Clinical, microbial, and experimental features associated with Grade C postoperative pancreatic fistula and pancreaticojejunostomy disruption.

BACKGROUND: Most postoperative pancreatic leaks after pancreatoduodenectomy (PD) remain clinically insignificant biochemical leaks, whereas a minority progress to life-threatening Grade C postoperative pancreatic fistula (POPF). The biological events underlying this progression remain poorly understood. We investigated the clinical, microbial, and immunological mechanisms associated with severe POPF progression. METHODS: Of 642 consecutive patients assessed between March 2022 and March 2024, 74 were excluded and 568 were included in the clinical cohort. Risk factors for Grade C POPF were evaluated using univariable analysis and Firth penalized multivariable logistic regression. For translational analyses, peritoneal fluid from 20 patients with biochemical leak and 20 patients with Grade C POPF underwent 16S ribosomal RNA (rRNA) sequencing and multiplex cytokine profiling. Human pancreatic tissues from two Grade C POPF patients and three histologically normal controls were evaluated descriptively. Experimental evaluation used a rat model combining pancreatic and intestinal fistulas to simulate enteric contamination relevant to pancreaticojejunostomy leakage. RESULTS: Among 568 included patients, 20 (3.5%) developed Grade C POPF. Pancreaticojejunostomy disruption occurred in 13 of 20 Grade C patients and 17 of 548 non-Grade C patients (65.0% vs. 3.1%, P<0.001). In Firth penalized multivariable logistic regression, disruption remained strongly associated with Grade C POPF [odds ratio (OR) 21.84, 95% confidence interval (CI): 7.12-72.63, P<0.001]. Compared with biochemical leak, Grade C POPF was associated with lower peritoneal microbial diversity, differential representation of several genera, and higher concentrations of interferon-gamma (IFN-γ), interleukin-6 (IL-6), interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α). The small human tissue series descriptively showed inflammatory injury and CD68+ iNOS+ macrophage accumulation. In rats, pancreatic leakage plus enteric contamination aggravated pancreatic injury and inflammation; antibiotic treatment attenuated these findings but could not isolate microbiota depletion from direct antibacterial or anti-inflammatory effects. CONCLUSIONS: Grade C POPF was strongly associated with pancreaticojejunostomy disruption and was accompanied by peritoneal microbial and inflammatory differences compared with biochemical leak. The animal model simulated key features of pancreaticojejunostomy leakage and supports the possibility that enteric contamination contributes to inflammatory aggravation.