Lipidomic Profiling of Lung Pleural Effusion Identifies Unique Metabotype for EGFR Mutants in Non-Small Cell Lung Cancer

Abstract

Cytology and histology forms the cornerstone for the diagnosis of non-small cell lung cancer (NSCLC) but obtaining sufficient tumour cells or tissue biopsies for these tests remains a challenge. We investigate the lipidome of lung pleural effusion (PE) for unique metabolic signatures to discriminate benign versus malignant PE and EGFR versus non-EGFR malignant subgroups to identify novel diagnostic markers that is independent of tumour cell availability. Using liquid chromatography mass spectrometry, we profiled the lipidomes of the PE of 30 benign and 41 malignant cases with or without EGFR mutation. Unsupervised principal component analysis revealed distinctive differences between the lipidomes of benign and malignant PE as well as between EGFR mutants and non-EGFR mutants. Docosapentaenoic acid and Docosahexaenoic acid gave superior sensitivity and specificity for detecting NSCLC when used singly. Additionally, several 20- and 22- carbon polyunsaturated fatty acids and phospholipid species were significantly elevated in the EGFR mutants compared to non-EGFR mutants. A 7-lipid panel showed great promise in the stratification of EGFR from non-EGFR malignant PE. Our data revealed novel lipid candidate markers in the non-cellular fraction of PE that holds potential to aid the diagnosis of benign, EGFR mutation positive and negative NSCLC.

Figure 1

Principal component analysis scores plots for (A) benign (n = 30) and malignant lung PE (n = 36), and the PE of (B) EGFR mutant (n = 19) and non-EGFR mutant (n = 17) collected from the malignant patients. Green, blue and red circles represent the benign, malignant non-EGFR mutant and malignant EGFR mutant PE respectively. (C) Heat map of differential lipid metabolites derived from individual pairwise comparisons between benign, non-EGFR mutant and EGFR mutant PE samples. All represented species are statistically significant (VIP > 1, p-value < 0.05, fold change (FC) ≥ 1.5) for at least one of the pairwise comparisons. Metabolites are grouped according to their lipid classes.

Figure 2. Diagnostic performance of lipid markers in discriminating pleural effusion from malignant NSCLC patients (n = 41) from benign subjects (n = 30).

ROC curves of malignant versus benign subjects for individual PE lipid markers in the class of (A) fatty acids (B) sphingolipids. (C) ROC curves of malignant versus benign subjects for an optimal combination of 4 lipid malignancy markers from SVM modelling.

Figure 3

Dot plots describing the relative levels of (A) FA(22:6), (B) FA(20:5) in benign (green), non-EGFR mutant (blue) and EGFR mutant (red) PE samples. p-value calculated based on Mann-Whitney U test, where *denotes p < 0.05, **denotes p < 0.01, ***denotes p < 0.001. ROC curves of non-EGFR mutant versus EGFR mutant subjects for individual PE lipid markers in the class of (C) fatty acids and (D) phospholipids. (E) ROC curves of non-EGFR mutant versus EGFR mutant subjects for an optimal combination of 7 lipid malignancy markers from SVM modelling.