Increased Fusobacterium relative abundance was not associated with MSI status in mucinous CRC in the TCGA dataset
From the TCGA dataset, we included 72 cases (12%) of mucinous and 522 cases (88%) of non-mucinous CRC. The clinical and pathologic characteristics of the included patients are summarized in Table 1. We found mucinous tumors were more likely to be proximal (P < 0.001) and had a higher incidence of MSI compared to the non-mucinous CRC group (P < 0.001). The two cohorts also demonstrated significant differences when categorized by Consensus Molecular Subtype (P < 0.001). Mucinous tumors were overrepresented in CMS1 (immune) (26.4% 11.7%) and CMS4 (mesenchymal) (31.9% v 23%) categories and underrepresented in the CMS2 (canonical) group. Following an investigation into whether the relative abundance (RA) of Fusobacterium at the genus taxonomic rank differed between mucinous and non-mucinous CRC, we found a trend, albeit not statistically significant, whereby patients with mucinous CRC trended to have higher Fusobacterium relative abundance compared to patients with non-mucinous CRC (P = 0.07).
Table 1
Clinical characteristics of the patients of the TCGA-COAD-READ cohort. CRC colorectal cancer, AJCC American Joint Committee on Cancer, CMS consensus molecular subtypes, IQR inter-quartile range.Categorical data reported as n (%). Continuous data reported as median (IQR). aData not available in full cohort: n in parentheses = number with data available
| | Mucinous CRC (n = 72) | Non-mucinous CRC (n = 522) | p-Value |
Age | | 67(52-77.5) | 68(58–76) | 0.611 |
Sex | Male | 37(51.4%) | 241(46.2%) | 0.48 |
| Female | 35(48.6%) | 281(53.8%) | |
Tumor Location(n = 579)a | Colon | 58(81.7%) | 366(72%) | 0.115 |
| Rectum | 13(18.3%) | 142(28%) | |
Stage (n = 574)a | AJCC 1 | 10(14.1%) | 90(17.9%) | 0.571 |
| AJCC 2 | 28(39.4%) | 189(37.6%) | |
| AJCC 3 | 25(35.2%) | 147(29.2) | |
| AJCC 4 | 8(11.3%) | 77(15.3%) | |
T Stage(n = 593)a | T1 | 2(2.8%) | 18(3.5%) | 0.528 |
| T2 | 9(12.5%) | 90(17.3%) | |
| T3 | 50(69.4%) | 358(68.7%) | |
| T4 | 11(15.3%) | 55(10.6%) | |
N Stage(n = 591)a | N0 | 39(54.2%) | 297(57.2%) | 0.528 |
| N1 | 16(22.2%) | 128(24.7%) | |
| N2 | 17(23.6%) | 94(18.1%) | |
M Stage(n = 525)a | M0 | 51(86.4%) | 389(83.5%) | 0.693 |
| M1 | 8(13.6%) | 77(16.5%) | |
Resection Margin(n = 438)a | R0 | 51(96.2%) | 381(99%) | 0.157 |
| R1/R2 | 2(3.8%) | 4(1%) | |
Lymphovascular Invasion(n = 535)a | Yes | 22(33.8%) | 197(41.9%) | 0.269 |
| No | 43(66.2%) | 273(58.1%) | |
Perineural Invasion(n = 230)a | Yes | 6(22.2%) | 54(26.6%) | 0.800 |
| No | 21(77.8%) | 149(73.4%) | |
Vascular Invasion(n = 514)a | Yes | 12(20%) | 115(25.3%) | 0.459 |
| No | 48(80%) | 339(74.7%) | |
Microsatellite Status(n = 553)a | MSI | 21(31.8%) | 68(14%) | < 0.001 |
| MSS | 45(68.2%) | 419(86%) | |
CMS(n = 536)a | CMS1 | 19(26.4%) | 61(11.7%) | < 0.001 |
| CMS2 | 4(5.6%) | 234(44.8%) | |
| CMS3 | 20(27.8%) | 55(10.5%) | |
| CMS4 | 23(31.9%) | 120(23%) | |
Next, we sought to investigate the relationship between Fusobacterium abundance and MSI status. In keeping with pre-existing evidence, when we restricted our analysis to non-mucinous CRC cases, we found MSI tumors to be strongly associated with increased prevalence of Fusobacterium (P < 0.001) (Fig. 1 (A)). No statistically significant association was evident between Fusobacterium relative abundance and MSI status in the mucinous cohort (P = 0.190, Fig. 1 (A)).
