Fusobacterium’s link to colorectal neoplasia sequenced: A systematic review and future insights

Table 1 Studies examining association between Fusobacteria and colorectal neoplasia

Authors	Year	Cohort information	Specimen type	Detection method	Relation to CRC location	CRC features	Fusobacteria sequencing depth and associations with colorectal neoplasia
Ahn et al[25]	2013	United States cohort: 47 CRCs and 94 healthy controls. Matched for age, sex, BMI and hospital	Stool from cases and controls	16S rRNA sequencing	-	-	Genus level: Fusobacterium rDNA was significantly more detected in of CRCs (31.9%) vs of controls (16%)
Vogtmann et al[26]	2016	United States cohort: 52 CRCs and 52 healthy controls, recruited between 1985-1987, matched by sex and BMI. Controls did not have a colonoscopy evaluation to rule out large polyps French validation cohort: 53 CRCs and 83 controls (61 healthy colons and 27 small adenomas) recruited from 2004-2006	Stool from cases and controls	Whole-genome shotgun sequencing Compared to16S rRNA sequencing from Ahn et al[25] study	-	-	Genus level: Whole genome analysis: Fusobacterium rDNA was significantly more detected in CRCs (76.9%) vs controls (48.1%). 16S rRNA sequencing: Fusobacterium rDNA was significantly more detected in CRCs (31.9%) vs controls (16%) Genus level: Fusobacterium rDNA was significantly more detected in CRCs (10.08%) vs controls (0.01%)
Gao et al[27]	2015	Chinese cohort: 31 CRCs and 30 healthy controls	Fresh-frozen tissue from cancer, adjacent non-cancerous tissue and normal mucosa samples at the time of surgery/colonoscopy and after colonoscopy bowel preparation	16S rRNA sequencing	Genus level: Fusobacterium rDNA was more detected in distal CRC compared to proximal CRC	-	Genus level: Higher relative percentage of Fusobacterium rDNA copies (relative to other bacterial rDNA) in CRCs (9.58%) vs adjacent non-cancerous tissues (0.57%)
Park et al[28]	2016	Korean cohort: 8 TAs, 10 SSA/Ps and 8 CRCs	Fresh-frozen tissue after colonoscopy bowel preparation	16S rRNA sequencing	All CRCs were distal	-	Phylum level: Fusobacterium rDNA was detected in 37.5% of TAs, 50% of SSA/Ps and 100% of CRCs. Higher relative percentage of Fusobacterium rDNA copies in CRC tissue (33.8%) vs TA (4.3%) and SSA (1.9%). No difference in relative concentration of Fusobacterium rDNA copies between TAs and SSA/Ps
Feng et al[29]	2015	Austrian cohort: 46 CRCs, 44 advanced adenomas and 57 healthy controls. Ages between 45-86 yr, both genders and White race	Stool from cases and controls	Metagenomics Whole-genome shotgun sequencing	-	-	Genus level: Higher relative percentage of Fusobacterium rDNA copies in CRCs vs Carcinoma in situ. Higher relative percentage of Fusobacterium rDNA copies in CRCs vs advanced adenomas vs controls Genus level: No association between relative percentage of Fusobacterium rDNA copies and anthropometric measures or diet (meat, fiber, vegetables and fruit intake)
Burns et al[30]	2015	United States cohort: 44 CRCs	Fresh-frozen tissue from cancer and adjacent non-cancerous tissue at the time of surgery and after bowel preparation	16S rRNA sequencing	-	-	Species level: No specific species identification. Higher relative concentration of Fusobacterium rDNA copies in CRCs vs adjacent non-cancerous tissues. Correlated enrichment with Providencia Species level: No specific species identification. Tumor microbiome was enriched with genes encoding virulence and toxin proteins that were associated with and dependent on the presence of Fusobacterium and Providencia
