MSACL 2018 US Abstract

Topic: Metabolomics

In vitro Volatile Compound Bacterial Profiling Using GC-MS and their Potential Role for the Diagnosis of Oesophago-Gastric Cancer

Mina Adam (Presenter)
Imperial College London UK

Bio: I joined Professor George Hanna and Professor Julian Marchesi’s research group in November 2015 as a clinical PhD research fellow at Imperial College London. I completed my undergraduate medical education (MBBCH) honours degree at Cairo University in 2010. In 2013, I commenced my postgraduate specialist residency program in General and Gastrointestinal Oncology Surgery. This enabled me to accomplish my Master’s degree and commence my research. My current work is centred on the influence of the microbiome and its effect on volatile organic compounds produced from human Gastro-Oesophageal mucosal tissue samples using different mass spectrometry platforms.

Authorship: Mina Adam (1), Sacheen Kumar (1), Andrea Romano (1), Julian Marchesi (1), George B Hanna (1)
(1) Imperial College London, London, UK

Short Abstract

The potential role of bacteria in Oesophago-Gastric(OG) tumorigenesis and their influence on Volatile Organic Compounds (VOCs) produced within the upper gastrointestinal tract is currently unknown. The detection and quantification of VOCs has great potential in terms of disease diagnosis. In this study, Gas Chromatography Mass Spectrometry has been utilised for the identification of VOCs from bacteria that are present in OG cancer. Data demonstrated the presence of specific predominant bacteria in cancer patients producing acetic acid, phenol, acetaldehyde and 2-pentene. Similar compounds have previously been identified at increased concentrations in exhaled breath and the headspace of tissue biopsies from OG cancer patients. These findings support further culturing investigations to understand the role of microbiome with VOCs and their potential diagnostic and therapeutic significance.

Long Abstract

Introduction

The National Oesophago-Gastric (OG) Cancer Audit identified that there were 10,996 patients diagnosed with oesophago-gastric cancer between April 2012 and March 2013 in the United Kingdom. Worldwide, there are an estimated 482,300 new oesophageal cancer cases per annum. The prognosis of oesophago-gastric cancer is poor in Western countries as a result of the paucity of alarm symptoms in early stages of the disease; this results in late clinical presentation and associated delays in the institution of treatment. The development of non-invasive, accurate biomarkers to determine oesophago-gastric cancer risk could help facilitate earlier diagnosis and potentially improve patient survival.

The potential role of bacteria in Oesophago-gastric tumorigenesis and their influence on Volatile Organic Compounds (VOCs) produced within the upper gastrointestinal tract is currently unknown. Bacterial colonisation of the stomach by the organism Helicobacter Pylori has a well-known link to oesophago-gastric cancer risk. Beyond the presence of this organism in certain individuals it has been demonstrated that the stomach microbiome is dominated by organisms from the bacterial phyla of Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Each of these bacteria have been identified within the stomach at a genus level, this increasing knowledge of bacterial presence within the stomach may provide a potential mechanism of VOC production in these patients, by bacterial action upon enzymes found within the gastric juice of cancer patients. The bacterial constitution of the stomach can also vary in the presence of gastric cancer, and this variation could also contribute towards increased VOC production as demonstrated by our group in tissue biopsies of these cancer patients as well as exhaled breath. The aim of this study is to identify the predominant microbial species within Oesophago-Gastric adenocarcinoma and their potential role in the production of VOCs.

Methods

Cancer samples were collected from Helicobacter Pylori negative patients undergoing surgical resection of gastric adenocarcinoma. Patients who had a normal OGD test were included in the “healthy” cohort group. Tissue samples were collected from gastric malignancy and matched healthy mucosa from the same patients. Samples were immediately placed in liquid nitrogen and stored at -80°C prior to analysis. During DNA extraction tissue samples were processed using the PowerLyzer DNA Isolation kit. Successfully extracted DNA then underwent real time quantitative PCR to amplify the 16S gene followed by metataxonomic 16S sequencing analysis on the Illumina MiSeq platform. Bacteria, which have the potential to produce VOCs of interest, were identified using the KEGG dataset and other available literature.

Comparison of bacterial species within cancers and non-cancer samples revealed higher abundance of specific bacteria in the cancer cohort. Each of these bacteria were then anaerobically grown in vials using the appropriate growth media and incubated at 37°C. Blank samples with uninoculated media were used as controls to eliminate any VOCs released from growth medium. Bacterial late exponential and static growth stage was initially determined to allow volatile emissions reach maximum. Gas Chromatography Mass Spectrometry (GC-MS) was the preferred method for organic compound analysis owing to its high resolution, high sensitivity and standardized method for machine setup and data analysis. The MS instrument was tuned and volatile standards were tested as a quality control reference before starting experiments. Headspace analysis was undertaken via solid phase micro-extraction (SPME) using a carboxen/polydimethysiloxane SPME fibre in 20ml screw-top glass vials with a PTFE septum. SPME extraction was performed at 60°C with intermittent agitation at 500rpm. Volatiles were collected in the absence of airflow, after 24 and 48 hours of incubation followed by direct release into a heated gas chromatography injector. Supervised and unsupervised statistical modelling was performed with incorporation of clinical metadata.

Results

Samples were classified into cancer and healthy groups following histopathological confirmation. Comparison of dominant bacterial phyla within cancer and non-cancer samples using univariate statistical analysis revealed higher abundance of Firmicutes (p<0.075) in cancer samples. Further genus level analysis revealed the presence of a number of Lactobacillus Fermentum, Escherichia Coli and Streptococcus Salivarius in the cancer cohort. VOCs including acetic acid, phenol, acetaldehyde and 2-pentene were present in the headspace of those predominant bacteria. These compounds have previously been identified at increased concentrations in the exhaled breath and headspace of tissue biopsies from OG cancer patients.

Conclusions & Discussion

This is the first study to explore the abundance of specific microbial phyla within tumour biopsies of patients with OG cancer and how they can potentially play an important role in tumorigenesis. VOCs detected within bacterial headspace are similar to compounds that have been found in tissue biopsies, biofluids and exhaled breath from cancer patients across several mass spectrometry platforms. These preliminary findings support further culturing investigations to understand the role of microbiome in VOC production and their potential diagnostic and therapeutic significance in OG cancer.


References & Acknowledgements:

1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69–90.

2. Arvanitakis C, Nikopoulos A, Giannoulis E, Theoharidis A, Georgilas V, Fotiou H, et al. The impact of early or late diagnosis on patient survival in gastric cancer in Greece. Hepatogastroenterology. 1992 Aug;39(4):355–7.

3. Mohamed A Farag, Geun Cheol Song, Yong-Soon Park2, Bianca Audrain, Soohyun Lee, Jean-Marc Ghigo, Joseph W Kloepper & Choong-Min Ryu. Biological and chemical strategies for exploring inter-and intra-kingdom communcation mediated via bacterial volatile signals. 2017 June

4. Kumar S, Huang J, Abbassi-Ghadi N, Mackenzie HA, Veselkov KA, Hoare JM, Lovat LB, Spanel P, Smith D, Hanna GB. Mass spectrometric analysis of exhaled breath for the identification of volatile organic compound biomarkers in esophageal and gastric adenocarcinoma. Ann Surg 2015


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