A pilot study using metagenomic sequencing of the sputum microbiome suggests potential bacterial biomarkers for lung cancer

RESEARCH ARTICLE A pilot study using metagenomic sequencing of the sputum microbiome suggests potential bacterial biomarkers for lung cancer Simon J. S. Cameron1, Keir E. Lewis2,3, Sharon A. Huws1, Matthew J. Hegarty1, Paul D. Lewis3, Justin A. Pachebat1, Luis A. J. Mur1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Institute of Biological, Environmental and Rural Sciences, Edward Llywd Building, Penglais Campus, Aberystwyth, Ceredigion, United Kingdom, 2 Department of Respiratory Medicine, Prince Phillip Hospital, Hywel Dda University Health Board, Llanelli, United Kingdom, 3 College of Medicine, Swansea University, Swansea, United Kingdom * Abstract OPEN ACCESS Citation: Cameron SJS, Lewis KE, Huws SA, Hegarty MJ, Lewis PD, Pachebat JA, et al. (2017) A pilot study using metagenomic sequencing of the sputum microbiome suggests potential bacterial biomarkers for lung cancer. PLoS ONE 12(5): e0177062. https://doi.org/10.1371/journal. pone.0177062 Editor: Nancy Lan Guo, West Virginia University, UNITED STATES Received: October 23, 2015 Accepted: April 23, 2017 Published: May 25, 2017 Copyright: © 2017 Cameron et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Lung cancer (LC) is the most prevalent cancer worldwide, and responsible for over 1.3 million deaths each year. Currently, LC has a low five year survival rates relative to other cancers, and thus, novel methods to screen for and diagnose malignancies are necessary to improve patient outcomes. Here, we report on a pilot-sized study to evaluate the potential of the sputum microbiome as a source of non-invasive bacterial biomarkers for lung cancer status and stage. Spontaneous sputum samples were collected from ten patients referred with possible LC, of which four were eventually diagnosed with LC (LC+), and six had no LC after one year (LC-). Of the seven bacterial species found in all samples, Streptococcus viridans was significantly higher in LC+ samples. Seven further bacterial species were found only in LC-, and 16 were found only in samples from LC+. Additional taxonomic differences were identified in regards to significant fold changes between LC+ and LC-cases, with five species having significantly higher abundances in LC+, with Granulicatella adiacens showing the highest level of abundance change. Functional differences, evident through significant fold changes, included polyamine metabolism and iron siderophore receptors. G. adiacens abundance was correlated with six other bacterial species, namely Enterococcus sp. 130, Streptococcus intermedius, Escherichia coli, S. viridans, Acinetobacter junii, and Streptococcus sp. 6, in LC+ samples only, which could also be related to LC stage. Spontaneous sputum appears to be a viable source of bacterial biomarkers which may have utility as biomarkers for LC status and stage. Data Availability Statement: All relevant data are within the paper and its Supporting Information files Funding: BBSRC (UK) support (BBS/E/W/ 10964A01A) allowed the metagenomic sequencing to be carried out. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction Lung cancer is the most prevalent cancer in the world with 1.3 million deaths recorded each year [1]. Lung cancers are classified into various subtypes reflecting their cytology and cellular origins. The main sub-divisions are non-small-cell lung carcinoma (NSCLC) and small-cell lung carcinoma (SCLC). The overall five year survival rate for lung cancer has improved very PLOS ONE | https://doi.org/10.1371/journal.pone.0177062 May 25, 2017 1 / 17 Microbiome biomarkers for lung cancer little over the last 30 years, with only 15% of patients living for five or more years after initial diagnosis [2]. These poor survival rates are primarily due to its late detection, with two thirds of patients diagnosed at a stage where chemotherapy and lung thoracotomy is less likely to be successful [3]. The main risk factor for the development of lung cancer is tobacco smoking, but genetic predisposition also plays a major role [4]; possibly explaining why not all smokers develop the lung condition [5]. A history of previous lung disease such as chronic obstructive pulmonary disease (COPD), chronic bronchitis, tuberculosis and pneumonia has been associated with an increased risk of developing lung cancer [6]. Interestingly, in the “never smokers” group a significantly increased risk of lung cancer was observed only in patients with a previous history of pneumonia and tuberculosis. Such observations suggest that microbial changes–possibly linked to inflammatory events–could be an independent risk factor associated with certain types of risk cancer [7]. Since the link between Helicobacter pylori and gastric cancer was identified [8], the possible links between the host and its microbiome, in terms of response, exacerbation or even the initiation of carcinogenesis are receiving increased attention. Changes in the bacterial loads for key species, for example, have been linked to oral squamous carcinoma, colorectal cancer and oesophageal cancer [9]. Within the context of lung cancer, a link between H. pylori seropositivity and risk of lung cancer has been investigated through the use of serum samples from patients with lung cancer and age-matched controls [10]. Although, no correlation was reported, it did show that a number of people with lung cancer tested seropositive for H. pylori and there is a possibility it could be present in the lung cancer microbiome. The use of serum in this study highlights how the microbiome-cancer links have been investigated using cancers, such as oral [11–14] and colorectal [15–17] where sampling can be minimally invasive. However, the enclosed nature of the lung complicates sample collection and has involved sampling using bronchoalveolar lavage fluids (BAL), tissue from excised lungs obtained during transplantation surgery [18], or indirectly through serum [10]. In our previous study, we have used sputum to suggest chemical biomarkers linked to lung cancer. Sputum is a complex of mucus, microorganisms, cellular debris and other particles trapped in the lungs by mucus. It provides a non-invasive method of obtaining upper bronchial tract samples that also involves minimal patient discomfort [19]. The production of sputum is a symptom of inflammatory lung airway diseases such as lung cancer, COPD, asthma, and cystic fibrosis, it is often used to provide insight into the underlying malignancies [20]. Indeed, conditions such as asthma [21], COPD [18, 22, 23] and cystic fibrosis [24] have used microbial profiling techniques to reveal potentially importan (...truncated)