WO2015085225A1 - Signatures de biomarqueur lipidique pour diagnostic de lésion pulmonaire - Google Patents

Signatures de biomarqueur lipidique pour diagnostic de lésion pulmonaire Download PDF

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WO2015085225A1
WO2015085225A1 PCT/US2014/068882 US2014068882W WO2015085225A1 WO 2015085225 A1 WO2015085225 A1 WO 2015085225A1 US 2014068882 W US2014068882 W US 2014068882W WO 2015085225 A1 WO2015085225 A1 WO 2015085225A1
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galcer
glc
cer
lung
control sample
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PCT/US2014/068882
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Hector De Leon
Stephanie Boue
Julia HOENG
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Hector De Leon
Stephanie Boue
Hoeng Julia
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Publication of WO2015085225A1 publication Critical patent/WO2015085225A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2570/00—Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G01N2800/122—Chronic or obstructive airway disorders, e.g. asthma COPD

Definitions

  • the present invention relates to lipid biomarkers that are useful for the diagnosis of lung injury, such as emphysema.
  • the present invention also relates to methods of diagnosing or prognosing lung injury, such as emphysema.
  • the present invention further relates to methods of treating or preventing lung injury by inhibiting the expression or activity of genes related to lipid metabolism or transport.
  • the present invention also relates to methods of identifying compounds useful for treating or preventing lung injury.
  • Emphysema is a lung condition that occurs when the alveoli are gradually destroyed, rendering an emphysema patient progressively short of breath.
  • Emphysema is part of a group of diseases collectively called Chronic Obstructive Pulmonary Disease (COPD) and is characterized by parenchymal destruction, loss of alveolar attachments and decrease in elastic recoil.
  • COPD chronic Obstructive Pulmonary Disease
  • COPD is the third leading cause of death in the U.S. and is projected to be the fourth leading cause of death worldwide by 2030.
  • over €20 billion is spent treating COPD, while approximately $18 billion is spent in the U.S.
  • Emphysema can be caused by long-term exposure to lung irritants in the environment, such as air pollution, chemical fumes, dust and tobacco smoke.
  • emphysema Because long-term exposure to lung irritants is typically required, most individuals who suffer from emphysema are more than 40 years old when symptoms first present. Such symptoms include an ongoing cough or a cough that produces an excess of mucus; shortness of breath, especially with physical activity; wheezing and chest tightness. Early detection of emphysema can be difficult because symptoms slowly worsen over time, and most affected individuals do not notice early symptoms because they are mild or easy to correct by lifestyle adjustment.
  • Emphysema is typically diagnosed by signs and symptoms, including medical history, family history and test results. Diagnostic tests for emphysema include lung function tests, such as spirometry, including lung volume
  • the diagnostic tests for emphysema require the disease to have progressed to the point that lung function is moderately affected.
  • the present invention is directed to lipid biomarkers for classifying, diagnosing or grading emphysema.
  • a first aspect of the invention provides a method of diagnosing an individual as being at risk for or having lung injury comprising detecting the level of two or more lipid biomarkers in a test sample obtained from the individual; and comparing the level of the two or more lipid biomarkers in the test sample to the level of the two or more lipid biomarkers in a control sample, wherein, if the level of the two or more lipid biomarkers is different in the test sample than in the control sample, then the individual suffers from or is at risk of having lung injury.
  • the two or more lipid biomarkers may independently be selected from a sterol, a diradylglycerol, an eicosanoid, a glycerophosphocholine, a glycerophosphoethanolamine, a glycerophosphoglycerol, a glycerophosphoinositol, a glycerophosphoserine, an acidic glycosphingolipid, a ceramide, a neutral glycosphingolipid, a phosphosphingolipid, and a sphingoid base.
  • the sterol is selected from cholesterol ester (CE)(14:0); CE(15:0); CE(16:0); CE(16: 1); CE(17:0); CE(17: 1); CE(18:0); CE(18: 1); CE(18:2); CE(18:3); CE(20:3) CE(20:4); CE(20:5); CE(22:0);
  • CE(22:5); CE(22:6); and CE(24:2) preferably CE(20:4) or CE(22:5).
  • the eicosanoid may be selected from 5-HEPE; 5-HETE; 5-oxoETE; 5,6-
  • DHET 6-keto-PGFl alpha; 8-HETE; 8,9-DHET; 9-HODE; 11-HETE; 11,12- DHET; 12-HEPE; 12-HETE; 12-oxoETE; 13-HODE; 13-HOTrE; 14,15-DHET;
  • 15-HEPE 15-HETE; arachidonic acid; docosahexaenoic acid; eicosapentaenoic acid; Prostaglandin D2 (PGD 2 ); Prostaglandin E2 (PGE 2 ); PGF2alpha; TXB 2 ; and
  • TXB 3 preferably, 5-HETE; 8,9-DHET; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD 2 ; PGE 2 ; or TXB 3 .
  • the glycerophosphocholine may be selected from phosphatidylcholine
  • PC PC(14:0/14:0); PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18:1); PC(16:0/16:0);
  • PC(18: l/20:4); PC(18: l/22:6); PC(18:2/18:2); and PC(18:2/20:4) preferably,
  • the glycerophosphoglycerol is selected from phosphatidylglycerol (PG)(16:0/16:0); PG(16:0/18: 1); PG(16:0/18:2);
  • the glycerophosphoethanolamine is selected from
  • PE phosphatidylethanolamine
  • the ceramide (Cer) may be selected from Cer(dl8:0/16:0);
  • the neutral glycosphingolipid is selected from glucosyl/galactosyl Cer (Glc/GalCer)(dl8:0/16:0); Glc/GalCer(dl 8:0/18:0); Glc/GalCer(dl8:0/20:0); Glc/GalCer(dl 8:0/22:0); Glc/GalCer(dl 8:0/24:0);
  • Gb3 globotriaosylceramide
  • the neutral glycosphingolipid is selected from Glc/GalCer(dl8:0/16:0); Glc/GalCer(dl 8:0/24:0); Glc/GalCer(dl8:l/16:0);
  • the acidic glycosphingolipid may be selected from GMl(dl8:l/16:0); GMl(dl8:l/24:0); GMl(dl8:l/24:l); GM3(dl8:l/16:0); GM3(dl8:l/18:0);
  • the sphingoid base may be selected from sphingosine- 1 -phosphate (SlP)(dl8:l); SlP(dl8:2); sphinganine-1 -phosphate (SAlP)(dl8:0); sphinganine
  • SPA sphingosine
  • the lipid biomarker is selected from 5-HETE; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD2; PC(16: 1/16:1);
  • the individual suffers from or is at risk of having lung injury if the level of one or more of PGD2; PGE2; PC(14:0/14:0);
  • LacCer(dl 8: 1/24: 1) is higher in the test sample than in the control sample, and wherein the test sample and the control sample are obtained from a large airway, such as a bronchial biopsy, or the lung, such as a lung biopsy, including a biopsy of the parenchyma.
  • the individual suffers from or is at risk of having lung injury if the level of one or more of PC(16: 1/18: 1); PC(18:1/18: 1);
  • Glc/GalCer(dl8:0/24: l); and Cer(dl 8:0/18:0) is higher in the test sample than in the control sample, and wherein the test sample and the control sample are blood samples.
