Distal Gastrectomy with Billroth II Reconstruction Is Associated with Oralization of Gut Microbiome and Intestinal Inflammation: a Proof-Of-Concept Study

Distal Gastrectomy with Billroth II Reconstruction Is Associated with Oralization of Gut Microbiome and Intestinal Inflammation: a Proof-Of-Concept Study

Ann Surg Oncol (2021) 28:1198–1208 https://doi.org/10.1245/s10434-020-08678-1 ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS Distal Gastrectomy with Billroth II Reconstruction is Associated with Oralization of Gut Microbiome and Intestinal Inflammation: A Proof-of-Concept Study Angela Horvath, PhD1, Augustinas Bausys, MD2,3,6 , Rasa Sabaliauskaite, PhD4, Eugenijus Stratilatovas, MD, PhD2, Sonata Jarmalaite, PhD4, Burkhard Schuetz, PhD5, Philipp Stiegler, MD, PhD6, Rimantas Bausys, MD, PhD2,3, Vanessa Stadlbauer, MD, PhD1, and Kestutis Strupas, MD, PhD3 1Department of Gastroenterology and Hepatology, Medical University of Graz, Graz, Austria; 2Department of Abdominal Surgery and Oncology, National Cancer Institute, Vilnius, Lithuania; 3Clinic of Gastroenterology, Nephrourology and Surgery, Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, Vilnius, Lithuania; 4National Cancer Institute, Vilnius, Lithuania; 5Biovis Diagnostik, Limburg, Germany; 6Department of Transplantation Surgery, Medical University of Graz, Graz, Austria ABSTRACT Results. Microbiome oralization following SGB2 was Background. Subtotal gastrectomy with Billroth II defined by an increase in Escherichia–Shigella, Entero- reconstruction (SGB2) results in increased gastric pH and coccus, Streptococcus, and other typical oral cavity diminished gastric barrier. Increased gastric pH following bacteria (Veillonella, Oribacterium, and Mogibacterium) PPI therapy has an impact on the gut microbiome, abundance. The fecal calprotectin was increased in the intestinal inflammation, and possibly patient health. If SGB2 group [100.9 (52.1; 292) vs. 25.8 (17; 66.5); similar changes are present after SGB2, these can be rel- p = 0.014], and calprotectin levels positively correlated evant for patient health and long-term outcomes after with the abundance of Streptococcus (rs = 0.639; padj = surgery. The aim of the study is to investigate whether 0.023). Gastrointestinal symptoms in SGB2 patients were SGB2 is associated with specific changes in gut micro- associated with distinct taxonomic changes of the gut biome composition and intestinal inflammation. microbiome. Patients and Methods. This cross-sectional proof-of- Conclusions. SGB2 is associated with oralization of the concept study includes patients after SGB2 (n = 14) for gut microbiome; intestinal inflammation and microbiome early gastric cancer and their nongastrectomized in-house changes were associated with gastrointestinal symptoms. relatives as controls (n = 8). Fecal microbiome composi- These novel findings may open gut microbiome as a new tion, intestinal inflammation (fecal calprotectin), gut target for therapy to improve quality of life and general permeability (DAO, LBP, sCD14), systemic inflammation patient health in long-term survivors after SGB2. (CRP) markers, and gastrointestinal symptoms are inves- tigated. This study is registered at ClinicalTrials.gov (NCT03418428). Gastrectomy is the only potentially curative treatment option for gastric cancer (GC), one of the most common malignancies worldwide.1,2 Most patients with non- metastatic GC require total or subtotal gastrectomy with Vanessa Stadlbauer and Kestutis Strupas have share senior extended lymph node dissection. The method to reconstruct authorship. the gastrointestinal (GI) tract integrity after subtotal gas- trectomy (SG) remains controversial, while Billroth I (B1), Ó The Author(s) 2020 Billroth II (B2), or Roux-en-Y (RY) are all available and First Received: 12 February 2020; acceptable methods.3 Irrespective of type of reconstruction, Published Online: 5 June 2020 SG results in serious anatomical and physiological changes A. Bausys, MD in the GI tract, including increase in gastric pH due to e-mail: [email protected] Gut Microbiome After Distal Gastrectomy 1199 reduced secretion of the gastric acid.4–6 Therefore, there is history of gastric surgery were included in the control a strong rationale to expect alterations that are typical for group. The exclusion criteria for the participants were as gastric acid suppression in the gut microbiome following follows: (1) chemotherapy or radiotherapy 12 months prior SG. to inclusion, (2) gastric stump cancer, (3) usage of antibi- Changes in the gastric and distal GI microbiome fol- otics, pro-, pre-, or synbiotics, H2-blocker, or PPI 1 month lowing gastric acid suppression have been proposed by prior to inclusion, (4) history of any other gastrointestinal studies investigating the impact of proton pump inhibitors tract resections than SGB2, (5) recurrence of gastric can- (PPI) on the microbiome.7–10 PPI intake alters the com- cer, and (6) current