The long-term survival following oesophagectomy for oesophageal cancer has doubled from 25% to 50% in the last 30 years. A multi-centre cohort study, the Lasting Symptoms after Esophageal Resection (LASER), found that 67% of OC survivors suffered from a long-term symptom affecting their health-related quality of life (HRQL), mainly linked to gastrointestinal (GI) symptoms. Despite this, OC survivors remain unresearched despite the significant effect on HRQL. Mechanisms underlying GI symptoms observed post-oesophagectomy remain poorly explored.

The anatomical changes and major reconstruction performed during an oesophagectomy is known to be associated with a change in the gut microbiome. The hypothesis that gut microbiome dysbiosis (perturbation from that perceived to represent a healthy microbiome) is associated with GI symptoms is supported by limited evidence in small intestinal bacterial overgrowth (SIBO). SIBO remains poorly understood and treated. SIBO is diagnosed using hydrogen and methane breath tests without a good understanding of underlying mechanisms of how these volatiles are produced.

We aim to improve our understanding by exploring the effect of gut microbiome dysbiosis in a more systematic approach. Multi-omic analysis allows the study of metabolic functionality and the phenotypic expression of the microbiome. Furthermore, a systems biology approach that integrates metabolomic and metagenomic data from biofluids would provide a vertical mechanistic framework underpinning the metabolic production of volatile organic compounds (VOCs) to identify breath biomarkers in a wider scale than the current hydrogen and methane breath tests.

Aims:

1. Identify underlying metabolic pathways associated with microbiome derived metabolites in saliva, stool, and breath.

2. Identify microbiome dysbiosis in saliva and stool associated with GI symptoms.

3. Develop a breath test to detect volatile biomarkers associated with microbiome dysbiosis.

4. Propose a strategy for treatment of long-term symptoms.

Methods:

MAPLES is a multicentre cross-sectional study that will investigate disease free patients with oesophageal adenocarcinoma following multimodality treatment. 100 patients have been recruited, all >1 year after an oesophagectomy. Patients are considered disease free if they have a CT with no evidence of recurrence within the last 6 months or more than 5 years post oesophagectomy. Patients were invited to attend an outpatient clinic along with the stool sample and fasted for at least 4 hours for collection of saliva, urine and breath and complete validated HRQL questionnaires.

Metagenomic sequencing will be analysed using Illumina HiSeq sequencer on saliva and stool samples. Metabonomic analysis will be carried out using Nuclear Magnetic Resonance Spectroscopy (NMR) and Ultra-performance liquid chromatography mass spectrometry (UPLC-MS) on saliva, stool, plasma, and urine samples. Volatolomic analysis will be analysed using mic-polar, polar and two-dimensional gas chromatography-time of flight mass spectrometry (GC-MS-TOF) on breath, saliva, blood, urine, and stool samples.

Bioinformatics analysis will link identified microbiota and their functional pathways with their identified end metabolites and long-term GI symptoms. This will be performed using in-house bioinformatic pipelines that exist for integrative analysis of multi-omics data, existing open access resources (e.g. MetaboAnalyst), or customised R scripts (e.g. MixOmics).