  • the individual suffers from or is at risk of having lung injury if the level of one or more of arachidonic acid; PC(16:0/18:0);
  • PC(18:0/18.1); Cer(dl8: l/18:0); LacCer(dl8.1/18:0) and LacCer(dl 8.1/20.0) is lower in the test sample than in the control sample, and wherein the test sample and the control sample are obtained from a large airway, such as a bronchial biopsy, or the lung, such as a lung biopsy, including a biopsy of the parenchyma.
  • the individual suffers from or is at risk of having lung injury if the level of one or more of PC(16:0/18:0); PC(16:0/20:4);
  • Cer(dl8: l/16:0); Cer(dl8:l/18:0); and Cer(dl8: l/18: l) is lower in the test sample than in the control sample, and wherein the test sample and the control sample are blood samples.
  • the individual suffers from or is at risk of having lung injury if the level of three or more lipid biomarkers is different in the test sample than in the control sample. In some embodiments, the individual suffers from or is at risk of having lung injury if the level of four or more lipid biomarkers is different in the test sample than in the control sample. In some embodiments, the individual suffers from or is at risk of having lung injury if the level of five or more lipid biomarkers is different in the test sample than in the control sample. In some embodiments, the individual suffers from or is at risk of having lung injury if the level of ten or more lipid biomarkers is different in the test sample than in the control sample.
  • the method may further comprise detecting the level of the two or more lipid biomarkers in the control sample.
  • the test sample may be selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • the test sample is obtained from a large airway of the individual, such as a bronchial biopsy, or the lung of the individual, such as a lung biopsy, including a biopsy of the parenchyma.
  • control sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • the control sample may be obtained from a large airway or lung of an individual not affected with lung injury, such as from a bronchial biopsy or a lung biopsy of the individual not affected with lung injury.
  • the control sample is obtained, prior to the onset of lung injury, from the individual at risk for or having the emphysema.
  • the control sample is obtained from an individual that does not suffer from lung injury.
  • the level of the two or more lipid biomarkers in the test sample and the level of the two or more lipid biomarkers in the control sample may be detected by mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, dual polarization interferometry or chromatography.
  • the mass spectrometry is electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, or atmospheric pressure chemical ionization mass spectrometry.
  • the chromatography is thin layer chromatography, solid-phase extraction
  • HPLC high performance liquid chromatography
  • hydrophilic interaction liquid chromatography hydrophilic interaction liquid chromatography
  • ultra-performance liquid chromatography ultra-performance liquid chromatography
  • the lung injury is emphysema or COPD.
  • a method of diagnosing an individual as being at risk for or having lung injury comprising
  • the two or more lipid biomarkers are independently selected from a sterol, a diradylglycerol, an eicosanoid, a glycerophosphocholine, a
  • glycerophosphoinositol a glycerophosphoserine
  • an acidic glycosphingolipid a ceramide
  • a neutral glycosphingolipid a phosphosphingolipid
  • a phosphosphingolipid a sphingoid base
  • sterol is selected from cholesterol ester (CE)(14:0); CE(15:0); CE(16:0); CE(16: 1); CE(17:0); CE(17: 1);
  • the sterol is CE(20:4) or CE(22:5).
  • the eicosanoid is selected from 5-HEPE; 5-HETE; 5-oxoETE; 5,6-DHET; 6-keto- PGFlalpha; 8-HETE; 8,9-DHET; 9-HODE; 11-HETE; 11,12-DHET; 12-HEPE; 12-HETE; 12-oxoETE; 13-HODE; 13-HOTrE; 14,15-DHET; 15-HEPE; 15-HETE; arachidonic acid; docosahexaenoic acid; eicosapentaenoic acid; Prostaglandin D2 (PGD 2 ); Prostaglandin E2 (PGE 2 ); PGF2alpha; TXB 2 ; and TXB 3 .
  • eicosanoid is selected from 5-HETE; 8,9-DHET; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD 2 ; PGE 2 ; and TXB 3 .
  • glycerophosphocholine is selected from phosphatidylcholine (PC)(14:0/14:0);
  • glycerophosphocholine is selected from PC(16:0/16:0); and PC(16: 1/16: 1).
  • the glycerophosphoglycerol is selected from phosphatidylglycerol (PG)(16:0/16:0); PG(16:0/18: 1); PG(16:0/18:2); PG(18:1/18: 1); PG(18: 1/18:2); and PG(18:2/18:2).
  • glycerophosphoglycerol is selected from PG(18: 1/18: 1), PG(18: 1/18:2) and PG(18:2/18:2).
  • PE PE(16:0/16:0); PE(16:0/18: 1); PE(16:0/18:2); PE(16:0/20:4); PE(16:0/22:4); PE(18:0/18:0); PE(18:0/20:4); PE(18:0/22:4); PE(18:1/18: 1); PE(18: l/20:4); and PE(22:6/22:6).
  • glycerophosphoethanolamine is selected from PE(16:0/16:0); PE(16:0/18: 1); PE(16:0/18:2); and PE(16:0/20:4).
  • ceramide is selected from Cer(dl8:0/16:0); Cer(dl 8:0/18:0); Cer(dl8:0/18: l);
  • Glc/GalCer (dl8:0/16:0); Glc/GalCer(dl 8:0/18:0); Glc/GalCer(dl8:0/20:0); Glc/GalCer(dl8:0/22:0); Glc/GalCer(dl 8:0/24:0); Glc/GalCer(dl 8:0/24: 1);
  • lactosylCer (LacCer)(dl 8:0/16:0); LacCer(dl8: l/16:0); LacCer(dl8: l/18:0); LacCer(dl8:l/20:0); LacCer(dl 8: 1/22:0); LacCer(dl 8: 1/23:0);
  • glycosphingolipid is selected from Glc/GalCer(dl8:0/16:0);
  • glycosphingolipid is selected from GMl(dl8:l/16:0); GM3(dl8:l/16:0); and GM3(dl8:l/24:0).
  • sphingoid base is selected from sphingosine-1 -phosphate (SlP)(dl8:l);
  • lipid biomarker is selected from 5 -HETE; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD2; PC(16: 1/16: 1); PE(16:0/16:0); PE(16:0/18:2); PE(16:0/20:4);
  • SPH(d20: 1) is higher in the test sample than in the control sample, and wherein the test sample and the control sample are obtained from a large airway or a lung.
  • Cer(dl8: l/18:0); and Cer(dl8: l/18: l) is lower in the test sample than in the control sample, and wherein the test sample and the control sample are blood samples.
  • test sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • test sample is obtained from a large airway or a lung of the individual.
  • test sample is obtained from a bronchial biopsy or a lung biopsy.
  • control sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • control sample is obtained from a large airway or a lung of an individual not affected with lung injury.
  • control sample is obtained from a bronchial biopsy or a lung biopsy of the individual not affected with lung injury.
  • control sample is obtained from the individual at risk for or having the emphysema prior to onset of the lung injury.
  • control sample is obtained from an individual that does not suffer from lung injury.
  • MALDI desorption/ionization
  • chromatography is thin layer chromatography, solid-phase extraction chromatography, high performance liquid chromatography (HPLC), hydrophilic interaction liquid chromatography, or ultra-performance liquid chromatography.
