Compositions, methods and kits for diagnosis of lung cancer
The present invention provides methods for identifying biomarker proteins that exhibit differential expression in subjects with a first lung condition versus healthy subjects or subjects with a second lung condition. The present invention also provides compositions comprising these biomarker proteins and methods of using these biomarker proteins or panels thereof to diagnose, classify, and monitor various lung conditions. The methods and compositions provided herein may be used to diagnose or classify a subject as having lung cancer or a non-cancerous condition, and to distinguish between different types of cancer (e.g., malignant versus benign, SCLC versus NSCLC).
1. A method of scoring a pulmonary nodule in a subject comprising:
(a) assaying the expression of a plurality of proteins comprising determining the protein expression level of at least each of ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN and COIA1_HUMAN from a biological sample obtained from the subject by selected reaction monitoring mass spectrometry of proteolytically digested fragments of the plurality of proteins contacted to labeled synthetic peptide fragments, wherein the determining step comprises detecting peptide transitions comprising at least ALQASALK (SEQ ID NO:25) (401.25, 617.4), AVGLAGTFR (SEQ ID NO:26) (446.26, 721.4), GFLLLASLR (SEQ ID NO:27) (495.31, 559.4), LGGPEAGLGEYLFER (SEQ ID NO:28) (804.4, 1083.6), and VEIFYR (SEQ ID NO:29) (413.73, 598.3);
(b) calculating a score from the protein expression of at least each of ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1 HUMAN and COIA1_HUMAN from the biological sample determined in step (a);
wherein said score is determined as P s =1/[1+exp(−α−Σ i=1 5 β i *{hacek over (I)} i,s −γ*{hacek over (I)} COIA1 *{hacek over (I)} FRIL )], where {hacek over (I)} i,s is Box-Cox transformed and normalized intensity of transition i in said sample (s), β i is the corresponding logistic regression coefficient, α is a panel-specific constant, and γ is a coefficient for the interaction term.
2. The method of claim 1 , wherein the subject has a lung condition.
3. The method of claim 1 , wherein the pulmonary nodule is 30 mm or less.
4. The method of claim 3 , wherein the pulmonary nodule is between 8-30 mm.
5. The method of claim 2 , wherein said lung condition is cancer or a non-cancerous lung condition.
6. The method of claim 2 , wherein said lung condition is non-small cell lung cancer.
7. The method of claim 5 , wherein said non-cancerous lung condition is chronic obstructive pulmonary disease, hamartoma, fibroma, neurofibroma, granuloma, sarcoidosis, bacterial infection or fungal infection.
8. The method of claim 1 , wherein the subject is a human.
9. The method of claim 1 , wherein said biological sample is tissue, blood, plasma, serum, whole blood, urine, saliva, genital secretions, cerebrospinal fluid, sweat, excreta, or bronchoalveolar lavage.
10. The method of claim 1 , wherein assaying the expression of a plurality of proteins further comprises determining the protein expression level of at least one of PEDF_HUMAN, MASP1_HUMAN, GELS_HUMAN, LUM_HUMAN, C163A_HUMAN and PTPRJ_HUMAN.
11. The method of claim 1 , wherein determining the protein expression level of at least each of ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN and COIA1_HUMAN comprises fragmenting each protein to generate at least one peptide.
12. The method of claim 11 , wherein the proteins are fragmented by trypsin digestion.
13. The method of claim 12 , further comprising providing a synthetic, modified, heavy peptides corresponding to each peptide generated from each of ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN and COIA1_HUMAN.
14. The method of claim 13 , wherein at least one of the synthetic peptides has an isotopic label attached.
15. The method of claim 1 , wherein the expression of a plurality of proteins is performed by liquid chromatography-selected reaction monitoring/mass spectrometry (LC-SRM-MS).
16. The method of claim 11 , wherein a transition for each peptide is determined by liquid chromatography-selected reaction monitoring/mass spectrometry (LC-SRM-MS).
17. A method of determining that a pulmonary nodule in a subject is not lung cancer, comprising:
(a) contacting a biological sample obtained from the subject with a proteolytic enzyme to produce peptide fragments from a panel of proteins present in the biological sample, wherein the panel comprises ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN and COIA1_HUMAN;
(b) combining the produced peptide fragments from the panel from step (a) with labeled, synthetic peptide fragments which correspond to the produced peptide fragments from the panel;
(c) performing selected reaction monitoring mass spectrometry to measure the abundance of the peptide fragments from step (b), wherein the measuring step comprises detecting the peptide transitions comprising at least ALQASALK (SEQ ID NO:25) (401.25, 617.4), AVGLAGTFR (SEQ ID NO:26) (446.26, 721.4), GFLLLASLR (SEQ ID NO:27) (495.31, 559.4), LGGPEAGLGEYLFER (SEQ ID NO:28) (804.4, 1083.6), and VEIFYR (SEQ ID NO:29) (413.73, 598.3);
(d) calculating a score based on the peptide fragment measurements of step (c) and one protein-protein interaction comprising peptide fragments produced from FRIL_HUMAN and COIA1_HUMAN, wherein said score is determined as P s =1/[1+exp(−α−Σ i=1 5 β i *{hacek over (I)} i,s −γ*{hacek over (I)} COIA1 *{hacek over (I)} FRIL )], where {hacek over (I)} i,s is Box-Cox transformed and normalized intensity of transition i in said sample (s), β i is the corresponding logistic regression coefficient, α is a panel-specific constant, and γ is a coefficient for the interaction term; and
(e) ruling out lung cancer for the human subject if the score in step (d) is closer to 0.0 than to 1.0.
18. The method of claim 17 , further comprising:
(f) Performing image monitoring on the subject for at least a one-year period if the pulmonary nodule is determined not to be lung cancer.