nongastric malignancies. position and increases the diversity of the gastric microbiome.7 In the distal GI tract that is naturally rich in Stool Sample Collection and Sequencing microbes, the microbial diversity decreases after PPI intake.8–10 Moreover, the fecal microbiome shows To evaluate the gut microbiome, fresh stool samples increased levels of predominantly oral bacteria, such as were collected from the study participants and immediately Streptococcus, Veillonella, Rothia,orOribacterium,as stored at - 80 °C until the DNA extraction. DNA was well as an increase of potential pathogens, such as Ente- extracted with the MagNA Pure LC DNA Isolation Kit III rococcus, Escherichia–Shigella,orHaemophilus, after PPI (Bacteria, Fungi) (Roche, Mannheim, Germany) according therapy. At the same time, autochtonous and beneficial to the manufacturer’s instructions. Hypervariable region bacteria, including Faecalibacterium, Ruminococcaceae, V1–V2 was amplified (primers: 27F-AGAGTTT- and Lachnospiraceae, decrease significantly.8,9,11–13 GATCCTGGCTCAG; R357-CTGCTGCCTYCCGTA) Recently, the increase in oral bacteria in the stool of and sequenced using Illumina Miseq technology (Illumina, patients with liver cirrhosis was linked to intestinal Eindhoven, the Netherlands), as previously published.18 inflammation, gut barrier disruption, and 3-year mortal- Sequencing data are made publicly available in the ity.14 The described alterations in the microbial National Center for Biotechnology Information (NCBI) composition were partly attributed to the loss of the gastric sequence read archive (accession no. PRJNA592441). acid barrier.15 Since gastric pH increases after SG with B2 reconstruction (SGB2), we hypothesize that similar alter- Processing of Sequence Data ations of the microbiome might occur in gastrectomized patients. This study investigates whether SGB2 is associ- Raw sequencing data were processed using QIIME 2 ated with specific increased gastric pH-related changes in tools on a local Galaxy instance (https://galaxy.medunigra gut microbiome composition and intestinal inflammation. z.at/).19 Denoising (primers removing, quality filtering, correcting errors in marginal sequences, removing chimeric PATIENTS AND METHODS sequences, removing singletons, joining paired-end reads, and dereplication) was done with DADA2.20 Taxonomy Study Participants was assigned based on Silva 132 database release at 99% operational taxonomic unit level with a naı¨ve Bayes Patients older than 18 years with a history of subtotal classifier. gastrectomy for early gastric cancer (EGC) were included in the study group (SGB2 group). The EGC was defined as Laboratory Assessment invasive gastric cancer that invades no more deeply than the submucosa, irrespective of lymph node metastasis.16 Enzyme-linked immunsorbent assay (ELISA) was used EGC patients were selected to avoid the potential impact of to measure fecal calprotectin, serum diamine oxidase the disease or intensive adjuvant chemotherapy on the gut (DAO) (both: Immundiagnostik, Bensheim, Germany), microbiome. All patients underwent open subtotal gas- lipopolysaccharide binding protein (LBP, Hycult Biotech, trectomy with a D2 lymphadenectomy as described in the Uden, the Netherlands), and soluble CD14 (sCD14, R&D fourth version of the Japanese Gastric Cancer Treatment Systems, Minneapolis, MN). guidelines 17 at the National Cancer Institute, Vilnius, Lithuania. Following resection, the gastrointestinal tract Gastrointestinal Symptoms integrity was reconstructed by the antecolic end to side gastrojejunostomy on a long loop with a handsewn anas- The Lithuanian versions of the European Organization tomosis (Billroth II). Braun’s side to side jejunostomy was for Research and Treatment (EORTC) Quality of Life performed in all cases approximately 30 cm below gas- Questionnaire Core 30 (QLQ-C30) and gastric cancer- trojejunostomy. Patient’s in-house relatives without a specific module—EORTC QLQ-STO22—were used to 1200 A. Horvath et al. assess patient’s quality of life. For the analysis, the answers Microbiome Composition to the questions on the abdominal discomfort, diarrhea, and abdominal bloating were dichotomized into ‘‘symptoms’’ In total, 2,042,502 sequencing reads were generated. and ‘‘no symptoms’’ categories. Gastrointestinal symptoms After denoising, an average of 39,085 (min: 24,549; max: were associated with the microbiome composition. 49,361) reads per sample were available for analysis. Alpha diversity assessed by Shannon index after rarefica- Statistical Analysis tion (sampling depth: 24,549 reads) was significantly decreased in gastrectomy patients compared with controls For statistical analysis of microbiome compositions, the (p = 0.025, Fig. 2a). Median bacterial richness quantified web-based application Calypso (version 8.84) was by Chao1 index was comparable between groups employed. Features were normalized by

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