Fusobacterium nucleatum Is Associated with Tumor Characteristics, Immune Microenvironment, and Survival in Appendiceal Cancer

Abstract

Emerging evidence highlights the role of the tumor microbiome, including Fusobacterium nucleatum (Fn), in a wide range of gastrointestinal cancers. Fn purportedly contributes to tumorigenesis by activating oncogenic pathways and modulating immune responses. Although the prevalence and impact of Fn has been extensively studied in colorectal cancer, no previous systematic or in situ studies have been performed in appendiceal cancer (AC). The aim of this study was to evaluate the prevalence and association of Fn density in AC with clinical factors and oncologic outcomes. Archival tissue from 54 patients with AC was assessed for Fn density using RNA in situ hybridization. Clinicopathological variables were obtained for each case through electronic medical record review, and the immune microenvironment was characterized in each case using immunohistochemistry to quantify CD3+ and CD8+ T lymphocytes and M1-/M2-like tumor-associated macrophages. In AC, Fn density was associated with patient age, tumor grade, and histologic subtype. Fn was negatively associated with CD3+ and CD8+ T lymphocytes and positively associated with M2-like TAMs in low-grade AC. Interestingly, tumor Fn content was associated with better overall and progression-free survival, even when controlling for tumor grade. In this exploratory study, we found that Fn is prevalent in AC. Fn is associated with a number of clinical, pathologic, immunologic, and prognostic variables in AC that are distinct from the corresponding observed associations in colorectal cancer. Further research is warranted to validate these findings and explore the mechanistic contributions of Fn to AC pathogenesis or immune response.

Keywords: Fusobacterium nucleatum; appendiceal cancer; immune microenvironment; survival outcomes; tumor microbiome.

Figure 1

RNA-in situ hybridization with Fn-specific probe. (A) Negative control specimen of small bowel tissue injected with PBS. (B) Positive control of small bowel tissue injected with undiluted resuspended Fn culture. (C,D) Individual Fn cells adherent to neoplastic mucosal tissue from patients with mucinous adenocarcinoma and low-grade appendiceal mucinous neoplasm, respectively. (E,F) Clustered Fn forms from patients with high grade goblet cell adenocarcinoma and a premalignant serrated sessile lesion of the appendix, respectively.

Figure 2

Co-localized staining of Fn and immune cells in appendiceal neoplasms. Staining for Fn (upper left), CD3+ lymphocytes (upper middle), CD8+ lymphocytes (upper right), M1-like macrophages (right middle) and M2-like macrophages (lower right) in a goblet cell adenocarcinoma primary appendiceal tumor. The red arrows point to the identified target cells in each panel, respectively.

Figure 3

F. nucleatum is prevalent and widely variable in tissue tumor content among cases of appendiceal cancer. Histogram of Fn density among examined cases demonstrates a right-skewed distribution with high-Fn-density outliers.

Figure 4

Intra-tumoral Fn density and patient age in appendiceal cancer. A log-linear relationship was observed between tumor tissue Fn content and increasing age.

Figure 5

Association of intra-tumoral Fn density with tumor histologic type, grade, and location (primary vs. peritoneal metastases) in AC. Weak evidence for variation on the basis of histologic type and grade was found (p value shown for Kruskal–Wallis equality of populations rank test), whereas no measurable difference was obtained between primary and metastatic lesions. LAMN, low grade appendiceal mucinous neoplasm; mAC, mucinous adenocarcinoma; gcAC, goblet cell adenocarcinoma; srcAC, signet ring cell adenocarcinoma; AC NOS, adenocarcinoma not otherwise specified; G1, Grade 1; G2, Grade 2; G3, Grade 3.

Figure 6

Intra-tumoral Fn density and the AC immune microenvironment. Overall, tumor Fn content was negatively associated with intra-tumoral lymphocyte density (upper left) and positively associated with tumor-associated macrophage (TAM) density (lower left). These relationships were most pronounced in high-grade tumors for lymphocytes (upper right) and low-grade tumors for TAMs (lower right).

Figure 7

Kaplan–Meier curves for progression and survival in AC, stratified by intra-tumoral Fn density. Fn was significantly associated with improved survival in AC (Table 2), a finding that was grade independent. A trend toward improved progression-free survival was also observed on multivariable analysis (Table 2). For progression-free and overall survival analysis, 49 patients had outcome metrics available at the time of analysis. On each graph, the blue line and the orange line represent the <40th percentile and the >40th percentile groups, respectively, determined by the CuttOff Finder as described by Budczies et al [24].