Viljoen et al[31]	2015	South African cohort: 55 CRCs. 70.4% mixed ancestry, 14.8% White, 11.1% African, equal gender, 7 MSI-high (4 CRCs with Lynch syndrome), 3 MSI-low. 41.5% received chemo-radiation prior to sample collection. 18 FFPE CRCs that are MSI high (2 CRCs with Lynch syndrome)	Fresh-frozen tissue from cancer and adjacent non-cancerous tissue at the time of surgery and after bowel preparation. FFPE samples after bowel preparation	16S rRNA sequencing Metagenomics	No association between number of Fusobacterium rDNA copies in CRC tissue and colon vs rectum location	Association between higher number of Fusobacterium rDNA copies in CRC tissue and MSI-H status	Species level: No specific species identification. Fusobacterium rDNA was detected in 82% of CRCs and 81% adjacent non-cancerous tissues with concurrent presence in 80% of paired CRC and adjacent tissue. Higher number of Fusobacterium rDNA copies in CRCs vs adjacent non-cancerous tissues.Species level: No specific species identification. Higher number of Fusobacterium rDNA copies in CRC tissue was associated with African race and age < 60. Species level: No specific species identification. Higher number of Fusobacterium rDNA copies in CRC tissue correlated with pks-positve E. coli in normal tissue; but no correlation with Enteropathogenic Escherichia coli, Streptococcus gallolyticus, Enterococcus faecalis, Enterotoxigenic Bacteroides fragili or afaC-positive E. coli
Zhou et al[32]	2016	Chinese cohort: 97 CRCs and 48 healthy controls. Age and sex matched	Fresh-frozen tissue from cancer, adjacent non-cancerous tissue and normal mucosa samples at the time of surgery/colonoscopy and after bowel preparation	16S rRNA sequencing	No association between relative percentage of Fusobacterium rDNA copies in CRC tissue and colon vs rectum location	No association between relative percentage of Fusobacterium rDNA copies in CRC tissue and CEA, P53, EGFR, Ki67, CA199 or CRP	Species level: No specific species identification. Higher number of Fusobacterium rDNA copies correlated positively with presence of chronic inflammation in CRC tissue Species level: No specific species identification. Fusobacterium rDNA was detected in 72.16% in CRCs vs 67.01% of adjacent non-cancerous tissues, both higher than controls. Species level: Higher number of Fusobacterium rDNA copies correlated positively with that of Enterotoxigenic Bacteroides fragilis, E. faecalis in CRC tissue when compared to adjacent non-cancerous tissue
Dejea et al[33]	2014	United States cohort: 30 CRCs, 6 adenomas and 22 healthy controls Malaysian cohort: 21 CRCs and 1 adenoma.	Fresh-frozen, formalin fixed tissue from tumors (adenomas and cancers), adjacent normal tissue and normal mucosa samples at the time of surgery/colonoscopy after bowel preparation	16S rRNA sequencing	Similar relative percentage of Fusobacterium rDNA copies (≥ 25%) in CRC tissue proximal to hepatic flexure vs more distal CRC	-	Phylum level: Fusobacterium rDNA was detected in 32% of CRC, none of the matched normal tissue or healthy controls
Marchesi et al[34]	2011	Netherlands cohort: 6 CRCs	Fresh-frozen tissue from cancer and adjacent non-cancerous tissue at the time of surgery and after bowel preparation	16S rRNA sequencing	-	-	Genus level: Fusobacterium rDNA was more detected with also higher relative percentage of Fusobacterium rDNA copies in CRC compared to adjacent non-cancerous tissue
Thomas et al[35]	2016	Brazilian cohort: 18 rectal cancers (no prior neoadjuvant therapy) and 18 healthy controls	Fresh-frozen tissue from cancer and normal mucosa samples at the time of surgery/colonoscopy and after bowel preparation	16S rRNA sequencing	Rectal cancers only	-	Species level: No specific species identification. Higher number of Fusobacterium rDNA copies in rectal cancers compared to normal controls
Wang et al[36]	2012	Chinese cohort: 46 CRCs and 56 healthy controls	Stool, prior to bowel preparation	16S rRNA sequencing	-	-	Genus level: Higher relative percentage of Fusobacterium rDNA copies in CRC tissue compared to controls
Gao et al[37]	2017	Chinese cohort: 65 CRC patients	Fresh-frozen tissue from cancer and matched adjacent non-cancerous tissue at the time of surgery after bowel preparation	16S rRNA sequencing	Genus level: Fusobacterium rDNA was more detected in distal CRC compared to proximal CRC	No association between relative percentage of F. nucleatum rDNA copies in CRC tissue and presence of K-ras mutation	Phylum level: Fusobacterium rDNA was significantly more detected in CRC (8.5%) compared to matched normal tissue (4.13%)