  • Figure 1A provides representative images of lung tissue from cigarette smoke (CS)-exposed, Sham, and Cessation animals at the 2-, 3-, and 7-month time points. Tissues were stained with haematoxylin and eosin (H&E).
  • Figure IB provides a representative image of multinucleated giant cells found in lung tissue from CS-exposed animals. Tissues were stained with alcian blue periodic acid Schiff reagent (AB-PAS).
  • Figure 2 provides histopathological findings and histomorphometry in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months.
  • B The number of multinucleated giant cells are shown as the mean numbers ⁇ SEM.
  • Figure 3 provides pulmonary lipid profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung lipid species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 4 provides relative percentage differences in lung molecular PCs and PGs concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05,
  • Figure 5 A provides relative percentage differences in lung molecular PCs and PGs concentrations between mice exposed to smoke (CS) and fresh air
  • FIG. 5B provides relative percentage differences in lung molecular concentrations of PEs between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months.
  • Figure 5C provides relative percentage differences in plasma molecular concentrations of PCs between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 6 provides pulmonary ceramide lipid species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular ceramide species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 7 provides pulmonary glucosyl/galactosyl ceramide lipid species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular glucosyl/galactosyl ceramide species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 8 provides pulmonary lactosyl ceramide lipid species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular lactosyl ceramide species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 9 provides pulmonary GM1 ganglioside species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular GM1 ganglioside species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 10 provides pulmonary GM3 ganglioside species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular GM3 ganglioside species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 11 provides pulmonary sphingoid base species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular sphingoid base species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05,
  • Figure 12 provides pulmonary globotriaosylceramide species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular globotriaosylceramide species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 13 provides pulmonary cholesterol ester species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular cholesterol ester species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 14 provides plasma cholesterol ester species profiles in mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in plasma molecular cholesterol ester species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05,
  • Figure 15 provides a CONSORT diagram for the clinical study.
  • the flow chart of the study shows the number of subjects enrolled in the study and the number of completers indicating reasons for subject recruitment, as recommended by the Consolidated Standards of Reporting Trials guidelines.
  • Figure 16 shows median relative differences in lipid concentrations categorized by lipid class in the serum of healthy, current smokers (CS) compared to never-smokers (NS), former smokers (FS) compared to current smokers and to never-smokers, respectively.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 17 provides median relative differences in diradylglycerol (DAG) concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 18 provides box-whisker plots and scatter grams of individual diradylglycerol concentrations in the serum of current smokers and never-smokers, respectively.
  • Panel A represents the log-transformed results for DAG(16:0/18: 1), and panel B those for DAG(18: 1/18: 1) serum levels in nmol/mL.
  • Box-whisker plots reflect the first and third quartile (lower and upper boundary of box, respectively), median (green line), and minimum and maximum values (lower and upper whisker, respectively) for the corresponding study group.
  • Outliers are represented by open circles.
  • Serum lipid concentrations are further represented by dots, where red dots indicate study subjects taking lipid-modifying drugs and blue dots those who do not.
  • Figure 19 provides median relative differences in
  • PC glycerophosphatidylcholine
  • Figure 20 provides median relative differences in triacylglycerol (TAG) concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively.
  • TAG triacylglycerol
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 21 provides median relative differences in lactosylceramide concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 22 provides median relative differences in glucosyl/galactosyl ceramide concentrations in the serum of healthy, current smokers (CS) compared to never-smokers (NS), former smokers (FS) compared to current smokers and to never-smokers, respectively.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05.
  • Figure 23 provides median relative differences in
  • PE glycerophosphoethanolamine
  • Figure 24 provides median relative differences in eicosanoid (EICO) concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively.
  • EICO eicosanoid
  • Figure 25 provides median relative differences in ceramide (Cer) concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 26 shows median relative differences in glycophosphosphingolipid (SM) concentrations in the serum of healthy, current smokers (CS) compared to never-smokers (NS), former smokers (FS) compared to current smokers and to never-smokers, respectively.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05.
  • Figure 27 shows median relative differences in sterol (CE) concentrations in the serum of healthy, current smokers (CS) compared to never-smokers (NS), former smokers (FS) compared to current smokers and to never-smokers, respectively.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 28 shows median relative differences in lipid concentrations categorized by lipid class in the serum of smokers with mild COPD (COPD) compared to never-smokers (NS), former smokers (FS) and current smokers (CS), respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 29 shows median relative differences in sterol (CE) concentrations in the serum of smokers with mild COPD (COPD) compared to never-smokers (NS), former smokers (FS) and current smokers (CS), respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom.
  • Figure 30 provides box-whisker plots and scattergrams of individual sterol concentrations in the serum of current smokers and never-smokers, respectively.
  • Panel A represents the results for CE(16:0), panel B those for CE(18:2), panel C those for CE(19:0), and panel D represents the log-transformed results for CE(20:5) serum levels in nmol/mL.
  • Box-whisker plots reflect the first and third quartile (lower and upper boundary of box, respectively), median (green line), and minimum and maximum values (lower and upper whisker, respectively) for the corresponding study group.
  • Outliers are represented by open circles.
  • Serum lipid concentrations are further represented by dots, where red dots indicate study subjects taking lipid-modifying drugs and blue dots those who do not.
  • Figure 31 shows median relative differences in phosphosphingolipid concentrations in the serum of smokers with mild COPD (COPD) compared to never-smokers (NS), former smokers (FS) and current smokers (CS), respectively.
  • COPD COPD
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 32 provides median relative differences in
  • glycerophosphatidylcholine concentrations in the serum of smokers with mild COPD COPD
  • COPD COPD
  • NS never-smokers
  • FS former smokers
  • CS current smokers
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • biomarker refers to a characteristic whose presence, absence or level indicates a biological state. Typically, the properties of biomarkers indicate a normal process, a pathogenic process or a response to a pharmaceutical or therapeutic intervention.
  • a biomarker can be a cell, a gene, a gene product, an enzyme, a hormone, a protein, a peptide, an antibody, a nucleic acid molecule, a metabolite, a lipid, a free fatty acid, cholesterol or some other chemical compound.
  • a biomarker can be a morphologic biomarker (for example, a histological change, DNA ploidy, malignancy-associated changes in the cell nucleus and premalignant lesions) or a genetic biomarker (for example, DNA mutations, DNA adducts and apoptotic index).
  • a morphologic biomarker for example, a histological change, DNA ploidy, malignancy-associated changes in the cell nucleus and premalignant lesions
  • a genetic biomarker for example, DNA mutations, DNA adducts and apoptotic index
  • COPD Chironic Obstructive Pulmonary Disease
  • COPD refers to a complex disease that results in progressive loss of lung function.
  • COPD is typically characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways.
  • COPD can include the occurrence of chronic bronchitis or emphysema, both of which result in airway narrowing.
  • Clinically, COPD is typically detected by limited airflow in lung function tests.
  • COPD is typically irreversible and gets progressively worse over time. Symptoms of COPD include chronic cough, chronic sputum production, dyspnea, rhonchi, wheezing, chest tightness, tiredness and decreased airflow in lung function tests.
  • COPD ulcerative colitis
  • BODE index is a scoring system that measures FEV1, body-mass index, 6-minute walk distance, and a modified MRC (Medical Research Council) dyspnea scale to estimate outcomes in COPD.