Mucinous status and elevated Fusobacterium relative abundance were both independently found to impact immune cell expression in the TCGA CRC cohort
Overall, mucinous tumors were associated with a significantly greater expression ratio of total immune cells to epithelial/stromal cells in the TCGA dataset (P < 0.001) (Table 2, Fig. 1 (B)). Specifically, the mucinous cohort were associated with significantly greater CD8 + lymphocyte (P = 0.018), regulatory T-cell (P < 0.001), and M2 macrophage (P = 0.003) expression (Table 2, Fig. 1(B)).
Table 2
Immune cell expression in the TCGA cohort according to mucinous status and fusobacterium relative abundance as computed using the quanTIseq package.
Cell Type | Level | Coefficient | Lower 95% CI | Upper 95% CI | p-value |
Epithelium/Stroma | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium(high) | -3.70 -1.30 1.8 | -5.80 -2.60 -1.60 | 1.70 0.06 5.10 | < 0.001 0.061 0.297 |
CD4 + Lymphocytes | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium(high) | 0.40 0.42 -0.30 | -0.19 0.04 -1.20 | 0.98 0.79 0.65 | 0.182 0.032 0.538 |
CD8 + Lymphocytes | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium (high) | 0.42 0.14 -0.36 | 0.07 -0.08 -0.92 | 0.76 0.37 0.20 | 0.018 0.207 0.204 |
T Regs | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium (high) | 1.1 0.39 0.12 | 0.46 <-0.01 -0.87 | 1.70 0.78 1.10 | < 0.001 0.053 0.807 |
Dendritic Cells | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium (high) | -0.02 0.03 -0.04 | -0.15 -0.05 -0.25 | 0.11 0.12 0.17 | 0.744 0.458 0.713 |
B Cells | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium (high) | 0.28 0.14 -0.54 | -0.03 -0.06 -1.00 | 0.59 0.35 -0.04 | 0.078 0.159 0.035 |
M1 Macrophage | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium (high) | 0.63 0.73 -0.39 | -0.18 0.2 -1.7 | 1.40 1.30 0.93 | 0.129 0.007 0.565 |
M2 Macrophage | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium (high) | 0.96 -0.35 <-0.01 | 0.44 -0.69 -0.85 | 1.5 -0.01 0.85 | < 0.001 0.040 0.990 |
Natural Killer Cells | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium (high) | -0.03 -0.09 0.01 | -0.41 -0.34 -0.62 | 0.36 0.16 0.63 | 0.899 0.484 0.976 |
Neutrophils | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium (high) | 0.31 -0.16 -0.25 | -0.96 -0.80 -1.90 | 1.00 0.49 1.40 | 0.951 0.635 0.756 |
Tumors with high Fusobacterium relative abundance were found to be associated with significantly greater expression of CD4 + lymphocytes (P = 0.031) and M1 macrophages (P = 0.006), whilst M2 macrophages (P = 0.043) were under-expressed in this group (Table 2, Fig. 1 (B)). Evaluation of the mucinous cohort in isolation, found significant under-expression of B-cells (P = 0.035) in patients with elevated levels of Fusobacterium (Table 2, Fig. 1 (B)).
Elevated Fusobacterium prevalence is associated with better outcomes in mucinous CRC in the TCGA dataset
Existing evidence has linked Fusobacterium prevalence with prognostic outcomes in CRC. To examine its precise impact with regards to mucinous tumours, we compared outcomes between patients with high and low Fusobacterium burden, in both mucinous and non-mucinous cohorts in isolation (Fig. 2 (A,B)). When we restricted our analysis to non-mucinous CRC patients, we found high Fusobacterium prevalence did not appear to significantly impact DFS (HR 1.24, 95% CI 0.84 to 1.82, likelihood ratio test P = 0.29, logrank P = 0.28, Fig. 2 (B)), DSS (HR 1.62, 95% CI 0.96 to 2.73, likelihood ratio test P = 0.08, logrank P = 0.07, Fig. 2 (B)) or OS (HR 1.27, 95% CI 0.82 to 1.97, likelihood ratio test P = 0.29, logrank P = 0.29, Fig. 2 (B)). However, analysis of patients with mucinous CRC demonstrated patients with elevated Fusobacterium prevalence trended to have more favourable clinical outcomes, specifically with reference to DSS(HR 0.24, 95% CI 0.05 to 1.14, likelihood ratio test P = 0.04, logrank P = 0.052, Fig. 2(A)) where outcomes were significantly better in patients with a high Fusobacterium burden.
Fusobacterium prevalence in rectal cancer tumor microarray validation cohort
The BRCC cohort included 15 cases (26%) of mucinous and 43 cases (74%) of non-mucinous rectal cancer, with 66% of the cohort having underwent neoadjuvant chemoradiotherapy. 14% (n = 2) of the mucinous cohort were MSI-high compared to 2% (n = 1) of the non-mucinous group. Further clinical and pathologic characteristics of the included patients are summarised in Table 3.