Allali et al[38]	2015	United States and Spanish cohorts: 90 CRC patients	Fresh-frozen tissue from cancer and adjacent non-cancerous tissue at the time of surgery after bowel preparation	16S rRNA	-	United States cohort: Higher relative percentage of Fusobacterium rDNA copies in both CRC and matched normal tissue in the right colon and splenic flexure vs left colon and sigmoid colon Spanish cohort: Higher relative percentage of Fusobacterium rDNA copies in CRC tissue in the left colon	Phylum level: Higher relative percentage of Fusobacterium rDNA copies in CRC vs matched normal tissue in Spanish cohort but not United States cohort. Higher relative percentage of Fusobacterium rDNA copies in matched adjacent non-cancerous tissue of the United States cohort compared to matched adjacent non-cancerous tissue of the Spanish cohort
Zackular et al[39]	2014	United States and Canadian cohort: 30 CRC, 30 TA and 30 healthy controls	Frozen fecal samples prior to colonoscopy and bowel preparation.	16S rRNA	No relation between relative percentage of Fusobacterium rDNA copies in CRC tissue to CRC location	-	Genus level: Higher relative percentage of Fusobacterium rDNA copies in CRC compared to both adenoma and healthy controls
Zeller et al[40]	2014	French cohort (population F): 53 CRC, 42 TAs, and 61 healthy control patients. German cohort (Population G): 38 CRC patients. German, Danish and Spanish cohorts (Population H): 297 IBD and healthy controls. German cohort (48 CRC patients at the time of CRC surgery)	Populations F and G: Stool prior to colonoscopy bowel preparation Population H: Stool German cohort: CRC and matched normal tissue	16S rRNA	-	-	Species level: Higher relative percentage of Fusobacterium rDNA copies in CRC compared to controls Subspecies level: F. nucleatum ssp. vincentii and F. nucleatum ssp. animalis are predominant in CRC tissue
Castellarin et al[41]	2012	Canadian cohort: 99 CRCs	Frozen tissue from cancer and adjacent non-cancerous tissue at the time of surgery after bowel preparation	Metagenomics F. nucleatum quantitative PCR	No association with proximal vs distal CRC location	Association between higher relative percentage of F. nucleatum rDNA copies in CRC and tumor involvement of more than 50% of the colon circumference	Subspecies level: Higher relative percentage of F. nucleatum subsp. Nucleatum rDNA copies in CRCs compared to matched normal tissues Species level: Confirmed in vitro invasion of F. nucleatum into human epithelial colonic cells Species level: No association between F. nucleatum and history of treatment or patient age
Chen et al[42]	2017	Chinese cohort: 14 CRCs 98 FFPE CRC	Frozen tissue at the time of surgery after bowel preparation FFPE CRC tissue after bowel preparation	16S rRNA FISH F. nucleatum-targeted probe	Higher frequency of F. nucleatum rDNA detection in proximal CRC than distal location	-	16s rRNA: Phylum level: Fusobacterium was a dominant phylum in CRC. FISH analysis. Species level: F. nucleatum rDNA was detected in 62.2% of FFPE CRC tissues No. difference in patients gender or age between CRCs that are F. nucleatum positive (detected) or absent
McCoy et al[43]	2013	United States Cohort: 10 CRCs, 48 adenomas and 67 healthy controls	Fresh-frozen normal rectal biopsies from adenoma and controls after bowel preparation Frozen tissue from CRC and adjacent non-cancerous tissue at the time of surgery after bowel preparation	F. nucleatum quantitative PCR 16S rRNA	-	Association between high number F. nucleatum rDNA copies in tissue and IL-6, IL-10, IL-17 and TNF-α in adenoma cases but no similar associations were seen in controls	Species level: Higher number of F. nucleatum rDNA copies seen in adenoma cases vs controls Species level: No association between F. nucleatum rDNA copy numbers and adenomas size/number Phylum level: Increased number of Fusobacterium rDNA copies in CRCs compared to matched normal tissues
