Lipidomic profiling of human serum enables detection of pancreatic cancer

Abstract

Pancreatic cancer has the worst prognosis among all cancers. Cancer screening of body fluids may improve the survival time prognosis of patients, who are often diagnosed too late at an incurable stage. Several studies report the dysregulation of lipid metabolism in tumor cells, suggesting that changes in the blood lipidome may accompany tumor growth. Here we show that the comprehensive mass spectrometric determination of a wide range of serum lipids reveals statistically significant differences between pancreatic cancer patients and healthy controls, as visualized by multivariate data analysis. Three phases of biomarker discovery research (discovery, qualification, and verification) are applied for 830 samples in total, which shows the dysregulation of some very long chain sphingomyelins, ceramides, and (lyso)phosphatidylcholines. The sensitivity and specificity to diagnose pancreatic cancer are over 90%, which outperforms CA 19-9, especially at an early stage, and is comparable to established diagnostic imaging methods. Furthermore, selected lipid species indicate a potential as prognostic biomarkers.

Fig. 1. Overview of study design for the differentiation of PDAC patients (T, red) from normal healthy controls (N, blue) and pancreatitis patients (Pan, green) based on the lipidomic profiling of human serum using various mass spectrometry-based approaches.

a Phase I (discovery) for 364 samples (262 T + 102 N) divided into training (213 T + 79 N) and validation (49 T + 23 N) sets measured by UHPSFC/MS, shotgun MS (LR), and MALDI-MS. b Phase II (qualification) for 554 samples (444 T + 98 N + 12 Pan) divided into training (328 T + 82 N + 12 Pan) and validation (116 T + 16 N) sets measured by UHPSFC/MS, shotgun MS (LR and HR), and RP-UHPLC/MS at 3 different laboratories. c Phase III (verification) for 830 samples (546 T + 262 N + 22 Pan) divided into training (430 T + 246 N + 22 Pan) and validation (116 T + 16 N) sets measured by UHPSFC/MS for samples obtained from four collection sites.

Fig. 2. Effect of gender separation on the quality of OPLS-DA models used for the differentiation of human serum samples obtained from PDAC patients (T) and healthy controls (N) for the training set using UHPSFC/MS in the Phase I.

a Both genders. b Males. c Females. d Specificity, sensitivity, and accuracy for individual models. Source data are provided as a Source Data file.

Fig. 3. Results for the Phase I obtained in lab 1. Individual samples are colored according to tumor (T) stage: T1 - yellow, T2 - orange, T3 - red, T4 - rose, and Tx - brown (information about the stage is not available).

OPLS-DA for males measured a with UHPSFC/MS and c with shotgun MS for the training set (104 T + 30 N). OPLS-DA for females measured with b UHPSFC/MS and d shotgun MS for the training set (157 T + 49 N). ROC curves for males (M) and females (F) in training (Tr.) and validation (Va.) sets: e UHPSFC/MS, and f shotgun MS. Box plots for molar concentration in human serum from PDAC patients (T) and healthy controls (N) for males (M) and females (F): g SM 41:1 measured by UHPSFC/MS, h SM 41:1 measured by shotgun MS (LR), for both box plots for males (104 T and 30 N) and females (109 T and 49 N), i SHexCer 41:1(OH) measured by MALDI-MS, and j SHexCer 40:1(OH) measured by MALDI-MS, for both box plots for males (15 T and 14 N) and females (18 T and 19 N). In each box plot, the centerline represents the median, the bounds represent the 1st and 3rd quartile and whiskers span 1.5 fold inter-quartile range from the median. Source data are provided as a Source Data file.

Fig. 4. Comparison of Phase II results obtained at three different laboratories using four mass spectrometry-based approaches.

Box plots of lipid concentrations normalized to the NIST reference material for samples obtained from PDAC patients (443 T) and healthy controls (95 N) of both genders including both validation and training sets: a SM 41:1, b LPC 18:2, and c Cer 41:1 for UHPSFC/MS (Method 1), shotgun MS (LR) (Method 2), shotgun MS (HR) (Method 3), and RP-UHPLC/MS (Method 4). In each box plot, the centerline represents the median, the bounds represent the 1st and 3rd quartile and whiskers span 1.5 fold inter-quartile range from the median. The RSD of the concentrations obtained by four methods for each sample d SM 41:1, e LPC 18:2, and f Cer 41:1. Color annotation: light blue—control females, blue—control males, red—cancer females, and dark red—cancer males. ROC curves for males (M) and females (F) in training (Tr.) and validation (Va.) sets: g UHPSFC/MS, h shotgun MS (LR), i shotgun MS (HR), and j RP-UHPLC/MS. Source data are provided as a Source Data file.

