disclaimer: The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Additional Contributions: We would like to thank the participants and staff of the Nurses’ Health Study and the Health Professionals Follow-up Study for their valuable contributions, as well as the following state cancer registries for their help: Alabama, Arizona, Arkansas, California, Colorado, Connecticut, Delaware, Florida, Georgia, Idaho, Illinois, Indiana, Iowa, Kentucky, Louisiana, Maine, Maryland, Massachusetts, Michigan, Nebraska, New Hampshire, New Jersey, New York, North Carolina, North Dakota, Ohio, Oklahoma, Oregon, Pennsylvania, Rhode Island, South Carolina, Tennessee, Texas, Virginia, Washington, Wyoming.

Additional Information: We use HUGO (Human Genome Organisation)-approved official symbols for genes and gene products, including APC, BRAF, CACNA1G, CD3, CD8, CDKN2A, CRABP1, FOXP3, IGF2, KRAS, MLH1, NEUROG1, PIK3CA, PTPRC, RUNX3, SLCO2A1, and SOCS1, all of which are described at http://www.genenames.org.

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