Increased Fusobacterium tumoural abundance affects immunogenicity in mucinous colorectal cancer and may be associated with improved clinical outcome

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 summarised in Table 1. We found mucinous tumours 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 categorised by consensus molecular subtype (p < 0.001). Mucinous tumours were over-represented in CMS1 (immune) (26.4% vs 11.7%) and CMS4 (mesenchymal) (31.9% vs 23%) categories and under-represented 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)

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 tumours to be strongly associated with an increased abundance of Fusobacterium (p < 0.001) (Fig. 1A). No statistically significant association was evident between Fusobacterium relative abundance and MSI status in the mucinous cohort (p = 0.19, Fig. 1A).

Mucinous status and elevated Fusobacterium relative abundance were both independently found to impact composition of immune cells in the TCGA CRC cohort

Overall, mucinous tumours were associated with a significantly greater ratio of total immune cells to epithelial/stromal cells in the TCGA dataset (p < 0.001) (Table 2, Fig. 1B). Specifically, the mucinous cohort were associated with significantly greater proportions of CD8 + lymphocytes (p = 0.018), regulatory T-cells (p < 0.001), and M2 macrophages (p = 0.003) (Table 2, Fig. 1B).

Table 2 Immune cell expression in the TCGA cohort according to mucinous status and fusobacterium relative abundance as computed using the quanTIseq package

Tumours with high Fusobacterium relative abundance were found to be associated with significantly greater proportions of CD4 + lymphocytes (p = 0.031) and M1 macrophages (p = 0.006), whilst M2 macrophages (p = 0.043) were under-represented across this group (Table 2, Fig. 1B). Evaluation of the mucinous cohort in isolation, found a significant reduction in the proportion of B-cells (p = 0.035) in patients with elevated Fusobacterium relative abundance (Table 2, Fig. 1B).

Elevated Fusobacterium prevalence is associated with better outcomes in mucinous CRC in the TCGA dataset

Existing evidence has linked Fusobacterium abundance 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 relative abundance, in both mucinous and non-mucinous cohorts in isolation (Fig. 2A, B). When we restricted our analysis to non-mucinous CRC patients, we found high Fusobacterium relative abundance 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. 2B), DSS (HR 1.62, 95% CI 0.96 to 2.73, likelihood ratio test p = 0.08, logrank p = 0.07, Fig. 2B) or OS (HR 1.27, 95% CI 0.82 to 1.97, likelihood ratio test p = 0.29, logrank p = 0.29, Fig. 2B). However, univariate Cox regression models demonstrated how mucinous CRC patients with elevated Fusobacterium relative abundance 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. 2A).

We investigated the association between Fusobacterium relative abundance at higher resolution, namely at the species level, with mucinous status and clinical outcome (Sup. Fig. 1). In line with previous literature reports, we observed that F. nucleatum was the most abundant species (average 1.17%, 95% CI 0.00 to 10.60%), both across the whole unselected patient population and by mucinous status (mucinous: average 1.67%, 95% CI 0.00 to 11.46% vs. non-mucinous: average 1.10%, 95% CI 0.00 to 10.14%), (Sup. Fig. 1A-B). Fusobacterium periodonticum (average 0.15%, 95% CI 0.00% to 1.11%), Fusobacterium necrophorum (average 0.07%, 95% CI 0.00 to 0.22%), Fusobacterium gonidiaformans (average %, 95% CI 0.00 to 0.37%) and Fusobacterium mortiferum (average 0.04%, 95% CI 0.00 to 0.20%) were amongst the species with the highest mean relative abundance (Sup. Fig. 1A). When restricting the analysis to mucinous CRC patients, we observed an enrichment for Fusobacterium necrophorum (mucinous: average 0.18%, 95% CI 0.00 to 1.30% vs. non-mucinous: average 0.06%, 95% CI 0.00 to 0.21%) species (Sup. Fig. 1A).

Next, we sought to investigate the association between the relative abundance of species from the Fusobacterium genus with clinical outcome (OS, DSS, DFS, Sup. Fig. 1C). Univariate Cox regression models fitted on the whole unselected patients population revealed that patients with high F. nucleatum relative abundance have worse OS (HR 1.57, 95% CI 1.05–2.36, p = 0.03) and DSS (HR 1.87, 95% CI 1.16–3.03, p = 0.01), (grey-shaded panels, Sup. Fig. 1C). Furthermore, Cox regression models fitted with an interaction term capturing the differential impact of species abundance by mucinous status confirmed the findings at the genus taxonomic rank (light-red-shaded panels, Sup. Fig. 1C). High species relative abundance is associated with more favourable clinical outcomes in the mucinous subpopulation. In contrast, the reverse is observed in the non-mucinous subpopulation whereby high species relative abundance is associated with worse clinical outcomes.

Fusobacterium abundance in rectal cancer tumour 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)

Fusobacterium abundance 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 abundant in mucinous as opposed to non-mucinous RC; however, this trend fell short of statistical significance (p = 0.070) (Fig. 3A).

Fig. 3figure 3

Fusobacterium abundance and mucinous status were found to impact immune cell expression in RC in our BRCC cohort. A Box and whisker blots depicting Fusobacterium abundance according to mucinous status. Statistical significance was evaluated using a Kruskal–Wallis test and the p-value is reported. B Image derived from the GeoMx platform of a mucinous core depicting pancytokeratin (Blue), Fusobacterium (Green) and Syto 13 (Red). C Box and whisker plots depicting specific immune cell counts; according to mucinous status and Fusobacterium relative abundance (high and low) within the BRCC RC cohort. Statistical significance was evaluated using Kruskal–Wallis tests. * indicates a statistically significant difference between Fusobacterium high and Fusobacterium low cohorts. # indicates a statistically significant difference between mucinous and non-mucinous cohorts

Mucinous status and elevated Fusobacterium abundance are associated with increased proportions of immune cells in our rectal cancer tumour microarray validation cohort

Next, we looked to determine an association between mucinous status and Fusobacterium abundance with immune cell populations 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. 3C).

Table 4 Immune cell expression in the BRCC cohort according to mucinous status and fusobacterium relative abundance

Tumours with high Fusobacterium abundance were found to be associated with a significantly greater proportion of B cells (p = 0.031) (Table 4, Fig. 3C).

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