Table 3
Clinical characteristics of the patients of the BRCC cohort. RC rectal cancer, AJCC American Joint Committee on Cancer .Categorical data reported as n (%). Continuous data reported as median (IQR). aData not available in full cohort: n in parentheses = number with data available
| | Mucinous RC (N = 15) | Non-Mucinous RC(N = 43) | p Value |
Male | | 53.3% (8) | 58.1% (25) | 0.75 |
Age | Median (IQR) | 71 (29–81) | 70(42–89) | 0.10 |
Stage | AJCC 1 | 13.3% (2) | 11.6% (5) | 0.17 |
| AJCC 2 | 60.0% (9) | 34.8% (15) | |
| AJCC 3 | 26.0% (4) | 53.4%(23) | |
| AJCC4 | 0.0% (0) | 0.0%(0) | |
T Stage | Tis-T1-T-2 | 20.0% (3) | 25.5% (11) | 0.28 |
| T3 | 60.0% (9) | 65.1% (28) | |
| T4 | 20.0% (3) | 9.3% (4) | |
N Stage | N0 | 73.3% (11) | 48.8% (21) | 0.52 |
| N1 | 6.7% (1) | 41.8%(18) | |
| N2 | 20.0% (3) | 9.3%(4) | |
M Stage | M0 | 100% (15) | 100% (43) | NA |
| M1 | 0.0% (0) | 0.0% (0) | |
Neoadjuvant CRT(n = 56)a | | 53.3% (8) | 69.8% (30) | 0.32 |
Adjuvant CRT (n = 52)a | | 53.3% (8) | 39.5% (17) | 0.26 |
MSI (n = 55)a | | 14.3%(2) | 2.4% (1) | 0.09 |
KRAS (n = 55)a | Mutant | 35.7%(5) | 17.1%(7) | 0.15 |
BRAF (n = 54)a | Mutant | 7.7%(1) | 2.4% (1) | 0.38 |
LVI (n = 57)a | | 6.7%(1) | 19%(8) | 0.26 |
Perineural Invasion(n = 56)a | | 14.3%(2) | 11.9%(5) | 0.82 |
Extramural Invasion(n = 56)a | | 7.1%(1) | 19.0%(8) | 0.29 |
Fusobacterium burden was quantified at a patient level and compared between patients with mucinous and non-mucinous RC. We again observed a trend whereby, Fusobacterium was more prevalent in mucinous as opposed to non-mucinous RC, however this trend fell short of statistical significance (P = 0.070) (Fig. 3(A)).
Mucinous status and elevated Fusobacterium colonisation association with immune cell expression in rectal cancer tumor microarray validation cohort
Next, we looked to determine an association between mucinous status and Fusobacterium prevalence with immune cell expression in our BRCC rectal cancer cohort. Mucinous rectal tumours were associated with significantly greater CD8 + lymphocyte (P = 0.022), regulatory T-cell (P = 0.047), and B-cell (P = 0.025) counts (Table 4, Fig. 3(C)).
Table 4
Immune cell expression in the BRCC cohort according to mucinous status and fusobacterium relative abundance.
Cell Type | Level | Coefficient | Lower 95% CI | Upper 95% CI | p-value |
Epithelium/Stroma | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium(high) | 0.02 -0.16 0.25 | -0.12 -0.07 -0.37 | 0.12 0.17 0.16 | 0.914 0.384 0.491 |
CD4 + Lymphocytes | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium(high) | 0.16 0.24 -0.04 | -0.02 -0.02 -0.04 | 0.01 0.01 0.03 | 0.349 0.202 0.950 |
CD8 + Lymphocytes | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium (high) | 0.27 0.31 -0.25 | -0.02 -0.02 -0.02 | 0.01 0.02 0.04 | 0.022 0.097 0.491 |
T Regs | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium (high) | 0.36 0.14 0.13 | -0.02 -0.01 -0.02 | 0 0.01 0.02 | 0.047 0.454 0.754 |
B Cells | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium (high) | 0.41 0.41 -0.42 | -0.01 -0.01 -0.01 | <-0.01 0 0.04 | 0.025 0.029 0.228 |
Other Immune cells | Mucinous Status(mucinous) Fusobacterium (high) Mucinous Status(mucinous): Fusobacterium (high) | 0.30 0.51 -0.17 | -0.04 -0.03 -0.04 | 0.01 -0.01 0.06 | 0.103 0.006 0.662 |
Tumors with high Fusobacterium relative abundance were found to be associated with significantly greater expression of B cells (P = 0.031) (Table 4, Fig. 3(C)). Again, in our survival analysis, patients with mucinous RC and elevated Fusobacterium colonisation trended to have better DFS and OS, however in this smaller cohort, the improved outcomes were not found to be statistically significant (Supplementary Fig. 1)