  • control sample refers to a sample against which a test sample is compared in order to diagnose, prognose, classify or grade the test sample.
  • a control sample may be healthy tissue or may be a well-characterized sample, such as from an individual suffering from COPD, including but not limited to, GOLD stage 1, GOLD stage 2, GOLD stage 3, or GOLD stage 4 COPD.
  • a control sample can be analyzed concurrently with or separately from the test sample, including before or after analyzing the test sample.
  • the data from the analysis of a control sample may be stored, e.g., in a computer readable medium or in a manual, for comparison against test samples analyzed in the future, or as data for training network-based or machine-learning methods.
  • a control sample may be developed as a medical standard for comparison. For example, analysis of control samples has developed medical standards for normal fed and fasted blood glucose levels; normal, at risk, and hypertensive blood pressures, and normal resting heart rates.
  • the term "control sample” includes samples that provided a medical standard. Accordingly, a test sample may be compared against a medical standard generated from control samples. For example, production of a variant of a lipid may be indicative of a change medical condition. Alternatively, a change in production level of a lipid may be indicative of a change in medical condition.
  • a control sample may be lung tissue, such as tissue obtained by biopsy from a healthy individual, or some other sample.
  • a control sample may be sputum, saliva, bronchial washing, bronchial aspirates, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • Tissue specimens such as those obtained by biopsy, may be fixed (e.g., formaldehyde-fixed paraffin-embedded (FFPE)).
  • FFPE formaldehyde-fixed paraffin-embedded
  • the control sample may be obtained from a tissue bank.
  • the control sample may also be obtained from a cadaver or an organ donor.
  • fatty acid refers to a carboxylic acid with a long aliphatic chain, which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from triglycerides or phospholipids. When they are not attached to other molecules, they are known as "free" fatty acids.
  • FEV1 force expiratory volume in one second
  • FEV1 refers to the volume of air that can forcibly be blown out in one second, after full inspiration.
  • Average values for FEV1 in healthy individuals depend on sex and age and have been well-characterized in the art.
  • FEV1 and the FEV1 to FVC ration (FEV1/FVC) are used clinically to grade COPD. In healthy adults
  • FEV1/FVC should be approximately 75-80%.
  • obstructive diseases such as myeloma
  • FEV1 is diminished because of increased airway resistance to expiratory flow. While the FVC may be decreased as well, due to the premature closure of airway in expiration, FEV1 is typically more affected because of the increased airway resistance, so the FEV1/FVC ratio reflects the degree of airway closure compared to lung volume.
  • FVC force vital capacity
  • the term "individual” refers to a vertebrate, preferably a mammal.
  • the mammal can be, without limitation, a mouse, a rat, a cat, a dog, a horse, a pig, a cow, a non-human primate or a human.
  • the individual is a human.
  • the term "individual at risk for lung injury” refers to an individual who is predisposed to lung injury, such as COPD, including emphysema. Predisposition to lung injury may be due to one or more genetic or environmental factors. For example, an individual related to a COPD patient is more likely to get COPD than an individual who is not related to a COPD patient. Further, exposure to environmental factors such as radon gas, asbestos, tobacco smoke, and air pollution can increase the risk for lung injury and predispose an individual to lung injury.
  • the term "individual having a lung injury” or “individual suffering from injury” refers to an individual experiencing progressive loss of lung function, typically characterized by alveoli destruction.
  • Lung injury can be bronchial or emphysematous and may be detected by analyzing clinical, functional, and radiological findings or detecting relevant biomarkers.
  • lipid refers to a class of organic compounds that are fatty acids or their derivatives and are, typically, insoluble in water but soluble in organic solvents. Lipids may be divided into eight categories: fatty acids, glycero lipids, glycerophospho lipids, sphingo lipids, saccharo lipids, polyketides sterol lipids and prenol lipids. Fatty acids and fatty acid derivatives may be identified using a notation giving the number of carbon atoms and of double bonds (separated by a colon).
  • palmitic acid which has sixteen carbon atoms and no double bonds
  • oleic acid which has eighteen carbons and one double bond
  • Some lipids comprise a head group with one or more fatty acid tails.
  • phosphatidylcholines comprise a choline head groups and two fatty acid tails, one saturated and one unsaturated. Such lipids may be identified by their fatty acid tails.
  • PC(16:0/18: 1) refers to a phosphtidylcholine lipid with a palmitic acid tail and an oleic acid tail.
  • lipid signature refers to a group of lipids produced by a cell or a tissue, whose combined production pattern may be indicative of, e.g., a normal state, an at-risk state, a diseased state, a treated state or a recovery state.
  • a lipid signature may be characterized by which lipids are produced or at what level each lipid is produced.
  • Lipid signatures are particularly useful in diagnosing, prognosing, classifying or grading complex diseases states, which result from the combination of several genetic and environmental factors.
  • the lipid signatures disclosed herein may be used, e.g., for the diagnosis, prognosis, classification and/or grading of lung injury, such as emphysema, in an individual.
  • MALDI-TOF matrix-assisted laser
  • Time-of-flight (TOF) mass spectrometry refers to a method in which an ion's mass-to-charge ratio is determined via the time that it takes an ionized particle to reach a detector at a known distance.
  • saturated refers to a compound, such as a fatty acid, that has no double or triple bonds or ring. In saturated hydrocarbons, every carbon atom is attached to two hydrogen atoms, except those at the ends of the chain, which bear three hydrogen atoms.
  • :0 refers to a saturated fatty acid. For example, 16:0 (palmitic acid) refers to a saturated fatty acid comprising sixteen carbon atoms.
  • test sample refers to a sample obtained from an individual at risk for, having or suffering from lung injury.
  • a test sample may be any sample suspected of containing or exhibiting a biomarker.
  • test sample is analyzed and compared to a control sample, including medical standards developed from control samples, to diagnose, prognose, classify or grade lung injury in the individual.
  • a test sample may be obtained from lung tissue, such as tissue obtained by biopsy from a tumor, or other biological tissue.
  • a test sample may be sputum, saliva, bronchial washing, bronchial aspirates, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • Tissue specimens, such as those obtained by biopsy may be fixed (e.g., formaldehyde-fixed paraffin-embedded (FFPE)).
  • FFPE formaldehyde-fixed paraffin-embedded
  • the term "unsaturated” refers to a compound, such as a fatty acid, that contains carbon-carbon double bonds or triple bonds.
  • a chain of carbons such as a fatty acid
  • a double or triple bond will cause a kink in the chain.
  • Unsaturated fats tend to be liquid at room temperature, rather than solid, due to the kinks in the chain, which prevent the molecules from packing closely together to form a solid. These fats are typically called oils and are present in fish and plants.
  • the degree of unsaturation refers to the number of double and triple bonds in the fatty acid. In certain fatty acid nomenclature, the number following the colon refers to a saturated fatty acid.
  • 18: 1 (oleic acid) refers to a fatty acid comprising eighteen carbon atoms and one double bond (i.e., one degree of unsaturation).
  • 18:2 (linoleic acid) refers to a fatty acid comprising eighteen carbon atoms and two double bonds (i.e., two degrees of unsaturation).
  • lipid biomarkers useful for diagnosing, prognosing, classifying or grading lung injury, such as COPD, including emphysema.