Fukugaiti et al[44]	2015	Brazilian cohort: 7 CRCs and 10 healthy controls	Stool, prior to bowel preparation	16S rRNA sequencing	-	-	Species level: Both F. nucleatum and Clostridium difficile had higher number of rDNA copies in stool of CRC patients when compared to controls
Warren et al[45]	2013	Canadian cohort: 65 CRCs	Frozen tissue from cancer and adjacent non-cancerous tissue at the time of surgery after bowel preparation	Metatran-scriptomics	-	-	Species level: Higher relative percentage of F. nucleatum rDNA copies in CRC compared to matched normal tissue Genus level: Co-occurrence of F. nucleatum with Campylobacter (in vitro co-aggregation with C. showae) and Leptotrichia in CRC tissue Genus level: Higher relative percentage of Fusobacterium rDNA copies in tumor tissue was correlated with host immune response genes and oncogenes
Ito et al[46]	2015	Japanese cohort: 138 Microvesicular HPs, 129 SSAs, 102 TSAs, 131 adenomas and 544 CRCs with matched adjacent non-cancerous tissue as well as 20 healthy controls	FFPE CRC tissue after bowel preparation	F. nucleatum quantitative PCR	No relation between F. nucleatum detection or higher number of rDNA copies in CRC and CRC location (Rectum to splenic flexure vs Transverse colon to cecum) Gradual increase in percentage of SSAs that are F. nucleatum positive from sigmoid colon to cecum; no similar finding seen for TA, TSA and HP	High number of F. nucleatum rDNA copies in CRC was associated with MLH1 methylation, CMP-high status and MSI-high status. No association between detection or number of F. nucleatum rDNA copies in CRC to KRAS mutation, PIK3A mutation or miRNA 31 expression	Species level: F. nucleatum rDNA was detected in 56% of CRCs. Higher number of F. nucleatum rDNA copies in CRC tissue compared to matched normal tissue. F. nucleatum rDNA was not detected in 17/20 of healthy controls; no significant difference in number of F. nucleatum rDNA copies between matched normal tissue and healthy controls Species level: F. nucleatum rDNA detected in 24% of HPs, 35% of SSAs, 30% of TSAs and 33% of TAs. No difference in frequency of F. nucleatum rDNA detection between these groups Species level: F. nucleatum rDNA more frequently detected in CRCs compared to polyps after adjustment for confounders Species level: High number of F. nucleatum rDNA copies in CRC was positively associated with large tumor size
Nosho et al[47]	2016	Japanese cohort: 511 CRCs	FFPE CRC tissue after bowel preparation	F. nucleatum quantitative PCR	-	F. nucleatum rDNA presence in CRC was associated with MSI high status	Species level: F. nucleatum rDNA was present in 8.6% of CRCs
Mima et al[48]	2015	United States cohort: 598 CRCs from the Nurses’ Health Study and Health Professionals Follow-up Study	FFPE CRC tissue after bowel preparation	F. nucleatum quantitative PCR	-	High number F. nucleatum rDNA copies in CRC tissue was associated with lower CD3+ T-cells density. No association with CD8+, CD45RO+, or FOXP3+ T-cells density in CRC. No significant association with Crohn’s-like histology, or tumor infiltrating lymphocytes	Species level: F. nucleatum rDNA was more frequently detected in CRCs (13%) compared to matched normal tissue (3.4%)
Kostic et al[49]	2012	Spanish, United States and Vietnamese cohort: 95 CRCs	Frozen tissue from cancer and adjacent non-cancerous tissue at the time of surgery after bowel preparation	Whole genome sequencing 16S rRNA F. nucleatum quantitative, PCR	No association with CRC location	No association with CRC purity, inflammation, necrosis, and vascularization	Species level: Higher relative percentage of Fusobacterium rDNA copies in CRC compared to matched normal tissue Detected species: F. nucleatum (most dominant species), F. necrophorum, F. mortiferum, and F. perfoeten. Species level: Higher relative percentage of Fusobacterium rDNA copies in Spanish vs US/Vietnamese cohorts Species level: No association with age, gender, ethnicity
