Characterisation of microbiota in saliva, bronchoalveolar lavage fluid, non-malignant, peritumoural and tumour tissue in non-small cell lung cancer patients: a cross-sectional clinical trial

Abstract

Background: While well-characterised on its molecular base, non-small cell lung cancer (NSCLC) and its interaction with local microbiota remains scarcely explored. Moreover, current studies vary in source of lung microbiota, from bronchoalveolar lavage fluid (BAL) to tissue, introducing potentially differing results. Therefore, the objective of this study was to provide detailed characterisation of the oral and multi-source lung microbiota of direct interest in lung cancer research. Since lung tumours in lower lobes (LL) have been associated with decreased survival, characteristics of the microbiota in upper (UL) and lower tumour lobes have also been examined.

Methods: Using 16S rRNA gene sequencing technology, we analysed microbiota in saliva, BAL (obtained directly on excised lobe), non-malignant, peritumoural and tumour tissue from 18 NSCLC patients eligible for surgical treatment. Detailed taxonomy, diversity and core members were provided for each microbiota, with analysis of differential abundance on all taxonomical levels (zero-inflated binomial general linear model with Benjamini-Hochberg correction), between samples and lobe locations.

Results: Diversity and differential abundance analysis showed clear separation of oral and lung microbiota, but more importantly, of BAL and lung tissue microbiota. Phylum Proteobacteria dominated tissue samples, while Firmicutes was more abundant in BAL and saliva (with class Clostridia and Bacilli, respectively). However, all samples showed increased abundance of phylum Firmicutes in LL, with decrease in Proteobacteria. Also, clades Actinobacteria and Flavobacteriia showed inverse abundance between BAL and extratumoural tissues depending on the lobe location. While tumour microbiota seemed the least affected by location, peritumoural tissue showed the highest susceptibility with markedly increased similarity to BAL microbiota in UL. Differences between the three lung tissues were however very limited.

Conclusions: Our results confirm that BAL harbours unique lung microbiota and emphasise the importance of the sample choice for lung microbiota analysis. Further, limited differences between the tissues indicate that different local tumour-related factors, such as tumour type, stage or associated immunity, might be the ones responsible for microbiota-shaping effect. Finally, the "shift" towards Firmicutes in LL might be a sign of increased pathogenicity, as suggested in similar malignancies, and connected to worse prognosis of the LL tumours.

Trial registration: ClinicalTrials.gov ID: NCT03068663. Registered February 27, 2017.

Keywords: Bronchoalveolar lavage; Lobe location; Lower lobe tumour; Lung cancer; Lung microbiota; Non-malignant lung tissue; Non-small cell lung cancer; Peritumoural lung tissue; Saliva.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Diversity of the salivary and four lung microbiota. a Beta diversity of salivary and lung microbiota represented by non-metric multidimensional scaling (NMDS) based on weighted and unweighted UniFrac distances. Alpha diversity of saliva and four lung samples assessed by b number of observed OTUs, c Faith’s phylogenetic diversity, and d Shannon diversity. Statistical significance of difference in beta diversity was assessed with adonis function (vegan) with 999 permutations. Statistical significance of difference in alpha diversity was assessed with Kruskal-Wallis followed by, where appropriate, Man-Whitney U test with BH correction for multiple comparison. *: p ≤ 0.05, **: p ≤ 0.01. BAL - bronchoalveolar lavage fluid, KW – Kruskal-Wallis test, LUNG.DP - non-malignant distal piece, LUNG.PT - peritumoural tissue, LUNG. T – tumour, uwUF – unweighted UniFrac, wUF – weighted UniFrac

Fig. 2

Relative abundance and prevalence of the four lung and salivary microbiota. a Each tree represents the taxonomical composition of one sample type. The colour and node size correspond to taxon abundance. All taxa with abundance higher than 0.001% are shown, and percentage is noted for all taxa with abundance higher than 0.01%. Number of samples within which the taxon was detected is noted within branches. Maximal number of samples is 17 for saliva and non-malignant tissue, 16 for tumour, 15 for BAL, and 14 for peritumoural tissue (Table 1). Synthetic presentation of the most abundant taxa was provided on b phylum and c genus level. BAL - bronchoalveolar lavage fluid, LUNG.DP - non-malignant distal piece, LUNG.PT - peritumoural tissue, LUNG. T - tumour