  • the lipid biomarkers may independently be selected from a sterol, a diradylglycerol, an eicosanoid, a glycerophosphocholine, a
  • glycerophospho inositol a glycerophosphoserine, an acidic glycosphingo lipid, a ceramide, a neutral glycosphingolipid, a phosphosphingolipid, and a sphingoid base.
  • the sterol is selected from cholesterol ester (CE)(14:0); CE(15:0); CE(16:0); CE(16: 1); CE(17:0); CE(17: 1); CE(18:0);
  • CE(22:5); CE(22:6); and CE(24:2) preferably CE(20:4) or CE(22:5).
  • CE(20:4) or CE(22:5) are downregulated by CS.
  • the eicosanoid may be selected from 5-HEPE; 5-HETE; 5-oxoETE; 5,6-
  • DHET 6-keto-PGFl alpha; 8-HETE; 8,9-DHET; 9-HODE; 11-HETE; 11,12- DHET; 12-HEPE; 12-HETE; 12-oxoETE; 13-HODE; 13-HOTrE; 14,15-DHET;
  • 15-HEPE 15-HETE; arachidonic acid; docosahexaenoic acid; eicosapentaenoic acid; Prostaglandin D2 (PGD 2 ); Prostaglandin E2 (PGE 2 ); PGF2alpha; TXB 2 ; and
  • TXB 3 preferably, 5-HETE; 8,9-DHET; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD 2 ; PGE 2 ; or TXB 3 .
  • the eicosanoid is be selected from 5-HETE; 8,9-DHET; 11,12-DHET; 14,15-DHET; arachidonic acid; or TXB 3 .
  • the eicosanoid may be TXB 3 .
  • the eicosanoid is not PGD 2 or PGE 2 .
  • the glycerophosphocholine may be selected from phosphatidylcholine
  • PC PC(14:0/14:0); PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18:1); PC(16:0/16:0);
  • PC(18: l/20:4); PC(18: l/22:6); PC(18:2/18:2); and PC(18:2/20:4) preferably, PC(14:0/14:0); PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18: 1); PC(16:0/16: 1); or
  • the glycerophosphocholine is PC(16:0/16:0) or
  • PC(16: 1/16: 1) which are both upregulated in lung tissue following CS exposure.
  • PC 18:0/20:4; 18: 1/20:4; and 18:2/20:4 are downregulated in plasma following CS exposure.
  • the glycerophosphoglycerol is selected from phosphatidylglycerol (PG)(16:0/16:0); PG(16:0/18: 1); PG(16:0/18:2);
  • PG(18:2/18:2) are upregulated in lung tissue following CS exposure.
  • the glycerophosphoethanolamine is selected from phosphatidylethanolamine (PE)( 16:0/16:0); PE(16:0/18:1); PE(16:0/18:2);
  • PE(16:0/18:1); PE(16:0/18:2); and PE(16:0/20:4) are upregulated in lung tissue following CS exposure.
  • the ceramide (Cer) may be selected from Cer(dl8:0/16:0);
  • the ceramide may be selected from
  • the neutral glycosphingolipid is selected from glucosyl/galactosyl Cer (Glc/GalCer)(dl8:0/16:0); Glc/GalCer(dl 8:0/18:0);
  • Glc/GalCer (dl8:l/26:l); lactosylCer (LacCer)(dl8:0/16:0); LacCer(dl8:l/16:0);
  • Gb3 globotriaosylceramide
  • the neutral glycosphingolipid is selected from Glc/GalCer(dl8:0/16:0); Glc/GalCer(dl 8:0/22:0); Glc/GalCer(dl 8:0/24:0);
  • the neutral glycosphingolipid is selected from Glc/GalCer(dl8:0/16:0);
  • the acidic glycosphingolipid may be selected from GMl(dl8: l/16:0);
  • the acidic glycosphingolipid is selected from GMl(dl8: l/16:0); GM3(dl8: l/16:0); and GM3(dl8:l/24:0), which are each upregulated in lung tissue following CS exposure.
  • the GM1 class of lipids i.e., "Sum(Gl)”
  • the GM3 class of lipids i.e., "Sum(G3)
  • a change in Sum(Gl) or Sum(G3) may indicate severe lung injury.
  • the sphingoid base may be selected from sphingosine-1 -phosphate (SlP)(dl8: l); SlP(dl8:2); sphinganine-1 -phosphate (SAlP)(dl8:0); sphinganine (SPA)(dl8:0); SPA(d20:0); sphingosine (SPH)(dl6: l); SPH(dl8: l); SPH(dl8:2); and SPH(d20: 1).
  • the sphingoid base may be selected from sphingosine-1 -phosphate (SlP)(dl8: l); SlP(dl8:2); sphinganine-1 -phosphate (SAlP)(dl8:0); sphinganine (SPA)(dl8:0); SPA(d20:0); sphingosine (SPH)(dl6: l); SPH(dl8
  • the SIP class of lipids i.e., "Sum(SlP)" are upregulated in lung tissue following CS exposure.
  • the SA1P class of lipids i.e., "Sum(SAlP)" are upregulated in lung tissue following CS exposure.
  • the SPA class of lipids are upregulated in lung tissue following CS exposure.
  • the SPH class of lipids i.e., "Sum(SPH)" are upregulated in lung tissue following CS exposure.
  • the sphingoid base may be selected from Sum(SlP); Sum(SAlP); Sum(SPA); and Sum(SPH).
  • the sphingoid base is not a sphingosine-1 -phosphate.
  • the lipid biomarker is selected from CE(20:4);
  • the lipid biomarker is selected from CE(20:4); CE(22:5); 5-HETE; 8,9-DHET;
  • the lipid biomarker is not PGD 2 or PGE 2 . In some embodiments, the lipid biomarker is not a sphingosine-1 -phosphate.
  • the lipid biomarker is selected from 5-HETE; 5,6-
  • DHET 8-HETE; 11,12-DHET; 12-HEPE; 12-oxoETE; 14,15-DHET; arachidonic acid; eicosapentaenoic acid; PGD2; PC(14:0/14:0); PC(14:0/16:0); PC(14:0/18: 1);
  • the lipid biomarker is selected from 5 -HETE; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD2; PC(16: 1/16: 1); PE(16:0/16:0); PE(16:0/18:2); PE(16:0/20:4); Cer(dl8: l/26: 1); Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl 8: 1/23:0);
  • lipid biomarkers have increased production in an sample from an individual suffering from or at risk for lung injury compared to a control sample.
  • lipid biomarkers 20 or more, 25 or more, 30 or more, or 35 or more of the lipid biomarkers have increased production in an sample from an individual suffering from or at risk for lung injury compared to a control sample.
  • the lipid biomarkers that are up- regulated in an individual suffering from or at risk for lung injury may be selected from PGD2; PGE2; PC(14:0/14:0); PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18: 1);
  • Lipid biomarkers such as PC(16: 1/18: 1); PC(18:1/18:1); Glc/GalCer(dl8:0/24:l); or Cer(dl 8:0/18:0), may be up-regulated in blood samples from an individual suffering from or at risk for lung injury.