Flanagan et al[50]	2014	Czech cohort: 49 CRCs German cohort: 45 CRCs. Irish cohort:	Frozen tissue from cancer, adjacent non-cancerous tissue at the time of surgery after bowel preparation. Stool 28 CRCs and 52 TAs Stool from 7 CRCs, 24 TAs patients (10 adenoma with HGDs, 12 TVAs and 2 adenomas) and 25 healthy controls	F. nucleatum quantitative PCR	No association between relative percentage of F. nucleatum rDNA copies in CRC and colon vs rectum location	Association between higher relative percentage of F. nucleatum rDNA copies in CRC tissue and TP53 mutation in the Irish cohort (Small sample size) Association between higher relative percentage of F. nucleatum rDNA copies in CRC tissue with KRAS mutation No association between F. nucleatum and BRAF mutation. No association between F. nucleatum and CRC grade	Species level: Higher relative percentage of F. nucleatum rDNA copies in CRC and HGD compared to matched normal tissue. Similar relative percentage of F. nucleatum rDNA copies in TA, TVA compared to their respective matched normal tissue Species level: Increased relative percentage of F. nucleatum rDNA copies during the adenoma-carcinoma progression in cancerous and matched normal tissue (TA to TVA to HGD to CRC) Species level: Increased relative percentage of F. nucleatum rDNA copies in stool of CRC patients compared to adenomas. Stool relative percentage of F. nucleatum rDNA copies is similar between adenoma and healthy controls patients. No significant correlation in relative percentage of F. nucleatum rDNA copies between disease tissue (CRC and adenomas) and stool samples from same patient
Wu et al[51]	2013	Chinese cohort: 19 CRCs and 20 healthy controls. Matched for age, sex and body mass index	Stool	16S rRNA sequencing			Species level: Higher relative percentage of Fusobacterium rDNA copies compared to controls. Several species involved: F. nucleatum, F. periodonticum, F. necrophorum, F. ulcerans, F. varium, and F. gonidiaformans
Li et al[52]	2016	Chinese cohort: 101 CRC patients	Fresh-frozen tissue from cancer and adjacent non-cancerous tissue at the time of surgery after bowel preparation	F. nucleatum quantitative PCR	-	-	Species level: Increased relative percentage of F. nucleatum rDNA copies in 87.13% of CRCs compared to controls Higher relative percentage of F. nucleatum rDNA copies in CRC compared to controls
Mira-Pascual et al[53]	2015	Spanish cohort: 7 CRC, 8 TA, 7 healthy controls	Frozen fecal samples prior to colonoscopy and bowel preparation Biopsies from normal rectal mucosa of controls and neoplasm of cases after bowel preparation	F. nucleatum quantitative PCR	-	-	Species level: F. nucleatum rDNA more frequently present in fecal and tissue samples of tumor group (CRCs and polyps) compared to controls
Amitay et al[54]	2017	German cohort of patients aged 50 years old and above: 46 CRC, 113 advanced adenomas (TA > 1 cm in size, TVA, or with HGD), 110 adenomas, and 231 healthy controls	Frozen fecal samples prior to colonoscopy and bowel preparation. Median time between collection and storage was 7 d	F. nucleatum quantitative PCR	-	-	Subspecies level: Fusobacterium rDNA was more frequently present in CRC (54.3%) than advanced adenoma (23.9%), TA (23.6%) and healthy controls (25.1%) (P < 0.001) rDNA sequence of F. periodonticum was more detected in CRC compared to controls (P = 0.003). No difference in detection of rDNA of Fusobacterium simiae, F. nucleatum ssp. nucleatum, F. nucleatum ssp. animalis, F. nucleatum ssp. vincentii and F. nucleatum ssp. polymorphum between CRC and controls No significant difference between relative concentration of F. nucleatum rDNA copies in advanced adenomas/TAs vs Controls
