Methods for diagnosing a colorectal cancer (crc) health state or change in crc health state, or for diagnosing risk of developing crc or the presence of crc in a subject

Abstract

The present invention relates to the diagnosis of colorectal and ovarian cancers (CRC and OC, respectively). The present invention describes the relationship between endogenous small molecules and CRC or OC. Specifically, the present invention relates to the diagnosis of CRC and OC through the measurement of vitamin E isoforms and related metabolites. The present invention also relates to diagnostic markers identified in said method. The present invention relates to the underlying case and pre-symptomatic phases of CRC, the diagnosis of various stages and severity of CRC, the early detection of CRC, monitoring and diagnosing the effect of therapy on CRC and OC health states.

1 . (canceled) 
     
     
         2 . An analytical method for diagnosing a patient's colorectal cancer (CRC) health state, or change in CRC health state, or for diagnosing risk of CRC or the presence of CRC in a patient, comprising the steps of:
 a) performing a mass spectrometry assay on at least one blood sample from said patient using a Fourier transform ion cyclotron resonance, time of flight, magnetic sector, quadrupole or triple quadrupole mass spectrometer to obtain a collision induced dissociation (CID) MS/MS fragmentation pattern for one or more than one metabolite marker and to obtain quantifying data for said one or more than one metabolite marker;   b) generating a result of said mass spectrometry assay, said result comprising at least the presence or absence of a decrease in the level of said one or more than one metabolite marker in said blood sample based on a comparison of said quantifying data for said one or more than one metabolite marker to corresponding data obtained for one or more than one reference blood sample; and   c) assigning the patient as having or not having colorectal cancer (CRC), or having or not having a change in CRC health state, or having or not having a risk of CRC,   wherein the one or more than one metabolite marker comprises one or more molecule selected from the molecular formulae consisting of: C2H 50 O 4 , C2H 50 O 5 , C 28 H 52 O 5 , C 32 H 58 O 6 , C 36 H 64 O 6  and C 36 H 66 O 6 ,   the metabolite having the molecular formula of C 28 H 50 O 4  being characterized by a CID MS/MS fragmentation pattern using N 2  as collision gas and analyzed under negative ionization comprising the following daughter ions: 449, 405, 431, 417, 413, 399, 387, 371, 281, and 277,   the metabolite having the molecular formula of C 28 H 50 O 5  being characterized by a CID MS/MS fragmentation pattern using N 2  as collision gas and analyzed under negative ionization comprising the following daughter ions: 465, 403, 447, 433, 421, 405, 349, 297, 279, 241, 223, and 185,   the metabolite having the molecular formula of C 28 H 52 O 5  being characterized by a CID MS/MS fragmentation pattern using N 2  as collision gas and analyzed under negative ionization comprising the following daughter ions: 467, 423, 449, 431, 405, 389, 297, 279, 263, 215, 187, 169, and 141,   the metabolite having the molecular formula of C 32 H 58 O 6  being characterized by a CID MS/MS fragmentation pattern using N 2  as collision gas and analyzed under negative ionization comprising the following daughter ions: 537, 519, 501, 493, 491, 475, 459, 315, 297, 285, 281, 239, and 237,   the metabolite having the molecular formula of C 36 H 64 O 6  being characterized by a CID MS/MS fragmentation pattern using N 2  as collision gas and analyzed under negative ionization comprising the following daughter ions: 591, 573, 559, 556, 527, 415, 313, 297, 295, 175, 149, 115, 113, 95, and 59, and   the metabolite having the molecular formula of C 36 H 66 O 6  being characterized by a CID MS/MS fragmentation pattern using N 2  as collision gas and analyzed under negative ionization comprising the following daughter ions: 593, 575, 557, 550, 532, 529, 515, 315, 313, 297, 277, 215, 201, and 171,   wherein a decrease in the level of said one or more than one metabolite marker in the blood sample from the patient relative to a reference blood sample indicates that the patient has CRC, a change in CRC, or is at risk of CRC.   
     
     
         3 . The method of  claim 2 , wherein the mass spectrometer is equipped with a chromatographic system. 
     
     
         4 . The method of  claim 2 , wherein the blood sample is a whole blood sample, a subfraction of whole blood, a blood serum sample, or a blood plasma sample. 
     
     
         5 . The method of  claim 2 , wherein a liquid/liquid extraction is performed on the sample whereby non-polar metabolites are dissolved in an organic solvent and polar metabolites are dissolved in an aqueous solvent. 
     
