Pseudomyxoma peritonei: is disease progression related to microbial agents? A study of bacteria, MUC2 AND MUC5AC expression in disseminated peritoneal adenomucinosis and peritoneal mucinous carcinomatosis

Abstract

Background and aims: Pseudomyxoma peritonei (PMP) is characterized by peritoneal tumors arising from a perforated appendiceal adenoma or adenocarcinoma, but associated entry of enteric bacteria in the peritoneum has not been considered as a cofactor. Because Gram-negative organisms can upregulate MUC2 mucin gene expression, we determined whether bacteria were detectable in PMP tissues.

Methods: In situ hybridization was performed on resection specimens from five control subjects with noninflamed, nonperforated, non-neoplastic appendix and 16 patients with PMP [six with disseminated peritoneal adenomucinosis (DPAM) and 10 with peritoneal mucinous carcinomatosis (PMCA)]. Specific probes were designed to recognize: (1) 16S rRNA common to multiple bacteria or specific to H. pylori; (2) H. pylori cagA virulence gene; or (3) MUC2 or MUC5AC apomucins. Specimens from one patient with PMCA were examined by ultrastructural immunohistochemistry. Bacterial density and apomucin expression were determined in four histopathological compartments (epithelia, inflammatory cells, stroma, and free mucus).

Results: Enteric bacteria were detected in all specimens. Bacterial density and MUC2 expression were significantly (p < 0.05) higher in PMCA than in DPAM and controls and were highest in free mucin. MUC2 was also expressed in dysplastic epithelia and in associated inflammatory cells. MUC2 expression was significantly correlated with bacterial density.

Conclusions: Multiple enteric bacteria are present in PMP, and bacterial density and MUC2 expression is highest in the malignant form of PMP. Based on these observations, we propose that the bacteria observed in PMP may play a role in the mucinous ascites and perhaps promote carcinogenesis.

FIG. 1

Multilobulated polypoid mucoid accumulation surrounded by the lamina propria, in the vicinity of lymphoid aggregate in a DPAM specimen (A); one goblet cell containing mucin granules in a PMCA specimen (B); glandular epithelia with mucosal hyperplasia associated with aggregates of lymphocytes, eosinophils and macrophages in a DPAM specimen (C).

FIG. 2

ISH detection of TNCB (arrows) in the vicinity of two cells typical of signet cell carcinoma (dashed arrows) (A), in the cellular wall of goblet cell (B), in the wall of capillaries and in close association with erythrocytes (insert) (C), and inside inflammatory cell (D).

FIG. 3

(A-D): ISH detection of H. pylori 16S rRNA expression (in blue) in the thin membrane limiting mucoid cavities (A), inside the cytoplasm of columnar mucussecreting cell (B), inside a goblet cell (C), and in the lumen of a blood vessel (D). (E-G): FISH detection of H. pylori 16S rRNA (E, red) and cagA (F, green) and co-localization of both (G). Insert: Insert: higher magnification illustrating merged expression of 16S rRNA (red) and cagA (green) in the core of H. pylori (yellow), with halo of 16S rRNA expression (red, surrounding it), and cagA (green, at one end of the bacterium and extending onto flagelli. (H-J): FISH detection of MUC2 expression in mucoid deposits (green) and nuclei stained with dapi (blue) (H) and of MUC5AC expression (red), in the connective tissue containing mucoid accumulation (I), with merge demonstrating separate expression of MUC5AC and MUC2 and the absence of co-localization (no yellow stain) (J).

FIG. 4

Semi-thin section photographed under light microscopy after toluidine blue staining and corresponding transmission electron microscopy picture. (A) pools of mucin with floating islands of malignant epithelium dissecting through the desmoplastic muscularis propria (100×) and columnar epithelial cells (black arrow) and luminal aggregates of bacteria. The area of the black rectangle in A is then observed under immersion (1000×) and illustrated in B, showing the morphology of columnar epithelial cells (black arrow in A) and the presence of luminal aggregates of bacteria. The area of the white rectangle in B is then observed in the next section under transmission electron microscopy and immunogold preparation and illustrated in C, showing a single H. pylori surrounded by cross, oblique, and longitudinal sections of connective tissue collagen fibrils. Most gold particles are localized in the homogeneous cytoplasmic portion (88%) but a few particles (12%) appear to label the emergence of a flagellum from the bacterium (thick arrow) or cross sections of flagellae (thin arrow).