Yu et al[55]	2015	Chinese cohort: 42 CRCs, 47 TAs and 52 healthy controls	Stool Left colonic biopsies	F. nucleatum quantitative PCR16S rRNA	-	-	Species level: Relative percentage of F. nucleatum rDNA copies per sample gradually increased from healthy control to TAs and to CRC. Results seen in both stool and tissue samples
Ye et al[56]	2017	United States cohort: 25 CRC patients	Fresh-frozen tissue from CRC and adjacent non-cancerous tissue at the time of surgery after bowel preparation	F. nucleatum quantitative PCR16S rRNA	-	Increased Chemokine (C-C motif) ligand 20 (CCL20) chemokine expression in all stages of CRC suggesting it is an early event in carcinogenesis. F. nucleatum ssp. Animalis induced CCL20 cytokine expression in CRC cell lines and monocytes. Monocytes are activated and migrate in the presence of F. nucleatum ssp. Animalis, this effect is inhibited by blocking CCL20 No control bacteria used in this experiment	Genus level: Increased relative percentage of Fusobacterium rDNA copies in CRC tissue vs normal matched tissue Species level: CRC samples contained F. periodonticum, F. canifelinum, F. varium, F. simiae, and F. nucleatum. F. nucleatum was the most frequently detected among Fusobacterium species Subspecies level: F. nucleatum ssp. Animalis most dominant among F. nucleatum subspecies in CRC samples
Chen et al[57]	2012	Chinese cohort: 46 CRCs and 56 healthy controls. BMI range 20-24, matched by sex	Stool and fecal swabs from cases and controls prior to bowel preparation Fresh-frozen tissue from cancer and adjacent non-cancerous tissue from cases at the time of surgery after bowel preparation	16S rRNA sequencing	-	-	Species level: Increased relative concentration of F. varium rDNA copies in fecal swabs of CRCs compared to controls Genus level: Increased relative concentration of Fusobacterium rDNA copies in CRC tissues vs stool specimens (4.97% vs 0.47%, P < 0.001) Genus level: Unifrac PCA analysis found no difference in microbial composition of cancers and adjacent non-cancerous tissues
Kasai et al[58]	2016	Japanese cohort: 9 CRCs (3 invasive and 6 carcinoma in adenoma), 50 TAs and 49 healthy controls	Stool prior to colonoscopy bowel preparation	16S rRNA sequencing	-	-	Species level: Increased relative percentage of F. varium rDNA copies in carcinoma in adenomas vs not detected in controls
Tahara et al[61]	2014	United States cohort: 149 CRCs and 89 adjacent tissues	Fresh-frozen CRC and adjacent non-cancerous tissue	Pan Fusobacterium and F. nucleatum quantitative PCR	-	Association with CIMP-high CRC, wild type TP53, MLH1 methylation, MSI-high and CHD7/8 mutation positivity	Genus and species level: Fusobacterium and F. nucleatum rDNA were more frequently detected in CRCs (74.3% and 52.3% respectively) compared to adjacent normal appearing mucosa (52.8% and 30.3% respectively). Higher relative percentage of Fusobacterium and F. nucleatum rDNA in CRC compared to normal appearing mucosa
Yazici et al[62]	2017	United States cohort: CRC (97 African Americans and 56 Whites). Healthy controls (100 African Americans and 76 Whites)	Fresh-frozen CRC, adjacent non-cancerous tissue and normal mucosa samples at the time of surgery/colonoscopy and after bowel preparation	F. nucleatum quantitative PCR 16S rRNA	-	-	Genera level: Fusobacterium was most abundant sulfidogenic bacteria identified in the study. No difference in relative concentrations of Fusobacterium rDNA copies between normal mucosa of cases and controls. Increased sulfidogenic bacteria in African Americans compared to non-Hispanic whites