Fig. 3

Differential abundance between lung and salivary microbiota and their core composition. a Coloured nodes and branches in each tree represent the taxa with significantly different abundance between two compared microbiota. The colour intensity is proportional to log2-fold change in abundance in the favour of the sample with the same colour. Taxa names are shown in the common legend tree below comparisons. Statistical significance was assessed by zero-inflated general linear model using Wald’s test (DESeq2), with p-value threshold of α ≤ 0.05 after BH correction. b Core microbiota determined as OTUs present in 100% of each sample for one sample type. The colour represents relative abundance on transformed log4 scale. c Average value of sum of abundances of the core OTUs and of other OTUs per each subject in each sample types. BAL - bronchoalveolar lavage fluid, LUNG.DP - non-malignant distal piece, LUNG.PT - peritumoural tissue, LUNG. T – tumour, OTU – outer taxonomic unit

Fig. 4

Diversity and predominant taxa in lung samples from upper and lower tumour lobes. Beta diversity found significantly different between peritumoural tissue from upper and lower lobes based on both a weighted (wUF) and b unweighted (uwUF) UniFrac distances. c Significantly different beta diversity based on wUF between peritumoural tissue and tumour in the upper lobe. d Weighted and e unweighted UF distances between samples coming from the same patient (i.e. paired distances) compared between upper and lower tumour lobes. The facet name represents the referent sample (e.g. “distance to BAL”) to which were calculated the distances noted on x-axis (e.g. “from LUNG.T”). Smaller distance indicates increased similarity. Alpha diversity for four lung samples between upper and lower tumour lobe assessed by f Faith’s phylogenetic diversity, g number of observed OTUs, and h Shannon diversity. i Most abundant phyla in each of the microbiota samples if the tumour is found in upper or lower lobes. Significance of difference in beta diversity was assessed with adonis function (vegan, 999 permutations). Statistical significance in alpha diversity and paired distances was assessed with Kruskal-Wallis followed by, where appropriate, Man-Whitney U test with BH correction for multiple comparison. *: p ≤ 0.05, **: p ≤ 0.01. BAL - bronchoalveolar lavage fluid, LUNG.DP - non-malignant distal piece, LUNG.PT - peritumoural tissue, LUNG. T - tumour

Fig. 5

Differential abundance between upper and lower tumour lobes in salivary and lung microbiota. Each tree represents taxa with significantly different abundance relative to sample’s origin (for lung) in the upper or lower tumour lobe. For saliva, the comparison shows the significant difference in salivary microbiota between patients with tumour either in upper or lower lobe. Coloured nodes and branches represent the taxa with significantly different abundance and the intensity is proportional to log2-fold change in abundance in the favour of the lobe noted with the same colour. Statistical significance was assessed by zero-inflated general linear model using Wald’s test (DESeq), with p-value threshold of α ≤ 0.05 after BH correction. Bar chart shows the relative abundance of taxa noted in the taxonomical trees. BAL - bronchoalveolar lavage fluid, LUNG.DP - non-malignant distal piece, LUNG.PT - peritumoural tissue, LUNG. T - tumour

Fig. 6

Comparison of abundance between salivary and lung microbiota relative to tumour lobe location. The two parts of the figure represent comparison in abundance between samples linked to upper (U) lobes in the upper part of the figure and to lower (L) lobes in the lower part of the figure. Coloured nodes and branches represent taxa with significantly different abundance between the two compared samples. The colour intensity is proportional to log2-fold change in abundance in the favour of the sample with the same colour. Statistical significance was assessed by zero-inflated general linear model using Wald’s test (DESeq), with p-value threshold of α ≤ 0.05 after BH correction. BAL - bronchoalveolar lavage fluid, L – lower lobe, LUNG.DP - non-malignant distal piece, LUNG.PT - peritumoural tissue, LUNG. T – tumour, U – upper lobe