Fig. 5. Results for the lipidomic profiling of male serum samples from PDAC patients (T) and healthy controls (N) in Phase III.

a PCA for the training set (219 T + 122 N). b OPLS-DA for the training set (219 T + 122 N). Individual samples are colored according to tumor (T) stage: T1 - yellow, T2 - orange, T3 - red, T4 - rose, and Tx - brown (information about the stage is not available). c Sensitivity (red), specificity (blue), and accuracy (green) for the training (219 T + 122 N) and validation (56 T + 6 N) sets. d S-plot for the training set with the annotation of most upregulated (red) and downregulated (blue) lipid species. e Heat map for both training and validation sets (275 T + 128 N) using the lipid species concentrations [nmol/mL]. f OPLS-DA for early stages T1 + T2, age aligned (mean age is 65 ± 4 years for N and 67 ± 4 for T), and number aligned (39 T + 39 N). This graph includes both genders. Source data are provided as a Source Data file.

Fig. 6. Results for the lipidomic profiling of female serum samples from PDAC patients (T) and healthy controls (N) in Phase III.

a PCA for the training set (211 T + 124 N). b OPLS-DA for the training set (211 T + 124 N). Individual samples are colored according to their tumor (T) stage: T1 - yellow, T2 - orange, T3 - red, T4 - rose, and Tx - brown (information about the stage is not available). c Sensitivity (red), specificity (blue), and accuracy (green) for training and validation sets. d S-plot for the training set with the annotation of most upregulated (red) and downregulated (blue) lipid species. e Heat map for both training and validation sets (271 T + 134 N) using the lipid species concentrations [nmol/mL]. Source data are provided as a Source Data file.

Fig. 7. Results for the lipidomic profiling in Phase III and investigating the influence of cancer stage, age, and diabetes mellitus.

a ROC curves for males (M) and females (F) in training (Tr.) and validation (Va.) sets. Box plots of lipid molar concentrations normalized to the NIST reference material for: b SM 41:1 and c Cer 41:1. Only samples with known tumor (T) stage classification were included, where early stage (T1 and T2, 24 males and 30 females) and late stages (T3 and T4, 174 males and 176 females) are summarized and compared to samples of healthy controls (128 males and 134 females) and pancreatitis patients (13 males and 9 females). Comparison of age intervals for control (blue) and cancer (red) samples (d) SM 41:1 and e LPC 18:2. Box plot investigating the influence of diabetes (f) SM 41:1. In each box plot, the centerline represents the median, the bounds represent the 1st and 3rd quartile and whiskers span 1.5 fold inter-quartile range from the median. Comparison of the ROC curves for the samples investigated in Phase III for both genders (g) red - CA 19-9, blue - lipidomics, green - combination of CA 19-9 and lipidomics, and purple - CancerSeek. h Sensitivity (red) and specificity (blue) for CA 19-9, lipidomics, combination of CA 19-9 and lipidomics, and CancerSeek. Source data are provided as a Source Data file.

Fig. 8. Results for the lipidomic profiling of human serum samples for PDAC patients (T) and healthy controls (N) including both genders in Phase III.

Influence of surgery on the lipidomic profile: a OPLS-DA for females (211 T + 124 N) using the training set with highlighted samples before (green, n = 13) and after (orange, n = 10) surgery. Box plots of molar lipid concentrations for paired samples collected before and after surgery for both genders (2 males and 10 females): b SM 41:1, and c LPC 18:2. Box plots for paired samples collected before (n = 22) and after treatment (n = 22 for collection 1, n = 12 for collection 2, n = 7 for collection 3, n = 4 for collection 4) for both genders using molar concentrations: d SM 41:1, e LPC 18:2. In each box plot, the centerline represents the median, the bounds represent the 1st and 3rd quartile and whiskers span 1.5 fold inter-quartile range from the median. OPLS-DA models only for subjects before any treatment or surgery separately for f males (83 T + 122 N) and g females (72 T + 124 N). Source data are provided as a Source Data file.

Fig. 9. Potential of selected lipids for the survival prognosis in Phase II measured by UHPSFC/MS.

Kaplan–Meier Survival plots for: a LPC 18:2 (n = 128 for binary code 0, and n = 72 for binary code 1), b Cer 36:1 (n = 89 for 0, and n = 111 for 1), and c PC 32:0 (n = 99 for 0, and n = 101 for 1) together with the two-sided log-rank test p-value. d Cox proportional-hazards model for CA 19-9, PC 32:0, PC O-38:5, LPC 18:2, Cer 36:1, Cer 38:1, Cer 42:2, and SM 42:2. The forest plot illustrates the 95% confidence intervals of the hazard ratios and the corresponding log rank test p values for every parameter are presented. Hazard ratios > 1 indicate poorer survival. Lipid species concentrations normalized to the NIST reference material obtained for all samples in Phase II were converted into the binary code, whereby 0 was set for c < median and 1 for c > median (the median of concentrations was calculated for each lipid species including all samples). Source data are provided as a Source Data file.

Fig. 10. Network visualization of the most dysregulated lipid species in PDAC for data from Phase III.

Graphs show lipidomic pathways with clustering into individual lipid classes for a males, and b females using the Cytoscape software (http://www.cytoscape.org). Circles represent the detected lipid species, where the circle size expresses the significance according to p-value, while the color darkness defines the degree of up/downregulation (red/blue) according to the fold change. The most discriminating lipids are annotated. Source data are provided as a Source Data file.