  • lipid biomarkers such as PC(16: 1/18: 1); PC(18:1/18:1); Glc/GalCer(dl8:0/24:l); or Cer(dl 8:0/18:0
  • lipid biomarkers such as PC(16: 1/18: 1); PC(18:1/18:1); Glc/GalCer(dl8:0/24:l); or Cer(dl 8:0/18:0
  • lipid biomarkers such as
  • LacCer(dl 8: 1/23:0); LacCer(dl8:l/24:0); or LacCer(dl8:l/24:l), may be up- regulated in a large airway, such as in a sample from a bronchial biopsy, or a lung, for example in a sample from a lung biopsy, including a biopsy of the parenchyma, of an individual suffering from or at risk for lung injury.
  • SAlP(dl8:0); SPA(dl8:0); SPH(dl6:l); SPH(dl8:l); SPH(dl8:2); or SPH(d20:l) is up-regulated in a large airway, such as in a sample from a bronchial biopsy, or a lung, for example in a sample from a lung biopsy, including a biopsy of the parenchyma, of an individual suffering from or at risk for lung injury.
  • SPA(dl8:0); SPH(dl6: l); SPH(dl8: l); SPH(dl8:2); and SPH(d20: l) is up- regulated in a large airway, such as in a sample from a bronchial biopsy, or a lung, for example in a sample from a lung biopsy, including a biopsy of the parenchyma, of an individual suffering from or at risk for lung injury.
  • At least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 45 of lipid biomarkers have decreased production in an sample from an individual suffering from or at risk for lung injury compared to a control sample.
  • 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, or 35 or more of the lipid biomarkers have decreased production in an sample from an individual suffering from or at risk for lung injury compared to a control sample.
  • the lipid biomarkers that are down- regulated in an individual suffering form or at risk for lung injury may be selected from PC(16:0/18:0); PC(16:0/20:4); PC(16:0/22:6); PC(18:0/18.1); PC(18:0/18:2); PC(18:0/20:4); PC(18:0/22:6); PC(18: 1/18:2); PC(18: l/20:4); PC(18:2/18:2); PC(18:2/20:4); Glc/GalCer(dl8:0/22:0); Glc/GalCer(dl8:l/16:0);
  • Lipid biomarkers such as PC(16:0/18:0); PC(16:0/20:4);
  • Cer(dl8: l/16:0); Cer(dl8: l/18:0); or Cer(dl8: l/18: l), may be down-regulated in blood samples from an individual suffering from or at risk for lung injury.
  • lipid biomarkers such as arachidonic acid; PC( 16:0/18:0);
  • PC(18:0/18.1); Cer(dl8: l/18:0); LacCer(dl8.1/18:0) or LacCer(dl 8.1/20.0), may be down-regulated in a large airway, such as in a sample from a bronchial biopsy, or a lung, for example in a sample from a lung biopsy, including a biopsy of the parenchyma, of an individual suffering from or at risk for lung injury.
  • the lipid biomarker are up-regulated to a certain degree in a sample from an individual suffering from or at risk for lung injury compared to a control sample.
  • each up-regulated lipid biomarker may, independently, be up-regulated at least 1.5-fold, at least 2-fold, at least 2.5- fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 100-fold, at least 1,000-fold or more compared to the control sample.
  • the lipid biomarkers are down-regulated to a certain degree in a sample from an individual suffering from or at risk for lung injury compared to a control sample.
  • each down-regulated lipid biomarker may, independently, be down-regulated at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 100-fold, at least 1,000-fold or more compared to the control sample.
  • the lipid biomarkers of the invention may be used in methods of diagnosing, prognosing, classifying or grading lung injury in biological sample or an individual.
  • the lung injury may be COPD, including emphysema.
  • One aspect of the invention provides a method of diagnosing, classifying or grading lung injury in an individual at risk for or suffering from a lung injury.
  • the method comprises classifying a test sample as injured or non- injured, such as emphysematous or non-emphysematous or COPD or non-COPD.
  • the method comprises measuring the levels of at least 2 lipid biomarkers described above in a test sample; and comparing those measurements to the level of the at least two lipid biomarker in a control sample to obtain a classification of the test sample as injured or non-injured, such as emphysematous or non-emphysematous or COPD or non-COPD.
  • the levels of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, or at least 150 lipid biomarkers described above are measured.
  • the methods of the invention comprise obtaining a test sample from an individual, determining the absence, presence or level of one or more of the lipid biomarkers described above in the test sample, comparing said absence, presence or level to the absence, presence or level of the same lipid biomarker(s) in a control sample, and diagnosing the individual as having or being at risk for lung injury based on the comparison.
  • the invention provides a method for monitoring the progress or recovery of a lung injury in an individual, said method comprising determining at suitable time intervals in one or more samples taken from said individual differential production levels of the lipid biomarkers described above.
  • the invention provides a method for monitoring the progress or recovery of a lung injury treatment in an individual, said method comprising determining at suitable time intervals before, during, or after therapy (for example, at different time points during the treatment) in one or more samples taken from said individual differential production levels of the lipid biomarkers described above.
  • the invention provides a method for monitoring lung injury in an individual resulting from exposure to air-borne pollutants, said method comprising determining at suitable time intervals in one or more samples taken from said individual differential production levels of the lipid biomarkers described above.
  • the invention provides a method for monitoring changes in the severity of a lung injury in an individual, said method comprising determining at suitable time intervals before, during, or after changing the method or pattern of nicotine consumption (for example, at different time points during smoking cessation or switching from a combusted tobacco product, e.g., cigarette, to a heated tobacco product or an electronic cigarette) in one or more samples taken from said individual differential production levels of the lipid biomarkers described above.
  • the individual is a cigarette smoker; the individual is a former cigarette smoker; the individual was a cigarette smoker who has stopped smoking cigarette for at least 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 36 month(s) prior to the measurements; the individual is or the individual was a cigarette smoker who has switched to using a heated tobacco product or a nicotine- containing product which can include an electronic cigarette or a nicotine patch, for at least 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 36 month(s) prior to the measurements instead of smoking cigarette.
  • the method comprises detecting the level of at least 2 lipid biomarkers described above in a test sample obtained from the individual; and comparing the level of the at least 2 lipid biomarkers in the test sample to the level of the at least 2 lipid biomarkers in a control sample. In some embodiments, if the level of the at least 2 lipid biomarkers is different in the test sample than in the control sample, then the individual suffers from or is at risk for lung injury. In some embodiments, the level of the at least 2 lipid biomarkers is higher in the test sample than in the control sample. Optionally, the level of the lipid biomarkers is lower in the test sample than in the control sample.
  • the method further comprises detecting the level of the lipid biomarkers in the control sample.
  • the levels of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, or at least 150 lipid biomarkers are detected.
  • the test sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • the test sample is obtained from a large airway of the individual, such as a bronchial biopsy, or the lung of the individual, such as a lung biopsy, including a biopsy of the parenchyma of the individual.
  • control sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • control sample is obtained from a large airway or a lung of an individual not affected with a lung injury, such as from a bronchial biopsy or a lung biopsy of the individual not affected with the lung injury.
  • control sample is obtained from the individual at risk for or having lung injury prior to onset of the lung injury.
  • the control sample is obtained from an individual that does not suffer from a lung injury.
  • the individual suffers from or is at risk of having lung injury if the level of one or more of PGD2; PGE2; PC(14:0/14:0);
  • LacCer(dl 8: 1/24: 1) is higher in the test sample than in the control sample, wherein the test sample and the control sample are obtained from a large airway, such as a bronchial biopsy, or the lung, such as a lung biopsy, including a biopsy of the parenchyma.
