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

Abstract

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.

Fig 1. Alpha diversity group means.

Alpha diversity measures of species richness, as calculated by the MG-RAST analysis pipeline, show no significant difference between either positive or negative lung cancer groups. This suggests that any changes to the lung microbiome as a result of a malignancy are not large-scale community shifts.

Fig 2. Principal component analysis of taxonomic and functional classifications.

PCA plots and biplots, to identify factors leading to observed groupings, created using the MetaboAnalyst platform using normalised percentage abundance of (A and B) bacterial species and (C and D) level 3 functional alignments. LC- samples are indicated by red symbols and LC+ by green symbols. Coloured areas indicate 95% confidence intervals of PCA groupings as calculated by MetaboAnalyst. For biplots, red-letter annotations show the factor contributing to the observed separation.

Fig 3. Significant fold changes in species abundance from negative to positive for lung cancer.

Using the online features of MetaboAnalyst 2.0, significant fold changes, as determined by t-Tests with P values <0.05, were identified. Five species, from three genera, were all higher in positive lung cancer samples, with Granulicatella adiacens and Streptococcus intermedius showing the highest change.

Fig 4. Significant fold changes in levels 2 and 3 functions from negative to positive lung cancer.

Using MetaboAnalyst 2.0, significant fold changes of Level 2 (grey bars) and 3 (black bars) functional alignments, as determined through t-Tests with P values <0.05, were identified. A total of four Level 2 functional alignments were higher in positive lung cancer, alongside seven Level 3 functions. Three Level 3 functional alignments were lower in positive lung cancer samples.

Fig 5. Regression analysis suggests importance of G. adiacens in positive lung cancer samples.

Species regression analyses were completed, and those with an R² value of greater than 80% were plotted to identify differing relationships between negative and positive lung cancers. This type of relationship was shown to exist between G. adiacens and six other species, with a strong positive relationship present in positive lung cancer samples, and no correlation evident within negative lung cancer samples. Normalised percentage abundances are shown on x and y axes.