Park et al[69]	2017	South Korean cohort: 160 MSI-high CRC. Excluded rectal carcinoma post neoadjuvant chemotherapy	FFPE CRC tissue after bowel preparation	F. nucleatum quantitative PCR	No association between relative concentrations of F. nucleatum rDNA copies in CRC and proximal versus distal/rectal CRC location	Association between high relative concentrations of F. nucleatum rDNA in CRC and BRAF mutation, CDKN2A (P16) promoter hypermethylation, tumor-infiltrating pan-macrophage density and CD68+ macrophages in tissue when compared with F. nucleatum-low/negative CRCs No association between high relative concentration of F. nucleatum rDNA in CRC tissue and CRC infiltrating CD3+ lymphocyte; PD-L1 expression status; Kras mutation; expression of MLH1, MSH2, MSH6 or PMS; CIMP status; or MLH 1 methylation when compared to CRCs that were F. nucleatum-low/negative	Species level: Of the MSI-high CRCs, 9% had high F. nucleatum rDNA relative concentrations, 58% had low F. nucleatum rDNA relative concentrations, and 33% of CRCs had no detected F. nucleatum in the tissue
Wei et al[70]	2016	Chinese cohort: 180 CRCs, all stages, median follow up 47 months	Fresh-frozen tissue from cancer and adjacent non-cancerous tissue at the time of surgery after bowel preparation	F. nucleatum quantitative PCR	No association between relative concentration of F. nucleatum rDNA copies in CRC and CRC location in colon vs rectum	High relative concentration of F. nucleatum rDNA copies in CRC was associated with high TNF-α, MMP9, NF-κB, β-catenin, CTNNB, KRAS and BRAF expression as well as lower MLH1 expression No association between relative concentration of F. nucleatum rDNA copies in CRC and COX1 or COX2 protein levels	Species level
Mima et al[72]	2016	United States cohort: 1,102 CRCs from the Nurses’ Health Study and Health Professionals Follow-up Study. Median follow up of 10.7 years	FFPE CRC tissue after bowel preparation	F. nucleatum quantitative PCR	Percentage of CRCs with high number of F. nucleatum rDNA copies increased gradually from rectum (2.5%) to cecum (11%) with a linear trend (P < 0.0001)	-	Species level
Mima et al[73]	2015	United States cohort: 1069 CRCs from the Nurses’ Health Study and Health Professionals Follow-up Study. Median follow up of 10.7 years	FFPE CRC tissue after bowel preparation	F. nucleatum quantitative PCR	Association with proximal CRCs location	Association between high number of F. nucleatum rDNA copies in CRCs and poor tumor differentiation Association between high number of F. nucleatum rDNA copies in CRCs and MSI-high CRC independent of CIMP and BRAF status Association between high number of F. nucleatum rDNA copies in CRC and MLH1methylation	Species level:
Yoon et al[78]	2017	North Korean cohort: 6 CRCs, 6 TAs, 6 SSAs and 6 healthy controls. Equal male female distribution	Normal rectal mucosa after bowel preparation	16S rRNA	-	-	Species level: F. nucleatum found in only one SSA patient rectal biopsy
Kostic et al[79]	2013	United States and United Kingdom cohorts: 27 CRCs, 28 TAs and 31 healthy controls	Fresh-frozen tissue from adenomas and adjacent normal tissue at the time of colonoscopy after bowel preparation Stool	Fusobacterium quantitative PCR	-	-	Species level: No specific species identified Fusobacterium detected in 48% of adenomas. Increased number of Fusobacterium rDNA copies in adenomas compared to matched normal tissue Species level: No specific species identified Higher Fusobacterium detection and number of copies in stool from CRC and adenoma compared to controls

Table 2 Studies looking at Fusobacterium associations with colorectal cancer stage and prognosis

Authors	Sample size	CRC depth of invasion	CRC lymph nodes metastasis	CRC metastatic disease	CRC stage	CRC prognosis
Viljoen et al[31]	South African cohort: 55 CRCs.	-	-	-	Association between higher number of F. nucleatum rDNA copies and late stage CRC (stage III and IV compared to stage I and II)	-
Zhou et al[32]	Chinese cohort: 97 CRCs	No association with relative percentage of Fusobacterium rDNA copies	No association with relative percentage of Fusobacterium rDNA copies	No association with relative percentage of Fusobacterium rDNA copies	No association with relative percentage of Fusobacterium rDNA copies	-