  • the individual suffers from or is at risk of having lung injury if the level of one or more of PC(16: 1/18: 1); PC(18:1/18: 1);
  • Glc/GalCer(dl8:0/24: l); and Cer(dl 8:0/18:0) is higher in the test sample than in the control sample, wherein the test sample and the control sample are blood samples.
  • the individual suffers from or is at risk of having lung injury if the level of one or more of arachidonic acid; PC(16:0/18:0);
  • PC(18:0/18.1); Cer(dl8: l/18:0); LacCer(dl8.1/18:0) and LacCer(dl 8.1/20.0) is lower in the test sample than in the control sample, wherein the test sample and the control sample are obtained from a large airway, such as a bronchial biopsy, or the lung, such as a lung biopsy, including a biopsy of the parenchyma.
  • the individual suffers from or is at risk of having lung injury if the level of one or more of PC(16:0/18:0); PC(16:0/20:4);
  • Cer(dl8: l/16:0); Cer(dl8:l/18:0); and Cer(dl8: l/18: l) is lower in the test sample than in the control sample, wherein the test sample and the control sample are blood samples.
  • Detection of the lipid biomarkers described herein in a test sample or a control sample may be performed by any method known in the art.
  • the methods of the invention rely on the detection of the presence or absence of lipid biomarker, or the qualitative or quantitative assessment of either over- or under-production of a lipid biomarker in a population of cells or a tissue in a test sample relative to a standard (for example, a control sample).
  • the level of one or more lipid biomarkers in the test sample and the level of one or more lipid biomarkers in the control sample may be detected by mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, dual polarization interferometry or chromatography.
  • the mass spectrometry is electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, or atmospheric pressure chemical ionization mass spectrometry.
  • the chromatography is thin layer chromatography, solid-phase extraction
  • HPLC high performance liquid chromatography
  • hydrophilic interaction liquid chromatography hydrophilic interaction liquid chromatography
  • ultra-performance liquid chromatography ultra-performance liquid chromatography
  • the lung injury is emphysema or COPD.
  • Example 1 Exposure of mice to cigarette smoke [0110] The study design included 3 groups of C57BL/6 mice: Sham (fresh air- exposed), CS (exposed to mainstream smoke from 3R4F, a reference cigarette from the University of Kentucky and smoking cessation. Animals from the Sham and CS groups were exposed to fresh air and cigarette smoke, respectively, for up to seven months. To model the effects of smoking cessation, animals from the cessation group (CESS) were first exposed to CS for 2 months and then switched to filtered air for 5 additional months.
  • CESS cessation group
  • mice were bred under specific pathogen-free conditions were obtained from Charles River, USA and were 8-10 weeks old at exposure initiation. Mice were individually identified by the subcutaneous implantation of transponders and were housed and whole- body exposed in the animal laboratory under specific pathogen-free conditions. Random allocation of mice to experimental groups was conducted prior to exposure. Animals were fed a standard chow diet (T2914C irradiated rodent diet, Harlan). Filtered tap water was supplied ad libitum and changed daily.
  • NACLAR National Advisory Committee For Laboratory Animal Research
  • mice were observed daily for mortality, morbidity, and signs of injury. Body weight was measured twice per week during the exposure period. The number of animals analyzed for each endpoint is given in Table 1.
  • carboxyhemoglobin (COHb) levels were determined as a marker of smoke uptake at 3, 4 and 6 months.
  • mice were exposed in whole-body exposure chambers to mainstream cigarette smoke (CS) or fresh air (sham) for a period of two, three, or seven months.
  • CS mainstream cigarette smoke
  • sham fresh air
  • CS-uptake was confirmed by monitoring blood carboxyhemoglobin (COHb) at 3 time points and nicotine and cotinine
  • the left lung was instilled with 4% paraformaldehyde in PBS (pH 7.4) and fixed for 24 hours before standard paraffin-embedding, sectioning and staining. Briefly, a small cannula (1.6 mm diameter) was gently introduced into the trachea and the proximal left bronchus and fixed to the latter with a ligature.
  • Fixative was delivered to the left lung by gravity at 15 cm water pressure. After the instillation procedure was completed and the lungs were filled, the cannula was carefully removed and the ligature tightened to prevent fixative flowing out of the lung. The lung was then fixed by immersion in the same fixation solution for 24 hours. Histopathological evaluation was performed at 5 different levels of the lung, displaying the central and peripheral aspects of the parenchyma. For each lung section, a separate evaluation was performed. Mean scores of the 5 data sets were calculated for each animal and for each endpoint. The mean values per animal were used for the exposure group- based analysis.
  • H&E haematoxylin and eosin
  • ABS alcian blue periodic acid schiff s
  • BA resorcin fuchsin
  • Unpigmented and pigmented macrophages are free cells in the alveolar lumen not showing any cytoplasmatic pigmentation or containing fine-granular, brownish to yellow cytoplasmic pigmentation, which may be due to CS particles inhaled, respectively.
  • Pigmented macrophage nests consist of multiple macrophages in the alveolar lumen clustered in small groups within adjacent alveoli.
  • Multinucleated giant cells are very large macrophages (size ranging from 18 to 28 ⁇ ) that are positively stained after AB-PAS incubation (Figure IB), likely due to uptake of excessive amounts of surfactant produced at the alveolar surface in response to CS inhalation.
  • CS-exposure was associated with increased mean chord length (Figure 2C), increased destructive index (defined as the percentage of emphysematous tissue over normal tissue, Figure 2D), and fewer bronchiolar attachments (Figure 2E) relative to sham-exposed animals from month two onwards.
  • Some destruction of the lung tissue could be observed with increasing severity in the sham group, likely due to aging.
  • the damage caused by the initial two month of CS-exposure is not reversible, as the post-cessation mice show disease progression. Progression rate is however slower post-cessation than in continuously exposed mice.
  • Microlab Star robot Gangliosides were extracted according to the method described by Fong et al (Fong et al, 2009, Lipids 44, pp. 867-874) with minor modifications, and eicosanoids were extracted as described by Deems et al (Deems et al, 2007, Methods in Enzymol, 432, pp.59-82).
  • lipid extracts were analyzed using a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 5500) equipped with a robotic nanoflow ion source (NanoMate HD).
  • QTRAP 5500 a hybrid triple quadrupole/linear ion trap mass spectrometer equipped with an ultra-high pressure liquid chromatography (UHPLC) system (CTC HTC PAL autosampler and Rheos Allegro pump) using a multiple reaction monitoring (MRM) -based method in negative ion mode.
  • UHPLC ultra-high pressure liquid chromatography
  • MRM multiple reaction monitoring
  • the mass spectrometry data files were processed using LipidViewTM VI .1 and MultiQuantTM 2.0 Software (Ab Sciex, Massachusetts, USA) to generate a list of lipid names and peak areas. Lipids were normalized to their respective internal standard and the tissue weight. The concentrations of molecular lipids are presented as nmol/mg wet tissue for lung samples. For statistical analysis, a Wilcoxon rank-sum test was conducted for each lipid for comparing the study groups. Monte-Carlo estimation of exact p-values was performed. Multiple testing was controlled with FDR q-values.