     
         6 . The method of  claim 5 , wherein the extracted samples are analyzed by positive or negative electrospray ionization, positive or negative atmospheric pressure chemical ionization, or combinations thereof. 
     
     
         7 . The method of  claim 5 , wherein the extracted samples are analyzed by MS/MS transition. 
     
     
         8 . The method of  claim 5 , wherein the extracted samples are analyzed by extracted ion current (EIC) chromatography and MS/MS transition. 
     
     
         9 . The method of  claim 2 , wherein said one or more than one reference sample is from one or more CRC-negative humans. 
     
     
         10 . The method of  claim 2 , further comprising
 performing a mass spectrometry assay on a blood sample from said patient to obtain quantifying data for one or more than one internal standard molecule; and   obtaining a ratio for each of the levels of said one or more than one metabolite marker to the level obtained for the one or more than one internal standard molecule;   wherein step (b) comprises generating said result based on a comparison of each ratio to one or more corresponding ratios obtained for the one or more than one reference blood sample.   
     
     
         11 . The method of  claim 7 , wherein at least one MS/MS transition is identified for the metabolite having the molecular formula of C 28 H 50 O 4 , said at least one MS/MS transition being selected from the group consisting of 449.4/405.4, 449.4/431.4, 449.4/417.3, 449.4/387.4, 449.4/371.3, 449.4/281.3, 449.4/277.3 and combinations thereof. 
     
     
         12 . The method of  claim 7 , wherein the MS/MS transition for the metabolite having the molecular formula of C 2 H 50 O 5  is 465.4/403.4. 
     
     
         13 . The method of  claim 7 , wherein at least one MS/MS transition is identified for the metabolite having the molecular formula of C 28 H 52 O 5 , said at least one MS/MS transition being selected from the group consisting of 467.4/423.4, 467.4/449.4, 467.4/481.4, 467.4/405.4, 467.4/389.4, 467.4/297.3, 467.4/279.3, 467.4/263.3, 467.4/215.2, 467.4/187.16, 467.4/169.2, 467.4/141.2 and combinations thereof. 
     
     
         14 . The method of  claim 7 , wherein at least one MS/MS transition is identified for the metabolite having the molecular formula of C 32 H 58 O 6 , said at least one MS/MS transition being selected from the group consisting of 537.5/519.5, 537.5/501.5, 537.5/491.4, 537.5/457.5, 537.5/459.4, 537.5/315.3, 537.5/297.3, 537.5/285.1, 537.5/239.2, 537.5/237.2, 537.5/475.5 and combinations thereof. 
     
     
         15 . The method of  claim 7 , wherein at least one MS/MS transition is identified for the metabolite having the molecular formula of C 36 H 64 O 6 , said at least one MS/MS transition being selected from the group consisting of 591.5/573.5, 591.5/559.5, 591.5/555.5, 591.5/527.4, 591.5/415.4, 591.5/313.3, 591.5/297.3, 591.5/295.3, 591.5/175.1, 591.5/149.1, 591.5/113.0, 591.5/115.0 and combinations thereof. 
     
     
         16 . The method of  claim 7 , wherein at least one MS/MS transition is identified for the metabolite having the molecular formula of C 36 H 66 O 6 , said at least one MS/MS transition being selected from the group consisting of 593.6/575.6, 593.6/557.5, 593.6/549.5, 593.6/531.5, 593.6/529.4, 593.6/515.4, 593.6/315.3, 593.6/313.3, 593.6/297.3, 593.6/277.3, 593.6/215.1, 593.6/201.1, 593.6/171.1 and combinations thereof. 
     
     
         17 . The method of  claim 10 , wherein the internal standard molecule is cholic acid. 
     
