Neutral Desorption Extractive Electrospray Ionization Mass Spectrometry Analysis Sputum for Non-Invasive Lung Adenocarcinoma Detection

Figures & data

Table 1 Characteristics of Study Participants

Characteristics	LAC*	Control Group (Con)
		Ben^#	Hea^&
Number	76	28	39
Age (mean ± SD)	60.1 ± 8.7	63.4 ± 9.4	58.6 ± 9.8
Gender (Male/Female)	32/44	13/15	17/22
Smoking Index > 400	23.70%	25.00%	23.10%
Clinical Cancer Stage
I	21
II	41
III	14
Pathological Diagnosis
Bronchiectasis		12
Hamartoma		9
Inflammatory Myofibroblastic Tumor		7

Abbreviations: *LAC, lung adenocarcinoma; ^#Ben, benign disease; ^&Hea, healthy individuals

Table 2 Significant Metabolites Detected by ND-EESI-MS

Metabolite	m/z	AUC	VIP^a	p.value^b	FDR q-value	Fold Change (LAC/Con)
Hydroxyphenyl lactic acid	m/z 181.07	0.80	1.22	1.08E-08	4.53E-08	1.67
Phytosphingosine	m/z 318.29	0.79	1.29	1.15E-07	4.01E-07	0.55
N-Nonanoylglycine	m/z 214.15	0.79	1.74	1.88E-09	1.97E-08	2.07
Sphinganine	m/z 302.38	0.78	1.08	1.06E-08	4.53E-08	0.68
S-Carboxymethyl-L-cysteine	m/z 180.04	0.77	1.02	8.67E-09	4.53E-08	0.70
Ornithine	m/z 131.07	0.74	1.04	2.89E-07	8.68E-07	1.55
Succinic acid	m/z 116.93	0.72	0.95	2.13E-06	5.58E-06	1.44
L-Fucose	m/z 165.09	0.72	0.88	6.69E-06	1.17E-05	0.72
L-Carnitine	m/z 142.09	0.72	0.73	5.26E-06	1.17E-05	0.79
Acetylphenylalanine	m/z 415.21	0.69	1.01	5.86E-06	1.17E-05	0.69
Caprylic acid	m/z 145.18	0.68	0.89	6.41E-06	1.17E-05	0.63
5-Hydroxymethyluracil	m/z 141.01	0.68	0.52	2.89E-04	4.65E-04	1.20
Linoleic acid	m/z 303.23	0.67	0.96	4.61E-04	6.05E-04	1.56
L-Lactic acid	m/z 89.02	0.67	1.04	5.33E-04	6.58E-04	1.68
N-Succinyl-L-glutamate-5-semialdehyde	m/z 254.06	0.66	0.85	9.35E-04	0.00109	1.41
5-Methyl tetrahydrofolate	m/z 231.12	0.66	0.82	3.10E-04	4.65E-04	1.39
Hexanoyl carnitine	m/z 258.17	0.66	0.82	3.44E-04	4.81E-04	1.39
Tetradecanoyl carnitine	m/z 394.29	0.65	0.82	0.001734	0.00182	0.67
Propionylcholine	m/z 321.29	0.63	0.66	0.001174	0.00129	1.24

Notes: ^aVariable importance in projection (VIP) values obtained from the partial least squares-discriminant analysis model, ^bp values calculated using Student’s t-test.

Abbreviations: LAC, lung adenocarcinoma; Con, control group; FDR, false discovery rate; AUC, area under the curve.

Figure 1 Typical ND-EESI-MS spectra in positive mode of human sputum samples of a lung adenocarcinoma (LAC) patient and a control group.

Figure 2 Heat map of significantly changed metabolites detected by ND-EESI-MS for comparison of LAC/Con.

Figure 3 Differential characteristics of enhanced biomarker panel with 5 differential metabolites (AUC > 0.75 and VIP > 1) detected by ND-EESI-MS for distinguishing between lung adenocarcinoma patients (LAC, n = 76) and controls (Con, n = 67). (A) Log₁₀- transformed relative abundance of 5 differential metabolites for comparison between LAC and Con. (B) QC normalized relative abundance data were log₁₀-transformed and Pareto scaled. Score plot of the OPLS-DA model for discrimination between LAC and Con. [R²X (cum)= 0.301, R²Y (cum)= 0.228, Q² (cum)= 0.494]. (C) QC normalized relative abundance data were log₁₀-transformed and Pareto scaled. ROC curve illustrating the classification performance of the 5-metabolite OPLS-DA model for distinguishing between LAC and Con. [AUROC = 0.917, 95% CI: 0.861–0.965, sensitivity: 0.9, specificity: 0.8]. (D) QC normalized relative abundance data were log₁₀-transformed and Pareto scaled. External validation of OPLS-DA model with 50% sample holdout: cross-validation classification accuracy of 87.9% using a PLS-DA classifier.

Figure 4 The metabolome view of pathway enrichment analysis comparing LAC patients and controls. (A) Overview of the most important metabolomic changes observed by ND-EESI-MS in sputum of lung adenocarcinoma patients, 1: Sphingolipid metabolism, 2: Tyrosine metabolism, 3: Ubiquinone and other terpenoid-quinone biosynthesis, 4: Arginine and proline metabolism, 5: Linoleic acid metabolism. (B) Metabolic network showing significant differences in sphingolipid metabolism, fatty acid metabolism, carnitine synthesis and Warburg effect, etc.