  • PC phosphatidylcholine
  • PC-0 and PC-P PC plasmalogen
  • PG phosphatidylglycerol
  • PE phosphatidylethanolamine
  • PC( 16:0/16: 1) Figure 5 A
  • the median concentration difference for these species was up to 240% (p ⁇ 0.01), while in the majority of PC molecules little or no change in concentration in response to CS exposure was observed throughout the experiment.
  • the upregulation of PGs was driven mainly by minor species PG(18:1/18: 1), PG(18:1/18:2) and PG(18:2/18:2), upregulated up to 400%
  • LacCer demonstrated a similar pattern as other ceramides, namely that long-chain Gb3 species are most significantly upregulated (Figure 12).
  • Smoke exposure elevated the levels of saturated/mono-unsaturated cholesterol esters in the lung, while levels of poly-unsaturated species were downregulated (Figure 13).
  • Polyunsaturated cholesterol ester species, especially CE20:4 and CE22:5, in the plasma were also down-regulated by cigarette smoke ( Figure 14).
  • the study used a parallel-group, case-controlled study design in order to determine the differential expression of molecular and physiological biomarkers in subjects with COPD (COPD) when compared to healthy (no COPD) current smokers (CS), healthy (no COPD) former smokers (FS) and healthy never-smokers (NS).
  • COPD COPD
  • CS healthy current smokers
  • FS healthy (no COPD) former smokers
  • NS healthy never-smokers
  • AEs Adverse Events
  • concomitant medication details were also recorded on an ongoing basis in the subjects' eCRF.
  • the inclusion and exclusion criteria for this study can be found in Table 4 at www.clinicaltrials.gov using identifier NCT01780298.
  • Subjects attended the center for visit 3 within 3 to 14 days after visit 2. Eligibility was reassessed against the inclusion/exclusion criteria prior to any other procedures and subjects underwent the procedures as indicated in Table 4. Subjects attended the study center for visit 4 within 3 to 14 days after visit 3 and underwent the procedures as described in Figure 15. A follow-up telephone call was made to subjects within 3 to 10 days after visit 4 to record adverse events (AEs) and concomitant medication details and give smoking cessation advice to current smokers. Table 4. Demographics, smoking history, spirometric parameters and cardiorespiratory vital signs across the study's evaluable population. Data are presented as median ran e .
  • BMI Body mass index
  • FEV1 forced expiratory volume in 1 second
  • Lipids were extracted using a modified Folch lipid extraction procedure (Ekroos, K. (2008). Unraveling Glycerophospholipidomes by Lipidomics. (F. Wang, Ed.) (pp. 369-384). Totowa, NJ: Humana Press) performed on a Hamilton Microlab Star robot. Extract samples were spiked with known amounts of non- endogenous synthetic internal standards.
  • lipid extracts were analyzed using a QTRAP 5500 hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems) equipped with a robotic nanoflow ion source (NanoMate HD, Advion Biosciences) according to Stahlman and colleagues (Stahlman M, Ejsing CS, Tarasov K, Perman J, Boren J, Ekroos K (2009) High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry.
  • QTRAP 5500 hybrid triple quadrupole/linear ion trap mass spectrometer Applied Biosystems
  • SM sphingomyelins
  • DAG diradylglycerols
  • TAG triacylglycerols
  • Targeted eicosanoid and sphingolipid lipidomics were performed using UHPLC (CTC Analytics AG)-coupled QTRAP 5500 mass spectrometry using multiple reaction monitoring (MRM) in positive ion mode.
  • the MS data files were processed using LipidViewTM 1.1 (AB Sciex) or MultiQuantTM 2.0 (AB Sciex) for generating a list of lipid names and peak areas. Lipids were normalized to their respective internal standard (the peak area of the endogenous lipid was divided by the peak area of the corresponding internal standard) and sample volume, yielding concentrations of molecular lipids in ⁇ .
  • the differences and relative differences between the groups were estimated using Hodges-Lehmann estimator (the median value of the cross-pairwise differences between individuals of the two groups). A rank- sum Wilcoxon test was performed to calculate p values. Differences with a p value below 0.05 were considered statistically significant.
  • lipidomics results at the lipid class level are summarized in Figure 16 and indicate small but significant increases in serum diradylglycerols (DAG), neutral glycosphingolipids (LacCer), glycerophospholipids including glycerophosphocholines (PC) and
  • PE glycerophosphoethanolamines
  • TAG triglycerides
  • glycerophosphatidylcholine molecules were generally higher in the serum of current smokers compared to that of never-smokers resulting in an overall up- regulation of this lipid class in disease-free smokers. While not significantly different, serum levels of PC(18:0/20:5) increased noticeably in former smokers relative to current smokers, while the levels of all other members of this lipid class affected by smoking decreased following smoking cessation and mostly returned to levels observed in never-smokers ( Figure 16 and Figure 19).
  • Serum TAG levels were also significantly higher in current compared to never-smokers (Figure 16). This was mainly due to a concerted increase in palmitoleic acid- and oleic acid-containing glycerol triesters in the serum of current smokers ( Figure 20). However, with the exception of the stearic acid ester
  • TAG(52: 1) serum TAG levels exhibited no significant differences when comparing current with former and former with never-smokers, respectively, indicating that potential smoking-related changes in TAG profiles may be reversible upon smoking cessation.
  • Glc/GalCer(dl8:l/18:0) and Glc/GalCer(dl 8: 1/24: 1) were also significantly elevated in the serum of current relative to never-smokers. With the exception of Glc/GalCer(dl8: l/16:0) and Glc/GalCer(dl8: l/18:0), most of Glc/GalCer(dl8: l/16:0) and Glc/GalCer(dl8: l/18:0), most
  • PE glycerophosphoethanolamines
  • two - PE(16:0/20:4) and PE(18: 1/18: 1) - were significantly elevated in the serum of current smokers relative to never-smokers ( Figure 16 and Figure 23). While levels of both molecules were decreased in former smokers compared to current smokers, only the difference in serum PE(16:0/20:4) levels was found to be significant.
  • serum levels of all affected PE species in former smokers were very similar to those seen in never-smokers, suggesting that the effect of cigarette smoke exposure may be reversible upon cessation.
  • SM(dl 8: 1/24: 1) did not, potentially reflecting irreversible smoking-related effects on the serum lipidome.
  • Lipidomics analysis in serum was also performed to examine the effects of the development of mild COPD (GOLD stages I and II; COPD vs. CS, COPD vs. NS). Lipidomics results at the lipid class level are summarized in Figure 28 and indicate small but significant decreases in serum sterols (CE) and
  • SM glycosphingophospho lipids

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Abstract

La présente invention concerne des biomarqueurs lipidiques qui sont utiles pour diagnostiquer, classifier et pronostiquer une lésion pulmonaire, telle qu'un emphysème. L'invention concerne en outre des procédés de diagnostic utilisant ces biomarqueurs lipidiques. L'invention concerne en outre des gènes de traitement de lipides qui sont utiles pour traiter, diagnostiquer, classifier et pronostiquer une lésion pulmonaire, telle qu'un emphysème. L'invention concerne en outre des procédés thérapeutiques par inhibition de ces gènes ou des protéines pour lesquels ils codent.
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