Zackular et al[39]	United States and Canadian cohort: 30 CRC, 30 TA, 30 healthy controls	-	-	-	No association with relative percentage of F. nucleatum rDNA copies in CRC	-
Castellarin et al[41]	Canadian cohort: 99 CRCs	-	Association between relative percentage of F. nucleatum rDNA copies in CRC and regional lymph nodes metastasis	-	No association with relative percentage of F. nucleatum rDNA copies in CRC	No association to between relative percentage of F. nucleatum rDNA copies in CRC and CRC overall survival
Chen et al[42]	Chinese cohort: 98 CRCs	-	No association with presence of F. nucleatum rDNA in CRC	-	-	-
Ito et al[46]	Japanese cohort: 544 CRCs	-	-	-	No association with detection or number of F. nucleatum rDNA copies	No association between detection or number of F. nucleatum rDNA copies and CRC overall survival-unknown follow up period
Nosho et al[47]	Japanese cohort: 511 CRCs	-	-	-	No association with detection of F. nucleatum rDNA in CRC	No association between F. nucleatum rDNA presence in CRC and CRC-specific survival-unknown follow up period
Mima et al[48]	United States cohort: 598 CRCs.	-	-	-	-	No relation between F. nucleatum rDNA copies in CRC and CRC-specific survival or CRC overall survival- unknown follow up period
Flanagan et al[50]	Czech, German and Irish cohorts: 122 CRCs	-	-	-	No association with relative percentage of F. nucleatum rDNA copies	Higher relative percentage of F. nucleatum rDNA copies was associated with shorter CRC overall survival within 3-5 years follow up (HR = 19.96, 95%CI: 1.42-281.42) (no adjustment for other confounders)
Li et al[52]	Chinese cohort: 101 CRC	No association	Association between relative percentage of F. nucleatum rDNA copies in CRC and lymph nodes metastasis	-	No association with relative percentage of F. nucleatum rDNA copies in CRC	-
Amitay et al[54]	German cohort: 46 CRC	-	-	-	Relative percentage of F. nucleatum rDNA copies in CRC was associated with advanced stage [stage I vs II (P = 0.012) and stage I vs III (P = 0.042)]	-
Park et al[69]	South Korean cohort: 160 MSI-high CRC.	-	No association with F. nucleatum rDNA detection or number of copies	-	No association with F. nucleatum rDNA detection or number of copies	No association between F. nucleatum rDNA detection or number of copies and disease-free survival
Wei et al[70]	Chinese cohort: 180 CRCs	Association between high relative percentage of F. nucleatum rDNA copies in CRC and depth of invasion	Association between high relative percentage of F. nucleatum rDNA copies in CRC and lymph nodes metastasis	-	-	High relative percentage of F. nucleatum rDNA copies in CRC was associated shorter CRC overall survival within 3 years follow up [HR = 1.993 (1.024 to 3.879)] High relative percentage of F. nucleatum rDNA copies in CRC was associated with shorter CRC disease-free survival within 3 years follow up [HR = 1.829 (1.000 to 3.345)]
Yu et al[71]	Chinese cohort: 88 CRCs	-	F. nucleatum rDNA was more frequently detected in metastatic lymph nodes of proximal vs distal CRC F. nucleatum detected in 100% of metastatic lymph nodes compared to 40% of lymph nodes without metastasis (P < 0.001)	-	-	-
Mima et al[73]	United States cohort: 1069 CRCs	Association between number of F. nucleatum rDNA copies in CRC and higher pT of the TNM staging	No association with number of F. nucleatum rDNA copies in CRC	No association with number of F. nucleatum rDNA copies in CRC	No association with number of F. nucleatum rDNA copies in CRC	High number of F. nucleatum rDNA copies in CRC was associated with shorter CRC-specific survival within 10.7 years follow up [HR = 1.58 (1.04 to 2.39)] for F. nucleatum-high vs F. nucleatum-negative CRCs]. (Multivariable models included CRC stage, age, sex, year of diagnosis, family history of CRC, CRC location, MSI status, CIMP status, KRAS status, BRAF, PIK3CA and CRC LINE-1 methylation.) No association between F. nucleatum rDNA copies in CRC and CRC overall mortality

CRC: Colorectal cancer; CIMP: CpG island methylator phenotype.