     
         18 . An analytical method for diagnosing a patient's colorectal cancer (CRC) health state, or change in CRC health state, or for diagnosing risk of CRC or the presence of CRC in a patient, comprising the steps of:
 a) performing a mass spectrometry assay on at least one blood sample from said patient using a Fourier transform ion cyclotron resonance, time of flight, magnetic sector, quadrupole or triple quadrupole mass spectrometer to obtain a collision induced dissociation (CID) MS/MS fragmentation pattern for one or more than one metabolite marker and to obtain quantifying data for said one or more than one metabolite marker;   b) generating a result of said mass spectrometry assay, said result comprising at least the presence or absence of a decrease in the level of said one or more than one metabolite marker in said blood sample based on a comparison of said quantifying data for said one or more than one metabolite marker to corresponding data obtained for one or more than one reference blood sample; and   c) assigning the patient as having or not having colorectal cancer (CRC), or having or not having a change in CRC health state, or having or not having a risk of CRC,   wherein the one or more than one metabolite marker comprises one or more molecule selected from the molecular formulae consisting of: C 2 H 48 O 4  and C 28 H 48 O 5      the metabolite having the molecular formula of C 28 H 48 O 4  being characterized by a CID MS/MS fragmentation pattern using N 2  as collision gas and analyzed under negative ionization comprising the following daughter ions: 447, 385, 429, 403, and 279,   the metabolite having the molecular formula of C 28 H 48 O 5  being characterized by a CID MS/MS fragmentation pattern using N 2  as collision gas and analyzed under negative ionization comprising the following daughter ions: 463, 419, 445, 401, 383, 315, 297, and 241,   wherein a decrease in the level of said one or more than one metabolite marker in the blood sample from the patient relative to a reference blood sample indicates that the patient has CRC, a change in CRC, or is at risk of CRC.   
     
     
         19 . The method of  claim 18 , wherein the extracted samples are analyzed by MS/MS transition. 
     
     
         20 . The method of  claim 18 , wherein at least one MS/MS transition is identified for the metabolite having the molecular formula of C 28 H 48 O 4 , said at least one MS/MS transition being selected from the group consisting of 447.4/385.4, 447.4/429.4, 447.4/403.4, 447.4/279.2 and combinations thereof. 
     
     
         21 . The method of  claim 18 , wherein at least one MS/MS transition is identified for the metabolite having the molecular formula of C 28 H 48 O 5 , said at least one MS/MS transition being selected from the group consisting of 463.4/419.4, 463.4/445.3, 463.4/401.3, 463.4/383.3, 463.4/315.3, 463.4/297.3, 463.4/241.2, 463.4/223.2 and combinations thereof. 
     
     
         22 . An analytical method for detecting a colorectal cancer (CRC) disease marker, comprising the steps of:
 providing at least one blood sample from a patient; and   performing a mass spectrometry assay on the at least one blood sample using a Fourier transform ion cyclotron resonance, time of flight, magnetic sector, quadrupole or triple quadrupole mass spectrometer to obtain a collision induced dissociation (CID) MS/MS fragmentation pattern for one or more than one metabolite marker and to obtain quantifying data for said one or more than one metabolite marker;   wherein the one or more than one metabolite marker comprises one or more molecule selected from the molecular formulae consisting of: C 28 H 50 O 4 , C 28 H 50 O 5 , C 28 H 52 O 5 , C 32 H 58 O 6 , C 36 H 64 O 6  and C 36 H 66 O 6 ,   the metabolite having the molecular formula of C 28 H 50 O 4  being characterized by a CID MS/MS fragmentation pattern using N 2  as collision gas and analyzed under negative ionization comprising the following daughter ions: 449, 405, 431, 417, 413, 399, 387, 371, 281, and 277,   the metabolite having the molecular formula of C 28 H 50 O 5  being characterized by a CID MS/MS fragmentation pattern using N 2  as collision gas and analyzed under negative ionization comprising the following daughter ions: 465, 403, 447, 433, 421, 405, 349, 297, 279, 241, 223, and 185,   the metabolite having the molecular formula of C 28 H 52 O 5  being characterized by a CID MS/MS fragmentation pattern using N 2  as collision gas and analyzed under negative ionization comprising the following daughter ions: 467, 423, 449, 431, 405, 389, 297, 279, 263, 215, 187, 169, and 141,   the metabolite having the molecular formula of C 32 H 58 O 6  being characterized by a CID MS/MS fragmentation pattern using N 2  as collision gas and analyzed under negative ionization comprising the following daughter ions: 537, 519, 501, 493, 491, 475, 459, 315, 297, 285, 281, 239, and 237,   the metabolite having the molecular formula of C 36 H 64 O 6  being characterized by a CID MS/MS fragmentation pattern using N 2  as collision gas and analyzed under negative ionization comprising the following daughter ions: 591, 573, 559, 556, 527, 415, 313, 297, 295, 175, 149, 115, 113, 95, and 59, and   the metabolite having the molecular formula of C 36 H 66 O 6  being characterized by a CID MS/MS fragmentation pattern using N 2  as collision gas and analyzed under negative ionization comprising the following daughter ions: 593, 575, 557, 550, 532, 529, 515, 315, 313, 297, 277, 215, 201, and 171.