No Unique Microbiome Detected in Pancreatic Cyst Fluid

As in other tumors, inflammation is considered a critical risk factor for the progression of intraductal papillary mucinous neoplasms (IPMNs). Two research teams recently proposed the pancreas and cyst fluid harbor a unique population of bacteria—a "pancreatic cyst microbiome"—that interact with each other and the human host to drive the inflammatory response. This notion requires a complete rethinking of early cyst and tumor development in the pancreas.

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However, these studies did not rule out the possibility of DNA contamination by analyzing data from negative controls of the sampling environment or by repeatedly processing and sequencing the same samples.

Marie-Madlen Pust, PhD, a fellow in the Center for Computational and Integrative Biology at Massachusetts General Hospital, Ramnik J. Xavier, MD, PhD, director of the Center for the Study of IBD at Mass General and Kurt J. Isselbacher professor of Medicine in the Field of Gastroenterology at Harvard Medical School, Carlos Fernandez-del Castillo, MD, director of the Pancreas and Biliary Surgery Program at Mass General and professor of Surgery at Harvard Medical School, and colleagues addressed these deficiencies in a study published in Gut. They found no microbiome within IPMNs and concluded antibiotic therapy is unlikely to have any benefit in the disease.

Initial Investigation

The researchers conducted 16S rRNA gene sequencing on 338 cyst fluid samples obtained from 190 patients:

• 145 had an IPMN, including 42 who had invasive carcinoma within the IPMN
• 21 had other cystic pancreatic neoplasms
• 16 had pancreatic ductal adenocarcinoma and cystic degeneration
• 8 had pseudocysts or other pancreatic cystic lesions

All those samples were obtained with sterile technique during surgery, directly from the cystic lesion to avoid any passage through the gastrointestinal tract. In addition, 19 negative control samples were obtained from the operating room itself with a sterile syringe.

A subset of samples was re-processed and re-sequenced up to three times with the same wet-lab procedure to avoid kit, environmental, and cross-contamination.

The team failed to uncover a unique pancreatic cyst microbiome in any of the fluid samples that deviated from the microbiome signature of the negative controls. The quantity of microbial DNA was, by definition, insufficient to indicate the presence of a complex microbial community. Furthermore, most DNA was attributable to contamination from the sampling and processing procedures.

Further Validation

The current methodology might not rule out the presence of an ultra-low-biomass microbial community. To account for that, the researchers spiked 20 additional samples and five additional negative controls with known concentrations of bacterial cells not typically part of the human microbiome. There were no significant differences between cyst fluid samples and controls in diversity estimates or microbial community composition.

Outlier Signals

Even though no pancreatic microbiome was detected, the team did unravel a bacterial signature in a small subset of patients, with and without the spike-in strategy. This signature was not characterized by higher taxonomic diversity but rather by increased dominance of a gut-associated microbe. The discovery of potential bacterial infection within pancreatic pathologies is not unexpected.

Clinical Relevance

The finding that no pancreatic cyst microbiome existed in the cohort emphasizes the need to explore alternative explanations for the role of inflammation in early cyst and tumor development, to aid development of potential preventive or therapeutic interventions.

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